Subtilisin

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:

01 August 2006 - 16 March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Analysis of the different concentrations not conducted under GLP.

Analytical monitoring:

yes

Details on sampling:

- Concentrations (nominal): Range finding test used concentrations 1.1, 10.7 and 106.8 mg enzyme concentrate dry matter/L. The definitive test concentrations were 6.7, 13.4, 26.7, 53.4 and 106.8 mg enzyme concentrate dry matter/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

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 (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

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

150-200 mg CaCO3/L

Test temperature:

15 ± 2°C

pH:

6.15-7.26

Dissolved oxygen:

73-104 % air saturation value

Conductivity:

330 µS/cm

Nominal and measured concentrations:

The nominal and measured concentrations for the definitive test were:
nominal: 6.7, 13.4, 26.7, 53.4 and 106.8 mg enzyme concentrate dry matter/L
measured: 3.44, 10.9, 22.4, 52.6, 142 mg enzyme concentrate dry matter/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 high-grade 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 (nominal): 1.1, 10.7 and 106.8 mg enzyme concentrate dry matter/L
- Results used to determine the conditions for the definitive study: Results from range finding study.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

15.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% c.l.: 11.3 - 21.3 mg enzyme concentrate dry matter/l

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

52.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC10

Effect conc.:

10.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality (fish)

Details on results:

- Nominal results:
96-h LC50: 18.9 mg enzyme concentrate dry matter/L (95%-CI: 14.2;25.2)
96-h LC10: 13.4 mg enzyme concentrate dry matter/L
96-h LC100: 53.4 mg enzyme concentrate dry matter/L
- 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 the two lowest concentrations became acclimatised and from 48 hours onwards, all appeared normal
- Mortality of control: 0
- Abnormal responses: treatment related sub-lethal effects at nominal concentrations of 26.7 and 106.8 mg enzyme concentrate dry matter/L .

For the two highest concentrations the mean recovery was calculated based on samples 0 hours fresh and 24 hours old preparations. The 72 hours fresh and 96 hours old preparations were not sampled because all of the fish had died.

The measurements of water quality (temperature, pH, concentrations of dissolved oxygen and
total hardness) remained within acceptable limits throughout the study.

Reported statistics and error estimates:

LC50 values were calculated using the SAFEStat LD50 application (SAS 8.2) and the nominal and mean measured concentrations.

Sublethal observations / clinical signs:

Effects on pigmentation, respiration and co-ordination were observed. During the initial four hours of exposure all fish (including those in the control) exhibited hyperventilation. This was attributed to the stringent conditions employed in the test, with the fish taking longer to settle at the low pH after their addition to the vessels and therefore was not considered to be treatment-related. From 24 hours onwards, the fish in the control and test groups at the 2 lowest concentrations became acclimatised to the test conditions and from 48 hours onwards, all appeared normal.

At 26.7 to 106.8 mg enzyme concentrate dry matter/L (nominal concentration), the fish exhibited other sub-lethal effects (lethargy, loss of co-ordination and coughing), which were attributed to exposure to the test substance.

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 for Subtilisin, batch PXI48489 (measured) was 15.6 mg enzyme concentrate dry matter/L (95% confidence limits, 11.3 and 21.3 mg enzyme concentrate dry matter/L) corresponding to 8.2 mg active enzyme protein/L.
≤10% mortality based on measured 10.9 mg enzyme concentrate dry matter/L (equivalent to 5.7 mg active enzyme protein/L). Conducted under semi-static (daily renewal) exposure conditions.

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 6.7, 13.4, 26.7, 53.4 and 106.8 mg/L based on enzyme concentrate dry matter. 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 6.7 mg enzyme concentrate dry matter/L, where the measured level was 65%. In the samples of freshly prepared media remaining at 72 hours (6.7 to 26.7 mg enzyme concentrate dry matter/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 13.4 mg enzyme concentrate dry matter/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 18.9 mg enzyme concentrate dry matter/L; measured 15.6 mg enzyme concentrate dry matter/L (95% confidence limits, 11.3 and 21.3 mg enzyme concentrate dry matter/L), equivalent to 8.2 mg aep/L.

100% mortality: nominal 53.4 mg enzyme concentrate dry matter/L; measured 52.6 mg enzyme concentrate dry matter/L.

≤10% mortality: nominal 13.4 mg enzyme concentrate dry matter/L; measured 10.9 mg enzyme concentrate dry matter/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 10.7 mg enzyme concentrate dry matter/L (nominal), corresponding to 5.6 mg aep/L.

Conducted under semi-static (daily renewal) exposure conditions. Validity criteria were met.

Description of key information

The 96-hour LC50 for Subtilisin, batch PXI48489 (measured) was 15.6 mg enzyme concentrate dry matter/L (95% confidence limits, 11.3 and 21.3 mg enzyme concentrate dry matter/L) corresponding to 8.2 mg active enzyme protein/L.
≤10% mortality based on measured 10.9 mg enzyme concentrate dry matter/L (equivalent to 5.7 mg active enzyme protein/L). Conducted under semi-static (daily renewal) exposure conditions.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC50

Remarks:

enzyme concentrate dry matter

Effect concentration:

15.6 mg/L