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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate EU test method and in compliance with GLP.

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to
EU Method C.3 (Algal Inhibition test)
according to
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
Identification EPYRRON-OPL
Molecular formula C23H35NO2
Molecular weight 357.53
CAS Number 159325-45-8
Description Cream powder with lumps (determined at WIL Research Europe B.V.)
Batch DGJ524K1NAT
Purity/Composition 85-95%
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 15 January 2014 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface). No correction was made for the purity/composition of the test substance.

The batch of EPYRRON-OPL tested was a cream powder with lumps with a purity of 85-95% and the test substance was not completely soluble in test medium at the loading rates initially prepared.

In the combined limit/ range-finding test preparation of test solutions started with individual loading rates of 1.0, 10 and 100 mg/l. In the final test preparation of test solutions started with an loading rate of 100 mg/l. A 15-minute treatment with ultrasonic waves was applied and was followed by a 2 day-period of magnetic stirring. The obtained dispersions were subsequently filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove the undissolved fraction of test substance. In the final test the lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions for both experiments were all clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species Pseudokirchneriella subcapitata, strain: NIVA CHL 1

Source In-house laboratory culture.

Reason for selection This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species

Study design

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

Test conditions

Details on test conditions:
EPYRRON-OPL Solutions containing 1.0, 3.2, 10, 32 and 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg/l.

Controls Test medium without test substance or other additives.

Replicates 6 replicates of the control,
3 replicates of each test concentration,
1 replicate of the solution containing 10% of the filtrate without algae,
1or 2 replicate of each test concentration for sampling purposes.

Test procedures and conditions
Test duration 72 hours

Test type Static

Test vessels 100 ml, all-glass, containing 50 ml of test solution

Medium M2

Cell density An initial cell density of 1 x 104 cells/ml.

Illumination Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of
83 to 88 E.m-2.s-1.

Incubation Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Statistically significant reduction of growth rate was found at every concentration tested (Bonferroni t test, α = 0.05). However, reduction lower than 10% is considered not biologically significant and so was the effect observed at the lowest concentration (8.6%). A reduction of approximately 30% was observed at TWA concentrations ranging from 0.065 to 1.0 mg/l. At the highest concentration a reduction of 92% was observed.

Also inhibition of yield was statistically significant at every concentration tested and ranged from 33% at the lowest concentration to 100% at the highest concentration tested.

Microscopic observations at the end of the revealed that algae cells were similar to the control at the TWA concentration of 0.016 mg/l, whereas at 0.065 to 1.0 mg/l cells were smaller.

Applicant's summary and conclusion

Validity criteria fulfilled:

The EC50 for growth rate reduction (72h-ERC50) for Epyrron was 1.4 mg/l with a 95% confidence interval ranging from 1.3 to 1.6 mg/l, based on TWA concentrations, and by read across the EC50 for growth reduction for Epyrrol is considered similar.
Executive summary:

Under the conditions of the present study withPseudokirchneriella subcapitata, EPYRRON-OPL reduced growth rate and inhibited the yield of this fresh water algae species significantly at all of the concentrations tested.


The EC10for growth rate reduction (72h-ERC10) was 0.018 mg/l with a 95% confidence interval ranging from 0.0095 to 0.033 mg/l, based on TWA concentrations.


The EC50for growth rate reduction (72h-ERC50) was 1.4 mg/l with a 95% confidence interval ranging from 1.3 to 1.6 mg/l, based on TWA concentrations.


The EC10for yield inhibition (72h-EYC10) was 0.0072 mg/l and was considered an estimate as it was obtained by extrapolation.


The EC50for yield inhibition (72h-EYC50) was 0.029 mg/l with a 95% confidence interval ranging from 0.016 to 0.054 mg/l.

By read across the EC50 for growth reductions and yield inhibitions for Epyrrol are considered similar.