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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:
04 - 06 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 Apr 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
National GLP Compliance Monitoring Authority, Govt. of India (22 Jul 2016)
Analytical monitoring:
yes
Remarks:
HPLC
Details on sampling:
- Concentrations: All treatment levels and the control after 0 and 48 h.
- Sampling method: 10 mL were sampled from each replicate and mixed together for each group at 0 and 48 h. The samples were divided into two equal portions of 20 mL. One portion was used for test concentration analysis and the second portion was stored.
- Sample storage conditions before analysis: -20 ± 5 °C
Vehicle:
yes
Remarks:
acetone
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Four stock solutions (10, 17, 29, and 83 mg/mL) were prepared by dissolving respective test item amounts (10 - 83 mg) in 1 mL acetone. 10 µL of each stock solution was diluted to 100 mL with reconstituted water. For the control, 1000 µL blank medium was diluted to 100 mL with reconstituted water.
- Differential loading: Yes
- Controls: 1000 µL blank medium in 100 mL reconstitued water
- Chemical name of vehicle: Acetone (the test item was insoluble in reconstituted water)
- Concentration of vehicle in test medium: 10 µL acetone were diluted to 100 mL with reconstituted water
- Evidence of undissolved material: In the preliminary range-finding study, precipitation of test item was observed up to a concentration of 16.66 mg/L, while no precipitation was observed at 8.33 mg/L. Therefore, 8.33 mg/L was selected as the highest test concentration.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: MicroBio Test Inc, Mariakerke, Belgium
- Age of daphnids at test start: < 24 h
- Feeding during breeding: Unicellular green algae Pseudokirchneriella subcapitata
- Feeding during test: No

ACCLIMATISATION
- Acclimation period: Gravid females were acclimatised to the test conditions for a min. of 48 h.
- Acclimation conditions: Same as test (19.5 °C, 8.50 mg/L dissolveg oxygen, pH 7.36 and 16 h light/8 h dark photoperiod, 1340 lux, total hardness (CaCO3): 190.4 mg/L)
- Type and amount of food: Unicellular green algae Pseudokirchneriella subcapitata

METHOD FOR PREPARATION AND COLLECTION OF DAPHNIDS:
First instar daphnids (< 24 h) were separated from the adults in a culture of daphnids derived from one single mother daphnid. This culture had previously been acclimatised to the study conditions. The daphnids were collected with a micropipette (100 - 1000 µL).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
184.8 mg/L CaCO3
Test temperature:
19.6 - 20.4 °C
pH:
7.61 - 7.99
Dissolved oxygen:
7.91 - 9.01 mg/L
Nominal and measured concentrations:
Control, 1.00, 1.70, 2.90, 4.90, and 8.30 mg/L (nominal)
-- , 1.13, 1.83, 3.18, 5.29, and 8.84 mg/L (measured after 48 h)
Details on test conditions:
TEST SYSTEM
- Test vessel: 600 mL glass beakers filled with 100 mL test media
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water according to OECD 202
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen content in reconstituted water was measured at 0 and 48 h with a Multi Portable meter. Total hardness was measured once during acclimitisation by titrimetic method.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 1330 lux (0 h), 1310 lux (24 h), and 1340 lux (48 h)
- Other: The whole study was conducted in a specially designed low temperature water bath preset to a water temperature of 20.0 °C and a slow water flow (by a motor) to achieve uniform temperature. The test vessels were placed in the water bath and held in position with special clamps in the chamber.

EFFECT PARAMETERS MEASURED:
- Immobilization and behavioural symptoms: after 0, 24, and 48 h

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- Test concentrations: Control (0.0 mg/L test item), vehicle control, 0.01 (0.0 mg/L test item), 0.01, 0.10, 1.00, and 8.33 mg/L
- Results used to determine the conditions for the definitive study: Yes, the percent immobility were 100% at 8.33 mg/L and 0% in all other treatments.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Lethargy was observed after 24 h (at 4.9 and 8.3 mg/L) and 48 h (at 4.9 mg/L).
- Mortality of control: 0%
- Other adverse effects control: No signs of desease, stress or trapping at the surface of water were observed in any of the replicates of the control group.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50 (48 h): 0.26 mg/L
- Other: The reference test dates from 10 - 14 Jun 2017
Reported statistics and error estimates:
Probit analysis

VALIDITY CRITERIA

The study fulfilled the validity criteria laid down by the guideline (Table 1).

Table 1: Validity criteria for OECD 202.

Criterion from the guideline

Outcome

Validity criterion fulfilled

In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.

Immobilization was 0% in the control

 

 

Yes

The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.

The dissolved oxygen concentration was ≥ 7.91 mg/L in the control and test vessels at the end of the test.

Yes

 

 

BIOLOGICAL RESULTS

Results are summarized in Table 2. The derived EC50 (48 h) was 4.7 mg/L and the NOEC (48 h) was 1.7 mg/L (nominal).

Table 2. Immobility data after 0, 24 and 48 h.

Nominal test concentration

[mg/L]

n° of replicates

n° of daphnids

immobility n° and % at

0 h

24 h

48 h

%

%

%

Control

4

20

0

0

0

0

0

0

vehicle control

4

20

0

0

0

0

0

0

1.0

4

20

0

0

0

0

0

0

1.7

4

20

0

0

0

0

0

0

2.9

4

20

0

0

0

0

1

5

4.9

4

20

0

0

0

0

11

55

8.3

4

20

0

0

4

20

20

100

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

OECD guideline, GLP compliant study. Daphnia magna were exposed to the test substance for 48 hours during the test. % of immobility and abnormal behaviours were recorded.

48h EC50 value of 4.7 mg/L was calculated.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
4.7 mg/L

Additional information