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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 2017 - 27 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
EC No. 2016/266
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Completely soluble in test medium up to 100 mg/L.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control, at t=0, t=24h and t=72h
- Sampling method: 1.8 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: in a freezer (≤-15°C) at the analytical laboratory of the Test Facility
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition method: A 100 mg/L stock solution of test item in test medium was prepared by applying a 21-minute period of ultrasonic waves to accelerate dissolution of the test item. This stock solution was used as highest test concentration and lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.
- After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10E04 cells/mL.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: 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.
- Age of inoculum (at test initiation): Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10E04 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
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
22 - 23°C
pH:
start: 8.0 - 8.1
end: 8.2
Nominal and measured concentrations:
- Nominal concentrations: 0.10, 1.0, 10 and 100 mg/L (combined limit/range-finding test)
- Measured concentrations: only samples taken from the control and the highest concentration, at t=0, t=24h and t=72h, were analysed. At test start and throughout the test the measured concentrations of the 100 mg/L test solution were at the level of nominal (98% on all occasions). Therefore, the effect parameters were expressed based on the analytically confirmed nominal concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 1 x 10E04 cells/mL
- Control end cells density: 188 x 10E04 cells/mL (mean)
- No. of vessels per concentration (replicates): 6 replicates of the highest (limit) concentration; 3 replicates of the lower concentrations
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (M2; according to OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO
- Intervals of water quality measurement: pH - at the beginning and at the end of the test. Temperature - continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 83 to 90 µE.m-2.s-1.
- Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density (t=0, 24h, 48h, 72 h)
- 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 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and extra replicates as a background for the treated solutions. Algal cell densities were measured at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period. Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.
- Other: Appearance of the cells - at the end of the final test microscopic observations were performed on the control and the limit concentration to observe for any abnormal appearance of the algae.


TEST CONCENTRATIONS
Combined limit/range-finding test: 0.1, 1.0, 10 and 100 mg/L
Based on the results a definitive study was not required.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (performed February 2017)
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
Remarks on result:
other: Nominal test concentration confirmed by analysis.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Nominal test concentration confirmed by analysis.
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
Remarks on result:
other: Nominal test concentration confirmed by analysis.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (unusual cell shape, colour differences, flocculation, adherence to test vessels, aggregation of algal cells): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed algal cells when compared to the control.
- Other: No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
- Water parameters (pH and temperature) remianed within the limits prescribed by the test guidelines.
Results with reference substance (positive control):
In the latest sensitivity check, performed in the period 13 - 16 February 2017, an was determined. The sensitivity of the algal culture was within the range determined with the historical data collected at the testing laboratory (0.82 - 2.3 mg/L).

- Results with reference substance valid? yes
- EC50: 72h-ErC50 = 1.2 mg/L (growth rate inhibition) and 72h-EyC50 = 0.43 mg/L (yield inhibition)
- Both EC50 values are within the historical ranges observed for growth rate and yield inhibition.
- Other: Dose-response test - test concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L

Acceptability of the test:

- In the controls, cell density increased by an average factor of at least 16 within 3 days (188).

- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (12%).

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (1.4%).

In addition:

- Constant test conditions (pH and temperature) were maintained throughout the study period.

- The concentration of the substance was satisfactorily maintained throughout the study period.

The study met all validity criteria and is therefore considered valid.

Validity criteria fulfilled:
yes
Conclusions:
The 72h-ErC50 of the substance to the freshwater algae Pseudokirchneriella subcapitata was determined to be >100 mg/L, based on analytically confirmed nominal test concentrations.
The 72h-ErC10 and 72h-NOErC were >100 mg/L and 100 mg/L, respectively.
Executive summary:

In a study performed in accordance with OECD 201 (2011) and according to GLP principles, the toxicity of the substance to the freshwater algae Pseudokirchneriella subcapitata was investigated. In a combined limit/range-finding test algae were exposed for 72 hours to an untreated control and nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L (control and 100 mg/L - 6 replicates; 0.10, 1.0 and 10 mg/L - 3 replicates).

As the substance was completely soluble in test medium up to 100 mg/L, a stock solution of the substance at 100 mg/L was prepared (applying a 21 minute period of ultrasonication to accelerate dissolution of the test item). This stock solution was used as highest test concentration and lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Samples for analytical confirmation of actual exposure concentrations were taken at t = 0, 24 and 96 hours from all test levels, but only the control and the highest concentration samples from these sampling times were analysed. The measured concentration in the highest test concentration was found to be at the level of the nominal concentration throughout the test (i.e. 98% of nominal). Since the test concentration remained stable during the test (101% of initial) the effect parameters were reported in terms of the analytically confirmed nominal concentration.

No significant differences in cell densities, and thus growth rate inhibition rates and yield inhibition, were recorded between the test concentrations and the control group. Therefore, the 72h-ErC50 of the substance to Pseudokirchneriella subcapitata is concluded to be >100 mg/L, based on analytically confirmed nominal test concentrations. The 72h-ErC10 and 72h-NOErC values are >100 mg/L and 100 mg/L, respectively.

The study met the OECD validity criteria.

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information