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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:
14-03-1990 till 29-08-1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Activated sludge was used as inoculum; Ammonium chlroide was omitted from the medium to prevent nitrification; The test substance was tested adsorbed on silica gel, the test was prolonged
Principles of method if other than guideline:
A few minor deviations from the protocol for the closed bottle test were introduced:
- Instead of effluent/extract/mixture, activated sludge was used as an inoculum. The inoculum was taken from an activated sludge plant. The sludge was preconditioned to reduce the endogenous respiration rates (aeration of 200 mg dry weight for 1 week)
- The sludge was diluted to a concentration in the BOD bottles of 2 mg DW/litre.
- Ammonium chloride was omitted from the medium to prevent nitrification.
- The closed bottle test was prolonged by measuring the course of oxygen decrease in the bottles of day 28 using a special funnel which fitted exactly into the BOD bottle. The oxygen electrode was then put into the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After the withdrawal of the oxygen electrode the medium collected was put back into the bottle and closed.
- The test compound is a poorly soluble substance in water and therefore the test substance was first dissolved in dichloromethane. The test subsance in dichloromethane (0.56 ml) was added to 2g silica gel (100-200 mesh) weighed in a glass petri dish. The solvent was allowed to evaporate by placing the petri dish in a ventilated hood for 3 hour, and the entire contents were then transformed to the BOD bottle.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Obtained from an activated sludge plant treating predominantly domestic wastewater. 200 mg Dry Weight (DW/litre) sludge was preconditioned by aerating for a period of 1 week. The test was performed in 290 ml BOD (Biological Oxygen Demand) bottles.
Duration of test (contact time):
126 d
Initial conc.:
2 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
other: ratio between biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD)
Details on study design:
The sodium acetate was added to the bottles using a stock solution of 1.0 g/litre. The sodium acetate was added to the bottles using a stock solution of 1.0 g/litre. In the closed bottle test, Redicote N422 was added to an aqueous solution of mineral salts and exposed to relatively low numbers of microorganisms (the inoculum was a 200 mg dry weight/litre sample of active sludge from a local water treatment works collected and aerated for one week before the test) under aerobic conditions for a period of 28 days. The test concentration of Redicote N422 was 2 mg/litre. Control vessels comprised mineral solution alone, inoculated nutrient solution alone, inoculated nutrient solution alone with silica gel, and inoculated mineral solution with the reference material sodium acetate (6.7 mg/litre). The pH of the medium at day 28 was 7.5.
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
12
Sampling time:
5 d
Parameter:
% degradation (O2 consumption)
Value:
33
Sampling time:
15 d
Parameter:
% degradation (O2 consumption)
Value:
36
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
38
Sampling time:
42 d
Parameter:
% degradation (O2 consumption)
Value:
36
Sampling time:
70 d
Details on results:
The test substance is partly biodegraded in the closed bottle test (28 days) and should therefore not be classified as readily biodegradable.
In the prolonged test the biodeg percentage does not further increase indicating that a possible intermediate is formed. The lack of total biodegradability does not mean that the test substance or a possible intermediate is recalcitrant in nature. The stringency of the test procedures could account for the recalcitrance of the substance.
The test is valid based on the oxygen consuption of the control with sodium acetate and the endogenous respiration of 0.4 mg/L.
Results with reference substance:
The day 28 results for the degradation of sodium acetate reference material (90% BOD/ThOD) and for cumulative O2 consumption (4.7 mg/ O2 litre) fulfil the validity criteria.

Oxygen consumption in the closed bottle test in the presence of the test substance and sodium acetate

Time (days)

5

15

28

42

70

98

126

154

Reaction product of tall oil fatty acid and aminoethylpiperazine (mg O2/litre)

0.7

1.9

2.1

2.2

2.1

2.0

2.0

-

Sodium acetate (mg O2/litre)

3.6

4.3

4.7

-

-

-

-

-

 

Percentage biodegradation of the test substance and sodium acetate in the closed bottle test

Time (days)

5

15

28

42

70

98

126

154

Reaction product of tall oil fatty acid and aminoethylpiperazine (% BOD/ThOD)

12

33

36

38

36

34

34

-

Sodium acetate (% BOD/ThOD)

