SRC TR-12-032

HPV3 TOXICITY TO ALGAE TEST PROTOCOL REVIEW

Benzene, 1-chloro-4-(trifl uoromethyl)-, CASRN 98-56-6

Prepared by:

Teresa Manyin, William L. Richards, and Gary Diamond

Chemical, Biological and Environmental Center SRC, Inc.
7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP-W-09-027
Task FG007.2.009.006

Submitted to:

OCSPP/OPPT/RAD (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 Wo rk Assignment Manager

March 6, 2012HPV3 Toxicity to Algae Test Protocol Review CASRN 98-56-6

1 HPV3 Toxicity to Algae Test Protocol Revi ew: Benzene, 1-chloro-4 -(trifluoromethyl)-,
CASRN 98-56-6

The test rule, “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 FR 65385, October 21, 2011), requires C6 aquatic toxicity testing for benzene, 1-chloro-4-(trifluoromethyl)- (CAS RN 98-56-6). The C6 requirement consists of a Toxicity to Plants (Algae) Test following ASTM guideline E 1218-04. A protocol fo r this test, to be conducted by CiToxLAB Hungary, Ltd., was subm itted by Greenwich Chemical Consulting on behalf of the PCBTF ( para -chlorobenzotrifluoride) Testing Consortium. The letter from Greenwich Chemical Consulting that accompanied th is protocol indicated that EPA had granted the PCBTF Testing Consortium’s request to conduc t this test using OECD test guideline (TG)
201. 1

The test protocol was reviewed for complia nce with OECD TG 201. The physicochemical properties of the test substance, which may neces sitate special considerations in the aquatic testing process, were also c onsidered in this evaluation.

The submitted protocol, “Growth Inhibition Test with Benzene, 1-chloro-4-(trifluoromethyl)- on Algae ( Pseudokirchneriella subcapitata )”, is considered adequate to satisfy the algae toxicity testing requirement with the following stipulations:

1. The final study report should in clude the composition of the test substance, including identities and concentrations of major ingred ients and major impurities. The purity of the
test substance must be ≥ 99%.
2. The final study report should pr ovide evidence that the study va lidity criteria, specified in section 1.2 of the submitted protocol (p. 5), were satisfied.
3. Analytical measurements of test substance concentration should be conducted for each test concentration, rather than only the highest and lowest te st concentrations (p. 9). According to OECD TG 201, “analysis of a ll test concentrations... is recommended where concentrations are unlikely to remain within 80-120% of nomina l”. Given that the test substance is highly volatile, there is a st rong possibility that test concentrations will
decrease to levels be low 80% of nominal.
4. The test protocol specifies that “[f]or vol atile... substances, add itional samplings for analysis at 24 hour intervals during the expos ure period will be used in order to better
define loss of the test substanc e” (p. 9). This statement is applicable to the test rule chemical, benzene, 1-chloro-4-(trifluoromet hyl)-, which has an estimated Henry’s law
constant of 3.47 x 10 -2 atm-m 3 /mole (3.52 x 10 3 Pa-m 3 /mole), 2 indicating that the chemical is very volatile. The study schedul e in the submitted protocol (p. 16) should be
revised to indicate that an alytical sampling for measur ement of test substance concentrations will also occur on days 1 and 2 (i.e., at 24 and 48 hours after initiation of exposure), rather than only on days 0 and 3.
5. OECD TG 201 indicates that extra replicat es may be needed to accommodate additional samplings for analytical measurements at 24-h our intervals. To allow for the additional

1 Request granted via email from P. Campanella to J. Massingale on February 9, 2012.

2 Henry’s law constant estimated using HenryWin v3.20 (Bond method) in EPI Suite v4.10.HPV3 Toxicity to Algae Test Protocol Review CASRN 98-56-6

