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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well conducted report carried out to GLP with analysis certificate and chemical analysis. Lacking information on analytical method calibration, standard curve etc. Reliable except for incorrect CAS number reported. An, amendment required to receive a reliable with restrictions score. Amendment from study director received (29/4/09) reliability score adjusted accordingly.
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)
Deviations:
yes
Remarks:
see field below (principles of methof if oether than guideline)
Principles of method if other than guideline:
A few minor deviations from the protocol of the SCAS test were introduced:
- the fill and draw procedure was performed only six times per week instead of daily;
- to maintain a constant pH in the SCAS unit, 1 ml of a concentrated phosphate buffer (1.6 M, pH = 7) was added six times a week;
- effluent samples were filtered using Schleicher and Schiill membranes (cellulose nitrate) with pores of 8 pm so that the test substance suspension
passed through while the sludge was filtered.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: See Below
Details on inoculum:
Secondary activated sludge (1992.03.27) and primary settled sewage were collected from the WWTP Nieuwgraaf in Duiven. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic sewage. The primary settled sewage was collected weekly and stored at -20°C until required \
(minor deviation from the Guidelines). 150 ml of secondary activated sludge containing approximately 2 g suspended solids (DW) per litre was used
as an inoculum for each unit.
Duration of test (contact time):
84 d
Initial conc.:
33.3 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Remarks:
(NPOC analysis)
Details on study design:
The SCAS test was performed according to the EEC. OECD and IS0 Test Guidelines. The test was performed in diffused light at 20-25°C.
Each SCAS unit was filled with 150 ml of activated sludge and the aeration was started. After 23 hours the aeration was stopped and the sludge was
allowed to settle for 45 minutes. Before settling it was necessary to clean the walls of the units to prevent the accumulation of solids above the level of the liquid. A separate brush was used for each unit to prevent crosscontamination. The tap was opened and 100 ml of the supernatant liquor
withdrawn. Subsequently, a sample of primary settled sewage (99 ml) and concentrated phosphate buffer (1 ml) were added to the sludge remaining
in each SCAS unit. Aeration was started anew. At this stage no test material was added and the units were fed daily with primary settled sewage.
At day 0 the individual settled sludges were mixed and 50 ml of the resulting composite sludge was added to each unit. 94 ml of primary settled sewage, 5 ml deionized water and 1 ml ofconcentrated phosphate buffer were added to the control unit and 94 ml of primary settled sewage, 1 ml of
concentrated phosphate buffer and 5 ml of the test compound stock solution to the test unit. Aeration was started again and continued for 23 hours. The above fill and draw procedure was repeated 6 times per week throughout the test. Supernatant drawn off was analysed for non-purgeable
organic carbon (NPOC). The NPOC values were used to follow the removal of the test substance for a few months. Only at the start (two
weeks) of the test the NPOC in the supernatant liquor was daily determined. The next period a less frequent analysis was performed.
Reference substance:
other: No reference used.
Preliminary study:
A Ready biodegredability test was performed prior to the SCAS test. As no biodegredation was observed the SCAS test was chosen to follow up the
initial closed bottle test result.
Test performance:
No specific quality criteria reported. However Temperature and pH were sufficiently monitored and control SCAS test was run in parallel.
Parameter:
% degradation (DOC removal)
Value:
16
Sampling time:
84 d
Details on results:
Substance is not biodegraded in the SCAS Test.

% Removal

Time (Days)

NPOC CONTROL

NPOC TEST

REMOVAL %

-8

19.8

25.4

0

-7

21.2

25.4

0

-6

19.0

22.1

0

-5

19.5

18.4

0

4

18.8

10.2

0

2

16.8

11.1

0

1

14.0

16.1

0

0

15.0

13.5

0

1

15.5

15.1

98

2

14.2

16.1

76

3

14.1

22.2

56

4

13.2

28.6

35

6

17.8

34.9

39

7

14.1

38.1

24

8

13.5

39.5

20

9

12.6

45.4

20

10

12.7

42.5

20

11

13.5

41.5

26

13

16.4

45.5

38

14

15.1

43.5

26

15

14.0

45.2

28

17

13.4

26.2

23

20

14.1

41.2

32

22

15.5

40.1

46

24

15.3

39.1

23

27

15.5

42.3

24

29

15.0

42.8

20

31

14.1

41.1

24

34

13.8

40.0

20

36

13.8

42.8

20

38

12.3

44.8

16

41

12.7

36.0

13

43

12.5

39.3

25

45

14.9

40.7

32

48

13.9

42.2

32

50

14.1

41.5

29

52

14.4

43.5

23

55

14.6

41.7

20

57

12.4

37.5

13

59

11.3

38.8

14

63

12.4

39.7

22

64

11.8

39.0

26

66

10.6

36.3

25

69

11.3

39.1

22

71

10.8

36.0

16

73

11.2

38.6

11

76

10.5

38.0

10

78

10.1

37.1

15

80

11.0

39.3

13

83

11.5

37.2

17

84

11.9

36.7

18

 

