Amylase, gluco-

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:

20 December 2005 - 02 November 2006

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 227.9 mg enzyme concentrate dry matter/L
- Sampling method: Triplicate samples, of 5 ml, were taken from control and the test stock solution (including the “no
algae” level) at 0 hours and from culture vessels at 72 hours (replicates pooled) for analysis. The samples were not filtered to remove algal cells before analysis. Additional samples were taken from flasks containing the “no algae” cultures at 72 hours (replicates pooled).
- Sample storage conditions before analysis: frozen at - 20°C in the dark

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A sample of the test material weighing 1584 mg was dispersed directly in 1 litre of algal medium to give the nominal test concentration of 227.9 mg enzyme concentrate dry matter/L.
- Controls: medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae & Protozoa, SAMS Research Services Ltd, Argyll, Scotland.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24°C

pH:

7.2 - 7.9

Nominal and measured concentrations:

A preliminary range finding study was conducted with nominal test concentrations of 1.04, 10.4, 103.6 and 227.9 mg enzyme concentrate dry matter/L.
Definitive study:
Nominal: 227.9 mg enzyme concentrate dry matter/L
Measured: 238.3 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): loosely stoppered
- Material, size, headspace, fill volume: glass, 250 mL, fill volume:100 mL
- Initial cells density: control: 12087 cells/mL; 220 mg TOS/L: 15073 cells/mL
- Control end cells density: 1348333 cells/mL
- No. of vessels per concentration (replicates): 6
- 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/deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7350-7540 Lux provided by fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] samples were taken at 24, 48 and 72 hours and the cell densities determined by direct counting using a haemacytometer (Improved Neubauer). Cell densities at 0 hours were to low to be counted using a haematocytometer and therefore estimation was conducted using a Coulter® Multisizer II particle counter.

TEST CONCENTRATIONS
- Preliminary test: Nominal test concentrations of 1.0, 10, 100 and 220 mg TOS/L.
- Results used to determine the conditions for the definitive study: yes

- Test concentrations in definitive study: 220 mg TOS/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 227.9 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.:

227.9 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): no
- Any stimulation of growth found in any treatment: no

Validity criteria fulfilled:

yes

Conclusions:

Glucoamylase, batch PPY24900 did not inhibit the growth of Pseudokirchneriella subcapitata, Strain No. CCAP 278/4 at a concentration of 227.9 mg enzyme concentrate dry matter/L under the conditions of this test.
Both ErC50 and EbC50 (0 - 72 hours) were > 227.9 mg enzyme concentrate dry matter/L (equivalent to > 103.7 mg active enzyme protein/L).
The no-observed-effect concentration NOEC was 227.9 mg enzyme concentrate dry matter/L (equivalent to 103.7 mg active enzyme protein/L).

Executive summary:

A study was conducted to assess the inhibitory effect of Glucoamylase, batch PPY24900 on the growth of the unicellular green alga Pseudokirchneriella subcapitata, Strain No. CCAP 278/4. Six algal cultures, with an initial cell count of approximately 1 x 10⁴ cells/mL, were exposed to Glucoamylase, batch PPY 24900 at a nominal test concentration of 227.9 mg enyzme concentrate dry matter/L. Mean measured concentration was 238.4 mg enzyme concentrate dry matter/L. These cultures, together with an untreated control group of six replicates, were incubated in a Gallenkamp illuminated orbital incubator under continuous illumination at temperatures in the range of 24 ± 1°C for 72 hours. Cell numbers were counted daily to monitor growth.

Verification of test concentrations was performed based on enzyme activity. The amount of measured enzyme (activity/g) was then converted to mg enzyme concentrate dry matter/L in order to calculate the achieved measured concentrations. Mean measured concentrations ranged from 119% of nominal at 0 hours (fresh), 92.5% at 72 hours (expired). The samples contaning no algae ranged from 120% at 0 hours (fresh, no algae), and 70% at 72 hours (expired, no algae). In the ‘no algae’ flasks it is considered that enzyme exposure to light energy would be greater with no algal cells to absorb light resulting in further enzyme degradation.

Glucoamylase, batch PPY24900 did not inhibit the growth of Pseudokirchneriella subcapitata, Strain No. CCAP 278/4 at a concentration of 227.9 mg enzyme concentrate dry matter/L under the conditions of this test.

Both ErC50 and EbC50 (0 - 72 hours) were > 227.9 mg enzyme concentrate dry matter/L (equivalent to > 103.7 mg active enzyme protein/L).

The no-observed-effect concentration NOEC was 227.9 mg enzyme concentrate dry matter/L (equivalent to 103.7 mg active enzyme protein/L).

Description of key information

Glucoamylase, batch PPY24900 did not inhibit the growth of Pseudokirchneriella subcapitata, Strain No. CCAP 278/4 at a concentration of 227.9 mg enzyme concentrate dry matter/L under the conditions of this test.

Both ErC50 and EbC50 (0 - 72 hours) were > 227.9 mg enzyme concentrate dry matter/L (equivalent to > 103.7 mg active enzyme protein/L).

The no-observed-effect concentration NOEC was 227.9 mg enzyme concentrate dry matter/L (equivalent to 103.7 mg active enzyme protein/L).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

103.7 mg/L

Additional information

NOEC based on active enzyme protein.