Land use and occupation and pesticide contamination assessment in honey from Africanized honey bees (Hymenoptera: Apidae) using gas chromatography-mass spectrometry

Abstract

We examined pesticide contamination in honey from Africanized honey bees in two different seasons, and evaluated the concentration and incidence of these compounds in georeferenced apiaries to provide relevant information for food safety and environmental quality. The land use and occupation of the region were evaluated by selecting 15 apiaries and georeferencing three randomly selected colonies of Africanized honey bees within each apiary. Honey samples were collected during two seasons, and three organochlorine pesticides (p.p`-DDE, Mitotane and Bicyclo[2.2.1]) and four organophosphate pesticides (Ethoprophos, Methyl Parathion, Chlorpyrifos and Fenchlorphos) were analyzed in the honey using gas chromatography-mass spectrometry. 80% of the apiaries showed a predominance of agricultural land use in the foraging areas of the bees. There was a higher concentration of pesticides in samples collected during Season I compared to Season II. There were differences in the proportion of samples contaminated with pesticides between seasons. Some honey samples showed concentrations of the chlorpyrifos pesticide above the levels recommended by Brazilian legislation. Pesticide contamination of honey is not limited to specific application season, indicating the persistent presence and easy availability of pesticides, including banned pesticides not sold or prescribed by agronomists, potentially smuggled from neighboring countries.

Key words
Apis mellifera; bee products; environmental pollution; food safety; phytosanitary products

INTRODUCTION

Bees collect floral resources for the survival of the colony, and therefore require them to be pure and free of contaminants. Environments with pesticides can contaminate the honey and pollen collected by bees, making them unsuitable for human consumption. Exposure to pesticides can pose serious risks to bee health, including death, which can affect the production and quality of honey (Gierer et al. 2019).

Bees can come into contact with pesticides indirectly, either through contaminated air during their travels, by carrying the pesticide on their bodies, or by collecting water, nectar, or pollen that contains residues of agrochemicals (Tette et al. 2016, Goñalons & Farina 2018).

Due to the potential risk of pesticide residues in food and the environment, constant monitoring of the concentration levels of these substances is necessary. An important and reliable way to perform this analysis is through the study of bee products, as well as the behavior and residues adhered to the body of these insects (Dively et al. 2015, Dolezal et al. 2016, Fisher & Rangel 2018, Leite et al. 2018, Belsky & Joshi 2019, Nocelli et al. 2019, Oliveira et al. 2020, Milone & Tarpy 2021, Rani et al. 2021, Silva et al. 2021).

The use of organochlorine and organophosphate pesticides in agriculture and their presence in honey has drawn the attention of the health authorities (Bandini & Spisso 2017). Therefore, studies on honey contamination have been conducted in various parts of the world, aiming to quantify different pesticides (Silva et al. 2015, Hungerford et al. 2021, Mukiibi et al. 2021, Kędzierska-Matysek et al. 2022, Boakye et al. 2023, Jaramillo-Zárate & Londoño-Giraldo 2023). This analysis has become essential for monitoring environmental quality and food safety.

Recent studies have discussed the global trend of increasing pesticide consumption due to the expansion of agriculture, the growing demand for food due to population growth, and the rise in pest and disease infestations in crops (Sharma et al. 2020, Rani et al. 2021). In the Western region of Paraná, monoculture is deeply ingrained in the production of crops such as corn, wheat, cassava, and soybeans. In Brazil, soybean and corn crops are the ones that use the most pesticides.

The soybean crop in the Paraná state cannot be cultivated from June 15th to September 15th, which is known as the “sanitary void,” in order to delay the development of Asian rust in the region (SEAB 2007). After this period, when planting is allowed, the application of pesticides intensifies due to crop management practices. Flowering occurs over a 60-day period, with different stages of development, and each producer determines the planting period as well as the pesticide to be used (Embrapa 2014).

The soybean sanitary void is an important measure to reduce bee exposure to pesticides, and that pesticide residue levels in honey can vary depending on local conditions, including agricultural practices and the presence of other crops. Our hypothesis is that bees from apiaries located in areas close to agricultural environments are more vulnerable to contact with pesticide residues, and that the presence of contaminants in honey may be associated with the period of bee production and land use and occupation.

The aim of this study was to investigate pesticide contamination in honey from Africanized Apis mellifera honey bees in two different seasons, assessing the concentration and incidence of these compounds in georeferenced apiaries. Additionally, we sought to investigate whether there is an association between the occurrence of contaminated honey samples and land use and occupation around the apiary, as well as the harvest season, in order to provide relevant information for food safety and environmental quality.

