AN-2:

A FLOW-THROUGH LIFE-CYCLE TOXICITY TEST 

WITH THE CLADOCERAN (Daphnia magna) 

FINAL REPORT 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-120 

U.S. EPA OPPTS NUMBER 850.1300 

OECD GUIDELINE 211 

ASTM Standard E 1193-97 

AUTHORS: 

Tui Minderhout, Ph.D. 

Timothy Z. Kendall, M.S. 

Sean P. Gallagher, B.S. 

Henry O. Krueger, Ph.D. 

STUDY INITIATION DATE: January 2, 2012 

STUDY COMPLETION DATE: June 21, 2012 

SUBMITTED TO: 

Albemarle Corporation 

451 Florida Street 

Baton Rouge, LA 70801 

Wildlife International, Ltd. 

8598 Commerce Drive 

Easton, Maryland 21601 USA 

1-410-822-8600 

Page 1 of 59 
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STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 

No claim of confidentiality, on any basis whatsoever, is made for any information contained in this 

study. I acknowledge that the information not designated as within the scope of FIFRA section 10(d) 

(1)(A), (B) or (C) and which pertains to a registered or previously registered pesticide is not entitled 

to confidential treatment and may be released to the public, subject to the provisions regarding 

disclosure to multinational entities under FIFRA (10)g. 

 Company: ________________________________(Typed Name) 

 Company Agent: ________________________________(Typed Name) 

 Title: ________________________________ 

 Signature:______________________________Date:______________________ 



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TABLE OF CONTENTS

Title/Cover Page ..................................................................................................................................... 1 

Statement of No Data Confidentiality Claims ........................................................................................ 2 

Good Laboratory Practice Compliance Statement ................................................................................. 3 

Quality Assurance Statement ................................................................................................................. 4 

Report Approval ..................................................................................................................................... 5 

Table of Contents ................................................................................................................................... 6 

Summary ................................................................................................................................................ 9 

Introduction .......................................................................................................................................... 10 

Objective .............................................................................................................................................. 10 

Experimental Design ............................................................................................................................ 10 

Materials and Methods ......................................................................................................................... 11 

 Test Substance ......................................................................................................................... 11 

 Test Organism .......................................................................................................................... 11 

 Dilution Water ......................................................................................................................... 12 

 Test Apparatus ......................................................................................................................... 13 

 Preparation of Test Concentrations ......................................................................................... 14 

 Analytical Sampling ................................................................................................................ 14 

14C AN-2 Stock Analysis ......................................................................................................... 14 

 Analytical Method ................................................................................................................... 15 

 Environmental Conditions ....................................................................................................... 15 

 Biological Observations and Measurements ............................................................................ 16 

 Statistical Analyses .................................................................................................................. 16 

Results and Discussion ......................................................................................................................... 17 

 Measurement of Test Concentrations ...................................................................................... 17 

 Water Chemistry Measurements .............................................................................................. 18

 Survival and Clinical Observations ......................................................................................... 18 

 Reproduction ........................................................................................................................... 19 

 Growth ..................................................................................................................................... 20 

Conclusions .......................................................................................................................................... 21 

References ............................................................................................................................................ 22 
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TABLE OF CONTENTS (Continued)

TABLES AND FIGURES 

Table 1. Measured Concentrations of 14C AN-2 in stock Solutions Analyzed by LSC .................. 23 

Table 2. Measured Concentrations of 14C AN-2 in Pretest Diluter Verification Samples .............. 24 

Table 3. Measured Concentrations of 14C AN-2 in Test Solution Samples Analyzed by LSC ...... 25 

Table 4. Means and Ranges of Water Quality Measurements Taken During the 21-Day 

 Exposure to 14C AN-2 ....................................................................................................... 27 

Table 5. Summary of Survival, Reproduction and Growth of Daphnia magna

 Exposed to 14C AN-2 for 21 Days .................................................................................... 28 

APPENDICES 

Appendix 1. Dilution Water Chemistry Parameters Measured During the 4-Week 

 Period Immediately Preceding the Test .................................................................... 29 

Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. 

 Well Water ................................................................................................................ 30 

Appendix 3. Analysis of 14C AN-2 in Freshwater ......................................................................... 32 

3.1 Analytical Method Outline for the Analysis of 14C AN-2 

 in Freshwater ............................................................................................................ 33 

3.2 Typical Counting Conditions for Liquid Scintillation Counting .............................. 34 

3.3 Calculations for Sample Quantitation ....................................................................... 35 

Appendix 4. Temperature of Water in the Test Chambers During Exposure to 14C AN-2 ............ 36 

Appendix 5. Dissolved Oxygen Concentration of Water in the Test Chambers 

 During Exposure to 14C AN-2 ........................................................................................ 37 

Appendix 6. pH of Water in the Test Chambers During Exposure to 14C AN-2 ............................ 38 
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TABLE OF CONTENTS (Continued)

APPENDICES (Continued) 

Appendix 7. Specific Conductance, Hardness and Alkalinity in the Negative Control 

 and Highest Concentration Treatment Group During Exposure to 14C AN-2 ........... 39 

Appendix 8. Mortality and Observations of First-Generation Daphnids Exposed to 

 14C AN-2 for 21 Days ............................................................................................... 40 

Appendix 9. Neonate Production by First-Generation Daphnids Exposed to 

 14C AN-2 for 21 Days ............................................................................................... 48 

Appendix 10. Length and Dry Weight of Surviving First-Generation Daphnids 

 after Exposure to 14C AN-2 for 21 Days ................................................................... 50 

Appendix 11. Changes to Protocol .................................................................................................. 58 

Appendix 12. Personnel Involved in the Study ............................................................................... 59 
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SUMMARY

SPONSOR: Albemarle Corporation 

TITLE: AN-2: A Flow-Through Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna) 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-120 

TEST DATES: Experimental Start (OECD): March 8, 2012 

 Experimental Start (EPA): March 19, 2012 

 Biological Termination: April 9, 2012 

 Experimental Termination: April 25, 2012 

LENGTH OF EXPOSURE: 21 Days 

TEST ORGANISM: Cladoceran (Daphnia magna) 

SOURCE OF TEST ORGANISMS: Wildlife International, Ltd. 

 Easton, Maryland 21601 

AGE OF TEST ORGANISMS: Neonates (< 24 hours old at initiation) 

TEST CONCENTRATIONS: Nominal Mean Measured 

 Negative Control < LOQ 

 Solvent Control < LOQ 

 1.6 ng/L 1.4 ng/L 

 3.1 ng/L 2.4 ng/L 

 6.3 ng/L 5.5 ng/L 

 13 ng/L 12 ng/L 

 25 ng/L 21 ng/L 

 50 ng/L 43 ng/L 

RESULTS: Based on mean measured test concentrations: 

 No-Observed-Effect-Concentration: 43 ng/L 

 Lowest-Observed-Effect-Concentration: greater than 43 ng/L 

 Maximum Acceptable Toxicant Concentration: greater than 43 ng/L 

 21-Day EC50 (Immobility): greater than 43 ng/L 

 95% Confidence Interval: not calculated 

 21-Day EC50 (Reproduction): greater than 43 ng/L 

 95% Confidence Interval: not calculated 
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INTRODUCTION

This study was conducted by Wildlife International, Ltd. for Albemarle Corporation at the 

Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the 

definitive test was conducted from March 19 to April 9, 2012, with dry weight measurements 

completed on April 11, 2012. Raw data generated by Wildlife International, Ltd. and the final report 

are filed under Project Number 471A-120 in archives located on the Wildlife International, Ltd. site. 

OBJECTIVE 

The objective of this study was to determine the effects of AN-2 on the survival, growth and 

reproduction of the cladoceran, Daphnia magna, during a 21-day exposure period under flow-through 

test conditions. 

EXPERIMENTAL DESIGN 

Daphnids were exposed to a geometric series of six test concentrations, a negative control 

(dilution water) and a solvent control (0.025 mL/L HPLC-grade dimethylformamide). Two replicate 

test chambers were tested for each treatment and control group. Each replicate contained two 

compartments with five daphnids, resulting in a total of 20 daphnids in each treatment and control 

group. Nominal test concentrations were selected in consultation with the Sponsor based on 

exploratory range finding toxicity data and the solubility limit in the test system. The nominal test 

concentrations were 1.6, 3.1, 6.3, 13, 25 and 50 ng AN-2 /L. Mean measured test concentrations were 

determined from samples of test water collected from each treatment and control group at test 

initiation, at approximately weekly intervals during the test and at test termination. 

Delivery of the test substance to the test chambers was initiated seven days prior to the 

introduction of the daphnids to the test water in order to achieve equilibrium of the test substance in 

the test chambers. At test initiation, neonate daphnids were impartially assigned to exposure 

chambers. First-generation daphnids were observed daily during the test for mortality, the onset of 

reproduction, and clinical signs of toxicity. Following the onset of reproduction, the numbers of 

second-generation daphnids were counted three times per week (i.e., Monday, Wednesday and 

Friday), and at test termination (Day 21). Body lengths and dry weights of the surviving 

first-generation daphnids were measured at the end of the exposure period. Observations of the 

effects of AN-2 on survival, reproduction and growth were used to determine the no-observed-effect 
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concentration (NOEC), the lowest-observed-effect concentration (LOEC), and the maximum 

acceptable toxicant concentration (MATC). EC50 values were determined based on reproduction and 

on first-generation immobility at test termination. 

MATERIALS AND METHODS 

The study was conducted according to the procedures outlined in the protocol, “AN-2: A 

Flow-Through Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna)”. The protocol was 

based on procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 211: 

Daphnia magna Reproduction Test (1); the U.S. Environmental Protection Agency Series 850 - 

Ecological Effects Test Guidelines, OPPTS Number 850.1300: Daphnid Chronic Toxicity Test (2); 

and ASTM E 1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests (3). 

Test Substance 

The 14C radiolabelled test substance used to prepare the test solutions for the study was 

received from Quotient Bioresearch on May 23, 2011, and was assigned Wildlife International, Ltd. 

identification number 10063, upon receipt. The radiolabelled test substance was stored under frozen 

conditions. The test substance, a liquid, was identified as: 4,4’-Methylenebis (2,6-di-tert-butyl[U14C]phenol); Batch number CFQ41115. The test substance had a reported specific activity of 

129 μCi/mg or 55 mCi/mmol, based on gravimetric analysis, and a radiochemical concentration of 

968 μCi/mL. 

