Chemosphere 239 (2020) 124809

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Carcinogenic and non-carcinogenic risk assessment of trace elements and POPs in honey from Shirak and regions of

Davit Pipoyan a, Stella Stepanyan a, Meline Beglaryan a, Seda Stepanyan a, * Shushanik Asmaryan a, Azatuhi Hovsepyan a, Nicolo Merendino b, a Center for Ecological Noosphere Studies of NAS RA, Abovyan 68, 0025, Yerevan, Armenia b Department of Ecological and Biological Sciences (DEB), Tuscia University, Largo Dell’Universita snc, 01100, Viterbo, Italy highlights

Honey is a highly nutritious natural product widely produced and consumed in Shirak and Syunik regions of Armenia. The quality of honey varies with the environmental conditions, and honey can contribute to human health or threaten it. This is the first attempt to carry out dietary exposure assessment of honey in mining and non-mining regions of Armenia. Carcinogenic risk assessment showed that CR value of Ni and As exceeded the threshold risk level in both regions. article info abstract

Article history: Honey is a highly nutritious natural product widely produced and consumed by people in Shirak and Received 3 May 2019 Syunik regions of Armenia. Unlike Shirak, Syunik is under the impact of mining industry. Since the Received in revised form environmental pollution can adversely impact the safety of honey and entail a probable risk to human 2 September 2019 health, it is important to evaluate the presence of potentially toxic trace elements in honey samples from Accepted 6 September 2019 both regions and draw comparisons. This study assesses the dietary exposure to trace elements and Available online 9 September 2019 persistent organic pollutants through the intake of honey for the first time among people in Shirak and Handling Editor: Jian-Ying Hu Syunik regions. 24-hour dietary recall method was used to investigate the consumption of honey. The presence of seven trace elements (Pb, Cd, As, Hg, Cu, Zn, and Ni) and persistent organic pollutants Keywords: (hexachlorocyclohexane (HCH), dichlorodiphenyltrichloroethane (DDT) and its metabolites) were Honey determined in honey samples using atomic absorption spectrometry and gas chromatography-mass Persistent organic pollutant spectrometry, respectively. In several samples, the concentrations of Cu were above the maximum Risk allowable level. Non-carcinogenic risk values did not exceed the acceptable level, while carcinogenic risk Trace element values for Ni and As exceeded the risk level of 10 6 in both regions. Moreover, among the persistent organic pollutants, only the concentration of DDT in honey from Shirak was above the European Union maximum residue level. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction nutritional value as well as antibacterial, probiotic and other characteristics (Naccari et al., 2014). It includes different sugars, Honey obtained from nectar plants is widely consumed for its organic acids, essential elements and vitamins (Alimentarius, 2001). However, the quality of honey varies substantially with the environmental conditions, and depending on it, honey can

