CiToxLAB Hungary Ltd.

Veszprém - Szabadságpuszta

FINAL REPORT

Growth inhibition Test with

Benzene, 1-chloro-4-(trifluoromethyl)-

on Algae (Pseudokirchneriella subcapitata)

Study code: 12/056-022AL

Study Director: Krisztina Sipos, M.Sc.

19 March 2013



Study code: 12/056-022AL Final Report Page 5 of 23

GENERAL INFORMATION

STUDY TITLE : Growth inhibition test with

Benzene, 1-chloro-4-(trifluoromethyl)-

on Algae (Pseudokirchneriella subcapitata)

TEST ITEM : Benzene, 1-chloro-4-(trifluoromethyl)-

SPONSOR : PCBTF Testing Consortium

c/o Greenwich Chemical Consulting, Inc.

Address: 3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

USA

STUDY MONITOR : Mr. John V. Massingale

Greenwich Chemical Consulting, Inc.

Address: 3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

USA

TEST FACILITY : CiToxLAB Hungary Ltd.

(Formerly known as LAB Research Ltd. before 1st September 

2011)

Address: H-8200 Veszprém, Szabadságpuszta

Hungary

Phone: 36 88 545-300

Fax: 36 88 545-301

STUDY DIRECTOR : Krisztina Sipos, M.Sc.

QUALITY ASSURANCE : Szabolcs Gáty, M.Sc.

RESPONSIBLE PERSON FOR

ANALYTICAL MEASUREMENTS : Zsolt Sárvári, M.Sc.
Study code: 12/056-022AL Final Report Page 6 of 23

TECHNICAL STAFF : Ecotoxicological staff

START OF EXPERIMENT : 04 December 2012

END OF EXPERIMENT : 07 December 2012

BASIS OF STUDY:

– OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga 

and Cyanobacteria, Growth Inhibition Test", adopted 23rd March, 2006.

– EPA Ecological Effects Test Guidelines, OPPTS 850.5400, Algal Toxicity, Tiers I 

and II, EPA 712-C-96-164, April 1996

– Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the 

purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the 

European Parliament and of the Council on the Registration, Evaluation, 

Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.3 

(published in the Official Journal of the European Union L 220 of 24 August 2009)
Study code: 12/056-022AL Final Report Page 7 of 23

CONTENTS

Page

1. SUMMARY 8

2. INTRODUCTION 9

3. MATERIALS AND METHODS 9

3.1. TEST ITEM, CONTROLS AND VEHICLE 9

3.2. EXPERIMENTAL ORGANISMS 11

3.3. TEST CONDITIONS 11

3.4. DESCRIPTION OF THE TEST PROCEDURE 12

UNTREATED CONTROL 13

3.5. OBSERVATIONS 14

3.6. ANALYTICAL MEASUREMENTS 14

3.7. RESULTS EVALUATION 14

3.8. ARCHIVES 17

3.9. DISTRIBUTION OF THE FINAL REPORT 17

3.10.DEVIATIONS FROM THE STUDY PLAN 17

3.11.REFERENCES 18

4. RESULTS 19

4.1. VALIDITY 19

4.2. CONCENTRATION OF THE TEST ITEM 19

4.3. CELL NUMBERS 19

4.4. AVERAGE SPECIFIC GROWTH RATES 20

4.5. AREAS UNDER THE GROWTH CURVES 21

4.5. YIELD 21

5. CONCLUSION 22

APPENDICES 23

Test Conditions 1 of Appendix 1

Cell Number (x 104 cell/mL) 1 of Appendix 2

Analytical Investigation of Main Study (Methods and Results) 1 – 4 of Appendix 3

Composition of OECD Medium 1 of Appendix 4

Copy of the GLP Certificates 1 - 2 of Appendix 5

Copy of the Certificate of Analysis 1 of Appendix 6
Study code: 12/056-022AL Final Report Page 8 of 23

1. SUMMARY

The effect of Benzene, 1-chloro-4-(trifluoromethyl)- test item was assessed on algal 

growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum 

capricornutum), over an exposure period of 72 hours.

This study was performed based on the U.S. Toxic Substances Control Act (TSCA) 

Section 4 Test Rule and the regulations set forth at 40 CFR 799.5089.

Because no toxic effect was observed at the solubility level of the test item in the 

performed preliminary tests, only this test concentration (100 % v/v saturated solution)

at the solubility level of the test item in the test medium and one control group were

tested in a limit test in the definitive study.

