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Improving ready biodegradability testing of fatty amine derivatives
Chemosphere 73 (2008) 506–510
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Chemosphere j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / ch e m o s p h e r e
Improving ready biodegradability testing of fatty amine derivatives C.G. van Ginkel a,*, C. Gancet b, M. Hirschen c, M. Galobardes d, Ph. Lemaire e, J. Rosenblom f a
Akzo Nobel Technology and Engineering, P.O. Box 5136, 6802 EC Arnhem, Netherlands ARKEMA, P.O. Box 34, Lacq, 64170 Artix, France c Clariant Produkte (Deutschland) GmbH, 65926 Frankfurt am Main, Germany d Kao Chemicals Europe S.L. Puig dels Tudons 10, 08210 Barcelona, Spain e Total Fluides, 51 Esplanade de general de Gaulle, La defense 10, 92907 Paris, France f Akzo Nobel Surfactants AB, S-444 85 Stenungsund, Sweden b
a r t i c l e
i n f o
Article history: Received 13 March 2008 Received in revised form 11 June 2008 Accepted 12 June 2008 Available online 31 July 2008 Keywords: OECD ready biodegradability tests Fatty amine derivatives Toxicity Bioavailability
a b s t r a c t This study assesses the biodegradation potential of a number of fatty amine derivatives in tests follow ing the OECD guidelines for ready biodegradability. A number of methods are used to reduce toxicity and improve the bioavailability of the fatty amine derivatives in these tests. Alkyl-1,3-diaminopropanes and octadecyltrimethylammonium chloride are toxic to microorganisms at concentrations used in OECD ready biodegradability tests. The concentration of these fatty amine derivatives in the aqueous phase can be reduced by reacting humic, or lignosulphonic acids with the derivatives or through the addition of silica gel to the test bottles. Using these non-biodegradable substances, ready biodegradability test results were obtained with tallow-1,3-diaminopropane and octadecyltrimethylammonium chloride. Dem onstration of the ready biodegradability of the water-insoluble dioctadecylamine under the prescribed standard conditions is almost impossible due to the limited bioavailability of this compound. However, ready biodegradability results were achieved by using very low initial test substance concentrations and by introducing an organic phase. The contents of the bottles used to assess the biodegradability of diocta decylamine were always mixed. False negative biodegradability results obtained with the fatty amine derivatives studied are the result of toxic effects and/or limited bioavailability. The aids investigated there fore improve ready biodegradability testing. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Numerous studies have shown that the most important process reducing environmental concentrations of organic chemicals is biodegradation. Biodegradation is mediated by microorganisms participating in the recycling of elements such as carbon, nitrogen and sulphur. When degradation of man-made chemicals does not occur, accumulation must perforce take place. This insight resulted in the development of biodegradability tests for regulatory pur poses. Of the tests developed the ready biodegradability tests, designed to provide limited opportunity for biodegradation to occur, are the most widely used. Organic compounds passing these tests are assumed to biodegrade rapidly and completely in all aer obic ecosystems and biological treatment plants. A negative result in a ready biodegradability test does not demonstrate that the compound is persistent in the environment. Rather, such a result means that establishing (ready) biodegradability will require more comprehensive testing.
* Corresponding author. Tel.: +31 26 3662634; fax: +31 26 3662528. E-mail address: kees.vanginkel@akzonobel.com (C.G. van Ginkel) . 0045-6535/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.06.037
The Organization for Economic Cooperation and Development (OECD) has published guidelines for ready biodegradability tests such as the Sturm test, the MITI test and the Closed Bottle test. Ready biodegradability tests assess growth-linked biodegradation under aerobic conditions because the test substance is added as sole carbon and energy source. The use of carbon dioxide forma tion and oxygen depletion as measures necessitates the use of high initial test substance concentrations. For poorly water-soluble and toxic substances, these specified high test substance concentra tions are controversial because the test results are an unreliable predictor of the environmental fate of substances present in the lg l¡1 range. An initial concentration as low as 0.5 mg/l of the test substances can be applied in the Closed Bottle test, which is very low compared to the initial concentrations used in other ready bio degradability tests. The Closed Bottle test is therefore considered the most promising test for toxic and poorly water-soluble com pounds. Fatty amine derivatives are an important class of anthropo genic organic compounds containing both hydrophobic and pos itively charged hydrophilic moieties. The hydrophobic group is generally a hydrocarbon chain of 10–20 carbon atoms strongly affecting the water-solubility and toxicity. The apparent non biode gradability of fatty amine derivatives caused by the toxicity of the
C.G. van Ginkel et al. / Chemosphere 73 (2008) 506–510
test substance was noted first by Dean-Raymond and Alexander (1977). Reduction of the toxicity of fatty amine derivatives in ready biodegradability tests has been achieved through the addition of linear alkylbenzenesulphonates, and silica gel (Larson, 1983; van Ginkel, 1995). The biodegradation of fatty amine derivatives may also be restricted by their limited bioavailability, which is asso ciated with the hydrophobic nature of long alkyl chains and the positive charge. Especially fatty amine derivatives with long alkyl chains tend to strongly adsorb, thus becoming poorly available for microorganisms generally located in the water phase (van Ginkel et al., 2003). Use of inert cariers and emulsifiers was proposed to solve problems encountered in assessing the ready biodegradabil ity of poorly water-soluble substances (Gerike, 1984; Handley et al., 2002). Efroyson and Alexander (1991) used an organic phase to improve the bioavailability in biodegradability studies. The pres ent investigation was deemed necessary in the light of conflicting results obtained with fatty amine derivatives (Swisher, 1987). A jus tifiable outcome of ready biodegradability tests requires improve ment of the methodology. To this end, this paper describes the challenges presented by ready biodegradability testing of dioctadecylamine, octadecyltrim ethylammonium salt, and alkyl-1,3-diaminopropanes. In addition, information on the bioavailability in tests, and possible inhibitory effects have been obtained by chemical analyses and toxicity tests, respectively. 2. Materials and methods
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2.4. Closed Bottle test The Closed Bottle test was performed according to the Test Guidelines (OECD, 1992). The nutrient medium of the Closed Bot tle 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. Ammonium chloride was omit ted from the medium to prevent nitrification. The activated sludge was always diluted to 2 mg DW (dry weight) l¡1 in the closed bot tles. The tests were performed in 0.3 l BOD (biological oxygen demand) bottles with glass stoppers. Use was made of three bot tles containing only inoculum, and three bottles containing the test substance and inoculum. The initial test substance concentra tions in the bottles were 2.0, or 0.5 mg l¡1. The bottles containing a magnetic bar were closed and incubated in the dark on magnetic stirrer plates (150 rpm). The biodegradation was measured by monitoring the course of the oxygen decrease in the bottles with a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently the oxy gen 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 elec trode, the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, 1992). Controls with humic acid, lignosul phonic acid, or silica gel but without test substance were always included.
