Document vbm69M1mdyd71kq6Vgg7O1xY

PFOS: A TOXICITY TEST TO DETERMINE THE EFFECTS OF THE TEST SUBSTANCE ON SEEDLING EMERGENCE OF SEVEN SPECIES OF PLANTS FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-1 10 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 0.Krueger, Ph.D. Raymond L. Van Hoven, Ph.D. STUDY INITIATION DATE: November 12,2001 STUDY COMPLETION DATE: May 30,2003 SUBMITTED TO: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 136 wildlifeXnternationat: Ltd. PROJECTNO.: 454-110 GOOD LABORATORY PRACTICE COMPLIANCESTATEMENT SPONSOR 3M Corporation TITLE: PFOS: A Toxicity Test to Determine the Effixts of the Test Substance On Seedling Emergenceof !kven Spccics of Plants WILDLIFE INTERNATIONAL,LTD. PROJECTNO.: 454-110 STUDY COMPLETION: May 30,2003 This study was conducted in compliancewith Good Laboratory Practice Standards aspublished by the U.S.EnvironmentalProtectionA j p c y in 40 CFR Part 160, 17August 1989;OECD Principles of Good Laboratory Practice (ENVNCICHEM(98)17);and Japan MAFF,59 NohSan, NotificationNo. 3850,Agricultural ProductionBureau,lOAugust 1984,with the following exception: The stability of the test substance under storage conditions at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Jo R. Porch SPONSOR REPRESJ2NTATIVE: *- -2- Wildlife Zntemational..Ltd. PROJECTNO.: 454-1 10 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as pul)lished by the U.S.Environmental Protection Agency io 40 CFR Part 160, 17 August 1989; OECD Prhciples of Good Laboratory practice (ENVMC/CHEM(98)17);and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all aud& and inspixtiom and the dates that any findings were reported to the Study Dircctor and Laboratory Managemerit were as follows: ACTIVITY Test DATE CONDUCTED mtioNovnember 12,2001 Observations November 19,2001 Height and Weight Measurements, Plant Tissue Collection December 3,200 1 Matrix Fortifications Jan- 24,2002 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT November 12,2001 November 15.2001 November 19,2001 November 20,2001 December 3,2001 Dcccmber 6,2001 January 24,2002 January 25,2002 Ana'ytica1 Data and Dran Report Biological Data and Draft Report November 5-8, 11,2002 September 13-20,2002 November 11,2002 November 25,2002 September20,2002 May 28,2003 Final Repon May 27-28,2003 All inspectionswere study-basedunless otherwisenoted. May 28,2003 May :30,2003 Quality Assurance'Representatintative 5-30-03 Date -3- WildlifeIntmutional, Ltd. PROJECT NO.: 454-110 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Toxicity Test to Determine the Effects of the Tcst Substanceon Seedling Emergence of Seven Species of Plants WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-110 STUDY DIRECTOR: S u e of Terrestrial Plant and Insect Studies *a ! - & CHEMISTRY PRINCIPAL INVESTIGATOR: b y m d .VanHoven,Ph.D. Date scwts it WILDLIFE INTERNATIONAL.LTD. MANAGEMENT: Director OF A a c ToxicologylTerrestrial Plantsand Insects Director of Chemistry -4- PROJECT .NO. 454-110 TABLE OF CONTENTS TitleKover 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 ExperimentalDesign................................................................................................................................... 9 Materials and Methods.............................................................................................................................. 10 Test Substance.............................................................................................................................. 10 Preparation and Soil Incorporation of Test Substance................................................................. 10 Test Species ................................................................................................................................. 10 Test Soil ....................................................................................................................................... 11 Planting of Seeds.......................................................................................................................... 11 Watering of Seedlings.................................................................................................................. 12 Environmental Conditions ........................................................................................................... 12 Pesticide and Metal Screening of Well Water and Soil ................................................................ 12 Observations and Measurements .................................................................................................. 12 Soil Sampling and Analysis ......................................................................................................... 13 Tissue Sampling and Analysis ...................................................................................................... 13 Data Analyses .............................................................................................................................. 14 Results ...................................................................................................................................................... 15 Analytical Chemistry ................................................................................................................... 15 Biological Results ........................................................................................................................ 18 Conclusions............................................................................................................................................... 19 References................................................................................................................................................. 20 -5- PROJECT .NO. 454-110 Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 TABLE OF CONTENTS (continued) TABLES Seedling Condition Rating System ....................................................................................... 21 Effects of PFOS on Seedling Emergence. Survival. Shoot Weight. and Height in a 21- Day Seedling Emergence Test with Alfalfa .......................................................................... 22 Effects of PFOS on Seedling Emergence. Survival. Shoot Weight. and Height in a 21- Day Seedling Emergence Test with Flax .............................................................................. 23 Effects of PFOS on Seedling Emergence. Survival. Shoot Weight. and Height in a 21- Day Seedling Emergence Test with Lettuce ......................................................................... 24 Effects of PFOS on Seedling Emergence. Survival. Shoot Weight. and Height in a 21- Day Seedling Emergence Test with Onion ........................................................................... 25 Effects of PFOS on Seedling Emergence. Survival. Shoot Weight. and Height in a 21- Day Seedling Emergence Test with Ryegrass....................................................................... 26 Effects of PFOS Seedling Emergence. Survival. Shoot Weight. and Height in a ;!l-Day Seedling Emergence Test with Soybean ............................................................................... 27 Effects of PFOS Seedling Emergence. Survival. Shoot Weight. and Height in a ;!l-Day Seedling Emergence Test with Tomato.................................................................................. 28 Observed NOEC and Calculated ECx Estimates for PFOS on Seedling Emergence. Survival. Shoot Weight. and Height in a 21-Day Seedling Emergence Test ........................ 29 APPENDICES Appendix 1 Personnel Involved in the Study ................................................................................ 30 Appendix 2 Changes to the Protocol ............................................................................................. 31 Appendix 3 Certificate of Analysis ............................................................................................... 32 Appendix 4 The Analysis of PFOS In Soil and Seven Species of Plants In Support of Wildlife International.Ltd.ProjectNo.. 454-110................................................................... 35 Appendix 5 EnvironmentalConditions......................................................................................... 77 Appendix 6 Test Results. Alfalfa................................................................................................... 83 Appendix 7 Test Results. Flax....................................................................................................... 90 -6- PROJECT .NO. 454-110 Appendix 8 Appendix 9 Appendix 10 Appendix 11 Appendix 12 Appendix 13 Appendix 14 TABLE OF CONTENTS (continued) Test Results. Lettuce.................................................................................................. 97 Test Results. Onion.................................................................................................. 104 Test Results. Ryegrass ............................................................................................. 111 Test Results. Soybean .............................................................................................. 118 Test Results. Tomato ............................................................................................... 125 Bulk Soil Characterization....................................................................................... 132 Results of Water and Soil Pesticide Screening........................................................ 133 -7- PROJECT NO.: 454-110 SUMMARY WILDLIFE INTERNATIONAL,LTD. PROJECT NO: 454-110 TEST SUBSTANCE: Perflourooctanesulfonate,Potassium Salt (PFOS) STUDY TITLE: PFOS: A Toxicity Test to Determine the Effects of the Test Substance on Seedling Emergence of Seven Species of Plants GUIDELINES: OECD Guideline for Testing of Chemicals, Proposal for Revision of Guideline 208: Terrestrial Non-Target Plant Tests OPPTS 850.4100 (Public Draft) OPPTS 850.4225 (Public Draft) NOMINAL TEST LEVELS: 0 (Control), 3.91, 15.6,62.5,250, 1000 mg a.i./kg dry soil TEST DATES: STUDY INITIATION: Experimental Start (OECD): Experimental Start (EPA): Experimental Termination: STUDY COMPLETION: November 12,2001 November 12,2001 November 12,2001 August 28, 2002 May 30,2003 LENGTH OF TEST: Emergence Portion: 2 1 days Extended Growth Portion: up to 205 days (varies by species) TEST SPECIES: Alfalfa (Medicago sativa), Flax (Linum usitatissimum), Lettuce (Lactuca sativa), Onion (Allium cepa),Ryegrass (Loliumperenne), Soybean (Glycine max), Tomato (Lycopersicon esculentum) RESULTS: Species Common name (Latin name) Family Relative Sensitivity ECZ5(mg a.i./kg) Endpoint Lettuce (Lactuca sativa) Ryegrass (Loliurnperenne) Tomato (Lycopersicon esculenturn) Onion (Allium cepa) Alfalfa (Medicago sativa) Flax (Linum usitatissimurn) Soybean (Glycine man) 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 \Yeight 160 Shoot Weight -8- PROJECT NO.: 454-110 INTRODUCTION This seedling emergence study was conducted for 3M Corporation at the Wildlife International, Ltd. greenhouse facility in Easton, Maryland. The in-life portion of the test was conducted from November 12, 2001 to June 5, 2002. Raw data generated at Wildlife International, Ltd., the study protocol, and a copy of the final report were filed in the archives located on the Wildlife International, Ltd. site. Key personnel involved in the study are listed in Appendix 1. PURPOSE The purpose of the study was to determine the effects of Perflourooctanesulfonate,Potassium Salt (PFOS) on the seedling emergence and growth of seven species of plants. EXPERIMENTAL DESIGN There were two parts to this study. The first part was a twenty-one day seedling emergence test. The second part, an extended growth period, followed immediately. The experimental design for the overall study consisted of a negative control and five treatment groups, with fbur 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 non- GLP rangefinding test, and are not necessarily indicative of expected environmental concentrations. A control group, which received no test substance incorporation, was maintained 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 assigpment of plant condition scores were made. Fresh weights were conducted on all replicates containing more than one living seedling. Where possible, one seedling from each replicate was then left to grow until plants produced fruit (i.e., the extended growth period), whereupon, plants and fruit were clipped and weighed independently. -9- PROJECT NO.: 454-110 MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A, Toxicity Test to Determine the Effects of the Test Substance on Seedling Emergence of Seven Species of Plants." Changes to the approved protocol are listed in Appendix 2. The methods used in conducting this study were based upon procedures specified in the OECD Proposal for Rtwision 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 3 1,2006. 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. Approximatelly 1000 g was removed from a measured 70 kg of bulk soil and mixed with weighed test substance for each test concentration. The remaining 69 kg of bulk soil was placed into soil mixer, and the 1 kg of`soil with test substance was added. The constituents were then mixed for ten minutes in order to prepare the test soil for each treatment group. Soils were mixed from lowest to highest concentration to avoid cross-contamination. The negative control soil was prepared prior to the treatment groups, -butin the same manner, except no test substance was added. At the completion of mixing, the test s80ilswere sampled to provide material for analytical confirmation of the test concentrations. Analytical samples were stored at ambient room conditions for up to four days after their collection until they were processed for analysis. Test Species The common and scientific names for the seven species tested, the seed source, and their approximate planting depths are listed below: - 10 - PROJECT NO.: 454-110 Test Species I Variety: Alfalfa (Medicago sativa) I None Given Flax (Linum usitatissimum) /None Given Lettuce (Lactuca sativa) I Summertime Onion (Allium cepa) I Texas Grano Ryegrass (Loliumperenne)l Manhattan 3 Soybean (Glycine mar) I Green Envy Tomato (Lycopersiconesculenturn)I Rutgers Seed Source: Frontier Natural Products, Norway, IA ,USA Arrowhead Mills, Hereford, TX, USA Territorial Seed Co., Cottage Grove, OR, USA Territorial Seed Co., Cottage Grove, OR, USA Meyer Seed Co., Baltimore, MD, USA Johnny's Selected Seeds, Albion, ME, USA Meyer Seed Co.,Baltimore, MD, USA -Planting Depth 6mm 6 mm 6mm 6mm 6mm 20 mm 6mm 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 2!1% 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 arid leave ten uniform holes for planting. One indiscriminatelyselected seed was then planted in each hole, for a total of ten seeds in each pot. Personnel then closed the holes by slightly depressing the soil surface with their fingers. - 11 - PROJECT NO.: 454-110 Watering of Seedlings Water lost through transpiration and evaporation was replaced by subirrigation with well water from the greenhouse facility. Seedlings were subirrigated as needed during the test to minimize the potential for the leaching of the test substance through the soil. Subirrigation trays were iilled 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 (14ppendix 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 o:r absence of possible signs of phytotoxicity such as necrosis, leaf wrinkle, chlorosis, plant lodging or plant stunting. Each emerged seedling was then assigned a numerical score (see Table 1) that described the plant condition (4). Condition score is a subjective or qualitative assessment that determines whether damage is slight, moderate, or severe. A score of 10 does not mean that 10% of the plant is showing the effect (e.g. chlorosis), merely that the severity of the effect (e.g. chlorosis) is very slight. -12- PROJECT NO.: 454-110 On day 21, seedling height was measured to the nearest whole centimeter from the surfiice of the soil to the tip of the tallest leaf (alfalfa, flax, lettuce, onion, and ryegrass) or to the apical meristem (soybean and tomato). All living seedlings except one per replicate (when available) were then clipped at soil level, combined, and weighed within 5 minutes of clipping. The total weight of the shoots was divided by the number of seedlings weighed in order to calculate the mean