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Environmental fate & pathways

Biodegradation in water: screening tests

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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:
Due to the similarity between the two enzymes, similar results are expected for xanthan lyase.
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 4 g/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%.

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
Justification for type of information:
Due to the similarity between the two enzymes, similar results are expected for xanthan lyase.
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
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 (10 mL) 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 30 mg/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: 8.5 g/L; di-potassium hydrogen phosphate: 21.75 g/L; di-sodium monohydrogen phosphate dihydrate: 33.4 g/L; ammonium chloride: 0.5 g/L) plus 1 mL magnesium sulphate heptahydrate (22.5 g/L) plus 1 mL calcium chloride dihydrate (36.4 g/L) plus 1 mL iron(III)chloride hexahydrate (0.25 g/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: 30 mg/L

TEST SYSTEM
Test flasks containing a final volume of 500 mL were added magnetic stirrers and each bottle fitted with an electrolytic cell assembly (containing the electrolyte, 1M copper sulphate solution, and the CO2 absorber, 5 mL 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
Parameter:
% degradation (O2 consumption)
Value:
ca. 10
Sampling time:
1 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 60
Sampling time:
5 d
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.

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:
November 2005 - March 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to the similarity between the two enzymes, similar results are expected for xanthan lyase.
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 (25 mL) 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: 30 mg/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
Parameter:
% degradation (CO2 evolution)
Value:
ca. 10
Sampling time:
2 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
63
Sampling time:
6 d
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 CO2 production by the control mixtures (79.2 and 72.6 mg CO2) fulfill the validity criteria for this test.

The mean cumulative CO2 production 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 CO2 production (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.

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 June - 4 October 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to the similarity between the two enzymes, similar results are expected for xanthan lyase.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The study report included a current certificate of GLP compliance for the test facility.
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): Aeration tank at Worlingworth sewage treatment works (Suffolk, UK)
- Preparation of inoculum for exposure: aliquots (25 mL) 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: 30 mg/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: 20 to 24°C
- pH: 7.4 ± 0.2
- 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
Parameter:
% degradation (CO2 evolution)
Value:
ca. 10
Sampling time:
2 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
60
Sampling time:
7 d
Parameter:
% degradation (CO2 evolution)
Value:
102
Sampling time:
29 d
Results with reference substance:
The degradation of the reference substance had achieved 64% of its TCO2 after 6 days of incubation and 79% 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 102% 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 was performed in accordance with GLP.

The results obtained for the degradation of sodium benzoate (reference substance) and for cumulative CO2 production by the control mixtures (81.4 mg CO2 ) fulfil the validity criteria for this test.

The mean cumulative CO2 production by mixtures containing the test substance at 10 mg C/L was equivalent to appr. 10% of the TCO2 after 2 days, 60% on Day 7 and 102% 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.

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:
4 February - 30 July1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to the similarity between the two enzymes, similar results are expected for xanthan lyase.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: Activated sewage sludge bacteria
Details on inoculum:
- Source of inoculum/activated sludge: The aeration stage of the HRC Limited sewage treatment plant, treating predominantly domestic sewage.
- Preparation of inoculum for exposure: A sample of activated sewage sludge was collected from the sewage treatment plant on 31 March 1992. The sample was filtered through coarse filter paper (first 200 mL discarded) and used on the day of collection.
- Concentration of sludge: 0.5 mL of inoculum per litre dilution water
Duration of test (contact time):
28 d
Initial conc.:
90 mg/L
Based on:
test mat.
Initial conc.:
35 mg/L
Based on:
other: Sodium benzoate, Fisons A.R. Grade, Batch 2923430L, Purity 99%
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of test medium: Dilution water up to litre, plus 1 mL nutrient solutions (a)-(f): (a) potassium dihydrogen phosphate: 8.5 g/L; di-potassium hydrogen phosphate: 21.75 g/L; di-sodium monohydrogen phosphate dihydrate: 33.4 g/L; ammonium chloride: 20 g/L, (b) magnesium sulphate heptahydrate: 22.5 g/L, (c) calcium chloride: 27.5 g/L, (d) iron(III)chloride hexahydrate: 0.25 g/L, (e) Mangan sulphate tetrahydrate: 39.9 mg/L, boric acid: 57.2 mg/L, zinc sulphate heptahydrate: 42.8 mg/L, ammonium molybdate tetrahydrate: 36.85 mg/L, Iron-chelate: 100 mg/L and (f) Yeast extract: 15 mg/100 mL; plus 0.5 mL inoculum; plus sufficient test substance to give 5-40 mg/L final volume.
- Test temperature: 22 ± 1°C
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2 controls, 2 test substances and 2 standard substances
- Method used to create aerobic conditions: Aeration by magnetic stirrers and compressed air via narrow bore glass tubes.
- Measuring equipment: IONICS TC/TOC Analyser model 555.
- Test performed in open system: yes

