Ifi" Thisis to certify that the thesis entitled FATE OF ACYLANILIDES IN SOILS AND POLYBROMINATED BIPHENYLS (PBB'S) IN SOILS AND PLANTS _ . .presented by Sheng-Fu Joseph Chou has been accepted towards fulfillment of the requirements for Ph. D. Soil Science degree in ‘72 4 Major professor Date [Liz/[K /‘ 7f Ff??? 0-7639 JUL 1 a 1439:: 'LQJL 1 31994 ."7' " . "3Qfi FATE OF ACYLANILIDES IN SOILS AND POLYBROMINATED BIPHENYLS (PBB'S) IN SOILS AND PLANTS By Sheng-Fu Joseph Chou A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1977 ABSTRACT FATE OF ACYLANILIDES IN SOILS AND POLYBROMINATED BIPHENYLS (PBB'S) IN SOILS AND PLANTS By Sheng-Fu Joseph Chou Biodegradation by soil microorganisms accounts for the dissipation of alachlor [2-chloro—2',6'-diethy1-N-(methoxymethyl)acetanilide], demethoxymethylalachlor (2-chloro-2',6'-diethy1acetanilide), and Antor herbicideTM [2-chloro—N—(2',6'—diethylpheny1)—N—methy1(ethylcarboxy- late)acetamide] in soil. The half-life of alachlor in several soils was between 7 to 14 days, while the half-lives of demethoxymethylalachlor and Antor were approximately 2.7 and 3.0 days, respectively. Rate of 14002 production and disappearance of 14C-alachlor increased with in- creasing temperatures until temperatures greater than 300C were reached. Organic matter additions to soil failed to increase rates of alachlor degradation. In the field studies alachlor was non-mobile and exhibited half-lives similar to laboratory determined values. The alachlor concen- tration in soil at yellow nutsedge emergence was approx. 0.2 ppmw. The rate of disappearance of 14C-ring and 14C-carbonyl labeled Antor was identical, which suggests that most of the metabolic products from Antor retain the chloroacetyl moiety. I found at least four major alachlor metabolites in hexane-acetone extracts of soil. Two were identified as 2-chloro—2'6'-diethylacetanilide and l—chloro-acetyl—Z,3-dihydro-7— ethylindole. Most of the added radioactivity in alachlor and Antor could not be recovered as 14002 or in benzene-isopropanol and hexane-acetone extracts. This unrecovered radioactivity ranged from 86 to 94% after 30 days of Sheng-Fu Joseph Chou incubation. Most of the alachlor and Antor remained in soil as inter— mediates which were tightly bound to or incorporated in soil organic matter. However, 51 and 62% of the label were recovered by a humic acid extractant, Na4P207, from alachlor and Antor amended soils, respec— tively. The majority of 140 label was found in the small molecular weight fraction (mw<500). Autoradiograms showed at least four alachlor and six Antor metabolites in this fraction. All appear to be polar. The flame retardant, PBB, was found to be comprised of 2,2',4,4' S,5'-hexabromobiphenyl as the major component, two isomers of penta- bromobiphenyl, three additional isomers of hexabromobiphenyl, and two isomers of heptabromobiphenyl. The PBB's were extremely persistent with no isomer showing any consistent disappearance in soil incubations of up to one year. Soil incubation utilizing 14C-hexa- and heptabromobi- phenyls, the two major isomers in PBB, showed that less than 0.2% of the l4C—PBB was degraded to 14CO after 12 months. Soils incubated with 2 products of l4C-PBB which had undergone photodegradation showed only minor conversion to 14C02. Gas chromatographic analysis showed equal, 84%, recovery of PBB in sterilized and non-sterilized soil after one year incubation. Autoradiograms of corn and soybean seedlings grown in hydroponic solutions showed no translocation of 14C-PBB (a) from 14C-PBB treated solutions to plant tops or (b) within the leaf from 14C-PBB treated spots on the upper leaf surface. A significant portion of the 14C-PBB associated with the roots was removed when the roots were dipped in acetone. Three root craps (radishes, carrots, and onions) were grown in two soils, one high in clay and organic matter and the other low. The soils were treated with a mixture of fireMaster BP6 (PBB) and 14C-PBB Sheng—Fu Joseph Chou to achieve final concentrations of 100 ppmw and 100 ppbw. The extent of PBB associated with the roots from the high PBB treatment ranged from approximately 50 to 500 ng/g plant tissue for plants grown in Spinks loamy sand soil and 30 to 120 ng/g plant tissue for plants grown in Brookston clay loam soil. No PBB was found with roots from the lower PBB treatment in Brookston soil. To my wife, Mei-In, for her encouragement and patience. To my parents and friends, Dr. and Mrs. R. L. Cook, for their moral support. ii ACKNOWLEDGEMENTS I am deeply indebted to Dr. James M. Tiedje for his encouragement and brillant guidance and his invaluable suggestions to make this manu- script understandable. I would also like to cite the invaluable criticisms of the members of my guidance committee: Drs. B. G. Ellis, D. Penner, A. R. Wolcott, and J. L. Lockwood. I am also thankful to Dr. A. R. Wolcott for use of the gas chromato- graph and Dr. D. Penner for teaching me the radioautographic techniques. My appreciation is extended to Dr. Lee W. Jacobs for his contri- bution of ideas in the greenhouse and laboratory aspects of this study. The assistance of Frank Vicini and Alice Marczewski in the analysis of greenhouse grown plants is very much appreciated. A special thanks is due to Sue Knoll for the task of typing the many revisions of this manuscript. I owe too a sepcial thanks to my friends who helped in one way or another for this study. The financial assistance from the Regional Research Project NC-96, and Biomedical Sciences Support Grants, NIH., are acknowledged. iii TABLE OF CONTENTS Page LIST OF TABLES O O O O O O O O O O O O O O O O O O C O O O O O O 0 Vi LIST OF FIGURES. O O O O I O I O O O C O O O O O O O O O O O O O O V111 CHAPTER I. BIODEGRADATION OF ¥fl0 ACYLANILIDE HERBICIDES, ALACHLOR AND ANTOR IN SOILS . . . . . . . . . . . |._a Introduction. . . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . . Soil incubations . . . . . . . . . . . . . . . Field studies. . . . . . . . . . . . . . . . . Bioassay study . . . . . . . . . . . . . . Extraction and analyses. . . . . . . . . . . . Results and Discussion. . . . . . . . . . . . . Literature Cited. . . . . . . . . . . . . J-‘mO‘UT-bUJWH N CHAPTER II. CHARACTERIZATION OF POLAR AND HUMIC-BOND SOlh METABOLITES OF ALACHLOR AND ANTOR HERBICIDE . . . 26 Introduction. . . . . . . . . . . . . . . . . . . . 26 Materials and Methods . . . . . . . . . . . . . . . 27 Substrates . . . . . . . . . . . . . . . . . . 27 Soil incubation. . . . . . . . 1 . . . . . . . 28 Molecular weight fractionation C-metabolites 29 Thin-layer chromatographic study of metabolites 29 Binding of alachlor metabolites to soil and soil organic matter. . . . . . . . . . . . . . 30 Results and Discussion. . . . . . . . . . . . . . . 31 Binding of fungal alachlor metabolites to soil 39 Literature Cited. . . . . . . . . . . . . . . . . . 44 CHAPTER III. CHARACTERIZATION OF POLYBROMINATED BIPHENYLS AND THEIR PERSISTENCE IN SOILS O O O O O O O O O O O O I O O O 46 Introduction. . . . . . . . . . . . . . . . . . . . 46 Materials and Methods . . . . . . . . . . . . . . . 47 PBB substrates . . . . . . . . . . . . . . . . 47 Soil incubations . . . . . . . . . . . . . . . 47 Analyses . . . . . . . . . . . . . . . . . . . 49 Pure culture studies . . . . . . . . . . . . . 52 Results and Discussion. . . . . . . . . . . . . . . 52 Literature Cited. . . . . . . . . . . . . . . . . . 74 iv CHAPTER IV. PLANT UPTAKE OF POLYBROMINATED BIPHENYLS (PBB'S). Introduction. . . Materials and Methods . Results and Discussion. Literature Cited. Page 76 76 76 79 90 LIST OF TABLES CHAPTER I 1. Evidence for biological degradation of alachlor in soil; effect of Ste ilization and anaerobic conditions on recovery of C-alachlor after incubation in Spinks soil . 2. Half-lives of alachlor in several Michigan and Iowa soils. 3. Bioassay of yellow nutsedge tuber on alachlor amended soil 4. Percent of added 14C recovered as 14CO and in hexane- acetone extracts after incubation in Brookston soil. . CHAPTER II 1. Percent of added radioactivity recovered from 14C-alachlor after incubation in Brookston soil . . . . . . . . . . . . 2. Perc t of added radioactivity recovered from 14C-alachlor and C—demethoxymethylalachlor after 20 days incubation in Sp inks 8011 O I O I O O O O O C O O I O O O O O O O l4 l4 3. Distribution ii C following 30 days incubation of C- alachlor and C—Antor herbicide in Brookston soil . . . . 4. Percent of 14C that could be extracted from sterilized Spinks and Brookston Soils to which C-alachlor metabolites produced by fungi had been added . . . . . . . . . . . . . 5. Loss in extractability of alachlor and alachlor metabolites following incubation with humic acid powder. . . . . . . . 6. Loss in extractability of alachlor and alachlor metabolites following incubation with freshly extracted humic and fulvic aCid O O O O O O O O O O O O I O O O O O O O O O O O O O 0 CHAPTER III 1. Recovery of six major PBB isomers from soil by using diff- erent SOlvents O O O O O O O O O O O O O O O O O O O O O O 2. Recovery of six major PBB isomers after incubation of 4 ppmw in Brookston soil. . . . . . . . . . . . . . . . . . . . . 3. F ratios for comparison of recoveries of six PBB isomers at zero time and after 24 weeks of incubation. . . . vi Page 9 ll 18 19 32 33 35 4O 41 42 50 60 61 CHAPTER IV 1. Page Recovery of two major isomers (hexa— and heptabromo- biphenyls) after incubation of 0.71 ppmw in Brookston sandy loam 8011 O O O O O O O O O O O C O I O O O O O O O 63 Recovery of PBB isomers after incubation of 0.4 ppmw in Brookston soil. . . . . . . . . . . . . . . . . . . . . . 