 

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
other: Not inherently biodegradable in SCAS Test.
Conclusions:
The test was conducted with only minor modifications to the guideline. Although the study lacked some detail regarding the chemical analysis for
a GLP report. An analysis certificate and sufficient purity information were present. Test substance was not degraded in the SCAS Test.
Some of the substance was removed by absorbtion onto sludge. This study can be considered reliable with restrictions
Executive summary:

Well conducted report carried out to GLP with analysis certificate and chemical analysis to the appropriate guideline. Lacking information on analytical method calibration, standard curve etc. Further reliable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP test according to guidelines. Some methodology not described. further reliable.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Minor adaptions to protocol
Principles of method if other than guideline:
Activated Sludge was used as innoculum.The sludge was preconditioned to reduce endogenous respiration rates. Ammonium chloride was ommitted from the medium to prevent nitrification. The test was also prelonged.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: See below for details.
Details on inoculum:
Secondary activated sludge was obtained from the RZWI Nieuwgraaf in Duiven . The RZWI Nieuwgraaf is an activated
sludge plant treating predominantly domestic wastewater.
Duration of test (contact time):
162 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test was performed in 280 ml BOD (Biological Oxygen Demand) bottles. The closed bottle test was performed according to the EEC/OECD
test guidelines. With slight modifications ( listed above ).The pH was measured using a pH meter, Ankersmit A141. Stocks of 1.0g/l were made for
both reference substance and test substance. Results were handled appropriately.
Full explination of methods not given.
Reference substance:
other: Sodium Acetate
Preliminary study:
No information regarding prelimanary Studies.
Test performance:
The validity of the test is shown by the oxygen consumption in the control bottle with sodium acetate and an
endogenous respiration of 0.6 mg/litre. The pH of the medium at the end of the test period (28 days) was 6.9.
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
162 d
Details on results:
Test chemical is not biodegraded in the closed bottle test.
Results with reference substance:
90% degredation after 28 days.

Study type likely not suitable for substance due to toxicity determination of biodegredation was not possible. This study therefore is not definative evidence that the substance is not biodegradable

.

A more representative result could be achieved by using a method that releases the chemical slowly into solution. Reliable with restrictions score may be given. Lack of biodegradation may therefore be due to toxicity of the test cornpound because of the endogenous respiration was inhibited by triethylenetetramine. Furthermore, the stringency of the test procedures could account for the recalcitrance of triethylenetetramine in the closed bottle test. For these reasons, lack of biodegradation does not mean that triethylenetetramine is recalcitrant in nature

Validity criteria fulfilled:
yes
Interpretation of results:
other: Not readily Biodegradable.
Conclusions:
The test was carried out reliably without major restrictions in the methodology. The substance was not readily biodegradable in this test. Optimization of the test method or alternative method to reduce toxicity to inoculum may yield a higher degredation result. It can be concluded that this
substance is not readily biodegradable in the closed bottle test of this type however greater biodegredation may be found using a more suitable
Silica gel (Slow Release) method for example.
Executive summary:

Test was conducted reliably according to guidelines. Lacking information over calculation of theoretical oxygen demand. Guideline was suitably followed. Although no certificate of analysis was included, purity was reported. Study was conducted to GLP.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Reliability 2 possible with more test substance information. Methodology of test carried out reliably.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Minor adaptions to protocol
Principles of method if other than guideline:
Activated Sludge was used as innoculum.The sludge was preconditioned to reduce endogenous respiration rates. Ammonium chloride was ommitted from the medium to prevent nitrification. The test was also prelonged.
GLP compliance:
no
Remarks:
Guidelines were however followed.
Oxygen conditions:
aerobic
Inoculum or test system:
other: See below for details.
Details on inoculum:
Secondary activated sludge was obtained from the RZWI Nieuwgraaf in Duiven . The RZWI Nieuwgraaf is an activated
sludge plant treating predominantly domestic wastewater.
Duration of test (contact time):
> 100 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test was performed in 280 ml BOD (Biological Oxygen Demand) bottles. The closed bottle test was performed according to the EEC/OECD
test guidelines. With slight modifications ( listed above ).The pH was measured using a pH meter, Ankersmit A141. Stocks of 1.0g/l were made for
both reference substance and test substances. Data was calculated according to guideline. Full explination of methods not given.
Reference substance:
other: Sodium Acetate
Preliminary study:
No information regarding prelimanary Studies.
Test performance:
The validity of the test is shown by the oxygen consumption in the control bottle with sodium acetate and an endogenous respiration
of 0.6 mg/litre. The pH value of the media at day 28 was 6.9.
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
120 d
Remarks on result:
other: For All Tested Substances
Details on results:
Test chemicals were not biodegraded in the closed bottle test.
Results with reference substance:
> 90% degredation after 28 days.

Due to toxicity to innoculum biodegredation could not be determined with the method used. It is therefore impossible to test triethylenetetramine (TETA), tetraethylenepentamine (TEPA), pentaethylenehexamine (PEHA), higher ethylenepolyamines (HEPA) and N-aminoethylpiperazine (AEP) in the standard closed bottle test due to the toxicity of the test compounds and the "high" initial concentration in the test. However, the slow release of polyethylene amines from a carrier prevents high initial concentrations and may provide a more realistic scenario for these type of substances for future tests. More identification information is required to receive a reliable with restrictions score in current state a reliability score is not possible despite test methodolgy being reliably conducted.

Validity criteria fulfilled:
no
Remarks:
Substance ID's require conformation
Interpretation of results:
other: Not readily Biodegradable.
Conclusions:
The test was carried out reliably without major restrictions. The substances were not readily biodegradable in these tests. Optimization of the test
method or alternative method to reduce toxicity to innoculum may yield a higer degredation result. It can be concluded that this substance is not
readily biodegradable in the closed bottle test of this type, however greater biodegredation may be found using a more suitable Silica gel
method for example. Although the methodology of this test was carried out reliably,lack of substance identification for the substances tested
make a reliability score unassignable. Klimisch 4
Executive summary:

Test conducted according to appropriate guideline. Multiple substances were tested and reported together lacking identification information on the indervidual substances. However general quality criteria was reported and methodolgy followed can be considered reliable.Lacking information over calculation of theoretical oxygen demand. Guideline suitably followed. Certificate of analysis for the substances tested were not included, purity and Batch/lot number were missing from substance information, Study was not conducted to GLP. Study type likely not to be suitable for substance as toxicity made determination of biodegredation not possible.

Endpoint:
biodegradation in water: screening tests
Type of information:
other: Research Study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Well conducted test and useful endpoint. However lack of substance ID means that a reliability score cannot be assigned. A reliability score 2 ispossible with missing ID Data.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Work was conducted as part of a research report to help understand possible environmental behaviour of substances concerned. A Guideline was
therefore not followed. The test was based on determining if the test substances could be used as a sole source of nitrogen by microbes. This was
conducted in the presence of different carbon sources with TETA to determine the most appropriate carbon source, and then all test substances were testing using the carbon source with the best result. No nitrogen was present in the medium. The test chemical used was therfore the only available
source of nitrogen growth of microbes and removal of TETA using chemical analysis was investigated.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: See Below
Details on inoculum:
Activated sludge used as inocula were obtained from an activated sludge plant, treating predominantly domestic waste water (WWPP Nieuwgraaf,
Duiven).
Initial conc.:
0.1 g/L
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Media
All experiments were performed using a nutrient medium containing per litre: 1550 mg K2HP04, 850 mg NaH2P04, 100 mg MgS0,.7H20, 10 mg N
aEDTA, 10 mg FeS0,7H20, 10 mg CaCI, and 100 p1 trace elements solution of Vishniac and Santer.