* Corresponding author. Department of Ecological and Biological Sciences (DEB), contribute to human health or threaten it. Industrial pollution and Laboratory of Cellular and Molecular Nutrition, Tuscia University, Largo arsenic-based pesticides are just one of the few sources of dell’Universita snc, 01100, Viterbo, Italy. contamination with pollutants that lead to serious impairment of E-mail addresses: [email protected] (D. Pipoyan), stella.stepanyan@cens. honey quality (Pisani et al., 2008). Contamination with trace ele- am (S. Stepanyan), [email protected] (M. Beglaryan), seda.stepanyan@ ments is a worldwide concern due to their non-biodegradability, cens.am (S. Stepanyan), [email protected] (S. Asmaryan), aza. [email protected] (A. Hovsepyan), [email protected], [email protected] toxicity, persistence, and accumulative behavior (Zhang et al., (N. Merendino). 2018). Even though trace elements are beneficial for human https://doi.org/10.1016/j.chemosphere.2019.124809 0045-6535/© 2019 Elsevier Ltd. All rights reserved. 2 D. Pipoyan et al. / Chemosphere 239 (2020) 124809 health at low concentrations, they can have adverse effects at high subsamples) of multi-floral honey were collected and analyzed. The concentrations (Cicchella et al., 2010). Moreover, trace element decomposition of honey was carried out in the ARM ISO-IEC 17025- intake by humans through the food chain can have toxic, neuro- 2005 accredited laboratory of the Center for Ecological-Noosphere toxic, and carcinogenic effects (EU, 2002). Among them, As, Pb, and Studies of National Academy of Sciences of Armenia. Ni have the most relevance to potential human health issues For determining the contents of non-volatile chemicals, honey (Albanese et al., 2010). In addition to trace elements, pesticides like has been exposed to high thermal degradation according to AOAC DDT and HCH are also associated with cancer risks and are 985.01 method (Fredes and Montenegro, 2006). Whereas, for persistent in the environment. The International Agency for determining the contents of As and Hg, honey was exposed to acid Research on Cancer has classified DDT as Group 2 A probable hu- decay according to AOAC 920.180 method (Fredes and Montenegro, man carcinogen (IARC, 2018) and HCH as Group 2B possible human 2006). carcinogen (IARC, 1987). According to number of studies, ingestion is the major exposure 2.3. Digestion of samples route to trace elements for population, therefore, studying the contents of trace elements in food and analysing the carcinogenic Samples were digested with nitric (HNO3), sulfuric (H2SO4), and and non-carcinogenic effects on human health is of high impor- perchloric (HClO4) acids. The digestion program was carried out tance (Pipoyan et al., 2018a; Ru et al., 2013). accordingly - 500 W/5 min up to 180 C, 0 W/2 min, and then The main aim of this study is to evaluate the concentrations of 500 W/10 min at 180 C. Then, the samples were cooled and seven trace elements (Pb, Cd, As, Hg, Cu, Zn and Ni) and persistent filtered. Eventually, the filtrates were moved to 50 ml Erlenmeyer organic pollutants (DDT, DDE, DDD and HCH) in honey samples flasks. from Shirak and Syunik regions of Armenia and carry out dietary risk assessment of these contaminants. Both regions are actively 2.4. Analysis of trace elements and POPs involved in honey production. Since Syunik is the region where Armenia's biggest mining center is located (Pipoyan et al., 2018b), The levels of seven trace elements (Pb, Cd, As, Hg, Cu, Zn, and Ni) while Shirak is the region farthest from it, it was also of interest to were determined with atomic absorption spectrometer (Perki- compare the results of dietary exposure assessment from two re- nElmer AAnalyst 800 high-performance AAS, US). Solution prepara- gions. Previously, no risk assessment to health through honey tion was made with double distilled deionized water. The glassware consumption has been conducted there. Hence, this is the first ever was cleaned by 10% HNO3. The instrument was calibrated with attempt to carry out dietary exposure assessment of honey in both standard solution of appropriate element. Blank standards from mining and non-mining regions of Armenia. As part of this study, PerkinElmer authorized distributor were used. The solutions were population consumption patterns were investigated, the levels of diluted appropriately for calibration. The variation coefficients of trace elements and persistent organic pollutants (POPs) in honey replicate analysis were determined (Pipoyan et al., 2018a). were determined. Afterwards, non-carcinogenic (target hazard For detecting the contents of organic pollutants in honey, the quotient (THQ), and carcinogenic risks (CR) were estimated. The samples were extracted in accordance with EPA 3546 (US EPA, present study covers only adults (aged from 18 to 70) that consume 2007a, 2007b), using Start E (Milestone) microwave extractor. Af- honey, since no data was available regarding the honey consump- terwards, the decomposition was done using gas chromatograph tion of people under the age of 18. mass spectrometer according to EPA 608, EPA 8081 (US EPA, 1984; 2007a, 2007b). GC confirmatory analysis was carried out on a GC- 2. Materials and methods MS (Trace GC Ultra; Thermo Electron Corporation, USA) supplied with automatic split/splitless injector (COMBI PAL; CTC ANALYTICS 2.1. Study site choice AG) and a mass selective detector (Trace DSQ; Thermo Electron Corporation, USA). The study covers Shirak (40500N43550E) and Syunik (39150N 46150E) regions of Armenia (Fig. 1). Shirak is in the north of the 2.5. Quality assurance and quality control country, while Syunik is the southernmost region and is under the influence of mining industry. Appropriate quality assurance procedures were carried out for Syunik's territory is characterized as a natural copper-enriched the reliability of the results. For error minimization, the determi- biogeochemical province; thus, it is a major contributor in the in- nation of trace element and POP levels was performed with three dustrial sector of the country (Saghatelyan et al., 2012). This region replications. For the accuracy of the results, repeated analysis of stands out for its diversity of natural elements. It is the largest samples against Standard Reference Materials for all trace elements mining center of Armenia and includes and mines, was made (SRM 1570a). The obtained results were within ±2% of operating already over half a century. Mining production consti- the certified values, suggesting that the results were precise. tutes 90.2% of the total industrial production of the region and As part of quality assurance, proficiency testing (PT) was con- substantially impacts the ecological situation in Syunik (Muradyan ducted (PTeFCe773 was used). The proficiency testing of all the and Asmaryan, 2015). As reported previously, highly probable POPs was successful. Certified reference materials for pesticides contamination risks of several fruits and vegetables are common were purchased from LGC accredited by the United Kingdom among Syunik's communities, which are out of state control Accreditation Service (UKAS) for the provision of PT schemes (Pipoyan et al., 2018a). against ISO/IEC 17043 standard. The limit of detection (LOD) for Pb and Hg was 3.0E-04 mg/kg, 2.2. Sample collection and treatment for Cd and As were and 1.67E-04 and 2.00E-04 mg/kg respectively, while for Cu, Zn, and Ni 5.00E-03 mg/kg, with blank values reading Honey sampling was done from March to June 2016 according to as 0 mg/kg for all the trace elements in deionized water. The limit of the international standards and included 5 villages from Syunik and quantification (LOQ) for Pb, As, and Hg was 1.00E-03, for Cd and Zn 5 from Shirak (Alimentarius, 2001). From each village, honeys from were 5.00E-04 and 6.00E-02 mg/kg, while for Cu and Ni 4.00E- three main producers were sampled, to ensure sample represen- 02 mg/kg. tativeness. In total, 30 composite samples (each including 5 For persistent organic pollutants (DDT, DDE, DDD and HCH) LOD D. Pipoyan et al. / Chemosphere 239 (2020) 124809 3

