4.... . r. .11.}; . . . PS“. . 4 Hun-QJY'? 9. V»; . 7 I! (I I... :5; $.33}: 8. i 2. 5.! 33 . . s. ). ‘ 311.; fi 5. 1:53-15 3. . 3 4.1.4.1.. t. .2 . t I. .13: . I. x. .3. .1115...- I Ll. 32.3.2.3; :3 .1717... . .43.. 1 .. . . . .. . 3:?! .1. :95 1.3.5:... 3. l . “RU-«Igg- .13 2.3L. . 0.. 1...; 5 .mWfiWJmkil I . . . in}... 2’ 3!... $3.? . J. I .39 t 1 :3: L..21§.P€.ml 1 8.: 1.1 n .. Li‘l : . . : 11:33.... 3.4521... . : it a"... v31: 3 1.9555131... $4509.? 3 .3535}! 3‘“...ng :’-.lu I! ...§..Lnfi. 5;. 4...»...3 x... 1 5.11.... L .. I. I I: athr..t:.f.4\ I)“ 3.. THESiS '2 .2 2500 LIBRARY Michigan State University This is to certify that the dissertation entitled PART ONE: DEVELOPMENT OF ANALYTICAL METHODS FOR THE DETERMINATION OF SELECTED “ORGANIC PESTICIDES” PART TWO: DDT AND IT'S METABOLITES IN SOIL AND AIR AT AN AGRICULTURAL SITE NEAR SOUTH HAVEN, MICHIGAN: DETERMINATION AND IMPLICATIONS presented by 0 meme \lmlemw has been accepted towards fulfillment of the requirements for a ( P h‘ D degree in 'Etmlc\o‘;\1 \E‘WUWQHMQ ML \DK‘\ CU\O 3‘ "MW \ AM M ajor prorfLsshr) Dateé’ilq')cl‘? MSU is an Afflrmatiw Action/Equal Opportunity Institution 0- 12771 PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 cJCIRCIDateDue.p65-p.15 PART ONE DEVELOPMENT OF.ANALYTICAL METHODS FOR THE DETERMINATION OF SELECTED “ORGANIC PESTICIDES” PART TWO DDT AND IT’S METABOLITES IN SOIL AND AIR AT AN AGRICULTURAL SITE NEAR.SOUTH HAVEN, MICHIGAN: DETERMINATION AND IMPLICATIONS By CHRISTINE VANDERVOORT A.DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Entomology/Environmental Toxicology 1999 ABSTRACT PART ONE DEVELOPMENT OF ANALYTICAL METHODS FOR THE DETERMINATION OF SELECTED “ORGANIC PESTICIDES” By CHRISTINE VANDERVOORT Part one of this research was conducted to develop a multiple pesticide analytical residue method for the determination of the “Organic Pesticides”, Nicotine, Pyrethrum, Rotenone, and Warfarin. This research determined the optimum detection and chromatographic conditions for the analytical determination of these four pesticides in a multiresidue scheme. The desire to achieve a multiresidue method that was efficient (accurate and precise) and economical yielded a method that did not meet desired lower limit of detection. The four pesticides had physical properties quite different from each other which required the method to change pH from neutral to basic and then to an acidic pH. The second part of the method development work involved pesticides that did not have individual residue analytical methods that were previously published. The pesticides analyzed were a-terthienyl, azadiractin, ryanodine, and veratridine. The chemical and physical properties of these four pesticides had many similarities and thus facilitated their simultaneous extraction, separation, and detection. PART TWO DDT AND IT’S METABOLITES IN SOIL AND AIR AT AN AGRICULTURAL SITE NEAR SOUTH HAVEN, MICHIGAN: DETERMINATION AND IMPLICATIONS By CHRISTINE VANDERVOORT The second part of this research involved analysis of air and soil samples to determine the spatial distribution of DDT and it’s metabolites (sum of DDT) in Southwestern Michigan. Historically, levels of DDT have been elevated in this geographical region. The levels were found to be easily quantified with gas chromatography (GC) during the sampling period from April 1998 to August 1998. Historically this site has been under extensive fruit and vegetable farming and received high inputs of DDT in the past. The research supports the postulate that the elevated levels of DDT in the area are due to volatilization from the soil. The soil samples from the site had DDT levels elevated above outlying areas. The ratio of DDT to its metabolites also supports the view that the DDT was from past spraying before the 1973 ban in the United State of DDT. Calculations were determined fi'om the residue data to estimate the time for the soil to dissipate the residues and it was found to range from a few years to thousands of years. ACKNOWLEDGMENTS I would like to thank my major professor Dr. Matthew J. Zabik for his guidance and support during my education at Michigan State University. I am also grateful to my committee members, Dr. Larry G. Olsen, Dr. Jerry Cash, Dr. Donald Penner, and Dr. Edward Walker for serving on my guidance committee and their critical review of this manuscript. The education I received from Dr. Zabik was very obviously in analytical chemistry but also in putting many years of learning into a complete learning and understanding. I learned how to take a challenge and find an answer . Finally, I would like to thank all of my family and friends for their continued support and confidence in me. My whole family was educated along with me. I look forward to helping them in their endeavors. iv TABLE OF CONTENTS PART ONE ANALYTICAL METHOD DEVELOMENT . . . . . . . . . . 1 INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . l OBJECTIVES. . . . . . . . . . . . . . . . . . . . . . . . 8 OBJECTIVE I . . . . . . . . . . . . . . . . . . 8 OBJECTIVE II. . . . . . . . . . . . . . . . . . 9 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . 10 ORGANIC FARMING & FOOD INDUSTRY. . . . . . . . 10 CHARACTERISTICS AND USES OF THE PESTICIDES . . ll TERTHIENYL. . . . . . . . . . . . . . . . ll AZADIRACTIN . . . . . . . . . . . . . . . 12 NICOTINE. . . . . . . . . . . . . . . . . l4 PYRETHRUM . . . . . . . . . . . . . . . . 15 ROTENONE. . . . . . . . . . . . . . . . . 17 RYANIA. . . . . . . . . . . . . . . . . . 20 SABADILLA . . . . . . . . . . . . . . . . 22 WARFARIN. . . . . . . . . . . . . . . . . 24 ANALYTICAL METHOD DEVELOPMENT. . . . . . . . . 26 GENERAL CONSIDERATION FOR ANALYTICAL METHOD DEVELOPMENT . . . . . . . . . . . . . . . 33 RESEARCH . . . . . . . . . . . . . . . . . . . . . . . . 36 METHOD I - NICOTINE, WARFARIN, ROTENONE, AND PERMETHRIN. . . . . . . . . . . . . . . . . . . . . 36 FUTURE RESEARCH . . . . . . . . . . . . . . . 54 METHOD II - SABADILLA, a-TERTHIENYL, RYANIA, AND AZADIRACTIN. . . . . . . . . . . . . . . . . . . . 55 RESULTS AND CONCLUSIONS . . . . . . . . . . . 65 APPENDICES A - F. . . . . . . . . . . . . . . . . . . . 67 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . . 76 PART TWO DDT CONCENTRATIONS IN THE SOIL AND AIR OF SOUTH HAVEN, MICHIGAN. . . . . . . . . . . . . . . . 83 INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . 83 OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . 83 OBJECTIVE III . . . . . . . . . . . . . . . . 83 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . 85 PERSISTENCE IN THE ENVIRONMENT . . . . . . . 85 TOXICITY . . . . . . . . . . . . . . . . . . 9O ATMOSPHERIC TRANSPORT. . . . . . . . . . . . 91 SOIL AND SEDIMENT CONCENTRATIONS . . . . . . 94 DEGRADATION OF DDT . . . . . . . . . . . . . 95 RESEARCH . . . . . . . . . . . . . . . . . . . . . . . 97 SAMPLE COLLECTION. . . . . . . . . . . . . . 97 ANALYTICAL PREPARATION AND CLEANING. . . . . 102 EXTRACTION . . . . . . . . . . . . . . . . . 102 SILICA GEL COLUMN CHROMATOGRAPHY . . . . . . 103 GAS CHROMATOGRAPHY . . . . . . . . . . . . . 103 RESULTS AND CONCLUSIONS. . . . . . . . . . . . . . . . 105 w AIR SAMPLES. SOIL DATA. CONCLUSIONS OF SOIL IMPACT ON AIR CONCENTRATIONS FUTURE WORK .APPENDICES G - R. BIBLIOGRAPHY. Wi 105 158 163 .166 .168 .197 LIST OF TABLES Table 1.0 Solubility of Solvents Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Maximum Absorbance at Specific Wavelengths Vapor Pressure Absorbance Maximas 37 (nm)37 38 4O Analyte Absorbance Sums at Given Wavelengths . 41 Resolution between Peaks Theoretical plate and HETP values. Linear Regression for Standards. Percent Recoveries for Fortified Samples LOD and LOQ Values Solubility of Solvents Maximum Absorbance at Specific Wavelengths Resolution of Between Peaks. Theoretical plate and HETP values. Linear Regression for Standards. Percent Recoveries for Fortified Samples LOD and LOQ Values Projects that have Measured DDT and it's Isomers in South Haven, MI. Properties of DDT, DDE, DDD, and Kelthane Sampling schedule for 1998 Air and Soil Samples Soil Data of Summed DDT Concentrations viii 43 44 46 . 52 52 55 (nm)56 57 61 64 88 89 98 159 Table 3.4 Table 3.5 Table 3.6 Amount of EDDT found in one hectare of soil 25.4 cm deep . . . . . . . . . . . . . . . . 160 Percent Loss of EDDT per year from one hectare for the two Locations. . . . . . . . 161 The percent loss per year and Amount of Years to Reduce the Soil Burden to Zero Concentration per Metabolite. 162 ix Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 1.10 LIST OF FIGURES d-Terthienyl Molecular Structure Azadiractin Molecular Structure. Nicotine Molecular Structure Pyrethrum Molecular Structure. Rotenone Molecular Structure Ryania Molecular Structure Sabadilla Molecular Structure Warfarin Molecular Structure Nicotine Standard Curve. Pyrethrum Standard Curve Rotenone Standard Curve. Warfarin Standard Curve. d—Terthienyl Standard Curve. Azadiractin Standard Curve Ryanodine Standard Curve 12 13 15 17 19 21 .23 25 47 48 48 49 59 59 60 Figure 2.3 Veratridine Standard Curve 60 Figure 3.0 DDT Degradation Pathway 88 Figure 3.1 General Geographical Area of the Study. 99 Figure 3.2 Localized Sampling Map. . 100 Figure 3.3 Concentration of Vapor Phase o,p’—DDE at SH at Site A . . . . 106 Figure 3.4 Concentration of Vapor Phase p,p'-DDE at SH at Site A . . . . . 106 Figure 3.5 Concentration of Vapor Phase o,p'-DDD at SH at Site A 107 Figure 3.6 Concentration of Vapor Phase p,p'-DDD at SH at Site A. 107 Figure 3.7 Concentration of Vapor Phase o,p’-DDT at SH at Site A . 108 Figure 3.8 Concentration of Vapor Phase p,p’-DDT at SH at Site A . 108 Figure 3.9 Concentration of Vapor Phase Kelthane at SH at Site A . 109 Figure 3.10 Concentration of Vapor Phase o,p'—DDE at SH at Site B 110 Figure 3.11 Concentration of Vapor Phase p,p’-DDE at SH at Site B 110 Figure 3.12 Concentration of Vapor Phase o,p'—DDD at SH at Site B 111 Figure 3.13 Concentration of Vapor Phase p,p'-DDD at SH at Site B 111 Figure 3.14 Concentration of Vapor Phase o,p'-DDT at SH at Site B 112 Figure 3.15 Concentration of Vapor Phase p,p'—DDT at SH at Site B . . . . 112 xi Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 3.23 3.30 Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration of Vapor B . of Vapor C of Vapor of Vapor of Vapor of Vapor of Vapor of Vapor C. of Vapor at CLM at Site D Concentration of Vapor at CLM at Site D Concentration of Vapor at CLM at Site D Concentration of Vapor at CLM at Site D Concentration of Vapor at CLM at Site D Concentration of Vapor at CLM at Site D Concentration of Vapor at CLM at Site D Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Kelthane o,p'—DDE p,p’-DDE o,p'-DDD p,p’-DDD o,p’-DDT p,p’-DDT Kelthane o,p'-DDE p,p'—DDE o,p'-DDD p,p'-DDD o,p'—DDT p,p'-DDT Kelthane All Analytes for South Haven Site A mi 113 114 114 115 115 116 116 117 118 118 119 119 120 120 121 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 3.41 3.42 3.43 Vapor Phase. All Analytes Vapor Phase. All Analytes Vapor Phase. All Analytes Vapor Phase. Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site Concentration at SH at Site for for for xiii South Haven Site B South Haven Site C Coloma Site D o,p’-DDE Particulate p,p’—DDE Particulate o,p'-DDD Particulate p,p'-DDD Particulate o,p’-DDT Particulate p,p’-DDT Particulate Kelthane Particulate o,p’—DDE Particulate p,p’-DDE Particulate o,p’-DDD Particulate p,p’-DDD Particulate o,p’-DDT Particulate 122 122 123 123 124 124 125 125 126 126 127 128 128 129 129 130 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 3.53 3.55 Concentration of at SH at Site B. Concentration of at SH at Site B. Concentration of at SH at Site C. Concentration of at SH at Site C. Concentration of at Site C. Concentration of at Site C. . . . Concentration of at SH at Site C. Concentration of at SH at Site C. Concentration of at SH at Site C. Concentration of at CLM at Site D Concentration of at CLM at Site D Concentration of at CLM at Site D Concentration of at CLM at Site D Concentration of at CLM at Site D Concentration of at CLM at Site D Concentration of at CLM at Site D mv p,p’-DDT Particulate Kelthane Particulate o,p’-DDE Particulate p,p’-DDE Particulate o,p'-DDD Particulate p,p'-DDD Particulate o,p'-DDT Particulate p,p'-DDT Particulate Kelthane Particulate o,p’-DDE Particulate p,p'-DDE Particulate o,p'-DDD Particulate p,p'-DDD Particulate o,p’—DDT Particulate p,p’-DDT Particulate Kelthane Particulate 130 131 132 132 at SH 133 at SH 133 134 134 135 136 136 137 137 138 138 139 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 3.63 3.65 A11 Analytes for South Haven Site A Particulate. All Analytes for South Haven Site B Particulate. All Analytes for South Haven Site C Particulate. . . . . All Analytes for Coloma Site D Particulate. Sum of Site A Sum of Site B Sum of Site C Sum of Site D Site A All Analytes for South Haven Particulate & Vapor Phase All Analytes for South Haven Particulate & Vapor Phase All Analytes for South Haven Particulate & Vapor Phase All Analytes for South Haven Particulate & Vapor Phase Correlation of Temperature with Concentration of EDDT. Site B Correlation of Temperature with Concentration of ZDDT. 140 140 141 141 142 142 143 143 144 145 Temperature Correlation with Concentration of ZDDT for all air samples. Average Vapor Phase DDT & Metabolites Dispersion for All Sites at SH & CLM Average Particulate Phase DDT & Metabolites Dispersion for All Sites at SH & CLM. Site A DDT & Metabolite Ratios for Vapor + Particulate. Site B DDT & Metabolite Ratios for Vapor + Particulate 145 146 147 150 .151 Figure Figure Figure Figure Figure Figure Figure .78 .79 .80 .81 .82 .83 .84 Site C DDT & Metabolite Ratios for Vapor + Particulate Site D DDT & Metabolite Ratios for Vapor + Particulate . . . All Sites DDT & Metabolite Ratios Amount Site A Amount Site B Amount Site C Amount Site D of Sum of DDT Moving per Day. . of Sum of DDT Moving per Day of Sum of DDT Moving per Day . . . . . . of Sum of DDT Moving per Day off off off off .152 .153 .154 .156 .156 157 157 Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix LIST OF APPENDICES SOP FOR DETERMINATION OF NICOTINE IN TOMATO, POTATO PEEL,, EGGPLANT, AND GREEN PEPPER. . . . . . . . . . . . 67 SOP FOR DETERMINATION OF PYRETHRUM IN POTATO. . . . . . . . . . . . . . . . . 69 SOP FOR DETERMINATION OF ROTENONE IN LETTUCE & TOMATO. . . . . . . . . . . . 71 SOP FOR DETERMINATION OF WARFARIN IN CORNMEAL. . . . . . . . . . . . . . . . 73 SOP FOR DETERMINATION OF Nicotine, Rotenone, Pyrethrum, and Warfarin IN CROPS . . . . . . . . . . . . . . . . . 74 SOP FOR DETERMINATION OF a-Terthienyl, Azadiractin, Ryanodine, and Veratridine (aARV) IN APPLES . . . 75 Method III - DDT, DDD, DDE and Kelthane - Air — Cleaning of Tools. . . . . . 167 Method III — DDT, DDD, DDE and Kelthane - Air - Reagent Preparation . . . . .168 Method III — DDT, DDD, DDE and Kelthane - Air - Extraction of XADZ resins and Quartz fiber filters (QF). . . . . . .170 Method III - DDT, DDD, DDE, AND Kelthane - Soil — Reagent Prep. . . . . . . . .173 Concentration Data Combined for Vapor & Particulate for All Sites . . . . . . 175 .Appendix L — Concentration Data Combined for Vapor Phase for All Sites . . . . . . . . . 179 Appendix M — Concentration Data Combined for Particulate Phase for all Sites . . . 183 Appendix N - Sample Volumes, Pressure, and Temperature Data for all Sites. . . . 187 Appendix O - Soil Concentration Data Site A. . . . 190 Appendix P - Wind Speed and Direction for Sample Days at SH. . . . . . . . . . . . . . 193 Appendix Q - Wind Speed and Direction for Sample Days . . . . . . . . . . . . . . . . .195 Appendix R - Soil Texture and Moisture Content. . .196 xflfi PART ONE DEVELOPMENT OF ANALYTICAL METHODS FOR THE DETERMINATION OF SELECTED “ORGANIC PESTICIDES” INTRODUCTION Natural compounds with pesticidal activity are being considered and used as replacements and enhancements to the present synthetic jpesticide arsenal. Thousands of secondary plant compounds are being isolated and tested for biological activity in ‘Natural Product” Laboratories. Secondary' plant compounds, very' likely, developed. their biological activities 1J1 response 1K) plant 3pests. The toxicological and environmental properties of these novel compounds are many times a mystery due to limited testing. Natural pesticides may be a mixture of several biologically active constituents as in Sabadilla which contains several alkaloids. The mixtures may have synergistic and additive effects that cause major complication to determining absolute responsibility of a biological action to one chemical. The regulatory hurdles to bring a product of this nature to market may be prohibitive if handled as a synthetic pesticide. Natural pesticides play an important role in nature and to humans. Through their manipulation and exploitation humans may find important and effective uses of natural products. Continued research will provide a valuable group of chemicals for known and as of yet unknown uses. The use of cultural pest control techniques known collectively as ‘organic farming” produces food that has a demand by a certain segment of consumers. Organic farming, projected to be responsible for 1.5% of domestic food and fiber production. with annual growth rates of about 20% since 1990 (Mahoney, 1998). Many farmers are adopting organic production methods because of problems with pesticides such as resistance and environmental concerns. Although producers and consumers of organic foods have a variety of motives for their beliefs in this alternative type of farming technology, both are concerned about the possible health hazards of pesticide residues and. believe ‘organic farming” eliminates this risk. 11 popular national magazine ‘Self” wrote an article on organic produce with comments to readers' questions such as this: ‘Organic produce is sold locally and in season, as a result, it tends to be fresher, retaining more “vitamins and.:minerals than conventionally grown. produce shipped from far away. All else being equal, organic tomatoes, for example, are as nourishing as regular ones- minus those pesky pesticides.” (Sullivan, 1998). Conventionally grown produce offers the same nutritional value if brought to market immediately. Organic produce is under more stress than conventional produce due to more disease control and fewer alleochemicals produced as protectants against the pathogens. This article offers scientifically backed information rather than emotional hype. Federal laws concerning ‘organic farming” have been introduced but no action has been taken, so presently there are no national regulations that control this group. December 16, 1997 a draft proposal for organic farming regulations was put out for comment. Agriculture Secretary Dan Glickman received 200,000 comments to the draft proposal. The initial organic regulation proposal for organic farming approved foods from engineered crops, crops that had biosolids applied (municipal sludge), and irradiated foods to fall under the organic label. The majority of comments opposed foods from engineered crops, biosolids application, and irradiated food under the organic food production label. Glickman concluded that those practices would not be included in the organic rule (Glickman, 1998). The public comment to proposed regulations