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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:
From October 07, 2015 to October 23, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Analytical samples taken at 0 h (initial value) from fresh test solutions and after 48 h from aged test solutions from control, 4.27, 9.39, 20.7, 45.5, 100 and 220 mg/L were analysed to determine the total organic carbon content (TOC analysis).
Vehicle:
no
Details on test solutions:
- Test Medium:
Elendt M4 medium was used as test medium. At test initiation the pH-value of the control (untreated test medium) was 7.94, the dissolved oxygen concentration was 8.8 mg/L and the total hardness was 13°dH (232 mg/L as CaCO3).

- Test substance concentrations:
The necessary amount of the test substance for preparing the stock solution was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. Foaming was observed. The lower test solutions were prepared by dilution of the stock solution with test medium. Defined volumes of the prepared solution were transferred to each test vessels. The test solution volume was 50 mL per test vessel. The concentrations in the test were spaced by a factor of 2.2. The test concentrations were 0, 4.27, 9.39, 20.7, 45.5, 100 and 220 mg/L .
Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna STRAUS, Clone V
- Source: Federal Environmental Agency in Berlin/Germany.
- Breeding: Daphnia magna was bred as single culture (one Daphnid per 100 mL) in Elendt M4 medium. The pH-value of the aerated water was within a range of 6.0 – 9.0. The dissolved oxygen was above 60% saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH. The animals were fed with single cell green algae (Desmodesmus subspicatus, former Scenedesmus subspicatus) at least three times a week. The Daphnids were reared at a temperature of 20±2°C in a climatic chamber with 16 h of illumination and 8 h of darkness. The medium was changed three times per week. A pipette was used to separate the young daphnids from the adults. Freshly hatched daphnids max. 24 h old were used for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
swimming ability and the immobilisation rate
Hardness:
13°dH (232 mg/L CaCO3)
Test temperature:
19.0 – 21.6°C
pH:
7.79 – 8.02
Dissolved oxygen:
8.2 – 8.9 mg/L
Nominal and measured concentrations:
Nominal: 0, 4.27, 9.39, 20.7, 45.5, 100 and 220 mg/L.
TOC content nominal (56 %) (based on the TOC of the test substance of 56 %): 2.39, 5.56, 11.6, 25.5, 56.0 and 123 mg/L



Details on test conditions:
Test procedure: static
Duration: 48 h
Exposure to light: 16 h photoperiod daily
Feeding: none
Test vessels: four 100 mL glass beakers per concentration, each filled with 50 mL and one additional replicate for physico-chemical parameters without test organisms
Loading: 10 mL of test solution for each organism
Aeration: none
Number of animals: 20 per concentration in 4 replicates of 5 organisms
Reference substance (positive control):
yes
Remarks:
potassium dichromate (99.95% w/w pure), batch no. 112403J
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 220 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: -
Details on results:
Biological Results:
After 24 h of exposure no immobilisation above the allowed control immobilisation was observed in the control and in all test substance concentrations (4.27, 9.39, 20.7, 45.5, 100 and 220 mg/L).

After 48 h of exposure no immobilisation was observed in the control and the test substance concentrations 4.27 and 9.39 mg/L. 5% of the daphnids were immobilised at test substance concentration 20.7 mg/L. 30% of the daphnids were found immobile at 45.5 mg/L and 25% at 100 mg/L. At the highest test substance concentration of 220 mg/L 45% of the daphnids were immobile.

Analytical Results:
TOC analysis showed that the test substance was stable in the upper concentration levels throughout the period of the test. The mean initial measured content of TOC was 89% of the nominal concentration. The mean measured content of TOC in aged test solution was 69% of the nominal concentration.

The total hardness (as CaCO3) of the untreated control was determined to be 13°dH (232 mg/L CaCO3); the mean pH-value of the untreated control was determined to be 7.84±0.08, the mean temperature of the control and all test substance concentrations was measured to be 20.0±1.1°C and the mean oxygen concentration was determined to be 8.6±0.2 mg/L.
Results with reference substance (positive control):
The toxicity of the reference substance potassium dichromate was tested at 1.0 and 2.0 mg/L with 20 test organisms per test concentration around the same time period of the study. The results indicate an EC50 (24 h) of the reference substance potassium dichromate between 1.0 and 2.0 mg/L. Since the results are in accordance with the requirements of the OECD Guideline 202 and fall within the historical data generated with the reference substance at the testing facility, the daphnids were suitable for the determination of the toxicological effects of the test substance.
Reported statistics and error estimates:
The 24 and 48 h EC50 are the estimated concentrations where 50% of the daphnids were immobilised after 24 and 48 h, respectively. No statistical determination was performed as less than 50% immobilisation was observed in the highest test substance concentration after 24 and 48 h. The NOEC was established based on the highest concentration at which the immobilisation is not higher than the allowed control immobilisation (≤10 % immobilisation).

