Spectroscopic Characterization of Soil Organic Matter

19 Spectroscopic Characterization of Soil Organic Matter R. BARTHA and T.-S. HSU

Downloaded by UNIV OF LEEDS on January 16, 2018 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0029.ch019

Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, N. J. 08903

In studies concerning the environmental fate of pesticides and other man-made chemicals, it has been noted repeatedly that some of these compounds or the products of their partial degradation "disappear." They do so in the sense that they become undetectable by the conventional techniques of residue analysis, yet radiotracer evidence negates their mineralization, i.e. their complete conversion to mineral constituents. It is now clear that in some of these cases chemical reactions take place between humic substances and the introduced compounds, leading to complexed and often immobilized residues. Such chemical binding of residues was observed primarily in soil and in sediment s, but similar reactions can take place in natural waters containing dissolved humic substances. While absorption by clay minerals and humic substances can also lead to a temporary immobilization of residues, this phenomenon can be distinguished from covalent binding by the fact that it can be overcome by exhaustive solvent extraction or by ion exchange techniques. In contrast, covalently boundresidues can be released only by relatively severe treatment, e.g. hydrolysis by strong acid or a l k a l i , and such treatment may irreversibly alter both the residue and its binding s i t e . Some of the bound residues can not be released at all in a chemically recognizable form by currently available procedures. For the above reasons, a nondestructive technique for bound residue analysis and for the study of the nature of the chemical attachment is clearly desirable. It is the aim of this brief review to assess the potential usefulness of various spectrometric techniques for this purpose. Some ongoing studies in our laboratory on the covalent binding of chloroaniline residues to humus are discussed in this context. Origin, Classification and Composition of Humic Substances. The soil organic matter derives from remains of plants, animals and microbes. Humic substances are defined as that portion of the soil organic matter than has undergone sufficient 258

Kaufman et al.; Bound and Conjugated Pesticide Residues ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


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transformation to render the parent material unrecognizable. Humic materials are present in mineral s o i l s t y p i c a l l y at less than 5% by weight. They are generated not only in s o i l s but a l s o in aquatic environments; a d d i t i o n a l humic material may be added to natural waters by leaching from s o i l s (1,2). The genesis of humic material is a two stage process in v o l v i n g the predominantly m i c r o b i a l degradation of o r g a n i c polymers to monomeric c o n s t i t u e n t s such as phenols, quinones, amino a c i d s , sugars, e t c . , and the subsequent polymerization of these due to spontaneous chemical r e a c t i o n s , a u t o x i d a t i o n and o x i d a t i o n catalyzed by m i c r o b i a l enzymes such as o x i d a s e s , p o l y phenol oxidases and peroxidases (j3,.|0 . The humic material is in a dynamic s t a t e o f e q u i 1 i b r i u m , i t s synthesis being compen sated f o r by gradual m i n e r a l i z a t i o n of the e x i s t i n g m a t e r i a l . According to t h e i r s o l u b i l i t y c h a r a c t e r i s t i c s , humic sub stances can be f r a c t i o n a t e d into f u l v i c a c i d ( s o l u b l e in a l k a l i , not p r e c i p i t a t e d by acid) humic a c i d ( s o l u b l e only at a l k a l i n e pH) and humin ( i n s o l u b l e in a l k a l i ) (5). To none of these f r a c t i o n s can a d e f i n i t e chemical s t r u c t u r e be a s s i g n e d , all three being randomly assembled i r r e g u l a r polymers. The main differences between f u l v i c and humic acids are the lower mole c u l a r weight, higher oxygen to carbon r a t i o , and higher r a t i o of a c i d i c f u n c t i o n a l groups per weight of the former as compared to the l a t t e r , but the spectrum is continuous and the d i v i d i n g l i n e is a r b i t r a r y . Molecular weights range from around 700 to 300,000, t o t a l a c i d i t i e s from 485 to 1,420 meq/100g. Humin is regarded as a s t r o n g l y bound complex o f f u l v i c and humic acids to mineral material rather than a c l a s s o f compounds by i t s e l f (6,7). The a l c o h o l - s o l u b l e hymatomelanic a c i d , a minor compound p r e v i o u s l y suspected to be an a r t i f a c t of e x t r a c t i o n , now appears to be a genuine f r a c t i o n c o n s i s t i n g o f e s t e r i f i e d o r methylated humic acids(8). Since humic compounds are random polymers, we cannot hope to learn t h e i r exact chemical s t r u c t u r e . At b e s t , we can e s t a b l i s h type s t r u c t u r e s , e . g . representative sequences o f interconnected atoms w i t h i n the humic a c i d molecule. The theories on humic type s t r u c t u r e s are c o n t r o v e r s i a l and subject to constant re v i s i o n and refinement. The perhaps most accepted current theory v i s u a l i z e s an aromatic " c o r e " c o n s i s t i n g of s i n g l e and condensed aromatic, h e t e r o c y c l i c and, perhaps, q u i n o i d a l (3) r i n g s , linked and c r o s s - l i n k e d by carbon-carbon, e t h e r , amino, and azo bonds. The rings bear a v a r i e t y o f functional groups, the more prominent of which are c a r b o x y l , phenolic hydroxyl and carbonyl groups. Attached to t h i s core are amino a c i d s , p e p t i d e s , sugars and phenols, which form further cross l i n k a g e s . The r e s u l t is a three-dimensional sponge-like s t r u c t u r e that r e a d i l y absorbs water, ions and organic molecules in an exchangeable manner and, in a d d i t i o n , may chemically bind s u i t a b l e compounds to i t s r e a c t i v e f u n c t i o n a l groups (9,10,Π,]2,12,Ιί*) . As a consequence, v i r t u a l l y all natural organic compounds and apparently a l s o numerous man-made chemicals can occur in bound or absorbed



