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Surfactant Properties and Biodegradation of Polyethoxylates from Phenolic Lipids

John H.P. Tyman* and Ian E. Bruce
Department of Chemistry, Brunel University, Uxbridge, Middlesex, UB8 3PH United Kingdom

ABSTRACT: The surface tension of aqueous solutions of cardanol and cardol polyethoxylates derived from technical cashew nut-shell liquid were examined to determine their relative surfactancy. The compositions with the greatest surface tension reduction, or optimal surfactancy, were selected for biodegradation testing, in terms of total organic carbon, using a soil inoculum. The experimental surfactants in this study were compared with commercial t-nonyl polyethoxylate and glucose as reference materials. Over a period of 28 d, cardanol polyethoxylate and its saturated analog were degraded to 17 and 25%, respectively, and cardol polyethoxylate and its saturated analog were degraded approximately half that extent (37 and 46%). t-Nonylphenyl polyethoxylate remained substantially undegraded (77%), whereas glucose was almost completely degraded (5%). Paper no. S1373 in JSD 7, 169173 (April 2004). KEY WORDS: Alkylphenol polyethoxylates, biodegradation of phenols, surface tension reduction, surfactancy.

Technical cashew nut-shell liquid (CNSL), obtained by the industrial processing of phenolic lipids in the natural material Anacardium occidentale, which contains principally anacardic acid 1, cardol 2, and 2-methylcardol 3. Decarboxylation gives cardanol 4, together with unchanged cardol, as shown in Scheme 1. The polyethoxylates 5 and 6, depicted in Scheme 2, were obtained by the reaction of cardanol and cardol with ethylene oxide (1). t-Nonylphenol polyethoxylate 7 is shown for comparison. In each case, they contain a profile of oligomers in which m ranges from 1 to about 48. The ethoxylates were deemed ideal candidates for further surfactant and biodegradation studies, specifically, by first determining the optimal number of ethoxylate (EO) groups required for surfactancy, as judged by their relative reduction of the surface tension of water. With this information, biodegradation studies were carried out on the sample in each series exhibiting the highest surfactancy. Studies on the environmental occurrence of alkylphenols and alkylphenol ethoxylates (2) and their persistence in
*To whom correspondence should be addressed. E-mail: jhptyman@hotmail.com Abbreviations: CNSL, cashew nut-shell liquid; DOC, dissolved organic carbon; EO, ethoxylate; OECD, Organization for Economic Cooperation and Development; TOC, total organic carbon.
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aquatic environments have both been reviewed extensively (3). Commercial alkylphenol polyethoxylates have received criticism on environmental and toxicological grounds (4), and although subsequent studies have suggested their accumulation is not signicant (5), there also have been indications that they could be gradually phased out (6). Recent work (7) has indicated the continued existence of marine pollution with alkylphenol polyethoxylates. Studies on the response of activated sludge to variations in the composition of feed effluent (8) have indicated that alkyl ethoxylateadapted biomass was more capable of adaptation to alkylphenol ethoxylate than the reverse. Overall, there are conflicting views on the fate and effect of the present class of alkylphenol polyethoxylates in the environment. Since approximately 90% of the alkylphenol ethoxylates in the European Union are based on the petrochemical intermediate t-nonylphenol, it is of interest to examine the use of natural alkylphenols, based on the supposition that a biosynthesized compound might prove to be more biodegradable and environmentally acceptable than t-nonylphenol and its polyethoxylates. Also, an ethoxylate from a natural alkylphenol would necessarily require useful surfactant properties, and in this regard, there has been little research on the surfactant activity of such products. As a result, both the surfactancy and biodegradability of the polyethoxylates from the phenolic lipids cardanol 4 and cardol 2 were studied in the present work. The choice of biological mediumnamely, whether to choose one based on activated sludge or one of soil origin in the several test methods used for determination of the

