Evaluation of an enzyme immunoassay forthe detection of the insect growth regulatorfenoxycarb in environmental and biologicalsamples†

Hong TM Le,1 Ferenc Szurdoki2‡ and Andras Szekacs1*1Plant Protection Institute, Hungarian Academy of Sciences, PO Box 102, H-1525 Budapest, Hungary2Department of Entomology, University of California, 1 Shields Avenue, Davis, CA 95616, USA

Abstract: A competitive enzyme-linked immunosorbent assay (ELISA) for fenoxycarb was adapted for

quantitative detection of this insect growth regulator in various environmental, agricultural, food and

biological matrices. Environmental samples were taken from soil and surface waters in Hungary. The

ELISA enabled fenoxycarb detection in surface waters in the 1.1–125ng ml�1 concentration range

without sample cleanup. In contrast, soil produced a strong matrix effect due to humic acids and other

soil components. Several fruit homogenates and commercial fruit juices (eg apple, pear, grape) were

analyzed by the ELISA. The assay was found to be suitable for analysis of fenoxycarb in fruit juices

diluted 1:40. Biological samples included insect, fish and bovine tissues. The ELISA was applied to

detect fenoxycarb in various biological matrices from larvae of the silkworm, Bombyx mori L. The

assay proved useful for the analysis of haemolymph diluted 1:10 or at higher dilutions. Fat body and

whole body homogenates, however, caused severe matrix effects. Fenoxycarb was detected in liver

homogenates (diluted 1:40) from fish treated with various doses of fenoxycarb, and the concentrations

determined correlated with the applied doses. The method was used to analyze spiked bovine urine

samples diluted 1:10 or at greater dilutions. Fenoxycarb content determined by the ELISA in water and

fruit juice samples was validated using GC-MS with solid-phase microextraction (SPME) sample

preparation. The results of these studies demonstrated both the value and limitations of the assay when

used for monitoring fenoxycarb in environmental, food and biological samples.

# 2003 Society of Chemical Industry

Keywords: fenoxycarb; ELISA; water, soil, food; fruit juice; biological tissues; Bombyx mori; haemolymph; fishliver; bovine urine

1 INTRODUCTIONFenoxycarb (ethyl 2-(4-phenoxyphenoxy)ethylcarba-

mate; Insegar; Fig 1, 1), a broad activity spectrum

insect growth regulator (IGR) is used in stored

products, forestry, agriculture and as a public health

insecticide.1–4 The compound exhibits its biological

activity over a wide range of insect pests, except for

thrips5 and ticks,6 and it has been used in integrated

pest management practices.1,7,8 The main mode of

action of fenoxycarb and its proinsecticide analogues9

is to mimic the action of juvenile hormone (JH),

although the compound has been shown to exert more

complex effects in the insect hormonal system.10–12

The fate of fenoxycarb in the ecosystem and its

effects on non-target organisms have been studied in

depth.13,14 Although it is much more selective than

conventional insecticides, and causes no harmful

effects to ruminants (eg sheep) at registered agricul-

tural doses,15 fenoxycarb can exert adverse effects

against some beneficial and non-target insects,16

Figure 1. The chemical structures of fenoxycarb (1) and its haptenicderivatives (2, 3) applied in the optimized ELISA systems. The aminogroups of the haptens were utilized to link the molecule to carrier proteins.