SAMPLING
- Sampling frequency: day 0, 1, 3, 7, 14, 21, 27 and 28
- Sampling method: Samples (ca. 20 mL) were withdrawn from each vessel and filtered through Sartorius Minisart NML, 0.45 µm disposable filters, discarding the first 10 mL.
- Sample storage before analysis: Samples were analysed immediately.

CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculated test medium
- Standard substance: Sodium Benzoate
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (DOC removal)
Value:
28
Sampling time:
3 d
Parameter:
% degradation (DOC removal)
Value:
57
Sampling time:
7 d
Parameter:
% degradation (DOC removal)
Value:
74
Sampling time:
14 d
Parameter:
% degradation (DOC removal)
Value:
78
Sampling time:
21 d
Key result
Parameter:
% degradation (DOC removal)
Value:
91
Sampling time:
28 d
Results with reference substance:
Sodium benzoate achieved 74 % degradation within 3 days and 103% degradation within 28 days.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
Under the conditions of the test SP 491 Peroxidase was found to be ultimatelly biodegradable.
Executive summary:

Peroxidase, SP 491, was assessed for ready biodegradability according to OECD Guideline 301E and EEC Directive 67/548 Annex V C.3 as published 84/449/EEC, and in compliance with GLP.

Peroxidase, SP 491 attained 91% biodegradation within 28 days. The pass level of 70% was not reached within 10 days of exceeding the 10% level, thus the test substance cannot be termed as readilly biodegradable.

The reference substance attained 99% biodegradation within 28 days, thereby confirming the suitability of the inoculum and the culture conditions.

In conclusion, Peroxidase SP 491 was found to be ultimately biodegradable under the conditions of the test.

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:
May 19 to October 17, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to the similarity between the two enzymes, similar results are expected for xanthan lyase.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: Activated sewage sludge bacteria
Details on inoculum:
- Source of inoculum/activated sludge: The aeration stage of the Anglian sewage treatment plant, Godmanchester, treating predominantly domestic sewage.
- Preparation of inoculum for exposure: A sample of activated sewage sludge was collected from the sewage treatment farm on 19 May 1994. The sample was allowed to settle for approximately 30 min and supernatant filtered through Whatman GFA coarse filter paper (first 200 mL discarded). The filtrate was maintained under aeration and used within 24 hours of collection.
- Concentration of sludge: 0.5 mL of inoculum per litre dilution water
Duration of test (contact time):
28 d
Initial conc.:
1 000 mg/L
Based on:
test mat.
Initial conc.:
34 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of test medium: 1000 mL dilution water plus 1 mL nutrient solutions (a)-(f): (a) potassium dihydrogen phosphate: 8.5 g/L; di-potassium hydrogen phosphate: 21.75 g/L; di-sodium monohydrogen phosphate dihydrate: 33.4 g/L; ammonium chloride: 0.5 g/L), (b) magnesium sulphate heptahydrate (22.5 g/L), (c) calcium chloride (27.5 g/L), (d) iron(III)chloride hexahydrate (0.25 g/L), (e) Mangan sulphate tetrahydrate (39.9 mg/L), boric acid (57.2 mg/L), zinc sulphate heptahydrate (42.8 mg/L), ammonium molybdate tetrahydrate (36.85 mg/L), Jern-chelate: 100 mg/L and (f) Yeast extract 15 mg/100 mL; plus 0.5 mL inoculum; plus sufficient test substance to give 5-40 mg/L final volume. This is diluted to 2 litres with dilution water.
- Test temperature: 21 ± 1°C
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2 controls, 2 test substances and 2 standard substances
- Method used to create aerobic conditions: Aeration by magnetic stirrers and compressed air via narrow bore glass tubes.
- Measuring equipment: IONICS TC/TOC Analyser model 555.
- Test performed in open system: yes