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form in humic substances; even a c t i v e enzymes were r e c e n t l y recovered in humus-bound form (15). The r i n g structures that serve as b u i l d i n g blocks of the humic a c i d core may o r i g i n a t e from the m i c r o b i a l degradation of l i g n i n or may be synthesized by various microorganisms from other carbon s u b s t r a t e s . D i r e c t l y or a f t e r o x i d a t i o n to quinones the p h e n o l i c compounds condense with amino acids in a process that can be modelled in v i t r o . Substances c l o s e l y resembling humic acids were s y n t i ï e s i z e d by enzymatic o x i d a t i o n of phenol mixtures in the presence of amino acids or peptides (16,17)* In a d d i t i o n , simple mixtures such as m e t h y l g l y o x a l - g l y c i n e and g l u c o s e - g l y c i n e were reported to form, upon heat a c t i v a t i o n , substances resembling humic acids (Jjf). Spectrometry o f Humic Substances. In an e f f o r t to e l u c i d a t e t h e i r chemical s t r u c t u r e , spect r o m e t r y techniques were a p p l i e d e x t e n s i v e l y to both supposedly " i n t a c t 1 1 and to i n t e n t i o n a l l y modified ( e . g . methylated o r acetylated) humic compounds. These s t u d i e s are i n s t r u c t i v e as to the p o s s i b i l i t i e s and l i m i t a t i o n s of spectrometric techniques as a p p l i e d to humic m a t e r i a l s . They were r e c e n t l y reviewed by S c h n i t z e r (18) and w i l l be discussed here b r e i f l y . Only a l i m i t e d body of l i t e r a t u r e e x i s t s at t h i s time on the primary concern of t h i s d i s c u s s i o n i . e . on spectrometric studies of humus-bound p e s t i c i d e residues. V i s i b l e and U l t r a v i o l e t (UV) S p e c t r a . These spectra ref l e c t t r a n s i t i o n s between e l e c t r o n i c energy l e v e l s and a r e , for p r a c t i c a l purposes, confined to the wavelength range of 200 to 800 nm. The p r i n c i p a l chromophores are conjugated double (or t r i p l e ) bonds both in a l i p h a t i c and in aromatic carbon compounds (19). The broad and overlapping nature of the absorption bands does not allow much information to be obtained by t h i s technique on the f i n e s t r u c t u r e of such large and complex molecules as the humic compounds. The usefulness of v i s i b l e and UV spectra is l a r g e l y confined to the c h a r a c t e r i z a t i o n of monomeric degradation products o f humic compounds. O v e r a l l decreases in conjugated double bonds occur during o x i d a t i o n , c a t a l y t i c h y d r o g é n a t i o n , and other treatments that lead to extensive depolymerization of humic substances, and these can be detected by a decreased absorption in the v i s i b l e and UV range. The comparative absorption of humic compounds in the v i s i b l e range is b e l e i v e d to be c o r r e l a t e d to the degree o f condensation o f t h e i r aromatic n u c l e i , hence darker c o l o r i n d i c a t e s a greater abundance of condensed n u c l e i (2). Infrared (IR) Spectra. The energy l e v e l s of most molecular v i b r a t i o n s f a l l into the energy range o f IR r a d i a t i o n . IR spectra contain a great deal of rather s p e c i f i c information