69

83

90

-

-

-

-

-

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
N-[2-(piperazin-1-yl)ethyl]C18-insaturated-alkylamide was partly biodegraded (30-40% BOD/ThOD) under conditions of the closed bottle test, and a degradation plateau was attained after 28 days. N-[2-(piperazin-1-yl)ethyl]C18-insaturated-alkylamide N-[2-(piperazin-1-yl)ethyl]C18-insaturated-alkylamide was partly biodegraded (30-40% BOD/ThOD) under conditions of the closed bottle test, and a degradation plateau was attained after 28 days. N-[2-(piperazin-1-yl)ethyl]C18-insaturated-alkylamide should therefore not be classified as readily biodegradable under the conditions of the test.
Executive summary:

Reaction product of tall oil fatty acid and aminoethylpiperazine (CAS number:92062 -17 -4) is partly biodegraded (30 -40%) in the closed bottle test (OECD 301D) which was performed under GLP conditions. The test substance should therefore not be classified as readily biodegradable.

During the prolonged phase of the study no further biodegradation was observed indicating that a possible intermediate is formed. Recalcitrance in the closed bottle test does not mean that the substance is recalcitrant in nature because of the stringency of the test procedures.

The test is valid as show by the endogenous respiration and total mineralization of the reference compound: sodium acetate.

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:
Study performed in GLP lab but not under GLP conditions
Justification for type of information:
Publication in which it explained why N-[2-(piperazin-1-yl)ethyl]C18-unsatured-alkylamide (CAS number: 1228186-18-2) was found to be not readily biodegradable using standard OECD 301D test conditions.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Activated sludge at a concentration of 2 mg dry weight/L was used as inoculum instead of an effluent/extract mixture and ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (van Ginkel and Stroo 1992).
GLP compliance:
no
Remarks:
But tests are performed in GLP laboratory
Specific details on test material used for the study:
The N-(1-ethylpiperazine) tall oil amide used in this study was identical to the substance used in the STP simulation study (OECD 303A).

Other Chemicals:
N-[3-(Dimethylamino)propyl] cocoamide, N-[3- (dimethylamino)propyl] hydrogenated tallow amide, N-[3-(dimethylamino)propyl] rapeseed amide and N-[3-(bis(2-hydroxyethyl)amino)propyl] cocoamide were provided by AkzoNobel Surface Chemistry AB. Synperonic PE/P105 was obtained from Croda.
N-Methyl-N-ethanoate dodecanamide, N,N-dibutyldodecanamide, N,N-dimethyldodecanamide, N,N-dimethyl-1,3-propanediamine, oleate, dodecanoate, citrate, N-(2-aminoethyl)piperazine, hexanoamide, dodecanamide, oleamide, acetate and stearate were obtained from Sigma Aldrich.
All other chemicals used were of reagent grade.

Deionized water containing no more than 0.01 mg L-1 copper was prepared in a water purification system.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
The activated sludge used as inoculum for the closed bottle tests and the enrichment cultures was collected from an aeration tank of a municipal wastewater treatment plant located in Duiven, the Netherlands.
This plant consists of mechanical and biological stages for the treatment of predominantly domestic wastewater.
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
For the closed bottle tests and metabilisation of the different substrates
Details on study design:
Besides to closed bottle tests enrichment cultures were used to evaluate the biodegradation route.

Enrichment, isolation and characterization
Selective enrichments, using N-[3-(dimethylamino) propyl] cocoamide or N-(1-ethylpiperazine) tall oil amide as sole source of carbon and energy, were performed at 30 °C in a 1.5 L fermentor with a working volume of 1 L (Applikon, Schiedam, the Netherlands). The impeller speed was 400 rpm and the pH was maintained at 6.8 with a solution of 50 g/L Na2HPO4, using a pH-electrode connected to a pH controller (ADI 1020, Applikon, Schiedam, the Netherlands). Mineral salts medium amended with N-[3-(dimethylamino)propyl] cocoamide or N-(1-ethylpiperazine) tall oil amide was supplied with a dilution rate of 10 day-1 to the fermentor using a peristaltic pump (Meyvis & Co., Bergen op Zoom, the Netherlands). After 1 week of operation the dilution rate of the enrichment on N-[3-(dimethylamino) propyl] cocoamide was stepwise increased during a 30 days period to 1 day-1. Repeated batch subculturing with N-[3-(dimethylamino)propyl] cocoamide and N-(1-ethylpiperazine) tall oil amide was started after 40 days, using the fermentor enrichment cultures as inoculum. Microorganisms enriched on N-[3-(dimethylamino)propyl] cocoamide were also used as inoculum for repeated batch subculturing on N,N-dimethyl-1,3-propanediamine. After growth was obtained dilutions of the batch subcultures were streaked on agar plates and subsequently one colony was streaked repeatedly to purity. Analyses to identify all three strains were carried out by DSMZ (Braunschweig, Germany) using fatty acid methyl ester analysis, substrate profiling and 16S rRNA sequencing.

