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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 2019-01-28 to 2019-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h and t=48 h
Volume: 1.8 mL from the approximate centre of the test vessels
Storage: 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. Additionally, reserve samples of 1.8 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
Preparation of test solutions started with the highest concentration of 100 mg/L applying 15 minutes of magnetic stirring to accelerate dissolution of the test item in 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.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
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. Characteristics Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding
Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an allglass 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.
Culture medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
180 mg/L (expressed as CaCO3)
Test temperature:
18-22°C
pH:
6.2-8.0
Dissolved oxygen:
8.8-9.1 mg/L
Nominal and measured concentrations:
0.10, 1.0, 10 and 100 mg/L
Details on test conditions:
Test Concentrations
Test item: 0.10, 1.0, 10 and 100 mg/L
Control: Test medium without test item or other additives.

Test Procedure and Conditions
Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass
Test medium: 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.
Number of daphnids: Control and highest test concentration: 20 per test group. Lower concentrations: 10 per concentration
Loading: 5 per vessel containing 50 mL of test solution.
Light: A daily photoperiod of 16 hours.
Feeding: No feeding
Aeration: No aeration of the test solutions was applied.
Introduction of daphnids: Within 15 minutes after preparation of the test solutions.


Measurements and Recordings
Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all test concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No immobility was observed in the control and at any of the test concentrations throughout the exposure period.
Results with reference substance (positive control):
The 24h-EC50 in this reference test with K2Cr2O7 was within the expected range of 0.6 and 2.1 mg/L as specified in ISO International Standard 6341, October 2012. The 48h-EC50 was within the expected range of 0.28 and 0.75 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
The 24h-EC50 was 0.84 mg/L with a 95% confidence interval between 0.73 and 0.97 mg/L.
The 48h-EC50 was 0.47 mg/L with a 95% confidence interval between 0.41 and 0.55 mg/L

In conclusion, the sensitivity of this batch of Daphnia magna was in agreement with ISO International Standard 6341, October 2012 and the historical data collected at Charles River Den Bosch.
Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the 48h-EC50 for Daphnia magna exposed to the test item was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L.
Executive summary:

The objective of the study was to evaluate the tets item for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours based on the OECD guideline No. 202, 2004.

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a nominal concentration of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to nominal concentrations of 0.10, 1.0 and 10 mg/L, in the combined range-finding test. 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 the highest test concentration were analysed. The measured concentration was at 102-104% relative to the nominal concentration of 100 mg/L throughout the test. Based on this result, effect parameters were expressed in terms of analytically confirmed nominal concentrations.  

No immobility was observed in the control and at any of the test concentrations throughout the exposure period.

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

In conclusion, the 48h-EC50 for Daphnia magna exposed to the test item was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L.

Description of key information

The 48h-EC50 for Daphnia magna exposed to the test item was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L.

Key value for chemical safety assessment

Additional information

The objective of the study was to evaluate the tets item for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours based on the OECD guideline No. 202, 2004.

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a nominal concentration of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to nominal concentrations of 0.10, 1.0 and 10 mg/L, in the combined range-finding test. 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 the highest test concentration were analysed. The measured concentration was at 102-104% relative to the nominal concentration of 100 mg/L throughout the test. Based on this result, effect parameters were expressed in terms of analytically confirmed nominal concentrations.  

No immobility was observed in the control and at any of the test concentrations throughout the exposure period.

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

In conclusion, the 48h-EC50 for Daphnia magna exposed to the test item was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L.