SRC TR-14-006

HPV3 TOXICITY TO ALGAE STUDY PLAN REVIEW (FINAL)

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

Prepared by:

Amy Nguyen, Jennifer Rayner, Teresa Manyin, and William L. Richards

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

March 6, 2014
HPV3 Toxicity to Algae Study Plan Review (Final) CASRN 98-56-6

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HPV3 Toxicity to Algae Study Plan 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 1218-04ε1. A study plan for this test, to be 

conducted by Harlan Laboratories, Ltd., was submitted on January 9, 2014 by Greenwich 

Chemical Consulting, Inc. on behalf of the PCBTF (para-chlorobenzotrifluoride) Testing 

Consortium. EPA previously granted the PCBTF Testing Consortium’s request to conduct the 

test using OECD test guideline (TG) 2011.

The study plan was reviewed for compliance with OECD TG 201. Recommendations outlined 

in OECD Series on Testing and Assessment Number 23: Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures (OECD, 2000) were also considered 

because the test item has physicochemical properties (i.e., high volatility and relatively low water 

solubility)2that necessitate special modifications to the aquatic toxicity testing process.

The submitted study plan, “Benzene, 1-chloro-4-(trifluoromethyl)-, CASRN 98-56-6, paraChlorobenzotrifluoride (PCBTF): Algal Growth Inhibition Test”, is considered adequate to 

satisfy the toxicity to algae testing requirement with the following stipulations and modifications:

1. The final study report must 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. Section 1.1 (Guidelines/Regulations, p. 7) of the study plan states that the test methods 

are designed to be compatible with OECD TG 201 and Method C.3 of Commission 

Regulation (EC) No. 761/2009. Since the principles and procedures of OECD (2000) 

need to be followed in several sections of this study plan (e.g., for minimizing 

volatilization of test substance during test solution preparation, exposure, and sampling),

this guideline should have been cited in Section 1.1 of the study plan and must be cited in 

the final study report. 

3. The test substance has a low solubility limit in water (29 mg/L at 25°C)2. A previous 

toxicity to algae study for CASRN 98-56-6, submitted by the PCBTF Testing 

Consortium, reported an initial (time = 0 hours) measured test concentration of 7.04 mg/L 

 

1 Request granted via email from P. Campanella (EPA) to J. Massingale (Greenwich Chemical Consulting) on 

February 9, 2012 (available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-

0174 as of March 6, 2014).

2 CASRN 98-56-6 is highly volatile, as indicated by an estimated Henry’s Law Constant of 3.47 x 10-2

atm-m

3

/mole 

at 25°C (U.S. EPA. 2012. Estimation Programs Interface SuiteTM (EPI Suite) for Microsoft® Windows, v4.11. U.S. 

Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/

exposure/pubs/episuitedl.htm as of March 6, 2014). The water solubility of CASRN 98-56-6 is 29 mg/L at 25°C 

(The Merck Index – An Encyclopedia of Chemicals, Drugs, and Biologicals. 2006. Edited by M.J. O’Neil. 

Whitehouse Station, NJ: Merck and Co., Inc., p. 352. As cited by the U.S. National Library of Medicine’s 

Hazardous Substances Data Bank (HSDB)).
HPV3 Toxicity to Algae Study Plan Review (Final) CASRN 98-56-6

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for a “saturated solution” 3. Another toxicity to algae study for CASRN 98-56-6, 

submitted by Finchimica S.p.A. on behalf of KOWA American Corporation (a member 

of the PCBTF Testing Consortium), reported an initial (time = 0 hours) measured test 

concentration of 27.4 mg/L for the highest nominal concentration of 40.0 mg/L4. For a 

toxicity to algae study to be considered adequate, EPA requires that concentrations up to 

the limit of solubility of the test item (or a limit concentration of 100 mg/L, whichever is 

lower) must be tested if the EC50 is less than the solubility limit (or 100 mg/L, whichever 

is lower). Section 3.4 (Dosage, p. 10) of the study plan states that pre-experiments will 

be performed to determine the solubility and stability of the test item in the test water. 

