DIETHYLTOLUENE DIAMINE (DETDA): 

A 48-HOUR STATIC ACUTE TOXICITY TEST 

WITH THE CLADOCERAN (Daphnia magna) 

FINAL REPORT 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-122 

OECD GUIDELINE 202 

U.S. EPA OPPTS NUMBER 850.1010 

AUTHORS: 

Daniel S. Brougher, M.S. 

Timothy Z. Kendall, M.S. 

Sean P. Gallagher, B.S. 

Henry O. Krueger, Ph.D. 

STUDY INITIATION DATE: January 26, 2012 

STUDY COMPLETION DATE: March 20, 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 44 
Wildlife International, Ltd. 

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STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 

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

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

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

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

disclosure to multinational entities under FIFRA (10)g. 

 Company: ________________________________(Typed Name) 

 Company Agent: ________________________________(Typed Name) 

 Title: ________________________________ 

 Signature:______________________________Date:______________________ 



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TABLE OF CONTENTS

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

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

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

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

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

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

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

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

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

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

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

 Test and Reference Substances ............................................................................................... 10 

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

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

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

 Preparation of Test Concentrations ......................................................................................... 12 

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

 Analytical Method ................................................................................................................... 12 

 Environmental Conditions ....................................................................................................... 13 

 Observations ............................................................................................................................ 14 

 Statistical Analyses .................................................................................................................. 14 

Results and Discussion ......................................................................................................................... 15 

 Measurement of Test Concentrations ...................................................................................... 15 

 Observations and Measurements ............................................................................................. 15

Conclusions .......................................................................................................................................... 16 

References ............................................................................................................................................ 17 
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TABLE OF CONTENTS (Continued) 

TABLES AND FIGURES 

Table 1. Measured Concentrations of Diethyltolunene Diamine (DETDA) 

in Test Water Samples... .................................................................................................. 18 

Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers ........................ 19 

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

 in the Dilution Water at Test Initiation ............................................................................. 20 

Table 4. Cumulative Immobility and Observations ........................................................................ 21 

Table 5. EC50 Values ..................................................................................................................... 22 

Figure 1. Concentration-Response Curve (48-Hour Immobility Data) ............................................ 23 

APPENDICES

Appendix 1. Chemical Characterization ...................................................................................... 24 

Appendix 2. Dilution Water Chemistry Parameters Measured During the 

 4-Week Period Immediately Preceding the Test ..................................................... 28 

Appendix 3. Analyses of Pesticides, Organics and Metals in Wildlife 

 International, Ltd. Well Water ................................................................................ 29 

Appendix 4. The Analysis of Diethyltoluene Diamine (DETDA) in Freshwater ........................ 31 

4.1 Analytical Method Flowchart for the Analysis of Diethyltoluene 

 Diamine (DETDA) in Freshwater ........................................................................... 32 

4.2 Typical HPLC Operational Parameters ................................................................... 33

4.3 Analytical Stocks and Standards Preparation.......................................................... 34

4.4 Example Calculations for a Representative Sample ............................................... 35

4.5 Quality Control Samples of Diethyltoluene Diamine (DETDA) in Freshwater ..... 36

4.6 Representative Calibration Curve for Diethyltoluene Diamine (DETDA) ............. 37

4.7 Representative Chromatogram of a Low-level Diethyltoluene 

Diamine (DETDA) Calibration Standard ................................................................ 38

4.8 Representative Chromatogram of a High-level Diethyltoluene 

Diamine (DETDA) Calibration Standard ................................................................ 39

4.9 Representative Chromatogram of a Matrix Blank Sample ..................................... 40

4.10 Representative Chromatogram of a Matrix Fortification Sample ........................... 41 

4.11 Representative Chromatogram of a Test Sample .................................................... 42 

Appendix 5. Changes to Protocol ................................................................................................ 43 

Appendix 6. Personnel Involved in the Study ............................................................................. 44 
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SUMMARY 

SPONSOR: Albemarle Corporation 

TITLE: Diethyltoluene Diamine (DETDA): A 48-Hour Static Acute Toxicity Test with the 

Cladoceran (Daphnia magna) 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-122 

