Document BBDwGnK9nx8n3OyBar80JxdJ

/3f PFO S: A TO X IC ITY TEST TO DETER M IN E TH E EFFEC TS O F TH E TEST SUBSTANCE O N SEED LIN G EM ERG ENCE O F SEVEN SPEC IES O F PLA N TS 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 SU BM ITTED TO: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 W ildlife International, L td 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 136 002193 W ildlife International. Ltd. PROJECT NO.: 454-110 GOOD LABORATORY PRACTICE COM PLIANCE STATEM ENT SPONSOR: 3M Corporation TITLE: PFOS: A Toxicity Test to Determine the Effects o f the Test Substance On Seedling Emergence o f Seven Species o f 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 die U .S. Environmental Protection Agency in 40 CFR Part 1 6 0 ,1 7 August 1989; OECD Principles o f Good Laboratory Practice (ENV/M C/CHEM (98)17); and Japan MAFF, 59 NobSan, Notification No. 3850, Agricultural Production Bureau,10 August 1984, with die following exception: The stability o f 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 002194 W ildlife International L td PROJECT NO.: 454-110 QUALITY ASSURANCE STATEM ENT 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 o f Good Laboratory Practice (ENV/M C/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification N o. 3850, Agricultural Production Bureau, 10 August 1984. The dates o f all audits and inspections and the dates d u t any finding 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 M atrix 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 December 6,2001 January 24,2002 January 25,2002 November 11,2002 November 25,2002 September 20,2002 May 28,2003 Final Report May 27-28,2003 May 28,2003 All inspections were study-based unless otherwise noted. May 30,2003 41 w________ Jam es H . Colem an, B.S. Q uality A ssurance Representative Sr3o-o3 D ate -3 - 002195 W ildlife International, Ltd. PROJECT NO.: 454-110 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Toxicity Test to Determine the Effects o f the Test Substance on Seedling Emergence o f Seven Species o f Plants WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-110 STUDY DIRECTOR: John^C Porch isor o f Terrestrial Plant and Insect Studies 3b Date I CHEMISTRY PRINCIPAL INVESTIGATOR: jW U ,. Van Hoven, Ph.D. i f / j j / a3 Date WILDLIFE INTERNATIONAL. LTD. MANAGEMENT: Director o f Aquatic Toxicology/Temestrial Plants and Insects Director o f Chemistry -4 - 00Z19G W llA life 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 002197 -5 - W ilA life Internationa]. L td PROJECT NO.: 454-110 TABLE OF CONTENTS (c o n tin u e d ) 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 Appendix Appendix Appendix Appendix Appendix Appendix Appendix A P P E N D IC E S 1 Personnel Involved in the Study.................................................................................. 30 2 Changes to the Protocol................................................................................................31 3 Certificate of Analysis..................................................................................................32 4 The Analysis of PFOS In Soil and Seven Species of Plants In Support of Wildlife International, Ltd. Project No.: 454-110................................................................... 35 5 Environmental Conditions............................................................................................77 6 Test Results, Alfalfa......................................................................................................83 7 Test Results, Flax..........................................................................................................90 -6- U0C198 W ild life 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 002199 -7 - W ilrli/e International. L td PROJECT NO.: 454-110 SUM M ARY 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 (Liman 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- - O . ^ o o W ilA life International. L id PROJECT NO.: 454-110 INTR O D U C TIO N 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. EXPERIM ENTAL DESIG N 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, w hich received no test substance incorporation, w as 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. 002201 -9 - W ild life International L id PROJECT NO.: 454-110 M A TER IA LS AND M ETH O DS 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 co n c en tra tio n . T h e re m a in in g 69 kg o f bulk so il w as p la c e d in to soil m ix e r, a n d th e 1 kg o f so il w ith 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: 002202 -10- W ild life International; LteL 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, MO, USA Soybean (Glycine max) I Green Envy Johnny's Selected Seeds, Albion, ME, USA Tomato {Lycopersicon esculentum) / 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. 002203 -li - W ild life International Ltrl PROJECT NO.: 454-110 Watering o f 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. 002204 - 12- W ild liie InternationalL td 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 o f 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- 002205 M ilA life International' L id 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 o f 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: Snecies Alfalfa Flax Lettuce Onion Ryegrass Soybean Tomato Date o f 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 follow s: Seedling Emergence: The number o f emerged seedlings per ten planted seeds in each pot. Survival: The number o f emerged seedlings in each pot that were living at test termination per ten planted seeds. Shoot Weight: The mean shoot weight o f 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, 002206 -14- 'WiltUife InternationalfL 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 (/K0.05). 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 NLIN procedure of SAS version 8 (5). ECX values (i.e. EC2s and ECS0) 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 Rq0[(log(ECx)-log(C))/a +ZX] O O l R0 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(ECJ = the logarithm of the predicted ER giving an x percentage o f decrease in the biological parameter vs. the control Z,, = the normal deviate above which x percentage of the area of the standard normal distribution lies, o = the standard deviation o f the normal distribution Effects on survival w ere designated as L C , values, 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 o f 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.iVkg, corresponding to 92.3%, 71.2%, 81.3%, 110%, and 99.8% of the nominal concentrations, respectively (Appendix 4.19). PFOS measured -15- 002207 ilellife International?L id 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.iA g treatment levels had mean measured values of 1.29, 3.56, 16.2, 157 and 474 mg a.iAg, corresponding to 33.0%, 22.8%, 25.9%, 62.8% and 47.4% o f the nominal concentrations, respectively at test termination. A representative ion chromatogram of an artificial soil test sample is presented in Appendix 4.21. Dav 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 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. 002208 - 16- AAfildlifeIntemaiionalfLid. 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 (ms a.i./kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (me a.i./kg dry weight) in: Veaetation 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 (me a.iAe) 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 (me a.i./ke) 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 o f 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 <4.7 O.SO <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 (mg a.i./kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg drv 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- 002209 W ilA lifeInternational Lid. PROJECT NO.: 454-110 Soybean - Total days of exposure = 67 Nominal Soil Concentration (me a.i./ke') 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 (me a.i./ke) 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, EC*, and LC* for 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 cepd) Ryegrass (Lolium perenne) Dicots: Alfalfa (Medicago sativa) Flax (Linum usitatissimum) Lettuce (Lactuca sativa) Soybean (Glycine max) Tomato (Lycopersicon escuientum) LOEC 250 250 21-day Emergence NOEC ECy ECjo 21-day Survival LOEC NOEC e c 25 (All units mg a-i./kg)________ 6 2 .5 5 0 .8 208 6 2 .5 15.6 47.1 62.5 203 344 250 62.5 174 EC 57.3 310 1000 1000 1000 >1000 1000 250 250 250 1000 250 372 399 393 >1000 311 745 599 564 >1000 474 250 250 250 >1000 62.5 62.5 62.5 62.5 1000 15.6 251 144 257 >1000 68.7 452 226 386 >1000 105 002210 -18- W ild life International. Ltd. PROJECT NO.: 454-110 Species Monocots: Onion (.Allium cepa) Ryegrass (Lolium perenne) Dicots: Alfalfa (Medicago sativa) Flax (Linum usitatissimum) Lettuce (Lactuca sativa) Soybean (Glycine max) Tomato (Lycopersicon esculentum) LOEC 62.5 15.6 21-da> Height NOEC EC* 15.6 29.1 3.91 46.3 21-day Shoot Weight EC LOEC I NOEC ECjj (All units 46.5 15.6 3.91 12.9 131 15.6 3.91 7.51 EC 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 E C j j (mg a.i7kg) 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 o f the test. -19- 002211 W ild lile International; L id PROJECT NO.: 454-110 REFERENCES 1 OECD Guideline for Testing of Chemicals. 1998. Guideline fo r 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. T alb ert 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 fo r the Determination o f Perjluorooctanesulfonate, 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 fo r the Determination o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues. -20- W ilA liie International' LtA. 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. 002213 -21 - W ilA life International LtA. 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 Concentration (mg a.iVkg) Number o f 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) Control 8.75 1.50 9.00 1.15 9.00 1.15 8.75 0.96 0.12 0.013 Height (cm) (% Reduction) (Mean SD) 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 (20%) 7.25 1.71 (19%) 7.25 1.71 (19%) 6.25 1.71* (29%) 0.03 0.008* (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, p<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%) 002214 -22- ilAlife*International' LtrL 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 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) Control 7.25 1.26 7.25 1.26 7.25 1.26 7.25 1.26 Shoot Weight (g) (% Reduction) (Mean SD) 0.19 0.029 Height (cm) (% Reduction) (Mean SD) 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 (0%) 7.25 1.71 (0%) 7.25 1.71 (0%) 3.25 1.50* (55%) 0.02 0.009* (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, 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. 8.7 0.90 (3%) 8.8 0.80 (2%) 8.2 0.54 (8%) 1.3 0.35* (86%) N/A 002215 -23 - W ilA life International LtcL 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 Concentration (mg a.iAg) Number o f Emerged Seedlings (% Reduction) Day 7 Day 15 Day 21 (Mean SD) (Mean SD) (Mean SD) Seedling Survival (% Reduction) (Mean SD) Shoot Weight (8) (% Reduction) (Mean SD) Height (cm) (% Reduction) (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 (-3%) 9.00 0.82 (-3%) 9.00 0.82 (-3%) 9.00 0.82 (-3%) 0.25 0.051* (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 (3%) 8.50 1.29 (3%) 8.50 1.29 (3%) 8.50 1.29 (3%) 0.05 0.019* (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%) 00221S -24- W ild life International L id 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 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) Control 8.00 0.82 8.50 1.29 8.75 1.50 8.50 1.29 Shoot Weight (8) (% Reduction) (Mean SD) 0.10 0.006 Height (cm) (% Reduction) (Mean SD) 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 (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. 