2 samplings at 24 and 48 hours afte r initiation of expos ure, two additional replicates will be required at each test concentr ation. Only one replicate at each test concentration is required for analytical measurement at each sampling time; however, the replicate must be discarded after analytical sampling occurs because the removal of a water sample will disrupt the test system by increasing headsp ace and allowing the volatile test substance to dissipate. Therefore, two additional repl icates are required to accommodate the two added analytical sampling times (one each at 24 and 48 hours). The re plicate used for the 72-hour analytical sampling may also be meas ured for algal cell concentration because this sampling occurs at the termination of the exposure period.
6. The protocol should follow the guidance pr ovided in OECD Series on Testing and
Assessment Number 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 3 Section 3.4: Volatile substan ces. Since Henry’s law constant for benzene, 1-chloro-4-(trifluoromethyl)- is estimated to be greater than 100 Pam 3 /mole, more than 50% of the substance coul d be lost from the water phase within 3-4
hours 4 if the test method is not altered to reduce the loss of test substance due to
volatilization. OECD recommendations for tes ting of volatile subs tances include the following:

a. “As a general rule test vessels should be sealed during preparation and exposure and the headspace be kept to a minimum.” However, the “use of a sealed exposure system in the algal growth inhi bition test will result in culture growth being limited by CO 2 depletion and increasing pH”. To prevent such limitation of algal growth in a sealed test system, the protocol should be modi fied to provide an adequate source of CO 2 using the principles presen ted in the following methods:
i. The gaseous CO 2 can be replaced by adding additional sodium bicarbonate to the medium (e.g., Mayer et al., 2000). 5 If this method is used, the inoculum cell density should be reduced and pH should be adjusted to 7.0
with HCl before initiation of the test.
ii. A CO 2 -enriched headspace can be used (e.g., Halling-Sørensen et al., 1996) 6

. iii. The test can be conducted in a flow-through system capable of maintaining the supply of CO 2 while avoiding a headspace (e.g., Tjeerdema and Singer, 1991). 7

Note that the duration of th e algae test must be 72 hours, rather than the 48-hour tests used in Mayer et al. (2000) and Ha lling-Sørensen et al. (1996). To prevent
CO 2 depletion, it may be necessary to further reduce inoculum cell density and/or

3 Organisation for Economic Co-operation and Development, Environment Directorate, Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology. ENV/JM/MONO(2000)6. December 15, 2000.

4 Mackay, D. 1992. Multimedia Environmental Models, Lewi s Publishers (as cited in OECD Series on Testing and Assessment Number 23).

5 Mayer, P., Nyholm, N., Verbruggen, E.M.J., Hermens, J.L.M ., and Tolls, J. 2000. Algal growth inhibition test in filled, closed bottles for volatile and sorptive materials. Environmental Toxicology and Chemistry 19: 2551-2556.

6 Halling-Sørensen, B., Nyholm, N., an d Baun, A. 1996. Algal toxicity tests with volatile and hazardous compounds in air-tight test flasks with CO 2 enriched headspace. Ch emosphere 32: 1513-1526.

7 Tjeerdema, R.S., and Singer, M.M. 1991. Closed flow-through aquatic toxicity testing and microscopic organisms: Not necessarily incompatible. Marine Pollution Bulletin 22: 59-61.HPV3 Toxicity to Algae Test Protocol Review CASRN 98-56-6

3 increase the sodium bicarbonate concentr ation. As stated in OECD Series on Testing and Assessment Number 23: “Whateve r system is adopted it is advisable to establish that acceptable control culture growth can be achieved before starting
the test. A test with a reference substa nce should also be performed to confirm
that the result is consistent with that obtained in a conventional test system.”
b. Additionally, “test concen trations should, where po ssible, be prepared individually by addition of test substance directly to the test vessels rather than by
dilution of a stock solution.”

Regardless of the methods employed to reduce lo ss of test substance due to volatilization, analytical measurements should be conducted on samples obtained at 24-hour intervals. As stated in OECD TG 201, if the measured concentra tions deviate from either the nominal or initial measured concentration by more than 20%, the calculated EC 50 s should be based on geometric
mean measured concentrations.