Sedolisin

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

29 January - 31 January 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD and EC test guidlines, but the test concentrations were not verified.

Justification for type of information:

Enzymes are of the same sub-sub class and similar toxicological properties are expected.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Oxygen concentration dropped to < 3.0 mg O2/L.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in test water to give an initial stock solution of 1000 mg/L (equivalent to 943 mg enzyme concentrate dry matter/L). Serial dilutions of this stock solution were prepared to give the desired series of exposure levels.
- Controls: dilution medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: Laboratory culture originating from a strain supplied by the Institute National de Recherche Chimique Appliqué (IRChA), France.
- Age at study initiation (mean and range, SD): less than 24 hours old.
- Method of breeding: Parthenogenesis ensured by stock culture conditions.
- Feeding during test: no feeding during test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

171 mg CaCO3/L

Test temperature:

nominal: 20 ± 2°C
measured: 21°C

pH:

measured: 7.4 - 8.2

Dissolved oxygen:

measured: 2.1 - 8.7 mg O2/L

Nominal and measured concentrations:

Nominal concentrations: 5.3, 9.4, 17.0, 30.2, 52.8, 94.3, 169.7, 301.8, 528.1 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar
- Type (delete if not applicable): loosely covered with aluminium foil
- Material, size, headspace, fill volume: glass, fill volume 200mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
Biomass loading rate: 20 mL medium per organism.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water filtered, dechlorinated and softened by passage through an Elga® water purification system. The water passes through a high pressure sand filter to remove colloids and particulate matter and a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passes through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water are then remixed in the ratio of approximately 1:1 to give a supply with the desired water hardness. Batches of softened water were "aged" with aeration for at least 12 hours prior to use by recirculating through a gravel filter bed.
- Alkalinity: HCO3 210mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.
The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 hours after the start of the study.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

60.4 mg/L

Nominal / measured:

nominal

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 53.8 - 68.8 mg enzyme concentrate dry matter/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

30.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

mobility

Details on results:

- Mortality of control: No control animals were immobilised

Validity criteria fulfilled:

yes

Remarks:

Oxygen concentration dropped to < 3.0 mg O2/L at 48 hours at concentrations 301.8 and 528.1 mg enzyme concentrate dry matter/L, however, the study is still considered to be valid.

Conclusions:

The 48-hour EC50 of the test substance for the immobilisation of Daphnia magna was nominal 60.4 mg enzyme concentrate dry matter/L corresponding to 12.4 mg active enzyme protein/L.

Executive summary:

The acute toxicity of subtilisin to Daphnia magna was assessed under static exposure conditions, with renewal of the media after 24 hours. The study was conducted in accordance with EC and OECD Guidelines, and in compliance with GLP.

Groups of twenty, 1st instarDaphnia(less than 24 hours old) were exposed for 48 hours to 9 concentrations of subtilisin dissolved in water. The incidence of immobilisation was recorded for each test and control group at 24 hours and 48 hours and the following values determined:

48-hour EC50 (95% c.l.): 60.4 mg enzyme concentrate dry matter/L (53.8 & 68.8) or 12.4 mg active enzyme protein/L

NOEC at 24 and 48h: 30.2 mg enzyme concentrate dry matter/L

All concentrations are given in terms of nominal concentration.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

03-04-2017 to 03-06-2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Remarks:

Oxygen content was not measured.

