SRC TR

- 13

- 051

HPV 3 TEST RULE DATA ADEQUACY REVIEW ( REVISED FINAL ) :

TOXICITY TO ALGAE

Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- , CASRN 98

- 56

- 6

Prepared by:

Amy Nguyen, Julie Klotzbach, William Richards, and Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027 Task FG007.3.009.006

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

August 9 , 2013HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

i TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT

................................ .............................. 1
B. EVALUATING DATA ADEQUACY

................................ ................................ ..................... 1

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

........ 2

D. DATA FOR SIDS ENDPOINTS

................................ ................................ .............................. 5

D

- 1. Toxicity to Aquatic Plants

................................ ................................ ................................ . 5HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission (dated April 18, 2013 ) from Greenwich Chemical Consulting, Inc. on behalf of the PCBTF ( para

- chlorobenzotrifluoride) Testing Consortium that includes data from toxicity testing of b enzene, 1

- chloro

- 4

- (trifluoromethyl)

- (CASRN 98

- 56

- 6). These data were received by EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 FR 65 385 , October 21, 2011; referred to herein as HPV3; available online at http://www.regulations.gov/#!documentDetail; D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0086 as of July 15 , 2013 )

. The HPV3 test rule requires C6 aquatic toxicity testing for CASRN 98

- 56

- 6. The C6 requirement consists of toxicity to plants (algae) testing. No other toxicity testing for CASRN 98

- 56

- 6 is required by the HPV3 test rule.

A study plan for a toxicity to algae test was submitted by Greenwich Chemical Consulting on behalf of the PCBTF Testing Consortium on February 17, 2012 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0108 as of August 9, 2 013). EPA reviewed the submitted study plan and provided comments and recommendations for conducting an acceptable study to the sponsor

.

In its April 18, 2013 submission, Greenwich Chemical Consulting, Inc. supplied data on behalf of the PCBTF Testing Consortium from a toxicity to algae test

. The study report reviewed herein, Growth Inhibition Test with Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- on Algae ( Pseudokirchneriella subcapitata ), is available online at http://www.regulations.gov/#!document Detail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0141 as of July 15 , 2013.

These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this rep ort.

B. EVALUATING DATA ADEQUACY

The submitted data are evaluat ed for adequacy in Section D. The study was summarized according to a standard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether the data were adequate to satisfy the C6 testing requirement for CASRN 98

- 56

- 6, study details were compared to OECD TG 201

. 1 Recommendations outlined in OECD Series on Testing and Assessment Number 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000)

w ere also considered because the test item has physicochemical properties (i.e., high volatility and rela tively low water solubility) 2 that necessitate special considerations in the aquatic testing
process.

1 Although the HPV3 test rule specifies that algae toxicity testing should be conducted according to ASTM guideline E 1218

- 04 ε1 , EPA previously granted a request from the PCBTF Testing Consortium to follow OECD TG 201 instead (request granted via email from P. Campanella to J. Massingale on February 9, 2012).
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 July 16, 2013). The wat er solubility of CASRN 98

- 56

- 6 is 29 mg/L at 25°CHPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

2 C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

The study submitted by Greenwich Chemical Consulting, Inc

. on behalf of the PCBTF Testing Consortium in the submission dated April 18, 2013 is in adequate to satisfy the algae toxicity (C6) test ing requirement promulgated in the final HPV3 test rule for CASRN 98

- 56

- 6

. As a result, a revised toxicity to algae test needs to be conducted, using appropriate methods to prevent the deficiencies discussed below.

The study submitted by Greenwich Chemical Consulting, Inc. is considered inadequate for the following reasons :

(1) The test was not conducted at the solubility lim it of the test item

. The measured concentration of test item of 7.04 mg/L at the start of exposure is approximately 4

- fold lower than the reported water solubility of 29 mg/L at 25 ° C 1

. A significant portion of the test item appears to have been lost during preparation of the limit test solution , which had a nominal loading rate of 100 mg/L. Loss of test item may have been due to the contribution of several factors, including the following:

a. I ncubation of the stock solution at 30 ° C for 24 hours p rior to equilibration for 24 hours at 20 ° C

. By elevating the temperature to 30 ° C, the rate of volatilization of the test substance became increased. To reduce the risk of volatilization, EPA recommends that the stock solution be prepared and equilibrated at the same temperature that is used during the algae test (22

- 23 ° C). b. The presence of possible headspace in the mixing flask. The amount of test item lost to volatilization is expected to be directly proportional to the amount of headspace in the flask. The study report did not specify the headspace volume. EPA recommends that headspace be minimized in the mixing flask.