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about t n e inner s t r u c t u r e and the f u n c t i o n a l groups of an organ i c molecule and, consequently, IR spectrometry is one of the preferred tools of s t r u c t u r a l i d e n t i f i c a t i o n . The most useful wavelength range f o r the chemist l i e s between 2.5 and 16 μ o r , as more commonly expressed, between the wave numbers 4,000 and 625 c m ' 1 . The s t r e t c h i n g and bending v i b r a t i o n s of various functional groups have t h e i r c h a r a c t e r i s t i c wave number regions. While the molecular environment of the f u n c t i n a l groups has a marked in fluence on the exact p o s i t i o n of the absorption maxima, it is frequently p o s s i b l e to assign an absorption band to a s p e c i f i c functional group even if i t s molecular environment is not known with c e r t a i n t y (19,20). It is t h i s p a r t i c u l a r feature of the IR spectra that makes them q u i t e useful in the study of humic substances. The main IR absorption bands of i n t e r e s t are summarized by Stevenson and B u t l e r (6). The low energy o r M f i n g e r p r i n t " region (1500-625 cm" 1 ) is r e l a t i v e l y f e a t u r e l e s s in humic compounds because of the overlapping inner v i b r a t i o n s of the large and complex humic molecules. In the higher evergy r e g i o n , the 3,300 cm"* band is assigned to the 0-H s t r e t c h i n g of Η-bonded OH groups, the 2,900 cm^band to the C-H s t r e t c h i n g of a l i p h a t i c carbon c h a i n s , the 1,720 cm" 1 band to the C=0 s t r e t c h i n g of carboxyl and ketone groups, the 1,610 cm" 1 band to the C-C v i b r a t i o n s of aromatic rings with a c o n t r i b u t i o n a l s o from the C=0 s t r e t c h i n g o f Η-bonded ketone groups, and the 1,250 cm" 1 band to the C-0, s t r e t c h i n g and OH deformation o f carboxyl groups. A c e t y l a t i o n introduces a strong new band o f 1,375 cm" 1 due to the C-C deformation o f the a d d i t i o n a l a c e t y l groups (Figure 1). Methylation of humic substances increases the i n t e n s i t y of 2,900, 1,720 and 1,250 cm" 1 bands due to the in crease o f C-H,C=0 and C-0 s t r e t c h i n g s , r e s p e c t i v e l y . The normally small band at 1,450 c m " 1 , due to the C-H deformation of methyl groups, a l s o increased. S p e c i f i c a c e t y l a t i o n , methylation and IR spectrometry of the so modified humic compounds lends support to the presence of quinones in f u l v i c and humic acids (9). The need for b r e v i t y has n e c e s s i t a t e d here the over s i m p l i f i c a t i o n of a h i g h l y complex s u b j e c t , and for q u a l i f i c a t i o n for the above statements a d d i t i o n a l references should be con sulted (2J,22,23,24). IR spectra were u t i l i z e d in the study of metal ion i n t e r actions with humic substances (25). A decrease o f the 1,725 c m ' 1 band ( c a r b o x y l i c C,0) and increases of the 1,600 and 1,400 c m * 1 bands (carboxylate) i n d i c a t e d the formation o f an i r o n - c a r b o x y l ate) complex. The decomposition of oxygen-containing f u n c t i o n a l groups during the gradual p y r o l y s i s o f humic material was a l s o followed by changes in the IR spectra (26).


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Figure 1. IR spectra of original, acetylated,and methylated humic acids. After Ref. 18

E l e c t r o n Spin Resonance (ESR) S p e c t r a . Referred t o a l s o as e l e c t r o n paramagnetic resonance (EPR) these s p e c t r a r e f l e c t energy absorption due t o magnetic s p i n resonance o f unpaired electrons. The " f r e e r a d i c a l s " o f o r g a n i c compounds contain such unpaired e l e c t r o n s . Under the influence o f an external magnetic f i e l d , microwave e x c i t a t i o n allows t r a n s i t i o n s t o higher energy l e v e l s t o be observed, as i n d i c a t e d by absorption o f the microwave r a d i a t i o n . The t r a n s i t i o n energies a r e under the in fluence o f the molecular environment o f the free r a d i c a l , and thus contain useful information about it (27). T y p i c a l l y , free r a d i c a l s a r e h i g h l y l a b i l e and s h o r t - l i v e d intermediates o f some chemical r e a c t i o n s , i n c l u d i n g some b i o l o g i c a l l y c a t a l y z e d o x i dations. In c e r t a i n condensed aromatic systems and q u i n o i d a l s t r u c t u r e s f r e e r a d i c a l s may become s t a b i l i z e d . It is a t y p i c a l feature o f humic substances that they r e g u l a r l y c o n t a i n s t a b i l i z e d free r a d i c a l s , presumably in form of semiquinones (28,29, .30>JLl)* T n e ESR-signal is one o f the arguments in favor f o r the presence o f q u i n o i d a l s t r u c t u r e s in humic s u b s t r a n c e s . For a f u l v i c a c i d a s p i n count o fO.58X 10 per g was measured, and from t h i s value it was estimated that one semiquinone r a d i c a l


