Bound and Conjugated Pesticide Residues

In the course of studying the decomposition of radioactive, foreign organic compounds in soil, we invariably find radioactivity associated with the so...
0 downloads 0 Views 976KB Size
20 Classification of Bound Residues Soil Organic Matter:

Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 9, 2018 at 13:19:12 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Polymeric Nature of Residues in Humic Substance R. W. MEIKLE, A. J. REGOLI, and N. H . KURIHARA Dow Chemical U.S.A., Ag-Organics Research, 2800 Mitchell Drive, Walnut Creek, Calif. 94598 D. A. LASKOWSKI Dow Chemical U.S.A., Ag-Organics Department, Midland, Mich. 48640

In the course of studying the decomposition of radioactive, foreign organic compounds in soil, we invariably find radioactivity associated with the soil organic matter. This associated radioactivity has generally not been identified because of the d i f f i c u l t y of working with the material. As the incubation time for the organic compound in soil increases, the amount of radioactivity in the soil organic matter also generally increases. Consequently, it has become increasingly important to have some notion of how this radioactivity is combined structurally with the soil organic matter. The organic matter of soils consists of a mixture of plant and animal products in various stages of decomposition, of substances synthesized biologically and/or chemically from the breakdown products, and of microorganisms and small animals and their decomposing remains. To simplify this very complex system, organic matter is usually divided into two groups: (a) nonhumic substances and (b) humic substances. Nonhumic substances include compounds of known chemical characteristics. To this class of compounds belong carbohydrates, proteins, peptides, amino acids, fats, waxes, resins, pigments and other low-molecular-weight organic substances. In general, these compounds are relatively easily attacked by microorganismsinthe soil and have a relatively short survival rate. The bulk of the organic matter in most soils consists of humic substances. These are amorphous, brown or black, hydrophilic, acidic, polydisperse substances of molecular weights ranging from several hundreds to tens of thousands. Based on their solubility in a l k a l i and acid, humic substances are usually divided into three main fractions: (a) humic acid (HA), which is soluble in dilute alkaline solution but is precipitated by acidification of the alkaline extract; (b) f u l v i c acid (FA), which is that humic fraction which remains in the aqueous acidified solution, i.e., it is s o l uble in both acid and base; and (c) the humic fraction that cannot be extracted by dilute base and acid, which is referred to as humin. 272

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

20.

MEIKLE ET AL.

273

Bound Residues in Soil Organic Matter

There is i n c r e a s i n g evidence t h a t the chemical s t r u c t u r e and prope r t i e s o f the humin f r a c t i o n a r e s i m i l a r t o those o f HA, and t h a t i t s i n s o l u b i l i t y a r i s e s from the firmness w i t h which it combines w i t h i n o r g a n i c soil and water c o n s t i t u e n t s . Data a v a i l a b l e a t t h i s time suggest t h a t s t r u c t u r a l l y the three humic f r a c t i o n s are s i m i l a r to each o t h e r , but t h a t they d i f f e r in molecular weight, u l t i m a t e a n a l y s i s , and f u n c t i o n a l group content. The FA f r a c t i o n has a lower molecular weight but a higher content o f oxygen-containing f u n c t i o n a l groups per u n i t weight than do HA and the humin f r a c t i o n . While the f r a c t i o n a t i o n scheme is a r b i t r a r y — the f r a c t i o n s a r e s t i l l m o l e c u l a r l y heterogeneous — it has nonetheless been w i d e l y accepted. The a b i l i t y o f s y n t h e t i c c r o s s - l i n k e d polydextran g e l s t o separate molecules by t h e i r molecular s i z e has become i n c r e a s i n g l y important in the study o f polymeric substances ( 1 ) . This r e p o r t d e s c r i b e s the use o f Sephadex® g e l s t o f r a c t i o n a t e r a d i o a c t i v e humic substances e x t r a c t e d from soil a f t e r the soil has been incubated w i t h ^ j r a d i o a c t i v e d i t a l i m f o s f u n g i c i d e , 0 , 0 - d i e t h y l phthalimido-1C-phosphonothioate. Experimental Three soil samples were used in t h i s study and t h e i r p h y s i c a l p r o p e r t i e s are d e s c r i b e d in Table I . Mechanical analyses were c a r r i e d out u s i n g the hydrometer method ( 2 ) . Soil pH was measured in water a t a 1:1 soil:solution r a t i o w i t h a g l a s s e l e c t r o d e assembly ( 3 ) . Organic matter content of soil was determined u s i n g a wet combustion method ( 4 ) . The moisture content of the s o i l s a t 1/3 bar t e n s i o n was a l s o determined ( 5 ) . TABLE I Some p r o p e r t i e s o f the s o i l s used in the study o f d i t a l i m f o s decomposition. Sand, %

