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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 June - 23 August 2016
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: UGe-RS Kilo 1
Color: Yellow
Odor: Not specific
Storage conditions: room temperature
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiring date: 12.12.2018
Correction factor: 1.07
Specific details on test material used for the study:
Batch No.: UGe-RS Kilo 1
Appearance: Yellow powder
Expiration date: 12 December 2018
Storage: Room temperature

Study design

Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 27 July 2016 (seven days before the main test). The prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 22 ± 2 oC, for about 7 days.
Origin:
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 with shaking and again centrifuged. This procedure was repeated twice.
Preparation of Activated Sludge Inoculum:
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with reconstituted water (see Section 5.4) and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.81, just before use the pH was: 7.26. A pH adjustment of activated sludge inoculum was not performed.
Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium, reconstituted water
Pre-conditioning of Activated Sludge Inoculum:
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 108-109/L; therefore on the day of the test this inoculum was diluted 1000 x with reconstituted water to reach the necessary 105-106 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improves the precision of the method. ) for 7 days (from July 27 to August 03, 2016) at the test temperature (the actual temperature: 20.4 – 22.0 oC). 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 inoculum was not pre-adapted to the test chemical.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Stock Solutions
In purified, deionised water analytical grade salts were added to prepare the following stock solutions:
A) Solution: KH2PO4 8.50 g
K2HPO4 21.75 g
Na2HPO4 x 12H2O 67.16 g2
NH4Cl 0.50 g
Water ad 1000 mL
B) Solution: CaCl2 x 2 H2O 18.20 g
Water ad 500 mL
C) Solution: MgSO4 x 7 H2O 11.25 g
Water ad 500 mL
D) Solution: FeCl3 x 6 H2O 0.125 g
Water ad 500 mL3
(The “D” stock solution was prepared on the day of the reconstituted water preparation and was not further stored).
Ratio of Ingredients
1-1 mL of the stock solutions A - D) were combined and filled to a final volume of 1000 mL with water (purified deionised)4
2 This concentration corresponds to 33.4 g of Na2HPO4 x 2H2O per liter as stated in the guideline. . 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 8.27 mg/L. The pH of the reconstituted water was 7.44.
Environmental Conditions
The test was carried out in a controlled environment room (during the preparation, aeration and incubation of the reconstituted water, preparation of test bottles (units), during the formulation, oxygen and pH measuring) at a temperature of 22 ± 2°C according to the guideline. The actual temperature range was 20.6 - 21.9 oC.
The test flasks were incubated in incubator at 22 ± 2 °C, in the dark. During the incubation (28 days) of the test units the temperature range was the following: 20.0-20.2 oC.
During the pre-conditioning of activated sludge inoculum the temperature was 20.4-22.0 oC.
Temperature was measured continuously using min/max thermometer (in controlled environment room) or built-in thermometer (in incubator) and noticed at least once a day.
The oxygen concentration of test water (reconstituted water) was in the range of 8-9 mg/L. It was measured at the start of the test and found to be 8.27 mg/L.
The pH was checked prior study start and found to be 7.44. A pH adjustment was considered as not necessary.
The test conditions were measured with suitable instruments and documented in the raw data.
Equipments:
Large glass tank (volume:~30 L) and
Large glass bottles (volume: 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 ± 2 oC with a temperature deviation of ±1 oC) environment room (and/or incubator) with thermometer with exclusion of light,
Balance, Centrifuge.
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.
The Test Groups
1.) Test Item (flasks 1a and 1b):
Based on the measured chemical oxygen demand (COD) of 1.95 mg O2/mg test item, and based on the results of a preliminary experiment, at the start of the test a suitable volume (50 mL) of stock solution was thoroughly mixed into the respective volume (ad. 5000 mL) of aqueous test medium (corresponding to 3.0 mg/L test item concentration, respectively a COD of about 3.0 x 1.95 = 5.85 mg O2/L).
2.) Procedure Control, Sodium benzoate (flasks 2a and 2b):
Based on the theoretical oxygen demand (ThODNH3) of sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), the 50 mL stock solution (300 mg/L) of sodium benzoate was thoroughly mixed into the respective volume (ad. 5000 mL) of aqueous test medium (corresponding to 3.0 mg/L reference item concentration5
More studies run in parallel and the corresponding procedure control was common. , respectively a ThODNH3 of about 3.0 x 1.67 = 5.01 mg O2/L).
3.) Inoculum Control (flasks 3a and 3b):
Only filtered inoculum was added to the aqueous test medium.
Microbial inoculum (2.0 mL per litre) was added to each preparation bottle.
More studies run in parallel and the corresponding inoculum control was common.
4.) Toxicity Control (flasks 4a and 4b):
Test (50 mL) and reference item (50 mL) stock solutions were mixed into the aqueous test medium (ad. 5000 mL) corresponding to the 3.0 mg/L test item concentration [chosen based on the calculated COD of the test item and the preliminary experiment] and to 3.0 mg/L concentration of the reference item.
In general: microbial inoculum (2.0 mL per litre) was added to each preparation bottle.
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
Supplier: Reanal, Budapest Hungary, Manufacturer: ACRÕS, Lot No.: A0347324 Retest Date: May 2019