c. Prolonged or vigorous shaking of the mixing flask

. Agitation of a solution encourages volatilization. EPA suggests that the testing lab consider stirring, rather than shaking, the stock solution during the mixing process. Alternatively, if the lab uses a shaking method again, EPA suggests a low

- energy shaking method, rather than vigorous shaking. d. Exposure to air during filtration of the test solution. The study report did not indicate whether the solution was exposed to air during filtration; this would have provided another opportunity for loss of tes t substance by volatilization. Unless the testing lab cannot prevent exposure to air during filtration, EPA suggests that the lab consider using centrifugation to separate the undissolved test substance from the water fraction. Headspace in centrifuge co ntainers should be minimal

. e. The study report did not detail the method by which the mixing flask was sealed. The testing lab should ensure that the mixing flask is tightly sealed and that the seal is not permeable to air.

( 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 Medicin e’s Hazardous Substances Data Bank (HSDB)).
1 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 H azardous Substances Data Bank (HSDB).HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

3 Regardless of the methods chosen by the testing lab for preparing the test item solution, the final concentration of the saturated solution should be approximately 29 mg/L ; the testing lab should conduct analytical measurement of the test substance concentration in the stock solution to c onfirm that maximum water solubility has been achieved . It is important that the testing lab validates its methods for achieving a saturated test solution before algae toxicity testing is initiated.

(2) The study report states that the study was performed using a closed system in accordance with OECD recommendations for highly volatile substances. As stated in OECD Series on Testing and Assessment Number 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, “As a general r ule vessels should be sealed during preparation and exposure and the headspace kept to a minimum.” This guidance

was also specified in EPA’s review of the sponsor’s study plan.

However, the test vessel stopper s used to prevent volatilization loss w ere described in the study report as “air

- permeable”. Therefore, the test system was not closed, allowing significant volatilization loss over the course of the exposure period. Loss of test item from the test vessels is demonstrated by the decline in measu red concentrations , which

were 7.04, 3.00, 3.15, and 1.26 mg/L at 0, 24, 48, and 72 hours , respectively

. The
p ercentage of test item lost was approximately 57, 55, and 82 % at 24, 48, and 72 hours, respectively, relative to time 0. The testing lab should use stoppers that are not permeable to air in order to limit test item losses through volatilization.

It is also possible that some of the test item was lost to volatilization if the test solutions were exposed to air during sampling of algae for cell c ounts at 24 and 48 hours. Unless the testing lab is able to obtain a small sample of algae for determination of cell counts without opening the test vessels and exposing the test solutions to air, the number of replicates should be increased to allow for 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). For a limit test, measurements of cell density from 6 replicates are necessary at each time point. For a full (not a limit) test,
measurements of cell density from 3 replicates at each test concentration are necessary at each time point.

(3) The report is m issing important experimental information including detailed methods for preparation of the test item solution, results of statistical analyses of data at 24 and 48 hours, and an explicit description of the “modified” test methods used for rang e

- find ing
st udy 2. This additional information should be included in the study report. A reduction in algal biomass of 14%, relative to controls, was observed in the limit test solution at 24 hours; although the study report does not indicate whether this reduction is statistically significant, the magnitude of th e observed reduction suggests that the test substance has toxic effects, which are expected to be come more apparent when the test is repeated at the water solubility limit.HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

4 In conclusion , the submitter needs to repeat the toxicity to aquatic plants (algae) test, implementing correct procedures and reporting as described above to avoid the noted deficiencies.HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

5 D. DATA FOR SIDS ENDPOINTS

D

- 1 . Toxicity to Aquatic Plants

Summary of endpoint: Not adequate

Study 1: Toxicity to Pseudokirchneriella subcapitata ( Sipos , 2013)

Title: Growth inhibition test with benzene, 1

- chloro

- 4

- (trifluoromethyl)

- on algae Pseudokirchneriella subcapitata )

This study is: Adequate Not Adequate

This study is considered in adequate for the following reasons: (1) The test was not conducted at the solubility limit. The measured concentration of test item of 7.04 mg/L at the start of exposure is approximately 4

- fold lower than the reported water solubility of 29 mg/L at 25 ° C 1

. (2) The study report states that the study was performed in a closed system in accordance with OECD recommendations for highly volatile substances. However, the test vessel stoppers used to prevent volatilization loss w ere described as “air

- permeable”. Therefore, the test system was not closed, a llowing significant loss of test item by volatilization over the course of the exposure period. Loss of test item from the test vessels is demonstrated by the decline in measured concentrations during the test period

. The decline in test item concentration during the exposure suggests volatilization was occurring through the air

- permeable stoppers. It is also possible that some of the test item in algal test chambers was lost to volatilization if the test solutions we re exposed to air during sampling of algae for cell counts at 24 and 48 hours. (3) The report is missing important experimental information including detailed methods for preparation of the test item solution, results of statistical analyses of data at 24 an d 48 hours, and an explicit description of the “modified” test methods used for range

finding study 2

.