2.1. Materials 2.5. Methods to reduce toxicity in the biodegradability tests Dioctadecylamine (CAS No. 112-99-2), octadecyltrimethylam monium chloride (CAS No. 112-03-8) and silicone oil AR 20 were purchased from Fluka Chemika, Buchs, Switzerland. The alkyl1,3-diaminopropanes used were provided by Akzo Nobel Surfac tants, Stenungsund, Sweden and CECA, Paris, France. The purity of the alkyl-1,3-diaminopropanes was determined to be >95%. Lignosulphonic acid, sodium salt and humic acid, sodium salt were purchased from Sigma–Aldrich, Zwijndrecht, Netherlands. The polyalkoxylate alkylphenol (Agnique BP NP 1530) was obtained from Cognis Corp. Cincinatti Oh, US. Silica Gel 100 (0.2–05 mm) was obtained from Merck, Darmstadt, Germany. Deionized water containing no more than 0.01 mg l¡1 Cu was prepared in a water purification system. 2.2. Activated sludge Activated sludge was collected from aeration tanks of a waste water treatment plant located in Duiven, Netherlands and Abidos 64, France. These plants treat wastewater of which >80% is from domestic sources. The activated sludge was collected one week prior to the initiation of the tests, and preconditioned through con tinuous aeration during this period to allow minimization of the endogenous respiration (van Ginkel and Stroo, 1992). 2.3. Toxicity tests Toxicity tests were performed with either Vibrio fischeri (Microtox®) or with activated sludge. Acute toxicity to V. fischeri was determined according to the EN ISO 11348-3 (1998) standard using a Microbics M500 luminescence analyzer. Tests to determine inhibition of the respiration of activated sludge were carried out according to EN ISO 8192 (1986). The medium used contained the following ingredients per 1 litre of deionized water: 16 g pep tone, 11 g beef extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 · 2H2O, 0.2 g MgSO4 · 7H2O, 2.8 g K2HPO4. Oxygen consumption of the sludge was monitored electrochemically.
Each bottle contained 2 g of silica gel to reduce the toxicity to the microorganisms. Addition of silica gel made agitation of the contents of the bottles problematic because the rotation of the stir rer bars was complicated. Both humic acid and lignosulphonic acid were added at a concentration of 2 mg l¡1 in the bottles. 2.6. Methods to enhance bioavailability of water-insoluble compounds Accurate administration at concentrations ranging from 0.5 to 2.0 (standard) mg l¡1 in the bottles was accomplished by first dissolving dioctadecylamine in dichloromethane. The test sub stance in dichloromethane (approximately 10 ml) was directly added to bottles. The solvent was allowed to evaporate by plac ing the bottles on a roller bank in a ventilated hood for 24 h to obtain an even distribution of the test substance on the walls of the bottles. Suspensions of dioctadecylamine were obtained by adding 0.1 g of the test substance and 0.1, 0.25, or 0.5 g of polyalkoxylate alkylphenol in 20 ml of deionized water. These mixtures were ultra sonified for 20 min at 200 W using a Vibra cell ultrasonic proces sor (Sonics Vibra Cell Newtown, CT USA) giving suspensions. The suspensions were diluted in mineral salts medium containing acti vated sludge to give a test substance concentration of 2.0 mg l¡1 in the bottles. Dioctadecylamine was also dissolved in silicone oil AR 20 at a concentration of 0.2 g l¡1. Oil-in-water emulsions were prepared by mixing 10 ml of the silicone oil and 10 ml of water contain ing 1.0 g l¡1 of the polyalkoxylate alkylphenol. An emulsion was obtained by magnetically stirring the water and oil mixture in a beaker glass for 10 min at 600 rpm. This emulsion was made up to a litre with mineral salts medium containing activated sludge. This procedure results in a silicone oil concentration of 1.0% and a pol yalkoxylate alkylphenol concentration of 10 mg l¡1 in the bottles. The test substance concentration was 2.0 mg l¡1.
