SRC TR-14-007

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL):

TOXICITY TO ALGAE

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

Prepared by:

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

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

March 6, 2014
Final HPV3 Test Rule Data Adequacy Review (March 2014) 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.............................................................................................. 3

D-1. Toxicity to Aquatic Plants ................................................................................................. 3

APPENDIX: Test Guideline Source Material to Support Reliability Ratings Assigned to 

Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements .............. 9
Final HPV3 Test Rule Data Adequacy Review (March 2014) CASRN 98-56-6

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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 December 20, 2013 by Finchimica S.p.A., on behalf of KOWA 

American Corporation (one of several member companies of the PCBTF (parachlorobenzotrifluoride) 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 March 6, 2014). 

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 December 20, 2013 submission from Finchimica S.p.A. contained the following study 

report:

 “4-Chloro-α,α,α-trifluorotoluene (PCBTF): Toxicity to green algae Pseudokirchneriella 

subcapitata determined in a growth inhibition study”, available online at 

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

March 6, 2014.

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 substance 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 March 6, 2014). The water solubility of CASRN 98-56-6 is 29 mg/L at 25°C 

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

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

Hazardous Substances Data Bank [HSDB]).
Final HPV3 Test Rule Data Adequacy Review (March 2014) CASRN 98-56-6

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

The study submitted by Finchimica S.p.A. on behalf of the KOWA American Corporation (a 

member company of the PCBTF Testing Consortium), in the submission dated December 20, 

2013 is inadequate to satisfy the algae toxicity (C6) testing requirement promulgated in the final 

HPV3 test rule for CASRN 98-56-6. This study is not adequate for the following reasons:

(1) This study failed to identify a discrete EC50 because inhibition of algal growth rate and 

yield at the highest test concentration was less than 50% in the definitive test. The study 

only determined that the EC50 (based on geometric mean measured concentrations) was 

greater than the highest test concentration, 0.41 mg/L, which is only ~1.4% of the 

reported water solubility limit of the test substance (29 mg/L at 25°C). Therefore, the test 

failed to satisfy guideline requirements to test up to a maximum concentration of 

100 mg/L or the solubility limit of the test substance (whichever is lower) if an EC50

cannot be identified at a lower concentration.

(2) Test concentrations declined to undetectable levels during the first 24 hours of the 

exposure period. The measured concentrations of test substance at 0 hours were 5.73, 

10.4, 13.6, 19.7, and 27.4 mg/L for nominal concentrations of 7.9, 11.9, 17.8, 26.7, and 

40.0 mg/L, respectively. However, test substance concentrations were below the limit of 

detection (LOD = 100.9 μg/L) at 24, 48, and 72 hours for all nominal concentrations.

(3) The test substance is highly volatile and the experiment was not conducted using 

procedures intended to minimize loss of the test substance through volatilization. The 

decline in test concentrations to undetectable levels during the exposure period suggests 

that nearly all of the test substance was lost from solution by volatilization. To minimize 

loss of a volatile test substance from test media, vessels must be sealed with an airtight 

stopper and contain no headspace. The test vessel stoppers in this study were described 

as “air-permeable”; therefore, the test system was not closed, allowing significant loss of 

test substance by volatilization over the course of the exposure period. Furthermore, a 

substantial amount of headspace was present in the exposure vessels; the 100-mL glass 

flasks test vessels were filled with 50 mL of test media, which was inoculated with an 

unspecified volume of algal culture. 

(4) There was no evidence that the solubility of the test substance in the growth medium was 

determined prior to initiation of the algae test; the study report did not provide a value for 

the solubility limit in the growth medium.

The algal growth inhibition test needs to be repeated, using appropriate methods to prevent

the deficiencies discussed above.
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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 (Noe et al., 2013)

Title: 4-Chloro-α,α,α-trifluorotoluene (PCBTF): Toxicity to green algae Pseudokirchneriella 

subcapitata determined in a growth inhibition study

This study is: Adequate Not Adequate

This study is not adequate for the following reasons:

(1) This study failed to identify a discrete EC50 because inhibition of algal growth rate and 

yield at the highest test concentration was less than 50% in the definitive test. The study 

only determined that the EC50 (based on geometric mean measured concentrations) was 

greater than the highest test concentration, 0.41 mg/L, which is only ~1.4% of the 

reported water solubility limit of the test substance (29 mg/L at 25°C). Therefore, the test 

failed to satisfy guideline requirements to test up to a maximum concentration of 

100 mg/L or the solubility limit of the test substance (whichever is lower) if an EC50

cannot be identified at a lower concentration.

