Glucosidase, β-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2011 - 12 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Deviations are considered not to have affected the validity and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate included in report

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Lot/batch No.: PPC31776
- Expiration date of the lot/batch: 07 December 2020
- Stability under test conditions: Stable for at least 24 hours at room temperature.
- Storage condition of test material: Frozen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and 100 mg TOS/L
- Sampling method: At the start of the definitive test, three samples (15 mL) were taken from the freshly-prepared control and test media. After 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Additional samples were also taken from a flask containingCellulase at a nominal concentration of 100 mg TOS/L but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.
- Sample storage conditions before analysis: frozen

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance (878 mg) was added directly to algal culture medium (1 L) to provide the test medium at a nominal concentration of 100 mg TOS/L. An aliquot (8.6 mL) of the algal inoculum was added to a portion (800 mL) of the test medium to give an initial cell density of 1 x 104 cells/mL. An aliquot (100 mL) of the appropriate inoculated test medium was added to each of the test vessels.
- Controls: Medium

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland
- Method of cultivation: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 9.3 x 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

21.8-24.1°C

pH:

7.54-7.96

Nominal and measured concentrations:

nominal: 0 and 100 mg TOS/L
At the start of the test, enzyme recovery was 104% of the nominal value. After 72 hours, the recovery decreased to 90% of nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): Flasks were loosely plugged with with foam bungs.
- Material, size, fill volume: glass, 250 mL, 100 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 1372083 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes (sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: nominally 4440 to 8880 lux provided by 6 x 30 W "cool white" 1 metre fluorescent tubes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell densities measured using a haemocytometer. The estimate of cell numbers in each sample was based on the mean of four consecutive counts. The presence of any abnormal cell numbers in each sample was also noted during screening of each test level.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations:1, 10, 100 mg TOS/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS (Total Organic Solids)

Basis for effect:

other: growth rate, biomass and yield

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 52.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

aep (active enzyme protein)

Basis for effect:

other: growth rate, biomass and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 52.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

aep

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 52.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

aep

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 52.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

aep

Basis for effect:

other: yield

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: at 100 mg TOS/L there was a significant stimulation at 24 to 48h, but no significant stimulation after 72h of exposure.

Reported statistics and error estimates:

The mean coefficient of variation (CoV) for daily growth rates in control cultures ranged between 5.87 and 8.18 during the definitive test and the CoV for the average specific growth rates of the control culture was 1.46 during the 72h period.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, Cellulase was not found to be acutely toxic to Pseudokirchneriella subcapitata at a nominal concentration of 100 mg TOS/L equivalent to 52.1 mg aep/L.
Consequently, the 72-hour EbC50, ErC50 and EyC50 values for Cellulase could not be calculated but must be >100 mg TOS/L equivalent to 52.1 mg aep/L and the “no observed effect concentration” was ≥ 100 mg TOS/L equivalent to 52.1 mg aep/L.

Executive summary:

The effect of Cellulase on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex IV to Commission Regulation (EC) No 761/2009 (O.J. No. L220/36, 2009) Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006).

Six algal cultures, with an initial nominal cell density of 1 x 10^4 cells/mL, were exposed to Cellulase at a nominal concentration of 100 mg TOS (Total Organic Solids)/L or 52.1 mg active enzyme protein (aep)/L. The test medium was prepared in OECD medium by the direct addition of the test substance to the

dilution medium. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 21.8 to 24.1 °C for 72 hours.

At the request of the Sponsor, the test concentration was verified by analysis of the enzyme concentration, which was performed at the Sponsor’s laboratory.

Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve, growth rate and yield.

The following values were derived from the data:

	Nominal Cellulase concentration (mg TOS/L)	Nominal Cellulase concentration (mg aep/L)
Area under the growth curve EbC50 (72 h)	>100	>52.1
Average specific growth rate ErC50 (0 - 72 h)	>100	>52.1
Yield EyC50 (0 - 72 h)	>100	>52.1
“No observed effect concentration”	≥ 100	≥ 52.1