Toxicity of Insecticides with Different Modes of Action to Apis Mellifera Larvae (Hymenoptera, Apidae)

During the last decade, there has been an increase in pollinator declines, and pesticides have been identified as a major cause worldwide (Goulson et al., 2015; Woodcock et al., 2017). The pesticide usage pattern in India is alarming because most of which are insecticides (51%), which affect insect pollinators, followed by herbicides (19%), fungicides and bactericides (33%), and other types such as rodenticides and nematicides (FAO, 2018). Managed honeybee colonies are periodically moved in the vicinity of agricultural fields to increase honey production and improve crop pollination. Since insect pollinators collect nectar and pollen from many crops, they can be exposed to pesticide residues from contact with pollen or foraging for nectar, which can then be transported to the colony. Contamination is not only observed in adults but also in larvae and beehive products, potentially leading to adverse effects to the bees (Doublet et al., 2014; Sharma et al., 2022). Honey bee larvae are orally exposed when they consume pollen and nectar; therefore, the potential for chronic toxicity occurs at the brood stage, early life stages are more sensitive to specific contaminants compared to adult stages (Atkins, 1986; Rortais, et al., 2005). Few toxicity studies evaluate the risk of pesticides to honey bee larvae, but approaches to larvae exposure need to be considered for honey bee larvae (Fantke et al., 2018).

Therefore, we chose fipronil and lambda-cyhalothrin, the two most frequently detected pesticides in the hive at concentrations found in pollen and bee bread. Fipronil belongs to the phenyl pyrazole group (C₁₂H₄C_l2F₆N₄OS) and is the first product of this group to be introduced for pest control and widely used in India against, thrips, termites, beetles, caterpillars that infest crops such as potato, corn, soy and onion. Lambda-cyhalothrin, another class of insecticide, comes under pyrethroids and is used on various crops, such as almonds, apples and cherries (Epstein et al., 2000). Pyrethroid insecticides have been widely detected at the global scale (Tang et al., 2018) and are often associated with honey bee deaths (Zhou et al., 2011).

In 1981, an in vitro rearing methodology was proposed as a potential risk assessment tool for testing the toxicity of pesticides to worker bee larvae (Wittmann & Engels, 1981), and since then several in vitro rearing protocols with acceptable survival success have been developed (Vandenberg & Shimanuki, 1987; Peng et al., 1992; Oomen et al., 1998). The most recommended method was by Oomen et al. (1998), an in-hive method in which experimental bees comprise free-flying colonies are artificially contaminated by affixing a one-litre syrup feeder to the hive for twenty-four hours. The method is not reproducible, particularly if the test product is stored in the combs and not immediately dispensed to the brood by nurse bees. In addition, the method provides no quantitative data because it is impossible to measure the product consumed by larvae, and it cannot be used as a laboratory test.

Another method is the vitro method developed by Aupinel et al. (2005, 2007) and adopted by OECD (2016), later revised in 2021, in which 3^rd larval stage is shifted to laboratory conditions in culture plates and fed artificial diets. Acute exposure was performed on the 4^th day after grafting. Despite standardization, current in vitro rearing methods suffer variable survival rates, and OECD guidelines specify a minimum of 70% survival to adult emergence in the untreated controls for the test to be considered valid. In the present study, a method that mimics realistic exposure scenarios of honey bee larvae is used, in which the larva is left in the hive, marked the area with pins (60 larvae) and treated with different concentrations of test chemicals, therefore, simulating realistic exposure scenarios for honey bee larvae. All other protocols followed are in accordance with OECD (2015) requirements for larval testing.

Keeping in view the harmful effects of pesticides to honey bees and other pollinators, the present study evaluates the effects of fipronil and lambda-cyhalothrin on larvae of Apis mellifera colonies kept in field conditions.

