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Article J. Braz. Chem. Soc., Vol. 00, No. 00, 1-10, 2019
Printed in Brazil - ©2019 Sociedade Brasileira de Química
http://dx.doi.org/10.21577/0103-5053.20190066
*e-mail: snixdorf@uel.br
Pesticide Determination in Water Samples from a Rural Area by Multi-Target
Method Applying Liquid Chromatography-Tandem Mass Spectrometry
Mariana B. Almeida,a Tiago B. Madeira,a Lycio S. Watanabe,a Paulo Cesar Melettib and
Suzana Lucy Nixdorf *,a
aDepartamento de Química, Universidade Estadual de Londrina (UEL),
Rod. Celso Garcia Cid/Pr, 445, km 380, Campus Universitário, 86057-970 Londrina-PR, Brazil
bDepartamento de Ciências Fisiológicas, Universidade Estadual de Londrina (UEL),
Rod. Celso Garcia Cid/Pr, 445, km 380, Campus Universitário, 86057-970 Londrina-PR, Brazil
The rises of toxic effects caused by pesticides are of concern. However, Brazilian legislation still
needs scientific subsides to improve the water quality requirements. This can be attributed in part to
the few existing studies showing the occurrence and levels of multi-residue pesticides. Therefore,
the objective of this study was to investigate the presence and the residue-levels of pesticides
in surface, ground and drinking water of Tibagi River micro-basin in Paraná State, Brazil. Data
obtained over a year for the physical-chemical parameters, screening and quantification, made by
liquid chromatography-tandem mass spectrometry (LC-MS/MS), confirmed the contamination by
several pesticides. Alarming concentrations of diuron and imazethapyr in a permanent preservation
area and in groundwater were observed. The absence of legislation for most pesticides under study
makes difficult the prohibition of their use and the control of their residue-levels on the environment.
The results draw attention to further discussion and engagement around the pesticide regulations.
Keywords: residue level, environmental regulation, water quality requirements, diuron,
imazethapyr
Introduction
Brazilian agriculture in the past few years has been
growing and using an indiscriminate amount of pesticides
to increase the crop yield becoming the main world’s
market.1-5 During 2013, the most commercialized pesticides
were glyphosate and atrazine, both herbicides. However,
fungicides, insecticides, acaricides, nematicides, growth
regulators, repellents, and biocides are also commonly
used.6
It is recognized that the agronomic intensification
associated with the use of pesticides increases the
environmental impacts, usually observed through physico-
chemical and microbiological parameters in water
samples, for example. Consequently, the toxic effects on
animals and humans, occupationally exposed, and to the
environment also increase.4,7 Beyond the intensive use of
different classes of pesticides, the lack of information and
technical assistance in rural areas also contribute to soil
and water contamination by the rural workers, as a result
of the incorrect discharge of residues during the clean-up
of equipment and plastic containers on the environment.8
The pesticides disseminated throughout the environment
have complex behavior, going through a variety of
physics, chemistry and biological processes that generate
metabolites and degradation products.9 Regardless of the
concentrations, these products can present higher toxicity
than the original compounds and may have a straight
correlation with the increasing number of diseases, like
cancer.10-14
Besides pesticides, the presence of other emerging
contaminants in the environment, such as industrial
compounds, pharmaceuticals, personal care products and
disinfection by-products, results in negative effects in living
organisms due to the non-target effect and contributes to
increase surface and groundwater pollution.15,16 Recent
studies have shown that the exposure of parents before
a child’s birth increases the risk of infant leukemia and
Parkinson’s disease to 70%.17,18
At the South of Brazil, the Paraná State is known for the
intensive production and commercialization of pesticides,
occupying the third position according to the report made
Pesticide Determination in Water Samples from a Rural Area by Multi-Target
Method Applying Liquid Chromatography-Tandem Mass Spectrometry
Mariana B. Almeida,a Tiago B. Madeira,a Lycio S. Watanabe,a
Paulo Cesar Melettib and Suzana Lucy Nixdorf *,a
Pesticide Determination in Water Samples from a Rural Area by Multi-Target Method J. Braz. Chem. Soc.
