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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:
10 November 2016 to 08 December 2016
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Before the preparation of the respective test solutions with the test material, an aqueous stock solution of the test item with a concentration of 75 mg/L was prepared and diluted accordingly. During the preparation of the test solutions they were mixed by mechanical stirring to ensure a good dispersion. The test item concentration in the test solutions was 7.5 mg/L.
Taking into account that purity of the test item is only 82.0 wt%, the chosen test material concentration was based on the measured chemical oxygen demand (COD):

0.80 ±0.013 mg O2/mg test item and on the performed 14-d preliminary test.
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
TEST SYSTEM
- Source of inoculum/activated sludge: Secondary effluent, microorganisms from a domestic waste water treatment plant in Veszprém, Hungary.
- Preparation of inoculum for exposure: The secondary effluent used for this study was allowed to settle for an hour, then the decanted effluent was aerated until use (not later than an hour).
- Concentration of sludge: Not specified, however 0.5 mL of microbial inoculum was added to each preparation flask.
Duration of test (contact time):
28 d
Initial conc.:
7.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
Stock solutions
In deionised water analytical grade salts were added to prepare the following stock solutions:

a) Solution:
- KH2PO4: 2.125 g
- K2HPO4: 5.4375 g
- Na2HPO4 x 12H2O: 16.795 g
- NH4Cl: 0.125 g
- Deionised water: ad 250 mL

b) Solution:
- MgSO4 x 7H2O 5.625 g
- Deionised water ad 250 mL

c) Solution:
- CaCl2 x 2H2O 9.10 g
- Deionised water ad 250 mL

d) Solution:
- FeCl3 x 6H2O 0.25 g
- Deionised water ad 1 000 mL

Adequate amount of the stock solutions a) - d) were combined and filled up with deionised water to the appropriate final volume (ratio of composition referring to 1 mL of the stock solutions a) - d) filled up with deionised water to a final volume of 1 000 mL according to the guideline).

- Test temperature: The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ± 2 °C according to guideline. The test flasks were placed into an incubator and kept at 21.0 - 22.8 °C, in the dark. The temperature was measured on weekdays during the experiment.
- pH: The pH value of the test water was checked prior start of the experiment. The pH of the test water was 7.26.
- pH adjusted: No
- Aeration of dilution water: The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature.
- Continuous darkness: Yes
- Other: The oxygen concentration of test water was 8.9 mg/L at the start of the test.

TEST SYSTEM
- Culturing apparatus: Type and Size: BOD bottles (300 mL) with special neck and glass stoppers.
- Number of culture flasks/concentration: 10 bottles containing the test item and inoculum.
Based on the measured chemical oxygen demand (COD) of 0.80 mg O2/mg test item, test item stock solution* (corresponding to 25.35 mg of test material) was thoroughly mixed into 3.38 litres of aqueous test medium (corresponding to 7.5 mg/L test item, with a COD of about 6.00 mg O2/L).
* The concentration of the stock solution was: 75 mg/L.
- Other: Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.

SAMPLING
The COD (chemical oxygen demand) of the test item was determined in the analytical department of the test facility using Lovibond® COD Measuring System.
Because of 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 total oxidised nitrogen (nitrate and nitrite) concentrations were determined after each oxygen measurement.
The analysis was carried out with Lovibond MaxiDirect portable photometer using Nitrite LR tablet reagent, and Vario NitraX Reagent Set.

Measurement of Oxygen: The incubation period of the closed bottle test was 28 days.
The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.

Measurement of Temperature: Temperature was measured continuously and registered mostly on each working day, but at least each day of oxygen consumption measurement during the experiment.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 bottles containing only inoculum. Only filtered inoculum was added to 3.40 litres of aqueous test medium.
- Toxicity control: 10 bottles containing the test item, reference item and inoculum. Test item stock solution* (338 mL) and reference item stock solution** (33.8 mL) were mixed into 3.38 litres of aqueous test medium corresponding to 7.5 mg/L test item (COD of 6.00 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L).
* The concentration of the test item stock solution was: 75 mg/L.
** The concentration of the reference item stock solution was: 360 mg/L.
- Procedure Control: 10 bottles containing the reference item (sodium benzoate) and inoculum.
Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution* corresponding to 12.240 mg of Sodium benzoate was mixed into 3.40 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L).
* The concentration of the stock solution was: 360 mg/L.

CALCULATION OF RESULTS
Calculation of BOD: The BOD (mg O2 per mg test item) expected after each period was calculated as follows:

(mg O2/L of T.i. and/or R.i. - mg O2 /L of i.control) / mg T.i. and/or R.i./L in flask

where:
T.i. = test item
R.i. = reference item,
i.control = inoculum control


Calculation of Biodegradation %: The percentage biodegradation of the test item and of the reference item was calculated as follows:

BOD(mg O2/mg T.i. or R.i.) / COD (mg O2/mg T.i.) or ThOD (mg O2/mg R.i.)

where:
T.i. = test item
R.i. = reference item,
i.control = inoculum control
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
2.5
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of the test material reached a mean of 2.5 % after 28 days based on the measured COD of the test item.
According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore the test item is considered not readily biodegradable.
The test item can be assumed to be not inhibitory at the applied concentration level of 7.5 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 31.4 % biodegradation was noted within 14 days and 38.3% biodegradation after 28 days of incubation.
Key result
Parameter:
COD
Value:
2.5 other: %
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 65.8 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

Percentage Biodegradation in Different Time Intervals During the Exposure Period of 28 Days

Treatment

Concentration

(mg/L)

Flask No

Percent of biodegradation after n days of exposure

7

14

21

28

Test Material

7.5

1a

-1.7

0.8

3.3

0.8

1b

-1.7

4.2

5.0

4.2

Mean

-1.7

2.5

4.2

2.5

Reference Item

3.6

2a

60.0

67.5

83.3

89.2

2b

58.3

64.2

86.7

87.5

Mean

59.2

65.8

85.0

88.3

Toxicity Control

Test item: 7.5

Reference Item: 3.6

4a

27.0

31.0

38.0

39.1

4b

26.3

31.8

37.3

37.6

Mean

26.7

31.4

37.6

68.3

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of the test, the percentage biodegradation of the test material reached a mean of 2.5 % after 28 days based on the measured COD of the test item. The test item is therefore considered not readily biodegradable.
Executive summary:

A study was conducted to evaluate the ready biodegradability of the test material in accordance with the standardised guidelines OECD 301D, EU Method C.4 -E and EPA OPPTS 835.3110 under GLP conditions.

The test item was investigated for its ready biodegradability in a Closed Bottle Test over a period of 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.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the test material reached a mean of 2.5 % after 28 days based on the measured COD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore the test item is considered not readily biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 65.8 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 31.4 % biodegradation was noted within 14 days and 38.3 % biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 7.5 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

Under the conditions of the study, the test item is considered not readily biodegradable.

Description of key information

Under the conditions of the test, the percentage biodegradation of the test material reached a mean of 2.5 % after 28 days based on the measured COD of the test item.  The test item is therefore considered not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A study was conducted to evaluate the ready biodegradability of the test material in accordance with the standardised guidelines OECD 301D, EU method C.4 -E and EPA OPPTS 835.3110 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test item was investigated for its ready biodegradability in a Closed Bottle Test over a period of 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.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the test material reached a mean of 2.5 % after 28 days based on the measured COD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore the test item is considered not readily biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 65.8 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 31.4 % biodegradation was noted within 14 days and 38.3 % biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 7.5 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

Under the conditions of the study, the test item is considered not readily biodegradable.