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Studies on bound 14C-prometryn residues in soil and plants
Chemosphere,Vol.ll,No.8,pp Printed in Great Britain
771-795,1982
OO45-6535/82/O80771-25503.00/O O 1 9 8 2 Pergamon Press Ltd.
STUDIES ON BOUND 14C-PROI.IETRYIIRESIDUES IH SOIL AND PLANTS
Si~ahamat U. Khan Chemistry and Biology Researcll I n s t i t u t e Researcll Branch, A g r i c u l t u r e Canada Ottawa, Ontario Canada KIA OC~
ABSTRACT A i~igh-teuperature d i s t i l l a t i o n
technique ~sas developed for de~eruining and cneL~ically
i d e n t i f y i n g tile bound (nonextractable) residues of 14C-prometryn in an organic soil and plants.
A considerable portion of the bound 14C residues in the incubated organic soil was
i d e n t i f i e d as prometryn.
These residues ',lere absorbed by plants gro,m iJ~ the s o i l .
iluno-
and di-N-dealkylated metabolites of prometryn ~Jere present in the plant bound 14C residues and a major p o r t i o n of bound residues as associated ~lith l i g n i n . were also released from soil by microbes.
Soil-bound 14C residues
The bound 14C residues in soil were associated
u i t n ~luuin, humic acid, and f u l v i c acid f r a c t i o n s .
T,leruoanalytical me~i~ods were used to
obtained i n f o r ~ a t i o n on the nature and l o c a t i o n o f 14C bound residues in s o i l and humic .:.lateri al s.
Presented at tile F i r s t FAO/IAEA Research Coordination fleeting on Isotopic Tracer-Aided Studies of Bound Pesticide Residues in S o i l , Plants and Food, San Jos@, Costa Rica, i~ovember 30 - December 9, 1981.
Che~listry and Biology Research I n s t i t u t e Contribution No. / . ~ 0 ~
771
772
INTRODUCTION To assess the environmental significance of pesticide residues in soil and plants one must distinguish between two types of residues: and those that are not extractable with solvents.
those that are extractable with solvents, Boundpesticide residues are basically
those residues remaining in soil or plants after exhaustive solvent extraction.
These
residues would escape detection in the analytical procedures conventionally used in residue analysis. The significance of bound pesticide residues is chiefly addressed in terms of their bioavailability, both in amounts and forms of uptake. Theseand other considerations were topics of a research conference sponsored by the Division of Pesticide Chemistry, American Chemical Society (19).
Thus in soil and plants, bound pesticide residues may constitute a
potential environmental problem for the follm~ing reasons: I.
The nature and/or identity of bound residues of a pesticide expected to be present in soil or plants is not known.
2.
L i t t l e is known about the significance of bound residues in terms of their b i o a v a i l a b i l i t y , t o x i c i t y and accumulative nature.
3.
Conventional analytical methods may not detect these type of residues, thus underestimating the soil or plant burden of total pesticide residues.
4.
The environmental fate of bound residues is not known.
In view of the widespread interest and concern for bound pesticide residues in soil and plants, we co~aenced, in 1978, a project on bound prometryn [2-(methylthio)-4,6bis(isopropylamino)-s_-triazine]
residues an organic soil and their uptake by plants.
The
herbicide prometryn is widely used for control of broad leaf weeds and grasses in vegetable crops grown on organic soils.
The l a t t e r occur widely in eastern Canada and are of great
economic value to vegetable crops and t u r f production.
Our investigations on bound residues
were mainly concerned with the following three objectives: I.
Measurementand chemical identification.
2.
Distribution, nature and characteristics.
3.
Release and biological a v a i l a b i l i t y .
Data reported in this paper are based on research efforts in our laboratory at the chemistry and biology Research Institute, Agriculture Canada, Ottawa, Canada.
773
BOUND RESIDUES ANALYSIS In most of the studies reported in the l i t e r a t u r e , quantification of 14C bound residues in soil or plants has been achieved by total combustion. The soil or plant material after exhaustive solvent extraction is combusted to transform 14C residues into is trapped in basic solvents. counting.
14C02, which
The r a d i o - a c t i v i t y is then determined by s c i n t i l l a t i o n
This technique is limited to the quantitative determination of 14C bound
residues and cannot be used to determine the chemical foma of tile bound residues. Drastic extractive procedures that destroy the structure of soils or plants by s o l u b i l i z i n g the materials, and strong acid or base hydrolysis techniques have also been utilized.
However, these methods often result in the destruction of bound residues i d e n t i t y .
Recently, a novel technique was developed in our laboratory to dete~aine and chemically i d e n t i f y 14C bound residues in an organic soil (23)
The technique involved higll
temperature d i s t i l l a t i o n (HTD) of the extracted soil to release bound residues using a Lindberg Tube furnace (Fig. l ) .
An air-dried soil sample containing bound residues was
placed in a porcelain boat and inserted in the middle of the quartz tube.
One end of the
tube was closed with a swagelok, while the other end was connected with a series of traps containing suitable solvents.
The furnace was heated gradually from the room temperature to
about 80O°C and maintained at this temperature for about 15 minutes. sweep gas at a flow rate of 50 ml/min.
Helium was used as a
At the end of the experiment, the collection U-tube
(trap I I ) was thoroughly washed with solvents.
The quartz tube was also washed with solvents
and this washing combined with the f i r s t trapping solution (trap I ) .
Radioactivity of the
material in d i f f e r e n t traps was determined by s c i n t i l l a t i o n counting.
The material in traps
I , I I , and I I I was processed as depicted in Fig. 2 and f i n a l l y analyzed by gas chromatography for general i d e n t i f i c a t i o n and quantification, and with gas chromatography - mass spectrometry for specific i d e n t i f i c a t i o n . In one of our experiments, a moist organic soil (45.4%C) treated with uniforlaly 14C-ring labeled prometryn was incubated aerobically in the dark for one year. The soil was then exhaustively extracted with solvents and air-dried.
The soil was then
again moistened ( f i e l d capacity), incubated for about a week and exhaustively extracted with solvents in order to exclude any possible release of r a d i o a c t i v i t y . remaining in the soil amounted to 57.4% of the total applied 14C.
The bound residues
774
Swtihn ~ ~s: P~it~elS~r~~l
Q~W
°° '
Furnace ..~___
Moveable Quartz "fu~Tgelo k
eatmg ape He Gas, Flow Rate 50 ml/min
/Methanol ~ Trap I Trap II ] Trap Ill 25% HCL U-Tubein in Methanol Dry Ice AcetoneMixture if ~[ Hydroxideof • ~Hyamine IOX :
Trap IV
Figure I .
Apparatus used f o r high-teLlperature d i s t i l l a t i o n
of saJlples (23).
Analysis of the soil sample by HTD technique revealed that more than h a l f (54%) of the t o t a l bound residue was i n the forT,1 of proL1etryn, the reL1ainder constituted hydroxy propazine (
8%) and u n i d e n t i f i a b l e methanol soluble r,l a t e r i a l
(18%).
During the d i s t i l l a t i o n
about 2~o
of the bound residue ~las decor,lposed to 14C02. IJhen a i r or nitrogen were used as the sueep gases, considerably lower recovery of the compounds and a greater decomposition to 14C02 occurred.
Our attempts to eliminate completely or reduce s i g n i f i c a n t l y the then,lal
decomposition of 14C bound residues to 14C02 during d i s t i l l a t i o n
have not been
successful • The HTD technique described above has been successfully used in our laboratory to detemine 14C bound residues in oat plants grown in 14C-prometryn treated s o i l
(22). Tile
dried root and shoot samples a f t e r exhaustive e x t r a c t i o n with chloroform and methanol-water were analyzed by the HTD technique.
GC and GC-{'IS Analyses of the d i s t i l l a t e s
indicated tile
presence of a r,lono-N-dealkylated compound, namely, 2-(methylthio)-4-amino-6( i s o p r o p y l a ~ H n o ) - s - t r i a z i n e , and traces of 2 - ( m e t h y l t h i o ) - 4 , 6 - d i a m i n o - s - t r i a z i n e .
775
Heat to 800 C with He flow at 50 ml/min
Tra~) IV Evaporate methanol and add water Scintillation counting Extract with ether I F 1 Organic phase Aqueous phase
Wash with methanol
Concentrate
Concentrate
Evaporate and dis solve in methanol
Adjust oH 9-10 and extract with ether
Organic phase
I
Evaporate and dissolve m methanol
,;
i [~
C o m b i n e and concentrate to a small volume
Aqueous phase
i
Discard
i
1
]
f 3 Evaporate to just dryness, redissolve Scintillation counting in chloroform and chromatograph on acidic A1203 column pre-washed with chloroform ]_ r~ 7 Elute with chloroform Elute with methanol ~ 1 ---3 I i- - - - ] Scintillation Evaporate Scintillation Concentrate to a small count mg to dryness counting volume Redissolve in 10% acetone in hexane and chromatograph on acidic A1203 column prewashed with hexane I ,~
.
Elute with 5% acetone in hexane Discard
Figure 2.
i
Elute with 25% acetone in hexane G LC
Methvlate I Evaporate to iust dryness, redissolve m 10% acetone in hexane and chromatograph on acidic A1203 cotumn prewashed with hexane
F
L
E~ute with 25% acetone in hexane G LC
]
Elute with 5% acetone in hexane Discard
Schematic diagram for the analysis of none×tractable
(bound) residues (23),
776
Recently ~e used the HTD technique f o r d e t e m i n i n g 14C bound residues in hu~ic substances.
The soil containing bound (nonextractable) 14C residues was f r a c t i o n a t e d i n t o
huuic substances by a l k a l i e x t r a c t i o n follo~led by acid p r e c i p a t i o n . material
Freeze dried huuic
(50 r.lg) containing 14C bound residue w1as subjected to HTD and tile F.laterial in
d i f f e r e n t traps analyzed (Fig. 2).
I t was observed t h a t a considerable portion of 14C
residues in huuic materials ~las cor.lprised of the parent herbicide and i t s uonodealkylated product (25). ~lhile our studies ~lere in progress (22,23), Balba et a l .
