RECYCLING NURSERY RUNOFF: UNDERSTANDING PLANT SENSITIVITY TO NU TRIENTS AND RESIDUAL PESTICIDES By Shital Poudyal A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Horticulture Doctor of Philosophy 2020 ABSTRACT RECYCLING NURSERY RUNOFF: UNDERSTANDING PLANT SENSITIVITY TO NUTRIENTS AND RESIDUAL PESTICIDES By Shital Poudyal Runoff g enerated from landscape nursery operations contains agrochemicals such as pesticides and fertilizers, which, if released off - site, may pollute the environment. Nursery producers are increasingly interested in alternatives to using freshwater for irrigation due to i ncreased environmental awareness and reduced water availability. As a result, some progressive nursery growers are already adopting the practice of retaining and recycling nursery runoff water for irrigation . While retaining and recycling runoff m ay be a practical solution, growers' concerns about the potential negative impact of residual pesticides on crop growth and quality still impede its adoption. Therefore the objectives of my studies were to reduce the concentration of nutrients in runoff wa ter and to evaluate the impact of irrigating with recycl ed runoff water on growth and physiology of nursery crops . The first study was to identify minimum phosphorus concentration required for the optimum morphological and physiological performance in thre e common woody ornamental taxa; Hydrangea quercifolia (Queen of hearts), Cornus obliqua (Redtwig dogwood) and Physocarpus opulifolius (Seward). The optimum phosphorus concentrations for growth and photosynthetic biochemistry ranged between 4 and 7 mg·L - 1 , depending on taxa. For the second study, I investigated the response of com mon landscape nursery plants to residual pesticide commonly found in nursery runoff. H ydrangea paniculata (Limelight), Cornus obliqua (Powell Gardens) , Hosta (Gold Standard) were exposed to low residual concentrations of isoxaben, chlorpyrifos and oxyfluorfen, simulating irrigation with nursery runoff. Exposure to o xyfluorfen produced phytotoxicity symptoms (visual leaf damage), while chlorpyri fos and isoxaben did not produce phyto toxicity. Among the three taxa, H. paniculata was the most sensitive species, and C. obliqua was the most resistant. Therefore the effects of pesticides were pesticide - specific and taxa - specific. For the third study, I investigated whether phytotoxicity in response to residual herbicide exposure was dependent on the growth stage of plants. In this study, H. paniculata plants were exposed to a low residual concentration of oryzalin and oxyfluorfen at the various growth stages , starting shorty after bud - break . Residual herbicide exposure had more impact on growth and photosynthetic physiology at early growth stages ; however, the recovery rate of those plants was also rapid. For my final study, I conducted three - year field research replicating an actual nursery grower practice of recycling nursery water. S ix ornamental species were irrigated with recycled water obtained from a nursery bed receiving ten different pesticides. In addition, the efficacy of woodchip bioreactors to reduced pesticides in water was also tested. Results from this study established the possibility of using recycled water to irrigate ornamentals plants such as Hydrangea macrophylla (Let's dance blue jangles), Hydrangea paniculata (Limelight), Thuja occidentalis (American Pillar), Juniperus horizontalis (Blue rug), Hydrangea arborescens (Invincibelle Spirit II®) and Rosa sp . (Oso Easy Double Red®) without impacting the growth and physiology of th ose plants. W oodchips bioreactor was also found to be effective in remediating pesticides from wat er. The result s of three greenhouse studies and a field study together provide new information on reducing the concentration of nutrients and pesticides in nursery runoff water and demonstrate the possibility of recycling nursery runoff. The findings of th is dissertation are vital in solving the emerging problem of agrochemical pollution and water scarcity that is currently faced by nursery growers. iv Dedicated to Suzzen for her relentless support and to my altruistic mom v ACKNOWLEDGEMENTS I am deeply thankful to Dr. Bert Cregg for his mentorship, love, and support throughout my Ph.D. program and beyond. I am lucky to have someone like you as an advisor. I am also thankful to my committee members Drs. Tom Fernandez, Dr. Jim Owen, and Dr. Thomas Sharkey, who significantly contributed to my personal and professional development. Thank you, Dr. Damon Abdi, Dana Ellision, Dan Kort, and Deborah Trelstad, it was awes ome working with you guys . Also, thank you to the entire team of CleanwateR3. I learned a lot. It is a challenge working with water lines at >100 PSI; thank you to the entir e team at Hort Farm for reducing pressure to create a working environment, both for water lines and for me. I would also like to thank all the faculty members, staff, and friends from the Department of Horticulture who directly and indirectly help me during every step of my program. I would also like to thank my funding agencies USDA - SCR I, project GREEEN, and Michigan Department of Agriculture and Rural Dev elop ment. my wife, Suzzen, for the sacrifices she had to make. I am indebted to you. I also thank my mom for believing in me and all my family members for their support. I am also grateful to Ujjwal Karki, Victor Karthik, Rajiv Paudel, Anurag Dawadi, the entire Nepalese community, and MSU OISS for making me feel at home. Finally, I would l ike to thank everyone who helped me complete this journey of my life. I will always remember you. vi TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ......................... ix LIST OF FIGURES ................................ ................................ ................................ ....................... xi SECTION I ................................ ................................ ................................ ................................ ..... 1 IRRIGATING NURSERY CROPS WITH RECYCLED RUN - OFF: A REVIEW OF POTENTIAL IMPACTS OF PESTICIDE S ON PLANT GROWTH AND PHYSIOLOGY (Literature review) ................................ ................................ ................................ ....................... 1 Abstract ................................ ................................ ................................ ................................ ....... 3 1. Introduction ................................ ................................ ................................ ............................. 4 1.1. Increase interest in water capture and reuse in nurseries ................................ .................. 5 1.2. Examples of water capture and recycle in nurseries ................................ ......................... 6 1.3. Concerns about crop safety ................................ ................................ ............................... 7 2. Pesticides used in nursery production and potential for crop damage ................................ .... 9 2.1. Herbicides ................................ ................................ ................................ ....................... 10 2.2. Insecticides ................................ ................................ ................................ ..................... 10 2.3. Fungicides ................................ ................................ ................................ ....................... 12 3. Properties of p esticides that may affect phytotoxicity of runoff ................................ ........... 12 3.1. Solubility ................................ ................................ ................................ ........................ 13 3.2. Adsorption ................................ ................................ ................................ ...................... 13 3.3. Persistence ................................ ................................ ................................ ...................... 14 3.4. Volatility ................................ ................................ ................................ ......................... 14 4. Factors affecting the potential for crop injury ................................ ................................ ....... 15 4.1. Pesticide ................................ ................................ ................................ .......................... 15 4.2. Plant sensitivity ................................ ................................ ................................ ............... 16 4.3. Pesticide dose and exposure ................................ ................................ ........................... 17 4.4. Growth stages and plant parts ................................ ................................ ......................... 18 5. Mitigating risks from residual pesticides in recycled runoff ................................ ................. 19 5.1. Pesticide dependent reduction ................................ ................................ ........................ 19 5.2. Constructed wetlands and vegetative buffer ................................ ................................ ... 20 5.3. Sand Filters ................................ ................................ ................................ ..................... 21 5.4. Activated carbon filters (ACF) and filter socks ................................ .............................. 21 6. Conclusion ................................ ................................ ................................ ............................. 22 APPENDIX ................................ ................................ ................................ ............................... 24 LITERATURE CITED ................................ ................................ ................................ ............. 29 SECTION II ................................ ................................ ................................ ................................ .. 42 PHOSPHORUS REQUIREMENT FOR BIOMASS ACCUMULATION IS HIGHER COMPARED TO PHOTOSYNTHETIC BIOCHEMISTRY FOR THREE ORNAMENTAL SHRUBS ................................ ................................ ................................ ................................ ... 42 Abstract ................................ ................................ ................................ ................................ ..... 44 1. Introduction ................................ ................................ ................................ .......................... 46 2. Materials and Methods ................................ ................................ ................................ .......... 49 vii 2.1. Exp erimental Setup ................................ ................................ ................................ ......... 49 2.2. Phosphorus treatments ................................ ................................ ................................ .... 50 2.3. Growth measurements ................................ ................................ ................................ .... 50 2.4. Phosphorus partitioning ................................ ................................ ................................ .. 51 2.5. Physiological measurements ................................ ................................ ........................... 52 2.6. Statistical analysis ................................ ................................ ................................ ........... 53 3. Results ................................ ................................ ................................ ................................ ... 54 3.1. Morphological res ponse to phosphorus concentration ................................ ................... 54 3.2. Partitioning of applied phosphorus ................................ ................................ ................. 55 3.3. Photosynthetic response to phosphorus concentration ................................ ................... 56 3.4. Correlation among morpho - physiological variables ................................ ...................... 57 4. Discussion ................................ ................................ ................................ ............................. 58 4.1. Morphological response to phosphorus concentration ................................ ................... 58 4.2. Fate of applied phosphorus ................................ ................................ ............................. 59 4.3 Physiological performance in response to phosphorus concentration ............................. 59 5. Conclusion ................................ ................................ ................................ ............................. 61 APPENDIX ................................ ................................ ................................ ............................... 63 LITERATURE CITED ................................ ................................ ................................ ............. 76 SECTION III ................................ ................................ ................................ ................................ . 82 DOSE - DEPENDENT PHYTOTOXICITY OF PESTICIDES IN SIMULATED NURSERY RUNOFF ON LANDSCAPE NURSERY PLANTS ................................ ................................ 82 Abstract ................................ ................................ ................................ ................................ ..... 84 1. Introduction ................................ ................................ ................................ ........................... 85 2. Materials and Methods ................................ ................................ ................................ .......... 89 2.1. Plant Material and Treatments ................................ ................................ ........................ 89 2.2. Physiological Measurements and Growth ................................ ................................ ...... 91 2.3. Statistical Analysis ................................ ................................ ................................ ......... 92 3. Resul ts ................................ ................................ ................................ ................................ ... 93 3.1. Leaf Visual Injury and Growth in Response to Pesticide Treatment ............................. 93 3.2. Physiological Performance in Response to Pesticide Treatments ................................ .. 94 3.3. Pesticide Absorption ................................ ................................ ................................ ....... 95 4. Discu ssion ................................ ................................ ................................ ............................. 96 4.1. Growth and Physiology ................................ ................................ ................................ .. 96 4.2. Pesticide Absorption ................................ ................................ ................................ ....... 99 5. Concl usions ................................ ................................ ................................ ......................... 101 APPENDIX ................................ ................................ ................................ ............................. 104 LITERATURE CITED ................................ ................................ ................................ ........... 114 SECTION IV ................................ ................................ ................................ .............................. 120 SENSITIVITY OF HYDRANGEA TO RESIDUAL HERBICIDES IN RECYCLED IRRIGATION VARIES WITH PLANT GROWTH STAGE ................................ ................ 120 Abstract ................................ ................................ ................................ ................................ ... 122 1. Introduction ................................ ................................ ................................ ......................... 124 2. Materials and methods ................................ ................................ ................................ ........ 128 2.1. Plant material and treatments ................................ ................................ ....................... 128 viii 2.2. Assessment of physiological and morphological effect of herbicide ........................... 130 2.3. Statistical analysis ................................ ................................ ................................ ......... 132 3. Results ................................ ................................ ................................ ................................ . 133 3.1. Morphological responses to herbicide exposure ................................ .......................... 133 3.2. Physiological responses to herbicide exposure ................................ ............................ 135 3.3. Final evaluation ................................ ................................ ................................ ............ 136 3.4. Evaluation of flowers ................................ ................................ ................................ .... 137 4. Discussion ................................ ................................ ................................ ........................... 137 4.1. Morphological response depends on the growth stage of plant ................................ .... 137 4.2. Physiological measurements provide a rapid indicator of herbicide damage and recovery ................................ ................................ ................................ ................................ ............. 140 4.3. Flowers were not damaged by residual oryzalin and oxyfluorfen ................................ 141 4.4. Leaf visual injury takes the longest to recover ................................ ............................. 142 5. Conclusion ................................ ................................ ................................ ........................... 143 APPENDIX ................................ ................................ ................................ ............................. 14 5 LITERATURE CITED ................................ ................................ ................................ ........... 154 SECTION V ................................ ................................ ................................ ................................ 161 EFFECT OF RESIDUAL PESTICIDES IN RECYCLED NURSERY RUNOFF ON GROWTH AND PHYSIOLOGY OF ORNAMENTAL SHRUBS ................................ ....... 161 Abstract ................................ ................................ ................................ ................................ ... 163 1. Introduction ................................ ................................ ................................ ......................... 164 2. Materials and Methods ................................ ................................ ................................ ........ 168 2.1 Field layout and water treatments ................................ ................................ .................. 168 2.2 Plant evaluation ................................ ................................ ................................ ............. 172 2.3 Pesticide sampling ................................ ................................ ................................ ......... 174 2.4 Statistical analysis ................................ ................................ ................................ .......... 175 3. Resul ts ................................ ................................ ................................ ................................ . 175 3.1. Pesticide concentration in water ................................ ................................ ................... 175 3.2 Plant response to water treatments ................................ ................................ ................ 178 4. Discussion ................................ ................................ ................................ ........................... 181 4.1 Residual pesticide in recycled water varies by compound ................................ ............ 181 4.2 Woodchip bioreactor can reduce pesticide concentration ................................ ............. 182 4.3 Recycled water can be used to irrigate ornamental shrubs ................................ ............ 183 5. Conclusion ................................ ................................ ................................ ........................... 187 APPENDIX ................................ ................................ ................................ ............................. 189 LITERATURE CITED ................................ ................................ ................................ ........... 199 ix LIST OF TABLES Table I - 1. Pesticides detected in irrigation runoff and retention ponds from container nursery production sit es. Reported concentrations are for the maximum amount detected and always occurred during first flush of runoff. ................................ ................................ ............................ 25 Table I - 2. Common herbicides applied in nurseries and their effect on plants .......................... 27 Table II - 1. Root and shoot dry weight (g) of P. opulifolius H. quercifolia C. obliqua separations were carried out using Fisher Least Significant Difference (LSD) post hoc test when appropriate. Means within a taxon that are followed by same letters are not significantly d ifferent at given p values. ................................ ................................ ................................ ........................... 67 Table II - 2. Partitioning of applied phosphorus to leachate, leaf, stem and root, including the amou nt of P stored in the substrate for P. opulifolius H. quercifolia and C. obliqua Difference (LSD) post - hoc test. Means that are followed by same letters are not significantly different given p value ................................ ................................ ................................ .................. 68 Table II - 3. Pearson's correlation coefficient for P. opulifolius , H. quercifolia C. obliqua Leaf size (total leaf area per plant/ leaf number per plant), P% in leaf is phosphorus percent in leaf by weight, V cmax is maximum velocity of rubisco for carboxylation, J is the rate of photosynthetic electron transport for RuBP regeneration, TPU is triose phosphate use and is the light - adapted fluorescence. ................................ ................................ ................................ .................... 74 Table II - 4. Breakdown of micronutrients analysis with elements and concentration. ................ 75 Table III - 1. Mean total dry biomass (g) for Hydrangea paniculata Cornus obliqua Hosta irrigated for three months with simulated runoff containing oxyfluorfen, isoxaben, or chlorpyrifos. Means within a column followed by the same letter for a given taxon are not different at p < 0.05. Post - hoc mean separation was done using the Fisher least significance difference (LSD) test. ................................ ................................ .... 107 Table IV - 1. Flow chart for herbicide exposure. ................................ ................................ ........ 146 Table IV - 2. Total dry above - ground biomass (TDB), leaf visual rating (VR), SPAD index (SPAD), growth index (GI), photosynthes is (A) and light - adapted chlorophyll fluorescence H. paniculata exposed to either oryzalin (8 mg/L) or oxyfluorfen (0.02 mg/L) at various growth stages (GS) for ten days. GS+5 received herbicide exposure five days after initiation of growth, g GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 days af ter initiation of growth. Mean separations for each herbicide were carried out using Least Significant Difference (LSD) x post - hoc test. Means within each herbicide that are followed by the same letters are not significantly different at given p - values. ................................ ................................ .................... 153 Table V 1. Pesticides application rates (express as g a.i./ha), concentration of pesticide solution (expressed as g a.i./L), total amount of solution sprayed (expressed as liter) and pesticide concentration in recycled runoff (RR) water and remediated recycled runoff (RRR) water (expressed as µg/L) water during the three year study period. Each water source was sampled twice after each application. First samples were collected a day after pesticide application and the last sample were collected 10 to 15 days after pesticide application. Samples for pesticide concentration were not co llected in 2017. ................................ ................................ .................. 192 xi LIST OF FIGURES Figure II - 1. Growth index (A) and total dry biomass (B) of P. opulifolius H. quercifolia C. obliqua concentration. Non - linear regression curves (logistic growth curves) are plotted for both GI and TDB. Standard errors of the means are denoted as vertical lines on the curves. .......................... 64 Figure II - 2. Leaf number per plant, leaf size, and total leaf area per plant for P. opulifolius C. obliqua H. quercifolia separations for each taxa were carried out using Least Significant Difference (LSD) post - hoc test. Means within a taxon that are fol lowed by same letters are not significantly different at p=0.05. ................................ ................................ ................................ ................................ ....................... 65 Figure II - 3. Representative plants for each P concentration o f P. opulifolius H. quercifolia C. obliqua - 1 for 6 months in the greenhouse. ................................ ................................ ................................ .......... 66 Figure II - 4. Root - to - Shoot (R:S) ratio of P. opulifolius H. quercifolia and C. obliqua were carried out using Fishe r Least Significant Difference (LSD) post - hoc test and presented as inset table. Means within a taxon indicated by the same letter are not different at the given p value. Standard errors are denoted as vertical lines on the curves. ................................ ......................... 70 Figure II - 5. Response of photosynthesis to increasing internal carbon dioxide concentration (A/Ci Curve) for P. opulifolius H. quercifolia C. obliqua - linear model of rectangular hyperbola. R - squared values for all models for all three taxa were above 0.96. ................................ ................................ ................................ ................................ ....................... 71 Figure II - 6. Maximum velocity of rubisco for carboxylation ( V cmax ) (A); rate of photosynthetic electron transport for RuBP regeneration ( J ) (B), and triose phosphate use ( TPU ) (C) of P. opulifolius H. quercifolia C. obliqua response to phosphorus concentration. Values of A/Ci Curves were analyzed based on equations provide by Sharkey (2016) to generate V cmax , J and TPU for each replication. Fisher Least Significant Difference (LSD) was used to compare means among phosphorus fertilization levels and presented as inset table. Means within a taxon indicated by the same letter are not different a t given p - value. Standard errors are denoted as vertical lines on the curves. ................................ . 72 Figure II - 7. Light - response to increasing phosphorus concentration for P. opulifolius H. quercifolia C. obliqua phosphorus fertilization levels and p resented in as inset table. Means within a taxon indicated by the same letter are not different at given p value. Standard errors are denoted as vertical lines on the curves. ................................ ................................ ................................ ................................ ..... 73 xii Figure III - 1. Mean leaf visual rating of Hydrangea paniculata Cornus obliqua Hosta five concentrations of oxyfluorfen for three months. Visual rating was based on a scale of 1 to 10 (10 = no injury to 1 = dead plant). Means within a taxon followed by the same letter are n ot different at p < 0.05. Mean separation was by the Fisher least significance difference (LSD) test. ................................ ................................ ................................ ................................ ..................... 105 Figure III - 2. Images of application irrigated for three months. ................................ ................................ ........................ 106 Figure III - 3. Mean chlorophyll index (CI) of Hydrangea paniculata Cornus obliqua of oxyfluorfen applied for three months. CI for Hydrangea followed quadratic regression while the CI of Cornus decreased linearly. ................................ ................................ ........................... 108 Figure III - 4. Mean light - Hydrangea paniculata Cornus obliqua Hosta in response to simulated Hydrangea and Cornus both followed quadratic regression, while regression of Hosta was not significant at p < 0.05. ................................ ................................ ................................ ................ 109 Figure III - 5. Carbon dioxide response (A/Ci) curve of Hydrangea paniculata Cornus obliqua Hosta concentrations of oxyfluorfen (Oxy) for three months. ................................ .............................. 110 Figure III - 6. Mean Vcmax (maximum rate of RUBISCO for carboxylation) and J (rate of electron transport for RuBP regeneration) of Hydrangea paniculata Cornus obliqua Hosta runoff containing five concentrations of oxyfluorfen for three months. Means within a taxon followed by the same letter are not different at p < 0.05. Mean separation was by the Fisher least significance difference (LSD) test. ................................ ................................ ................................ ................................ .............. 111 Figure III - 7. Concentration of oxyfluorfen ( A ), isoxaben ( B ) ,and chlorpyrifos ( C ) in leaves for Hydrangea paniculata Cornus obliqua Hosta plants following irrigation with simulated runoff containing five different concentrations of oxyfluorfen, isoxaben, and chlorpyrifos applied for three months. Means within a taxon followed by the same letter are not diff erent at p < 0.05. Post - hoc mean separation was done using the Fisher least significance difference (LSD) test. ................................ ................................ ..................... 112 Figure III - 8. Concentration of oxyfluorfen ( A ), isoxaben ( B ), and chlorpyrifos ( C ) in the stem for Hydrangea paniculata Cornus obliqua oxyfluorfen ( D ), isoxaben ( E ), and chlorpyrifos ( F ) in root for Hydrangea paniculata Cornus obliqua Hosta following irrigation with simulated runoff containing five c oncentrations of oxyfluorfen (Oxy), isoxaben (Iso), and chlorpyrifos (Chl), applied for three months. Means within a taxon followed by the same letter are xiii not different at p < 0.05. Post - hoc mean separation was done using the Fisher least significance diff erence (LSD) test. Bar graphs for treatment are missing when the residual pesticide concentration is very low (zero or close to zero). ................................ ................................ ....... 113 Figure IV - 1. Relative growth index of H. paniculata top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (GS) GS+5 received herbicide exposure five days after initiation of growth, GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 rec eived herbicide exposure 35 separations for each herbicide were carried out using Least Significant Difference (LSD) post - hoc test. Means within each herb icide across all growth stages that are followed by the same letters are not significantly different at p=0.05. ................................ ................................ .......................... 147 Figure IV - 2. Representative herbicide damage immediately after the end of oxyfluorfen exposure (A) and ten days after the end of oryzalin exposure (B). Plants were exposed to oxyfluorfen or oryzalin at growth stage (GS), GS+15 for ten days. Both plants received a score of seven out of ten for leaf visual rating. ................................ ................................ ................................ ............. 148 Figure IV - 3. Leaf visual rating of H. paniculata (8 mg/L; top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (GS) GS+5 received herbicide exposure five days after initiation of growth, GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 separations for each herbicide were carried out using Least Significant Difference (LSD) post - hoc test. Means within each herbicide across all growth stages that are followed by the same letters are not significantly different at p=0.05. ................................ ................................ .......................... 149 Figure IV - 4. Relative SPAD index of H. paniculata top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbic ide exposure at various stages of plant growth. Growth stage (GS) GS+5 received herbicide exposure five days after initiation of growth, GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after i nitiation of growth and GS+35 received herbicide exposure 35 separations for each herbicide were carried out using Least Significant Difference (LSD) post - hoc test. Means within each herbicide across all growth stages that are followed by the same letters are not significantly different at p=0.05. ................................ ................................ .......................... 150 Figure IV - 5. Relative net photosynthesis of H. paniculata mg/L; top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (GS) GS+5 received herbicide exposure five days after initiation of growth, GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 days afte r initiation of growth. Standard errors of the means are denoted by vertical xiv (LSD) post - hoc test. Means within each herbicide across all growth stages that a re followed by the same letters are not significantly different at p=0.05. ................................ ................................ . 151 Figure IV - 6. Percent reduction in light - adapted fluorescence of H. paniculata response oryzalin (8 mg/L; top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (GS) GS+5 received he rbicide exposure five days after initiation of growth, GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 days after initiation of growth. Standard errors of the means are denoted by Difference (LSD) post - hoc test. Means within each herbicide across all growth stages that are followed by the same letters are not significantly different at p=0.05. ................................ ....... 152 Figure V - 1. Lay out of the field study. The plant evaluation bed had four rows and three irrigation zones in each row. Each row had all three water treatment zones that were randomly assigned. Irrigation treatments were water either from raw groundwater (RGW), recycled ru noff (RR) from the collection reservoir or remediation recycled runoff (RRR) from the collection reservoir. Figure is not to the scale. ................................ ................................ ................................ ........................ 190 Fig ure V - 2 . Weekly reference potential evapotranspiration (Weekly ref. PET) and weekly precipitation during the treatment application period at the research site from 2017 to 2019. Source: Michigan State University EnviroWeather: https://mawn.ge o.msu.edu/station.asp?id=msu ..... 191 Figure V - 3 . Growth index of six ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water. Recycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled run off water was the recycled runoff water that passed through a heat - expanded shale and woodchip bioreactor system. Means within a taxon that are followed by same letters are not significantly different at p=0.05. Standard errors of the means are denoted by - hoc test. ................... 193 Figure V - 4 . Shoot weight of six ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water in 2018 and 2019. Recycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that passed through a heat - expanded shale and woodchip bioreactor system . Means within a taxon that are followed by same letters are not significantly different at p=0.05. (LSD) post - hoc test. ................... 194 Figure V - 5 . Leaf weight, stem weight and root weight of six ornamental taxa irrigated with raw groundwa ter (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water in 2018. Recycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the reg ion. Remediated recycled runoff water was the recycled runoff water that passed through a heat - expanded shale and xiv woodchip bioreactor system. Means within a taxon that are followed by same letters are not significantly different at p=0.05. Standard errors (LSD) post - hoc test. ................................ ................................ ................................ .................... 195 Figure V - 6 . Net photosynthesis and Light - adapted fluorescence of four ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water in year 2018 and 2019. Re cycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that passed through a he at - expanded shale and woodchip bioreactor system. Means within a taxon that are followed by same letters are not significantly different at p=0.05. Standard errors of the means are denoted by Difference (LSD) post - hoc test. ................................ ................................ ................................ .. 196 Figure V - 7 . Dark - adapted fluorescence of different ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water. Recycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that passed through a heat - expanded shale and woodchip bioreactor system. Means within a taxon that are followed by same letters are not significantly di fferent at p=0.05. ................................ ................................ ................................ . 197 Figure V - 8 . Chlorophyll SPAD index of four different ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water. Recycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated rec ycled runoff water was the recycled runoff water that passed through a heat - expanded shale and woodchip bioreactor system. Means within a taxon that are followed by same letters are not significantly different at p=0.05. Standard errors of the means are de (LSD) post - hoc test. ................................ ................................ ................................ .................... 