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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-04-22 till 2009-05-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Version / remarks:
adopted May 12, 1981
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Inoculum or test system:
other: activated sludge, domestic, non adapted; lake and river water; compost
Details on inoculum:
TEST SYSTEM: Inoculum from specially grown non adapted micro-organisms. The micro-organisms were taken from sources as specified below.
SOURCES
Activated sludge: Municipal sewage treatment plant, D-31137 Hildesheim; Municipal sewage treatment plant, D-31157 Sarstedt
Waste water: Municipal sewage treatment plant, D-31137 Hildesheim
Compost: Composting plant, D-31137 Hildesheim
River: Innerste, D-31157 Sarstedt
Lake: Giftener See, D-31157 Sarstedt, OT Giften; Fishing lake, D-31157 Sarstedt, OT Heisede; Mesocosm of test facility
Receipt: 2009-03-11
Reasons for the selection: Activated sludge from the sewage plant at Hiidesheim and Sarstedt is well suited as it receives predominantly municipal sewage and hardly industrial chemical waste. Freshly collected non adapted water from different sampling places as wett as compost acc. to the guideline.
Preparation: Same volumes of each source were filled together (activated sludge washed twice with autoclaved tap water before) on 2009-03-11. This mixture was allowed to settle and the supernatant was decanted and used as inoculum.
Keeping: The inoculum was stirred and aerated continuously. It was fed every working day. Therefore the inoculum was allowed to settle and one third of the supernatant was decanted and filled up with nutrient solution acc. to the guideline.
Inoculation: 25 mL/L supernatant of the inoculum were used to initiate inoculation.
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 302 C
- Additional substrate: no
- Test temperature: 25.0 -26.0 °C
- pH: 6.20 - 6.93
- pH adjusted: no
- Suspended solids concentration: 1.66 g/L corresponding to 41.5 mg/L in the test vessels
- Continuous darkness: yes
- Dispersion treatment: Continuous stirring

TEST SYSTEM
- Culturing apparatus: Brown glass bottles (volume 500 mL)
- Number of culture flasks/concentration: 3
- Measuring equipment: OxiTop® OC110 controller, WTW; OxiTop® measuring heads, WTW
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: bottles closed with OxiTop® measuring heads

SAMPLING
- Sampling frequency: at start and end of test
- Sampling method: HPLC-DAD
- Sample storage before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (inoculum, medium)
- Blank control: yes (test item, medium)
- Functional control: yes (reference item, inoculum, medium)

STATISTICAL METHODS: Excel, MICROSOFT CORPORATION; Achat OC, WTW GMBH; SigmaPlot (Windows), SPSS CORPORATION
Reference substance:
benzoic acid, sodium salt
Test performance:
The study was performed according to OECD Guideline 302 C and GLP principles. The validity criteria were fulfilled according to the guideline:
• The percentage degradation of the functional control reached the pass level of 65 % after 3 days (validity criterion: 65 % after 14 days).
• The difference of extremes of replicate values of removal of the test item at the end of the test was less than 20 %.
• The recovery rate in the blank at test end was 98 % (validity criterion: > 10 %).
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
Colony forming units (CFU) of the MITI-inoculum were determined at test start by standard dilution plate count: 1.8 x 109 CFU/L. The concentration of suspended solids of the inoculum at test start was 1.66 g/L corresponding to 41.5 mg/L in the test vessels.
No elimination / abiotic degradation of the test item occurred in the blank. The recovery rate in the blank at test end was 98 %. The validity criterion that the recovery rate in the blank must be > 10% was fulfilled.
The biodegradation of the test item in all test item replicates during 28 days was 0 %.
Results with reference substance:
The functional control reached the pass level > 65 % after 3 days and came to a biodegradation rate of 82 % after 28 days. The validity criterion that the degradation should be > 65 % after 14 d was fulfilled.