2
by the Ministries of Health in 2016 and of Agriculture in
2018.19 The discrepancy might be related to the presence of
36 industries of pesticides in the state. These large numbers
of industries associated with agricultural activities indicate
the need for severe environmental monitoring. The concern
is related to the possibility of surface and groundwater
contamination due to the presence of agrochemicals and
the incorrect discharge of wastewater, which might affect
the drinking water quality. Although many countries treat
and distribute water to the population, in rural areas the
water might come directly from natural sources, such as
mines or wells without adequate treatment.
The water quality requirements are not a consensus
and can follow national and international guidelines.
In general, all the guidelines evaluate odor, taste, color,
turbidity, conductivity, pH and concentration of organic and
inorganic compounds, although compounds of interest and
their maximum allowed values may vary for each country
according to their use.2,20-22
Currently, Brazil has monitoring programs of pesticides
such as the National Monitoring Water Quality for Human
Consumption Program (VIGIAGUA), available on the
Monitoring Information on Water Quality for Human
Consumption System (SISAGUA). However, according
to the legislation, less than 10% of the pesticides in use
are monitored.2,21,23 Recently, the Pesticides law (No. 7802
of 1989) is undergoing several changes, which is causing
many divergences between businessmen, environmentalists
and medical entities.24 One of the most controversial items
includes permission for products classified as “acceptable
risk”, prohibiting only those classified as “unacceptable
risk”.
The Brazilian legislation already has several laws
regarding known toxic compounds without allowed limit
of concentration for drinking water. On the other hand,
the European Union (EU) has no target pesticides, but
is more restricted regarding the total concentration that
should be less than 0.50 µg L-1 for drinking water. This
fact emphasizes the need to review the matter of pesticide
monitoring in the national legislation.25,26 Despite
the discrepancy between national and international
regulations, accordingly to Avelhan and Zylbersztajn,27
Brazil has the most advanced and restricted law
concerning environmental protection, but its enforcement
has several loopholes.
Regardless of the law and its application, some areas are
well known by the suspect of pesticides contamination, as
the case of a rural area in the Northern region of Paraná State.
During the years of 2002 and 2006, the Public Ministry
realized some investigations after suspecting poor water
quality due to the non-natural taste and smell and the
high fish mortality.28 However, several years later the
problem persists, and little has been done to protect this
area. Thus, considering the lack of scientific evidence and
scarce data of pesticide multi-residue contamination in
this region, the aim of the present study was to evaluate
physico-chemical parameters and pesticides of the Tibagi
River micro-basin water over a year. We hope this study
will contribute providing scientific subsidies to promote
the protection needed to this area by regulatory agencies.
It is further expected that the data generated will serve as
a basis for broadening the range of monitored pesticides
besides reducing their residue levels in the environment.
Experimental
Study area and samples
The studied rural area is located in the city of Arapongas
at the North of Paraná State (23.49S, 51.42W).The study
area is recognized for its intense agricultural activity
(mainly soy, wheat and corn) and to be close to an
agrochemical industry. The water bodies present in this
region are part of micro-basins, which constitute the basin
of the Tibagi River. According to resolution No. 357/2005
from the Brazilian’s National Council for the Environment
(Conselho Nacional do Meio Ambiente, CONAMA), this
river is classified as class 2.21 Its tributaries and springs,
although not cataloged, must belong to the same class, or
to a more protected class (class 1, or special class, the latter
one designated for water mines). These water sources have
being used by the population in plant cultivation, for animal
breeding and for human consumption.
This region has a humid subtropical climate, and
although there is no defined dry season, rains are more
intense in the warmer months. Thus, considering the
possibility of rainy periods and also the seasonal agricultural
activity and pesticides uses, four collection campaigns were
carried out in 9 sampling points during the year 2015:
March (M), June (J), September (S) and December (D).
The nine sampling sites were chosen taking into account
the relevance and the presence of crops, proximity to the
agrochemical industry, presence of water well, proximity to
populated area, and the ease of access to streams potentially
receiving runoff and effluents (Figure 1, and Table S1 in
the Supplementary Information (SI) section).