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is present f o r every 44,000 carbon atoms (28). The o r i g i n of the free r a d i c a l s is as i n t r i g u i n g as t h e i r p e r s i s t e n c e , and the most p l a u s i b l e theory is that they were formed during and preserved s i n c e the o r i g i n a l b i o l o g i c a l o x i dation that lead to the formation of the humic compound. The b i o l o g i c a l e f f e c t o f these s t a b i l i z e d free r a d i c a l s and t h e i r p o t e n t i a l to i n i t i a t e polymerization reactions or the binding of p e s t i c i d e residues to humic m a t e r i a l s are v i r t u a l l y v i r g i n areas awaiting experimental work. Nuclear Magnetic Resonance (NMR) S p e c t r a . The nuclei of c e r t a i n atoms e x h i b i t a magnetic s p i n momentum. The most important o f these atoms are 7-H,13-C.,19-F and 31-P. When placed in a homogeneous magnetic f i e l d and e x c i t e d with radiowaves, energy t r a n s i t i o n s , as evidenced by radiowave a b s o r p t i o n , take place. A g a i n , the molecular environment of the proton influences the resonance energy r e s u l t i n g in a resonance s h i f t r e l a t i v e to the a r b i t r a r y reference point o f the t r i m e t h y l s i l a n e (TMS) s i g n a l (19). NMR spectrometry is a powerful tool f o r the exp l o r a t i o n o f the immediate chemical environment of a proton (32) that has been d e f i n i t e l y u n d e r u t i l i z e d in the study of soil organic matter. The l i m i t e d s o l u b i l i t y of the humic compounds in the s u i t a b l e deuterated solvents presents a problem, but the r a p i d l y i n c r e a s i n g s e n s i t i v i t y o f the instruments is l i k e l y to improve t h i s s i t u a t i o n . To date the NMR-technique was a p p l i e d only to hydrogen protons in humic a c i d . Barton and S c h n i t z e r (33) investigating a low molecular weight methylated f u l v i c a c i d noted the absence of aromatic and o l e f i n i c protons. This s u r p r i s i n g r e s u l t seems to i n d i c a t e that p r a c t i c a l l y all hydrogens are replaced by s u b s t i t u e n t s on the aromatic core of humic substances. Felbeck (34) a p p l i e d NMR spectrometry to products of hydrogenolysis of soil organic matter and noted the lack of deuterium-exchangeable protons on nitrogen atoms, and a l s o noted a prevalence of methylene peaks over methyl and methine. The l a t t e r f i n d i n g was i n d i c a t i v e o f a low degree of branching o f the carbon c h a i n s , at l e a s t in the material modified by h y d r o g e n o l y s i s . General L i m i t a t i o n s o f Spectroscopic C h a r a c t e r i z a t i o n o f Humic Substances. Spectroscopic methods work best in the c h a r a c t e r i z a t i o n of homogeneous substances of small or i n t e r mediate molecular s i z e . Under such circumstances spectra are sharp, reasonably s i m p l e , and can be i n t e r p r e t e d with r e l a t i v e ease. In case of humic substances the i n d i v i d u a l molecules are large and complex and d i s s i m i l a r to each o t h e r . The overlap of a multitude of absorption bands r e s u l t s in broad areas o f absorption rather than in d i s t i n c t absorption maxima. For t h i s reason, the s p e c t r a contain a s e v e r e l y reduced amount of useful information and, in a d d i t i o n , the i n t e r p r e t a t i o n o f the r e s i d u a l information is fraught with complexity. S o l u b i l i t y of humic


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compounds in s u i t a b l e solvents at high enough concentrations may present t e c h n i c a l problems. Molecular s i z e and a consequent lack of v o l a t i l i t y l a r g e l y prevents the a p p l i c a t i o n of mass s p e c t r o metry to i n t a c t humic compounds and, t h e r e f o r e , d i s c u s s i o n of mass spectra was omitted here. However, e s p e c i a l l y in combina t i o n with gas chromatography, t h i s technique can be very useful in c h a r a c t e r i z a t i o n of degradation or p y r o l y s i s products.


