Lipase, triacylglycerol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2003 - 4 February 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in report

Analytical monitoring:

yes

Details on sampling:

- Nominal concentrations: 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L

- Sampling method: Triplicate 5 mL samples were taken from the control and each test concentration at 0 and 72 hours (fresh media), and 24 and 96 hours (expired media)
- Sample storage conditions before analysis: Frozen until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Individual weighings of the test material were dispersed directly in diluent water to give nominal test concentrations of 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L.
- Controls: Medium

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Donnington Fish Farm, Gloucestershire, UK
- Length at study initiation (mean) : 4.2 ± 0.35 cm
- Weight at study initiation (mean wet weight): 1.11 ± 0.29 g
- Feeding during test: no

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): Same as for the test.
- Type and amount of food: Commercial fish food fry pellet; Fish were fed 2% of their body weight.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): No medication given during the holding period and mortalities were recorded as below 1% in the 7 days before the definitive test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

145-206 mg/L as CaCO3

Test temperature:

13-14°C

pH:

7.0 - 7.9

Dissolved oxygen:

8.5 - 9.9 mg O2/L

Salinity:

-

Nominal and measured concentrations:

nominal: 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L
Mean measured: 7.5, 27.4, 78.7, 218.2, 479.3 mg enzyme concentrate dry matter/L

In samples of freshly prepared media, enzyme recovery ranged from 61 to 177% of the nominal value at 0 hours, 51-191% of nominal at 24 hours, 84-206% of nominal at 72 hours and from 57 to 187% of nominal at 96 hours.

The actual measured concentration was generally higher than the expected concentration of the Lipase enzyme protein in the solutions. The only exception is the dose solution 11.9 mg enzyme concentrate dry matter/L which is lower than expected and 26.2 mg enzyme concentrate dry matter/L, which equaled the expected concentration.
The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to some minor discrepancy from expected results. The standard curve in the ELISA runs were within the acceptance criteria set for the method and therefore accepted.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium
- Material, fill volume, depth: glass, 20L, 19 cm
- Aeration: yes
- Renewal rate of test solution (frequency): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.4 g bodyweight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered, dechlorinated and softened by passage through Vivendi water purification system. The water passes through a high grade activated carbon filter to remove chlorine and organic contaminants. A proportion of the supply then passes through a water softener before final reverse osmosis treatment. These two supplies are then re-mixed in the ratio of approximately 2 parts reverse osmosis treated water to 1 part carbon filtered water to give the desired water hardness.
- Total organic carbon: 3 mg/L
- Particulate matter: 290 mg/L
- Metals: <0.02 mg/L
- Pesticides: <0.01 µg/L
- Chlorine: 0.02 to 0.04 mg/L
- Alkalinity: CaCO3 97 mg/L
- Ca/mg ratio: 14.25
- Conductivity: 480 µS/cm

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations on mortality at 15 minutes and 2,4, 24, 48, 72 and 96 hours and in addition subjective assessment were made on type and incidence of sub-lethal effects compared with control fish.
The criteria for death employed in this study were absence of respiratory movement and absence of response to physical stimulation of the caudal peduncle.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 0.1, 1.2 and 12.4 mg enzyme concentrate dry matter/L
- Results used to determine the conditions for the definitive study: yes, results of range finding indicated that the LC50 was likely to be >12.4 mg enzyme concentrate dry matter/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 262.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other:

Remarks:

enzyme concentrate dry matter

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

54.8 mg/L

Nominal / measured:

nominal

Conc. based on:

other:

Remarks:

enzyme concentrate dry matter

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: At nominal cocnentrations of 119.2 and 262.3 mg enzyme concentrate dry matter/L coughing, swimming at the base of the tank and resting on the base of the tank was observed.
- Mortality of control: no
- Other adverse effects control: no

Sublethal observations / clinical signs:

The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to some minor discrepancy from expected results. The standard curve in the ELISA runs were within the acceptance criteria set for the method and therefore accepted. The results are based on the nominal concentrations - which are the lower values - as a worse case.

Validity criteria fulfilled:

not specified

Conclusions:

No mortality in either the control or the test groups was noted throughout the exposure period, therefore the EC50 could not be calculated. Under the conditions of the test, the 96 hour LC50 value for Lipase, batch PPW 21180 with rainbow trout was > 262.3 mg enzyme concentrate dry matter/L equivalent to 37.4 mg active enzyme protein (aep)/L.
The 'no-observed effect concentration' for Lipase, batch PPW 21180 with rainbow trout was 54.8 mg enzyme concentrate dry matte/L, equivalent to 7.8 mg aep/L.
The values are based on the nominal concentrations - which are the lower values - as a worse case.

Executive summary:

A study was performed to assess the acute toxicity of Lipase, batch PPW 21180 to rainbow trout (Oncorhynchus mykiss) under semi-static conditions.

Groups of seven juvenile fish were exposed to a range of concentrations, nominally 11.9, 26.2, 54.8, 119.2 and 262.3 mg enzyme concentrate dry matter/L of Lipase, batch PPW 21180 dispersed in water. Mean measured concentrations were 7.5, 27.4, 78.7, 218.2, 479.3 mg enzyme concentrate dry matter/L. Observations were made on the numbers of dead fish and the incidence of sub-lethal effects after 15 minutes and 2, 4, 24, 48, 72 and 96 hours exposure.

Verification of test concentrations was performed based on enzyme activity. Measured concentrations ranged from 61-177% of nominal at 0 hours, 51-191% of nominal at 24 hours, 84-206% of nominal at 72 hours and from 57 -187% of nominal at 96 hours. At the three highest nominal test concentrations, 54.8, 119.2 and 262.3 mg enzyme concentrate dry matter/L, the initial measured concentrations were relatively high, between 136 and 206% of nominal. With the exception of the 72 to 96 hour period at 11.9 mg enzyme concentrate dry matter/L, the enzyme appeared very stable throughout the period between medium renewal (maintained 84 to 111% of initial concentration). The slight loss of enzyme activity during the 72 to 96 hour period at 11.9 mg enzyme concentrate dry matter/L, 68% recovery, was thought to be due to biodegradation caused by the accumulation of bacteria which would naturally occur in the test system over the course of the test. The effect of biodegradation was insufficient to affect the higher test concentrations. The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to some minor discrepancy from expected results. The standard curve in the ELISA runs were within the acceptance criteria set for the method and therefore accepted. However, as a worse-case scenario, the lower values from the nominal concentrations will be used.

No mortality in either the control or the test groups was noted throughout the exposure period, therefore the EC50 could not be calculated. The the 96 hour LC50 value for Lipase, batch PPW 21180 with rainbow trout was > 262.3 mg enzyme concentrate dry matter/L equivalent to 37.4 mg active enzyme protein (aep)/L.

The 'no-observed effect concentration' for Lipase, batch PPW 21180 with rainbow trout was 54.8 mg enzyme concentrate dry matte/L, equivalent to 7.8 mg aep/L.

Description of key information

No mortality in either the control or the test groups was noted throughout the exposure period, therefore the EC50 could not be calculated. Under the conditions of the test, the 96 hour LC50 value for Lipase, batch PPW 21180 with rainbow trout was > 262.3 mg enzyme concentrate dry matter/L equivalent to 37.4 mg active enzyme protein (aep)/L.

The 'no-observed effect concentration' for Lipase, batch PPW 21180 with rainbow trout was 54.8 mg enzyme concentrate dry matte/L, equivalent to 7.8 mg aep/L.

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