Amides, C18-unsatd., N-[2-(1-piperazinyl)ethyl]

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study (experimental phase) was conducted from 2020-10-14 to 2021-05-10.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Synonym: 14C labelled fatty acids, C18-unsaturated, diamide with 1-piperazineethanamine
Radiochemical purity: 98.0 % (HPLC with radio detection)
Specific activity: 2148 MBq/mmol (LC-MS)
Specific concentration: 5.30 MBq/mL (Volumetric analysis by LSC)
Appearance: Clear, colourless solution in methanol
Structure / Location of the label: C18H33O-NH-C(radiolabelled)H2-CH2-(C4H8N2)-C18H33O
Molecular formula: C42H79N3O2
Labelled molecular weight : 659.96 g/mol @ 58.1 mCi/mmol
Stability under test conditions: Not applicable
Expiry date: Not specified, stability (radiochemical purity) was verified by synthesis laboratory prior to start of the study
Recommended storage: below - 70 °C

Test Facility Actions
Receipt: 2020-10-06
Retention sample: Not retained, due to its radioactivity and small sample amount
Storage Conditions: - 20 ± 2°C, dark, in tightly closed original container

Applied Radioactivity
The measured activity was in the range of 97.6 - 102.7% (mean 99.8%) of the calculated activity. The calculations of percentage residual activity in the test item replicates was done based on the nominal concentration.

Applied Radioactivity per Replicate
10 µg/L
Volume per replicate [mL] 100
Stock solution [kBq/790µL] 536
Applied volume stock solution [µL] per replicate 5.4
Calc. activity per replicate [kBq] 3.25
100 µg/L
Volume per replicate [mL] 100
Stock solution [MBq/377 µL] 2.0
Applied volume stock solution [µL] per replicate 6
Calc. activity per replicate [kBq] 32.55


Radioactivity at Test Start
Repl. kBq/ Repl % AR*
P1 10 µg/L 3222.1 99.0
P2 3252.7 99.9
P29 100 µg/L 31778.7 97.6
P42 33412.2 102.7
Mean 99.8
* related to nominal concentration

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

Surface Water
The sampling site was selected with respect to the regional biological and chemical water quality maps (interactive online version) of the Lower Saxony Water Management, Coastal Defence and Nature Conservation Agency (German: NLWKN).
Detailed information about the sampling site, sampling conditions and physico-chemical characteristics of the surface water are given below.

Sampling Site, Sampling Conditions and Water Characteristics:
Parameter
Sampling site location: Am Anger, 30982 Pattensen, Germany
Coordinates: 52˚11’39.419'' N 9˚47’7.49'' E
Sampling date & time: 2020-10-09 /9:40 h
Weather conditions: Dry, cloudy
Depth of collection: Surface (upper 0 – 20 cm)
Appearance : Clear, colourless
Water Temperature [°C]: 13.1
pH value: 7.35
O2 concentration [mg O2/L]: 8.89
DOC [mg/L]: 1.25
TOC [mg/L]: 2.47
TIC [mg/L]: 53.26
NO2-N [mg/L]: 0.08
NO3-N [mg/L]: 2.78
NH3-N [mg/L]: 0.25
Total N [mg/L]: 3.56
Ortho-P [mg/L]: < 0.05
Total P [mg/L]: < 0.05
Suspended matter*: 15.1 mg/L*
20.1 mg/L#

* determined with membrane filter with pore size 0.45 µm
# determined with membrane filter with pore size 0.20 µm
< value below measuring range

Duration of test (contact time):

90 d

Initial conc.:

100 µg/L

Based on:

test mat.

Initial conc.:

10 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

Test System: Natural surface water (field fresh sampled) of the river ‘LEINE’.
The sampling site was selected with respect to the regional biological and chemical water quality maps (interactive online version) of the Lower Saxony Water
Management, coastal Defence and Nature Conservation Agency (German: NLWKN).
Reasons for the selection: The surface water meets the recommendations of the guideline.
Collection / Sampling: The water was collected from the surface of the river (approx. upper 20 cm water) at an undisturbed recess. Furthermore, temperature, pH value and O2 concentration
of the water were measured at field sampling.
Handling: The water was clear and colourless. Little floating matter was removed by filtration.
Acclimation: Until test start the surface water was acclimated for 5 days under test conditions at 12 ± 2 °C in the dark with aeration.

