## CiToxLAB Hungary Ltd.

Veszprém

- Szabadságpuszta



# FINAL REPORT

Growth inhibition Test with

Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6]

(hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7])

on Algae ( Pseudokirchneriella subcapitata )

S tudy code: 11/ 3 39

- 022AL

Study Director: Krisztina Sipos, M.Sc.

1 9 March 2013Study code: 11/339

- 022AL Final Report Page 5 of 2 8

GENERAL INFORMATION

STUDY TITLE : Growth inhibition test with Ethanedioic acid,

hydrate (1:2 ) [CAS RN 6153

- 56

- 6] (hydrated

form of ethanedioic acid [CAS RN 144

- 62

- 7])

on Algae ( Pseudokirchneriella subcapitata )

TEST ITEM : Ethanedioic acid, hydrate (1:2)

[CAS RN 6153

- 56

- 6] (hydrated form of

ethanedioic acid [CAS RN 144

- 62

- 7])

SPONSOR S : Univa r USA Inc.

Address: 17425 NE Union Hill Road

Redmond, WA 98052

USA

Telephone: +1 425 889 3400

Facsimile: +1 425 889 3760

: WEGO Chemical and Mineral Corp.

Address: 239 Great Neck Road

Great Neck, NY 11021

USA

Telephone: +1 516 487 3510

Facsimile: +1 516 487 3794

S TUDY MONITOR : Mr. John V. Massingale

Greenwich Chemical Consulting, Inc.

Address: 3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

USA

Telephone: +1 561 578 8110

Facsimile: +1 561 578 8120

E

- mail: j ohn@grcci.com

TEST FACILITY : CiToxLAB Hungary Ltd

.

Address: H

- 8200 Veszprém, Szabadságpuszta

Hungary

Phone: 36 88 545

- 300

Fax: 36 88 545

- 301Study code: 11/339

- 022AL Final Report Page 6 of 2 8

STUDY DIRECTOR : Krisztina Sipos, M.Sc.

QUALITY ASSURANCE : Éva Makovi

- Fábián, B.Sc.

RESPONSIBLE PERSON FOR

ANALYTICAL MEASUREMENTS : Zsolt Sárvári

, M.Sc.

TECHNICAL STAFF : Ecotoxicological staff

START OF EXPERIMENT : 19 November 2012

END OF EXPERIMENT : 06 December 2012

BASIS OF STUDY :

– OECD Guideline for Testing of Chemicals, Section 2, No. 20 1: "Freshwater Alga

and Cyanobacteria, Growth Inhibition Test", adopted 2 8 th July , 20 11

.

– 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 Unio n L 220 of 24 August

2009)

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

and II, EPA 712

- C

- 96

- 164, Adopted April 1996

This study is being performed based on the U.S. Toxic Substances Control Act

(TSCA) Section 4 Test Rule and the regulations set forth at 40 CFR 799.508 7

.Study code: 11/339

- 022AL Final Report Page 7 of 2 8

CONTENTS

Page

1. SUMMARY 8

2. INTRODUCTION 10

3. MATERIALS AND METHOD S 10

3.1. TEST ITEM AND CONTRO LS AND VEHICLE 10

3.2. EXPERIMENTAL ORGANISMS 12

3.3. TEST CONDITIONS 13

3.4. DESCRIPTION OF THE T EST PROCEDURE 14

3.5. OBSERVATIONS 15

3.6. ANALYTICAL MEASUREME NTS 15

3.7. RESULTS EVALUATION 16

3.8. ARCHIVES 18

3.9. DISTRIBUTION OF THE FINAL REPORT 19

3.10

. REFERENCES 19

3.11

. DEVIATIONS FROM THE STUDY PLANS 20

4. RESULTS 21

4.1. VALIDITY 21

4.2. CELL NUMBERS 22

4. 3

. MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS 23

4. 4

. AVERAGE SPECIFIC GRO WTH RA TES 23

4. 5

. AREAS UNDER THE GROW TH CURVES 25

4. 6

. YIELD 26

5. CONCLUSION 27

APPENDICES 2 8

Test Conditions 1

- 2 of App endix 1

Cell Number 1

- 2 of A ppendix 2

Concentration / Effect relationship Graphs of the Test Item 1

- 2 of Appendix 3

Composition of OECD medium 1 of Appendix 4

Analytical Investigation of Main Study (Methods and Results) 1

- 4 of Appendix 5

Copy of the GLP Certificate 1 of Appendix 6

Copy of the Certificate of Analysis 1 of Appendix 7Study code: 11/339

- 022AL Final Report Page 8 of 2 8

1. SUMMARY

The effect of test item was assessed on algal growth using the unicellular green alga

Pseudokirchneriella subcapitata , over an exposure period of 72 h ours.

Because significant toxic response was observed during the preliminary range

- finding

test, five test concentrations in a geometric series (factor 2.0 ) and one control were

tested in the main experiment (definitive test, Phase 1)

.