The corresponding measured geometric mean concentration (based on the analytical 

measurements) was 3.0 mg/L.

The test design included eight replicates (six for observations and two replicates for 

analytical measurements) at test concentration and six replicates for the untreated 

control.

Statistical comparisons of biomass, average specific growth rates and yield in control 

and in treated groups were carried out using analysis of variance (ANOVA) and 2 

Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item were determined directly from the 

raw data (because of the lack of toxic effects).

All validity criteria were met during this experiment.

Under the conditions of this algal growth inhibition test the observed endpoints 

for the effect of Benzene, 1-chloro-4-(trifluoromethyl)- were the following:

The 72h EbC50 value (biomass): > 100 % v/v saturated solution

The 72h ErC50 value (growth rate): > 100 % v/v saturated solution

The 72h EyC50 value (yield): > 100 % v/v saturated solution

The No-Observed Effect Concentration (NOEC): 100 % v/v saturated solution

The Lowest Observed Effect Concentration (LOEC): > 100 % v/v saturated solution

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.
Study code: 12/056-022AL Final Report Page 9 of 23

2. INTRODUCTION

The purpose of the study was to determine the effect of the test item Benzene, 1-

chloro-4-(trifluoromethyl)- on the growth of the unicellular green algal species 

Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum).

Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to a 

concentration at saturation over several generations under defined conditions to 

demonstrate that the test item has no influence on the growth of Pseudokirchneriella 

subcapitata up to at least this test concentration.

The algal growth in relation to a control culture was determined over a fixed period of 

72 hours. The method of application and the test species Pseudokirchneriella 

subcapitata was recommended by the test guidelines.

3. MATERIALS AND METHODS

3.1. TEST ITEM, CONTROLS AND VEHICLE

3.1.1. Test item

Name: Benzene, 1-chloro-4-(trifluoromethyl)-

Synonym: PCBTF

CAS Number: 98-56-6

Batch/Lot number: 20111028

Molecular formula: C7H4ClF3

Molecular weight: 180.56

Appearance: Clear colourless liquid

Purity: 99.69%

Manufacture date: 28 October 2011

Expiry date: 28 October 2013

Storage conditions: Room temperature

The test item of a suitable chemical purity was supplied by the Sponsor. All precautions

required in the handling and disposal of the test item were outlined by the Sponsor. 

These documents are part of the raw data. Identification of the test item was performed 

in the Central Dispensary of CiToxLAB Hungary Ltd. on the basis of the information 

provided by the Sponsor.
Study code: 12/056-022AL Final Report Page 10 of 23

3.1.2. Dilution and Preparation of Testing Solutions

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

a saturated solution method (WAF method according 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).

3.1.3. Reference Control

For the evaluation of the quality of the algae and validation of the experimental 

conditions, Potassium dichromate is tested at least twice a year to demonstrate 

satisfactory test conditions.

The date of the last study (Study Code: 11/357-022AL) with the reference item 

Potassium dichromate is (Batch Number: 0769128): 16 - 19 January 2012. 

The 72h ErC 50 : 0.98 mg/L, (95 % confidence limits: 0.91 – 1.05 mg/L) 

The 72h EbC 50 : 0.70 mg/L, (95 % confidence limits: 0.65 – 0.76 mg/L)

The 72h EyC 50 : 0.68 mg/L, (95 % confidence limits: 0.62 – 0.73 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 

8692).

3.1.4. Untreated Control

The dilution water (OECD medium) was used without of addition of the test item.

3.1.5. Dilution Water

Reconstituted algal growth medium (OECD medium, according to OECD 201) was 

used as dilution water for both the range finding and definitive tests.

The composition of the growth medium and the chemicals used are given in Appendix 4.
Study code: 12/056-022AL Final Report Page 11 of 23

3.2. EXPERIMENTAL ORGANISMS

Species: Pseudokirchneriella subcapitata

(formerly known as Selenastrum capricornutum)

Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; 

ATCC 22662)

Source: The algae were supplied by the SAG: Collection of Algal 

Cultures, Inst. Plant Physiology, University of Göttingen, 

GERMANY. Cultured under standardised conditions (see 

OECD 201) in the Ecotoxicological Laboratory of 

CiToxLAB Hungary Ltd.