Spectrometric Studies

on P e s t i c i d e Residue - Humus i n t e r a c t i o n s .

The reduced a c t i v i t y o f many preemergence h e r b i c i d e s in high humus s o i l s (35.36) is a general i n d i c a t i o n of the a b i l i t y of humic compounds to bind man-made chemicals by various mechanisms. Such b i n d i n g t y p i c a l l y leads to increased p e r s i s t e n c e conbined with immobilization and decreased b i o l o g i c a l a c t i v i t y , but there are marked exceptions to t h i s r u l e . D i a l k y l phthalates (37,38) and DDT (39) were reported to be m o b i l i z e d by absorption to or complexing with water s o l u b l e f u l v i c a c i d s . The absorption mechanisms of h e r b i c i d e s to humic compounds was r e c e n t l y subject to a l u c i d review by Stevenson {j). He l i s t s ion exchange, Η - b o n d i n g , van der Vaals forces and c o o r d i n a t i o n through a metal ion as the prevalent modes o f attachment. The molecular s t r u c ture of h e r b i c i d e s determines the predominant mechanism, but more than one absorption mechanism may act on the same h e r b i c i d e .

humic acid

diquat

Figure 2. Charge transfer complex of diquat with humic acid. After Ref.40 B i p y r i d y l i u m H e r b i c i d e s . Herbicides of cat i o n i c n a t u r e , such as the two b i p y r i d y l i u m h e r b i c i d e s paraquat and diquat are bound by ion exchange r e a c t i o n s . This type o f binding was s u c c e s s f u l l y i n v e s t i g a t e d by Khan (40,41) u s i n g IR spectrometry. Based on the s h i f t o f C-H o u t - o f - p l a n e bending v i b r a t i o n s (from 815 cm" 1 to 825 cm" 1 f o r paraquat, from 792 cm"*1 to 765 cm*"1 for d i q u a t , r e s p e c t i v e l y ) he deduced the formation of a charge t r a n s f e r complex (Figure 2). Less b a s i c h e r b i c i d e s may undergo s i m i l a r reactions due to protonation (40).


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CI Figure 3. Attachment of s-triazines to humic acid by charge transfer and hydrogen bonding mechanisms. After Ref. 42

5-Trîazînes. H - b o n d î n g may take place between C=0 groups of the humic compounds and the secondary amino groups o f s - t r i a z i n e s (Figure 3 ) . Evidence f o r t h i s type of bonding was obtained from IR-spectra by S u l l i v a n and Felbeck (42). These workers reacted e t h a n o l i c s o l u t i o n s of humic acids with various t r i a z i n e h e r b i cides. Carbonyl absorption (1720 c m " 1 ) was reduced in every case and in a d d i t i o n , the bands at 2,900 c m " 1 (C-H) and 3,300 cm""1 (-0H) were reduced to varying degrees. New absorption bands appeared at 1,625 c n T 1 (C00~ of humic a c i d and/or C=N of the s - t r i a z i n e s ) and at 1,390 cm'" 1 ( i n d i c a t i v e of a s a l t of a carboxylic acid). From these data it was concluded that the primary binding s i t e s of the humic a c i d are carboxyl groups with a c o n t r i b u t i o n from phenolic h y d r o x y l s . Since in some cases the reduction in carbonyl groups (1,720 c m " 1 ) was not accompanied by an increase o f the C00" (1,625 cm" 1 ) and carboxyl s a l t (1,390 c m " 1 ) bands, it was suggested that C « 0 from quinones may have a l s o served as a binding s i t e . As there was no evidence for involvement of any other p o r t i o n o f the h e r b i c i d e molecule, the amino group was proposed as the a c t i v e binding s i t e of the s-triazines. A d d i t i o n a l work on the binding of s - t r i a z i n e s by other techniques (14,43) tended to support the above c o n c l u s i o n s . P o t e n t i a l A p p l i c a t i o n s . Aside of the reviewed IR work, we are not aware of studies on h e r b i c i d e absorption by humic material which used spectrometric techniques as t h e i r primary research tool. In the l i g h t of widespread speculations that free r a d i c a l s may play a r o l e in h e r b i c i d e residue b i n d i n g , the lack of studies on quenching o f humus ESR s i g n a l s by p e s t i c i d e residues is rather surprising. Another spectrometric technique that has great p o t e n t i a l in bound p e s t i c i d e residue research is the 13~C NMR (44),



