SRC TR-09-057

ORPHAN CHEMICAL DATA ADEQUACY REVIEW AND ROBUST SUMMARY ADEQUACY 

REQUIREMENTS 

Fatty acids, tall-oil, reaction products with diethylenetriamine, acetates

Prepared by:

Emily Cohen, Christopher Janson and Joan D. Garey

Environmental Science Center

Syracuse Research Corporation

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-04-019

Task FA411.4.409.004

Submitted to:

OPPT/RAD/HPVCB (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Kathleen C. Kennedy, Project Officer

David Brooks, Work Assignment Manager

January 16, 2009
Orphan Chemical Data Adequacy Review and Robust Summary Adequacy Requirements 

Chemical Name:

Fatty acids, tall-oil, reaction products with diethylenetriamine, acetates, CAS No. 68153-60-6

January 2009

Submitted to:

High Production Volume Chemicals Branch 

Risk Assessment Division 

Office of Pollution Prevention and Toxics 

Environmental Protection Agency 

WAM: David Brooks 

1200 Pennsylvania Avenue, NW 

Washington, DC 20460-0001

Prepared By:

SRC 

Environmental Science Center 

2451 Crystal Drive, Suite 804 

Arlington, VA 22202
ACUTE TOXICITY TO AQUATIC VERTEBRATES

Summary of endpoint: Adequate

Title: Acute toxicity to fish

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: (CAS No. 68153-60-6)

Remarks: Project 02-088-2

METHOD

Method/guideline followed: PARCOM. “Protocols on Methods for Testing of Chemicals Used in 

Offshore Oil Industry. Part B Protocol for a Fish Acute Toxicity Test”. 1995

Test type: Static acute with a 48 hour renewal

GLP (Y/N): Yes

Year study performed: 2003

Species/Strain: Sheepshead minnow, Cyprinodon variegatus

Analytical monitoring: None, nominal concentrations

Exposure time: 96 hours

Number of animals/sex/dose: 10/dose

Statistical Methods: Trimmed Spearman-Karber method.

Concentration: 0 (control), 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L 

Remarks: Deionized water based test substance stocks introduced into glass, rectangular 21-L aquaria 

containing 10 liter of synthetic seawater. Synthetic seawater was prepared using deionized laboratory 

water and Hawaiian Marinemix artificial salts. An identical vessel containing only seawater was 

designated the control. Ten sheepshead minnows were randomly placed into each vessel for a total of ten 

fish per concentration. Fish mean total length and weight were 1.1 cm and 0.017 g, respectively. Fish 
were not fed for 24 hours prior to exposure. Six nominal test concentrations (0.5, 1.0, 2.0, 4.0, 8.0 and 

16.0 mg/L) were evaluated. Temperature was maintained at nominal 20 +/- 2 C and photoperiod at 16 

hours light: 8 hours dark.

Mortality counts and behavioral observations were made every 24 hours during the 96 hour study. 

Dissolved oxygen, pH, salinity and temperature were recorded initially and every 24 hours thereafter for 

all surviving concentrations. Test vessels were not aerated. Test solutions were renewed at 48 hours.

A 2.3 mg/L 3,5-dichlorophenol reference study was conducted concurrently with this study.

RESULTS

Remarks: The 96 hour LC50 was determined to be 5.3 (4.6-6.1) mg/L using the Trimmed SpearmanKarber method. The no observed effect concentration (NOEC) at 96 hours was 4.0 mg/L. The minimum 

concentration causing 100% mortality was 8.0 mg/L. No mortalities were noted in the control. Normal 

behavior was noted at all times and in all concentrations up to 4.0 mg/L. The one mortality noted in the 

1.0 mg/L at 96 hours was not deemed to be toxicant related. Sheepshead minnow exposure to 2.3 mg/L 

3,5-dichlorophenol demonstrated 50% survival at 96 hours; the acceptable range is 20 to 80 percent.

