Sodium ethylenesulphonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Arrival in facility: 15 February 2021
Stability: 6 months
Internal code: 21/6733

Analytical monitoring:

yes

Remarks:

The method used is based on Reversed Phase chromatography using acetonitrile/buffer as mobile phase and direct injection with UV detection.

Details on sampling:

Chemical analysis of the test item concentration in the test solutions was performed to determine the exposure concentrations of the test item during the exposure period. Substance specific analysis was performed at the test site biochemA GmbH.
The samples were filled directly into 2 mL glass vials and stored in the freezer at ≤ -18 °C. After 0 h, 24 h, 48 h and 72 h exposure, the additional vessels for chemical analysis of NC, A, B, C, D and E were sampled: 2 samples of 1 mL per treatment group. Of each sampled treatment, one of the analytical samples from 0 h, 24 h, 48 h and 72 h was sent to the analytical laboratory at the test site biochemA GmbH for chemical analysis, in a frozen state and one transfer using an insulated box with thermal packs. The remaining samples were stored as retain samples in the freezer at ≤ -18 °C until finalisation of the study. Detailed information of the analytical method is given in the analytical phase report.
Additionally, TOC analysis of the test solutions was performed to determine the exposure concentrations of the test item during exposure period. The Non Purgable Organic Carbon [NPOC] method (sparging with oxygen after acidification) was applied with a total carbon analyser (Shimadzu TOC-L with autosampler ASI-L) using a non-dispersive infrared (NDIR) detector. The TOC attributable to the test substance in the test item solutions was calculated by subtraction of the mean value of the control (NC) measurements.
After 0 h, 24 h, 48 h and 72 h exposure, the additional vessels for TOC analysis (without test organisms in order to not disturb the TOC analysis) of NC, A, B, C, D and E were sampled: 4 samples of 8 mL per treatment group. Of the 4 samples per treatment and sampling time, 2 samples were analysed directly after sampling using the TOC-analyser and 2 samples were stored as retain samples in the freezer at ≤ -18 °C until finalisation of the study.

Vehicle:

no

Test type:

static

Water media type:

freshwater

Remarks:

The purpose was to investigate the effects of the test item on the growth of freshwater algae Raphidocelis subcapitata. A four day old pre-culture was used as inoculum in the test and algae were exposed to five test item concentrations for 72 hours.

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature during the exposure was 22.0 – 22.9 °C (required: 21 - 24 °C, constant within ± 2 °C)

pH:

The pH was 7.6 – 7.8 in the control and 7.6 – 7.9 in the test item treatment (required: increase ≤ 1.5).

Nominal and measured concentrations:

Nominal test item concentration [mg/L]: NC: 0; A: 25; B: 50; C: 100; D: 200; E: 400

Details on test conditions:

The purpose of this study was to investigate the effects of the test item on the growth of freshwater algae Raphidocelis subcapitata. A four day old pre-culture was used as inoculum in the test. The algae were exposed to five concentrations of the test item in OECD TG 201 medium for 72 h.
A static test design was applied with an accompanying TOC analysis and an additional substance specific chemical analysis to determine the test item concentrations in the test vessels. The algal
biomass was determined after 0 h, 24 h, 48 h and 72 h exposure by measuring the surrogate parameter chlorophyll fluorescence. In the present study, five nominal concentrations of 25 – 400 mg/L test item were used. A preliminary range finding test without GLP was performed before the start of this GLP-study. Nominal concentrations of 12, 60 and 300 mg/L test item were tested and resulted in inhibition of 3.4 %, 4.4 % and 9.7 % for growth rate and 14.3 %, 17.7 % and 34.7 % for yield (72 h), respectively.
The test was performed with OECD TG 201 medium according to OECD 201 (2006), prepared as following:
Solution A1: Ammonium chloride p.a. (NH4Cl 1.5 g), Magnesium chloride p.a. (MgCl2 × 6 H2O 1.2 g), Calcium chloride dihydrate e.p. (CaCl2 × 2 H2O 1.8 g ), Magnesium sulfate heptahydrate p.a. (MgSO4 × 7 H2O 1.5 g), Potassium dihydrogen phosphate p.a. (KH2PO4 0.16 g), dissolved in 1000 mL ultrapure water
Solution A2: Iron (III) chloride hexahydrate p.a. (Fe(III)Cl3 × 6 H2O 0.064 g), Sodium-EDTA dihydrate (Na2EDTA × 2 H2O 0.100 g), dissolved in 1000 mL ultrapure water
Solution A3: Boric acid p.a. (H3BO3 0.185 g), Manganese chloride tetrahydrate p.a. (Mn(Cl2) × 4 H2O 0.415 g), Sodium molybdate dihydrate p.a. (Na2MoO4 × 2 H2O 0.007 g), Zinc chloride p.a. (ZnCl2 0.003 g), Cobalt chloride hexahydrate p.a. (CoCl2 × 6 H2O 0.0015 g), Copper chloride dihydrate p.a. (CuCl2 × 2 H2O 0.00001 g), dissolved in 1000 mL ultrapure water (for ZnCl2, CoCl2 and CuCl2, higher concentrated solutions were prepared and the corresponding volumes used for A3).
Solution A4: Sodium hydrogen carbonate p.a. (NaHCO3 50 g), dissolved in 1000 mL ultrapure water
Solutions A1 – A3 were autoclaved, and solution A4 was sterile-filtered (pore diameter 0.2 μm).
A 10-fold concentrated medium was prepared by using 100 mL of stock solution A1 as well as 10 mL of stock solutions A2, A3 and A4, filling up to 1000 mL with ultrapure water and aerating for 30 min. The 1-fold concentrated medium was prepared by diluting 325 mL of the 10-fold concentrated medium with 2925 mL ultrapure water.

