Disodium oxalate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 11, 2015 to November 10, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Oxalic acid dihydrate

Cas Number:

6153-56-6

Molecular formula:

C2O4H2.2H2O

IUPAC Name:

Oxalic acid dihydrate

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 15000900
- Expiration date of the lot/batch: May 20, 2017

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations selected were 1, 2.70, 7.29, 19.68 and 53.14 mg/L
- Sampling method: control, low (1 mg/L), intermediate (7.29 mg/L) and high (53.14 mg/L) test concentration samples were analysed by HPLC
- Sample storage conditions before analysis:room temperature

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): OECD Medium

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): ATCC 22662 (Procured from LGC Promochem India Pvt. Ltd, Bangalore 560 058)
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: OECD medium

Pre- Culture:
- Pre-cullture period: 3 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

72 hours

Test conditions

Test temperature:

Room temperature

pH:

8.1

Nominal and measured concentrations:

The concentrations selected were 1, 2.70, 7.29, 19.68 and 53.14 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Conical flask
- Type (delete if not applicable): closed
- Initial cells density: 26,425 cells/mL
- Control end cells density: 3020833.33 cells/mL
- Photoperiod: Continuous light
- Light intensity: 6580 – 6760 Lux
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber;
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.7

- Range finding study
- Test concentrations: 1, 10, 25, 50 and 100 mg/L concentration

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC10

Remarks:

12.61 mg/L

Effect conc.:

> 11.69 - < 13.61 mg/L

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

24 h

Dose descriptor:

EC50

Remarks:

37.03 mg/L

Effect conc.:

> 35.64 - < 38.46 mg/L

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

EC10

Remarks:

7.04 mg/L

Effect conc.:

> 6.48 - < 7.64 mg/L

Basis for effect:

biomass

Effect concentrations 4

Key result

Duration:

24 h

Dose descriptor:

EC50

Remarks:

25.39 mg/L

Effect conc.:

> 24.38 - < 26.45 mg/L

Basis for effect:

biomass

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

EC10

Remarks:

18.89 mg/L

Effect conc.:

> 18.01 - < 19.81 mg/L

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

31.26 mg/L

Effect conc.:

> 30.25 - < 32.31 mg/L

Basis for effect:

growth rate

Effect concentrations 7

Key result

Duration:

48 h

Dose descriptor:

EC10

Remarks:

6.22 mg/L

Effect conc.:

> 5.76 - < 6.71 mg/L

Basis for effect:

biomass

Effect concentrations 8

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

20.48 mg/L

Effect conc.:

> 19.69 - < 21.3 mg/L

Basis for effect:

biomass

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

7.55 mg/L

Effect conc.:

> 7.06 - < 8.08 mg/L

Basis for effect:

growth rate

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

20.58 mg/L

Effect conc.:

> 19.83 - < 21.35 mg/L

Basis for effect:

growth rate

Effect concentrations 11

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

5.56 mg/L

Effect conc.:

> 5.14 - < 6.01 mg/L

Basis for effect:

biomass

Effect concentrations 12

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

19.14 mg/L

Effect conc.:

> 18.39 - < 19.92 mg/L

Basis for effect:

biomass

Details on results:

Based on the test results,
The ErC50 of OXALIC ACID observed for test period of '24' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 37.03 mg/L, with lower confidence limit of 35.64 mg/L and the upper confidence limit of 38.46 mg/L.
The EyC50 of OXALIC ACID observed for a test period of '24' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 25.39 mg/L, with lower confidence limit of 24.38 mg/L and the upper confidence limit of 26.45 mg/L.
The ErC50 of OXALIC ACID observed for test period of '48' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 31.26 mg/L, with lower confidence limit of 30.25 mg/L and the upper confidence limit of 32.31 mg/L.
The EyC50 of OXALIC ACID observed for a test period of '48' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 20.48 mg/L, with lower confidence limit of 19.69 mg/L and the upper confidence limit of 21.30 mg/L
The ErC50 of OXALIC ACID observed for test period of '72' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 20.58 mg/L with lower confidence limit of 19.83 mg/L and the upper confidence limit of 21.35 mg/L.
The EyC50 of OXALIC ACID observed for a test period of '72' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 19.14 mg/L with lower confidence limit of 18.39 mg/L and the upper confidence limit of 19.92 mg/L.

