# 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

# W ildlife International, Ltd. Project Number 471A-120

- 2 -

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 d esignated 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:______________________

# W ildlife International, Ltd. Project Number 471A-120

- 6 -

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 ......................................................................................... 1 4

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

14 

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

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

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

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

Statistical Anal yses ......................................................................................................... ......... 16

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

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

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

Survival and Clinical Observations ......................................................................................... 1 8

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

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

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

References .................................................................................................................... ........................ 22

# W ildlife International, Ltd. Project Number 471A-120

- 7 -

TABLE OF CONTENTS (Continued)

TABLES AND FIGURES

Table 1. Measured Concentrations of 14 

# C AN-2 in stock Solutions Analyzed by LSC .................. 23

Table 2. Measured Concentrations of 14 

# C AN-2 in Pretest Diluter Verification Samples .............. 24

Table 3. Measured Concentrations of 14 

# C 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 14 

# C AN-2 ....................................................................................................... 27

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

Exposed to 14 

# C 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 Me tals in Wildlife International, Ltd.

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

Appendix 3. Analysis of 14 

# C AN-2 in Freshwater ......................................................................... 32

3.1 Analytical Method Outline for the Analysis of 14 

# C AN-2

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

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

3.3 Calculations for Samp le Quantitation ....................................................................... 35

Appendix 4. Temperature of Water in the Test Chambers During Exposure to 14 

# C AN-2 ............ 36

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

During Exposure to 14 

# C AN-2 ........................................................................................ 37

Appendix 6. pH of Water in the Test Chambers During Exposure to 14 

# C AN-2 ............................ 38

# W ildlife International, Ltd. Project Number 471A-120

- 8 -

TABLE OF CONTENTS (Continued)

APPENDICES (Continued)

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

and Highest Concentration Treatment Group During E xposure to 14 

# C AN-2 ........... 39

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

14 

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

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

14 

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

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

after Exposure to 14 

# C AN-2 for 21 Days ................................................................... 50

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

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

# W ildlife International, Ltd. Project Number 471A-120

- 9 -

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

# W ildlife International, Ltd. Project Number 471A-120

- 10 -

INTRODUCTION

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

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

definitive test was conducted from March 19 to Ap ril 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 th e 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 tota l of 20 daphnids in each treatment and control

group. Nominal test concentrations were sel ected 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 ch ambers was initiated seven days prior to the

introduction of the daphnids to the test water in orde r to achieve equilibrium of the test substance in

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

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

reproduction, and clinical signs of toxicity. Fo llowing 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 e nd of the exposure period. Observations of the

effects of AN-2 on survival, reproduction and growth were used to determine the no-observed-effect

# W ildlife International, Ltd. Project Number 471A-120

- 11 -

concentration (NOEC), the lowest-observed-e ffect concentration (LOEC), and the maximum

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

on first-generation immobility at test termination.

MATERIALS AND METHODS

The study was conducted according to the proce dures 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 Guidelin es 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 14 

# C radiolabelled test substance used to pr epare 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 radi olabelled test substance was stored under frozen

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

129 μ Ci/mg or 55 mCi/mmol, based on gravimetric anal ysis, and a radiochemical concentration of

968 μ Ci/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 inve rtebrates 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 mainta ined by Wildlife International, Ltd., Easton,

Maryland.

Adult daphnids were cultured in water from th e 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

# W ildlife International, Ltd. Project Number 471A-120

- 12 -

525Aplus pH meter. Dissolved oxygen concentrations were  7.7 mg/L (  86% of saturation),

measured with a Thermo Orion Mode l 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 suspen sion of the freshwater green alga, Pseudokirchneriella

subcapitata . Daphnids were fed two or three times per da y 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 pe r 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 neonat es 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 wa ter 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 moderate ly-hard water. The specific conductance,

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

test are presented in Appendix 1. The results of periodic analyses performed to measure the

# W ildlife International, Ltd. Project Number 471A-120

- 13 -

concentrations of selected organic and inorgani c 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 con centration of the test substance, a solvent control and a negative

control (dilution water) to test chambers. Syri nge 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 diluti on water prior to delivery to the test chambers.

The flow of dilution water into each mixing ch amber 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 soluti ons or solvent to the mixing chambers were

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

chambers were calibrated prior to the test and ca librated/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 th e 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 th e 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 wate r temperature throughout the test period. Test

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

in two test compartments suspended in each of tw o 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 concentra tion and replicate designation.

# W ildlife International, Ltd. Project Number 471A-120

- 14 -

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 dimethyl formamide (DMF) at a nominal concentration of

0.064, 0.124, 0.252, 0.52, 1.0 and 2.0 μg/mL. Th e 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 pla ced in the syringe pump every 4 to 7 days during

the study.

The five test substance stock solutions were in jected 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 concen trations. 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 concentra tion 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 di luter were collected for

analysis on Day –11 to confirm the stock concentra tions, 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 treatme nt 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.

14 

# C AN-2 Stock Analysis

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

transferred to scintillation vials containing approxi mately 15 mL of Ultima Gold XR scintillation

cocktail, and the solutions were analyzed by 

# liquid scintillation counting (LSC) (Table 1).

# W ildlife International, Ltd. Project Number 471A-120

- 15 -

Analytical Method

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

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

samples were verified by liquid scintillation counti ng (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 ad dition to the test samples, a 13 mL sample of

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

radioactivity. Measured concentrations of 14 

# C-AN-2 are presented in Table 3. A method flowchart is

presented in Appendix 3.1 and in strument 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. Temper ature also was monitored continuously in one

negative control test chamber using a Fulscope ER/C r ecorder. 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,

# W ildlife International, Ltd. Project Number 471A-120

- 16 -

and at the end of the test. Measurements typi cally 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 t est 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 Shimad zu 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 we re 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 th e water column, uncoordinated swimming or cessation of feeding).

