Indian Journal of Biotechnology Vo l I, July 2002, pp 292-297

Comparison of ELISA and GC Methods to Detect DDT Residues in Water Samples

B E Amitarani *, Akmal Pasha, Putte Gowda, T R Nagendraprasad and N G K Karanth

Pesticide Residue Analysis and Abatement Laboratory, Department of Food Protectants & Infestation Control , Central Food Technolog ica l Research In stitute, Mysore 570 Ot3 , India

Received / 2 Novell/ber 200 / ; accepted 8 February 2002

ELISA and GC methods were used to analyse DDT residues in about 30 water samples collected from different talukas of Mandya District of Karnataka. Polyclonal antibody based immunoassay developed at CFTRI, Mysore, performed well to detect the DDT residues. The minimum detectable level of DDT by ELISA was one part per billion (ppb) in the water samples tested. The insecticide residue ranged from 1 to 20 ppb. Experiments also revealed no matrix effect and hence did not require any prior clean-up. The pH of the water did not interfere in the assay. The ELISA method validated in the present work is specific to DDT. The results of ELISA with respect to DDT residues were found to be comparable to values obtained from the GC analysis of the water samples. The water samples could be directly used for ELISA test, thereby making the analysis quick, simple and cost effective.

Keywords: DDT residues, ELISA, Ge, water samples

Introduction DDT [1, 1'-(2,2,2-dichloro diphenyl trichloro­

ethy lene)] has been ex tensively studied for its toxicity. Monitoring of ubiquitous environmental pollutants such as DDT, has always been considered important for controlling human exposure. As compared to several other countries, the higher body burden of pesticides in Indian population and in drinking water is indicative of hi gher exposure to these chemicals (Jani et aI, 1998). India is one of the major point source countries in the tropical belt and a reason for the global contamination by organochlorines. DDT constituted a major portion of the total pesticide use in Indi a (Gupta, 1991). Pesticide pollutants are hydrophobic in nature, get adsorbed on soil/sediment and accumulate in the tissues of organisms. Bottom sediments and soil is the ultimate sink for pesticide pollutants , which are brought into aquatic system through surface run-off and atmospheric fall-out.

In India, DDT was routinely sprayed to control mosquitoes to eradicate malaria in most of the irrigated lands growing rice and sugarcane. There are chances for DDT to contaminate the different water sources.

*Author for correspondence: Tl'l: +91 -0821-514760, 516802 ; Fax: 91- 0821- 517233. E-mail-amitharani @yahoo.com

DDT is generally analysed by gas chromatography (GC) with electron capture detector, mass metry or High Performance Liquid Chromatography (HPLC) (Dikshit et ai, 1990) . Each of these methods, which needs pesticide ex tract ion, clean-up and concentration of the extract by well-trained personnel, is labour intensive, time-consuming and ex pensive, making the method unsuitable in environmental studies and export sample ana lysis. With the increasing demand for pesticide res idue analysis certification in export houses, quarantine department and textile trade industries, there is a need to develop simple, quick, cost effecti ve and sensi ti ve assays to detect pesticide residues. Under these circumstances, immunoassays are fast being developed and becoming popular.

Immunoassays are now routinely used in clinical analysis of proteins, hormones and drugs , and developed both as screening tool s and as quantitati ve analytical methods for pesticide residues in the environment (Jung et aI, 1989; Wratten & Feng, 1990; Ferguson et ai, 1993; Skerritt, 1994 ; Skerritt & Amitarani, 1996; Karanth et ai, 1998). ELISA is fast gaining ground as one of the quick analytical methods for analysis/screening of pesticide residues in foods, soil, water etc.

Considering the use of DDT in India for the eradication of malaria, the presen t study was undertaken to monitor DDT in water samples from

AMITARANI el al.: DDT RESIDUES ANALYSIS IN WATER SAMPLES BY ELISA AND GC 293

different sources in a highly irrigated agricu ltural district. The study covers the effect of the matrices in the water samples on the DDT assay and validates the ELISA data with GC analysis. This is first report regarding comparative study of ELISA (competitive) and GC as a detection method for residues of DDT in different sources of water samples.