The test substance was completely soluble in synthetic seawater. The chemical and physical parameters 

monitored during the study were all within normal range: pH 7.8 – 8.0, dissolved oxygen 6.9 – 7.6 ppm, 

temperature 18.4 – 20.3 C and salinity 32 – 34 ppt

Cumulative 96 hour mortality

Control = 0

0.5 mg/L = 0

1.0 mg/L = 1

2.0 mg/L = 0

4.0 mg/L = 0

8.0 mg/L = 10

16.0 mg/L = 10

CONCLUSION

The 96 hour, static acute renewal toxicity of the test substance to the sheepshead minnow was 5.3 mg/L. 

The corresponding NOEC value is 4.0 mg/L

RELIABILITY CODE

1 - The test is considered valid since the 2.3 mg/l 3,5-dichlorophenol reference toxicity study was within 

acceptable parameters. Temperature, dissolved oxygen and pH were also within test guidelines.
REFERENCES

Static acute renewal toxicity to the Sheepshead Minnow (Cyprinodon variegates) (2006)

Disk 3: 8(d)86070000012 (OECD) (page 3)
ACUTE TOXICITY TO AQUATIC INVERTEBRATES

Summary of endpoint: Inadequate

Title: Acute toxicity to invertebrates

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity to invertebrates endpoint as freshwater species are the 

preferred test subjects.

TEST SUBSTANCE

Identity: CAS No. 68153-60-6

Remarks: Project O-7002F

METHOD

Method/guideline followed: PARCOM. Ring Test Protocol “Acute Toxicity to the Marine Copepod 

Acartia tonsa” & ISO/CD 14669: “Determination of Acute Lethal Toxicity to Marine Copepods”.

Test type: Static acute

GLP (Y/N): Yes

Analytical monitoring: None, nominal concentrations in seawater

Year study performed: 2002

Species/strain: Acartia tonsa

Exposure time: 48 hours

Concentrations: 0 (control), 0.1, 0.5, 2.5, 10.0 mg/L

Remarks: Acartia tonsa eggs used for this study were obtained from the University of Copenhagen, 

Laboratory of Marine Biology, Helsingar, Denmark on April 12, 2002 and stored in the refrigerator. Test 

animals were cultured in small batches and the age of the animals used in this study was

28 days. The test substance was dissolved in filtered and aerated seawater. The concentration of this stock 

corresponded with the highest concentration of product tested (10 mg/l). The stock was mixed in a 1 liter

Erlenmeyer flask on a magnetic stirrer (~100 rpm) for 2 – 4 hours. The flask was wrapped in aluminum 

foil to avoid exposure to light. Five Acartia tonsa were randomly placed into each replicate (4) for a total 

of twenty copepods per concentration. Four nominal test concentrations (0.1, 0.5, 2.5, and 10.0 mg/l) and 
a control were evaluated. Temperature was maintained at a nominal 20 +/- 2.4 C with a photoperiod of 

16 hours light: 8 hours dark.

Mortality counts were made every 24 hours during the 48 hour study. Water chemistry was also 

performed at this time. Test vessels were not aerated. A 1.0 mg/l 3,5- dichlorophenol reference study was 

conducted concurrently with this study.

RESULTS

The 48-hour LC50 was determined to be 2.8 mg/l; confidence limits are not available, using the Logistic 

Regression Model (JMP Program, version 3, 1998). The 48 hour NOEC was 0.5 mg/l while the LC10 and 

LC90 were respectively 2.3 and 3.4 mg/l. The minimum concentration causing 100% mortality was 10.0 

mg/l. No mortalities were noted in the control. Acartia tonsa exposure to 1.0 mg/l 3,5-dichlorophenol 

demonstrated 60% survival at 48 hours; the acceptable range is 20 to 80 percent.

The test substance was completely soluble in seawater. The chemical and physical parameters monitored 

during the study were all within normal range: 48 hour control dissolved oxygen was greater than 4 mg/l 

(i.e., 7.3 mg/l) and the temperature range during the study was 19.4 - 20.4 C within the test guideline of 

20 ± 2.4 oC.

