A 48-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST

WITH THE CLADOCERAN ( Daphnia magna )

FINAL REPORTSTATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any inform ation contained in this study on the basis of its

falling within the scope of FIFRA section 10(d)(1)(A), (B) or (C).

Company: _________________________ (Typed Name)

Company Agent: _________________________ (Typed Name)

Title: _________________________

Signature: ________________________________ Date: ____________

### GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

### QUALITY ASSURANCE STATEMENT

This study was examined for compliance with Good Laboratory Practice Standards as

published by the U.S. Environmental Protection Agency (40 

### CFR Parts 160 and 792, 17 August

1989); OECD Principles of Good Laboratory Practice (ENVIMCICHEM (98) 17); and Japan MAFF

(1 

### 1 NohSan, Notification No. 6283, Agricultural Production Bureau, 1 October 1999). The dates of

all inspections and audits and the dates that any findings were reported to the Study Director and

Laboratory Management were as follows:

ACTIVITY: DATE REPORTED TO:



### DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT:

Protocol June 27,2005 June 27,2005 July 5, 2005

Test Substance Preparation August 1,2005 August 1,2005 August 4,2005

Observations and Temperature August 4,2005 August 4,2005 August 9,2005

Measurements

Data and Draft 

### Report August 22,2005 August 22,2005 August 23,2005

Final Report August 26,2005 August 26,2005 August 26,2005

All inspections were study-based unless otherwise noted.

~ualib ~ssurkce Program Supervisor

### -5-



### REPORT APPROVAL

SPONSOR:TABLE OF CONTENTS

Title Page..................................................................................................................... ........................... 1

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

Good Laboratory Practice Co mpliance Statement ................................................................................. 3

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

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

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

Summary ........................................................................................................................ ........................ 8

Introduction ................................................................................................................... ......................... 9

Objective ...................................................................................................................... .......................... 9

Experimental Design ............................................................................................................ .................. 9

Materials and Methods .......................................................................................................... ................. 9

Test Substance................................................................................................................ ......... 10

Test Organism ................................................................................................................. ........ 10

Dilution Water................................................................................................................ ......... 11

Test Apparatus................................................................................................................ ......... 11

Preparation of Test Concentrations ......................................................................................... 11

Analytical Sampling........................................................................................................... ..... 12

Environmental Conditions...................................................................................................... . 12

Observations.................................................................................................................. .......... 13

Conditions for the Validity of the Test.................................................................................... 13

Statistical Analyses.......................................................................................................... ........ 13

Results and Discussion......................................................................................................... ................ 14

Measurement of TOC in Test Solutions.................................................................................. 14

Observations and Measurements............................................................................................. 14

Conclusions .................................................................................................................... ...................... 14

Refere nces ..................................................................................................................... ....................... 15TABLE OF CONTENTS (Continued)

TABLES

Table 1 . Measurements of Total Organic Carbon in Test Solution Samples.................................. 16

Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers........................ 17

Table 3. Specific Conductance, Hardness, Alkalinity and Total Organic Carbon

Measured in Dilution Water at Test Initiation .................................................................. 18

Table 4 . Cumulative Mortality, Immobility and Observations....................................................... 19

Table 5 . EL50 Values .................................................................................................................. ... 20

APPENDICES

Appendix 1. Specific Conductance, Hardness, Alkalinity and pH of Well Water

Measured During the 4-Week Period Immediately Preceding the Test.................. 21

Appendix 2. Analyses of Pesticides, Organics and Metals in

Well Water .............................................................................................................. 22

Appendix 3. Changes to Protocol ................................................................................................ 24

Appendix 4. Personnel Involved in the Study ............................................................................. 25INTRODUCTION

This study was conducted by . for

quatic toxicology facility in Easton, Maryland. The in-life phase of the definitive

toxicity test was conducted from August 2 to 4, 2005. Raw data generated by

Ltd. and a copy of the final report are filed under Project in archives located on

the

OBJECTIVE

The objective of this study was to evaluate the acute toxicity of the test substance to the

cladoceran, Daphnia magna , during a 48-hour exposure period unde r static-renewal test conditions.

