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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2014.03.17 to 2014.03.17
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:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
- Source of inoculum/activated sludge: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 18 March 2014 (on the day of the test).
- Preparation of inoculum for exposure: 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 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 reconstituted water and then aerated until use (in this study 100 mL inoculum was prepared).
- 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: Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial conc.:
25 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: According to guideline
- Test temperature: During the preparation, aeration and incubation of the reconstituted water and at the activated sludge inoculum preparation and incubation, the temperature was 21.9-23.1 °C. During the incubation (28 days) of the test units the temperature range was the following: 20.8-22.9 °C.
- pH: The pH was checked prior study start and found to be 7.80.
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Winkler bottles with glass stoppers
- Number of culture flasks/concentration:
10 (+2 reserve) bottles containing the test item and inoculum
10 (+2 reserve) bottles containing the reference item and inoculum (procedure control)
10 (+2 reserve) bottles containing only inoculum (inoculum control)
10 (+2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)
- Method used to create aerobic conditions: aeration system.
- Measuring equipment:
Large glass tank (volume:~30 L),
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 analyzer
Temperature controlled (22±2°C) environment room with thermometer with exclusion of light,
Balance, Centrifuge, Ultrasonic bath.
- Measurement of Oxygen: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method].
Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.
- Measurement of total oxidized N: (nitrite and nitrate): Because of the nitrogen content of the test item, samples for nitrate and nitrite analysis were taken from all vessels (of test item, inoculum control and toxicity control group) and the oxidized nitrogen (nitrate and nitrite) concentrations were measured after each oxygen measurement.
- Measurement of Temperature: Temperature was measured continuously using min/max thermometer and noticed daily.


SAMPLING
- Sampling frequency : Nitrate and nitrite ion concentration were determined in test solutions at 0 , 7th, 14th, 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: In tightly closed container, under dry conditions at room temperature

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only filtered inoculum (10 mL) was added to the aqueous test medium (5000 mL).
- Toxicity control: Test (500 mL) and reference item (50 mL) stock solutions were mixed into the aqueous test medium (5000 mL) corresponding to the investigated test item concentration of 2.5 mg/L [chosen based on the preliminary information about the test item and based on its ThODNO3] and to 3.6 mg/L concentration of the reference item (ThODNH4 of 6.012 mg O2/L).

Reference substance:
benzoic acid, sodium salt
Preliminary study:
The concentration was chosen based on the preliminary test results and based on the theoretical oxygen demand of 2.65 mg O2/ mg test item (ThODNO3 -calculated according to equation given in the guidelines) of the test item. The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore the biodegradability of the test item in the main test was calculated based on its ThODNH4 of 2.35 mg O2/ mg test item.
In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.6 mg/L (as procedure control), inoculum control and toxicity control were investigated.
Test performance:
All validity criteria of the study were met.
Key result
Parameter:
% degradation (O2 consumption)
Value:
24.8
Sampling time:
28 d
Details on results:
- Biodegradation of the test Item: The percentage biodegradation of the test substance reached a mean of 24.8 % after 28 days based on its ThODNH4. Therefore the test item can be considered to be not ready biodegradable.
Key result
Parameter:
BOD5
Value:
0.61 other: mg O2/mg T.i
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to a mean of 67.6 % after 14 days, and to a mean of 71.9 % after 28 days of incubation, based on ThODNH4.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
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. The test item was exposed 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 item was investigated at the concentration of 2.5 mg/L. The concentration was chosen based on the preliminary test results and based on the theoretical oxygen demand of 2.65 mg O2/ mg test item (ThODNO3 -calculated according to equation given in the guidelines) of the test item. The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore the biodegradability of the test item in the main test was calculated based on its ThODNH4 of 2.35 mg O2/ mg test item. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.6 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of the test item reached a mean of 24.8 % after 28 days based on its ThODNH4. The reference item sodium benzoate was sufficiently degraded to a mean of 67.6 % after 14 days, and to a mean of 71.9 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 38.3 % biodegradation was noted within 14 days and 44.6 % biodegradation after 28 days of incubation. The test item is considered to be not ready biodegradable but inherently biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window.

The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

Description of key information

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
Type of water:
freshwater

Additional information

The purpose of this study was to determine the ready biodegradability of the test item. The test item was exposed 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 item was investigated at the concentration of 2.5 mg/L. The concentration was chosen based on the preliminary test results and based on the theoretical oxygen demand of 2.65 mg O2/ mg test item (ThODNO3 -calculated according to equation given in the guidelines) of the test item. The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore the biodegradability of the test item in the main test was calculated based on its ThODNH4 of 2.35 mg O2/ mg test item. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.6 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of the test item reached a mean of 24.8 % after 28 days based on its ThODNH4. The reference item sodium benzoate was sufficiently degraded to a mean of 67.6 % after 14 days, and to a mean of 71.9 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 38.3 % biodegradation was noted within 14 days and 44.6 % biodegradation after 28 days of incubation. The test item is considered to be not ready biodegradable but inherently biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.