agronomy

Article
What Can Insects Tell Us about the Use of Pesticides?
The Detection and Quantification of Chlorpyrifos via QuEChERS
and HEADSPACE-SPME Methods Using GC/MS/MS
Cristian Valdés 1,2 , Carlos M. Silva-Neto 3 , Claudio Salas 4 , María Teresa Muñoz-Quezada 5 , Maribel Mamani 6
and Rodrigo O. Araujo 7, *

1 Centro de Investigación de Estudios Avanzados del Maule, Vicerrectoría de Investigación y Postgrado,

Universidad Católica del Maule, Avenida San Miguel 3605, Talca 3466706, Chile; cvaldesv@ucm.cl
2 Escuela de Ingeniería en Biotecnología, Centro de Biotecnología de los Recursos Naturales (Cenbio),
Universidad Católica del Maule, Talca 3466706, Chile
3 Instituto Federal de Goiás, Rua Dona Sanduca, s/n—Residencial Barravento, Goiania 74594-111, GO, Brazil;
carloskoa@gmail.com
4 Instituto de Investigaciones Agropecuarias, INIA Inthiuasi, Colina San Joaquín s/n, La Serena 1700000, Chile;
claudio.salas@inia.cl
5 School of Public Health, Faculty of Medicine, Universidad de Chile, Avenida Independencia 939,
Santiago 8320000, Chile; mtmunoz@uchile.cl
6 Laboratorio de Bioprocesos, Universidad Católica del Maule, Avenida San Miguel 3605, Talca 3466706, Chile;
maribel.viviana@gmail.com
7 Laboratorio de Entomología General y Aplicada, Centro de Investigación de Estudios Avanzados del Maule,
Universidad Católica del Maule, Talca 3466706, Chile
* Correspondence: araujorodrigodeoliveira@gmail.com

Abstract: The global use of pesticides, exceeding 4 million tons annually, poses substantial threats
to public health and the environment. Numerous studies emphasize the correlation between pes-
ticide exposure, genotoxicity, and neurobehavioral effects, which particularly impact children and
Citation: Valdés, C.; Silva-Neto, C.M.;
agricultural workers. Recent research underscores the health risks associated with moderately toxic
Salas, C.; Muñoz-Quezada, M.T.;
pesticides like chlorpyrifos. Chlorpyrifos contamination in crops, a result of extensive pesticide use
Mamani, M.; Araujo, R.O. What Can
since the 1960s, raises significant concerns regarding human health and the environment. Given its
Insects Tell Us about the Use of
Pesticides? The Detection and
potential atmospheric volatilization from crops, alternative detection methods are imperative. This
Quantification of Chlorpyrifos via study employs gas chromatography coupled with mass spectrometry (GC/MS) in MS/MS mode,
QuEChERS and HEADSPACE-SPME focusing on detecting chlorpyrifos in Chilean lettuce, using insects as indicators. Two extraction
Methods Using GC/MS/MS. methods, micro-QuEChERS and HEADSPACE-SPME, were compared, with HEADSPACE-SPME
Agronomy 2024, 14, 21. demonstrating superior sensitivity (6.77 ng/mg per sample vs. 3.99 ng/mg per sample) and offering
https://doi.org/10.3390/ time- and solvent-saving advantages. Additionally, HEADSPACE-SPME preserves samples for future
agronomy14010021 research, enhancing its utility. The study confirms the presence of pesticide residues in insects from
Academic Editor: Xinzhong Zhang lettuce crops, with 52.3% displaying chlorpyrifos when the SPME method was used. Predatory
and parasitoid insect families like Braconidae and Ichneumonidae show high pesticide loads. The
Received: 7 November 2023
findings of this study emphasize the widespread contamination of chlorpyrifos in lettuce crop insects
Revised: 19 December 2023
and highlight the efficacy of SPME for detection, suggesting its broader applicability for evaluating
Accepted: 19 December 2023
pesticide residues in horticultural systems. This optimized method holds promise for advancing
Published: 21 December 2023
chlorpyrifos contamination detection in agroecosystems, contributing to environmental monitoring
and food security.