65 Percent of original 14C evolved as 14CO from 14C-PBB amended Brookston soil. . . . . . . . .2. . . . . . . . . 66 Percent of added 14C—extracted from UV-irradiated l4C- PBB amended Brookston soil. . . . . . . . . . . . . . . . 67 PBB found associated with radish, carrot and onion roots after 6, 9, and 10 weeks, respectively, of growth in PBB contaminated 8011 O O O O O O O O O O O O O O O I O O O O 89 vii CHAPTER I 1. LIST OF FIGURES 4 Effect of incubatizn temperature in recovery of 1 C from alachlor as CO and in benzene-isopropanol extracts after 13 days incubation in Spinks soil . . . . Alachlor remaining in Brookston soil after incubation under field or laboratory conditions. A - Field treated soil incubated in flasks in lab, field or sterilized. B - Field soils treated with two concentrations of herbi- cide and rainfall they experienced . . . . . . . . . . . Distribution of alachlor in Brookston soil profile 50 days after spray application of the herbicide to the 8011 surface 0 O O O O O O O O I O O O O O O O O O O Autoradiogram of alachlor metabolites from hexane- acetone extract. 1 - alachlor metabolites produced by Chaetomium; 2 - alachlor standard; 3 - demethoxy- methylalachlor standard; 4, 5, 6, and 7-hexane- acetone extracts after 0, 10, 20, and 30 days of incubation in Brookston soil, respectively. Identified spots: a - demethoxymethylalachlor; b - l-chloroacethyl- 72,3-dihydro—7-ethylindole; c - alachlor. . . . . . . . CHAPTER II 1. Autoradiograms of alachlor and Antor metabolites found in sodium pyrophosphate extracts of soil. A, non—polar solvent system; B, polar solvent system. 1 & 8, fungal metabolites of alachlor; 2 & 7, fungal metabolites of Antor. Pyrophosphaig extracts of so}; after 30 days incubation of' 3, C—alachlor; 4, C- methoxymethyl- alachlor; 5, C-ring labeled Antor; 6, C-carbonyl labeled Antor. . . . . . . . . . . . . . . . . . . . . CHAPTER III 1. Gas chromatogram showing the six major components of PBB (peak 1 to 6) and their identity, and the three minor com- ponents (peaks a to c). The major component has the isomeric structure shown . . . . . . . . . . . . . . Mass spectra of the six major components of PBB; m/e of base peak is identified . . . . . . . . . . . . 60 MHz proten NMR spectrum of the major isomer (6 BrI) of fireMaster BP—6 . . . . . . . viii Page 10 l4 17 22 38 53 54 57 Page C-13 NMR spectrum of the major isomer (6 BrI) of fireMaster BP-6 O O O O C O O O O C O O O O C O O C O O I O I O O O 59 Auto diogram of TLC plate showing 14C-PBB standard and C in extracts after incubation in soil . . . . . . 70 Autoradiogramlgf TLC platEAShowing 14C—PBB standard, UV-iradiated C-PBB and C in extracts after incubation in soil . . . . . . . . . . . . . . . . . . . 72 CHAPTER IV 1. Soybean plants (top) and autoradiographfi (bottom). A, 7 days after leaves were treated thh C-PBB's. B and C are after root exposure to C-PBB's for 3 and 7 days, respectively . . . . . . . . . . . . . . . . . . 82 Soybean plants (top) and autoradipgraphs (bottom). A and B are after root exposure to C—PBB's for 4 and 8 days, respectively. C is identical to A except that roots were dipped in acetone prior to autizadiography. D, 8 days after leaves were treated with C-PBB's . . . 84 Corn plants (top) and autoradiographi4(bottom). A, 2 days after leaves were treated4with C-PBB's. B and C are after root exposure to C-PBB's for 3 and 7 days, respectively . . . . . . . . . . . . . . . . . . . 86 Corn plants (top) and autoradiggraphs (bottom). A and B are after root exposure to C-PBB's for 4 and 8 days, respectively. C is identical to A except that roots were dipped in acetone prior to autoradiography. D, 8 days after leaves were treated with C—PBB's. . . . . 88 ix CHAPTER I BIODEGRADATION OF TWO ACYLANILIQH HERBICIDES, ALACHLOR AND ANTOR HERBICIDE , IN SOILS Introduction Alachlor [2-chloro—2',6'-diethy1-N—(methoxymethyl)acetanilide] and AntorTM [2-chloro—N-(2',6'-diethy1pheny1)-N-methyl(ethylcarboxylate)- acetamide] are preemergence herbicides. Alachlor is now in wide spread use for control of annual grasses, redroot pigweed (Amaranthus retro- flexus L.) and yellow nutsedge (Cyperus esculentus L.) in corn (Zea mays L.) and soybeans [Glycine max (L) Merr.] (Armstrong, et al., 1973; Wax, et al., 1972). Antor is a selective herbicide with activity against many annual grasses and some broadleaved weeds. Hercules, Inc., is attempting to obtain registration for use of Antor herbicide on sugar beets, cotton, soybeans, corn, wheat and some vegetable crops. Despite the importance of the acylanilide class of herbicides little information on degradation and fate of these compounds has been published. Tiedje and Hagedorn (1975) found degradation of alachlor by a common soil fungus, Chaetomium globosum. The products were chloride and four identifiable organic metabolites: 2-chloro-2',6'-diethyl- acetanilide (DMM or demethoxymethylalachlor); 2,6-diethy1—N-(methoxy- methyl)aniline; 2,6—diethylaniline, and l-chloroacetyl-Z,3-dihydro—7- ethylindole. McGahen and Tiedje (1977) also found the degradation of Antor by the same soil fungus and identified five metabolites: Antor herbicide is a trademark of Hercules Chemical Corp. Alachlor is the common name of a herbicide marked by Monsanto Chemical Corp. under the trademark of Lasso. 2-chlor-N-(2',6'-diethylphenyl)acetamide, 2—hydroxy1-N—(2'-ethyl-6'- vinylphenyl)-N—methyl(ethylcarboxylate)acetamide, N-methyl(ethyl- carboxylate)—2,3-dihydro-7-ethylindole, 2-chloro-N—(2'-ethyl-6'-vinyl- phenyl)-N-methyl(ethylcarboxylate)acetamide, and N—2',6'-diethy1phenyl)— a-(ethylcarboxylate)imine. The degradation of alachlor by another soil fungus (Smith and Phillips, 1975; Chahal, et al., 1976) and in a model ecosystem has been reported (Yu, et al., 1975), but in neither case were metabolites identified. Hargrove and Merkel (1971) found that acid-catalyzed degradation of alachlor in soil, under conditions of low humidity and high temperature, resulted in the formation of demethoxymethylalachlor. However, this intermediate did not accumulate under more natural soil conditions. Beestman and Deming (1974) reported that microbial degrada- tion was the major route of alachlor dissapation in soil with half-lives ranging from 2 to 14 days for several soils. Taylor (1972) found that alachlor degradation in soil was not accompanied by mineralization of the aromatic portion of the herbicide. These reports for alachlor are consistent with the relatively rapid rate of degradation of other acylanilide herbicides (Bartha, 1971; Chisaka and Kearney, 1970; Kauf- man, et al., 1971; Kaufman and Blake, 1973), though in several cases degradation was not complete (Bartha, 1971; Chisaka and Kearney, 1970). This investigation was initiated to determine the rate and extent of alachlor and Antor degradation in soil and to identify any metabolite intermediates. Materials and Methods Soil incubations The degradation of alachlor was studied in Brookston sandy loam (pH, 7.08; OM, 3.38%), Conover silt loam (pH, 7.04; OM, 2.03%), Spinks loamy sand (pH, 6.29; OM, 1.25%), Muscatine loam (pH, 5.24; OM, 5.31%), and Tama clay loam (pH, 5.83; OM, 3.47%) surface soils obtained from plots on Michigan State University experimental farms and an Iowa farm. The soils had never received application of alachlor. The soils were freshly collected and allowed to dry to approximately 12-15% moisture before use. Fifty grams of soil that had passed through a 2 mm sieve was placed in 250 ml Erlenmeyer flasks. One milliliter of a 100 ppm solution of filter sterilized l4C-ring labeled alachlor (1.73 mCi/m mole) was distributed dropwise on the soil (equivalent to 1.8 kg/ha). The amended soil in each flask was then moistened by adding 12 ml of sterilized distilled water, sealed and incubated at 25°C for 0, 10, 20, 30, 40, and 50 days. Replicate flasks were sampled at the indicated time periods. This study was repeated in the Spinks, Tama, and Muscatine soils with similar experimental techniques. The half-life (t%) was calculated as a first order decay constant. For studies to determine the importance of microbial catalysis in degradation the incubation design was similar but the 14C-alachlor amended Spinks soil was incubated at 8, 18, 30, and 50°C; or under anaerobic conditions. Other flasks were autoclaved then reinoculated with a 2-ml soil suspension after 1 day, or sterilized with 10 m1 pro- pylene oxide. After sterilization 2.5 ml of 100 ppm filter-sterilized 14C-alachlor solution (0.02 uCi) was added. In an attempt to stimulate cometabolism, triplicate flasks of Spinks soil were amended with a solution of readily available carbon (0.1% glucose, 0.1% glycine, 0.1% acetate, and 0.1% peptone). Comparison of the degradation rate of alachlor, demethoxymethyl- alachlor, and Antor herbicide was conducted in the Brookston soil. Three milliliters of 100 ppm 14C-ring-labeled alachlor (1.73 mCi/m mole), l4C-ring-labeled Antor (1.21 mCi/m mole), ll'C-carbonyl-labeled Antor (0.09 mCi/m mole) were distributed dropwise on 30 g soil. Five milliliter of distilled water was used to moisten the soil. Incubation was in the dark at 28°C for 0, 5, 10, 20, and 30 days. Four flasks of each treatment were extracted after each incubation period. Respired 14CO was trapped in 1 m1 of 1N NaOH which was contained 2 in a disposable 2 m1 beaker suspended above the soils. It was assumed that recovery of the liberated 14CO2 in the alkali trap was substantially complete since equivalent levels of NaH14C0 14002 trapped within 8 h. Field studies 3 added to soils showed 95% Alachlor solution was sprayed on 3 x 15 square meter plots at rates 2.24 