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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 2017 - 22 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
July 17, 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
TOXI-COOP ZRT., 8230 Balatonfüred, Arácsi út 97., Hungary
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sewage plant for domestic sewage in Balatonfüred, Hungary, on 17 February 2017 (six days before the main test)
- Preparation of inoculum for exposure:
The activated sludge used was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with mineral medium and then aerated under test conditions (for 6 days) until use. The pH of the activated sludge inoculum after preparation was 7.69, just before use: 7.28.
- Pretreatment: The inoculum was not pre-adapted to the test chemical.
- Concentration of sludge: Microbial inoculum (2.0 mL per litre test medium) was added to each preparation bottle.
- Water filtered: yes
- Type and size of filter used: cotton wool
Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
test mat.
Initial conc.:
6.32 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Stadndard mineral medium was used. The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature (pH: 7.32; dissolved oxygen concentration: 8.97 mg/L)
- Test temperature: During the incubation (28 days) of the test units the temperature range was the following: 20.0 - 20.1 °C. During the preparation, aeration and incubation of the mineral medium the temperature was 20.7 - 21.3 °C.
- pH: 7.32
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Winkler bottles with special neck and glass stoppers
- Number of culture flasks:
10 containing test item and inoculum
10 containing reference item and inoculum
10 containing only inoculum (control)
10 containing reference item and inoculum (toxicity control)
- Method used to create aerobic conditions: Aeration system
- Measuring equipment:
Large glass tank (volume: ~30 L) and
Large glass bottles (volume: 5 L),
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 analyser
Temperature controlled (22 ± 2 °C) environment room and incubator with thermometer with exclusion of light,
Balance, Centrifuge.
Oxygen measurement: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method].
Temperature measurement: Temperature was measured continuously using a built-in thermometer of the thermostat and noted once or twice a day.

SAMPLING
- Sampling frequency: Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28; Nitrate and nitrite ion concentrations in the test solutions were determined on the 7th, 21st and 28th day of the test.
- Sampling method: The nitrite and nitrate concentrations were determined using photometric method with nitrite and nitrate cell test (Merck).
- Sample storage before analysis: The samples were stored in freezer until determination of nitrate and nitrite. The 7-day, 21-day and 28-day samples were analysed directly after oxygen measurements.

CONTROL AND BLANK SYSTEM
- Inoculum control: Only filtered inoculum (10 mL) was added to the aqueous test medium (ad. 5000 mL)
- Toxicity control: Test (50 mL) and reference item (50 mL) stock solutions were mixed into the aqueous test medium (ad. 5000 mL) corresponding to the test item concentration of 4.0 mg/L [chosen based on the preliminary experiment] and to 3.0 mg/L concentration of the reference item.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The test item solubility, behavior, and toxicity were tested in a 14-day preliminary experiment. In the preliminary experiment the test item was investigated at the concentration of 5 mg/L. No toxic effect of the test item was found at this investigated concentration.
Test performance:
All validity criteria of the study were met.
Key result
Parameter:
% degradation (O2 consumption)
Value:
48.6
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of the test item reached a mean of 48.6 % after 28 days based on its chemical oxygen demand (COD). The pass level for ready biodegradability is the removal of 60 % COD in a 10-day window. Under the test conditions of the present study a significant level of test item biodegradation was noticed; however, the value remained far below the pass level; therefore, the test item was considered to be not ready biodegradable.
Results with reference substance:
The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. (The percentage degradation of the reference item was 80.9 % on the 14th day.)

Results of Nitrite and Nitrate measurements

The measured quantities of nitrite in the inoculum control, test item and toxicity control samples were below the LOQ in the 0-day, 7-day and 14-day samples.

In the 21-day inoculum control samples in average 0.70 mg/L, in the 28-day inoculum samples in average 0.79 mg/L nitrite was detected. Similarly to the inoculum control samples, the nitrite concentrations in the 21-day and 28-day test item samples showed a slight increasing tendency.

In the 21-day and 28-day toxicity control samples the nitrite concentrations were less than 0.03 mg/L.

The nitrate concentration of the samples was less than 0.4 mg/L on day 0, day 7 and day 14; but in the 21-day inoculum samples in average 0.7, in the 28-day inoculum control samples in average 0.9 mg/L nitrate was detected. In the 21-day test item samples the nitrate concentration was in average 0.9 mg/L. In the 28-day test item samples in average 1.4 mg/L nitrate was determined. In the 21-day and 28-day toxicity control samples the nitrate concentrations were less than 0.4 mg/L. Nitrite and nitrate were measured even in the inoculum control samples. The oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population. Therefore, any correction of the measured dissolved oxygen concentrations was considered as not necessary or not possible. The measured relatively higher nitrite-nitrate concentration values were caused likely by a technical effect (possible discoloration of the solutions and/or turbidity).

Results of Toxicity Control

In the toxicity control a mean of 55.3 % biodegradation was noted within 14 days and a mean of 55.8 % biodegradation was determined after 28 days of incubation.

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
Under the test conditions the percentage biodegradation of the test item reached a mean of 48.6 % after 28 days based on its chemical oxygen demand (COD). 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 test item can be considered to be not ready biodegradable but inherently biodegradable.
Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item after exposure to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test was conducted according OECD TG 301 D (Closed bottle test).

The test item concentration of 4.0 mg/L was chosen based on the preliminary test results. The chemical oxygen demand (COD) of this multi-constituent test item (1.58 mg O2/mg test item) was determined at the start of the test. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated.

The parallel running analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. Therefore the biodegradability value of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed.

The reference item sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 55.3 % biodegradation was noted within 14 days and 55.8 % biodegradation after 28 days of incubation.

Under the test conditions the percentage biodegradation of the test item reached a mean of 48.6 % after 28 days based on its chemical oxygen demand (COD). 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 test item can be considered to be not ready biodegradable but inherently biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to section 13 for "Read-Across justification".
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
% degradation (O2 consumption)
Value:
48.6
Sampling time:
28 d

Description of key information

Under the test conditions the percentage biodegradation of the calcium salt of the target substance reached a mean of 48.6 % after 28 days based on its chemical oxygen demand (COD). 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 test item can be considered to be not ready biodegradable but inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

The ready biodegradability was addressed with read-across approach to the calcium salt of the target substance.

The purpose of this study was to determine the ready biodegradability of the analouge substance after exposure to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test was conducted according OECD TG 301 D (Closed bottle test).

The test item concentration of 4.0 mg/L was chosen based on the preliminary test results. The chemical oxygen demand (COD) of this multi-constituent test item (1.58 mg O2/mg test item) was determined at the start of the test. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated.

The parallel running analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. Therefore the biodegradability value of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed.

The reference item sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 55.3 % biodegradation was noted within 14 days and 55.8 % biodegradation after 28 days of incubation.

Under the test conditions the percentage biodegradation of the test item reached a mean of 48.6 % after 28 days based on its chemical oxygen demand (COD). 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 test item can be considered to be not ready biodegradable but inherently biodegradable.