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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:
2017-07-14 to 2017-08-09
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 (Alga, Growth Inhibition Test)
according to guideline
EU Method C.3 (Algal Inhibition test)
according to guideline
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment
Version / remarks:
Guidance number 23, December 14, 2000.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
- Analytical purity: 99.5% (based on UPLC)
- Source and lot/batch No.of test material: I17BB0589
- Expiration date of the lot/batch: 12 February 2018 (retest date)

- Storage condition of test material: In refrigerator (2-8°C) in container flushed with argon
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: not reported

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: Samples for possible analysis were taken from all test concentrations and the control at t=0, t=24h and t=72h. 2.0 mL of volume was taken from the approximate centre of the test vessels. In addition, the filter containing undissolved residue was kept for possible analysis. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: The samples were stored in a freezer. Additionally, reserve samples of 2.0 mL were taken for possible analysis.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to an expected low water solubility of the test item, saturated solutions (SS) were prepared. The preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring at room temperature to ensure maximum dissolution of the test item in the test medium. This resulted in a clear and colourless dispersion that contained undissolved floating material and precipitate. The obtained mixture was filtered through a 0.45 μm membrane filter (Whatman; RC55) to remove the undissolved fraction. The filter was pre-conditioned with a small volume of test solution that was discarded. After adjusting the pH with 1N HCL (Merck, Darmstadt, Germany; combined limit/range-finding test: from 9.4 to 8.3; final test: from 9.7 to 8.2), the resulting SS was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were 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 10^4 cells/mL.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal stock cultures were started by inoculating growth medium 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. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

- Acclimation period: not relevant (except pre-culture 3 days before start of the test)

Study design

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

Test conditions

24 mg CaCO3/L
Test temperature:
22 - 23°C
Dissolved oxygen:
not reported
not relevant
not reported
Nominal and measured concentrations:
Combined limit/range-finding test:
nominal test concentrations: 1.0 10 and 100% of the SS prepared at 100 mg/L
measured test concentration t = 0 h: 0.37 and 38 mg/L
measured test concentration t = 72 h: 1% SS (nominal ): 0.127 mg/L %, 10 (nominal): not measured and 100% SS (nominal): 13.9 mg/L

Final test:
nominal test concentrations: 1.0, 3.2, 10, 10, 32, 100 % of the SS prepared at 100 mg/L
measured test concentration t = 0 h: 0.325, 1.1, 3.22, 3.29, 10.6, 33.2 mg/L
measured test concentration t = 72 h: 0.0951, 0.354, 1.12, 1.15, 3.75, 11.9 mg/L

Details on test conditions:
- Test vessel: 100 mL all-glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Initial cells density: 10,000 cells/mL
- Control end cells density: 153.8 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3

- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 78 to 86 µE/m2/s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: At the beginning, 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 immersion probe (path length = 20mm).
- Effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: x3.2
- Range finding study: yes
- Test concentrations: 1.0 and 10% of the SS (setup as combined range finder/limit test)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological relevance
Key result
72 h
Dose descriptor:
Effect conc.:
6.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
95% confidence interval ranging from 6.7 to 7.1 mg/L.
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- 72-h EC50 (yield) = 6.9 mg T003641/L (95% C.L. 6.7-7.1 mg/L)
- 72-h EC10 (growth rate) = 0.84 mg T003641/L (95% C.L. 7.9-8.9 mg/L)
- 72-h EC10 (yield) = 0.46 mg T003641/L (95% C.L. 0.44-0.48 mg/L)
- 72-h NOEC (yield) < 0.19 mg T003641/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 0.99 mg/L (95% C.L. 0.97 to 1.0 mg/L)
- 72-h EC50 (yield) = 0.37 mg/L (95% C.L. 0.43 to 1.1 mg/L)
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure 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:
A 72-h growth inhibition test with the unicellular green algae Pseudokirchneriella subcapitata was performed with the test substance T003641 according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had a significant inhibitory effect on the growth of Pseudokirchneriella subcapitata up to and including a TWA concentration of 0.48 mg/L, after the test period of 72 hours and based on biological relevance. The EC50 for growth rate inhibition (72h-ERC50) was 6.9 mg/L with a 95% confidence interval ranging from 6.7 to 7.1 mg/L. The results of the test can be considered reliable without restrictions.