Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

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:

25 September- 3 October 2017

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, Annex 5 corrected 28 July 2011

Deviations:

yes

Remarks:

The expiry date of the test item was prolonged based on a new certificate of analysis provided by the sponsor. This has no influence on the results of this study, as the experimental work was completed before the initial expiry date of the test item.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266

Deviations:

yes

Remarks:

The expiry date of the test item was prolonged based on a new certificate of analysis provided by the sponsor. This has no influence on the results of this study, as the experimental work was completed before the initial expiry date of the test item.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test medium, test item and algae (three replicates per concentration)
- Blank control (Bn); containing test medium and algae (six replicates)
- Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution. A 100 mg/l (of main component; i.e. 209 mg/l of test item) stock solution was prepared by adding the test item to sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware and stirred for 30 minutes. The respective dilutions of this stock solution were then prepared with sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) and sterile glassware. 100 ml of each test solution were added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
CHECK, Steht sonst in jedem Bericht!!!Pure Algal medium, also sterile filtered into sterile test flasks and inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls. CHECK!!!!
For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
- Evidence of undissolved material: Not relevant. Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from an axenic slope culture
- Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- Control of sensitivity: twice per year, with potassium dichromate

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

Min: 22.0; Max: 23.2; Mean: 22.5

pH:

6.7 - 7.7

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

The concentrations of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%)) in the test media were measured by HPLC analysis at the beginning and after 24, 48 h and 72 h of exposure. These analyses revealed that the concentrations decreased over the 72 h test period and did not remain within 80-120% of the nominal concentration. The measured concentrations at the beginning of the test were 99.2, 31.4, 9.73, 2.99 and 0.979 mg/l;
and 95.1, 27.6, 8.28, 2.35 and 0.719 mg/l (e.g. 96, 88, 85, 79 and 73% of the initial value) after the 72 h test period.
Therefore, the effective concentrations ErCx/EyCx were assessed based on the geometric mean of the measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm), capped with air permeable stoppers
- Test medium: Recommended OECD medium
- Preculture: Exponentially growing culture of Desmodesmus subspicatus
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity: the light intensity amounts to about 3000 lux which corresponds to about 40 µE•m-2•s-2
- Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units; all test solutions contain the same initial biomass of test alga)

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of the main component (i.e. 2.09, 20.9 and 209 mg/l of the test item) was performed. The HPLC determinations indicate that the test concentrations are decreasing, as they did not remain in the recommended 80-120% range of the nominal concentration and/or of the initial concentration.

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.

DEVIATIONS FROM THE GUIDELINES
The CAS no. given in the study plan was not correct. It was therefore revised in the report.
The expiry date of the test item was prolonged based on a new certificate of analysis provided by the sponsor. This has no influence on the results of this study, as the experimental work was completed before the initial expiry date of the test item.

Reference substance (positive control):

not required

Remarks:

Control of sensitivity: None – the culture is re-purchased annually

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.08 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Remarks:
- The stock solution for the 1st definitive test was prepared with 100 mg/l of the test item instead of 100 mg/l of the main component. Hence, the test concentrations were too low and the test had to be repeated in a 2nd definitive test. Only the results of the 2nd definitive test are reported here.

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: yes (growth rate and yield)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 82% at 100 mg/l, 75% at 31.6 mg/l and 21% at 10.0 mg/l. No significant effects were observed at 3.16 and at 1.00 mg/l as compared to the blank control.
Based on these effects and the measured concentrations, the median effect concentration with respect to growth rate (ErC50/0–3 days) of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) to Desmodesmus subspicatus was calculated to be 17.8 mg/l (95% confidence limits: 16.4–19.4 mg/l).
The low effect concentration with respect to growth rate ErC10 was calculated to be 4.70 mg/l (95% confidence limits: 3.87–5.51 mg/l).
The no observed effect concentration (NOErC) with respect to growth rate was determined to be 2.39 mg/l.

