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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:
key study
Study period:
24 Jan 1990 - 12 Jul 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
A detailed description of the methodology is not given. Test type not entirely suitable for this substance.
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 inoculum. The sludge was preconditioned to reduce endogenous respiration rates. Ammonium chloride was ommitted from the medium to prevent nitrification. The test period was prolonged.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: Secondary activated sludge
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. This lack of biodegradation may be due to toxicity of the test cornpound because of the endogenous respiration is inhibited by triethylenetetramine.
Results with reference substance:
90% degredation after 28 days.

Lacking information over calculation of theoretical oxygen demand. Guideline was suitably followed. Although no certificate of analysis was included, purity was reported. Batch/lot number was missing from substance information. Study was conducted to GLP. Study type likely not suitable for substance due to toxicity to inoculum . Determination of biodegredation was not possible. This study is therefore not definative evidence that this substance not biodegradable.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test was carried out reliably without major restrictions. 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 carrier method with Silica gel method for example.
Executive summary:

Test conducted according to appropriate guideline. Quality criteria were reported, sufficient substance information was given and methodolgy followed can be considered reliable. Batch/lot number not reported. Due to toxicity to inoculum biodegredation in the closed bottle test was not observed. An alternative method to reduce the toxicity to inoculum would be more applicable to this substance. The study is valid with the restrictions mentioned.

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: Data on substance identity lacking
Remarks:
Methodology of test carried out reliably (guideline followed). Lacking substance information and test method not optimal for substances tested.
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 period was prolonged.
GLP compliance:
no
Remarks:
Guidelines were however followed.
Oxygen conditions:
aerobic
Inoculum or test system:
other: Secondary activated sludge
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 explanation of methods not given.
Reference substance:
other: Sodium Acetate
Preliminary study:
No information regarding preliminary 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/L. 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.

Study type likely not to be suitable for substance as toxicity made determination of biodegradation not possible.

However general quality criteria was reported and methodology followed can be considered reliable. 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:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test was carried out reliably without major restrictions. Optimization of the test method or alternative method to reduce toxicity to innoculum may yield a higher degradation 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 "carrier" 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.Lacking information over calculation of theoretical oxygen demand. Guideline was 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.With suitable substance ID information reliable with restrictions score is possible.

Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003-07-23 to 2003-08-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD Guideline study with valid positive, negative, and toxicity controls.
Qualifier:
according to guideline
Guideline:
OECD Guideline 306 (Biodegradability in Seawater)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
anaerobic
Inoculum or test system:
natural water: marine
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Seawater was collected from Tor Bay, Devon, UK.
- Storage length: 7 days
- Pretreatment: Water was filtered to 10 um and aerated for 7 days prior to nutrient addition to the following concentrations: 8.5 mg/L KH2PO4; 21.75 mg/L K2HPO4; 33.3 mg/L Na2HPO4.2H2O; 0.5 mg/L; NH4Cl; 22.5 mg/L MgSO4.7H2O; 27.5 mg/L CaCl2.6H2O; 0.25 mg/L FeCl3.6H2O; and 0.40 mg/L EDTA disodium salt. The final medium was aerated for 2.5 hours before use and the initial (Day 0) dissolved oxygen was determined with a YSI meter.
- Water filtered: yes
- Type and size of filter used, if any: 10 µm
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
3 mg/L
Based on:
test mat.
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 281 mL
- Composition of medium: 8.5 mg/L KH2PO4; 21.75 mg/L K2HPO4; 33.3 mg/L Na2HPO4.2H2O; 0.5 mg/L; NH4Cl; 22.5 mg/L MgSO4.7H2O; 27.5 mg/L CaCl2.6H2O; 0.25 mg/L FeCl3.6H2O; and 0.40 mg/L EDTA disodium salt in seawater.
- Test temperature: 20 +/- 2 °C
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Glass reagent bottles with ground glass stoppers
- Number of culture flasks/concentration: 3
- Measuring equipment: YSI meter

SAMPLING
- Sampling frequency: 5, 9, 15, 20 and 28 days
- Sampling method: Dissolved oxygen measurement

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes, with test substance at 2 mg/L plus reference substance at 1.4 mg/L
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
< 30
Sampling time:
28 d
Remarks on result:
other: 2 mg/L - Without nitrification
Parameter:
% degradation (O2 consumption)
Value:
< 15
Sampling time:
28 d
Remarks on result:
other: 2 mg/L - With nitrification
Parameter:
% degradation (O2 consumption)
Value:
< 20
Sampling time:
28 d
Remarks on result:
other: 3 mg/L - Without nitrification
Parameter:
% degradation (O2 consumption)
Value:
< 10
Sampling time:
28 d
Remarks on result:
other: 3 mg/L - With nitrification
Parameter:
% degradation (O2 consumption)
Value:
< 15
Sampling time:
28 d
Remarks on result:
other: 4 mg/L - Without nitrification
Parameter:
% degradation (O2 consumption)
Value:
< 7
Sampling time:
28 d
Remarks on result:
other: 4 mg/L - With nitrification
Details on results:
With nitrification, triethylene tetramine ThOD = 1.67 mg O2/mg
Without nitrification, triethylene tetramine ThOD = 3.40 mg O2/mg
Results with reference substance:
Sodium benzoate attained a mean biodegradation of 69% after 5 days and 75% after 28 days. The time to achieve 50% of the final degradation was 3 days, which is within the range required by OECD Guideline 306.
Validity criteria fulfilled:
yes
Remarks:
OECD Guideline 306 criteria fulfilled.
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance showed no significant biodegradation after 28 days at any concentration tested. (< 15% if nitrification is not included, < 7% if nitrification is included, based on the most sensitive test vessels used).
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-03-27 - 1992-06-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted report carried out to GLP with analysis certificate and chemical analysis. Lacking information on analytical method calibration, standard curve etc. Further reliable.
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)
Deviations:
yes
Remarks:
see field "Principles of method if other 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: Secondary activated sludge and primary settled sewage
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.:
32.9 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 ISO 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:
20
Sampling time:
84 d
Remarks on result:
other: Removed by adsorbtion only
Details on results:
No biodegredation was measured in the SCAS test.