MATERIALS AND METHODS

Study site

The vegetation in the study area, located in the municipality of Santa Helena, Paraná, Brazil, is characterized by legal reserve areas and riparian forest along the Itaipu reservoir, as well as commercial agricultural plantations, mainly soybean (Glycine max L), wheat (Triticum aestivum L.), and corn (Zea mays L.).

The study was conducted in two distinct periods: the first spanned from September 1st to November 15th, and the second extended from November 16th to January 3rd. These specific timeframes were selected due to agricultural practices predominant in the region. Typically, soybean and corn cultivation dominate the area, except during the “sanitary void” period, occurring between July 15th to September 15th. During this period, the soil is dedicated to winter crops like wheat and oats (SEAB 2007). Soybean planting begins in mid-September (Embrapa 2014), aligning with the first study period. The duration of the crop’s flowering period varies from 4 to 6 weeks (McGregor 1976), with periods of 16 days (Chiari et al. 2005) and 28 days (Chiari et al. 2008) observed in experiments in the northern region of Paraná.

The forested areas in the region were predominantly reforested after the formation of the Itaipu Reservoir, with the planting of native and exotic species in a strip of up to 200 m from the shore, throughout the basin of the flooded stretch of the Paraná River, including the Santa Helena Biological Refuge. The forested areas in the municipality are mainly composed of species from the Fabaceae, Lauraceae, Malvaceae, and Boraginaceae families, including several beekeeping species identified through floristic surveys (Kipper et al. 2010, Camargo et al. 2014, Tonelli et al. 2022) and melissopalynology analysis (Moraes et al. 2019, Sereia et al. 2011, Sekine et al. 2013).

Land use and occupation

A sample (n = 15) of apiaries was randomly selected from a poll of 45 apiaries associated with the Agrofamiliar Solidary Cooperative (COOFAMEL) in the municipality of Santa Helena, Paraná, Brazil (24°51’37” S and 54°19’58” W; 258 m a.s.l), ensuring that there was no overlap in the selection of areas. At the outsed of the study during the first season, three colonies of Africanized honey bees were randomly selected in each apiary and georeferenced by the midpoint between them for the study of land use and occupation. These points were incorporated into the Geographic Information System (GIS) Spring 5.0.6, and a radius of 1.5 km was drawn around each apiary (an area of 706.5 ha), which corresponds to the maximum foraging distance of Africanized honey bees for food collection (Wolff et al. 2008), totaling 10,597.5 ha evaluated by images. We used a color composition in the images, Red, Green, and Blue (RGB), defining four classes for the evaluations: forested areas (FA), agricultural and pasture areas (AA), water areas (WA), and built-up areas (BA) (Figure 1).

Figure 1
Distribution of apiaries in the municipality of Santa Helena, Paraná, Brazil, delineated in the area corresponding to the action radius of Africanized honey bees.

Sampling for honey

The colonies within each apiary were used as individual sampling units for the honey analysis. An individual honey sample was collected from each of the three randomly selected colonies in every apiary (n = 15), resulting in three samples per apiary for each season. The samples underwent individual analysis during two distinct harvest seasons within the same year: Season I occurred from September 1st to November 15th, and Season II spanned from November 16th to January 3rd. This resulted in 41 and 45 observations for the first and second periods, respectively. It’s important to note that during Season I, honey collection was not feasible from three colonies, resulting in n=41. Notably, Season I coincided with the soybean “sanitary void” occurring between September 1st and November 15th, marked by planting restrictions, while Season II, from November 16th to January 3rd, did not involve any planting restrictions.

Before the start of each study period, in each of the three selected standard-size colonies within every chosen apiary, a frame with fresh beeswax foundation was inserted. This measure aimed to prevent potential contamination by residues of wax and previous honey traces. During each apiary visit, one frame was extracted from each hive to collect approximately 250 g of honey for analysis. The subsample was carefully taken from this single comb to ensure a representative portion for further laboratory examination. After extraction, new prefabricated frames equipped with fresh beeswax foundation were promptly inserted. This detailed procedure was implemented to ensure the collection of samples indicative of honey maturity, as confirmed by the observation of an 80% sealed honeycomb area, a recognized criterion signaling honey ripeness, within the hives of Africanized honey bees. Subsequently, the gathered samples were securely stored in appropriately labeled sterile plastic containers for futher analysis.