(2) Test concentrations declined to undetectable levels during the first 24 hours of the 

exposure period. The measured concentrations of test substance at 0 hours were 5.73, 

10.4, 13.6, 19.7, and 27.4 mg/L for nominal concentrations of 7.9, 11.9, 17.8, 26.7, and 

40.0 mg/L, respectively. However, test substance concentrations were below the limit of 

detection (LOD = 100.9 μg/L) at 24, 48, and 72 hours for all nominal concentrations.

(3) The test substance is highly volatile and the experiment was not conducted using 

procedures intended to minimize loss of the test substance through volatilization. The 

decline in test concentrations to undetectable levels during the exposure period suggests 

that nearly all of the test substance was lost from solution by volatilization. To minimize

loss of a volatile test substance from test media, vessels must be sealed with an airtight 

stopper and contain no headspace. The test vessel stoppers in this study were described 

as “air-permeable”; therefore, the test system was not closed, allowing significant loss of 

test substance by volatilization over the course of the exposure period. Furthermore, a 

substantial amount of headspace was present in the exposure vessels; the 100-mL glass 

flasks test vessels were filled with 50 mL of test media, which was inoculated with an 

unspecified volume of algal culture.

(4) There was no evidence that the solubility of the test substance in the growth medium was 

determined prior to initiation of the algae test; the study report did not provide a value for 

the solubility limit in the growth medium.
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TEST SUBSTANCE 

Identity: Benzene, 1-chloro-4-(trifluoromethyl)- (referred to as 4-chloro-α,α,α-trifluorotoluene

[PCBTF] in the study report); CASRN 98-56-6

Remarks: Purity: 99.3%; Batch No. 11/2012; impurities (all present at < 1%) included 2-

chloro-benzotrifluoride (0.15%), 3-chloro-benzotrifluoride (0.55%), dichloroderivatives (< 

0.01%), and water (76 ppm).

METHOD

Method/guideline followed: OECD TG 201 (2011) “Freshwater Algae and Cyanobacteria,

Growth Inhibition Test” and OECD Series on Testing and Assessment Number 23 (2000) 

“Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”

Test type: Toxicity to Plants (Algae)

GLP compliant?: Yes

Year study was performed: 2013

Test species: Freshwater green algae (Pseudokirchneriella subcapitata); cultured in-house, 

originally obtained from the Institute of Plant Physiology at the University of Göttingen, located 

in Göttingen, Germany.

Analytical monitoring: Yes

Exposure duration: 72 hours

Concentrations tested: Nominal concentrations: 7.9, 11.9, 17.8, 26.7, and 40.0 mg/L (corrected 

for purity of test substance); Geometric mean measured concentrations: 0.28, 0.32, 0.34, 0.37, 

and 0.41 mg/L 

Test method/conditions remarks: 

Preparation of Test Solutions:

A 40.0 mg/L stock solution (nominal concentration adjusted for purity of 99.3%) was prepared 

by adding 35.5 μL of the test substance, corresponding to 47.4 mg (based on a density of 1.3361 

g/mL), to a 1-liter bottle filled with 1170 mL of reconstituted water. “The bottle was tightly 

closed to prevent test item evaporation. The obtained solution appeared transparent with oily 

droplets on the bottom of the bottle, therefore it was mixed for two hours by the mean[s] of a 

magnetic stirrer in dark conditions to achieve the total solubilisation of test item. After stirring, 

the solution appeared homogenous and transparent.” [Noe et al. 2013 p. 12] The remaining test 

concentrations were prepared by diluting aliquots of the 40.0 mg/L stock solution in 

reconstituted water.
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Algal Culture Conditions: 

“The algae were cultured in a climatic chamber at 24°C ± 2°C under continuous uniform 

illumination in the range 4440-8880 Lux in the spectral range 400-700 nm.” [Noe et al. 2013, p. 

