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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-02-1992 and 30-07-1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Version / remarks:
OECD Guideline No. 301E and EEC Directive 67/548 Annex V C.3 as published in 84/449/EEC.
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Active enzyme protein of peroxidase (EC no. 232-668-6, CAS no. 9001-05-2, EC name Peroxidase, Enzyme class no. 1.11.1.7)
Molecular formula:
Not applicable, see remarks.
IUPAC Name:
Active enzyme protein of peroxidase (EC no. 232-668-6, CAS no. 9001-05-2, EC name Peroxidase, Enzyme class no. 1.11.1.7)
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
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:
solid: crystalline
Details on test material:
- Lot/batch No.: PPX 3806
- Expiration date of the lot/batch: 04 March 1994

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
46.7 mg/L
Based on:
DOC
Initial conc.:
90 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Standard substance: Sodium benzoate.
Innoculum pre-treatment: The aerated sample was filtered through a coarse filter paper (first approximate 200 mL discarded) and used on the day of collection.
Usage rate: 0.5 mL of inoculum per litre of dilution water.
Vessels: 2 litre glass beakers.
Test concentrations: (a) Inoculated test medium. (b) 90 mg/L test substance. (c) 35 mg/L standard substance. Each in duplicate.
Preparation of cultures: The dissolved organic carbon (DOC) of each stock solution was determined and sufficient material added to 2 litres medium to give a nominal initial value of ≈45 mgC/L for the test substance and ≈20 mgC/L for the standard substance.
Aeration: By magnetic stirrers and compressed air via narrow bore glass tubes.
Temperature: 22 ± 1°C.
Lighting: The test vessels were shielded from light.
Evaporation: Losses of evaporation were made good by the addition of deionised water immediately prior to sampling.
Sampling: Samples (≈ 20 mL) were withdrawn from each vessel and filtered through Sartorius Minisart® NML, 0.45 µm disposable filters, discarding the first 10 mL. The samples were analysed immediately. Samples were taken on Days 0, 1, 3, 7, 14, 21, 27 and 28.
Analysis: Measurement of dissolved organic carbon (DOC) was conducted using an Ionics TC/TOC Analyser Model 555. DOC analysis was carried out in duplicate for each sample.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
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
Key result
Parameter:
% degradation (DOC removal)
Value:
91
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
The reference substance - sodium benzoate - had 74% degradation by day 3 and 103% degradation by day 28.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Peroxidase attained 91% biodegradation within 28 days. The "pass" level of 70% was not reached within 10 days of exceeding the 10% level. However, results should be interpreted with caution as enzymes are multi-component substances, which may imply that the 10-day window criterion is difficult to accomplish. Therefore, peroxidase can be classified as readily biodegradable.
Executive summary:

Biodegradability of peroxidase was tested using OECD Guideline No. 301E and EEC Directive 67/548 Annex V C.3 as published in 84/449/EEC under GLP. Criterion for assessment was Reduction in dissolved organic carbon (DOC). The testing concentrations were (a) Inoculated test medium, (b) 90 mg/L test substance, (c) 35 mg/L standard substance.

The dissolved organic carbon (DOC) of each stock solution was determined and sufficient material added to 2 litres medium to give a nominal initial value of ≈45 mgC/L for the test substance and ≈20 mgC/L for the standard substance. The biodegradation of Peroxidase and the standard substance is shown below:

% Biodegradation

Day       Test substance       Standard

0               0                            0

1            <10>                    <14>

3              28                          74

7              57                          90

14            74                          89

21            78                          99

27            86                         106

28            91                         103

Peroxidase attained 91 % biodegradation within 28 days. The "pass" level of 70% was not reached within 10 days of exceeding the 10% level. However, results should be interpreted with caution as enzymes are multi-component substances, which may impy that the 10-day window criterion is difficult to accomplish. Therefore, peroxidase can be classified as readily biodegradable.

The standard substance, sodium benzoate, attained 103 % biodegradation within 28 days, thereby confirming the suitability of the inoculum and the culture conditions.