The nominal concent rations of test item used in the definitive test (phase 1) were:

6.25; 12.5; 25; 50 and 10 0 mg /L

. The corresponding measured geometric mean test

item concentrations were: 4

. 5 ; 7

. 9 ; 19

. 7 ; 44 .9 and 89

. 7 mg/L.

Test concentrations was analytically determined at the start and at the end of the

experiment. As the analytically measured concentration deviated more than 20 per

cent from the nominal in most case s , the biological results are based on the measured

geometric mean concentration s

.

The test design includ ed three replicates at test concentration s and six replicates for

the untreated control (Phase 1)

.

Because there was evidence of marked change in the pH of the test water after

addition of the test substance, and the pH was below 6 in the definitive test (Phase 1)

at the two highest concentration levels of 50 and 100 mg/L , a complementary study

(Phase 2) was carried out, adjusting the pH of the stock solution to that of the dilution

water before addition of the test substance. We extend ed the study to prov ide more

data to allow a better interpretation.

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

Bonferroni t

- Test and Dunnett’s Test ( **α** = 0.05) by TOXSTAT software.

The E

r C 50, E b C 50 and E

y C 50 values of the test item were determined using Probit

analysis by SPSS PC+ software.Study code: 11/339

- 022AL Final Report Page 9 of 2 8

With respect to the inhibitory effect of the test item, the 0

- 72 h average specific

growth rates, yield and areas w ere significantly different from that of the

control group in the examined concentration range of 19.7

– 89.7 mg/L

( measured ). The overall NOEC was determined as 7

. 9 mg/L ( measured ); the

overall LOEC was determined as 19.7 mg/L ( measured ). The biological r esults

are summarised in the following table:

Table 1: Influence of Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated

form of ethanedioic acid [CAS RN 144

- 62

- 7]) on the Growth of

Pseudokirchneriella subcapitata

Parameter

(0

- 72 h) Growth rate ( r)

[mg/L] Yield (y)

[mg/L] Biomass (b)

[mg/L]

Calculation based on measured geometric mean concentrations

EC 50 27.17 24.28 24.28

95 % conf. limits 25.21

– 29.70 22.70

– 26.49 22.70

– 26.49

NOEC 7.9 7.9 7.9

LOEC 19.7 19.7 19.7

Based on the results of the performed definitive and complementary

experiments the observed effect can be considered to be related to the acid

character of the test item.Study code: 11/339

- 022AL Final Report Page 10 of 2 8

2. INTRODUCTION

The purpose of the study was to determine the effect of the test item 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

various concentrations of the test item over several gener ations under defined

conditions.

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.

VALI DITY CRITERIA OF THE STUDY :

- The cell concentration in the untreated control cultures should be increased by a

factor of at least 16 within three days (72 hours)

, corresponding to a specific

growth rate of 0.92 day

- 1

- The mean coefficient of variation for s ection

- by

- section specific growth rates

(days 0

- 1, 1

- 2 and 2

- 3, for 72

- h tests) in the control cultures must not exceed

35%.

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

period in replicate control cultures must not e xceed 7%.

3. MATERIALS AND METHOD S

3.1. TEST ITEM AND CONTROLS AND VEHICLE

3.1.1. Test item

Name: Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6]

(hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7])

Chemical name: Oxalic acid, hydrate

Synonym: Ox alic acid

Batch number: YP1

- 20120528C

CAS number: 6153

- 56

- 6

Molecular Formula: C 2 H 2 O 4 ×2H 2 O

Molecular Weight: 126.1 g/mol (dihydrate form)

Purity: 99.69 %

Description of the test item: White crystals

Manufacture date: May 28, 2012

Retest date: May 27, 2013

Storage condition: controlled room temperature (15

- 25oC)

Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses

and face mask) were applied to assure personnel health and

safety.Study code: 11/339

- 022AL Final Report Page 11 of 2 8

The test item of a suitable chemical purity was s upplied 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 Hunga ry Ltd

. on the basis of the

information provided by the Sponsor.

3.1. 2

. Dilution and Preparation of Testing Solutions

An amount of 100 mg test item was dissolved in one litre vehicle (OECD medium) by

mechanical dispersion in order to give the 100 mg/L test concentration. The test

solutions were prepared by the appropriate diluting of this stock solution and distributed

into test vessels prior to introduction of algae (see Table 2 below)

.

Table 2 : Preparation of test solutions from stock solution (Phase 1)

Nominal c oncentration

[mg/L] Amount of stock solution

(mL) Amount of OECD Medium

(mL)

100 .0 1000

–

50 .0 2 0 0 q.s. ad 4 00

25 .0 1 00 q.s. ad 4 00

12.5 50 q.s. ad 4 00

6.25 25 q.s. ad 4 00

q.s. ad = quantum sufficiat ad (a sufficient quantity to make)

Table 3 : Preparation of test solutions from stock solution (Phase 2)

Nominal c oncentration

[mg/L] Amount of stock solution

(mL) Amount of OECD Medium

(mL)

100.0 1000

–

50.0 350 q.s. ad 70 0

q.s. ad = quantum sufficiat ad (a sufficient quantity to make )

In the complementary experiment (Phase 2) the pH of the stock solution was a djust ed

and then the appropriate dilution was prepared and distributed into test vessels prior

to introduction of algae.