was to tighten restrictions on livestock feed, limit antibiotics, reduce acceptable levels of pesticide residues and give small farmers more authority. A revised national regulation was supposed to come out for comment later in 1998. Regulation for ‘organic farming” have been defined in a few states along with organic grower groups that regulate what may be used to produce ‘organic” food. Most organic growers use the 10% rule, which says a food can be regarded as organic if it has 10% or less residues of the EPA tolerance. This also qualifies most conventional foods as ‘organid’. Organic farming follows a rule that soil must not have had pesticides applied in the previous 36 months. With the ubiquitous nature of DDT, dieldrin, and other organochlorine chemicals, can the soil ever be free of pesticides? Residues of DDT and its metabolites occur in detectable quantities in California soils (Odermatt, 1993). ‘Organic” labels without certification is merely an unverified manufacturer's packaging claim (Smillie, 1999). ‘Certified organic,” does not mean pesticide free, chemical free, minimally processed or more nutritious. A. certified. organic facility'1must. present inspectors with documentation to track the production of the raw agricultural product. The perceptions of organic farming versus high input farming bring concerns for health and economics. Cflaims made about ‘Organic food” include: ‘Organic food” has been grown, without toxic pesticides or artificial fertilizers, grown in soil whose humus content was increased by the additions of organic matter, grown in soil whose mineral content was increased with application of natural mineral fertilizers, has not been treated with preservatives, hormones, antibiotics, etc (Steffan, 1971). The conventional farmer relies on machinery and chemicals to increase productivity and maintain profitability or off farm inputs. The organic farmer and the conventional farmer both must maintain profitability to continue in their businesses. The organic farmer usually receives a premium price for organically grown food, where as the high input farmer produces disease and insect free produce and generally of higher quantity and quality. The approach to crop protection for the farmer has been a systematic one, were the organic farmer will try to rely on biological rather than chemical control when possible. .An example being, in response to a fungal attack on a crop on a conventional farm the control measure will be pesticides. An organic farmer will look at the nutritional status and stress on the crop and its ability to resist the disease to an extent that the yields are not greatly diminished. The organic agriculturist has a philosophy with many commonalties to other organic agriculturists but to confound that they also have many alternative philosophies. The important holistic nature of the organic farm implies interactions between crops, soil, animals, and the social structure of the family. Many of the theories in organic farming have not been stated in clear scientific terms but rather in social and emotional terms. Some of the common elements are balanced crop rotation, green cover, animal byproducts, shallow plowing, no synthetic pesticides, and pest control through biological and avoidance techniques. The conventional farmer also uses similar techniques to the organic farmer with the exception of synthetic pesticide use. The sustainable agriculture movement has many sound practices such as cover crops to supply essential nutrients like nitrogen and provide a natural habitat for beneficial predators. The cover crops such as vetch, peas, and clover also reduce soil erosion and hinders emergence of weeds. When soil under conventional farming has synthetic fertilizers substituted with organic fertilizer the soil maintains better tilth, i.e. texture, and their ability to retain moisture is improved (Steffan, 1971), although organic fertilizers do not insure nominal levels of nutrients. Sustainable agriculture runs been 1J1 response to environmental and social costs that have come from enormous yields from conventional farming such as large petroleum, pesticide, eumi nutrient inputs from. off the farm and this causes reliance on government subsidies and bank loans. Generally organic farmers have smaller farms which rely on farm resources rather than off farm, even though organic farmers also must rely on bank loans to remain profitable. Many of the beliefs of organic farmers have not been scientifically tested. Claims of zero pesticide residues in fresh or processed foods must be viewed with incredulity since Ix) extensive residue studies (n1 active ingredients have been conducted. The assumption that organic chemicals degrade rapidly has a major problem associated with it, in that the applications have no regulated preharvest interval, number of applications, application rates, or formulations or active ingredients. ‘Natural” pesticides may be applied at harvest and at high rates but they are not required to have residue analysis for the active ingredients or metabolites of toxicological concerns. The need for a precise determination of the residues applied to organic products will allow assurance to the consumer that they are truly consuming safe food. This proposed research will investigate the development of analytical methods for the determination of selected ‘organic” pesticides on and in foods. The mere unorthodoxy of organic farming should not be used to automatically reject this technology; but research programs should be conducted to evaluate the magnitude and fate (If the active ingredients found 1J1 ‘natural” pesticides in fresh and pmocessed agricultural products to better understand the safety of ‘organic” foods. OBJECTIVES Objective—I The research. focus ‘was CH1 the development of methodology for detecting residues of natural organic chemicals applied by organic farming techniques. The initial stage involved analysis of natural pesticides, which have published standard residue methods and their integration into a jpossible multi residue scheme. The target chemicals for this stage were nicotine, rotenone, pyrethrums, and warfarin. The hypothesis is that all of the chemicals can be analyzed using one multi residue method to achieve accurate, precise, and ug/g residue values. The null hypothesis is that the chemicals can not be analyzed at sufficiently low levels to determine typical quantities of pesticide on or in food through a multi residue method. Objective-II The second stage of the research involved the development of residue methods for the ‘natural” pesticides for' which there are not available sensitive (ug/g or ng/g) and selective published analytical methods. Analytical methods were developed for the following active ingredients: d-terthienyl (marigold), azadirachtin (neem), ryanodine & dehydroryanodine (ryania), cevine, sabadine, cevadine, and veratridine (sabadilla). The hypothesis was that all of the chemicals can be analyzed using one multi residue method to achieve accurate, precise, and ug/g residue values. The null hypothesis is that the chemicals can not be analyzed at sufficiently low levels to determine typical quantities of pesticide on or in food through a multi residue method. IJHNERAHIHUBIREVUJMN Pesticide analysis involves large quantities of organic solvent use and waste. The focus in recent years has been to reduce laboratory-generated waste and lower the detection limits. Several techniques have been examined to reduce waste and generate lower levels of detection of the target analyte. Supercritical fluid extraction and solid phase extraction have had some success. The supercritical equipment has been expensive and thus inaccessible to some laboratories. Solid phase extraction (SPE) has been used in cleanup steps and this provides some reductbmn in solvent extraction over solvent partitioning. SPE may be used in isolation of analytes from each other and then elution of only a select chemical. ORGANIC FARMING & FOOD INDUSTRY Organic foods have annual growth rates around 20 % (Mahoney, 1998). The market appears to be headed for continued growth. CHganic foods are great for pmocessing because they don't need to be cosmetically perfect as in the fresh fruit market. William Breene, professor emeritus, University of Minnesota says, ‘It should be remembered that IO neither can it be proven that they are healthier nor can they said to be pesticide free”, (Mahoney, 1998). The organic foods account for about 1.5% of domestic food sales and they have had a growth rate of about 20% since 1990 for a total revenue of $4 billion (Mahoney, 1998). The natural foods market has many claims that don't have traditional efficacy testing. A ‘Natural” product KOLESTOP® has ‘phytosterols (plant sterols) that are found. in the fat- soluble fractions of plants and are effective in improving circulating lipid to reduce risk of coronary heart disease. Chemically similar to cholesterol, phytosterols inhibit the absorption of cholesterol. Phytosterol consumption in humans under a wide range of study conditions has been shown to reduce plasma total and low density lipoprotein (LDL) cholesterol. Most studies report no effect of phytosterol administration in high density lipoprotein (HDL) cholesterol or triglyceride levels (Jones, et al.., 1997). This product has many claims without the traditional FDA review. CHARACTERISTICS AND USES OF THE PESTICIDES a-Terthienyl Chemistry and Source: Terthienyls are released from the roots of growing Asteraceaes (marigolds) and have shown a strong nematicidal activity. a-Terthienyl's molecular formula is shown in Figure 1.0. H Pharmacology: Marigolds secrete toxic compounds of a a- terthienyl type into the soil, which kills nematodes. To be effective marigolds must be planted as a solid crop and grown for 90 days to begin secreting d-terthienyl to reduce the nematode population. Marigolds also act as a trap crop. Nematodes enter their roots but are unable to complete their life cycle and die without reproducing. 8 Figure 1.0 a-terthienyl Molecular Structure Formulation: Plant-derived products are not sold commercially for control of nematodes. iflua actual plant can be planted as the controlling agent. .Azadirachtin (Neem) Chemistry and Source: Azadirachtin has been derived from the seeds of the neem tree, Azadirachta indica, which has a wide distribution throughout Asia and Africa (Merck Index, Eleven Edition, 1989). Azadirachtin's molecular formula is shown in Figure 1.1 Pharmacology: The observation that the desert locust did not eat the leaves of the neem tree and another closely related tree species, led to the isolation and identification of azadirachtin in 1967. Since then, azadirachtin has been shown to have repellent, antifeedent, and/or growth regulating' insecticidal activity against a large number of insect species and some mites. It has also been reported to act as a repellent to nematodes. Neem extracts have also been used in medicines, soap, toothpaste CH3 Figure 1.1 Azadirachtin Molecular Structure and cosmetics (Author unknown http://www.calchemico. com/neemnhtml). Toothpaste 'with. neem. extracts has claims that they prevent tooth decay and periodontal disease. Formulation: The most common commercial formulations of neem is Neemix, which is available for fruit tree, and lists leafminers, mealybugs, aphids, fruit flies, caterpillars and 13 psylla. as insects that will run: feed. on ‘treated trees. Azadirachtin has shown good activity against spotted tentiform leafminer in tests in past years, but the formulation that was available at that time was somewhat phytotoxic. In insecticide trials in 1992 with another azadirachtin product called Margosan-O, the product showed good activity against leafhopper. Margosan-O does not include a label for fruit crops, however. Azadirachtin has a relatively short environmental life and. a low :mammalian toxicity (rat oral LDw >10,000 mg/kg). It can be used up to and including the day of harvest, with reentry permitted without protective clothing after the spray has dried. It has toxicity to fish and aquatic invertebrates Nicotine Chemistry and Source: Nicotine has a tertiary amine composed of pyrrolidine and pyridine rings found in dried leaves of Nicotiana tabacum and N. rustica. The extract is a colorless to pale yellow, oily liquid that is very hygroscopic and turns brown on exposure to air or light. Nicotine has two sz, pKl at 6.16 (15 ° C) and pKz at 10.96 (Merck Index, Eleven Edition, 1989). Nicotine forms salts in acids and double salts with many metals and acids. Nicotine’s molecular formula has been given in Figure 1.2. Pharmacology: .Nicotine functions mainly as an1 excitatory l4 stimulus in the central and peripheral nervous system. 'Transmission. at the :neuromuscular junction. is associated with increased cation conductance. K+ is allowed to leave the post-junction. area as rkf enters. The peripheral nervous system nicotine reacts like acetylcholine at ganglion and neuromuscular sites. Nicotine's toxic action is due to both stimulation and blocking of autonomic ganglia and skeletal muscles at the neuromuscular junction. Figure 1.2 Nicotine Molecular Structure Formulation: Black Leaf 40 is a 40% nicotine sulfate formulation that may be sprayed or added to hydrated lime and spread as a dust (Cook, 1998). Pyrethrum Chemistry and Source: This compound will be produced in the flowers of Chrysanthemum cinerariaefolium and was the forerunner of iflua synthetic pyrethroid insecticides. The active insecticidal ingredients are obtained from the flowers. There are four active ingredients, Cinerin I, 15 Cinerin II, Jasmolin I, Jasmolin II (Casida, 1995). Pharmacology: Pyrethrum run; a relatively non—toxic relationship to humans and other mammals, although the dust produces allergy attacks in people who are allergic to ragweed pollen. The acute oral LDw ranged from 750 to 1000 mg/kg (Extoxnet, 1994). Pyrethrum has been shown to be toxic to fish, but ‘relatively” non-toxic to honey bees. Natural pyrethrums are contact poisons which act on the nervous system to cause a ‘knockdown” that causes the insect not to be able to move or fly away. To assure a lethal dose, pyrethrum was often sprayed with other synergists and insecticides. Pyrethrum's molecular formula is shown in Figure 1.3 Formulation: There are not nearly as many commercially available formulations of this chemical as there are for rotenone, but it has availability as an emulsifiable concentrate, in combination with rotenone, or alone as a wettable powder. Pyrethrum cost the least expensive of these four materials. Depending on the rate 16 R = CH3 for Pyrethrin l R = COOCH3 for Pyrethrin ll HZCH=CHCH=CH2 CH3 '1 CO H CH3 Figure 1.3 Pyrethrum Molecular Structure used, it may be less expensive than many synthetic insecticides. It also may be synergized by piperonyl butoxide (PBO). Pyrethrum is labeled for use against a large number of pests. An addendum to the label for one formulation. of pyrethrum. showed. it to be moderately to highly' effective (6l-lOO% control) against the following pests cu? fruit: grape leafhopper, potato leafhopper, leaf curl plum aphid, blueberry flea beetle, blueberry thrips and blueberry sawfly. It may be used efficaciously against cranberry fruitworm and also will be quickly broken down in the environment and may be used up to and including the day of harvest. Rotenone Chemistry and Source: Rotenone has been extracted from the root of various plants of the Derris or Lonchocarpus species from Southeast Asia, Central and South America. The molecular formula for Rotenone, CBHzfik ({2R-(2a,6aa,12aa)]- l7 1,212,12a-tetrahydro-8,9-dimethoxy-2-(1-=methylethenyl)[1] benzopyranol[3,4-b]furo[2,3—h][1]benzopyran-6(6aH)—one) has been determined. Derris root has long been used as a fish poison and its insecticidal properties were known to the Chinese long before it was first isolated in 1895. Formulated product may be available as at least 118 formulated products from a large number of manufacturers. Rotenone's molecular formula may be found in Figure 1.4 Pharmacology: Rotenone's selectivity has been found to be a non-systemic contact and stomach poison. Site of action may be in electron transport chain (The Pesticide Manual, Tenth Edition, 1994). It may be synergized by the addition of PBO, which also comes from botanical material. Rotenone is less expensive than synthetic insecticides, but is moderately priced for a botanical. It was the most commonly mentioned of the botanicals in pre-synthetic literature and has shown that it was at least somewhat effective against a large number of insect pests. These include: pear psylla, strawberry leafroller, European corn borer, European apple sawfly, cherry fruit fly, apple maggot, cranberry fruitworm, raspberry' fruitworm, pea aphid (with similarity to rosy apple aphid), European red mite and two—spotted spider mite, codling moth, plum curculio, Japanese beetle and tarnished plant bug; 'Unfortunately, Rotenone has shown toxicity to ladybird beetles and predatory mites. But, it has been shown 18 to be non-toxic to syrphid flies that feed 0n aphids, and to honeybees. Rotenone is rapidly degraded in sunlight, lasting a week or less. Of the botanicals mentioned here, rotenone has the most toxicity to humans and other mammals. The acute oral LDM, ranges from 12-2000 mg/kg in various animals (Extoxnet, 1993). In small doses it may 1x2 irritating or numbing to mucous membranes. Use as a potent piscicide has been known because of the high toxicity to fish, having been commonly used as a fish poison. Toxicity has also been shown to occur in birds and pigs. Formulation: A recent regulatory development CH3 Figure 1.4 Rotenone Molecular Structure illustrates the tenuous situatMMi of many manor-use materials and may end up rendering rotenone unavailable for use on many crops. According to a USDA news release and as quoted in the Federal Register (July 20, 1995), the Rotenone Task Force has announced that it plans to delete all the agricultural uses from rotenone labels because of the cost of reregistration; these uses include all tree 19 fruits and small fruits. The registrants plan to madntain rotenone uses for fish control and flea/tick/mite control on dogs and cats. They will reconsider their plans for deletion if someone shows a willingness to develop the necessary data for reregistration. Ryania Chemistry and Source: A product of the roots and stems of Ryania speciosa of Trinidad, ryania acts as both a stomach and contact poison on target insects. It was found that Ryanodine was the most expensive of the materials covered in this research, and also was not as readily available as rotenone or pyrethrum. Ryanodine, the active ingredient, was formulated as a wettable powder and labeled for use against the codling moth in apples. It has also shown to be toxic to the European corn borer and may control cranberry fruitworm. In tests it provided excellent control of a pest complex comprising codling :moth, oriental fruit moth and lesser appleworm. It also controlled aphids, white apple leafhopper and spotted tentiform leafminer. It has been shown to be more persistent than rotenone or pyrethrum and also more selective. Generally' it has not been found to be very harmful to pest predators and parasites, but