Proteinase, Aspergillus acid

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August - 24 November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

except for the analysis of the test material

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Subtilisin (EC no. 232-752-2, CAS no. 9014-01-1, EC name: Subtilisin, Enzyme Class no. 3.4.21.62)

IUPAC Name:

Active enzyme protein of Subtilisin (EC no. 232-752-2, CAS no. 9014-01-1, EC name: Subtilisin, Enzyme Class no. 3.4.21.62)

Constituent 2

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Substance type: UVCB
- Physical state: liquid
- Storage condition of test material: deep freezer (ca. -20°C) in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Range finding test used concentrations 1, 10 and 100 mg TOS/L. The definitive test concentrations were 6.25, 12.5, 25, 50 and 100 mg TOS/L.
- Sampling method:Triplicate samples (10mL) 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

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The test media were freshly prepared each day during the definitive test. As the test substance was supplied as a frozen liquid, an aliquot of the test substance was thawed on each occasions and discarded after use. At each concentration the test substance (1.54, 3.08, 6.16, 12.33 or 24.66 g) was dispersed in diluent water (5L). The contents of the flask were gently inverted thirty times to mix contents before being poured into test vessel (glass aquarium) and the volumes adjusted to 18 litres with diluent water.
- Controls: Medium

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Strain: Oncorhoynchus mykiss
- Source: commercial fish farm in the UK
- Length at study initiation (length definition, mean): 5.84cm (4-6cm)
- Weight at study initiation (mean): 1.73g
- Feeding during test: no


ACCLIMATION
- Acclimation period: 11 days
- Acclimation conditions (same as test or not): the fish were gradually acclimated to the conditions of the study by gradual replacement of the holding tank water with pH adjusted diluent water.To maintain the levels of dissolved oxygen in the holding tank, the water was gently aerated, which resulted in an increase in pH levels between the additions of water. Each day during the seven day period before the definitive test, the pH of the holding water was returned to approximately 6.1-6.3 by the addition of pH adjusted diluent water.
- Type and amount of food: commercial fish food (TROUW (UK) Ltd., Nutra Fry 02); amount equivalent to 1% of the total wet-weight in the holding tank
- Feeding frequency: daily until 24 hours before exposure
- Health during acclimation (any mortality observed): no

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

150-200 mg CaCO3/L

Test temperature:

15 ± 2°C

pH:

6-6.5

Dissolved oxygen:

73-104 % air saturation value

Nominal and measured concentrations:

The nominal and measured concentrations for the definitive test were:
nominal: 6.25, 12.5, 25, 50 and 100 mg TOS/L
measured: 3.22, 10.2, 21.0, 49.2, 133 mg TOS/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium
- Material, size, headspace, fill volume: glass, depth 17 cm, 18 litres of medium,
- Aeration: aeration provided via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: initial static loading of 0.67 g body weight/litre

- 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: 0.82 mg/L
- Chlorine: 0.05 mg/L (free chlorine 0.03 mg/L)
- Conductivity: 330 µS/cm
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: to approximately 6-6.5
- 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 approximately 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: 2
- Range finding study
- Test concentrations: 1, 10 and 100 mg TOS/L
- Results used to determine the conditions for the definitive study: Results from range finding study.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

14.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: total organic solids (TOS)

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% c.l.: 10.6 - 19.9 mg TOS/l

Effect concentrations 2

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

17.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL 13.3-23.5

Effect concentrations 3

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

8.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mortality (fish)

Effect concentrations 4

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

other: visual observation for lethal and treatment-related-effects

Remarks on result:

other: based on the findings of the range finding test

Effect concentrations 5

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

other: visual observation for lethal and treatment-related-effects

Remarks on result:

other: based on the findings from the range finding test

Effect concentrations 6

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

mortality (fish)

Effect concentrations 7

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

49.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: TOS

Basis for effect:

mortality (fish)

Effect concentrations 8

Duration:

96 h

Dose descriptor:

LC10

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

mortality (fish)

Effect concentrations 9

Duration:

96 h

Dose descriptor:

LC10

Effect conc.:

10.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: TOS

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: During the initial four hours of exposure all fish including those in the control exhibited hyperventilation. This was attributed to the stringent test conditions employed in the test, with the fish taking longer to settle at the low pH after their addition to the vessel and therefor not considered treatment related. From 24 hours onwards, the fish in the control and test groups at 6.25 and 12.5 mg TOS/L became acclimatised and from 48 hours onwards, all appeared normal
- Mortality of control: 0
- Abnormal responses: treatment realated sub-lethal effects at 25 and 100 mg TOS/L

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

The 96-hour LC50 value for the test substance was nominally 17.7 mg TOS/L, with 95% confidence limits of 13.3 and 23.5 mg TOS/L. Based on the initial measured levels, the 96-hour LC50 was 14.6 mg TOS/L (95% confidence limits, 10.6 and 19.9 mg TOS/L) corresponding to 8.2 mg aep/L.
The no-observed-effect concentration (NOEC) was not clearly identified during the definitive test but based on the findings of a range finding test, the NOEC was 10 mg TOS/L (nominal) corresponding to 4.6 mg aep/L.

Executive summary:

An acute toxicity test to rainbow trout (Oncorhynchus mykiss) was conducted to determine the effect of the test substance on fish. The study was conducted in accordance with OECD and EC guidelines, and in compliance with GLP.

Groups of seven juvenile fish were exposed to subtilisin, dispersed in water at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L based on Total Organic Solid (TOS). The test media were prepared by direct addition of the test substance to diluent water (dechlorinated tap water, hardness ca. 180 mg/L as CaCO3); because the activity of the active component of the enzyme was reduced under alkaline conditions, the pH of the diluent water was adjusted to approximately 6.5 before use.

The test concentrations were verified based on the determination of the enzyme by ELISA. At the start of the test, the mean measured concentrations in samples of freshly prepared media ranged between 108 and 154% of their nominal values except at 6.25 mg TOS/L, where the measured level was 65%. In the samples of freshly prepared media remaining at 72 hours (6.25 to 25 mg TOS/L), the measured levels were lower, ranging between 45 and 72% of nominal. The reason for the difference between these two batches of media was not identified although an error during formulation of the test media was not supported by the raw data.

In samples of expired (24-hours-old) media, the measured levels were substantially maintained (ranging between 77 and 115% of their starting values) except on one occasion at 12.5 mg TOS/L where only 41%. Since the analytical results indicate that there was some variability in the exposure concentrations, the study results have been calculated in terms of both the nominal and mean measured concentrations.

Observations of the fish were made after approximately 2, 4, 24, 48, 72 and 96 hours of exposure. Based on these findings the following values have been estimated:

96-hour LC50: nominal 17.7 mg TOS/L; measured 14.6 mg TOS/L (8.2 mg aep/L)

100% mortality: nominal 50 mg TOS/L; measured 49.2 mg TOS/L

≤10% mortality: nominal 12.5 mg TOS/L; measured 10.2 mg TOS/L

The no-observed-effect concentration (NOEC) was not clearly identified during the definitive test but based on the findings of a range finding test, the NOEC was 10 mg TOS/L (nominal).