The sources of water at each sampling point (SP) were
collected as follows: 1, 3, 4, 5, 6 and 7, from stream or
river, 2 from a mine, 8 from public supply and 9 from
groundwater (water well). Although the major crops in
this area are soy, wheat and corn, crops of tomatoes and
eucalyptus were also noticed around the sampling points
Almeida et al. 3Vol. 00, No. 00, 2019
(SP) 2 and 5, respectively (Figure 1). Most samples were
collected from surface water bodies, being the sampling
point SP 5 the only one under a permanent preservation
area. The tap water sample (SP 8) has been treated
according to the Brazilian requirements of sanitation and
potability with limits established by the Ministry of Health
ordinance No. 2914/2011.23
1 L of water sample was collected in each sampling
point in a clean amber glass bottle. Samples were kept under
refrigeration (4 °C) until further processing.
Physico-chemical parameters
Water quality based on the physico-chemical parameters
was evaluated according to the resolution No. 357/2005
from CONAMA.21 In natura samples were characterized in
terms of: color at λ = 455 nm (Nanocolor® Vis Macherey-
Nagel GmbH & Co. KG, Düren, Germany), conductivity
(Digimed DM-3P, Campo Grande, Brazil), pH (pH/Ion
Meter 781, Metrohm, Switzerland) and turbidity (Hach
2100Q portable turbidimeter, Hach Corporation, United
States).
Sample preparation for pesticide analysis
In natura water samples were vacuum filtered through a
0.22 µm cellulose ester membrane (Millipore, USA) before
solid phase extraction (SPE). The SPE procedure was done
using Sep Pak C18 Cartridges (500 mg) (Waters, Milford,
MA, USA) previously conditioned with 4 mL of methanol
(HPLC grade, J. T. Baker, Center Valley, PA, USA) and
equilibrated with 4 mL of ultrapure water (Milli-Q®,
Millipore, USA). 250 mL of the water sample were passed
through the cartridges using a vacuum manifold system.
After washing with 2 mL of ultrapure water, the cartridges
were dried under a vacuum and kept frozen (−12 °C)
until further analysis.29 The elution was performed with
2 mL of methanol, and the eluate was diluted 10 times
in ultrapure water before liquid chromatography-tandem
mass spectrometry (LC-MS/MS) analysis. Preconcentration
factor reached 12.5 times.
Standards
Considering the agricultural activity and the use of
the pesticides due to the crop areas, analytical standards
of ametryn, atrazine, azoxystrobin, carbendazim, diuron,
hexazinone, imazaquin, imazethapyr, imidacloprid,
propiconazole, tebuconazole and tebuthiuron (purity
between 96.5 and 99.9%, Sigma Aldrich, Madrid, Spain)
were used to prepare a stock solution of each pesticide at a
concentration of 400.00 mg L-1 using methanol as solvent.
Mixed standard solutions were prepared in methanol-water
(10:90, v/v) in concentration range of 0.10-375.00 µg L-1
and used to obtain analytical curves and further analysis
of validation steps.
Liquid chromatography conditions
The LC-MS/MS method was performed by an Waters
ACQUITY™ UPLC® I-Class system (Manchester, UK)
consisting of a binary pump with solvent manager, mobile
phase degasser, autosampler with flow-through needle
containing sample manager and column heater. Mobile
phase was composed of solvent A (water) and solvent B
(methanol) both with 0.1% formic acid. A linear gradient
elution was employed: 5% B (0-0.24 min), 5-95% B
(0.25-7.74 min), 95% B (7.75-8.50 min) and 5% B
(8.51-10.00 min). The injection volume for each sample
was 1.0 µL in full loop injection mode and the sample
manager was kept at 4 °C. Chromatographic separation
was achieved at 0.45 mL min-1 flow rate using a Waters
UPLC® BEH C18 column (2.1 mm × 50 mm, 1.7 µm,
Waters, Milford, MA, USA) maintained at 40 °C.
Figure 1. (a) Map location of the area under study at Paraná State and (b) an aerial view of the 9 points of water sampling collections.
Pesticide Determination in Water Samples from a Rural Area by Multi-Target Method J. Braz. Chem. Soc.
4
Mass spectrometry conditions
Mass detection was performed using a tandem quadrupole
mass spectrometer, Waters ACQUITY® TQD (Manchester,
UK), equipped with an electrospray ionization interface
(ESI). The method for the screening of 402 pesticides was
performed based on Morphet and Hancock.30 The presence
or absence of the analyte defined was made by checking
the existence of 2 transitions in the selective reaction
monitoring (SRM) mode, in which the first transition is
used for quantitation, while the second transition is applied
to confirm the presence of the pesticide in the sample. Ion
ratio was also performed. According to the compounds found
during the screening step, 12 pesticides were evaluated due
to relevance and availability of the analytical standards.