The final study report must include data from the pre-experiments on the solubility and 

stability of the test item in the test water. This is particularly important for solubility 

since these measurements will demonstrate whether the test concentrations reached the 

limit of solubility in the test water.

4. EPA agrees that the test should be performed in a closed system with no headspace. The 

study plan (Section 3.5, Materials, p. 10) indicates that glass stoppers will be used to seal 

test vessels; however, this type of stopper does not necessarily form an airtight seal. EPA 

recommends the use of a method for sealing the vessels that has been shown to be 

airtight. For example, Mayer et al. (2000) sealed test vessels with Teflon®-faced silicone 

septum screw caps. Preliminary tests should be conducted to demonstrate that CASRN 

98-56-6 loss is minimized when the selected sealing method is used.5 

5. To successfully conduct an algal toxicity test in a closed system, several modifications to 

standard guideline methods (i.e., OECD TG 201) must be made, as described by Mayer et 

al. (2000):

a. The initial algal cell density must be reduced to decrease the demand for CO2. 

Section 3.7 (Study Design, p. 11) of the study plan indicates that the initial cell 

density will be 2-5 x 103cells/mL. EPA recommends an initial density of no 

more than 3 x 103cells/mL.

b. Algal growth media must be enriched with additional sodium bicarbonate to 

provide a source of CO2 for algal growth. Appendix 1 of the study plan indicates 

that the final concentration of sodium bicarbonate in the algal growth media will 

be “up to 500 mg/L”, but does not indicate a minimum concentration. EPA 

recommends that at least 300 mg NaHCO3/L be added to algal growth medium.

 

3

Sipos, K. 2013. Growth inhibition test with benzene, 1-chloro-4-(trifluoromethyl)- on algae (Pseudokirchneriella 

subcapitata). Performing Laboratory: CiToxLAB Hungary Ltd., Veszprém, Szabadságpuszta, Hungary. Study No. 

12/056-022AL. Sponsor: PCBTF Testing Consortium, c/o Greenwich Chemical Consulting, Palm Beach Gardens, 

FL. (Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-

0141 as of March 6, 2014).

4 Noe, F., Dini, R., and Desiree, G. 2013. 4-Chloro-α,α,α-trifluorotoluene (PCBTF): Toxicity to green algae 

Pseudokirchneriella subcapitata determined in a growth inhibition study. ChemService S.r.l. Controlli e Ricerche, 

Novate Milanese, Italy. Study No. CH-095/2013. Sponsor: Finchimica S.p.A., Manerbio, Italy. (Study report 

available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0224 as of March 

6, 2014.)

5

In the previously submitted toxicity to algae study by Noe et al. (2013), in which no precautions were taken to 

prevent volatilization during the exposure period, concentrations of CASRN 98-56-6 at all nominal test 

concentrations had declined to undetectable levels within the first 24 hours of the exposure period and were below 

the analytical limit of detection (0.1009 mg/L) for the remainder of the test. These results demonstrate the highly 

volatile nature of CASRN 98-56-6 and reinforce the importance of implementing appropriate procedures to 

minimize volatilization of this test substance.
HPV3 Toxicity to Algae Study Plan Review (Final) CASRN 98-56-6

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c. The pH of the algal growth media must be adjusted to 7.0 after the additional 

sodium bicarbonate is added to the media. Appendix 1 of the study plan indicates 

that the pH will be adjusted to 7.5 prior to adding sodium bicarbonate. This 

procedure must be modified such that pH is adjusted to 7.0 after adding sodium 

bicarbonate.

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

used by Mayer et al. (2000). According to OECD (2000) (Section 3.4, p. 28), regarding 

the use of modified procedures for volatile substances, “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 system. The test result 

should be reported with the reference substance.” Therefore, a preliminary test to 

establish acceptable control culture growth using the modified test procedures should be 

performed prior to the study. In addition, a preliminary test of the reference chemical to 

validate the modified test procedures is recommended prior to the preliminary and 

definitive tests of the test rule chemical. 