Growth medium, cultivation, and growth conditions
The mineral salts medium used for isolation and growth of the bacteria contained per litre 1.55 g K2HPO4, 0.85 g NaH2PO4, 0.5 g NH4Cl, 0.1 g MgSO4.7H2O, 0.01 g Na2H2EDTA.H2O, 0.01 g FeSO4.7H2O and 0.1 mL of a trace solution described by
Vishniac and Santer (1957).
Agar plates used for isolation were incubated at 30 °C and contained beside the mineral salts medium 15 g/L bacteriological agar and 1 g/L of the substrates: N-[3-(dimethylamino)propyl] cocoamide, N,N-dimethyl-1,3-propanediamine or dodecanoate.
For the isolation of a microorganism degrading N-(1-ethylpiperazine) tall oil amide agar plates with 1 g/L dodecanoate were used because it was not possible to make agar plates containing N-(1-ethylpiperazine) tall oil amide.
All three isolated strains were grown and maintained in batch cultures using 1 L flasks with 200 mL of the mineral salts medium spiked with 1 g/L N-[3-(dimethylamino)propyl] cocoamide, 1 g/L N,Ndimethyl-1,3-propanediamine or 1 g/L N-(1-ethylpiperazine)
tall oil amide, respectively.

Growth experiments
with the isolates were performed in 100 mL flasks using 20 mL of mineral salts medium supplemented with different substrates (1 g/L). In the batch cultures containing N-[3-(dimethylamino)propyl] cocoamide, N-(1-ethylpiperazine) tall oil amide
and several of the other growth substrates the initial substrate concentration was toxic to the isolates. To reduce the substrate concentration in the water phase and herewith the toxicity, 16 g/L silica gel was added (van Ginkel et al. 1992). Growth was observed by measuring the increase in turbidity over time with a Hach Ratio XR turbidimeter (Hach Lange GmbH, Dusseldorf, Germany). Moreover, cell number increase was routineously determined using phase contrast microscopy. All batch cultures were shaken at 100 rpm in an orbital incubator at 30 °C.
Continuous growth experiments with the isolates were performed in the fermentor systems used for the selective enrichments. At steady state conditions the mineral salts medium amended with 1 g/L N-[3-(dimethylamino)propyl] cocoamide or 1 g/L N-(1-
ethylpiperazine) tall oil amide was dosed at a dilution rate of 1 day-1, the temperature was 30 °C and the pH was 6.8.

Washed cell suspensions and cell-free extracts
Cell suspensions of the continuous cultures were harvested by centrifugation at 10,000 g for 10 min at 4 °C, washed three times with 15 mM phosphate buffer, pH 7.0 or for the preparation of cell-free extract with 50 mM Tris/HCl buffer, pH 9.0. The washed cell suspensions were stored at 4 °C. Cell breakage of washed cell suspensions was achieved by sonication. Debris and whole cells were removed by centrifugation (30,000 g for 10 min at 4 °C) and the supernatant containing the cell free extract was used immediately in the enzyme assay. For the preparation of cell free extracts of acetate and dodecanamide grown cells, cells were harvested from a 2 L batch culture at the end of the exponential growth phase.