(Received 5 May 2002; revised version received 14 October 2002; accepted 25 October 2002)

* Correspondence to: Andras Szekacs, Plant Protection Institute, Hungarian Academy of Sciences, PO Box 102, H-1525 Budapest, HungaryE-mail: aszek@nki.hu† One of a collection of papers on various aspects of current research on pest management in Hungary, collated by Dr Istvan Ujvary‡ Current address: Minerva Biotechnologies Corp, Rosenstiel Building, 6th Floor, 415 South Street, Waltham, MA 02453, USAContract/grant sponsor: Hungarian Ministry of Education; contract/grant number: OMFB 02193/1999Contract/grant sponsor: Hungarian Research Fund (OTKA); contract/grant number: T032232

# 2003 Society of Chemical Industry. Pest Manag Sci 1526–498X/2003/$30.00 410

Pest Management Science Pest Manag Sci 59:410–416 (online: 2003)DOI: 10.1002/ps.656

aquatic arthropods17–20 and fish.2,3,21 It has also been

implicated in the production of non-spinning syn-

drome of the silkworm, Bombyx mori L.3,11,22,23 As a

consequence, analysis of this IGR in environmental

material is becoming an important issue.

Traditional analytical methods for fenoxycarb, such

as TLC, HPLC24–28 and GC,24,27,29–32 require costly

extraction and clean-up procedures.24,25 Enzyme-

linked immunosorbent assays (ELISA) provide a

simple, cost-effective alternative to overcome the

limitations of traditional pesticide residue analysis

techniques. Since such a rapid analysis method for

insect and other biological tissues would also aid

studies on the mode of action and clearance of

fenoxycarb, ELISA systems for its detection have

been developed in our laboratories33,34 and by other

research groups.22,35 In the present paper we discuss

the application of the ELISA systems that we have

developed for fenoxycarb to environmental samples

and various biological tissues.

2 MATERIALS AND METHODS2.1 ReagentsFenoxycarb was extracted from a commercially avail-

able 250g kg�1 WP (Insegar 25WP, Syngenta, Basel,

Switzerland) using chloroformþethyl acetate (1þ1;

by volume). Gelatin from bovine skin was obtained

from Reanal Rt (Budapest, Hungary) and other

chemicals were purchased from Aldrich Chemical

Co (Milwaukee, WI, USA). Biological reagents and

immunobiologicals were purchased from Sigma

Chemical Co (St Louis, MO, USA) or from ICN

ImmunoBiological (Lisle, IL, USA), except for goat

anti rabbit immunoglubulin (IgG) conjugated to

horseradish peroxidase (HRP) that was obtained from

BioRad Laboratories (Hercules, CA, USA). Solvents

used were of analytical grade, and were purchased

from Merck (Darmstadt, Germany) or Aldrich

Chemical Co. Standard solutions of fenoxycarb and

related compounds were prepared in methanol and

stored in a freezer.

2.2 ApparatusELISA experiments were carried out in 96-well poly-

styrene microplates purchased from Nunc (Roskilde,

Denmark, cat No 442404). Absorbances in the wells

of the microplates were read on an iEMS spectro-

photometric microplate reader (Labsystems, Helsinki,

Finland). The microplate reader was controlled and

data were evaluated in a computer using the software

package Ascent provided by the same manufacturer.

GC-MS analyses were carried out on a Saturn 2000

workstation (Varian, Walnut Creek, CA, USA)

2.3 ELISAHaptenic compounds and protein conjugates were

prepared and ELISA systems were developed as

reported previously.34 ELISA tests were performed

on 96-well microplates using the appropriate BSA-

conjugate as sensitizing antigen diluted in carbonate

buffer (0.1M, pH 9.6; 100ml per well). Conjugates

were immobilized by incubating the plates at 4°C for

12h, and wells were blocked by incubation at 4°C for

1h with a solution of gelatin (from bovine skin;

10g litre�1) in 0.01M phosphate-buffered saline

(PBS; 8g sodium chloride litre�1; pH 7.4; 150ml perwell) at 4°C for 1h. The plate was washed with PBS

containing Tween 20 surfactant (PBST 0.2; 2ml

litre�1), and samples or standard solutions diluted in

PBS buffer containing Tween 20 (PBST 0.05; 0.5ml

litre�1) and aqueous methanol (5ml litre�1) were

added to the wells (50ml per well), followed by the

antiserum diluted in PBST 0.05 (50ml per well). Plateswere incubated at 37°C for 1h, washed with PBST

0.2, and the enzyme-labelled second antibody (goat

anti-rabbit IgG conjugated to HRP) was added at a

dilution of 1:12000 in PBST 0.05 (100ml per well).