has been shown to be somewhat toxic to the predators Atractotomus maliand 20 Diaphnocoris spp. It may also be used up to 24 hours before harvest. Ryania's molecular formula is shown in Figure 1.5. Pharmacology: Ryania's insecticidal properties act as a stomach poison and ryania often depresses the insects feeding initially, so that it undergoes a long period of inactivity before death. It has residual properties longer than the other botanicals. Relative to rotenone, ryania has a moderate toxicity in acute or chronic oral toxicity Figure 1.5 Ryania Molecular Structure testing done in mammals; this was partly why much attention has been given to this insecticide in recent years. The acute oral LDSO of ryania ranges from 750 to 1200 mg/kg, less toxic than rotenone and slightly more toxic than pyrethrum. It is toxic to fish. Formulation: Ryan 50 a product of the roots and stems of Ryania speciosa of Trinidad. It has also been shown to be toxic to the European corn borer and may control cranberry fruitworm. In recent tests it provided excellent control of a pest complex comprising codling moth, oriental fruit moth 21 and. lesser' appleworm. It. also controlled. aphids, white apple leafhopper and spotted tentiform leafminer. Rotenone has been. found. to 1x2 more persistent than rotenone or pyrethrum and also more selective. It generally has not been harmful to pest predators and parasites, but it has been found to be somewhat toxic to some minor predatory mites. It may be used up to 24 hours before harvest. Sabadilla Chemistry and Source: The source of sabadilla was found to be the seed of a.txopical lily, veratrum Sabadilla and V3 Officinale that contains several toxic alkaloids (Grieve, 1995). The alkaloids of toxicological importance are cevadine, veratridine, cevine, and sabadine. Sabadilla’s molecular formula may is shown in Figure 1.6. Pharmacology: In previous articles about botanical insecticides printed in Scaffolds (Kain, 1995), it was stated that sabadilla was not toxic to honeybees. However, the information provided by different sources since then has been ambiguous. Some say, that it is relatively non-toxic to honeybees and others (including the manufacturer) say it has been found to be toxic. The confusion may lie in the fact that sabadilla has shown to be toxic to honeybees on contact, but without any residual activity. In the interest of playing it safe (especially given the current state of 22 bee health), it would probably be best to consider sabadilla a hazard to honeybees and follow all necessary precautions to prevent their exposure to the material. Sabadilla has been shown to be less toxic to mammals than rotenone or pyrethrum; the acute oral LDw was determined to be greater than 4000 mg/kg. OCH3 CH30 Figure 1.6 Sabadilla Molecular Structure Formulation: There are very few commercial formulations of this material. It may be found as a dust that may also be added to water and sprayed, but clogging of the nozzles may occur. It will control potato leafhopper and is somewhat effective. Sabadilla mixed with lime or sulfur or dissolved in kerosene to provide a base to facilitate application (Douglas, 1996). Sabadilla was found to be moderately priced for a botanical (similar to rotenone). It has little 23 effect on predators/parasitoids, except for the predatory mite Typholdromus pyri, to which it was extremely toxic in recent tests by Joe Kovach (Kain, 1995). Sabadilla may be used up to 24 hours before harvest. Apple is the only deciduous tree fruit crop specifically' mentioned on the label of the one product found registered for use in New York State. Warfarin Chemistry and Source: Warfarin has a colorless, crystalline structure and a formulation of CAHL504(4-hydroxy-3-(3-oxo- l-phenylbutyl)-2H-1-benzopyran—2-one). The anticoagulant ability of warfarin was discovered and reported in 1944 and by 1952 was registered for use in the United States. Warfarin’s molecular formula is shown in Figure 1.7. Pharmacology: Warfarin inhibits normal function of Vitamin K in blood coagulation. With continuous exposure severe bleeding and death occur (Warfarin, 1995). The LDso of various animals ranged from 1 tx> 1200 mg/kg (Extoxnet, 1995). Warfarin poisoning symptoms include mucus membrane bleeding, hematomas in joints, cerebral hemorrhage leading to paralysis and death. 24 O / CHCHZCOCHa 0H CsHs Figure 1.7 Warfarin Molecular Structure Formulation: The compound comes as ready-to-use bait, concentrate, powder, liquid concentration, and various powders and dust formulations. 25 .ANALYTICAL METHOD DEVELOPMENT FOR LIQUID CHROMATOGRAPHY Reverse Phase Liquid. Chromatography' (RPLC) has been modified by many different sorptive materials and mobile phases since the initial chromatography columns. The separation. of analytes by' differential migration from a narrow application zone in a porous sorptive medium. Column chromatography, thin layer chromatography (TLC), and paper chromatography are subdivisions of solution chromatography based on the sorptive material. The analytes move due to solution-solid adsorption and partition distributions. Analytes move by zone migration which has a fraction of the of the mobile phase velocity, R. At the molecular level each molecule adsorbs to the stationary phase and its migration will be stopped while other molecules move on. Each molecule goes through a stop and go path but statistically all the molecules that are the same are moving at the same theoretical velocity. Since each molecule has a specific velocity which includes ta (time adsorbed) and ta (time desorbed) then R is shown by: R = (ta)/( ta + td) The effect of separation depends on the solute migration to regions of lower concentration, eddy diffusion or differential path tortuosity, and the propensity of a gaussian concentration profile to pmecede. (Hue result of 26 the above phenomena is an initial separation of analytes and also a band broadening of individual analytes. Temperature can be used to effect the resolution of compounds because diffusion has temperature dependency. Adsorption depends on the phenomenon of molecule being held on the surface of a solid support. A molecule adsorbed to the surface of an absorbent will have a potential energy, Pe, due to intermolecular forces holding it there, and a kinetic energy, Kg, due to vibrational movement. Dfluni Ke exceeds Pe the molecule will leave the surface and the molecules move on. EC depends on mass, shape, and temperature which contribute to the band broadening effect seen in chromatography. Adsorption is a reversible process and characterized by weak forces. Examples of adsorption chromatography are columns which utilize adsorbents such as calciuni carbonate, silica gel, aluminum. oxide, charcoal, etc. and ether, carbon tetrachloride, alcohols, acetone, and water as solvents. Partitioning will determine the equilibrium distribution of an analyte between two immiscible solvents. Partition chromatography has been seen with column, paper, and thin layer chromatography. The reverse phase notation refers 1x3 the solid phase part of aa non-polar stationary compound such as silicone oil, Cm, or paraffin. The mobile phase will be relatively polar, as compared to the 27 stationary phase, solvent such an; methanol, acetonitrile, and water mixtures. Reverse phase separations are useful for nonpolar hydrocarbons because the nonpolar compounds will differentially be retarded as they move with the mobile phase. In a typical Cm column the anayltes partition with the column’s octadecyl molecules through Van der Waals forces. The more polar compounds travel through the column faster due to weaker interaction with the octadecyl column packing material. A typical order of elution from the column would be strong Lewis acids (carboxylic acids), weak Lewis acids (alcohols, phenols), strong Lewis bases (amines), weak. Lewis bases (ethers, aldehydes, ketones), permanent dipoles (CHCL3), induced dipoles (CCL4), then aliphatic hydrocarbons. The mobile phase for RP-Cm column should typically be a mixture of water with methanol or acetonitrile. Varying the water content of the mobile phase can optimize the separation. As the organic content of the mobile phase increases the retention time of the analytes is decreased. The organic molecules spend more time in the organic phase as polarity decreases. Chromatography deals with the separation of chemicals, which then need to be detected by another technique. Many suitable detectors are available such as spectrometers, mass detectors, and refractive index detectors. The molecule's chemical and physical characteristics provide modes of 28 detection and from them determine the appropriate means of detection. The molecules absorb in the UV region of the electromagnetic (EM) spectrum makes them suitable for UV detection. Electromagnetic waves, as its name implies, are composed of two components: a oscillating electric field anui a oscillating magnetic field mutually perpendicular to each other. The two waves are in phase. The EM spectrum has wavelengths from 10"14 to 107 m and the UV spectrum from 10‘8 (vacuum UV-lO nm) to 3.5 x 10” m (Near UV-350 nm). EM radiation composition will be of discrete photons of energy. The photon energy can be quantized by the equation: E = hV’= hC/A E represents energy in joule (J), V for frequency (5”), 11 for Planck’s constant (6.63 x 10'34 J5), A for wavelength hm), and c for the speed of light (3 x 108 m/s). Spectroscopy deals with the interaction of EM radiation with sample material. As the radiation enters into the sample the beam may reflect, refract within the sample, scatter, absorb the radiation or be transmitted through the sample. A sample that was stimulated by the input of energy in the form of EM radiation will have absorbed energy and then be iJiaa higher energy state or cause expulsion of an electron and be ionized. The results of photon absorption 29 by the sample leads to a reduction in the intensity of the EM radiation being transmitted through the sample. Relaxation of an excited species can occur by emission of a photon (photoluminescence) or release of kinetic energy. 'UV radiation absorption causes electronic excitationd The actual amount of energy absorbed for a change in electrons Energy State may be related by: (E1 "' E0) = hV In organic molecules there are three general types of electrons; sigma bonded electrons, pi electron bonds, and n electrons. Sigma electrons form high-energy bonds and [RI radiation will not have sufficient energy to excite sigma bonded electron. Sigma bonds are found in saturated bonds and this feature makes these compounds ideal solvents for UV absorption spectroscopy. Pi bonded electrons are found in aromatic and conjugated compounds. N electrons are nonbonding electron pairs found in N, O, S, or halogen compounds and these electrons can be excited by UV absorption” The absorbed energy necessary to cause an electronic excitation varies slightly due the different vibrational and. rotational. molecular energy levels which leads to an absorption band where an atomic spectra is a sharp line. 30 For an electronic transition to occur the electron must not change its spin orientation as it goes from an unexcited state to an excited one. A compound that does not change spin orientation is called a singlet and one that changes is a triplet. The triplet conversion is a forbidden transition and occurs very rarely. When a molecule emits energy from the singlet state to ground it is referred to as fluorescence. Singlet-triplet transitions occur rarely and they are called inter system crossing. When an electron return from a triplet state to ground a phosphorescence emission occurs. A.singlet to ground occurs in about 10'8 seconds and triplet to ground 10’2 to 100 seconds. To determine if a molecule will absorb in the UV range a transition from a bonding or lone-pair orbital to an unfilled non-bonding or anti-bonding orbital must be available. The chromophore (electrons responsible for the absorption) :must kxa identified. Some examples of chromophores are ketones that have a n to 1t* transition which are examples of a lone-pair of electrons on oxygen goes to the an anti-bonding orbital. UV spectroscopy can be used for both qualitative and quantitative analysis. For qualitative identification a scan is done and compared to knowns for shape and specific spectral absorbance areas. 'UV absorption spectra overlap considerably for different compounds so this is not a 31 decisive technique for identifying a compound. Once the compound has been identified UV may be a powerful tool for quantitative analysis. Absorption follows definite physical laws. Transmittance has been defined as the ratio of I1 (intensity of radiation leaving the sample) to I0 (intensity of radiation entering the sample): T: I1/Io and may be related to absorbance by: A = -log T = abc Absorptivity will be represented by a, b which will be path length, and c the concentration. These relationships show the logarithmic relationship between transmittance and concentration and the linear' relationship between absorbance and concentration. These relationships hold true for dilute solutions. UV absorption is sensitive 100 ppb to 1 ppm. 32 GENERAL CONSIDERATIONS FOR.ANALYTICAL METHOD DEVELOPMENT IMethod. development will involve finding common solvents, columns, and detection methods. The method development process shall 1x3 approached according tx> the following sections: 0 Establish Criteria for the Method 0 lMethod Development The establishment of criteria will pertain to the proposed MDL of about 1.0 ppm being sought. The recoveries will be done with a variation in precision of less than 30% of the mean and accuracy between 60% and 130%. The validation will determine the working concentration range, to include the level of quantitation (LOQ) and approximately 10 X this value to encompass anticipated residues found. The standard curve range will be determined for general shape, ie. linear, exponential, or polynomial fit and the upper range of the curve. The acceptance criteria for goodness of fit of the curve, I}, will be greater than 0.95. A minimum validation data set will include: 0 Two samples at the MDL run concurrently with control samples. 0 Two samples fortified at the maximum concentration of the validation range run concurrently with control samples. 33 0 One reagent blank. The method will include lists of equipment, materials and reagents, the stepwise procedure used to execute the method, a summary of the validation results, the appropriate validation data, representative chromatograms and a discussion of the results. When considering extraction of the sample the technique will consider the nature of the sample. To determine the correct extraction method the sample matrix and analytes are considered. When using a SPE column the analytes interact. with. the jpacking' material and. are preferentially retained or eluted. The analyte will absorb to the packing' material and the contaminants will pass through or alternately the analyte will pass through and the contaminates will be retained on the column. Several packing phases are available for specific purposes. Normal Phase, Reversed. Phase, Ion-Pairing, and Ion-Exchange packings are available. Selection of the proper SPE tube involves knowing: 0 Degree of contamination 0 Sample complexity o .Analyte concentration range 0 .Analyte solubility in solvents 0 Strength of analyte/sorbent interaction 34 0 Sample volume SPE tubes come in several sizes, from 1 ml to 60 ml, to assure optimal sample extraction and cleanup capabilities. SPE tubes are conditioned prior to sample introduction to activate the packing material according to the packing material and compounds of interest. Sample volumes from microliters to liters may be added to the SPE tube. Reverse phase packings lose their extraction efficiency and sample recoveries go down as the volume of sample increases because the packing material loses its activation brought on by preconditioning the column. The column was washed after sample introduction with a solvent in which the analyte has a low solubility. The wash volume typically should be about the same volume as the tube. The analyte was then eluted off the column with a solvent that has a strong affinity for it. The sample was then reduced or brought to volume for injection onto the analytical instrument. 35 RESEARCH METHOD I - NICOTINE, WARFARIN, ROTENONE, AND PYRETHRUM The initial research was initiated by looking at the individual pesticides, their chemical, physical properties, and referenced methods found in Appendices A-D. These four chemicals were chosen because of their frequency of use in organic farming systems. Solubility was the first parameter looked at. The analyte of interest was put into individual test tubes and different solvents were added to evaluate the solubility. The results are given in Table 1.0: Each test tube was shaken and then evaluated for precipitate or phase separation in the tube. All four chemicals showed solubility in methanol, acetonitrile, and acetone. Reference data agreed with the experimental data. The choice of the solvent was made also in conjunction with mobile phase (HPLC) and gas chromatography (GC) compatibility. Methanol was chosen as the solvent because of solubility of the analyte, ultraviolet (UV) and visible absorption properties, and volatility concerns. 