SRC TR

- 15

- 039

HPV3 TEST RULE DATA ADEQUACY REVIEW ( FINAL ):

TOXICITY TO ALGAE

Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- , CASRN 98

- 56

- 6

Prepared by:

Megan Nelson , William L. 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 FG007.4.009. 006

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460

Christine Edwards, EPA Administrative Contracting Officer Debra Milligan, Alternate EPA Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager

June 30 , 2015Final HPV 3 Test Rule Data Adequacy Review (June 2015) CASRN 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

................................ ................................ .............................. 3

D

- 1. Toxicity to Aquatic Plants

................................ ................................ ................................ . 3Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a toxicity to algae test for benzene, 1

- chloro

- 4

- (trifluoromethyl)

- (CASRN 98

- 56

- 6) submitted on May 13, 2015 by Greenwich Chemical Consulting, Inc.

, on behalf of the PCBTF (

para

- chlorobenzotrifl uoride) Testing Consortium, to EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 FR 65385, 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 June 30, 2015 ).

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.

The May 13, 2015 submission from Greenwich Chemical Consulting, Inc. contained the following stu dy report:

 “ Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- CAS RN 98

- 56

- 6 (para

- Chlorobenzotrifluoride (PCBTF)): Algal Growth Inhibition Test ”, available online at http: //www.regulations. gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0308 as of June 30 , 2015

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

B. EVALUATING DATA ADEQUACY

The submitted data are evaluated 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 Difficul t Substances and Mixtures (2000) were also considered because the test substance has physicochemical properties (i.e., high volatility and relatively 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 June 30, 2015 ). 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. 2 006. 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 ] ).Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

2 C. SUMMA RY 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 Ma y 13, 2015 is adequate to satisfy the algae toxicity (C6) testing require ment promulgated in the final HPV3 test rule for CASRN 98

- 56

- 6

.
There are no outstanding HPV 3 test rule requirement s for toxicity testing of CASRN 98

- 56

- 6

.Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

3 D. DATA FOR SIDS ENDPOINTS

D

- 1

. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1 : Toxicity to Pseudokirchneriella subcapitata (Vryenhoef, 2015)

Title: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- CAS RN 98

- 56

- 6 ( para

- Chlorobenzotrifluoride (PCBTF)): Algal Growth Inhibition Test

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- (CASRN 98

- 56

- 6 ), also known as Parachlorobenzotrifluoride

- 900N

.

Remarks: Clear colorless liquid , free of suspended material; Batch No. 2014

- 02

- 08 ; Purity: 99.77%.

METHOD

Method/guideline followed: OECD TG 201 (2006) “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” referenced as Method C.3 of Commissi on Regulation (EC) No. 761/2009 and OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, No. 23 (2000).

Test type: Toxicity to plants

– algae (static)

GLP compliant ?: Yes

Year st udy was performed: 201 4

- 2015

Te st species: Green algae ( Pseudokirchneriella subcapitata ); strain: CCAP 278/4; obtained from the Culture Collection of Algae and Protozoa (CCAP) of SAMS Research Services Ltd, Argyll, Scotland.

Analytical monitoring: Yes

Exposure duration: 72 hours

Concentrations tested: Range

- Finding Test: Nominal concentrations: 0 (control), 0.10, 1.0, 10

, and 100 % v/v saturated solution; equivalent to 0

- Hour measured concentration s of 0.067, 0.60, 5.0

, and 50 mg /L. Definitive Test: Nomi nal concentrations: 0 (control), 1.0, 3.2, 10, 32

, and 100% v/v saturated solution; equivalent to 0

- Hour measured concentrations of 0.37, 0.84, 3.7 , 16

, and 51 mg/L.Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

4 Test method/conditions remarks: Culture medium for the range

- finding and definitive test s

was prepared using reverse osmosis purified deionized water. “ Due to the volatile nature of the test item, testing was conducted in completely filled (i.e., no headspace), stoppered test vessels in order to minimize possible losses due to volatilization... [I]in order to prevent inhibition of growth due to the restric tion of gaseous exchange, additional sodium bicarbonate [500 mg/L] was added to the culture medium to provide a source of carbon dioxide for algal growth.” [ Vryenhoef 2015, p. 7 ] “ At the request of the Sponsor the pH of the culture medium was adjusted to pH 7.0 after the addition of the sodium bicarbonate.” [ Vryenhoef 2015, p. 13 ] The culture medium used for the first range

