DIETHYLTOLUENE DIAMINE (DETDA): 

A 96-HOUR STATIC ACUTE TOXICITY TEST 

WITH THE FATHEAD MINNOW (Pimephales promelas) 

FINAL REPORT 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-123 

OECD GUIDELINE 203 

U.S. EPA OPPTS NUMBER 850.1075 

AUTHORS: 

Daniel S. Brougher, M.S. 

Timothy Z. Kendall, M.S. 

Sean P. Gallagher, B.S. 

Henry O. Krueger, Ph.D. 

STUDY INITIATION DATE: February 17, 2012 

STUDY COMPLETION DATE: March 26, 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



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TABLE OF CONTENTS

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

Good Laboratory Practice Compliance Statement ................................................................................. 2 

Quality Assurance Statement ................................................................................................................. 3 

Report Approval ..................................................................................................................................... 4 

Table of Contents ................................................................................................................................... 5 

Summary ................................................................................................................................................ 7 

Introduction ............................................................................................................................................ 8 

Objective ................................................................................................................................................ 8 

Experimental Design .............................................................................................................................. 8 

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

 Test Substance ........................................................................................................................... 9 

 Test Organism ............................................................................................................................ 9 

 Dilution Water ......................................................................................................................... 10 

 Test Apparatus ......................................................................................................................... 10 

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

 Analytical Sampling ................................................................................................................ 11 

 Analytical Method ................................................................................................................... 11 

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

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

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

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

 Measurement of Test Concentrations ...................................................................................... 14 

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

Conclusions .......................................................................................................................................... 15 

References ............................................................................................................................................ 16 
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TABLE OF CONTENTS (Continued) 

TABLES AND FIGURES 

Table 1. Measured Concentrations of Diethyltoluene Diamine (DETDA) in Freshwater Samples . 17 

Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers ......................... 18 

Table 3. Specific Conductance, Hardness and Alkalinity in the Dilution Water 

 at Test Initiation .................................................................................................................. 20 

Table 4. Cumulative Mortality and Observations ............................................................................. 21 

Table 5. LC50 Values ....................................................................................................................... 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 96-Hour Static Acute Toxicity Test with the 

Fathead Minnow (Pimephales promelas) 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-123 

TEST DATES: Experimental Start (OECD): February 20, 2012 

 Experimental Start (EPA): February 20, 2012 

 Exposure Termination: February 24, 2012 

 Experimental Termination: February 24, 2012 

LENGTH OF EXPOSURE: 96 Hours 

TEST ORGANISM: Fathead Minnow (Pimephales promelas) 

SOURCE OF TEST ORGANISMS: Chesapeake Cultures 

 Hayes, VA 23072 

AGE OF TEST ORGANISMS: Juveniles 

MEASUREMENTS OF 10 

NEGATIVE CONTROL FISH: Mean Wet Weight: 0.27 g Range: 0.23 – 0.32 g 

 Mean Total Length: 3.0 cm Range: 2.8 – 3.2 cm 

TEST CONCENTRATIONS: Nominal Mean Measured 

 Negative Control < LOQ 

 Solvent Control < LOQ 

 7.5 mg a.i./L 7.2 mg a.i./L 

 15 mg a.i./L 15 mg a.i./L 

 30 mg a.i./L 29 mg a.i./L 

 60 mg a.i./L 70 mg a.i./L 

 120 mg a.i./L 106 mg a.i./L 

RESULTS: Based on mean measured test concentrations: 

 96-Hour LC50: > 106 mg a.i./L 

 No-Mortality Concentration: 15 mg a.i./L 

 No-Observed-Effect Concentration: 15 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 20, 2012 to February 24, 2012. Raw data generated 

by Wildlife International, Ltd. and the final report are filed under Project Number 471A-123 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 fathead minnow (Pimephales promelas) during a 96-hour exposure period under static 

test conditions. 

EXPERIMENTAL DESIGN 

Fathead minnows were exposed to a geometric series of five test concentrations and a negative 

control (dilution water) for 96 hours under static conditions. Two replicate test chambers were 

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

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 7.5, 15, 

30, 60 and 120 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, the approximate mid-point and the end of the test. 

