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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:
2018
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Remarks:
Gas Chromatographic (GC)
Details on sampling:
Sampling for Chemical Analysis:
- Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations
- Control and test solutions were sampled in duplicate at the start of exposure and after 48 hours; the duplicate samples were kept separately as a reserve; the nominal volume per sample was 10 mL. mixed with 10 mL of acetonitrile; the volume of each sample was recorded
- After sampling and before shipment, all samples were stored in amber glass bottles in the dark at a temperature of ≤ -18°C; a record was kept for each sample
Vehicle:
no
Details on test solutions:
Preparation of Stock and Test Solutions:
- A stock solution was prepared at a nominal loading rate of 100 mg/L by adding 100.15 mg of the test item to 1000 mL of the test medium, stirred (500 rpm) for 20 minutes at ambient temperature and the pH was adjusted from 9.1 to 8.2 using 1 M HCl; visual inspection of the flask showed a clear solution without Tyndall effect
- The stock solution was prepared once to prepare the test solutions
- The volume of the stock solution was large enough to prepare the limit concentration and all analytical samples
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM:
- Species: Daphnia magna STRAUS (clone M10), a water flea
- Origin: The organisms were originally supplied by KU Leuven, Belgium and cultured at ECT Oekotoxikologie GmbH since December 22, 2011
- Age of parental daphnids: less than 35 d
- Age of the daphnids at the beginning of the test: less than 24 h

HOLDING CONDITIONS:
- Material of stock vessel: Glass
- Volume of medium per stock vessel: 1.8 L
- Depth of medium in the stock vessel: approximately 14.5 cm
- Number of daphnids kept as stock per culture vessel (batch size): approximately 20
- Separation of adult and young daphnids: 2-5 times per week
- Medium: Elendt medium M4
- Renewal of medium: twice per week
- Temperature: within required ranges (20 +/- 2°C)
- Photoperiod (light/dark) 16/8 h
- Light intensity: within required ranges (50 to 1000 lx)
- Aeration: None
- Food: algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
- Feeding frequency: 4 times per week
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
14.2 °dH (254 CaCO3 mg/L)
Test temperature:
20.0 - 20.7 °C
pH:
7.9 - 8.2
Dissolved oxygen:
8.0 - 8.3 mg/L
Salinity:
n.a.
Conductivity:
no data
Nominal and measured concentrations:
100 mg/L nominal concentration equals approx. 61 (mg test item/L) measured concentration after 48 hours of exposure.
Details on test conditions:
EXPOSURE CONDITIONS:
- Test units: 100-mL glass beakers, closed by watch glass
- Volume of test solution per test vessel: 50-60 mL
- Test medium: Elendt Medium M4
- Number of daphnids per test vessel: 5
- Adjustment of pH in the medium: none
- Application of test item: test item dissolved in Medium M4 applied once at the beginning of the test
- Aeration of test vessels: none
- Test concentrations (nominal): Control; 100 mg test item/L
- Number of replicates per concentration: 4
- Number of replicates in the controls: 4
- Renewal of the test solution during the test period: static, no renewal
- Feeding during exposure: none
- Temperature (target): 18 to 22°C, constant within a range of 2°C
- Light intensity (target): 50 to 1000 lx
- Light regime: 16 h light; 8 h dark
- Biological parameter: immobility

WATER QUALITY MEASUREMENTS:
At start of the test, after temperature adaptation of the test solutions, and at the end of the test the following parameters were measured and recorded in one replicate test vessel of the control and the highest test concentration:
- Temperature,
- pH,
- Dissolved oxygen content in % of air saturation value (ASV) and in mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 68.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The measured initial test item concentration was 68.4 mg/L. Since the concentration remained stable at 88.9% of the measured initial concentration, the biological endpoints are expressed based on the mean measured initial concentrations (68.4 mg/L).
Results with reference substance (positive control):
The most recent reference test with potassium dichromate performed in a separate study (Study No. IDA1711; November 2017) resulted in an EC50(24 h) of 0.74 mg/L.

According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L. The toxicity of the reference item is within this range. Therefore, the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.
Reported statistics and error estimates:
In this limit test no immobility was observed in the control or in limit concentration of 100 mg test item/L. A statistical evaluation for differences between treatment and control was therefore not required and was not performed.
Validity criteria fulfilled:
yes
Remarks:
immobilisation and other abnormalities in the controls did not exceed 10% by the end of the test; dissolved oxygen concentration remained above 3 mg/L throughout the exposure period
Conclusions:
No toxic effects against daphnia were observed at a limit test concentration of 100 mg/L (EC0 (48h) >= 100 mg/L) under exposure conditions. It is therefore concluded that the EC50 of the test item is higher than the limit concentration tested (nominal: 100 mg test item/L). This corresponds to a measured initial test item concentration of 68.4 mg/L.
Executive summary:

The study was performed according to Method C.2 ‘Acute toxicity for Daphnia’ (1992), described in the Council Regulation (EC) No 92/69, which is equivalent to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilization Test'.  Daphnids were exposed to the test item added to water at a limit test concentration of nominally 100 mg/L for a period of 48 hours under static conditions. Immobilization rates were recorded at 24-hour intervals. During the test period the test solutions were not aerated. The daphnids were not fed. Those animals not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile. The data obtained were analysed in order to demonstrate that the EC50 is higher than the limit concentration. To verify the nominally applied concentrations, samples were taken from the test solutions and analytically measured. The samples designated for chemical analysis were shipped to the analytical test site and analysed under the responsibility of the principal investigator for analysis. No toxic effects against daphnia were observed at a limit test concentration of 100 mg/L (EC0 (48h) >= 100 mg/L) under exposure conditions, thus no statistical analysis was required to determine the EC50.

It is concluded that the EC50 of the test item is higher than the limit concentration tested (nominal: 100 mg test item/L). This corresponds to a measured initial test item concentration of 68.4 mg/L. All validity criteria were fullfilled. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute Daphnia study.

Description of key information

No toxic effects against daphnia were observed at a limit test concentration of 100 mg/L (EC0 (48h) >= 100 mg/L) under exposure conditions. It is therefore concluded that the EC50 of the test item is higher than the limit concentration tested (nominal: 100 mg test item/L). This corresponds to a measured initial test item concentration of 68.4 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
68.4 mg/L

Additional information

"Should read: EC 50 > 68.4 mg/L (The result is based on measured concentration)"

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