weight per plant. The one plant per replicate, when available, which was not sacrificed was allowed to1 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 arid 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 2 1, 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 - PROJECT NO.: 454-110 from each of three replicate pots. Three additional plant tissue samples were taken from the single plant remaining in each replicate, when available, for each species at each test concentration at test termination of the extended growth period. Plants from the fourth replicate in each group, when present, were used to determine the moisture content of plant tissue at the end of each portion of the test (21-day and extended growth). Samples were stored frozen until analysis. Chemical analysis of the plant tissue collected in this study was performed by Wildlife International, Ltd. (Appendix 4). The test substance was used to prepare calibration standards. Dates of test termination for each species are listed below: Species Alfalfa Flax Lettuce Onion Ryegrass Soybean Tomato Date of Test Termination April 2,2002 February 14,2002 January 18,2002 January 18,2002 June 5,2002 January 18,2002 February 14,2002 Data Analyses Statistical analyses were used to aid in the evaluation of effects of test substance application on seedling emergence, survival, shoot weight, and height. These variables were defined for statistical analysis as follows: Seedling Emergence: The number of emerged seedlings per ten planted seeds in each pot. Survival: The number of emerged seedlings in each pot that were living at test termination per ten planted seeds. Shoot Weight: The mean shoot weight of sacrificed living emerged seedlings in each pot. Height: The average height of living emerged seedlings in each pot. Test data were evaluated to determine the no-observed-effect-concentration(NOEC) and lowestobservable-effect-concentration (LOEC) for condition and growth. The NOEC is defined as the maximum test substance concentration that shows no adverse phytotoxic effects and below which no phytotoxic effects are manifested. The LOEC is defined as the lowest test substance concentration used in the study that shows an adverse effect on a variable of interest. Mean seedling emergence, - 14- PROJECT NO.: 454-110 survival, weight, and height of the control and treatment groups were compared with Dunnett's t-test, using the DUNNETT option of the GLM (general linear model) procedure of SAS version 8 (5). Significance was determined at the level of 0.05 (p<0.05). Dunnett's test was used to aid in establishing the NOEC by determining which treatment groups differed significantly from the control group. Statistical analyses for species also included the determination of effect rates (EC estimates) and their confidence limits using the non-linear regression analysis of Bruce and Versteeg (6) when reductions in test endpoints among one or more treatment groups were 25% or more relative to control means. Analyses were conducted using the NLIN procedure of SAS version 8 (5). EC, values (i.e. EC25and ECSo)were defined as the test substance application rater; that caused an x% change in the treatment group mean emergence, dry weight, or height relative to the control group. EC, estimates were calculated using nominal test concentrations and treatment group mean values with the following equation: I Ro * @[(log(EC,)-log(C))/o +Z,] 0 0 R= { I Ro c=o where R = the predicted biotic response at concentration C RO = the predicted biotic response for controls @[ 1 log(EC.J = the cumulative area under the standard, normal distribution = the logarithm of the predicted ER giving an x percentage of decrease in the biological parameter vs. the control zx = the normal deviate above which x percentage of the area of the standard normal distribution lies. 0 = the standard deviation of the normal distribution Effects on survival were designated as LC, values, and were calculated using the method described above. If the fit of the data to the regression model was poor, or if confidence intervals were not calculated, the EC, estimates were calculated using linear interpolation (7). RESULTS Analytical Chemistry Artificial Soil. Samples of artificial soil collected on Days 0 and 21, and at test termination were submitted and analyzed for PFOS. All negative control artificial soil samples were <:LOQ for PFOS. Day 0 analyses of the 3.91, 15.6, 62.5, 250, and 1000 mg a.i./kg treatment levels `had mean measured values of 3.61, 11.1, 50.8, 276, and 998 mg a.i./kg, corresponding to 92.3%, 71.2?/0,81.3%, 1lo%, and 99.8% of the nominal concentrations, respectively (Appendix 4.19). PFOS measured - 15 - PROJECT NO.: 454-110 values for test termination samples are presented in Appendix 4.20. The 3.91, 15.6, 62.5, 250 and 1000 mg a.i./kg treatment levels had mean measured values of 1.29, 3.56, 16..2, 157 and 474 mg a.i./kg, corresponding to 33.0%, 22.8%, 25.9%, 62.8% and 47.4% of the nominal conce:ntrations, respectively at test termination. A representative ion chromatogram of an artificial soil test sample is presented in Appendix 4.2 1. Dav 2 1 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. -16- PROJECT NO.: 454-110 Nominal Soil Concentration Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) Vegetation Fruit c2.7 4.4 6.2 4.4 4.2 4.4 11 16 None living None living Nominal Soil Concentration Measured PFOS Concentration (mg a.i./kg dry weight) (mg a.i./k& Vegetation Fruit Control <2.4 c0.12 3.91 5.0 0.23 15.6 19 14 3 I 62.5 I 55 I 2~ .6. I ~~ 250 Insufficient sample Insufficient sample 1000 None living None living Nominal Soil Concentration (mn a.i./k& Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) Vegetation Fruit <5.5 Not applicable 8.6 Not applicable 11 Not applicable 42 Not applicable Insufficient sample Not applicable None living Not applicable Nominal Soil Concentration (mg a.i./kg) Control 3.91 1c L 62.5 250 1000 Measured PFOS Concentration (mg a.i.ikg dry weight) Vegetation Fruit <4.7 <4.7 3.1 11 ?? Insufficient sample None living None living Insufficient sample None living None living Ryegrass - Total days of exposure = 205 I - n: -17- PROJECT NO.: 454-110 Soybean - Total days of exposure = 67 Nominal Soil Concentration Measured PFOS Concentration (mg a.i./kg dry weight) in: (me: a.i./ke:) Vegetation Fruit Control <6.0 <0.18 3.91 16 1.4 I 15.6 I 36 I 0.87 I 250 1 in 3.2 I 1000 I None living I None living I Tomato - Total days of exposure = 94 Nominal Soil Concentration (me: a.i.kg) Control 3.91 15.6 62.5 250 1000 Measured PFOS Concentration (mg a.i./kg dry weight) in: Vegetation Fruit <7.6 4.47 <7.6 34 1.1 50 Insufficient sample Insufficient sample None living None livin 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 summarizedby species in Tables 2 through 8. Complete results are presented by species in Appendices 6 through 12. Species Monocots: Onion (Allium cepa) Ryegrass (Lolium perenne) Dicots: Alfalfa (Medicago sativa) Flax (Linum usitatissimum) Lettuce (Lactuca sativa) Soybean (Glycine max) Tomato (Lycopersicon esculentum) LOEC I I I 21-day Emergence NOEC EC25 ECso LOEC I 2 1-day NOEC ISurviva'l E C (All units 3a.i.kg) 250 62.5 50.8 208 62.5 15.6 47.1 250 62.5 203 344 250 62.5 174 2T 57.3 310 1000 250 372 745 250 6i!.5 25 1 452 1000 250 399 599 250 6i1.5 144 226 1000 250 393 564 250 62.5 257 3 86 >loo0 1000 >IO00 >loo0 >loo0 1000 >loo0 >loo0 1000 250 311 474 62.5 15.6 68.7 105 - 18- PROJECT NO.: 454-110 Species Monocots: Onion (Allium cepa) Ryegrass (Loliumperenne) Dicots: Alfalfa (Medicago sativa) Flax (Linum usitatissimum) Lettuce (Lactuca sativa) Soybean (Glycine mar) Tomato (Lycopersicon esculentum) LOEC I I 21-day Height NOEC ECZ5 I ECS0 LOEC I21-da +-CN Shoot Wei ht (All unit: ig a.i./kg) 62.5 15.6 29.1 46.5 15.6 3.91 12.9 28.1 15.6 3.91 46.3 131 15.6 3.91 7.511 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 8.9;! 20.1 250 62.5 284 464 250 62.5 160 326 62.5 15.6 22.1 93.9 62.5 15.6 1 l.:7 28.5 CONCLUSIONS The relative sensitivities and most sensitive endpoints for the seven test species in response to soil- incorporated PFOS based on 21-day results are listed below: Species Common name (Latin name) Family -1 - Relative Sensitivity ECZ5(mg a.i./kg) - Endpoint Lettuce (Lactuca sativa) Ryegrass (Loliumperenne) Tomato (Lycopersicon esculentum) Onion (Allium cepa) Alfalfa (Medicago sativa) Flax (Linum usitatissimum) Soybean (Glycine mar) 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 - There were no additional findings of phytotoxicity at the termination of the extended growth portion of the test. -19- PROJECT NO.: 454-110 REFERENCES 1 OECD Guideline for Testing of Chemicals. 1998. Guideline for Testing of Chemicals, Proposal for Revision of 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 Toxicily, Tier I1 (Seedling Emergence). 4 Frans, Robert E. and Ronald E. Talbert. 1977. Design of Field Experiments and the Measurement and Analysis of Plant Responses. Pages 15-23 in B. Truelove, ed. Research Methods in Weed Science. Southern Weed Science Society, Auburn University, Alabama. 5 SAS Institute, Inc. 1999. SAS Proprietary Software Version 8 Cary, NC, SAS Institute, Inc. 6 Bruce, Robert D. and Donald J. Versteeg. 1992. A Statistical Procedure for Modeling Continuous Toxicity Data. Environmental Toxicology and Chemistty. 11: 1485-1494. 7 Norberg-King, T.J. 1993. A Linear Interpolation Method for 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 Validationfor the Determination of Per-uorooctanesulfonate, Potassium Salt (PFOS) in ArtiJiciaI 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 Validationfor the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues. -20- Table 1 Seedling Condition Rating System PROJECT NO.: 454-1 10 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 doubtfu'l ~~ 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 arid Analysis of Plant Responses. Pages 15-23 in B. Truelove, ed. Research Methods in Weed Science. Southern Weed Science Society, Auburn University, Alabama. -21 - PROJECT NO.: 454-110 Table 2 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 2 1-Day Seedling - Emergence Test with ALFALFA P Q Test Number of Emerged Seedlings Seedling Shoot Weight Height Concentration (mg a.i./kg) Day 7 (Mean f SD) (% Reduction) Day 15 (Mean f SD) Day 21 (Mean f SD) Survival (% Reduction) (Mean f SD) (g)l (% Reduction) (Mean =tSD) (cm) (% Reduction) (Mean f SD) Control 8.75 f 1.50 9.00f 1.15 9.00f 1.15 8.75 f 0.96 0.12 f 0.013 6.5 f 0.64 3.91 8.25 f0.50 8.50 f0.58 8.50 f 0.58 8.25 f 0.50 0.1 1 f 0.033 (6%) (6%) (6%) (6%) (4%) 15.6 7.75 f 1.50 (11%) 8.00 f 1.41 (11%) 8.00 f 1.41 (1 1%) 8.00 f 1.41 (9%) 0.10 f 0.022 (15%) 62.5 7.00f 1.15 8.00 f 1.15 8.00f 1.15 8.00f 1.15 0.10 f 0.010 (20%) (11%) (11%) (9%) (11%) 250 7.00 f 1.41 7.25 f 1.71 7.25 f 1.71 6.25 f 1.71* 0.03 f 0.008* (20%) (19%) (1 9%) (29%) (78%) 1000 2.50 f 2.89* 2.50 f 2.89* 3.25 f 3.30* 1.50f 1.73* 0.02 f 0.008* (71%) (72%) (64%) (83%) (87%) * Treatment group mean is significantlydifferent from the control mean (Dunnett's test,p<0.05). Mean f SD - Treatment group mean plus or minus one standard deviation. 6.4 f 0.75 (2%) 5.4 f 0.76 (1 6%) 6.1 f 0.90 (6%) 2.8 f 0.52* (57%) 1.0 f o.oo* (85%) P -22- PROJECT NO.: 454-1 10 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 Day 7 (Mean f SD) ("hReduction) Day 15 (Mean f SD) Day 21 (Mean f SD) Seedling Survival ("hReduction) (Mean f SD) Shoot Weight (g> (% Reduction) (Mean 2 SD) Height (cm) (ohReduction) (Mean f SD) Control 7.25 f 1.26 7.25 f 1.26 7.25 f 1.26 7.25 f 1.26 0.19 f 0.029 8.9 f 0.27 3.91 6.50 f 1.00 7.00 f 1.41 7.25 f 1.50 6.75 f 1.26 0.18 f 0.022 (1 0%) (3%) (0%) (7%) (8%) 15.6 8.00 f 0.82 (-10%) 8.00 f 0.82 (-10%) 8.00 f 0.82 (-10%) 8.00 f 0.82 (- 10%) 0.18 f 0.023 (1 0%) 62.5 8.50 f 1.00 (-17%) 8.50 f 1.00 (-17%) 8.50 f 1.00 (-17%) 8.50 f 1.00 (-17%) 0.16 f 0.017 (1 8%) 250 7.25 f 1.71 7.25 f 1.71 7.25 f 1.71 3.25 f 1.50* 0.02 f 0.009* (0%) (0%) (0%) (55%) (91%) 1000 O* O* O* O* NIA ( 100%) (1 00%) (1 00%) (1 00%) * 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 f SD - Treatment group mean plus or minus one standard deviation. 8.7 f 0.90 (3%) 8.8 f 0.80 (2%) 8.2 f 0.54 (8%) 1.3 f 0.35* (86%) NIA - 23 - PROJECT NO.: 454-110 Table 4 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling - Emergence Test with LETTUCE Test Number of Emerged Seedlings Seedling Shoot Weight Height Concentration (mg a.i./kg) Day 7 ("YO Reduction) Day 15 Day 21 Survival ("YO Reduction) (9 (% Reduction) (cm) ("YO Reduction) (Mean f SD) (Mean f SD) (Mean f SD) (Mean f SD) (Mean :k SD) (Mean f SD) Control 8.75 f 0.50 8.75 f 0.50 8.75 f 0.50 8.75 f 0.50 0.38 f 0.060 6.4 f 0.5 1 3.91 9.00 f 0.82 (-3%) 9.00 f 0.82 (-3%) 9.00 f 0.82 (-3%) 9.00 f 0.82 (-3%) 0.25 f0.051* (35?6) 5.0 f 0.56* (23%) 15.6 8.75 f 1.50 9.00f 1.15 9.00f 1.15 9.00f 1.15 0.24 f0.081* 5.0 f 1.07* (0%) (-3%) (-3%) (-3%) (36%) (23%) 62.5 8.50 f 1.29 8.50 f 1.29 8.50 f 1.29 8.50 f 1.29 0.05 f 0.019* 2.6 f 0.43* (3%) (3%) (3%) (3%) (88?6) (59%) 250 8.00 f 1.41 8.00 f 1.41 8.25 f 1.26 6.75 f 0.96* 0.01 f 0.006* 1.O f0.06* (9%) (9%) (6%) (23%) (98%) (84%) 1000 1.25 f 1.26* 1.25 f 1.26* 1.25f 1.26* 0.50 f 0.58* 0.00 f o.ooo* 1.0 f o.oo* -- (86%) (86%) (86%) (94%) (100%) * Treatment group mean is significantly different from the control mean (Dunnett's test,p<0.05). Mean f SD - Treatment group mean plus or minus one standard deviation. (84%) - 24 - PROJECT NO.: 454-110 Table 5 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day !3eedling -- - Emergence Test with ONION Test Number of Emerged Seedlings Seedling Shoot Weight Height Concentration (mg a.i./kg) Day 7 (% Reduction) Day 15 Day 21 Survival (% Reduction) (8) (% Reduction) (cm) (% Reduction) (Mean f SD) (Mean f SD) (Mean f SD) (Mean f SD) (Mean f SD) (Mean f SD) Control 8.00 f 0.82 8.50 f 1.29 8.75 f 1.50 8.50 f 1.29 0.10 f 0.006 8.1 f 0.55 3.91 8.5M 1.29 9.00 f 1.41 9.25 f 0.96 9.00f 1.15 0.09 f 0.015 (-6%) (-6%) (-6%) (-6%) (1 5%) 15.6 7.75 f 0.50 (3%) 8.25 f 0.50 (3%) 8.25 f 0.50 (6%) 8.00 f 0.82 (6%) 0.07 f 0.006* (3 11%) 62.5 5.00 f 2.00* (38%) 6.25 f 2.63 (26%) 6.25 f 2.63 (29%) 3.25 f 0.50* (62%) 0.02 f 0.019* (77") 250 2.00 f 2.45* 4.00 f 3.16* 4.00 f 3.16* O* N/A (75%) (53%) (54%) (1 00%) 1000 (10O0*%) (10O0*%) (10O0*%) (10O0*%) N/A * P 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 f SD - Treatment group mean plus or minus one standard deviation. 7.3 f 0.49 (9%) 7.3 f 0.15 (10%) 2.6 f 0.83* (68%) N/A N/A - 25 - PROJECT NO.: 454-110 Table 6 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling - Test Emergence Test with RYEGRASS Number of Emerged Seedlings Seedling -- - Shoot Weight Height Concentration (mg a.i.ikg) Day 7 (Mean f SD) ("hReduction) Day 15 (Mean f SD) Day 21 (Mean f SD) Survival ("hReduction) (Mean f SD) (9) ("7'0Reduction) (Mean :t SD) (cm) (% Reduction) (Mean f SD) Control 7.75 f0.96 8.00 f 0.82 8.00 f 0.82 8.00 f 0.82 0.12 f 0.025 16.9f 0.69 3.91 8.50 f 1.00 9.25 f 0.96 9.25 f 0.96 9.25 f 0.96 0.11 f 0.013 15.4f 1.28 (-10%) (-16%) (-16%) (- 16%) (1 2?G) (9%) 15.6 7.50 f 1.91 9.00 f 0.82 9.00 f 0.82 9.00 f 0.82 0.07 f 0.014* 13.7 f 0.77* (3%) (-13%) (-13%) (-13%) (39%) (1 9%) 62.5 7.50 f 0.58 8.25 f 0.96 8.50 f 1.29 8.25 f 0.96 0.08 f 0.014* 13.6 f 0.92* (3%) (-3%) (-6%) (-3%) (32%) (1 9%) 250 4.00 f 1.41* 5.75 f 1.26* 5.75 f 1.26* 5.25 f 2.22* 0.01 f 0.006* 3.7 f 0.97* (48%) (28%) (28%) (34%) (91%) (78%) 1000 O* 0.25 f 0.50* 0.75 f0.50* 0.75 f0.50* 0.03 f 0.010* 2.0 f o.oo* -- (100%) (97%) (91%) (91%) (76%) * Treatment group mean was significantly different from the control mean (Dunnett's test, p<O.O5) Mean f SD - Treatment group mean plus or minus one standard deviation. (88%) -26 - PROJECT NO.: 454-110 Table 7 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 21-Day Seedling - - Emergence Test with SOYBEAN Test Number of Emerged Seedlings Seedling Shoot Weight Height Concentration (mg a.i./kg) Day 7 (Mean f SD) (% Reduction) Day 15 (Mean f SD) Day 21 (Mean f SD) Survival (% Reduction) (Mean f SD) (g:) (% Reduction) (Mean :k SD) (4 I(% Reduction) (Mean f SD) Control 9.75 f 0.50 1o.ow0.00 10.00 f 0.00 10.00 f 0.00 3.63 f 0.583 28.3 f 4.17 3.91 9.75 f 0.50 (0%) 9.75 f 0.50 (3%) 9.75 f 0.50 (3%) 9.75 f 0.50 (3%) 3.64 f 0.236 (0%) 26.8 f 3.17 (5%) 15.6 9.50 f 0.58 (3%) 9.50 f 0.58 (5%) 9.50 f 0.58 (5%) 9.50 f 0.58 (5%) 3.63 f 0.330 (0%) 26.9 f 3.31 (5%) 62.5 10.00 f 0.00 10.00 f 0.00 10.00 f 0.00 10.00 f 0.00 4.00 f 0.302 (-3%) (0%) (0%) (0%) (- 10%) 30.7 f 2.51 (-8%) 250 10.00 f 0.00 10.00 f 0.00 10.00 f 0.00 9.75 f 0.50 2.07 f 0.3 10* 22.5 f 2.26* (-3%) (0%) (0%) (3%) (43?