Fathead minnows were impartially assigned to exposure chambers at test initiation. Observations 

of mortality and other signs of toxicity were made approximately 2, 24, 48, 72 and 96 hours after test 

initiation. Cumulative percent mortality observed in the treatment groups was used to determine LC50 

values at 24, 48, 72 and 96 hours. The no-mortality concentration and the no-observed-effect 

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

observation data. 
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MATERIALS AND METHODS 

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

Diamine (DETDA): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales 

promelas)”. The protocol was based on procedures outlined in the OECD Guidelines for Testing of 

Chemicals, Guideline 203: Fish, Acute Toxicity Test (1); U.S. Environmental Protection Agency Series 

850 – Ecological Effects Test Guidelines, OPPTS Number 850.1075: Fish Acute Toxicity Test, 

Freshwater and Marine (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 used to prepare the test solutions 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: Diethyltoluene Diamine (DETDA); Lot 

number 4500051816-011110; CAS number 68479-98-1. The test substance contained 97.3% active 

ingredient and had an expiration date of March 3, 2013. The test substance was characterized by the 

Sponsor under Good Laboratory Practices and the characterization report is provided in Appendix I. 

Test Organism 

The fathead minnow (Pimephales promelas) was selected as the test species for this study. This 

species is representative of an important group of aquatic vertebrates and was selected for use in the test 

based upon past history of use in the laboratory. Fathead minnows used in the test were received as 

juveniles from Chesapeake Cultures of Hayes, VA. Identification of the species was verified by the 

supplier. 

All fish used in the test were juveniles from the same source and year class, and the length of the 

longest fish measured was no more than twice the length of the shortest. The average total length of 

10 negative control fish measured at the end of the test was 3.0 cm, with a range of 2.8 to 3.2 cm. The 

average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 

0.27 grams, with a range of 0.23 to 0.32 grams. Loading was defined as the total wet weight of fish per 

liter of test water and was 0.18 g fish/L. 
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The fish were held for at least 14 days prior to the test 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 21.4 to 23.5C, measured with a 

liquid-in-glass thermometer. The pH of the water ranged from 8.3 to 8.5, measured with a Thermo Orion 

Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were 8.1 mg/L (93% of saturation), 

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

Daily during the holding period, except during periods of fasting prior to testing, the fish were 

fed a commercially-prepared diet supplied by Sera, North America, supplemented with brine shrimp 

nauplii (Artemia sp.) supplied by Brine Shrimp Direct, Ogden, Utah. The fish were not fed for at least 

two days prior to the test or during the test. 

During the 2-week period prior to the test the fish showed no signs of disease or stress. At test 

initiation, fathead minnows were collected from the culture tank and impartially distributed one and two 

at a time to the test chambers until each contained 10 fish. 

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. 

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

alkalinity and pH of the well water during the four-week period immediately preceding the test are 

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

selected organic and inorganic constituents in the well water are presented in Appendix 2. 

Test Apparatus 

Test chambers were 25-L stainless steel aquaria filled with approximately 15 L of test water. The 

depth of the test water in a representative test chamber was 17 cm. The chambers were indiscriminately 
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positioned by treatment group in two temperature-controlled water baths to maintain the target water 

temperature throughout the test period. All test chambers were labeled with the project number, test 

concentration and replicate designation. 

Preparation of Test Concentrations 

Individual test solutions were prepared directly in each test chamber. All test solutions were 

adjusted to 100% active ingredient during preparation, based on the test substance purity (97.3%). 

Solutions were prepared at nominal concentrations of 7.5, 15, 30, 60 and 120 mg a.i./L by mixing 

calculated amounts of test substance into dilution water with a final volume of 15 L. The test substances 

were weighed into tared 100 mL glass beakers. Approximately 60 mL of the pre-measured 15 L dilution 

water was added, and the beakers were sonicated for 2 – 40 minutes. Using a portion of the pre-measured 

dilution water, each test solution was then rinsed into its appropriate 25 L test chamber, and stirred with a 

top down mixer for 2-3 minutes. The negative control solution was dilution water only. 

