Amylase, α-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1992 to November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

Description of key information

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

EC10 or NOEC for freshwater algae:

2.6 mg/L

Additional information

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