36 Table 1.0 Solubility of Solvents Chemical Methanol .Acetone Acetonitrile Water Nicotine Soluble Soluble Soluble Soluble Pyrethrum If Soluble Soluble Slightly Insoluble & II Soluble Rotenone Soluble Soluble Slightly Slightly Soluble Soluble ‘Warfarin Soluble Soluble Soluble Soluble (Alkaline) Obviously’ the material must be soluble in the solvent, invisible to the detector and nonreactive with the method. Additionally, volatility of the solvent must be considered because it could have detrimental effects on the concentration of the standards due to evaporation over time. The maximum absorbances of solvents under consideration are given in Table 1.1. Table 1.1 Maximum Absorbance at Specific Wavelengths (nm) Solvent nm/A nm/A nm/A nm/A nm/A Mathanol 205/1.0 225/.16 250/.02 BOO/.005 400/.005 .Acetontrl 190/1.0 205/.1 225/.01 250/.005 350/.005 Water 254/.001 - — — - .Acetone 330/1.0 340/.06 350/.01 375/.005 400/.005 Methanol and acetonitrile had the most favorable UV absorbance maxima (ie. below 205 nm). Volatility was looked at in terms of solvent storage of standards. Table 1.2 shows the vapor pressure of 37 solvents under consideration. Acetonitrile and methanol had favorable vapor pressures for decreasing solvent loss in standards over time in storage. Hexane and other nonpolar solvents were not used because the analytes were insoluble in them. Table 1.2 Vapor Pressure Solvent vapor Pressure (Torr) Methanol 125 Acetonitrile 88.8 @ 25° C .Acetone 184.5 Hexane 120 solvents under consideration. Acetonitrile and methanol had favorable vapor pressures for decreasing solvent loss in standards over time in storage. Hexane and other nonpolar solvents were not used because the analytes were insoluble in them. The HPLC, GC, and capillary electrophoresis (EC) chromatographic aspects of the method were investigated, with particular attention to compatibility with the instruments. The flame ionization detector (FID) was considered for GC applications since it detects most carbon based molecules. Each analyte showed minimal response at greater than 100 ppm levels. This level was too high for residue work, which should be 10‘2 to 10” times the 100 ppm 38 level. Nicotine was easily detected using the nitrogen/phosphorus detector (NPD) on the 5890 Hewlett Packard GC with a Carbowax column. EC was abandoned as a chromatographic technique due to overlapping peaks and drifting during runs. The analytes being pH dependent were influenced. by the temperature, which was not held constant. EC could be promising if only the slightly polar chemicals, Warfarin and Nicotine were considered. HPLC was investigated as to mobile phase compatibility with the analyte and analyte separation from the mobile phase and other analytes, initially along with the wavelength for combined best detection. Ultraviolet (UV) and visible (Vis) absorbance maximas were determined for each analyte using a Gilford UV/Vis ‘Spectrometer. The absorbance maximas are given in Table 1.3. The wavelength was given relative to the strongest absorbing wavelength as 1.00. The spectra for each individual analyte were taken to determine optimum. wavelength” The choice of a common wavelength for all four chemicals was determined as a summation of absorbances over all the analytes at a particular wavelength. TNue most favorable wavelength for the simultaneous detection of all the analytes was 280 nm. 39 Table 1.3 Absorbance Maximas Chemical 7» (nm) / A 2» (nm) / A 1 (nm) / A Nicotine 215 / 0.95 260 / 0.93 265 / 1.00 Pyrethrum I 209 / 0.89 222 / 1.00 240 / 0.89 Pyrethrum II 209 / 0.89 222 / 1.00 240 / 0.89 Rotenone 226 / 0.95 241 / 0.94 290 / 1.00 Warfarin 215 / 1.00 285 / 0.40 309 / 0.41 They are given in Table 1.4. The 280 nm was selected because the sum of the absorbances was 1.72, which was the greatest sum. Various solvents were tried to optimize the resolution between the analytes and achieve the best peak shapes. The problem with optimizing was achieving satisfactory results for all four analytes. Nicotine has a basic character with pK1 at 6.16 (15 ° C) and pKz at 10.96 while warfarin has a slightly acidic molecule. As long as the mobile phase was organic with a neutral pH the molecules 40 Table 1.4 Analyte Absorbance Sums at Given Wavelengths Chemical Retention 227 nm 254 nm 280 nm 295 Time nm Nicotine 1.75 0.17 0.67 0.67 0.17 Pyrethrum I 3.00 0.12 0.04 0.02 - Pyrethrum II 3.30 0.04 0.06 0.06 - Rotenone 2.90 0.40 0.40 0.17 0.20 Warfarin 1.85 0.30 0.16 0.80 0.17 Response Sum 1.03 1.33 1.72 0.54 stayed non-ionic. Ionic molecules would not be retained by the C18 column and be eluted with or before the solvent peak. Methanol was found to be the best solvent for the standards and as the mobile phase for the HPLC due to all the chemicals were soluble in it and the solvent front on the HPLC did not interfere with the analytes as they came off the detector. When the solvents and mobile phases were mixed the column would have conditioning problems and not recover to a stable baseline in time for the analyte to elute. Buffered mobile phases such as a phosphate buffer at ~pH 10 worked well with nicotine but not warfarin. The desire to use one mobile phase to alleviate the need for reconditioning the column. between analyte injections was chosen, except for Nicotine had to be chromatographed 4i separately with a Nafiuxn buffered acetonitrile mobile phase. The three analytes, Warfarin, Rotenone, and Pyrethrum had retention times of 1.72, 2.92, and 3.00 minutes respectively. Nicotine was determined with Nafiflmh buffered acetonitrile mobile phase to maximize recoveries and will be considered at later. The resolution between peaks may be found in Table 1.5 and calculated by the following formula: R8 = (v2 - v1)/{(w2 + w1)* 0.5} Peak resolution has the ratio of the difference between two peaks retention times, vm of analyte n divided by the average, wn peak width for peak n. The resolution between Warfarin and Rotenone represent totally resolved peaks in that the tangents of the peaks to the baseline of the chromatogram do not intersect, i.e. non overlapping lines. This is not the case with Rotenone and Pyrethrum. The use of different wavelengths provides for sufficient separation via wavelength and chromatographic separation. Rotenone detection was at 254 nm and Pyrethrum at 227 nm for increased sensitivity after initial method development work at 280 nm. Part of the problem with separation of Rotenone and Pyrethrum has to do with the fact that Pyrethrum has several different fractions. The slightly 42 different molecular formulas had subtle differences on the partitioning while traveling through the column. The slight differences brought about broadening of the analytical peak which in turn cause peak overlap. values of Rs 2 1 represent ‘totally separated” peaks. Table 1.5 Resolution between Peaks Chemical Retention wavelength. ‘Width Resolution Time (min) (mm) (min) R‘ Warfarin 1.72 280 0.32 - Rotenone 2.92 254 0.64 2.50 Pyrethrum. 3.00 227 0.79 0.11 Column efficiency can be calculated by determining height equivalent to one theoretical plate (HETP). HETP was determined by dividing the length of the column by the number of theoretical plates. Theoretical plates represent a concept of the number of partitioning steps an analyte would go through as it traverses the length of the column. The larger N would represent the better efficiency because more partitioning occurred. Theoretical plates were determined for the analytes by the following formula: 43 N = 16(tr/Wb)2 In the above formula t, represents retention time and Wb equals the peak width at the baseline. The values of HETP and N is shown in Table 1.6. Warfarin and Pyrethrum had the best HETP, which imply that, the choice of mobile phase and column provide good chromatographic conditions for these analytes. The column was a Brownlee Laboratory reverse phase C18 (RP-C18) Table 1.6 Theoretical plate and HETP values Chemical Retention Peak Column Theoretical HE TP Time Width length Plates (min) (min) (mm) Warfarin 1.72 0.48 250 364.17 0.69 Rotenone 2 . 92 1 . 94 250 55 . 10 4 . 54 Pyrethrum 3.00 0.81 250 344.77 0.73 Spheri-IO column, 250 mm X 4.6 mm. A Waters 501 HPLC pump with a Rheodyne 7125 injector was used for the analysis. Detection was with a Milton Roy variable wavelength Spector Monitor® 3100 model connected to a Spectra—Physics SP4270 integrator. The RP-C18 column was chosen because of its versatility with many organic compounds and ease in functioning over a pH range of pH 2 to pH 7. A concern with this column was degradation by hydrolysis of the silica matrix. It also degrades under basic conditions that are 44 preferred for the Nicotine analysis. Once the analytes are separated from each other the concentration was determined from the UV absorbance measurements. QUANTITATION The analyte was isolated (by HPLC) and put into a solution. with a IKHI UV" absorbing solvent. Calibration curves are prepared by plotting absorbance vs concentration or transmittance \ns concentration and.ai linear regression line determined. and tflua unknown concentration calculated from the regression line. UV detection can be coupled with chromatography using a flow through cell and isolation of different fractions (If the sample. If" background interferences exist extracting the sample without an analyte would allow for subtraction of unwanted absorbance. Nicotine was run with a basic NazHPO4 buffered acetonitrile (60:40) mobile phase on a DevelosilTM ODS-UG Speri-S, 150 1mm X (4.6 mmi column. The DevelosilTM ODS-UG. provides stability at high pH values. The mobile phase was pH adjusted to about pH 10. The pump, detector and integrator are the same as used for the previous three analytes. Standard curves were run for all of the analytes, they are shown in Figures 1.16 — 1.19. The results of the linear regression are given in Table 1.7. The regression line does 45 not use (0,0) as a point in the equation the line. Each line was determined with three to four standards within a typical concentration range to be used for validation. Nicotine was done with a NafiHKh buffered acetonitrile mobile phase at ~ pH 10. IN: {#1 10 Nicotine will have some ionization due to the pKns. The single ionization will be to the extent that of A/HA” will be 0.1096 or about 90% ionized. The double ionization will be to 0.014% double ionized. The extraction was started with individual chemicals and then when consistent recoveries were obtained the method was combined with another chemical. Table 1.7 Linear Regression for Standards Chemical y-intercept x-coefficient rf Nicotine 1000000 971232 0.975 Pyrethrum. —95663 65896 1.000 Rotenone 430717 129359 0.997 Warfarin 4046 147895 1.000 Calibration curves were prepared by plotting absorbance vs concentration and a linear regression line determined. The unknown concentration was then calculated from the regression line. UV detection can be coupled with the chromatography using a flow through cell. If 46 background interferences exist extracting of unwanted absorbance. Standard curves were run for all of the analytes, they are shown in Figures 1.8 - 1.11. The results of the linear regression are given in Table 1.7. The regression line does not use (0,0) as a point in the equation line. Each line was determined with three to four standards within a typical concentration range to be used for validation. The extraction was started with individual chemicals and when consistent recoveries were obtained another analyte would be added to the method to coextract. Nicotine Standard Curve (Naomamm Figure 1.8 Nicotine Standard Curve 47 Pyrethrum Standard Curve 8000000 6000000 4000000 2000000 Absorbance Figure 1.9 Pyrethrum Standard Curve Rotenone Standard Curve Absorbance PPm Figure 1.10 Rotenone Standard Curve 48 Absorbance Warfarin Standard Curve 2000000 1 500000 1 000000 500000 0 ppm Figure 1.11 Warfarin Standard Curve Warfarin and Rotenone were the first two chemicals to be extracted together. The method followed may be found below: 1. 2. Weighed 10 g of sample into a separatory funnel. Added 50 ml of hexane and 30 ml of saturated NaCl solution. to the separatory funnel. Shook for two minutes and let phases separate. Put the hexane layer through 5 g of NaZSO4 and a glass wool plug filled funnel. Collected the hexane layer into a turbo-vap tube. Repeat the addition of 50 m1 of hexane two more times. Rinse the separatory funnel with hexane and add to the Na2S04 filled funnel. Rinse the NaZSO4 filled funnel with hexane and add to turbo vap tube. Reduce the volume of hexane to ~ 0.5 ml and take it up with methanol to 2ml for HPLC analysis. HPLC analysis will be done at 1 ml/min with methanol as the mobile phase on a C-18 column. A wavelength of 254 nm and 280 nm for detection of Rotenone and Warfarin respectively was used. The method provided modest recoveries CM? 34%-54% for Warfarin and good recoveries for Rotenone of 86%-115%. Since Warfarin has a slight acidic nature there was a loss 49 of warfarin to the aqueous phase. Adjusting the pH to less than 5.5 with an aqueous solution of dilute HCl was added to the method after the first hexane extraction and then repeat the extraction with 50 ml of fresh hexane. This brought the recoveries for Warfarin up to 63%-68%. The recoveries were low for typical residue work but showed promise with the added concern of introducing two more chemicals through the same method. Work to improve the recoveries 'will In; continued. later' with. the addition. of other solvents for increased extraction recoveries. Pyrethrum was the next chemical added to the extraction method. The results again showed good promise with recoveries Ibetween 80% - 117% with. the combined. method. Nicotine was added to the method next with recoveries of 17% 33%. The original method for Nicotine is given in Appendix A. The method was reviewed to review the pH concerns that Nicotine has due to its two basic nitrogen atoms. Initially the sample was cleaned with an acidic water wash and then brought to a basic pH with 10 N NaOH until pH was between 8 and 9 and then 50 ml of dichloromethane was added. The dichloromethane portion was saved and the aqueous portion was again extracted with dichloromethane and the extracts were combined. This would combine nicely with the method for the first three chemicals. The first method with all four chemicals included a basic extraction by adjusting the pH 50 with concentrated NaOH until the pH was greater than 12. The extraction was repeated with 80 ml of fresh hexane. The recoveries were up to 102% at 6 ug/ml spike level. At lower levels time baseline noise interfered to aa greater extent relative to the peak of interest. The final method may be found in Appendix F. In this method the sample is first extracted at a neutral pH to obtain.tflua neutral species with dichloromethane. NaCl is added to the aqueous phase to push nonionic molecules out of the aqueous phase into the dichloromethane. At a neutral pH Nicotine’s speciation has a ratio of 6:94:0.01 of the double ionized, single ionized, to the nonionized species respectively. This accounts for the poor recoveries at the lower pHs for Nicotine. The aqueous phase was then made basic to assure that Nicotine would be nonionic and extractable by the hexane. The speciation at pH 12 of Nicotine was 91.6:8.4 Nicotine to single ionized Nicotine. The aqueous phase was then acidified to assure Warfarin would be protonated to allow for preferred residence concentration to be in the organic solvent. The overall recoveries for the combined .method are given in Table 1.8. The four chemicals showed wide ranges of recoveries within each chemical. Warfarin though it had 51 Table 1.8 Percent Recoveries for Fortified Samples Chemical Percent Recovery Spike Range Mean (n=2) Concentration % (us/m1) Nicotine 83.8 8.0 66-102 Pyrethrum 98.5 5.0 80-117 Rotenone 100.5 4.9 86-115 Warfarin 65.5 2.0 63-68 a smaller range from 63%-68% of recoveries had the lowest mean recovery' of 65.5%, attributable to the pH changes during extraction. At each step the analyte was lost to some degree. The chemicals were evaluated for Limit of Detection (LOD) and Limit of quantitation (LOQ) and the results is shown in Table 1.9. The LCD represents the lowest Table 1.9 LCD and LOQ values Chemical LCD LOQ ug/ml ug/g Nicotine 1.19 8.0 Pyrethrum 0.81 5.0 Rotenone 0.5 4.9 Warfarin 0.5 2.0 52 concentration of a standard detected with the UV detector of a standard. injected. into the IHNKZ. LOQ represents the lowest concentration of a spiked sample put through the extraction method, then injected into the HPLC and detected and quantified with acceptable recoveries. In the multimethod the level of detection of l ug/g was not achieved but could be lowered if done as individual methods (non published data from Dr. Matthew Zabik’s laboratory). 53 FUTURE RESEARCH The partitioning between the aqueous phase was favored for each chemical by the different pHs and NaCl additions. In choosing chemicals to analyze together for future research the pK; would preferably be closer and the chemicals would all be neutrals, bases, or acids. The combination of the three has been quite troublesome and the recoveries were not especially good for all chemicals. Another point of consideration was the mode of detection in that the wavelengths of maximum detection varied for the four chemicals. This could be easily evaluated by the use of a diode array detector for multiple simultaneous wavelength detection. Further work could also be done in trying solid phase extraction (SPE) to eliminate the large volumes of solvent required to achieve modest recoveries. The solvent savings both in purchases and in disposal cost are a driving force in laboratories. The reduced exposure to hazardous solvents would be another advantage of SPE. 54 METHOD II - SABADILLA, a-TERTHIENYL, RYANIAV AND .AZADIRACHTIN The research started. with evaluation of each chemical's solubility in organic solvents and Absorptivity to UV-Vis radiation for detection. Table 2.0 shows the solvents of choice for each chemical. Table 2.0 Solubility of Solvents Chemical Hexane Methanol Acetone a-Terthienyl Soluble Soluble Soluble .Azadirachtin Insoluble Soluble Soluble Ryanodine Insoluble Soluble Soluble veratridine Insoluble Soluble Soluble Methanol was chosen as the solvent of choice because of both solubility of the analyte and lower volatility compared to acetone. The UV-Vis maximas are given in Table 2.1 for the chemicals. Sabadilla has a composition of over 30 alkaloids with two of primary toxicological concern. Strong UV chromophores exist only for Veratridine so that will be the component analyzed for with the HPLC-UV detection system available (Zang, 1997). The other alkaloids can be detected using HPLC-MS for detection. 55 Ryania's active ingredients (n5 toxicological concern are Ryanodine and Dehydroryanodine and the standard used was composed of both constituents. Azadirachtin has a very poor UV maximum in that it occurs very close to the wavelength were solvents also absorb electromagnetic radiation. Table 2.1 Maximum Absorbance at Specific Wavelengths (nm) Chemical A (nm) /.A 1 (nm) /.A 1 (nm) /.A a-Terthienyl 224 / 0.81 252 / 0.91 350 / 1.00 .Azadirachtin 217 / 0.72 232 / 1.00 NA Ryanodine 210 / 0.30 269 / 1.00 NA veratridine 239 / 1.00 271 / 0.93 298 / 0.74 The initial HPLC work was done at 254 nm with methanol at 1.0 ml/min. Resolution was calculated using the equation on page 47 in the text. Values are given in Table 2.2. Ryanodine, ‘Azadirachtin and Veratridine overlap at concentrations greater than about 2 ug/ml, which can be determined by the low resolution between the three chemicals. Column efficiency was evaluated by looking at HETP and theoretical plates. The results are given in Table 2.3. Azadirachtin and a-Terthienyl did have the best theoretical plate counts, which allows for better separation efficiency. Slowing down the mobile phase to 0.5 ml/min 56 provided better resolution but poorer recoveries due to the peaks flatten out at the slower flow rate. Table 2.2 Resolution of Between Peaks Chemical Retention Wavelength Width Resolution Time (min) (nm) ““1“ R“ Ryanodine 3.24 260 0.70 - .Azadirachtin 3.46 260 0.50 0.37 ‘Veratridine 3.60 260 0.80 0.22 a—Terthienyl 4 . 77 260 0 . 75 1 . 51 The individual UV-Vis spectra for each analyte were done and ultraviolet (UV) and visible (Vis) absorbance maximas were determined for each analyte using a Gilford UV/Vis Spectrometer. HPLC retention time Table 2.3 Theoretical plate and HETP values Chemical Retention Peak Column Theoret HETP Time Width length ical (min) (min) (mm) Plates a—Terthienyl 4 .77 0.75 250 647 0 .39 .Azadirachtin 3.46 0.50 250 766 0.33 Ryanodine 3.24 0.70 250 343 0.73 ‘Veratridine 3.60 0.80 250 324 0.77 data for calculating resolution, theoretical plates, and HETP was determined from the chromatograms run on the. This 57 data was run at 254 nm on a HPLC system, this will be found described later in the text in detail. The standard curves are in Figures 2.0-2.3. 