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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:
03 September 2018 - 07 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Appearance: Brown viscous liquid
- Storage condition of test material: At room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control
- Sampling method: 1.8 mL from the approximate centre of the test vessels, at t=0 h and t=48 h.
- Sample storage conditions before analysis: 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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to test medium. The test solution with highest concentration of 100 mg/L (corrected for purity) (in the combined limit/range-finding test and 2nd full test) and 1.0 mg/L (in the 1st full test) was prepared by applying one day of magnetic stirring to accelerate dissolution of the test item in 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.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Age at study initiation: <24 hours
- Source: In-house laboratory culture with a known history
- Method of breeding: cyclical parthenogenesis under specified breeding conditions
- Age of parental stock: >2 weeks
- Feeding during test: no
- 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.

CULTIVATION
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 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.

Study design

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

Test conditions

Hardness:
180 mg/L (expressed as CaCO3)
Test temperature:
19°C
pH:
At t = 0 h: 7.2-8.0
At t = 48 h: 7.6-8.3
Dissolved oxygen:
At t = 0 h: 8.7-9.6 mg/L
At t = 48 h: 9.1-9.2 mg/L
Nominal and measured concentrations:
Only relevant concentrations from the 2nd full (final) test are reported here.

Nominal concentrations: 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32, 100 mg/L
Measured concentrations: 0.0324, 0.112, 0.366, 1.15, 3.68, 11.2, 35.9, 110 mg/L at start of the test (101-115% relative to nominal) and concentrations remained remained stable throughout the test. Based on these results, effect parameters were expressed in terms of analytically confirmed nominal concentrations.
See Table 1 in 'Any other information on results' for more details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass, with 50 mL test solution
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnids: 39 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium (prepared with water purified by reverse osmosis)
- Culture medium different from test medium: yes, M7
- Intervals of water quality measurement: Dissolved oxygen and pH: at beginning and end of the test, for all concentrations and the control. Temperature: continuously in a temperature control vessel, beginning at the start of the test.

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

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): At 24 hours and at 48 hours.

COMBINED LIMIT/RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, the expected EC50 was around nominally 0.1 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Nov 2018)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 0.080 - 0.17 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
0.21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 0.14 - 0.33 mg/L
Details on results:
- Behavioural abnormalities: none reported
- Mortality of control: none
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
- No immobility was observed in the control group throughout the test. At the end of the test, a dose-related increase of immobility was observed at all concentrations tested, up to total immobility at analytically confirmed nominal concentrations of 1.0 mg/L and higher. These results were in agreement with the results of the combined limit/range-finding test.
Results with reference substance (positive control):
The 48h-EC50 was 0.40 mg/L with a 95% confidence interval between 0.33 and 0.48 mg/L.
The actual responses in this reference test with K2Cr2O7 were generally within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.28 and 0.90 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.
Reported statistics and error estimates:
The EC50 values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding analytically confirmed nominal test item concentrations using the maximum likelihood estimation method.

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Table 1: 2nd Full (Final) Test: Test Samples

Time of sampling
[hours]

Concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

0

n.d.

n.a.

 

 

0.032

0.0324

101

 

 

0.10

0.112

112

 

 

0.32

0.366

114

 

 

1.0

1.15

115

 

 

3.2

3.68

115

 

 

10

11.2

112

 

 

32

35.9

112

 

 

100

110

110

 

48

0

n.d.

n.a.

n.a.

 

0.032

0.0334

104

103

 

0.10

0.108

108

96

 

0.32

0.359

112

98

 

1.0

1.09

109

95

 

3.2

3.50

109

95

 

10

11.1

111

99

 

32

35.2

110

98

 

100

107

107

97

n.d. Not detected.

n.a. Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the 2nd Full (Final) Test

Time (h)

Replicate

 

 

Nominal conc. (mg/L)

Control

0.032

0.10

0.32

1.0

3.2

10

32

100

0

A

5

5

5

5

5

5

5

5

5

B

5

5

5

5

5

5

5

5

5

C

5

5

5

5

5

5

5

5

5

D

5

5

5

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

20

20

20

 

 

 

 

24

A

0

0

5

2

5

5

5

5

5

B

0

1

1

4

5

5

5

5

5

C

0

0

1

3

4

5

5

5

5

D

0

1

2

1

4

5

5

5

5

Total immobilised

0

2

9

10

18

20

20

20

20

Effect %

0

10

45

50

90

100

100

100

100

 

 

 

 

 

 

 

 

 

48

A

0

0

5

4

5

5

5

5

5

B

0

1

2

5

5

5

5

5

5

C

0

0

1

4

5

5

5

5

5

D

0

2

2

4

5

5

5

5

5

Total immobilised

0

3

10

17

20

20

20

20

20

Effect %

0

15

50

85

100

100

100

100

100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see section 'Overall remarks'
Conclusions:
48h-EC50 for Daphnia magna exposed to the substance was 0.12 mg/L based on analytically confirmed, purity corrected nominal concentrations (95% confidence interval between 0.080 and 0.17 mg/L).