Licheninase

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

August 1992 to November 1993

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Method: The test substance was dissolved in sterile nutrient medium to give an initial stock solution of 200 mg/L. This stock solution was further diluted with sterile nutrient medium to produce a series of solutions exactly twice the concentration of the intended exposure levels. 150 mL of algal preculture was mixed with 150 mL of each of these solutions to give final test series.
- Controls: medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Centre of Algae & Protozoa c/o Freshwater Biological Association, Cumbria, UK
- Method of cultivation:
Pre-culture: Sterile nutrient medium was inoculated from a master culture and incubated under continuous illumination (ca. 7000 lux) and stirring (orbital shaker) at 24 ± 1°C to give an algal suspension in log phase growth.
Culture conditions: Conical flasks each containing 100 mL test or control culture were loosely stoppered and placed at random in a Gallenkamp Illuminated Orbital Incubator. The cultures were incubated, without media renewal for 72 hours under continuous illuminatioon of approximately 7000 lux. The temperature was maintained at 24 ± 1°C and gaseous exchange and suspension of the algal cells was ensured by the action of the orbital shaker oscillating at 100 cycles per minute.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.9 - 24.2°C

pH:

7.5 - 10.2

Nominal and measured concentrations:

Nominal: 0, 6.25, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks (250 mL)
- Type (delete if not applicable): loosely stoppered
- Material, size, fill volume: glas, 250 mL, 100 mL
- Initial cells density: 8.2*10^4 cells/mL
- Control end cells density: 2.4*10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse-osmosis purified water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux provided by 7 x 30 W "warm white" 1 metre fluorescent tubes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm using 4 cm cell path cuvettes in a Cecil 373 Series 2 Spectrophotometer. The cell densities of the control cultures at initiation and at termination were determined by direct counting with the aid of a haemocytometer (Improved Neubauer).


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 6.25, 12.5, 25, 50 and 100 mg/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 22-26 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 42-57 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities in the control cultures or any of the test cultures apart from the two highest exposure levels (50 and 100 mg/L), where cells were observed to be smaller than average and clumped.
- Any stimulation of growth found in any treatment: no

Validity criteria fulfilled:

yes

Conclusions:

Alpha-amylase is inhibitory to the growth of Desmodesmus subspicatus at concentrations in excess of 12.5 mg/L corresponding to 1.3 mg active enzyme protein (aep)/mL. The EbC50 (72h) value is 24 mg/L and the ErC50 (24-72h) is 49 mg/L corresponding to 2.5 and 5.2 mg aep/L, respectively.

Executive summary:

A study was conducted to assess the inhibitory effect of alpha-amylase on the growth of the unicellular green alga Desmodesmus subspicatus, Strain No. CCAP 276/20.

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/458 Annex VIII, Part C (87/302/EEC).

Algal cultures exposed to five test concentrations of alpha-amylase plus one untreated control were incubated on an orbital shaker under continuous illumination at 24 ± 1°C for 72 hours. Growth was monitored daily by measuring the absorbance of each culture at 665 nm.

The following values were derived from the data:

E_bC₅₀(72h):       24 mg/l corresponding to 2.5 mg active enzyme protein (aep)/L

E_rC₅₀(24-72h):      49 mg/l corresponding to 5.2 mg aep/L

NOEC 72h for AUC (Area under the curve): 12.5 mg/L corresponding to 1.3 mg aep/L

NOEC for the 24 -72 hour growth rate: 25 mg/L corresponding to 2.6 mg aep/L

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 September 2016 to 27 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Principles of method if other than guideline:

Only nominal concentrations.

GLP compliance:

no

Specific details on test material used for the study:

Name: Licheninase
Batch nr.: PPB41588
Received: 18 July 2016
Condition: Frozen

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dilution in media
- Controls: ISO medium

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): MicroBiotests Inc. The algal growth inhibition bioassay has been developed by the research teams at the Laboratory for Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium.
- Method of cultivation: No culturing, the Algaltoxkit makes use of microalgae which are immobilised in a special matrix in which they survive for several months without losing their viability. After de-immobilization and transfer into an adequate algal culturing medium, the microalgae resume their growth immediately.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

According to the formula of the OECD Guideline 201 "Alga, Growth Inhibition Test" and the ISO/DIS Guideline 8692 "Water Quality - Freshwater Algal Growth Inhibition Tests with Unicellular Green Algae".