58 Terthienyl Standard Curve Absorbance Figure 2.0 a—Terthienyl Standard Curve Azadiractin Standard Curve Absorbance Figure 2.1 Azadirachtin Standard Curve S9 Ryanodine Standard Curve Absorbance PPm Figure 2.2 Ryanodine Standard Curve veratridine Standard Curve Absorbance Figure 2.3 Veratridine Standard Curve 60 Table 2.4 Linear Regression for Standards Chemical y-intercept x-coefficient r2 a—Terthienyl 5904 99516 0.99 .Azadirachtin -216618 621894 0.98 Ryanodine 281345 246666 0.99 ‘Veratridine 30288 301965 0.89 All of the standard curves had good linearity with r2 greater than 0.98 with the exception of Veratridine. The standard curves for each analyte are in Figures 2.8 — 2.11. The Veratridine standard curve had a showed log relationship with concentration, i.e. Response = 1n concentration. This also shows that Veratridine should be evaluated with a linear standard curve only over a small concentration range to avoid non-linearity. Solid. phase extraction (SPE) was considered at for extraction purposes with this set of chemicals due to their hydrophobic and nonionic characteristics. These characteristics make them good for SPE in that a common solvent could be used to elute them. SPE has generally attractive characteristics as an analytical extraction method due to the ease of handling, time saving alternative to liquid/liquid extraction, significantly reduced solvent 61 usage, and also may be used to remove interference compounds. The method given in Appendix F was followed. The sample extract was put on the 6 m1 Bakerbond SPE Octadecyl (Cm) Reversed Phase column as a mixture of all four analytes in methanol with 6 replications. The SPE columns were attached to a vacuum manifold to provide a uniform flow rate of the solvents. The spike amounts ranged from 0.36- 1.08 pig for Ryanodine, 0.28-0.84 ug for Veratridine, and 0.02—0.06 ug for both d-Terthienyl and Azadirachtin. After elution of the analyte the sample was analyzed using HPLC. The HPLC column was a Brownlee Laboratory reverse phase Cm (RP-C13) Spheri-lO column, 250 mm X 4.6 mm. A Waters 501 HPLC pump with a Rheodyne 7125 injector was used for the analysis. Detection was with a Milton Roy variable wavelength Spector Monitor®> 3100 model connected to a Spectra-Physics SP4270 integrator. Recoveries of the analysis are given in Table 2.5. The recoveries using the integrator calculated area produced recoveries greater than 130% for Ryanodine, Azadirachtin, and a-Terthienyl. This was due to high background relative to the analyte. Using the height of the peak for the previously mentioned three analytes the recoveries were less than 130%. 62 Table 2.5 Percent Recoveries for Fortified Samples Chemical Integrator Hand.Measured Best Area Height Measurement a-Terthienyl 151 73 73 .Azadirachtin 232 111 111 Ryanodine 194 93 93 veratridine 119 152 119 Overall Mean i 177 i 48 108 i 32 100 i 18 Std Dev Veratridine showed the opposite effect to recoveries. To optimize the recoveries of Veratridine was be measured with height and the other three the integrator value was used. The LOD and LOQ values for the chemicals is shown in Table 2.6. Similar the previous method when the analytes are analyzed by themselves the LOQ can be reduced. 63 Table 2.6 LOD and LOQ Values Chemical LOD LOQ ug/ml 119/g d-Terthienyl 5-0 0-02 .Azadirachtin 0.625 0.02 Ryanodine 1.8 0.36 veratridine 3.4 0.28 64 RESULTS AND CONCLUSIONS Further work could be done with HPLC/MS that would differentiate the mass and structural form present, so analytes that elute very close on a HPLC column could be known. Biological samples are complex and require more separation than water samples using capillary electrophoresis (CE) could also be employed. The essential part of a complex systems has to do with finding more than one way of separating compounds. The different forms of separation may act differently, ie. ionic compounds could be isolated from nonpolar compounds with CE even if they absorbed at the same wavelength. Organic pesticides are essentially the same as conventional synthetic pesticides and should be treated accordingly. Oral LDw of Nicotine and Rotenone have values of 60 mg/kg in rats and Malathion has a value of 5500 mg/kg. Malathion is under Environmental Protection Agency (EPA) review under FQPA (Food Quality Protection Act) at this time because of its human toxicity. The very reason that some plants develop chemical arsenals against pest should assure you that organically derived chemicals have similar and if not more potent toxicological actions both to humans and other living organisms. 65 The government regulates food safety by two major federal laws which are administered by EPA. for Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and Health and Human Services/Food and Drug Administration (HHS/FDA) the Federal Food, Drug, and Cosmetic Act (FDCA). FDCA establishes tolerances for pesticide residues in food and tolerances are enforced by HHS/FDA for most foods and US Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) for meat, poultry, and some egg products. Food safety should follow consistent regulations for pesticides and additives to assure both a safe exposure level through eating and environmental sources have a minimal impact for non target organisms. To foster the naive attitude that organic foods are safer than conventional foods will ironically be a disservice to the very people you intend to protect from pesticide and additive exposure. Increasing assurance of a safe food will be through recognizing and educating the public of chemical toxicity without differentiation of chemical source. 66 APPENDIX.A SOP FOR DETERMINATION OF NICOTINE IN CROPS RESPONSIBLE PERSON: Chris Vandervoort SCOPE: This SOP will provide for quantitation of residues of nicotine in crops matrices REFERENCES: Sheen, Shuh, Detection of Nicotine in Foods and Plant Material, Journal of Food Science, 53:5:1988:p1572-1573. TERMINOLOGY: GC = Gas Chromatography HAZARDS & PRECAUTIONS: 1. Weigh 10 g of sample and mix with 30 ml water, 1 ml of 3 N HCl, and 30 g (NHHZSCM, Heat in steam bath for 30 minutes with stirring. Cool slightly and filter through Whatman # 1 filter with vacuum. Transfer to a 250 ml separatory funnel with approximately 2, 3-4 mu portions of saturated (NHDZSO4. Add 50 ml of dichloromethane and shake for at least 30 seconds. Discard dichloromethane portion. Add 10 N NaOH til pH is between 8 and 9. Add 50 ml of dichloromethane and shake for at least 1 minute. Save dichloromethane into a 125 ml separatory funnel. Repeat with fresh 50 ml of dichloromethane and combine extracts. Add 5 ml 1 N HZSO4 and shake for at least 1 minute. Discard the dichloromethane. Transfer the aqueous phase to a Turbo—Vap tube with 2-3 ml of water and add to tube. Add 1 g NaZSO4 to the tube. and place in a 90 ° C water bath with N2 to remove dichloromethane. Cool and add 10 N NaOH til pink color persists in phenolphthalein. (kxxl and add 67 sufficient Nafiflh to saturate. Add 1 ml of benzene or (toluene). 6. GLC analysis of nicotine analytical parameters. 20 m Carbowax .25 mm column NP detector. 68 .APPENDIX B SOP FOR DETERMINATION OF PYRETHRUM IN CROPS TITLE: Determination of Pyrethrum in Crops RESPONSIBLE PERSON: Chris Vandervoort SCOPE: This SOP will provide for quantitation of residues of pyrethrum in crop matrices REFERENCES: Fujie, G.H. and O.H. Fullmer. (1978) Determination of Cis- and trans-pyrethrum residues in plant, animal, and soil matrices by gas chromatography. J. Agric. Food Chem., 26, p 395-398. TERMINOLOGY: GC = Gas Chromatography Extraction .Place 10 g sample into a 250 Erlenmeyer flask and add 50 ml of a 2:1 mixture of hexane:isopropanol. .Blend for ~ 3 minutes and pour off the solvent through a Buchner funnel with Whatman # 4 filter into a 250 ml separatory funnel. .Add an additional 50 m1 of 2:1 mixture and blend for ~ 1 minute. .Add ~ 2 g of Celite and filter through the Buchner funnel and rinse the filter cake with 25 ml of hexane. Add 125 ml of 10 % NaCl to separatory funnel and shake for ~ 1 minute. .Transfer the aqueous layer to another separatory funnel. .Add 100 ml of 10 % NaCl to the first separatory funnel and shake for ~ 1 minute. Transfer lower aqueous phase to the second separatory funnel and drip the solvent through anhydrous sodium sulfate into a round bottom flask. Rinse separatory funnel with an additional 5 m1 of hexane and drip through anhydrous sodium sulfate. Add an additional 50 ml of hexane to second separatory funnel and shake for ~ 1 minute. Discard aqueous layer and drip hexane through anhydrous sodium sulfate. 69 . Reduce volume < 1 ml, add hexane to 10 ml. Florisil Cleanup Prepare a 1 cm i.d. glass column with 2 g of Florisil activated at 135‘°C and topped with 1 cm of anhydrous sodium sulfate. Prewash column with 20 mfl. the south west were DDT may still be used illegally (Muir, 1993) may contribute to the DDT in the north. Helton conducted a study before DDT was banned in 1967 and 1968 and found DDT to be present in all atmospheric samples taken from nine 93 areas throughout the United States (Stanley, 1971). Air samples collect 1989 to 1990 showed 160 fold increase in levels of EDDT in samples collected from tropical Asia, where DDT is still used for agriculture and vector control, than from the Bering Straits (Iwata, 1993). The corresponding samples taken from adjacent ocean water had a 6.4 fold concentration factor. This data suggests that extensive usage is still occurring in tropical Asia. Another confirmation of the transport theory relies on the ratio of p,p’—DDT to p,p'-DDE (T/E) in that commercial products contain only a small portion of DDE and the T/E ratios differ due to sample location in the globe. Low ratios of T/E are seen in the North Pacific and North Atlantic basins compared to samples from the tropical Asian areas being sprayed. p,p’—DDT will be converted to p,p'—DDE due to UV absorption and metabolism by organisms during and before transport. Soil and Sediment Concentrations Trends in ZDDT deposition and use can be traced in sediments from reservoirs due to their increased sediment rates over natural lakes (Van Metre, 1997). Van Metre’s study showed high ranges of ZDDT of 27 to 74 ug/kg in the sediment samples for 1965 to reductions of up to 93% in samples from 1990. The temporal concentration trends show a correlation to the use and nationwide ban in 1972 of DDT. 94 The two of the reservoirs had overall 58 to 78% of the EDDT due to DDE. A soil study conducted in California in 1985 looked at speciation of the DDT resides in soils as a function of the EDDT (Odermatt,et al.., 1993). The results showed a range for DDT residues of 0 to 80%, DDD of 0 to 35%, and DDE of 15 to 100%. The mean ratios were 39% DDT, 8% DDD, and 58% DDE. The ratios support that the residues are ifixmi historical applications. These ratios were not looked at for the isomeric ratios, which would have been of interest when, back calculating to the formulated product. DDT as a group of contaminants would preferentially stay in the organic phase of soil and when bound unavailable for UV and microbial degradation. Degradation of DDT The formulated technical mixture nominally contains 14.9% o,p’—DDT, 77.1% p,p’—DDT, 0.1% o,p’-DDE, 4.0% pnpfl- DDE, 0.1% o,p’-DDD, and 0.3% p,p'—DDD. A mixture analyzed 40 years later in our laboratory contains 22% o,p'—DDT, 70% p,p'-DDT, 4% o,p’—DDE, 3% p,p’-DDE, 0.6% o,p’-DDD, and 0% p,p'—DDD. A paper written by MUller-Herold, 1996, looked at the dominant contributions to decay for DDT and found a rapid decay in the atmosphere and a high solubility in soil. The global limiting lifetime of DDT was calculated to 95 be 83 days, using the weighted average of 16 years in the soil, 1 year in water, and 7.4 days in the atmosphere. 96 RESEARCH Sample Collection The research samples were obtained from Coloma, Michigan and South Haven, Michigan. Coloma was considered to In; a control (uncontaminated site) and South Haven had the historically high ZDDT residues. The sample schedule is given in Table 3.2. A map of the general geographical area is shown in Figure 3.1 and a localized map for the sampling sites is shown in Figure 3.2. At South Haven there were 3 PUF samplers and Coloma had one. Sample A was on the south end of a notill corn field and B and C were collocated on the east side of the same corn field. Sample D was from Coloma which was a grass covered vacant parcel. A field blank was an air canister and quartz fiber filter sample that was opened at the sample site, put into the sampler, immediately removed and closed, then brought back. to the laboratory' for analysis. A. trip .blank is similar to the field blank but it was not opened in the field. The purpose of these samples was to assess any possible contamination from unexpected sources. Continuous wind speed, wind direction, and temperature were collected at Coloma and the South Haven sites. 97 Table 3.2 Sampling schedule for 1998 Air and Soil Samples Run Date Adr Sample Soil Field Trip 4/14/98 X X 4/20/98 4/26/98 5/02/98 5/04/98 5/06/98 5/08/98 5/10/98 5/12/98 5/14/98 5/16/98 5/19/98 5/25/98 5/31/98 6/06/98 6/12/98 6/18/98 6/24/98 6/30/98 7/06/98 7/12/98 7/18/98 7/24/98 7/30/98 8/05/98 8/11/98 8/17/98 8/19/98 8/23/98 X x>4x:x> 0.1 pm for the particulate fraction of the air sample. Following the filter was a polyurethane foam plug (PUF) then nominally 10 g; of XADZ resin followed by another PUF. fNua PUF and XADZ were cleaned following a 7 day cleaning cycle with multiple organic solvents before being put IJNK) the sample thimbles for field collection (see the actual procedure in Appendix G). The soil was sampled at the beginning and the end of the study. Samples were taken adjacent to the air sampling equipment and at three depths within the soil. Each soil core was nominally 10 inches and after being brought to the laboratory it was divided into three equal portions. Each portion was about 3.3 inches, and after dividing, the sample sections were mixed to obtain a homogenous sample. At each site a duplicate was taken for determining precision. Along the field's diagonals a sample was taken at SH. Each transect was a random composite of about 15 individual soil samples collected from SE-NW and SW-NE corners of the corn field to obtain a sample that represented the whole field. l0] After collection the samples were placed in a cooler at < (PC and transported to the laboratory within 12 hours for continued storage at nominal —20T3 until extraction to reduce any further degradation or chemical changes before extraction. The samples were analyzed within seven days from sampling to decrease any changes that may occur during storage. Analytical Preparation and Cleaning Initially all the material to be used in the analytical portion of the research had to be cleaned to remove interfering material to give a sufficiently clean background for nanogram to picogram detection of the analytes. The complete procedure may be found in Appendices G and H. All organic reagents used were pesticide-grade. Extraction Extraction took two days for each sample set. Each sample set contained at least one concurrent spiked sample. The air samples were extracted as the vapor phase and the particulate phase separately. The procedures were similar except for the size of glassware and proportion of solvent needed for extraction. The samples were placed in soxhlet extractor with 50:50 acetone/hexane and extracted for 18 to 24 hours. The detailed extraction procedure is; shown in Appendix I. l02 The soil samples were extracted following a similar procedure as the air and the detailed procedure is shown in Appendix J. Silica Gel Column Chromatography Following extraction, the extracts were cleaned up on a 4% deactivated silica gel column to remove interfering contaminants and the volume reduced for gas chromatography. The gas chromatography analyte peaks had consistent retention times and good baseline separation so that not all of the samples were put through a silica column. If the resulting chromatograph had co-eluting peaks that could not be separated with the Hewlett-Packard (HP) software by manual integration then the extract was put through a silica gel column. Gas Chromatography The samples were reduced to about 2ml volume for GC analysis with a Turbo—Vap evaporator and further diluted after the first injection if needed to reduce concentrations at the GC. The GC was a 5890 Series II Hewlett-Packard (HP) equipped with a Ni63 electron capture detector using HP 3365 ChemStation for data acquisition and reporting. The IBM compatible computer had a dual channel interface to connect the ChemStation software to the GC. The injector was set at 250°C and the detector at 350 0C. The oven was initially 103 holds at 1000C for one minute and then began ramped at 1 C’C/minute to 240°C, at this time all of the analytes had eluted off the column. The column was cleaned by ramping at 10°C/minute to 280°C. The total run time was 144 minutes. The GC column was a J & W Scientific DB-5 column with an internal diameter of 0.25 mm with a 0.1 um film, 30 m long and the analytes were off the column in 108 minutes. 104 RESULTS AND CONCLUSIONS The vapor and particulate fractions were analyzed separately' to differentiate ‘metabolic ratios in the air samples that may have occured. The soil samples were then analyzed in relationship to the air samples. .Air Samples The air monitoring data provided detectable residue of all of time analytes during the study. It was determined that the Coloma (CLM) site was not a true control due to the presence of analyte residues. The residue levels found at CLM were significant but generally not elevated above the South Haven (SH) site. The magnitude of values measured at SH were consistent from year to year and independent of the research team conducting the analysis determined from data provided by Michigan Department of Environmental Quality. The sample concentrations may be found in Appendix K. Each site can be viewed for the total analytes per sample in vapor phase, particulate phase, and total air sample (Vapor + Particulate) in Figures 3.3 to 3.70. Also note that the y axis for sites A, B, and C are greater than D and are all different. Generally the (Hid site can 1x3 considered less impacted than the SH site with about a two fold difference in residue levels 105 k b picogram: per cu nmfler Concentration of o,p'-DDE at Site A Figure 3.3 A Concentration of Vapor Phase o,p’-DDE at SH Site h b picograms per cu Concentration of p,p'-DDE at Site A . w , . wm .0- . ea . 392%“.9 ‘SSSEESHSE i511. “@3354 WWW .. . .v Weaeéem a... “as. 52*. mg. .0“.ng We... a. a. ”C “333. w: ~ m w, v.- : - . .maéizwa . x . w. M.,-M...“ . a . -. . w ,Aefieemm . ‘ w: . . . 4 3N,“ '~ Figure 3.4 A Concentration of Vapor Phase p,p’-DDE at SH Site 106 Concentration of o,p'-DDD at Site A bic picogram: per cu Q Q Q [s Figure 3.5 Concentration of Vapor Phase o,p'-DDD at SH at Site A Concentration of p,p'-DDD at Site A II: are; . , . 5:25:11. . _ .( . .§.(< ...“' .‘..".-. ' b ' < (.4243? . I P 453mg§§4§m§m , ~ 4.54 .~ 44...)“. ..4 23$, . 4 .4“, 4 4 M: ““544 , . W. ~>.-. - x“ u. ‘sv ‘6 . are Hem-nu - . * v. - MM» “an we 242.-x4- :t memeszw. - 4. 3.44.44 Ry \w4 44¢ \44 _ 4:“: 2e .m‘e—e‘. 4.4.243? ~ 4 .....4444-12. ”4.4.4.14“ mw 4*...- m :4 Anew... ‘ . < v.“ IL "4% '. we weenie: :4 picogram: per cu meter 4/1 4/98 42808 5H2B8 5/26/98 SENS. 63338 7flm8 7Q1N8 BMNS 8H8B8 Da 8 Figure 3.6 Concentration of Vapor Phase p,p’-DDD at SH at Site A 107 .9. a 3 U h an. “3 EE 2 u o .2 fl- Concentration of o,p'-DDT at Site A Figure 3.7 Site A Concentration of Vapor Phase o,p'-DDT at SH at bk 2 S picograms per cu nnmr . ”SE . - imssm :«eessseeeszs . 