SCHEME 1
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SCHEME 2

biodegradability of polyethoxylates is important. In our work, we elected to adopt a European Economic Community procedure based on a shake flask system rather than to use activated sludge, as is generally applied in the United States. Some parameters of the Organization for Economic Cooperation and Development (OECD) screening test for the biodegradation of surfactants with regard to inoculum, aeration, feed, and media were examined; we concluded that no inoculum was needed to begin the biodegradation process and that it was possible to use standard mineral microelements (9). Although the dilute bacterial media present in the shake flask systems do not simulate sewage treatment plant conditions, they are useful for the initial screening of a variety of substances and are adaptable to procedures involving radiolabeled materials. A variety of chemical and spectroscopic techniques have been used for the analytical determination of polyethoxylates. One of the many procedures in biodegradation studies has been to determine the rate and extent of reduction of dissolved organic carbon (DOC), which involves a filtration step, on samples for analysis or those of total organic carbon (TOC), in which samples are not ltered. The most reliable and accurate method is probably the use of a carbon analyzer such as an inductively coupled plasma spectrometer. However, we found that, at the very low concentrations used in our experiments, the errors encountered were too great. An alternative and simpler chemical method for determining TOCone consisting of oxidation of the carbon present with acidic dichromate and absorption of the resulting carbon dioxide in barium hydroxidewas found to be satisfactory in this study. To determine biodegradability, a low concentration of microorganisms and a maximum duration of 28 d (designed to screen substances) was used. Next, TOC analyses were carried out on samples withdrawn periodically. Substances passing this test with greater than 70% removal of DOC were assumed to be extensively degraded during sewage treatment in practice and were likely to be removed rapidly from bulk water. It was of interest to compare the susceptibility of different types of aromatic substitution in phenols described in recent biodegradation studiesoften with a wide variety of specic organismswith the phenolic structures of the polyethoxylates in the present work. Such work is of interest in charting the relationship between organic structure and the biological activity of different classes of organisms. Thus, biodegradation studies with organic pollutants have been mainly concerned with bacteria, fungi, and yeasts. In an exJOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 7, NO. 2 (APRIL 2004)

amination of other types of organic polluntants, namely, eukaryotic algae such as Ochromonas danica (10), phenol was found to degrade more readily than p-cresol, although higher homologs were not examined. By contrast, with thermophilic microorganisms such as Bacillus thermoglucosidasius, nonoxidized aromatic compounds were not biodegraded, and phenol and m-cresol exhibited the highest degradation. This occurred to a much greater extent than the biodegradation of o- and p-cresol, (11). Even though the studies with this group of microorganisms have not included higherchain-length compounds, the effect of m-substitution in the present phenolic polyethoxylates among other biological organisms could nevertheless be of potential interest. In a study on the biodegradation of different phenols in river water (12) at mild temperatures, at neutral pH, and with excess oxygen, the relative order of biodegradation was found to be resorcinol > phenol > o-cresol > o-chlorophenol. The higher rate for 1,3-substitution may again have significance for longer-chain compounds. The 1,3,4, namely, partly m-substitution in lignans, was found to be susceptible (13) to a basidomycete, white rot fungus (Lentinula edodes). More generally, enzymatic systems have been investigated. Thus, the removal of phenolic pollutants in wastewaters by soybean peroxidase has been studied (14), and the advantages of enzyme-based, compared with physicochemical, treatment have been indicated. The biodegradation of phenol in coke wastewater has been examined with enzymes from Pseudomonas sp. immobilized on flat polyamide membranes (15). In studies reminiscent of the use of specific organisms to biotransform dedicated compounds to organic intermediates, the use of a recombinant strain of E. coli P260 has been examined for the controlled biodegradation of aromatic compounds and subsequent synthesis (16) of a variety of organic compounds. Some sources (17) have commented on the occurrence of phenolic and nonphenolic estrogenic mimics in the environment. Indeed, the long-term effects upon the living world and the environment of the great range of presentday chemicalsvery largely derived from petrochemical sourcesremain relatively unknown. In this connection, the greater use of natural resources in chemical production (18) would appear to be timely.

EXPERIMENTAL PROCEDURES
Materials. Technical CNSL was obtained from 3M Research Ltd. (Harlow Essex, United Kingdom). The separation of cardanol and cardol from technical CNSL, their polyethoxylation

