Lipase, triacylglycerol

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:

5 September 2003 - 4 February 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in report

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L
- Sampling method: Samples have been taken at 0 and 48h.
- Sample storage conditions before analysis: Frozen

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 262.3 mg enzyme concentrate dry matter/L. Serial dilutions of this stock solution were prepared with test medium to give the nominal test concentrations of 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L
- Controls: Test medium

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

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20- 21°C

pH:

7.5-7.8

Dissolved oxygen:

6.1-9.1 mg O2/L

Nominal and measured concentrations:

nominal: 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L
Measured: 0.8, 1.7, 3.3, 54.8, 280.2 mg enzyme concentrate dry matter/L
The measured results from the dose solutions were not as expected. The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to minor discrepancies from expected results.

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

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 262.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 262.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

The measured results from the dose solutions were not as expected. The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to minor discrepancies from expected results.

The values are based on the nominal concentrations - which are the lower values - as a worse case.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, Lipase, batch PPW 21180 was not found to be acutely toxic to Daphnia magna up to 262.3 mg enyzme concentrate dry matter/L (equivalent to 37.4 mg active enzyme protein/L) and thus no 48-hr EC50 value could be determined and must be over the highest tested concentration. NOEC was 262.3 mg enyzme concentrate dry matter/L (equivalent to 37.4 mg active enzyme protein/L). The values are based on the nominal concentrations - which are the lower values - as a worse case.

Executive summary:

The acute toxicity of Lipase, batch PPW 21180 to Daphnia magna was assessed under static exposure conditions. Groups of twenty, first instar daphnids, less than 24 hours old, were exposed to Lipase, batch PPW 21180 dispersed in Elendt M4 medium for 48 hours at nominal concentrations of 11.9, 26.2, 54.8, 119.2 and 262.3 mg enzyme concentrate dry matter/L. Geometric mean measured concentrations were 0.8, 1.7, 3.3, 54.8 and 280.2 mg enzyme concentrate dry matter/L. Verification of test concentrations was performed based on enzyme activity. The amount of measured enzyme (ng/g) was then converted to enzyme concentrate dry matter 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 nominal testing concentration, 280.2 mg enzyme concentrate dry matter/L, had sufficient measurable enzyme activity (69%) after 48 hours exposure. The measured results from the dose solutions were not as expected. The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to minor discrepancies from expected results. The values are based on the nominal concentrations - which are the lower values - as a worse case.

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 nominal concentration.

No immobilisation in either the control or the test group was noted throughout the exposure period, therefore the EC50 could be calculated because these values lay above that of the highest test concentration.

48-hr EC50:                                        > 262.3 mg enzyme concentrate dry matter/L (equivalent to > 37.4 mg active enzyme protein/L)

“No-observed effect concentration”:    262.3 mg TOS/L (equivalent to 37.4 mg aep/L)

Description of key information

Under the conditions of the test, Lipase, batch PPW 21180 was not found to be acutely toxic to Daphnia magna up to 262.3 mg enyzme concentrate dry matter/L (equivalent to 37.4 mg active enzyme protein/L) and thus no 48-hr EC50 value could be determined and must be over the highest tested concentration. NOEC was 262.3 mg enyzme concentrate dry matter/L (equivalent to 37.4 mg active enzyme protein/L). The values are based on the nominal concentrations - which are the lower values - as a worse case.