SRC TR-12-032 

HPV3 TOXICITY TO ALGAE TEST PROTOCOL REVIEW 

Benzene, 1-chloro-4-(trifluoromethyl)-, 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 Work Assignment Manager 

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

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HPV3 Toxicity to Algae Test Protocol Review: 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)- (CASRN 98-56-6). The C6 requirement consists of a Toxicity to 

Plants (Algae) Test following ASTM guideline E 1218-04. A protocol for this test, to be 

conducted by CiToxLAB Hungary, Ltd., was submitted by Greenwich Chemical Consulting on 

behalf of the PCBTF (para-chlorobenzotrifluoride) Testing Consortium. The letter from 

Greenwich Chemical Consulting that accompanied this protocol indicated that EPA had granted 

the PCBTF Testing Consortium’s request to conduct this test using OECD test guideline (TG) 

201.1 

The test protocol was reviewed for compliance with OECD TG 201. The physicochemical 

properties of the test substance, which may necessitate special considerations in the aquatic 

testing process, were also considered 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 include the composition of the test substance, including 

identities and concentrations of major ingredients and major impurities. The purity of the 

test substance must be ≥ 99%. 

2. The final study report should provide evidence that the study validity 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 test concentrations (p. 9). 

According to OECD TG 201, “analysis of all test concentrations... is recommended 

where concentrations are unlikely to remain within 80-120% of nominal”. Given that the 

test substance is highly volatile, there is a strong possibility that test concentrations will 

decrease to levels below 80% of nominal. 

4. The test protocol specifies that “[f]or volatile... substances, additional samplings for 

analysis at 24 hour intervals during the exposure period will be used in order to better 

define loss of the test substance” (p. 9). This statement is applicable to the test rule 

chemical, benzene, 1-chloro-4-(trifluoromethyl)-, which has an estimated Henry’s law 

constant of 3.47 x 10-2 atm-m3

/mole (3.52 x 103 Pa-m3/mole),2 indicating that the 

chemical is very volatile. The study schedule in the submitted protocol (p. 16) should be 

revised to indicate that analytical sampling for measurement 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 replicates may be needed to accommodate additional 

samplings for analytical measurements at 24-hour 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

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samplings at 24 and 48 hours after initiation of exposure, two additional replicates will be 

required at each test concentration. 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 headspace and allowing the volatile test substance to 

dissipate. Therefore, two additional replicates are required to accommodate the two 

added analytical sampling times (one each at 24 and 48 hours). The replicate used for the 

72-hour analytical sampling may also be measured for algal cell concentration because 

this sampling occurs at the termination of the exposure period. 

6. The protocol should follow the guidance provided 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 substances. Since Henry’s law constant 

for benzene, 1-chloro-4-(trifluoromethyl)- is estimated to be greater than 100 Pam3

/mole, more than 50% of the substance could be lost from the water phase within 3-4 

hours4

 if the test method is not altered to reduce the loss of test substance due to 

volatilization. OECD recommendations for testing of volatile substances 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 inhibition test will result in culture growth 

being limited by CO2 depletion and increasing pH”. To prevent such limitation of 

algal growth in a sealed test system, the protocol should be modified to provide an 

adequate source of CO2 using the principles presented in the following methods: 

i. The gaseous CO2 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 CO2-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 CO2 while avoiding a headspace (e.g., 

Tjeerdema and Singer, 1991).7

Note that the duration of the algae test must be 72 hours, rather than the 48-hour 

tests used in Mayer et al. (2000) and Halling-Sørensen et al. (1996). To prevent 

CO2 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, Lewis 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., and Baun, A. 1996. Algal toxicity tests with volatile and hazardous 

compounds in air-tight test flasks with CO2 enriched headspace. Chemosphere 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

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increase the sodium bicarbonate concentration. As stated in OECD Series on 

Testing and Assessment Number 23: “Whatever 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 substance should also be performed to confirm 

that the result is consistent with that obtained in a conventional test system.” 

b. Additionally, “test concentrations should, where possible, 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 loss 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 concentrations deviate from either the nominal or initial 

measured concentration by more than 20%, the calculated EC50s should be based on geometric 

mean measured concentrations.