3

. 1. 3

. Untreated Control

Algal growth medium was ino culated with algal cells (without test item) and was

examined in parallel to the test item concentration

.Study code: 11/339

- 022AL Final Report Page 12 of 2 8

3.1. 4

. Reference C ontrol

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

conditions, Potassium dichromate is t ested 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 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)

3.1. 5

. Dilution Water

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

used as dilution water for the ran ge finding, definitive test and also for the

complementary study

.

The composition of the growth medium and the chemicals used are given in Appendix 4

.

3. 2

. EXPERIMENTAL ORGANIS MS

Species: Ps eudokirchneriella subcapitata

(formerly known as Selenastrum capricornutum )

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

ATCC 2266 2 )

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

Cultures, Inst. Plant Physiology, U n iversity of Göttingen

, GERMANY

. Cultured under standardised conditions (see

OECD 201) in the Ecotoxicological Laboratory of

CiTox LAB 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

10 4 cells/mL.Study code: 11/339

- 022AL Final Report Page 13 of 2 8

Pre

- culturing: The pre

- culture was intended to give an amount of alga

suspe nsion 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 10 7 cells/mL.

3. 3

. TEST CONDITIONS

Temperature

Culture temperature was checked at the beginning of the study and each day

thereafter in a fl ask filled with water, in the climatic chamber. In addition, temperature

was continuously measured (with a min/max thermometer) within the climate

chamber. The temperature was in the range of 2 2

. 1

– 2 2

. 3 °C (Phase 1) and in the

range of 2 2

. 5

– 2 2

. 8 °C (Pha se 2) m easured in the flask and between 2 1

. 8 and

2 2

. 9 ° C (Phase 1) and between 2 2

. 1 and 23. 3 ° C (Phase 2) measured within the

climate chamber.

pH

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

each concentration. The pH of the control medium was not increased by more than

1 .5 units during the test

. The range of the pH was 3

. 57

– 8

. 69 during the definitive

test (Phase 1) and was 7. 70

– 9. 20 during the complementary study (Phase 2)

.

Light Intensity

The algal culture fl asks were continuously illuminated. The light intensity at the

position occupied by algal culture flasks during the test was about 8 116 lux

( equivalent to 110 **μ** E/m 2 /s) , which was ensured with fluorescent lamps (with a

spectral range of 400

- 700 nm). The dif ferences 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.Study code: 11/339

- 022AL Final Report Page 14 of 2 8

3. 4

. DESCRIPTION OF THE T EST PROCEDURE

The exposure time was 72 hours. The test was started ( Day 0 ) by inoculation of a

biomass of approximately 10 4 algal cells per mL test medium in both of the definitive

and complementary studies.

The test was performed with three replicates per test conce n tration and six replicates

in the control group in the definitive test (Phase 1) and with six replicates per test

conce n tration and in the control group in the complementary study (Phase 2)

. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were

continuously shaken by a laboratory orbital shaker to keep algae in suspension . The

flasks were co v er ed with air

- permeable stoppers.

3. 4 .1. Preliminary Range Finding Test

A concentration range

- finding test was conducted to determine the approximate

to xicity 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. The test was performed with two replicates per eac h test

conce n tration and three replicates in the control group.

During the formulation procedure the test solutions were prepared by the method

described above (3.1.2.).

The concentration levels used and results (72 h) of the preliminary range

- finding test

are summarised in the following table.

Table 4 : Results of the Preliminary Range

- Finding Test

Nominal concentrations

[mg/L] Untreated

control 0. 1 1 .0 10.0 100 .0

Average of cell number

at 72 hours (x 10 4 cell/mL) 70.67 68.50 68.00 68.00 1.00

3. 4 .2. Te st item concentrations in the Definitive Test (Phase 1)

Because significant toxic response was observed in the preliminary range

- finding test,

five test concentrations in a geometric series (factor 2.0) and one control w ere tested

in the definitive test (Phase 1)

.

The following nominal concentrations were tested: 6.25, 12.5

, 25

, 50 and 100 mg /L.

The measured geometric mean test item concentrations were: 4

. 5 ; 7

. 9 ; 19

. 7 ; 44 .9 and

89

. 7 mg/L.Study code: 11/339

- 022AL Final Report Page 15 of 2 8

As the analytically measured concentration deviated more tha n 20 per cent from the

nominal in most case s , the biological results are based on the measured geometric

mean concentration s

.

The analytically measured test item concentrations are detailed in Appendix 5

.

