Document jgKKNKr6zKKd86yjnDwEm4Nxk

PFOS: A TOXICITY TEST TO DETERMINE THE EFFECTS OF THE TEST SUBSTANCE ON SEEDLING EMERGENCE OF SEVEN SPECIES OF PLANTS FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-110 Environmental Laboratory Project Number U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.4100 and 850.4225 and OECD Guideline for Testing of Chemicals Proposal for Revision of Guideline 208: Terrestrial Non-Target Plant Tests AUTHORS: Andrew J. Brignole John R. Porch Henry O. Krueger, Ph.D. Raymond L. Van Hoven, Ph.D. STUDY INITIATION DATE: November 12, 2001 STUDY COMPLETION DATE: May 30, 2003 SUBMITTED TO: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, L td 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 o f 136 W ildlife International. Ltd. PROJECT NO.: 454-110 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Toxicity Test to Determine the Effects ofthe Test Substance On Seedling Emergence of Seven Species of Plants WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-110 STUDY COMPLETION: May 30, 2003 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM(98)17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau,10 August 1984, with the following exception: The stability of the test substance under storage conditions at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: SPONSOR REPRESENTATIVE: Date -2 - W ildlife International. Ltd, PROJECT NO.: 454-110 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM(98)17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all audits and inspections and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY Test Substance Preparation Observations DATE CONDUCTED November 12,2001 November 19, 2001 Height and Weight Measurements, Plant Tissue December 3, 2001 Collection Matrix Fortifications January 24,2002 Analytical Data and Draft Report November 5-8, 11, 2002 Biological Data and Draft Report September 13-20,2002 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT November 12, 2001 November 15,2001 November 19, 2001 November 20,2001 December 3,2001 Decomber, 2001 January 24, 2002 January 25, 2002 November 11, 2002 November 25, 2002 September 20,2002 May 28, 2003 Final Report May 27-28,2003 All inspections were study-based unless otherwise noted. May 28, 2003 May 30,2003 Tames H. Coleman, B.S. Quality Assurance Representative _____ 5- Z - o 3 Date -3 - W ildlife International. Ltd. PROJECT NO.: 454-110 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Toxicity Test to Determine the Effects of the Test Substance on Seedling Emergence of Seven Species of Plants WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-110 STUDY DIRECTOR: Date WILDLIFE INTERNATIONAL. LTD MANAGEMENT: Director of Aquatic Toxicology/Terrestrial Plants and Insects Director of Chemistry Date -4 - WilA liie International' L id PROJECT NO.: 454-110 TABLE OF CONTENTS Title/Cover Page......................................................................................................................................... 1 Good Laboratory Practice Compliance Statement...................................................................................... 2 Quality Assurance Statement...................................................................................................................... 3 Report Approval.......................................................................................................................................... 4 Table of Contents........................................................................................................................................ 5 Summary..................................................................................................................................................... 8 Introduction................................................................................................................................................. 9 Purpose..................................................................................................................... 9 Experimental Design................................................................................................ 9 Materials and Methods............................................................................................. 10 Test Substance............................................................................................. 10 Preparation and Soil Incorporation of Test Substance................................................................. 10 Test Species................................................................................................ 10 Test Soil...................................................................................................... 11 Planting of Seeds........................................................................................ .................................11 Watering of Seedlings................................................................................ .................................12 Environmental Conditions........................................................................................................... 12 Pesticide and Metal Screening of Well Water and Soil............................................................... 12 Observations and Measurements................................................................................................. 12 Soil Sampling and Analysis........................................................................ 13 Tissue Sampling and Analysis..................................................................................................... 13 Data Analyses.............................................................................................................................. 14 Results...................................................................................................................................................... 15 Analytical Chemistry................................................................................... 15 Biological Results........................................................................................................................ 18 Conclusions............................................................................................................... 19 References................................................................................................................................................. 20 -5 - WilA liie International. L id PROJECT NO.: 454-110 TABLE OF CONTENTS (continued) TABLES Table 1 Seedling Condition Rating System...................................................................................... 21 Table 2 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21Day Seedling Emergence Test with Alfalfa......................................................................... 22 Table 3 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21Day Seedling Emergence Test with Flax............................................................................. 23 Table 4 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21Day Seedling Emergence Test with Lettuce........................................................................ 24 Table 5 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21Day Seedling Emergence Test with Onion.......................................................................... 25 Table 6 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21Day Seedling Emergence Test with Ryegrass..................................... .................................26 Table 7 Effects of PFOS Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with Soybean.............................................................................. 27 Table 8 Effects of PFOS Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with Tomato................................................................................ 28 Table 9 Observed NOEC and Calculated ECx Estimates for PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test....................... 29 APPENDICES Appendix 1 Personnel Involved in the Study................................................................................. 30 Appendix 2 Changes to the Protocol.............................................................................................. 31 Appendix 3 Certificate of Analysis................................................................................................ 32 Appendix 4 The Analysis of PFOS In Soiland Seven Species of Plants In Support of Wildlife International, Ltd. Project No.: 454-110.................................................................. 35 Appendix 5 Environmental Conditions.......................................................................................... 77 Appendix 6 Test Results, Alfalfa.................................................................................................... 83 Appendix 7 Test Results, Flax........................................................................................................ 90 -6 - Wildliie International, L id PROJECT NO.: 454-110 TABLE OF CONTENTS (continued) Appendix 8 Test Results, Lettuce...................................................................................................97 Appendix 9 Test Results, Onion................................................................................................... 104 Appendix 10 Test Results, Ryegrass.............................................................................................. I l l Appendix 11 Test Results, Soybean............................................................................................... 118 Appendix 12 Test Results, Tomato................................................................................................125 Appendix 13 Bulk Soil Characterization....................................................................................... 132 Appendix 14 Results of Water and Soil Pesticide Screening........................................................133 -7 - Wildlife International' L id PROJECT NO.: 454-110 SUMMARY WILDLIFE INTERNATIONAL, LTD. PROJECT NO: 454-110 TEST SUBSTANCE: Perflourooctanesulfonate, Potassium Salt (PFOS) STUDY TITLE: PFOS: A Toxicity Test to Determine the Effects of the Test Substance on Seedling Emergence of Seven Species of Plants GUIDELINES: OECD Guideline for Testing of Chemicals, Proposal for Revision of Guideline 208: Terrestrial Non-Target Plant Tests OPPTS 850.4100 (Public Draft) OPPTS 850.4225 (Public Draft) NOMINAL TEST LEVELS: 0 (Control), 3.91, 15.6, 62.5, 250, 1000 mg a.i./kg dry soil TEST DATES: STUDY INITIATION: Experimental Start (OECD): Experimental Start (EPA): Experimental Termination: STUDY COMPLETION: November 12, 2001 November 12, 2001 November 12, 2001 August 28, 2002 May 30, 2003 LENGTH OF TEST: Emergence Portion: 21 days Extended Growth Portion: up to 205 days (varies by species) TEST SPECIES: Alfalfa (Medicago sativa), Flax (Linum usitatissimum), Lettuce (Lactuca sativa), Onion (Allium cepa), Ryegrass (Lolium perenne), Soybean (Glycine max), Tomato (Lycopersicon esculentum) RESULTS: Species Common name (Latin name) Lettuce (Lactuca sativa) Ryegrass (Lolium perenne) Tomato (Lycopersicon esculentum) Onion (Allium cepa) Alfalfa (Medicago sativa) Flax (Linum usitatissimum) Soybean (Glycine max) Family Compositae Gramineae Solanaceae Liliaceae Leguminosae Linaceae Leguminosae Relative Sensitivity EC25 (mg a.i./kg) 6.79 7.51 11.7 12.9 53.3 81.6 160 Endpoint Height Shoot Weight Shoot Weight Shoot Weight Shoot Weight Shoot Weight Shoot Weight -8- Wildlife International, L td PROJECT NO.: 454-110 INTRODUCTION This seedling emergence study was conducted for 3M Corporation at the Wildlife International, Ltd. greenhouse facility in Easton, Maryland. The in-life portion of the test was conducted from November 12, 2001 to June 5, 2002. Raw data generated at Wildlife International, Ltd., the study protocol, and a copy of the final report were filed in the archives located on the Wildlife International, Ltd. site. Key personnel involved in the study are listed in Appendix 1. PURPOSE The purpose of the study was to determine the effects of Perflourooctanesulfonate, Potassium Salt (PFOS) on the seedling emergence and growth of seven species of plants. EXPERIMENTAL DESIGN There were two parts to this study. The first part was a twenty-one day seedling emergence test. The second part, an extended growth period, followed immediately. The experimental design for the overall study consisted of a negative control and five treatment groups, with four replicate pots in each group. To begin the twenty-one day portion of the test, ten seeds were planted in each pot. Test concentrations of PFOS were made by soil incorporation to each treatment group prior to the planting of seeds. The nominal test substance concentrations were 3.91, 15.6, 62.5, 250, and 1000 mg of PFOS per kilogram of dry soil (mg a.i./kg). These rates were selected based on the results of a nonGLP rangefinding test, and are not necessarily indicative of expected environmental concentrations. A control group, which received no test substance incorporation, was m aintained concurrently. Seeds were impartially assigned to growth pots on the day of test initiation. The replicate pots were placed in a randomized block design on a greenhouse table after planting. Observations of emergence were made on Days 7, 15, and 21. On day 21, observations of height, and assignment of plant condition scores were made. Fresh weights were conducted on all replicates containing more than one living seedling. Where possible, one seedling from each replicate was then left to grow until plants produced fruit (i.e., the extended growth period), whereupon, plants and fruit were clipped and weighed independently. -9 - Wildlife International' Ltd. PROJECT NO.: 454-110 MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A Toxicity Test to Determine the Effects of the Test Substance on Seedling Emergence of Seven Species of Plants." Changes to the approved protocol are listed in Appendix 2. The methods used in conducting this study were based upon procedures specified in the OECD Proposal for Revision of Guideline 208: Terrestrial Non-Target Plant Tests (1) and the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Numbers 850.4100 (2) and 850.4225 (3). Test Substance The test substance was received from 3M Corporation on October 29, 1998, and was assigned Wildlife International, Ltd. identification number 4675. The test substance was a white powder identified as FC-95; lot number 217; with a reanalysis date of August 31, 2006. Information provided by the Sponsor indicated a test substance purity of 86.9%. The test substance was stored at ambient room temperature. Preparation and Soil Incorporation of Test Substance The test soil was prepared by mixing PFOS into bulk test soil with a measured soil moisture of 15%. Test substance for treatment groups 3.91, 15.6, 62.5, 250, and 1000 mg a.i./kg was prepared by weighing five known weights (0.2690, 1.1, 4.3, 17.1, and 68.5 g) of PFOS. Approximately 1000 g was removed from a measured 70 kg of bulk soil and mixed with weighed test substance for each test concentration. The remaining 69 kg o f bulk soil was placed into soil mixer, and the 1 kg o f soil with test substance was added. The constituents were then mixed for ten minutes in order to prepare the test soil for each treatment group. Soils were mixed from lowest to highest concentration to avoid cross-contamination. The negative control soil was prepared prior to the treatment groups, but in the same manner, except no test substance was added. At the completion of mixing, the test soils were sampled to provide material for analytical confirmation of the test concentrations. Analytical samples were stored at ambient room conditions for up to four days after their collection until they were processed for analysis. Test Species The common and scientific names for the seven species tested, the seed source, and their approximate planting depths are listed below: - 10- Wildlife Intemational. Lid PROJECT NO.: 454-110 Test Species / Variety: Seed Source: Alfalfa (Medicago sativa) / None Given Frontier Natural Products, Norway, IA , USA Flax (Linum usitatissimum) /None Given Arrowhead Mills, Hereford, TX, USA Lettuce (Lactuca sativa) / Summertime Territorial Seed Co., Cottage Grove, OR, USA Onion (Allium cepa) / Texas Grano Territorial Seed Co., Cottage Grove, OR, USA Ryegrass (Lolium perenne)/ Manhattan 3 Meyer Seed Co., Baltimore, MD, USA Soybean (Glycine max) / Green Envy Johnny's Selected Seeds, Albion, ME, USA Tomato (Lycopersicon esculentum) i Rutgers Meyer Seed Co., Baltimore, MD, USA Planting Depth 6 mm 6 mm 6 mm 6 mm 6mm 20 mm 6 mm These species were chosen because they represent ecologically important families, and are readily cultivated test organisms that are widely used in research. An additional consideration was to select species which could be grown to fruiting relatively quickly, while maintaining a reasonable plant size. Seeds were selected from a single size class within each species in order to reduce the potential for bias from differing seed sizes. Seeds used in this study were not treated with fungicides, insecticides or repellents prior to test initiation. Test Soil The soil used for the test represented a loam soil, and was composed of kaolinite clay, industrial quartz sand, and peat mixed in a 2:25:1 ratio (w:w:w). Crushed limestone was added to buffer the pH of the soil, and a slow-release fertilizer was added to provide nutrients essential for plant growth. A sample of soil representative of that used in this study was sent to Agvise Laboratories, Inc., in Northwood, North Dakota, for analysis of the particle size distribution and organic matter content of the soil (Appendix 13). The soil was determined to consist of 49% sand, 30% silt, and 21% clay, with an organic matter content of 2.1%. The soil pH was measured by Wildlife International Ltd. to be 7.79. A copy of the complete report from Agvise Laboratories, Inc. was filed in the archives at Wildlife International, Ltd. along with the raw data for this study. Planting of Seeds Seeds were planted in plastic pots (approximately 16 cm in diameter and 12 cm deep) on the day of test substance application. A template was used to gently compact the soil and leave ten uniform holes for planting. One indiscriminately selected seed was then planted in each hole, for a total of ten seeds in each pot. Personnel then closed the holes by slightly depressing the soil surface with their fingers. -11 - Wildliie International. L id PROJECT NO.: 454-110 Watering of Seedlings Water lost through transpiration and evaporation was replaced by subirrigation with well water from the greenhouse facility. Seedlings were subirrigated as needed during the test to minimize the potential for the leaching of the test substance through the soil. Subirrigation trays were filled to a predetermined depth to help standardize the amount of water delivered to each tray. The days on which watering occurred are listed in Appendix 5. Environmental Conditions The environmental conditions (light intensity, temperature, and relative humidity) during the test are summarized in Appendix 5. The temperature within the greenhouse was controlled with a Wadsworth MicroStep S/A Environmental Control System. Artificial lighting was used to supplement natural sunlight in order to provide a uniform 14-hour photoperiod. The light intensity, temperature, and relative humidity within the greenhouse were