The parameters used for external quantitation method were
optimized individually for each one of the 12 pesticides
under study (Table 1).
For both methods (screening and quantitation), the ion
source was operated at 120 °C with a capillary voltage
of 1.0 kV and extraction cone voltage of 3.0 V. Nitrogen
was employed for both the dissolvent (800 L h−1) and cone
(5 L h−1) kept at 400 °C. Acquisition mode chosen was
SRM at an argon collision gas pressure of 3.5 × 10−3 mbar.
Data acquisition and processing were achieved by using
MassLynx™ 4.1 software (Waters, Manchester, UK).
Validation process
Before the analysis of the pesticides on the samples, the
method was validated with respect to accuracy, precision,
linearity and limits of detection (LOD) and quantitation
(LOQ) following the EU Guidelines.31 The analytical range
evaluated was from 0.01 to 50.00 µg L-1. The accuracy
of the method was accomplished by recovery studies,
performed by spiking known amounts of the standard
mixture (n = 3) with low (0.25 µg L-1), medium (1.00 µg L-1)
and high (50.00 µg L-1) concentration levels of pesticides
in ultrapure water. Accuracy was calculated as the percent
ratio between the found and known concentrations.
Table 1. Mass spectrometry parameter of the quantified pesticides in water bodies
tR / min Pesticide Molecular formule Precursor ion / m/z Product ion / m/z Cone voltage / V Collision energy / eV
2.50 carbendazim C9H9N3O2192.1 132.10 33 28
160.10 33 18
3.27 imidacloprid C9H10ClN5O2256.1 175.10 34 20
209.10 34 15
4.65 imazethapyr C15H19N3O3290.11 69.05 42 28
86.08 42 26
4.97 hexazinone C12H20N4O2253.1 71.00 35 30
171.10 35 16
5.05 imazaquim C17H17N3O3312.2 86.20 40 28
267.20 40 20
5.11 tebuthiuron C9H16N5OS 229 116.00 36 26
172.00 36 18
5.42 azoxystrobin C22H17N3O5404 329.00 28 30
372.00 28 15
5.48 ametryn C9H17N5S 228.1 68.10 38 36
186.10 38 18
5.62 atrazine C8H14ClN5216.1 96.10 39 23
174.10 39 18
5.81 diuron C9H10Cl2N2O 233 46.30 34 14
72.10 34 18
6.36 propiconazole C15H17Cl2N3O2342 69.00 46 22
159.00 46 34
7.24 tebuconazole C16H22ClN3O 308 70.10 40 22
125.00 40 40
tR: retention time; m/z: mass-to-charge ratio.
Almeida et al. 5Vol. 00, No. 00, 2019
The precision measurement of three different levels of
concentration (0.01, 1.00 and 10.00 µg L-1, n = 6) was also
performed and has been determined as percent coefficient
of variation based on relative standard deviation (RSD in
percentage). The limits of detection and of quantitation
were also determined by the signal-to-noise (S/N) ratio.
Statistical analysis
A multivariate statistical technique was chosen to
evaluate the large number of data obtained by quantitation
of pesticides and physico-chemical analysis. The principal
component analysis (PCA) allowed the reduction of the
data set size, supporting the interpretation without loss
of information. A 3D plot with factor scores were used to
obtain a better view of principal component (PC) 1, 2 and
3. All the statistical analysis was accomplished through
Statistica 8.0 software.32
Results and Discussion
The resolution No. 357/2005 from CONAMA establishes
characteristics and concentrations of substances for water
bodies according to the destination of these resources.
In addition to appearance, organoleptic characteristics
and microbiological parameters, this resolution specifies
values for physico-chemical parameters and also maximum
concentration limits for various substances, such as oil
and its derivate, nutrients, metals and some pesticides,
among other compounds.21 Unfortunately, only a few
agrochemicals are listed in the resolution, and does not
represent the most frequently used and detected pesticides
in that area.
