1,3-Propanediamine, N-[3-((C11-14, C13-rich)oxy)propyl]- branched acetate

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:

08-04-2010 - 08-06-2010

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:

Two minor deviations from the guidelines of the Closed Bottle test were introduced; a) ammonium chloride was omitted from the medium to prevent
oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), b) humic acid (16.0 mg/L) was added to reduce the toxicity of the test substance in the test.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge (02-04-2010) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992).

Duration of test (contact time):

60 d

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stock solutions
Deionized water used in the Closed Bottle test contained per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate and the test substance were added to the bottles using stock solutions of 1.0 g/L.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and 16.0 mg/L humic acid, 10 bottles containing inoculum, test substance and humic acid (16.0 mg/L), and 6 bottles containing sodium acetate and inoculum. The concentrations of the test substance and sodium acetate in the bottles were 1.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. The bottles with the test substance and the bottles with humic acid were placed on magnetic stirrer plates (600 rpm). The bottles contained magnetic bars in the bottles. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992)

Calculation of the results

Calculation of endogenous respiration
The endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
Mc is the mean oxygen level in the control bottle inoculated with activated sludge.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of ,N-(3-(tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and N-(3-(tridecyloxy)propyl)-1,3-propane diamine branchedand sodium acetate were calculated from their molecular formulae and molecular weights
The calculated theoretical oxygen demand of N-(3-(tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and N-(3-(tridecyloxy)propyl)-1,3-propane diamine branched is 2.7 mg/mg.
The theoretical oxygen demand of sodium acetate is 0.8 mg/mg.

Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference compound bottles were calculated as follows:
Oxygen consumptionn (mg/L) by test substance = Mch - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or ch = the mean oxygen level in the control bottles inoculated with activated sludge n days after the start of the test (C). The control of the test substance contained humic acid (CH).
Mt or a = the mean oxygen concentration in the bottles containing the test compound (t) or the reference compound, sodium acetate (a), and inoculated with activated sludge n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test compound and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

8

Sampling time:

28 d

Remarks on result:

other: in the presence of humic acid

Parameter:

% degradation (O2 consumption)

Value:

63

Sampling time:

60 d

Remarks on result:

other: in the presence of humic acid

Details on results:

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-(3-(tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and
N-(3-(tridecyloxy)propyl)-1,3-propane diamine branched to microorganisms degrading acetate is not relevant. Humic acid was added to the bottles with N-(3-(tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and N-(3-(tridecyloxy)propyl)-1,3-propane diamine branched because this substance is toxic to the competent bacteria. Inhibition of the endogenous respiration of the inoculum by the test substance in the presence of humic acid was not observed. 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 7.0 at the start of the test. The pH of the medium at day 28 was 7.0 (control), 7.1 (control with humic acid and test).
Temperatures were within the prescribed temperature range of 22 to 24°C.

Validity criteria fulfilled:

yes

Remarks:

endogenous respiration of <1.5 mg/L; differences between replicates at day 28 < 20%; sodium acetate was degraded 74% after 14 days.; oxygen concentrations >0.5 mg/L in all bottles during the test period.

Conclusions:

Test performed under GLP according guidelines with (minor) acceptable deviations, meeting all validity criteria N-(3-(Tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and N-(3-(tridecyloxy)propyl)-1,3-propane diamine branched is biodegraded 8% at day 28 in the Closed Bottle test, and should therefore notbe classified as readily biodegradable.
In the prolonged Closed Bottle test N-(3-(tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and N-(3-(tridecyloxy)propyl)-1,3-propane diamine branched is degraded 63% at day 60. The biodegradation percentage found at day 60 in this enhanced biodegradability test shows that N-(3-(tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and N-(3-(tridecyloxy)propyl)-1,3-propane diamine branched is not persistent.

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. The test was prolonged because the pass level was not reached at Day 28. N-(3-(Tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and N- (3-(tridecyloxy)propyl)-1,3-propane diamine branched did not cause a reduction in the endogenous respiration in the presence of humic acid. The test substance is therefore considered to be non-inhibitory to the inoculum. N-(3- (Tridecyloxy)propyl-1,3-propane diamine, branched monoacetate and N-(3- (tridecyloxy)propyl)-1,3-propane diamine branched was biodegraded 8% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test (enhanced biodegradability testing) a biodegradation percentage of 63 was achieved at day 60. Hence this substance should be classified as not persistent. The test is valid as shown by an endogenous respiration of 0.9 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 74% 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.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

january 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

acceptable deviatons

Principles of method if other than guideline:

Instead of an effluent/extract mixture, activated sludge was used as an inoculum.