continuously monitored during the test with a Campbell CR-10 datalogger. Pesticide and Metal Screening of Well Water and Soil The well water and soil used for plant studies are analyzed periodically for pesticide and metals. No analytes were measured at levels that were expected to have an impact on the study (Appendix 14). Reports for the latest analyses are stored in the archives at the Wildlife International, Ltd. site in Easton, Maryland. Observations and Measurements Observations on Days 7 and 15 were conducted to document seedling emergence. Observations on Day 21 were made to document seedling emergence and growth, and to determine the condition of individual seedlings. Observations consisted of noting whether emergence had or had not occurred, and assessing the condition of each seedling. Emergence was defined as the presence of visible plant tissue at the surface of the soil. Seedling condition was described by noting the presence or absence of possible signs of phytotoxicity such as necrosis, leaf wrinkle, chlorosis, plant lodging or plant stunting. Each emerged seedling was then assigned a numerical score (see Table 1) that described the plant condition (4). Condition score is a subjective or qualitative assessment that determines whether damage is slight, moderate, or severe. A score of 10 does not mean that 10% of the plant is showing the effect (e.g. chlorosis), merely that the severity of the effect (e.g. chlorosis) is very slight. -12- Wildlife International. L id PROJECT NO.: 454-110 On day 21, seedling height was measured to the nearest whole centimeter from the surface of the soil to the tip of the tallest leaf (alfalfa, flax, lettuce, onion, and ryegrass) or to the apical meristem (soybean and tomato). All living seedlings except one per replicate (when available) were then clipped at soil level, combined, and weighed within 5 minutes of clipping. The total weight of the shoots was divided by the number of seedlings weighed in order to calculate the mean weight per plant. The one plant per replicate, when available, which was not sacrificed was allowed to grow for participation in the extended growth test. Alfalfa, flax, soybean, and tomato were grown until fruit production. Onions were grown until an enlarged bulb was evident. Lettuce was grown until adequate leaf tissue was available for analysis. Ryegrass seed production was anticipated, but was not observed. Soil Sampling and Analysis On the day of test soil preparation (November 12, 2001), three soil samples were collected from each treatment group to verify the test concentrations and determine the homogeneity of the test substance in the carrier (soil). One sample was collected from the control group. Day 0 samples were collected from the soil from each test group remaining after pots were filled for planting. On day 21 and at termination of the last test species, two soil samples were collected from the treatment groups to verify and determine homogeneity of the test substance in the soil. One sample was collected from the control group on both sampling dates. These samples were collected from test pots. Day 21 samples were collected by removing a small portion of soil from the surface and placing it in the sample container. Test termination samples were collected on June 5, 2002 using a. soil core sampler which removed a cylindrical core of the soil from the surface to near the bottom of the test pot. Since the test was terminated on different days for the various species, the test termination soil samples were collected from the pots containing ryegrass, which was the last species to terminate. Samples were stored at ambient room conditions until analysis was begun on August 14, 2002. Chemical analysis of the soil used in this study was performed by Wildlife International, Ltd. (Appendix 4). The test substance was used to prepare calibration standards. Tissue Sampling and Analysis On Day 21, December 3, 2001, following observations of emergence, and measurements of height and fresh weight, three samples of plant tissue were taken from each species at each test concentration, and analyzed for PFOS. The three samples were obtained from a composite of tissue -13 - Wildliie Intema tional. Lid. PROJECT NO.: 454-110 from each of three replicate pots. Three additional plant tissue samples were taken from the single plant remaining in each replicate, when available, for each species at each test concentration at test termination of the extended growth period. Plants from the fourth replicate in each group, when present, were used to determine the moisture content of plant tissue at the end of each portion of the test (21-day and extended growth). Samples were stored frozen until analysis. Chemical analysis of the plant tissue collected in this study was performed by Wildlife International, Ltd. (Appendix 4). The test substance was used to prepare calibration standards. Dates of test termination for each species are listed below: Species Alfalfa Flax Lettuce Onion Ryegrass Soybean Tomato Date of Test Termination April 2, 2002 February 14, 2002 January 18, 2002 January 18, 2002 June 5, 2002 January 18, 2002 February 14, 2002 Data Analyses Statistical analyses were used to aid in the evaluation of effects of test substance application on seedling emergence, survival, shoot weight, and height. These variables were defined for statistical analysis as follows: Seedling Emergence: The number of emerged seedlings per ten planted seeds in each pot. Survival: The number of emerged seedlings in each pot that were living at test termination per ten planted seeds. Shoot Weight: The mean shoot weight of sacrificed living emerged seedlings in each pot. Height: The average height o f living emerged seedlings in each pot. Test data were evaluated to determine the no-observed-effect-concentration (NOEC) and lowestobservable-effect-concentration (LOEC) for condition and growth. The NOEC is defined as the maximum test substance concentration that shows no adverse phytotoxic effects and below which no phytotoxic effects are manifested. The LOEC is defined as the lowest test substance concentration used in the study that shows an adverse effect on a variable of interest. Mean seedling emergence, - 14- AAAildlife International. L id PROJECT NO.: 454-110 survival, weight, and height of the control and treatment groups were compared with Dunnett's t-test, using the DUNNETT option of the GLM (general linear model) procedure of SAS version 8 (5). Significance was determined at the level of 0.05 (p<0.05). Dunnett's test was used to aid in establishing the NOEC by determining which treatment groups differed significantly from the control group. Statistical analyses for species also included the determination of effect rates (EC estimates) and their confidence limits using the non-linear regression analysis of Bruce and Versteeg (6) when reductions in test endpoints among one or more treatment groups were 25% or more relative to control means. Analyses were conducted using the NLESi procedure of SAS version 8 (5). ECX values (i.e. EC25 and EC50) were defined as the test substance application rates that caused an x% change in the treatment group mean emergence, dry weight, or height relative to the control group. ECXestimates were calculated using nominal test concentrations and treatment group mean values with the following equation: f Ro 0>[(log(ECx)-log(C))/a +ZX] C>0 *= 4 l C=0 where R = the predicted biotic response at concentration C R0 = the predicted biotic response for controls . <t>[ ] = the cumulative area under the standard, normal distribution log(ECx) = the logarithm of the predicted ER giving an x percentage of decrease in the biological parameter vs. the control Zx = the normal deviate above which x percentage of the area of the standard normal distribution lies. cr = the standard deviation of the normal distribution Effects on survival w ere designated as LCXvalues, and w ere calculated using the m ethod described above. If the fit of the data to the regression model was poor, or if confidence intervals were not calculated, the ECXestimates were calculated using linear interpolation (7). RESULTS Analytical Chemistry Artificial Soil. Samples of artificial soil collected on Days 0 and 21, and at test termination were submitted and analyzed for PFOS. All negative control artificial soil samples were <LOQ for PFOS. Day 0 analyses of the 3.91, 15.6, 62.5, 250, and 1000 mg a.i./kg treatment levels had mean measured values of 3.61, 11.1, 50.8, 276, and 998 mg a.i./kg, corresponding to 92.3%, 71.2%, 81.3%, 110%, and 99.8% of the nominal concentrations, respectively (Appendix 4.19). PFOS measured -15 - WiJcUi/eIniema tional. Lid. PROJECT NO.: 454-110 values for test termination samples are presented in Appendix 4.20. The 3.91, 15.6, 62.5, 250 and 1000 mg a.i./kg treatment levels had mean measured values of 1.29, 3.56, 16.2, 157 and 474 mg a.i./kg, corresponding to 33.0%, 22.8%, 25.9%, 62.8% and 47.4% of the nominal concentrations, respectively at test termination. A representative ion chromatogram of an artificial soil test sample is presented in Appendix 4.21. Day 21 Samples. The results of soil and tissue analyses of samples collected on Day 21 were not considered representative of the actual levels of PFOS in soil and tissue, and are therefore not reported. The measured levels of PFOS in soil samples were generally well above nominal. The apparently high concentrations were thought to be an artifact resulting from two factors. First, the process of subirrigation during the test was thought to concentrate PFOS near the soil surface of test pots. Second, the samples were collected by removing a small amount of soil from the top of the soil profile. In addition, tissue samples collected on Day 21 were thought to have been inadvertently contaminated during collection. Due to the small size of the seedlings on Day 21, and the apparently high levels of PFOS at the soil surface, it was determined that soil particles were attached to the seedlings and biased the analytical results. Plant Tissues. Samples of fruit tissue from five species of plant (alfalfa, flax, onion, soybean, and tomato) grown in negative control and PFOS-treated artificial soils were collected at test termination (for a given species) and analyzed for PFOS residues. Samples of vegetative tissue from seven species of plant (alfalfa, flax, lettuce, onion, ryegrass, soybean, and tom ato) grown in negative control and PFOS-treated artificial soils were collected at experimental termination (for a given species) and analyzed for PFOS residues. Measured PFOS concentrations are presented in Appendices 4.22 - 4.33. Representative ion chromatograms of plant fruit and vegetative tissue test samples are presented in Appendices 4.34 and 4.35, respectively. - 16- Wildliie Intemaiional. Lid. PROJECT NO.: 454-110 The results of tissue analyses are summarized by species in the following tables. Alfalfa - Total days of exposure =141 Nominal Soil Concentration (mg a.i./kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) in: Vegetation Fruit <2.7 <6.4 6.2 <6.4 4.2 <6.4 11 <6.4 16 <6.4 None living None living Flax - Total days of exposure = 94 Nominal Soil Concentration (mg a.i./kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) in: Vegetation Fruit <2.4 <0.12 5.0 0.23 19 1.4 55 2.6 Insufficient sample Insufficient sample None living None living Lettuce - Total days of exposure = 67 Nominal Soil Concentration (mg a.i./kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) in: Vegetation Fruit <5.5 Not applicable 8.6 Not applicable 11 Not applicable 42 Not applicable Insufficient sample Not applicable None living Not applicable Onion - Total days of exposure = 67 Nominal Soil Concentration ima a.i./kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) in: Vegetation Fruit <4.7 0.50 <4.7 3.1 11 22 Insufficient sample Insufficient sample None living None living None living None living Ryegrass - Total days of exposure = 205 Nominal Soil Concentration (ma a.i./ka) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) in: Vegetation Fruit <3.3 Not applicable 8.2 Not applicable 31 Not applicable 49 Not applicable 66 Not applicable None living Not applicable - 17- Wildliie Internaiional. Lid. PROJECT NO.: 454-110 Soybean - Total days of exposure = 67 Nominal Soil Concentration ("me a.i./kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) in: Vegetation Fruit <6.0 <0.18 16 1.4 36 0.87 63 1.2 110 3.2 None living None living Tomato - Total days of exposure = 94 Nominal Soil Concentration (tUg.a.i./kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) in: Vegetation | Fruit <7.6 <0.47 <7.6 <0.47 34 1.1 50 0.95 Insufficient sample Insufficient sample None living None living Biological Results The LOEC, NOEC, ECX, and LCXfor the various parameters of each of the seven species are summarized in the tables below. Results of the test are summarized by species in Tables 2 through 8. Complete results are presented by species in Appendices 6 through 12. Species Monocots: Onion (Allium cepa) Ryegrass (Lolium perenne) Dicots: Alfalfa (Medicago sativa) Flax (Linunt usitatissimum) Lettuce (Lactuca sativa) Soybean (Glycine max) Tomato (Lycopersicon esculentum) LOEC 250 250 21-day Emergence NOEC e c 25 e c 50 21-day Survival LOEC NOEC e c 25 (All units mg a-i-/kg)________ 62.5 50.8 208 62.5 15.6 47.1 62.5 203 344 250 62.5 174 e c 50 57.3 310 1000 250 372 745 250 62.5 251 452 1000 250 399 599 250 62.5 144 226 1000 250 393 564 250 62.5 257 386 >1000 1000 >1000 >1000 >1000 1000 >1000 >1000 1000 250 311 474 62.5 15.6 68.7 105 - 18- Wildliie International. L id PROJECT NO.: 454-110 Species Monocots: Onion (Allium cepa) Ryegrass (Loliutn perenne) Dicots: Alfalfa (Medicago sativa) Flax (Linum usitatissimum) Lettuce (Lactuca sativa) Soybean (Glycine max) Tomato (Lycopersicon esculentum) 21-day Height LOEC NOEC e c 25 62.5 15.6 29.1 15.6 3.91 46.3 e c 50 21-day Shoot Weight LOEC NOEC e c 25 (All units mg a.i./kg) 46.5 15.6 3.91 12.9 131 15.6 3.91 7.51 e c 50 28.1 53.8 250 62.5 102 249 250 62.5 53.3 146 250 62.5 97.6 140 250 62.5 81.6 119 3.91 <3.91 6.79 39.9 3.91 <3.91 8.92 20.1 250 62.5 284 464 250 62.5 160 326 62.5 15.6 22.1 93.9 62.5 15.6 11.7 28.5 CONCLUSIONS The relative sensitivities and most sensitive endpoints for the seven test species in response to soilincorporated PFOS based on 21-day results are listed below: Species Common name (Latin name) Family Relative Sensitivity EC25 (mg a.i./kg) Endpoint Lettuce (Lactuca sativa) Ryegrass (Lolium perenne) Tomato (Lycopersicon esculentum) Onion (Allium cepa) Alfalfa (Medicago sativa) Flax (Linum usitatissimum) Soybean (Glycine max) Compositae Gramineae Solanaceae Liliaceae Leguminosae Linaceae Leguminosae 6.79 Height 7.51 Shoot Weight 11.7 Shoot Weight 12.9 Shoot Weight 53.3 Shoot Weight 81.6 Shoot Weight 160 Shoot Weight There were no additional findings of phytotoxicity at the termination of the extended growth portion of the test. -19- W ild liie International L td PROJECT NO.: 454-110 REFERENCES 1 OECD Guideline for Testing of Chemicals. 1998. Guideline for Testing o f Chemicals, Proposalfo r Revision o f Guideline 208: Terrestrial Non-Target Plant Tests. Organization for Economic Cooperation Development 2 U.S. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test Guidelines {draft), OPPTS Number 850.4100: Terrestrial Plant Toxicity, Tier I (Seedling Emergence). 3 U.S. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test Guidelines {draft), OPPTS Number 850.4225: Terrestrial Plant Toxicity, Tier II (Seedling Emergence). 4 Frans, Robert E. and Ronald E. Talbert. 1977. Design of Field Experiments and the Measurement and Analysis of Plant Responses. Pages 15-23 in B. Truelove, ed. Research Methods in Weed Science. Southern Weed Science Society, Auburn University, Alabama. 5 SAS Institute, Inc. 1999. SAS Proprietary Software Version 8 Cary, NC, SAS Institute, Inc. 6 Bruce, Robert D. and Donald J. Versteeg. 1992. A Statistical Procedure for Modeling Continuous Toxicity Data. Environmental Toxicology and Chemistry. 11: 1485-1494. 7 Norberg-King, T.J. 1993. A Linear Interpolation Method fo r Sublethal Toxicity: The Inhibition Concentration (ICp) Approach (Version 2.0). U.S. Environmental Protection Agency, Environmental Research Laboratory, Duluth, Minnesota. 8 Wildlife International, Ltd. 2003. Project No. 454C-120. Van Hoven, R. L., MacGregor, J.A., and Nixon, W. B. Final Report. Analytical Method Validation for the Determination o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Artificial Soil. 9 Wildlife International, Ltd. 2001. Project No. 454C-125. Van Hoven, R. L., MacGregor, J.A., and Nixon, W. B. Final Report. Analytical Method Validation for the Determination o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues. -20- MAildliie International. L td Table 1 Seedling Condition Rating System PROJECT NO.: 454-110 Rating 0 10 20 30 40 50 60 70 80 90 100 Category No Effect Slight Effect Moderate Effect Severe Effect Complete Effect Description No noticeable effect Effect barely noticeable Some effect, not apparently detrimental Effect more pronounced, not obviously detrimental Effect moderate, plants appear able to recover More lasting effect, recovery somewhat doubtful Lasting effect, recovery doubtful Heavy injury, loss of individual leaves Plant nearly destroyed, a few surviving leaves Occasional surviving leaves Death of entire plant Rating scale adapted from: Frans, Robert E. and Ronald E. Talbert. 1977. Design of Field Experiments and the Measurement and Analysis of Plant Responses. Pages 15-23 in B. Truelove, ed. Research Methods in Weed Science. Southern Weed Science Society, Auburn University, Alabama. -21 - Wildliie InternaiionalfL id PROJECT NO.: 454-110 Table 2 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with ALFALFA Test Number of Emerged Seedlings Concentration (% Reduction) (mga.i./kg) Day 7 Day 15 Day 21 _____________ (Mean SD)______(Mean SD)______(Mean SD) Seedling Shoot Weight Height Survival (g) (cm) (% Reduction) (% Reduction) (% Reduction) (Mean SD)_____(Mean SD)_____ (Mean SD) Control 8.75 1.50 9.00 1.15 9.00 1.15 8.75 0.96 0.12 0.013 6.5 0.64 3.91 8.25 0.50 (6%) 8.50 0.58 (6%) 8.50 0.58 (6%) 8.25 0.50 (6%) 0.11 0.033 (4%) 15.6 7.75 1.50 (11%) 8.00 1.41 (11%) 8.00 1.41 (11%) 8.00 1.41 (9%) 0.10 0.022 (15%) 62.5 7.00 1.15 (20%) 8.00 1.15 (11%) 8.00 1.15 (11%) 8.00 1.15 (9%) 0.10 0.010 (11%) 250 7.00 1.41 7.25 1.71 7.25 1.71 6.25 1.71* 0.03 0.008* (20%) (19%) (19%) (29%) (78%) 1000 2.50 2.89* (71%) 2.50 2.89* (72%) 3.25 3.30* (64%) 1.50 1.73* (83%) 0.02 0.008* (87%) * Treatment group mean is significantly different from the control mean (Dunnett's test, /><0.05). Mean SD - Treatment group mean plus or minus one standard deviation. 6.4 0.75 (2%) 5.4 0.76 (16%) 6.1 0.90 (6%) 2.8 0.52* (57%) 1.0 0.00* (85%) -22- Wildliie International. L id PROJECT NO.: 454-110 Table 3 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with FLAX Test Number of Emerged Seedlings Concentration (% Reduction) (mga.i./kg) Day 7 Day 15 Day 21 _____________ (Mean SD)______(Mean SD)______(Mean SD) Seedling Shoot Weight Height Survival (g) (cm) (% Reduction) (% Reduction) (% Reduction) (Mean SD)_____ (Mean SD)______ (Mean SD) Control 7.25 1.26 7.25 1.26 7.25 1.26 7.25 1.26 0.19 0.029 8.9 0.27 3.91 6.50 1.00 7.00 1.41 7.25 1.50 6.75 1.26 0.18 0.022 (10%) (3%) (0%) (7%) (8%) 15.6 8.00 0.82 (-10%) 8.00 0.82 (-10%) 8.00 0.82 (-10%) 8.00 0.82 (-10%) 0.18 0.023 (10%) 62.5 8.50 1.00 (-17%) 8.50 1.00 (-17%) 8.50 1.00 (-17%) 8.50 1.00 (-17%) 0.16 0.017 (18%) 250 7.25 1.71 7.25 1.71 7.25 1.71 3.25 1.50* 0.02 0.009* (0%) (0%) (0%) (55%) (91%) 1000 0* (100%) 0* (100%) 0* (100%) 0* (100%) N/A * Treatment group mean is significantly different from the control mean (Dunnett's test, ;;<0.05). N/A - Not applicable since there were no surviving plants at measurement. Mean SD - Treatment group mean plus or minus one standard deviation. 