Fig. 1. The map of study area. and LOQ were 6.00E-05 mg/kg and 6.00E-04 mg/kg, respectively. studied regions were 70 and 60 kg), AT is the time over which the dose is averaged. For evaluating the carcinogenic risk, AT of 70 years (25550 days) was considered (US EPA, 1999). The EDI values of each 2.6. Data collection and analysis contaminant were summed to get the value of cumulative daily intake. From July to October 2017, the consumption of honey was investigated through 24-h dietary recall (24HR) method. 24HR is a structured interview during which individual recalls actual food 2.6.2. Non-carcinogenic risk assessment intake for the past 24 h (EFSA, 2009a, 2009b, 2009c). The recalled To assess human health risks posed by chronic exposure, target day was taken as the beginning of the day when the respondent hazard quotient was calculated according to the formula below (US wakes up until waking up on the next day. The main advantage of EPA, 1991b): 24HR is that the person is asked for more detailed information THQ ¼ EDI=RfD (2) when needed. The 24HR recall is the main dietary assessment method used in where RfD is the daily intake reference dose (mg/kg of body weight Europe (EFSA, 2009a, 2009b, 2009c). This method was chosen for per day), an estimate of a daily exposure to the human population the present study since the literacy rate in the studied regions is that is likely to be without an appreciable risk of deleterious effects relatively low, and this method is considered to be especially during a lifetime (US EPA, 1993). The RfD values for Pb and Hg were suitable for people with low literacy rate (EFSA, 2009a, 2009b, taken as 0.0035 and 0.00054 mg/kg/bw/day (EFSA, 2010, 2012), 2009c). The information was collected through personal inter- using provisional tolerable weekly intake (PTWI) for Pb, tolerable view, using pre-designed forms. To get more accurate information weekly intake (TWI) for Hg and dietary reference intake for Cu on the amount of honey consumed, the interviewer used images (ATSDR, 2004). The RfD for Ni, Zn, As, and Cd were 0.02, 0.3, 0.0003 and household measures, such as the spoon with which the and 0.001 mg/kg/bw/day (US EPA, 1989a; 1991a; 1991b, 2005). respondent ate the honey. SPSS software (SPSS Ins., version 11) was Since, for inorganic As in food commodities (other than fish and used for data entry and analysis. The survey results showed that seafood) 70% was considered as best reflecting overall average of among all participants only 201 males and females at the age of total arsenic (EFSA, 2009c), in this study the RfD for inorganic e 18 70 from Shirak and Syunik regions consumed honey. arsenic were used in THQ calculation. The RfD value for DDT is 0.0005 mg/kg/bw/day (US EPA, 1987). 2.6.1. Estimated daily intake (EDI) Estimated daily intake (EDI) was assessed with the following 2.6.3. Carcinogenic risk assessment formula (US EPA, 1997): Carcinogenic risk for trace elements (Pb, Cd, As and Ni) and POPs (DDT, DDD, DDE, HCH) was estimated according to the formula EDI ¼ Cx IRxEFxED=ðBwxATÞ (1) below (US EPA, 2005): where C is the mean concentration of contaminant (mg/kg), IR is CR ¼ SFxEDI (3) the rate of ingestion of food (kg/day), EF is the frequency of expo- sure (365 days/year), ED is the duration of exposure (63.6 and 69.7 where CR is a carcinogenic risk for a lifetime, while SF is a slope years for males and females respectively, taking into account mean factor for estimating the probability of an individual developing life expectancy from 8 years of age). In the denominator, BW is the cancer from exposure to the contaminant for a lifetime. SF for Pb, body weight (kg) (mean body weights for males and females in Cd, As and Ni are 0.0085, 0.38, 1.5 and 1.7 (mg/kg/day) 1. The value 4 D. Pipoyan et al. / Chemosphere 239 (2020) 124809 for Pb is according to US EPA, while for other elements the literature tailing repository discharges into Voghchi river and pollutes the source was used (Real et al., 2017; US EPA, 1991a). The SF value for agroecosystems irrigated by the river's water (Tepanosyan et al., DDT is 0.34 (mg/kg/day) 1 (US