The retention of hydrophobic organic compounds by humic acid

G-eochimica et Cosmochimica Acta, 1972,Vol. 36, pp. 746 to 764. Pergamon Prees.Printedin NorthernIreland

The retention of hy~o~hobic organic ~o~po~n~ by humic acid* S. U. KHAN? and &I. Soil Rssesrch

Institute,

SCHNITZER

Canada Department of Agriculture, Ottawa, Ontario

(Received2 November 1971; acceptedir, revisedform25 Januaq

1971)

Abghact-A humic acid, extracted from the Ah horizon of a Black Chernozemsoil was found to fkx or strongly retain significant,amounts of hydrophobic organic compounds. Only approximately 8 per cent of the total alkanes and fatty acids occurring in the humic acid could be removed by exhaustive extraction with organic solvents. Another 19 and 11 per cent, of the two classes of compounds could be removed by organic solvents after methylation of the humic acid, but extraction of the bulk of these compounds rec@red extensive ohromatographic separations of the rnethy~at~ humic acid. Alkanes and fatty acids were ~t~ated to account for up to O-34 and 0.47 per cent of the weight of the origin& humic acid, respect&sly. Other hydrophobic organic compounds extracted were dialkyl phthalates, some of which are toxic, and di-set-butyl adipate, which were estimated to account for about 1 per cent of the initial weight of the humic acid. The organic esters were shown not, to be artifacts but are considered to be of biosynthetic origin. The data reported herein show that 100 g of humic acid can firmly retain up to 2 g, and possibly more, of hydrophobic organic compounds by a mechanism that most likely involves adsorption on external surfaces and in internal voids of a molecuIar sieve-type structural arrangement. The adsorption behaviour of humic substances for organic compounds, some of which may be toxic pollutants, should be of interest to those concernedwith environmental and geochemicalproblems. INTRODUCTION

substances are widely ~stributed in nature, occurring in soils, lakes (ISHIWATAM, 1969), rivers (LAHAR, 1968) and in the sea (RASHID and KINU, 1969). The principal humic fractions are humio acid (HA), which is soluble in base but insoluble in acid, and fulvic acid (PA), which is soluble in both base and acid. While much research has been done on interactions between the principal humic fractions and inorganic substances often associated with humic materials such as metal ions, hydrous oxides and clay minerals, little is known about reactions of humic substances with hydrophobic organic compounds that occur in soils and waters. Previous communications from this laboratory (OGNER and SCHNITZER, 1970a ; 1970b; SCHNITZER and OGNER, 1970; KHAN and SCHNITZER, 1971a, b) were concerned with the isolation of alkanes, fatty acids and dialkyl phthalates from a water-soluble FA. Less than 10 per cent of the alkanes and fatty acids and only traces of the dialkyl phthalates could be removed from the l?A by exhaustive extraction with organic solvents. To isolate the bulk of these compounds, it was necessary to methylate the PA and to undertake extensive chromatographic separations. Thus, the PA was capable of not only firmly retaining these hydrophobic compounds, but could in addition make them soluble in water and so increase their mobility in aquatic environments. The adsorption behavior of FA should therefore be of interest to geochemists and to those concerned with the preservation of the environment, HUMIC

* Contribution No. 398. t Present address: Research Station, Canada Department of Saskatchewan. 746