(4), reported the successful
release of t i g h t l y complexed 3 - c h l o r o a n i l i n e and 3 , 4 - d i c l ~ l o r o a n i l i n e froa plant l i g n i n s using a s i u i l a r technique.
I t appears t h a t the HTD technique developed in our laboratory could
also be used f o r the analyses of other bound pesticide residues in s o i l and plants. BOUND RESIDUES IN SOIL Bound pesticide residues in s o i l are the focal p o i n t of several recent studies in various laboratories.
Table 1 suFx,larizes some of the work reported during the past few years on
bound residues in d i f f e r e n t s o i l s f o r a nullber of pesticides.
In most of these studies,
radiolabeled pesticides ~lere used and the bound r a d i o a c t i v i t y ~#as detemined by cor.lbusting the extracted soil to produce 14C02, which was then quantitated by s c i n t i l l a t i o n counting.
The bound residues expressed as % of applied pesticide ranged from 7 to 90%.
A
s i g n i f i c a n t quantity of bound residues is being "any amount of unextractable residue, greater than 10% expressed as the parent co,.Ipound, reuaining one year a f t e r a single treazment" (J). Therefore, i n s i t u a t i o n s ~here s i g n i f i c a n t concentrations of bound pesticide residues occur i n s o i l , the t o t a l pesticide residues in s o i l may be underestiuated as the bound residues w i l l not be detected in routine a n a l y s i s . Foruation of bound residues have been shown to increase ~.lith incubation t i , l e .
In our
studies i t ~las observed t h a t the amounts of the extractable 14C residues recovered frou the uniforl.~ly 14C-ring labeled prometryn treated organic s o i l decreased over an incubation period of 150 days (Fig. 3). s o i l bound 14C residues.
This in t u r n , corresponded to an increase in the f o m a t i o n of
Thus by the end of the incubation period (150 days), extractable
14C residues decreased to 36.5% w h i l e the bound 14C residues (deten.lined by cor.lbustion to 14C02) increased to 43.0% of the i n i t i a l l y
added 14C.
The t o t a l r a d i o a c t i v i t y
recovered at the end of 150 days auounted to about 80% of t h a t i n i t i a l l y
applied.
SiLlilarly,
the r a d i o a c t i v i t y recovered by HTD of samples increased ~ i t h incubation time and by the end
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of 150 days amounted to about 40% of the i n i t i a l l y added 14C. However, the amounts of 14C recovered by HTD were slightly lower than those obtained by combustion to 14C02 (Fig. 3).
GC examination of the methanol-extractable residues from the incubated soil
indicated that the total amounts of prometryn extracted decreased from 95 to 23% of the i n i t i a l l y applied herbicide, whereas that of hydroxypropazine (calculated in terms of prometryn) increased from 1.7 to 9.4% over the incubation period.
Furthermore, analyses of
tile high temperature d i s t i l l a t e s by GC indicated that the amounts of bound prometryn residues increased from 0.2 to 19.4% of the i n i t i a l l y applied herbicide.
Thus, the total recovery of
prometryn residues (extractable plus bound) at the end of an incubation period of 150 days was about 52% of the applied herbicide.
The disappearance of radiocarbon added to the soil
could not be accounted for only by the extractable and bound 14C residues (Fig. 3).
It
appears that the loss of radiocarbon up to about 20% due to volatilization, possibly due to evolution of 14C02 and volatile degradation products, occurred during the incubation period. BOUND RESIDUES IN HUI41C SUBSTANCES Recently, we determined the distribution of bound 14C residues among various humic fractions of an organic soil containing 57.4% bound 14C of the total i n i t i a l l y applied 14C-prometryn (25).
The method of extraction, separation and purification of humic
materials outlined in Fig. 4 is analogous to that described by Schnitzer and Khan (32).
The
yield of humin, humic acid (HA) and fulvic acid (FA) amounted to 0.67, 0.04 and 0.12 g/g soil, respectively. Proportions of total bound 14C in humin, HA and FA fractions were 57, I I and 26%, respectively (Fig. 4).
The incorporation of 14C bound residues into humic
materials observed in our study is consistent with those reported for other pesticides described earlier.
The 14C residues were concentrated in the humin fraction (Fig. 4).
Furtherhlore, only a small amount of 14C (3.3%) was released in solution by solubilizing the mineral fraction from humin by the HCI-HF treatment. The average molecular weight of bound 14C residues associated with humin and IIA appeared to be greater than 12000 since very l i t t l e 14C was dialyzable. The presence of 14C bound residues in FA fraction (26%) is of special interest.
The l a t t e r is a naturally occurring water soluble low molecular weight
polyelectrolyte and is considered to be the dominant soluble organic fraction present in the soil solution under f i e l d conditions.
I t is also well known that FA is present in many
surface waters and imparts a yellow to brown color in natural water (7,32).
I t is l i k e l y
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Extractable and bound 14C residues in an organic soil during a 150-day incubation period, after soil treatment with uniformly 14C-ring labeled prometryn at 12.4 mg/kg (0.047 uCi/g). Curve E, extracted 14C; curve Bl, bound 14C determined by combusting soil to 14C02; curve B2, bound 14C determined by high-temperature distillation technique; and curve T, total of extractable (E) and bound (Bl) (23).
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that bound residues associated with the soluble FA fraction may become bioavailable to both plants and exposed aqueous or soil fauna. I t was observed that a considerable proportion of 14C residues in humic materials was comprised of the parent herbicide, i . e . prometryn, and i t s monodealkylated product namely, 2-(methylthio)-4-amino-6-(isopropylamino)-s_-triazine
(Table 2).
However, FA fraction
contained an appreciable amount of 2-hydroxy analogue of prometryn [2-hydroxy-4,6bis(isopropylamino)-s--triazine]. Alkaline extraction with O.IN NaOHunder nitrogen, widely used for the extraction of humic materials from s o i l , was thought to hydrolyze or chemically degrade the herbicide residues in soil.
However, our experiments revealed nearly
quantitative recoveries of unchangedprometryn from aqueous solutions when the herbicide was extracted with O.IN NaOH (pH 13).
Hitching and Roberts (17) used 1.25 N aqueous NaOH
solution at 80°C for 2 h for extraction of humic materials containing bound residues and were able to identify small amounts of the total radioactivity in HA.
Table 2.
BoundResidues in Humic Materials (25)
Fraction
Prometryn ppm
Humic acid F u l v i c acid Humin
Hydroxypropazine ppm
0.3 0.3 1.9
0.I 0.9 NDa
Deisopropylprometryn ppm 0.2 0.I I.I
aNondetectable, <0.01 ppm. MECHANISM OF BOUND RESIDUES FORMATION The general consensus appears to be that in the formation of bound residues, p e s t i c i d e or i t s metabolites are chemically bonded i n t o the soil organic matter.
However, our recent data
provide evidence that support the contention that physical binding may also play an important role in the formation of soil bound residues. Isothermal heating has been used for i n v e s t i g a t i n g the mechanism of thermal decomposition of organic matter (31).
I t was thought that thermoanalytical methods applied to soil and
humic materials containing bound residues may provide useful information on the nature of p e s t i c i d e binding. The high temperature d i s t i l l a t i o n
apparatus described e a r l i e r was used f o r isothermal
782
heating (23).
An air dried soil sample (200-300 mg) containing 14C bound residues, was
placed in a porcelain boat and inserted in the middle of the quartz tube preheated to the desired temperature. Helium or a i r was used as purge gas at a flow rate of 40-50 ml/min. The sample was heated at the desired temperatures isothermally for a period t i l l release of 14C was observed in the methanol and CO2 traps (23).
no further
In preliminary
experiments i t was observed that 45 min. and 60 min. heating was adequate for a maximum release of 14C from the soil under a stream of a i r and helium, respectively.
In some cases
samples were also heated under oxygen stream and covered by a combustion catalyst (CuO-Al203).
At the end of the heating period the radioactivity of the trapping
solutions was determined by l i q u i d s c i n t i l l a t i o n counting. In other experiments a freeze dried humin sample (50 mg) containing 14C bound residues was heated isothermally under a stream of air, nitrogen or helium as described above for a fixed period of 60 min. Preliminary experiments indicated that heating for a longer period did not increase the 14C release.
At the end of the experiment the char resulting from
heating was removed and analyzed for C, H, N and S. The radioactivity of the trapping solutions was also
Oxygenwas calculated by difference. determined. The yields of HA and FA
obtained from soil by the fractionation procedure used in this study were not adequate to allow for the thermal analysis of these materials. The thenaal profiles of 14C bound residues of soil in a i r and helium streams are shown in Fig. 5.
The principal reactions occurring under air and helium during the isothermal
heating of soil containing 14C bound residues differed in that ( i ) 14C was released over a broader temperature range under helium, ( i i ) the release of 14C under helium occurred and complete decomposition took place at temperatures that were lO0-200°C higher than under a i r , and ( i i i ) all the bound 14C released was decomposed to 14CO2 in air stream, whereas in helium stream a portion of bound 14C released (up to 26%) remained undecomposed as evidenced by'the presence of radioactivity in the methanol trap.
From these observations
i t appears that the release of bound 14C was associated with the therlaal decomposition of the organic matter fractions of the soil.
Under oxygen (data not shown) or a i r the
conditions were more conducive for a faster organic matter decomposition than under helium. Thus, the release of bound 14C on heating under helium was considerably slower than under air.
783
The themaal profile of bound 14C residues in humin under air, helium or nitrogen ~as similar to those obtained for soil. temperature is sho~n in Fig. 6. the CO2 trap.
The amount of 14C released from humin as a function of
In an air stream nearly all the radioactivity was found in
However, heating under helium or nitrogen resulted in release of 14C, part
of which(up to 35%) was also trapped in methanol. While very l i t t l e 14C was released under air at 150°C, extensive release occurred between 200-300°C (=90%) reaching I00% at 500°C or higher (Fig. 6).
On the other hand while under helium or nitrogen
radioactivity was released at 150°C about half of the total bound 14C was released at 325°C-350°C and by 700°C the recovery of 14C in the trapping solutions was nearly quantitative.