EPA, 1987). Carcinogenic risk of less 2018). Yet, the levels of Pb did not exceed the maximum accept- than 1 10 6 was taken as the acceptable level for humans (US able level (1.00E-01 mg/kg) set by Commission Regulation or the EPA, 1999). level for honey imported to EU (8.00E-02 mg/kg) in any of the It should be noted that EDI or THQ calculations were not per- studied sites (APEDA, 2008; EC, 2005). In comparison with the formed in case of the samples that had n/d values (less than LOD). research results reported by Bilandzic et al. (2011) and Ru et al. This is because the number of those samples exceeded 60% and as (2013), Pb concentrations detected in this study were much lower mentioned by the Report of a Joint FAO/WHO Consultation only if than those in Croatia (6.52E-02 mg/kg) and China 8.58E-02 mg/kg). fewer than 60% of results are less than the LOD or LOQ, then a The concentrations of Cd ranged from 6.00E-04 to 3.10E-03 mg/ reasonable estimate of the mean can probably be obtained by kg. The highest levels were detected in honey samples from Ver- setting all n/d and n/q (non-quantified) results to LOD/2 or LOQ/2, ishen village of Syunik region where the mining industry is located. respectively (WHO , 2008). It did not exceed the maximum allowable level for export of honey to the European Union (8.00E-03 mg/kg) or the level set by Eurasian Economic Union (5.00E-02 mg/kg) (APEDA, 2008; EAEU, 2.7. Statistical analysis 2011). In comparison to other studies, Cd contents in Syunik were lower than those in Zhejiang province of China (4.20E-03 mg/kg) Student's t-test and ANOVA analysis were performed. Since and in Siena County of Italy (3.91E-03 mg/kg) (Pisani et al., 2008; Ru there was no statistically significant difference between male and et al., 2013). female consumption, the same value (16.2 g) was used for both The concentrations of As ranged from 5.00E-03 to 4.80E-02 mg/ genders as the average daily consumption rate for honey. Statistical kg. All the values were within limits set by EAEU (5.00E-01 mg/kg) tests were done with Microsoft Excel and SPSS software (SPSS Ins., and were close to the values detected in China (1.30E-02) and version 22.0). Croatia (1.97E-02 mg/kg) (Bilandz˅ic et al., 2011; EAEU, 2011; Ru et al., 2013). The concentrations of Hg (0e1.00E-03 mg/kg) detec- 3. Results and discussions ted in this study were also relatively close to those in Croatia (2.70E-03 mg/kg) and China (1.65E-03 mg/kg) and did not exceed 3.1. Levels of trace elements and POPs in honey the maximum contaminant level for honey exports to EU (1.00E- 02 mg/kg). The concentrations of seven trace elements in honey were The concentrations of Cu ranged from 9.00E-02 to determined and presented in Table 1. The results were compared 1.86Eþ00 mg/kg. There were violations reported in both regions, with the maximum levels of EU, maximum allowable concentra- but the values in Syunik were generally higher relative to those in tions of Customs Union Technical Regulation (EAEC) where Shirak. This can be attributed to the presence of tailing repository in Armenia is a member state, as well as with the study results of Syunik. The Cu content in Norashenik (1.86Eþ00 mg/kg) exceeded different countries. the maximum acceptable level suggested by EU (1.00Eþ00 mg/kg) As shown in Table 1, concentrations of Pb ranged from 2.30E-03 almost twice (APEDA, 2008). Even though Cu is an essential to 4.50E-02 mg/kg. The highest concentration was reported in element for human body, its excessive intake can seriously damage honey samples from Syunik region, which can be explained by the human health (Chi et al., 2007). Hence, the results reported in this presence of mines and tailing repositories nearby. It has been village shall warrant concerns. Another essential element to health demonstrated through systematic multipurpose geochemical is Zn, which also can have adverse effects on human health at studies that due to mining activities, the accessory elements such as exceedingly high concentrations (Amin et al., 2013; EFSA, 2014). In Pb and Zn pollute the soil of the region. The water of Artsvanic

