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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
48 h
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(15α)-3,17-Dioxoandrost-4-en-15-yl acetate
EC Number:
Cas Number:
Molecular formula:
(15α)-3,17-Dioxoandrost-4-en-15-yl acetate
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
The test organism was bred and held in Elendt M4 medium which was also used for preparing of test item solutions. Young daphnids aged less than 24 hours at the start of the experiment were exposed to the test item for a period of 48 hours and acute toxic effect displayed as immobilization of test organism recorded at 24 and 48 hours after the start of test. The stock solution was prepared by measuring 80.06 mg of test item into 1 000 ml Elendt M4 growth medium. The suspension was stirred and ultrasonicated till complete dilution. After stirring the stock solution was clear and colourless. The concentration of stock solution was 80.06 mg/L.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Test species: Daphnia magna Straus
Age: < 24 hours neonates
Origin: National Institute of Environmental Health
Date of arriving: 16 May 2013
Culture conditions: The test species is maintained in Elendt M4 medium with culture conditions at 21±1 °C and a light/dark cycle of 16/8 hours with light intensities of less than 1000 Lux. The cultures are fed with centrifugalized green alga (Pseudokirchneriella subcapitata) cells daily at weekdays.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

Nominal and measured concentrations:
The toxic property of test item was investigated at seven concentrations which were prepared by different dilutions of stock solution of test item. Elendt M4 medium was used for dilution of stock solution. In the test the following nominal concentrations were applied: 80.06; 40.03; 20.02; 12.01; 8.01; 4.00 and 2.40 mg/L.
The concentration of test item was measured at each concentration level at start and at the end of the test by a validated HPLC method. The geometric means of measured concentrations were determined. The following actual concentrations were calculated from the measured values in the test: 81.54; 40.77; 20.27; 12.24; 8.09; 4.12 and 2.45 mg test item/L.
Details on test conditions:
Selection of young daphnids: On the first day of the test, all of juvenile daphnids were removed from the selected breeding cultures by sieving. Juvenile daphnids were disposed. Next day the neonates (which are younger than 24 hours) were separated from the parental culture by filtering and were transferred into test vessels.
Preparation of stock solution: The preparation of stock and test solutions was based on the preliminary non GLP range finding test [9]. As the Range Finding Test was not performed in compliance with the GLP-Regulations it is excluded from the Statement of Compliance in the final report, but the raw data of this test are archived under the study code of present study. For the stock solution 80.06 mg of test item was measured into 1 000 ml Elendt M4 medium. The suspension was stirred and ultrasonicated till complete dissolution. After stirring the stock solution was clear and colourless. The concentration of stock solution was 80.06 mg/L.
Preparation of test solutions: Seven dilution levels were prepared by mixing appropriate volume of Elendt M4 medium and stock solution of the test item. The maximum separation factor between of test vessels was 2.00. The following table shows the preparation of the different dilution levels. The volume of the prepared test solutions was 300 ml.
Test procedure: The test was maintained under static conditions for a period of 48 hours. At the start of the study each test vessels contained 50 ml of test solutions and five young daphnids. In the test four replicates total of 20 animals at each test concentration level were investigated. The test vessels were covered with plastic lids and were kept in thermostat at 21 ± 1 °C with a 16/8 hours light/dark cycle. Each control vessels containing five young daphnids in Elendt M4 medium were prepared in four replicates and kept under the same static conditions. The number of immobilised and abnormally behaved daphnids was recorded after 24 and 48h. A daphnia is considered to be immobile if it is not able to swim within 15 seconds after a gentle agitation of the test vessel.
The daphnids were not fed during the test.

Results and discussion

Effect concentrationsopen allclose all
48 h
Dose descriptor:
Effect conc.:
7.23 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Key result
48 h
Dose descriptor:
Effect conc.:
11.61 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
The effective concentrations (ECx) were calculated with Probit analysis using linear max. Likelihood regression. The EC10, EC20 and EC50 were calculated for immobility at both 24 h and 48 h period.
The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) of the test item were determined with Fisher’s exact binominal test with Bonferroni correction by ToxRat® software. The results were not conclusive.

Applicant's summary and conclusion

Validity criteria fulfilled:
Acute toxic effect of Acetoxy-AD was investigated in Daphnia sp. Acute Immobilisation test (according to OECD 202 guideline). The EC50 was 11.61 mg/L at 48 h. In this Daphnia sp., Acute Immobilisation Test for Acetoxy-AD the results indicated that the test item is hazardous to the aquatic environment and classified into the third acute toxic hazardous category [GHS Acute aquatic hazard Category Acute III. – 48 hr EC50 (for Crustacea) > 10 mg/L but ≤ 100 mg/L]