12]

The algal maintenance culture medium and test medium were the same, consisting of 

reconstituted water. The medium was prepared according to OECD TG 201 by adding analytical 

grade salts to deionized water with a conductivity of < 5 μScm-1. The prepared medium had a 

pH of 8.1 ± 0.2. 

Experimental Procedures:

Exponentially growing algal cultures were exposed to the test substance under static conditions 

for 72 hours. Controls were treated under similar conditions in the absence of test substance. 

Testing was conducted using 6 replicates for the control and 3 replicates for each of the test 

concentrations. The test vessels consisted of 100-mL capacity, conical glass flasks capped with 

air-permeable stoppers. 

At the start of the test, 50 mL of the prepared test solutions were poured into the exposure 

vessels and inoculated with algae to obtain a starting density of approximately 104cells/mL. 

“The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test 

in order to ensure that the assay was performed with [an] exponentially growing algal 

population. The cell density of the inoculum was around 106cells/mL.” [Noe et al. 2013, p. 13]

During the test, the algae were incubated in a climatic chamber, with continuous illumination 

(light intensity of 5220 to 5401 Lux) and shaking. Using an electronic particle counter, cell 

densities were determined from samples taken from each replicate at 24, 48, and 72 hours. 

“The measured [algal density] values were used to calculate the percentage inhibition of cell 

growth as yield and as growth rate in comparison to the negative control.” [Noe et al. 2013, p. 

15]

Temperature under the light was continuously monitored during the exposure period with a data 

logger. The pH was measured in each freshly prepared test solution at the beginning of the test 

and in each test vessel at the end of the exposure period (i.e., 72 hours).

Analytical Monitoring: 

Test substance concentrations were analytically determined from samples collected at the 

beginning of the test (i.e., 0 hours) and at 24-hour intervals during the test (i.e., 24, 48, and 72 

hours). 

“10 mL samples of test item solutions and negative control [solutions] were collected from 

freshly prepared solutions at time 0 and at the end of the test period (72 hours) after removing 

algal cells by centrifugation. Duplicate samples of the test item solutions and the negative 

control were taken at the indicated times from the test solutions, but only one sample [per test 

concentration] was analysed, while the other[s] were stored as retain[ed] samples to be analysed 
Final HPV3 Test Rule Data Adequacy Review (March 2014) CASRN 98-56-6

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only if necessary. At time 24 and 48 hours... an aliquot of 14 mL (10 mL for analysis and 4 mL 

as retained sample) for each concentration [was also collected].” [Noe et al. 2013, p. 17]

The analyses were performed according to an internally validated analytical method with a high 

performance liquid chromatography (HPLC) instrument coupled with a diode array detector at a 

wavelength of 220 nm. The limit of detection (LOD) of the analytical method was 100.9 μg/L, 

and the limit of quantification was 252.2 μg/L. 

Evaluation of Data and Statistical Analysis:

“The statistical analysis was carried out by the means of CETIS elaboration software v1.026D; at 

each observation time the ErC10, ErC20,[and] ErC50 [or EyCx] time were assessed by a Linear 

Interpolation analysis, while the NOEC was assessed by Bonferroni Adj t method.” [Noe et al. 

2013, p. 26]

Validation Criteria:

The results of the test were considered valid if the following criteria were met:

 “A test is not considered acceptable if the cell density in the control cultures has not 

increased by a factor of at least 16 within the test period.

 The coefficient of variation of daily growth rates in the control cultures must not exceed 

35%.

 The coefficient of variation of average growth in replicate control cultures must not 

exceed 7%.” [Noe et al. 2013, p. 18]

RESULTS

Endpoint value(s): 

The following values were based on geometric mean measured concentrations: 

72-hour EC50 (growth rate and yield) > 0.41 mg/L

72-hour NOEC (growth rate and yield) = 0.41 mg/L

Results remarks: 

Measured concentrations of the test substance in freshly prepared test solutions ranged from 

68.5–87.1% of nominal concentrations at test initiation; measured concentrations at 0 hours were 

~5.73, 10.4, 13.6, 19.7, and 27.4 mg/L at nominal concentrations of 7.9, 11.9, 17.8, 26.7, and 

40.0 mg/L, respectively. The measured test concentrations could not be determined at 24, 48, or 

72 hours because the test concentrations were below the limit of detection (LOD = 100.9 μg/L). 

