A 48-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST 

WITH THE CLADOCERAN (Daphnia magna) 

FINAL REPORT 
 

 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 

No claim of confidentiality is made for any information 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 Compliance 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 

References ............................................................................................................................................ 15 
 

 

TABLE 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 ............................................................................. 25 
 

 

 

 

 

 

 

 

 

 
 

 

INTRODUCTION

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 under 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 (dilution 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 rates 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. Daphnids 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 chambers 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 Guideline for Testing of Chemicals, 202: Daphnia sp. Acute 

Immobilization Test and Reproduction Test (1); U.S. EPA Series 850 – Ecological Effects Test 
 

 

Guidelines 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 number 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 invertebrates 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 from cultures maintained by 

Ltd., Easton, Maryland. 

Adult daphnids were cultured in water from the 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, measured 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 from four individual adult daphnids that had 

produced at least one previous brood. At test initiation, 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 transfers 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 μm, 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 μm 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, and 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 substance 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 WAFs were allowed to settle for approximately one 

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

and added to the appropriate test chamber. 
 

 

At test initiation, the test solutions were cloudy white in appearance, increasing in intensity 

with increasing test substance concentration, with 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 refrigerated, 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 chambers. 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 off 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 after 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 Orion 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 Instrument Model 33 Salinity-Conductivity-Temperature 

meter. Hardness and alkalinity measurements were 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 determine 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 toxicity or abnormal behavior also 

were evaluated. Observations were made approximately 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 interpretation of the mortality, immobility and observation data. 
 

 

RESULTS AND DISCUSSION 

Measurement of TOC in Test Solutions 

Results of analyses for TOC in the test solutions 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 content increased with increasing test substance 

concentration, and was generally consistent over the test period. 

Observations and Measurements 

Measurements of temperature, dissolved oxygen and 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 were 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 negative control group and in the 6.3 mg/L treatment group 

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

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

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

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

observed to have particulate matter attached to their 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 was 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 CaCO3) 

 

Alkalinity 174 

 (mg/L as CaCO3) 

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 in 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 
 

 

Appendix 4 

Personnel Involved in the Study 

The following key . personnel were involved in the 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