8.7 0.90 (3%) 8.8 0.80 (2%) 8.2 0.54 (8%) 1.3 0.35* (86%) N/A -23 - Wildliie International. Lid. PROJECT NO.: 454-110 Table 4 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with LETTUCE Test Number of Emerged Seedlings Concentration (% Reduction) (mga.i./kg) Day 7 Day 15 Day 21 _____________ (Mean SD)______(Mean SD)_____ (Mean SD) Seedling Shoot Weight Height Survival (g) (cm) (% Reduction) (% Reduction) (% Reduction) (Mean SD)_____ (Mean SD)_____ (Mean SD) Control 8.75 0.50 8.75 0.50 8.75 0.50 8.75 0.50 0.38 0.060 6.4 0.51 3.91 9.00 0.82 9.00 0.82 9.00 0.82 9.00 0.82 0.25 0.051* (-3%) (-3%) (-3%) (-3%) (35%) 15.6 8.75 1.50 (0%) 9.00 1.15 (-3%) 9.00 1.15 (-3%) 9.00 1.15 (-3%) 0.24 0.081* (36%) 62.5 8.50 1.29 8.50 1.29 8.50 1.29 8.50 1.29 0.05 0.019* (3%) (3%) (3%) (3%) (88%) 250 8.00 1.41 8.00 1.41 8.25 1.26 6.75 0.96* 0.01 0.006* (9%) (9%) (6%) (23%) (98%) 1000 1.25 1.26* 1.25 1.26* 1.25 1.26* 0.50 0.58* 0.00 0.000* (86%) (86%) (86%) (94%) (100%) * Treatment group mean is significantly different from the control mean (Dunnett's test, p<0.05). Mean SD - Treatment group mean plus or minus one standard deviation. 5.0 0.56* (23%) 5.0 1.07* (23%) 2.6 0.43* (59%) 1.0 0.06* (84%) 1.0 0.00* (84%) - 24- Wildliie Iniema iional. Lid. PROJECT NO.: 454-110 Table 5 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with ONION Test Number of Emerged Seedlings Concentration (% Reduction) (mga.i./kg) Day 7 Day 15 Day 21 _____________ (Mean SD)______(Mean SD)______ (Mean SD) Seedling Shoot Weight Height Survival (g) (cm) (% Reduction) (% Reduction) (% Reduction) (Mean SD)_____ (Mean SD)_____ (Mean SD) Control 8.00 0.82 8.50 1.29 8.75 1.50 8.50 1.29 0.10 0.006 8.1 0.55 3.91 8.50 1.29 9.00 1.41 9.25 0.96 9.00 1.15 0.09 0.015 (-6%) (-6%) (-6%) (-6%) (15%) 15.6 7.75 0.50 (3%) 8.25 0.50 (3%) 8.25 0.50 (6%) 8.00 0.82 (6%) 0.07 0.006* (31%) 62.5 5.00 2.00* (38%) 6.25 2.63 (26%) 6.25 2.63 (29%) 3.25 0.50* (62%) 0.02 0.019* (77%) 250 2.00 2.45* 4.00 3.16* 4.00 3.16* 0* (75%) (53%) (54%) (100%) N/A 1000 0* (100%) 0* (100%) 0* (100%) 0* (100%) N/A * Treatment group mean is significantly different from the control mean (Dunnetf s test, /><0.05). N/A - Not applicable since there were no surviving plants at measurement. Mean SD - Treatment group mean plus or minus one standard deviation. 7.3 0.49 (9%) 7.3 0.15 (10%) 2.6 0.83* (68%) N/A N/A -25 - Wildliie International. L id PROJECT NO.: 454-110 Table 6 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with RYEGRASS Test Number of Emerged Seedlings Concentration (% Reduction) (mg a.i./kg) Day 7 Day 15 Day 21 _____________ (Mean SD)______(Mean SD)______(Mean SD) Seedling Shoot Weight Height Survival (g) (cm) (% Reduction) (% Reduction) (% Reduction) (Mean SD)_____(Mean SD)_____ (Mean SD) Control 7.75 0.96 8.00 0.82 8.00 0.82 8.00 0.82 0.12 0.025 16.9 0.69 3.91 8.50 1.00 9.25 0.96 9.25 0.96 9.25 0.96 0.11 0.013 15.4 1.28 (-10%) (-16%) (-16%) (-16%) (12%) (9%) 15.6 7.50 1.91 (3%) 9.00 0.82 (-13%) 9.00 0.82 (-13%) 9.00 0.82 (-13%) 0.07 0.014* (39%) 13.7 0.77* (19%) 62.5 7.50 0.58 (3%) 8.25 0.96 (-3%) 8.50 1.29 (-6%) 8.25 0.96 (-3%) 0.08 0.014* (32%) 13.6 0.92* (19%) 250 4.00 1.41* 5.75 1.26* 5.75 1.26* 5.25 2.22* 0.01 0.006* 3.7 0.97* (48%) (28%) (28%) (34%) (91%) (78%) 1000 0* (100%) 0.25 0.50* (97%) 0.75 0.50* 0.75 0.50* (91%) (91%) 0.03 0.010* (76%) * Treatment group mean was significantly different from the control mean (Dunnett's test, p<0.05) Mean SD - Treatment group mean plus or minus one standard deviation. 2.0 0.00* (88%) - 26- Wildliie International. L id PROJECT NO.: 454-110 Table 7 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with SOYBEAN Test Number of Emerged Seedlings Concentration (% Reduction) (mga.i./kg) Day 7 Day 15 Day 21 _____________ (Mean SD)______ (Mean SD)______(Mean SD) Seedling Shoot Weight Height Survival (g) (cm) (% Reduction) (% Reduction) (% Reduction) (Mean SD)_____ (Mean SD)______(Mean SD) Control 9.75 0.50 10.00 0.00 10.00 0.00 10.00 0.00 3.63 0.583 28.3 4.17 3.91 9.75 0.50 (0%) 9.75 0.50 (3%) 9.75 0.50 (3%) 9.75 0.50 (3%) 3.64 0.236 (0%) 26.8 3.17 (5%) 15.6 9.50 0.58 (3%) 9.50 0.58 (5%) 9.50 0.58 (5%) 9.50 0.58 (5%) 3.63 0.330 (0%) 26.9 3.31 (5%) 62.5 10.00 0.00 10.00 0.00 10.00 0.00 10.00 0.00 4.00 0.302 (-3%) (0%) (0%) (0%) (-10%) 30.7 2.51 (-8%) 250 10.00 0.00 10.00 0.00 10.00 0.00 9.75 0.50 2.07 0.310* 22.5 2.26* (-3%) (0%) (0%) (3%) (43%) (21%) 1000 9.75 0.50 (0%) 10.00 0.00 (0%) 10.00 0.00 (0%) 10.00 0.00 (0%) 0.57 0.047* (84%) 4.1 0.39* (85%) * Treatment group mean is significantly different from the control mean (Dunnett's test, p<0.05). Mean SD - Treatment group mean plus or minus one standard deviation. - 27- Wild life International' L id PROJECT NO.: 454-110 Table 8 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with TOMATO Test Concentration (mg a.i./kg) Number of Emerged Seedlings (% Reduction) Day 7 Day 15 Day 21 (Mean SD)______(Mean SD)______(Mean SD) Seedling Survival (% Reduction) (Mean SD) Shoot Weight (g) (% Reduction) (Mean SD) Height (cm) (% Reduction) (Mean SD) Control 6.50 1.91 9.00 0.82 9.25 0.96 9.25 0.96 0.35 0.143 5.2 0.75 3.91 6.50 1.73 8.00 1.15 8.25 1.50 8.25 1.50 0.40 0.081 (0% ) ( 11%) ( 11%) ( 11%) (-14%) 5.6 0.31 (-7%) 15.6 5.25 2.99 (19%) 8.25 1.71 (8%) 8.25 1.71 ( 11%) 8.00 1.41 (14%) 0.28 0.108 (19%) 4.8 0.94 (9%) 62.5 5.50 1.91 (15%) 8.25 1.71 (8%) 8.50 1.73 (8%) 6.75 0.96* (27%) 0.08 0.029* (79%) 2.6 0.14* (50%) 250 1.25 1.50* 7.25 0.96 7.25 0.96 0.75 0.96* 0.03 N/A* (8 1 %) (19%) (22%) (92%) (91%) 2.0 0.00* (62%) 1000 0* 0.75 1.50* 1.00 1.41* 0* ( 100%) (92%) (89%) ( 100%) N/A N/A * Treatment group mean is significantly different from the control mean (Dunnett's test, p<0.05). N/A - Not applicable since there were no surviving plants at measurement. Mean SD - Treatment group mean plus or minus one standard deviation. -28- Wildliie International. Lid. PROJECT NO.: 454-110 Table 9 Observed NOEC and Calculated ECx Estimates for PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test Species E n d p o in t (N O E C ) (m g ai/kg) Estim ate Lower 95% CL (m g ai/kg) Upper 95% CL A lfalfa E m ergence (250) Survival (62.5) e c 25 E C 50 372 745 l c 25 251 LC50 452 202 541 178 364 688 > 10 0 0 353 563 H eight (62.5) e c 25 E C 50 102 249 26.8 106 391 584 W eight (62.5) e c 25 E C 50 53.3 146 4.12 27.3 690 783 Flax E m erg en ce (250) Survival (62.5) e c 25 E C 50 399 599 l c 25 I-C 5 0 144 226 126 402 103 160 461 641 177 368 H eight (62.5) e c 25 9 7 .6 EC50 140 81.3 125 117 158 W eight (62.5) e c 25 8 1 .6 e c 50 119 46.0 79.4 145 178 Lettuce Em ergence (250) e c 25 393 E C 50 564 300 474 515 671 Survival (62.5) L C 25 L C50 257 386 220 344 301 433 H eight (0 .0 ) e c 25 6 .7 9 E C 50 39.9 <3.91 3.88 226 410 W eight (0.0) e c 25 8.92 E C 50 20.1 <3.91 5.26 58.3 77.1 O n io n E m ergence (62.5) Survival (15.6) H eight (15.6) W eight (3.91) e c 25 E C 50 50.8 208 L C 25 L C 50 47.1 57.3 e c 25 29.1 EC 50 46.5 e c 25 12.9 E C 50 28.1 12.2 <3.91 <3.91 17.5 <3.91 8.94 <3.91 1.99 195 644 >1000 188 904 242 >1000 396 1- Confidence intervals could not be determined. Species E n d p o in t (NOEC) (m g ai/kg) Estim ate Lower 95% CL (m g ai/kg) Upper 95% CL Ryegrass Em ergence (62.5) Survival (62.5) H eig h t (3.91) E C 25 E C 50 203 344 l c 25 L C50 174 310 e c 25 E C 50 46.3 131 131 254 107 223 4.63 28.8 313 465 281 432 464 597 W eight (3.91) e c 25 E C 50 7.51 53.8 Soybean E m ergence (1000) e c 25 > 1 0 0 0 EC50 >1000 Survival (1000) L C 25 L C50 >1000 >1000 H eight (62.5) E C 25 E C 50 284 464 W eight (62.5) e c 25 E C 50 160 326 Tom ato E m ergence (250) e c 25 E C 50 311 474 Survival (15.6) l c 25 L C 50 68.7 105 <3.91 <3.91 172 333 69.1 189 208 360 45.0 77.8 >1000 >1000 - - _ - 468 648 372 561 464 625 105 143 H eight (15.6) EC25 E C 50 22.1 93.9 <3.91 10.3 >1000 852 W eight (15.6) e c 25 E C 50 11.7 28.5 <3.91 <3.91 217 213 - 29- W ildliie International. Lid\ PROJECT NO.: 454-110 Appendix 1 Personnel Involved In the Study The following key personnel were involved in the conduct or management of this study: (1) Henry O. Krueger, Ph.D., Director of Aquatic Toxicology/Terrestrial Plants and Insects (2) John R. Porch, Supervisor of Terrestrial Plant and Insect Studies (3) Andrew J. Brignole, Biologist (4) Raymond L. Van Hoven, Ph.D., Scientist (5) Willard B. Nixon, Ph.D., Director of Chemistry - 30- W ildliie International' L id PROJECT NO.: 454-110 Appendix 2 Changes to Protocol This study was conducted according to the approved protocol with the following changes: 1 The protocol was amended to include procedures regarding day 21 data and soil sample collection as well as extended growth portion of the test. 2 The protocol was amended to include procedures for data and soil sample collection at test termination. 3 The soil was composed of kaolinite clay, industrialized quartz sand, and peat mixed in a 2:25:1 ratio with regards to weight. The soil consisted of 49% sand, 30% silt, and 21% clay. 4 Observations of emergence were made on Day 15 rather than 14. 5 The sponsor's representative was changed. -31 - Wildlife IntemationalfLid. PROJECT NO.: 454-110 Appendix 3 Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS,Lot217 Reference #: SD-018 __________ _________ Purity: 86.9%____________________ Test Name Specifications Purity1 Result 86.9% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. N itrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids 5(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis6: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.005 wt.Avt.% 2. 0.001 wt.Avt.% 3. 1.439 wt./wt.% 4. 6.849 wt.Avt.% 5. <0.001 wt.Avt.% 6. 0.005 wt.Avt.% 7. <0.001 wt.Avt.% 1.93 wt.Avt.% 8.41 wt.Avt.% None Detected 0.33 wt.Avt.% None Detected Not Applicable3 1. <0.015 wt.Avt.% 2. 0.59 wt.Avt.% 3. <0.040 wt.Avt.% 4. < 0 .0 0 9 w tV w t.% 5. <0.006 wt.Avt.% 6. <0.007 wt.Avt.% 7. 8.76 wt.Avt.% 1. <0.1 wt.Avt.% 2. <0.1 wt.Avt.% 3. 0.10 wt.Avt.% 4. 0.28 wt.Avt.% 1. 12.48 wt.Avt.% 2. 0.244 wt.Avt.% 3. 1.74 wt.Avt.% 4. 8.84 wt.Avt.% 5. 54.1 wt.Avt.% COA023-018A Page 1 of 3 - 32- Wild liie International? L id PROJECT NO.: 454-110 Appendix 3 (continued) Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 `Purity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.09% Purity = 100% - 13.09% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. "Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfbr determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity.5 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid theoretical value calculations based on the empirical formula, CgFnSOj'K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A -33 - Page 2 of 3 ild life International' Ltd. PROJECT NO.: 454-110 Appendix 3 (continued) Certificate of Analysis Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 www.cenirelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wt/wt % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. Prepared By: Scientist, Centre Analytical Laboratories fo //! /o Date :Reviewed By: q L m f U tc t Jota Flaherty / Date Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 o f 3 - 34- W ild liie Interna tional. Lid. PROJECT NO.: 454-110 Appendix 4 The Analysis of PFOS In Soil and Seven Species of Plants In Support of Wildlife International, Ltd. Project No.: 454-110 -35 - Wildliie International' Lid. PROJECT NO.: 454-110 INTRODUCTION Soil and plant tissue (vegetative and fruit) samples were collected from a plant toxicity study designed to determine the effects of Perfluorooctanesulfonate, Potassium Salt (PFOS) to seven species of plants. This study was conducted by Wildlife International, Ltd. for 3M Corporation and identified as Project Number 454-110. The chemistry phase of this study was conducted at the Wildlife International, Ltd. analytical chemistry facility in Easton, Maryland using high performance liquid chromatography with triple quadrupole mass spectrometric detection (HPLC/MS/MS). Samples were prepared and analyzed between November 15, 2001 and August 28,2002. MATERIALS AND METHODS Test Substance The test substance used for this study was Wildlife International, Ltd. identification number 4675. The test substance was used to prepare calibration and matrix fortification samples. Analytical Methodology Artificial Soil Submitted artificial soil samples were analyzed for PFOS following procedures documented in Wildlife International, Ltd. study number 454C-120 entitled "Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Artificial Soil" (8). The entire submitted soil sample was blended for approximately two minutes prior to sub-sampling the requisite 10-g aliquot for the analytical determ ination. The sub-sam ples were extracted with methanol, agitated for a minimum of 30 minutes on a gyratory shaker table at approximately 250 rpm, and vacuum filtered with qualitative filter paper. The retained soil was triple rinsed with methanol. The filtrate was then transferred to a 200-mL volumetric flask and brought to volume with methanol. Approximately 20 milliliters of each sample was transferred to a separate vial or tube and centrifuged for approximately 5 minutes at 2000 rpm. Dilutions into the calibration range of the HPLC/MS/MS methodology were performed with a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure water. Samples were then analyzed by direct injection. Concentrations of PFOS were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 3000LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a -36- Wildlife International. L id PROJECT NO.: 454-110 Keystone Betasil C]8column (50 mm x 2.0 mm, 3 pm particle size) fitted with a Keystone Javelin Cig guard column (20 mm x 2.0 mm). A method flowchart for PFOS determinations in artificial soil is provided in Appendix 4.1 and the instrument parameters are summarized in Appendix 4.2. Plant Tissue Submitted plant tissue samples were analyzed for PFOS following procedures documented in Wildlife International, Ltd. study number 454C-125 entitled "Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues" (9). Vegetative (i.e. stems and leaves) and fruit tissues were processed and analyzed using the same procedures. The entire submitted plant sample was manually shredded, cut, and/or blended as appropriate. If sufficient sample quantity was available, one-gram aliquots of each homogenate were weighed into vials for analytical determination. Numerous study samples (i.e. Day 21 vegetative tissues) were quantity limited. Therefore, the following criterion was implemented: If a given replicate (A, B, or C) for a test level contained >0.5 g of tissue, the replicate was individually analyzed. If two or more replicates contained <0.5 g, the replicates were composited. Composited replicates were analyzed when the composite weight was >0.5 g. Plant sub-samples were extracted in the vials with ten milliliters of methanol. Extraction consisted of manual shaking for approximately one minute followed by approximately 30 minutes of agitation on a gyratory shaker table set at approximately 250 rpm. The sub-samples were then centrifuged for approximately ten minutes at approximately 2000 rpm. Dilutions into the calibration range of the HPLC/MS/MS methodology were performed with a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure water. Samples were then analyzed by direct injection using the same instrumental conditions as described for the determination of PFOS in soil (Appendix 4.2). A method flowchart for the determination of PFOS in plant tissues is provided in Appendix 4.3. Primary and Secondary Stock Solutions All primary and secondary stock preparations were adjusted for the purity of the test substance (86.9%). Details on the preparation of the stock solutions of PFOS are provided in Appendix 4.4. The primary and secondary stocks were used for preparation of calibration standards and matrix fortification samples. -37- W ild life International' Lid. PROJECT NO.: 454-110 Calibration Standards and Calibration Curves Calibration standards of PFOS, prepared in 50:50 methanol: NANOpure water by appropriate dilution of the 0.00100 pg a.i./ pL stock solution of PFOS in methanol (Appendix 4.4), were analyzed with each soil and plant tissue sample set. For soil analyses, PFOS calibration standards ranged in concentration from 1.00 to 10.0 pg a.i./L. For plant tissue analyses, PFOS calibration standards ranged in concentration from 0.400 to 5.00 pg a.i./L. The calibration standard series was injected at the beginning and end of each run, and one standard was injected, at a minimum, after every five samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using peak area responses versus the respective concentrations of the calibration standards. Typical calibration curves for PFOS determinations in artificial soil and plant tissues are presented in Appendices 4.5 and 4.6, respectively. Representative ion chromatograms of low- and high-level PFOS calibration standards are presented in Appendices 4.7 and 4.8. The concentrations of PFOS in the samples were determined by substituting peak area responses into the applicable linear regression equation. Concentrations were calculated on a dry weight basis using moisture determination data submitted with the samples. An example of the calculations for a representative artificial soil sample is included in Appendix 4.9. Limits o f Quantitation Artificial Soil The method limit of quantitation (LOQ) in artificial soil was 1.18 mg a.i./kg on a dry- weight basis and was calculated as the product of the lowest calibration standard (1.00 pg a.i./L = 0.00100 mg a.i./L), the weight/volume dilution factor of the matrix blank samples (1000) divided by the percent solids for the test system soil (84.9%). Plant Tissue - Fruit Method LOQs in plant fruit tissues were reported on a dry-weight basis and were calculated for each plant fruit species, except alfalfa, as the product of the low-level calibration standard concentration, the weight/volume dilution factor of the matrix blank samples, divided by the percent solids determined for the fruit species negative control samples. The presence of -38- Wildlife International. L id PROJECT NO.: 454-110 contamination and/or interfrent matrix species in alfalfa fruit tissue necessitated a unique LOQ definition relative to the other plant fruit species. For alfalfa fruit, the LOQ was calculated on a dry-weight basis as the maximum mg PFOS/kg equivalent concentration observed in the negative control extract rounded upward to the nearest tenth mg a.i./kg value (see Limits of QuantitationPlant Tissue- Vegetative). Plant Tissue - Vegetative Method LOQs in plant vegetative tissues were reported on a dry weight basis and were calculated as the maximum mg PFOS/kg equivalent concentration observed in the negative control extracts for each plant vegetative tissue species, rounded upward to the nearest tenth mg a.i./kg value. As was the case for alfalfa fruit, unidentified matrix interferences were often evident in selected negative control samples at levels above the low standard equivalent LOQ definition (see Limits of Quantitation, Plant Tissue - Fruit). Further, the magnitude of the matrix interference was highly variable within a set of negative controls for a given vegetative tissue species, thereby negating use of an averaged background correction approach. Matrix Fortifications