“Since the analytical recoveries of test item during the test period (in the fresh and spent test item 

solutions) were lower than the range 80%-120% ([average of] 75.7% in fresh solution and lower 

than the L.O.D. in the spent solution), the geometric mean of measured concentrations was 

calculated... and the toxic endpoints were calculated in terms of [the] geometric mean of test 

item measured concentrations.” [Noe et al. 2013, p. 17]

To calculate the geometric mean measured concentrations, the LOD was used as the measured 

value at 24, 48, and 72 hours, since all measurements at these time points were below the LOD. 
Final HPV3 Test Rule Data Adequacy Review (March 2014) CASRN 98-56-6

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The resulting geometric mean measured concentrations were ~0.28, 0.32, 0.34, 0.37, and 0.41 

mg/L at nominal concentrations of 7.9, 11.9, 17.8, 26.7, and 40.0 mg/L, respectively. 

The average cell density at 72 hours was ~293, 332, 389, 378, 342, and 379 x 104cells/mL at 

nominal concentrations of 7.9, 11.9, 17.8, 26.7, and 40.0 mg/L, respectively. Algal growth rate 

and yield were not inhibited by test substance exposure at any concentration. 

The pH (for all treated and control vessels combined) ranged from 7.62 to 7.91 at test initiation 

and from 9.31 to 9.60 at test termination. The pH in the control increased by 1.4 units from 0 to 

72 hours. The pH in treated test vessels increased by 1.5–1.9 units from 0 to 72 hours. 

Temperature under the light ranged from 23.3 to 24.1 °C, with a mean value of 24.0 °C. 

Validation Criteria:

The test was considered valid because it met the validation criteria, as indicated by the following:

 The cell density in the control cultures increased by a factor of 293, which is greater than 

the minimum requirement of 16. 

 The coefficient of variation of daily growth rates in the control cultures was 25.6%, 

which is less than the allowed maximum of 35%. 

 The coefficient of variation of average growth in control replicates was 1.2%, which is 

less than the allowed maximum of 7%. 

CONCLUSIONS

The 72-hour EC50 values for inhibition of algal growth rate and yield were > 0.41 mg/L. The 72-

hour NOEC for inhibition of algal growth rate and yield was 0.41 mg/L. 

STUDY RELIABILITY 

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

(1) This study failed to identify a discrete EC50 because inhibition of algal growth rate and 

yield at the highest test concentration was less than 50% in the definitive test. The study 

only determined that the EC50 (based on geometric mean measured concentrations) was 

greater than the highest test concentration, 0.41 mg/L, which is only ~1.4% of the 

reported water solubility limit of the test substance (29 mg/L at 25°C). Therefore, the test 

failed to satisfy guideline requirements to test up to a maximum concentration of 

100 mg/L or the solubility limit of the test substance (whichever is lower) if an EC50

cannot be identified at a lower concentration.

(2) Test concentrations declined to undetectable levels during the first 24 hours of the 

exposure period. The measured concentrations of test substance at 0 hours were 5.73, 

10.4, 13.6, 19.7, and 27.4 mg/L for nominal concentrations of 7.9, 11.9, 17.8, 26.7, and 

40.0 mg/L, respectively. However, test substance concentrations were below the limit of 

detection (LOD = 100.9 μg/L) at 24, 48, and 72 hours for all nominal concentrations.

(3) The test substance is highly volatile and the experiment was not conducted using 

procedures intended to minimize loss of the test substance through volatilization. The 

decline in test concentrations to undetectable levels during the exposure period suggests 

that nearly all of the test substance was lost from solution by volatilization. To minimize
Final HPV3 Test Rule Data Adequacy Review (March 2014) CASRN 98-56-6

8

loss of a volatile test substance from test media, vessels must be sealed with an airtight 

stopper and contain no headspace. The test vessel stoppers in this study were described 

as “air-permeable”; therefore, the test system was not closed, allowing significant loss of 

test substance by volatilization over the course of the exposure period. Furthermore, a 

substantial amount of headspace was present in the exposure vessels; the 100-mL glass 

flasks test vessels were filled with 50 mL of test media, which was inoculated with an 

unspecified volume of algal culture. 

