SRC TR-11-019

HPV 2 TEST RULE DATA ADEQUACY REVIEW

1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4:

Toxicity to Aquatic Plants Study

Prepared by:

Teresa Manyin and Joan D. Garey

Chemical Biological Environmental Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.1.009.001

Submitted to:

OPPT/RAD/HPVCB (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

February 25, 2011
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HPV2 Test Rule Data Adequacy Review

1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4:

Toxicity to Aquatic Plant Study

February 2011

Submitted to:

High Production Volume Chemicals Branch 

Risk Assessment Division 

Office of Pollution Prevention and Toxics 

Environmental Protection Agency 

WAM: Meena Sonawane

Alternate WAM: David Brooks

1200 Pennsylvania Avenue, NW 

Washington, DC 20460-0001

Prepared By:

SRC, Inc. 

Chemical, Biological, and Environmental Center 

2451 Crystal Drive, Suite 804 

Arlington, VA 22202
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TOXICITY TO AQUATIC PLANTS

Summary of endpoint: Inadequate

Title: Determination of the effect of KM200 on the growth of the freshwater green alga 

Selenastrum capricornutum (OECD 201 and EU C.3 Guidelines)

This study is: Adequate Not Adequate

The study is invalid because: (1) No analytical measures of the test substance were obtained 

during testing. An appropriate method for performing analytical measurements of the test 

substance was not used. The test needs to be redone using an appropriate method. (2) The purity 

of the test substance was less than 100% and information on the composition of the remaining 

percentage of the test substance was not provided. (3) It is not clear that the reported nominal 

concentrations take into account the purity of the test substance.

TEST SUBSTANCE 

Identity: 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4

Remarks: Purity: 70-90%; trade name: KM200; clear liquid; Batch no. L11381

METHOD

Method/guideline followed: OECD Guideline 201 and EU C.3 Guidelines

Test type: Static toxicity test to freshwater algae

GLP compliant?: Yes

Year study was performed: 2002

Test species: Green algae (Pseudokirchneriella subcapitata, formerly Selenastrum 

capricornutum); culture supplied by CCAP, The Freshwater Biological Association, Cumbria, 

England.

Analytical monitoring: No (Analytical monitoring was attempted, but results were not 

reported.)1

Exposure duration: 70.5 hours

 1

Analytical monitoring was attempted by the study authors. However, due to inconsistent results from the chemical 

analyses, the measured concentrations of test substance were not reported by the study authors. The measurements 

were considered to be unreliable. See pg. 6 of this document, section on Study Reliability, for more information.
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Concentrations tested: Nominal: 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L2; measured 

concentrations were not reported. However, no indication is provided as to whether the 

concentrations take into account the 70-90% purity of the test substance.

Test method/conditions remarks: After a range-finding test, a definitive growth inhibition test 

was conducted. The test medium was prepared in ultra pure water and was sterilized by 

micropore filtration. The prepared medium contained 150 mg NaHCO3/L and had a hardness of 

24.3 mg CaCO3/L. Test solutions were prepared in medium at nominal concentrations of 0, 3.2, 

10, 18, 32, 56, 101, and 322 mg/L.

At test initiation, an inoculum of 1.0 mL of an algal suspension containing 8 x 105 cells/mL was 

added to 200-mL flasks containing 100 mL of test solution, yielding an initial cell density of 8 x 

103 cells/mL. The test was carried out in duplicate, with four controls containing algae only and 

a single set of flasks containing test substance without algae (blanks) at each nominal 

concentration. All flasks were incubated at 23 ± 2 °C and shaken at approximately 200 rpm on 

an orbital shaker. The flasks were illuminated using fluorescent lamps at a light intensity of 60-

120 μmol/s/m2 (measured with a Bottemanne Weather Instruments Photosynthetic Radiometer 

RA200 Q). 

The initial cell density was calculated from the measured density in the algal pre-culture used for 

the inoculation, divided by the appropriate dilution factor. Algal densities (cells/mL) and algal 

biovolume (μm3/mL) were determined after 23.5, 47, and 70.5 hours using an electronic particle 

counter (Coulter Multisizer IIe). Measured values were corrected for background values in the 

appropriate blanks. The mean measured values were used for calculation of effect levels.

The pH was measured at the start (in medium without algae) and after 70.5 hours in selected 

cultures. The morphology of the algae was examined visually with the aid of a microscope at the 

start and end of the test. The light intensity at two different culture positions was measured at 

the start of the test.

Samples for chemical analysis of test substance concentrations were taken at the start of the test 

from control series without algae and at the end of the test from series with algae. Chemical 

analysis was based on an HPLC method using a fluorescence detector and an external standard. 

The test substance was fluorogenically labeled with a mixed o-phtalaldehyde/mercaptoethanol 

reagent before HPLC analysis. 