Differential thermo gravimetry of humic materials showed elimination of all
COOH + OH groups between 200°C-400°C and decomposition of humic 'nuclei' at 450°-550°C
I00-
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Releaseof bound 14C from soil on isotherlaal heating under (o) air, and (m) helium (25).
784
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TEMPERATURE C
Figure 6.
(32).
Release of bound 14C from humin on isothemnal heating under (o) a i r , ( • ) h e l i u m , and ( • ) nitrogen (25).
Thus, one may assume that s t a b i l i t y of bound pesticide residues is due to their l i n k
with phenolic OH or COOHgroups of soil organic matter or humic materials.
For example,
chemically s t a b i l i z i n g reactions between phenolic hydroxyls and free aro~natic amino groups from the metabolites of herbicides have been postulated.
Thus, under these conditions the
presence of unchanged parent molecules of pesticides in the bound residues is not expected. However, our study demonstrated that in addition to small amounts of hydroxy and dealkylated analogues, a considerable portion of the bound residues in the organic soil and humic fractions was present in the form of prometryn.
Thus, conceivably in addition to the
chemical binding of the metabolized pesticide, physical binding of the parent ~nolecule or i t s derivative also play an important role in the formation of soil bound residues.
I t has been
suggested that humic materials are made of phenolic and benzenecarboxylic acids joined by llydrogen bonds to form a molecular sieve type polymeric structure of considerable s t a b i l i t y (32).
One of the characteristics of the structure is that i t contains voids or holes of
d i f f e r e n t molecular dimensions which can trap or f i x organic molecules, such as pesticides.
785
From the thermoanalytical data i t appears tilat the thermal decouposition of organic matter or humic substances weakens the structure by eliminating the functional groups and eventually decomposing the 'nuclei'
This in turn pen.lits tile release of bound residues t~lat are
trapped in the structure.
Further support for such a uechanism comes frou tlme methylation
study discussed l a t e r . Schnitzer and Hoffman (31) used the atouic H/C vs. O/C graphical-statistical r e l a t i o n ship of Van Krevelen (37) to study tlle principal reactions occurring during the thermogravil;letry of soil organic matter under nitrogen,
i t ~las thought that this approac~l
uay y i e l d some valuable information on the relationship bet~een the release of bound 14C and reaction processes occurring during heating of the huuic ~laterials. vs. O/C ratios are plotted for humin heated under nitrogen.
In Fig. 7 atomic H/C
I t appears that the r,min
reaction of humin up to 350°C ~as dehydration (31) ~#hich uas accoupanied by the elii~lination of about half of the bound 14C. The next reaction up to 500°C could be ascribed to dehydrogenation ~,lhich resulted in another 40% release of bound 14C. F i n a l l y , betvleen 500-700°C the reaction processes involving both dehydration and dehydrogenation (31) could have taken place accompanied by the release of the remaining 14C. Methylati on of HA and Humin: The HA or humin sauple ~las suspended in uethanol and ,,lethylated ~lith freshly prepared diazor,lethane repeatedly for several days u n t i l tlme 14C released in solution remained constant. washed ~#ith methanol several tiues.
Tile mixture was filZered and the sauple residue
The coubined f i l t e r a t e was concentrated to small
volume and analyzed by l i q u i d s c i n t i l l a t i o n counting.
The dried solid residue ~as coubusted
to 14CO2 for deten.lining the reuaining total 14C residues. Exhaustive methylation of humin and hui,lic acid resulted in about 25-30% release of the i n i t i a l l y bound 14C. i.lethylation reduces hydrogen bonding betueen tile 'binding units' of humic materials (32).
Thus, the molecular sieve-like structural arrangement in humic
material is ueakened. The bound pesticide residues, adsorbed on i t s surface and trapped in internal voids, may then be released as indicated in our study. PHOTOCHEI41CAL STABILITY OF BOUNDRESIDUES I t became of interest to determine ~lhether photodegradation under UV l i g h t could be a process for the release of bound 14C residues.
Soil samples (l gm) containing 14C bound
residues v#ere irradiated with UV l i g h t using a 15}I General Electric 45 cr,1 germicidal lamp (253.7 nm). The suspension (Soil:Ilater = 1:600) uas placed 15 cu belo~ the la,.ip, continuously
-/86
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Atomic H/C vs. O/C ratios for humin containing 14C bound residues
Figure 7.
heated under nitrogen.
Numbers in parenthesis show the percentage
of 14C released at different temperatures (25).
stirred with a magnetic s t i r r e r and irradiated for 216 h.
Preliminary experiments indicated
that under the experimental conditions described UV i r r a d i a t i o n of soil suspension for a longer period or at higher water contents did not result in any further release of 14C in solution.
At the end of the experiment the soil suspension was centrifuged and the soil
residue was exhaustively extracted with methanol.
The aqueous supernatant was combined witl~
the methanol extract and concentrated to a small volume on a rotary evaporator.
An aliquot
of this was analyzed by l i q u i d s c i n t i l l a t i o n counting while the remaining portion was evaporated to dryness.
The material was then dissolved in chloroform and processed for gas
chromatographic and mass spectrometric analyses.
Residual methanol from the extracted soil
was allowed to evaporate by a i r drying the soil sample and a portion of i t was combusted to 14C02 to determine the remaining total 14C. The control, consisted of soil containing 14C bound residues, was also processed as described above except the suspension was kept in dark.
i
: ~P.~elimtnary experiments indicated that the decomposition of prometryn in water when exposed to UV l i g h t was complete in about 3 h.
The herbicide was converted quantitatively to
787
i t s hydroxy analogue.
I t was observed that the s t a b i l i t y of bound 14C residues in soil
suspension was affected by UV l i g h t .
Thus, at the conclusion of the exposure period of 216 h
tile extractable radioactivity accounted for about 30% of the total bound 14C i n i t i a l l y present in the s o i l .
UV irradiation for a longer period did not increase the amounts of
extractable 14C. Only up to 3-5% or less of the total bound 14C was recovered as extractable in the dark.
GC and GC-MSanalyses indicated that methanol extract of the UV
irradiated soil suspension contained mainly hydroxypropazine. Thus, i t appears that UV l i g h t caused the release of some of the bound prometryn residues from soil which were subsequently decomposed to the hydroxy analogues. For comparative purposes, soil samples f o r t i f i e d with prometryn (24 ppm) were also processed under the experimental conditions described above. The herbicide in the soil suspensions was readily decomposed under the influence of UV light.
Thus, at the end of a 72 h exposure, 75% of the material i n i t i a l l y used to f o r t i f y
soil sample was extractable in the form of hydroxypropazine. Although 70% of the bound 14C in soil remained unextractable even after prolonged exposure, the fact that UV l i g h t caused the release of 30% bound 14C should be of concern in detemaining the environmental s t a b i l i t y of bound residues. In other experiments, HA or humin sample (lO mg) was suspended in IO ml d i s t i l l e d water, stirred and i r r i d i a t e d with UV l i g h t for 96 h as described before. Concurrently samples were also run in the dark. The suspension was centrifuged, solid residue washed with water and the aqueous phases combined. The solid residue after air drying was combusted to 14C02 for determining the 14C remaining in the bound form. The aqueous phase was concentrated to a small volume and analyzed by liquid s c i n t i l l a t i o n counting. Exposure of aqueous solutions of humic acid or humin containing bound 14C residues to UV l i g h t for 96 h resulted in 6.1 and 1.2% extractable 14C, respectively.
The relatively
small release of 14C from bound form was unexpected since hulaic materials have been shown to act as photosensitizers for nonabsorbing pesticides (21).
Prolonged exposure of humin or
humic acid to UV l i g h t did not s i g n i f i c a n t l y increase the release of bound 14C. The concentration of extractable 14C a f t e r UV i r r a d i a t i o n was too low to be analyzed by GC. These observations therefore suggest that bound residues in humin or humic acid f r a c t i o n s of organic matter undergo to very l i t t l e
p h o t o l y t i c degradation.
MICROBIOLOGICAL RELEASE OF BOUND RESIDUES The question of the potential release and
b i o l o g i c a l a v a i l a b i l i t y of soil bound residues
788
has been addressed in several studies.
Recently, we investigated the microbiological release
of bound residues from an organic soil treated
with 14C-prometryn (24).
Extracted soil
containing 14C bound residues uas incubated in a llarburg apparatus ~#ith a l i q u i d inoculum obtained from an untreated and unextracted control soil (Fig. 8).
The control consisted of
untreated soil exhaustively extracted ~ith the same solvents as those used for reuoving tlie extractable residues fro~:q the treated s o i l .
The control sample ~vas also placed in Warburg
vessel and inocului:1 added. KOII (20%) was used in the center wells of the vessels to trap 14CO2 and the temperature maintained at 20°C. The loss of radiocarbon due to evolution of 14CO2 during incubation period of 22 days ~as negligible froi.~ the soil containing 14C bound residues.
The extractable and unextrac
table (bound) radioactivity in the soil after incubation with an inoculum for 22 days amounted to 27.0 and 71.6%, respectively, of the total 14C bound residues (Fig. 8). indicated that microbes released part of the 14C bound residues from the s o i l .
This
In a
preliminary experiF.lent, an aliquot of the soil containing bound residues was ~]ixed with s t e r i l i z e d d i s t i l l e d water, allotted to incubate for about 3 weeks and then exhaustively extracted u i t h methanol.
Analysis of the extracted material revealed negligible auounts of
r a d i o a c t i v i t y ( <1% of the t o t a l ) , indicating that 14C residues were s t i l l bound to the soil uhen no inoculum ~as added. Examination of the extractable residues indicated the presence of prometryn, hydroxypropazine and a small amount of the p a r t i a l l y N_-dealkylated compound, 2-(methylthio)-4-amino-6-(isopropylamiao)-s-triazine .
The results suggest tilat
the microbes i n i t i a l l y released the bound prometryn, ~hich was then degraded in the incubated soil to hydrolyzed and dealkylated products.
Analysis of the bound residues revealed tile
presence of prometryn and small amounts of hydroxypropazine and mono-N_-deaIKylated prometryn.