TEST SUBSTANCE

Identity: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- (synonym: PCBTF ) ; CASRN 9 8

- 56

- 6

Remarks: 99.69% purity; Clear, colorless liquid ; Batch No. 20111028

1 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 Hazardo us Substances Data Bank (HSDB).HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

6 METHOD

Method/guideline followed: OECD TG 201 (2006 ; “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” )

, Method C.3 of Commission Regulation (EC) No. 761/2009 (identical to OECD TG 201), and OPPTS 850.5400 ( “Algal Toxicity, Tiers I and II” )

Test type: Toxicity Test to Freshwater Algae (Static Conditions)

GLP compliant?: Yes

Year study was performed: 201 2

Test species: Green algae ( Pseudokirchneriella subcapitata )

, strain number 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)

, were obtained from SAG: Collection of Algal Cultures, Inst. Plant Physiology, at the University of Göttingen, located in Göttingen, Germany

. Analytical monitoring: Yes

, reported only for the definitive test (at 0 , 24, 48, and 72 hours)

Exposure duration: 72 hours

Concentrations tested: Range

- finding test 1 (nominal): 0, 0.1, 1.0, 10, and 100 mg/L Range

- finding test 2 (nominal): 0, 0.1, 1.0, 10, 50, and 100 mg/L Definitive test: Nominal loading rate : 0 and 100 mg/L

. G eometric mean measured concentration of the nominal 100 mg/L solution during the 72

- hour definitive test : 3.0 mg/L

.

Test method/conditions remarks: Preparation of Test Item Solution s : For preparation of the stock solution of the test item , the study author states that “ b ecause the test item is very poorly soluble in water, a test solution was prepared using a saturated solution method (WAF method 1 according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mi xtures, OECD No. 23). A supersaturated test item solution (nominally 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the experiment in very carefully sealed Erlenmeyer flasks. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. Any non

- dissolved test materials were removed by filtration through a fine (0.22 μm) filter to give the 100 % v/v s aturated solution (loading rate 100 mg/L).” [Sipos 2013, p. 10]

1 The author erroneously refers to this test item preparation procedure as a “WAF (water accommodated fraction)” method and refers to “Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures ”

, OECD Series on Testing and Assessment Number 23. However, the referenced guidance describes a WAF as “aqueous media containing only the fraction of multi

- component substance s that is dissolved and/or present as a stable dispersion or emulsion”. Therefore, the term “WAF” applies only to multi

- component substances in which one or more of the components is not fully soluble in water. Because the test item was not a multi

- component substance, the use of the term “WAF” is not appropriate in this context.HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

7 However, the measured concentration of 7.04 mg/L at the start of exposure is approximately 4

- fold lower than the water solubility of 29 mg/L at 25 ° C reported by the Merck Index 1

. Therefo r e, the limit test was not conducted at the solubility limit. The solution described above is not a saturated solution. I t appears that a significant portion of the test item was lost during preparation of the test item solution. The test item is highly volatile and may have been lost to air during the process described above. It is likely that the higher temperature used during the initial incubation (30 ° C)

, possible flask headspace , shaking of the flask

, and filtration of the te st solution contributed to the significant loss of test item from solution. The study report did not provide any additional information on these methods . Therefore, the following aspects are unclear: (1) the reason for using a temperature of 30 ° C rather than the test temperature of 22

- 23 ° C (a higher temperature would have increased the rate of volatilization) ; (2) the amount of headspace that may have been present during test solution preparation (a larger amount of headspace would allow a greater loss of test substance due to volatilization); (3) method of shaking (vigorous shaking would have encouraged volatilization); and (4) whether filtration was conducted in a manner that minimized the exposure of the test solution to air ( to prevent los s via volatilization)

. Reconstituted algal growth medium (OECD medium, prepared according to OECD TG 201) was used for both the range

- finding and definitive tests. In the range

- finding test s , test item concentrations below the nominal 100 mg/L concentration were prepared by dilution of the stock solution with growth medium

. Growth medium without the addition of test item was used for untreated controls.