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2.7. Analyses A blade tensiometer (Kruess K9 tensiometer) was used to mea sure the surface tension of a series of alkyl-1,3-diaminepropanes concentrations. Dilutions from 0.4 to 100 mg l¡1 were prepared from a 1 g l¡1 mother solution in C.4-E medium. To assess the adsorption of silica gel, 100 ml of each dilution was added to closed flasks containing various amounts of silica. Flasks were then stirred overnight on roller devices and measurements were carried out the following day. The dissolved oxygen concentrations were determined electro chemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, Netherlands). The pH was measured using a Knick 765 calimatic pH meter (Elektroni sche Messgerate GmbH, Berlin, Germany). The temperature was measured and recorded with a thermocouple connected to a data logger. The dry weight (DW) of the inoculum was determined by filtering 50 ml of the activated sludge over a preweighed 12 lm Schleicher and Schuell filter. This filter was dried for 1.5 h at 104 °C and weighed after cooling. DW was calculated by subtracting the weight of the filters and by dividing this difference by the filtered volume. 3. Results and discussion 3.1. Toxicity It is fairly obvious that there may be problems assessing the biodegradability of chemicals that are toxic to microorganisms in ready biodegradability tests. Their biodegradation potential will depend on the concentration the test substance and the susceptibil ity of the competent microorganisms. Most fatty amine derivatives are toxic to microorganisms and the mechanism of toxicity is most likely membrane disruption. It is impossible to assess the inhibition of microorganisms capa ble of degrading the fatty amine derivatives present in the inoc ulum of ready biodegradability tests. Nevertheless, the effect of fatty amine derivatives on microbial activity was investigated in a number of toxicity tests to determine the possible influence on the biodegradation at concentrations applied in ready biodegrad ability tests. In the activated sludge respiration test, the EC50-3h of coco-1,3-diaminopropane was 3.0 mg l¡1. For tallow-1,3-diamino propane, an EC50 value of 6.0 mg l¡1 was measured. EC50 (15 min) luminescence inhibition values of 3.0 mg l¡1 were determined for both tallow-1,3-diaminopropane, and coco-1,3-diaminopropane. Finally, the effects of coco-1,3-diaminopropane, and tallow-1,3diaminopropane were assessed by measuring the oxygen con sumption of the activated sludge used as inoculum in the Closed Bottle test for one week at various concentrations of the test sub stance. With tallow-1,3-diaminopropane, inhibition of the endoge nous respiration was detected at all concentrations tested (Fig. 1). Coco-1,3-aminopropane was less toxic to activated sludge. Higher oxygen consumption was detected at coco-1,3-diaminopropane concentrations of 0.5 and 1.0 demonstrating acclimatization of the sludge to coco-1,3-diaminopropane within one week. At the highest concentrations tested, i.e. 4.0 and 8.0 mg l¡1, inhibition of the respiration was still found after one week (data not shown). All these results indicate that toxic effects resulting in reduced bio degradation of the alkyl-1,3-diaminopropanes cannot be excluded especially in ready biodegradability tests using high initial test sub stance concentrations. The presence of silica gel, humic acid and lignosulphonic acid may prevent inhibition of the microbial activity of the activated sludge used as inoculum. Addition of these substances may result in adsorption of the test substance on the silica gel or in the for mation of non-toxic complexes. The influence of the addition of
Fig. 1. Oxygen consumption by activated sludge in bottles in the absence (j) and in presence of 0.5 (h), 2.0 (d) and 8.0 (s) mg l¡1 tallow-1,3-diaminopropane. Each point represents the mean (±SD) of three replicates.
silica gel on the concentration of coco-1,3-diaminopropane was measured through the surface tension. This simple analytical method not requiring extraction was chosen because it reflects the aqueous (bioavailable) fraction. At a nominal concentration of 10 mg l¡1 coco-1,3-diaminopropane, addition of 0.22 g l¡1 of sil ica gel resulted in a coco-1,3-diaminopropane concentration of 2.0 mg/l. A decrease in concentration from 10 to below 1 mg l¡1 was found in the presence of 6.7 g l¡1 of silica gel (Fig. 2). Compa rable results were obtained with humic and lignosulphonic acids and other alkyl-1,3-diaminopropanes. These results demonstrate that a decrease of the concentration of tallow-1,3-diaminopropane to a non-toxic level through the addition of silica gel, humic and lignosulphonic acids can be achieved. Indeed, additional oxygen consumption was noted in the pres ence of tallow-1,3-diaminopropane after one week in the pres ence of silica gel and after two weeks in the presence humic and lignosulphonic acids, which corresponded to biodegradation per centages of approximately 20. After 28 days >60% biodegradation was achieved with silica gel. The results obtained with humic and
Fig. 2. Surface tension of coco-1,3-diaminopropane solutions in the presence of 2.2 (j) and 6.7 (e) g l¡1 of silica gel and without silica gel (h).
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Fig. 3. Biodegradation of octadecyltrimethylammonium chloride in the Closed Bot tle tests without additions (j) and with 2 g silica per bottle (h), 2.0 mg l¡1 lignosul phonic acid (s) and 2.0 mg l¡1 humic acid (d).
l ignosulphonic acids were lower. Addition of these acids might make the test substance less bioavailable. Octadecyltrimethylammonium chloride is also toxic to micro organisms (data not shown). Biodegradation of octadecyltrime thylammonium chloride was, as a result of this toxicity, was not observed in the Sturm test (Larson, 1983). Biodegradation was also not achieved in a standard Closed Bottle test (Fig. 3). Octadecylt rimethylammonium chloride was therefore also tested in the pres ence of silica gel, lignosulphonic acid and humic acid. Fig. 3 shows that silica gel is the best aid available to reduce the inhibitory effects of octadecyltrimethylammonium chloride. Positive results with hexadecyltrimethylammonium chloride were also obtained with the help of silica gel by nine out of ten laboratories (Painter et al., 2003). The mean biodegradation of hexadecyltrimethylam monium chloride found in Carbon Dioxide Headspace tests carried out by these laboratories was 75%. 3.2. Bioavailability Whereas the degradation of water-soluble compounds can be determined in any of the ready biodegradability tests, methods for testing poorly water-soluble compounds are limited. The BODIS test has been presented as a possibility (Gerike, 1984; ISO 14593, 2005). However, the introduction of a gas phase in the test vessel of the BODIS test requires higher initial test substance concentrations compared to the Closed Bottle test. The Closed Bottle test allows ini tial test substance concentrations as low as 0.5 mg l¡1, at which con centration a distinction between the background respiration and oxygen consumption in the presence of the test substance is still possible. Mixing the content of test flasks is important to increase the mass transfer of an poorly water-soluble substance to the com petent microorganisms. This is easily achieved in the Closed Bot tle test with the use of magnetic stirrer bars. In the Closed Bottle test, water-insoluble test substances are usually applied without taking into account possible unequal distribution of the test sub stance on the wall of the bottle. A more even distribution of the test substance on the walls of the bottles can be achieved by using a roller bank. Using the poorly water-soluble dioctadecylamine, linear curves were obtained in the Closed Bottle test (Fig. 4). Limited bioavail ability is expected to result in linear instead of logarithmic bio degradation curves. In general it has been reported that the mass
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Fig. 4. Percentage of biodegradation of dioctadecylamine versus time in Closed Bottle tests. The initial dioctadecylamine concentrations were 2 mg l¡1 (j,h) and 0.5 mg l¡1 (d). Closed legends indicate mixing of the content of the bottles.
