SRC TR-13-051

HPV3 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, 2013
HPV3 Chemical Data Adequacy Review (Revised Final) CASRN 98-56-6

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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 ................................................................................................. 5
HPV3 Chemical Data Adequacy Review (Revised Final) CASRN 98-56-6

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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 benzene, 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 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 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, 2013). 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 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 Difficult Substances and Mixtures (2000)

were also considered because the test item has physicochemical properties (i.e., high volatility 

and relatively low water solubility)2that 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 1218-04ε1, 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 water solubility of CASRN 98-56-6 is 29 mg/L at 25°C 
HPV3 Chemical Data Adequacy Review (Revised Final) CASRN 98-56-6

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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 inadequate to satisfy the algae toxicity 

(C6) testing 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 limit 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. Incubation of the stock solution at 30°C for 24 hours prior 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 test 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 containers 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 Medicine’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 

Hazardous Substances Data Bank (HSDB).
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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 confirm 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 rule 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 stoppers used to prevent volatilization loss were 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 measured concentrations, which 

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

percentage 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 counts 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 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. 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 the observed reduction suggests that the test substance has 

toxic effects, which are expected to become more apparent when the test is repeated at 

the water solubility limit.
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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.
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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 inadequate 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 were described as “air-permeable”. 

Therefore, the test system was not closed, allowing 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 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 rangefinding study 2. 

TEST SUBSTANCE 

Identity: Benzene, 1-chloro-4-(trifluoromethyl)- (synonym: PCBTF); CASRN 98-56-6

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 

Hazardous Substances Data Bank (HSDB).
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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: 2012

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. Geometric 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 Solutions: 

For preparation of the stock solution of the test item, the study author states that “because the test 

item is very poorly soluble in water, a test solution was prepared using a saturated solution 

method (WAF method1according to the Guidance Document on Aquatic Toxicity Testing of 

Difficult Substances and Mixtures, 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 saturated 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 substances 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. 
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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 Index1. 

Therefore, the limit test was not conducted at the solubility limit. The solution described above 

is not a saturated solution. It 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 test 

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 loss 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 tests, 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 study author stated that since the test item is highly volatile, the study was performed in a 

closed system following OECD recommendations. However, the test vessel stoppers were

described as “air-permeable”. Given that volatilization can occur through an air-permeable 

stopper, the test system was not a closed system. This conclusion is supported by the timerelated 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 NaHCO3 (to replace the gaseous CO2), the pH 

[was] adjusted to 7.0 by addition of HCl, and the resulting CO2 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 addition 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 Difficult 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 algae 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.)
HPV3 Chemical Data Adequacy Review (Revised Final) CASRN 98-56-6

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cell count... was determined by microscopic method[s] and this cell suspension was diluted with 

algal growth medium to 107cells/mL.” [Sipos 2013, p. 11]

“The test was started (0 hours) by inoculation of [test solutions with] a biomass of approximately 

3 x 103algal 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 Test Procedure:

Two range-finding tests were conducted to determine appropriate concentrations for the 

definitive test. The 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 differences 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 104cells/mL to 3 x 103cells/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 analytical monitoring was conducted in the range-finding 

tests. 

Definitive Test Procedure:

Results of the range-finding tests showed no treatment-related effects on the average number of 

cells at 72 hours. Therefore, the only concentration 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 hours 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 103

cells/mL follows the method described by Mayer et al. (2000; see above). 

Although OECD TG 201 recommends an initial biomass of 5 x 103- 104cells/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 103cells/mL is appropriate when testing 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; 

available 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).
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algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect 

any abnormal appearance 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 all 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] Concentrations 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, water temperature in the climate 

chamber was continuously measured. pH was recorded at the beginning 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/m2/s).

The following calculations and statistical 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 values 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-]Sample t-test (α = 0.05) by TOXSTAT software.” [Sipos 2013, p. 16] The results 

of these statistical comparisons were reported only for the 72-hour time point. No 

statistics were presented for the 24- or 48-hour observations.

“The EC50 values of the test item and their confidence limits were not calculated, due to the lack 

of toxic effects.” [Sipos 2013, p. 16]

Validation criteria were not stated in the methods section of the study report. However, the 

results section of the study report (Sipos 2013, pp. 19, 25) includes data demonstrating that algal 

growth in control cultures satisfied OECD TG 201 validation criteria.
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RESULTS

Endpoint value(s): The following results were determined based on the geometric mean of the 

measured concentrations over 72 hours1:

Growth Rate:

72-hr ErC50 > 3.0 mg/L

72-hour NOEC = 3.0 mg/L; 72-hour LOEC > 3.0 mg/L 

Biomass:

72-hr EbC50 > 3.0 mg/L

72-hour NOEC = 3.0 mg/L; 72-hour LOEC > 3.0 mg/L 

Yield:

72-hr EyC50 > 3.0 mg/L

72-hour NOEC = 3.0 mg/L; 72-hour LOEC > 3.0 mg/L 

Results remarks: 

Range-Finding Test Results:

The study author reported that no toxic effects on cell number were observed at any 

concentration tested in the range-finding tests. 

Definitive Test 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 level 

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 (twosample 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 percent inhibition of growth rate during 0-24, 0-48, and 0-72 hours was 6.2%, 2.2%, 

and 0.5%, respectively, relative to the controls.

The percent 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 20%. The study report states that the EC50 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 concentration of 3.0 mg/L is markedly lower than 

the solubility limit of 29 mg/L at 25°C reported in the Merck Index. 
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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 8.46.

Algal growth in control cultures satisfied OECD TG 201 validation criteria:

Cell density in control cultures increased by a factor of 182x within 72 hours1. (OECD 

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

hours.)

The mean coefficient of variation for section-by-section specific growth rates (days 0-1,

1-2, and 2-3) in the control cultures was 22.29%. (OECD TG 201 states that the mean 

coefficient of variation for section-by-section growth rates must not exceed 35%.)

The coefficient of variation of average specific growth rates during the whole test period 

(days 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 for growth rate, biomass, and yield, based on the 

geometric mean of measured concentrations:

The 72-hr EC50 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 “based 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 EC50

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 item2. 

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 is 182, based on an initial cell density of 3 x 103cells/mL and final cell density of 54.5 x 

104cells/mL. (54.5 x 104cells/mL ÷ 3 x 103cells/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).
HPV3 Chemical Data Adequacy Review (Revised Final) CASRN 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 were described 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 measured concentrations, which were 7.04, 3.00, 3.15, and 1.26 mg/L at 0, 

24, 48, and 72 hours, respectively. The percentage 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. 

REFERENCES

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 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).