Silt, %

Clay, %

Organic carbon, %

Soil moisture content at 1/3 bar tension

pH

Loam, D a v i s , California

46

35

18

O.86

21.75

6.4

Sandy Loam, No. Dakota

66

22

12

2.2

22.52

7.3

S i l t y Clay Loam, Geneseo, Illinois

14

54

32

4.2

26.31

5.8

Soil t e x t u r a l classification and source

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

274

BOUND

AND

C O N J U G A T E D PESTICIDE

RESIDUES

These s o i l s were incubated w i t h r a d i o a c t i v e d i t a l i m f o s and then analyzed f o r t h i s compound and i t s decomposition products a t a p p r o p r i a t e times. The r e s u l t s of t h i s study w i l l be r e p o r t e d elsewhere. The s o i l s were first e x t r a c t e d w i t h a c i d i f i e d ether to remove e x t r a c t a b l e r a d i o a c t i v e compounds, r i n s e d w i t h water, and d r i e d at ambient temperature. Humic substances were obtained from the e x t r a c t e d s o i l s by shaking 2-g. soil samples f o r 18 hours a t ambient temperature w i t h 3g. of DOWEX® A - l c h e l a t i n g r e s i n (sodium form) of 50 t o 100 mesh and 25 ml. of water. The t o t a l nominal c a p a c i t y of the r e s i n was 2.6 meq./3g. The soil suspensions were c e n t r i f u g e d a t 12,000 χ G. A l i q u o t s of these r a d i o a c t i v e s o l u t i o n s were examined by g e l chromatography, s e p a r a t i o n i n t o humin-humic a c i d - f u l v i c a c i d f r a c t i o n s , and d i a l y s i s . G e l chromatography. The p o l y d e x t r a n g e l s (Sephadex G-50 and G-100) were prepared as recommended by the manufacturer ( 6 ) . The column used was 2.6 χ 70 cm. The v o i d volume (V ) was determined e m p i r i c a l l y by u s i n g Blue Dextran 2000 (Pharmacia). V as shown on the f i g u r e s i n d i c a t e s the first excluded Blue Dextran f r a c t i o n . The t o t a l bed volume (V ) was obtained by water c a l i b r a t i o n of the column b e f o r e packing trie bed. V f o r the G-50 columns was 248 ml and f o r the G-100 column, 266 ml. The g e l d i d not compact d u r i n g e l u t i o n . F i v e - m l . f r a c t i o n s of column e f f l u e n t were c o l l e c t e d . The f l o w r a t e was maintained a t O.5 ml/ min. The b u f f e r systems used as e l u a n t were O.025M sodium borate (pH 9.1) f o r the G-50 g e l and O.1M sodium hydroxide f o r the G-100 gel. The f r a c t i o n a t i o n ranges of the g e l s are r e p o r t e d (6) to be as f o l l o w s : G-50, s o l u t e s w i t h molecular weights from 500 to 10,000; c o r r e s p o n d i n g l y f o r G-100, s o l u t e s w i t h molecular weights from 1,000 to 100,000. These v a l u e s are based on c a l i b r a t e d dextrans (Pharmacia). Over a c o n s i d e r a b l e range, the e l u t i o n volume (V ) of a p o l y ­ mer from a dextran g e l column is approximately a l i n e a r f u n c t i o n of the l o g a r i t h m of the molecular weight ( 2 j j B , 2 , 1 0 ) . The g e l columns were c a l i b r a t e d f o r molecular weight u s i n g samples of c a l i b r a t e d dextrans (Dextran T®, Pharmacia). Dextran in the e l u t e d f r a c t i o n s was determined by the method of Dubois, et a l (11). A l i n e a r r e g r e s s i o n of I n molecular weight on the " g e l a f f i n i t y c o n s t a n t " (K ) allowed the c a l c u l a t i o n of apparent molecular weights of eYuted r a d i o a c t i v i t y from the e l u t i o n volume (V ). The constant, Κ , is d e f i n e d by Laurent and K i l l a n d e r ( 9 ) and it is r e l a t e S to V as f o l l o w s : Κ — e av (V - V ) / ( V -V ). Κ is independent of column geometry and packing d e n s i t y . TrîYs constant we d e f i n e as the " g e l a f f i n i t y constant" where i t s magnitude bears a d i r e c t r e l a t i o n s h i p to the a f f i n i t y of the e l u t e d molecule f o r the g e l . e