Justification of species: The species of Pseudokirchneriella subcapitata used, 

being a fast-growing species, is convenient for culturing 

and testing and is a recommended species by relevant 

guidelines.

Initial cell number: The initial cell number in the test cultures was 

3 x 103 cells/mL.

Pre-culturing: The pre-culture was intended to give an amount of alga 

suspension suitable for the inoculation of test cultures. 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 cell count of above culture was determined by 

microscopic method and this cell suspension was diluted with 

Algal Growth Medium to 107 cells/mL.

3.3. TEST CONDITIONS

3.3.1. Parameters in the Study

Temperature

Culture temperature was checked at the beginning of the study and every 24 hours in a 

flask filled with water, in the climatic chamber. In addition, water temperature was 

continuously measured (with a min/max thermometer) within the climate chamber. The 

temperature was in the range of 22.2 – 22.9 °C measured in the flask and between 22.0

and 23.1 °C measured within the climate chamber.

pH

The pH was checked at the beginning and at the end of the study, in the control and in 

test solution. The average pH of the control medium was not increased by more than

1.5 units during the test. The pH of the test solutions containing the test material and 

controls were in the same range, it was 7.00 at the start and the range of the pH was 

8.36 – 8.46 at the end of the study.
Study code: 12/056-022AL Final Report Page 12 of 23

Light Intensity

The algal culture flasks were continuously illuminated. The light intensity at the 

position occupied by algal culture flasks during the test was about 8209 lux (equivalent 

to 111 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 

400-700 nm). The differences in light intensity between the test vessels did not exceed 

± 15 % and therefore provided equal conditions for each test vessel.

The data of test conditions are detailed in Appendix 1.

3.4. DESCRIPTION OF THE TEST PROCEDURE

Taking into account that the test item is a highly volatile substance the study was

performed in a closed system followed the OECD recommendations.

A simple filled closed bottle test with low algal densities and bicarbonate enrichment 

was performed to minimize the loss of test substance from solution.

The link between algal growth, the dissolved CO2 concentration and the pH of the algal 

medium is crucial for reproducible algal growth inhibition tests. To prevent such 

limitation of algal growth in a sealed test system, the study was performed based on the 

test procedure set forth in Mayer et al., 2000:

The algae medium was enriched with 300 mg/L NaHCO3 (to replace the gaseous CO2),

the pH will be adjusted to 7.0 by addition of HCl, and the resulting CO2 concentration

was sufficient to support algal growth in the absence of headspace.

The test was started (0 hours) by inoculation of a biomass of approximately 3 x 103

algal cells per mL test medium. (This slightly lower level did not affect the performance 

of the test.)

Volumes of 60 mL algal suspension per replicate, in steril plastic flasks, were

continuously shaken by a laboratory orbital shaker. The flasks were covered with airpermeable stoppers. Each test vessel were uniquely identified with at least study code, 

test group and replicate.

Because the test item is not stable over the 72h exposure period, additional samplings 

for analysis at 24 hour intervals were used in order to better define loss of the test 

substance during the exposure period.

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.
Study code: 12/056-022AL Final Report Page 13 of 23

3.4.1. Preliminary Range Finding Test

Two concentration range-finding tests were conducted to determine the approximate 

toxicity of the test item so that appropriate test concentrations can be selected for use 

in the definitive test. Algal cells were exposed to each concentration of the test item 

plus a control, for 72 hours using the standard method according to the Study Plan in 

the first case, while a modified method was used in the second case (see details in 

Section 3.4.). The first test was performed with two replicates per each test 

concentration and three replicates in the control group, while the second test 

(modified) was performed with three replicates per each test concentration and also in 

the control group.

For the test concentration with nominal loading rates of 100 mg/L the required amount 

of test item was dispersed/dissolved into the test medium and handled as described in 

Sections 3.1.2 and 3.4. For the lower test concentrations a dilution of the 100 % v/v 

saturated solution were prepared. The results (72 h) of the preliminary tests are 

summarised in the following tables.