198 1 SECTION I IRRIGATING NURSERY CROPS WITH RECYCLED RUN - OFF: A REVIEW OF POTENTIAL IMPACTS OF PESTICIDES ON PLANT GROWTH AND PHYSIOLOGY (Literature review) 2 Irrigating Nursery Crops with Recycled Run - Off: A Review of Potential Impacts of Pesticides on Plant Growth and Physiology Shital Poudyal 1 and Bert M. Cregg 1, 2 , * 1 Department of Horticulture, Michigan State University, 1066 Bogue St. , East Lansing, MI 48824 2 Department of Forestry, Michigan State University, 408 Wilson Rd., East Lansing, MI 48824 * Corresponding author : cregg@msu.edu This s ection h as b een p ublished in HortTechnology (doi: 10.21273/HORTTECH04302 - 19) 3 Abs t ract Interest in capturing and reusing runoff from irrigation and rainfall in container nurseries is increasing due to water scarcity and water use regulations. However, grower concerns related to contaminants in runoff water and other issues related to water s afety are potential barriers to the adoption of water capture and reuse technologies. In this review, we discuss some of the key concerns associated with potential phytotoxicity from irrigating container nursery crops with recycled runoff. The concentratio n of pesticides in runoff water and retention ponds is orders of magnitude lower than typical crop application rates, therefore the risk of pesticide phytotoxicity from irrigation with runoff water is relatively low. Nonetheless, some pesticides, particula rly certain herbicides and insecticides, can potentially affect crops due to prolonged chronic exposure. Pesticides with high solubility, low organic adsorption coefficients, and long persistence have the greatest potential for crop impact as they are the most likely to be transported with runoff from container pads. Potential impact on plant growth or disruption of physiological processes differs among pesticides and sensit ivity of individual crop plants. Growers can reduce risks associated with residual p esticides in recycled irrigation water by adopting best management practices (e.g., managing irrigation to reduce pesticide runoff, reducing pots spacing during pesticide application, use of vegetative filter strips) that reduce the contaminant load reachi ng containment basins as well as adopting remediation strategies that can reduce pesticide concentration in recycled water. Keywords : Herbicides, Insecticides, Phytotoxicity, N ursery management 4 1 . Introduction Increasing water demand is exacerbating water scarcity worldwide. In a list of top 10 global risk factors published by World Economic Forum, inadequate water supply was the top risk factor in terms of impact and the eighth risk factor in terms of likelihood (World Economic Forum, 2 015) . Agriculture is the major use of freshwater withdrawals and accounts for 69% of global water withdrawal (FAO AQUASTAT, 2014) . Fischer et al. (2007) estimates water requirements for irrgiation to increase by 45% globally in next 60 years. Many parts of the United States are facing increasing drought severity and frequency. As a result, surface water sources are declining in many areas. For example, water flow in the Colorado River, which supplies water to around 40 million people, could diminish by 35% by 2050 (Johnson, 2013; Rajagopalan et al., 2009; Wines, 2014). With increasing drought and possible decline in water availability, horticultural producers need to find ways to improve irrigation efficiency including reusing runoff for irrigation. In 201 5, irrigation accounted for 37% of total water use in the U.S. (Maupin, 2018) . Horticulture is a major sector of U.S. agriculture and is expanding. In 2014, the sales of all horticultural crops in U.S. was estimated to be $13.8 billion, a $2 billion increa se from 2009 ( U.S. Department of Agriculture, National Agricultural Statistics Service, 2016) . Ample irrigation is a crucial requirement for many horticultural crops, which has led to a continuous increase in water consumption since 2004 (Fulcher et al., 2 016) . In 2013, 556,490 acres out of 661,862 acres (84%) of horticulture operations were irrigated. These operations consumed 223 billion gallons of water, 65% of which was applied as overhead sprinkler irrigation (Vilsack and Reilly, 2013) . Irrigation effi ciency can be particularly low in container nursery production. In overhead irrigation systems, up to 80% of water applied may be lost as runoff, depending on container spacing and container size (Beeson and Knox, 1991; Mathers et 5 al., 2005) . However, over head or sprinkler irrigation remains the most common, reliable, and economical method of irrigating plants. A c ommon nursery recommendation is to schedule irrigation to achieve a 15% leaching fraction (i.e., 15% of applied irrigation is leached through the bottom of containers). Allowing a portion of irrigation water to flow out of containers prevents the often more liberal, such as applying 0.75 inch of irrigat ion per day, resulting in significant over - watering and runoff (Bailey et al., 1999; Warsaw et al., 2009) . 1.1 . Increase interest in water capture and reuse in nurseries Container nursery production is one of the most intensive agricultural systems in ter ms of resource inputs. Nurseries produce high - value specialty crops in a relatively short period. To produce marketable plants and reduce the risk of crop failure, growers rely heavily on irrigation, fertilizers and pesticides. This can lead to significant losses of nitrogen (N), phosphorus (P) and potassium (K) through runoff ( Andersen and Hansen, 2000; Broschat, 1995; Colangelo and Brand, 2001) . Elevated concentration of N and P in water can cause eutrophication, dead zones and algal blooms (Conley et al. , 2009; National Oceanic and Atmospheric Administration, 2017) . Pesticide runoff in nursery production is also a common concern (Mangiafico et al., 2008, 2009) . A 10 - year survey of major streams and ground water found 97% of stream water and 61% of shallow ground water near agricultural areas had one or more pesticides present (Gilliom et al., 2006) . Pesticides, even at low concentrations, may be detrimental to aquatic and terrestrial life. With rising water scarcity and increasing water pollution, the U.S. Environmental Protection Agency along with state and local regulatory agencies are limiting groundwater withdrawals for agriculture and, in some cases, mandating runoff capture and reuse (Beeson et al., 2004; Fulcher et al., 2016) . Moreover, regulations a imed at reducing fertilizer and pesticide runoff will likely increase (Lin et 6 al., 2009) . With this changing scenario, the nursery industry is obliged to consume less fresh water (Fulcher et al., 2016) and look for ways to capture and reuse nursery runoff. 1.2 . Examples of water capture and recycle in nurseries Capturing runoff water on - site in a containment or retention pond is often the best way to prevent potential environmental problems associated with nursery runoff (Fain et al., 2000) . In nurseries that capture runoff, collected water may be recycled to irrigate plants, either with or without treatment. Capturing and recycling nursery runoff water protects water sources, reduces water costs and provides a constant water supply (Wilson and Broembsen, 2015) . Initial investment cost to build recycled water systems can be high but are often subsidized by various agencies, which can offset the initial investment cost in a few years. For example, a major nursery in California was able to recover the cost of a water recycling system within one year based on savings associated with purchasing less water (Pitton et al., 2018). Therefore, more nurseries are capturing and recycling runoff water. In a survey of 24 greenhouses and nurseries across 11 states, nurser ies met 33% of their daily water requirement during peak irrigation demand using recycled water (Meador et al., 2012) . In a survey of 65 nurseries in Ventura County, California, the number of nurseries collecting runoff doubled in just 2 years (from 2004 t o 2006), indicating a rapid adoption rate of runoff capture (Mangiafico et al., 2010) . In a recent survey of 60 nursery and greenhouse producers in Virginia, 51 (77%) said that they would capture and collect runoff water (Mack et al., 2017) . Similarly, in a 1998 survey of 24 nurseries on the Alabama coastline, 75% of the nurseries captured runoff in some way (Fain et al., 2000) . Larger nurseries recycled 68% of total water applied while smaller nurseries, if they had a recycling pond, recycl ed 98% of their water (Fain et al., 2000) . Out of 58 west - central Florida nurseries surveyed in a workshop in 2000, 20 reported collecting runoff (Gisele et al., 2006) . Likewise, in a survey of 192 nursery growers 7 across the U.S., 43% said they used water from retention ponds as source of irrigation but were still concerned about the water quality (White et al., 2013) . 1.3 . Concerns about crop safety Capturing and reusing nursery runoff can assure water security for nurseries and protect water resources, but its safety in terms of crop health is a concern for growers. This hinders adoption of water capturing and recycling technologies. Potential proble ms with irrigating nursery crops with runoff include water quality, introduction (or re - introduction) of fungal pathogens and potential damage to crops from contaminants, particularly pesticides, that may be present in runoff. Issues associated with pathog ens in recycled water are discussed elsewhere in this issue (Parke et al., 2019). In this review, we will consider the potential for pesticides in runoff to impact the growth and physiology of nursery crops. 1.3.1 . Water quality Maintaining water qualit y is crucial for nursery producers. Electrical conductivity (EC), pH and alkalinity are major factors in determining irrigation water quality, but these factors may fluctuate in containment ponds. Nursery runoff water may have higher pH, EC and alkalinity than recommended (Lu et al., 2006a; Zhang and Hong, 2017) . This may be due to leaching of soluble salts from containers or microbial activities in the pond. In a study on evaluating water quality of runoff flowing to nine different containment basins, pH o f runoff was usually higher than the recommended pH of 6.8 (Copes et al., 2017) . In a study on nutrients leaching at different irrigation and fertilizer rates, EC levels often fluctuated in leachate and were occasionally above 1 dS · m - 1 which is slightly hi gher than recommended rate (< 1 dS · m - 1 ) for irrigation water (Million et al., 2007; Will and Faust, 2010) . 8 1.3.2 . Pathogens (diseases) Pathogens are usually the main concern when managing nursery runoff water. The presence of pathogens even in one product ion area can infest an entire collection pond; if water from the infected ponds is reused, inoculum can spread over an entire nursery. Plant pathogens are frequently detected in nursery runoff water and collection ponds (Bush et al., 2003; Ghimire et al., 2011; Junker et al., 2016; MacDonald, 1994; Pottorff and Panter, 1997; Werres et al., 2007), but this does not always translate to infection of plant material. Along with reusing contaminated nursery runoff water, reuse of dirty pots, lack of proper draina ge and contact of pots with contaminated ground are the most common reasons behind the spread of pathogens such as Phytophthora sp. and Pythium sp. (Kong and Richard son, 2004; Parke et al., 2008). 1.3.3 . Pesticides Apart from water quality and diseases, gr owers are also concerned about pesticides when considering reuse of runoff water. Parween et al. (2016) extensively evaluated the effect of pesticides on grasses and agronomic crops. In this review, we evaluate how nursery crops are impacted by presence of residual pesticides in irrigation water. Most of the research reviewed relates to nursery production, but where information was limited, we reviewed other, related agricultural production systems. When pesticides are applied in container nurseries, wide p lant spacing may result in pesticide deposition to non - target areas between the plant containers. For example, in a study on methiocarb application efficiency on weeping fig ( Ficus benjamina ) and lady palm ( Rhapis excelsa ), 16% to 30% of pesticide granules landed in the spaces between containers (Wilson et al., 2005). Subsequent overhead irrigation can carry pesticides in runoff that ultimately is captured in 9 retention ponds. Pesticide runoff has been reported in various nursery operations (Keese et al., 19 94; Riley, 2003; Wilson et al., 1996; ). In a study by Gilliam et al. (1992), 80% of applied granular herbicide landed off - target when empty 2.8 - L containers were spaced at 30 cm apart. Typically, only a small fraction of pesticides leach out of containers because of high pesticide retention properties, particularly adsorption, of most soilless substrates. Hence, the largest portion of pesticide runoff is due to pesticide application to non - target areas (Roseth and Haarstad, 2010) . Properties of pesticides such as solubility, volatility and adsorption as well as nursery management practices such as irrigation method and timing, crop spacing and ground cover determine the quantity of pesticides in runoff water that eventually reach retention ponds. Briggs et al. (1998a) , found isoxaben, thiophanate - methyl, trifluralin and chlorpyrifos in runoff water from container production systems. Species vary in their sensitivity to pesticides (i.e., a pesticide safe for one species may be potentially detrimental for othe r species), therefore plant sensitivity should be considered when decisions related to water recycling are made (Baz and Fernandez, 2002; Fernandez et al. 1999; Lu et al., 2006b; Moorman, 2011; Straw et al., 1996) . Some of the common pesticides found in ru noff water and retention ponds, along with their concentr ations, are listed in Table 1. 2 . Pesticides used in nursery production and potential for crop damage In general, pesticides are compounds designed to control pests that damage crops. In nursery crops, the most widely used pesticides are herbicides, insecticides and fungicides. Nematicides and rodenticides may also be applied in certain cases. Pesticides a re usually carbon based compounds and have functional groups that target specific sites in animal and/or plant metabolism to kill or inhibit their performance. 10 2.1 . Herbicides Herbicides are often the greatest concern among contaminants in nursery runoff because the pesticide target and crop are the same type of organism - plants. Herbicides can be pre - emergence or post - emergence. Pre - emergence herbicides are commonly used in container nurseries and are applied to prevent seed germination and weed emergenc e. Post - emergence herbicides are used to kill established weeds. If used on container plants, many post - emergent herbicides may injure established crop plants along with target weeds hence, they are rarely applied in containers except to control weeds in n on - crop areas - walkways, aisles and ditches (Atland, 2014; Robbins and Boyd, 2011) . Mode of action refers to the specific mechanisms by which herbicides kill or suppress weeds. More than 20 different modes of action have been documented for commercially available herbicides (Duke and Dayan, 2015); out of those, herbicides with six different modes of action are commonly used in nurseries (Table 2) ( Robbins and Boyd, 2011). The term site of action is more specific and defines where specifically an herbicide makes an impact (Table 2). For example, mode of action of oxyfluorfen is cell membrane disruption and its site of action is protoporphyrinogen oxidase (PPO) inhibition (Ross and Childs, 1996; University of Wisconsin - Extension, 2013) . 2.2 . Insecticides Al though the mode of action of insecticides targets insect metabolism, some insecticides can affect plants as well. Insecticides can reduce plant growth, mainly by inhibiting chlorophyll formation and interfering with photosynthetic reactions (Mishra et al., 2008; Parween et al., (2011a, 2011b, 2012) . Chlorpyrifos, a commonly used insecticide, can alter chlorophyll concentration, affect leaf sugar content, inhibit chlorophyll formation and degrade chlorophyll 11 (Parween et al., 2011a, 2011b; Prasad et al., 2015 ). Insecticides belonging to the pyrethroid family and organophosphate family can stress plants, triggering production of free radicals (Bashir et al., 2007; Parween et al., 2012). Free radicals are highly reactive molecules that can damage cell membranes. Carotenoid content increased when plants were exposed to pyrethroid and organophosphate, as a response to an increase in free radicals ( Prasad et al., 2015) . Pyrethroid and organophosphate also produce reactive oxygen species and cause lipid/membrane pero xidation ( Prasad et al., 2015) . Superoxide dismutase, an antioxidant enzyme in plants, increased when plants were exposed to these pesticides (Parween et al., 2012; Prasad et al., 2015) . Deltamethrin, a pyrethroid, inhibits formation of spindle fibers duri ng cell division thus producing abnormal cells (Chauhan et al., 1986) . Pyrethroids may also reduce photosynthetic light use (Rózsavölgyi and Horváth, 2008) . Insecticides from organophosphate and carbamate families can inhibit nitrification, as nitrifying b acteria are sensitive to these insecticides (Lin et al., 1972) . Imidacloprid can reduce seed germination (Dubey and Fulekar, 2011; Stevens et al., 2008) and deltamethrin can extend the vegetative cycle of plants (Fidalgo et al., 1993) . Pyriproxyfen, fipron il, imidacloprid and thiamethoxam reduce the phosphorus solubilization activity of rhizosphere bacteria causing reduction in phosphorus available for plant uptake (Ahemad and Khan, 2011) . Indole acetic acid (IAA) production from rhizosphere bacteria is als o reduced by these pesticides, that may lead to reduction in cell elongation and growth (Ahemad and Khan, 2012) . Oxydemeton - methyl and pirimicarb when combined with fungicides, mancozeb and flusilazol, reduced photosynthesis by inhibiting phosphorylation and adenosine triphosphate (ATP) formation (Untiedt and Blanke, 2004) . Hence, insecticide presence in retention ponds has the potential to induce phytotoxicity. 12 2.3 . Fungicides Nursery managers often apply various fungicides to protect crops from a wide ra nge of fungal diseases. While fungicides may be effective at controlling fungal diseases, they may also be phytotoxic to sensitive plants. (Chase, 2010; Getter, 2015). Fungicides may contain inorganic ingredients, such as copper and sulfur, or organic comp ounds, like metalaxyl or triflumizole. Both inorganic and organic compounds can induce phytotoxic effects on plants (Petit et al., 2012; Tjosvold et al., 2005). Fungicides may affect plant growth and performance by directly inhibiting photosynthesis or by degradation of ribulose 1,5 - bisphosphate (RuBP) carboxylase (Van Assche and Clijsters, 1990). They also may slow regeneration of RuBP, reduce stomatal conductance, lessen stomatal opening and degrade photosystems (Nason et al., 2007; Xia et al., 2006). Fun gicides can also oxidize and destroy membranes. Destroying membranes leads to reduction of electron transport reactions, altered source sink relations and reduction in pigments such as chlorophyll a, chlorophyll b and carotenoids (Benton and Cobb, 1997; Sa ladin et al., 2003; Vinit - Dunand et al., 2002). Fungicides have also been reported to reduce photochemical efficiency through the reduction in photochemical quenching (Dias, 2012). There are a few instances of increased photosynthesis and growth in respons e to relatively low doses of fungicide treatment (Saladin et al., 2003; Untiedt and Blanke, 2004). 3 . Properties of pesticides that may affect phytotoxicity of runoff Pesticides differ in their physical and chemical properties. These properties, ultimately, determine the potential for compounds to move with runoff water and cause phytotoxicity. Some of the basic properties of pesticides and how those properties determin e the fate and potential 13 phytotoxicity of various pesticide compounds are considered below (adapted f rom National Pesticide Center). 3.1 . Solubility Solubility is the ability of a pesticide compound to dissolve in a solvent (water). It is measured in milli gram of a compound per liter (mg·L - 1 ) of solvent or parts per million (ppm). Pesticides with solubility lower than 10 mg·L - 1 are considered to have low water solubility, while pesticides with solubility higher than 1000 mg·L - 1 are highly water soluble. Hig hly soluble pesticides are likely to dissolve and move with runoff water to containment ponds where their concentration may build up. Pesticides like acephate (818,000 mg·L - 1 ), glyphosate (1,050,000 mg·L - 1 ) and mefenoxam (8400 mg·L - 1 ) are highly water - solu ble and may accumulate in retention ponds. 3.2 . Adsorption Adsorption or sorption coefficient (K d ) is a measure of how well compounds bind to soil particles. However, K d does not take into consideration soil organic matter, which is the main sorbent of pesticides in container substrates. Therefore, the organic carbon - water coefficient (K oc ) is used to estimate pesticide adsorption of container media (Wauchope et al., 2002) . Pesticides with a higher adsorption coefficient adsorb to substrate p articles and ground surface. Hence, they are less likely to move with runoff water compared to compounds with a low K oc . Within retention ponds, pesticides with high K oc may also bind to sediment and are less likely to move with recycled irrigation. Bifen thrin (K oc = 131,000 to 302,000), chlorpyrifos (K oc = 7,000 to 25,000) and oxyfluorfen (K oc = 8900) have high adsorption coefficients and are less likely to move with runoff 14 water. Regardless, some pesticides such as oxyfluorfen can cause crop damage even at a very low concentration, 0.01 mg·L - 1 (Poudyal et al., 2018). 3.3 . Persistence Pesticide degradation occurs via various factors such as light, water, chemicals, microbes or plants. The half - life (DT 50 ) refers to the time required for a pesticide to red uce to half of the concentration initially applied. The half - life of pesticides varies depending on environmental conditions. Based on half - life, pesticides are classified as non - persistent (DT 50 < 30 d), moderately persistent (DT 50 = 31 - 90 d), and persist ent (DT 50 > 90 d) (Deer, 2004). Persistent pesticides have a greater potential to remain longer in retention ponds. With other factors remaining constant, a pesticide with a longer half - life (persistent) has a higher potential to cause phytotoxic effect on plants; although pesticides with short half - lives (non - persistent) can require frequent re - application that can increase their concentration in retention ponds. Pesticides such as isoxaben (2 to 6.6 months), oryzalin (1.4 to 4.4 months) and oxyfluorfen (1 to 6 months) may have long half - lives so, even after months they can still be present in retention ponds. 3.4 . Volatility Volatility is a measure of the potential of a compound to evaporate and is usually measured in millimeters of mercury (mm Hg). Pesticides with vapor pressure < 0.000001 mm Hg are less volatile whereas, pesticides with vapor pressure > 0.01 mm Hg are highly volatile. During pesticide applications, vapor from volatile pesticides may quickly drift to nearby non - target plants and may cause immediate phytotoxicity on sensitive species. Highly volatile pesticides are less likely to end up in runoff and be transported to a containment pond. Pesticides like chlorpyrifos (0.00002 mm Hg) and triflumizole ( 0.0000014 mm Hg) are volatile and may drift to nearby areas causing 15 injury to sensitive species. The volatility of pesticides, while important, is not constant and differs with environmental conditions and interactions with other chemicals. For example, vo latilization may reduce the half - life of chlorpyrifos in surface water to as short as 0.3 d to 3.2 d. However, in a retention pond, this may extend to 1 to 2 months due to slower microbial transformation and lower pH of collected water (Meikle et al., 1983 ; Leistra et al., 2006; Lu et al., 2006a) . 4 . Factors affecting the potential for crop injury Pesticide concentrations are low in nursery runoff water and even lower in retention ponds compared to labeled application rates. For comparison, the recommended rate of isoxaben application is around 4500 mg·L - 1 (calculated using label rate), and the concentration found in retention pond is around 0.055 mg·L - 1 ( Wilson et al., 1996). A similar pattern is true for most pesticides, but irrigating with runoff can resu lt in chronic plant exposure to pesticides. Long - term exposure to low concentrations of residual pesticides has potential to cause crop injury. This potential for damage depends on a series of factors including plant sensitivity, pesticide type, pesticide dose, length of pesticide exposure, and growth stage when plants are exposed. 4.1 . Pesticide Certain pesticides may be more likely to cause plant injury than others. Baz and Fernandez (2002) observed that isoxaben (4 ppm) was more damaging to semi - aquati c woody nursery plants than oryzalin (4 ppm), both in terms of growth and photosynthetic responses. Similarly, isoxaben (5 ppm) had a greater impact on growth and photosynthetic parameters of semi - aquatic herbaceous perennials compared to oryzalin (5 ppm) (Fernandez et al., 1999). In a large phytotoxicity trial by Mathers et al. (2012) , phytotoxic damage was observed on rose ( Rosa sp . ) when sprayed with isoxaben (2.25 lb/acre) + oryzalin (0.8 qt/acre) but exposure to indaziflam (38.1 lb /acre ) did not 16 cause damage . In the same study, combined application of flumioxazin (0.612 fl oz/acre) and oryzalin (25.85 fl oz /acre ) injured compact euonymus ( Euonymus alatus ) and common purple lilac ( Syringa sp . ), but dimethenamid - P ( 13.4 fl oz /acre) + pendimethalin (0.04 q t /acre ) did not induce phytotoxicity on compact euonymus (Mathers et al., 2012) . In a fungicide study, ghent azalea ( Rhododendron daviesi ) exhibited phytotoxicity resembling sunburn and leaf lesions after 1 week of treatment with a sulfur - based fungicide ( 19 ppm) but other fungicides, propiconazole (0.088 mL·L - 1 ) and trifloxystrobin (0.15 mL·L - 1 ), did not produce phytotoxicity (Vea and Palmer, 2017a). In the same study, copper sulfate pentahydrate (0.655 mL·L - 1 ) produced phytotoxic damage on flowering dogwood ( Cornus florida ) but chlorothalonil (0.36 mL·L - 1 ) was completely safe (Vea and Palmer, 2017a). In a dose - response study including isoxaben (0 to 1.4 ppm), oxyfluorfen (0 to 0.02 ppm) and chlorpyrifos (0 to 0.4 ppm), panicle hydrangea ( Hydrangea pan iculata ), silky dogwood ( Cornus obliqua ) and hosta ( Hosta sp . each pesticides. Out of three pesticides, only oxyfluorfen (0.005 to 0.02 ppm) caused phytotoxic damage (Poudyal et al., 2018). Neem oil extract (7 m L·L - 1 ) reduced photosynthesis and growth of gerbera daisy ( Gerbera sp ), but insecticide abamectin (1.51 ppm) had no effect (Spiers et al., 2008). Insecticides such as cinnamaldehyde (1.494 mL·L - 1 ) and pyrethrin (1.164 mL·L - 1 ) were phytotoxic on spanish lav ender ( Lavandula stoechas ), oregano ( Origanum vulgare ), rosemary ( Rosmarinus officinalis Hypericum perforatum ), woolly thyme ( Thymus pseudolanuginosus ) and nutmeg thyme ( Thymus praecox ), but insecticides such as capsaicin (62.25 mL·L - 1 ) and azadirachtin (3.75 ppm) were safe on those species (Cloyd and Cycholl, 2002). 4.2 . Plant sensitivity Plants often differ in their sensitivity to pesticides. In an evaluation trial of isoxaben (0.045 lb/acre) and pendimethalin (2 lb/acre), both compoun ds reduced plant height for winter 17 creeper ( Euonymus fortune Ilex crenata oxyfluorfen and isoxaben were app lied separately with irrigation water at either 1 or 10 ppm. gardenia ( Gardenia jasminoides radicans ). Oryzalin (10 ppm) and oxyfluorfen (10 ppm) reduced shoot fresh weight of fountain grass ( Pennisetum rupeli ) but did not affect shoot fresh weight of daylily ( Hemerocallis hybrid study, when dwarf gardenia and fountain grass were irrigated with 0.01, 0.1 and 1 ppm of oryzalin; 1 ppm reduced root and shoot weight for fountain grass but did not affect dwarf gardenia (Bhandary et al., 1997b). Isoxaben, even at the label recommended rate, caused serious phytotoxic damage on foxglove ( Digitalis purpurea ), purpl e coneflower ( Echinacea purpurea ), Stachys byzantine ) and false spirea ( Astilbe sp.) but was safe on statice ( Limonium latifolium ), little bluestem ( Schizachyrium scoparium ) and red maple ( Acer rubrum ) (Vea and Palmer, 2017b). Application of o xyfluorfen + oryzalin (12 lb/acre) on woolly yarrow ( Achillea tomentosa ) cause stunted growth (70% to 80% growth reduction) but woolly thyme ( Thymus pseudolanuginosus ) was not a ffected (Staats et al., 1998). 4.3 . Pesticide dose and exposure Although pesti cide concentrations in retention ponds are low compared to application rates, continuous irrigation even with low doses may build up and cause phytotoxic responses. Miticides such as phosmet (applied at 0.6, and 1.2 g·L - 1 ) and vinyl dimethyl phosphate (app lied at 0.6, 1.2, and 2.4 g·L - 1 ) had dose - dependent effects on flowers of various cultivars of chrysanthemum ( Chrysanthemum sp . ) damage was only seen with at higher doses (Poe , 1970) . In a study by Bhandary et al. (1997a) , 1 ppm of oryzalin reduced fresh s hoot weight of fountain grass by 13.5% 18 while 10 ppm reduced growth by 92.7%. Similarly, 1 ppm of oxyfluorfen reduced fresh shoot weight of fountain grass by 11.4% compared to 31.2% by 10 ppm of oxyfluorfen. In the same study, isoxaben at 1 ppm and 10 ppm i ncreased root phytotoxicity (based on a 0 to 10 rating) by 37% and 56%, respectively. Indaziflam (herbicide) was applied at 200, 400, and 800 lb/acre to different ornamental plants and the application was repeated after 4 weeks . In smooth hydrangea ( Hydrangea arborescens big leaf hydrangea ( Hydrangea macrophylla judd viburnum ( Viburnum X Juddii ) increasing the dose of pesticide increased phytotoxic damage, and repeated applications further exacerbated the damage (Mathers et al., 2012) . Isoxaben application at the recommended rate (0.5 to 1 lb/acre) had no phytotoxic effect on butterfly bush ( Buddleia davidii ) or hairawn muhly ( Muhlenbergia capillaris ), but Isoxaben application produced phytotoxic symptoms when the dose was increased to 2 lb/acre (Vea and Palmer, 2017b) . 4.4 . Growth stages and plant parts Sensitivity of nursery crops to pesticides is likely to vary depending on the growth stage of the plants at the time of application. However, research detailing th e phytotoxic response of plants to pesticide application at various growth stages is limited and results have been variable. Generally, pesticide labels suggest avoiding pesticide application at the seedling or younger stage. In a study by Richardson (1972 ) , phytotoxicity in sugarcane was higher when 2,4 - D (3.3 kg/ha) was applied at later stages of growth. However, in sunflower, application of the herbicide (flumioxazin, 30 g/ha) at early stages of growth caused greater photosynthesis reduction and slower r ecovery from damage compared to application at later stages (Jursik et al., 2013) . Chlordimeform (1.2 g·L - 1 ), an insecticide, produced phytotoxic damage on younger leaves of 19 chrysanthemum but had no effect on older leaves. Cyhexatin (0.14 g·L - 1 ), a miticid e, produced phytotoxic damage on flowers of chrysanthemum, but foliage was not affected (Poe, 1970) . 5 . Mitigating risks from residual pesticides in recycled runoff As indicated in the foregoing discussion, residual pesticides in recycled runoff can poten tially damage nursery crops under certain conditions. Fortunately, there are several ways to reduce the risk of phytotoxicity arising from irrigation with recycled runoff. A complete discussion of mitigation technologies is be yond the scope of this review. Below we present a few examples of techniques to reduced or remediate pesticides in runoff. For a more complete review of mitigation technologies, readers are referred to a review paper by Majsztrik et al. (2017) and to the Clean WateR 3 website ( www.clean water3.org/ ) . 5.1 . Pesticide dependent reduction The potential for a pesticide to be present in runoff depends on the formulation of the pesticide. In a study on the effect of formulation on runoff of isoxaben and trifluralin, concentration of isoxaben in runoff was 25% to 61% greater when the product was applied as a granular formulation compared to a spray application (Briggs et al., 2002a) . For trifluralin, formulation di d not affect runoff concentration (Briggs et al., 2002a) . The type of nursery bed liner can also affect pesticide runoff. When comparing nursery bed flooring, plastic ground cover had the greatest isoxaben runoff, followed by landscape fabric and then grav el. Runoff loss from both granular and sprayable formulation was higher for plastic and fabric compared to gravel (Wilson et al., 1995) . Irrigation design is also effective in controlling pesticide runoff. Cyclic irrigation, where total water for each day is applied in short intermittent cycles, usually has less pesticide runoff compared to continuous irrigation (Briggs et al., 1998b) . Using pesticides with low solubility, whenever 20 possible, can also reduce the concentration of pesticides in runoff (Riley, 2003) . Plant shape and size, container size and spacing can also influence pesticide runoff. Minimizing plant spacing when applying pesticides can reduce non - target application. Herbicide application loss was 23%, 51% and 80% when spacing was 0, 8 and 12 i nches between containers, respectively (Gilliam et al., 1992) . Highly soluble and less volatile pesticides such as isoxaben and thiophanate - methyl have greater runoff potential compared to less soluble pesticides such as chlorpyrifos and trifluralin (Brigg s et al., 1998a) . 5.2 . Constructed wetlands and vegetative buffer A constructed wetland is a marsh designed to hold and treat runoff, while vegetative buffers are usually narrow strips of land established with plants in the path of runoff. Both of these sy stems reduce pesticide concentration through adsorption, microbial degradation, volatilization, infiltration and plant uptake (Newman, 2010) . In a review of pesticide removal using constructed wetlands, pesticides such as organochlorine (97% removal) and o rganophosphate (94% removal) were almost completely removed while pyrethroid removal was around 80% ( Vymazal and . Pesticide removal efficiency of constructed wetlands and vegetative buffer increases with pesticide K oc value and runoff rete ntion time (Stearman et al., 2003; Vymazal and . Both vegetative buffers and constructed wetland systems can reduce pesticide concentration in runoff anywhere from 50% to 99% (Otto et al., 2016) and are particularly effective at removing py rethroids (Bennett et al., 2005; Budd et al., 2009) . Runoff retention time for pesticide removal may vary from few hours to days depending upon the properties of pesticides 21 5.3 . Sand Filters Well - engineered sand filter syste ms have great potential to remove runoff pesticides (Hedegaard and Albrechtsen, 2015) . A rapid sand filter can remove pesticides like mecoprop (MCPP), bentazone and glyphosate with a success rates varying from 7 to 85% (Hedegaard and Albrechtsen, 2014) . Re moval of pesticides such as trifluralin, fenitrothion and endosulfan have also been achieved using sand filters (Aslan, 2005) . The microbial community on the top layer of slow sand filter can degrade pesticides, thus, creating an effective pesticide remova l system (Escolà Casas and Bester, 2015; Samuelsen et al., 2017) . 5.4 . Activated carbon filters (ACF) and filter socks Activated carbon is a positively charged substrate that can adsorb polar or negatively charged pesticides. Efficacy of carbon filters dep ends on the type of carbon filter material used, temperature and flow rate of the filtration system. Activated carbon filters effectively remove agricultural pesticides (Hetrick et al., 2011; Jusoh et al., 2011; Kabashima et al., 2004; Mart n - Gullón and F ont, 2001) as runoff water passes through the carbon filter. Removal efficiency may be as high as 99.5% for organic compounds and negatively charged ions (Majsztrick et al., 2017) . Carbon filters with granular activated carbon completely removed acephate a nd paclobutrazol when the contact time was 64 s. The same system also removed bifenthrin, chlorpyrifos, imidacloprid and glyphosate by 72.2%, 89%, 85.3% and 99% respectively (Grant et al., 2019). Filter socks are long tubes made of mesh material that are c ommonly employed to intercept sediment carried in runoff. In a low - flow system, fill material such as wood chips, can be used to remove pesticides from runoff water, but in a high - flow system, filter socks may not be very effective (Majsztrick et al., 2017 ; Roseth and Haarstad, 2010) . Different substrates like pine bark, 22 sphagnum moss, peat, sand and compost are effective filler material for pesticide removal. In a study testing the efficacy of substrate for removal of 21 different pesticides, pine bark was the most efficient and removed nearly 100% for 20 different pesticides. Peat removed nearly 100% of 16 different pesticides (Roseth and Haarstad, 2010) . Substrates with high adsorption coefficient such as pine bark will have higher removal efficiency (Roseth and Haarstad, 2010) . In a study by Shipitalo et al. (2010) , filter socks filled with compost were effective in removing sediments and agrochemicals such as alachlor (18% removal) and glyphosate (5% removal) from surface runoff. Along with above listed strategies, additional best management practices can help manage pesticides in nursery runoff (Southern Nursery Association, 2013) . 