Table 1: Biodegradation [%] of the test item in comparison to the funcional control

Date

[d]

Biodegradation [%]

Functional Control

Test item

35 mg/L

30mg/L

R

P1

P2

P3

28.04.2009

0

0

0

0

0

29.04.2009

1

48

0

0

0

30.04.2009

2

58

0

0

0

01.05.2009

3

68

0

0

0

02.05.2009

4

70

0

0

0

03.05.2009

5

72

0

0

0

04.05.2009

6

82

0

0

0

05.05.2009

7

79

0

0

0

06.05.2009

8

82

0

0

0

07.05.2009

9

82

0

0

0

08.05.2009

10

79

0

0

0

09.05.2009

11

79

0

0

0

10.05.2009

12

79

0

0

0

11.05.2009

13

79

0

0

0

12.05.2009

14

82

0

0

0

13.05.2009

15

82

0

0

0

14.05.2009

16

82

0

0

0

15.05.2009

17

82

0

0

0

16.05.2009

18

82

0

0

0

17.05.2009

19

82

0

0

0

18.05.2009

20

84

0

0

0

19.05.2009

21

82

0

0

0

20.05.2009

22

87

0

0

0

21.05.2009

23

82

0

0

0

22.05.2009

24

84

0

0

0

23.05.2009

25

82

0

0

0

24.05.2009

26

82

0

0

0

25.05.2009

27

87

0

0

0

26.05.2009

28

82

0

0

0

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item must be regarded as not inherently biodegradable after 28 days (bidegradation: 0%).
Executive summary:

The inherent biodegradability and the primary degradation of the test item were determined in the Modified MITI Test (II) with a non adapted activated inoculum for a period of 28 days. The study was conducted from 2009-04-28 to 2009-05-27 according to OECD guideline 302 C. The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 58.2 mgO2/L per test vessel. The oxygen was depleted by respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation is therefore expressed as the percentage BOD depletion and was calculated for each study day. The primary degradation was followed by specific test item analysis at test start and test end. The mean oxygen depletion in the inoculum control was 0.0 mgO2/L on day 28. In order to check the activity of the test system sodium benzoate was used as functional control. The functional control reached the pass level > 65 % after 3 days and came to a biodegradation rate of 82 % after 28 days. The biodegradation of the test item in comparison to the inherent degradable functional control was 0%. In all test item replicates no biodegradation occurred during 28 days (degradation: 0 %). No elimination / abiotic degradation of the test item occurred in the blank. The recovery rate in the blank at test end was 98 %. The validity criteria of the guideline are fulfilled. The test item must be regarded as not inherently biodegradable after 28 days. The primary biodegradation / elimination was 0 %.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2007-04-03 to 2007-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
July 17,1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
July 31, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge, micro organisms from a domestic waste water treatment plant was supplied by the sewage plant Rossdorf, Germany. 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 tap water 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 1.5 g dry material per litre were mixed with test water and then aerated until use. The washed activated sludge was pre-incubated for two days under aeration.
Duration of test (contact time):
28 d
Initial conc.:
191 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: standard medium according to OECD 301F
- Additional substrate: no
- Test temperature: 22°C
- pH: 7.6 (measured at the start of the test); 7.5 - 7.8 (measured at the end of the test)
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration: 1.5 g dry material/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of 500 mL
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD-Sensor-System, Aqualytic, 63231 Neu Isenburg, Germany
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: Evolved carbon dioxide was absorbed in an aqueous solution (45 %) of potassium hydroxide

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (sludge and test water)
- Abiotic control: yes (test item and test water)
- Toxicity control: yes (test item, reference item, sludge and test water)
- Procedure control: yes (reference item, sludge and test water)
Reference substance:
benzoic acid, sodium salt
Test performance:
Validity Criteria of the Study:
Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 16.2 mg 02/L and thus not greater than 60 mg 02/L within 28 days as required by the test guideline.
pH-Value: The pH-value of the test item flasks at the end of the test was within the range of pH 6.0 - 8.5 as required by the test guideline.
Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (>60 %) within 14 days as required by the test guideline.
The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation.
Test Item: The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window should be less than 20 %.
Since the biodegradation did not reach the pass criterion of 60% degradation and no 10 day-window could be determined, the validity criterion was not applicable.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
Under the test conditions no biodegradation of test item could be observed. The percentage degradation did not exceed 60 % within the 10-day window and after 28 days of incubation. The test item is considered not readily biodegradable.
In the toxicity control containing both, test item and reference item 40% biodegradation was noted within 14 days and 42 % biodegradation was determined after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge micro organisms because degradation was > 25% within 14 days.
The oxygen demand in the abiotic control was zero; no correction of the test item degradation rates had to be done.
Results with reference substance:
The reference Item sodium benzoate was sufficiently degraded to 70 % after 4 days and to 92% after 28 days of incubation. The percentage degradation of the reference item confirms the suitability of the used activated sludge inoculum.