kg/ha and 4.48 kg/ha. Two adjacent unsprayed plots were used as control. After 0, 2, 7, 14, and 22 days post application, four replicate soil samples were collected from each plot. Each replicate sample was a composite of two surface soil samples, 2 cm deep by 10 cm diameter, taken by a shallow can. To compare field with laboratory degradation, surface soil from the 2.24 kg/ha treated plot was used in laboratory incubations. The field soil was mixed, sieved, placed in Erlenmeyer flasks and moistened as above. Control flasks were sterilized with propylene oxide. Half of the non-sterile flasks were put back in the field and the other half were incubated in the dark in the laboratory at 30°C. Three replicate flasks were sampled on each date. Fifty days following alachlor application, soil samples were collect- ed by soil probe (3.45 cm ID) from the 4.48 kg/ha plot and divided into 3.8 cm depth increments. Four replicates were used for analysis and each replicate was a composite of four cores. Bioassay study Surface soil samples were collected from control and 4.48 kg/ha alachlor-treated plots 19 days after alachlor application in the manner previously described. Clay pots were filled with 6 cm of control soil. Six yellow nutsedge tubers, collected from the control plot, were placed on the soil surface. The tubers were covered with 2.5 cm of alachlor treated soil. Other assays were of treated soil diluted by 1 part and 2 parts control soil. A11 pots were placed in the growth chamber under a regime of 12 h fluorescent light (64500 lux) at 29°C and 12 h of dark at 21°C. The pots were located in a randomized design with four replicates. Moisture levels in the soil were maintained by weighing the pots and adding the necessary water daily. After 39 days in the growth chamber (58 days after alachlor applied) when the yellow nutsedge shoots broke through the soil surface in the non-diluted treatment, samples of the top 2.5 cm of soil was collected by a No. 7 stainless steel cork borer. Four replicates each comprised of three composites were used for extrac- tion and GLC analysis of residual alachlor. After 50 days in the growth chamber all shoots were harvested and their dry weights determined. Extraction and analyses Alachlor was extracted from the soil with three 50-ml portions of benzene-isopropanol (2:1, v/v). For the first extraction the solvent- soil mixture was allowed to stand in the flask overnight and then shaken with rotary shaker at 250 rpm for 30 min. After decanting the solvent, the second and third extraction were made by shaking solvent and soil at 250 rpm for 30 min prior to decanting. Anhydrous NaZSO4 was added to the combined extracts to remove water. The combined extracts were concentrated to 50 or 100 ml prior to analysis on Beckman GC-S gas chromatograph equipped with an electron capture detector. A glass column of 1.83 m by 3 mm ID and containing 1.5% OV-l7/1.95% QF-l on 60/80 mesh Chromosorb Q was used. The inlet, column, and detector temperatures were 220°, 2000, and 250°C, respectively. The carrier gas (He) flow was 85 m1/min. Soil samples from the bioassay study were extracted by the same procedure except that 1.5 ml distilled water was added in each flask before the extraction. The combined 60—m1 benzene-isopropanol extract was condensed to 10 m1 prior to analysis. The field samples were placed into 500 ml Erlenmeyer flasks and 50 ml distilled water added. Three 100-ml portions of benzene-isopropanol (2:1, v/v) were used to extract alachlor following the same methods previously described except that all slurries were shaken on rotary shaker at 250 rpm for 50 min. The combined extracts were concentrated to 20 ml. Two milliliter of each extract was gently blown dry with N2 and the residue redissolved in an appropriate amount of Freon 113 prior to analysis on Perkin—Elmer 900 gas chromatograph equipped with a flame ionization detector. A stainless steel column of 1.8 M by 3 mm 0. D. and containing 3% SE-30 on 60/80 mesh Chromosorb Q was used. The inlet, 0, and 250°C respectively. column, and detector temperatures were 220°, 170 The carrier gas (He) flow was 30 ml/min. In studies of comparison of degradation rates all soil samples were extracted with three 40-m1 portions of hexane-acetone (9:1, v/v) since I had found that this solvent mixture reduced the amount of soil organic matter extracted yet recovered as much of the acylanilides. The extracts were analyzed on the Beckman GC-5 gas chromatograph equipped with a glass column containing 2% SE-30 on 100/120 mesh Gas-Chrom Q column. 0 The inlet, column, and detector temperature were 230°, 200 , and 270°C respectively. The carrier gas flow was 40 m1/min. The 14C-label was radioassayed by a Packard Tri-Carb Liquid Scintil- lation Spectrometer, Model 3310. One milliliter of the benzene- isopropanol extract was radioassayed in 15 ml of a scintillation solution containing 4 g PPO and 0.5 g POPOP/l of toluene, while the hexane- acetone extract was radioassayed in 15 ml Bray's solution (1960). The 14CO2 trapped in luN,NaOH was counted in Bray's solution containing 4% Cab-O—Sil. All counts were corrected for quenching by external standard- ization and machine efficiency. For the thin-layer chromatographic study of metabolites, the hexane- acetone extracts were concentrated to dryness and redissolved in 0.5 m1 chloroform. Samples (10 ul) were spotted on 250 u pre-coated silica gel 60 F-254 plates (EM Laboratory, Elmsford, NY). TLC plates were developed in benzene-methanol (95:5, v/v) (Yu, et al., 1975) and subsequently examined after autoradiography. Results and Discussion Evidence supporting biodegradation as the major mechanism for alachlor disappearance is summarized in Table l and Figure 1. Soil sterilized by two different methods, propylene oxide and autoclaving, showed only a 19% loss of alachlor during incubation compared to a 72% loss in non-sterile soil. Also reinoculation of autoclaved soil caused an increase in degradation rate as would be expected of a microbiological mechanism. Exclusion of 02 also slowed alachlor disappearance as would be expected for most biological reactions though slight anaerobic degrada- tion may have occurred (~10%). The temperature effect on degradation (Figure 1) yields a pattern expected for biologically and not chemically catalyzed reactions since the rate of both 14CO2 production and alachlor disappearance dropped after 30°C, probably due to denaturation of enzymes. As noted in these and other sterile control treatments in this paper, a low rate of non-biologically catalyzed alachlor disappearance always occurred. Generalizing from these studies, it appears that about 20% of the disappearance is non—biological while 80% is biological. The greater quantity of 14C extracted than alachlor extracted (GLC analysis) shown in both Table 1 and Figure 1 suggests that alachlor metabolites were also extracted. The half-lives (t%) of alachlor in several soils are reported in Table 2. They ranged between 7 to 14 days which is in agreement with the 2 to 14 day values reported by Beestman and Deming (1974). The slightly slower rate of degradation for experiment II in Spinks soil may have been due to the lower temperature and/or the more inactive state of the microflora due to energy depletion in soils collected in summer. Table 1. Evidence for biological degradation of alachlor in soil; ngect of sterilization and anaerobic conditions on recovery of C- alachlor after incubation in Spinks soil. Treatment Incubation Incubation % of original recovereda temp. (0C) time (day) Alachlor by 14C extracted GLC None — 0 94.6 97.8 None 30 13 28.5 45.1 Propylene oxide — O 90.6 95.3 Propylene oxide 30 13 82.5 86.6 Autoclaved - 0 95.2 97.3 Autoclaved 30 13 80.1 84.7 Autoclaved, reinoculated 3O 13 45.1 61.6' Anaerobic 24-27 13 70.2 81.5 a Mean of three or four replicates. 1.5 1.2 O \O O O\ % of 14C evolved as 14CO2 0.3 0.0 10 q 100 o 14 M CD2 (0 +3 0 14 m C extracted r- f" "" 60 fi H o .4 S m at m _. 'd ala or “>40 56: by GLC ;§’ H (H 0 ER _ -20 o l J l l 0 o 10 20 3o 40 50 Incubation temperature ( C) 14C from Figure 1. Effect of infflbation temperature n .nuamp Eu N ou nowusnfiuumav maaeammm coaumuuamoaoo m a.aa > mo~.ma aama ..e=muamz «saws ms.a .eamaa .coumaooum m.oH > moo.w cama ..ashlzmz mn\wx qw.~ .vawam .coumxooam N.m a> mmm.m aama .amz ammaa .eamaa .aoamaooam q.m om mmm.m sama .ams ammaa .nma .aoumMOOHm o.m ma o.oa mama .aasm aa noumaoOLm m.m ma o.a mama .amm a soumaooem m.ma ma o.m mama .auo amzoav maaumomaz m.aa ma o.m mama .uoo amsoav mama m.~a ma o.m mama .ummm aa .maaamm q.a ma o.~ mama .amm a .maaamm s.¢a ma o.a mama .nmm am>oaoo amamev auov assume mwaalmamm .asmu coaquSUGH Haom ca .oaou vmuomaaoo Hwom mama coaumwuommv mamamm cam Haom .mHaom mSOH paw cmwaSUaz Hmum>wm Gm aoazomam mo mm>malmamm .a magma 12 The latter mechanism has been reported to be important to rates of EDTA biodegradation (Tiedje, 1977). In previous experiments I could not enrich organisms using alachlor as the sole carbon source. Since biodegradation does occur in soil and by consitutive enzymes in a soil fungus (McGahen, 1977), a cometabolic degratory mechanism seemed the likely explanation for soil degradation. However, organic matter additions to Spinks soil failed to increase rates of alachlor degradation. After 20 days of incubation only 0.13% of the original 14C—alachlor was evolved as 14CO2 when available carbon was added compared to 2.6% in non-amended soil. These carbon substrates apparently allowed rapid growth of microorganisms that were incapable of degrading alachlor. A similar result was observed when glucose additions to Spinks soil failed to increase the rate of glyphosate degradation (Moshier, 1977). Tiedje and Hagedorn (1975) had found that the presence of carbon in the medium did not enhance the rate of alachlor degradation by resting cells of Chaetomium globosum. The losses of alachlor in field studies are shown in Figure 2B. The degradation of alachlor in both treatments followed the same pattern and rate. By the 22nd day 2.02 ppmw and 4.18 ppmw of alachlor were recovered from 2.24 kg/ha and 4.48 kg/ha treated plots, respectively. The half-lives were 10.5 to 12.7 days, respectively (Table 2). This confirms that a rapid rate of degradation also occurred in the field though it was slightly slower than for laboratory conditions. This was probably due to the more restrictive moisture and temperature conditions in the field. In any case the general similarity between field and laboratory results prove the validity of the flask incubation for assess- ing degradation in soils. 