Immobility was defined as a lack of movement, ex cept 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, We dnesday and Friday during the test. The body

length and the dry weight of each surviving first-ge neration 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.

# W ildlife International, Ltd. Project Number 471A-120

- 17 -

Negative control and solvent control data fo r each parameter were compared using an

appropriate statistical test. No significant differe nces 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-va riable data, while reproduction and growth data

were considered continuous-variable data. Discret e-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-var iable 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 treatm ents 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 pr oduced no significant trea tment-related effects on

survival, reproduction or growth. The LOEC was define d as the lowest test co ncentration that produced

a significant treatment-related effect on survival, re production 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-genera tion 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 initia tion and termination appeared clear and colorless,

with no evidence of precipitation observed in any control or treatment solution.

# W ildlife International, Ltd. Project Number 471A-120

- 18 -

Samples of the stock solutions being deliv ered to the diluter system had measured

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

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

samples collected to verify the diluter system prio r 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 treat ment group on Day 21 was suspect (the recovery was

177%), additional sample was collected from this trea tment 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 collect ed 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 a nd pH in the test chambers are summarized in

Table 4, and individual measuremen ts are presented in A ppendices 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 sp ecific conductance, hardness and alkalinity in the

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

measurements are presented in Appendix 7. M easurements 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 di lution 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 da ys of exposure, survival in the negative control

# W ildlife International, Ltd. Project Number 471A-120

- 19 -

and solvent control groups were 75 and 95%, respec tively. There was statistically no significant

difference in survival between the negative and solv ent 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 decr ease 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 greate r 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 treatme nt 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. Wh ile 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 dap hnids 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 survivin g 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 th e test, indicating that there was no apparent delay

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

neonates in the negative control and solvent cont rol 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.

# W ildlife International, Ltd. Project Number 471A-120

- 20 -

Adult daphnids in the negative and solvent c ontrol 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 daphni ds 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 weight s of surviving first-generation daphnids are

presented in Table 5. Individual growth measuremen ts 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 c ontrol 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, resp ectively, and mean dry weights of 0.76, 0.60, 0.74,

0.76, 0.63 and 0.60 mg, respectively. Dunnett’s t est 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 redu ctions 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 th e 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 (le ngth and dry weight) was 43 ng/L, and the LOEC

was greater than 43 ng/L.

# W ildlife 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 su rvival, reproduction or growth at concentrations

≤ 43 ng/L. Consequently, the NOEC, based on surv ival, 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 repr oduction were both greater than 43 ng/L, the

highest concentration tested.

