Environ. Sci. Techno/. 1995,29, 2301-2304
Persistence of DDT and Nature of Bound Residues in Soil at Higher Attiiude
TABLE 1
Climatic Conditions of Dharmsala (Elevation 1211 m Above Sea level) in Himachal Pradesh, India temp (“C) month
DILEEP K. SINGH* AND HARI C. AGARWAL Department of Zoology, University of Delhi, Delhi, 110007, India
The persistence, metabolism, and binding of I4Clabeled and unlabeled p,p‘- DDTin soil a t higher altitude were studied for 1 year under field conditions a t Dharmsala in Himachal Pradesh, India (elevation 1211 m above sea level, 32’15’ N latitude and 76’15’ E longitude). Hollow poly(viny1 chloride) cylinders (17.5 cm L a n d 10 cm id.) open a t both ends were pushed into the ground, and the soil in each of them was treated with 8 pCi of [ 1 4 CI-p,p’-DDT and 10 mg of unlabeled p,p’-DDT. At every sampling time, three cylinders were dug out, and the soil in them was analyzed. About 64% of DDT was lost from these in 1 year. The halflife of DDT was 250 days. At zero time, p,p’-DDT accounted for 98% of the extractable residues, which gradually declined to about 29% in 1 year. The residues consisted mainly of p,p’-DDT and smaller proportions of p,p’-DDE, p,p’-DDD, and DDMU. p,p‘DDE was the major metabolite and accounted for 24% of the extractable residues after 365 days. DDD and DDMU accounted for a maximum of 6.4% and 2.5% in extractable residues after 35 and 305 days, respectively. Initially, the bound (unextractable) residues were very small but increased gradually to a maximum of 8.7% after 1 year. Soil-bound residues could be released from the soil by sulfuric acid without affecting the chemical nature of the residues. The chemically released residues consisted mainly of p,p’-DDT and smaller proportions of p,p’-DDE, p,p‘DDD, and DDMU.
Introduction DDT [l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane] is a relatively persistent organochlorine insecticide, and its residues have been detected in almost all abiotic and biotic components of the environment. It dissipates faster in tropical and subtropical regions than in temperate regions. Its degradationleads to the formation of DDE[1,1-dichloro2,2-bis(p-chlorophenyl)ethylene],DDD [l,1-dichloro-22bis(p-chlorophenyl)ethane],DDMU [l-chloro-2,2-bis(pchlorophenyl)ethylene], and bound residues ( 1 - 4 ) . The formation of bound residues is considered important for pesticide retention (3 and may temporarily reduce the harmful effects of pesticide residues in soil ( 1 , 6). An
0013-936X/95/0929-2301$09.00/0
E 1995 American Chemicai Society
October 20, 1992 November December January 1993 February March April May June July August September October
max
min rainfall (mm)
28.5 12.6 19.1 18.6 20.7 28.8 34.5 37.6 25.2 28.5 27.5 27.3
relative humidity
2.3 4.2 5.8 15.5 22.8 20.4 18.5 20.6 18.8 12.2
(YO)
max
min
5.1
85
49
89 45.5 233.9 1.4 70.1 188.6 752.8 344.8 471.0
86 86 80 68 60 69 98 87 96 67
29 37 37 33 30 48 84 75 80 44
0.0
important mechanism for the release of bound residues from soil is microbial action (7, 8). These may be released gradually from the soil and in time increase the insecticide load of the soil (6). The bound residues so released may be available for uptake by the biota, Le., plants (9, 10) and earthworms ( 1 1 ) . Initial data on the persistence of DDT indicate that it is persistent in soil in the plains, yet the literature is almost silent on its fate at higher altitudes where climatic conditions and vegetation are different from those in the plains. The temperatures may be much lower at higher altitude than in the plains and may be closer to the conditions in temperate climates. Therefore, the persistence, metabolism, and nature of bound residues of DDT in soil at higher altitude in a subtropical region were investigated for 1 year.
