Honey as a bioindicator of environmental organochlorine insecticides contamination

Abstract Honey is a suitable matrix for the evaluation of environmental contaminants including organochlorine insecticides. The present study was conducted to evaluate residues of fifteen organochlorine insecticides in honey samples of unifloral and multifloral origins from Dir, Pakistan. Honey samples (5 g each) were extracted with GC grade organic solvents and then subjected to Rotary Evaporator till dryness. The extracts were then mixed with n-Hexane (5 ml) and purified through Column Chromatography. Purified extracts (1μl each) were processed through Gas Chromatograph coupled with Electron Capture Detector (GC-ECD) for identification and quantification of the insecticides. Of the 15 insecticides tested, 46.7% were detected while 53.3% were not detected in the honey samples. Heptachlor was the most prevalent insecticide with a mean level of 0.0018 mg/kg detected in 80% of the samples followed by β-HCH with a mean level of 0.0016 mg/kg detected in 71.4% of the honey samples. Honey samples from Acacia modesta Wall. were 100% positive for Heptachlor with a mean level of 0.0048 mg/kg followed by β-HCH with a mean level of 0.003 mg/kg and frequency of 83.3%. Minimum levels of the tested insecticides were detected in the unifloral honey from Ziziphus jujuba Mill. Methoxychlor, Endosulfan, Endrin and metabolites of DDT were not detected in the studied honey samples. Some of the tested insecticides are banned in Pakistan but are still detected in honey samples indicating their use in the study area. The detected levels of all insecticides were below the Maximum Residue Levels (MRLs) and safe for consumers. However, the levels detected can cause mortality in insect fauna. The use of banned insecticides is one of the main factors responsible for the declining populations of important insect pollinators including honeybees.

products to evaluate the risk posed by these chemicals to honeybees and public health.
The present study was conducted to evaluate the levels of fifteen organochlorine insecticides in honey samples of different floral origins at Dir Upper and Dir Lower, Pakistan. This study is the first of its kind in the study area and the detected levels of organochlorine insecticides in honey samples will act as indicator of the illegal use of these banned pesticides in the ambient environment as well as indicator of the level of risk posed by these contaminants to honeybees and public health.

Sampling
Raw honey samples of different floral origins (n = 35) were collected during 2017 and 2018 at managed and natural apiaries and local markets of District Dir Upper and Dir Lower, Khyber Pakhtunkhwa province, Pakistan. Common honeybee species found in the study area is Apis mellifera L. The honey samples were classified into six categories based on the floral origin (as shown in Table 1). In the present study, fifteen organochlorine insecticides were studied in honey samples which are tabulated (as shown in Table 2).

Preparation of standards and stock solutions
Pesticide standards, organic solvents and chemicals used were of GC grade (Merck, Germany). For stock solutions, 100 μg/mL reference standards were individually prepared in n-hexane in a 100 mL of volumetric flask. For working solutions, 5 mL of stock solution was diluted in 50 mL of n-hexane for the preparation of 10 μg/mL of individual standard solution. Analysis of organochlorine insecticides in honey samples was carried out according to previously described methods by Jimenez et al. (1998), Choudhary and Sharma (2008) and Malhat et al. (2015).

