Quantitative determination of diuron in ground and surface water by time-resolved fluoroimmunoassay: Seasonal variations of diuron, carbofuran, and paraquat in an agricultural area

Article abstract of DOI:10.1021/jf063442o

The aim of this research is to develop an ultrasensitive time-resolved fluorescence immunoassay (TR-FIA) for herbicide diuron in water samples. This method appears to be a promising approach, instead of conventional analytical techniques, in the screening

Full text of DOI:10.1021/jf063442o

J. Agric. Food Chem. 2007, 55, 3823−3828 3823
Quantitative Determination of Diuron in Ground and Surface
Water by Time-Resolved Fluoroimmunoassay: Seasonal
Variations of Diuron, Carbofuran, and Paraquat in an
Agricultural Area
MARIA A. BACIGALUPO* AND GIACOMO MERONI
Istituto di Chimica del Riconoscimento Molecolare, CNR, Via M. Bianco 9, Milan 20131, Italy
The aim of this research is to develop an ultrasensitive time-resolved fluorescence immunoassay
(TR-FIA) for herbicide diuron in water samples. This method appears to be a promising approach,
instead of conventional analytical techniques, in the screening procedure of organic pollutants because
it is simple, rapid, and specific, and it does not require sample preconcentration or cleanup. Lanthanide
chelate used as label allows to achieve sensitivity even 10 times higher than most of the other
techniques. It has been applied to monitoring diuron contamination in specimens collected along a
year in an agricultural area. The water specimens were collected monthly from lake, well, and irrigation
ditch in the agricultural area south of Milan. Assay was performed using diuron-specific polyclonal
antibody raised in sheep; as fluorescent marker, we used rabbit antisheep IgG conjugated with a
chelating molecule complexed with Eu3+. The compound 4-(3-(3,4-dichloro-phenyl)-1-methyl-ureido)-
butyric acid (CPD) was synthesized and conjugated with bovine serum albumin (BSA) to prepare a
solid phase. Sensitivity achieved was 20 ng L-1 below the European Community limits. Paraquat
(PQ) and carbofuran (CF) presence in the same samples has been also evaluated in a similar
way, using immunoassays with time-resolved revelation systems. Diuron concentration shows a
peak coinciding with a peak of carbofuran during summer periods. The peak of diuron was
65 pg/mL in June and 180 pg/mL in September in ditch and lake water samples, respectively;
carbofuran concentration was higher than diuron in all samples: a carbofuran peak was revealed in
September and October resulting in 87 ng/mL. Herbicide paraquat was not detectable in any assayed
sample.
KEYWORDS: Diuron; urea herbicides; immunoassay; europium chelate; time-resolved fluorescence;
pesticides
INTRODUCTION
transformations, it exhibits 200 days half-life, while in water
(in which hydrolysis or photolysis is major route of degradation)
it has 90 days half-life (4). The reduction of dissolved oxygen
concentration induced by diuron led to decomposition of
photosynthetic microorganisms and modification of microbial
flora of aquatic environment. Diuron is highly toxic to aquatic
invertebrates (5); by preventing algal cellular reproduction,
diuron indirectly limited the release of dissolved organic
substances by phytoplankton (6, 7). Furthermore, even if
low lethality is observed in adult fishes, dose-dependent
delay is present in hatch (8). In addition, genotoxic effects
on plant chromosomes such as inhibition of mitotic index
and induction of chromosome aberrations have also been
reported (9).
The herbicide diuron is a substituted urea compound used to
control a wide variety of annual and perennial weeds or as
antifouling biocides (1, 2). This compound inhibits the photo-
synthesis by blocking electron transport, through preventing
the CO2 fixation and limiting the adenosine triphosphate
production. It is very soluble in water and very stable in soil
(3): in soil, in which the processes of degradation are biotic
The herbicide PQ (1,1′-dimethyl-4,4′-bipyridinium) is a
quaternary nitrogen compound, active as redox drug inhibiting
the reduction of NADP to NADPH during photosynthesis. It is
used as a nonselective contact herbicide. It is known to generate
superoxide anions in mitochondria and cytosol of yeast and
* To whom correspondence should be addressed. Tel: ++39-02-
28500023. Fax: ++39-02-28901239. E-mail: mariangela.bacigalupo@
icrm.cnr.it.
10.1021/jf063442o CCC: $37.00
© 2007 American Chemical Society
Published on Web 04/13/2007