EXPERIMENTAL DESIGN

Daphnids were exposed to a geometric series of five water accommodated fractions (WAF)

of the test substance and a negative control (dilu tion water). Two replicate test chambers were

maintained in each treatment and control group, with ten daphnids in each test chamber for a total of

20 daphnids per concentration. Nominal loading rat es were selected in consultation with the Sponsor

based on the results of an exploratory range finding toxicity test. Nominal WAF loading rates

selected were 6.3, 13, 25, 50 and 100 mg/L. Daphni ds were transferred to newly prepared test

solutions after 24 hours. Samples of test water were collected at test initiation, prior to and following

renewal at 24 hours, and at test termination for analysis of total organic carbon (TOC).

Daphnids were impartially assigned to test cham bers at test initiation. Observations of

mortality, immobility and other signs of toxicity were made approximately 3, 24 and 48 hours after

test initiation. Cumulative percent mortality and immobility observed in the treatment groups were

used to determine EL50 values at 24 and 48 hours. The no-mortality/immobility level and the noobserved-effect level were determined by visual interpretation of the mortality, immobility and

observation data. The results of the study were based on the nominal WAF loading rates.

MATERIALS AND METHODS

The study was conducted according to the procedures outlined in the protocol, A 48-Hour

Static-Renewal Acute Toxicity Test with the Cladoceran ( Daphnia magna ). The protocol was based

on procedures outlined in the OECD Guide line for Testing of Chemicals, 202: Daphnia sp. Acute

Immobilization Test and Reproduction Test (1); U.S. EPA Series 850 – Ecological Effects TestGuidelines OPPTS Number 850.1010: Aquatic Invertebrate Acute Toxicity Test (2); and ASTM

Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with

Fishes, Macroinvertebrates and Amphibians (3).

Test Substance

The test substance was received from on April 19, 2005. It was assigned

. identification num ber 7161 upon receipt and was stored under ambient

conditions. The test substance, a liquid, was identified as Tall Oil Fatty Acid, Lot number 8488/035.

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 and ease of culturing in the laboratory. Daphnid neonates used in the

test were less than 24 hours old and were obtained fro m cultures maintained by

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. The adult daphnids used to supply neonates for the test were held

for 21 days prior to collection of the juveniles for testing. The adults showed no signs of disease or

stress during the holding period. During the 14-day period immediately preceding the test, water

temperatures ranged from 19.6 to 20.4oC, measured with a hand-held liquid-in-glass thermometer.

The pH of the water ranged from 8.1 to 9.1, measu red with a Fisher Scientific Accumet Model 915

pH meter. Dissolved oxygen ranged from 8.2 to 9.0 mg/L ( ≥ 91% of saturation), measured with a

Yellow Springs Instruments Model 51B dissolved oxygen meter.

Neonate daphnids were obtained for testing fro m four individual adult daphnids that had

produced at least one previous brood. At test initia tion, the juvenile daphnids were collected from the

cultures and indiscriminately transferred one and two at a time to transfer chambers (e.g., 10 mL glass

beakers) until each chamber contained ten daphnids. Each group of daphnids then was transferred to

an indiscriminately assigned test chamber. All tr ansfers were made below the water surface using

wide-bore pipettes. Daphnids in the cultures were fed daily a mixture of yeast, Cerophyll ® and trout

chow, as well as a suspension of the freshwater green alga, Selenastrum capricornutum . The adults

were fed prior to test initiation, but neonates were not fed during the test.Dilution Water

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

40 meters deep located on the 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 well water was passed through a sand filter to remove particles greater than

approximately 25 μ 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 μ m and passed through an ultraviolet

sterilizer to remove microorganisms and fine particles. The results of periodic analyses performed to

measure the concentrations of selected organic and inorganic constituents in the well water used by

re presented in Appendix 2.

Test Apparatus

Test chambers were 250-mL glass beakers containing 250 mL of test water. The depth of the

test water in a representative chamber was 6.4 cm. Test chambers were labeled with the project

number, test concentration and replicate, and were indiscriminately positioned in a temperaturecontrolled environmental chamber set to maintain the desired test temperature.