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

 Experimental Start (EPA): February 8, 2012 

 Biological Termination: February 10, 2012 

 Experimental Termination: February 10, 2012 

LENGTH OF EXPOSURE: 48 Hours 

TEST ORGANISM: Cladoceran, Daphnia magna

SOURCE OF TEST ORGANISMS: Wildlife International, Ltd. cultures 

 Easton, Maryland 21601 

AGE OF TEST ORGANISMS: Neonates <24 hours at test start 

TEST CONCENTRATIONS: Nominal Mean Measured 

 Negative Control < LOQ 

 0.25 mg a.i./L 0.24 mg a.i./L 

 0.50 mg a.i./L 0.49 mg a.i./L 

 1.0 mg a.i./L 0.98 mg a.i./L 

 2.0 mg a.i./L 1.9 mg a.i./L 

 4.0 mg a.i./L 3.9 mg a.i./L 

 8.0 mg a.i./L 7.8 mg a.i./L 

RESULTS: Based on mean measured test concentrations: 

 48-Hour EC50: 5.8 mg a.i./L 

 95% Confidence Interval: 4.7 – 7.6 mg a.i./L 

 No-Immobility Concentration: 1.9 mg a.i./L 

 No-Observed-Effect Concentration: 0.98 mg a.i./L 
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INTRODUCTION

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

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

definitive toxicity test was conducted from February 8, 2012 to February 10, 2012. Raw data 

generated by Wildlife International, Ltd. and the final report are filed under Project Number 

471A-122 in archives located on the Wildlife International, Ltd. site. 

OBJECTIVE 

The objective of this study was to determine the acute effects of Diethyltoluene Diamine 

(DETDA) on the cladoceran, Daphnia magna, during a 48-hour exposure period under static test 

conditions. 

EXPERIMENTAL DESIGN 

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

(dilution water) for 48 hours under static conditions. Two replicate test chambers were maintained in 

each treatment and control group, with 10 daphnids in each test chamber, for a total of 20 daphnids 

per concentration. Nominal test concentrations were selected in consultation with the Sponsor based 

on the results of exploratory range finding toxicity data. Nominal test concentrations selected were 

0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg Diethyltoluene Diamine (DETDA) active ingredient (a.i.)/L. Mean 

measured test concentrations were determined from samples of test water collected from each 

treatment and control group at the beginning and end of the test. 

Daphnids were impartially assigned to exposure chambers at test initiation. Observations of 

immobility and other signs of toxicity were made approximately 2.5, 24 and 48 hours after test 

initiation. Cumulative percent immobility observed in the treatment groups was used to determine 

EC50 values at 24 and 48 hours. The no-immobility concentration and the no-observed-effect 

concentration (NOEC) were determined by visual interpretation of the immobility and biological 

observation data. 

MATERIALS AND METHODS 

The study was conducted according to the procedures outlined in the protocol, 

“Diethyltoluene Diamine (DETDA): A 48-Hour Static Acute Toxicity Test with the Cladoceran 
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(Daphnia magna)”. The protocol was based on procedures outlined in the OECD Guidelines for 

Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test (1); U.S. 

Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines, OPPTS Number 

850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids (2); and ASTM Standard 

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

Macroinvertebrates and Amphibians (3). 

Test and Reference Substances 

The test substance used to prepare the test solutions, calibration standards, and the analytical 

matrix fortification samples for the study was received from Albemarle Corporation on September 21, 

2011. It was assigned Wildlife International, Ltd. identification number 10232 upon receipt and was 

stored under ambient conditions. The test substance, a liquid, was identified as: Diethyltolunene 

Diamine (DETDA); Lot number - 4500051816-011110; CAS number – 68479-98. The test substance 

contained 97.27% active ingredient and had an expiration date of March 3, 2013 (Appendix 1). 

Test Organism 

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

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

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

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

Maryland. 

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

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

temperatures in the cultures ranged from 19.7 to 21.1oC, measured with a hand-held, liquid-in-glass 

thermometer. The pH of the water ranged from 8.2 to 8.5, measured with a Thermo Orion Benchtop 

4 Star Plus pH meter. Dissolved oxygen concentrations were 7.1 mg/L (78% of saturation), 

measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter. 

Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media and trout chow 

(YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata. The 

adults were fed prior to test initiation, but neonates were not fed during the test. 
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The five adult daphnids used to supply neonates for the test were held for at least 14 days 

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

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

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

were produced during the holding period. At test initiation, the juvenile daphnids were collected from 

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

chamber contained 10 daphnids. Each group of daphnids then was transferred to an indiscriminately 

assigned test chamber to initiate the test. All transfers were made below the water surface using 

wide-bore pipettes. 

Dilution Water 

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

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

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

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

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

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

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

preceding the test are presented in Appendix 2. The results of periodic analyses performed to 

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

presented in Appendix 3. 