Materials and Methods

Materials The general glass wares and other plastic wares,

required for analyzing DDT in the samples by ELISA and GC, were procured from the local market.

Chemicals. All the fine chemicals were procured from Sigma, USA. General solvents and salts of high purity were obtained locally. High binding immunoassay plates were purchased from Nunc, Denmark. The Hapten, polyclonal antibody and HRP­pesticide conjugate for DDT assay were produced at CFTRI, Mysore using New Zealand rabbits. The rabbits were procured from the Animal House, CFTRI and maintained under good laboratory conditions at the Central Animal Facility , CFTRT. The rabbits were fed pellets and water ad libitum.

Water samples. About 5-10 water samples were collected from different areas from Mandya District, Karnataka, amounting to a total of 30 samples. The samples were collected from the following areas: Srirangapatna (Samples S I to S 10), Nagamangala (N I to Ns), K.R. Pet (K I to Ks), Pandavapura (PI to P9) and Malavalli (M I)' The water samples were from sources like borewell, lake and channels. The water from these sources are also used for drinking purpose in and around Mandya District, Karnataka, which is about 40-50 kms away from the host institute. The samples were stored at 4°C before use and analyzed within 24 hrs of collection.

Methods 1. Hapten design and sYllthesis. The Hapten used in

the present study was DDT-OH conjugated to ovalbumin. The details of the synthesis and design have been described in Amitarani et al (2001).

2. Antibody production. Rabbits were used for the production of antibody according to the method described in Amitarani et al (200 I).

3. Pesticide dilution. DDT was dissolved in methanol to get a 1000-ppm solution (I mg/ml). It was serially diluted with distilled water from a 1000 ppm

stock to get final concentrations of 10, 3.3 , 0.3, 0.1, 0.03, 0.0 I and 0 ppm. Pesticide dilutions were made using solvents, which were further diluted by 1110 in phosphate buffered saline with 0.5% fish gelatin (PBS-FG) before loading onto the plate. Pesticide dilution must be done in glass tubes as pesticides stick to plastic surfaces. Each pesticide dilution from 0 to 10 ppm was added in duplicate to the antibody-coated plate. The antibody was coated in carbonate buffer (PH 9.6) at a concentration of I Ilg/IOO Ill/well and left overnight at room temperature. The plate was emptied the next day and washed with buffer (PH 7) and blocked with 1% BSA solution (ISO Ill) and left for 1 hr incubation at room temperature. The BSA solution was then removed and the plate tapped dry on a towel/tissue paper and used to perform the assay.

4. HRP-pesticide conjugate dilution. It was prepared by dissolving the HRP in phosphate buffer, cooled in an ice bath and the active ester dissolved in dry DMF, shaken and allowed to sit at 8°C overnight, dialysed three times against PBS and stored at 8°C. Equal quantities of HRP and Hapten were conjugated in distilled water in the presence of EDC, dialysed against PBS and used. HRP conjugate was diluted in SO mM PBS-FG. A stock of Y2 k was made in SO mM PBS-FG. 100 III of this 1/2 k stock was added to the 15 ml of SO mM PBS-FG to get final working concentration of 1/300k.

Procedure to Load the Plate Each 100 III of pesticide and HRP-conjugate

(l/300k) were added in duplicate to the plate wells . The pesticide and HRP were allowed to bind with the antibody for 1 hr at room temperature. The plate was then washed with wash buffer three times to remove the excess pesticides/HRP and the plate tapped dry. Then the substrate chromogen (tetramethyl benzidine) solution was added (1 SO Ill) and allowed for 30 min for the colour development. The reaction was stopped after 30 min by adding SO III of the stop solution (2.5N H2S04). The absorbance was read at 450 nm on a plate reader. A standard graph was plotted using the 00 vs Concentration on a semi log graph.

