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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2017 - May 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
according to guideline
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
Physical state/Appearance: white solid
Batch: 80100005
Purity: 99.80%
Expiry Date: 13 October 2018
Storage Conditions: room temperature in the dark

Study design

Oxygen conditions:
Inoculum or test system:
sewage, domestic (adaptation not specified)
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 24 April 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Preparation of the inoculum:
The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.8 g/L prior to use.
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:
- Composition of medium: Mineral medium detailed in OECD TG 301 (1992, page 10 of 62).
- Test temperature: 22 – 24 °C
- pH: 7.4 ± 0.2 (n = 18, mean ± SD)
- pH adjusted: yes, if required.
- Aeration of dilution water: Yes, overnight prior to adding the test and reference item.
- Suspended solids (ss) concentration: 30 mg ss/L
- Continuous darkness: yes
Test followed a duplicate test design for inoculated and procedure controls and test item. Only one replicate was used for toxicity control.
The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.
Inorganic Carbon (IC) and IC/Total Carbon (TC) Ratio:
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. All samples were analyzed for IC immediately. The remainder of all samples were frozen for further analysis if required. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29. The samples were analyzed for IC using a Shimadzu TOC-LCSH TOC analyzer. Samples (135 μL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
Samples (30 mL) were removed from the inoculum control and test item vessels on Day 0 and filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. The samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer. Samples (50 μL) were injected into the TC and IC channels of the TOC analyzer. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in triplicate.
Calculation followed the OECD TG 301 (1992).
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge . Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.
% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 86% biodegradation after 14 days and 97% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Table 1. Percentage Biodegradation Values Throughout the Experiment.


       % Biodegradation

   Procedure Control  Test Item  Toxicity Control
 0  0  0  0
 2  50  0  27
 6  67  0  29
 8  81  2  34
 10  82  14  38
 14  86  5  43
 21  98  12  53
 28  90  20  65
 29  97  20  59

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2. Procedure Control and Test Item degradation values are a mean calculated from two replicate vessels.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The total CO2 evolution in the inoculum control vessels on Day 28 was 29.14 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines. The IC content of the test item suspension in the mineral medium at the start of the test was 0% which was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% (inoculum controls 7%, procedure controls 0% and test item 10%) and hence satisfied the validation criterion given in the OECD Test Guidelines.
Executive summary:

The biodegradation of the test item (WS-23) was assessed according to the OECD TG 301B. The test item cannot be considered to be readily biodegradable as only 20% biodegradation was attained after 28 days. This did not meet the criteria of attaining equal to or greater than 60% biodegradation as given in OECD Guideline No. 301B.