Selection and extraction of analytes

Standard Restek analytes of organochlorine (OCCs) and organophosphate (OPPs) pesticides were used for selection and extraction. Ten grams of each honey sample were weighed directly into 50 mL falcon tubes, initially diluted in 5 mL of distilled water and 25 mL of PA ethyl acetate, and constantly stirred for 1 min on a magnetic stirrer. They were then centrifuged at 2500 rpm for 10 min, the supernatant was reserved, and the sample was subjected to reextraction with 25 mL of ethyl acetate in the centrifuge for 10 min. The supernatant was saved, and the sediment was discarded. The collected extracts were combined and concentrated in a rotary evaporator under reduced pressure at 60 °C and 80 rpm. The resulting residue was solubilized in 5 mL of ethyl acetate for subsequent cleaning (adapted from Rissato et al. 2006). Florisil cartridges activated with 5 mL of acetone were used for cleaning. The samples were solubilized in 5 mL of ethyl acetate and filtered through a PTFE 0.45 μm membrane. To recover the target analytes (mobile phase for pesticide separation with solvents), 10 mL of hexane/ethyl acetate (1:1, v/v) were added directly to the Florisil cartridges with the filtrate, and the solvent with pesticides was saved. The resulting extract was subjected to concentration under evaporation, and the residue was solubilized in 1 mL of ethyl acetate, stored in an Eppendorf tube, and kept at a temperature of -5 °C for subsequent chromatographic analysis by GC-MS (Gas Chromatography–Mass Spectrometry).

Chromatographic analyses

Analyses were carried out on a Shimadzu QP2010-Plus GC/MS instrument equipped with an automatic sampler and electronic flow control, using an injection volume of 1 μL in splitless mode for 0.50 min and a 50/ 1 split at 250 ºC with 70 eV ionization energy. The temperature was increased from 80 ºC to 280 ºC at a rate of 5 ºC/ min, and held at 280 ºC for 15 min, totaling 41.5 min.

Working solutions were prepared from stock solutions, at concentrations of 500 μg/ kg for OCCs and 1000 μg/kg for OPPs, diluted in ethyl acetate and containing all studied pesticides (multiresidue). Appropriate volumes of each stock solution were transferred to a 10 mL volumetric flask containing a small volume of ethyl acetate, and completed with the same solvent. The mixture of working solutions (1 μg/ mL) was stored in amber vials to prevent contamination and evaporation.

Compound identification was confirmed by their respective retention index values, obtained from the Shimadzu and NIST libraries, using Shimadzu software. Analytical curves were established, and chromatograms were obtained in several segments for at least three ions in the specific ion monitoring (SIM) acquisition mode using electron ionization (EI) at 70 eV. A single chromatographic run was performed for OCCs and OPPs pesticide standards.

After analytical adjustments, five recovery tests were carried out on samples stored in 1 mL of ethyl acetate, ensuring the efficiency of the extraction method with recovery levels between 70 and 98%. All samples were analyzed in triplicate.

The validated OPPs pesticides were: Ethoprophos (ETOP) (72%); Methyl Parathion (METPAR) (98%); Chlorpyrifos (CHLO) (79%); and Fenchlorphos (FENC) (70%). For OCCs analytes, the recovered residues were p.p`-DDE (DDE) (76%), Mitotane (p’ DDT) (MITOT) (82%), and Bicyclo[2.2.1] (BICYC) (74%).

Data analysis

Mean, standard deviation and maximum, and minimum values for the concentrations of the evaluated pesticides were calculated, as well as the proportions associated with land use and occupation.

The maximum likelihood method was used to determine estimators of unknown parameters of the model. Canonical link functions g(µ) = ln(µ/1-µ) (binomial distribution), g(µ) = µ (normal distribution), and g(µ) = ln(µ) (Poisson distribution and negative binomiail) were adopted for the variables that showed better aderence. We used analysis of deviance (ANODE) to fit the model:

$$\eta =g(\mu )=\mu +E_i+A_j+E\ast A_j+{\epsilon }_{ijk}$$

in which:

µ = general mean effect,

E_i = effect of ith date (i = 1 and 2),

A_j = effect of jth apiary (j = 1,...,15),

E*A_ij = date ith and apiary jth interaction effect

ε_ijk = random error associated with each observation.

We used the likelihood ratio test for selecting models. The significance of the coeficientes was evaluated using the Wald (χ²) statistic. Confidence intervals of 95% were constructed for the proportion of pesticide incidence in honey.

For samples that were found to be outside the limits established by current legislation, the statistical distributions that provided the best fit were the binomial distribution with standard error correction using a quasibinomial model for METPAR and the normal distribution for CHLO and DDE. The logLik values were -41.0, 26.4, and 25.7 for METPAR, CHLO, and DDE, respectively.

For the characteristics of pesticide incidence (%) in Africanized honey bees honey, the statistical distributions that provided the best fit were binomial with correction of standard errors by a quasibinomial model (ETOP, METPAR, and FENC) and normal (CHLO, DDE, MITOT, and BICYC). The values found for the logarithm of the likelihood function (logLik) were -24.5 (ETOP), -41.0 (METPAR), -29.6 (FENC), 26.4 (CHLO), 6.99 (DDE), -2.58 (MITOT), and -22.5 (BICYC).

