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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:
20 April to 28 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 202. All validity criteria were fulfilled
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- test substance storage: protected from light
- Vapour pressure: 1.51 Pa (calculated by sponsor); 5.8 Pa at 20°C (determined by WIL Research Europe); 11 Pa at 25°C (determined by WIL Research Europe)
- Solubility in water: 0.667 g/L at 20°C (determined at WIL Research Europe B.V.)
- Stability in water: Stable at pH 7
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the blank-control according to the schedule below.
Frequency: At the start of the test and after 24 hours from the freshly prepared solutions; At the first renewal (t=24h) and the end of the test from the 24-hour old solutions
Volume: 3.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates were taken from vessel A.
Additionally, reserve samples of 3.0 ml were taken for possible analysis. If not used, these samples were stored in a freezer 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:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Preparation of test solutions started with a stock solution of 100 mg/L applying 15-21 minutes of magnetic stirring. The lower test concentrations were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless.
- Controls: Test medium without test substance or other additives.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- 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 selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Age at study initiation: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them 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.
- Feeding: Daily, a suspension of fresh water algae.
- Feeding during test: No feeding
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
20-21 °C
pH:
7.7 (0 h); 7.7-8.0 (48 h)
Dissolved oxygen:
8.9-9.0 (0 h); 8.8-8.9 (48 h)
Salinity:
No data
Nominal and measured concentrations:
10, 17, 30, 52 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, closed airtight with minimum headspace (i.e. containing 60 mL test medium)
- Type: Closed
- Aeration: No aeration of the test solutions.
- Renewal rate of test solution: Semi-static with renewal of test solutions after 24 hours.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Loading rate: 5 per vessel containing 60 mL of test solution
- Introduction of daphnids: Within 1 hour after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Medium: Adjusted ISO medium; M7, as prescribed by Dr. Elendt-Schneider

OTHER TEST CONDITIONS
- Photoperiod: No light

EFFECT PARAMETERS MEASURED
- Immobility (including mortality) At 24 and 48 hours.
- pH and dissolved oxygen: At the beginning, after 24 hours of exposure (fresh and old solutions) and at the end of the test, for all concentrations and the control.
- Temperature of medium: Continuously in a temperature vessel. In addition, of fresh and old medium upon refreshment and at the end of the test.

RANGE-FINDING STUDY
- The study started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and concentrations of 0.10, 1.0, 10 and 100 mg/L. Test procedure and conditions were similar to those applied in the final test, except for the volume of the test vessels and test medium (50 mL containing 50 mL test solution).
- Results used to determine the conditions for the definitive study: No immobility was observed at and below10 mg/L, while all daphnids became immobile at 100 mg/l within 24 hours. Therefore, the expected EC50 was between 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L)
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95%-confidence interval between 32 and 43 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval between 18 and 23 mg/L
Details on results:
Immobility:
- No immobility was observed at 10 and 17mg/L (24 h); at 10 mg/L (48 h).
- At 24 h, 29, 85 and 100% immobility were observed at 30, 52 and 100 mg/L, respectively.
- At 48 h, 20, 81 (95)*, 100 and 100% immobility were observed at 17, 30, 52 and 100 mg/L, respectively.

* At an analytically confirmed nominal concentration of 30 mg/L, daphnids were trapped at the surface at the end of the test in two of the four vessels. The mobility of these daphnids was not recorded. Therefore, the EC50 was calculated twice: once assuming all daphnids trapped at the surface were mobile and once assuming all daphnids trapped at the surface were immobile. The resulting EC50 values are comparable. In a worst-case approach, the lower EC50 should be considered the endpoint of this study.
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 48 h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at WIL Research Europe.
The 24h-EC50 was 0.72 mg/L with a 95% confidence interval between 0.64 and 0.82 mg/L.
The 48h-EC50 was estimated to correspond with 0.36 mg/L with 10% effect at 0.32 mg/L and 100% effect at 0.56 mg/L.
Reported statistics and error estimates:
Calculation of EC50:
The 24 and 48h-EC50-value was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method.
ToxRat Professional v 2.10.05 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 6.1.3/1: Number of introduced daphnids and incidence of immobility in the final test

 

Time (h)

Replicate

Nominal concentration (mg/L)*

Control

10

17

30

52

100

0

A

5

5

5

5

5

5

B

5

5

5

6

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

21

20

20

24

A

0

0

0

1

4

5

B

0

0

0

2

4

5

C

0

0

0

1

5

5

D

0

0

0

2

4

5

Total immobilised

0

0

0

6

17

20

Effect %

0

0

0

29

85

100

48

A

0

0

0

5

5

5

B

0

0

0

5 (1)