transfer rate of poorly water-soluble organic compounds into the aqueous phase is the rate-limiting step in their degradation (Stucki and Alexander, 1987; Alexander, 1994). Methods improving the bio availability are therefore essential for the assessment of the biode gradability. Mixing of the content of the closed bottles enhanced the biodegradability of dioctadecylamine through increased mass transfer. Biodegradation percentages of 22 at day 28 and 65 at day 84 were achieved. In the standard Closed Bottle test (without mix ing), 8%, 33%, and 61% biodegradation were reached at day 28, 84 and 140, respectively (Fig. 4). Mixing is a normal phenomenon in both aquatic environments and the technosphere and therefore a legitimate improvement of the test procedure. Limitation of the biodegradability may be reduced further by decreasing the initial test substance concentration. The biodegradability was therefore also tested at 0.5 mg l¡1 of dioctadecylamine. Accurate assessment of the biodegradability of dioctadecylamine in a Closed Bottle test is still possible at concentrations below 2.0 mg l¡1 by e.g. increas ing the number of replicates. The mean oxygen concentrations and standard deviations in six control bottles and six bottles with 0.5 mg l¡1 dioctadecylamine at day 28 were 7.82 ± 0.15 and 6.53 ± 0.15. The observed difference of the mean values assessed with t-statistics was significantly different at the 99% confidence level. Using the difference, 63% biodegradation was achieved at day 28 (Fig. 4). A concentration of 0.5 mg l¡1 is still several orders of magnitude higher than the levels predicted to occur in the envi ronment and wastewater treatment plants. The bioavailability of dioctadecylamine may be improved using an organic phase and/or surfactants. These surfactants should be non-toxic, be able to enhance solubilization of the test substance and preferably be non-biodegradable. In nature microbial growth on water-insoluble organic compounds as for instance hydrocar bons, is often accompanied by the appearance of biosurfactants. Addition of surfactants to increase the biodegradation in ready bio degradability tests can therefore be regarded as a natural phenome non. Polyalkoxylate alkylphenols are known for their recalcitrance. However, limited degradation of the polyalkoxylate alkylphenol used was found. The use of this surfactant should therefore be lim ited to 10 mg l¡1 in the bottles resulting in only minor additional oxygen consumption. A “suspension” of dioctadecylamine and the polyalkoxylate alkylphenol could be prepared. Administration of dioctadecylamine as “suspension” did not result in biodegradation of dioctadecylamine. Both increased and decreased biodegradation
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alkyl-1,3-diaminopropanes. Methods to increase the bioavailabil ity of the poorly water-soluble dioctadecylamine are also effective. The contradictory results obtained are inevitable due to the varied interactions between the fatty amine derivatives and (in)organic substances and the different properties of the fatty amine deriva tives. Conflicting results in ready biodegradability tests are always treated by only considering positive results as valid. Mixing the contents of the bottles, low concentrations of the test substance and use of aids such as surfactants probably results in conditions mimicing the environment better than the conditions in a stan dard ready biodegradability test. The methods described therefore improve biodegradability testing. Acknowledgement Financial support for the research described in this paper has been provided by APAG. References ¡1
Fig. 5. Biodegradation of dioctadecylamine (2.0 mg l ) in Closed Bottle test with sil icone oil as a second phase. The biodegradation was tested in the absence (j) and presence of 10 mg l¡1 of polyalkoxylate alkylphenol (h). Addition of polyalkoxylate alkylphenol resulted in the formation of an emulsion.
in the presence of surfactants have been reported (Volkering et al., 1998). The solvent used as organic phase has to be non-toxic and nonbiodegradable. Silicone oil is not biodegradable nor does it contain biodegradable impurities. The introduction of silicone oil as an organic phase resulted in a ready biodegradability result i.e. 65% at day 28. Use of silicone oil therefore not only enabled an accurate administration of the test substance but also enhanced the bioavail ability of the test substance. The density of the silicone oil used was slightly higher than the density of water, which could result in loss of the oil with test substance from the bottles during closure. Silicone oil attached to the electrode could also lead to an unin tended loss of the test substance from the bottles. The use of sili cone oil therefore required special measures such as agitation of the content of the bottles during closure and the possibility to sac rifice bottles. Addition of the polyalkoxylate alkylphenol resulted in formation of a oil-in-water emulsion. A ready biodegradability result was also achieved with this emulsion (Fig. 5). Increased bio availability through the introduction of an organic phase has also been observed in other biodegradation studies (Efroyson and Alex ander, 1991; Ascon-Cabrera and Lebeault, 1993). 4. Conclusions Demonstration of the ready biodegradability of fatty amine derivatives is often impossible because these substances inhibit growth of microorganisms. Use of lignosulphonic acid and humic acid and especially silica gel overcome difficulties in demonstrating the ready biodegradability of alkyltrimethylammonium salts and
Alexander, M., 1994. Biodegradation and Bioremediation. Academic Press, San Diego, USA. Ascon-Cabrera, M., Lebeault, J-M., 1993. Selection of xenobiotic-degrading micro organisms in a biphasic aqueous–organic system. Appl. Environ. Microbiol. 59, 1717–1724. Dean-Raymond, D., Alexander, M., 1977. Bacterial metabolism of quaternary ammo nium compounds. Appl. Environ. Microbiol. 33, 1037–1041. Efroyson, R.A., Alexander, M., 1991. Biodegradation by an Arthrobacter species of hydrocarbons partitioned into an organic solvent. Appl. Environ. Microbiol. 57, 1441–1447. Gerike, P., 1984. The biodegradability testing of poorly water soluble compounds. Chemosphere 13, 169–190. Handley, J.W., Mead, C., Rausina, G.A., Waid, L.J., Gee, J.C., Herron, S.J., 2002. The use of inert carriers in regulatory biodegradation tests of low density poorly watersoluble substances. Chemosphere 48, 529–534. ISO 11348-3, 1998. Water quality – determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) – part 3: method using freeze-dried bacteria. Geneva, Switzerland. ISO 14593, 2005. Water quality – evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – method by analysis of inorganic carbon in sealed vessels (CO2 headspace test) No. 14593, Geneva, Switzer land. ISO 8192, 1986. Water quality – test for inhibition of oxygen consumption by acti vated sludge. Geneva, Switzerland. Larson, R.J., 1983. Comparison of biodegradation rates in laboratory screening test studies with rates in natural water. Residue Rev. 85, 159–171. OECD Guideline for testing of chemicals, 1992. Degradation and accumulation, No. 301: Ready biodegradability (1992). Paris, Cedex, France. Painter, H.A., Reynolds, P., Comber, S., 2003. Application of the headspace CO2 method (ISO 14593) to the assessment of the ultimate biodegradability of sur factants: results of a calibration exercise. Chemosphere 50, 29–38. Swisher, R.D., 1987. Surfactant Biodegradation, second ed. Marcel Dekker Inc., New York, US. van Ginkel, C.G., 1995. Biodegradation of cationic surfactants. In: Karsa, D.R., Porter, M.R. (Eds.), Biodegradation of Surfactants. Blackie Academic & Professional, Lon don, United Kingdom, pp. 183–203. van Ginkel, C.G., Stroo, C.A., 1992. Simple method to prolong the Closed Bottle test for the determination of the inherent biodegradability. Ecotoxicol. Environ. Safe. 24, 319–327. van Ginkel, C.G., Hoenderboom, A.M., van Haperen, A., Geurts, M.G.J., 2003. Assess ment of the biodegradability of dialkyldimethylammonium salts in flow through systems. Environ. Sci. Health (Part A) 38 (9), 1825–1835. Volkering, F., Breure, A.M., Rulkens, W.H., 1998. Microbiological aspects of surfac tant use for biological soil remediation. Biodegradation 8, 401–417.