Q

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

20.

MEIKLE

ET AL.

Bound Residues in Soil Organic Matter

275

F r a c t i o n a t i o n of soil e x t r a c t s . A t r a d i t i o n a l f r a c t i o n a t i o n of the soil r e s i n e x t r a c t s w i t h a l k a l i i n t o humic a c i d ( p r e c i p i t a t e d from a l k a l i n e s o l u t i o n by a c i d ) , f u l v i c a c i d (that p a r t o f the a l k a l i n e s o l u t i o n not p r e c i p i t a t e d by a c i d ) , and humin (organic m a t e r i a l not s o l u b l e in a l k a l i ) was performed as described by S c h n i t z e r and Kahn (12). D i a l y s i s o f soil e x t r a c t s . The soil r e s i n e x t r a c t s were a l s o submitted t o d i a l y s i s a g a i n s t running tap water in c e l l u l o s e acetate a t ambient temperature. R e s u l t s and D i s c u s s i o n Of the l a r g e number o f e x t r a c t a n t s that have been t e s t e d , d i l u t e aqueous sodium hydroxide has been the most commonly used and q u a n t i t a t i v e l y the most e f f e c t i v e reagent f o r e x t r a c t i n g humic substances from s o i l s . When the incubated s o i l s l i s t e d in Table I were e x t r a c t e d w i t h hot IN sodium hydroxide s o l u t i o n or w i t h DOWEX A - l c h e l a t i n g r e s i n and water, a f t e r first being e x t r a c t e d w i t h a c i d i f i e d ether t o remove e x t r a c t a b l e r a d i o a c t i v e compounds, we found that the two e x t r a c t i o n methods were e q u a l l y e f f i c i e n t a t removing r a d i o a c t i v e humic substances. These r e s u l t s are shown in Table I I . TABLE I I E x t r a c t i o n of r a d i o a c t i v i t y from s o i l s u s i n g DOWEX A - l Resin and IN sodium h y d r o x i d e -

Chelating

Soil t e x t u r a l classification and source Loam, D a v i s ,

Incubation c o n d i t i o n s time,days temp,°C

R a d i o a c t i v i t y , % in..— resin NaOH extract extract

California

56

15

95

82

40

35

76

80

33

25

82

88

Loam, D a v i s , California Sandy Loam, No. Dakota S i l t y Clay Loam, Geneseo, I l l i n o i s

175

15 91 85 Ave. 86 84 a/ S o i l s had been incubated w i tSt'd. h d i t ea rl ri om rf o s - 4 C (5 ppm) and sub2 sequently e x t r a c t e d w i t h ether/O.IN HC1 (1.5/1.0 v / v ) . b/ These values a r e % o f that present in soil a f t e r a c i d i f i e d ether e x t r a c t i o n . The i n i t i a l v a l u e s were 37%, 31%, 35% and 30% o f the a p p l i e d r a d i o a c t i v i t y in the s o i l s as l i s t e d in the table.

