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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:
2017-05-18 to 2017-06-28
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 subsances 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: 96.3% (based on chromatographic purity GC)
- Source and lot/batch No.of test material: A16EB1463
- Expiration date of the lot/batch: 12 September 2017 (retest 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: 0.2 g/L
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. 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.
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 and its degradation products, a saturated solution (SS) was prepared.The preparation of test solutions started with a loading rate of 100 mg/L applying one day of magnetic stirring at room temperature to ensure full degradation of the test item to its metabolites. The obtained aqueous mixture was left to stabilize for approximately 20 minutes where after the SS was taken out by siphoning. The SS was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- 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: 4 days before the start of the range-finding test and 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
final test:
t=0h: 6.8 - 7.3
t=72h: 7.4 - 7.8
Dissolved oxygen:
not applicable
not relevant
not relevant
Nominal and measured concentrations:
nominal test concentrations: 1.0, 10 and 100% SS (1.0% SS not analysed)
measured test concentration t = 0 h: 8.4 and 88 mg/L
measured test concentration t = 72 h: 6.16 and 84.8 mg/L

Final test:
nominal test concentrations: 0, 10, 18, 32, 56, 100 % of the SS prepared at 100 mg/L
measured test concentration t = 0 h: 0.0051, 9.09, 16.2, 28.9, 49.7, 92.6 mg/L
measured test concentration t = 72 h: 0.028, 6.53, 12.9, 24.3, 46.5, 88.3 mg/L

The lowest concentration of 9.1 mg/L decreased to 72% of initial at the end of the test, while the four higher concentrations remained stable (80-95% of initial). Given these results, the effect parameters were based on the TWA concentrations.
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: 101.1 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: yes, the obtained filtrate of the 100% saturated solution was set with 1M HCl from 8.1/8.2 to 6.7/6.8.
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 60 to 120 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 (path length = 20mm).
- Effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: 3.2
- 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.:
81 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L. 76-87 mg/L
Key result
72 h
Dose descriptor:
Effect conc.:
27 mg/L
Nominal / measured:
meas. (TWA)
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) = 32 mg T000749/L (95% C.L. 31-33 mg/L)
- 72-h EC10 (growth rate) = 34 mg T000749/L (95% C.L. 29-39 mg/L)
- 72-h EC10 (yield) = 11 mg T000749/L (95% C.L. 11-12 mg/L)
- 72-h NOEC (yield) = 8.1 mg T000749 /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 or inhibition of yield (Step-down Jonckheere-Terpstra 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 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).
Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T000749 according to the OECD guideline 201 (GLP conditions). The analytically confirmed nominal EC50 for growth rate inhibition (72h-ErC50) was 81 mg/L with a 95% confidence interval ranging from 76 to 87 mg/L and the NOEC was 27 mg/L. The results of the test can be considered reliable without restrictions. It can be concluded that the test substance is not harmful to algae.

Description of key information

Based on the OECD 201 guideline study (Bouwman, 2017) with the unicellular green algae Pseudokirchneriella subcapitata, it can be concluded that the test item had an inhibitory effect on the growth and yield of Pseudokirchneriella subcapitata up to and including the test concentration of 8.1 mg/L, after the test period of 72 hours. The 72-h ErC50 was 81 mg/L (calculated as a time weighted average concentration) with a 95% confidence interval ranging from 76 to 87 mg/L. The results of the test can be considered reliable without restrictions.

Key value for chemical safety assessment

EC50 for freshwater algae:
81 mg/L
EC10 or NOEC for freshwater algae:
8.1 mg/L

Additional information