13 Figure 2. Alachlor remaining in Brookston soil after incubation under field or laboratory conditions. A-Field treated soil incu- bated in flasks in lab, field or sterilized. B-Field soils treated with two concentrations of herbicide and rainfall they experienced. 14 (u!) ugoa (nu/6x) Jomaolo {o uogmuueauog I!) do 0.. on >03 mm Aux—.3 2:2 cot—33:. ON 2 N— a - 20:. is 03.... 2.0.30... Xx «— (ded) Jogqaop Jo uououueauog 15 Evidence for absence of extensive leaching of alachlor in field soils is shown in Figure 3. After this 50 day period during which 8 cm of rain fell, 92.1% of the alachlor was recovered from the top 3 cm of soil. Because the data indicated rapid alachlor degradation, the minimum effective soil concentration was determined by bioassay and GLC analysis. The results are summarized in Table 3. Sixty—nine days after alachlor application the dry weight of yellow nutsedge shoots was only 20.7 mg for treated soil, compared to the control pots with 10 times this biomass. At 58 days when the yellow nutsedge shoots began to emerge, I found only 0.22 ppmw alachlor remained in the soil. At this low concentration the inhibition was probably released so that the shoots of yellow nutsedge started to elongate. Armstrong et al. (1973) found that an alachlor concentration of 3.7 x 10-6 M (1 ppm) still effectively inhibited yellow nutsedge growth in petri dish culture. However, alachlor does not inhibit sprouting of yellow nutsedge tubers. Knowing the minimum effec- tive concentration and the half-life of degradation one should be able to predict the approximate date of weed emergence, or alternatively to adjust the concentration applied to achieve the desired length of inhibi- tion. In this field study this calculation would predict 5% half-lives or 60 days, which was about when weed emergence in the field was noted. The losses of 14C-ring labeled alachlor and demethoxymethylalachlor (a postulated alachlor metabolite), and 14C-ring and l4C-carbonyl labeled Antor herbicide following incubation in soil are shown in Table 4. For all three chemicals, the quantity of 14CO2 produced was minor relative to decomposition of the substrate. The 14C extracted was either the same as (for Antor) or slightly greater than (for alachlor and DMM) the 16 Figure 3. Distribution of alachlor in Brookston soil profile 50 days after spray application of the herbicide to the soil surface. 17 I p b _ _ F _ c6 «.0 a... 25:53 .o_:ou_u ao :o_.u::oo:oo o.m _. 0.: mfi (W0) Hldaa a.» 18 Table 3. Bioassay in yellow nutsedge tubers on alachlor amended soil. Treatments Weight of shoot Alachlor Shoot at 69 days at time emergence of shoot elongation by GLC (mg)a (ppmw) (days since treatment) Control 285 1:117.1 NDb 5 treated soil + % control soil 61.8 + 20.9 ND ND 2/3 treated soil + 1/3 control soil 49.2 :_9.4 ND ND Treated soil 20.6 i 9.7 0.22 i 0.02 58 a All treatments were significantly different from each other (<0.05). b ND = not determined. l9 .Haom w om\wn oom mmB mommm mumuumnsm U .mmmmamam 00 hp uomauxm odoumomlmamxma Scum mmum>ooma mumuumpsm mo uamuumm a .mmumuaaamu usom no can: m m.N o.m 5.N o.m mwfialmamm «.0m m.Hm H.o m.Hm N.mm H.o H.mm H.Hm 0.0 m.mw 5.0m H.o mHHHmumlom m.o O.N m.¢ m.o N.N H.N ¢.H N.HH O.q m.q N.mH N.N om O.H N.N m.m ¢.H m.N m.H N.N m.NH w.N H.¢H N.mN m.H ON H.© o.w H.H H.5 5.5 «.0 m.m «.mH Om.H H.N¢ w.Hm m.o OH N.om ¢.wm w.o m.5m o.mm wN.o N.qm w.¢¢ «.0 5.qo «.05 m.o m o.mm «.mm o w.ma m.mm o m.om m.mm o H.mm m.mm o o oAmmmmm Hmuou mo NV Ammmv UNUUNHUNG UQUUNHUNG VUUUNHUNm UUUUQHUNO oau noqa Nooaa oau -usa Nooqa oae nosa Nouaa noau noqa Nouqa waau uouq< mmHmnmalahdonumo Houc< meoanIwafim 22a mmamnmalwdfim “canomam mmamnmafwdmm coaumASUGH .Haom doumxooum cm coaumnsocm amumm muomuuxm accumumloamxmz aw mam Nov mm moum>ooma um mommm mo unmoumm .q oHan «H «a 20 quantity of parent compound recovered by GLC. This indicates that most of the metabolites were not extractable. All parent compounds had virtually disappeared after 30 days incubation. The short half-life of DMM (2.7 days) compared to that for alachlor (8.0 days) explains why large quantities of this possible intermediate would not accumulate. The large quantity of this compound found by other investigators (Hargrove and Merkle, 1971) must have occurred because the high temperature (46°C) severely inhibited biological activities. The degradation rate of Antor was approximately three times that of alachlor. The 14C extracted and the parent substrate recovered were identical for the 14C—ring and 14C-carbonyl-labeled Antor at each sampl- ing date. This suggests that most of the metabolic products of Antor retain the chloroacetyl side chain. The 14C in extracts in excess of the parent herbicide (Table l, 4 and Figure l) was thought to be due to the presence of metabolites. Hexane-acetone extracts of soil were examined by TLC-autoradiography for the presence metabolites. The autoradiogram shown in Figure 4 revealed at least four metabolites, two of which could be identified as (a) 2- chloro-Z',6'-diethy1aniline (DMM) and (b) l-chloroacetyl-Z,3-dihydro-7- ethylindole. Identification was based on identical mobilities to the fungal produced standards on TLC and by GLC. Since it is possible that the indoline ring closed in the heated GC inlet, the indoline peak was collected from the effluent of the GLC columns and run on TLC beside the uninjected material. The mobilities were identical indicating no alterna- tion in structure by GLC conditions. This material separated by GLC was also used as the standard on TLC to locate the indoline spot. Figure 4. 21 Autoradiogram of alachlor metabolites from hexane—acetone extract. 1 - alachlor metabolites produced by Chaetomium; 2 - alachlor standard; 3 - demethoxymethyl alachlor standard; 4, 5, 6, and 7 - hexane-acetone extracts after 0, 10, 20, and 30 days of incubation in Brookston soil, respectively. Identified spots: a - demethoxymethyl- alachlor; b - l-chloroacetyl-Z,3-dihydro-7-ethylindole; c - alachlor. 23 The two identified products appear to be the only soil metabolites incommon with fungal metabolites. The fungus produces at least three additional products not found in soil, while the soil produces two not formed by the fungus. No metabolites of Antor could be found by similar TLC analyses. This is consistent with finding no l4C extracted in excess of that attributable to the herbicide (Table 4). Polar metabolites of both alachlor and Antor could be recovered from soil by using the humic acid extractant, 0.1 M Na4P207. The binding of these intermediates to soil organic matter and their characterization are discussed elsewhere (Chapter II). 10. 11. 12. 24 Literature Cited Armstrong, F. F., W. F. Meggitt, and D. Penner. 1973. Yellow nutsedge control with alachlor. Weed Sci. 21:354-357. Bartha, R. 1971. Fate of herbicide—derived chloroaniline in soil. J. Agri. Food Chem. 19:385-387. Beestman, G. B., and J. M. Deming. 1974. Dissipation of acetanilide herbicides from soils. Agron. J. 66:308-311. Bray, G. A. 1960. A simple efficient liquid scintillation counter. Anal. Biochem. 1:279-285. Chahal, D. S., I. S. Bans, and S. L. Chopra. 1976. Degradation of alachlor [Z—chloro-N-(methoxymethy1)-2',6'-diethy1acetanilide] by soil fungi. Plant and Soil. 45:689-692. Chisaka, H. and P. C. Kearney. 1970. Metabolism of propanil in soils. J. Agri. Food Chem. 18:854-858. Hargrove, R. S. and M. G. Merkle. 1971. The loss of alachlor from soil. Weed Sci. 19:652—654. Kaufman, D. D., J. R. Plimmer, and J. Iwan. 1971. Microbial degradation of propachlor. Amer. Chem. Soc. Abst. l62nd Meeting, Washington, D. C. Aer. Kaufman, D. D. and J. Blake. 1973. Microbial degradation of several acetamide, acylanilide, carbamate, toluidine and urea pesticides. Soil Biol. Biochem. 5:297-308. McGahen, L. L. and J. M. Tiedje. 1977. Degradation of two new acylanilide herbicides, Antor and Dual by the soil fungus, Chaetomium globosum. submitted to J. Agri. Food Chem. McGahen, L. L. 1977. Metabolism of two new acylanilide herbi- cides, Antor [2-chlor05N7(2',6'-diethy1pheny])—N-methyl(ethyl- carboxylate)-acetamide] and Dual [2—chloro-Nf(2'—ethy1—6'-methyl- phenyl)fiN7(2-methoxy-l-methylethyl)acetamide] by the soil fungus, Chaetomium globosum. M.S. thesis, Michigan State University, E. Lansing, Mich. pp. 39. Moshier, L. J. 1977. Factors affecting N:(phosphoromethyl)glycine (Glyphosate) activity in turfgrass and alfalfa [Medicago sativa (1)] Seedling environments and degradation in the soil. Ph.D. thesis, Michigan State Univ., E. Lansing, Mich. pp. 85. 13. 14. 15. 16. 17. 18. 25 Smith, A. E. and D. V. Phillips. 