The EC values with respect to growth rate (ErC values) were calculated by means of a parametric 

model, assuming exponential growth and a constant error per measurement. The EC values with 

respect to area under the growth curve (EbC values) were calculated by the method provided in 

OECD Guideline 201. The values were calculated by linear interpolation of a plot of the 

percentage reduction in growth (IA) against the log concentration of the test substance. The 

NOEC was determined as the highest concentration at which no statistically significant inhibition 

was observed. The effect was considered significant if the average inhibition of the growth rate 

 2 The nominal concentrations were reported as 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L in section 2.5.3 and Annex 

D of the study report, but were reported as 0, 3.3, 10, 18, 33, 57, 102, and 326 mg/L in section 2.5.1 of the study 

report.
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was higher than 5%. Statistical significance was determined with a one-tailed t-test (α = 5%).

RESULTS

Endpoint value(s): Based on nominal concentrations reported: ErC50 = 62 mg/L; EbC50 = 33 

mg/L; NOEC (growth rate) = 18 mg/L; LOEC (growth rate) = 32 mg/L

Results remarks: The temperature and light intensity were measured to be 23.9-24.6 °C 

(average of 24.0 °C) and 87-115 μmol/s/m2, respectively. At the start of the test, the pH of each 

flask was found to be 8.4. At the end of the test, the pH range was 8.2-8.3 in the absence of algal 

cells and 8.2-9.1 in the presence of algal cells.

Due to inconsistent results from the chemical analyses of test substance concentrations, the 

measured concentrations in test solutions were not reported by the study authors. Therefore, the 

effect levels were determined using nominal concentrations.

After 70.5 hours of exposure, the mean cell density was 76.6, 80.0, 80.1, 76.3, 34.6, 12.4, 1.8, 

and 1.3 x 104 cells/mL at nominal concentrations of 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L, 

respectively. The mean percent reduction in growth rate was -7, -8, -4, 49, 80, 98, and 99% at 

nominal concentrations of 3.2, 10, 18, 32, 56, 101, and 322 mg/L, respectively. Calculated 

biomass values were 65.8, 65.7, 64.7, 59.4, 36.9, 7.0, 0.8, and 0.3 x 106 μm3/mL at nominal 

concentrations of 3.2, 10, 18, 32, 56, 101, and 322 mg/L, respectively. Microscopic inspection 

of the morphology of algal cells revealed no abnormal cells in treated cultures at the end of the 

test.

Based on nominal concentrations, the ErC50 was found to be 62 mg/L, with a 95% confidence 

interval of 55 to 70 mg/L. The ErC10 and ErC90 were 31 and 120 mg/L, respectively. The EbC50

was found to be 33 mg/L, with a 95% confidence interval of 32 to 56 mg/L. The EbC10 and 

EbC90 were 21 and 79 mg/L, respectively. Based on inhibition of growth rate, the NOEC was 

found to be 18 mg/L.

CONCLUSIONS

A significant reduction in the growth rate of Pseudokirchneriella subcapitata was observed at a 

nominal test substance concentration of 32 mg/L. The EC50 was found to be 62 mg/L based on 

inhibition of growth rate and 33 mg/L based on reduction in biomass.

STUDY RELIABILITY

[3] Not reliable. The study is not reliable for the following reasons: (1) No reliable analytical 

measures of the test substance were obtained during testing. The analytical method attempted

for measuring test substance concentrations during the toxicity test was not appropriate because 

it relied on rapid hydrolysis of the test substance and, therefore, analytical testing using an 
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appropriate method was not conducted;3 (2) the composition of 10-30% of the test substance 

(described as 70-90% pure) is not defined, suggesting that the test substance may contain 

impurities; (3) it is not clear if the nominal concentrations have been adjusted to account for 

purity of the test substance, such that the concentrations of the test substance reported in the 

study may be overestimated by as much as 30%; hence, the EC50s, NOECs, and LOECs may be 

overestimated by as much as 30%; and (4) the definitive test used only two replicates per test 

concentration, whereas OECD Guideline 201 recommends three replicates per test concentration. 

REFERENCE

Oldersma, H. and C.T. Bowmer. 2002. Determination of the effect of KM200 on the growth of 

the freshwater green alga Selenastrum capricornutum (OECD 201 and EU C.3 Guidelines). 

TNO Study Number 02-4064/02. Submitted by Troy Corporation.

 3

Although the study report states that the test substance is unstable in water and, therefore, the test solutions were 

undefined mixtures of the test substance and hydrolysis products from the onset of testing, the U.S. EPA 

Reregistration Eligibility Decision for Grotan (HHT) indicates that the hydrolysis half-life of CASRN 4719-04-4 is 

50 and 302 days at pH 7 and 9, respectively. Therefore, the hydrolysis of the test substance is not expected to be 

rapid under the test conditions described for the algae study. 
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Summary of Endpoints

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Toxicity to Aquatic Plants Yes No Yes