Acetylglucosaminidase, β-

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April 2011 - 28 September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Justification for type of information:

Same sub-sub class of enzymes, therefore, similar results are expected for beta-N-acetylhexosaminidase.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

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

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Worlingworth sewage treatment works (Suffolk, UK).
- Preparation of inoculum for exposure: At the time of collection the sludge was sieved (1 mm2 mesh)then transported to the laboratory and left to stand for approximately 30 minutes to allow the sewage solids to settle. A portion of the supernatant was removed and the sludge aerated until required.
The concentration of suspended solids in a homogenised sample was determined before the start of the test. Aliquots (10mL) of the sludge were filtered through dried and preweighed Whatman GF/C filters, which were then dried agian at approximatley 105°C for one hour, allowed to cool in a desiccator and reweighed. Herafter the mixed liquor suspended solids content of the sludge was determined and the volume required to give a solids level of 30mg/L in test cultures were calculated. This was added to bottles one day before test initiation to allow a period of ageing.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

50 mg/L

Based on:

COD

Initial test substance concentration 2

Initial conc.:

357 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral Salts Medium: 10 mL Stock solution 1 (potassium dihydrogen phosphate: 8.5g/L; di-potassium hydrogen phosphate: 21.75g/L; di-sodium monohydrogen phosphate dihydrate: 33.4g/L; ammonium chloride: 0.5g/L) plus 1mL magnesium sulphate heptahydrate (22.5 g/L) plus 1mL calcium chloride dihydrate (36.4 g/L) plus 1mL iron(III)chloride hexahydrate (0.25g/L) was diluted in tap water to a final volume of 1 litre.
- Test temperature: 21.4 to 23.1°C
- pH: 7.2 to 7.8
- pH adjusted: to 7.4 ± 0.2 with 5M HCl
- Suspended solids concentration: 30mg/L


TEST SYSTEM
Test flasks containing a final volume of 500mL were added magnetic stirrers and each bottle fitted with an electrolytic cell assembly (containing the electrolyte, 1M copper sulphate solution, and the CO2 absorber, 5mL of 2M potassium hydroxidel) and connected to the computer controlled system. A magnetic stirrer was set to give a vortex on each mixture. The test was conducted in a thermostatically water bath at a temperature of 22 ± 2°C
- Number of culture flasks/concentration: 2 blank-control and 2 test, 1 reference, 1 inhibition assay, 1 ATU control and 1 ATU test
- Measuring equipment: Co-ordinated Environmental Services (CES) Ltd. automated respirometer and associated software was used to monitor cumulative amount of oxygen consumed by the mixtures. A record of the cumulative oxygen demand made by each cell was printed at, typically, hourly intervals.


CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculated Mineral Salts Medium alone
- Toxicity control: Test substance (50 mg O2/L) plus reference substance (50 mg O2/L) in inoculated mineral medium
- Reference substance: Sodium benzoate in inoculated mineral medium at 50 mg O2/L
- Allylthiourea (ATU) control: Inoculated mineral salts medium plus ATU (11.6 mg/L)

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

ca. 10

Sampling time:

1 d

% Degradation 2

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 60

Sampling time:

5 d

% Degradation 3

Parameter:

% degradation (O2 consumption)

Value:

129

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

The blank corrected oxygen demanded by the culture containing the reference substance (sodium benzoate) achieved 66% of the ThOD after 3 days of incubation and 96% by Day 28.
In the presence of cellulase degradation of sodium benzoate had achieved 64% by Day 2.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Cellulase was found to be readily biodegradable in the 'Ready Biodegradability, Manometric Respirometry Test' (OECD 301F).

Executive summary:

The ready biodegradability of Cellulase was assessed in accordance with OECD Procedure 301F ‘Ready Biodegradability, Manometric Respirometry Test’, adopted 17 July 1992 and in compliance with GLP. The main biodegradability test was preceded by chemical oxygen demand (COD) analysis as the theoretical oxygen demand of the test substance could not be calculated.

Cellulase was added to two bottles containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 50 mgO₂/L. Control groups comprised three cultures; two containing inoculated mineral salts medium alone, and one containing inoculated mineral salts medium plus the reference substance sodium benzoate (50 mgO₂/L). An additional mixture containing sodium benzoate and Cellulase (both at 50 mgO₂/L) was established in order to assess the potential inhibitory effects of the test substance on the microbial inoculum. Allylthiourea was added to one control culture and to one culture containing the test substance in order to assess nitrification processes by the test substance. The test system comprised of an automated system for oxygen (O₂) generation and the cultures were stirred and held in a thermostatically-controlled water bath.

The COD of Cellulase was found to be 0.14 mgO₂/mL.

The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 16.48 mgO₂/500 mL or 66% of the ThOD (25 mgO₂/500 mL) after 3 days of incubation. In the presence of Cellulase, degradation of sodium benzoate had achieved 64% by Day 2. Cumulative levels of oxygen consumption by the controls after 28 days (13.47 and 12.74 mgO₂/500 mL, equivalent to 26.94 and 25.48 mgO₂/L) were considered to be acceptable for this assay system. These results confirm that Cellulase was not inhibitory to the activity of the microbial inoculum and that the test was valid.

Mean oxygen consumption in biotic mixtures containing Cellulase was equivalent to 10% of the COD value (25 mgO₂/500 mL) after approximately 1 day, 60% after approximately 5 days and 129% at the end of the test (Day 28).  A degradation plateau was observed by approximately Day 10.

Substances are considered to be readily biodegradable in this type of test if oxygen consumption is equal to or greater than 60% of the COD value of the test mixtures within ten days of the consumption achieving 10%. Therefore, Cellulase was considered to be readily biodegradable under the conditions of this test.