6 . Conclusion Managing water resources is a major concern for nursery growers throughout the U.S. Growers in many re gions face the prospect of increasing scrutiny and regulation by environmental agencies. Therefore, nursery growers are looking for alternative ways to meet crop water demand. Capturing and reusing runoff water may be an option to cope with water shortages and environmental regulations, but the risk associated with pesticide phytotoxicity may hinder grower adoption of recycle and reuse technologies. Pesticides can cause phytotoxic responses by interfering with metabolic processes of plants including inhibit ing tubulin formation, inhibiting chlorophyll formation, penetrating lipid membranes and more. This interference leads to reduction in plant photosynthesis and growth. However, pesticide concentrations in remediation ponds is typically several orders of ma gnitude lower than the application rate, due to dilution from irrigation and rainwater (Camper et al., 1994), greatly reducing potential p hytotoxicity to nursery crops. Nursery growers need to be cautious when irrigating with captured runoff that may conta in 23 herbicides and certain insecticides. Herbicides such as oxyfluorfen may be phytotoxic at very low concentration. Plants, such as rose, may be sensitive to numerous pesticides. Even with low pesticide concentrations, repeated application may build up pes ticides and cause phytotoxic symptoms. Among pesticides, herbicides typically pose the greatest risk because of the similarity between pest controll ed (weeds) and the crop plant. Nonetheless, insecticides and fungicides may also affect nursery crops; fung icides containing copper sulfate may cause phytotoxic responses. Other factors to consider include pesticide solubility, adsorption potential and half - life of pesticides. Pesticides with high solubility, low adsorption to organic matter and a long half - lif e are more problematic as they have a higher tendency to be carried in runoff and also degrade very slowly. The growth stage of plants is also important when considering potential phytotoxicity. Exposure to pesticides at early stages of growth may have gre ater potential for injury but research with growth stage and phytotoxicity is very limited with nursery crops. The best strategy to prevent pesticide phytotoxicity is to minimize pesticide movement to retention ponds. Reducing container spacing during pes ticide application can reduce off - target pesticide loss. Using less soluble pesticides will lower the potential for pesticides to be in recycled irrigation water. Creating vegetative buffer zones and using filter socks to trap pesticides reduces pesticide movement and accelerates their degradation. All of these techniques lower the concentration of pesticides ending up in retention ponds. Therefore, capture and reuse of runoff for irrigation may be a viable and sustainable option for nursery growers, helpin g them deal with water sc arcit y and environmental issu es. 24 APPENDIX 25 APPENDIX Table I - 1 . Pesticides detected in irrigation runoff and retention ponds from container nursery production sites. Reported concentrations are for the maximum amount detected and always occurred during first flush of runoff. Pesticide detected Concentration z Water sampled Citation Metolachor 7.8 ± 3.6 ppm Irrigation runoff Mahnken et al., 1999 Simazine 2.2 ± 0.7 ppm Irrigation runoff Trifluralin 0.08 ppm Irrigation runoff Wilson et al., 1996 Isoxaben 0.75 ppm Irrigation runoff Trifluralin 5.00 ppb Containment pond Isoxaben 55.0 ppb Containment pond Oxyfluorfen 4.9 ppb Irrigation runoff Goodwin et al., 2001 Oryzalin 43 ppb Irrigation runoff Oxyfluorfen < 1.00 ppb Containment pond Oryzalin < 1.00 ppb Containment pond Bifenthrin 10.6 ppb Runoff near production area Kabashima et al., 2004 Cis - permethrin 24.6 ppb Runoff near production area Trans - permethrin 4.4 ppb Runoff near production area Bifenthrin 0.03 ppb Irrigation runoff Mangiafico et al., 2009 Chlorpyrifos 0.12ppb Irrigation runoff Diazinon 0.02 ppb Irrigation runoff Trifluralin 0.17 ppm Irrigation runoff Warsaw et al., 2012 Metalaxyl 2.19 ppm Irrigation runoff Oxyfluorfen < 0.10 ppm Containment pond Riley et al., 1994 Pendimethalin 4 ppb Containment pond Oxyfluorfen < 0.01 ppm Retention pond Camper et al, 1994 Pendimethalin 4 ppb Retention pond Oryzalin 0.16 ppm Retention pond Chlorpyrifos 1.59 ppb Water entering retention pond Mangiafico et al., 2008 26 Table I - 1 Diazinon 17.4 ppb Water entering retention pond Bifenthrin 20.6 ppb Water entering retention pond Cypermetherin 2.00 ppt Water entering retention pond Thiophanate - methyl 1.64 ppm Irrigation runoff Briggs et al., 2002b Metalaxyl 61.0 ppm Irrigation runoff Chlorothalonil 0.95 ppm Irrigation runoff Isoxaben 2.20 ppm Irrigation runoff Briggs et al., 2003 Oryzalin 3.80 ppm Irrigation runoff z 1 ppm = 1 mg·L - 1 , 1 ppb = 1 µg·L - 1 , 1 ppt = 1 pg ·L - 1 27 Table I - 2 . Common herbicides applied in nurseries and their effect on plants Site of action WSSA group Z Effect on plants Common name Citation 5 - enolpyruvyl - shikimate - 3 - phosphate (ESPS) synthase inhibitors 9 These herbicides are absorbed through foliage and translocated through phloem. They inhibit synthesis of 5 - enolpyruvylshikimate - 3 - phosphate (EPSP). EPSP is a key enzyme in shikimic acid pathway that produces amino acids; tryptophan, tyrosine and phenylalan ine. Inhibition of this enzyme cease essential amino acid formation, causing plant death Glyphosate (Shaner, 2006) TIR1 (transport inhibitor response 1) auxin receptor/ synthetic auxin 4 These herbicides are absorbed through foliage and root. They trigger production of 9 - cis - epoxycarotenoid dioxygenase (NCED) which in turn up - regulates abscisic acid production. Abscisic acid causes senescence, inhibition of cell division and elongation and stomatal closure. These herbicides also trigger 1 - aminocyclopropane - 1 - carboxylic acid (ACC) biosynthesis, which cause senescence related symptoms. Dicamba, 2 - 4 D (Grossm ann, 2007, 2010) Photosystem II inhibitors 5 In photosystem II, electron flows through the number of sites, including the movement from Q A to Q B (plastoquinones) which is mediated by D1 protein. Herbicides with this mode of action bind with D1 protein of thylakoid in electron transport chain blocking the movement of electron through plastoquinones (Q A to Q B ). This ceases the production of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and (ATP) and reduces the rate of photosynthesis. Choked electron transport chain also cause oxidative stress leading to lipid membrane de struction and disintegration. These class of herbicides are absorbed through root and shoot. Simazine, Atrazine (Nakaji ma et al., 1996; Lambre va et al., 2014) 28 Table I - 2 Protoporphyri nogen oxidase (PPO) inhibitors 14 Protoporphyrinogen oxidase (PPO) enzyme oxidizes protoporphyrinogen IX to protoporphyrin IX. Protoporphyrin IX is the precursor for chlorophyll and heme synthesis. PPO inhibitor blocks synthesis of PPO enzyme leading to reduction in chlorophyll formation. This ultimately reduces photosynthesis. Increased accumulation of protoporphyrinogen IX, in presence of light, reacts with molecular oxygen to produce reactive oxygen species (ROS). ROS are very unstable and destroy cell membranes causing cell leakage. Ox yfluorfe n (Lee and Duke, 1994) Long - chain fatty acid (LCFA) inhibitors 15 This class of herbicide inhibits formation of long - chain fatty acid (LCFA) by reducing incorporation of stearic acid, malonic and acetate in the chain. It also inhibits formation of enzymes required for elongation of LCFA. LCFA inhibitors also inhibit LCFA incorporation into cell wall. LCFA being essential component of plasma membrane, inhibiting its formation will kill cell and plants. Acetochlor, Metolachlo r (Schmal fuß et al., 1 998; Matthes et al., 1998) Microtubule assembly inhibitors 3 This class of herbicide binds with free tubulin. Tubulin synthesizes microtubules, which is essential for cell division. When herbicide binds to tubulin, tubulin cannot synthesize microtubules; hence, the cell is arrested in dividing stage. Symptoms of inj ury include swollen root tips. Pendimetha lin, Trifluralin, Oryzalin (Sandma nn et al., 1980) Z WSSA = Weed Science Society of America 29 LITERATURE CITED 30 LITERATURE CITED Ahemad, M. and M.S. Khan. 2011. Effects of insecticides on plant - growth - promoting activities of phosphate solubilizing rhizobacterium Klebsiella sp. strain PS19. Pesticide Biochem. Physiol. 100:51 56. Ahemad, M. and M.S. Khan. 2012. Effects of pesticides on plant growth promoting traits of Mesorhizobium strain MRC4 . J. Saudi Soc. Agr. Sci. 11:63 71. Al - Khatib, K. 2019. Herbicide symptoms. Univ. California, Div. Agr. Natl. Resources. 13 Mar. 2019. . Andersen, L. a nd C.W. Hansen. 2000. Leaching of nitrogen from container plants grown under controlled fertigation regimes. J. Environ. Hort. 18:5 - 8. biodenitrification and sand filter system. Process Biochem. 40:417 424. Atland, J. 2014. Weed Control in herbaceous perennial container production. Greenhouse Prod. News. 9. 5 May. 2019. . Bailey, D., T. Bilderback, and D. Bir. 1999. Water considerations for container production of plants. North Carolina Coop. Ext. Serv. 557. 8 May 2019. < https://content.c es.ncsu.edu/water - considerations - for - container - production - of - plants > . Bashir, F., Mahmooduzzafar, T.O. Siddiqi, and M. Iqbal. 2007. The antioxidative response system in Glycine max (L.) Merr. exposed to deltamethrin, a synthetic pyrethroid insecticide. En viron. Pollution 147:94 100. Baz, M. and R.T. Fernandez. 2002. Evaluating woody ornamentals for use in herbicide phytoremediation. J. Amer. Soc. Hort. Sci. 127:991 997. Beeson, R.C., M.A. Arnold, T.E. Bilderback, B. Bolusky, S. Chandler, H.M. Gramling, J .D. Lea - Cox, J.R. Harris, P.J. Klinger, H.M. Mathers, J.M. Ruter, and T.H. Yeager. 2004. Strategic vision of container nursery irrigation in the next ten years 1 . J. Environ. Hort. 22:113 115. Beeson, R.C., and G.W. Knox. 1991. Analysis of efficiency of overhead irrigation in container production. HortScience. 26:848 850. Bennett, E.R., M.T. Moore, C.M. Cooper, S. Smith, F.D. Shields, K.G. Drouillard, and R. Schulz. 2005. Vegetated agricultural drainage ditches for the mitigation of pyrethroid - associated runoff. Environ. Toxicology. Chem. 24:2121 7. Benton, J.M., and A.H. Cobb. 1997. The modification of phytosterol profiles and in vitro photosynthetic electron transport of Galium aparine L. (cleavers) treated with the fungicide, 31 epoxiconazole. Plant Growth Regulat. 22:93 100. Bhandary, R.M., T. Whitwell, and J. Briggs. 1997a. Growth of containerized landscape plants is influenced by herbicides residues in irrigation water. Weed Technol. 11:793 797. Bhandary, R., T. Whitwell, J.A. Briggs, and R.T. Fernandez. 1997b. Influence of surflan (oryzalin) concentrations in irrigation water on growth and physiological processes of Gardenia jasminoides radicans and Pennisetum rupelli . J. Environ. Hort. 15:169 172. Briggs, J.A., M.B. Riley, and T. Whitwell. 1998a. Quanti fication and remediation of pesticides in runoff water from containerized plant production. J. Environ. Quality. 27:814 820. Briggs, J.A., T. Whitwell, R.T. Fernandez, and M.B. Riley. 2002a. Formulation effects on isoxaben and trifluralin in runoff water f rom container plant nurseries. Weed Sci. 50:536 541. Briggs, J.A., T. Whitwell, R.T. Fernandez, and M.B. Riley. 2002b. Effect of integrated pest management strategies on chlorothalonil, metalaxyl, and thiophanate - methyl runoff at a container nursery. J. Am er. Soc. Hort. Sci. 127:1018 1024. Briggs, J.A., T. Whitwell, and M.B. Riley. 2003. Effect of delayed irrigation on isoxaben and oryzalin runoff from a container nursery. Weed Sci. 51:463 470. Briggs, J., T. Whitwell, M.B. Riley, and T. Lee. 1998b. Cyclic irrigation and grass waterways combine to reduce isoxaben losses from container plant nurseries. J. Environ. Hort. 16:235 238 Broschat, T.K. 1995. Nitrate, phosphate, and potassium leaching from container - grown plants fertilized by several methods. HortScience 30:74 77. wetlands in pesticide removal from tailwaters in the Central Valley, California. Environ. Sci. Technol. 43:2925 30. Bush, E.A., C. Hong, and E.L. Stromberg. 2003. Fluctuations of phytophthora and pythium spp . in components of a recycling irrigation system. Plant Dis. 87(12):1500 1506. Camper, N.D., T. Whitwell, R.J. Keese, and M.B. Riley. 1994. Herbicide levels in nursery containment pond water and sediments. J. Environ. Hort. 12:8 12. Case, L.T., H.M. Mathers, and A.F. Senesac. 2005. A review of weed control practices in container nurseries. HortTechnology. 15:535 545. Chase, A. 2010. Testing for phytotoxicity. Greenhouse. Grower. 2. 8 May 2019. . Chauhan, L.K.S., T.S. Dikshith, and V. Sundararaman. 1986. Effect of deltamethrin on plant cells I. Cytological effects on the root meristems of Allium cepa. Mutation. Re s. 171:25 32 30.. Clean WateR 3 . 2019. Clean wateR 3 reduce, remediate and recycle. 8 May 2019. < https://www.cleanwater3.org/default.asp > Cloyd, R.A. and N.L. Cycholl. 2002. Phytotoxicity of selected inse cticides on greenhouse - grown herbs. HortScience 37:671 672. Colangelo, D.J. and M.H. Brand. 2001. Nitrate leaching beneath a containerized nursery crop receiving trickle or overhead irrigation. J. Environ. Quality 30:1564 1574. Conley, D.J., H.W. Paerl, R. W. Howarth, D.F. Boesch, S.P. Seitzinger, K.E. Havens, C. Lancelot, and G.E. Likens. 2009. Controlling eutrophication: Nitrogen and phosphorus. Science. 80:323. Copes, W.E., H. Zhang, P.A. Richardson, B.E. Belayneh, A. Ristvey, J. Lea - Cox, and C. Hong. 201 7. Nutrient, pH, alkalinity, and ionic property levels in runoff containment basins in Alabama, Louisiana, Maryland, Mississippi, and Virginia ornamental plant nurseries. HortScience 52:641 648. Deer, H. 2004. Pesticide adsorption and half - life. Utah State Univ. Ext. 10. 8 May 2019. . Dias, M.C. 2012. Phytotoxicity: An overview of the physiological responses of plants exposed to fungicides. J. Bot. 2012:1 4. Dubey, K.K. and M.H. Fulekar. 2011. Effect of pesticides on the seed germination of Cenchrus setigerus and pennisetum pedicellatum as monocropping and co - cropping system: Implications for rhizospheric bioremediation. Romanian Biotechnol. Lett. 16:5909 591 8. Duke, S.O. and F.E. Dayan. 2015. Discovery of new herbicide modes of action with natural phytotoxins. p. 79 92. In Discovery and Synthesis of Crop Protection Products. Amer. Chem. Soc. Escolà Casas, M. and K. Bester. 2015. Can those organic micro - pollutants that are recalcitrant in activated sludge treatment be removed from wastewater by biofilm reactors (slow sand filters)? Sci. Total Environ. 506 507:315 322. Fain, G.B., C.H. Gilliam, K.M. Tilt, J.W. Olive, and B. Wallace. 2000. Survey of b est management practices in container production nurseries. J. Environ. Hort. 18:142 144. FAO AQUASTAT. 2014. Water withdrawn by sector, around 2007. Food Agr. Organization Aquastat. 5 Nov. 2018. . Fernandez, R. T., T. Whitw ell, M. B. Riley and C. R. Bernard. 1999. Evaluating semiaquatic herbaceous perennials for use in herbicide phytoremediation. J.Amer. Soc. Hort. Sci. 124:539 544. 33 Fidalgo, F., I. Santos, and R. Salema. 1993. Effects of deltamethrin on field grown potato pl ants: Biochemical and ultrastructural aspects. Ann. Bot. 72:263 267. Fischer, G., F.N. Tubiello, H. van Velthuizen, and D.A. Wiberg. 2007. Climate change impacts on irrigation water requirements: Effects of mitigation, 1990 - 2080. Technol. Forecast. Soc. Ch ange 74:1083 1107. Fulcher, A., A. V. LeBude, J.S. Owen, S.A. White, and R.C. Beeson. 2016. The next ten years: Strategic vision of water resources for nursery producers. HortTechnology 26:121 132. Getter, K. 2015. Plant phytotoxicity in the greenhouse. Mi chigan State Univesity Ext. 8 May 2019. . Ghimire, S.R., P.A. Richardson, P. Kong, J. Hu, J.D. Lea - Cox, D.S. Ross, G.W. Moorman, and C. Hong. 2011. Distribution and diversity of Phytopht hora species in nursery irrigation reservoir adopting water recycling system during winter months. J. Phytopathol. 159:713 719. Gilliam, C.D., D.C. Fare, and A. Beasley. 1992. Nontarget herbicide losses from application of granular ronstar to container nur series. J. Environ. Hort. 10:175 176. Gilliom, R.J., J.E. Barbash, C.G. Crawford, P. A. Hamilton, J.D. Martin, N. Nakagaki, L.H. Nowell, J.C. Scott, P.E. Stackelberg, G.P. Thelin, and D.M. Wolock. 2006. Pesticides in the 1992 2001.U.S. Geological Survey Circular 1291 Gisele, S., T. Yeager, and D. Haman. 2006. Survey of container nursery irrigation practices in west - central Florida: An educational opportunity. HortTechnology Ext. Educ. Methods 16:5707 5710. Goodwin, P.B. and S. Beach. 2001. Oxadiazon, oryzalin, and oxyfluorfen residues in container plant nurseries. HortScience 36:900 904. Grant, G.A., P.R. Fisher, J.E. Barrett, and P.C. Wilson. 2019. Removal of agrichemicals from water using granular activated carbon filtr ation. Water, Air, Soil Pollution 230:7. Grossmann, K. 2007. Auxin herbicide action: lifting the veil step by step. Plant Signaling and Behavhavior. 2:421 3. Grossmann, K. 2010. Auxin herbicides: Current status of mechanism and mode of action. Pest Mgt. Sc i. 66:113 120. Hedegaard, M.J. and H. J. Albrechtsen. 2014. Microbial pesticide removal in rapid sand filters for drinking water treatment - Potential and kinetics. Water Res. 48:71 81. Hedegaard, M.J. and H. J. Albrechtsen. 2015. Removal of pesticides wit h filter sand from biological rapid sand filters. IWA specialized conference biofilms in drinking water systems from treatment to tap . 2015:236 237. 8 May 2019. < http://orbit.dtu.dk/files/116665774/Removal_of_pesticides_with_filter_sand_from_biologi 34 cal_rapid_sand_filters.pdf > Hetrick, J., R.J. Pisigan, E. Behl, and C. Peck. 2011. Incorporation of water treatment effects on pesti cide removal and transformations in drinking water exposure assessments. United States Environ. Protection. Agency. 8 May 2019.. Johnson, T. 2013. 10 cities that could run out of water. The weather channel. 12. 8 May 2019. . Junker, C., P. Goff, S. Wagner, S. Werres, F. Biological, and F. Jk i. 2016. Occurrence of Phytophthora species in commercial nursery production. Plant Health. Progress. 17:64 75. Jursik, M., K. Hamouzova, J. Andr, and J. Soukup. 2013. Effect of different adjuvants on phytotoxicity of flumioxazin to sunflower in different growth stages. Romanian. Agricultural. Res. 30:335 372. Jusoh, A., W.J.H. Hartini, N. Ali, and A. Endut. 2011. Study on the removal of pesticide in agricultural run off by granular activated carbon. Bioresources. Technol. 102:5312 5318. Kabashima, J.N., S. J. Lee, D.L. Haver, K.S. Goh, L.S. Wu, and J. Gan. 2004. Pesticide runoff and mitigation at a commercial nursery site. p. 34. In Pesticide Decontamination and Detoxification ACS Symposium Series; American Chemical Society: Washington DC, 2003. Keese, R.J ., N.D. Camper, T. Whitwell, M.B. Riley, and P.C. Wilson. 1994. Herbicide runoff from ornamental container nurseries. J. Environ. Qual. 23:320 324. Kong, P., and P.A. Richardson. 2004. Pythium and recycled irrigation water. Greenhouse. Prod. News 5. 8 May 2019. . Lambreva, M.D., D. Russo, F. Polticelli, V. Scognamiglio, A. Antonacci, V. Zobnina, G. Campi, and G. Rea. 2014. Structure/function/dynamics of photosystem II plastoquinone binding sites. Current Protein Peptide Sci. 15:285 95. Lee, H.J., and S.O. Duke. 1994. Protoporphyrinogen ix - oxidizing activities involved in the mode of action of peroxidizing herbicides. J. Agr. Food Chem. 42:2610 2618. Leistra, M., J.H. Smelt, J. Hilbrand Weststrate, F. Van Den Berg, and R. Aalderink. 2006. Volatilization of the pesticides chlorpyrifos and fenpropimorph from a potato crop. Environ. Sci. Technol. 40:96 102. Lin, L., Z. - Q. Deng, and D.D. Gang. 2009. Nonpoint source pollution. Water Environ. Res. 81:1996 2018. Lin, S.C., B.R. Funke, and J.T. Schulz. 1972. Effects of some organophosphate and carbamate 35 insecticide on nitrification and legume growth. Plant and soil 37:489 496. Lu, J., L. Wu, J. Newman, B. Faber, and J. Gan. 2006a. Degradation of pesticides in nurs ery recycling pond waters. J. Agr. Food Chem. 54:2658 2663. Lu, J., L. Wu, J. Newman, B. Faber, D.J. Merhaut, and J. Gan. 2006b. Sorption and degradation of pesticides in nursery recycling ponds. J. Environ. Qual. 35:1795 1802. MacDonald, J.D. 1994. Occurr ence of Phytophthora species in recirculated nursery irrigation effluents. Plant Dis. 78:607. Mack, R., J.S. Owen, A.X. Niemiera, and J. Latimer. 2017. Virginia nursery and greenhouse grower survey of best management practices. HortTechnology 27:386 392. M ahnken, G.E., W.A. Skroch, R.B. Leidy, and T.J. Sheets. 1999. Metolachlor and simazine in surface runoff water from a simulated container plant nursery. Weed Technol. 13:799 806. Majsztrik, J.C., R.T. Fernandez, P.R. Fisher, D.R. Hitchcock, J. Lea - Cox, J.S . Owen, L.R. Oki, and S.A. White. 2017. Water use and treatment in container - grown specialty crop production: A review. Water Air Soil Pollution 228:151. Mangiafico, S.S., J. Gan, L. Wu, J. Lu, J.P. Newman, B. Faber, D.J. Merhaut, and R. Evans. 2008. Deten tion and recycling basins for managing nutrient and pesticide runoff from nurseries. HortScience 43:393 398. Mangiafico, S.S., J. Newman, D.J. Merhaut, J. Gan, B. Faber, and L. Wu. 2009. Nutrients and Pesticides in Stormwater runoff and soil water in production nurseries and citrus and avocado groves in California. HortTechnology 19:360 367. Mangiafico, S.S., J. Newman, M. Mochizuki, D. Zurawski, D.J. Merhaut, and B. Faber. 2010. Nurseries surveyed in Southern California adopt best practices for water quality. California. Agr. 64. 8 May 2019. . Mart n - Gullón, I. and R. Font. 2001. Dynamic pesticide removal with activated carbon fibers. Water Res. 35:516 520. Mathers, H., L. Case, M. Bigger, P. Gordon, and L. Gie se. 2012. Yearly research summary report 2012 Ornamental Research. Ohio State Univ. Ext. 8 May 2019. < https://www.mathersenvironmental.com/wp - content/ uploads/2016/06/Research - Summary - 2012.pdf > Mathers, H.M., T.H. Yeager, and L.T. Case. 2005. Improving irrigation water use in container nurseries. HortTechnology 15:8 12. Matthes, B., J. Schmalfuß, and P. Böger. 1998. Chloroacetamide mode of action, II: In hibition of very long chain fatty acid synthesis in higher plants. Verlag der Zeitschrift für Naturforschung. 53c: 1004 1011. 36 Maupin, M.A. 2018. Summary of estimated water use in the united states in 2015. USGS fact Sheet 6. 8 May 2019. . Meador, D.P., P.R. Fisher, P.F. Harmon, N.A. Peres, M. Teplitski, and C.L. Guy. 2012. Survey of physical, chemical, and microbial water quality in greenhouse and nursery irrigation water. HortTechnology 22:778 786. Meikle, R.W., N.H. Kur ihara, and D.H. Devries. 1983. Chlorpyrifos: The photodecomposition rates in dilute aqueous solution and on a surface, and the volatilization rate from a surface. Arch. Environ. Contamination. Toxicology 12:189 193. Million, J., T. Yeager, and J. Albano. 2 007. Consequences of excessive overhead irrigation on runoff during container production of Sweet Viburnum . J. Environ. Hort. 25:117 125. Mishra, V., G. Srivastava, S.M. Prasad, and G. Abraham. 2008. Growth, photosynthetic pigments and photosynthetic acti vity during seedling stage of cowpea ( Vigna unguiculata ) in response to UV - B and dimethoate. Pestic. Biochem. Physiol. 92:30 37. Moorman, G.W. 2011. Phytotoxicity. Penn State Ext. 8 May 2019. . Nakajima, Y., S. Yosh ida, and T.A. Ono. 1996. Differential effects of urea/triazine - type and phenol - type photosystem II inhibitors on inactivation of the electron transport and degradation of the D1 protein during photoinhibition. Plant Cell Physiol. 37:673 680. Nason, M.A., J . Farrar, and D. Bartlett. 2007. Strobilurin fungicides induce changes in photosynthetic gas exchange that do not improve water use efficiency of plants grown under conditions of water stress. Pest Mgt. Sci. 63:1191 - 1200. National pesticide center. 13 Mar. 2019. < http://npic.orst.edu/>. Newman, J. 2010. Manage runoff with vegetated buffers. Nurs. Mgt. 8 May 2019. NOAA. 2017. What is eutrophication? Natl. Ocean Serv. 10. 8 May 2019.. Otto, S., S.E. Pappalardo, A. Cardinali, R. Masin, G. Zanin, and M. Borin. 2016. Vegetated ditches for the mitigation of pesticides runoff in the Po valley. PLoS One 11:e0153287 Parke, J., C. Lewis, and N. Grunwald. 2008. Tracing the path of pathogens. Digger. 12. 8 May 2019. . Parke, J. L., N. Redekar, J. Eberhart. 2019. Hazard analysis for Phytophthora spp. in nurseries: Three case studies. HortTechnology (In press). Parween, T., S. Jan, and T. Fatma. 2011a. Alteration in nitrogen metabolism and plant growth during different developmental stages of green gram ( Vigna radiata L.) in response to 37 chlorpyrifos. Acta Physiol. Plant. 33:2321 2328. Parween, T., S. Jan, and T. Fatma. 2012. Evaluation of oxidative stress in Vigna radiata L. in response to chlorpyrifos. Intl. J. Environ. Sci. Technol. 9:605 612. Parween, T., S. Jan, S. Mahmooduzzafar, and T. Fatma. 2011b. Assessing the impact of chlorpy rifos on growth, photosynthetic pigments and yield in Vigna radiata L. at different phenological stages. African J. Agr. Res. 6:4432 4440. Parween, T., S. Jan, S. Mahmooduzzafar, T. Fatma, and Z.H. Siddiqui. 2016. Selective effect of pesticides on plant A review. Critical Rev. Food Sci. Nutr. 56:160 179. Petit, A.N., F. Fontaine, P. Vatsa, C. Clément, and N. Vaillant - Gaveau. 2012. Fungicide impacts on photosynthesis in crop plants. Photosynth. Res. 111:315 326. Pitton, B.J.L., C.R. Hall, D.L. Haver, S.A. Wh ite, and L.R. Oki. 2018. A cost analysis for using recycled irrigation runoff water in container nursery production: A Southern California nursery case study. Irr. Sci. 36:217 226. Poe, S.L. 1970. Evaluations Of pesticides for phytotoxicity on chrysanthemu m flowers. Florida Agricultural Experiment Stations Journal. 3702:469 471. Pottorff, L.P. and K.L. Panter. 1997. Survey of Pythium and Phytophthora spp. in irrigation water used by Colorado commercial greenhouses. HortTechnology 7:153 155. Poudyal, S., B.M. Cregg, and T.R. Fernandez. 2018. Overcoming barriers to use of nursery run - off water: understanding plant sensitivity to residual pesticides. HortScience 53(9S):124. (Abstr.) Prasad, S.M., A. Singh, and P. Singh. 2015. Physiological, biochemical and g rowth responses of Azolla pinnat a to chlorpyrifos and cypermethrin pesticides exposure: a comparative study. Chem. Ecol. 31:285 298. Prather, T.S., J.M. Ditomaso, and J.S. Holt. 2000. Herbicide resistance: definition and management strategies. Univ. Califo rnia. Div. Agr. Natl. Resources. Publication 8012. 8 May 2019. < https://anrcatalog.ucanr.edu/pdf/8012.pdf >. Rajagopalan, B., K. Nowak, J. Prairie, M. Hoerling, B. Harding, J. Barsugli, A. Ray, and B . Udall. 2009. Water supply risk on the Colorado River: Can management mitigate? Water Resources Res. 45: W08201. Reade, J.P.H. and A.H. Cobb. 2002. Herbicides: Modes of action and metabolism. p. 134 170. In R.E.L. Naylor, (ed.). Weed Management Handbook. Blackwell Science Ltd, Oxford, UK. Regan, R. and R. Ticknor. 1987. Application of isoxaben and pendimethalin to container - grown broadleaved evergreen shrubs. Ornamentals Northwest Arch. 11. Richardson, F.E. 1972. Critical growth stages for 2,4 - D phytotoxi city to sugarcane. In Proc. of 38 the South African Sugar Technoligists' Assoc. 6:168 175. Riley, M.B. 2003. Herbicide losses in runoff of containerized plant production nurseries. HortTechnology 13(1):16 21. Riley, M.B., R.J. Keese, N.D. Camper, T. Whitwell and P.C. Chris. 1994. Pendimethalin and oxyfluorfen residues in pond water and sediment from container. Weed Technol. 8:299 303. Robbins, J., and J. Boyd. 2011. Weed control in container nurseries. Univ. Arkansas, Div. Agr. 7. FSA6123. 8 May 2019. . Roseth, R. and K. Haarstad. 2010. Pesticide runoff from greenhouse production. Water Sci. Technol. 61:1373 1381. Ross, M.A. and D.J. Childs. 1996. Herbicide mode - of - action summary. Purdue Univ. Coop. Ext. 4. 8 May 2019. . Rózsavölgyi, T. and F. Horváth. 2008. Effect of pyrethroid insecticides on the photosynthetic activity of pea mesophyll protoplasts. Acta Biol Szeged 52:233 235. Saladin, G., C. Magné, and C. Clément. 2003. Effects of fludioxonil and pyrimethanil, two fungicides used against Botrytis cinerea , on carbohydrate physiology in Vitis vinifera L. Pest Mgt. Sci. 59:1083 1092. Samuelsen, E.D., N. Badawi, O. Nybroe, S.R. Sørensen, and J. Aamand. 2017. Adhesion to sand and ability to mineralise low pesticide concentrations are required for efficient bioaugmentation of flow - through sand filters. Appl. Microbiol. Biotechnol. 101:411 421. Sandmann, G., P.M. Bramley, and P. Böger. 1980. The inhibitory mode of action of the pyridazinone herbicide norflurazon on a cell - free carotenogenic enzyme system. Pestic. Biochem. Physiol. 14:185 191. Schmalfuß, J., B. Matthes, P. Mayer, and P. Böger. 1998. Chloroacetamide mode of action, I: Inhibition of very long chain fatty acid synthesis in Scenedesmus acutus . Zeitschrift für Naturforsch. 53c:995 1003. Shaner D. 2006. An overview of glyphosate mode of action: Why is it such a great herbicide? In North Central Weed Science Society Proceedings. 61:94. Shipitalo, M.J., J. V. Bonta, E.A. Dayton, and L.B. Owens. 2010. Impact of grassed waterways and compost filter socks on the quality of surface runoff from corn fields. J. Environ. Qual. 39:1009 1018. Southern Nursery Association. 2013. Best management practices: Gui de for producing nursery crops. Third. Southern Nursery Association, Inc. GA. 29 Mar. 2019. . 39 Spiers, J.D., T. Davies, C. He, K.M. Heinz, C.E. Bogran, and T.W. Starman. 2008. Do insecticide affect plant growth and developm ent? Greenhouse. Grower. 2:1 3. Staats, D., D. Hillock, and J.E. Klett. 1998. Weed control and phytotoxicity of preemergence herbicides applied to container - grown herbaceous plant. HortTechnology 8:325 328.. Stearman, G.K., D.B. George, K. Carlson, and S. Lansford. 2003. Pesticide removal from container nursery runoff in constructed wetland cells. J. Environ. Qual. 32:1548 1556. Stevens, M.M., R.F. Reinke, N.E. Coombes, S. Helliwell, and J. Mo. 2008. Influence of imidacloprid seed treatments on rice germina tion and early seedling growth. Pest Mgt. Sci. 64:215 222. Straw, N.A., N.J. Fielding, and A. Walters 1996. Phytotoxicity of insecticides used to control aphids on Sitka spruce, Picea sitchensis (Bong.) Carr. Crop Protection 15:451 459. Tjosvold, S., D. Chambers, and S. Koike. 2005. Evaluation of fungicides for the control of Phytophthora ramorum infecting rhododendron, camellia, viburnum and pieris. Sudden Oak Death Second Science Symposium: The State of Our Knowledge, January 18 - 21, 200 5, Monterey, California. 1. 8 May 2019. . University of Wisconsin - Extension. 2013. Corn and Soybean Herbicide Chart. Univ. Wisconsin. 1. 8 May 2019. < http://www.soybeanresearchinfo.com/pdf_docs/weeds_herbicide_MOA.pdf > Untiedt, R. and M.M. Blanke. 2004. Effects of fungicide and insecticide mixtures on apple tree canopy photosynthes is, dark respiration and carbon economy. Crop Protection. 23:1001 1006. U.S. Department of Agriculture, National Agricultural Statistics Service. 2016. U.S. Horticulture in 2014: Results from the 2014 Census of Horticultural Specialities. ACH12 - 33. Van As sche, F. and H. Clijsters. 1990. Effects of metals on enzyme activity in plants. Plant. Cell Environ. 13:195 - 206. Vea, E., and C. Palmer. 2017a. IR - 4 Ornamental horticulture program powdery mildew efficacy. The IR - 4 Project. 8 May 2019. < http://www.ir4pro ject.org/about - environmental - horticulture/environmental - horticulture - research - summaries>. Vea, E., and C. Palmer. 2017b. IR - 4 Ornamental horticulture program isoxaben crop safety. The IR - 4 Project. 8 May 2019. . Vilsack, T., and J.T. Reilly. 2013. 2012 Census of agriculture. Farm and Ranch Irrigation Survey (2013). USDA, Natl. Agr. Stat. Serv. 8 May 2019. Vinit - Dunand, F., D. Epron, B. Alaoui - Sossé, and P.M. Badot. 2002. Effects of copper on growth and on photosynthesis of mature and expanding leaves in cucumber plants. Plant Sci. 163: 53 58. nová. 2015. The use of constructed wetlands for removal of pesticides from agricultural runoff and drainage: A review. Environ. Intl. 75:11 20. Warsaw, A.L., R.T. Fernandez, B.M. Cregg, and J.A. Andresen. 2009. Container - grown ornamental plant growth and w ater runoff nutrient content and volume under four irrigation treatments. HortScience 44:1573 1580. Warsaw, A.L., R. Thomas Fernandez, D.R. Kort, B.M. Cregg, B. Rowe, and C. Vandervoort. 2012. Remediation of metalaxyl, trifluralin, and nitrate from nursery runoff using container - grown woody ornamentals and phytoremediation areas. Ecol. Eng. 47:254 263. Wauchope, R.D., S. Yeh, J.B.H.J. Linders, R. Kloskowski, K. Tanaka, B. Rubin, A. Katayama, W. Kördel, Z. Gerstl, M. Lane, and J.B. Unsworth. 2002. Pesticide soil sorption parameters: Theory, measurement, uses, limitations and reliability. Pest Mgt. Sci. 58:419 445. Werres, S., S. Wanger, T. Brand, K. Kaminski, and D. Seipp. 2007. Survival of Phytophthora ramorum in recirculating irrigation water and subsequent infection of rhododendron and viburnum. Plant Dis. 91:1034 1044. White, S., J. Owen Jr, J. Majsztrik, T. Fernandez, P. Fisher, C. Hall, T. Irani, J. D. Lea - Cox, J. P. Newman, and L. Oki. 2013. Grower identified priorities for water research in ornamental crops. Southern Nursery Association Research Conference Vol. 57:299 301. Will, E. and J.E. Faust. 2010. Irrigation water quality for greenhouse production. Univ. Tennessee Agr. Ext. Serv. PB 1617. Wilson, S.K. and S. von Broembsen. 2015. Capturing and re cycling irrigation runoff as a pollution prevention measure. Oklahoma State Univ. Ext. BAE - 1518. 8 May 2019. < http://pods.dasnr.okstate.edu/docushare/dsweb/Get /Document - 7408/BAE - 1518web.pdf >. Wilson, C., P. Strimple, S. Wilson, and J. Albano. 2005. Nontarget deposition of methiocarb applied to a foliage plant staging area. Bul. Environ. Contamination Toxicology 74:509 517. Wilson, P.C., T. Whitwell, and M.B. Ril ey. 1995. Effects of ground cover and formulation on herbicides in runoff water from miniature nursery sites. Weed Sci. 43:671 677. Wilson, C., T. Whitwell, and M.B. Riley. 1996. Detection and dissipation of isoxaben and trifluralin in containerized plant nursery runoff water. Weed Sci. 44:683 688. Wines, M. 2014. Colorado river drought forces a painful reckoning for states. New York Times.29 Mar. 2019. . World Economic Forum. 2015. Global Risks 2015. World Econ. Forum, Geneva Switz. REF: 090115. 8 May 2019. < http://www3.weforum.org/docs/WEF_Global_Risks_2015_Report15.pdf >. Xia, X.J., Y.Y. Huang, L. Wang, L.F. Huang, Y.L. Yu, Y.H. Zhou, and J.Q. Yu. 2006. Pesticides - induced depression of photosynthesis was alleviated by 24 - epibrassinolide pretreatment in Cucumi s sativus L. Pestic. Biochem. Physiol. 86:42 - 48. Zhang, H., and C. Hong. 2017. The pH factor. Nurs. Mangement 6. 8 November 2018. . 42 SECTION II PHOSPHORUS REQUIREMENT FOR BIOMASS ACCUMULATION IS HIGHER COMPARED TO PHOTOSYNTHETIC BIOCHEMISTRY FOR THREE ORNAMENTAL SHRUBS 43 Phosphorus requirement for biomass accumulation is higher compared to photosynthetic biochemistry for three ornamental shrubs Shital Poudy al 1,* , James S. Owen Jr. 2 , Thomas D. Sharkey 3 , R.T. Fernandez 1 , and Bert Cregg 1,2 1 Department of Horticulture, 1066 Bogue Street, Michigan State University, East Lansing, MI 48824 2 USDA Agricultural Research Service, Application Technology Research, 1680 Madison Avenue, Wooster, OH, USA 44691 3 Department of Biochemistry and Molecular Biology, Michigan State University, 612 Wilson Rd, Rm 210 East Lansing, MI, USA 48824 4 Department of Forestry, Michigan State University, 480 Wilson Road, East Lansing, MI, USA 48824 * Corresponding author : cregg @msu.edu This section has been submitted to Scientia Horticulturae 44 Abstract Ornamental nursery producers grow a variety of plant species and rely heavily on water and nutrient applications to maximize plant growth and quality. We conducted the current study to understand the morpho - physiological basis of plant response to phosphorus (P) concentration and identify optimum phosphorus concentratio n required for three common woody ornamental taxa; Hydrangea quercifolia Bartr . Cornus obliqua Physocarpus opulifolius Maxim. with a complete nutrient solution that varied only in P concentration (0, 1, 2, 4, 6, 8 mg·L - 1 ). For total dry biomass growth, the optimum P concentration was close to 7 mg·L - 1 for all three ta xa. However, P. opulifolius required less phosphorus for maximum growth index (plant height + plant width in two directions) compared to H. quercifolia and C. obliqua . Phosphorus concentration below 4 mg·L - 1 reduced leaf size and resulted in greater partit ioning of biomass and phosphorus to root growth. Analysis of responses of photosynthesis to intercellular carbon dioxide ( A/Ci curves) indicated a continuous increase in photosynthetic parameters to increasing phosphorus concentrations. Rate of rubisco for carboxylation ( V cmax ), RuBP regeneration rate (J) and rate of triose phosphate use ( TPU ) limited photosynthesis in phosphorus - deficient plants for all three taxa. However, P requirement for photosynthesis biochemistry was less compared to growth. Light - ha concentrations for growth and photosynthetic biochemistry ranged between 4 and 7 mg·L - 1 , depending on taxa. These P concentrations are lower than common recomme ndations and less than the amounts provided by typical commercial fertilizers. Application of phosphorus above 7 mg · L - 1 is above that needed for growth and physiological function and could contribute to phosphorus runoff from nurseries. 45 Keywords : A/Ci curves; gas exchange; nursery management; phosphorus partitioning 46 1. Introduction Horticulture is a large and economically significant industry, both in the U.S. and around the world. In the U.S., the sale of horticultural crops was worth $13.8 billion in 2014 (United States Department of Agriculture National Agricultural Statistics Service, 2016). Landscape nursery production is one of the major sectors of horticulture and requires frequent, usually daily, irrigation and continuous additions of mineral nutrients when the crops are actively growing. For container - grown ornamentals, growers seek to optimize irrigation by applying enough water to meet evapotranspiration loss while leaching out deleterious accumulated soluble salts. A commonly cited best man agement practice (BMP) is to irrigate to the level that results in 10 - 20% leaching of applied water (leaching fraction) (Bilderback et al., 2013) although even this recommendation may lead to over - irrigation (Pershey et al., 2015). Nonetheless, in practice , application of water often exceeds the BMP, and a large portion of irrigation water may be lost as agrichemical laden irrigation return flow (Warsaw et al., 2009; Danelon et al., 2010). Container nursery crops in the U.S. are typically grown in soil - less media, composed primarily of softwood bark. Phosphorus leaching is higher in soil - less media (pine bark, sphagnum peat, vermiculite or sand) in comparison to regular soil (Broschat, 1995) due to inherent phosphorous load from the bark itself along with po or retention due to low anion exchange capacity and preferential flow through the porous substrate when irrigated (Owen et al., 2008; Fields et al., 2014). Thus, 30 - 60% of applied P is commonly leached when using bark based substrate (Newman, 2014). This l eachate and resultant irrigation return flow can pollute surface and groundwater systems (United States Environmental Protection Agency, 2005, 2016). Water quality concerns associated with nursery and greenhouse irrigation return flow, such as eutrophicat ion and algal blooms, are primarily related to nitrogen and P present in runoff water 47 (Conley et al., 2009; Paerl, 2009; Fulcher et al., 2016). In 2014, P runoff was the primary cause of harmful algal blooms (HABs) in Lake Erie that left more than half a m illion people without drinking water (Michalak et al., 2013; Watson et al., 2016). Lowering P fertilization is an option for nursery growers to protect water resources, but lowering P below the sufficiency threshold will reduce plant growth and quality. Fo r container - grown ornamentals, 5 to 10 mg·L - 1 of substrate extractable P is often considered the target level for optimum plant growth (Broschat and Klock - Moore, 2000; Ristvey et al., 2004; Zhang et al., 2004; Bilderback et al., 2013) but many liquid and c ontrol release fertilizers commonly used in the landscape nursery trade provide 15 to 50 mg·L - 1 of P when applied at labeled rates (Broschat, 1995; Soti et al., 2015). Phosphorus requirements of plants vary by taxa, and recent studies suggest the possibili ty of reducing P below 10 mg·L - 1 without compromising plant growth and quality (Shreckhise et al., 2018, 2019b). For example, achieved maximum shoot dry weight at 4.7 m g·L - 1, and 2.9 mg·L - 1 of P, respectively. For Lantana - 1 was sufficient for optimum growth at the reproductive stage of the plant (Kim and Li, 2016). In contrast, Impatiens hawkeri Bull. `Paradise Violet', and Catharanthus roseus `Pacifica Red' Vinca grown in soil - less substrate had maximum growth and shoot dry weight at 31 mg·L - 1 and 23 mg·L - 1 of P application (Whitcher et al., 2005). Increasing P also increases th e shoot - to - root ratio by increasing shoot mass (Biddinger et al., 2019), as seen in L. camara, where increasing P from 3 mg·L - 1 to 5 mg·L - 1 increased shoot - root ratio (Kim and Li, 2016). The impacts of P availability on growth reflect the integration of its effect on physiological processes, particularly photosynthetic biochemistry. Phosphorus is an essential plant nutrient that is present in plants in various membranes, nucleic acids, and energy compounds (Armstrong, 48 1999). The effects of P deficiency on growth may be due to both source and sink limitations on photosynthesis (Pessarakli, 2005). Phosphorus is required for numerous physiological processes, including light reactions and Calvin - Benson cycle of photosynthesis (Brooks, 1986; Poorter et al., 201 0). Phosphorus deficiency reduces photosynthesis by limiting RuBP regeneration (Fredeen et al., 1990), inhibiting ATP synthesis (Carstensen et al., 2018), inhibiting enzymes required in the Calvin - Benson cycle (Rao and Terry, 1989) and reducing stomatal co nductance (Martins et al., 2015). Phosphorus deficiency also reduces chlorophyll fluorescence (Nowak and Stroka, 2001) by lowering the efficiency of PSII, and damage the photosynthetic apparatus by increasing the production of free radicals (Xu et al., 200 7). Physiol ogical impacts in response to P are predominantly determined by the severity of P deficiency and the length of P starvation (Terry and Ulrich, 1973; Xu et al., 2007). Improving our understanding of the interrelationships between P effects on photosynthetic biochemistry and plant productivity may provide additional insights in optimizing P fertilization to maximize growth while minimizing adverse environmental impacts. T o date, holistic studies optimizing P fertilization for maximum physiological and morphological performance of plants, and quantifying benefits of lowering P in terms of P runoff are still lacking. Therefore, the goal of our study was to investigate the fe asibility of reducing P fertilization without reducing crop growth or quality and linking the benefits of lowering P to water quality preservation. Our specific objectives were to (1) determine the effect of P on photosynthesis (A/Ci curves, and light - adap ted fluorescence) and morphological responses (growth index, total dry weight, root - shoot ratio, leaf number, and leaf size) in three different ornamental plant taxa, (2) identify the type of photosynthetic limitation that may result from P deficiency, and (3) categorize P partitioning to plant growth and P runoff. 