Test Apparatus 

Test chambers were 250-mL glass beakers filled with approximately 230 mL of test water. 

The depth of the test water in a representative chamber was 5.5 cm. The chambers were 

indiscriminately positioned by treatment group in a temperature-controlled environmental chamber to 

maintain the target water temperature throughout the test period. All test chambers were labeled with 

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

A primary stock solution was prepared by mixing a calculated amount of test substance in 

2.0 L of dilution water at a nominal concentration of 8.0 mg a.i./L, the highest concentration tested. 

The stock solution was adjusted to 100% active ingredient during preparation, based on the test 

substance purity (97.3%). The stock solution was sonicated for approximately 2 minutes and mixed 

by inversion at least 20 times. Aliquots of the primary stock solution were proportionally diluted 

with dilution water to prepare five additional test solutions at nominal concentrations of 0.25, 0.5, 1.0, 

2.0, and 4.0 mg a.i./L. The solutions were mixed by inversion and approximately 250 mL of solution 

was placed in each of two replicate test chambers per treatment group. The negative control solution 

was dilution water only. 

Analytical Sampling 

Samples were collected from the batches of test solution prepared for each treatment and 

control group at the beginning of the test and from each test chamber at 48 hours (± 1 hour) to 

measure concentrations of the test substance. The samples (~20mL) were collected at mid-depth, 

placed in glass vials, and processed immediately for analysis. 

Analytical Method 

The method used for the analysis of DETDA in the freshwater samples was based upon 

methodology developed by Wildlife International, Ltd. The analytical method consisted of diluting 

the samples with freshwater, as necessary, and analyzing by high performance liquid chromatography 

with variable wavelength detection at 220 nm. 

Concentrations of DETDA in the samples were determined using an Agilent Series 

1100/1200 High Performance Liquid Chromatograph (HPLC) equipped with an Agilent Series 1100 

Variable Wavelength Detector. Chromatographic separations were achieved using a YMC-PACK 

ODS AM column (150 mm x 4.6 mm, 3 m particle size). A method flowchart is provided in 

Appendix 4.1, and typical instrumental parameters are summarized in Appendix 4.2. 

Calibration standards of DETDA were prepared for the analysis of the test samples, ranging 

in concentration from 0.150 to 1.50 mg a.i./L, using a stock solution of DETDA in acetonitrile 

(Appendix 4.3). Fresh calibration standards were prepared and analyzed with each sample set. 
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Linear regression equations were generated using the sum of the peak area responses versus the 

respective concentrations of the calibration standards. The concentration of DETDA in the samples 

was determined by substituting the sum of the peak area responses of the samples into the applicable 

linear regression equation. The peak areas of the 2 components of DETDA were summed in order to 

derive the concentration of DETDA expressed as "a.i.". An example of the calculations for a 

representative sample is included in Appendix 4.4. 

The method limit of quantitation (LOQ) was defined as 0.150 mg a.i./L, calculated as the 

product of the concentration of the lowest calibration standard (0.150 mg a.i./L) and the dilution 

factor of the matrix blank samples (1.00). Two matrix blank samples were analyzed to determine 

possible interferences. No interferences were observed at or above the LOQ during the sample 

analyses (Appendix 4.5). 

Samples of freshwater were fortified at 0.250, 0.750 and 8.00 mg a.i./L using stock solutions 

of DETDA in acetonitrile (Appendix 4.3), and were analyzed concurrently with the test samples. The 

measured concentrations for the matrix fortification samples ranged from 96.2 to 100% of fortified 

concentrations (Appendix 4.5). 

A representative calibration curve is presented in Appendix 4.6. Representative 

chromatograms of low and high-level calibration standards are presented in Appendices 4.7 and 4.8, 

respectively. Representative chromatograms of a matrix blank sample and a matrix fortification 

sample are presented in Appendices 4.9 and 4.10, respectively. A representative chromatogram of a 

test sample is presented in Appendix 4.11. 

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

each test chamber at the beginning and end of the test and at approximately 24 hours during the test 

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

a container of water placed adjacent to the test chambers, using a Fulscope ER/C Recorder. 

Dissolved oxygen and pH were measured in each test chamber at the beginning and end of 

the test and at approximately 24 hours during the test. 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. 

Hardness, alkalinity, specific conductance and total organic carbon (TOC) in the dilution 

water at the beginning of the test were measured. Hardness and alkalinity measurements were made 

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. TOC was measured using a Shimadzu Model TOC-VCSH total 

organic carbon analyzer based on procedures in Standard Methods for the Examination of Water and 

Wastewater (4). 