3 .4.3. Test item concentrations in the Complementary Study (Phase 2)

Because there was evidence of marked change in the pH of the test water after

addition of the test substance, and the pH was below 6 in the definitive test at the two

highest concentration levels of 50 and 100 mg/L , a complementary study was carried

out, adjusting the pH of the stock solution to that of the dilution water before

addition of the test substance. The pH was adjusted through the addition of NaOH.

The complementary study was carried out using only the two highest concentration s

of 50 and 100 mg/L , where the pH values were below the accepted range in the

definitive test

. The measured geometric mean test item concentrations were: 34.2 and

97

. 2 mg/L.

The test was performed with six replicates per test conce n tration and in the cont rol

group.

The analytically measured test item concentrations are detailed in Appendix 5

.

3. 5

. OBSERVATIONS

The cell numbers were determined at 24, 48 and 72 hours after the treatment 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 24 h , 48 h and 72 h) to detect any abnormal appearance of the algae.

3.6. ANALYTICAL MEASUREME NTS

Analytical measurement was performed at the co ntrol and at the applied test

concentration levels at the beginning and at the end of the test s (Phase 1 and 2)



### .

The samples were analysed by HPLC system.

Methods and results of test item concentration analysis are described in Appendix 5

.Study code: 11/339

- 022AL Final Report Page 16 of 2 8

3. 7

. RESUL TS 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 concentration of test item which results in a 50 %

reduction of bio mass (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 concentration of test item which results in a 50 %

reduction of yield (y) relative to the control

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 owest O bserved E ffect C oncentration) the lowest test

concentration a t 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 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

n t N

n N

**− −** 

**=**

Where ln (N

n ) = natural logarithm of measured number of cells/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 testStudy code: 11/339

- 022AL Final Report Page 17 of 2 8

The percentage inhibition of growth rate (% I

**μ** ):

100 I % t

**⋅ −** 

**=**

c c

**μ μ**  **μ**

**μ** %

Where % I

**μ** = percent inhibition in aver age 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

Where N 0 = nominal number of cells/mL at time t 0 (start of the te st)

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 test

The percentage inhibition of area (% I A ):

% 100

**⋅ −** 

**=**

c t c

A A A I

A %

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

A

c = mean area of the control

A

t = mean area o f 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 c alculated.

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 under the growth curve (biomass) , average specific growth rate and y ield 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.Study code: 11/339

- 022AL Final Report Page 18 of 2 8

3. 7

. 5

. Statistical Analysis

The section

- by

- section specifi c 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 grow th 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 Exc el for Windows software (Microsoft Co./One

Microsoft Way/Redmond, WA 98052

- 6399).

The E

r C 50, E b C 50 and E

y C 50 values of the test item were determined using Probit

analysis by SPSS PC+ software.

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

Bonferroni t

- Test and Dunnett’s Test ( **α** = 0.05) by TOXSTAT software.

For the determination of the LOEC and NOEC, the calculated mean biomass, growth

ra tes and yield at the test concentrations were tested on significant differences to the

control values by Bonferroni t

- Test and Dunnett’s Test

.

3. 8

. ARCHIVES

The study documents:

- study plan and amendment s

,

- all raw data,

- sample of test item,

- stud y report and any amendment

- correspondence

are stored according to the Hungarian GLP and to the CiTox LAB Hungary Ltd.

SOP

- s in the archives of CiTox LAB Hungary Ltd. 8200 Veszprém, Szabadságpuszta,

Hungary

.

After the retention time has elapsed all the arch ived 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.Study code: 11/339

- 022AL Final Report Page 19 of 2 8

3. 9

. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PD F file

Archive: 1x original, bound

3. 1 0

. REFERENCES

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

Alga and Cyanobacteria, Growth Inhibition Test", adopted 2 8 th July , 20 11

.

2. Commission Regulation (EC) No 761/2009 of 23 J uly 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,

Authori sation 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 f or

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 corresp onds 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.Study code: 11/339

- 022AL Final Report Page 20 of 2 8

3.1 1

. DEVIATION S FROM THE STUDY PLAN

Concerning: Statistical evaluation

According to the Study Plan: Statistical comparisons of biomass, average

specific growth rates and yield in control and in

treated groups will be carried out using analysis of

va riance (ANOVA) and Bonferroni t

- Test ( **α** = 0.05) by TOXSTAT software

Deviation: Statistical comparisons of biomass, average

specific growth rates and yield in control and in

treated groups wer e carried out using analysis of

variance (ANOVA) and Bonferroni t

- Test and

Dunnett’s Test ( **α** = 0.05) ( **α** = 0.05) by

TOXSTAT software

Reason: Technical

Presumed effect on the study: noneStudy code: 11/339

- 022AL Final Report Page 21 of 2 8

4. RESULTS

4.1. VALIDITY

4.1.1. Definitive test (Phase 1)

T he cell density in the control cultures increased by a factor of 63 .17 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 21.47 %.

