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)

Study code: 11/339-022AL

Study Director: Krisztina Sipos, M.Sc.

19 March 2013



Study code: 11/339-022AL Final Report Page 5 of 28

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])

SPONSORS : Univar 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

STUDY 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: john@grcci.com

TEST FACILITY : CiToxLAB Hungary Ltd.

Address: H-8200 Veszprém, Szabadságpuszta

Hungary

Phone: 36 88 545-300

Fax: 36 88 545-301
Study code: 11/339-022AL Final Report Page 6 of 28

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. 201: "Freshwater Alga 

and Cyanobacteria, Growth Inhibition Test", adopted 28th July, 2011.

– 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)

– 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 799.5087.
Study code: 11/339-022AL Final Report Page 7 of 28

CONTENTS

Page

1. SUMMARY 8

2. INTRODUCTION 10

3. MATERIALS AND METHODS 10

3.1. TEST ITEM AND CONTROLS AND VEHICLE 10

3.2. EXPERIMENTAL ORGANISMS 12

3.3. TEST CONDITIONS 13

3.4. DESCRIPTION OF THE TEST PROCEDURE 14

3.5. OBSERVATIONS 15

3.6. ANALYTICAL MEASUREMENTS 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 GROWTH RATES 23

4.5. AREAS UNDER THE GROWTH CURVES 25

4.6. YIELD 26

5. CONCLUSION 27

APPENDICES 28

Test Conditions 1 - 2 of Appendix 1

Cell Number 1 - 2 of Appendix 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 7
Study code: 11/339-022AL Final Report Page 8 of 28

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 hours.

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 concentrations of test item used in the definitive test (phase 1) were: 

6.25; 12.5; 25; 50 and 100 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 cases, the biological results are based on the measured 

geometric mean concentrations.

The test design included three replicates at test concentrations 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 extended the study to provide 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 ErC50, EbC50 and EyC50 values of the test item were determined using Probit 

analysis by SPSS PC+ software.
Study code: 11/339-022AL Final Report Page 9 of 28

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 

(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 results 

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

EC50 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 28

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 generations 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.

VALIDITY 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 section-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 exceed 7%.

3. MATERIALS AND METHODS

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: Oxalic acid

Batch number: YP1-20120528C

CAS number: 6153-56-6

Molecular Formula: C2H2O4×2H2O

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 28

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.

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 concentration

[mg/L]

Amount of stock solution 

(mL)

Amount of OECD Medium 

(mL)

100.0 1000 –

50.0 200 q.s. ad 400

25.0 100 q.s. ad 400

12.5 50 q.s. ad 400

6.25 25 q.s. ad 400

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

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

Nominal concentration

[mg/L]

Amount of stock solution 

(mL)

Amount of OECD Medium 

(mL)

100.0 1000 –

50.0 350 q.s. ad 700

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

In the complementary experiment (Phase 2) the pH of the stock solution was adjusted

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 inoculated 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 28

3.1.4. 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)

3.1.5. Dilution Water

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

used as dilution water for the range 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 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 

104 cells/mL.
Study code: 11/339-022AL Final Report Page 13 of 28

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

Temperature

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

thereafter in a flask 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 22.1 – 22.3 °C (Phase 1) and in the 

range of 22.5 – 22.8 °C (Phase 2) measured in the flask and between 21.8 and 

22.9 °C (Phase 1) and between 22.1 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 flasks were continuously illuminated. The light intensity at the 

position occupied by algal culture flasks during the test was about 8116 lux 

(equivalent to 110 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.
Study code: 11/339-022AL Final Report Page 14 of 28

3.4. DESCRIPTION OF THE TEST PROCEDURE

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

biomass of approximately 104 algal cells per mL test medium in both of the definitive 

and complementary studies.

The test was performed with three replicates per test concentration and six replicates 

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

concentration 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 covered with air-permeable stoppers.

3.4.1. Preliminary Range Finding Test

A concentration range-finding test was 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. The test was performed with two replicates per each test 

concentration 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 104

cell/mL) 70.67 68.50 68.00 68.00 1.00

3.4.2. Test 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 were 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 28

As the analytically measured concentration deviated more than 20 per cent from the 

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

mean concentrations.

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 concentrations 

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 concentration and in the control 

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

3.6. ANALYTICAL MEASUREMENTS

Analytical measurement was performed at the control and at the applied test 

concentration levels at the beginning and at the end of the tests (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 28

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

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
Study code: 11/339-022AL Final Report Page 17 of 28

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

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.
Study code: 11/339-022AL Final Report Page 18 of 28

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 ErC50, EbC50 and EyC50 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 

rates 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 amendments,

- all raw data,

- sample of test item,

- study 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.
Study code: 11/339-022AL Final Report Page 19 of 28

3.9. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PDF file

Archive: 1x original, bound

3.10. REFERENCES

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

Alga and Cyanobacteria, Growth Inhibition Test", adopted 28th July, 2011.