Copyright: © 2023 by the authors. Keywords: agriculture; agronomy; agroecosystem; entomology; horticulture; integrated pest management
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons 1. Introduction
Attribution (CC BY) license (https://
Pesticides are extensively employed worldwide for pest control, primarily within the
creativecommons.org/licenses/by/
4.0/).
agricultural sector [1]. Annually, more than 4 million tons of active ingredients are used

Agronomy 2024, 14, 21. https://doi.org/10.3390/agronomy14010021 https://www.mdpi.com/journal/agronomy

Agronomy 2024, 14, 21 2 of 12

in commercially available pesticides [2]. Individuals face direct exposure to pesticides in

occupational, agricultural, and domestic settings, as well as indirect exposure through
environmental matrices such as air, water, soil, and food [3].
Contamination from pesticides is a widespread issue with far-reaching consequences
for both public health and the environment. Human exposure to these chemicals primarily
occurs through dermal contact, oral ingestion, and inhalation. Individuals who encounter
pesticides in substantial amounts, either directly or indirectly, are at risk of experiencing
acute poisoning symptoms such as nausea, headaches, muscle weakness, cramps, vomiting,
and, in severe cases, loss of consciousness, cardiac arrest, and even death. Furthermore,
prolonged exposure to lower-to-moderate pesticide doses can lead to the development
of chronic diseases, significantly impacting individuals’ overall quality of life and well-
being [4–6].
In Latin America, as evidenced by several studies [7], a link has been observed between
exposure to organophosphate and carbamate pesticides, as well as various classes of
pesticides, and the presence of genotoxicity biomarkers and adverse neurobehavioral
effects. This phenomenon is particularly notable among children and agricultural workers.
Recent research examining the health risk index associated with pesticide exposure [2]
revealed that certain low-toxicity pesticides produce hazardous burdens comparable to
those of high-toxicity pesticides due to their extensive use. Approximately 24% of the
global population resides in areas near locations with pesticide applications that exceed
acceptable levels, and 32% of the population could surpass acceptable intake limits for
these chemicals.
An additional, an indirect consequence of intensive pesticide management is local
environmental contamination, which can have significant repercussions on agricultural
production [8]. While a majority of studies on this subject concentrate on its implications
for human health or crop vitality, there exists a less explored yet equally consequential
dimension to consider: the influence of high-intensity pesticide management on the com-
position and functionality of the insect community surrounding economically vital crops.
These agricultural systems are critically dependent on ecosystem services encompassing
pollination, nutrient cycling, soil enhancement, and pest control [9]. Intensive agriculture,
characterized by its heightened reliance on agrochemical inputs, has been demonstrated to
markedly erode these indispensable services [10].
One of the most frequently utilized and controversial organophosphorus agrochemi-
cals is chlorpyrifos [6]. Its extensive application in agriculture has led to cases of poisoning,
potentially resulting in the death of non-target species upon exposure. Furthermore, it
contaminates and exerts adverse effects on various aspects, including plant development,
aquatic life, soil quality, livestock, wildlife, and human health [11,12].
In response to this issue, several strategies have been explored to monitor its presence
in the environment, regulate its use, and explore alternative options. It is important to note,
however, that these initiatives are still in their early stages or may prove to be economically
burdensome, particularly in countries with moderate-to-low incomes [13,14].
In Chile, chlorpyrifos ranks among the top-selling insecticides [15]. Its presence has
been identified through comprehensive monitoring efforts, encompassing air monitor-
ing [16–18], water quality assessments [19], honeybee investigations [20], and residue
analyses in food products [21–27], and even in the urinary biomarkers of children [28,29].
The results of all these studies point to high levels of chlorpyrifos residues in the air,
vegetables, and fruits, along with elevated concentrations of chlorpyrifos metabolites in urine.
In Chile, the exempt Resolution 5810/2022 prohibits specific pesticide brands con-
taining chlorpyrifos either within a two-year timeframe or until the depletion of existing
pesticide stocks. Additionally, Resolution 7128/2022 outlines Ministry of Agriculture
guidelines for the 2022–2026 period, focusing on sustainable forestry and agricultural
development, food security, and sovereignty [30,31]. Consequently, the development of
an efficient technique for promptly detecting chlorpyrifos residues in agricultural areas
exposed to these pesticides is essential.
Agronomy 2024, 14, 21 3 of 12

These findings highlight the importance of conducting research that evaluates both
direct and indirect pesticide exposure, employing efficient and expeditious methods on a
broader scale.
The employment of solid-phase microextraction (SPME) stands out as a swifter ap-
proach that eliminates the need for solvents when compared to other techniques, such as
solid-phase extraction (SPE), which has been reported to be more efficient for pesticide
extraction [32].
The solid-phase microextraction (SPME) technique, combined with mass spectrometry
(GC/MS), has been employed for the analysis of volatile components in Tenebrio molitor
(Coleoptera: Tenebrionidae) and Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)
larvae. These studies have revealed the presence of aldehydes, alkaloids, esters, ketones,
aromatic ketones, fatty acids, and terpenes which were detected 24 and 48 h after the
application of the insecticides fluthrin, thiamethoxam, and acetamiprid [14].
Nevertheless, there is a dearth of information regarding the application of analyti-
cal techniques, such as chromatography coupled with mass spectrometry, for assessing
pesticides in insects. Hence, we assert that this study, presented below, is innovative in
addressing this research gap.
The present study aimed to identify and quantify traces of pesticides, including
chlorpyrifos, absorbed by insects in lettuce crops managed using both traditional and
integrated pest management practices in the Coquimbo Region of Chile. Moreover, the
study sought to assess the efficiency of detecting pesticide residue in these insect samples
using a new SMPE injection technique in comparison to the conventional micro-QuEChERS
method. These samples were subsequently analyzed using GC/MS/MS.

