Pullulanase

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1995-02-13 to 1995-06-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Enzymes and other proteins are polymers built of different combinations of the 20 common amino acids. The sequence and length of the amino acids in the polymer differ between enzymes, and this determines the 3-dimensional structure, the activity and specificity of the enzyme. The physico-chemical characteristics of enzymes are mainly dependent on the amino acids building the enzyme. Since all enzymes are built up of a combination of the same 20 common amino acids, the physical and chemical characteristics will be very similar across different enzymes as well as the biodegradability.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in the report

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Domestic waste water treatment plant : ARA Ergolz II, Füllinsdorf, Switzerland
- Preparation of inoculum for exposure: The sludge was washed three times with tap water and an amount corresponding to 4g/L dry material (± 10%) was mixed with Sörensen buffer solution pH7 and then aerated prior to incubation. An amount of 0.5 mL sludge (filtered over cotton wool) was added to 1000 mL test medium.

Duration of test (contact time):

ca. 28 d

Initial conc.:

44 mg/L

Based on:

test mat.

Initial conc.:

>= 18.2 - <= 19 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: according to OECD Guideline No. 301 E
- Test temperature: 19.7 - 21.5°C
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2


SAMPLING
- Sampling frequency: day 0 (treatment day) , 7, 14, 21, 27 and 28
- Sampling method: Per sampling interval, two flasks of the samples containing the test article or the reference compound, one flask of the inoculum blank and one flask of the toxicity control were taken and analysed for DOC in triplicate.


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: Aniline and the test compound were dissolved in the test medium at concentrations of 45 mg/L SP 703 and 26 mg/L aniline corresponding to an actually measured concentration of 39.3 mg DOC/L

Reference substance:

aniline

Preliminary study:

Test substance concentration was based on the DOC-values from a pre-test.

Key result

Parameter:

% degradation (DOC removal)

Value:

99

Sampling time:

28 d

Key result

Parameter:

% degradation (DOC removal)

Value:

95

Sampling time:

7 d

Results with reference substance:

The reference compound aniline reached complete biodegradation during the first seven exposure days. At the end of the test, aniline was readily biodegraded by an average of 95.1%.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Alpha-amylase was found to be readily biodegradable in the 'Modified OECD Screening Test' (OECD 301E).

Executive summary:

Alpha-amylase was tested for ready biodegradability in the '28 -day Modified OECD Screening Test'. The test was performed according to OECD 301E and in compliance with GLP.

The concentration of DOC (Disssolved Organic Carbon) in the test flasks containing alpha-amylase decreased by an average of 95.0 %, 92.5 % and 93.8% within 7, 14 and 21 days of exposure, respectively. At the end of the test (day 28), alpha-amylase was biodegraded by an average of 99.0%. Consequently, alpha-amylase was found to be readily biodegradable.

The reference compound Aniline reached complete biodegradation during the first seven exposure days. At the end of the test (day 28) Aniline was readily biodegraded by an average of 95.1%.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Enzymes and other proteins are polymers built of different combinations of the 20 common amino acids. The sequence and length of the amino acids in the polymer differ between enzymes, and this determines the 3-dimensional structure, the activity and specificity of the enzyme. The physico-chemical characteristics of enzymes are mainly dependent on the amino acids building the enzyme. Since all enzymes are built up of a combination of the same 20 common amino acids, the physical and chemical characteristics will be very similar across different enzymes as well as the biodegradability.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Aeration tank at Worlingworth sewage treatment works (Suffolk, UK)
- Preparation of inoculum for exposure: aliquots (25mL) of a homogenised sample were filtered through dried (approximately 105°C) and pre-weighed Whatman GF/C filter papers. Filters were dried and re-weighed. The solids in the sludge determined and an appropriate volume used to inoculate control and test vessels.
- Initial cell/biomass concentration: 30mg/L
- Water filtered: yes
- Type and size of filter used, if any: Whatman GF/C

Duration of test (contact time):

29 d

Initial conc.:

10 mg/L

Based on:

ThIC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:Composition of Mineral Salts Medium was consistant with the medium described in OECD test guideline 301 (301B).
- Test temperature: 22.2 to 23.8°C
- pH: 7.4 - 7.6
- pH adjusted: yes
- Aeration of dilution water: Inoculum was aerated prior to test initiation.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes (test performed in amber glass bottles)

TEST SYSTEM
- Number of culture flasks/concentration: Duplicate test flasks used.
- Method used to create aerobic conditions: Vessels were continuously flushed with air (air has been treated before to remove carbon dioxide)
- Details of trap for CO2 and volatile organics if used: 3 Dreschel bottles in series, each containing 0.025N, nominal barium hydroxide (110 mL) were connected to the air outlet from each test vessel.


SAMPLING
- Sampling frequency: daily during the first week of the test and hereafter on day 10, 14, 21, 28 and 29
- Sampling method: 20 mL samples in duplicate were taken from the Dreschler bottle nearest to the test vessel, which was removed and replaced.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 10

Sampling time:

2 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

63

Sampling time:

6 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

90

Sampling time:

29 d

Results with reference substance:

The degradation of the reference substance, sodium benzoate, had reached 60% of its theoretical CO2 production (TCO2) after 6 days of incubation and 76% of its TCO2 after 29 days of incubation.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test substance was considered to be readily biodegradable under the conditions of this test. The test substance reached 90% degradation by the end of the 29-day test period.

Executive summary:

A ready biodegradability test (OECD test 301B, Modified Sturm Test) was performed to determine the biodegradability characteristics of the test substance.

The study was conducted according to OECD and EC test guidelines, and in compliance with GLP.

The results obtained for the degradation of sodium benzoate (reference substance) and for cumulative CO₂production by the control mixtures (79.2 and 72.6 mg CO₂) fulfill the validity criteria for this test.

The mean cumulative CO₂production by mixtures containing the test substance at concentrations based on a dose concentration equivalent to 10 mg C/L reached approx 10% of the theoretical CO₂production (TCO₂) after 2 days, 63% on Day 6 and 90% by the end of the test on Day 29.

On this basis, the test substance was considered readily biodegradable under the conditions of the test.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

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:

The study was performed in accordance with Directive (EC) 440/2008, C.4-D, 'Determination of ready biodegradability, Manometric respirometry' and OECD proceudre 301F 'Ready biodegradability, Mamometric Respirometry Test', adopted 17 July 1992 and in compliance with GLP.

Justification for type of information:

Enzymes and other proteins are polymers built of different combinations of the 20 common amino acids. The sequence and length of the amino acids in the polymer differ between enzymes, and this determines the 3-dimensional structure, the activity and specificity of the enzyme. The physico-chemical characteristics of enzymes are mainly dependent on the amino acids building the enzyme. Since all enzymes are built up of a combination of the same 20 common amino acids, the physical and chemical characteristics will be very similar across different enzymes as well as the biodegradability.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in report

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 conc.:

50 mg/L

Based on:

COD

Initial conc.:

357 mg/L

Based on:

test mat.

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:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 10

Sampling time:

1 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 60

Sampling time:

5 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

129

Sampling time:

28 d

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.

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 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.

Description of key information

Pullulanase is readily biodegradable.

 

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Biodegradability studies according to OECD 301 test guidelines have been performed with a wide variety of industrial enzymes among those alpha-amylase, glucoamylase and cellulase, which are glycosidases like pullulanase. Alpha-amylase, glucoamylase and cellulase were found to be readily biodegradable, but also industrial enzymes belonging to other enzyme classes are generally found to be readily biodegradable. Thus pullulanase can be considered readily biodegradable.

The physico-chemical properties of enzymes including logKow are very similar . They are further proteins built up of amino acids and the type, order and number of the amino acids in the polymer differs between enzymes, determining the 3-dimensional structure, the activity and specificity of the individual enzyme type. Because all enzymes are built up of the same amino acids the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other non-proteolytic enzymes should be fully applicable.

The overall conclusion is that pullulanase is readily biodegradable.