The plate was incubated at 37°C for 1h, washed with

PBST 0.2, and enzymatic activity was detected using a

chromogenic substrate solution (100ml per well) con-taining o-phenylenediame (OPD; 320mg ml�1) and

hydrogen peroxide (0.3ml litre�1). Following incu-

bation with the substrate at room temperature, the

enzymatic reaction was stopped by adding 2M sulfuric

acid to the wells (50ml per well), and absorbancies

were read immediately at 492nm. To prepare stan-

dard calibration curves for fenoxycarb, the methanolic

stock solution was diluted with appropriate volumes of

PBST 0.05 to give a dilution series. Sigmoid standard

curves were calculated from absorbance data measures

using the Rodbard equation.36

2.4 Application of the ELISA method onenvironmental and biological samples2.4.1 Surface waterWater samples tested included distilled water, tap

water and various surface water samples [water from

the River Danube and surface, lake and river water

samples collected throughout Hungary by the Soil

Conservation and Plant Hygiene Service (SCPHS)].

Within the scope of a national monitoring programme,

197 surface water samples were received in annual

sampling campaigns from SCPHS, 37 raw drinking

water samples (prior to chlorination) were received

from Wedeco Ltd (Vac, Hungary), and 4 tap water

and purified water samples were additionally used as

controls. Tap water and water from the Danube

contained no floating particles so no filtration step was

necessary prior to analysis. Water samples were used

without any purification or dilution; their pH was

adjusted to 7.4 (surface water samples were slightly

alkaline, their pH ranged from 8.1 to 9.1). Standard

dilution series of fenoxycarb, starting at 5000ng ml�1

concentration, were prepared in these neutralized

water samples, and the fenoxycarb content was

detected by competitive ELISA.

2.4.2 SoilSoil samples were subjected to solvent extraction

Pest Manag Sci 59:410–416 (online: 2003) 411

ELISA for fenoxycarb in environmental and biological samples

according to the recommended FAO/WHO protocol

allowing 87–95% recoveries in a single-step metha-

nolic extraction without sample clean-up. Briefly,

methanol (50ml) was added to the soil (20g), and

the mixture was agitated on an orbital shaker (IKA

GmbH, Staufen, Germany) for 1h, after which the

extract was decanted and filtered. (Because soil

particles and humic acid subsided well, centrifugation

was not necessary.) To avoid solvent effects, metha-

nolic extracts were diluted with PBS, and standard

curves of fenoxycarb were obtained for the diluted

extracts. As a control experiment to these determina-

tions, the fenoxycarb content of spiked samples was

examined in the humic acid fraction of the same soil.

Spiked humic acids were extracted similarly to the soil

samples.

2.4.3 ProduceFruit samples were prepared for analysis according to

the recommended FAO/WHO protocol: the fruit

sample from organically grown produce (100g) was

homogenized in a laboratory blender (Ika GmbH,

Staufen, Germany) in PBS (20ml), the homogenate

was centrifuged (30min at 13000g), filtered, and the

pH of the filtrate was adjusted to 7.4 with aqueous

sodium hydroxide solution (0.1M). Fruit juices (apple,

pear, grape) were obtained from commercial sources.

Apple juice (14% fruit content, 11% dry weight), pear

juice (35% fruit content, 12.5% dry weight), grape

juice (14% fruit content, 11% dry weight) all from

Elma Rt (Ersekhalma, Hungary) were filtered and

diluted with PBS prior to analysis.

