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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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Definitive test: nominal concentrations: 0.3, 0.6, 1.2, 2.4 and 4.8 mg/L and TWA concentrations: 0, 0.2, 0.43, 0.9, 2.0, and 3.8 mg/L respectively; controls: Test medium without test substance or other additives. Spacing factor determined from range-finding test and physico-chemical properties of the substance.
- Sampling method: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule:
1. At the start of the test and after 24 hours from the freshly prepared solutions. and
2. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions, volume: 15 ml from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were used on day of sampling; reserve samples were stored in a freezer until analysis (≤ -15°C). Reserve samples of 15 ml were taken for possible analysis if needed.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): < 24 hours.
- Method of breeding: Parthenogenesis
- Feeding during test: No.
- Food type: Not applicable
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
renewal of the medium at 24 h
Hardness:
The ISO medium hardness: 180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3
Test temperature:
The temperature continuously measured in a temperature control vessel varied between 19.8 and 20.3°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
pH:
Test conditions remained within the limits prescribed by the guideline; pH: 7.8-8.6, not varying by more than 1.5 unit at the end of the test.
Dissolved oxygen:
Test conditions remained within the limits prescribed by the guideline; oxygen: >= 3 mg/l at the end of the test.
Nominal and measured concentrations:
Range-finding test: 0.05, 0.5 and 5.0 mg/l ; plus Control containing no test substance or other additives.
Definitive test: nominal concentrations: 0.3, 0.6, 1.2, 2.4 and 4.8 mg/L and TWA concentrations: 0, 0.2, 0.43, 0.9, 2.0, and 3.8 mg/L respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 ml
- Type (delete if not applicable): closed without headspace; semi-static with renewal at 24 hours.
- Material, size, headspace, fill volume: Glass- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel containing 50 ml of test solution
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis (RO-water, GEON Waterbehandeling, The Netherlands); used to prepare ISO Medium M7
- Culture medium different from test medium: No, ISO Medium M7, renewed by 50% twice per week.
OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 16 hours light; 8 hours dark photoperiod daily
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 1.8 to 2.4 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 0.9 to 2.0 mg/L
Details on results:
- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations:- Mortality of control: No mortalities.
- Other adverse effects control: None.
- Abnormal responses: None.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 0.90 mg/l. 48h-EC50 was 0.43 mg/l with a 95% confidence interval between 0.39 and 0.48 mg/l
- Other: 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 test organism was in agreement with the historical data.

Table 1. pH and oxygen (mg/l) concentrations during the final test

TWA

concentration

(mg/l) #

Start (t=0 h)

t=24h (old)

t=24h (fresh)

End (t=48 h)

pH

O2

pH

O2

pH

O2

pH

O2

Control

7.8

9.4

8.2

9.0

8.1

8.8

7.9

8.8

0.20

7.8

9.3

8.2

8.5

8.1

8.9

7.9

8.7

0.43

7.8

9.3

8.2

8.6

8.1

8.9

7.9

8.8

0.90

7.9

9.3

8.1

8.5

8.1

8.9

7.9

8.8

2.0

7.8

9.2

8.1

8.6

8.0

8.8

7.9

8.8

3.8

7.8

9.2

8.1

8.6

8.0

8.8

7.9

8.7

# test item

 

Table 2. Number of introduced daphnids and incidence of immobility in the final test

Time (h)

Replicate

TWA concentration test item (mg/l) and number immobile

Control

0.20

0.43

0.90

2.0

3.8

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0

0

0

2

5

B

0

0

0

0

3

5

C

0

0

0

0

2

5

D

0

0

0

0

3

5

Total immobilised

0

0

0

0

10

20

Effect %

0

0

0

0

50

100

 

 

 

 

 

 

48

A

0

0

1

1

5 (4)

5

B

0

0

0

0

5

5

C

0

0

0

0

5

5

D

0

0

0

0

5

5

Total immobilised

0

0

1

1

20

20

Effect %

0

0

5

5

100

100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions.

These organisms were reimmersed into the respective solution before recording of mobility.

Validity criteria fulfilled:
yes
Conclusions:
The test substance 48h-EC50 was 1.3 mg/l (95% CL: 0.90 – 2.0 mg/l) based on TWA concentrations.
Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines. Preparation of test solutions started with the highest concentration, i.e. 5.0 mg/L for the range-finding and 4.8 mg/L for the final test. A magnetic stirring period of 15-16 minutes was applied to ensure complete homogenisation. The resulting solution was used as highest test concentration and used to prepare the lower test concentrations by subsequent dilution in test medium. A final EC50 test was performed with a range based on the result of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to nominal test substance concentrations of 0.30, 0.6, 1.2, 2.4 and 4.8 mg/L. The total exposure period was 48 hours with renewal of test solutions after 24 hours. Samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions at the start of the test and at the beginning of the second renewal period (after 24 hours of exposure) and from 24-hour spent solutions at the end of the first renewal period and at the end of the test (after 24 and 48 hours of exposure, respectively). Analysis of the samples taken during the final test showed that measured concentrations in freshly prepared solutions were between 73 and 92% relative to nominal. In the 24-hour spent solutions it was observed that measured concentrations had decreased by 7 to 31% below to the initial measured concentrations. Based on these results, the average exposure concentrations were calculated to correspond with 0.20, 0.43, 0.90, 2.0 and 3.8 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The 48h-EC50 based on average exposure concentrations was 1.3 mg/L with 5% immobility at 0.90 mg/L and 100% immobility at 2.0 mg/L.

Description of key information

EC50-48h (invertebrates) = 1.3 mg/L (95% CL: 0.90 – 2.0 mg/L) based on TWA concentrations, 48-hour, freshwater, OECD 202, 2014

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.3 mg/L

Additional information

Key study: OECD TG 202, 2014 : The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines. Preparation of test solutions started with the highest concentration, i.e. 5.0 mg/L for the range-finding and 4.8 mg/L for the final test. A magnetic stirring period of 15-16 minutes was applied to ensure complete homogenisation. The resulting solution was used as highest test concentration and used to prepare the lower test concentrations by subsequent dilution in test medium. A final EC50 test was performed with a range based on the result of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to nominal test substance concentrations of 0.30, 0.6, 1.2, 2.4 and 4.8 mg/L. The total exposure period was 48 hours with renewal of test solutions after 24 hours. Samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions at the start of the test and at the beginning of the second renewal period (after 24 hours of exposure) and from 24-hour spent solutions at the end of the first renewal period and at the end of the test (after 24 and 48 hours of exposure, respectively). Analysis of the samples taken during the final test showed that measured concentrations in freshly prepared solutions were between 73 and 92% relative to nominal. In the 24-hour spent solutions it was observed that measured concentrations had decreased by 7 to 31% below to the initial measured concentrations. Based on these results, the average exposure concentrations were calculated to correspond with 0.20, 0.43, 0.90, 2.0 and 3.8 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The 48h-EC50 based on average exposure concentrations was 1.3 mg/L with 5% immobility at 0.90 mg/L and 100% immobility at 2.0 mg/L.