Licheninase

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:

1992-08-17 to 1993-06-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Given the similarity between the two enzymes, this study can be used as read-across for licheninase.REPORTING FORMAT FOR THE CATEGORY APPROACH

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

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 10000 mg/L. Serial dilutions of this stock solution were prepared with test water to give the desired series of exposure levels.
- Controls: test water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna Straus
- Source: Institute National de Recherche Chimique Appliqué (IRChA), France
- 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

Hardness:

171 mg CaCO3/L

Test temperature:

21 °C

pH:

7.7 - 8.1

Dissolved oxygen:

4.8 - 8.6 mg O2/L

Nominal and measured concentrations:

nominal. 320, 560, 1000, 1800, 3200 and 5600 mg/L

Details on test conditions:

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


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 move 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. Chlorine levels remained below detection limit at all times.
- Culture medium different from test medium: no


OTHER TEST CONDITIONS
- Photoperiod: 16 hours light: 8 hours dark


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4 900 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 3900 - 6200 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

519 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

138 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1700-2300 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

212 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

106 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Details on results:

- Mortality of control: 0

Reported statistics and error estimates:

EC50 values and 95% confidence limits were calculated according to the method of Thompson and Weil (Thompson, W.R. & Weil, C.S., 1952, Biometrics 8; 51-54).
The NOEC (no-observed-effect concentration) is the highest concentration at and below which the incidence of immobilisation is equal or less than 10%.

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 value for alpha-amylase with Daphnia magna is 2000 mg test material/L corresponding to 212 mg active enzyme protein/L.

Executive summary:

A study was performed to assess the acute toxicity of alpha-amylase to Daphnia magna.

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202, and in compliance with GLP.

Groups of 20, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to six concentrations of the test substance dissolved in water. The incidence of immobilisation was recorded for each test and control group at 24 and 48 hours and the following values determined:

EC₅₀ (24h): 4900 mg/L corresponding to 519 mg active enzyme protein/L

EC₅₀(48h): 2000 mg/L corresponding to 212 mg active enzyme protein/L

NOEC (48h): 1000 mg/L corresponding to 106 mg active enzyme protein/L

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 July 2016 to 12 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

28 July 2011.

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

- Concentrations: 6.25, 12.5, 25, 50 and 100 mg TOS/L
- Sampling method: All test material were diluted with test media to the final concentration directly on the plate.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Appropriate concentrations were made by dilution.
- Controls: ISO water

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

ACCLIMATION
- Acclimation period: The ephippia are stored in darkness, at 5°C (±2°C) to keep their viability. When the ephippia are placed under the illumination and temperature conditions (i.e. 6.000 lux and 20-22°C), the eggs develop in about 3 days of time into neonates which can then be used immediately for the toxicity tests.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

ISO medium. CaCl2.2H2O = 294 mg/L

Test temperature:

19-21 °C

pH:

7.0 - 8.0

Dissolved oxygen:

Within limits

Salinity:

Within limits

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg TOS/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
- Fill volume: 10 mL
- Aeration: The Standard Freshwater must be aerated for at least 15 minutes prior to using it.
- No. of organisms per vessel: 5
- 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

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: The daphnids were stored in the incubator at 20°C, in darkness.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 12.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 12.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

mobility

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.

Reported statistics and error estimates:

Not necessary in this case as no animals died.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, licheninase, batch PPB41588 was not found to be toxic to Daphnia magna at a nominal concentration up to 100 mg TOS/L after 48h exposure. Thus no 48h EC50 could be calculated and must be >100 mg TOS/L (>12.2 mg aep/L).

Executive summary:

The purpose of this study was to screen the potential toxic effect of licheninase, batch PPB41588 in Daphnia magna. The test system was DAPHTOXKIT F^TM magna supplied by MicroBioTests Inc.

Twenty daphnids, four replicates of five animals per well, were exposed in each control and test group. Daphnids were considered to be immobile if they were unable to swim within 15 seconds following gentle touch with a pin. The number of mobile and immobile daphnids were counted approximately 24 and 48 hours after the start of exposure. The daphnids were exposed for 48 hours to the below concentrations of the test item and a blank control.

Concentrations tested: 6.25, 12.5, 25, 50 and 100 mg TOS/mL.

EC50: calculated/estimated 50% effect concentration with respect to immobility.

No Observed Effect Concentration: NOEC is derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% is considered significant.

The results obtained in this investigation appear in the table below. No toxicity of the test item towards Daphnia magna was observed:

Test substance	EC50 conc. 48 hours	NOEC conc. 48 hours
Licheninase PPB41588	>100 mg TOS/L (>12.2 mg aep/L)	>100 mg TOS/L (>12.2 mg aep/L)

Description of key information

For crustacean Daphnia magna:

Under the conditions of the test, licheninase, batch PPB41588 was not found to be toxic to Daphnia magna at a nominal concentration up to 100 mg TOS/L after 48h exposure. Thus, no 48h EC50 could be calculated and must be >100 mg TOS/L (>12.2 mg/L aep).

The 48h EC50 value for alpha-amylase with Daphnia magna was 2000 mg test material/L corresponding to 212 mg active enzyme protein/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

212 mg/L