7.3 0.49 (9%) 7.3 0.15 (10%) 2.6 0.83* (68%) N/A N/A 002217 -25 - W 'ildliff*International' L id PROJECT NO.: 454-110 Table 6 Effects o f PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling Emergence Test with RYEGRASS 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) Control 7.75 0.96 8.00 0.82 8.00 0.82 8.00 0.82 0.12 0.025 Height (cm) (% Reduction) (Mean SD) 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%) 2.0 0.00* (88%) * 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. 002218 - 26 - ilcHifeInternational 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 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) Control 9.75 0.50 10.000.00 10.00 0.00 10.00 0.00 Shoot Weight (8) (% Reduction) (Mean SD) 3.63 0.583 Height (cm) (% Reduction) (Mean SD) 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, j k O.05). Mean S D - Treatment group mean plus or minus one standard deviation. 002219 -27- W ild liie InternationalL td 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.iA g) Number of Emerged Seedlings (% Reduction) Day 7 Day 15 Day 21 (Mean SD) (Mean SD) (Mean SD) Seedling Survival (% Reduction) (Mean SD) Control 6.50 1.91 9.00 0.82 9.25 0.96 9.25 0.96 Shoot Weight (8) (% Reduction) (Mean a SD) 0.35 0.143 Height (cm) (% Reduction) (Mean SD) 5.2 0.75 3.91 15.6 62.5 250 1000 6.50 1.73 (0%) 8.00 1.15 (11%) 8.25 1.50 (11%) 8.25 1.50 (11%) 0.40 0.081 (-14%) 5.6 0.31 (-7%) 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%) 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%) 1.25 1.50* (81%) 7.25 0.96 (19%) 7.25 0.96 (22%) 0.75 0.96* (92%) 0.03 N/A* (91%) 2.0 0.00* (62%) 0* (100%) 0.75 1.50* (92%) 1.00 1.41* (89%) 0* (100%) 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. N/A Q02220 -28- W ileUife Interna iionalfLirL 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 gai/kg) E stim ate Lower Upper 95% CL 95% CL (m gai/kg) A lfalfa E m ergence (250) Survival (62.5) e c 372 HC'so 745 LC 251 L C 452 202 541 178 364 688 >1000 353 563 H eig h t (62.5) EC EC 102 249 26.8 106 391 584 W eight (62.5) E C 53.3 E C jo 146 4.12 27.3 690 783 Flax E m ergence (250) EC 399 E C jo 599 126 402 461 641 Survival (62.5) L C 144 LC 226 103 160 177 368 H eig h t (62.5) E C 97.6 E C jo 140 81.3 125 117 158 W eight (62.5) E C 81.6 EC50 119 46.0 79.4 145 178 Lettuce Em ergence (250) E C 393 E C jo 564 300 474 515 671 Survival (62.5) LC 257 LC, 386 220 344 301 433 H eig h t (0 .0 ) E C 6.79 EC, 39.9 <3.91 3.88 226 410 O n io n W eight (0 .0 ) E m ergence (62.5) Survival (15.6) H eig h t (15.6) EC E C jo 8.92 2 0 .1 E C 50.8 E C jo 208 L C 25 LC 47.1 57.3 E C 29.1 E C jo 46.5 <3.91 5.26 1 2 .2 <3.91 <3.91 17.5 <3.91 8.94 58.3 77.1 195 644 >1000 188 904 242 W eight (3.91) E C 12.9 E C jo 28.1 <3.91 1.99 >1000 396 1- Confidence intervals could not be determined. Species E n d p o in t (NOEC) (m gai/kg) Estim ate Lower 95% CL (m gai/kg) Upper 95% CL Ryegrass Emergence (62.5) EC 203 E C jo 3 4 4 131 254 313 465 Survival (62.5) L C 174 LCjo 310 107 223 281 432 H eig h t (3.91) E C 46.3 E C jo 131 4.63 28.8 464 597 W eight (3.91) E C 7.51 E C jo 53.8 Soybean E m ergence (1 0 0 0 ) EC E C jo >1000 >1000 <3.91 <3.91 \ - >1000 >1000 - Survival (1 0 0 0 ) LC L C jo >1000 >1000 - - H eig h t (62.5) EC 284 E C jo 46 4 172 333 468 648 W eight (62.5) E C 160 EC*, 326 69.1 189 372 561 T o m a to Emergence (250) E C 311 E C jo 4 7 4 208 360 464 625 Survival (15.6) LC 68.7 L C jo 105 45.0 77.8 105 143 H eig h t (15.6) EC EC 2 2 .1 93.9 <3.91 10.3 >1000 852 W eight (15.6) E C 11.7 E C jo 2 8 .5 <3.91 <3.91 217 213 002221 -29- W ilfU ife International' L td PROJECT NO.: 454-110 Appendix 1 Personnel Involved In the Study The following key personnel were involved in the conduct or management o f 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 002222 -30- W iltU iie In tern ation al 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. 002223 -31 - W ildlife international,. L id PROJECT NO.: 454-110 Appendix 3 Certificate of Analysis INTERIM CERTIFICATE OFANALYSIS R evision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFO S,Lot217 Reference#: SD-018 Teat Name Purity1 Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium3 3. Nickel 6. boo 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. Nitrate 3. Nitrite 6. Phosphate 7. Sulfate4 Organic A dds1(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Specifications - < tri i ,, " ` ' White Crystalline Powder ' ** ' i , ' l, U '> v 4 / 1 ') - ' 5 >< - 3 5 j , i v"s^ < h^ ^ " i<> > > " "* ' ; > , * * >> v* : :r::r ''i: -il:; ::i:Vf:f ^ j:i|i ;E-?i3S>!! *;i * < * J3 > tt - ,,<'\ > k ** - *4 * ' > *** V ' 4i ^* 3 ^ * AV ? !jiiil:ri- : : :j ; :^ :;<: j " :::>i 1. Theoretical Value =17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Remit 86.9% Conforms Positive 1. 0.005 wt/wt.% 2. 0.001 wt/wt.% 3. 1.439 wt/wt.% 4. 6.849 wt/wt.% 5. <0.001 wt./wt.% 6. 0.005 wt/wt.% 1 7. <0.001 wt/wt.% | 1.93 wt/wt.% 8.41 wt/wt.% None Detected 0.33 wt /wt.% None Detected Not Applicable1 1. <0.015 wt/wt.% 2. 0.59 wt/wt.% 3. <0.040 wt/wt.% 4. <0.009 w t/w t.% 5. <0.006 wt/wt.% 6. <0.007 wt/wt.% 7. 8.76 wt/wt.% 1. <0.1 wt/wt.% 2. <0.1 wt/wt.% 3. 0.10 wt/wt.% 4. 0.28 wt/wt.% 1. 12.48 wt/wt.% 2. 0.244 wt/wt.% 3. 1.74 wt/wt.% 4. 8.84 wt/wt.% 5. 54.1 wt/wt.% COA023-018A Page 1of3 002224 -32- W ildli/e International' Lid. PROJECT NO.: 454-110 Appendix 3 (continued) Certificate of Analysis INTERIM CERTIFICATE OFANALYSIS C en tre A n a ly tica l L ab oratories C O A R eferen ce #: 0 2 5 -0 1 8A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 `Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+oiganic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.09% Purity = 100% - 13.09% = 86.9% 1Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfar in the sample appears to be converted to SO and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this inteipretation. Based on the results, the SO is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropemanoic acid Pentafluoropropanoic acid 'Theoretical value calculations based on the empirical formula, CFnSOi'K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A - 33- Page 2 o f3 002225 W irlli/e international' L id PROJECT NO.: 454-110 Appendix 3 (continued) Certificate of Analysis Centre Analytical Laboratories. Inc. 3048 Research brivs Stats Collage, R 416801 www.canlralab.oam Phone: (814) 231-8032 Pax: (814) 231-1253 or (814) 231-1580 INTERIMCERTIFICATE OFANALYSIS Revision 3 C eatre A nalytical Laboratories COA R eference #: 023-018A LC/MS Purity Profile: Impurity 4 C5 C6 C7 Total w t/w t.% TI2 03 4.% 1.14 0.41 Note: The C4 and C6 values wen calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the avenge result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using die average result from the C6 and C8 standard curves. Scientist, Centre Analytical laboratories ReviewedBy: n i m fLJtd John Flaherty 7 Date Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 of3 002226 -34- W iltU ife International 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 002227 - 35- W ilJlifeIni&maiionalfLid 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 o f 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 determination. The sub-samples 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 o f 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 002228 - 36- W ilAUfe International L id PROJECT NO.: 454-110 Keystone Betasil Ct8column (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- 002229 W ilAJifp 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 o f 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 mga.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- 002230 W iltilife International.Liei 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 o f 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.iVkg 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 w ith blending for approxim ately tw o m inutes. A 10-g aliq u o t of 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 002231 - 39- ilAlife*International L id PROJECT NO.: 454-110 4.10). Sample measured concentrations were not corrected for the mean procedural recoveries. A representative ion chromatogram o f 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.iAg (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.iA g treatment levels had mean measured values of 1.29, 3.56,16.2,157 and 474 mg a.iA g, 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. 002232 -40- ildlife*International Ltd. 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. 002233 -41 - W ildlift* International' Liei 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 o f PFOS stock solution in methanol with a gas-tight syringe. Allow to air dry. The matrix blank is unfortified soil matrix. 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. Weigh approximately 10.0 grams of each study sample into a tared weigh boat. Record weights. Transfer to 8 oz. French square bottles. 4 For each sample, measure 100 mL of methanol with a graduated cylinder and transfer into the French square bottle. 4 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. 4 Transfer approxim ately 20 m L o f each sample into a separate glass centrifuge tube or scintillation vial and cap. Centrifuge samples for approximately 5 minutes at 2000 rpm. 4 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. 4 Ampulate and submit sample for HPLC/MS/MS analysis. 002234 -42- W ildlifeInternationalLid 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 Clg (50 mm x 2.0 mm, 3-pm particle size) Keystone Javelin Q gcartridge (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 002235 -43- W iltUiieInternational.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. 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. 4 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 o f one minute. 1 Place samples on gyratory shaker table and shake at a setting of 250 rpm for approximately thirty minutes. Centrifuge the vials at approximately 2000 rpm for approximately 10 minutes. 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 o f 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. 4 Transfer an aliquot of each sample to an autosampler vial and submit for HPLC/MS/MS analysis. 