6. EPA previously proposed a separate set of replicates, to be used for analysis of cell 

counts and analytical measurement of the test item concentration, for each observation 

time point (i.e., 24, 48, and 72 hours).6 In the cover letter from Greenwich Chemical 

Consulting, Harlan Laboratories comments that “The use of the additional replicates that 

EPA suggests would mean that in the case of a full test there would be 12 replicate 

vessels per test concentration (and control) i.e. a total of 72 vessels. If the test was 

conducted as a limit test then the total number of vessels would still be 48. This would 

make the test too cumbersome and prone to error...” Harlan suggested a compromise, 

although Harlan’s proposed study design was not clearly stated in the submitted study 

plan. To reduce the number of replicate test vessels, EPA would consider it acceptable to 

reduce the number of replicates to 5 per test concentration:

a. One replicate will be kept sealed (with no prior sampling of test solutions) until 24 

hours, at which time samples will be taken for both analysis of cell counts and test 

item concentration; this replicate will then be discarded.

b. One replicate will be kept sealed (with no prior sampling of test solutions) until 48 

hours, at which time samples will be taken for both analysis of cell counts and test 

item concentration; this replicate will then be discarded.

c. Three replicates will be kept sealed (with no prior sampling of test solutions) until 72 

hours, at which time samples will be taken for both analysis of cell counts and test 

item concentration.

Therefore, each of the replicates containing treated test solutions will be used only once 

for sampling before being discarded, because the process of removing a sample of test 

solution is expected to allow release of the highly volatile test substance from the test 

vessels. 

 

6

“HPV3 Test Rule Data Adequacy Review (Revised Final): Toxicity To Algae, Benzene, 1-chloro-4-

(trifluoromethyl)-, CASRN 98-56-6”, available online at http://www.regulations.gov/#!documentDetail;D=EPAHQ-OPPT-2009-0112-0190 as of March 6, 2014.
HPV3 Toxicity to Algae Study Plan Review (Final) CASRN 98-56-6

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For the negative control, 9 replicates should be used:

a. Three replicates will be kept sealed (with no prior sampling of test solutions) until 24 

hours, at which time samples will be taken for analysis of cell counts; these replicates 

will then be discarded.

b. Three replicates will be kept sealed (with no prior sampling of test solutions) until 48 

hours, at which time samples will be taken for analysis of cell counts; these replicates 

will then be discarded.

c. Three replicates will be kept sealed (with no prior sampling of test solutions) until 72 

hours, at which time samples will be taken for both analysis of cell counts and test 

item concentration.

Samples collected at 24 and 48 hours will be used to (1) monitor the section-by-section 

specific growth rates (as recommended in OECD TG 201 and ASTM E1218ε1) and (2) to 

provide information describing the loss of the test substance from solution (as 

recommended for volatile test substances in item 37 of OECD TG 201). If a limit test is 

conducted, the number of replicates sampled at 72 hours for the limit concentration and 

negative control would be increased from three to six. EPA recommends that cell 

densities at 0 hours be determined by “electronic particle counting of the preculture and 

division by the dilution factor”, as described by Mayer et al. (2000); therefore, additional 

replicates would not be necessary for sampling at 0 hours.

7. Section 3.7 (Study Design, p. 11) of the study plan indicates that at least five 

concentrations of CASRN 98-56-6 will be tested, whereas Section 3.4 (Dosage, p. 10) of 

the study plan states only that “The test item solution will be prepared as a saturated 

solution using an initial test item dispersion of 100 mg/L” and does not indicate the 

concentrations that will be tested or how these concentrations will be prepared. OECD 

(2000) (Section 3.4, Volatile Substances, p. 27) states that test concentrations [of volatile 

substances] should “be prepared individually by addition of test substance directly to the 

test vessels rather than by dilution of a stock solution.” To be compatible with this 

guidance from OECD (2000), each of the “at least five” concentrations of CASRN 98-56-

6 for testing shall be prepared in different vessels and the final study report shall modify

the language of Section 3.4 of the study plan to describe the preparation of different test 

solution concentrations in different vessels. The highest test concentration should be 

prepared as a saturated solution as already described in paragraph 3 of Section 3.4 of the 

study plan. The target values of lower test concentrations should be based on the 

measured concentration of test substance in the saturated solution (after undissolved test 

substance has been removed) in combination with experimental data already available on 

the solubility of the test substance in test water (see item 3 of this study plan review).