Table 1 The contents of trace elements in honey.

Mean contents of trace elements, mg/kg

Region Village Mean/SD Pb Cd As Hg Cu Zn Ni

Shirak M 2.70E-02 2.00E-03 n/d 1.00E-03 5.95E-01 9.60E-01 8.49E-01 SD 2.00E-03 1.00E-03 e 6.00E-04 1.60E-01 2.80E-01 1.41E-01 M 3.50E-03 2.00E-03 5.00E-03 n/d 5.60E-01 4.60E-01 4.20E-01 SD 1.00E-03 5.77E-05 2.00E-03 e 1.10E-01 2.10E-01 9.00E-02 M 2.85E-02 1.00E-03 n/d n/d 3.00E-01 2.32Eþ00 3.10E-01 SD 3.00E-03 1.00E-03 ee8.00E-02 4.00E-02 1.10E-01 Aygabats M 9.00E-03 2.20E-03 1.90E-02 n/d 1.13Eþ00 1.93Eþ00 3.11E-01 SD 1.00E-03 5.00E-04 5.00E-02 e 2.10E-01 1.10Eþ00 8.00E-02 M 2.00E-02 6.00E-04 2.80E-02 n/d 1.90E-01 5.10E-01 4.10E-01 SD 4.00E-03 1.00E-04 9.00E-03 e 2.00E-02 1.60E-01 1.10E-01 Syunik Norashenik M 3.10E-02 2.00E-03 n/d n/d 1.86Eþ00 5.00E-01 4.10E-01 SD 6.00E-03 1.00E-04 ee3.10E-01 1.40E-01 2.10E-01 M 4.10E-02 2.10E-03 3.10E-02 n/d 6.40E-01 4.20E-01 2.98E-01 SD 8.00E-03 1.00E-03 9.00E-03 e 1.90E-01 1.10E-01 1.30E-01 M 2.30E-03 3.10E-03 4.80E-02 n/d 2.50E-01 1.85Eþ00 2.98E-01 SD 1.00E-03 1.00E-03 1.90E-01 e 1.60E-01 6.40E-01 1.20E-01 M 4.00E-03 7.00E-04 9.00E-03 n/d 1.24Eþ00 6.70E-01 2.40E-01 SD 1.00E-03 1.00E-04 1.20E-02 e 1.60E-01 1.60E-01 1.10E-01 Syunik M 4.50E-02 1.00E-03 2.90E-02 n/d 9.00E-02 5.60E-01 6.10E-01 SD 6.00E-03 1.00E-03 1.90E-01 e 8.00E-02 2.10E-01 1.20E-01

Note: Data are the mean (M) of n ¼ 3 replicates, n/d-not detected. D. Pipoyan et al. / Chemosphere 239 (2020) 124809 5 contrast to Cu, the values of Zn detected in honey samples from DDD, and HCH levels were not detected at all (Table 2). The Syunik and Shirak regions (4.20E-01-2.32Eþ00 mg/kg) were within detected content (9.70E-01 mg/kg) exceeded EU maximum residue maximum permissible limit established by FAO/WHO limit of 5.00E-02 mg/kg (EC, 2005) around 20 times and was higher (1.00Eþ01 mg/kg) (FAO/WHO, 2003). The highest contents of Zn than the level reported in Europe (4.40E-01 mg/kg) (Ravoet et al., were detected in Artik village, equal to 2.32Eþ00 mg/kg, thus 2015). people with Zn deficiency might consider consuming honey from In total, among all the trace elements only the concentrations of there. Cu exceeded the maximum acceptable levels. Meanwhile, among The concentrations of Ni ranged from 2.40E-01 to 8.49E-01 mg/ the POPs, only DDT levels exceeded the maximum residue limit. kg. There are no established maximum levels for Ni in food (EFSA, DDT levels were observed in Shirak region only, while higher trace 2015). However, the comparison with literature shows that the element levels were reported in Syunik region. values obtained in this study are relatively high since Ni concen- trations in honey were 1.30E-04 - 8.80E-04 mg/kg in Turkey 3.2. Estimated daily intake (EDI) of trace elements and DDT (Altundag et al., 2015), and 1.80E-03 - 1.10E-01 mg/kg in Iran (Sobhanardakani and Kianpour, 2016). While the source of In order to carry out health risk assessment, the concentration contamination in Syunik can be attributed to mining industry, the data was combined with ingestion rate data. The estimated daily case in Shirak is more interesting. Contamination source there intake of trace elements and DDT through honey was calculated might be the pollution of river due to the presence of iron (Table 3) and compared with the health-based guidance values and molybdenum ore deposits nearby. Although they have never (HBGVs) established by international organizations. been exploited, the drainage waters from those deposits were EDI values of all the trace elements and DDT were within their found to contain high levels of Ni, As, Cr, etc. (RM LLC/EPA NGO, reference values. EDIs of Hg were the lowest and were mostly equal 2014). to 0 mg/kg/bw/day both in Syunik and Shirak regions. EDI values of Among the POPs, DDT levels were detected in honey samples Pb, Cd, As, and Cu were generally higher in Syunik than in Shirak. from only one site (Aygabats village of Shirak region), while DDE, This can be explained by the presence of mining industrial center in

Table 2 The contents of POPs in honey.