Contents lists available at ScienceDirect
Chemosphere j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / ch e m o s p h e r e
Improving ready biodegradability testing of fatty amine derivatives C.G. van Ginkel a,*, C. Gancet b, M. Hirschen c, M. Galobardes d, Ph. Lemaire e, J. Rosenblom f a
Akzo Nobel Technology and Engineering, P.O. Box 5136, 6802 EC Arnhem, Netherlands ARKEMA, P.O. Box 34, Lacq, 64170 Artix, France c Clariant Produkte (Deutschland) GmbH, 65926 Frankfurt am Main, Germany d Kao Chemicals Europe S.L. Puig dels Tudons 10, 08210 Barcelona, Spain e Total Fluides, 51 Esplanade de general de Gaulle, La defense 10, 92907 Paris, France f Akzo Nobel Surfactants AB, S-444 85 Stenungsund, Sweden b
a r t i c l e
i n f o
Article history: Received 13 March 2008 Received in revised form 11 June 2008 Accepted 12 June 2008 Available online 31 July 2008 Keywords: OECD ready biodegradability tests Fatty amine derivatives Toxicity Bioavailability
a b s t r a c t This study assesses the biodegradation potential of a number of fatty amine derivatives in tests follow ing the OECD guidelines for ready biodegradability. A number of methods are used to reduce toxicity and improve the bioavailability of the fatty amine derivatives in these tests. Alkyl-1,3-diaminopropanes and octadecyltrimethylammonium chloride are toxic to microorganisms at concentrations used in OECD ready biodegradability tests. The concentration of these fatty amine derivatives in the aqueous phase can be reduced by reacting humic, or lignosulphonic acids with the derivatives or through the addition of silica gel to the test bottles. Using these non-biodegradable substances, ready biodegradability test results were obtained with tallow-1,3-diaminopropane and octadecyltrimethylammonium chloride. Dem onstration of the ready biodegradability of the water-insoluble dioctadecylamine under the prescribed standard conditions is almost impossible due to the limited bioavailability of this compound. However, ready biodegradability results were achieved by using very low initial test substance concentrations and by introducing an organic phase. The contents of the bottles used to assess the biodegradability of diocta decylamine were always mixed. False negative biodegradability results obtained with the fatty amine derivatives studied are the result of toxic effects and/or limited bioavailability. The aids investigated there fore improve ready biodegradability testing. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Numerous studies have shown that the most important process reducing environmental concentrations of organic chemicals is biodegradation. Biodegradation is mediated by microorganisms participating in the recycling of elements such as carbon, nitrogen and sulphur. When degradation of man-made chemicals does not occur, accumulation must perforce take place. This insight resulted in the development of biodegradability tests for regulatory pur poses. Of the tests developed the ready biodegradability tests, designed to provide limited opportunity for biodegradation to occur, are the most widely used. Organic compounds passing these tests are assumed to biodegrade rapidly and completely in all aer obic ecosystems and biological treatment plants. A negative result in a ready biodegradability test does not demonstrate that the compound is persistent in the environment. Rather, such a result means that establishing (ready) biodegradability will require more comprehensive testing.