2. Materials and Methods

2.1. Study Area
The research was conducted within the Coquimbo Region, Chile, which is one of the
primary regions for leafy vegetable production in the country, alongside the Valparaíso
and Metropolitan Regions. This region is distinguished by its balanced distribution of
integrated pest management (approximately 55%) and traditional management practices
(approximately 45%) [33].
This balance makes it an ideal setting for conducting a comparative analysis. Seven
specific sampling sites were selected within the Coquimbo Region of Chile. Detailed data
are provided in Table 1.

Table 1. Locations and characteristics of sampling points.

Average Insecticide Field

Geographical
Place ID Pest Management Method Applications per Productive
Coordinates
Lettuce Crop Cycle Area (ha)
Experimental field
1 Pan de Azúcar, 30◦ 04′ 27.7′′ S 71◦ 14′ 34.7′′ W IPM * and habitat management 2–3 8
INIA Intihuasi
Mario Yáñez (AR
2 30◦ 00′ 34.3′′ S 71◦ 14′ 30.0′′ W IPM * and habitat management 2–3 8
INIA Intihuasi)
Rafael La Paz (AR
3 29◦ 59′ 56.9′′ S 71◦ 15′ 22.5′′ W IPM * and habitat management 2–3 4
INIA Intihuasi)
Exclusive and scheduled
4 High pesticide load 1 30◦ 04′ 25.0′′ S 71◦ 15′ 56.2′′ W 7–9 1200
use of insecticides
Exclusive and scheduled
5 High pesticide load 2 30◦ 04′ 20.7′′ S 71◦ 15′ 12.8′′ W 7–9 400
use of insecticides
Exclusive and scheduled
6 High pesticide load 3 1 30◦ 03′ 07.1′′ S 71◦ 15′ 02.1′′ W 7–9 20
use of insecticides
Exclusive and scheduled
7 High pesticide load 4 30◦ 00′ 47.6′′ S 71◦ 14′ 24.2′′ W 7–9 60
use of insecticides
* IPM = integrated pest management.
Agronomy 2024, 14, 21 4 of 12

Due to legal constraints, the actual names of companies that apply traditional manage-
ment practices were replaced with designations such as “high pesticide load 1–4”.

2.2. Insect Collection

Insect sampling was conducted for 14 months, from February 2021 to March 2022,
using yellow pans. The sampling method was adapted from Bellamy et al. [9] to suit the
lettuce crops under study. Two sites were sampled within each of the seven lettuce crop
fields. The sites were selected along a transect running from (1) the middle of the farm,
referred to as the “inside site”, and (2) 30 m from the edge of the lettuce crop. At each site,
five replicate yellow pan traps were placed, spaced at least 5 m apart, in accordance with
Sutherland [34] and Brown and Matthews [35]. The selection of a site 30 m from the crop
edge was made to minimize potential variations due to edge effects in fields of different
sizes, thus serving as a standardized point of comparison across the selected lettuce crop
fields. The “inside” point was approximately 100 m from the farm’s edge. Traps were left
in position at each site for 24 h periods.
Yellow pan traps are passive collection tools that operate on a premise that lever-
ages the attractive properties of the color as an attractant for flying insects, especially
Hymenoptera, Coleoptera, Lepidoptera, Diptera, and Hemiptera [36]. The yellow color is
known to be highly effective at attracting a wide range of insect groups, including herbivo-
rous pests [37], parasitoid wasps [38], and pollinators [39]. The pan traps used were bright
yellow plastic bowls, measuring 20 cm in diameter and 3 cm deep, positioned just above
ground level. Within each trap was 300 mL of a solution composed of 1/3 propylene glycol
and 2/3 water.

