Proteinase, Aspergillus acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-03-2017 to 05-01-2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

For industrial enzymes, the PNEC (predicted no effect concentration) value for enzymes in general is considered the highest dose tested for a given endpoint in studies conducted and published by the enzyme industry. The one exception is for some proteases and oxidoreductases, where effects may be observed at lower concentrations. Similarly, although the algal growth inhibition study with sedolisin was not conducted GLP, the 48-hour algal growth inhibitory effect concentration (EC50) for sedolisin was greater than the limit dose of 100 mg/L. The weight-of-evidence indicates that enzymes in general are not or of very low ecotoxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Only nominal concentrations.

GLP compliance:

no

Remarks:

See *justification for type of information'

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of sedolisin (EC no. 848318-58-1, CAS no. 814-114-0, EC name Sedolisin, Enzyme class no 3.4.21.100 )

Molecular formula:

Not available

IUPAC Name:

Active enzyme protein of sedolisin (EC no. 848318-58-1, CAS no. 814-114-0, EC name Sedolisin, Enzyme class no 3.4.21.100 )

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Lot/batch No.: PPF38268
- Expiration date of the lot/batch: 22-april-2025
- Storage condition of test material: Frozen

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations: 10, 30, 50, 70, 100 TOS mg/L.

Test solutions

Vehicle:

no

Details on test solutions:

According to the MicroBiotests Inc SOP.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23°C +/- 2 °C

pH:

8.1 +/- 0.2

Dissolved oxygen:

Media was saturated with air before use

Nominal and measured concentrations:

Nominal concentrations

Details on test conditions:

TEST SYSTEM
- 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

GROWTH MEDIUM
- Standard medium used: yes.
- 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
- 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

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 24.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 10 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

No significant growth inhibition and no abnormalities were observed.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Endopeptidase, batch PPF38268 was tested in concentrations 10, 30, 50, 70, 100 TOS mg/L. It was found to inhibit growth rate at all concentrations tested after 72h exposure, whereas no significant inhibition was observed after 24 and 48h exposure. Inhibition of the growth rate at 72h was found to be approximately 3% at 10 mg TOS/L, 34 % at 30 mg TOS/L, 30% at 50 mg TOS/L, and 12 % at 100 mg TOS/L indicating only slightly toxicity towards algal growth. There was no dose-response relationship and because of the low observed toxicity towards algal growth no ErC50 value could be estimated and must be > 100 mg TOS/L (> 24.7 mg aep/L). The NOEC value was determined to be < 10 mg TOS/L (< 2.5 mg aep/L).

Executive summary:

The purpose of this study was to screen for toxicity of endopeptidase, batch PPF38268 towards algae. A 72h algal growth inhibition test was performed in long cell test vials with Pseudokirchneriella subcapitata. Algae are supplied as de-immobilized algal beads. The Algal tox kit 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 (I%) of algal growth rate compared to the control.

IC50: Calculated/estimated 50% growth inhibition compared to control algae.

Endopeptidase, batch PPF38268 was found to inhibit growth rate at all concentrations tested after 72h exposure, whereas no significant inhibition was observed after 24 and 48h exposure. Inhibition of the growth rate at 72h was found to be approximately 3% at 10 mg TOS/L, 34 % at 30 mg TOS/L, 30% at 50 mg TOS/L, and 12 % at 100 mg TOS/L indicating only slightly toxicity towards algal growth. There was no dose-response relationship and because of the low observed toxicity towards algal growth no ErC50 value could be estimated and must be > 100 mg TOS/L (> 24.7 mg aep/L). The NOEC value was determined to be < 10 mg TOS/L (< 2.5 mg aep/L).