Observations 

All organisms were observed periodically to determine the number of immobile organisms in 

each treatment group. 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 2.5, 24 and 48 hours 

after test initiation. 

Statistical Analyses 

The immobility data were analyzed using the computer program of C. E. Stephan (5). The 

program was designed to calculate the EC50 value and the 95% confidence interval by probit 

analysis, the moving average method, and binomial probability with nonlinear interpolation (6, 7, 8). 

In this study, probit analysis was used to calculate the 48-hour EC50 value and the 95% confidence 

interval. Since there was <50% immobility at 24 hours, the 24-hour EC50 value, as well as the 
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no-immobility concentration and NOEC, were determined by visual interpretation of the immobility 

and observation data. 

RESULTS AND DISCUSSION 

Measurement of Test Concentrations 

Nominal concentrations selected for use in this study were 0.25, 0.5, 1.0, 2.0, 4.0 and 

8.0 mg a.i./L. Test solutions in the test chambers at these nominal concentrations appeared clear and 

colorless during the test, with no evidence of precipitation observed. 

Results of analyses to measure concentrations of Diethyltoluene Diamine (DETDA) in the 

test solution samples collected during the test are presented in Table 1. Measured concentrations of 

the samples ranged from approximately 95 to 98% of nominal. When measured concentrations of the 

samples were averaged, the mean measured test concentrations for this study were 0.24, 0.49, 0.98, 

1.9, 3.9 and 7.8 mg a.i./L, representing 96, 98, 98, 95, 98 and 98% of nominal concentrations, 

respectively. The results of the study were based on the mean measured concentrations. 

Observations and Measurements 

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

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

Dissolved oxygen concentrations remained 8.5 mg/L (93% of saturation) throughout the test. 

Measurements of pH ranged from 8.1 to 8.5. The measurements of hardness, alkalinity, specific 

conductance and TOC in the dilution water at test initiation were typical of Wildlife International, 

Ltd. well water (Table 3). Light intensity at test initiation was 617 lux at the surface of the water of 

one representative test chamber. 

Daily observations for immobility and signs of toxicity during the test are presented in 

Table 4. Daphnia in the negative control groups appeared normal throughout the test. All daphnids 

in the 0.24, 0.49, and 0.98 mg a.i./L treatment groups also appeared normal throughout the test, with 

no immobility or overt signs of toxicity observed. Percent immobility at test termination in the 

3.9 and 7.8 mg a.i./L treatment groups was 25 and 70%, respectively. Three daphnids in the 1.9 mg 

a.i./L treatment group appeared lethargic at test termination, while the remaining daphnids in this 

treatment group appeared normal throughout the test. Lethargic daphnids were observed in the 
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3.9 mg a.i./L treatment group at test termination, and lethargic daphnids were observed in the 7.8 mg 

a.i./L treatment group at both the 24-hour interval and at test termination. The no-immobility 

concentration was 1.9 mg a.i./L and the NOEC was 0.98 mg a.i./L. EC50 values at 24 and 48 hours 

were determined from the immobility data and are shown in Table 5. A graph of the 

concentration-response curve is included in Figure 1. 

CONCLUSIONS 

The cladoceran, Daphnia magna, was exposed for 48 hours under static conditions to six 

mean measured concentrations of Diethyltoluene Diamine (DETDA) ranging from 0.24 to 

7.8 mg a.i./L. The 48-hour EC50 value was 5.8 mg a.i./L, with a 95% confidence interval of 4.7 to 

7.6 mg a.i./L. The slope of the concentration-response curve was 4.616. The no-immobility 

concentration was 1.9 mg a.i./L and the NOEC was 0.98 mg a.i./L. 
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REFERENCES 

1 Organisation for Economic Cooperation and Development. 2004. OECD Guidelines for 

Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test. Adopted 13 April 

2004. 

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

Guidelines (draft), OPPTS Number 850.1010: Aquatic Invertebrate Acute Toxicity Test, 

Freshwater Daphnids. 

3 American Society for Testing and Materials. 2007. ASTM Standard E729-96: Standard 

Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, 

and Amphibians. 

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

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

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

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

Personal communication. 

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

London. 