Ammonium chloride was omitted from the medium to prevent nitrification

The closed bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing the BOD bottle

Lilaflot D 817 M is toxic to microorganisms. Therefore, Lilaflot D 817 M was also tested in the presence of silica gel (2 g/l) and humic acid (10 mg/l)to reduce the concentration of the test compound in the water phase.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge plant RWZI Nieuwgraaf in Duiven, treating predominantly domestic wastewater
- Preparation of inoculum for exposure: The sludge was preconditioned to reduce endogenous respiration rates. To this end, the sludge (200 mg dry wt/liter) was aerated for a period of 7 days. The sludge was diluted to a concentration in the biochemical oxygen demand (BOD) bottles of 2 mg dry wt/liter.

-Acclimatized sludge was produced in a semicontinuous activated sludge (SCAS) unit. In the
SCAS unit activated sludge was exposed to a 50 mg/L Lilaflot D 817 M complexed with 50
mg/L humic acid and the non-purgeable organic carbon (NPOC) was analysed to determine
the carbon removal. The test was performed according to modified EU Test Guidelines [EU, 1988: 'Official Journal of the European Communities', L133, 1988.05.30, Part C:Methods for the determination of ecotoxicity. Biodegradability Modified SCAS test]

Duration of test (contact time):

112 d

Initial conc.:

2 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.
- Test temperature: 20 - 22 °C
- pH: 6.8 - 7.0
- pH adjusted: no
- Aeration of mineral medium: yes
- Suspended solids concentration: sludge DW in BOD bottle = 2 mg/l
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: incubator
- Number of culture flasks/concentration:
3 bottles containing only unadapted inoculum = inoculum blank
3 bottles containing unadapted inoculum + test substance =test

3 bottles containing only unadapted inoculum + humic acid = inoculum blank
3 bottles containing unadapted inoculum + test substance + humic acid =test

3 bottles containing only unadapted inoculum + silica gel = inoculum blank
3 bottles containing unadapted inoculum + test substance + silica gel =test

3 bottles containing only adapted inoculum + humic acid= inoculum blank
3 bottles containing test substance + adpated inoculum + humic acid =test

- Method used to create aerobic conditions: aeration with pressured air
- Measuring equipment: oxygen meter


SAMPLING
- Sampling frequency: day 0, 7, 14, 21 ,28, 42, 49, 56, 63, 77, 84, 98, 112
- Sampling method: the bottles from day 0 were used also for measuring the other days (by using a special funel to collect the disipated medium)


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

CALCULATIONS:
THOD Lilaflot 817 M = 2.7 mg/mg ; measured COD Lilaflot 817 M = 2.1 mg/mg

Oxygen consumption (mg/l) (BOD) = mean oxygen concentration (mg/l) inoculum blank - mean oxygen concentration (mg/l) test
Biodegradation (%) = BOD/COD *100




T

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

112 d

Remarks on result:

other: unacclimatized sludge, no silica, no humic acid

Parameter:

% degradation (O2 consumption)

Value:

67

Sampling time:

112 d

Remarks on result:

other: unacclimatized sludge, with silica gel

Parameter:

% degradation (O2 consumption)

Value:

65

Sampling time:

98 d

Remarks on result:

other: unaclimatized sludge, with humic acid

Parameter:

% degradation (O2 consumption)

Value:

61

Sampling time:

56 d

Remarks on result:

other: acclimatized sludge, with humic acid

Details on results:

Inhibition of the endogenous respiration of the inoculum by the test substance was
detected in the bottles during a period of 112 days. The biodegradation was therefore
inhibited due to the "high" initial concentration of the test compound. In the presence of
silica gel and humic acid no inhibition of the endogous respiration was noted. The pH of
the media was 7 at the start of the test. The pH of the medium at day 28 was 6.8. Temperatures
ranged from 20 to 22°C. The validity of the tests is demonstrated by oxygen
concentrations >0.5 mg/L in all bottles during the test period.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Although this test is not performed under GLP and not all the raw data are reported the test seems to be ok (of good quality).
To reduce the toxicity of Lilaflot D 817 M silica gel or humic acid were added. Humic acid gave the best result. Lilaflot D 817 M was biodegraded 5% at
day 28 in the Closed Bottle test with unacclimated sludge, and should therefore not be classified as readily biodegradable. Since, in the prolonged Closed Bottle test the test compound was biodegraded >6O % at last day, it should be classified as inherently and completely (ultimately) biodegradable.

Acclimatized sludge was produced in an SCAS unit. Lilaflot D 817 M complexed with humic acid
was removed immediately in the SCAS unit (data not given) probably due to adsorption.
Lilaflot D 817 M was spiked for two months. The sludge from this SCAS unit was used as
inoculum in the Closed Bottle test. Due to acclimatization the lag period in the Closed Bottle test was reduced.
This result clearly indicates that Lilaflot D 817 M will be degraded in low loaded biological treatment systems.

Based on the chemical structure (highly branched alkyl chain) it can not be expected that
Lilaflot D 817 M is readily biodegradable. Usually the biodegradation of surfactants with
branched alkyl chains is initiated by the oxidation of the hydrophylic part . This is
probably also true for Lilaflot D 817 M. The first growth phase found in the Closed Bottle
test with acclimated sludge is probably caused by oxidation of the hydrophilic part. The
second growth phase probably represents the oxidation of the branched alkyl moiety at a
low rate .