SAMPLING
- Sampling frequency: day 0, 4, 7, 11, 14, 18, 21, 25 and 28
- Sampling method: Samples (ca. 20 mL) were withdrawn from each vessel and filtered through Sartorius Minisart NML, 0.45 µm disposable filters, discarding the first 10 mL.
- Sample storage before analysis: Samples were analysed immediately or stored at -20°C prior to analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculated test medium
- Standard substance: Sodium Benzoate
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (DOC removal)
Value:
67
Sampling time:
4 d
Key result
Parameter:
% degradation (DOC removal)
Value:
85
Sampling time:
11 d
Parameter:
% degradation (DOC removal)
Value:
91
Sampling time:
18 d
Parameter:
% degradation (DOC removal)
Value:
98
Sampling time:
28 d
Results with reference substance:
Sodium benzoate achieved 88 % degradation within 4 days and 99% degradation within 28 days.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the test SP 472 Terminox was found to be readily biodegradable.
Executive summary:

Catalase, SP 472 Terminox, was assessed for ready biodegradability according to OECD Guideline 301E and EEC Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 4-B, and in compliance with GLP.

Catalase, SP 472 Terminox achieved 67% biodegradation within 4 days and 98% biodegradation within 28 days. The pass level of 70% was reached within 10 days of exceeding the 10% level.

The reference substance attained 99% biodegradation within 28 days, thereby confirming the suitability of the inoculum and the culture conditions.

In conclusion, SP 472 Terminox was found to be readily biodegradable under the conditions of the test.

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:
23 December 2006 - 17 March 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to the similarity between the two enzymes, similar results are expected for xanthan lyase.
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): Worlingworth sewage treatment works, which treats predominantly domestic waste.
- Preparation of inoculum for exposure: aliquots (25 mL) of a homogenised sample were filtered through dried (approximately 105°C) and pre-weighed Whatman GF/C filter papers. Filters were dried for at least one hour, allowed to cool and re-weighed. The solids in the sludge determined and an appropriate volume used to inoculate control and test vessels.
- Initial cell/biomass concentration: 30 mg/L
- Water filtered: yes
- Type and size of filter used, if any: Whatman GF/C
Duration of test (contact time):
29 d
Initial conc.:
30 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: 22.1-23.8°C
- pH: 7.3 - 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 for inoculum blank and test substance. Single flask was used for reference alone and reference with 10 mg DOC/L test substance.
- 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 (100 mL) were connected to the air outlet from each test vessel.

SAMPLING
- Sampling frequency: on day 1, 2, 3, 4, 5, 6, 8, 9, 14, 21, 28 and 29
- Sampling method: 20 mL samples in duplicate were taken from the Dreschel bottle nearest to the test vessel, which was removed and replaced.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
19
Sampling time:
2 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
64
Sampling time:
8 d
Parameter:
% degradation (CO2 evolution)
Value:
85
Sampling time:
29 d
Results with reference substance:
The degradation of the reference substance, sodium benzoate, had reached 64% of its theoretical CO2 production (TCO2) after 5 days of incubation and 83% of its TCO2 after 29 days of incubation.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Asparaginase, PPV 24743, was considered to be readily biodegradable under the conditions of this test. The test substance reached 85% 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 (69.3 and 66.0 mg CO2) fulfill the validity criteria for this test.

The mean cumulative CO2production by mixtures containing Asparaginase, PPV 24743 at concentrations based on a dose concentration equivalent to 10 mg C/L reached approx 10% of the theoretical CO2 production (TCO2) after 2 days, 64% on Day 8 and 85% by the end of the test on Day 29.

On this basis, Asparaginase PPV, 24743 was considered readily biodegradable under the conditions of the test.

Description of key information

Xanthan lyase 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. All enzymes regardless the enzyme class were found to be readily biodegradable.

The physico-chemical properties of enzymes including logPow 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 enzymes should be fully applicable.

The overall conclusion is that xanthan lyase is readily biodegradable.