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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-03-05 - 1996-04-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Qualifier:
according to
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Qualifier:
according to
Guideline:
ISO 7827 (Evaluation in an Aqueous Medium of the "Ultimate" Aerobic Biodegradability of Organic Compounds - Method by Analysis of Dissolved Organic Carbon (DOC))
Principles of method if other than guideline:
GLP-guideline study; only one replicate with test substance was considered in the assessment of the degradation
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Dimethylpiperazin
- Analytical purity: > 99%
- Lot/batch No.: Abl. Nr. 32-1659
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge taken from a laboratory WWTP fed with domestic and synthetic sewage.
Duration of test (contact time):
28 d
Initial conc.:
32 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: inorganic medium according to guideline
- Test temperature: ambient
- pH: no data
- pH adjusted: no
- Aeration of dilution water: no data
- Continuous darkness: yes
- Other:


TEST SYSTEM
- Culturing apparatus: Shaken flasks cultured under aerobic conditions and constant temperature
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: continuous aeration as prescribed by the guideline
- Measuring equipment: DOC analyser (Shimadzu TOC 500 and TOC 5000)
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: yes
- Other:


SAMPLING
- Sampling frequency: on days 0, 1, 3, 5, 7, 11, 14, 17, 21, 24, 27, 28
- Sample storage before analysis: no data


CONTROL AND BLANK SYSTEM
- Inoculum blank: BC, 2 replicates
- Abiotic sterile control: PC, 2 replicates
- Toxicity control: RS, 2 replicates
- Other: inhibition control (IH, 2 replicates); adsorption control (AC, 2 replicates)


STATISTICAL METHODS: not applicable
Reference substance:
aniline
Key result
Parameter:
% degradation (DOC removal)
Value:
90
Sampling time:
28 d
Details on results:
At test termination (28 days) the substance was degraded by ca. 90 % in replicate No. 1 (PS1), whereas in the second replicate (PS2) no degradation was observed after 28 days. In PS2 the first significant degradation was measured after 32 days (19%), followed by 88% and 92% after 35 and 36 days, respectively. Therefore, a second test using the activated sludge taken from PS1 was performed. In this test with adapted activated sludge a biodegradation of > 90 % within 6 days was recorded.
Results with reference substance:
Degradation of control substance:
94 % after 7 day(s)

Degradation kinetics:

Day

Elimination (%)

Toxicity control

Inhibition control

Abiotic control

Adsorption control

Test substance

PS1

PS2

0

0

0

0

0

0

0

1

4

-4

0

3

3

7

3

4

0

5

2

-1

-1

5

60

44

0

-5

-2

-3

7

94

43

2

-11

-3

-3

11

96

44

-2

 

-5

0

14

101

47

-1 *

 

1

6

17

102

44

-7

 

-16

0

21

98

35

-6

 

9

-8

24

93

42

-4

 

90

-5

27

94

41

-7

 

90

-6

28

96

48

-10

 

89

-11

32

 

 

 

 

 

19

35

 

 

 

 

 

88

36

 

 

 

 

 

92

* 1 mL of HgCl2stock solution was added

Degradation results of the additional test (PS3) with adapted inoculum:

-8% after 1 day

98% after 6 days

93% after 7 days

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
With batch PS1, the 10-days window was met. At the end of the test, after 28 days, the test substance was degraded to 89%. The degradation of the reference substance aniline was 94 % after 7 days. Elimination of the test substance by abiotic processes or adsorption was less than 10 % after 28 and 7 days, respectively. The degradation in the inhibition control after 14 days was > 35%. Considering a longer test duration, results of PS1 and PS2 would have differed by < 20%.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-04-17 - 2008-05-15
Reliability:
2 (reliable with restrictions)
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Dimethylpiperazin
- Analytical purity: 100%
- Lot/batch No.: 1901-2/2005
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- test item concentration: 100 mgL
- Reference item: aniline (100 mg/L)
- Activated sludge: 30 mg/L
- Temperature: 25±1 °C
- Duration: 28 days

TEST SYSTEM
- Number of culture flasks/concentration: 6
Reference substance:
aniline
Key result
Parameter:
% degradation (DOC removal)
Value:
5.6
Sampling time:
28 d
Results with reference substance:
The reference substance was degraded at least 60% within 9 days.

 Vessel n° Sample description            BOD (mg)
     7th day  14th day  21st day  28th day
 1 2 water + test item 0.0 0.0  0.0  0.0 
 2  sludge + test item  2.1 3.5  4.9  5.5 
 3  sludge + test item  2.0 3.3  4.8  5.9 
 4  sludge + test item  2.3 3.3  4.8  5.6
 5  control blank  3.7 6.3  7.6  7.9 
 6  sludge + aniline  63.7 75.4  77.6  78.3 
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In this DOC removal test conducted according to OECD guideline 301C, no biodegradation of the test substance was observed after 28 days. Test performance was demonstrated to be good (as shown by the results with the reference item and the toxicity control) and all validity criteria were met. The results of the test can be considered reliable. The test substance is not readily biodegradable.

Description of key information

The key study was performed equivalent to OECD guideline 301C under GLP conditions (Huntsman, 2008). Only 5.6 % degradation was observed with the test substance after 28 days (DOC removal). The results support the conclusion that the biodegradation of the test substance is slow. Therefore, the test substance is considered not readily biodegradable. Adaptation of the micro-organisms is an important proces for the degradation rate of the test substance in the environment. Results from an OECD 301B study (BASF, 1996) indicate that the test substance show enhanced biodegradability but under acclimated conditions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One study describing a ready biodegradability test was identified for the substance. The study of BASF (1996) describes a 28-days DOC die-away test and is performed according to OECD 301A. The study was assigned a Klimisch reliability score of 2 (reliable with restictions) as only one replicate with the test substance was considered in the assessment of the biodegradation of the substance. The test substance was degraded by ca. 90 % in replicate No. 1 (PS1) and the 10-day window was met, whereas in the second replicate (PS2) no degradation was observed after 28 days. In PS2 the first significant degradation was measured after 32 days (19%), followed by 88% and 92% after 35 and 36 days, respectively. Therefore, a second test using the activated sludge taken from PS1 was performed. In this test with adapted activated sludge a biodegradation of > 90 % within 6 days was recorded. The reference substance (aniline) showed up to 94 % degradation after 7 days. Over 35 % of a control substance was degraded after 15 days in the presence of the test substance. Therefore, it can be concluded that the test results are valid. In nonadapted sludge from domestic sewage treatment plants, the degradation of the substance is very slow, with lagphases of more than 30 days, while in inoculum mixed with adapted sludge the lag phases are much shorter. The 28-day DOC die-away test concluded that 1,4-dimethylpiperazine is readily biodegradable.

In another study, the test substance was tested according to MITI (I) (OECD 301C) (Huntsman, 2008). The study was assigned a klimisch score of 2. Activated sludge was used as inoculum (30 mg/L), pH 7. The test concentration was 100 mg/L, and the reference substance used was aniline. After 14 days, 3.3% of the test substance was biodegraded, compared to > 60% of the reference substance. After 28 days, 5.6% of the substacce was degraded. However, the results support the conclusion that the biodegradation of the test substance is slow under non acclimated, aerobic conditions.