8. The study plan does not describe the equipment and procedures that will be used to 

minimize loss of test substance by volatilization during the steps required to prepare 

various concentrations of CASRN 98-56-6 in test vessels into which algae will be 

inoculated. Section 3.4 of OECD (2000) recommends the use of sealed vessels with 

minimal or zero headspace during test solution preparation and the use of syringe pumps 

to dispense solutions of volatile substances into sealed test vessels. The final study report 

shall describe the methods and equipment that are used to minimize volatilization of test 

substance during the preparation of test solutions, transfer of test solutions to test vessels, 

and inoculation of the vessels with algae suspensions.
HPV3 Toxicity to Algae Study Plan Review (Final) CASRN 98-56-6

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9. Section 3.4 (Dosage, p. 10) of the study plan indicates that “Pre-experiments will be 

performed to determine... the suitability of either filtration or centrifugation to remove 

excess undissolved test item” but does not indicate the precautions that will be taken to 

minimize test substance loss by volatilization during the process of removal of 

undissolved test substance. OECD (2000) (Section 3.1.1, pp. 20-21) recommends the use 

of centrifugation for this purpose since “[f]iltration through a membrane filter... [is] less 

widely advocated because of the potential for losses due to adsorption onto the filter 

matrix.” If filtration were to be used, Section 3.1.1 of OECD (2000) indicates that 

methods to prevent or reduce adsorption to the filters, such as preconditioning the filters

with solutions of the test substance, could be considered. However, unless the testing 

laboratory can provide evidence that CASRN 98-56-6 is not lost during filtration by 

adsorption to the filter matrix, the removal of excess undissolved test item shall be 

conducted by centrifugation and the centrifugation containers shall have no headspace to 

minimize loss of test substance by volatilization. The final study report must provide a 

justification for, or validation of, the separation technique that includes descriptions of

the methods and equipment that were used to minimize loss of test substance from 

solution during the removal of excess undissolved test item from the saturated solution.

10. Section 3.8.3.2 (Storage and Sample Preparation, p. 13) of the study plan states that 

samples will be analyzed immediately or stored and, if the test substance is unstable, 

measures will be taken to maintain stability until analysis. Given that CASRN 98-56-6 is 

highly volatile, EPA notes that sampling and pooling of test solutions for analysis of test 

substance concentration must be conducted using methods and equipment that will 

minimize loss of the test substance from solution. Samples must be collected into tightlysealed vessels with no headspace. Measures to maintain test substance stability and 

minimize volatility during collection, transfer, storage, and processing (including removal 

of algae by centrifugation prior to analysis) of samples shall be documented in the final 

study report. Also, methods of sample collection and storage shall be validated, as 

recommended by Section 4.2, p. 42 of OECD (2000), to show whether or not 

concentrations of test substance diminish prior to analysis; evidence of this validation 

shall be included in the final study report.

REFERENCES

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. 

OECD. 2000. “OECD Series on Testing and Assessment Number 23: Guidance document on 

aquatic toxicity testing of difficult substances and mixtures”. Organisation for Economic Cooperation and Developmental, ENV/JM/MONO(2000)6. Available at http://www.oecdilibrary.org/environment/guidance-document-on-aquatic-toxicity-testing-of-difficult-substancesand-mixtures_9789264078406-en as of March 6, 2014.

“Study Plan: Benzene, 1-chloro-4-(trifluoromethyl)-, CASRN 98-56-6, parachlorobenzotrifluoride (PCBTF): Algal Growth Inhibition Test.” Harlan Laboratories Ltd.,

Shardlow, Derbyshire, UK. Study No. 41304731. Sponsor: PCBTF Testing Consortium, c/o 
HPV3 Toxicity to Algae Study Plan Review (Final) CASRN 98-56-6

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Greenwich Chemical Consulting, Palm Beach Gardens, FL. Available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0226 as of March 

6, 2014.