Test temperature:

23 ± 2°C

pH:

Within limits.

Dissolved oxygen:

Not measured.

Nominal and measured concentrations:

The algae were exposed for 72 hours to the following nominal concentrations of the test substance: 6.25, 12.5, 25, 50 and 100 mg TOS/L.

Details on test conditions:

A 72h algal growth inhibition test is performed in long cell test vials with Pseudokirchneriella subcapitata. Algae are supplied as de-immobilized algal beads. The Algal toxkit test follows the prescriptions of the OECD "Algal growth inhibition test" (Guideline 201) and the ISO "Water Quality - Freshwater Algal Growth Inhibition Tests with Unicellular Green Algae" (ISO Standard 8692).
Endpoint of the study is inhibition of algal growth rate.
The algae were exposed for 72 hours to the below concentrations of the test substances at the dose concentrations of 6.25, 12.5, 25, 50 and 100 mg TOS/L.
Algal cell density was measured after 24, 48 and 72 h exposure with a spectrophotometer at a wavelength of 670 nm and algal growth rate calculated according to ISO Standard 8692.

TEST SYSTEM
- Test vessel: 25 mL calibrated flask
- Material, size, headspace, fill volume: 25 mL
- Initial cells density: 1*10^4 algae/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes.
- Detailed composition if non-standard medium was used: According to the formula of the OECD Guideline 201 "Alga, Growth Inhibition Test" and the ISO/DIS Guideline 8692 "Water Quality - Freshwater Algal Growth Inhibition Tests with Unicellular Green Algae".

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 72 h
- Light intensity and quality: 10000 lux for sideway illumination or min. 3000-4000 lux for bottom illumination

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer (670 nm).

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 12.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 12.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

No significant growth inhibition and no abnormalities were observed.

Validity criteria fulfilled:

yes

Conclusions:

Licheninase batch PPB41588 was not toxic to Pseudokirchneriella subcapitata as no significant growth inhibition was observed up to a concentration of 100 mg TOS/L. Thus, no 72h ErC50 could be calculated, and should be >100 mg TOS/L (>12.2 mg aep/L).

Executive summary:

A 72h algal growth inhibition test is performed in long cell test vials with Pseudokirchneriella subcapitata. Algae are supplied as de-immobilized algal beads. The Algal toxkit test follows the prescriptions of the OECD "Algal growth inhibition test" (Guideline 201) and the ISO "Water Quality - Freshwater Algal Growth Inhibition Tests with Unicellular Green Algae" (ISO Standard 8692).

Endpoint of the study is inhibition of algal growth rate.

Average specific growth rate = ErC50 (0 - 72 h).

“No observed effect concentration”.

The algae were exposed for 72 hours to the below concentrations of the test substances.

 

Test compound	Exposure concentration mg TOS /L
	5	4	3	2	1
Licheninase, batch PPB41588	100	50	25	12.5	6.25

 

Algal cell density was measured after 72 h exposure with a spectrophotometer at a wavelength of 670 nm and algal growth rate calculated according to ISO Standard 8692.

Test substances	ErC50 (72h) Concentration in  mg TOS/L	NOEC (72h) Concentration in mg TOS/L
Licheninase, batch PPB41588	n.d	>100

n.d.: not detected 

Conclusion:

Licheninase batch PPB41588 was not toxic to Pseudokirchneriella subcapitata as no significant growth inhibition was observed up to a concentration of 100 mg TOS/L. Thus, no 72h ErC50 could be calculated, and should be >100 mg TOS/L (>12.2 mg aep/L).

 

Description of key information

For algae:

- Licheninase batch PPB41588 was not toxic to Pseudokirchneriella subcapitata as no significant growth inhibition was observed up to a concentration of 100 mg TOS/L. Thus, no 72h ErC50 could be calculated, and should be >100 mg TOS/L (>12.2 mg/L aep).

- Alpha-amylase is inhibitory to the growth of Desmodesmus subspicatus at concentrations in excess of 12.5 mg/L corresponding to 1.3 mg active enzyme protein (aep)/mL. The EbC50 (72h) value is 24 mg/L and the ErC50 (24-72h) is 49 mg/L corresponding to 2.5 and 5.2 mg aep/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.2 mg/L