General Test Methods: The s tudy author stated that since the test item is highly volatile

, the study was performed in a closed system follow ing OECD recommendations. However, the test vessel stopper s were described as “ air

- permeable ”

. Given that volatili zation can occur through an air

- permeable stopper , the test system was not a closed system . This conclusion is supported by the time

related decrease in test item concentration over the 72

- hour incubation period (7.04 mg/L at time 0 to 1.25 mg/L at 72 hours)

. “The algae medium was enriched with 300 mg/L NaHCO 3 (to replace the gaseous CO 2 ), th e pH [was] adjusted to 7.0 by addition of HCl, and the resulting CO 2 concentration was sufficient to support algal growth in the absence of headspace.” 2 [Sipos 2013, p. 12]

“The pre

- culture was incubated under the conditions of the study in an aerated algal growth medium and used when still exponentially growing (after an incubation period of 3 days). The

1 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).
2 The addit ion of supplemental sodium bicarbonate to the medium and adjustment of pH to 7 follows the method described by Mayer et al. (2000), which is referenced by OECD Series on Testing and Assessment Number 23 (“Guidance Document on Aquatic Toxicity Testing of Di fficult Substances and Mixtures”) as a potential method for testing toxicity of volatile substances to algae using a closed system. Therefore, the addition of sodium bicarbonate and adjustment of pH to 7 are appropriate procedures to follow when testing a lgae in a closed system. (Mayer, P., Nyholm, N., Verbruggen, E.M.J., Hermens, J.L.M., and Tolls, J. 2000. An algal growth inhibition test in filled, closed bottles for volatile and sorptive materials. Environmental Toxicology and Chemistry 19: 2551

- 2556 .)HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

8 cell count... was determined by microscopic method[s] and this cell suspe nsion was diluted with algal growth medium to 10 7 cells/mL.” [Sipos 2013, p. 11]

“ The test was started (0 hours) by inoculation of [test solutions with] a biomass of approximately 3 x 10 3 algal cells per mL test medium

... 1 Volumes of 60 mL algal suspension per replicate, in steril[e] plastic flasks, were continuously shaken by a laboratory orbital shaker. The flasks were covered with air

- permeable stoppers

. ” [Sipos 2013, p. 12]

Range

- finding T est Procedure: Two range

- finding tests were conducted to determine appropriate concentrations for the d efinitive test. T he study report states that the first range

- finding test was conducted according to the “Study Plan”, but the second range

- finding test used a “modified plan”. The study report does not identify the specific differences between the “Study Plan” and the “modified plan” , but these differe nces appear to include the following: enrichment of the test medium with additional (300 mg/L) sodium bicarbonate, adjustment of pH to 7.0, reduction of the initial biomass from 1 x 10 4 cells/mL to 3 x 10 3 cells/mL , and reduction of the test flask volume from 100 mL to 60 mL. 2

In the first range

- finding test, Pseudokirchneriella subcapitata were exposed to a series of nominal loading rates of 0, 0.1, 1.0, 10, and 100 mg/L for 72 hours , using three replicates for the control group and two replicates for each test concentration

. In the second range

- finding test, algal cells were exposed to nominal loading rates of 0, 0.1, 1.0, 10, 50, and 100 mg /L for 72 hours using three replicates for the control group and each test concentration

. The report did not indicate whether a nalytical monitoring was conducted in the range

- finding
test s.

Definitive T

est Procedure: Results of the range

- finding tests showed no treatment

- related effects on the average number of cells at 72 hours. Therefore, the only concentr ation of test item evaluated in the definitive test

was a nominal concentration of 100 mg/L

. “The test was performed with eight replicates (six for observations and two replicates for analytical measurements) per test concentration and six replicates in the control groups.” [Sipos 2013, p. 12]

“The cell numbers were determined at 24, 48 and 72 h ours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the

1 The use of an initial biomass of 3 x 10 3 cells/mL follows the method described by Mayer et al. (2000; see above). Although OECD TG 201 recommends an initial biomass of 5 x 10 3

- 10 4 cells/mL for Pseudokirchneriella subcapitata , OECD Series on Testing and Assessment Number 23 (“Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”) suggests a reduced inoculum cell density when using a closed system. Therefore, an initial biomass of 3 x 10 3 cells/mL is appropriate when tes ting algae in a closed system.
2 These differences were deduced by comparing the study plan for algal toxicity testing submitted to EPA by Greenwich Chemical Consulting on behalf of the PCBTF Testing Consortium (submission dated February 17, 2012; availabl e online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0108 as of July 12, 2013) with the methods presented in Section 3.4 of the study report of Sipos (2013).HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