5

5

C

0

0

3

4

5

5

D

0

0

1

3 (2)

5

5

Total immobilised

0

0

4

17 (20)

20

20

Effect %

0

0

20

85 (95)

100

100

 

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. The mobility of these organisms was not recorded. Total immobilised between brackets is the total number of daphnids immobilised assuming daphnids trapped at the surface were immobile (total immobilised not between brackets is the number of daphnids immobilised assuming daphnids trapped at the surface were mobile)

* Analytically confirmed nominal concentration

 

Measured concentrations

Analysis of the samples taken at the start and end of the two 24-hour renewal periods showed measured concentrations representing 87-107% of nominal. Therefore, further results were based on analytically confirmed nominal concentrations.

Experimental conditions:

The test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the protocol (18-22 °C, constant within 2 °C).

Acceptability of the test

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.

The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the 48 h-EC50 was 21 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 18 and 23 mg/L).
Executive summary:

Study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the test substance to Daphnia magna, under semi-static conditions. 

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 10, 17, 30, 52 and 100 mg/L. The total exposure period was 48 hours, with renewal of test solutions after 24 hours. Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the two renewal intervals. Before definitive test, combined limit/range-finding test was conducted and the percent immobilization was 0, 0, 0 and 100% at 0.10, 1.0, 10 and 100 mg/L, respectively. 

 

Analysis of the samples taken at the start and end of the two 24-hour renewal periods showed measured concentrations representing 87-107% of nominal. Therefore, further results were based on analytically confirmed nominal concentrations.

 

No immobility was observed at 10 and 17mg/L (24 h); at 10 mg/L (48 h). At 24 h, 29, 85 and 100% immobility were observed at 30, 52 and 100 mg/L, respectively. At 48 h, 20, 85 (95)*, 100 and 100% immobility were observed at 17, 30, 52 and 100 mg/L, respectively.

 

As daphnids trapped at the surface at 30 mg/l at the end of the test were not assessed for mobility, EC50 values were calculated twice: once assuming the daphnids trapped at the surface were all mobile and once assuming the daphnids trapped at the surface were all immobile. The resulting EC50 values are comparable. In a worst-case approach, the lower EC50 should be considered the most appropriate endpoint value and was used in this assessment. 

*the figure in brackets represents immobility if all the floaters at 30 mg/L were taken as immobile.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Under the test conditions, the 48 h-EC50 was 21 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 18 and 23 mg/L) and using the lowest of the 2 EC50s calculated.

Description of key information

OECD Guideline 202, EU Method C.2, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 21 mg/L (95% CL: 18-23 mg/L) based on analytically confirmed nominal concentrations

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
21 mg/L

Additional information

One key study is available (WIL, 2015) to assess the 48h acute toxicity of the registered substance to Daphnia magna, under semi-static conditions, according to OECD Guideline 202 and EU Method C.2 with GLP statement.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 10, 17, 30, 52 and 100 mg/L. The total exposure period was 48 hours, with renewal of test solutions after 24 hours. Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the two renewal intervals (method GC). Before definitive test, combined limit/range-finding test was conducted and the percent immobilization was 0, 0, 0 and 100% at 0.10, 1.0, 10 and 100 mg/L, respectively.   

Analysis of the samples taken at the start and end of the two 24-hour renewal periods showed measured concentrations representing 87-107% of nominal. Therefore, further results were based on analytically confirmed nominal concentrations.

 No immobility was observed at 10 and 17mg/L (24 h); at 10 mg/L (48 h). At 24 h, 29, 85 and 100% immobility were observed at 30, 52 and 100 mg/L, respectively. At 48 h, 20, 85 (95)*, 100 and 100% immobility were observed at 17, 30, 52 and 100 mg/L, respectively. As daphnids trapped at the surface at 30 mg/l at the end of the test were not assessed for mobility, EC50 values were calculated twice: once assuming the daphnids trapped at the surface were all mobile and once assuming the daphnids trapped at the surface were all immobile. The resulting EC50 values are comparable. In a worst-case approach, the lower EC50 should be considered the endpoint of this study. 

*the figure in brackets represents immobility if all the floaters at 30 mg/L were taken as immobile.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 Under the test conditions, the 48 h-EC50 was 21 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 18 and 23 mg/L).