Results with reference substance (positive control):

Growth Inhibition study was performed in Freshwater green Algae (Pseudokirchneriella subcapitata) in accordance with guideline OECD-201 with Potassium Dichromate. The concentrations used were 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L.
Initially on ‘0’ day approximately 18250 algal cells/ml were inoculated in conical flask containing OECD test medium. The volume of OECD test medium was 100 ml each in control and treatment groups. 6 replicates were maintained for control and 3 replicates for each test concentration. Prior to exposure, pre-culture was maintained for a period of 3 days.
On day of exposure algal cells were observed microscopically and cell count was carried out approximately at ‘24’, ‘48’ and ‘72’ hours after exposure with Haemocytometer. Microscopic observations revealed that the algal cells were normal appearance and no abnormality of the algal cell was observed on exposure to the test item.
Average Specific Growth Rate:
The percent inhibition on the average growth rate at the end of ‘72’ hour was 0.03%, 67.13%, 71.90%, 76.58% and 86.93% in test concentrations of 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L respectively.
From the average specific growth rate recorded with the different test concentrations viz., 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L the concentration that inhibits 50% growth (ErC50) of Freshwater green Algae (Pseudokirchneriella subcapitata) was calculated to be 1.20 mg/L, with lower confidence limit of 1.15 mg/L and upper confidence limit of 1.26 mg/L.
The lowest observed effect concentration (LOEC) was calculated as 0.5 mg/L and the no observed effect concentration (NOEC) was recorded as 0.2 mg/L respectively.
Yield:
The percent inhibition on the yield at the end of ‘72’ hour was 0.07%, 54.13%, 92.35%, 93.82% and 99.95% in test concentrations of 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L respectively.
From the yield recorded with the different test concentrations viz., 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L, the concentration that inhibits 50% growth (EyC50) of Freshwater green Algae (Pseudokirchneriella subcapitata) was recorded as 0.67 mg/L, with lower confidence limit of 0.65 mg/L and upper confidence limit of 0.69 mg/L.
The lowest observed effect concentration (LOEC) was calculated as 0.5 mg/L and the no observed effect concentration (NOEC) was recorded as 0.2 mg/L respectively.
Based on the test results,
The ErC50 of Potassium Dichromate observed for a period of ‘72’ hour in freshwater alga (Pseudokirchneriella subcapitata) was calculated as 1.20 mg/L, with lower confidence limit of 1.15 mg/L and upper confidence limit of 1.26 mg/L.
The EyC50 of Potassium Dichromate observed for a period of ‘72’ hour in freshwater alga (Pseudokirchneriella subcapitata) was calculated as 0.67 mg/L, with lower confidence limit of 0.65 mg/L and upper confidence limit of 0.69 mg/L.

Reported statistics and error estimates:

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for growth rate were recorded as 19.68 mg/L and 53.14 mg/L respectively (24 hours)
The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for growth rate were recorded as 1 mg/L and 2.7 mg/L respectively (48 hours)
The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for yield were recorded as 1 mg/L and 2.70 mg/L respectively (72 hours)
The biomass in the control culture increased exponentially by a factor of more than 16 within the ‘72’ hour test period. This corresponds to a specific growth rate of 1.07 day – 1
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3 for ‘72’-hours test) in the control cultures was 1.4 %.
The coefficient of variation of average specific growth rate during the whole test period in replicate control cultures did not exceed 7%.

Any other information on results incl. tables

NOEC AND LOEC – AVERAGE GROWTH RATE
	24 hr	48 hr
NOEC	19.68 mg/L	1 mg/L
LOEC	53.14 mg/L	2.7 mg/L

NOEC AND LOEC – YIELD
	24 hr	48 hr
NOEC	19.68 mg/L	1 mg/L
LOEC	53.14 mg/L	2.7 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the test results,
The ErC50 of OXALIC ACID observed for test period of '24' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 37.03 mg/L, with lower confidence limit of 35.64 mg/L and the upper confidence limit of 38.46 mg/L.
The EyC50 of OXALIC ACID observed for a test period of '24' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 25.39 mg/L, with lower confidence limit of 24.38 mg/L and the upper confidence limit of 26.45 mg/L.
The ErC50 of OXALIC ACID observed for test period of '48' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 31.26 mg/L, with lower confidence limit of 30.25 mg/L and the upper confidence limit of 32.31 mg/L.
The EyC50 of OXALIC ACID observed for a test period of '48' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 20.48 mg/L, with lower confidence limit of 19.69 mg/L and the upper confidence limit of 21.30 mg/L
The ErC50 of OXALIC ACID observed for test period of '72' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 20.58 mg/L with lower confidence limit of 19.83 mg/L and the upper confidence limit of 21.35 mg/L.
The EyC50 of OXALIC ACID observed for a test period of '72' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 19.14 mg/L with lower confidence limit of 18.39 mg/L and the upper confidence limit of 19.92 mg/L

Executive summary:

Growth Inhibition study was conducted inFreshwater green Algae(Pseudokirchneriella subcapitata)in accordance withOECD-201. Guideline with OXALIC ACID. Thetestconcentrationswere selectedas1, 2.70, 7.29, 19.68 and 53.14 mg/L.Test item was dissolved in the testmedium, as per OECD 201. 

Initially on ‘0’ day,approximately 26425 cells/mLwereinoculated inaconical flask containingthe testmedium.The volume oftest medium was 100 mLin controlandeachtreatmentflasks.Sixreplicates were maintained for controland 3 replicates for eachtestconcentration.