* Corresponding author. Tel.: +31 26 3662634; fax: +31 26 3662528. E-mail address: kees.vanginkel@akzonobel.com (C.G. van Ginkel) . 0045-6535/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.06.037
The Organization for Economic Cooperation and Development (OECD) has published guidelines for ready biodegradability tests such as the Sturm test, the MITI test and the Closed Bottle test. Ready biodegradability tests assess growth-linked biodegradation under aerobic conditions because the test substance is added as sole carbon and energy source. The use of carbon dioxide forma tion and oxygen depletion as measures necessitates the use of high initial test substance concentrations. For poorly water-soluble and toxic substances, these specified high test substance concentra tions are controversial because the test results are an unreliable predictor of the environmental fate of substances present in the lg l¡1 range. An initial concentration as low as 0.5 mg/l of the test substances can be applied in the Closed Bottle test, which is very low compared to the initial concentrations used in other ready bio degradability tests. The Closed Bottle test is therefore considered the most promising test for toxic and poorly water-soluble com pounds. Fatty amine derivatives are an important class of anthropo genic organic compounds containing both hydrophobic and pos itively charged hydrophilic moieties. The hydrophobic group is generally a hydrocarbon chain of 10–20 carbon atoms strongly affecting the water-solubility and toxicity. The apparent non biode gradability of fatty amine derivatives caused by the toxicity of the
C.G. van Ginkel et al. / Chemosphere 73 (2008) 506–510
test substance was noted first by Dean-Raymond and Alexander (1977). Reduction of the toxicity of fatty amine derivatives in ready biodegradability tests has been achieved through the addition of linear alkylbenzenesulphonates, and silica gel (Larson, 1983; van Ginkel, 1995). The biodegradation of fatty amine derivatives may also be restricted by their limited bioavailability, which is asso ciated with the hydrophobic nature of long alkyl chains and the positive charge. Especially fatty amine derivatives with long alkyl chains tend to strongly adsorb, thus becoming poorly available for microorganisms generally located in the water phase (van Ginkel et al., 2003). Use of inert cariers and emulsifiers was proposed to solve problems encountered in assessing the ready biodegradabil ity of poorly water-soluble substances (Gerike, 1984; Handley et al., 2002). Efroyson and Alexander (1991) used an organic phase to improve the bioavailability in biodegradability studies. The pres ent investigation was deemed necessary in the light of conflicting results obtained with fatty amine derivatives (Swisher, 1987). A jus tifiable outcome of ready biodegradability tests requires improve ment of the methodology. To this end, this paper describes the challenges presented by ready biodegradability testing of dioctadecylamine, octadecyltrim ethylammonium salt, and alkyl-1,3-diaminopropanes. In addition, information on the bioavailability in tests, and possible inhibitory effects have been obtained by chemical analyses and toxicity tests, respectively. 2. Materials and methods
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2.4. Closed Bottle test The Closed Bottle test was performed according to the Test Guidelines (OECD, 1992). The nutrient medium of the Closed Bot tle 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. Ammonium chloride was omit ted from the medium to prevent nitrification. The activated sludge was always diluted to 2 mg DW (dry weight) l¡1 in the closed bot tles. The tests were performed in 0.3 l BOD (biological oxygen demand) bottles with glass stoppers. Use was made of three bot tles containing only inoculum, and three bottles containing the test substance and inoculum. The initial test substance concentra tions in the bottles were 2.0, or 0.5 mg l¡1. The bottles containing a magnetic bar were closed and incubated in the dark on magnetic stirrer plates (150 rpm). The biodegradation was measured by monitoring the course of the oxygen decrease in the bottles with a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently the oxy gen 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 elec trode, the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, 1992). Controls with humic acid, lignosul phonic acid, or silica gel but without test substance were always included.
2.1. Materials 2.5. Methods to reduce toxicity in the biodegradability tests Dioctadecylamine (CAS No. 112-99-2), octadecyltrimethylam monium chloride (CAS No. 112-03-8) and silicone oil AR 20 were purchased from Fluka Chemika, Buchs, Switzerland. The alkyl1,3-diaminopropanes used were provided by Akzo Nobel Surfac tants, Stenungsund, Sweden and CECA, Paris, France. The purity of the alkyl-1,3-diaminopropanes was determined to be >95%. Lignosulphonic acid, sodium salt and humic acid, sodium salt were purchased from Sigma–Aldrich, Zwijndrecht, Netherlands. The polyalkoxylate alkylphenol (Agnique BP NP 1530) was obtained from Cognis Corp. Cincinatti Oh, US. Silica Gel 100 (0.2–05 mm) was obtained from Merck, Darmstadt, Germany. Deionized water containing no more than 0.01 mg l¡1 Cu was prepared in a water purification system. 2.2. Activated sludge Activated sludge was collected from aeration tanks of a waste water treatment plant located in Duiven, Netherlands and Abidos 64, France. These plants treat wastewater of which >80% is from domestic sources. The activated sludge was collected one week prior to the initiation of the tests, and preconditioned through con tinuous aeration during this period to allow minimization of the endogenous respiration (van Ginkel and Stroo, 1992). 2.3. Toxicity tests Toxicity tests were performed with either Vibrio fischeri (Microtox®) or with activated sludge. Acute toxicity to V. fischeri was determined according to the EN ISO 11348-3 (1998) standard using a Microbics M500 luminescence analyzer. Tests to determine inhibition of the respiration of activated sludge were carried out according to EN ISO 8192 (1986). The medium used contained the following ingredients per 1 litre of deionized water: 16 g pep tone, 11 g beef extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 · 2H2O, 0.2 g MgSO4 · 7H2O, 2.8 g K2HPO4. Oxygen consumption of the sludge was monitored electrochemically.
Each bottle contained 2 g of silica gel to reduce the toxicity to the microorganisms. Addition of silica gel made agitation of the contents of the bottles problematic because the rotation of the stir rer bars was complicated. Both humic acid and lignosulphonic acid were added at a concentration of 2 mg l¡1 in the bottles. 2.6. Methods to enhance bioavailability of water-insoluble compounds Accurate administration at concentrations ranging from 0.5 to 2.0 (standard) mg l¡1 in the bottles was accomplished by first dissolving dioctadecylamine in dichloromethane. The test sub stance in dichloromethane (approximately 10 ml) was directly added to bottles. The solvent was allowed to evaporate by plac ing the bottles on a roller bank in a ventilated hood for 24 h to obtain an even distribution of the test substance on the walls of the bottles. Suspensions of dioctadecylamine were obtained by adding 0.1 g of the test substance and 0.1, 0.25, or 0.5 g of polyalkoxylate alkylphenol in 20 ml of deionized water. These mixtures were ultra sonified for 20 min at 200 W using a Vibra cell ultrasonic proces sor (Sonics Vibra Cell Newtown, CT USA) giving suspensions. The suspensions were diluted in mineral salts medium containing acti vated sludge to give a test substance concentration of 2.0 mg l¡1 in the bottles. Dioctadecylamine was also dissolved in silicone oil AR 20 at a concentration of 0.2 g l¡1. Oil-in-water emulsions were prepared by mixing 10 ml of the silicone oil and 10 ml of water contain ing 1.0 g l¡1 of the polyalkoxylate alkylphenol. An emulsion was obtained by magnetically stirring the water and oil mixture in a beaker glass for 10 min at 600 rpm. This emulsion was made up to a litre with mineral salts medium containing activated sludge. This procedure results in a silicone oil concentration of 1.0% and a pol yalkoxylate alkylphenol concentration of 10 mg l¡1 in the bottles. The test substance concentration was 2.0 mg l¡1.