002236 -44- W iltU ile International'L 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-tnL 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 (pga.i7pL) 0.00100 0.00100 0.00100 0.00100 0.00100 Calibration Standards for Determination of PFOS in Soil Aliquot (m 100 250 500 750 1000 Final Volume im m 100 100 100 100 100 Standard Concentration (PR a.i./E) 1.00 2.50 5.00 7.50 10.0 Stock Concentration Cue a.i./uLl 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 (pga.i./L) 0.400 0.500 1.00 2.50 3.50 5.00 002237 -45- W ild life international. Liei PROJECT NO.: 454-110 Appendix 4.5 Typical Calibration Curve for PFOS Determinations in Artificial Soil PFOB 499.0-> 99.1 No Internal Staratati Weighted (1/x) Intercept 1818.5138 Slope - 10974958.0000 Correlation Coed. - 0.99798 Area 002238 -46- W ild life Interna H anoi L id PROJECT NO.: 454-110 Appendix 4.6 Typical Calibration Curve for PFOS Determinations in Plant Tissues PROS 499.0*>99.1 No Internal Standard W eighted (1/x) Intercept - 27.4030 Slope 3429.6262 Correlation Coeft. = 0.99864 Area 002239 -47- W ildlife International,Lid. PROJECT NO.: 454-110 Appendix 4.7 Representative Chromatogram of a Low-Level PFOS Calibration Standard PF0S_1 STD 0.400 u0 a lA 4675A-01 ID -26 4.98 Hi 1 period pros No Internal Standard Use Area 1: 4.97 MRM. 298 scans 499.0->99.1 Noise Three. Quant Thres. 2.0 0.5 Min. Width 12 M ult Width 10 Base. Width 40 RT Win. (sacs) 10 Smooth Expected RT 2 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 10Oi 90 BO 70 80 50 40 30 20 10 intensity: 1700 cps 40 62 79 110 145164 41 81 121 161 0.69 1.36 03 2.70 227 . _287_r 21 ' 241 ' 281 Scan 3.37 4.04 4.71 Time Nominal concentration: 0.400 pg a.i./L. - 48- Q O SL & ifO W flifp International' L iei PROJECT NO.: 454-110 Appendix 4.8 Representative Chromatogram of a High-Level PFOS Calibration Standard PFOS 5 STD 0.0100 mg aJJL 467SA-01 ID -20 4.98 in 1 period p re s No Internal Standard Use Area 1: 4.97 MRM. 298 scans 499.0->99.1 Notes Thres. 30.0 Quant Titras. Mn. Width Mutt. Width Bass. Width 1.0 3 6 40 KT Win. (secs) 20 Smooth Expected RT 1 4.01 Area 109807 Height 11092 Start Time 3.89 End Time 4.36 Integration Width 0.87 Retention Time Integration Type &95 A -V B Mon, Nov 10, 2001 12:41 intensity: 10000 cps Nominal concentration: 10.0 pg a.i./L. 002241 - 49- W ilcttifeInternationalLid PROJECT NO.: 454-110 Appendix 4.9 Example Calculations for a Representative Sample The analytical Tesult 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: PA-b C= m 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 die final sample solution was calculated as: ,, 28656- 1818.5138 C -- 10974956 0.002445 mg a.i./L The measured concentrations of PFOS in the artificial soil sample determined as follows: Concentration PFOS (mg a . i ^ ) - ^Solids) where C = Concentration (mg a.i./L) as determined above Ve = Extraction Volume (100 mL) Vji = First Initial Volume (100 mL) Vj2 = Second Initial Volume (0.0500 mL) Vn = First Final Volume (200 mL) Vq = Second Final Volume (50.0 mL) W = Weight of extracted sample (10.0 g) ,,- , 0.002445 x 100 x 200 x 50.0 Concentration PFOS (mg a.i./kg) - 1Q0 x l0Q x q.0500 x 0.849 Concentration PFOS (mg a.iVkg) = 57.60 PFOS (mg a.i./Kg) in sample Percent of Nominal Concentration = PFOS (mg a.i./Kg) nominal x 100 57.60 : 62.5 x 100 = 92.2% Calculated with HPLC/MS/MS instrument software: MacQuan, version 1.6. 002242 -50- W ildlifeInternaionalrLid 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.iA g ) Mean Percent Recovery M A B-1 M A B-3 MAS - 1 M A S-7 M A S-2 MAS- 8 M A S-3 M A S-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 < 1.18J <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. ^The method limit o f quantitation (LOQ) in artificial soil was 1.18 mg a.i./kg on a dry-weight basis and was calculated as the product o f the lowest calibration standard (0.00100 mg a.iTL) and the weigjit/volume dilution factor o f the matrix blank samples (1000) divided by the percent solids for the test system soil (84.9%). 002243 -51 - W ilAliif*InternationalLtd PROJECT NO.: 454-110 Appendix 4.12 Representative Chromatogram of an Artificial Soil Matrix Fortification Sample PFOSJ 464-110- 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 NoteaThiaa. Quant Thru. 30.0 1.0 Min. Width 3 6 . Width RT Win. (aat *) Smooth 40 20 1 Expected RT 4.01 Area 51987 Height 5360 S tilt Time 3.65 End Time 4.32 Integration Width 0.67 Retention Time 3.97 Integration Type k -V B intensity. 10000 cp i Sample Identification: 454-110-MAS-2. Nominal Concentration: 118 mg a.i./kg (dry-weight basis). 002244 - 53- W ilAIife International 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) Fortified5 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 Alfalfa Matrix Blank Alfalfa Fortification Flax Matrix Blank Flax Fortification Flax Fortification Flax Fortification Termination Termination Termination Termination Termination Termination 0.0 50.0 0.0 0.0495 0.503 47.9 3.094 50.4 <0.040 0.0581 0.521 44.4 8.434 138 <0.12 0.170 1.52 130 101 117 104 92.7 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 Onion Matrix Blank Onion Fortification Onion Fortification Onion Fortification Soybean Matrix Blank Soybean Fortification Soybean Fortification Soybean Fortification Termination Termination Termination Termination Termination Termination Termination Termination 0.0 0.0490 0.467 47.9 0.0 0.0500 0.496 50.0 <0.040 0.0477 0.451 51.4 <0.040 0.0508 0.478 47.9 <0.42 0.494 4.68 533 <0.18 0.222 2.09 209 97.3 96.6 107 -, 102 96.4 95.7 tom-f-MAB-1 tom-f-MAS-1 tom-f-MAS-2 tom-f-MAS-3 Tomato Matrix Blank Tomato Fortification Tomato Fortification Tomato Fortification Termination Termination Termination Termination 0.0 0.0498 0.495 47.8 <0.040 0.0468 0.427 42.3 <0.47 0.554 5.05 501 93.9 86.3 88.6 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. JThe method limit of quantitation (LOQ) in fruit tissue was calculated as the product of the lowest calibration standard (0.000400 mg a.iVL) and the weight/volume dilution factor of the matrix blank sample. 'Fortified 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.iA g. 002245 -54- W ilA life International L id PROJECT NO.: 454-110 Appendix 4.14 Representative Chromatogram of a Plant Fruit Tissue Matrix Blank Sample PF0S.8 454-110- SOY-F-MAB-1 4.M In 1 period p ro s No Internal Standard UeeArea 1: 4.97 MRM. 296 scans 499.0-> 99.1 Nolee Three. 2.0 Quant Three. 0.5 M n. Width 12 M ull Width SO B o i. Width 80 RT Win. (secs) 20 Smooth Expected RT 2 3.75 Ana 0 Height 0 Start Time End Time Integration Width Retention Time Inlsyi'JittOF) Typ* 0.00 0.00 0.00 0.00 Tue. Mar 26, 2002 14:06 100i 90 80 70 60 50 40 30i 20 io 0` Intensity: 4000 cps 1 i 51 41 0.69 81 81 1.36 133155 121 161 2.03 70 197 224 265 2 d l ' 241 ' 281 Scan 3.37 4.04 4.71 Tima Sample Identification: 454-110-SOY-F-MAB-1 (soybean matrix). The arrow indicates the retention time of PFOS. 002246 -55 - W ilcUiie International L iei PROJECT NO.: 454-110 Appendix 4.15 Representative Chromatogram of a Plant Fruit Tissue Matrix Fortification Sample PFOS 11 454-110- SOY-F-MAS-4 4.98 in 1 period PPOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.099.1 Noise Ihres. 2.0 Quant Dues. 0.5 Mb. Width 12 Mud. Width 10 Base. Width 80 HT Win. (secs) 20 Smooth 2 Expected HT 3.75 Area 16586 Height 1396 Start Time End Time Integration Width Retention Time Integration Type 3.55 4.42 0.B7 3.77 A - VB Tue, Mar 28, 2002 14:24 lOOi 90 80 70 60 SO 40 30 20 10 16 0J 40 61 85 41 81 0.69 1.36 116 121 2.03 157 161 2.70 intensity: 4000 cps 2 i15 1l 19ft Jf t 21 241 r 261 Scan 3.37 4.04 4.71 Time Sample Identification: 454-110-SOY-F-MAS-3 (soybean matrix). Nominal Concentration: 50.0 mg a.i./kg (wet-weight basis). 002247 - 56- W iltUifcInternaiionaIrLid 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 Recovered1 alf-v-MAB-1 alf-v-MAS-3 Alfalfa Matrix Blank Alfalfa Fortification Termination Termination 0.0 48.1 <0.84 44.2 <2.7 142 91.9 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 Flax Matrix Blank Flax Fortification Lettuce Matrix Blank Lettuce Fortification Lettuce Fortification Onion Matrix Blank Onion Fortification Ryegrass Matrix Blank Ryegrass Fortification Termination Termination Termination Termination Termination Termination Termination Termination Termination 0.0 48.8 0.0 0.502 50.4 0.0 50.1 0.0 49.5 <0.72 47.5 <0.46 0.536 48.8 <0.42 44.1 <1.8 54.3 <2.4 158 <5.5 6.37 579 <4.7 492 <3.3 100 97.3 _ 107 96.7 .. 88.0 __ 110 soy-v-MAB-1 soy-v-MAS-2 Soybean Matrix Blank Soybean Fortification Termination Termination 0.0 49.3 <2.4 48.4 <6.0 119 98.1 tom-v-MAB-1 tom-v-MAS-3 Tomato Matrix Blank Tomato Fortification Termination Termination 0.0 48.8 < 1.4 49.9 <7.6 281 102 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The method limit o f 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. 002248 - 57- W ilAliie*InternationalLtd PROJECT NO.: 454-110 Appendix 4.17 Representative Chromatogram of a Plant Vegetative Tissue Matrix Blank Sample P fO S .S 454-110- MAB-1 Sun, Jan 27, 2002 08:05 4.88 In 1 period PfC6 No Internal Standard UaaAraa 1: 4.87 MRM, 298 acana 499.0->B8.1 Nolee Thro*. 2.0 Quant T h ru . 0.5 Min. Width 12 M ult Width 10 Bata. Width 40 RT WW (M O ) 10 Smooth Expected RT 2 4.05 Area 887 Haight 69 Start Tima End Tima Integration Width 3.62 4.04 0.42 Ratentlon Tima Integration Typo 3.82 A BB intensity: 1700 cps Sample Identification: 454-110-LET-V-MAB-l (lettuce matrix). The arrow indicates the retention time of PFOS. 002249 - 58- ilAife International' L id PROJECT NO.: 454-110 Appendix 4.18 Representative Chromatogram of a Plant Vegetative Tissue Matrix Fortification Sample PFO SIO 454-110- MAS-3 Sun, Jan 27. 2002 06:17 4.96 In 1 period p ro s No M enial Standard Um Area 1: 4.97 MRM. 298 teens 499.0->99.1 Nolle Thiet. Quant Three 2.0 0.5 Mn. Width 12 Mult. Width Baie. Width RTWIn. (aeci) Smooth Expected RT 10 40 10 2 4.05 Area 6660 Height 671 Start Time End Time 3.62 4.19 Integration Width 0.57 Retention Time Integration Type 3.67 A -V B 100-1 9080 70 60 50 40 30 20 10 13 0J 4 41 0.69 106 131 162 81 121 i l l 1.36 2.03 2.70 intensity: 1700 cps 231 1I 1 9Q1 A ^ -5 6 1 201 241 ' 281 Scan 3.37 4.04 4.71 Time Sample Identification: 454-110-LET-V-MAS-3 (lettuce matrix). Nominal Concentration: 50.4 mg a.i./kg (wet-weight basis). 002250 - 59- W iltUifeInternationalLid 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.iA g ) Measured1 Percent Recovered1 Mean Measured (mg a.i./kg) Mean Percent Recovery 01 0.0 02 3.91 03 3.91 04 3.91 < 1.182 3.43 2.83 4.56 -- <1.18 87.7 3.61 72.4 117 92.3 05 15.6 06 15.6 07 15.6 08 62.5 09 62.5 10 62.5 11.1 11.2 86.4J 50.0 44.7 57.6 71.1 72.0 554J 80.0 71.6 92.2 11.1 50.8 71.2 81.3 11 250 12 250 13 250 415 166 276 110 244 97.5 170 67.9 14 1000 15 1000 16 1000 1070 983 942 107 998 99.8 98.3 94.2 Overall Mean = 92.5 Standard Deviation 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. ^ e method limit quantitation (LOQ) in artificial soil was 1.18 mg a.iVkg on a dry-weight basis and was calculated as the product o f the lowest calibration standard (0.00100 mg a.iTL) and the weight/volume dilution factor o f 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 002251 W ilcUiieInternationalLid PROJECT NO.: 454-110 Appendix 4.20 Termination Recoveries for PFOS in Artificial Soil Sample Number Nominal (454-110-) Concentration 28 0.0 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 PFOS Concentration (mg a.iA g) Measured1 < 1.182 1.21 1.36 4.91 2.21 16.3 16.1 153 161 432 515 Percent Recovered1 - 31.0 34.7 31.5 14.2 26.1 25.8 61.4 64.5 43.2 51.5 Mean Measured (mg a.iA g ) <1.18 1.29 3.56 16.2 157 474 Mean Percent Recovery 33.0 22.8 25.9 62.8 47.4 Overall Mean = 38.4 Standard Deviation = 16.4 CV = N= 43% 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. ^The method limit quantitation (LOQ) in artificial soil was 1.18 mg a.iA g on a dry-weight basis and was calculated as the product o f the lowest calibration standard (0.00100 mg a.iVL) and the weight/volume dilution factor o f the matrix blank samples (1000) divided by the percent solids for the test system soil (84.9%). 