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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-04-2017 to 03-06-2017
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 proteases and oxidoreductases, where effects may be observed at lower concentrations. Similarly, although the daphnia immobilisation test with Sedolisin was not conducted GLP, the 48h effect concentration (EC50) for Sedolisin was greater than the limit dose of 100 mg TOS/L. The weight-of-evidence indicates that enzymes in general are not ecotoxic.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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'

Test material

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: 6.25, 12.5, 25, 50, 100 mg TOS/L

Test solutions

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

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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
20-25°C
Nominal and measured concentrations:
6.25, 12.5, 25, 50, 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: open
- 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

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: mg TOS/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 24.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 100 other: mg TOS/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 24.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, endopeptidase, batch PPF38268 was found not to be toxic to Daphnia magna at a nominal concentration up to 100 mg TOS/L (24.7 mg aep/L) after 48h exposure. Thus, no 48h EC50 could be calculated and must be > 100 mg TOS/L (> 24.7 mg aep/L).
Executive summary:

The purpose of this study was to screen endopeptidase, batch PPF38268, 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 endopeptidase, batch PPF38268 was performed as a screening with a broad range of concentrations 6.25, 12.5, 25, 50, 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, endopeptidase, batch PPF38268 was found not to be toxic to Daphnia magna at a nominal concentration up to l00 mg TOS/L (24.7 mg aep/L) after 48h exposure. Thus, no 48h EC50 could be calculated and must be > 100 mg TOS/L (> 24.7 mg aep/L).