# W ildlife 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. 20 th 

# 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.

# W ildlife International, Ltd. Project Number 471A-120

- 23 -

Table 1

Measured Concentrations of 14 

# C-AN-2 in Stock Solutions Analyzed by LSC

Sample

Type Sample

Number

(471A-120-) Nominal

AN-2

Concentration

( μ g/mL) Specific

Activity

(dpm/ μ g) Total [ 14 

# C]

Found

(dpm) AN-2

Equivalents

Found 1



# ( μ g/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

# W ildlife 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 14 

# C

Found

(dpm) AN-2

Equivalents

Found 1,2,3



# (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 / μ g.

2 

# AN-2 Equiv. = ({[Total dpm found - background cont ribution)/Sample volume]}/ Specific Activity) x 1000

3 

# LOQ = {[(Total dpm found / Sample Volume ]}/Specific Activity) x 1000= 0.873 ng/L.Wildlife International, Ltd.Project Number 471A-120- 25 -Table 3Measured Concentrations of14C AN-2 in Test Solution Samples Analyzed by LSCNominalAN-2Concentration(ng/L)SampleNumber(471A-120-)SamplingInterval(Day)SampleVolume(L)SpecificActivity(dpm/g)Total[14C]Found(dpm)AN-2EquivalentsFound(ng/L)1,2,3PercentofNominalMeanAN-2Equivalents(ng/L)MeanPercent ofNominalBackground ----------07142122--------------------24.3125.2834.3024.1924.67---------------------- --Negative Control(0.0)1917250714210.2000.2000.2000.20028638028638028638028638029.3725.6629.9724.03< LOQ< LOQ< LOQ< LOQ---------- --Solvent Control(0.0)21018260714210.2000.2000.2000.20028638028638028638028638026.3228.9427.8127.74< LOQ< LOQ< LOQ< LOQ---------- --1.6 311192733071421*220.2000.2000.2000.2000.20028638028638028638028638028638094.30104.2699.06185.98124.381.221.381.132.821.7476.486.270.71771091.4 881AN-2 Equivalents = ({[Total dpm found - background contribution)/Sample volume]}/Specific Activity) x 10002The calculated limit of quantitation (LOQ) was 0.873 ng/L.3Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.* Day 21 sample is excluded from the meancalculation due to high recovery. An additional sample was taken on Day 22 and willbe used in the calculation.Wildlife International, Ltd.Project Number 471A-120- 26 -Table 3 (Continued)Measured Concentrations of14C-AN-2 in Test Samples Analyzed by LSCNominalAN-2Concentration(ng/L)SampleNumber(471A-120-)SamplingInterval(Day)SampleVolume(L)SpecificActivity(dpm/g)Total[14C]Found(dpm)AN-2EquivalentsFound(ng/L)1,2,3PercentofNominalMeanAN-2Equivalents(ng/L)MeanPercent ofNominal3.1 41220280714210.2000.2000.2000.200286380286380286380286380186.09184.37156.49141.412.822.782.132.0591.189.668.866.02.4 776.3 51321290714210.2000.2000.2000.200286380286380286380286380347.46348.55332.56337.605.645.645.215.4789.689.682.786.95.5 8713 61422300714210.2000.2000.2000.200286380286380286380286380748.18693.56681.32657.7712.611.711.311.197.289.886.985.112 9225 71523310714210.1000.1000.1000.100286380286380286380286380629.64629.83612.85636.3221.121.120.221.484.584.480.885.521 8450 81624320714210.05000.05000.05000.0500286380286380286380286380649.51685.01625.03621.2943.746.141.341.787.392.182.583.443 861AN-2 Equivalents = ({[Total dpm found - background contribution)/Sample volume]}/Specific Activity) x 10002The calculated limit of quantitation (LOQ) was 0.873 ng/L.3Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 27 -Table 4Means and Ranges of Water Quality Measurements Taken During the 21-Day Exposure to14C AN-2Mean MeasuredConcentration(ng/L)Mean ± Std. Dev. and Range of Measured Parameters1Temperature2(°C)Dissolved Oxygen3(mg/L)pHHardness4(mg/L as CaCO3)Alkalinity4(mg/L as CaCO3)Conductivity4(S/cm)Negative Control 19.9 ± 0.20 8.3 ± 0.20 8.1 ± 0.05 134 ± 10 177 ± 1 367 ± 7(19.7 – 20.2)) (7.9 – 8.6) (8.1 – 8.2) (124 – 144) (176 – 178) (361 – 375)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 ± 1 374 ± 7(19.6 – 20.2) (6.4 – 8.4) (8.0 – 8.2) (140 – 144) (178 – 180) (365 – 381)1Total organic carbon measured in the dilution water attest initiation and termination was <1 mg C/L.2Temperature monitored continuously during the test ranged from approximately 19 to 20C, measured to the nearest 1C.3A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20oC in freshwater.4-- = no measurements scheduled.Wildlife International, Ltd.Project Number 471A-120- 28 -Table 5Summary of Survival, Reproduction and Growth ofDaphnia magnaExposed to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)Percent AdultSurvival1,2Mean No. NeonatesPer Reproductive Day± Std. Dev.1, 3Mean Length± Std. Dev.(mm)Mean Dry Weight± Std. Dev.1(mg)Negative Control75 8.1 ± 0.53 4.6 ± 0.06 0.63 ± 0.11Solvent Control95 8.7 ± 1.3 4.5 ± 0.05 0.69± 0.033Pooled Control85 8.4 ± 1.0 4.5 ± 0.05 0.66 ± 0.0741.490 8.9 ± 1.2 4.6 ± 0.06 0.76 ± 0.102.490 6.7 ± 1.1 4.3 ± 0.10*, 40.60 ± 0.0135.5100 8.1 ± 0.74 4.4 ± 0.08*, 40.74 ± 0.0461295 6.9 ± 1.1 4.4 ± 0.00*, 40.76 ± 0.0952190 7.7 ± 1.7 4.4 ± 0.05*, 40.63 ± 0.0554390 8.3 ± 1.6 4.5 ± 0.13 0.60± 0.039*Indicates a statistically significant difference in mean total length from the pooled control(Dunnett’s one-tailed test, p≤0.05).1There were no statistically significant differences in survival (using Fisher’s Exact test, p > 0.05), in reproduction or in mean dry weight (using Dunnett’sone-tailed test, p > 0.05) from the pooled controls.221-day EC50 for survival: >43 ng/L321-day EC50 for reproduction: >43 ng/L4The statistically significant differences in mean total lengthnoted at 2.4, 5.5, 12 and 21 ng/L treatment levels were not considered to be biologicallymeaningful since the reductions were slight and did not follow adose response pattern. In addition, the mean length of the daphnids in these treatmentgroups were within the ranges of the mean length of daphnids in the historical control data (3.6 to 6.3 mm).

# W ildlife 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 CaCO 3 

# ) (N = 4)

Alkalinity 178 176 – 178

(mg/L as CaCO 3 

# ) (N = 4)

pH 8.1 8.0 – 8.1

(N = 4)

# W ildlife 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 Laborator ies on samples collected on December 28, 2011.

# W ildlife 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 Laborator ies on samples collected on December 28, 2011.

# W ildlife International, Ltd. Project Number 471A-120

- 32 -

Appendix 3

The Analysis of 14 

# C AN-2 in Freshwater

# W ildlife International, Ltd. Project Number 471A-120

- 33 -

Appendix 3.1

Method Outline of the Analysis of 14 

# C-AN-2 in Freshwater



# METHOD OUTLINE FOR THE ANALYSIS OF

14 

# C-AN-2 IN FRESHWATER

Rinse separatory funnels and roundbot tom flasks with dichloromethane.



Using a tiltapetter, add 100 mL of dichlorome thane 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 separator y 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 approxim ately 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.

# W ildlife 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: 14 

# C-UG

QUENCH INDICATOR: tSIE/AEC

EXT. STD. TERMINATOR: Count

QUENCH SET: 14 

# C-UG

# W ildlife International, Ltd. Project Number 471A-120

- 35 -

Appendix 3.3

Calculations for Sample Quantitation

Sample Quantitation

The equivalent concentrations of 14 

# C-AN-2 (ng/L) were calculated from the following equation:

Equivalent Concentration

(ng/L) of 14 

# C-AN-2 = 

# 1000 x (dpm/μg) activity Specific x (L) volume Sample (dpm) ion concentrat background found dpm Total



Limit of Quantitation for Freshwater



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

LOQ (ng/L)

= 1000 x (dpm/μg) 86380 2 x (L) 200

. 0 (dpm) 50

LOQ (ng/L) = 0.873 ng/L

The calculated limit of quantitation (LOQ) was 0.873 ng/L

# W ildlife International, Ltd. Project Number 471A-120

- 36 -

Appendix 4



# Temperature of Water in the Test Chambers During Exposure to 14 

# C AN-2

Mean Measured

Concentration

(ng/L) Replicate Temperature (  C)

Day 0 Day 7 Day 14 Day 21

Negative Control A 1 

# 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 measuremen ts. Temperature monitored continuously in one control test chamber ranged from 19 to

