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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 - 4 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2009)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
(2004)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (blank control) and 100 mg/L were analyzed.
- Frequency: at t=0 h, t=24 h and t=72 h
- Volume: 4.5 mL
- Storage: Samples were stored in a freezer until analysis. In the analytical report it was confirmed that samples were stable when stored in the freezer (see IUCLID Chapter 8 for all details).
Vehicle:
no
Details on test solutions:
- Method: Preparation of test solutions started with a nominal concentration of 100 mg/L applying a 5-minute treatment period with ultrasonic waves to accelerate the dissolving of the test substance in the test medium. The pH of this concentration was adjusted from 3.5 to 6.5 using 1N NaOH. The lower test concentrations were prepared by subsequent dilutions of the 100 mg/L concentration in test medium.
- Controls: Test medium without test substance or other additives (The pH was adjusted from 8.2 to 6.6 using 1N HCl)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The final test solutions were all clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Species: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation:
Algae stock cultures were started by inoculating growth medium (M1) 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.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2, according to OECD 201) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
- Start of the test: 22.4°C.
- During the test: 21.9 - 22.9°C
pH:
- Start of the test: 6.5 - 6.6
- End of the test: 7.3
Nominal and measured concentrations:
- Nominal concentrations (combined limit/range-finding test): 0 (blank control), 0.1, 1.0, 10 and 100 mg/L
- Measured concentrations
0 h: 0* (blank control) and 97.0 mg/L (100 mg/L)
24h: 0* (blank control) and 98.9 mg/L (100 mg/L)
72 h: 0* (blank control) and 100 mg/L (100 mg/L)

*) In the chromatograms, a small peak was observed at the retention time of FDCA. This response was due to carry-over from a previous injection. FDCA was therefore not considered to be present in the nominal 0 mg/L samples.
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass with a capacity of 100 mL and containing 50 mL of test solution
- Type: Capped vessels
- Initial cells density: 1x10^4 cells/ml
- No. of vessels per concentration (replicates): 6 replicates for the highest test concentration and 3 replicates for each lower test concentration
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes, M2 prepared according to OECD 201.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap-water purified by reverse osmosis
- Culture medium different from test medium: Yes (M1). Three days before the start of the test the algal stock culture were 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 in the control and the highest test concentration. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were made on the highest test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 72 to 79 μE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (path length = 20 mm). Algal medium was used as blank. Measurements were performed after 0, 24, 48 and 72 h of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x10
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Measured concentrations: Analysis of the samples taken from the highest test concentration showed that the actual concentration was in agreement with nominal at the start of the test (97%) and remained stable during the test period (100% of nominal at the end of the test). Given these results, effect parameters were based on nominal concentrations.

- Biological results: Statistically significant differences were recorded between the values for growth rate and yield at the highest test concentration when compared to the control group (2 Sample t-Test, α = 0.05). However, the effects were not considered to be biologically relevant because they were less than 10%. The EC50 for yield inhibition (EYC50: 0-72h) was >100 mg/L and NOEC was 100 mg/L.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

- Acceptability of the test:
1. In the control, cell density increased by an average factor of >16 within 2 days;
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%;
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.
Results with reference substance (positive control):
- Potassium dichromate: EC50 for growth rate reduction (ErC50: 0-72h) was 1.9 mg/L (95% confidence interval ranging from 1.3 to 2.7 mg/L). Test was performed in June 2011.
Validity criteria fulfilled:
yes
Conclusions:
The 72h-EC50 for growth rate reduction and yield inhibition was >100 mg/L and the 72h-NOEC was 100 mg/L.
Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to FDCA at the following nominal concentrations: 0 (blank control), 0.1, 1.0, 10 and 100 mg/L. The EC50 for growth rate reduction was >100 mg/L and the 72h-NOEC was 100 mg/L, based on analytically confirmed nominal concentrations. The study is considered to be reliable without restrictions.

Description of key information

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to FDCA. The EC50 for growth rate reduction was >100 mg/L and the 72h-NOEC was 100 mg/L, based on analytically confirmed nominal concentrations. The study is considered to be reliable without restrictions.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to FDCA at the following nominal concentrations: 0 (blank control), 0.1, 1.0, 10 and 100 mg/L. The EC50 for growth rate reduction was >100 mg/L and the 72h-NOEC was 100 mg/L, based on analytically confirmed nominal concentrations. The study is considered to be reliable without restrictions.