(R*,R*)-1,4-dimercaptobutane-2,3-diol

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 - 31 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

(R*,R*)-1,4-dimercaptobutane-2,3-diol

EC Number:

222-468-7

EC Name:

(R*,R*)-1,4-dimercaptobutane-2,3-diol

Cas Number:

3483-12-3

Molecular formula:

C4H10O2S2

IUPAC Name:

1,4-disulfanylbutane-2,3-diol

Specific details on test material used for the study:

Batch no.: 44606300 / 50774
Storage conditions: 2–8°C, under nitrogen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test media at the start of the test as well as after 24 hours in the fresh and old media and after 48 hours of exposure. In the fresh test media, the samples were taken from the freshly prepared test solutions as well as from the pure test water serving as control. In the aged test media, aliquots from the respective replicates were pooled before sampling.
All samples were diluted with acetonitrile containing 1.0% conc. phosphoric acid to obtain a composition of sample:acetonitrile (9:1; v:v) containing 0.1% conc. phosphoric acid. Subsequently, the samples were stored deep-frozen (at about -20 °C) until analysis or analysed directly without storage. 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 vessels were set up:
- Test solution (Tn); containing test water with test item (four replicates with five Daphnia each)
- Blank control (Bn); containing pure test water (four replicates with five Daphnia each)
- Since the test item is soluble, the test solutions were prepared by respective dilutions of a suitable stock solution, which was prepared by adding 100 mg/l of the test item to test water and stirring until dissolution. The respective dilutions of this stock solution were then prepared with test water. Pure test water served as blank controls.
The test vessels were filled with 50 ml of the respective test medium. Daphnia, aged less than 24 hours and already acclimatised to the test water, were 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.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISMS
- Test organism: Daphnia magna Straus, supplied by IES Ltd., 4108 Witterswil, Switzerland
- Breeding: The daphnids are cultivated in Elendt M4 medium at 18–22 °C, controlled at ±1 °C
- Illumination: 16 h per day, about 550-1100 lux, source: overhead white fluorescent tubes
- Medium: Reconstituted water (Elendt M4 medium)
- Feed: Suspension of Desmodesmus subspicatus in Elendt M4 medium with an optical density OD6
80 of about 12 units. This results in a final concentration of about 0.1 units from Monday to Thursday
and 0.4 units on Fridays in the culture vessel.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

2.5 mM (based on the sum of Ca2+ and Mg2+), Carbonate Alkalinity: 0.8 mM

Test temperature:

18–22°C, controlled at ±1 °C

pH:

7.0 to 7.7

Dissolved oxygen:

The minimum dissolved oxygen concentration in the controls and the test vessels at the end of each 24 h-period was >=8.1 mg O2/l (required >=3 mg O2/l).

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

The concentrations of the test item (DTT) as well as its oxidation product (ox. DTT) in the test media were analysed by HPLC with UV detection in samples from the test start as well as in the samples after 24 hours (fresh and old test media) and 48 hours of exposure.
The test item was correctly dosed in both 24-hour test intervals, but degradation set in very quickly. After 24 hours of exposure, DTT seems to have completely transformed into ox. DTT in all test concentrations with ox. DTT recoveries based on nominal test item concentrations of 93-103%.
Based on the results of the non-GLP preliminary tests, the test item itself as well as its degradation products have been shown to exhibit toxic properties. As it is not possible to distinguish the exact proportions to which each species contributed and also taking into account combined toxicity effects, the effective concentrations ECx were assessed based on the nominal concentrations of the test item.

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, five per vessel
- Age: Less than 24 h
- Light: 116 hours photoperiod per day, about 550–1100 lux, 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 1.5 units in one test.
- Feed: The Daphnia are not fed during the test
- Test type: Semi-static exposure conditions; renewal of test medium after 24 h of exposure
- 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 of the test item molecule (DTT) as well as the formation of a degradation product (oxidised DTT) was performed (two replicates for each of the test concentration and the control; five daphnids per replicate).

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.