Oxidation rate by washed cell suspensions
Oxygen uptake was measured with a Biological Oxygen Monitor (Yellow Springs Instruments, Yellow Springs, Ohio), which consisted of an electrode and a water-jacketed vessel (5 mL). Washed cell suspensions were incubated in the vessel at 30 °C for at least five minutes to allow determination of the endogenous respiration rate. Subsequently, 0.1 mL of a 1 g/L substrate solution or suspension was injected, and the substrate-dependent respiration rate was determined. As dodecanamide has a very low water solubility, the water solubility was increased by preparing a suspension of 1 g/L dodecanamide with ethylene oxide/propylene oxide block copolymer, synperonic PE/P105 (1:1 w/w).

Enzyme assay
The amidase reaction was assayed (modified from Denger et al. 2008) discontinuously at 30 °C in 50 mM Tris/HCl buffer, pH 9.0, including 2.1 mM N-[3-(dimethylamino)propyl] cocoamide or 2.1 mM dodecanamide. The reaction was started with the addition of crude cell extract (34 and 239 µg protein/mL for cells incubated with N-[3-(dimethylamino) propyl] cocoamide and grown on N-[3- (dimethylamino)propyl] cocoamide and acetate, respectively; 89 µg protein/mL was used for cells grown on N-[3-(dimethylamino)propyl] cocoamide and incubated with dodecanamide) and stopped again by addition of 0.1 mL of 3 M HCl. Samples (1 mL) were taken at intervals and the formation of N,N dimethyl-1,3-propanediamine or ammonium was measured by cat-ion chromatography.
Preliminary study:
The study from C.G. van Ginkel from 1990 can be considered as the preliminary study.
Key result
Parameter:
% degradation (O2 consumption)
Value:
70
Sampling time:
28 d
Remarks on result:
other:
Remarks:
Biodegradability of N-[3- (Dimethylamino)propyl] cocoamide
Key result
Parameter:
% degradation (O2 consumption)
Value:
34
Sampling time:
28 d
Remarks on result:
other:
Remarks:
Biodegradability of N-(1-Ethylpiperazine) tall oil amide
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Remarks on result:
other:
Remarks:
Biodegradability of N-(2-aminoethyl)piperazine
Key result
Parameter:
% degradation (O2 consumption)
Value:
84
Sampling time:
11 d
Remarks on result:
other:
Remarks:
Biodegradability of N,N-dimethyl-1,3-propanediamine
Details on results:
The biodegradation of N-[3-(dimethylamino)propyl] cocoamide, N-(1-ethylpiperazine) tall oil amide and their hydrolysis products, N,N-dimethyl-1,3-propanediamine and N-(2-aminoethyl)piperazine was assessed in Closed Bottle tests inoculated with activated sludge (Fig. 2). N-[3-(Dimethylamino)propyl] cocoamide reached 70 %biodegradation thereby fulfilling the pass criterion of >=60% within 28 days. N-[3-(Dimethylamino)propyl] cocoamide is therefore regarded as readily biodegradable.
The ready biodegradability of N,N-dimethyl-1,3-propanediamine underpins the ready biodegradability N-[3-(dimethylamino)propyl] cocoamide.
N-(1-Ethylpiperazine) tall oil amide is degraded partially in the closed bottle test, as indicated by only 34 % biodegradation achieved (Fig. 2). Inability of microorganisms to degrade N-(2-aminoethyl)piperazine is most likely the cause of this incomplete degradation. Biodegradation of both N-[3-(dimethylamino)propyl] cocoamide and N-(1-ethylpiperazine) tall oil amide started without a lag-phase indicating fast hydrolysis of the amide bonds and subsequent degradation of the fatty acids formed (Fig. 2).

Results from Enrichment

Strains PK1, PK2 and PK3, were isolated from activated sludge using continuous cultures and subsequent batch sub-culturing. Strain PK1 was enriched and isolated using N-[3-(dimethylamino)propyl]

cocoamide as the sole source of carbon and energy.

Strain PK2 was enriched in a fermentor fed with N-[3 -(dimethylamino)propyl] cocoamide followed by repeated batch sub-culturing using N,N-dimethyl- 1,3-propanediamine as growth substrate. Agar plates with N,N-dimethyl-1,3-propanediamine were used to isolate this strain.

N-(1-Ethylpiperazine) tall oil amide was used as growth substrate for the enrichment of strain PK3, which was subsequently streaked to purity using agar plates containing dodecanoate.