/0) (2 1 %) 1000 ___p_ 9.75 f 0.50 10.00 f 0.00 10.00 f 0.00 10.00 f 0.00 0.57 f 0.047* 4.1 f 0.39* - (0%) (0%) (0%) (0%) (84%) * Treatment group mean is significantlydifferent from the control mean (Dunnett's test,~ ~ 0 . 0 5 ) . Mean f SD - Treatment group mean plus or minus one standard deviation. - (85%) -27 - PROJECT NO.: 454-110 Table 8 Effects of PFOS on Seedling Emergence, Survival, Shoot Weight, and Height in a 2 1-Day Seedling Emergence Test with TOMATO Test Concentration (mg a.i.kg) Number of Emerged Seedlings Day 7 (% Reduction) Day 15 Day 21 (Mean f SD) (Mean f SD) (Mean f SD) P Seedling Shoot Weight Survival ("! Reduction) (g) (% Reduction) (Mean f SD) (Mean SD) Height (cm) (% Reduction) (Mean f SD) Control 6.50 f 1.91 9.OM 0.82 9.25 f 0.96 9.25 f 0.96 0.35 f 0.143 5.2 f 0.75 3.91 6.50 f 1.73 (0%) 8.00f 1.15 (1 1%) 8.25 f 1.50 (11%) 8.25 f 1.50 (11%) 0.40 f 0.081 (-14%) 5.6 f0.31 (-7%) 15.6 5.25 f 2.99 (1 9%) 8.25 f 1.71 (8%) 8.25 f 1.71 (11%) 8.00 f 1.41 (14%) 0.28 f 0.108 ( 19%) 4.8 f 0.94 (9%) 62.5 5.50 f 1.91 8.25 f 1.71 8.50 f 1.73 6.75 f 0.96* 0.08 f 0.029* 2.6 f 0.14* (15%) (8%) (8%) (27%) (79%) (50%) 250 1.25 f 1.50* 7.25 f 0.96 7.25 f 0.96 0.75 f 0.96* 0.03 f N/A* (81%) (19%) (22%) (92%) (91%) 2.0 f o.oo* (62%) - --- 1000 (10O0*%) 0.75(9f2%1.)50* 1.O(O8f9%1.)41* (10O0*%) Nl.4 NIA * Treatment group mean is significantly different from the control mean (Dunnett's test, p<0.05)1. NIA - Not applicable since there were no surviving plants at measurement. Mean f SD - Treatment group mean plus or minus one standard deviation. - 28 - 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 Endpoint (NOEC) (mg aikg) Estimate Lower upper 95%CL 95%CL (mg aikg) Alfalfa Emergence ECzs 372 202 (250) EC50 745 541 688 >1000 Survival E 2 5 251 178 353 (62.5) E 5 0 452 364 563 Height ECz5 102 26.8 391 (62.5) ECso 249 106 584 Weight ECz5 53.3 4.12 690 (62.5) ECjo 146 27.3 783 Flax Emergence EC25 399 126 461 (250) ECso 599 402 64 1 Survival Lc25 144 103 177 (62.5) E 5 0 226 160 368 Height ECz5 97.6 81.3 117 (62.5) EC5o 140 125 158 Weight ECzs 81.6 46.0 145 (62.5) EC5o 119 79.4 178 Lettuce Emergence ECZS 393 3 00 515 (250) ECso 564 474 67 1 Survival Lc25 257 220 301 (62.5) Lcso 386 344 433 Height EC25 6.79 <3.91 226 (0.0) EC5o 39.9 3.88 410 Weight ECz5 8.92 <3.91 58.3 (0.0) ECso 20.1 5.26 77.1 Onion Emergence EC25 50.8 12.2 195 (62.5) ECso 208 <3.91 644 Survival (15.6) E 2 5 47.1 57.3 <3.91 17.5 >loo0 188 Height EC25 29.1 <3.91 904 (15.6) ECso 46.5 8.94 242 Weight (3.91) Ec25 12.9 ECso 28.1 <3.91 1.99 >loo0 396 ' - Confidence intervals could not be determined. Species Endpoint WEC) (mg aikg) Ryegrass Emergence (62.5) Survival (62.5) Height (3.91) Weight (3.91) Soybean Emergence ( 1000) Survival Tomato Height (62.5) Weight (62.5) Emergence (250) Survival (15.6) Height (15.6) Weight (15.6) Estimate Lower Upper 95%CL 95%CL (mg iaikg) Ec25 203 131 313 ECso 344 254 465 Lc25 174 107 281 LCso 310 223 432 ECzs 46.3 4.63 464 ECso 131 28.8 597 EC25 7.51 ECso 53.8 ECz5 >lo00 ECso >lo00 LCzs >IO00 LCso >IO00 <3.91 <3.91 -' - - >1000 >1000 ECzs 284 172 468 ECso 464 333 648 ECzs 160 69.1 372 ECso 326 189 561 ECz5 311 208 464 EC50 474 3 60 625 E 2 5 68.7 45.0 105 Lcso 105 77.8 143 EC25 22.1 EC50 93.9 <3.91 10.3 > 1000 852 ECzs 11.7 <3.91 217 ECso 28.5 <3.91 213 - 29 - PROJECT NO.: 454-110 Appendix 1 Personnel Involved In the Study The following key personnel were involved in the conduct or management of this study: Henry 0.Krueger, Ph.D., Director of Aquatic Toxicology/TerrestrialPlants and Insects John R. Porch, Supervisor of Terrestrial Plant and Insect Studies Andrew J. Brignole, Biologist Raymond L. Van Hoven, Ph.D., Scientist Willard B. Nixon, Ph.D., Director of Chemistry - 30 - 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 proceduresregarding day 21 data m d 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, industrializedquartz sand, and peat mixed in a 2:25: 1 ratio with regards to weight. The soil consisted of 49% sand, 30% silt, and 2 1% clay. 4 Observations of emergence were made on Day 15 rather than 14. 5 The sponsor's representative was changed. -31 - PROJECT NO.: 454- 110 Appendix 3 Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS, Lot 217 Reference #: SD-018 Result 86.9% I Elemental Analysis6: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulhr 5 . Fluorine COA023-018A Theoretical Value = 5.95% Theoretical Value = 60% Positive 1. 0.005 wt./wt.% 2. 0.001 wt./wt.% 3. 1.439 wt./wt.% 4. 6.849 wt./wt.% 1 5 . <0.001 wt./wt.% 6. 0.005 wt./wt.% 7. <0.001 wt./wt.% ~ 1.93 wt./wt.% ~ 8.41 wt./wt.% , I None Detected 0.33 wt./wt.% i None Detected Not Applicable' 1. <0.015 wt./wt.% 2. 0.59 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5 . <0.006wt./wt.% 6 . c0.007wt./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.% Page 1 of 3 - 32 - PROJECT NO.: 454-110 Appendix 3 (continued) Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/3 1/00 Expiration Date: 08/31/01 Storage Conditions: Frozen 5-10"C Re-assessment Date: 08/3 1/01 IPurity = 100%- (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0,59%+NMR impurities, 1.93%+0rganicacid 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. 3Purityby DSC is generally not applicable to materials of low punty. No endotherm was observed for this sample. 4Sulhrin the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulhr determination in the elemental analysis, lending confidenceto this interpretation. Based on the results, the so4 is not considered an impurity. 'TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoreticalvalue calculationsbased on the empirical formula, CgF17SO<K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-0 18A - 33 - Page 2 of3 PROJECT NO.: 454-110 Appendix 3 (continued) Certificate of Analysis Centre Analytical laboratories, Inc 3048 Research Drive State Colkga, PA 16801 www.cenlrehb.com Phone: (814) 231-8032 Fax:(814) 231-1253or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference # 023-018A LClMS Purity Profile: Impurlty d w t . Yo I CS I 1.33 I C6 4.72 ~ c7 1.14 Total 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the CA and C6 standard curves. Likewise,the C7 value was calculatedusing the averageresult h m the C6 and CS standard curves. Prepared By: Scientist, Centre Analytical Laboratories &/o I Date 0hmM ReviewedBy: ,J6hnFlaherty / Date LaboratoryManager,CentreAnalytical Laboratories COA023-01SA Page 3 of 3 - 34 - PROJECT NO.: 454-110 Appendix 4 The Analysis of PFOS In Soil and Seven Species of Plants In support of Wildlife International,Ltd. Project No.: 454-110 - 35 - PROJElCT NO.: 454-1 10 INTRODUCTION Soil and plant tissue (vegetative and fruit) samples were collected from a plant toxicity study designed to determine the effects of Perfluorooctanesulfonate, Potassium Salt (PFOS) to seven species of plants. This study was conducted by Wildlife International, Ltd. for 3M Corporation and identified as Project Number 454-110. The chemistry phase of this study was conducted at the Wildlife International, Ltd. analytical chemistry facility in Easton, Maryland using high performance liquid chromatography with triple quadrupole mass spectrometric detection (HPLC,MS/MS). Samples were prepared and analyzed between November 15,2001 and August 28,2002. MATERIALS AND METHODS Test Substance The test substance used for this study was Wildlife International, Ltd. identification number 4675. The test substance was used to prepare calibration and matrix fortification samples. Analytical Methodolonv Artijicial 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 of each sample was transferred to a separate vial or tube and centrifuged for approximately 5 minutes at 2000 rpm. Dilutions into the calibration range of the HPLC/MS/MS methodology were performed with a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure@water. Samples were then an(a1yzedby 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 Spectrome:ter equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieve,d using a -36- PROJElCT NO.: 454-1 10 Keystone Betasil CIScolumn (50 mm x 2.0 mm, 3 pm particle size) fitted with a Keystoine Javelin CISguard 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 stu4y 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 20.5 g of tissue, the replicate was individually analyzed. If two or more replicates contained <0.5 g, the replicates were composited. Compositedreplicates were analyzed when the composite weight was,20.5 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 `werethen 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 an,alyzedby 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 PFQS are provided in Appendix 4.4. The primary and secondary stocks were used for preparation of calibration standards and matrix fortification samples. - 37 - PROJBCT NO.: 454-110 Calibration Standards and Calibration Curves Calibration standards of PFOS, prepared in 5050 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 F'FOS was utilized to monitor PFOS in all calibration and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using peak area responses versus the respective concentrations of the calibration standards. Typical calibration curves for PFOS determinations in artificial soil and plant tissues are presented in Appendices 4.5 and 4.6, respectively. Representative ion chromatograms of low- and high-level PFOS calibration standards are presented in Appendices 4.7 and 4.8. The concentrations of PFOS in the samples were determined by substituting peak area responses into the applicable linear regression equation. Concentrations were calculated on a dry weight basis using moisture determination data submitted with the samples. An example of the calculations for a representative artificial soil sample is included in Appendix 4.9. Limits of Quantitation Artijicial Soil The method limit of quantitation (LOQ) in artificial soil was 1.18 mg a.i./kg on a dryweight basis and was calculated as the product of the lowest calibration standard (1.OO 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 h i t 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 weightkolume dilution factor of the matrix blank samples, divided by the percent solids determined for the fruit species negative control samples. The presence of - 38 - PROJECT NO.: 454-1 10 contamination andor interferent matrix species in alfalfa h i t tissue necessitated a unique LOQ definition relative to the other plant h i t species. For alfalfa fruit, the LOQ was calculiated on a dry-weight basis as the maximum mg PFOSkg equivalent concentration obstmed in the negative control extract rounded upward to the nearest tenth mg a.i./kg value (see Limits of QuantitationPlant Tissue- Vegetative). Plant Tissue - Vegetative Method LOQs in plant vegetative tissues were reported on a dry weight basis and were calculated as the maximum mg PFOS/kg equivalent concentration observed in the negative control extracts for each plant vegetative tissue species, rounded upward to the nearest tenth mg a.i./kg value. As was the case for alfalfa h i t , 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 ma&nitudeof 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 Artijkial 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 homogenized with blending for approximately two minutes. A 10-g aliquot of each homogenized 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 ('4ppendix -39- PROJECT NO.: 454-110 4.10). Sample measured concentrations were not corrected for the mean procedural recoveries. A representative ion chromatogram of a matrix fortification sample in artificial soil is presented in Appendix 4.12. Plant Tissues Selected aliquots (approximately 1.OO g) of negative control plant tissues were fortified with appropriate stock solutions of PFOS prepared in methanol using a gas-tight syringe.. A matrix blank was prepared with unfortified negative control plant tissue. Fruit and vegetative plant tissue samples were fortified at target nominal concentrations of 0.050, 0.50, and 50.0 mg a.i./kg (wet weight basis) and analyzed concurrently with the samples to determine mean procedural recoveries. In fruit tissue, the mean procedural recoveriles 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 chromalograms 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 <LO() 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%, 1'10%, 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 rng a.i./kg treatment levels had mean measured values of 1.29,3.56, 16.2, 157 and 474 mg a.i./kg, corresponding to 33.0%, 22.8%, 25.9%, 62.8% and 47.4% of the nominal concentrations, respectively. A representative ion chromatogram of an artificial soil test sample is presented in Appendix 4.2:l. -40 - PROJElCT NO.: 454-1 10 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 terminat-ion(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 h i t and vegetative tissue test samples are presented in Appendices 4.34 and 4.35, respectively. -41 - PROJECT NO.: 454-110 Appendix 4.1 Analytical Method Flowchart for the Processing of PFOS in Soil Prepare QC samples in soil matrix from the appropriate methanol stocks. Weigh approximately 20.0 g of soil matrix for each QC into a tared weigh boat. Record weights. Transfer to 8-oz. French square glass bottles. Fortify each soil sample with the appropriate volume of PFOS stock solution in methanol with a gas-tight syringe. Allow to air dry. The matrix blank is unfortified soil matrix. 1 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. 1 Weigh approximately 10.0 grams of each study sample into a tared weigh boat. Record weights. Transfer to 8 oz. French square bottles. 1 For each sample, measure 100 mL of methanol with a graduated cylinder and transfer into the French square bottle. 1 Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. .1 Vacuum filter with qualitative filter paper and rinse retained soil 3 times with methanol into filtrate. -1 Transfer the filtrate into a 200-mL, volumetric flask and bring to volume with methanol. Mix well with several repeat inversions. 1 Transfer approximately20 mL of each sample into a separate glass centrifuge tube or sointillation vial and cap. Centrifuge samples for approximately 5 minutes at 2000 rpm. 3. 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. 1 Ampulate and submit sample for HPLC/MS/MS analysis. -42 - PROJECT NO.: 454-110 Appendix 4.2 -INSTRUMENT: Typical HPLC/MS/MS Operational Parameters -- Hewlett-Packard Model 1 100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass SpectrometerOperated in multiple ion reaction monitoring (MRM) mode. ION SOURCE: Perkin-Elmer TurboIonSpray ANALYTICAL COLUMN: Keystone Betasil CI8(50 mm x 2.0 mm, 3-pm particle size) GUARD COLUMN: Keystone Javelin CIScartridge (20 mm x 2 nxn) OVEN TEMPERATURE: 40C STOP TIME: 5.00 min FLOW RATE: 250 pL/min MOBILE PHASE: 30% NANOpure@Water with 0.1% Formic ,4cid: 70% Methanol INJECTION VOLUME: 5.0 pL PFOS PEAK RETENTION TIME: PFOS MONITORED MASS: Approximately 4 minutes 499.0 amu + 99.1 amu -43 - PROJECT NO.: 454-1 10 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 appropriatenumber of 1-g aliquots of each homoge:nateinto a tared vial. 1 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. .1 For each sample, measure 10.0 mL of methanol with a glass Class A volumetric pipette. Cap the vials and shake manually for a minimum of one minute. 1 Place samples on gyratory shaker table and shake at a setting of 250 rpm for approximatelythirty minutes. .1 Centrifuge the vials at approximately 2000 rpm for approximately 10 minutes. Prepare appropriate dilutions of study and QC samples to bring concentrations with:in the calibration range of the PFOS LCMS methodology: Partially fill Class A volumetric flasks with 50%: 50% methanol/ NANOpure@water dilution solvent. Add appropriate volume of sample with a gas-tight syringe and bring to volume with dilution solvent. Process matrix 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. .1 Transfer an aliquot of each sample to an autosampler vial and submit for HPI,C/MS/MS analysis. -44- PROJE'CTNO.: 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-niL final vollume 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 lug a.i./uL) 0.00 100 0.00100 0.00100 0.00100 0.00100 Calibration Standards for Determination of PFOS in Soil Aliquot 0 100 250 500 750 1000 Final m Volume 100 100 100 100 100 Standard Concentration lug a.i./L) 1.oo 2.50 5 .OO 7.50 10.0 Stock Concentration lug a.i./uLI 0.00100 0.00100 0.00100 0.00100 0.00100 0.00100 Calibration Standards for Determination of PFOS in Plant Tissues Aliquot 0 40.0 50.0 100 250 350 500 Final Volume 0 100 100 100 100 100 100 Standard Concentration tug a.i./L) 0.4QO 0.500 1.oo 2.50 3.50 5 .OO -45 - PROJECT NO.: 454-110 Appendix 4.5 Typical Calibration Curve for PFOS Determinations in Artificial Soil FKS 499.0->99.1 No Internal Standard Weighted (llx) Intercept = 1818.5138 Slope = 10974956.0000 Correlation Coeff. = 0.99798 Area -46 - PROJECT NO.: 454-110 Appendix 4.6 Typical Calibration Curve for PFOS Determinations in Plant Tissues rn 499 Weighted b 9 9 . 