Agriculture, Regina,

746

S. U. &IAN

and M. SCHNITZER

especially if the hy~ophobic organio compo~ds are toxic ~llutants. To explain the relatively firm retention of hy~ophobic organic compounds, it was postulated on the basis of extensive chemical investigations (OGRER and SCHNITZER, 1971; KHAN and SCENITZER, 1971a; SCHNTTZER, 1971), that FA has a molecular sieve-type structure, made up of phenolic and benzenecarboxylic acids, and that this structure can adsorb inorganic as well as organic compounds of the proper molecular dimensions on its outer surface and in internal voids. HA, in contrast to FA, is wnterinsoluble, has a higher molecular weight and fewer oxygen-containing functional groups (CO,H, OH and C==O) per unit weight. We were interested in learning whether HA, which quantitatively is the major humic fraction in many soils and sediments, could also firmly retain hydrophobic organict compounds and whether the molecular sieve-type structure that we had proposed for FA was also applicable to HA. We were also concerned with obtaining information on the qualitative and quantitative distribution of alkanes, fatty acids and esters in HA, the nature of their association with HA, and on their likely origins. EXPERIMENTAL METHODS HA The HA originated from a soil sample which was taken from the undisturbed Ah horizon of a Black Chernozem soil (pH = 6.4, C = 6.1 per cent), developed on clay loam glacial till in Central Alberta, Canada. The sample was air-dried to pass a l-mm sieve. Methods of extraction, separation and purification of the HA were identical to those described elsewhere (KHAN and SOWDEN, 1971). The purified HA contained, on a dry, ash-free basis, 56.4 per cent C, 5.5 per cent H, 4.1 per cent N, 1.1 per cent S and 33.0 per cent 0. Fictional group analysis showed 4.5 meq CO,H, 2.1 meq phenolic OH, 2.8 meq alcoholic OH, 4-5 meq C = 0 and O-3meq OCHa per g of HA. The extracted and purified HA accounted for 27.5 per cent of the organic matter in the original soil sample. Ext~ractionof alkanes artd fatty acids One hundred g of air-dried HA (ash content = 0.9 per cent) was extracted for 24 hr in a Soxhlet extractor 6rst with 1,000 ml of n-hexane, then with 1,000 ml of benzene and finally with 1,000 ml of ethyl acetate. The three extracts weighed 0.036,0+027 and 0.192 g, respectively, and were found, by i.r. analysis, to contain both alkanes and fatty acids. To separate the two types of compounds, each extract was dissolved in 3 per cent EOH-methanol. The basic solution was extracted with n-hexane to remove alkanes (and possibly lipids) (0.021 g), then acidified and extracted with ra-hexaneto remove fatty acids (0.106 g). Normal alkanes (0.002 g} were separated from branched-cyclic alkanes (0.01 g) by refluxing with a 5 A molecular sieve (O’CONNORet aZ., 1962) in dry isooctane for 72 hr. Fatty acids were first methylated with diazomethane; the resulting esters were separated by three consecutive urea adductions (SWERN, 1964) into straight chain (O*OlOg) and branched-cyclic (0+055g) fatty acid methyl esters. The remaining HA (99.0 g) was then suspended in methanol and methyla~ repeatedly with diazomethane until the OCH, content remained constant at 17% per cent. The methylated HA, was extracted with 1,OOOml of n-hexane, the extract was saponified (PARKER, 1969) and separated, as described above, into straight chain alkanes (0.008 g) and branched-cyclic alkanes (0.026 g) and into straight chain fatty acids (0.018 g) and branched-cyclic fatty acids (O-014g). The methylated HA was then extracted with 4 x 1,000 ml of benzene. The benzene soluble material (15-24 g) was chromatographed over neutral dnminum oxide (activity 1, 750.0 g) and eluted with 1,000 ml of n-hexane (fraction A, O-278g), 1,000 ml of benzene (fraction B, 0,266 g), 1,000 ml of benzene-ethyl acetate (9: 1) (fraction C, 0.945 g), 1,000 ml of benzene-ethyl acetate (1:l) (fraction D, 1*356g), 1,OOOml of ethyl acetate (fraction E, 0.341 g) and f&ally with 1,000 ml of methanol (fraction D, P-3000 g). Each fraction was 6rst separated by preparative