002252 -61- AAfiltUifeInternationalLid 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- 4.98 in 1 period PTO6 No Internal Standard U te Area 1: 4.97 MRM. 298 cans 499.0-99.1 Nolee Three. 30.0 Quant Three. M K Width MutL Width Baae. Width RT Win. (aeca) Smooth 1.0 3 6 40 20 1 Expected RT 4.01 Area 28656 Height 2916 Start Time End Time Integration Width 3.72 4.39 0.67 Retention Time 3.99 Integration Type A BB intenaity: 10000 cps Sample Identification: 454-110-10. Nominal Concentration: 62.5 mg a.iA g (dry-weight basis). 002253 -62- iltilife International' Lid. 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.iA g ) Measured1 Mean Measured (mg a.iA g ) alf-v-19 alf-v-20 alf-v-21 0.0 0.0 0.0 < 2 .7 2 < 2 .7 < 2 .7 < 2 .7 alf-v-22 alf-v-23 alf-v-24 3.91 3.91 3.91 < 2 .7 <2.7 6.16 6.16 alf-v-25 alf-v-26 alf-v-27 15.6 15.6 15.6 5.66 2.74 <2.7 4.2 alf-v-28 alf-v-29 alf-v-30 62.5 62.5 62.5 12.9 11.2 9.66 11.1 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 reported on a dry-weight basis. T h e method limit o f quantitation (LOQ) in alfalfa vegetative tissue (2.7 mg a.iA g on a dry-weight basis) was calculated as the maximum mg a.i7kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.iA g . *No sample was collected due to mortality at this concentration. 002254 -63- W ild liie International,L td PROJECT NO.: 454-110 Appendix 4.23 Termination Measured Values for PFOS in Alfalfa Fruit Tissue Number (454-110-) Sample Nominat Exposure PFOS Concentration (mg a.iA g ) Measured1 Mean Measured (mg a.iA g ) alf-f-l3 alf-f-23 alf-f-33 alf-f-44 alf-f-54 alf-f-6 0.0 0.0 0.0 3.91 3.91 3.91 < 6.4J .. -- < 6 .4 < 6 .4 < 6 .4 alf-f-73 alf-f-83 alf-f-93 15.6 15.6 15.6 < 6 .4 < 6 .4 alf-f-l 03 alf-f-l l 3 alf-f-l 23 62.5 62.5 62.5 < 6 .4 < 6 .4 alf-f-l 33 alf-f-l 43 alf-f-l 53 alf-f-l 6s alf-f-l 73 alf-f-l 8s 250 250 250 1000 1000 1000 < 6 .4 -- -- < 6 .4 .. 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2The method limit o f quantitation (LOQ) in alfalfa fruit tissue (6.4 mg a.iA g 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.iA g. 1A composite sample was made o f all samples collected at this concentration. 4There was insufficient sample weight (<0.5 g) available for analysis. 5No sample was collected due to mortality at this concentration. 002255 -64- W ilA life InternA iinnal LiiL PROJECT NO.: 454-110 Appendix 4.24 Termination Measured Values for PFOS in Flax Vegetative Tissue Number (454-110-) Sample Nominal Exposure PFOS Concentration (mg a.iA g ) Measured' Mean Measured (mg a.iA g) flx-v-19 flx-v-20 flx-v-21 0.0 0.0 0.0 < 2.42 < 2 .4 < 2 .4 < 2 .4 fix-v-22 flx-v-23 flx-v-24 3.91 3.91 3.91 4.56 4.95 6.43 3.85 flx-v-25 flx-v-26 flx-v-27 15.6 15.6 15.6 13.1 18.8 26.4 17.0 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 62.5 62.5 62.5 250 250 250 1000 1000 1000 54.7 55.0 72.7 37.5 _ -- -- .. __ -- -- 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary s lig h tly . R e s u lts re p o rte d o n a d ry -w e ig h t b a sis. 2The method limit o f quantitation (LOQ) in flax vegetative tissue (2.4 mg a.i A g on a dry-weight basis) was calculated as the maximum mg a.iA g equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.iA g. 3No sample was collected due to mortality at this concentration. 4 There was insufficient sample weight (<0.5g) available for this analysis. 002256 - 65- W ildlifeIntemaiionalrLid 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) Measured1 Mean Measured (mg a.iA g ) flx-f-1 flx-f-2 flx-f-3 flx-f-4 flx-f-5 flx-f-6 flx-f-7 flx-f-8 flx-f-9 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 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 < 0.122 <0.12 <0.12 0.217 0.277 0.195 1.07 0.622 2.41 1.26 2.20 4.23 _ -- - mm -- <0.12 0.230 1.36 2.56 .. 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary s lig h tly . R e s u lts re p o rte d o n a d ry -w e ig h t b a sis. 2The method limit o f 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.i7L) and the weight/volume dilution factor o f 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.S g) available fot analysis. 002257 -66- WiltU iie In tern a tio n a l L id 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.iA g ) Measured1 Mean Measured (mg a.iA g ) let-v-19 let-v-20 let-v-21 0.0 0.0 0.0 < 5 .5 2 <5.5 < 5 .5 < 5 .5 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 iet-v-344 let-v-354 let-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 12.2 8.63 6.16 7.52 15.5 10.6 7.06 9.26 39.8 41.9 58.9 27.0 _ -- -- . ~ -- 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. R e s u lts re p o rte d o n a d ry -w e ig h t b a sis. 2The method limit o f quantitation (LOQ) in lettuce vegetative tissue (5.5 mg a.i A g on a dry-weight basis) was calculated as the maximum mg a.i A g equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. ' There was insufficient sample weight (<0.5 g) available for analysis. 4No samples were collected due to mortality at this concentration. 002258 - 67- W ildlifeInternationalLid PROJECT NO.: 454-110 Appendix 4.27 Termination Measured Values for PFOS in Onion Vegetative Tissue Number (454-110-) oni-v-19 oni-v-20 oni-v-21 oni-v-22 oni-v-23 oni-v-24 Sample Nominal Exposure 0.0 0.0 0.0 3.91 3.91 3.91 PFOS Concentration (mg a.iA g) Measured1 < 4 .7 J < 4 .7 <4.7 <4.7 < 4 .7 <4.7 Mean Measured (mg a.iA g ) <4.7 <4.7 oni-v-25 oni-v-26 oni-v-27 oni-v-283 oni-v-293 oni-v-304 15.6 15.6 15.6 62.5 62.5 62.5 5.86 15.4 <4.7 -- - 10.6 _ oni-v-314 oni-v-324 oni-v-334 oni-v-344 oni-v-354 oni-v-364 250 250 250 1000 1000 1000 -- -- __ -- -- 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2The method limit o f quantitation (LOQ) in onion vegetative tissue (4.7 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.iA g equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.iA g . 3There was insufficient sample weight (<0.5 g) available for analysis. 4No sample was collected due to mortality or lack o f emergence at this concentration. -68- 002259 "WiltilifeInternational.Lid 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.iVkg) Measured1 Mean Measured (mg a.ijlcg) oni-f-1 oni-f-23 oni-f-3 oni-f-4 oni-f-5 oni-f-63 0.0 0.0 0.0 3.91 3.91 3.91 0.517 -- 0.487 5.81 0.453 -- 0.502 3.13 oni-f-7 oni-f-8 oni-f-9 oni-f-103 oni-f-113 oni-f-124 oni-f-134 oni-f-144 oni-f-154 oni-f-164 oni-f-174 oni-f-184 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 1000 1000 1000 < 0.422 19.7 24.6 _ -- - -- - _ -- -- 22.2 __ __ Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. R e su lts re p o rte d o n a d ry -w e ig h t b a sis. 2The method limit o f quantitation (LOQ) in onion fruit tissue was 0.42 mg a.i./kg on a dry-weight basis and was calculated as the product o f the lowest calibration standard (0.000400 mg a.i./L) and the weight/volume dilution factor o f 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 o f fruit production at this concentration. 002260 - 69- W iJdJi/e?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-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.iA g) 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.iA g) <3.3 8.18 31.4 48.7 66.1 .. 'M easured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results re p o rte d o n a d ry -w e ig h t b a sis. 2The method limit o f 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.iA g. 3No sample was collected due to mortality or lack o f emergence at this concentration. 002261 - 70- AATilrUiff*International'Lid. 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.iA g) Measured1 Mean Measured (mg a.iA g ) 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-363 250 250 250 1000 1000 1000 64.2 114 175 103 -- - 1 Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. R e su lts re p o rte d o n a d ry -w e ig h t b a sis. 2The method limit o f quantitation (LOQ) in soybean vegetative tissue (6.0 mg a.i A g on a dry-weight basis) was calculated as the maximum mg a.iA g equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.iA g. 3 No sample was collected due to mortality at this concentration. 002262 -71 - W ilAlifeInternaiionalLid. 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.iA g) 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 3.91 3.91 3.91 0.947 0.339 2.91 1.40 soy-f-7 soy-f-8 soy-f-9 soy-f-10 soy-f-11 soy-f-12 15.6 15.6 15.6 62.5 62.5 62.5 0.464 1.63 0.528 1.14 1.14 1.33 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 -- -- -- 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results re p o rte d o n a d ry -w e ig h t b a sis. 2The method limit o f quantitation (LOQ) in soybean fruit tissue was 0.18 mg a.iVkg on a dry-weight basis and was calculated as the product o f the lowest calibration standard (0.000400 mg a.iVL) and the weight/volume dilution factor o f 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. 002263 - 72- W ilctliUInternational L id 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.iA g ) Measured* Mean Measured (m ga.iAg) tom-v-19 tom-v-20 tom-v-21 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-3l' tom-v-324 tom-v-334 tom-v-344 tom-v-354 tom-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 < 7 .6 2 < 7 .6 < 7 .6 <7.6 < 7 .6 < 7 .6 12.5 27.9 61.1 70.9 44.5 34.9 .. - -- -- < 7 .6 < 7 .6 33.8 50.1 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results re p o rte d o n a d ry -w e ig h t b a sis. 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.iA g. ' There was insufficient sample weight (<0.5 g) available for analysis. 4N o sample was collected due to mortality or lack o f emergence at this concentration. 002264 - 73- W ildliie International Ltd\ 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.iA g ) Measured1 Mean Measured (mg a.iA g ) tom-f-1 tom-f-2 tom-f-3 tom-f-43 tom-f-5 tom-f-6 0.0 0.0 0.0 3.91 3.91 3.91 < 0.472 <0.47 <0.47 _ <0.47 <0.47 <0.47 <0.47 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-17* tom-f-184 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 1000 1000 1000 <0.47 1.51 0.580 0.946 <0.47 <0.47 -- -- ,. -- -- 1.05 0.946 __ .. Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results re p o rte d o n a d ry -w e ig h t b a sis. 2The method limit o f quantitation (LOQ) in tomato fruit tissue was 0.47 mg a.i A g on a dry-weight basis and was calculated as the product o f the lowest calibration standard (0.000400 mg a.iA ) and the weight/volume dilution factor o f 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 o f emergence at this concentration. 002265 - 74- W ildliff*International'Liei PROJECT NO.: 454-110 Appendix 4.34 Representative Chromatogram of a Plant Fruit Tissue Test Sample p ro s _ 2 9 454-110- SQY-F-15 4.98 In 1 period pros No Internai Standan) Use Area 1: 4.07 MRM, 298 scans 499.0*>99.1 Noise Tines. 2.0 Quant Hues. 0.5 Mki. WHh 12 MulL Width 10 Base. Width 80 RT Win. (secs) 20 Smooth 2 Expected RT 3.75 Aree 20080 Height 1699 Start Time End Time Integration Width Retention Time Integration Type 3.48 4.37 0.89 3.72 A - VB Tu*. Mar 28. 2002 16:14 Intensity: 4000 cps Sample Identification: 454-110-SOY-F-15 (soybean matrix). 002266 - 75- W ileUifeInternational,. L id PROJECT NO.: 454-110 Appendix 4.35 Representative Chromatogram o f a Plant Vegetative Tissue Test Sample PFOS.34 454-110- LET-V-28 4.98 In 1 period PROS No Internal Standard UeeAiea 1: 4.97 MRM, 288 scans 409.0->08.1 Noise TTires. 