Analytical Sampling 

Samples were collected from each test chamber of each treatment and control group at the 

beginning of the test and at 48 and 96 hours (± 1 hour) to measure concentrations of the test substance. 

The samples 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 3.1, and 

typical instrumental parameters are summarized in Appendix 3.2. 
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Calibration standards of DETDA were prepared for the analysis of the test samples, ranging in 

concentration from 3.00 to 30.0 mg a.i./L, using a stock solution of DETDA in acetonitrile (Appendix 3.3). 

Fresh calibration standards were prepared and analyzed with each sample set. 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 3.4. 

The method limit of quantitation (LOQ) was defined as 3.00 mg a.i./L, calculated as the product of 

the concentration of the lowest calibration standard (3.00 mg a.i./L) and the dilution factor of the matrix 

blank samples (1.00). Three matrix blank samples were analyzed to determine possible interferences. No 

interferences were observed at or above the LOQ during the sample analyses (Appendix 3.5). 

Samples of freshwater were fortified at 7.50, 20.0, and 120 mg a.i./L using a stock solution of 

DETDA in acetonitrile (Appendix 3.3), and were analyzed concurrently with the test samples. The 

measured concentrations for the matrix fortification samples ranged from 81.7 to 102% of fortified 

concentrations (Appendix 3.5). 

A representative calibration curve is presented in Appendix 3.6. Representative chromatograms 

of low and high-level calibration standards are presented in Appendices 3.7 and 3.8, respectively. 

Representative chromatograms of a matrix blank sample and a matrix fortification sample are presented in 

Appendices 3.9 and 3.10, respectively. A representative chromatogram of a test sample is presented in 

Appendix 3.11. 

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 two representative test chambers from each water bath at test 

initiation using a SPER Scientific Model 840006C light meter. 
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The target test temperature during the study was 22 ± 1oC. Temperature was measured in each 

test chamber at the beginning and end of the test and at approximately 24-hour intervals during the test 

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

control test chamber using a Fulscope ER/C Recorder, which was verified prior to test initiation with a 

liquid-in-glass thermometer. 

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

test and at approximately 24-hour intervals 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 pH meter. 

Hardness, alkalinity and specific conductance in the dilution water were measured at the 

beginning of the test. 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. 

Observations 

All organisms were observed periodically to determine the number of mortalities in each 

treatment group. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were 

evaluated. Observations were made approximately 2, 24, 48, 72 and 96 hours after test initiation. 

Statistical Analyses 

In this study, there was less than 50% mortality in any of the Diethyltoluene Diamine (DETDA) 

treatment groups during the test which precluded the statistical calculation of LC50 values at 24, 48, 72 

and 96 hours. Due to the method used to calculate the 96-hour LC50 value, the slope of the dose 

response curve could not be calculated. Since there was <50% mortality at 24, 48, 72 and 96 hours, the 

96-hour LC50 value, as well as the no-mortality concentration and NOEC, were determined by visual 

interpretation of the mortality and observation data. 
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RESULTS AND DISCUSSION 

Measurement of Test Concentrations 

Nominal concentrations selected for use in this study were 7.5, 15, 30, 60 and 120 mg a.i./L. 

During the test, the negative control, 7.5, 15, 30 and 60 mg a.i./L test solutions were clear and colorless in 

appearance at test initiation. The 120 mg a.i./L test solution was clear and had a slight purple color at test 

initiation. At test termination, the negative control, 7.5, and 15 mg a.i./L test solutions were clear and 

colorless in appearance, while the 30, 60 and 120 mg a.i./L were clear and purple in color with purple 

color intensity increasing with increasing concentration. 

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 74 to 139% of nominal. When measured concentrations of the 

samples were averaged, the mean measured test concentrations for this study were 7.2, 15, 29, 70 and 

106 mg a.i./L, representing 96, 100, 97, 117 and 89% 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 22 ± 1oC range established for the test. 

Dissolved oxygen concentrations remained ≥6.1 mg/L (≥70% of saturation) throughout the test. 