The retention of hydrophobic organic compounds by humic acid

747

TLC on activated aluminum oxide plates (15O’C for 18 hr) with toluene-ethyl acetate (1: 1) as solvent. Both alkanes and fatty acid methyl eaters were found by exposure to I, vapour to ooncentrate in the upper halves of the TLC plates. The two types of compounds were removed from the plates and rerun by TLC on A&O* using toluene-ethyl acetate (2: 1) as solvent. Following removal from the TLC plates, the materials were separated into alkanes and fatty acids by the procedures desaribed above. Fractions A and B yielded 0.086 g of straight chain alkanes, 0.040 g of cyclic-branched alkanes and 0,138 g of straight chain fatty acids. Fractions C, B, E and F were found to be free of alkanes and fatty acids. Tests for the presence of unsaturated alkanes and fatty acids by TLC on 10 per cent silver nitrate-silica gel plates (MORRIS, 1964) with ?a-hexane as solvent were negative. The fractions failed to show absorption in the U.V. region nor was C==C stretching at 1646-1660 cm- l, typical of unconjugated linear olefins (SILVERSTEINand BASSILCR, 1967),detectable in the i-r. spectra. Rlaa2ysis of allcanes and fatty aca-ds Qualitative and quantitative analyses of 7a-alkanes and n-fatty acid methyl esters were performed on a Hewlett-Packard model 402 gas chromatograph, equipped with a flame ionization detector and a 1800 x 4 mm glass cohunn packed with 5 per cent SE-30 on Chromosorb W H&IDS, 60 to 80 mesh. The cohunn was programed from 90 to 290% at a rate of 7.5% per min, and then maintained at 290% until all compounds had eluted. For optional identification, the gas c~omato~phic separation of knowns and unknowns was also investigated on a IS00 x 4 mm glass ctolumn packed with 3 per cent Carbowax 20 M on Chromosorb W HMDS, 60 to 80 mesh. The eohmm was programmed from 90 to 250% at a rate of 7.5% per mm and maintained at 250°C until all compounds were eluted. The carrier gas was helium which was used at a flow rate of 75 ml/mm Tentative identitioation of each peak was first made by comparing retention times of unknowns with those of known n-alkanes and n-fatty acid methyl esters analysed under the same gas ehromatographio conditions. For more definitive ideutifloation small amounts of known n-alkanes and B-fatty acid methyl esters were added to the unknown mixture and increases in peak height were observed for each component. The gas ~~oma~~phic identification of each n-alkane and n-fatty acid methyl ester was confirmed by combined gas chromate. graphic-mass spectrometrio analysis. Quantitative estimations were made with the aid of a Hewlett-Packard model 3370 A integrator, connected to the gas chromatograph. i.r. spectra of the unmethylated and methylated HA were reoorded on KBr pellets (1 mg IIA per 400 mg KBr); spectra of alkanes and fatty acid methyl esters were taken as smears between NaCl plates. All solvents were purified by distillation through high efficiency columns, and solvent blanks were found to be negligible. Following two successive TLC separations on AlsOs-coated plates, fractions D, E and F were further separated by TLC on silica gel with toluene-ethyl acetate (3 : l), and then by preparative gas chromatography, using the same column and column packing as that employed for the separation of n-alkanes and n-fatty acid methyl esters. Materials representing the major gas chromatographic peaks were eluted from the gas chromatographic column, collected in capillary tubes and analysed by i.r. (as micro.KBr pellets on a Beckman i.r..12 spectrophotom. eter equipped with a beam condenser) and by mass s~etrometric analysis on a CEC model 21-490 mass spectrometer, using a heated direct inlet probe. The unknowns were identified by comparing their mass and micro-i.r. spectra and gas chromatographic retention times with those of standards of known structures. In addition to dialkyl phthalates, appreciable amounts of di-sec.-butyl adipate were also isolated and identified.

RESULTS AND DISCUSSION AlkCUZt?S

Total yields of ailcanesextracted from both the ~methyla~d and the methylated HA amounted to 173.64mg or O-17 per cent of the original HA (Table 1). Since

748

s. U. KHAN aad i%f. &!HNITZER Table 1. Yields of alkanes Mg extracted from 100 g of HA

Type of alkane

Unmethylated HA

Methylated HA

Methylated J&I + ehromatographic separations

Normal Branched-cyclic Total

2.45 X0.76 13.20

7.67 25.76 33.43

86.81 40.20 127.01

preliminary experiments with known alkanes and fatty acids had shown that at least 50 per cent of the starting material was lost during the extensive fractionation and purification procedures that were employed in this investigation, it is likely that