2.4.4 Biological tissuesTissue samples from the silkworm, Bombyx mori L,

received from. H Fugo and SG Dedos (Department of

Environmental Science and Resources, Tokyo Uni-

versity of Agriculture and Technology, Tokyo, Japan)

included haemolymph, fat body and whole body

homogenates from larvae at various stages of develop-

ment. Haemolymph samples were used in the ELISA

at dilutions of 1:10 or at higher dilutions, spiked with

fenoxycarb between 1.22 and 5000ng ml�1 in PBS,

without sample purification. Fat body and whole body

homogenates were diluted 1:5 or 1:10 with PBS,

spiked with fenoxycarb as above, centrifuged

(12000rev min�1) to remove insoluble components,

and then subjected to ELISA analysis.

Fish liver samples were kindly provided by colla-

borators at the Department of Biochemistry at Szeged

University, Hungary. Both sexes of carp (Cyprinuscarpio L) were raised at natural daily photoperiods in

thermostated tap water aeriated 24h before the experi-

ments. Individual fish (600–800g each) were treated

intraperitoneally with fenoxycarb (0–10mg kg�1) dis-

solved in sunflower oil, using stock solutions of 0.1,

0.5, 2.0 and 20mg ml�1 concentration. Control fish

received pure sunflower oil. The fish were then kept

in separate tanks for 36h at either 14(�2)°C or

20(�2)°C. The microsomal fractions of the liver of

the subject animals, prepared accorning to Forlin,37

were frozen in liquid nitrogen, and were stored at

�80°C until used.

2.5 Detection of fenoxycarb by GC-MSFenoxycarb was detected in water and fruit juice

samples spiked with fenoxycarb using gas chroma-

tography with a mass spectrometric detector

(GC-MS). Spiked samples were prepared for GC-MS

analysis by solid phase microextraction (SPME).

Thus, 4ml portions of each sample were directly

extracted by SPME using a 65-mm-thick carbowax/

divinylbenzene (CW/DVB) fibre. SPME fibers and the

holder assembly were purchased from Supelco

(Bellefonte, PA, USA). Extraction time by immersion

of the SPME fiber was 20min at room temperature

with stirring by means of a magnetic stirrer. After

extraction, sample desorption from fibre was carried

out at 250°C by direct isothermal injection into the

GC system. GC-MS conditions were as follows: fused-

silica column CP-Sil 8 CB (Chrompack, Middleburg,

The Netherlands), 0.25mm film thickness, 30m�0.25mm ID; injection mode splitless; injector tem-

perature 230°C; column temperature programmed

from 80°C (held for 1min) to 300°C at a rate of 20°Cmin�1. Helium was used as carrier gas, pressure

0.097MPa; ionization current, 350mA; electron

energy, 70eV. The ion trap was scanning in EI mode

from 40 to 650amu. The selected ions for quantitation

of fenoxycarb were those at 116 and 88amu, the two

most abundant molecule ions from the mass spectrum.

3 RESULTS AND DISCUSSION3.1 Competitive inhibition ELISAs for fenoxycarbTwo optimized, immobilized antigen-based ELISA

systems were used for the determination of fenoxy-

carb: a hapten-homologous and a hapten-heterologous

immunoassay.34 The ELISAs were based on two

haptenic derivatives of fenoxycarb (2 and 3, Fig 1):

these amino compounds were diazotized and then

conjugated to proteins by azo coupling.34 Hapten

conjugates to various proteins, eg haemocyanin from

keyhole limpet (KLH) or thyroglobulin (TYG), were

used as immunogens, while bovine serum albumin

(BSA) conjugates were applied as coating antigens in

the ELISA. (Protein conjugates are referred to by the

number of the hapten and the abbreviation of the

carrier protein, eg, 2-BSA.) Assay performance of

the systems were characterized by IC50 values and the

lower limit of detection (LOD) determined in the

immunoassays. The IC50 value represents the con-

centration of the target analyte resulting in a 50%

decrease in the maximal corrected assay signal in the

competitive ELISA system. The LOD value is defined

as the analyte concentration reducing the mean blank

assay signal by three standard deviations of the blank

reading. The two optimized ELISA systems used in

the present study included a hapten-homologous

(coating: 1mg ml�1 of 3-BSA; antiserum: anti-3-

412 Pest Manag Sci 59:410–416 (online: 2003)

HTM Le, F Szurdoki, A Szekacs

TYG(3) (antiserumNo 4960) at a dilution of 1:4000),

and a hapten-heterologous system (coating:

2.5mg ml�1 of 2-BSA; antiserum: anti-3-TYG(3) at a

dilution of 1:2000), allowed high sensitivity detec-

tion of fenoxycarb (IC50 values of 2.7(�1.6) and

1.1(�0.6)ng ml�1, and LOD values of 0.2 and

0.11ng ml�1, respectively). The cross-reactivity (de-

fined as the percentage ratio of the IC50 value of a

given compound and that of fenoxycarb in the same

ELISA system) pattern of the immunoassays has been

studied before, and the two assays were found highly

selective for fenoxycarb.34

3.2 Application of the ELISA method on varioussamples3.2.1 Water samplesAssay performance was tested in various water samples

including drinking water, tap water, lake (Lakes

Balaton and Velencei, smaller ponds and water reser-

voirs) and river (Rivers Danube and Tisza, smaller

watercourses), as well as surface water samples

collected in agricultural, rural and national park areas

throughout Hungary. Standard curves in competitive

inhibition ELISA experiments were established with

fenoxycarb in each type of the above water samples

artificially contaminated with fenoxycarb. Results

indicated unchanged sensitivities and curve shapes in

comparison with standard curves obtained in distilled

water and assay buffer. Standard curves obtained in

tap water and water from the River Danube are

depicted in Fig 2(A).

3.2.2 Soil extractsSoil samples subjected to solvent extraction resulted in

methanolic extracts that were analyzed by ELISA after

dilution 1:20 with PBS. As seen from Fig 2(A), a

rather strong matrix effect was seen in this diluted soil

extract, making it practically impossible to establish a

standard inhibition curve with fenoxycarb. In order to

assess which soil component might have been respon-

sible for such a strong matrix effect, a methanolic

extract of humic acids (extracted similarly to soil

samples) was also tested for matrix effects. The

detrimental effect of humic acids on the standard

inhibition curve with fenoxycarb is apparent (Fig

2(A)), but much weaker than that of the soil extract.

Thus, it appears that matrix influences in soil extracts

are not caused only by humic acid content. On the

basis of these experiments, the ELISA system in its

present state is not applicable to the analysis of soil

extracts, which would require additional sample clean-

up prior to immunoanalysis.

3.2.3 Fruit samplesThe ELISA systems were applied to several fruits that

are commonly treated with fenoxycarb to control

insect pests. Thus, filtered homogenates and juices of

apple, pear and grape were analyzed by the ELISA.

Fruit samples homogenized in PBS or fruit juices

obtained from commercial sources were filtered,

diluted with PBS as necessary and were subjected to

ELISA. Strong matrix effects were observed in both

apple and pear homogenates. Matrix effects were

probably related to natural fibre content of the sample,

as indicated by the fact that a somewhat better

standard curve was established in apple homogenate:

the higher the fibre content in the fruit homogenate,

the stronger was the matrix effect observed. Matrix

effects were alleviated by removing fibre by centrifuga-

tion. In contrast, spiked fenoxycarb content was

readily detectable from commercial fruit juices at 1:5

or higher dilutions in apple juice and at 1:10 or higher

dilutions in grape juice (Fig 2(B)). A slight matrix

effect was seen in pear juice at a dilution of 1:10, but

this effect was eliminated at higher dilution (1:40)

using the hapten-heterologous ELISA system. Matrix

effects were similar, although somewhat more pro-

nounced in the hapten-homologous ELISA. Matrix

effects (and therefore, the dilution required for

detection of the analyte) correlate with the fibre

content of the fruit juice.