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B O U N D A N D C O N J U G A T E D PESTICIDE

RESDDUES

13-C emits a r e l a t i v e l y weak NMR s i g n a l , but modern instrumenta t i o n using the F o u r i e r transformation p r i n c i p l e and computerprocessed m u l t i p l e scannings to f i l t e r out random n o i s e , have enormously increased the s e n s i t i v i t y and t i m e - e f f i c i e n c y o f the NMR instruments, and made t h e i r a p p l i c a t i o n f o r routine s t r u c t u r al i n v e s t i g a t i o n s p o s s i b l e . The natural abundance of 13"C is low (1.1%) but a r t i f i c i a l l y enriched 13"C compounds are becoming a v a i l a b l e for research. NMR-spectrometry of humic compounds with 13-C enriched bound p e s t i c i d e residues would give extremely useful information not otherwise o b t a i n a b l e , s i n c e it would e l u c i d a t e the actual b i n d i n g environment. In case of 14-C l a b e l i n g , t h i s is to be deduced i n d i r e c t l y from degradation s t u d i e s , g r e a t l y i n c r e a s i n g the danger o f a r t i f a c t s , s i d e r e actions and, consequently, erroneous or ambiguous r e s u l t s . The cost and l i m i t e d a v a i l a b i l i t y of s u i t a b l e NMR instruments is s t i l l an o b s t a c l e ; one that hopefully w i l l diminish with time. Çh11 ο roan i 1 i ne Res i dues. Covalent bond formation between p e s t i c i d e residues and humic compounds was not covered in Stevenson's review (7) and we have some ongoing work to report in t h i s area although, to d a t e , spectrometric techniques played only a minor r o l e in t h i s i n v e s t i g a t i o n . The b i o d é g r a d a t i o n of phenylami de h e r b i c i d e s r e s u l t s in release of a n i l i n e moieties (45,46,47). Studies with 14-C labeled 3,4-dichloroani1ine (DCA) and î^chToroani1ine, compounds representative o f the a n i l i n e moieties of several phenylamide h e r b i c i d e s , showed that these c h l o r o a n i 1 i n e s are subject to absorption as well as to covalent binding in soil ( 4 8 ) . The mineral part of soil plays only a minor r o l e in a b s o r p t i o n , the greater p o r t i o n o f the a n i l i n e s becomes attached to the soil organic matter. The nature of the non-covalent b i n d i n g was not i n v e s t i g a t e d in d e t a i l , but the b a s i c character of the a n i l i n e s suggests ion exchange and hydrogen bonding mechanisms. This r e v e r s i b l e a b s o r p t i o n , by b r i n g i n g the a n i l i n e molecules in intimate contact with the humic a c i d molecules is probably of importance a l s o for the subsequent covalent b i n d i n g . At 5 ppm a p p l i c a t i o n r a t e , 85-90% of the a p p l i e d a n i l i n e is c o v a l e n t l y bound w i t h i n 5 days. About 50% o f the bound DCA is released in unchanged form a f t e r a c i d or a l k a l i n e h y d r o l y s i s ; 50% of the r a d i o a c t i v i t y remains attached to the humic compounds. We have no d i r e c t evidence to prove that t h i s attached r a d i o a c t i v i t y s t i l l represents i n t a c t c h l o r o ani l i n e , but we b e l i e v e t h i s to be a reasonable assumption. To e l u c i d a t e the nature of both the hydrolyzable and the non-hydrolyzable a n i l i n e b i n d i n g , we compared IR-spectra of DCA, of humic a c i d and o f the DCA-humic a c i d complex (Figure 4 ) . The complex contained less than O.25% DCA by weight, and no obvious changes in spectrum, as compared to the untreated humic a c i d , could be d i s c e r n e d . Being unsuccessful in t h i s p r e l i m i n a r y spectrometric approach, we turned to model reactions in order to gain i n s i g h t i n t o the p o s s i b l e mechanisms o f the b i n d i n g .



19.

