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 (C12H4Cl2F6N4OS) 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 3rd larval stage is shifted to laboratory conditions in culture plates and fed artificial diets. Acute exposure was performed on the 4th 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
Colonies of
Technical-grade chemicals were used in all experiments (>95% purity) sourced from sigma-Aldrich, and acetone was purchased from Merck Life Sciences. Treatments were administered using a 0 to 100 μl Eppendorf micropipette and a single 2 μl injection of the test solution at the bottom of each chosen comb cell. Larvae were given only acetone in the control treatment.
Experiments were conducted in 2020 and 2021. In order to determine the LD50 range, a preliminary trial was conducted with concentrations of the test chemicals with a mortality rate of 20–80% (Tab. 1). As both test chemicals had low water solubility, organic solvent e.g. acetone @1% was used to prepare test solutions instead of water according to the guidelines of OECD Guidance document, 214 (OECD, 2016). In the control treatment, larvae received a solvent (acetone) equal to that administered in insecticidal treatment.
Insecticides and doses evaluated
Serial No. | Insecticide | Doses (Adult) Oral toxicity | Source |
---|---|---|---|
1 | Fipronil (96.7% Purity) |
0.005 μg, 0.01 μg 0.18 μg, 0.22 μg 0.30 μg, 0.38 μg |
Sigma-Aldrich |
2 | Lambda-cyhalothrin (96.7% Purity) |
0.28 μg, 0.55 μg 0.85 μg, 1.10 μg 1.50 μg, 1.70 μg |
Sigma-Aldrich |
Three colonies were used to test the different concentrations of each insecticides (n=60 larvae). Combs with treated and untreated larvae were tested for survival up to capping. The presence of standard (i.e., pearly white) larvae was taken as evidence of indication of survival. The larval mortality was calculated in % by comparing the number of bees that died during larval stage (day 3 to day 8) to the number of larvae on Day 3 when dosing started. The larval mortality was inferred from empty cells, larva that was immobile or did not react to the contact of the paintbrush or larvae with slight colour changes (i.e., brownish) was noted as dead (Gashout H.A, 2009)
Six doses of each insecticide that resulted in larval mortality in the range of 20 to 80% or close to this range were selected for calculating LD50 values. The mortality due to each insecticide was corrected using Abbott's correction (Abbott, 1925) as follows:
Corrected mortality data and its fiducial limits were calculated by probit regression analysis (Finney, 1971) to calculate LD50 values using OPSTAT software (Sheron et al., 1998).
The average observed mortality was 1.66% in control in fipronil, whereas in lambda-cyhalothrin it was 13.33%, which is within the acceptable range observed for control mortality as reported by OECD (2016) using the in-vitro larval rearing method where cumulative larval mortality from day 3 to day 8 should be less than 15% across all replicates.
The data on the larval mortality of
Comparison between the larval observed mortality (OM %) and corrected mortality (CM %) for each tested concentration (Conc μg/larva) of fipronil using a probit analysis
0.005 | 0.699 | 60 | 12 | 20.00 | 18.64 | −0.891 | −0.958 |
0.01 | 1.000 | 60 | 22 | 36.66 | 35.59 | −0.369 | −0.395 |
0.18 | 1.255 | 60 | 29 | 48.33 | 47.45 | −0.064 | 0.082 |
0.22 | 1.342 | 60 | 36 | 60.00 | 59.32 | 0.236 | 0.245 |
0.30 | 1.477 | 60 | 40 | 66.66 | 66.09 | 0.415 | 0.497 |
0.38 | 1.580 | 60 | 49 | 81.66 | 81.35 | 0.891 | 0.689 |
0.00 | 0.00 | 60 | 1 | 1.66 | - | - | - |
Regression equation Y = 1.8692× + 2.7337
Heterogeneity test
χ2 cal = 0.673
χ2 tab = 12.592
LD50(μg) = 0.163
Fiducial limit (μg) = 0.113 and 0.234
The data on larval mortality of
Comparison between the larval observed mortality (OM %) and corrected mortality (CM %) for each tested concentration (Conc μg/larva) of lambda-cyhalothrin using a probit analysis
0.28 | 1.447 | 60 | 14 | 23.33 | 11.53 | −1.198 | −1.521 |
0.55 | 1.740 | 60 | 18 | 30.00 | 19.23 | −0.869 | −0.581 |
0.85 | 1.929 | 60 | 35 | 58.33 | 51.92 | 0.048 | 0.024 |
1.10 | 2.041 | 60 | 40 | 66.66 | 61.53 | 0.293 | 0.383 |
1.50 | 2.176 | 60 | 46 | 76.66 | 73.07 | 0.615 | 0.815 |
1.70 | 2.230 | 60 | 57 | 95.00 | 94.23 | 1.574 | 0.989 |
0.00 | 0.00 | 60 | 8 | 13.33 | - | - | - |
Regression equation Y = 3.2087× - 1.1693
Heterogeneity test
χ2 cal = 0.022
χ2 tab = 9.488
LD50(μg) = 0.83
Fiducial limit (μg) = 0.667 and 1.047
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
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
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