SRC TR

- 1 4

- 006

HPV 3 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 .009.00 6

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 , 201 4HPV3 Toxicity to Algae Study Plan Review ( Final ) CASRN 98
- 56
- 6

1 HPV 3 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 C 6 aquatic toxicity testing for b enzene, 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 ε1 . A study plan for this test , to be conducted by Harlan Laboratories , Ltd., was submitted on January 9, 2014 by Greenwich Chemical Consu lting , 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) 201 1

. 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) 2 that necessitate special modifications to the aquatic toxicity testing process.
Th e submitted study plan , “Benzene, 1

- c hloro

- 4

- (trifluoromethyl)

- , CASRN 98

- 56

- 6, para Chlorobenzotrifluoride (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. S ection 1.1 ( Guidelines/Regulations , p. 7) of the s tudy p lan 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 S ection 1.1 of the study plan and must be cited in t he 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 a n 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 M arch 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 Prot ection 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

2 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 a n initial (time = 0 hours) measured test concentration of 27.4 mg/L for the highest nominal concentration of 40.0 mg/L 4

. F or 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 EC 50 is less than the solubility limit (or 100 mg/L , whichever is lower )

. S ection 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 wate r. 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 headspac e. The study plan (Section 3.5, Materials, p. 10) indicate s 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 h as 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 i s 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 CO 2 . Section 3.7 ( Study Design , p. 11) of the study plan indicates that the initial cell density will be 2

- 5 x 10 3 cells/mL. EPA recommends an initial density of no more than 3 x 10 3 cells/mL

. b. Algal growth media must be enriched w ith additional sodium bicarbonate to provide a source of CO 2 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 co ncentration. EPA recommends that at least 300 mg NaHCO 3 /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 M arch 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 m g/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 volatiliza tion of this test substance.HPV3 Toxicity to Algae Study Plan Review ( Final ) CASRN 98
- 56
- 6

3 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 co nfirm 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 proced ures 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 cond ucted 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 pl an. 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 th e 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=EPA

HQ

- OPPT

- 2009

- 0112

- 0190 as of March 6 , 2014.HPV3 Toxicity to Algae Study Plan Review ( Final ) CASRN 98
- 56
- 6

4 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 replicate s 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 E1218 ε1 ) and (2) to provide information describing the loss of the test substance from solution (as recommended for volatile test substances i n 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 “e lectronic 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 in dicates that at least five concentrations of CASRN 98

- 56

- 6 will be tested , whereas S ection 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 t est item dispersion of 100 mg/L ” and does not indicate the concentrations that will be tested or how these concentrations w ill be prepared

. OECD (2000) ( S ection 3.4, Volatile S ubstances, p. 27) states that test concentrations [of volatile substances] should “be prepared individuall y 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 9 8

- 56

- 6 for testing shall be prepared in different vessels and the final study report shall modify the language of S ection 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 satu rated solution as already described in paragraph 3 of S ection 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 solutio n (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 mini mize 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 o f 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

5 9. S ection 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 vola tilization 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 te st substance , c ould be considered. However, u nless the testing laboratory can provide evidence that CASRN 98

- 56

- 6 is not lost during filtration by adsorp tion to the filter matrix, the removal of excess undissolved test item shall be conducted by centrifugation and the centrifugation con tainers shall have no headspace to minimize loss of test substance by volatilization. The final study report mu st provide a justification for, or validation of , the separation technique that include s descriptions o f 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. S ection 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 tightly

sealed vessels with no headspace. M easures 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 concentr ations of test substance diminish prior to analysis ; evidence of this validation shall be included in the final study repo rt.
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 Co

operation and Developmental, ENV/JM/MONO(2000)6. Available at http://www.oecd

ilibrary.org/environment/guidance

- document

- on
- aquatic

- toxicity

- testing

- of

- difficult

- substances

and

- mixtu res_9789264078406

- en as of March 6 , 2014. “Study Plan: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- , CASRN 98

- 56

- 6, para chlorobenzotrifluoride (PCBTF): Algal Growth Inhibition Test .” Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41304731. Sp onsor: PCBTF Testing Consortium, c/oHPV3 Toxicity to Algae Study Plan Review ( Final ) CASRN 98
- 56
- 6

6 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.