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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:
2017-10-06 to 2017-11-09
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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 number 23, December 14, 2000.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Analytical purity: >99.0% (based on chromatographic purity GC: 99.8%)
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the lot/batch: 2018-02-09 (retest date)
- Purity test date: 2017-04-19 (certificate of analysis release date)

- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: not indicated
Analytical monitoring:
Details on sampling:
- Sampling method: Samples were taken before the start of the test and after 72 hours from each test medium and from the control. Additionally, reserve samples of 2.0 mL were taken for possible analysis. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer until analysis.
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 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. The resulting SS was used as the highest test concentration. The pH of the highest concentration and the test medium were adjusted to 6.5 from 3.6 and 8.0, respectively with either 1 M HCl or 1 M NaOH . Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
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.

- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless
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)
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
24 mg CaCO3/L
Test temperature:
22-23 °C
t=0h: 6.6
t=72h: 7.6-8.2
Dissolved oxygen:
not applicable
not relevant
not relevant
Nominal and measured concentrations:
nominal test concentrations: 1.0, 10 and 100% SS (1% SS not analysed)
measured test concentration t = 0 h: 9.53, 96.7 mg/L
measured test concentration t = 72 h: 9.67, 90.9 mg/L

Final test:
nominal test concentrations: 10, 18, 32, 56 and 100% of the SS prepared at 100 mg/L
measured test concentration t = 0 h: n.d., 8.40, 14.8, 27.0, 27.4, 46.7, 84.3 mg/L
measured test concentration t = 72 h: 0.013, 8.38, 15.0, 27.1, 28.2, 47.9, 87.0 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: 239.4 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

- 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 86 to 89 µE.m-2.s-1)

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 (pathlength = 10mm).
- Effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: 1.0, 10 and 100% 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
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
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) = >100 mg 4-nitrobenzene-1-sulfonylchloride/L
- 72-h EC10 (growth rate) = >100 mg 4-nitrobenzene-1-sulfonylchloride/L
- 72-h EC10 (yield) = 27 mg 4-nitrobenzene-1-sulfonylchloride/L (95% C.L. 23-29 mg/L)
- 72-h NOEC (yield) = 10 mg 4-nitrobenzene-1-sulfonylchloride/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 1.1 mg/L (95% C.L. 1.1 to 1.1 mg/L)
- 72-h EC50 (yield) = 0.365 mg/L (95% C.L. 0.35 to 0.36 mg/L)
Reported statistics and error estimates:
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 nominal concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).
Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance 4-nitrobenzene-1-sulfonylchloride according to the OECD guideline 201 (GLP conditions). It can be concluded that the EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded the nominal concentration of 100 mg/L. The 72h-NOEC for growth rate inhibition was 10 mg/L when based on statistical significance and 100 mg/L when based on biological relevance. The results of the test can be considered reliable without restrictions.

Description of key information

The study of Tobor-Kaplon (2018), investigating the acute toxicity of 4-nitrobenzene-1-sulfonylchloride to algae according to OECD guideline 201, was considered as the key study for endpoint coverage.

The EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded the nominal concentration of 100 mg/L. The 72h-NOEC for growth rate inhibition was 10 mg/L when based on statistical significance and 100 mg/L when based on biological relevance.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information