7 Thompson, W.R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. 

8 Stephan, C.E. 1977. “Methods for Calculating an LC50”, Aquatic Toxicology and Hazard 

Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 

65-84. 
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Table 1

Measured Concentrations of Diethyltoluene Diamine (DETDA) in Test Water Samples 

Nominal Test 

Concentration 

(mg a.i./L) 

Sample 

Number 

(471A-122-) 

Sampling 

Time 

(Days)

Measured 

Concentration 

(mg a.i./L)1 

Percent 

of 

Nominal2

Mean 

Measured 

Concentration 

(mg a.i./L) 

Mean 

Measured 

Percent of 

Nominal

Negative Control 

(0.0) 

1 0

2 

2 

< LOQ

< LOQ 

< LOQ 

--

-- 

-- 

-- --

0.25 2 0

2 

2 

0.239

0.239 

0.239 

95.7

95.6 

95.4 

0.24 96

0.50 3 0

2 

2 

0.493

0.484 

0.484 

98.6

96.8 

96.8 

0.49 98

1.0 4 0

2 

2 

0.989

0.966 

0.971 

98.9

96.6 

97.1 

0.98 98

2.0 5 0

2 

2 

1.87

1.91 

1.92 

93.7

95.3 

95.8 

1.9 95

4.0 6 0

2 

2 

3.92

3.86 

3.88 

97.9

96.5 

97.1 

3.9 98

8.0 7 0

2 

2

7.91

7.74 

7.80

98.9

96.8 

97.5

7.8 98

1

 The limit of quantitation (LOQ) was 0.150 mg a.i./L, calculated as the product of the concentration of the 

lowest calibration standard (0.150 mg a.i./L) and the dilution factor of the matrix blank samples (1.00). 2 Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.
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Table 2 

Temperature, Dissolved Oxygen and pH of Water in the Test Chambers 

Mean 

Measured 

Concentration 

(mg a.i./L) Replicate 

0 Hour 24 Hours 48 Hours 

Temp.1

(°C) 

DO2

(mg/L) pH 

Temp.1

(°C) 

DO2

(mg/L) pH 

Temp.1

(°C) 

DO2

(mg/L) pH 

 

Negative A 19.3 8.7 8.1 20.4 8.7 8.4 20.4 8.6 8.5 

Control B 19.2 8.7 8.1 20.3 8.8 8.4 20.4 8.6 8.5 

 

0.24 A 19.1 8.8 8.2 20.3 8.8 8.4 20.3 8.6 8.5 

 B 19.2 8.8 8.2 20.2 8.8 8.4 20.2 8.6 8.5 

0.49 A 19.3 8.7 8.2 20.3 8.8 8.4 20.3 8.6 8.5 

 B 19.3 8.8 8.2 20.3 8.8 8.4 20.3 8.6 8.5 

0.98 A 19.2 8.8 8.2 20.3 8.8 8.4 20.3 8.5 8.5 

 B 19.2 8.8 8.2 20.3 8.8 8.4 20.3 8.6 8.5 

1.9 A 19.4 8.7 8.2 20.3 8.8 8.4 20.3 8.5 8.5 

 B 19.3 8.8 8.2 20.3 8.8 8.4 20.3 8.7 8.5 

3.9 A 19.4 8.9 8.2 20.3 8.7 8.4 20.3 8.7 8.5 

 B 19.3 8.9 8.2 20.3 8.7 8.4 20.3 8.7 8.5 

 

7.8 A 19.4 8.8 8.2 20.4 8.7 8.4 20.2 8.8 8.5 

 B 19.3 8.8 8.2 20.3 8.7 8.4 20.3 8.8 8.5 

 

1 Manual temperature measurements. Temperature monitored continuously during the test ranged from 

approximately 20 to 20.5oC, measured to the nearest 0.5oC. 2

 A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20.0oC in freshwater. 
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Table 3 

Specific Conductance, Hardness, Alkalinity and Total Organic Carbon 

in the Dilution Water at Test Initiation 

Parameter Day 0 

Specific Conductance 340 

 (μS/cm) 

 

Hardness 140 

 (mg/L as CaCO3) 

 

Alkalinity 180 

 (mg/L as CaCO3) 

Total Organic Carbon <1 

 (mg C/L) 
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Table 4 

Cumulative Immobility and Observations 

Mean Measured 

Concentration 

(mg a.i./L) Rep. 