(4) There was no evidence that the solubility of the test substance in the growth medium was 

determined prior to initiation of the algae test; the study report did not provide a value for 

the solubility limit in the growth medium.

REFERENCE

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

green algae Pseudokirchneriella subcapitata determined in a growth inhibition study. 

ChemService S.r.l. Controlli e Ricerche, Novate Milanese, Italy. Study No. CH-095/2013. 

Sponsor: Finchimica S.p.A., Manerbio, Italy. Study report available online at 

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

6, 2014.

Text in quotes is taken directly from Noe et al. (2013).
Final HPV3 Test Rule Data Adequacy Review (March 2014) CASRN 98-56-6

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APPENDIX: Test Guideline Source Material to Support Reliability Ratings Assigned to 

Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements

This appendix provides additional information regarding deficiencies of the toxicity to algae 

study, which is considered inadequate to satisfy the test rule requirement. For each deviation 

from relevant test guidelines (i.e., standard test guidelines purportedly followed by the study 

according to the study report), quotes of relevant passages from the guidelines are supplied, 

followed by a brief summary of the quoted material and, where appropriate, an interpretation by 

a qualified scientist. Emphasis has been added to quotes (in the form of italics) by the reviewer.

Study: Toxicity to Pseudokirchneriella subcapitata (Noe et al., 2013)

Relevant Guidelines: OECD TG 201 (2011) “Freshwater Alga and Cyanobacteria, Growth 

Inhibition Test” and OECD Series on Testing and Assessment Number 23 (2000) “Guidance 

Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”. The test 

substance has physicochemical properties (i.e., high volatility and relatively low water 

solubility)1that necessitate special considerations in the aquatic testing process.

[A] Deviation 1: This study failed to identify a discrete EC50 because inhibition of algal growth 

rate and yield at the highest test concentration was less than 50% in the definitive test. The study 

only determined that the EC50 (based on geometric mean measured concentrations) was greater 

than the highest test concentration, 0.41 mg/L, which is only ~1.4% of the reported water 

solubility limit of the test substance (29 mg/L at 25°C). Therefore, the test failed to satisfy 

guideline requirements to test up to a maximum concentration of 100 mg/L or the solubility limit 

of the test substance (whichever is lower) if an EC50 cannot be identified at a lower 

concentration.

OECD TG 201, p. 1, Section “Principle of the test”, item 3 states: “The purpose of this 

test is to determine the effects of a substance on the growth of freshwater microalgae and/or 

cyanobacteria...”; and item 6 states: “The test endpoint is inhibition of growth, ... the 

concentration bringing about a specified x % inhibition of growth rate (e.g. 50%) is 

determined and expressed as the ErCx (e.g. ErC50).”

OECD TG 201, p. 7, “Description of the method”, Sub-section “Limit test”, item 42 

states: “Under some circumstances, e.g. when a preliminary test indicates that the test 

substance has no toxic effects at concentrations up to 100 mg/L or up to its limit of solubility 

in the test medium (whichever is the lower), a limit test involving a comparison of responses 

in a control group and one treatment group (100 mg/L or a concentration equal to the limit of 

solubility), may be undertaken. It is strongly recommended that this be supported by analysis 

of the exposure concentration.”

OECD TG 201, p. 11, Section “Data and reporting”, Sub-section “Test report”, item 61 

states: “The test report must include the following:” and sub-item “Results” states: 

 

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

atm-m

3

/mole 

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

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

exposure/pubs/episuitedl.htm as of March 4, 2014). The water solubility of CASRN 98-56-6 is 29 mg/L at 25°C 

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

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

Hazardous Substances Data Bank (HSDB)).
Final HPV3 Test Rule Data Adequacy Review (March 2014) CASRN 98-56-6

10

“estimates of toxicity for response variables e.g., EC50, EC10, EC20 and associated confidence 

intervals.”

OECD 23, p. 20, Section “Media preparation and exposure systems for difficult test 

substances” states: “Hence, while the target should be to achieve exposure concentrations 

up to the aqueous solubility of the test substance, it may be necessary to utilise higher nominal 

concentrations to achieve it.”