Measurements of pH ranged from 8.2 to 8.4 during the test. The measurements of hardness, alkalinity 

and specific conductance in the dilution water at test initiation were typical of Wildlife International, Ltd. 

well water (Table 3). Light intensity at test initiation was 695 lux at the surface of the water of one 

representative test chamber. 

Daily observations of mortality and other signs of toxicity observed during the test are presented 

in Table 4. All fish in the negative control group appeared normal throughout the test. All fathead 

minnows in the 7.2, and 15 mg a.i./L treatment groups also appeared normal throughout the test, with no 

mortalities or overt signs of toxicity observed. Signs of toxicity observed among fish in the 29, 70 and 

106 mg a.i./L treatment groups included surfacing, loss of equilibrium and lethargy. Percent mortality in 

the 29, 70 and 106 mg a.i./L treatment groups at test termination was 5, 0 and 20%, respectively. While 
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there was no mortality at the 70 mg a.i./L test concentration, signs of toxicity were noted in the 29 mg 

a.i./L treatment group and the single mortality at this concentration could not be precluded as treatment 

related. The no-mortality concentration and the NOEC were 15 mg a.i./L. LC50 values at 24, 48, 72 and 

96 hours were determined from the mortality data and are shown in Table 5. 

CONCLUSIONS 

Fathead minnows (Pimephales promelas) were exposed for 96 hours under static conditions to 

five mean measured concentrations of Diethyltoluene Diamine (DETDA) ranging from 7.2 to 

106 mg a.i./L. The 96-hour LC50 value was >106 mg a.i./L, the highest concentration tested. Fathead 

minnows exposed to Diethyltoluene Diamine (DETDA) at concentrations ≤15 mg a.i./L appeared normal 

with no mortality or overt signs of toxicity. The 96-hour no-mortality concentration and the NOEC were 

15 mg a.i./L. 
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REFERENCES 

1 Organisation for Economic Cooperation and Development. 1992. OECD Guidelines for 

Testing of Chemicals, Guideline 203: Fish, Acute Toxicity Test. Adopted 17 July 1992. 

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

(draft), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine. 

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. 
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Table 1

Measured Concentrations of Diethyltoluene Diamine (DETDA) in Freshwater Samples 

Nominal Test 

Concentration 

(mg a.i./L) 

Sample 

Number 

(471A-123-) 

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 

2 

13 

14 

25 

26 

0

0 

2 

2 

4 

4 

< LOQ

< LOQ 

< LOQ 

< LOQ 

< LOQ 

< LOQ 

--

-- 

-- 

-- 

-- 

-- 

-- --

7.5 3 

4 

15 

16 

27 

28 

0

0 

2 

2 

4 

4 

7.32

7.64 

6.70 

7.03 

7.21 

7.58 

97.5

102 

89.3 

93.8 

96.1 

101 

7.2 96

15 5 

6 

17 

18 

29 

30 

0

0 

2 

2 

4 

4 

14.8

15.5 

13.8 

14.4 

14.9 

15.5 

99.0

104 

91.9 

96.1 

99.5 

104 

15 100

30 7 

8 

19 

20 

31 

32 

0

0 

2 

2 

4 

4 

28.6

30.0 

27.4 

28.0 

29.1 

30.0 

95.4

99.9 

91.4 

93.3 

97.0 

100 

29 97

60 9 

10 

21 

22 

33 

34 

0

0 

2 

2 

4 

4 

83.2

61.8 

74.0 

59.2 

80.4 

62.5 

139

103 

123 

98.6 

134 

104 

70 117

120 11 

12 

23 

24 

35 

36 

0

0 

2 

2 

4 

4

95.2

120 

89.6 

116 

95.4 

122

79.3

99.9 

74.7 

96.8 

79.5 

102

106 89

1

 The limit of quantitation (LOQ) was 3.00 mg a.i./L, calculated as the product of the concentration of the 

lowest calibration standard (3.00 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 

Nominal 

Concentration 

(mg a.i./L) Replicate 

0 Hour 24 Hour 

Temp.1

(oC) 

