## CiToxLAB Hungary Ltd.

Veszprém

- Szabadságpuszta



# FINAL REPORT

Growth inhibition Test with

B enzene , 1

- chloro

- 4

- (trifluoromethyl)

- on Algae ( Pseudokirchneriella subcapitata )

Study code: 12 / 056

- 022AL

Study Director: Krisztina Sipos, M.Sc.

1 9 March 201 3Study code: 12/056

- 022AL Final Report Page 5 of 2 3

GENERAL INFORMATION

STUDY TITLE : Growth inhibition test with

B enzene

, 1

- chloro

- 4

- (trifluoromethyl)

- on Algae ( Pseudokirchneriella subcapitata )

TEST ITEM : B enzene

, 1

- chloro

- 4

- (trifluoromethyl)

-

SPONSOR : PCBTF Testing Consortium

c/o Greenwich Chemical Consulting, Inc.

Address: 3300 PGA Bouleva rd, Suite 635

Palm Beach Gardens, FL 33410

USA

S TUDY 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

. ( Forme rly 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 2 3

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 23 rd 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 Ju ly 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,

Authoris ation 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 2 3

CONTENTS

Page

1. SUMMARY 8

2. INTRODUCTION 9

3. MATERIALS AND METHOD S 9

3.1. TEST ITEM, CONTROLS AND VEHICLE 9

3.2. EXPERIMENTAL ORGANIS MS 11

3.3. TEST CONDITIONS 11

3.4. DESCRIPTION OF THE T EST PROCEDURE 12

UNTREATED CONTROL 13

3.5. OBSERVATIONS 14

3.6. ANALYTICAL MEASUREME NTS 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

3.1 1

. REFERENC ES 18

4. RESULTS 19

4.1. VALIDITY 19

4.2. CONCENTRATION OF THE T EST ITEM 19

4.3. CELL NUMBERS 19

4.4. AVERAGE SPECIFIC GRO WTH RATES 20

4.5. AREAS UNDER THE GROW TH CURVES 21

4.5. YIELD 21

5. CONCLUSION 22

APPENDICES 2 3

Tes t Conditions 1 of Appendix 1

Cell Number (x 10 4 cell/mL) 1 of A ppendix 2

Analytical Investigation of Main Study (Methods and Results) 1

– 4 o f Appendix 3

Composition of OECD Medium 1 of Appendix 4

Copy of the GLP Certificate s 1

- 2 of Appendix 5

Copy o f the Certificate of Analysis 1 of Appendix 6Study code: 12/056

- 022AL Final Report Page 8 of 2 3

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 test s , 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 measur ed 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

- Tes t ( **α** = 0.05) by TOXSTAT software.

The E

r C 50, E b C 50 and E

y C 50 value s 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 o f Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- were the following:

The 72h E b C 50 value (biomass): > 100 % v/v saturated solution

The 72h E

r C 50 value (growth rate): > 100 % v/v saturated solution

The 72h E

y C 50 value (yield): > 100 % v/v saturated solution

The N o

- 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 effe ct at saturation ( 3.0 mg/L, measured) ; the EC 50 results and the LOEC are higher than the solubility level of the test item in the

test medium.Study code: 12/056

- 022AL Final Report Page 9 of 2 3

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 satu ration 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 det ermined 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 METHOD S

3.1. TEST ITEM, CONTROLS AND VEHICLE

3.1.1. Test item

Name: Benz ene, 1

- chloro

- 4

- (trifluoromethyl)

- Synonym: PCBTF

CAS Number: 98

- 56

- 6

Batch/Lot number: 20111028

Molecular formula: C 7 H 4 ClF 3 Molecular weight: 180.56

Appearance: Clear colourless liquid

Purity: 99.69%

Manufacture date: 28 October 2011

Expiry date: 28 Octob er 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 CiTox LAB Hungary Ltd

. on the basis of the information

provided by the Spo nsor.Study code: 12/056

- 022AL Final Report Page 10 of 2 3

3.1. 2

. Dilution and Preparation of Testing Solutions

Because the test item is very poorly s oluble in water

, a test solution was prepared using

a saturated solution method (WAF method according to the G uidance Document on

Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23). A

supersaturated test item solution (nominally 10 0 mg/L) was prepared by

dispersing/dissolving the amount of test item into the t est m edium ( 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

material s were removed by filtrat ion 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

F or 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 r eference item

Potassium dichromate is ( Batch Number: 0769128) : 16

- 19 January 2012

.

The 72h E

r C 50 : 0.98 mg/L, (95 % confidence limits: 0.91

– 1.05 mg/L)

The 72h E b C 50 : 0.70 mg/L, (95 % confidence limits: 0.65

– 0.76 mg/L)

The 72h E

y C 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 a lgal g rowth m edium ( OECD medium, according to OECD 201) was

used as dilution water for both the ran ge 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 2 3

3. 2

. EXPERIMENTAL ORGANIS MS

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 Al gal

Cultures, Inst. Plant Physiology, Un iversity 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 subcapit ata 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 10 3 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 p eriod 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 10 7 cells/mL.