49 2. Materials and Methods 2.1 . Experimental Setup The experiment was conducted in a research greenhouse at Michigan State University, East Lansing, MI, USA . H. quercifolia C. obliqua Raf. P. opulifolius Wine® ninebark) grown in 11.36 L containers were used for the study. All the plants were planted as liners from 10 - cm diameter pl ug cells on May 6, 2016, in a mixture of aged pine bark (85% volume) and peat moss (15% volume) (Renewed Earth LLC, Otsego, MI, USA) without any amendments. Before the current study, plants were grown outdoors under typical nursery practices for the region ; 19 mm of daily overhead irrigation and top - dressed with controlled - release fertilizer (19:1.75:6.65; N:P:K) with micronutrients (5 - Plants were brought into an unheated hoop - house covered with 0.15 mm p oly film on the October 28, 2016, where they received partial chilling outdoors. Pots were carefully checked for residual fertilizer prills, which were removed even though it was already past the six months fertilizer release period. Plants were then moved to a walk - in cooler (6 º C) for five weeks to complete their chilling requirement. Plants were brought into the greenhouse on January 11, 2017 and observed until bud - break. The temperature in the greenhouse was set to 22°C for 18 hours (6:00 to 24:00) and 20°C for the remaining 6 hours. Supplemental lighting was provided by high - pressure sodium lamps in the greenhouse that automatically turned on when the photosynthetic photon flux density was lower than 440 µmol·m - 2 ·s - 1 and provided a 16 - h photoperiod. 50 2. 2 . Phosphorus treatments Fertigation started on January 26, 2017. Phosphorus was applied as potassium phosphate (potassium phosphate dibasic, Sigma - Aldrich, St. Louis, Missouri) mixed in irrigation water (fertigation). Plants received one of six P treatmen t concentrations (0, 1, 2, 4, 6, or 8 mg·L - 1 ). Each treatment had six individual plant replications per taxa. After the emergence of leaves, each plant was hand watered with a solution consisting 100 mg·L - 1 of nitrogen (urea), 60 mg·L - 1 of potassium (muria te of potash), 80 mg·L - 1 of micr onutrients (Table 4 - Micromax® micronutrients, ICL Fertilizers, Dublin, Ohio) and the designated rate of P. The fertilizer solutions were precisely measured for each irrigation event and completely dissolved in irrigation w ater before application. After each fertigation event, leachate was collected in saucers placed under each container and measured for volume. Irrigation amounts were routinely adjusted to account for changes in plant water use during the study, targeting a 15 - 20% leaching fraction (leached volume/nutrient solution applied). During early growth (February to March 2017), fertigation was less frequent (weekly or two times a week), beginning April 2017, plants started growing vigorously; therefore, fertigation frequency was increased (once every other day to every day). The experiment was carried out for six months, until July 17, 2017. 2.3 . Growth measurements Growth index (GI; the average value of the sum of plant height and two perpendicular widths) of each p lant was measured at the end of the study on July 10, 2017. Following the final growth measurements, all leaves were detached from the stems of each plant, counted, and leaf area was determined using a leaf area meter (LI - 3100, LI - COR Inc., Lincoln, NE USA ). Leaf size (cm 2 leaf - 1 ) was calculated as total plant leaf area / number of leaves per plant. Leaves, stems, and 51 roots were dried and weighed. Root and shoot dry weights were summed to calculate total dry biomass (TDB), and the root - to - shoot ratio was c alculated by dividing root mass with shoot mass. 2.4 . Phosphorus partitioning Our experimental protocol allowed us to develop a P budget for each plant. To estimate the amount of actual P applied and P leached, fertilizer solutions and leachates were anal yzed for total phosphorus content using flow injection analysis digestion method in Lachat (Model: QuikChem 8500 series 2 with Total Nitrogen and Total Phosphorus manifolds) at seven different times during the course of study. Tissue P concentration in lea ves and stems was determined from each plant, while P concentration in roots was determined from three (out of six) subsamples for each treatment. Samples for tissue P concentration were sent to a commercial plant laboratory (Waters Agricultural Laboratori es, Camilla, GA USA) for analysis where plant tissues were analyzed using the wet digestion method combined with the inductively coupled plasma (ICP) method (Cunniff and Association of Official Analytical Chemists International, 1995) . This approach allowe d us to formulate a P budget based on the amount applied, taken up by the plant, and the amount leaving the container as leachate. P applied = P uptake + P leached + P substrate Where: P applied = total phosphorus applied during the experiment P uptake = phosphorus taken up by each plant during the experiment, calculated as the sum of phosphorus in leaves, stems, and roots - initial phosphorus in stems and roots. P leached = total phosphorus leached from each container 52 P substrate = total phosphorus stored in the substrate at the end of the study calculated as; total phosphorus applied (phosphorus taken up by plants + total phosphorus leached from the container). 2.5 . Physiological measurements A portable photosynthesis system (LI - 6400 XT, LI - COR, Inc., Lincoln, NE) equipped with a fluorescence chamber head (LI - 6400 - 40, LI - COR, Inc., Lincoln, NE) was used to measure photosynthesis and light - watering during the entire course of physio logical measurements; hence, they did not show any sign of water stress. 2.5.1. A/Ci curves A section of a fully expanded healthy leaf was enclosed in the fluorescence chamber head at either the 3 rd or 4 th node from the top for P. opulifolius and C. obliqu a and at the 1 st or 2 nd node for H. quercifolia . Photosynthetically active radiation (PAR) in the chamber was set to 1500 µmol·m - 2 ·s - 1 . The block temperature on the chamber head was set to 25°C and the reference CO 2 was varied, starting at 400 ppm, gradual ly decreased to 0 ppm and then increased to 800 ppm (400,300,200,100, 50,0, 300,400,600 and 800 ppm) (Singh et al., 2013) . Photosynthesis values at various internal carbon dioxide concentration ( Ci ) were used to generate A/Ci curves, using the non - linear r ectangular hyperbola model developed by Archontoulis and Miguez (2015) , Where y = photosynthesis, x = Intercellular CO 2 concentration, Yasym = Asymptotic value of Y, a = initial slope of curve at low x levels (<200 ppm) and Rd = dark respiration. 53 Data from A/Ci curves were used to estimate V cmax (the maximum velocity of rubisco for carboxylation), J (rate of photosynthetic electron transfe r for RuBP regeneration) and TPU (triose phosphate use) values, using the non - linear equation provided as an Excel spreadsheet by Sharkey (2016) . Curves used to generate values for V cmax , J, and TPU were visually observed for the best fit to minimize error s. In some cases, where the calculator estimated an unrealistic value of day respiration (Rd) and mesophyll conductance (gm), Rd was constrained to < 6 µmol·m - 2 ·s - 1 and gm was constrained to < 3 µmol·m - 2 ·s - 1 ·Pa - 1 . 2.5.2 Chlorophyll fluorescence Quantifying light - adapted chlorophyll fluorescence ( ) can determine the rate of electron transport or the efficiency of PSII (Murchie and Lawson, 2013) ; therefore, a section of a healthy leaf (similar as above), of each plant, was enclosed in the same chamber with 1500 µmol·m - 2 ·s - 1 of PAR, 400 ppm CO 2 , and 40 - 60% humidity at 25°C. After approximately 5 minutes of acclimatization, was measured (LI - 6400XT, LI - COR Inc., Lincoln, NE, USA) to de termine the efficiency of PSII. 2.6 . Statistical analysis All data were analyzed separately for each taxa using SAS software (SAS Institute Inc., Cary, NC, USA). A logistic growth curve was used to determine optimum P fertilization for GI and TDB (Archontoulis and Miguez, 2015; Shreckhise et al., 2018) , Where, y = either GI or TDB, c = asymptote of the curve, a = growth rate and b = inflection point of maximum growth rate. 54 The equation for the logistic growth curve was differentiated to find the point of asymptotic deceleration for GI and TDB (Mischan et al., 2011) using the fourth order derivative adapting the process of Shreckhise et al. (2018) . For all other comparisons, data were analyzed using one - way ANOVA. Post - hoc mean nce (LSD). Correlations among morpho - physiological variables were analyzed u sing Pearson correlation test. 3. Results 3.1. Morphological response to phosphorus concentration Plant growth response to P concentration varied by taxa (Fig. 1a and 1b). Growth Index (GI) and Total Dry Biomass (TDB) were lowest for plants not receiving any P. The response of GI and TDB to P concentration followed a logistic growth curve model. Using a fourth - order derivative for each model we determined the optimum P concentratio n for each taxon. For P. opulifolius and H. quercifolia ; optimum P for GI was achieved at 3.44 and 6.3 mg·L - 1 , respectively (Fig. 1a). Optimum P concentration for GI for C. obliqua could not be calculated within the range of phosphorus that we applied, usi ng the logistic growth curve. Optimum P for TDB was achieved at 6.94, 6.76 and 6.54 mg·L - 1 of phosphorus for P. opulifolius , H. quercifolia, and C. obliqua ; respectively (Fig. 1b). Leaf number (LN), leaf size (LS; total leaf area per plant /number of leaves per plant), and total leaf area per plant (TLA) increased with increasing P concentration (Fig. 2). P. opulifolius and C. obliqua had maximum leaf numbers at 8 mg · L - 1 of P while H . quercifolia had maximum leaf number at 4 mg · L - 1 of P. Phosphorus concentrations below 4 mg·L - 1 reduced LS, while those 55 above 4 mg·L - 1 did not affect LS on any of the taxa (Fig 2). Total leaf area per plant for C. obliqua and H. quercifolia reached a maxi mum at 6 mg·L - 1 of phosphorus while that for P. opulifolius was maximum at 8 mg·L - 1 of P. Visual symptoms of P deficiency, i.e., shorter internodes, purpling of leaves, and smaller leaf sizes were observed in all three taxa at 0 and 1 mg·L - 1 of P (Fig. 3). Increasing P concentration increased root and shoot growth for all three taxa. Roots had maximum dry weight at 6 mg·L - 1 of P for all three taxa. Maximum shoot dry weight was achieved at 6 mg·L - 1 of P for P. opulifolius and H. quercifolia and at 8 mg·L - 1 of P for C. obliqua (Table 1). Root - to - shoot ratio for P. opulifolius decreased from 0.93 to 0.52 when P concentration was increased from 0 mg·L - 1 to 2 mg·L - 1 , further increases in P concentration did not decrease root - to - shoot ratio. For H. quercifolia and C. obliqua root - to - shoot ratio decreased from 0.55 to 0.33 and 0.7 to 0.37, respectively, when the P concentration was increased from 0 mg·L - 1 to 4 mg·L - 1 , further increase in P concentration did not decrease root - to - shoot ratio (Fig 4). 3.2. Partitio ning of applied phosphorus In order to assess the fate of P applied, we compared the total amount of P in each fraction (P in leaves, stems, roots, and leachate). Non - substrate P (P in leaf, stem, root, and leachate) was higher than the total P applied fo r 0 mg·L - 1 of P treatment for all three taxa. For all plants receiving 1 mg·L - 1 of P or more, the non - substrate P was lower than total P applied; hence some P should have been stored in the substrate. Leaves accounted for the largest fraction of P taken up by plants, except for P. opulifolius at the 0 and 1 mg·L - 1 concentrations, for which P content in roots accounted for > 50% of total plant P (Table 2). 56 For all three taxa, increasing P concentration increased the total amount of P output as leachate and P in the substrate (Table 2). Increasing P concentration from 0 to 2 mg·L - 1 increased P allocation to leaves, but beyond 4 mg·L - 1 of P, P allocation to le aves decreased (Table 2). Increasing P concentration increased P partition to leachate. For example, increasing P concentration from 1 mg·L - 1 to 8 mg·L - 1 increased P fraction into leachate from 10% to 17% respectively and total P leached increased by 91% f or P. opulifolius , 59% for H. quercifolia and 70% for C. obliqua when P concentration was increased from 6 mg·L - 1 to 8 mg·L - 1 (Table 2). 3.3. Photosynthetic response to phosphorus concentration A/Ci curves were modeled with a non - linear model of a rectang ular hyperbola (R - squared > 0.96 for all three taxa). For all three taxa, increasing P increased net photosynthesis. Increases in photosynthesis associated with P concentration were consistently greater at higher values of Ci (> 300 ppm) (Fig 5). For P. o pulifolius , V cmax - 1 of P and reached a plateau at 2 mg·L - 1 of P (Fig. 6a). For H. quercifolia , V cmax - 1 - 1 of P. For C. obliqua , 0 mg·L - 1 of P had the lowest V cmax while P concentrati - 1 of P did not show a significant difference in carboxylation efficiency (Fig 6a). Photosynthetic limitation by the rate of electron transport was also evident at lower P concentrations. P. opulifolius and H. quercifolia had lowest electron - 1 of P, and the lowest electron transport rate for C. obliqua was at 0 mg L - 1 of P. P. opulifolius and C. obliqua had maximum electron - 1 of P, while H. quercifolia had maximum electron transport rate at mg·L - 1 of P (Fig 6b). Phosphorus concentration also affected photosynthesis limitation as a result of TPU but was less sensitive compared to V cmax and J . For all three taxa, TPU was lowest at the 57 - 1 - 1 of P. Therefore, increasing phosphorus from 2 to 8 mg·L - 1 did not increase TPU (Fig 6c) . Light - adapted fluorescence ( ) reached maximum levels at relatively low P concentrations for all three taxa (Fig. 7). Increasing P to 1 mg·L - 1 to a maximum for H. quercifolia and C. obliqua and 2 mg · L - 1 of P maximized fluorescence for P. opulifolius . 3.4. Correlation among morpho - physiological variables For all three taxa, TDB correlated (p<0.05) with P percentage in leaf (r = 0.87 P. opulifoli us ; r= 0.44 H. quercifolia ; r= 0.65 C. obliqua ) and average leaf size (r = 0.85 P. opulifolius ; r=0.91 H. quercifolia ; r=0.81 C. obliqua ) (Table 3). Biomass productivity (TDB) correlated well (r = > 0.56) with parameters related to photosynthetic biochemis try such as V cmax , J and TPU for all taxa (Table 3). Correlation order of TDB with those physiological parameters for all three taxa were in the order V cmax > J > TPU . Biomass productivity also correlated (r = > 0.45) with quantum efficiency of PS II ( ) for all three taxa but was weaker compared to photosynthetic biochemistry (Table 3). Foliar P concentration was only correlated with parameters related to photosynthetic biochemistry ( V cmax , J, TPU and ) for P. opulifolius and C. obliqua . Root - to - shoot ratio was negatively correlated with TDB for all taxa (r = - 0.73 P. opulifolius ; r = - 0.73 H. quercifolia ; r = - 0.62 C. obliqua ) and with P concentration in leaf for P. opulifolius (r= - 0.78) and C. obliqua (r = - 0.66) (Table 3). 58 4. Discuss ion Plant productivity is the integrated result of leaf surface area accretion, net photosynthetic activity, and allocation of photosynthate to plant organs. In the current study, P concentration affected all aspects of plant productivity. 4.1. Morphologic al response to phosphorus concentration For all three taxa, optimum P concentration required for maximum GI and maximum dry mass accumulation varied but was always less than 7 mg·L - 1 . This is consistent with recent observations that growth of woody ornamen tals may be maximized at 2.9 to 4.7 mg·L 1 (Shreckhise et al., 2018) . Leaf size for all three taxa followed similar trend as GI and TDB, as leaf size increased with P up to 4 mg·L - 1 with no further increase at higher P concentrations. Phosphorus concentrat ion of 8 mg·L - 1 for P. opulifolius and 6 mg·L - 1 for C. obliqua and H. quercifolia produced maximum total leaf area per plant. Hence, P fertilization is required for leaf expansion and growth. Increases in P concentration was reported to increase leaf area in common bean, sunflower ( Helianthus annuus ) and white clover ( Trifolium repens ) (Lynch et al., 1991; Rodríguez et al., 1998; Høgh - Jensen et al., 2002) . Root - to - shoot ratio was maximum at 1 mg · L - 1 of P for all three taxa and minimum at 2 mg · L - 1 of P for P. opulifolius , and at 4 mg · L - 1 of P for H. quercifolia and C. obliqua . Thus, these results reveals the effect of phosphorus on carbon · L - 1 ), phosphorus will be utilized more for root growth but when P · L - 1 ) it will be more readily used for shoot growth, since P acquisition is easily obtained from the labile pool of P in the substrate. Root - to - shoot ratio had a negative correlation to TDB and P % in leaf. Therefore, decreas ing root - to - shoot ratio was primarily because of increase in shoot growth. 59 4.2. Fate of applied phosphorus Combined P in leachate and plant tissue was greater than total P applied for treatment receiving 0 mg·L - 1 of P as plants used P stored in the substrate when external phosphorus was not supplied. Ristvey et al., (2007), observed a similar response in container - grown azalea ( Rhododendron week - 1 . The total amount of P i n leaves and stems increased with increasing P concentration (to 8 mg·L - 1 for H. quercifolia and C. obliqua and to 6 mg·L - 1 for P. opulifolius ), but according to our model, P application beyond 6.94 mg·L - 1 did not increase GI, TDB or any physiological perf ormance in any of the taxa. Therefore, the increase in tissue P content when P is applied above this concentration indicates luxury consumption; i.e, absorption and storage of P beyond the current plant requirement, which also has been observed in a wide r ange of plant species including container - grown plants (Ristvey et al., 2007) and forest trees (Lawrence, 2001) . Increasing P concentration increased P loss in leachate and total amount of P in the substrate. Similar observation was made in other studies w here increasing P application increased phosphorus loss from the system (Ristvey et al., 2007; Shreckhise et al., 2018, 2019a) . Therefore, increasing P beyond the optimum requirement would have no benefit on growth and physiological processes but could inc rease the amount of phosphorus in runoff. Hence, we would not recommend application of phosphorus over the optimum requirement of 6.94 mg·L - 1 for P. opulifolius 6.76 mg·L - 1 for H. quercifolia and 6.54 mg·L - 1 for C. obliqua . 4.3 Physiological performance i n response to phosphorus concentration To fix one molecule of CO 2 , in Calvin - Benson cycle, three molecules of ortho - phosphate (PO 4 ) are required (Walker and Robinson, 1978) ; therefore P deficiency can limit photosynthesis. 60 In our study increasing P concen tration, up to a point, increased net CO 2 assimilation for a wide range of intercellular CO 2 concentrations (0 to 600 ppm) in all three taxa. Similar increases in net assimilation with increasing P concentration was observed in sunflower ( Helianthus annuus L. cv Asmer), maize ( Zea mays L. cv Eta) (Jacob and Lawlor, 1991) and pine seedling (Loustau et al., 1999) . Photosynthesis in light - saturated conditions can be rubisco limited, RuBP limited, or TPU limited; and a well - constructed carbon dioxide response ( A/Ci ) curve can be used to determine the type of limitation (Farquhar et al., 1980; Sharkey, 2016) . For H. quercifolia and C. obliqua , P concentrations < 4 mg·L - 1 reduced carboxylation rate. For those two taxa, photosynthesis at lower P concentrations (< 4 mg·L - 1 ) was reduced partly because of the limited supply of rubisco enzyme. For P. opulifolius , rubisco restricted photosynthesis only at < 2 mg·L - 1 of phosphorus. The rate of RuBP regeneration may also limit photosynthesis in phosphorus deficient plants. For P. opulifolius and C. obliqua , photosynthesis was limited by RuBP regeneration at phosphorus concentrations < 2 mg·L - 1 . For H. quercifolia RuBP regeneration limited photosynthesis at < 4 mg·L - 1 of phosphorus. At higher rates of photosynthesis, export of carbon compounds from Calvin - Benson cycle slows down, causing a reduction in photosynthesis (Yang et al., 2016) also referred to as TPU limited photosynthesis. In our study, 2 mg · L - 1 of P was sufficient to overcome the limitation caused by TPU for all t hree taxa. Therefore, photosynthesis limitations caused by rubisco and RuBP regeneration were more sensitive compared to the limitation caused by TPU . This sensitivity is also further verified by the correlation analysis of TDB with V cmax , J and TPU . Phosp horus deficiency has also been observed to reduce V cmax and J max for several other taxa (Loustau et al., 1999; Lin et al., 2009; Singh et al., 2013) . In contrast to the parameters of the Calvin - Benson cycle, light utilization by plants of all three taxa wa s less affected by P concentration. For all three taxa, plants that received no P had lower compared to plants that received P, and 1 mg · L - 1 of 61 P for H. quercifolia and C. obliqua and 2 mg · L - 1 of P for P. opulifolius was sufficient to maximize . Other studies have observed no reduction in chlorophyll content and light harvesting capacity at low phosphorus rates (Brooks, 1986; Campbell and Sage, 2006) . In our study, light harvesting capacity was reduced when no P was supplied, but a low rate o f P was sufficient for optimum functioning of photosystem II. 5. Conclusion For all three taxa, GI and TDB were the parameters that were most sensitive to P application thus, needed higher P concentrations compared to other morphological parameters and ph ysiological variables. Analysis of A/Ci curves indicated a broader response to P concentration compared to light - adapted chlorophyll fluorescence, thus, suggesting an overall photosynthetic response to be phosphorus driven more by photosynthetic biochemist ry rather than light harvesting reactions. When compared among all three taxa, reduction in carboxylation rate (rubisco limited) was the main reason for reduction in photosynthesis followed by the rate of electron transport (RuBP regeneration) then by trio se phosphate use ( TPU ). Overall, GI and TDB were optimized at approximately 7 mg · L - 1 of P for all three taxa, which is much lower than those in water - soluble fertilizers or P release rate of controlled - release fertilizers that are commonly available and u sed in the nursery industry. Therefore, nursery growers may be able to reduce P fertilization without reducing crop growth. Even a slight reduction in phosphorus rates over a long period can substantially reduce total phosphorus runoff. For example, if P c oncentration were lowered, from 8 mg·L - 1 to 6 mg·L - 1 , leachate P concentration would be reduced by 59 - 91% depending on taxa grown. Reducing P in irrigation return flow can 62 morphological processes across many ornamental taxa. 63 APPENDIX 64 APPENDIX Figure II - 1 . Growth index (A) and total dry biomass (B) of P. opulifolius H. quercifolia C. obliqua phosphorus concentration. Non - linear regression curves (logistic growth curves) are plotted for both GI and TDB. Standard errors of the means are denoted as vertical lines on the curves. 65 Figure II - 2 . Leaf number per plant, leaf size, and total leaf area per plant for P. opulifolius C. obliqua H. quercifolia phosphorus concentration. Standard er separations for each taxa were carried out using Least Significant Difference (LSD) post - hoc test. Means within a taxon that are followed by same letters are not significantly different at p=0.05. 66 Figure II - 3 . Representative plants for each P concentration of P. opulifolius H. quercifolia C. obliqua - 1 for 6 months in the greenhouse. 67 Table II - 1 . Root and shoot dry weight (g) of P. opulifolius H. quercifolia C. obliqua separ ations were carried out using Fisher Least Significant Difference (LSD) post hoc test when appropriate. Means within a taxon that are followed by same letters are not significantly different at given p values. P (mg·L - 1 ) P. opulifolius H. quercifolia C. ob liqua Root Shoot Root Shoot Root Shoot 0 10.09c 10.94d 7.75c 14.22d 8.70e 12.8d 1 10.09c 12.78d 6.70c 12.86d 11.21de 19.28d 2 12.70c 24.39c 10.70c 25.44c 13.26cd 28.10d 4 17.59b 36.83b 15.01b 45.17b 21.49bc 55.98c 6 18.22ab 44.30a 17.31ab 52.84ab 30.87a 74.06b 8 21.88a 47.42a 19.39a 58.68a 25.9ab 83.19a p - value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 68 Table II - 2 . Partitioning of applied phosphorus to leachate, leaf, stem and root, including the amount of P stored in the substrate for P. opulifolius H. quercifolia C. obliqua Fi sher Least Significant Difference (LSD) post - hoc test. Means that are followed by same letters are not significantly different given p value . P (mg·L - 1 ) Total - P input (mg) = Total P in leachate (mg) Total P in leaf (mg) Total P in stem (mg) Total P in root (mg) Total P in substrate (mg) Percent of P in leachate Percent of P in leaf Percent of P in stem Percent of P in root Percent of P in substrate 0 0.58 = 1.44d 3.84d 1.31c 6.49b - 12.50d - - - - - 1 22.53 = 2.31cd 5.53d 1.72c 4.44b 8.53c 10.25 24.55 7.63 19.71 37.86 2 40.31 = 3.62c 12.69c 5.06c 7.51b 11.43bc 8.98 31.48 12.55 18.63 28.36 4 78.99 = 11.71b 24.20b 11.57b 17.49a 14.02bc 14.82 30.64 14.65 22.14 17.75 6 119.04 = 18.43b 30.78a 18.80a 26.13a 24.9b 15.48 25.86 15.79 21.95 20.92 8 204.25 = 35.23a 36.70a 23.98a 29.37a 78.97a 17.25 17.97 11.74 14.38 38.66 p - value <0.0001 <0.0001 <0.0001 <0.0005 <0.0001 0 0.55 = 1.73f 5.45d 2.45bc 5.28bc - 14.36d - - - - - 1 21.04 = 2.31e 8.52d 1.36c 3.22c 5.65cd 10.97 40.49 6.46 15.21 26.85 2 37.64 = 3.37d 18.20cd 2.64bc 6.1761bc 7.27cd 8.95 48.35 7.01 16.37 19.31 4 73.73 = 5.68c 30.56bc 5.84ab 10.29ab 21.36bc 7.7 41.45 7.92 13.95 28.97 6 111.13 = 18.77b 37.11ab 6.28ab 10.37ab 38.60b 16.89 33.39 5.65 9.33 34.73 8 192.69 = 29.76a 48.0a 6.98a 16.24a 89.71a 15.44 25.95 3.62 8.43 46.56 p - value <0.0001 <0.0005 <0.05 <0.01 <0.0001 69 Table II - 2 C. obliqua 0 0.59 = 2.72d 4.87d 2.54d 0.69d - 10.23d - - - - - 1 21.91 = 2.67d 8.59cd 3.53cd 1.17d 5.94cd 12.19 39.2 16.12 5.36 27.12 2 39.19 = 3.37d 17.77c 5.77bcd 2.98cd 9.30bc 8.59 45.33 14.73 7.59 23.74 4 76.75 = 9.59c 33.82b 6.48bc 7.12c 19.75bc 12.49 44.06 8.44 9.28 25.71 6 115.65 = 20.02b 41.03b 8.13b 21.59a 24.89b 17.31 35.48 7.03 18.67 21.52 8 198.45 = 34.10a 58.51a 13.26a 16.24b 76.36a 17.18 29.48 6.68 8.18 38.48 p - value <0.0001 <0.0001 <0.0005 <0.0005 <0.0001 70 Figure II - 4 . Root - to - Shoot (R:S) ratio of P. opulifolius H. quercifolia C. obliqua separations were carried out using Fisher Least Significant Difference (LSD) post - hoc test and presented as inset table. Means within a taxon indicated by the same letter are not different at the given p value. Standard errors are denoted as vertical lines on the curves. 71 Figure II - 5 . Response of photosynthesis to increasing internal carbon dioxide concentration (A/Ci Curve) for P. opulifolius H. quercifolia C. obliqua ted as the mean of five replicates. All the curves followed non - linear model of rectangular hyperbola. R - squared values for all models for all three taxa were above 0.96. 72 Figure II - 6 . Maximum velocity of rubisco for carboxylation ( V cmax ) (A); rate of photosynthetic electron transport for RuBP regeneration ( J ) (B), and triose phosphate use ( TPU ) (C) of P. opulifolius H. quercifolia C. obliqua response to phosphorus c oncentration. Values of A/Ci Curves were analyzed based on equations provide by Sharkey (2016) to generate V cmax , J and TPU for each replication. Fisher Least Significant Difference (LSD) was used to compare means among phosphorus fertilization levels and presented as inset table. Means within a taxon indicated by the same letter are not different at given p - value. Standard errors are denoted as vertical lines on the curves . 73 Figure II - 7 . Light - response to increasing phosphorus concentration for P. opulifolius H. quercifolia C. obliqua phosphorus fertilization levels and p resented in as inset table. Means within a taxon indicated by the same letter are not different at given p value. Standard errors are denoted a s vertical lines on the curves. 74 Table II - 3 . Pearson's correlation coefficient for P. opulifolius H. quercifolia C. obliqua ratio, Leaf size (total leaf area per plant/ leaf number per plant), P% i n leaf is phosphorus percent in leaf by weight, V cmax is maximum velocity of rubisco for carboxylation, J is the rate of photosynthetic electron transport for RuBP regeneration, TPU is triose phosphate use and is the light - adapted fluorescence. Pe arson's correlation coefficient for P. opulifolius R/S ratio Leaf size P% in leaf V cmax J TPU TDB - 0.73*** 0.85*** 0.87*** 0.69*** 0.65*** 0.56** 0.45* R/S ratio - 0.74*** - 0.78*** - 0.60*** - 0.50** - 0.44* - 0.55*** Leaf size 0.75*** 0.62*** 0.58** 0.51** 0.42* P% in leaf 0.77*** 0.63*** 0.55** 0.48** V cmax 0.92*** 0.81*** 0.47* J 0.99*** 0.46* TPU 0.34 NS Pearson's correlation coefficient for H. quercifolia R/S ratio Leaf size P% in leaf V cmax J TPU TDB - 0.73*** 0.91*** 0.44* 0.77*** 0.75*** 0.68*** 0.51** R/S ratio - 0.68*** - 0.23 NS - 0.60*** - 0.56** - 0.52** - 0.52** Leaf size 0.44* 0.70*** 0.68*** 0.61*** 0.46** P% in leaf 0.31 NS 0.30 NS 0.24 NS 0.04 NS V cmax 0.92*** 0.89*** 0.44* J 0.98*** 0.47* TPU 0.47* Pearson's correlation coefficient for C. obliqua R/S ratio Leaf size P% in leaf V cmax J TPU TDB - 0.62*** 0.81*** 0.65*** 0.78*** 0.7*** 0.69*** 0.51** R/S ratio - 0.75*** - 0.66*** - 0.6** - 0.59** - 0.58** - 0.53** Leaf size 0.53** 0.7*** 0.61** 0.60** 0.61** P% in leaf 0.65*** 0.56** 0.57** 0.43* V cmax 0.91*** 0.91*** 0.70*** J 0.99*** 0.68*** TPU 0.66*** *** p - - - p - value > 0.05. 75 Table II - 4 . Breakdown of micronutrients analysis with elements and concentration . Micronutrients Source Amount in percentage Calcium (Ca) Calcium Carbonate 6.00% Magnesium (Mg) Magnesium carbonate 3.00% Sulfur (S) Copper, zinc ferrous and manganese sulphate 12.00% Boron (B) Sodium borate 0.10% Copper (Cu) Copper sulfate 1.00% Iron (Fe) Ferrous sulphate 17.00% Manganese (Mn) Manganese sulfate 2.50% Molybdenum (Mo) Sodium molybdate 0.05% Zinc (Zn) Zinc sulfate 1.00% 76 LITERATURE CITED 77 LITERATURE CITED Archontoulis, S. V., and F.E. Miguez. 2015. Nonlinear regression models and applications in agricultu ral research. Agron. J. 107(2): 786 798. Armstrong, D.L. 1999. Functions of Phosphorus in Plants. p. 6 7. In Armstrong, D.L. (ed.), Better Crops with Plant Food. Potash & Phosphate Institute. Biddinger, E.J., C. Liu, R.J. Joly, and K.G. Raghothama. 2019. Physiological and Molecular Responses of Aeroponically Grown Tomato Plants to Phosphorus Deficiency. J. Am. S oc. Hortic. Sci. 123: 330 333. Bilderback, T., C. Boyer, M. Chappell, G. Fain, D. Fare, C. Gilliam, B.E. Jackson, J. Lea - Cox, A.V. LeBude, A. Niemiera, J. Owen, J. Ruter, K. Tilt, S. Warren, S. White, T. Whitwell, R. Wright, and T. Yeager. 2013. Best Manage ment Practices: Guide for Producing Nursery Crops. 3rd ed. Southern Nursery Association, Acworth, GA. Brooks, A. 1986. Effects of Phosphorus Nutrition on Photosynthetic Quantum Yield and Amounts of some Calvin - cycle Metabolites in Spinach Leaves. Aust. J. Plant Physiol. 13: 221 237. Broschat, T.K. 1995. Nitrate, phosphate, and potassium leaching from container - grown plants fertilized by se veral methods. HortScience 30: 74 77. Broschat, T.K., and K.A. Klock - Moore. 2000. Root and shoot growth responses to phos phate fertilization in container - gr own plants. HortT echnology 10: 765 767. Campbell, C.D., and R.F. Sage. 2006. Interactions between the effects of atmospheric CO2 content and P nutrition on photosynthesis in white lupin ( Lupinus albus L.). Plant, Cell Envi ron. 29: 844 853. Carstensen, A., A. Herdean, S.B. Schmidt, A. Sharma, C. Spetea, M. Pribil, and S. Husted. 2018. The impacts of phosphorus deficiency on the photosynthetic electron transport chain. Plant Physiol. 177: 271 284. Conley, D.J., H.W. Paerl, R.W. Howarth, D.F. Boesch, S.P. Seitzinger, K.E. Havens, C. Lancelot, and G.E. Likens. 2009. Controlling eutrophication : Nitrogen and phosphorus . Science 80 - 323. Cunniff, P., and Association of Official Analytical Chemists International. 1995. Official Methods of Analysis of AOAC. 16th ed. Arlington, VA: The Association, ©1995. Danelon, M., A. Kachenko, J. McDonald, C. Rolfe, and B. Yiasoumi. 2010. Nursery industry water management best practice guidelines 2010 (A Kachenko, Ed.). Nurs. Gard. Ind. Aust. (Appendix 13). Farquhar, G.D., S. von Caemmerer, and J.A. Berry. 1980. A biochemical model of photosynthetic CO2 assimilation in lea ves of C3 species. Planta 149: 78 90 . 78 Fields, J.S., W.C. Fonteno, B.E. Jackson, J.L. Heitman, and J.S. Owen. 2014. Hydrophysi cal properties, moisture retention, and drainage profiles of wood and traditional components for greenhou se substrates. HortScience 49: 827 832 . Fredeen, A.L., T.K. Raab, I.M. Rao, and N. Terry. 1990. Effects of phosphorus nutrition on photosynthesis in Glycine max (L.) Merr. Planta 181: 399 405 . Fulcher, A., A. V. LeBude, J.S. Owen, S.A. White, and R.C. Beeson. 2016. The next ten years: Strategic vision of water resources for nursery producers. Horttechnology 26: 121 132. Høgh - Jensen, H., J.K. Schjoerring, and J.F. Soussana. 2002. The influence of phosphorus deficiency on growth and nitrogen fixation of whit e clover plants. Ann. Bot. 90: 745 753. Jacob, J., and D.W. Lawlor. 1991. Stomatal and meshophyll limitations of photosynthesis in phosphate deficient su nflower, maize and wheat plants. J. Exp. Bot. 42: 1003 1011 . Kim, H. - J., and X. Li. 2016. effects of phosphorus on shoot and root growth, partitioning, and phosphorus utilization efficiency in Lantana . HortScience 51: 1001 1009. Lawrence, D. 2001. Nitrogen a nd phosphorus enhance growth and luxury consumption of four secondary forest tree spe cies in Borneo. J. Trop. Ecol. 17(6): 859 869 . Lin, Z.H., L.S. Chen, R.B. Chen, F.Z. Zhang, H.X. Jiang, and N. Tang. 2009. CO2 assimilation, ribulose - 1,5 - bisphosphate carb oxylase/ oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP - test, of tea leaves in response to phosph orus supply. BMC Plant Biol. 9: 1 12. Loustau, D., M. Ben Brahim, J. - P. Gaudillère, and E. Dreyer. 1999. Photosynthetic respon ses to phosphorus nutrition in two - year - old maritime pin e seedlings. Tree Physiol. 19: 707 715 . Lynch, J., A. Läuchli, and E. Epstein. 1991. vegetative growth of the common bean in response to phosphorus nutrition. Crop Sci. 31: 380 387. Martins, L.E.C., F.A . Monteiro, and B.C. Pedreira. 2015. photosynthesis and leaf area of brachiaria brizantha in response to phosphorus and zinc nutrition. J. Plant Nutr. 38: 754 767 . Michalak, A.M., E.J. Anderson, D. Beletsky, S. Boland, N.S. Bosch, T.B. Bridgeman, J.D. Chaff in, K. Cho, R. Confesor, I. Daloglu, J. V. DePinto, M.A. Evans, G.L. Fahnenstiel, L. He, J.C. Ho, L. Jenkins, T.H. Johengen, K.C. Kuo, E. LaPorte, X. Liu, M.R. McWilliams, M.R. Moore, D.J. Posselt, R.P. Richards, D. Scavia, A.L. Steiner, E. Verhamme, D.M. Wright, and M.A. Zagorski. 2013. Record - setting algal bloom in Lake Erie caused by agricultural and meteorological trends consistent with expected future conditions. Proc. N atl. Acad. Sci. U. S. A. 110: 6448 6452. Mischan, M.M., S.Z. de Pinho, and L.R. de C arvalho. 2011. Determination of a point sufficiently close to the asymptote in nonlinear gro wth functions. Sci. Agric. 68: 109 114. Newman, J.P. 2014. Container nursery production and business management manual (JP 79 Newman, Ed.). University of California, Di vision of Agriculture and Natural Resources, S BN - 13: 978 - 1 - 60107 - 842 - 1, Oakland, CA. Nowak, J., and S. Stroka. 2001. The effect of phosphorus nutrition on growth, flowering and 566. In Maloupa, E., Gerasopoulos, D. (eds.), International Symposium on Growing Media and Hydroponics. Owen, J.S., S.L. Warren, T.E. Bilderback, and J.P. Albano. 2008. Phosphorus rate, leaching fraction, and substrate influence on influent quantity, effluent nut rient content, and response of a containerized woody o rnamental crop. HortScience 43: 906 912. Paerl, H.W. 2009. Controlling eutrophication along the freshwater marine continuum : Dual nutrie nt (N and P) reductions are essential. Estuaries and Coasts 32: 593 601 . Pershey, N.A., B.M. Cregg, J.A. Andresen, and R.T. Fernandez. 2015. Irrigating based on daily water use reduces nursery runoff volume and nutrient load without reducing growth of four conifers. HortScience 50: 1553 1561 . Pessarakli, M. 2005. Handbook o f Photosynthesis (M Pessarakli, Ed.). 2nd ed. Taylor & Francis. Poorter, H., Ü. Niinemets, A. Walter, F. Fiorani, and U. Schurr. 2010. A method to construct dose - response curves for a wide range of environmental factors and plant traits by means of a meta - analysis of phe notypic data. J. Exp. Bot. 61: 2043 2055. Rao, M., and N. Terry. 1989. Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet 1. Changes in Growth, Gas Exchange, and Calvin C ycle Enzymes. Plant Physiol 90: 814 819 . Ristve y, A.G., J.D. Lea - Cox, and D.S. Ross. 2004. Nutrient uptake, partitioning and leaching losses from container - nursery produc tion systems. Acta Hortic. 630: 321 328. Ristvey, A.G., J.D. Lea - Cox, and D.S. Ross. 2007. Nitrogen and Phosphorus Uptake Efficiency a nd Partitioning of Container - grown Azalea During Spring Growth. J. Am. Soc. Hortic. Sci. 132: 563 571. Rodríguez, D., M.M. Zubillaga, E.L. Ploschuk, W.G. Keltjens, J. Goudriaan, and R.S. Lavado. 1998. Leaf area expansion and assimilate production in sunflow er (Helianthus annuus L.) growing under low phosphor us conditions. Plant Soil 202: 133 147. Sharkey, T.D. 2016. What gas exchange data can tell us about photosynth esis. Plant, Cell Environ. 39: 1161 1163. Shreckhise, J.H., J.S. Owen, M.J. Eick, A.X. Niemiera, J.E. Altland, and S.A. White. 2019a. Dolomite and micronutrient fertilizer affect phosphorus fate in pine bark substrate used for containerized nursery crop production. Soil Sci. Soc. Am. J. 83: 1410 . S hreckhise, J.H., J.S. Owen, and A.X. Niemiera. 2018. Growth Response of Three Containerized Woody Plant Taxa to Varying Low Phosphorus Fertilizer C oncentrations. HortScience 80 53: 628 637. Shreckhise, J.H., J.S. Owen, and A.X. Niemiera. 2019b. Growth response of Hydrangea macrophylla and Ilex crenata cultivars to low - phosphorus controlled - release fertilizers. Sci. Hortic. (Amsterdam). 246: 578 588 . Singh, S.K., G. Badgujar, V.R. Reddy, D.H. Fleisher, and J.A. Bunce. 2013. Carbon dioxide diffusion across stomata and mesophyll and photo - biochemical processes as affected by growth CO2 and phosphorus nutrition in cotton. J. Plant Physiol. 170 : 801 813. Soti, P., A. Fleurissaint, S. Reed, and K. Jayachandran. 2015. Effect s of control release fertilizers on nutrient l eaching, palm growth and production cost. Agriculture 5: 1135 1145. Terry, N., and A. Ulrich. 1973. Effects of Phosphorus Deficiency on the Photosynthesis and Respiration of Leaves of Sugar Beet. Plant Physiol. 51(1): 43 47 . United States Department of Agriculture National Agricultural Statistics Service. 2016. Census of Horticultural Specialties (2014). 2012 Census Agric. 3(AC12 - SS - 3) . United States Environmental Protection Agency. 2005. Protecting Water Quality from Agricultural Runoff. Environ. Prot. Agency (EPA 841 - F - 05 - 001). United States Environmental Protection Agency. 2016. National Nonpoint Source Program - A catalyst for water quality improvements. Environ. Prot. Agency EPA 841 - R - 16 - 009. Available at https://www.epa.gov/sites/production/files/20 16 - 10/documents/nps_program_highlights_report - 508.pdf. Walker, D.A., and S.P. Robinson. 1978. Chloroplast and Cell A contemporary view of photosynthetic carbon assimil ation. Ber. Dtsch. Bot. Ges. 91: 513 526 . Warsaw, A.L., R.T. Fernandez, B.M. Cregg, and J. A. Andresen. 2009. Container - grown ornamental plant growth and water runoff nutrient content and volume under four irrigation treatments. HortScience 44: 1573 1580 . Watson, S.B., C. Miller, G. Arhonditsis, G.L. Boyer, W. Carmichael, M.N. Charlton, R. Confes or, D.C. Depew, T.O. Höök, S.A. Ludsin, G. Matisoff, S.P. McElmurry, M.W. Murray, R. Peter Richards, Y.R. Rao, M.M. Steffen, and S.W. Wilhelm. 