INTRODUCTION

Honeybees are important pollinators of a wide diversity of native crops and plants, the pollination carried out by these insects is necessary for 35% of crops intended for human consumption (^{Ollerton, 2017}). However, worldwide they have suffered a decrease in their populations, a phenomenon called Colony Collapse Disorder (CCD) (^{Brutscher et al., 2016}). The characteristics of the collapsed colonies are: the partial or total death of the colony with the presence of dead bees in or near the hive, the partial or total disappearance of the colony, with the abandonment of food reserves and the young and weakening of the colony through slow development during spring, under optimal conditions (^{Simon-Delso et al., 2014}).

Although various factors have been reported such as the probable causes of colony collapse, one of the most important is exposure to pesticides (^{Calatayud-Vernich et al., 2018}; ^{Sánchez-Bayo et al., 2016}; ^{Traynor et al., 2016}). Bees are exposed to pesticides when they search for nectar-polliniferous resources, especially if the colonies are located near agricultural areas (^{O’Neal et al., 2018}). Exposure to sub-lethal doses of pesticides has been shown to affect bee behavior (^{Balbuena et al., 2015}), foraging (^{Cresswell y Thompson, 2012}), its longevity (^{Wu et al., 2011}), thermoregulation (^{Tosi et al., 2016}); as well as their olfactory learning and memory (^{Lu et al., 2014}).

Pesticide residues can accumulate on bee bread, honey, and wax (^{Johnson et al., 2010}; ^{Lozano et al., 2019}); presenting in the latter the residual storage capacity of pesticides (^{Benuszak et al., 2017}). Therefore, from a contaminated honeycomb, the residues can be transferred to the stored honey, presenting a risk to consumers. Also, the consumption of "honey in honeycomb", as a food additive in fruit treatment, food supplement or as a flavoring, represents a health risk (^{Wilmart et al., 2016}).

In the semi-desert of northern Mexico, the presence of pesticides in low concentrations has been found in honey and wax samples (^{Alcántar-Rosales et al., 2016}), and a decrease of up to 2010 has been reported for the period from 2010 to 2017. 35% of bee colonies (^{SIAP, 2018}).

Therefore, the objective of this research was to determine and quantify the presence of pesticides in honey and wax samples from bee colonies, with and without a history of collapse.

MATERIAL AND METHODS

RESULTS

Pesticides detected in wax and honey from colonies BC, CA and SA. In wax, a greater diversity of pesticides (insecticides, fungicides, acaricides and herbicides) was found, compared to the honey samples (insecticides and fungicides). On the other hand, the greatest number of pesticides by groups was found in colonies with a history of collapse (20), followed by colonies without a history (19) and finally with those under collapse (7). This same behavior was observed when separating pesticides by category, finding in all cases a greater diversity in CA, followed by SA and finally by BC (Table 1).

Regarding the amount of pesticides, the insecticide acetamiprid was the only one detected in all the wax samples analyzed and was present in a greater amount in the colonies CA (0.686 mg kg^-1), followed by BC (0.402 mg kg^-1 ), and finally by SA (0.234 mg kg^-1) (Table 2). This same insecticide was present in all the honey samples, but in greater quantity in the BC colonies (0.633 mg kg^-1), followed by the SA (0.404 mg kg^-1), and finally the CA (0.266 mg kg^-1) (table 3).

On the other hand, the insecticide malathion was found in 11 wax samples from the colonies CB, CA and SA; however, the amounts were low. It should be added that cis permethrin was found only in six wax samples CA (0.087 mg kg^-1) and SA (0.002 mg kg^-1) (Table 2).

DISCUSSION

Diversity of pesticides in wax and honey. The wax samples presented a greater diversity of pesticides (insecticides, fungicides, acaricides and herbicides), compared to those of honey. In the same way ^{Johnson et al. (2010)}, report the occasional presence of pesticide residues in honey, since most pesticides are hydrophobic and can be transferred more easily through interactions of honey bees towards their wax (^{Calatayud-Vernich et al., 2018}). Therefore, pesticides can be found more frequently in wax than in honey; or, although the levels of pesticides in honey are low, they tend to contaminate the wax due to their lipophilic nature (^{Valdovinos-Flores et al., 2017}); as well as a low wax replacement in the hive and recycle to reintroduce. Therefore, highly hydrophobic and stable pesticides are the main factors for the storage of pesticides in wax (^{Calatayud-Vernich et al., 2018}).

From the total pesticides, insecticides were the main ones found in the wax samples, with 59.1%, which correspond mainly to organophosphates (chlorpyrifos, malathion, coumaphos, dimethoate and malaoxon), pyrethroids (deltamethrin, cis and trans permethrin) , neonicotinoids (acetamiprid and imidacloprid), and organochlorines (pentachlorophenol). Likewise, organophosphates (malaoxon dimethoate, methamidophos and omethoate), and neonicotinoids (acetamiprid and imidacloprid) were found in honey; representing 75.0% of the samples. The presence of insecticides in honey and beeswax represents the greatest risk for pollinating insects (^{Botías y Sánchez-Bayo, 2018}; ^{Ostiguy et al., 2019}); therefore, the detection and quantification of these in the samples analyzed reflect their high exposure to bees, causing irreversible damage in some colonies.