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

period (day 0

- 3) in the control cultures was 0

. 8 2 %.

4.1.2. Complementary study (Phase 2)

The cell density in the control cultures increased by a factor of 73.00 within three

days

.

The mean coefficient of variation for section

- by

- section specific growth rates

(da ys 0

- 1; 1

- 2; 2

- 3) in the control cultures was 17.16 %.

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

period (day 0

- 3) in the control cultures was 0.8 8 %.

All validity criteria were met, t herefore both of the definitiv e and complementary

studies can be considered as valid.Study code: 11/339

- 022AL Final Report Page 22 of 2 8

4. 2

. CELL NUMBER S

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

of determinations are listed in Appendix 2.

Figure 1 : Mean Values of Cell Number s Plott ed against Time (Phase 1)Study code: 11/339

- 022AL Final Report Page 23 of 2 8

Figure 2 : Mean Values of Cell Numbers Plotted against Time (Phase 2)

4. 3

. MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS

Thin cells were observed at the two highest examined concentrations of 50 and

100.0 mg/L (nominal), 48 and 72 hours after the start of the test in the definitive test

(Phase 1).

4. 4

. AVERAGE SPECIFIC GRO WTH RATES

4.4.1. Definitive test (Phase 1)

The results of the statistical evaluation (based on Bonferroni t

- Test; **α** =0.05) show

that the 0

- 72 h aver age specific growth rate was statistically significantly different

from the untreated control value at the concentration range of 19.7

– 89.7 mg/L

( measured ) , correspondingly the No Observed Effect Concentration (NOEC) was

determined as 7

. 9 mg/L ( measured )

. The 72 h E

r C 50 value was determined [ by Probit

analysis ( SPSS PC+ software) ] as 27 .17 mg/L (95 % confidence limits:

25.21

– 29.70 mg/L ).Study code: 11/339

- 022AL Final Report Page 24 of 2 8

Table 5 : Growth Rates ( **μ** ) and Percentage Inhibition of **μ** during the Test Period (Phase 1)

Measured

Concentration

[ mg/L ] Growth rate ( **μ** ) and % inhibition of **μ**

0

– 24 h 0

– 48 h 0

– 72 h

**μ** % **μ** % **μ** %

Control 0.0 680 + 0.0 0.0 565 0.0 0.05 76 0.0

4.5 0.0690 +

- 1.4 0.0 568

- 0.5 0.0578

- 0.4

7.9 0.0640 6.0 0.0573

- 1.4 0.0578

- 0.5

19.7 0.0498 * 26.9 0.0545 3.6 0.0549* 4.7

44.9 0. 0000* 100.0 0.0000* 100.0 0.0000* 100.0

89.7 0.0 000* 100.0 0.0 000* 100.0 0.0 000* 100.0

* : statistically significantly different compared to the control values (Bonferroni t

- Test; **α** = 0.05)

+ : at th is value the rounding of the EXCEL and TOXSTAT software was different. The table

contains the values calculated with EXCEL.

4.4.2. Complementary study (Phase 2)

The results of the statistical evaluation (based on Dunnett’s Test ; **α** =0.05) show that

the 0

- 72 h average specific growth rate was statistically s ignificantly different from the

untreated control value at the tested concentration s of 34.2 and 97.2 mg/L

(measured)

.

Table 6 : Growth Rates ( **μ** ) and Percentage Inhibition of **μ** during the Test Period (Phase

2)

Measured

Concentration

[ mg/L ] Growth rate ( **μ** ) and % inhibition of **μ**

0

– 24 h 0

– 48 h 0

– 72 h

**μ** % **μ** % **μ** %

Control 0.0 69 3 + 0.0 0.061 5 + 0.0 0.059 6 0.0

34.2 0.0 652 + 5.9 0.0 606 + 1.5 0.0 582 * 2.2

97.2 0.0 096* 86.1 0.0 556 * 9.5 0.0 563 * 5.5

* : statistically significantly different compared to the control v alues ( Dunnett’s Test ; **α** = 0.05)

+ : at this value the rounding of the EXCEL and TOXSTAT software was different. The table

contains the values calculated with EXCEL.Study code: 11/339

- 022AL Final Report Page 25 of 2 8

4. 5

. AREAS UNDER THE GROW TH CURVES

4.5.1. Definitive test (Phase 1)

The results of the statistical evaluation (based on Bonferroni t

- Test; **α** =0.05) show

that the 0

- 72 h areas w ere statistically significantly different from the untreated control

value at the examined concentration range of 19.7

– 89.7 mg/L (measured)

, correspondingly the N o Observed Effect Concentration (NOEC) was determined as

7.9 mg/L ( measured )

. The 72 h E b C 50 value was determined [ by Probit analysis

( SPSS PC+ software) ] as 24.28 mg/L (95 % confidence limits: 22.70

– 26.49 mg/L )

.