Materials and Methods Materials. Uniformlyphenyl ring-labeled [l4C1DDT[[l,l,ltrichloro-2,2-bis(p-chloro[14Clphenyl)ethanel (DuPont, Boston, MA) sp. act. 27.95 mCilmmol]was added to unlabeled p,p’-DDT (99+% pure, HPLC) for field application. All glassware was silanized before use. Methods. Field Treatment and Sampling. The experiment was conducted at Dharmsala (elevation 1211m above sea level, 32’15’ N latitude and 76’15’ E longitude) in Himachal Pradesh, India. A plot (4 m x 5 m) was prepared for the field experiment. The soil was sandy loam, rich in organic matter and slightly acidic (pH 6.3). The maximum temperature was 37.6 ‘C in summer, and the minimum was 2.3 “C in winter (Table 1). The relative humidity was high during the monsoon season (July-September). Total rainfall during experimental period was about 2202 mm. Photoperiod was maximum in June (about 13 h) and minimum in December (about 9 h). Thirty-three cylinders (17.5 cm L and 10 cm i.d.1 open at both ends were pushed down into the soil (1,6) leaving a 3-cm rim above ground to prevent runoff. The cylinders were left undisturbed for about 2 months under natural conditions for equilibration. The soil in the cylinders was treated on October 28, 1992, with 8 pCi of [14C]-p,p’-DDT and 10 mg of unlabeled p,p’-DDTin 10 mL of hexane. Three cylinders were dug out immediately after treatment to serve as the zero-time sample. The remaining cylinders were
VOL. 29, NO. 9.1995 /ENVIRONMENTAL SCIENCE & TECHNOLOGY m 2301
taken out in triplicate at various intervals up to 365 days after soil treatment. The cylinders were immediately covered with aluminum foil, sealed in polyethylene bags, and stored at -20 "C till analyses ( 1 ) . Extraction, Cleanup, and Combustion of Soil. Methods for extraction, cleanup, and combustion were followed as described by Agarwal et al. (I) and Singh and Agarwal (6). The top 10-cm soil column was carefully removed from each cylinder and air dried. Fifty-gram samples of airdried soil, in triplicate, were extracted by 3 vol of methanol in Soxhlet apparatus for 24 h (72 cycles). Extraction for longer periods did not yield any further radioactivity. Sulfuric acid was used for cleanup of the extract since it did not affect DDT, DDE, DDD, and DDMU. The total 14C radioactivity of unextracted and extracted soil was estimated by combusting three 500-mg replicates of soil from each cylinder in a Harvey biological oxidizer Model OX-400. Chemical Release of Bound Residues. Bound residues were released by treatment with concentrated sulfuric acid (6). The released residues were taken up in HPLC-grade methanol for analyses by HPLC. Analysis of Insecticide Residues. Thin-Layer Chromatography. DDT residues were analyzed according to the method described by Agarwal et al. ( 1 ) and Singh and Agarwal(6). The methanol extracts of soil were subjected to TLC analysis on pre-coated Baker-flex Silica gelIB2 plates. A 100 pL aliquot of cleaned, methanol-extracted samples was applied and developed in two solvent systems, hexane and hexane/chloroform/methanol,7:2:1 vlvlv. Standard DDT, DDE, DDD, and DDMU were run alongside for reference. The spots were visualized by rhodamine 6-G and iodine vapor. High-PerformanceLiquid Chromatography (HPLC). The HPLC method developed by Singh and Agarwal (6) was followed for separation, identification, and quantification of p,p'-DDT, p,p'-DDE,p,p'-DDD, and DDMU. A Shimadzu LC-4A liquid chromatograph attached to a C-R3A Chromatopac and SPS-2ASW detectorwithvariable wavelength was used. A CISRP Zorbax ODS column (25 cm L and 4.5 mm Ld.) was found to be most suitable when methanol was used as the eluting solvent at a flow rate of 0.5 mL/min. The absorbance was recorded at 240 nm at 0.16 aufs sensitivity. The retention times for DDD, DDT, DDMU, andDDEwere7.3&0.02,9.1f 0 . 