Few studies have evaluated how insecticides mainly belonging to phenyl pyrazole group (Fipronil) and pyrethroids (Lambda-cyhalothrin) affect the developmental phase of honey bee larvae. The results of this study demonstrate that fipronil and lambda-cyhalothrin negatively impact larval development. The results also showed that the impact on larval development is dose-dependent. This finding agrees with the funnel Hypothesis (Warne MS, 1995) which states that toxicity will tend towards concentration. The LD₅₀ obtained was 0.163 μg/larva. The results of the current experiments contradict with those of OECD (2017), which indicated that the LD₅₀ value of fipronil was 0.0218 μg/larva. Genetic variations and the approach used by OECD (2017) may cause the differences between our findings.

Other aspects of brood growth may also vary among colonies (Collins, 2004). Additionally, lineage-specific changes in susceptibility to stressors and brood temperature-related vulnerability to toxicants are taken into account (Medrzycki et al., 2010; Jensenet al., 2009). Furthermore, there have been no investigations of GABA receptors in A. mellifera larvae; most of the studies on GABA have been conducted on Drosophila sp. larvae (Enell et al., 2007). Dzitoyeva et al. (2005) stated that GABA receptors in Drosophila are necessary for optimal larval development, given that fipronil, a phenyl pyrazole pesticide, blocks aminobutyric acid (GABA) receptors and central nervous system excitement ultimately results in the death of the insect (Law, 2008). Similarly, Silva et al. (2015) conducted bioassay investigations of fipronil on honey bee larvae and reported necrosis in the head and thorax when bees were subjected to 5 ng and 20 ng a.i /larvae. Even though few larvae survived toxicity tests in our research, developmental failure from larva to pupa was observed and the presence of empty cells was probably the result of hygienic behaviour of house cleaning bees as they threw the affected abnormal larvae.

These findings are supported by Locke (1998) and Martins & Bitondi (2012) who investigated the effect of fipronil on bee fat tissue during postembryonic development and found that fipronil inhibited the synthesis of hexamerins, a protein involved in the transport of hormones, making it essential for metamorphosis. Comparing our result of LD₅₀ for larvae (0.83 μg/larvae) with those for adults of A. mellifera (0.909 μg/bee - Gough et al., 1984; European Food Safety-Authority, 2013), we noticed that larvae seemed more susceptible to lambda-cyhalothrin than adults. The present investigations are supported by the works of Micheletti and Soares (1993), Cruz-Landim (2009) and Suchail et al. (2001, 2004), who observed that honey bee larvae do not defecate until the pupal stage and then excrete a waste pellet known as meconium (Winston, 1987), and that metabolites may accumulate and thus increase the toxicity of the parent substance in larval stage. Concentrations of pesticides and metabolites inside brood tissues may result in constant pesticide stress (Wu, 2011). Regular avoidance behaviour and excretion of toxic wastes might contribute to decreased toxicity in the field (Desneux, 2007; Atkins, 1986).

It was also found that when larvae survived, they did not develop into pupae. This work was supported by the findings of Tuteja et al. (2022), who reported that lambda-cyhalothrin was highly toxic for 5–6 day old larvae, reduced capping stage to 6.67% and no larva survived until emergence at 12.5 ppm. Dai et al. (2010) similarly found that pyrethroids could reduce capping rate and extend the duration of the immature stage by causing delayed larval development. The observed mortality in the fipronil control group was 1.66%, while the lambda-cyhalothrin control group was 13.33%, both of which were well within the standards of the OECD 239 Guidance Document (2016).

In the present study, we established that exposure of honeybee larvae to fipronil and lambda-cyhalothrin at concentrations comparable to those reported in honeybee products might result in larval mortality and developmental failure. The mortality might have been caused by the solvent acetone and the high temperatures during the experiments. On the basis of LD₅₀ toxicity, fipronil and lambda-cyhalothrin proved toxic for larvae of A. mellifera. Considering the larval toxicity of fipronil and lambda-cyhalothrin in hives, the application during crop bloom is harmful to pollinators and should be limited to minimize its impact on pollinators. Studies on bee colonies should complement our findings in the next stage. The distribution and accumulation of fipronil, lambda-cyhalothrin and their metabolites in honey bees at various developmental stages must be investigated at colony level, as honey bees are eusocial insects. It can be evaluated through semi-field (Tier 2) as described by Schur et al., (2002) and field studies (Tier 3).