Cellulase

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 March 2011 - 13 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 4.7, 10.3, 22.8, 50.1, 110.1 mg enzyme concentrate dry matter/L
- Sampling method: Three, mid-vessel samples (15mL) of media were taken from the control and test vessels at 0 and 72 hours (fresh media) and at 24 and 96 hours (expired media) for analysis.
- 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: The test substance, which is stored frozen, was defrosted overnight at approximately 4°C. Once defrosted the sample will be gently mixed before weighing to ensure homogeneity. The test substance was dispersed in 1L diluent water (17.6g, 7.9g, 3.65g, 1.65g or 0.750 g) in a volumetriuc flask. The contents of each were shaken vigorousley before being poured into a test vessel (glass aquarium) and the volume adjusted to 20 litres with diluent water. Test media were freshly prepared on each day of the definitive test.
- 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: Commercial fish farm in the UK and were reared at the farm from eggs hatched in July 2011
- Length at study initiation (mean) : 4.92 cm
- Weight at study initiation (mean wet weight): 1.05g
- Feeding during test: no

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): The stock of fish were held in aerated supply of diluent water under flow -through conditions until required. During the 14-day period prior to the definitive test, temperatures remained within the range 14.2 to 14.4°C, pH values within the range 7.90 to 7.99, dissolved oxygen concentrations within the range 82 to 95% air saturation value (ASV) and total hardness within the range 153 to 175 mg/L as CaCO3.
- Type and amount of food: Commercial fish food (TROUW (UK) Ltd., Nutra Fry 02); Fish were fed an amount equivalent to between 1.5 and 3% of the total wet-weight of fish in the holding tank.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): No medication given during the holding period and no mortality occured during the 14 days before the efinitive test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

121-152 mg/L as CaCO3

Test temperature:

12.8 - 14.2°C

pH:

7.89 - 8.59

Dissolved oxygen:

77-108 % ASV

Salinity:

-

Nominal and measured concentrations:

nominal: 0, 4.7, 10.3, 22.8, 50.1 and 110.1 mg enzyme concentrate dry matter/L
In samples of freshly prepared media, enzyme recovery ranged between 67 and 102% of the nominal value and in samples of expired (24-hour-old) media between 71 and 110% of the nominal value.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium
- Material, fill volume, depth: glass, 20L, 18.5 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: 0.369 g bodyweight/litre

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered dechlorinated tap water blended with tap water that had been softened and subsequently treated by reverse osmosis to a hardness of 150-200 mg/L as CaCO3.
- Total organic carbon: 0.92 mg/L
- Particulate matter: <3.0 mg/L

- Organochlorine pesticides: < 0.04 µg/L
- Chloride: 13 mg/L
- Conductivity: 300 µS/cm (20°C)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Analysis of a sample taken in March 2011

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations on mortality and subjective assessments on the incidence and type of sub-lethal effects compared with control fish were made at approximately 2,4, 24, 48, 72 and 96 hours.
The criteria of death em

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 1.1, 11.0 and 110.1 mg enzyme concentrate dry matter/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 110.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

50.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: At 110.1 mg enzyme concentrate dry matter/L lethargy was observed with fish to be quiescent both at the surface of the media and at the base of the test vessel. Incidences of aggression were observed at lower exposure concentrations but are not considered to be a treatment related effect.
- Mortality of control: no
- Other adverse effects control: no

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the test, Cellulase, batch PPC31776 was not found to be acutely toxic to rainbow trout (Oncorhynchus mykiss). Consequently, the 96-hour LC50 value for Cellulase could not be calculated and must be >110.1 mg enzyme concentrate dry matter/L (equivalent to 52.1 mg active enzyme protein/L). The "no-observed effect concentration" was 50.1 mg enzyme concentrate dry matter/L (equivalent to 23.7 mg active enzyme protein/L).

Executive summary:

A study was performed to assess the acute toxicity of Cellulase, batch PPC31776 to rainbow trout (Oncorhynchus mykiss) under semi-static conditions with daily renewal of the media.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 1992) Part C, Method 1 “Acute Toxicity For Fish” and the OECD Guideline for Testing of Chemicals No. 203 “Fish, Acute Toxicity Test” (1992).

Groups of seven juvenile fish were exposed to Cellulase, dispersed in water at nominal concentrations of 4.7, 10.3, 22.8, 50.1 and 110.1 mg enzyme concentrate dry matter/L. The test media were prepared by direct addition of the test substance to water.

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

Observations of the fish were made after approximately 2, 4, 24, 48, 72 and 96 hours of exposure. After 96 hours, the highest concentration at which no mortality had occurred was 50.1 mg enzyme concentrate dry matter/L (equivalent to 23.7 mg active enzyme protein/L). At 110.1 mg enzyme concentrate dry matter/L (52.1 mg aep/L), mortality was recorded as 14% (one fish) and treatment-related effects were observed.

Based on these findings the following values have been estimated:

96-hour LC50 value: > 110.1 mg enzyme concentrate dry matter/L (equivalent to 52.1 mg aep/L)

“No observed effect concentration”: 50.1 mg enzyme concentrate dry matter/L (equivalent to 23.7 mg active enzyme protein/L).

Description of key information

Under the conditions of the test, Cellulase, batch PPC31776 was not found to be acutely toxic to rainbow trout (Oncorhynchus mykiss). Consequently, the 96-hour LC50 value for Cellulase could not be calculated and must be >110.1 mg enzyme concentrate dry matter/L (equivalent to 52.1 mg active enzyme protein/L). The "no-observed effect concentration" was 50.1 mg enzyme concentrate dry matter/L (equivalent to 23.7 mg active enzyme protein/L).