Mean contents of POPs, mg/kg

Region Honey origin (town/village) DDT DDD DDE HCH

Shirak Ashotsk n/d n/d n/d n/d Sarapat n/d n/d n/d n/d Artik n/d n/d n/d n/d Aygabats 1.156 ± 0.405 n/d n/d n/d Jrapi n/d n/d n/d n/d Syunik Norashenik n/d n/d n/d n/d Sisian n/d n/d n/d n/d Verishen n/d n/d n/d n/d Shaghat n/d n/d n/d n/d Syunik n/d n/d n/d n/d

Note: Data are the mean (M) of n ¼ 3 replicates, n/d-not detected.

Table 3 Estimated Daily Intake (EDI) of trace elements and DDT.

Region Honey origin (town/village) Male/Female EDI of trace elements, mg/kg/bw/day

Pb Cd As Hg Cu Zn Ni

Shirak Ashotsk M 6.25E-06 4.63E-07 e 2.31E-07 1.38E-04 2.22E-04 1.96E-04 F 7.29E-06 5.40E-07 e 2.70E-07 1.61E-04 2.59E-04 2.29E-04 Sarapat M 8.10E-07 4.63E-07 1.16E-06 e 1.30E-04 1.06E-04 9.72E-05 F 9.45E-07 5.40E-07 1.35E-06 e 1.51E-04 1.24E-04 1.13E-04 Artik M 6.60E-06 2.31E-07 ee6.94E-05 5.37E-04 7.17E-05 F 7.70E-06 2.70E-07 ee8.10E-05 6.26E-04 8.37E-05 Aygabats M 2.08E-06 5.09E-07 4.40E-06 e 2.62E-04 4.47E-04 7.20E-05 F 2.43E-06 5.94E-07 5.13E-06 e 3.05E-04 5.21E-04 8.40E-05 Jrapi M 4.63E-06 1.39E-07 6.48E-06 e 4.40E-05 1.18E-04 9.49E-05 F 5.40E-06 1.62E-07 7.56E-06 e 5.13E-05 1.38E-04 1.11E-04 Syunik Norashenik M 7.17E-06 4.63E-07 ee4.30E-04 1.16E-04 9.49E-05 F 8.37E-06 5.40E-07 ee5.02E-04 1.35E-04 1.11E-04 Sisian M 9.49E-06 4.86E-07 7.17E-06 e 1.48E-04 9.72E-05 6.90E-05 F 1.11E-05 5.67E-07 8.37E-06 e 1.73E-04 1.13E-04 8.05E-05 Verishen M 5.32E-07 7.17E-07 1.11E-05 e 5.79E-05 4.27E-04 6.90E-05 F 6.21E-07 8.37E-07 1.30E-05 e 6.75E-05 4.98E-04 8.05E-05 Shaghat M 9.26E-07 1.62E-07 2.08E-06 e 2.87E-04 1.55E-04 5.55E-05 F 1.08E-06 1.89E-07 2.43E-06 e 3.35E-04 1.81E-04 6.48E-05 Syunik M 1.04E-05 2.31E-07 6.71E-06 e 2.08E-05 1.30E-04 1.41E-04 F 1.22E-05 2.70E-07 7.83E-06 e 2.43E-05 1.51E-04 1.65E-04 EDI of DDT, mg/kg/bw/day Shirak Aygabats M 2.24E-04 F 2.62E-04 6 D. Pipoyan et al. / Chemosphere 239 (2020) 124809

Table 4 Target Hazard Quotient (THQ) of trace elements and DDT.

Region Honey origin (town/village) Male/Female THQ of trace elements

Pb Cd As Hg Cu Zn Ni

Shirak Ashotsk M 1.79E-03 4.63E-04 e 4.06E-04 1.38E-02 7.41E-04 9.82E-03 F 2.08E-03 5.40E-04 e 4.74E-04 1.61E-02 8.64E-04 1.15E-02 Sarapat M 2.31E-04 4.63E-04 3.86E-03 e 1.30E-02 3.55E-04 4.86E-03 F 2.70E-04 5.40E-04 4.50E-03 e 1.51E-02 4.14E-04 5.67E-03 Artik M 1.88E-03 2.31E-04 ee6.94E-03 1.79E-03 3.59E-03 F 2.20E-03 2.70E-04 ee8.10E-03 2.09E-03 4.19E-03 Aygabats M 5.95E-04 5.09E-04 1.47E-02 e 2.62E-02 1.49E-03 3.60E-03 F 6.94E-04 5.94E-04 1.71E-02 e 3.05E-02 1.74E-03 4.20E-03 Jrapi M 1.32E-03 1.39E-04 2.16E-02 e 4.40E-03 3.93E-04 4.74E-03 F 1.54E-03 1.62E-04 2.52E-02 e 5.13E-03 4.59E-04 5.54E-03 Syunik Norashenik M 2.05E-03 4.63E-04 ee4.30E-02 3.86E-04 4.74E-03 F 2.39E-03 5.40E-04 ee5.02E-02 4.50E-04 5.54E-03 Sisian M 2.71E-03 4.86E-04 2.39E-02 e 1.48E-02 3.24E-04 3.45E-03 F 3.16E-03 5.67E-04 2.79E-02 e 1.73E-02 3.78E-04 4.02E-03 Verishen M 1.52E-04 7.17E-04 3.70E-02 e 5.79E-03 1.42E-03 3.45E-03 F 1.77E-04 8.37E-04 4.32E-02 e 6.75E-03 1.66E-03 4.02E-03 Shaghat M 2.64E-04 1.62E-04 6.94E-03 e 2.87E-02 5.17E-04 2.78E-03 F 3.09E-04 1.89E-04 8.10E-03 e 3.35E-02 6.03E-04 3.24E-03 Syunik M 2.98E-03 2.31E-04 2.24E-02 e 2.08E-03 4.32E-04 7.06E-03 F 3.47E-03 2.70E-04 2.61E-02 e 2.43E-03 5.04E-04 8.24E-03 THQ of DDT Shirak Aygabats M 4.49E-01 F 5.24E-01