Preparation of Test Concentrations

Test solutions were prepared prior to test initiation on Day 0. New test solutions were

prepared in separate test chambers on Day 1, a nd all daphnids were transferred from old to new

solutions. Individual WAFs were prepared for each test concentration. Nominal WAF concentrations

were 6.3, 13, 25, 50 and 100 mg/L. The test subs tance was mixed directly with dilution water on a

weight:volume basis. Each 12-L WAF was stirred for approximately 24 hours in a 13.2 L pyrex

aspirator bottle using a Teflon ® -lined magnetic stir bar to create a vortex depth of approximately 30%

of the test solution height. After mixing, the WA Fs were allowed to settle for approximately one

hour. The test solutions were then drawn from sp igots placed near the bottom of the mixing vessels

and added to the appropriate test chamber.At test initiation, the test solutions were cl oudy white in appearance, increasing in intensity

with increasing test substance concentration, w ith visible particulate matter suspended in the

solutions. At test termination, the solutions ranged in appearance from slightly cloudy white to

cloudy white, with particulate matter suspended in solution and on the bottom of the test chambers.

Analytical Sampling

Samples of the test solutions were collected at approximately 0, 24 and 48 hours to measure

concentrations of total organic carbon (TOC). Samples were collected from the WAF preparation

vessels at test initiation and at 24 hours, and from pooled solution from each test chamber prior to

renewal at 24 hours and at test termination. The samples were collected from mid-depth into glass

vials, acidified with 2N HCl, and stored refrigera ted, as needed, prior to analysis. TOC was measured

using a Shimadzu Model TOC-5000 total organic carbon analyzer.

Environmental Conditions

Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone ® 50) were

used for illumination of the culture and test cham bers. A photoperiod of 16 hours of light and 8 hours

of darkness was controlled with an automatic timer. A 30-minute transition period of low light

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

intensity at test initiation was 446 lux at the surface of the water of one representative test chamber.

The target test temperature during the study was 20 ± 1oC. Temperature was measured in

each test chamber at test initiation, prior to and afte r renewal at 24 hours, and at test termination using

a liquid-in-glass thermometer. Temperature also was measured continuously in a container of water

placed adjacent to the test chambers using a Fulscope ER/C Recorder, which was verified prior to test

initiation with a liquid-in-glass thermometer. The pH and dissolved oxygen content of the water were

measured in each test chamber at test initiation, prior to and after renewal at 24 hours, and at test

termination. Hardness, alkalinity, specific conductance and total organic carbon (TOC) were

measured in the dilution water at test initiation. An additional dilution water sample was collected

following the test to confirm the TOC concentration due to contamination of the Day 0 sample.Light intensity was measured using a SPER Scientific Model 840006C light meter.

Dissolved oxygen was measured using a Thermo Or ion Model 850Aplus dissolved oxygen meter, and

measurements of pH were made using a Thermo Orion Model 525Aplus meter. Specific conductance

was measured using a Yellow Springs Instrume nt Model 33 Salinity-Conductivity-Temperature

meter. Hardness and alkalinity measurements we re made by titration based on procedures in

Standard Methods for the Examination of Water and Wastewater (4). TOC was measured using a

SHIMADZU Model TOC-5000 total organic carbon analyzer.

Observations

Observations were made periodically to dete rmine the numbers of dead and immobile

organisms. Immobility was defined as a lack of movement by the organism except for minor activity

of the appendages. The numbers of individuals exhibiting signs of t oxicity or abnormal behavior also

were evaluated. Observations were made approxi mately 3, 24 and 48 hours after test initiation.

Conditions for the Validity of the Test

The following criteria used to judge the validity of the test were met:

1. immobility of the daphnids in the negative control group did not exceed 10% by the end of

the test, and

2. the dissolved oxygen concentration was at least 60% of the air saturation value throughout

the test.

Statistical Analyses

The results of the test are based on the nominal WAF loading rates. There was less than 50%

mortality/immobility in the test substance treatment groups during this study, which precluded the

statistical calculation of EL50 values. Therefore, the 24 and 48-hour EL50 values were estimated to

be greater than the highest nominal WAF loading rate tested. The no mortality/immobility level and

the NOEL were determined by visual interpretati on of the mortality, immobility and observation data.RESULTS AND DISCUSSION

Measurement of TOC in Test Solutions

Results of analyses for TOC in the test soluti ons are presented in Table 1. Measurements of

TOC in the newly prepared WAF solution on Day 0 and Day 1 ranged from 2.2 to 34.3 mg C/L.