DO2

(mg/L) pH 

Temp.1

(oC) 

DO2

(mg/L) pH 

Temp.1

(oC) 

Negative Control A 21.5 8.7 8.3 21.6 7.6 8.2 21.7 

 B 21.6 8.4 8.3 21.7 7.5 8.3 21.8 

7.2 A 21.5 8.4 8.3 21.6 7.5 8.3 21.7 

 B 21.6 8.5 8.3 21.7 7.7 8.3 21.7 

15 A 21.5 8.3 8.3 21.7 7.7 8.3 21.7 

 B 21.6 8.3 8.3 21.7 7.5 8.3 21.7 

29 A 21.5 8.5 8.3 21.6 7.5 8.3 21.7 

 B 21.6 8.5 8.3 21.8 7.4 8.3 21.7 

70 A 21.5 8.5 8.3 21.6 7.4 8.3 21.6 

 B 21.6 8.4 8.3 21.7 7.5 8.3 21.7 

106 A 21.5 8.6 8.3 21.6 7.6 8.3 21.6 

 B 21.6 8.6 8.3 21.7 7.6 8.3 21.8 

1 Manual temperature measurements. Temperature monitored continuously during the test ranged from approximately 21.6 to 22.1°C, 

0.1°C. 

2 A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22oC in freshwater. 
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Table 2 (Continued) 

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

Nominal 

Concentration 

(mg a.i./L) Replicate 

72 Hour 96 Hour 

Temp.1

(oC) 

DO2

(mg/L) pH 

Temp.1

(oC) 

DO2

(mg/L) 

Negative Control A 21.7 7.2 8.2 21.7 6.8 

 B 21.8 7.1 8.3 21.7 6.6 

7.2 A 21.7 7.0 8.3 21.7 6.6 

 B 21.7 7.2 8.3 21.7 6.7 

15 A 21.7 7.3 8.3 21.7 6.7 

 B 21.8 7.5 8.3 21.7 6.8 

29 A 21.6 7.5 8.3 21.7 6.7 

 B 21.7 7.5 8.3 21.7 6.7 

70 A 21.6 7.4 8.3 21.7 6.7 

 B 21.7 7.4 8.3 21.7 6.6 

106 A 21.6 6.8 8.3 21.6 6.4 

 B 21.9 6.6 8.2 21.7 6.1 

1 Manual temperature measurements. Temperature monitored continuously during the test ranged from approximately 21.6 to 22

the nearest 0.1°C. 

2 A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22oC in freshwater. 
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Table 3

Specific Conductance, Hardness and Alkalinity 

in the Dilution Water at Test Initiation 

Parameter Day 0 

Specific Conductance 315 

 (μS/cm) 

 

Hardness 136 

 (mg/L as CaCO3) 

 

Alkalinity 172 

 (mg/L as CaCO3) 
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Table 4 

Cumulative Mortality and Observations 

Nominal 

Concentration 

(mg a.i./L) Rep. 

No. 

Exposed 

2 Hours 24 Hours 

No. Dead 1 Observations 2 No. Dead 1 Observations 2 No. D

Negative Control A 10 0 10 AN 0 10 AN 0 

 B 10 0 10 AN 0 10 AN 0 

7.2 A 10 0 10 AN 0 10 AN 0 

 B 10 0 10 AN 0 10 AN 0 

15 A 10 0 10 AN 0 10 AN 0 

 B 10 0 10 AN 0 10 AN 0 

29 A 10 0 10 AN 0 10 AN 0 

 B 10 0 10 AN 0 10 AN 1 

70 A 10 0 10 AN 0 7 AN; 3 C 0 

 B 10 0 10 AN 0 9 AN; 1 C 0 

106 A 10 0 10 AN 1 2 AN; 7 C 1 

 B 10 0 10 AN 1 5 A; 4 C 2 

1 Cumulative number of dead fish. 2 Observations: A = surfacing; AN = appear normal; C = lethargy; N = loss of equilibrium. 
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Table 4 (Continued) 

Cumulative Mortality and Observations 

Nominal 

Concentration 

(mg a.i./L) Rep. 