Daphnia/ 

Replicate 

~ 2.5 Hours 24 Hours 48 H

Number 

Immobile 1 Observations 2

Number Immobile

1 Observations 2

Number 

Immobile 1 

Negative Control A 10 0 5 AN; 5 Q, AN 0 6 AN; 4 Q, AN 0 

 B 10 0 5 AN; 5 Q, AN 0 7 AN; 3 Q, AN 0 

0.24 A 10 0 3 AN; 7 Q, AN 0 10 AN 0 

 B 10 0 7 AN; 3 Q, AN 0 10 AN 0 

0.49 A 10 0 10 Q, AN 0 10 AN 0 

 B 10 0 7 AN; 3 Q, AN 0 10 AN 0 

0.98 A 10 0 4 AN; 6 Q, AN 0 10 AN 0 

 B 10 0 4 AN; 6 Q, AN 0 9 AN; 1 Q, AN 0 

1.9 A 10 0 6 AN; 4 Q, AN 0 10 AN 0 5 A

 B 10 0 5 AN; 5 Q, AN 0 8 AN; 2 Q, AN 0 6 A

3.9 A 10 0 8 AN; 2 Q, AN 2 8 AN 2 1 

 B 10 0 6 AN; 4 Q, AN 3 3 AN; 4 Q, AN 3 

7.8 A 10 0 6 AN; 4 Q, AN 2 6 AN; 2 C 7 

 B 10 0 4 AN; 6 Q, AN 3 4 AN; 3 C 7 

1 Cumulative number of immobile daphnids. 2 Observed effects: AN = appear normal; C = lethargy; Q, AN = trapped at water surface but appear normal after gentle submersion; Q, C = trapped at 

lethargic after gentle submersion. 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Table 5 

EC50 Values 

Time 

EC50 

(mg a.i./L) 

95% Confidence Interval 

(mg a.i./L) 

Statistical 

Method 

24 Hours > 7.8 --1 NA 2

48 Hours 5.8 4.7 – 7.6 Probit method 3

1

 95% confidence limits could not be calculated with the immobility data obtained. 

2

 NA = not applicable; <50% immobility precluded statistical calculation of an EC50 value. 

3

 The EC50 value and 95% confidence limits were determined by the Probit method. 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Figure 1 

Concentration-Response Curve (48-Hour Immobility Data) 

Slope = 4.616

Mean, Measured Concentration (mg a.i./L)

0 2 4 6 8 10

Percent Immobility

0

10

20

30

40

50

60

70

80

90

100
Wildlife International, Ltd. 

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Appendix 1 

Chemical Characterization
Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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Appendix 2 

Dilution Water Chemistry Parameters Measured 

During the 4-Week Period Immediately Preceding the Test 

Parameter Mean Range 

Specific Conductance 353 332 – 378 

 (μS/cm) (N = 4) 

 

Hardness 147 144 – 156 

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

Alkalinity 178 176 – 180 

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

pH 8.2 8.0 – 8.7 

 (N = 4) 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 3 

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 < 0.0098 Gamma BHC – Lindane < 0.0098 

Alpha BHC < 0.0098 Gamma Chlordane < 0.020 

Alpha Chlordane < 0.0098 Guthion (Azinphos-methyl) < 5.9 

Beta BHC < 0.0098 HCB < 0.0098 

Bolstar < 2.0 Heptachlor < 0.0098 

Chlordane < 0.49 Heptachlor Epoxide < 0.0098 

Coumaphos < 2.9 Kepone < 0.20 

Delta BHC < 0.0098 Malathion < 2.9 

Demeton-O < 2.0 Merphos < 2.0 

Demeton-S < 2.9 Methoxychlor < 0.098 

Diazinon < 5.9 Methyl Parathion < 2.0 

Dichlorvos < 2.9 Mevinphos < 4.9 

Dieldrin < 0.020 Mirex < 0.25 

Disulfoton < 2.0 Naled < 5.9 

Dursban (Chlorpyrifos) < 2.9 o,p-DDD < 0.020 

Endosulfan I < 0.0098 o,p-DDE < 0.74 

Endosulfan II < 0.020 o,p-DDT < 0.020 

Endosulfan Sulfate < 0.020 p,p-DDD < 0.020 

Endrin < 0.020 p,p-DDE < 0.020 

Endrin Aldehyde < 0.098 p,p-DDT < 0.020 

Endrin Ketone < 0.020 Phorate < 2.0 

EPN < 2.0 Ronnel < 2.0 

Ethion < 2.0 Stirophos < 2.0 

Ethoprop < 2.9 Telodrin < 0.0098 

Ethyl Parathion < 2.0 Tokuthion < 2.0 

Famphur < 2.9 Toxaphene < 2.9 

Fensulfothion < 8.8 Trichloronate < 2.0 

Fenthion < 2.0 Trithion < 2.0 

 

1

 Analyses performed by Lancaster Laboratories on samples collected on December 29, 2010. 
Wildlife International, Ltd. 

