Esterase, aryl

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:

5 September 2003 - 4 February 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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 Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class no. 3.1.1.3)

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class no. 3.1.1.3)

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.: PPW 21180
- Expiration date of the lot/batch: 19 March 2012
- Stability under test conditions: The test material and dilutions in water (10% and 100%) are stable for at least 24 hours at room temperature
- Storage condition of test material: - 20⁰C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 10, 22, 46, 100 and 220 mg Total organic solids (TOS)/L
- Sampling method: Samples have been taken at 0 and 48h.
- 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 was dispersed in test medium to give a stock solution of 220 mg TOS/L. Serial dilutions of this stock solution were prepared with test medium to give the nominal test concentrations of 10, 22, 46, 100 and 220 mg TOS/L
- Controls: Test 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°C and a photoperiod of 16 hours light: 8 hours dark. Culture medium was renewed three times each week. Cultures were fed daily with a suspension of the unicellular green alga, Chlorella vulgaris. 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:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

20- 21°C

pH:

7.5-7.8

Dissolved oxygen:

6.1-9.1 mg O2/L

Nominal and measured concentrations:

nominal: 0, 10, 22, 46, 100 and 220 mg TOS/L
Measured: n.d., 0.7, 1.4, 2.8, 4.6 and 235 mg TOS/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jars
- Type (delete if not applicable): loosely covered
- Material, size, fill volume: glass, 250 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

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 and immobile Daphnia were counted 24 and 48 hours after the start of the study.

TEST CONCENTRATIONS
- Range finding study: no

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 235 mg/L

Nominal / measured:

meas. (geom. mean)

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.:

>= 37.4 mg/L

Nominal / measured:

meas. (geom. mean)

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.:

> 235 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: TOS

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 37.4 mg/L

Nominal / measured:

meas. (geom. mean)

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, Cutinase, PPW 21180 was not found to be acutely toxic to Daphnia magna at a mean measured concentration of 235 mg Total Organic Solids (TOS)/L equivalent to 37.4 mg active enzyme protein (aep)/L and thus no 48h EC50 value could be determined.

Executive summary:

The acute toxicity of Cutinase, PPW 21180 to Daphnia magna was assessed under static exposure conditions.

Groups of twenty, first instar Daphnia, less than 24 hours old, were exposed to Cutinase, PPW 21180 dispersed in Elendt M4 medium for 48 hours at nominal concentrations of 10, 22, 46, 100 and 220 mg TOS/L. Geometric mean measured concentrations were 0.70, 1.4, 2.8, 4.6 and 235 mg TOS/L. Nominal test concentrations are based on Total Organic Solid (TOS), of which the test material contained 10.4%. Verification of test concentrations was performed based on enzyme activity. The amount of measured enzyme (ng/g) was then converted to mg TOS/L in order to calculate the achieved measured concentrations. Measured concentrations ranged from 37 – 167% of nominal at 0 hours and from below the limit of detection (< 2%) – 69% of nominal at 48 hours. The reduction in enzyme activity over the 48 hour exposure period is thought to be due to digestion by the test organism. Only the highest testing concentration, 220 mg/L, had sufficient measurable enzyme activity (69%) after 48 hours exposure.

The numbers of immobilised daphnids were recorded for each test and control group after 24 and 48 hours and the following values were determined. All results are expressed in terms of geometric mean measured concentration.

No immobilisation in either the control or the test group was noted throughout the exposure period, therefore neither the no-observed effect concentration (NOEC) nor the EC50 could be calculated because these values lay above that of the highest test concentration.

48h EC50                                           > 235 mg Total Organic Solids (TOS)/L or > 37.4 mg active enzyme protein (aep)/L

“No-observed effect concentration”:   ≥ 235 mg TOS/L or 37.4 mg aep/L