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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:
2012-06-08 - 2012-07-14
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffcat ZR 50
- Physical state: liquid (pale yellow)
- Analytical purity: >99%
- Purity test date: 12.01.2011
- Lot/batch No.: PFW100207
- Expiration date of the lot/batch: not provided
- Storage condition of test material: room temperature in the dark
Analytical monitoring:
Details on sampling:
Duplicate samples for analytical verification of test substance concentrations were taken at 0 and 72 h.
Samples from replicates were pooled.
Samples were stored at -20°C prior to analysis.
Details on test solutions:
- 100 mg/L stock solution prepared through direct addition to culture medium.
- Other concentrations prepared by dilution.
- Stock and test solutions were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): 3-5 days, in exponential growth phase
- Method of cultivation: Master cultures were maintained in the lab under constant aeration and constant illumination at 21°C.
- Preculture: Sufficient master culture added to ca. 100 mL volumes of culture media to given initial cell density ca. 1000 cells/mL. Flasks kept under constant agitation (100-150 rpm) and constant illumination (24°C) until algal cell density was ca. 10000-100000 cells/mL (actually 302000 cells/mL).
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:
ca. 15 mg CaCO3/L
Test temperature:
24 +/- °C
7.4-9.9 at 0 h
8.0-8.2 at 72 h
pH variation in control < 1.5 pH units during the test (7.4 to 8.0)
Dissolved oxygen:
not applicable
not applicable
Nominal and measured concentrations:
Nominal: control, 6.25, 12.5, 25, 50, 100 mg/L
Measured concentrations were within 92 to 110% of nominal
Details on test conditions:
- Test vessel: glass conical flasks
- Type (delete if not applicable): stoppered with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: glass, 250 mL, 100 mL fill volume
- Initial cells density: 5000 cells/mL (nominal, mean measured = 6040)
- Control end cells density: 688000 cells/mL (mean measured)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: yes

- Source/preparation of dilution water: reverse osmosis purified deionised water, pH adjusted to 7.5 with 0.1N NaOH or HCl
- Culture medium different from test medium: no
- Intervals of water quality measurement during the test: pH was determined at 0 and 72 h in each test solution, temperature was recorded daily (incubator)

- Incubated in and INFORS Multitron Version 2 incubator, constantly shaken at approximately 150 rpm
- Adjustment of pH: no (only in non-spiked test medium)
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux, warm white lighting (380-730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density was determined at 0, 24, 48 and 72 h
- Determination of cell concentrations: Coulter Multisizer Particle Counter

- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations in range finding study: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
potassium dichromate
72 h
Dose descriptor:
Effect conc.:
33 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI = 29-37 mg/L
72 h
Dose descriptor:
Effect conc.:
12.5 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Growth rate-based:
72-h ErC10 = 16 mg/L
72-h ErC20 = 21 mg/L
72-h LOEC = 25 mg/L
72-h EbC10 = 7.7 mg/L
72-h EbC20 = 10 mg/L
72-h EbC50 = 17 mg/L (95% CI = 15-19 mg/L)
72-h NOEC = 6.25 mg/L
72-h LOEC = 12.5 mg/L
Results with reference substance (positive control):
72-h ErC50 = 1.4 mg/L (95% CI = 1.2-1.7 mg/L)
72-h EbC50 = 0.59 mg/L (95% CI = 0.53-0.65 mg/L)
72-h NOEC = 0.25 mg/L
These results were within normal ranges for this reference item.
Reported statistics and error estimates:
- ECx values were calculated using computerised interpolation using the Xlfit software package (IDBS). 95% confidence limits were calculated using the simplified method of evaluating dose-effect experiments (Litchfield and Wilcoxon, 1949).
- One-way ANOVA incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was used for determination of NOEC and LOEC values.
Validity criteria fulfilled:
In this study, the test substance's toxicity to growth of the unicellular freshwater alga Pseudokirchneriella subcapitata was observed during 72 h, yielding a growth-rate based EC50 of 33 mg/L and a NOEC of 12.5 mg/L. Measured test substance concentrations were close to nominal. The results of the test can be considered reliable.

Description of key information

A full study was performed under GLP conditions and according to OECD TG 201 (Huntsman, 2012). A NOEC of 12.5 mg/L and an EC50 of 33 mg/L, both based on growth rate, were found for the unicellular green alga selenastrum capricornutum (Pseudokirchneriella subcapitata) after an exposure of 72 hours (Huntsman, 2012).

Key value for chemical safety assessment

EC50 for freshwater algae:
33 mg/L
EC10 or NOEC for freshwater algae:
12.5 mg/L

Additional information

A full study was performed under GLP conditions and according to OECD TG 201. A NOEC of 12.5 mg/L and an EC50 of 33 mg/L, both based on growth rate, were found for the unicellular green alga Selenastrum capricornutum (Pseudokirchneriella subcapitata) after an exposure of 72 hours (Huntsman, 2012).