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

276

BOUND AND CONJUGATED PESTICIDE RESIDUES

Resin e x t r a c t i o n of soil r e s u l t s in e f f i c i e n t removal of p o l y v a l e n t c a t i o n s t h a t b i n d o r g a n i c substances in soil. T h i s i n c r e a ses the d l s p e r s i t y of humic substances and a l s o i n c r e a s e s t h e i r s o l u b i l i t y by d i s r u p t i n g the hydrogen bonds of the f i x e d m e t a l l i c c a t i o n s . Sodium hydroxide accomplishes much the same t h i n g but is a more severe reagent. Thus, e x t r a c t i o n of s o i l s w i t h a c h e l a t i n g r e s i n w i l l u s u a l l y r e s u l t in l e s s degradation t o soil organic matter (13). When a l i q u o t s of the r e s i n soil e x t r a c t s were submitted to g e l chromatography the r e s u l t s shown in F i g u r e s 1 to 5 were o b t a i n ed. In each case, a p o r t i o n of the r a d i o a c t i v e m a t e r i a l placed on the column was e l u t e d in two main f r a c t i o n s . The apparent molec u l a r weights and percent recovery based on a p p l i e d r a d i o a c t i v i t y are i n d i c a t e d on the f i g u r e s . I t is recognized t h a t the molecular weights shown in these f i g u r e s are o n l y approximate. Manufacturers use dextrans to c a l i b r a t e t h e i r p o l y d e x t r a n c r o s s - l i n k e d g e l s . I f the humic substance molecules are more asymmetric than the dextrans used f o r c a l i b r a t i o n , as seems l i k e l y , then any p a r t i c u l a r grade of g e l w i l l exclude lower molecular weight humic substances than the nominal v a l u e would i n d i c a t e . Put another way: For equal molecular weight substances, a h i g h e r degree of molecular asymmetry is e q u i v a l e n t to a l a r g e r s i z e . Thus, the apparent molecular weight v a l u e s in these f i g u r e s are probably h i g h . The evidence c l e a r l y i n d i c a t e s there are a t l e a s t two r a d i o a c t i v e polymer f r a c t i o n s in each of the soil samples. The North Dakota soil (Figure 3) appears to have f i v e a d d i t i o n a l r a d i o a c t i v e f r a c t i o n s but t h i s degree of s e p a r a t i o n would need c o n f i r m i n g . The range of apparent molecular weights f o r these polymer f r a c t i o n s is 2100 t o >10,000. However, when the North Dakota soil sample e x t r a c t was submitted to g e l chromatography u s i n g a g e l w i t h l e s s c r o s s - l i n k i n g , thus extending the e x c l u s i o n l i m i t of the g e l , it was found t h a t the h i g h molecular weight f r a c t i o n in the e x t r a c t could be assigned an apparent molecular weight >100,000 (Figure 5 ) . The apparent molecular weight range of humus is reported to be 600 to 300,000 (14). To f u r t h e r c h a r a c t e r i z e the r a d i o a c t i v e polymeric substances in the DOWEX A - l r e s i n e x t r a c t s of soil, a sample of the North Dakota soil e x t r a c t was separated i n t o f u l v i c a c i d , humic a c i d and humin u s i n g the t r a d i t i o n f r a c t i o n a t i o n scheme d e s c r i b e d by S c h n i t z e r and Kahn (12). The p r o p o r t i o n of r a d i o a c t i v i t y in humic a c i d to that in f u l v i c a c i d was 1.8:1.O. A hot, IN sodium hydroxide e x t r a c t i o n of t h i s same soil, f o l l o w e d by s e p a r a t i o n i n t o humic a c i d and f u l v i c a c i d , r e s u l t e d in a r a d i o a c t i v e humic a c i d - f u l v i c a c i d p r o p o r t i o n of O.6:1.O. In the one case, where soil was e x t r a c t e d w i t h a c h e l a t i n g r e s i n , the r a d i o a c t i v e humic a c i d f r a c t i o n was h i g h r e l a t i v e to the r a d i o a c t i v e f u l v i c a c i d ; in the other — e x t r a c t i o n w i t h hot sodium hydroxide — the r a d i o a c t i v e humic a c i d f r a c t i o n was low. The reason f o r t h i s r e v e r s a l is that hot sodium hydroxide causes g r e a t e r degradation of the humic a c i d polymers (high molecular weight) than does the c h e l a t i n g r e s i n . The r e s u l t i n g

