Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)

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:

10 March 2003 - 21 June 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP, quality criteria are met, lack of details on test material

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

yes

Remarks:

(item is adsorbed on silicagel)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge from the biological wastewater treatment plant in Abidos.
- Storage length: 1d
- Preparation of inoculum for exposure:
- Pretreatment: effluent is centifuged at approximately 20° C for 20 min. at 4000g. The base is washed in a volume of water to obtain a concentration factor of approximately 1 with respect to the sample. This inoculum is preconditioned (aeration) until flasks are seeded.
- Concentration of obtained sludge: 3.37 g/L dry weight
- Concentration of sludge in the test vessels: 30 mg dry material /l

Duration of test (contact time):

28 d

Initial conc.:

41.2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The study was performed according to OECD guideline 301F .

A measured volume (250 mL) of inoculated mineral medium, containing 41.2 mg/L of INIPOL002 with a theoretical oxygen demand of 2.97 mg O2/L as the sole source of organic carbon, is stirred in a closed flask at a constant temperature of 21 ° C for up to 28 days. The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas pressure in the respirometer flask. Degradation is followed by measuring the quantity of oxygen generated automatically by the automatic respirometer, the SAPROMAT. The CO2 generated by the ultimate biodegradation process is fixed by the soda lime in the cupel of the reactor. This results in a negative pressure which is detected by the manometer and communicated to the control unit. In return, the latter sends an electrical current into the oxygen generator which electrochemically generates a quantity of oxygen which is proportional to the strenght of the electrical current. The quantity of oxygen consumed by the test item (corrected for the quantity produced in the blank test with the inoculum) is expressed as a percentage of ThOD or COD.

The activity of the inoculum is controled by means of a reference item. Simultaneously, the posisble inhibiting effects of the test item are studied with respect to the inoculum, with a mixture of test and reference items, resulting in the following series:

Ft1, Ft2: + test item, in duplicate
Fb1, Fb2: blank, in duplicate
Fc1, Fc2: inoculum control, in duplicate
Fa1, Fa2: silica gel control, in duplicate
Fi: Inhibition

Reference substance:

benzoic acid, sodium salt

Preliminary study:

not applicable

Test performance:

- Difference of extremes of replicate values of the removal of test chemical at the plateau, at the end of the test, or at the end of the 10-d window, was less than 20%.
- Percentage biodegradation of the reference item reached a level of 83% by 14 days which is above the level of 60% for ready biodegradability.
- Oxygen uptake of the inoculum blank was about 12 mg O2/L in 28 days, which is below 60 mg O2/ L
- In the toxicity controls containing both the reference item and N-(oleyl)-1,3-propanediamine oleates, the percentage biodegradation reached 68% after 28 days. Since this value is higher than 25%, the test item is not inhibitory for inoculum.

Key result

Parameter:

% degradation (O2 consumption)

Value:

61

Sampling time:

28 d

Details on results:

The maximum degradation level of INIPOL 002 is 61% of the ThOD in 28 days. The latency phase for the degradation of the item (i.e. 10% mineralisation reached) is approximately 2 days, and the percentage of degradation after the time interval of 10 days after this latency phase is 53%.

Results with reference substance:

The percentage mineralisation of the reference item reached a level of 83% by 14 days.

Results and calculations, together with degradation curve are included in attached document. 

Validity criteria fulfilled:

yes

Remarks:

study quality criteria are met

Interpretation of results:

readily biodegradable

Conclusions:

N-(oleyl)-1,3-propanediamine oleates is readily biodegradable in an aerobic ready biodegradation test according to OECD TG 301F. The test item was mineralised by 61% in 28 days.

Executive summary:

The biodegradation of the test item was determined in an aerobic ready biodegradation test according to OECD TG 301F (manometric respirometry test) under GLP conditions (reliability 2). The test material (41.2 mg/L at start) was exposed to sewage sludge micro-organisms with culture medium in a closed flask at at a constant temperature of 21 ° C for 28 days.

