Proteinase, Bacillus subtilis metallo-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-05-2009 to 24-06-2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

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 proteases and oxidoreductases, where effects may be observed at lower concentrations.The protease bacillolysins EC50 was between 46 and 100 mg TOS/L. The weight-of-evidence indicates that enzymes in general are not or of low ecotoxicity.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

no

Remarks:

See'Justification for type of information'

Analytical monitoring:

no

Details on sampling:

- Concentrations: 4.6, 10, 21, 46, 100 mg TOS/L

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

Test temperature:

20-25°C

Nominal and measured concentrations:

4.6, 10, 21, 46, 100 mg TOS/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

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 46 - < 100 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

other: Total organic Solids

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 3.6 - < 7.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

aep

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

21 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

other: Total organic solids

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

aep

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, bacillolysin batch PPA23881 tested with Daphnia magna, revealed:
46 mg TOS/L (3.6 mg aep/L) < 48-h EC50 < 100 mg TOS/L (7.8 mg aep/L) (nominal concentrations).
Therefore, 48-h EC50 value was calculated to be > 46 mg TOS/L (3.6 mg aep/L). 48-h NOEC was calculated to be 21 mg TOS/L (1.6 mg aep/L).

Executive summary:

The purpose of this study was to screen bacillolysin batch PPA23881 for the test material concentration estimated to immobilise 50% of theDaphnia magnaafter 24 and 48 hours exposure. DAPHTOXKIT F™ magna was supplied by MicroBioTests Inc.

The testing was performed as a screening with a broad range of concentrations 4.6, 10, 21, 46, 100 mg TOS/L. 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, bacillolysin batch PPA23881 tested withDaphnia magna, revealed:

46 mg TOS/L (3.6 mg aep/L) < 48-h EC50 < 100 mg TOS/L (7.8 mg aep/L) (nominal concentrations).

Therefore, 48-h EC50 value was calculated to be > 46 mg TOS/L (3.6 mg aep/L). 48-h NOEC was calculated to be 21 mg TOS/L (1.6 mg aep/L).

Description of key information

Under the conditions of the test, bacillolysin batch PPA23881 tested with Daphnia magna, revealed:

46 mg TOS/L (3.6 mg aep/L) < 48-h EC50 < 100 mg TOS/L (7.8 mg aep/L) (nominal concentrations).

Therefore, 48-h EC50 value was calculated to be > 46 mg TOS/L (3.6 mg aep/L). 48-h NOEC was calculated to be 21 mg TOS/L (1.6 mg aep/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.6 mg/L