2.0 Quant Three. Win. Width 0.5 12 Mutt. Width 10 Base. Width 40 RT Win. (sees) 20 Smooth 2 Expected RT 4.05 ATM 6351 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 lO O i 90BO 70 60 50 40 30 20 10 0-1 0.68 1.36 2.03 2.70 3.37 Intensity: 1700 cps 251 4.04 4.71 Time Sample Identification: 454-110-LET-V-28 (lettuce matrix). 002^67 "WiMlrfeInternational.Lid. 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 16.7 2 8 .2 2 1 .0 29 6 2 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 -------------- r1 Indicates days on w hich all species w ere w atered. 30 73 3 Indicates days on which alfalfa, lettuce, ryegrass, and soybean trays were watered. 3 PA R - Photosynthetically 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 18.0 16.1 17.5 18.7 16.9 17.8 15.4 17.3 17.3 16.9 17.9 17.5 002268 - 77- W iltUileInternational'Lid PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Date Temperature ( C ) 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 12/27/01* 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 12/31/01" 14.8 27.2 18.6 9 31 20 01/01/025 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 0 1 /1 1 /0 2 16.1 16.1 23.9 19.6 25.9 19.4 28 58 43 19 61 43 01/12/02" 15.1 27.5 19.8 15 54 36 01/13/02 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' -------------- r 15.5 24.5 19.8 23 56 40 2 Indicates days on which soybean and tomato were watered. 3 Indicates days on which alfalfa, lettuce, ryegrass, soybean, and tom ato were watered. 4 Indicates days on w hich tom ato was watered. 3 Indicates days on w hich soybean was watered. ` Indicates days on which flax, lettuce, onion and ryegrass w ere watered. 7 Indicates days on which alfalfa, flax, lettuce, onion, and ryegrass were watered. * Indicates days on which all species watered except tomato. * Indicates days on which alfalfa, flax, lettuce, and onion were watered. 10 P A R - P h o to sy n th etically A ctiv e R adiation 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 002269 - 78- W ilfllifeInternationalLtd. PROJECT NO.: 454-110 Date 01/18/02 01/19/02 01/20/02' 01/21/022 01/22/023 01/23/023 01/24/02 01/25/02 01/26/02* 01/27/02* 01/28/02 01/29/02 01/30/02 01/31/023 02/01/023 02/02/02* 02/03/02* 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-- r Temperature (Q 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 2 6 .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 Mean Relative Humidity (%) 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 7 4 4 2 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 which alfalfa and ryegrass were watered. Indicates days on which all trays w ere watered. Indicates days on which 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 002270 - 79- 'WilfVi/eIntematnnaJr.tA. PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperatute (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 02/28/02' 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 /0 2 ' 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 !' Indicates days on w hich alfalfa and ryegrass w ere w atered. 2 Indicates days on which alfalfa were watered. 3 PA R - Photosynthetically Active 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 17.6 16.2 17.5 14.7 14.7 13.8 13.5 14.0 17.8 14.8 14.1 14.2 002271 -so- W iltUifeInternational L id PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperature (C) Relative Humidity (%) Date Minimum Maximum Mean 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 04/02/02* 16.7 25.8 21.4 21 74 43 04/03/02 16.5 30.9 22.0 32 80 51 04/04/021 15.9 25.3 20.3 16 51 28 04/05/02* 16.6 26.1 20.4 14 46 27 04/06/02* 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 04/09/02* 17.3 25.3 21.6 41 87 66 04/10/02* 17.1 25.4 21.1 29 89 55 04/11/02 17.0 25.9 21.0 29 66 47 04/12/02* 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 04/15/02* 19.8 30.0 23.7 49 84 70 04/16/02 20.2 29.7 24.7 54 87 71 04/17/02* 19.5 31.6 24.9 51 86 69 04/18/02 20.1 28.8 24.0 57 86 73 04/19/02* 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 04/22/02* 16.5 26.4 20.8 28 77 52 0 4 /2 3 /0 2 16.3 2 5 .S 2 0 .7 20 59 34 04/24/02* 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 04/29/02* 16.7 27.1 20.8 29 77 48 04/30/02* 16.5 25.7 21.1 29 77 47 05/01/02* 17.7 25.4 21.5 30 79 57 05/02/02 17.9 26.9 22.6 64 84 75 05/03/02* 17.1 25.5 21.3 20 82 41 ---0-5--/0--4--/0--2-- - 16.5 25.6 21.0 22 72 44 1 Indicates days on which alfalfa and ryegrass were watered. 1 Indicates days on which ryegrass w as watered. 3 PAR - 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 13.4 13.8 17.3 14.1 12.8 17.2 15.6 13.6 13.8 15.0 13.7 14.3 002272 -81 - WilA life In tern a tio n a l L id PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperature (C) Relative Humidity (%) Date Minimum Maximum Mean 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 05/10/02' 18.3 26.1 22.2 47 84 64 05/11/02' 17.8 25.6 21.6 20 67 47 05/12/02 18.8 27.4 23.3 41 86 67 05/13/02' 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 59 84 73 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 ' Indicates days on which ryegrass was watered. 3 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 002273 -82- W ilfllife Internaiional. L id PROJECT NO.: 454-110 Appendix 6.1 Alfalfa Emergence Day 7 T reatm ent Number o f Emerged Seedlings in Replicate: Group AB CD n M ean Std. Dev. Control 7 10 10 8 3.91 mg a.i /kg 8 9 8 8 15.6 mg a.i./kg 9 9 7 6 62.5 mg a.i./kg 6 6 8 8 250 mg a.i./kg 8 5 7 8 1000 mg a.i./kg 0 0 5 5 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 Day 15 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 1000m ga.i./kg 0 0 5 5 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 2.50 2.89 Day 21 T reatm ent Number o f Emerged Seedlings in Replicate: Group AB C D n M ean 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 002274 - 83- W ild life International! Lid\ Appendix 6.2 Mean Alfalfa Emergence on Day 21 PROJECT NO.: 454-110 o Data ----- Regression ------95% C o n i Int. 50% Inhibition ECjj 372.306 EC*> 745.418 Lower 95% Confidence Limit 201.512 Lower 95% Confidence Lim it 540.879 Curve Parameters Upper 95% Confidence Limit 687.860 R 8.4045 Upper 95% Confidence Lim it 1027.07 R 8.4045 0.4470 <r 0.4470 r2 0.97096 0.97096 002275 W ildlifeInternational,Ltd PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.iA g 250 mg a.iA g 1000 mg a.iA g Appendix 6.3 Alfalfa 21-Day Survival Day 21 Number o f Living Seedlings in Replicate: AB C D 8 9 10 8 89 8 8 99 8 6 77 9 9 84 6 7 01 14 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 o Data on --Regression ------95% Conf. Int. (sA 50% Inhibition EC,, 251.073 EC 452.272 Lower 95% Confidence Limit 178.402 Lower 95% Confidence Limit 363.580 Curve Param eters Upper 95% Confidence Limit 353.265 Ro 8.2801 Upper 95% Confidence Limit 562.730 R 8.2801 - 85- a 0.3790 0.99162 0.3790 0.99162 002276 W ilAlif*International.Lid. PROJECT NO.: 454-110 T reatm ent Group Control 3.91 mg a.iTkg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 6.4 Alfalfa Mean Seedling Fresh Weight, Day 21 A 0.11 0.10 0.08 0.11 0.03 - M ean W eight (g) for Replicate: BCD 0.10 0.10 0.12 0.16 0.13 0.09 0.12 0.08 0.11 0.10 0.09 0.12 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 ECa 53.2844 HC 146.049 0 200 400 600 800 1000 1200 Concentration (mg aJVkg) Lower 95% Confidence Limit 4.11623 Lower 95% Confidence Lim it 27.2521 Curve Parameters Upper 95% Confidence Limit 689.763 R 0.1144 Upper 95% Confidence Limit 782.528 R, 0.1144 0.6492 0.6492 0.90534 0.90534 002277 -86- W ilc iie International L id PROJECT NO.: 454-110 Appendix 6.5 Alfalfa Seedling Height on Day 21 T reatm ent Group Replicate Height (cm) for Plant Number: n 1 2 3 4 5 6 7 8 9 10 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 62.5 mg a.i./kg A B C D A B C D 58854454 3 9 5 8755559 4 9 2258357 4 8 67775 5 6 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.iJkg A B C D 3423432 3 2 2%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 M ean 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 002278 - 87- W ildliie InternationalL td PROJECT NO.: 454-110 T reatm ent 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 M ean 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 M ean 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 EC,, 102.447 ECso 249.058 Lower 95% Confidence Lim it 26.8287 Lower 95% Confidence Lim it 106.292 Curve Parameters Upper 95% Confidence Limit 391.201 Rq 6.2793 Upper 95% Confidence Limit 583.579 R 6.2793 -88- 0.5720 T3 0.93960 a 0.5720 r2 0.93960 002279 W ilrlhfeInternational'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 A B C D A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 90.N 0.0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 70.N 30.N 0.- 0.- 0.- 0.- 0.- 0.0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.0.- 30.N 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 9 8 6 /n 7 9 9 10 0 13 0 0 4 0 0 250 mg a.i.Ag A B C D 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 100.- 0.- 0.- 0.- 0.- 5 100.- 80.N 0.- 0.- 0.- 0.- 0.- /1 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 11 20 26 13 1000 mg a.i./kg A 0 B 0.- 1 0 C 100.- 100.- 100.- 100.- 60.N 5 92 D too.- 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 o f observed signs of toxicity. N - Necrosis 30.0 0.0 25.5 0.0 0.0 11.3 0.0 0.0 33.3 44.7 44.3 35.4 17.9 3 6 .0 002280 -89 W iltUifeInternationalLid PROJECT NO.: 454-110 Appendix 7.1 Flax Emergence Day 7 Treatment Number o f Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 7 9 6 7 4 7.25 1.26 3.91 mg a.i./kg 7 5 7 7 4 6.50 1.00 15.6 mg a.i./kg 8 9 7 8 4 8.00 0.82 62.5 mg a.i./kg 9 9 9 7 4 8.50 1.00 250 mg a.i./kg 8 9 5 7 4 7.25 1.71 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 Day 15 Treatment Num ber o f Emerged Seedlings in Replicate: Group AB C D it Mean Std. Dev. Control 7 9 6 7 4 7.25 1.26 3.91 mg a.i./kg 7 5 8 8 4 7.00 1.41 15.6 mg a .iA g 8 9 7 8 4 8.00 0.82 62.5 mg a.i./kg 9 9 9 7 4 8.50 1.00 250 mg a.iA g 8 9 5 7 4 7.25 1.71 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 Treatment Group Control 3.91 m ga.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i.Ag 1000 mg a.i./kg Day 21 Number o f Emerged Seedlings in Replicate: AB C D 79 6 7 85 8 8 89 7 8 99 9 7 89 5 7 00 0 0 W M ean Std. Dev. 4 7.25 1.26 4 7.25 1.50 4 8.00 0.82 4 8.50 1.00 4 7.25 1.71 4 0.00 0.00 002281 - 90- W ild life*In tern ation al L id Appendix 7.2 Mean Flax Emergence on Day 21 10 r 9+ PROJECT NO.: 454-110 Emergence (no.) Treatment Group * Treatm ent group m ean is significantly different from the control m ean (D unnett's test, p<0.05) The non-linear regression technique failed to generate useable results. Therefore, linear interpolation was used. ECjs 398.7069 Lower 95% Confidence Limit 126.1605 Upper 95% Confidence Limit 460.7759 Lower 95% Upper 95% ECso Confidence Limit Confidence Limit 599.1379 401.6809 640.5172 ECk and EC estimates calculated by linear interpolation using the ICPIN program (7). 00Z2S2 -91 - W iA liie InternationalL id PROJECT NO.: 454-110 Appendix 7.3 Flax 2 1-Day Survival T reatm ent 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 Treatment Group * Treatm ent group m ean is significantly different from the control mean (D unnett's te st,p<0.05) The non-linear regression technique failed to generate useable results. Therefore, linear interpolation was used. Effect Rate ECz, Effect Concentration 144.1964 Lower 95% Confidence Limit 103.4821 Upper 95% Confidence Limit 177.3710 EC 225.8929 159.6256 367.5893 ECs and EC*> estimates calculated by linear interpolation using the ICPIN program (7). 