The statistical distribution that provided the best fit for the characteristics expressed by the concentration of pesticides in Africanized honey bees honey was the negative binomial. For each of these characteristics, the values found for the logarithm of the likelihood function (logLik) were as follows: 30,815.8 (ETOP), 157,561.0 (METPAR), 4,339.1 (FENC), 24,912.2 (CHLO), 2,028.5 (DDE), 14,438.8 (MITOT), and 3,811.8 (BICYC). The values of the Pearsonχ ² or Deviance statistic, used to correct the underdispersion found after fitting the negative binomial distribution, were: Pearsonχ ² (ETOP) = 0.3058, Pearsonχ ² (METPAR) = 0.1960, Pearsonχ ² (FENC) = 0.6009, Deviance (CHLO) = 0.0837, Deviance (DDE) = 0.1710, Deviance (MITOT) = 0.1632, and Deviance (BICYC) = 0.2862, all with 82 degrees of freedom.

We conducted a Pearson correlation analysis using data from the concentration of residues found in honey and land use and occupation. The data obtained for each date in the pesticides analyses of the honey were examined with Non-metric Multidimensional Scaling (NMDS), employing Euclidean distance after chord transformation. After we built the dissimilarity matrix with the normalized data, we used the command “metaMDS” to generate random and interactive processes to find the best solution possible. The goodness of fit measured of the NMDS was evaluated according to “stress” and Shepard diagrams. The “R” statistical and programming environment version 4.2.1 (R Core Team 2022) was used.

RESULTS

We verified, through satellite images of the areas surrounding each of the 15 apiaries that, on average, 59% (±21%) of the ground is occupied by agricultural crops, pastures or exposed soil (AA), 25% (±13%) by riparian forests and forest fragments (AF), 10% (±12%) by water areas (WA), and 6% (±9%) by buildings (BA). Additionally, 80% (n=12) of the apiaries showed a predominance of agriculture, including pastures, in the foraging areas of the bees. Table I shows the land use and occupancy percentages for the four classes defined in the study, as well as the concentration of pesticide residues (in ppb) found in the three selected colonies in each of the 15 apiaries evaluated in each honey harvest seasons.

Table I
Percentage of land use and occupancy in relation to the foraging area of Africanized honey bees in each apiary (706.5 ha) and the concentration of pesticide residue (in ppb) found in the honey during two harvest seasons.

Apiary	La<LOD use a<LOD occupancy (%)a				Seasons 1 (ppb)b (01 Sept to 15 Nov)							Seasons 2 (ppb)b (16 Nov to 03 Jan)
	FA	AA	BA	WA	ETOP	METPAR	FENC	CHLO	DDE	MITOT	BICYC	ETOP	METPAR	FENC	CHLO	DDE	MITOT	BICYC
1	18	49	33	0	<LOD	163.65	35.85	60.47	<LOD	<LOD	0.9	<LOD	<LOD	23.59	<LOD	<LOD	<LOD	<LOD
2	35	29	0	36	<LOD	<LOD	23.6	<LOD	<LOD	105.80	60.56	<LOD	<LOD	35.22	54.06	<LOD	<LOD	<LOD
3	53	26	0	21	126.55	80.51	10.56	<LOD	<LOD	8.83	1.37	<LOD	83.69	<LOD	<LOD	<LOD	<LOD	4.93
4	13	83	4	0	<LOD	87.95	23.25	<LOD	<LOD	<LOD	<LOD	39.44	161.12	10.97	<LOD	<LOD	<LOD	<LOD
5	20	70	1	9	<LOD	79.96	12.1	55.2	<LOD	<LOD	<LOD	<LOD	85.65	<LOD	<LOD	<LOD	<LOD	<LOD
6	24	71	2	3	<LOD	<LOD	16.35	<LOD	<LOD	21.86	10.79	<LOD	160.23	10.99	<LOD	<LOD	<LOD	<LOD
7	10	87	3	1	<LOD	80.68	33.56	<LOD	<LOD	<LOD	<LOD	<LOD	80.49	<LOD	<LOD	<LOD	<LOD	<LOD
8	15	83	2	1	<LOD	80.63	22.95	<LOD	0.15	<LOD	2.86	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD
9	26	66	2	7	<LOD	120.54	32.65	<LOD	<LOD	<LOD	<LOD	41.33	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD
10	19	45	3	32	48.83	243.91	22.14	<LOD	<LOD	<LOD	0.73	<LOD	88.75	<LOD	<LOD	4.00	<LOD	<LOD
11	41	56	1	1	122.9	81.57	22.02	<LOD	4.37	<LOD	<LOD	86.88	91.73	10.79	<LOD	<LOD	<LOD	<LOD
12	15	59	24	2	39.30	81.34	<LOD	<LOD	<LOD	<LOD	<LOD	44.69	83.09	10.96	<LOD	<LOD	1.13	<LOD
13	24	62	4	10	142.54	124	16.42	<LOD	47.01	83.05	42.98	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD
14	47	22	10	22	63.51	242.77	16.31	<LOD	0.26	5.59	2.14	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD
15	21	74	4	1	87.65	163.28	<LOD	<LOD	<LOD	<LOD	1.16	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD
Total	-	-	-	-	631.28	1630.79	287.76	115.67	51.79	225.13	123.49	212.34	834.75	102.52	54.06	4.00	1.13	4.93