1 No Internal Standard I IX) Intercept = 27.4030 Slope = 3429.6262 Correlation Coeff. = 0.99864 Area Conc. ug a.i.R -47 - PROJECT NO.: 454-110 Appendix 4.7 Representative Chromatogram of a Low-Level PFOS Calibration Standard PFOS-1 STD 0.400 ug s.ilL 4675A-011 D-26 4.98 in 1 period Ros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mull. Width 10 Base. Width 40 RT Win. (secs) 1 0 Smooth 2 ExpectedRT 4.05 Area 1363 Height 139 Start Time 3.57 End Time 4.02 Integration Width 0.45 Retention Time InteOralion Type -3.80 A BB Sun, Jan 27, 2002 0723 1 Ob 908 07060 50. 40302e 1 intensity: 1700 cps 41 81 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Nominal concentration: 0.400 pg a.i./L. -48 - PROJECT NO.: 454-110 Appendix 4.8 Representative Chromatogramof a High-Level PFOS Calibration Standard PFOS-5 STD 0.0100 mg a.l./L 4675A-011 D-20 4.98 in 1 period Fls No Internal Standard Use Area 1: 4.97 MRM, 296 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. 1 .O Min. Width 3 Mult. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 ExpectedRT 4.01 Area 109807 Height 11092 Start Time 3.69 End Tim 4.36 Integration Width 0.67 Retention Time Integration Type -3.95 A VB Mon. Nov 19, 2001 1241 1 oc- 90. 8070. 60 50. 403020. 10. intensity: 10000 c:ps 2Q6 Nominal concentration: 10.0 pg a.i./L. -49- PROJECT NO.: 454-110 Appendix 4.9 Example Calculations for a Representative Sample The analytical result and percent recovery of PFOS in artificial soil for sample number 454-110-10, nominal concentration of 62.5 mg a.i./kg, was calculated using the following equations: c = -P A - b m where PA = peakarea m = slopeoftheline C = concentration b = y - axisintercept Using the appropriate regression data from the sample analysis sequence (Appendix 4.5), the concentration in the final sample solution was calculated as: 28656 - 1818.5138 = 10974956 = 0.002445 mg a.i./L The measured concentrations of PFOS in the artificial soil sample determined as follows: ConcentrationPFOS (mg C) x (V,) x (vfl) x (V, = (Wi (Vi,) (Vi2)x.(%Soids) where C = Concentration (mg a.i./L) as determined a.bove V, = Extraction Volume (100 mL) Vil = First Initial Volume (1 00 mL) Vi2 = Second Initial Volume (0.0500 mL) Vfl = First Final Volume (200 mL) V a = Second Final Volume (50.0 rnL) W = Weight of extracted sample (10.0 g) ConcentrationPFOS (mg a.i./kg) = 57.60 Percent of Nominal Concentration= PpFFOoSS((mm\ aa..i*iK./:K) innomsain:ml le x 100 ---5672.6.50 x 100= 92.2% Calculated with HPLCMSMS instrument software: MacQuan, version 1.6. -50- PROJECT NO.: 454-110 Appendix 4.10 Artificial Soil Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Number Sample Sampling PFOS Concentration (mg a.i./kg) Percent Mean Measured Mean Percent MAB-1 MAB - 3 Soil Matrix Blank Soil Matrix Blank Initiation 0.0 < 1.1g2 -- Termination 0.0 < 1.18 -- < 1.18 -- MAS- 1 Soil Matrix Fortification Initiation 2.36 2.24 95.1 2.12 89.8 MAS-7 Soil Matrix Fortification Termination 2.36 1.99 84.4 MAS - 2 Soil Matrix Fortification Initiation 118 108 91.3 106 89.8 MAS - 8 Soil Matrix Fortification Termination 118 104 88.1 MAS-3 Soil Matrix Fortification Initiation 1410 1270 90.0 1 ;!90 91.5 MAS-9 Soil Matrix Fortification Termination 1410 1310 93.1 P P Overall Mean = 90.3 - Standard Deviation = 3.78 cv = 4.19 N= 6 __.- 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2The method limit of quantitation (LOQ) in artificial soil was 1.18 mg a.i./kg on a dry-weight basis and was ca1culate:d as the product of the lowest calibration standard (0.00100mg a.i./L) and the weightholume dilution factor of the matrix blank samples (1000)divided by -= P -51 - PROJECT NO.: 454-110 Appendix 4.11 Representative Chromatogram of an Artificial Soil Matrix Blank Sample PFOS-7 454-1 10- MAB-1 Mon. Nov 19, 2001 12:53 4.98 in 1 period PFOS No internal Standard Use Area 1: 4.97 MRM,296 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. 1 .O Min. Width 3 Mull. Width 6 Base. Width 40 RTWin. (sacs) 20 Smooth 1 ExpectedRT 4.01 Area 0 Heighl 0 Start Time 0.00 End Time 0.00 integration Width 0.00 Retention Time 0.00 Integration Type 10090 80 70 60 50 40 30 20 ih 1" 0'12' inlensily: 10000 cp!; 1 50 75 96 121140 169 202 240 273 4'1 ' 8'1 ' 1 5 1 ' 181 ' 261 ' 2 i l ' 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample Identification:454-1IO-MAB-1. The arrow indicates the retention time of PFOS. - 52 - PROJECT NO.: 454-110 Appendix 4.12 Representative Chromatogram of an Artificial Soil Matrix Fortification Sample PF0s-g 454-1 10- MAS-2 Mon, NOV 19, 2001 13:05 4.98 in 1 period Fms No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. 1.o Min. Width 3 Mult. Wldth 6 Base. Width 40 RT Win. (sea) 2 0 Smooth 1 ExpeaedRT 4.01 h a 51907 Height 5360 start Time 3.65 End Time Integration Width Retention Time Integration Type 4.32 0.67 3.97 A - VE '09 9 8 7 6 5 4 3 2 1 intensity: 10000 cps 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample Identification: 454-110-MAS-2. Nominal Concentration: 118 mg a.i./kg (dry-weight basis). - 53 - PROJECT NO.: 454-110 Appendix 4.13 Fruit Tissue Matrix Blanks and FortificationsAnalyzed ConcurrentlyDuring Sample Analysis Number Sample Sampling Concentration (mg a.i.kg) Measured',' Wet-Weight Dry-WeigF Percent Basis __p Recovered' alf-f-MAB-l Alfalfa Matrix Blank Termination 0.0 alf-f-MAS-3 Alfalfa Fortification Termination 50.0 3.0g4 8.434 -- 50.4 138 101 flx-f-MAB-1 flx-f-MAS-1 flx-f-MAS-2 flx-f-MAS-3 Flax Matrix Blank Flax Fortification Flax Fortification Flax Fortification Termination Termination Termination Termination 0.0 0.0495 0.503 47.9 < 0.040 < 0.12 -- 0.0581 0.170 1 I7 0.521 1.52 104 44.4 130 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-l soy-f-MAS-2 soy-f-MAS3 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 < 0.42 -- 0.0477 0.494 97.3 0.45 1 4.68 96.6 51.4 533 107 < 0.040 < 0.18 -- 0.0508 0.222 102 0.478 2.09 96.4 47.9 209 95.7 tom-f-MAB-l tom-f-MAS- 1 tom-f-MAS-2 tom-f-MAS3 Tomato Matrix Blank Tomato Fortification Tomato Fortification Tomato Fortification Termination Termination Termination Termination 0.0 0.0498 0.495 47.8 < 0.040 < 0.47 -- 0.0468 0.554 93.9 0.427 5.05 86.3 42.3 501 88.6 P P 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 'The method limit of quantitation (LOQ) in fruit tissue was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weightholume dilution factor of the matrix blank sample. 'Fortified PFOS concentrationsare presented on a wet-weight basis. 4Themethod limit of quantitation (LOQ) in alfalfa h i t tissue (6.4 mg a.i./kg on a dry-weight basis = 2.3 mg a.i./kg on EL wet-weight basis) was calculated as the maximum mg PFOSkg equivalent concentration measured in negative control sample extracts rounded upward to the nearest tenth mg a.i.k =__ P - 54 - PROJECT NO.: 454-110 Appendix 4.14 Representative Chromatogram of a Plant Fruit Tissue Matrix Blank Sample PFOS-8 454-110- SOY-F-MAE-1 4.98 in 1 period PKE No Internal Standard Use Area 1: 4.97 MRM. 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mult. Wldth 50 Base. Width 80 RlWin.(secs) 20 Smooth 2 Rl 3.75 Area 0 Height 0 Start lime 0.00 End Time 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type Tue, Mar 26, 2002 14:06 O01 90 80. 70 60. 50. 4030. 20. 10 5 1 81 04.- ~ 133155 intensity: 4000 cps T 265 Sample Identification:454-1IO-SOY-F-MAB-1 (soybean matrix). The arrow indicates the retention time of PFOS. - 55 - PROJE'CTNO.: 454-110 Appendix 4.15 Representative Chromatogram of a Plant Fruit Tissue Matrix Fortification Sample 4.98 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM. 290 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Wath 12 Mull. Width 10 Base. Width 80 RT Win. (secs) 20 Smooth 2 ExpectedRT 3.75 Area 16586 Height 1396 Start Time 3.55 End Time 4.42 Integration Width Retention Time Integration Type 0.87 A -3.77 VB intensity: 4000 cps 10 44 225 3 2 1 0.69 1.36 2.03 2.70 3.37 4.04 4.71 T i m Sample Identification:454-110-SOY-F-MAS-3(soybean matrix). Nominal Concentration: 50.0 mg a.i./kg (wet-weight basis). - 56 - PROJECT NO.: 454-110 Appendix 4.16 Vegetative Tissue Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Number (454-110-) alf-v-MAB-1 alf-v-MAS-3 Sample T11p e Alfalfa Matrix Blank Alfalfa Fortification Concentration (mg a.i./kg) - Sampling Interval Fortified' P Termination 0.0 Termination 48.1 Measured',' --- Wet-Weight Dry-WeigF Basis Basis < 0.84 < 2.7 44.2 142 Percent Recovered' -- 91.9 flx-v-MAB-1 flx-V-MAS-3 Flax Matrix Blank Flax Fortification Termination 0.0 Termination 48.8 c 0.72 < 2.4 -- 47.5 158 97.3 let-v-MAB- 1 let-v-MAS-2 let-v-MAS-3 Lettuce Matrix Blank Lettuce Fortification Lettuce Fortification Termination Termination Termination 0.0 0.502 50.4 < 0.46 < 5.5 -- 0.536 6.37 107 48.8 579 96.7 oni-v-MAB-2 Onion Matrix Blank Termination 0.0 oni-v-MAS-5 Onion Fortification Termination 50.1 < 0.42 < 4.7 -- 44.1 492 88.0 rye-v-MAB-1 Ryegrass Matrix Blank Termination 0.0 rye-v-MAS-2 Ryegrass Fortification Termination 49.5 < 1.8 < 3.3 -- 54.3 100 1l o SOY-V-MAB-1 Soybean Matrix Blank Termination 0.0 SOY-V-MAS-2 Soybean Fortification Termination 49.3 < 2.4 < 6.0 -- 48.4 119 98.1 tom-v-MAB-1 Tomato Matrix Blank Termination 0.0 tom-v-MAS-3 Tomato Fortification Termination 48.8 < 1.4 < 7.6 -- 49.9 28 1 102 ~ 'Measured -PercentRecoveredcualted using MYQuan, version =e. Manzalculations may vary slightly. 'The method limit of quantitation (LOQ) in each vegetative tissue was calculated as the maximum mg PFOSkg equivalent concentration measured in negative control extracts for that tissue rounded upward to the nearest tenth mg a.i./kg. ' F o r t i f i ;e d _ P F O S ns e n t e d o - 57 - PROJECT NO.: 454-110 Appendix 4.17 Representative Chromatogram of a Plant Vegetative Tissue Matrix Blank Sample PFOS-8 454-1 10- MAE-1 Sun, Jan 27, 2002 08:05 m4.98 in 1 period No Internal Slandard Use Area 1: 4.97 MRM. 298 scans 499.0->99.1 NoiseThres. 2.0 QuantThres. 0.5 Min. Width 12 Mull. Width 10 Base. Width 40 RTWin. (secs) 10 Smooth 2 ExpectedRT 4.05 Area 687 Height 6 9 Start Time 3.62 ~ nTdim 4.04 Integration Width Retention Time Integration Type 0.42 -3.82 A BB loq intensity: 1700 cps I 5 3J4 2 1 3r\6% - . . . 41 0.69 1 98 . I 81 1.36 -' 145 169 . I I I 121 161 2.03 2.70 228 2 5 6 . 2 0. 1 3.37 241 4.04 2 8 1 SCUl 4.71 Time Sample Identification: 454-110-LET-V-MAB-1(lettuce matrix). The arrow indicates the retention time of PFOS. - 58 - PROJECT NO.: 454-110 Appendix 4.18 Representative Chromatogram of a Plant Vegetative Tissue Matrix Fortification Sample PFOS-10 454-1 10- MAS4 Sun. Jan 27, 2002 08:17 4.98 In 1 period wix No-hemal Standard Use Area intensity: 1700 CpS 1: 4.97 MRM, 298 scans 499.0->99.1 NoiseThres. 2.0 Quanl Thres. 0.5 Min. Width 12 Mult. Width 10 Bane.Width 40 RTWin.(secs) 1 0 Smooth 2 npXed RT 4.05 Area 6660 Height 671 Start Time 3.62 End lime 4.19 Integration Width Retention Time Integration Type 0.57 -3.87 A VB I 231 A 3 2 1 41 81 121 161 n 0.69 1.36 2.03 2.70 3.37 4.114 4.71 Time Sample Identification:454-110-LET-V-MAS3 (lettuce matrix). Nominal Concentration: 50.4 mg a.i./kg (wet-weight basis). - 59 - PROJECT NO.: 454-110 Appendix 4.19 Number 01 P Sample Nominal - - Day 0 Recoveries for PFOS in Artificial Soil PFOS Concentration (mg a.i./kg) Mean Percent Measured Jc&i. 0.0 < 1.1s2 -- 4.18 - Mean Percent Recovery -- 02 3.91 03 3.91 04 3.91 3.43 87.7 3.61 92.3 2.83 72.4 4.56 117 05 15.6 06 15.6 07 15.6 11.1 71.1 11.1 71.2 11.2 72.0 86.4' 5543 08 62.5 09 62.5 10 62.5 50.0 80.0 50.8 81.3 44.7 71.6 57.6 92.2 11 250 12 250 13 250 415 166 276 110 244 97.5 170 67.9 14 1000 1070 107 998 99.8 15 1000 983 98.3 16 1000 - 942 94.2 Overall Mean = 92.5 - Standard Deviation = 26.0 cv = 28% N= 14 P P - '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.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i.L) and the weightholume dilution factor of the matrix blank samples (1000) divided by the percent solids for the test system soil (84.9%). 3Statistical outlier. Data excluded from Mean Measured calculation. - - 60 - PROJECT NO.: 454-110 Appendix 4.20 Number 28 - - Termination Recoveries for PFOS in Artificial Soil P -- PFOS Concentration Sample (mg a.i./kg) Mean Mean Nominal Percent Measured Percent .i./kg) -Recovery 0.0 < 1.182 -- 4.18 -- 29 3.91 30 3.91 1.21 31.0 1 .:29 33.0 1.36 34.7 31 15.6 32 15.6 4.9 1 31.5 3.56 22.8 2.21 14.2 33 62.5 34 62.5 16.3 26.1 16.2 25.9 16.1 25.8 35 250 36 250 153 61.4 157 62.8 161 64.5 37 1000 432 43.2 474 47.4 - 38 1000 - 515 51.5 4 - Overall Mean = 38.4 -- -- Standard Deviation = 16.4 cv = 43% N= 10 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2Themethod limit quantitation (LOQ) in artificial soil was 1.18 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i./L) and the weight/volume dilution factor of the -61 - PROJECT NO.: 454-110 Appendix 4.21 Representative Chromatogram of an Artificial Soil Test Sample PFOS-22 10 Mon, Nov 19, 2001 14:23 454-1 10- 4.98 in 1 period PIUS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 NOIWmres. 30.0 Qumt Thres. 1.0 Min. width 3 Mull. Width 6 0are.Width 40 RT Win. (sea) 2 0 SmoOth 1 W d RT 4.01 Area 26856 Height 2916 stall Time 3.72 E M Time 4.39 Integration Width 0.67 Retention Time Integration Type -3.99 A BB 10 34 2 1 intensity: 1OOOCl cps 288 41 0.69 81 121 1.36 2.03 161 201 :241 2.70 3.37 4.04 2 8 1 !jCan 4.71 'rime Sample Identification: 454-110-10. Nominal Concentration: 62.5 mg a.i./kg (dry-weightbasis). - 62 - PROJECT NO.: 454-110 Appendix 4.22 - - Termination Measured Values for PFOS in Alfalfa Vegetative Tissue - _p _a _p PFOS Concentration Number Sample (mg a.i.kg) - - Mean bfeasured (rr- a.i./kg) alf-v-19 0.0 alf-v-20 0.0 alf-v-2 1 0.0 < 2.72 < 2.7 < 2.7 < 2.7 alf-v-22 3.91 alf-v-23 3.91 alf-v-24 3.91 < 2.7 6.16 < 2.7 6.16 alf-v-25 15.6 alf-v-26 15.6 alf-v-27 15.6 5.66 4.2 2.74 < 2.7 alf-v-28 62.5 alf-v-29 62.5 alf-v-30 62.5 12.9 11.2 9.66 11.1 alf-v-3 1 250 alf-v-32 250 alf-v-33 250 14.2 15.8 15.8 17.3 a1f-v-343 1000 alf-v-353 1000 alf-~-36~ -_ 1000 P sing MacQuan, version 1.6 software. Manual calculations ma,yvary slightly. Results reported on a dry-weight basis. 2The method limit of quantitation (LOQ) in alfalfa vegetative tissue (2.7 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the:nearest tenth - - - mg a.i./kg. 3No sample was collected due to mortality at this concentration. - - P - - 63 - PROJECT NO.: 454-110 Appendix 4.23 -- Termination Measured Values for PFOS in Alfalfa Fruit Tissue a PFOS Concentration Sample (mg a.i./kg) Mean Number Measured (mg a.i./kg) 0.0 0.0 0.0 a1f-f-4~ 3.91 a1f-f-5~ 3.91 alf-f-6 3.91 15.6 15.6 15.6 62.5 62.5 62.5 250 250 250 < 6.4* < 6.4 < 6.4 < 6.4 < 6.4 < 6..4 < 6..4 < 6.4 < 6.4 alf-f- 16' 1000 alf-f-175 1000 alf-f-lP 1000 e calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2 The method limit of quantitation (LOQ) in alfalfa fruit tissue (6.4 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg PFOSkg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i.kg. 3 A composite sample was made of all samples collected at this concentration. 