alkanes account for up to O-34 per cent of the weight of the HA. It is noteworthy that only 13.20mg or 7.6per cent of the total alkanes could be extracted from the unmethylated HA. Following methylation, organic solvents extracted another 19 per cent of the tota aIkanes, but the bulk of the total alkanes could be removed only after methylation and c~omato~ap~c separations by column and repeated thinlayer chromato~aphy on A&O,. The latter sorbent appeared to play an impo~nt role in the fra~ionation process by strongly retaining the methylat~ humic materials and so permitting the separation of the hydrophobic compounds by organic eluants (Table 1). This situation was similar to that encountered previously with FA (OQNER and SCHNITZER,1970b), where organic solvents extracted only 3 per cent of the total alkanes from unmethylated FA; following methylation of the FA and chromatographic separations, the remaining 97 per cent could be extracted, In contrast to normal alkanes, greater proportions of branched-cyclic hydrocarbons could be extracted from the unmethylated HA and from the methylated HA that had not been subjected to o~omatographic separations (Table If. A possible explanation for these findings is that the relatively bulky branched-cyc~c hydrocarbons were adsorbed on the external surfaces of the molecular sieve-like structure, while normal alkanes were held within internal voids of the sieve. Thus, branchedcyclic hydrocarbons were retained less strongly by the HA than were normal ones. Normal alkanes constituted approximately 56 per cent of the hydrocarbons that could be removed from the HA. An ir. spectrum for n-alkanes extracted from methylated HA, and typical for this class of compounds, is shown in Fig. 1, curve c. Weight distribution plots of n-alkanes isolated from the HA before methylation (Fig. 2a) and after methylation + chromatographic separations (Fig. 2b) show a range of n-alkanes from C,, to Cal, with considerable diiIerences in their distribution. While C-odd numbered hydrocarbons in the C,, to C,, range were the predom~ant alkanes, extracted from the unmethylated HA, the n-C,, alkane was the most prominent hydrocarbon removed from the HA after methylation + chromatographic separations. This alkane alone accounted for almost 30 per cent of the n-alkanes in this major fraction, The C-odd to C-even ratio of n-alkanes extracted from the unmethylated HA was l-45,but the ratio for a-alkanes extracted after methylation + chromatographic separations was only O-60.The overall C-odd to C-even ratio for all n-alkanes isolated was O-62.The last two ratios were strongly affected by the isolation of relatively large amounts of the n-C, alkane but would otherwise have been close to unity.

The retention of hydrophobic organic compounds by humic acid

749

r

Fig. 1. Infra-red speotra of (a) unmethylatod HA; (b) methylated HA; (c)Nalkanes extracted from HA after methylation + chromatographic separations; (d) m-fatty acid methyl esters extracted from IIA after methylation + chromatographic separations.

The general distribution of n-alkanes extracted from the unmethylated HA resembled those of pasture plants, soils and recent sediments (OR0 et al., 1965; STEVENSON, 1966; JONES, 1970). It is thus possible that these alkanes Oxnard from plant waxes on the surface vegetation. The d~tribution of a-alkanes isolated from the HA after methylation + chromatographic separations, howevex, was similar to that of microbial hydrocarbons which exhibit C-odd to C-even carbon atom ratios which are often less than unity and which have chain lengths greater than C,, (JONES,1969). The isolation of relatively large amounts of WC,, hydrocarbons in addition to substantial quantities of n-C&, M&,, WC&, ~z-C!~s, n-C&, and rt-C,, alkanes (see Fig. 2b) thus points to a microbial origin for these normal alkanes. As has been mentioned by JONES(1969), microbial hydrocarbon patterns depend on growth media, growth conditions and analytical methods. While effects of the latter on the hydrocarbon distribution reported herein are diflioult to assess, the results can be ~~rpre~d in the follo~g manner: hy~ooarbons ori~at~g from plant waxes were adsorbed “outside,” while n-alkanes coming from microbial sources were retained “inside” the molecular sieve-type HA structure.

S. U. KHAN and M. SCENITZER

760

Carbon

number

Fig. 2. Weight ~stribution of ~~.Eanes extracted from (a) ~ethyl~~ (b) HA after m%thylation + e~oma~~phic

Fatty

HA;

separations.

acids

The behavior of n-fatty acids in HA towards extraction by organic solvents paralleled that of n-alkanes. As shown in Table 2, only 10.45 mg or less than 7 per cent of the total n-fatty acids was extractable from the unmethylated HA, another 11 per cent could be exlzaoted after methylation; to extract the remaining 82 per oent of n-fatty acids, methylation of the HA, followed by chromatographic separations, was required. By contrast, almost 80 per cent of the bran~h~-cyclic fat&y acids could be removed from the ~ethyla~ HA, ~di~at~ that these acids were retained less tightly by the HA than were n-fat&y acids. Normal fatty acids constituted about 70 per cent of the total fatty acids extraofed, the remaining acids were branched-cyclic. In toto, 235.3 mg of fatty acids were extracted, accounting for up to O-47per cent of the initial HA, if losses during separation are again assumed to be about 50 per cent. An i.r. spectrum of n-fatty acids (as methyl esters), extracted from HA after methylation + chromatographic separations, and typical for this class of compounds, is shown in Fig. 1, curve d. Weight distribution curves for n-fatty acids (as methyl esters) isolated from the HA before and after methylation + c~omato~aphic separations are shown in Figs. 3a and 3b. Normal fatty acids removed from the ~methylated HA ranged from C,, to Cs8, with n-C,, and 12-C,, most prominent. Normal fatty acids isolated from the HA after methylation + chromatographic separations ranged from C,, to C&, with n-C,, and &?,, predominating. The C-even to C-odd ratio of the n-fatty acids isolated from unmethylated HA was 12.1, but the ratio for n-acids extracted from the HA after methylation + chromatographic separations was only 2.4; the overall ratio for all n-fatty acids extracted was 2.6. According to KVENVOLDEN(1966), even-carbonnumbered fatty acids in the C, to C,, range predominate in most living organisms, whereas C,, to C,, even-carbon-numbered acids are found p~cip&~y in waxes of insects and plank. These observations suggest two different sources for the n-fa&ty

