Polyethylene polyamine, pentaethylenehexamine 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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Some methodology not described. Test type was not entirely suitable for this substance due to toxicity biodegredation could not be determined.

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 was also prolonged.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge was obtained from the RZWI Nieuwgraaf in Duiven, The Netherlands. 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 1.0 g/l were made for both reference substance and test substance. Results were handled appropriately. Full explination of methods not given.

Reference substance:

acetic acid, sodium salt

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.

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. Study type likely not suitable for substance due to toxicity, determination of biodegredation was therefore 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. 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 method for example.

Executive summary:

Test carried out according to appropriate guidelines, missing some description of THOD methodology. Due to toxicity the biodegredation could not be determined for this substance. The test was conducted reliably but is not entirely applicable to this substance. Study can therefore be considered reliable with restrictions.

Reference 2

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

Deviations:

yes

Remarks:

see below, field principles of method if other etc

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:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge (1992.03.27) and primary settled sewage were collected from the WWTP Nieuwgraaf in Duiven, The Netherlands. 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.:

35 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:

18

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	22.5	0
-7	21.2	20.6	0
-6	19.0	18.9	0
-5	19.5	8.3	0
4	18.8	14.0	0
2	16.8	14.8	0
1	14.0	15.2	0
0	15.0	15.2	0
1	15.5	19.7	88
2	14.2	25.7	67
3	14.1	33.7	44
4	13.2	38.1	29
6	17.8	42.8	29
7	14.1	42.4	19
8	13.5	42.2	18
9	12.6	43.2	13
10	12.7	42.7	14
11	13.5	42.6	17
13	16.4	46.1	15
14	15.1	43.6	17
15	14.0	43.2	17
17	13.4	40.8	22
20	14.1	38.5	30
22	15.5	40.2	29
24	15.3	38.3	34
27	15.5	42.4	23
29	15.0	45.4	13
31	14.1	44.7	13
34	13.8	41.9	20
36	13.8	41.8	20
38	12.3	44.5	8
41	12.7	39.1	25
43	12.5	39.4	23
45	14.9	39.7	29
48	13.9	42.4	19
50	14.1	39.9	26
52	14.4	41.1	24
55	14.6	42.9	19
57	12.4	40.8	19
59	11.3	38.9	21
63	12.4	36.7	31
64	11.8	37.2	27
66	10.6	36.0	27
69	11.3	38.1	23
71	10.8	39.5	18
73	11.2	40.6	16
76	10.5	39.2	18
78	10.1	38.7	18
80	11.0	40.2	17
83	11.5	40.3	18
84	11.9	40.0	20

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Study can be considered reliable with restrictions. Biodegredation was not observed in the SCAS test.

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 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.

Reference 3

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. Methodology of test carried out reliably (guideline followed). Lacking substance information and test method 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

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 was also prolonged.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge was obtained from the RZWI Nieuwgraaf in Duiven, The Netherlands. The RZWI Nieuwgraaf is an activated sludge plant treating predominantly domestic wastewater.

Duration of test (contact time):

120 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:

acetic acid, sodium salt

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.

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.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 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 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:

The Test was 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 methodology followed can be considered reliable. Reliable with restrictions score is possible.

Reference 4

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: Essentially a well conducted test and useful endpoint. However lack of substance ID means that a reliability score cannot be assigned. A reliability score 2 is possible 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:

activated sludge, domestic, non-adapted

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

Based on:

test mat.

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:

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 chemicals tested in this case PEHA 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:

Research based tests with no guideline and minimal writup and no GLP accreditation. Lacking specific substance information. Analytical support was given for proof of principal for TETA. The other substances including PEHA were assessed by bacterial growth only. With sufficient ID information a higher reliability score could be given.

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 35 mg/L (DOC). Based on DOC removal a degradation of 18% 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.

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.