• ^aFA forested areas, AA = agricultural a<LOD pasture areas, WA = water areas and BA = built-up áreas.
  ^bETOP = Etoprophos, METPAR = Methyl Parathion, CHLO = Chlorpyrifos, FENC = Fenchlorphos, DDE = p.p`-DDE, MITOT = Mitotane and BICYC = Bicyclo[2.2.1]
  < LOD – below the detection limit.

Out of the 41 honey samples analyzed in Season I, 15% did not contain any pesticides. Meanwhile, 29%, 27%, 22%, 5%, and 2% of the samples presented one, two, three, four, and five pesticides, respectively. In Season II, out of the 45 honey samples analyzed, 56% had no pesticide residues, while 29%, 13%, and 2% exhibited one, two, and three pesticides, respectively. At the first station, 5% of the samples exceeded the maximum limits recommended by Brazilian legislation of 10 ppb for CLHO residues; at the second station, this percentage was 2%. For the other pesticides studied, there are no established limits.

The proportion of Africanized honey bees honey samples contaminated with FENC and BICYC in Season I was higher (P = 0.005 and P = 0.001, respectively) than in Season II (Table II).

Table II
Comparison between incidence proportion of pesticides in samples of Africanized honey bees honey collected in Season I (Sep 01 to Nov 15) and Season II (Nov 16 to Jan 03) and their confidence intervals (CI).

		Detections (ppb) Seasons 1
Pesticides a	Class	x/n b	Proportion (%) *	CI ** (95 %)
ETOP	Organophosphate	11/41	27	(14.22;42.94)
METPAR		18/41	44	(28.47;60.25)
FENC		24/41	59a	(42.11;73.68)
CHLO		2/41	5	(0.60;16.53)
DDE	Organochlorine	4/41	10	(2.72;23.13)
MITOT		5/41	12	(4.08;26.20)
BICYC		10/41	25a	(12.36;40.30)
		Detections (ppb) Seasons 2
Pesticides	Class	x/nb	Proportion (%)*	CI** (95%)
ETOP	Organophosphate	4/45	9	(2.48;21.22)
METPAR		11/45	24	(28.47;60.25)
FENC		9/45	20b	(9.58;34.60)
CHLO		1/45	2	(0.056;11.77)
DDE	Organochlorine	1/45	2	(0.056;11.77)
MITOT		1/45	2	(0.056;11.77)
BICYC		1/45	2b	(0.056;11.77)

• ^aETOP = Ethoprophos, METPAR = Methyl Parathion, CHLO = Chlorpyrifos, FENC = Fenchlorphos, DDE = p.p`- DDE, MITOT = p’ DDT and BICYC = Bicyclo[2.2.1].
  ^bx/ n = Ratio between the number of contaminated experimental units and the sample size. *Proportions followed by different lowercase letters in the column differ from each other, between seasons, by the lsmeans difference test, at a 5% probability level.
  **Confidence interval for the mean with a 95% probability.

Among the honey samples collected during Season I (n=41), the contamination percentages with ETOP, METPAR, FENC, CHLO, DDE, MITOT, and BICYC were 27%, 44%, 59%, 5%, 10%, 12%, and 25%, respectively. In the honey samples collected during Season II (n=45), the contamination percentages with ETOP, METPAR, and FENC were 9%, 24%, and 20%, respectively, while CHLO, DDE, MITOT, and BICYC were found in 2% of the honey samples. (Table II).

There was a higher concentration in parts per billion (ppb) of pesticides FENC (P = 0.006), CHLO (P = 0.03), MITOT (P = 0.0001), and BICYC (P = 0.009) in the samples collected during Season I compared to Season II. No differences were observed between seasons for ETOP (P = 0.12), METPAR (P = 0.06), and DDE (P = 0.28) (Table III).

Table III
Concentrations of pesticides detected in samples of Africanized honey bees honey during Season I (Sep 01 to Nov 15) and Season II (Nov 16 to Jan 03).