20  C, measured to the nearest 1oC.Wildlife International, Ltd.Project Number 471A-120- 37 -Appendix 5Dissolved Oxygen Concentration of Water in the Test Chambers During Exposure to14C AN-2Mean MeasuredConcentration(ng/L)ReplicateDissolved Oxygen Concentration (mg/L) by Day of Test1,20 2 4 7 9 11 14 16 18 21Negative Control A 8.5 -- 8.1 -- 8.4 -- 8.2 -- 7.9 --B -- 8.6 -- 8.2 -- 8.4 -- 8.3 -- 8.2Solvent Control A 8.2 -- 7.2 -- 8.0 -- 6.7 -- 6.8 --B -- 8.4 -- 6.9 -- 7.2 -- 7.8 -- 7.31.4 A 7.7 -- 7.4 -- 7.9 -- 6.0 -- 6.5 --B -- 8.4 -- 6.0 -- 6.8 -- 7.7 -- 6.52.4 A 8.3 -- 8.0 -- 8.1 -- 5.7 -- 5.9 --B -- 8.4 -- 7.5 -- 6.9 -- 7.7 -- 6.35.5 A 7.7 -- 7.7 -- 7.8 -- 5.6 -- 5.9 --B--8.3--6.5--6.4--7.7--6.312 A 7.9 -- 7.7 -- 8.0 -- 5.8 -- 6.0 --B--8.1--6.8--7.2--7.5--6.821 A 8.2 -- 7.8 -- 7.9 -- 5.8 -- 6.0 --B -- 8.5 -- 7.6 -- 6.5 -- 7.5 -- 6.643 A 7.6 -- 7.7 -- 8.0 -- 6.6 -- 6.4 --B -- 8.4 -- 7.4 -- 7.6 -- 7.6 -- 6.91A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20oC in freshwater.2-- = no measurement scheduled; additional measurements weretaken, when necessary, to better monitor dissolved oxygenconcentrations.

