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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
07. December 2015
Deviations:
yes
Remarks:
Details on devistions from EU Method C.3 have been reported in section "Any other information on materials and methods incl. tables".
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011
Deviations:
yes
Remarks:
Details on devistions from OECD Guideline 201 have been reported in section "Any other information on materials and methods incl. tables".
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch no. 170103
Appearance: White crystalline powder
Composition: Sodium 3-Sulfobenzoate
CAS No.: 17625-03-5
EINECS-No.: 241-602-5
Molecular formula: C7H5SO5Na
Molecular weight: 224.17 g/mol
Purity: > 99.0% (Titration with NaOH, Glass indicating electrode, Calomel reference electrode)
Homogeneity: totally homogeneous
Volatility: not applicable
Stability: H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: >1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date: 2017
Expiry date: 14. Feb. 2024
Storage: Room Temperature (20 ± 5°C), keep away from humidity
Analytical monitoring:
yes
Details on sampling:
Pre-Culture
For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 4 days under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 21.9 – 24.9 °C). The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of the culture was determined.

Performance of the Study
For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.398 mL) to achieve a cell concentration of 2.5 EXP03 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (0.696 mL). In this mixture, the pH-value was measured.
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The concentration of the test item in the test vessels was measured at the start and at the end of the test. Samples for the analytical determination at the end of the test were taken from the replicates without algae.
Vehicle:
yes
Remarks:
algal medium
Details on test solutions:
1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration
The concentrations to be tested are based on non GLP pre-tests
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Unicellular freshwater green alga.
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae

Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Hardness is not available, details on composition of stock solutions are reported in section "salinity".
Test temperature:
The temperature in the experiment was in the range 20.5 – 23.4 °C and; therefore, not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
pH:
Blank control pH = 7.7 (0 h) pH = 7.9 (72 h)
Nominal Concentration in mg/L = 1 pH = 7.7 (0 h) pH = 8.0 (72 h)
Nominal Concentration in mg/L = 3.2 pH = 7.7 (0 h) pH = 8.1 (72 h)
Nominal Concentration in mg/L = 10 pH = 7.7 (0 h) pH = 8.3 (72 h)
Nominal Concentration in mg/L = 32 pH = 7.7 (0 h) pH = 8.2 (72 h)
Nominal Concentration in mg/L = 100 pH = 7.7 (0 h) pH = 8.0 (72 h)
Dissolved oxygen:
Not determined.
Salinity:
Composition of the Solutions:
Stock Solution I
NH4Cl 1500 mg
MgCl2*6H2O 1200 mg
CaCl2*2H2O 1800 mg
MgSO4*7H2O 1500 mg
KH2PO4 160 mg
H2O deionised ad 1000 mL

Stock Solution II
FeCl3*6H2O 64 mg
Na2EDTA*2H2O 100 mg
H2O deionised ad 1000 mL

Stock Solution III
H3BO3 185 mg
MnCl2*4H2O 415 mg
ZnCl2 3 mg
CoCl2*6H2O 1.5 mg
CuCl2*2H2O 0.01 mg
Na2MoO4*2H2O 7 mg
H2O deionised ad 1000 mL

Stock Solution IV
NaHCO3 50 g
H2O deionised ad 1000 mL

Algal medium
Stock solution I 10.0 mL
Stock solution II 1.0 mL
Stock solution III 1.0 mL
Stock solution IV 1.0 mL
H2O deionised ad 1000 mL

Stock solutions I and III and the deionised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration. The algal medium corresponds to the OECD TG 201 medium.