	Detections (ppb) Season I (n = 41)
Pesticides a	Max *	Min *	Means * ± SD
ETOP	142.64	<LOD	42.08± 73.4
METPAR	243.91	<LOD	108.71± 123.0
FENC	35.85	<LOD	19.18a ± 17.0
CHLO	60.47	<LOD	7.71a ± 37.9
DDE	47.0	<LOD	2.64± 14.8
MITOT	105.8	<LOD	15.00a ± 55.5
BICYC	60.56	<LOD	8.23ª ± 31.0
	Detections (ppb) Season II (n = 45)
Pesticides	Max *	Min *	Mean * ± SD
ETOP	86.88	<LOD	14.15± 49.1
METPAR	161.12	<LOD	55.65± 103.7
FENC	35.22	<LOD	6.83b ± 13.9
CHLO	54.06	<LOD	3.60b ± 24.2
DDE	4.0	<LOD	0.26± 1.96
MITOT	1.13	<LOD	0.07b ± 0.51
BICYC	4.93	<LOD	0.32b ± 2.20

• ^aETOP = Ethoprophos, METPAR = Methyl Parathion, CHLO = Chlorpyrifos, FENC = Fenchlorphos, DDE = p.p`- DDE, MITOT = Mitotane and BICYC = Bicyclo[2.2.1].
  *Means followed by different lowercase letters between seasons, for each pesticide, differ from each other by the lsmeans difference test, at a 5% level of significance. SD = standard deviation. Max = maximum value. Min = below the detection limit (<LOD). The samples that had values below the detection limit were considered equal to zero for the calculation of the mean.

In Season I, we found a strong positive association between the presence of MITOT and BICYC pesticides in honey (r = 0.99, P < 0.001). In Season II, we observed a strong negative association between AF and AA variables (r = -0.80, P < 0.001), a moderate positive association between AW and AF (r = 0.51, P < 0.001), and a weak positive association between the amount of BICYC in honey and the AF of the apiaries (r = 0.33, P < 0.001). Additionally, we identified other significant (P < 0.01) but weak associations, such as the association between the AF of the apiaries and the amount of ETOP in honey (r = 0.43), between the AW of the apiaries and the presence of MITOT in honey (r = 0.31), and between the amount of BICYC in honey and the AW of the apiaries (r = 0.31). We also found weak positive associations between the presence of DDE and MITOT (r = 0.43) and the presence of DDE and BICYC (r = 0.40).

In general, during Season I, apiaries 4, 7, and 8 showed greater similarity among themselves and similar values for the amount of FENC in honey and AA. CHLO was found only in honey samples from apiaries 1 and 5. Apiaries 1, 9, 10, 12, 14, and 15 were more similar among themselves and presented similar values for BA and METPAR in the honey. Apiaries 3, 11, and 13 were more similar among themselves and presented similar values for ETOP and DDE. Apiaries 2 and 6 were more similar among themselves and presented similar values for MITOT and BICYC (Figure 2).

Figure 2
NMDS of the Chord-transformed distance matrix for the data from Season I (September 1st to November 15th). ETOP = Ethoprophos, METPAR = Methyl Parathion, CHLO = Chlorpyrifos, FENC = Fenchlorphos, DDE = p.p`- DDE, MITOT = Mitotane and BICYC = Bicyclo[2.2.1]. FA forested areas, AA = agricultural and pasture areas, WA = water areas and BA = built-up areas.

In Season II, apiaries 3, 4, 5, 6, 7, 10, 11, and 12 were more similar to each other and had similar values for MITOT, MET, DDE, ETOP, and BIOYC. Apiaries 1, 8, 9, 13, 14 and 15 were more similar to each other in terms of pesticide quantity in honey and AA, BA, and FA. Apiary 2 had a higher amount of CHLO (Figure 3).

Figure 3
NMDS of the Chord-transformed distance matrix for the data from Season II (November 16th to January 3rd). ETOP = Ethoprophos, METPAR = Methyl Parathion, CHLO = Chlorpyrifos, FENC = Fenchlorphos, DDE = p.p`- DDE, MITOT = Mitotane and BICYC = Bicyclo[2.2.1]. FA forested areas, AA = agricultural and pasture areas, WA = water areas and BA = built-up areas.

DISCUSSION

The predominance of 12 apiaries (80.00%) in agricultural and pasture areas (AA) for honey bees foraging is similar to the land use and occupation study of 30 apiaries in the municipality of Santa Helena, as reported by Camargo et al. (2014). In this study, an average of 21.17% of forested areas, 48.39% of agricultural areas, and 12.36% of pasture areas were found.

