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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
17 July 1992
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: crystalline

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, adapted
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
5.5.1 Environmental Conditions

The test was carried out in a controlled environment room (during the formulation and oxygen measuring) at a temperature of 22  2C according to the guideline. The test bottles were incubated in a controlled environment room at 22  2C, in the dark. Temperature was measured continuously using min/max thermometer and recorded every day.
During pre-conditioning of activated sludge inoculum the temperature was 20.0-20.9oC; during the preparation, aeration and incubation of the mineral medium, preparation of test bottles (units), the temperature was 20.4-20.9oC.
During the incubation (28 days) of the test units the temperature range was the following: 20.0-20.3oC.
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 9.35 mg/L.
The pH was checked prior study start and found to be 7.41. A pH adjustment was considered as not necessary.
The test conditions were measured with suitable instruments and documented in the raw data.

5.5.2 Equipments

Large glass tank (volume:~30 L) and
Large glass bottles (volume: at least 5L),
Narrow necked, Winkler bottles with glass stoppers,
Funnels and coarse filter papers,
Oxygen and pH meter with appropriate O2 and pH electrode,
Aeration system, Moisture analyzer
Temperature controlled (in the range of 22 ±2oC with a temperature deviation of ±1oC) incubator with thermometer with exclusion of light,
Balance, Centrifuge, Freezer,
Spectrophotometer (for nitrate, nitrite determination).

5.5.3 Test Units

Type and Size: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
Identification: Each test flasks were uniquely identified with study number, test group, days of measurement and replicate number.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
The pre-experiments on solubility of the test item, and the 9-day toxicity test was not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of present study.
The test item solubility, behavior, and toxicity were tested in a 9-day preliminary experiment. The test design was the same as described at the main experiment. In the preliminary experiment the test item was investigated at the concentration of 5.0 mg/L. No toxic effect of the test item was noticed at this investigated concentration level.
% Degradationopen allclose all
Key result
% degradation (O2 consumption)
ca. 41.2
Sampling time:
2 d
Key result
% degradation (O2 consumption)
ca. 60.9
Sampling time:
5 d
Key result
% degradation (O2 consumption)
ca. 64.4
Sampling time:
7 d
Key result
% degradation (O2 consumption)
ca. 67.3
Sampling time:
12 d
Key result
% degradation (O2 consumption)
ca. 67.7
Sampling time:
14 d
Key result
% degradation (O2 consumption)
ca. 68.2
Sampling time:
21 d
Key result
% degradation (O2 consumption)
ca. 108.2
Sampling time:
28 d

BOD5 / COD results

BOD5 / CODopen allclose all
Key result
ca. 60.9 other: %
Remarks on result:
other: 5th day
Key result
ca. 68.2 other: %
Remarks on result:
other: 21th day

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The test item is considered to be ready biodegradable, since the pass level for ready biodegradability that is the removal of 60 % ThODNH4 in a 10-day window was fulfilled.
Any correction of the BOD values with the observed nitrite and/or nitrate values was not performed.
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.
Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item
Ammonium Adipate. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 oC) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate (at a concentration of 3.0 mg/L) was tested simultaneously under the same conditions as the test item, and functioned as a procedure control (reference control). Additionally, inoculum (containing the filtered inoculum, only) and toxicity (containing both the test item and reference item) controls were examined.

The chosen test item concentration of 5.0 mg/L investigated in the main test was based on the calculated theoretical oxygen demand (ThODNH3assuming that no nitrification occurs) of 1.15 mg O2/mg test item, and based on theinformation about the toxicity of the test item (based on preliminary toxicity test results).

Under the test conditions ready biodegradation of this test item was noticed.

The percentage biodegradation of Ammonium Adipate reached a mean of 60.9 % on the 5thday of the test and 68.2 % after 21 days based on its ThODNH3(calculated based on the available chemical formula of the test item). The 10-day window criterion for ready biodegradation was unequivocally fulfilled, more than 60 % biodegradation of the test item (60.9 %, based on its ThODNH3)was observed already on the fifth day of the test.

In thistest the test item biodegradation curve reached its plateau approximately on the 7thday, from that day slight increases in biodegradability values occurred; however, for informative reason the test was not stopped before the 28thday.

The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred until the 21stday of the test. The measured quantities of nitrite and nitrate in the inoculum control, test item and toxicity control samples were below the LOQ in the 0-day, 7-day, 14-day and 21-day samples.The nitrate concentration of the 28-day samples was less than 0.4 mg/L (LOQ) as well.

However, the measured nitrite concentration was in average 0.51 mg/L in the 28-day inoculum samples and high nitrite concentrations, > 2.30 mg/L, above the measurement range were noticed in the 28-day test item samples.

According to the referred OECD 301 guideline (Annex V) the oxygen consumed in nitrate formation approximates 4.57x increase of nitrate-N concentration, and the oxygen consumed in nitrite formation is 3.43x increase of nitrite-N concentration.

In this study the change of the measured dissolved oxygen concentrations in the 28-day inoculum control bottles did not correspond to the calculated consumed oxygen of ammonium oxidation processes, but the dissolved oxygen concentration dropped radically in the 28-day test item bottles.

The test item biodegradation degree could allow an overgrowth of microbial population influencing the oxygen taken up or even the start ofa possible ammonium oxidationprocess that also affect the dissolved oxygen level.

Any correction of the measured 28-day dissolved oxygen concentrations was not possible in the case of test item samples, but the outlier high biodegradation level of the 28-day test item samples (without correction 108.2 %) was excluded from the evaluation. This exclusion has no effect on the final conclusion of the study.

The reference item Sodium benzoate was sufficiently degraded to a mean of 73.0 % after 14 days, and to a mean of 72.2 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.The biodegradability reached its plateau on about the 7thday and from this daythe slight changes were considered as being within the biological variability range of the applied test system.

In the toxicity control containing both, the test item and the reference item, a mean of 66.8 % biodegradation was noted within 14 days and 65.7 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).The biodegradability in the toxicity control reached its plateau on about the 7thday and from this daythe slight changes were considered as being within the biological variability range of the applied test system.