and Matrix Blanks Artificial Soil Selected 20.0-g aliquots of artificial soil matrix were fortified with the appropriate stock solutions of PFOS prepared in methanol using a gas-tight syringe. The fortified soils were then hom ogenized with blending for approxim ately two m inutes. A 10-g aliquot o f each hom ogenized fortified soil sample was weighed into a tared weigh boat and transferred to an 8-oz. Frenchsquare bottle for extraction. The matrix blanks were unfortified artificial soil. Along with the sample analyses, three matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix 4.10). A representative ion chromatogram of a matrix blank sample in artificial soil is presented in Appendix 4.11. Artificial soil samples were fortified at 2.36, 118, and 1410 mg a.i./kg (dry weight basis) and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 83.9%, 89.0% and 86.5%, respectively (Appendix - 39- Wildliie International. L td PROJECT NO.: 454-110 4.10). Sample measured concentrations were not corrected for the mean procedural recoveries. A representative ion chromatogram of a matrix fortification sample in artificial soil is presented in Appendix 4.12. Plant Tissues Selected aliquots (approximately 1.00 g) of negative control plant tissues were fortified with appropriate stock solutions of PFOS prepared in methanol using a gas-tight syringe. A matrix blank was prepared with unfortified negative control plant tissue. Fruit and vegetative plant tissue samples were fortified at target nominal concentrations of 0.050, 0.50, and 50.0 mg a.i./kg (wet weight basis) and analyzed concurrently with the samples to determine mean procedural recoveries. In fruit tissue, the mean procedural recoveries ranged from 86.3 to 117% of nominal concentrations (Appendix 4.13). Sample measured concentrations were not corrected for the mean procedural recoveries. Representative ion chromatograms of a matrix blank and a matrix fortification sample in a plant fruit tissue are presented in Appendices 4.14 and 4.15, respectively. In vegetative tissue, the mean procedural recoveries ranged from 88.0 to 110% of nominal concentrations (Appendix 4.16). Sample measured concentrations were not corrected for the mean procedural recoveries. Representative ion chromatograms of a matrix blank and a matrix fortification sample in a plant vegetative tissue are presented in Appendices 4.17 and 4.18, respectively. RESULTS Artificial Soil Samples of artificial soil collected on Days 0 and 21, and at test termination were submitted and analyzed for PFOS. All negative control artificial soil samples were <LOQ for PFOS. Day 0 analyses of the 3.91, 15.6, 62.5, 250, and 1000 mg a.i./kg treatment levels had mean measured values of 3.61, 11.1, 50.8, 276, and 998 mg a.i./kg, corresponding to 92.3%, 71.2%, 81.3%, 110%, and 99.8% of the nominal concentrations, respectively (Appendix 4.19). PFOS measured values for test termination samples are presented in Appendix 4.20. The 3.91, 15.6, 62.5, 250 and 1000 mg a.i./kg treatment levels had mean measured values of 1.29, 3.56, 16.2, 157 and 474 mg a.i./kg, corresponding to 33.0%, 22.8%, 25.9%, 62.8% and 47.4% of the nominal concentrations, respectively. A representative ion chromatogram of an artificial soil test sample is presented in Appendix 4.21. - 40- Wildliie International. L id PROJECT NO.: 454-110 Plant Tissues Samples of fruit tissue from five species of plant (alfalfa, flax, onion, soybean, and tomato) grown in negative control and PFOS-treated artificial soils were collected at test termination (for a given species) and analyzed for PFOS residues. Samples of vegetative tissue from seven species of plant (alfalfa, flax, lettuce, onion, ryegrass, soybean, and tomato) grown in negative control and PFOS-treated artificial soils were collected at experimental termination (for a given species) and analyzed for PFOS residues. Measured PFOS concentrations are presented in Appendices 4.22 4.33. Representative ion chromatograms of plant fruit and vegetative tissue test samples are presented in Appendices 4.34 and 4.35, respectively. -41 - AAAildliie International. L id PROJECT NO.: 454-110 Appendix 4.1 Analytical Method Flowchart for the Processing of PFOS in Soil Prepare QC samples in soil matrix from the appropriate methanol stocks. Weigh approximately 20.0 g of soil matrix for each QC into a tared weigh boat. Record weights. Transfer to 8-oz. French square glass bottles. Fortify each soil sample with the appropriate volume of PFOS stock solution in methanol with a gas-tight syringe. Allow to air dry. The matrix blank is unfortified soil matrix. I For each QC and test sample, transfer the entire contents from the 8-oz French square glass bottle to a blender. Homogenize each sample for approximately 2 minutes. Place the entire homogenate into a Ziploc bag. I Weigh approximately 10.0 grams of each study sample into a tared weigh boat. Record weights. Transfer to 8 oz. French square bottles. i For each sample, measure 100 mL of methanol with a graduated cylinder and transfer into the French square bottle. I Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. 4 Vacuum filter with qualitative filter paper and rinse retained soil 3 times with methanol into filtrate. 4, Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. Mix well with several repeat inversions. I Transfer approximately 20 mL o f each sample into a separate glass centrifuge tube or scintillation vial and cap. Centrifuge samples for approximately 5 minutes at 2000 rpm. Prepare appropriate dilution(s) to bring final concentration into the calibration range of the LCMS methodology. For all dilutions, use 50% methanol:50% NANOpure water dilution solvent, gas- tight syringes, and Class A volumetric glassware. I Ampulate and submit sample for HPLC/MS/MS analysis. -42- W ildliie Intema tonalfLid PROJECT NO.: 454-110 Appendix 4.2 Typical HPLC/MS/MS Operational Parameters INSTRUMENT: ION SOURCE: ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS PEAK RETENTION TIME: PFOS MONITORED MASS: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer Operated in multiple ion reaction monitoring (MRM) mode. Perkin-Elmer TurboIonSpray Keystone Betasil C18(50 mm x 2.0 mm, 3-pm particle size) Keystone Javelin Q 8cartridge (20 mm x 2 mm) 40C 5.00 min 250 pL/min 30% NANOpure Water with 0.1% Formic Acid: 70% Methanol 5.0 pL Approximately 4 minutes 499.0 amu -- 99.1 amu -43 - Wildlife International' Lid. PROJECT NO.: 454-110 Appendix 4.3 Analytical Method Flowchart for the Processing of PFOS in Plant Tissue Remove samples from frozen storage. Homogenize samples with manual shredding, cutting and/or blending, as appropriate. Weigh the appropriate number of 1-g aliquots of each homogenate into a tared vial. 4 Quality control samples are prepared from 1-g aliquots of homogenized negative control plant tissue. Fortify matrix fortification samples with the appropriate PFOS stock solution(s) in methanol using gas-tight syringe(s). The matrix blank is unfortified plant matrix. For each sample, measure 10.0 mL of methanol with a glass Class A volumetric pipette. Cap the vials and shake manually for a minimum of one minute. I Place samples on gyratory shaker table and shake at a setting of 250 rpm for approximately thirty minutes. 4 Centrifuge the vials at approximately 2000 rpm for approximately 10 minutes. i Prepare appropriate dilutions of study and QC samples to bring concentrations within the calibration range of the PFOS LCMS methodology: Partially fill Class A volumetric flasks with 50%: 50% methanol/ NANOpure water dilution solvent. Add appropriate volume of sample with a gas-tight syringe and bring to volume with dilution solvent. Process m atrix blank samples using the same dilution and aliquot volumes as for the lowest fortification level. Cap and mix well by several repeat inversions. Store the remaining original methanol extracts in the walk-in cooler. I Transfer an aliquot of each sample to an autosampler vial and submit for HPLC/MS/MS analysis. - 44- Wildlife InternationalfL id PROJECT NO.: 454-110 Appendix 4.4 Analytical Stocks and Standards Preparation A 10.0 pg a.i./pL primary stock solution of PFOS was prepared by weighing 1.1508 g of the test substance on an analytical balance. The test substance was brought to 100-mL final volume with methanol. Secondary stock solutions (1.00, 0.100, 0.0100, 0.00100, and 0.000100 pg a.i./pL) of PFOS in methanol were prepared from the primary stock by serial volumetric dilution. The calibration standards were prepared in 50% (v/v) methanol in NANOpure water. The following shows dilution schemes for the sets of calibration standards employed for determination of PFOS in soil and plant tissues: Stock Concentration (ug a.i./uL) 0.00100 0.00100 0.00100 0.00100 0.00100 Calibration Standards for Determination of PFOS in Soil Aliquot (pL) 100 250 500 750 1000 Final Volume (mL) 100 100 100 100 100 Standard Concentration (ug a.i./L) 1.00 2.50 5.00 7.50 10.0 Stock Concentration (ug a.i./uD 0.00100 0.00100 0.00100 0.00100 0.00100 0.00100 Calibration Standards for Determination of PFOS in Plant Tissues Aliquot (m 40.0 50.0 100 250 350 500 Final Volume (mL) 100 100 100 100 100 100 Standard Concentration (ug a.i./L) 0.400 0.500 1.00 2.50 3.50 5.00 - 45- y V ilclliie Iniem aiionalt Lid. PROJECT NO.: 454-110 Appendix 4.5 Typical Calibration Curve for PFOS Determinations in Artificial Soil PFOS 499.0->99.1 No Internal Standard Weighted (1/x) Intercept = 1818.5138 Slope = 10974956.0000 Correlation Coeff. = 0.99798 Area -46- Wildliie International, L td PROJECT NO.: 454-110 Appendix 4.6 Typical Calibration Curve for PFOS Determinations in Plant Tissues PFCS 4 9 9 .0 -> 9 9 .1 No Internal Standard Weighted (1/x) Intercept = 27.4030 Slope = 3429.6262 Correlation Coeff. = 0.99864 Area -47- Wild liie International. L id ! PROJECT NO.: 454-110 Appendix 4.7 Representative Chromatogram of a Low-Level PFOS Calibration Standard PFOS 1 STD 0.400 ug a.UL 4675A-01 ID-26 4.98 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. 2.0 0.5 Min. Width 12 Mult. Width 10 Base. Width 40 RT Win. (secs) 10 Smooth 2 Expected RT 4.05 Area 1363 Height 139 Start Time 3.57 End Time 4.02 Integration Width 0.45 Retention Time 3.80 Integration Type A-BB Sun, Jan 27, 2002 07:23 100-] 908070605040302010 intensity: 1700 cps 40 62 79 110 145164 41 81 121 161 0.69 1.36 2.03 2.70 227 i l A - J L 267 201 241 281 Scan 3.37 4.04 4.71 Time Nominal concentration: 0.400 pg a.i./L. -48- Wildlife International' L td PROJECT NO.: 454-110 Appendix 4.8 Representative Chromatogram of a High-Level PFOS Calibration Standard PFOS_5 STD 0.0100 mg a .IA 4675A-011D-20 4.98 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. 1.0 Min. Width 3 Mult. Width Base. Width 6 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.01 Area 109807 Height 11092 Start Time 3.69 End Time 4.36 Integration Width 0.67 Retention Time 3.95 Integration Type A - VB Mon. Nov 19, 2001 12:41 10Oi 908070 6050 intensity: 10000 cps 236 30 10 o-S --20T--4-T1---5-8-7-3- T- 109 147 180 41 81 121 161 201 0.69 1.36 2.03 2.70 3.37 281 Scan 4.71 Time Nominal concentration: 10.0 jug a.i./L. -49- Wildlife International? L id PROJECT NO.: 454-110 Appendix 4.9 Example Calculations for a Representative Sample The analytical result and percent recovery of PFOS in artificial soil for sample number 454-110-10, nominal concentration of 62.5 mg a.i./kg, was calculated using the following equations: where PA = m= C= b= peak area slope of the line concentration y - axis intercept Using the appropriate regression data from the sample analysis sequence (Appendix 4.5), the concentration in the final sample solution was calculated as: 28656- 1818.5138 10974956 0.002445 mg a.i./L The measured concentrations of PFOS in the artificial soil sample determined as follows: . .x (C) x (Ve) X (Vf,) x (Vg) Concentration PFOS (mg a.i./kg) - ^w ) x x (V_2) x (o/oSolids) where C = Concentration (mg a.i./L) as determined above Ve = Extraction Volume (100 mL) Vi, = First Initial Volume (100 mL) Vi2 = Second Initial Volume (0.0500 mL) Vf, = First Final Volume (200 mL) w =V Q = Second Final Volum e (50.0 mL) Weight of extracted sample (10.0 g) __ 0.002445x 100x200x50.0 Concentration PFOS (mg a.i./kg) = 1QQx 100 x 0.0500 x 0.849 Concentration PFOS (mg a.i./kg) = 57.60 . ,, . PFOS (mg a.i./Kg) in sample __ Percent of Nominal Concentration = PFOS (mg a.i./Kg) nominal x 100 = ^ y x 100 = 92.2% Calculated with HPLC/MS/MS instrument software: MacQuan, version 1.6. - 50- WildJi/e International. L id PROJECT NO.: 454-110 Appendix 4.10 Artificial Soil Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Number (454-110-) Sample Type Sampling Interval PFOS Concentration (mg a.i./kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./kg) Mean Percent Recovery MAB- 1 M A B-3 MAS - 1 MAS- 7 MAS- 2 MAS- 8 M AS- 3 MAS- 9 Soil Matrix Blank Soil Matrix Blank Soil Matrix Fortification Soil Matrix Fortification Soil Matrix Fortification Soil Matrix Fortification Soil Matrix Fortification Soil Matrix Fortification Initiation Termination Initiation Termination Initiation Termination Initiation Termination 0.0 0.0 2.36 2.36 118 118 1410 1410 < 1.182 < 1.18 2.24 1.99 108 104 1270 1310 - 95.1 84.4 91.3 88.1 90.0 93.1 < 1.18 -- 2.12 106 1290 89.8 89.8 91.5 Overall Mean = 90.3 Standard Deviation = 3.78 CV= 4.19 ___________________________________________________________________ N = 6____________________________ 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2The method limit of quantitation (LOQ) in artificial soil was 1.18 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i./L) and the weight/volume dilution factor of the matrix blank samples (1000) divided by the percent solids for the test system soil (84.9%). ___________________________ -51 - AAAild life Intem a iionalfL id PROJECT NO.: 454-110 Appendix 4.11 Representative Chromatogram of an Artificial Soil Matrix Blank Sample PFOS_7 454-110- MAB-1 Mon. Nov 19, 2001 12:53 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) 1.0 3 6 40 20 Smooth 1 Expected RT 4.01 Area 0 Height 0 Start Time End Time Integration Width Retention Time Integration Type 0.00 0.00 0.00 0.00 100i 90 80706050 403020 IO 12 50 75 96 121140 169 202 41 81 121 ' 161 ` 21 0.69 1.36 2.03 2.70 3.37 intensity: 10000 cps I 1 240 273 241 ' 281 Scan 4.04 4.71 Time Sample Identification: 454-110-MAB-l. The arrow indicates the retention time of PFOS. - 52- WildliigInternational. L id PROJECT NO.: 454-110 Appendix 4.12 Representative Chromatogram of an Artificial Soil Matrix Fortification Sample pros 9 454-110- MAS-2 Mon, Nov 19, 2001 13:05 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.01 Area 51987 Height 5360 Start nme 3.65 End nme 4.32 Integration Width 0.67 Retention nme 3.97 Integration Type A - VB intensity: 10000 cps Sample Identification: 454-110-MAS-2. Nominal Concentration: 118 mg a.i./kg (dry-weight basis). -53 - Wildlile Intenm tional. L id PROJECT NO.: 454-110 Appendix 4.13 Fruit Tissue Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Number (454-110-) Sample ______________________ Type Sampling Interval PFOS Concentration (mg a.i./kg) Fortified3 Measured1,2 __________________ Wet-Weight Dry-Weight Basis Basis Percent Recovered1 alf-f-MAB-1 alf-f-MAS-3 flx-f-MAB-1 flx-f-MAS-1 flx-f-MAS-2 flx-f-MAS-3 oni-f-MAB-1 oni-f-MAS-1 oni-f-MAS-2 oni-f-MAS-3 soy-f-MAB-1 soy-f-MAS-1 soy-f-MAS-2 soy-f-MAS-3 tom-f-MAB-1 tom-f-MAS-1 tom-f-MAS-2 tom-f-MAS-3 Alfalfa Matrix Blank Alfalfa Fortification Flax Matrix Blank Flax Fortification Flax Fortification Flax Fortification Onion Matrix Blank Onion Fortification Onion Fortification Onion Fortification Soybean Matrix Blank Soybean Fortification Soybean Fortification Soybean Fortification Tomato Matrix Blank Tomato Fortification Tomato Fortification Tomato Fortification Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination 0.0 50.0 0.0 0.0495 0.503 47.9 0.0 0.0490 0.467 47.9 0.0 0.0500 0.496 50.0 0.0 0.0498 0.495 47.8 3.094 50.4 < 0.040 0.0581 0.521 44.4 < 0.040 0.0477 0.451 51.4 < 0.040 0.0508 0.478 47.9 < 0.040 0.0468 0.427 42.3 8.434 138 <0.12 0.170 1.52 130 <0.42 0.494 4.68 533 <0.18 0.222 2.09 209 <0.47 0.554 5.05 501 101 -- 117 104 92.7 -- 97.3 96.6 107 -- 102 96.4 95.7 -- 93.9 86.3 88.6 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The method limit of quantitation (LOQ) in fruit tissue was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weight/volume dilution factor of the matrix blank sample. 3Fortified PFOS concentrations are presented on a wet-weight basis. 4The method limit of quantitation (LOQ) in alfalfa fruit tissue (6.4 mg a.i./kg on a dry-weight basis = 2.3 mg a.i./kg on a wet-weight basis) was calculated as the maximum mg PFOS/kg equivalent concentration measured in negative control sample extracts rounded upward to the nearest tenth mg a.i./kg.______ _________________________________________________________________________________ -54- W ildlife International L id PROJECT NO.: 454-110 Appendix 4.14 Representative Chromatogram of a Plant Fruit Tissue Matrix Blank Sample PFOS 8 454-110- SOY-F-MAB-1 4.98 in 1 period PFOS No internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 2.0 0.5 12 50 80 20 2 3.75 Area 0 Height 0 Start Time 0.00 End Time Integration Width Retention Time Integration Type 0.00 0.00 0.00 Tue, Mar 26, 2002 14:06 10O| 90807060504030 20 100- intensity: 4000 cps I 1 T 51 41 0.69 81 81 1.36 133155 J ill- " i 265 121 ' l i 21 ' 241 ' 281 Scan 2.03 2.70 3.37 4.04 4.71 Time Sample Identification: 454-110-SOY-F-MAB-l (soybean matrix). The arrow indicates the retention time of PFOS. -55- Wildliie International L id PROJECT NO.: 454-110 Appendix 4.15 Representative Chromatogram of a Plant Fruit Tissue Matrix Fortification Sample PFOSJ1 454-110- SOY-F-MAS-3 4.98 in 1 period PF06 No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mult. Width 10 Base. Width 80 RT Win. (secs) 20 Smooth 2 Expected RT 3.75 Area 16586 Height 1396 Start Time End Time Integration Width Retention Time Integration Type 3.55 4.42 0.87 3.77 A-VB Tue, Mar 26, 2002 14:24 Intensity: 4000 cps Sample Identification: 454-110-SOY-F-MAS-3 (soybean matrix). Nominal Concentration: 50.0 mg a.i./kg (wet-weight basis). - 56- Wildliie International L id PROJECT NO.: 454-110 Appendix 4.16 Vegetative Tissue Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Number (454-110-) Sample Type Sampling Interval PFOS Concentration (mg a.i./kg) Fortified3 Measured1,2 Wet-Weight Dry-Weight Percent Basis Basis Recovered12 alf-v-MAB-1 aIf-v-MAS-3 flx-v-MAB-1 flx-v-MAS-3 let-v-MAB-1 let-v-MAS-2 let-v-MAS-3 oni-v-MAB-2 oni-v-MAS-5 rye-v-MAB-1 rye-v-MAS-2 soy-v-MAB-1 soy-v-MAS-2 tom-v-MAB-1 tom-v-MAS-3 Alfalfa Matrix Blank Alfalfa Fortification Flax Matrix Blank Flax Fortification Lettuce Matrix Blank Lettuce Fortification Lettuce Fortification Onion Matrix Blank Onion Fortification Ryegrass Matrix Blank Ryegrass Fortification Soybean Matrix Blank Soybean Fortification Tomato Matrix Blank Tomato Fortification Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination Termination 0.0 48.1 0.0 48.8 0.0 0.502 50.4 0.0 50.1 0.0 49.5 0.0 49.3 0.0 48.8 <0.84 44.2 <0.72 47.5 <0.46 0.536 48.8 <0.42 44.1 < 1.8 54.3 <2.4 48.4 < 1.4 49.9 <2.7 142 <2.4 158 <5.5 6.37 579 <4.7 492 <3.3 100 <6.0 119 <7.6 281 91.9 _ 97.3 _ 107 96.7 _ 88.0 _ 110 __ 98.1 _ 102 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The method limit of quantitation (LOQ) in each vegetative tissue was calculated as the maximum mg PFOS/kg equivalent concentration measured in negative control extracts for that tissue rounded upward to the nearest tenth mg a.i./kg. 3Fortified PFOS concentrations are presented on a wet-weight basis._______________________________________________________ -57- Wildlife IntemationalfLid PROJECT NO.: 454-110 Appendix 4.17 Representative Chromatogram of a Plant Vegetative Tissue Matrix Blank Sample PFOS_8 454-110- MAB-1 Sun, Jan 27, 2002 08:05 4.98 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise litres. Quant Thres. Min. Width Mult. Width 2.0 0.5 12 10 Base. Width RT Win. (secs) Smooth Expected RT 40 10 2 4.05 Area 687 Height 69 Start Time End Time Integration Width Retention Time Integration Type 3.62 4.04 0.42 3.82 A-BB 10Oi 90B070605040302010o-l intensity: 1700 cps 145 169 IS.8 - A 8 , 258 241 281 Sein 4.04 4.71 Time Sample Identification: 454-110-LET-V-MAB-l (lettuce matrix). The arrow indicates the retention time of PFOS. -58- Wildliie International L id PROJECT NO.: 454-110 Appendix 4.18 Representative Chromatogram of a Plant Vegetative Tissue Matrix Fortification Sample PFOS 10 454-110- MAS-3 Sun. Jan 27, 2002 08:17 4.98 In 1 period Prue No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. 