Test Organism 

The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are 

representative of an important group of aquatic invertebrates and were selected for use in the test 

based upon past history of use in the laboratory. Daphnid neonates used in the test were less than 

24 hours old and were obtained from cultures maintained by Wildlife International, Ltd., Easton, 

Maryland. 

Adult daphnids were cultured in water from the same source and at approximately the same 

temperature as used during the test. During the 2-week period immediately preceding the test, water 

temperatures in the cultures ranged from 19.5 to 21.3oC, measured with a hand-held, liquid-in-glass 

thermometer. The pH of the water ranged from 8.2 to 8.6, measured with a Thermo Orion Model 
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525Aplus pH meter. Dissolved oxygen concentrations were 7.7 mg/L (86% of saturation), 

measured with a Thermo Orion Model 850Aplus dissolved oxygen meter. 

During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and 

trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella 

subcapitata. Daphnids were fed two or three times per day through Day 7 of the test and then were 

fed four times per day until the last day of the test. At each feeding, each test chamber was fed 

0.75 mL of YCT and 1.5 mL of algae. This amount of feed is equal to approximately 

0.7 mg C/daphnid/day. While this amount of feed exceeds the OECD guideline recommended 

amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed 

in the flow-through system to support acceptable reproduction rates. 

The six adult daphnids used to supply neonates for the test were held for at least 17 days prior 

to collection of the juveniles for testing, and had each produced at least one previous brood. Adult 

daphnids in the culture had produced an average of at least three young per adult per day over the 

7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were 

produced during the holding period. To initiate the test, the juvenile daphnids were collected from 

the cultures and indiscriminately transferred one or two at a time to transfer chambers until each 

chamber contained 5 daphnids. Each group of neonates then was impartially assigned to a control or 

treatment group and the neonates were transferred to the test compartments to initiate the test. All 

transfers were made below the water surface using wide-bore pipettes. 

Dilution Water 

The water used for culturing and testing was freshwater obtained from a well approximately 

40 meters deep located on the Wildlife International, Ltd. site. The well water was passed through a 

sand filter to remove particles greater than approximately 25 m, and pumped into a 37,800-L storage 

tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 m 

to remove fine particles and was passed through an ultraviolet (UV) sterilizer. 

The well water is characterized as moderately-hard water. The specific conductance, 

hardness, alkalinity and pH of the well water during the four-week period immediately preceding the 

test are presented in Appendix 1. The results of periodic analyses performed to measure the 
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concentrations of selected organic and inorganic constituents in the well water are presented in 

Appendix 2. 

Test Apparatus 

The toxicity test was conducted using an exposure system consisting of a continuous-flow 

diluter used to deliver each concentration of the test substance, a solvent control and a negative 

control (dilution water) to test chambers. Syringe pumps (Harvard Apparatus, Massachusetts) were 

used to deliver test substance stock solutions or solvent to impartially assigned mixing chambers 

where the stocks or solvent were mixed with dilution water prior to delivery to the test chambers. 

The flow of dilution water into each mixing chamber was controlled using rotameters and was 

adjusted to provide approximately 9 volume additions of test water in each test chamber per day. 

After mixing, the flow from each mixing chamber was split to deliver test water to two replicate test 

chambers. 

The syringe pumps used to deliver stock solutions or solvent to the mixing chambers were 

calibrated prior to the test. The rotameters used to control the flow of dilution water to the mixing 

chambers were calibrated prior to the test and calibrated/verified approximately weekly during the 

test. The proportion of the test water that was split into each replicate test chamber was checked prior 

to the test and approximately weekly during the test to ensure that flow rates varied by no more than 

± 10% of the mean flow rate for the two replicates. Delivery of test solutions to the test chambers 

was initiated seven days prior to the introduction of the test organisms to the test water in order to 

achieve equilibrium of the test substance. The general operation of the exposure system was checked 

visually at least once on the first and last days of the test and at least two times per day during the test. 

The delivery system and the test chambers were placed in a temperature-controlled 

environmental chamber to maintain the target water temperature throughout the test period. Test 

chambers were 9-L glass aquaria filled with approximately 8 L of test water. The daphnids were held 

in two test compartments suspended in each of two test chambers. Test compartments were 300 mL 

glass beakers, approximately 6.5 cm in diameter and 12 cm in height. Nylon mesh screens covered two 

holes on opposite sides of each test compartment to permit test solution to flow in and out of the 

compartment. The depth of the test water in a representative compartment was 8.7 cm, while the 

depth of water in a representative test chamber was 18.5 cm. All test chambers were labeled with the 

project number, test concentration and replicate designation. 
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Preparation of Test Concentrations 

Individual stock solutions were prepared for each of the six concentrations tested, and were 

prepared once during the study. Each test stock solution was prepared by mixing a calculated amount 

of radiolabelled AN-2 into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 

0.064, 0.124, 0.252, 0.52, 1.0 and 2.0 μg/mL. The stock solutions were mixed by inversion. The 

stock solutions at 0.064 to 1.0 μg/mL appeared clear and colorless, while the stock solution at 

2.0 μg/mL appeared clear with a slight purple tint. Stock solutions were stored refrigerated in glass 

amber bottles, and aliquots of each stock were placed in the syringe pump every 4 to 7 days during 

the study. 

The five test substance stock solutions were injected into the diluter mixing chambers at a 

rate of 2.5 μL/minute where they were mixed with dilution water delivered at a rate of 

100 mL/minute to achieve the desired test concentrations. The negative control received dilution 

water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing 

chamber for the solvent control. The concentration of DMF in the solvent control and all AN-2 

treatment groups was 0.025 mL/L. 

Analytical Sampling 

Samples were collected from each treatment and control group five days prior to the start of 

the test after conditioning the diluter for two days. Water samples also were collected from 

alternating replicate test chambers in each treatment and control group at the beginning of the test, at 

approximately weekly intervals during the test and at the end of the test to measure concentrations of 

the test substance. Samples of the stock solutions being delivered to the diluter were collected for 

analysis on Day –11 to confirm the stock concentrations, after the stocks were prepared. Additional 

samples of test solutions were collected on Day 22 to confirm concentration of the 1.6 ng/L treatment 

level when the analytical result of this treatment level on Day 21 was suspect. All test solution 

samples were collected from mid-depth, placed in separatory funnels, and processed immediately for 

analysis by LSC. 

14C AN-2 Stock Analysis

Aliquots of the 0.064, 0.124, 0.252, 0.52, 1.0 and 2.0 μg/mL radioactive stocks were 

transferred to scintillation vials containing approximately 15 mL of Ultima Gold XR scintillation 

cocktail, and the solutions were analyzed by liquid scintillation counting (LSC) (Table 1). 
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Analytical Method

The analytical method used to analyze the test samples was based upon methodology 

developed by Wildlife International, Ltd. The concentrations of the test substance in freshwater 

samples were verified by liquid scintillation counting (LSC). Samples were extracted two times with 

dichloromethane, evaporated to dryness and reconstituted using acetonitrile. Extracts were 

transferred into scintillation vials containing 13 mL of Ultima Gold XR scintillation cocktail and 

submitted for analysis by LSC. A Packard 2500TR Liquid Scintillation Analyzer was used to 

determine disintegrations per minute (dpm). In addition to the test samples, a 13 mL sample of 

Ultima Gold XR scintillation cocktail was analyzed at each interval to determine background 

radioactivity. Measured concentrations of 14C-AN-2 are presented in Table 3. A method flowchart is 

presented in Appendix 3.1 and instrument parameters are presented in Appendix 3.2. The 

calculations for a sample quantitation are included in Appendix 3.3. 

Environmental Conditions 

Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that 

emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a 

photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light 

intensity was provided when lights went on and off to avoid sudden changes in lighting. Light 

intensity was measured at the water surface of one representative test chamber at the beginning of the 

test using a SPER Scientific Model 840006C light meter. 

The target test temperature during the test was 20 ± 1°C. Temperature was measured in each 

test chamber at the beginning of the test, approximately weekly during the test, and at the end of the 

test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one 

negative control test chamber using a Fulscope ER/C recorder. The recorder was verified prior to test 

initiation and approximately weekly during the test using a liquid-in-glass thermometer. 

Dissolved oxygen was measured in one replicate test chamber of each treatment and control 

group at the beginning of the test, approximately three times per week during the test, and at the end 

of the test. Measurements typically rotated between the two replicates in each treatment or control 

group at each measurement interval. Measurements of pH were made in one replicate test chamber of 

each treatment and control group at the beginning of the test, approximately weekly during the test, 
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and at the end of the test. Measurements typically rotated between the two replicates in each 

treatment or control group at each measurement interval. Dissolved oxygen was measured using a 

Thermo Orion 850Aplus dissolved oxygen meter, and measurements of pH were made using a 

Thermo Orion 525Aplus pH meter. 

Hardness, alkalinity and specific conductance were measured in alternating replicates of the 

negative control (dilution water) and the highest test concentration at the beginning of the test, 

approximately weekly during the test and at the end of the test. Hardness and alkalinity were 

measured by titration based on procedures in Standard Methods for the Examination of Water and 

Wastewater (4). Specific conductance was measured using an Acorn Series Model CON6 

Conductivity-Temperature meter. Total organic carbon (TOC) in the dilution water at the beginning 

and end of the test was measured using a Shimadzu Model TOC-VCSH total organic carbon analyzer, 

based on procedures in Standard Methods for the Examination of Water and Wastewater (4). 

Biological Observations and Measurements 

Observations of each first-generation daphnid were made daily during the test. At these 

times, the numbers of immobile daphnids were recorded along with any clinical signs of toxicity (e.g., 

inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). 

Immobility was defined as a lack of movement, except for minor spontaneous random movement of 

the appendages. The presence of eggs in the brood pouch, aborted eggs, males or ephippia also were 

recorded daily. With the onset of reproduction, neonates produced by the first-generation daphnids 

were counted and then discarded every Monday, Wednesday and Friday during the test. The body 

length and the dry weight of each surviving first-generation daphnid were measured at the end of the 

test. 

Statistical Analyses 

Test endpoints analyzed statistically for first-generation daphnids were survival, 

reproduction, and growth (length and dry weight). Reproduction was based on the number of live 

young produced per reproductive day. Reproductive days were defined as the number of days that the 

adult daphnid was alive from the day the first brood was released from any adult daphnid in the test 

until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended 

on the last day it was alive. 
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Negative control and solvent control data for each parameter were compared using an 

appropriate statistical test. No significant differences between the control groups were found for any 

parameter tested (p > 0.05). Therefore, the control data for all parameters were pooled for 

comparison with the treatment groups. 