2 , 10.36i0.01,and10.54 i 0.03 min, respectively. Recovery Experiments. The efficiency of different analytical procedures used was determined using samples of 14C-labeledtest compound at every step. The efficiency of Soxhlet extraction of soil with methanol was 98%. Drying the samples in vacuo by Buchner flash rotary evaporator reduced the recovery by 3%. The methanol/hexane partitioning further reduced the recovery by 3-5%. The efficiency of the tissue oxidizer was 99+%. The data were corrected for recovery. Radioassay. A Packard 2000 CA liquid scintillation spectrometer with automatic quench correction facilitywas used. The scintillation cocktail was prepared according to the modification of Chaudhari andYadav (12)of the method of White (13). Analysis of Data. The zero-time samples were taken as 100%. Each value represents a mean of nine replicates. The quantities of residues of DDT and its metabolites were estimated from the disintegrations per minute recovered and the specific activityofthe parent compound. Unlabeled residues were also estimated from peak areas in HPLC 2302 ENVIRONMENTAL SCIENCE 5 TECHNOLOGY / VOL. 29. NO. 9,1995
TABLE 2
Total, Extractable, and Bound Residues of DDT in Soil at Dharmsala" days after treatment
total residues (,ug/g of soil)
extractable residues bg/g of soil)
bound residues hg/g of soil)
0 7 21 35 65 95 125 180 245 305 365
12.05 i 0.20 10.22 i 0.52 7.86 i 0.12 7.28 i 0.21 5.46 i 0.05 5.43 i 0.06 5.28 i 0.17 4.70 i 0.01 4.47 i 0.01 4.48 It 0.07 4.38 i 0.08
11.79 i 0.05 10.02 i 0.03 7.59 i 0.05 6.83 i 0.14 5.11 i 0.03 4.76 i 0.05 4.66 i 0.04 3.95 i 0.22 3.69 i 0.16 3.62 i 0.14 3.40 0.03
0.01 i 0.0 0.18 5 0.05 0.29 i 0.07 0.37 i 0.01 0.47 i 0.01 0.52 i 0.00 0.63 i 0.01 0.69 i 0.05 0.74 i 0.04 0.91 i 0.01 1.01 i 0.02
a Soil was treated with 8uCi of 14C-labeledand 10 mg of unlabeled DDT per cylinder. Total and bound residues were estimated by
combusting the soil in a biological oxidizer.
20 0
Total residues Extract. residues
W--.
Bound r e s i d u e s
..-.--. .--m
0
3-:
0-0
m-
M --
00
I60
240
I
I
c 320
-
I
c
Days ( a f t e r soil t r e o t m e n t ;
FIGURE 1. Dissipation of DDT in soil at Dharmsala.
analyses. Each sample was chromatographed twice, and each value was an average of 18 analyses. Total and bound residues were estimated by combusting the unextracted soil and methanol-extracted soil, respectively.
Results and Discussion The total 14C activity present in zero-time samples was 174.53 x lo5dpm. The concentration declined from 12.05 pglg soil (dry weight) at zero time to 4.38 pglg soil after 1year (Table2). The dissipation seems to follow a biphasic pattern (Figure 1). In the first 3 weeks, the loss of radiocarbon was rapid; it was about 35% in 21 days. The residues apparently do not get adsorbedlbound strongly and get dislodged by various physical processes including vaporization (1, 6). The dissipation of DDT was faster at higher altitude than in the plains in Delhi ( 4 , 6). This may be attributed to higher W radiation, high rainfall, and soil moisture at higher altitude. These factors may favor a rapid rate of volatilization/co-distillation. In the second phase (21-365 days), the rate of dissipation was much slower possibly due to a low concentration of pesticide, high organic content of the soil, and formation of soil-bound residues (6). Half-Life. The half-life [TI,*]of [l4C1DDTwas 34 days in the first phase and 409 days in the second phase; the overall Tl,2 was 250 days. In previous studies conducted in the plains at Delhi, the overall half-life was found to range from 319 to 343 ( 1 ) and from 234 to 317 ( 4 ) days. It is reasonable to assume that factors such as soil moisture, soil temperature,more intense Wradiation, slightly acidic
