Pesticide Residues in Honey from Stingless Bee Melipona Subnitida (Meliponini, Apidae)

Pesticides are widely used to protect agricultural plantations from pests, and thus improve crop production. However, inappropriate use may result in residues in human foods such as vegetables (Madureira et al., 2012), fruits (Oliveira et al., 2012), meat (Oliveira et al., 2018), fish (Oliveira et al., 2015), eggs (Hildmann et al., 2015), milk (Oliveira et al., 2014) and honey from the honeybee Apis mellifera (Pacífico da Silva et al., 2015). People consuming these contaminated foods are thus inadvertently exposed to residual pesticides, which increase the occurrence of such toxic effects as carcinogenesis, immunologic disorders and neurological disturbances (Kotsonis & Burdock, 2013).

Beyond the risk for consumers of contaminated honey, environmental contamination by pesticides affects the health of the bees, impairing their populations. In fact, pesticides including OPP may interfere with their cognitive abilities, behavior and immune responses against pathogens, and at high concentrations, OPP may even prove fatal (Rortais et al., 2005; Pacífico da Silva et al., 2015; Pacífico da Silva et al., 2016). The reduction in bee population may affect agricultural production, because about 35% of crops depend on flower pollination by animals (Klein et al., 2007), and this dependence is increasing worldwide (Aizen et al., 2008; Aizen & Harder, 2009). A number of bee species are known to act as pollinators but considerably vary in the plants they pollinate. Some pollinating bee species include the honeybee A. mellifera and bees from the Meliponini tribe (Maia-Silva et al., 2012).

The insects from the Meliponini tribe, Apidae family, are a large group of highly eusocial stingless bees (Camargo & Pedro, 1992; Pedro, 2014). Several species, mostly from the Melipona genus, are used for beekeeping i.e., meliponicultur). One such species kept in Brazil is the Melipona subnitida Ducke, popularly known as “Jandaíra,” with an average annual production of 2.5 L of honey per colony and maximum production of 5.6 L per colony (Cortopassi-Laurino et al., 2006). The honey from the Melipona species is commercialized as a natural medicine for children, the elderly and the sick (Jones, 2013; Carvalho, Martins, & Mourão, 2014). Thus, this honey must be inspected for the possible presence of residual pesticides that might affect consumers.

The present study aimed to determine the occurrence of pesticide residues in the honey of M. subnitida. For this, the simultaneous detection of 116 analytes in the honey samples was performed using a sensitive chromatographic method by liquid chromatography-tandem mass spectrometry (LC/MS-MS) (Souza Tette et al., 2016).

The evaluation of the presence of pesticides in M. subnitida honey revealed that twenty-five samples (71.4%) were contaminated by some amount of pesticide (Tab. 1). From these twentyfive positive samples, twenty-four (96.0%) had just one pesticide detected, and just one (4.0%) had three pesticides. The detected compounds were the organophosphate pesticides (OPP) chlorpyrifos-methyl (2 samples, 5.71% of all tested samples), monocrotophos (24 samples, 68.6%) and trichlorfon (6 samples, 17.1%). Residual levels exceeding the maximum residue levels (MRL; EC, 2008; EC, 2012; EC, 2018) was found for monocrotophos in 68.6% of tested samples and trichlorfon in 5.71%. On the other hand, none of the tested samples showed chlorpyrifos-methyl levels over the MRL.

Of the twenty-one samples collected in the countryside, fifteen (71.4%) were positive for some amount of pesticide, one (4.76%) for chlorpyrifos-methyl, fourteen (66.7%) for monocrotophos, and two (9.52%) for trichlorfon. Within the fourteen samples from urban areas, ten (71.4%) showed some amount of pesticide, one (7.14%) for chlorpyrifos-methyl, ten (71.4%) for monocrotophos and four (28.6%) for trichlorfon. A comparison of the frequencies of pesticides between both areas showed no significant difference (P > 0.05, Fisher's Exact Test from count data).