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2.7. Analyses A blade tensiometer (Kruess K9 tensiometer) was used to mea sure the surface tension of a series of alkyl-1,3-diaminepropanes concentrations. Dilutions from 0.4 to 100 mg l¡1 were prepared from a 1 g l¡1 mother solution in C.4-E medium. To assess the adsorption of silica gel, 100 ml of each dilution was added to closed flasks containing various amounts of silica. Flasks were then stirred overnight on roller devices and measurements were carried out the following day. The dissolved oxygen concentrations were determined electro chemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, Netherlands). The pH was measured using a Knick 765 calimatic pH meter (Elektroni sche Messgerate GmbH, Berlin, Germany). The temperature was measured and recorded with a thermocouple connected to a data logger. The dry weight (DW) of the inoculum was determined by filtering 50 ml of the activated sludge over a preweighed 12 lm Schleicher and Schuell filter. This filter was dried for 1.5 h at 104 °C and weighed after cooling. DW was calculated by subtracting the weight of the filters and by dividing this difference by the filtered volume. 3. Results and discussion 3.1. Toxicity It is fairly obvious that there may be problems assessing the biodegradability of chemicals that are toxic to microorganisms in ready biodegradability tests. Their biodegradation potential will depend on the concentration the test substance and the susceptibil ity of the competent microorganisms. Most fatty amine derivatives are toxic to microorganisms and the mechanism of toxicity is most likely membrane disruption. It is impossible to assess the inhibition of microorganisms capa ble of degrading the fatty amine derivatives present in the inoc ulum of ready biodegradability tests. Nevertheless, the effect of fatty amine derivatives on microbial activity was investigated in a number of toxicity tests to determine the possible influence on the biodegradation at concentrations applied in ready biodegrad ability tests. In the activated sludge respiration test, the EC50-3h of coco-1,3-diaminopropane was 3.0 mg l¡1. For tallow-1,3-diamino propane, an EC50 value of 6.0 mg l¡1 was measured. EC50 (15 min) luminescence inhibition values of 3.0 mg l¡1 were determined for both tallow-1,3-diaminopropane, and coco-1,3-diaminopropane. Finally, the effects of coco-1,3-diaminopropane, and tallow-1,3diaminopropane were assessed by measuring the oxygen con sumption of the activated sludge used as inoculum in the Closed Bottle test for one week at various concentrations of the test sub stance. With tallow-1,3-diaminopropane, inhibition of the endoge nous respiration was detected at all concentrations tested (Fig. 1). Coco-1,3-aminopropane was less toxic to activated sludge. Higher oxygen consumption was detected at coco-1,3-diaminopropane concentrations of 0.5 and 1.0 demonstrating acclimatization of the sludge to coco-1,3-diaminopropane within one week. At the highest concentrations tested, i.e. 4.0 and 8.0 mg l¡1, inhibition of the respiration was still found after one week (data not shown). All these results indicate that toxic effects resulting in reduced bio degradation of the alkyl-1,3-diaminopropanes cannot be excluded especially in ready biodegradability tests using high initial test sub stance concentrations. The presence of silica gel, humic acid and lignosulphonic acid may prevent inhibition of the microbial activity of the activated sludge used as inoculum. Addition of these substances may result in adsorption of the test substance on the silica gel or in the for mation of non-toxic complexes. The influence of the addition of
Fig. 1. Oxygen consumption by activated sludge in bottles in the absence (j) and in presence of 0.5 (h), 2.0 (d) and 8.0 (s) mg l¡1 tallow-1,3-diaminopropane. Each point represents the mean (±SD) of three replicates.
silica gel on the concentration of coco-1,3-diaminopropane was measured through the surface tension. This simple analytical method not requiring extraction was chosen because it reflects the aqueous (bioavailable) fraction. At a nominal concentration of 10 mg l¡1 coco-1,3-diaminopropane, addition of 0.22 g l¡1 of sil ica gel resulted in a coco-1,3-diaminopropane concentration of 2.0 mg/l. A decrease in concentration from 10 to below 1 mg l¡1 was found in the presence of 6.7 g l¡1 of silica gel (Fig. 2). Compa rable results were obtained with humic and lignosulphonic acids and other alkyl-1,3-diaminopropanes. These results demonstrate that a decrease of the concentration of tallow-1,3-diaminopropane to a non-toxic level through the addition of silica gel, humic and lignosulphonic acids can be achieved. Indeed, additional oxygen consumption was noted in the pres ence of tallow-1,3-diaminopropane after one week in the pres ence of silica gel and after two weeks in the presence humic and lignosulphonic acids, which corresponded to biodegradation per centages of approximately 20. After 28 days >60% biodegradation was achieved with silica gel. The results obtained with humic and
Fig. 2. Surface tension of coco-1,3-diaminopropane solutions in the presence of 2.2 (j) and 6.7 (e) g l¡1 of silica gel and without silica gel (h).
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Fig. 3. Biodegradation of octadecyltrimethylammonium chloride in the Closed Bot tle tests without additions (j) and with 2 g silica per bottle (h), 2.0 mg l¡1 lignosul phonic acid (s) and 2.0 mg l¡1 humic acid (d).