2.0 0.5 Min. Width 12 Mult. Width 10 Base. Width 40 RT Win. (secs) Smooth Expected RT 10 2 4.05 Area 6660 Height 671 Start Time Endnme Integration Width 3.62 4.19 0.57 Retention Time 3.87 Integration Type A - VB 100-] 908070 6050 40 30- 1013 o- 46 41 0.69 intensity: 1700 cps 1I06I 13I1 81 121 1.36 2.03 162 161 ' 2.70 231 1I IA A201 201 241 3.37 4.04 281 Scan 4.71 Time Sample Identification: 454-110-LET-V-MAS-3 (lettuce matrix). Nominal Concentration: 50.4 mg a.i./kg (wet-weight basis). -59- Wild lifo Intem a tion ai L id PROJECT NO.: 454-110 Appendix 4.19 Day 0 Recoveries for PFOS in Artificial Soil Sample Number Nominal (454-110-) Concentration PFOS Concentration (mg a.i./kg) Measured1 Percent Recovered1 Mean Measured (mg a.i./kg) Mean Percent Recovery 01 0.0 02 3.91 03 3.91 04 3.91 05 15.6 06 15.6 07 15.6 08 62.5 09 62.5 10 62.5 11 250 12 250 13 250 14 1000 15 1000 16 1000 < 1.182 3.43 2.83 4.56 11.1 11.2 86.43 50.0 44.7 57.6 415 244 170 1070 983 942 - 87.7 72.4 117 71.1 72.0 5543 80.0 71.6 92.2 166 97.5 67.9 107 98.3 94.2 <1.18 3.61 11.1 50.8 276 998 92.3 71.2 81.3 110 99.8 Overall Mean == Standard Deviation == 92.5 26.0 CV == N == 28% 14 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2The method limit quantitation (LOQ) in artificial soil was 1.18 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i./L) and the weight/volume dilution factor of the matrix blank samples (1000) divided by the percent solids for the test system soil (84.9%). Statistical outlier. Data excluded from Mean Measured calculation.__________________ -60- Wild life International' L id PROJECT NO.: 454-110 Appendix 4.20 Termination Recoveries for PFOS in Artificial Soil Sample Number Nominal (454-110-) Concentration PFOS Concentration (mg a.i./kg) Measured1 Percent Recovered1 Mean Measured (mg a.i./kg) Mean Percent Recovery 28 0.0 < 1.182 - <1.18 - 29 3.91 30 3.91 31 15.6 32 15.6 33 62.5 34 62.5 35 250 36 250 37 1000 38 1000 1.21 31.0 1.29 33.0 1.36 34.7 4.91 31.5 3.56 22.8 2.21 14.2 16.3 26.1 16.2 25.9 16.1 25.8 153 61.4 157 62.8 161 64.5 432 43.2 474 47.4 515 51.5 Overall Mean = 38.4 Standard Deviation = 16.4 CV = 43% ______________________________________ ________________ N = 10_________________________________ 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2The method limit quantitation (LOQ) in artificial soil was 1.18 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i./L) and the weight/volume dilution factor of the m atrix b la n k sam p les (1 0 0 0 ) d iv id ed b y th e p ercen t so lids fo r th e test sy stem soil (8 4 .9 % ). -61 - Wildliie International. Ltd. PROJECT NO.: 454-110 Appendix 4.21 Representative Chromatogram of an Artificial Soil Test Sample PFOS_22 10 Mon, Nov 19, 2001 14:23 454-110- 4.9B in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Hires. Min. Width Mult. Width 30.0 1.0 3 6 Base. Width RT Win. (secs) Smooth Expected RT 40 20 1 4.01 Area 28656 Height 2916 Start Time End Time Integration Width 3.72 4.39 0.67 Retention Time Integration Type 3.99 A-BB intensity: 10000 cps Sample Identification: 454-110-10. Nominal Concentration: 62.5 mg a.i./kg (dry-weight basis). -62- Wild liie International? L id PROJECT NO.: 454-110 Appendix 4.22 Termination Measured Values for PFOS in Alfalfa Vegetative Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) alf-v-19 alf-v- 2 0 alf-v- 2 1 alf-v- 2 2 alf-v-23 alf-v-24 alf-v-25 alf-v-26 alf-v-27 alf-v-28 alf-v-29 alf-v-30 0 .0 0 .0 0 .0 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 < 2.72 <2.7 <2.7 <2.7 <2.7 6.16 5.66 2.74 <2.7 12.9 9.66 1 1 .1 <2.7 6.16 4.2 1 1 .2 alf-v-31 alf-v-32 alf-v-33 alf-v-343 alf-v-353 alf-v-363 250 250 250 1000 1000 1000 14.2 15.8 15.8 17.3 --_ _ -- -- 'Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results rep o rted on a d ry -w eig h t basis. 2The method limit of quantitation (LOQ) in alfalfa vegetative tissue (2.7 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. 3No sample was collected due to mortality at this c o n c e n t r a t i o n . ______________________________________________ -63 - Wildlile International' L td PROJECT NO.: 454-110 Appendix 4.23 Termination Measured Values for PFOS in Alfalfa Fruit Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) alf-f-1 3 alf-f-2 3 alf-f-33 0.0 0.0 0.0 < 6.42 <6.4 alf-f-44 alf-f-54 alf-f- 6 alf-f-73 alf-f-8 3 alf-f-93 alf-f-1 0 3 alf-f-1 1 3 alf-f-1 2 3 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 __ -- <6.4 <6.4 <6.4 <6.4 <6.4 <6.4 alf-f-133 alf-f-143 alf-f-153 alf-f-165 alf-f-175 alf-f-185 250 250 250 1000 1000 1000 <6.4 __ -- -- <6.4 1 Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. R esu lts re p o rte d o n a d ry -w eig h t b asis. 2 The method limit of quantitation (LOQ) in alfalfa fruit tissue (6.4 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg PFOS/kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. 3 A composite sample was made of all samples collected at this concentration. 4 There was insufficient sample weight (<0.5 g) available for analysis. 5 No sample was collected due to mortality at this concentration.____________________________________ -64- Wildliie International. L id PROJECT NO.: 454-110 Appendix 4.24 Termination Measured Values for PFOS in Flax Vegetative Tissue Number (454-110-) flx-v-19 flx-v-20 flx-v-21 flx-v-22 flx-v-23 flx-v-24 flx-v-25 flx-v-26 flx-v-27 flx-v-28 flx-v-29 flx-v-30 flx-v-313 flx-v-324 flx-v-334 flx-v-343 flx-v-353 flx-v-363 Sample Nominal Exposure 0.0 0.0 0.0 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 1000 1000 1000 PFOS Concentration (mg a.i./kg) Measured' < 2.412 <2.4 <2.4 4.56 6.43 3.85 13.1 26.4 17.0 54.7 72.7 37.5 _ -- - _ -- -- Mean Measured (mg a.i./kg) <2.4 4.95 18.8 55.0 __ _ 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slig h tly . R esu lts re p o rte d o n a d ry -w eig h t b asis. 2The method limit of quantitation (LOQ) in flax vegetative tissue (2.4 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. 3No sample was collected due to mortality at this concentration. 4 There was insufficient sample weight (<0.5g) available for this analysis._______________ ________________ -65 - Wildliie International; L id PROJECT NO.: 454-110 Appendix 4.25 Termination Measured Values for PFOS in Flax Fruit Tissue Sample Number (454-110-) Nominal Exposure PFOS Concentration (mg a.i./kg) Measured' Mean Measured (mg a.i./kg) flx-f-1 flx-f-2 flx-f-3 0 .0 0.0 0.0 < 0.1212 <0.12 <0.12 <0.12 flx-f-4 flx-f-5 flx-f-6 3.91 3.91 3.91 0.217 0.277 0.195 0.230 flx-f-7 flx-f-8 flx-f-9 15.6 15.6 15.6 1.07 0.622 2.41 1.36 flx-f-10 flx-f-11 flx-f-12 flx-f-133 flx-f-144 flx-f-154 flx-f-163 flx-f-173 flx-f-183 62.5 62.5 62.5 250 250 250 1000 1000 1000 1.26 2.20 4.23 _ -- - -- -- 2.56 _ __ 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slig h tly . R esu lts rep o rted on a d ry -w eig h t b asis. 2The method limit of quantitation (LOQ) in flax fruit tissue was 0.12 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weight/volume dilution factor of the matrix blank samples (100) divided by the percent solids for the negative control fruit tissue (34.3%). 3No sample was collected due to mortality at this concentration. 4 There was insufficient sample weight (<0.5 g) available for a n a l y s i s . ______________ -66- Wildliie Iniema iionaltLtcL PROJECT NO.: 454-110 Appendix 4.26 Termination Measured Values for PFOS in Lettuce Vegetative Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) let-v-19 let-v-20 let-v-21 let-v-22 let-v-23 let-v-24 let-v-25 let-v-26 let-v-27 let-v-28 let-v-29 let-v-30 let-v-313 let-v-323 let-v-333 let-v-344 let-v-354 let-v-364 0.0 0.0 0.0 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 1000 1000 1000 < 5.52 <5.5 <5.5 12.2 6.16 7.52 15.5 7.06 9.26 39.8 58.9 27.0 _ -- - -- -- -- <5.5 8.63 10.6 41.9 .. _ 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. R esu lts rep o rted on a d ry -w eig h t basis. 2The method limit of quantitation (LOQ) in lettuce vegetative tissue (5.5 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. 3There was insufficient sample weight (<0.5 g) available for analysis. 4No samples were collected due to mortality at this concentration._______________________________________________ -67- A/Vildliie International. L td PROJECT NO.: 454-110 Appendix 4.27 Termination Measured Values for PFOS in Onion Vegetative Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg oni-v-19 oni-v-20 oni-v-21 oni-v-22 oni-v-23 oni-v-24 0.0 0.0 0.0 3.91 3.91 3.91 < 4.72 <4.7 <4.7 <4.7 <4.7 <4.7 <4.7 <4.7 oni-v-25 oni-v-26 oni-v-27 oni-v-283 oni-v-293 oni-v-304 oni-v-314 oni-v-324 oni-v-334 oni-v-344 oni-v-354 oni-v-364 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 1000 1000 1000 5.86 15.4 <4.7 _ -- - __ -- - _ -- -- 10.6 _ __ __ 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. R e s u lts reported on a dry-weight basis. 2The method limit of quantitation (LOQ) in onion vegetative tissue (4.7 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. 3There was insufficient sample weight (<0.5 g) available for analysis. 4No sample was collected due to mortality or lack of emergence at this c o n c e n t r a t i o n . ________________ -68- Wild life International' L td! PROJECT NO.: 454-110 Appendix 4.28 Termination Measured Values for PFOS in Onion Fruit Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) oni-f-1 oni-f-23 oni-f-3 oni-f-4 oni-f-5 oni-f-63 oni-f-7 oni-f-8 oni-f-9 oni-f-103 oni-f-113 oni-f-124 0.0 0.0 0.0 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 0.517 -- 0.487 5.81 0.453 -- < 0.422 19.7 24.6 __ -- -- 0.502 3.13 22.2 ... oni-f-134 oni-f-144 oni-f-154 oni-f-164 oni-f-174 oni-f-184 250 250 250 1000 1000 1000 __ . . . -- __ -- --- 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. R esu lts rep o rted o n a d ry -w eig h t basis. 2The method limit of quantitation (LOQ) in onion fruit tissue was 0.42 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weight/volume dilution factor of the matrix blank samples (100) divided by the percent solids for the negative control fruit tissue (9.65%). 3There was insufficient sample weight (<0.5 g) available for analysis. 4No sample was collected due to mortality or lack of fruit production at this c o n c e n t r a t i o n . ___________________ -69- Wildliie International L id PROJECT NO.: 454-110 Appendix 4.29 Termination Measured Values for PFOS in Ryegrass Vegetative Tissue Number (454-110-) rye-v-19 rye-v-20 rye-v-21 rye-v-22 rye-v-23 rye-v-24 rye-v-25 rye-v-26 rye-v-27 rye-v-28 rye-v-29 rye-v-30 rye-v-31 rye-v-32 rye-v-33 rye-v-343 rye-v-353 rye-v-3 63 Sample Nominal Exposure 0.0 0.0 0.0 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 1000 1000 1000 PFOS Concentration (mg a.i./kg) Measured1 < 3.32 <3.3 <3.3 7.13 8.70 8.70 13.8 49.0 <3.3 42.5 67.0 36.7 31.9 37.5 129 _ -- -- Mean Measured (mg a.i./kg) <3.3 8.18 31.4 48.7 66.1 __ Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results rep o rted on a d ry -w eig h t basis. 2The method limit of quantitation (LOQ) in ryegrass vegetative tissue (3.3 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. No sample was collected due to mortality or lack of emergence at this concentration.________________________________ -70- W ildlile International, L id PROJECT NO.: 454-110 Appendix 4.30 Termination Measured Values for PFOS in Soybean Vegetative Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) soy-v-19 soy-v-20 soy-v-21 0.0 0.0 0.0 < 6.02 <6.0 <6.0 <6.0 soy-v-22 soy-v-23 soy-v-24 3.91 3.91 3.91 17.7 15.6 10.0 19.1 soy-v-25 soy-v-26 soy-v-27 soy-v-28 soy-v-29 soy-v-30 15.6 15.6 15.6 62.5 62.5 62.5 29.3 35.8 48.9 29.3 63.9 63.3 70.7 55.4 soy-v-31 soy-v-32 soy-v-3 3 soy-v-343 soy-v-3 53 soy-v-3 63 250 250 250 1000 1000 1000 64.2 114 175 103 __ -- -- - ' Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. R esu lts rep o rted on a d ry -w eig h t basis. 2The method limit of quantitation (LOQ) in soybean vegetative tissue (6.0 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. 3No sample was collected due to mortality at this concentration.________________________________________________ -71 - Wildlife IniemationalfL td PROJECT NO.: 454-110 Appendix 4.31 Termination Measured Values for PFOS in Soybean Fruit Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) soy-f-1 soy-f-2 soy-f-3 0.0 0.0 0.0 < 0.182 <0.18 <0.18 <0.18 soy-f-4 soy-f-5 soy-f-6 soy-f-7 soy-f-8 soy-f-9 soy-f-10 soy-f-11 soy-f-12 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 0.947 0.339 2.91 0.464 1.63 0.528 1.14 1.14 1.33 1.40 0.874 1.20 soy-f-13 soy-f-14 soy-f-15 soy-f-163 soy-f-173 soy-f-183 250 250 250 1000 1000 1000 1.57 3.21 5.56 2.49 -- __ - Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results rep o rted on a d ry -w eig h t basis. 2 The method limit of quantitation (LOQ) in soybean fruit tissue was 0.18 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weight/volume dilution factor of the matrix blank samples (100) divided by the percent solids for the negative control fruit tissue (22.9%). 3No sample was collected due to mortality at this concentration.___________________________________________ _______ -72- WiJdli/e Intema tional Lid PROJECT NO.: 454-110 Appendix 4.32 Termination Measured Values for PFOS in Tomato Vegetative Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) tom-v-19 tom-v-20 tom-v-21 0.0 0.0 0.0 < 7.62 <7.6 <7.6 <7.6 tom-v-22 tom-v-23 tom-v-24 tom-v-25 tom-v-26 tom-v-27 tom-v-28 tom-v-29 tom-v-30 tom-v-313 tom-v-324 tom-v-334 tom-v-344 tom-v-354 tom-v-364 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 1000 1000 1000 <7.6 <7.6 <7.6 12.5 27.9 61.1 70.9 44.5 34.9 __ -- - _ -- -- <7.6 33.8 50.1 _ _ Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results rep o rted o n a d ry -w eig h t basis. 2The method limit of quantitation (LOQ) in tomato vegetative tissue (7.6 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. 3There was insufficient sample weight (<0.5 g) available for analysis. 4No sample was collected due to mortality or lack of emergence at this concentration._________________________________ -73 - Wildliie International. L id PROJECT NO.: 454-110 Appendix 4.33 Termination Measured Values for PFOS in Tomato Fruit Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) tom-f-1 tom-f-2 tom-f-3 tom-f-43 tom-f-5 tom-f-6 tom-f-7 tom-f-8 tom-f-9 tom-f-10 tom-f-11 tom-f-12 tom-f-133 tom-f-144 tom-f-154 tom-f-164 tom-f-174 tom-f-184 0.0 0.0 0.0 3.91 3.91 3.91 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 1000 1000 1000 < 0.472 <0.47 <0.47 _ <0.47 <0.47 <0.47 1.51 0.580 0.946 <0.47 <0.47 _ -- - _ -- -- <0.47 <0.47 1.05 0.946 __ -- 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results rep o rted on a d ry -w eig h t basis. 2The method limit of quantitation (LOQ) in tomato fruit tissue was 0.47 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weight/volume dilution factor of the matrix blank samples (100) divided by the percent solids for the negative control fruit tissue (8.45%). 3There was insufficient sample weight (<0.5 g) available for analysis. 4No sample was collected due to mortality or lack of emergence at this concentration.__________ ______________________ -74- WildliG International' L id PROJECT NO.: 454-110 Appendix 4.34 Representative Chromatogram of a Plant Fruit Tissue Test Sample PFOS_29 454-110- SOY-F-15 Tue, Mar 26, 2002 16:14 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Hires. 2.0 Quant Thres. 0.5 Min. Width 12 Mult. Width 10 Base. Width 80 RT Win. (secs) 20 Smooth 2 Expected RT 3.75 Area 20080 Height 1699 Start Time End Time Integration Width Retention Time Integration Type 3.48 4.37 0.89 3.72 A - VB 100-1 908070605040302010o4 intensity: 4000 cps 222 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample Identification: 454-110-SOY-F-15 (soybean matrix). -75 - Wildliie International' Lid\ PROJECT NO.: 454-110 Appendix 4.35 Representative Chromatogram of a Plant Vegetative Tissue Test Sample PFOS_34 454-110- LET-V-28 4.9S in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mult. Width 10 Base. Width 40 RT Win. (secs) 20 Smooth 2 Expected RT 4.05 Area 5351 Height 521 Start Time 3.92 End Time 4.49 Integration Width 0.57 Retention Time 4.20 Integration Type A - BB Sun, Jan 27, 2002 10:41 intensity: 1700 cps Sample Identification: 454-110-LET-V-28 (lettuce matrix). 76- - Wildli/e International' L id PROJECT NO.: 454-110 Appendix 5 Environmental Conditions Temperature (C) Date Minimum Maximum Mean Relative Humidity (%) Minimum Maximum Mean 11/12/01' 15.9 26.8 19.7 11 32 23 11/13/01 16.3 26.8 19.8 13 41 27 11/14/01' 16.4 26.2 20.1 17 72 39 11/15/01 16.4 27.0 20.5 20 63 39 11/16/01 16.6 28.8 21.0 21 63 42 11/17/01' 16.4 25.5 19.9 27 63 44 11/18/01' 16.3 26.7 20.1 21 54 38 11/19/01 16.4 26.6 20.5 30 81 50 11/20/01 16.3 26.7 19.9 13 81 40 11/21/01' 16.2 27.0 19.7 12 51 33 11/22/01 16.3 28.0 20.2 14 47 32 11/23/01' 16.4 26.6 20.4 16 57 34 11/24/01 16.7 25.0 20.8 46 75 60 11/25/01 18.0 25.9 21.2 42 83 64 11/26/01' 16.7 26.7 20.9 31 85 59 11/27/01 16.5 24.9 20.4 35 75 53 11/28/01' 17.9 26.1 20.9 47 80 66 11/29/01 17.7 25.6 21.0 49 81 68 11/30/01 18.2 26.3 21.5 54 85 70 12/01/01 16.8 28.5 21.3 36 83 61 12/02/012 16.4 26.8 20.0 22 55 39 12/03/01 16.4 32.0 20.9 19 55 37 12/04/01 16.5 26.8 20.6 20 57 40 12/05/01' 16.7 29.4 21.3 17 59 40 12/06/01 1 6 .7 2 8 .2 2 1 .0 29 62 47 12/07/01' 16.8 25.5 20.4 33 68 50 12/08/01 16.7 24.3 19.9 28 71 47 12/09/01 16.6 27.2 20.0 16 71 38 12/10/01 16.4 24.5 19.6 23 53 36 12/11/01' 16.4 26.1 19.9 31 68 49 12/12/01' 16.3 25.3 19.6 29 66 48 12/13/01 16.6 24.2 19.9 47 79 60 12/14/01 16.8 24.0 20.3 54 80 70 12/15/01' 16.4 26.7 19.8 16 77 34 12/16/01 16.1 27.2 19.6 13 38 27 12/17/01 16.4 25.3 19.6 30 73 1 Indicates days on w hich all species w ere w atered. 2 Indicates days on w hich alfalfa, lettuce, ryegrass, and soybean trays w ere watered. 3 P A R - P hotosynthetically A ctive Radiation 43 Light Intensity Moles PAR3 14.9 19.8 19.7 18.6 17.9 17.4 18.9 18.0 20.3 19.9 18.9 19.3 18.3 17.6 18.7 17.3 17.5 17.5 17.3 18.2 16.1 17.8 18.1 18.5 1 8 .0 16.1 17.5 18.7 16.9 17.8 15.4 17.3 17.3 16.9 17.9 17.5 -77- W dettile International, L id . PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperature (C) Date Minimum Maximum Mean Relative Humidity (%) Minimum Maximum Mean 12/18/01* 16.5 27.3 19.8 16 79 43 12/19/01' 16.4 26.4 19.7 19 53 34 12/20/01 16.2 27.6 19.5 10 54 25 12/21/01' 16.1 26.4 19.4 11 33 20 12/22/01 16.1 27.5 19.4 11 32 24 12/23/01' 15.3 26.4 19.6 14 57 30 12/24/01' 16.2 26.7 19.5 14 62 35 12/25/01' 15.3 28.3 19.3 9 29 21 12/26/01' 15.3 27.3 19.2 12 31 21 12/27/012 15.1 25.9 19.0 10 32 21 12/28/01 14.8 25.4 18.7 16 40 28 12/29/01 14.8 25.6 18.9 15 37 25 12/30/013 14.8 27.3 18.6 8 24 17 12/31 / 0 14 14.8 27.2 18.6 9 31 20 01/01 /02s 14.7 27.0 18.7 9 26 20 01/02/02 14.8 28.0 18.5 11 40 25 01/03/02 14.9 23.1 17.9 20 37 28 01/04/02 14.9 27.4 18.6 10 41 25 01/05/02 14.9 27.3 19.1 11 46 29 01/06/026 15.0 24.4 18.4 24 55 40 01/07/024 14.8 21.9 18.1 30 54 42 01/08/02 14.8 26.5 18.6 11 45 28 01/09/025 14.9 24.4 18.9 20 51 35 01/10/027 16.1 23.9 19.6 28 58 43 01/11/02 16.1 25.9 19.4 19 61 43 01/12/024 15.1 27.5 19.8 15 54 36 01/13/028 16.2 27.7 19.9 12 57 34 01/14/029 16.1 25.5 19.8 17 61 36 01/15/02 15.4 27.7 20.0 17 62 39 01/16/02 15.2 27.9 19.6 15 52 35 01/17/02' 15.5 24.5 19.8 23 56 40 1 Indicates days on w hich all species w ere w atered. 