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Appendix 3 (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 13.0 

Antimony < 0.0200 Manganese < 0.0050 

Arsenic < 0.0200 Mercury < 0.00020 

Barium < 0.0050 Nickel < 0.0100 

Beryllium < 0.0050 Nitrate Nitrogen 0.55 

Bromide < 2.5 Nitrite Nitrogen < 0.50 

Cadmium < 0.0050 Potassium 6.77 

Calcium 35.1 Selenium < 0.0200 

Chloride 3.5 Silver < 0.0050 

Chromium < 0.0150 Sodium 18.5 

Cobalt < 0.0050 Sulfate 5.6 

Copper < 0.0100 Thallium < 0.0300 

Fluoride < 0.50 Vanadium < 0.0050 

Iron < 0.200 Zinc < 0.0200 

Lead < 0.0150 

1

 Analyses performed by Lancaster Laboratories on samples collected on December 29, 2010. 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 4 

The Analysis of Diethyltoluene Diamine (DETDA) in Freshwater 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 4.1 

Analytical Method Flowchart for the Analysis of Diethyltoluene Diamine (DETDA) in Freshwater 

METHOD FLOWCHART FOR THE ANALYSIS OF 

DETDA IN FRESHWATER 

Prepare calibration standards in freshwater using volumetric flasks and gas-tight syringes. 



Prepare QC samples in freshwater using volumetric flasks, volumetric pipettes, culture tubes 

and gas-tight syringes. The matrix blank will be freshwater. 



Dilute samples with freshwater (as needed) using volumetric flasks, 

volumetric pipettes, culture tubes and gas-tight syringes. 



Transfer to autosampler vials and submit for HPLC/UV analysis. 
Wildlife International, Ltd. 

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Appendix 4.2 

Typical HPLC Operational Parameters 

INSTRUMENT: Agilent Model 1100/1200 High Performance Liquid 

Chromatograph (HPLC) with an Agilent Series 1100 

Variable Wavelength Detector 

ANALYTICAL COLUMN: YMC-PACK ODS-AM Column (150 mm x 4.6 mm, 3 μm 

particle size) 

OVEN TEMPERATURE: 40C 

STOP TIME: 14 minutes 

FLOW RATE: 1.000 mL/minute 

SOLVENT A: 

SOLVENT B: 

 

0.1% H3PO4

CH3CN 

GRADIENT PROFILE: Time Flow 

 (min) %A %B (mL/min) 

 0.01 90.0 10.0 1.000 

 1.00 90.0 10.0 1.000 

 9.00 50.0 50.0 1.000 

10.00 50.0 50.0 1.000 

10.10 90.0 10.0 1.000 

14.00 90.0 10.0 1.000 

INJECTION VOLUME: 100.00 μL 

DETDA RETENTION TIME: Component 1: Approximately 5.3 minutes 

Component 2: Approximately 6.0 minutes 

PRIMARY ANALYTICAL 

WAVELENGTH: 220 nm 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 4.3 

Analytical Stocks and Standards Preparation 

A stock solution of DETDA test substance was prepared by weighing 0.1030 grams (weight corrected 

for purity) of the test substance on an analytical balance. The test substance was transferred to a 

100-mL class A volumetric flask, and brought to volume using acetonitrile. The primary stock 

solution (1.00 mg a.i./mL) was diluted in acetonitrile to prepare a 0.100 mg a.i./mL stock solution. 

The 1.00 and 0.100 mg a.i./mL stock solutions were used to prepare the fortification samples. The 

0.100 mg a.i./mL stock solution was used to prepare the calibration standards in freshwater. The 

following shows the dilution scheme for a set of calibration standards for the test samples: 

Stock 

Concentration 

(mg a.i./mL) 

Aliquot 

(L)

Final

Volume 

(mL) 

Standard 

Concentration 

(mg a.i./L) 

0.100 

0.100 

0.100 

0.100 

0.100 

15.0

45.0 

80.0 

120 

150 

10.0

10.0 

10.0 

10.0 

10.0 

0.150 

0.450 

0.800 

1.20 

1.50 
Wildlife International, Ltd. 

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Appendix 4.4 

Example Calculations for a Representative Sample 

The analytical result and percent recovery for sample number 471A-122-4, with a nominal 

concentration of 1.0 mg a.i./L, were calculated using the following equations: 

Concentration of DETDA in sample (mg a.i./L) = Sum of peak areas - (y-intercept)

Slope x dilution factor 

Percent of nominal concentration = Measured concentration of sample (mg a.i./L)