Measurements of TOC in the 24-hour old solutions collected from the test chambers on Day 1 and

Day 2 ranged from <1 to 33.1 mg C/L. TOC c ontent increased with increasing test substance

concentration, and was generally consistent over the test period.

Observations and Measurements

Measurements of temperature, dissolved oxygen a nd pH of the water in each test chamber are

presented in Table 2. Water temperatures were within the 20 ± 1 ° C range established for the test.

Dissolved oxygen concentrations remained ≥ 7.1 mg/L ( ≥ 79% of saturation) throughout the test.

Measurements of pH ranged from 8.2 to 8.4. The measurements of hardness, alkalinity and specific

conductance in the dilution water at test initiation we re typical of well

water (Table 3). TOC in the dilution water was confirmed to be <1 mg C/L.

Daily observations for mortality, immobility and signs of toxicity during the test are

presented in Table 4. All daphnids in the negativ e control group and in the 6.3 mg/L treatment group

appeared normal throughout the exposure period. Pe rcent mortality/immobility in the 13, 25, 50 and

100 mg/L treatment groups was 5, 0, 25 and 35%, respec tively, at test termination. Lethargic daphnia

were observed in the 13, 25, 50 and 100 mg/L treat ment groups, generally increasing in number with

increasing concentration. Most daphnids in th e 13, 25, 50 and 100 mg/L treatment groups were

observed to have particulate matter attached to th eir appendages at test termination. Based on the

single immobile daphnid in the 13 mg/L treatment group, the no-mortality/immobility level was

considered to be 6.3 mg/L. The NOEL also w as 6.3 mg/L. EL50 values at 24 and 48 hours were

estimated to be greater than 100 mg/L, the highest nominal WAF loading rate tested (Table 5).

CONCLUSIONS

The cladoceran, Daphnia magna , was exposed for 48 hours under static-renewal test

conditions to five nominal water accommodated fractions (WAF) of ranging

from 6.3 to 100 mg/L. The 48-hour EL50 value was >100 mg/L, the highest nominal WAF loading

rate tested. The no mortality/immobility level and the NOEL were both 6.3 mg/L.REFERENCES

1 Organisation for Economic Cooperation and Development . 1984. Guideline 202: Daphnia

sp. Acute Immobilization Test and Reproduction Test . OECD Guideline for Testing of

Chemicals. Updated Guideline, adopted April 1984.

2 U.S. Environmental Protection Agency. 1996. Aquatic Invertebrate Acute Toxicity Test,

Freshwater Daphnids. Series 850- Ecological Effects Test Guidelines ( draft ), OPPTS Number

850.1010.

3 ASTM Standard E729-96. 1996. Standard Guide for Conducting Acute Toxicity Tests on Test

Materials with Fishes, Macroinvertebrates, and Amphibians . American Society for Testing and

Materials.

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

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

Association. Water Pollution Control Federation, New York.Table 3

Specific Conductance, Hardness, Alkalinity and Total Organic Carbon

Measured in Dilution Water at Test Initiation

Parameter Day 0

Specific Conductance 320

(μmhos/cm)

Hardness 136

(mg/L as CaCO 3 )

Alkalinity 174

(mg/L as CaCO 3 )

Total Organic Carbon <1 1

(mg C/L)

1 The dilution water sample collected at test initiation was contaminated during

handling. An additional sample was collected following the test to confirm TOC

levels in the dilution water were <1 mg C/L.Appendix 2

Analyses of Pesticides, Organics and Metals inAppendix 4

Personnel Involved in the Study

The following key . pers onnel were involved in th e conduct or management

of this study:

1. Henry O. Krueger, Ph.D., Director of Aquatic Toxicology/Terrestrial Plants & Insects

2. Cary A. Sutherland, Laboratory Supervisor

3. Susan J. Palmer, Senior Biologist

4. Amy S. Blankinship, Biologist

5. Abul Siddiqui, Scientist