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

Bound Residues in Soil Organic Matter

MEIKLE ET AL.

60

1

40

I DU"

15% = 0 IC av mw , >10,000 / 2% g Κ = O.65 ^ av . ^mw = 3200

U uU

j

y * |] IP

y «•

U

UL'

20 +

ν

ν ο

79 ι

t

t

1

158 237 E l u t i o1n volume 1-(ml)

316

Figure 1. Elution diagram for radioactive polymersinchelating resin extract of Davis, Calif,soil(15°C): Sephadex G-50, O.025M sodium borate, pH 9.1

16% Κ =0 av mw = >10,000

t

70

140 210 280 E l u t i o n volume (ml)

Figure 2. Elution diagram for radioactive polymersinchelating resin extract of Davis, Calif,soil(35°C): Sephadex G-50, O.025M sodium borate, pH 9.1

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

278

BOUND AND CONJUGATED PESTICIDE RESIDUES

12%

•9

6%

138 207 276 E l u t i o n volume (ml)

Figure S. Elution diagram for radioactive polymersinchelating resin extract of North Dakotasoil(25°C): Sephadex G-50, O.25M sodium borate, pH 9.1

80

160 240 320 E l u t i o n volume (ml)

Figure 4. Elution diagram for radioactive polymersinchelating resin extract of Illinoissoil(15°C): Sephadex G-50, O.025M sodium borate, pH 9.1

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

20.

MEIKLE

ET AL.