Table 1.: Results of the first Preliminary Test

Nominal loading rate

[% v/v saturated solution]

Untreated

control 0.1 1.0 10.0 100.0

Average of cell number

at 72 hours (x 104

cell/mL) 79.33 76.00 75.00 73.00 71.50

Table 2.: Results of the second Preliminary Test 

Nominal loading rate

[% v/v saturated solution]

Untreated

control 0.1 1.0 10.0 50.0 100.0

Average of cell number

at 72 hours (x 104

cell/mL) 54.00 57.00 56.67 56.33 56.00 53.33

3.4.2. Test item concentrations in the Definitive Test

Because no toxic effect was observed at the solubility level of the test item in the 

performed preliminary tests, only this test concentration (100 % v/v saturated solution)

at the solubility level of the test item in the test medium and one control group were

tested in a limit test in the definitive study.
Study code: 12/056-022AL Final Report Page 14 of 23

3.5. OBSERVATIONS

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 algal cells in each concentration and in the control was 

performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

3.6. ANALYTICAL MEASUREMENTS

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.

The samples were analysed by HPLC system.

Methods and results of test item concentration analysis are described in Appendix 3.

3.7. RESULTS EVALUATION

3.7.1. Definitions

Cell Density: the number of cells per mL

Growth: the increase of cell density over the test period

Biomass (b): the actual number of cells per volume of medium (cells/mL) 

calculated as the area under the growth curve (A)

Yield (y): the cell density at the end of the test minus the starting cell density

Average Specific 

Growth Rate (μ): the increase in cell density per time unit

EbC50: the calculated concentration of test item which results in a 50 % 

reduction of biomass (b) relative to the control

ErC50: the calculated concentration of test item which results in a 50 % 

reduction of growth rate (μ) relative to the control

EyC50: the calculated concentration of test item which results in a 50 % 

reduction of yield (y) relative to the control
Study code: 12/056-022AL Final Report Page 15 of 23

NOEC: (No Observed Effect Concentration) the highest test concentration 

at which no significant inhibition of growth is observed relative to 

the control

LOEC: (Lowest Observed Effect Concentration) the lowest test 

concentration at which a significant inhibition of growth is observed 

relative to the control

3.7.2. Calculation of Average Specific Growth Rate

Concentration-effect relationship was calculated by comparing growth rates in control, 

test cultures in the following way.

The average specific growth rate (μ) for individual cultures are calculated from the 

following relationship:

μ

0

) 0 ln( ) ln(

t nt

N n N

−

− =

Where ln (Nn) = natural logarithm of measured number of cells/mL at time tn

ln (N0) = natural logarithm of measured number of cells/mL at time t0

t0 = time (hour) of the beginning of the test

tn = time (hour) of nth measurements after the beginning of the test

The percentage inhibition of growth rate (% Iμ):

% I 100 t ⋅ − = c

c

μ

μ μ

μ %

Where % Iμ = percent inhibition in average specific growth rate

μc = mean growth rate of the control

μt = mean growth rate of test concentration t

3.7.3. Calculation of Area Under the Growth Curve

( ) 2

2 ( ) 22

2

1

1 0

2 1

1 2 0

1

1 0

−

− −

⋅ − + − ⋅ − +

+ − = ⋅ + n n

n n

t t

N N N

t t

N N N

t

N N A

Where N0 = nominal number of cells/mL at time t0 (start of the test)

N1 = mean measured number of cells/mL at t1 (24 hours)

N2 = mean measured number of cells/mL at t2 (48 hours)

Nn = mean measured number of cells/mL at tn

t1 = time of first measurement after start of the test

t2 = time of second measurement after start of the test

tn = time of nth measurement after start of the test
Study code: 12/056-022AL Final Report Page 16 of 23

The percentage inhibition of area (% IA):

% ⋅100 − = c

c t

A

A A I A %

Where % IA = percent inhibition in area under the growth curve

Ac = mean area of the control

At = mean area of test concentration t

3.7.4. Calculation of Yield

Yield is calculated as the biomass at the end of the test minus the starting biomass for each 

single vessel of controls and treatments. For each test concentration and control, mean yield 

values were calculated.

Percent inhibition in yield (% Iy):

100

y

y y Iy ⋅ − = c

c i

%

where: yc = mean value for yield in the control group

yi = mean value for yield for the test concentration

Area under the growth curve (biomass), average specific growth rate and yield were 

calculated for each test flask. Then the mean area under the growth curve, the growth 

rate and mean yield were determined as arithmetic mean value over all test flasks per 

treatment.

3.7.5. Statistical Analysis

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) and to demonstrate exponential growth for the entire study period.

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). 

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

the lack of toxic effects.