Experimental Procedure
14C-labelled Test Item: [ethylamino-1-14C]Compound A
Nominal test item concentration: 10 µg/L and 100 µg/L
Application solution: 536 kBq in 790 µL methanol (10 µg/L)
2.0 MBq in 0.377 mL methanol (100 µg/L)
Activity in the test solution: 3.25 kBq/100 mL (10 µg/L)
32.55 kBq/100 mL (100 µg/L)
Pre-treatment: None
Blank Control: Surface water with the respective amount solvent but without test and/or reference item.
Sterile control: Sterilized surface water with the [ethylamino-1-14C]Compound A at test concentrations. For sterilization, the water was autoclaved prior to application.
Solvent control: Surface water and reference item, treated with the same amount of methanol as used for the application of the test item.

Test Procedure
Duration: 90 days
Application: At test start (application), appropriate volumes of the test item application solutions were pipetted directly into each replicate (test medium).
Test vessels: 150 mL gastight glass bottles
250 mL bottles for replicates for metabolite identification.
14CO2 Traps: Internal traps containing 1.5 mL 5 mol/L NaOH solution were used for trapping 14CO2.
Volatile Transformation Products: Trapping of volatiles was not necessary.
Test medium: Natural surface water of the river ‘Leine’.
Volume of the test medium: 100 mL
150 mL (replicates for metabolite identifcation)
Replicates:
Test item replicates (14C-labelled): Duplicates per sampling and per concentration. 22 replicates were prepared for each concentration.
Sterile controls: Duplicates per sampling and per concentration. 16 replicates were prepared for each concentration.
Blank controls: Single, in total 10 replicates were prepared.
Reference control and solvent control replicates: Duplicates per sampling. 6 replicates were prepared for each group.
Replicates for metabolite identification (non-labelled): 4 test item replicates and two control replicates were prepared.

Incubation: Nominal: 12 ± 2 °C in the dark
Actual: 11.4 -13.2 °C in the dark
Agitation: Continuous stirring at approx. 120 rpm to maintain particles and microorganisms in suspension and to facilitate oxygen transfer from the headspace to the liquid.


Type and Frequency of Measurements
Temperature: The incubation temperature was recorded continuously throughout the test.
pH and Oxygen: Measurements of pH and oxygen concentration in the test system (Blank control) were done in parallel to each sampling.

Mineralization and Transformation (Primary Degradation)
Sampling for determination of transformation (LSC and combination of HPLC, fraction collector and offline LSC) was carried out directly after application and at 8 additional sampling points for the test item replicates.
Sampling was done on day 0, 2, 5, 6, 7, 15, 27, 41, 61 and 90.
The results of the sampling on day 5 indicated losses due to adsorption. Therefore, an additional sampling was done on day 6 and the sampling procedures were modified/optimized. The results of day 6 were not included in the evaluation. The optimized sampling was done from day 7 on for all samplings.
The sampling points were chosen to enable the establishment of the pattern of decline of the test item.
Two test item replicates per test item concentration were harvested at each sampling time for determination of transformation and mineralization.
Residual total activity in the test solution, dissolved 14CO2 and evolved 14CO2 were determined by LSC.
The amount of test item and degradation products in the test solution, biomass extracts and vessel surface extracts were determined by combination of HPLC, fraction collector and offline LSC.

Sterile controls were analysed at the beginning and at the end of the study. An additional analysis was done at day 12 to assess the adsorption behaviour of the test item.

Mineralisation of reference: For the reference control and solvent control sampling was done item on day 2, 6 and 15 (only reference control). Residual activity in the test solution, dissolved 14CO2 and evolved 14CO2 was determined by LSC.
 
