Subtilisin

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 November 2007 - 5 December 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

ELISA method did not show good recovery for freshly prepared stock suspension. Reliability is questionable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

1998

Deviations:

yes

Remarks:

Since bicine control did not fulfil the validity criteria, the test was performed without addition of bicine to the test medium but with adjustment with HCl of pH to approx. 6.0 at test start and renewals. This has no impact on the validity of the study.

GLP compliance:

yes

Remarks:

except for the analysis of the test material

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.002, 0.007, 0.02, 0.07, 0.22 and 0.74 mg enzyme concentrate dry matter/L
- Sampling method: Duplicate samples of 2 x 10 mL in Nunc-Immuno Minisorp Tubes were collected from the stock solutions and from the test solutions 0.07, 0.22 and 0.74 mg enzyme concentrate dry matter/L. The subsamples were frozen to -20°C immediately after collection.
- Sample storage conditions before analysis: Frozen at appr. -20°C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions of 100 mg/L of the test batch were prepared by weighing 25 mg and adding it to 250 mL of test medium. This procedure was followed at the beginning of the test and at each renewal of test medium, i.e. every second day during the test period. The stock solutions were used for preparing a dilution series of the test product in the test medium.
- Controls: medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Source: Langedam, Birkerød
- Age at study initiation: Less than 24 hours
- Feeding during test
- Food type: alga Pseudokirchneriella subcapitata
- Amount: appr. 0.2 mgC/animal/day until day 8, herafter 0.4 mgC/animal/day
- Frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

Nominal: 19.5 ± 0.1°C

pH:

6.0-7.9

Dissolved oxygen:

98-100%

Nominal and measured concentrations:

Nominal: 0.07, 0.22 and 0.74 mg enzyme concentrate dry matter/L (the three highest concentrations).
Measured: Subtilisin could not be detected in expired/old media. In freshly prepared media the measured concentrations ranged between 4 and 41.6% of the expected concentration.
The estimated three top test concentrations were: 0.005, 0.022 and 0.11 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flask
- Material, size, headspace, fill volume: glass, 120-mL, fill volume 80 mL
- Renewal rate of test solution (frequency/flow rate): every second day
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

OTHER TEST CONDITIONS
- Adjustment of pH: to pH 6.2 ± 0.2 using either HCl or NaOH
- Photoperiod: 16 hours light: 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Number of offspring, mortality among parent animals, time at which the first offspring were present, aborted broods and dead larvae

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 0.07, 0.22 and 0.74 mg enzyme concentrate dry matter/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.002 - < 0.005 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

reproduction

Remarks on result:

other: Nominal: 0.022 mg enzyme concentrate dry matter/L.

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.022 - < 0.11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

reproduction

Remarks on result:

other: Nominal: between 0.22 and 0.74 mg enzyme concentrate dry matter/L.

Details on results:

The time integrated mean value of 7% for "Subtilisin, batch PBI48037" was used for the calculation of NOEC.
EC10 was estimated to be between 0.0016 mg enzyme concentrate dry matter/L (nominal 0.022 mg enzyme concentrate dry matter/L) and 0.0053 mg enzyme concentrate dry matter/L (nominal 0.07 mg enzyme concentrate dry matter/L).
- Mortality of parent animals (nominal concentrations/ mg enzyme concentrate dry matter/L):
control: 1
(conc. 1) 0.002: 1
(conc. 2) 0.007: 1
(conc. 3) 0.02: 3
(conc. 4) 0.07: 1
(conc. 5) 0.22: 2
(conc. 6) 0.74: 9
- Average number of live offspring produced per day per live female: control: 88.2; (1): 70; (2): 67.2; (3): 74.5; (4): 56.9; (5): 51.3; (6): 56.
- Number of males and females (parental): 10 females per group
- Time to first brood release or time to hatch: 9-10 days

Validity criteria fulfilled:

yes

Conclusions:

The time integrated mean value of 7% for Subtilisin, batch PBI48037 was used for the calculation of NOEC. The data processing showed that 21-day NOEC was 0.0016 mg enzyme concentrate dry matter/L. The 21-d EC10 was estimated to be between 0.0016 mg/L (nominal 0.022 mg enzyme concentrate dry matter/L) and 0.0053 mg enzyme concentrate dry matter/L (nominal 0.07 mg enzyme concentrate dry matter/L), corresponding to values 0.79 and 2.7 μg active enzyme protein/L. The 21-day EC50 was estimated to be between 0.022 mg enzyme concentrate dry matter/L and 0.11 mg enzyme concentrate dry matter/L. Media renewal every second day.