Survival data was considered to be discrete-variable data, while reproduction and growth data 

were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square 

and Fisher’s Exact test to identify treatment groups that showed a statistically significant difference 

(p  0.05) from the control. All continuous-variable data were evaluated for normality using 

Shapiro-Wilk’s and for homogeneity of variance using Levene’s (α = 0.01). When the data passed the 

assumptions of normality and homogeneity, those treatments that were significantly different from the 

control means were identified using Dunnett’s test (α = 0.05). All statistical tests were performed using 

a personal computer with SAS (5) software. 

The results of the statistical analyses were used to aid in the determination of the NOEC, LOEC 

and MATC. However, scientific judgement was used to determine if statistical differences were 

biologically meaningful, and if the data followed a concentration-dependent response. The NOEC was 

defined as the highest test concentration that produced no significant treatment-related effects on 

survival, reproduction or growth. The LOEC was defined as the lowest test concentration that produced 

a significant treatment-related effect on survival, reproduction or growth. The MATC was calculated as 

the geometric mean of the NOEC and LOEC. EC50 values were determined based on reproduction 

and on the immobility observed in the first-generation daphnids at the end of the test (6, 7). 

RESULTS AND DISCUSSION 

Measurement of Test Concentrations 

Nominal concentrations selected for use in the study were 1.6, 3.1, 6.3, 13, 25 and 50 ng/L. 

During the course of the test, the appearance of the test solutions at these nominal concentrations was 

observed in both the diluter mixing chambers, where test substance stocks and dilution water were 

combined prior to delivery to the test chambers, and in the test chambers. The test solutions in the 

mixing chambers and test chambers at test initiation and termination appeared clear and colorless, 

with no evidence of precipitation observed in any control or treatment solution. 
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Samples of the stock solutions being delivered to the diluter system had measured 

concentrations that ranged from 100 to 105% of nominal concentrations, confirming the 

concentrations being delivered to the diluter system (Table 1). The measured concentrations of 

samples collected to verify the diluter system prior to the test ranged from 76.9 to 96.7% of nominal 

concentrations (Table 2). Samples of the test solutions collected during the test had measured 

concentrations that ranged from 66.0 to 177% of nominal concentrations (Table 3). Since the 

analytical result of the sample at 1.6 ng/L treatment group on Day 21 was suspect (the recovery was 

177%), additional sample was collected from this treatment group to confirm the test concentration on 

Day 22. The results of the sample collected on Day 22 confirmed the test concentration; the result of 

the sample for the 1.6 ng/L treatment group collected on Day 21 was excluded from the calculation of 

mean measured test concentration. When the measured concentrations of test solution samples 

collected on Days 0, 7, 14, 21 and 22 of the test were averaged for each treatment group, the mean 

measured test concentrations were 1.4, 2.4, 5.5, 12, 21 and 43 ng/L, which represented 88, 77, 87, 92, 

84 and 86% of nominal concentrations, respectively. The results of the study were based on the mean 

measured concentrations. 

Water Chemistry Measurements 

Measurements of temperature, dissolved oxygen and pH in the test chambers are summarized in 

Table 4, and individual measurements are presented in Appendices 4, 5 and 6, respectively. Water 

temperatures were within the 20 ± 1C range established for the test. Dissolved oxygen 

concentrations remained ≥62% of saturation (5.6 mg/L). Measurements of pH ranged from 8.0 to 

8.2 during the test. Weekly measurements of specific conductance, hardness and alkalinity in the 

negative control water and in the highest test concentration are summarized in Table 4, and individual 

measurements are presented in Appendix 7. Measurements of specific conductance, hardness and 

alkalinity were comparable between the control and treatment group and did not appear to be 

influenced by AN-2 concentration. TOC in the dilution water at test initiation and termination was 

<1 mg C/L (Table 4). Light intensity at test initiation was 171 lux at the surface of the water of one 

representative test chamber. 

Survival and Clinical Observations 

A summary of survival among the first-generation daphnids is presented in Table 5. Daily 

observations are presented in Appendix 8. After 21 days of exposure, survival in the negative control 
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and solvent control groups were 75 and 95%, respectively. There was statistically no significant 

difference in survival between the negative and solvent control groups. Therefore, the control data 

were pooled for comparison with the treatment groups. Survival in the 1.4, 2.4, 5.5, 12, 21 and 

43 ng/L treatment groups at test termination was 90, 90, 100, 95, 90 and 90%, respectively. Fisher’s 

Exact test indicated that no statistically significant decrease in survival was detected in any of the AN-2 

treatment groups in comparison to the pooled control (p > 0.05). Consequently, the NOEC for survival 

was 43 ng/L and the LOEC was greater than 43 ng/L. Based on the immobilities observed in the 

treatment groups, the 21-day EC50 value was greater than 43 ng/L, the highest concentration tested. 

Daphnids in the 1.4, 2.4, 5.5, 12, 21 and 43 ng/L treatment groups that survived to test 

termination generally appeared normal. During the test pale, small and/or lethargic daphnids were 

observed in the 1.4, 2.4, 12, 21 and 43 ng/L treatment groups. However, the numbers of sublethal 

effects noted in the 1.4, 12 and 21 ng/L treatment groups were infrequent. At test termination, 

approximately 33% of surviving first-generation daphnids in the 43 ng/L treatment group appeared 

pale in comparison to the control organisms. While the discoloration of the daphnids in the 43 ng/L 

treatment group were not analyzed statistically, it is important to note that the condition appeared to 

follow the dose-response pattern. All surviving daphnids in the 2.4 and 5.5 ng/L treatment groups 

were normal in appearance at test termination. 

Reproduction 

A summary of neonate production by surviving first-generation daphnids is presented in 

Table 5. A complete listing of the number of neonates counted on each observation day is provided 

in Appendix 9. The first day of brood production in the negative and solvent control replicates and in 

all AN-2 treatment replicates was Day 7, 8 or 9 of the test, indicating that there was no apparent delay 

in the onset of production at any AN-2 concentration tested. With the exception of two immobile 

neonates in the negative control and solvent control groups and three immobile neonates in the 

43 ng/L treatment group on Day 9 or 11 of the test, no immobile neonates were noted in the 1.4, 2.4, 

5.5, 12 and 21 ng/L treatment groups during the test (Appendix 9). No aborted or shed eggs were 

present in any of the control or treatment groups. No males or ephippia were produced during the 

test. 
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Adult daphnids in the negative and solvent control groups produced an average of 8.1 and 

8.7 live young per reproductive day, respectively. There was no significant difference in reproduction 

between the negative and solvent control groups. Therefore, the control data were pooled for 

comparison with the treatment groups. Adult daphnids in the 1.4, 2.4, 5.5, 12, 21 and 43 ng/L 

treatment groups produced an average of 8.9, 6.7, 8.1, 6.9, 7.7 and 8.3 live young per reproductive 

day, respectively. Dunnett’s test indicated there were no statistically significant decreases in mean 

neonate production in any of the AN-2 treatment groups in comparison to the pooled control (p > 0.05). 

 Consequently, the NOEC for reproduction was 43 ng/L and the LOEC was greater than 43 ng/L. 

Based on the reproduction observed in the treatment groups, the 21-day EC50 value was greater than 

43 ng/L, the highest concentration tested. 

Growth 

Summaries of the mean lengths and dry weights of surviving first-generation daphnids are 

presented in Table 5. Individual growth measurements are provided in Appendix 10. Daphnids in the 

negative and solvent control groups averaged 4.6 mm in length and 0.63 mg in dry weight, while 

daphnids in the solvent control group averaged 4.5 mm in length, and 0.69 mg in dry weight, 

respectively. There were no significant differences in the growth parameters between the negative 

and solvent control groups. Therefore, the control data were pooled for comparison with the 

treatment groups. Daphnids in the 1.4, 2.4, 5.5, 12, 21 and 43 ng/L treatment groups had mean 

lengths of 4.6, 4.3, 4.4, 4.4, 4.4 and 4.5 mm, respectively, and mean dry weights of 0.76, 0.60, 0.74, 

0.76, 0.63 and 0.60 mg, respectively. Dunnett’s test indicated there were no statistically significant 

decreases in mean dry weight in any of the AN-2 treatment groups in comparison to the pooled 

control (p > 0.05) but statistically significant reductions were noted in mean total length in the 

2.4, 5.5, 12 and 21 ng/L treatment groups when compared to the pooled controls (p ≤ 0.05). Since the 

reductions in mean total length of daphnids in the 2.4, 5.5, 12 and 21 ng/L treatment groups did not 

follow a dose response pattern, the slight differences noted were not considered to be biologically 

meaningful. In addition, the mean length of the daphnids in the 2.4, 5.5, 12 and 21 ng/L treatment 

groups were within the ranges of the mean length of daphnids in the historical control data (3.6 to 

6.3 mm). Consequently, the NOEC for growth (length and dry weight) was 43 ng/L, and the LOEC 

was greater than 43 ng/L. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 21 - 

CONCLUSIONS 

The cladoceran, Daphnia magna, was exposed to AN-2 at mean measured concentrations of 

1.4, 2.4, 5.5, 12, 21 and 43 ng/L under flow-through conditions for 21 days. There were no 

statistically significant treatment-related effects on survival, reproduction or growth at concentrations 

≤43 ng/L. Consequently, the NOEC, based on survival, reproduction and growth, was 43 ng/L. The 

LOEC was greater than 43 ng/L and the MATC was calculated to be greater than 43 ng/L. The 

21-day EC50 values for adult immobility and reproduction were both greater than 43 ng/L, the 

highest concentration tested. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 22 - 

REFERENCES

1. Organization for Economic Cooperation and Development. 2008. OECD Guidelines for 

Testing of Chemicals, Guideline 211: Daphnia magna Reproduction Test. Adopted 3 October 

2008. 

2. U.S. Environmental Protection Agency. 1996. Series 850 – Ecological Effects Test 

Guidelines (draft), OPPTS Number 850.1300: Daphnid Chronic Toxicity Test. 

3. American Society for Testing and Materials. 2004. ASTM Standard E1193-97: Standard 

Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests. 

4. APHA, AWWA, WEF. 1998. Standard Methods for the Examination of Water and 

Wastewater. 20th Edition, American Public Health Association. American Water Works 

Association. Water Environment Federation, Washington, D.C. 