2 Indicates days on w hich soybean and tom ato were watered. 3 Indicates days on w hich alfalfa, lettuce, ryegrass, soybean, and tom ato were watered. 4 Indicates days on w hich tom ato was watered. 5 Indicates days on w hich soybean w as watered. 6 Indicates days on w hich flax, lettuce, onion and ryegrass w ere w atered. 7 Indicates days on w hich alfalfa, flax, lettuce, onion, and ryegrass w ere w atered. 8 Indicates days on w hich all species w atered except tom ato. 9 Indicates days on w hich alfalfa, flax, lettuce, and onion were w atered. 10 P A R - P h o to sy n th e tic a lly A ctiv e R a d ia tio n Light Intensity Moles PAR10 18.3 17.1 20.1 20.3 19.6 17.9 17.0 19.6 19.9 20.2 17.6 17.9 21.8 22.2 20.0 19.2 18.8 19.7 20.5 16.6 17.4 19.3 18.4 17.5 16.8 19.2 21.0 18.4 19.4 20.7 18.6 78- - W ild life International. L id PROJECT NO.: 454-110 Date 01/18/02 01/19/02 01/20/021 01/21/022 01/22/023 01/23/023 01/24/02 01/25/02 01/26/024 01/27/024 01/28/02 01/29/02 01/30/02 01/31/023 02/01/023 02/02/024 02/03/024 02/04/023 02/05/02 02/06/023 02/07/02 02/08/02 02/09/02 02/10/02 02/11/02 02/12/02 02/13/02 02/14/02 02/15/02 02/16/02 02/17/022 02/18/02 02/19/02 02/20/02 ---0-2--/2--1-/-0--2-- T Temperature (C) Minimum Maximum 14.9 26.8 14.7 22.9 14.7 26.2 15.1 24.7 15.1 27.8 15.5 24.3 16.4 25.5 15.1 27.6 15.1 27.6 15.3 27.3 16.4 29.1 16.3 27.2 16.8 30.7 16.7 24.4 16.2 29.4 15.1 28.2 15.0 26.4 14.4 26.7 14.4 27.8 15.0 25.3 15.2 24.1 15.0 26.5 15.0 26.7 15.2 26.8 15.1 26.4 15.0 26.0 15.0 25.9 15.0 30.8 15.0 25.1 16.4 27.6 14.9 28.0 14.5 27.2 14.9 26.5 16.5 25.8 16.4 26.7 Appendix 5 (continued) Environmental Conditions Relative Humidity (%) Mean Minimum Maximum Mean 19.9 12 51 33 19.2 29 58 41 19.3 17 61 41 19.5 30 67 50 19.9 15 59 39 19.7 38 78 54 20.4 47 84 64 19.8 14 84 46 19.9 14 63 43 19.9 21 67 44 20.9 18 71 51 20.8 25 78 51 21.7 31 78 55 20.0 42 76 57 20.8 33 78 55 19.7 12 49 30 19.7 15 56 36 18.9 14 58 35 18.9 10 43 24 19.5 14 55 33 19.4 32 62 48 19.8 17 69 43 19.9 19 67 44 20.2 33 79 55 19.8 15 74 42 19.6 14 55 35 19.7 11 53 30 19.6 11 52 30 19.6 20 56 39 20.8 11 55 33 19.9 14 51 31 19.5 12 57 30 20.0 12 56 33 20.7 27 75 47 20.6 21 78 48 Indicates days on w hich alfalfa and ryegrass were watered. Indicates days on w hich all trays w ere w atered. Indicates days on w hich alfalfa, flax, ryegrass, and tom ato were watered. PA R - Photosynthetically A ctive Radiation Light Intensity Moles PAR5 20.1 17.4 14.3 17.8 21.3 17.9 17.7 20.6 21.1 19.9 19.6 18.2 19.2 18.2 17.5 22.9 20.3 17.3 23.4 20.9 17.6 21.4 20.6 17.2 23.3 19.5 22.2 24.6 18.1 23.7 21.5 24.8 22.7 18.4 15.6 -79- Wild life International. Lid. PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperature (C) Relative Humidity (%) Date Minimum Maximum Mean Minimum Maximum Mean 02/22/021 15.4 26.6 20.2 16 60 37 02/23/02 15.2 27.9 19.8 17 53 37 02/24/02 15.1 27.0 19.9 13 58 33 02/25/02 15.0 26.7 20.0 13 68 40 02/26/02 16.3 26.7 20.7 22 67 46 02/27/02 14.5 25.8 19.3 15 56 33 02/28/021 14.5 28.2 19.5 12 46 28 03/01/02* 14.8 27.3 19.8 11 75 36 03/02/02 15.1 24.6 19.6 28 82 57 03/03/02 15.7 25.1 20.1 34 89 61 03/04/022 14.7 27.0 19.0 12 39 26 03/05/022 14.5 25.9 19.0 13 57 30 03/06/02' 15.2 25.7 19.9 18 56 36 03/07/02' 16.2 26.1 20.6 17 62 34 03/08/02 16.2 27.2 21.1 19 76 43 03/09/02 16.6 31.2 22.5 32 77 57 03/10/02 15.9 28.5 20.6 8 74 28 03/11/022 15.0 26.3 19.5 11 44 24 03/12/02' 15.1 25.1 19.9 25 65 40 03/13/02 16.5 24.1 20.3 44 71 55 03/14/02' 16.6 27.0 20.8 29 70 51 03/15/02 16.5 27.0 21.8 42 73 56 03/16/02' 16.4 30.5 21.6 31 78 54 03/17/02 16.2 24.1 19.9 31 63 44 0 3 /1 8/021 1 6 .2 2 4 .6 19.8 39 67 55 03/19/02 16.3 26.2 20.2 24 65 43 03/20/022 16.4 23.9 20.1 44 73 55 03/21/02 15.1 25.9 20.1 23 72 41 03/22/022 14.1 27.0 19.0 11 34 20 03/23/02 14.9 25.5 19.8 14 43 26 03/24/02' 15.6 25.7 20.5 14 53 31 03/25/02' 16.8 25.8 20.6 23 55 38 03/26/022 16.9 25.2 20.6 37 82 52 03/27/02 16.4 26.5 20.1 25 83 45 03/28/022 16.0 25.2 20.2 16 55 35 03/29/02 16.9 25.4 20.9 29 64 43 1 Indicates days on w hich alfalfa and ryegrass w ere watered. 2 Indicates days on w hich alfalfa were watered. 3 PA R - Photosynthetically A ctive Radiation Light Intensity Moles PAR3 22.6 20.2 24.5 19.0 16.9 22.7 26.2 25.5 17.1 16.8 19.8 13.8 14.8 16.1 16.3 19.1 26.6 14.0 18.2 18.4 14.0 14.0 17.0 18.9 1 7 .6 16.2 17.5 14.7 14.7 13.8 13.5 14.0 17.8 14.8 14.1 14.2 -80- ildliie IntemationalfLtcL PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperature (C) Date Minimum Maximum Mean Relative Humidity (%) Minimum Maximum Mean 03/30/02' 16.8 29.7 22.3 23 78 49 03/31/02 16.6 25.4 20.5 37 76 57 04/01/02 16.6 25.4 20.6 18 74 43 04/02/022 16.7 25.8 21.4 21 74 43 04/03/02 16.5 30.9 22.0 32 80 51 04/04/022 15.9 25.3 20.3 16 51 28 04/05/022 16.6 26.1 20.4 14 46 27 04/06/022 15.2 25.9 20.3 13 48 27 04/07/02 15.7 26.1 20.4 13 64 32 04/08/02 16.9 25.4 21.1 28 73 49 04/09/022 17.3 25.3 21.6 41 87 66 04/10/022 17.1 25.4 21.1 29 89 55 04/11/02 17.0 25.9 21.0 29 66 47 04/12/022 17.4 25.6 21.2 44 85 62 04/13/02 17.0 28.0 22.1 53 89 71 04/14/02 18.7 27.5 23.1 57 88 71 04/15/022 19.8 30.0 23.7 49 84 70 04/16/02 20.2 29.7 24.7 54 87 71 04/17/022 19.5 31.6 24.9 51 86 69 04/18/02 20.1 28.8 24.0 57 86 73 04/19/022 19.5 30.3 23.5 55 86 72 04/20/02 19.6 28.5 23.0 56 86 70 04/21/02 16.7 25.4 20.7 34 79 56 04/22/022 16.5 26.4 20.8 28 77 52 0 4 /2 3 /0 2 16.3 2 5 .5 2 0 .7 20 59 34 04/24/022 17.3 26.1 21.2 20 64 39 04/25/02 16.4 24.9 20.9 30 70 53 04/26/02 16.4 25.4 20.8 19 69 43 04/27/02 16.8 25.5 21.0 28 77 51 04/28/02 17.5 26.7 21.9 61 88 75 04/29/022 16.7 27.1 20.8 29 77 48 04/30/022 16.5 25.7 21.1 29 77 47 05/01/022 17.7 25.4 21.5 30 79 57 05/02/02 17.9 26.9 22.6 64 84 75 05/03/022 17.1 25.5 21.3 20 82 41 05/04/02 16.5 25.6 21.0 22 72 44 1 Indicates days on w hich alfalfa and ryegrass w ere w atered. 2 Indicates days on w hich ryegrass w as watered. 3 P A R - Photosynthetically A ctive Radiation Light Intensity Moles PAR3 18.5 18.6 14.0 15.5 14.9 14.3 14.3 14.5 14.8 14.8 14.9 14.0 14.3 16.6 15.5 14.5 16.1 14.2 15.1 14.5 16.1 14.5 18.7 18.0 1 3 .4 13.8 17.3 14.1 12.8 17.2 15.6 13.6 13.8 15.0 13.7 14.3 -81 - W"ild liie International' L id PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperature (C) Date Minimum Maximum Mean Relative Humidity (%) Minimum Maximum Mean 05/05/02 17.3 25.5 21.3 33 72 56 05/06/021 17.7 25.5 21.8 34 72 55 05/07/02 18.5 27.2 22.7 51 85 71 05/08/02 18.2 28.0 22.3 41 83 62 05/09/02 17.8 25.5 21.9 47 88 67 05/10/021 18.3 26.1 22.2 47 84 64 05/11/021 17.8 25.6 21.6 20 67 47 05/12/02 18.8 27.4 23.3 41 86 67 05/13/021 18.6 29.2 23.7 57 87 74 05/14/02' 16.7 26.2 20.9 23 58 39 05/15/02 16.7 26.0 21.3 26 65 44 05/16/02' 17.7 26.0 22.4 36 66 56 05/17/02' 20.3 28.7 23.3 48 78 65 05/18/02 16.7 25.2 21.3 30 84 55 05/19/02 16.6 25.8 21.1 19 62 40 05/20/02' 17.4 25.4 21.2 19 59 38 05/21/02' 17.4 26.0 21.1 22 49 36 05/22/02 17.3 26.5 21.3 23 57 41 05/23/02' 17.4 25.6 21.3 35 73 52 05/24/02 17.7 25.4 22.4 42 76 63 05/25/02' 18.4 25.8 22.3 48 82 64 05/26/02 18.0 26.9 23.0 58 87 74 05/27/02 19.3 27.6 23.7 61 84 72 05/28/02' 19.4 27.9 23.7 60 83 72 0 5 /2 9 /0 2 19.6 2 8 .0 2 3 .5 5 9 84 7 3 05/30/02' 18.7 28.1 23.6 62 87 74 05/31/02' 20.1 28.9 25.1 66 87 77 06/01/02 22.0 27.6 24.9 59 90 74 06/02/02' 20.1 29.2 24.0 35 90 63 06/03/02 19.2 25.8 22.5 38 68 54 06/04/02' 20.2 26.7 23.3 55 81 68 06/05/02 21.9 30.5 26.2 ------------------- i 1 Indicates days on w hich ryegrass w as watered. 2 PA R - Photosynthetically A ctive Radiation 62 91 76 Light Intensity Moles PAR2 13.8 12.8 16.1 15.5 16.9 14.2 13.6 13.8 13.9 14.0 13.5 13.5 14.0 16.6 13.1 13.8 14.5 13.7 13.1 13.3 14.5 13.5 14.4 14.4 15.3 14.5 14.2 13.5 14.3 14.5 14.7 14.1 -82- W ild life International. Lid. PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i /kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 6.1 Alfalfa Emergence _____ Day 7_____ Number o f Emerged Seedlings in Replicate: AB C D 7 10 10 8 89 8 8 99 7 6 66 8 8 85 7 8 00 5 5 n Mean Std. Dev. 4 8.75 1.50 4 8.25 0.50 4 7.75 1.50 4 7.00 1.15 4 7.00 1.41 4 2.50 2.89 D ay 15 Treatment Number o f Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 3.91 mg a.i./kg 15.6 mg a.i./kg 8 8 9 10 9 9 10 9 8 8 8 6 4 9.00 1.15 4 8.50 0.58 4 8.00 1.41 62.5 mg a.i./kg 7 7 9 9 4 8.00 1.15 250 mg a.i./kg 9 5 7 8 4 7.25 1.71 1000 mg a.i./kg 0 0 5 5 4 2.50 2.89 Day 21 Treatment Number o f Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 8 10 10 8 3.91 mg a.i./kg 8 9 9 8 15.6 mg a.i./kg 9 9 8 6 62.5 mg a.i./kg 7 7 9 9 250 mg a.i./kg 9 5 7 8 1000 mg a.i./kg 0 1 5 7 4 9.00 1.15 4 8.50 0.58 4 8.00 1.41 4 8.00 1.15 4 7.25 1.71 4 3.25 3.30 -83- Wild liie Intem a tional. L id Appendix 6.2 Mean Alfalfa Emergence on Day 21 PROJECT NO.: 454-110 o Data ------- Regression ........ 95% Conf. Int. 50% Inhibition e c 25 372.306 E C 5, 745.418 Concentration (mg a.i./kg) Curve Parameters Lower 95% Confidence Lim it 201.512 Upper 95% Confidence Lim it 687.860 Ro 8.4045 Lower 95% Confidence Lim it 540.879 Upper 95% Confidence Lim it 1027.07 Ro 8.4045 0.4470 0.97096 0.4470 0.97096 -84- W ild liie International. L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 6.3 Alfalfa 21-Day Survival Day 21 Number o f Living Seedlings in Replicate: A BC D 8 9 10 8 89 8 8 99 8 6 77 9 9 84 6 7 01 1 4 n 4 4 4 4 4 4 Mean 8.75 8.25 8.00 8.00 6.25 1.50 Std. Dev. 0.96 0.50 1.41 1.15 1.71 1.73 10 w6a C/i up a (ZI Data Regression 95% Conf. Int. 50% Inhibition e c 25 2 5 1 .0 7 3 E C 5O 4 5 2 .2 7 2 200 400 600 800 1000 1200 Concentration (mg a.i./kg) Lower 95% C onfidence Lim it 178.402 Lower 95% C onfidence Lim it 3 6 3 .5 8 0 Curve Parameters Upper 95% C onfidence Lim it 3 5 3 .2 6 5 Ro 8.2801 Upper 95% C onfidence Lim it 5 6 2 .7 3 0 Ro 8.2801 a 0.3790 a 0.3790 r2 0.99162 r2 0.99162 -85 - Wildliie International? L id PROJECT NO.: 454-110 Appendix 6.4 Alfalfa Mean Seedling Fresh Weight, Day 21 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg _________ M ean Weight (g) for Replicate: AB C D 0.11 0.10 0.12 0.13 0.10 0.10 0.16 0.09 0.08 0.12 0.08 0.11 0.11 0.10 0.09 0.12 0.03 0.01 0.03 0.03 - 0.02 0.02 0.01 n 4 4 4 4 4 3 Mean 0.12 0.11 0.10 0.10 0.03 0.02 Std. Dev. 0.013 0.033 0.022 0.010 0.008 0.008 o Data ^ ---Regression -----95% Conf. Int. 50% Inhibition e c 25 5 3 .2 8 4 4 E C 50 146.049 0 200 400 600 800 1000 1200 Concentration (mg a.i./kg) Lower 95% C onfidence Lim it 4 .1 1 6 2 3 Lower 95% C onfidence Lim it 27.2521 Curve Parameters Upper 95% C onfidence Lim it 6 8 9 .7 6 3 Ro 0.1144 Upper 95% C onfidence Lim it 7 8 2 .5 2 8 Ro 0.1144 CT 0.6492 a 0.6492 r2 0 .9 0 5 3 4 r2 0 .9 0 5 3 4 -86- Wildlife International. L id PROJECT NO.: 454-110 Appendix 6.5 Alfalfa Seedling Height on Day 21 Treatment Group Replicate Height (cm) for Plant Number: 1 2 3 4 5 6 7 8 9 10 Mean Control A 6 6 6 3 6 12 6 4 8 B 5 3 4 8 4 7 5 5 11 9 C 7 4 8 8 6 6 8 8 6 8 10 D 10 5 6 10 5 6 5 10 8 3.91 mg/kg A B C D 5587595 4 8 7 5 10 4 6 8 6 7 11 9 11 10 6 5 5 7 3 8 8 6946744 4 8 15.6 mg a.i./kg A B C D 58854454 3 58755559 4 2258357 4 67775 5 9 9 8 6 62.5 mg a.i./kg A B C D 5655756 7 6 5 5 8 7 4 12 7 64364579 2 9 8 5 9 8 10 7 4 6 6 9 250 mg a.i./kg A B C D 3423432 3 222 2 14 2 3 5 4 442332 2 8 4 6 7 1000 mg a.i./kg A B1 C1 D 111 1 The symbol indicates that the seedling either did not emerge or died prior to measurement. 0 1 1 4 6.1 5.8 6.9 7.1 6.0 7.1 6.9 5.5 5.1 5.9 4.5 6.2 5.6 6.7 5.1 7.0 3.0 2.0 3.2 2.9 1.0 1.0 1.0 Std. Dev. 2.64 2.49 1.37 2.42 1.77 2.26 2.70 1.85 1.76 1.69 2.20 0.98 0.79 2.69 2.15 1.94 0.76 0.00 1.47 0.90 0.00 87- - AAAild liie Intem a tonal. L id. PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 6.6 Alfalfa Mean Seedling Height on Day 21 Mean Height (cm) for Replicate: AB C D 6.1 5.8 6.9 7.1 6.0 7.1 6.9 5.5 5.1 5.9 4.5 6.2 5.6 6.7 5.1 7.0 3.0 2.0 3.2 2.9 1.0 1.0 1.0 n 4 4 4 4 4 3 Mean 6.5 6.4 5.4 6.1 2.8 1.0 Std. Dev. 0.6 0.8 0.8 0.9 0.5 0.0 o Data ------ Regression -----95% Conf. Int. ........50% Inhibition e c 25 102.447 E C so 2 4 9 .0 5 8 Lower 95% C onfidence Lim it 2 6 .8 2 8 7 Lower 95% C onfidence Lim it 106.292 Curve Parameters Upper 95% C onfidence Lim it 391.201 Ro 6 .2 7 9 3 Upper 95% C onfidence Lim it 5 8 3 .5 7 9 Ro 6 .2 7 9 3 -88- a 0 .5 7 2 0 a 0 .5 7 2 0 r2 0 .9 3 9 6 0 r2 0 .9 3 9 6 0 Wildliie International' Lid. PROJECT NO.: 454-110 Appendix 6.7 Alfalfa Seedling Condition, Day 21 Treatment Group Replicate Condition (score.sign)1for Plant Number: n Mean Std. Dev. 1 2 3 4 5 6 7 8 9 10 Control 3.91 mg a.i./kg A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 B 100.- 0.- 50.N 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 15 33.7 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 C 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 11 33.3 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg A B C D A B C D A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 90.N 0.- 9 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 70.N 30.N 0.- 0.- 0.- 0.- 0.- 0.- 8 0.- 0.- 0.- 0.- 0.- 0.- 6 10 30.0 0 0.0 13 25.5 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- */7 0 0.0 0.- 30.N 0.- 0.- 0.- 0.- 0.- */7 4 11.3 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 11 33.3 100.- 0.- 0.- 0.- 0.- 5 20 44.7 100.- 80.N 0.- 0.- 0.- 0.- 0.- V/ 26 44.3 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 13 35.4 1000 mg a.i./kg A 0 B 0.- 1 0 C 100.- 100.- 100.- 100.- 60.N 5 92 D 100.- 100.- 100.- 80.N 60.N 0.- 80.N 7 74 'The symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity of observed signs of toxicity. N - Necrosis 17.9 3 6 .0 -89- Wildliie International' Lid\ PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 7.1 Flax Emergence Day 7 Number o f Emerged Seedlings in Replicate: AB C D 79 6 7 75 7 7 89 7 8 99 9 7 89 5 7 00 0 0 n 4 4 4 4 4 4 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Day 15 Number o f Emerged Seedlings in Replicate: AB C D 79 6 7 75 8 8 89 7 8 99 9 7 89 5 7 00 0 0 n 4 4 4 4 4 4 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg D ay 21_________ Number o f Emerged Seedlings in Replicate: A BC D 79 6 7 85 8 8 89 7 8 99 9 7 89 5 7 00 0 0 n 4 4 4 4 4 4 Mean 7.25 6.50 8.00 8.50 7.25 0.00 Std. Dev. 1.26 1.00 0.82 1.00 1.71 0.00 Mean 7.25 7.00 8.00 8.50 7.25 0.00 Std. Dev. 1.26 1.41 0.82 1.00 1.71 0.00 Mean 7.25 7.25 8.00 8.50 7.25 0.00 Std. Dev. 1.26 1.50 0.82 1.00 1.71 0.00 -90- Wild life International. L id PROJECT NO.: 454-110 Appendix 7.2 Mean Flax Emergence on Day 21 10 - , 9 8 7 6 5 4 3 2 1 0 * v -kv <5&/ . y v> ^ Treatment Group * Treatment group mean is significantly different from the control mean (Dunnett's test,y><0.05) The non-linear regression technique failed to generate useable results. Therefore, linear interpolation was used. e c 25 398.7069 Lower 95% Confidence Limit 126.1605 Upper 95% Confidence Limit 460.7759 S OLU Lower 95% Confidence Limit Upper 95% Confidence Limit 599.1379 401.8809 640.5172 EC25 and EC50estimates calculated by linear Interpolation using the ICPIN program (7). -91 - Wild liie International Ltd. PROJECT NO.: 454-110 Appendix 7.3 Flax 21-Day Survival Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Day 21 Number o f Living Seedlings in Replicate: AB C D 79 6 7 75 8 7 89 7 8 99 9 7 14 4 4 00 0 0 n Mean Std. Dev. 4 7.25 1.26 4 6.75 1.26 4 8.00 0.82 4 8.50 1.00 4 3.25 1.50 4 0.00 0.00 sV , <0/ Treatment Group * Treatment group mean is significantly different from the control mean (Dunnett's test, p<0.05) The non-linear regression technique failed to generate useable results. Therefore, linear interpolation was used. Effect Rate e c 25 Effect Concentration 144.1964 Lower 95% Confidence Limit 103.4821 Upper 95% Confidence Limit 177.3710 E C 5o 225.8929 159.6256 367.5893 EC25 and EC50 estimates calculated by linear Interpolation using the ICPIN program (7). -92- AAIildlife International' Liei PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 7.4 Flax Mean Seedling Fresh Weight, Day 21 Mean Weight (g) for Replicate: AB C D 0.16 0.19 0.19 0.23 0.21 0.19 0.16 0.17 0.21 0.16 0.16 0.17 0.17 0.17 0.14 0.17 0.01 0.02 0.02 n 4 4 4 4 3 0 Mean 0.19 0.18 0.18 0.16 0.02 Std. Dev. 0.029 0.022 0.023 0.017 0.009 o Data ------ Regression -----95% Conf. Int. ........50% Inhibition e c 25 81.5831 E C 50 118.796 Lower 95% C onfidence Lim it 46.0151 Lower 95% C onfidence Lim it 7 9 .3 7 8 0 Concentration (mg a.iVkg) Curve Parameters Upper 95% C onfidence Lim it 144.644 Ro 0 .1 8 3 3 Upper 95% C onfidence Lim it 177.828 Ro 0 .1 8 3 3 -93 - <7 0.2420 CT 0 .2 4 2 0 r2 0 .9 9 0 0 0 r2 0 .9 9 0 0 0 W ildliie International. L id PROJECT NO.: 454-110 Appendix 7.5 Flax Seedling Height on Day 21 Treatment Replicate Height (cm) for Plant Number: 1 2 3 4 5 6 7 8 9 10 Control A 5 9 9 9 8 10 10 7 B . 5 9 9 9 12 10 11 12 5 9 C 11 11 8 9 9 5 6 D 10 8 11 7 10 9 9 7 3.91 mg a.i./kg A B C D 8 8 10 9 10 10 10 9 11 9 9 10 8 11 6 8 1 11 11 9 7 8 3 8 10 9 9 7 5 8 7 15.6 mg a.i./kg A 9 10 8 11 10 9 11 10 8 B . 9 10 8 9 8 11 9 9 9 9 C 9 8 11 11 6 9 2 7 D 6 8 8 10 9 7 8 10 8 62.5 mg a.i./kg A . 5 8 10 10 8 10 11 7 8 9 B . 11 12 9 10 9 5 13 9 1 9 C . 9 8 8 11 10 3 5 6 10 9 D 9 3 7 9 9 10 7 7 250 mg a.i./kg A B C D 1 2 11 1 111 1 12 2 2 1 4 4 4 1000 mg a.i./kg A B C D The symbol indicates that the seedling either did not emerge or died prior to measurement. 0 0 0 0 Mean 8.6 9.1 8.8 9.1 9.3 9.6 8.1 7.7 9.8 9.1 8.0 8.3 8.6 8.8 7.8 7.7 1.0 1.3 1.0 1.8 Std. Dev. 1.72 2.62 2.23 1.35 0.95 0.89 3.40 2.29 1.04 0.93 3.16 1.39 1.88 3.70 2.64 2.36 0.50 0.00 0.50 -94- Wildlile International' L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 7.6 Flax Mean Seedling Height on Day 21 Mean Height (cm) for Replicate: AB C D 8.6 9.1 8.8 9.1 9.3 9.6 8.1 7.7 9.8 9.1 8.0 8.3 8.6 8.8 7.8 7.7 1.0 1.3 1.0 1.8 n 4 4 4 4 4 0 Mean 8.9 8.7 8.8 8.2 1.3 Std. Dev. 0.27 0.90 0.80 0.54 0.35 o Data ------ Regression - - - - 95% Conf. Int. 50% Inhibition e c 25 9 7 .6 3 3 8 E C 5q 140.411 Lower 95% C onfidence Lim it 81.3392 Lower 95% C onfidence Lim it 124.825 Curve Parameters Upper 95% C onfidence Lim it 117.166 Ro 8.7913 Upper 95% C onfidence Lim it 157.943 Ro 8.7913 -95 - a 0.2340 a 0 .2 3 4 0 r2 0 .9 9 9 3 8 r2 0 .9 9 9 3 8 W ildliie International' L id PROJECT NO.: 454-110 Appendix 7.7 Flax Seedling Condition, Day 21 Treatm ent R e p lica te Condition (score.sign)1 for Plant N um ber: 12 3 4 5 6 7 8 9 10 M ean Std. Dev. C o n tro l A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 B . 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 40.U SC 9 4 C 0.- 0.- 0.- 0.- 0.- 0.- 6 0 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 3.91 m g a.i./kg A B C D 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 13 0.- 0.- 0.- 0.- 0.- 5 0 0.- 0.- 0.- 0.- 80.LC 0.- 0.- 0.- 8 10 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 13 15.6 m g a.i./kg A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 B . 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 C 0.- 0.- 0.- 0.- 0.- 0.- 80.U SC 7 11 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 62.5 m g a.i./kg A . 