2016. The re - eutrophication of Lake Erie: Harmful algal blooms and hypoxia. Harmful Algae 56: 44 66. Whitcher, C.L ., M.W. Kent, and D.W. Reed. 2005. Phosphorus concentration affects new guinea impatiens and vinca in recirculating subirrigation. HortScience 40: 2047 2051. Xu, H.X., X.Y. Weng, and Y. Yang. 2007. Effect of phosphorus deficiency on the photosynthetic chara cteristics of rice plant s. Russ. J. Plant Physiol. 54: 741 748 . Yang, J.T., A.L. Preiser, Z. Li, S.E. Weise, and T.D. Sharkey. 2016. Triose phosphate use limitation of photosynthesis: short - term and long - term effects. Planta 243: 687 698. 81 Zhang, D., R.E. Mor an, and L.B. Stack. 2004. Effect of phosphorus fertilization on growth and flowering of Scaevola aemula R. Br. 1728 1731. 82 SECTION II I DOSE - DEPENDENT PHYTOTOXICITY OF PESTICIDES IN SIMULATED NURSERY RUNOFF ON LANDSCAPE NURSERY PLANTS 83 Dose - dependent phytotoxicity of pesticides in simulated nursery runoff on landscape nursery plants Shital Poudyal 1,* , R.T. Fernandez 1 , James S. Owen Jr. 2 and Bert Cregg 1,2 1 Department of Horticulture, 1066 Bogue Street, Michigan State University, East Lansing, MI 48824 2 USDA Agricultural Research Service, Application Technology Research, 1680 Madison Avenue, Wooster, OH, USA 44691 3 Department of Biochemistry and Molecular Biology, Michigan State University, 612 Wilson Rd, Rm 210 East Lansing, MI, USA 48824 4 Department of Forestry, Michigan State University, 480 Wilson Road, East Lansing, MI, USA 48824 * Corresponding author: cregg @msu.edu This section has been published in Water; https://doi.org/10.3390/w11112354 84 Abstract Managers of ornamental nurseries are increasingly reusing runoff water as an irrigation source, but residual pesticides in recycled water may result in plant phytotoxicity on crop plants. Our study focu sed on understanding the responses of container - grown landscape plants to residual n, chlorpyrifos, and oxyfluorfen (0, 0.15, 0.35, 0.7, and 1.4 mg/L of isoxaben; 0, 0.05, 0.1, 0.2, and 0.4 mg/L of chlorpyrifos; and 0, 0.005, 0.01, 0.015, and 0.02 mg/L of oxyfluorfen) applied as overhead irrigation. After three months of application, we assessed the dry weight biomass, growth, and parameters related to photosynthetic physiology (soil plant analysis development (SPAD) chlorophyll index, light - adapted chlorophyll fluorescence, and photosynthesis carbon dioxide response (A/Ci) curves. We als o sampled the plant leaf, stem, and root tissues for residual pesticides. The effects of the pesticides were pesticide - specific and taxa - specific. Exposure to oxyfluorfen resulted in visible injury in all three taxa and reduced total biomass, chlorophyll i ndex, and photosynthesis in Hydrangea and Hosta. All three taxa absorbed and retained pesticide in leaf and stem tissues. Growers should follow best management practices to reduce exposure from irrigation with runoff, particularly for herbicides with post - emergent activity. Keywords : nursery runoff; isoxaben; oxyfluorfen; chlorpyrifos; photosynthesis; A/Ci curve 85 1. Introduction Horticulture is a major industry in the U.S. In 2014, the sale of floriculture, nursery, and specialty crops were worth $13.8 bil lion, up by 18% since 2009 (United States Department of Agriculture National Agricultural Statistics Service, 2016) . For container nurseries , irrigation is often applied based on general rules of thumb , such as 19 mm of water per day. These application rat es often greatly exceed plant water needs and result in substantial runoff (Warsaw et al., 2009; Danelon et al., 2010) . In a nursery with 4 L containers placed six inches apart, up to 80% of applied water may be lost as runoff (Mathers et al., 2005) . Furth ermore, f requent pesticide application is common among nursery producers. Therefore, runoff generated from container nurseries may contain various pesticides, and if released without treatment, surface water contamination and toxicity to aquatic life can o ccur (Keese et al., 1994; Lao et al., 2008; Warsaw et al., 2012) . Due to the significant freshwater use by the nursery industry and the environmental problems associated with runoff, water regulations for nurseries are becoming more stringent. To cope with new regulations and ensure water security, the captur e and reus e of runoff water is increasing among nursery growers (Brown, 2002; Schmitz et al., 2013; Wilson and Broembsen, 2015) . While capturing and reusing runoff may be a practical solution to reduce contaminants in neighboring s of residual pesticide on crop growth and quality may impede its adoption (Wilson and Broembsen, 2015) as nursery growers 86 report evidence of pesticide phytotoxicity when runoff water is used for irrigation (personal communication with growers). In the current study , we examined the impacts of isoxaben, oxyfluorfen, and chlorpyrifos on three widely cultivat ed nursery crops. We selected these compounds for study because they are commonly used in the nursery trade and represent different modes of action. Mor e over , all three pesticides may be found in nursery runoff , and if present at higher concentrations, can injure nursery plants (Bhandary et al., 1997; Briggs et al., 1998) . Isoxaben ( c ommon tradenames Gallery ® , Snapshot ® ) is a pre - emergence herbicide that works by inhibiting cell wall biosynthesis in dividing cells, causing stunted plants. Various nursery pl ants are susceptible to this herbicide. Isoxaben at 5 mg/L reduced plant height, leaf emergence , and photosynthesis in Canna generalis (canna), Pontaderia cordata (pickerel weed), and Iris (charjoys Jan) (Fernandez et al., 1999) . Isoxaben at 10 mg/L also r educed the root visual appearance scale in Pennisetum rupeli (fountain grass) and Hemerocallis hybrid (daylily) a s well as the fresh root weight in Ilex crenata Hellers holly) (Bhandary et al., 1997) , but isoxaben application at 1.1 kg a.i./ha a lone did not injure six different container - grown ornamental grass species (Neal and Senesac, 1991) , nor did it affect plant height in Ilex crenata (Japanese holly). Chlorpyrifos ( c ommon tradenames Dursban ® , ffect plants by inhibiting the activity of enzymes for growth and development , causing smaller plants (Parween et al., 2011a) . Chlorpyrifos (525 mg/L) induced membrane disintegration through lipid peroxidation and also increased 87 superdimutase (SOD) activit y in Vigna radiata (Parween et al., 2012) . Chlorpyrifos at 30 mg/L also reduced growth and biomass in Azolla pinnata (Prasad et al., 2015) . Oxyfluorfen ( c ommon tradename Goal) is a widely used pre and post - emergence herbicide in container nursery productio n. It is mostly used for controlling broadleaf weeds and annual grasses (Dow AgroSciences, 2014) . Oxyfluorfen acts by inhibiting the synthesis of protoporphyrinogen oxidase (Lee and Duke, 1994) . Oxyfluorfen at 1 g/ha, when applied as a post - emergence herbicide to control weeds in sunflower, produced severe phytotoxicity on sunflower (Jursik et al., 2011) . Oxyfluorfen is not recommended for use in sunflower , but has been found to be safe on eight d ifferent container - grown ornamental crops at 0.9 kg a.i./ha including red - osier dogwood ( Cornus sericea ), cranberry cotoneaster ( Cotoneaster apiculata ), European cranberry viburnum ( Viburnum opulus ( Forsythia intermedia Redera helix ), green luster holly ( Ilex crenata Pachysandra terminalis ), and Browni yew ( Taxus x media (Vea and Palmer, 2009) . Application of oxyfluorfen at 0.07 kg a.i./ha as a foliar spray produced severe phytotoxicity on Euonymus fortunei (Colorata) (Horowitz et al., 1989) . Pesticides in runoff water from nurseries are usually diluted with other water sources and therefore occur at relatively low concentrations. However, frequent, of ten daily, irrigation with nursery runoff creates the potential for chronic low - dose exposure to an array of pesticides that may have phytotoxic effects. Pesticides that cause phytotoxicity usually interfere with 88 physiological and biochemical processes in non - target plants. Many of these effects are related to photosynthetic function , and measuring these responses can indicate the extent of physiological damage caused by pesticides (Krugh and Miles, 1996; Spiers et al., 2008; Parween et al., 2011b; Vinet an d Zhedanov, 2012) . A novel aspect of our approach in the current study was to examine the potential impacts of chronic, low dose application of pesticides on physiological responses of nursery crops. Advances in portable photosynthesis systems have simplif ied the measurement of key photosynthetic responses such as A/Ci curves that can provide insights into photosynthetic reactions that may be early indicators of phytotoxic responses. For example, the maximum carboxylation rate of r ibulose - 1,5 - bisphosphate c arboxylase oxygenase (RuBisCO) (Vcmax) may limit photosynthesis at lower (0 to 200 ppm) intercellular carbon dioxide concentration s , and the rate of electron transfer for r ibulose 1,5 - bisphosphate (RuBP) regeneration (J) may limit photosynthesis at higher (>300 ppm) intercellular carbon dioxide concentration s (Veeraswamy et al., 1993; Sharkey et al., 2007; Parween et al., 2011b; Sharkey, 2016) A few studies have described the effects of isoxaben, chlorpyrifos, and oxyfluorfen on various plants (Lal et al., 1987; Neal and Senesac, 1991; Salihu et al., 1999; Parween et al., 2011a) , but the impact of residual (low) concentrations of these compounds on common landscape nursery plants receiving pesticides for an entire growing season has not been documented. Phyt otoxicity of isoxaben, oxyfluorfen, and chlorpyrifos may vary depending on the plant taxa irrigated, the concentration of pesticide in water , and the duration of the pesticide appli cation and growers 89 particularly lack information on the long - term phytotoxi city caused by these pesticides in runoff water. They are also unaware of the severity of phytotoxicity caused by these pesticides on common landscape nursery plants. Therefore, understanding the impacts of prolonged exposure to low doses of these pesticid es may provide insights into the safe use of recycled runoff for irrigation and ultimately encourage the reuse of runoff water among nursery growers. Therefore, the objectives of this study were to evaluate the morphological and physiological effects of va rious concentrations of isoxaben, chlorpyrifos , and oxyfluorfen on three commonly cultivate d container - grown landscape nursery plants, Hydrangea paniculata , , and Cornus obliqua G 2. Materials and Methods 2.1. Plant Material and Treatments This study was conducted in a greenhouse at the Michigan State University Horticulture Teaching and Research Center (HTRC) located in Holt, Michigan, USA. We used Limelight Hydrangea ( Hydrangea paniculata , Re d Rover ® silky dogwood ( Cornus obliqua G , and Gold Standard Hosta ( Hosta L black plastic containers for our study. We planted Hydrangea and Cornus plants as liners in spring 2017 in pine bark and peat moss subs trate (80:20; v olume: v olume). Th e se two plants were grown outdoors at the HTRC and received standard nursery culture including 19 mm of daily overhead irrigation and controlled release fertilizer ( 19:4:8 N : P 2 O 5 : K 2 O with micronutrients, 5 6 months, Harrell 's LLC, 90 Lakeland, FL, USA ) applied as a top - dressing. In early December 2017, Hydrangea and Cornus plants , along with bulbs of Hosta plants , were placed in a walk - in cooler at 6 °C for five weeks to complete their chilling requirement s before they were brought into the greenhouse on January 8, 2018. The temperature in the greenhouse was set to 22 °C, and a sodium lamp provided 16 - h of photoperiod. Different concentrations of isoxaben, oxyfluorfen, or chlorpyrifos were applied as overhea d irrigation mixed in irrigation water. We selected five different concentrations of each pesticide as treatments (0, 0.15, 0.35, 0.7 , and 1.4 mg/L of isoxaben; 0, 0.05, 0.1, 0.2 , and 0.4 mg/L of chlorpyrifos; and 0, 0.005, 0.01, 0.015 , and 0.02 mg/L of ox yfluorfen). Pesticide rates were based on pesticide residues reported in nursery retention ponds (Riley et al., 1994; Briggs et al., 2003; Mangiafico et al., 2009) and the solubility of the pesticides in water. A black 100 - L covered plastic tank was used a s a stock tank for each treatment. A calculated amount of each pesticide was dissolved in 100 L of water to achieve the desired concentration. A sump pump was used to agitate the pesticide solution and apply the pesticide solution as overhead irrigation on plants. An irrigation distribution test for treatment zones had a distribution uniformity of 89.73%. Pesticide solutions were freshly prepared two to three times a week. Each pesticide treatment consisted of three taxa and six replications per taxa. Treat ments began once all the plants had produced a new flush of growth (8 February 2018). We applied pesticide treatments with each irrigation event that varied from once every three days to every day , depending on plant water use. Pesticide treatments continu ed for three months. 91 2.2. Physiological Measurements and Growth A portable photosynthesis system (LI - 6400 XT, Li - Cor, Inc., Lincoln, NE, USA) mounted with a leaf chamber fluorometer (LI - 6400 - 40, Li - Cor, Inc., Lincoln, NE, USA) was used to develop A/Ci curv es and light - adapted fluorescence for all three taxa. A section of fully mature leaf on either the third or fourth node from the top for Cornus and Hydrangea and on the first or second node for Hosta was used for all physiological measurements. Photosynthe tically active radiation (PAR) in the chamber was set to 1500 µmol m s . The block temperature was set to 25 °C, and the reference CO 2 , supplied by a 12 g CO 2 cartridge (LI - 6400 XT, Li - Cor, Inc., Lincoln, NE, USA), was varied, starting at 0 ppm to 800 p pm (0, 50, 100, 200, 300, 400, 500, 600, and 800 ppm). Net photosynthesis (A) values at various intercellular carbon dioxide concentrations (Ci) were used to generate carbon dioxide response (A/Ci) curves. Data from the A/Ci curves were used to estimate Vc max and J values using the non - linear equation provided by Sharkey (2016) . For light - adapted chlorophyll fluorescence measurements, a section of a fully mature leaf of each plant was enclosed in the LI - 6400 - 40 chamber with 1500 µmol m s of PAR, 400 ppm of CO 2 , and 40 60% humidity at 25 °C. We acclimatized the leaf for 5 min , after which we measured light - adapted fluorescence (LI - 6400xt Instruction Manual, version 6, Li - Cor Inc., Lincoln, NE, USA) to determine the efficiency of photosystem II. SPAD leaf chlorophyll index was also measured on three fully expanded leaves per plant on either the second or third node for Hydrangea and Cornus by using a portable SPAD meter (SPAD - 502; Minolta C orporation, Ltd., Osaka, Japan). The 92 v ariegated golden leaf color of Hosta produced unrealistic values of the SPAD index; hence, we did not measure the SPAD index for those plants. We examined the leaves of each plant and scored them for visible pesticide injury based on a rating system on a scale of one to ten, with ten being a healthy leaf without damage, and one being a dead leaf. After scoring plants for visible symptoms, the leaves, stems, and roots were harvested, dried in an oven (45 °C), and weighed. All of the dry weights were combined to determine the total d ry biomass (TBD). Samples of dried leaves, stem , and roots were sent to a commercial laboratory (Brookside Labs, Laboratories, Inc., New Bremen, OH, USA) to determine the residual levels of isoxaben, chlorpyrifos, and oxyfluorfen in the tissue. The QuEChER S technique was used to extract all tissue samples. Oxyfluorfen and chlorpyrifos were quantified using g as chromatography mass spectrometry (GC - MS), and isoxaben was quantified using l iquid c hromatography tandem mass spectrometry (LC - MS/MS) (Raina, 2011) . 2.3. Statistical Analysis All statistical analyses were carried out using SAS (version 9.4; SAS Institute, Inc., Cary, NC, USA). Regression analysis in SAS was carried out for the chlorophyll index and light - adapted sticide treatments. For the total dry biomass (TDB), visual leaf injury, and residual pesticide concentration in the leaves, stems, and roots, we analyzed data using one way ANOVA for each species and pesticide. Vcmax and J estimates derived from A/Ci curv es were also analyzed by using one way ANOVA for each tax on and each pesticide treatment. 93 Any data that did not meet the assumption of homogeneity of variance were transformed prior to statistical analysis. 3. Results 3.1. Leaf Visual Injury and Growth in Response to Pesticide Treatment Leaf visual injury on plants was observed only for oxyfluorfen applications ( Figure 1). Exposure to isoxaben or chlorpyrifos did not result in any visible damage to the taxa tested (data not shown). For oxyfluorfen, 0 and 0. 005 mg/L did not produce any visual symptoms on any taxa. Increasing the oxyfluorfen dose to 0.01 mg/L produced leaf injury on Hydrangea and Hosta , but not on Cornus . Leaf injury was visible on Cornus plants only at the maximum dose (0.02 mg/L) of oxyfluor fen application. Visible leaf injury on Hydrangea and Hosta increased with an increasing dose of oxyfluorfen (Figure 1). Visible symptoms of oxyfluorfen exposure included leaf browning and smaller leaves (Figure 2). Oxyfluorfen application also reduced TDB for Hydrangea , but not for Hosta and Cornus (Table 1). For Hydrangea , increasing oxyfluorfen to 0.015 mg/L did not reduce the TDB, but further increase in dose reduced TDB. For Hosta , the decrease in the TBD was linear but was not statistically significan t (Table 1). Exposure to isoxaben or chlorpyrifos in irrigation water did not affect the TDB of any of the taxa tested, except for Hosta , where the application of isoxaben first reduced TDB (till 0.7 mg/L) and then the biomass increased (1.4 mg/L). 94 3.2. Ph ysiological Performance in Response to Pesticide Treatments Irrigating Hydrangea and Cornus plants with simulated runoff containing oxyfluorfen reduced the SPAD chlorophyll index of leaves. For Cornus , the SPAD chlorophyll index decreased linearly with increasing oxyfluorfen concentration , while the SPAD index for Hydrangea decreased rapidly at oxyfluorfen concentrations above 0.01 mg/L (Figure 3). Isoxaben and chlorpyrifos did not affect the SPAD index in any of the three taxa (data not shown). Chlorpyr ifos and oxyfluorfen did not affect the light - adapted fluorescence in any of the taxa (data not shown). Isoxaben reduced light - adapted fluorescence for Hydrangea and Cornus , but did not affect the Hosta (Figure 4). In both Hydrangea and Cornus , , with further increases in isoxaben concentration (Figure 4). Irrigating with simulated runoff containing oxyfluorfen affected the photosynthetic rates of Hosta and Hydrangea (Figure 5). Exposure to oxyfluorfen reduced photosynthesis rates in Hosta when oxyfluorfen concentration in irrigation water was 0.01 mg/L or higher . For Hydrangea , exposure to oxyfluorfen decreased photosynthetic rates only when ox yfluorfen concentrations in irrigation were 0.015 mg/L or more. Exposure to oxyfluorfen in irrigation water did not affect photosynthesis in Cornus . Irrigation with water containing isoxaben slightly reduced photosynthesis for Hosta at concentrations of 0. 07 mg/L or above (Figure 6). Isoxaben did not reduce photosynthesis in Hydrangea and Cornus (Figure 6). Chlorpyrifos did not affect the photosynthesis of any of the taxa tested (data not shown). Reduction in Vcmax and J 95 were only seen for oxyfluorfen appli cation (Figure 7). Oxyfluorfen limited photosynthesis in Hydrangea by reducing Vcmax and J at concentration s of 0.015 m/L or above , and in Hosta by reducing Vcmax and J at a concentration of 0.01 mg/L or above (Figure 7). 3.3. Pesticide Absorption Leaf pes ticide concentration for the pesticides increased with increasing dose (Figure 8). However, taxa varied in their uptake and retention of each pesticide. For oxyfluorfen, Hydrangea had maximum absorption and retention in leaves, followed by Hosta , and then Cornus (Figure 8a). However, for isoxaben and chlorpyrifos, Cornus absorbed the highest amount, followed by Hydrangea and then by Hosta (Figure 8b,c). Isoxaben absorption and retention in leaves were consistently lower in all three taxa when compared to ox yfluorfen and chlorpyrifos. Stem and roots also absorbed and retained pesticides (Figure 9a f). For all three pesticides, pesticide residues were always present in the stem (Figure 9a c). The order of pesticide residue concentration in stem was chlorpyrifo s > oxyfluorfen > isoxaben. Hosta plants do not have a true stem, therefore, we did not conduct a stem pesticide analysis in Hosta . Fine roots of Hydrangea absorbed and retained oxyfluorfen and isoxaben, but not chlorpyrifos (Figure 9d f). For Hydrangea , absorption of oxyfluorfen was greater when compared to isoxaben. Hosta absorbed and retained all three pesticides, but unlike Hydrangea , its pesticide root retention order was chlorpyrifos > isoxaben > oxyfluorfen (Figure 9d f). For Cornus , oxyfluorfen w as not retained in fine roots, but fine roots absorbed and retained chlorpyrifos and oxyfluorfen (Figure 9d f). 96 4. Discussion 4.1. Growth and Physiology The potential for crop injury from residual pesticides can be a barrier for nursery operators to re - use runoff for irrigation. Additionally, unrealized reductions in crop growth from diluted, persistent pesticides can reduce profits by increasing production time or reducing plant quality. Leaves absorb pesticides that are applied to foliage, often producing leaf injury (Stevens and Baker, 1987) . Visible injury is of concern to nursery producers, even if growth is not affected, because aesthetic appearance is important in marketing ornamental plants. Pesticide injury to leaves depends on the dose and type of pesticide used (Poudyal and Cregg, 2019) . In this study, oxyfluorfen produced dose - dependent visible injury in all three taxa. Exposure to 0.02 mg/L of oxyfluorfen reduced visual leaf rating in Hydrangea , Hosta , and Cornus by 56.7%, 37.5%, and 18.4% when c ompared to the untreated control, respectively. Isoxaben and chlorpyrifos at the rates we used did not produce any visible injury, and hence irrigation with runoff containing these compounds can be considered relatively safe for use on these taxa. Oxyfluor fen works as a pre - emergent and post - emergence contact herbicide, therefore it is not surprising that it caused the greatest visible injury. When applied to leaves , oxyfluorfen inhibits chlorophyll formation in addition to causing lipid peroxidation and me mbrane degradation (Lee and Duke, 1994) . In contrast, isoxaben is a pre - emergence herbicide that blocks germination (Heim et al., 1990) , and chlorpyrifos is an insecticide. The sensitivity of plants to oxyfluorfen in this study is consistent 97 with observati ons by nursery growers who have reported crop damage following exposure to oxyfluorfen when runoff water was used as irrigation (personal communication). Oxyfluorfen application also produced leaf injury on r ice ( Oryza sativa ) (Priya et al., 2017) , yelloww ood ( Cladrastis kentukea ) (Mathers, N/A ) , and sunflower (Jursik et al., 2011) . Lactofen , a herbicide with a similar mode of action to oxyfluorfen, also produced leaf injury on soybean leaves (Wichert and Talbert, 1993) . Oxyfluorfen (0.02 mg/L) reduced the T DB of Hydrangea by 21.5% and the TBD of Hosta by 43.1%. The leaf is where photosynthesis, a process to convert light, CO 2 , and water to food, takes place , and photosynthesis governs plant growth (Kirschbaum, 2011) . In our study, leaf injury from oxyfluorfen was observed primarily in Hydrangea and Hosta . For Cornus , leaf injury was only observed at the maximum dose (0.02 mg/L) of oxyfluorfen. This leaf injury in Hydrangea ultimately led to a reduction in TDB for Hyd rangea . Isoxaben and chlorpyrifos did not injure leaves; hence, healthy growth was seen in plants receiving those pesticides. Effects of exposure to pesticides in simulated runoff irrigation on photosynthetic parameters largely reflected sensitively as see n in visible injury to leaves. Isoxaben and chlorpyrifos did not affect the SPAD chlorophyll index, but oxyfluorfen reduced chlorophyll index for Hydrangea and Cornus . Oxyfluorfen is a protoporphyrinogen oxidase (PPO) inhibitor, and in the presence of ligh t, this herbicide produces reactive oxygen species that break down chlorophyll and organelle membranes (Sherwani et al., 2015) . Chlorophyll fluorescence can be an early indicator of pesticide damage and has been used to predict herbicide damage for various taxa (Silva 98 et al., 2014; Wang et al., 2018) . In our study, however, oxyfluorfen or chlorpyrifos did not affect light - adapted chlorophyll fluorescence on any taxa. Although exposure to isoxaben in runoff did not affect the SPAD chlorophyll index, it did s lightly reduce the light - adapted fluorescence for Cornus and Hydrangea . In a phytoremediation study by Fernandez et al. (1999), isoxaben also reduced chlorophyll fluorescence in canna, pickerel weed, and iris (Fernandez et al., 1999) . Photosynthesis and in tercellular carbon dioxide response curves (A/Ci) can be used to determine the photosynthetic capacity of plants (Singh et al., 2013) and the shape of the A/Ci curve is generally determined by the capacity of r ubisco for carboxylation (Vcmax) (at lower Ci rates < 200 ppm) and the rate of RuBP regeneration (J) (at higher Ci rates, >300 ppm) (Sharkey et al., 2007; Dinh et al., 2017) . Visual observations of the A/Ci curve indicated oxyfluorfen concentrations of 0.015 and 0.02 mg/L reduced the carboxylation cap acity of RUBISCO and the rate of electron transport for RuBP regeneration for Hydrangea . For Hosta , oxyfluorfen rates of 0.01 mg/L or higher reduced those parameters. These visual observations were also statistically confirmed by calculating Vcmax and J va lues. Oxyfluorfen reduced Vcmax and J values both for Hydrangea and Hosta , therefore reduction in rate of photosynthesis in both of those taxa was by the decrease in carboxylation capacity of RUBISCO enzyme (at lower Ci) and reduction in the rate of electr on transport for RuBP regeneration (at higher Ci). Oxyfluorfen did not limit photosynthetic rates, Vcmax, and J for Cornus at any concentrations. Even though isoxaben is a pre - emergent herbicide, it slightly reduced photosynthesis in Hosta when the levels of isoxaben 99 were 0.7 mg/L or higher , but unlike oxyfluorfen, reduction in Vcmax and J was not observed. The lack of response in Vcmax and Jmax confirm ed the reduction in photosynthe s i s to be minimal; therefore, it never translated to a decrease in growth. The decline in photosynthesis by oxyfluorfen corresponded well to the leaf injury. Photosynthetic response to pesticide exposure was more sensitive when compared to the TDB response to pesticide exposure. 4.2. Pesticide Absorption Oxyfluorfen was absorbed and retained in leaves for all three taxa, and the absorption increased with increasing dose. Even though oxyfluorfen was retained on the leaves of all three taxa, leaf injury varied. Hydrangea had the maximum leaf injury , followed by Hosta , which is also supported by the fact that Hydrangea had the highest leaf retention of oxyfluorfen followed by Hosta . Taxa vary in their tolerance to oxyfluorfen, which is mainly governed by pesticide absorption, pesticide degradation inside leaves , and the affinity of the target sites (sites inside plants where herbicide binds to produce response) to herbicide (Chun et al., 2001) . For Cornus to be tolerant, either most of the absorbed oxyfluorfen must have degraded inside the leaves or were stopped from reaching the tar get site. Isoxaben sensitivity is taxa - specific (Schneegurt et al., 1994 ) , and a wide range of plants are tolerant to lower concentrations of post applied isoxaben (Wehtje et al., 2006) . In the current study, isoxaben absorption and retention in leaves wer e dose - dependent and similar across taxa. Among the pesticides investigated, isoxaben was least absorbed and retained, which may be because the leaf absorption of isoxaben is very low, and isoxaben is 100 minimally translocated beyond the application point (Sc hneegurt et al., 1994 ; Wehtje et al., 2006) . Isoxaben also did not produce visible symptoms or reduce growth. The mode of action for isoxaben is the inhibition of cell wall biosynthesis, which is dose - dependent (Heim et al., 1990) . Our range of doses for i soxaben may not have been high enough to produce phytotoxicity. In a study by Heim et al. (1993) , variation in the sensitivity of Agrostis palustris to isoxaben was associated with decreased sensitivity of isoxaben binding sites , which might have also occu rred in our study. Fernandez et al. (1999) , found that isoxaben reduced photosynthesis in three monocot species, while a slight reduction in photosynthesis for monocot - Hosta was also observed in our study. Isoxaben application did not reduce photosynthesis in Hydrangea and Cornus as isoxaben response is taxa - specific (Willoughby et al., 2003) . Similar to oxyfluorfen and isoxaben, absorption and retention of chlorpyrifos in leaves was also dose - dependent and increased with increasing dose. Chlorpyrifos may enter inside plant tissue through leaves or roots, and its absorption and retention vary wi thin species (Lu et al., 2014) . In a study by Fan et al. ( 2013 ) , chlorpyrifos was absorbed and retained in the leaves of six different leafy vegetables with retention concentration varying within species (Fan et al., 2013) . In our study, absorption and ret ention of chlorpyrifos did not affect growth and physiological performance on any of the taxa. In wheat, root application of chlorpyrifos led to the accumulation of chlorpyrifos in root and shoots, but growth was not affected (Copaja et al., 2014) . Wheat a nd rapeseed also absorbed chlorpyrifos that was mixed in irrigation water, but growth was not affected 101 in either taxon (Wang et al., 2007) . Chlorpyrifos does not have specific sites of action in plants but may produce phytotoxicity depending on dose, howev er, the dose of chlorpyrifos that we applied was not enough to produce any morphological or physiological symptoms in the three taxa that we tested. In our study, the type and concentration of pesticides absorbed and retained in fine roots varied dramatically among the taxa. Isoxaben concentrations in stem and fine roots were lower when compared to chlorpyrifos or oxyfluorfen because isoxaben is less m obile in plants, and up to 99% of isoxaben applied may be adsorbed by pine bark (Schneegurt et al., 1994 ; Wehtje et al., 2006) . Application of all three pesticides also resulted in the accumulation of those pesticides in the stem, which may either be throu gh root absorption, translocation from the leaves , or both (Duke , 1990; Schneegurt et al., 1994 ; Chun et al., 2001) . 5. Conclusions Phytotoxicity due to pesticide exposure from runoff irrigation depends on the plant type, type of pesticide applied, and con centration of pesticide. In our study, 0.01 mg/L was the threshold level of oxyfluorfen to produce leaf visual injury in Hydrangea and Hosta , while 0.02 mg/L of oxyfluorfen was required to induce phytotoxicity i n Cornus . Irrigation with simulated runoff co ntaining isoxaben and chlorpyrifos were comparatively safe for all three taxa tested. Isoxaben caused a slight reduction in PSII efficiency , but neither isoxaben or chlorpyrifos affected dry weight biomass, photosynthetic biochemistry, or caused visible le af injury as oxyfluorfen did. This 102 response likely reflects the fact that oxyfluorfen is an herbicide that has post - emergent activity and therefore has the potential to affect sensitive plants following prolonged low - dose exposure. The other pesticides exa mined in this study are an insecticide (chlorpyrifos) and an herbicide without post - emergent activity (isoxaben), which may be less likely to impact plant growth and physiological function. Among the three taxa, Hydrangea was most sensitive , followed by Ho sta , and then by Cornus . Differences among taxa in their sensitivity to oxyfluorfen may also be due in part to differences in plant uptake and translocation. The taxa that were most affected by oxyfluorfen exposure, Hydrangea and Hosta , also had the highes t leaf residual concentrations of that compound. Growth impacts of pesticide exposure in irrigation water are also linked to physiological function. Pesticides had more significant effect on photosynthesis compared to growth. The results of this study esta blish the potential of using runoff water containing isoxaben and chlorpyrifos. However, consideration should be made on the concentration of pesticides in runoff and plant taxa irrigated. As with all nu r sery research, a limitation of the current study is that we only considered three taxa, whereas most commercial nurseries produce dozens, if not hundreds , of different types of plants. We specifically selected taxa that had shown sensitivity to pesticides in similar studies, but it is possible that some tax a may have lower thresholds for pesticide impacts. Likewise, in addition to the three pesticides studied, other compounds including mefenoxam, oryzalin, glyphosate, acephate, and bifenthrin may be found in nursery retention ponds (Poudyal and Cregg, 2019) . We suggest that researchers conduct similar studies with other commonly used pesticides in a nursery and also determine the pesticide sensitivity of the plants 103 that are different from ours. Th eir research in combination with ours will provide a stronger b ase for the adoption of irrigation practice using runoff water. 104 APPENDIX 105 APPENDIX Figure III - 1 . Mean leaf visual rating of Hydrangea paniculata Cornus obliqua Hosta five concentrations of oxyfluorfen for three months. Visual rating was based on a scale of 1 to 10 (10 = no injury to 1 = dead plant). Means within a taxon followed by the same letter are not different at p < 0.05. Mean separation was by the Fisher least significance difference (LSD) test. 106 Figure III - 2 . application irrigated for three months. 1 07 Table III - 1 . Mean total dry biomass (g) for Hydrangea paniculata Cornus obliqua G , and Hosta runoff containing oxyfluorfen, isoxaben , or chlorpyrifos. Means within a column followed by the same letter for a given taxon are not different at p < 0.05. Post - hoc mean separation was done using the Fisher l east s ignificance d ifference (LSD) test. Concentration (mg/L) Oxyfluorfen Hydrangea Cornus Hosta 0 135.71ab 189.65a 31.58a 0.005 152.97a 180.68a 30.23a 0.01 153.11a 188.44a 23.63a 0.015 130.65ab 210.92a 22.72a 0.02 106.49b 207.20a 17.93a Concentration (mg/L) Chlorpyrifos Hydrangea Cornus Hosta 0 135.71a 189.65a 31.58a 0.05 139.71a 166.40a 29.76a 0.1 138.25a 213.63a 41.32a 0.2 138.62a 197.22a 32.38a 0.4 143.45a 194.87a 23.71a Concentration (mg/L) Isoxaben Hydrangea Cornus Hosta 0 135.71a 189.65a 31.58a 0.15 140.63a 225.85a 19.11bc 0.35 155.07a 198.92a 16.54c 0.7 161.66a 211.75a 13.05c 1.4 141.34a 186.94a 27.20ab 108 Figure III - 3 . Mean chlorophyll index (CI) of Hydrangea paniculata Cornus obliqua G concentrations of oxyfluorfen applied for three months. CI for Hydrangea followed quadratic regression while the CI of Cornus decreased li nearly. 109 Figure III - 4 . Mean light - Hydrangea paniculata Cornus obliqua G Hosta runoff containing five diff Hydrangea and Cornus both followed quadratic regression, while regression of Hosta was not significant at p < 0.05. 110 Figure III - 5 . Carbon dioxide response (A/Ci) curve of Hydrangea paniculata Cornus obliqua G Hosta concentrations of oxyfluorfen (Oxy) for three months. 111 Figure III - 6 . Mean Vcmax (maximum rate of RUBISCO for carboxylation) and J (rate of electron transport for RuBP regeneration) of Hydrangea paniculata Cornus obliqua G Hosta with simulated runoff containing five concentrations of oxyfluorfen for three months. Means within a taxon followed by the same letter are not different at p < 0.05. Mean separation was by the Fisher l east s ignificance d ifference (LSD) test. 112 Figure III - 7 . Concentration of oxyfluorfen ( A ), isoxaben ( B ) , and chlorpyrifos ( C ) in leaves for Hydrangea paniculata Cornus obliqua G Hosta following irrigation with simulated runoff containing five different concentrations of oxyfluorfen, isoxaben, and chlorpyrifos applied for three months. Means within a taxon followed by the same letter are not different at p < 0.05. Post - hoc mean separation was done using the Fishe r l east s ignificance d ifference (LSD) test. 113 Figure III - 8 . Concentration of oxyfluorfen ( A ), isoxaben ( B ) , and chlorpyrifos ( C ) in the stem for Hydrangea paniculata Cornus obliqua of oxyfluorfen ( D ), isoxaben ( E ) , and chlorpyrifos ( F ) in root for Hydrangea paniculata Cornus obliqua G Hosta following irrigation with simulated runoff containing five concentrations of oxyfluorfen (Oxy), isoxaben (Iso) , and chlorpyrifos (Chl), applied for three months. Means within a taxon followed by the same letter are not different at p < 0.05. Post - hoc mean separation was done using the Fisher l east s ignificance d ifference (LSD) test. Bar graphs for treatment are missing when the residual pesticide concentration is very low (zero or close to zero). 114 LITERATURE CITED 115 LITERATURE CITED Bhandary, R.M., T. Whitwell, and J. Briggs. 1997. Growth of containerized landscape plants is influenced by herbicides residues in irrigation water. Weed Technol. 11: 793 797. Briggs, J.A., M.B. Riley, and T. Whitwell. 1998. Quantification and remediation o f pesticides in runoff water from containerized plant produ ction. J. Environ. Qual. 27: 814 820 . Briggs, J.A., T. Whitwell, and M.B. Riley. 2003. Effect of delayed irrigation on isoxaben and oryzalin runoff from a co ntainer nursery. Weed Sci. 51 : 463 470 . B rown, K. 2002. Water scarcity: Forecasting the future with spotty data. Science. 80 : 926 927. Chun, J.C., H.J. Lee, S.J. Lim, S.E. Kim, and J.O. Guh. 2001. Comparative absorption, translocation, and metabolism of foliar - applied oxyfluorfen in wheat and bar ley. Pestic. Biochem. Physiol. 70: 118 125. Copaja, S. V., R. Vergara, and H.R. Bravo. 2014. Bioavailability of Chlorpyrifos in Wheat Plants ( Triticum aestivum ). Agric. Sci. 05 : 660 667. Danelon, M., A. Kachenko, J. McDonald, C. Rolfe, and B. Yiasoumi. 2010. Nursery industry water management best practice guidelines 2010 (A Kachenko, Ed.). Nurs. Gard. Ind. Aust. (Appendix 13). Dinh, T.H., K. Watanabe, H. Takaragawa, M. Nakabaru, and Y. Kawamitsu. 2017. Photosynthetic response and nitrogen use efficiency of su garcane under drought stress conditions with different nitrogen application levels. Plant Prod. Sci. 20: 412 422 . Dow AgroSciences. 2014. Specimen Label: Goal 2XL. Available at https://www.corteva.us/products - and - solutions/crop - protection/goal - 2xl.html . Duke, S. 0. 1990. Overview of Herbicide Mechanisms of Action . Environ. Health Perspect. 87: 263 271 . Fan, S., F. Zhang, K. Deng, C. Yu, S. Liu, P. Zhao, and C. Pan. 201 3. Spinach or amaranth contains highest residue of metalaxyl, fluazifop - p - butyl, chlorpyrifos, and lambda - cyhalothrin on six leaf vegetables upon open field applicati on. J. Agric. Food Chem. 61: 2039 2044. Fernandez, R.T., T. Whitwell, M.B. Riley, and C.R. Bernard. 1999. Evalu ating semiaquatic herbaceous perennials for use in herbicide phytoremediation . J. Am. Soc. Horti c. Sci. 116 124: 539 544 . Heim, D.R., L.A. Bjelk, J. James, M.A. Schneegurt, and I.M. Larrinua. 1993. Mechanism of isoxaben tolerance in Agrostis palustris var. penncross. J. E xp. Bot. 44: 1185 1189 . Heim, D.R., J.R. Skomp, E.E. Tschabold, and I.M. Larrinua. 1990. Isoxaben inhibits the synthesis of acid insoluble cell wall materials in Arabidopsis thaliana. Plant Physiol. 93 : 695 700 . Horowitz, M., C.. Elmore, and D. Boquist. 1989. Directed application of Goal (Oxyfluorfen) to container grown Euonymus, to minimize phytotoxicity and le aching. J. Environ. Hortic. 7:1 7 21. Jursik, M., J. Andr, J. Holec, and J. Soukup. 2011. Efficacy and selectivity of p ost - emergent applicationof flumioxazin and oxyfluorfen in sunflowe r. Plant, Soil Environ. 57: 532 539. Keese, R.J., N.D. Camper, T. Whitwell, M.B. Riley, and P.C. Wilson. 