Reference substance (positive control):

yes

Remarks:

potassium dichromate: ErC50 (72 h) of 1.06 mg/L (CL 95 %: 0.96 – 1.16 mg/L; interlaboratory test result for ErC50: mean of 1.19 mg/L with SD of 0.27 mg/L) and an EyC50 (72 h) of 0.46 mg/L (CL 95 %: 0.44 – 0.47 mg/L).

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 400 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

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:

EC10

Effect conc.:

148.9 mg/L

95% CI:

> 64.4 - < 344.1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

239.5 mg/L

95% CI:

> 138.5 - < 400

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

55.4 mg/L

95% CI:

> 36.4 - < 84.1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Results with reference substance (positive control):

potassium dichromate: ErC50 (72 h) of 1.06 mg/L (CL 95 %: 0.96 – 1.16 mg/L; interlaboratory test result for ErC50: mean of 1.19 mg/L with SD of 0.27 mg/L) and an EyC50 (72 h) of 0.46 mg/L (CL 95 %: 0.44 – 0.47 mg/L).

Summary of results: As the measured test item concentrations are within ± 20 % of the nominal concentrations, according to OECD 201 (2006), all results are given in relation to the nominal test item concentrations.

 

(Lowest/No Observed) Effect Concentration	Nominal test item concentration [mg/L]
95 % Confidence Limits (CL)	Yield (72 h)	Growth rate (72 h)
EC50	239.5	> 400
CL	138.5 – 400.0	n.d.
EC10	55.3	148.9
CL	36.4 – 84.1	64.4 – 344.1
LOEC	200	200
NOEC	100	100

n.d.: not determined due to mathematical reasons or inappropriate data

The test is valid according to OECD Test Guideline 201 (23 March 2006).

The measured mean TOC concentrations in the test item treatments were 80.5 – 105.4 % of the nominal TOC concentrations. In the control, the test item was not detected applying the substance specific chemical analysis. The measured test item concentrations in the test item treatments were 99.18 – 108.3 % of the nominal concentrations.

 

Results of the R. subcapitata Growth Inhibition Test

Algae cell concentration: The algae cell concentrations in the control (NC) and the test item treatments are presented in Figure 1 (see Illustration).

Cell morphology: The morphology of the algal cells in the test item treatments as well as in the control showed no obvious abnormality.

Yield: The inhibition of the yield in the test item treatment, in relation to the control, is presented in Table 1.

Table 1: Inhibition of yield after 24 h, 48 h and 72 h exposure

Nominal test item concentration [mg/L]	Inhibition of yield [%]
	24 h	48 h	72 h
NC	--	--	--
25	-3.5	-24.3	-57.9
50	11.5	10.8	7.9
100	26.6	17.6	7.7
200	40.8	45.3	45.9
400	35.3	48.0	52.0

 

6.2.4 Growth rate

The inhibition of the specific growth rate in the test item treatment, in relation to the control, is presented in Table 2.

Table 2: Inhibition of growth rate after 24 h, 48 h and 72 h exposure

Nominal test item concentration [mg/L]	Inhibition of growth rate [%]
	24 h	48 h	72 h
NC	--	--	--
25	-2.6	-8.0	-11.5
50	6.5	3.7	2.4
100	17.2	6.6	2.4
200	27.9	19.9	14.8
400	23.7	21.6	17.7

 

Effect concentrations: The effect concentrations after 72 h exposure are calculated with the statistical software ToxRat Professional 3.3.0. As the measured test item concentrations are within ± 20 % of the nominal concentrations, according to OECD 201 (2006), all results are given in relation to the nominal test item concentrations.