3824 J. Agric. Food Chem., Vol. 55, No. 10, 2007
Bacigalupo and Meroni
antibody raised in sheep (obtained using diuron conjugated to thyro-
globulin as an immunogen) was purchased from Guildhay Ltd (Guild-
ford, U.K.).
Apparatus. A single-photon-counting time-resolved fluorometer
(1232 DELFIA Fluorometer; Wallac, Turku, Finland) was used to
measure fluorescence.
mammalian cells leading to the formation of several reactive
oxygen species (10, 11).
The pesticide CF (2,3-dihydro-2,2-dimethyl-7-benzofuranyl-
methylcarbamate) is worldwide used to control soil and leaf-
feeding insects and nematodes. CF toxicity is very high in
human and birds, as it is a potent inhibitor of cholinesterase by
binding to the serine residues (12). It is widely used and can be
found as pollutant in the air, water, soil, and foods.
Determination of pesticide residues in water from field
application is generally restricted to the area around the site of
application. However, contamination may be more extensive
and may reach aquifer-confining systems, which requires large-
scale monitoring programs.
The hazard is most commonly associated with long-term
exposure to these contaminants. In particular, as for all
pesticides, it is necessary to evaluate the exposure to diuron in
the general population, especially in occupationally exposed
people and residents in contaminated sites.
Therefore, several analytical approaches such as liquid
chromatography, gas chromatography, mass spectrometry (13-
19), electrophoresis (20, 21), and immunoassay (22-24)
have been described for detection of this compound. Conven-
tional methods consist of separative techniques to isolate the
target compound from other more concentrated compounds.
Interferent peaks in the chromatograms and noisy baseline must
be removed to allow sensitivity, which is lower than im-
munological methods.
Therefore, in conventional approach, the samples must be
singly processed, which is laborious and time-consuming. As
the number of controls required increases, the screening of many
samples becomes very expensive. Furthermore, each step of an
analytical process introduces errors into the final result. Im-
munological methods appear to be a promising approach for
the development of a screening procedure. These methods cover
a wide concentration range and do not require preconcentration
or cleanup. They are simple, rapid, sensitive, and specific.
Furthermore, many samples can be analyzed simultaneously in
a short incubation time.
Specimen Collection. The surface water specimens were collected
from Fagnana lake (Buccinasco, Milan, Italy), from an irrigation ditch,
and from a well in the agricultural area located south of Milan. Sampling
was made monthly from September 2005 to July 2006 at the same
places and under the same conditions. The water samples were collected
in glass vials, were filtered, and were stored at 4 °C until use.
Syntheses. 1. BCPDA and IgG-(BCPDA)n. Europium chelator 4,7-
bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCP-
DA) was synthesized from 2,9-dimethyl-4,7-diphenyl-[1,10]phenan-
throline in a three-step procedure as described in detail by Evangelista
et al. (26). Rabbit antisheep IgG-(BCPDA)n conjugate was prepared
using 2 mg of affinity-purified IgG dissolved in 2 mL of 0.1 mol L-1
sodium carbonate buffer (pH 9). A solution of 500 µg of BCPDA in
100 µL of ethanol was then added, and the mixture was incubated for
30 min at room temperature. The labeled IgG was separated on
Sephadex G-50 column, eluting with 0.05 mol L-1 Tris-HCl buffer
(pH 7.3). The value for n (=36) was assessed as the absorbance of
BCPDA at 325 nm; ꢀ ) 1.52 × 104 M-1 cm-1
.
2. CPD Hapten. 4-(3-(3,4-Dichloro-phenyl)-1-methyl-ureido)-butyric
acid (CPD) was obtained according to the method described for
isoproturon (27) with the following modifications. A 4-methylamino-
butyric acid hydrochloride solution was prepared by adding 8.5
mmol of this compound to 10 mL of water containing 18.5 mmol
sodium hydroxide; 8.4 mmol of 3,4-dichlorophenylisocyanate was
added to the solution under fume hood and was stirred for 2 h at
room temperature at which time the solution pH was 11. The solution
was filtered, and the filtrate was acidified to pH 3 by adding 2
mol L-1 HCl. The resulting precipitate was filtered, was washed with
H2O till pH 5, and then was dried over CaCl2 to yield 890 mg of
product. This compound was chromatographically unequivocal on
silica gel F254 TLC plates; Rf ) 0.8 (methanol:chloroform 1:4); mp
149-151 °C.
For instance, in the assay described in this paper, a single
operator can analyze about 100 water samples simultaneously
within 2 h including the reading time. If lanthanide chelates
are used as labels, the immunoassay results are even 10 times
more sensitive than most of the other techniques. Lanthanide
chelates show narrow and strong emission bands around 600
nm and an exceptionally long decay time (up to a millisecond
instead of nanoseconds of most molecules), which allow the
elimination of the high background of the fluorescent labels.
In this paper, we reported a time-resolved fluoroimmunoassay
(TR-FIA) to determine diuron concentrations in ground and
surface water.
Thus, this study has a twofold objective. The first goal is to
develop a method in which we combine advantages of im-
munological methods and sensitivity of time-resolved fluorescent
revelation system; the second objective is to monitor seasonal
variations of diuron concentrations in ground and surface water
in an agricultural area south of Milan.
3. BSA-CPD. Fifteen micromoles of CPD was dissolved in 400 µL
anhydrous dimethylformamide and was cooled to 4 °C before the
addition of 5 µL tri-n-butylamine. After 10 min, 2 µL isobutylchloro-
formate was added, and the solution was left for 20 min at 4 °C before
being added to 8 mg BSA dissolved in 2.5 mL 50% dimethylformamide
in aqueous 0.9% NaCl. The pH was adjusted to 8.5 using 1 N NaOH,
and the reaction mixture was left overnight at 4 °C before being eluted
on Sephadex G-50 with 0.05 M NH4HCO3 at room temperature,
lyophilized, and stored at 4 °C.
Preparation of Solid Phase. Polystyrene microtiter wells were
coated overnight at 27 °C with 200 µL of 0.1 M carbonate buffer, pH
9.0, containing 10 µg/mL of BSA-CPD conjugate. The microtiter wells
were washed with carbonate buffer, and a second coat was made with
250 µL of 2% BSA solution in the same buffer for 4 h at 27 °C. The
wells were washed five times with 0.05 M Tris-HCl buffer pH 7.5
containing 0.9% NaCl and 0.05% NaN3 and were stored dry at 4 °C
until use.
Antibody Titer Evaluation. The antibody was assayed by incubation
in wells coated with the BSA-CPD compound: 100 µL of serial
dilutions of antiserum in buffer 0.05 M Tris-HCl buffer pH 7.5
containing 0.9% NaCl (1:250, 1:2500, and 1:25 000) was applied to
the coated wells, and the antibody bound to the solid phase was detected
by means of rabbit antisheep immunoglobulin G conjugated to BCPDA.
After washing, 150 µL of dissociation solution containing 4 mol L-1
urea, 1% sodium dodecylsulfate, and 10-6 mol L-1 Eu3+ was added to
MATERIALS AND METHODS
Materials. All of the chemicals, including standard diuron and the
diuron-related cross-reacting compounds (linuron; chlortoluron: iso-
proturon; 3,4-dichloroaniline; metobromuron; chlorosulfuron: meto-
lachlor), bovine serum albumin (BSA), and rabbit antisheep IgG were
obtained from Sigma-Aldrich (Milan, Italy); diuron-specific polyclonal