1994. Her bicide runoff from ornamental container nurseries. J. Environ. Qual. 23: 320 3 24. Kirschbaum, M.U.F. 2011. Does enhanced photosynthesis enhance growth? Lessons learned from CO2 enrichmen t studies. Plant Physiol. 155: 117 24 . chemicals on terrestrial plants using chlorophyll fluorescence. Environ. Toxicol. Chem. 15: 495 500. Lal, S., D.M. Saxena, and R. Lal. 1987. Effects of DDT, fenitrothion and chlorpyrifos on growth, photosynthesis and nitrogen fixation in Anabaena (Arm 310) and Aulosira fertilissima . Agr ic. Ecosyst. Environ. 19: 197 209. Lao, W.J., G. Arye, F. Ernst, Y.P. Xu, S. Bondarenko, D. Haver, J. Kabashima, and J. Gan. 2008. Reduction of pyrethroid runoff from a commerci al nursery. ACS Symp. Ser. 991: 428 446. Lee, H.J., and S.O. Duk e. 1994. Protoporphyrinogen IX - oxidizing activities involved in the mode of action of peroxidizing herbicides. J. Agric. Food Chem. 42: 2610 2618. Lu, M.X., W.W. Jiang, J.L. Wang, Q. Jian, Y. Shen, X.J. Liu, and X.Y. Yu. 2014. Persistence and dissipation of chlorpyrifos in brassica chinensis, lettuce, celery, asparagus lettuce, eggplant, and pepper in a greenhou se (Y Zhang, Ed.). PLoS One 9: e100556 . Mangiafico, S.S., J. Newman, D.J. Merhaut, J. Gan, B. Faber, and L. Wu. 2009. Nutr ients and 117 pesticides in stor mwater runoff and soil water in production nurseries and citrus and avocado groves in California. Horttechnology 19: 360 367 . Mathers, H. Herbicide injury. Michigan State Univ . Coll. Agric. Nat. Resour. Available a https://www.canr.msu.edu/uploads/files/6 - 2 3%20Nursery%20grower%20checklist%20TOM%20herbicide%20injury.pdf)Available at https://www.canr.msu.edu/uploads/files/6 - 23 Nursery grower checklist TOM herbicide injury.pdf (verified 23 September 2019). Mathers, H.M., T.H. Yeager, and L.T. Case. 2005. Improv ing irrigation water use in container nurseries. Horttechnology 15: 8 12. Neal, J.C., and A.F. Senesac. 1991. Preemergent Herbicide Safety in Container - grown Ornamen tal Grasses. HortScience 26: 157 159 . Parween, T., S. Jan, and T. Fatma. 2011a. Alteration in nitrogen metabolism and plant growth during different developmental stages of green gram ( Vigna radiata L.) in response to chlorpyrif os. Acta Physiol. Plant. 33: 2321 2328 . Parween, T., S. Jan, S. Mahmooduzzafar, and T. Fatma. 2011b. Assessing the impact o f chlorpyrifos on growth, photosynthetic pigments and yield in Vigna radiata L. at different phenological stag es. African J. Agric. Res. 6: 4432 4440 . Parween, T., S. Jan, S. Mahmooduzzafar, and T. Fatma. 2012. Evaluation of oxidative stress in Vigna radiat a L. in response to chlorpyrifos. Int. J. Environ. Sci. Technol. 9: 605 612. Poudyal, S., and B.M. Cregg. 2019. Irrigating nursery crops with recycled run - off: A review of the potential impact of pesticides on plant growth and physiology. Horttechnology 29: 716 729 . Prasad, S.M., A. Singh, and P. Singh. 2015. Physiological, biochemical and growth responses of Azolla pinnata to chlorpyrifos and cypermethrin pesticides exposure: A compar ative study. Chem. Ecol. 31: 285 298. Priya, S.R., C. Chinnusamy, M.P. Artha nar, and P. Janaki. 2017. Carryover effect and plant injury from oxyfluorfen herbicide applied in transplante d rice. Int. J. Chem. Stud. 5: 535 539 . Raina, R. 2011. Chemical Analysis of Pesticides Using GC/MS, GC/MS/MS, and LC/MS/MS. In Stoytcheva, M. (ed.) , Pesticides - Strategies for Pesticides Analysis. InTech. Riley, M.B., R.J. Keese, N.D. Camper, T. Whitwell, P. Chris, M.B. Riley, R.J. Keese, N.D. 118 Camper, T.E.D. Whitwell, and P.C. Wilson. 1994. Pendime thalin and oxyfluorfen residues in pond water and se diment from container. Weed Technol. 8: 299 303. Salihu, S., J.F. Derr, and K.K. Hatzios. 1999. Differentia l response of ajuga ( Ajuga reptans ), w intercreeper ( Euonymus fortunei ), and d warf b urning b ush ( Euonymus alatus to root - and shoot - applied isoxaben . Weed Technol. 13: 685 690. Schmitz, C., H. Lotze - campen, D. Gerten, J.P. Dietrich, B. Bodirsky, A. Biewald, and A. Popp. 2013. Blue water scarcity and the economic impacts of future agricultural trade and demand. Water Resour. Res. 49: 3601 3617. Schneegurt, M.A., J.L. Roberts, L.A . Bjelk, and B.C. Gerwick. 1994 . Postemergence Activity o f Isoxaben. Weed Technol. 8: 183 189 . Sharkey, T.D. 2016. What gas exchange data can tell us about photosynthesis. Plant, Cell Environ. 39 : 1161 1163. Sharkey, T.D., C.J. Bernacchi, G.D. Farquhar, and E.L. Singsaas. 2007. Fitting photosynthetic carbon dioxide response curves for C3 leav es. Plant, Cell Environ. 30: 1035 1040. Sherwani, S.I., I.A. Arif, and H.A. Khan. 2015. Modes of action of different classes of herbicid es . InTech. Silva, F.B., A.C. Costa, R.R. Pereira Alves, and C.A. Megguer. 2014. Chlorophyll fluorescence as an indicator of cellular damage by glyphosate herbicide in Raphanus sativus L. plants. Am. J. Plant Sci. 05 :2509 2519 . Singh, S.K., G. Badgujar, V. R. Reddy, D.H. Fleisher, and J.A. Bunce. 2013. Carbon dioxide diffusion across stomata and mesophyll and photo - biochemical processes as affected by growth CO2 and phosphorus nutrition in co tton. J. Plant Physiol. 170: 801 813. Spiers, J.D., T. Davies, C. He , K.M. Heinz, C.E. Bogran, and T.W. Starman. 2008. Do insecticide affect plant growth an d development? Greenh. Grow. 2: 1 3. Stevens, P.J.G., and E.A. Baker. 1987. Factors affecting the foliar absorption and redistribution of pesticides. 1. Properties of le af surfaces and their interactions with sp ray droplets. Pestic. Sci. 19: 265 281 . United States Department of Agriculture National Agricultural Statistics Service. 2016. Census of Horticultural Specialti es (2014). 2012 Census Agric. 3. AC12 - SS - 3. Vea, E., a nd C. Palmer. 2009. IR - 4 ornamental horticulture program oxyfluorfen crop safety. 119 Veeraswamy, J., T. Padmavathi, and K. Venkateswarlu. 1993. Effect of selected insecticides on plant growth and mycorrhizal development in sorghum. Agric. Ecosyst. Environ. 43 : 337 343. Vinet, L., and A. Zhedanov. 2012. Crop Stress and its Management: Perspectives and Strategies (B Venkateswarlu, AK Shanker, C Shanker, and M Maheswari, Eds.). Springer Netherlands, Dordrecht. Wang, L., X. Jiang, D. Yan, J. Wu, Y. Bian, and F. Wan g. 2007. Behavior and fate of chlorpyrifos introduced into soil crop systems by irrigation. Chemosphere 66: 391 396 . Wang, P., H. Li, W. Jia, Y. Chen, and R. Gerhards. 2018. A fluorescence sensor capable of real - time herbicide effect monitoring in greenhous e s and the field. Sensors 18:3771. Warsaw, A.L., R.T. Fernandez, B.M. Cregg, and J.A. Andresen. 2009. Container - grown ornamental plant growth and water runoff nutrient content and volume under four irrigati on treatments. HortScience 44: 1573 1580 . Warsaw, A .L., R. Thomas Fernandez, D.R. Kort, B.M. Cregg, B. Rowe, and C. Vandervoort. 2012. Remediation of metalaxyl, trifluralin, and nitrate from nursery runoff using container - grown woody ornamentals and phytoremediatio n areas. Ecol. Eng. 47: 254 263 . Wehtje, G. , C.H. Gilliam, M.E. Miller, and J.E. Altland. 2006. Foliar vs. Roo t Sensitivity of Hairy Bittercress ( Cardamine hirsuta ) to Isoxaben. Weed Technol. 20: 326 333. Wichert, R.A., and R.E. Talbert. 1993. Soybean [ Glycine max (L.)] r espons e to lactofen. weed Sci. 41: 23 27 . Willoughby, I., D. Clay, and F. Dixon. 2003. The effect of pre - emergent herbicides on germination and early growth of broadleaved species used fo r direct seeding. Forestry 76: 83 94. Wilson, S.K., and S. Von Broembsen. 2015. Capturing and rec ycling irrigation runoff as a pollution prevention measure. Oklahoma State Univ. Ext. 1518(4). 120 SECTION I V SENSITIVITY OF HYDRANGEA TO RESIDUAL HERBICIDES IN RECYCLED IRRIGATION VARIES WITH PLANT GROWTH STAGE 121 Sensitivity of Hydrangea to residual herbicides in recycled irrigation varies with plant growth stage Shital Poudyal 1, * , James S. Owen Jr. 2 , R. Thomas Fernandez 1 , and Bert Cregg 1,3, * 1 Department of Horticulture, 1066 Bogue Street, Michigan State University, East Lansing, MI 48824 2 USDA Agricultural Research Service, Application Technology Research, 1680 Madison Avenue, Wooster, OH, USA 44691 3 Department of Biochemistry and Molecular Biology, Michigan State University, 612 Wilson Rd, Rm 210 East Lansing, MI, USA 488 24 4 Department of Forestry, Michigan State University, 480 Wilson Road, East Lansing, MI, USA 48824 * Corresponding author:cregg@msu.edu ; poudyals@msu.edu 122 Abstract Recycling irrigation runoff is a viable option to achieve sustainability in horticultu ral production systems, but residual herbicides present in recycled water may be phytotoxic. The sensitivity of plants to residual herbicides may vary depending on the growth stage of the plant. Therefore, if sensitive growth stages are avoided, the risk a ssociated with using recycled water may be reduced. Here, we quantified the effect of residual oryzalin and oxyfluorfen exposure at various growth stages of Hydrangea paniculata. Exposure to both herbicides reduced plant growth, leaf visual rating, SPAD in dex, net photosynthesis and light - adapted fluorescence of H. paniculata. Herbicide injury was higher for plants exposed to herbicides at early growth stages; however, the recovery rate of those plants was also rapid. For oxyfluorfen, leaf damage was less n oticeable as plants continued to produce healthy new growth immediately after the end of exposure but for oryzalin, even newly formed leaves developed herbicide injury, therefore leaf damage continued to progress before recovering. Physiological measuremen ts such as SPAD index, net photosynthesis and light - adapted fluorescence responded more quickly compared to growth index and leaf visual rating hence provided an earl y indicator of plant recovery. It is best to avoid early growth stages when irrigating wit h recycled water that may contain herbicides. However, damage caused by residual herbicide exposure at all growth stages was transient, and plants recovered over time. When assessing herbicide damage and recovery, physiological measurements such as net 123 pho tosynthesis and light - adapted fluorescence can provide rapid insights on instant plant performance. Keywords : Oryzalin; Oxyfluorfen; Nursery; Ornamental crop; Phytotoxicity 124 1. Introduction Production of container - grown ornamental nursery plants is an intensive horticultural system that requires frequent inputs of water and agrochemicals to produce visually appealing plants. Irrigation in nurseries often generates substantial amounts of runoff and up to 70 - 80% of applied water may be lost from nursery p roduction areas. (Beeson and Knox, 1991; Fain et al., 2000; Poudyal and Cregg, 2019) . Runoff generated from nurseries often contains various agrochemicals, which, if released without remediation, may degrade neighboring ecosystems. Public awareness of non - p oint source pollution is growing, and so are the regulations to reduce irrigation return flow. Several states including California, Florida, Texas, Oregon, and Maryland restrict water discharge from nurseries, and other states will likely follow. (Oki and W hite, 2012; Fulcher et al., 2016) . As water security, accountability and costs associated with withdrawals from primary water sources are rising (Rodell et al., 2018; de Amorim et al., 2018) , recycling return flow is becoming environmentally sustainable an d economically viable (Fulcher et al., 2016; Ferraro et al., 2017; Pitton et al., 2018) . Therefore, nursery growers in states with and without mandatory return flow capture are starting to recycle water f or irrigating ornamental crops. Recycling nursery re turn flow for irrigation conserves water and can improve water security but it also holds some degree of risk to growers. Residual pesticides in recycled water may be phytotoxic to sensitive crops (Poudyal et al., 2019; Poudyal and Cregg, 2019) , and some g rowers report evidence of phytotoxicity associated with pesticides (personal communication). 125 Chronic, low - dose exposure to pesticides in irrigation water can result in reduced plant growth, chlorosis, leaf distortion and other visible plant injury. For exa mple, pendimethalin (2.24 kg a.i./ha) reduced plant width in heather ( Calluna vulgaris L.) and isoxaben 0.05 kg a.i./ha reduced plant height in wintercreeper euonymus ( Euonymus fortunei Turcz.), when applied as overhead spray (Regan and Ticknor, 1987) . Gly phosate residue in the rhizosphere reduced growth and biomass production in sunflower ( Helianthus annuus L.) (Tesfamariam et al., 2009) and the application of imazapyr and triclopyr for weed management in power transmission lines reduced germination rate an d vegetative growth of non - target plants; yarrow ( Achillea millefolium L.) and fireweed ( Chamerion angustifolium L.) (Isbister et al., 2017) . The concentration of pesticides in recycled water is orders of magnitude lower compared to standard application rates, but still may cause sub - lethal effects on plants. Sensitivity of plants and their capacity to overcome injury may depend on the growth st ages of plants (Follak and Hurle, 2004) . Most leaves in young plants or actively growing shoots are new and have thinner cuticles compared to mature plants and shoots (Jursik et al., 2013; Rouse and Dittmar, 2013) , hence young plants and new shoots are mor e prone to phytotoxicity compared to matured plants and shoots, but may not always be the case (Richardson, 1972) . Phytotoxic symptoms produced by short term exposure to pesticides are either reversible or irreversible (Follak and Hurle, 2004) , and the lat ter is of most concern to growers. Peach seedlings sprayed with simazine at 3 mg/L and terbacil at 3 mg/L showed excellent recovery from the damage but the seedlings sprayed with oryzalin at 6 126 mg/L did not recover (Lourens et al., 1989) . Trimec Classic (2 ,4 - D + MCPA + dicamba) and glyphosate at 1.6 kg a.i/ha were applied as overhead spray in rose plants and the plants were evaluated for pesticide related injury. Injury by Trimec recovered after 11 weeks of exposure but the injury caused by glyphosate did n ot recover. Peach did not recover from the phytotoxicity cause by oryzalin (6 mg/L) throughout the study period (Gonzalez and Karlik, 1999) . Herbicides commonly used in container nursery production, including oryzalin and oxyfluorfen, are often found in nu rsery return flow (Keese et al., 1994; Riley et al., 1994; Goodwin and Beach, 2001) . Oryzalin is a pre - emergent herbicide belonging to the dinitroaniline family; it binds to free tubulin and restricts the formation of microtubules, arresting cells in the d ividing stage, but when the herbicide is washed off the new microtubules reappear. After exposure to oryzalin, younger cells show quick recovery and reassembly of microtubules while older cells take longer to recover (Wasteneys and Williamson, 1989) . Oxyfl uorfen is a protoporphyrinogen oxidase (PPO) inhibitor and is applied as both pre - and post - emergent herbicide. Photo - oxidative damage caused by oxyfluorfen can reduce net photosynthesis ( A ) and chlorophyll fluorescence (Sharma et al., 1989) . Oxyfluorfen al so causes disturbances in mitotic cell division, producing clastogenic effects and C - mitotic effects (Dragoeva et al., 2012) . Plants may recover from phytotoxic damage caused by oxyfluorfen depending upon the length of exposure and time available for recovery. Complete recovery from phytotoxicity in rice was seen just a month after oxyfluorfen exposure (Priya et al., 2017). Pla nt injury associated with oxyfluorfen exposure is 127 often more acute when plants are exposed to oxyfluorfen at early growth stages compared to late growth stages (Akey and Machado, 1985; Nosratti et al., 2017) . In order to manage risks associated with recyc led water for irrigation, we need to develop an improved understanding of the basis of plant injury from chronic low - dose pesticide exposure. Recycling return flow water for irrigation is a viable option and, if the sensitive growth stages are avoided, the risk associated with irrigation from recycled water can be minimized. Quantifying chlorophyll fluorescence and A of plants exposed to the herbicide can reveal physiological herbicide injury (Moreland et al., 1972; Sharma et al., 1989; Krugh and Miles, 199 6; Baker, 2004; PAN et al., 2009) and can be used to monitor herbicidal stress in plants. In addition to physiological performance; growth, visual appearance, and flower quality are also essential attributes of ornamental plants as customers are more likel y to buy visually appealing plants. Therefore morphological assessments, in addition to physiological performance, can provide a complete picture of herbicide injury in plants. This study was focused on (1) quantifying the physiological and morphological e ffects of residual oryzalin and oxyfluorfen in simulated recycled water at various growth stages of Hydrangea paniculata Siebold. (Limelight), (2) identifying variation in sensitivity among growth stages of plants to residual herbicide exposure, and (3) de termining time required to recover from herbicide damage. We used Hydrangea paniculata as it is one of the most popular shrub in the U.S. - and are sensitive to residual herbicide in irrigation water (Poudyal et al., 2 019) . 128 2 . Materials and methods 2.1. Plant material and treatments This study was conducted in a greenhouse at the Michigan State University Horticulture Teaching and Research Center (HTRC) located in Holt, Michigan, USA (42.67° N, 84.48° W). Hydrangea pani culata 20; Volume: Volume) were used for our study. Starter plants from 10 cm plugs (liners) of H. paniculata were planted on May 24, 2018, and grown outdoors in 11.3 L plastic con tainers at the HTRC and received 19 mm of daily overhead irrigation and medium recommended dose (60 g per container) of controlled - release fertilizer (18 5 8; N - P 2 O 5 - K 2 O with micronutrients, 5 6 months, ICL Specialty fertilizers, Summerville, SC, USA) appl ied as a top - dressing. Plants were brought into an unheated plastic hoop house on October 26, 2018, and leaves were allowed to senesce and the plants to go dormant. All plants were pruned consistently, leaving only three shoots of 10 cm length per plant. All the plants were brought into the greenhouse on January 15, 2019, and were fertilized with 60 g of the same fertilizer as mentioned above and irrigated via a drip irrigation system. The temperature in the greenhouse was set to 23°C and plants received n atural light. Buds began to sprout on plants on January 27, 2019, and by Feb 6, 2019, all the plants had visible leaves on at least six different nodes. As all the plants had initiated growth by Feb 6, 2019, this day was . 129 Plants were assigned at random to two treatment groups; one set receiving simulated recycled irrigation containing 0.02 mg/L of oxyfluorfen (Goal 2XL; Dow AgroSciences LLC, Indianapolis, IN) and the other set receiving simulated recycled irrigation containing 8 mg/L of oryzalin (Surflan AS; United Phosphorus Inc., King of Prussia, PA). We prepared the desired concentration of oxyfluorfen and oryzalin solution by dissolving the appropriate amount of each herbicide in 50 liters of water. Two 100 L black plastic tanks were used to prepare and store herbicide solution. A submersible sump pump was used to agitate the herbicide solution and to manually apply herbicide solution as overhead irrigation on all the leaves of the plant and on the subst rate. Herbicide solution was applied daily with an irrigation wand (Yardworks® Front Trigger Red 7 - Pattern Nozzle, Model Number: 56715) and lasted for a minute. Herbicide solutions were freshly prepared two times a week. We selected 0.02 mg/L of oxyfluorfe n and 8 mg/L of oryzalin as herbicide treatments as these are the maximum concentrations reported in nursery irrigation return flow or retention reservoirs (Keese et al., 1994; Riley et al., 1994; Briggs et al., 2003) . Each treatment group was further div ided into five sub - groups, with five individual plants (replication) per sub - group. One sub - group of plants served as an untreated control; the remaining four groups received herbicide exposure at four different growth stages, i.e., five days after initiat ion of growth (GS+5; maximum of two nodes per branch), 15 days after initiation of growth (GS+15; maximum of five nodes per branch), 25 days after initiation of growth (GS+25; maximum 130 of seven nodes per branch) and 35 days after initiation of growth (GS+35 ; maximum of nine nodes per branch). A flow chart for herbicide exposure is described in Table 1. Plants were temporarily isolated with foam panels during the spraying process to avoid cross - contamination and then put back in place. After ten days of conti nuous exposure, plants were returned to the drip irrigation system and observed for damage and recovery over the course of the next 20 days and also at the end of the study at 65 days. Each time the plants were evaluated for treatment responses, simultaneo us observations of plants from the contro l group were also carried out. In addition to the plants mentioned above, three sets of H. paniculata with three plants per set were grown separately until bloom under drip irrigation following a similar management strategy as previous mentioned plants. When flower panicles were approaching complete bloom, flowers on the first set were sprayed with oxyfluorfen, flowers on the second set were sprayed with oryzalin using the same application rates, durations, and meth ods as for the whole - plant exposure experiments above. A third set of three plants were allowed to bloom and acted as a control. 2.2. Assessment of physiological and morphological effect of herbicide Herbicide injury was assessed for each treatment group at the end of each ten - day herbicide exposure period. Injury was also assessed on 10 and 20 days after cessation of herbicide exposure to determine plant recovery. One final assessment was conducted at the end of the study i.e. 65 days after first leaf eme rgence. On each assessment of phytotoxicity, control plants were assessed simultaneously to compare with the herbicide exposure group. 131 At each assessment, leaves were examined for the visible damage (e.g., discoloration, stunting, and curling) and scored on the scale of zero (all dead leaves) to ten (no leaf damage). Growth index (GI; an average of plant height and two perpendicular width s) was measured on each plant. A portable photosynthesis system (LI - 6400 XT, Li - Cor, Inc., Lincoln, NE) mounted with a leaf chamber fluorometer (LI - 6400 - 40, Li - Cor, Inc., Lincoln, NE) was used to measure A and light - adapted fluorescence ( ). A section of a fully mature leaf on either the 3 rd or 4 th node from the shoot of each plant was used for physiological measurem ents. Photosynthetically active radiation (PAR) in the chamber was set to 1500 µmol m - 2 s - 1 , block temperature was set to 25°C, and 400 ppm of CO 2 was supplied, and relative humidity in the chamber varied between 40 60%. Each leaf was then acclimatized for five minutes. We first measured A and then . We also measured the SPAD index (leaf chlorophyll index) on three leaves per plant on either the 3 rd or 4 th node from the top using a portable SPAD meter (SPAD - 502; Minolta corporation, Ltd., Osaka, Japan). At the end of the study, plants were harvested and dried in an oven (45°C), to determine total above - ground biomass (TDB; the weight of leaves and stem after drying in an oven at 45°C for three days). Flowers were assessed for herbicide phytotoxici ty on nine additional plants. After ten days of herbicide exposure, panicles were left to completely bloom (for 12 days) and then were assessed 132 for their GI (average of height and two perpendicular widths), total flower mass (TFM; the weight flower after d rying in an oven at 45°C for 3 days) and visual injury (on the scale of zer o to ten). 2 .3. Statistical analysis The experiment was conducted as a completely randomized design with five replications per treatment. All the statistical analyses were carried out using SAS (version 9.4; SAS Institute, Inc., Cary, NC). Before analysis, all the data except visual injury were converted to percentage based on means of the control. The mean value of control was assumed to be 100 percent and the variation in control was also calculated based on mean value of 100 percent. For each herbicide, we analyzed visual leaf injury, and percentage change in GI, A, , and SPAD index using one way ANOVA. Final data after 65 days of the initiation of growth for TDB, visual le af injury, GI, A, , and SPAD index were analyzed, using one way ANOVA. Preliminary analyses indicated significant differences for some evaluation parameters between herbicides and growth stage × herbicide interactions; therefore, data were analyzed separately for each herbicide. Fisher's least significant difference post - hoc mean separation was carried out for ANOVA with p - value of 0.05 or less to determine a difference within treatments, for each herbicide. Any data that did not meet the assumption of homogeneity of variance were transfor med before statistical analysis. 133 3. Results 3.1. Morphological responses to herbicide exposure Exposure to simulated recycled irrigation containing oryzalin reduced GI of H. paniculata compared to plants that were no t exposed (Fig. 1). The largest reduction in GI (20%) was observed immediately after the end of oryzalin exposure, when plants were exposed at the earliest growth stage i.e., GS+5. However, plants exposed to oryzalin at GS+5 recovered quickly compared to o ther growth stages. Oryzalin exposure at GS+15, GS+25, and GS+35 resulted between 9 to 12% reduction in GI immediately after the end of the exposure. Even with lower reduction compared to GS+5, GI of plants exposed at GS+5, GS+15, and GS+25, was still lowe r compared to control plants, 20 days from the end of oryzalin exposure. Reduction in GI caused by oxyfluorfen exposure was similar to that of oryzalin. Reduction in GI, immediately after the end of oxyfluorfen exposure, was highest (32%) when plants were exposed to oxyfluorfen at GS+5 and least (7.5%) when plants were exposed to oxyfluorfen at near maturity i.e., GS+35. GI of plants receiving oxyfluorfen exposure at GS+25 recovered completely in 20 days after the end of exposure and GI in plants receiving oxyfluorfen exposure at GS+35 recovered completely just in ten days after the end of oxyfluorfen exposure. Plants receiving oxyfluorfen exposure at GS+5 and GS+15 did not recover completely even after 20 days from the end of oxyfluorfen exposure (Fig. 1). The location of leaf injury was similar for both herbicides and occurred on younger leaves towards the tip of the stem. In contrast, the type of damage caused by each herbicide was different. 134 Oryzalin exposure distorted leaf shape and produced random yello w patches in leaves while oxyfluorfen exposure reduced leaf size and caused complete or interveinal necrosis (Fig 2). Immediately after the end of oryzalin exposure, plants exposed at GS+5 had the lowest leaf visual rating (7.4), while plants exposed at al l other growth stages had lower but similar (8.2 to 8.8) leaf damage (Fig. 3). Plants did not recover from leaf injury immediately after end of oryzalin exposure. Instead, leaf visual rating declined from one day after the end of exposure to 10 days after the end of exposure. Leaf injury across all growth stages started recovering (by growth of healthy new leaves) rapidly and was only 10% lower compared to control on the 20 th day after the end of oryzalin exposure (Fig. 3). Leaf injury for oxyfluorfen expos ure followed a similar pattern as GI. Immediately after the end of oxyfluorfen exposure, the lowest leaf visual rating (4.8) was observed on plants exposed to oxyfluorfen at GS+5, whereas leaf visual rating was highest (8) on plants exposed to oxyfluorfen at GS+25. Unlike oryzalin, plants exposed to oxyfluorfen at all growth stages started recovering (by growth of healthy new leaves) immediately after the end of oxyfluorfen exposure. After 20 days from the end of oxyfluorfen exposure, plants exposed at GS+5 had lowest leaf injury, while plants exposed at GS+35 had maximum leaf injury. However, leaf visual rating for plants receiving oxyfluorfen exposure at all growth stages was still 6 to 13% lower compared to control even on the 20 th day after the end of o x yfluorfen exposure (Fig. 3). 135 3.2 . Physiological responses to herbicide exposure Exposure to both herbicides reduced SPAD chlorophyll index. SPAD index was reduced on plants across all growth stages on the 1 st and the 10 th day after the end of herbicide e xposure. However, on the 20 th day after the end of the herbicide exposure SPAD index of exposed plants was similar to control plants regardless of when plants were exposed to herbicide (Fig. 4). Exposure to each herbicide at all growth states reduced A at one or ten days after exposure, or both (Fig. 5). However, A recovered to the same level as non - exposed plants for all plants regardless of exposure dates or herbicide by day 10 or 20 (Fig. 5). Net photosynthesis did not decrease immediately after the end of oryzalin exposure on GS+5 and GS+25 plants. However, A was consistently lower across all growth stages on the 10 th day from the end of oryzalin exposure. The largest reduction in A (36%) occurred when plants were exposed at GS+15, immediately after the end of oryzalin exposure. However, plants receiving oryzalin exposure across all growth stages had similar A compared to control, 20 days after the end of oryzalin exposure. For oxyfluorfen exposure, reduction in A was observed for all growth stages, immed iately after the end of oxyfluorfen exposure. However unlike oryzalin, plants across all the growth stages slowly and progressively recovered in next 10 days, at which time the A of plants exposed to oxyfluorfen and control was similar. Oryzalin exposure at GS+15 and GS+35 immediately reduced but for GS+5 and GS+25 was not reduced immediately. 10 days after the end of oryzalin exposure 136 was still lower on GS+35 and was further lowered for GS+5 but for GS+15 was fully recovere d. Reduction in was never observed on GS+25. However 20 day after the end of oryzalin exposure for all growth stages was similar to that of control (Fig. 6). Oxyfluorfen exposure at all growth stages, except GS+25, immediately reduced (Fig. 6). However 10 days after the end of oxyfluorfen exposure completely recovered for three out of four growth stages, except GS+25, which completely recovered by 20 days of the end of oxyfluorfen exposure (Fig. 6). Overall recovery was slightly faster for ox yfluorfen compared to oryzalin. 3.3 . Final evaluation Plants receiving oryzalin exposure at different growth stages had similar TDB, GI, SPAD index, A , and at 65 days after the emergence of first leaf. They only differed in the leaf visual rating. Leaves injury did not recover completely on plants receiving oryzalin exposure at GS+15, GS+25 and GS+35. Sixty - five days after the emergence of the first leaf, le af visual rating was lowest for plants in treatment group GS+35 and GS+25 (Table 2). Sixty - five days after the emergence of the first leaf, plants receiving oxyfluorfen exposure at GS+5 and GS+15 had 31% and 15% lower TDB compared to control. Plants receiv ing oxyfluorfen at all other growth stages had TDB similar to that of control. In contrast, the leaf visual rating was lower for plants receiving oxyfluorfen exposure at later growth stages. Leaf visual rating for GS+35, GS+25 and GS+15 was reduced by 17%, 8% and 4%, respectively. GI, SPAD index, 137 A and at the end of the vegetative stage completely recovered in all the plants exp osed to oxyfluorfen (Table 2). 3.4. Evaluation of flowers GI, TFM, and visual rating of flowers of H. paniculata were not affected (p>0.05) by exposure to either oxyfluorfen or oryzalin. GI for control, oryzalin and oxyfluorfen were 8.86 ± 0.2 g, 7.19 ± 0.38 g and 7.41 ± 0.18 g respectively. TFM for control, oryzalin and oxyfluorfen were 17.49 ± 0.56 cm, 17.21 ± 0.56 cm and 1 7.41 ± 43 cm respectively and visual rating for flowers receiving any of three treatments were 10 out of 10. 4. Discussion 4.1. Morphological response depends on the growth stage of plant Studies evaluating the effect of herbicides at specific leaf stages of weed and crops are common (Roe and Buchman, 1963; Klingaman et al., 1992) . However, researchers acknowledge that studies concerning herbicide sensitivity at varying stages of plant are comparatively rare (Shim et al., 2003) . Some herbicides may injure younger leaves, while others may produce damage on older leaves (Kuk et al., 2006; Yoon et al., 2011) . In our study, we increased the duration of herbicide exposure but reduced the concentration of herbicide compared to general herbicide application practi ce in order to simulate irrigation with recycled water. Both oxyfluorfen at 0.02 mg/L and oryzalin at 8 mg/L produced phytotoxicity in H. paniculata and injury was primarily 138 observed in younger and growing leaves for all four growth stages that we tested. However, the maximum morphological damage occurred for GS+5 plants; hence it was the most sensitive growth stage for both herbicides. Younger leaves adsorb, retain and translocate higher concentration of herbicides because of thinner cuticle and wax layer compared to mature leaves (Akey and Machado, 1985; Zhu et al., 2018) and higher number of exposed leaves due to lesser canopy density in younger leaves increase pesticide interception (Sellers et al., 2003) . Antioxidant capacity of leaves is known to incre ase herbicide resistance and is relatively low in younger leaves (Moustaka et al., 2015; Nobossé et al., 2018) . All leaves in GS+5 plants were young, rapidly growing, and had open canopy at the time of herbicide exposure; therefore, they sustained maximum herbicide damage. Plants that received herbicide at GS+15, GS+25 and GS+35, had some mature leaves that were increasingly tolerant to residual concentration of herbicide resulting in lower herbicide injury. Dithiopyr, a similar herbicide as oryzalin, produ ced a greater reduction in growth when applied at early growth stages (McCullough et al., 2014) and oxyfluorfen injury was also found to be higher on plants exposed to oxyfluorfen at early growth stages compared to late growth stages (Akey and Machado, 198 5; Nosratti et al., 2017) . In our study, after the end of herbicide exposure, leaf visual rating on plants exposed to oxyfluorfen started to recover immediately. This is consistent with oxyfluorfen mode of action as it is minimally translocated from the ap plication site and mostly works as a contact herbicide (Chun et al., 2001) . In contrast, for plants exposed to oryzalin, herbicide damage increased from one to ten days after the end of exposure as oryzalin is readily absorbed and sometimes translocated fr om newly growing leaves 139 (Appleby and Valverde, 1989; Sterling, 1994) . Therefore, even after the end of oryzalin exposure, oryzalin absorbed and retained in leaves was affecting newly forming and enlarging leaves. Another reason for the difference in recove ry may be due to the mode of action of these herbicides. Oxyfluorfen produces reactive molecules that disrupt cell membranes and cause cell death; this reaction is immediate in the presence of light (Kunert et al., 1985; Anatra - Cordone et al., 2005) . while oryzalin restricts the formation of microtubules that do not produce immediate visual injury or other effects (Hugdahl and Morejohn, 1993) . Oxyfluorfen has minimal impact on cell division and growth while the mode of action for oryzalin is predominantly r elated to cell division and growth; therefore, the effect of oryzalin is delayed and persists longer. The effect of sub - lethal dose of herbicide may vary depending on the growth stage of plants (Boutin et al., 2014) . In our study maximum visual damage was observed when plants were exposed to herbicide at early growth stage and similar to our finding, soybean plants also had a maximum visual injury at early growth states when exposed to a sub - lethal dose of 2,4 - D (Scholtes et al., 2019) . Recovery in GI and l eaf visual rating was rapid in plants exposed to herbicides at GS+5, because cell multiplication and growth are rapid at early vegetative stages compared to other stages of plant growth (Van De Sande - Bakhuyzen and Alsberg, 1927; Goudriaan and Van Laar, 199 4) . 140 4 .2. Physiological measurements provide a rapid indicator of herbicide damage and recovery In our study, physiological measurements (SPAD index, A and ) responded to herbicide exposure. Oxyfluorfen directly reduces chlorophyll formation but oryzalin does not have a direct impact on chlorophyll, and this was evident in our study through SPAD index. The reduction in the SPAD index was higher for oxyfluorfen compared to oryzalin, during the early growth stage i.e., GS+5 (statistical comparison n ot shown). Physiological measurements such as A and may be used as early indicators of herbicide damage (Yanniccari et al., 2012; Wang et al., 2018) . In our study, both A and fluorescence parameters had a faster and greater response to herbicide c ompared to visual injury and growth, at later growth stages from GS+15 to GS+35 and GI and leaf visual rating had greater response at early growth stage i.e., GS+5. For oryzalin, leaf visual rating was lowest ten days after the end of the exposure, but A a nd had already started to recover. The increase in A and was followed by visual leaf recovery evident on the 20 th day after the end of oryzalin exposure. Thus leaf damage by herbicides such as oryzalin that do not produce immediate visible damage can be identified quickly by using physiological tools and those tools can be used as early indicators for herbicide damage, preferably at later growth stages when visible damage take some time to appear . In contrast to our result, exposure to oryz alin did not reduce A for dwarf gardenia ( Gardenia jasminoides Pennisetum rupelli Steud.) 141 probably because the concentration used was eight times lower compared to ours (Bhandary et al., 1997) . As discussed earlier, r ecovery from injury associated with oxyfluorfen exposure was faster than recovery from oryzalin and was obvious during physiological evaluations. For oxyfluorfen, both A and completely recovered from herbicide damage as early as ten days after the end of exposure and was followed by morphological recovery. Thus physiological tools can also be efficiently applied to detect herbicide recovery in addition to herbicide damage. Physiological recovery of plants from oryzalin exposure was slower compared t o oxyfluorfen. Physiological parameters such as A and were same or lower 10 day after the end of oryzalin exposure compared to a day after the end of oryzalin exposure, expect on GS+15 for . Overall, plants exposed to oryzalin took somewhere from 10 to 20 days for A and to completely recover. However, this was still quicker than recove ry of GI and visible symptoms. 4.3 . Flowers were not damaged by residual oryzalin and oxyfluorfen Both oxyfluorfen and oryzalin exposure did not produc e any effect on flowers in H. paniculata . Oxyfluorfen mode of action requires the presence of chlorophyll within the chloroplast, in flowers (petals), there are chromoplasts instead of chloroplast which is the main reason behind the resistance of flowers t o oxyfluorfen (Thomson and Whatley, 1980; Lysenko and Varduny, 2013) . Stomatal density in flowers is lower compared to leaves (Zhang et al., 2018) and lower stomatal density also reduces herbicide penetration and damage. Thus exposing flowers to these herb icides did not produce injury. Oryzalin application impacts flower morphology at a 142 cellular level by swelling the tip of conical cells, changing the epidermal cell angle and producing shorter cells (Ren et al., 2017) but was not observed at the mo rphologic al scale in our study. 