Neonicotinoid Analysis in Sunflower (Helianthus annuus) Honey Samples Collected around Tekirdag in Turkey

In recent years, the widespread use of neonicotinoids in agricultural areas has caused environmental pollution due to its lower toxicity to mammals. Honey bees, which are considered as biological indicators of environmental pollution, can carry these pollutants to the hives. Forager bees returning from sunflower crops that have been treated with neonicotinoids treated sunflower fields cause residue accumulation in the hives, which reason colony-level adverse effects. This study analyses neonicotinoid residues in sunflower (Helianthus annuus) honey sampled by beekeepers from Tekirdag province. Honey samples have been subjected to liquid-liquid extraction methods before liquid chromatography-mass spectrometry (LC-MS/MS). The method validation was carried out to fulfill all the necessary requirements of procedures SANCO/12571/2013. Accuracy was in the range of 93.63–108.56%, for recovery in the range of 63.04–103.19%, and for precision in the range 6.03–12.77%. Detection and quantification limits were determined according to the maximum residue limits of each analyte. No neonicotinoid residues were found above the maximum residue limit in the sunflower honey samples analysed.


Introduction
Neonicotinoids are insecticides that show nicotine-like stimulating efects by binding to nicotinic acetylcholine receptors in the CNS. Neonicotinoids are more toxic to insects because they bind more tightly and irreversibly to nicotinic acetylcholine receptors in insects than in mammals [1,2].
Neonicotinoid-type plant protection products are widely used in the agricultural feld, especially for preserving seeds due to their less toxic efect on mammals; they also have unintended consequences on nontarget organisms like agrarian pollinators [3]. Pollution is also detected in the felds where neonicotinoid-treated seeds are planted and in the application area's surface or public drinking water sources [4].
Commercial beekeepers move millions of honeybee colonies to sunfower felds to get sunfower honey during the blooming sessions. During these visits, honey bees carry possible pesticide residues in the environment to the hives by means of nectar and pollen. Neonicotinoids can afect the bee's ability to fy for feeding purposes, such as learning and remembering ways to reach food sources. Terefore, it can be considered as part of the important role in colony collapse disorder [5].
Te neonicotinoids have been restricted in the frst Europe due to the adverse environmental efects resulting from a severe decrease in honey bees, insect populations, and bird species and numbers. Te European Union established maximum residue limits (MRLs) for acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam in the range of 10-200 ng·k −1 [6].
Te importance of sunfower honey in the global honey trade is diferent; its favour is not strong dominantly. Terefore, it blends well with other honey types, and it is one of the most suitable and economic honey for commercial blending. Honey, which must comply with EU standard norms, must be reliable regarding food safety and public health. High pesticide concentrations can cause high mortality in bees, loss of colonies, and honey production unsuitable for food safety [7].
For public health, pesticides in honey and other food have become a severe health and safety checkpoint worldwide, and demands for detecting chemicals that may pose an environmental risk have increased in recent years. Successful results have been obtained in analysing multiple residues of antibiotics and pesticides in honey using liquid chromatography-mass spectrometry.
Tis study aims to determine neonicotinoid residues in sunfower honey collected from 33 diferent beekeepers around Tekirdag province. According to the results obtained, a risk assessment will be made in terms of public health. Te study is the frst to investigate neonicotinoid pesticide residues in honey around Tekirdag. It is thought that it will importantly contribute to databases in this regard.

S 4 -Standard
Working Solution (100 ng·mL −1 Each). 0.1 mL of each S 2 -standard working solution was taken and placed in a 10 ml measuring fask. Sufcient methanol was added up to the mark. All standard working solutions were stored at 4-6°C. Tis prepared solution was used to defne reference standards in the MS detector.

Reference Standard Solution Mix for the Spike.
Spiking solutions were prepared at 10 ng·mL −1 for clothianidin, dinotefuran, nitenpyram, and thiamethoxam, 50 ng·mL −1 for acetamiprid and imidacloprid, and 200 ng·mL −1 for thiacloprid, according to the levels of maximum residue limit (MRL) in the honey, respectively (Table 1). One mL of clothianidin, dinotefuran, nitenpyram, and thiamethoxam S 4 working solution, 5 mL of acetamiprid and imidacloprid S 4 working solution, and 0.2 mL of thiacloprid S 2 working solution were put into a 10 mL measuring balloon and flled with methanol up to the mark.

Internal Standard Solution.
Clothianidin-d 3 was used as an internal standard at a 10 ng·mL −1 .

Mobile Phase A.
Acetonitrile was used as a mobile phase A.
2.2.7. Mobile Phase B. 2 mL of acetic acid were placed in a 1 L fask and the reagent diluted with water to the marked line. Te mobile phases were degassed in an ultrasonic bath for 15 min.