Nominal concentration of sample (mg a.i./L) x 100 

Sum of Peak Areas = 206.48758 

Y-Intercept = -1.0083 

Slope = 209.9061 

Dilution Factor = 1.00 

Concentration of DETDA in sample (mg a.i./L) = 206.48758 + 1.0083

209.9061 x 1.00 

Concentration of DETDA in sample (mg a.i./L) = 0.989 

Percent of nominal concentration = 0.989 mg a.i./L

1.0 mg a.i./L x 100 

Percent of nominal concentration = 98.9% 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 4.5 

Quality Control Samples of DETDA in Freshwater 

Sample 

Number 

(471A-122-) 

Sample 

Type 

Fortified 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L)1

Percent 

Recovery2

MAB-1 

MAB-2 

MAS-1 

MAS-2 

MAS-3 

MAS-4 

MAS-5 

MAS-6 

Matrix Blank 

Matrix Blank 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

0.0 

0.0 

0.250 

0.750 

8.00 

0.250 

0.750 

8.00 

< LOQ 

< LOQ 

0.250 

0.753 

7.70 

0.251 

0.752 

7.99 

-- 

-- 

99.9 

100 

96.2 

100 

100 

99.9 

 Mean = 

Standard Deviation = 

CV = 

99.3 

1.54 

 1.55% 

1

 The limit of quantitation (LOQ) was 0.150 mg a.i./L, calculated as the product of the 

concentration of the lowest calibration standard (0.150 mg a.i./L) and the dilution factor of the 

matrix blank samples (1.00). 

2

 Results were generated using Excel 2000 in the full precision mode. Manual calculations 

may differ slightly. 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 4.6 

Representative Calibration Curve for DETDA 

0

50

100

150

200

250

300

350

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600

(mg a.i./L)

Peak area counts

 Slope = 209.9061; Y-Intercept = -1.0083; R2

 = 0.99996 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 4.7 

Representative Chromatogram of a Low-level DETDA Calibration Standard 

0 2 4 6 8 10 12 min

mAU 

-10

0

10

20

30

40

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\001-0101.D) 5.347 5.971

Nominal concentration: 0.150 mg a.i./L. The arrows indicate the approximate retention times for the 

components of DETDA. Component 1 retention time is 5.3 minutes and Component 2 retention time 

is 6.0 minutes.
Wildlife International, Ltd. 

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Appendix 4.8 

Representative Chromatogram of a High-level DETDA Calibration Standard 

0 2 4 6 8 10 12 min

mAU 

0

20

40

60

80

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\005-0501.D) 5.347 5.980

Nominal concentration: 1.50 mg a.i./L. The arrows indicate the approximate retention times for the 

components of DETDA. Component 1 retention time is 5.3 minutes and Component 2 retention time 

is 6.0 minutes.
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 4.9 

Representative Chromatogram of a Matrix Blank Sample 

0 2 4 6 8 10 12 min

mAU 

-10

0

10

20

30

40

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\006-0601.D)

Sample number 471A-122-MAB-1. The arrows indicate the approximate retention times for the 

components of DETA. Component 1 retention time is 5.3 minutes and Component 2 retention time is 

6.0 minutes.
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 4.10 

Representative Chromatogram of a Matrix Fortification Sample 

0 2 4 6 8 10 12 min

mAU 

-10

0

10

20

30

40

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\007-0701.D) 5.361 5.986

Sample number 471A-122-MAS-1; 0.250 mg a.i./L nominal concentration. The arrows indicate the 

approximate retention times for the components of DETDA. Component 1 retention time is 5.3 

minutes and Component 2 retention time is 6.0 minutes.
Wildlife International, Ltd. 

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Appendix 4.11 

Representative Chromatogram of a Test Sample 

0 2 4 6 8 10 12 min

mAU 

-10

0

10

20

30

40

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\013-1401.D) 5.356 5.985

Sample number 471A-122-4; Day 0; 1.0 mg a.i./L nominal concentration. The arrows indicate the 

approximate retention times for the components of DETDA. Component 1 retention time is 5.3 

minutes and Component 2 retention time is 6.0 minutes.
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 5 

Changes to Protocol 

This study was conducted in accordance with the approved Protocol. 
Wildlife International, Ltd. 

 Project Number 471A-122 

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Appendix 6 

Personnel Involved in the Study 

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

of this study: 

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

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

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

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

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

6. Daniel S. Brougher, M.S., Senior Biologist 

7. Jessica M. Griebel, B.S., Biologist