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 B . 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 C . 0.- 0.- 0.- 0.- 0.- 40.N 0.- 0.- 0.- 9 4 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 250 m g a.i./kg A B C D 100.- 100.- 100.- 100.- 100.- 100.- 100.- 90.U SC 8 . 100.- 100.- 100.- 100.- 100.- 50.USC 90.U SC 70.U SC 90.U SC 9 100.- 90.U SC 80.U SC 80.U SC 80.U SC 5 100.- 100.- 100.- 80.U SC 80.U SC 80.U SC 80.U SC 7 99 89 86 89 1000 m g a.i./kg A 0 B0 C0 D0 The symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity of observed signs of toxicity. USC - Unshed Seed Coat, LC - Leaf Curl, N - Necrosis 0.0 13.3 0.0 0.0 35.4 0.0 28.3 35.4 0.0 0.0 30.2 0.0 0.0 0.0 13.3 0.0 3.5 17.6 8.9 10.7 -96- Wild life Intem a tionalfL tcL PROJECT NO.: 454-110 A ppendix 8.1 Lettuce Emergence ___________________________________ Day 7______ Treatment Group Number of Emerged Seedlings in Replicate: AB C D n Mean Std. Dev. Control 3.91 mg a.i./kg 9 8 8 9 9 9 4 8.75 0.50 9 10 4 9.00 0.82 15.6 m g a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg 10 9 10 3 7 7 7 0 8 10 4 8.75 1.50 8 10 4 8.50 1.29 7 8 4 8.00 1.41 1 1 4 1.25 1.26 Day 15 Treatment Number of Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. C ontrol 9 8 9 9 4 8.75 0.50 3.91 m g a.i./kg 8 9 9 10 4 9.00 0.82 15.6 m g a.i./kg 10 8 8 10 4 9.00 1.15 62.5 mg a.i./kg 9 7 8 10 4 8.50 1.29 250 mg a.i./kg 10 7 7 8 4 8.00 1.41 1000 mg a.i./kg 3 0 1 1 4 1.25 1.26 Day 21 Treatment Number of Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 3.91 m g a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg 9 8 10 9 10 3 8 9 8 7 8 0 9 9 4 8.75 0.50 9 10 4 9.00 0.82 8 10 4 9.00 1.15 8 10 4 8.50 1.29 7 8 4 8.25 1.26 1 1 4 1.25 1.26 -97- Wildliie International, L td A ppendix 8.2 M ean Lettuce Em ergence on D ay 21 PROJECT NO.: 454-110 o Data Regression - - - - 95% Conf. Int. 50% Inhibition Concentration (mg a.i7kg) e c 25 3 9 3 .3 6 9 e c 50 5 6 4 .0 2 7 Lower 95% C onfidence Lim it 3 0 0 .1 9 3 Lower 95% C onfidence Lim it 4 7 4 .0 2 4 Curve Parameters Upper 95% C onfidence Lim it 5 1 5 .4 6 6 Ro 8 .8 1 2 6 Upper 95% C onfidence Lim it 6 7 1 .1 2 0 Ro 8 .8 1 2 6 a 0.2320 a 0.2320 r2 0 .9 9 6 3 2 r2 0 .9 9 6 3 2 -98- Wildliie International' L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 8.3 Lettuce 21-Day Survival Day 21 Number of Living Seedlings in Replicate: AB C D 98 9 9 89 9 10 10 8 8 10 97 8 10 86 7 6 10 0 1 n 4 4 4 4 4 4 Mean 8.75 9.00 9.00 8.50 6.75 0.50 Std. Dev. 0.50 0.82 1.15 1.29 0.96 0.58 o Data ------Regression - - - - 95% Conf. Int. 50% Inhibition e c 25 2 5 7 .2 7 6 e c 50 386.011 Lower 95% C onfidence Lim it 220.141 Lower 95% C onfidence Lim it 3 4 3 .7 9 5 Curve Parameters Upper 95% C onfidence Lim it 3 0 0 .6 7 7 Ro 8 .8 1 6 6 Upper 95% C onfidence Lim it 433.411 Ro 8 .8 1 6 6 -99- CT 0 .2 6 1 3 a 0 .2 6 1 3 r2 0 .9 9 7 1 9 r2 0 .9 9 7 1 9 Wild liie Intem a iiona! L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 8.4 Lettuce Mean Seedling Fresh Weight, Day 21 Mean Weight (g) for Replicate: AB C D 0.46 0.38 0.32 0.38 0.21 0.28 0.20 0.30 0.32 0.13 0.27 0.26 0.06 0.03 0.06 0.03 0.01 0.01 0.02 0.00 0.00 0.00 n 4 4 4 4 4 2 Mean 0.38 0.25 0.24 0.05 0.01 0.00 Std. Dev. 0.06 0.05 0.08 0.02 0.01 0.00 o Data ------ Regression - - - 95% Conf. Int. 50% Inhibition 0 200 400 600 800 1000 1200 Concentration (mg a.i./kg) e c 25 8 .9 2 4 8 3 E C 50 2 0 .1 4 1 9 Lower 95% C onfidence Lim it 1 .3 6 4 9 0 Lower 95% C onfidence Lim it 5 .2 6 2 6 0 Curve Parameters Upper 95% C onfidence Lim it 58.3445 Ro 0.3466 Upper 95% C onfidence Lim it 77.1081 Ro 0 .3 4 6 6 CT 0.5242 CT 0.5242 r2 0 .9 4 7 2 2 r2 0.94722 -100- W ildliie International' Ltd\ PROJECT NO.: 454-110 Appendix 8.5 Lettuce Seedling Height on Day 21 Treatment Group Replicate Height (cm) for Plant Number: n 1 2 3 4 5 6 7 8 9 10 Control A B C D 67895687 8 9 8968456 5 8 4 5 4 8 5 6 10 5 6 9 67866665 7 9 3.91 mg a.i./kg A 65544 57 5 8 B 55677754 4 9 C 66346333 4 9 D 7 4 4 3 7 6 3 4 7 5 10 15.6 mg a.i./kg A 4 5 6 7 4 6 5 6 8 10 10 B 4435353 2 8 C 47664 58 4 8 D 6 4 5 6 3 4 5 5 7 2 10 62.5 mg a.i./kg A 55222232 2 9 B 322322 5 7 C 54 5 12 3 2 2 8 D 2 3 2 1 1 2 2 2 3 2 10 250 mg a.i./kg A B C D 12 1 111 1 1 8 11111 1 6 111111 1 7 11111 1 6 1000 mg a.i./kg A 1 B C D1 The symbol indicates that the seedling either did not emerge or died prior to measurement. 1 0 0 1 Mean 7.1 6.4 5.9 6.3 5.1 5.6 4.2 5.0 6.1 3.6 5.5 4.7 2.8 2.7 3.0 2.0 1.1 1.0 1.0 1.0 1.0 1.0 Std. Dev. 1.27 1.77 1.96 0.87 0.99 1.24 1.39 1.63 1.85 1.06 1.51 1.49 1.30 1.11 1.51 0.67 0.35 0.00 0.00 0.00 - 101 - MAildli/e Intema tional. Lid. PROJECT NO.: 454-110 Appendix 8.6 Lettuce Mean Seedling Height on Day 21 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg ________ Mean Height (cm) for Replicate: AB C D 7.1 6.4 5.9 6.3 5.1 5.6 4.2 5.0 6.1 3.6 5.5 4.7 2.8 2.7 3.0 2.0 1.1 1.0 1.0 1.0 1.0 1.0 n 4 4 4 4 4 2 Mean 6.4 5.0 5.0 2.6 1.0 1.0 Std. Dev. 0.51 0.56 1.07 0.43 0.06 0.00 o Data ------ Regression - - - - 95% Conf. Int. ....... 50% Inhibition e c 25 6 .7 9 3 6 0 EC50 3 9 .8 7 4 9 Lower 95% C onfidence Lim it 0 .2 0 3 9 9 Lower 95% C onfidence Lim it 3.87972 Curve Parameters Upper 95% C onfidence Lim it 226.204 Ro 6 .5 0 2 5 Upper 95% C onfidence Lim it 4 0 9 .7 3 2 Ro 6 .5 0 2 5 -102- a 1.1396 a 1.1396 r2 0 .9 4 2 9 4 r2 0 .9 4 2 9 4 Wildlife International' LtcL PROJECT NO.: 454-110 Appendix 8.7 Lettuce Seedling C ondition, D ay 21 T reatm en t R eplicate C ondition (score.sign) 1 for Plant Number: 1 2 3 4 5 6 7 8 9 10 M ean C on trol A B C D 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .0 .0 .- 0 .- 9 0 .- 8 0 .- 9 0 .- 9 0 0 0 0 3.91 m g a.i./kg A B C D 0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .0 .0 .- 0 .- 8 0 .- 9 0 .- 9 0 .- 10 0 0 0 0 15.6 m g a.i./kg A B C D 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .- 0 .- 0 .- 0 .- 0 .- 0 .0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 10 0 80 .- 0 .- 0 80 .- 0 .- 0 0 .- 4 0 .N 10 4 6 2 .5 m g a.i./kg A 90 .- 0 .- 5 0 .N 3 0 .N 0 .- 50.N 5 0 .N 5 0 .N 4 0 .N 30 B 4 0 .N 4 0 .N 40.N 4 0 .N 4 0 .N 3 0 .N 2 0 .N 7 36 8C 0 .- 2 0 .N 0 .- 0 .- 50.N 3 0 .N 5 0 .N 50.N 25 D 4 0 .N 4 0 .N 4 0 .N 4 0 .N 3 0 .N 30.N 4 0 .N 5 0 .N 3 0 .N 50.N 10 39 2 5 0 m g a.i./kg 10A 1 0 0 .- 1 0 0 .- 80.N 0 .- 4 0 .N 0 .- 6 0 .N 0 .- 6 0 .N 0 .- 44 8B 1 0 0 .- 1 0 0 .- 0 .- 40.N 0 .- 0 .- 3 0 .N 4 0 .N 39 C 6 0 .N 0 .- 30.N 4 0 .N 4 0 .N 4 0 .N 0 .- 7 30 8D 1 0 0 .- 1 0 0 .- 3 0 .N 8 0 .N 0 .- 4 0 .N 50.N .L C 3 0 .N 54 1 0 0 0 m g a.i./kg A B C D 1 0 0 .- 1 0 0 .- 8 0 .N 1 0 0 .90.U SC 3 0 1 1 93 100 90 The symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity of observed signs of toxicity. N - Necrosis, USC - Unshed Seed Coat, LC - Leaf Curl Std. Dev. 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 1 2 .6 23.5 7.9 23.3 7.4 4 2 .0 4 1 .6 22.4 36.2 11.5 - 103 - Wildlife International' L td PROJECT NO.: 454-110 Appendix 9.1 Onion Emergence Treatment Day ? Number of Emerged Seedlings in Replicate: Group AB CD n Mean Std. Dev. Control 8 7 8 9 4 8.00 0.82 3.91 mg a.i./kg 10 8 9 7 4 8.50 1.29 15.6 mg a.i./kg 8 8 7 8 4 7.75 0.50 62.5 mg a.i./kg 4 8 4 4 4 5.00 2.00 250 mg a.i./kg 5 3 0 0 4 2.00 2.45 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 Day 15 Treatment Number of Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 8 7 9 10 4 8.50 1.29 3.91 mg a.i./kg 10 10 9 7 4 9.00 1.41 15.6 mg a.i./kg 8 9 8 8 4 8.25 0.50 62.5 mg a.i./kg 5 10 6 4 4 6.25 2.63 250 mg a.i./kg 5 8 2 1 4 4.00 3.16 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 Day 21 Treatment Number of Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 8 7 10 10 4 8.75 1.50 3.91 mg a.i./kg 10 10 9 8 4 9.25 0.96 15.6 mg a.i./kg 8 9 8 8 4 8.25 0.50 62.5 mg a.i./kg 5 10 6 4 4 6.25 2.63 250 mg a.i./kg 5 8 2 1 4 4.00 3.16 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 - 104- AAAild lile International' L id PROJECT NO.: 454-110 A ppendix 9.2 M ean O nion Em ergence on D ay 21 12 n F T10 V(sJ I OJD L. I T II A oN ' I . Wr `V *' b / v- <>v Treatment Group c / *Treatment group mean is significantly different from the control mean (Dunnett's test, p<0.05). The non-linear regression technique failed to generate useable results. Therefore, linear interpolation was used. Effect Rate EC25 Effect C o n ce n tra tio n 50.7750 Lower 95% C o n fid e n ce Limit 12.1515 Upper 95% C o n fid e n ce Limit 194.5350 208.3333 -25.0000 644.2308 EC25 and EC50 estimates calculated by linear interpolation using the ICPIN program (7). 0LUs -105 - Wild life Intem a tignai. L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 9.3 Onion 21-Day Survival D ay 21 Number of Living Seedlings in Replicate: AB C D 8 7 9 10 10 10 8 8 79 8 8 34 3 3 00 0 0 00 0 0 n 4 4 4 4 4 4 Mean 8.50 9.00 8.00 3.25 0.00 0.00 Std. Dev. 1.29 1.15 0.82 0.50 0.00 0.00 o Data ------ Regression -----95% Conf. Int. 50% Inhibition E C 25 4 7 .0 9 7 7 ECso 57.2928 Lower 95% C onfidence Lim it 0 .9 9 2 3 8 Lower 95% C onfidence Lim it 17.4622 Curve Parameters U pper 95% C onfidence Lim it 2 2 3 4 .6 0 Ro 8 .5 0 0 0 Upper 95% C onfidence Lim it 187.975 Ro 8 .5 0 0 0 -106- a 0.1262 a 0.1262 r2 0.99445 r2 0 .9 9 4 4 5 Wildlife International. Lid. PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 9.4 Onion Mean Seedling Fresh Weight, Day 21 Mean Weight (g) for Replicate: AB C D 0.10 0.09 0.10 0.10 0.06 0.09 0.09 0.10 0.06 0.06 0.08 0.07 0.02 0.05 0.03 0.00 n 4 4 4 4 0 0 Mean 0.10 0.09 0.07 0.02 Std. Dev. 0.006 0.015 0.006 0.019 o Data ------ Regression -----95% Conf. Int. ........50% Inhibition h c 25 12.9092 E C 5o 2 8 .1 1 2 5 0 200 400 600 800 1000 1200 Concentration (mg a.i7kg) Lower 95% C onfidence Lim it 0 .1 6 1 1 0 Lower 95% C onfidence Lim it 1 .9 9 4 3 4 Curve Parameters Upper 95% C onfidence Lim it 1034.67 Ro 0 .0 9 5 7 U pper 95% C onfidence Lim it 3 9 6 .3 6 9 Ro 0.0957 CT 0.5011 C 0.5011 r2 0.98693 r2 0.98693 -107- Wildliie International? L td PROJECT NO.: 454-110 Appendix 9.5 Onion Seedling Height on Day 21 Treatment G roup Replicate Height (cm) for Plant Number: 1 2 3 4 5 6 7 8 9 10 Control A 13 4 8 9 9 5 13 8 5 B 877878 8 7 C 7 7 10 7 9 7 13 8 8 9 D 2 7 7 8 8 10 10 14 8 2 10 3.91 mg a.i./kg A 7 6 7 6 7 8 7 7 7 5 10 B 8 8 9 5 7 8 8 8 7 7 10 C 8899777 8 8 D 899 8 187 8 8 15.6 mg a.i./kg A B C D 5 6 5 11 8 8 9 12 3 8 8 8 6 7 8 6 9 14 6 8 1 8 7 4 8579839 8 7 9 8 8 62.5 mg a.i./kg A B C D 32 4 3 452 1 4 315 3 112 3 250 mg a.i./kg A B C D 0 0 0 0 1000 mg a.i./kg A B C D The symbol indicates that the seedling either did not emerge or died prior to measurement. 0 0 0 0 Mean 8.6 7.6 8.4 7.6 6.7 7.5 7.9 7.3 7.4 7.3 7.1 7.1 3.0 3.0 3.0 1.3 Std. Dev. 3.25 0.53 2.01 3.60 0.82 1.08 0.83 2.60 2.23 2.40 3.80 2.10 1.00 1.83 2.00 0.58 -108- Wildliie InternationalrL id PROJECT NO.: 454-110 Appendix 9.6 Onion Mean Seedling Height on Day 21 Treatment _________M ean Height (cm) for Replicate: Group A B C D n Control 8.6 7.6 8.4 7.6 4 3.91 mg a.i./kg 6.7 7.5 7.9 7.3 4 15.6 mg a.i./kg 7.4 7.3 7.1 7.1 4 62.5 mg a.i./kg 3.0 3.0 3.0 1.3 4 250 mg a.i./kg . ... 0 1000 mg a.i./kg_______ .___________.___________ .___________ .___________ 0 Mean 8.1 7.3 7.3 2.6 Std. Dev. 0.55 0.49 0.15 0.83 o Data Regression - - - 95% Conf. Int. ...... 50% Inhibition ecm 2 9 .0 7 3 7 E C 50 4 6 .5 0 5 0 Lower 95% C onfidence Lim it 0 .9 3 4 7 6 Lower 95% C onfidence Lim it 8 .9 4 1 2 9 Curve Parameters Upper 95% C onfidence Lim it 9 0 4 .0 6 6 Ro 7 .6 9 8 5 Upper 95% C onfidence Lim it 241.824 Ro 7.6985 -109- a 0.3025 a 0.3025 r2 0 .9 8 6 0 0 r2 0 .9 8 6 0 0 W ildliie International. L id PROJECT NO.: 454-110 Appendix 9.7 Onion Seedling Condition, Day 21 Treatment Replicate Condition (score.sign)1for Plant Number: 1 2 3 4 5 6 7 8 9 10 Mean Control A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 100.- 10 10 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 3.91 mga.i./kg A 0.- 0.- 0.- 40.CL 0.- 0.- 0.- 0.- 0.- 0.- 10 4 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 C 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 11 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 15.6 mg a.i./kg A 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 13 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 D 0.- 0.- 0.- 0.- 0.- 60.N 0.- 0.- 8 8 62.5 mg a.i./kg A 100.- 100.- 0.- 0.- 0.- 5 40 B 100.- 100.- 100.- 100.- 100.- 100.- 0.- 0.- 0.- 0.- 10 60 C 100.- 100.- 100.- 0.- 0.- 0.- 6 50 D 100.- 0.- 0.- 0.- 4 25 250 mg a.i./kg A 100.- 100.- 100.- 100.- 100.- 5 100 B 100.- 100.- 100.- 100.- 100.- 100.- 100.- 100.- 8 100 C 100.- 100.- 2 100 D 100.- 1 100 1000 mg a.i./kg A 0 B0 C0 D0 T h e "." symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity of observed signs of toxicity. N - Necrosis, CL - Chlorosis Std. Dev. 0.0 0.0 31.6 0.0 12.6 0.0 33.3 0.0 35.4 0.0 0.0 21.2 54.8 51.6 54.8 50.0 0.0 0.0 0.0 -110- Wildlife IntemaiionalfL id PROJECT NO.: 454-110 Appendix 10.1 Ryegrass Emergence Day 7 Treatment Number o f Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 7 9 7 8 4 7.75 0.96 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 9 8 7 9 6 7 97 10 6 88 4 8.50 1.00 4 7.50 1.91 4 7.50 0.58 250 mg a.i./kg 6 4 3 3 4 4.00 1.41 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 D ay 15 Treatment Number o f Emerged Seedlings in Replicate: AB C D n Mean Std. Dev. Control 3.91 mg a.i./kg 8 9 9 10 7 10 8 8 4 8.00 0.82 4 9.25 0.96 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg 9 7 6 1 9 8 4 0 10 8 99 76 00 4 9.00 0.82 4 8.25 0.96 4 5.75 1.26 4 0.25 0.50 Day 21 Treatment Number o f Emerged Seedlings in Replicate: Group AB C D H Mean Std. Dev. Control 8 9 7 8 3.91 mg a.i./kg 9 10 10 8 15.6 mg a.i./kg 9 9 10 8 62.5 mg a.i./kg 7 8 9 10 250 mg a.i./kg 6 4 7 6 1000 mg a.i./kg 1 1 0 1 4 8.00 0.82 4 9.25 0.96 4 9.00 0.82 4 8.50 1.29 4 5.75 1.26 4 0.75 0.50 - Ill - W ildlife International' L id Appendix 10.2 Mean Ryegrass Emergence on Day 21 PROJECT NO.: 454-110 o Data ---- Regression - - - 95% Conf. Int. .......50% Inhibition Concentration (mg a.i./kg) e c 25 2 0 2 .6 7 5 E C 50 3 4 3 .6 3 7 Lower 95% C onfidence Lim it 131.220 Lower 95% C onfidence Lim it 2 5 3 .8 0 5 Curve Parameters Upper 95% C onfidence Lim it 3 1 3 .0 4 0 Ro 8 .7 2 2 3 Upper 95% C onfidence Lim it 4 6 5 .2 6 5 Ro 8.7223 a 0 .3 4 0 0 <J 0.3400 r2 0.98361 r2 0.98361 - 112- A/Vildlife International. L td PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 10.3 Ryegrass 21-Day Survival _______ Day 21________ Number o f Living Seedlings in Replicate: AB C D 89 7 8 9 10 10 8 9 9 10 8 78 9 9 62 7 6 11 0 1 n 4 4 4 4 4 4 Mean 8.00 9.25 9.00 8.25 5.25 0.75 Std. Dev. 0.82 0.96 0.82 0.96 2.22 0.50 o Data ------ Regression - - - - 95% Conf. Int. 50% Inhibition e c 25 173.500 ECso 3 1 0 .0 9 9 Lower 95% C onfidence Lim it 107.029 Lower 95% C onfidence Lim it 2 2 2 .8 4 4 Curve Parameters Upper 95% C onfidence Lim it 2 8 1 .2 5 5 Ro 8.7020 Upper 95% C onfidence Lim it 4 3 1 .6 1 8 Ro 8 .7 0 2 0 -113 - a 0.3740 a 0 .3 7 4 0 r2 0 .9 8 4 0 9 r2 0 .9 8 4 0 9 Wildliie International? L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 10.4 Ryegrass Mean Seedling Fresh Weight, Day 21 Mean Weight (g) for Replicate: AB C D 0.12 0.09 0.12 0.16 0.09 0.12 0.12 0.11 0.07 0.06 0.09 0.08 0.07 0.07 0.10 0.09 0.01 0.02 0.01 0.01 0.03 0.04 0.02 n 4 4 4 4 4 3 Mean 0.12 0.11 0.07 0.08 0.01 0.03 Std. Dev. 0.025 0.013 0.014 0.014 0.006 0.010 o Data ------ Regression - - - - 95% Conf. Int. ...... 50% Inhibition e c 25 7 .5 0 5 8 5 E C 50 53.8022 Lower 95% C onfidence Lim it 0 .0 0 3 8 6 0 1 1 3 Lower 95% C onfidence Lim it 0 .4 1 6 2 0 Curve Parameters Upper 95% C onfidence Lim it 14594.86 Ro 0 .1 2 6 2 Upper 95% C onfidence Lim it 6953.44 Ro 0 .1 2 6 2 -114- a 1.26881 a 1.2681 r2 0 .9 2 0 2 4 r2 0.83214 A/Vildliie International. L td PROJECT NO.: 454-110 Appendix 10.5 Ryegrass Seedling Height on Day 21 G roup Replicate Height (cm) for Plant Number: n 1 2 3 4 5 6 7 8 9 10 Control A 15 15 21 20 16 16 11 20 8 B 10 15 14 22 17 19 12 19 19 9 C 17 18 11 22 20 19 18 7 D 18 18 19 15 14 19 16 13 8 3.91 mg a.i./kg A 13 17 15 16 12 20 16 17 12 9 B 18 17 18 20 18 21 18 15 21 6 10 C 18 16 9 15 14 6 21 13 17 17 10 D 12 16 14 14 10 15 15 19 8 15.6 mg a.i./kg A 12 13 13 9 19 12 12 14 17 9 B 19 19 11 21 8 9 14 9 5 9 C 19 14 16 13 19 13 8 14 11 11 10 D 14 13 21 12 19 12 14 12 8 62.5 mg a.i./kg A B C D 9 5 16 18 17 15 12 20 9 6 10 13 15 14 17 20 12 19 16 4 15 16 18 15 15 12 10 17 13 13 7 15 19 7 8 9 9 250 mg a.i./kg A B C D 45645 3 6 54 2 262222 5 7 4 2 14 3 2 6 1000 mg a.i./kg A 2 B2 C D2 The symbol indicates that the seedling either did not emerge or died prior to measurement. 1 1 0 1 Mean 16.8 16.3 17.9 16.5 15.3 17.2 14.6 14.4 13.4 12.8 13.8 14.6 13.1 13.0 15.0 13.4 4.5 4.5 3.0 2.7 2.0 2.0 2.0 Std. Dev. 3.37 3.87 3.44 2.33 2.65 4.34 4.40 2.67 2.96 5.72 3.49 3.46 4.74 4.54 4.77 3.61 1.05 0.71 1.73 1.21 - 115 - Wildlife Iniemational. L id PROJECT NO.: 454-110 Appendix 10.6 Ryegrass Mean Seedling Height on Day 21 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg _________M ean Height (cm) for Replicate: AB C D 16.8 16.3 17.9 16.5 15.3 17.2 14.6 14.4 13.4 12.8 13.8 14.6 13.1 13.0 15.0 13.4 4.5 4.5 3.0 2.7 2.0 2.0 2.0 n 4 4 4 4 4 3 Mean 16.9 15.4 13.7 13.6 3.7 2.0 Std. Dev. 0.69 1.28 0.77 0.92 0.97 0.00 o Data ..... --Regression -----95% Conf. Int. ....... 50% Inhibition e c 25 4 6 .3 5 5 4 E C 50 1 3 1 .0 9 9 Lower 95% C onfidence Lim it 4 .6 3 1 2 7 Lower 95% C onfidence Lim it 2 8 .7 7 4 0 Curve Parameters Upper 95% C onfidence Lim it 4 6 4 .0 8 8 Ro 16.2180 Upper 95% C onfidence Lim it 5 9 7 .1 7 3 Ro 16.2180 -116- a 0 .6 6 9 3 a 0 .6 6 9 3 r2 0.92846 r2 0 .9 2 8 4 6 AA ildliie International? Ltd. PROJECT NO.: 454-110 Appendix 10.7 Ryegrass Seedling Condition, Day 21 Treatment Replicate Condition (score.sign)1for Plant Number: 1 2 3 4 5 6 7 8 9 10 Mean Control A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 80 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 3.91 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 15.6 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 90 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 62.5 mg a.i./kg A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 D 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 10 250 mg a.i./kg A B C D 10.N 0.- 10.N 10.N 10.N 0.- 6 100.- 100.- 0.- 0.- 4 30.N 0.- 20.N 20.N 20.N 40.N 0.- 7 0.- 50.N 90.N 0.- 0.- 0.- 6 7 50 19 23 1000 mg a.i./kg A 10.N 1 10 B 30.N 1 30 C0 D 0.- 1 0 The symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity of observed signs of toxicity. N - Necrosis Std. Dev. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 31.6 5.2 57.7 14.6 38.3 -117- W ildlife International' L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 11.1 Soybean Emergence _____ Day 7 cNumber o f Emerged Seedlings in Replicate: AB D 10 10 10 9 10 10 9 10 9 10 10 9 10 10 10 10 10 10 10 10 9 10 10 10 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Day 15 Number o f Emerged Seedlings in Replicate: AB C D 10 10 10 10 10 10 9 10 9 10 10 9 10 10 10 10 10 10 10 10 10 10 10 10 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Day 21 Number o f Emerged Seedlings in Replicate: AB C D 10 10 10 10 10 10 9 10 9 10 10 9 10 10 10 10 10 10 10 10 10 10 10 10 n Mean Std. Dev. 4 9.75 0.50 4 9.75 0.50 4 9.50 0.58 4 10.00 0.00 4 10.00 0.00 4 9.75 0.50 n Mean Std. Dev. 4 10.00 0.00 4 9.75 0.50 4 9.50 0.58 4 10.00 0.00 4 10.00 0.00 4 10.00 0.00 n Mean Std. Dev. 4 10.00 0.00 4 9.75 0.50 4 9.50 0.58 4 10.00 0.00 4 10.00 0.00 4 10.00 0.00 -118- Wild liie International? L id Appendix 11.2 Mean Soybean Emergence on Day 21 PROJECT NO.: 454-110 Emergence (no.) . ty' <$V <o' V aF V V Treatment Group No treatment group mean is significantly different from the control mean (Dunnettt's test, />>0.05). No regression was conducted due to the lack o f effects. - 119- Wild liie International' L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 11.3 Soybean 21-Day Survival Day 21 Number o f Living Seedlings in Replicate: AB C D 10 10 10 10 10 10 9 10 9 10 10 9 10 10 10 10 9 10 10 10 10 10 10 10 12 n 4 4 4 4 4 4 10 8 -- u 9 ?im 3 Vk m Mean 10.00 9.75 9.50 10.00 9.75 10.00 Std. Dev. 0.00 0.50 0.58 0.00 0.50 0.00 & G w *> $0 b' & 0? Treatment Group N o treatment group mean is significantly different from the control mean (Dunnett's test, /?