Collection of Samples.
During sunfower honey harvest, honey samples were collected from supers at each of the 33 stationary apiaries in July/August of 2015 from 10 diferent district centres of Tekirdag (40°58′41″N, 27°30′42″E). All samples were confrmed to be sunfower honey by pollen analysis. [14] was used for the honey samples. Briefy, two grams of each honey sample were weighed into 15 mL polypropylene centrifuge tubes and an internal standard solution (100 μL) was added to the tubes. Te mixed standard spiking solutions were added (50, 100, 150, and 200 μL) to control the quality of the samples. 0.5 mL of acetonitrile and 2.0 mL of dichloromethane were placed in each tube. Te tubes were mixed by vortex for 1 minute, incubated in an ultrasonic bath for 10 minutes, returned to the vortex for 1 minute, and centrifuged at 2, 500g for 5 minutes, 6 mL of supernatant was then removed using a pipette and transferred into glass tubes. Te organic fraction was evaporated to dryness in a stream of nitrogen at 40°C within a water bath. Two mL of mobile phase was added onto the dry residue and mixed by vortex for two minutes. Te result was fltered into an autosampler vial using a 0.2 µm syringe flter.

Instrumentation.
Analyses were performed on AB Sciex 3200 QTRAP brand/model high-pressure liquid chromatography-mass spectrometry equipment controlled by Analyst 1.6.1 software. An Agilent Poroshell 120 SB: C18 2.7 µm 100 × 3.0 mm column was used for chromatographic separation. Acetonitrile (A) and water acidifed with 0.2% acetic acid were used as the mobile phase. Te linear gradient mobile phase; 0-1 min 80% A,   Table 2. A capillary voltage of 5500 V, nebulizer gas of 7 psi, curtain gas of 30 psi, heater gas of 50 psi, and collision gas of 50 psi were set. Te temperature of the TurboIonSprey module was set at 400°C.
Ionization was performed in positive ion mode using the electrospray ionization (ESI) module.
3.1.1. Specifcity/Selectivity. Blank samples were analysed by loading diferent standard substances; no interference was observed in the retention times. It was concluded that the analysis method was suitable for selectivity/sensitivity. Te chromatogram obtained from loading at the MRL level is shown in Figure 1.

Linearity.
To determine the linearity of the method, six parallel analyses were performed using four diferent concentration points at 0.5, 1, 1.5, and 2 MRL levels in accordance with the MRL level in honey. Calibration curves for each standard substance were created. Te r 2 value in the calibration curve of each standard item was found to be between 0.9908 and 0.9984 (Table 1).

Limit of Detection (LOD) and Limit of Quantifcation (LOQ).
To determine the limit of detection and the limit of quantifcation, 10 parallel analyses were performed at 0.5 MRL. Te results obtained are shown in Table 1.

Decision Limit (CC α ) and Detection Capability (CC β ).
Te decision limit (CC α ) and detection capability (CC β ) were calculated using the results obtained from the study linearity and are shown in Table 1.
3.1.5. Accuracy. Te accuracy was calculated using the study linearity and recovery results shown in Table 3.