Fungicides in wax (27.3%) and honey (25.0%), were the second class of pesticides found with the highest presence. ^{Botías y Sánchez-Bayo (2018)} point out that some fungicides can increase the toxicity of insecticides by reducing the detoxification capacity of bees. Colony fungicide residues have also been found to be related to the prevalence of disease in bees (^{Simon-Delso et al., 2014}). Furthermore, it is suggested that the effect of fungicides on pollinators is not due to direct toxicity, but due to the alteration of the microbiome present in the pollen and nectar of the treated and / or contaminated plants that the bees feed on and their own bacterial flora (^{VanEngelsdorp et al., 2009}); which has important consequences on the nutrition and health status of bees.

Finally, herbicides (9.1%) and acaricides (4.5%) are the pesticides with the lowest presence in the wax samples analyzed. Herbicides do not represent acute toxicity to pollinating insects (^{Botías y Sánchez-Bayo, 2018}); however, its use indirectly affects bees, because they eliminate large numbers of wild plants and reduce floral diversity, which is the main source of food (^{Bohnenblust et al., 2016}); the low presence of herbicides in the samples analyzed can be attributed to this.

In the case of acaricides, which are used to control Varroa destructor, they can act additively or synergistically with insecticide residues in bee colonies (^{Johnson et al., 2013}); however, in our case, the acaricides found in the samples was 4.5% of the total pesticides, which is why these possibly cause minor adverse effects in the bee colonies.

Diversity of pesticides in wax and honey from colonies BC, CA and SA. Colonies with a history of collapse had a greater diversity of pesticides (20), compared to colonies without a history (19), and colonies with a collapse (7); This is also reflected by disaggregating pesticides into insecticides, fungicides, acaricides and herbicides; except for the latter two cases, where there was no presence in BC, and the diversity was similar in the CA and SA colonies.

^{Alcántar-Rosales et al. (2016)}, report similar data in honey and wax regarding the reduced diversity of pesticides for the colonies that collapsed, finding two pesticides (neonicotinoids and organophosphates) in the honey samples, and four (organophosphates, benzimidazole, pyrethroids and derivatives of pyridine) in the wax; however, these are in low concentrations. In relation to this, the wax contaminated with pesticides, being in contact with the developing egg until the bee emerges, can cause sub-lethal effects in worker bees, mainly affecting larval development and longevity of bees. (^{Wu et al., 2011}); being able to cause indirect effects in the colony, such as premature changes in the role of bees and in foraging activity. For the present case, the colonies that collapsed presented the least diversity of pesticides, but in high quantities; possibly showing that the presence of insecticides and fungicides affected bees, causing their collapse.

It is also important to highlight that the presence of pesticides is due to the management of the hives (stationary and migratory), as well as the location of the apiary, among other factors (^{Ostiguy et al., 2019}). For our case, in the three groups (CA, SA and BC), the majority of hives are mobilized, and it has been reported that the mobilization of hives intended for pollination causes greater exposure to pesticides (^{Traynor et al., 2016}). In our case, beekeepers mobilize them, for two reasons: the search for flowering, and when they are leased for crop pollination; this management can cause stress to bees, making them more susceptible to pesticide poisoning (^{Sánchez-Bayo et al., 2016}).

The location of the apiaries is also an important factor, due to the effect that the surrounding vegetation will have on the hives. Intensified agriculture has been shown to cause loss of natural habitats; therefore, intensive cultivation and, in general, the lack of plant biodiversity limit the quantity of food, which causes a decrease in abundance and richness of pollinators; as well as an impact on the health of honey bees (^{Kovács-Hostyánszki et al., 2017}).

In our case, the surrounding vegetation at the time of sampling and the crops that are mostly planted are as follows: for the BC group, the predominant vegetation was pinabete (Tamarix spp.); and the crops close to the apiaries were corn (Zea mays), alfalfa (Medicago sativa) and cotton (Gossypium spp.). For the CA group, the predominant vegetation was pinabete (Tamarix spp.) and mesquite (Prosopis laevigata), with crops of maize (Z. mays), sorghum (Sorghum vulgare), cotton (Gossypium spp.), Alfalfa (M. sativa), watermelon (Citrullus lannatus), melon (Cucumis melo), chili (Capsicum annuum), and squash (Cucurbita pepo). Finally, for the SA group, the main predominant vegetation was mesquite (P. laevigata) and pinabete (Tamarix spp.), with crops of alfalfa (M. sativa), corn (Z. mays), sorghum (S. vulgare) and cotton (Gossypium spp.). This gives evidence that the hives in the region are widely exposed to pesticides, which means that agricultural areas contribute to the high presence of pesticides in the hive products (^{Traynor et al., 2016}).

The greatest diversity of pesticide residues was found in honey and wax in the CA colonies, it could be related to the amount of crops near the colonies where the samples were taken; however, the amount of pesticides was less compared to collapsed colonies with the presence of high amounts of pesticides in honey and wax. This was possibly due to the fact that these colonies presented greater exposure to the treated crops, or due to a recent application of pesticides during the colonies' stay with respect to CA and SA; since they presented lower levels of pesticides, even being in the same region.