5. The SAS System for Windows. 1999 - 2001. Version 8.2. SAS Institute Inc., Cary, North 

Carolina. 

6. Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. 

Personal communication. 

7. Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, 

London. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 23 - 

Table 1 

Measured Concentrations of 14C-AN-2 in Stock Solutions Analyzed by LSC 

Sample 

Type 

Sample 

Number 

(471A-120-) 

Nominal 

AN-2 

Concentration 

(μg/mL) 

Specific 

Activity 

(dpm/μg) 

Total [14C] 

Found 

(dpm) 

AN-2 

Equivalents 

Found1

(μg/mL) 

Percent 

of 

Nominal 

Background -- -- -- 29.84 -- -- 

Stock S-1 0.064 286380 1868.27 0.0642 100 

Stock S-2 0.124 286380 3585.94 0.124 100 

Stock S-3 0.252 286380 7450.33 0.259 103 

Stock S-4 0.52 286380 15296.70 0.533 103 

Stock S-5 1.0 286380 29941.60 1.04 104 

Stock S-6 2.0 286380 60254.40 2.10 105 

1

 AN-2 Equivalents = ({[(Total dpm found – background contribution)/Sample Volume]}/Specific Activity). 

Sample Volume = 0.100 mL 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 24 - 

Table 2 

Measured Concentrations of AN-2 in Pretest Diluter Verification Samples 

Nominal 

Concentration 

(ng/L) 

Sample 

I.D. 

(471A-120-) 

Sampling 

Time 

(Day) 

Sample 

Volume 

(L) 

Total 14C 

Found 

(dpm) 

AN-2 

Equivalents 

Found1,2,3 

(ng/L) 

Percent 

of 

Nominal 

Background -- -5 -- 23.94 -- -- 

Negative Control PT-1 -5 0.200 28.80 < LOQ -- 

Solvent Control PT-2 -5 0.200 26.98 < LOQ -- 

1.6 PT-3 -5 0.200 94.38 1.23 76.9 

3.1 PT-4 -5 0.200 176.69 2.67 86.0 

6.3 PT-5 -5 0.200 372.98 6.09 96.7 

13 PT-6 -5 0.200 697.53 11.8 90.5 

25 PT-7 -5 0.100 636.31 21.4 85.5 

50 PT-8 -5 0.0500 596.95 40.0 80.0 1 The specific activity is 286380 dpm/μg. 2 AN-2 Equiv. = ({[Total dpm found - background contribution)/Sample volume]}/Specific Activity) x 1000 3 LOQ = {[(Total dpm found / Sample Volume]}/Specific Activity) x 1000= 0.873 ng/L. 
Wildlife International, Ltd. Pr

- 25 - 

Table 3 

Measured Concentrations of 14C AN-2 in Test Solution Samples Analyzed by LSC 

Nominal 

AN-2 

Concentration 

(ng/L) 

Sample 

Number 

(471A-120-) 

Sampling 

Interval 

(Day) 

Sample 

Volume 

(L) 

Specific Activity 

(dpm/g) 

Total 

[14C] 

Found 

(dpm) 

AN-2 

Equivalents 

Found 

(ng/L)1,2,3

Percent 

of 

Nominal 

Mea

AN

Equiva

(ng/

Background -- 

-- -- -- -- 

0 7 

14 21 22 

-- -- -- -- -- 

-- -- -- -- -- 

24.31 25.28 34.30 24.19 24.67 

-- -- -- -- -- 

-- -- -- -- -- 

-- 

Negative Control 

 (0.0) 

1 9 

17 25 

0 7 

14 21 

0.200 0.200 0.200 0.200 

286380 286380 286380 286380 

29.37 25.66 29.97 24.03 

< LOQ < LOQ < LOQ < LOQ 

-- -- -- -- 

-- 

Solvent Control 

 (0.0) 

2 

10 18 26 

0 7 

14 21 

0.200 0.200 0.200 0.200 

286380 286380 286380 286380 

26.32 28.94 27.81 27.74 

< LOQ < LOQ < LOQ < LOQ 

-- -- -- -- 

-- 

1.6 3 

11 19 27 33 

0 7 

14 

 21* 

22 

0.200 0.200 0.200 0.200 0.200 

286380 286380 286380 286380 286380 

94.30 

104.26 

99.06 

185.98 124.38 

1.22 1.38 1.13 2.82 1.74 

76.4 86.2 70.7 

177 109 

1.4 

1 AN-2 Equivalents = ({[Total dpm found - background contribution)/Sample volume]}/Specific Activity) x 1000 2 The calculated limit of quantitation (LOQ) was 0.873 ng/L. 3 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly. 

* Day 21 sample is excluded from the mean calculation due to high recovery. An additional sample was taken on Day 22 and will be used 
Wildlife International, Ltd. Pr

- 26 - 

Table 3 (Continued) 

Measured Concentrations of 14C-AN-2 in Test Samples Analyzed by LSC 

Nominal 

AN-2 

Concentration 

(ng/L) 

Sample 

Number 

(471A-120-) 

Sampling 

Interval 

(Day) 

Sample 

Volume 

(L) 

Specific Activity 

(dpm/g) 

Total 

[14C] 

Found 

(dpm) 

AN-2 

Equivalents 

Found 

(ng/L)1,2,3

Percent 

of 

Nominal 

Mea

AN

Equiva

(ng/

3.1 4 

12 20 28 

0 7 

14 21 

0.200 0.200 0.200 0.200 

286380 286380 286380 286380 

186.09 184.37 156.49 141.41 

2.82 2.78 2.13 2.05 

91.1 89.6 68.8 66.0 

2.4 

6.3 5 

13 21 29 

0 7 

14 21 

0.200 0.200 0.200 0.200 

286380 286380 286380 286380 

347.46 348.55 332.56 337.60 

5.64 5.64 5.21 5.47 

89.6 89.6 82.7 86.9 

5.5 

13 6 

14 22 30 

0 7 

14 21 

0.200 0.200 0.200 0.200 

286380 286380 286380 286380 

748.18 693.56 681.32 657.77 

12.6 11.7 11.3 11.1 

97.2 89.8 86.9 85.1 

12 

25 7 

15 23 31 

0 7 

14 21 

0.100 0.100 0.100 0.100 

286380 286380 286380 286380 

629.64 629.83 612.85 636.32 

21.1 21.1 20.2 21.4 

84.5 84.4 80.8 85.5 

21 

50 8 

16 24 32 

0 7 

14 21 

0.0500 0.0500 0.0500 0.0500 

286380 286380 286380 286380 

649.51 685.01 625.03 621.29 

43.7 46.1 

41.3 41.7 

87.3 92.1 82.5 83.4 

43 

1 AN-2 Equivalents = ({[Total dpm found - background contribution)/Sample volume]}/Specific Activity) x 1000 2 The calculated limit of quantitation (LOQ) was 0.873 ng/L. 3 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 27 - 

Table 4 

Means and Ranges of Water Quality Measurements Taken During the 21-Day Exposure to 14C AN-

Mean Measured 

Concentration 

(ng/L) 

Mean ± Std. Dev. and Range of Measured Parameters 1

Temperature 2

(°C) 

Dissolved Oxygen 3

(mg/L) pH 

Hardness 4

(mg/L as CaCO3) 

Alkalinity 4

(mg/L as CaCO

Negative Control 19.9 ± 0.20 8.3 ± 0.20 8.1 ± 0.05 134 ± 10 177 ± 1 

 (19.7 – 20.2)) (7.9 – 8.6) (8.1 – 8.2) (124 – 144) (176 – 178) 

Solvent Control 19.9 ± 0.18 7.5 ± 0.61 8.1 ± 0.08 -- -- 

 (19.7 – 20.2) (6.7 – 8.4) (8.0 – 8.2) -- -- 

1.4 20.0 ± 0.21 7.1 ± 0.84 8.0 ± 0.05 -- -- 

 (19.7 – 20.3) (6.0 – 8.4) (8.0 – 8.1) -- -- 

2.4 20.0 ± 0.19 7.3 ± 1.01 8.1 ± 0.10 -- -- 

 (19.8 – 20.2) (5.7 – 8.4) (8.0 – 8.2) -- -- 

5.5 20.0 ± 0.19 7.0 ± 0.95 8.1 ± 0.06 -- -- 

 (19.7 – 20.2) (5.6 – 8.3) (8.0 - 8.1) -- -- 

12 20.0 ± 0.23 7.2 ± 0.82 8.1 ± 0.08 -- -- 

 (19.7 – 20.3) (5.8 – 8.1) (8.0 – 8.2) -- -- 

21 20.0 ± 0.24 7.2 ± 0.94 8.1 ± 0.08 -- -- 

 (19.8 – 20.4) (5.8 – 8.5) (8.0 – 8.2) -- -- 

43 19.9 ± 0.24 7.4 ± 0.62 8.1 ± 0.08 141 ± 2 180 ± 1 

 (19.6 – 20.2) (6.4 – 8.4) (8.0 – 8.2) (140 – 144) (178 – 180) 

1 Total organic carbon measured in the dilution water at test initiation and termination was <1 mg C/L. 2 Temperature monitored continuously during the test ranged from approximately 19 to 20C, measured to the nearest 1C. 3 A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20oC in freshwater. 4 -- = no measurements scheduled. 
Wildlife International, Ltd. Pr

- 28 - 

Table 5 

Summary of Survival, Reproduction and Growth of Daphnia magna Exposed to 14C AN-2 for 21 Day

Mean Measured 

Concentration 

(ng/L) 

Percent Adult 

Survival 1,2

Mean No. Neonates 

Per Reproductive Day 

± Std. Dev. 1, 3

Mean Length 

± Std. Dev. 

(mm) 

Me

± 

Negative Control 75 8.1 ± 0.53 4.6 ± 0.06 0

Solvent Control 95 8.7 ± 1.3 4.5 ± 0.05 0

Pooled Control 85 8.4 ± 1.0 4.5 ± 0.05 0

1.4 90 8.9 ± 1.2 4.6 ± 0.06 02.4 90 6.7 ± 1.1 4.3 ± 0.10*, 4 05.5 100 8.1 ± 0.74 4.4 ± 0.08*, 4 0

12 95 6.9 ± 1.1 4.4 ± 0.00*, 4 021 90 7.7 ± 1.7 4.4 ± 0.05*, 4 043 90 8.3 ± 1.6 4.5 ± 0.13 0

* Indicates a statistically significant difference in mean total length from the pooled control (Dunnett’s one-tailed test, p≤0.05). 1 There were no statistically significant differences in survival (using Fisher’s Exact test, p > 0.05), in reproduction or in mean dry weight (u

one-tailed test, p > 0.05) from the pooled controls. 