In this method, greater the concentration of the pesticide, lesser the colour is produced at the end of the test.

Matrix Effect Studies

Organic solvents are used to extract the pesticides from the food sample. Often, along with the pesticide,

294 INDIAN J BlOTECHNO L, JULY 2002

vari ous constituents of the food/water like carbohydrates, pigments, fa ts, mineral sa lts are also ex trac ted. These constituents are ca lled matrices and can interfere in the ELISA. Thus, there is need to remove such matrices from the samples before perfo rming the assay.

Matrix effect of water 0 1/ DDT assay. The water sampl es to be analysed were Ll sed direc tl y in the preparation of a matri x curve and a graph was prepared where 1000 ppm pes ti cide solution was diluted with the samples instead of di stilled water. If there is no matri x interference, the matri x curve will run parallel to the standard curve with di stilled water. In the presence of matri x interference, the curve shifts to the right.

Analysis of DDT Residues in Water Samples by Immunoassay

The sampl es stored at 4°C were analyzed within 24

hrs of co llect ion. 100 III of water sample was added d irec tl y to the well and concentrati on of DDT in these samples was read against the di stilled water standard graph (positi ve contro l).

Analysis of DDT Residues in Water Samples by GC

The samples were analysed by a modifi ed method (Dikshit et ai, 1990). In a separating fl as k, 5 ml of water sample was taken. To thi s, 5 ml of hexane was added. The fl ask was shaken well to shi ft the pesticide fro m the water sample to the hexane layer, whi ch was co llec ted in the test tube and a llowed to evaporate to

dryness. The dried res idue was stored at 4°C. The

res idue was dissolved in 20 III of hexane and I III of the sample injected . The pesticide ex trac tion was according to the method o f EPA .

Estimation of DDT Using GC

DDT was estimated using GC (GC 8000 seri es Fi sons instruments) equi pped with a 63N electron capture detector connected to a digi tal venturies data processor. A 183 cmx5. 1 cm stainless steel column packed with 10% OY- 17 (non po lar vinyl sili con) on chromosorb-w was used and the temperatures were kept as : injec tor, 250°; column : 230°; and detec tor, 300°C. The carri er gas was N2 (99.9 % pure) with a fl ow rate of 30-mllmin . DDE and DDT standards were di ssolved separate ly in GLC grade hexane and di ffe rent concentrations were inj ected. A standard graph was prepared by plott ing DDT and DOE

concentrations agai nst the square root of the peak areas.

Estimation of DDT and DDE in the Water Samples byGC

The cleaned and d ried res idues from water samples of different pl aces were di ssolved in hexane (20 Ill ),

o f whi ch I III was inj ec ted into the GC at the standard conditi ons desc ribed above for reso lving DDT and DO E. Thi s method permitted the es timati on of DDT and its metabolite ODE simultaneously without prior separati on of the two from the extracted mi xture.

Q llantificatio Il

Peak areas and retenti on time of DDT and its metabolite were co mpared with respective standards injected after every 3 samples.

Results

I. Standard Graph of DDT by ELISA In the DDT standard graph by ELISA (Fig. 1), the

ICso value was 10 ppb. In the competitive assay, absorbance is in versely proportional to the pes ti c ide concentration.

II. Matrix Effect on DDT Assay

On analyzing different samples by ELIS A, matrix effec t was seen only in two muddy samples (Fig. 2). The pH of the samples ranged from 6-8 and had no ad verse effect on the assay.

III. Analysis of DDT Residues ill Water Samples (a) By ELISA. ELISA could detect DDT res idues in

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AM ITARANI el al.: DDT RESIDUES ANALYSIS IN WATER SAMPLES BY ELISA AND GC 295

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water samples. The minimum residue detected was I ppb and the maximum was 20 ppb in the samples.