171 PROPERTIES AND BIODEGRADATION OF POLYETHOXYLATES

with EO, and their characterization as surfactants have been described (1). t-Nonylphenol polyethoxylate, obtained from S.C. Johnson and Son, Inc. (Racine, WI) was of commercial origin. Reaction of the phenolic lipids, cardanol, and cardol (shown in Scheme 2) with EO was carried out in each case until an average of m = 48 EO groups/mol was added. In the characterization studies of these polyethoxylates, 16 samples were collected in the course of the polyethoxylation reactions and submitted to high-performance liquid chromatography and nuclear magnetic resonance spectroscopic examination. In each case, these same 16 samples were then also examined for their surfactant and biodegradation properties. The ve polyethoxylates, namely, cardanol polyethoxylate 5 and its saturated analog 5 (n = 0), and cardol polyethoxylate 6 and its saturated analog 6 (n = 0), were rst used to determine the species yielding the greatest reduction in the surface tension of water. Then, in each case, this optimal sample was submitted to biodegradation studies and compared with t-nonylphenol polyethoxylate 7. Surfactant property determinations. The reduction in the surface tension of distilled water (measured as Nm1), by 1% aqueous solutions of cardanol and cardol polyethoxylates and by saturated versions of both polyethoxylates and of t-nonylphenol polyethoxylate, was determined at 24C by the use of a du Noy tensiometer (19). In this method a circular ring of radius r attached by a single wire at diametrically opposite points rests horizontally and is completely wetted on the surface of the solutes. The force, f, required to detach it from the opposing vertical action of the surface tension, (at zero contact angle), is given by f = 4r . The equipment is operated manually. Biodegradation experiments. The method used was based on a modified OECD screening test for biodegradability (20), an alternative approach to using activated sludge. The test compound, in a standard nutrient medium at a concentration of 100 mg/L, was inoculated with a low concentration of microorganisms from a mixed population and aerated at constant temperature for no more than 28 d. Biodegradation was measured by TOC analysis. Control flasks were run in parallel to determine TOC blanks, and the procedure was checked in duplicate with a reference compound of known biodegradability, namely, glucose at 100 mg/L. (i) Inoculation. Soil (100 g) (fertile, not sterilized, and free of herbicides and fertilizers) was suspended in water (1000 mL) and stirred, and the suspension was allowed to settle for 30 min. The supernatant liquid was filtered through coarse lter paper, the rst 200 mL was discarded, and the remainder was aerated immediately until used. The inoculum was used on the day of collection. Materials and reference substances were evaluated in duplicate. The nutrient solution (200 cm3) containing the required TOC concentration of the test compound (100 mg/L) was treated with the inoculum (0.1 cm3). Controls with no test substance but containing the inoculum were also run. During the experiment, a suitable volume (10 cm3) of

the test material, the reference, and the control were withdrawn in each case, but not filtered, and the TOC was determined in duplicate from samples in two reaction flasks tted with loose metal caps that did not impede airow. The asks were gently agitated in a shaker shielded from light for 28 d at 25C, and samples were withdrawn for TOC determination every 4 d. (ii) TOC analysis. The acidic dichromate method was used (21). The carbon dioxide that evolved was trapped by standard aqueous barium hydroxide, which was then titrated with standard acid.

RESULTS AND DISCUSSION

Surfactancy. Although surfactancy may be assessed in a variety of ways, such as by measuring the foaming ability of aqueous solutions, or their oil-solubilization characteristics, the reduction in surface tension (Nm1) of water by dilute solutions of polyethoxylates was used in this study to determine the optimal average ethoxylate chain length of the various samples. The particular ethoxylate sample in each of the series yielding the greatest reduction of surface tension was then considered the best candidate for a biodegradation study. The reductions in surface tension of 1% solutions of cardanol, cardol, and 3-pentadecylphenol polyethoxylates, respectively, are shown in Figure 1. For purposes of comparison, a 1% solution of common commercial t-nonylphenol polyethoxylate 7, with an average of 8 EO groups, gave a reduction in surface tension of 33 Nm1 . From these results, the samples of cardanol polyethoxylate 5, cardol polyethoxylate 6, and 3-pentadecyl polyethoxylate 5 (n = 0), with an average of 13.4 EO, 10.3 EO, and 14.8 EO groups, respectively, exhibited the greatest reductions in surface tension among the 16 samples examined in each class. Cardanol polyethoxylate and its saturated analog appeared to be similar in surfactancy, as expected, with sidechain unsaturation conferring slightly improved properties.

FIG. 1. Variation in the reduction in surface tension of water by cardanol, 3-pentadecylphenol, and cardol polyethoxylates with the average number of ethylene oxide (EO) groups/mole.

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By contrast, cardol polyethoxylate 5 exhibited a relatively smaller reduction in surface tension and, consequently, would be expected to be a less effective surfactant. Although technical CNSL itself might prove a useful surfactant upon polyethoxylation, the removal of cardol appears to be benecial for achieving the highest potential surfactancy. A comparison of the three compounds depicted in Figure 1 revealed that t-nonylphenol polyethoxylate 7 was comparable in surfactancy with cardanol polyethoxylate and that the sample with an average of 8 EO groups was considered to be an appropriate reference material in biodegradation work. Biodegradation studies. As shown in Figures 2 and 3, the percentages of initial TOC of cardanol polyethoxylate and 3-pentadecylphenol polyethoxylate remaining after 28 d were 17 and 25%, respectively, compared with 77% for t-nonyl polyethoxylate. The biodegradation of cardanol polyethoxylate was comparable to that of the reference material glucose. In contrast, the corresponding results for cardol polyethoxylate and saturated cardol polyethoxylate were 37 and

