Registration Dossier

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 April 2016 - 19 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23.
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Remarks:
3 November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- physical appearance: brown coloured, brittle solid
- storage conditions: at room temperature
Specific details on test material used for the study:
- Solubility in water: 4.21 g/L at 20°C (project 511838). This should be considered a maximum value, as it might have been biased by the presence of undissolved material.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 and t=48
Volume: 1.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis (storage stability was of samples under these conditions was demonstrated in project 511838, see section 8).
In addition, the filter used to prepare the WAF at 100 mg/L was retained for possible analysis of the residue.

- At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Additionally, reserve samples of 1.0 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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: The test item was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity composition of the test item. All test concentrations were prepared separately and started with loading rates ranging from 1.0 to 100 mg/L applying three days of magnetic stirring to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were filtered through a 0.45 μm membrane filter where after the clear and colourless Water Accommodated Fractions (WAFs) were used for testing.
- Controls: test medium without test item or other additives (blank-control)

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture with a known history. At least third generation, obtained by a cyclical 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: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started 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. After 7 days of cultivation half of the medium was renewed twice a week.
- Medium other than test medium: yes, M7
- Feeding: daily, a suspension of fresh water algae.

Study design

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

Test conditions

Hardness:
180 mg CaCO3/L
Test temperature:
20-21 °C
pH:
7.6 - 8.0 throughout the test
Dissolved oxygen:
8.3 - 9.2 throughout the test
Nominal and measured concentrations:
Nominal test concentrations: WAFs prepared at nominal rates of 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L
Measured initial test concentrations: 0.197, 0.825, 1.55, 2.10, 2.72, 3.32 and 3.72 mg/L for WAFs at 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L respectively for Final test 2. Measured concentrations were based on one component of the test item.
Effect concentrations were based on measured initial concentrations in Final test 2 since the test item proved to remain stable in test medium during the test period (94-113% of intial measured concentrations). It should be noted that, as the QC samples analyzed together with the samples of Final test 2 exceeded the criterion for recovery of 70-110%, the analyzed concentrations of the test samples might be overestimated (see recoveries in 'Details on analytical methods').
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

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours, daily

EFFECT PARAMETERS MEASURED: mobility and mortality at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning and at the end of the test, for all concentrations and the control. Temperature of the medium: continuously in a temperature control vessel beginning at the start of the test.

RANGE-FINDING STUDY
- Test concentrations: WAFs prepared at 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, at t=48, all daphnids in the WAFs at 10 and 100 mg/L were immobilized. No significant immobility was observed in the WAF at 1.0 mg/L. The expected EC50 was between WAFs at 1.0 and 10 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (July 2016)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
4.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
- No biological, behavioural or other abnormalities were observed.

- Analysis showed measured concentrations at t=48 h were 94-113% of initial measured concentrations. Because test concentrations proved to remain stable during the test period, results were based on initial measured concentrations. Since the test material had a low solubility, EL50 values were also raported.
- Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 48h-EC50: 0.39 mg/L (95%-cI: 0.33-0.44 mg/L)
- Other: results fell within the historical range.
Reported statistics and error estimates:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1 Number of introduced daphnids and incidence of immobility in Final test 1

Time

Replicate

Test item WAF at x mg/L

 

 

Control

1.0

1.8

3.2

5.6

10

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

1

 

B

0

0

0

0

0

0

 

C

0

0

0

0

1

0

 

D

0

0

0

0

0

0

 

Total Immobilised

0

0

0

0

1

1

 

Effect %

0

0

0

0

5

5

48

A

0

0

0

0

0

1

 

B

0

0

0

0

0

0

 

C

0

0

0

0

01

0

 

D

0

0

0

0

0

0

 

Total Immobilised

0

0

0

0

0

1

 

Effect %

0

0

0

0

0

0

Table 2 Number of introduced daphnids and incidence of immobility in Final test 2

Time

Replicate

Test item WAF at x mg/L (measured concentration)

 

 

Control

1.0 (0.20)

2.2 (0.74)

4.6 (1.7)

10 (2.0)

22 (2.4)

46 (3.2)

100 (3.7)

0

A

5

5

5

5

5

5

5

5

 

B

5

5

5

5

5

5

5

5

 

C

5

5

5

5

5

5

5

5

 

D

5

5

5

5

5

5

5

5

 

Total introduced

20

20

20

20

20

20

20

20

24

A

0

0

0

2

3

2

2

4

 

B

0

0

0

2

4

3

4

3

 

C

0

0

0

2

3

5

4

4

 

D

0

0

0

3

3

3

5

5

 

Total Immobilised

0

0

0

9

13

13

15

16

 

Effect %

0

0

0

45

65

65

75

80

48

A

0

0

0

3

5

5

5

5

 

B

0

0

0

2

5

5

5

5

 

C

0

0

0

4

5

5

5

5

 

D

0

0

0

4

5

5

5

5

 

Total Immobilised

0

0

0

13

20

20

20

20

 

Effect %

0

0

0

65

100

100

100

100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall Remarks' section for details
Conclusions:
The 48h-EC50 was 1.5 mg/L based on measured initial concentrations based on one component of the test item (95% confidence interval between 1.4 and 1.7 mg/L). The 48h-EL50 was 4.2 mg/L (95% confidence interval between 3.7 and 4.9 mg/L).