Table 7 : Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Measured

Concentration

[ mg/L ] Area under the Growth Curves (A) and Percentage Inhibition of A

0

– 24 h 0

– 48 h 0

– 72 h

A % A % A %

Control 50 .0 0.0 2 70 .0 0.0 1 186 .0 0.0

4.5 52.0

- 4.0 276.0

- 2.2 1 208.0

- 1.9

7.9 44.0 12.0 264.0 2.2 1200.0

- 1.2

19.7 28.0 * 44.0 208.0 * 23.0 972.0* 18.0

44.9 0.0* 100.0 0.0* 100.0 0.0* 100.0

89.7 0.0 * 100.0 0.0* 100.0 0.0 * 100.0

* : statistically significantly different compared to the control values (Bonferroni t

- T est; **α** = 0.05)

4.5.2. Complementary study (Phase 2)

The results of the statistical evaluation (based on Dunnett’s Test ; **α** =0.05) show that

the 0

- 72 h areas were statistically significantly different from the untreated control

value at the tested concent rations of 34.2 and 97.2 mg/L (measured)

.

Table 8 : Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Measured

Concentration

[ mg/L ] Area under the Growth Curves (A) and Percentage Inhibition of A

0

– 24 h 0

– 48 h 0

– 72 h

A % A % A %

Control 52 .0 0.0 322 .0 0.0 1404 .0 0.0

34.2 46.0 11.5 300.0 6.8 1292.0 * 8.0

97.2 4 .0* + 92.3 170.0* 47.2 1014.0* 27.8

* : statistically significantly different compared to the control values ( Dunnett’s Test ; **α** = 0.05)

+ : at this value th e rounding of the EXCEL and TOXSTAT software was different. The table

contains the values calculated with EXCEL.Study code: 11/339

- 022AL Final Report Page 26 of 2 8

4. 6

. YIELD

4.6.1. Definitive test (Phase 1)

The results of the statistical evaluation (based on Bonferroni t

- Test; **α** =0.05) show

that the 0

- 72 h yield was statistically significantly different from the untreated control

value at the tested concentration range of 19.7

– 89.7 mg/L (measured)

, correspondingly the No Observed Effect Concentration (NOEC) was determined as

7.9 mg/L ( measured )

. Th e 72 h E

y C 50 value was determined [ by Probit analysis

( SPSS PC+ software) ] as 2 4

. 28 mg/L (95 % confidence limits: 2 2

. 70

– 26

. 49 mg/L ).

Table 9 : Yield (Y) and Percentage Inhibition of Y during the Test Period

Measured Concentration

[ mg/L ] Yield (Y) and % inhibition of Y

0

– 72 h

Y %

Control 62.2 0.0

4.5 63.3

- 1.9

7.9 63.3

- 1 .9

19.7 51.0 * 18.0

44.9 0.0 * 100.0

89.7 0 .0 * 100. .0

* : statistically significantly different compared to the control values (Bonferroni t

- Test; **α** = 0.05)

4.6.2. Complement ary study (Phase 2)

The results of the statistical evaluation (based on Dunnett’s Test ; **α** =0.05) show that the 0

- 72 h yield was statistically significantly different from the untreated control value at the

tested concentrations of 34.2 and 97.2 mg/L (meas ured)

.

Table 10 : Yield (Y) and Percentage Inhibition of Y during the Test Period

Measured Concentration

[ mg/L ] Yield (Y) and % inhibition of Y

0

– 72 h

Y %

Control 72 .0 0.0

34.2 65.3 * 9.3

97.2 56.8 * 13.0

* : statistically significantly different co mpared to the control values ( Dunnett’s Test ; **α** = 0.05)Study code: 11/339

- 022AL Final Report Page 27 of 2 8

5

. CONCLUSION

The effect of Ethanedioic acid test item was assessed on algal growth using the

unicellular green alga Pseudokirchneriella subcapitata ( Selenastrum capricornutum)

,

over an exposure period of 72 hours.

With respect to the inhibitory effect of the test item, the 0

- 72 h average specific

growth rates, yield and areas were significantly different from that of the

control group in the examined concentration range of 19.7

– 89.7 mg/L

(mea sured). The overall NOEC was determined as 7

. 9 mg/L (measured); the

overall LOEC was determined as 19.7 mg/L (measured). The biological results

are summarised in the following table:

Table 1: Influence of Ethanedioic acid on the Growth of Pseudokirchneri ella subcapitata

Parameter

(0

- 72 h) Growth rate (r)

[mg/L] Yield (y)

[mg/L] Biomass (b)

[mg/L]

Calculation based on measured geometric mean concentrations

EC 50 27.17 24.28 24.28

95 % conf. limits 25.21

– 29.70 22.70

– 26.49 22.70

– 26.49

NOEC 7.9 7.9 7.9

LOEC 19.7 19.7 19.7

Based on the results of the performed definitive and complementary

experiments the observed effect can be considered to be related to the acid

character of the test item.Study code: 11/339

- 022AL Final Report Page 28 of 2 8



## APPENDICESStudy code: 11/339

- 022AL Final Report Page 1 of Appendix 1

APPENDIX 1

TEST CONDITIONS

Table 11 : pH

- Values in the Test Media at the Start and End of the Test (Phase 1)