46%, respectively, confirming that linear hydrophobic chains are highly benecial to biodegradation and that certain types of unsaturation appear advantageous. Our results are in agreement with earlier studies showing that certain types of branching in the chain of alkyl polyethoxylates have a negative effect on their biodegradability. Also, the presence of two ethoxylate side chains in cardol polyethoxylate might partly explain its slower biodegradation compared with that of cardanol polyethoxylate. The inferior surfactancy and biodegradability of cardol polyethoxylate, as compared with cardanol polyethoxylate, would seem to justify separation of the component phenols in technical CNSL prior to ethoxylation. A cost/benet study covering the full range of uses of these polyethoxylates could prove useful in this regard, bearing in mind that technical CNSL from different regional sources contains approximately 20% cardol. The nature of the organic material constituting the TOC after 28 d has not been identified. By analogy with other work (22), it seems probable that it constitutes the carboxymethyl derivative of cardanol, cardanol itself, or further metabolic degradation products. For t-nonylphenol polyethoxylate, biological degradation of the side chain by both aerobic and anaerobic pathways has been described (3, p. 41), leading to t-nonylphenoxyacetic acid from 2-tnonylphenoxy ethanol. One could reasonably conclude that natural phenolic lipids behave similarly. Although it would indeed be of interest to identify the metabolites by high-performance liquid chromatographymass spectrometry or other techniques, this analysis was not performed in the present work. Concern has been expressed regarding the use of t-nonyl polyethoxylates because of their slow and incomplete biodegradation. The surfactants derived from natural cashew phenols, having improved biological compatibility as described in the present work, would appear to be an attractive alternative class of surfactants.

FIG. 2. Biodegradation of cardanol, 3-pentadecyl phenol, and tnonylphenol polyethoxylates. TOC, total organic carbon; for other abbreviation see Figure 1.

ACKNOWLEDGMENTS
We thank S.C. Johnson and Son Inc. for financial support. Dr. S. Smith, Department of Biology, is thanked for help with the biodegradation experiments. This paper is part 37b of the Long-Chain Phenols series. Part 37a appeared as Synthesis and Characterization of Polyethoxylate Surfactants Derived from Phenolic Lipids (JSD 6, 291297, 2003).

REFERENCES
1. Tyman, J.H.P., and I.E. Bruce, Synthesis and Characterization of Polyethoxylate Surfactants Derived from Phenolic Lipids, J. Surfact. Deterg. 6:291 (2003). 2. Bennie, D.T., Review of the Environmental Occurrence of Alkylphenols and Alkylphenol Ethoxylates, Water Qual. Res. J. Canada 34:79 (1999). 3. Maguire, R.J., Review of the Persistence of Nonylphenol and Nonylphenol Ethoxylates in Aquatic Environments, Water Qual. Res. J. Canada 34:37 (1999). 4. Marcomini, A., B. Pavoni, A. Sfriso, and A.A. Orio, Persistent Metabolites of Alkylphenol Polyethoxylates in the Marine Environment, Chemosphere 17:853 (1988).

FIG. 3. Biodegradation of cardol and saturated cardol polyethoxylates and glucose. For abbreviations see Figures 1 and 2.
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15. Bohdziewicz, J., Biodegradation of Phenols by Enzymes from Pseudomonas sp. Immobilised onto Ultracentrifuge Membranes, Process Biochem. (Oxford) 33:811 (1998). 16. Ferror Munoz, E., and A. Gibello Prieto, Process for the Biodegradation of Aromatic Compounds and Synthesis of Pigments, Colorants, Alcaloids, and Polymers with the Use of the Recombinant Strain Escherichia coli P260, European Patent 0857779 (1998). 17. Sumpter, J.P., S. Jobling, and C.R. Tyler, Estrogenic Substances in the Aquatic Environment and Their Potential Impact on Animals, Particularly Fish, Soc. Exp. Biol. Semin. Ser. 57:205 (1996). 18. Tyman, J.H.P., The Role of Biological Raw Materials in Synthesis, in Studies in Natural Products Chemistry, edited by Atta-urRahman, Elsevier, New York, 1995, Vol. 17, p. 601. 19. Taylor, H.S., and A.T. Taylor, Elementary Physical Chemistry, 3rd edn., Van Nostrand, New York, 1944, p. 140. 20. Council Directive of March 31st, 1982, on the Approximation of the Laws of the Member States Relating to Methods of Testing the Biodegradability of Nonionic Surfactants and Amending Directive 73/404/EEC/82/242.EEC, Official J. Eur. Commun. 109 (1982). 21. Huddleston, R.L., and R.C. Alldred, Evaluation of Detergent by Using Activated Sludge, J. Am. Oil Chem. Soc. 41:732 (1964). 22. Watson, J.S., D.M. Jones, and R.P.J. Swannell, Formation of Carboxylic Acids During Biodegradation of Crude Oil, In situ Bioremediation of Petroleum Hydrocarbons and Other Organic Compounds, edited by B.C. Allerman and A. Leeson, Batelle Press, Columbus, OH, 1999, p. 239.

[Received May 21, 2003; accepted November 12, 2003]

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