I t is possible that some of tlle metabolites formed during the incubation period
may have become a part of the bound portion of the residues in the s o i l . PLAINT UPTAKEOF BOUIID RESIDUES Pesticide residues absorbed and translocated in plant tissues may be present in tl~ree possible forms: ( i ) freely extractable residues, ( i i ) extractable conjugates bound to natural co~ponents of plants, and ( i i i ) , constituents.
unextractable or bound residues incorporated into tile plant
The l a t t e r may be considered as analogous to the bound residues in s o i l .
789
14any studies using radiolabeled compounds have revealed that a considerable portion of the pesticide residue may become bound in the plant.
Since bound residues in plants cannot
be removed by conventional solvent extraction techniques designed to extract and detect the parent compound and certain metabolites, more attention should be directed towards evaluating their residual nature in plants.
SOIL CONTAININGl iCA BOUNDRESIDUES
| Incubation with soil inoculum for 22 days in a Warburg vessel
I
I--
1
KOH
SOIL
(~co 2 )
L
=3.Ol:r
Exhaustive extraction with methanol
Not Extractable (Bound residues) 71.6%
HTD
i
GC
Extractable 27.0%
GC
I
GC-MS
GCI-MS
Figure 8.
Schematic diagram for the analysis of extractable and bound residues in soil incubated with inoculum (24).
Recently, we investigated the potential uptake of 14C bound residues by oat plants from an organic soil treated with 14C-prometryn (22).
Soil containing 14C bound residues
uixed with washed silica sand (soil :sand : 1:I .25) were used to grow oat plants in a controlled envirom~ntal chamber. The pots were watered each day as necessary and f e r t i l i z e d with standard Hoagland's nutrient solution during the growing season. After 21 days the plants were harvested and roots and shoots were separated. The analytical procedure used for the extraction, isolation, and determination of 14C residues in plant tissues is sho~m in Fig. 9. The crop of oat plants removed 0.53% of the previously soil bound !4C in the soil-sand mixture used for growing the plants in this experiment. The shoots contained 69.8% of the
790
total plant radioactivity whereas the remaining radioactivity (30.2%) was present in the roots.
Table 3 shows the extractable and unextractable (bound) 14C residues in the plant
tissues as deten,lined according to the scheme depicted in Fi~. 9.
Amounts of total
extractable 14C residues were larger in roots, but were smaller in shoots accounting for 75.0% and 51.1%, respectively, of the total 14C residue.
The chlorofona extracts (nonpolar
residues) contained a very small proportion of 14C, which constituted
only a trace amount
of unchanged prometryn ( <0.05 ppm) as indicated by GC analysis. Ilost of the methanol water soluble 14C residues (polar residues) were present in the shoot and root samples in the conjugated form. This was indicated by the absence of any free metabolites in the eluate obtained by eluting XAD-2 column with methanol (Fig. 9).
However, hydrolysis of this eluate
resulted in the release of a compound which after methylation with diazomethane was identified as the methoxy derivative of hydroxypropazine [2-methoxy-4,6-bis(isopropyl -amino)-s-triazi ne]. The data demonstrate that some of the radioactivity absorbed by plants from the soil containing bound 14C residues became again bound (unextractable) in the plant tissues. Plant bound 14C residues were smaller in roots and greater in shoots amounting to 19.9% and 40.2%, respectively, of the total 14C recovered from each tissue (Table 3).
GC analyses of
the HTD d i s t i l l a t e s indicated the presence of a mono-N_-dealkylated compound namely, 2-(methylthio)-4-al~ino-6-(isopropylamino)-s-triazine,
and traces of 2-(methyltlHo)
-4,6- di amino-s_-triazi ne. The extracted plant tissue containing bound 14C l i k e l y contained mainly lignin, carbohydrate, and denatured protein.
I t became of interest to deterl.~ine the distribution of
plant bound 14C in these fractions (Table 3).
Tlle lignin extracted by the procedure
described in Fig. 9 comprises free and bound lignin.
The l a t t e r is bound to the carbohydrate
of the cell wall and could only be extracted by boiling ~ith dioxane-HCl under N2.
Tlle
data show that the total lignin (free + bound) from roots and shoots contained I0.5% and 28.5%, respectively, of the total 14C bound residues in the plant tissue.
The remaining
solid residue of roots and shoots which presumably consists of mainly carbohydrate and protein contained relatively smaller amounts of 14C residues (4.7% and 7.5% of the total 14C bound residues in roots and shoots, respectively). About 2% of the total 14C was present in the aqueous supernatant, which may be due to the hydrolysis products and can be regarded as extractable residues.
I t is l i k e l y that most of the 14C associated with the
791
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793
l i g n i n or solid residues fraction ~las comprised of 2-(methyltilio)-4-amino-6-(isopropylah~ino) -s-triazine.
The l a t t e r was identified as the major product in the HTD d i s t i l l a t e s of plant
tissues. I t has been shown that l i g n i n is the r:~ajor bound residue fraction of aniline and chloroaniline compounds in plants.
In a recent study, S t i l l et al. (36) investigated the
molecular nature and identity of bound 3-chloroaniline and 3,4-dichloroaniline residues in rice plants grown hydroponically. The procedure used for fractionation of root tissues was similar to that described in Fig. 9. l i g n i n fraction fro~.~ the roots.
More than 40% of the 14C was present in the isolated
Theseworkers suggested that chloroanilines may be bonded
covalently to lignin via 1,6 addition to a quinone ~,lethide intermediate during the lignin synthesis.
The e-carbon in the l i g n i n side chain ( i . e . the benzylic carbon) was considered
to be the uost l i k e l y chloroaniline nitrogen binding site (36). SUMMARY The use of 14C labeled pesticides has made us aware of the existence of bound residues in soil and plants.
These residues would escape detection by the conventional analytical
methods thereby resulting in an underestimation of the soil or plant burden of total pesticide residues. The methodology for the analysis of bound pesticide residues is s t i l l in the development stage. The total 14C bound residues in soil and plants are usually estimated by combustion of the extracted material to yield 14C02. The high temperature d i s t i l l a t i o n technique may provide a possible means for the chemical identification of bomld residues.
However, the application of this technique to bound residue analysis of other
pesticides has not been f u l l y explored. Contrary to the general consensus that the bound residues become an integral part of tile matrix without recognizable relationship to the original pesticide, data presented in this paper demonstrate that a considerable portion of such residues in soil may also comprise the parent molecule, The organic fraction of a soil appears to have the potential for for~in~ bound residues ~ith pesticides or products arising from their degradation.
I t is suggested
that in addition to chemical binding, pesticides or their metabolites are also firmly retained by organic fractions (humic materials) by a process that more l i k e l y involves adsorption on external surfaces and entrapment in the internal voids of a molecular sieve-type structural arrangement. The l a t t e r may be considered analogous to the "clathrate compounds".
794
Evidence is presented to demonstrate the b i o a v a i l a b i l i t y of soil bound 14C residues to oat plants.
Furthen:1ore, i t is shoun that soil microbes can potentially release 14C bound
residues frou s o i l .
A uajor portion of bound peszicide residues in plant tissues uay be
associated ~vith lignin.
Thus, lignin fon.~ation in plants may serve as a syste~.1 for plants to
store pesticide residues by incorporating them into insoluble lignin. REFERENCES I.
~.Ibrosi, D., P.C. Kearney, and J.A.
ilacchia, J. Agr. Food Cheu. 25, 342 (1977a).
2.
~.ibrosi, D., P.C. Kearney, and J.A. ilacchia,
3.
Anon, "Registration of Pesticides in the United States - Proposed Guidelines"
J. Agr. Food Chem. 25, 868 (1977b).
Federal Register, 40, 123 (1975). 4.
Balba, H.II., G.G. S t i l l , and E.R. I1ansager, J. Assoc. Off. Anal. Che~.l. 62, 237 1979).
5.
Bartha, R.,
6.
Chisaka, H., and P.C. Kearney, J. Agr. Food Chem. I__~8, 854 (1970).
7.
Christman, R.F., and R.A. I,linear,
J. Agr. Food Chem. 19, 385 (1971).
In "organic compounds in aquatic environments
S.D. Faust and J.V. Hunter, Eds., Marcel Dekker, Inc., New York. p. l l 9 (1971). 8.
Fuhr, F., and II. :iittelstaedt,
J. Agr. Food Chem. 28, 122 (1980).
9.
Fuhrenann, T.II., and E.P. Lichtenstein,
J. Agr. Food Chem. 26, 605 (1978).
lO. Golab, T., and ',I.A. Althaus, ~leed Sci. 23___, 165 (1975). I I . Golab, T. and I'I.E. Amundson, Environ, Qual. Safety Suppl. I i I . 258 (1975). 12. Golab, T., II.A. Althaus, and H.L. IIooten,
J. Agr. Food Chem., 2_7_7,163 (1979).
13. Golab, T., C.E. Bishop, A.L. Donoho, J.A. (ianthey, and L.L. Zornes, Bioche~;~. Pilysiol. 5, 196 (1975). 14. Helling, C.S., and A.E. Krivonak,
J. Agr. Food Chem. 26, I156 (1978a).
15. Helling, C.S., and A.E. Krivonak,
J. Agr. Food Chem. 26, I164 (1978b).
16. H i l l , I.R.,
ACS Symposium Series 29, 358 (1975).
17. Hitchings, E.J., and T.R. Roberts,
Pestic.Sci. lO, l (1979)
18. Katan, J., T.~I. Fuhremann, and E.P. Lichtenstein,
Science 193, 892 (1976).
19. Kaufuan, D.D., G.G. S t i l l , G.D. Paulson, and S.K. Bandal, Ed., "Bound and Conjugated Pesticide Residues", ACS Syuposium Series 29, (1976). 20. Kazano H., P.C. Kearney, and D.D. Kaufman, J. Agr. Food Chem. 20, 975 (1972). 21. Khan, S.U., "Pesticides in the Soil Environuent", Elsevier Scientific Publishing Co., Amsterdam, (1980a), pp. I04-I08.