The predominance of agricultural areas around the sampled apiaries in Santa Helena, the beekeeping plant species present in forested areas are more important for honey production by bees than cultivated plants. In a study conducted by Camargo et al. (2014), in beehives located in areas with high and low apiary density in the municipalities of Marechal Cândido Rondon and Santa Helena, Paraná, the authors observed a higher honey production in the overcrowded area of apiaries in Santa Helena. In the sampled apiaries, where agricultural areas (48.39%) and pasture (12.36%) predominated in relation to forested areas (21.17%), the authors concluded that the abundant availability of beekeeping flora in forested areas, confirmed by floristic surveys, was responsible for this greater production.

Moraes et al. (2019) studied the presence of pollen types in honey samples from Santa Helena and Terra Roxa, in Paraná. While Santa Helena had a higher amount of pollen grains from species such as Hovenea dulcis (38.68%), Eucalyptus sp. (12.55%), Parapiptadenia rígida (8.00%), Leucaena leucocephala (6.99%), and Glycine max (4.20%) compared to the total pollen in all samples, Terra Roxa had a higher amount of pollen from species such as G. max (36.55%), Mimosa scabrella (15.46%), Eucalyptus sp. (7.74%), Mikania sp. (7.04%), and P. rigida (5.14%). Although Terra Roxa is predominantly agricultural, the samples still contained tree species, mainly from Legal Reserve areas, whose pollen grains appear in the honey. However, the predominant pollen grains reflect the land use and occupation of the municipality, as confirmed by previous floristic surveys.

We found higher concentrations of the pesticides ETOP and METPAR in honey produced by Africanized honey bees in both evaluated seasons compared to the other pesticides investigated. Among the investigated pesticides, FENC, CHLO, MITOT and BICYC showed higher concentrations during Season I compared to Season II. This could be attributed to late september, coinciding with the onset of soybean management. The presence of these same pesticides in higher concentrations in honey samples collected during Season I compared to Season II may be explained by their use in other crops. Additionally, the presence of ETOP, METPAR, and DDE in honey samples collected in both seasons indicates the use of these pesticides in different crops throughout the year, as observed in the studied areas.

Cosmann & Drunkler (2012) found that the most commercially sold pesticides in Paraná are mainly intended for soybean and corn crops. CHLO was among the most sold for corn, while methamidophos and acephate were the most employed in soybean. The same authors highlighted that organophosphates, such as the ones mentioned, are the leading cause of intoxication in the state.

The presence of METPAR and ETOP at high and medium concentrations in both seasons may indicate the excessive use of these pesticides, probably in soybean and corn crops, considering that these are summer crops in the region. These crops are frequently visited by bees (Malerbo-Souza & Silva 2011, Chiari et al. 2013), which may have led to the high contamination levels observed in the samples. However, it is important to note that the active ingredient METPAR was banned for use in pesticide products in Brazil starting in 2015, as determined by Anvisa’s Resolution RDC 56 of December 11, 2015 (ANVISA 2016).

The detection of other active ingredients at lower concentrations in honey samples from both seasons indicates their use. Some of these compounds, such as DDE and BICYC, are products of DDT degradation, which are highly harmful and prohibited. They persist in the environment for 2 to 15 years and can be harmful to human health even in small concentrations (ATSDR 2002). It is important to note that the harmful effects of low doses of pesticides are often underestimated, disregarding their sublethal effects, which may be present in honey and other foods (Desneux et al. 2007, Yang et al. 2008). While lethal effects are more easily observable and can lead to product registration loss, many sublethal effects on the physiological and behavioral processes of bees can affect the population of these insects (Leite et al. 2018)

Several studies (Freitas & Pinheiro 2010, Freitas & Pinheiro 2012, Giacoppo et al. 2014, Cavalcante et al. 2016, Barboza et al. 2018) have shown the harmful effects of different types of pesticides on bees, affecting their physiological and behavioral functions. For example, pesticides such as carbamates and organophosphates can inhibit acetylcholinesterase and impair bees’ communication with food sources (Carvalho et al. 2009). METPAR, a widely used product in Brazil in extensive soybean areas, has a persistent residual effect and can be harmful to bees (Freitas & Pinheiro 2012).

We found higher concentrations of pesticides such as FENC, CHLO, MITOT, and BICYC in honey samples during Season I when soybean is planted and germinates in the region. This can be explained by the intensive use of pesticides to control pests during this time. It is likely that apiaries with higher percentages of contaminated samples shared a common source of contamination, probably from agricultural areas and pastures, which were transported by nectar and wind. Honey is highly hygroscopic and can easily absorb contaminants.

As mentioned earlier, FENC has been banned for use in agricultural areas since 2010, according to Brazilian legislation (ANVISA 2019). Therefore, its presence in honey samples may be related to the improper use of the product in urban areas near apiaries. It is important to note that the improper use of pesticides, even in urban areas, can result in environmental contamination and affect the health of bees and other pollinators.

