Dehydratase, carbonate

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:

26 September 2022 to January 11 2023

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Experiment 2 only one replicate for the treatment groups.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Experiment 2 only one replicate for the treatment groups.

Principles of method if other than guideline:

Only nominal concentrations.

GLP compliance:

yes

Remarks:

non-GLP study

Analytical monitoring:

no

Details on sampling:

- Concentrations:
Experiment 1: 42.3, 84.6, 169.2, 338.3, 676.6 mg enzyme concentrate dry matter/L.
Experiment 2: 8.4, 25.1, 75.2, 225.5, 676.6 mg enzyme concentrate dry matter/L.

Vehicle:

no

Details on test solutions:

According to the MicroBiotests Inc SOP.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): MicroBiotests Inc. The algal growth inhibition bioassay has been developed by the research teams at the Laboratory for Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium.
- Method of cultivation: According to the MicroBiotests Inc. SOP.
Algae are supplied as de-immobilized algal beads.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

As described in OECD Guideline 201and the ISO/DIS Guideline 8692.

Test temperature:

As described in OECD Guideline 201and the ISO/DIS Guideline 8692.

pH:

7.2-7.6

Nominal and measured concentrations:

Nominal concentrations

Details on test conditions:

TEST SYSTEM
Experiment 1:
- Test vessel: 25 mL calibrated flask
- Material, size, headspace, fill volume: 25 mL
- Initial cells density: 1*10^4 algae/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

Experiment 2:
Due to precipitations seen in experiment 1, the batch was sterile filtered in experiment 2. 3x 100 mL vials for the controls, 1X 100 mL vials for each test concentration. Vials were loosely covered with parafilm and were under constant shaking.

GROWTH MEDIUM
- Standard medium used: yes. An amount of 250 mg sodium bicarbonate will be added to the algae medium, before preparing the test concentrations, to account for the CO2 quenching mechanism of Carbonic anhydrase.
- Detailed composition if non-standard medium was used: As described in OECD Guideline 201and the ISO/DIS Guideline 8692.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 72 h
- Light intensity and quality: 10000 lux for sideway illumination or min. 3000-4000 lux for bottom illumination

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

59.4 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Details on results:

In experiment 1, there were a lot of precipitates from the batch present, and it was difficult to distinguish under the microscope between algae cells and precipitates. The batch was sterile filtered in experiment 2, and the beakers were under constant shaking and containing 250 mg sodium bicarbonate. This helped with algae cells counting. Triplicates for the control group and single replicates for the treatment groups.

Validity criteria fulfilled:

yes

Conclusions:

The exposure of R. subcapitata to Carbonic anhydrase, batch PPE93985 was toxic to algae based on its algaestatic, CO2 quenching mode of action. 250 mg sodium bicarbonate and constant shaking were introduced to the test system. 72-hr EC50 was 59.4 mg enzyme concentrate dry matter, equivalent to 8.8 mg active enzyme protein/L. The study is non-GLP and analytical measurements have not been undertaken.

Executive summary:

The purpose of this study was to screen for toxicity of Carbonic anhydrase, batch PPE93985 towards algae. A 72h algal growth inhibition test was performed in long cell test vials with R. subcapitata, followed by a second experiment in 100 mL vials by constant shaking. Algae are supplied as de-immobilized algal beads. The Algal toxkit test follows the prescriptions of the OECD "Algal growth inhibition test" (Guideline 201) and the ISO "Water Quality - Freshwater Algal Growth Inhibition Tests with Unicellular Green Algae" (ISO Standard 8692). Endpoint of the study is percent inhibition of algal growth rate compared to the control.

Due to precipitations seen in experiment 1, the batch was sterile filtered in experiment 2. 3x 100 mL vials for the controls, 1X 100 mL vials for each test concentration. Vials were loosely covered with parafilm and were under constant shaking. Concentrations used were:
Experiment 1: 42.3, 84.6, 169.2, 338.3, 676.6 mg enzyme concentrate dry matter/L.
Experiment 2: 8.4, 25.1, 75.2, 225.5, 676.6 mg enzyme concentrate dry matter/L.

The exposure of R. subcapitata to Carbonic anhydrase, batch PPE93985 was toxic to algae based on its algaestatic, CO₂ quenching mode of action. 250 mg sodium bicarbonate and constant shaking were introduced to the test system in experiment 2. 72-hr EC50 was 59.4 mg enzyme concentrate dry matter, equivalent to 8.8 mg active enzyme protein/L. The study is non-GLP and analytical measurements have not been undertaken.

Description of key information

The exposure of R. subcapitata to Carbonic anhydrase, batch PPE93985 was toxic to algae based on its algaestatic, CO2 quenching mode of action. 250 mg sodium bicarbonate and constant shaking were introduced to the test system. 72-hr EC50 was 59.4 mg enzyme concentrate dry matter, equivalent to 8.8 mg active enzyme protein/L. The study is non-GLP and analytical measurements have not been undertaken.

Key value for chemical safety assessment

EC50 for freshwater algae:

59.4 mg/L