- finding and definitive test s contained concentrations similar to those recommended by OECD TG 201 1 , with 3.27 μg ZnCl 2 /L, 1.43 μg CoCl 2 ·6H 2 O/L, 0.012 μg CuCl 2 ·2H 2 O/L, and 7.26 μg Na 2 MoO 4 ·2H 2 O/L

.

Preliminary Media Preparation Trial: Due to the physicochemical properties of the test substance 2

, “ a media preparation trial was conducted in order to determine the solubility of the test item under test conditions ”. [ Vryenhoef 2015, p. 13 ] To compare methods for saturated solution preparation, “[a] nominal amount of test item ( 1 090 mg) was dispersed, in duplicate, in 10.9 liters of culture medium [resulting in a loading rate of 100 mg/L] with the aid of magnetic stirring at a rate such that a vortex was formed which represented approximately 5% of the overall media column height for periods of either 24 or 48 hours. After stirring samples were tak en for chemical analysis after the following pretreatments:

• Centrifugation at 10000 g for 30 minutes • Centrifugation at 40000 g for 30 minutes • Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre

- con dition the filter) • Filtration through a 0.2 μ m Sartorius Sartopore filter (approximately 2 liters discarded in order to pre

- condition the filter) ” [ Vryenhoef 2015, p. 34 ]

Based upon analytical concentrations found for solutions prepared by centrifugation (9.90 to 19.0 mg/L) and filtration (54.2 to 59.0 mg/L ) by the pretreatments above

, solutions of test substance for the algae studies were “ prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stir red for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μ m Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 57 mg/L, the limit of wate r solubility of this Test item under test conditions. ” [ Vryenhoef 2015, p. 34 ]

1 The concentrations specified by OECD TG 201 are 3 μg ZnCl 2 /L, 1.5 μg CoCl 2 ·6H 2 O/L, 0.01 μg CuCl 2 ·2H 2 O/L, and 7 μg Na 2 MoO 4 ·2H 2 O/L.

2 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 ] ). In add ition, 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 Age ncy, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/ exposure/pubs/episuitedl.htm as of June 30, 2015 ).Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

5 Range

- Finding Test Procedure: Freshwater algae ( 2 replicates/concentration; initial cell density of 3 x 10 3 cells/mL ) were exposed to nominal concentration s of 0 (control), 0.1

, 1.0, 10, or 100% v/v saturated solution for 72 hours.

Preparation of Test Solutions: To prepare the test solutions, an amount of test material (1100 mg) was added to 11 liters of test medium. The solution was stirred with a magnetic stirrer for 24 hours at a rate that produced a 5% vortex in the solution. “A fter 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approxima te 2 liters discarded in order to pre

- condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (900 mL ) of each of the stock solution s was separately inoculated with algal suspension (2.7 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution (equivalent to 0

- Hour measured concentrations of 0.06 [7] , 0.60, 5.0 and 50 m g/L).” [ Vryenhoef 2015, pp. 13

- 14 ]

Inoculation of Test Culture s and Environmental Conditions: “The test was conducted in 250 mL glass conical flasks each completely filled (i.e.

, no headspace) with test preparation and sealed with a ground glass stopper to reduce evaporation and minimize losses due to the test items volatile nature. Two replicate flasks were used for each control and test concentration.” [ Vryenhoef 2015, p. 14 ]

“The control group was maintained under identical conditions but not exposed to the test item.” [ Vryenhoef 2015, p

. 14 ]

Sealed test vessels were incubated at 24 ± 1°C under continuous lighting of approximately 7000 lux and constantly shaken at approximately 150 rpm for 72 hours

. Concentrations of test substance were measured in sample s collected at 0 and 72 hours from each test concentration

. C ell density in each flask was measured at 72 hours using a Coulter Multisizer Particle Counter. Thre e cell counts were conducted for each flask.