Table 1: Results of the test, 24 h values

Concentration

Test substance

Control

4.27

9.39

20.7

45.5

100

220

mg/L

immobilised daphnids after 24 h

Group 1

0

0

0

0

0

0

0

Group 2

0

0

0

0

0

0

0

Group 3

0

0

0

0

0

0

0

Group 4

0

0

0

0

0

0

0

Σ

0

0

0

0

0

0

0

%

0

0

0

0

0

0

0

Table 2: Results of the test, 48 h values

Concentration

Test substance

Control

4.27

9.39

20.7

45.5

100

220

mg/L

immobilised daphnids after 48 h

Group 1

0

0

0

1

1

1

2

Group 2

0

0

0

0

2

0

3

Group 3

0

0

0

0

2

3

2

Group 4

0

0

0

0

1

1

2

Σ

0

0

0

1

6

5

9

%

0

0

0

5

30

25

45

Analytical results

Table 3: TOC measurements of the test substance during the test

Test substance nominal [mg/L]

TOC content

Sampling [h]

TOC [mg/L]

TOC [%]

nominal

(56%) [mg/L]1

Measured in test solution

Measured in test solution (control corrected)

Control

0

0 (fresh)

2.3

-

-

48 (aged)

2.0

-

-

4.27

2.39

0 (fresh)

5.0

2.7

113

48 (aged)

2.8

0.8

33

9.93

5.56

0 (fresh)

6.5

4.2

76

48 (aged)

5.0

3.0

54

20.7

11.6

0 (fresh)

11.7

9.4

81

48 (aged)

10.1

8.1

70

45.5

25.5

0 (fresh)

23.7

21.4

84

48 (aged)

21.8

19.8

78

100

56.0

0 (fresh)

52.5

50.2

90

48 (aged)

52.6

50.6

90

220

123

0 (fresh)

113

111

90

48 (aged)

114

112

91

LOQ = 0.3 mg/L,

1= based on the TOC of the test substance of 56%,

- = not calculable

Validity criteria fulfilled:
yes
Remarks:
According to the OECD 202 guideline this study can be regarded as valid, as in the control not more than 10 % of animals were immobilised and the dissolved oxygen concentration was ≥ 3 mg/L at the end of the test in control and test vessels.
Conclusions:
The EC50 (48 h) for immobilisation was determined to be >220 mg/L (nominal). The corresponding NOEC (48 h) was 20.7 mg/L (nominal).
Executive summary:

A study was conducted to determine the acute toxicity of the test substance to Daphnia magna according to OECD Guideline 202, in compliance with GLP. Four replicates of 5 organisms each were exposed to the test substance at nominal concentrations of 0, 4.27, 9.39, 20.7, 45.5, 100 and 220 mg/L under static conditions for 48 h. Immobility was recorded at 24 and 48 h. Samples were taken at 0 h (initial value) from fresh test solutions and after 48 h from aged test solutions to determine total organic carbon content (TOC analysis). The mean initial measured content of TOC was 89% of the nominal concentration. The mean measured content of TOC in aged test solution was 69% of the nominal concentration. The immobilty observed in the control was found to be within the acceptability criteria. After 48 h of exposure, no immobilisation was observed in the control and the test substance concentrations of 4.27 and 9.39 mg/L. At 20.7, 45.5, 100 and 220 mg/L, immobilisation was 5, 30, 25 and 45% of the Daphnids, respectively. Hence, under the study conditions, the EC50 (48 h) was determined to be >220 mg/L (nominal) (Zawadsky C, 2015).

Description of key information

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance to Daphnia magna according to OECD Guideline 202, in compliance with GLP. Four replicates of 5 organisms each were exposed to the test substance at nominal concentrations of 0, 4.27, 9.39, 20.7, 45.5, 100 and 220 mg/L under static conditions for 48 h. Immobility was recorded at 24 and 48 h. Samples were taken at 0 h (initial value) from fresh test solutions and after 48 h from aged test solutions to determine total organic carbon content (TOC analysis). The mean initial measured content of TOC was 89% of the nominal concentration. The mean measured content of TOC in aged test solution was 69% of the nominal concentration. The immobilty observed in the control was found to be within the acceptability criteria. After 48 h of exposure, no immobilisation was observed in the control and the test substance concentrations of 4.27 and 9.39 mg/L. At 20.7, 45.5, 100 and 220 mg/L, immobilisation was 5, 30, 25 and 45% of the Daphnids, respectively. Hence, under the study conditions, the EC50 (48 h) was determined to be >220 mg/L (nominal) (Zawadsky C, 2015).