Statistics/Evaluation
The measurements included determination of total organic 14C and direct and indirect determination of 14CO2. Furthermore abiotic degradation losses due to sorption are taken into account and the distribution of total organic 14C was calculated.
The mass balance is expressed in percentage of the applied initial concentration for each sampling time and replicate.
The DT50 and DT90 trigger endpoint determination was calculated based on the recommendations of the Guidance Document on Estimating Persistence and Degradation Kinetics from Environmental Fate Studies on Pesticides in EU Registration (FOCUS).  

Reference substance:

aniline

Remarks:

Radiochemical purity: 99.2%; Specific activity : 110 mCi/mmol

Compartment:

abiotic control measured at end of test

% Total extractable:

>= 97.6

% Recovery:

97.6

Compartment:

abiotic control measured at end of test

% Total extractable:

>= 87.8

% Recovery:

87.8

Compartment:

biologically active treatment at end of test

Sampling date:

2021

% Total extractable:

>= 85.9

% Non extractable:

>= 3.9

% CO2:

>= 20.9

% Recovery:

110.6

Compartment:

biologically active treatment at end of test

Sampling date:

2021

% Total extractable:

>= 79.7

% Non extractable:

>= 3.9

% CO2:

>= 27.9

% Recovery:

106.5

Parent/product:

parent

Compartment:

water

Key result

% Degr.:

>= 94.6

Parameter:

radiochem. meas.

Sampling date:

2020

Sampling time:

61 d

Parent/product:

parent

Compartment:

water

Key result

% Degr.:

>= 97.1

Parameter:

radiochem. meas.

Sampling date:

2021

Sampling time:

90 d

Parent/product:

parent

Compartment:

water

Key result

% Degr.:

>= 97.3

Parameter:

radiochem. meas.

Sampling date:

2020

Sampling time:

61 d

Parent/product:

parent

Compartment:

water

Key result

% Degr.:

>= 98

Parameter:

radiochem. meas.

Sampling date:

2021

Sampling time:

90 d

Key result

Compartment:

natural water: freshwater

DT50:

4.82 d

Type:

other: The transformation of [ethylamino-1-14C]Compound A followed a bi-phasic pattern, DTx values were calculated with the DFOP kinetics

Temp.:

12 °C

Remarks on result:

other: The NER were as a worst-case regarded as not degraded [ethylamino-1-14C]Compound A and included in the kinetic calculations

Key result

Compartment:

natural water: freshwater

DT50:

4.07 d

Type:

other: The transformation of [ethylamino-1-14C]Compound A followed a bi-phasic pattern, DTx values were calculated with the DFOP kinetics

Temp.:

12 °C

Remarks on result:

other: The NER were as a worst-case regarded as not degraded [ethylamino-1-14C]Compound A and included in the kinetic calculations.

Transformation products:

yes

Remarks:

Three metabolites (M1, M2 and M3) were detected from day 5 on. M1 was predominately detected in the test solution, whereas M2 and M3 were solely detected in the extracts of the biomass and test vessel surface.

No.:

#2

No.:

#3

No.:

#1

Details on transformation products:

In parallel to the transformation of [ethylamino-1-14C]Compound A, three metabolites (M1, M2 and M3) were detected from day 5 on. M1 was predominately detected in the test solution, whereas M2 and M3 were solely detected in the extracts of the biomass and the test vessel surface. M2 and M3 were transient metabolites, the highest concentrations were determined at day 7. 7.8% AR (10 µg/L) and 12.1% AR (100 µg/L) of M2 and 6.2% AR (10 µg/L) and 8.2% AR (100 µg/L) of M3 were determined. Thereafter the concentration was decreasing continuously, and was ≤0.5% AR for both metabolites at 100 µg/L. At 10 µg/L the concentration was < LOQ from day 41 on. The formation phase of the terminal metabolite M1 lasted until day 27, thereafter the metabolite remained in a concentration range of 73% AR – 82% AR.

