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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:
24 Aug 2019 - 27 Sep 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all groups.
Frequency: at t=0 h and t=48 h
Volume: 2.0 mL from the approximate centre of the test vessels.
Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- method: Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. To this end, the according test item amounts were weighed onto watch glasses and added to flasks containing 1 L medium. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning and used as test concentrations.
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna, at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Strain/clone: Straus, 1820.
- Method of breeding: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the culture: 4 weeks.
- Renewal of the culture: After 7 days of cultivation, half of the medium twice a week.
- Feeding: Daily, a suspension of fresh water algae.
- Validity of the 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.
- Age at study initiation: Less than 24 hours old, from parental daphnids of more than two weeks old.
- Feeding during test: No


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/l expressed as CaCO3
Test temperature:
between 20 and 21°C
pH:
7.6 - 8.3
Dissolved oxygen:
8.4 - 9.0 mg/L
Salinity:
not applicable
Conductivity:
not aplicable
Nominal and measured concentrations:
Nominal concentrations:
-WSFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L.

Measured concentrations:
- Samples taken from all test concentrations and the control were analysed by following the mono-ester (m/z 209.1) and di-ester (m/z 321.1).
- The measured concentrations of the mono-ester were at the level of nominal throughout the exposure period, i.e. were at 97-105% of nominal at the start and end of exposure.
- The measured concentrations of the di-ester were 0.94, 2.4, 3.8, 5.4 and 7.7 mg/L at the start of the test, respectively to the loading rates of 10, 18, 32, 56 and 100 mg/L. The concentrations remained stable, i.e. were at 95-103% relative to initial at the end of the test.
- Based on these results, effect parameters were expressed as initially measured concentrations of the di-ester, since the lower measured concentrations represent the worst case scenario.

Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass.
- Volume of solution: 50 mL
- Aeration: No aeration of the test solutions was applied.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following salts (analytical grade) were added to tap water
purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L
The hardness of test medium expressed as CaCO3: 180 mg/L.
- Culture medium different from test medium: yes, for culturing M7 medium is used.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A daily photoperiod of 16 hours.

EFFECT PARAMETERS MEASURED:
24h-EC50, 48h- EC50

RANGE-FINDING STUDY
- Test concentrations: WSFs prepared at loading rates of 1.0, 10, and 100 mg/L.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
Intitial measured concentration of di-ester (m/z 321.1).
Basis for effect:
mobility
Details on results:
- Immobility of control: No immobility was observed in the control throughout the exposure period.
- Other adverse effect control: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Results with reference substance valid: The study generally met the acceptability criteria prescribed
by the study plan and was considered valid.
- Dose-response test: concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L.
- ECx:
The 24h-EC50 was 0.80 mg/L with a 95% confidence interval between 0.68 and 0.89 mg/L.
The 48h-EC50 was 0.55 mg/L with a 95% confidence interval between 0.50 and 0.62 mg/L.
Reported statistics and error estimates:
The EC50 could not be determined using a regression method. Instead, the 24 and 48h EC50 values were calculated by applying the Spearman-Karber procedure (non-linear; without trimming) on the percentages of affected daphnids and the logarithms of the corresponding initially measured test item concentrations.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 2          
Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)

Replicate

The substance; Loading rate (mg/L)

Control

10

18

32

56

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

 5#

5

2

B

0

0

0

5

5

4

C

0

0

0

5

5

2

D

0

0

0

5

5

0

Total immobilised

0

0

0

20

20

8

Effect %

0

0

0

100

100

40

 

 

 

 

 

 

48

A

0

0

1

5

5

4

B

0

0

0

5

5

5

C

0

0

0

5

5

5

D

0

0

0

5

5

5

Total immobilised

0

0

1

20

20

19

Effect %

0

0

5

100

100

95

#Microscopic observation revealed no test item attached to the daphnids.

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the 48h-EC50 for Daphnia magna exposed to the substance was 3.0 mg/L based on measured concentrations of di-ester (95% confidence interval between 2.8 and 3.2 mg/L).
Executive summary:

The short-term toxicity of the substance to aquatic invertebrates was determined in a daphnia acute study according to OECD guiudeline No. 202. In addition, procedures were based on the test method described in the OECD series on testing and assessment number 23, 2019.

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WSFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The total exposure period was 48 hours. Samples taken from all test concentrations and the control were analysed by following the mono-ester (m/z 209.1) and di-ester (m/z 321.1). The measured concentrations of the mono‑ester were at the level of nominal throughout the exposure period, i.e. were at 97 – 105% of nominal at the start and end of exposure. The measured concentrations of the di-ester were 0.94, 2.4, 3.8, 5.4 and 7.7 mg/L at the start of the test, respectively to the nominal loading rates of 10, 18, 32, 56 and 100 mg/L. The concentrations remained stable, i.e. were at 95 – 103% relative to initial at the end of the test. Based on these results, effect parameters were expressed as initially measured concentrations of the di-ester, since the lower measured concentrations represent the worst case scenario. At the end of the test, a dose-related increase of immobility was observed at WSFs individually prepared at a loading rate of 18 mg/L and higher, reaching 95% immobility at the three highest test concentrations. In conclusion, the 48h, EC50 for Daphnia magna exposed to the substance was 3.0 mg/L based om measured concentrations (95% confidence interval between 2.8 an 3.2 mg/L).

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

Description of key information

Daphnia Magna, 48 -hours, test medium not adjusted, EC50 = 3.0 mg/L (95% confidence interval between 2.8 and 3.2 µg/L) (values based on measured initial di-ester concentrations)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
3 mg/L

Additional information

The short-term toxicity of the substance to aquatic invertebrates was determined in a daphnia acute study according to OECD guiudeline No. 202. In addition, procedures were based on the test method described in the OECD series on testing and assessment number 23, 2019.

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WSFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all test concentrations and the control were analysed by following the mono-ester (m/z 209.1) and di-ester (m/z 321.1). The measured concentrations of the mono‑ester were at the level of nominal throughout the exposure period, i.e. were at 97 – 105% of nominal at the start and end of exposure. The measured concentrations of the di-ester were 0.94, 2.4, 3.8, 5.4 and 7.7 mg/L at the start of the test, respectively to the nominal loading rates of 10, 18, 32, 56 and 100 mg/L. The concentrations remained stable, i.e. were at 95 – 103% relative to initial at the end of the test. Based on these results, effect parameters were expressed as initially measured concentrations of the di-ester, since the lower measured concentrations represent the worst case scenario.

At the end of the test, a dose-related increase of immobility was observed at WSFs individually prepared at a loading rate of 18 mg/L and higher, reaching95% immobility at the three highest test concentrations.

In conclusion, the 48h, EC50for Daphnia magna exposed to the substance was 3.0 mg/L based om measured concentrations (95% confidence interval between 2.8 an 3.2 mg/L).

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

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