# W ildlife International, Ltd. Project Number 471A-120

- 38 -

Appendix 6



# pH of Water in the Test Chambers During Exposure to 14 

# C 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.

# W ildlife International, Ltd. Project Number 471A-120

- 39 -

Appendix 7



# Specific Conductance, Hardness and Al kalinity in the Negative Control

and Highest Concentration Treatment Group During E xposure to 14 

# C 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 CaCO 3 

# ) 128 140 124 144

Alkalinity

(mg/L as CaCO 3 

# ) 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.

14 

# C AN-2 Treatment Group

Parameter Test Day

0 7 14 21

Specific Conductance

(μS/cm) 381 365 371 378

Hardness

(mg/L as CaCO 3 

# ) 140 140 140 144

Alkalinity

(mg/L as CaCO 3 

# ) 180 180 180 178

Mean Measured

Concentration (ng/L) 43

Replicate A B A BWildlife International, Ltd.Project Number 471A-120- 40 -Appendix 8Mortality and Observations of First-Generation Daphnids Exposed to14C AN-2 for 21 DaysDay ofTest1Negative ControlReplicate A1 Replicate A2 Replicate B1 Replicate B2No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations30 0 5 AN 0 5 AN 0 5 AN 0 5 AN1 0 5 AN 0 5 AN 0 5 AN 0 5 AN2 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN3 0 5 AN 2 3 C 0 5 AN 0 5 AN4 (T) 0 5 AN 3 -- 0 5 AN 0 5 AN5 0 5 AN+ 0 -- 0 3 AN; 2 AN+ 0 1 AN; 4 AN+6 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+7 (T) 0 5 AN+ 0 -- 0 5 AN+; P 0 5 AN+; P8 0 5 AN+; P 0 -- 0 5 AN+ 0 5 AN+9 (T) 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+10 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+11 (T) 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+12 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+13 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+14 (T) 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+15 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+16 (T) 0 3 AN+; 2 AN 0 -- 0 5 AN+ 0 5 AN+17 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+18 (T) 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+19 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+20 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+21 0 5 AN+ 0 -- 0 5 AN+ 0 5 AN+1T = Surviving adult daphnids were transferred to clean test compartments.2Imm. = Immobile.3Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy.Wildlife International, Ltd.Project Number 471A-120- 41 -Appendix 8 (Continued)Mortality and Observations of First-Generation Daphnids Exposed to14C AN-2 for 21 DaysDay ofTest1Solvent ControlReplicate A1 Replicate A2 Replicate B1 Replicate B2No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations30 0 5 AN 0 5 AN 0 5 AN 0 5 AN1 0 5 AN 0 5 AN 0 5 AN 0 5 AN2 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN3 0 1 V, C; 4 AN 0 5 AN 0 5 AN 0 5 AN4 (T) 0 1 D, C; 4 AN 0 5 AN 0 5 AN 0 5 AN5 0 1 D, C; 4 AN 0 4 AN; 1 AN+ 0 3 AN; 2 AN+ 0 4 AN; 1 AN+6 1 D 2 AN; 2 AN+ 0 3 AN; 2 AN+ 0 1 AN; 4 AN+ 0 4 AN; 1 AN+7 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+8 0 4 AN+; P 0 5 AN+; P 0 5 AN+; P 0 5 AN+; P9 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+10 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+11 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+12 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+13 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+14 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+15 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+16 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+17 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+18 (T) 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+19 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+20 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+21 0 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+1T = Surviving adult daphnids were transferred to clean test compartments.2Imm. = Immobile.3Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) in comparison to controlorganism; V = trapped on the screen or to the silicone sealant in the test compartment.Wildlife International, Ltd.Project Number 471A-120- 42 -Appendix 8 (Continued)Mortality and Observations of First-Generation Daphnids Exposed to14C AN-2 for 21 DaysDay ofTest11.4 ng/LReplicate A1 Replicate A2 Replicate B1 Replicate B2No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations30 0 5 AN 0 5 AN 0 5 AN 0 5 AN1 0 5 AN 0 5 AN 0 5 AN 0 5 AN2 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN3 0 5 AN 0 5 AN 0 5 AN 0 5 AN4 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN5 0 2 AN; 3 AN+ 0 4 AN; 1 AN+ 0 3 AN; 2 AN+ 0 3 AN; 2 AN+6 0 1 AN; 4 AN+ 0 2 AN; 3 AN+ 0 2 AN; 3 AN+ 0 5 AN+7 (T) 0 1 AN; 4 AN+; P 0 5 AN+ 0 5 AN+; P 0 1 AN ; 4 AN+; P8 0 1 AN; 4 AN+ 0 5 AN+; P 0 5 AN+ 0 5 AN+9 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+10 0 5 AN+ 0 5 AN+ 1 D+ 4 AN+ 0 5 AN+11 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 5 AN+12 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 5 AN+13 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 5 AN+14 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 5 AN+15 0 5 AN+ 0 5 AN+ 0 4 AN+ 1 D+ 4 AN+16 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 4 AN+17 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 4 AN+18 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 3 AN+; 1 AN19 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 3 AN; 1 AN+20 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 4 AN+21 0 1 D+; 4 AN+ 0 5 AN+ 0 4 AN+ 0 4 AN+1T = Surviving adult daphnids were transferred to clean test compartments.2Imm. = Immobile.3Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; D = discoloration (pale) in comparison to control organism.Wildlife International, Ltd.Project Number 471A-120- 43 -Appendix 8 (Continued)Mortality and Observations of First-Generation Daphnids Exposed to14C AN-2 for 21 DaysDay ofTest12.4 ng/LReplicate A1 Replicate A2 Replicate B1 Replicate