795
22. Khan, S.U.,
J. Agr. Food Cheu. 2_88,1096 (1980b).
23. Khan, S.U., and H.A. Hamilton,
J. Agr. Food Chem. 2_88,126 (1980).
24. Khan, S.U., and K.C. Ivarson,
J. Agr. Food Chem. 29, 1301 (1981).
25. Khan, S.U.,
J. Agr. Food Chem. 30, 175 (1982).
26. Lichtenstein, E.P., J. Katan, and B.N. Anderegg, J. Agr. Food Che~.l. 25, 43 (1977). 27. Lichtenstein, E.P., T.T. Lian9, and T.W. Fuhremann, J. Agr. Food Chem. 2_66,948 (1978).
28. I.|urtily, N.B.K., D.D. Kaufuan and G.F. Fries,
J. Environ. Sci. Health, Bl4, l (1979).
29. Ogawa, K., N. Tsuda, F. Yamauchi, I. Yamaguchi, and T. Misato,
J. Pestic. Sci. l ,
219 (1976). 30. Roberts, T.R., and M.E. Standen, Pestic. Sci. 12, 285 (1981). 31. Schnitzer, M., and J. Hoffman, Geochemica et Cosmochimica Acta., 29, 859 (1965). 32. Schnitzer, 14., and S.U. Khan, Humic Substances in the Environnent. New York: Marcel Dekker (1972). 33. Smith, R.A., W.S. Belles, Kei-Wei Shen, and %~.G. Woods, Pestic. Biocher.~. Physiol. 3_, 278 (1973). 34. Smith, A.E., and D.C.G. Muir,
WeedResearch 20, 123 (1980).
35. Spillner, C.J., J.R. DeBaun, and J.J. 14enn, J. Agr. Food Chem. 27_, I054 (1979). 36. S t i l l , G.G., H.M. Balba, and E.R. I1ansager, J. Agr. Food Chem. 29, 739 (1981). 37. Van Krevelen, D.W., Fuel, 2__99,269 (1950). 38. Viswanathan, R., J. Scheunert, J. Kohli, W. Klein and F. Korte,
J. Environ.
Sci. Health, B13, 243 (1978). 39. Wheeler, W.B., G.D. Stratton, R.R. Twilley, L.T. Ou, D.A. Carlson, and J.11. Davidson,
J. Agr. Food Chem. 2_]7, 702 (1979).
(Received in Germany
iO May 1982)
771-795,1982
OO45-6535/82/O80771-25503.00/O O 1 9 8 2 Pergamon Press Ltd.
STUDIES ON BOUND 14C-PROI.IETRYIIRESIDUES IH SOIL AND PLANTS
Si~ahamat U. Khan Chemistry and Biology Researcll I n s t i t u t e Researcll Branch, A g r i c u l t u r e Canada Ottawa, Ontario Canada KIA OC~
ABSTRACT A i~igh-teuperature d i s t i l l a t i o n
technique ~sas developed for de~eruining and cneL~ically
i d e n t i f y i n g tile bound (nonextractable) residues of 14C-prometryn in an organic soil and plants.
A considerable portion of the bound 14C residues in the incubated organic soil was
i d e n t i f i e d as prometryn.
These residues ',lere absorbed by plants gro,m iJ~ the s o i l .
iluno-
and di-N-dealkylated metabolites of prometryn ~Jere present in the plant bound 14C residues and a major p o r t i o n of bound residues as associated ~lith l i g n i n . were also released from soil by microbes.
Soil-bound 14C residues
The bound 14C residues in soil were associated
u i t n ~luuin, humic acid, and f u l v i c acid f r a c t i o n s .
T,leruoanalytical me~i~ods were used to
obtained i n f o r ~ a t i o n on the nature and l o c a t i o n o f 14C bound residues in s o i l and humic .:.lateri al s.
Presented at tile F i r s t FAO/IAEA Research Coordination fleeting on Isotopic Tracer-Aided Studies of Bound Pesticide Residues in S o i l , Plants and Food, San Jos@, Costa Rica, i~ovember 30 - December 9, 1981.
Che~listry and Biology Research I n s t i t u t e Contribution No. / . ~ 0 ~
771
772
INTRODUCTION To assess the environmental significance of pesticide residues in soil and plants one must distinguish between two types of residues: and those that are not extractable with solvents.
those that are extractable with solvents, Boundpesticide residues are basically
those residues remaining in soil or plants after exhaustive solvent extraction.
These
residues would escape detection in the analytical procedures conventionally used in residue analysis. The significance of bound pesticide residues is chiefly addressed in terms of their bioavailability, both in amounts and forms of uptake. Theseand other considerations were topics of a research conference sponsored by the Division of Pesticide Chemistry, American Chemical Society (19).
Thus in soil and plants, bound pesticide residues may constitute a
potential environmental problem for the follm~ing reasons: I.
The nature and/or identity of bound residues of a pesticide expected to be present in soil or plants is not known.
2.
L i t t l e is known about the significance of bound residues in terms of their b i o a v a i l a b i l i t y , t o x i c i t y and accumulative nature.
3.
Conventional analytical methods may not detect these type of residues, thus underestimating the soil or plant burden of total pesticide residues.
4.
The environmental fate of bound residues is not known.
In view of the widespread interest and concern for bound pesticide residues in soil and plants, we co~aenced, in 1978, a project on bound prometryn [2-(methylthio)-4,6bis(isopropylamino)-s_-triazine]
residues an organic soil and their uptake by plants.
The
herbicide prometryn is widely used for control of broad leaf weeds and grasses in vegetable crops grown on organic soils.
The l a t t e r occur widely in eastern Canada and are of great
economic value to vegetable crops and t u r f production.
Our investigations on bound residues
were mainly concerned with the following three objectives: I.
Measurementand chemical identification.
2.
Distribution, nature and characteristics.
3.
Release and biological a v a i l a b i l i t y .
Data reported in this paper are based on research efforts in our laboratory at the chemistry and biology Research Institute, Agriculture Canada, Ottawa, Canada.
773
BOUND RESIDUES ANALYSIS In most of the studies reported in the l i t e r a t u r e , quantification of 14C bound residues in soil or plants has been achieved by total combustion. The soil or plant material after exhaustive solvent extraction is combusted to transform 14C residues into is trapped in basic solvents. counting.
14C02, which
The r a d i o - a c t i v i t y is then determined by s c i n t i l l a t i o n
This technique is limited to the quantitative determination of 14C bound
residues and cannot be used to determine the chemical foma of tile bound residues. Drastic extractive procedures that destroy the structure of soils or plants by s o l u b i l i z i n g the materials, and strong acid or base hydrolysis techniques have also been utilized.
However, these methods often result in the destruction of bound residues i d e n t i t y .
Recently, a novel technique was developed in our laboratory to dete~aine and chemically i d e n t i f y 14C bound residues in an organic soil (23)
The technique involved higll
temperature d i s t i l l a t i o n (HTD) of the extracted soil to release bound residues using a Lindberg Tube furnace (Fig. l ) .
An air-dried soil sample containing bound residues was
placed in a porcelain boat and inserted in the middle of the quartz tube.
One end of the
tube was closed with a swagelok, while the other end was connected with a series of traps containing suitable solvents.
The furnace was heated gradually from the room temperature to
about 80O°C and maintained at this temperature for about 15 minutes. sweep gas at a flow rate of 50 ml/min.
Helium was used as a
At the end of the experiment, the collection U-tube
(trap I I ) was thoroughly washed with solvents.
The quartz tube was also washed with solvents
and this washing combined with the f i r s t trapping solution (trap I ) .
Radioactivity of the
material in d i f f e r e n t traps was determined by s c i n t i l l a t i o n counting.
The material in traps
I , I I , and I I I was processed as depicted in Fig. 2 and f i n a l l y analyzed by gas chromatography for general i d e n t i f i c a t i o n and quantification, and with gas chromatography - mass spectrometry for specific i d e n t i f i c a t i o n . In one of our experiments, a moist organic soil (45.4%C) treated with uniforlaly 14C-ring labeled prometryn was incubated aerobically in the dark for one year. The soil was then exhaustively extracted with solvents and air-dried.
The soil was then
again moistened ( f i e l d capacity), incubated for about a week and exhaustively extracted with solvents in order to exclude any possible release of r a d i o a c t i v i t y . remaining in the soil amounted to 57.4% of the total applied 14C.
The bound residues
774
Swtihn ~ ~s: P~it~elS~r~~l
Q~W
°° '
Furnace ..~___
Moveable Quartz "fu~Tgelo k
eatmg ape He Gas, Flow Rate 50 ml/min
/Methanol ~ Trap I Trap II ] Trap Ill 25% HCL U-Tubein in Methanol Dry Ice AcetoneMixture if ~[ Hydroxideof • ~Hyamine IOX :
Trap IV
Figure I .
Apparatus used f o r high-teLlperature d i s t i l l a t i o n
of saJlples (23).
Analysis of the soil sample by HTD technique revealed that more than h a l f (54%) of the t o t a l bound residue was i n the forT,1 of proL1etryn, the reL1ainder constituted hydroxy propazine (
8%) and u n i d e n t i f i a b l e methanol soluble r,l a t e r i a l
(18%).
During the d i s t i l l a t i o n
about 2~o
of the bound residue ~las decor,lposed to 14C02. IJhen a i r or nitrogen were used as the sueep gases, considerably lower recovery of the compounds and a greater decomposition to 14C02 occurred.
Our attempts to eliminate completely or reduce s i g n i f i c a n t l y the then,lal
decomposition of 14C bound residues to 14C02 during d i s t i l l a t i o n
have not been
successful • The HTD technique described above has been successfully used in our laboratory to detemine 14C bound residues in oat plants grown in 14C-prometryn treated s o i l
(22). Tile
dried root and shoot samples a f t e r exhaustive e x t r a c t i o n with chloroform and methanol-water were analyzed by the HTD technique.
GC and GC-{'IS Analyses of the d i s t i l l a t e s
indicated tile
presence of a r,lono-N-dealkylated compound, namely, 2-(methylthio)-4-amino-6( i s o p r o p y l a ~ H n o ) - s - t r i a z i n e , and traces of 2 - ( m e t h y l t h i o ) - 4 , 6 - d i a m i n o - s - t r i a z i n e .