Metabolite Idetification
An authentic standard of (Piperazin-1-yl)acetic acid was used for the method implementation. The mass spectrometric analysis indicated that the ions m/z = 56.1 and 99.0 are daughter ions of this compound under collision induced dissociation (MS/MS) in the mass spectrometer. The daughter ion m/z = 99.0 results from the elimination of formic acid whereas the daughter ion m/z = 56.0 results from the cleavage of the piperazine ring. No mass shift for these daughter ions occur in case of the 14C-isotopologue of (Piperazin-1-yl)acetic acid. This compound elutes approximately at a retention time of 3.15 min as figured out during the method implementation. (Piperazin-1-yl)(1-14C)acetic acid is not present in test item samples on day 0 and sterile control samples on day 0 and 5 whereas this compound was detected in the test item samples from day 5. (Piperazin-1-yl)(1-14C)acetic acid eluted at a retention time of 3.15 min and was detected by the mass transition 147.0 > 99.0. The mass transition 145.0 > 99.0 from the residual unlabelled (Piperazin-1-yl)acetic acid in the test item was not detected. M1 had an amount of approximately 20% of AR (see Table 20) and therefore, the mass transition of the unlabelled (Piperazin-1-yl)acetic acid was below the LOD due to the isotope ratio 14C/12C of 12:1. In addition, this result was confirmed by analysis of blank controls and test item samples from day 71 of the metabolite identification study. (Piperazin-1-yl)acetic acid was absent in the control blanks but was detected by the mass transitions 145.0 > 99.0 and 145.0 > 56.0 in the test item samples.
 
Results – M2 and M3
Metabolite M2 and M3 are isomers and the result of the cleavage of the amide bond either that of the secondary amide or the tertiary amide. Samples from the test vessel extracts of the main GLP study with [ethylamino-1-14C]Compound A were used for the analysis of M2 and M3. Structure elucidation for these metabolites are based on mass transitions originating from the 14C-label. M2 eluted with a relative retention time of 0.74 and M3 with 0.76. Both metabolites are characterised by the mass transition 396.3 > 310.3. M3 is characterised by further mass transitions 396.3 > 379.3 and 396.3 > 115.1. The mass transition 396.3 > 379.3 was used for the differentiation between the two isomers. The transition 396.3 > 379.3 shows the elimination of ammonia. Elimination of ammonia is a characteristic collision induced fragmentation of primary amines under MS/MS conditions. This finding is in accordance to the proposed chemical structure of M3.

Residues:

yes

Remarks:

The maximum NER were 7.5% AR on day 27 (10 µg/L) and 5.2% AR on day 61 (100 µg/L). At test end 3.9% NER were determined in both concentrations.

Details on results:

Oxygen Concentration and pH during the Study
Measurements of pH and oxygen concentration in the test system (blank control) of the high concentration was done in parallel to each sampling. The oxygen concentration was in the range 6.19 – 9.52 mg O2/L and the pH was in the range 6.88 – 7.68. The results are given in Table 9.

Table 9: Oxygen Concentration and pH during the Study
Day O2 Day O2
mg/L pH mg/L pH
0 8.99 7.68 16 6.73 6.88
2 7.86 7.40 27 7.31 6.94
5 6.19 7.06 41 8.78 7.03
6 6.89 7.09 61 7.88 7.01
7 6.65 7.03 90 9.52 7.12


Transformation and Mineralization of [ethylamino-1-14C]Compound A
Degradation of [ethylamino-1-14C]Compound A was fast and started within two days after application. The degradation progressed steadily and only 12.8% applied radioactivity (AR) (10 µg/L) and 12.5% AR (100 µg/L) [ethylamino-1-14C]Compound A were determined after 7 days. At test end 2.0% AR (10 µg/L) and 2.9% AR (100 µg/L) were determined.

Formation of 14CO2 started in parallel to the transformation of [ethylamino-1-14C]Compound A, 5.3 % 14CO2 (10 µg/L) and 3.3 % 14CO2 (100 µg/L) were determined on day 15. At test end 27.9 % 14CO2 (10 µg/L) and 20.9 % 14CO2 (100 µg/L) were determined ).