TABLE 3
DDT and Its Metabolites in Soil at Dharmsalaa days after treatment
0 7 21 35 65 95 125 180 245 305 365 a
ODD (pg/g of soil)
DDT (pg/g of soil)
0.05 f 0.0 0.03 f 0.0 0.21 f 0.0 0.41 f 0.0 0.04 f 0.0 0.04 f 0.0 0.04 f 0.0 0.07 f 0.02 0.10 f 0.01 0.08 f 0.00 0.12 f 0.01
11.43 f 0.1 9.86 f 0.29 6.26 f 0.06 5.51 f 0.03 4.16 f 0.04 4.15 f 0.07 4.27 f 0.01 3.16 f 0.03 3.02 f 0.07 2.86 f 0.02 2.83 f 0.04
0.09 0.06 0.09 0.08 0.09 0.08 0.10 0.09
f 0.0 f 0.0 f 0.01 f 0.02 f 0.01 f 0.02 f 0.01 f 0.01
DDE hg/g soil) 0.33 0.55 0.59 0.37 0.62 0.74 0.87 0.88 0.94 0.90 0.96
f 0.0 f 0.01 f 0.0 f 0.03 f 0.01 f 0.05 f 0.03 f 0.01 f 0.01 f 0.0 f 0.04
Experimental conditions are the same as in Table 2.
TABLE 4
Soil-Bound Residues at Dharmsala Released Chemicallf days after treatment
total bound residues (pg/g soil)
released bound residues ( p g / g soil)
YO recovery
7 21 35 65 95 125 180 365
0.18 i 0.05 0.29 f 0.07 0.37 f 0.01 0.47 f 0.01 0.52 f 0.00 0.63 f 0.01 0.69 0.05 1.01 f 0.02
0.14 f 0.04 0.22 f 0.05 0.32 f 0.14 0.39 i 0.00 0.38 f 0.00 0.57 f 0.04 0.47 f 0.02 0.86 f 0.01
81.0 f 0.1 78.3 f 0.3 86.7 f 2.0 84.4 f 0.7 73.2 f 0.5 91.2 f 0.9 69.1 f 0.3 85.4 f 1.1
a
DDMU bg/g of soil)
*
Experimental conditions are the same as in Table 2.
pH of the soil, and higher rainfall may account for a lower half-life of DDT at the higher altitudes of Dharmsala than in the plains in Delhi. Aganval et al. (11,Edwards (21, Cliath and Spencer (141, and Spencer (15) reported that volatilization is a major pathway for the fast dissipation of organochlorines from the soil. Framer et al. (16) and Nash (17) showed that in spite of low vapor pressure the organochlorines are lost from the soil by volatilization.Once some pesticide is removed from the soil surface, the loss of remaining pesticide by volatilizationlco-distillation becomes a diffusion controlled process. Extractable Residues. Dissipation of extractable residues followed a diphasic curve similar to that of the total residues. 1 year after DDT treatment, the extractable residues accounted for about 28% of the initially applied DDT (Table 2). These residues were shown to consist of p,p‘-DDT; p,p’-DDE, p,p’-DDD, and p,p’-DDMU by TLC and HPLC. DDT declined from 98% of the extractable residues at zero time to 28.8%in 1year. DDE was the main
metabolite of DDT in soil that increased to 24% of the initial value in 1 year. DDD and DDMU showed the maximum values of 6.4% and 2.53% in 35 and 305 days, respectively (Table 3). The extractable residues accounted for the bulk ofthe total DDT residues in soil. These represent the major component of the total DDT residues that are lost by the processes such as volatilization/co-distillation and mineralization. It is this portion of the residues that is responsible for the adverse effects of DDT on the environment. Bound Residues. Soil-bound residues constitute a significant proportion of the total residues. They cannot be extracted from the soil with organic solvents and therefore cannot be detected or estimated except by application of radioisotopic techniques. The formation of bound residues makes the pesticide residues unavailable to the soil biota. This may be a method of immobilizing a pesticide residue molecule, which can be gradually degraded by different methods (18). In the present study, bound residues constituted only a small fraction of the total pesticide present in the soil and may represent residues bound to the soil organic matter. At zero time, the amount of bound residues was very small (0.01 pg/g). It gradually increased with time reaching a maximum of 8.7%in 1year. Similar data for bound residues of DDT in soil were reported from Delhi ( 1 , 4, 6). At the end of the year, about 36.3% of the initially applied DDT was present in the soil of which more than 23% was in the bound form. During the second phase of dissipation (21 days after DDT treatment), the rate of dissipation was very slow and the proportion of bound residues in total residues increased gradually. It is probable that bound residues may be contributing to the slow rate of dissipation of DDT (5). It has been reported that DDT has higher affinity for hydrophobic sites of the organic matter such as fats, waxes, resins, aliphatic side