Deviations from the nominal weighted loads were less than 5 %
Conductivity:
Not determined.
Nominal and measured concentrations:
Mean of Measured Concentrations

Nominal Concentration Test (mg/L) = 1
Mean Measured Concentration t=0 h (mg/L) = 0.88
Mean Measured Concentration t=72 h (mg/L) = 1.02

Nominal Concentration Test (mg/L) = 3.2
Mean Measured Concentration t=0 h (mg/L) = 3.07
Mean Measured Concentration t=72 h (mg/L) = 3.22

Nominal Concentration Test (mg/L) = 10
Mean Measured Concentration t=0 h (mg/L) = 9.52
Mean Measured Concentration t=72 h (mg/L) = 10.15

Nominal Concentration Test (mg/L) = 32
Mean Measured Concentration t=0 h (mg/L) = 31.30
Mean Measured Concentration t=72 h (mg/L) = 33.09

Nominal Concentration Test (mg/L) = 100
Mean Measured Concentration t=0 h (mg/L) = 96.35
Mean Measured Concentration t=72 h (mg/L) = 105.73
Details on test conditions:
Treatments tested: 1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration. The concentrations to be tested are based on non GLP pre-tests.
Replicates

Treatments:
3 vessels, filled with 45 ±1 mL test solution and algae.
1 vessels, filled with 45 ±1 mL test solution without algae for analytical determination.

Blank Control:
6 vessels, each filled with 45 ±1 mL algal medium and algae.
1 vessel filled with 45 ±1 mL algal medium without algae for analytical determination.
Duration: 72 hours.
Lighting: within the specified range (4440 – 8880 Lux).
Temperature: 20.5 – 23.4 °C.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Results with reference substance (positive control):
72h ErC50 = 0.66 mg/L (95% confidence interval 0.64 – 0.69 mg/L)
72h EyC50 = 0.40 mg/L (95% confidence interval 0.38 – 0.41 mg/L)

Details on findings

Cell Numbers

The cell numbers were determined with an electronic particle counter. The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table.

Table. Cell Number/mL.

Nominal Concentrationin mg/L

Parameter

Cell Number/mL

0 h

24 h

48 h

72 h

Blank control

Mean

2540

8400

53230

269533

SD

0

2856

3982

23607

1

Mean

2540

7133

43667

328973

SD

0

570

6405

55829

3.2

Mean

2540

8760

67053

391680

SD

0

1041

3061

4014

10

Mean

2540

8920

76167

384133

SD

0

1716

566

21113

32

Mean

2540

7433

52767

281727

SD

0

889

8250

55185

100

Mean

2540

8967

55973

259167

SD

0

1529

2089

12826

SD = Standard Deviation

Light Intensity

The light intensity is checked monthly and recorded in a control chart. The lighting corresponded to the specified range of 4440 - 8880 lux.

pH values

In the following table, the pH values measured at the start and the end of the test are stated.

Table. pH values.

Nominal Concentrationin mg/L

0 h

72 h

Blank control

7.7

7.9

1

7.7

8.0

3.2

7.7

8.1

10

7.7

8.3

32

7.7

8.2

100

7.7

8.0


Microscopical Observations

In the following table, the appearance of the algae at the end of the test is stated.

Table. Microscopical Observations.

Nominal Concentrationin mg/L

Normal and Healthy Appearance of the Algae

Blank control

Yes

1

Yes

3.2

Yes

10

Yes

32

Yes

100

Yes

 

Analytical Determination

At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC.

The QC samples were measured in duplicate at the beginning and end of each sequence.

The measured concentrations lay between 88 % and 106 % of the nominal concentrations. Therefore, the determination of the results was based on the nominal concentrations (see OECD Guidance Doc. No.23).

The measured concentrations for treatment and blank control are given in the following tables.

Table. Raw Data and Measured Concentrations.

Nominal
Concentration Test Item

Area
 t = 0 h

Area
 t = 72 h

Dilution Factor

Measured

Concentration
 t = 0 h

Measured

Concentration
 t = 72 h

mg/L

 

 

 

mg/L

mg/L

QC 10 mg/L

6.4760 

6.3678 

--

10.000

10.000

QC 10 mg/L

6.4666 

6.2938 

--

10.000

10.000

Blank control

n.a.

n.a.

--

0.00

0.00

Blank control

n.a.

n.a.