2.3. Sample Distribution for Analysis

The insects’ masses ranged from 20 to 200 mg depending on the available sample.
In all cases, the sample was evenly divided for both the QuEChERS and HEADSPACE-
SPME analyses. HPLC-grade ethyl acetate and standard chlorpyrifos were purchased from
MERCK; the QuEChERS kit was purchased from Agilent.
Solid-phase microextraction (SPME): To extract volatile compounds from the insect
(Table 1), every sample was weighed before the extraction method (direct SPME or micro-
QuEChERS). The extraction of volatile compounds from the free space of the samples was
performed using polydimethylsiloxane/carboxen/divinylbenzene (PDMS/CAR/DVB) at
a temperature of 105 ◦ C. The extraction time for the fiber was 40 min, based on reported
work related to pesticide detection in larvae [14].
Micro-QuEChERS: A QuEChERS method was adapted [40] at the microgram level.
The protocol was as follows: The sample was dried at 50 ◦ C for 12 h and subsequently
homogenized using a mortar. The weighed content was deposited in an Eppendorf tube
to which 1 mL of ethyl acetate was added and shaken for one minute. QuEChERS Fi-
nal Polish EMR-Lipid® (25–250 mg) was introduced and stirred for 1 min. The tube was
centrifuged at 12,000 rpm for 4 min at room temperature. Subsequently, 600 uL of the super-
natant was extracted and transferred to a new Eppendorf tube containing anhydrous MgSO4:
PSA (12–120 mg). The contents were agitated for 1 min and then centrifuged at 12,000 rpm for
4 min at room temperature. The resulting supernatant was filtered for a GC/MS/MS analysis.
GC/MS/MS Chromatographic Conditions: Thermo TSQ DUO equipment (Thermo Fischer
Scientific, Waltham, MA, USA) was used in Selective Reaction Monitoring (SRM) mode. Helium
served as the mobile phase with a flow rate of 1 mL/min, and the injector temperature was
maintained at 230 ◦ C. The RTX5-ms column was utilized under the following conditions: an
initial temperature of 60 ◦ C for 2 min followed by a temperature increase to 180 ◦ C at a rate of
20 ◦ C/min which was held for 2 min, and a further temperature increase to 270 ◦ C at a rate of
10 ◦ C/min which was held for 2 min. The mass spectrometer system was operated in electron
ionization (EI) mode at 70 eV. The ion source and transfer line temperatures were both 280 ◦ C.
For quantifying the pesticide chlorpyrifos, the SRM method was optimized using the
“AUTO-SRM” function, allowing for the selection of appropriate energies for the fragmen-
Agronomy 2024, 14, 21 5 of 12

tation of “parent” ions and monitoring “daughter” ions. Specifically, the 169 m/z ion
was used for qualification, while the 79 m/z ion was selected for quantification (Table 2).
The qualitative/quantitative data were processed using Chromeleon software version 7.2.10.

Table 2. Energy conditions optimized for SRM.

Precursor (m/z) Product (m/z) Collision Energy (eV)

97 79 10
197 169 10

The QuEChERS extraction was standardized with the pesticide chlorpyrifos (1 ng/mg
of insect) using the spiked method, achieving an average recovery of 95.5% in pesticide-free
insects with an RSD of less than 2% (Table 3).

Table 3. Recovery of chlorpyrifos using QuEChERS.

Extraction Recovery Rate (%)

1 96.1
2 96.8
3 95.4
4 94.3
5 94.8
6 96.1

The HEADSPACE-SPME extraction technique is based on the release of the pesticide

from the surface of the insect rather than the entire insect as in a “bulk” method (such as
QuEChERS). Therefore, it depends on the adsorption and desorption relationship of these
compounds on the insect’s surface. We propose using different masses of chlorpyrifos in
the HEADSPACE-SPME vial and constructing a calibration curve that allows for a good
correlation as the volatile pesticide will be analyzed under the same incubation conditions
as the insects themselves.
In the case of the micro-QuEChERS technique, the limit of detection (LOD) was
0.9 ng/g, and the limit of quantification (LOQ) was 2.3 ng/g, whereas for HEADSPACE-
SPME, the LOD was 2.6 ng/g, and the LOQ was 12 ng/g.
For statistical comparison between the two pesticide detection methods used in this
research, a paired t-test was performed with a statistical significance level set at 99%.
This method of analysis is suitable for experiments in which both methods are applied
to the same materials. The analysis focused on comparing the following parameters:
the percentage of samples in which pesticides were detected (detection efficiency), the
total number of pesticides found in all samples (including those without detection), and
the number of pesticides in samples in which detections were made. The analyses were
performed using “Past 4.09” software, and bar graphs were generated to visualize mean
values and standard deviation variations for each analyzed variable. It is important to
note that the quantity of pesticide detected per entomological family was not subjected
to a statistical comparison due to the limited number of sample repetitions per group for
meaningful comparisons.