TABLE 5
Chemically Released DDT and Its Metabolites from Bound Residues in Soila days after treatment 7 21 35 65 95 125 180 365 a
DDD (pg/g soil)
DDT (pg/g soil)
f 0.00 f 0.00 f 0.00 f 0.00 f 0.00 f 0.00 f 0.00 f 0.00
0.13 40.005 0.19 f 0.008 0.27 f 0.003 0.31 f 0.004 0.30 f 0.007 0.43 f 0.00 0.35 f 0.00 0.62 f 0.01
0.004 0.010 0.020 0.010 0.010 0.020 0.020 0.030
DDMU (pglg soil)
0.01 0.02 0.02 0.03
f 0.00 f 0.00 f 0.00 f 0.00
DDE (pg/g soil) 0.003 f 0.0 0.008 f 0.0 0.030 f 0.0 0.060 f 0.0 0.050 f 0.01 0.100 f 0.01 0.080 f 0.00 0.170 f 0.01
Exuerimental conditions are the same as in Table 2.
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2303
chains on humic and fulvic acids (19,201and lignin-derived materials, and a few polar groups (21). Another reason for the persistence of residual compounds in soil may be traping in soil micropores (22). It is possible that, after long periods of time or under suitable soil conditions, this bound form may be released in the environment due to volatilization/ co-distillation and decomposition (23) and may adversely affect the biota (6). The bound residues so released now become available for degradation and uptake by the biota. Indeed such an uptake by plants has been documented (3, 9, 10). For this reason, it has become important to study the nature of soil-bound residues. Early attempts using a high-temperature distillation technique have been partially successful in the identification of released residues (24). Here, the method developed by Singh and Agarwal(6) was followed. The sulfuric acid treatment of the soil was found to be suitable as it released up to 91.2% of the bound residues (Table 4) and did not alter the nature of residues. HPLC analysis of the released residues showed the presence of p,p’-DDT, p,p’-DDE, p,p’-DDD, and DDMU. Seven days after treatment, p,p‘-DDT accounted for 95%, which decreased to 72.5%in 1year (Table 5). p,p’-DDE gradually increased to 20.5% in a year while p,p’-DDD increased to a maximum of 6.6% in 21 days. A small amount of DDMU was also recorded 95 days after treatment (Table 5).
Conclusions The above results show that the half-life of DDT (250 days) in soil was shorter at the higher altitude of Dharmsala in soil than at lower altitudes in the plains of Delhi (319-343 days). This may be attributed to a slightly acidic pH of the soil, more intense W radiation, co-distillation at reduced vapor pressues, and comparatively high rainfall in Dharmsala than in plains in Delhi. It is interesting to note that even though the soil and air temperatures were lower in Dharmsala, the T1/2of the DDT was lower. Further studies on the reasons for this lower half-life at higher altitude may be of interest. Here, the extractable residues contain a detectable amount of DDMU whereas none could be found in the soils in Delhi. The composition of soil-bound DDT residues very closely reflects the composition of the extractable residues similar to that reported by Singh and Aganval(6) and Zayed et al. (25). It is therefore quite likely that p,p’-DDT, p,p’-DDE, p,p’-DDD, and DDMU may be gradually released from the soil and in time increase the insecticide load of the soil. Thus, the binding of DDT with soil may at best lead only to a temporary nonavailability of the residues to the biota.
