Amylase, gluco-

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:

20 December 2005 - 02 November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Range finding test used concentrations 1.04, 10.4, 103.6 and 227.9 mg enzyme concentrate dry matter/L. The definitive test concentration was 227.9 mg enzyme concentrate dry matter/L.
- Sampling method: Triplicate samples (5mL) of media were taken from the control and each test vessel at 0 and 72 hours (fresh media) and at 24 and 96 hours (expired media) for analysis.
- Sample storage conditions before analysis: frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A sample of the test material weighing 31.68 g was dispersed directly in 20 litres of diluent water to give the nominal test concentration of 227.9 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: Westacre Trout Farm, Norfolk, UK
- Length at study initiation (length definition, mean, range and SD): mean standard length: 4.9cm (SD = 0.34cm)
- Weight at study initiation (mean and range, SD): mean weight: 1.89g (SD = 0.45g)
- Feeding during test: no

ACCLIMATION
- Acclimation period: 12 day
- Acclimation conditions (same as test or not):The stock of fish was obtained from the supplier on 15 February 2006 and acclimatised to test conditions from this date. Holding water temperature remained within the range 13 to 14°C and dissolved oxygen within the range 8.3 to 9.2 mg O2/L in the 12-day period immediately before the study with a 16 hour light: 8 hour dark photoperiod.
- Type and amount of food: The fish were fed 2% of their body weight daily with commercial fry pellet.
- Feeding frequency: daily until 24 hours before exposure, but food was not given during the 24-hour period immediately before exposure or during the exposure period itself.
- Health during acclimation (any mortality observed): No mortality

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

147-173 mg CaCO3/L

Test temperature:

13-14°C

pH:

7.0-7.8

Dissolved oxygen:

7.1-8.2 mg O2/L

Nominal and measured concentrations:

range finding: nominal: 1.04, 10.4, 103.6 and 227.9 mg enzyme concentrate dry matter/L; no measurements
limit test:
nominal concentrations: 0 and 227.9 mg enzyme concentrate dry matter/L
Measured concentrations:
Mean: 250.7 mg enzyme concentrate dry matter/L (Measured concentrations ranged from 117.5% of nominal at 0 hours and 24 hours, 102.9% of nominal at 72 hours and 102.5% of nominal at 96 hours). The test substance could not be detectet in control samples.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria
- Material, size, headspace, fill volume: glass, size: 24x45x24 cm, fill volume: 20 litres
- Aeration: supplementary aeration through narrow bore glass tubes.
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.95 g bodyweight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for the study was laboratory tap water filtered, dechlorinated and softened by passage through a Vivendi water purification system. The water was passed through a highgrade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply was then passed through a water softener before final reverse osmosis (RO) treatment to produce a highly purified water supply. These two supplies were then remixed in the appropriate ratio to give a laboratory supply with the desired water hardness
- Total organic carbon: 5 mg/L
- Chlorine: 0.01-0.05 mg/L (free chlorine 0.00 - 0.02 mg/L)
- Conductivity: 500 uS/cm
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light: 8 hours dark

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Range finding study
- Test concentrations: 1.04, 10.4, 103.6 and 227.9 mg enzyme concentrate dry matter/L (preliminary test)
- Results used to determine the conditions for the definitive study: results from range finding study were used.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 227.9 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.:

227.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

other: visual observation for lethal and treatment-related effects

Details on results:

- Behavioural abnormalities: no
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Validity criteria fulfilled:

not specified

Conclusions:

No mortality was observed following the exposure of rainbow trout to the test substance at a concentration of 227.9 mg enzyme concentrate dry matter/L. Due to the low toxicity of the test material, the 96-hour LC50 value of the substance was unable to be calculated. Therefore, the 96-hour LC50 value for the test substance with rainbow trout was determined to be > 227.9 mg enzyme concentrate dry matter/L (equivalent to >103.7 mg active enzyme protein/L).
The no-observed effect concentration (NOEC) was 227.9 mg enzyme concentrate dry matter/L (equivalent to 103.7 mg active enzyme protein/L).

Executive summary:

A study was performed to assess the acute toxicity of Glucoamylase, batch PPY 24900 to rainbow trout (Oncorhynchus mykiss) under semi-static conditions.

The study was conducted in accordance with OECD and EC guidelines, and in compliance with GLP. A group of ten juvenile fish were exposed to a single test concentration, nominally 227.9 mg enyzme concentrate dry matter/L of Glucoamylase, batch PPY 24900 dispersed in dechlorinated water.

Verification of test concentrations was performed based on enzyme activity. The amount of measured enzyme (AGU/g) was then converted to mg enzyme concentrate dry matter/L in order to calculate the achieved measured concentrations. Measured concentrations ranged from 117.5% of nominal at 0 hours and 24 hours, 102.9% of nominal at 72 hours and 102.5% of nominal at 96 hours). The test substance could not be detectet in control samples. These results confirmed that the nominal exposure concentration was achieved and maintained satisfactorily for the 24-hour duration between each medium renewal. Mean measured concentration was 250.7 mg enzyme concentrate dry matter/L. The results were based on nominal concentrations as a worse-case.

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.

No mortality was observed following the exposure of rainbow trout to the test substance at a concentration of 227.9 mg enzyme concentrate dry matter/L. Due to the low toxicity of the test material, the 96-hour LC50 value of the substance was unable to be calculated. Therefore, the 96-hour LC50 value for the test substance with rainbow trout was determined to be > 227.9 mg enzyme concentrate dry matter/L (equivalent to >103.7 mg active enzyme protein/L).

The no-observed effect concentration (NOEC) was 227.9 mg enzyme concentrate dry matter/L (equivalent to 103.7 mg active enzyme protein/L).

Description of key information

No mortality was observed following the exposure of rainbow trout to the test substance at a concentration of 227.9 mg enzyme concentrate dry matter/L. Due to the low toxicity of the test material, the 96-hour LC50 value of the substance was unable to be calculated. Therefore, the 96-hour LC50 value for the test substance with rainbow trout was determined to be > 227.9 mg enzyme concentrate dry matter/L (equivalent to >103.7 mg active enzyme protein/L).

The no-observed effect concentration (NOEC) was 227.9 mg enzyme concentrate dry matter/L (equivalent to 103.7 mg active enzyme protein/L).