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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 SEP 2021 - 08 NOV 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 266/2016, Annex Part C, C.3: „Freshwater Alga and Cyanobacteria, Growth Inhibition Test“

Version / remarks:

published in the Official Journal of the European Union L 54 of 01 March 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 23rd March, 2006 (Annex 5 corrected: 28 July 2011).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-UV method

Details on sampling:

- Concentrations: 100 mg/L nominal concentration
- Sampling method: Based on the results of the preliminary test, only a single treatment concentration at saturation (i.e. solubility limit of the test item in the test medium) and an untreated control group were tested in a limit test.
The test item is poorly water soluble material. In order to achieve the maximum solubility level in the test medium the test solution was prepared as follows: a supersaturated solution (1000 mg/L nominal loading) was prepared by dispersing/dissolving the appropriate test item amount into the test medium (OECD medium). This solution was agitated by magnetic stirrer for at least two days and then the non-dissolved test material was separated by filtration through a membrane filter (pore size: 0.22 μm) in order to obtain the saturated test solution. Untreated control ran in parallel in the test. Since the test item has a high absorptive potential (log Pow = 8.22) also, the vessels used for the preparation of the test solution as well as the test vessels was preconditioned with the respective test solution for at least two days.
- Sample storage conditions before analysis: After the formulation procedure, algal cells were immediately introduced into the test solutions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item is poorly water soluble material. In order to achieve the maximum solubility level in the test medium the test solution was prepared as follows: a supersaturated solution (1000 mg/L nominal loading) was prepared by dispersing/dissolving the appropriate test item amount into the test medium (OECD medium). This solution was agitated by magnetic stirrer for at least two days and then the non-dissolved test material was separated by filtration through a membrane filter (pore size: 0.22 μm) in order to obtain the saturated test solution. Untreated control ran in parallel in the test. Since the test item has a high absorptive potential (log Pow = 8.22) also, the vessels used for the preparation of the test solution as well as the test vessels was preconditioned with the respective test solution for at least two days.
- Controls:
Untreated Control: The dilution water (OECD Medium; see section 5.5) without addition of test item was used as untreated control solution.
Reference Control: For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany

ACCLIMATION
- Culturing media and conditions (same as test or not): The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 3 days. The algal cultures used in this study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted algal growth medium (OECD Medium, according to OECD 201) was used as dilution water in the experiment.

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

The temperature was between 22.4 and 23.4 °C measured in the flask and in the range of 22.1 – 23.9 °C measured within the climate chamber.

pH:

The range of the pH was 7.86 – 9.09 during the experiment. The pH of the control medium did not increase by more than 1.5 units during the test.

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentration: < LOQ (i.e. <0.05 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass Erlenmeyer flasks of ~250 mL volume filled with 100 mL test medium. Each test vessel was uniquely identified with study number, test group and replicate.
- Type (delete if not applicable): closed (the test flasks were covered with air-permeable stoppers.)
- Aeration: no
- Initial cells density: The initial cell density was about 10^4 cells/mL, in each test flask.
- No. of vessels per concentration (replicates): six replicates
- No. of vessels per control (replicates): six replicates

GROWTH MEDIUM
- Standard medium used: yes Reconstituted algal growth medium (OECD Medium, according to OECD 201) was used as dilution water in the experiment.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations in the prepared algal growth medium.

OTHER TEST CONDITIONS
- Adjustment of pH: The test was performed without pH adjustment.
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: 7516 lux, which was ensured with fluorescent lamps (spectral range of 400 - 700 nm).

TEST CONCENTRATIONS
- Range finding study: Algal cells were exposed to the treatment and a control group for 72 hours using three replicates, respectively.
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of the preliminary test, only a single treatment concentration at saturation (i.e. solubility limit of the test item in the test medium) and an untreated control group were tested in a limit test.