The retention of hydrophobic organic compounds by humic acid

751

Table 2. Yields of fatty acids Mg extracted from 100 g of HA Type of fatty acid

Unmethylated HA

Normal Branched-cyclic Total

10.45 54.55 65.00

Methyl&d

HA

Methyl&d HA + chromatographic separations 138.01 0 138.01

18.08 14.25 32.33

acids : those isolated from the unmethylated HA (adsorbed “outside”) may have been of microbiological origin, whereas many of the n-fatty acids extracted from the HA after methyl&ion + chromatogmphic separations (adsorbed “inside”) could have come from plant waxes. Thus, the situation for n-fatty acids appeared to be the reverse of that for n-slkanes. Reasons for the differing adsorption behavior of n-alkanes and rz-fatty acids are not well understood at this time. It is possible that the distribution of the acids is, in part, a function of the separation techniques that were used; also, the origin of the fatty acids is fraught with uncertainties. Additional research is required to clarify these points. The results do, however, confirm earlier findings by SCHNITZERand OGNER (1970) that humic substances retain highermolecular-weight n-fatty acids more tightly than they do lower-molecular-weight ones. The proposal that most n-fatty acids retained by the HA originated from plant waxes is in accord with findings of MEINSCHEIN and KENNY (1957) on the sources of C,, to C,, n-fatty acids in soils. &h%m&p

between n-fatty acids and n-alkanes isolatedfrom HA

Since fatty acids are widely distributed in both plants and animals and are structurally similar to alkanes, the suggestion has been made (KVENVOLDEN, 1966)

,

a

Carbon

number

Fig. 3. Weight distribution of n-fatty acids (as methyl esters) extracted from (a) rmmethylated HA; (b) HA after methyl&ion + chromatogmphic separations.

752

S.

U. KHANand M.

SCHNITZEB

that in nature m-fatty acids are decarboxylated and reduced to form n-alkanes.

P~ra~e~m in ~stribution of n-fatty acids and B-alkanes, indicative of 8 direct prec~sor-product relationship, has been observed in ancient sediments (K~~o~DE~, 1966). Such relationship, however, does not exist between n-fatty acids and %&anes extracted from both unmethylated and methylated HA. In a previous investigation, SCHNITZER and OQNER (1970) also failed to find a direct precursorproduct relationship between the two types of compounds extracted from FA. Dialkyl phthalates were also isolated from methylated HA after chromatography. It is noteworthy that extraction with organic solvents failed to remove detectable amounts of these compounds from unmethylated HA. As shown in Table 3,349*5 mg of dialkyl phthalates were isolated from methylated DA, accounting for 0.7 per cent of the weight of the initisl material. especially notewo~hy is the isolation of relatively large amounts of toxic (RADEVAand DINOEVA,1966; HABERMANet al., 1967) bis(2-ethylhexyl) phthalate and benzyl-butyl phthalate. Another ester isolated in relatively large amounts (179.3 mg) was di-sec.-butyl &dip&e. Including the latter compound, the phthalates and the adipate accounted for up to 1 per cent of the original HA. Phthal~tes and adipates are used in industry as plasticizers, lubricants and in the m~ufact~e of alkyd resins and dyes. In earlier investigations OCINERand SCHNITZER(1970) and KXGLNand SCHNXTZER (1971a, b) isolated small amounts of dialkyl phthalates from methyl&ted FA and from low-molecular weight FA fractions that had been separated by gel filtration. It was at first suspected that the phthalates were contaminants that had interacted with the PA during the extraction and pur~~ation procedure. Hot toluene extracts of reagent bottles, ion-exchange resin and of the liquid pha+seof the gas chromatographie column failed to show any significant amounts of phthalates. Also, the air in the laboratory and the reagent blanks were practically free of phthalstes. As to possible origins of the phthalates, it is noteworthy that they have been reported to oocur in plants (HAYASHIetal., 1967), petroleum (BREQER,1966) and in fungal metabolites ( SUGIYAMA et CCL,X966) so that a bios~thetic origin in the soil c&mot be excluded. The extent to which humic substances csn adsorb dialkyl phthalates wa,srecently demonstrated by MYATS~DA and SCHNITZER (197 l), who showed that one number-average molecular weight (950 g) of PA could “complex” (maintain in aqueous solution) 4 moles (1,560 g) of bis(2-ethylehexyl) phthalate, while two number-average molecular weights of FA could “complex” 3 moles (990 g) of dicyclohexyl phthal~te, and one numberaverage molecular weight of FA “complexed” 1 mole (278 g) of dibutyl phthalate. i.r. spectra of untreated PA, pure phthalates, and of the “complexes” failed to provide indications for the occurrence of chemical reactions between FA and the phthalates. Reactions between HA and hydrophobic organic compounds