GLP compliance:

no

Remarks:

See 'Justification for type of information'

Analytical monitoring:

no

Details on sampling:

- Concentrations: 7.5, 14.9, 29.8, 59.6, 119.2 mg enzyme concentrate dry matter/L

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dilution in media (Standard Freshwater ISO 6341).
- Controls: Standard Freshwater ISO6341.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna Straus
- Source: MicroBioTests Inc. Daphnia magna have been developed by the research teams at the Laboratory for Biological Research in Aquatic Pollution (LABRAP) at the Ghent University in Belgium.
- Age at study initiation (mean and range, SD): less than 24 hours
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20-25°C

Nominal and measured concentrations:

Nominal concentrations: 7.5, 14.9, 29.8, 59.6, 119.2 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
The test was conducted according to DAPHTOXKIT F MAGNA Crustacean Toxicity Screening Test for Freshwater with a test system supplied by MicroBioTests Inc.
http://www.microbiotests.be/SOPs/Daphtoxkit%20magna%20F%20SOP%20-%20A5.pdf

- Test vessel: The bioassays are conducted in disposable multiwell test plates with 30 test wells.
- Type: open
- Material, size, headspace, fill volume: 10 mL
- Aeration: The Standard Freshwater (ISO medium) must be aerated for at least 15 minutes prior to using it.
- No. of organisms per vessel: 20 (5 daphnia per well)
- No. of vessels per concentration and control (replicates): Each plate is provided with 4 wells for the controls and 4 wells (A,B,C,D) for each toxicant concentration.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium, formula according to ISO 6341

Composition :
vial 1 : NaHCO3 (129.5 mg - dissolved in 2 L = 67.75 mg/L)
vial 2 : CaCl2.2H2O (588 mg - dissolved in 2 L = 294 mg/L)
vial 3 : MgSO4.7H2O (246.5 mg - dissolved in 2 L = 123.25 mg/L)
vial 4 : KCl (11.5 mg - dissolved in 2 L = 5.75 mg/L)

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 119.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 119.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

mobility

Details on results:

Details on results
- Mortality of control: 0
- Other adverse effects control: No
- Abnormal responses: No
- Effect concentrations exceeding solubility of substance in test medium: No
Daphnia were considered to be immobile if they were unable to swim within 15 seconds following gentle touch of the animal with a pin.

Validity criteria fulfilled:

yes

Remarks:

Oxygen content was not measured.

Conclusions:

Under the conditions of the test, Sedolisin, batch PPF38268 was found not to be toxic to Daphnia magna at a nominal concentration up to 119.2 mg enzyme concentrate dry matter/L (24.7 mg aep/L) after 48h exposure. Thus, no 48h EC50 could be calculated and must be >119.2 mg enzyme concentrate dry matter/L (equivalent to > 24.7 mg active enzyme protein/L).

Executive summary:

The purpose of this study was to screen Sedolisin, batch PPF38268, for the test material concentration estimated to immobilise 50% of the Daphnia magna after 24 and 48 hours exposure. DAPHTOXKIT F™ magna was supplied by MicroBioTests Inc. The testing of the Sedolisin, batch PPF38268 was performed as a screening with a broad range of concentrations 7.5, 14.9, 29.8, 59.6, 119.2 mg enzyme concentrate dry matter/L. The acceptance criterion for a valid test is that the spontaneous immobility in the control is below or equal to 10%.

Under the conditions of the test, Sedolisin, batch PPF38268 was found not to be toxic to Daphnia magna at a nominal concentration up to 119.2 mg enzyme concentrate dry matter/L (equivalent to 24.7 mg active enzyme protein/L) after 48h exposure. Thus, no 48h EC50 could be calculated and must be > 119.2 mg enzyme concentrate dry matter/L (equivalent to > 24.7 mg active enzyme protein/L).

Description of key information

Under the conditions of the test, Sedolisin, batch PPF38268 was found not to be toxic to Daphnia magna at a nominal concentration up to 119.2 mg enzyme concentrate dry matter/L (equivalent to 24.7 mg active enzyme protein/L) after 48h exposure. Thus, no 48h EC50 could be calculated and must be >119.2 mg enzyme concentrate dry matter/L (>24.7 mg active enzyme protein/L).

Subtilisin, batch PPA 4046 was used as a read-across and the 48-hour EC50 of the test substance for the immobilisation of Daphnia magna was nominal 60.4 mg enzyme concentrate dry matter/L corresponding to 12.4 mg active enzyme protein/L.