The mass balance at test start and from day 7 until test end (day 90) was in the range of 91.9 – 106.5% for 10 µg/L and 86.5 – 110.6% for 100 µg/L.
The mass balance was < 80% at day 2 and day 5. Analysis of the test vessel surface and biomass showed that significant amounts of [ethylamino-1-14C]Compound A, M2 and M3 were adsorbed at the biomass and test vessel surface. From day 7 on biomass and test vessel surface were analysed additionally and the mass balance was > 90% until test end.

Results with reference substance:

After 6 days mineralisation of the reference item Aniline sulphate, [14C(U)]- was ≥ 70% in the reference control and > 50% in the solvent control. The high mineralization (formation of 14CO2) indicates that the surface water contained an active microbial population. Details are given in Table 10.

Table 10:        Mineralization of Aniline Sulphate, [¹⁴C(U)]- in the Reference Control

Concentration	AR (test start)	Exposure Day	Reference Control
			% AR		% Mineralisation^1)
	kBq/Repl		Repl. 1	Repl. 2	Repl. 1	Repl. 2
Reference Control	14.14	2	64.7	61.6	35.3	38.4
		6	27.2	24.9	72.8	75.1
		15	22.8	20.9	77.2	79.1
Solvent Control	14.14	2	66.5	66.2	33.5	33.8
		6	48.6	47.5	51.4	52.5

1) calculated from residual activity in test solution (after acidification) and applied radioactivity (100% - %AR)

10 µg/L: Distribution of Total 14C Radioactivity as % of AR

Sampling Day	0	2	5	7	15	27	41	61	90
Total Radioactivity Test Solution	99.5	78.3	49.9	56.0	69.2	62.3	64.5	62.7	70.2
Biomass Extract aqueous	-	-	-	0.7	0.6	0.3	0.7	0.7	0.3
Biomass Extract organic	-	-	-	6.7	15.9	3.3	3.8	3.4	2.3
NER	-	-	-	4.6	4.3	7.5	4.7	5.9	3.9
Test Vessel Surface	-	-	-	19.1	4.5	5.9	1.9	2.2	1.9
14CO2	-	0.0	0.3	4.9	5.3	15.0	20.3	25.0	27.9
Mass Balance	99.5	78.3	50.2	91.9	99.8	94.4	95.8	100.0	106.5

 

100 µg/L: Distribution of Total 14C Radioactivity as % of AR

Sampling Day	0	2	5	7	15	27	41	61	90
Total Radioactivity Test Solution	100.1	80.6	44.4	45.5	60.5	71.9	70.0	66.9	80.9
Biomass Extract aqueous	-	-	-	0.9	1.4	0.3	0.5	1.1	0.4
Biomass Extract organic	-	-	-	13.8	11.4	5.0	5.5	5.6	2.8
NER	-	-	-	4.3	4.3	3.6	4.4	5.2	3.9
Test Vessel Surface	-	-	-	20.2	10.0	2.9	1.6	1.2	1.8
14CO2	-	0.01	0.09	1.9	3.3	7.9	13.6	21.6	20.9
Mass Balance	100.1	80.6	44.5	86.5	90.90	91.6	95.6	101.6	110.6

 