002283 - 92- W ilAlife* Inif?mationalTLiei PROJECT NO.: 454-110 T reatm ent Group Control 3.91 mg a.i./kg 15.6m ga.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 M ean W eight (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 --R e g r e s s io n 95% Conf.Int. 50% Inhibition ECa 81.5831 EC 118.796 Lower 95% Confidence Lim it 46.0151 Lower 95% Confidence Limit 79.3780 Concentration (mg a.iA g ) Curve Param eters Upper 95% Confidence Limit 144.644 R 0.1833 Upper 95% Confidence Limit 177.828 R. 0.1833 - 93- a 0.2420 o 0.2420 r1 0.99000 r* 0.99000 002284 W ilAhff*InfernfjtinnniLtrl PROJECT NO.: 454-110 Appendix 7.5 Flax 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 5 9 9 9 8 10 10 7 5 9 9 9 12 10 11 12 5 9 , 11 11 8 9 9 5 6 10 8 11 7 10 9 9 7 3.91 mg a.iTkg A B C D 8 8 10 9 10 10 10 7 . 9 11 9 9 10 S , 8 11 6 8 1 11 11 9 8 7 8 3 8 10 9 9 7 15.6 mg a.i./kg A B C D 9 10 8 11 10 9 11 10 8 9 10 8 9 8 11 9 9 9 9 . 9 8 11 11 6 9 2 7 6 8 8 10 9 7 8 10 8 62.5 mg a.i./kg A B C D 5 8 10 10 8 10 11 7 8 11 12 9 10 9 5 13 9 1 9 8 8 11 10 3 5 6 10 9 3 7 9 9 10 7 9 9 9 7 250 mg a.i./kg A B C D 11 2 11 1 4 111 1 4 12 2 2 4 1000 mg a.iA g 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 002285 -94- W iltilife International. L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mga.iAg 15.6 mg a.iAg 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 ECa 97.6338 ECio 140.411 Concentration (mg a.i7kg) Lower 95% Confidence Lim it 8 0 392 Lower 95% Confidence Lim it 124.825 Curve Param eters Upper 95% Confidence Limit 117.166 Ro 8.7913 Upper 95% Confidence Limit 157.943 R 8.7913 -95- <x 0.2340 t2 0.99938 0.2340 0.99938 002286 VtfildliieInternationalLid PROJECT NO.: 454-110 Appendix 7.7 Flax Seedling Condition, Day 21 Treatm ent Group R e p lica te Condition (score.sign)' for Plant N um ber Std. n Mean Dev. 12 3 4 5 6 7 8 9 10 Control 3.91 m g a .iA g 15.6 m g a .iA g 62.3 m g a-iA g 250 m ga.iA g 1000m ga.iA g A B C D A B C D A B C D A B C D A B C D A B C D 0.- 0.- 0 - 0- 0- 0- 0 - 7 0 0.0 0 - 0.- 0.- 0.- 0 - 0- 0- 0 - 40.U SC 9 4 13.3 0.- 0 - 0- 0- 0- 0 - 6 0 0.0 0.- 0.- 0 - 0- 0- 0- 0 - 7 0 0.0 100.- 0.- 0.- 0- 0- 0- 0- 0 - 8 13 35.4 0 - 0 - 0 - 0 - 0 - S 0 0.0 0 - 0.- 0.- 0 - 80.LC 0- 0- 0 - 8 10 28.3 100.- 0.- 0.- 0- 0- 0- 0- 0 - 8 13 35.4 0.- 0.- 0.- 0 - 0- 0- 0- 0 - 8 0 0.0 0.- 0.- 0.- 0.- 0 - 0- 0- 0- 0 - 9 0 0.0 0.- 0.- 0 - 0- 0- 0 - 80. U SC 7 11 30.2 0.- 0.- 0.- 0 - 0- 0- 0- 0 - 8 0 0.0 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 0-- 0.- 0.- 0.- 0 - 40.N 0- 0- 0 - 9 4 13.3 0.- 0.- 0 - 0- 0- 0- 0 - 7 0 0.0 100.- 100.- 1 0 0 - 1 0 0 - 100- 100- 100- 90.USC 8 99 3.5 100.- 100.- 100.- 1 0 0 - 1 0 0 - 50.USC 90.USC 70.U SC 90.U SC 9 89 17.6 10 0 - 90.USC 80.USC 80.USC 80.USC 5 86 8.9 100.- 1 0 0 - 1 0 0 - 80.U SC 80.USC 80.USC 80.USC 7 89 10.7 0 0 0 0 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 002287 - 96- W ilAlife*InternationalLid. PROJECT NO.: 454-110 Appendix 8.1 Lettuce Emergence Treatment .B S E l Number ofEmerged Seedlings in Replicate: Group A B C D n Mean Std. Dev. Control 9 8 9 9 4 8.75 0.50 3.91 mga.i./kg 8 9 9 10 4 9.00 0.82 15.6 mg a.iA g 62.5 mg a.i./kg 250 mg a.iA g 10 9 10 7 7 7 8 10 4 8.75 1.50 8 10 4 8.50 1.29 7 8 4 8.00 1.41 1000 mg a.i./kg 3 0 1 1 4 1.25 1.26 Day 15 Treatment Number of Emerged Seedlings in Replicate: Group A B C D n Mean Std. Dev. Control 9 8 9 9 4 8.75 0.50 3.91 mg a.i./kg 8 9 9 10 4 9.00 0.82 15.6 mg a.i./kg 10 8 8 10 4 9.00 1.15 62.5 mg a.iA g 9 7 8 10 4 8.50 1.29 250 mg a.iA g 10 7 7 8 4 8.00 1.41 1000 mg a.iA g 3 0 1 1 4 1.25 1.26 Day 21 Treatment Number of Emerged Seedlings in Replicate: Group AB CD n Mean Std. Dev. Control 9 8 9 9 4 8.75 0.50 3.91 mg a.i./kg 8 9 9 10 4 9.00 0.82 15.6 mg a.iA g 10 8 8 10 4 9.00 1.15 62.5 mg a.iA g 250 mg a.iA g 1000 mg a.i./kg 9 10 3 7 8 0 8 10 4 8.50 1.29 7 8 4 8.25 1.26 1 1 4 1.25 1.26 002288 - 97- W iltUifeInternationalLid Appendix 8.2 Mean Lettuce Emergence on Day 21 PROJECT NO.: 454-110 o Data ----- Regression --- - 95% Conf.Int. 50% Inhibition E C jj 393.369 EC 564.027 Lower 95% Confidence Lim it 300.193 Lower 95% C onfidence Limit 474.024 Curve Parameters Upper 95% Confidence Limit 515.466 Ro 8.8126 0.2320 T3 0.99632 Upper 95% ,, , ,, ,,, Ro o r C onfidence L im it_____________________________________________________________________ 671.120 8.8126 0.2320 0.99632 0022&9 - 98- W iltU ife International' L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mga.i./kg 15.6mga.i./kg 62.5 mg a.i./kg 250 mg a.iTkg 1000 mg a.i./kg Appendix 8.3 Lettuce 21-Day Survival Day 21 Number of Living Seedlings in Replicate: AB C D 98 89 10 8 97 86 10 99 9 10 8 10 8 10 76 01 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 EC 257.276 EC 386.011 Lower 95% Confidence Lim it 220.141 Lower 95% Confidence Lim it 343.795 Curve Parameters Upper 95% Confidence Limit 300.677 Re 8.8166 Upper 95% Confidence Limit 433.411 Re 8.8166 -99- 0.2613 a 0.2613 0.99719 r2 0.99719 002$90 W ilA liie InternaHemal?L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.iAg 15.6 mg a.iAg 62.5 mg a.iAg 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 .iA g ) EC,s 8.92483 ECso 20.1419 Lower 95% Confidence Lim it 1.36490 Lower 95% Confidence Lim it 5.26260 Curve Parameters Upper 95% Confidence Limit 58.3445 R 0.3466 Upper 95% Confidence Lim it 77.1081 Ro 0.3466 a 0.5242 o 0.5242 r2 0.94722 0.94722 -100- 002291 W ilAlife*Ini&rnaiianal'Lid\ 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 Mean 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 6554457 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 4766458 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 12 111 1 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 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 - 002292 W ilA life International L id PROJECT NO.: 454-110 Treatment Group Control 3.91 mga.i./kg 15.6 mg a.i./kg 62.5 mg a.iTkg 250 mg a.L/kg 1000 mg a.i./kg Appendix 8.6 Lettuce Mean Seedling Height on Day 21 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 it 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 EC,, 6.79360 EC 39.8749 Concentration (mg a.i7kg) Lower 95% Confidence Limit 0.20399 Lower 95% Confidence Lim it 3.87972 Curve Parameters Upper 95% Confidence Limit 226.204 Re 6.5025 Upper 95% Confidence Lim it 409.732 Re 6.5025 -102- 0 1.1396 r2 0.94294 0 1.1396 i2 0.94294 002293 W ildlifeIntematinnalLid PROJECT NO.: 454-110 Appendix 8.7 Lettuce Seedling Condition, Day 21 Treatm ent R e p lica te Condition (score.sign)' for Plant Num ber. 1 2 3 4 5 6 7 8 9 10 M ean C o n tro l A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0 - 8 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 3.91 m g a .iA g 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.- 0.- 0.- 0.- 9 0 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 13.6m ga.iykg 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 0.- 0.- 8 0 0.- 0.- 8 0 0.- 40.N 10 4 62.5 m g a .iA g A 0.- 0.- 50.N 30.N 0.- 50.N 50.N 50.N 40.N 9 30 B 40.N 40.N 40.N 40.N 40.N 30.N 20.N 7 36 C 0 - 20.N 0.- 0.- 50.N 30.N 50.N 50.N 8 25 D 40.N 40.N 40.N 40.N 30.N 30.N 40.N 50.N 30.N 50.N 10 39 250 mg a.iA g A 100.- 100.- S0.N 0.- 40.N 0.- 60.N 0.- 60.N 0.- 10 44 B 100.- 100.- 0.- 40.N 0.- 0.- 30.N 40.N 8 39 C 60.N 0.- 30.N 40.N 40.N 40.N 0.- 7 30 D 100.- 100.- 30.N 80.N 0.- 40.N 50.N.LC 30.N 8 54 lOOOm ga.i./kg A 100.- 100.- 80.N 3 93 B0 C 100.- 1 to o D 90.USC 1 90 'The symbol indicates that the seedling did not emerge. A score o f 0 indicates a normal seedling, while a score o f 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity o f 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 12.6 23.5 7.9 23.3 7.4 42.0 41.6 22.4 36.2 11.5 0Q2Z94 -103- W ilAliieInternational'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 9.1 Onion Emergence Day 7 Number ofEmerged Seedlings in Replicate: AB C D 87 8 9 10 8 9 7 88 7 8 48 4 4 53 0 0 00 0 0 Treatment Group Control 3.91 mga.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.iAg 1000 mg a.i./kg Day 15 Number of Emerged Seedlings in Replicate: A BC D 8 7 9 10 10 10 89 9 8 7 8 5 10 6 4 58 2 1 00 0 0 Treatment Group Control 3.91 mg a.iAg 15.6 mg a.iAg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.iAg Day 21 Number ofEmerged Seedlings in Replicate: AB C D 87 10 10 10 10 9 8 89 8 8 5 10 6 4 58 2 1 00 0 0 n Mean Std. Dev. 4 8.00 0.82 4 8.50 1.29 4 7.75 0.50 4 5.00 2.00 4 2.00 2.45 4 0.00 0.00 n Mean Std. Dev. 4 8.50 1.29 4 9.00 1.41 4 8.25 0.50 4 6.25 2.63 4 4.00 3.16 4 0.00 0.00 n Mean Std. Dev. 4 8.75 1.50 4 9.25 0.96 4 8.25 0.50 4 6.25 2.63 4 4.00 3.16 4 0.00 0.00 -104- 002295 W ilcUife International Lti Appendix 9.2 Mean Onion Emergence on Day 21 12 10 PROJECT NO.: 454-110 TQ) 5 s' & b/ <> & ^ #' Treatment Group ^ <$>' Treatm ent group m ean is significantly different from the control m ean (D unnett's test, p<0.05). The non-linear regression technique failed to generate useable results. Therefore, linear interpolation was used. Effect Rate Effect Concentration Lower 95% Confidence Limit Upper 95% Confidence Limit EC 50.7750 12.1515 194.5350 EC 208.3333 -25.0000 644.2308 EC a and ECso estimates calculated by linear interpolation using the ICPIN program (7). -105- 002296 W 'ileUifeInternationalLtd PROJECT NO.: 454-110 T reatm ent Group Control 3.91 mg a .iA g 15.6m ga.i7kg 62.5 mg a.iA g 250 mg a.iA g 1000 mg a.i./kg Appendix 9.3 Onion 21-Day Survival Day 21 Number o f Living Seedlings in Replicate: AB C D 8 7 9 10 10 10 79 8 8 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 I EC 47.0977 EC 57.2928 Lower 95% Confidence Limit 0.99238 Lower 95% Confidence Limit 17.4622 Curve Parameters Upper 95% Confidence Limit 2234.60 Rq 8.5000 Upper 95% Confidence Limit 187.975 Ro 8.5000 -106- o 0.1262 r1 0.99445 o 0.1262 r1 0.99445 002297 W ilAlifeInternationalLid PROJECT NO.: 454-110 Treatment Group Control 3.91 mga.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.iAg Appendix 9.4 Onion Mean Seedling Fresh Weight, Day 21 Mean Weight (g) for Replicate: A BCD 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 EC 12.9092 EC> 28.1125 0 200 400 600 800 1000 1200 Concentration (mg a.iA g) Lower 95% Confidence Lim it 0.16110 Lower 95% Confidence Lim it 1.99434 Curve Parameters Upper 95% Confidence Limit 1034.67 Ro 0.0957 Upper 95% Confidence Limit 396.369 Ro 0.0957 -107- a 0.5011 r2 0.98693 0 0.5011 i2 0.98693 002298 W ilAlitaInternationalLid PROJECT NO.: 454-110 Appendix 9.5 Onion Seedling Height on Day 21 T reatm ent Group Replicate Height (cm) for Plant Number: 1 2 3 4 5 6 7 8 9 10 M ean Control A B. 13 4 8 9 9 5 13 8 877878 8 5 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 m ga.