Physico-chemical analysis
Class 2 rivers and streams, as the water bodies under
study, may be used without water treatment for irrigation
and livestock, but for human consumption, adequate
treatment is required. However, some of the physico-
chemical parameters analyzed showed values above the
legal limits, highlighting conductivity, which was the
parameter with more outliers according to the preconized
standards (Table 2).
Conductivity is a physico-chemical parameter that
may be used as an indirect measurement of pollutant
concentrations since it depends on the dissolved ions in water,
such as phosphate, nitrate and chloride ions, and common
contaminants from crop areas.33,34 Despite its importance,
no limit is described in the legislation. Nevertheless, it is
Table 2. Data from 9 different water sources in 4 sample collections during a year by month (M, J, S and D) regarding the color, conductivity, pH, and turbidity
Parameters Month Sampling point (SP)
123456789
Color / (mg Pt L-1)
M 39.82 13.55 39.82 25.49 39.82 18.32 20.71 11.16 13.55
J 56.53 25.49 30.26 35.04 54.14 30.26 30.26 23.10 25.49
S 104.20a37.43 32.65 56.53 99.52a30.26 42.20 32.65 37.43
D 226.09a130.56a111.46a230.86a467.28a123.40a123.40a101.91a187.88a
Conductivity / (µS cm-1)
at 25 °C
M 93.90 82.10 146.80b325.00b2.90 28.80 4.20 138.50b421.00b
J 98.00 85.00 150.50b296.00b2.50 28.10 4.10 134.50b409.00b
S 76.40 72.60 133.20b269.00b1375.00b31.00 3.50 120.30b344.00b
D 81.60 73.20 96.90 219.00b1286.00b30.50 3.40 131.20b360.00b
pH
M 6.04 6.19 5.91a6.30 6.92 6.38 6.43 7.08 6.84
J 5.96a6.28 5.87a6.32 6.65 7.02 6.45 7.00 6.70
S 6.10 5.60a6.53 6.67 6.83 7.44 6.81 7.36 7.29
D 6.86 5.73a5.78a6.90 6.89 6.82 6.45 7.03 6.93
Turbidity / NTU
M 5.69 3.78 0.46 4.52 6.66 2.15 1.04 0.18 1.90
J 3.63 0.54 0.39 1.59 5.49 0.57 0.67 0.22 0.30
S 6.72 1.35 0.43 4.48 8.90 0.76 0.54 0.27 0.65
D 20.20 2.02 0.55 19.40 93.20 1.76 1.05 0.17 4.11
M: March; J: June; S: September; D: December; adata values higher than the legislation limit (resolution 357/2005 from the Brazilian National Council
for the Environment (CONAMA)); limits according to the resolution No. 357/2005 from CONAMA: color < 75.00 mg Pt L-1, pH value between 6.00
and 9.00, and turbidity < 40.00 NTU for class 1 and < 100 NTU for class 2 and 3; blimits preconized by Brazilian National Health Foundation (Fundação
Nacional de Saúde (FUNASA):32 conductivity < 100.00 µS cm-1.
Pesticide Determination in Water Samples from a Rural Area by Multi-Target Method J. Braz. Chem. Soc.
6
known that natural water receiving domestic or industrial
effluent, on the other hand, can present conductivity above
than 1000.00 µS cm-1.33 A study performed in two rivers at
the Rio Grande do Sul State showed conductivity values
lower than 90 µS cm-1 for samples considered as natural
waters.35 Values higher than 100.00 µS cm-1, as in cases of
SP 3, SP 4 and SP 9 with results in the range of 120.30 to
421.00 µS cm-1, may indicate environmental impact in natural
water. SP 5 conductivity stands out due to the raised levels
observed in the samplings in September and December
(Table 2) with an increase more than 600 times the value of
1200.00 µS cm-1, pointing out pollution.
All December samplings showed color values higher
than 75.00 mg Pt L-1 probably due to the rainy period
(59.8 mm of precipitation) that increases the amount of
available organic matter and suspended solids.