The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas pressure in the respirometer flask by using an automatic respirometer, the SAPROMAT. The CO2 generated by the ultimate biodegradation process is fixed by the soda lime in the cupel of the reactor. The quantity of oxygen consumed by the test item (corrected for the quantity produced in the blank test with the inoculum) is expressed as a percentage of ThOD. Under the test conditions, mineralisation of the test substance reached 51% within 11 days and 61% at the end of the test (28 days). The level of mineralisation reached 53% ThOD at the end of the 10-d window (day 12). The test substance did not inhibit the micro-organisms as shown by the toxicity control flask. It can be concluded that the test substance is readily biodegradable. 

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-03-2008 17-04-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

acceptable deviations

Principles of method if other than guideline:

Ammonium chloride was omitted from the medium to prevent nitrification.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Details on inoculum:

River water was sampled at 13-03-2008 from the river Rhine near Heveadorp, The Netherlands. The river water was preconditioned to reduce the endogenous respiration rates (one week in total). Small particles were removed by sedimentation after 1 day aeration.

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The nutrient medium of the closed bottle test contained per litre of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O.
- The closed bottles were filled with river water and medium at a ratio of 1:1.
- Test temperature: 22-23°C
- pH: 7.3 - 7.0
- pH adjusted: no
- Aeration of mineral medium / river water: yes
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: incubator
- Number of culture flasks/concentration:
10 bottles containing only inoculum = inoculum blank
10 bottles containing only inoculum + 1 g silica gel/bottle = inoculum blank (silica)
10 bottles containing test substance , inoculum and 1 g silica gel / bottle =test
6 bottles containing sodium acetate and inoculum = reference control
- Method used to create aerobic conditions: aeration with pressured air
- Measuring equipment: oxygen meter


SAMPLING
- Sampling frequency: day 0, 7, 14, 21 ,28, 42
- Sampling method: the bottles from day 0, 7, 14 , 21 and 28 were discarded after the oxygen measurement (reference control is measured on day 0,7 and 14) (two duplicate bottle for each measurement)


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: no
- Reference control: yes



CALCULATIONS:
THOD N-oleyl-1,3-diaminopropane = 3.0 mg/mg
THOD sodiumacetate = 0.8 mg/mg

Oxygen consumption (mg/l) (BOD) = mean oxygen concentration (mg/l) inoculum blank - mean oxygen concentration (mg/l) test (or reference)
inoculum blank with silica gel is used for calculations of test
inoculum blank is used for calculations of referecence control

Biodegradation (%) = BOD/THOD *100

Key result

Parameter:

% degradation (O2 consumption)

Value:

66

Sampling time:

28 d

Details on results:

ThOD
The calculated theoretical oxygen demand of N-oleyl-1,3-diaminopropane is 3.0 mg/mg. This theoretical oxygen demand is calculated by assuming formation ofammonium, carbon dioxide and water. The theoreticai oxygen demand of sodium acetate is 0.8 mg/mg.

Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined because possible toxicity of N,oleyl-1,3-diaminopropane to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected.

Test conditions
The pH of the media was 7.3 at the start of the test. The pH of the medium at day 28 was 7.0. Temperatures ranged from 22 to 23°C.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 1.0 mg/L at day 28 (Table I). Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 81. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

The Closed Bottle test
N-oleyl-1,3-diaminopropane is biodegraded 66% at day 28 in the Closed Bottle test (Table 2), and should therefore be classified as readily biodegradable. Over 60% biodegradation was not achieved within a period of 10 days immediately following the attainment of 10% biodegradation. N-oleyl-1,3-diaminopropane is a mixture of alkyl-1,3-diaminopropanes and primary alkylamines. The degradation curve will therefore be the sum of a number of growth curves. The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a "standard" growth curve in ready biodegradability
tests. The time-window should be ignored as a pass fail criterion because of these scientific reasons.

Results with reference substance:

Sodium acetate was fully biodegraded within 14 days.