Executive summary:

A Daphnia magna reproduction test has been performed according to OECD guideline 211, and in compliance with GLP. The test performed was a semi-static test with medium renewal every second day. Freshly prepared and 48 hour-old test media were analysed for test material. No test material could be detected in 48 hour-old test media indicating fast degradation of the test substance in the media. Thus it was not possible to make an accurate estimation of the exposure concentrations. However, estimates of exposure concentration have been made based on time-weighted mean.

The data processing showed that 21-d NOEC was 0.0016 mg enzyme concentrate dry matter/L (nominal 0.022 mg enzyme concentrate dry matter/L), corresponding to 0.79 μg active enzyme protein/L.

The 21-d EC10 was estimated to be between 0.0016 mg/L (nominal 0.022 mg enzyme concentrate dry matter/L) and 0.0053 mg enzyme concentrate dry matter/L (nominal 0.07 mg enzyme concentrate dry matter/L), corresponding to values 0.79 and 2.7 μg active enzyme protein/L.

The 21-d EC50 was estimated to be between 0.022 mg enzyme concentrate dry matter/L (nominal 0.22 mg enzyme concentrate dry matter/L) and 0.11 mg enzyme concentrate dry matter/L (nominal 0.74 mg enzyme concentrate dry matter/L).

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

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 211 (Daphnia magna Reproduction Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations (nominal): 0, 0.034, 0.11, 0.34, 1.1 and 3.4 mg enzyme concentrate dry matter/L
- Sampling method: From each treatment vessel samples have been taken on at least two occasions prior to the addition of Daphnia magna to the test system and on at least 4 occasions during the study.
- Sample storage conditions before analysis: Samples have been analysed directly after collection in order to avoid degradation of the test material.

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Source: From a culture maintained within Charles River bred by acyclical parthogenesis and as such will be female.
-Age at study initiation: less than 24 hours
- Feeding during test
- Food type: microalga Chlorella vulgaris
- Amount: 0.1 - 0.2 mg C/Daphnia/day

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

181 - 299 mg CaCO3/L

Test temperature:

18-22°C

pH:

7.33 - 7.86

Dissolved oxygen:

71.9 - 99.4%

Nominal and measured concentrations:

Nominal: 0.034, 0.11, 0.34, 1.1 and 3.4 mg enzyme concentrate dry matter/L.
Measured: 0.01, 0.02, 0.06, 0.57 and 1.14 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass
- Type (delete if not applicable): open
- Material, size, fill volume: glass, ca. 2 L
- Type of flow-through : A single glass tank (22 cm x 15 cm X 12 cm, length x width x height) for each treatment with an overflow hole providing ca. 2 litre of liquid capacity. Suspended within this tank were 4 glass chambers, constructed from 8 cm long glass cylinders (5 cm internal diameter) with a fine mesh stainless steel screen attached to the base.
- Renewal rate of test solution (flow rate): 5- 10 volume changes in a 24h period
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
- Total organic carbon: <=2 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- Light intensity: < 1600 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

RANGE-FINDING STUDY
- Test concentrations: Nominal: 0.001, 0.01, 0.11 and 1.05 mg enzyme concentrate dry matter/L; Measured: < 0.004, 0.095, 1.377 mg enzyme concentrate dry matter/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

reproduction

Remarks on result:

not determinable

Key result

Duration:

21 h

Dose descriptor:

NOEC

Effect conc.:

1.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

reproduction

Details on results:

- Behavioural abnormalities: No
- Observations on body length and weight: At the end of the test, all surviving D. magna were of similar size. However, between Days 0 and 11 the 0.57 mg enzyme concentrate dry matter/L treatment group D magna appeared smaller than the controls. Between Days 0 and 13, D. magna at 1.14 mg enzyme concentrate dry matter/L also appeared smaller than the controls. No ephippia were recorded throughout the test period.
- Other biological observations: No
- Other adverse effects control: No
- Immobilisation of control: 1 (5%)
- 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: None

The number of immobile adult D. magna recorded during the reproduction test was 1 (5%) in the control and in the 0.01 mg enzyme concentrate dry matter/L treatment group; 3 (15%) in the 0.06 mg enzyme concentrate dry matter/L treatment group; and 5 (25%) in the 0.57 mg enzyme concentrate dry matter/L treatment group.

Validity criteria fulfilled:

yes

Conclusions:

The 21-day EC50 (reproduction) of Savinase, batch PPA25057 could not be calculated due to low levels of inhibition. 21-d NOEC (reproduction) was 1.14 mg enzyme concentrate dry matter/L (equivalent to 0.324 mg active enzyme protein/L). Test was conducted under flow-through conditions.