Determination of Diuron by Time-Resolved Fluoroimmunoassay
J. Agric. Food Chem., Vol. 55, No. 10, 2007 3825
Table 1. Percentage of Cross-Reactivity of Diuron-Related Compounds
with Diuron-Specific Antibody Calculated at 50% of Fluorescence
Signal Reduction
Figure 1. Dose−response curve and precision profile (percent coefficient
of variation) for TR-FIA of diuron. F and F0 are expressed as cps. Each
point represents the mean of eight determinations in duplicate.
Figure 2. Parallelism of the assay expressed as logit−log function for
well, ditch, and lake samples spiked with different amount of diuron. F
and F0 are expressed as cps (means of three determinations run in
duplicate).
fluorescence intensity in comparison with 100% of saturated solid-
phase fluorescence was chosen as working titer.
TR-FIA. TR-FIA was performed with the diuron standard dissolved
in tap water. A hundred microliters of serial dilutions of standard
solution between 1 pg and 100 ng was transferred in duplicate into
polystyrene microwells coated with BSA-CPD conjugate. Specific
antibody was diluted at a working titer of 1:7500 in buffer 0.05 M
Tris-HCl solution, pH 7.5, containing 0.9% NaCl and 0.2% BSA; 50
µL of antibody solution was added to all of the wells other than that
used for the blank evaluation, to which the same volume of buffer was
added. Assay was performed directly in the water by applying 100 µL
of samples in duplicate instead of the standards. Matrix effect was
assayed by recovery evaluation of 2, 10, 100, and 1000 pg of standard
diuron added to 100 µL of well, ditch, and lake water (winter month
samples) with undetectable diuron concentration. The wells were
washed, and 150 µL of buffer containing an excess of rabbit antisheep
IgG labeled with BCPDA was added and incubated for 30 min. After
each well. Fluorescence was measured after 10 min at the excitation
wavelength of 345 nm. The delay time was 400 µs after excitation, the
emitted light being read at 615 nm. The dilution giving 50% of