4 There was insufficient sample weight ( < O S g) available for analysis. - 64 - PROJECT NO.: 454-110 Appendix 4.24 - - Termination Measured Values for PFOS in Flax Vegetative Tissue PFOS Concentration Sample (mg a.i./kg) Number Mean Measured kg) flx-v- 19 0.0 flx-v-20 0.0 flx-v-2 1 0.0 < 2.4' < 2.4 < 2.4 < 2.4 flx-v-22 3.91 flx-V-23 3.91 flx-V-24 3.91 4.56 4.95 6.43 3.85 flx-V-25 15.6 flx-V-26 15.6 flx-V-27 15.6 13.1 18.8 26.4 17.0 flx-V-28 62.5 flx-V-29 62.5 flx-V-30 62.5 54.7 55.0 72.7 37.5 flx-v-3 1 250 fl~-v-32~ 250 flx-v-334 250 1000 1000 -1000 de - using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 'The method limit of quantitation (LOQ) in flax vegetative tissue (2.4 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentrationmeasured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. - 3 No sample was collected due to mortality at this concentration. There was insufficient sample weight (<0.5g) available for this analysis. - 65 - PROJECT NO.: 454-110 Appendix 4.25 - Termination Measured Values for PFOS in Flax Fruit Tissue I __b. -- PFOS Concentration Sample (mg a.i./kg) Mean Number __ Measured (mg a.i.kg> flx-f- 1 0.0 flx-f-2 0.0 flx-f-3 0.0 < 0.122 < 0.12 < 0.12 < 0.12 flx-f-4 3.91 flx-f-5 3.91 flx-f-6 3.91 flx-f-7 15.6 flx-f-8 15.6 flx-f-9 15.6 flx-f- 10 62.5 flx-f-11 62.5 flx-f- 12 62.5 0.217 0.277 0.195 1.07 0.622 2.41 1.26 2.20 4.23 0.230 1.36 2.56 flx-f-1 33 250 flx-f- 144 250 flx-f-154 250 flx-f-1 63 1000 flx-f-1 73 1000 - - -- flx-f-183 1000 were calculated using MacQuan, version I .6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2 The method limit of quantitation (LOQ) in flax fruit tissue was 0.12 mg a.i./kg on a dryweight basis and was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weightholume dilution factor of the matrix blank samples (100) divided by the percent solids for the negative control fruit tissue (34.3%). No sample was collected due to mortality at this concentration. -66- PROJECT NO.: 454-110 Appendix 4.26 Termination Measured Values for PFOS in Lettuce Vegetative Tissue Number (454- 1 IO-) Sample Nominal Exposure - _ _ = PFOS Concentration (mg a.i./kg) Measured' Mean Measured (mg>a.i./kg) let-v- 19 0.0 let-v-20 0.0 let-v-21 0.0 < 5s2 < 5.5 < 5.5 <: 5.5 let-v-22 3.91 let-v-23 3.91 let-v-24 3.91 12.2 8.63 6.16 7.52 let-v-25 15.6 let-v-26 15.6 let-v-27 15.6 15.5 '10.6 7.06 9.26 let-v-28 62.5 let-v-29 62.5 let-v-3 0 62.5 39.8 41.9 58.9 27.0 let-v-3 I 250 let-v-3~~ 250 let-~-33~ 250 1000 1000 1000 ' Measured values were calculated using MacQuan, version 1.6 software. Manual calculations rnay vary slightly. Results reported on a dry-weight basis. 2 The method limit of quantitation (LOQ) in lettuce vegetative tissue (5.5 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. There was insufficient sample weight (<OS g) available for analysis. 4Nosamples were collected due to mortality at this concentration. P - 67 - PROJECT NO.: 454-1 10 Appendix 4.27 -- Termination Measured Values for PFOS in Onion Vegetative Tissue PFOS Concentration Sample (mg a.i./kg) Mean Number Measured oni-v-19 0.0 oni-v-20 0.0 oni-v-2 1 0.0 < 4.72 < 4.7 < 4.7 < 4.7 oni-v-22 3.91 oni-v-23 3.91 oni-v-24 3.91 < 4.7 < 4.7 < 4.7 < 4.7 oni-v-25 15.6 oni-v-26 15.6 oni-v-27 15.6 62.5 62.5 62.5 5.86 10.6 15.4 < 4.7 -- oni-v-3i 4 250 oni-v-3~~ 250 oni-~-33~ 250 1000 1000 1000 ' Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 'The method limit of quantitation (LOQ) in onion vegetativetissue (4.7 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentrationmeasured 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. N4o s a m p l e was collected due to mortality or lack of emergence at this concentration. -P - -68 - PROJECT NO.: 454-110 Appendix 4.28 P Number (454-1 10- - Termination Measured Values for PFOS in Onion Fruit Tissue P PFOS Concentration Sample (mg a.i./kg) Nominal Exposure Measured' Mean Measured (mg s oni-f-1 0.0 0ni-f-2~ 0.0 oni-f-3 0.0 0.517 -- 0.487 0.502 oni-f-4 3.91 oni-f-5 3.91 oni-f-63 3.91 5.81 3.13 0.453 oni-f-7 15.6 oni-f-8 15.6 oni-f-9 15.6 oni-f- 1o3 62.5 oni-f-113 62.5 oni-f- 1z4 62.5 < 0.42' 22.2 19.7 24.6 oni-f-13~ 250 oni-f-14~ 250 oni-f- 1s4 250 oni-f- 164 1000 oni-f-17~ 1000 oni-f-1g4 P 1000 P P -- I Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2 The method limit of quantitation (LOQ) in onion fruit tissue was 0.42 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weight/volume dilution factor of the matrix blank samples (100) divided by the percent solids for the negative control fruit tissue (9.65%). - There was insufficient sample weight (<OS g) available for analysis. 4No sample was collected due to mortality or lack of fruit production at this concentration. P -- - 69 - PROJECT NO.: 454-1 10 Appendix 4.29 Termination Measured Values for PFOS in Ryegrass Vegetative Tissue Number Sample PFOS Concentration (mg a.i./kg) Mean Measured (me a.i./kg) rye-v- 19 0.0 rye-v-20 0.0 rye-v-2 1 0.0 < 3.32 < 3.3 < 3.3 <: 3.3 rye-v-22 3.91 rye-v-23 3.91 rye-v-24 3.91 7.13 8.18 8.70 8.70 rye-v-25 15.6 rye-v-26 15.6 rye-v-27 15.6 13.8 .3 1.4 49.0 < 3.3 rye-v-28 62.5 rye-v-29 62.5 rye-v-30 62.5 42.5 48.7 67.0 36.7 rye-v-3 1 250 rye-v-32 250 rye-v-33 250 31.9 156.1 37.5 129 r~e-v-34~ 1000 r~e-v-35~ 1000 rye-~-36~ 1000 ' Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2 The method limit of quantitation (LOQ) in ryegrass vegetative tissue (3.3 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest - 70 - PROJECT NO.: 454-110 Appendix 4.30 - Termination Measured Values for PFOS in Soybean Vegetative Tissue PFOS Concentration Sample (mg a.i./kg) Number - Mean Measured soy-v- 19 0.0 soy-v-20 0.0 soy-v-2 1 0.0 < 6.0' < 6.0 < 6.0 < 6.0 soy-v-22 3.91 soy-v-23 3.91 soy-v-24 3.91 17.7 15.6 10.0 19.1 soy-v-25 15.6 SOY-V-26 15.6 soy-v-27 15.6 29.3 35.8 48.9 29.3 SOY-V-28 62.5 soy-v-29 62.5 soy-v-30 62.5 63.9 63.3 70.7 55.4 soy-v-3 1 250 soy-v-32 250 soy-v-33 250 64.2 I14 175 103 soy-v-343 1000 s0y-v-3s3 1000 ' - s o y - ~ - 3 6 ~ 1000 P P Measured values were calculated using MacQuan, version 1.6 software. Manual calculations 'may vary slightly. Results reported on a dry-weight basis. *Themethod limit of quantitation (LOQ) in soybean vegetative tissue (6.0 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentrationmeasured in negative control extracts rounded upward to A the nearest tenth mg a.i./kg. at this concentration. P P P -71 - PROJECT NO.: 454-110 Appendix 4.31 - Number soy-f- 1 soy-f-2 soy-f-3 - -- Termination Measured Values for PFOS in Soybean Fruit Tissue PFOS Concentration Sample (mg a.i./kg) Mean Measured (n 0.0 < 0.1g2 < 0.18 0.0 < 0.18 0.0 < 0.18 soy-f-4 3.91 soy-f-5 3.91 SOY-f-6 3.91 0.947 1.40 0.339 2.9 1 soy-f-7 15.6 soy-f-8 15.6 soy-f-9 15.6 soy-f-10 62.5 soy-f-1 1 62.5 soy-f-12 62.5 0.464 1.63 0.528 1.14 1.14 1.33 0.874 1.20 soy-f- 13 250 soy-f-14 250 soy-f- 15 250 1.57 3.21 5.56 2.49 soy-f-163 soy-f-173 soy-f-183 1000 1000 1000 I Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. The method limit of quantitation (LOQ) in soybean fruit tissue was 0.18 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the weighthlume diluticln factor of the matrix blank samples (100) divided by the percent solids for the negative control fruit tissue (22.9%). - 72 - PROJECT NO.: 454-110 Appendix 4.32 - - - -- Termination Measured Values for PFOS in Tomato Vegetative Tiissue a PFOS Concentration Sample (mg a.i./kg) - Mean - Number (454-1lo-) Measured' - Measured (rig a.i./kg) tom-v- 19 0.0 tom-v-20 0.0 tom-v-2 1 0.0 < 7.6' < 7.6 < 7.6 < 7.6 tom-v-22 3.91 tom-v-23 3.91 tom-v-24 3.91 < 7.6 < 7.6 < 7.6 < 7.6 tom-v-25 15.6 tom-v-26 15.6 tom-v-27 15.6 12.5 33.8 27.9 61.1 tom-v-2 8 62.5 tom-v-29 62.5 tom-v-3 0 62.5 70.9 50.1 44.5 34.9 tom-v-3 1 250 t0m-v-32~ 250 t0m-v-33~ 250 t0m-v-34~ 1000 tom-v-3s4 1000 tom-~-36~ 1000 <culations -- may vary slightly. Results reported on a dry-weight basis. 'The method limit of quantitation (LOQ) in tomato vegetative tissue (7.6 mg a.i./kg on a dry-weight basis) was calculated as the maximum mg a.i./kg equivalent concentration measured in negative control extracts rounded upward to the nearest tenth mg a.i./kg. 3 There was insufficient sample weight (<0.5 g) available for analysis. 4Nosample was collected due to mortality or lack of emergence at this concentration. P -- -73 - PROJECT NO.: 454-110 Appendix 4.33 -Termination Measured Values for PFOS in Tomato Fruit Tissue PFOS Concentration Sample (mg a.i./kg) - Number Mean Measured a.i./kg) tom-f-1 0.0 tom-f-2 0.0 tom-f-3 0.0 t0m-f-4~ 3.91 tom-f-5 3.91 tom-f-6 3.91 tom-f-7 15.6 tom-f-8 15.6 tom-f-9 15.6 tom-f-10 62.5 tom-f-1 1 62.5 tom-f-12 62.5 < 0.472 < 0.47 < 0.47 -- < 0.47 < 0.47 < 0.47 1.51 0.580 0.946 < 0.47 < 0.47 < 0.47 < 0.47 1 .os 0.946 tom-f-13' 250 tom-f-1 4 ~ 250 tom-f- 1s4 250 tom-f-164 tom-f-174 tom-f-1g4 1000 1000 1000 Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Results reported on a dry-weight basis. 2 The method limit of quantitation (LOQ) in tomato fruit tissue was 0.47 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.000400mg a.i./L) and the weightholume dilution factor ofthe matrix blank samples (100) divided by the percent solids for the negative control h i t tissue (8.45%). - There was insufficient sample weight (<0.5 g) available for analysis. No sample was collected due to mortality or lack of emergence at this concentration. P -- - 74 - PROJElCTNO.: 454-110 Appendix 4.34 Representative Chromatogram of a Plant Fruit Tissue Test Sample PFOS-29 454-1 10- SOY-F-15 Tue. Mar 26, 2002 1634 4.98 in I period Fl6 No lntemal Standard Use Area intensity: 4000 cps 1: 4.97 MRM, 298 scans 499.0->99.1 NoiseThres. 2.0 Quant Thres. 0.5 Min. Width 12 Mult. Width 10 Base. Width 80 RTWin. (secs) 20 Smooth 2 Expected UT 3.75 Area 20080 Height 1699 Start Time 3.40 End Time 4.37 Integration Width Retention Time Integration Type 0.89 A 3- .72 VB 222 4 3 2 1 1 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample Identification: 454-110-SOY-F-15(soybean matrix). - 75 - PROJECT NO.: 454-110 Appendix 4.35 RepresentativeChromatogramof a Plant Vegetative Tissue Test Sample PFOS-34 454-1 10- LET-V-28 Sun, Jan 27. 2002 10:41 4.98 in 1 period rn No Internal Standard Use Area intensity: 1700 cps 1: 4.97 MRM, 298 scans 499.0->99.1 NdseThres. 2.0 Quant Thres. 0.5 Min. Width 12 Mutt. Width 10 Base. width 40 RTWur. (sea) 2 0 Smooth 2 ExpededRT 4.05 Area 5351 Height 521 Start Time 3.92 End Time 4.49 Integration Width 0.57 Retention Time lnttqration Type 4.20 A - BB 251 A 2 1 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample Identification: 454-110-LET-V-28(lettuce matrix). - 76 - PROJECT NO.: 454-110 Appendix 5 Environmental Conditions Temperature ("C) Relative Humidity ("A) Date Minimum Maximum Mean 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 ll/14/Ol1 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 11/18~011 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 ll/21/Ol1 16.2 27.0 19.7 12 51 33 ' 11122101 16.3 11/23/01 16.4 11124101 16.7 28.0 20.2 26.6 20.4 25.0 20.8 14 47 32 16 57 34 46 75 60 ' 11/25/01 18.0 11/26/01 16.7 1 112710 1 16.5 25.9 21.2 26.7 20.9 24.9 20.4 42 83 64 31 85 59 35 75 53 11/28/01' 17.9 11/29/01 17.7 26.1 20.9 25.6 21 .o 47 80 66 49 81 68 11/30/01 18.2 26.3 21.5 54 85 70 1210 110 1 16.8 28.5 21.3 36 83 61 12/02/01 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 12/05/01 16.7 12/06/0 1 16.7 26.8 20.6 29.4 21.3 28.2 21.0 20 57 40 17 59 40 29 62 47 12/07/01 I 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 ' 12111/01' 16.4 1211210 1 16.3 12113101 16.6 26.1 19.9 25.3 19.6 24.2 19.9 31 68 49 29 66 48 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 12116/01 16.1 27.2 19.6 13 38 27 ' 12117101 16.4 25.3 19.6 30 73 43 Indicates days on which all species were watered. ' Indicates days on which alfalfa, lettuce, ryegrass, and soybean trays were watered. PAR - Photosynthetically Active Radiation 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 - 77 - PROJECT NO.: 454-110 Appendix 5 (continued) Date ' 12/18/01 12/19/01 Temperature ("C) Minimum Maximum 16.5 27.3 16.4 26.4 Environmental Conditions Relative Humidity (%) Mean Minimum Maximum Mean 19.8 16 79 43 19.7 19 53 34 ' 12/20/01 16.2 1212 1/01 16.1 ' 12/22/01 16.1 12/23/01 15.3 27.6 19.5 26.4 19.4 27.5 19.4 26.4 19.6 10 54 25 11 33 20 11 32 24 14 57 30 12/24/01 16.2 26.7 19.5 14 62 35 12/25/01 I 15.3 28.3 19.3 9 29 21 12/26/0 1 I 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 12/30/01 14.8 27.3 18.6 8 24 17 12/31/014 14.8 27.2 18.6 9 31 20 01/01/02~ 14.7 27.0 18.7 9 26 20 0 1/02/02 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 0 1/06/026 15.0 24.4 18.4 24 55 40 0 1/07/024 14.8 21.9 18.1 30 54 42 0 1/08/02 14.8 26.5 18.6 11 45 28 0 1/09/025 14.9 24.4 18.9 20 51 35 01/10/02~ 16.1 23.9 19.6 28 58 43 01/11/02 16.1 25.9 19.4 19 61 43 0 1112102~ 15.1 27.5 19.8 15 54 36 01/13/028 16.2 27.7 19.9 12 57 34 0It14/029 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/I 7/02' 15.5 24.5 19.8 23 56 40 'I Indicates days on which all species were watered. * Indicates days on which soybean and tomato were watered. Indicates days on which alfalfa, lettuce, ryegrass, soybean, and tomato were watered. Indicates days on which tomato was watered. Indicates days on which soybean was watered. ' Indicates days on which flax, lettuce, onion and ryegrass were watered. 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. lo PAR - Photosynthetically Active Radiation Light Intensity Moles PAR'' 18.3 17.1 20.1 20.3 19.6 17.9 17.0 19.6 19.9 20.2 17.6 17.9 21.8 22.2 20.0 19.2 18.8 19.7 20.5 16.6 17.4 19.3 18.4 17.5 16.8 19.2 21.0 18.4 19.4 20.7 18.6 -78- PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temuerature PC) ~I Relative Humidity_(%. ) , Date Minimum Maximum Mean Minimum Maximum Mean 01118/02 14.9 26.8 19.9 12 51 33 01119/02 14.7 22.9 19.2 29 58 41 01/20/02' 14.7 26.2 19.3 17 61 41 0 112 1/02* 15.1 24.7 19.5 30 67 50 0 1122102~ 15.1 27.8 19.9 15 59 39 01/23/023 15.5 24.3 19.7 38 78 54 0 1/24/02 16.4 25.5 20.4 47 84 64 0 1/25/02 15.1 27.6 19.8 14 84 46 0 1/26/024 15.1 27.6 19.9 14 63 43 01/27/024 15.3 27.3 19.9 21 67 44 0 1/28/02 16.4 29.1 20.9 18 71 51 0 1/29/02 16.3 27.2 20.8 25 78 51 0 1/30/02 16.8 30.7 21.7 31 78 55 0 1131/023 16.7 24.4 20.0 42 76 57 0210 I 102~ 16.2 29.4 20.8 33 78 55 02/02/02~ 15.1 28.2 19.7 12 49 30 02/03/024 15.0 26.4 19.7 15 56 36 02/04/02' 14.4 26.7 18.9 14 58 35 02/05/02 14.4 27.8 18.9 10 43 24 02/06/023 15.0 25.3 19.5 14 55 33 02/07/02 15.2 24.1 19.4 32 62 48 02/08/02 15.0 26.5 19.8 17 69 43 02/09/02 15.0 26.7 19.9 19 67 44 02110102 15.2 26.8 20.2 33 79 55 02/11/02 15.1 26.4 19.8 15 74 42 02112/02 15.0 26.0 19.6 14 55 35 02113/02 15.0 25.9 19.7 11 53 30 02114/02 15.0 30.8 19.6 11 52 30 02115/02 15.0 25.1 19.6 20 56 39 02116/02 16.4 27.6 20.8 11 55 33 02117/022 14.9 28.0 19.9 14 51 31 02118/02 14.5 27.2 19.5 12 57 30 02/19/02 14.9 26.5 20.0 12 56 33 02/20/02 16.5 25.8 20.7 27 75 47 ' 02121/02 16.4 26.7 20.6 21 78 48 Indicates days on which alfalfa, flax, onion, ryegrass and tomato were watered. * Indicates days on which alfalfa and ryegrass were watered. Indicates days on which all trays were watered. Indicates days on which alfalfa, flax, ryegrass, and tomato were watered. PAR - Photosynthetically Active Radiation Light Intensity Moles PAR'- 20.1 17.4 14.3 17.8 21.3 17.9 17.7 20.6 21.1 19.9 19.6 18.2 19.2 18.2 17.5 22.9 20.3 17.3 23.4 20.9 17.6 21.4 20.6 17.2 23.3 19.5 22.2 24.6 18.1 23.7 21.5 24.8 22.7 18.4 15.6- - 79 - PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperature ("C) Relative Humidity (%) Date Minimum Maximum Mean Minimum Maximum Mean 02/22/02' 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 02128/02' 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 0311 1/02' 15.0 26.3 19.5 11 44 24 03/12/02' 15.1 25.1 19.9 25 65 40 03113/02 16.5 24.1 20.3 44 71 55 03114/02' 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 0311 8/02' 16.2 24.6 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 0312 1/02 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 which alfalfa and ryegrass were watered. Indicates days on which alfalfa were watered. PAR - 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 - 80 - PROJECT NO.: 454-110 Appendix 5 (continued) Environmental Conditions Temperature ("C) Relative Humidity ("A) Date Minimum Maximum Mean Minimum Maximum Mean 03/30/02' 16.8 29.7 22.3 23 78 49 0313 1/02 16.6 04101102 16.6 25.4 20.5 25.4 20.6 37 76 57 18 74 43 04/02/022 16.7 04/03/02 16.5 25.8 21.4 30.9 22.0 21 74 43 32 80 51 04/04/022 15.9 25.3 20.3 16 51 28 04/05/022 16.6 26.1 20.4 14 46 27 04/06/022 15.2 25.9 20.3 13 48 27 04/07/02 15.7 26.1 20.4 13 64 32 04/08/02 16.9 25.4 21.1 28 73 49 04/09/022 17.3 04110/022 17.1 25.3 21.6 25.4 21.1 41 87 66 29 89 55 0411 1/02 17.0 04112/022 17.4 25.9 21.0 25.6 21.2 29 66 47 44 85 62 04113/02 17.0 28.0 22.1 53 89 71 04/14/02 18.7 04/15/022 19.8 27.5 23.1 30.0 23.7 57 88 71 49 84 70 04116/02 20.2 04/17/022 19.5 29.7 24.7 31.6 24.9 54 87 71 51 86 69 04118/02 20.1 28.8 24.0 57 86 73 04119/022 19.5 04/20/02 19.6 30.3 23.5 28.5 23.0 55 86 72 56 86 70 04121102 16.7 25.4 20.7 34 79 56 04/22/022 16.5 26.4 20.8 28 77 52 04/23/02 16.3 25.5 20.7 20 59 34 041241022 17.3 04/25/02 16.4 04/26/02 16.4 04/27/02 16.8 04/28/02 17.5 04/29/022 16.7 26.1 21.2 24.9 20.9 25.4 20.8 25.5 21.0 26.7 21.9 27.1 20.8 20 64 39 30 70 53 19 69 43 28 77 51 61 88 75 29 77 48 04/30/022 16.5 25.7 21.1 29 77 47 05/01/022 17.7 25.4 21.5 30 79 57 05/02/02 17.9 26.9 22.6 64 84 75 05/03/022 17.1 25.5 21.3 20 82 41 05/04/02 ' 16.5 Indicates days on which 25.6 alfalfa and 21.0 ryegrass were watered. 