Table 1: Percentage Biodegradation of test item, reference item and toxicity control

Time

Percentage Biodegradation [%]1

(Days)

Test item

Sodium benzoate

Toxicity control

1

0

0

36

15

2

0

0

49

21

3

0

0

59

27

4

-2

-2

70

32

5

-2

-2

76

35

6

-2

-2

79

36

7

-3

-3

82

38

8

-4

-4

83

38

9

-4

-4

84

39

10

-4

-4

85

39

11

-4

-4

86

40

12

-5

-5

87

40

13

-5

-5

88

40

14

-5

-5

89

40

15

-6

-6

89

41

16

-6

-6

89

41

17

-6

-6

89

41

18

-6

-6

89

41

19

-7

-7

90

41

20

-7

-7

91

42

21

-7

-7

91

42

22

-7

-7

91

42

23

-7

-7

92

42

24

-7

-7

92

42

25

-7

-8

92

42

26

-7

-7

91

42

27

-7

-7

93

42

28

-7

-8

92

42

 

1based on ThODNH4:

ThODNH4of Test item: 1.830 mg/mg

ThODNH4of sodium benzoate: 1.666 mg/mg

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Under the test conditions no biodegradation of test item could be observed. The percentage degradation did not exceed 60 % within the 10-day window and after 28 days of incubation. The test item is considered not readily biodegradable.
Executive summary:

The biodegradation of the test item was investigated in a manometric respirometry test according to OECD 301F and EU Method C.4 -D. The domestic, non adapted sludge was used. The biodegradation was followed by the oxygen uptake of the micoorganisms during exposure for 28 days. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item and fundtioned as procedure control. Under the test conditions no biodegradation of test item could be observed. The percentage degradation did not exceed 60 % within the 10-day window and after 28 days of incubation. The test item is considered not readily biodegradable. The reference item sodium benzoate was sufficiently degraded to 70 % after 4 days and to 92% after 28 days of incubation. The percentage degradation of the reference item confirms the suitability of the used activated sludge inoculum. In the toxicity control containing both, test item and reference item 40% biodegradation was noted within 14 days and 42 % biodegradation was determined after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge micro organisms because degradation was > 25% within 14 days. The oxygen demand in the abiotic control was zero; no correction of the test item degradation rates had to be done.

Description of key information

No biodegradation of test item could be observed. The percentage degradation did not exceed 60 % within the 10-day window and after 28 days of incubation. The test item is considered not readily biodegradable (reference 5.2.1 -1).

The test item must be regarded as not inherently biodegradable after 28 days (bidegradation: 0%, reference 5.2.1 -2).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The biodegradation of the test item was investigated in a manometric respirometry test according to OECD 301F and EU Method C.4 -D. The domestic, non adapted sludge was used. The biodegradation was followed by the oxygen uptake of the micoorganisms during exposure for 28 days. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item and fundtioned as procedure control. Under the test conditions no biodegradation of test item could be observed. The percentage degradation did not exceed 60 % within the 10-day window and after 28 days of incubation. The test item is considered not readily biodegradable. The reference item sodium benzoate was sufficiently degraded to 70 % after 4 days and to 92% after 28 days of incubation. The percentage degradation of the reference item confirms the suitability of the used activated sludge inoculum. In the toxicity control containing both, test item and reference item 40% biodegradation was noted within 14 days and 42 % biodegradation was determined after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge micro organisms because degradation was > 25% within 14 days. The oxygen demand in the abiotic control was zero; no correction of the test item degradation rates had to be done (reference 5.2.1 -1).

The inherent biodegradability and the primary degradation of the test item were determined in the Modified MITI Test (II) with a non adapted activated inoculum for a period of 28 days. The study was conducted from 2009-04-28 to 2009-05-27 according to OECD guideline 302 C. The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 58.2 mgO2/L per test vessel. The oxygen was depleted by respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation is therefore expressed as the percentage BOD depletion and was calculated for each study day. The primary degradation was followed by specific test item analysis at test start and test end. The mean oxygen depletion in the inoculum control was 0.0 mgO2/L on day 28. In order to check the activity of the test system sodium benzoate was used as functional control. The functional control reached the pass level > 65 % after 3 days and came to a biodegradation rate of 82 % after 28 days. The biodegradation of the test item in comparison to the inherent degradable functional control was 0%. In all test item replicates no biodegradation occurred during 28 days (degradation: 0 %). No elimination / abiotic degradation of the test item occurred in the blank. The recovery rate in the blank at test end was 98 %. The validity criteria of the guideline are fulfilled. The test item must be regarded as not inherently biodegradable after 28 days. The primary biodegradation / elimination was 0 % (reference 5.2.1 -2).