Acknowledgments We sincerelythank the International Atomic Energy Agency, Vienna, for the partial support of this work as a part of
2304 m ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 9, 1995
Research Contract 5624IRBIR2. The award of Junior and Senior Research Fellowships of University Grants Commission and Research Associateship by University of Delhi to D.K.S. is greatfully acknowledged. Thanks are due to Mr. Y. C. Katoch of Dharmsala for assistance in setting up the experiment.
Literature Cited (1) .:ganval, H. C.; Singh, D. K.; Sharma, V. B. J. Enuiron. Sci. Health 1994, B29 (11, 73. (2) Edwards, C. A. Residue Rev. 1966, 13, 83. (3) IAEA TECDOC-476. In Isotope techniques for studying the fate of persistent pesticides in tropics, !AM: Vienna, 1988. (4) Samuel, T.; Agarwal, H. C.; Pillai, M. K. K. Pestic. Sci. 1988, 18, 1129. (5) Kaufman, D. D. In Bound and conjugated pesticide residues; Kaufman, D. D., Still, G. G., Paulson, G. D., Bandal, S. K., Eds.; ACS Symposium Series 29; American Chemical Society: Washington, DC, 1976; p 1. (6) Singh, D. K.; Aganval, H. C.J. Agric. Food Chem. 1992, 40, 1713. (7) KhanS. U.; Iverson, K. C. 1. Agric. Food Chem. 1981, 29, 923. ( 8 ) Rack, K. D.; Lichtenstein, E. P. J. Agric. Food Chem. 1985,33,938. (9) Khan, S. U. 1. Agric. Food Chem. 1980, 28, 1096. (10) Verma, A.; Pillai, M. K. K. Soil B i d . Biockem. 1991, 23, 347. (11) Fuhremann, T. W.; Lichtenstein, E. P. J. Agric. Food Ckem. 1978, 25, 605. (12) Chaudhri, B. N.; Yadav, H. S. Indian J. Med. Res. 1969,57, 1287. (13) White, D. R. Int. 1. Appl. Radiat. Isot, 1965, 19, 49. (14) Ciiath, M. M.; Spencer, W. F. Enuiron. Sci. Technol. 1972,6,910. (15) Spencer,W. F. Pestic. Res. J. 1991, 3, 1. (16) Farmer, W. J.; Ique, K.; Spencer, W. F.; Martin, I. P. SoilSci. SOC. Am. Proc. 1972, 36, 443. (17) Nash, R. G. J. Agric. Food Chem. 1983, 31, 210. (18) Kearney, P. C. In Bound and conjugated pesticide residues; Kaufman, D. D., Stili, G. G., Paulson, G. D., Bandal, S. K., Eds.; ACS Symposium Series 29; American Chemical Society: Washington, DC, 1976; p 378. (19) Ballard, T. M. Soil Sci. SOC.Amer. Proc. 1971, 35, 145. (20) Stevenson, F. J. In Bound and conjugated pesticide residues;
(21) (22) (23)
(24) (25)
Kaufman, D. D., Still, G. G., Paulson, G. D., Bandal, S. K., Eds.; ACS Symposium Series 29; American Chemical Society: Washington, DC, 1976; p 180. Walker, A.; Crawford, D.V. Inlsotopesandradiation insoilorganic matter studies; IAEA: Vienna, 1968; p 91. Steinberg, S. M.; Pignatello, J. J.; Sawhney, B. L. Enuiron. Sci. Tecknol. 1987, 21, 1201. Hamaker, J. W.; Goring, C. A. I. In Boundandconjugatedpesticide residues; Kaufman, D. D., Still, G. G., Paulson, G. D., Bandal, S. K., Eds.; ACS Symposium Series 29; American Chemical Society: Washington, DC, 1976; p 219. Khan, S. U. 1. Agric. Food Ckem. 1982, 30, 175. Zayed, S. M. A. D.; Mostafa, I. Y.; El-Arab, A. E. J. Environ. Sci. Health 1994, B29 (11, 169.
Received for review December 23, 1994. Revised manuscript received June 12, 1995. Accepted June 13, 1995.@ ES9407756 *Abstract published in Advance ACS Abstracts, August 1, 1995.