3. Results
A total of 68 out of 130 samples were detected using SPME-HEADSPACE, while
64 samples were detected using the micro-quEChERS method.
A comprehensive analysis was conducted on 130 samples of insects captured and sepa-
rated according to family level and examined through GC/MS, using both the HEASPACE-
SPME and micro-QuEChERS techniques. The results of the sample analysis by SPME
extraction are shown in Table 4. In this analysis, it was observed that chlorpyrifos was
detected in 48.46% of the sample population when employing the HEADSPACE-SPME
Agronomy 2024, 14, 21 6 of 12

method, whereas it was identified in 43.84% when using the Micro-QuEChERS technique.
Three ranges of chlorpyrifos content were established: low (<0.1 ng chlorpyrifos/mg insect),
medium (0.1–1 ng chlorpyrifos/mg insect), and high (>1 ng chlorpyrifos/mg insect).

Table 4. Comparison of chlorpyrifos levels in samples analyzed through SPME and QuEChERS
extraction techniques at high, medium, and low concentrations.

SMPE (ng of QuEChERS (ng of

Insect Families N N QuEChERS
Chlorpyrifos/mg of Insect) Chlorpyrifos/mg of Insect)
High 31 15.03 22 12.71
Agromyzidae - - 1 2.11
Aleyrodidae 1 5.06 1 12.18
Aphididae - - 1 1.24
Apidae 1 1.96 1 2.15
Braconidae 3 20.14 1 45.26
Diptera 1 3.55 - -
Cicadellidae 3 23.91 2 11.06
Diptera 1 12.73 - -
Gelechidae 4 15.83 2 19.17
Hemerobiidae 1 49.16 1 23.26
Ichneumonidae 2 18.85 2 18.21
Lygaeidae 1 1.75 1 2.30
Muscidae 2 1.06 - -
Mycetophilidae 1 2.72 - -
Mymaridae - - 1 4.36
Mycetophilidae 1 13.39 1 6.98
Nitidulidae 1 1.88 1 1.34
Psyllidae 1 56.71 1 31.25
Pteromalidae 3 21.96 2 22.70
Sciaridae 3 2.08 2 1.90
Tenebrionidae - - 1 1.22
Syrphidae 1 9.73 - -
Medium 36 0.42 21 0.46
Agromyzidae 2 0.51 1 0.88
Aleyrodidae 1 0.34 - -
Anthocoridae 1 0.77 - -
Anthomidae 3 0.44 4 0.24
Aphididae 1 0.63 - -
Apidae 1 0.73 1 0.18
Calliphoridae - - 1 0.33
Cicadellidae 1 0.26 1 0.53
Farmicidae - - 1 0.62
Gelechidae - - 1 0.98
Lygaeidae 1 0.27 1 0.60
Muscidae 2 0.30 3 0.42
Nitidulidae 2 0.58 1 0.88
Noctuidae 2 0.14 3 0.15
Plutellidae 1 0.29 - -
Psyllidae 1 0.61 - -
Pteromalidae - - 1 0.21
Sarcophagidae 1 0.65 1 0.99
Staphylinidae 2 0.24 - -
Tenebrionidae 1 0.33 - -
Syrphidae - - 1 0.87
Gelechidae - - 1 0.98
Lygaeidae 1 0.27 1 0.60
Muscidae 2 0.30 3 0.42
Nitidulidae 2 0.58 1 0.88
Agronomy 2024, 14, 21 7 of 12

Table 4. Cont.

SMPE (ng of QuEChERS (ng of

Insect Families N N QuEChERS
Chlorpyrifos/mg of Insect) Chlorpyrifos/mg of Insect)
Noctuidae 2 0.14 3 0.15
Plutellidae 1 0.29 - -
Psyllidae 1 0.61 - -
Pteromalidae - - 1 0.21
Sarcophagidae 1 0.65 1 0.99
Staphylinidae 2 0.24 - -
Tenebrionidae 1 0.33 - -
Syrphidae - - 1 0.87
Low 9 0.04 14 0.03
Agromyzidae - - 1 0.09
Anthomidae 1 0.04 1 0.006
Aphididae - - 1 0.02
Apidae 1 0.003 1 0.005
Calliphoridae 1 0.04 1 0.05
Chironomidae 1 0.09 1 0.002
Gelechidae 2 0.05 2 0.08
Muscidae 1 0.01 1 0.03
Mycetophilidae - - 1 0.02
Nitidulidae 2 0.06 3 0.01
Staphylinidae - - 1 0.02
Agronomy 2024, 14, x FOR PEER REVIEW 8 of 13
Total 63 7.56 57 5.09