Bound Residues in Soil Organic Matter

279

decomposition products have a lower molecular weight and tend to f r a c t i o n a t e as f u l v i c a c i d s . Dormar (15) has shown that e x t r a c t i o n of organic matter w i t h c h e l a t i n g r e s i n provides humic substances w i t h minimum a l t e r a t i o n . The humic and f u l v i c a c i d f r a c t i o n s separated from the DOWEX A - l r e s i n e x t r a c t o f the North Dakota soil were each submitted t o g e l chromatography and the r e s u l t s appear in Figures 6 and 7. We see h i g h molecular weight r a d i o a c t i v e m a t e r i a l in the humic a c i d f r a c t i o n and it comprises the major p a r t of the moveable r a d i o ­ a c t i v i t y in t h i s f r a c t i o n . The lower molecular weight r a d i o ­ a c t i v e m a t e r i a l appears in the moveable p o r t i o n of the f u l v i c a c i d f r a c t i o n w i t h some overlap o f 2300 Dalton polymers i n t o the humic a c i d f r a c t i o n . Thus the molecular weight d i s t r i b u t i o n of r a d i o a c t i v e f r a c t i o n s in the soil e x t r a c t s f o l l o w s the p a t t e r n expected f o r f r a c t i o n a t i o n of humic substances. When a l i q u o t s o f the DOWEX A - l r e s i n soil e x t r a c t s of each soil were d i a l y z e d through cellophane ( c e l l u l o s e acetate) an average 53% o f the r a d i o a c t i v e m a t e r i a l was r e t a i n e d by the membrane. That p o r t i o n of the n o r t h Dakota soil e x t r a c t r e t a i n e d by the cellophane membrane was submitted t o g e l chromatography using g e l G-50. The r e s u l t s are shown in Figure 8. We see that the r a d i o a c t i v e polymers w i t h Κ >0, apparent molecular weight, 10,000 were r e t a i n e d and appear in Figure 8 (see Figure 3 f o r comparison). This is another demons­ t r a t i o n that a p o r t i o n of the r a d i o a c t i v i t y in the r e s i n e x t r a c t s of soil is a s s o c i a t e d w i t h n o n - d i a l y z a b l e , h i g h molecular weight humic substances. The recovery o f r a d i o a c t i v e m a t e r i a l from the Sephadex g e l columns v a r i e d from 17% to 31% of that put on the column. I n the case o f the d i a l y s i s experiment, only 17% of the r a d i o a c t i v i t y a p p l i e d t o the column appear in the e l u a t e as a s i n g l e peak in F i g u r e 8. Apparently a l a r g e p a r t (83%) o f the high molecular weight (>10,000) r a d i o a c t i v e m a t e r i a l is in some way s t r o n g l y adsorbed by the g e l . When t h i s g e l was removed from the column and segments were assayed f o r r a d i o a c t i v i t y , 93% of the r e t a i n e d a c t i v i t y was found in the first i n c h and 100% in the first 5 inches. Sephadex gels are known to adsorb some p r o t e i n s (16), aromatic and h e t e r o c y c l i c compounds (17), and humum molecules (14). This phenomenon probably accounts f o r the low recovery o f r a d i o a c t i v e m a t e r i a l from the g e l columns used in our work. ^ I t has been shown in our work w i t h d i t a l i m f o s - C / s o i l that the s p e c i f i c r a d i o a c t i v i t y of the humus f r a c t i o n s , as dpm/mg. o f carbon, bears an i n v e r s e r e l a t i o n s h i p to molecular weight. The data showing the r e l a t i o n s h i p are reproduced as Table I I I . These changes in the s p e c i f i c a c t i v i t y of the soil organic carbon f r a c t i o n s are c o n s i s t e n t w i t h the concept that the more s o l u b l e f r a c t i o n s have a more r a p i d turnover. Thus, if humin represents organic carbon that is formed and broken down more s l o w l y than f u l v i c a c i d , f o r example, then a s m a l l e r p r o p o r t i o n of the t o t a l carbon of the humin w i l l be "new" carbon c o n t a i n i n g

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

BOUND AND CONJUGATED PESTICIDE RESIDUES

16%

11%

a

57

114 171 228 E l u t i o n volume (ml)

Figure 5. Elution diagram for radioactive polymersinchelating resin extract of North Dakotasoil:Sephadex G-100,O.1Msodium hydroxide

K = O.85 av m = 2300

60 +

40 4-

49

98 147 196 E l u t i o n volume (ml)

Figure 6. Elution diagram for radioactive humic acid from chelating resin extract of North Dakota soil: Sephadex G-50,O.025Usodium borate, pH 9.1

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

Bound Residues in Soil Organic Matter

60

g. ο

4.

9% 8% Κ O.88 Κ = 1.03 av av mw = 2100 raw = 1700 χ

40

,

/

120 180 E l u t i o n volume (ml) Figure 7. Elution diagram for radioactive fulvic acid from chelat­ ing resin extract of North Dakota soil: Sephadex G-50, O.025M sodium borate, pH 9.1

50

100 150 E l u t i o n volume (ml)

200

Figure 8. Elution diagram for radioactive polymers retained by membrane after dialysis of chefoting resin extract of North Dakota soil: Sephadex G-50,O.025M sodium borate, pH 9.1

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

282

BOUND

AND

C O N J U G A T E D PESTICIDE

RESIDUES

TABLE I I I S p e c i f i c a c t i v i t i e s , dpm/mgC, f o r f r a c t i o n a t e d soil organic matter a f t e r i n c u b a t i o n of s o i l s w i t h d i t a l i m f o s C.