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 2 

Sample t-Test (α = 0.05) by TOXSTAT software.
Study code: 12/056-022AL Final Report Page 17 of 23

3.8. ARCHIVES

The study documents:

- study plan and amendment,

- all raw data,

- sample of test item,

- study report, study phase report and any amendment

- correspondence

are stored according to the Hungarian GLP and to the CiToxLAB Hungary Ltd. SOP-s 

in the archives of CiToxLAB Hungary Ltd. 8200 Veszprém, Szabadságpuszta, 

Hungary.

After the retention time has elapsed all the archived materials listed above will be 

returned to the Sponsor or retained for a further period if agreed by a contract. 

Otherwise the materials will be discarded.

3.9. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PDF file

Archive: 1x original, bound

3.10. DEVIATIONS FROM THE STUDY PLAN 

There were no deviations encountered in the course of the test.
Study code: 12/056-022AL Final Report Page 18 of 23

3.11. REFERENCES

1. OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga 

and Cyanobacteria, Growth Inhibition Test", adopted 23rd March, 2006.

2. Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the 

purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the 

European Parliament and of the Council on the Registration, Evaluation, 

Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.3 

(published in the Official Journal of the European Union L 220 of 24 August 

2009)

3. EPA Ecological Effects Test Guidelines, OPPTS 850.5400, Algal Toxicity, Tiers I 

and II, EPA 712-C-96-164, Adopted April 1996

4. Directive 2004/10/EC of the European Parliament and of the Council of 11 

February 2004

5. OECD Principles of Good Laboratory Practice, adopted by Council on 26th 

November 1997 [C(97)186/Final], Environment Directorate, Organisation for 

Economic Cooperation and Development, ENV/MC/CHEM(98)17, Paris 1998

6. Hungarian Good Laboratory Practice Regulations: 9/2001 (III. 30) EÜM-FVM joint 

decree of the Minister of Health and the Minister of Agriculture and Regional 

Development which corresponds to the OECD GLP ENV/MC/CHEM (98) 17

7. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series 

on Testing and Assessment No. 23, Paris September 2000.

8. Mayer, P., Nyholm, N., Verbruggen, E.M.J., Hermens, J.L.M., and Tolls, J. 

2000Algal growth inhibition test in filled, closed bottles for volatile and sorptive 

materials. Enviromental Toxicology and Chemistry 19: 2551-2556.
Study code: 12/056-022AL Final Report Page 19 of 23

4. RESULTS

4.1. VALIDITY

The cell density in the control cultures was increased by a factor of 54.50 within three days.

The mean coefficient of variation for section-by-section specific growth rates 

(days 0-1; 1-2; 2-3) in the control cultures was 22.29 %.

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

period (day 0-3) in the control cultures was 0.70 %.

All validity criteria were met, therefore the study can be considered as valid.

4.2. CONCENTRATION OF THE TEST ITEM

The nominal concentration of test item used in the main experiment was 100 % v/v 

saturated solution. The corresponding measured geometric mean test item 

concentration was 3.0 mg/L.

Test concentration was analytically determined at the beginning of the tests and 24-

hour intervals thereafter.

Biological results are related to the geometric mean of the measured test item 

concentrations (see Table 3.).

Table 3: Calculation of exposure concentrations

Nominal 

concentration

Measured concentrations 

(mg/L) Geometric mean

start 24h 48h 72h/end mg/L

Control n.d. n.d. n.d. n.d. -

100 % v/v 7.04 3.00 3.15 1.26 3.0

n.d. – not detected

4.3. CELL NUMBERS

The cell number in each flask was determined at the 24th, 48th, 72nd hours. The results 

of determinations are detailed in Appendix 2.

The shape of the algal cells growing was obviously not affected.
Study code: 12/056-022AL Final Report Page 20 of 23

Figure 1: Mean Values of Cell Numbers Plotted against Time

4.4. AVERAGE SPECIFIC GROWTH RATES

The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that 

the 0-72 h average specific growth rate was not statistically significantly different from 

the untreated control value, accordingly the No Observed Effect Concentration (NOEC)

was determined as 100 % v/v saturated solution (3.0 mg/L, measured).