Cumulative 48 Hour Mortality

Control = 0

0.1 mg/l = 0

0.5 mg/l = 0

2.5 mg/l = 5

10.0 mg/l = 20

CONCLUSIONS

The 48-hour static acute LC50 of the test substance to Acartia tonsa is 2.8 mg/l. The corresponding 

NOEC value is 0.50 mg/l. These test results are based upon nominal concentrations of the test substance 

in seawater.

RELIABILITY CODE

3 – not valid

REFERENCES

AnalyCen Ecotox AS, “Toxicity Test Results with Acartia tonsa for the Test substance (2002)

Disk 3: 8(d)86070000012 (OECD) (page 10)

TOXICITY TO AQUATIC PLANTS
Summary of endpoint: Adequate

Title: Toxicity to marine algae

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: CAS No. 68153-60-6

Remarks: Project 02-088-1

METHOD

Method/guideline followed: ISO Draft International Standard 1994. ISO/DIS 10253.2, ISO/TC 147/SC

5, 1994 -05-17: Water Quality – Marine algal growth inhibition test with Skeletonema costatum and 

Phaeodactylum tricornutum. ISO Central Secretariat.

Test Type: Static acute growth inhibition

GLP (Y/N): Yes

Analytical monitoring: Electronic particle counting (Coulter Counter Multisizer 3 with computer 

interface/related software)

Year study performed: 2003

Species/Strain: Skeletonema costatum, UTex #LB2308

Element basis: 10,000 cells/mL inoculums, initial cell density 1,501,390 cells/mL, 6.660 mL inoculum/L 

of nutrient seawater media

Exposure period: 72 hours

Statistical methods: Linear regression analysis

Concentration: 0.04, 0.07, 0.13, 0.24, 0.75, 1.3, 2.4, 4.3 mg/L

Remarks: The inoculum used in this study was extracted from four day-old stock culture. The 

Skeletonema costatum (UTEX #LB 2308) culture used in this study was obtained from stock maintained 

in the natural seawater algal assay medium obtained from the original culture from the Department of 

Biology’s Culture Collection of Algae at the University of Texas at Austin, Austin, TX 78712. This strain 
was used by Petrolite during a PARCOM Ring Test and its growth and response was found to be 

comparable to other strains used. ESG incubated the stock cultures under the same environmental 

conditions as those used in the definitive test.

Method of cultivation: Stock subcultures were transferred into 250 mL Erlenmeyer flasks containing fresh 

medium 1-2 times per week to provide 3±1 day old cultures for assay inoculations. 

Controls: Six control vessels were prepared with culture medium and inoculum but no test substance. A 

single concentration series of blanks (media and test substance without inoculum) including a blank 

control (medium only) were also employed.

An Eberbach Shaker power unit (#6140), located in a Forma walk-in environmental chamber (set at 20 

C), was employed and continuously illuminated by 6000 – 10,000 lux of light provided by overhead coolwhite fluorescent lights. Flasks, 250 mL in size, were randomly placed on the power unit and rotated 

continuously at 100 +/- 5 oscillations per minute. Random placement of the Erlenmeyers was determined 

using an Excel software program. The flasks were repositioned randomly each day to minimize spatial 

differences in the chamber. The temperature was measured and recorded at zero, 24, 48, and 72 hours in 

each of three strategically located temperature control flasks

Test temperature range: Protocol stipulates 20±2 degrees Celsius (19.8-21.2 degrees C. measured)

Dilution water source: The natural seawater algal assay medium was prepared with seawater obtained 

from Marine Biological Laboratory (MBL), Aquatic Resource Division, Woods Hole, MA. MBL filtered 

the water to 0.8 μm prior to shipping. Upon receipt, the seawater was placed in an environmental chamber 

set at 20 C or lower. When needed for media preparation, the water was removed from storage and 

filtered to 0.2 μm. The salinity was determined and recorded. The salinity of the water used in this test 

was found to be 31 ppt.

pH algal medium: Nutrients obtained from Aquaculture Supply, Dade City, FL and a commercially 

prepared trace sodium silicate nutrient concentrate were used to prepare the algal medium f/2 plus silica 

formulation recommended by Guillard (1975). Following the addition of nutrients, the pH of the medium 

was checked and adjusted if necessary. The pH of the media used in this test was 8.0. The medium was 

stored at 20 C or lower until needed for testing.

