Subtilisin

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2012 - February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate included in report

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.034, 0.11, 0.34, 1.1 and 3.4 mg enzyme concentrate dry matter/L.
- Sampling method: duplicate aliquots (ca. 1mL) were removed from one replicate tank at each concentration on Days -4, -1, 4, 11, 18, 25 and 32.
- Sample storage conditions before analysis: frozen at - 20°C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Due to the nature of the test item, it was not possible to add it to the test system in the state it was supplied. The test item was therefore refined to remove any inert particulate material. Multiple small aliquots of Subtilisin, batch PPA25057 were removed and centrifuged to allow the determination of solid content. The bulk test item was then homogenised by stirring on a magnetic stirrer, followed by centrifugation to separate the solid and liquid components of the test item. The supernatant was then removed and frozen in batches for use on study. Using the known density (1.034533 g/mL) and solid content (0.04587 g/mL), a conversion factor was calculated and applied to all dose calculations to allow the study to be dosed at relative nominal concentration.
Preparation of stock solutions:
Stock solutions of Subtilisin, batch PPA 25027 in 0.2 µm filtered deionised water were prepared daily throughout the pre-equilibration and test periods. Solutions were prepared by serial dilution (1:10 v/v) of two primary stock solutions of Subtilisin, batch PPA 25057 (nominally 510 mg/mL and 159.3 mg/mL). The primary stock solutions were prepared in a 5 mL volume by dissolving a weighed amount of Subtilisin, batch PPA 25057 in 0.2 µm filtered deionised water (range: 2549.53–2555.58 mg to prepare the 510 mg/mL stock solution and 795.58–796.93 mg to prepare the 159.3 mg/mL stock solution). Stock solutions were added to syringes, and the loaded syringes added to the infusion pumps at ca. 24 h intervals. Stock solutions were maintained in an incubator set at 4°C.

- Controls: Medium

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Strain: Pimephales promelas
- Source: Charles River
POST-HATCH FEEDING
- Start date: newly hatched
- Type/source of feed: Artemia salina nauplii (brine shrimp, first instar larvae) less than 24 hours old
- Start date: 1 week after hatch
- Type/source of feed: Nauplii less than 48 hours old

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Hardness:

68.8 - 88.0 mg CaCO3/L

Test temperature:

24.1-25.8°C

pH:

6.33-7.38

Dissolved oxygen:

74.5 - 96.2%

Conductivity:

212 - 241 μS/cm

Nominal and measured concentrations:

Nominal: 0.034, 0.11, 0.34, 1.1, 3.4 mg enzyme concentrate dry matter/L
Measured: 0.007, 0.021, 0.15, 0.44, 1.49 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Test tanks (external dimensions 22 cm length x 13 cm height x 15 cm width) constructed from glass plates sealed together with non-toxic aquarium silicone adhesive were used for the test. An overflow hole, covered with a stainless steel mesh screen to prevent any loss of fry, was positioned at a height to provide a capacity of ca. 3 L.
Egg incubation chambers used in the test were 8 cm long glass cylinders (5 cm internal diameter) with a fine stainless steel mesh attached to the bottom with non-toxic aquarium silicone adhesive. The vessels were suspended in the test tanks by stainless steel wire, which in turn, was attached to a bar which oscillated upwards and downwards at a rate of 2-4 rpm per minute (controlled by a cam shaft mounted on an electric motor). This motion ensured that the embryos were continuously exposed to a gentle flow of water. Each test tank contained one oscillating incubation chamber.