3.2.4 Insect tissuesOne of the prime fields of the possible applications of

the fenoxycarb ELISA system is the determination of

fenoxycarb in insect-based biological matrices. Moni-

toring fenoxycarb content in the haemolymph of the

silkworm, B mori, is of particular importance, because

this insect shows great sensitivity to even minute

amounts of fenoxycarb in its sole food, mulberry

leaves.12,22 The applicability of the optimized ELISA

systems was evaluated on various tissues of B morilarvae, including haemolymph, whole body homoge-

nate and fat body tissue. Figure 2(C) displays standard

curves obtained in haemolymph and whole body

homogenate at various dilutions with PBS. Results

indicate that haemolymph samples can be analyzed by

ELISA at dilutions of 1:10 or greater, while a strong

matrix effect relative to the standard curve detected in

buffer is seen in whole body homogenate even at

higher dilution and after centrifugation. The standard

curve obtained in fat body homogenate appears to be

undistorted in shape, but with a strong shift towards

higher fenoxycarb contents (decreased sensitivity).

This is likely to be due to strong physicochemical

binding of the lipophilic fenoxycarb to lipid compo-

nents of the tissue. Even stronger matrix effects were

seen when the method was applied to the fat body

tissue of the insect, where practically no detectable

standard could be obtained. Results indicate that the

immunoassay is applicable to insect haemolymph at

dilutions at or above 1:10 with PBS, but more

lipophilic samples, fat body or whole body homo-

genates, exert severe matrix effects.

3.2.5 Fish liver samplesFenoxycarb content was detected in various fractions

of fish liver extracts of untreated (control) animals and

those treated with various doses of the IGR. All three

fractions, full liver sample homogenate, and first and

Pest Manag Sci 59:410–416 (online: 2003) 413

ELISA for fenoxycarb in environmental and biological samples

second supernatant in its fractionated extraction were

subjected to ELISA determination. In control experi-

ments (fish untreated or treated with unspiked oil) no

matrix effects were seen when liver homogenate

samples were diluted above 1:40 with PBST 0.05,

and fenoxycarb was not detected in these control

samples. In contrast, fenoxycarb was detected in all

three liver tissue fractions in all treated animals.

Results (Fig 3) indicate no statistically significant

differences among fractions of individual liver

samples, but display a consistent rise in fenoxycarb

content as the treatment dosage increased.

3.2.6 Bovine blood and urineFor metabolism studies in animals, it may be necessary

to monitor fenoxycarb levels inside the body (eg blood

levels) as well as those discharged from the body

through urination. For this purpose, matrix effects on

ELISA performance were tested in bovine blood and

urine. Urine samples were tested crude, and diluted

1:10 and 1:100 in PBS. Analysis results, seen in Fig

2(D), indicate that matrix component in bovine urine

caused a detrimental effect on the assay signal that had

to be diluted out. Standard curves recorded with urine

diluted 1:10 and 1:100 with PBS did not differ

Figure 2. Standard curves for fenoxycarb in the optimized hapten-homologous ELISA system in various samples. (A) Standard curves obtained in (&—&) tapwater, (* - - *) water from the river Danube, (~ � � �~) diluted methanolic soil extract, (! - � - !) diluted methanolic extract of humic acids. (B) Standard curvesobtained in (* � � � *) assay buffer, (& - � - &) grape juice undiluted, (& --- &) diluted 1:5 and (~—~) 1:10. (C) Standard curves obtained in hemolymph fromL5D5 instar larvae of Bombyx mori (& - � - &) undiluted, (& - - &) diluted 1:10 and (*—*) 1:100. (D) Standard curves obtained in (* � � � *) buffer, (& - � - &)bovine urine undiluted, (& --- &) diluted 1:10 and (~—~) 1:100. Assay parameters: (A, B, D) 2-BSA as coating antigen at 1mg ml�1; or (C) 3-BSA at2.5mg ml�1; blocking: 1% gelatin in PBS; anti-2-TYG(3) serum at a dilution of 1:2000; anti-IgG-HRP at a dilution of 1:12000. Assays were carried out in triplicatesin a single microtitre plate in each set.