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 8998 187 8 8 15.6 mg a.i./kg A B C D 5 6 5 11 8 8 9 7 12 3 8 8 8 6 7 8 6 9 9 14 6 8 1 8 7 4 8 8579839 8 8 62.5 mg a.i./kg A B C D 32 4 452 1 315 112 3 4 3 3 250 mg a.iA g 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 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- 002299 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.iAg 1000 mg a.iAg Appendix 9.6 Onion Mean Seedling Height on Day 21 Mean Height (cm) for Replicate: AB C D 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 .. .. . n 4 4 4 4 0 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 E C jj 29.0737 EC 46.5050 Lower 95% Confidence Lim it 0.93476 Curve Param eters Upper 95% Confidence Limit 904.066 Ro 7.6985 Lower 95% Upper 95% C on fid en ce L im it______________C onfidence L im it 8.94129 241.824 Ro 7.6985 - 109- 0.3025 0 0.3025 0.98600 r2 0.98600 002300 W ilAlifelniem ational' L id PROJECT NO.: 454-110 Appendix 9.7 Onion Seedling Condition, Day 21 Treatment Group Replicate Condition (score.sign)1for Plant Number n Mean 1 2 3 4 5 6 7 8 9 10 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 mg a.iA g 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 15.6m ga.iA g 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 oO B 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.iA g 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 C .0 D0 `The symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of lOO indicates a dead seedling. Intermediate scores are assigned to indicate the relative seventy of observed signs o f 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- 002301 W ild life International L id PROJECT NO.: 454-110 Appendix 10.1 Ryegrass Emergence Day 7 Treatment Number of Emerged Seedlings in Replicate: Group A B C D n Mean Std. Dev. Control 7 9 7 8 4 7.75 0.96 3.91 mg a.i./kg 15.6mga.i./kg 9 8 9 6 97 10 6 4 8.50 1.00 4 7.50 1.91 62.5 mg a.iVkg 7 7 8 8 4 7.50 0.58 250 mg a.iAg 6 4 3 3 4 4.00 1.41 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 Day 15 Treatment Number of Emerged Seedlings in Replicate: Group A B C D n 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 9 250 mg a.i./kg 6 4 7 6 1000 mg a.i./kg 1 0 0 0 4 8.00 0.82 4 9.25 0.96 4 9.00 0.82 4 8.25 0.96 4 5.75 1.26 4 0.25 0.50 Day 21 Treatment Number of Emerged Seedlings in Replicate: Group AB CD n Mean Std. Dev. Control 3.91 mg a.iAg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg 8 9 9 7 6 1 9 10 9 8 4 1 78 10 8 10 8 9 10 76 01 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 002302 -111 W ilfllife International L td Appendix 10.2 Mean Ryegrass Emergence on Day 21 PROJECT NO.: 454-110 Data Regression 95% Conf. Int. 50% Inhibition EC 202.675 EC 343.637 Lower 95% Confidence Limit 131.220 Lower 95% Confidence Lim it 253.805 Curve Parameters Upper 95% Confidence Limit 313.040 R. 8.7223 Upper 95% Confidence Limit 465.265 Ro 8.7223 a 0.3400 a 0.3400 r> 0.98361 r* 0.98361 -112- 002303 W iltU iie Interna tionalrLidl 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.iVkg lOOOmg a.i./kg Appendix 10.3 Ryegrass 21-Day Survival Day 21 Number of 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 EC 173.500 EC 310.099 Lower 95% Confidence Lim it 107.029 Lower 95% Confidence Lim it 222.844 Curve Parameters Upper 95% Confidence Limit 281.255 Ro 8.7020 Upper 95% Confidence Limit 431.618 Ro 8.7020 - 113- 0.3740 0.98409 0.3740 0.98409 002304 W ild life InfernationaL LitL 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.iA g 1000 mg a.i./kg Appendix 10.4 Ryegrass Mean Seedling Fresh Weight, Day 21 Mean Weight (g) for Replicate: A BCD 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 fresh Weight (g) o Data Regression ----95% Conf. Int. 50% Inhibition E C 25 7.50585 ECso 53.8022 Concentration (mg aJ7kg) Lower 95% Confidence Lim it 0.003860113 Lower 95% Confidence Limit 0.41620 Curve Parameters Upper 95% Confidence Limit 14594.86 R 0.1262 Upper 95% Confidence Limit 6953.44 Ro 0.1262 - 114- a 1.26881 a 1.2681 r1 0.92024 0.83214 002305 W ilciliie fniemationaL L id PROJECT NO.: 454-110 Appendix 10.5 Ryegrass Seedling Height on Day 21 Treatment Group Replicate Height (cm) for Plant Number: ft 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 15.6mga.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.iAg A B C D 45645 3 6 .54 2 262222 5 7 42 14 3 2 6 1000 mg a.iAg 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- 002306 W ilAlife* International L id PROJECT NO.: 454-110 Treatment Group Control 3.91 m g a .iA g 15.6 mg a .iA g 62.5 mg a.iA g 250 mg a.iA g 1000m ga.iA g Appendix 10.6 Ryegrass Mean Seedling Height on Day 21 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 -------R e g re s s io n 95% Cnf.Int. - 50% Inhibition ECU 46.3554 EC 131.099 Concentration (mg a J A g ) Lower 95% Confidence Lim it 4.63127 Lower 95% Confidence Limit 28.7740 Curve Param eters Upper 95% Confidence Lim it 464.088 Ro 16.2180 Upper 95% Confidence Lim it 597.173 Ro 16.2180 -116- 0 0.6693 a 0.6693 r2 0.92846 r3 0.92846 002307 AAAiltliieInternational^Ltd PROJECT NO.: 454-110 Appendix 10.7 Ryegrass Seedling Condition, Day 21 Treatment Group Replicate Condition (score.sign)1for Plant Number: n Mean 1 2 3 4 5 6 7 8 9 10 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.iA g 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.- to 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 m ga.iA g 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.iA g 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.iAg A B C D 30.N 10.N 0.- 0.- 20.N 0.- 50.N 10.N 100.20.N 90.N 10.N 100.20.N 0.- 10.N 0.40.N 0.- 0.0.0.0.- 6 4 7 6 7 50 19 23 1 0 0 0m ga.iA g A 10.N i 10 B 30.N i 30 C0 D 0.- i 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- 002308 W ild life InternationoL L id PROJECT NO.: 454-110 Appendix 11.1 Soybean Emergence Day 7 Treatment Number ofEmerged Seedlings in Replicate: Group A B C D n Mean Std. Dev. Control 10 10 10 9 4 9.75 0.50 3.91 mg a.i./kg 10 10 9 10 4 9.75 0.50 15.6 mg a.i./kg 9 10 10 9 4 9.50 0.58 62.5 mg a.iAg 10 10 10 10 4 10.00 0.00 250 mg a.i./kg 10 10 10 10 4 10.00 0.00 1000 ms a.i./kg 9 10 10 10 4 9.75 0.50 Day 15 Treatment Number ofEmerged Seedlings in Replicate: Group A B C D n Mean Std. Dev. Control 10 10 10 10 4 10.00 0.00 3.91 mg a.i./kg 10 10 9 10 4 9.75 0.50 15.6 mg a.i./kg 9 10 10 9 4 9.50 0.58 62.5 mg a.i./kg 10 10 10 10 4 10.00 0.00 250 mg a.i./kg 10 10 10 10 4 10.00 0.00 1000 mg a.i./kg 10 10 10 10 4 10.00 0.00 Day 21 Treatment Number of Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 10 10 10 10 4 10.00 0.00 3.91 mg a.i./kg 10 10 9 10 4 9.75 0.50 15.6 mg a.i./kg 9 10 10 9 4 9.50 0.58 62.5 mg a.i./kg 10 10 10 10 4 10.00 0.00 250 mg a.i./kg 10 10 10 10 4 10.00 0.00 1000 mg a.i./kg 10 10 10 10 4 10.00 0.00 -118- 002309 W iieilife International L id Appendix 11.2 Mean Soybean Emergence on Day 21 PROJECT NO.: 454-110 Em ergence (no.) / Treatment Group N o treatm ent group m ean is significantly different from the control m ean (D unnettt's test, p>0.05). N o regression was conducted due to the lack o f effects. -119- 002310 W ildlife Intem a iional. L id PROJECT NO.: 454-110 T reatm ent Group Control 3.91 mg a .iA g 15.6 mg a .iA g 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.iA g 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 n 4 4 4 4 4 4 M ean 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 & v , ks' & Treatment Group / N o treatm ent group m ean is significantly different from the control m ean (D unnett's test, p X ).05). No regression was conducted due to the lack o f effects. - 120 - 002311 W ileUife International LieL PROJECT NO.: 454-110 Treatment Group Control 3.91 mga.iAg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 11.4 Soybean Mean Seedling Fresh Weight, Day 21 Mean Weight (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 ECy 160.325 EC 325.762 Concentration (mg a.iJkg) Carve Parameters Lower 95% Confidence Lim it 69.1194 Upper 95% Confidence Limit 371.878 Ro 3.7487 Lower 95% Confidence Limit 189.234 Upper 95% Confidence Limit 560.661 - 121 - o 0.4565 i* 0.95631 0.4565 0.95631 002312 W ilA liie Internaiioiml. L td 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 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 3.91 mga.i./kg 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 15.6 mg a.i./kg 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 C1 22 24 17 18 20 20 21 16 22 14 10 D 21 25 24 23 25 23 29 26 29 23 10 1000 ms a.iA s A 2 1 3 3 4 8 5 2 5 3 10 B 4 3 6 4 6 4 3 4 4 3 10 C 3 5 3 4 6 3 8 5 6 2 10 D 5 4 3 5 4 4 4 5 6 3 10 The symbol indicates that the seedling either did not emerge or died prior to measurement 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- 002313 W ild life Interna tionalfLiei 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.i7kg 1000 mg a.i./kg Appendix 11.6 Soybean Mean Seedling Height on Day 21 Mean 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 EC 284.053 EC 464.195 Lower 95% C onfidence Limit 172.346 Lower 95% Confidence Lim it 332.583 Curve Param eters Upper 95% Confidence Limit 468.274 Ro 28.1676 Upper 95% Confidence Limit 647.888 28.1676 -123- o 0.3162 I* 0.97727 0.3162 0.97727 002314 W ilA life International LieL PROJECT NO.: 454-110 Appendix 11.7 Soybean Seedling Condition, Day 21 T reatm ent Group R e p lica te 1 2 Condition (score.sign)' for Plant Number: 3 4 3 67 8 n Mean 9 10 C o n tro l A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 B 0.- 0.- 0 - 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 3.91 m g a.i A g A B C D 0.- 0.0.- 0.- 0.0.- 0.- 13.6 m g a.iA g A B C D 0.0.- 0.0.- 0.- 0.- 62.3 m g a.i A g A B C D 0.- 0.0.- 0.0.- 0.0.- 0.- 230 m g a.i A g A 100.- 0.- B 0.- 0.- C 20.LC 0.- D 0.- 0.- 0.- 0.0.- 30.LC 0.- 0.0.- 0.- 0.0.0.0.- 0- 0.- 0.- 0.- 0 - 10 0 0.- 0.- 0.- 0.- 0.- 10 3 0.- 0.- 0.- 0.- 0.- 9 0 0.- 0.- 0.- 0.- 0 - 10 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.- 0.- 0.- 0.- 0.- 0.- 40. LC 0.- 10 4 0.- 0.- 0.- 0.- 0.- 30.LC 0.- 0.- 10 3 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.- 20.CL 0.- 0- 0.- 0.- 0.- 0.- 10 12 0.- 0.- 0.- 0.- 90.N 0.- 20.L C 0.- 1 0 11 0.- 0.- 20.CL 0.- 0.- 0.- 0.- 10.LC 1 0 5 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 1000 m g a.iA g A 50.SC 60.SC 40.N 50.N 40.N 60.LC 40.N 50.N 40.LC 50.N 10 48 B 50.N 70.SC.N 30. LC 40.LC.N 20.LC 50.N 50.N 60.N 30.LC 50.N 10 45 C 80.N 70.N 60.USC 70.S C N 60.SC.N 50.SC.N 40.LC 40.LC 40.LC 90.SC 10 60 D 4 0 .L C 50.N 70.N 60.N 50.N 30. LC 40.N 50.USC 60.N 60.N 10 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 Std. Dev. 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 12.6 9.5 0.0 31.6 28.5 8.5 0.0 7.9 15.1 17.6 12.0 002315 W ilAM PIntpmnHarm1.L id PROJECT NO.: 454-110 Appendix 12.1 Tomato Emergence Day 7 Treatment Number o f Emerged Seedlings in Replicate: Group AB CD /I 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 AB C D n Mean Std. Dev. Control 10 9 9 8 4 9.00 0.82 3.91 mg a.i7kg 9 7 7 9 4 8.00 1.15 15.6m ga.L /kg 62.5 mg a.i./kg 250 mg a.iA g 1000 mg a.i./kg 9 10 6 3 8 6 8 0 10 6 89 87 00 4 8.25 1.71 4 8.25 1.71 4 7.25 0.96 4 0.75 1.50 Day 21 Treatment Number o f Emerged Seedlings in Replicate: Group AB C D n Mean Std. Dev. Control 10 10 9 8 4 9.25 0.96 3.91 mg a.i./kg 9 7 7 10 4 8.25 1.50 15.6 mg a.i./kg 9 8 10 6 4 8.25 1.71 62.5 mg a.i./kg 10 6 9 9 4 8.50 1.73 250 mg a.i./kg 6 8 8 7 4 7.25 0.96 1000 mg a.i./kg 3 1 0 0 4 1.00 1.41 - 125- 002316 W iltUifeInternationalLid Appendix 12.2 Mean Tomato Emergence on Day 21 PROJECT NO.: 454-110 o Data -------R e g re s s io n 95% Conf.Int. 50% Inhibition ECa 310.670 EC 474.133 Concentration (mg a.iJkg) Lower 95% C onfidence Limit 207.874 Lower 95% Confidence Limit 359.832 Curve Parameters Upper 95% Confidence Limit 464.408 R 8.5640 Upper 95% Confidence Limit 624.741 Ro 8.5640 o 0.2721 0 0.2721 r2 0.98557 r2 0.98557 -126- 002317 W ilAlifeInternationalLid 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.iVkg Appendix 12.3 Tomato 21-Day Survival Day 21 Number o f 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 1--R e g r e s s io n - - 95% Conf. Int. 50% Inhibition E C jj 68.6594 EC*> 105.463 Concentration (mg aJVkg) Curve Parameters Lower 95% Confidence Lim it 45.0091 Upper 95% Confidence Limit 104.761 R* 8.5031 Lower 95% Confidence Lim it 77.7857 Upper 95% Confidence Limit 142.988 Ro 8.5031 -127- a 0.2763 <T 0.2763 i3 0.98988 r3 0.98988 002318 W ilA life 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 12.4 Tomato Mean Seedling Fresh Weight, Day 21 M ean 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 . fre sh W eight (g) o Data Regression - - - - 95% Conf. hit. 50% Inhibition EC 11.7031 EC 28.5102 Concentration (mg a J A g ) Curve Parameters Lower 95% Confidence Limit 0.63139 Upper 95% Confidence Limit 216.920 Ro 0.3874 Lower 95% Confidence Limit 3.80978 Upper 95% Confidence Limit 213.354 Ro 0.3874 -128- 0.5734 0.5734 r* 0.93492 r1 0.93492 002319 W ild life International LteL PROJECT NO.: 454-110 Appendix 12.5 Tomato 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 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.iAg 62.5 mg a.i./kg A B C D A B C D 67323544 7 9 . 