22 72 44 ' Indicates days on which ryegrass was watered. PAR - Photosynthetically Active Radiation Light Intensity Moles P,4R3 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 - 81 - PROJECT NO.: 454-1 10 Appendix 5 (continued) Environmental Conditions TemDerature(."c,) Relative Humidity (%) Date Minimum Maximum Mean Minimum Maximum Mean 05/05/02 17.3 05/06/02' 17.7 05/07/02 18.5 05/08/02 18.2 05/09/02 17.8 05110/02' 18.3 0511 1/02' 17.8 25.5 21.3 25.5 21.8 27.2 22.7 28.0 22.3 25.5 21.9 26.1 22.2 25.6 21.6 33 72 56 34 72 55 51 85 71 41 83 62 47 88 67 47 84 64 20 67 47 05/12/02 18.8 27.4 23.3 41 86 67 05113/02' 18.6 05114/02' 16.7 05115/02 16.7 05/16/02' 17.7 05/17/02' 20.3 29.2 23.7 26.2 20.9 26.0 21.3 26.0 22.4 28.7 23.3 57 87 74 23 58 39 26 65 44 36 66 56 48 78 65 05118/02 16.7 05119/02 16.6 05/20/02' 17.4 0512 1/02' 17.4 25.2 21.3 25.8 21.1 25.4 21.2 26.0 21.1 30 84 55 19 62 40 19 59 38 22 49 36 05/22/02 17.3 05/23/02' 17.4 05/24/02 17.7 26.5 21.3 25.6 21.3 25.4 22.4 23 57 41 35 73 52 42 76 63 05/25/02' 18.4 25.8 22.3 48 82 64 05/26/02 05/27/02 05/28/02' 05/29/02 05/30/02' 0513 1/02' 0610 1/02 06/02/02' 06/03/02 06/04/02' 06/05/02 ' 18.0 26.9 23.0 19.3 27.6 23.7 19.4 27.9 23.7 19.6 28.0 23.5 18.7 28.1 23.6 20.1 28.9 25.1 22.0 27.6 24.9 20.1 29.2 24.0 19.2 25.8 22.5 20.2 26.7 23.3 2~~ 1.9 30.5 26.2 Indicates days on which ryegrass was watered. PAR - Photosynthetically Active Radiation 58 87 74 61 84 72 60 83 72 59 84 73 62 87 74 66 87 77 59 90 74 35 90 63 38 68 54 55 81 68 62 91 76 Light 1nte:nsity Moles PAR^ 13.8 12.8 16.1 15.5 16.9 14.2 13.6 13.8 13.9 14.0 13.5 13.5 14.0 16.6 13.1 13.8 14.5 13.7 13.1 13.3 14.5 13.5 14.4 14.4 15.3 14.5 14.2 13.5 14.3 14.5 14.7 14.1 - 82 - PROJECT NO.: 454-110 Appendix 6.1 Alfalfa Emergence P Day 7 -P Treatment Number of Emerged Seedlings in Replicate: Group A B C D n Mean Std. Dev. Control 7 10 10 8 4 8.75 1.50 3.91 mg a.i /kg 8 9 8 8 4 8.25 0.50 15.6 mg a.i./kg 9 9 7 6 4 7.75 1.50 62.5 mg a.i./kg 6 6 8 8 4 7.00 1.15 250 mg a.i./kg 8 5 7 8 4 7.00 1.41 1000 mg a.i./kg 0 0 5 5 4 2.50 2.89 Treatment Number of Eme GrouAp_B r g e -d See d l in C g s in R e p l i cat D e : n Mean - Std. Dev. Control 8 10 10 8 4 9.00 1.15 3.91 mg a.i./kg 8 9 9 8 4 8.50 0.58 15.6 mg a.i./kg 9 9 8 6 4 8.00 1.41 62.5 mg a.i./kg 7 7 9 9 4 8.00 1.15 250 mg a.i./kg 9 5 7 8 4 7.25 1.71 1000mg a.i./kg 0 0 5 5 4 2.50 2.89 Treatment Number of Emerged Seedlings in Replicate: GrouD A B C D n Mean Std. Dev. Control 8 10 10 8 4 9.00 1.15 3.91 mg a.i./kg 8 9 9 8 4 8.50 0.58 15.6 mg a.i./kg 9 9 8 6 4 8.00 1.41 62.5 mg a.i./kg 7 7 9 9 4 8.00 1.15 250 mg a.i./kg 9 5 7 8 4 7.25 1.71 1000 mg a.i./kg 0 1 5 7 4 3.25 3.30 - 83 - Appendix 6.2 Mean Alfalfa Emergence on Day 2 1 PROJECT NO.: 454-110 o Data Regression . - - - - . 95% Conf. Int. 50% Inhibition 2 -- 01 , , , , i , I , , 1 , I , , /I I I I , 1 ' ' ' ' 1 I ' ' ' 0 200 400 600 800 1000 1200 Concentration(mg siJkg) Curve Parameters ECzs 372.306 Lower 95% Confidence Limit 201.512 Upper 95% Confidence Limit 687.860 Ro 8.4045 0 0.4470 12 0.97096 EGO 745.418 Lower 95% Confidence Limit 540.879 Upper 95% Confidence Limit 1027.07 Ro 8.4045 rs 0.4470 12 0.97096 - 84 - PROJElCT NO.: 454-1 10 Appendix 6.3 Alfalfa 21-Day Survival ~ Day21 Treatment Number of Living Seedlings in Replicate: - Gr A B C D n Control 8 9 10 8 4 3.91 mg a.i./kg 8 9 8 8 4 15.6 mg a.i./kg 9 9 8 6 4 62.5 mg a.i./kg 7 7 9 9 4 250 mg a.i./kg 8 4 6 7 4 1000 mg a.i./kg 0 1 1 4 4 -- Mean 8.75 8.25 8.00 8.00 6.25 1S O Std. Dev. P 0.96 0.50 1.41 1.15 1.71 1.73 10 , I :----..- n 7 d E W 6 I- -I .\ f 5 :- a 4 I- v1 3 :- 2 I- :- o1 ! I I I l l 8 ~ / / % I ~ , 8 8 8 I 8 1 8 I ~ # ? # I ~ # / / I 0 200 400 600 800 1000 1200 Concentration (mg aiJkg) Regression 95% Conf. Int. 50% Inhibition EC25 251.073 EC50 452.212 Lower 95% Confidence Limit 178.402 Lower 95% Confidence Limit 363.580 Curve Parameters Upper 95% Confidence Limit 353.265 Ro 8.2801 Up.p.er 95% Confidence Limit 562.130 Ro 8.2801 0 0.3790 0 0.3790 2 0.99162 1' 0.99162 -85 - PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 6.4 Alfalfa Mean Seedling Fresh Weight, Day 21 Mean Weight (8) for Replicate: A B C D n 0.1 1 0.10 0.12 0.13 4 0.10 0.10 0.16 0.09 4 0.08 0.12 0.08 0.11 4 0.1 1 0.10 0.09 0.12 4 0.03 0.01 0.03 0.03 4 0.02 0.02 0.01 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 0.20 -t 5 n v M 0.15 .e .\ 0.00 I , * - I 0 ---...._._ I I I I ' I , 200 400 0 ' ' ' I 1 ' ' ' I ' I ' ' , ' ' ' ' 600 800 1000 1200 Concentration(mg aiJkg) o Data --Regression ._...9_5%- Conf. Int. 50% Inhibition EC2s 53.2844 ECso 146.049 Lower 95% Confidence Limit 4.1 1623 Lower 95% Confidence Limit 27.2521 Curve Parameters Upper 95% Confidence Limit 689.763 Rl 0.1 144 Upper 95% Confidence Limit 782.528 Rl 0.1 144 cr 0.6492 c 0.6492 ? 0.90534 ? 0.90534 - 86 - PROJECT NO.: 454-110 Appendix 6.5 Alfalfa Seedling Height on Day 2 1 Treatment Group Replicate Height (cm) for Plant Number: n Mean Std. Dev. 1 2 3 4 5 6 7 8 9 10 Control A .666361264 8 6.1 2.64 B .5348475511 9 5.8 2.49 C 7 4 8 8 6 6 8 8 6 8 10 6.9 1.37 D . 1 0 5 6 1 0 5 6 5 10 8 7.1 2.42 3.91 m g k g A .55875954 8 6.0 1.77 B . 7 5 10 4 6 8 6 7 11 9 7.1 2.26 C . 11106 5 5 7 3 8 8 6.9 2.70 D .69467444 8 5.5 1.85 15.6 mg a.i./kg A .588544543 9 5.1 1.76 B .587555594 9 5.9 1.69 C .22583574 8 4.5 2.20 D .677755 6 6.2 0.98 62.5 mg a.i./kg A .5655756. 7 5.6 0.79 B .65587412 7 6.7 2.69 C .643645792 9 5.1 2.15 D .8598107466 9 7.0 1.94 250 mg a.i.kg A B C D .34234323 8 3.0 0.76 .2222 4 2.0 0.00 .142354 6 3.2 1.47 .4423322 7 2.9 0.90 1000mg a.i./kg A 0 B .1 1 1 .o C . 1 1 1.o D .1111 4 1.o 0.00 The "." symbol indicates that the seedling either did not emerge or died prior to measurement. - 87 - PROJECT NO.: 454-110 Treatment Grou 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 SeedlingHeight on Day 21 Mean Height (cm) for Replicate: A C D n 6.1 5.8 6.9 7.1 4 6.0 7.1 6.9 5.5 4 5.1 5.9 4.5 6.2 4 5.6 6.7 5.1 7.0 4 3.0 2.0 3.2 2.9 4 1.o 1.o 1.o 3 Mean 6.5 6.4 5.4 6.1 2.8 1.o - Std. Dev. 0.6 0.8 0.8 0.9 0.5 0.0 200 400 600 800 1000 -2 Concentration(mg ai./kg) EC25 102.447 ECso 249.058 Lower 95% Confidence Limit 26.8287 Lower 95% Confidence Limit 106.292 Curve Parameters Upper 95% Confidence Limit 391.201 Ro 6.2793 Upper 95% Confidence Limit 583.579 Ro 6.2793 - 88 - 0 Data Regression ..._..9.5% Conf. Int. 510% Inhibition CJ 0.5720 0 0.5720 12 0.93960 ? 0.93960 PROJECT NO.: 454-110 Appendix 6.7 Alfalfa Seedling Condition, Day 2 1 Treatment Group Replicate ~ 1 2 ~~ Condition (scoreign)' for Plant Number: 3 4 5 6 7 8 II Mean DSetdv.. 9 10 p - Control 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 I5 33.7 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- I O 0 0.0 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- CI 0 0.0 3.91 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- I!, 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- c: 0 0.0 0 0.0 11 33.3 0 0.0 15.6 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 90.N 0.- 51 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- $1 . 70.N 30.N 0.- 0.- 0.- 0.- 0.- 0.- 8 0.- 0.- 0.- 0.- 0.- 0.- ti 10 30.0 0 0.0 13 25.5 0 0.0 62.5 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 . 0.- 30.N 0.- 0.- 0.- 0.- 0.- 7 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 41 0 0.0 4 11.3 0 0.0 0 0.0 250 mg a.i./kg A B C D 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 . 100.- 0.- 0.- 0.- 0.- 3 . 100.- 80.N 0.- 0.- 0.- 0.- 0.- 7 . 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- c: 11 33.3 20 44.7 26 44.3 13 35.4 1000 mg a.i./kg A 0 B 0.- 1 0 C . 100.- 100.- 100.- 100.- 60.N 5; 92 17.9 D . 100.- 100.- 100.- . 80.N 60.N 0.- 80.N 7 74 36.0 '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 - 89 - PROJECT NO.: 454-110 Appendix 7.1 Flax Emergence - Treatment Number of Emerged Seedlings in Replicate: Group A B C D P Control 7 9 6 7 3.91 mg a.i./kg 7 5 7 7 15.6 mg a.i./kg 8 9 7 8 62.5 mg a.i./kg 9 9 9 7 250 mg a.i./kg 8 9 5 7 1000 mg a.i./kg 0 0 0 0 n ____p 4 4 4 4 4 4 Mean 7.25 6.50 8.00 8.50 7.25 0.00 d Std. Dev. 1.26 1.oo 0.82 1.oo 1.71 0.00 - 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.ikg 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 1000mg a.i./kg Day 15 - - - Number of Emerged Seedlings in Replicate: A B - P C D n P 7 9 6 7 4 7 5 8 8 4 8 9 7 8 4 9 9 9 7 4 8 9 5 7 4 0 0 0 0 4 Number of Emerged Seedlings in Replicate: A B C D 7 9 6 7 8 5 8 8 8 9 7 8 9 9 9 7 8 9 5 7 0 0 0 0 n P 4 4 4 4 4 4 -- -Mean 7.25 7.00 8.00 8.50 7.25 0.00 - Std. Dev. 1.26 1.41 0.82 1.oo 1.71 0.00 - - . Mean 7.25 Std. Dev. 1.26 7.25 1S O 8.00 0.82 8.50 1.oo 7.25 1.71 0.00 0.00 - 90 - PROJECT NO.: 454-110 Appendix 7.2 Mean Flax Emergence on Day 2 I 9 L - 8 7 * 0 E 6 5 4 3 2 1 0 I T l- * Treatment Group * Treatment group mean is significantly different from the control mean (Dunnett's test,p<O.[D5) The non-linear regression technique failed to generate useable results. Therefore,linear interpolationwas used. EC25 398.7069 Lower 95% Confidence Limit 126.1605 Upper 95% Confidence Limit - 460.7759 ECso Lower 95% Confidence Limit - Upper 95% Confidence Limit - - 599.1 379 401.8809 640.5172 - E& and ECsoestimates calculated by linear interpolation using the ICPIN program (7). -91 - PROJECT NO.: 454-110 P Treatment Group Control 3.91 mg a.i./kg 15.6mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 7.3 Flax 21-Day Survival P Day21 p - Number of Living Seedlings in Replicate: A C D 7 9 6 7 -- n B Mean Std. Dev. 4 7.25 1.26 7 5 8 7 4 6.75 1.26 8 9 7 8 4 8.00 0.82 9 9 9 7 4 8.50 1.oo 1 4 4 4 4 3.25 1S O 0 0 0 0 4 0.00 0.00 Treatment Group * Treatment group mean is significantly different from the control mean (Dunnett's test, p<0.05) The non-linear regression technique failed to generate useable results. Therefore, linear interpolation was used. Effect Rate ECzs Effect Concentration 144.1964 Lower 95% Confidence Limit 103.4821 Upper 95% Confidence Limit 177.3710 EC50 225.8929 159.6256 367.5893 EC25 and EC50 estimatescalculatedby linear interpolation using the ICPIN program (7). - 92 - PROJECT NO.: 454-110 - Treatment Group - Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 7.4 Flax Mean Seedling Fresh Weight, Day 2 1 Mean Weight (8) for Replicate: A B C D n 0.16 0.19 0.19 0.23 4 0.21 0.19 0.16 0.17 4 0.21 0.16 0.16 0.17 4 0.17 0.17 0.14 0.17 4 0.01 0.02 0.02 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 0.25 0.20 n M 0.15 v B."2 M 0.10 E h 0.05 0.00 200 -0.05 ECzs 81.5831 ECso 118.796 Lower 95% Confidence Limit 46.015 1 Lower 95% Confidence Limit 79.3780 400 600 800 1000 Concentration (mg niJkg) Curve Parameters Upper 95% Confidence Limit 144.644 Ro 0.1833 upper 95% Confidence Limit 177.828 Ro 0.1833 - -- o Data Regression _ _ . _95.%- .Conf. Int. 50% Inhibition (z 0.2420 0 0.2420 l.2 0.99000 13 0.99000 -93 - PROJECT NO.: 454-110 Appendix 7.5 Flax Seedling Height on Day 21 ~ Treatment Group Replicate Height (cm) for Plant Number: n Mean Std. Dev. 12345678910 Control A . 5 9 9 9 8 10 10 7 8.6 1.72 B . 5 9 9 9 12101112 5 9 9.1 2.62 C . 11118 9 9 5 6 8.8 2.23 D . 108117109 9 7 9.1 1.35 3.91 mg a.i./kg A B C D . 8 8 10 9 10 10 10 7 9.3 0.95 . 9 11 9 9 10 5 9.6 0.89 .811681111198 8.1 3.40 .783810997 7.7 2.29 15.6 mg a.i./kg A . 9 10 8 11 10 9 11 10 8 9.8 1.04 B .910898119999 9.1 0.93 C . 9 811116 9 2 7 8.0 3.16 D . 6 8 8 10 9 7 8 10 8 8.3 1.39 62.5 mg a.i./kg A . 5 8 1010 8 1011 7 8 9 8.6 1.88 B . 1 1 1 2 9 1 0 9 5 13 9 1 9 8.8 3.70 C .9881110356109 7.8 2.64 D .937991077 7.7 2.36 250 mg a.i./kg A B C D .1I 1.o .21114 1.3 0.50 .11114 1.o 0.00 .12224 1.8 0.50 1000mg a.i./kg A .o B .a C .o D .a The "." symbol indicates that the seedling either did not emerge or died prior to measurement. - 94 - PROJECT NO.: 454-110 Appendix 7.6 - - P 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 A __c 8.6 9.3 9.8 8.6 1.o Flax Mean Seedling Height on Day 2 1 - Mean Height (cm) for Replicate: B C D - P n Mean P 9.1 8.8 9.1 4 8.9 9.6 8.1 7.7 4 8.7 9.1 8.0 8.3 4 8.8 8.8 7.8 7.7 4 8.2 1.3 1 .o 1.8 4 1.3 0 - Std. Dev. 0.27 0.90 0.80 0.54 0.35 10 , 1 -- o Data Regression _ - . - _95_%.Conf. Int. 50% Inhibition 200 400 600 800 1000 -2 Concentration (mgaiJkg) EC25 97.6338 EC50 140.41 1 Lower 95% Confidence Limit 81.3392 Lower 95% Confidence Limit 124.825 Curve Parameters Upper 95% Confidence Limit 117.166 Rl 8.7913 Upper 95% Confidence Limit 157.943 Rl 8.7913 1 00 CY 0.2340 d 0.2340 ? 0.99938 ? 0.99938 - 95 - PROJECT NO.: 454-110 Appendix 7.7 Flax Seedling Condition, Day 2 1 TrGearotmupent Replicate 1 - 2 3 Condition (score.sign)' for Plant Number: 4 5 6 7 8 _t 9 _p.- Std. 10 -Mean Dev. Control 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.- 7 0 0.0 0.- 4o.usc 9 4 13.3 0.- 0.- 6 0 0.0 0.- 0.- 7 0 0.0 3.91 mg a.i./kg A B C D 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 13 35.4 0.- 0.- 0.- 0.- 0.- 5 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 15.6 mg a.i./kg A B C D 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.- 8O.USC 7 11 30.2 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 62.5 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 40.N 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 0.- 9 0 0.0 0.- 9 4 13.3 0.- 7 0 0.0 250 mg a.i./kg A B C D 100.- 100.- 100.- 100.- 100.- 100.- 100.- 9 o . u s c 8 99 3.5 100.- 100.- 100.- 100.- 100.- 5 o . u s c 9 o . u s c 7 o . u s c 9 o . u s c 9 89 17.6 100.- 9 o . u s c 8O.USC 8O.USC 8O.USC 5 86 8.9 100.- 100.- 100.- 8O.USC 8O.USC 8O.USC 8O.USC 7 89 10.7 1000 mg a.i./kg A 0 B 0 C 0 D 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. USC - Unshed Seed Coat, LC -Leaf Curl, N - Necrosis - 96 - PROJE'CTNO.: 454-1 10 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 8.1 Lettuce Emergence -Day 7 Number of Emerged Seedlingsin Replicate: A B C D n P 9 8 9 9 4 8 9 9 10 4 10 7 8 10 4 9 7 8 10 4 10 7 7 8 4 3 0 1 1 4 --- Mean Std. Dev. 8.75 0.50 9.00 0.82 8.75 1.50 8.50 1.29 8.00 1.41 1.25 1.26 P 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 1000mg a.i./kg Day 15 P - Number of Emerged Seedlingsin Replicate: A B C D 9 8 9 9 8 9 9 10 10 8 8 10 9 7 8 10 10 7 7 8 3 0 1 1 --- n Mean Std. Dev. 4 8.75 0.50 4 9.00 0.82 4 9.00 1.15 4 8.50 1.29 4 8.00 1.41 4 1.25 1.26 - Treatment Group P Control 3.91 mg a.i./kg 15.6mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg 1 Day 21 - - Number of Emerged Seedlingsin Replicate: A B C D __e 9 8 9 9 8 9 9 10 10 8 8 10 9 7 8 10 10 8 7 8 3 0 1 1 -- n 4 4 4 4 4 4 Mean 8.75 9.00 9.00 8.50 8.25 1.25 P Std. Dev. 0.50 0.82 1.15 1.29 1.26 1.26 - 97 - Appendix 8.2 Mean Lettuce Emergence on Day 2 1 PROJECT NO.: 454-1 10 10 o Data -Regression --. - - - - .- - 95% Conf Int. < 50?6 Inhibition 2 0 200 400 600 800 1000 1200 Concentration (mgaiJkg) EC25 393.369 EC50 564.027 Lower 95% Confidence Limit 300.193 Lower 95% Confidence Limit 474.024 Curve Parameters Upper 95% Confidence Limit 515.466 Rl 8.8126 Upper 95% Confidence Limit 671.120 Rl 8.8126 0 0.2320 0 0.2320 ? 0.99632 rz 0.99632 - 98 - PROJECT NO.: 454-110 Appendix 8.3 Lettuce 21-Day Survival - Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000mg a.i./kg - Number of Living Seedlings in Replicate: A B C D 9 8 9 9 8 9 9 10 10 8 8 10 9 7 8 10 8 6 7 6 1 0 0 1 n Mean P 4 8.75 4 9.00 4 9.00 4 8.50 4 6.75 4 0.50 - Std. Dev. 0.50 0.82 1.15 1.29 0.96 0.58 112 , 10 2 0 0 200 400 600 800 1000 Concentration (mg s i J k g ) ECzs 257.276 EGO 386.01 1 Lower 95% Confidence Limit 220.141 Lower 95% Confidence Limit 343.795 Curve Parameters Upper 95% Confidence Limit 300.677 Ra 8.8166 Upper 95% Confidence Limit 433.411 Rl 8.8166 1 o Data --Regression - - .- - - . 95% Conf. Int. 50??Inhibition 1200 Is 0.2613 0 0.2613 12 0.99719 8 0.99719 -99- PROJE'CTNO.: 454-110 Appendix 8.4 - - Treatment Grou 3 Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a i l k g 1000 mg a.i./kg p A 0.46 0.21 0.32 0.06 0.01 0.00 Lettuce Mean Seedling Fresh Weight, Day 2 1 - - Mean Weight (g) for Replicate: B C D n -- - 0.38 0.32 0.38 4 Mean 0.38 0.28 0.20 0.30 4 0.25 0.13 0.27 0.26 4 0.24 0.03 0.06 0.03 4 0.05 0.01 0.02 0.00 4 0.01 0.00 2 0.00 --Std. Dev. 0.06 0.05 0.08 0.02 0.01 0.00 3p~ 0.4 .s-f 0.3 4 0.2 0.1 0.0 0 200 400 600 800 1000 Concentration(mg aiJkg) EC25 8.92483 ECso 20.1419 Lower 95% Confidence Limit 1.36490 Lower 95% Confidence Limit 5.26260 Curve Parameters Upper 95% Confidence Limit 58.3445 Ro 0.3466 upper 95% Confidence Limit 77.108 1 Ro 0.3466 0 Data -Regression - - - - .