OECD 23, p. 20, Section 3.1 “Poorly water-soluble substances”, Sub-section 3.1.1 

“Preliminary experiment” states: “A preliminary experiment should be considered for all 

studies with poorly water-soluble substances to determine the maximum dissolved 

concentration that can be achieved under test conditions, which is defined as the saturation 

concentration, and the preparation conditions that are required to achieve it. The results of the 

preliminary experiment will form a basis of and justification for the media preparation 

procedures adopted for the toxicity tests and a reference point (i.e. water solubility under test 

conditions) against which the test results can be evaluated.” 

Summary of Quoted Material: A primary objective of a toxicity to algae test is to determine a 

discrete EC50 value. The test should be conducted at exposure concentrations up to the solubility 

limit of the test substance. Interpretation: If a discrete EC50 is not determined because 

insufficient (< 50%) inhibition of growth rate and yield occurred at the highest concentration 

tested and this concentration is below the limit of solubility in the test medium (and the limit of 

solubility is below 100 mg/L), the test is not considered acceptable because it fails to provide 

useful information for assessment of toxicity to algae. 

[B] Deviation 2: Test concentrations declined to undetectable levels during the first 24 hours of 

the exposure period. The measured concentrations of test substance at 0 hours were 5.73, 10.4, 

13.6, 19.7, and 27.4 mg/L for nominal concentrations of 7.9, 11.9, 17.8, 26.7, and 40.0 mg/L, 

respectively. However, test substance concentrations were below the limit of detection (LOD = 

100.9 μg/L) at 24, 48, and 72 hours for all nominal concentrations.

OECD TG 201, p. 6, Section “Description of the method”, Sub-section “Measurements 

and analytical determinations”, item 36 states: “Provided an analytical procedure for 

determination of the test substance in the concentration range used is available, the test 

solutions should be analysed to verify the initial concentrations and maintenance of the 

exposure concentrations during the test.”

OECD 23, p. 15, Section 1 “Preliminary assessment of stability of test substance” states: 

“Modifications to media preparation and exposure systems may be required where exposure 

concentrations of a test substance are likely to decline significantly over the test period. In 

OECD guidelines for aquatic toxicity testing a decline in concentration of 20% is considered 

sufficient to warrant consideration of measures to reduce the decline. Data providing an 

indication of the stability of the test substance under the test conditions should therefore be 

obtained before commencing testing.”

OECD 23, pp. 16-17, Section 1.2 “Preliminary stability study” states: “The possibility of 

losses during sampling, sample treatment and analysis must be considered. Where samples 

are stored before analysis, losses during sample storage should also be assessed.”

OECD TG 201, pp. 2-3, Section “Applicability of the test”, item 13 states: “For testing of 

substances that are volatile, strongly adsorbing, coloured, having a low solubility in water or 

substances that may affect the availability of nutrients or minerals in the test medium, certain 
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modifications of the described procedure may be required (e.g., closed system, conditioning of 

the test vessels). Guidance on some appropriate modification is given in (2) (3) and (4).” 

(2) - International Organisation for Standardisation (1998). ISO/DIS 14442. Water quality –

Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, 

metals and waste water. 

(3) - OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances 

and Mixtures. Environmental Health and Safety Publications. Series on Testing and 

Assessment, no. 23. Organisation for Economic Co-operation and Development, Paris.

(4) - International Organisation for Standardisation (1998). ISO 5667-16 Water quality –

Sampling – Part 16: Guidance on Biotesting of Samples.

OECD 23, p. 20, Section “Media preparation and exposure systems for difficult test 

substances” states: “All reasonable efforts should be made to achieve true test solutions 

containing dissolved concentrations of the test substance that are stable and close to target.” 

and “The media preparation and exposure methods should be evaluated prior to commencing 

testing to ensure that the required quantities of test medium can be produced and the required 

concentrations can be achieved and maintained.”

OECD 23, p. 27, Section 3.4 “Volatile substances” states: “Losses of volatile substances 

from test media during preparation and exposure can be minimised using relatively 

straightforward modifications to procedures. As a general rule vessels should be sealed 

during preparation and exposure and the headspace kept to a minimum. Likewise, test 

concentrations should, where possible, be prepared individually by addition of test substance 

directly to the test vessels rather than by dilution of a stock solution. Systems with zero 

headspace should be used where it is not possible to analyse exposure concentrations. 