Syunik (Tepanosyan et al., 2018). Both for males and females, the (THQ) was estimated, indicating human health risks posed by highest EDI values of Pb, Cd, As and Cu were reported in Syunik, chronic exposure (Table 4, Fig. 2) (see Fig. 3). Verishen and Norashenik villages. The highest EDI values of Zn and THQ values for Pb were lower than one in all of the studied sites, Ni were detected in Artik and Ashotsk villages, respectively. As with the highest value reported among females in Syunik region noted, none of the values exceeded the reference values provided (3.47E-03). The THQ values for Cd, As, and Cu were also low, with by EFSA, US EPA and ASTDR (ATSDR, 2004; EFSA, 2009a, 2009b, the maximum value detected among females in Syunik region 2009c; 2010; 2012, 2014; 2015; US EPA, 1991a). Moreover, the EDI (8.37E-04, 4.32E-02, and 5.02E-02 respectively). The THQ values for values of DDT did not exceed the US EPA tolerable daily intake (TDI) Hg, Zn, and Ni were far below one in all of the studied sites, indi- of 5.00E-04 mg/kg/bw/day. cating no risks of chronic-toxic effects through honey consumption. THQ value of DDT equaled to 4.49E-01 and 5.24E-01 among 3.3. Non-carcinogenic risk assessment males and females of Aygabats (Shirak), respectively. Since DDT was not detected in any of the honey samples from Syunik region, there For non-carcinogenic risk assessment target hazard quotient were no adverse health effect risks induced by DDT in Syunik.

Fig. 2. THQ for trace elements in honey samples from Shirak region. D. Pipoyan et al. / Chemosphere 239 (2020) 124809 7

Fig. 3. THQ for trace elements in honey samples from Syunik region.

Overall, non-carcinogenic risk of trace elements in honey was 3.4. Carcinogenic risk assessment higher among people in Syunik rather than Shirak region, which is explained by the presence of the mining industry in Syunik. In Depending on the exposure dose, several trace elements can general, the risk values among females were higher than among have carcinogenic effects. There are three cancer risk levels set by males in both regions. In comparison to a similar study conducted US EPA and these are expressed as one chance in a million, one in Italy, the THQ values of Pb and Cd in the present study were chance in ten thousand, and one chance in thousand lifetime considerably higher than the values in Sicily (5.1E-06 and 3.57E-07, exposure. Overall, CR < 10 6 is the safe limit, CR of 10 6-10 4 is the respectively), thus, indicating higher non-carcinogenic risks for acceptable cancer risk range depending on the exposure circum- Armenians vis-a-vis Italians (Naccari et al., 2014). Moreover, the stances, while CR > 10 4 is the threshold limit and is considered as values presented were much higher than those reported in unacceptable (US EPA, 1989b). Moreover, CR > 10 3 is the moderate neighboring country, Iran, where honey consumption was found to risk level which raises serious health safety concerns. The CRs of Pb, have no potential significant health risks (Sobhanardakani and Cd, As, and Ni were estimated and presented in Table 5(see Figs. 4 Kianpour, 2016). and 5) The CR values of Pb ranged from 0 to 8.04E-08 for males and from 5.26E-09 to 1.03E-07 for females, thus, the values were within

Table 5 Carcinogenic risk of trace elements and DDT.