- - !)5% Conf. Int. 50% Inhibition 1200 0 0.5242 (J 0.5242 12 0.94722 I 0.94722 - 100 - PROJECT NO.: 454-110 Appendix 8.5 Lettuce SeedlingHeight on Day 21 Treatment Group Replicate Height (cm) for Plant Number: n 12345678910 Control A .6789568789 B .896845658 C . 4 5 4 8 5 6 10 5 6 9 D .6786666579 3.91 mg a.i./kg A .655445758 B .5567775449 C .6634633349 D 744376347510 15.6 mg a.i./kg A 4 5 6 7 4 6 5 6 8 10 10 B .443535328 C .476645848 D 6 4 5 6 3 4 5 5 7 2 10 62.5 mg a.i./kg A .5522223229 B .3223225 7 C ,545123228 D 2 3 2 1 1 2 2 2 3 2 10 250 mg a.i./kg A B C D .121111118 .1111116 .11111117 .1111116 1000mg a.i./kg A .1I B .o C .o D .I1 The "." symbol indicates that the seedling either did not emerge or died prior to measurement. Mean 7.1 6.4 5.9 6.3 5.1 5.6 4.2 5.0 6.1 3.6 5.5 4.7 2.8 2.7 3.0 2.0 1.1 1.o 1.o 1.o 1.o 1.o 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 - PROJECT NO.: 454-110 Treatment Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i.kg Appendix 8.6 Lettuce Mean Seedling Height on Day 2 1 Mean Height (cm) for Replicate: 7.1 6.4 5.9 6.3 4 5.1 5.6 4.2 5.0 4 6.1 3.6 5.5 4.7 4 2.8 2.7 3.0 2.0 4 1.1 1.o 1.o 1.o 4 1.o 1.o 2 - Std. Dev. 6.4 0.51 5 .O 0.56 5.0 1.07 2.6 0.43 1.o 0.06 1.o 0.00 ,12 I 0 Data Regression - - - - .- - 95% Conf. Int. 50% Inhibition EC25 6.79360 EC50 39.8749 Concentration(mg a.iJkg) Lower 95% Confidence Limit 0.20399 Lower 95% Confidence Limit 3.87972 Curve Parameters Upper 95% Confidence Limit 226.204 Ro 6.5025 Upper 95% Confidence Limit 409.732 Ro 6.5025 I 0 1.1396 (T 1.1396 - 102 - z 0.94294 12 0.94294 PROJECT NO.: 454-1 10 Appendix 8.7 Lettuce Seedling Condition, Day 2 1 Treatment Group Replicate 1 Control A B C D Condition (scoresign)' for Plant Number: 2 3 4 5 6 7 8 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.- --n Mean Std. Dev. 9 10 0.- 0.- 9 0 0.0 0.- 0.- 8 0 0.0 0.- 0.- 9 0 0.0 0.- 0.- 9 0 0.0 3.91 mg a.i./kg A B C D 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.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 15.6 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- IO 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 40.N 10 4 12.6 62.5 mg a.i./kg A 0.- 0.- 50.N 30.N 0.- 50.N 50.N 50.N 40.N 9 30 23.5 B 40.N 40.N 40.N 40.N 40.N 30.N 20.N 7 36 1.9 C 0.- 20.N 0.- 0.- 50.N 30.N 50.N 50.N 8 25 23.3 D 40.N 40.N 40.N 40.N 30.N 30.N 40.N 50.N 30.N 50.N 10 39 1.4 250 mg a.i./kg A 100.- 100.- 80.N 0.- 40.N 0.- 60.N 0.- 60.N 0.- 10 44 42.0 B 100.- 100.- 0.- 40.N 0.- 0.- 30.N 40.N 8 39 41.6 C 60.N 0.- 30.N 40.N 40.N 40.N 0.- 7 30 22.4 D 100.- 100.- 30.N 80.N 0.- 40.N 50.N,LC 30.N 8 54 36.2 1000 mg a.i./kg A 100.- 100.- 80.N 3 93 11.5 B 0 L 100.- I 100 D 9o.usc I 90 'The "." symbol indicates that the seedling did not emerge. A score of 0 indicates a normal seedling, while a score of 100 indicates a dead seedling. Intermediate scores are assigned to indicate the relative severity of observed signs of toxicity. N -Necrosis, USC - Unshed Seed Coat, LC - Leaf Curl - 103 - PROJECT NO.: 454-110 Appendix 9.1 Onion Emergence P Treatment Number of Emerged Seedlings in Replicate: P Group _A. B C D n P Control 8 7 8 9 4 3.91 mg a.i./kg 10 8 9 7 4 15.6 mg a.i./kg 8 8 7 8 4 62.5 mg a.i./kg 4 8 4 4 4 250 mg a.i./kg 5 3 0 0 4 1000 mg a.i./kg 0 0 0 0 4 P Treatment P GBrou Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Day 15 Number A of Emerged Seedlings C - in Replicate: D n 8 7 9 10 4 10 10 9 7 4 8 9 8 8 4 5 10 6 4 4 5 8 2 1 4 0 0 0 0 4 Mean 8.00 8.50 7.75 5.00 2.00 0.00 - Std. Dev. 0.82 1.29 0.50 2.00 2.45 0.00 Mean 8.50 9.00 8.25 6.25 4.00 0.00 Std. Dev. 1.29 1.41 0.50 2.63 3.16 0.00 - Treatment Number of Emerged Seedlings in Replicate: n Mean Std. Dev. Control 8 7 10 10 4 8.75 1.50 3.91 mg a.i./kg 10 10 9 8 4 9.25 0.96 15.6mg a.i.kg 8 9 8 8 4 8.25 0.50 62.5 mg a.i./kg 5 10 6 4 4 6.25 2.63 250 mg a.i./kg 5 8 2 1 4 4.00 3.16 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 - 104- PROJElCTNO.: 454-110 Appendix 9.2 Mean Onion Emergence on Day 2 1 12 10 T 8 2 c v I- 8 0 86 E 4 2 * I 0 Treatment Group *Treatment group mean is significantly different from the control mean (Dunnett's test, p<0.015). 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 L.imit EC25 50.7750 12.1515 194.5350 EC50 208.3333 -25.0000 644.2308 ECZ5and ECsoestimates calculated by linear interpolation using the ICPIN program (7). - 105 - PROJECT NO.: 454-110 Appendix 9.3 Onion 21-Day Survival Treatment Number of Living Seedlings in Replicate: Group A B C D n P Control 8 7 9 10 4 3.91 mg a.i./kg 10 10 8 8 4 15.6 mg a.i./kg 7 9 8 8 4 62.5 mg a.i./kg 3 4 3 3 4 250 mg a.i./kg 0 0 0 0 4 1000 mg a.i./kg 0 0 0 0 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 12.0 10.0 8.0 6.0 4.0 2.0 0.0 -2.0 ECzs 47.0977 EGO 57.2928 Concentration(mgPiJkg) Lower 95% Confidence Limit 0.99238 Curve Parameters Upper 95% Confidence Limit 2234.60 Ra 8.5000 Lower 95% Confidence Limit 17.4622 Upper 95% Confidence Limit 187.975 Ro 8.5000 - 106 - o Data Regression - .- - - - - 95% Conf. Int. 1 50% Inhibition I 0 0.1262 0 0.1262 ? 0.99445 1.2 0.99445 PROJECT NO.: 454-1 10 Appendix 9.4 Onion Mean Seedling Fresh Weight, Day 2 1 P P A Treatment Grou Mean Weight (8) for Replicate: B C D n Control 0.10 0.09 0.10 0.10 4 3.91 mg a.i./kg 0.06 0.09 0.09 0.10 4 15.6 mg a.i./kg 0.06 0.06 0.08 0.07 4 62.5 mg a.i./kg 0.02 0.05 0.03 0.00 4 250 mg a.i./kg 0 1000 mg a.i./kg 0 P - Mean 0.10 0.09 0.07 0.02 Std. Dev. I 0.006 0.0 15 0.006 0.019 0.25 , 1 5 I A1A U.IU y 1 '\ , Q) R .&\. Regression I 95% Conf. Int. I 50% Inhibition 0.00 I 0 200 400 600 800 Concentration(mga.iJkg) 1000 1:!00 EC25 12.9092 ECso 28.1 125 Lower 95% Confidence Limit 0.16110 Lower 95% Confidence Limit 1.99434 Curve Parameters Upper 95% Confidence Limit 1034.67 Ro 0.0957 ..UDDel' 95% Confidence Limit 396.369 Ro 0.0957 rn 0.501 1 0 0.5011 12 0.98693 f 0.98693 - 107 - PROJECT NO.: 454-110 Appendix 9.5 -Treatment Group P Control Replicate - 1 - - - Onion SeedlingHeight on Day 21 Height (cm) for Plant Number: n 2 3 4 5 6 7 8 9 10 g. A .13489951388 B .87787887 C 77107971388. 9 D 2 7 7 8 8 1 0 1 0 1 4 8 2 IO 3.91 mg a.i./kg A 7 6 7 6 7 8 7 7 7 510 B 889578887710 C .889977788 D .89981878 8 15.6 mg a.i./kg A .565118897 B .12388867869 C .9146818748 D .857983988 62.5 mg a.i./kg A B C D .3243 .45214 .3153 .1123 250 mg a.i./kg A B C D .o .o .o .o 1000 mg a.i./kg A .o B .o C .o D .o The "." symbol indicates that the seedling either did not emerge or died prior to measurement. P -- Mean Std. Dev. 8.6 3.25 7.6 0.53 8.4 2.01 7.6 3.60 6.7 0.82 7.5 1.08 7.9 0.83 7.3 2.60 7.4 2.23 7.3 2.40 7.1 3.80 7.1 2.10 3 .O 1.oo 3.0 1.83 3.0 2.00 1.3 0.58 - 108 - PROJElCT NO.: 454-1 10 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 9.6 Onion Mean SeedlingHeight on Day 21 Mean Height (cm) for Replicate: A B C D n 8.6 7.6 8.4 7.6 4 6.7 7.5 7.9 7.3 4 7.4 7.3 7.1 7.1 4 3.0 3.0 3.0 1.3 4 0 0 Mean 8.1 7.3 7.3 2.6 Std. Dev. 0.55 0.49 0.15 0.83 18 13 -2 -7 ' Concentration (mg aiJkg) EC25 29.0737 ______~ EC50 46.5050 Lower 95% Confidence Limit 0.93476 Lower 95% Confidence Limit 8.94129 Curve Parameters U.uu.er 95% Confidence Limit 904.066 Ro 7.6985 Upper 95% Confidence Limit 241.824 Ra 7.6985 o Data -Regression - - - - .- - 95?6 Conf. Int. J 0 0.3025 0 0.3025 IJ 0.98600 12 0.98600 - 109 - PROJECT NO.: 454-110 Appendix 9.7 Onion SeedlingCondition, Day 21 Treatment Group Replicate Condition (score.sign)' for Plant Number: n Mean DSetdv.. 1 2 3 4 5 6 7 8 9 10 Control A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 0.0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 100.- IO 10 31.6 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 3.91 mg a.i./kg A 0.- 0.- 0.- 40.CL 0.- 0.- 0.- 0.- 0.- 0.- 10 4 12.6 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- IO 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 A 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 13 35.4 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 D 0.- 0.- 0.- 0.- 0.- 60.N 0.- 0.- 8 8 21.2 62.5 mg a.i./kg A 100.- 100.- 0.- 0.- 0.- 5 40 54.8 B 100.- 100.- 100.- 100.- 100.- 100.- 0.- 0.- 0.- 0.- 10 60 51.6 C . 100.- 100.- 100.- 0.- 0.- 0.- 6 50 54.8 D 100.- 0.- 0.- 0.- 4 25 50.0 250 mg a.i./kg A 100.- 100.- 100.- 100.- 100.- 5 100 0.0 B 100.- 100.- 100.- 100.- 100.- 100.- 100.- 100.- 8 100 0.0 C 100.- 100.- 2 100 0.0 D 100.- I 100 1000 mg a.i./kg A 0 B 0 C 0 D 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, CL - Chlorosis - 110 - 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 9 9 9 7 4 8.50 1.oo 15.6 mg a.i./kg 8 6 10 6 4 7.50 1.91 62.5 mg a.i./kg 7 7 8 8 4 7.50 0.58 250 mg a.i./kg 6 4 3 3 4 4.00 1.41 1000 mg a.i./kg 0 0 0 0 4 0.00 0.00 Treatment Number of Emerged Seedlings in Replicate: Group A B C D n Mean Std. Dev. Control 8 9 7 8 4 8.00 0.82 3.91 mg a.i./kg 9 10 10 8 4 9.25 0.96 15.6 mg a.i./kg 9 9 10 8 4 9.00 0.82 62.5 mg a.i./kg 7 8 9 9 4 8.25 0.96 250 mg a.i./kg 6 4 7 6 4 5.75 1.26 1000 mg a.i./kg 1 0 0 0 4 0.25 0.50 Treatment Dav 21 Number of Emerged Seedlings in Replicate: Control 8 9 7 8 4 3.91 mg a.i./kg 9 10 10 8 4 15.6 mg a.i./kg 9 9 10 8 4 62.5 mg a.i./kg 7 8 9 10 4 250 mg a.i./kg 6 4 7 6 4 1000 mg a.i./kg 1 1 0 1 4 8.00 9.25 9.00 8.50 5.75 0.75 Std. Dev. P 0.82 0.96 0.82 1.29 1.26 0.50 - 111 - Appendix 10.2 Mean Ryegrass Emergence on Day 2 1 PROJBCT NO.: 454-1 10 I--. 12 ...-- 10 \ --\, .. \ ---. 0 Data --Regression .- - - - - . 95% Conf. Int. 50?4 Inhibition 0 200 400 600 800 1000 1200 Concentration(mga i l k g ) EC25 202.675 EC50 343.637 Lower 95% Confidence Limit 13 1.220 Lower 95% Confidence Limit 253.805 Curve Parameters Upper 95% Confidence Limit 3 13.040 Ra 8.7223 Upper 95% Confidence Limit 465.265 Ra 8.7223 rs 0.3400 rs 0.3400 1-2 0.98361 ? 0.98361 - 112- PROJECT NO.: 454-110 Appendix 10.3 Ryegrass 21-Day Survival Treatment Number of Living Seedlings in Replicate: Group A B C D n Control 8 9 7 8 4 3.91 mg a.i./kg 9 10 10 8 4 15.6 mg a.i./kg 9 9 10 8 4 62.5 mg a.i./kg 7 8 9 9 4 250 mg a.i./kg 6 2 7 6 4 1000mg a.i./kg 1 1 0 1 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 12 10 g- 8 W E6 'E 3 m4 2 0 0 ECzs 173.500 ECso 3 10.099 o Data Regress ion - .- - - - . 95% Conf. Int. 50% Inhibition 200 400 600 800 1000 1200 Concentration(mgai./kg) Lower 95% Confidence Limit 107.029 Lower 95% Confidence Limit 222.844 Curve Parameters upper 95% Confidence Limit 281.255 Ro 8.7020 UD.D.er 95% Confidence Limit 431.618 Ro 8.7020 I3 0.3740 (s 0.3740 rz 0.98409 f 0.98409 - 113 - PROJECT NO.: 454-110 Appendix 10.4 P 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.ikg 0.20 af, n 29 0.15 Ej M .I g 0.10 E 0.05 0.00 - -- Ryegrass Mean SeedlingFresh Weight, Day 21 P - - -- Mean Weight (g) for Replicate: A B C D n Mean Std. Dev. 0.12 0.09 0.12 0.16 4 0.12 0.025 0.09 0.12 0.12 0.11 4 0.1 1 0.013 0.07 0.06 0.09 0.08 4 0.07 0.014 ,0.07 0.07 0.10 0.09 4 0.08 0.014 0.01 0.02 0.01 0.01 4 0.01 0.006 0.03 0.04 0.02 3 0.03 0.010 o Data Regressicin .- - - - .- 95% Conf. Int. 50% Inhibition 800____ .____. 1.__ 0_ 0_0 __. 1200 EC25 7.50585 ECso 53.8022 95% Confidence Limit 0.0038601 13 Lower 95% Confidence Limit 0.41620 Curve Parameters Upper 95% Confidence Limit 14594.86 Ro 0.1262 upper 95% Confidence Limit 6953.44 Ro 0.1262 - 114 - 0 1.26881 CY 1.2681 12 0.92024 rz 0.83214 PROJECT NO.: 454-110 Appendix 10.5 P Treatment Grnun Replicate Ryegrass Seedling Height on Day 2 1 Height (cm) for Plant Number: 1234 5 6789 --- n Mean Std. Dev. 10 Control . A 15 15 21 20 16 16 11 20 .8 16.8 3.37 B . 10 15 14 22 17 19 12 19 19 9 16.3 3.87 . C . 17 18 11 22 20 19 18 7 17.9 3.44 D 18 18 19 15 14 19 16 13 8 16.5 2.33 3.91 mg a.i./kg A . 13 17 15 16 12 20 16 17 12 9 15.3 2.65 B 18 17 18 20 18 21 18 15 21 6 10 17.2 4.34 . C 18 16 9 15 14 6 21 13 17 17 IO 14.6 4.40 D 12 16 14 14 10 15 15 19 8 14.4 2.67 . 15.6 mg a.i./kg A 12 13 13 9 19 12 12 14 17 9 13.4 2.96 B . 19 19 11 21 8 9 14 9 5 9 12.8 5.72 C 19 14 16 13 19 13 8 14 11 11 10 13.8 3.49 D . 14 13 21 12 19 12 14 12 8 14.6 3.46 62.5 mg a.i./kg A . 9 5 16 18 17 15 12 7 13.1 4.74 B . 20 9 6 10 13 15 14 17 8 13.0 4.54 C . 20 12 19 16 4 15 16 18 15 9 15.0 4.77 D . 15 12 10 17 13 13 7 15 19 9 13.4 3.61 250 mg a.i./kg A B C D .4564536 4.5 1.05 .542 4.5 0.7 1 .26222257 3 .O 1.73 .4214326 2.7 1.21 1000mg a.i./kg A .21 2.0 B .21 2.0 C .o D .21 2.0 The "." symbol indicates that the seedling either did not emerge or died prior to measurement. - 115 - PROJECT NO.: 454-1 10 Treatment Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 10.6 Ryegrass Mean SeedlingHeight on Day 21 Mean Height (cm) for Replicate: 16.8 16.3 17.9 16.5 4 15.3 17.2 14.6 14.4 4 13.4 12.8 13.8 14.6 4 13.1 13.0 15.0 13.4 4 4.5 4.5 3.0 2.7 4 2.0 2.0 2.0 3 Mean 16.9 15.4 13.7 13.6 3.7 2.0 Std. Dev. 0.69 1.28 0.77 0.92 0.97 0.00 o Data -Regression .......95% Ibnf. Int. 50% hhibition -5 1 EC25 46.3554 EGO 131.099 30 I Concentration(mg aiJkg) Lower 95% Confidence Limit 4.63127 Lower 95% Confidence Limit 28.7740 Curve Parameters Upper 95% Confidence Limit 464.088 Ro 16.2180 up.p.er 95% Confidence Limit 597.173 - KO 16.2180 0 0.6693 0 0.6693 1.2 0.92846 f 0.92846 - 116 - PROJECT NO.: 454-1 10 Appendix 10.7 Ryegrass Seedling Condition, Day 2 1 Treatment Group Replicate Condition (scoresign)' for Plant Number: 1 2 3 4 5 6 7 8 9 10 P 8 - Std. Mean Dev. Control 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 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 0.0 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 3.91 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 15.6 mg a.i.kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 62.5 mg a.i.kg A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 7 0 0.0 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 8 0 0.0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 D 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 10 31.6 250 mg a.i.kg A B C D 10.N 0.- 1O.N 10.N 1O.N 0.- 6 7 5.2 100.- 100.- 0.- 0.- 4 50 57.7 30.N 0.- 20.N 20.N 20.N 40.N 0.- 7 19 14.6 0.- 50.N 90.N 0.- 0.- 0.- 6 23 38.3 1000 mg a.i.kg A B 10.N I 10 30.N I 30 C 0 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 - 117 - PROJECT NO.: 454-1 10 Appendix 11.1 Soybean Emergence P - Day 7 - Treatment Group - Number of Emerged Seedlings in Replicate: A B C D P n P Control 10 10 10 9 4 3.91 mg a.i./kg 10 10 9 10 4 15.6 mg a.i./kg 9 10 10 9 4 62.5 mg a.i./kg 10 10 10 10 4 250 mg a.i./kg 10 10 10 10 4 1000 mg a.i./kg 9 10 10 10 4 p - -- Mean 9.75 Std. Dev. 0.50 9.75 0.50 9.50 0.58 10.00 0.00 10.00 0.00 9.75 0.50 P Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Day 15 - Number of Emerged Seedlings in Replicate: - A B C D P 10 10 10 10 10 10 9 10 9 10 10 9 10 10 10 10 10 10 10 10 10 10 10 10 - n Mean 4 10.00 4 9.75 4 9.50 4 10.00 4 10.00 4 10.00 - Std. Dev. 0.00 0.50 0.58 0.00 0.00 0.00 , Treatment Group Control 3.91 mg a.i./kg 15.6mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000mg a.i./kg Day 21 - Number of Emerged Seedlings in Replicate: A B C D n P 10 10 10 10 4 10 10 9 10 4 9 10 10 9 4 10 10 10 10 4 10 10 10 10 4 10 10 10 10 4 -- Mean 10.00 Std. Dev. 0.00 9.75 0.50 9.50 0.58 10.00 0.00 10.00 0.00 10.00 0.00 - 118 - Appendix 11.2 Mean Soybean Emergence on Day 2 1 T PROJECT NO.: 454-110 Treatment Group No treatment group mean is significantly different from the control mean (Dunnettt's test, p>0.05). No regression was conducted due to the lack of effects. - 119 - PROJECT NO.: 454-110 Treatment P Control 3.91 mg a.i./kg 15.6mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 11.3 Soybean 21-Day Survival 7 -- Number of Living Seedlings in Replicate: n Mean Std. Dev.- 10 10 10 10 4 10.00 0.00 10 10 9 10 4 9.75 0.50 9 10 10 9 4 9.50 0.58 10 10 10 10 4 10.00 0.00 9 10 10 10 4 9.75 0.50 10 10 10 10 4 10.00 0.00 t- l2 10 T r 8 6 4 2 0 Treatment Group No treatment group mean is significantly different kom the control mean (Dunnett's test, p0.105). No regression was conducted due to the lack of effects. - 120 - PROJECT NO.: 454-1 10 - Treatment Grou Control 3.91 mg a i l k g 15.6 mg a.i.1kg 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: A C D n 3.42 4.46 3.55 3.10 4 3.75 3.92 3.49 3.41 4 3.94 3.59 3.80 3.18 4 3.81 3.71 4.12 4.37 4 2.36 2.11 1.63 2.18 4 0.62 0.60 0.53 0.54 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 -1 EC25 160.325 ECso 325.762 200 400 600 800 1000 Concentration (mg a.iJkg) Curve Parameters Lower 95% Confidence Limit 69.1194 Upper 95% Confidence Limit 371.878 Ro 3.7487 Lower 95% Confidence Limit 189.234 Upper 95% Confidence Limit 560.661 Ro 3.7487 - 1 0 Data Regress ion ..- . .