B2No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations30 0 5 AN 0 5 AN 0 5 AN 0 5 AN1 0 5 AN 0 5 AN 0 5 AN 0 5 AN2 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN3 0 5 AN 0 5 AN 0 5 AN 0 5 AN4 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN5 0 3 AN; 2 AN+ 0 3 AN; 2 AN+ 0 1 AN; 4 AN+ 0 3 AN; 2 AN+6 0 3 AN; 2 AN+ 0 2 AN; 3 AN+ 0 5 AN+ 0 1 AN; 4 AN+7 (T) 0 2 AN; 3 AN+ 0 2 AN; 3 AN+ 0 5 AN+; P 0 5 AN+8 0 1 AN; 4 AN+; P 0 5 AN+; P 0 5 AN+ 0 5 AN+; P9 (T) 0 2 AN; 3 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+10 0 2 AN; 3 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+11 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 1 D+ 4 AN+12 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 4 AN+13 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 4 AN+14 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 4 AN+15 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 4 AN+16 (T) 0 5 AN+ 0 5 AN+ 0 1 D, C+; 4 AN+ 0 4 AN+17 0 5 AN+ 0 5 AN+ 1D+ 4 AN+ 0 4 AN+18 (T) 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 4 AN+19 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 4 AN+20 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 4 AN+21 0 5 AN+ 0 5 AN+ 0 4 AN+ 0 4 AN+1T = Surviving adult daphnids were transferred to clean test compartments.2Imm. = Immobile.3Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) in comparison to the controlorganism.Wildlife International, Ltd.Project Number 471A-120- 44 -Appendix 8 (Continued)Mortality and Observations of First-Generation Daphnids Exposed to14C AN-2 for 21 DaysDay ofTest15.5 ng/LReplicate A1 Replicate A2 Replicate B1 Replicate B2No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations30 0 5 AN 0 5 AN 0 5 AN 0 5 AN1 0 5 AN 0 5 AN 0 5 AN 0 5 AN2 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN3 0 5 AN 0 5 AN 0 5 AN 0 5 AN4 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN5 0 3 AN; 2 AN+ 0 4 AN; 1 AN+ 0 2 AN; 3 AN+ 0 2 AN; 3 AN+6 0 5 AN+ 0 5 AN+ 0 1 AN; 4 AN+ 0 1 AN; 4 AN+7 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+; P 0 5 AN+; P8 0 5 AN+; P 0 5 AN+; P 0 5 AN+ 0 5 AN+9 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+10 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+11 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+12 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+13 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+14 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+15 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+16 (T) 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+17 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+18 (T) 0 1 AN; 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+19 0 1 AN; 4 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+20 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+21 0 5 AN+ 0 5 AN+ 0 5 AN+ 0 5 AN+1T = Surviving adult daphnids were transferred to clean test compartments.2Imm. = Immobile.3Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production.Wildlife International, Ltd.Project Number 471A-120- 45 -Appendix 8 (Continued)Mortality and Observations of First-Generation Daphnids Exposed to14C AN-2 for 21 DaysDay ofTest112 ng/LReplicate A1 Replicate A2 Replicate B1 Replicate B2No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations30 0 5 AN 0 2 V, AN; 3 AN 0 3 V, AN; 2 AN 0 3 V, AN; 2 AN1 0 5 AN 0 5 AN 0 5 AN 0 5 AN2 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN3 0 5 AN 0 5 AN 0 5 AN 0 5 AN4 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN5 0 1 AN; 4 AN+ 0 3 AN; 2 AN+ 0 5 AN 0 4 AN; 1 AN+6 0 5 AN+ 0 2 AN; 3 AN+ 0 5 AN+ 0 2 AN; 3 AN+7 (T) 0 5 AN+; P 0 5 AN+ 0 5 AN+ 0 5 AN+; P8 0 5 AN+ 0 5 AN+; P 0 5 AN+ 0 5 AN+9 (T) 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+; P 0 1 AN; 4 AN+10 0 5 AN+ 0 2 D+, 3 AN+ 0 5 AN+ 0 1 AN; 4 AN+11 (T) 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 5 AN+12 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 5 AN+13 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 5 AN+14 (T) 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 5 AN+15 0 5 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 5 AN+16 (T) 0 1 AN; 4 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 5 AN+17 0 5 AN+ 0 1 D+; 1 D, C+; 3 AN+ 0 5 AN+ 0 5 AN+18 (T) 0 5 AN+ 0 1 D+; 1 D, C+; 3 AN+ 0 1 AN; 4 AN+ 0 1 AN; 4 AN+19 0 5 AN+ 0 1 D+; 1 D, C+; 3 AN+ 0 1 AN; 4 AN+ 0 1 AN; 4 AN+20 0 5 AN+ 1 D+ 1 D+; 3 AN+ 0 5 AN+ 0 5 AN+21 0 5 AN+ 0 1 D+; 3 AN+ 0 5 AN+ 0 5 AN+1T = Surviving adult daphnids were transferred to clean test compartments.2Imm. = Immobile.3Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) in comparison to the controlorganism; V = trapped on the screen or to the to silicone sealant in test compartment.Wildlife International, Ltd.Project Number 471A-120- 46 -Appendix 8 (Continued)Mortality and Observations of First-Generation Daphnids Exposed to14C AN-2 for 21 DaysDay ofTest121 ng/LReplicate A1 Replicate A2 Replicate B1 Replicate B2No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations30 0 5 AN 0 5 AN 0 5 AN 0 3 V, AN; 2 AN1 0 5 AN 0 5 AN 0 5 AN 0 5 AN2 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN3 0 5 AN 0 5 AN 0 5 AN 0 5 AN4 (T) 0 5 AN 0 5 AN 0 5 AN 0 5 AN5 0 3 AN; 2 AN+ 0 3 AN; 2 AN+ 0 1 AN; 4 AN+ 0 4 AN; 1 AN+6 0 5 AN+ 0 5 AN+ 0 1 AN; 4 AN+ 0 4 AN; 1 AN+7 (T) 0 5 AN+; P 0 5 AN+; P 0 5 AN+ 0 5 AN+8 0 5 AN+ 0 5 AN+ 0 5 AN+; P 0 5 AN+; P9 (T) 0 2 D, C+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+10 0 2 D, C+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+11 (T) 0 1 D, C+; 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+12 1+ 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+13 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+14 (T) 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+15 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+16 (T) 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+17 0 1 D+; 3 AN+ 0 5 AN+ 0 1 D+; 4 AN+ 0 