Executive summary:

Lilaflot D 817 M caused reduction in the endogenous respiration during the test period of 112 days. The test substance is therefore considered to be very toxic to the inoculum. In order to prevent inhibitory effects of Lilaflot D 817M, tests were performed with the addition of humic acid and silica gel. Finally a Closed Bottle test with acclimatized sludge and humic acid was performed. Lilaflot D 817 M in the presence of humic acid was biodegraded 5 % at day 28 in the Closed Bottle test inoculated with unacclimated sludge. Hence this compound should not be classified as readily biodegradable. In the prolonged Closed Bottle test with humic acid this compound was biodegraded 65% at day 98. The biodegradation reached 61% at day 56 of a test with humic acid inoculated with acclimated sludge. These results demonstrate that this test compound should be classified as inherently biodegradable. The percentages reached at the last days of the test show that Lilaflot D 817 M is completely biodegraded. Based on the chemical structure (highly branched alkyl chain) it can not be expected that Lilaflot D 817 M is readily biodegradable. The result with the acclimated sludge clearly indicates that this compound will be removed by biodegradation in low loaded biological treatment systems and that the half-live of the compound in natural acclimated ecosystems is approximately 30 days.

Description of key information

N-[3-(tridecyloxy)propyl]-1,3-propane diamine, branched monoacetate and N-[3-(tridecyloxy)propyl]-1,3-propane diaminebranched is an etheramine surfactant which was found to be toxic to the activated sludge bacteria used for the inoculum. To mitigate this toxicity 16 mg/L Humic acid found to be necessary.

In the ready test, the test substance is not found to be readily biodegradable as only 8% biodegradation was observed after 28 days. After 60 days however 63% biodegradation was observed indicating that the substance is inherently and ultimately biodegradable and is based on these observations not considered to be persistent in the aquatic compartment.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Type of water:

freshwater

Additional information

N-[3-(tridecyloxy)propyl]-1,3-propane diamine,branchedmonoacetate and N-[3-(tridecyloxy)propyl]-1,3-propane diaminebranchedis an etheramine surfactant characterized by a hydrophobic moiety i.e. tridecyl and a structurally related hydrophilic moiety of dipropylenetriamine linked together with an ether bond. Biodegradation of surfactants refers to the reduction in complexity of the chemicalthrough metabolic activity of microorganisms.If a surfactant is to serve as a carbon and energy source for aerobic microorganisms then it has to be converted into a form that can enter the central metabolism of microorganisms. Normally this involves converting the surfactant into one, or more, low molecular weight intermediates of the tricarboxylic acid (TCA) cycle or compounds that feed into it. Many of these conversions are described in pathways forsurfactants (van Ginkel, 2007).Although micro-organisms capable of degrading surfactants are immensely diverse, the central metabolism (b-oxidation and TCA cycle) is remarkably similar. Kluyver and Donker (1920) first described this similarity known as the unity of biochemistry. This unity is the key to justification of the use of read-across.

Based on the structure of 1,3-propanediamine, N[3-(tridecyloxy)propyl]-, branched the following biodegradation pathways is most plausible: an attack on the hydrophilic part of the molecule followed by the degradation of the alkyl chain. A structurally related compound of the hydrophilic moiety dipropylene triamineis metabolised through ß-alanine, which feeds into the TCA cycle. Dipropylene triamine(N-(3-aminopropyl)-1,3-propanediamine) is readily biodegradable (van Ginkel et al, 2009; Rothkopf and Bartha, 1984). The biodegradability of the hydrophilic moiety is expected to be comparable with dipropylentriamine. The biodegradability of the branched alkyl chains are expected to be NOT readily biodegradable because of branching. Branched alcohols are known to be inherently biodegradable.

N-[3-(tridecyloxy)propyl]-1,3-propane diamine,branchedmonoacetate and N-[3-(tridecyloxy)propyl]-1,3-propane diaminebranchedis therefore expected to be not readily biodegradable. Indeed,N-[3-(tridecyloxy)propyl]-1,3-propane diamine,branchedmonoacetate and N-[3-(tridecyloxy)propyl]-1,3-propane diaminebranchedwas biodegraded 8% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test (enhanced biodegradability testing) a biodegradation percentage of 63 was achieved at day 60 (AkzoNobel, 2010). This result could only be achieved by using a high concentration of humic acid (van Ginkel et al, 2010).N-[3-(tridecyloxy)propyl]-1,3-propane diamine,branchedmonoacetate and N-[3-(tridecyloxy)propyl]-1,3-propane diaminebranchedshould be classified as NOT persistent because of theprolonged Closed Bottle test (enhanced test) result and the biodegradability of structurally related substances.