3425423 5 8 54534678 7 9 55745 7 6 22332 334 8 33223 3 6 t 33422 2 6 3223223 7 250 mg a.iVkg A B C D 22 2 .0 0 2 1 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- 002320 ilAifa International L iei PROJECT NO.: 454-110 Treat ment Gr oup Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.iA g 1000 mg a.i./kg Appendix 12.6 Tomato Mean Seedling Height on Day 21 Me a n Hei ght ( cm) for Replicate: A BCD 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% Conf. Int. 50% Inhibition ECa 22.1411 EC 93.8642 Concentration (mg a J A g ) Lower 95% Confidence Limit 0.45165 Lower 95% C onfidence Limit 10.3348 Curve Parameters Upper 95% Confidence Limit 1085.43 Rf 5.5594 Upper 95% Confidence Limit 852.315 R 5.5594 -130- 0.9299 0.9299 r2 0.91119 r2 0.91119 002321 W iA life International. L td PROJECT NO.: 454-110 Appendix 12.7 Tomato Seedling Condition, Day 21 T re atm en t G roup R ep licate C o n d itio n (sc o re.sig n )1 fo r P la n t N u m b er: S td . n M ean D ev. T 1 2 3 4 5 6 7 S 9 10 C o n tro l A 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 1 0 0 B 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 1 0 0 C 0 .- 0 .- 0 .- 0 - 0 .- 0 .- 0 .- 0 .- 0 .- 9 0 D 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 8 0 3 .9 1 m g a .i A g A B C D 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 9 0 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 7 0 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 7 0 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 1 0 0 1 5 .6 m g a .iA g A B C D 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 9 0 4 0 .N 0 .- 0 .- 0 .- 3 0 .N 4 0 .N 0 .- 0 .- 8 14 1 0 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 0 .- 1 0 10 0 .- 0 - 0 .- 1 0 .L C 0 .- 0 .- 6 2 6 2 .5 m g a-i A g A B C D 1 0 0 .- 5 0 .N 1 0 0 .1 0 0 .- 5 0 .N 1 0 0 .0 .- 4 0 .N 1 0 0 .1 0 .N 3 0 .N 0 .0 .- 3 0 .N 5 0 .N 0 .- 4 0 .N 2 0 .N 1 0 0 .2 0 .N 0 .2 0 .N 5 0 .N 0 .0 .- 2 0 .N 5 0 .N 2 0 .N 4 0 .N 2 0 .N 0 .2 0 .N 7 0 .N 1 0 0 .- 10 6 9 9 52 10 50 36 o o o 2 5 0 m g a .i A g A B C D 1 0 0 .1 0 0 .- 1 0 0 .1 0 0 .1 0 0 .- 1 0 0 .1 0 0 .1 0 0 .1 0 0 .- 1 0 0 .1 0 0 .1 0 0 .- 1 0 0 .1 0 0 .1 0 0 .1 0 0 .- 1 0 0 .1 0 0 .1 0 0 .1 0 0 .- 7 0 .N 1 0 0 .1 0 0 .1 0 0 .- 7 0 .N 1 0 0 .1 0 0 .8 0 .N 6 8 8 7 90 100 100 97 tO O O m g a .iA g -------------------r z r r n i A B C D 1 0 0 .- 1 0 0 .1 0 0 .- 3 100 1 100 0 0 1AA indicates a d ead seedling. In term ediate scores are assigned to indicate th e relative sev erity o f ob serv ed sig n s o f toxicity. N - N ecrosis, L C - L eaf C url 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 1 9 .2 3 1 .6 4 .1 2 9 .7 1 1 .0 4 4 .2 3 7 .5 1 5 .5 0 .0 0 .0 7 .6 0 .0 -131 - 002322 W ilAlifeInternational'Lid PROJECT NO.: 454-110 Appendix 13 Bulk Soil Characterization AOVISB Soil Characterisation Report Submitting firm Protocol or Study Mo Sample ID. Trial ID. Date Received Date Reported WILDLIFE INT. LTD. MR QHS-01-04 MR 10- 1-01 10- 11-2001 XSVXSB Lab Mb 01- 1274 Percent Sand 49 Percent Silt 30 Percent Clay 21 DSDR Textural Class (hydrometer method) Loam Bulk Density (disturbed) gm/cc Cation Exchange Capacity (meq/100 g) 1.04 9.0 % Moisture at 1/3 Bar 23.8 Percent Organic Matter 2.1 pH in 1:1 soil .-water ratio 5.0 rMa sMo iwiy no B Nofthwood HD5U67 OOSMMOO FAXm0H7-<0U cnyft igH><mhwwwiH mm HBRNpfeafM ibiam Base Saturation Data (-Hrvn Calcium Magneeium Sodium Potassium Hydrogen Percent sen 33.4 14.8 5.3 4.6 41.9 600 160 110 160 38 ted in compliance of 40 CFR Part 160. /o -//-/ Robert Deutsch 1 Date Soil Scientist/Analytical Investigator Agricultural "testing -132- 002323 W ild liie InternationalLtd. PROJECT NO.: 454-110 Appendix 14 Results o f Water and Soil Pesticide Screening W ildlife International, Ltd. W ell W ater: Pesticides and O rganics Comoonent A cep h a te A lachlor Aldicarb sulfone Aldicarb sulfoxide A ldrin A lpha-B H C A m etryne A trazine A zin p h o s-eth y l A zinphos-m ethyl B eta-B H C Bifenox Bitertanol B rom acil B rom ophos-m ethyl Bromoxynil octanoic acid ester Captafol Carbaryl 3-hydroxy Carbofuran Carbofuran Carbophenothion cis-C h lo rd an e trans-Chlordane C h lordim eform C h lo rfen so n tra n s-C h lo rfe n v in p h o s C hlorobenzilate C h lo ro n eb C h lo ro p ro p h am Chloropropylate C h lo ro x u ro n C h lorpy rifo s-eth y l Chlorpyrifos-m ethyl C h lo rth al Coumaphos C ro to x y p h o s Cyanazine Cyfluthrin I Cypermethrin 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 M easured Concentration (ppb o rn a it) <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 Component D iazinon D ichlobenil Dichloran D ichlorvos Diclofop methyl D icofol D icrotophos Dieldrin D im ethoate D io x ath io n D iphenam id D ip h en y lam in e D isulfoton D iuron Endosulfan I Endosulfan II Endrin Endrin ketone EPN E th a lflu ralin Ethion Ethoprop E th o x y q u in Etridiazole F enam iphos F enarim ol Fenobucarb Fenpropathrin Fensulfothion Fenthion Fentrothion Fluzifop-P-butyl Fonofos H eptachlor Heptachlor epoxide H exachlorobenzene Isazophos lsofenphos L ep to p h o s Lindane Linuron M alathion M etalaxyl M ethamidophos M ethidathion M ethiocarb 'A nalyses perform ed by Exygen R esearch on samples collected on July 24,2001. M easured Concentration (ppbornjt/jt) <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 - 002324 W iA life International' LtcL PROJECT NO.: 454-110 Appendix 14 (continued) Results of Water and Soil Pesticide Screening W ildlife International, Ltd. W ell W ater: Pesticides A nd O rganics (Page 2) C om ponent M cthomyl M ethoxychlor M ethyl parathion M etolachlor M etribuzin cis-M cvinphos M irex M onocrotophos M yclobutanil 1-Napthol N apropam ide N itrapyrin N o rflu razo n O xadiazon Oxamyl O x yfluorfen Paraoxon Parathion P endem ethalin Pentachloronitrobenzene cis-P erm ethrin Perthane Phorate Phosalone P hospham idon Piperalin P irim icarb P irim iphos-ethyl P irim iphos-m ethy! Profcnfos PTofluoralin M easured Concentration (p p b o rn 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 P ronam ide Propanil Propargite Propham Propoxur Pyrethrin I Q uinalphos Quinom ethionate Q u izalo fo p -e th y l Ronnel S im azine S im etryn Sulprofos Terbacil Terbufos Tetrachlorovinphos Tetradifon T h io b e n c a rb T h io b e n d a z o le Thionazm THPI Tilt I T ilt 11 T ridem efon Trifluralin Trimethyl carbamate Vegedex VinclozoHn M easured Concentration (p p b o rn g /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 Alum inum A rsenic Beryllium C adm ium C a lc iu m C hrom ium C o b a lt Copper Iron M agnesium (p p m o r m g/L)_________________________________________________ ( p p m o rm g /L ) < 0.2 < 0 .0 1 < 0 .0 0 5 < 0 .0 0 5 <50 <0.01 M anganese M ercury M olybdenum Nickel Potassium Selenium < 0 .0 1 5 < 0 .0 0 0 2 < 0.015 <5 <50 < 0.005 <5 < 0 .0 2 5 <5 <50 Silver Sodium Z in c < 0 .0 1 <50 < 0.02 'A nalyses perform ed by Exygen R esearch on sam ples collected on July 24,2 0 0 1 . U02325 W iU lifp International' L id PROJECT NO.: 454-110 Appendix 14 (continued) Results of Water and Soil Pesticide Screening Component W ildlife International, Ltd. G reenhouse Soil: Pesticides and O rganics M easured Concentration (ppb ornn/it) Component M easured Concentration (ppb or ng/g) Acephate A lachlor Aldicarb sulfone Aldicarb sulfoxide A ldrin A lp h a-B H C A m etryne A trazin e A zinphos-ethyl A zm phos-m ethyl B eta-B H C Bifenox Bitertanol B rom acil B rom ophos-m ethyl Bromoxynil octanoic acid ester C a p ta fo l Carbary) 3-hydroxy Carbofuran C a rbofinan Carbophenothion cis-C h lo rd an e tran s-C h lo rd an e C hlordim eform Chlorfenson tran s-C h lo rfen v in p h o s C h lo ro b en zilate Chloroneb Chloropropham Chloropropylate Chloroxuron C h lorpy rifo s-eth y l C hlorp y rifo s-m eth y l C h lo rth al Coumaphos Crotoxyphos Cyanazine Cyfluthrin I Cypermethrin 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 ich lo b en il Dichloran Dichlorvos Diclofop methyl Dicofol D icro to p h o s Dieldrin D im ethoate D io x ath io n D iphenam id D iphenylam ine D isulfoton Diuron Endosulfan 1 Endosulfan II Endrm Endrin ketone EPN Ethalfluralin Ethion Ethoprop Ethoxyquin Etridiazole F enam iphos F enarim ol Fenobucarb Fenpropathrm Fensulfothion Fenthion Fentrothion Fluzifop-P-butyl Fonofos H ep tachlor Heptachlor epoxide H ex ach lo ro b en zen e Isazophos Isofenphos Leptophos L in d a n e Linuron M alathion M etalaxyl M ethamidophos 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 'Analyses perform ed by Exygen Research on samples collected on July 24,2001. -135- 002326 W ilAliff* international L id PROJECT NO.: 454-110 Appendix 14 (continued) Results of Water and Soil Pesticide Screening C om ponent W ildlife International, Ltd. Greenhouse Soil: Pesticides And O rganics (Page 2) M easured Concentration (p p b o rn g /g ) Component M easured Concentration (ppborng/g) M ethomyl M ethoxychlor Methyl parathion M etolachlor M etribuzm cis-M evinphos M irex M onocrotophos M yclobutanil 1-Napthol N apropam ide N itrap y rin N o rflurazon O xadiazon Oxamyl Oxyfluorfen Paraoxon Parathion Pendem ethalin Pentachloronitrobenzene cis-P erm ethrin Perthane Phorate Phosalone P hospham idon Piperalin P irim icarb P irim iphos-ethyl P irim iphos-m ethyl Profen fos 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 P rom ecarb P rom etryne P ronam ide Propanil Propargite Propham Propoxur Pyrethrin I Q uinalphos Quinom ethianate Q u izalo fo p -e th y l Ronnel Sim azine S im etryn Sulprofos T erb ac il Terbufos T etrach lo ro v in p h o s Tetradifon Thiobencarb T h io b e n d a z o le T h io n a z in THPI Tilt I T ilt II T ridem efon Trifluralin Trimethyl carbamate Vegedex V indozolin <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 Alum inum A rsen ic Beryllium C ad m iu m C a lc iu m C hrom ium C o b a lt Copper Iron M agnesium (ppm or mg/Ka) <15300 < 3 .8 3 < 1.91 < 1 .9 1 <7660 10.7 <1910 < 9 .5 7 <76600 <1470 M etals M anganese M ercury M olybdenum Nickel Potassium Selenium Silver Sodium Z in c (ppm or m g/Kg) <230 1.17 1.68 <1910 <76600 < 1 .9 1 < 3 .8 3 < 1910 13.6 `A nalyses p erfo rm ed b y E xygen R esearch on sam ples collected on July 2 4 ,2 0 0 1 . -136- U0327