Definitive Test Procedure: Freshwater algae ( 5 replicates per test concentration, 9 replicates for the control; initial cell density of 3 x 10 3 cells/mL) were exposed to nominal concentration s of 0 (control), 1.0, 3.2, 10, 32 or 1 00% v/v saturated solution for 72 hours.

Preparation of Test Solutions: To prepare the test solutions, an amount of test material (1100 mg) was added to 11 liters of test medium. The solution was with a magnetic stirrer for 24 hours at a rate that produced a 5% vortex in the solution. “After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 lit ers discarded in order to pre

- condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 32, 10, 3.2 and 1.0 % v/v saturated solution. An aliquot (2 liters ) o f each of the stock solution s was separately inoculated with 5.2 mL of algal suspension to give the required test concentrations of 1.0, 3.2,Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

6 10 , 32 and 100% v/v saturated solution (equivalent to 0

- H our measured concentrations of 0.37, 0.84, 3.7, 16 and 51 mg/L).” [ Vryenhoef 2015, p

. 14 ]

Inoculation of Test Culture s and Environmental Conditions:

“ As in the range

- finding test 250 mL glass conical flasks each completely filled (i.e.

, no headspace) with test solution were used. Of the five test replicates prepared at the start of the test: (a) one replicate was kept sealed (with no prior sampling) until 24 hours, at which time samples were taken for both cell density determination and chemical analysis; this replicate was then discarded; (b) one test replic ate was kept sealed (with no prior sampling) until 48 hours, at which time samples were taken for both cell density determination and chemical analysis; this replicate was then discarded; (c) three test replicates were kept sealed (with no prior sampling) until 72 hours, at which time samples were taken for both cell density determination and chemical analysis. Therefore, each of the test replicates containing treated test solutions was used only once for sampling before being discarded.” [ Vryenhoef 2015, p. 15 ]

“Of the nine control replicates prepared at the start of the test: (a) three replicates were kept sealed (with no prior sampling) until 24 hours, at which time samples were taken for cell density determination then discarded; (b) three samples were kept sealed (with no prior sampling) until 48 hours, at which time samples were taken for cell density determination then discarded; and (c) three replicates were kept sealed (with no prior sampling) until 72 hours, at which time samples were taken for bo th cell density determination and chemical analysis.” [ Vryenhoef 2015, p. 15 ]

“The control group was maintained under identical conditions but not exposed to the test item.” [ Vryenhoef 2015, p. 15 ]

“Pre

- culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.16 x 10 6 cells per mL. Inoculation of 2 liters of test medium with 5.2 mL of this algal suspension gave an initial nominal cell density of 3 x 10 3 cells per mL and had no significant dilution effect on the final test concentration.” [ Vryenhoef 2015, p. 15 ]

Sealed test vessels were incubated at 24 ± 1°C under continuous lighting of approximately 7000 lux and constantly shaken at approximately 150 rpm for 72 hours. The pH was measured in the control and test vessels at test initiation and after the 72

- hour exposure

.
Analytical Monitoring: “Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.” [ Vryenhoef 2015, p. 16 ] In addition, “ [a] sample of the vessel sacrificed for cell density determination at 24 and 48 hours was taken for chemical analy sis.” [ Vryenhoef 2015, p. 1 6 ] Analytical measurements were conducted using gas chromatography with an electron capture detector.

Evaluation of Cell Densities and Appearance: C ell density in each flask was measured using a Coulter Multisizer Particle Counter at 0, 24, 48, and 72 hours. Three cell counts were conducted for each flask. Microscopic examinations wereFinal HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

7 conducted on samples of algal cells from each test and control vessel (replica tes pooled) at the end of the test.

Statistical Analysis: “One way analysis of variance incorporating Bartlett’s test for homogeneity of variance

... and Dunnett’s multiple comparison procedure for comparing several treatments with a control

... was carried ou t on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control

groups. All statistical analyses were performed using the SAS computer softw are package

... ” [ Vryenhoef 2015, p. 18 ]

Acceptability Criteria: The test was considered acceptable if t he following criteria were met:

 “ The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

 The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the tests (days 0

- 1, 1

- 2 and 2

- 3) must not exceed 35%.

