Amines, polyethylenepoly-, tetraethylenepentamine fraction

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-01-24 - 1990-07-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP test according to guidelines. Validility criteria reported. Some methodology not described. Test type not entirely suitable for this substance due to toxicity.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Minor adaptions to protocol

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. The sludge was preconditioned to reduce endogenous respiration rates. Ammonium chloride was ommitted from the medium to prevent nitrification.

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 1.0 g/l were made for both reference substance and test substance. Results were handled appropriately.
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/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.

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 under GLP. Study type likely not suitable for substance due to toxicity, determination of biodegradation was not possible. This study is therefore not definative evidence that this substance is not biodegradable.

Due to toxicity to inoculum biodegradation in the closed bottle test was not observed. An alternative method to reduce the toxicity to inoculum would be more applicable to this substance.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test was carried out reliably without major restrictions. 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 degradation result. It can be concluded that this substance is not readily biodegradable in the closed bottle test of this type however greater biodegradation may be found using a more suitable Silica gel method for example. The study is valid with the restrictions mentioned.

Executive summary:

Test conducted according to appropriate guideline. Quality criteria were reported, sufficient substance information was given and methodology followed can be considered reliable. Batch/lot number was also reported.

Reference 2

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:

documentation insufficient for assessment

Remarks:

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

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

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. The sludge was preconditioned to reduce endogenous respiration rates. Ammonium chloride was ommitted from the medium to prevent nitrification.

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 1.0 g/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 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.

Validity criteria fulfilled:

no

Remarks:

Substance ID's require confirmation

Interpretation of results:

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 inoculum 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. Non GLP. Multiple substances were tested and reported together lacking identification information on the indervidual substances. However general quality criteria was reported and methodology followed can be considered reliable. 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. Lacking information over calculation of theoretical oxygen demand. Certificate of analysis for the substances tested were not included, purity and Batch/lot number were missing from substance information.

Reference 3

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:

guideline study with acceptable restrictions

Remarks:

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 below (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 µm 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.:

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 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 of concentrated 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:

17

Sampling time:

84 d

Remarks on result:

other: Removal by adsorpton to sludge only

Details on results:

Test substance was not biodegraded.

Results with reference substance:

No reference substance reported. Control used for comparison/ calculation of removal.

% Removal

Time (Days)	NPOC CONTROL	NPOC TEST	REMOVAL %
-8	19.8	26.2	0
-7	21.2	21.4	0
-6	19.0	20.2	0
-5	19.5	11.2	0
4	18.8	17.4	0
2	16.8	16.5	0
1	14.0	15.1	0
0	15.0	16.4	0
1	15.5	21.9	81
2	14.2	29.7	53
3	14.1	34.5	39
4	13.2	37.9	26
6	17.8	44.8	19
7	14.1	40.3	21
8	13.5	43.6	10
9	12.6	42.6	10
10	12.7	43.1	9
11	13.5	40.5	19
13	16.4	46.9	8
14	15.1	44.9	11
15	14.0	45.4	6
17	13.4	40.8	18
20	14.1	38.4	27
22	15.5	39.1	29
24	15.3	38.1	32
27	15.5	35.5	40
29	15.0	41.9	19
31	14.1	37.3	30
34	13.8	38.6	26
36	13.8	43.8	10
38	12.3	41.8	11
41	12.7	41.1	15
43	12.5	39.6	19
45	14.9	40.3	24
48	13.9	41.6	17
50	14.1	41.2	19
52	14.4	39.8	24
55	14.6	44.4	11
57	12.4	38.3	22
59	11.3	40.3	13
63	12.4	40.5	16
64	11.8	38.5	20
66	10.6	36.5	22
69	11.3	40.9	11
71	10.8	39.9	13
73	11.2	39.4	15
76	10.5	39.5	13
78	10.1	38.0	16
80	11.0	39.5	14
83	11.5	38.6	19
84	11.9	39.0	19

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

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.

Reference 4

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, The Netherlands).

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 K2HPO4, 850 mg NaH2PO4, 100 mg MgSO,.7H2O, 10 mg NaEDTA, 10 mg FeSO,7H2O, 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.

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:

not measured/tested

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

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 33.3 mg/L (DOC). Based on DOC removal a degradation of 17% 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.

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 degradation of the test substance.