2 21-day EC50 for survival: >43 ng/L 3 21-day EC50 for reproduction: >43 ng/L 4 The statistically significant differences in mean total length noted at 2.4, 5.5, 12 and 21 ng/L treatment levels were not considered to be bi

meaningful since the reductions were slight and did not follow a dose response pattern. In addition, the mean length of the daphnids in th

groups were within the ranges of the mean length of daphnids in the historical control data (3.6 to 6.3 mm). 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 29 - 

Appendix 1 

Dilution Water Chemistry Parameters Measured 

During the 4-Week Period Immediately Preceding the Test 

Parameter Mean Range 

Specific Conductance 354 335 – 367 

 (μS/cm) (N = 4) 

 

Hardness 141 140 – 144 

 (mg/L as CaCO3) (N = 4) 

Alkalinity 178 176 – 178 

 (mg/L as CaCO3) (N = 4) 

pH 8.1 8.0 – 8.1 

 (N = 4) 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 30 - 

Appendix 2 

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water 

Pesticides and Organics 1

Component 

Measured Concentration 

(μg/L) Component 

Measured Concentration 

(μg/L) 

Aldrin < 0.0097 Gamma BHC – Lindane < 0.0097 

Alpha BHC < 0.0097 Gamma Chlordane < 0.019 

Alpha Chlordane < 0.0097 Guthion (Azinphos-methyl) < 5.8 

Beta BHC < 0.0097 HCB < 0.0097 

Bolstar < 1.9 Heptachlor < 0.0097 

Chlordane < 0.48 Heptachlor Epoxide < 0.0097 

Coumaphos < 2.9 Kepone < 0.19 

Delta BHC < 0.0097 Malathion < 2.9 

Demeton-O < 1.9 Merphos < 1.9 

Demeton-S < 2.9 Methoxychlor < 0.097 

Diazinon < 5.8 Methyl Parathion < 1.9 

Dichlorvos < 2.9 Mevinphos < 4.8 

Dieldrin < 0.019 Mirex < 0.24 

Disulfoton < 1.9 Naled < 5.8 

Dursban (Chlorpyrifos) < 2.9 o,p-DDD < 0.019 

Endosulfan I < 0.0097 o,p-DDE < 0.73 

Endosulfan II < 0.019 o,p-DDT < 0.019 

Endosulfan Sulfate < 0.019 p,p-DDD < 0.019 

Endrin < 0.019 p,p-DDE < 0.019 

Endrin Aldehyde < 0.097 p,p-DDT < 0.019 

Endrin Ketone < 0.019 Phorate < 1.9 

EPN < 1.9 Ronnel < 1.9 

Ethion < 1.9 Stirophos < 1.9 

Ethoprop < 2.9 Telodrin < 0.0097 

Ethyl Parathion < 1.9 Tokuthion < 1.9 

Famphur < 2.9 Toxaphene < 2.9 

Fensulfothion < 8.7 Trichloronate < 1.9 

Fenthion < 1.9 Trithion < 1.9 

 

1

 Analyses performed by Lancaster Laboratories on samples collected on December 28, 2011. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 31 - 

Appendix 2 (Continued) 

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water 

Metals 1

Component 

Measured Concentration 

(mg/L) Component 

Measured Concentration 

(mg/L) 

 

Aluminum < 0.200 Magnesium 13.2 

Antimony < 0.0200 Manganese < 0.0050 

Arsenic < 0.0200 Mercury < 0.00020 

Barium < 0.0050 Nickel < 0.0100 

Beryllium < 0.0050 Nitrate Nitrogen < 0.50 

Bromide < 2.5 Nitrite Nitrogen < 0.50 

Cadmium < 0.0050 Potassium 7.00 

Calcium 34.9 Selenium < 0.0200 

Chloride 4.5 Silver < 0.0050 

Chromium < 0.0150 Sodium 19.0 

Cobalt < 0.0050 Sulfate 5.7 

Copper < 0.0100 Thallium < 0.0300 

Fluoride 0.85 Vanadium < 0.0050 

Iron < 0.200 Zinc < 0.0200 

Lead < 0.0150 

1

 Analyses performed by Lancaster Laboratories on samples collected on December 28, 2011. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 32 - 

Appendix 3 

The Analysis of 14C AN-2 in Freshwater 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 33 - 

Appendix 3.1 

Method Outline of the Analysis of 14C-AN-2 in Freshwater 

METHOD OUTLINE FOR THE ANALYSIS OF 

14C-AN-2 IN FRESHWATER 

Rinse separatory funnels and roundbottom flasks with dichloromethane. 



Using a tiltapetter, add 100 mL of dichloromethane to each sample. Stopper and shake each 

sample, with venting, for approximately one minute. Allow the organic and 

aqueous layers to separate. Drain the dichloromethane (lower) layer 

into a roundbottom flask. 



Add 100 mL of dichloromethane to separatory funnel containing the aqueous fraction. 

Shake and partition as described above and combine each extract in its 

respective roundbottom flask. 



Rotary evaporate each sample to approximately 0.50 – 1.0 mL using a water bath 

maintained at ~40°C. Do not rotary evaporate to dryness. 



Evaporate the samples to dryness under a gentle stream of nitrogen. 



Rinse the roundbottom flask with several aliquots of acetonitrile. Transfer the rinsates to a 

scintillation vial. Add approximately 13 mL of Ultima Gold XR and analyze by LSC. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 34 - 

Appendix 3.2 

Typical Counting Conditions for Liquid Scintillation Counting 

INSTRUMENT: Packard 2500TR Liquid Scintillation Analyzer 

COUNTING TIME: 3.00 minutes 

DATA MODE: DPM 

NUCLIDE: 14C-UG 

QUENCH INDICATOR: tSIE/AEC 

EXT. STD. TERMINATOR: Count 

QUENCH SET: 14C-UG 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 35 - 

Appendix 3.3 

Calculations for Sample Quantitation 

Sample Quantitation 

The equivalent concentrations of 14C-AN-2 (ng/L) were calculated from the following equation: 

Equivalent Concentration 

(ng/L) of 14C-AN-2 = 

Sample volume (L) x Specific activity (dpm/μg) x 1000

Total dpm found  background concentration (dpm)

Limit of Quantitation for Freshwater 

The limit of quantitation (LOQ) was calculated from the following equation: 

LOQ (ng/L) 

 = x 1000

0.200 (L) x 286380 (dpm/μg)

50 (dpm)

LOQ (ng/L) = 0.873 ng/L 

The calculated limit of quantitation (LOQ) was 0.873 ng/L 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 36 - 

Appendix 4 

Temperature of Water in the Test Chambers During Exposure to 14C AN-2 

Mean Measured 

Concentration 

(ng/L) Replicate 

Temperature (C) 

Day 0 Day 7 Day 14 Day 21 

Negative Control A1 19.8 19.7 20.0 20.2 

 B 19.7 19.8 19.9 20.2 

 

Solvent Control A 19.8 19.8 20.0 20.2 

 B 19.8 19.7 20.0 20.1 

1.4 A 19.8 19.7 20.0 20.1 

 B 19.9 19.7 20.1 20.3 

2.4 A 19.8 19.8 20.1 20.2 

 B 19.9 19.8 20.2 20.2 

5.5 A 19.8 19.8 20.1 20.2 

 B 19.7 19.8 20.1 20.1 

12 A 19.7 19.9 20.2 20.2 

 B 19.8 19.9 20.2 20.3 

21 A 19.9 19.8 20.1 20.4 

 B 19.8 19.8 20.2 20.3 

43 A 19.8 19.6 20.0 20.2 

 B 19.8 19.6 20.0 20.2 

1

 Manual temperature measurements. Temperature monitored continuously in one control test chamber ranged from 19 to 

20C, measured to the nearest 1oC. 
Wildlife International, Ltd. Pr

- 37 - 

Appendix 5 

Dissolved Oxygen Concentration of Water in the Test Chambers During Exposure to 14C AN-2 

Mean Measured 

Concentration 

(ng/L) Replicate 

Dissolved Oxygen Concentration (mg/L) by Day of Test 1,2

0 2 4 7 9 11 14 16 1

Negative Control A 8.5 -- 8.1 -- 8.4 -- 8.2 -- 7

 B -- 8.6 -- 8.2 -- 8.4 -- 8.3 

Solvent Control A 8.2 -- 7.2 -- 8.0 -- 6.7 -- 6

 B -- 8.4 -- 6.9 -- 7.2 -- 7.8 

1.4 A 7.7 -- 7.4 -- 7.9 -- 6.0 -- 6

 B -- 8.4 -- 6.0 -- 6.8 -- 7.7 

2.4 A 8.3 -- 8.0 -- 8.1 -- 5.7 -- 5

 B -- 8.4 -- 7.5 -- 6.9 -- 7.7 

5.5 A 7.7 -- 7.7 -- 7.8 -- 5.6 -- 5

 B -- 8.3 -- 6.5 -- 6.4 -- 7.7 

12 A 7.9 -- 7.7 -- 8.0 -- 5.8 -- 6

 B -- 8.1 -- 6.8 -- 7.2 -- 7.5 

21 A 8.2 -- 7.8 -- 7.9 -- 5.8 -- 6

 B -- 8.5 -- 7.6 -- 6.5 -- 7.5 

43 A 7.6 -- 7.7 -- 8.0 -- 6.6 -- 6

 B -- 8.4 -- 7.4 -- 7.6 -- 7.6 

1 A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20oC in freshwater. 2 -- = no measurement scheduled; additional measurements were taken, when necessary, to better monitor dissolved oxyg

concentrations. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 38 - 

Appendix 6 

pH of Water in the Test Chambers During Exposure to 14C AN-2 

Mean Measured 

Concentration 

(ng/L) Replicate 

pH 1

Day 0 Day 7 Day 14 Day 21 

 

Negative Control A 8.1 -- 8.1 -- 

 B -- 8.1 -- 8.2 

 

Solvent Control A 8.2 -- 8.0 -- 

 B -- 8.1 -- 8.1 

1.4 A 8.1 -- 8.0 -- 

 B -- 8.0 -- 8.0 

2.4 A 8.2 -- 8.0 -- 

 B -- 8.1 -- 8.0 

5.5 A 8.1 -- 8.0 -- 

 B -- 8.1 -- 8.0 

12 A 8.2 -- 8.0 -- 

 B -- 8.1 -- 8.1 

21 A 8.2 -- 8.0 -- 

 B -- 8.1 -- 8.1 

43 A 8.2 -- 8.0 -- 

 B -- 8.1 -- 8.1 

 1

 -- = no measurement scheduled. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 39 - 

Appendix 7 

Specific Conductance, Hardness and Alkalinity in the Negative Control 

and Highest Concentration Treatment Group During Exposure to 14C AN-2 

Negative Control 

Parameter 

Test Day 

 0 1 7 14 21 1

 

Specific Conductance 

(μS/cm) 

375 361 361 370 

Hardness 

(mg/L as CaCO3) 

128 140 124 144 

 

Alkalinity 

(mg/L as CaCO3) 

176 178 178 176 

 

Replicate A B A B 

1

 TOC measured in the dilution water at test initiation and termination was <1mg C/L. 