(b) Standard graph of DDT by Cc. DDT and DOE, as shown in the standard graph (Fig. 3), cou ld be resol ved easi I y and had a retention ti me of 19.47 and 13 .2 min , respective ly.

Estimation of DDT and Its Metabolite The GC-ECD analys is showed that the least

detectahle quantity was 500 pg for both the molecules under tk ilbove explained condit ions of analys is and the RI for DDT and ODE were 20lh and 151h mill, respectively. The relationship between the concentration and square root of the peak area was found to be linear. The concentration of DDT and DDE res idues are shown in Fig. 4.

Comparison of GC and ELISA as Analytical Tools f or Pesticide Residues in Water

GC and ELISA were comparable (Fig. 5) having good correlation, r= O. 98.

Discussion The water samples co ll ected for thi s study from

different sources are used by the local peop le for drinki ng, cattle rearing and irrigation. The residues found in the different water samples were DDT (1-20 ppb) and DOE ( 1-6 ppb) . The detected DDT and DDE residues were within the minimum dai ly intake range in water (Anonymous, 1986). Th us, it can be concluded that the water is safe for consumption and does not have any adverse effect if consumed/used for various acti viti es .

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INDIAN J BIOTECHNOL, JULY 2002

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AMITARANI et al.: DDT RES IDUES AN ALYSIS IN WATER SAMPLES BY ELISA AND GC 297

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The present study indicates that analysis by ELISA is comparable to GC and can be used for the analysis of DDT residues in water samples. ELlSA has more advantage over GC in that the sample could be used directly without clean-up . By ELISA, a ll the 30 samples could be analysed within two days , whereas by GC, the whole process takes about 15 days. The DDT residues found were within the permissible limits and were fit for drinking and other uses.

Acknowledgement The authors are thankful to the Director, CFfRI ,

Mysore, for his encouragement. The authors are also thankful to Dr Srinivas , DHO, Mysore, for sharing the data on the use of DDT in various taluks of Mandya District for the control of malaria.

References Amitarani BE e/ ai, 200l.Application of ELISA-A quick ,

simple. inex pensive and sensiti ve assay method to analyse DDT residues in grapes. Asian J En viroll Biotechnol Microbial. 3, 167- 17 1.

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Dikshit T S S et ai, 1990. Res idues of DDT and HCH in major sources of drinking water in Bhopal, Indi a. UN Bul/ Environ Con/alii Toxicol. 45. 389-393.

Ferguson B S e/ ai, 1993. Pesticide testing by enzyme immunoassay at trace levels in environmental and ag ri cultural samples. Sci Total Environ , 132,4 15-125.

Gupta Y P, 1991. The Illustrated Weekl y of India. Jan 12-13.

Jani J P et ai, 1998. Dichl orod iphenyl trichloroethyl and hexachlorocyc lohexane in human adipose ti ssue of the Indian popUlation. Scaml J Work Environ Heal/h, 14,20 1-204.

Jung F et ai, 1989. Use of immunochemical tec hniques for the analysis of pesticides. Pestic Sci, 26,303-317.

Karanth N G K et ai, 1998. Seeking agric ultural produce free of pesticide residues. in Australi an Centre fo r In ternational Agricultural Research (ACIAR) Proc No. 85. Pp 263.

Skerritt J H, 1994. Fi eld immunoassays for pesticide residues in agrochemical immunoanal ysis. in New Frontiers in Agrochemical Immunoassay, edi ted by D A Kurtz, J H Skerritl & L H StankeI'. AOAC Internati onal , Arlington. V A, USA. Pp 93-104.

Skerritt J H & Amitnran i B E, 1996. Immunoassay fo r res idue anal ys is food safety .ill ACS Symp Ser 62 1, Chapter 3. Pp 2 1-29.

Wratten S J & Feng P C, 1990. Pes ti cide immunoassay. in , Development and applications of immunoassay for food analys is, edited by J H Ritlenborg. Elsev ier Applied Science, New York . Pp 20 1-220.