- Renewal rate of test solution (frequency/flow rate): ca. 5 tank replacements every 24 hours
- No. of fertilized eggs/embryos per vessel: 20 per egg chamber
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted fresh water was used as the test and culture medium. This was prepared at Charles River, with high grade salts and reverse osmosis grade water. The salts were dissolved into a volume of deionised water (500 L) and this solution was metered into a flow of deionised water, to produce reconstituted freshwater based on the formula recommended by OECD Guideline 203.
- Total organic carbon: 2.073 - 3.160 mg/L
- Particulate matter: 0.00
- Alkalinity: 15 mg CaCO3/L
- Conductivity: 202 µS/cm
- Intervals of water quality measurement: Alkalinity, hardness and conductivity once every 7 days and TOC and particulate matter at the beginning and at the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: light/dark period 16:8hours
- Light intensity: 521 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Hatching success: Incubation chambers in each tank were observed daily and the number of dead, diseased or infertile embryos were recorded and discarded to reduce the risk of microbial contamination to surviving embryos. In the definitive test, Day 0 post-hatch was determined to be the day on which 95% of the embryos in the control had hatched (Study Day 4).
Fry: Daily observations of mortality, appearance, behaviour and any abnormal effects were recorded.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.063 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

adult mortality

Key result

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality

Key result

Duration:

32 d

Dose descriptor:

EC50

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality

Details on results:

- Other results:
NOEC on hatching: ≥ 1.5 mg enzyme concentrate dry matter/L.
NOEC for body length, weight and time to 95% hatch: 0.15 mg enzyme concentrate dry matter/L
EC5, EC10 and EC20 on mortality: 0.032, 0.06 and 0.144 mg enzyme concentrate dry matter/L respectively.

- Mortality/survival at embryo, larval, juvenile, and adult stages: A significant effect on the larval mortality was observed at concentrations ≥0.64 mg/L (nominal ≥5 mg/L)
- Days to hatch or time to release of young: no effects
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): no effects
- Observations on body length and weight of young and/or exposed parents at one or more time periods: a significant inhibition of the growth rate was observed at 0.16 mg/L (nominal 2 mg/L)
- Number of healthy fish at end of test: control: 48 ; control: 52; 0.5 mg/L: 43; 1.0 mg/L: 49; 2.0 mg/L: 42; 5.0 mg/L: 33 ; 10 mg/L: 24

Validity criteria fulfilled:

yes

Remarks:

The hardness of the dilution water deviated by more than 10% of the average during the course of the definitive test. This has no impact on the validity of the test.

Conclusions:

The EC50 value of Subtilisin, batch PPA25057, measure on mortality was estimated to be 0.74 mg enzyme concentrate dry matter/L (corresponding to 0.21 mg active enzyme protein/L). The EC5, EC10 and EC20 values, measured on mortality were estimated to be 0.032, 0.063, 0.15 and 0.74 mg enzyme concentrate dry matter/L, respectively.

It is concluded that the NOEC of Subtilisin, batch PPA 25057, on hatching success in fathead minnows is ≥1.49 mg enzyme concentrate dry matter/L.

Mortality, body length and weight and time to 95% hatch appear to be the most sensitive parameters in this early life stage test of the fathead minnow, with the NOEC for Subtilisin, batch PPA 25057 being 0.15 mg enzyme concentrate dry matter/L (measured as/equivalent to 0.042 mg active enzyme protein/L). Conducted under continuous flow conditions.

Executive summary:

The effects of prolonged exposure to Subtilisin, batch PPA 25057 on the early life stages of the fathead minnow (Pimephales promelas) were assessed over embryo development, hatching and for 28 days post-hatch, in accordance with OECD Guideline 210 (2013).

 

Based on the results of a range finding test, the definitive test was conducted under continuous flow conditions, with embryos (< 24 h old) exposed to the following nominal concentrations: 0.034, 0.11, 0.34, 1.1 and 3.4 mg enzyme concentrate dry matter/L of Subtilisin, batch PPA 25057. A control was also included. Four replicate tanks were tested at each concentration.

 

Analysis of test samples taken during the exposure period indicated that measured concentration of Subtilisin, batch PPA 25057 were not maintained within ±20% of nominal over the test period. Due to the nature of the test item and the analytical method results are reported in mg of active enzyme protein per litre of test solution (mg AEP/L). The geometric mean measured concentrations were determined to be 0.01, 0.02, 0.15, 0.44 and 1.49 mg enzyme concentrate dry matter Subtilisin, batch PPA 25057/L for the respective nominal concentrations of 0.034, 0.11, 0.34, 1.1 and 3.4 mg enzyme concentrate dry matter/L.

