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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5th April - 3rd May 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
Specific details on test material used for the study:
Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): ETE Barueri (Sabesp)
- Concentration of sludge: 0.02 mg/L suspended solids
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Preparation of stock solutions:
The stock solution of test item (1000 mg/L) was prepared by weighing 0.1000 g using an analytical balance and diluting with 100.0 mL of deionised water.
The stock solution of reference substance (1000 mg/L) was prepared by weighing 0.1000 g of sodium benzoate using an analytical balance and diluting with 100.0 mL of deionised water.

Preparation of test solutions:
The stock solutons were constantly mixed during the preparation of the test solutions. The test solutions were prepared in synthetic mineral medium.

To verify the ready biodegradability of the test item, a solution of 2.00 mg/L was prepared.

To evaluate the activity of the microorganisms present in the water, a solution of 2.00 mg/L with the reference substance, sodium benzoate was prepared.

To evaluate if the test item was toxic to the microorganisms, a toxicity control comprising 2.00 mg/L of the test item and 2.00 mg/L of the reference substance was prepared.

A blank control, comprising only the synthetic mineral medium and the innoculum of microorganisms, was included in the test to eliminate the consumption of oxygen by interfering substances.

Ten flasks were prepared for each treatment. Two flasks were used for each reading day.


The concentration of dissolved oxygen (mg O2/L) and the pH of all the solutions were determined a the start of the test (day 0). On days 7, 14, 21 and 28, two flasks of each test solution were removed for analysis of the dissolved oxygen concentration by determining biochemical oxygen demand (BOD). The water temperature was checked daily.

Determination of the chemical oxygen demand (COD) of the samples:
The COD analysis of thesamples was performed by the closed reflux method followed by colorimetric determination, according to the Standard methods for the examination of Water and Wastewater (APHA et al., 2005) and the equipment manufacturer specifications (HACH, 1993).

The sample was homogenized and an aliquot was diluted in deionized water, providing a stock solution with adequate concentration for analysis. This preparation was performed in duplicate and each prepared solution an aliquot was taken and added to a flask containing the reagents for acid digestion. The tubes were placed in a digestion tube at 150 ° C for 2 hours. A blank control with deionized water was treated the same way. After cooling, a spectrophotometer was used to measure the absorbance of the solutions present in the tubes to determine the COD of the sample.

Treatment of results:

For each day of analysis the oxygen consumption was calculated as the difference in the consumption of oxygen of the blank control and the test sample solutions and the result was expressed as in mg O2/mg of test substance:

BOD = (mg O2 sample - mg O2 blank)/ mg sample

The % biodegradation was calculated as:

% Biodegradation = BOD x 100/COD
Reference substance:
benzoic acid, sodium salt
% degradation (O2 consumption)
Sampling time:
7 d
% degradation (O2 consumption)
Sampling time:
14 d
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
It was verified that there was consumption of 1.08 mg/L of oxygen in the blank control, lower than the limit of 1.50 mgO2/L.
The percentage of biodegradability obtained for the reference substance was 70%, above the limit of 60% established by the test standard.
The residual concentration of oxygen in the treatments (sample and reference) was above 4.41 mgO2/L.
All of the Test validation criteria were addressed (OECD, 1992).
Results with reference substance:
The percentage of biodegradability obtained for the reference substance was 70% after 28 days

Oxygen consumption (mg O2/ mg sample):

 Solution  Day 7  Day 14  Day 21  Day 28
 Reference substance  1.38  1.30  1.19  1.17
 Test sample  0.38  0.45  0.60  0.57
 Toxicity control  0.75  0.86  0.84  0.79
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
Under the conditions of this study, the test item was readily biodegradble.

Description of key information

Based on a study performed according to OECD 301D, the substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:

Additional information