Table 3: Effect concentrations for yield and growth rate at the end of the test

(Lowest/No Observed) Effect Concentration	Nominal test item concentration [mg/L]
95 % Confidence Limits (CL)	Yield (72 h)	Growth rate (72 h)
EC50	239.5	> 400
CL	138.5 – 400.0	n.d.
EC20	91.5	337.9
CL	60.9 – 137.7	133.8 – 862.5
EC10	55.3	148.9
CL	36.4 – 84.1	64.4 – 344.1
LOEC	200	200
NOEC	100	100

n.d.: not determined due to mathematical reasons or inappropriate data

 

Test parameters

- The pH was 7.6 – 7.8 in the control and 7.6 – 7.9 in the test item treatment (Table 4; required: increase ≤ 1.5).

- The temperature during the exposure was 22.0 – 22.9 °C (required: 21 - 24 °C, constant within ± 2 °C). 

- The lighting was continuous with an intensity of 93.1 µE m-² s-1 ± 6.0 % (required: 60 – 120 µE m-² s-1).

Table 4: pH after 0 h and 72 h exposure

Nominal test item concentration [mg/L]	0 h	72 h
NC	7.8	7.6
25	7.8	7.6
50	7.9	7.6
100	7.9	7.6
200	7.9	7.7
400	8.0	7.7

 

Results of the TOC analysis: The measured mean TOC concentrations in the test item treatments were 80.5 – 105.4 % of the nominal TOC concentrations (Table 5). As the measured mean TOC concentrations are within ± 20 % of the nominal TOC concentrations, according to OECD 201 (2006), all results are given in relation to the nominal test item concentrations.

Table 5: Analytically measured TOC concentrations after 0 h, 24 h, 48 h and 72 h exposure

Nominal test item concentration [mg/L]	Sample time [h]	Measured TOC conc. [mg/L]*	Mean measured TOC conc. [mg/L]	Mean TOC conc. NC-corrected [mg/L]	Recovery rate (C-content test item: 5.845 %) [%]	Geometric mean recovery rate [%]
NC	0	0.516	0.78	--	--	--
		1.035
	24	0.577	0.88	--	--
		1.186
	48	0.724	0.96	--	--
		1.191
	72	0.927	1.29	--	--
		1.663
25 (A)	0	1.916	1.92	1.15	78.4	80.5
		1.926
	24	2.057	2.03	1.15	78.4
		1.999
	48	2.211	2.22	1.26	86.6
		2.234
	72	2.482	2.45	1.15	78.9
		2.412
50 (B)	0	3.435	3.50	2.73	93.3	92.4
		3.569
	24	3.469	3.52	2.64	90.4
		3.576
	48	3.817	3.73	2.77	95.0
		3.648
	72	3.974	3.95	2.66	91.0
		3.935
100 (C)	0	6.338	6.32	5.55	94.9	95.3
		6.309
	24	6.338	4.74	5.36	91.8
		6.151
	48	6.636	6.73	5.77	98.7
		6.822
	72	6.818	6.90	5.60	95.9
		6.978
200 (D)	0	13.19	13.06	12.3	105	105.4
		12.93
	24	13.23	13.15	12.3	105
		13.07
	48	13.24	13.39	12.4	106
		13.53
	72	13.64	13.58	12.3	105
		13.52
400 (E)	0	24.30	24.19	23.4	100	99.5
		24.08
	24	23.76	24.10	23.2	99.3
		24.43
	48	24.40	24.22	23.3	99.5
		24.04
	72	24.43	24.48	23.2	99.2
		24.53

Limit of Quantification (LOQ): 0.128 mg/L

 

Results of the substance specific chemical analysis: In the control, the test item was not detected. The measured test item concentrations in the test item treatments were 99.18 – 108.3 % of the nominal concentrations. As the measured test item concentrations are within ± 20 % of the nominal concentration, according to OECD 201 (2006), all results are given in relation to the nominal test item concentration.