10 µg/L: [ethylamino-1-14C]Compound A and Metabolite 1, 2 and 3 as % of AR

Sampling Day	0	2	5	7	15	27	41	61	90
[ethylamino-1-14C] Compound A
Test Solution	101.1	99.7	63.5	4.2	< LOQ	4.8	< LOQ	< LOQ	< LOQ
Biomass Extract organic	-	-	-	2.8	7.8	2.7	1.9	1.7	1.1
Testvessel Extract	-	-	-	7.9	2.2	2.6	0.8	1.0	0.9
Total	101.1	99.7	63.5	12.8	10.1	10.1	2.7	2.7	2.0
M1
Test Solution	-	-	36.5	54.3	76.6	62.8	73.0	73.3	77.7
Biomass Extract organic	-	-	-	< LOQ	<LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Testvessel Extract	-	-	-	0.7	0.5	0.7	< LOQ	< LOQ	< LOQ
Total	-	-	36.5	55.0	76.8	63.5	73.0	73.3	77.7
M2
Biomass Extract organic	-	-	-	1.8	2.7	0.6	0.7	1.2	0.4
Testvessel Extract	-	-	-	6.0	1.5	1.5	0.5	0.5	0.5
Total	-	-	-	7.8	4.2	2.1	1.2	1.8	0.9
M3
Biomass Extract organic	-	-	-	2.0	3.2	0.7	0.8	0.8	0.5
Testvessel Extract	-	-	-	4.2	0.8	0.7	0.2	0.3	0.2
Total	-	-	-	6.2	4.0	1.5	1.1	1.0	0.8

Italic values: Values < LOQ, values calculated from total radioactivity and test item/metabolite distribution at day 15 (biomass) and day 27 (test vessel surface)

 

100 µg/L: [ethylamino-1-14C]Compound A and Metabolite 1, 2 and 3 as % of AR

Sampling Day	0	2	5	7	15	27	41	61	90
[ethylamino-1-14C] Compound A
Test Solution	92.9	95.4	79.7^1	4.5	3.9	1.9	2.0	1.8	< LOQ
Biomass Extract organic	-	-	-	3.5	5.2	3.0	2.9	2.9	2.1
Testvessel Extract	-	-	-	4.6	4.4	1.4	0.9	0.7	0.8
Total	92.9	95.4	79.7	12.5	13.5	6.3	4.7	5.4	2.9
M1
Test Solution	-	-	20.3	36.9	58.6	73.0	79.1	73.6	81.4
Biomass Extract organic	-	-	-	0.1	0.1	< LOQ	0.1	< LOQ	< LOQ
Testvessel Extract	-	-	-	2.4	1.8	0.6	0.5	0.3	0.6
Total	-	-		39.4	60.5	73.6	79.6	74.0	82.0
M2
Biomass Extract organic	-	-	-	4.3	2.0	0.3	0.4	0.3	0.3
Testvessel Extract	-	-	-	7.8	2.3	0.3	< LOQ	< LOQ	< LOQ
Total	-	-	-	12.1	4.4	0.6	0.4	0.3	0.3
M3
Biomass Extract organic	-	-	-	3.7	2.7	0.4	0.4	0.4	0.2
Testvessel Extract	-	-	-	4.5	1.8	0.5	0.1	< LOQ	< LOQ
Total	-	-	-	8.2	4.6	0.9	0.5	0.4	0.2

1) corrected for mass balance loss

Validity criteria:

Mass balance at the end of the test should be 90 - 110 %

Observed value:

91.9 – 106.5% for 10 µg/L and 86.5 – 110.6% for 100 µg/L at test start, from day 7 until test end (day 90).
< 80% at day 2 and day 5, losses due to adsorption on test vessel surface

Validity criteria fulfilled:

yes

Validity criteria:

Mineralisation of the reference item Aniline

Observed value:

> 70 % Mineralisation after 6 days

Validity criteria fulfilled:

yes

Conclusions:

• Degradation of [ethylamino-1-14C]Compound A was fast and started within two days after application. At test end 2.0% AR (10 µg/L) and 2.9% AR (100 µg/L) were determined.

• In parallel to the transformation of [ethylamino-1-14C]Compound A, three metabolites (M1, M2 and M3) were detected from day 5 on. M1 was predominately detected in the test solution, whereas M2 and M3 were solely detected in the extracts of the biomass and the test vessel surface. M1 was identified as (Piperazin-1-yl)acetic acid (section 13.6.4). M2 and M3 were transient metabolites (section 13.6.5).

• Formation of 14CO2 and NER started in parallel to the transformation of [ethylamino-1-14C]Compound A, 5.3 % 14CO2 (10 µg/L) and 3.3 % 14CO2 (100 µg/L) were determined on day 15. At test end 27.9 % 14CO2 (10 µg/L) and 20.9 % 14CO2 (100 µg/L) were determined. The maximum NER were 7.5% AR on day 27 (10 µg/L) and 5.2% AR on day 61 (100 µg/L). At test end 3.9% NER were determined in both concentrations.