Introduction
Honey is a complex matrix of at least 181 constituents used worldwide for nutritional and therapeutic purposes (Alvarez-Suarez et al., 2010). Honey should be free of chemical contamination for human use but like many other foods, it is prone to environmental contaminants. Determination of insecticide residues in bee products is necessary for ensuring safety to consumers and bee populations (Fernández et al., 2002). Honeybees and their products can be effectively used as bioindicators of environmental pollution from miticides (Fell and Cobb, 2009), trace and heavy metals and metalloids (Zhelyazkova, 2012;Yaqub et al., 2020;Lazarus et al., 2021), polychlorinated biphenyls (Santos et al., 2021), DDT (Cervera-Chiner et al., 2020;Freitas et al., 2021) and various pesticides (Alghamdi et al., 2020;Choi et al., 2020;Bramlitt, 2021;Rodríguez-Martínez et al., 2021). Honeybees fly several kilometers all around from their apiaries and have access to a large area picking pesticides through various pathways like collection of contaminated nectar, pollen and resins (Tosi et al., 2018), through contact with contaminated crops and plants, inhalation from contaminated air, ingestion with polluted water and through direct application of miticides and pesticides (Bogdanov, 2006;Colin et al., 2004).
Pesticides play main role in the decline of honeybee populations at individual as well as colony levels (Potts et al., 2010;Sánchez-Bayo et al., 2016;Sánchez-Bayo and Wyckhuys, 2019). Sublethal doses of pesticides in honeybees affect their behavior and immune system (Desneux et al., 2007), reproduction and learning (Wu et al., 2011;Williamson and Wright, 2013), locomotion , and homing flight .
Use of organochlorine insecticides started in the 1940s and have played a key role in public health and agriculture sector (Ruiz-Toledo et al., 2018). Due to the adverse effects of organochlorine insecticides in humans and other fauna, they were started banning worldwide in the beginning of 1970s including Pakistan, but their residues and impact still prevail due to their persistence and longer half-lives in the environment which range from few days to several years (Faheem et al., 2015;Jayaraj et al., 2016).
Majority of pesticides reported to date from Pakistan in milk, fruits, vegetables, fish meal and cottonseed samples are chlorinated and exceeds the Maximum Residue Level (MRL) (Tariq et al., 2007). Organochlorine insecticides have also been reported in breast milk and human serum samples of cancer patients from Pakistan (Khwaja et al., 2013;Attaullah et al., 2018Attaullah et al., , 2019. The presence of organochlorine insecticides is a matter of concern for public health and for the populations of insect fauna. The ongoing decline of important insect pollinators particularly honeybees is mainly associated with the indiscriminate use of insecticides. In Pakistan, very little attention is given to the role of insecticides and their impact on the insect pollinators, human health and environmental hazards. There is a dire need to evaluate the residues of various contaminants particularly the commonly used organochlorine insecticides in beehive Helium was used as the carrier gas while make-up gas was Nitrogen at 120 kPa. Chromatograms of the samples were compared with the standard chromatograms for identification and quantification of the individual analytes. Recovery and sensitivity experiments were conducted by adding known volumes of the tested pesticides in triplicate at various fortification levels. Blank samples were also processed for finding out the differences, if any. The average recovery percentages were determined for the fortified samples. Limits of detection (LODs) and limits of quantification (LOQs) were determined as the concentration of pesticide producing a peak with signal to noise ratio (S/N) of 3/1 and 10/1 respectively.

Data analysis
Data was calculated by using Microsoft Excel (Version 2016) and presented as percentage, mean, standard deviation, range and confidence intervals of the organochlorine insecticides detected in honey samples of different floral origins.

Extraction
Honey sample (5 g each) was mixed with 10 mL of methanol-distilled water (30:70 v/v) and then homogenized to reduce its viscosity. A mixture of n-hexane and ethyl acetate (10 mL each) at (50:50 v/v) was added and agitated for 20 minutes followed by centrifugation at 3000 rpm for 10 minutes. The supernatant was collected in a separator flask and the residues were re-extracted twice with 10 mL of ethyl acetate. Rotary evaporator was used for the evaporation of solvent till dryness at 65 °C. The residues were dissolved in 5 mL of ethyl acetate.

Clean-up
The 5 mL extract of each sample was purified by passing through a column containing 0.5 g silica, 1 g anhydrous sodium sulphate and 2 g of activated Florisil. The column was pre-washed with n-hexane (10 mL) and then 5mL extract of each sample was passed one by one for purification from impurities. Each time the columns were thoroughly flushed with n-hexane (20 mL) before running the new sample. The eluate of each sample was concentrated to dryness in centrifuge tubes and each was re-dissolved in ethyl acetate (1 mL) and stored at -20 °C.

