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Toxicity to aquatic algae and cyanobacteria

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:

From January 30, 2012 to February 02, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Specific details on test material used for the study:

The test substance was a clear green to brown highly viscous liquid.

Analytical monitoring:

yes

Details on sampling:

Determination of the concentrations, Oudhoff, 2012.
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the Analytical Report (APPENDIX 4) (Oudhoff, 2012).
Frequency: at t=0 h, t=24 h and t=72 h. Volume: 3 mL from the approximate centre of the test vessels. Storage: samples were stored in a freezer until analysis. At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with individually prepared loading rates of 1.0, 10 and 100 mg/L applying a 1-hour period of magnetic stirring followed by a 0.5-hour settlement period. The clear and colourless Water Accommodated Fractions (WAFs) were subsequently siphoned and used as test solutions. 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):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Strain: NIVA CHL 1. Source: in-house laboratory culture. Reason for selection: this system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species. Stock culture: algae stock cultures were started by inoculating growth medium 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. Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. Stock culture medium: M1; according to the NPR 6505. Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use. Pre-culture medium M2; according to the OECD 201 Guideline.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

-

Post exposure observation period:

no

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

21.5 - 22.6

pH:

8.1 +/- 0.2

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Measured and calculated concentrations

Details on test conditions:

Test vessels. 100 ml, all-glass, containing 50 ml of test solution. Medium: M2. Cell density: an initial cell density of 1 x 10E04 cells/ml. Illumination: continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 67 to 74 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.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (Potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

other: NOErC

Effect conc.:

6.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: highest dose tested

Remarks:

This corresponds to a nominal concentration of 100 mg/L.

Key result

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

> 6.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: highest dose tested

Remarks:

This corresponds to a nominal concentration of 100 mg/L.

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 6.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: highest dose tested

Remarks:

This corresponds to a nominal concentration of 100 mg/L.

Duration:

72 h

Dose descriptor:

other: EYC10

Effect conc.:

< 6.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield inhibition

Remarks on result:

other: there was 17% inhibition at the highest dose tested

Remarks:

This corresponds to a nominal concentration of 100 mg/L.

Key result

Duration:

72 h

Dose descriptor:

other: EYC50

Effect conc.:

> 6.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield inhibition

Remarks on result:

other: highest dose tested

Remarks:

This corresponds to a nominal concentration of 100 mg/L.

Details on results:

The test substance inhibited the yield of this fresh water algae species significantly at a TWA concentration of 6.8 mg/l being the highest concentration tested. No reduction of growth rate was recorded at any of the concentrations of the test substance tested.
The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition (EYC50: 0-72h) were beyond the range tested, i.e. exceeded a TWA concentration of 6.8 mg/L. The NOEC for growth rate reduction was 6.8 mg/L.
Due to the very low solubility of the test substance in water, concentration levels that might be toxic for algae could not be reached.

Results with reference substance (positive control):

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher. The EC50 for growth rate reduction (ERC50: 0-72h) was 1.8 mg/L with a 95% confidence interval ranging from 1.4 to 2.4 mg/L. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range. The EC50 for yield inhibition (EYC50: 0-72h) was 0.69 mg/L with a 95% confidence interval ranging from 0.50 to 0.95 mg/L. The historical ranges of the 72h-EC50 for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Samples:

Analysis of the sample taken from the highest concentration at the start of the test showed a measured concentration of 11 mg/L. The actual concentration dropped to 60% and further to 51% of the initial after 24 and 72 hours of exposure, respectively. Similar results were obtained for the highest WAF incubated without algae. The Time Weight Average concentration was calculated to be 6.8 mg/L.

Reduction of growth rate and inhibition of yield:

No significant differences were recorded between the values for growth rate at any of the test concentrations when compared to the control group. Yield was significantly inhibited (17%) in the WAF prepared at a loading rate of 100 mg/L, i.e. at a TWA concentration of 6.8 mg/L (Bonferroni t-test, α = 0.05). Note that solutions were slightly hazy from t=24h in the replicates of the highest WAF. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72h ErC50 (growth rate) and EYC50 (yield inhibition) (Pseudokirchneriella subcapitata) of the test substance were determined to be above the TWA concentration of 6.8 mg/L (being the highest concentration tested). As the values were beyond the range tested, i.e. exceeded a TWA concentration of 6.8 mg/L, the NOEC for growth rate reduction was 6.8 mg/L.

Executive summary:

A study was conducted to determine the toxicity of the test substance to aquatic algae according to OECD Guideline 201, EU Method C.3 and ISO 8692. Pseudokirchneriella subcapitata were exposed for 72 h to WAFs prepared at loading rates of 0 (control), 1.0, 10 and 100 mg/L. Cell densities were evaluated at 0, 24, 48 and 72 h. Samples for analytical dose verification were taken from all dose groups at test start, then after 24 and 72 h. A positive control (potassium dichromate) was included in the study. Analysis of the sample taken from the highest concentration at the start of the test showed a measured concentration of 11 mg/L. The actual concentration dropped to 60% and further to 51% of the initial after 24 and 72 h of exposure, respectively. Similar results were obtained for the highest WAF incubated without algae. The Time Weight Average (TWA) concentration was calculated to be 6.8 mg/L. No significant differences were recorded between the values for growth rate at any of the test concentrations when compared to the control group. Yield was significantly inhibited (17%) in the WAF prepared at a loading rate of 100 mg/L (6.8 mg/L (TWA)). Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control. Under the study conditions, the 72h ErC50 (growth rate) and EYC50 (yield inhibition) for Pseudokirchneriella subcapitata were determined to be > 6.8 mg/L (TWA). Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher (Tobor-Kaplon, 2012).

Description of key information

The 72h ErC50 (growth rate) and EYC50 (yield inhibition) for Pseudokirchneriella subcapitata were determined to be > 6.8 mg/L (TWA; corresponding to nominal concentrations of 100 mg/L).

Additional information

A study was conducted to determine the toxicity of the test substance to aquatic algae according to OECD Guideline 201, EU Method C.3 and ISO 8692. Pseudokirchneriella subcapitata were exposed for 72 h to WAFs prepared at loading rates of 0 (control), 1.0, 10 and 100 mg/L. Cell densities were evaluated at 0, 24, 48 and 72 h. Samples for analytical dose verification were taken from all dose groups at test start, then after 24 and 72 h. A positive control (potassium dichromate) was included in the study. Analysis of the sample taken from the highest concentration at the start of the test showed a measured concentration of 11 mg/L. The actual concentration dropped to 60% and further to 51% of the initial after 24 and 72 h of exposure, respectively. Similar results were obtained for the highest WAF incubated without algae. The Time Weight Average (TWA) concentration was calculated to be 6.8 mg/L. No significant differences were recorded between the values for growth rate at any of the test concentrations when compared to the control group. Yield was significantly inhibited (17%) in the WAF prepared at a loading rate of 100 mg/L (6.8 mg/L (TWA)). Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control. Under the study conditions, the 72h ErC50 (growth rate) and EYC50 (yield inhibition) for Pseudokirchneriella subcapitata were determined to be > 6.8 mg/L (TWA). Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher (Tobor-Kaplon, 2012).