Exposure vessel type: Test vessels were 250 mL Erlenmeyer flasks fitted with 23-45 mm air permeable 

Jaece foam plugs. Four replicates per test concentration, no aeration. 

Stock solutions: Stock concentration of test material (50 mg/mL) prepared in deionized water and added 

in the appropriate amounts to 50 mL algal media achieve each nominal test concentration.

Water chemistry pH: 0 hour: Control (8.01), 0.04 (8.04), 0.07 (8.04), 0.13 (8.06), 0.24 (8.08), 0.42 (8.07), 

0.75 (8.08), 1.3 (8.09), 2.4 (8.10), 4.03 (8.11)
72 hour: Control (8.45), 0.04 (8.80), 0.07 (8.56), 0.13 (8.72), 0.24 (8.43), 0.42 (8.77), 0.75 (8.05), 1.3 

(8.02), 2.4 (8.00), 4.03 (8.00)

Light levels and quality: Continuous illumination by 6,000 – 10,000 lux of light provided by overhead 

cool-white fluorescent lighting. Readings of 7051, 8954, and 8008 lux were measured at three spatially 

random positions on shaker table.

RESULTS

Remarks: 

Element value: 

Biomass (mg/L)

24 hr EC50 = 0.49

48 hr EC50 = 0.56

72 hr EC50 = 0.58

72 hr EC90 = 0.75

72 hr NOEC = 0.04

Growth rate (mg/L)

24 hr EC50 = 0.49

48 hr EC50 = 0.69

72 hr EC50 = 0.74

72 hr EC90 = 0.87

72 hr EC50 = 0.42

Control response: satisfactory

Statistical results: satisfactory, R-squared 95+(24, 48, 72 hr data sets)

Percent biomass inhibition (%) per concentration (mg/L) observations:

24 hour: 0.04(0), 0.07(44.9), 0.13(0), 0.24(0), 0.42(6.9), 0.75(87.4), 1.3(114), 2.4(118), 4.03(176)

48 hour: 0.04(0), 0.07(4.4), 0.13(0), 0.24(0), 0.42(0), 0.75(94.0), 1.3(102), 2.4(105), 4.03(110)

72 hour: 0.04(0), 0.07(12.2), 0.13(0), 0.24(0), 0.42(1.7), 0.75(90.6), 1.3(101), 2.4(101), 4.03(101)

Percent growth rate inhibition (%) per concentration observations (mg/L):

24 hour: 0.04(0), 0.07(44.9), 0.13(0), 0.24(0), 0.42(6.9), 0.75(87), 1.3(114), 2.4(118), 4.03(176)

48 hour: 0.04(0), 0.07(22.8), 0.13(0), 0.24(0.2), 0.42(0), 0.75(94.9), 1.3(101), 2.4(103), 4.03(100)

72 hour: 0.04(0), 0.07(0.81), 0.13(0), 0.24(0), 0.42(0), 0.75(56.8), 1.3(104), 2.4(114), 4.03(95.6)

CONCLUSIONS

The test substance is biologically active toward Skeletonema; 72 hr EC50 < 1.0 mg/L
RELIABILITY CODE

1 - The test was valid since the control cell density increased by a factor of more than 16 in 72 hr. This 

increase corresponds to a growth rate of 0.04 h-1. The pH did not vary by more than 1.0 unit during the 

test.

REFERENCES

“Acute Toxicity of _ to the Saltwater Diatom, Skeletonema costatum (72 hr Exposure)”. (2006)

Disk 3: 8(d)86070000012(OECD) (page 5)
Summary of Endpoints

Endpoint Study Information 

Provided

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Daphnia Yes No Yes

Toxicity to Aquatic Plants Yes Yes No

Health Effects

Acute Toxicity No No Yes

Repeated-Dose Toxicity No No Yes

Genetic Mutation No No Yes

Chromosomal Aberrations No No Yes

Toxicity to Reproduction No No Yes

Developmental Toxicity No No Yes