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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:
From 13 November 2015 to 23 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
(2011)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2009)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Solubility in water: completely miscible
- Stability in water: stable
Analytical monitoring:
yes
Details on sampling:
- Concentrations sampled: all test concentrations (and the control), at t=0 and t=72h
- Volume: 2.4 mL
- At the end of the exposure period, the replicates were pooled at each concentration before sampling
- Sample storage conditions before analysis: in a freezer
Vehicle:
no
Details on test solutions:
The test substance was completely miscible in test medium at the concentrations tested. No correction was made for the purity/composition of the test substance. Preparation of test solutions started with a concentration of 100 mg/L (combined limit/range-finding test) and 10 mg/L (final test), respectively. No special treatment other than careful mixing was necessary to completely dissolve the test substance in medium. Lower test concentrations were prepared by subsequent dilutions in test medium. All final test solutions were clear and colourless. 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 10^4 cells/mL.
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: In-house laboratory culture
- 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 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:
21 - 22°C
pH:
7.9 -8.1
Nominal and measured concentrations:
0.10, 0.32, 1.0, 3.2 and 10 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 188.1 x 10^4 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per concentration without algae (replicates): 1

GROWTH MEDIUM: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

TEST MEDIUM: M2; according to OECD 201, formulated using Milli-RO water

OTHER TEST CONDITIONS
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 83 to 88 μE.m-2.s-1
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

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, at t=24h, at t=48h and at t=72h, cell densities were determined by spectrophotometric measurements of samples at 680 nm using a spectrophotometer with immersion probe (path length = 20 mm). Algal medium was used as blank.
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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Combined limit/range-finding test:
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
-- At 100 mg/L, there was 100% inhibition of growth rate
-- At 10 mg/L, there was 80% inhibition of growth rate
-- At 1.0 mg/L, there was 35% inhibition of growth rate
Reference substance (positive control):
yes
Remarks:
(sensitivity check using potassium dichromate)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.84 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 2.67 - 3.03 mg/L; Concentrations remained stable during the 72-hour exposure period (102-105% of initial).
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.32 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 0.28-0.37 mg/L
Details on results:
- Measured test substance concentrations: At the start of the test, the actual test concentrations were 0.11, 0.42, 1.2, 3.7 and 12 mg/L. The concentrations remained stable during the 72-hour exposure period (102-105% of initial). As the initial concentrations partly deviated by more than 20% of nominal, the initial measured concentrations were used for determining the effect concentrations.
- Growth rate inhibition was found to be statistically significant for all exposure concentrations. However, exposure to 0.42 mg/L and less did not cause more than 10% effect, and were considered biologically irrrelevant. The NOEC for this endpoint was thus 0.42 mg/L. As the NOEC is higher than the EC10, it is not assigned as a key result.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
In the latest sensitivity check, performed in the period 14 - 17 December 2015, an 72h-EC50 of 1.5 mg/L for growth rate inhibition 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).

Inhibition of growth rate:

- 12 mg/L: 77.5%

- 3.7 mg/L: 58.4%

- 1.2 mg/L: 32.5%

- 0.42 mg/L: 6.8% (with exclusion of 1 replicate)

- 0.11 mg/L: 4.1%

Validity criteria fulfilled:
yes
Conclusions:
In a study performed in accordance with OECD 201 (2011) and according to GLP principles, an 72h-EC50 of 2.84 mg/L for growth rate was determined for the substance, based on initial measured concentrations. The 72h-EC10 for growth rate was determined to be 0.32 mg/L.
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. A final test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control and three replicates per group were exposed to nominal concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L. Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. At the start of the test, the actual test concentrations were 0.11, 0.42, 1.2, 3.7 and 12 mg/L. The concentrations remained stable during the 72-hour exposure period (102-105% of initial). As the initial concentrations partly deviated by more than 20% of nominal (and remained stable during the exposure period), the initial measured concentrations were used for determining the effect concentrations. The study met the acceptability criteria. Growth rate inhibition was found to be statistically significant for all exposure concentrations. Under the conditions of the present study, the 72h-EC50 and 72h-EC10 for growth rate were 2.84 and 0.32 mg/L, respectively.

Description of key information

In a study performed in accordance with OECD 201 (2011) and according to GLP principles, an 72h-EC50 of 2.84 mg/L for growth rate was determined for the analogue substance, based on initial measured concentrations. The 72h-EC10 for growth rate was determined to be 0.32 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
2.84 mg/L
EC10 or NOEC for freshwater algae:
0.32 mg/L

Additional information

In a study performed in accordance with OECD 201 (2011) and according to GLP principles, the toxicity of the analogue substance to the freshwater algae Pseudokirchneriella subcapitata was investigated. A final test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control and three replicates per group were exposed to nominal concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L. Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. At the start of the test, the actual test concentrations were 0.11, 0.42, 1.2, 3.7 and 12 mg/L. The concentrations remained stable during the 72-hour exposure period (102-105% of initial). As the initial concentrations partly deviated by more than 20% of nominal (and remained stable during the exposure period), the initial measured concentrations were used for determining the effect concentrations. The study met the acceptability criteria. Growth rate inhibition was found to be statistically significant for all exposure concentrations. Under the conditions of the present study, the 72h-EC50 and 72h-EC10 for growth rate were 2.84 and 0.32 mg/L, respectively.