 

The hatching success of embryos in individual replicates ranged from 75-100%. Hatching success in the control group was 92.5%. Statistical analysis indicated no significant differences in hatching success between the treatment groups and the control group. As there was no difference in hatching success between the treatment groups and the control; the no observed effect concentration (NOEC) for hatching success is concluded to be ≥1.49 mg enzyme concentrate dry matter/L. The corresponding lowest observed effect concentration (LOEC) is concluded to be >1.49 mg enzyme concentrate dry matter/L.

 

Hatching commenced 2 days after the initiation of the test in all treatment groups. Time to 95% hatch in the control group and three lowest concentrations was Day 4. In the two highest concentration groups, time to 95% hatch was Day 3. The NOEC for time to hatch is concluded to be 0.15 mg enzyme concentrate dry matter/L; the corresponding LOEC is concluded to be 0.44 mg enzyme concentrate dry matter/L. 

 

Survival of fry at the end of the exposure phase was compared to the number of fry successfully hatched and released in individual replicates, and ranged from 0-100%. Survival of fry in the control group was 100%. Statistical analysis indicated a significantly higher mortality in the two highest treatment groups than in the control. There was no significant difference in mortality between the three lowest treatment groups and the control. The EC₅₀ value was estimated to be 0.74 mg enzyme concentrate dry matter/L (equivalent to 0.21 mg active enzyme protein/L). The EC₅, EC₁₀ and EC₂₀ values were estimated to be 0.032, 0.063 and 0.15 mg enzyme concentrate dry matter/L, respectively.

The NOEC for mortality is concluded to be 0.15 mg enzyme concentrate dry matter/L; the corresponding LOEC is concluded to be 0.44 mg enzyme concentrate dry matter/L.

 

Fry length and body weight were measured at the end of the test. Statistical analysis indicated no significant differences in length and body weight between the treatment groups and the control. The NOEC is concluded to be 0.15 mg enzyme concentrate dry matter/L for both body lengths and body weights; the corresponding LOEC is concluded to be 0.44 mg enzyme concentrate dry matter/L.

 

Based on the above results, mortality, body length and weight, and time to 95% hatch appear to be the most sensitive parameters in the early life stage test of the fathead minnow, with the NOEC for Subtilisin, Batch PPA 25057 being 0.15 mg enzyme concentrate dry matter/L.

 

Test solution quality parameters measured throughout the test were in the following ranges; temperature: 24.7-27.1°C, pH: 6.33-7.38 and dissolved oxygen: 74.5-96.2% ASV.

 

Validity criteria for the test was met, as the hatching success in the controls was greater than 70% (92.5%); survival of fry in the control group was greater than 75% (100%); dissolved oxygen concentration in each of the test tanks was above 60% ASV for the duration of the test (≥74.5% ASV) and the water temperature did not differ by more than ± 1.5°C between test tanks. As such, this test is regarded as valid.

 

 

Endpoint	Parameter	Value (mg enzyme concentrate dry matter/L)	Value (mg active enzyme protein/L)
Mortality	EC5	0.032	0.009
	EC10	0.063	0.017
	EC20	0.15	0.041
	EC50	0.74	0.210
	NOEC	0.15	0.042
	LOEC	0.44	0.126
Hatch	NOEC	≥1.49	≥0.426
	LOEC	≥1.49	≥0.426
Time to 95% hatch	NOEC	0.15	0.042
	LOEC	0.44	0.126
Fry Length	NOEC	0.15	0.042
	LOEC	0.44	0.126
Fry Weight	NOEC	0.15	0.042
	LOEC	0.44	0.126

 

 

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 September 2007 - 5 November 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to the low recovery of the exposure concentration, the study must be regarded as supporting study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Except for analytical analysis of the test concentrations