14C AN-2 Treatment Group 

Parameter 

Test Day 

0 7 14 21 

 

Specific Conductance 

(μS/cm) 

381 365 371 378 

 

Hardness 

(mg/L as CaCO3) 

140 140 140 144 

 

Alkalinity 

(mg/L as CaCO3) 

180 180 180 178 

 

Mean Measured 

Concentration (ng/L) 43 

Replicate A B A B 
Wildlife International, Ltd. Pr

- 40 - 

Appendix 8 

Mortality and Observations of First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Day of 

Test 1

Negative Control 

Replicate A1 Replicate A2 Replicate B1 

No. Imm.2 Observations 3 No. Imm. 2 Observations 3 No. Imm. 2 Observations 3 No

0 0 5 AN 0 5 AN 0 5 AN 1 0 5 AN 0 5 AN 0 5 AN 

 2 (T) 0 5 AN 0 5 AN 0 5 AN 

3 0 5 AN 2 3 C 0 5 AN 

 4 (T) 0 5 AN 3 -- 0 5 AN 

5 0 5 AN+ 0 -- 0 3 AN; 2 AN+ 6 0 5 AN+ 0 -- 0 5 AN+ 

 7 (T) 0 5 AN+ 0 -- 0 5 AN+; P 

8 0 5 AN+; P 0 -- 0 5 AN+ 

 9 (T) 0 5 AN+ 0 -- 0 5 AN+ 

10 0 5 AN+ 0 -- 0 5 AN+ 

 11 (T) 0 5 AN+ 0 -- 0 5 AN+ 

12 0 5 AN+ 0 -- 0 5 AN+ 13 0 5 AN+ 0 -- 0 5 AN+ 

 14 (T) 0 5 AN+ 0 -- 0 5 AN+ 

15 0 5 AN+ 0 -- 0 5 AN+ 

 16 (T) 0 3 AN+; 2 AN 0 -- 0 5 AN+ 

17 0 5 AN+ 0 -- 0 5 AN+ 

 18 (T) 0 5 AN+ 0 -- 0 5 AN+ 

19 0 5 AN+ 0 -- 0 5 AN+ 20 0 5 AN+ 0 -- 0 5 AN+ 21 0 5 AN+ 0 -- 0 5 AN+ 

1 T = Surviving adult daphnids were transferred to clean test compartments. 2 Imm. = Immobile. 3 Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy. 
Wildlife International, Ltd. Pr

- 41 - 

Appendix 8 (Continued) 

Mortality and Observations of First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Day of 

Test 1

Solvent Control 

Replicate A1 Replicate A2 Replicate B1 

No. Imm.2 Observations 3 No. Imm. 2 Observations 3 No. Imm. 2 Observations 3 No

0 0 5 AN 0 5 AN 0 5 AN 1 0 5 AN 0 5 AN 0 5 AN 

 2 (T) 0 5 AN 0 5 AN 0 5 AN 

3 0 1 V, C; 4 AN 0 5 AN 0 5 AN 

 4 (T) 0 1 D, C; 4 AN 0 5 AN 0 5 AN 

5 0 1 D, C; 4 AN 0 4 AN; 1 AN+ 0 3 AN; 2 AN+ 6 1 D 2 AN; 2 AN+ 0 3 AN; 2 AN+ 0 1 AN; 4 AN+ 

 7 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 

8 0 4 AN+; P 0 5 AN+; P 0 5 AN+; P 

 9 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 

10 0 4 AN+ 0 5 AN+ 0 5 AN+ 

 11 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 

12 0 4 AN+ 0 5 AN+ 0 5 AN+ 13 0 4 AN+ 0 5 AN+ 0 5 AN+ 

 14 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 

15 0 4 AN+ 0 5 AN+ 0 5 AN+ 

 16 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 

17 0 4 AN+ 0 5 AN+ 0 5 AN+ 

 18 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 

19 0 4 AN+ 0 5 AN+ 0 5 AN+ 20 0 4 AN+ 0 5 AN+ 0 5 AN+ 21 0 4 AN+ 0 5 AN+ 0 5 AN+ 

1 T = Surviving adult daphnids were transferred to clean test compartments. 2 Imm. = Immobile. 3 Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) 

organism; V = trapped on the screen or to the silicone sealant in the test compartment. 
Wildlife International, Ltd. Pr

- 42 - 

Appendix 8 (Continued) 

Mortality and Observations of First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Day of 

Test 1

1.4 ng/L 

Replicate A1 Replicate A2 Replicate B1 

No. Imm.2 Observations 3 No. Imm. 2 Observations 3 No. Imm. 2 Observations 3 No

0 0 5 AN 0 5 AN 0 5 AN 1 0 5 AN 0 5 AN 0 5 AN 

 2 (T) 0 5 AN 0 5 AN 0 5 AN 

3 0 5 AN 0 5 AN 0 5 AN 

 4 (T) 0 5 AN 0 5 AN 0 5 AN 

5 0 2 AN; 3 AN+ 0 4 AN; 1 AN+ 0 3 AN; 2 AN+ 6 0 1 AN; 4 AN+ 0 2 AN; 3 AN+ 0 2 AN; 3 AN+ 

 7 (T) 0 1 AN; 4 AN+; P 0 5 AN+ 0 5 AN+; P 

8 0 1 AN; 4 AN+ 0 5 AN+; P 0 5 AN+ 

 9 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 

10 0 5 AN+ 0 5 AN+ 1 D+ 4 AN+ 

 11 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 

12 0 5 AN+ 0 5 AN+ 0 4 AN+ 13 0 5 AN+ 0 5 AN+ 0 4 AN+ 

 14 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 

15 0 5 AN+ 0 5 AN+ 0 4 AN+ 

 16 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 

17 0 5 AN+ 0 5 AN+ 0 4 AN+ 

 18 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 

19 0 5 AN+ 0 5 AN+ 0 4 AN+ 20 0 5 AN+ 0 5 AN+ 0 4 AN+ 21 0 1 D+; 4 AN+ 0 5 AN+ 0 4 AN+ 

1 T = Surviving adult daphnids were transferred to clean test compartments. 2 Imm. = Immobile. 3 Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; D = discoloration (pale) in comparison 
Wildlife International, Ltd. Pr

- 43 - 

Appendix 8 (Continued) 

Mortality and Observations of First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Day of 

Test 1

2.4 ng/L 

Replicate A1 Replicate A2 Replicate B1 

No. Imm.2 Observations 3 No. Imm. 2 Observations 3 No. Imm. 2 Observations 3 No

0 0 5 AN 0 5 AN 0 5 AN 1 0 5 AN 0 5 AN 0 5 AN 

 2 (T) 0 5 AN 0 5 AN 0 5 AN 

3 0 5 AN 0 5 AN 0 5 AN 

 4 (T) 0 5 AN 0 5 AN 0 5 AN 

5 0 3 AN; 2 AN+ 0 3 AN; 2 AN+ 0 1 AN; 4 AN+ 6 0 3 AN; 2 AN+ 0 2 AN; 3 AN+ 0 5 AN+ 

 7 (T) 0 2 AN; 3 AN+ 0 2 AN; 3 AN+ 0 5 AN+; P 

8 0 1 AN; 4 AN+; P 0 5 AN+; P 0 5 AN+ 

 9 (T) 0 2 AN; 3 AN+ 0 5 AN+ 0 5 AN+ 

10 0 2 AN; 3 AN+ 0 5 AN+ 0 5 AN+ 

 11 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 

12 0 5 AN+ 0 5 AN+ 0 5 AN+ 13 0 5 AN+ 0 5 AN+ 0 5 AN+ 

 14 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 

15 0 5 AN+ 0 5 AN+ 0 5 AN+ 

 16 (T) 0 5 AN+ 0 5 AN+ 0 1 D, C+; 4 AN+ 

17 0 5 AN+ 0 5 AN+ 1D+ 4 AN+ 

 18 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 

19 0 5 AN+ 0 5 AN+ 0 4 AN+ 20 0 5 AN+ 0 5 AN+ 0 4 AN+ 21 0 5 AN+ 0 5 AN+ 0 4 AN+ 

1 T = Surviving adult daphnids were transferred to clean test compartments. 2 Imm. = Immobile. 3 Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) 

organism. 
Wildlife International, Ltd. Pr

- 44 - 

Appendix 8 (Continued) 

Mortality and Observations of First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Day of 

Test 1

5.5 ng/L 

Replicate A1 Replicate A2 Replicate B1 

No. Imm.2 Observations 3 No. Imm. 2 Observations 3 No. Imm. 2 Observations 3 No

0 0 5 AN 0 5 AN 0 5 AN 1 0 5 AN 0 5 AN 0 5 AN 

 2 (T) 0 5 AN 0 5 AN 0 5 AN 

3 0 5 AN 0 5 AN 0 5 AN 

 4 (T) 0 5 AN 0 5 AN 0 5 AN 

5 0 3 AN; 2 AN+ 0 4 AN; 1 AN+ 0 2 AN; 3 AN+ 6 0 5 AN+ 0 5 AN+ 0 1 AN; 4 AN+ 

 7 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+; P 

8 0 5 AN+; P 0 5 AN+; P 0 5 AN+ 

 9 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 

10 0 5 AN+ 0 5 AN+ 0 5 AN+ 

 11 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 

12 0 5 AN+ 0 5 AN+ 0 5 AN+ 13 0 5 AN+ 0 5 AN+ 0 5 AN+ 

 14 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 

15 0 5 AN+ 0 5 AN+ 0 5 AN+ 

 16 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 

17 0 5 AN+ 0 5 AN+ 0 5 AN+ 

 18 (T) 0 1 AN; 4 AN+ 0 5 AN+ 0 5 AN+ 

19 0 1 AN; 4 AN+ 0 5 AN+ 0 5 AN+ 20 0 5 AN+ 0 5 AN+ 0 5 AN+ 21 0 5 AN+ 0 5 AN+ 0 5 AN+ 

1 T = Surviving adult daphnids were transferred to clean test compartments. 2 Imm. = Immobile. 3 Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production. 
Wildlife International, Ltd. Pr

