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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:
supporting study
Study period:
02 Jul - 02 Aug 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: ISO Standard 14593: Water quality - Evaluation of the ultimate aerobic biodegradability of organic compounds in an aqueous medium - Method by analysis of inorganic carbon in sealed vessels
Deviations:
yes
Remarks:
Minimum temperatures below the guideline range of 22 ± 2°C were recorded on days 1, 2 and 3.
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 835.3120 (Sealed Vessel Carbon Dioxide Production Test)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A sample of activated sludge was collected from one of the return lines at Burley Menston sewage treatment works (Yorkshire water) which treats predominantly domestic sewage.
- Storage conditions: On arrival in the laboratory, the sample was aerated by means of a compressed air supply.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
DOC
Initial conc.:
1.39 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: 10 mL/L solution (1) and 1 mL/L of each of solutions (2), (3) and (4) which were composed as follows: (1) 8.4999 g/L KH2PO4, 21.75 g/L K2HPO4, 33.3995 g/L Na2HPO4*2H2O, 0.4996 g/L NH4Cl, (2) 36.4004 g/L CaCl2*2H2O, (3) 22.5014 MgSO4*7H2O, (4) 0.25 g/L FeCl3*6H2O and one drop concentrated HCl
- Test temperature: 18.8 - 19.5 °C on the first three days, then 20.2 - 22.7 °C
- Suspended solids concentration in the medium: 4 mg/L
- Shaking: continuous shaking at 135 rpm
- Amount of sludge in the medium: 0.59 mL/L

TEST SYSTEM
- Culturing apparatus: "125 mL" glass serum bottles with a capacity of approx. 160 mL, sealed with rubber stoppers and aluminium crimp caps.
- Liquid/headspace ratio: 2:1
- Number of culture flasks/concentration: 5

SAMPLING
- Sampling frequency: Sampling was carried out on day 28
- Sampling method: 1 mL 7 M NaOH was injected through the rubber seal of each incubation flask and flasks were shaken for a further hour. Then, triplicate 1 mL samples of the liquid phase were drawn through the rubber plugs of each bottle using a gas-tight syringe.
- Sample storage before analysis: instant analysis

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 5 bottles
- Reference: yes, 5 bottles
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (inorg. C analysis)
Value:
88
St. dev.:
1
Sampling time:
28 d
Remarks on result:
other: percentage of theoretical inorganic carbon yield
Results with reference substance:
The reference substance attained 96 ± 3% degradation after 28 days, expressed as percentage of theoretical inorganic carbon yield.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Jun - 05 Aug 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
adopted 2006
Qualifier:
according to
Guideline:
other: ISO 14593, adopted 1999
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Five days before test start, a sample of activated sludge was obtained from the aeration tank of Worlingsworth sewage treatment works, which treats predominantly domestic waste.
- Preparation of inoculum for exposure: On the day of collection, the sample was passed through a sieve with a mesh of 1 mm2. A sub-sample (500 mL) was removed and centrifuged at 3000 rpm for 1 min and the supernatant removed. The sample was then made up to volume with mineral salts medium and centrifuged. This procedure was repeated twice and the sample was aerated until required.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD guideline 301 was used.
- Solubilising agent: 0.094 mg Tween 85 was added to each vial
- Test temperature: 21.1 - 23.0 °C
- pH: 7.5
- Aeration of dilution water: The test vessels with medium were aerated for 5 days until test start.
- Suspended solids concentration: 4 mg/L culture medium

TEST SYSTEM
- Culturing apparatus: 160 mL Wheaton vials containing 100 mL of culture
- Number of culture flasks/concentration: 26 vials
- Measuring equipment: OI Model 1010 Carbon Analyser with autosampler

SAMPLING
- Sampling frequency: Days 0, 3, 7, 8, 10, 14, 21 and 28
- Sampling method: Injection of 7 M NaOH, shaking for 1 h, transfer of content to carbon analyser vials which were immediately sealed.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 26 vials
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
76.5
Sampling time:
28 d
Details on results:
- Degradation kinetics: The test substance attained 29.9, 50.6, 52.1, 61.6, 66.3 and 76.5% degradation after 3, 7, 8, 10, 14 and 28 days, respectively.
- At the end of the 10 day window, more than 60% degradation was reached.
Results with reference substance:
Sodium benzoate attained 91.2% after 14 days and 93.6% after 21 days.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable

Description of key information

Ready biodegradable: 76.5% (CO2 evolution) in 28 days (OECD 310)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Ready biodegradability was assessed in two tests with domestic activated sludge as inoculum. The key study (Dickinson, 2008) was carried out according to OECD guideline 310 and GLP. Based on CO2 evolution, a degradation rate of 76.5% was determined after 28 days of incubation (the 10-day window was met). The biodegradation assessment is further supported by a non-GLP study carried out according to ISO Standard 14593 (Bealing, 2001).After 28 days of incubation, the substance attained 88% degradation based on CO2 evolution.

In conclusion, 2-ethylhexyl benzoate (CAS No. 5444-75-7) can be considered readily biodegradable according to OECD criteria.