2 D+; 3 AN+18 (T) 0 1 D+; 3 AN+ 0 1 AN; 4 AN+ 1 D+ 4 AN+ 0 1 D; 1 D+; 3 AN+19 0 1 D+; 3 AN+ 0 1 AN; 4 AN+ 0 4 AN+ 0 1 D; 1 D+; 3 AN+20 0 1 D+; 3 AN+ 0 5 AN+ 0 4 AN+ 0 2 D+; 3 AN+21 0 1 D+; 3 AN+ 0 5 AN+ 0 4 AN+ 0 2 D+; 3 AN+1T = Surviving adult daphnids were transferred to clean test compartments.2Imm. = Immobile.3Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) in comparison to the controlorganism; V = trapped on the screen or to the to silicone sealant in test compartment.Wildlife International, Ltd.Project Number 471A-120- 47 -Appendix 8 (Continued)Mortality and Observations of First-Generation Daphnids Exposed to14C AN-2 for 21 DaysDay ofTest143 ng/LReplicate A1 Replicate A2 Replicate B1 Replicate B2No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations3No. Imm.2Observations30 0 5 AN 0 5 AN 0 5 AN 0 5 AN1 0 5 AN 0 5 AN 0 5 AN 0 5 AN2 (T) 0 1 V, C; 4 AN 0 5 AN 0 5 AN 0 5 AN3 0 5 AN 0 5 AN 0 5 AN 0 5 AN4 (T) 0 1 S, D; 4 AN 0 5 AN 0 5 AN 0 5 AN5 0 1 S, D,C; 3 AN; 1 AN+ 0 3 AN; 2 AN+ 0 3 AN; 2 AN+ 0 4 AN; 1 AN+6 0 1 S, D,C; 3 AN; 1 AN+ 0 2 AN; 3 AN+ 0 5 AN+ 0 5 AN+7 (T) 0 1 S, D,C; 4 AN+ 0 5 AN+ 0 5 AN+ 1+ 4 AN+8 0 1 S, C, D; 4 AN+; P 0 5 AN+; P 0 5 AN+; P 0 4 AN+; P9 (T) 0 1 S, C, D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 4 AN+10 0 1 S, C, D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 4 AN+11 (T) 0 1 S, D+; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 4 AN+12 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 4 AN+13 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 4 AN+14 (T) 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 4 AN+15 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 5 AN+ 0 4 AN+16 (T) 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 1 D+; 4 AN+ 0 4 AN+17 0 2 D+; 3 AN+ 0 1 D; 1 D+; 3 AN+ 0 1 D+; 4 AN+ 0 1 AN; 3 AN+18 (T) 0 2 D+; 3 AN+ 0 1 D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 4 AN+19 0 2 D+; 3 AN+ 0 1 D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 4 AN+20 1 D+ 1 D+; 3 AN+ 0 1 D; 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 1 AN; 3 AN+21 0 1 D+; 3 AN+ 0 2 D+; 3 AN+ 0 2 D+; 3 AN+ 0 1 D+; 3 AN+1T = Surviving adult daphnids were transferred to clean test compartments.2Imm. = Immobile.3Observations: AN = appear normal; + = eggs present in brood pouch; P = onset of production; C = lethargy; D = discoloration (pale) in comparison to the controlorganism; S = small; V = trapped on the screen or to the silicone sealant in the test compartment.Wildlife International, Ltd.Project Number 471A-120- 48 -Appendix 9Neonate Production by First-Generation Daphnids Exposed to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)Rep.Number of Live Neonates Produced by Test Day1,2No. LiveNeonatesProducedNumber ofReproductiveDays3No. LiveNeonatesProduced PerReproductive DayMean No.Neonates PerReproductive Day± Std. Dev.47 9 11 14 16 18 21Negative Control A1 0 37 134 (1) 152 0 110 156 589 75 7.9 8.1 ± 0.53A2 -- -- -- -- -- -- -- -- -- --B1 9 85(1) 63 134 130 59 100 580 75 7.7B2 18 57 134 165 51 141 85 651 75 8.7Solvent Control A1 0 51 107 154 93 82 128 615 60 10.3 8.7 ± 1.3A2 0 78 76 144 77 103 149 627 75 8.4B1 0 74(2) 88 160 36 144 169 671 75 8.9B2 0 46 59 133 102 64 129 533 75 7.11.4 A1 5 74 106 199 50 121 138 693 75 9.2 8.9 ± 1.2A2 0 94 24 142 155 38 101 554 75 7.4B1 2 71 92 152 94 184 61 656 63 10.4B2 15 7 58 181 128 146 48 583 68 8.62.4 A1 0 71 36 105 101 69 133 515 75 6.9 6.7 ± 1.1A2 0 83 27 104 66 35 78 393 75 5.2B1 5 45 123 143 29 117 76 538 70 7.7B2 0 83 69 115 78 21 80 446 64 7.01Neonate 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.3Reproductive 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 thetest until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last dayit was alive.4Calculated using SAS System for Windows; manual calculations may differ slightly.-- = no neonate produced due to 100% immobility of the adult daphnid.Wildlife International, Ltd.Project Number 471A-120- 49 -Appendix 9 (Continued)Neonate Production by First-Generation Daphnids Exposed to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)Rep.Number of Live Neonates Produced by Test Day1,2No. LiveNeonatesProducedNumber ofReproductiveDays3No. LiveNeonatesProduced PerReproductive DayMean No.Neonates PerReproductive Day± Std. Dev.47 9 11 14 16 18 215.5 A1 0 85 50 170 132 133 83 653 75 8.7 8.1 ± 0.74A2 0 82 30 164 156 58 84 574 75 7.7B1 7 76 105 175 74 133 91 661 75 8.8B2 1 74 77 127 49 88 135 551 75 7.312 A1 15 45 112 124 59 129 151 635 75 8.5 6.9 ± 1.1A2 0 78 81 124 82 23 80 468 73 6.4B1 0 106 0 129 174 17 50 476 75 6.3B2 12 75 31 135 120 44 54 471 75 6.321 A1 1 87 62 127 81 145 68 571 65 8.8 7.7 ± 1.7A2 11 46 128 152 42 189 112 680 75 9.1B1 0 64 87 116 29 118 122 536 71 7.5B2 0 58 27 62 109 40 103 399 75 5.343 A1 0 91 21 108 140 28 94 482 73 6.6 8.3 ± 1.6A2 0 82(2) 105 138 41 157 116 639 75 8.5B1 0 87(1) 59 150 109 62 103 570 75 7.6B2 0 55 137 124 0 161 146 623 60 10.41Neonate 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.3Reproductive 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 thetest until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last dayit was alive.4Calculated using SAS System for Windows; manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 50 -Appendix 10Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)ReplicateDaphnidNumberIndividualLength(mm)1ReplicateMeanLength(mm)2TreatmentMean Length± Std. Dev.(mm)3IndividualDry Weight(mg)1ReplicateMeanDry Weight(mg)2TreatmentMean Dry Weight± Std. Dev.(mg)3Negative Control A1 1 4.4 4.6 4.6 ± 0.058 0.54 0.53 0.63 ± 0.112 4.6 0.603 4.6 0.644 4.7 0.475 4.7 0.42A2 1 -- -- -- --2 -- --3 -- --4 -- --5 -- --B1 1 4.6 4.5 0.82 0.752 4.4 0.663 4.4 0.954 4.5 0.655 4.6 0.67B2 1 4.6 4.6 0.63 0.622 4.3 0.543 4.8 0.824 4.7 0.435 4.6 0.671-- = immobility prior to test termination.2Calculated using Excel in full precision; manual calculations may differ slightly.3Calculated using SAS System for Windows; manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 51 -Appendix 10 (Continued)Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)ReplicateDaphnidNumberIndividualLength(mm)1ReplicateMeanLength(mm)2TreatmentMean Length± Std. Dev.