775
Heat to 800 C with He flow at 50 ml/min
Tra~) IV Evaporate methanol and add water Scintillation counting Extract with ether I F 1 Organic phase Aqueous phase
Wash with methanol
Concentrate
Concentrate
Evaporate and dis solve in methanol
Adjust oH 9-10 and extract with ether
Organic phase
I
Evaporate and dissolve m methanol
,;
i [~
C o m b i n e and concentrate to a small volume
Aqueous phase
i
Discard
i
1
]
f 3 Evaporate to just dryness, redissolve Scintillation counting in chloroform and chromatograph on acidic A1203 column pre-washed with chloroform ]_ r~ 7 Elute with chloroform Elute with methanol ~ 1 ---3 I i- - - - ] Scintillation Evaporate Scintillation Concentrate to a small count mg to dryness counting volume Redissolve in 10% acetone in hexane and chromatograph on acidic A1203 column prewashed with hexane I ,~
.
Elute with 5% acetone in hexane Discard
Figure 2.
i
Elute with 25% acetone in hexane G LC
Methvlate I Evaporate to iust dryness, redissolve m 10% acetone in hexane and chromatograph on acidic A1203 cotumn prewashed with hexane
F
L
E~ute with 25% acetone in hexane G LC
]
Elute with 5% acetone in hexane Discard
Schematic diagram for the analysis of none×tractable
(bound) residues (23),
776
Recently ~e used the HTD technique f o r d e t e m i n i n g 14C bound residues in hu~ic substances.
The soil containing bound (nonextractable) 14C residues was f r a c t i o n a t e d i n t o
huuic substances by a l k a l i e x t r a c t i o n follo~led by acid p r e c i p a t i o n . material
Freeze dried huuic
(50 r.lg) containing 14C bound residue w1as subjected to HTD and tile F.laterial in
d i f f e r e n t traps analyzed (Fig. 2).
I t was observed t h a t a considerable portion of 14C
residues in huuic materials ~las cor.lprised of the parent herbicide and i t s uonodealkylated product (25). ~lhile our studies ~lere in progress (22,23), Balba et a l .
(4), reported the successful
release of t i g h t l y complexed 3 - c h l o r o a n i l i n e and 3 , 4 - d i c l ~ l o r o a n i l i n e froa plant l i g n i n s using a s i u i l a r technique.
I t appears t h a t the HTD technique developed in our laboratory could
also be used f o r the analyses of other bound pesticide residues in s o i l and plants. BOUND RESIDUES IN SOIL Bound pesticide residues in s o i l are the focal p o i n t of several recent studies in various laboratories.
Table 1 suFx,larizes some of the work reported during the past few years on
bound residues in d i f f e r e n t s o i l s f o r a nullber of pesticides.
In most of these studies,
radiolabeled pesticides ~lere used and the bound r a d i o a c t i v i t y ~#as detemined by cor.lbusting the extracted soil to produce 14C02, which was then quantitated by s c i n t i l l a t i o n counting.
The bound residues expressed as % of applied pesticide ranged from 7 to 90%.
A
s i g n i f i c a n t quantity of bound residues is being "any amount of unextractable residue, greater than 10% expressed as the parent co,.Ipound, reuaining one year a f t e r a single treazment" (J). Therefore, i n s i t u a t i o n s ~here s i g n i f i c a n t concentrations of bound pesticide residues occur i n s o i l , the t o t a l pesticide residues in s o i l may be underestiuated as the bound residues w i l l not be detected in routine a n a l y s i s . Foruation of bound residues have been shown to increase ~.lith incubation t i , l e .
In our
studies i t ~las observed t h a t the amounts of the extractable 14C residues recovered frou the uniforl.~ly 14C-ring labeled prometryn treated organic s o i l decreased over an incubation period of 150 days (Fig. 3). s o i l bound 14C residues.
This in t u r n , corresponded to an increase in the f o m a t i o n of
Thus by the end of the incubation period (150 days), extractable
14C residues decreased to 36.5% w h i l e the bound 14C residues (deten.lined by cor.lbustion to 14C02) increased to 43.0% of the i n i t i a l l y
added 14C.
The t o t a l r a d i o a c t i v i t y
recovered at the end of 150 days auounted to about 80% of t h a t i n i t i a l l y
applied.
SiLlilarly,
the r a d i o a c t i v i t y recovered by HTD of samples increased ~ i t h incubation time and by the end
777
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778
of 150 days amounted to about 40% of the i n i t i a l l y added 14C. However, the amounts of 14C recovered by HTD were slightly lower than those obtained by combustion to 14C02 (Fig. 3).
GC examination of the methanol-extractable residues from the incubated soil
indicated that the total amounts of prometryn extracted decreased from 95 to 23% of the i n i t i a l l y applied herbicide, whereas that of hydroxypropazine (calculated in terms of prometryn) increased from 1.7 to 9.4% over the incubation period.
Furthermore, analyses of
tile high temperature d i s t i l l a t e s by GC indicated that the amounts of bound prometryn residues increased from 0.2 to 19.4% of the i n i t i a l l y applied herbicide.
Thus, the total recovery of
prometryn residues (extractable plus bound) at the end of an incubation period of 150 days was about 52% of the applied herbicide.
The disappearance of radiocarbon added to the soil
could not be accounted for only by the extractable and bound 14C residues (Fig. 3).
It
appears that the loss of radiocarbon up to about 20% due to volatilization, possibly due to evolution of 14C02 and volatile degradation products, occurred during the incubation period. BOUND RESIDUES IN HUI41C SUBSTANCES Recently, we determined the distribution of bound 14C residues among various humic fractions of an organic soil containing 57.4% bound 14C of the total i n i t i a l l y applied 14C-prometryn (25).
The method of extraction, separation and purification of humic
materials outlined in Fig. 4 is analogous to that described by Schnitzer and Khan (32).
The
yield of humin, humic acid (HA) and fulvic acid (FA) amounted to 0.67, 0.04 and 0.12 g/g soil, respectively. Proportions of total bound 14C in humin, HA and FA fractions were 57, I I and 26%, respectively (Fig. 4).
The incorporation of 14C bound residues into humic
materials observed in our study is consistent with those reported for other pesticides described earlier.
The 14C residues were concentrated in the humin fraction (Fig. 4).
Furtherhlore, only a small amount of 14C (3.3%) was released in solution by solubilizing the mineral fraction from humin by the HCI-HF treatment. The average molecular weight of bound 14C residues associated with humin and IIA appeared to be greater than 12000 since very l i t t l e 14C was dialyzable. The presence of 14C bound residues in FA fraction (26%) is of special interest.
The l a t t e r is a naturally occurring water soluble low molecular weight
polyelectrolyte and is considered to be the dominant soluble organic fraction present in the soil solution under f i e l d conditions.
I t is also well known that FA is present in many
surface waters and imparts a yellow to brown color in natural water (7,32).
I t is l i k e l y
779
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-
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-
~
-:
J
-~
TIE + B l )
\ \\
i
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~
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Time
Figure 3.
" "
100
" ~3
of i n c u b a t i o n {days)
Extractable and bound 14C residues in an organic soil during a 150-day incubation period, after soil treatment with uniformly 14C-ring labeled prometryn at 12.4 mg/kg (0.047 uCi/g). Curve E, extracted 14C; curve Bl, bound 14C determined by combusting soil to 14C02; curve B2, bound 14C determined by high-temperature distillation technique; and curve T, total of extractable (E) and bound (Bl) (23).
780
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781
that bound residues associated with the soluble FA fraction may become bioavailable to both plants and exposed aqueous or soil fauna. I t was observed that a considerable proportion of 14C residues in humic materials was comprised of the parent herbicide, i . e . prometryn, and i t s monodealkylated product namely, 2-(methylthio)-4-amino-6-(isopropylamino)-s_-triazine
(Table 2).
However, FA fraction
contained an appreciable amount of 2-hydroxy analogue of prometryn [2-hydroxy-4,6bis(isopropylamino)-s--triazine]. Alkaline extraction with O.IN NaOHunder nitrogen, widely used for the extraction of humic materials from s o i l , was thought to hydrolyze or chemically degrade the herbicide residues in soil.
However, our experiments revealed nearly
quantitative recoveries of unchangedprometryn from aqueous solutions when the herbicide was extracted with O.IN NaOH (pH 13).
Hitching and Roberts (17) used 1.25 N aqueous NaOH
solution at 80°C for 2 h for extraction of humic materials containing bound residues and were able to identify small amounts of the total radioactivity in HA.
Table 2.
BoundResidues in Humic Materials (25)
Fraction
Prometryn ppm
Humic acid F u l v i c acid Humin
Hydroxypropazine ppm
0.3 0.3 1.9
0.I 0.9 NDa
Deisopropylprometryn ppm 0.2 0.I I.I
aNondetectable, <0.01 ppm. MECHANISM OF BOUND RESIDUES FORMATION The general consensus appears to be that in the formation of bound residues, p e s t i c i d e or i t s metabolites are chemically bonded i n t o the soil organic matter.
However, our recent data
provide evidence that support the contention that physical binding may also play an important role in the formation of soil bound residues. Isothermal heating has been used for i n v e s t i g a t i n g the mechanism of thermal decomposition of organic matter (31).
I t was thought that thermoanalytical methods applied to soil and
humic materials containing bound residues may provide useful information on the nature of p e s t i c i d e binding. The high temperature d i s t i l l a t i o n
apparatus described e a r l i e r was used f o r isothermal
782
heating (23).
An air dried soil sample (200-300 mg) containing 14C bound residues, was
placed in a porcelain boat and inserted in the middle of the quartz tube preheated to the desired temperature. Helium or a i r was used as purge gas at a flow rate of 40-50 ml/min. The sample was heated at the desired temperatures isothermally for a period t i l l release of 14C was observed in the methanol and CO2 traps (23).
no further
In preliminary
experiments i t was observed that 45 min. and 60 min. heating was adequate for a maximum release of 14C from the soil under a stream of a i r and helium, respectively.