Chronic exposure of bees to pesticides at sublethal doses can affect neurological functions, such as memory and behavior; symptoms that may occur before the collapse of the hive (^{Lu et al., 2014}); In addition to this, exposure to pests, diseases, poor nutrition, or the interaction between pesticides and pathogens, contribute to the mortality of bee colonies (^{Broadrup et al., 2019}), which possibly occurred in BC colonies.

Amount of pesticides in honey and wax samples. The insecticide found in all the honey and wax samples was acetamiprid, which was also the one that had the highest amount on average, both in honey (0.385 mg kg^-1) and in wax (0.316 mg kg^-1) ; exceeding the Maximum Residue Limits (MRLs) of the European Union (EU) (0.05 mg kg^-1). Data reported by ^{Gaweł et al. (2019)} in honey differ from ours, whose concentrations are low and range from 0.001 to 0.13 mg kg^-1. Also, ^{Da Silva et al. (2015)}, report an average of 0.0025 mg kg^-1.

Studies carried out by ^{El Hassani et al., (2008)} indicate that the consumption of this pesticide at sublethal doses of 0.1 µg/bee affects its behavior and olfactory learning, due to its immunosuppressive effect (^{Di Prisco et al., 2013}), causing a greater susceptibility to infection by the microsporidium Nosema (^{Broadrup et al., 2019}). Furthermore, the immune weakening favors the spread of the Varroa mite in the honey bee colonies, which is a source of virus transmission (Di Prisco et al., 2016); such as the Deformed Wings Virus (DWV), the Israeli Acute Paralysis Virus (IAPV), the Acute Paralysis Virus (ABPV) and the Kashmir Virus (KBV) (^{Belsky y Joshi, 2019}; ^{Brutscher et al., 2016}). The combination of these diseases with neonicotinoid insecticides, contribute to the collapse of the hives (^{Sánchez-Bayo et al., 2016}). Furthermore, acetamiprid can present synergistic effects when combined with other pesticides (^{Wang et al., 2019}), which possibly explains the collapse of the colonies in this region; in addition, they probably had a longer time of exposure to pesticides.

The second pesticide in frequency was Malathion, which was found in 11 wax samples with levels from 0.005 to 0.041, and an average of 0.015 mg kg^-1; figure not exceeding the EU MRLs of 0.05 mg kg^-1. For the north of Mexico ^{Valdovinos-Flores et al. (2017)}, report the presence of Malathion in 100% of the wax samples, with levels ranging from 0.006 to 1,532 mg kg^-1, with an average of 0.018 mg kg^-1. Malathion, used in agriculture as an insecticide and acaricide and for the control of urban pests, has low persistence and high toxicity in insects (^{Toxnet, 2019}).

The results of this study demonstrate a high incidence of organophosphate Malathion in northern Mexico, and although it has low persistence, it is of high incidence in the samples analyzed. This is probably explained by the application of organophosphate near the hives before the samples were collected, since, as previously mentioned, most of the hives move around in search of flowering and are located mainly near agricultural crops. .

Finally, cis- permethrin was found less frequently (six wax samples), but in greater quantity (0.087 mg kg^-1); however, the EU does not specify its MRL. Similar data are reported by ^{Johnson et al. (2010)}, with values of 0.133 mg kg^-1. However, this insecticide is highly toxic to bees, with a topical LD50 of 0.024 µg/bee ^{(Piccolomini et al., 2018)}. Prolonged exposure of pyrethroids can affect cellular and humoral immunity; as well as the decrease in immunity in bees (^{Qi et al., 2019}). Permethrin is mainly used as an insecticide and acaricide for the treatment of forest use seeds, and for vector control (^{Toxnet, 2019}); and its wide use may be the reason for its greater quantity and presence in the samples analyzed, since some samples came from the proximity of agricultural areas.

It is important to note that La Comarca Lagunera has been a benchmark in terms of cotton planting, forage cultivation for cattle, and also horticultural production is on the rise (SIAP, 2019); making it a region where a great diversity of pesticides have been used, many of which have a residual effect (^{Vargas-González et al., 2016}). Therefore, inadequate agricultural practices and the inefficient use and management of pesticides (^{Esquivel-Valenzuela et al., 2019}) have created a serious public health problem, due to poisoning by agrochemicals, as well as for the environment; highlighting the damage caused to beekeeping by its effect on the collapse of hives.

CONCLUSIONS

The greatest amount of pesticides were found in the wax of the colonies with a history of collapse; thus they also present the greatest diversity (insecticides, fungicides, acaricides and herbicides). The presence of pesticides in honey and wax from the colonies under, with, and without a history of collapse may be the consequence of the phytosanitary treatments used in agriculture, so that their presence may be influenced by the origin of the sample, since that the radius of bees’ action is up to ten kilometers. However, our data does not allow us to affirm that the presence of pesticides is the main or only cause of colony collapse; and therefore, it is required to continue with this type of research to determine the factors that affect the health of honey bees, such as the presence of pesticides, parasites and diseases in the region.