 The coefficients of variation of the average specific growth rate in replicate control cultures must not exceed 7%.” [ Vryenhoef 2015, p. 18 ]

RESULTS

Endpoint value(s): Based on the 0

- Hour measured test concentrations in the definitive test :

Response Variable EC 50 (mg/L) 95% Confidence Limits (mg/L) No Observed

Effect Concentration (NOEC) (mg/L) Lowest

Observed Effect Concentration (LOEC) (mg/L) Chronic

Value (ChV) (mg/L)

Growth Rate 6.2 4.8

– 8.1 0.84 3.7 1.76
Yield 2.2 1.8

– 2.6 0.84 3.7 1.76

Results remarks: Range

- Finding Test Results: Cell densities and m ean percentage values for in hibition of growth rate and yield relative to the control at each nominal test substance concentration were as follows:

Nominal Concentrations (% v/v Saturated Solution) Mean Cell Densities (cells per mL) Inhibition Values (%)

0 Hours 72 Hours a Growth Rate Yield

Control (0) 3.00E+03 5.62E+05

- - 0.10 3.00E+03 5.76E+05 0 [2] b
1.0 3.00E+03 5.14E+05 3 9
10 3.00E+03 7.03E+04 40 88Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

8 100 3.00E+03 3.31E+03 99 100

a Cell densities at 72 hours are the means of two replicates per concentration.

b [Increase in growth rate or yield compared to controls]

“The results showed no effect on growth at the test concentrations of 0.10 and 1.0% v/v saturated
solution. However, growth was observed to be reduced at 10 and 100% v/v saturated s olution.” [ Vryenhoef 2015, p. 19 ]

“ B ased on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.” [ Vryenhoef 2015, p. 19 ]

Measurements of pH and temperature during the range

- finding tests were not provided in the study report. At 0 hours, measured test substance concentrations were 0.067, 0.60, 5.0, and 50 mg/L for nominal concentrations of 0.1, 1.0, 10, and 100% v/v saturated solution, respectively. At 72 hours, measu red test substance concentrations were 0.059, 0.44, 4.1, and 48 mg/L for nominal concentrations of 0.1, 1.0, 10, and 100% v/v saturated solution, respectively. Test concentrations at 72 hours were 74% to 97% of the initial measured concentrations.

“The analytical monitoring was not validated prior to the analysis of the range

- finding samples; full validation was later conducted at a suitable concentration.” [ Vryenhoef 2015, p. 3 9 ]

Definitive Test Results:
A t 0 hours , measured test substance concentrations were 0.37, 0.84, 3.7, 16 , and 51 mg/L for the 1.0, 3.2, 10, 32

, and 100% v/v saturated solution test preparations, respectively. “ There was no significant change in the measured concentrations at 24, 48 and 72 hour s (85% to 98% of the 0

- Hour measured concentrations) and so the results [i.e., the toxicity values] are based on 0

- Hour measured test concentrations only as recommended in Section 39 of the OECD Test Guideline No. 201.” [ Vryenhoef 2015, p. 1 9 ] “The method was considered to be sufficiently accurate and precise for the purposes of this test. The test sample results were not corrected for recovery.” [ Vryenhoef 2015, p. 3 9 ] The limit of quantification (LOQ) was 0.0099 mg/L.

Over the 72

- Hour exposure , growth rate and yield were affected by the presence of the test substance

. For growth rate inhibition, “[t]here were no statistically significant differences between the control, 0.37 and 0.84 mg/L test concentrations (P≥0.05), however, all other test

c oncentrations were significantly differen t (P<0.05) and, therefore, the ‘No Observed Effect Concentration’ (NOEC) based on growth rate was 0.84 mg/L. Correspondingly the ‘Lowest Observed Effect Concentration’ (LOEC) based on growth rate was 3.7 mg/L.” [ Vr yenhoef 2015,

p. 20 ] For inhibition of yield, “[t]here were no statistically significant differences between the control, 0.37 and 0.84 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P< 0.05) and, therefore the ‘No Observed Effect Concentration’ (NOEC) based on yield was 0.84 mg/L. Correspondingly the ‘Lowest Observed Effect Concentration’ (LOEC) based on yield was 3.7 mg/L.” [ Vryenhoef 2015, p. 20 ]Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