Table 4: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Test group

Growth rate μ and % inhibition of μ

0–24 h 0–48 h 0–72 h

μ % μ % μ %

Control 0.0903 0.0 0.0753 0.0 0.0722 0.0

100 % v/v saturated solution 0.0847 6.2 0.0737 2.2 0.0719 0.5
Study code: 12/056-022AL Final Report Page 21 of 23

4.5. AREAS UNDER THE GROWTH CURVES

The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 

0-72 h areas were not statistically significantly different from the untreated control value, 

accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % v/v 

saturated solution (3.0 mg/L, measured).

Table 5: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group

Areas under the Growth Curves (A) and % inhibition of A

0–24 h 0–48 h 0–72 h

A % A % A %

Control 28.4 0.0 187.2 0.0 968.0 0.0

100 % v/v saturated solution 24.4 14.1 169.2 9.6 922.0 4.8

4.5. YIELD

The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 

0-72 h yield was not statistically significantly different from the untreated control value, 

accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % v/v 

saturated solution (3.0 mg/L, measured).

Table 6: Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group

Yield (Y) and % inhibition of Y (0-72 h)

Y %

Control 54.2 0.0

100 % v/v saturated solution 52.7 2.8
Study code: 12/056-022AL Final Report Page 22 of 23

5. CONCLUSION

The effect of Benzene, 1-chloro-4-(trifluoromethyl)- test item was assessed on algal 

growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum 

capricornutum), over an exposure period of 72 hours at the highest achievable 

concentrations of test item.

Under the conditions of this algal growth inhibition test the observed endpoints 

for the effect of Benzene, 1-chloro-4-(trifluoromethyl)- were the following:

The 72h EbC50 value (biomass): > 100 % v/v saturated solution

The 72h ErC50 value (growth rate): > 100 % v/v saturated solution

The 72h EyC50 value (yield): > 100 % v/v saturated solution

The No-Observed Effect Concentration (NOEC): 100 % v/v saturated solution

The Lowest Observed Effect Concentration (LOEC): > 100 % v/v saturated solution

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.
Study code: 12/056-022AL Final Report Page 23 of 23

APPENDICES
Study code: 12/056-022AL Final Report Page 1 of Appendix 1

APPENDIX 1

TEST CONDITIONS

Table 7: pH-Values in the Test Media at the Start and End of the Test

Test Group pH-values

Start End

Control 7.00

8.46

8.42

8.40

8.40

8.38

8.44

100 % v/v saturated solution 7.00

8.36

8.41

8.42

8.44

8.45

8.40

Table 8: Temperature in the Climate Chamber and in the Test Media 

during the Test

Parameter Exposure Time

Day 0 Day 1 Day 2 Day 3

temperature (oC) measured in flask 22.2 22.5 22.5 22.9

min / max temperature (oC)

of the climate chamber

max 23.1 22.8 22.7 23.0

min 22.2 22.0 22.2 22.0
Study code: 12/056-022AL Final Report Page 1 of Appendix 2

APPENDIX 2

CELL NUMBER

Table 9: Cell Number (x 104 cell/mL)

Test group

Number of cells

0 h 24 h 48 h 72 h

Control

0.3 2 10 52

0.3 3 11 56

0.3 3 11 56

0.3 3 12 52

0.3 2 11 56

0.3 3 12 55

Mean 0.3 2.67 11.17 54.50

SD 0.0 0.5 0.8 2.0

100 % v/v saturated 

solution

0.3 2 10 56

0.3 3 11 52

0.3 2 9 53

0.3 3 11 54

0.3 2 11 52

0.3 2 10 51

Mean 0.3 2.33 10.33 53.00

SD 0.0 0.5 0.8 1.8
Study code: 12/056-022AL Final Report Page 1 of Appendix 3

APPENDIX 3

ANALYTICAL INVESTIGATIONS OF THE STUDY

(METHODS AND RESULTS)

1. Principle of the Analytical Method

Concentration of Benzene, 1-chloro-4-(trifluoromethyl)- in the alga test solutions was 

determined during the test.

The samples were taken from the test solutions containing the test item during the test and 

one sample from the control at the start and at the end. 

The samples were analysed by an HPLC-UV method described in details in the analytical 

method validation of Benzene, 1-chloro-4-(trifluoromethyl)- (12/056-316AN). 

