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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July to 25 August 2004
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
ASTM Guideline No.: E 1720-95; ISO/DIS Guideline No.: 14593; OPPTS Guideline No.: 835.3120
Principles of method if other than guideline:
ASTM Guideline No.: E 1720-95; ISO/DIS Guideline No.: 14593; OPPTS Guideline No.: 835.3120
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 3
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
not specified
Specific details on test material used for the study:
Ethyl phenols, Lot No.: 13 Jan 2004, purity 98.8%, received from Chemtran Services USA

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The activated sludge was obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachusetts.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
All solutions were prepared using purified reagent grade water (meeting ASTM Type II ® requirements) obtained with a Sybron/Barnstead NANOpure II system. The filter-sterilized water typically shows greater than 16.7 Mohm-cm resistivity and less than 1 mg/L total organic carbon, which is the detectable limit established by Springbom Smithers. The mineral salts medium was prepared using this purified reagent water. All chemicals were at least reagent grade and were obtained from commercial sources.
Activated sludge was collected on 28 July 2004 and transported to Springbom Smithers. Upon arrival at Springbom Smithers, the sludge was passed through a 2-mm stainless-steel metal screen and then centrifuged in four 1-L bottles for 10 minutes at 1500 rpm. The supernatant from each bottle was decanted and the sludge was kept in the bottles. Tap water was used to wash the sludge twice. The moisture content of the activated sludge was determined using an Sartorius Model MA-30 moisture balance to be 95.44%. Activated sludge (0.4386 g) was added to 2000 mL of test medium to give a solids concentration of 10 mg solids/L. The inoculum was stirred for ten minutes using a stir plate and Teflon-coated stir bar, sonicated for five minutes and filtered through glass wool prior to use.
The test design consisted of individual 20-mL clear glass crimp cap vials containing 13.5 mL of aqueous test medium. he test system was housed in the dark, in a walk-in environmental chamber set to maintain a temperature of 22 ± 2 °C. The daily temperature recorded in the environmental chamber during the study ranged from 19.6 to 21.9 °C. Test vessels were covered with aluminium foil and were mixed by swirling on days 2, 5, 7, 14, 21 and 28. To minimize bias, replicates were prepared, placed in the incubation chamber, and sampled randomly.
A 10 mg C/mL sodium benzoate stock solution was prepared by placing 1.7316 g of sodium benzoate in a 100-ml volumetric flask and bringing to volume with purified reagent water.
Eighty one (81) test vessels were established: 27 vials for the test substance, 27 vials for the reference substance (sodium benzoate) and 27 vials for the inoculum control. Test medium containing activated sludge microorganisms was added to each test vessel. Test vessels were dosed with the test substance or reference substance. Control vessels contained 13.5 mL of inoculated test medium only. Test solutions were prepared as described below:
Test solutions for ethyl phenols were prepared by adding 12.5 µL of test substance ( density = 1.02 g/mL) to 1000 mL of mineral media, resulting in a final concentration of 10 mg C/mL. An aliquot of this solution (13.5 mL) was then added to each of the 27 test vessels.
Sodium benzoate control solutions were prepared by adding 0.4 mL of the 10.0 mg C/mL stock solution to 400 mL of mineral media, resulting in a final concentration of 10 mg C/mL. An aliquot of this solution ( 13.5 mL) was added to each of the 27 test vessels.
After all of the test vessels had been filled, the vessels were scaled with butyl rubber septa and aluminum crimp caps, and wrapped in aluminium foil. The test vessels were then placed in the environmental chamber and incubated in the dark for 28 days.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
Ultimate biodegradation of the reference substance, sodium bcnzoatc, initially increased and
peaked on day 10 at 94.9%, then began to decrease until test termination (85.8% on day 28) due to continued C02 evolution in the inoculum controls. Sodium benzoate, therefore can be
considered "readily biodegradable" under the OECD criterion since > 60% C02 was produced within a 10-day window after reaching 10% C02 production. In addition, the rapid and
extensive biodegradation of sodium benzoate confirmed the presence of an active and viable
microbial population and confirmed system integrity.

Any other information on results incl. tables

Table 1: Cumulative CO2 (mg/L) evolved from the test vessels during the 28-day headspace biodegradation study

 Carbon Dioxide Evolved: mg/L  
 Test days
Test vessel24710142128
Inoculum control1.061.584.955.437.7010.169.02
Std. Dev.0.650.570.620.780.620.920.03
Sodium benzoate28.1631.6535.6139.4940.8142.5439.05
Std. Dev.0.490.331.620.560.830.971.31
Ethyl phenols10.1626.6230.5536.4938.8040.5939.79
Std. Dev.0.660.221.221.390.830.171.54

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
Ethyl phenols can be classified as "readily biodegradable" under conditions of the test.
Executive summary:

This study was performed to determine the potential for biodcgradation of ethyl phenols in water by the carbon dioxide evolution method based on the ASTM E 1720-95 Sealed Vessel, the ISO/DIS-14593 Headspace, and the OPPTS 835.3120 C02 Evolution Biodegradation Test guidelines.

Test vessels were incubated aerobically in the dark for a period of 28 days.

The mean biodegradation value for ethyl phenols at test termination was 87.0% of theoretical, indicating that biodegradation occurred. Furthermore, the biodegradation value for ethyl phenols on day 7 was 73. 9%. Since the biodegradation of ethyl phenols reached > 60% within the first 7 days, ethyl phenols can be considered "readily biodegradable" according to the OECD guidelines.

In addition, the rapid and extensive biodegradation of sodium benzoate confirmed the presence of an active and viable microbial population and confirmed system integrity.