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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Aug 2018 - 07 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Adopted April 13, 2004.
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
UPLC-MS/MS
Details on sampling:
Sampling for Analysis of Test Concentrations:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below:
Frequency: at t=0 h and t=48 h
Volume: 1.8 mL from the approximate centre of the test vessels
Storage : Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additionally, reserve samples of 1.8 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
The batch of 3-Methyl Sulfolane tested was a clear colourless to pale yellow liquid and completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with the highest concentration of 100 mg/L applying a 15- minute period of magnetic stirring to accelerate dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure. Any residual volumes were discarded.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test system:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test, young daphnids with an age of < 24 hours were selected from parental daphnids older than two weeks.

Breeding:
Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
The temperature continuously measured in a temperature control vessel was maintained at 20°C during the test
pH:
pH t=0h : 7.9
pH t=48h: 8.1
Dissolved oxygen:
O2 t=0h : 8.6
O2 t=48h: 8.8
Nominal and measured concentrations:
Nominal concentrations: 0, 0.10, 1.0, 10 and 100 mg/L.
Measured concentrations:
t(0hr): n.d., 0.108, 1.04, 9.86, 101 mg/l
t(48hr): n.d., 0.107, 1.04, 9.74, 101 mg/L

n.d. Not detected.
Details on test conditions:
Test Concentrations:
- 3-Methyl Sulfolane: 0.10, 1.0, 10 and 100 mg/L.
- Controls: Test medium without test item or other additives.

Test Procedure and Conditions:
- Test duration: 48 hours
- Test type: Static
- Test vessels: 60 mL, all-glass.
- Medium:
The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
- CaCl2.2H2O: 211.5 mg/L
- MgSO4.7H2O: 88.8 mg/L
- NaHCO3: 46.7 mg/L
- KCl: 4.2 mg/L
- Number of daphnids: Control and highest concentration: 20 per test group. Intermediate concentrations: 10 per concentration
- Loading: 5 per vessel containing 50 mL of test solution.
- Light: 16 hours photoperiod daily
- Feeding: No feeding
- Aeration: No aeration of the test solutions was applied.
- Introduction of daphnids: 22 minutes after preparation of the test solutions.

Measurements and Recordings:
- Immobility (including mortality): At 24 hours and at 48 hours.
- pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
- Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Effect parameters measured:
Immobility: animals not able to swim within 15 seconds after gentle agitation of the test vessel, even if they can still move their antennae.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Measured concentration:
Samples taken from the control and all test concentrations were analysed. The concentrations measured in all test concentrations were at the level of the corresponding nominal concentrations throughout the test, i.e. were 97-108% of the nominal concentration at the start and at the end of the test. Based on these results, the nominal exposure concentrations were used to determine the effect parameters.

Immobility:
No immobility was observed in the control and any of the test concentrations throughout the exposure period.

Acceptability of the Test:
1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Results with reference substance (positive control):
The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.53 and 0.72 mg/L.
Reported statistics and error estimates:
No EC50 could be calculated because the test item proved to be non-toxic (EC50 > maximum concentration tested).

Number of Introduced Daphnids and Incidence of Immobility in the Test

 

Time (h)

Replicate

3-Methyl Sulfolane; Nominal conc. (mg/L)

Control

0.10

1.0

10

100

0

A

5

5

5

5

5

B

5

5

5

5

5

C

5

 

 

 

5

D

5

 

 

 

5

Total introduced

20

10

10

10

20

24

A

0

0

0

0

0

B

0

0

0

0

0

C

0

 

 

 

0

D

0

 

 

 

0

Total immobilised

0

0

0

0

0

Effect %

0

0

0

0

0

 

 

 

 

 

48

A

0

0

0

0

0

B

0

0

0

0

0

C

0

 

 

 

0

D

0

 

 

 

0

Total immobilised

0

0

0

0

0

Effect %

0

0

0

0

0

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
No immobility was observed in the control and any of the test concentrations throughout the exposure period. The 48h-EC50 for Daphnia magna exposed to 3-Methyl Sulfolane was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.
Executive summary:

A combined limit/range-finding test was performed with daphnia magna according to the OECD 202 guideline and GLP. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a nominal concentration of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to nominal concentrations of 0.10, 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Samples taken from the control and all test concentrations were analysed. The concentrations measured in all test concentrations were at the level of the corresponding nominal concentrations throughout the test, i.e. were 97-108% of the nominal concentration at the start and at the end of the test. Based on these results, the nominal exposure concentrations were used to determine the effect parameters. No immobility was observed in the control and any of the test concentrations throughout the exposure period. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EC50 for Daphnia magna exposed to 3-Methyl Sulfolane was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

Description of key information

A combined limit/range-finding test was performed with daphnia magna according to the OECD 202 guideline and GLP. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a nominal concentration of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to nominal concentrations of 0.10, 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Samples taken from the control and all test concentrations were analysed. The concentrations measured in all test concentrations were at the level of the corresponding nominal concentrations throughout the test, i.e. were 97-108% of the nominal concentration at the start and at the end of the test. Based on these results, the nominal exposure concentrations were used to determine the effect parameters. No immobility was observed in the control and any of the test concentrations throughout the exposure period. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EC50 for Daphnia magna exposed to 3-Methyl Sulfolane was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

The 48h-EC50 for Daphnia magna exposed to 3-Methyl Sulfolane was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.