No. 

Exposed 

72 Hours 96 Hours 

No. Dead 

1 Observations 2 No. Dead 1 Observation

Negative Control A 10 0 10 AN 0 10 AN 

 B 10 0 10 AN 0 10 AN 

7.2 A 10 0 10 AN 0 10 AN 

 B 10 0 10 AN 0 10 AN 

15 A 10 0 10 AN 0 10 AN 

 B 10 0 10 AN 0 10 AN 

29 A 10 0 10 AN 0 7 AN; 3 C 

 B 10 1 9 AN 1 6 AN; 3 C 

70 A 10 0 3 A; 2 AN; 5 C 0 3 A; 1 AN; 6 

 B 10 0 1 A; 5 AN; 4 C 0 1 A; 4 AN; 5 

106 A 10 1 5 A; 4 C 1 6 A; 3 C 

 B 10 3 7 A 3 7 A 

1 Cumulative number of dead fish. 2 Observations: A = surfacing; AN = appear normal; C = lethargy; N = loss of equilibrium. 
Wildlife International, Ltd. 

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Table 5 

LC50 Values 

Time 

LC50 

(mg a.i./L) 

95% Confidence Interval 

(mg a.i./L) 

Statistical 

Method 

24 Hours > 106 --1 NA 2

48 Hours > 106 --1 NA 2

72 Hours > 106 --1 NA 2

96 Hours > 106 --1 Binomial test 3

1

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

2

 NA = not applicable; <50% mortality precluded statistical calculation of an LC50 value. 

3

 An upper 95% confidence limit could not be calculated. At a confidence level of 95%, the 

binomial test shows that the LC50 is above 106 mg a.i./L. 
Wildlife International, Ltd. 

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Appendix 1 

Chemical Characterization
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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 362 347 – 378 

 (μS/cm) (N = 4) 

 

Hardness 144 144 – 144 

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

Alkalinity 178 176 – 180 

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

pH 8.3 8.1 – 8.7 

 (N = 4) 
Wildlife International, Ltd. 

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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. 
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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. 

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Appendix 4 

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

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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 

DIETHYLTOLUENE DIAMINE (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. 
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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: 25.00 μL 

APPROXIMATE DETDA 

RETENTION TIMES: 

Component 1: 5.0 minutes 

Component 2: 5.7 minutes 

PRIMARY ANALYTICAL 

WAVELENGTH: 220 nm 
Wildlife International, Ltd. 

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Appendix 4.3 

Analytical Stocks and Standards Preparation 

 A stock solution of Diethyltoluene Diamine (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 used to prepare the fortification samples and 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) 

1.00 

1.00 

1.00 

1.00 

1.00 

30.0 

50.0 

100 

200 

300 

10.0 

10.0 

10.0 

10.0 

10.0 

3.00 

5.00 

10.0 

20.0 

30.0 
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Appendix 4.4 

Example Calculations for a Representative Sample 

The analytical result and percent recovery for sample number 471A-123-5, with a nominal 

concentration of 15 mg a.i./L, were calculated using the following equations: 

Concentration of Diethyltoluene Diamine (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 = 720.71421 

Y-Intercept = 2.8258 

Slope = 48.3656 

Dilution Factor = 1.00 

Concentration of Diethyltoluene Diamine (DETDA) in sample (mg a.i./L) 

 = 

720.71421 - 2.8258

48.3656 x 1.00 

Concentration of Diethyltoluene Diamine (DETDA) in sample (mg a.i./L) = 14.8 

Percent of nominal concentration = 14.8 mg a.i./L

15 mg a.i./L x 100 

Percent of nominal concentration = 99.0%* 

* Results were generated using Excel 2000 in full precision mode. Manual calculations may differ 

slightly. 
Wildlife International, Ltd. 