Executive summary:

• Degradation of [ethylamino-1-14C]Compound A was fast and started within two days after application. At test end 2.0% AR (10 µg/L) and 2.9% AR (100 µg/L) were determined.


• In parallel to the transformation of [ethylamino-1-14C]Compound A, three metabolites (M1, M2 and M3) were detected from day 5 on. M1 was predominately detected in the test solution, whereas M2 and M3 were solely detected in the extracts of the biomass and the test vessel surface. M1 was identified as (Piperazin-1-yl)acetic acid (section 13.6.4). M2 and M3 were transient metabolites (section 13.6.5).


• Formation of 14CO2 and NER started in parallel to the transformation of [ethylamino-1-14C]Compound A, 5.3 % 14CO2 (10 µg/L) and 3.3 % 14CO2 (100 µg/L) were determined on day 15. At test end 27.9 % 14CO2 (10 µg/L) and 20.9 % 14CO2 (100 µg/L) were determined. The maximum NER were 7.5% AR on day 27 (10 µg/L) and 5.2% AR on day 61 (100 µg/L). At test end 3.9% NER were determined in both concentrations.

 

• Data of Kinetic Fit and DTx Values of [ethylamino-1-14C]Compound A and Metabolites M1, M2 and M3 at 12 °C

10 µg/L
		[ethylamino-1-14C]Compound A	M1	M2	M3
Model		DFOP	SFO	SFO	SFO
Chi-square		29.9	7.34	38.5	13.8
DT50	Days	4.07	211	1.23	7.05
DT90	Days	46.9	700	4.07	24.9

100 µg/L
		[ethylamino-1-14C]Compound A	M1	M2	M3
Model		DFOP	SFO	SFO	SFO
Chi-square		33.4	4.22	5.3	7.63
DT50	Days	4.82	> 10^5	2.13	3.97
DT90		26.5	> 10^5	7.08	13.2

 

Description of key information

The main conclusion of the aerobic mineralization testing of [ethylamino-1-14C]Compound A (radiolabeled constituent fatty acids, C18-unsaturated, diamide with 1-piperazineethanamine ) which is a main constituent from N-[2-(piperazin-1-yl)ethyl]C18-unsatured-alkylamide (AA-AEP, CAS No 1228186-18-2) in water is that [ethylamino-1-14C]Compound A is rapidly degraded with a very short half-life of 4.1/4.8 days at test concentration of 10/100 µg/L but that as expected the mineralisation of the AA-AEP is not complete as (Piperazin-1-yl)acetic acid is remaining after 90 days of testing at 12°C.

The main conclusion of Sewage Treatment Plant simulation testing is that AA-AEP is removed  for >99.99% removed from waste water in the test unit using specific chemical analysis from day 44 to 48 quantifying octadecenyl amide of N-(2-aminoethyl)piperazine as representative component. These analyses demonstrate that the removal of AA-AEP is complete. The AA-AEP concentrations in sludge of the reactor sampled on days 47 and 48 were 0.88 and 0.80 mg/L. The mean removal percentage of AA-AEP from the influent through adsorption onto sludge was therefore 0.04 -0.044%. This percentage demonstrates that tall oil, reaction products with N-(2-aminoethyl)piperazine are almost completely removed by biodegradation. The CAS test demonstrates that tall oil, reaction products with N- (2-aminoethyl)piperazine almost completely removed from the wastewater by biodegradation in conventional biological wastewater treatment plants. The STP simulation study is performed with a limited mass balance as evaporation can be excluded from the mass-balance equation.  

Key value for chemical safety assessment

Half-life in freshwater:

4.5 d

at the temperature of:

12 °C

Additional information

The aerobic mineralization of [ethylamino-1-14C]Compound A in surface water was determined in a simulation biodegradation test over a test period of 90 days at 12 °C according to OECD guideline 309 under GLP conditions. 