Results and discussion

Preliminary study:
The test item solubility, behavior, and toxicity were tested in a 14-day preliminary toxicity experiment. The test design was the same as described at the main experiment.
In the preliminary toxicity test the test item was investigated at 4 mg/L concentration and any toxic effect of the test item was not noticed 35.3 % biodegradability occurred within 14 days in the toxicity control group.
Test performance:
The chosen test item concentration of 3.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests, and based on the measured chemical oxygen demand (COD) of 1.95 mg O2/ mg test item of the test item.
Under the test conditions ready biodegradation of this test item was not noticed, the percentage biodegradation of Yellow LF 6911 reached a mean of 22.7 % after 28 days based on its COD (the highest value of 23.2 % was noticed on the 21st day of the test and the variations slight changes were considered as being within the biological variability range of the applied test system).
The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred (the slight changes in nitrite concentration in the 28-day samples were caused likely by a technical effect), therefore the biodegradability value of the test item was calculated based on its COD; any correction, based on the nitrite and/or nitrate content was not performed.
The reference item Sodium benzoate was sufficiently degraded to a mean of 67.6 % after 14 days, and to a mean of 78.0 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.
In the toxicity control containing both, the test item and the reference item, a mean of 40.0 % biodegradation was noted within 14 days and 40.4 % 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).
% Degradation
Key result
Parameter:
% degradation (O2 consumption)
Value:
> 22 - <= 23.5
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of Yellow LF 6911 reached a mean of 22.7 % after 28 days based on its COD. The highest biodegradability value of 23.2 % was noticed on the 21st day of the test and the variations slight changes were considered as being within the biological variability range of the applied test system. The test item can be considered to be not ready biodegradable.

Biodegradation of the Reference Item
The reference item Sodium benzoate was sufficiently degraded to a mean of 67.6 % after 14 days, and to a mean of 78.0 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Biodegradation of the Toxicity Control
In the toxicity control containing both, the test item and the reference item, a mean of 40.0 % biodegradation was noted within 14 days and 40.4 % 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).

BOD5 / COD results

BOD5 / COD
Key result
Parameter:
COD
Value:
1.95 g O2/g test mat.
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 78.0 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Any other information on results incl. tables

Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

mg O2/L after n days of exposure

[mg/L]

No.

0

7

14

21

28

Test item

 

1a

8.28

6.34

6.16

5.82

5.66

3.0

1b

8.26

6.45

5.95

5.71

5.73

 

mean

8.27

6.40

6.06

5.77

5.70

Reference item

 

2a

8.24

4.62

3.60

3.15

3.13

3.0

2b

8.25

4.33

3.79

3.61

3.07

 

mean

8.25

4.48

3.70

3.38

3.10

Inoculum control

3a

8.27

7.18

7.08

7.11

7.03

3b

8.27

7.41

7.12

7.13

7.02

mean

8.27

7.30

7.10

7.12

7.03

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

8.25

3.55

2.86

2.93

2.85

4b

8.24

3.31

2.62

2.41

2.39

mean

8.25

3.43

2.74

2.67

2.62

 

Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

Percent of biodegradation after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

 

1a

16.5

16.2

22.4

23.5

3.0

1b

14.3

19.5

23.9

22.0

 

mean

15.4

17.9

23.2

22.7

Reference item

 

2a

52.9

69.4

78.8

77.3

3.0

2b

58.9

65.8

69.8

78.7

 

mean

55.9

67.6

74.3

78.0

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

34.3

38.9

38.4

38.3

4b

36.5

41.0

43.1

42.4

mean

35.4

40.0

40.8

40.4

Nitrate Concentrations

Analytical occasions

Measured nitrate concentration (mg/L) in the test flasks

1a

1b

3a

3b

4a

4b

0 day

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

7thday

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

14thday

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

21stday

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

28thday

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

Remark:             1a, 1b, 3a, 3b, 4a and 4b mean the flask numbers.

 

 

 

Nitrite Concentrations

 

Analytical occasions

Measured nitrite concentration (mg/L) in the test flasks

 

 

1a

1b

3a

3b

4a

4b

 

 

0 day

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

7thday

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

14thday

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

21stday

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

28thday

0.07

0.15

0.09

0.09

0.09

0.05

 

Remark:             1a, 1b, 3a, 3b, 4a and 4b mean the flask numbers.

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Remarks:
biodegradation of 22.7 % was reached after 28 days
Conclusions:
The test item was considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is 60 % of COD.
The measured quantities of nitrate in the inoculum control, test item and toxicity control samples were below the LOQ throughout the study.
The measured quantities of nitrite in the inoculum control, test item and toxicity control samples were below the LOQ in the 0-21st day samples and the nitrite quantities detected on the 28th day samples did not account for any correction of the oxygen consumption values.
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 Yellow LF 6911. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. 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 3.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests, and based on the measured chemical oxygen demand (COD) of 1.95 mg O2/ mg test item of the test item. Under the test conditions ready biodegradation of this test item was not noticed, the percentage biodegradation of Yellow LF 6911 reached a mean of 22.7 % after 28 days based on its COD (the highest value of 23.2 % was noticed on the 21st day of the test and the variations slight changes were considered as being within the biological variability range of the applied test system). The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred (the slight changes in nitrite concentration in the 28-day samples were caused likely by a technical effect), therefore the biodegradability value of the test item was calculated based on its COD; any correction, based on the nitrite and/or nitrate content was not performed. The reference item Sodium benzoate was sufficiently degraded to a mean of 67.6 % after 14 days, and to a mean of 78.0 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 40.0 % biodegradation was noted within 14 days and 40.4 % 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).