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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2017 - 01 November 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011
according to guideline
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Details on test material:
Physical appearance: colourless to pale yellow liquid
Storage conditions: at room temperature protected from light, container flushed with nitrogen.
Specific details on test material used for the study:
- The test item is insoluble in water.
- Specific gravity / density: 1.059 (20ºC); 1.057 (25ºC)

Sampling and analysis

Analytical monitoring:
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, t=24 h and t=72 h
Volume: 2.0 mL
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 with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Details on test solutions:
- Method: Test solutions were prepared at a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for two hours (combined limit/range-finding test) or one hour (final test). The clear and coloursless Saturated Solution (SS) was collected by means of siphoning and used as either the highest test concentration (combined limit/range-finding test) or a stock solution (final test). Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis)) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C, until the pre-culture started.

- 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium as used in the Final test: Standard M2 medium, according to OECD 201 Guideline.

Study design

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

Test conditions

24 mg CaCO3/L
Test temperature:
22-24 °C
At the start of the test: 8.2 - 8.3
At the end of the test: 8.2 - 8.5
Nominal and measured concentrations:
Nominal concentrations: 0.10, 0.32, 1.0, 1.0 (without algae), 3.2, 10 and 32% of a saturated solution prepared at 100 mg/L
Measured concentrations (TWA): 0.064, 0.22, 0.71, 0.81, 2.4, 7.6 and 26 mg/L, respectively to nominal concentrations stated above.
For measured concentrations per time point, see table 1.
Details on test conditions:
- Test vessel: 100 mL all glass vessels, containing 50 mL of test solution.
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 297 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- In addition: 1 or 2 replicates of the highest concentration without algae; 1 or 2 replicates of each test concentration for sampling purposes.

- Medium used: M2 medium

- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.
- Intervals of water quality measurement: pH measured at beginning and end of test; temperature of medium continuously measured in a temperature control vessel.

- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity of 89 µE.m-2.s-1.

- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank.

- Test concentrations: 1.0, 10 and 100% of the SS prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, a 64.9% inhibition of growth was observed in the 10% SS test solution, while inhibition of growth was only 4.8% in the 1.0% SS test solution. Therefore, the EC50 was expected to be between 1.0 and 10% of a SS prepared at a loading rate of 100 mg/L.
Reference substance (positive control):
Potassium Dichromate (test performed Sept 2017).

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
5.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 5.2-5.4
Key result
72 h
Dose descriptor:
Effect conc.:
0.22 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: Yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a concentration of 2.4 mg/L (TWA) when compared to the control.

- Test concentrations remained stable during the first 24 hours of exposure (92-96% of initial measured concentrations) but decreased to 48-87% of initial after 72 hours of exposure. Based on these results, TWA concentrations were calculated and used to determine the effect parameters.
- Samples taken from the solutions containing 1.0% of the SS with and without algae showed that test item concentrations were similar during the first 24-hours of exposure. However, while the solution with algae decreased to 64% of initial at the end of the test, the solution without algae remained more stable and contained 85% of initial at the end of the test. Therefore, it was expected that the presence of algae slightly affected the test item concentration in solution.

- The 72h-NOErC was 0.22 mg/L, based on statistical significance. Based on biological relevance (inhibition >10%), the 72h-NOErC was determined to be 0.71 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Tested concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- 72-h ErC50: 1.1 mg/L
- Other: the effect parameter was within the historical range of the test facility.
Reported statistics and error estimates:
NOEC and EC50: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
ECx values: probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
Based on the results of a toxicity study towards algae, performed according to OECD guideline 201 and GLP principles, the 72h-ErC and the 72h-NOErC of the test substance were determined to be 5.3 and 0.22 mg/L, respectively (based on TWA concentrations).