9 algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appeara nce of the algae.” [Sipos 2013, p. 14]

“Analytical measurement was performed on the control at the beginning and at the end of the test, and on the samples containing the test material at the beginning of the tests and 24

- hour intervals thereafter. In a ll cases, determination of test substance concentrations was only performed on one replicate vessel at each test concentration from the additional sample in order to better define loss of the test substance during the exposure period. The replicates were discarded after analytical sampling.” [Sipos 2013, p. 14] C oncentrations of test item were analyzed by high performance liquid chromatography with UV detection ( LOQ: 0.1 μg/mL)

. Culture temperatures were recorded at the beginning of the study and at 24

- hour intervals in a
water

- filled flask placed in the climatic chamber. In addition, w ater temperature in the climate chamber was continuously measured. pH was recorded at the begi nning and end of the study. The algal culture flasks were illuminated continuously with fluorescent lamps (spectral range of 400 to 700 nm) with a light intensity of approximately 8209 lux (equivalent to 111 μE/m 2 /s).

The following calculations and stati stical analyses were performed : “The section

- by

- section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test [days 0

- 1, 1

- 2 and 2

- 3])

... to demonstrate exponential growth for the entire study period.” [Sipos 2013, p. 16] “The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean value s and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052

- 6399).” [Sipos 2013, p. 16] “Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and [ two

- ] S ample t

- t est (α = 0.05) by TOXSTAT software.” [Sipos 2013, p. 16] The results of these statistical comparisons w ere reported only for the 72

- hour time point. No statistics were presented for the 24

- or 48

- hour observations.

“The EC 50 values of the test item and their confidence limits were not calculated, due to the lack of toxic effects.” [Sipos 2013, p. 16]

Validation c riteria were not stated in the methods section of the study report. However, t he results section of the study report (Sipos 2013, p p. 19 , 25 ) includes data demonstrating that a lgal growth in control cultures satisfied OECD TG 201 validation criteria

.HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

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- 6

10 RESULTS

Endpoint value(s): The following results were determined based on the geometric mean of the measured concentrations over 72 hours 1 :

Growth Rate : 72

- hr E r C 50 > 3.0 mg/L 72

- hour NOEC = 3.0 mg/L; 72

- hour LOEC > 3.0 mg/L

Biomass : 72

- hr E b C 50 > 3.0 mg/L 72

- hour NOEC = 3.0 mg/L; 72

- hour LOEC > 3.0 mg/L

Yield: 72

- hr E y C 50 > 3.0 mg/L 72

- hour NOEC = 3.0 mg/L; 72

- hour LOEC > 3.0 mg/L

Results remarks:

Range

- F inding T

est Results: The study author reported that n o toxic effects on cell number were observed at any concentration tested in the ra nge

- finding test s

. Definitive T

est Results: Measured concentrations of the test item at the nominal concentration of 100 mg/L were 7.04, 3.00, 3.15, and 1.26 mg/L at 0, 24, 48, and 72 hours , respectively

. The time

- related decrease in test item concentration is consistent with loss of test material due to volatilization. The geometric mean of the measured concentrations was 3.0 mg/L. The test item was below the l evel of detection in the control sample at each time point

. Effects on growth rate, biomass, and yield are summarized as follows: No statistically significant differences between the control and treatment groups (two

sample t

- test; α = 0.05) were observed for parameters of growth rate, biomass, and yield for the 72

- hour exposure period

. Since the study report does not indicate whether differences were statistically significant at 24 and 48 hours, it is unclear whether there

was significant evidence of toxicity of the test item at these time points. The p ercent inhibition of g rowth rate during 0

- 24

, 0

- 48 , and 0

- 72 hour s was 6.2%, 2.2%, and 0.5%, respectively, relative to the controls. The p ercent inhibition of biomass during 0

- 24, 0

- 48, and 0

- 72 hours was 14.1%, 9.6%, and 4.8%, respectively, relative to the controls.