--

0.00

0.00

1.0

0.6195 

0.7429 

--

0.906

1.090

1.0

0.5922 

0.6478 

--

0.863

0.944

3.2

1.9877 

2.1159 

--

3.025

3.203

3.2

2.0482 

2.1365 

--

3.119

3.235

10

6.1928 

6.5627 

--

9.538

10.048

10

6.1707 

6.6943 

--

9.504

10.251

32

4.0644 

4.2847 

5

31.208

32.708

32

4.0869 

4.3846 

5

31.383

33.477

100

6.2702 

6.8361 

10

96.582

104.689

100

6.2408 

6.9708 

10

96.126

106.762

QC 10 mg/L

6.4940 

6.7403 

--

10.000

10.000

QC 10 mg/L[T1] 

6.5269 

6.7240 

--

10.000

10.000

 

Table. Mean of Measured Concentrations and % of Nominal Concentration.

Nominal
Concentration Test Item

Mean Measured Concentration
 t = 0 h

Mean Measured Concentration
 t = 72 h

% of Nominal
t = 0 h

% of Nominal

t = 72 h

mg/L

mg/L

mg/L

%

%

1

0.88

1.02

88

102

3.2

3.07

3.22

96

101

10

9.52

10.15

95

101

32

31.30

33.09

98

103

100

96.35

105.73

96

106

 

Growth Rate, Yield

From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table.

Table. Growth Rate µ, Yield.

Nominal Concentration in mg/L

Parameter

Growth Rate (0-72 h) [day-1]

Yield (0-72 h)
[Cell Concentration/mL]

Blank control

Mean

1.55

266993

SD

0.03

23607

1

Mean

1.62

326433

SD

0.06

55829

3.2

Mean

1.68

389140

SD

0.00

4014

10

Mean

1.67

381593

SD

0.02

21113

32

Mean

1.56

279187

SD

0.07

55185

100

Mean

1.54

256627

SD

0.02

12826

SD = Standard Deviation

 

Inhibition

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

Table. Inhibition Values.

Nominal concentration in mg/L

% Inhibition

Growth Rate (0-72 h)

Yield (0-72 h)

Blank control

0

0

1

-4.14

-22.26

3.2

-8.09

-45.75

10

-7.65

-42.92

32

-0.72

-4.57

100

0.79

3.88

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

 

Validity criteria fulfilled:
yes
Conclusions:
Sodium hydrogen m-sulphonatobenzoate has not exhibited any significant inhibition of algal growth. NOEC 72 h of sodium 3-sulfobenzoate, both for growth rate and yield, has been determined ≥ 100 mg/L.
Executive summary:

Determination of the toxicity of sodium 3-sulfobenzoate towards Desmodesmus subspicatus has been performed according to OECD 201 resp. EU C.3. One valid experiment was performed. No Significant inhibition of algal growth was observed.

At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC. The measured concentrations at the beginning and the end lay between 88 % and 106 % of the nominal concentrations. Therefore, the determination of the results was based on the nominal concentrations (OECD Guidance Doc. No.23).

The 72h-EC50values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.

The pH of the blank control should not fluctuate by more than 1.5 units. The change was 0.2 units in the blank control.

All validity criteria were met.

No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

The following results for the test itemsodium 3-sulfobenzoatewere determined:

Table. Results of the test item.

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

≥ 100 mg/L

> 100 mg/L

> 100 mg/L

> 100 mg/L

Yield

≥ 100 mg/L

> 100 mg/L

> 100 mg/L

> 100 mg/L

Description of key information

Determination of the toxicity of sodium 3-sulfobenzoate towards Desmodesmus subspicatus has been performed according to OECD 201 resp. EU C.3.One valid experiment was performed. No Significant inhibition of algal growth was observed.

At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC. The measured concentrations at the beginning and the end lay between 88 % and 106 % of the nominal concentrations. Therefore, the determination of the results was based on the nominal concentrations (OECD Guidance Doc. No.23).

The 72h-EC50values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.

The pH of the blank control should not fluctuate by more than 1.5 units. The change was 0.2 units in the blank control.

All validity criteria were met.

No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

The following results for the test itemsodium 3-sulfobenzoatewere determined:

Table. Results of the test item.

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

≥ 100 mg/L

> 100 mg/L

> 100 mg/L

> 100 mg/L

Yield

≥ 100 mg/L

> 100 mg/L

> 100 mg/L

> 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

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