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.037, 0.074, 0.15, 0.37 and 0.74 mg enzyme concentrate dry matter/L
- Sampling method: duplicate subsamples were taken at the start of the analysis and hereafter as a minimum once every week during the 33-day test period. Subsamples were collected in 12 mL Nunc-Immuno Minisorp Tubes
- Sample storage conditions before analysis: frozen at - 20°C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions of the test by weighing out respectively 40, 80, 160, 400 and 800 mg of the test batch and adding it to a 2 litre of the test medium. Stock solutions were prepared every second day throughout the test period and placed in box with ice to keep the temperature below 10°C.
- Controls: Medium

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Strain: Danio rerio
- Source: Credo fish, Nørresundby, Denmark and purchased from Hørsholm Dyrehandel, Hørsholm, Denmark

POST-HATCH FEEDING
- Start date: newly hatched
- Type/source of feed: TetraMin®Baby (Tetra GmbH, Melle)
- Start date: 2 days-old
- Type/source of feed: TetraMin®Baby (Tetra GmbH, Melle) supplemented with newly hatched Artemia salina nauplii
- Start date: 2 weeks after hatch
- Type/source of feed: only Artemia salina nauplii

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Test temperature:

24.4 - 25.7°C

pH:

6.9-7.4

Dissolved oxygen:

79-100%

Nominal and measured concentrations:

Nominal: 0.037, 0.074, 0.15, 0.37 and 0.74 mg enzyme concentrate dry matter/L
Measured: 0.001, 0.0031, 0.012, 0.047 and 0.092 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 6-L glass aquaria
- Material, size, headspace, fill volume: glass, size: 6L, 3L fill volume
- Renewal rate of test solution (frequency/flow rate): 4 times daily
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Freshly produced synthetic medium was used in the test. The medium was prepared from Millipore water according to the ISO Standard 7346/3/.

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted with 1M HCl to be below 7.
- Photoperiod: light/dark period 14:10 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : embryonic development, hatching, survival, abnormal appearance and behavior were observed at least once a day. During weekends the recordings were only made once.

VEHICLE CONTROL PERFORMED: no

Reference substance (positive control):

no

Duration:

33 d

Dose descriptor:

EC50

Effect conc.:

> 0.092 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality

Remarks:

larval

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality

Remarks:

larval

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

0.047 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mortality

Remarks:

larval

Details on results:

-No effects were obtained for the following effect parameters: Time to 50% hatch, embryonic mortality, malformation, growth (length and weight) and swimming behavior.
NOEC and LOEC for embryonic mortality, time to 50% hatching, larval malformation and behavior and larval growth (weight and length) were ≥ 0.092 mg enzyme concentrate dry matter/L (nominal: ≥ 0.74 mg enzyme concentrate dry matter/L), and >0.092 mg enzyme concentrate dry matter/L, respectively.
- Mortality/survival at embryo, larval, juvenile, and adult stages: A significant effect (LOEC) on the larval mortality was observed at concentrations ≥0.047 mg enzyme concentrate dry matter/L (nominal ≥0.37 mg enzyme concentrate dry matter/L)
- Days to hatch or time to release of young: Days 1-2.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): no effects
- Observations on body length and weight of young and/or exposed parents at one or more time periods: a significant inhibition of the growth rate (length) was observed at 0.012 mg enzyme concentrate dry matter/L (nominal 0.15 mg enzyme concentrate dry matter/L) .

- Number of healthy fish at end of test: control: 48 ; control: 52; 0.0.37 mg enzyme concentrate dry matter/L: 43; 0.074 mg enzyme concentrate dry matter/L: 49; 0.15 mg enzyme concentrate dry matter/L: 42; 0.37 mg enzyme concentrate dry matter/L: 33 ; 0.74 mg enzyme concentrate dry matter/L: 24.

