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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:
2016-01-08 to 2016-03-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 06 January 2016 (7 days before 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 test water (see below) and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.62, just before use: 6.87. A pH adjustment of activated sludge inoculum was not performed.

- Pretreatment: Pre-conditioning (06-13 January 2016) consisted of aerating (2 L/minute) activated sludge (in mineral medium, reconstituted water) for 7 days at the test temperature (the actual temperature range was 20.5-22.9 °C). During the aeration 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, usually 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum fell in the range of 108/L; therefore on the day of the test this inoculum was diluted adequately with reconstituted water to reach the necessary cell concentration (guideline proposal of 104-106 approx. cells/L). After preparation the sludge was filtered through cotton wool. Pre-conditioning improved the precision of the test methods by reducing blank values. The inoculum was not pre-adapted to the test chemical.
Nutrient agar: Supplier: MERCK;
Batch Number: VM664850,
Expiry date: 05 November 2019

Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: In water (purified deionised) analytical grade salts were added to prepare the following stock solutions:
a) KH2PO4, 8.50 g; K2HPO4, 21.75 g; Na2HPO4 x 12H2O 67.16 g; NH4Cl 0.50 g; Water ad. 1000 mL
b) CaCl2 x 2H2O 18.20 g; Water ad. 500 mL;
c) MgSO4 x 7 H2O 11.25 g; Water ad. 500 mL
d) FeCl3 x 6H2O 0.125 g; Water ad. 500 mL
(The “d” stock solution was prepared on the day of the reconstituted water preparation and was not further stored). 1-1 mL of the stock solutions a - d) were combined and filled to a final volume of 1000 mL with deionised water (aqua purificata). The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was checked and found 8.22 mg/L. The pH of the reconstituted water was 7.22.

- Test temperature: During the preparation, aeration and incubation of the reconstituted water and at the activated sludge inoculum preparation and incubation, the temperature was 20.2-20.8 °C. During the incubation (28 days) of the test units the temperature range was the following: 20.0-20.4 °C.

- pH: The pH was checked prior study start and found to be 7.22. pH adjustment was considered as not necessary.
- pH adjusted: no
- Continuous darkness: yes
- Oxygen Concentration of Test Water: About 8-9 mg/L (was measured at the start of the test and found to be 8.22 mg/L).

- Other: 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 flasks were incubated in a controlled incubator at 22 ± 2°C, in the dark.
Temperature was measured continuously using min/max thermometer and noticed daily

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and 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)

- Measuring equipment: 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.

SAMPLING
- Sampling frequency: Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.
- Sampling method: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method].

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only filtered inoculum (10 mL) was added to the aqueous test medium (5000 mL). More studies run in parallel and the corresponding inoculum control was common.
- Toxicity control: Test and reference item stock solutions (50-50 mL) were mixed into the aqueous test medium (ad. 5000 mL) corresponding to the test item concentration of 3.0 mg/L [chosen based on the calculated ThODNH4 of the test item and the preliminary experiment] and to 3.0 mg/L concentration of the reference item.
- Other: Procedure Control: Sodium benzoate. Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), at the start of the test a stock solution of Sodium benzoate (50 mL) was mixed into (ad. 5000 mL) the aqueous test medium (corresponding to 3.0 mg/L reference item, respectively a ThODNH4 of about 3.0 x 1.67= 5.01 mg O2/L). More studies run in parallel and the corresponding procedure control was common.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
yes
Test performance:
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. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item and functioned as a procedure control.
This study is recognised by the test guidelines and provides a rational basis to assess the ready biodegradation properties of the test item when incubated with activated sludge.
Key result
Parameter:
% degradation (O2 consumption)
Value:
61.1
Sampling time:
28 d
Details on results:
The test item does not contain N. For this reason, total oxidised Nitrogen (nitrate and nitrite) concentrations were not measured after each oxygen measurement.
Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5 %), even if oxidation of the ammonium N in the medium occurs erratically as between test and blank vessels.

