1,2-Ethandiylbis(oxy-2,1-ethandiyl)-dimethansulfonat

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:

8 - 14 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

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:

no

Test guideline 2

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:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

Cas Number:

80322-82-3

Molecular formula:

C8H18O8S2

IUPAC Name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

1,2-Ethandiylbis(oxy-2,1-ethandiyl)-monomethanesulfonat

IUPAC Name:

1,2-Ethandiylbis(oxy-2,1-ethandiyl)-monomethanesulfonat

Test material form:

liquid

Details on test material:

Appearance: Dark yellow liquid
Batch No.: 01110008
Storage: -5 – 40 °C, container tightly closed, protected from light

Specific details on test material used for the study:

Batch no.: 01110008
Storage conditions: 2–8°C, protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test media including the control at the start of the test (without algae), after 24, 48 and 72 hours of exposure (containing algae). For sampling at the start of the test the samples were taken from the freshly prepared test solutions as well as from the pure test water serving as control. For sampling at 24, 48 and 72 hours, the replicates per treatment were pooled prior to sampling. These samples were centrifuged at 4500 g for 10 min to separate the algae.

All samples were diluted with acetonitrile to obtain a composition of sample:acetonitrile (6:4; v:v). Subsequently, the samples were stored deep-frozen (at about -20 °C) until analysis. In non-GLP preliminary experiments, the test item proved to be stable under these storage conditions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn): containing test water, test item and algae (three replicates per test concentration)
- Control (Bn): containing test water and algae (six replicates)
- Since the test item is readily soluble in test water, the test solutions were prepared by respective dilutions of a stock solution. A stock solution with a concentration of 100 mg/l was prepared by adding the test item to test water in sterile glassware and stirring it for 15 minutes until dissolution. The respective dilutions of this stock solution were then prepared with test water and sterile glassware. 100 ml of each test media were added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.

For the exposure, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per minute. The test flasks were repositioned daily during the exposure.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Test organism: Raphidocelis subcapitata (SAG Nr. 61.81), supplied by the SAG Culture Collection of Algae at Göttingen University, 37073 Göttingen, Germany.
- Culture: 250 ml flask containing 100 ml of OECD medium inoculated with an exponentially growing preculture of Raphidocelis subcapitata.
- Illumination: Continuous, 4440 – 8880 lux ± max. 15% variation, source: Osram Fluora L 18W/77 and LEDVANCE Cool light, 10 W, 4000 K, 1000 lm
- Temperature: 21–24 °C maintained at ±2 °C in a thermo-controlled room.
- Control of sensitivity: For evaluation of the algal quality and the test procedure, potassium dichromate is tested as a positive control twice a year.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

The calculated water hardness of the test water is 24.2 mg/l as CaCO3.

Test temperature:

21–24 °C maintained at ±2 °C in a thermo-controlled room.

pH:

7.8 - 8.7

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

The concentrations of Triethylene glycol dimethanesulfonate in the test media were determined by HPLC with evaporative light scattering detection in the samples from the test start as well as in the samples after 24, 48 and 72 hours of exposure.

These analyses showed that the test concentrations were correctly dosed and remained within 80–120% of the nominal value over the whole exposure period. Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item.

Based on the results of this non-GLP range finding test the following test item concentrations were selected for the definitive test: 100, 56.0, 32.0, 18.0 and 10.0 mg/l.

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: Reconstituted water (OECD medium) prepared according to the test guidelines was used for algal cultivation and testing.
- Preculture: Exponentially growing liquid culture of Raphidocelis subcapitata kept under the same environmental conditions as in the test.
- Illumination: Source: Osram Fluora L 18W/77 and LEDVANCE Cool light, 10 W, 4000 K, 1000 lm
- Light intensity and homogeneity: Continuous, 4440 – 8880 lux ± max. 15% variation. The test flasks were repositioned daily during the exposure
- 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: Approximately 5’000 cells/ml; all test media contained the same initial density of algal cells.

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 test item was performed (three replicates for the control and two replicates per test concentration).

Based on the results of this non-GLP range finding test the following test item concentrations were selected for the definitive test: 100, 56.0, 32.0, 18.0 and 10.0 mg/l.

EFFECT PARAMETERS MEASURED
- Biomass: Algal biomass is determined at 24, 48 and 72 hours of exposure using a spectrophotometer at 680 nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). To this end, aliquots of 5 ml are removed from each test flask. At test start (0 hours) the nominal value is applied.

Reference substance (positive control):

yes

Remarks:

Control of sensitivity: Potassium dichromate is tested as a positive control twice a year.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

21.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

AVERAGE SPECIFIC GROWTH RATE
With respect to the endpoint average specific growth rate the following inhibitory effects were observed compared to the control: 5% at 100 mg/l, 5% at 56.0 mg/l, 3% at 32.0 mg/l and 2% at 18.0 mg/l nominal concentration. No significant adverse effects were observed at 10.0 mg/l nominal concentration.