FRACTION I n c r e a s i n g molecular weight ( f u l v i c a c i d ) humic a c i d )

Soil Sample Davis 15°

soil,

Davis 35°

soil,

> (humin)

2080

2074

467

2162

1904

481

No. Dakota soil, 25°

641

582

281

Illinois soil, 15°

638

281

46

Formation of humic substances in soil is a dynamic process o c c u r r i n g through the a c t i o n of microbes on p l a n t m a t e r i a l (18). Macromolecules are formed at the expense of carbohydrates of p l a n t o r i g i n . These macromolecules i n c l u d e b a c t e r i a l gums, a l g i n i c a c i d , p e c t i c a c i d , and other l e s s w e l l - d e f i n e d polymeric c a r b o x y l i c a c i d s . Aromatic polyphenols formed by way of o x i d a t i o n of quinones can condense w i t h amino a c i d s to u l t i m a t e l y g i v e h u m i c - l i k e substances. Basidiomicetes as w e l l as other microscopic f u n g i have been found to degrade l i g n i n to form appreciable^ amounts of humic a c i d - l i k e polymers (19). P h e n o l i c u n i t s from C-labeled phenolase l i g n i n have been shown to be i n c o r p o r a t e d i n t o f u n g i - s y n t h e s i z e d polymers (20). The general consensus appears to be t h a t there is a genetic r e l a t i o n between the v a r i o u s humic substances. F u l v i c a c i d is considered to represent poly-condensation m a t e r i a l formed from s i m p l e r molecules. C o n t i n u a t i o n of p o l y m e r i z a t i o n and chemical m o d i f i c a t i o n leads to the l e s s s o l u b l e humic a c i d and e v e n t u a l l y to i n s o l u b l e humin, thought to have the h i g h e s t molecular weight and most r e s i s t a n t s t r u c t u r e . The e a r l i e r , and probably more r a p i ^ y formed, f u l v i c a c i d s w i l l be c l o s e r to e q u i l i b r i u m w i t h the C p o o l of simpler and s m a l l e r molecules than w i l l m a t e r i a l s f a r t h e r down the sequence and would, t h e r e f o r e , have a h i g h e r s p e c i f i c acj£vity. During t h i s sequence of r e a c t i o n s the incorporated C becomes an i n t e g r a l p a r t of the molecular s t r u c t u r e without r e c o g n i z a b l e r e l a t i o n s h i p to the parent molecule from which it is d e r i v e d . The r a t e of humin degradation is very slow (21)• Sorenson (22) s t u d i e d the degradation of l a b e l e d glucose and c e l l u l o s e in

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

20.

MEIKLE ET

AL.

Bound Residues in Soil Organic Matter

283

three s o i l s . A f t e r a r a p i d i n i t i a l b r e a ^ o w n , h a l f - l i v e s of 5 to 9 years were r e p o r t e d f o r the remaining C to be degraded. These data imply t h a t , even w i t h r e a d i l y metabolized compounds, i n c o r ­ p o r a t i o n i n t o humic substances occurs and l i m i t s the extent to which complete degradation to C0~ proceeds. L i k e w i s e , p e s t i c i d e molecules degrade and the products u l t i m a t e l y become i n c o r p o r a t e d in humic m a t e r i a l s . These macromolecules so formed are i n d i s t i n ­ guishable from those d e r i v e d from carbon compounds n a t u r a l to soil. Other authors have demonstrated the formation of humin from r e a d i l y decomposable o r g a n i c compounds (23,24). In summary, we have shown that when an o r g a n i c compound i n c o r p o r a t e d in soil is decomposed, a p a r t of the decomposition products u l t i m a t e l y become a s s o c i a t e d w i t h the soil organic m a t e r i a l . These products are sometimes r e f e r r e d to as "bound m a t e r i a l " . In r e a l i t y a l a r g e p a r t of the soil o r g a n i c matter can be s o l u b i l i z e d w i t h reagents such as hot aqueous sodium hydroxide or DOWEX A - l c h e l a t i n g r e s i n and water. The l a t e r is p r e f e r r e d because it is much l e s s d e s t r u c t i v e to the organic matter. F u r t h e r , we have shown t h a t a p a r t of the decomposition products are combined w i t h the e x t r a c t e d o r g a n i c m a t e r i a l in such a way t h a t the products are an i n t e g r a l p a r t of the p o l y ­ molecular s t r u c t u r e of the o r g a n i c m a t e r i a l . F i n a l l y , we have shown t h a t the f r a c t i o n s of soil organic m a t e r i a l , commonly known as f u l v i c a c i d , humic a c i d and humin, c o n t a i n i n c o r p o r a t e d decomposition products. These macromolecules can be separated i n t o r a d i o a c t i v e f r a c t i o n s having apparent molecular weights ranging from 2100 to >100,000. LITERATURE CITED 1. 2.