Table 6: Analytically determined test item concentrations after 0 h, 24 h, 48 h and 72 h exposure

Nominal test item concentration [mg/L]	Sample	Sample time [h]	Determined concentration [mg/L]	Recovery [%] nominal
NC	1600-NC1-0h	0	No Peak	--
	1600-NC1-24h	24	No Peak	--
	1600-NC1-48h	48	No Peak	--
	1600-NC1-72h	72	No Peak	--
25 (A)	1600-A1-0h	0	26.35	105.4
	1600-A1-24h	24	27.08	108.3
	1600-A1-48h	48	26.17	104.7
	1600-A1-72h	72	26.26	105.0
50 (B)	1600-B1-0h	0	51.82	103.6
	1600-B1-24h	24	52.08	104.2
	1600-B1-48h	48	51.60	103.2
	1600-B1-72h	72	52.35	104.7
100 (C)	1600-C1-0h	0	103.1	103.1
	1600-C1-24h	24	102.6	102.6
	1600-C1-48h	48	104.3	104.3
	1600-C1-72h	72	103.9	103.9
200 (D)	1600-D1-0h	0	202.3	101.2
	1600-D1-24h	24	204.9	102.5
	1600-D1-48h	48	207.2	103.6
	1600-D1-72h	72	209.0	104.5
400(E)	1600-E1-0h	0	397.2	99.30
	1600-E1-24h	24	396.9	99.23
	1600-E1-48h	48	396.7	99.18
	1600-E1-72h	72	401.5	100.4

Limit of Detection (LOD): 2.18 mg/L

Limit of Quantification (LOQ): 7.26 mg/L

 

Criteria of validity:

- The biomass (cell concentration) in the control has increased by a factor of 54.9 and therefore ≥ 16 during the test period. 

- The mean coefficient of variation for section-by-section growth rate (day 0 – 1, 1 – 2 and 2 – 3) in the control is 17.1 % and therefore ≤ 35 %.

- The coefficient of variation of average specific growth rate during the test period in the control replicates is 4.0 % and therefore ≤ 7 %.

The test is valid according to OECD Test Guideline 201 (23 March 2006).

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for Sodium vinyl sulphonate 30% based on growth rate (72 h) was found being > 400 mg/L; the NOEC was found being 100 mg/L based on growth rate and yield according to OECD Test Guideline 201 (23 March 2006) ( Test organism: Raphidocelis subcapitata).

Executive summary:

Summary of results: As the measured test item concentrations are within ± 20 % of the nominal concentrations, according to OECD 201 (2006), all results of this Growth Inhibition Test with R. subcapitata are given in relation to the nominal test item concentrations.

(Lowest/No Observed) Effect Concentration	Nominal test item concentration [mg/L]
95 % Confidence Limits (CL)	Yield (72 h)	Growth rate (72 h)
EC50	239.5	> 400
CL	138.5 – 400.0	n.d.
EC10	55.3	148.9
CL	36.4 – 84.1	64.4 – 344.1
LOEC	200	200
NOEC	100	100

n.d.: not determined due to mathematical reasons or inappropriate data

The measured mean TOC concentrations in the test item treatments were 80.5 – 105.4 % of the nominal TOC concentrations. In the control, the test item was not detected applying the substance specific chemical analysis. The measured test item concentrations in the test item treatments were 99.18 – 108.3 % of the nominal concentrations. The test is valid according to OECD Test Guideline 201 (23 March 2006).

Description of key information

In a new OECD 201 study (Growth Inhibition Test with R. subcapitata) the ErC50 (based on active substance following 96 hours exposure) was found being >400 mg/l. The LOEC was found being 200 mg/l and the NOEC was set to 100 mg/l.

In another study, performed with a multi-constituent substance, containing sodium ethylene sulphonate, in a 72-h algal growth inhibition test with Pseudokirchnerella subcapitata the EC50 value based on growth rate was determined to be greater than 100 mg/L for 25.09 % active. The overall NOEC was determined to be 0.3 mg/L. All results were based on nominal concentrations. Apparently, the impurity profile of the multi-constituent substance is decisive for algal growth and thus, this study was disregarded for assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

400 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

In a new OECD 201 study (Growth Inhibition Test with R. subcapitata) the ErC50 (based on active substance following 96 hours exposure) was found being >400 mg/l. The LOEC was found being 200 mg/l and the NOEC was set to 100 mg/l.

The measured mean TOC concentrations in the test item treatments were 80.5 – 105.4 % of the nominal TOC concentrations. In the control, the test item was not detected applying the substance specific chemical analysis. The measured test item concentrations in the test item treatments were 99.18 – 108.3 % of the nominal concentrations. The test is valid according to OECD Test Guideline 201 (23 March 2006). As the measured test item concentrations are within ± 20 % of the nominal concentrations, according to OECD 201 (2006), all results of this Growth Inhibition Test with R. subcapitata are given in relation to the nominal test item concentrations.

In another study, performed with a multi-constituent substance, containing sodium ethylene sulphonate, in a 72-h algal growth inhibition test with Pseudokirchnerella subcapitata the EC50 value based on growth rate was determined to be greater than 100 mg/L for 25.09 % active. The overall NOEC was determined to be 0.3 mg/L. All results were based on nominal concentrations. Apparently, the purity profile is decisive and thus, this study was disregarded for assessment.