Soil

BARTHA AND H s u

4000

2500

267

Organic Matter

1800

1400 1200

800 750

KKX) 900

FREQUENCY, CM*

1

Figure 4. IR spectra of 3,4-dichloroaniline (DCA), humic acid (H), and their complex (H-DCA) (recordedinKBr)

Under ambient conditions we obtained hydrolyzable binding o f chloroani1ines to aldehydes and to qui nones in form o f a n i l s and ani1inoquinones, r e s p e c t i v e l y (Figure 5 ) . For the nonhydrolyzable binding o f a n i l i n e s , on t h e o r e t i c a l b a s i s , a number o f reactions (Figure 6) can be suggested from the

R-C=O

•

Η Ν-0?α 2

* aldehyde

5• (Mjuinone DCA

\&

.

f00m

in

water

DCA

R-C=N-^CI w

3.4-dicMoroonil

sœ- Çr^^A^' 2-iOCA).

2,5-di(DCA)-

quinone

quinone

Figure 5. Reactions of 3,4-dichloroaniline (DCA) with aldehydes and p-quinone


BOUND

A N D CONJUGATED

PESTICIDE

RESIDUES


a v a i l a b l e chemical l i t e r a t u r e (kS). We are now in the process of t e s t i n g some o f the more relevant reactions as models f o r the nonhydrolyzable attachment o f DCA to humic a c i d . Our p r e l i m i n a r y r e s u l t s are encouraging. Spectrometric methods w i l l undoubtedly be o f great use in the c h a r a c t e r i z a t i o n of these model r e a c t i o n products, and armed with s p e c i f i c i n f o r -

Heterocyclic Chemistry

Figure 6. Chemical reactions that lead to non-hydrolyzable attachment of antlines (49)


19.

BARTHA AND HSU

Soil Organic Matter

269


mation we may be more successful in the spectrometric i n v e s t i gation of DCA-humic a c i d complexes in the f u t u r e . While much work remains to be done, we b e l i e v e that the phenomenon of the c o v a l e n t l y bound a n i l i n e residues may resemble a very f a m i l i a r but in i t s d e t a i l s s t i l l obscure natural process of ammonia and amino a c i d attachment to humus that occurs both in hydrolyzable and in non-hydrolyzable forms (50), The mechanisms by which phenoxazines are formed from 4-methylcatechol and ammonia (Figure 7,A), and phenazines are formed from quinones and ammonia (Figure 7,B) r e s u l t i n g in h e t e r o c y c l i c nonhydrolyzable nitrogen (50 have very obvious analogies to the

Figure 7. Reactions of 4-methykatechol (A) and of p-quinone (B) with ammonia, leading to non-hydrolyzable incorporation of nitrogen into phenoxazine (A) and phenazine (B) type compounds. After Ref. 51

type o f reactions we propose for ani1ine b i n d i n g . If we look at DCA as ammonia tagged by a s t a b l e and e a s i l y recognizable chlorophenyl r i n g , we can imagine that our a p p l i e d research on DCA binding to humus may eventually c o n t r i b u t e to the understanding of much more fundamental aspects of soil chemistry. LITERATURE CITED.

1.

2.

Swain, F. M. "Geochemistry of Humus". In: Organic Geochemistry. (I.A. Berger, ed.) pp. 81-147, Pergamon Press, New York, 1963. Kononova, M. M. "Soil Organic Matter." Pergamon Press, New York, 1966.


270

3. 4. 5.


6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28. 29. 30. 31. 32.