[ethylamino-1-14C]Compound A is the radiolabeled version of fatty acids, C18-unsaturated, diamide with 1-piperazineethanamine which is a main constituent from N-[2-(piperazin-1-yl)ethyl]C18-unsatured-alkylamide, EC No 629-767-5 (AA-AEP, CAS No 1228186-18-2).

The degradation of [ethylamino-1-14C]Compound A was fast and started within two days after application. At test end 2.0% AR (10 µg/L) and 2.9% AR (100 µg/L) were determined.

• In parallel to the transformation of [ethylamino-1-14C]Compound A, three metabolites (M1, M2 and M3) were detected from day 5 on. M1 was predominately detected in the test solution, whereas M2 and M3 were solely detected in the extracts of the biomass and the test vessel surface. M1 was identified as (Piperazin-1-yl)acetic acid. M2 and M3 were transient metabolites 

Formation of 14CO2 and NER started in parallel to the transformation of [ethylamino-1-14C]Compound A, 5.3 % 14CO2 (10 µg/L) and 3.3 % 14CO2 (100 µg/L) were determined on day 15. At test end 27.9 % 14CO2 (10 µg/L) and 20.9 % 14CO2 (100 µg/L) were determined. The maximum NER were 7.5% AR on day 27 (10 µg/L) and 5.2% AR on day 61 (100 µg/L). At test end 3.9% NER were determined in both concentrations.

The main conclusion of the aerobic mineralization testing of [ethylamino-1-14C]Compound A in surface water is that [ethylamino-1-14C]Compound A is rapidly degraded with a very short half-life of 4.1 - 4.8 days  (10/100 µg/L) but as expected the mineralisation of the AA-AEP is not complete as (Piperazin-1-yl)acetic acid is remaining after 90 days of testing at 12°C.

 

In addition to simulation testing of the biodegradation in water and sediment, simulation testing of the fate of the registered substance (AA-AEP) in an STP was performed using a CAS test setup.

The continuous activated sludge (CAS) test was performed according to ISO Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. Tall oil, reaction products with N-(2-aminoethyl)piperazine were exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test. Tall oil, reaction products with N-(2-aminoethyl) piperazine were spiked at a nominal influent concentration of 50 mg/L (36.5 mg/L carbon; calculated) for a period of 48 days and included a control fed with domestic wastewater only. The immediate high removal percentages can be attributed to adsorption and probably biodegradation. The mean removal percentage of tall oil, reaction products with N-(2-aminoethyl)piperazine calculated over 15 measurements obtained from day 34 to 48 of the test was 89 +/-1% (95% confidence interval). These organic carbon removal percentages in excess of the pass level of 80% indicate that the test substance is completely removed. An accurate assessment of the removal of tall oil, reaction products with N-(2- aminoethyl)piperazine was established with specific analyses. The method (LCMS/ MS) for the determination of tall oil, reaction products with N-(2- aminoethyl)piperazine was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), recovery and specificity. The mean removal percentage of tall oil, reaction products with N-(2-aminoethyl)piperazine in the test unit was assessed with the specific analysis from day 44 to 48 was >99.999% using octadecenyl amide of N-(2-aminoethyl)piperazine as representative component. These analyses demonstrate that the removal of tall oil, reaction products with N-(2-aminoethyl)piperazine is complete. Tall oil, reaction products with N-(2-aminoethyl)piperazine concentrations in the sludge of the reactor sampled on days 47 and 48 were 0.88 and 0.80 mg/L. The mean removal percentage of tall oil, reaction products with N-(2-aminoethyl)piperazine from the influent through adsorption onto sludge assessed in two samples was therefore 0.04 -0.044%. This percentage demonstrates that tall oil, reaction products with N-(2-aminoethyl)piperazine are almost completely removed by biodegradation. The CAS test demonstrates that tall oil, reaction products with N- (2-aminoethyl)piperazine almost completely removed from the wastewater by biodegradation in conventional biological wastewater treatment plants.