- 45 - 

Appendix 8 (Continued) 

Mortality and Observations of First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Day of 

Test 1

12 ng/L 

Replicate A1 Replicate A2 Replicate B1 

No. Imm.2 Observations 3 No. Imm. 2 Observations 3 No. Imm. 2 Observations 3 No

0 0 5 AN 0 2 V, AN; 3 AN 0 3 V, AN; 2 AN 1 0 5 AN 0 5 AN 0 5 AN 

 2 (T) 0 5 AN 0 5 AN 0 5 AN 

3 0 5 AN 0 5 AN 0 5 AN 

 4 (T) 0 5 AN 0 5 AN 0 5 AN 

5 0 1 AN; 4 AN+ 0 3 AN; 2 AN+ 0 5 AN 6 0 5 AN+ 0 2 AN; 3 AN+ 0 5 AN+ 

 7 (T) 0 5 AN+; P 0 5 AN+ 0 5 AN+ 

8 0 5 AN+ 0 5 AN+; P 0 5 AN+ 

 9 (T) 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+; P 

10 0 5 AN+ 0 2 D+, 3 AN+ 0 5 AN+ 

 11 (T) 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

12 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 13 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

 14 (T) 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

15 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

 16 (T) 0 1 AN; 4 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

17 0 5 AN+ 0 1 D+; 1 D, C+; 3 AN+ 0 5 AN+ 

 18 (T) 0 5 AN+ 0 1 D+; 1 D, C+; 3 AN+ 0 1 AN; 4 AN+ 

19 0 5 AN+ 0 1 D+; 1 D, C+; 3 AN+ 0 1 AN; 4 AN+ 20 0 5 AN+ 1 D+ 1 D+; 3 AN+ 0 5 AN+ 21 0 5 AN+ 0 1 D+; 3 AN+ 0 5 AN+ 

1 T = Surviving adult daphnids were transferred to clean test compartments. 2 Imm. = Immobile. 3 Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) 

organism; V = trapped on the screen or to the to silicone sealant in test compartment. 
Wildlife International, Ltd. Pr

- 46 - 

Appendix 8 (Continued) 

Mortality and Observations of First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Day of 

Test 1

21 ng/L 

Replicate A1 Replicate A2 Replicate B1 

No. Imm.2 Observations 3 No. Imm. 2 Observations 3 No. Imm. 2 Observations 3 No

0 0 5 AN 0 5 AN 0 5 AN 1 0 5 AN 0 5 AN 0 5 AN 

 2 (T) 0 5 AN 0 5 AN 0 5 AN 

3 0 5 AN 0 5 AN 0 5 AN 

 4 (T) 0 5 AN 0 5 AN 0 5 AN 

5 0 3 AN; 2 AN+ 0 3 AN; 2 AN+ 0 1 AN; 4 AN+ 6 0 5 AN+ 0 5 AN+ 0 1 AN; 4 AN+ 

 7 (T) 0 5 AN+; P 0 5 AN+; P 0 5 AN+ 

8 0 5 AN+ 0 5 AN+ 0 5 AN+; P 

 9 (T) 0 2 D, C+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 

10 0 2 D, C+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 

 11 (T) 0 1 D, C+; 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 

12 1+ 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 13 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 

 14 (T) 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 

15 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 

 16 (T) 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 

17 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 

 18 (T) 0 1 D+; 3 AN+ 0 1 AN; 4 AN+ 1 D+ 4 AN+ 

19 0 1 D+; 3 AN+ 0 1 AN; 4 AN+ 0 4 AN+ 20 0 1 D+; 3 AN+ 0 5 AN+ 0 4 AN+ 21 0 1 D+; 3 AN+ 0 5 AN+ 0 4 AN+ 

1 T = Surviving adult daphnids were transferred to clean test compartments. 2 Imm. = Immobile. 3 Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) 

organism; V = trapped on the screen or to the to silicone sealant in test compartment. 
Wildlife International, Ltd. Pr

- 47 - 

Appendix 8 (Continued) 

Mortality and Observations of First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Day of 

Test 1

43 ng/L 

Replicate A1 Replicate A2 Replicate B1 

No. Imm.2 Observations 3 No. Imm. 2 Observations 3 No. Imm. 2 Observations 3 No

0 0 5 AN 0 5 AN 0 5 AN 1 0 5 AN 0 5 AN 0 5 AN 

 2 (T) 0 1 V, C; 4 AN 0 5 AN 0 5 AN 

3 0 5 AN 0 5 AN 0 5 AN 

 4 (T) 0 1 S, D; 4 AN 0 5 AN 0 5 AN 

5 0 1 S, D,C; 3 AN; 1 AN+ 0 3 AN; 2 AN+ 0 3 AN; 2 AN+ 6 0 1 S, D,C; 3 AN; 1 AN+ 0 2 AN; 3 AN+ 0 5 AN+ 

 7 (T) 0 1 S, D,C; 4 AN+ 0 5 AN+ 0 5 AN+ 

8 0 1 S, C, D; 4 AN+; P 0 5 AN+; P 0 5 AN+; P 

 9 (T) 0 1 S, C, D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

10 0 1 S, C, D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

 11 (T) 0 1 S, D+; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

12 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 13 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

 14 (T) 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

15 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 

 16 (T) 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 1 D+; 4 AN+ 

17 0 2 D+; 3 AN+ 0 1 D; 1 D+; 3 AN+ 0 1 D+; 4 AN+ 

 18 (T) 0 2 D+; 3 AN+ 0 1 D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 

19 0 2 D+; 3 AN+ 0 1 D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 20 1 D+ 1 D+; 3 AN+ 0 1 D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 21 0 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 

1 T = Surviving adult daphnids were transferred to clean test compartments. 2 Imm. = Immobile. 3 Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) 

organism; S = small; V = trapped on the screen or to the silicone sealant in the test compartment. 
Wildlife International, Ltd. Pr

- 48 - 

Appendix 9 

Neonate Production by First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Mean Measured 

Concentration 

(ng/L) Rep. 

Number of Live Neonates Produced by Test Day1,2 No. Live 

Neonates 

Produced 

Number of 

Reproductive

Days 3

No. L

Neona

Produce

Reproduct

7 9 11 14 16 18 21 

Negative Control A1 0 37 134 (1) 152 0 110 156 589 75 7.9 

 A2 -- -- -- -- -- -- -- -- -- --  B1 9 85(1) 63 134 130 59 100 580 75 7.7  B2 18 57 134 165 51 141 85 651 75 8.7 

Solvent Control A1 0 51 107 154 93 82 128 615 60 10.3 

 A2 0 78 76 144 77 103 149 627 75 8.4  B1 0 74(2) 88 160 36 144 169 671 75 8.9  B2 0 46 59 133 102 64 129 533 75 7.1 

1.4 A1 5 74 106 199 50 121 138 693 75 9.2 

 A2 0 94 24 142 155 38 101 554 75 7.4  B1 2 71 92 152 94 184 61 656 63 10.4  B2 15 7 58 181 128 146 48 583 68 8.6 

2.4 A1 0 71 36 105 101 69 133 515 75 6.9 

 A2 0 83 27 104 66 35 78 393 75 5.2  B1 5 45 123 143 29 117 76 538 70 7.7  B2 0 83 69 115 78 21 80 446 64 7.0 

1 Neonate production was counted approximately 3 times per week following the onset of reproduction. 2 (#) = number of neonates observed to be immobile; if not indicated following neonate number then none were immobile.

3 Reproductive days were defined as the number of days that the adult daphnid was alive from the day the first brood was released from 

test until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last day it was alive. 

4 Calculated using SAS System for Windows; manual calculations may differ slightly. 

 -- = no neonate produced due to 100% immobility of the adult daphnid. 
Wildlife International, Ltd. Pr

- 49 - 

Appendix 9 (Continued) 

Neonate Production by First-Generation Daphnids Exposed to 14C AN-2 for 21 Days 

Mean Measured 

Concentration 

(ng/L) Rep. 

Number of Live Neonates Produced by Test Day1,2 No. Live 

Neonates 

Produced 

Number of 

Reproductive

Days 3

No. L

Neona

Produce

Reproduct

7 9 11 14 16 18 21 

5.5 A1 0 85 50 170 132 133 83 653 75 8.7 

 A2 0 82 30 164 156 58 84 574 75 7.7  B1 7 76 105 175 74 133 91 661 75 8.8  B2 1 74 77 127 49 88 135 551 75 7.3 

12 A1 15 45 112 124 59 129 151 635 75 8.5 

 A2 0 78 81 124 82 23 80 468 73 6.4  B1 0 106 0 129 174 17 50 476 75 6.3  B2 12 75 31 135 120 44 54 471 75 6.3 

21 A1 1 87 62 127 81 145 68 571 65 8.8 

 A2 11 46 128 152 42 189 112 680 75 9.1  B1 0 64 87 116 29 118 122 536 71 7.5  B2 0 58 27 62 109 40 103 399 75 5.3 

43 A1 0 91 21 108 140 28 94 482 73 6.6 

 A2 0 82(2) 105 138 41 157 116 639 75 8.5  B1 0 87(1) 59 150 109 62 103 570 75 7.6  B2 0 55 137 124 0 161 146 623 60 10.4 

1 Neonate production was counted approximately 3 times per week following the onset of reproduction. 2 (#) = number of neonates observed to be immobile; if not indicated following neonate number then none were immobile.

3 Reproductive days were defined as the number of days that the adult daphnid was alive from the day the first brood was released from 

test until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last day it was alive. 

4 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 50 - 

Appendix 10 

Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to 14C AN-2 for 21 D

Mean Measured 

Concentration 

(ng/L) Replicate 

Daphnid 

Number 

Individual 

Length 

 (mm) 1

Replicate 

Mean 

Length 

(mm) 2

Treatment 

Mean Length 

± Std. Dev. 