2. Equipment and Chemicals

2.1. Apparatus

HPLC system: Merck-Hitachi LaChrom HPLC system

D-7000 Interface, No.: 1442-122

L-7100 HPLC pump, No. : 1516-030

L-7200 Autosampler, No.: 1406-005

L-7400 UV Detector, No.: 1502-017

L-7614 Degasser, No.: 14412YA0500

L-7360 Column Oven, No.: 00107295

Balance: BP221S Sartorius, No.: 11809117

Ultrasonic bath: Elmasonic S300H, ELMA, No.: 010890105

Water purification system: MILLIPORE, DIRECT Q3, FOMNO 7334I

Refrigerator: Zanussi, No.: ZLKI-262

Freezer: Zanussi, No.: 1154801265
Study code: 12/056-022AL Final Report Page 2 of Appendix 3

2.2. Test Item

Name: Benzene, 1-chloro-4-(trifluoromethyl)-

Synonym: PCBTF

CAS Number: 98-56-6

Batch/Lot number: 20111028

Molecular formula: C7H4ClF3

Molecular weight: 180.56

Appearance: Clear colourless liquid

Purity: 99.69%

Manufacture date: 28 October 2011

Expiry date: 28 October 2013

Storage conditions: Room temperature

2.3. Other materials

Ultra pure water (ASTM Type I): prepared by Direct-Q 3 system, Millipore

Acetonitrile: HPLC grade, Sigma-Aldrich, Batch: SZBC300SV

3. Method of analysis of test item concentration

Samples were analysed by the following HPLC-UV method:

Column: Zorbax Eclipse Plus C8 150 × 4.6 mm, 5μm

Mobil Phase: ACN : water = 7:3

Flow: 1.0 ml/min.

Injection volume: 10 μl

Temperature: 25 °C

Detector: UV at 220 nm 

Retention time: 4.6 min ± 10%
Study code: 12/056-022AL Final Report Page 3 of Appendix 3

4. Results of the Method Validation

Table 1: Method validation results (12/056-316AN)

Selectivity No interfering component was 

observed

Reinjection repeatability

(7 injections) CV% ≤ 2.5 %

Linear range 0.1 – 20 μg/ml

Limit of Quantification 0.1 μg/ml

Recovery 15 %

Precision 22 %

Stability of the samples At least 20 hours in the autosampler

Stock solution stability At least 7 days at 5 ± 3 °C

5. Measured data

5.1. Calibration 

Table 2: Data of the regression lines

Date of measurement Intercept Slope Correlation 

coefficient (r2

)

04 December 2012 588 19901 1.000

05 December 2012 65 19806 1.000

06 December 2012 -173 19509 1.000

07 December 2012 163 20122 1.000

Study code: 12/056-022AL Final Report Page 1 of Appendix 4

APPENDIX 4

COMPOSITION OF OECD MEDIUM

Separate stock solutions were first prepared in deionised water. The growth medium was 

prepared by adding an appropriate volume of these different stock solutions to deionised 

water in order to achieve the final concentrations.

Stock solution Substance Final concentration in the 

prepared growth medium

Stock solution 1

(macro nutrients)

NH4Cl 15.0 mg/L

MgCl2 × 6 H2O 12.0 mg/L

CaCl2 × 2 H2O 18.0 mg/L

MgSO4 × 7 H2O 15.0 mg/L

KH2PO4 1.6 mg/L

Stock solution 2

(iron)

FeCl3 × 6 H2O 64.0 μg/L

Na2EDTA × 2H2O 100.0 μg/L

Stock solution 3

(trace elements)

H3BO3 185.0 μg/L

MnCl2 × 4 H2O 415.0 μg/L

ZnCl2 3.0 μg/L

CoCl2 × 6 H2O 1.5 μg/L

CuCl2 × 2 H2O 0.01 μg/L

Na2MoO4 × 2 H2O 7.0 μg/L

Stock solution 4

(bicarbonate) NaHCO3 50.0 mg/L
Study code: 12/056-022AL Final Report Page 1 of Appendix 5

APPENDIX 5

COPY OF THE GLP CERTIFICATES

The facility name until 1st September 2011 was LAB Research Ltd. From that date, the registered name has been 

CiToxLAB Hungary Ltd., this information has been transmitted to the GLP competent authority. The above GLP 

certificate is valid for this facility (now known as CiToxLAB Hungary Ltd.) until the certificate expires (16 

December 2012.)
Study code: 12/056-022AL Final Report Page 2 of Appendix 5

COPY OF THE GLP CERTIFICATES
Study code: 12/056-022AL Final Report Page 1 of Appendix 6

COPY OF THE CERTIFICATE OF ANALYSIS