The pH value is one of the most important physico-
chemical parameters evaluated in this work since the
persistence and the partitioning of the pesticides molecules
in the aqueous media is directly related to it.35 The pH
range observed was 5.6 to 7.44, of which six samples
were considered outliers accordingly to the limit range
(6.00-9.00).21 In this pH range, several compounds still persist
on their non-ionized form due to the pKa value, promoting
their association with the organic matter available.36
Regarding the turbidity, a water of natural source
usually ranges from 3.00 to 500.00 nephelometric turbidity
units (NTU), while in drinking water, the turbidity should
be lower than 1.00 NTU. The turbidity of the samples
ranged from 0.17 to 93.20 NTU. Since the classification
that the rivers are class 2, all the samples can be considered
as in accordance with the quality standards for this
parameter. However, SP 5 (D) presented a worrisome value
of 93.20 NTU, higher than all the other samples and close
to the allowed limit.
Another requirement for class 2 is the virtual absence
of substances that provide taste or odor. All the samples
collected, except the SP 8 (tap water), had a non-natural
smell, a fact that could be used to classify the sampling
points as unfit for use. However, these waters have been
continuously used by the local population for vegetables,
fruit and grain irrigation, for breeding animals, and also
for human consumption.
Pesticide screening
Most of the standard water and wastewater treatments
do not eliminate certain substances, such as pesticides. For
this reason, if the drinking water is obtained from surface
and groundwater, the pesticide residues presented in the
sources can also reach and expose humans.2
Due to the strong agricultural activity, the evaluation
of the presence of pesticides as main contaminants was
performed firstly by a qualitative screening method for
402 different pesticides.30 Despite the selectivity of SRM
mode, it is common to get false positive or negative results
due to matrix interferent effect and slight changes in
retention time.37 After a meticulous data analysis, only the
compounds that presented both transitions in the duplicate
analysis were considered as a positive result. Therefore, the
presence of at least 26 different pesticides in the samples
was confirmed. Samples SP 4, SP 5, SP 7 and SP 9 were
the most contaminated points in all the samplings (data not
shown). SP 5 showed more than 15 different compounds by
sampling. The total ion chromatogram (TIC) of SP 5 for the
four collections (Figure 2) also shows the different profiles
Figure 2. Total ion chromatogram at the screening of pesticides acquired in SRM mode for SP5.
Almeida et al. 7Vol. 00, No. 00, 2019
of each month and the different intensities of compounds
present in this sample at the screening stage.
Quantitative method validation
Among the pesticides confirmed during the screening
step, a quantitative method to evaluate 12 pesticides was
developed and optimized (Table 1). The results are present
in Table S2 in the SI section. Linearity was achieved
presenting coefficients of determination (R2) higher than
0.99 for all studied pesticides in a concentration range
of 0.01 to 50.00 µg L-1. Precision was assessed based on
values found in the real water samples, and all the studied
compounds presented relative standard deviations lower
than 20%. The accuracy of the method calculated through
the 3-level recovery on low, medium and high standard
concentrations (0.25, 1.00 and 50.00 µg L-1, respectively)
were within the acceptable levels of 70 to 120%. LOQ
was considered as the lowest concentration point on a
calibration curve,38 at 0.01 µg L-1 for all pesticides, except
for the imazethapyr (LOQ = 0.05 µg L-1) and azoxystrobin
(LOQ = 0.10 µg L-1).
The method was considered feasible and reliable for the
target pesticides and the obtained values were in accordance
with the EU Guidelines.31
Pesticides quantification in samples
Pesticide quantification was performed after method
validation in the 36 samples, which were analyzed in
triplicate with RSDs under 20% for each pesticide. The
total concentration obtained of pesticides per sample is
summarized in Table 3, with values ranging from 0.28 to
20.63 µg L-1. For individual results of pesticide per sample
and collection period (M, J, S and D) presented as mean ±
standard deviation see SI section, Tables S3 to S6.
The presence or absence of a pesticide in water can
be related to its persistence or movement throughout the
soil. The physico-chemical properties of each pesticide
(half-life sorption potential (Koc), and solubility in water)
can be used to calculate the partitioning index known
by groundwater ubiquity score (GUS). The GUS index
can indicates the trend of mobility to water bodies and
groundwater in extremely low, low, moderate and high
displacement, although it does not take into account the
local environmental conditions.36,39,40 Accordingly, to
the GUS index, the compounds under study presented
moderate (propiconazole < diuron < azoxystrobin <
ametryn < tebuconazole) and high (carbendazim < atrazine
< imidacloprid < hexazinone < imazaquim < tebuthiuron <
imazethapyr) potential for movement.39,40 Nevertheless, the
concentrations obtained during the sample analysis showed
a behavior different than expected for some compounds,
such as diuron.