1975. Degradation of alachlor by Rhizoctonia solani. Agron. J. 67, 347-349. Taylor, R. 1972. Degradation of alachlor [2-chloro-2',6'-diethyl- N-(methoxymethyl)acetanilide] in soils and by microorganisms. M.S. thesis, Michigan State Univ., E. Lansing, Mich. pp. 25. Tiedje, J. M. and M. L. Hagedorn. 1975. Degradation of alachlor by a soil fungus, Chaetomium globosum. J. Agri. Food Chem. 23, 7 7-810 Tiedje, J. M. 1977. Influence of environmental parameters on EDTA biodegradation in soils and sediments. J. Environ. Quality. 6, 21-25 0 Wax, L. M., E. W. Stoller, F. W. Slife, and R. N. Anderson. 1972. Yellow nutsedge control in soybeans. Weed Sci. 20, 194-200. Yu, C. C., G. M. Booth, D. J. Hansen, and J. R. Larsen. 1975. Fate of alachlor and propachlor in a Model ecosystem. J. Agri. Food Chem. 23, 877-879. CHAPTER II CHARACTERIZATION OF POLAR AND HUMIC-BOUND SOIh METABOLITES OF ALACHLOR AND ANTOR HERBICIDE Introduction Acylanilide herbicides are rapidly degraded by microorganisms in soil but their aromatic portion is persistent. Chou and Tiedje (1975) found that the amount of 14CO2 evolved from 14C-ring labeled alachlor in soils averaged only 4.1% of the original label after 50 days of incuba- tion. After this incubation 82% of the added label could not be extract— ed from soil with benzene—isopropanol or accounted for as 14CO Similar 2. results were observed for Antor. This suggests that polar and/or bound metabolites are the major fate of acylanilide herbicides in soils. Hsu and Bartha (1974a, 1974b, 1976) have shown that binding of acylanilide- derived residues with soil organic matter does occur. However, informa- tion of this type has not been published for the N-alkyl substituted acylanilides. Because most of the herbicide material could not be recovered, this study was undertaken to characterize the fate of the metabolite. Evidence for polar and bound intermediates is presented. 26 27 Materials and Methods Substrates Alachlor was obtained from City Chemical Co. and was purified as described by Tiedje and Hagedorn (1975). The final purity was 99.9% as determined by GLC. Uniformly 14C-ring-labeled alachlor (specific activity, 1.73 mCi/m mole) was supplied by Monsanto Chemical Co., St. Louis, M0. The label purity was 99.7% by thin-layer chromatography. 2-chloro-2',6'-diethylacentanilide (demethoxymethylalachlor) was prepared by hydrolysis of alachlor in 5 N HCl according to the procedure of Hargrove and Merkle (1971). The purity of the final product was 99.9% by GLC (Tiedje and Hagedorn, 1975). 14C-ring-labeled demethoxy- methylalachlor was prepared from l4C-ring-labeled alachlor in the same manner, the specific activity was 0.05 mCi/m mole. Antor was obtained as a formulation from Hercules, Inc., Wilmington, Dela., and was purified by the same method used for alachlor. This procedure was repeated until the Antor purity was 99.9% by GLC. 14C- ring- (specific activity 1.21 mCi/m mole) and 14C-carbonyl-labeled Antor (0.09 mCi/m mole) were also provided by Hercules, Inc. The carbonyl label was located in the chloroacetyl moiety. 14 C-alachlor and 14C-Antor metabolites were prepared by incubating these substrates with resting cells of Chaetomium globosum as previously described (Tiedje and Hagedorn, 1975; McGahen and Tiedje, 1977). The incubation was long enough so that most of the substrate was metabolized. The metabolites were extracted with CHC13, the solvent removed, and the metabolites redissolved in water-ethanol (9:1, v/v). The activity of this alachlor metabolite solution used for binding experiments was 6 28 nCi/ml; this solution was filter sterilized before use. Metabolites used as TLC standards were extracted in CHCl3 and concentrated prior to use. Soil incubation Incubation studies were conducted in 250 ml Erlenmeyer flasks which contained 30 g of soil that had passed through a 2 mm sieve. The soils were stored in large sealed jars at 4°C and never allowed to air-dry before use. These herbicide materials were dissolved in water solution (usually 100 ppm) and distributed dropwise on the soil surface. Final substrate concentrations were 5 to 10 ug/g soil. Distilled water was added as necessary to moisten the soil to within 60 to 80% of water holding capacity. All soils were incubated sealed, in the dark at 280C. The respired 14CO2 was trapped in 1 m1 1 N NaOH which was contained in a disposable 2 m1 beaker suspended above the soils. Incubations were terminated by extraction of the soil with either benzene-isopropanol (2:1, v/v) or hexane-acetone (9:1, v/v) (Chapter I). Three successive extractions were used as this was found to entirely remove the substrate. The effectiveness of more rigorous extractants at removing residual 4C materials was examined using soils which had been extracted with benzene-isopropanol. The following extractants were used: H20, p- dioxane, dimethylformamide (DMF), tetrahydrofuran (THF)—ethanol (3:1, v/v), 0.1 M Na-EDTA, 0.1 M Na4P207 (pH=7.0) and 0.5 N NaOH. All soil samples were shaken in the extractant for 24 h on a rotary shaker. An additional set of soil samples was extracted by refluxing with CHCl3 for 24 h. The soil was removed by centrifugation or filtration through Whatman No. 1 filter paper. The soluble 14C was assayed by liquid 29 scintillation counting (Chapter I). For extractants that removed colored humic material, the solution was combusted by wet oxidation (Allison, et al., 1965), the 14CO trapped and counted. The 14C remain- 2 ing in soil after extraction was determined by the above combustion method on 5 g of air—dried soil. Four replicate samples were used for each extractant. Molecular weight fractionation of 14C-metabolites Brookston soil incubated with 14C-alachlor and 14C-Antor for 30 days was extracted with hexane-acetone to remove non-polar substrate and metabolites, and re-extracted with Na to remove polar and bound 4P2°7 metabolites. The latter solution was fractionated according to molecular weight by ultrafiltration using an Amicon Diaflo cell, Model 52. The membranes (with their nominal molecular weight cut offs) and the sequences of use were: XM-300 (300,000 mw), XM-50 (50,000 mw), UM-2 (1,000 mw), and UMr05 (500 mw). The procedure employed was similar to that of Ogura (1974) and Wheeler (1976) who successfully used the method to fractionate dissolved organic matter in estuarine waters. The Diaflo cell was operated at the lowest pressure necessary to achieve the desired flow of 0.4 and 0.1 m1/min. Fifty milliliters of the pyrophosphate extract was fractionated until approximately 3 ml remained above the filter. Two 10 m1 portions of distilled water were used to wash the concentrate above each filter. The filtrate and concentrate were assayed by liquid scintil- lation counting to determine the total 14C in each fraction. Thin-layer chromatographic study of metabolites The last fraction from the Diaflo filtration (mw< 500) was extract- ed with three 50—ml portions of diethyl ether. Anhydrous NaZSO4 was added to the combined ether extracts to remove water. The extracts were 30 concentrated to 0.5 ml and 10 ul was spotted on a 250 u pre-coated silica gel 60 F-254 plate (EM laboratory, Elmsford, NY). Alachlor and Antor metabolites produced by Chaetomium globosum were also spotted on the plates as standards. The sodium pyrophosphate extract from a 14C- demethoxymethylalachlor amended soil was also extracted with ether and analyzed on the same plate. TLC plates were developed in a non-polar solvent system-~benzene-methanol (95:5, v/v) (Yu, et al., 1975) and a polar solvent system-—l-butanol-glacial acetic acid-water (12:3:5, v/v) (Armstrong, et al., 1973). After development plates were examined for 14C spots by autoradiography with Kodak No—Screen X-ray film. Binding of alachlor metabolites to soil and soil organic matter l4C-alachlor metabolites and 14C-alachlor were incubated with soil and soil organic matter fractions to determine their binding capacity. For the soils experiments 30 g of propylene oxide sterilized Spinks and Brookston soils was incubated with 3 ml of alachlor metabolites. After the indicated incubation periods the soils were extracted with three 40 m1 portions of hexane-acetone. The 14C extracted was counted by liquid scintillation counting. Humic acid was obtained by shaking Brookston soil with 0.1 M Na4P207 (pH 7.0) under N2 for 24 h, centrifuging out the soil, adding HCl until pH 2 was reached, and centrifuging to recover the humic acid pellet. The pellet was washed three times with distilled water and the humic acid freeze-dried. The humic powder was added to a flask and 1 ml of l4C-metabolite and l4C-alachlor solutions added. The flasks were flushed With N2, sealed and incubated. After the indicated incubation periods the flasks were extracted with four 20-m1 portions of hexane- acetone (8:2, v/v). The extracted 14C was counted as above. 