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Ecotoxicological information

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:
29 Jan 2016 - 09 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: off-white powder
- Storage condition of test material: At room temperature
Specific details on test material used for the study:
Solubility in water: 5.4 mg/L (20°C)
Stability in water: yes

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Concentrations: 0, 4.6, 10, 22, 46 and 100 mg/L
Sampling method: Singular 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: 2.0 mL from the approximate centre of the test vessels.
Storage: samples were stored in a freezer until analysis
The filter containing the undissolved residue was kept for possible analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: preparation of test solutions started with the highest test concentration of 100 mg/L applying 2 days of magnetic stirring to ensure maximum dissolution of the test item in the test medium. The resulting dispersion was filtered through a 0.45 μm membrane filter (Whatman; RC55) to remove the undissolved test item, which resulted in a clear and colourless saturated solution (SS). The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. Note that weighing and formulation of test solutions were performed protected from light as much as possible and that the control group received a similar treatment (i.e. filtration).
- Controls: test medium without test item or other additives
- Evidence of undissolved material: no
Controls: test medium without test item or other additives.
Evidence of undissolved material: no, all final test solutions were clear and colourless.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820
- Source: in-house laboratory culture with a known history, least third generation, obtained by acyclical parthenogenesis under specified breeding conditions. 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.
- Age at study initiation: young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Cultivation method: Each batch was started with newborn daphnids, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. Cultures were renewed after 7 days of cultivation half of the medium twice a week.
- Feeding during test: no

Breeding conditions:
- Temperature of the medium: 18-22°C
- Feeding: daily, a suspension of fresh water algae.
- Breeding medium same as test medium: no, M7

ACCLIMATION: no

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
180 mg/L CaCO3
Test temperature:
Between 19.4 and 20.4°C throughout the 2nd final test
pH:
Between 8.0 and 8.2 throughout the 2nd final test
Dissolved oxygen:
Between 8.7 and 9.1 mg O2/L throughout the 2nd final test
Nominal and measured concentrations:
Based on the results of the combined limit/range-finding test the expected EC50 ranged between concentrations obtained in 10 and 100% of the SS.
Nominal concentrations: 4.6, 10, 22, 46 and 100 mg/L
Analysis of the samples showed that concentrations were 77 to 97% of initial. Consequently, average measured concentrations were calculated.
Mean measured concentrations (2nd final test): 0.10, 0.25, 0.57, 1.2 and 2.9 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill-volume: 80 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity: none; the study was performed in the dark

EFFECT PARAMETERS MEASURED: Mobility (including mortality) after 48 hours. In addition, dissolved oxygen and pH of test medium were measured at the beginning and at the end of the test. Temperature of the medium was measured and controlled continuously from the start of the test.

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, based on the result, the expected EC50 ranged between concentrations obtained in 10 and 100% of the SS.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (December 2015)

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 2.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
The first final test was repeated due to the fact that a dose-response effect was absent and consequently no reliable EC50 could be calculated. Above shown results are from the second final test.

- No biological, behavioural or other abnormalities observed
- Effect concentrations exceeding solubility of substance in test medium: no

All test conditions were maintained within the limits prescribed by the study plan.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 24h-EC50: 0.69, 95%CI (0.59, 1.2)
- 48h-EC50: 0.38, 95%CI (0.33, 0.43)

Any other information on results incl. tables

Table 1            Measured concentrations versus nominal concentrations

Broomdex

(%SS)

Measured concentration (mg/L)

Average measured (mg/L)

t=0h

t=48h

4.6

0.114

0.095

0.10

10

0.287

0.222

0.25

22

0.621

0.532

0.57

 46

1.27

1.23

1.2

100

2.94

2.81

 2.9

 

Table 2            Number of introduced daphnids and incidence of immobilityin the 2ndfinal test

Time (h)

Replicate

Measured concentration Broomdex (mg/L)

Control

0.10

0.25

0.57

1.2

2.9

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

0

0

B

0

0

0

0

0

0

C

0

0

0

0

0

0

D

0

0

0

0

0

1

Total immobilised

0

0

0

0

0

1

Effect %

0

0

0

0

0

5

 

 

 

 

 

 

48

A

0

0

1

3

4

4

B

0

0

0

2

4

5

C

0

0

1

3

3

4

D

0

0

0

4

4

5

Total immobilised

0

0

2

12

15

18

Effect %

0

0

10

60

75

90

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
1) In the control, no daphnids became immobilised or showed other signs of disease or stress. 2) The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Conclusions:
The 24h-EC50 could not be determined since it was > 2.9 mg/L. The 48h-EC50 was 0.65 mg/L based on average measured concentrations (95% confidence interval between 0.48 and 0.88 mg/L).