Percentage Biodegradation:
Under the test conditions the percentage biodegradation of the test item reached a mean of 61.1 % after 28 days based on its calculated ThODNH4.
In the test 28.7 % biodegradation was noticed on the 7th day, 50.8 % on the 14th day and 59.7 % on the 21st day of the test. The pass level for ready biodegradability is the removal of 60 % ThODNH4 in a 10-day window. In this test the threshold for ready biodegradability, the removal of 60 % of ThODNH4 was reached at about the 21st day; however the 10-day window criterion was additionally not fulfilled. The 10 % of ThODNH4 threshold was reached before the 7th day of the test. Based on the above results the test item was considered to be ready biodegradable not fulfilling the 10-day criterion.

Biodegradation of Toxicity Control
Percentage Biodegradation:
In the toxicity control containing both, the test item and the reference item, a mean of 52.5 % biodegradation was noted within 14 days and a mean of 61.3 % biodegradation was determined after 28 days of incubation.
Key result
Parameter:
ThOD
Value:
1.89 other: mg O2/mg test item
Results with reference substance:
Biodegradation of Reference Item
Percentage Biodegradation:
The reference item Sodium benzoate was sufficiently degraded to a mean of 72.4 % after 14 days, and to a mean of 83.8 % after 28 days of incubation, based on ThODNH4.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test item is considered to be ready biodegradable not fulfilling the 10-day criterion, since the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window.
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. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item and functioned as a procedure control. This study is recognised by the test guidelines and provides a rational basis to assess the ready biodegradation properties of the test item when incubated with activated sludge.

The test item was investigated at the concentration of 3.0 mg/L. The concentration was chosen based on the preliminary test results and based on the theoretical oxygen demand of 1.89 mg O2/ mg test item (ThODNH4, calculated according to equation given in the guidelines, assuming that no nitrification occurs). 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. All validity criteria of the study were met.

Under the test conditions significant level of test item biodegradation was noticed. The percentage biodegradation of the test item reached a mean of 61.1 % after 28 days based on its ThODNH4. While the threshold for ready biodegradability (60 % biodegradation based on the ThODNH4) was reached on about the 21st day of the test, the additionally needed 10-day window criterion was not fulfilled; therefore the test item can be considered as ready biodegradable not fulfilling the 10-day window criterion.

The reference item sodium benzoate was sufficiently degraded to a mean of 72.4 % after 14 days, and to a mean of 83.8 % after 28 days of incubation, based on ThODNH4.

In the toxicity control containing both, the test item and the reference item, a mean of 52.5 % biodegradation was noted within 14 days and 61.3 % biodegradation after 28 days of incubation.

Description of key information

The test item is considered to be ready biodegradable not fulfilling the 10-day criterion, since the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window

Additional information

Biodegradation in water screening test:

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. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item and functioned as a procedure control. This study is recognised by the test guidelines and provides a rational basis to assess the ready biodegradation properties of the test item when incubated with activated sludge. The test item was investigated at the concentration of 3.0 mg/L. The concentration was chosen based on the preliminary test results and based on the theoretical oxygen demand of 1.89 mg O2/ mg test item (ThODNH4, calculated according to equation given in the guidelines, assuming that no nitrification occurs). 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. All validity criteria of the study were met. Under the test conditions significant level of test item biodegradation was noticed. The percentage biodegradation of the test item reached a mean of 61.1 % after 28 days based on its ThODNH4. While the threshold for ready biodegradability (60 % biodegradation based on the ThODNH4) was reached on about the 21st day of the test, the additionally needed 10-day window criterion was not fulfilled; therefore the test item can be considered as ready biodegradable not fulfilling the 10-day window criterion. The reference item sodium benzoate was sufficiently degraded to a mean of 72.4 % after 14 days, and to a mean of 83.8 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 52.5 % biodegradation was noted within 14 days and 61.3 % biodegradation after 28 days of incubation.