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.
Study code: 11/339-022AL Final Report Page 20 of 28

3.11. DEVIATIONS 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 

variance (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 were 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: none
Study code: 11/339-022AL Final Report Page 21 of 28

4. RESULTS

4.1. VALIDITY

4.1.1. Definitive test (Phase 1)

The 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 0.82 %.

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 

(days 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 0.88 %.

All validity criteria were met, therefore both of the definitive and complementary 

studies can be considered as valid.
Study code: 11/339-022AL Final Report Page 22 of 28

4.2. CELL NUMBERS

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

of determinations are listed in Appendix 2.

Figure 1: Mean Values of Cell Numbers Plotted against Time (Phase 1)
Study code: 11/339-022AL Final Report Page 23 of 28

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 GROWTH 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 average 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 ErC50 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 28

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 0.0680+ 0.0 0.0565 0.0 0.0576 0.0

4.5 0.0690+ -1.4 0.0568 -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 0.0000* 100.0 0.0000* 100.0 0.0000* 100.0

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

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 significantly different from the 

untreated control value at the tested concentrations 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 0.0693

+ 0.0 0.0615+ 0.0 0.0596 0.0

34.2 0.0652+ 5.9 0.0606+ 1.5 0.0582* 2.2

97.2 0.0096* 86.1 0.0556* 9.5 0.0563* 5.5

* : statistically significantly different compared to the control values (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 28

4.5. AREAS UNDER THE GROWTH 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 were statistically significantly different from the untreated control 

value at the examined 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 EbC50 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 0.0 270.0 0.0 1186.0 0.0

4.5 52.0 -4.0 276.0 -2.2 1208.0 -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-Test; 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 concentrations 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 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 26 of 28

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). The 72 h EyC50 value was determined [by Probit analysis

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

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 compared to the control values (Dunnett’s Test; α = 0.05)
Study code: 11/339-022AL Final Report Page 27 of 28

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 

(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 results 

are summarised in the following table:

Table 1: Influence of Ethanedioic acid 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

EC50 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 28

APPENDICES
Study 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

Table 12: pH-Values in the Test Media at the Start and End of the Test (Phase 2)

Nominal

Concentration

[mg/L]

Measured

Concentration 

[mg/L]

pH-values

Start End

Control 0.0 7.82

9.16

9.10

9.20

9.16

9.11

9.13

50 34.2 7.80

9.07

9.06

9.10

9.02

9.04

9.05

100 97.2 7.70

9.00

9.04

9.08

9.00

9.02

9.00
Study 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 22.3 22.1 22.3 22.2

min / max temperature (oC)

of the climate chamber

max 22.7 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 22.4 22.2 22.1
Study code: 11/339-022AL Final Report Page 1 of Appendix 2

APPENDIX 2

CELL NUMBER

Table 15: Cell Number (x 104 cell/mL) determined in the Main Experiment (Phase 1)

Nominal

Concentration

[mg/L]

Measured

Concentration

[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 cells
Study code: 11/339-022AL Final Report Page 2 of Appendix 2

Table 16: Cell Number (x 104 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 3.8
Study 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 Ethanedioic acid on Biomass
Study code: 11/339-022AL Final Report Page 2 of Appendix 3

Figure 4: Effect of Ethanedioic acid on Yield
Study 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 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: 11/339-022AL Final Report Page 1 of Appendix 5

APPENDIX 5

ANALYTICAL INVESTIGATIONS OF MAIN STUDY

(METHODS AND RESULTS)

1. Principle of the Analytical Method

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 beginning 

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 

of 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-7400 UV Detector, No.: 1374-025

L-7614 Degasser, No.: 144116A0094

L-7360 Column Oven, No.: 00118060

Balances: 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-262
Study 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: C2H2O4×2H2O

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 (15-25oC, < 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 concentration

Samples were analysed by the following HPLC-UV method:

Column: Zorbax SB-C8 250 × 4.6 mm, 5μm

Mobil Phase: 50 mM KH2PO4 (pH:2.1) : ACN 9:1

Flow: 1.0 ml/min.

Injection volume: 10 μl

Temperature: 25 °C

Detector: UV at 220 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: Method validation 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 regression lines

Date of measurement Slope Intercept Correlation 

coefficient (r2

)

19 November 2012 2772 564 1.000

22 November 2012 2964 486 1.000

Study code: 11/339-022AL Final Report Page 1 of Appendix 6

APPENDIX 6

COPY OF THE GLP CERTIFICATE
Study code: 11/339-022AL Final Report Page 1 of Appendix 7

APPENDIX 7

COPY OF THE CERTIFICATE OF ANALYSIS