Recovery.
To determine recovery, analysis was performed according to the blank fortifed sample at levels 0.5, 1, 1.5, and 2 MRL shown in Table 3.
According to the sunfower honey samples analysis, the MRL value was not detected for any neonicotinoid residue. Te data under the maximum residue levels have not been evaluated. Te results of the analysis of the honey samples are shown in Table 4.  Tables 1 and 3. Te matrix-matched curves showed good linearity (r 2 > 0.99) for all the analytes. Te concentrations of the analytes were obtained directly from the matrix calibration curve with the use of internal standards. Te selectivity of the method was found to be gratifying with no interference peaks from endogenous compounds in the retention time of the target analytes in honey samples. Precision, expressed as the repeatability, gave the RSD values in agreement with the SANCO criteria of RSD ≤20%. Te RSD, were in the range of 6.3-12.77% for honey samples.      Satisfactory average recoveries were calculated used of the internal standards. Te average recovery result ranged 63.04-103.19% for honey samples, and is in accordance with the SANCO validation guideline of recovery, which should be in the range of 60-140%. Te results of analysis of honey samples: no neonicotinoid pesticide residues were detected above the maximum residue limits in honey samples collected from Tekirdag province and its surroundings. Among the possible reasons, a neonicotinoid pesticide type drug is not used in and around Tekirdag. Tis may have been caused by agricultural producers' avoidance of the use of neonicotinoid pesticides, as some countries in Europe have banned or restricted the use of neonicotinoid pesticides.
Previous studies published about the confrmation method and validation of the residues of neonicotinoids in honey are summarised below.
In a study by Kavanagh et al. in Irish honey samples, imidacloprid was found to be the most common neonicotinoid (found in 13.43% of honey samples), followed by clothianidin (12.40%) and thiacloprid (11.37%). Tey concluded that the frequency of imidacloprid in honey samples may not be limited to its use in the agricultural feld but may also occur due to its presence in a range of commercial products used in sports and recreational lawn care products, herb care homes, home gardens, and locally public parks [17].
In Austria, acetonitrile extraction and dispersive solidphase extraction (QuEChERS type) were used in Tanner and Czerwenka's analytical method to detect neonicotinoid residues in honey. Residues of acetamiprid, thiacloprid, and thiamethoxam were detected in Austrian honey samples; however, no sample exceeded the maximum residue limits. Flower honey samples contained more neonicotinoid residues than forest honey samples [18]. It is seen that the level of neonicotinoid pesticide residues in honeys of Austria detected in this study are below the maximum residue limits in line with the results obtained from our study.
Ligor et al. developed a method using QuEChERS extraction and UHPLC/UV to determine neonicotinoid residues in honey samples. Te method was applied to honey collected from Poland and other countries. 53 honey samples were analysed, and neonicotinoids were detected at concentrations higher than the LOQ in 19 honey samples from Australia (3 samples), Brazil (1 sample), Italy (1 sample), and Poland (12 samples). No neonicotinoid residues were detected in the Turkish honey sample [19]. Te absence of neonicotinoid residues in the analysis of honey samples from Turkey seems to be in line with the result of our study.
In the study by Woodcock, we evaluated the efectiveness of this policy in reducing the risk of exposure to honeybees by collecting 130 honey samples from beekeepers in the UK before (2014: N � 21) and after (2015:  International Journal of Analytical Chemistry N � 109) the enactment of the moratorium. Neonicotinoids were present in approximately half of the honey samples taken before the moratorium and in more than one-ffth of the honey samples taken after the moratorium. Clothianidin was the most frequently detected neonicotinoid [20]. A 3-year feld study was conducted in France from 2002 to 2005 to examine pesticide residues found in colonies and honeybee (Apis mellifera L.) colony health by Chauzat et al. No pesticide residues were detected in 12.7% of the sampling periods. It was reported that no statistical relationship was found between colony mortality and pesticide residues. Imidacloprid residues were frequently detected in pollen, honey, and honeybee samples [21].
Mrzlikar et al. developed a reliable analytical method using two extraction techniques (SPE, QuEChERS) and LC-MS/MS (SRM) for fve neonicotinoids in 51 honey samples collected between 2014 and 2016. Despite being banned in the country in 2011, residues of acetamiprid and thiacloprid were detected in low contamination [12].
An average of 8.2 ng·g −1 clothianidin and 17.2 ng·g −1 thiamethoxam were detected in 68% and 75% of honey samples, respectively, from hives located 30 km from Saskatchewan City in Canada. Moreover, clothianidin was found in >50% of bee and pollen samples. Imidacloprid was detected in ∼30% of honey samples [11].
In a study by Han et al., a total of 94 honey samples were selected from the Chinese market, based on the production region and sales volume in 2020. Neonicotinoids and their metabolites were detected in 97.9% of honey samples. Acetamiprid, thiamethoxam, and imidacloprid were the top three neonicotinoids in honey with detection frequencies of 92.6%, 90.4%, and 73.4%, respectively [22].
A study conducted in North America from 2007 to 2008 examined the efects of pesticides on the health of bee colonies. 1% of 208 wax samples, 17.7% of 350 pollen samples, and 0.0% of 140 honey samples were detected as having imidacloprid residues [23].
Residues of neonicotinoids were investigated in honey, pollen, and bee samples sampled in Greece between 2011 and 2013, while any residue did not detect in the honey samples. However, 0.7-14.7 ng·g −1 clothianidin in bee samples in 2011, 6.1-69.04 ng·g −1 in pollen samples, and 2.7-39.9 ng·g −1 was detected in 2012 bee samples and 308.3-1273 ng·g −1 clothianidin in pollen samples [24]. Te absence of neonicotinoid residues in the analysis of honey samples from Turkey seems to be in line with the result of our study.
Residues of neonicotinoid products restricted in the European Union were not found in honey samples from Tekirdag on the European side of Turkey. According to the results of the study, we can say that the use of neonicotinoid products has decreased in our country.

Conclusions
According to the analysis results of 33 sunfower honey samples collected from around and Tekirdag province are free of any neonicotinoid residues exceeding the maximum residue limits were detected. It was understood that in further studies, more honey samples should be analysed as well as other hive products.

Data Availability
Te datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Disclosure
An earlier version of the manuscript has been presented as a preprint in the following link: https://www.researchsquare. com/article/rs-1683983/v1 [25]. Te funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Conflicts of Interest
Te authors declare that they have no conficts of interest.