In some cases
samples were also heated under oxygen stream and covered by a combustion catalyst (CuO-Al203).
At the end of the heating period the radioactivity of the trapping
solutions was determined by l i q u i d s c i n t i l l a t i o n counting. In other experiments a freeze dried humin sample (50 mg) containing 14C bound residues was heated isothermally under a stream of air, nitrogen or helium as described above for a fixed period of 60 min. Preliminary experiments indicated that heating for a longer period did not increase the 14C release.
At the end of the experiment the char resulting from
heating was removed and analyzed for C, H, N and S. The radioactivity of the trapping solutions was also
Oxygenwas calculated by difference. determined. The yields of HA and FA
obtained from soil by the fractionation procedure used in this study were not adequate to allow for the thermal analysis of these materials. The thenaal profiles of 14C bound residues of soil in a i r and helium streams are shown in Fig. 5.
The principal reactions occurring under air and helium during the isothermal
heating of soil containing 14C bound residues differed in that ( i ) 14C was released over a broader temperature range under helium, ( i i ) the release of 14C under helium occurred and complete decomposition took place at temperatures that were lO0-200°C higher than under a i r , and ( i i i ) all the bound 14C released was decomposed to 14CO2 in air stream, whereas in helium stream a portion of bound 14C released (up to 26%) remained undecomposed as evidenced by'the presence of radioactivity in the methanol trap.
From these observations
i t appears that the release of bound 14C was associated with the therlaal decomposition of the organic matter fractions of the soil.
Under oxygen (data not shown) or a i r the
conditions were more conducive for a faster organic matter decomposition than under helium. Thus, the release of bound 14C on heating under helium was considerably slower than under air.
783
The themaal profile of bound 14C residues in humin under air, helium or nitrogen ~as similar to those obtained for soil. temperature is sho~n in Fig. 6. the CO2 trap.
The amount of 14C released from humin as a function of
In an air stream nearly all the radioactivity was found in
However, heating under helium or nitrogen resulted in release of 14C, part
of which(up to 35%) was also trapped in methanol. While very l i t t l e 14C was released under air at 150°C, extensive release occurred between 200-300°C (=90%) reaching I00% at 500°C or higher (Fig. 6).
On the other hand while under helium or nitrogen
radioactivity was released at 150°C about half of the total bound 14C was released at 325°C-350°C and by 700°C the recovery of 14C in the trapping solutions was nearly quantitative.
Differential thermo gravimetry of humic materials showed elimination of all
COOH + OH groups between 200°C-400°C and decomposition of humic 'nuclei' at 450°-550°C
I00-
9O
BO
70-
60.
5O
40-
~o-
k'o.
I0-
200
Figure 5.
4~
6~
8~
Releaseof bound 14C from soil on isotherlaal heating under (o) air, and (m) helium (25).
784
0
e
200
400
600
8oo
TEMPERATURE C
Figure 6.
(32).
Release of bound 14C from humin on isothemnal heating under (o) a i r , ( • ) h e l i u m , and ( • ) nitrogen (25).
Thus, one may assume that s t a b i l i t y of bound pesticide residues is due to their l i n k
with phenolic OH or COOHgroups of soil organic matter or humic materials.
For example,
chemically s t a b i l i z i n g reactions between phenolic hydroxyls and free aro~natic amino groups from the metabolites of herbicides have been postulated.
Thus, under these conditions the
presence of unchanged parent molecules of pesticides in the bound residues is not expected. However, our study demonstrated that in addition to small amounts of hydroxy and dealkylated analogues, a considerable portion of the bound residues in the organic soil and humic fractions was present in the form of prometryn.
Thus, conceivably in addition to the
chemical binding of the metabolized pesticide, physical binding of the parent ~nolecule or i t s derivative also play an important role in the formation of soil bound residues.
I t has been
suggested that humic materials are made of phenolic and benzenecarboxylic acids joined by llydrogen bonds to form a molecular sieve type polymeric structure of considerable s t a b i l i t y (32).
One of the characteristics of the structure is that i t contains voids or holes of
d i f f e r e n t molecular dimensions which can trap or f i x organic molecules, such as pesticides.
785
From the thermoanalytical data i t appears tilat the thermal decouposition of organic matter or humic substances weakens the structure by eliminating the functional groups and eventually decomposing the 'nuclei'
This in turn pen.lits tile release of bound residues t~lat are
trapped in the structure.
Further support for such a uechanism comes frou tlme methylation
study discussed l a t e r . Schnitzer and Hoffman (31) used the atouic H/C vs. O/C graphical-statistical r e l a t i o n ship of Van Krevelen (37) to study tlle principal reactions occurring during the thermogravil;letry of soil organic matter under nitrogen,
i t ~las thought that this approac~l
uay y i e l d some valuable information on the relationship bet~een the release of bound 14C and reaction processes occurring during heating of the huuic ~laterials. vs. O/C ratios are plotted for humin heated under nitrogen.
In Fig. 7 atomic H/C
I t appears that the r,min
reaction of humin up to 350°C ~as dehydration (31) ~#hich uas accoupanied by the elii~lination of about half of the bound 14C. The next reaction up to 500°C could be ascribed to dehydrogenation ~,lhich resulted in another 40% release of bound 14C. F i n a l l y , betvleen 500-700°C the reaction processes involving both dehydration and dehydrogenation (31) could have taken place accompanied by the release of the remaining 14C. Methylati on of HA and Humin: The HA or humin sauple ~las suspended in uethanol and ,,lethylated ~lith freshly prepared diazor,lethane repeatedly for several days u n t i l tlme 14C released in solution remained constant. washed ~#ith methanol several tiues.
Tile mixture was filZered and the sauple residue
The coubined f i l t e r a t e was concentrated to small
volume and analyzed by l i q u i d s c i n t i l l a t i o n counting.
The dried solid residue ~as coubusted
to 14CO2 for deten.lining the reuaining total 14C residues. Exhaustive methylation of humin and hui,lic acid resulted in about 25-30% release of the i n i t i a l l y bound 14C. i.lethylation reduces hydrogen bonding betueen tile 'binding units' of humic materials (32).
Thus, the molecular sieve-like structural arrangement in humic
material is ueakened. The bound pesticide residues, adsorbed on i t s surface and trapped in internal voids, may then be released as indicated in our study. PHOTOCHEI41CAL STABILITY OF BOUNDRESIDUES I t became of interest to determine ~lhether photodegradation under UV l i g h t could be a process for the release of bound 14C residues.
Soil samples (l gm) containing 14C bound
residues v#ere irradiated with UV l i g h t using a 15}I General Electric 45 cr,1 germicidal lamp (253.7 nm). The suspension (Soil:Ilater = 1:600) uas placed 15 cu belo~ the la,.ip, continuously
-/86
022 C C 4< I*~ IaCl
#
~0 o
cIs,
OB-
g
SOCS3%14CI
4 ~ C 184%14C)
500"C '93% 14C~
o~
O2
. . . . .
~ 0
q O4
2
o!6
,~
~ T O M I C O, C R A T I O
Atomic H/C vs. O/C ratios for humin containing 14C bound residues
Figure 7.
heated under nitrogen.
Numbers in parenthesis show the percentage
of 14C released at different temperatures (25).
stirred with a magnetic s t i r r e r and irradiated for 216 h.
Preliminary experiments indicated
that under the experimental conditions described UV i r r a d i a t i o n of soil suspension for a longer period or at higher water contents did not result in any further release of 14C in solution.
At the end of the experiment the soil suspension was centrifuged and the soil
residue was exhaustively extracted with methanol.
The aqueous supernatant was combined witl~
the methanol extract and concentrated to a small volume on a rotary evaporator.
An aliquot
of this was analyzed by l i q u i d s c i n t i l l a t i o n counting while the remaining portion was evaporated to dryness.
The material was then dissolved in chloroform and processed for gas
chromatographic and mass spectrometric analyses.
Residual methanol from the extracted soil
was allowed to evaporate by a i r drying the soil sample and a portion of i t was combusted to 14C02 to determine the remaining total 14C. The control, consisted of soil containing 14C bound residues, was also processed as described above except the suspension was kept in dark.
i
: ~P.~elimtnary experiments indicated that the decomposition of prometryn in water when exposed to UV l i g h t was complete in about 3 h.
The herbicide was converted quantitatively to
787
i t s hydroxy analogue.
I t was observed that the s t a b i l i t y of bound 14C residues in soil
suspension was affected by UV l i g h t .
Thus, at the conclusion of the exposure period of 216 h
tile extractable radioactivity accounted for about 30% of the total bound 14C i n i t i a l l y present in the s o i l .
UV irradiation for a longer period did not increase the amounts of
extractable 14C. Only up to 3-5% or less of the total bound 14C was recovered as extractable in the dark.
GC and GC-MSanalyses indicated that methanol extract of the UV
irradiated soil suspension contained mainly hydroxypropazine. Thus, i t appears that UV l i g h t caused the release of some of the bound prometryn residues from soil which were subsequently decomposed to the hydroxy analogues. For comparative purposes, soil samples f o r t i f i e d with prometryn (24 ppm) were also processed under the experimental conditions described above. The herbicide in the soil suspensions was readily decomposed under the influence of UV light.
Thus, at the end of a 72 h exposure, 75% of the material i n i t i a l l y used to f o r t i f y
soil sample was extractable in the form of hydroxypropazine. Although 70% of the bound 14C in soil remained unextractable even after prolonged exposure, the fact that UV l i g h t caused the release of 30% bound 14C should be of concern in detemaining the environmental s t a b i l i t y of bound residues. In other experiments, HA or humin sample (lO mg) was suspended in IO ml d i s t i l l e d water, stirred and i r r i d i a t e d with UV l i g h t for 96 h as described before. Concurrently samples were also run in the dark. The suspension was centrifuged, solid residue washed with water and the aqueous phases combined. The solid residue after air drying was combusted to 14C02 for determining the 14C remaining in the bound form. The aqueous phase was concentrated to a small volume and analyzed by liquid s c i n t i l l a t i o n counting. Exposure of aqueous solutions of humic acid or humin containing bound 14C residues to UV l i g h t for 96 h resulted in 6.1 and 1.2% extractable 14C, respectively.