BOUND AND CONJUGATED PESTICIDE RESIDUES

Haider, K. and Martin, J. P. Soil. Biol. Biochem. (1970) 2:145-156. Martin, J. P. and Haider, K. Soil Sci. (1971) 111:54-63. Stevenson, F. J. In: C. A. Black (ed.) "Methods of Soil Analysis" pp. 1409-1421, American Society of Agronomy, Madison, 1965. Stevenson, F. J. and Butler, J. H. In: Organic Geochemistry - Methods and Results. (G. Eglington and M. T. J. Murphy, eds.) pp. 534-557, Springer, New York 1969. Stevenson, F. J. Bioscience (1972) 22:643-650. Tan, Κ. H. Soil Sci. Soc. Amer. Proc. (1975) 39:70-73. Mathur, S. P. Soil Sci. (1972) 113:136-139. Felbeck, G. T. J r . Adv. Agron. (1965) 17:327-368. Felbeck, G. T. J r . Soil Sci. (1971) 111:42-48. Flaig, W. Soil Sci. (1971) 111:19-33. Haworth, R. D. Soil Sci. (1971) 111:71-78. Hayes, M.H.B. Res. Rev. (1970) 32:131-174. McLaren, A. D., Pukite, A. H., and Barshad, I. Soil Sci. (1975) 119:178-180. Haider, Κ., Frederick, L. R. and Flaig, W. Plant and Soil (1965) 22:49-64. Ladd, J. N. and Butler, J. H. A. Austral. J. Soil Res. (1966) 4:41-54. Schnitzer, M. In:"Soil Biochemistry" Vol. 2 (A. D. McLaren and J. Skujins, eds. pp. 60-95, Dekker, New York, 1971. Williams, D. H. and Fleming, I. "Spectroscopic Methods in Organic Chemistry" McGraw H i l l , London 1973. Nakanishi, K. "Infarared Absorption Spectroscopy Practical." Holden-Day, San Francisco, 1962. Stevenson, F. J. and Goh, Κ. M. Soil Sci.(1972) 113:334-345. Schnitzer, M. and Skinner, I. M. Soil. Sci. (1965) 99:278-284. Stevenson, F. J. and Goh, Κ. M. Soil Sci. (1974) 117: 34-41. Schnitzer, M. Soil Sci. (1974) 117:94-102. Schnitzer, M. and Skinner, S. I. M. Soil Sci. (1963) 96:86-93. Schnitzer, M. and Hoffman, I. Soil Sci. Soc. Amer. Proc. (1964) 28:520-525. Steel ink, C. and T o l l i n , G. In: Soil Biochemistry (A. D. McLaren and G. Peterson, eds.) pp. 147-169, Marcel Dekker, New York, 1967. Schnitzer, M. and Skinner, S. I. M. Soil Sci. (1969) 108:383-390. Atherton, Ν. M., Cranwell, P. Α., Floyd, A. J., and Haworth, R. D. Tetrahedron (1967) 23:1653-1667. Cheshire, M.V. and Cranwell, P.A. J. Soil Sci. (1972)23:424-430. Haworth, R. D. Soil Sci. (1971) 111:71-79. Bible, R. H. J r . "Interpretation of NMR Spectra" Plenum Press. New York, 1965.


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33. 34. 35. 36. 37.


38. 39. 40. 41. 42. 43. 44.

45.

46.

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AND

Hsu

Soil

Organic Matter

271

Barton, D. H. R. and Schnitzer, M. Nature (1963) 198:217218. Felbeck, G. T. J r . Soil Sci. Soc. Amer. Proc. (1965) 29:48-55. Bailey, G. W. and White, J. L. J. Agr. Food Chem. (1964) 12:324-333. Upchurch, R. P. Res. Rev. (1966) 16:46-85. Matsuda, K. and Schnitzer, M. Bull. Env. Contam. Toxicol. (1971) 6:200-204. Ogner, G. and Schnitzer, M. Science (1970) 170:317-318. Ballard, T. M. Soil Sci. Soc. Amer. Proc. (1971) 35:145-147. Khan, S. U. Can. J. Soil Sci. (1973) 53:199-204. Khan, S. U. J. Env. Qual. (1974) 3:202-206. Sullivan, J. D. Jr. and Felbeck, G. T. J r . Soil Sci. (1968) 106:42-52. L i , G.-C. and Felbeck, G. T. J r . Soil Sci. (1972) 113:140-148. Levy, G. C. and Nelson, G. L. "Carbon 13 Nuclear Magnetic Resonance for Organic Chemists" Wiley-Interscience, New York, 1972. Geissbühler, H. In: Degradation of Herbicides. (P. C. Kearney and D. D. Kaufman, eds.) pp. 79-111, Dekker, New York, 1969. Herrett, R. A. In: Degradation of Herbicides. (P. C. Kearney and D. D. Kaufman, eds.) pp. 113-145, Dekker, New York, 1969. Bartha, R. and Pramer, D. Adv. Appl. Microbiol. (1970) 13:317-341. Hsu, T.-S. and R. Bartha. Soil Sci. (1974) 116:444-452. Joule, J. A. and Smith, G. F. "Heterocyclic Chemistry" van Nostrand Reinhold, London, 1972. Nömmik, H. Plant and Soil (1970) 33:581-595. Lindbeck, M. R. and Young, J. L. Anal. Chim. Acta (1965) 32:73-80.

ACKNOWLEDGEMENT: This paper of the Journal S e r i e s , New Jersey A g r i c u l t u r a l Experiment S t a t i o n , New Brunswick, N.J., was supported by RR,NE-63 and Hatch funds.


Spectroscopic Characterization of Soil Organic Matter

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