Mucorpepsin

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

20 July 2020 to 08 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Based on a GLP study with hydrolase beta-N-acetylhexosaminidase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted: 23 March 2006
Annex 5 corrected: 28 July 2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

Only the highest concentration could be measured, as the other concentrations fell below the assay detection limit. Therefore, nominal concentrations were used.

Details on sampling:

Only the highest concentration could be measured, as the other concentrations fell below the assay detection limit. Therefore, nominal concentrations were used. Samples were prepared in the presence and absence of algae.
- Concentrations: 260 mg enzyme contrate dry matter/L (equivalent to 100 mg active enzyme protein/L)
- Sample storage conditions before analysis: During the duration of the test, the samples were treated the same as the test concentrations. Thereafter, the samples were stored frozen until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dilution in medium
- Controls: ASTM medium

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Original cultures obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
- Acclimation period: Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 10^3 cells/mL.
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

ASTM medium

Test temperature:

23-24⁰C

pH:

7.7-8.6 in control,
7.3-8.6 in test concentrations

Details on test conditions:

TEST SYSTEM
- Test vessels: 250 ml conical flasks containing 100 ml test solution and loosely stoppered.
- Type: Polyurethane foam bungs.
- Aeration: None. Gaseous exchange and suspension of algal cells maintained by orbital shaker.
- Initial cells density: 5*10^3 cells/mL
- Control end cells density: 5.79*10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Method of initiation: Serial dilutions with the test substance were prepared and inoculated with algae suspension to achieve the desired concentration.

GROWTH MEDIUM
- Standard medium used: ASTM medium

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Photoperiod: Continuous (approximately 7000 lux).
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Light microscope. It was not possible to monitor algal cells using a Coulter® Multisizer Particle Counter due to dispersed material interfering with the Coulter counts.

TEST CONCENTRATIONS
- Concentrations: 16.3, 32.6, 65.2 130.4 and 260.7 mg enzyme concentrate dry matter/L.

Medium renewal: None.
Duration of exposure: 72 hours.
Measurement of growth: Light microscope. It was not possible to monitor algal cells using a Coulter® Multisizer Particle Counter due to dispersed material interfering with the Coulter counts.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

101.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enyzme concentrate dry matter

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 260.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

65.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enyzme concentrate dry matter

Basis for effect:

growth rate

Details on results:

Mean cell density of control @ 0 h: 0.5 x 10^3 cells/mL
Mean cell density of control @ 72 h: 5.79*10^5 cells/mL

- Exponential growth in the control: yes
- Observation of abnormalities:
- Unusual cell shape: No
- Color differences: No
- Flocculation: No
- Adherence to test vessels: no
- Aggregation of algal cells: Cell clumping was observed to be present in the test cultures at 32.6, 65.2, 130.4 and 260.7 mg enzyme concentrate dry matter/L.
- Any stimulation of growth found in any treatment: Stimulation was observed at the two lowest concentrations 16.3, 32.6 mg enzyme concentrate dry matter/L.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Raphidocelis subcapitata to beta-N-acetylhexosaminidase revealed an ErC50 > 260.7 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). NOEC for both growth rate and yield was 65.2 mg enzyme concentrate dry matter/L and LOEC was 130.4 mg enzyme concentrate dry matter/L. Yield EbC50 was 101.7 mg enzyme concentrate dry matter/L.

Executive summary:

A study was performed to assess the effect of beta-N-acetylhexosaminidase on the growth of the green algae Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.

At the request of the Sponsor, Raphidocelis subcapitata was exposed to aqueous dispersions of the test item at nominal concentrations of 16.3, 32.6, 65.2, 130.4 and 260.7 mg enzyme concentrate dry matter/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at temperature of 24 ± 1 °C.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a hemocytometer and light microscope.

Analysis of the test preparations was conducted by the Sponsor. Only the highest concentration could be measured, as the other concentrations fell below the assay detection limit. Therefore, nominal concentrations were used in this study. The results for the highest concentration were within 20% of the nominal concentrations. Similar results were seen for samples with and without algae for the highest concentration.

Exposure of Raphidocelis subcapitata to beta-N-acetylhexosaminidase revealed an ErC50 > 260.7 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). NOEC for both growth rate and yield was 65.2 mg enzyme concentrate dry matter/L and LOEC was 130.4 mg enzyme concentrate dry matter/L. Yield EbC50 was 101.7 mg enzyme concentrate dry matter/L.

Description of key information

Mucorpepsin has not been tested with algae, but another hydrolase was used as read-across.

Exposure of Raphidocelis subcapitata to beta-N-acetylhexosaminidase revealed an ErC50 > 260.7 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). NOEC for both growth rate and yield was 65.2 mg enzyme concentrate dry matter/L and LOEC was 130.4 mg enzyme concentrate dry matter/L. Yield EbC50 was 101.7 mg enzyme concentrate dry matter/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

62.5