- - 95% Conf. Int. 50% Inhibition 0 0.4565 Is 0.4565 12 0.95631 12 0.9563 1 - 121 - PROJECT NO.:454-1 10 Appendix 1 1.5 Soybean Seedling Height on Day 2 1 Treatment Group Control -- Replicate Height (cm) for Plant Number: n 1 2 3 4 5 6 7 8 9 10 Mean Std. Dev. A 25 28 28 25 24 28 25 14 31 35 10 26.3 5.46 B 32 39 38 34 27 33 31 34 39 19 10 32.6 6.10 C 31 29 24 33 30 33 29 35 29 36 1 0 30.9 3.51 D 18 13 13 23 23 24 33 30 30 28 10 23.5 7.04 3.91 mg a.i./kg A 33 36 33 27 28 35 31 24 29 31 10 30.7 3.74 B 27 25 26 21 34 22 37 28 21 38 IO 27.9 6.37 C . 22 30 30 27 25 20 13 24 21 9 23.6 5.36 D 28 29 19 30 26 20 33 15 29 21 1 0 25.0 5.85 15.6 mg a.i./kg A . 29 31 29 32 26 27 32 41 25 9 30.2 4.76 B 20 21 24 29 29 25 30 15 29 24 10 24.6 4.88 C 28 33 23 30 29 28 32 31 27 31 10 29.2 2.90 D . 23 24 24 22 19 25 27 20 28 9 23.6 2.96 62.5 mg a.i./kg A 23 28 29 33 28 19 32 25 28 28 10 27.3 4.11 B 34 31 32 36 31 31 35 38 13 34 10 31.5 6.92 C 34 38 31 32 31 28 29 23 32 29 IO 30.7 3.95 D 32 36 33 29 31 35 37 33 35 32 10 33.3 2.45 250 mg a.i./kg A . 20 22 21 28 27 17 28 28 12 9 22.6 5.70 B 29 31 20 20 19 21 4 27 31 29 10 23.1 8.27 C' 22 24 17 18 20 20 21 16 22 14 1 0 19.4 3.10 D 21 25 24 23 25 23 29 26 29 23 10 24.8 2.62 1000 mg a.i./kg A 2 1 3 3 4 8 5 2 5 3 IO 3.6 2.01 B 4 3 6 4 6 4 3 4 4 3 IO 4.1 1.10 C 3 53463856210 4.5 1.84 D 543544456310 4.3 0.95 The "." symbol indicates that the seedling either did not emerge or died prior to measurement. - 122 - PROJECT NO.: 454-110 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 11.6 Soybean Mean Seedling Height on Day 2 1 Mean Height (cm) for Replicate: A B C D n 26.3 32.6 30.9 23.5 4 30.7 27.9 23.6 25.0 4 30.2 24.6 29.2 23.6 4 27.3 31.5 30.7 33.3 4 22.6 23.1 19.4 24.8 4 3.6 4.1 4.5 4.3 4 _3_ - Mean 28.3 Std. Dev. 4.17 26.8 3.17 26.9 3.31 30.7 2.51 22.5 2.26 4.1 0.39 40 35 30 25 Y 2 20 M .d a 15 10 5 0 0 0 Data Regression ..- - - - - 95% 'Conf. Int. 500/0 lnhibition 200 400 600 800 1000 1200 Concentration(mg ai./kg) EC25 284.053 ECso 464.195 Lower 95% Confidence Limit 172.346 Lower 95% Confidence Limit 332.583 Curve Parameters Upper 95% Confidence Limit 468.274 Ro 28.1676 Upper 95% Confidence Limit 647.888 Ro 28.1676 - 123 - 0 0.3162 Is 0.3162 12 0.97727 12 0.97727 PROJECT NO.: 454-110 Appendix 11.7 Soybean Seedling Condition, Day 2 1 Treatment Replicate Condition (scoresign)' for Plant Number: Std. Group - Mean Dev. 1 2 3 4 5 6 7 8 9 10 Control A 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 B 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 C 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 3.91 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 0.- 0.- 0.- 30.LC 0.- 0.- 0.- 0.- 13.- 0.- 10 3 9.5 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 15.6 mg a.i.kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- IO 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 9 0 0.0 62.5 mg a.i.kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 0.- 0.- 0.- 0.- 40.LC 0.- 10 4 12.6 0.- 0.- 0.- 30.LC 0.- 0.- 10 3 9.5 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 250 mg a.i.kg A 100.- 0.- B 0.- 0.- C 20.E 0.- D 0.- 0.- 0.- 20.CL 0.- 0.- 0.- 0.- 0.- 0.- 10 12 31.6 0.- 0.- 0.- 0.- 90.N 0.- 20.LC 0.- 10 11 28.5 0.- 0.- 20.CL 0.- 0.- 0.- 10.- 1 O . E 10 5 8.5 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 10 0 0.0 1000 mg a.i.kg A 50.SC 60.SC 40.N 50.N 40.N 6 0 . E 40.N 50.N 4 0 . w 50.N 10 48 7.9 B 50.N 70.SC,N 3 0 . E 40.LC,N 20.w 50.N 50.N 60.N 3 0 . E 50.N 10 45 15.1 C 80.N 70.N 6O.USC 70.SC.N 60.SC.N ,50.SC..N 4 0 . K 4 0 . E 4 0 . E 90.SC 10 60 17.6 ' The D 40.LC "." symbol indicates 50.N that the 70.N seedling did 60.N 50.N not emerge. A score 30. of 0 E 40.N indicates a 5O.USC 60.N 60.N normal seedling, while a sccire 10 of 100 51 12.0 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 - 124 - PROJECT NO.: 454-110 Control 9 3.91 mg a.i./kg 6 15.6 mg a.i./kg 6 62.5 mg a.i./kg 8 250 mg a.i./kg 0 1000 mg a.i./kg 0 Appendix 12.1 Tomato Emergence 7 5 5 6 5 9 2 9 4 4 6 4 3 2 0 0 0 0 - Mean Std. Dev. P 4 6.50 1.91 4 6.50 1.73 4 5.25 2.99 4 5.50 1.91 4 1.25 1S O 4 0.00 0.00 Treatment Group P - --- Number of Emerged Seedlings in Replicate: A B C D n Mean Control 10 9 9 8 4 9.00 3.91 mg a.i./kg 9 7 7 9 4 8.00 15.6 mg a.i./kg 9 8 10 6 4 8.25 62.5 mg a.i./kg 10 6 8 9 4 8.25 250 mg a.i./kg 6 8 8 7 4 7.25 1000mg a.i./kg 3 0 0 0 4 0.75 Std. Dev. 0.82 1.15 1.71 1.71 0.96 1S O 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 221 - Number of Emerged Seedlings in Replicate: C A B _L D P 10 10 9 8 9 7 7 10 9 8 10 6 10 6 9 9 6 8 8 7 3 1 0 0 - -- -- n Mean 4 9.25 4 8.25 4 8.25 4 8.50 4 7.25 4 1.oo - Std. Dev. 0.96 1.50 1.71 1.73 0.96 1.41 - 125 - Appendix 12.2 Mean Tomato Emergence on Day 2 1 PROJECT NO.: 454-110 0 Daka Regression - - - - - - . 9.5%Conf. Int. 50% Inhibition 0 200 400 600 800 1000 1200 Concentration (mg eiJkg) EC25 310.670 ECso 474.133 Lower 95% Confidence Limit 207.874 Lower 95% Confidence Limit 359.832 Curve Parameters Upper 95% Confidence Limit 464.408 Ro 8.5640 Upper 95% Confidence Limit 624.741 Ro 8.5640 rs 0.2721 0 0.2721 Iz 0.98557 12 0.98557 - 126 - PROJECT NO.: 454-1 10 Treatment Group Control 3.91 mg a.i./kg 15.6 mg a.i./kg 62.5 mg a.i./kg 250 mg a.i./kg 1000 mg a.i./kg Appendix 12.3 Tomato 21-Day Survival - Number of Living Seedlings in Replicate: A B C D n P 10 10 9 8 4 9 7 7 10 4 9 8 9 6 4 8 6 6 7 4 2 0 0 1 4 0 0 0 0 4 - Mean 9.25 8.25 8.00 6.75 0.75 0.00 - Std. Dev. 0.96 1S O 1.41 0.96 0.96 0.00 -2 EC25 68.6594 ECso 105.463 - o Data Re,gression - - - - .- - 95?hConf. Int. 50% Inhibition 7 7 200 400 600 800 1000 Concentration(mg aiJkg) Curve Parameters Lower 95% Confidence Limit 45.0091 Upper 95% Confidence Limit 104.761 Ro 8.5031 LOWU 95% Confidence Limit 77.7857 Upper 95% Confidence Limit 142.988 Ra 8.5031 0 0.2763 G 0.2763 rz 0.98988 12 0.98988 - 127 - PROJElCT NO.: 454-1 10 Appendix 12.4 P 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 Tomato Mean Seedling Fresh Weight, Day 2 1 P Mean Weight (8) for Replicate: B A C D n 0.3 1 0.23 0.56 0.3 1 4 0.42 0.29 0.49 0.40 4 0.30 0.13 0.39 0.32 4 0.06 0.12 0.07 0.06 4 0.03 1 0 --- Mean 0.35 0.40 0.28 0.08 0.03 Std. Dev. 0.143 0.081 0.108 0.029 0.90 0.80 - 0.60 n M 0.50 2 .y 0.40 0.30 f 0.20 & 0.10 -00..0100 $JA\ .___..-_.-_~_~---4-(_3_0-___-__-__-__ -_-_6-0_-0'__--.-8-0.0- 1000 -0.20 4 Concentration (mga.iJkg) Curve Parameters EC2.5 11.703 1 Lower 95% Confidence Limit 0.63139 Upper 95% Confidence Limit 216.920 Ro 0.3874 EC50 28.5102 Lower 95% Confidence Limit 3.80978 upper 95% Confidence Limit 213.354 Ro 0.3874 0 Data Regres2; ion . . - . . - -95% Calnf. Int. 50% Inhibition 0 0.5734 0 0.5734 ? 0.93492 ? 0.93492 - 128 - 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 .8665666659 D .473495658 3.91 mg a.i./kg A .7564768569 B .75753747 C .56356577 D 565857626610 15.6mg a.i./kg A .6732354479 B .342542358 C .5453467879 D .5574576 62.5 mg a.i./kg A .22332.3348 B .3322336 C .3342226 D .3223223.7 250 mg a.i./kg A B C D .222 .o .o .21 1000mg a.i./kg A .o B .o C .o D .o The "." symbol indicates that the seedling either did not emerge or died prior to measurement. 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 - PROJECT NO.: 454-110 Appendix 12.6 Tomato Mean SeedlingHeight on Day 21 v Treatment Mean Height (cm) for Replicate: GrouAp B C D n Control 5.3 4.2 6.0 5.4 4 3.91 mg a.i./kg 6.0 5.4 5.3 5.6 4 15.6 mg a.i./kg 4.6 3.5 5.4 5.5 4 62.5 mg a.i./kg 2.8 2.7 2.7 2.4 4 250 mg a.i./kg 2.0 2.0 2 1000 mg a.i./kg 0 Mean 5.2 5.6 4.8 2.6 2.0 - Std. Dev. _.- 0.75 0.3 1 0.94 0.14 0.00 12 o Data -Regression .- - - - - - 959h Conf. Int. 50% Inhibition -4 EC25 22.141 1 ECso 93.8642 Concentration(mg eiJkg) Lower 95% Confidence Limit 0.45165 Lower 95% Confidence Limit 10.3348 Curve Parameters Upper 95% Confidence Limit 1085.43 Ro 5.5594 Upper 95% Confidence Limit 852.3 15 Ro 5.5594 I 0 0.9299 0 0.9299 12 0.91119 12 0.91 119 - 130 - PROJElCT NO.: 454-110 Appendix 12.7 TrGearotmupent Replicate T 1 Tomato Seedling Condition, Day 2 1 -- Condition (score.sign)' for Plant Number: 2 3 4 5 6 7 8 9 10-- Mean Control 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.- 9 0 D 0.- 0.- 0.- 0.- 0.- 0.- 0:- 0.- 8 0 3.91 mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0:- 0.- 9 0 0.- 0.- 0.- 0.- 0.- 0:- 0.- 7 0 0.- 0.- 0.- 0.- 0.- 0:- 0.- 7 0 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0." 0.- 10 0 15.6mg a.i./kg A B C D 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0." 0.- 9 0 . 40.N 0.- 0.- 0.- 30.N 40.N 0:- 0.- 8 14 100.- 0.- 0.- 0.- 0.- 0.- 0.- 0.- 0:- 0.- 10 10 0.- 0.- 0.- 1O.LC 0.- 0.- 6 2 62.5 mg a.i./kg A B C D 100.- 50.N 50.N 40.N 30.N 50.N 100.- 50.N 50.N 0.- 10 52 0.- 0.- 20.N 0.- 20.N 20.N 6 10 100.- 100.- 100.- 0.- 40.N 0.- 0.- 40.N 70.N 9 50 100.- 0.- 10.N 30.N 20.N 20.N 20.N 20.N 100.- 9 36 250 mg a.i./kg A B C D 100.- 100.- 100.- 100.- 70.N 70.N 6 90 . 100.- 100.- 100.- 100.- 100.- 100.- 100.- 100.- 8 100 100.- 100.- 100.- 100.- 100.- 100.- 100.- 100.- 8 100 . 100.- 100.- 100.- 100.- 100.- 100.- 80.N 7 97 1000 mg a.i./kg A 100.- 100.- 100.- 3 100 ' B 100.- I 100 C .o D .o. 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, LC - Leaf Curl - Std. Dev. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 19.2 31.6 4.1 29.7 11.0 44.2 37.5 15.5 0.0 0.0 7.6 0.0 - 131 - PROJECT NO.: 454-110 Appendix 13 Bulk Soil Characterization A ML A B O RHA T O REI E S AGVISE Soil Characterization R e p o r t Suknitting firm = WILDLIFE INT. LTD. Protocol or Study No = NA Sample ID. E GHS-01-04 Trial ID. =NA Date Received = 10-1-01 Date Reported = 10-11-2001 W I S E Lab NO 01- 1274 Percent Sand 49 Percent Silt 30 Percent Clay 21 USDA Textural Class (hydrmeter method) Loam Bulk Density (disturbed) sm/CC 1.04 Cation Exchange Capacity (meq/lOO g) 9 .o t. Moisture at 1/3 Bar 23.8 Percent Oryanic Matter 2.1 pH in 1:l soi1:water ratio 5.0 Base Saturation Data Cation Percent ppm calcium Magnesium sodium Potassium Hyrogen 33.4 600 14.8 160 5.3 110 4.6 160 41.9 38 T h e R tjesp were cyplpted in compliance of 40 CFR Part 160. Date Soil Scientist/Analytical Investigator - 132 - PROJECT NO.: 454-110 Appendix 14 Results of Water and Soil Pesticide Screening Wildlife International, Ltd. Well Water: Pesticides and Organics Component Acephate Alachlor Aldicarb sulfone Aldicarb sulfoxide Aldrin Alpha-BHC Ametryne Atrazine Azinphos-ethyl Azinphos-methyl Beta-BHC Bifenox Bitertanol Bromacil Bromophos-methyl Bromoxynil octanoic acid ester Captafol Carbaryl 3-hydroxy Carbofuran Carbofuran Carbophenothion cis-Chlordane transChlordane Chlordimeform Chlorfenson trans-chlorfenvinphos Chlorobenzilate Chloroneb Chloropropham Chloropropylate Chloroxuron Chlorpyrifos-ethyl Chlorpyrifos-methyl Chlorthal Coumaphos Crotoxyphos C yanazine Cyfluthrin I Cypermethrin I o,p'-DDD o,p'-DDE p,p'-DDD p,p'-DDE o,p'-DDT p,p'-DDT DEF Demeton-0 Measured Concentration (ppb or ng/g) < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 250 < 50 < 50 Component Diazinon Dichlobenil Dichloran Dichlorvos Diclofop methyl Dicofol Dicrotophos Dieldrin Dimethoate Dioxathion Diphenamid Diphenylamine Disulfoton Diuron Endosulfan I Endosulfan I1 Endrin Endrin ketone EPN Ethalfluralin Ethion Ethoprop Ethoxyquin Etridiazole Fenamiphos Fenarimol Fenobucarb Fenpropathrin Fensulfothion Fenthion Fentrothion Fluzifop-P-butyl Fonofos Heptachlor Heptachlor epoxide Hexachlorobenzene Isazophos Isofenphos Leptophos Lindane Linuron Malathion Metalaxyl Methamidophos Methidathion Methiocarb `Analyses performed by Exygen Research on samples collected on July 24,2001. Measured Concentration (ppb or ng/g) < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50 - 133 - PROJECT NO.: 454-110 Appendix 14 (continued) Results of Water and Soil Pesticide Screening Wildlife International, Ltd. Well Water: Pesticides And Organics (Page 2) Component Methomyl Methoxychlor Methyl parathion Metolachlor Metribuzin cis-Mevinphos Mirex Monocrotophos Myclobutanil 1-Naptho1 Napropamide Nitrapyrin Norflurazon Oxadiazon Oxamyl Oxyfluorfen Paraoxon Parathion Pendemethalin Pentachloronitrobenzene cis-Permethrin Perthane Phorate Phosalone Phosphamidon Piperalin Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Profenfos Profluoralin Measured Concentration (ppb or ng/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 C 50 < 50 Component Promecarb Prometryne Pronamide Propanil Propargite Propham Propoxur Pyrethrin I Quinalphos Quinomethionate Quizalofop-ethyl Ronnel Simazine Simetryn Sulprofos Terbacil Terbufos Tetrachlorovinphos Tetradifon Tbiobencarb Tbiobendazole Thionazin THPI Tilt I Tilt I1 Tridemefon Trifluralin Trimethyl carbamate Vegedex Vinclozolin Metals Measured Concentration (ppb or n d 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 Aluminum Arsenic Beryllium Cadmium Calcium Chromium Cobalt Copper Iron Magnesium @pm or mg/L) < 0.2 < 0.01 < 0.005 < 0.005 < 50 < 0.01 <5 < 0.025 <5 < 50 Manganese Mercury Molybdenum Nickel Potassium Selenium Silver Sodium Zinc (ppm or mg/LJ < 0.015 < 0.0002 < 0.015 <5 < 50 < 0.005 < 0.01 < 50 < 0.02 'Analyses performed by Exygen Research on samples collected on July 24,2001. - 134 - PROJECT NO.: 454-110 Appendix 14 (continued) Results of Water and Soil Pesticide Screening Component Wildlife International, Ltd. Greenhouse Soil: Pesticides and Organics Measured Concentration (ppb or ng/g) Component Measured Concentration (ppb or np/g:i Acephate < 50 Diazinon < 50 Alachlor < 50 Dichlobenil < 50 Aldicarb sulfone < 50 Dichloran < 50 Aldicarb sulfoxide < 50 Dichlorvos < 50 Aldrin < 50 Diclofop methyl < 50 Alpha-BHC < 50 Dicofol < 50 Ametryne < 50 Dicrotophos < 50 Atrazine < 50 Dieldrin < 50 Azinphos-ethyl < 50 Dimethoate < 50 Azinphos-methyl < 50 Dioxathion < 50 Beta-BHC Bifenox < 50 Diphenamid < 50 < 50 Diphenylamine < so Bi tertanol < 50 Disulfoton < 50 Bromacil < 50 Diuron < 50 Bromophos-methyl < 50 Endosulfan I < 50 Bromoxynil octanoic ac:id ester < 50 Endosulfan I1 < 50 Captafol < 50 Endrin < 50 Carbaryl < 50 Endrin ketone < 50 3-hydroxy Carbofuran < 50 EPN < 50 Carbofuran < 50 Ethalfluralin < 50 Carbophenothion < 50 Ethion < 50 cis-Chlordane < 50 Ethoprop < 50 trans-Chlordane < 50 Ethoxyquin < 50 Chlordimeform < 50 Etridiazole < 50 Chlorfenson < 50 Fenamiphos < 50 trans-Chlorfenvinphos < 50 Fenarimol < 50 Chlorobenzilate < 50 Fenobucarb < 50 Chloroneb < 50 Fenpropathrin < 50 Chloropropham < 50 Fensulfothion < 50 Chloropropylate < 50 Fenthion < 50 Chloroxuron < 50 Fentrothion < 50 Chlorpyrifos-ethyl < 50 Fluzifop-P-butyl < 50 Chlorpyrifos-methyl < 50 Fonofos < 50 Chlorthal < 50 Heptachlor < 50 Coumaphos < 50 Heptachlor epoxide < 50 Crotoxyphos < 50 Hexachlorobenzene < 50 Cyanazine < 50 Isazophos < 50 Cyfluthrin I < 50 Isofenphos < 50 Cypermethrin I < 50 Leptophos < 50 o,p'-DDD o,p'-DDE < 50 Lindane < 50 < 50 Linuron < so p,p'-DDD < 50 Malathion < 50 p,p'-DDE < 50 Metalaxyl < 50 o,p'-DDT p,p'-DDT < 50 Methamidophos < 50 < 250 Methidathion < so DEF < 50 Methiocarb < 50 Demeton-0 < 50 `Analyses performed by Exygen Research on samples collected on July 24,2001. - 135 - PROJECT NO.: 454-1 10 Appendix 14 (continued) Results of Water and Soil Pesticide Screening Component Wildlife International, Ltd. Greenhouse Soil: Pesticides And Organics (Page 2) Measured Concentration (ppb or ng/g) Component Measured Concentration (ppb or ng/g) Methomyl < 50 Promecarb < 50 Methoxychlor < 250 Prometryne < 50 Methyl parathion < 50 Pronamide < 50 Metolachlor < 50 Propanil < 50 Metribuzin < 50 Propargite < 50 cis-Mevinphos < 50 Propham < 50 Mirex < 50 Propoxur < 50 Monocrotophos < 50 Pyrethrin I < 50 Myclobutanil < 50 Quinalphos < 50 1 -Naptho1 < 50 Quinomethionate < 50 Napropamide < 50 Quizalofop-ethyl < 50 Nitrap yrin < 50 Ronnel < 50 Norflurazon < 50 Simazine < 50 Oxadiazon < 50 Simetryn < 50 Oxamyl < 50 Sulprofos < 50 Oxyfluorfen < 50 Terbacil < 50 Paraoxon < 50 Terbufos < 50 Parathion < 50 Tetrachlorovinphos < 50 Pendemethalin < 50 Tetradifon < 50 Pentachloronitrobenzene < 50 Thiobencarb < 50 cis-Permethrin < 50 Thiobendazole < 50 Perthane < 50 Thionazin < 50 Phorate < 50 THPI < 50 Phosalone < 50 Tilt I < 50 Phosphamidon < 50 Tilt I1 < 50 Piperalin < 50 Tridemefon < 50 Pirimicarb < 50 Trifluralin < 50 Pirimiphos-ethyl < 50 Trimethyl carbamate < 50 Pirimiphos-methyl < 50 Vegedex < 50 Profen fos < 50 Vinclozolin < 50 Profluoralin < 50 Aluminum Arsenic Beryllium Cadmium Calcium Chromium Cobalt Copper Iron Magnesium (ppm or mg/Kg) < 15300 < 3.83 < 1.91 < 1.91 < 7660 10.7 < 1910 < 9.57 < 76600 < 1470 Metals Manganese Mercury Molybdenum Nickel Potassium Selenium Silver Sodium Zinc (ppm or mg/KI;) :* 230 1.17 1.68 < 1910 < ?6600 < 1.91 < 3.83 < 1910 13.6 'Analyses performed by Exygen Research on samples collected on July 24,2001. - 136 -