Reference substance (positive control):

yes

Remarks:

Acute reference test with potassium dichromate conducted twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

34.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

59.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 24 hours of exposure, the following immobilisation rates were observed: 90% at 100 mg/l and 35% at 50 mg/l. No immobilised daphnids were observed at all lower test concentrations or in the control.
The 24-h EC50 of 1,4-Dithiothreitol to Daphnia magna was calculated to be 59.8 mg/l (95% confidence interval: 49.9–72.0 mg/l). These ECx values were computed using a linear regression.
The no observed effect concentration (NOEC) after 24 hours was determined to be 25.0 mg/l.

After 48 hours of exposure, the following immobilisation rates were observed: 100% at 100 and 50 mg/l. No immobilised daphnids were observed at all lower test concentrations or in the control.
The 48-h EC50 of 1,4-Dithiothreitol to Daphnia magna was calculated to be 34.8 mg/l (95% confidence interval: 31.2–38.9 mg/l). These ECx values were computed using a linear regression.
The no observed effect concentration (NOEC) after 48 hours was determined to be 25.0 mg/l.

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 concentration EC50 for 24 and 48 hours was estimated based on the concentration tested and immobilisation observed.

The no observed effect concentration (NOEC) is the highest test concentration for which the test item is observed to have an immobilisation rate not exceeding 10%. Thus, immobilisation rates >10% are considered to be significant.

Applicant's summary and conclusion

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 1,4-Dithiotreitol (CAS no. 3483-12-3) to Daphnia magna was determined in a 48 hour semi-static test according to OECD guideline 202. The median effect concentrations (EC50) of 1,4-Dithiotreitol on Daphnia magna were estimated to be 59.8 mg/l and 34.8 mg/l nominal concentration after 24 and 48 h of exposure, respectively. The NOEC values were determined to be 25 mg/l each nominal concentration after 24 and 48 h of exposure, respectively. The results of the test can be considered reliable without restriction.

Executive summary:

The acute toxicity of 1,4-Dithiothreitol (CAS no. 3483-12-3) to Daphnia magna was investigated according to the OECD guideline 202 under semi-static exposure conditions over a period of 48 hours.

The test item 1,4-Dithiothreitol is solid, 99.3% m/m pure and soluble in test water up to at least 100 mg/l. Consequently, the test solutions were prepared by respective dilutions of a stock solution with nominal test item concentrations of 100, 50.0, 25.0, 12.5 and 6.25 mg/l.

The concentrations of the test item (DTT) as well as its oxidation product (ox. DTT) in the test media were analysed by HPLC with UV detection in samples from the test start as well as in the samples after 24 hours (fresh and old test media) and 48 hours of exposure. The test item was correctly dosed in both 24-hour test intervals, but degradation set in very quickly. After 24 hours of exposure, DTT seems to have completely transformed into ox. DTT in all test concentrations with ox. DTT recoveries based on nominal test item concentrations of 93-103%. The test item itself as well as its degradation products have been shown to exhibit toxic properties in non-GLP preliminary tests. Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item, as it is not possible to distinguish the exact proportions to which each species contributed and also taking into account combined toxicity effects.

After 24 hours of exposure, the following immobilisation rates were observed: 90% at 100 mg/l and 35% at 50 mg/l. No immobilised daphnids were observed at all lower test concentrations or in the control.

After 48 hours of exposure, the following immobilisation rates were observed: 100% at 100 and 50 mg/l. No immobilised daphnids were observed at all lower test concentrations or in the control.

The biological test results of 1,4-Dithiothreitol to Daphnia magna after 24 and 48 hours of exposure are summarised in the following table based on the nominal concentrations:

Parameter	Immobilisation (0-24 h) [mg/l]	Immobilisation (0-48 h) [mg/l]
EC50	59.8	34.8
NOEC	25.0	25.0

In conclusion, the 1,4-Dithiothreitol (CAS no. 3483-12-3) had toxic effects on Daphnia magna. The 48-hour EC50 was calculated to be 34.8 mg/l nominal concentration.

All validity criteria were fulfilled.