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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-02-09 to 1995-08-30
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
change of study monitor in april 1995
GLP compliance:
yes (incl. certificate)
Certificate included in study report
Analytical monitoring:
Details on sampling:
- Concentrations: 0 and 100 mg TOS/L
- Sampling method: Three, mid-vessel samples (10 mL) of media were taken from the control and test vessel at 0 and 72 hours (fresh media) and at 24 and 96 hours (expired media) for analysis.
- Sample storage conditions before analysis: Samples were stored frozen at -20°C
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The test substance (19.14 g) was dispersed in diluent water (2L) in a volumetric flask. The contents of the flask were shaken vigorously before being poured into a test vessel (glass aquarium). The volume in the test vessel was then adjusted to a final volume of 20 litres with diluent water.
- Controls: diluent water
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Commercial fish farm in the UK and were reared at the farm from eggs hatched in January 2010.
- Length at study initiation (length definition, mean, range and SD): mean total lenght 5.9 cm
- Weight at study initiation (mean and range, SD): mean wet weight 1.89 g

- Acclimation period: 7 days (range finding) and 14 days (definitive test)
- Type and amount of food: commercial fish food (TROUW (UK) Ltd.; Nutra Fry 02)
- Health during acclimation (any mortality observed): No medication was given during the holding period and no mortality occured the 14 days before the definitive test.
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
148 - 156 mg/L as CaCO3
Test temperature:
13.1 - 14.4°C
6.13 - 6.17
Dissolved oxygen:
84 - 111 % ASV (air saturation value)
Nominal and measured concentrations:
nominal: 0 and 100 mg/L
measured: fresh samples were between 30 and 70% of the nominal value
test media taken at the end of the 24 hour renewal periods were between 59 and 105% of the nominal value
Details on test conditions:
- Test vessel: glass aquarium
- Fill volume: 20 L test medium
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): 24h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

- Source/preparation of dilution water: The water used to hold the fish and for the study was laboratory tap water, dechlorinated and softened by passage through a water purification system. It was passed through a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passed through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water were then remixed to give a supply with the desired water hardness. This water was then held in an intermediate tank where it was equilibrated to the test temperature and gently aerated before being supplied to the holding and test areas.

- Photoperiod: 16 hours light: 8hours dark with periods of subdued light at the beginning and end of each light phase.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were observed after 2, 24, 48, 72 and 96 hours test duration for symptoms of intoxication and mortality.

- Range finding study: 1, 10 and 100 mg TOS/L
- Results used to determine the conditions for the definitive study: yes. No adverse were observed during range finding study and thus a limit test was performed.
Reference substance (positive control):
96 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
other: Total Organic Soilids (TOS)
Basis for effect:
Key result
96 h
Dose descriptor:
Effect conc.:
22.2 mg/L
Nominal / measured:
Conc. based on:
act. ingr.
active enzyme protein
Basis for effect:
Details on results:
- Behavioural abnormalities: no
- Mortality of control: 0
- Other adverse effects control: One fish exhibited darkened pigmentation at a nominal concentration of 100 mg TOS/L after
24 hours exposure which was evident throughout the test; but this was not considered to be of biological relevance.
- Abnormal responses: no
Validity criteria fulfilled:
not specified
The No-observed-effect concentration was the highest exposure concentration of 100 mg TOS/L corresponding to 22.2 mg active enzyme protein/L.
Executive summary:

A study was performed to assess the acute toxicity of Xylanase, PPQ 30189 to rainbow trout (Oncorhynchus mykiss) under semi-static conditions with daily renewal of the media. The active component of the test substance is an enzyme, which was known to be pH sensitive with reduced enzymatic activity under alkaline conditions. In order to maintain the stability of the enzyme the dilution water used in the study was adjusted to pH 6 before use.

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).

A group of ten juvenile fish was exposed to Xylanase, PPQ 30189 dispersed in water at a nominal concentration of 100 mg TOS (Total Organic Solids)/L; the test medium was 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, no mortality or significant sub-lethal effects on the fish were observed at a nominal concentration of 100 mg TOS/L (22.2 mg aep/L). Based on these findings the following values have been estimated: 96-hour LC50 value : >100 mg TOS/L (22.2 mg aep/L) “No observed effect concentration” : 100 mg TOS/L (22.2 mg aep/L).

Description of key information

Xylanase was not toxic at the tested concentration of 100 mg TOS/L (22.2 mg aep/L) and thus the NOEC was determined to be 100 mg/L (22.2 mg aep/L).The LC50 value could not be determnined due to the absence of toxicity of the test compound at the tested concentration.

Key value for chemical safety assessment

Additional information

The acute toxicity of xylanase to Rainbow Trout (Oncorhynchus mykiss) was determined in a 96 hour semistatic test with a daily test medium renewal according to the OECD Guideline No. 203 and in compliance with GLP. 

A limit test was performed to demonstrate that the test substance has no toxic effect on the test animals up to the concentration of nominal 100 mg test substance/L corresponding to 22.2 mg active enzyme protein (aep)/L.

The 96h NOEC was determined to be 100 mg/L corresponding to 22.2 mg aep/L (nominal). The 96h LC50 value could not be determined due to the absence of toxicity of the test substance but is clearly higher than 100 mg/L (22.2 mg aep/L)(nominal).