414 Pest Manag Sci 59:410–416 (online: 2003)

HTM Le, F Szurdoki, A Szekacs

statistically from that recorded in assay buffer,

indicating that fenoxycarb can be detected quantita-

tively in diluted urine samples. A more severe matrix

effect was seen in bovine blood, when both blood

platelets in full blood and immunoglobulins in the

serum seem to interfere with assay performance.

3.3 Validation of the ELISA by instrumentalanalytical (GC-MS) methodsFenoxycarb concentrations determined by ELISA

were validated in spiked water and fruit juice samples

using GC-MS with SPME for sample extraction.

Because none of the 238 water samples, including

drinking water, tap water, lake water and river water,

as well as surface water samples collected in 2000 and

2001 in agricultural, rural and national park areas

throughout Hungary, contained fenoxycarb, positive

field samples could not be used for assay validation.

Therefore, representative surface water samples spiked

with fenoxycarb were subjected to both GC-MS and

ELISA analyses. The GC retention time of fenoxycarb

(10.7min) allowed a rapid and convenient method of

analysis. Spiked fenoxycarb content (eleven concen-

trations between 0 and 55ng ml�1) detected by GC-

MS in water and diluted (1:40) fruit juice samples

were cross-validated and correlated with correspond-

ing concentrations detected by the ELISA systems.

The results of this validation, based on five calibration

points in water, three points in diluted apple juice and

three points in diluted grape juice, indicate good

correlation (conc(GC�MS)=1.021 conc(ELISA)þ0.384,

n =11, r2=0.976), and the regression slope is very

close to 1, verifying correct detection by both ELISAs.

None the less, a small intercept of the regression line,

and the consequent fact that the regression line runs

close to, but continuously above, the diagonal,

indicates a slight overestimation by the ELISA systems

relative to the GC-MS method.

4 CONCUSIONSEvaluation of our recently developed ELISA system to

detect fenoxycarb in environmental and biological

samples spiked with the analyte demonstrated that the

assay is applicable to surface waters without sample

cleanup or dilution. The ELISA was also suitable for

the detection of spiked fenoxycarb in diluted apple,

grape and pear juices (dilutions 1:5, 1:10 and 1:40,

respectively), haemolymph (diluted 1:10) from B morilarvae, and diluted (1:10) bovine urine. Furthermore,

the immunoassay detected fenoxycarb in liver

homogenates of fish treated with various doses of this

IGR. Therefore, the ELISA method can be utilized to

complement or replace instrumental analytical

methods to analyze fenoxycarb in these matrices.

Further work is required to eliminate severe matrix

effects caused by soil extracts, fruit homogenates,

lipophilic insect tissues and bovine blood. The assay

offered high sensitivity similar to or better than those

of other ELISAs for fenoxycarb.22,35 Using this

immunoassay, studies on the mode of action and

clearance of fenoxycarb in B mori haemolymph are in

progress.38,39

ACKNOWLEDGEMENTSWe thank H Fugo and SGDedos at the Department of

Environmental Science and Resources, Faculty of

Agriculture, Tokyo University of Agriculture and

Technology (Tokyo, Japan) for tissue samples of the

silkworm, and M Abraham and A Deri at the

Department of Biochemistry at Szeged University,

(Szeged, Hungary) for treated and control carp liver

samples. The authors wish to express their sincere

appreciation to pesticide analysts at SCPHS

(Hungary) for providing surface water samples.

Special thanks are due to L Gyorfi (SCPHS

Budapest), G Karoly (SCPHS Csopak Station) and

E Majzik (SCPHS Velence Station) for their expert

assistance. This work was supported by Hungarian

research grants OMFB 02193/1999 by the Hun-

garian Ministry of Education and T032232 by the

Hungarian Research Fund (OTKA).

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