3.

4.

5.

6.

7. 8.

Altgelt, Κ. H., and Segal, L., "Gel Permeation Chromatography", Dekker, New York (1971). Day, P. R., Particle fractionation and particle-size analysis, pages 545-566 in C. A. Black (ed), "Methods of Soil Analysis", Amer. Soc. of Agron., Inc., Madison, Wisc. (1965). Peech, Μ., Hydrogen-ion activity, pages 914-925 in C. A. Black (ed), "Methods of Soil Analysis", Amer. Soc. of Agron., Inc., Madison, Wisc. (1965). Allison, L. E., Organic Carbon, pages 1367-1378 in C. A. Black (ed), "Methods of Soil Analysis", Amer. Soc. of Agron. Inc., Madison, Wisc. (1965). Richards, L. Α., Physical condition of water in soil, pages 131-137 in C. A. Black (ed), "Methods of Soil Analysis", Amer. Soc. of Agron., Inc., Madison, Wisc. (1965). Anon., "Sephadex-gel f i l t r a t i o n in theory and practice", Pharmacia Fine Chemicals, Inc., 800 Centennial Ave., Piscataway, N. J. 08854. (1966). Carnegie, P. R., Nature (1965) 206, 1128. Andrews, P., Biochem. J. (1965), 96, 595.

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

284

9. 10. 11. 12. 13.

14. 15. 16 17. 18. 19.

20. 21

22. 23. 24.

BOUND AND CONJUGATED PESTICIDE RESIDUES

Laurent, T. C., and Killander, J., J. Chromatog. (1964), 14, 317. Carnegie, P. R., Biochem. J. (1965) 95, 9P. Dubios, M., G i l l e s , Κ. Α., Hamilton, J. Κ., Rebers, P. Α., and Smith, F., Anal. Chem. (1956), 28, 350. Schnitzer, Μ., and Kahn, S. U., "Humic Substances in the Environment", p. 17, Dekker, New York, (1972). Bremner, J. Μ., Organic nitrogen in s o i l s , pages 93-149 in W. V. Bartholomew and F. E. Clark (eds), "Soil Nitrogen", Amer. Soc. Agron., Madison, Wisc. (1965). Gjessing, E. T., Nature (1965), 197, 1091. Dormaar, J. F., Bull. Ass. Fr. Etude Sol (1973), (2), 71-9. Chem. Abstr. (1974), 80, 107113t. Glazer, Α. Ν., and Wellner, D., Nature (1962), 194, 862. Gelotte, B., J. Chromatography (1960), 3, 330. McLaren, A. D., Science (1961), 141, 3586. Hurst, Η. M. and Burger, W. Α., Lignin and Humic Acids, pages 260-286 in "Soil Biochemistry", McLaren, A. D., and Peterson, G. H. (eds), Dekker, New York (1967). Martin, J. P., and Haider, Κ., Soil Sci. (1971), 111, 54. Stevenson, I. L., Biochemistry of Soil, Page 242, in "Chemistry of the Soil No. 160", Bear, F. E. (ed), Reinhold Publ. Corp., New York (1964). Sorenson, L. H., Soil Biol. Biochem. (1972), 4, 245. Chekalar, Κ. I., and Illyuvieva, V. P., Pochvovedenie No. 5, pp. 40-5 (1962). Sinha, Μ. Κ., Plant and Soil (1972), 36, 283.

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