Nominal

Concentration

[ mg/L ] Measured

Concentration

[ mg/L ] pH

- values

Start End

Control 0.0 7.87 8.50

8.21

8.43

8.27

8.36

8.39

6.25 4.5 7.72 8.65

8.69

8. 57

12.5 7.9 7.54 8.62

8.54

8.60

25 19.7 7. 00 8. 47

8. 42

8. 53

50 44.9 4.64 4.90

4.81

4.84

100 89.7 3.57 3.77

3.63

3.64

Tabl e 12 : pH

- Values in the Test Media at the Start and End of the Test (Phase 2)

Nominal

Concentrati on

[ mg/L ] Measured

Concentration

[mg/L] pH

- values

Start End

Control 0.0 7.8 2 9. 16

9. 10

9. 20

9. 16

9. 11

9. 13

50 34.2 7.8 0 9.07

9.06

9.10

9.02

9.04

9.05

100 97.2 7. 70 9 .0 0

9 .0 4

9.08

9.00

9.02

9 .0 0Study code: 11/339

- 022AL Final Report Page 2 of Appendix 1

Table 13 : Temperature in the Climate Chamber and in the Test Media

during the Test (Phase 1)

Parameter Exposure Time

Day 0 Day 1 Day 2 Day 3

temperature (oC) measured in flask 2 2

. 3 2 2

. 1 22. 3 22. 2

min / max temperature (oC)

of the climate cham ber max 2 2

. 7 22

. 9 22

. 7 22. 8

min 22. 0 21.8 22. 0 22. 1

Table 14 : Temperature in the Climate Chamber and in the Test Media

during the Test (Phase 2)

Parameter Exposure Time

Day 0 Day 1 Day 2 Day 3

temperature (oC) measured in flask 22. 5 22.7 22.8 22. 7

min / max temperature (oC)

of the climate chamber max 22.9 23. 0 23. 1 23.3

min 22. 3 2 2.4 22.2 22. 1Study code: 11/339

- 022AL Fin al Report Page 1 of Appendix 2

APPENDIX 2

CELL NUMBER

Table 1 5 : Cell Number (x 10 4 cell/mL) determined in the Main Experiment (Phase 1)

Nominal

Concentration

[ mg/L ] Measured

Co ncentration

[ mg/L ] Number of cells

0 h 24 h 48 h 72 h

Control 0.0 1 6 15 63

1 4 12 60

1 6 15 62

1 5 16 63

1 5 16 65

1 5 17 66

Mean 1.00 5.17 15.17 63.17

SD 0.0 0.8 1.7 2.1

6.25 4.5 1 4 14 63

1 6 17 66

1 6 15 64

Mean 1.00 5.33 15.33 64.33

SD 0.0 1.2 1.5 1.5

12.5 7.9 1 5 15 63

1 5 16 65

1 4 16 65

Mean 1.00 4.67 15.67 64.33

SD 0.0 0.6 0.6 1.2

25 19.7 1 3 14 50

1 4 14 54

1 3 13 52

Mean 1.00 3.33 13.67 52.00

SD 0.0 0.6 0.6 2 .0

50 44.9 1 1 1 * 1 *

1 1 1 * 1 *

1 1 1 * 1 *

Mean 1.00 1.00 1.00 1.00

SD 0.0 0.0 0.0 0.0

100 89.7 1 1 1 * 1 *

1 1 1 * 1 *

1 1 1 * 1 *

Mean 1.00 1.00 1.00 1.00

SD 0.0 0.0 0.0 0.0

* Thin cellsStudy code: 11/339

- 022AL Fin al Report Page 2 of Appendix 2

Table 1 6 : Cell Number (x 10 4 cell/mL) determined in the Complementary Experiment

(Phase 2)

Nominal

Concentration

[ mg/L ] Measured

Concentration

[mg/L] Number of cells

0 h 24 h 48 h 72 h

Control 0.0 1 4 20 74

1 6 20 76

1 5 18 70

1 6 17 70

1 5 19 72

1 6 21 76

Mean 1.00 5.33 19.17 73.00

SD 0.0 0.8 1.5 2.8

50 34.2 1 5 20 72

1 5 18 67

1 4 17 63

1 6 19 66

1 4 18 64

1 5 18 66

Mean 1.00 4.83 18.33 66.33

SD 0.0 0.8 1.0 3.1

100 97.2 1 1 14 51

1 2 15 59

1 1 15 60

1 1 16 62

1 1 12 57

1 2 15 58

Mean 1.00 1.33 14.50 57.83

SD 0.0 0.5 1.4 3.8Study code: 11/339

- 022AL Final Report Page 1 of Appendix 3

APPENDIX 3

CONCENTRATION /EFFECT RELATIONSHIP GRAPHS

OF THE TEST ITEM

Figure 2 : Effect of Ethanedioic acid on Average Specific Growth Rate

Figure 3 : Effect of Et hanedioic acid on BiomassStudy code: 11/339

- 022AL Final Report Page 2 of Appendix 3

Figure 4 : Effect of Ethanedioic acid on YieldStudy code: 11/339

- 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 add ing an appropriate volume of these different stock solutions to deionised

water in order to achieve the final concentrations.

