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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 June 2003 - 8 September 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate included in 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): Oakley sewage treatment works ( UK).
- Preparation of inoculum for exposure: Aliquots (25 mL) of homogenised sample were filtered through dried and pre-weighed Whatman GFC filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solids concentration of 30 mg/L.
Duration of test (contact time):
28 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: Mineral Salts Medium: 10 mL Stock solution 1 (potassium dihydrogen phosphate: 8.5g/L; di-potassium hydrogen phosphate: 21.75 g/L; di-sodium monohydrogen phosphate dihydrate: 33.4 g/L; ammonium chloride: 0.5 g/L) was mixed with 800 mL dilution water (Tap water softened and treated by reverse osmosisn and then purified) and then adding 1 mL magnesium sulphate heptahydrate (22.5 g/L), 1 mL calcium chloride dihydrate (36.4 g/L) and 1 mL iron(III)chloride hexahydrate (0.25 g/L) for each litre of water required for the test.
- Test temperature: 20.1 - 23.8°C
- pH: 7.6 at the start
- pH adjusted: to 7.4 ± 0.2 with 5M HCl
- Aeration of dilution water: air-saturated
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Number of culture flasks/concentration: 2 control and 2 test substance, 1 reference, and 1 toxicity control
- Method used to create aerobic conditions: Test vessels were continuously flushed for 29 days with treated air.
- Test performed in closed vessels: yes
- Details of trap for CO2 : The air outlet from each vessel was connected to three Dreschel bottles in a series, each containing 0.025N, nominal barium hydroxide (100 mL).
- Other: The residual concentrations of barium hydroxide in the bottle nearest to the test vessels were determined at intervals by duplicate titration of 20 mL samples with hydrochloric acid (0.05N), using phenolphtalein indicator. Following the removal of the first Dreschler bottle in a series, the second was connected to the test vessel and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Mineral Salts Medium
- Toxicity control: Test substance (10 mg C/L) plus reference substance (10 mg C/L) in inoculated mineral medium
- Reference substance: Sodium benzoate in inoculated mineral medium at 10 mg C/L
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
10
Sampling time:
2 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
62
Sampling time:
6 d
Parameter:
% degradation (CO2 evolution)
Value:
105
Sampling time:
29 d
Results with reference substance:
The degradation of sodium benzoate achieved 65% of its TCO2 after 6 days and 88% after 29 days. Also in the presence of the test substance lipase, PPW 21880, sodium degradation of benzoate achieved 65% of its TCO2 after 6 days.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Lipase, PPW 21880 was found to be readily biodegradable in the CO2 evolution test.
Executive summary:

Introduction

The ready biodegradability of Lipase, PPW 21180 was assessed in the CO2Evolution test (Modified Sturm Test) Procedure C.4-C of the Annex to Directive 93/69/EEC, OECD Procedure 301B and US Environmental Protection Agency (EPA), Office of Prevention, Pesticides and Toxic Substances, (OPPTS) Method 835.3110 (m) (adopted January 1998).

 

RESULTS

Mean cumulative CO2production by mixtures containing Lipase, PPW 21180 was equivalent to 10% of the theoretical value by Day 2 and 62% on Day 6. Substances are considered to be readily biodegradable in this test if CO2production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. Therefore, Lipase, PPW 21180 can be considered to be readily biodegradable.

 

By the end of the test on Day 29, cumulative CO2evolution was >100% of the theoretical level (replicates 109% and 102%, mean 105%). However, as Lipase, PPW 21180 had achieved the pass criterion for ready biodegradation within a four-day period, this imprecision in the test system is not considered to have affected the interpretation of this investigation.

CONCLUSION

Lipase, PPW 21180 can be considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 1990 - 4 January 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
activated sludge, domestic (adaptation not specified)
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: The aerated sample was filtered through a coarse filter paper (first approximately 200 mL discarded) and used on day of collection.
- Concentration of sludge: 0.5 mL of inoculum per litre dilution water
Duration of test (contact time):
28 d
Initial conc.:
250 mg/L
Based on:
test mat.
Initial conc.:
ca. 40 mg/L
Based on:
ThIC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition oftest 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: 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) and (f) Yeast extract 15 mg/100 mL; plus 0.5 mL inoculum plus sufficient test substance. This is diluted to 2 litres with dilution water.
- Test temperature: 22 ± 1°C
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2 controls, 2 test substance and 2 reference substance
- 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 directly


CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculated test medium


Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (DOC removal)
Value:
44
Sampling time:
1 d
Key result
Parameter:
% degradation (DOC removal)
Value:
79
Sampling time:
3 d
Parameter:
% degradation (DOC removal)
Value:
92
Sampling time:
14 d
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
28 d
Results with reference substance:
Sodium benzoate achieved 79 % degradation within 3 days and 95% degradation within 28 days.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the test Lipase SP 400 was found to be readily biodegradable.
Executive summary:

Lipase SP 400 was assessed for ready biodegradability according to OECD Guideline 301E and EEC Directive 67/548 Annex V C.3 as published in 84/449/EEC, and in compliance with GLP.

SP 400 achieved 79% biodegradation within 3 days and 99% biodegradation within 28 days. The pass level of 70% was thus reached within 10 days of exceeding the 10% level.

The reference substance attained 95% biodegradation within 28 days, thereby confirmiung the suitability of the inoculum and the culture conditions.

In conclusion, SP 400 was found to be readily biodegradable under the conditions of the test.

Description of key information

Lipase was found to be readily biodegradable according to OECD test guideline 301. Lipase was found to be readily biodegradable according to OECD test guideline 301.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The ready biodegradability of two different lipase batches was assessed in accordance OECD Procedure 301B and 301E, and in compliance with GLP.

Both batches were more than 90% degraded within 28 days and the ten day window was fulfilled.

In addition, the results with the reference substance were within acceptance criteria and no inhibitory effects have been observed.

From these two studies it can be concluded that lipase is readily biodegradable.