The 72-hour EC50 based on average specific growth rate inhibition of Triethylene glycol dimethanesulfonate to Raphidocelis subcapitata was estimated to be >100 mg/l. The EC10 was estimated to be >100 mg/l.

The LOEC with respect to average specific growth rate was determined to be 18.0 mg/l according to a Williams’ multiple t-test (one-sided smaller, a = 0.05). The corresponding NOEC is therefore 10.0 mg/l.

YIELD
With respect to the endpoint yield, the following inhibitory effects were observed compared to the control: 24% at 100 mg/l, 24% at 56.0 mg/l, 15% at 32.0 mg/l and 9% at 18.0 mg/l nominal concentration. No significant adverse effects were observed at 10.0 mg/l nominal concentration.

The 72-hour EC50 based on yield inhibition of Triethylene glycol dimethanesulfonate to Raphidocelis subcapitata was estimated to be >100 mg/l. The EC10 was calculated to be 21.2 mg/l (95% confidence interval: 10.8–29.9 mg/l). These ECx were computed using a linear regression with a logit link function and a maximum likelihood algorithm (slope = 1.79, intercept = -4.57, degrees of freedom = 13, p(Chi²) = 1.00, r2 = 0.716, p(F) = <0.001).

The LOEC with respect to average specific growth rate was determined to be 18.0 mg/l according to a Williams’ multiple t-test (one-sided smaller, a = 0.05). The corresponding NOEC is therefore 10.0 mg/l.

OBSERVATIONS AND ENVIRONMENTAL CONDITIONS
The microscopic examination of the algal cells at the end of the test showed no abnormal appearance of the algae growing at the nominal test concentration of 100 mg/l. The appearance of the algal cells was not affected by the test item up to at least this concentration.

No remarkable observations were made concerning the appearance of the test media.

The pH value in the control replicates changed by 0.7 units. The light intensity was maintained at 5360 lux ± 8%. The temperature remained at 22°C during the whole exposure period. All values were within the guideline
ranges.

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® Pro.

Any other information on results incl. tables

Validity of the test:

 

Validity criterion (applies to control cultures)	Required value	Achieved value
Average specific growth rate [d-1]	>=0.92	1.83
CV of average specific growth rate over 3 days [%]	<=7	0.6
CV of section-by-section specific growth rates [%]	<=35	4.4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See table in section "any other information on results incl. tables".

Conclusions:

The acute toxicity of Triethylene glycol dimethanesulfonate (CAS no. 80322-82-3) to the green alga Raphidocelis subcapitata 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 Triethylene glycol dimethanesulfonate to the green alga Raphidocelis subcapitata was estimated to be >100 mg/l. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 10.0 mg/l. The results of the test can be considered reliable without restriction.

Executive summary:

The growth inhibitory effects of Triethylene glycol dimethanesulfonate (CAS no. 80322-82-3) to the green alga Raphidocelis subcapitata were investigated according to the OECD guideline 201 over a period of 72 hours.

The test item is liquid, 94.3% (area) pure and soluble in test water (OECD medium) up to at least 100 mg/l. Consequently, the test  solutions were prepared by respective  dilutions of a stock solution. The nominal concentrations tested were 100, 56.0, 32.0, 18.0 and 10.0 mg/l.

The concentrations of Triethylene glycol dimethanesulfonate in the test media were determined by HPLC with evaporative light scattering detection in the samples from the test start as well as in the samples after 24, 48 and 72 hours of exposure. These analyses showed that the test concentrations were correctly dosed and remained stable within 80–120% of the nominal value over the  exposure period. Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item over the entire 72 hours test period.

With respect to the endpoint average specific growth rate, the following inhibitory effects were observed compared to the control: 5% at 100 mg/l, 5% at 56.0 mg/l, 3% at 32.0 mg/l and 2% at 18.0 mg/l nominal concentration. No significant adverse effects were observed at 10.0 mg/l nominal concentration.

With respect to the endpoint yield, the following inhibitory effects were observed compared to the control: 24% at 100 mg/l, 24% at 56.0 mg/l, 15% at 32.0 mg/l and 9% at 18.0 mg/l nominal concentration. No significant adverse effects were observed at 10.0 mg/l nominal concentration.

The results of the average specific growth rate and yield inhibition of Triethylene glycol dimethanesulfonate to the green alga Raphidocelis subcapitata are summarised in the following table based on the nominal concentrations of the test item:

 

Parameter (0-72 h)	Growth rate [mg/l]	Yield [mg/l]
EC50	>100	>100
EC10	>100	21.2
LOEC	18.0	18.0
NOEC	10.0	10.0

The 72 h EC50 value based on average specific growth rate inhibition of Triethylene glycol dimethanesulfonate (CAS no. 80322-82-3) on the green alga Raphidocelis subcapitata was >100 mg/l and the EC10 was >100 mg/l based on nominal concentrations of the test item.

All validity criteria were fulfilled.