Stock solution Substance Final c oncentration 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

MnCl 2 × 4 H 2 O 415.0 **μ** g/L

ZnC l 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: 11/339

- 022AL Final Report Page 1 of Appendix 5

APPENDIX 5

ANALYTICAL INVESTIGA TIONS OF MAIN STUDY

(METHODS AND RESULTS )

1. Principle of the Analytical Metho d

Concentration of Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of

ethanedioic acid [CAS RN 144

- 62

- 7]) in the test solutions was determined at the begin ning

and at the end of the test.

The samples were taken from the test solutions containing the test item and one sample from

the control at both sampling time.

The samples were analysed by an HPLC

- UV method described in details in the analytical

method validation of Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form

o f ethanedioic acid [CAS RN 144

- 62

- 7]) (11/339

- 316AN)

.

2. Equipment and Chemicals

2.1. Apparatus

HPLC system: Merck

- Hitachi LaChrom HPLC system

D

- 7000 Interface, No.: 1126

- 175

L

- 7100 HPLC pump, No. : 1388

- 056

L

- 7200 Autosampler, No.: 1380

- 033

L

- 7 400 UV Detector, No.: 1374

- 025

L

- 7614 Degasser, No.: 144116A0094

L

- 7360 Column Oven, No.: 00118060

Balance s : BP221S Sartorius, No.: 11809117

L2200P Sartorius, No.:38100037

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

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

pH meter: SevenEasy S20 Mettler Toledo, No.:1231025119

Refrigerator: Zanussi, No.: ZLKI

- 262Study code: 11/339

- 022AL Final Report Page 2 of Appendix 5

2.2. Test Item

Name: Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated

form of ethanedioic acid [CAS RN 144

- 62

- 7 ])

Chemical name: Oxalic acid, hydrate

CAS number: 6153

- 56

- 6

Batch/Lot number: YP1

- 20120528C

Molecular formula: C 2 H 2 O 4 ×2H 2 O

Molecular weight: 126.1 (dihydrate form)

Appearance: White crystals

Purity: 99.69 %

Manufacture date: May 28, 2012

Expiry date: May 27, 2013

Storage conditions: Controlled room temperature (15

- 25 o C, < 70 RH%)

2.3. Other materials

Ultra pure water (ASTM Type I): prepared by Direct

- Q 3 system, Millipore

Acetonitrile : HPLC grade, VWR , Batch: 11Z3738

HPLC grade, Sigma

- Aldrich, Batch: SZBC300SV

Potassium dihydrogen phosphate: reagent, Lach

- Ner, Batch: PP/2010/02241

Phosphoric acid 85%: reagent, Lach

- Ner, Batch: PP/2011/12163

3. Method of analysis of test item c oncentration

Samples were analysed by the following HPLC

- UV method :

Co lumn: Zorbax SB

- C 8 2 50 × 4.6 mm, 5μm

Mobil Phase : 50 mM KH 2 PO 4 (pH:2.1) : ACN 9 : 1

Flow: 1.0 ml/min.

Injection volume: 10 μl

Temperature: 25 °C

Detector: UV at 2 2 0 nm

Retention time: 2

. 5 min ± 10%Study code: 11/339

- 022AL Final Report Page 3 of Appendix 5

4. Results of the Method Validation

Table 1: Metho d va lidation results (11/339

- 316AN )

Selectivity No interfering component was

observed

Reinjection repeatability

(7 injections) CV% ≤ 1.6 %

Linear range 1

– 100 μg/ml

Limit of Quantification 1 μg/ml

Recovery 100

- 106 %

Accuracy 0

- 6 %

Precision 1

– 4 %

Stability of the test item in Alga Test

Medium (3 days) 94

- 100 %

Stability of the samples At least 19 hours in the autosampler

Stock solution stability At least 4 days at 5 **±** 3 °C

5. Measured data

5.1. Calibration

Table 2: Data of the re gression lines

Date of measurement Slope Intercept Correlation

coefficient (r 2 )

19 November 2012 2772 564 1.000

22 November 2012 2964 486 1.000Study code: 11/339

- 022AL Final Report Page 1 of Appendix 6

APPENDIX 6

COPY OF THE GLP CERTIFICATEStudy code: 11/339

- 022AL Final Report Page 1 of Appendix 7

APPENDIX 7

COPY OF THE CERTIFIC ATE OF ANALYSIS