Glucosidase, β-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2011 to 12 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in report

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)

Constituent 2

Reference substance name:

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

Molecular formula:

Not available. See remarks.

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.: PPC31776
- Expiration date of the lot/batch: 07 December 2020
- Stability under test conditions: Stable for at least 24 hours at room temperature.
- Storage condition of test material: Frozen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and 100 mg Total organic solids (TOS) per litre
- Sampling method: At the start from the test, three samples (15 mL) were taken from the freshly prepared control and test media. After 48 hours, the contents of the test vessels from each group were pooled and further three samples (15 mL) were taken for analysis.
- Sample storage conditions before analysis: Frozen

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance (878 mg) was dispersed in dilution medium (1L) in a volumetric flask. The contents of the flask were shaken before poured in to the test vessels.
- Controls: Medium

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Magna
- Strain: Straus
- Source: Institute National de Recherche Chimique Appliqué (IRChA), France
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: Stock cultures are maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature controlled laboratory at nominally 20 ± 2°C. A photperiod of 16 hours light: 8 hours dark, with periods of subdued light at the beginning and end of each light phase. Culture medium was renewed three times each week. Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day. Culture conditions ensure that the stock animals reproduce by parthenogenesis. The day before the start of the test, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the egg-bearing adult Daphnia were removed from the culture vessels and held in a separate holding vessel. These animals were used in the test.
- Feeding during test: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

270 mg/L as CaCO3

Test temperature:

20.4-20.8°C

pH:

7.61-8.05

Dissolved oxygen:

40-99% ASV (air saturation value)

Nominal and measured concentrations:

nominal: 0 and 100 mg TOS/L
At the start of the test, enzyme recovery was 89% of the nominal value. After 48 hours the recovery decreased to 41% of nominal value.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass dishes
- Type (delete if not applicable): loosely covered
- Material, size, fill volume: glass, 120 mL, 100 mL
- Aeration: no
- 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: deionised, reverse osmosis water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark with periods of subdued light at the beginning and at the end of each light phase

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daphnia were considered immobile if they were unable to swim within approximmately 15 seconds following gentle agitation of the test vessel. The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 hours after the start of the study.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1, 10 and 100 mg TOS/L
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS (total organic solids)

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 52.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (aep)

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 52.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

aep

Basis for effect:

mobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, Cellulase was not found to be acutely toxic to Daphnia magna at a nominal concentration of 100 mg TOS/L equivalent to 52.1 mg aep/L.

Executive summary:

The acute toxicity of Cellulase to Daphnia magna was assessed under static exposure conditions. The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Commission Regulation (EC) No 440/2008 Part C, Method 2 “Daphnia sp. Acute Immobilisation Test” and the OECD Guideline for Testing of Chemicals No. 202, “Daphnia Acute Immobilisation Test” (2004).

A group of twenty Daphnia, less than 24 hours old, was exposed for 48 hours to Cellulase at a nominal concentration of 100 mg TOS (Total Organic Solids)/L equivalent to 52.1 mg aep (active enzyme protein)/L. The test medium was prepared in Elendt M4 medium by the direct addition of the test substance to the dilution medium.

At the request of the Sponsor, the test concentration was verified by analysis of the enzyme concentration, which was performed at the Sponsor’s laboratory.

Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours.

No immobilisation or adverse effects on the Daphnia were noted at a nominal concentration of 100 mg TOS/L.

Based on these findings the following values have been estimated:

48-hour EC50 value :                                   >100 mg TOS/L or > 52.1 mg aep/L

“No observed effect concentration” :            ≥ 100 mg TOS/L or 52.1 mg aep/L