(mm)3IndividualDry Weight(mg)1ReplicateMeanDry Weight(mg)2TreatmentMean Dry Weight± Std. Dev.(mg)3Solvent Control A1 1 4.6 4.6 4.5 ± 0.050 0.70 0.64 0.69 ± 0.0332 4.8 0.803 4.5 0.474 4.6 0.615 -- --A2 1 4.6 4.5 0.61 0.692 4.4 0.653 4.6 0.754 4.6 0.655 4.5 0.79B1 1 4.4 4.5 0.79 0.712 4.4 0.703 4.6 0.744 4.5 0.595 4.5 0.73B2 1 4.6 4.5 0.70 0.712 4.5 0.603 4.6 0.704 4.6 0.855 4.4 0.711-- = mortality prior to test termination.2Calculated using Excel in full precision; manual calculations may differ slightly.3Calculated using SAS System for Windows; manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 52 -Appendix 10 (Continued)Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)ReplicateDaphnidNumberIndividualLength(mm)1ReplicateMeanLength(mm)2TreatmentMean Length± Std. Dev.(mm)3IndividualDry Weight(mg)1ReplicateMeanDry Weight(mg)2TreatmentMean Dry Weight± Std. Dev.(mg)31.4 A1 1 4.7 4.5 4.6 ± 0.058 0.73 0.66 0.76 ± 0.102 4.5 0.513 4.6 0.814 4.3 0.555 4.4 0.69A2 1 4.4 4.5 0.73 0.682 4.5 0.643 4.4 0.594 4.6 0.785 4.4 0.67B1 1 4.7 4.6 0.86 0.842 4.4 0.713 4.8 0.874 4.5 0.925 -- --B2 1 4.7 4.6 0.92 0.862 4.5 0.813 4.5 0.794 4.5 0.925 -- --1-- = mortality prior to test termination.2Calculated using Excel in full precision; manual calculations may differ slightly.3Calculated using SAS System for Windows; manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 53 -Appendix 10 (Continued)Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)ReplicateDaphnidNumberIndividualLength(mm)1ReplicateMeanLength(mm)2TreatmentMean Length± Std. Dev.(mm)3IndividualDry Weight(mg)1ReplicateMeanDry Weight(mg)2TreatmentMean Dry Weight± Std. Dev.(mg)32.4 A1 1 4.3 4.4 4.3 ± 0.10 0.64 0.58 0.60 ± 0.0132 4.4 0.573 4.4 0.554 4.5 0.595 4.4 0.54A2 1 4.4 4.2 0.54 0.592 4.3 0.593 4.2 0.844 4.0 0.555 4.1 0.44B1 1 4.2 4.3 0.75 0.612 4.3 0.533 4.3 0.704 4.4 0.485 -- --B2 1 4.4 4.4 0.69 0.602 4.4 0.593 4.5 0.584 4.3 0.565 -- --1-- = mortality prior to test termination.2Calculated using Excel in full precision; manual calculations may differ slightly.3Calculated using SAS System for Windows; manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 54 -Appendix 10 (Continued)Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)ReplicateDaphnidNumberIndividualLength(mm)1ReplicateMeanLength(mm)2TreatmentMean Length± Std. Dev.(mm)3IndividualDry Weight(mg)1ReplicateMeanDry Weight(mg)2TreatmentMean Dry Weight± Std. Dev.(mg)35.5 A1 1 4.4 4.5 4.4 ± 0.08 0.77 0.75 0.74 ± 0.0462 4.5 0.863 4.6 0.724 4.5 0.635 4.5 0.75A2 1 4.4 4.4 0.74 0.802 4.4 0.883 4.6 0.694 4.4 0.855 4.3 0.84B1 1 4.2 4.4 0.64 0.732 4.3 0.553 4.4 0.814 4.3 0.765 4.6 0.91B2 1 4.2 4.3 0.88 0.692 4.4 0.673 4.3 0.594 4.5 0.635 4.3 0.661-- = mortality prior to test termination.2Calculated using Excel in full precision; manual calculations may differ slightly.3Calculated using SAS System for Windows; manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 55 -Appendix 10 (Continued)Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)ReplicateDaphnidNumberIndividualLength(mm)1ReplicateMeanLength(mm)2TreatmentMean Length± Std. Dev.(mm)3IndividualDry Weight(mg)1ReplicateMeanDry Weight(mg)2TreatmentMean Dry Weight± Std. Dev.(mg)312 A1 1 4.3 4.4 4.4 ± 0.00 0.80 0.67 0.76 ± 0.0952 4.2 0.753 4.5 0.754 4.2 0.525 4.6 0.55A2 1 4.4 4.4 0.69 0.792 4.6 0.803 4.3 0.844 4.2 0.835 -- --B1 1 4.5 4.4 0.89 0.882 4.1 0.933 4.4 0.774 4.4 0.905 4.5 0.89B2 1 4.3 4.4 0.64 0.702 4.4 0.753 4.4 0.794 4.3 0.735 4.4 0.611-- = mortality prior to test termination.2Calculated using Excel in full precision; manual calculations may differ slightly.3Calculated using SAS System for Windows; manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 56 -Appendix 10 (Continued)Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)ReplicateDaphnidNumberIndividualLength(mm)1ReplicateMeanLength(mm)2TreatmentMean Length± Std. Dev.(mm)3IndividualDry Weight(mg)1ReplicateMeanDry Weight(mg)2TreatmentMean Dry Weight± Std. Dev.(mg)321 A1 1 4.6 4.5 4.4 ± 0.05 0.77 0.64 0.63 ± 0.0552 4.6 0.713 4.2 0.494 4.5 0.595 -- --A2 1 4.4 4.4 0.49 0.552 4.5 0.473 4.3 0.594 4.5 0.545 4.3 0.66B1 1 4.5 4.4 0.68 0.682 4.6 0.613 4.3 0.784 4.3 0.665 -- --B2 1 4.3 4.4 0.69 0.642 4.7 0.663 4.5 0.724 4.4 0.525 4.3 0.621-- = mortality prior to test termination.2Calculated using Excel in full precision; manual calculations may differ slightly.3Calculated using SAS System for Windows; manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-120- 57 -Appendix 10 (Continued)Length and Dry Weight of Surviving First-Generation Daphnids After Exposure to14C AN-2 for 21 DaysMean MeasuredConcentration(ng/L)ReplicateDaphnidNumberIndividualLength(mm)1ReplicateMeanLength(mm)2TreatmentMean Length± Std. Dev.(mm)3IndividualDry Weight(mg)1ReplicateMeanDry Weight(mg)2TreatmentMean Dry Weight± Std. Dev.(mg)343 A1 1 4.0 4.3 4.5 ± 0.13 0.61 0.61 0.60 ± 0.0392 4.5 0.703 4.4 0.564 4.3 0.595 -- --A2 1 4.3 4.4 0.62 0.542 4.4 0.573 4.4 0.614 4.5 0.475 4.3 0.45B1 1 4.5 4.5 0.82 0.632 4.5 0.533 4.4 0.474 4.4 0.575 4.5 0.78B2 1 4.3 4.6 0.65 0.612 4.6 0.673 4.6 0.584 4.7 0.565 -- --1-- = mortality prior to test termination.2Calculated using Excel in full precision; manual calculations may differ slightly.3Calculated using SAS System for Windows; manual calculations may differ slightly.

# W ildlife 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 protoc ol 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 unaccep table 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.

# W ildlife International, Ltd. Project Number 471A-120

- 59 -

Appendix 12



# Personnel Involved in the Study

The following key Wildlife International, Lt d. 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