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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:
08 Feb 2019 - 16 Apr 2019
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
Report Date:
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:
solid: particulate/powder
Details on test material:
- Appearance: Off-white powder
- Storage condition of test material: In refrigerator protected from light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control
- Sampling method: 1.8 mL test solution were retrieved (by pipette) 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 of substance to test medium.
The test item was completey soluble in the test medium at all concentrations tested. Therefore, preparation of test solutions started with the highest concentration of 100 mg/L (corrected for purity) applying a 15 minute period of ultrasonic waves followed by one hour of magnetic stirring to accelerate the dissolution of the test item in test 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.

The above described procedure was applied in all tests performed (thus the combined limit/range-finding test as well as the final tests).

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
- Method of breeding: cyclical parthenogenesis under specified breeding conditions
- Age of parental stock: > 2 weeks
- Age at test initiation: < 24 hours
- 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: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed 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 - 20°C
pH:
At start (t=0 h): 7.7-8.0
At end (t=48 h): 8.1-8.2
Dissolved oxygen:
At start (t=0 h): 9.2-9.5 mg/L
At end (t=48 h): 8.8-9.0 mg/L
Nominal and measured concentrations:
Nominal: control and 4.6, 10, 22, 46 and 100 mg/L (corrected for purity)
Measured: The measured concentrations were at the level of nominal (i.e. 97 – 100% relative to nominal concentrations) throughout the test. Effect parameters were therefore expressed in terms of analytically confirmed nominal concentrations.
See Table 1 in 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- Feeding: no feeding
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnids: Within 45 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium prepared with tap water purified by Reverse Osmosis and the following salts: CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L
- Culture medium different from test medium: yes (M7)
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: 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 h and at 48 h.

RANGE-FINDING STUDY
- Combined Limit/Range-Finding Test concentrations: control and nominal concentrations of 0.1, 1.0, 10 and 100 mg/L (corrected for purity)
- Results used to determine the conditions for the definitive study: yes, expected EC50 between 10 and 100 mg/L (corrected for purity).
Reference substance (positive control):
yes
Remarks:
potassium dichromate (performed Jan 2019)

Results and discussion

Effect concentrations
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: nominal concentration corrected for purity
Details on results:
- Behavioural abnormalities: none observed in the control group.
- Mortality of control: none
- Analytical measurements: Measured concentrations were at the level of nominal throughout the test. Considering that the measured concentrations were comparable to the concentrations in the preceding tests, it was concluded that factors other than exposure to the test item caused the increased degree of immobiliziation in the combined limit/range-finding test. It was consequently decided to base the effect parameters on the results of the full tests.
- No immobility was observed in the control and the four lowest test concentrations throughout the final test. At the highest test concentration, a single daphnid was observed to be immobile at 24 and 48 hours of exposure. This was considered to be biologically not relevant, as up to 10% immobility are allowed for the control group. See Table 2 in 'Any other information on results' for full details on immobility.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Nominal concentrations: 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
- The 48h-EC50 was 0.47 mg/L with a 95% confidence interval between 0.41 and 0.55 mg/L.
- The 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 0.25 and 0.75 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with with ISO International Standard 6341, October 2012 and the historical data collected at the Test Facility (during the last 10 years).
Reported statistics and error estimates:
not applicable
The 24 and 48h-EC50-values could not be determined because the observed effects were below 50%.

Any other information on results incl. tables

Table 1: 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.

 

 

4.6

4.50

98

 

 

10

9.74

97

 

 

22

21.8

99

 

 

46

45.6

99

 

 

100

100

100

 

48

0

n.d.

n.a.

n.a.

 

4.6

4.46

97

99

 

10

9.68

97

99

 

22

21.3

97

97

 

46

45.6

99

100

 

100

100

100

100

n.d. Not detected.

n.a.  Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the First Full Test

Time (h)

Replicate

Nominal conc. (mg/L)

Control

4.6

10

22

46

100

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

3

B

0

0

0

0

1 (1)

3

C

0

0

0

0

0

2

D

0

0

0

0

0

1 (1)

Total immobilised

0

0

0

0

1

9

Effect %

0

0

0

0

51

45

 

 

 

 

 

 

48

A

0

0

0

0

0

4

B

0

0

0

0

1 (1)

1

C

0

0

0

0

0

0 (3)

D

0

0

0

0

0

1 (3)

Total immobilised

0

0

0

0

1

6

Effect %

0

0

0

0

51

30

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

1It should be noted that10% immobility is allowed in the control, and therefore effects of10% were considered biologically not relevant.

Table 3: Number of Introduced Daphnids and Incidence of Immobility in the Final Test (Second Full Test)

Time (h)

Replicate

Nominal conc. (mg/L)

Control

4.6

10

22

46

100

0

A

5

5

5

5

0

0

B

5

5

5

5

0

0

C

5

5

5

5

0

0

D

5

5

5

5

0

0

Total introduced

20

20

20

20

0

0

0

0

24

A

0

0

0

0 (1)

0

0

B

0

0

0

0

0

0

C

0

0

0

0 (2)

0

0

D

0

0

0

0

0

1

Total immobilised

0

0

0

0

0

1

Effect %

0

0

0

0

0

51

 

 

 

 

 

 

48

A

0

0

0

0

0

0 (1)

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

51

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

1It should be noted that 10% immobility is allowed in the control, and therefore effects of 10% were considered biologically not relevant.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 48h-EC50 of the substance for Daphnia magna was beyond the range of concentrations tested, i.e. exceeded an analytically confirmed nominal exposure concentration of 100 mg/L (corrected for purity).