Executive summary:

This study was designed to determine the effect of Savinase, batch PPA25057 on the reproductive capacity of Daphnia magna. The study was conducted over a 21 day period under flow through conditions. Four replicate vessels, contained within a tank of test solution, were prepared for each treatment group, each containing five D. magna neonates. The study was performed to meet the requirements of OECD Guideline 211 (1998).

Based on the results of a range finding test, the reproduction test was conducted under flow through conditions in Elendt M4 medium, with neonates being exposed to the following nominal concentrations of Savinase, batch PPA25057: 0.034, 0.11, 0.34, 1.1 and 3.36 mg enzyme concentrate dry matter/L. A control of untreated Elendt M4 medium was also included. As the test item is an unstable biological preparation, the method of exposure was selected to maximise the stability of Savinase, batch PPA25057 under test conditions.

 

Analysis of test samples taken during the exposure period indicated that measured concentrations of Savinase, batch PPA25057, were not maintained within ±20% of nominal over the test period due to the unstability of the test substance in aquatic media and thus results are based on measured and not nominal concentrations. Due to the nature of the test item and the analytical method results are reported as mg active enzyme protein (AEP)/L. The geometric mean measured concentrations of AEP in Savinase, Batch PPA 25057, were calculated equivalent to 0.01, 0.02, 0.06, 0.57 and 1.14 mg enzyme concentrate dry matter/L for the nominal concentrations of 0.034, 0.11, 0.34, 1.1 and 3.36 mg enzyme concentrate dry matter/L, respectively.

 

The number of immobile adult D. magna recorded during the reproduction test was 1 (5%) in the control and in the 0.01 mg enzyme concentrate dry matter/L treatment group; 3 (15%) in the 0.06 mg enzyme concentrate dry matter/L treatment group; and 5 (25%) in the 0.57 mg enzyme concentrate dry matter/L treatment group.

 

The total number of offspring produced by surviving D. magna at each concentration and the mean number of offspring produced per surviving D. magna, over the test period, were as follows:

 

Parameter	Geometric Mean Measured Concentration (mg enzyme concentrate dry matter/L)
	Control	0.01	0.02	0.06	0.57	1.14
Total Number of Offspring Produced*	1294	1477	1540	1362	957	1222
Mean Number of Offspring per Surviving D. Magna*	67	78	77	76	64	61

* Note: values exclude immobile D. magna observed during the test period. 

 

Offspring from adults which were observed to be immobile during the reproduction period were omitted from the calculation of total number of offspring and mean number of offspring per D. magna. The first offspring were observed on Day 8 in the control, and the three lowest concentrations; reproduction was first observed on Day 9 in the two highest concentrations.

 

Statistical analysis was conducted to confirm the effect Savinase, batch PPA25057 had on D. magna reproduction. The EC₅₀ for reproduction at Day 21 could not be estimated due to low levels of inhibition. The NOEC for reproduction at Day 14 and Day 21 was 0.06 and 1.14 mg enzyme concentrate dry matter/L, respectively.

 

During the test, test solution quality parameters were within the following ranges: pH: 7.33‑7.86; temperature: 19.3-20.8°C and dissolved oxygen concentration: 71.9-99.4% of air saturation value.

 

Validity criteria for a valid test were met: control mortality in the adult D. magna was ≤20% (5%); the mean number of offspring produced per control adult was ≥60 (67) and the coefficient of variation around the mean number of offspring produced per control adult was ≤25% (6.79%).

Description of key information

• (Reliability:2) The time integrated mean value of 7% for Subtilisin, batch PBI48037 was used for the calculation of NOEC. The data processing showed that 21-day NOEC was 0.0016 mg enzyme concentrate dry matter/L. The 21-d EC10 was estimated to be between 0.0016 mg/L and 0.0053 mg enzyme concentrate dry matter/L, corresponding to values 0.79 and 2.7 μg active enzyme protein/L. The 21-day EC50 was estimated to be between 0.022 mg enzyme concentrate dry matter/L and 0.11 mg enzyme concentrate dry matter/L. Media renewal every second day.


• The 21-day EC50 (reproduction) of Savinase, batch PPA25057 could not be calculated due to low levels of inhibition. 21-d EC10 (reproduction) was 0.51 mg enzyme concentrate dry matter/L, equivalent to 0.145 mg active enzyme protein/L. 21-d NOEC (reproduction) was 1.14 mg enzyme concentrate dry matter/L (equivalent to 0.324 mg active enzyme protein/L). Test was conducted under flow-through conditions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.51 mg/L