1 The geometric mean, calculated based on the measured concentrations (mg/L) at 0, 24, 48, and 72 hours, was reported as required by OECD TG 201 because measured concentrations differed relative to nominal concentrations by more than 2 0%. The study report states that the EC 50 values are “>100 % v/v saturated solution” , the NOEC is

“ 100 % v/v saturated solution ”, and the LOEC is “>100 % v/v saturated solution” ; however, the use of the term “saturated solution” is inappropriate because the mean measured concent ration of 3.0 mg/L is markedly lower than the solubility limit of 29 mg/L at 25°C reported in the Merck Index.HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

11 The percent inhibition of yield during 0

- 72 hours was 2.8% relative to the control

. (Percent inhibition of yield was not provided at 24 or 48 hours.)

Microscopic examination of the algal cells in control and test cultures at 24, 48, and 72 hours after test initiation revealed no morphological abnormalities.

During the test, daily temperature measurements ranged from 22. 2 to 22.9 ° C and continuous temperature measurements ranged from 22 .0 to 23.1 ° C. The pH at the beginning of the test was 7.00; pH measurements taken at the end of the test ranged from 8.36 to 8.4 6

. Algal growth in control cultures satisfied OECD TG 201 validation criteria: C ell density in control cultures increased by a factor of 182x within 72 hours 1

. (OECD TG 201 states that cell density should increase by a minimum factor of 16 within 72 hours

. ) T he mean coefficient of variation for section

- by

- section specific growth rates (day s 0

- 1

, 1

- 2 , and 2

- 3) in the control culture s was 22.29%

. (OECD TG 201 states that the mean coefficient of variation for section

- by

- section growth rates must not exceed 35%

. ) T he coefficient of variation of average specific growth rates during the whole test period (day s 0

- 3) in the control cultures was 0.70%

. (OECD TG 201 states that the mean coefficient of variation of average specific growth rates must not exceed 7 %

. )

CONCLUSIONS

The following results were determined f or growth rate, biomass, and yield, based on the geometric mean of measured concentrations : The 72

- hr EC 50 was > 3.0 mg/L. The 72

- hour NOEC and LOEC were 3.0 and > 3.0 mg/L, respectively.

The study author concluded that “ b ased on the results of this study, the test item Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- had no toxic effect at saturation (3.0 mg/L, measured); the EC 50 results and the LOEC are higher than the solubility level of the test item in the test medium.” [Sipos 2013, p. 22] However, the mean measured concentration of 3.0 mg/L is markedly lower than the reported solubility limit of 29 mg/L at 25 ° C for the test item 2

.

STUDY RELIABILITY

[ 3 ] Not Reliable. This study is considered “ not reliable ” for the following reasons:

(1) The test was not conducted at the solubility limit. The measured concentration of test item of 7.04 mg/L at the start of exposure is approximately 4

- fold lower than the reported

1 The study report erroneously states that the cell density increased by a factor of 54.50 during the 72

- hour test period. The correct factor i s 182, based on an initial cell density of 3 x 10 3 cells/mL and final cell density of 54.5 x 10 4 cells/mL. (54.5 x 10 4 cells/mL ÷ 3 x 10 3 cells/mL = 182)
2 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).HPV

3 Chemical Data Adequacy Review (Revised Final ) CAS RN 98

- 56

- 6

12 water solubility of 29 mg/L at 25 ° C 1 . A significant portion of the test item appears to have been lost during preparation of the test item solution

, possibly due to the contribution of several factors, including incubation of the stock solution at 30 ° C for 24 hours, presence of headspace in the mixing flask, use of a seal that may have been permeable to air, prolonged or vigorous shaking of the flask, and exposure to air during filtration of the test solution.

(2) The study report states that the study was performed in a closed system in accordance with OECD recommendations for highly volatile substances. However, the test vessel stoppers used to prevent volatilization loss w ere described as “air

- permeable”. Therefore, the test system was not closed, allowing significant volatilization loss over the course of the exposure period. Lo ss of test item from the test vessels is demonstrated by the decline in measured concentrations , which were 7.04, 3.00, 3.15, and 1.26 mg/L at 0, 24, 48, and 72 hours , respectively

. The p ercentage of test item lost was approximately 57, 55, and 82 % at 24, 48, and 72 hours, respectively, relative to time 0. The decline in test item concentration during the exposure suggests volatilization was occurring through the air

- permeable stoppers. It is also possible that some of the test item in algal test chambers was lost to volatilization if the test solutions were exposed to air during sampling of algae for cell counts at 24 and 48 hours.

(3) The report is missing important experimental information including detailed methods for preparation of the test item solution , results of statistical analyses of data at 24 and 48 hours

, and an explicit description of the “modified” test methods used for range

- finding study 2

.

R EFERENCE S

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

. Text in quotes is taken directly from Sipos (2013).

1 The M erck 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).