4-M-=ei'_-v4a.i:$m.~ me» “we ‘42 ~ .- Concentration of p,p'-DDT at Site A I _' . ,... ‘i‘ ..i- 4 IA Ase“ ~ ' ”. sis :2? mass . VA“ . .. L4,... 4 _" 3.4- iii-Ask. SMEB Figure 3.8 Site A Concentration of Vapor Phase p,p’-DDT at SH at 108 Concentration of Kelthane at Site A g 1200 ...}, § 1mm 8... 800 n3 600 E2 400 g‘ 200 .2 o D. Q Q Q Q Q Q Q Q Q Q 8 S S 8 3 8 g S 3 8 t E E Q Q Q B Q co S V V? m m Q B Q Date Figure 3.9 Concentration of Vapor Phase Kelthane at SH at Site A 109 Concentration of o,p'-DDE at Site B k picogram: per on near Figure 3.10 Concentration of Vapor Phase o,p’—DDE at SH at Site B Concentration of p,p'-DDE at Site B k b $511255 fix “afizfiiigfip ' . 44.. .244 .. s. u. v» . “.6 . 344.432.444.424 44.333. 42.4.44... picogram: per cu nnmr Figure 3.11 Concentration of Vapor Phase p,p’-DDE at SH at Site B “0 k b picograms per cu Q Q V ,_ \ v Concentration of o,p'-DDD at Site B agassaaai aeieessei VIDID (O N Q Dam Figure 3.12 Concentration of Vapor Phase o,p'-DDD at SH at Site B Concentration of p,p'-DDD at Site B .3 2000 = g 1500 g‘é 1000 5, 500 O .2 o a. Q Q Q Q Q Q Q 8 Q g i i i. i 3 E E :2 ? g E m w o N n o E [hm Figure 3.13 Site B Concentration of Vapor Phase p,p’-DDD at SH at 1” Concentration of o,p'-DDT at Site B It: fl = U h 8 a a O .2 fl. ”8°333838” §a§°estii4i§ ecsS°$NS°a Date Figure 3.14 Concentration of Vapor Phase o,p'-DDT at SH at Site B Concentration of p,p'-DDT at Site B k b picograms per cu nehr Dam Figure 3.15 Concentration of Vapor Phase p,p’—DDT at SH at Site B 112 Concentration of Kelthane at Site B .2 A a 0 b a U) E E a O .2 4 S 8 8 g 8 E 3 g ... t Q C Q «a Q Is Q to S V V ID In (D 1‘ Q Date Figure 3.16 Concentration of Vapor Phase Kelthane at SH at Site B 113 Concentration of o,p'-DDE at Site C bk picogramstper cu . i E E E 3 Figure 3.17 Concentration of Vapor Phase o,p’-DDE at SH at Site C Concentration of p,p'-DDE at Site C hm picograms per cu [kn Figure 3.18 Concentration of Vapor Phase p,p’-DDE at SH at H4 Concentration of o,p'-DDD at Site C £1000 3800 I- 85600 §§4oo a 2m) 0 .2 o aQQQRDQQQQQQ evam°$tfi°a Date Figure 3.19 Concentration of Vapor Phase o,p’-DDD at SH at Ic .o 5 3 a E D O 2 ii egaSPS“S”s Dam Figure 3.20 Concentration of Vapor Phase p,p’-DDD at SH at Site C H5 picogram: per cubic Concentration of o,p'-DDT at Site C QQQ iii: 3“”; Figure 3.21 Concentration of Vapor Phase o,p’-DDT at SH at Site C Concentration of p,p'-DDT at Site C 2 1200 .9 4 amaze main"? 2. 8 1000 2‘s." 4. a a 8°° m "' 600 E E 400 g 2M) '5. 0 Date Concentration of Vapor Phase p,p’-DDT at SH at 116 Concentration of Kelthane at Site C k §§§§§ picograms per cu near Q S. Q Is 8/1 8/98 Da Figure 3.23 Concentration of Vapor Phase Kelthane at SH at Site C H7 Concentration of o,p'-DDE at Site D 03:0 ..oe nEEuooE -DDE at CLM I f Vapor Phase o,p 101'! O Concentrat Figure 3.24 Site D Concentration of p,p'-DDE at Site D .22: 63:9 .3: mEEuooE te DDE at CLM I ion of Vapor Phase p,p Figure 3.25 Concentrat Site D 118 k b picograms per cu Concentration of o,p'-DDD at Site D “v“ . 4. . Use . .5 ' . 233‘. 9 a > we. \‘n‘i 7/21 l98 BMQB; 8/1 8/98 Figure 3.26 Site D k b "Bur picograms per cu Concentration of p,p'-DDD at Site D t \‘N‘AK’. 4.» x we- i4 . x} \ ~. t4 . 4 ... 4x44444433: . ‘ " Av 4 Figure 3.27 Site D 119 Concentration of Vapor Phase o,p’-DDD at CLM Concentration of Vapor Phase p,p’-DDD at CLM Concentration of o,p'-DDT at Site D gsoo 3 an 8.54% 03300 53200 g 100 .2 o o m o w o w o o o o FvfimoghsaB [hm Figure 3.28 Concentration of Vapor Phase o,p'-DDT at CLM Site D Concentration of p,p'-DDT at Site D k b plcograms per cu nmmr Figure 3.29 Concentration of Vapor Phase p,p’-DDT at CLM Site D ”0 Concentration of Kelthane at Site D §§§§§ picogram: per cubic N o 8 Figure 3.3 Site D 0 Concentration of Vapor Phase Kelthane at CLM 121 Sum of All Analytes-Site A-Vapor Phase 16 a = U i. a? E a o i. .3 0 em a w w o o o o o a o 3v$m°¢ohrst Duh Figure 3.31 All Analytes for SH Site A Vapor Phase Sum of All Analytes-Site B-Vapor Phase II: .9 = U i h 3: . n 2 6000 - 14$“? E2 4000 " 2:94er .2 o a Q Q Q 8 Q B Q a Q g t S! E Q to Q :4 Q 00 t V 1' In In (D B Q [hm Figure 3.32 All Analytes for SH Site B Vapor Phase 122 Sum of All Analytes-Site C-Vapor Phase IC a = u h 8. n E E u o e assesses... C Q : Q p Q s Q n E v v m m o n w Dam Figure 3.33 All Analytes for SH Site C Vapor Phase Sum of All Analytes-Site D—Vapor Phase 16 a = o 6 e n E E on o 3 e D Q Q Q Q “3 Q 00 Q Q Q Q g Q Q Q Q Q Q Q E Q S w Q R Q a E v- v u: m o n. m Figure 3.34 All Analytes for CLM Site D Vapor Phase n3 Concentration of o,p'-DDE Particulate at Site A IC 120 b 8 80 60 40 20 plccgrams per cu nnmr -0 QQQQQQQOQO iiiASiiiii eSaSPSNS”a Figure 3.35 Concentration of o,p’-DDE Particulate at SH at Site A Concentration of p,p'-DDE Particulate at Site A ic b picograms per cu meter Figure 3.36 Concentration of p,p’-DDE Particulate at SH at Site A 124 Concentration of o,p'-DDD Particulate at Site A 2 .e 3 3 8 z a O 2 fl. QQQQQDQOQQ iiiiiiiiii ages S"S‘°a Date Figure 3.37 Concentration of o,p’-DDD Particulate at SH at Site A Concentration of p,p'-DDD Particulate at Site A k picogram: per cu Figure 3.38 Concentration of p,p’—DDD Particulate at SH at Site A ns Concentration of o,p'-DDT Particulate at Site A g 500 = . o 400 ...-...... - -- .- 8 v.»'a§5§é§§f ‘& 5W 3 E 200 “515‘?” " E ‘. 3* 100 . 44 a2: 4_ ‘.4 1' .2 0 Iii-@2546 44 44 a. Da 8 Figure 3.39 Concentration of o,p’-DDT Particulate at SH at Site A Concentration of p,p'-DDT Particulate at Site A é 5m) 2 400 gfiwk . $533324» 44. 4.. 4 g- I- 300 me- 4 3 meme: .-a E 2 mm a%&% a 100 o '2 o Figure 3.40 Concentration of p,p’—DDT Particulate at SH at Site A D6 Concentration of Kelthane Particulate at Site A K: 120 100 80 60 b picograms per cu "war 40 20 0 Figure 3.41 Concentration of Kelthane Particulate at SH at Site A 127 Concentration of o,p'-DDE at Site 8 picogram: per cubic meter ‘5 ‘b q g b} ‘8 b. Figure 3.42 Concentration of o,p’—DDE Particu Site B late at SH at Concentration of p,p'-DDE Particulate at Site B 3 a a 3 a E a o 3 a. ””””83333 $$$$§mQFQ~ CQSQthQ'w ext-am to Is [hm 8/1 8/98 Figure 3.43 Concentration of p,p’-DDE Particulate at SH at Site B I28 k b picogram: per cu "mar Concentration of o,p'-DDD Particulate at Site B 100 so 60 4o 20 0 [hm Figure 3.44 Concentration of o,p’-DDD Particulate at SH at Site B Concentration of p,p'-DDD Particulate at Site B .2 a picograms per cu nnmr 'x zzxxaxaxxle 44444444444 .4; 44:04 V: x s w w w o o w o o g a 3 3 g $ % 3 S E .— 3 co E Q S Q m Q N Q m E v v m m o n m Dam 3.45 Concentration of p,p'-DDD Particulate at SH at n9 Concentration of o,p'-DDT Particulate at Site B It: .n :I o h 8. n E E 3 .2 n'oococcuoaooocoooooco egsegsgsgg {Gigo‘l'rsfi'cct vvm-n to is co Figure 3.46 Concentration of o,p’-DDT Particulate at SH at Site B Concentration of p,p'-DDT Particulate at Site B g 500 3 400 8.5 300 Egzoo 5. 100 8 ‘3. o Figure 3.47 Concentration of p,p’-DDT Particulate at SH at Site B 130 Concentration of Kelthane Particulate at Site B IC .o 3 v- 500 35 400 g g 300 2 mm g 100 .5 o Figure 3.48 Concentration of Kelthane Particulate at SH at Site B 131 Concentration of o,p'-DDE Particulate at Site C g 350 g 300 - 250 35 200 1% . g E 150 '. i.e.?“ - 5' 50 Mg .2 o ' ‘ fl. Figure 3.49 Concentration of o,p’—DDE Particulate at Site C Concentration of p,p'-DDE Particulate at Site C u 250 .. '3 0 2m) . 8. a 150 ”a ‘: 4* . 4 a g 100 “242* ., . ~ .. ., 44 .. we «at: g ‘4 at? " {5&2} i3 . “aims .- 44 :-..\‘ ' an 50 W44 4 ' 4 m4. 4 warm: 0 - fiagagitnfiggigfifi ~ ~ ‘ o Lizfit-‘Wi’m ‘“'4'44:="444=:44 4 454 4*. * a 0 Figure 3.50 Concentration of p,p’-DDE Particulate at SH at Site C 132 Concentration of o,p'-DDD Particulate at Site C k 5‘ b w“ “w. s -. -. we“ wk - was “ “Six" mmw . “ 80 60 40 20 H . m . w \.%%m% w.-. w picograms per cu meter 4/1 4/98 4/28/98 5H2N8 6/23/98 721B8 8/1 8/98 Figure 3.51 Concentration of o,p’-DDD Particulate at SH at Site C Concentration of p,p'-DDD Particulate at Site C bk N 8 3 U h 2.“ - 3 ’s:-.§;.: g, 50 3‘? O 'K, .2 o O. a a °° a 8 8 a a a 8 :5 a is. g i g E E : E N Q ; v E In ‘0 (O n B Data Figure 3.52 Concentration of p,p’-DDD Particulate at SH at Site C 133 picograms per cubic Concentration of o,p'-DDT Particulate at Site C co m (D w 0 0 6 Q 0 s g a g a g a. g g s a s .. g ~ s .. a Date Figure 3.53 Site C Concentration of o,p’-DDT Particulate at SH at k b picograms per cu Concentration of p,p'-DDT Particulate at Site C . . W. .‘ 4% a . ~ . . m. , '."N\'>"-"_ .. ""'\'P.».'9"il: "\‘I “q. 2mm. _ _ figflfim 1% - “m aka ~ ~.~ ‘- . .w. ~$ - Figure 3.54 Site C Concentration of p,p’-DDT Particulate at SH at 134 k b §§§§§§ picogram: per cu nnmr o's‘ Concentration of Kelthane Particulate at Site C 8 93° 8 8 8 8 3 ‘° 8 8 v: 8 g 8 E E S 3 v m m m h a Date Figure 3.55 Site C Concentration of Kelthane Particluate at SH at US Concentration of o,p'-DDE Particulate at Site D 3 25 ii 20 E 15 E E 10 g 5 .3. 0 Figure 3.56 Concentration of o,p’-DDE Particulate at CLM Site D Concentration of p,p'-DDE Particulate at Site D g 500 0 4M) I- 8 5. 300 E E 200 g 100 8 '5 0 [kn Figure 3.57 Concentration of p,p’—DDE Particulate Site D 136 at CLM Concentration of o,p'-DDD Particulate at Site D IC . A». b \3‘Q\\'.\\-. t. ~23}. .~ picograms per cu a a w a m o w w w w a to $ to S 3 E 3 3 $ t E S Q 0 Q R Q B t V v m m w s o [kw Figure 3.58 Concentration of o,p’-DDD Particulate at CLM Site D Concentration of p,p'-DDD Particulate at Site D It; .o 5 u a m E E a O 2 n. ”E”88”8838 gwgogntwiw Efltfloflnfio: vvmm (D N no [hm Figure 3.59 Concentration of p,p’-DDD Particulate at CLM Site D 137 Concentration of o,p'-DDT Particulate at Site D k b picogram: per cu Figure 3.60 Concentration of o,p’-DDT Particulate at CLM Site D Concentration of p,p'-DDT Particulate at Site D k b picograms per cu Figure 3.61 Concentration of p,p’—DDT Particulate at CLM Site D 138 Concentration of Kelthane Particulate at Site D K: .9 3 U a U E 2 a O .2 n. comcoooooowomco :SSS”$“S”B Date Figure 3.62 Concentration of Kelthane Particulate at CLM Site D 139 Sum of All Analytes—Site A-Particulate picogram per cubic meter Figure 3.63 All Analytes for SH Site A Particulate Sum of All Analytes—Site B—Particulate % E 2 .o 8 a E S w“ .06“ <9 .96 «9 a? «i .o‘ «9 .68 Date Figure 3.64 All Analytes for SH Site B Particulate l40 Sum of All Analytes-Site-C—Particulate E 700 ,_, 600 3 5m) §4oo 300 3' 200 .... E100 §§x§m sun _ gflf § 0 nfi§§fi§fi§a 3 co co co co co co co no no no a Q Q g g \ g Q Q g g v co F : s3 2 m 3 m E s: a : 1’ 1' In In (D N no Date Figure 3.65 All Analytes for SH Site C Particulate Sum of All Analytes-Site-D-Particulate g, iv °' 3 3 g $ 3 m E .— § 2 a g a g ‘° g " S °° a Date Figure 3.66 All Analytes for CLM Site D Particulate 141 Sum of All Analytes at Site A-Vapor 8. Particulate a: E 10000.0 ‘ f _. may 2 3000 0 $5“ ...afifim 0 6000.0 < m 8. 40000 5 2000.0 gt 0.0 — 9; 9: <2: ‘6 a a n- 9 co 9 9 \9" e \03’ \{a \03’ 0* .0“) 6,6” 455* «>9 a") «Q «0 ‘6‘“ a“) Date Figure 3.67 Sum of All Analytes for South Haven Site A Particulate & Vapor Phase Sum of All Analytes at Slte B—Vapor 8. Partlcluate 1 2000.0 1 0000.0 8000.0 6000.0 picogram per cubic meter A O O O O Dam Figure 3.68 Sum of All Analytes for South Haven Site B Particulate & Vapor Phase 142 Sum of All Analytes at Site C—Vapor & Particulate 14000.0 picogram per cubic meter m 0 O p O Figure 3.69 Sum of All Analytes for South Haven Site C Particulate & Vapor Phase Sum of All Analytes Site D- Vapor 8. Particulate ° 5000 0 .- . . «:5 MW... at Wig “W, 0 4000 0 ammmfii ”aetkifimm “”9“ - - w... n. n. - ma ' I- 3. akfifsf’igfiug (K r em; 0 |. . whit“ ‘ “.2. $3053“ §t§& no 3000.0 " . «a ... fl ' '. st<~< W s 2 2000.0 8 1 m o x \ nu.» Isvu U! - y. ”MN-:2 {\ m 0 M‘ttzwttg 0 mafia...- 0 me“. ~ 00 ‘ D. Date Figure 3.70 Sum of All Analytes for South Haven Site D Particulate & Vapor Phase 143 The concentration in the air of ZDDT should show a direct relationship to the temperature of the environment. A 10 degree increase in temperature gives approximately a 3- 4 times increase in volatility. Figures 3.71 to 3.72 show the relationship of concentration to atmospheric temperature for Site A and Site B. The temperature data for all sites is shown in Appendix L. Figure 3.73 shows the relationship of temperature to concentration for all of the data with the calculated exponential correlation equation. The equation found was: Concentration = 896e0‘1287Te"“"m‘t“‘re A r2 of 0.8128 was found, indicating a good correlation of the data. This information helps in the validation of the entire process from sampling to analytical detection Site A Con'elation of Temperature with Concentration of Sum of DDT pkwgmmtnr cubic meter Temperature Degrees Centigrade Figure 3.71 Site A Correlation of Temperature with Concentration of ZDDT I44 Site B Correlation of Temperature with Concentration of Sum of DDT w. .w .9. .x-.. \mhx tuwamx-é‘ wages? {20"- picogram per cubic meter 2 Temperature D Figure 3.72 Site B Correlation of Temperature with Concentration of ZDDT Temperature Correlation with Concentration of Sum of DDT “M 40000.0 .- . x‘ 35000.0 . ‘:.. ~ ma. 30000.0 25000.0 picogram per cubic meter to O O O .0 O FVNOMOQSIIWQ r' ‘- NN ‘_‘._ Temperature (Centigrade) Figure 3.73 Temperature Correlation with Concentration of ZDDT for all air samples 145 The dispersion of DDT and it’s metabolites in the vapor phase (Figure 3.74) shows o,p’-DDE and p,p'—DDE at greater than 50% of the total ZDDT with o,p’-DDD and p,p’-DDD at 17% and o,p’—DDT and p,p’—DDT at 17%. The particulate phase shows o,p’—DDD and p,p’-DDD at greater than 66% of the total ZDDT, o,p’-DDE and p,p’—DDE at 12% and o,p'—DDT and p,p’-DDT at 16%. Average Vapor Phase DDT 8. Metabolite Dispersion for All Sites at SH & CLM Kenhane 8% p.p'-DDD 12% Figure 3.74 Average Vapor Phase DDT & Metabolite Dispersion for All Sites at SH & CLM 146 Average Particulate DDT & Metabolite Dispersion for All Sites at SH & CLM ophDDE . Kelthane 3% p,péSDE O p.p'-DDD 63% Figure 3.75 Average Particulate DDT & Metabolites Dispersion for All Sites at SH & CLM 147 The particulate has the major portion of time residue, 66, in DDD and the vapor phase has themajor portion in DDE at 58%. This indicates that the residues are aged because the parent DDT is a smaller percent of the EDDT. One aspect for determining the age of the DDT residues is through. the ratic» of it's :metabolites to the parent compound and that data can be seen in Figures 3.74 to 3.80. The site specific pie charts for percent the DDT and it’s metabolites are found in Figures 3.76 to 3.79 and Figure 3.80 represents all sites combined. DDE was found at 2.4 times the concentration of DDT and 4.8 times the concentration of DDD in the vapor phase. These ratios indicate that the DDT has changed from the initial concentration were DDT composes 95% (in the formulated product) of the ZDDT to 26% (of the environmental sample over all sites and locations). Using the half—life equation to calculate expected concentrations of DDE after legal application ceased in 1973, it was found that DDT degraded according to the soil half-life of 16 years. From our laboratory data 100% DDT goes to 27% DDT (normalizing the 95% formulated product to 100% for clarity of calculation) and follows the equation: log C = log CO — kt/2.303 The results show a half-life of 13.2 years (actual calculation is shown in Appendix Q) which indicates both 148 soil, air, and water were contributing to the degradation of DDT to obtain a hybrid half-life between 1 year in water, 16 years in soil, and 7.4 days in the atmosphene. The half- life supports the idea that the controlling factor in degradation of DDT was soil and thus the atmospheric concentration are a result of the soil burden volatilizating during the warm weather and when there is no snow cover. 149 Site A DDT 8. Metabolite Ratios for Vapor + Particulate Keflhane p.p-DDT 5% ””305 19% Figure 3.76 Site A DDT & Metabolite Ratios for Vapor + Particulate HO Site 8 DDT 8. Metabolite Ratios for Vapor + Particulate Kenhane npDUT 5% 12% Figure 3.77 Site B DDT & Metabolite Ratios for Vapor + Particulate IN Site C DDT 8. Metabolite Ratios for Vapor + Particulate Kenhane 11% o,p-DDE Figure 3.78 Site C DDT & Metabolite Ratios for Vapor + Particulate 52 Site D DDT 8. Metabolite Ratios for Vapor + Particulate Kenhane QDDDE Figure 3.79 Site D DDT & Metabolite Ratios for Vapor + Particulate B3 All Sites DDT & Metabolite Ratios Kenhane 7% ‘ Figure 3.80 All Sites DDT & Metabolite Ratios The data show a 2.5 fold greater concentration of EDDT in the vapor phase over the particulate phase. The ZDDT concentration shows a positive correlaion with increasing temperature and this could account for the increased levels of BDDT in the vapor phase compared to the particulate phase. The amount of DDT and metabolites brought into the air column was used to determine the amount of ZDDT that moved off the sites during the sampling period to the atmosphere for long range transport. The air sample was taken from an effective height of 1 m above the soil surface. Over 24 hours the sample collected an average volume of 328 m3. The basic assumption for these calculations was that the air was sampled from 1 m above to l m. below the sampler inlet. To determine the average amount of chemical moved away from one hectare of the field the following equations were used: U4 A = Cx 20000 A is amount of EDDT in a hectare volume 2m high C is concentration of the EDDT in pg/m3 20000 is volume above a hectare that is 2m high by 100m long by 100m wide # Sweeps = windvelocity(meter/day)(%00m) # Sweeps of a hectare per day Wind velocity converted to meters per day 100m is the length of one side of a square hectare AmowyDay = Ax# Sweeps Amount per Day represents the amount of the ZDDT moving off a hectare per day. The resultant average wind speed for the sampling date is shown in Appendix N for determination of direction of movement of the ZDDT. The amount of ZDDT moving off of one hectare was calculated from the data and is shown in Figure 3.81 and Figure 3.84 for all sites. The average value of ZDDT moving off of Site A was 98 nngay, Site B was 194 mg/day, Site C was 136 mg/day and Site D was 53 mg/day. The contribution from the soil ZDDT burden to the total air concentration can be seen from this data. It shows a greater than 2 fold increase in ZDDT at site SH to CLM. BS SiteA h 8'300 $200 .9100 EC Ewgwgwoooon §8§w$8§8§§ a.a$‘°$"$‘°a Date Amount of Sum of DDT Moving off per Day at Figure 3.81 Amount of EDDT Moving Off Site A per Day milligrams per day Amount of Sum of DDT Moving off per Day at Site 8 WWW; 0°” 50‘ Figure 3.82 Amount of ZDDT Moving Off Site B per Day l56 Amount of Sum of DDT Moving off per Day at Site C ......... r ... ‘ ... . ..- ... ............. . . u c . ........ we» to 833 we .3 ES 3on 836 3on 8a :m 833 mm: 5. >2. .8: mEEm==E Date Figure 3.83 Amount of ZDDT Moving Off Site C per Day Amount of Sum of DDT Moving off per Day at Site D me {w vaB wQ FNR omRK QQMNB @908 waQm Date Amount of EDDT Moving Off Site D per Day Figure 3.84 l57 Soil Data The soil data is shown in Appendix M for p,p'-DDT, o,p’-DDT, p,p’—DDE, o,p’-DDE, p,p’—DDD, o,p'-DDD, and Kelthane. The data was summed for all DDT for each site and at each section of the sample (top, middle, and bottom), the result is shown in Table 3.3. The middle third showed the highest residues of 57% followed by the top at 36%, and the bottom third had 8% of the total concentration. The middle section was 1.6 times more concentrated then the top section and the middle was 7.1 times more concentrated than the bottom. Site BC showed the greatest residues at 4.3 ug/g average in the mid sampling level, with Site A at 4.1 ug/g average in the mid sampling level, and Site D at 0.4 ug/g average ill the mid samplling level. TUNE data shows that Site BC had 1.8 times as much EDDT as Site A and 7.4 times the amount found at Site D. This would imply with the greater burden of EDDT being in the soil at SH then the increased air concentrations would also be at SH. This was found to be the case, as can be seen in Figures 3.3 to 3.70 and verified in the data found in Appendix K. 158 a" . {T Table 3.3 Soil Data of Summed DDT Concentrations Sample Top Middle Bottom % Site Site Subsample Subsample Subsamplee Contamina- (ng/gi (ng/g) (“g/g) tn" A. 1126 4067 0.22 33% BC 4081 4360 793 59% D 377 442 400 8% % Position 36% 57% 8% Contaminati on The total amount of ZDDT in the soil that was sampled was used to determine the net loss of EDDT from the total soil burden. The amount of EDDT in the soil was determined by taking the average soil concentration times the volume of soil in one hectare 10 inches deep times the average bulk density of sandy loam soil (1.5g/cm5. The weight of one hectare of soil 25.4 cm deep was found to be 3.81 x 106 kg. Table 3.4 shows the maximum, average and minimum amount of the ZDDT in the soil at the two sites. The SH soil averaged 5.5 times more EDDT, on a calculated basis, as the CLM site. 159 Table 3.4 Amount of EDDT found in one hectare of soil 25.4 cm deep Site High (kg/ha) Average Low (kg/ha) (kg/ha) SH 111.6 79.4 47.2 CLM NA 14.5 NA NA.- Not Available The percent loss due to volatilization from the soil was calculated to determine and estimate the time needed to move all of the DDT out of the soil profile. The following equation was used for these calculations: ‘VoIi/IovedOfl / Year 2 AmountMovedOff / Year x 100% flflafldewnkn Table 3.5 shows the results using average and high (worst case scenarios) for levels of ZDDT to disappear per year from the soil profile. The range for SH goes from 0.04% to 0.95% per year ZDDT loss from the soil. (314 has a range from 0.13% to 1.4% per year loss from the soil. If these numbers are consistent for the years to come a theoretical calculation for time before soil burden of the ZDDT goes to zero would be 100% divided by the percent loss per year. The calculation predicts 2500 years at the worst case (slowest degradation) to 105 years for the average case at SH. For CLM the numbers range from 769 years to 71 years for the soil to be free of ZDDT. 160 Table 3.5 Percent Loss of ZDDT per year from one hectare for the two Locations Site Concentration Soil Burden Per Location Level SH SH SH CLM High Average LOW’ .Average .A High 0.13 0.18 0.31 NA Average 0.03 0.04 0.07 NA B High 0.40 0.57 0.95 NA. .Average 0.06 0.09 0.15 NA C High 0.18 0.25 0.43 NA. .Average 0.04 0.06 0.10 NA. D High NA NA NA 1 . 4 Average NA NA NA 0.13 The metabolites were then looked at separately for percent loss per year and the amount of time to reduce the entire soil burden to zero under consistent conditions as found during the 1998 sampling period. These calculation is shown in Table 3.6. o,p'-DDE had the shortest time for complete dissipation at 4 years, and p,p’-DDT had the longest at 6958 years. Although the numbers are derived from assumptions (consistent atmospheric and land use conditions) 11) facilitate the calculation time trends show that the ZDDT will continue to txeaa long term contaminant and available for exposure to the environment and animal life. 16] Table 3.6 The percent loss per year and Amount of Years to Reduce the Soil Burden to Zero concentration per Metabolite Metabolite Percent Loss per Years to Soil Year Burden to Zero Concentration o,p’—DDE 24.4 4 p,p'—DDE 0.09 1081 o,p’-DDD 0.34 298 p,p’-DDD 1.16 86 o,p’-DDT 0.19 526 p,p'-DDT 0.01 6958 Kelthane 0.16 636 162 Conclusions of Soil Impact on.Air Concentrations SH shows a 2 fold and greater air concentration level over CLM which reflect the fact that the soil burden at SH was almost 6 times more concentrated than CLM. Another factor that shows the soil contributes heavily to the air concentration can be seen with the correlation (r2 of 0.81) of concentration with temperature. If the atmospheric concentration was coming from the air alone then heating should cause lower concentrations due ix) volume expansion and dilution of the contaminant. This was not seen in the data, evident by the temperature to concentration correlation of r2 of 0.81. The soil data supports the theory long term residence in the soil because the highest concentration was in the mid section of the soil sample at both sites. This would indicate downward movement of the contaminants rather then fresh deposition. This site has been under notil farming for several years. The overall ratios of DDT to the metabolites indicate the DDT has degraded following a tug of 13.2 years. The vapor phase concentration will generally be 5 to 10 fold more concentrated than the particulate phase. There may be many implications with long term availability of contaminants that can be seen with the 163 \A‘n calculations ix) zero concentration levels being 1J1 several years to thousands of years. We have seen with many previous studies of ZDDT, concentration levels in wildlife will continue to bioaccumulate and compounds of toxicological concern such as DDT will continue to create symptoms in organisms that are exposed. Since DDT has been so persistent, the organisms that die will release the contaminants for future uptake by others and there is a recycling of old contaminants. Levels of DDT continue to persist in the Great Lakes and other bodies of waters and with present trends of long- term degradation there will continue to be hundreds of years before levels are diminished. The ambient levels that are seen in the US are now from applications many years ago and/or from long distance transport. There is evidence of feminization of snapping turtles, alligators, and panther occurring at sites contaminated with p,p'-DDE (de Solla,et al.., 1998). The continued evidence of endocrine disruption from organochlorine contaminants suggests the impact due to DDT will continue. Great Lake (GL) fish consumers were compared to non-GL fish consumers and p,p'-DDE, p,p’—DDT, and o,p’-DDT were detected in all subjects and only DDE was detected in the control group (Anderson, et al.., 1998) in blood samples. o,p’-DDT has induced growth of breast tumors by estrogenic I64 inhibitory action in human breast cancer cells (Verma, 1998) . 165 FUTURE WORK The data supports the persistence of residues many years after legal use in the US has been discontinued due to the ratios of DDT to the metabolites both in the soil and air samples. In future studies the ratio of p,p'-DDT to the metabolites should be looked at to determine the movement and age of the residues. A study under consideration now will look at the same site and include three additional sites in areas considered to not be highly impacted with ZDDT, determined by soil sampling results conducted during the fall of 1998 and winter/spring of 1999. The samplers will also be set collocated with one of the samplers set to collect air from the prevailing wind direction. The two will be compared to find if there does exist a directional component that should be considered to determine the source of the ZDDT. This data should help discriminate between the amount of long range transport that has contributed to the elevated levels found in South Haven, MI and old EDDT from past inputs. 166 .APPENDIX G METHOD III - DDT, DDD, DDE, AND KELTHANE - AIR A» wNi—i LAJNl—i £001.5me U'lubUJNi—i Glassware cleaning: MeOH or CHfifls rinse if dirty before soap wash Soap wash If still dirty 50:50 HZSO4:HNO3 soak overnight Tap water rinse DI water rinse Dry Muffle 450° C for four hours with foil (always use dull side of foil towards glass) on open ends Cool & Store Stainless Steel tools: Soap wash Tap water rinse DI water rinse Dry Wrap in foil & Store Pasteur pipettes & vials: Wrap glass in foil or place in a beaker and cover with foil Muffle at 4500 C for four hours Teflon: Sonicate for 15 minutes in CHMHQ Place in 70"C oven for two hours Store in sealed jar Glass Wool: Put in beaker and cover with foil Muffle at 4500 C for four hours Store 167 .APPENDIX H METHOD III - DDT, DDD, DDE, AND KELTHANE -.AIR Reagent Preparation A. Sodium Sulfate (Nafiflh): 1. Put Nafifih in beaker and muffle at 4500 C for four hours 2. Store in 100 ° C oven B. XADZ: )0Ub is and absorptive polyaromatic resin used for sample preparation and fractionation. In this study it was used as an absorptive sampling device. It has a high affinity for absorption of hydrophobic compounds up to 20,000 molecular weight. 1 Day 1 ii 1. Place XAID in extractor plugged with glass wool 2L Rinse with tap water many times, stirring to remove foam and small particles. Z3.Rinse with small amount of MeOH three times to remove the water l4.A.dd 500 ml of MeOH to 1 l flask 5. Add 20 boiling chips 6.Assemble soxhlet '7.Turn on heater (60-65 setting) 8 9 1 . Turn on chilled water .Cover soxhlet with foil 0.Extract for 24 hours Day 2 . Turn off and cool 15-30 minutes . Flush MeOH from soxhlet as possible. .Add 500 ml of acetone to 1 l flask .Add 20 boiling chips . Turn on heater (45 setting) .Cover soxhlet with foil .Extract for 24 hours \immwai—i Day 3 . Turn off and cool 15-30 minutes . Flush Acetone from soxhlet as possible. . Add 500 ml of hexane to 1 l flask .Add 20 boiling chips . Turn on heater (40-45 setting) .Cover soxhlet with foil .Extract for 24 hours \lmUWiDWNi-i 168 \JONUisbLDNi-i mQCNU'Ib WNi-‘l \10NU'isbbJNi—i QWNH 0" Day 4 . Turn off and cool 15-30 minutes . Flush Hexane from soxhlet as possible. .Add 500 ml of CHJHJ to l l flask ..Add 20 boiling chips Turn on heater (40-50 setting) . Cover soxhlet with foil .Extract for 24 hours Day 5 . Turn off and cool 15—30 minutes Flush CHNHQ from soxhlet as possible. Wait 15 minutes Add 100 ml of hexane to l l flask. Wait 15 minutes and flush. Repeat at least 3 times, until the level in the siphon tube is the same as in the soxhlet. ..Add 500 ml of hexane to a 1 l flask .Add 20 boiling chips . Turn on heater (40-45 setting) . Cover soxhlet with foil .Extract for 24 hours. Flushing may need to be induced before it flushes on its own. Day 6 Turn off and cool 15-30 minutes . Flush hexane from soxhlet as possible. .Add 500 ml of 1:1 acetonezhexane to a 3 l flask .Add 20 boiling chips . Turn on heater (40-45 setting) .Cover soxhlet with foil .Extract for 24 hours Day 7 Turn off and cool 15-30 minutes . Flush Acetone/Hexane from soxhlet as possible. . Pour XAID in a beaker and dry overnight at 65° C .Store in amber bottle in freezer at - 20° C for up to three months . Keep subsample in separate jar for checking lab blank and matrix spike Quartz Fiber Filters (QF): QF are quartz fiber filters placed on the front of the air sampler to collect the particulate fraction of the sample. The nominal particle size collected was > 0.1 um (Monosmith, 1996). l. 2. Each QF is wrapped in aluminum foil and muffled at 450 ° C for four hours Stored in freezer inside a plastic bag 169 APPENDIX I Extraction of XAD; resins and Quartz fiber filters (QF) 1. Supplies: Soxhlet extractor (55/50 & 24/40) Condenser (55/50) 500 ml Round Bottom (RB) Pipettes Boiling chips Acetone Hexane F- Standards CHfiHQ squirt bottle MeOH squirt bottle Glass wool Foil . Heating mantle H 2. XAD2 and QF Extraction: Day 1 Sample set will have samples & one blank, duplicate, & recovery QA sample Label date of collection, sample type, and extraction start day Thoroughly rinse inside of each piece of glassware in contact with the sample first with MeOH then followed by CH2C12 Add 5—6 clean Teflon chips to RB Pour 175 ml of acetone & 175 ml of hexane into RB Put Glass wool into siphon tube Transfer sample to soxhlet extractor: Either XALb or QF Rinse container to remove all XADZ Spike samples at this time onto the XADler QF Turn on the heating mantles to ~45 Turn on water Cover soxhlet with foil Extract for 18 to 24 hours 3. Remove solvent: Day 2 Cool for 15 -20 minutes Pour off solvent and store dark cool place Remove XADZ & boiling chips Rotary evaporate or Turbo-Vap to 2-5 ml Add 75 ml of hexane and take to 2-5 ml Add 75 ml of hexane and take to 2 ml W0 4. Silica Column Chromatography: Supplies: Hexane MeOH CH2C12 Glass wool N32504: 4 % deactivated silica .Activation/Deactivation of silica Put silica in beaker with foil at 100° C Put thermostat to 300° C keep in oven over night Turn oven to 100° C, don't remove silica until the oven has cooled Put silica on counter for ~5—10 minutes Put silica into a desiccator for 2 hours Deactivate with 4 % w/v with DI Shake for about 15 minutes Silica may be stored for 3 days in a stoppered flask Item QF XADz Silica 4-6 g 4-6 g Column size 3.5 inches 3.5 inches N82804 0.5 inches 0.5 inches Elution volume 25 ml 1°t hexane 25 ml 1St (1°C & 2"°l 2nd 1: l hexane fraction) hexane:CHfiflg 2nd 1:1 hexane:CHfiHQ Switching volume 4 ml 4 ml Elution volume 30 ml 3rd MeOH 30 ml 3rd (3rd fraction) MeOH Place ~ 1 cm Glass wool in column Fill column with hexane and add silica in a hexane slurry, tamp column to pack silica 171 Add NaZSO4 Wash column with 25 ml of hexane !!NEVER LET COLUMN GO DR!!! Add sample Elute 1°t fraction with 25 ml of hexane at ~ 1 drip/second Add 4 ml of 1:1 hexane:CHfifl¢ switching solvent Elute the 2m’fraction with 25 ml of 1:1 hexane:CHfiflQ Add 4 ml of MeOH switching solvent Elute the 3rd fraction with 30 ml of MeOH Compound Fraction p,p'-DDE 1st o,p-DDE 1°t p,p'-DDD 2nd o,p-DDD 2nd p,p’-DDT 2nd o,p-DDT 2nd Kelthane 2nd or 3rd Reduce volume to ~ 2 ml with N2 Put in 2 m1 GC autosampler vials and now ready for the GC. 172 APPENDIX J METHOD III - DDT, DDD, DDE, AND KELTHANE - SOIL Reagent Preparation Sodium Sulfate (N32804): 1” Put Nafiflh in beaker wash with CHfiflg and muffle at 400° C for four hours .2.Store in 100 ° C oven Extraction of soil 1. 2. Supplies: Soxhlet extractor (55/50 & 24/40) Condenser (55/50) 500 ml Round Bottom (RB) Thimbles Pipettes Boiling chips Acetone Hexane Standards (fibClz squirt bottle MeOH squirt bottle Glass wool Foil Heating mantle Extraction: Determine percent water by putting weigh about 10 g of soil and place in a oven 100 ° C over night. Reweigh the soil and determine the weight loss and calculate the percent weight loss. Mix 10 g of soil with 10 g anhydrous Nafixh and place in a extraction thimble Place 300 ml of 1:1 Acetone/Hexane in 500 ml RB with 1-2 clean boiling chips Extract for 16-24 hours at 4—6 cycles/hour Drain the extract through ~ 10 g of anhydrous Nafifih into a Turbo-Vap tube Add ~ 10 ml of hexane to Turbo—Vap tube to remove acetone and reduce volume to ~ 10 ml Silica Column Chromatography: Supplies: Hexane MeOH W3 CH2C12 Glass wool anhydrous Nafiflh dried at 400 ° C for 4 hours 4 % deactivated silica 100-200 mesh .Activation/Deactivation of silica Put silica in beaker with foil at 160° C, in oven over night Put silica on counter for ~5-10 minutes Put silica into a desiccator for 2 hours Deactivate with 3.3 % w/v with DI Shake for about 15 minutes Silica may be stored for 3 days in a stoppered flask Store in a desiccator Place ~ 1 cm Glass wool in 1 cm diameter column Fill column 3 g of silica Top with 2-3 cm of anhydrous Nafifih Wash column with 10 ml of hexane !!NEVER LET COLUMN GO DR!!! Don’t collect first hexane, stop when almost to top of anhydrous Nazsoq Add sample, rinse with 1-2 ml hexane twice Elute 1St fraction with 80 ml of hexane at ~ 1 drip/second Elute the 2m‘fraction with 50 ml of 1 hexane Add 4 ml of CHfifle switching solvent Elute the 3rd fraction with 15 ml of CHJHJ The CH2C12 (3rd fraction) must be changed to hexane for CC Reduce volume to ~ 0.5 ml with Turbo—Vap Add 10 ml of hexane Reduce volume to ~ lml with stream of N2 Put in 2 ml GC autosampler vials and now ready for the CC. 174 APPENDIX K Concentration Data Combined for vapor & Particulate for All Sites Concentration Total Date o,p-DDE p,p-DDE o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kelthane Sit. A 99/1113 pg/m3 pg/m3 p9/m3 pg/m3 pg/m3 pg/ma 4/14/98 0 453 1.7 38.7 143.6 121.5 0 4/20/98 0 461.3 182.3 209.9 121.5 113.3 0 4/26/98 8.2 27.2 0 0 10.9 27.2 0 5/2/98 517.2 204 0 4160.9 54.6 158 0 5/4/98 0 827.7 70.6 0 175.1 440.7 0 5/6/98 1104.8 331.4 2.8 36.8 0 155.8 0 5/8/98 644.1 1149.7 65 62.1 135.6 53.7 0 5/10/98 312.5 869.3 8.5 0 110.8 508.5 65.3 5/12/98 0 579.4 0 70.6 273.5 167.6 0 5/14/98 6409.2 1418.5 30.8 52.3 292.3 443.1 0 5/16/98 421.8 716.8 0 0 112.1 286.1 0 5/19/98 162.8 308.1 0 0 0 101.7 0 5/25/98 450.7 305.9 0 26.3 0 6.6 0 6/1/98 0 552 0 0 118.5 156.1 37.6 6/6/98 110.7 384.4 13 645 166.1 19.5 0 6/12/98 0 947.7 0 0 393.8 249.2 0 6/18/98 561.3 1107.4 0 0 417.2 239.3 0 6/24/98 0 1489.9 0 0 439.6 399.3 573.8 6/30/98 0 656.5 0 255.3 82.1 240.1 97.3 7/6/98 0 506.2 0 0 0 139.8 105.6 7/13/98 0 666.7 0 0 0 397.3 0 7/18/98 0 273.7 0 0 0 200 0 7/24/98 0 212 O 0 0 144.9 0 7/30/98 0 284.8 0 0 162.3 0 29.8 8/5/98 0 352.2 0 0 59.7 323.9 210.7 8/11/98 0 4187.2 13 7 0 913.2 1292.2 1064 8/17/98 0 1383.6 188.4 1373.3 763.7 804.8 366.4 8/23/98 0 959.1 348 1576 450.3 482.5 403.5 W5 Concentration Data Combined for vapor & Particulate for All Sites Concentration Total Date o,p-DDE p,p-DD]: o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kelthane SHaIB revm3 pehm! pqhfi! pghfi! pghm3 pghfi! pehfii 4/14/98 0 899.7 25.8 74.5 140.4 289.4 0 4/20/98 0 157.9 91.4 285.3 52.6 52.6 0 4/26/98 0 0 0 0 26.9 0 0 5/2/98 1470.4 647.9 0 3143.7 583.1 281.7 0 5/4/98 0 610.3 83.1 157.6 151.9 452.7 0 5/6/98 1868.9 774.9 31.3 0 151 378.9 0 5/8/98 1360 3217.1 0 0 111.4 220 0 5/10/98 258.7 456.4 23.3 29.1 72.7 186 90.1 5/12/98 24.6 815.4 9.2 43.1 261.5 310.8 0 5/14/98 6741.2 2644.1 47.1 76.5 450 923.5 0 5/16/98 582.4 2600 0 120.6 411.8 897.1 908.8 5/19/98 545.2 670.6 0 0 151.6 498.5 0 5/25/98 609.2 190.8 0 221.5 40 153.8 0 5/31/98 420.1 1381.9 0 163.2 402.8 937.5 246.5 6/6/98 0 484.9 0 0 177.3 0 210.7 6/12/98 6/18/98 686.1 1664.2 0 0 54.7 594.9 138.7 6/24/98 0 2985.3 0 0 584.6 1136 860.3 6/30/98 0 1123.3 0 0 133.3 420 73.3 7/6/98 0 1566.3 0 0 2336.6 563.1 576.1 7/13/98 0 1094.3 0 0 154.7 562.3 0 7/18/98 0 560.6 0 0 0 155.3 1227 7/24/98 26467 3106.6 2459.6 0 0 606.6 0 7/30/98 0 1044.6 0 0 583.6 252.8 713.8 8/5/98 0 143.9 0 0 0 214 110.7 8/11/98 0 840.9 0 0 155.8 490.3 87.7 8/17/98 0 923.3 0 1303.1 641.1 756.1 355.4 8/23/98 0 1441.6 353.9 1535.7 571.4 1048.7 0 176 Concentration Data Combined for vapor & Particulate for All Sites Concentration Date Site c P9/m3 Total o,p-DD! p,p-DD! o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kelthane pg/n3 p9/m3 pg/ms pg/ms pg/mS palms 4/14/98 0 881.8 43.2 0 118.2 377.5 60.5 4/20/98 0 279.5 90.4 432.9 158.9 169.9 0 4/26/98 0 47 0 0 5.5 19.3 0 5/2/98 61.3 119.8 0 0 0 103.1 0 5/4/98 0 1463.1 79.5 0 267 698.9 0 5/6/98 2389.5 848.8 32 0 162.8 427.3 0 5/8/98 724.9 426.9 0 0 60.2 126.1 0 5/10/98 709.5 930.2 16.8 0 139.7 318.4 