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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Mar 2006 (corrected Jul 2011)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Single Samples for analysis were taken from all test concentrations and the control.
Frequency: at t= 0, 24 and 72 h
Volume: 0.2 mL from the approximate centre of the test vessels. For sampling after 24 hours of exposure, one extra replicate was prepared. At the end of the exposure period, the replicates were pooled before sampling.
Storage: Not applicable, samples were analysed on the day of sampling
Details on test solutions:
The test item was not completely soluble at the loading rate of 100 mg/L. Thus, preparation of the test solution started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to reach the maximum dissolution of the test item in medium. Thereafter, the Water Accommodated Fraction (WAF) was collected by means of filtration through a 0.45 µm membrane filter (Supor®, Pall Corporation) and used as test concentration. The test solution was clear and colorless at the end of the preparation procedure. No correction was made for the purity/composition of the test item.
The method of preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
- Controls: Test medium without test item or other additives
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Green Algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (OECD medium as described in OECD TG 201) at a cell density of 1 x 10E4 cells/mL.
- Method of cultivation: For algae stock cultures, growth medium (prepared according to the NPR 6505 (Nederlandse Praktijk Richtlijn no. 6505) was inoculated with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (light intensity 60 to 120 µE/m2/s at 400 to 700 nm) at 21-24 °C in a climate room.

- Acclimation period: three days (pre-culturing)
- Culturing media and conditions (same as test or not): culturing media different, conditions same
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
0.24 mM (24 mg CaCO3/L)
Test temperature:
22 - 23 °C
8.2 – 8.4
Nominal and measured concentrations:
nominal loading rate: 100 mg/L (WAF)
measured test concentrations with and without algae: < LOQ (0.001 mg/L)
Details on test conditions:
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution, covered with perforated aluminium caps
- Type (delete if not applicable): open
- Aeration: continuous shaking
- Initial cells density: 1 x 10E4 cells/mL
- Control end cells density: 155 10E4 cells/mL
- No. of vessels per concentration (replicates): 6 plus 2 without algae
- No. of vessels per control (replicates): 6

- Standard medium used: yes (OECD medium)

- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Culture medium different from test medium: yes
- Intervals of water quality measurement: pH was measured in the control and the WAF at the beginning and at the end of the test. Temperature was measured continuously in a temperature control vessel.

- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: 89 to 90 µE.m-2.s-1, TLD-lamps

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density, recorded at 24-hour intervals.
- Determination of cell concentrations:
test start (t=0h): counting chamber, thereafter spectrophotometer

- Range finding study
- Test concentrations: combined limit/range-finding study testing WAFs prepared at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no significant differences were recorded between the values for growth rate or yield at any of the WAFs when compared to the control group.
Reference substance (positive control):
potassium dichromate
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
WAF prepared at a loading rate of 100 mg/L
Key result
72 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
nominal loading rate (WAF)
Basis for effect:
growth rate
Remarks on result:
WAF prepared at a loading rate of 100 mg/L
Details on results:
Under the conditions of the present study, no inhibition of growth rate or inhibition of yield was recorded.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: not significant

All test solutions were clear and colorless at the end of the preparation procedure.

For details see attached document "S-600 - Cell densities, growth rates and yield.pdf"
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-hour ErC50: 0.90 mg/L with a 95% confidence interval between 0.88 and 0.93 mg/L (historical data are between 0.82 and 2.3 mg/L).
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional®, Version 3.2.1 (ToxRat® Solutions GmbH).
For determination of the NOELR and the ELx 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 loading rate compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, one-sided, smaller).
The ECx-values could not be determined because the observed effects were below 10%.
Validity criteria fulfilled:
Increase of biomass >16 after 72 hours; mean coefficient of variation for section-by-section specific growth rate <35%; coefficient of variation for average specific growth rate <7%.

Description of key information

effect values for Pseudokirchneriella subcapitata (OECD 201):

0-72 h average specific growth rates: EL50: > 100 mg/L WAF, NOELR: ≥ 100 mg/L WAF

Key value for chemical safety assessment

Additional information

EL50 for freshwater algae: > 100 mg/L

NOELR for freshwater algae: ≥ 100 mg/L

The results indicate that the test material is not toxic to algae up to the limits of its solubility (< 1 µg/L in test water) and a NOEC and thus a PNEC cannot be determined.