Within the legislated organic compounds, atrazine
is the only pesticide which was evaluated in this study,
with a maximum limit level of 2.00 µg L-1.21 The SP 9 (J)
sample presented the highest concentration of atrazine
concentration (1.40 µg L-1). At the same time, imazethapyr
was present in a concentration of 6.54 µg L-1 in SP 7 (J)
and diuron at 12.59 µg L-1 in SP 7 (D). Diuron is one of
the most widely used herbicides in Brazil, and although
it is persistent in soil due to its low solubility in water,
several studies have been made to measure its concentration
in water bodies and to evaluate its toxicity in different
organisms.9,41 A study performed with samples collected
from two rivers from Mato Grosso do Sul State, an area
with a strong agricultural activity obtained concentrations
of diuron lower than 0.01 µg L-1.15 The lowest concentration
of diuron in the present work was 0.16 µg L-1. The presence
of atrazine, carbendazim, imidacloprid, hexazinone and
tebuthiuron in the Mato Grosso do Sul State was also
evaluated by the researchers, with concentrations lower than
0.1 µg L-1.15 Effective concentrations (EC, 50%) of diuron
were reported in the literature at the same range of those
found in SP 5, 7 and 9 (1.03-12.59 µg L-1).42
Researchers also have been studying imazethapyr and
tebuconazole concentrations in surface water surrounded
by rice crop areas, where the maximum concentrations
reported for these compounds were 0.326 and 0.015 µg L-1,
Table 3. Total concentration of the 12 pesticides quantified by LC-MS/MS in water bodies of a rural area studied at north Paraná State
Month of collection Total pesticide concentration / (µg L-1), n = 3
SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 SP 7 SP 8 SP 9
March (M) 0.29 0.44 0.31 1.62a12.54a0.28 11.26a 0.28 2.14a
June (J) 0.45 1.04a0.41 1.59a12.95a0.28 19.29a 0.31 2.72a
September (S) 0.50a 0.64a0.40 1.57a 6.33a0.45 15.70a 0.33 2.52a
December (D) 1.55a 0.85a0.38 1.30a 8.52a0.28 20.63a 0.28 3.55a
LC-MS/MS: liquid chromatography-tandem mass spectrometry; atotal concentration of pesticides that exceeds the allowed limit established by European
Union (< 0.50 µg L-1).25 Data summarized from the sum of the individual detailed results (Tables S3-S6 in the SI section).
Pesticide Determination in Water Samples from a Rural Area by Multi-Target Method J. Braz. Chem. Soc.
8
respectively.43 Those concentrations were lower than
the observed in our study, that reached 6.54 µg L-1 for
imazethapyr (SP 7 in June) and 0.80 µg L-1 for tebuconazole
(SP 4 in September). Comparing the results reported in the
literature with those obtained in the present paper, several
samples had higher concentrations of pesticides, endorsing
the contamination suspect.
Considering the EU guidelines for water quality
standards for consumption, the maximum residue limit
(MRL) for the total active ingredients is of 0.50 µg L-1 of
pesticides, including metabolite degradations and reaction
products.25,26 With regards to this, at least 58% of the
samples analyzed in our study have a total concentration
of pesticides higher than the recommended. However, it
is important to highlight that only 12 pesticides of those
26 confirmed compounds in the screening stage were
quantified, which means that the total concentration of
pesticides can be even higher.
Overall results and final considerations
PCA analysis (Figure 3) was carried out in order
to evaluate the contribution of all the variables under
study (pH, conductivity, color, turbidity and pesticide
concentrations) taking into account the SP location and the
period of collection (M, J, S and D), allowing a reduction of
the data matrix size.44 The variables with high importance
in the multivariate analysis model were diuron, imazaquim,
imazethapyr and color, while tebuthiuron, hexazinone and
azoxystrobim presented less importance.
