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Environmental fate & pathways

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:
From May 17 to June 14, 2019
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
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
according to
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
according to
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: crystalline
Specific details on test material used for the study:
Test Item name: Dehydrocholic Acid
Batch No.: 2018110457
Appearance: White microcrystalline powder
Expiry date: 30 November 2023
Storage: Room temperature (15 - 25 ºC)

Study design

Oxygen conditions:
not specified
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge, microorganisms from a domestic waste water treatment plant.
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 10 May 2019 (seven days before the test). The prepared activated sludge was continuously aerated (2L/minute) at the test temperature of 22±2 oC, for about 7 days.
Preparation of Activated Sludge Inoculum:
The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed (5.603 g wet weight), dried and the ratio of wet sludge to dry weight (0.4727 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (5 g dry weight that was equivalent to 59.27 g wet sludge) was suspended in mineral medium (Section 5.4; ad. 1000 mL) to yield a concentration equivalent to about 5 g per litre (on dry weight basis). The prepared activated sludge inoculum was aerated under test conditions (for 7 days) until use.
The pH of the activated sludge inoculum after preparation was 7.44, just before use the pH was: 7.31. A pH adjustment of activated sludge inoculum was not performed.
Pre-conditioning of Activated Sludge Inoculum:
Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium ) for 7 days (from May 10 to 17, 2019) at test temperature (the actual temperature: 20.0 – 20.7 oC). Before use the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of the different, 10-1, 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates.
Plates were incubated at 37 °C for 24 hours. The viable cell number of the cultures was determined by these plating experiments by manual colony counting.
The approximately cell count of aerated inoculum was ~108-109/L; therefore, before the test the inoculum was further diluted 10 000 x with mineral medium to reach the necessary ~104 – 106 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning (see above) improves the precision of the method.
The inoculum was not pre-adapted to the test chemical
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2.5 mg/L
Based on:
other: solubility
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test was carried out in a controlled environment room (during the preparation, aeration and incubation of the mineral medium, preparation of test bottles (units), during the formulation, oxygen and pH measuring) at a temperature of 22  2 C according to the guideline. The actual temperature range was 20.1 - 20.9 °C.
The test bottles were incubated in an incubator at 22  2 C, in the dark. During the incubation (28 days) of the test units the temperature range was 20.0-20.6 °C.
During the pre-conditioning of activated sludge inoculum, the temperature was 20.0-20.7 oC.
Temperature was measured continuously using min/max thermometer (in controlled environment room) or built-in thermometer (in incubator) and recorded at least once a day.
The oxygen concentration of test water (mineral medium) was in the range of 8-9 mg/L. It was measured at the start of the test and found to be 8.81 mg/L.
The pH was checked prior study start and found to be 7.42; further pH adjustment was considered as not necessary.
The test conditions were measured with suitable instruments and documented in the raw data.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
% degradation (O2 consumption)
Sampling time:
12 d
% degradation (O2 consumption)
Sampling time:
21 d
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
The percentage biodegradation of Dehydrocholic Acid reached a mean of 60.5 % after 28 days based on its ThODNH3. The highest biodegradability value of 62.3 % was obtained on the 21st day of the test. The test item biodegradation value reached its plateau at about the 12th day (where 57.4 % biodegradation was noticed) and from this day only slight changes were observed. Under the test conditions the test item biodegradation reached the pass level for ready biodegradability (the removal of 60 % of ThODNH3); however, did not fulfill the 10-day window criterion, therefore, the test item is considered to be not readily biodegradable.
In the toxicity control containing both, the test item and the reference item, a mean of 57.0 % biodegradation was noted within 14 days and 56.6 % biodegradation after 28 days of incubation. The biodegradation curve reached its plateau about on the 12th day, from that day the slight changes remained within the biological variability range of the applied test system. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

BOD5 / COD results

Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to a mean of 69.3 % after 14 days, and to a mean of 72.9 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable, but failing 10-day window
The test item is considered to be not readily biodegradable. According to the test guidelines the pass level for ready biodegradability is 60 % of ThODNH3 within the 10-day window.
In this study total oxidised nitrogen (nitrate and nitrite) concentration determination was not performed after the oxygen measurements.
According to the test guidelines the test item can be assumed as not inhibitory on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.
The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.