Reference substance (positive control):

yes

Remarks:

Name: Potassium dichromate (K2Cr2O7) Batch No.: K50498364 CAS No.: 7778-50-9 Expiry Date: 31 July 2023 Supplier: MERCK KGaA

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield, growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield, growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield, growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield, growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield, growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield at the treatment group (saturated test solution; equivalent to 100 mg/L nominal concentration) was not statistically significantly different from the untreated control.
Accordingly, the 72-h NOEC based on growth rate and yield was determined at least as the saturation concentration (equivalent to 100 mg/L nominal concentration). The 72-h LOEC and EC50 values were determined to be higher than saturation (equivalent to > 100 mg/L nominal concentration).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
The 72h ErC50: 0.62 mg/L (95 % confidence limits: 0.59 – 0.65 mg/L)
The 72h EyC50: 0.33 mg/L (95 % confidence limits: 0.32 – 0.34 mg/L)

Reported statistics and error estimates:

The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates in the treatment group and for the untreated control.

Validity of the Test:

- Cell density in the control cultures increased exponentially by a factor of more than 16 within 72 hours. Cell density increased by a factor of 123.7, this corresponds to a specific growth rate of 1.61 day^-1.
- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures did not exceed 35 %. The mean CV for section-by-section specific growth rates: 10.1 %.
- The coefficient of variation (CV) of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % in test. The CV for average specific growth rate (day 0-3): 1.6 %

Validity criteria fulfilled:

yes

Conclusions:

In this 72-hour algal growth inhibition test with Raphidocelis subcapitata the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the EC50 and the LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L. All validity criteria were met.

Executive summary:

This toxicity to aquatic algae and cyanobacteria study was performed according to OECD Guideline 201 under GLP compliance. Based on the results of a non-GLP preliminary test Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata, Strain number: 61.81 SAG) were tested in a limit test using only this concentration level and a control in order to demonstrate that the test item is not toxic to algae up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates in the treatment group and for the untreated control.

All validity criteria were met and therefore, the study was considered to be valid. The measured concentrations of the test item treated group were below the LOQ (i.e. < 0.05 mg/L) at each analytical occasion. Test item was not detected in the control samples. The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield in the treatment group (saturated solution, equivalent to 100 mg/L nominal concentration) was not statistically significantly different from the control. Accordingly, the 72-h NOEC (for growth rates and yield) was determined to be at least the saturation concentration (i.e. 100% v/v saturated solution, equivalent to 100 mg/L nominal concentration) and the LOEC and the EC50 values were determined to be higher than saturation.

In conclusion, in this 72-hour algal growth inhibition test with Raphidocelis subcapitata the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the EC50 and the LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L.

Description of key information

Key, toxicity to aquatic algae and cyanobacteria, OECD 201, static, limit-test, GLP: EC₅₀ (72h) > 100 mg/L nominal, analytical concentration <LOQ (0.05 mg/L) for Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

This toxicity to aquatic algae and cyanobacteria study was performed according to OECD Guideline 201 under GLP compliance. Based on the results of a non-GLP preliminary test Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata, Strain number: 61.81 SAG) were tested in a limit test using only this concentration level and a control in order to demonstrate that the test item is not toxic to algae up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates in the treatment group and for the untreated control.

All validity criteria were met and therefore, the study was considered to be valid. The measured concentrations of the test item treated group were below the LOQ (i.e. < 0.05 mg/L) at each analytical occasion. Test item was not detected in the control samples. The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield in the treatment group (saturated solution, equivalent to 100 mg/L nominal concentration) was not statistically significantly different from the control. Accordingly, the 72-h NOEC (for growth rates and yield) was determined to be at least the saturation concentration (i.e. 100% v/v saturated solution, equivalent to 100 mg/L nominal concentration) and the LOEC and the EC50 values were determined to be higher than saturation.

In conclusion, in this 72-hour algal growth inhibition test with Raphidocelis subcapitata the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the EC50 and the LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L.