4.4 . Leaf visual injury takes the longest to recover Oryzalin did not reduce TDB at the end of vegetative growth stage which possibly is because oryzalin had lower leaf damage and less reduction in the SPAD index compared to oxyfluo rfen. At the end of the vegetative growth stage (65 days after leaf initiation), GI for oxyfluorfen exposure was the same across all growth stages but TDB was lower for GS+5 and GS+15 (Table 2). Therefore oxyfluorfen exposure at early growth stages (GS+5 a nd GS+15) will increasing radial growth but reduce plant density. Reduction in TDB caused by oxyfluorfen application at early growth stages did not recover even after 50 days of oxyfluorfen exposure but exposure at later growth did not reduce final TDB. Si milarly, in other studies, oxyfluorfen application in strawberry to control broadleaf weeds produced transient foliar injury that usually did not translate to yield loss (Daugovish et al., 2008) and oryzalin application at 1 mg/L did not reduce root and sh oot weight in dwarf gardenia (Bhandary et al., 1997) . Visual leaf injury for plants exposed to both herbicides at GS+15, GS+25 and GS+35 were still present at the end of the vegetative growth stage, and the leaf injury from oxyfluorfen exposure was higher (4 - 13%) compared to oryzalin (5 - 10%) exposure. Therefore leaf injury needs the longest time to recover compared to other morphological and physiological parameters. In other studies, oryzalin applications at various rates on sweet potato produced sustained leaf distortion 143 (<10 %) and plant stunting (<12 %) (Chaudhari et al., 2018) and oxyfluorfen produced lasting leaf injury in cabbage ( Brassica oleracea L.), tomato ( Lycopersicon esculentum Mill.), cucumber ( Cucumis sativus L.), and lettuce ( Lactuca sativa L.) (Grabowski and Hopen, 1985) . However leaf injury produced by herbicide at early growth stages may completely recover. Visual leaf injury caused by oryzalin application, immediately after transplanting, in sweet potato reversed and did not translate to a reduction in yield (Chaudhari et al., 2018) and foliar injury in broccoli produced by post - emergence application of oxyfluorfen completely recovered in late - maturing varieties, while early maturing varieties had sustained foliar injury and yield loss (F arnham and Harrison, 1995) . Similarly, in our study visual leaf injury for GS+5 completely recovered while visual leaf injury cause by herbicide exposure at late r growth stage did not recover. 5. Conclusion Residual herbicides such as oxyfluorfen or oryzal in present in recycled water may produce sub - lethal effects on woody ornamentals when used for irrigation. Young and growing leaves are more susceptible to herbicidal injuries compared to mature leaves. Early growth stages of plants have a higher ratio of young to mature leaves and therefore are more prone to herbicide damage. Leaf injury from some herbicides will immediately begin to recover while leaf injury from others will continue to increase before starting to recover. Physiological measurements of he rbicide damage can be assessed earlier compared to morphological measurements, particularly for herbicides that do not produce damage immediately after exposure, and can reflect immediate plant 144 performance. Physiological measurements are more sensitive to herbicide injury at later growth stages while morphological measurement may be sensitive at early growth stages. Hence, those tools can be used as an early indicator of damage and recovery. Damage caused by herbicides such as oxyfluorfen that directly dest roy photosynthesis apparatus is more severe and may permanently reduce TDB if plants are exposed at early growth stages. Flowers were not affected by 0.02 mg/L of oxyfluorfen and 8 mg/L or oryzalin exposure because of the differences in cell structure comp ared to leaves. The limitation of our study is the use of only one plant taxon and two herbicides; results may be different if different taxa or herb icides with a different mode of action are used. 145 APPENDIX 146 APPENDIX Table IV - 1 . Flow chart for herbicide exposure. 147 Figure IV - 1 . Relative growth index of H. paniculata top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (GS) GS+5 received herbicide exposure five days after initiation of growth, GS+15 rece ived herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 days after initiation of growth. Standard errors of the means are denoted by vertical Significant Difference (LSD) post - hoc test. Means within each herbicide across all growth stages that are followed by the same letters are not significantly different at p=0.05. 148 Figure IV - 2 . Representative herbicide damage immediately after the end of oxyfluorfen exposure (A) and ten days after the end of oryzalin exposure (B). Plants were exposed to oxyfluorfen or oryzalin at growth stage (GS), G S+15 for ten days. Both plants received a score of seven out of ten for leaf visual rating. 149 Figure IV - 3 . Leaf visual rating of H. paniculata or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (GS) GS+5 received herbicide exposure five days after initiation of growth, GS+15 rece ived herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 days after initiation of growth. Standard errors of the means are denoted by vertical Mean separations for each herbicide were carried out using Least Significant Difference (LSD) post - hoc test. Means within each herbicide across all growth stages that are followed by the same letters are not significantly different at p=0.05. 150 Figure IV - 4 . Relative SPAD index of H. paniculata top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (G S) GS+5 received herbicide exposure five days after initiation of growth, GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 da ys after initiation of growth. Standard errors of the means are denoted Significant Difference (LSD) post - hoc test. Means within each herbicide across all growth stages that are followed by the same letters are not significantly different at p=0.05. 151 Figure IV - 5 . Relative net photosynthesis of H. paniculata mg/L; top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (GS) GS+5 received herbicide exposure five days after initiation of growth, GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbic ide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 days after initiation of growth. Standard errors of the means are denoted Signi ficant Difference (LSD) post - hoc test. Means within each herbicide across all growth stages that are followed by the same letters are not significantly different at p=0.05. 152 Figure IV - 6 . Percent reduction in light - adapte d fluorescence of H. paniculata response oryzalin (8 mg/L; top) or oxyfluorfen (0.02 mg/L; bottom) following 10 days of herbicide exposure at various stages of plant growth. Growth stage (GS) GS+5 received herbicide exposure five days after initiation of growth, GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 days after initiation of growth. Standard errors of th carried out using Least Significant Difference (LSD) post - hoc test. Means within each herbicide across all growth stages that are followed by the same letters are not signi ficantly different at p=0.05. 153 Table IV - 2 . Total dry above - ground biomass (TDB), leaf visual rating (VR), SPAD index (SPAD), growth index (GI), photosynthesis (A) and light - adapted chlorophyll fluorescence H. pan iculata exposed to either oryzalin (8 mg/L) or oxyfluorfen (0.02 mg/L) at various growth stages (GS) for ten days. GS+5 received herbicide exposure five days after initiation of growth, g GS+15 received herbicide exposure 15 days after initiation of growth, GS+25 received herbicide exposure 25 days after initiation of growth and GS+35 received herbicide exposure 35 days after initiation of growth. Mean separations for each herbicide were ca rried out using Least Significant Difference (LSD) post - hoc test. Means within each herbicide that are followed by the same letters are not significantly different at given p - values. Oryzalin exposure GS TDB (g) VR SPAD GI (cm) A Fv'/Fm' Control 143.85 10.00a 36.96 96.47 15.97 0.60 GS + 5 124.61 9.70ab 37.22 92.60 16.18 0.59 GS + 15 126.85 9.50b 35.30 91.40 14.79 0.60 GS + 25 119.85 9.30bc 36.64 89.00 15.21 0.60 GS + 35 127.65 9.00c 35.12 90.27 15.54 0.58 p - value NS <0.0005 NS NS NS NS Oxyfluorfen exposure GS TDB (g) VR SPAD GI (cm) A Fv'/Fm' Control 128.72a 10.00a 37.12 91.80 16.52 0.56 GS + 5 88.65c 10.00a 36.78 88.20 15.99 0.53 GS + 15 104.46bc 9.60b 35.16 91.07 15.20 0.55 GS + 25 108.60abc 9.20c 36.90 89.20 15.74 0.52 GS + 35 128.37ab 8.70d 37.72 92.73 15.46 0.54 p - value <0.05 <0.0005 NS NS NS NS 154 LITERATURE CITED 155 LITERATURE CITED Akey, W.C., and V.S. Machado. 1985. Response of onion ( Allium cepa ) to oxyfluorfen during early seedling devel opment. Can. J. Plant Sci. 65 : 357 362 . de Amorim, W.S., I.B. Valduga, J.M.P. Ribeiro, V.G. Williamson, G.E. Krauser, M.K. Magtoto, and J.B.S.O. de Andrade Guerra. 2018. The nexus between water, energy, and food in the context of the global risks: An analy sis of the interactions between food, water, and energy security. E nviron. Impact Assess. Rev. 72: 1 11. Anatra - Cordone, M., C. King, J. Klotzbach, and P.R. Durkin. 2005. Oxyfluorfen - Human Health and Ecological Risk Assessment - Final Report USDA, Forest Serv ice. USDA, For. Serv. (SERA TR 05 - 43 - 26 - 03b)Available at www.sera - inc.com (verified 19 December 2019). Appleby, A.P., and B.E. Valverde. 1989. Behav ior of dinitroaniline herbicides in plants . Weed Technol. 3: 198 206. Baker, N.R. 2004. Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possi bilities. J. Exp. Bot. 55: 1607 1621 . Beeson, R.C., and G.W. Knox. 1991. Analysis of Efficiency of Overhead Irrigation in Container Production. Hortscience 26: 848 850 . Bhandary, R., T. Whitwell, J.A. Briggs, and R.T. Fernande z. 1997. Influence of Surflan (Oryzalin ) Concentrations in Irrigation Water on Growth and Physiological Processes of Gardenia jasminoides radicans and Pennisetum rupelli. J. Environ. Horti c. 15: 169 172. Boutin, C., B. Strandberg, D. Carpenter, S.K. Mathiassen, and P.J. Thomas. 2014. Herbicide impact on non - target plant reproduction: What are the toxicological and ecological implications? Environ. Pollut. 185: 295 306 . Briggs, J.A., T. Whitwel l, and M.B. Riley. 2003. Effect of delayed irrigation on isoxaben and oryzalin runoff from a cont ainer nursery. Weed Sci. 51: 463 470 . Chaudhari, S., K.M. Jennings, and S.L. Meyers. 2018. Response of Sweetpotato to Oryzalin Application Rate a nd Timing. Weed Technol. 32: 722 725. Chun, J.C., H.J. Lee, S.J. Lim, S.E. Kim, and J.O. Guh. 2001. Comparative absorption, translocation, and metabolism of foliar - applied oxyfluorfen in wheat and barley. P estic. Biochem. Physiol. 70: 118 125. 156 Daugovish, O., S.A. Fennimore , and M.J. Mochizuki. 2008. Integration of Oxyfluorfen into Strawberry ( Fragaria×ananassa ) Weed Manageme nt Programs. Weed Technol. 22: 685 690. Dragoeva, A., V. Koleva, N. Hasanova, and S. Slanev. 2012. Cytoto xic and genotoxic effects of diphenyl - ether herb icide GOAL (Oxyfluorfen) using the Allium cepa test. Res. J. Mutagen. 2: 1 9. Fain, G.B., C.H. Gilliam, K.M. Tilt, J.W. Olive, and B. Wallace. 2000. Survey of best management practices in container production nurseries . J . Environ. Hort. 18: 142 144 . Farnham , M.W., and H.F. Harriso n. 1995. Response of Broccoli ( Brassica oleracea ) cultivars to post - transplant oxyfluorfen . Weed Technol. 9: 385 391. Ferraro, N., D. Bosch, J. Pease, and J.S. Owen. 2017. Costs of capturing and recycling irrigation water in containe r nurseries. HortScience 52: 258 263 . Follak, S., and K. Hurle. 2004. Recovery of non - target plants affected by airborne bromoxynil - octanoate and metribuzin. Weed Res. 44: 142 147. Fulcher, A., A. V. LeBude, J.S. Owen, S.A. White, and R.C. Beeson. 2016. The next ten years: Strategic vision of water resources for nursery producers. Horttechnology 26: 121 132. Gonzalez, M.P., and J. Karlik. 1999. Evaluation of herbicides for phytotoxicity to rose plants and ef ficacy. J. Environ. Hort 17: 164 167 . Goodwin, P.B., a nd S. Beach. 2001. Oxadiazon, oryzalin, and oxyfluorfen residues in container pl ant nurseries. HortScience 36: 900 904. Goudriaan, J., and H.H. Van Laar. 1994. Modelling Potential Crop Growth Processes. Springer Netherlands, Dordrecht. Grabowski, J.M., and H.J. Hopen. 1985. Phytotoxic Effect of Oxyfluorfen Vaporization. Weed Sci. 33: 306 309 . Hugdahl, J.D., and L.C. Morejohn. 1993. Rapid and reversible high - affinity binding of the dinitroaniline herbicide oryzalin to tubulin from Zea mays L. Plant Physiol. 10 2: 725 740 . Isbister, K.M., E.G. Lamb, and K.J. Stewart. 2017. Herbicide toxicity testing with non - target boreal pl ants: The s ensitivity of Achillea millefolium L. and Chamerion angustifolium L. to Triclopyr and Im azapyr. Environ. Manage. 60: 136 156. Jursik , M., K. Hamouzova, J. Andr, and J. Soukup. 2013. Effect of different adjuvants on 157 phytotoxicity of flumioxazin to sunflower in different growth stages. Rom. Agric. Res. 30 : 365 372. Keese, R.J., N.D. Camper, T. Whitwell, M.B. Riley, and P.C. Wilson. 1994. Herbici de runoff from ornamental container nurs eries. J. Environ. Qual. 23: 320 324. Klingaman, T.E., C.A. King, and L.R. Oliver. 1992. Effect of Application Rate, Weed Species, and Weed Stage of Growth on Imaze thapyr Activity. Weed Sci. 40: 227 232. Krugh, chemicals on terrestrial plants using chlorophyll fluorescence. Environ. Toxicol. Chem. 15: 495 500. Kuk, Y.I., J. - S. Shin, H. Il Jung, J.O. Guh, S. Jung, and N.R. Burgos. 2 006. Mechanism of paraquat tolerance in cucumber leaves of various ages. Weed Sci. 54: 6 15. Kunert, K.J., C. Homrighausen, H. Böhme, and P. Böger. 1985. Oxyfluor fen and lipid peroxidation: protein damage as a phytotoxic consequence . Weed Sci. 33: 766 770 . L ourens, A.F., A.H. Lange, and F.J. Calitz. 1989. Phytotoxicity of pre - emergence herbicides to peach seedlings ( Prunus persica ). South African J. Plant Soil 6: 97 102. Lysenko, V., and T. Varduny. 2013. Anthocyanin - dependent anoxygenic photosynthesis in colou red flower petals? Sci. Rep. 3: 3373. McCullough, P.E., D.G. de Barreda, S. Sidhu, and J. Yu. 2014. Dithiopyr behavior in smooth crabgrass ( Digitaria ischaemum ) as influenced by growth stage and temperature . Weed Sci. 62: 11 21 . Moreland, D.E., F.S. Far mer, and G.G. Hussey. 1972. Inhibition of photosynthesis and respiration by substituted 2,6 - dinitroaniline herbicides. I. Effects on chloroplast and mitochondrial activities. pestic. biochem. physiol . 2: 342 353 . Moustaka, J., G. Tanou, I.D. Adamakis, E.P. Eleftheriou, and M. Moustakas. 2015. Leaf age - dependent photoprotective and antioxidative response mechanisms to paraquat - induced oxidative stress in A rabidopsis tha liana . Int. J. Mol. Sci. 16: 13989 14006. Nobossé, P., E.N. Fombang, and C.M.F. Mbofung. 2018. Effects of age and extraction solvent on phytochemical content and antioxidant activity of fresh Moringa oleifera L . leaves. Food Sci. Nutr. 6: 2188 2198. Nosratti, I., S. Mahdavi - Rad, H. Heidari, and M. Saeidi. 2017. Differential tolerance of pumpkin 158 species to bentazon, metribuzin, trifluralin, and oxyfluorfen. Planta Daninha 35:e017165650. Available at https://www.totallan dscapecare.com/landscaping/hot - hydrangeas/ (verified 17 February 2020). Oki, L.R., and S.A. White. 2012. Ecological approaches used in nurseries to treat water. UC Nur s. Floric. Alliance News 15: 1 6 . PAN, H., X. LI, X. XU, and S. GAO. 2009. Phytotoxicity o f four herbicides on Ceratophyllum demersum, Vallisneria natans and Elodea nut tallii . J. Environ. Sci. 21: 307 312 Pitton, B.J.L., C.R. Hall, D.L. Haver, S.A. White, and L.R. Oki. 2018. A cost analysis for using recycled irrigation runoff water in container nursery production: a Southern California nursery c ase study. Irrig. Sci. 36: 217 226 . Poudyal, S., and B.M. Cregg. 2019. Irrig ating nursery crops with recycled run - off : A review of the potential impact of pesticides on plant growth and physiology . Hortt ec hnology 29(6): 716 729 . Poudyal, S., B. Cregg, T.R. Fernandez, and J. Owen. 2019. Dos e - dependent phytotoxicity of pesticides in simulated nursery runoff on landscape nursery plants . Water 11: 2354. Priya, S.R., C. Chinnusamy, M.P. Arthanar, and P. Janaki. 20 17. Carryover effect and plant injury from oxyfluorfen herbicide applied in transplante d rice. Int. J. Chem. Stud. 5: 535 539 . Regan, R., and R. Ticknor. 1987. Application of isoxaben and pendimethalin to container - grown broadleaved evergreen shrubs. Ornamentals Northwest Arch. 11(1). Ren, H., X. Dang, X. Cai, P. Yu, Y. Li, S. Zhang, M. Liu, B. Chen, and D. Lin. 2017. Spatio - temporal orientation of microtubules controls conical cell shape in Arabidopsis thaliana petals (D Oppenh eimer, Ed.). PLOS Genet. 13: e1006851 Richardson, F.E. 1972. Critical Growth Stages For 2,4 - D Phytotoxicity To Sugarcane In South Africa. Proc. South A frican Sugar Technol. Assoc. 6: 168 176. Riley, M.B., R.J. Keese, N.D. Camper, T. Whitwell, P. Chris, M.B. Riley, R.J. Keese, N.D. Camper, T.E.D. Whitwell, and P.C. Wilson. 1994. Pendimetha lin and oxyfluorfen residues in pond water and sediment fr om container. Weed Technol. 8: 299 303. 159 Rodell, M., J.S. Famiglietti, D.N. Wiese, J.T. Reager, H.K. Beaudoing, F.W. Landerer, and M.H. Lo. 20 18. Emerging trends in global freshwater availability. Nature 557: 651 659. Roe, E., and R. Buchman. 1963. Effect of herbicide, dosage, and volume on hazel brush at different foliar stages. For. Sci. 9: 477 484. Rouse, C.E., and P.J. Dittmar. 2013. Factors A ffecting Herbicide Use in Fruits and Vegetables. Univ. Florida, Inst. F ood Agric. Sci. Ext. HS1219 1 4. Scholtes, A.B., B.P. Sperry, D.B. Reynolds, J.T. Irby, T.W. Eubank, L.T. Barber, and D.M. Dodds. 2019. Effect of soybean growth stage on sensitivity to s ublethal rates of dicamba and 2,4 - D. Weed Technol. 33: 555 561 . Sellers, B.A., R.J. Smeda, and W.G. Johnson. 2003. Diurnal fluctuations and leaf angle reduce glufosinate efficacy. Weed Technol. 17: 302 306. Sharma, D., R. Bhardwaj, and V. Maheshwari. 1989. I nhibition of photosynthesis by oxyfluorfen. Curr. Sci. 58: 1334 1336. Shim, S.I., B.M. Lee, E.I. Ryu, and B.H. Kang. 2003. Response of leaf acetolactate synthase from different leaf positions and seedling ages to sulfonylurea herbicide. Pes tic. Biochem. Phy siol. 75: 39 46. Shrubs: In - demand. 2018. Gard. Cent. Mag.Available at https://www.gardencentermag.com/article/shrubs - in - demand - plants - june - 2018/ (verified 17 February 2020). Sterling, T.M. 1994. Mechanisms of Herbicide Absorption Across Plant Membranes and Accumulation i n Plant Cells. Weed Sci. 4: 263 276 . Tesfamariam, T., S. Bott, I. Cakmak, V. Römheld, and G. Neumann. 2009. Glyphosate in the rhizosphere - Role of waiting times and different glyphosate binding forms in soils for phytotoxicity to non - targe t pl ants. Eur. J. Agron. 31: 126 132. Thomson, W.W., and J.M. Whatley. 1980. Development of Nongreen Plastids. Annu. Rev. Pl ant Physiol. 31: 375 394 . Van De Sande - Bakhuyzen, H.L., and C.L. Alsberg. 1927. The growth curve in ann ual plants. Physiol. Rev. 7: 151 187 . Wang, P., H. Li, W. Jia, Y. Chen, and R. Gerhards. 2018. A fluorescence sensor capable of real - time herbicide effect monitoring in greenhous es and the field. Sensors 18:3771 . 160 Wasteneys, G. o., and R.E. Williamson. 1989. Reassembly of microtubules in Nite lla tasmanica : assembly of cortical microtubules in branching clusters and its relevance to steady - state microtubule asse mbly. J. Cell Sci. 93: 705 714. Yanniccari, M., E. Tambussi, C. Istilart, and A.M. Castro. 2012. Glyphosate effects on gas exchange and chlorophyll fluorescence responses of two Lolium perenne L. biotypes with differential herbicide sensitivity. Plant Phys iol. Biochem. 57: 210 217 . Yoon, J.Y., J.S. Shin, D.Y. Shin, K.H. Hyun, N.R. Burgos, S. Lee, and Y.I. Kuk. 2011. Tolerance to paraquat - me diated oxidative and environmental stresses in squash ( Cucurbita spp. ) leaves of various ages. P estic. Biochem. Physiol. 99: 65 76. Zhang, F.P., M.R. Carins Murphy, A.A. Cardoso, G.J. Jordan, and T.J. Brodribb. 2018. Similar geometric rules govern the distr ibution of veins and stomata in petals, sepals and leaves. New Phytol. 219: 1224 1234 . Zhu, X., Y. Zhang, Z. Du, X. Chen, X. Zhou, X. Kong, W. Sun, Z. Chen, C. Chen, and M. Chen. 2018. Tender leaf and fully - expanded leaf exhibited distinct cuticle structure and wax lipid composition in Camellia sinensi s cv Fuyun 6. Sci. RepoRTS | 8: 14944 . 161 SECTION V EFFECT OF RESIDUAL PESTICIDES IN RECYCLED NURSERY RUNOFF ON GROWTH AND PHYSIOLOGY OF ORNAMENTAL SHRUBS 162 Effect of residual pesticides in recycled nursery runoff on growth and physiology of ornamental shrubs Shital Poudyal 1, *, Damon E. Abdi 1 , James S. Owen Jr. 2 , R. Thomas Fernandez 1 and Bert Cregg 1,4 1 Department of Horticulture, 1066 Bogue Street, Michigan State Universi ty, East Lansing, MI 48824 2 USDA Agricultural Research Service, Application Technology Research, 1680 Madison Avenue, Wooster, OH, USA 44691 3 Department of Biochemistry and Molecular Biology, Michigan State Un iversity, 612 Wilson Rd, Rm 210, East Lansing, MI, USA 48824 4 Department of Forestry, Michigan State University, 480 Wilson Road, East Lansing, MI, USA 48824 * Corresponding author: poudyals@msu.edu 163 Abstract Nursery runoff may contain pesticide residues which , if released off - site, could impair surrounding ecosystems. As a solution, nursery growers can retain runoff water on - site and recycle retained water to irrigate plants. However, concerns related to potential phytotoxicity caused by residual pesticides in recycled water discourage growers from recycling water. To evaluate plant quality irrigated with recycled water, we conducted a three - year field study simulating a commercial nursery growing practice. Irrigation treatments were applied to six ornamental t axa grown in a nursery production bed. Irrigation treatments were raw groundwater from the onsite well (control), water recycled from a separate nursery bed containing plants that were treated with nine pesticides regularly over the growing seasons, and re cycled water from the nursery bed that had been remediated using heat - expanded shale aggregates and woodchip bioreactors. Plants receiving recycled water (runoff water with and without remediation) did not produce pesticide - related visual injury. However, result for growth index, chlorophyll SPAD index, dark - adapted fluorescence, and shoot biomass were irregular among raw groundwater and recycled water; for most instances, pesticides in recycled water did not reduce any of those parameters. Net photosynthes is and light - adapted fluorescence were similar for raw groundwater and recycled water. Results from this study demonstrate the possibility of using recycled for irrigation of woody ornamental shrubs. Keywords: Nursery sustainability; Specialty crops, Irri gation return flow, Remediated water, Bioreactors 164 1. Introduction Unsustainable withdrawal and luxuriant use of raw groundwater are common water - related problems around the globe, including the U.S., where irrigation accounts for 38% of freshwater withdrawal (Dieter et al., 2015; Boretti and Rosa, 2019) . Water availabili ty is decreasing and so is the total water use in U.S. but the demand for freshwater and water use in many sectors of agriculture is increasing (United States Geological Survey, 2015; Rodell et al., 2018) . Concerns regarding water scarcity are particularly acute for container production of nursery crops, which is an intensive system that requires relatively high inputs of water. Reduced water availability has created restrictive regulations and is forcing container - crop producers to look for alternatives to using raw groundwater (Beeson et al., 2004; Fulcher et al., 2016) . Nurseries growers also rely heavily on agrochemicals. These agrochemicals may collect in runoff water and can be transported from the production area, potentially contaminating surrounding ecosystems. Green industry is admired for its ecological contribution and nursery being part of green industry, nursery producers are interested towards implementing strategies that can alleviate environmental consequences of their production systems (Man giafico et al., 2008; Wilson and Broembsen, 2015; Fulcher et al., 2016; Majsztrik et al., 2017) . To address those problems, it is becoming more common for nursery growers to capturing and recycling nursery runoff (Fain et al., 2000; Meador et al., 2012; Ma ck et al., 2017) . Although recycling runoff water may be a sustainable solution, agrochemicals and pathogens present in recycled water could impact plant quality and health, creating risk for growers 165 (Poudyal and Cregg, 2019) . Disease infestation is the pr imary concern of nursery growers when using recycled water as recycled water may disseminate plant pathogens (Pottorff and Panter, 1997; Hong et al., 2003) . However, technologies such as UV radiation, slow sand filtration, crushed brick filtration, chlorin ation, and ozone treatments can help to limit the spread of plant diseases in nurseries (Stewart - Wade, 2011; Nyberg et al., 2014; Younis et al., 2019) . In addition to plant diseases, growers are also concerned about crop damage from pesticides in recycled water. Pesticides such as acephate, isoxaben, bifenthrin, prodiamine, glyphosate, triflumizole, mefenoxam, thiophanate - methyl, and chlorpyrifos are commonly applied in nurseries (United States Department of Agriculture and National Agricultural Statistics Service, 2011; Poudyal and Cregg, 2019) and have been found in nursery runoff (Poudyal and Cregg, 2019) . Herbicides are of particular concern because they directly impact plant growth and physiology; isoxaben destroys cellular membrane and structures, prod iamine hinders cell division and multiplication and glyphosate inhibit the production of essential plant enzymes (Amrhein et al., 1980; Heim et al., 1990; Brosnan et al., 2014) . In addition, insecticides such as acephate, chlorpyrifos and bifenthrin cause indirect phytotoxicity by disrupting the physiological process and by producing reactive oxygen species (Spiers et al., 2006; Parween et al., 2016) . The effect of fungicides on plant growth is not fully explored but instances of both positive and negative responses of plants toward fungicides are evident (Tjosvold et al., 2005; Dias, 2012; Petit et al., 2012 ) . The occurrence of phytotoxicity and its severity also depends on the concentration of pesticides and frequency of exposure, a pesticide that is safe at lower concentrations may produce phytotoxic symptoms if the 166 concentration is increased (Veeraswamy et al., 1993; Bhandary et al., 1997a) . The concentration of pesticides in recycled water are often substantially lower compared to application rates becau se of dilution caused by the volume of water in the receiving reservoir, subsequent irrigation, pesticide adsorption, microbial degradation, hydrolysis, photodegradation and volatilization (Wilson et al., 1996; Lu et al., 2006; Poudyal and Cregg, 2019) . Th erefore reduced concentrations of pesticide in the recycled water compared to the general application rates pave its possibility for reuse. Nonetheless, irrigation with recycled water can potentially result in chronic, low - dose exposure of plants to residu al pesticides and may reduce plant quality. Technologies such as vegetative waterways, constructed wetlands, sediment traps and sand filtration have effectively been used to remove pesticides from runoff water (Briggs et al., 1998; Stearman et al., 2003; K abashima et al., 2004; Warsaw et al., 2012; Hedegaard and Albrechtsen, 2014) . In addition to those technologies, woodchip bioreactors are also gaining popularity for its potential to reduce contaminants, including pesticides, in recycled water. Woodchips a dsorb pesticides and also host a wide range of microbial organisms capable of degrading pesticides (Morillo et al., 2017; Abatenh et al., 2017; Abdi et al., 2020) ; furthermore, they are inexpensive and easily available. Thus wood chip bioreactors could be used for the remediation of pesticides in recycled water. There are numerous scientific studies related to the remediation of pesticide in a laboratory or small scale (Morillo et al., 2017) , but growers need rapid, production - scale remediation systems to h andle their water treatment requirements. In our study, we built a 2 - stage 167 bioreactor system and evaluated their potential to lower pesticide concentration in runoff water. Documenting the effect of residual pesticides on growth and quality of ornamental s hrubs is crucial for nursery growers to implement recycling of nursery runoff. Therefore, we conducted a trial to determine the response of nursery plants to irrigation with recycled runoff (RR) water that was collected from an experimental nursery of cont ainer - grown plants. The nursery plot was managed based on standard commercial nursery practices for the region, including multiple pesticide applications each season. In addition to evaluating plants irrigated with RR, we also evaluated plants irrigated wi th remediated recycled runoff (RRR) water and raw groundwater (RGW). Remediated recycle runoff water was RR water that had been remediated through 2 - stage bioreactor and RGW was the water from local well (control). The objective of the study was to assess the growth, physiology and quality of ornamental shrubs irrigated with the different water sources. To achieve our objective, we conducted a three - year field research using conventional management practices. For an ornamental nursery grower, plant growth a nd quality are of utmost importance as consumer buying preferences are based on plant quality (Khachatryan and Choi, 2017) . Therefore, when evaluating the suitability of recycled water (refer to both RR water and RRR water) for ornamental plants, it is vit al to assess plant quality in addition to plant growth and performance. Plant visual assessment, chlorophyll SPAD index, growth index and plant biomass can be used to assess plant quality (Grieve and Poss, 2010; Furtini Neto et al., 2015) . In addition, phy siological measurements such as photosynthesis, light - adapted fluorescence and dark - adapted 168 fluorescence can reflect plant health and identify pesticide stress (Petit et al., 2012 ; Silva et al., 2014; Wang et al., 2018) . 2. Materials and Methods 2.1 Fiel d layout and water treatments Field studies were conducted at Michigan State University Horticulture Teaching and - 2019. In each year we compared the responses of container - grown nursery plants to irrigation from three irrigations sources; RR, RRR and RGW. A layout of the experimental design is provided in Fig. 1. 2.1.1 . Irrigation water sources A plant evaluation bed (12.5 m x 25 m) and a runoff bed (runoff bed; 12.5 m x 25 m) were built 50 m apart. They were slightly sloped to facilitate runoff drainage and capture. The runoff bed was first topped with black impermeable pond liner (1.15 mm thick) and then with landscape fabric/weed barrier. At the lower end of the runoff bed, a runoff collection reservoir (P1) capable of holding 4000 L of water was dug to capture runoff. A pond liner was also installed on P1 to restrict water infiltration. Adjacent to runoff bed six 2 - stage bioreactors were built to remediate a portion of runoff water from P1. A single bioreactor consisted of an open - top box (1.2 m x 2.4 m x 1.2 m), lined internally with a pond liner and half - filled with hardwood woodchips (average 169 woodchip volume of 18.2 cm 3 ). Two 1.2 m long polyvinyl chloride tubes (10 cm diame ter) filled with heat - expanded shale aggregate (Haydite grade B; majority particle size 0.95 cm, Digeronimo Aggregates LLC, Ohio) were placed on the top of the box. Adjacent to the bioreactors, a remediated runoff collection reservoir (P2) able to hold 400 0 L of water was also constructed and lined internally with a pond liner. 2.1.2 . Runoff generation zone 988 woody ornamentals plants of various shrub taxa grown in an 11.3 L black plastic containers filled with pine bark and peat moss substrate (80:20; Vo lume: Volume) were transferred to the runoff bed (pot to pot spacing: 0.53m) on 12 May 2017, 24 May 2018, and 25 May 2019, and fertilized with 50 g of slow - release fertilizer (Osmocote blend; 18:2.2:6.6 N:P:K with micronutrients, 8 - 9 months, Product code # A90177, ICL Specialty fertilizers, Summerville, SC, USA). The plant tax a grown in the runoff bed varied among years but included common nursery shrubs such as Deutzia gracilis ® ) , Hydrangea paniculata Siebo (Fire light and lime light) , Hydrangea arborescens L. , Hydrangea macrophylla , Weigela florida ne & Roses®) , Spiraea japonica Berberis thunbergii , Continus Coggygria Black) , Potentilla fruiticosa (Happy face) , Rosa Sp 170 (Oso easy double re) and Rosa x Hansa replicate commercial nursery practices. Plants in the runoff bed were watered using overhead sprinkler irrigation delivering 19 mm of irrigation daily. Pesticides were applied at recommended label rates to the runoff bed using a 1.2 m overhead spray boom with 4 fla t - fan nozzles during the growing season in each year (Table 1), except for glyphosate which was sprayed using backpack sprayer to avoiding direct contact to plants in the container. A gas - powered pump delivered 25 L of pesticides and 5 L of herbicide for e ach application event (Table 1). Pesticide application was scheduled on a day with no rain forecast. Herbicide (isoxaben, prodiamine or glyphosate) was applied early in the morning (8 am) and then the bed was irrigated. After irrigation, we waited for the runoff to cease and then the insecticides and/or fungicides were applied in the bed as a tank mixture . The day after pesticide application and thereafter, regular irrigation was resumed. Irrigation water leaving the runoff bed was collected in P1. A fracti on of water from P1 was pumped to the bioreactors, where it first passed through the heat - expanded shale aggregates in the PVC pipes which then flowed into the woodchips in the bioreactor. Water from the bioreactors was then collected in P2. Raw groundwate r was the water obtained from the farm well, RR water was the water collected from runoff bed in P1, without any RRR water was the water from P1 that went through the bioreactors and was collected on P2. In this study, recycled water refers to water both f rom P1 (RR water) and P2 (RRR water). 171 2.1.3 . Plant evaluation zone The plant evaluation bed was divided into 12 irrigation zones, each 2.4 m x 4.8 m. The plant evaluation bed had four rows serving as blocks and three zones within each block. Each irrigati on zone received raw groundwater (RGW) or RR water or RRR water. Each zone in the plant evaluation bed had six different plant taxa and eight replication per taxon. Starter plants from 10 cm plugs (liners) of Hydrangea macrophylla jangles), Hydrangea paniculata Thuja occidentalis L. Juniperus horizontalis Hydrangea arborescens nvincibelle Spirit II®) and Rosa sp . L. transplanted in an 11.3 L black plastic container filled with pine bark and peat moss media (80:20; Volume: Volume) and moved to the plant evaluation bed and spaced 0.53 m apart (pot to pot). In 2017, plants that were transplanted as liners on August 27, 2016, were used. Those plants were brought to the overwintering hoop after approximately two month of growth outside, on October 2 0, 2016, and plants underwent dormancy. Those plants were moved to the plant evaluation bed on 12 May 2017. Plants used in the 2018 and 2019 studies were transplanted from liners on 24 May 2018 and 25 May 2019, respectively. Every year after moving plants to the plant evaluation bed, plants were fertilized with 50 g of slow - release fertilizer (Osmocote blend; 18:5:8 N:P 2 O 5 :K 2 O with micronutrients, 8 - 9 months, Product code # A90177, ICL Specialty fertilizers, 172 Summerville, SC, USA) per plant. Fertilizer was a pplied on 17 May 2017, 8 June 2018, and 22 June 2019. Pest infestation did not occur in the plant evaluation bed hence we did not apply pes ticide on plant evaluation bed. 2.2 Plant evaluation Plant evaluation bed received irrigation treatments from 1 , August 2017, to 10 September 2017; from 05 July 2018, to 25 August 2018; and from 16 July 2019, to 25 September 2019. Precipitation and reference evapotranspiration for the duration of the study is provided in Fig. 2. Irrigation treatments were applied a s overhead irrigation each morning using an automated irrigation timer (5 am for RR water, 5:30 am for RRR water and from 7:30 am for RGW), as those times were most likely to have the lowest wind speeds each day. All plants on the plant evaluation bed were evaluated for pesticide - related visual injuries (PVI) three to seven days following each pesticide application to plants on the runoff bed. Potential com pletely dead plant and 10 being a healthy plant). After the completion of the treatment period for each year, plants in all three treatment groups were compared based on growth index (GI), PVI, chlorophyll SPAD index, dark - adapted fluorescence ( Fv/Fm ), lig ht - adapted fluorescence ( ), photosynthesis ( A ) and dry biomass. All measurements were conducted immediately after the end of irrigation treatment on plant evaluation bed. Growth Index, PVI and Fv/Fm were 173 measured on all eight replications per taxa i n all 12 zones but shoot and root biomass, A , and chlorophyll SPAD were measured only on a subsample of four plants per taxa in each zone. GI was calculated as the average of plant height and two perpendicular widths for all six species. Chlorophy ll SPAD index was measured using a portable SPAD meter (SPAD - 502; Minolta corporation, Ltd., Osaka, Japan) as an average of three leaves per plant. It was only measured for four out of six species excluding, T. occidentalis , and J. horizontalis , because of their overlapping scale - like leaf structure. Fv/Fm was measured using a portable fluorometer (OS30p+; Opti - Sciences, Inc., Hudson, NH, US) on fully matured leaves at the 3 rd or 4 th node from the top after acclimatizing those leaves with a dark - adaption ki t (Opti - Sciences, Inc., Hudson, NH, US) for 30 minutes, J. horizontalis was excluded from the measurement in 2017, but in 2018 and 2019, all six species were measured. In 2018 and 2019, and A were measured on a fully mature leaf on either the 3 rd o r 4 th node from the apex. We measured and A on four out of six species similar to the chlorophyll SPAD index, excluding T. occidentalis and J. horizontalis . A portable photosynthesis system (LI - 6400 XT, Li - Cor, Inc., Lincoln, NE) mounted with a lea f chamber fluorometer (LI - 6400 - 40, Li - Cor, Inc., Lincoln, NE) was used for the measurements. Photosynthetically active radiation (PAR) in the chamber was set to 1500 µmol m - 2 s - 1, block temperature was set to 25°C and 400 ppm of CO2 was supplied. The relat ive humidity in the chamber varied between 40 60%. Each leaf was acclimatized for five minutes before measuring A followed by measuring on the same leaf. In 2018 and 2019, after all the non - destructive 174 measurements, plants were harvested and dried in an oven at 50°C and weighed to determine dry biomass. Both shoot and root biomass were measured in 2018, but in 2019 we only measured shoot biomass . 2.3 Pesticide sampling Pesticide samples were collected in 2018 and 2019. Pesticide samples for both RR water and RRR water were collected one day prior and one day after pesticide applications to the runoff bed. Raw groundwater was sampled for pesticides a total of six times, three times in 2018 and three times in 2019. Main water lines running from P1 and P2 to plant evaluation bed were tapped near plant evaluation bed to collect respective water samples. RGW samples were also collected from main water lines suppling RGW to plant evaluation bed (Fig. 1). All water samples were collected in 50 ml amber vials and were immediately frozen. The frozen samples were then sent to a ISO 17025 accredited commercial laboratory (Brookside Laboratories, Inc., New Bremen, OH, USA) to determine pesticide concentrations in RR water, RRR water and RGW. For all compounds othe r than glyphosate, the analysis was performed on an LC - MS/MS using direct aqueous injection. Glyphosate was analyzed through direct aqueous injection but the analysis method followed EPA 547 using HPLC and hypochorite + OPA post - column derivatization with fluorescence detection. 