31 The humic-fulvic complex was collected as above except not fraction- ated by pH precipitation. Salts were removed by dialysis against water containing Dowex 50W-X8 H—form. Four days later 30 ml of the humic complex was placed in a flask and 2 m1 of 14C—metabolites and -alachlor were added. The flasks were flushed with N2, sealed and incubated in an anaerobic glove box. After incubation the flasks were extracted with three 50—ml portions of diethyl ether and the 14C extracted counted as above. All data from binding experiments are means of triplicate flasks. Results and Discussion The mass balance of 14C-alachlor products at different stages of decomposition is shown in Table 1. Very little of the 140 was respired to CO2 and only minor quantities could be recovered by organic solvent extraction. The major portion of the label, 86% at 30 days, required a more rigorous extractant for removal or could not be removed at all. The growth in the unaccounted for fraction relative to other fractions was greatest in the later stages of incubation, indicating that substrate and intermediate products were being slowly converted to the resistant soil humin fraction. Three successive extractions with benzene-isopropanol 1 h after addition to soil was found to remove >97% of alachlor and demethoxymethyl- alachlor and was assumed to remove other hydrophobic intermediates as well. Following this exhaustive extraction other polar and more rigorous extractants were used (Table 2). Water, which should remove polar l4 intermediates, recovered only 10% of the residual C. DMF and dioxane, which should not extensively alter soil organic matter though do extract 32 l4C-alachlor Table 1. Percent of added radioactivity recgvered from after incubation in Brookston soil . Incubation Evolved as Hexane-acetone Na4P207 Unaccounted time 14CO2 extract extract for (daYS) (7.) 0 0.0 98.4 - 1.6 10 0.5 48.0 39.0 12.5 20 1.1 23.9 51.5 23.5 30 1.4 12.4 51.3 34.9 a pH of extracting solution was 11.5. 250 ug alachlor/30 ug soil; data are the means of four replications. 33 .uouoanuuomm coaumaaaucaoo masvaa >3 momommo 5Huuoaam mucouuxm o .Nw.m¢ mo3 comumsnsoo mo hoaoaommmo onu mmo5omooowmou Nov mam moo ou moumsnaoo ouoouuxm «H m .mo5ommooamou mam moamouu wooed onu mam doauomfixo uoB he moumanaoo ooa conuoo afiaowuo Hmom Hosmfimom o .oamaoou Haom mo coauoouuxo cu uoaua maoauoouuxo o>ammooo=m oouse n .maowuoowamou know no oaooa onu ouo ouom "Hwom w: om\ououumnso w: omN o . . . . 5 N o . . 0 mm m 5m m mm 052 H ov o m oz o NH m 5 Hoaaooao m.~m m.mm m.ma ease a.aa m.a aanumeaxosumamm a.am a.am a.oa some a.aa m.~ m.mm m.ma a.aa exsaamumaomu m.aa s.a a.mm 0.44 a.aa emamxoaeum a.aa m.~ m.w5 a.am m.m~ mmzn a.am n.~ m.~m a.aa a.aa mHocoSuolmmH m.HN o.~ ©.mo 5.mq o.N~ mAz H.0V menmloz m.mm m.~ a.am a.om a.am caz a.oV aommsmz o.ma m.a w.qm 5.mm «.mq oAz m.ov mooz m.mN m.~ poacooa< mouo>ooou mouosnEoo mouoouuxm ucoauooam uoouuxo moo mouoaumnsm a Homomoumomm o«H kua>fiuoo0flmou HouOH locouaon osmamou Hmom aH cH .Hwom oxaaam cw coauonooam mmom ow wouwo uoanooamaknuoaaxonuoaomloca mam Hoanooaoloqa Scum mouo>ooou mua>fiuoo0Hmou mommo mo ucoouom .N oanme 34 a portion of it, extracted additional l4C. THF and EDTA which can alter the structure of organic matter was no more effective than DMF in remov- ing residual 14C. The humus extractants, Na4P207 and NaOH, recovered the most l4C. Because of the high pH of the NaOH extraction, substantial chemical alteration of the humus is thought to occur. These changes are far less severe with the pyrophosphate extractant if buffered at pH 7.0 and carried out under an inert atmosphere. In addition I found that the NaOH solution decomposed about 50% of added alachlor under the extractive regime while pyrophosphate had no effect. Thus, pyrophosphate was used as the humus extractant in subsequent studies. The inability of water and other polar solvents to remove much of the 14C indicates that most of the metabolites were bound to organic matter. This binding to organic matter is also shown by the significant quantities of 14C removed by the humus extractant (PYIOphosphate) and the large quantity remaining after this extraction, presumably in the humin fraction. The presence of label in the humin fraction was confirm- 14 14 ed by recovery of C as CO2 by combustion of the soil. Demethoxymethylalachlor, the alachlor metabolite (Chapter I), was converted somewhat more readily to 14CO2 though most of the products were associated with the organic matter, as was the case for the parent herbicide. This similar fate is consistent with DMM being an important alachlor intermediate in soil. Antor, a structurally related acylanilide herbicide, had a similar fate. As shown in Table 3, little 14C was recovered as 14CO2 and by the organic solvent extraction while most appeared bound to organic matter. If herbicide metabolites were bound to organic matter the 14C material extracted with humus could be expected to be associated with 35 .mxmoam ouoofiaaauu mo maooa ouo ouom “Haom m om\ououumn:m w: com o a.aa 0.m m.a m.a 0.a a.am 0.N m.a aaaaonamuv pouq< a.0m m.a m.a 0.a m.a a.am m.a m.a amaauv “0000 0.0m N.~ m.a 0.a a.N m.am a.aa 0.a amaaav H02002 aav 00mv 00m 00 000a 000a 00 000w0m 000.0m 00 000.00m 000.000A N00 a 00 mmumuumsam .u3 Hoe 5n uoouuxo rodmaoz mo coaquOHuooum Moouuxo uoouuxo q Ommqoz occuoum oucoEuoopu HHoxHo 5n mouoouuxo osmaoou Hfiom Ioaoxom .Hfiom aoumxooum aw omauwnuo: acua——<:l}o 68d!“ 58!“) scam I 2 Figure l. Ni- MINUTES Gas chromatogram showing the six major components of PBB (peaks 1 to 6) and their identity, and the three minor components (peaks a to c). The major component has the isomeric structure shown. Peak l Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 54 54B” I Pentobtomo (I) ' L'V'UVIVU‘I'T“ ArrerTI—‘T'I'I'U'U'I'I'YVIVU Pentobromo (II) Hexobromo (I) Hexobromo (II) /' 628 Hexobromo (m) Vjt'vrvrIIVII[VIVIVjY'UIIfTIV'Y Ujvyu‘ ‘d 500 550 600 650 m/e Figure 2. Mass spectra of the six major components of PBB; m/e of base peak is identified. 706 Heptobromo 700 I'V'I'ITF'I‘I'V‘ q Relative Intensity 55 heptabromobiphenyl (M+ 700). The major component, 6 Br(I), was pre- viously known to be a hexabromobiphenyl from gc-ms analysis of PBB's, but mass spectrometry provides no structural information. It can be seen from Figure 3 that the proton nmr spectrum shows the presence of two distinct peaks of equal intensity; thus the major isomer has a symmetrical structure. The major component was identified by carbon-13 nmr to be 2,2',4,4',5,5'-hexabromobiphenyl (Figure l) on the following evidence: 6c, ppm from TMS: C—1, 140.3, biphenyl linkage; C-2, 122.4, 2 Br; C-3, 136.7, 2 H; C-4, 125.9, 2 Br; C-5, 123.8, 2 Br; C-6, 134.8, 2 H (Figure 4). The hydrogen shifts were confirmed by a Gated Decoupling. The three minor components which are shown in the glc trace in Figure 1 but which were not quantitated in these studies were identified by gc- mass spectrometry as follows: a, hexabromobiphenyl; b, heptabromo- biphenyl; and c, unknown but suspected to be octabromobiphenyl. The recovery of PBB components after incubation in Brookston soil is shown in Table 2. It is clear that the PBB persisted for one-half year in soil with only slight disappearance of three of the components and no disappearance of the other three. The same observation of persis- tence holds for incubations with added available organic matter, under anaerobic conditions, and for Spinks soil as is summarized in Table 3. Since Spinks soil is more coarse textured and contains less organic matter may account for the inactivity of this soil for PBB degradation. The persistence of these PBB compounds is consistent with the evidence reported for PCB compounds which shows that the more heavily chlorinated members (penta or greater) are resistant to degradation though many lesser chlorinated components are metabolized (Ahmed and Focht, 1973; Baxter 25 al., 1975; Tucker gt al., 1975). 56 Figure 3. 60 MHz proton NMR spectrum of the major isomer (6 BrI) of fireMaster BP-6. 57 O -4--.— 0wv'v—tv '0 P 1411 .c————.---. . n F, r‘r’>b'* lib, IIII’ 1441114 111111111 11‘1‘ ‘11 . . 0 m. . . ._. .1, _ ,_ .. ..... H. . .2... a .n m . I mt... ._.: . 2?. .. n . . . . a .a. m .1 t. m . a _ H , u a . . a a... _a ... m.... m .r :.. _ u. .,._ . _ — a u . . .. . , ._.. _ _. m .... ..u . ._ ’0 . .U a. J . . _ .1 o o I. .fli. ...... _ ..._.... H .. 21:2... I.“ I“ .. . a . .t a .. 4. _ a: . . H." m ”T D h P bl. p .I b 7. FL p p pr..- . .b.» hu— 0 D F P V F _ b P F by, H b Lilr r F P D ~ 58 Figure 4. C-l3 NMR spectrum of the major isomer (6 BrI) of fireMaster BP’6. 59 On ow om azmmve 00 00. on. o3 ooa om— ? . 1%.éssé} iééga; ._:? sages 6O .am0.v m 00 amaaaou aazums ouaoao>wsoo mo mowcmm n u.n.o« om.c5 om.05 om.¢c £5.00 no.mc om.5o 0N oo.m5 mw.mc ono.H5 LN.50 no.mo mo.N5 NH o5.m5 mm.q5 nom.o5 oo.~m nm.mc mo.05 o o5.o5 m~.q5 om.~m om.mw oo.5w mq.q5 o oH.m5 mo.H5 oH.mw o5.mw oo.wm mm.05 ouozmmoau am a aaa am 0 aa am 0 a an 0 aa an m a um m amxmmsv «ucoEmcoEm Hocmwauo mo ucoouomiwmmm mo zuo>ooom oEHu soauonsocH .HHom :oumxooam ca Sam q no coauonsoca uoumo muoaomm mmm Hohms me mo 5uo>ooom .a magma 6 .uouuoa cacomuo mo uaoamcoa< + .AHC.V m um uaouamacwao ooooaoommomaa {as .Amo.V m um uamuamacwao ooaouoomaomaa as .AOH.V m um ucmuawwcwao oocouooamomaa « xAMH.©V AOH.O V a¢o.o V Amq.o V AN0.0 V 55¢.H V Hfiom mxcfiam Amm.oV «esaom.qu «Awa.o V 550.0 V «seAmN.qu AqN.N V +20 + coumxooum 20 + «Am5.©V ktkAmN.¢NV Aom.N V 50H.H V «*«Amm.OmV AwH.