3826 J. Agric. Food Chem., Vol. 55, No. 10, 2007
Bacigalupo and Meroni
Figure 3. (a) Concentrations of diuron (ng/mL) in the ditch water samples in different months of 2006 (bars correspond to the standard deviations). (3b)
Concentrations of diuron (ng/mL) in the lake water samples in different months of 2006 (bars correspond to the standard deviations).
determined by calculating the minimum amount of diuron that
could be significantly distinguished from zero (mean binding
at zero dose at 3 times the standard deviation (SD)). This value,
calculated from three curves prepared in duplicate, was 2 pg/
well. Evaluation of recovery in different water matrixes was
carried out in well, ditch, and lake samples taken in winter
(January) with undetectable diuron amounts. Samples were
spiked with 10, 100, 1000, and 10 000 pg of diuron: the three
curves resulted parallel to the standard curve obtained in tap
water (Figure 2).
Seasonal Variations in Water Samples. Because of its
widespread use in agriculture, its solubility in water diuron
should be assayed in agricultural countries to verify seasonal
variations during to the application periods. We used the
described assay to evaluate diuron concentration monthly in
2006, between September and July, in agricultural country
near Milan. The presence of pesticide carbofuran and
herbicide paraquat was also evaluated. Analytical results are
summarized in graphs reported below. Concentrations of diuron
are reported in Figure 3a and Figure 3b, for ditch and lake
samples. In well samples, concentration was below detection
limits.
Figure 4. Concentrations of carbofuran (ng/mL) in water samples of lake,
ditch, and well in different months in 2006 (bars correspond to the standard
deviations).
washing, 150 µL of dissociation solution was added to each well and
fluorescence was measured as described in Antibody Titer Evaluation
section.
Paraquat (PQ) and Carbofuran (CF) Analyses. The method
described for diuron determination shows characteristics like those
described for PQ. PQ analysis was performed in the same water samples
using TR-FIA with the same europium chelate as described in detail
in the literature (25).
Seasonal profile of carbofuran concentration in Fagnana lake,
ditch, and well is shown in Figure 4. Variations in different
summer months should be explained by taking into account
the rainfall data of the studied period. The occurrence of
rainfall facilitates the transport of pesticides from the site of
application to the nearest water courses and afterward to the
lake. Both the diuron and carbofuran concentrations in ditch
raise a peak in June and July, while in lake concentrations they
increase in the following months (September and October), after
the rainfall.
CF determination was carried out by immunoassay with Tb3+
/
dipicolinic acid complex as time-resolved revelation systems, according
to the previously published method (28).
RESULTS AND DISCUSSION
Antibody Specificity. Specificity of the polyclonal antibody
was evaluated by assaying the cross-reactions of diuron-related
compounds, calculated at 50% of fluorescent signal reduction
against the 100% of standard diuron. As reported in Table 1,
linuron shows a high cross-reaction value; the other structurally
related compounds did not interfere in the assay.
Calibration Graph and Validation. The antibody dilution
giving 50% of fluorescence intensity in comparison with 100%
of saturated solid-phase fluorescence was chosen as the working
titer. Dose-response curve is shown in Figure 1. The graph
was obtained by averaging eight individual curves normalized
by reporting fluorescence values as % F/F0 where F was the
mean of counts/s (cps) for each standard and F0 was the mean
of cps for zero diuron concentration; blank value (3100 ( 278
cps) was subtracted.
Herbicide paraquat was not detectable in any assayed sample,
which suggests that it was not applied in the considered area.
On the other hand, also diuron concentration is very low. As it
is present in very high dilution in aqueous samples, diuron is a
good model for the application of the described method. In these
conditions, the assay by common instrumental methods is
impractical, because all conventional techniques are less sensi-
tive than required. These techniques need complex sample
treatments. The successful use of extraction and cleanup methods
depends on many parameters, such as the choice of solid phase
and polarity of the solvents, and requires expert technicians.
Therefore, internal standard controls are necessary to monitor
the recovery. Furthermore, individual procedure must be used
for each sample. Immunoassays are a valid alternative. However,
even if immunoassays are rather widely used in clinical
Assay sensitivity was good as shown by a high slope of
calibration curve. The detection limit of the method was

Determination of Diuron by Time-Resolved Fluoroimmunoassay
J. Agric. Food Chem., Vol. 55, No. 10, 2007 3827
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the assayed concentrations, TR-FIA was validated by determin-
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in different samples. The obtained values showed a good assay
performance. The specificity always proved satisfactory with
the exception of linuron cross-reactivity. In the presence of
linuron, TR-FIA will thus overstimate the diuron. For samples
showing high values of diuron concentrations, it should be
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ACKNOWLEDGMENT
The authors are grateful to Dr. G. Morano, Municipal Education
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JF063442O