Validity criteria fulfilled:

yes

Conclusions:

The LOEC of Subtilisin, batch PBI48037 for larval mortality were 0.047 mg enzyme concentrate dry matter/L (equivalent to 0.024 mg active enzyme protein/L). The NOEC for larval mortality was estimated to be 0.012 mg enzyme concentrate dry matter/L. The lowest observed effect level (LOEC) for larval mortality of the test substance was nominally 0.37 mg enzyme concentrate dry matter/L, which was estimated to be 0.047 mg enzyme concentrate dry matter/L (measured). LC50 ≥0.092 mg enzyme concentrate dry matter/L (highest concentration). Performed under flow-through conditions.

Executive summary:

Subtilisin, batch PBI48037 was tested for lethal and sublethal effects on early life stages of Zebra fish (Danio rerio) according to the procedures described in the OECD Test Guideline No. 210 and conducted in compliance with GLP.

The test material was tested at following nominal concentrations: 0, 0.037, 0.074, 0.15, 0.37 and 0.74 mg enzyme concentrate dry matter/L.

The test was carried out in a flow-through test system with 6 liter glass aquaria each containing 3 liter of test solutions. Effect parameters observed during the 33 -day test period included time to hatch, mortality, behavior and growth of early life stages of zebra fish.

The actual test concentrations were analysed by specific ELISA analysis. The analysis showed recoverys from 0 to 32 % indicating fast degradation of the test compound. Samples were kept frozen until analysis and the freeze thawing process can also have contributed to the loss of enzymatic activity.

No effects were obtained for the following effect parameters: Time to 50% hatch, embryonic mortality, malformation, growth (length and weight) and swimming behavior.

Significant effect (LOEC) on the larval mortality was observed at estimated concentrations ≥0.047 mg enzyme concentrate dry matter/L (nominal concentration ≥0.37 mg enzyme concentrate dry matter/L). The no-observed-effect-concentration (NOEC) for larval mortality was thus 0.012 mg enzyme concentrate dry matter/L (nominal 0.15 mg enzyme concentrate dry matter/L). LC50 ≥0.092 mg enzyme concentrate dry matter/L (highest concentration). 

Due to the low recovery of the exposure concentration, the study must be regarded as supporting study.

 

Effect parameter		NOEC (mg enzyme concentrate dry matter/L)	LOEC (mg enzyme concentrate dry matter/L)
Larval mortality		0.012	0.047
Embryonic mortality		≥0.092	≥0.092
Time to 50% hatch		≥0.092	≥0.092
Larval malformation and behaviour		≥0.092	≥0.092
Larval growth	Weight	≥0.092	≥0.092
	Length	≥0.092	≥0.092

 

Description of key information

• The EC50 value of Subtilisin, batch PPA25057, measure on mortality was estimated to be 0.74 mg enzyme concentrate dry matter/L (equivalent to 0.21 mg active enzyme protein/L). The EC10 value was 0.06 mg enzyme concentrate dry matter/L (equivalent to 0.017 mg active enzyme protein/L). NOEC on hatching success in fathead minnows is ≥1.49 mg enzyme concentrate dry matter/L. Mortality, body length and weight and time to 95% hatch appear to be the most sensitive parameters in this early life stage test of the fathead minnow, with the NOEC for Subtilisin, batch PPA 25057 being 0.15 mg enzyme concentrate dry matter/L (measured as/equivalent to 0.426 mg active enzyme protein/L). Conducted under continuous flow conditions.

 

• The LOEC of Subtilisin, batch PBI48037 for larval mortality was 0.047 mg enzyme concentrate dry matter/L (equivalent to 0.024 mg active enzyme protein/L). The NOEC for larval mortality was estimated to be 0.12 mg enzyme concentrate dry matter/L. The lowest observed effect level (LOEC) for larval mortality of the test substance was nominally 0.37 mg enzyme concentrate dry matter/L, which was estimated to be 0.047 mg enzyme concentrate dry matter/L (measured). LC50 ≥0.092 mg enzyme concentrate dry matter/L (highest concentration). Performed under flow-through conditions.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Remarks:

enzyme concentrate dry matter

Effect concentration:

0.06 mg/L

Additional information

The EC10 value of Subtilisin, batch PPA25057 was given as a kew value, since that is the study corresponding to the lowest active enzyme protein.