With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 99% at 100 mg/l, 98% at 31.6 mg/l and 62% at 10.0 mg/l. No significant effects were observed at 3.16 mg/l and at 1.00 mg/l as compared to the blank control.
Based on these effects and the measured concentrations, the median effect concentration with respect to yield (EyC50/0–3 days) of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) to Desmodesmus subspicatus was calculated to be 7.08 mg/l (95% confidence limits: 6.45–7.65 mg/l).
The low effect concentration with respect to yield EyC10 was calculated to be 3.11 mg/l (95% confidence limits: 2.29–3.78 mg/l).
The no observed effect concentration (NOEyC) with respect to yield was determined to be 2.39 mg/l.

Reported statistics and error estimates:

Statistical analysis was performed with respect to the no observed effect concentrations. The statistical analysis was conducted with the software ToxRat® Standard Version 3.2.1.

Validity criteria fulfilled:

yes

Remarks:

In the control cultures: average specific growth rate of 1.52 (>0.92)/ coefficient of variation of average specific growth rates: 1% (<=7%)/ mean coefficient of variation for section-by-section specific growth rates 18.9% (<=35%).

Conclusions:

The acute toxicity of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) (CAS no. 75314-26-0) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) to Desmodesmus subspicatus was estimated to be 17.8 mg/l based on the geometric mean of the measured concentrations. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 2.39 mg/l based on the geometric mean of the measured concentrations. The results of the test can be considered reliable without restriction.

Executive summary:

The growth inhibitory effects of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) to the green alga Desmodesmus subspicatus were investigated according to test guideline OECD 201, over a period of 72 h.

The test item Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) is a viscous liquid, 47.9% pure (main component CAS no. 75314-26-0) and is soluble in water. Consequently, the test solutions were prepared by respective dilutions of a stock solution.

The nominal concentrations were 100, 31.6, 10.0, 3.16 and 1.00 mg/l of the main component (CAS no. 75314-26-0). Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

The concentrations of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) in the test media were measured by HPLC analysis at the beginning and after 24, 48 h and 72 h of exposure. These analyses revealed that the test concentrations decreased over the 72-h test period (e.g. 96, 88, 85, 79 and 73% of the initial value) and did not remain within 80 - 120% of the nominal concentration. Therefore, the effective concentrations ErCx/EyCx were assessed based on the geometric mean of the measured concentrations.

With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 82% at 100 mg/l, 75% at 31.6 mg/l and 21% at 10.0 mg/l. No significant effects were observed at 3.16 and at 1.00 mg/l as compared to the blank control.

With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 99% at 100 mg/l, 98% at 31.6 mg/l and 62% at 10.0 mg/l. No significant effects were observed at 3.16 mg/l and at 1.00 mg/l as compared to the blank control.

The results of the growth rate (r) and yield (y) inhibition of the main component of Benzene, 1,1’- oxybis(methyl-, sulfonated, ammonium salt (ca 50% ) to the green alga Desmodesmus subspicatus are summarized in the following table showing EC values and 95% confidence limits (cl) based on the geometric mean of the measured concentrations:

Parameter, expressed as Measured concentration (mg/l)	ErC10	ErC50	NOErC
Estimate	4.70	17.8	2.39
Lower 95%-cl	3.87	16.4
Upper 95%-cl	5.51	19.4
Parameter, expressed as Measured concentration (mg/l)	EyC10	EyC50	NOEyC
Estimate	3.11	7.08	2.39
Lower 95%-cl	2.29	6.45
Upper 95%-cl	3.78	7.65

cl: confidence limit

The 72 h ErC50 value of the main component (CAS no. 75314-26-0) of Benzene, 1,1’- oxybis(methyl-, sulfonated, ammonium salt (ca 50%) on the green alga Desmodesmus subspicatus was 17.8 mg/l. This value is based on the geometric mean of the measured concentrations.

All validity criteria were fulfilled.

Description of key information

The acute toxicity of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) (CAS no. 75314-26-0) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) to Desmodesmus subspicatus was estimated to be 17.8 mg/l based on the geometric mean of the measured concentrations. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 2.39 mg/l based on the geometric mean of the measured concentrations. The results of the test can be considered reliable without restriction.

Key value for chemical safety assessment

EC50 for freshwater algae:

17.8 mg/L

EC10 or NOEC for freshwater algae:

2.39 mg/L