The The pesticide

pesticide detectionmethods
detection methods exhibited
exhibited variations
variationsin both the the
in both percentage of sam-
percentage of samples
ples containing detected pesticides and the quantity of pesticide residues
containing detected pesticides and the quantity of pesticide residues within the insects.within the in-
sects. The SMPE method detected pesticide residues in 49.34% of the samples, while the
The SMPE method detected pesticide residues in 49.34% of the samples, while the QuECh-
QuEChERS method detected them in 42.10% of the samples (p > 0.001).
ERS method detected
Regarding them in
the number 42.10% found
of residues of theinsamples (p >the
the samples, 0.001).
SMPE Regarding
method yieldedthe number
of residues found
an average in ng/mg
of 3.56 the samples,
across allthe
theSMPE
analyzedmethod
samples.yielded an average
In contrast, of 3.56 ng/mg
the QuEChERS
across all theproduced
method analyzed ansamples.
average ofIn contrast,
2.10 ng/mg perthesample,
QuEChERS method
including produced
those without an average of
pesticide
residues per
2.10 ng/mg (p > sample,
0.001). including those without pesticide residues (p > 0.001).
When When consideringonly
considering only the
the samples
sampleswith withpesticide residues,
pesticide the SMPE
residues, the method found found
SMPE method
an average of 6.77 ng/mg per sample, while the QuEChERS method identified only 3.99
an average of 6.77 ng/mg per sample, while the QuEChERS method identified only
ng/mg per sample (p > 0.001). A visual representation of these numerical differences can
3.99 be
ng/mg per sample (p > 0.001). A visual representation of these numerical differ-
observed in Figure 1.
ences can be observed in Figure 1.

(A) (B) (C)

Figure 1. (A) Percentage of samples with detected pesticides; (B) pesticide quantity in all samples
Figure 1. (A) Percentage of samples with detected pesticides; (B) pesticide quantity in all samples
(including non-detected samples) in ng/mg of sample; (C) pesticide amount in samples with pesti-
(including non-detected
cide detection (ng/mg samples)
of sample).in ng/mg of sample; (C) pesticide amount in samples with pesticide
detection (ng/mg of sample).
In different locations characterized by varying pesticide application loads, the detec-
tion of chlorpyrifos
In different exhibited
locations discrepancies
characterized byamong environments
varying pesticide and between methods.
application loads, the detec-
In high-pesticide-load
tion of environments,
chlorpyrifos exhibited the SMPE
discrepancies methodenvironments
among detected 15.03 ng/mg of insects,methods.
and between
followed by 12.71 ng/mg of insects for the QuEChERS method. In contrast, in the environ-
ment with a medium pesticide load, the values were 0.42 ng/mg insect for SMPE and 0.46
ng/mg insect for QuEChERS. In low-pesticide-load environments, the values were 0.04
ng/mg insect for SMPE and 0.03 ng/mg insect for QuEChERS.
The detection values of chlorpyrifos varied among entomological families. For exam-
ple, Psyllidae had 56.71 ng of chlorpyrifos per mg of insect, Hemerobiidae had 49.16 ng of
chlorpyrifos per mg of insect, and Cicadellidae had 23.91 ng of chlorpyrifos per mg of
Agronomy 2024, 14, 21 8 of 12

In high-pesticide-load environments, the SMPE method detected 15.03 ng/mg of insects,

followed by 12.71 ng/mg of insects for the QuEChERS method. In contrast, in the envi-
ronment with a medium pesticide load, the values were 0.42 ng/mg insect for SMPE and
0.46 ng/mg insect for QuEChERS. In low-pesticide-load environments, the values were
0.04 ng/mg insect for SMPE and 0.03 ng/mg insect for QuEChERS.
The detection values of chlorpyrifos varied among entomological families. For exam-
ple, Psyllidae had 56.71 ng of chlorpyrifos per mg of insect, Hemerobiidae had 49.16 ng
of chlorpyrifos per mg of insect, and Cicadellidae had 23.91 ng of chlorpyrifos per mg of
insects. Values for all entomological groups are provided in Table 4.
Usually, the SMPE method also presents different values from the QuEChERS method
among entomological families but does not necessarily present higher values. Due to the
low number of samples per entomological family, it was not possible to make a statisti-
cal comparison between the chlorpyrifos detection values among the groups. However,
in absolute terms, some families had higher detection values, particularly in locations
characterized by high pesticide application loads.