The results show that HA can retain or fix signi~cant amounts of hydrophobic organic compounds which can be removed by non-destructive methods. The behavior of HA in this respect is similar to that of FA except that the latter material is water-soluble while HA is insoluble in water, which would tend to limit the mobility of the “complexes” formed,

753

The retention of hydrophobic organic compounds by humic acid Table 3. Yields of dialkyl phthalates and sec.-butyl adipate isolated from HA after methylation + ebrom~to~ap~c separations Compound No.

Dialkyl phthaIates

Yield (mg)

Dibutyl phthalate Isobutyl phthalate Benzyl-butyl phthalate Bis(2ethyl hexyl) phthalate Dioctyl phthalate Dinonyl phthalats Dialkyl phthalate (not identified)

11.57 8.88 116.24 180.62 21.55 5-22 5.41

Di-sec.-butyl adipate

179.31

and SCHNITZER (1971), KIZ.U and SCHNITZER (1971a) and SCHNITZER have suggested that FA consists of a molecular sieve-type structure in which phenolic and benze~ecarboxyli~ acids are joined by hy~ogen-bond~g. Such a structure could adsorb-on its surface and in internal voids-organic and inorganic compounds of appropriate molecular dimensions. The structure appears to be weakened by methylation which reduces hydrogen-bonding between the “building stones,” but, in addition, extensive chromatographic separations are required before the adsorbed compounds can be isolated. To what extent this structural concept also applies to HA requires further investigation, but, judging from the tenacity with which HA retains hydrophobic organic compounds, a molecular sieve-like structural ~angement appears also to be valid for HA. Further support for such a structure comes from studies on the oxidative degradation of the HA. The alkaline permanganate oxidation of 100 g of methylated HA produced 9.7 g of phenol& acids and 14.7 g of benzenecarboxylic acids (KHAN and SCHNITZER,1972). By comparison, the alkaline permanganate oxidation of 100 g of methylated FA (KHAN and SCHITZER,1971a) yielded 8.9 g of phenolic acids and 11.5 g of benzene-carboxylio acids, suggesting the occurrence of similar chemical structures in the two materials. There are obvious implications here with regard to environmental pollution. Since toxic po~utant~ can be held very tightly by humic substances, it is likely that their concentrations in soils and sediments are often underestimated. On the other hand, humic substances may adsorb toxic pollutants in such a manner as to make them unavailable to plants and animals, so that the problem is less serious than one would expect it to be. Clearly, more information is needed on interactions of humic substances with toxic pollutants and on the effects of the ‘“complexes” so formed on the environment and on geochemical processes. OWER

(1971)

A~~~~~~g~~~nt~-~~e thank S. I. M. SKINNERfor mass speetrom%trieanalyses and J. G. D~SJARDINSfor technical assistance. REVERENCED I. A. (1966) Geochemistry of lipids. J. Amer. Oil Chmnist Sot. 43, 197-202. BAXERUANS., GUESSW. L., ROWAN D. F., BOWMANR. 0. and BOXER R. K. (1967) Techn. Pap., Reg. Techn. Conf., Sot. Plast. Eng., N.Y. Sect., read in Chem. Abatr. 67, 115358 y (1967). HIAYASHI S., ASAXAWAY., ISHIDAT. and MATSUU~AT. (1967) Phthalate esters of Cryptotaenia CanadensisDC. Var Japonica Makino (Wmbe~life~e). ~e~~~~~~~ Letters 50, 5061-5063. BRECER

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