¢ V UHnOHomcm .cowmxooum AHo.oV AHN.oV k««A5M.HmV «*«A5w.mqV «sxAmm.me «ssAoN.ooV Haom coumxooam km 5 HHH Hm 0 HH Hm 0 H Hm 0 HH Hm m H Hm m ufiwEquHH .GOfiumnsoafi mo oxooB «N Houmo moo oEHu ouou um mHoEomH mmm o «o moauo>ouou mo comfiuomaoo How moauou m .m magma 62 A difficulty in these studies is the lack of precision due to variability in quantity recovered among replications. This does not appear to be due to technique since ratios between peaks vary in a random manner among the extracted solutions. It is also evident from Table 2 that a substantial loss occurred in the glassware in the absence of soil. These analytical difficulties are also apparent in other reports (Babish gt al., 1975; Gutenmann and Liske, 1975). Given these difficulties, one can only interpret the highly significant and consis- tent trends in the data. Only the disappearance of the second pentabromo- biphenyl isomer (5 BrII) in the Brookston soil (Table 3) fulfills this condition. Whether this disappearance is due to microbial degradation, sorption, or masking was not resolved although some biodegradation seems possible since only a specific isomer showed a decrease and there is precedence for microbial metabolism of certain dichloro substituted aromatic rings of PCB compounds (Ahmed and Focht, 1973; Baxter gt al., 1975). It is feasible that certain dibromo substituted aromatic rings of pentabromobiphenyls could be attacked since biphenyl oxidizing bacteria require an adjacent 2-3 position to form the first biphenyl metabolite cis-2,3-dihydro-2,3—dihydroxybiphenyl (Gibson gt al., 1973). In pure culture studies, no PBB metabolites were found in biphenyl medium after 21 days of incubation. Apparently these two microorganisms can not degrade fireMaster BP-6. In the second part of the study the recovery of the two major PBB isomers (hexa— and heptabromobiphenyls) after various periods of incuba- tion in Brookston sterile and non-sterile soil is shown in Table 4. Whether analyzed by 14C or gas chromatography the data clearly show no detectable biodegradation after 1 year. It is also striking that the 63 Table 4. Recovery of the two major isomers (hexa- and heptabromobiphenyls) after incubation of 0.71 ppmw in Brookston sandy loam soil. % of the original amount of PBB recovereda Analysis sterilized 0 month 3 months 6 months 9 months 12 months 14 b C extracted yes 93.3 86.8 88.5 85.5 84.7 no 92.7 85.3 87.3 84.8 84.3 Hexa isomer by GLC yes 91.5C 87.0 89.5 83.8 85.6 no 90.9 86.2 85.4 87.8 84.9 Hepta isomer by GLC yes 89.6d 85.3 87.1 82.2 83.9 no 89.1 82.9 86.2 82.1 83.8 a Each value is the mean of four reps. , 14 A of total C recovered. % of the hexabromobiphenyl isomer recovered by GLC analysis. % of the heptabromobiphenyl isomer recovered by GLC analysis. 64 14C and GLC analysis of each peak showed virtually identical quantities on each date. GLC data of the recovery of non-14C labelled isomers is shown in Table 5. The only possible evidence for degradation is for the 5 BrI isomer, which does not confirm the previous suggestion which indicated that only the 5 BrII isomer might have been subject to slight biodegradation. Of interest is the significant loss in extractability with time of all isomers (Table 4 & 5) in the sterile as well as non- sterile treatments. Total 14C collected in 1N NaOH is shown in Table 6. Though slightly more label was trapped from non-sterilized soil, the amount of additional label volatilized in the viable treatment was insignificant. Soils incubated with photodegradation products of 14C-PBB showed enhanced though still only minor conversion to 14C02. Products created by the photo-decomposition of PBB by UV-irradiation are apparently more volatile than non-irradiated PBB as shown by the increase in 14C collected from sterilized soils, especially at the first sampling. The microbial activity which occurred in the non-sterilized treatments appeared to increase the amount of 14C volatilized, suggesting that some of the 14C- degradation products of UV-irradiation may be metabolized. PBB irradiat- ed by UV-light forms lower brominated biphenyls (Ruzo and Zabik, 1975). The extractability of the 14C photodegraded PBB is shown in Table 7. Much of the added material was not extracted, in marked contrast to PBB (Table 2). Apparently the photodegraded products are more reactive with the soil organic matter thereby preventing their extraction. The early loss of extractability (0 and 3 months) is supportive of this explanation. Table 5. 65 Recovery of PBB isomers after incubation of 0.4 ppmw in Brookston soil. a Incubation Percent of PBB isomers of original amendment recovered time 5 Br I g 5 Br II 6 Br II 6 Br III (months) s N-S‘ s N-S s N-S s N-S 0 87.1 86.3 89.4 88.0 83.4 84.6 79.4 80.7 3 80.3 79.2 78.2 76.3 80.5 80.0 74.7 73.7 6 77.4 79.5 75.7 73.7 81.3 80.6 71.3 70.6 9 78.9 75.4 76.1 74.5 82.5 79.3 72.1 70.2 12 77.6 71.4 72.7 70.3 79.9 78.5 70.7 71.4 a Each value is the mean of four replications. b S = sterilized soil. C N-S = non-sterilized soil. 66 Table 6. Percent of original 14C evolved as 14CO from 14C-PBB amended Brookston soil. 2 % of original 14C trapped in NaOHa Treatment Substrate 3 months 6 months 9 months 12 months Sterilized 14c-PBB 0.03 0.07 0.10 0.12 Non-sterilized 14C-PBB 0.04 0.08 0.14 0.17 Sterilized UV-14C—PBBb 4.43 5.72 6.17 6.76 Non-sterilized UV-14C-PBB 6.24 7.20 7.96 9.94 a Each value 1 b UV exposed s the mean of four replications. 14C-PBB. 67 Table 7. Percent of added 14C-extracted from UV-irradiated l4C-PBB amended Brookston soil. . 14 a A of C recovered in hexane-acetone extracts Treatment 0 month 3 months 6 months 12 months Sterilized 76.1 36.5 34.1 Non-sterilized 72.1 35.4 32.0 a Each value is the mean of four replications. 68 Since partially degraded PBB would not yield 14C02, we also examined soil extracts for other 14C products by TLC-autoradiography (Figure 5). The TLC system used was shown to separate the isomers of fireMaster (detection by UV). No intermediates of PBB degradation could be found. From the autoradiogram it is apparent that the two 14C-PBB isomers were the only 14C products in soil. The autoradiogram of UV-treated 14C—PBB extract from soil is shown in Figure 6. It also showed little difference between the original amendment and the extract from the soil incubation. The marked change in the PBB isomers due to the UV irradiation is clearly shown by this figure. Virtually none of the original isomers remains. Most of the label is at the solvent front, the position where lesser brominated forms would be expected to run. The label at the origin and the streaking indicates that some of the 14C-PBB products may have complexed with the soil organic matter. This is consistent with the low efficiency of extraction of these photolyzed products. Conclusion The potential hazards from PBB-contaminated soils are low since PBB's are not taken up by plants (Chapter IV) or leached to ground water (Filonow, pg 31., 1976) at concentrations expected to be present, and they are probably not volatilized due to their low vapor pressure (5.2 x 10-8 mm Hg at 25C, Jacobs, gg'a1., 1976); however, they may remain in the soils for many years because of their resistence to degradation. Only low levels of contamination are expected in soils on most of the exposed farms because of dilution, as we have found for the farms examin- ed (Jacobs_g£.§1., 1977). In certain rare and localized situations 69 Figure 5. extoradiogram of TLC plate showing 14C—PBB standard 3&2 C in extracts after incubation in soil. "1 and 6" C- PBB standard "2 and 3" extracts from sterilized soil after 6 and 12 months incubation, respectively, "4 and 5" extracts from non-sterilized soil after 6 and 12 months incubation, respectively. Figure 6. 71 Autoradiogram of TLC plage showing 14C-PBB standard, UV- irradiated C-PBB and Cain extracts after inigbation in soil. "1" UV-irradiated C-PBB standard, "2" C-PBB standard, "3 and 4" extracts from sterilized soil after 6 and 12 months incubation, respectively, "5 and 6" extracts from non-sterilized soil after 6 and 12 months incubation, respectively. 72 73 where high level contamination may have occurred, potential concern could arise from erosion of contaminated soils or manures into streams and the accumulation of PBB's in terrestrial and aquatic food chains. 10. ll. 12. 74 Literature Cited Ahmed, M., and D. D. Focht. Oxidation of polychlorinated biphenyls by Achromobacter PCB. Bull. Environ. Contam. Toxicol. 10, 70 (1973). Babish, J. G. W. H. Gutenmann, and G. S. Stoewsant. Polybrominated biphenyls: Tissue distribution and effect on hepatic microsomal enzymes in Japanese quail. J, Agri. Food Chem. 23, 879 (1975). Baxter, R. A., P. E. Gilbert, R. A. Lidgett, J. H. Mainprize, and H. A. Vodden. The degradation of polychlorinated biphenyls by microorganisms. Science of the Total Environment. 4, 53 (1975). Bray, G. A. A simple efficient Liquid Scintillation Counter. Anal. Biochem. 1:279 (1960). Carter, L. J. Michigan's PBB Incident: Chemical Mix-up Leads to Disaster. Science. 192, 240 (1976). De Vos, R. H. and E. W. Peet. Thin-layer chromatography of poly— chlorinated biphenyls. Bull. Environ. Contam. Toxicol. 6, 164 (1971). Filonow, A. B., L. W. Jacobs, and M. M. Mortland. Fate of poly- brominated biphenyls (PBB's) in soils. Retention of hexabromo- biphenyl in four Michigan soils. J. Agri. Food Chem. 24, 1201 (1976). Gibson, D. T., R. L. Roberts, M. C. Wells, and V. M. Kobal. Oxida- tion of biphenyl by Beijerinckia species. Biochem. Biophys. Res. Comm. 50, 211 (1973). Gutenmann, W. H., and D. J. Lisk. Tissue storage and excretion in milk of polybrominated biphenyls in ruminants. J. Agri. Food Chem. 23, 1005 (1975). Isleib, D. R., and G. L. Whitehead. Polybrominated biphenyls: An Agricultural Incident and its Consequences. I. The Agricultural effect of exposure. IXth annual conf. on Trace Substances in Environmental Health, Univ. of Missouri, Columbia, MD, June 10, (1975). Jacobs, L. W., S. F. Chou, and J. M. Tiedje. Fate of polybrominated biphenyls (PBB's) in soils. Persistence and plant uptake. J, Agri. Food Chem. 24, 1198 (1976). Jacobs, L. W., S. F. Chou, and J. M. Tiedje. Environmental implica- tions of PBB's in soils and waters. National PBB Workshop, Michigan State University, Oct. 24-25, 1977. I.ll.ll‘.lylll| 13. 14. 15. 16. 75 Moore, R. W., J. V. O'Conner, nd 8. D. Aust. Idenitrification of a major component of polybrominated biphenyl as 2,2'3,4,4',5,5'- heptabromobiphenyl. Submitted for publication (1977). Robertson, L. W., and D. P. Chynoweth. Anather halogenated hydro- carbon. Environ. 