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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
72 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 201 (Alga, Growth Inhibition 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 grown and hold in OECD medium which was used also for preparing test item solutions. The stock solution was prepared by measuring 20.14 mg of test item into 1 000 ml OECD growth medium. The suspension was stirred and ultrasonicated till complete dilution. The concentration of stock solution was 20.14 mg/L.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test species: Pseudokirchneriella subcapitata (Korshikov) F.Hindák (formerly known as Selenastrum capricornutum and Raphidocelis subcapitata)
Source: Culture Collection of Algae and Protozoa, Scottish Marine
Institute (
Batch: CCAP 278/4
Date of arriving: 27 October 2017

Study design

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

Test conditions

Test temperature:
The new seed cultures are kept under continuous illumination for five or seven days at 21 ± 2 °C.
The temperature inside the incubator at the test vessels was measured and registered continuously by Extech SD200 thermometer and datalogger. Throughout the test the temperature was nearly constant.
At the beginning of the test the pH values were measured in the remaining test solution of the concurrent test vessels at each dilution level. At the end of the test the pH values were measured in each test vessel. The pH of test solutions was not adjusted at the beginning of the test. The following table shows the measured pH values at the beginning and at the end of the test.
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. OECD medium was used for dilution of stock solution. In the test the following calculated concentrations were applied: 20.14; 12.08; 6.04; 3.02; 2.01; 1.01 and 0.81 mg/L.
Details on test conditions:
Preparation and breeding of inoculum culture: The inoculum culture was prepared four days before starting the test and it was bred in incubator at 21 ± 2 °C. At the end of incubation the cell density of the inoculum culture was determined with a Bürker chamber. The cell concentration was 1 694 444 cell/ml
Preparation of stock solution: The preparation of stock and test solutions was based on the preliminary non GLP range finding test. 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. The stock solution was prepared by measuring 20.14 mg of test item into 1 000 ml OECD growth medium. The suspension was stirred and ultrasonicated till complete dilution. The nominal concentration was 20.14 mg/L.
Preparation of test solutions: Seven test solutions of different concentration were prepared by mixing appropriate volume of OECD medium and stock solution of the test item. The maximum separation factor between of test vessels was 2.0. Two additional replicates were prepared for controlling the abiotic degradation of the test substance. These test vessels were not inoculated with alga inoculum culture. The following table shows the preparation of test solutions. The volume of the prepared dilution levels was 300 ml except the control solution which was 600 ml. The alga inoculum culture was diluted 340.91 times so the initial alga cell concentration in every dilution and the control levels were 4 970 cells/ml.
Preparation of test vessels: Three replicates were prepared at every test, two at the abiotic control and six at the control level. The vessels contained 75 ml test media.
Test procedure: The test vessels were closed with silicone stopper and kept in the Binder KBW 400 incubator. For the proper mass transfer of carbon dioxide glass tubes with filter were placed through silicone stoppers. The test was maintained under static conditions for a period of 72 hours. The temperature during the test remained in the 21 ± 2 °C range. The temperature data were harvested by Extech SD200 datalogger. The vessels were shaken and illuminated continuously in the incubator. The vessels were placed randomly into the incubator and were repositioned daily after sampling. One of the vessels of abiotic controls was taken into the incubator with a complete aluminium foil cover to check the stability of test item in the absence of light. This vessel was signed as 8/B (vessel No: 23). The abiotic control on light were signed as 8/A (vessel No: 22).
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
0.96 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
1.32 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
2.42 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The yield is a response variable of the biomass increase during the test which is calculated as the difference of the biomass at the start and at the end of the test. The effective concentrations for yield are signed as EyCx. The EyCx values were determined including the 95 % confidence interval with Probit analysis using linear max. Likelihood regression. The EyC10, EyC20 and EyC50 were calculated for at both 24, 48 and 72 h period.

Applicant's summary and conclusion

Validity criteria fulfilled:
Acute toxic effect of Acetoxy-AD was investigated in Freshwater Alga and Cyanobacteria, Growth Inhibition Test (according to OECD 201 guideline). The ErC50 was 2.42 mg/L at 72 h. According to the Council Regulation (EC) 440/2008 Acetoxy-AD is hazardous to the aquatic environment and classified into the second acute toxic hazardous category [GHS Acute aquatic hazard Category Acute II. – 72 h EC50 (for algae or other aquatic plants) > 1 mg/L but ≤ 10 mg/L].