Gas chromatographic analysis
The extracted eluate (1 μL each) was analyzed through Claurus Gas Chromatograph coupled with 63Ni Electron Capture Detector at Center for Environmental Studies, Pakistan Council for Scientific and Industrial Research Laboratories Complex, Karachi. DB-5 silica capillary column (30 m × 0.25 mm × 0.25 μm) was used with 1μL of sample volume in split-less mode. Temperature was programmed as: Detector (280 °C); Injector port (220 °C) at the rate of 10 °C/min, 150 °C held for 1 min to 210 °C held for 1 min with samples followed by β-HCH (0.003 mg/kg) detected in 83.3% of the samples (as shown in Table 3; see Figure 1 and 2). The least contaminated honey with minimum detected levels of organochlorine insecticides was Ziziphus jujuba honey with a mean level of β-HCH of 0.0003 mg/kg, detected in 66.6% of the samples followed by Heptachlor (0.0003 mg/kg) detected in 50% of the samples (as shown in Table 6; see Figure 1 and 2).
Honey from Acacia nilotica, Brassica campestris, Helianthus annuus and multifloral honey was found with moderate levels of the studied organochlorine insecticides (as shown in Table 4, 5, 7 and 8; see Figure 1 and 2). of the tested organochlorine insecticides including α-HCH, γ-HCH, Endrin, Endosulfan, DDD, DDE, DDT and Methoxychlor were not detected in the honey samples (as shown in Table 2, see Figure 1).
The most prevalent organochlorine insecticide was Heptachlor with a mean detected level of 0.0018 mg/kg, detected in 80% of the samples followed by β-HCH (0.0016 mg/kg), detected in 71.4% of the honey samples (as shown in Table 2).
Heptachlor was detected in 100% of the honey samples from Acacia modesta, Brassica campestris and multifloral honey (as shown in Tables 3, 4 and 5; see Figure 2). Honey from Acacia modesta was found with highest detected mean level of heptachlor (0.0048 mg/kg) detected in 100% of the   that these chemicals are still prevalent in the environment and may pose health hazards to public health and risk to honeybee populations. Concentrations of HCB, Heptachlor and Aldrin detected in the present study were lower than reported in honey from Portugal (Blasco et al., 2004), Turkey (Erdoğrul, 2007 and Ghana (Darko et al., 2017).
Mean level of β-HCH was 0.0016 mg/kg and detected in 71.4% of the honey samples (as shown in Table 2). It is in conformance with a previous report on β-HCH in honey (Wang et al., 2010). Endosulfan was detected in none of the tested honey samples although it has been recently reported in honey samples from other parts of Pakistan with levels exceeding MRL (Farooqi et al., 2017). In Romania, levels of HCHs and DDTs were detected in 50% and 25% of the honey samples respectively (Antonescu and Mateescu, 2001). β-HCH was the second most detected organochlorine insecticide in the present study while residues of DDTs were not detected in any of the honey samples. The honey samples were found positive for at least some of the organochlorine insecticides acting as indicator of the illegal use of these banned insecticides in the ambient environment. The levels of organochlorine insecticides were below MRLs and declared as safe for public health. However, the detected levels are enough to cause harm to honeybees' health. This may be one of the important factors responsible for the declining populations of honeybees and other insect pollinators in the study area. Honey with high contamination of organochlorine insecticides was from Acacia modesta followed by Brassica campestris, while the least organochlorine insecticide contaminated honey was from Ziziphus jujuba. Further studies are recommended to study a wider spectrum of insecticides not only in honey

Discussion
Fifteen organochlorine pesticides were studied in honey samples of different floral origins. The detected levels indicate the use of these pesticides in the ambient environment. If residues of these persistent pesticides are higher than the Maximum Residue Levels, then they may pose a risk to public health. Honeybees and other insect pollinators can be affected even in very low levels. MRL for most of the organochlorine insecticides in honey samples is 0.01 mg/kg while MRL for DDT and its metabolites is 0.05 mg/kg (Tette et al., 2016;European Union, 2006). Levels of the fifteen organochlorine insecticides in all honey samples were below the MRL and may not be harmful to consumers but may severely affect the health and physiology of honeybees. Pesticide residues in honey samples reported from Spain (García-Chao et al., 2010) and Turkey (Yavuz et al., 2010), exceeded the MRL and were higher than those obtained in the present study. Organochlorine insecticide with highest frequency of detection were mostly those also having higher mean levels (as shown in Table 2; see Figure 1). Due to the persistence of organochlorine insecticides and bioaccumulation in tissues, there is a possibility that they can build up to deleterious levels thereby causing toxicity to bees. Fipronil a chlorinated pesticide in sub-lethal doses has been confirmed causing severe damage to honeybee colonies (Aliouane et al., 2009). The sublethal levels of the detected pesticides in the present study may pose risk to honeybees and other insect pollinators.
Some of the recent studies from Pakistan have reported organochlorine insecticides in a variety of samples (Randhawa et al., 2016;Attaullah et al., 2018). This indicate Table 7. Levels of the detected organochlorine insecticides in honey from Acacia nilotica.  samples but also in pollens, propolis, royal jelly and bees wax. Ecofriendly pest control measures and Integrated Pest Management (IPM) techniques should be promoted as a better alternative to chemical control for the conservation of biodiversity including honeybees and for ensuring public health safety.