175 2.4 Statistical analysis The layout for the water treatments in plant evaluation bed followed a randomized complete block design. SAS (ver. 9.4) was used to conduct statistical analysis and post - hoc mean comparisons were made using 3. Results 3.1. Pesticide concentration in water Raw groundwater samples collected in 2018 and 2019 were below detection limits for all pesticides included in our sample protocol (data not shown). Ho wever, pesticide residues were observed for RR water and RRR water. Concentrations of pesticides found in RR water and RRR water for 2018 and 2019 are listed in Table. 1. Acephate was the only compound that was sprayed on each pesticide application. In 201 8, throughout growing season, acephate concentration varied from 150 µg/L to 1.5 µg/L in RR water that was sampled a day after application (DAA). For the RRR water 1 DAA, acephate concentration varied from 57.3 µg/L to 5.6 µg/L. Approximately 10 days after all four acephate application, the concentration of acephate in the RR water was reduced by 95% to 100% compared to acephate concentration in RR water on 1 DAA. However, for RRR water, the reduction in acephate concentration 10 DAA was only between 83% an d 55.76% compared to acephate concentration in RRR water 1 DAA. In 2019, the concentration of acephate in RR water 1 DAA, 176 was between 137.2 µg/L and 78.7 µg/L and for RRR water on the same day, pesticide concentration was between 24.9 µg/L and 0 µg/L. Unli ke 2018, no acephate residue was found in RR water, collected 15 DAA. On two out of four sample dates, acephate residues were higher in RRR water than in RR water collected 1 DAA. Chlorpyrifos was only sprayed once in 2018. The concentration of chlorpyrifo s in the RR water 1 DAA was 41.1 µg/L whereas chlorpyrifos residues in RRR water were below detection limits on the same day. Nine DAA chlorpyrifos concentration in RR and RRR water samples were below the detection limit. Chlorpyrifos was not applied in 20 19 due to changes in university regulations. In 2018, the concentration of isoxaben in RR water and RRR water 1 DAA was 58.5 µg/L and 179 µg/L, respectively. At 11 DAA, isoxaben concentration in RR water decreased by 80.68%, compared to 1 DAA and the conce ntration in RRR water decreased by 89%. Isoxaben concentration was consistently reduced on subsequent sampling dates for both RR water and RRR water. In 2019, isoxaben concentration in RR water and RRR water was 166.2 µg/L and 8.3 µg/L, respectively, when sampled 1 DAA. Fourteen DAA, isoxaben concentration in RR water was reduced by 96.7% compared to isoxaben concentration in RR water 1 DAA and isoxaben concentration in RRR water increased by 202.4% compared to isoxaben concentration in RRR water 1 DAA. 177 Gly phosate was sprayed twice in 2018. For the first application, glyphosate was not present in both RR water and RRR water 1 DAA. At 12 DAA, however, 100 µg/L of glyphosate was found in the RR water but residue in the RRR water was below detection. On the sec ond application, 1051 µg/L of glyphosate was detected in RR water and 18.3 µg/L of glyphosate detected in RRR water, 1 DAA. In 2019, glyphosate was only applied once. A day after application, glyphosate concentration in RR water was 1917.5 µg/L while the c oncentration in RRR water was 520.2 µg/L. Fifteen DAA and thereafter, glyphosate residue was not detected in both RR water and RRR water except 25.2 µg/L of glyphosate in RRR water 45 DAA. Thiophanate - methyl was sprayed twice in 2018. Thiophanate - methyl w as below detection limits for both RR and RRR water 1 DAA and 15 DAA following the first application. However, after the second application of thiophanate - methyl, 5.5 µg/L of thiophanate - methyl in RR water and 1 µg/L of thiophanate - methyl in RRR water were detected 1 DAA. In 2019, 11.9 µg/L and 1.5 µg L - 1 of thiophanate - methyl was found in RR water and RRR water 1 DAA. Thiophanate - methyl was below detection in all water sampled 15 DAA. In 2018, triflumizole was detected in both RR water (13 µg/L) and RRR w ater (0.4 µg/L) on 1 DAA. At 15 DAA, triflumizole concentration in RR and RRR water were 1.5 µg/L and 0.03 µg/L, respectively. By 20 DAA, triflumizole was still present in RR but not detected in RRR water. In 2019, 16.9 µg/L of triflumizole was detected in RR water sampled 1 DAA and 1.1 µg/L at 15 178 DAA (93.5% reduction). For RRR water, 1.1 µg/L of triflumizole was present at 1 DAA and not detectable at 15 DAA. Neither bifenthrin, which was sprayed once in 2018 and twice in 2019, nor prodiamine, which was sp rayed once in 2019, were detected in any water samples. In 2018, 2.5 µg/L and 0.06 µg/L of mefenoxam was detected in RR water 1 DAA and 10 DAA, respectively. In RRR water, the concentration of mefenoxam was 3.9 µg/L, 1 DAA and 1.8 µg/L (53.8% reduction) a t 10 DAA. Trace amount (0.3 µg/L) of mefenoxam was detected in both RR and RRR water at 20 DAA. In 2019, mefenoxam was sprayed twice. Mefenoxam residue was not detected in any water samples after the first application, however, after the second application mefenoxam was detected at 9.5 µg/L only in RR water 1 DAA. In a few instances pesticides were detected prior to application of the compound in a given season. This was observed for triflumizole in 2018 and isoxaben in 2019. 3.2 Plant response to water tre atments 3.2.1 . Growth index and Pesticide - related visual injury Irrigation source did not have a consistent effect on GI in the three years of the study. In 2017 water treatments did not affect GI for any of the six taxa. In 2018, water treatments did not affect GI for any of the six taxa except T. occidentalis , for which plants receiving RGW and RR 179 water had similar GI but the GI of plants receiving RRR water was higher compared to plant receiving RGW and RR water (Fig 3). Similar to T. occidentalis in 201 8, in 2019, GI of H. paniculata receiving RGW and RR did not differ but was lower compared to plants receiving RRR water. GI of J. horizontalis and Rosa sp. was similar for plants receiving RGW and RR water but plants receiving RRR water had lower GI compa red to plants receiving RGW and RR water. GI of T. occidentalis was higher for RGW compared to both RRR water and RR water (Fig 3). Although the GI of some species differed among water treatments, pesticide related visible injury did not occur on plants of any taxa during the three years of the study (data not shown). 3.2.2 . Dry biomass In 2018, water treatments did not affect the total shoot weight of plants of five of the six taxa (Fig. 4). However, for T. occidentalis, shoot weight was higher for plants irrigated with RRR water compared to RGW (Fig 4). On further dividing shoot weight to leaf weight and stem weight, water treatments did not affect leaf weight for four taxa, however, Rosa sp. irrigated with RGW had highe r leaf weight compared to RR water but not RRR water and T. occidentalis had higher leaf weight for RRR water compared to RGW but not RR water. Stem weight of five taxa was similar for all three water treatments; however, for T. occidentalis RRR water had higher stem weight compared to RR water. The root weight of all six taxa was similar for all three water treatments (Fig 5). In 2019, shoot weight of H. arborescens and H. paniculata was higher for plants irrigated with RGW and RRR water compared to RR wat er. Conversely, for H. macrophylla , 180 plants receiving RR water had higher shoot biomass compared to RGW and RRR water. For J. horizontalis, water treatments did not affect shoot biomass and for R. sp. and T. occidentalis RGW and RR water had similar shoot m ass (Fig 4) . 3.2.3 . Net photosynthesis and fluorescence Irrigation source did not affect A or (Fig 6). However, Fv/Fm was higher for plants irrigated with RR water compared to RGW and RRR water for H. arborescens in 2017 and for all six taxa in 2018. In 2019, irrigation source did not affect Fv/Fm for any of the six taxa (Fig 7). 3.2.4 . Chlorophyll SPAD index In 2017 and 2018, water treatments did not affect chlorophyll SPAD index of plants in any taxa except H. macrophylla in 2018, which was higher for plants receiving RRR water compared to RGW and RR water. In 2019, chlorophyll SPAD index of H. arborescens and H. macrophylla plants receiving RRR water was higher compared to RGW and RR water. In the same year fo r H. paniculata , chlorophyll SPAD index was higher for plants receiving RR water compared to RGW and RRR water but for Rosa sp. water treatments did not affect chlorophyll SPAD index (Fig 8). 181 4. Discussion 4 .1 Residual pesticide in recycled water varies by compound A cephate, glyphosate and mefenoxam all have a high water solubility of 850 g/L, 157 g/L and 8400 mg/L, respectively. After application, these pesticides readily mix with irrigation water and RR. Therefore concentrations of these pesticides were r elatively higher in recycled water 1 DAA, compared to the other six pesticides. Concentration of acephate and glyphosate in RR water were substantially reduced by 10 or 15 DAA because acephate has a short half - life <3 days and glyphosate half - life in water is somewhere from 7 to 14 days (Giesy et al., 2000; Mamy and Barriuso, 2005; Christiansen et al., 2011; Mesnage et al., 2015). However, mefenoxam is persistent with an average half - life of 58 days (Long Island Pesticide Pollution Prevention Strategy, 2015 ) and may persist longer than 20 days. Isoxaben, chlorpyrifos, triflumizole and thiophanate - methyl are moderately soluble in water with a solubility of 1 mg/L, 1.4 mg/L, 10.2 mg/L and 26 mg/L, respectively which is substantially lower compared to acephate , glyphosate and mefenoxam. Therefore maximum detected concentrations of isoxaben, chlorpyrifos, triflumizole and thiophanate - methyl in recycled water were 5 to 25 times lower compared to maximum detected concentration of acephate, glyphosate and mefenoxam , Isoxaben persisted longer than 10 DAA because it has a half - life of approx. six months (Rouchaud et al., 1999; Quali - Pro, 2011) . Traces of isoxaben detected in recycled water even before the isoxaben application in 2019, suggest that there was some carry 182 over effect of isoxaben from last year and occasional desorption of isoxaben from sediments in the reservoir (Walker, 1987) . Michigan has relatively colder temperature and less sunshine which can reduce the rate of photo and microbial degradation which an d may have accounted for isoxaben persistence and carry - over (Wilson et al., 1995; Camper et al., 2001) . Chlorpyrifos and thiophanate - methyl both have a short half - life of <15 days, in addition, chlorpyrifos is degraded by light and microbes (Soeda et al., 1972; Racke, 1993; Mandal et al., 2010; Mugni et al., 2016) hence both of those pesticides were only found in recycled water at 1 DAA. Triflumizole has a half - life of 18 days and is also readily degraded by microbes (Lewis, 2009) . Microorganism present in bioreactors can degrade pesticides (Abdi et al., 2020) , microorganisms in our bioreactors possibly degraded triflumizole as a result, triflumizole concentration in RRR water, even 1 DAA, was very low. In 2018, traces of triflumizole were detected before t riflumizole application both in RR water and RRR water reason for which could not be explained. Bifenthrin and prodiamine both have a very low (0.1 mg/L and 0.01 mg/L respectively) water solubility and tightly bind to soil organic matter (Koc : 131000 to 3.02000 and 80 to 471000 respectively), also prodiamine rapidly photodegrade (Weber, 1990; Fecko, 1999; Acuña, 2009) . Hence both pesticides were not found in RR and RRR water samples. 4.2 Woodchip bioreactor can reduce pesticide concentration For most of the pesticides, bioreactors reduced the concentration of pesticides in water. In the bioreactors, RR water first passed through heat - expanded shale aggregates. These aggregates 183 have a predominantly negative charge, and therefore can adsorb pest icides of the opposite charge and have been successfully used to remove pesticides (Fushiwaki and Urano, 2001; Woignier et al., 2015; Marican and Durán - Lara, 2018) . However, in a study by Abdi et al., 2019, heat - expanded shale did not reduce the concentrat ion of chlorpyrifos, oxyfluorfen or bifenthrin (Abdi et al., 2020) . After passing through heat - expanded shale aggregates, water then flowed into bioreactor tanks half - filled with woodchips. Woodchips facilitate microbial degradation by serving as hosts for microorganisms and can adsorb pesticide with higher organic adsorption coefficient, hence can be used as an inexpensive onsite remediation technique for the removal of pesticides (Brás et al., 1999; Rodriguez - Cruz et al., 2007; Ilhan et al., 2012) . Woodch ip bioreactors have successfully reduced concentrations of pesticides such as oxyfluorfen, chlorpyrifos, bifenthrin, acetochlor, atrazine and sulfamethazine (Ilhan et al., 2012; Ranaivoson et al., 2019; Abdi et al., 2020) . In our study, the concentration o f most of the pesticides was reduced by the bioreactors probably because of microbial degradation in woodchip media, pesticide adsorption by woodchips and by greater exposure of RR water for photodegradation and volatilization. 4.3 Recycled water can be used to irrigate ornamental shrubs Our results indicate that residual pesticides in recycled water had little to no impact on either growth index or plant dry weight of container nursery plants. In 2017, pesticides were applied in three different events an d plants were exposed when most of the vegetative growth for the season had already occurred. Also, the frequency of rainy days between first spray and the last 184 spray was in the order of 2017>2018>2019. The higher frequency of rainfall may have washed off the residual pesticide from plants and diluted pesticides in treatment water hence may be the reason behind no differences in growth for 2017 and 2018. In 2019, growth was higher for J. horizontalis and Rosa sp . plants irrigated with RGW compared to RRR wa ter but not RR water. The reason for reduced growth under irrigation with the RRR water is unclear. It is unlikely that residual pesticides reduced growth of plants irrigated with RRR water, as plants irrigated with RR, which generally had the highest pest icide concentrations, grew as good as plants irrigated with RGW. For all three years, the pesticide concentrations found in RR water and RRR water did not cause pesticide - related visual injury. Pesticide concentration in RR water and RRR water was also no t high enough to reduce shoot dry mass in 2018. In 2019, all three water treatments had similar shoot dry mass except shoot weight of Rosa sp. was reduced by RRR water and shoot weight of H. arborescens and H. paniculata was reduced by RR water. However, p lants receiving RR water also had higher shoot dry mass for H. macrophylla . These slight differences in 2018 and 2019 probably are because of variables other than irritation treatments. Similar to shoot weight, in most cases, RGW was not in any way superio r for leaf weight, stem weight and root weight in 2018 compared to RRR water and RR water. Pesticide related visual injury, GI and total dry biomass are dependent upon pesticide concentration in water. In a study by Huang et al., 2015, glyphosate applied a t the rate of 0.0866 kg a.i./ha d id not reduce plant height and dry weight in soybean but increasing the dose further reduced both, dry weight and plant height, and those reductions were 185 directly proportional to dose applied (Huang et al., 2015) . In anoth er study by Poudyal et al., chlorpyrifos at a residual dose of 0.4 mg/L and isoxaben at 1.4 mg/L did not produce leaf visual injury in H. paniculata , Cornus obliqua (Powell garden) and Hosta (Gold standard) (Poudyal et al., 2019) . Similarly, prodiamine at a residual concentration of 6 mg/L also did not produce any visible symptoms on Prunus persica (peach seedling) (Lourens et al., 1989) . However, herbicides at higher doses have seen to produce visual injury in a wide range of ornamental species (Mathers et al., 2012) . Oryzalin at 100 µg/L did not affect fresh root and shoot weight of Pennisetum rupelli (fountain grass), but increasing dose to 1000 µg/L reduced both root and shoot weight (Bhandary et al., 1997b) and insecticides such as malathion, permethrin and tetramethrin had dose - dependent effect on biomass production of Sitka spruce (Straw and Fielding, 1998) . In our study, the pesticide concentration in RR water and RRR water was probably very diluted by irrigation RR hence did not affect plant growth. When plants are grown in an open field, rain events may wash off pesticide residues from plant parts lower ing the risk of phytotoxicity. pesticides, particularly herbicides, can potentially interfere with those physiological processes (Huang et al., 2012; Silva et al., 2014; Wang et al., 2018) . Therefore physiological tools can be used to rapidly assess the physiological impact of pesticides (Petit et al., 2012 ; Wang et al., 2018; Sharma et al., 2019; Giménez Moolhuyzen et al., 2020) . If the effect of pesticide residue in RR water and remediate water were long - lasting and affected plant physiology, these physiological 186 parameters would likely reflect it. In the current study, h owever, there was little evidence that pesticides in the RR water or RRR water impacted physiological function following irrigation with recycled water. Various authors have reported a reduction in photosynthesis and fluorescence parameters by exposure to chlorpyrifos (Xia et al., 2006) , isoxaben (Fernandez et al., 1999; Poudyal et al., 2019) , acephate (Haile et al., 2009) and glyphosate (Huang et al., 2012) but the concentration they used in all the cases was higher than the pesticide concentration found i n RR water and RRR water in our study. Our protocol for physiological measurements was designed to assess potential injury associated with long - term chronic pesticide exposure. However, physiological measurements are plastic and can recover from short - term damage. It is possible that we did not measured reductions in photosynthesis and fluorescence measured immediately after pesticide applications. Nonetheless, the lack of growth impacts associated with RR irrigation suggests any perturbations in photosynth etic function, if they occurred, were minor and transient. Moreover, there were no reductions in chlorophyll SPAD index for plants receiving RR water or RRR water compared to RGW. Pesticides, mainly herbicides, may produce negative effect on plant growth and physiology. However, a sub - lethal or lower dose of some pesticides may have a positive impact on plants and has been documented in few studies; sub - lethal/lower dose of eleven different herbicide increased root and shoot growth in Avena sativa (oat) (W iedman and Appleby, 1972) , glyphosate application at lower that recommend doses stimulated plant growth in a range of plants 187 species from cereal crops to ornamentals (Velini et al., 2008) , lower concentration of chlorpyrifos improved growth and photosynthe tic parameters in Vigna radiata (mung bean) while higher concentration reduced both, growth and photosynthesis (Parween et al., 2011) and fungicides such as phthalimide and azoles improved growth and photosynthesis in various crops (N. and Türkyilmaz, 2003 ; Petit et a l., 2012 ) . In our study pesticides present in RR water and RRR water were thousands of fold lower compared to general application rates and may have promoted plant growth and physiology, at few instances, instead of hindering it. However, studi es on the effect of lower concentrations of pesticides on plant growth and physiology have not extensively published and further confirmation needs to be done before asserting the positive impacts of pesticides at lower concentrations. 5. Conclusion From the results of our study, we can group pesticides into three different groups based on the likelihood to be detected in recycled water. Pesticides with high detection possibility include acephate, glyphosate and mefenoxam, pesticides with moderate detectio n possibly include isoxaben, chlorpyrifos, triflumizole and thiophanate - methyl and pesticides with low detection possibility include bifenthrin and prodiamine. I n our study pesticide concentrat ion in nursery retention reservoir was thousands of time lower compared to typical application rates and was dependent on pesticide solubility, pesticide adsorption and pesticide persistence. Finding from our 188 study reveals the possibility of using recycled water for irrigation of various woody ornamental species witho ut impacting the growth and physiology of those plants. 189 APPENDIX 190 APPENDIX Fig ure V - 1 . Layout of the field study. The plant evaluation bed had four rows and three irrigation zones in each row. Each row had all three water treatment zones that were randomly assigned. Irrigation treatments were water either from raw groundwater (RGW), recycled runoff (RR) from the coll ection reservoir or remediation recycled runoff (RRR) from the collection reservoir. Figure is not to the scale. 191 Fig ure V - 2 . Weekly r eference potential evapotranspiration (Weekly r ef. PET) and weekly precipitation during the treatment application period at the research site from 2017 to 2019. Source: Michigan S tate University Enviro Weather: https://mawn.geo.msu.edu/station.asp?id=msu 19 2 Table V - 1 . Pesticides application rates (express as g a.i./ha), concentration of pesticide solution (expressed as g a.i./L), total amoun t of solution sprayed (expressed as liter) and pesticide concentration in recycled runoff (RR) water and remediated recycled runoff (RRR) water (expressed as µg/L) water during the three year study period. Each water source was sampled twice after each application. Fir st samples were collected a day after pesticide application and the last sample were collected 10 to 15 days after pesticide application. Samples for pesticide concentration were not collected in 2017. 193 Fig ure V - 3 . Growth index of six ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water. Recycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that passed through a heat - expanded shale and woodchip b ioreactor system. Means within a taxon that are followed by same letters are not significantly different at icant Difference (LSD) post - hoc test. 194 Fig ure V - 4 . Shoot weight of six ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water in 2018 and 2019. Recycled ru noff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that passed through a heat - expand ed shale and woodchip bioreactor system. Means within a taxon that are followed by same letters are not Mean separations for each taxon were carried out usin (LSD) post - hoc test. 195 Fig ure V - 5 . Leaf weight, stem weight and root weight of six ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water in 2018. Recycled runoff water was captured from a nursery bed managed according t o standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that passed through a heat - expanded shale and woodchip bioreactor system. Means within a taxon that are followed by same letters are not (LSD) post - hoc test. 196 Figure V - 6 . Net photosynthesis and Light - adapted fluorescence of four ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water in year 2018 and 2019. Recycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that passed through a heat - exp anded shale and woodchip bioreactor system. Means within a taxon that are followed by same letters are not significantly different at p=0.05. Standard errors of the sing - hoc test. 197 Fig ure V - 7 . Dark - adapted fluorescence of different ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water. Recycled runoff water was captured from a nursery bed managed according to standard nurse ry practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that passed through a heat - expanded shale and woodchip bioreactor system. Means within a taxon that are followe d by same letters are not significantly different at p=0.05. 198 Fig ure V - 8 . Chlorophyll SPAD index of four different ornamental taxa irrigated with raw groundwater (RGW), recycled runoff (RR) water and remediated recycled runoff (RRR) water. Recycled runoff water was captured from a nursery bed managed according to standard nursery practices, including fertilization and pesticide applications, for the region. Remediated recycled runoff water was the recycled runoff water that pas sed through a heat - expanded shale and woodchip bioreactor system. Means within a taxon that are followed by same letters are not Mean separations for each ta (LSD) post - hoc test. 199 LITERATURE CITED 200 LITERATURE CITED Abatenh, E., B. Gizaw, Z. Tsegaye, and M. Wassie. 2017. The role of microorganisms in bioremediatio n - A r evie w. Open J. Environ. Biol. 2: 038 046. Abdi, D.E., J.S. Owen, J.C. Brindley, A.C. Birnbaum, P.C. Wilson, F.O. Hinz, G. Reguera, J. - Y. Lee, B.M. Cregg, D.R. Kort, and R.T. Fernandez. 2020. Nutrient and pesticide remediation using a two - stage bioreactor - adsorp tive system under two hydraulic re tention times. Water Res. 170:1 15311 . Acuña, A.A. 2009. Dissipation and efficacy of pendimenthalin, prodiamine, dithiopyr and bensulide as affected by dose and application timing for crabgrass ( Digitaria sp ) control in a t urfgrass environment. The Ohio State Univ. Amrhein, N., J. Schab, and H.C. Steinrücken. 1980. The mode of action of the herbicide glyphos ate. Naturwissenschaften 67: 356 357. Beeson, R.C., M.A. Arnold, T.E. Bilderback, B. Bolusky, S. Chandler, H.M. Gramlin g, J.D. Lea - Cox, J.R. Harris, P.J. Klinger, H.M. Mathers, J.M. Ruter, and T.H. Yeager. 2004. Strategic Vision of Container Nursery Irrigation in the Next Ten Years 1. J. Environ. Hort 22: 113 115 . Bhandary, R.M., T. Whitwell, and J. Briggs. 1997a. Grow th of containerized landscape plants is influenced by herbicides residues in irrig ation water. Weed Technol. 11 : 793 797. Bhandary, R., T. Whitwell, J.A. Briggs, and R.T. Fernandez. 1997b. Influe nce of surflan (oryzalin ) concentrations in irrigation water on growth and physiological processes of Gardenia jasminoides radicans and Pennisetum rupelli . J. Environ. Hortic. 15: 169 172. Boretti, A., and L. Rosa. 2019. Reassessing the projections of the world water development r ep ort. npj Clean Water 2:15 . Brás, I.P., L. Santos, and A. Alves. 1999. Organochlorine pesticides removal by pinus bark sorption . Environ. Sci. Technol. 33: 631 634 . Briggs, J.A., M.B. Riley, and T. Whitwell. 1998. Quantification and remediation of pesticid es in runoff water from containerized plant produ ction. J. Environ. Qual. 27: 814 820 . Brosnan, J.T., E.H. Reasor, J.J. Vargas, G.K. Breeden, D.A. Kopsell, M.A. Cutulle, and T.C. Mueller. 2014. A putative prodiamine - resistant annual bluegrass ( Poa annua ) p o pulation is Controlled by Indaziflam. Weed Sci. 62: 138 144. Camper, N.D., J. - H. Kim, and M.B. Riley. 2001. Degradation of isoxaben in soils and an aqueous system. J. Environ. Sci. Heal. Part B 36:7 29 739 . Christiansen, A., J. Gervais, K. Buhl, and D. Stone. 2011. Acephate Technical Fact Sheet. Natl. Pestic. Inf. Center, Oregon State Univ. Ext. Serv. Available at 201 http://npic.orst.edu/factsheets/archive/acephatech.html (verified 12 February 2020). Dias, M.C. 2012. Phytotoxicity: An Overview of the Physio logical Responses of Plants Exposed to Fu ngicides. J. Bot. 2012: 1 4 . Dieter, C.A., M.A. Maupin, R.R. Caldwell, M.A. Harris, T.I. Ivahnenko, J.K. Lovelace, N.L. Barber, and K.S. Linsey. 2015. Estimated Use of Water in the United States in 2015 (Circular 144 1). U.S. Dep. Inter. U.S. Geol. Surv. cir1441. Fain, G.B., C.H. Gilliam, K.M. Tilt, J.W. Olive, and B. Wallace. 2000. Survey of Best Management Practices in Container Production Nurseries. J. Environ. Hort. 18: 142 144 . Fecko, A. 1999. Environmental fate of bifenthrin. Environ. Hazards Asse ss. Program, Sacramento, CA 12: 1 9 . Fernandez, R.T., T. Whitwell, M.B. Riley, and C.R. Bernard. 1999. Eva luating semiaquatic herbaceous perennials for use in herbicide phytoremediation . J. Am. Soc. Hortic. Sci. 124: 539 544 . Fulcher, A., A. V. LeBude, J.S. Owen, S.A. White, and R.C. Beeson. 2016. The next ten years: Strategic vision of water resources for nursery p roducers. Horttechnology 26: 121 132. Furtini Neto, A.E., K.V.F. Boldrin, and N.S. Mattson. 2015. N utrition and q uality in orname ntal plants. Ornam. Hortic. 21: 139 150 . Fushiwaki, Y., and K. Urano. 2001. Adsorption of pesticides and their biodegraded products on clay minerals and soils. J. Heal. Sci. 47: 429 432. Giesy, J.P., S. Dobson, and K.R. Solomon. 2000. ecotoxicological risk assessment for roundup® herbici de. p. 35 120. In Reviews of Environmental Contamination and Toxicology. Springer, New York, NY. Giménez Moolhuyzen, Blom, Lorenzo Mínguez, Cabello, and Crisol Martínez. 2020. Photosynt hesis inhibiting effects of pesticides on sw eet pepper leaves. Insects 11: 69 . ewaters. J. Plant Nutr. 33: 1579 1592. Haile, F.J., D.L. Kern s, J.M. Richardson, and L.G. Higley. 2009. Impact of Insecticides and Surfactant on Lettuce Physiology and Y ield. J. Econ. Entomol. 93: 788 794 . Hedegaard, M.J., and H.J. Albrechtsen. 2014. Microbial pesticide removal in rapid sand filters for drinking wate r treatment - Potential and kinetics. Water Res. 48: 71 81. Heim, D.R., J.R. Skomp, E.E. Tschabold, and I.M. Larrinua. 1990. Isoxaben inhibits the synthesis of acid insoluble cell wall materials in Arabidopsi s thaliana. Plant Physiol. 93: 695 700 . 202 Hong, C.X. , P.A. Richardson, P. Kong, and E.A. Bush. 2003. Efficacy of chlorine on multiple species of Phytophthora in recycled nursery irrig ation water. Plant Dis. 87: 1183 1189 . Huang, Y., K.N. Reddy, S.J. Thomson, and H. Yao. 2015. Assessment of soybean injury fro m glyphosate using airborne multispectral remote s ensing. Pest Manag. Sci. 71: 545 552. Huang, J., E.N. Silva, Z. Shen, B. Jiang, and H. Lu. 2012. Effects of glyphosate on photosynthesis, chlorophyll fluorescence and physicochemical propertiesof cogongrass ( Imperata cylindrical L.). Plant Omics 5: 177 183. Ilhan, Z.E., S.K. Ong, and T.B. Moorman. 2012. Herbicide and antibiotic removal by woodchip denitrification filters: Sorption processes. W ater. Air. Soil Pollut. 223: 2651 2662. Kabashima, J.N., S.J. Lee, D.L. Haver, K.S. Goh, L.S. Wu, and J. Gan. 2004. Pesticide Runoff and Mitigation at a Commercial Nursery Site. p. 34. In Pesticide Decontamination and Detoxification, ACS Symposium Series; American Chemical Society: Washington, DC, 2003. UTC. Khachatryan, H., and H.J. Choi. 2017. Factor s affecting consumer preferences and demand for ornamental plants . Univ. Florida, Inst. Food Agric. Sci . Ext. FE938: 1 5 . Lewis, S.T. 2009. Triflumizole (Terraguard, Procure) . EPA Pestic. Fact Sheet 10:228 Available at http://pmep.cce.cornell.edu/profiles/fung - nemat/tcmtb - ziram/triflumizole/fung - prof - triflumizole.html (verified 6 February 2020). Long Island Pesticide Pollution Prevention Strategy. 2015. Active ingredient data package metalaxyl & mefe noxam active ingredient. Dep. Environ. Conserv. New York, Bur. Pest Manag. 5(4) Available at https://www.dec.ny.gov/docs/materials_minerals_pdf/mefenoxamdata.pdf (verified 6 February 2020). Lourens, A.F., A.H. Lange, and F.J. Calitz. 1989. Phytotoxicity of pre - emergence herbicides to peach seedlings ( Prunus persica ). So uth African J. Plant Soil 6: 97 102. Lu, J., L. Wu, J. Newman, B. Faber, D.J. Merhaut, and J. Gan. 2006. Sor ption and degradation of pesticides in nursery recycling ponds. J. Environ. Qual. 35: 1795 . Mack, R., J.S. Owen, A.X. Niemiera, and J. Latimer. 2017. Virgin ia nursery and greenhouse grower survey of best management p ractices. Horttechnology 27: 386 392 . Majsztrik, J.C., R.T. Fernandez, P.R. Fisher, D.R. Hitchcock, J. Lea - Cox, J.S. Owen, L .R. Oki, and S.A. White. 2017. Wate r use and treatment in container - grown specialty crop production : A review . Water. Air. Soil Pollut. 228:151 . Mamy, L., and E. Barriuso. 2005. Glyphosate adsorption in soils compared to herbicides replaced with the introd uction of glyphosate resi stant crops. Chemosphere 61: 844 855. Mandal, S., S. Das, and A. Bhattacharyya. 2010. Dissipation study of thiophanate methyl residue 203 in/on grapes ( Vitis vinifera L.) in India. Bull. E nviron. Contam. Toxicol. 84: 592 595. Mangiafico, S.S., J. Gan, L. Wu, J. Lu, J.P. Newman, B. Faber, D.J. Merhaut, and R. Evans. 2008. Detention and recycling basins for managing nutrient and pesticide runoff fro m nurseries. HortScience 43: 393 398. Marican, A., and E.F. Durán - Lara. 2018. A review on pest icide removal through different processes. En viron. Sci. Pollut. Res. 25: 2051 2064. Mathers, H., L. Case, M. Bigger, P. Gordon, and L. Giese. 2012. Yearly Research Summary Report 2012 Ornamental Research Available at https://www.mathersenvironmental.com/wp - content/uploads/2016/06/Research - Summary - 2012.pdf (verified 27 March 2020 ). Meador, D.P., P.R. Fisher, P.F. Harm on, N.A. Peres, M. Teplitski, and C.L. Guy. 2012. Survey of Physical, Chemical, and Microbial Water Quality in Greenhouse and Nursery Irrigati on Water. Horttechnology 22: 778 786 . Mesnage, R., N. Defarge, J. Spiroux de Vendômois, and G.E. Séralini. 2015. Po tential toxic effects of glyphosate and its commercial formulations below regulatory limits. Food Chem. Toxicol. 84:133 15. Morillo, E., J. Villaverde, and D. Barcelo. 2017. Advanced technologies for the remediation of pesticide - contaminated soils. Sci. To t. Environ. 586:576 - 597. Mugni, H., A. Paracampo, P. Demetrio, M. Pardi, G. Bulus, A. Ronco, and C. Bonetto. 2016. Toxic ity persistence of chlorpyrifos in runoff from experimental soybean plots to the non - target amphipod Hyalella curvispina : Effect of Crop Management. Arch. E nviron. Contam. Toxicol. 70: 257 264. N., T., and B. Türkyilmaz. 2003. Physiologi cal effects of captan fungicide on pepper ( Capsicum annuum L.) Pla nt. Pakistan J. Biol. Sci. 6: 2026 2029 . Nyberg, E.T., S.A. White, S.N. Jeffers, and W.C. Bridges. 2014. Removal of plant pathogen propagules from irrigation runoff using slow filtration systems: Quantifying physical and biological components. Water. Air. Soil Pollut. 225: 1999 . Parween, T., S. Jan, S. Mahmooduzzafar, and T. Fatma. 2011. Assessi ng the impact of chlorpyrifos on growth, photosynthetic pigments and yield in Vigna radiata L. at different phenological stages . African J. Agric. Res. 6: 4432 4440 . Parween, T., S. Jan, S. Mahmooduzzafar, T. Fatma, and Z.H. Siddiqui. 2016. Selectiv e Effect of Pesticides on Plant - A Review. Crit. Rev. Food Sci. Nutr. 56: 160 179. Petit, A.N., F. Fontaine, P. Vatsa, C. Clémen t, and N. Vaillant - Gaveau. 2012 . Fungicide impacts on photosynthesis in crop p lants. Photosynth. Res. 111: 315 326 . Pottorff, L.P., and K.L . Panter. 1997. Survey of Pythium and Phytophthora spp. in irrigation 204 water used by Colorado commercial greenhouses. Horttechnology 7: 153 155. Poudyal, S., and B.M. Cregg. 2019. Irrigatin g nursery crops with recycled run - off: A review of the potential impact of pesticides on plant growth and physiology . Horttechnology 29: 716 729 . Poudyal, S., B. Cregg, T.R. Fernandez, and J. Owen. 2019. Dose - dependent phytotoxicity of pesticides in simulated nursery runoff on landscap e nursery plants. Water 11: 2354. Qua li - Pro. 2011. Isoxaben 75 WG Available at https://www.greenbook.net/quali - pro/quali - pro - isoxaben - 75 - wg . Racke, K.D. 1993. Environmental fate of chlorpyrifos. Rev. Environ. Contam. Toxicol. 131: 1 150. Ranaivoson, A., P. Rice, F. John, G.W. Feyereisen, and M . Dittrich. 2019. Acetochlor and A comparison of experimental results with model estimates. Int. J. Hydrol. 3 : 286 306 . Rodell, M., J.S. Famiglietti, D.N. Wiese, J.T. Reager, H.K. Beaudoing, F.W. Landerer, and M.H. Lo. 2018. Emerging trends in global freshwater availability. Natu re 557: 651 659. Rodriguez - Cruz, S., M.S. Andrades, M. Sanchez - Camazano, and M.J. Sanchez - Martin. 2007. Relationship between the adsorption capacity of pesticides by wood re sidues and the properties of woods and pestic ides. Environ. Sci. Technol. 41: 3613 3619 Rouchaud, J., O. Neus, M.C. Van Labeke, K. Cools, and R. Bulcke. 1999. Isoxaben and BAS 479 14H retention/loss from peat substrate of nursery plants. Weed Sci. 4 7: 602 60 7 . Sharma, A., V. Kumar, A.K. Thukral, and R. Bhardwaj. 2019. Response s of plants to pesticide tox icity: An o verview. Planta Daninha 37:e019184291 Silva, F.B., A.C. Costa, R.R. Pereira Alves, and C.A. Megguer. 2014. Chlorophyll fluorescence as an indicator of cellular damage by glyphosate herbicide in Raphanus sativus L. plants. Am. J. Plant Sci. 05: 2509 2519 . Soeda, Y., S. Kosaka, and T. Noguchi. 19 72. The fate of thiophanate - methyl fungicide and its metabolites on plant leaves and glass pl ates. Agric. Biol. Chem. 36: 931 936 . Spiers, J.D., F.T. Davies, C. He, C.E. Bográn, K.M. Heinz, T.W. Starman, and A. Chau. 2006. Effects of insecticides on gas exc hange, vegetative and floral development, and overall quality of gerbera. HortScience 41(3): 701 706. Stearman, G.K., D.B. George, K. Carlson, and S. Lansford. 2003. Pesticide Removal from Container Nursery Runoff in Constructed Wetland Cells. J. Environ. Q ual. 32: 1548 . Stewart - Wade, S.M. 2011. Plant pathogens in recycled irrigation water in commercial plant nurseries and greenhouses: Their detection a nd management. Irrig. Sci. 29: 267 297. 205 Straw, B.Y.N., and N. Fielding. 1998. Phytotoxicity of insecticides u sed to control aphids on sitka spruce. For. Authority, UK. 7: 7 10 . Tjosvold, S., D. Chambers, and S. Koike. 2005. Evaluation of fungicides for the control of Phytophthora ramorum infecting Rhododendron, Camellia, Viburnum and Pieris. Sudd. Oak Death Second Sci. Symp., Januar y 18 - 21, 2005, Monterey, Calif. United States Department of Agriculture and National Agricultural Statistics Service. 2011. Agricultural chemical use: Nursery and floriculture crops 2009. United States Geological Survey. 2015. Total Water Use in the United States. United States Geol. Surv. Water Sci. Sch. Available at https://www.usgs.gov/special - topic/water - science - school/science/total - water - use - united - states?qt - science_center_objects=0#qt - science_center_objects (verified 24 February 2020). Veeraswamy, J., T. Padmavathi, and K. Venkateswarlu. 1993. Effect of selected insecticides on plant growth and mycorrhizal development in sorghum. Agric. Ecosyst. Environ. 43: 337 343. Velini, E.D., E. Alves, M.C. Godoy, D.K. Meschede, R.T. Souza, a nd S.O. Duke. 2008. Glyphosate applied at low doses can stimulate plant growth. Pest Manag. Sci. 64: 489 496 . Walker, A. 1987. Evaluation of a simulation model for prediction of herbicide movement and persis tence in soil. Weed Res. 27: 143 152 . Wang, P., H. Li, W. Jia, Y. Chen, and R. Gerhards. 2018. A fluorescence sensor capable of real - time herbicide effect monitoring in greenhouses and the field. Sensors 18 :3771. Warsaw, A.L., R. Thomas Fernandez, D.R. Kort, B.M. Cregg, B. Rowe, and C. Vandervoort. 2012. R emediation of metalaxyl, trifluralin, and nitrate from nursery runoff using container - grown woody ornamentals and phytore mediation areas. Ecol. Eng. 47: 254 263 . Weber, J.B. 1990. Behavior of Dinitroaniline Herbicide s in Soils. Weed Technol. 4: 394 406. Wied man, S.J., and A.P. Appleby. 1972. Plant growth stimulation by sublethal concentration s of herbicides. Weed Res. 12: 65 74 . Wilson, S.K., and S. Von Broembsen. 2015. Capturing and recycling irrigation runoff as a pollution prevention measure. Oklahoma State Univ. Ext. 1518:4 . Wilson, P.C., T. Whitwell, and M.B. Riley. 1995. Effects of ground cover and formulation on herbicides in runoff water from miniatur e nursery sites. Weed Sci. 43: 671 677. Wilson, C., T. Whitwell, and M.B. Riley. 1996. Detec tion and diss ipation of isoxaben and trifluralin in containerized plant nursery runoff water . Weed Sci. 44: 683 688 . Woignier, T., L. Duffours, P. Colombel, and P. Dieudonné. 2015. Nanoporous clay with carbon sink and pesticide trapping properties . Eur. Phys. J. Spec. T op. 224: 1945 1962. 206 Xia, X.J., Y.Y. Huang, L. Wang, L.F. Huang, Y.L. Yu, Y.H. Zhou, and J.Q. Yu. 2006. Pesticides - induced depression of photosynthesis was alleviated by 24 - epibrassinolide pretreatment in Cucumis sativus L. Pe stic. Biochem. Physiol. 86: 42 48 Younis, B.A., L. Mahoney, W. Schweigkofler, and K. Suslow. 2019. Inactivation of plant pathogens in irrigation water runoff using a novel UV disinfection system . Eur. J. Plant Pathol. 153: 907 914