l ignosulphonic acids were lower. Addition of these acids might make the test substance less bioavailable. Octadecyltrimethylammonium chloride is also toxic to micro organisms (data not shown). Biodegradation of octadecyltrime thylammonium chloride was, as a result of this toxicity, was not observed in the Sturm test (Larson, 1983). Biodegradation was also not achieved in a standard Closed Bottle test (Fig. 3). Octadecylt rimethylammonium chloride was therefore also tested in the pres ence of silica gel, lignosulphonic acid and humic acid. Fig. 3 shows that silica gel is the best aid available to reduce the inhibitory effects of octadecyltrimethylammonium chloride. Positive results with hexadecyltrimethylammonium chloride were also obtained with the help of silica gel by nine out of ten laboratories (Painter et al., 2003). The mean biodegradation of hexadecyltrimethylam monium chloride found in Carbon Dioxide Headspace tests carried out by these laboratories was 75%. 3.2. Bioavailability Whereas the degradation of water-soluble compounds can be determined in any of the ready biodegradability tests, methods for testing poorly water-soluble compounds are limited. The BODIS test has been presented as a possibility (Gerike, 1984; ISO 14593, 2005). However, the introduction of a gas phase in the test vessel of the BODIS test requires higher initial test substance concentrations compared to the Closed Bottle test. The Closed Bottle test allows ini tial test substance concentrations as low as 0.5 mg l¡1, at which con centration a distinction between the background respiration and oxygen consumption in the presence of the test substance is still possible. Mixing the content of test flasks is important to increase the mass transfer of an poorly water-soluble substance to the com petent microorganisms. This is easily achieved in the Closed Bot tle test with the use of magnetic stirrer bars. In the Closed Bottle test, water-insoluble test substances are usually applied without taking into account possible unequal distribution of the test sub stance on the wall of the bottle. A more even distribution of the test substance on the walls of the bottles can be achieved by using a roller bank. Using the poorly water-soluble dioctadecylamine, linear curves were obtained in the Closed Bottle test (Fig. 4). Limited bioavail ability is expected to result in linear instead of logarithmic bio degradation curves. In general it has been reported that the mass
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Fig. 4. Percentage of biodegradation of dioctadecylamine versus time in Closed Bottle tests. The initial dioctadecylamine concentrations were 2 mg l¡1 (j,h) and 0.5 mg l¡1 (d). Closed legends indicate mixing of the content of the bottles.
transfer rate of poorly water-soluble organic compounds into the aqueous phase is the rate-limiting step in their degradation (Stucki and Alexander, 1987; Alexander, 1994). Methods improving the bio availability are therefore essential for the assessment of the biode gradability. Mixing of the content of the closed bottles enhanced the biodegradability of dioctadecylamine through increased mass transfer. Biodegradation percentages of 22 at day 28 and 65 at day 84 were achieved. In the standard Closed Bottle test (without mix ing), 8%, 33%, and 61% biodegradation were reached at day 28, 84 and 140, respectively (Fig. 4). Mixing is a normal phenomenon in both aquatic environments and the technosphere and therefore a legitimate improvement of the test procedure. Limitation of the biodegradability may be reduced further by decreasing the initial test substance concentration. The biodegradability was therefore also tested at 0.5 mg l¡1 of dioctadecylamine. Accurate assessment of the biodegradability of dioctadecylamine in a Closed Bottle test is still possible at concentrations below 2.0 mg l¡1 by e.g. increas ing the number of replicates. The mean oxygen concentrations and standard deviations in six control bottles and six bottles with 0.5 mg l¡1 dioctadecylamine at day 28 were 7.82 ± 0.15 and 6.53 ± 0.15. The observed difference of the mean values assessed with t-statistics was significantly different at the 99% confidence level. Using the difference, 63% biodegradation was achieved at day 28 (Fig. 4). A concentration of 0.5 mg l¡1 is still several orders of magnitude higher than the levels predicted to occur in the envi ronment and wastewater treatment plants. The bioavailability of dioctadecylamine may be improved using an organic phase and/or surfactants. These surfactants should be non-toxic, be able to enhance solubilization of the test substance and preferably be non-biodegradable. In nature microbial growth on water-insoluble organic compounds as for instance hydrocar bons, is often accompanied by the appearance of biosurfactants. Addition of surfactants to increase the biodegradation in ready bio degradability tests can therefore be regarded as a natural phenome non. Polyalkoxylate alkylphenols are known for their recalcitrance. However, limited degradation of the polyalkoxylate alkylphenol used was found. The use of this surfactant should therefore be lim ited to 10 mg l¡1 in the bottles resulting in only minor additional oxygen consumption. A “suspension” of dioctadecylamine and the polyalkoxylate alkylphenol could be prepared. Administration of dioctadecylamine as “suspension” did not result in biodegradation of dioctadecylamine. Both increased and decreased biodegradation
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alkyl-1,3-diaminopropanes. Methods to increase the bioavailabil ity of the poorly water-soluble dioctadecylamine are also effective. The contradictory results obtained are inevitable due to the varied interactions between the fatty amine derivatives and (in)organic substances and the different properties of the fatty amine deriva tives. Conflicting results in ready biodegradability tests are always treated by only considering positive results as valid. Mixing the contents of the bottles, low concentrations of the test substance and use of aids such as surfactants probably results in conditions mimicing the environment better than the conditions in a stan dard ready biodegradability test. The methods described therefore improve biodegradability testing. Acknowledgement Financial support for the research described in this paper has been provided by APAG. References ¡1
Fig. 5. Biodegradation of dioctadecylamine (2.0 mg l ) in Closed Bottle test with sil icone oil as a second phase. The biodegradation was tested in the absence (j) and presence of 10 mg l¡1 of polyalkoxylate alkylphenol (h). Addition of polyalkoxylate alkylphenol resulted in the formation of an emulsion.
in the presence of surfactants have been reported (Volkering et al., 1998). The solvent used as organic phase has to be non-toxic and nonbiodegradable. Silicone oil is not biodegradable nor does it contain biodegradable impurities. The introduction of silicone oil as an organic phase resulted in a ready biodegradability result i.e. 65% at day 28. Use of silicone oil therefore not only enabled an accurate administration of the test substance but also enhanced the bioavail ability of the test substance. The density of the silicone oil used was slightly higher than the density of water, which could result in loss of the oil with test substance from the bottles during closure. Silicone oil attached to the electrode could also lead to an unin tended loss of the test substance from the bottles. The use of sili cone oil therefore required special measures such as agitation of the content of the bottles during closure and the possibility to sac rifice bottles. Addition of the polyalkoxylate alkylphenol resulted in formation of a oil-in-water emulsion. A ready biodegradability result was also achieved with this emulsion (Fig. 5). Increased bio availability through the introduction of an organic phase has also been observed in other biodegradation studies (Efroyson and Alex ander, 1991; Ascon-Cabrera and Lebeault, 1993). 4. Conclusions Demonstration of the ready biodegradability of fatty amine derivatives is often impossible because these substances inhibit growth of microorganisms. Use of lignosulphonic acid and humic acid and especially silica gel overcome difficulties in demonstrating the ready biodegradability of alkyltrimethylammonium salts and
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