Syringe pumps can be used to dispense concentrated solutions of volatile substances (in water 

miscible carrier solvents, if required) into sealed vessels. Samples collected for analysis 

should be placed in zero headspace vials.”

OECD 23, p. 26, Section 3.3 “Substances toxic at low concentrations” states: “Where a 

measured concentration at the end of the exposure period is absent or where it indicates that 

the substance is not detected, the validity of the test should be reconfirmed.”

OECD TG 201, p. 12, Section “Data and reporting”, Sub-section “Test report”, item 61 

states: “The test report must include the following:” and sub-item “Results” states: 

“discussion of the results, including any influences on the outcome of the test resulting from 

deviations from this Guideline.”

Summary of Quoted Material: Measured concentrations of test substance should demonstrate 

maintenance of exposure concentrations. The stability of the test substance in the growth 

medium should be determined before study initiation so that measures may be taken to minimize 

a decline in test substance concentration during the test period. The possibility that test 

substance was lost from solution during sampling, handling of samples, and analysis of samples 

must be considered. Modifications of standard testing protocols may be required for test 

substances that are volatile and/or have low water solubility. Study authors should make all 

reasonable efforts to maintain stable measured test substance concentrations that are close to 

nominal concentrations. Losses of volatile substances from test media should be minimized by 

implementing modified test procedures. When measured test substance concentrations are below 

the analytical detection limit, the validity of the test should be reconfirmed. Unusual findings 

(e.g., large declines in test substance concentrations during the test) are to be addressed in the 
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study report. Interpretation: If test substance concentrations decline by 20% or more during the 

test, modification of test methods may be required to minimize this decline. When analyses 

indicate that test substance concentrations are below the detection limit, the validity of the study 

is of concern; as a result, the study results should be confirmed by repeating the toxicity test 

using appropriate methods.

[C] Deviation 3: The test substance is highly volatile and the experiment was not conducted 

using procedures intended to minimize loss of the test substance through volatilization. The 

decline in test concentrations to undetectable levels during the exposure period suggests that 

nearly all of the test substance was lost from solution by volatilization. To minimize loss of a 

volatile test substance from test media, vessels must be sealed with an airtight stopper and 

contain no headspace. The test vessel stoppers in this study were described as “air-permeable”; 

therefore, the test system was not closed, allowing significant loss of test substance by 

volatilization over the course of the exposure period. Furthermore, a substantial amount of 

headspace was present in the exposure vessels; the 100-mL glass flasks test vessels were filled 

with 50 mL of test media, which was inoculated with an unspecified volume of algal culture. 

OECD TG 201, pp. 2-3, Section “Applicability of the test”, item 13 states: “For testing of 

substances that are volatile, strongly adsorbing, coloured, having a low solubility in water or 

substances that may affect the availability of nutrients or minerals in the test medium, certain 

modifications of the described procedure may be required (e.g., closed system, conditioning of 

the test vessels). Guidance on some appropriate modification is given in (2) (3) and (4).” 

(2) - International Organisation for Standardisation (1998). ISO/DIS 14442. Water quality –

Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, 

metals and waste water. 

(3) - OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances 

and Mixtures. Environmental Health and Safety Publications. Series on Testing and 

Assessment, no. 23. Organisation for Economic Co-operation and Development, Paris.

(4) - International Organisation for Standardisation (1998). ISO 5667-16 Water quality –

Sampling – Part 16: Guidance on Biotesting of Samples.

OECD 23, pp. 27-28, Section 3.4 “Volatile substances” states: “Losses of volatile 

substances from test media during preparation and exposure can be minimised using relatively 

straightforward modifications to procedures. As a general rule vessels should be sealed 

during preparation and exposure and the headspace kept to a minimum. Likewise, test 

concentrations should, where possible, be prepared individually by addition of test substance 

directly to the test vessels rather than by dilution of a stock solution. Systems with zero 

headspace should be used where it is not possible to analyse exposure concentrations. 