In the present study, M. subnitida honey showed three OPP: monocrotophos, trichlorfon, and chlorpyrifos-methyl. The residual levels exceeded the maximum residue levels (MRL; EC, 2008; EC, 2012; EC, 2018) for monocrotophos (68.6% samples) and trichlorfon (5.71%). This contamination of the honey might be attributed to environmental contamination, due to inappropriate agricultural utilization of pesticides. In fact, in a region near where our study was conducted, the water was found to contain residual levels of pesticides including chlorpyrifos (Gama, Oliveira, & Cavalcante, 2013; Pinheiro et al., 2017) and monocrotophos (Pinheiro et al., 2017).

Earlier studies also aimed to search residual pesticide levels in honey in Brazil and found OPP. Pacifico da Silva et al. (2015) found nineteen different pesticides after testing fifty-three honey samples form the Rio Grande do Norte state, Northeastern Brazil. Seven of these pesticides were OPP: chlorpyrifos, ethion, paraoxon, parathion, phosalone, and sulfotep. On the other hand, Souza Tette et al. (2016) tested one hundred honey samples from five states of Brazil, but just the organophosphorus trichlorfon was found in one sample.

The frequencies of pesticides from colonies raised in an urban area and from the countryside were similar (71.4%). This finding indicates that both colonies had similar foraging patterns, which was reinforced by a pollen analysis of all tested honey samples showing about 52% of the amount of pollen from Mimosa tenuiflora (data not shown). In fact, M. subnitida bees can fly distances of up to 4,000 m to forage (Silva et al., 2014).

Due to their high toxicity, monocrotophos and trichlorfon as insecticides have not been not allowed to be used in Brazil since 2006 and 2010, respectively (ANVISA, 2006; ANVISA, 2010). However, the use of such illegal compounds as pesticides is frequent in Brazil due to the high difference in market price (Lemos, Carvalho, & Ortiz, 2018). Illegal commerce has health and environmental concerns, and in fact both monocrotophos and trichlorfon were found in honey in the present study in levels higher than MRL. Thus, this finding reinforces the risk to human health due to use of illegal compounds that might contaminate food.

OPP are a commonly used class of insecticides with hundreds of commercial compounds available worldwide. Its primary toxic mechanism is the inhibition of acetylcholinesterase (AChE), an esterase that hydrolyzes the neurotransmitter acetylcholine. Fatal acute poisoning is due to the over-stimulation of cholinergic receptors in the peripheral and central nervous systems. Some OPP may induce delayed polyneuropathy days after a single exposure (Costa, 2006). The residual levels of OPP found in honey in our study were too low to produce either of these two toxic effects, but the consumption of contaminated honey might contribute to chronic OPP exposure.

Some epidemiological studies in humans connected long-term exposure to low doses of OPP with the development of neuropsychological disturbances. Furthermore, studies using laboratory animals have shown neurobehavioral interferences by doses lower than those that induce cholinergic signs of poisoning (Ray & Richards, 2001). One possible mechanism is the induction of neuroinflammation by OPP that potentially results in neurodegeneration and neurodegenerative disorders (Banks & Lein, 2012; Nurulain et al., 2014). Monocrotophos was found to inhibit AChE activity and induce oxidative stress in the brain of rats exposed to doses lower than No Observed Effect Level (Karumuri et al., 2019). Thus, we hypothesize that people who continuously consume this contaminated honey might be at risk of developing these neuropsychological disturbances.

Another risk associated with chronic OPP exposure is the development of cancer (Mostafalou & Abdollahi, 2013). Several epidemiological studies conducted in farmers and commercial pesticide applicators have shown an increased risk for several types of cancer due to OPP exposure. Among the OPP compounds found in the honey samples in our study, an increased risk for development of non-Hodgkin lymphoma by exposure to chlorpyrifos, and trichlorfon (Waddell et al., 2001) and lung cancer by chlorpyrifos (Lee et al., 2004) were found.

In conclusion, the honey produced by the stingless bee M. subnitida may be contaminated by pesticides, which emphasizes the need for food monitoring before commercialization. The high frequency of positive samples (71.4%) found in our study is indicative of environmental contamination, probably due to the inappropriate agricultural utilization of pesticides.