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot	Oc	Oa
0	8.9	8.9	8.9	8.9
	8.9	8.9	8.9	8.9
Mean (M)	8.9	8.9	8.9	8.9
7	8.0	7.6	8.1	4.7
	8.3	7.0	8.3	4.6
Mean (M)	8.2	7.3	8.2	4.7
14	8.1	4.5	8.0	3.5
	7.9	4.3	7.9	3.7
Mean (M)	8.0	4.4	8.0	3.6
21	7.9	2.6	8.0
	8.0	2.5	8.0
Mean (M)	8.0	2.6	8.0
28	7.8	1.7	7.9
	7.8	2.0	7.8
Mean (M)	7.8	1.9	7.9

 

Table 1: dissolved oxygen concentrations (mg/L) in the closed bottles.

Ocs : Mineral nutrient solution without test material but with inoculum and silica gel.

Ot: Mineral nutrient solution with test material adsorbed onto silica gel (3.0 mg/L) and inoculum.

Oc: Mineral nutrient solution without test material but with inoculum.

Oa: Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum.

Validity criteria fulfilled:

yes

Remarks:

endogenous respiration of 1.0 mg/l; total mineralization of reference compound 81% at day 14; >0.5 mg/l O2 in all the bottles during the test; differences duplicates <20% at end test

Interpretation of results:

readily biodegradable

Conclusions:

N-oleyl-1,3-diaminopropane is biodegraded 66% at day 28 in the Closed Bottle test and should therefore be classified as readily biodegradable. Over 60% biodegradation was not achieved within a period of 10 days immediately following the attainment of 10% biodegradation. N-oleyl-1,3-diaminopropane is a mixture of alkyl-1,3-diaminopropanes and primary alkylamines. Moreover surfactants such as alkyl-1,3-diaminopropanes are degraded sequentially by a consortium of microorganisms (vanGinkel 1996). The degradation curve will therefore be the sum of a number of growth
curves. The 10-day time window criterion was developed on the assumption that a testsubstance is degraded according to a “standard” growth curve in ready biodegradability tests. The time-window should be ignored as a pass fail criterion because of these scientific
reasons.

Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. N-oleyl-1,3-diaminopropane in the presence of silica gel did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. N-oleyl-1,3-diaminopropane was biodegraded 66% at day 28 in the Closed Bottle test. Hence this substance should be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 81% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Description of key information

The registered substance was found readily biodegradable according to a GLP and OECD 301F study (Gancet, 2004). 

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The registered substance is a salt of one mole of N-[(9Z)-octadec-9 -en-1 -yl]propane-1,3 -diaminium compounded with two moles of (9Z)-octadec-9 -enoate (Oleic acid). The registered substance was found readily biodegradable in an OECD 301F test (Gancet, 2004) according to GLP. The result demonstrate 61% of biodegradation after 28 days.

 

The time window criterion was developed for pure substances on the assumption that a compound is degraded by a single organism according to a sigmoidal growth curve in ready biodegradation tests. The degradation curve of Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1 ) is the sum of the growth curves of oleate and the two moieties of (Z)-N-9-octadecenylpropane-1,3-diamine. The time-window should therefore be ignored as a pass or fail criterion and the registered substance is  therefore classified as readily biodegradable based on the biodegradation percentage of 61% at day 28.

According to the REACH guidance document R7b v4 page 210 “The 10-day window does not apply […] if the test substance is made of a composition of homologous constituents.” The registered salt is constituted by diamine and fatty acid with different chain length, therefore the 10 days windows does not apply. According to this result, it is possible to conclude that the registered substance made by structurally similar chemicals is readily biodegradable. 

 

The first chemical part of the registered salt, the Oleic acid (C18’ fatty acid) is predicted to be readily biodegradable according to the Biowin prediction (see QSAR supporting study).  

 

Further testing has been conducted on the other moiety (Z)-N-octadec-9-enylpropane-1,3-diamine (CAS 7173 -62 -8) as well as on an analogous substance (CAS 68155-37-3). Most studies found both substances readily biodegradable (Ginkel, 1990, 1994, 2008 ; Gancet, 2004 ; Hirschen, 2004). Only two studies failed to demonstrate ready biodegradability of the diaminium moiety or its analogue (Hoechst, 1992 ; Danneberg, 1993). These studies suffered some methodological deficiencies.

 

Taken altogether, the results allow to consider the registered substance and its both moiety are readily biodegradable.