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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
04-02-2010 - 13-04-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP according guidelines with (minor) acceptable deviations, meeting all validity criteria.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
See read across document in Chapter 13
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
acceptable deviations
Principles of method if other than guideline:
Minor deviations from the guidelines of the Closed Bottle test were introduced; a) ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river water was used as inoculum. One extension from the guidelines 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
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (04-02-2010).
The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992).
River water without particles used as inoculum was obtained by removing solids sedimented by
gravity. The particles were removed by sedimentation.
Duration of test (contact time):
60 d
Initial conc.:
2 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
The river water used in the Closed Bottle test was spiked 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 not added 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 river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle),
10 bottles containing river water, silica gel and test substance, and 6 bottles containing sodium acetate and river water. The concentrations
of the test substance and sodium acetate in the bottles were 2.0 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. 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 river water.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of tall oil diethylenetriamine imidazoline, and sodium acetate were calculated from their molecular formulae and
molecular weights. The calculated theoretical oxygen demand (ThOD) and Chemical Oxygen Demand
(COD) of tall oil diethylenetriamine imidazoline are both 2.7 mg/mg. The ThOD 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 = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or cs is the mean oxygen level in the control bottles filled with river water spiked with mineral salts with (cs) and without silica gel (c) n days after the start of the test.
Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a), present in river water spiked with mineral salts n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test substance 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) or Chemical Oxygen Demand (COD).
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
24
Sampling time:
28 d
Remarks on result:
other: 61% biodegradation at day 60
Details on results:
Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of tall oil diethylenetriamine imidazoline to microorganisms degrading acetate is not relevant. Inhibition of the endogenousrespiration of the inoculum by the test substance tested in the presence of silica gel was not detected. Inhibition of the biodegradation due to the "high" initial concentration of the test substance is therefore not expected.

Test conditions
The pH of the media was 8.0 at the start of the test. The pH of the medium at day 28 was 7.9 (control and test) and 7.8 (control with silica gel).
Temperatures were within the prescribed temperature range of 22 to 24°C.
Validity criteria fulfilled:
yes
Remarks:
endogenous respiration of 1.4 mg/L at day 28, differences of the replicate values at day 28 less than 20%. 87% biodegradation of the reference compound, at day 14; oxygen concentrations >0.5 mg/L in all bottles during the test period
Interpretation of results:
inherently biodegradable
Conclusions:
Test performed under GLP according guidelines with (minor) acceptable deviations, meeting all validity criteria.
Tall oil diethylenetriamine imidazoline was biodegraded 24% at day 28 in the Closed Bottle test. This test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test tall oil diethylenetriamine imidazoline was biodegraded 61% at day 60.
The biodegradation reached at the day 60 of the test demonstrates that this test substance 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. Tall oil diethylenetriamine imidazoline 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. Tall oil diethylenetriamine imidazoline was biodegraded 24% at day 28 in the Closed Bottle test. This test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test tall oil diethylenetriamine imidazoline was biodegraded 61% at day 60. The biodegradation reached at the day 60 of the test demonstrates that this test substance is not persistent. The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 87% 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

Based on the currently available data on hydrolysis and biodegradation are imidazolines quickly hydrolyzed to amidoamines under ambient conditions. The (micro-organism mediated) hydrolysis of these amidoamines is probably the rate determining step. After breaking the amido bond, the substance is completely degraded to H2O, CO2 and the starting polyethyleneamine, with exception of DETA which is also completely biodegraded.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

Imidazoline DETA

Fatty acid C18 unsaturated diethylenetriamine imidazoline may be biocidal to micro-organisms and consequently inhibitory in all ready biodegradability tests. Reduction of the toxicity of fatty amine derivatives in ready biodegradability tests has therefore been achieved through the addition silica gel (van Ginkel et al, 2008). For toxic substances, the specified high test substance concentrations in ready biodegradability tests are controversial because substances are present in the environment in the sub μg/L range. Fatty acid C18 unsaturated diethylenetriamine imidazoline tested in the presence of silica gel was biodegraded 24% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test Fatty acid C18 unsaturated diethylenetriamine imidazoline was biodegraded 61% at day 60 (Akzo Nobel, 2010a). The biodegradation reached at the day 60 demonstrates that Fatty acid C18 unsaturated diethylenetriamine imidazoline is ultimately biodegradable and not persistent. In another Closed Bottle test a biodegradation of 17 % was reached at day 28 (Arkema, 2009). 

Complete (ultimate) degradation of tall oil diethylenetriamine imidazoline was also found in a semi continuously fed activated sludge (SCAS) test (van Ginkel et al, 2010b). In this test removal percentages of organic carbon of >90% were achieved. Complete degradation of Fatty acids C18 unsat. diethylenetriamine imidazoline can also be concluded from the ready biodegradability of fatty acids and diethylenetriamine formed upon hydrolysis (Popp, 1977; van Ginkel et al 1995)

 

Imidazoline PEPA

In the semi-continuously-fed activated sludge (SCAS) test removal percentages of 100% were immediately accomplished for Fatty acids C18 unsaturated reaction products with polyethylenepolyamines. Removal percentages decreased to approximately 80% after a few weeks. This removal percentage was maintained throughout the remaining test period of more than 300 days. The ability of micro-organisms to completely biodegrade Fatty acids C18 unsaturated reaction products with polyethylenepolyamines may be demonstrated in a Closed Bottle test with adapted sludge from the SCAS unit (Table II).  In this test, 35 and 38% biodegradation was achieved after 28 and 56 days, respectively. The result in the SCAS test and Closed Bottle tests demonstrate that Fatty acids C18 unsaturated reaction products with polyethylenepolyamines are only partially degraded (van Ginkel et al, 2010c).

Biodegradation percentages of <60% were always found in ready biodegradability test also showing that Fatty acids C18 unsaturated reaction products with polyethylenepolyamines are not ultimately biodegradable (IVL 1983; van Ginkel et al 2010c). Partial degradation is attributed to the oxidation of the alkyl chains and the inability of microorganisms to grow on polyethylenepolyamines (necessary to acquire positive results in OECD test). The alkyl chains (fatty acids) linked with an amide bond to the polyethylenepolyamines is thought to be degraded after an initial biologically catalysed hydrolysis. Alkyl chains attached to the imidazoline ring are probably liberated as fatty acid after an initial chemical hydrolysis of the imidazoline ring followed by hydrolysis of the amide bond. Amide bonds are in general very easily cleaved by amidases present in the competent microorganisms. Fatty acids C18 unsaturated reaction products with polyethylenepolyamines are as a consequence readily converted into carbon dioxide, water and polyethylenepolyamines. Polyethylenepolyamines cannot be degraded in OECD 301 and 302 tests and are therefore considered to be persistent. It can however be concluded that Fatty acids C18 unsaturated reaction products with polyethylenepolyamines (parent compounds) are readily biodegradable. In other words, the alkyl chain of Fatty acids C18 unsaturated reaction products with polyethylenepolyamines is readily biodegradable.