Prior to the exposure,pre-culture was maintained for a period of3days. After the exposure on day ‘0’, algal cells were observed microscopically and the cell count was done atapproximately 24, 48 and 72 hoursby usingHaemocytometer. Microscopic observationrevealed if the cells were normal or abnormalinappearance postexposure tothe testitem.

Average Specific Growth Rate:

The percent inhibition on the average growth rate at the end of '24' hour was 0.15%, 3.12%, 5.16%, 11.87% and 76.19% in test concentrations of 1, 2.70, 7.29, 19.68 and 53.14 mg/L respectively.

From the average specific growth rate recorded with the different test concentrations viz., 1, 2.70, 7.29, 19.68 and 53.14 mg/L, the concentration that inhibited 50% growth (ErC50) of Freshwater green Algae (Pseudokirchneriella subcapitata) was calculated to be 37.03 mg/L, with lower confidence limit of 35.64 mg/L and the upper confidence limit of 38.46 mg/L.

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for growth rate were recorded as 19.68 mg/L and 53.14 mg/L respectively

The percent inhibition on the average growth rate at the end of '48' hour was -0.13%, 1.04%, 1.60%, 6.32% and 100.80% in test concentrations of 1, 2.70, 7.29, 19.68 and 53.14 mg/L respectively.

From the average specific growth rate recorded with the different test concentrations viz., 1, 2.70, 7.29, 19.68 and 53.14 mg/L, the concentration that inhibited 50% growth (ErC50) of Freshwater green Algae (Pseudokirchneriella subcapitata) was calculated to be 31.26 mg/L, with lower confidence limit of 30.25 mg/L and the upper confidence limit of 32.31 mg/L.

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for growth rate were recorded as 1 mg/L and 2.7 mg/L respectively

The percent inhibition on the average growth rate at the end of '72' hour were 0.06%, 4.72%, 10.07%, 39.27% and 142.23% in test concentrations of 1, 2.70, 7.29, 19.68 and 53.14 mg/L respectively.

From the average specific growth rate recorded with the different test concentrations viz., 1, 2.70, 7.29, 19.68 and 53.14 mg/L, the concentration that inhibited 50% growth (ErC50) of Freshwater green Algae (Pseudokirchneriella subcapitata) was calculated to be 20.58 mg/L, with lower confidence limit of 19.83 mg/L and the upper confidence limit of 21.35 mg/L.

 The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for growth rate were recorded as 1 mg/L and 2.70 mg/L respectively.

Yield:

The percent inhibition on the yield at the end of '24' hour was 0.41%, 7.43%, 11.97%, 25.59% and 91.21% in test concentrations of 1, 2.70, 7.29, 19.68 and 53.14 mg/L respectively.

From the yield recorded with the different test concentrationsviz.,1, 2.70, 7.29, 19.68 and 53.14 mg/L the concentration that inhibited 50% growth (EyC50) of Freshwater green Algae (Pseudokirchneriella subcapitata)was calculated as 25.39 mg/L, with lower confidence limit of 24.38 mg/L and the upper confidence limit of 26.45 mg/L.

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for yield were recorded as 19.68 mg/L and 53.14 mg/L respectively

The percent inhibition on the yield at the end of '48' hour was 0.02%, 8.90%, 14.03%, 33.79% and 99.31% in test concentrations of 1, 2.70, 7.29, 19.68 and 53.14 mg/L respectively.

From the yield recorded with the different test concentrationsviz.,1, 2.70, 7.29, 19.68 and 53.14 mg/L the concentration that inhibited 50% growth (EyC50) of Freshwater green Algae (Pseudokirchneriella subcapitata)was calculated as 20.48 mg/L, with lower confidence limit of 19.69 mg/L and the upper confidence limit of 21.30 mg/L

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for yield were recorded as 1 mg/L and 2.7 mg/L respectively

The percent inhibition on the yield at the end of '72' hour was 0.03%, 9.71%, 15.67%, 38.60% and 99.55% in test concentrations of 1, 2.70, 7.29, 19.68 and 53.14 mg/L respectively.

From the yield recorded with the different test concentrationsviz.,1, 2.70, 7.29, 19.68 and 53.14 mg/L, the concentration that inhibited 50% growth (EyC50) of Freshwater green Algae (Pseudokirchneriella subcapitata)was calculated as 19.14 mg/L, with lower confidence limit of 18.39 mg/L and the upper confidence limit of 19.92 mg/L.

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for yield were recorded as 1 mg/L and 2.70 mg/L respectively

Analytical verification of the test item in the exposure medium sample was carried out in the test item concentrations of1, 7.29 and 53.14 mg/L. The samples at '0' hour and '72' hour were analyzed. The mean recoveries data are given below:

·        '0' hour –94.83%, 95.96% and 94.32% for 1.0 mg/L, 7.29 mg/L and 53.14 mg/L respectively. 

'72' hour –92.82%, 95.06% and 93.81% for 1.0 mg/L, 7.29 mg/L and 53.14 mg/L respectively