Identification of enriched isolates

The profiles of the cellular fatty acids for PK1 and PK2 were both typical for the genus Pseudomonas. The results of the phenotypic tests confirmed these identifications. Partial sequences of the 16S rRNA of PK1 and PK2 showed 100 % similarity to Pseudomonas aeruginosa and Pseudomonas putida, respectively. The cellular fatty acid profile for strain PK3 resembled that of the species Aeromonas hydrophila. This identification was supported by 99.8 % similarity with A. hydrophila using the partial sequence of the 16S rRNA and the phenotypic tests.

Growth and oxydation rates

Growth of P. aeruginosa strain PK1 and A. hydrophila strain PK3 was determined in batch cultures containing different substrates. Both strains were able to grow on the amidoamines (secondary amides), N- [3-(dimethylamino)propyl] cocoamide, N-[3-(dimethylamino) propyl] rapeseed amide, N-[3-(dimethylamino) propyl] hydrogenated tallow amide, N-(1- ethylpiperazine) tall oil amide and N-[3-(bis(2- hydroxyethyl)amino)propyl] cocoamide. P. aeruginosa strain PK1 and A. hydrophila strain PK3 also supported growth on primary fatty acid amides dodecanamide and oleamide. Growth was also observed on citrate, acetate, dodecanoate, oleate and stearate. Strain PK1 and PK3 were not able to utilize the tertiary fatty acid amides N-methyl-N-ethanoate-dodecanamide, N,N-dibutyldodecanamide and, N,N-dimethyldodecanamide as sole source of carbon and energy. N,N-Dimethyl-1,3-propanediamine and N-(2-aminoethyl)piperazine did not support growth of both PK1 and PK3.

In batch cultures containing a nitrogen free mineral salts medium amended with 1 g/L of N-[3-(dimethylamino) propyl] cocoamide, growth of P. aeruginosa strain PK1 and P. putida strain PK2 was only observed when the medium was inoculated with both strains.

Organic carbon and nitrogen removal was determined in continuous cultures with strain PK1 growing on N-[3-(dimethylamino)propyl] cocoamide and strain PK3 utilizing N-(1-ethylpiperazine) tall oil amide. Hydrolysis of the amide bonds, followed by biodegradation of the released fatty acids would result in organic carbon removal percentages of 71 and 75 for N-[3-(dimethylamino)propyl] cocoamide and N-(1-ethylpiperazine) tall oil amide, respectively.

These removal percentages are close to the measured organic carbon removals of 67 % for N-[3-(dimethylamino) propyl] cocoamide and 71 % for N-(1-ethylpiperazine) tall oil amide. Organic nitrogen removal was negligible in the continuous cultures. This finding is in line with the observed accumulation of N,N-dimethyl- 1,3-propanediamine and N-(2-aminoethyl)piperazine during biodegradation of N-[3-(dimethylamino)propyl] cocoamide and N-(1-ethylpiperazine) tall oil amide, respectively. N,N-Dimethyl-1,3-propanediamine moiety of N-[3-(dimethylamino)propyl] cocoamide was

almost completely recovered (94 %) from the effluent of the continuous culture. A recovery of 84 % of N-(2- aminoethyl)piperazine from the effluent strongly indicates stoichiometric formation of this diamine

during degradation of N-(1-ethylpiperazine) tall oil amide. Oxidation of different substrates was studied with washed cell suspensions of N-[3-(dimethylamino)propyl] cocoamide grown P. aeruginosa strain PK1 (Table 1).