0 5/12/98 823.4 524.2 11.4 0‘ 94 208 0 5/14/98 8303.8 2660.8 50.1 56 413 985.3 0 5/16/98 452.8 2175.9 0 0 342 772 697.1 5/19/98 20.5 231 950.3 444.4 701.8 17.5 0 5/25/98 408.6 425.7 0 14.3 125.7 308.6 0 5/31/98 234.1 1075.1 0 106.9 306.4 774.6 216.8 6/6/98 183.9 554.8 6.5 0 90.3 0 345.2 6/12/98 0 2346.7 0 0 1186.7 986.7 0 6/18/98 1015.3 1993.9 0 0 896 737 978.6 6/24/98 0 2724.8 0 0 516.8 97.3 926.2 6/30/98 0 0 0 0 0 0 0 7/6/98 0 2211.5 0 0 3305.1 897.3 1695 7/13/98 0 1107.6 0 0 191 527.8 0 7/18/98 0 626.7 0 0 0 200 1183 7/24/98 0 931.3 0 0 20.6 408.9 852.2 7/30/98 0 558.6 0 0 248.3 110.3 0 8/5/98 0 134.6 0 0 134.6 128.4 474 8/11/98 0 104.6 0 0 0 70.8 147.7 8/17/98 0 897.5 0 0 378.1 473.5 254.4 8/23/98 0 2180.1 540.4 0 897.5 1360.2 767.1 177 Concentration Data Combined for vapor & Particulate for.All Sites Concentration Total Date o,p-DDE p,p-DD! o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kelthane sit. D p9/m3 pq/m3 pg/m3 pg/m3 pg/m3 99/1113 pg/ms 4/14/98 0 201.1 0 0 152.2 95.1 0 4/20/98 0 221.6 75.7 186.5 210.8 40.5 0 4/26/98 0 85.5 13 1497.4 51.8 54.4 0 5/2/98 72 85.3 0 0 80 722.7 0 5/4/98 0 516.2 27 370.3 113.5 210.8 0 5/6/98 217.4 236.4 0 356 54.3 92.4 0 5/8/98 186.8 986.3 0 0 159.3 313.2 0 5/10/98 116.5 411.9 0 173.4 243.9 113.8 0 5/12/98 611.4 274.5 0 0 59.8 95.1 0 5/14/98 1250 505.4 0 0 81.5 163 135.9 5/16/98 873.1 398.8 0 0 102.7 160.1 102.7 5/19/98 0 37.9 0 54.2 0 35.2 0 5/25/98 23.8 58.2 0 0 10.6 31.7 0 5/31/98 0 19 0 0 0 0 0 6/6/98 156.3 223.7 0 0 32.3 0 0 6/12/98 0 390.9 0 0 420.2 153.1 0 6/18/98 1381.8 48.5 0 0 0 0 0 6/24/98 0 876.9 0 0 83.1 181.5 1019 6/30/98 0 1724.6 0 0 142 576.8 202.9 7/6/98 1325.8 522.5 0 0 132 101.1 306.2 7/12/98 0 0 0 0 23.4 163.9 0 7/18/98 0 450.2 0 O 0 250.8 0 7/24/98 0 0 0 0 0 0 0 7/30/98 0 161 0 0 102.7 0 0 8/5/98 0 281.4 0 0 59.9 6 0 8/11/98 0 48 O 0 0 78.1 0 8/17/98 275.4 592.8 137.7 1038.9 524 559.9 134.7 8/23/98 0 1031.7 0 1584.5 500 489.4 778.2 178 z... ..me APPENDIX L Concentration Data Combined for Vapor Phase for All Sites Data 0 , p-DDI: p , p-DDE o , p-DDD p , p-DDD o , p-DDT p , p-DDT Kc]. than ng/ Sump ng/ Sump ng/ Samp ng/ Samp ng/ Samp ng/ Samp ng/ Sump Site A 4/14/98 0 164 0.6 14 52 44 0 4/20/98 0 131 28 0 34 18 0 4/26/98 0 0 0 0 4 0 0 5/2/98 180 71 0 0 19 55 0 5/4/98 0 293 12 0 62 138 0 5/6/98 353 116 0 13 0 53 0 5/8/98 208 405 23 22 48 11 0 m 97 306 3 0 39 174 23 5/12/98 0 197 0 24 93 55 0 5/14/98 2061 460 10 17 95 141 0 5/16/98 143 242 0 0 38 97 0 5/19/98 48 98 0 0 0 31 0 5/25/98 134 93 0 8 0 0 0 6/1/98 0 191 0 41 54 13 6/6/98 34 118 4 198 51 0 0 6/12/98 0 308 0 0 128 81 0 6/18/98 183 361 0 0 136 78 0 6/24/98 0 444 0 0 131 119 171 6/30/98 0 216 0 84 27 79 32 7/6/98 0 163 0 0 0 45 0 7/13/98 0 99 0 0 0 59 0 7/18/98 0 78 0 0 0 55 0 7/24/98 0 60 0 0 0 41 0 7/30/98 0 86 0 0 49 0 0 8/5/98 0 112 0 0 19 103 67 8/11/98 0 878 3 0 200 283 233 8/17/98 0 243 0 0 110 112 77 8/23/98 0 328 119 539 154 165 138 179 Concentration Data Combined for Vapor Phase for All Sites Data o,p-DDE p,p-DD]: o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kolthan ng/Samp ng/Sanp ng/Samp ng/Samp ng/Samp ng/Sunp ng/Samp Site 8 4/14/98 0 291 5 26 49 74 0 4/20/98 0 26 0 38 10 0 0 4/26/98 0 0 0 0 0 0 0 5/2/98 518 230 0 48 207 100 0 5/4/98 0 206 12 6 53 124 0 5/6/98 611 271 8 0 53 130 0 5/8/98 464 1126 0 0 39 77 0 5/10/98 71 157 8 10 23 53 31 5/12/98 0 265 3 14 85 99 0 5/14/98 2286 894 16 26 153 307 0 5/16/98 193 876 0 41 140 301 309 5/19/98 187 220 0 0 52 165 0 5/25/98 188 62 0 72 13 50 0 5/31/98 121 390 0 47 116 253 71 6/6/98 0 145 0 O 53 0 63 6/12/98 0 637 0 0 324 272 0 6/18/98 188 456 0 O 15 131 38 6/24/98 0 812 0 0 159 309 234 6/30/98 0 337 0 0 40 126 22 7/6/98 0 484 0 0 722 174 0 7/13/98 0 290 0 0 41 145 0 7/18/98 0 148 0 0 0 41 324 7/24/98 7199 845 669 0 0 165 0 7/30/98 0 281 0 0 157 68 192 8/5/98 0 39 0 0 0 58 30 8/11/98 0 259 0 0 48 151 27 8/17/98 0 265 0 0 98 117 71 8/23/98 0 444 109 473 176 203 0 180 Concentration Data Combined for Vapor Phase for All Sites Data o,p-DD! p,p-DD! o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kolthan ng/Samp ng/Samp ng/Samp nq/Samp ng/Samp ng/Samp ng/Samp Sit. C 4/14/98 0 267 10 0 41 111 0 4/20/98 0 70 0 93 50 44 0 4/26/98 0 9 0 0 2 0 0 5/2/98 22 43 0 0 0 15 0 5/4/98 0 515 16 0 94 222 0 5/6/98 710 269 3 0 51 138 0 5/8/98 239 149 O 0 21 44 0 5/10/98 220 333 6 0 50 112 0 5/12/98 289 184 4 0 33 71 0 5/14/98 2759 894 17 19 140 321 0 5/16/98 133 658 0 0 105 228 214 5/19/98 0 0 325 152 240 0 0 5/25/98 143 149 0 5 44 108 0 5/31/98 81 363 0 37 106 243 75 6/6/98 57 172 2 0 28 0 107 6/12/98 0 704 0 0 356 296 0 6/18/98 332 652 0 0 293 203 320 6/24/98 0 812 0 0 154 29 276 6/30/98 0 0 0 0 0 0 0 7/6/98 0 732 0 0 1094 297 344 7/13/98 0 319 0 0 55 151 0 7/18/98 0 188 0 0 0 60 355 7/24/98 0 271 0 0 6 119 188 7/30/98 0 162 0 0 72 32 0 8/5/98 0 44 0 0 44 42 155 8/11/98 0 0 0 0 0 23 0 8/17/98 0 254 0 0 107 134 72 8/23/98 0 702 174 0 289 323 247 181 Concentration Data Combined for Vapor Phase for All Sites Data o,p-DDE p,p-DDE o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kolthan ng/Samp ng/Samp ng/Samp ng/Sunp ng/Sulp ng/Samp ng/Samp Site D 4/14/98 0 74 0 0 56 35 0 4/20/98 0 82 0 0 71 0 0 4/26/98 0 26 2 60 13 21 0 5/2/98 27 21 0 0 0 267 0 5/4/98 0 191 10 11 42 78 0 5/6/98 78 87 0 0 20 30 0 5/8/98 68 359 0 0 58 113 0 5/10/98 43 152 0 64 90 42 0 5/12/98 213 96 0 O 22 25 0 5/14/98 451 177 0 0 30 45 50 5/16/98 286 132 0 0 34 53 34 5/19/98 0 0 0 20 0 6 0 5/25/98 9 21 0 0 4 12 0 5/31/98 0 0 0 0 0 0 0 6/6/98 58 83 0 0 12 0 0 6/12/98 0 120 0 0 129 47 0 6/18/98 456 16 0 0 0 0 0 6/24/98 0 285 0 0 27 59 331 6/30/98 0 595 0 0 49 199 70 7/6/98 472 186 0 O 47 36 109 7/12/98 0 0 0 0 7 49 0 7/18/98 0 0 0 0 0 0 0 7/24/98 0 0 0 0 0 0 0 7/30/98 0 47 0 0 30 0 0 8/5/98 0 94 0 0 20 2 0 8/11/98 0 16 0 0 0 26 0 8/17/98 92 198 46 347 104 97 45 8/23/98 0 293 0 450 142 139 221 182 .APPENEHD( Id Concentration Data Combined for Particulate Phase for All Sites Site A o,p-DD! p,p-DDE o,p-DDD p,p-DDD o,p-DDT p,p-DDT Xelthan Date ng/Sam ng/Sam ng/ Sam Ng/ Sam ng/Sam ng/Sam ng/Sam '7713537 0 0 0 4/20/98 36 3 76 4/26/98 10 0 5/2/98 1448 5/4/98 5/6/98 37 'EWSGE' 5/10/98 5/12/98 5/14/98 5/16/98 5/19/98 5/25/98 6/1/98 6/6/98 6/12/98 6/18/98 6/24/98 6/30/98 7/6/98 7/13/98 7/18/98 7/24/98 7/30/98 8/5/98 8/11/98 8/17/98 8/23/98 OOLOOO O H HN OW O H HN OLAJO N N OOOOOOOOOOOOOOOOOOO k0 OOOkOOOOOOOOOCOb—‘HOONHO (.0 b OOOOOOOOOOOOOOOOOOOOHWOOGDO U1 00 OOOOKOOOO 1—‘ ox H U‘ 01 b O ...: H OWOOOOOOOOOOOOOOOOOOOOOOOOOO H N OWOOOONKOOOOOOQONDOWNU‘WN030 Of—‘OOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOWCDO O O 183 Concentration Data Combined for Particulate Phase for All Sites Site 3 o,p-DD]: p,p-DDE o,p-DDD p,p-DDD o,p-DDT p,p-DDT Rolthan Dab. ng/ Sam ng/Sam ng/Sam Ng/Sam ng/Sam ng/Sam ng/Sun '4714798' 0 23 4 0 0 27 0 4/20/98 0 31 33 65 9 19 0 4/26/98 0 0 0 0 10 0 0 5/2/98 4 0 0 1068 0 0 0 5/4/98 0 7 17 49 0 34 0 5/6/98 45 1 3 o 0 3 0 5/8/98 12 o 0 0 0 0 0 5/10/98 18 0 0 0 2 11 0 5/12/98 8 0 0 0 0 2 0 5/14/98 6 5 0 0 0 7 0 5/16/98 5 8 0 0 0 4 0 5/19/98 0 10 0 o 0 6 0 5/25/98 10 0 0 0 0 0 0 5/31/98 0 8 0 o 0 17 0 6/6/98 0 0 0 0 0 0 0 6/12/98 0 0 0 0 o 0 0 6/18/98 0 0 0 0 0 32 0 6/24/98 0 0 0 0 0 0 0 6/30/98 0 0 0 0 0 0 0 7/6/98 0 0 0 0 0 0 178 7/13/98 0 0 0 0 0 4 0 7/18/98 0 0 0 0 0 0 0 7/24/98 0 0 0 0 o 0 0 7/30/98 0 0 0 0 0 0 0 8/5/98 0 0 0 0 0 0 0 8/11/98 0 0 0 0 0 0 0 8/17/98 0 0 0 374 86 100 31 8/23/98 0 o 0 0 0 120 o 184 Concentration Data Combined for Particulate Phase for All Sites Site C o,p-DDE p,p-DDE o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kalthan Date ng/Sam ng/Sam ng/Sam Ng/Sam ng/Sam ng/Sam ng/Sam 7Efifiafifir 0 39 5 0 0 20 21 4/20/98 0 32 33 65 8 18 0 4/26/98 0 8 0 0 0 7 0 5/2/98 0 0 0 0 0 22 0 5/4/98 0 0 12 0 0 24 0 5/6/98 112 23 8 0 5 9 0 5/8/98 14 0 0 0 0 0 0 5/10/98 34 0 0 0 0 2 0 5/12/98 0 0 0 0 0 2 0 5/14/98 56 8 o o 0 13 0 5/16/98 6 10 0 0 0 9 0 5/19/98 7 79 0 0 0 6 0 5/25/98 0 0 0 0 0 0 0 5/31/98 0 9 0 0 0 25 0 6/6/98 0 0 0 0 0 0 0 6/12/98 0 0 o 0 0 0 0 6/18/98 0 0 o 0 0 38 0 6/24/98 0 0 0 0 0 0 0 6/30/98 0 0 0 0 0 0 0 7/6/98 0 0 0 0 0 0 217 7/13/98 0 0 0 0 0 1 0 7/18/98 0 o 0 0 0 0 0 7/24/98 0 o 0 0 0 0 60 7/30/98 0 0 0 0 0 0 0 8/5/98 0 0 0 0 0 0 0 8/11/98 0 34 0 0 0 0 48 8/17/98 0 0 0 0 0 0 0 8/23/98 0 o 0 0 0 115 0 185 Concentration Data Combined for Particulate Phase for All Sites Site D o,p-DDE p,p-DDE o,p-DDD p,p-DDD o,p-DDT p,p-DDT Kolthan Date ng/ Sam ng/ Sam ng/Sam Ng/Sam ng/Sam ng/Sam ng/Sam =4714798' 0 0 0 0 0 0 0 4/20/98 0 o 28 69 7 15 0 4/26/98 0 7 3 518 7 0 0 5/2/98 0 11 0 0 30 4 0 5/4/98 0 0 0 126 0 0 0 5/6/98 2 0 0 131 0 4 0 5/8/98 0 0 0 0 0 1 0 5/10/98 0 o 0 0 0 0 0 5/12/98 12 5 0 o 0 10 0 5/14/98 9 9 0 0 0 15 0 5/16/98 3 0 0 0 0 0 0 5/19/98 0 14 0 0 0 7 0 5/25/98 0 1 o 0 o 0 0 5/31/98 0 7 0 o 0 0 0 6/6/98 0 0 0 0 0 0 0 6/12/98 0 0 0 0 0 0 0 6/18/98 0 0 0 0 0 0 0 6/24/98 0 0 o 0 0 0 0 6/30/98 0 0 0 0 0 0 0 7/6/98 0 o 0 0 0 0 0 7/12/98 0 0 0 0 0 0 0 7/18/98 0 149 0 0 0 83 0 7/24/98 0 0 0 0 o 0 0 7/30/98 0 0 0 o 0 0 0 8/5/98 0 0 0 0 0 0 0 8/11/98 0 o 0 o 0 0 0 8/17/98 0 0 0 o 71 90 0 8/23/98 0 0 0 0 0 0 0 186 Appendix N Sample Volume, Pressure, and Temperature Data for All Sites Pressure data collected at Holland Temp. data collected at each site: Coloma (260210014) and S. Haven (260050002) 3 Date Sampler Sampled Volume, m avg P, m avg T, No. Hg deg C 4/14/98 c 347 737 11 4/14/98 b 349 737 11 4/14/98 a 362 737 11 4/14/98 d 368 737 11 4/20/98 d 370 747 10 4/20/98 c 365 747 11 4/20/98 b 361 747 11 4/20/98 a 362 747 11 4/26/98 d 386 744 8 4/26/98 c 362 744 9 4/26/98 B 372 744 9 4/26/98 A 368 744 9 5/2/98 D 375 736 9 5/2/98 c 359 736 9 5/2/98 d 355 736 9 5/2/98 a 348 736 9 5/4/98 d 370 739 14 5/4/98 c 352 739 14 5/4/98 b 349 739 14 5/4/98 a 354 739 14 5/6/98 d 368 740 18 5/6/98 c 344 740 19 5/6/98 b 351 740 19 5/6/98 a 353 740 19 5/8/98 d 364 737 19 5/8/98 c 349 737 19 5/8/98 b 350 737 19 5/8/98 a 354 737 19 5/10/98 d 369 743 15 5/10/98 c 358 743 15 5/10/98 b 344 743 15 5/10/98 a 352 743 15 5/12/98 d 368 741 18 5/12/98 c 351 741 1 5/12/98 b 325 741 18 5/12/98 a 340 741 18 5/14/98 d 368 746 18 5/14/98 c 339 746 19 187 Date 5/14/98 5/16/98 5/16/98 5/16/98 5/16/98 5/19/98 5/19/98 5/19/98 5/19/98 5/25/98 5/25/98 5/25/98 5/25/98 5/31/98 5/31/98 5/31/98 6/1/98 6/6/98 6/6/98 6/6/98 6/6/98 6/12/98 6/12/98 6/12/98 6/12/98 6/18/98 6/18/98 6/18/98 6/18/98 6/24/98 6/24/98 6/24/98 6/24/98 6/30/98 6/30/98 6/30/98 6/30/98 7/6/98 7/6/98 7/6/98 7/6/98 7/12/98 7/13/98 7/13/98 7/13/98 7/18/98 7/18/98 Sampler' Sampled‘VOlume, m No. 00.9)0‘0CLO)U'OQWD‘OQQ’U‘OQDJ0'0QWU‘OQD’C‘OQ—mUOQDJU'OQQJU‘OQ—DJU‘OQQJ 325 331 307 340 339 369 342 343 344 378 350 325 304 368 346 288 346 371 310 299 307 307 300 286 325 330 327 274 326 325 298 272 298 345 316 300 329 356 331 309 322 299 288 265 297 331 300 188 3 avg P, mm Hg 746 743 743 743 743 744 744 744 744 743 743 743 743 736 736 736 740 747 747 747 747 735 735 735 735 744 744 744 744 744 744 744 744 737 737 737 737 745 745 745 745 746 744 744 744 744 744 avg T, deg C 19 22 22 22 22 22 22 22 22 13 13 13 13 20 20 20 15 10 10 10 10 22 21 21 21 24 24 24 24 27 27 27 27 21 21 21 21 23 24 24 24 20 23 23 23 23 23 Date 7/18/98 7/24/98 7/24/98 7/24/98 7/24/98 7/30/98 7/30/98 7/30/98 7/30/98 8/5/98 8/5/98 8/5/98 8/5/98 8/11/98 8/11/98 8/11/98 8/11/98 8/17/98 8/17/98 8/17/98 8/17/98 8/23/98 8/23/98 8/23/98 8/23/98 Sampler Sampled Volume, m N DJUOQwU‘OO—WUOQWU‘OQWC‘OQWUOQWO 285 334 291 272 283 292 290 269 302 334 327 271 318 333 325 308 219 334 283 287 292 284 322 308 342 189 3 avg P, mm Hg 744 747 747 747 747 745 745 745 745 747 747 747 747 746 746 746 746 745 745 745 745 741 741 741 741 avg T, deg C 23 21 21 21 21 21 22 22 22 22 22 22 22 21 21 21 21 23 23 23 23 27 27 27 27 APPENDIX 0 Soil Concentration Data Site A Soil Data in ug/g Site/Date Site A/East Site A/West épril T_op Middle Bottom 1122 Middle Bottom o,p-DDE 0 34 O 13 13 0 p,p-DDE 1 3044 0 1341 1434 0 o,p-DDD 0 194 0 66 71 0 p,p-DDD O 395 O 125 68 0 o,p-DDT 0 781 0 317 361 O p,p-DDT 3 6804 0 2575 2816 0 Kelthane 0 3369 0 1609 1873 0 Sept o,p-DDE 40 80 0 40 0 0 P,p-DDE 11700 1411 260 7750 0 100 o,p-DDD 480 760 0 320 0 0 P,p—DDD 210 150 0 120 0 0 o,p-DDT 3860 6170 20 2730 10 30 P,p-DDT 22180 232290 280 14920 50 180 Kelthane .1750 2400 20 920 0 20 190 Soil Concentration Data Site BC Site/Date Site BC/North Site BC/South Qril Top/dug Middle Bottom 192 Middle Bot/dug o,p-DDE 19.5 18 4 O 26 1 p,p-DDE 2563.5 2055 570 2101 3184 472.5 o,p-DDD 140 84 20. 95 149 14.5 p,p-DDD 308 59 28 204 521 24.5 o,p-DDT 673 463 129 528 709 101.5 p,p-DDT 5582.5 4012 1009 4040 6121 796 Kelthane 2878.5 0 427 0 0 198 Sept o,p-DDE 10 0.01 0 10 0 0 p,p-DDE 23350 22270 310 25700 4700 0 o,p-DDD 0 0 10 0 30 0 p,p-DDD 40 100 O 70 10 0 o,p-DDT 410 310 0 530 120 0 p,p-DDT 8410 7230 30 10500 2820 0 Kelthane 400 350 10 410 110 0 1m Soil Concentration Data Site D Site/Date Site D/East Site D/West firil Tpp Mid/dup Bottom T_op Middle Bottom o,p-DDE O 0 2 0 6 3 p,p-DDE 0 125.5 241 271 535 439 o,p-DDD 0 2 2 4 5 4 p,p—DDD 0 8.5 9 16 20 35 o,p-DDT 0 9 22 110 91 58 p,p-DDT 0 113.5 242 1096 840 528 Kelthane O 41 0 187 0 0 Sept o,p-DDE 10 20 30 0 10 20 p,p-DDE 2650 2350 1580 2320 2270 2770 o,p-DDD 10 10 10 10 20 30 p,p—DDD 20 10 10 20 20 40 o,p—DDT 500 390 40 270 550 970 p,p-DDT 3810 2570 1950 3110 4110 7550 Kelthane 70 50 20 60 130 260 192 APPENDIX P Wind Speed and Direction for Sample Days at SH Date Wind Speed (mph) Resultant Wind Direction 4/14/98 6.1 SSE 4/20/98 4.9 W 4/26/98 9.9 ENE 5/2/98 4.4 WNW 5/4/98 4.5 WNW 5/6/98 3.4 SSE 5/8/98 9.3 NE 5/10/98 7 NE 5/12/98 4.6 SSE 5/14/98 2.4 W 5/16/98 12.5 WSW 5/19/98 6.5 SSW OR WSW 5/25/98 8.3 WNW 6/1/98 4.8 WSW OR ESE 6/6/98 6 WNW 6/12/98 13.6 SW 6/18/98 5.2 ESE 6/24/98 6.3 sw 6/30/98 10.3 NW 7/6/98 5.4 SW 7/13/98 5 W 7/18/98 4 W 7/24/98 5 NW 7/30/98 6 NNW OR NW ‘8/5/98 4.6 E 8/11/98 5.8 NNE 8/17/98 5.5 SW 8/23/98 12.6 SW 193 Wind Speed and Direction for Sample Days at CLM Date Wind Speed (mph) Resultant Wind Direction 4/14/98 5 NW 4/20/98 3 N 4/26/98 8 ENE 5/2/98 3 W 5/4/98 3 NE 5/6/98 4 SSE 5/8/98 9 NE 5/10/98 6 NNW 5/12/98 7 NE 5/14/98 2 SSE 5/16/98 9 WSW 5/19/98 5 SW 5/25/98 5 WNW 6/1/98 4 W 6/6/98 10 NW 6/12/98 6 SW 6/18/98 6 SE 6/24/98 7 S 6/30/98 5 NNW 7/6/98 2 SSW 7/13/98 2 WNW 7/18/98 2 SE 7/24/98 4 NNW 7/30/98 4 NNW 8/5/98 3 ENE 8/11/98 5 N OR NNW 8/17/98 4 SW 8/23/98 8 SSW 194 APPENDIX Q Calculation of experimental half—life of DDT Known Data: log 27 = log 100 - (k x 25 years)/2.303 80 k = 0.052 So log 50 = log 100 — (0.052 x years)/2.303 Therefore years = 13.2 years = tuz 195 Appendix R Soil Texture and Moisture Content Sample name Soil Carbon Sand % Silt % Clay %:Mbistur*Type Depth* % e % A 110 SE 15 N 2 1.43 68.9 18.4 12.7 16.0 sandy loam A 110 SE 15 N M4 1.14 66.6 22.7 10.7 12.1 sandy loam A 110 SE 15 N B4 0.41 60.9 18.4 20.7 10.8 sandy clay loam A 15 N 105 of 2 1.73 64.9 22.4 12.7 16.0 sandy SE loam A 15 N 105 of M4 1.59 62.9 22.4 14.7 16.2 sandy SE loam A 15 N 105 of B4 0.42 80.9 24.4 14.7 12.9 sandy SE loam Coloma W 2 0.52 84.9 4.7 10.4 6.1 loamy sand Coloma M4 0.64 90.3 5.4 4.4 7.6 sand W Coloma B4 1.57 84.9 4.4 10.7 10.5 loamy W sand Coloma 50 E 2 1.55 81.2 7.9 10.9 13.4 loamy sand Coloma 50 E M4 0.85 84.5 9.2 6.4 11.2 loamy sand Coloma 50 E B4 0.64 86.5 8.2 5.4 8.0 loamy sand B/C 15 W 270 SE 2 1.72 72.9 14.4 12.7 16.3 sandy loam B/C 15 W 270 SE M4 1.22 78.9 8.4 12.7 10.5 sandy loam B/C 15 W 270 SE B4 0.77 74.9 10.4 14.7 9.2 sandy loam B/C 15 W 268 N 2 1.82 72.9 16.4 10.7 13.6 sandy of SE loam B/C 15 W 268 N M4 1.80 78.6 10.7 10.7 14.2 sandy of SE loam B/C 15 W 268 N B4 1.01 76.9 14.4 8.7 13.2 sandy of SE loam SW2NE Comp. 1.22 66.9 18.4 14.7 14.1 sandy loam SEZNW Comp. 1.25 70.9 14.4 14.7 12.7 sandy loam *2=top two inches M4=middle 4 inches B4=bottom 4 inches comp.=composite 196 Bibliography Anderson, H. A., et al. 1998. Profiles of Great Lake Critical Pollutants: A Sentinel Analysis of Human Blood and Urine. Environmental Health Prespectives. 106:5 279-289. De Solla, S. R., Bishopo, C. A., Van Der Kraak, G., Brooks, R. J. 1998. Impact of Organochlorine Contamination of Levels of Sex Hormones and External Morphology of Common Snapping Turtles (Chelydra serpentina serpentine) in Ontario, Canada. Environmental Health Prespectives. 106:5 253-260. Extoxnet. 1993. Pesticide Management Education Program. Cornell University. Ithaca, N.Y. Hippelein, M. and McLachlan, M. S. 1998. Soil/Air Partitioning of Semivolatile Organic Compounds. 1. Method Development and Influence of Physical—Chemical Properties. Environ. Sci. Techno., Vol. 32, No. 2. 310-316. Hoff, F. M., Muir, D. C. G., Grift, N. P. 1992. Annual Cycle of Polychlorinated Biphenyls and Organohalogen Pesticides in Air in Southern Ontario. 1. Air Concentration Data. Environ. Sci. Techno., Vol. 26, No. 2. 266-275. Iwata, H., Tanabe, S., Sakai, N., Tatsukawa, R. 1993. Distribution of Persistent Organochlorines in the Oceanic Air and Surface Seawater and the Role of Ocean on Their Global Transport and Fate. Environmental Science & Technology. 27. 6. 1080-1098. McConnell, L. L., Cotham, W. E., Bidleman, T. F. 1993. Environmental Science & Technology. 27. 1304-1311. Mischke, T., Brunetti, K., Acosta, V., Weaver, D., and Brown, M. 1985. Agricultural Sources of DDT Residues in California's Environment, Environmental Hazards Assessment Program Report. California Department of Food and Agriculture. Monosmith, C. L. and Hermanson, M. H. 1996. Spatial and Temporal Trends of Atmospheric Organochlorine Vapors in the Central and Upper Great Lakes. Environmental Science & Technology 30(12)3464-3472. 197 Muir, D. C. G., Segstro, M. D., Welbourn, P. M., Toom, D., Eisenreich, S. J., Macdonald, C. R., Welpdale, D. M. 1993. Patterns of Accumulation of Airborne Organochlorine Contminants in Lichens from the Upper Great Lakes Region of Ontario. Environmental Science & Technology. 27. 1201-1210. MUller—Herold, U. 1996. A Simple General Limiting Law for the Overall Decay of Organic Compounds with Global Pollution Potential. Environmental Science & Technology. 30. 586-591. Odermatt, J. R., Johnson, T. A. and Hummeldorf, R. G. 1993. Distribution of DDT Residues (DDT, DDD, and DDE) in California Soils. J. Soil Contamination. 2(4). Quensen III, J. F., Mueller, 5. A., Jain, M. K. , Tiedje, J. M. 1998. Reductive Dechlorination of DDE to DDMU in Marine Sediment Microcosms. Science. Vol.280. May 1, 1998. 722-724. Renner, R. 1998. “Natural ‘ Remediation of DDT, PCBs Debated. Environmental Science & Technology. 32. 15. 360A+ 363A. Spencer, W. F. and Cliath, M. M. 1990. Movement of Pesticides from Soil to the Atmosphere. Long Range Transport of Pesticides. 1-16. Stanley, C. W., Barney II, J. E., Helton, M. R., Yobs, A. R. 1971. Measurements of Atmospheric Levels of Pesticides. Environmental Science & Technology. Vol. 5. No. 5. 430-435. Swackhammer, D. L., McVeety, B. D., Hites, R. A. 1988. Environmental Science & Technology. 22. 664. VanMetre, P. C., Callender, E., Fuller, C. C. 1997. Historical Trends in Organochlorine Compounds in River Basins Identified Using Sediment Cores from Reservoirs. Environmental Science & Technology. 31. 8. 2339-2344. Verma, S. P., Goldin, B. R., Lin, P. S. 1998. The Inhibition of the Estrogenic Effects of Pesticides and Environmental Chemicals by Curcumin and Isoflavonoids. Environmental Health Perspectives. 106:12. 807—812. WHO. DDT and it’s Deriviatives. 1979. World Health Organization. 88-114. 198 (HI1111111111)((1111111)!)(1(I