The relatively
small release of 14C from bound form was unexpected since hulaic materials have been shown to act as photosensitizers for nonabsorbing pesticides (21).
Prolonged exposure of humin or
humic acid to UV l i g h t did not s i g n i f i c a n t l y increase the release of bound 14C. The concentration of extractable 14C a f t e r UV i r r a d i a t i o n was too low to be analyzed by GC. These observations therefore suggest that bound residues in humin or humic acid f r a c t i o n s of organic matter undergo to very l i t t l e
p h o t o l y t i c degradation.
MICROBIOLOGICAL RELEASE OF BOUND RESIDUES The question of the potential release and
b i o l o g i c a l a v a i l a b i l i t y of soil bound residues
788
has been addressed in several studies.
Recently, we investigated the microbiological release
of bound residues from an organic soil treated
with 14C-prometryn (24).
Extracted soil
containing 14C bound residues uas incubated in a llarburg apparatus ~#ith a l i q u i d inoculum obtained from an untreated and unextracted control soil (Fig. 8).
The control consisted of
untreated soil exhaustively extracted ~ith the same solvents as those used for reuoving tlie extractable residues fro~:q the treated s o i l .
The control sample ~vas also placed in Warburg
vessel and inocului:1 added. KOII (20%) was used in the center wells of the vessels to trap 14CO2 and the temperature maintained at 20°C. The loss of radiocarbon due to evolution of 14CO2 during incubation period of 22 days ~as negligible froi.~ the soil containing 14C bound residues.
The extractable and unextrac
table (bound) radioactivity in the soil after incubation with an inoculum for 22 days amounted to 27.0 and 71.6%, respectively, of the total 14C bound residues (Fig. 8). indicated that microbes released part of the 14C bound residues from the s o i l .
This
In a
preliminary experiF.lent, an aliquot of the soil containing bound residues was ~]ixed with s t e r i l i z e d d i s t i l l e d water, allotted to incubate for about 3 weeks and then exhaustively extracted u i t h methanol.
Analysis of the extracted material revealed negligible auounts of
r a d i o a c t i v i t y ( <1% of the t o t a l ) , indicating that 14C residues were s t i l l bound to the soil uhen no inoculum ~as added. Examination of the extractable residues indicated the presence of prometryn, hydroxypropazine and a small amount of the p a r t i a l l y N_-dealkylated compound, 2-(methylthio)-4-amino-6-(isopropylamiao)-s-triazine .
The results suggest tilat
the microbes i n i t i a l l y released the bound prometryn, ~hich was then degraded in the incubated soil to hydrolyzed and dealkylated products.
Analysis of the bound residues revealed tile
presence of prometryn and small amounts of hydroxypropazine and mono-N_-deaIKylated prometryn.
I t is possible that some of tlle metabolites formed during the incubation period
may have become a part of the bound portion of the residues in the s o i l . PLAINT UPTAKEOF BOUIID RESIDUES Pesticide residues absorbed and translocated in plant tissues may be present in tl~ree possible forms: ( i ) freely extractable residues, ( i i ) extractable conjugates bound to natural co~ponents of plants, and ( i i i ) , constituents.
unextractable or bound residues incorporated into tile plant
The l a t t e r may be considered as analogous to the bound residues in s o i l .
789
14any studies using radiolabeled compounds have revealed that a considerable portion of the pesticide residue may become bound in the plant.
Since bound residues in plants cannot
be removed by conventional solvent extraction techniques designed to extract and detect the parent compound and certain metabolites, more attention should be directed towards evaluating their residual nature in plants.
SOIL CONTAININGl iCA BOUNDRESIDUES
| Incubation with soil inoculum for 22 days in a Warburg vessel
I
I--
1
KOH
SOIL
(~co 2 )
L
=3.Ol:r
Exhaustive extraction with methanol
Not Extractable (Bound residues) 71.6%
HTD
i
GC
Extractable 27.0%
GC
I
GC-MS
GCI-MS
Figure 8.
Schematic diagram for the analysis of extractable and bound residues in soil incubated with inoculum (24).
Recently, we investigated the potential uptake of 14C bound residues by oat plants from an organic soil treated with 14C-prometryn (22).
Soil containing 14C bound residues
uixed with washed silica sand (soil :sand : 1:I .25) were used to grow oat plants in a controlled envirom~ntal chamber. The pots were watered each day as necessary and f e r t i l i z e d with standard Hoagland's nutrient solution during the growing season. After 21 days the plants were harvested and roots and shoots were separated. The analytical procedure used for the extraction, isolation, and determination of 14C residues in plant tissues is sho~m in Fig. 9. The crop of oat plants removed 0.53% of the previously soil bound !4C in the soil-sand mixture used for growing the plants in this experiment. The shoots contained 69.8% of the
790
total plant radioactivity whereas the remaining radioactivity (30.2%) was present in the roots.
Table 3 shows the extractable and unextractable (bound) 14C residues in the plant
tissues as deten,lined according to the scheme depicted in Fi~. 9.
Amounts of total
extractable 14C residues were larger in roots, but were smaller in shoots accounting for 75.0% and 51.1%, respectively, of the total 14C residue.
The chlorofona extracts (nonpolar
residues) contained a very small proportion of 14C, which constituted
only a trace amount
of unchanged prometryn ( <0.05 ppm) as indicated by GC analysis. Ilost of the methanol water soluble 14C residues (polar residues) were present in the shoot and root samples in the conjugated form. This was indicated by the absence of any free metabolites in the eluate obtained by eluting XAD-2 column with methanol (Fig. 9).
However, hydrolysis of this eluate
resulted in the release of a compound which after methylation with diazomethane was identified as the methoxy derivative of hydroxypropazine [2-methoxy-4,6-bis(isopropyl -amino)-s-triazi ne]. The data demonstrate that some of the radioactivity absorbed by plants from the soil containing bound 14C residues became again bound (unextractable) in the plant tissues. Plant bound 14C residues were smaller in roots and greater in shoots amounting to 19.9% and 40.2%, respectively, of the total 14C recovered from each tissue (Table 3).
GC analyses of
the HTD d i s t i l l a t e s indicated the presence of a mono-N_-dealkylated compound namely, 2-(methylthio)-4-al~ino-6-(isopropylamino)-s-triazine,
and traces of 2-(methyltlHo)
-4,6- di amino-s_-triazi ne. The extracted plant tissue containing bound 14C l i k e l y contained mainly lignin, carbohydrate, and denatured protein.
I t became of interest to deterl.~ine the distribution of
plant bound 14C in these fractions (Table 3).
Tlle lignin extracted by the procedure
described in Fig. 9 comprises free and bound lignin.
The l a t t e r is bound to the carbohydrate
of the cell wall and could only be extracted by boiling ~ith dioxane-HCl under N2.
Tlle
data show that the total lignin (free + bound) from roots and shoots contained I0.5% and 28.5%, respectively, of the total 14C bound residues in the plant tissue.
The remaining
solid residue of roots and shoots which presumably consists of mainly carbohydrate and protein contained relatively smaller amounts of 14C residues (4.7% and 7.5% of the total 14C bound residues in roots and shoots, respectively). About 2% of the total 14C was present in the aqueous supernatant, which may be due to the hydrolysis products and can be regarded as extractable residues.
I t is l i k e l y that most of the 14C associated with the
791
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l i g n i n or solid residues fraction ~las comprised of 2-(methyltilio)-4-amino-6-(isopropylah~ino) -s-triazine.
The l a t t e r was identified as the major product in the HTD d i s t i l l a t e s of plant
tissues. I t has been shown that l i g n i n is the r:~ajor bound residue fraction of aniline and chloroaniline compounds in plants.
In a recent study, S t i l l et al. (36) investigated the
molecular nature and identity of bound 3-chloroaniline and 3,4-dichloroaniline residues in rice plants grown hydroponically. The procedure used for fractionation of root tissues was similar to that described in Fig. 9. l i g n i n fraction fro~.~ the roots.
More than 40% of the 14C was present in the isolated
Theseworkers suggested that chloroanilines may be bonded
covalently to lignin via 1,6 addition to a quinone ~,lethide intermediate during the lignin synthesis.
The e-carbon in the l i g n i n side chain ( i . e . the benzylic carbon) was considered
to be the uost l i k e l y chloroaniline nitrogen binding site (36). SUMMARY The use of 14C labeled pesticides has made us aware of the existence of bound residues in soil and plants.
These residues would escape detection by the conventional analytical
methods thereby resulting in an underestimation of the soil or plant burden of total pesticide residues. The methodology for the analysis of bound pesticide residues is s t i l l in the development stage. The total 14C bound residues in soil and plants are usually estimated by combustion of the extracted material to yield 14C02. The high temperature d i s t i l l a t i o n technique may provide a possible means for the chemical identification of bomld residues.
However, the application of this technique to bound residue analysis of other
pesticides has not been f u l l y explored. Contrary to the general consensus that the bound residues become an integral part of tile matrix without recognizable relationship to the original pesticide, data presented in this paper demonstrate that a considerable portion of such residues in soil may also comprise the parent molecule, The organic fraction of a soil appears to have the potential for for~in~ bound residues ~ith pesticides or products arising from their degradation.
I t is suggested
that in addition to chemical binding, pesticides or their metabolites are also firmly retained by organic fractions (humic materials) by a process that more l i k e l y involves adsorption on external surfaces and entrapment in the internal voids of a molecular sieve-type structural arrangement. The l a t t e r may be considered analogous to the "clathrate compounds".
794
Evidence is presented to demonstrate the b i o a v a i l a b i l i t y of soil bound 14C residues to oat plants.
Furthen:1ore, i t is shoun that soil microbes can potentially release 14C bound
residues frou s o i l .
A uajor portion of bound peszicide residues in plant tissues uay be
associated ~vith lignin.
Thus, lignin fon.~ation in plants may serve as a syste~.1 for plants to
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(Received in Germany
iO May 1982)