 Project Number 471A-123 

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Appendix 4.5 

Quality Control Samples of Diethyltoluene Diamine (DETDA) in Freshwater 

Sample 

Number 

(471A-123-) 

Sample 

Type 

Fortified 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L)1

Percent 

Recovery2

MAB-1 

MAB-2 

MAB-3 

MAS-1 

MAS-2 

MAS-3 

MAS-4 

MAS-5 

MAS-6 

MAS-7 

MAS-8 

MAS-9 

Matrix Blank 

Matrix Blank 

Matrix Blank 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

Matrix Fortification 

0.0 

0.0 

0.0 

7.50 

20.0 

120 

7.50 

20.0 

120 

7.50 

20.0 

120 

< LOQ 

< LOQ 

< LOQ 

7.50 

20.1 

119 

6.89 

16.3 

110 

7.50 

20.4 

122 

-- 

-- 

-- 

99.9 

100 

99.0 

91.9 

81.7 

91.4 

99.9 

102 

102 

 Mean = 

Standard Deviation = 

CV = 

96.4 

6.78 

 7.03% 

1

 The limit of quantitation (LOQ) was 3.00 mg a.i./L, calculated as the product of the concentration of the lowest calibration 

standard (3.00 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. 

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Appendix 4.6 

Representative Calibration Curve for Diethyltoluene Diamine (DETDA)

0

200

400

600

800

1000

1200

1400

1600

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

(mg a.i./L)

Peak area counts

 Slope = 48.3656; Y-Intercept = 2.8258; R2

 = 0.99981 
Wildlife International, Ltd. 

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Appendix 4.7 

Representative Chromatogram of a Low-level Diethyltoluene Diamine (DETDA) Calibration 

Standard 

0 2 4 6 8 10 12 min

mAU 

0

25

50

75

100

125

150

175

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\001-0101.D) 4.989 5.694

Nominal concentration: 3.00 mg a.i./L. The arrows indicate the approximate retention times for the 

components of Diethyltoluene Diamine (DETDA). Component 1 retention time is 5.0 minutes and 

Component 2 retention time is 5.7 minutes.
Wildlife International, Ltd. 

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Appendix 4.8 

Representative Chromatogram of a High-level Diethyltoluene Diamine (DETDA) Calibration 

Standard 

0 2 4 6 8 10 12 min

mAU 

0

50

100

150

200

250

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\005-0501.D) 4.971 5.699 6.553

Nominal concentration: 30.0 mg a.i./L. The arrows indicate the approximate retention times for the 

components of Diethyltoluene Diamine (DETDA). Component 1 retention time is 5.0 minutes and 

Component 2 retention time is 5.7 minutes.
Wildlife International, Ltd. 

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Appendix 4.9 

Representative Chromatogram of a Matrix Blank Sample 

0 2 4 6 8 10 12 min

mAU 

0

20

40

60

80

100

120

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\006-0601.D)

Sample number 471A-123-MAB-1. The arrows indicate the approximate retention times for the 

components of Diethyltoluene Diamine (DETDA). Component 1 retention time is 5.0 minutes and 

Component 2 retention time is 5.7 minutes.
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Appendix 4.10 

Representative Chromatogram of a Matrix Fortification Sample 

0 2 4 6 8 10 12 min

mAU 

0

20

40

60

80

100

120

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\007-0701.D) 5.004 5.711

Sample number 471A-123-MAS-1; 7.50 mg a.i./L nominal concentration. The arrows indicate the 

approximate retention times for the components of Diethyltoluene Diamine (DETDA). Component 1 

retention time is 5.0 minutes and Component 2 retention time is 5.7 minutes. 
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Appendix 4.11 

Representative Chromatogram of a Test Sample 

0 2 4 6 8 10 12 min

mAU 

0

20

40

60

80

100

120

 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\014-1501.D) 5.022 5.731 6.569

Sample number 471A-123-5; Day 0; 15 mg a.i./L nominal concentration. The arrows indicate the 

approximate retention times for the components of Diethyltoluene Diamine (DETDA). Component 1 

retention time is 5.0 minutes and Component 2 retention time is 5.7 minutes. 
Wildlife International, Ltd. 

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Appendix 5 

Changes to Protocol 

 This study was conducted in accordance with the approved protocol. 
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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. Christian Disque, B.S., Biologist