(mm) 3

Individual 

Dry Weight 

 (mg) 1

Replicate 

Mean 

Dry Weig

(mg) 2

Negative Control A1 1 4.4 4.6 4.6 ± 0.058 0.54 0.53 

 2 4.6 0.60  3 4.6 0.64  4 4.7 0.47  5 4.7 0.42  A2 1 -- -- -- --  2 -- --  3 -- --  4 -- --  5 -- --  B1 1 4.6 4.5 0.82 0.75  2 4.4 0.66  3 4.4 0.95  4 4.5 0.65  5 4.6 0.67  B2 1 4.6 4.6 0.63 0.62  2 4.3 0.54  3 4.8 0.82  4 4.7 0.43  5 4.6 0.67 

1 -- = immobility prior to test termination. 2 Calculated using Excel in full precision; manual calculations may differ slightly. 3 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 51 - 

Appendix 10 (Continued) 

Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to 14C AN-2 for 21 D

Mean Measured 

Concentration 

(ng/L) Replicate 

Daphnid 

Number 

Individual 

Length 

 (mm) 1

Replicate 

Mean 

Length 

(mm) 2

Treatment 

Mean Length 

± Std. Dev. 

(mm) 3

Individual 

Dry Weight 

 (mg) 1

Replicate 

Mean 

Dry Weig

(mg) 2

Solvent Control A1 1 4.6 4.6 4.5 ± 0.050 0.70 0.64 

 2 4.8 0.80  3 4.5 0.47  4 4.6 0.61  5 -- --  A2 1 4.6 4.5 0.61 0.69  2 4.4 0.65  3 4.6 0.75  4 4.6 0.65  5 4.5 0.79  B1 1 4.4 4.5 0.79 0.71  2 4.4 0.70  3 4.6 0.74  4 4.5 0.59  5 4.5 0.73  B2 1 4.6 4.5 0.70 0.71  2 4.5 0.60  3 4.6 0.70  4 4.6 0.85  5 4.4 0.71 

1 -- = mortality prior to test termination. 2 Calculated using Excel in full precision; manual calculations may differ slightly. 3 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 52 - 

Appendix 10 (Continued) 

Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to 14C AN-2 for 21 D

Mean Measured 

Concentration 

(ng/L) Replicate 

Daphnid 

Number 

Individual 

Length 

 (mm) 1

Replicate 

Mean 

Length 

(mm) 2

Treatment 

Mean Length 

± Std. Dev. 

(mm) 3

Individual 

Dry Weight 

 (mg) 1

Replicate 

Mean 

Dry Weig

(mg) 2

1.4 A1 1 4.7 4.5 4.6 ± 0.058 0.73 0.66 

 2 4.5 0.51  3 4.6 0.81  4 4.3 0.55  5 4.4 0.69  A2 1 4.4 4.5 0.73 0.68  2 4.5 0.64  3 4.4 0.59  4 4.6 0.78  5 4.4 0.67  B1 1 4.7 4.6 0.86 0.84  2 4.4 0.71  3 4.8 0.87  4 4.5 0.92  5 -- --  B2 1 4.7 4.6 0.92 0.86  2 4.5 0.81  3 4.5 0.79  4 4.5 0.92  5 -- -- 

1 -- = mortality prior to test termination. 2 Calculated using Excel in full precision; manual calculations may differ slightly. 3 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 53 - 

Appendix 10 (Continued) 

Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to 14C AN-2 for 21 D

Mean Measured 

Concentration 

(ng/L) Replicate 

Daphnid 

Number 

Individual 

Length 

 (mm) 1

Replicate 

Mean 

Length 

(mm) 2

Treatment 

Mean Length 

± Std. Dev. 

(mm) 3

Individual 

Dry Weight 

 (mg) 1

Replicate 

Mean 

Dry Weig

(mg) 2

2.4 A1 1 4.3 4.4 4.3 ± 0.10 0.64 0.58 

 2 4.4 0.57  3 4.4 0.55  4 4.5 0.59  5 4.4 0.54  A2 1 4.4 4.2 0.54 0.59  2 4.3 0.59  3 4.2 0.84  4 4.0 0.55  5 4.1 0.44  B1 1 4.2 4.3 0.75 0.61  2 4.3 0.53  3 4.3 0.70  4 4.4 0.48  5 -- --  B2 1 4.4 4.4 0.69 0.60  2 4.4 0.59  3 4.5 0.58  4 4.3 0.56  5 -- -- 

1 -- = mortality prior to test termination. 2 Calculated using Excel in full precision; manual calculations may differ slightly. 3 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 54 - 

Appendix 10 (Continued) 

Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to 14C AN-2 for 21 D

Mean Measured 

Concentration 

(ng/L) Replicate 

Daphnid 

Number 

Individual 

Length 

 (mm) 1

Replicate 

Mean 

Length 

(mm) 2

Treatment 

Mean Length 

± Std. Dev. 

(mm) 3

Individual 

Dry Weight 

 (mg) 1

Replicate 

Mean 

Dry Weig

(mg) 2

5.5 A1 1 4.4 4.5 4.4 ± 0.08 0.77 0.75 

 2 4.5 0.86  3 4.6 0.72  4 4.5 0.63  5 4.5 0.75  A2 1 4.4 4.4 0.74 0.80  2 4.4 0.88  3 4.6 0.69  4 4.4 0.85  5 4.3 0.84  B1 1 4.2 4.4 0.64 0.73  2 4.3 0.55  3 4.4 0.81  4 4.3 0.76  5 4.6 0.91  B2 1 4.2 4.3 0.88 0.69  2 4.4 0.67  3 4.3 0.59  4 4.5 0.63  5 4.3 0.66 

1 -- = mortality prior to test termination. 2 Calculated using Excel in full precision; manual calculations may differ slightly. 3 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 55 - 

Appendix 10 (Continued) 

Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to 14C AN-2 for 21 D

Mean Measured 

Concentration 

(ng/L) Replicate 

Daphnid 

Number 

Individual 

Length 

 (mm) 1

Replicate 

Mean 

Length 

(mm) 2

Treatment 

Mean Length 

± Std. Dev. 

(mm) 3

Individual 

Dry Weight 

 (mg) 1

Replicate 

Mean 

Dry Weig

(mg) 2

12 A1 1 4.3 4.4 4.4 ± 0.00 0.80 0.67 

 2 4.2 0.75  3 4.5 0.75  4 4.2 0.52  5 4.6 0.55  A2 1 4.4 4.4 0.69 0.79  2 4.6 0.80  3 4.3 0.84  4 4.2 0.83  5 -- --  B1 1 4.5 4.4 0.89 0.88  2 4.1 0.93  3 4.4 0.77  4 4.4 0.90  5 4.5 0.89  B2 1 4.3 4.4 0.64 0.70  2 4.4 0.75  3 4.4 0.79  4 4.3 0.73  5 4.4 0.61 

1 -- = mortality prior to test termination. 2 Calculated using Excel in full precision; manual calculations may differ slightly. 3 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 56 - 

Appendix 10 (Continued) 

Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to 14C AN-2 for 21 D

Mean Measured 

Concentration 

(ng/L) Replicate 

Daphnid 

Number 

Individual 

Length 

 (mm) 1

Replicate 

Mean 

Length 

(mm) 2

Treatment 

Mean Length 

± Std. Dev. 

(mm) 3

Individual 

Dry Weight 

 (mg) 1

Replicate 

Mean 

Dry Weig

(mg) 2

21 A1 1 4.6 4.5 4.4 ± 0.05 0.77 0.64 

 2 4.6 0.71  3 4.2 0.49  4 4.5 0.59  5 -- --  A2 1 4.4 4.4 0.49 0.55  2 4.5 0.47  3 4.3 0.59  4 4.5 0.54  5 4.3 0.66  B1 1 4.5 4.4 0.68 0.68  2 4.6 0.61  3 4.3 0.78  4 4.3 0.66  5 -- --  B2 1 4.3 4.4 0.69 0.64  2 4.7 0.66  3 4.5 0.72  4 4.4 0.52  5 4.3 0.62 

1 -- = mortality prior to test termination. 2 Calculated using Excel in full precision; manual calculations may differ slightly. 3 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. Pr

- 57 - 

Appendix 10 (Continued) 

Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to 14C AN-2 for 21 D

Mean Measured 

Concentration 

(ng/L) Replicate 

Daphnid 

Number 

Individual 

Length 

 (mm) 1

Replicate 

Mean 

Length 

(mm) 2

Treatment 

Mean Length 

± Std. Dev. 

(mm) 3

Individual 

Dry Weight 

 (mg) 1

Replicate 

Mean 

Dry Weig

(mg) 2

43 A1 1 4.0 4.3 4.5 ± 0.13 0.61 0.61 

 2 4.5 0.70  3 4.4 0.56  4 4.3 0.59  5 -- --  A2 1 4.3 4.4 0.62 0.54  2 4.4 0.57  3 4.4 0.61  4 4.5 0.47  5 4.3 0.45  B1 1 4.5 4.5 0.82 0.63  2 4.5 0.53  3 4.4 0.47  4 4.4 0.57  5 4.5 0.78  B2 1 4.3 4.6 0.65 0.61  2 4.6 0.67  3 4.6 0.58  4 4.7 0.56  5 -- -- 

1 -- = mortality prior to test termination. 2 Calculated using Excel in full precision; manual calculations may differ slightly. 3 Calculated using SAS System for Windows; manual calculations may differ slightly. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 58 - 

Appendix 11 

Changes to Protocol 

This study was conducted in accordance with the approved protocol with the following 

change: 

1. The protocol was amended to change the test initiation and termination dates since the study 

was conducted at a later dates. The protocol was also amended to change the test 

concentrations to a higher test concentrations range since it was anticipated that the original 

test concentrations would result in an unacceptable analytical results during the study. 

2. The protocol is amended to remove the test substance WIL#7791 since the study was 

conducted using radiolabelled material only. 
Wildlife International, Ltd. 

 Project Number 471A-120 

- 59 - 

Appendix 12 

Personnel Involved in the Study 

The following key Wildlife International, Ltd. personnel were involved in the conduct or 

management of this study: 

1. Henry O. Krueger, Ph.D., Director of Aquatic, Plant and Insect Toxicology 

2. Willard B. Nixon, Ph.D., Director of Chemistry 

3. Sean P. Gallagher, B.S., Manager of Aquatic Toxicology 

4. Tui Minderhout, Ph.D., senior Biologist 

5. Timothy Ross, B.S., Laboratory Supervisor 

6. Timothy Z. Kendall, M.S., Supervisor 

7. Blake Young, M.S., Biologist