We observed that 44.00%, 5.00%, and 10.00% of honey samples collected during Season I contained METPAR, CHLO, and DDE, respectively. Thus, it is possible that these pesticides were used in concentrations, number of applications, and withholding periods different from the recommendations established by registration. This can lead to the accumulation of these substances in soil and groundwater, as observed by Carbo et al. (2008) and Hunt et al. (2016), further aggravating the environmental situation.

We detected the presence of honey samples with CHLO concentrations above the maximumm recommended limit set by legislation in both study periods. Thus, contamination of honey by CHLO may also be related to drift or contamination of the surrounding flora near the apiaries. It is important to note that the suspension and subsequent revocation of Resolution-RDC No. 206 of 08/23/04, which suspended the registration of products containing Chlorpyrifos, generated discussions and controversies regarding the risks associated with the use of this pesticide. Some studies indicate its high toxicity, both to humans and the environment, especially to bees (Kessler et al. 2015, Gradish et al. 2019). Therefore, the regulation and monitoring of the use of Chlorpyrifos are still relevant topics under discussion in the context of public policies and the scientific community. As for the other pesticides, there are no limits established by legislation.

According to Cosmann & Drunkler (2012), CHLO was among the best-selling insecticides for corn cultivation in the region of Cascavel, Paraná, Brazil. Currently, this product is applied directly to the leaves of corn, pasture, soybean, and wheat (ANVISA 2016), which are important agricultural areas in the region. Despite its release, according to the Brazilian Confederation of Beekeeping, this insecticide has been one of the main causes of bee mortality in Brazil.

Among the pesticide residues identified in honey, ETOP, FENC, MITOT, and BICYC are prohibited in agriculture according to Brazilian legislation (ANVISA 2019). It’s worth noting that these banned pesticides are not sold nor prescribed by agronomists, being smuggled products from neighboring countries. Despite their prohibition, these substances are still detected in honey samples, indicating their ilegal use in the region. Assessing the prevalence and potential risks posed by these illegal pesticides in commercially available honey would provide crucial insights into consumer exposure and guide regulatory interventions aimed at ensuring food safety.

The presence of ETOP and FENC in honey samples is concerning, as these pesticides are theoretically applied directly to the soil in urban areas and in crops such a banana, soybean, corn, and others to control nematodes and insects. The contact of these pesticides with bees can be reduced due to the three-day waiting period, however, there may still be risks to bees and other pollinators (Londres 2011). It is important to note that there is no tolerance limit established by current Brazilian legislation for these pesticides in honey, which makes monitoring and regulation of these products more difficult.

During Season I, some apiaries showed a higher correlation between AA and the presence of FENC, while in others the correlation was between BA and the presence of METPAR. The strongly positive correlation between MITOT and BICYC found in Season I explains a possible frequent and simultaneous use of these products. Ruiz-Toledo et al. (2018) evaluated the presence of pesticide residues in honey and pollen samples from A. mellifera colonies and found variation in the amount and identity of the pesticides detected in the diferente dates. It is worth noting that chronic exposure to low doses of various and multiple pesticides can have cumulative effects on the health of bees, both in adult and larval individuals (Grassl et al. 2018, Bommuraj et al. 2021, Almasri et al. 2022, Gao et al. 2022, Mohamed et al. 2023). When we compare the two periods, we generally observe that the occupation of the area near the apiary had less influence on the presence of contaminants in the honey than the season. In season I, with a higher presence of pesticides in the honey samples, AA showed a higher association with the amount of pesticides, however, in season II, although the amount of pesticides in the samples was lower, the type of occupation of the area around the apiary was quite variable. It should be considered that 80.00% of the apiaries are located in agricultural areas. This shows that nearby areas, even if they do not receive direct pesticide application, are not immune to contamination when close to agricultural areas subject to pesticide treatments.

MITOT and BICYC are compounds used in the production of veterinary drugs for bactericidal purposes (Priyanka et al. 2015) and are prohibited in agriculture (ANVISA 2019). The presence of these pesticides in honey may be related to the visits of bees to silage served in the trough for dairy cows, which is a common practice in the region. Silage is rich in sugars and is often used as a food source by bees.

Therefore, it is necessary to implement integrated pest management in crops based on the physiological knowledge and plant development, together with the peculiar practices of beekeeping activity, associated with the knowledge and biological characteristics of bees, including their foraging behavior, such as identifying times with higher frequency of visits.

ACKNOWLEDGMENTS

The authors wish to thank for the financial support Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) and Secretaria de Ciência, Tecnologia e Ensino Superior do Estado do Paraná (Programa Paranaense de Certificação de Produtos Orgânicos, processo 33/2012).

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Publication Dates

• Publication in this collection
  25 Oct 2024
• Date of issue
  2024

History

• Received
  14 May 2023
• Accepted
  23 June 2024