4. Discussion
Pesticide toxicity tests on insects are typically conducted by quantifying the pesticide
before exposure without confirming the actual dose reached or absorbed by the insect.
These tests are typically confined to controlled laboratory settings [41]. The purpose of
detecting pesticides in insects is to assess the efficacy of pesticides concerning both target
and non-target insects serving as a bioindicator. This approach aims to ascertain the actual
quantity of pesticide reaching insects and to elucidate the responses of various insect groups
to pesticide applications in crop environments.
The use of different entomological groups for pesticide detection has proven to be a
valuable strategy for verifying how pesticides affect the environment [42]. Tison et al. [43],
while looking for pesticide residues in Vespa velutina nests, found contamination in at
least 53% of the nests, with detection ranging from 0.5 to 19.5 ng/g (using the QuEChERS
extraction method coupled with HPLC-MS/MS). The authors noted that wasps, being
predators of honeybees (Apis mellifera), are at a higher risk of pesticide exposure due to
their predatory feeding habits.
Regarding the residual dynamics of pesticide residues in the environment, it is an-
ticipated that insect groups more exposed to pesticide applications would have a higher
residual load of pesticides. This perspective aligns with existing knowledge, such as the
accumulation of DDT in food webs [44], underlining the potential for predatory insects and
parasitoids to carry a significant pesticide burden.
The outcomes of this study affirm the presence of pesticide residues in insects collected
from lettuce crops, indicating their potential exposure to an array of agricultural chemicals
employed in these fields. A significant finding of this investigation is that 52.3% of the insect
samples displayed the presence of chlorpyrifos when the SPME method was used. In this
work, several entomological families such as Braconidae, Hemerobiidae, Ichneumonidae,
and Pteromalidae also showed high residual loads of pesticides. These families consist of
numerous predatory and parasitoid insect species. The strategy of employing functional or
phylogenetic groups of insects to understand the pathways of pesticides in the environment
is already a strategy employed, particularly for aquatic insects and bees [45]. With the
approach presented here, other groups of insects can also be considered for the evaluation
of pesticide residues in horticultural systems.
This result underscores the prevalence of chlorpyrifos contamination within the insect
community inhabiting lettuce crops. Moreover, the effective application of the SPME
technique in detecting chlorpyrifos is evident.
Additionally, the comparison between the SPME and QuEChERS methods (Table 4)
illustrates the distribution of the chlorpyrifos content in the samples. Categorizing the
chlorpyrifos content into low, medium, and high ranges provides a comprehensive under-
standing of contamination levels within the insect population. This differentiation helps
Agronomy 2024, 14, 21 9 of 12

assess the potential risks associated with the accumulation of chlorpyrifos in these insects.
Furthermore, it is revealed that other pesticide residues detected using the SPME method
exhibit higher detection proportions compared to those detected using the QuEChERS
method, with statistically significant differences (over 17% higher; p > 0.001). When exam-
ining the quantity of residues found in the samples, the SMPE method yielded a higher
average across all analyzed samples than the QuEChERS method, even in cases in which
no pesticide residues were present (almost 70% higher; p > 0.001).
These findings underscore the SMPE method’s capability to detect and quantify pesti-
cide residues with enhanced precision in terms of both detection and concentration. The
disparity in results between the two methods could be attributed to the intrinsic character-
istics of each extraction and analysis technique [32,40]. These results also underscore the
importance of carefully selecting the extraction method based on the study’s objectives and
the requirements for detection and quantification.
Ultimately, the choice of an appropriate extraction method can have significant im-
plications for the accuracy and reliability of results in large-scale environmental pesticide
residue monitoring studies [12,13].
The assessment of multiple extraction and analysis methods, as conducted in this study,
contributes to the advancement of scientific knowledge and the enhancement of techniques
employed in determining pesticide presence and concentration in the environment. This,
in turn, aims to develop more efficient and sustainable measures for insect pest control in
agricultural production [6].
Due to the toxicity of chlorpyrifos to humans, its detection is carried out in vegeta-
bles or samples of water and soil [46]. Among methods, the most widely used is the
QuEChERS method; however, like any method, it has advantages and disadvantages.
This method requires larger quantities of substrate for pesticide detection within the
method’s detection limits.
A way of testing the toxicity of pesticides to insects is to quantify the pesticide before
exposure without verifying the actual dose the insect received or absorbed [41], and an-
other alternative is to measure LD50 [47]; however, this requires an isolated population
of insects which does not represent a real environment of insect communities. Detecting
pesticides within insects themselves serves multiple purposes, including verifying a pes-
ticide’s effectiveness against both target and non-target insects, acting as a bioindicator
to ascertain the actual amount of pesticide reaching insects, and shedding light on how
different insect groups respond to pesticide applications in crops. While these assessments
are more commonly conducted on foods intended for human consumption due to method-
ological considerations, it is noteworthy to emphasize that there exists a specific number
of studies directly measuring the detection of pesticide levels in insects [48]. Therefore,
this approach presents several challenges, including determining the detectable concen-
tration of pesticides within an insect’s body and establishing the required sample size
for effective detection.
The results obtained through the SMPE extraction method provide a significant con-
tribution to compliance with global regulations regarding pesticide prohibition and re-
striction, especially in middle- and low-income countries [3]. The accurate detection and
quantification of pesticide residues, such as chlorpyrifos, is essential to ensuring food
safety, protecting human health, and preserving the environment. It is recognized as a
relevant proposition to reduce the health risk index due to direct and/or indirect exposure
to pesticides [2].
In particular, the capability of the SMPE method to detect and quantify pesticide residues,
as demonstrated in previous studies [32], could support the efforts of the Chilean Agricultural
and Livestock Service (SAG) in monitoring and regulating the presence of chlorpyrifos and
other banned pesticides in agricultural products and the environment [30,31]. Moreover, the
potential to employ more efficient and sensitive extraction methods, like SMPE, could
streamline the processes of detection and analysis, allowing for a timelier and more precise
response to non-compliance situations [13].
Agronomy 2024, 14, 21 10 of 12

