SRC TR

- 11

- 019

HPV 2 TEST RULE DATA ADEQUACY REVIEW

1,3,5

- Tr ia zine

- 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 .009.00 1

Submitted to:

OPPT/RAD/HPVCB (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan , Project Offic er Meena Sonawane, Work Assignment Manager David Brooks, Alternate Work Assignment Manager

February 25 , 201 12 HPV 2 Test Rule Data Adequacy Review

1,3,5

- Tr ia zine

- 1,3,5(2H,4H,6H)

- triethanol, CAS No. 4719

- 04

- 4 : Toxicity to Aquatic Plant Study

February 201 1

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

2 451 Crystal Drive, Suite 804 Arlington, VA 222023 TOXICITY TO AQUATIC PLANTS

Summary of endpoint: Ina dequate

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 substanc e 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 informa tion 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

- Tr ia zine

- 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

GL P 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 monitorin g: No (Analytical monitoring was attempted

, but results were not reported.) 1

Exposure duration: 7 0.5 hours

1 Analytical monitoring was attempted by the study authors. However, d ue 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.4 Concentrations tested: Nominal: 0, 3. 2 , 10, 18, 3 2 , 5 6 , 10 1 , and 3 22 mg/L 2 ; measured concentrations were not reported . However, no indication i s 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 p ure water and was sterilized by micropore filtration. The prepared medium contained 150 mg NaHCO 3 /L and had a hardness of 24.3 mg CaCO 3 /L

. Test solutions were prepared in medium at nominal concentrations of 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L.

A t test initiation, a n inoculum of 1.0 mL of an algal suspension containing 8 x 10 5 cells/mL was

added to 200

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

10 3 cells/mL. The test was carried out in duplicate

, with fo ur 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/m 2 (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 u sed 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 wer e 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 fluorogenical ly labeled with a mixed o

- phtalaldehyde/mercaptoethanol reagent before HPLC analysis.

The EC values with respect to growth rate (E

r C 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 (E b C 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 (I A ) 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 stud y 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.5 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 : E

r C 50 = 62 mg/L; E b C 50 = 33 mg/L; NOEC (growth rate) = 18 mg/L ; LOEC (growth rate) = 32 mg/L

Results rem arks: The temperature and light intensity were measured to be 23.9

- 24.6 °C (average of 24.0 °C) and 87

- 115 μmol/s/m 2 , 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 t he 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 , t he mean cell density was 76.6, 80.0, 80.1, 76.3, 34.6, 12.4, 1.8,

and 1.3 x 10 4 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 10 6 μm 3 /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 concentrati ons, t he E

r C 50 was found to be 62 mg/L, with a 95% confidence interval of 55 to 70 mg/L. The E

r C 10 and E

r C 90 were 31 and 120 mg/L, respectively. The E b C 50 was found to be 33 mg/L, with a 95% confidence interval of 32 to 56 mg/L. The E b C 10 and

E b C 90 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 m g/L. The EC 50 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 tes t 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 test ing using an6 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 concentratio ns have been adjusted to account for purity of the test substance, such that the concentration s of the test substance reported in the study may be over estimated by as much as 30% ; hence, the EC 50 s , NOECs, and LOECs may be overestimated by as much as 30%; a nd ( 4 ) the definitive test used only two replicates per test concentration, whereas OECD Guideline 201 recommends three replicates per test concentration

.

REFERENCE

Ol d ersma, H. and C.T. Bowm er. 2002. Determination of the effect of KM200 on the g row th of

the f reshwater g reen a lga Selenastrum capricornutum (OECD 201 and EU C.3 Guidelines).

TNO Study Number 02

- 4064/02. S ubmitted 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 . T herefore, the h ydrolysis of the test substance is not expected to be rapid under the test conditions described for the algae study.7 Summary of Endpoints

Endpoint Study Information

Available Acceptable Data Gap

Aquatic Effects

Toxici ty to Aquatic Plants Yes No Yes