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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:
24 - 27 Oct 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
according to
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Council Regulation EC No. 761/2009
GLP compliance:
yes (incl. certificate)
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Analytical monitoring:
Details on sampling:
- Concentrations: Control, 1.00, 3.16, 10.0, 31.6, and 100 mg/L (nominal)
- Sampling method: Samples were collected in fresh test solutions ( 0 h) and in old test solutions (72 h).
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler), if necessary.
Details on test solutions:
- Method: A stock solution of 100 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the solution was homogenous.
- Controls: Yes, consisting of test media without test item incubated under the same conditions as the test.
- Evidence of undissolved material: The test media were clear throughout the exposure phase.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: unicellular freshwater green alga
- Strain: SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): A 3 d old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 h per day. The culture medium was the nutrient medium Z, according to Lüttge et al. 1994.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
0.24 mmol Ca+Mg/L
Test temperature:
21.5 °C (mean)
0 h: 8.09 (control), 7.49 - 7.85 (treatments)
72 h: 9.45 (control), 7.88 - 9.10 (treatments)
Nominal and measured concentrations:
Control, 1.00, 3.16, 10.0, 31.6, and 100 mg/L (nominal)
< LOQ, 0.943, 3.05, 9.61, 30.5, and 93.6 mg/L (measured after 72 h)
Details on test conditions:
- Test vessel: Sterile Erlenmeyer flasks.
- Type: Sealed with cotton wool plugs.
- size, headspace, fill volume: Size: 250 mL; Fill volume: 100 mL
- Initial cell density: 6305 cells/mL
- Control end cell density: 1263285 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: Yes, OECD medium.

- Culture medium different from test medium: Same as test: OECD medium was used as dilution water.
- Intervals of water quality measurement: The pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

- Sterile test conditions: Yes.
- Photoperiod: 24 h light/d
- Light intensity and quality: 120 µE*m^-2*s^-1
- Other: The flasks were positioned randomly on a rotary shaker (shaking at approximately 70 rpm) and repositioned daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Chlorophyll-a fluorscence measured by fluorometer (Microplate Reader Chameleon V, excitation at 436 nm, emission at 685 nm) at 0, 24, 48 and 72 h
- Other: Microscopic evaluation of cell morphology at the start and end of exposure (72 h).

- Spacing factor for test concentrations: 3.16 (√10)
- Range finding study: Yes, preliminary non-GLP range-finding test.
- Test concentrations: 1, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, 88% growth rate inhibition was observed at a nominal concentration of 100 mg/L test item after 72 h.
Reference substance (positive control):
potassium dichromate
Key result
72 h
Dose descriptor:
Effect conc.:
23.9 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 11.3 -31.5 mg/L
Key result
72 h
Dose descriptor:
Effect conc.:
32.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 14.3 - 69.9 mg/L
Details on results:
- Exponential growth in the control (for algal test): Yes, cell growth increased 200-fold (specific growth rate 1.76 d^-1).
- Observation of abnormalities: No.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- ErC50 (72 h): 0.435 mg/L, 95% confidence interval 0.414 - 0.458 mg/L (potassium dichromate)
Reported statistics and error estimates:
ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 h were calculated by sigmoidal dose-response regression. NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOECL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The Shapiro-Wilk Test was used to test for normal distribution of data. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value is 0.05.


The measured concentrations of the test item were between 92% and 98% of the nominal value at the start of the exposure (0 h) and between 94% and 97% of the nominal value at the end of the exposure (72 h). This indicates that the test item concentration was successfully maintained throughout the duration of the test.

Since the measured test item concentrations were within ± 20% of the nominal concentration, the effect concentrations are based on nominal concentrations.


The test substance was found to inhibit the growth of the freshwater green alga P. subcapitata after 72 h. The derived NOEC values for both inhibition of growth rate and yield after 72 h were 10.0 mg/L, respectively.

Description of key information

ErC10 (72 h) = 23.9 mg/L (nominal, P. subcapitata, OECD 201)

ErC50 (72 h) = 32.1 mg/L, (nominal, P. subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
32.1 mg/L
EC10 or NOEC for freshwater algae:
23.9 mg/L

Additional information

There is one GLP study available investigating the effects of the substance on the growth of freshwater microalgae according to the OECD guideline 201.

In a static test, P. subcapitata was exposed to 5 nominal concentrations of the test item ranging from 1.0 – 100 mg/L for 72 h under controlled conditions. Negative controls were run in parallel and the test item concentration at the beginning and end of exposure was analytically verified by HPLC-DAD.

The test media were clear throughout the exposure period. The measured concentrations of the test substance in fresh media (0 h) ranged from 92 – 98% of the nominal values and from 94 – 97% of the nominal values in aged media (72 h). This indicates that the concentration of the test substance was successfully maintained throughout the duration of exposure. Since the measured concentrations were within the required ± 20%, all effect values are based on the nominal test item concentrations.

The test substance was found to inhibit the growth of the microalgae. However, no morphological abnormalities were observed. The derived NOErC was 10.0 mg/L and the LOErC was 31.6 mg/L. The corresponding effect concentrations and respective 95% confidence intervals are an ErC10 (72 h) value of 23.9 mg/L (11.3 – 31.5 mg/L) and an ErC50 (72 h) value of 32.1 mg/L (14.3 – 69.9 mg/L).