On the other hand, these results have significant implications for both agricultural
practices and environmental health. The detection of multiple pesticide residues indicates a
potential challenge in the management of pest control strategies [26] while simultaneously
minimizing the exposure of non-target species [2,30]. The prevalence of chlorpyrifos
residues in insects raises concerns about the possible transfer of these chemicals through
the food chain, which could impact both human and ecological health [20,22,23,27].
It is also relevant to underscore the significance of the SMPE method due to its capacity
to maintain sample integrity, a feature that sharply contrasts with the QuEChERS method,
which frequently leads to sample degradation. The preservation of sample integrity as-
sumes paramount importance, particularly when dealing with entomological specimens,
as it facilitates the replication of methodologies and supports a range of taxonomic and
ecological investigations. By preserving the structural integrity of insects, researchers can
accurately identify species, assess ecological interactions, and investigate the details of their
biology, thereby contributing substantially to the advancement of our understanding of the
natural world.
In summary, the findings of this study have the potential to significantly contribute
to the implementation and enforcement of regulations for the prohibition and restriction
of pesticide use on a global scale. Specifically, in the case of Chile, they could enhance the
capacity of the SAG to oversee exempt regulation 5810 [31]. This technological proposal
offers a new alternative to ensure proper pesticide management and to safeguard the health
and well-being of communities and the environment.

5. Conclusions
Based on the obtained results, we can infer that the SPME technique exhibits a greater
extraction capacity according to the outlined procedures. It is noteworthy that the SPME
technique holds a distinct advantage due to its lack of sample treatment procedures,
a step required in micro-QuEChERS, which necessitates a processing time of 2 to 3 h.
The utilization of automated SPME extraction leads to a reduction in instrumental error
associated with sample manipulation. Additionally, it allows for the potential reuse of the
sample for morphological analyses and insect characterization, if necessary, as the applied
heating temperature is insufficient to generate significant structural alterations in the visible
morphology of the insect. This preservation not only facilitates method replication but also
permits future taxonomic and ecological investigations regarding these specimens.
The identification of pesticide residues in insect samples from lettuce crops underscores
the complexity of pesticide exposure in agricultural systems. The significant presence of
chlorpyrifos, along with its distribution across various concentration ranges, as revealed
by the SPME method, emphasizes the ongoing need for the vigilant monitoring and
management of pesticide usage to mitigate its ecological and human health repercussions.

Author Contributions: Conceptualization, C.V., C.M.S.-N., C.S. and R.O.A.; methodology, C.V. and
R.O.A.; formal analysis, C.V.; investigation, C.V., C.M.S.-N., C.S., M.T.M.-Q., M.M. and R.O.A.;
resources, C.V. and R.O.A.; data curation, C.V., M.M. and R.O.A.; writing—original draft preparation,
C.V., M.T.M.-Q. and R.O.A.; writing—review and editing, C.V., C.M.S.-N., C.S., M.T.M.-Q. and R.O.A.;
project administration, R.O.A.; funding acquisition, R.O.A. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was supported by the “Agencia Nacional de Investigación y Desarrollo (ANID)”,
Chile, through the project “FONDECYT Iniciación” n◦ 11200014; the “Subsecretaría de Agricultura”,
Chile, and through the project “Programa de reducción de uso y riesgo de plaguicidas en la producción
comercial de hortalizas para la pequeña y mediana agricultura”, código INIA n◦ 502453-70.
Data Availability Statement: The data used in this research are not available for ethical reasons; any
requests for data availability should be directed to the author via correspondence.
Acknowledgments: The authors express their gratitude to Carlos Astudillo Oreste, Alejandro Andrés
Layana Salinas, Monika Victoria Maltés Ruiz, and Anibal Alberto Valencia Venegas for their contribu-
tions to various field activities related to this investigation.
Agronomy 2024, 14, 21 11 of 12

Conflicts of Interest: The authors declare no conflicts of interest.

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