% Removal

Time (Days)

NPOC CONTROL

NPOC TEST

REMOVAL %

-8

19.8

19.2

0

-7

21.2

21.4

0

-6

19.0

19.5

0

-5

19.5

9.3

0

4

18.8

10.8

0

2

16.8

16.8

0

1

14.0

14.5

 

0

15.0

15.5

 

1

15.5

23.2

77

2

14.2

33.5

41

3

14.1

39.0

24

4

13.2

43.2

9

6

17.8

47.9

9

7

14.1

48.4

0

8

13.5

47.2

0

9

12.6

45.4

0

10

12.7

42.5

9

11

13.5

41.5

15

13

16.4

45.5

12

14

15.1

43.5

14

15

14.0

45.2

5

17

13.4

26.2

61

20

14.1

41.2

18

22

15.5

40.1

25

24

15.3

39.1

28

27

15.5

42.3

19

29

15.0

42.8

16

31

14.1

41.1

18

34

13.8

40.0

20

36

13.8

42.8

12

38

12.3

44.8

1

41

12.7

36.0

29

43

12.5

39.3

19

45

14.9

40.7

22

48

13.9

42.2

14

50

14.1

41.5

17

52

14.4

43.5

12

55

14.6

41.7

18

57

12.4

37.5

24

59

11.3

38.8

16

63

12.4

39.7

17

64

11.8

39.0

17

66

10.6

36.3

22

69

11.3

39.1

16

71

10.8

36.0

23

73

11.2

38.6

17

76

10.5

38.0

16

78

10.1

37.1

18

80

11.0

39.3

14

83

11.5

37.2

22

84

11.9

36.7

25

 

 

 

 

 

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, this study can be considered reliable with this restriction. 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.
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: screening tests
Type of information:
other: Research Study
Adequacy of study:
supporting study
Study period:
1992-10-12
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Data on substance identity are lacking.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was conducted as part of a research report investigating the environmental fate of the tested substances. The study investigated the growth of microorganisms (activated sludge, domestic) in presence of different ethylene amines as the only source of nitrogen. A series of treatments containing different substances serving as carbon source was established. Break down of the test substances was followed by HPLC analysis.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
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 NaEDTA, 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)
Remarks on result:
other: 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.
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 in this case 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 by co-metabolic processes.
Executive summary:

Reliability 2 possible with more test substance information. Methodology of test carried out reliably (guideline followed). Lacking substance information and test method not optimal for substances tested.

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
Type of water:
freshwater

Additional information

The ready biodegradability of the substance was tested in a prolonged guideline test following OECD 301D (van Ginkel, 1990a). The oxygen consumption of secondary activated sludge exposed to an initial test substance concentration of 2 mg/L was followed for 162 days. At the end of the incubation period a degradation rate of 0% was determined. The lack of degradation might indicate a toxic or inhibiting effect of the substance to the activated sludge organisms. Comparable results were obtained in a second study conducted according to OECD 301D (van Ginkel, 1990b). The study investigated the ready biodegradability of several polyethylene amines. After a prolonged incubation period of 120 days no biodegradation based on the oxygen consumption of activated sludge organisms was determined. Therefore, the test substance is not readily biodegradable.

The inherent biodegradability of the substance was investigated in a study according to OECD guideline 302A (van Ginkel and Stroo, 1992). Secondary activated sludge and primary settled sewage were used as inoculum. The initial test substance concentration was 32.9 mg/L (DOC). Based on DOC removal a degradation of 20% was determined after 84 days. The observed partial removal of test substance was likely to be related to adsorption to activated sludge particles and not to biodegradation. Thus, inherent biodegradability could not be demonstrated.

The available data indicate that the substance is not readily or inherently biodegradable by activated sludge organisms.

In addition to the freshwater test a study on the biodegradation of the test substance in seawater is available. The test was conducted according to OECD guideline 306 (Barret, 2003). Test substance concentrations of 2, 3 and 4 mg/L respectively were added to natural sea water. The biodegradation of the substance was determined by measuring the oxygen consumption of the test medium. Inhibitory effects of the substance were not determined. After 28 days of incubation a degradation rate of <20% were recorded. Thus, a potential for primary biodegradation of the substance in the marine environment could not be demonstrated. However, the potential for degradation is lower in seawater compared to freshwater degradation tests (Guidance on information requirements and chemical safety assessment, Chapter R.7b; ECHA, 2017).

Furthermore data on the degradation of the test substance under nitrogen limited conditions are available (Kroon and van Ginkel, 1992). The study investigated the growth of microorganisms in presence of ethylene amines as the only source of nitrogen (test material concentration: 0.1 g/L). Different carbon sources were added to the test medium at a concentration of 0.1 g/L. After an incubation period of two weeks the microbial growth was determined by estimating the increase in turbidity compared to a control. The concentration of test substance was analytically verified by HPLC analysis. The results demonstrated a break down of the test substance under nitrogen limited conditions.

The available data indicate that the substance is not readily or inherently biodegradable by activated sludge organisms.