Table 1 Oxygen uptake rates for washed cell suspensions of P. aeruginosa strain PK1 grown on N-[3-(dimethylamino) propyl]  cocoamide at 30 °C

Substrate Oxygen consumption (nmol of O2 min-1 mg [dry wt] of cells-1)
N-[3-(Dimethylamino)propyl] cocoamide (a) 79
N-[3-(Dimethylamino)propyl] rapeseed amide (b) 54
N-[3-(Dimethylamino)propyl] hydrogenated tallow amide (c) 54
N-(1-Ethylpiperazine) tall oil amide (d) 50
N-[3-(Bis(2-hydroxyethyl) amino)propyl] cocoamide (a) 27
N-methyl-N-ethanoatedodecanamide (e) 0
N,N-dibutyldodecanamide (e) 

0

N,N-dimethyldodecanamide (e)  0
Dodecanamide (f)  6
Hexanoamide  5
Acetate  17
Dodecanoate  65
Citrate  0
Oleate  50
N-(2-Aminoethyl)piperazine  0
N,N-Dimethyl-1,3-propanediamine  0

Rates of oxygen uptake have been corrected for the endogenous oxygen uptake (9 nmol of O2 / min / mg [dry wt] of cells)

Alkyl chain length distributions specified by Karleskind (1996):

a: mainly C12 and C14

b: mainly C18 and C22

c: mainly C16 and C18

d: mainly C18

e: contains tertiary amide bond

f: Synperonic PE/P105 used for increasing the water solubility of dodecanamide did not influence the oxygen uptake rate.

Increased oxygen uptake rates were found with N-[3-(dimethylamino)propyl] alkylamides with coco-, rapeseed-, and hydrogenated tallow-alkyl chains. Strain PK1 was also capable of oxidizing N-(1-ethylpiperazine) tall oil amide, N-[3-(bis(2-hydroxyethyl) amino)propyl] cocoamide, hexanoamide, dodecanoamide, acetate, dodecanoate and oleate. No increase in the oxygen uptake rate was observed with citrate, N,N-dimethyl-1,3-propanediamine, N-(2-aminoethyl) piperazine, and the tertiary amides N-methyl- N-ethanoate-dodecanamide, N,N-dibutyldodecanamide and N,N-dimethyldodecanamide.

Amidase activity has been demonstrated in cellfree extracts P. aeruginosa strain PK1 grown on N-[3- (dimethylamino)propyl] cocoamide. Incubation of cell-free extract with N-[3-(dimethylamino)propyl]

cocoamide resulted in the formation of N,N-dimethyl-1,3-propanediamine (Fig. 3). Ammonium was liberated by this cell-free extract when incubated with dodecanamide. The specific enzyme activities with N- [3-(dimethylamino)propyl] cocoamide as substrate were 15.5 mkat/(kg protein) in extracts of N-[3 -(dimethylamino)propyl] cocoamide grown cells and 0.1 mkat/(kg protein) in extracts of acetate grown

cells (1 kat = 1 mol/s). The specific activity with dodecanamide as substrate was 0.6 mkat/(kg protein) in cell-free extracts of PK1 cells grown on N-[3- (dimethylamino)propyl] cocoamide.

Validity criteria fulfilled:
yes
Remarks:
in relation to the closed bottle tests
Interpretation of results:
inherently biodegradable
Remarks:
Parent is rapidly degraded.
Conclusions:
The aerobic biodegradation pathway for primary and secondary fatty acid amides involves an initial hydrolysis of the amide bond resulting in the formation of ammonium or (poly)amine and fatty acids. This hydrolytic reaction was found independent of the amine
structure and fatty acid chain. The initial point of attack and the broad substrate specificity of microorganisms degrading these fatty acid amides, allows read-across of ready biodegradability test results.
Ready biodegradability of a number of primary and secondary fatty acid amides has been demonstrated. Ready (ultimate) biodegradation of secondary fatty acid amides however depends on the biodegradability of the released amine. The inability of microorganisms to support growth of N-(2-aminoethyl)piperazine explains partial degradation of N-(1-ethylpiperazine) tall oil amide.

AA-AEP (CAS no 1228186-18-2) is thus rapidly biodegraded to N-aminoethylpiperazine and talloil carboxylic acid. The tall oil carboxylic acid is quickly degraded to CO2 and H2O and N-aminoethylpiperazine (CAS no 140-31-8) is not further degraded. The observed organic carbon removal percentages are good agreement with the theoretical percentages. A recovery of 84 % of N-(2-aminoethyl)piperazine from the effluent strongly indicates stoichiometric formation of this substance during degradation of N-(1-ethylpiperazine) tall oil amide.
Executive summary:

Abstract

To get insight in the biodegradation and potential read-across of fatty acid amides, N-[3 -(dimethylamino)propyl] cocoamide and N-(1-ethylpiperazine) tall oil amide were used as model compounds. Two bacteria, Pseudomonas aeruginosa PK1 and Pseudomonas putida PK2 were isolated with N-[3-(dimethylamino)propyl] cocoamide and its hydrolysis product N,N-dimethyl-1,3-propanediamine, respectively. In mixed culture, both strains accomplished complete mineralization of N-[3- (dimethylamino)propyl] cocoamide. Aeromonas hydrophila PK3 was enriched with N-(1-ethylpiperazine) tall oil amide and subsequently isolated using agar plates containing dodecanoate. N-(2-Aminoethyl) piperazine, the hydrolysis product of N-(1- ethylpiperazine) tall oil amide, was not degraded.

The aerobic biodegradation pathway for primary and secondary fatty acid amides of P. aeruginosa and A. hydrophila involved initial hydrolysis of the amide bond producing ammonium, or amines, where the

fatty acids formed were immediately metabolized. Complete mineralization of secondary fatty acid amides depended on the biodegradability of the released amine. Tertiary fatty acid amides were not transformed by P. aeruginosa or A. hydrophila. These strains were able to utilize all tested primary and secondary fatty acid amides independent of the amine structure and fatty acid. Read-across of previous reported ready biodegradability results of primary and secondary fatty acid amides is justified based on the broad substrate specificity and the initial hydrolytic attack of the two isolates PK1 and PK3.

Description of key information

The ready biodegradability of N-[2-(piperazin-1-yl) ethyl]C18 -unsaturated-alkylamide was evaluated in a closed bottle test (OECD 301D) under GLP conditions. Within 28 days 36% oxygen consumption was observed which did not further increase during the prolongation of the test up to 154 days and should therefore not be classified as readily biodegradable. Clarification of biodegradation route by Geerts et al (2014) shows that N-[2-(piperazin-1-yl) ethyl]C18 -unsaturated-alkylamide is partly biodegraded in the closed bottle test (28 days) and that N-(2-aminoethyl)piperazine (CAS no 140 -31 -8) is formed as metabolite. The 0% degradation observed for N-(2 -aminoethyl)piperazine (as presented in the SDS and disseminated dosser) confirms that N-[2-(piperazin-1-yl) ethyl]C18 -unsaturated-alkylamide is completely transformed into CO2, H2O and N-(2 -aminoethyl)piperazine.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

In the closed bottle test (OECD 301D) which was performed under GLP conditions, N-[2-(piperazin-1-yl) ethyl]C18-unsaturated-alkylamide was added to an aqueous solution of mineral salts and exposed to relatively low numbers of microorganisms (the inoculum was a 200 mg dry weight/litre sample of active sludge from a local water treatment works collected and aerated for one week before the test) under aerobic conditions for a period of 28 days. The test concentration of N-[2-(piperazin-1-yl) ethyl]C18-unsaturated-alkylamide was 2 mg/litre (sorbed to silica before the start of the test). Control vessels comprised mineral solution alone, inoculated nutrient solution alone, inoculated nutrient solution alone with silica gel, and inoculated mineral solution with the reference material sodium acetate (6.7 mg/litre). The pH of the medium at day 28 was 7.5.  The day 28 results for the degradation of sodium acetate reference material (90% BOD/ThOD) and for cumulative O2consumption (4.7 mg/ O2litre) fulfil the validity criteria. Oxygen consumption by the mixtures containing 2 mg/L of N-[2-(piperazin-1-yl) ethyl]C18 -unsaturated-alkylamide ranged from 0.7-2.0 mg/ O2 litre over the 154 day period. A degradation plateau at 36% O2 Oxygen consumption was attained after 28 days and N-[2-(piperazin-1-yl) ethyl]C18-unsaturated-alkylamide should therefore not be classified as readily biodegradable. The percentage does not further increase indicating that a metabolite is formed. Clarification of biodegradation route by Geerts et al (2014) shows that N-[2-(piperazin-1-yl) ethyl]C18 -unsaturated-alkylamide is partly biodegraded in the closed bottle test and that N-(2-aminoethyl)piperazine (CAS no 140 -31 -8) is formed as metabolite.