Method
The growth experiment was performed in a 1 litre serum bottle with 200 ml of nutrient media containing 0.1 g/l ethyleneamine
(TETA. HEPA, TEPA or PEHA) as sole nitrogen source and 1.0 g/l of a carbon source. The carbon sources tested were acetate, lactate,
(7 mM) to remove all nitrogen compounds. The headspace of the bottles was flushed with oxygen to prevent growth of nitrogen fixing bacteria.
The bottles were incubated at 30°C in the dark. After an incubation period of two weeks growth was determined by estimating the increase in
turbidity. A control containing a carbon source but no ethylene amine was incubated simultaneously to allow comparison for turbidity. The
utilization of TETA as nitrogen source was also determined by measuring the decrease of TETA.
Samples were taken at various times during the incubation. The samples withdrawn (25 ml) were filtered over an 8 pm filter and finally prepared for
HPLC analyses.
Reference substance:
not required
Remarks:
( For the goal of this research)
Preliminary study:
Research comprised of a preliminary test with TETA and 8 different carbon sources to establish the most appropriate carbon source to use in the
tests of TETA,TEPA,PEHA and HEPA. TETA concentration was monitored analytically with the method described above. Decrease in TETA
concentrations were seen in all cases however less TETA was broken down in the presence of starch as a carbon source.
Test performance:
Tests were checked for performance by comparison to the negative control. In the case of the first test decrease of TETA was determined analytically
to indicate test performance . (See results table above)
Details on results:
It can bee seen from the above table that TETA is almost completely broken down demonstrating TETA can be used as a nitrogen source by
environmentally occuring bacteria.
Results with reference substance:
No reference substance needed. Not a standard test.

Growth of microorganisms with varying nitrogen sources.

Nitrogen Source

Growth

Triethylenetetramine (TETA)

Tetraethylenepentamine (TEPA)

Pentaethylenehexamine (PEHA)

Higher ethylenepolyamines (HEPA)

 

++

++

++

++

 

++ Good Growth

Validity criteria fulfilled:
not applicable
Interpretation of results:
other: Likely to be biodegradable under environmental conditions
Conclusions:
Results suggest that in conditions where nitrogen is limiting TETA and all other chemical tested are likely to be broken down in the environment.
Although lacking GLP accreditation this study could achieve a reliable with restrictions score if substance information data can be confirmed. In
current state a reliability score is not assignable. Klimisch 4. However this report does still provide valuble evidence that microbes are capable of
biodegrading these substances via co metabolic processes.
Executive summary:

Lacking test substance information, GLP accreditation, methodology of test carried out reliably (guideline followed).

Reliability 2 possible with more test substance information.

Description of key information

 The substance is not readily or inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Two tests investigating the ready biodegradability of Amines, polyethylenepoly (PEPA) are available. One of the available studies was conducted as a prolonged guideline test following OECD 301D (Biodegradation of Higher Ethylene Amines, van Ginkel 1990). The biodegradation of the substance was investigated by comparing the theoretical oxygen demand with the measured oxygen consumption of activated sludge in presence of the test substance. The incubation period was prolonged to 162 days. At the end of the incubation period a degradation rate of 0% was determined. The substance is therefore not readily biodegradable according to OECD criteria. The results are supported by a second test following OECD 301D (Biodegradation of Various Polyethylene Amines, van Ginkel 1990). The study investigated the ready biodegradability of several polyethylene amines including PEPA. After a prolonged incubation period of 120 days no biodegradation based on the oxygen consumption was determined for PEPA.

In a study following OECD 302A the inherent biodegradability of PEPA was investigated (Removal of Higher Ethylene Polyamines in a SCAS Test, van Ginkel 1992). Secondary activated sludge and primary settled sewage were used as inoculum. The biodegradation rate was determined by following the removal of dissolved organic carbon (DOC) for 84 days. At test termination a degradation of 16% was reported. Thus inherent biodegradation was not observed.

Toxicity of PEPA to the competent microorganisms and the stringency of the test procedures could account for the recalcitrance of PEPA in the Closed Bottle test. Results obtained in the Semi-Continuous Activated Sludge (SCAS) test (inherent biodegradability test) also illustrate that PEPA is not completely (ultimately) biodegradable. It is unlikely that biodegradation of PEPA will occur in the environment.

However, co-metabolic transformation of PEPA has been demonstrated in batch experiments (Triethylenetetramine (TETA) as a sole source of nitrogen for microbial growth, van Ginkel 1992). In these experiments ethylene amines, including PEPA, were used as the only available nitrogen source for activated sludge microorganisms. After an incubation period of two weeks the microbial growth was determined by estimating the increase in turbidity compared to a control. The detected microbial growth indicates that PEPA can be utilized by microorganisms as nitrogen source in the environment.

Conclusion:

The available data indicate that the substance is not readily or inherently biodegradable.