>0.05). No regression was conducted due to the lack o f effects. -120- Wild liie Intem a tionalfL tcL PROJECT NO.: 454-110 Appendix 11.4 Soybean Mean Seedling Fresh Weight, Day 21 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg _________ M ean W eight (g) for Replicate: AB C D 3.42 4.46 3.55 3.10 3.75 3.92 3.49 3.41 3.94 3.59 3.80 3.18 3.81 3.71 4.12 4.37 2.36 2.11 1.63 2.18 0.62 0.60 0.53 0.54 n 4 4 4 4 4 4 Mean 3.63 3.64 3.63 4.00 2.07 0.57 Std. Dev. 0.583 0.236 0.330 0.302 0.310 0.047 o Data ---------- Regression - - ...... 95% Conf. Int. ............ 50% Inhibition e c 25 160.325 EC50 3 2 5 .7 6 2 Lower 95% C onfidence Lim it 6 9 .1 1 9 4 Lower 95% C onfidence Lim it 189.234 Curve Parameters Upper 95% C onfidence Lim it 3 7 1 .8 7 8 Ro 3 .7 4 8 7 U pper 95% C onfidence Lim it 560.661 Ro 3.7487 - 121 - CT 0.4565 CT 0.4565 r2 0.95631 r2 0.95631 Wildlife International' Lid! PROJECT NO.: 454-110 Appendix 11.5 Soybean Seedling Height on Day 21 Treatment Group Replicate Height (cm) for Plant Number: n 1 2 3 4 5 6 7 8 9 10 Control 3.91 mg a.i./kg 15.6 mg a.i./kg A 25 28 28 25 24 28 25 14 31 35 10 B 32 39 38 34 27 33 31 34 39 19 10 C 31 29 24 33 30 33 29 35 29 36 10 D 18 13 13 23 23 24 33 30 30 28 10 A 33 36 33 27 28 35 31 24 29 31 10 B 27 25 26 21 34 22 37 28 21 38 10 C 22 30 30 27 25 20 13 24 21 9 D 28 29 19 30 26 20 33 15 29 21 10 A 29 31 29 32 26 27 32 41 25 9 B 20 21 24 29 29 25 30 15 29 24 10 C 28 33 23 30 29 28 32 31 27 31 10 D 23 24 24 22 19 25 27 20 28 9 62.5 mg a.i./kg A 23 28 29 33 28 19 32 25 28 28 10 B 34 31 32 36 31 31 35 38 13 34 10 C 34 38 31 32 31 28 29 23 32 29 10 D 32 36 33 29 31 35 37 33 35 32 10 250 mg a.i./kg A 20 22 21 28 27 17 28 28 12 9 B 29 31 20 20 19 21 4 27 31 29 10 C 1 22 24 17 18 20 20 21 16 22 14 10 D 21 25 24 23 25 23 29 26 29 23 10 1000 mg a.i./kg A 2 1 3 3 4 8 5 2 5 3 B 4 36464344 3 C 3 53463856 2 D 543544456 3 The symbol indicates that the seedling either did not emerge or died prior to measurement. 10 10 10 10 Mean 26.3 32.6 30.9 23.5 30.7 27.9 23.6 25.0 30.2 24.6 29.2 23.6 27.3 31.5 30.7 33.3 22.6 23.1 19.4 24.8 3.6 4.1 4.5 4.3 Std. Dev. 5.46 6.10 3.51 7.04 3.74 6.37 5.36 5.85 4.76 4.88 2.90 2.96 4.11 6.92 3.95 2.45 5.70 8.27 3.10 2.62 2.01 1.10 1.84 0.95 - 122- Wild lile International. L id . PROJECT NO.: 454-110 Appendix 11.6 Soybean Mean Seedling Height on Day 21 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg _________M ean Height (cm) for Replicate: AB C D 26.3 32.6 30.9 23.5 30.7 27.9 23.6 25.0 30.2 24.6 29.2 23.6 27.3 31.5 30.7 33.3 22.6 23.1 19.4 24.8 3.6 4.1 4.5 4.3 n 4 4 4 4 4 4 Mean 28.3 26.8 26.9 30.7 22.5 4.1 Std. Dev. 4.17 3.17 3.31 2.51 2.26 0.39 o Data ------ Regression - - - 95% Conf. Int. 50% Inhibition 0 200 400 600 800 1000 1200 Concentration (mg a.i./kg) e c 25 2 8 4 .0 5 3 E C 50 4 6 4 .1 9 5 Lower 95% C onfidence Lim it 1 7 2 .3 4 6 Lower 95% C onfidence Lim it 332.583 Curve Parameters Upper 95% C onfidence Lim it 4 6 8 .2 7 4 Ro 2 8 .1 6 7 6 Upper 95% C onfidence Lim it 647.888 Ro 2 8 .1 6 7 6 -123 - CT 0.3162 CT 0.3162 r2 0.97727 r2 0.97727 W ildlife International. L id PROJECT NO.: 454-110 Appendix 11.7 Soybean Seedling Condition, Day 21 R eplicate 1 2 C ondition (score.sign) 1 for Plant Num ber: 3 4 5 67 8 n Mean 9 10 C on trol 10A 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 10B 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 10C 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 10D 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 3.91 m g a .i./k g 15.6 m g a.i./kg 62.5 m g a.i./kg A B C D A B C D A B C D 0 .- 0 .0 .- 0 .- 0 .0 .- 0 .- 0 .0 .- 0 .0 .- 0 .- 0 .- 0 .- 0 .0 .- 0 .0 .- 0 .0 .- 0 .- 0 .- 0 .0 .- 30.L C 0 .- 0 .0 .- 0 .- 0 .- 0 .0 .- 0 .0 .- 0 .0 .- 0 .- 0 .- 0 .0 .- 0 .0 .- 0 .0 .- 0 .- 0 .0 .0 .0 .- 0 .0 .0 .0 .- 0 .0 .0 .0 .- 100 .- 0 .- 0 .- 0 .- 0 .- 0 100 .- 0 .- 0 .- 0 .- 0 .- 3 90 .- 0 .- 0 .- 0 .- 0 .- 0 100 .- 0 .- 0 .- 0 .- 0 .- 0 90 .- 0 .- 0 .- 0 .- 0 .- 0 100 .- 0 .- 0 .- 0 .- 0 .- 0 100 .- 0 .- 0 .- 0 .- 0 .- 0 90 .- 0 .- 0 .- 0 .- 0 .- 0 100 .- 0 .- 0 .- 0 .- 0 .- 0 0 .- 0 .- 0 .- 4 0 .L C 0 .- 10 4 0 .- 0 .- 30.L C 0 .- 0 .- 10 3 100 .- 0 .- 0 .- 0 .- 0 .- 0 2 5 0 m g a.i./kg A 1 0 0 .- 0 .- B 0 .- 0 .- C 2 0 .L C 0 .- D 0 .- 0 .- 0 .- 2 0 .C L 0 .- 100 .- 0 .- 0 .- 0 .- 0 .- 12 100 .- 0 .- 0 .- 0 .- 9 0 .N 0 .- 2 0 .L C 0 .- 11 100 .- 0 .- 2 0 .C L 0 .- 0 .- 0 .- 0 .- 1 0 .LC 5 100 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 1 0 0 0 m g a.i./k g A B C D 5 0 .S C 5 0 .N 8 0 .N 4 0 .L C 60.SC 70.S C .N 7 0 .N 5 0 .N 4 0 .N 3 0 .L C 60.U SC 7 0 .N 50.N 40.LC .N 70.SC ,N 6 0 .N 4 0 .N 2 0 .L C 60.SC .N 50.N 6 0 .L C 5 0 .N 50.SC .N 3 0 .L C 4 0 .N 50.N 4 0 .L C 40.N 5 0 .N 6 0 .N 4 0 .L C 50.U SC 4 0 .L C 3 0 .L C 4 0 .L C 6 0 .N 5 0 .N 5 0 .N 90.SC 6 0 .N 10 10 10 10 48 45 60 51 1The symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity of observed signs of toxicity. N --Necrosis, LC --Leaf Curl, SC - Stem Curl, USC --Unshed Seed Coat, CL - Chlorosis 0 .0 0 .0 0 .0 0 .0 0 .0 9.5 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 1 2 .6 9.5 0 .0 31.6 28.5 8.5 0 .0 7.9 15.1 17.6 1 2 .0 -124- Wildlife International. L id PROJECT NO.: 454-110 Appendix 12.1 Tomato Emergence Day 7 Treatment Number o f Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 9 7 5 5 4 6.50 1.91 3.91 mg a.i./kg 6 6 5 9 4 6.50 1.73 15.6 mg a.i./kg 6 2 9 4 4 5.25 2.99 62.5 mg a.i./kg 8 4 6 4 4 5.50 1.91 250 mg a.i./kg 0 3 2 0 4 1.25 1.50 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 Day 15 Treatment Number o f Emerged Seedlings in Replicate: Group A BC D n Mean Std. Dev. Control 10 9 9 8 4 9.00 0.82 3.91 mg a.i./kg 9 7 7 9 4 8.00 1.15 15.6 mg a.i./kg 9 8 10 6 62.5 mg a.i./kg 10 6 8 9 250 mg a.i./kg 6 8 8 7 4 8.25 1.71 4 8.25 1.71 4 7.25 0.96 1000 mg a.i./kg 3 0 0 0 4 0.75 1.50 ___________________________________ Day 21 Treatment Number o f Emerged Seedlings in Replicate: Group A B C D Control 10 10 9 8 3.91 mg a.i./kg 9 7 7 10 15.6 mg a.i./kg 9 8 10 6 62.5 mg a.i./kg 10 6 9 9 250 mg a.i./kg 6 8 8 7 1000 mg a.i./kg 3 1 0 0 n Mean Std. Dev. 4 9.25 0.96 4 8.25 1.50 4 8.25 1.71 4 8.50 1.73 4 7.25 0.96 4 1.00 1.41 - 125 - Wildliie International' L id Appendix 12.2 Mean Tomato Emergence on Day 21 PROJECT NO.: 454-110 e c 25 3 1 0 .6 7 0 EC50 4 7 4 .1 3 3 Concentration (mg a.i./kg) Lower 95% C onfidence Lim it 2 0 7 .8 7 4 Lower 95% C onfidence Lim it 3 5 9 .8 3 2 Curve Parameters Upper 95% C onfidence Lim it 4 6 4 .4 0 8 Ro 8 .5 6 4 0 Upper 95% C onfidence Lim it 624.741 Ro 8 .5 6 4 0 CT 0.2721 c 0.2721 r2 0 .9 8 5 5 7 r2 0 .9 8 5 5 7 -126- WiJdli/e InternationalfLtd! PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 12.3 Tomato 21-Day Survival Day 21 Number of Living Seedlings in Replicate: AB C D 10 10 9 8 97 7 10 98 9 6 86 6 7 20 0 1 00 0 0 n 4 4 4 4 4 4 Mean 9.25 8.25 8.00 6.75 0.75 0.00 Std. Dev. 0.96 1.50 1.41 0.96 0.96 0.00 o Data ... - Regression - - - 95% Conf. Int. 50% Inhibition e c 25 6 8 .6 5 9 4 E C 50 105.463 Lower 95% C onfidence Lim it 45.0091 Lower 95% C onfidence Lim it 7 7 .7 8 5 7 Curve Parameters Upper 95% C onfidence Lim it 104.761 Ro 8.5031 Upper 95% C onfidence Lim it 142.988 Ro 8.5031 -127- a 0 .2 7 6 3 <T 0 .2 7 6 3 r2 0 .9 8 9 8 8 r2 0 .9 8 9 8 8 Wildlife International? Ltd. PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 12.4 Tomato Mean Seedling Fresh Weight, Day 21 Mean Weight (g) for Replicate: AB C D 0.31 0.23 0.56 0.31 0.42 0.29 0.49 0.40 0.30 0.13 0.39 0.32 0.06 0.12 0.07 0.06 0.03 n 4 4 4 4 1 0 Mean 0.35 0.40 0.28 0.08 0.03 Std. Dev. 0.143 0.081 0.108 0.029 o Data ------ Regression ......95% Conf. Int. ..... 50% Inhibition e c 25 11.7031 E C 50 2 8 .5 1 0 2 Lower 95% C onfidence Lim it 0 .6 3 1 3 9 Lower 95% C onfidence Lim it 3 .8 0 9 7 8 Curve Parameters Upper 95% C onfidence Lim it 2 1 6 .9 2 0 Ro 0 .3 8 7 4 Upper 95% C onfidence Lim it 2 1 3 .3 5 4 Ro 0 .3 8 7 4 -128- <T 0.5734 CT 0 .5 7 3 4 r2 0.93492 r2 0 .9 3 4 9 2 Wildliie International. L td PROJECT NO.: 454-110 Appendix 12.5 Tomato Seedling Height on Day 21 Treatment Replicate Height (cm) for Plant Number: 1 2 3 4 5 6 7 8 9 10 Control A 5 4 6 5 4 7 5 6 7 4 10 B 6 6 2 4 3 2 4 5 7 3 10 C 86656666 5 9 D 4734956 5 8 3.91 mg a.i./kg A 75647685 6 9 B 757537 4 7 C 563565 7 7 D 5 6 5 8 5 7 6 2 6 6 10 15.6 mg a.i./kg A B C D 67323544 7 9 3425423 5 8 54534678 7 9 55745 7 6 62.5 mg a.i./kg A B C D 22332 33 4 33223 3 33422 2 3223223 8 6 6 7 250 mg a.i./kg A B C D 22 2 0 0 21 1000 mg a.i./kg A B C D The symbol indicates that the seedling either did not emerge or died prior to measurement. 0 0 0 0 Mean 5.3 4.2 6.0 5.4 6.0 5.4 5.3 5.6 4.6 3.5 5.4 5.5 2.8 2.7 2.7 2.4 2.0 2.0 Std. Dev. 1.16 1.75 0.87 1.92 1.22 1.62 1.25 1.58 1.81 1.20 1.67 1.22 0.71 0.52 0.82 0.53 0.00 -129- Wildlile International' L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 12.6 Tomato Mean Seedling Height on Day 21 Mean Height (cm) for Replicate: AB C D 5.3 4.2 6.0 5.4 6.0 5.4 5.3 5.6 4.6 3.5 5.4 5.5 2.8 2.7 2.7 2.4 2.0 2.0 n 4 4 4 4 2 0 Mean 5.2 5.6 4.8 2.6 2.0 Std. Dev. 0.75 0.31 0.94 0.14 0.00 o Data ------ Regression -----95% C oni Int. 50% Inhibition e c 25 22.1411 E C 50 9 3 .8 6 4 2 Lower 95% C onfidence Lim it 0 .4 5 1 6 5 Lower 95% C onfidence Lim it 10.3348 Curve Parameters Upper 95% C onfidence Lim it 1085.43 Ro 5 .5 5 9 4 Upper 95% C onfidence Lim it 8 5 2 .3 1 5 Ro 5.5594 -130- a 0 .9 2 9 9 a 0 .9 2 9 9 r2 0 .9 1 1 1 9 r2 0 .9 1 1 1 9 Wildlife Iniema tional. Lid PROJECT NO.: 454-110 Appendix 12.7 Tomato Seedling Condition, Day 21 Treatm ent Group R eplicate C ondition (score.sign) for Plant Number: n M ean Std. Dev. T 1 2 3 4 5 6 7 8 9 10 C on trol 10A 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 10B 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 9C 0 .- 0 .- 0 .- 0 .- 0.- 0 .- 0 .- 0 .- 0 .- 0 8D 0.- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 3.91 m g a.i./kg A B C D 90 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 70 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 70 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 100 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 . - 0 15.6 m g a.i./kg A B C D 0 .- 0 .- 0 .- 0.- 0.- 0.- 0 .- 0.- 0.- 9 0 40.N 0.- 0.- 0.- 30.N 40.N 0.- 0.- 8 14 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 10 0.- 0.- 0.- 10.LC 0.- 0.- 6 2 62.5 m g a.i./kg A B C D 100.- 50.N 50.N 40.N 30.N 50.N 100.- 50.N 50. N 0.- 10 52 0.- 0.- 20.N 0.- 20.N 20.N 6 10 100.- 100.- 100.- 0.- 40.N 0.- 0.- 40.N 70.N 9 50 100.- 0.- 10.N 30.N 20.N 20.N 20.N 20.N 100.- 9 36 250 m g a.i./kg A B C D 100.- 100.- 100.- 100.- 70.N 70.N 6 90 100.- 100.- 100.- 100.- 100.- 100.- 100.- 100.- 8 100 100.- 100.- 100.- 100.- 100.- 100.- 100.- 100.- 8 100 100.- 100.- 100.- 100.- 100.- 100.- 80.N 7 97 1 0 0 0 m g a.i./k g A B c 100.- 100.- 100.- 3 100 100.- 1 100 D 1The symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity of observed signs of toxicity. N --Necrosis, LC - Leaf Curl 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 19.2 31.6 4.1 29.7 11.0 44.2 37.5 15.5 0 .0 0 .0 7.6 0 .0 -131 - AAAildliie International; L id PROJECT NO.: 454-110 Appendix 13 Bulk Soil Characterization B SSE la oratoriiT AGVISE Soil Characterization Report 604 Highway 15 p a Box 510 Northwood ND 56267 (700567-60 FA X (700 567-6013 email: agvise#polarcommom Homepage agviseUbsxocn Submitting firm = WILDLIFE INT. LTD. Protocol or Study No NA Sample ID. =6HS-01-04 Trial ID. NA Date Received = 10-1-01 Date Reported 10-11-2001 AGVISE Lab NO 01- 1274 Percent Sand 49 Percent Silt 30 Percent Clay 21 USDA Textural Class (hydrometer method) Loam Bulk Density (disturbed) gm/cc Cation Exchange Capacity (meq/100 g) 1.04 9.0 V Moisture at 1/3 Bar 23.8 Percent Organic Matter 2.1 pH in 1:1 soil:water ratio 5.0 Base Saturation Data P a M rn PercentI ppm Calcium 33.4 600 Magnesium 14.8 160 Sodium 5.3 110 Potassium 4.6 160 fw h n lHydrogen 41.9 38 Thepe tests were capnpleted in compliance of 40 CFR 1 //-/ Robert Deutsch 1 D/ate " Soil Scientist/Analytical Investigator A gricultural Testing -132- Wildliie International. Liei PROJECT NO.: 454-110 Appendix 14 Results of Water and Soil Pesticide Screening W ild life International, Ltd. W ell W ater: P esticid es and O rganics Com ponent A cephate A lachlor A ldicarb sulfone A ldicarb sulfoxide A ldrin A lpha-BH C A m etryne A tra zin e A zinphos-ethyl A zinphos-m ethyl Beta-BH C B ifenox B iterta n o l Brom acil Brom ophos-m ethyl B rom oxynil octanoic acid ester C ap tafol Carbaryl 3 -hydroxy Carbofuran C arbofuran C arbophenothion cis-C hlordane tran s-C h lord an e C hlordim eform C hlorfenson trans-C hlorfenvinphos C hlorobenzilate C h lo ro n eb C hloropropham C h lo ro p ro p y late C h loroxu ron C hlorpyrifos-ethyl C hlorpyrifos-m ethyl C h lo rth a l C oum aphos C roto x y p h o s C yanazine Cyfluthrin I Cyperm ethrin I o ,p '-D D D o ,p '-D D E p,p'-D D D p,p'-D D E o ,p '-D D T p ,p '-D D T DEF D em eto n -0 M easured C oncentration (ppb or ng/g) <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 < 50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <250 <50 <50 C om ponent D iazinon D ichlobenil D ich lo ra n D ichlorvos D iclofop m ethyl D icofol D icrotophos D ie ld r in D im ethoate D ioxathion D iphenam id D iphenylam ine D isulfoton D iu ron E ndosulfan I E n d o su lfa n II Endrin Endrin ketone EPN E th a lflu ra lin E th io n Ethoprop E th o x y q u in E trid ia zo le Fenam iphos Fenarim ol Fenobucarb Fenpropathrin F e n s u lfo th io n Fenthion F e n tro th io n F lu z ifo p -P -b u ty l F o n o fo s H eptachlor Heptachlor epoxide H exachlorobenzene Isazophos Isofenphos L ep top h os L in d a n e Linuron M alathion M etalaxyl M etham idophos M ethidathion M ethiocarb 'A nalyses perform ed by E xygen R esearch on sam ples collected on July 2 4 ,2 0 0 1 . M easured Concentration (ppb or ng/g) <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 < 50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 -133 - Wildlife International' L id PROJECT NO.: 454-110 Appendix 14 (continued) Results of Water and Soil Pesticide Screening W ild life International, Ltd. W ell Water: P esticid es A nd O rganics (P age 2 ) C om ponent M ethom yl M ethoxychlor M ethyl parathion M etolachlor M etribuzin cis-M evinphos M irex M onocrotophos M yclobutanil 1-N ap th ol Napropam ide N itra p y rin N orflurazon O xadiazon Oxamyl O xyflu orfen Paraoxon Parathion Pendem ethalin Pentachloronitrobenzene cis-Perm ethrin Perthane Phorate P h o sa lo n e Phospham idon P ip e ra lin Pirim icarb Pirim iphos-ethyl Pirim iphos-m ethyl Profenfos Profluoralin M easured Concentration (p p b orn g/g) <50 <250 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 C om ponent Prom ecarb P rom etryne Pronam ide Propanil Propargite Propham Propoxur Pyrethrin I Q u in a lp h o s Q uinom ethionate Q uizalofop-ethyl Ronnel Sim azine Sim etryn S u lp r o fo s T er b a c il T erb u fo s T etrach lorovin p h os T etra d ifo n T h io b e n c a r b T hiobendazole T h io n a z in THPI Tilt I T ilt II Tridem efon T riflu ra lin Trim ethyl carbam ate V egedex V inclozolin M easured Concentration (p pborng/g) <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 < 50 M etals A lum inum A rsenic B ery lliu m C adm ium C alcium Chrom ium C obalt Copper Iron M agnesium (ppm or m g/L ) < 0 .2 < 0 .0 1 < 0.005 < 0.005 <50 < 0 .0 1 <5 < 0.025 <5 <50 M anganese M ercury M olybdenum N ickel Potassium S e le n iu m Silver S o d iu m Z in c `A n a ly s e s p e rfo rm ed b y E x y g e n R ese a r ch o n s a m p le s c o lle c t e d o n J u ly 2 4 , 2 0 0 1 . (ppm or m g/L ) < 0.015 < 0 .0 0 0 2 < 0.015 <5 <50 < 0.005 < 0 .0 1 <50 < 0 .0 2 -134- Wildliie International. Lid. PROJECT NO.: 454-110 Appendix 14 (continued) Results of Water and Soil Pesticide Screening C om ponent W ild life International, Ltd. G reenhouse Soil: P esticid es and O rganics M easured C oncentration (p pborng/g) C om ponent M easured Concentration (ppb or ng/g) A cephate A lachlor Aldicarb sulfone Aldicarb sulfoxid e A ld rin A lpha-B H C Am etryne A tra zin e A zinphos-ethyl A zinphos-m ethyl Beta-BH C B ifenox B iterta n o l Brom acil Brom ophos-m ethyl B rom oxynil octanoic acid ester C ap tafol Carbaryl 3-hydroxy Carbofuran C arbofuran C arbophenothion cis-C hlordane tran s-C h lord an e Chlordim eform C hlorfenson tra n s-C h lo rfen v in p h o s C hlorobenzilate C hloroneb C hloropropham C h lo ro p ro p y la te C hloroxuron C hlorpyrifos-ethyl C hlorpyrifos-m ethyl C h lorth al C oum aphos C roto x y p h o s C yanazine C yfluthrin I Cyperm ethrin I o ,p '-D D D o ,p '-D D E p ,p '-D D D p ,p '-D D E o ,p '-D D T p ,p '-D D T DEF D em eton-O <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 < 50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <250 <50 <50 D iazinon D ichlobenil D ich lo ra n D ichlorvos D iclofop m ethyl D icofol D icrotophos D ie ld r in D im ethoate D ioxathion D iphenam id Diphenylam ine D isulfoton D iu ron Endosulfan I E n d o su lfa n II Endrin Endrin ketone EPN E th a lflu ra lin E th io n E th op rop E th o x y q u in E tr id ia z o le Fenam iphos Fenarim ol Fenobucarb Fenpropathrin F e n s u lfo th io n Fenthion F e n tro th io n F lu z ifo p -P -b u ty l Fonofos H ep tach lor Heptachlor epoxide H exachlorobenzene Isazophos Isofenphos L ep top h os L in d a n e Linuron M alathion M etalaxyl M etham idophos M ethidathion M ethiocarb <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 'A nalyses perform ed by E xygen R esearch on sam ples collected on July 2 4 ,2 0 0 1 . -135 - W ildliie International. L id PROJECT NO.: 454-110 Appendix 14 (continued) Results of Water and Soil Pesticide Screening C om ponent W ild life International, Ltd. G reenhouse Soil: P esticid es A nd O rganics (P age 2 ) M easured Concentration (ppb or n g/g) Com ponent M easured Concentration (ppb or ng/g) M ethom yl M ethoxychlor M ethyl parathion M etolachlor M etribuzin cis-M evinphos M irex M onocrotophos M yclobutanil 1-N apthol Napropam ide N itrap yrin N orflurazon O xadiazon Oxamyl O xyfluorfen Paraoxon Parathion Pendem ethalin Pentachloronitrobenzene cis-Perm ethrin Perthane Phorate P h o sa lo n e Phospham idon P ip e ra lin Pirim icarb Pirim iphos-ethyl Pirim iphos-m ethyl Profenfos Profluoralin <50 <250 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 Prom ecarb Prom etryne Pronam ide Propanil Propargite Propham Propoxur Pyrethrin I Q uinalphos Q uinom ethionate Q uizalofop-ethyl Ronnel Sim azine Sim etryn Sulprofos T er b a c il Terbufos T etra ch lo r o v in p h o s T etra d ifo n T h iob en carb T hiobendazole T hionazin THPI Tilt I T ilt II Tridem efon T riflu ra lin Trim ethyl carbam ate V eged ex V inclozolin <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 A lum inum A rsenic B eryllium C adm ium C alcium Chrom ium C obalt Copper Iron M agnesium (ppm or m g/K g) < 15300 < 3 .8 3 < 1.91 < 1.91 <7660 10.7 <1910 < 9 .5 7 < 76600 < 1470 M etals M anganese M ercury M olybdenum N ickel P o ta ssiu m S e le n iu m S ilv er S o d iu m Z in c (ppm or m g/K g) <230 1.17 1.68 < 1910 < 76600 < 1.91 < 3 .8 3 < 1910 13.6 'A nalyses perform ed b y E xygen R esearch on sam ples collected on July 2 4 , 2001. -136-