1,4-α-glucan branching enzyme

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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:

The alpha amylase study was used as a key study for read-across given the sensitivity of this enzyme towards aquatic organisms.

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: 5.5, 11, 22, 44, 87.9 mg enzyme concentrate dry matter/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: 5.5, 11, 22, 44, 87.9 mg enzyme concentrate dry matter/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

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 (44, 87.9 mg enzyme concentrate dry matter/L), where cells were observed to be smaller than average and clumped.
- Any stimulation of growth found in any treatment: no

Conclusions:

The toxicity of the test substance was not tested, however, a read-across was performed to Alpha-amylase.
Alpha-amylase, batch PPA4048 was inhibitory to the growth of Desmodesmus subspicatus. The ErC50 (24-72h) was 43.1 mg enzyme concentrate dry matter/L 5.2 mg active enzyme protein/L. NOEC for the 24 -72 hour growth rate was 22 mg enzyme concentrate dry matter/L. EbC50 (72h) was 21.1 mg enzyme concentrate dry matter/L.

Executive summary:

The toxicity of the test substance was not tested, however, a read-across was performed to Alpha-amylase.

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_rC₅₀(24-72h):         43.1 mg enzyme concentrate dry matter/L (equivalent to 5.2 mg active enzyme protein/L)

E_bC₅₀(72h):              21.1 mg enzyme concentrate dry matter/L

NOEC for the 24 -72 hour growth rate:               22 mg enzyme concentrate dry matter/L

NOEC 72h for AUC (Area under the curve):       11 mg enzyme concentrate dry matter/L

Description of key information

1,4 -alpha-glucan branching enzyme wa not tesed with algae, however, read-across was performed with a sensitive species to alpha-amylase.

Alpha-amylase, batch PPA4048 was inhibitory to the growth of Desmodesmus subspicatus. The ErC50 (24-72h) was 43.1 mg enzyme concentrate dry matter/L, equvalent to 5.2 mg active enzyme protein/L. NOEC for the 24 -72 hour growth rate was 22 mg enzyme concentrate dry matter/L. EbC50 (72h) was 21.1 mg enzyme concentrate dry matter/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.2 µg/L