3. 3

. TEST CONDITIONS

3. 3 .1. Parameters in the Study

Temperature

Culture temperature w as 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 2 2

. 2

– 2 2

. 9 °C meas ured in the flask and between 2 2

. 0

and 2 3

. 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 a nd

controls were in the same range

, it was 7. 0 0 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 2 3

Light Inten sity

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

position occupied by algal culture flasks during the test was about 8 209 lux (equivalent

to 1 11 **μ** E/m 2 /s) , which was ensured with fluorescent lamps (with a spectral ran ge 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 T EST PROCE DURE

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.

T he link between algal growth, the dissolved CO 2 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 s ealed 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 NaHCO 3 (to replace the gaseous CO 2 )

, the pH will be adjusted to 7.0 by addition of HCl, and the resulting CO 2 concentration

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

The test was started (0 hours) by inoculation of a biomass of approximately 3 x 10 3

algal cells per mL test medium. ( T his slightly lower level did not affect the perfo rmance

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 co v ered with air

permeable 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 conce n tration and six re plicates in the control

groups.Study code: 12/056

- 022AL Final Report Page 13 of 2 3

3.4.1. Preliminary Range Finding Test

Two concentration range

- finding test s w ere 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

conce n tration and three replicates i n the control group , while the second test

(modified) w as performed with three replicates per each test conce n tration 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 disp ersed/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 test s are

summarised in the follo wing table s

.

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 10 4 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 10 4 cell/mL) 54.00 57.00 56.67 56.33 56.00 53.33

3. 4

. 2

. Test item concentrations in the Definitive T est

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

performed preliminary test s , 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 2 3

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 obser vation of the algal cells in each concentration and in the control was

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

3.6. ANALYTICAL MEASUREME NTS

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 co ncentration 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 co ncentration 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 p e riod

Biomass (b) : the actual number of cells per volume of m e dium (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

E b C 50 : the calculated conce ntration of test item which results in a 50 %

reduction of biomass (b) relative to the control

E

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

reduction of growth rate (μ) relative to the control

E

y C 50 : the calculated concentrati on of test item which results in a 50 %

reduction of yield (y) relative to the controlStudy code: 12/056

- 022AL Final Report Page 15 of 2 3

NOEC : ( N o O bserved E ffect C oncentration) the highest test concentration

at which no significant inhibition of growth is observed relative to

the control

LOEC : ( L owes t O bserved E ffect C oncentration) the lowest test

concentration at which a si g nificant inhibition of growth is observed

relative to the control

3.7.2. Calculation of Average Specific Growth Rate

Concentration

- effect relationship was calculated by compar ing 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 n t N

n N

**− −** 

**=**

Where ln (N

n ) = natural logarithm of measured number of cell s/mL at time t

n ln (N 0 ) = natural logarithm of measured number of cells/mL at time t 0

t 0 = time (hour) of the beginning of the test

t

n = time (hour) of n th measurements after the beginning of the test

The percentage inhibition of growth rate (% I
**μ** ):

100 I % t

**⋅ −** 

**=**

c c

**μ μ**  **μ**

**μ** %

Where % I
**μ** = percent inhibition in average specific growth rate

**μ** c = mean growth rate of the control

**μ** t = mean growth rate of test concentration t

3.7.3. Calculation of Area Under the Growth Curve

) (
2 2 ) (
2 2

2 1 0 1

1 2 0 2 1

1 0 1

**− −**  **−**

**− ⋅**  **− +** 

**+**

**− ⋅**  **− +** 

**+**

**⋅ =**  n n n n t t N N N

t t N N N

t N N A

Wh ere N 0 = nominal number of cells/mL at time t 0 (start of the test)

N 1 = mean measured number of cells/mL at t 1 (24 hours)

N 2 = mean measured number of cells/mL at t 2 (48 hours)

N

n = mean measured number of cells/mL at t

n

t 1 = time of first measurement after start of the test

t 2 = time of second measurement after start of the test

t

n = time of n th measurement after start of the testStudy code: 12/056

- 022AL Final Report Page 16 of 2 3

The percentage inhibition of area (% I A ):

% 100

**⋅ −** 

**=**

c t c

A A A I

A %

Where % I A = percent inhibition in area under th e growth curve

A

c = mean area of the control

A
t = 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 (% I y ):

100

y y y I

y **⋅ −** 

**=**

c i c %

where: y c = mean value for yield in the control group

y

i = mean value for yield for the test concentration

Area und er 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 w as 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 EC 50 values of the test item and their confidence limits were not calculated , due to

the lack of toxic e ffects.

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 2 3

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 CiToxLA B 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 d iscarded.

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 2 3

3. 1 1

. R EFERENCES

1. OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga
and Cyanobacteria, Growth Inhibition Test", adopted 23 rd March, 2006.

2. Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the
purpose of its ad aptation 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 2 3

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 2 2

. 29 %.

