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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 September - 4 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test vessels were set up:
- Test solution (Tn); containing Daphnia medium with test item (four replicates per concentration with 5 Daphnia each)
- Blank control (Bn); containing pure Daphnia medium (four replicates with 5 Daphnia each)
- Since the test item is soluble, the test solution was prepared by respective dilutions of a stock solution (100 mg/l test item (i.e. 47.9 mg/l main component) dissolved in Daphnia medium) with aerated Daphnia medium. The resulting solution was used as the test solution in the toxicity test. Pure Daphnia medium served as blank controls.
- O2 concentration and pH were measured in the test medium for each concentration including the blank control. If the O2 concentration was too low (>5.0 mg/l required) the media was aerated by stirring. Afterwards, the media were filled into the test vessels (50 ml per vessel).
- Daphnia, aged less than 24 h and already acclimatized to the Daphnia medium, were then introduced into the test media, and the vessels were covered with a glass plate. The Daphnia were not fed during the test and the test vessels were not aerated.
- static exposure conditions
- Evidence of undissolved material: Not relevant. Since the test item is soluble, the test solution was prepared by respective dilutions of a stock solution (100 mg/l test item (i.e. 47.9 mg/l main component) dissolved in Daphnia medium) with aerated Daphnia medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISMS
- Test organism: Daphnia magna (Straus, 1820), derived from a healthy stock and not first brood progeny
- Breeding: Parental and young Daphnia held in 4 l glass aquaria (3 l medium) at 20±2°C
- Illumination: 16 h per day
- Medium: Continuously aerated Elendt M4 medium prepared with ultra-pure water (conductivity <1.5 µS/cm)
- Feed: Suspension of Desmodesmus subspicatus in Elendt M4 medium with an optical density OD680 of about 15 units

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

not reported

Test temperature:

18–22°C, controlled at ±1 °C

pH:

6.4 to 7.0

Dissolved oxygen:

The minimum dissolved oxygen concentration in the controls and the test vessels at the end of each 24h-period was >=7.3 mg O2/l (required >=3 mg O2/l).

Salinity:

not reported

Conductivity:

741 to 748 µS cm-1

Nominal and measured concentrations:

The concentrations of he main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%)) in the test medium were determined by HPLC analysis at the beginning and after 24 and 48 h of exposure.
These analyses revealed that the test concentrations were stable over the whole 48 h period and remained within 80-120% of the nominal concentration. The
measured concentrations of the main component at the beginning of the test were 21.1, 16.9, 13.5, 10.2, 8.50, 6.49 and 5.22 mg/l; and after 48 hours of exposure they were 21.7, 16.7, 13.2, 10.4, 8.44, 6.72 and 5.10 mg/l (i.e. respectively 103, 99, 98, 102, 99, 104 and 98% of the initial value).

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml beakers, all-glass, with 50 ml of test medium,covered with a glass plate to avoid evaporation and contamination of the test solutions with dust
- Test medium: Elendt M4 medium; prepared with ultra-pure water (conductivity <1.5 µS/cm)
- Number of Daphnia: 20 individuals per test concentration and for the blank, divided in 4 groups of 5 individuals
- Age: Less than 24 h
- Light: 16 h photoperiod a day, supplied by overhead white fluorescent tubes
- Temperature: 18–22 °C, controlled at ±1 °C
- pH: 6 to 9. The pH should normally not vary by more than 1.5units in one test.
- Feed: The Daphnia are not fed during the test
- Aeration: The test vessels were not aerated during the test.
- Test type: static exposure conditions
- Test duration: 48 h

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 whole test item) was performed. The HPLC determinations indicate that the test item concentrations are stable. Static conditions were therefore applied in the definitive test.

EFFECT PARAMETERS MEASURED
Observed immobility (inability to swim) of the daphnids: Observations of immobile Daphnia were made after 24 and 48 h of exposure. Any abnormal behaviour or appearance was reported.

DEVIATIONS OF THE GUIDELINES
The number of Daphnia was unnecessarily high (40 individuals per test concentration, 20 per vessel) and did not follow exactly the recommendations of TG OECD 202 (“at least 20 animals, preferably divided in four groups of five animals each”). According to the amended version, 20 individuals per test concentration and for the blank, were divided in 4 groups of 5 individuals.

A 2nd definitive test was run, to confirm whether the 48h-EC50 is higher, or lower than 10 mg/l of the 1st definitive test. Since the dose-response curve in the 1st definitive test was very steep (10% immobilization at 10 mg/l, and 100% at 20 mg/l), a smaller spacing factor (1.25) and more concentrations (7) were used in the 2nd definitive test. The test concentrations were: 19.5, 15.6, 12.5, 10.0, 8.00, 6.40 and 5.12 mg/l nominal concentration of the main component (corresponding to 40.8, 32.6, 26.1, 20.9, 16.7, 13.4 and 10.7 mg/l nominal concentration of the test item – considering a purity of 47.9%).