9 Nominal Concentrations (% v/v Satura ted Solution) Mean Cell Densities (cells per mL) Inhibition Values (%)

0 Hours 72 Hours a Growth Rate Yield

Control (0) 3.00E+03 5.96E+05

- - 1.0 3.00E+03 6.30E+05 [ 1 ] b [6 ] b
3.2 3.00E+03 5.32E+05 2 11
10 3.00E+03 1.35E+05 27 7 8
32 3.00E+03 5.99E+03 88 99
100 3.00E+03 5.65E+03 88 100

a Cell densities at 72 hours are the means of three replicates per concentration.

b [Increase in growth rate or yield compared to controls]

The pH of contro l cultures was 7.9 at 0 hours and 9.4 at 72 hours. For treated algal cultures , pH at 0 hours ranged from 7.4 to 7. 7 . A t 72 hours , pH was related to test substance concentration and rang ed from 9. 1 at the lowest concentration to 7.5 at the highest. “T emperature was maintained at 24 ± 1 °C throughout the test

. ” [ Vryenhoef 2015, p. 21 ]

Observations of the cultures: “After 72 hours there were no abnormalities detected in the control or test cultures at 0.37, 0.84, 16 and 51 mg/L, however some enlarged/swollen cells were observed to be present in the test cultures at 3.7 mg/L.” [ Vryenhoef 2015, p. 21 ]

Observations on Test Item Solubility: “At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72

- Hour test period all control, 0.37 and 0.84 mg/L test cultures were observed to be gree n dispersions. The 3.7 mg/L test cultures were observed to be pale green dispersions whilst the 16 and 51 mg/L test cultures were observed to be clear colorless solutions.” [ Vryenhoef 2015, p. 21 ] Acceptability Criteria: The definitive test met the follo wing criteria for acceptability prescribed by the protocol :

 “ [ T ] he cell concentration of the control cultures increased by a factor of 190 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a f

actor of 16 after 72 hours.” [ Vryenhoef 2015, p. 20 ]

 “The mean coefficient of variation for section by section specific growth rate for the control cultures was 22% and hence satisfied the validation criterion given in the OECD guideline which states the mean must not exceed 35%. ” [ Vryenhoef 2015, p. 21 ]

 “ The coefficient of variation for average specific growth rate for the control cultures over the test period (0

– 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this mu st not exceed 7% ” [ Vryenhoef 2015, p. 21 ]

CONCLUSIONS

Based on 0

- h our measured concentrations 1 , the 72

- hour EC 50 v alues for inhibition of growth and yield were 6.2 and 2.2 mg /L , respectively. T he 96

- hour NOEC and LOEC were 0.84 and 3.7

1 Compared with test substance concentrations measured at 0 hours in the definitive study, “ T here was no significant change in the measured concentrations at 24, 48 and 72 hours...” [Vryenhoef 2015, p. 19]; therefore, the study author based toxicity values on test concentrations measured at 0 hours.Final HPV3 Test Rule Data Adequacy Review (June 2015) CASRN 98

- 56

- 6

10 mg/L , respectively . Based on this information, the ChV was calculated by the reviewer to be 1.76 mg/L.

STUDY RELIABILITY

[ 1 ] Reliable with out restrictions

.

REFERENCE

Vryenhoef, H. 2015. Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- CAS RN 98

- 56

- 6 ( para

Chlorobenzotrifluoride (PCBTF)): Algal Growth Inhibition Test

. Harlan Laboratories Ltd., UK ; Laboratory Project No. 41304731 . Sponsor: PCBTF Testing Consortium c/o Greenwich Chemical Consulting, Inc.

, Palm Beach Gardens, FL

. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0308 as of June 30 , 2015

.
Text in quotes is taken directly from Vryenhoef (2015).