Syringe pumps can be used to dispense concentrated solutions of volatile substances (in water 

miscible carrier solvents, if required) into sealed vessels. Samples collected for analysis 

should be placed in zero headspace vials.” and Sub-section “Algal test” states: “The use of 

a sealed exposure system in the algal growth inhibition test will result in culture growth being 

limited by CO2 depletion and increasing pH. Consideration should therefore be given to 

reducing the inoculum cell density and adding additional sodium bicarbonate to the 

medium... Mayer et al. (2000) described a system using completely closed flasks with no gas 

phase in which gaseous CO2 is simply replaced with NaHCO3 and pH is adjusted with HCL.”
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Summary of Quoted Material: Modified test procedures may be required for highly volatile 

substances in accordance with OECD recommendations. Vessels that are used for preparation of 

test solutions and exposure should be sealed (with stoppers that are not permeable to air) and 

contain minimal headspace. When an algae test is conducted in a closed system, modifications 

of standard methods (e.g., reduction of initial cell density, addition of sodium bicarbonate, and 

pH adjustment), as described by Mayer et al. (2000), may be necessary to prevent limitation of 

algal growth due to depletion of CO2. Samples for test concentration analyses should be 

collected with minimal exposure to air (e.g., using syringe pumps) and placed in vials without 

headspace. Interpretation: To adequately conduct an algal inhibition test with a highly volatile 

test substance, test procedures must be modified to minimize losses during test solution 

preparation, exposure, and sample collection and analysis. 

[D] Deviation 4: There was no evidence that the solubility of the test substance in the growth 

medium was determined prior to initiation of the algae test; the study report did not provide a 

value for the solubility limit in the growth medium.

OECD TG 201, p. 2, Section “Information on the Test Substance”, item 10 states: “The 

water solubility, octanol water partition coefficient (Pow) and vapor pressure of the test 

substance should be known...”

OECD TG 201, p. 11, Section “Data and reporting”, Sub-section “Test report”, item 61 

states: “The test report must include the following:” and sub-item “Test substance” states: 

“physical nature and relevant physical-chemical properties, including water solubility limit.”

OECD 23, p. 20, Section 3.1 “Poorly water-soluble substances”, Sub-section 3.1.1 

“Preliminary experiment” states: “A preliminary experiment should be considered for all 

studies with poorly water-soluble substances to determine the maximum dissolved 

concentration that can be achieved under test conditions, which is defined as the saturation 

concentration, and the preparation conditions that are required to achieve it. The results of the 

preliminary experiment will form a basis of and justification for the media preparation 

procedures adopted for the toxicity tests and a reference point (i.e. water solubility under test 

conditions) against which the test results can be evaluated. In view of its importance the 

preliminary experiment should be fully reported.”

OECD 23, p. 25, Section 3.2 “Substances that are not acutely toxic at the limit of their 

water solubility” states: “It is important to recognise that the maximum achievable 

dissolved concentration of a substance in the test medium, i.e. saturation concentration, may 

not be the same as the water solubility of the substance as determined by, for example, OECD 

Guideline 105. Typically, the concentration will be less. It is also important to note that 

water solubility measurements made for regulatory purposes are usually made in distilled 

water (pH=6-9) and not test media (pH=7-8) and that differences in pH of the test media and 

distilled water may significantly affect the solubility, especially of ionised substances with a 

pKa between 5 and 9.”

Summary of Quoted Material: The solubility of the test substance in the growth medium 

should be determined prior to initiation of a toxicity to algae test. The solubility of the test 

substance must be provided in the study report. Interpretation: It is important to know the

solubility of the test substance in the growth media so that the appropriateness of the media 

preparation procedures can be assessed. Data describing test substance solubility in the growth 
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medium that are generated prior to the study are useful in optimizing the study design; without 

such data, an optimal design is difficult to achieve.

REFERENCES

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

green algae Pseudokirchneriella subcapitata determined in a growth inhibition study. 

ChemService S.r.l. Controlli e Ricerche, Novate Milanese, Italy. Study No. CH-095/2013. 

Sponsor: Finchimica S.p.A., Manerbio, Italy. Study report available online at 

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

6, 2014.

Mayer, P., Nyholm, N., Verbruggen, E.M.J., Hermens, J.L.M., and Tolls, J. 2000. Algal growth 

inhibition test in filled, closed bottles for volatile and sorptive materials. Environmental 

Toxicology and Chemistry 19: 2551-2556.