More similarities were expected within the collection
period due to the crop season, rain activity and runoff, than
for SP. However, in the 3D PCA plot, the opposite behavior
was observed, with groups formed by specific samples like
SP 4, SP 5 and 7, SP 9, whereas the other SP (1, 2, 3, 6 and
8) remained in the same group. This association indicates
that the contamination of these areas may have been
influenced by factors beyond the mere seasonal agricultural
activities of the area. SP 5 (M, J, S, D) and SP 7 (M, J, S,
D), for example, presented high concentrations of diuron
and imazethapyr in all the collections, compared to others
and remained as a single group. According to the GUS
index, imazethapyr has a higher potential of leachability
than diuron.39 Taken into account that the main use of diuron
is for the weed control in sugarcane or citrus cultivation
areas,9 crops that are not cultivated in the study area, the
high incidence of this herbicide on the water over all the
year is alarming.
In the screening step, the samples SP 4 and 9 were
considered as highly contaminated due to the presence
of several pesticides. After the multivariate analysis
(Figure 3), it was observed that the same sampling points
for the four collection campaigns were separated in some
isolated groups. This behavior showed to be consistent with
that previously observed, however, if it is considered the
total amount of the pesticides quantified per samples, SP 5
and 7 are more contaminated than SP 4 and 9. It is important
to consider that SP 5 is under a permanent preserved area,
which ideally should have less environmental impacts.
In the same way, SP 9 was collected from a groundwater
well, which is the source of water commonly used in many
cities and rural properties, almost without prior treatment
before consumption.
The only sample recommended for human
consumption that attends the EU guideline criteria,
considering the parameter evaluated, was SP 8 in all
campaigns. However, it is important to highlight that the
current quality standards from drinking water, surface
and groundwater, proclaimed by Brazilian legislation,
could be easier to meet if the water sources and spring
area were clean and preserved. On the other hand, the
current legislation still does not guarantee the water
quality regarding the absence of all pesticides in use and
in non-hazardous amounts to human health. According
to the article 11 of resolution No. 357/2005 from
CONAMA:21 “The public powers may, at any moment,
add other conditions and quality standards for a specific
water body or make them more restrictive due to local
conditions or based on scientific evidence”. In this way,
this study is expected to be a warning, contributing in
Figure 3. 3D view of principal component analysis (PCA) of water
samples collected in 4 different months (March, June, September and
December) of a year in 9 sampling points.
Almeida et al. 9Vol. 00, No. 00, 2019
the direction of public policies change, which need to be
more concerned about the environmental monitoring. In
addition, this work also shows the requirement of future
studies in the area using biological indicators through
ecotoxicological and toxicological tests, targets of our
next studies.
Conclusions
Evidences of “water bodies” contaminations in the
area were observed by the high conductivity values and
the presence of pesticides, with confirmation by the
screening and the quantitation of 12 pesticides in all the
studied samples, including drinking water (SP 8). High
concentrations of diuron and imazethapyr in surface
and groundwater drew more attention when compared
to other regions. However, the absence of legislation for
these compounds, like many others, makes difficult the
prohibition or control of the use and the residue level.
The high levels of contamination in a permanent
preserved area, water bodies and groundwater are
concerning, encouraging further assessment and monitoring
of this area, and bring subsidies for discussions on changes
in the parameters evaluated by the legislation and for
the management of natural resources by the competent
authorities.
Supplementary Information
Supplementary information is available free of charge
at http://jbcs.sbq.org.br as PDF file.
Acknowledgments
The authors are thankful to Ministério Público do
Trabalho do Paraná for their help in the area selection and
sampling and Waters Technologies of Brazil for the analysis
support. This study was financed in part by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior-Brasil
(CAPES)-Finance Code 001 for doctoral fellowships in
Chemistry-UEL. We would like to thank also CAPES
(project No. 88881.068504/2014-01, PROCAD/2013,
public notice 071/2013, process No. 3007/2014) for
financial support providing the chromatographic column
and (project No. AUX-PE-NANOBIOTEC-710/2009 -
process Nanobio 23/2008 No. 23038.019085/2009-14) for
the LC-MS/MS. Our gratitude also to Conselho Nacional de
Desenvolvimento Científico e Tecnológico- Brazil (CNPq)
by the fellowships - Bolsa Produtividade DT-2 Química
(Processo No. 309762/2017) and PIBIC.
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Submitted: November 13, 2018
Published online: April 18, 2019
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