17, 25 (1975). Ruzo, L. 0., and M. J. Zabik. Polyhalogenated biphenyls: photolysis of hexabromo and hexachlorobiphenyl in methanol solution. Bull. Environ. Contam. Toxicol. 13, 181 (1975). Tucker, E. S., V. W. Saeger, and 0. Hicks. Activated sludge primary biodegradation of polychlorinated biphenyls. Bull. Environ. Contam. Toxicol. 14, 705 (1975). CHAPTER IV PLANT UPTAKE OF POLYBROMINATED BIPHENYLS (PBB'S) Introduction PBB have entered Michigan soils from manures of farm animals fed PBB, from disposal of milk, carcasses and other produce, and from effluent and dust discharges of the PBB manufacturing plant. Since most insoluble halogenated hydrocarbons are not taken up or translocated by plants, one would predict PBB to behave similarly. However, because of the impor- tance of this conclusion to the quality of future Michigan food, I felt it prudent to thoroughly investigate this question. In my preliminary greenhouse studies I had shown no PBB in tops of orchard grass and carrots grown in soils amended with high levels of PBB (Jacobs et al., 1976). I did, however, find traces (20-40 ppb) of PBB associated with carrot roots. I also found no detectable PBB in plants collected from the most highly contaminated Michigan fields (Jacobs et al., 1977). This study was undertaken to evaluate PBB translocation from roots and leaves using 14C-PBB and to more thoroughly evaluate the association of PBB with root crops, carrots, radishes and onions. Materials and Methods Corn and soybean seedlings were grown in a coarse sand-vermiculite mixture with Hoagland's solution. At 3 weeks of age the seedlings were removed, the adhering particles washed off the roots, and the plants 76 77 placed in Hoagland's no. 1 hydroponic solution. For root uptake studies, l4C-PBB (hexa- and heptabromobiphenyl isomers only, see Jacobs et al., 1977) was added to the hydroponic solution to achieve a final PBB concen— tration of 100 ppb (1.42 uCi/L). To determine translocation from leaves, a 0.5 ul water drop containing 17 ng 14C-PBB (0.25 uCi) was spotted on the upper surface of a mature leaf. In the first trial, plants were exposed to PBB for 3 and 7 days, while in the second trial, plants were exposed for 4 and 8 days. After the indicated exposure periods the plants were removed from the hydroponic solutions and the roots were washed by dipping them into distilled water. In the second trial, part of the plant roots that were exposed to 14C-PBB were quickly dipped five times into an acetone bath and then washed with distilled water. All plants were then immediately frozen with crushed dry ice, freeze dried, and later examined by autoradiography. Radishes, carrots, and onions were grown in the greenhouse on Brookston clay loam (3.27 % org. C) and Spinks loamy sand (1.31 % org. C) soils amended with a mixture of 100 ppmw of fireMaster BP6 and 50 ppbw l4C-PBB (0.68 uCi/k soil). The soil was prepared by mixing 4.9 g of fireMaster BP6 and 2.35 mg 14C-PBB (33.1 uCi) in 100 ml acetone with 300 g of air-dried soil in a 500 m1 brown glass bottle. The soil was gently blown with N2 to remove excess acetone then mixed in a twin shell dry blender overnight at low speed. The PBB-treated soil was diluted with 48.7 kg of untreated soil in a mixer overnight to achieve the desired concentration of 100 ppmw. Fourty-eight grams of 100 ppmw soil was further diluted with 48 kg of untreated soil to achieve the 100 ppbw 78 concentration. From my previous experience this procedure gave a very satisfactory distribution of PBB in soil. PBB amended soil (5 kg) was placed in a plastic container lined with a polyethylene bag. The radish, carrot, and onion seeds were planted 1.25 cm below the soil surface and nutrient solutions were added as needed. The moisture levels of the soils were maintained at 15 and 10% for the Brookston and Spinks soils, respectively, by weighing the container and adding the required amount of distilled water daily. After 6, 9, and 10 weeks the radishes, carrots, and onions, respectively, were carefully removed from the container. The tubers or bulbs were washed with tap water then dried with paper towels. A few of the plants grown on the 100 ppmw PBB-treated soils were immediately frozen with liquid nitrogen, freeze-dried, and later examined by autoradiography. Plant roots were cut into small pieces and extracted with hexane- acetone (1:1 v/v) in a Waring blender using a glass mixing container. Three successive 100 ml portions of hexane-acetone were used to extract each plant sample and then combined. The hexane-acetone phase was separated, dried with anhydrous Na2804, and finally concentrated to 10 m1 on rotary flash-evaporator. This concentrate was passed through a Florisil column to remove interfering components by eluting with 200 ml benzene-hexane (1:1, v/v). The eluent was concentrated to 10 ml for gas chromatographic analysis. Concentrated extracts from the above samples were analyzed on a Beckman GC-5 gas chromatograph equipped with an electron capture detector (Filonow et al., 1976) and a 2% SE-30 on 100/120 mesh Gas-Chrom Q column operated at 250°C with a carrier flow of 40 ml/min. 79 Results and Discussion The hydroponic study and greenhouse experiment were designed to favor the greatest uptake of PBB. Corn and soybeans were selected for the hydroponic studies since both are major crops in Michigan. The results of the 14C uptake studies are shown in Figures 1, 2, 3, and 4. Autoradiograms of corn and soybean seedlings grown in the presence of l4C-PBB showed no translocation of PBB's. PBB was found concentrated at the roots, but no PBB was translocated to the plant tops. Due to the insolubility of PBB in water, I expected that the PBB would be primarily associated with the root surface. A significant portion of the 14C-PBB was removed when the roots were dipped in acetone (see Figure 2 and 4) as shown by the lighter autoradiograms. The autoradiograms also showed no movement of PBB within the leaf from the site of 14C—PBB application. Three root crops, radishes, carrots, and onions, were grown in two soils which differed greatly in organic matter and clay content. The extent of PBB uptake by roots was determined by autoradiography and by gas chromatographic analysis. No PBB uptake was shown on autoradiograms, however I did find trace amounts of PBB by gas chromatography associated with the roots (Table 1), because of the greater sensitivity of the latter method. The trace amounts of PBB found on the roots were probably associated with root surface. Iwata and Gunther (1974) found 97% of PCB residues in carrot roots in the peel. This observation has also been found for DDT and other organochlorine pesticides in soil in which carrots were grown. Carrot roots showed more PBB than radish or onion (Table 1). The plants grown on the sandier, low organic matter Spinks soil showed 80 higher PBB uptake than ones grown on the Brookston soil which had more clay and organic matter. This finding is consistent with the report of Filonow 3; al., (1976) who found that the adsorption of hexabromobiphenyl increased with increasing soil organic matter. From these results plus my previous results of greenhouse (Jacobs, et a1. 1976) and field studies (Jacobs et al., 1977) in which I found no PBB in plant tops, I conclude that PBB will not be transferred from contaminated soil to plant tops. Thus, recontamination of animals from feeds grown on contaminated soil will not likely occur from this source. Although root crops from very highly contaminated soil might contain traces of PBB. much of this PBB could probably be removed by peeling. 81 Figure l. Soybean plants (top) and autoradiographs (bottom). A, 7 days after leaves were treaigd with C-PBB's. B and C are after root exposure to C-PBB's for 3 and 7 days, respectively. 82 83 Figure 2. Soybean plants (top) and auigradiographs (bottom). A and B are after root exposure to C-PBB's for 4 and 8 days, respectively. C is identical to A except that roots were dipped in acetone prior to autoizdiography. D, 8 days after leaves were treated with C-PBB's. 84 85 Figure 3. Corn plants (top) and autoradiogzaphs (bottom). A, 7 days after leaves werelgreated with C-PBB's. B and C are after root exposure to C-PBB's for 3 and 7 days, respectively. 86 Figure 4. 87 Corn plants (top) and autoradiographs (bottom). A and B are after root exposure to C-PBB's for 4 and 8 days, respectively. C is identical to A except that roots were dipped in acetone prior to autoizdiography. D, 8 days after leaves were treated with C—PBB's. 88 89 Table 1. PBB found associated with radish, carrot and onion roots after 6, 9, and 10 weeks, respectively, of growth in PBB contaminated soil. PBB in plant roots, ng/gf Soil type PBB added Radishes Carrots Onions (ppm) Spinks loamy sand 0.1 7.2 20.4 nd+ 100 48.7 535 62.8 Brookston clay loam 0.1 nd nd nd 100 43.7 117 33.8 * 0n wet weight basis, each value is the mean of three replications. +nd = not detectable. 90 Literature Cited Filonow, A. B., L. W. Jacobs, and M. M. Mortland. Fate of poly- brominated biphenyls (PBB's) in soils. Retention of hexabromo- biphenyl in four Michigan soils. J. Agri. Food Chem. 24, 1201 (1976). Iwata, Y., F. A. Gunther, and W. E. Westlake. Uptake of PCB (Arochlor 1254) from soil by carrots under field conditions. Bull. Environ. Contam. Toxicol. 11, 523 (1974). Jacobs, L. W., S. F. Chou, and J. M. Tiedje. Fate of polybrominated biphenyls (PBB's) in soils. Persistence and plant uptake. J. Agri. Food Chem. 24, 1198 (1976). Jacobs, L. W., S. F. Chou, and J. M. Tiedje. Environmental implica- tions of PBB's in soils and waters. National PBB Workshop, Michigan State University, Oct. 24-25, 1977.