Mucorpepsin

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

20 July 2020 to March 11, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is another hydrolase with similar results to Mucorpepsin.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Please state version

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:

- 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 water
- Controls: Elendt M4 medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

Daphnia magna

Details on test organisms:

Common name: Daphnia magna
Strain: Straus
Source: In-house laboratory cultures. A clone of this species was originally supplied by the University of Sheffield, UK.
Age at study initiation: Gravid adults were isolated 24 hours prior to initiation of the test. Young daphnids produced overnight were used for testing.
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L CaCO3

Test temperature:

21-22°C

pH:

8.0-8.2 in control
7.3-8.2 in test concentrations

Dissolved oxygen:

8.6-8.8 mg O2/L in controls
6.1-8.8 mg O2/L

Nominal and measured concentrations:

16.3, 32.6, 65.2, 130.4 and 260.7 mg enzyme concentrate dry matter/L (nominal concentrations)
The sample 260.7 mg enzyme concentrate dry matter/L in Elendt M4 medium at 0 hours achieved 90.2% recovery, but fell below the limit of detection, and therefore, cannot be supported as a result. The respective sample in reverse osmosis water attained a recovery of 96.4%. The 48-hour old samples fell below the detection limit, which is close to the highest concentration.

Details on test conditions:

TEST SYSTEM
- Test vessels: 150 mL glass vessels each containing approximately 100 mL test solution.
- Experimental design: 20 animals per test concentration. 5 test concentrations plus 1 control. Duplicate samples were prepared for enzyme activity analysis and stored in the test concentrations.
- Type: covered to reduce evaporation
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium. Total hardness 250 mg/L as CaCO3.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
Method of initiation: Daphnia were placed in the vessels containing the test preparations.
Loading: 20 mL test solution per organism.
Photoperiod: 16 h light: 8 h dark with with 20 minute dawn and dusk transition periods.
Medium renewal: None.
Duration of exposure: 48 hours.
Criterion of effect: Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

EFFECT PARAMETERS MEASURED: The numbers of mobile, immobile and floating daphnia were counted approximately 24 and 48h after the start of the study.
TEST CONCENTRATIONS
16.3, 32.6, 65.2, 130.4 and 260.7 mg enzyme concentrate dry matter/L.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 260.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

260.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Details on results:

Details on results
- Mortality of control: 0
- Other adverse effects control: No
- Abnormal responses: No
- Effect concentrations exceeding solubility of substance in test medium: No

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to beta-N-acetylhexosaminidase based on nominal concentrations gave EC50 value of greater than 260.7 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). The No Observed Effect Concentration (NOEC) was the highest concentration tested, 260.7 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L).

Executive summary:

A study was performed to assess the acute toxicity of beta-N-acetylhexosaminidase to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

20 daphnids (4 replicates of 5 animals) were 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 for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized Daphnia was recorded after 24 and 48 hours.

Analysis of the test preparations at 0 and 48 hours were performed 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.

Exposure of Daphnia magna to beta-N-acetylhexosaminidase based on nominal concentrations gave EC50 value of greater than 260.7 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). The No Observed Effect Concentration (NOEC) was the highest concentration tested, 260.7 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Study period:

01.03.2018 - 23.03.2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

Remarks:

No analytical quantifications.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dilution in media (Standard Freshwater ISO 6341).
- Controls: Standard Freshwater ISO6341.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna Straus
- Source: MicroBioTests Inc. Daphnia magna have been developed by the research teams at the Laboratory for Biological Research in Aquatic Pollution (LABRAP) at the Ghent University in Belgium.
- Age at study initiation (mean and range, SD): less than 24 hours
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

225

Test temperature:

20-25°C

pH:

7.29 in the highest exposure concentration
Test media: 7.98

Nominal and measured concentrations:

21.3, 42.5, 85.0, 170.1, 340.2 mg enzyme concentrate dry matter/L nominal.

Details on test conditions:

TEST SYSTEM
The test was conducted according to DAPHTOXKIT F MAGNA Crustacean Toxicity Screening Test for Freshwater with a test system supplied by MicroBioTests Inc.
http://www.microbiotests.be/SOPs/Daphtoxkit%20magna%20F%20SOP%20-%20A5.pdf

- Test vessel: The bioassays are conducted in disposable multiwell test plates with 30 test wells.
- Type: closed
- Material, size, headspace, fill volume: 10 mL
- Aeration: The Standard Freshwater (ISO medium) must be aerated for at least 15 minutes prior to using it.
- No. of organisms per vessel: 20 (5 daphnia per well)
- No. of vessels per concentration and control (replicates): Each plate is provided with 4 wells for the controls and 4 wells (A,B,C,D) for each toxicant concentration.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium, formula according to ISO 6341

Composition :
vial 1 : NaHCO3 (129.5 mg - dissolved in 2 L = 67.75 mg/L)
vial 2 : CaCl2.2H2O (588 mg - dissolved in 2 L = 294 mg/L)
vial 3 : MgSO4.7H2O (246.5 mg - dissolved in 2 L = 123.25 mg/L)
vial 4 : KCl (11.5 mg - dissolved in 2 L = 5.75 mg/L)

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

398 mg/L

Nominal / measured:

nominal

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 42.5 mg/L

Nominal / measured:

nominal

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

mobility

Details on results:

Details on results
- Mortality of control: 0
- Other adverse effects control: No
- Abnormal responses: No
- Effect concentrations exceeding solubility of substance in test medium: No
Daphnia were considered to be immobile if they were unable to swim within 15 seconds following gentle touch of the animal with a pin.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the 48h EC50 value for mucorpepsin batch PPR 3583 was determined to 398 mg enzyme concentrate dry matter/L, equivalent to 117 mg active enzyme protein/L and the no-observed-effect concentration 42.5 mg enzyme concentrate dry matter/L, equivalent to 12.5 mg active enzyme protein/L.

Executive summary:

The purpose of this study was to screen mucorpepsin batch PPR3583 for the test material concentration estimated to immobilise 50% of the Daphnia magna after 24 and 48 hours exposure. DAPHTOXKIT F™ magna was supplied by MicroBioTests Inc. The testing of the mucorpepsin batch PPR3583 was performed as a screening with a broad range of concentrations 21.3, 42.5, 85.0, 170.1, 340.2 mg enzyme concentrate dry matter/L nominal. The acceptance criterion for a valid test is that the spontaneous immobility in the control is below or equal to 10%.

Under the conditions of the test, the 48h EC50 value for mucorpepsin batch PPR 3583 was determined to 398 mg enzyme concentrate dry matter/L, equivalent to 117 mg active enzyme protein/L and the no-observed-effect concentration 42.5 mg enzyme concentrate dry matter/L, equivalent to 12.5 mg active enzyme protein/L.

Description of key information

Under the conditions of the test, the 48h EC50 value for mucorpepsin batch PPR 3583 was determined to 398 mg enzyme concentrate dry matter/L, equivalent to 117 mg active enzyme protein/L and the no-observed-effect concentration 42.5 mg enzyme concentrate dry matter/L, equivalent to 12.5 mg active enzyme protein/L.

Read-across from beta-N-acetylhexosaminidase (another hydrolase) gave similar results.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

mg enzyme concentrate dry matter/L

Effect concentration:

398 mg/L