lysophospholipaseIUBMB 3.1.1.5

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

read-across based on grouping of substances (category approach)

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

Justification for type of information:

Read across: Due to the similarity of the two enzymes, it can be concluded that similar results will seen for lysophospholipase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in report

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 22, 46, 100 and 220 mg TOS/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 10, 22, 46, 100 and 220 mg TOS/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: 0, 10, 22, 46, 100 and 220 mg TOS/L
Mean measured: n.d., 6.3, 23, 66, 183 and 402 mg TOS/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.

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:0.104, 1.04 and 10.4 mg TOS/L
- Results used to determine the conditions for the definitive study: yes, results of range finding indicated that the LC50 was likely to be >10.4 mg TOS/L.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 402 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: Total organic solids (TOS)

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 68.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (aep)

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

66 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: TOS

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

11.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks:

aep

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: At nominal 100 and 220 mg TOS/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

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the test, the 96 hour LC50 value for cutinase PPW 21180 with rainbow trout was > 402 mg TOS/L or 68.3 mg active enzyme protein (aep)/L.
The 'no-observed effect concentration' for cutinase PPW 21180 with rainbow trout was 66 mg TOS/L or 11.2 mg aep/L.

Executive summary:

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

Groups of seven juvenile fish were exposed to a range of concentrations, nominally 10, 22, 46, 100 and 220 mg Total Organic Solid (TOS) /L of Cutinase, PPW 21180 dispersed in water. Mean measured concentrations were 6.3, 23, 66, 183 and 402 mg TOS/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.

Nominal test concentrations are based on 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 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, 46, 100 and 220 mg/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 10 mg TOS/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 10 mg TOS/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.

No mortality in either the control or the test groups was noted throughout the exposure period, therefore the EC50 could not be calculated. The following results are expressed in terms of mean measured concentration:

96 h LC50                                                            > 402 mg TOS/L or 68.3 mg active enzyme protein/L

‘No-observed effect concentration’ (NOEC)              66 mg TOS/L or 11.2 mg active enzyme protein/L

Description of key information

The short-term toxicity of lysophospholipase to fish was not conducted, however, it was conducted in a similar enzyme, cutinase.

Under the conditions of the test, the 96 hour LC50 value forcutinase, PPW 21180 with rainbow trout was >402 mg Total Organic Solids (TOS)/L or 68.3 mg active enzyme protein (aep)/L.
The 'no-observed effect concentration' for cutinase, PPW 21180 with rainbow trout was 66 mg TOS/L or 11.2 mg aep/L.

Given the similarity between the enzymes, it is expected that lysophospholipase will have similar short-term toxicity values to fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

68.3 mg/L