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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

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:
EC Number:
Cas Number:
Molecular formula:
Test material form:
solid: crystalline

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
The stock solution was prepared by measuring 99.99 mg of test item into 1 000 ml OECD growth medium. After 15 seconds ultrasonication and 24 hours stirring the suspension was filtered through 0.2 μm WhatmannTM ME24 filter. The first 50 ml filtrate was disposed. The concentration of the filtrate (stock solution) was 0.23 mg/L.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Data of test species:
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

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.
Nominal and measured concentrations:
The toxic property of test item was investigated at five 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: 0.23; 0.15; 0.12; 0.08 and 0.06 mg/L.
At each concentration level three replicates were used. In the case of control six replicates were applied. The initial alga concentration was 4 978 cells/ml. The vessels were closed with silicone stoppers with a filter for ensuring the proper air exchange. The vessels were kept in incubator at 21 ± 2 °C with continuous illumination. The treatments of control and test vessels were identical.
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 777 778 cell/ml
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. The stock solution was prepared by measuring 99.99 mg of test item into 1 000 ml OECD growth medium After 15 seconds ultrasonication and 24 hours stirring the suspension was filtered through 0.2 μm WhatmannTM ME24 filter. The first 50 ml filtrate was disposed. The concentration of the filtrate (stock solution) was 0.23 mg/L.
Preparation of test solutions: Five 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 1.5. 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 156.25 times so the initial alga cell concentration in every dilution and the control levels were 4 978 cells/ml.
Preparation of test vessels: Three replicates were prepared at every test 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.

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
0.29 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
48 h
Dose descriptor:
Remarks on result:
not determinable
Details on results:
According to the test results Cyclopropyl-AD-enolether has no toxic effect on alga growth rate even at its saturated concentration (0.23 mg/L). (The calculated value of EC10 is higher than the saturated concentration)

Applicant's summary and conclusion

Validity criteria fulfilled:
According to the Council Regulation (EC) 440/2008 Cyclopropyl-AD-enolether is not hazardous to the aquatic environment [72 h EC50 (for algae or other aquatic plants) > saturated concentration of Cyclopropyl-AD-enolether (0.23 mg/L)]. As the reported results are based on calculated concentration data which are gained out of a non-validated HPLC method, the toxicity results must be interpreted by precaution!