The coefficient of variation of average specific growth rates duri ng 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 experime nt w as 100 % v/v

saturated solution

. The corresponding measured geometric mean test item

concentration w as 3.0 mg/L.

Test concentration w as analytically determined at the beginning of the tests and 24

- hour intervals thereafter

.

Biological results are rela ted to the geometric mean of the me asured test item

concentrations (see Table 3 .)

.

Table 3 : Calcula tion 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 24 th , 48 th , 72 nd 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 2 3

Figure 1 : Mean Values of Cell Numbers Plotted against Time

4. 4

. AVERAGE SPECIFIC GRO WTH 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 Inhi bition 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 0.0 903 0.0 0.0 753 0.0 0.0 722 0.0

100 % v/v saturated solution 0.0 847 6.2 0.0 737 2 .2 0.0 719 0. 5Study code: 12/056

- 022AL Final Report Page 21 of 2 3

4. 5

. AREAS UNDER THE GROW TH 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 signi ficantly 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 2.8Study code: 12/056

- 022AL Final Report Page 22 of 2 3

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 Ben zene, 1

- chloro

- 4

- (trifluoromethyl)

- were the following:

The 72h E b C 50 value (biomass): > 100 % v/v saturated solution

The 72h E

r C 50 value (growth rate): > 100 % v/v saturated solution

The 72h E

y C 50 value (yield): > 100 % v/v saturated solution

The No

- Obs erved 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 EC 50 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 2 3



## APPENDICESStudy code: 1 2/056

- 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

T est 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. 0 0 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 2 2

. 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. 0Study code: 1 2/056

- 022AL Final Report Page 1 of Appendix 2

APPENDIX 2

CELL NUMBER

Table 9 : Cell N umber (x 10 4 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 1.8Study code: 1 2/056

- 022AL Final Report Page 1 of Appendix 3

APPENDIX 3

ANALYTICAL INVESTIGA TIONS 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 LaCh rom HPLC system

D

- 7000 Interface, No.: 1 442

- 1 22

L

- 7100 HPLC pump, No. : 1 516

- 0 30

L

- 7200 Autosampler, No.: 1 406

- 0 05

L

- 7400 UV Detector, No.: 1 502

- 0 17

L

- 7614 Degasser, No.: 14412YA0500

L

- 7360 Column Oven, No.: 00107295

Balance: BP221S Sartorius, No.: 1 1809117

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

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

Refrigerator: Zanussi, No.: ZLKI

- 262

Freezer: Zanussi, No.: 1154801265Study code: 1 2/056

- 022AL Final Report Page 2 of Appendix 3

2.2. Test Item

Name: Benzene, 1

- chloro

- 4

- (trifluoromethy l)

- Synonym: PCBTF

CAS Number: 98

- 56

- 6

Batch/Lot number: 20111028

Molecular formula: C 7 H 4 ClF 3 Molecular weight: 180.5 6

Appearance: Clear colourless liquid

Purity: 99.69%

Manufacture date: 28 October 2011

Expiry date: 28 October 2013

Storage conditions: Roo m 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 c oncentration

Samples were analysed by the fol lowing 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: 1 2/056

- 022AL Final Report Page 3 of Appendix 3

4. Results of the Method Valid ation

Table 1: Method va lidation results (12/056

- 316AN )

Selectivity No interfering component was

observed

Reinjection repeatability

(7 injections) CV% ≤ 2

. 5 %

Linear range 0. 1

– 2 0 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. Cal ibration

Table 2: Data of the regression lines

Date of measurement Intercept Slope Correlation

coefficient (r 2 )

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 1.000Study 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 growt h 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) NH 4 Cl 15.0 mg/L

MgCl 2 × 6 H 2 O 12.0 mg/L

CaCl 2 × 2 H 2 O 18.0 mg/L

MgSO 4 × 7 H 2 O 15.0 mg/L

KH 2 PO 4 1.6 mg/L

Stock solution 2

(iron) FeCl 3 × 6 H 2 O 64.0 **μ** g/L

Na 2 EDTA × 2H 2 O 100.0 **μ** g/L

Stock solution 3

(trace elements) H 3 BO 3 185.0 **μ** g/L

Mn Cl 2 × 4 H 2 O 415.0 **μ** g/L

ZnCl 2 3.0 **μ** g/L

CoCl 2 × 6 H 2 O 1.5 **μ** g/L

CuCl 2 × 2 H 2 O 0.01 **μ** g/L

Na 2 MoO 4 × 2 H 2 O 7.0 **μ** g/L

Stock solution 4

(bicarbonate) NaHCO 3 50.0 mg /LStudy code : 12/056

- 022AL Final Report Page 1 of Appendix 5

APPENDIX 5

COPY OF THE GLP CERT IFICATE S

The facility name until 1 st September 2 011 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. ) un til the certificate expires (16

December 2012. )Study code : 12/056

- 022AL Final Report Page 2 of Appendix 5

COPY OF THE GLP CERT IFICATE SStudy code: 12/056

- 022AL Final Report Page 1 of Appendix 6

COPY OF THE CERTIFIC ATE OF ANALYSIS