Reference substance (positive control):

yes

Remarks:

Acute reference test with potassium dichromate conducted twice a year

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

7.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

11.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

6.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

9.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

13.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

10.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No significant effects (<=10% immobilization) were observed at 10.0, 8.00, 6.40 and 5.12 mg/l or in the blank controls after 24 h of exposure and at 6.40 and 5.12 mg/l or in the blank controls after 48 h of exposure.
Therefore, the median effect concentrations (EC50) of of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) (CAS no. 75314-26-0) on Daphnia magna after 24 h of exposure was calculated to be 13.7 mg/l measured concentrations(95% confidence limits: 12.6–15.0 mg/l) and after 48 h of exposure it was calculated to be 11.4 mg/l measured concentrations (95% confidence limits: 10.4–12.6 mg/l).
The no observed effect concentration (NOEC) after 24 h of exposure was determined to be 10.2 mg/l measured concentrations and after 48 h of exposure it was determined to be 6.39 mg/l measured concentrations.

Results with reference substance (positive control):

Acute ref. test with K2Cr2O7 conducted twice a year. The EC50 value for the control of sensitivity for 24 h of exposure with K2Cr2O7 was estimated to be 0.89 mg/l (29.6.2016), which lies within the recomm. range of 0.6–2.1 mg/l acc. to OECD Guideline 202.

Reported statistics and error estimates:

The effective concentrations ECx were assessed based on the geometric mean of the measured concentrations of the test item.
No statistical analysis was performed.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria i.e. immobilization in the controls <=10% and O2 concentrations at the end of the test >=3 mg/L were fulfilled.

Conclusions:

The acute toxicity of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) (CAS no. 75314-26-0) to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. The median effect concentrations (EC50) of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) on Daphnia magna were estimated to be 13.7 mg/l measured concentration after 24 h and 11.4 mg/l measured concentration after 48 h of exposure. The NOEC values were determined to be 10.2 mg/l measured concentration after 24 h and 6.39 mg/l measured concentration after 48 h of exposure. The results of the test can be considered reliable without restriction.

Executive summary:

The acute toxicity of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) to Daphnia magna were investigated according to the test guideline OECD 202, under static exposure conditions over a period of 48 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 media were prepared by respective dilutions of a stock solution.

The nominal concentrations were 19.5, 15.6, 12.5, 10.0, 8.00, 6.40 and 5.12 mg/l of the main component (CAS no. 75314-26-0). 20 individuals, divided in 4 groups of 5 individuals, were used per test concentration and for the blank.

The concentrations of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) in the test medium were determined by HPLC analysis at the beginning and after 24 and 48 h of exposure. These analyses revealed that the test concentrations were stable over the whole 48 h period and remained within 80-120% of the nominal concentrations. However, instead of assessing the effective concentrations ECx on the nominal concentrations, they were assessed based on the measured concentrations (geometric mean of the respective time intervals), out of consistency with the algal growth inhibition test OECD 201 (ACH study no. A17-00075).

After 24 h, following immobilization rates were observed: 85% at 19.5 mg/l, 85% at 15.6 mg/l and 60% at 12.5 mg/l. No significant effects (<=10% immobilization) were observed at 10.0, 8.00, 6.40 and 5.12 mg/l or in the blank controls.

After 48 h, following immobilization rates were observed: 90% at 19.5 mg/l, 90% at 15.6 mg/l, 85% at 12.5 mg/l, 20% at 10.0 mg/l and 25% at 8.00 mg/l. No significant effects (<=10% immobilization) were observed at 6.40 and 5.12 mg/l or in the blank controls.

The results of the toxicity of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50% ) to Daphnia magna after 24 and 48 h of exposure are summarized in the following table, showing EC-values and 95% confidence limits (cl) based on the measured concentrations:

Parameter, expressed as Measured concentration (mg/l) 24 h of exposure	EC10	EC50	NOEC
Estimate	9.75	13.7	10.2
Lower 95%-cl	8.24	12.6
Upper 95%-cl	10.8	15.0
Parameter, expressed as Measured concentration (mg/l)  48 h of exposure
Estimate	7.37	11.4	6.39
Lower 95%-cl	6.09	10.4
Upper 95%-cl	8.34	12.6

cl: confidence limit

The 48 h EC50 value of the main component (CAS no. 75314-26-0) of Benzene, 1,1’-oxybis(methyl- , sulfonated, ammonium salt (ca 50%) to Daphnia magna was 11.4 mg/l, based on the measured

concentrations.

All validity criteria were fulfilled.

Description of key information

The acute toxicity of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) (CAS no. 75314-26-0) to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. The median effect concentrations (EC50) of the main component of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) on Daphnia magna were estimated to be 13.7 mg/l measured concentration after 24 h and 11.4 mg/l measured concentration after 48 h of exposure. The NOEC values were determined to be 10.2 mg/l measured concentration after 24 h and 6.39 mg/l measured concentration after 48 h of exposure. The results of the test can be considered reliable without restriction.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

11.4 mg/L