Region Honey origin (town/village) CR of trace elements

Males Females

Pb Cd As Ni Pb Cd As Ni

Shirak Ashotsk 4.83E-08 1.60E-07 e 3.03E-04 6.17E-08 2.04E-07 e 3.88E-04 Sarapat 6.26E-09 1.60E-07 1.58E-06 1.50E-04 8.00E-09 2.04E-07 2.02E-06 1.92E-04 Artik 5.09E-08 7.99E-08 e 1.11E-04 6.51E-08 1.02E-07 e 1.42E-04 Aygabats 1.61E-08 1.76E-07 5.99E-06 1.11E-04 2.06E-08 2.25E-07 7.66E-06 1.42E-04 Jrapi 3.57E-08 4.79E-08 8.83E-06 2.33E-04 4.57E-08 6.13E-08 1.13E-05 2.98E-04 Syunik Norashenik 5.54E-08 1.60E-07 e 1.47E-04 7.08E-08 2.04E-07 e 1.87E-04 Sisian 0.00Eþ00 1.68E-07 9.78E-06 1.07E-04 9.37E-08 2.15E-07 1.25E-05 1.36E-04 Verishen 4.11E-09 2.48E-07 1.51E-05 1.07E-04 5.26E-09 3.17E-07 1.94E-05 1.36E-04 Shaghat 7.15E-09 5.59E-08 2.84E-06 8.58E-05 9.14E-09 7.15E-08 3.63E-06 1.10E-04 Syunik 8.04E-08 7.99E-08 9.15E-06 2.18E-04 1.03E-07 1.02E-07 1.17E-05 2.79E-04 THQ of DDT Males Females Shirak Aygabats 6.93E-05 8.87E-05

Note: CR > 10 4 values are bolded. 8 D. Pipoyan et al. / Chemosphere 239 (2020) 124809

Fig. 4. CR for trace elements in honey samples from Shirak region.

the safe limit in all the studied sites. The CR of Cd also did not CR values of Ni exceeded not only the safe limit of 10 6, but also exceed the safe limit (4.79E-08 - 2.48E-07 for males and 6.13E -08 - the threshold limit of 10 4 (except for males from Shaghat village). 3.17E-07 for females). However, in case of As exposure through The highest CR value of Ni was obtained for females of Ashotsk honey consumption, the CR values exceeded the safe limit of 10 6, village (3.88E-04) and exceeded the threshold limit of 10 4 by indicating possible carcinogenic risk for people. This finding in- almost four times. dicates that even without considering other plausible rich sources The CR values of DDT for males (6.93E-05) and females (8.87E- of As (drinking water, rice and other grains, etc.), only with honey 05) of Aygabats village (Shirak region) exceeded the risk limit of consumption there is already possible carcinogenic risk. 10 6.

Fig. 5. CR for trace elements in honey samples from Syunik region. D. Pipoyan et al. / Chemosphere 239 (2020) 124809 9

Overall, among the studied trace elements only Ni was found to (accessed 04.12.18). ATSDR (Agency for Toxic Substances and Diseases Registry), 2004. Toxicological have possible carcinogenic effects as its CR values exceeded the fi 4 Pro le for Copper. Update. Agency for Toxic Substances and Diseases Registry, threshold cancer risk level (10 ) in all the studied sites (except for Centers for Disease Control, Atlanta, GA. Available at: https://www.atsdr.cdc. males in Shaghat). gov/toxprofiles/tp.asp?id¼206&tid¼37 (accessed 04.12.18). ˅    ˅ Ni and Ni compounds have been classified by IARC (2012) as Bilandz icc, N., Ðokicc, M., Sedak, M., Kolanovicc, B.S., Varenina, I., Konc urat, A., Rudan, N., 2011. Determination of trace elements in Croatian floral honey human carcinogens causing cancers of the lung, nasal cavity and originating from different regions. Food Chem. 128, 1160e1164. paranasal sinuses after inhalation. 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Commission Regulation (EC) No 396/2005 of the European Parliament and of the Council of 23 February 2005 on Maximum Residue Levels of Pesti- associated with local honey consumption. It is interesting that cides in or on Food and Feed of Plant and Animal Origin and Amending Council previous investigations carried out in Syunik region showed that Ni Directive 91/414/EEC. Available at: https://eur-lex.europa.eu/legal-content/EN/ ¼ & ¼ is present in agricultural farmlands and irrigation waters coming TXT/PDF/?uri CELEX:32005R0396 from EN (accessed 04.12.18). EFSA, 2009a. General principles for the collection of national food consumption from River Voghchi (Saghatelyan et al., 2013). Moreover, a research data in the view of a pan-European dietary survey. EFSA Journal 7 (12), 1435. conducted in Syunik indicated carcinogenic risks related to Ni EFSA Panel on Contaminants in the Food Chain (CONTAM), 2009b. Scientific opinion through food consumption (Pipoyan et al., 2018b). So, there might on cadmium in food. EFSA Journal 980, 1e139. Available at: http://www.efsa. europa.eu/sites/default/files/scientific_output/files/main_documents/980.pdf. be a link between the study results on Ni contents in soils and the EFSA, Panel on contaminants in the food chain (CONTAM), 2009c. Scientific opinion study results of honey contaminated with Ni. on arsenic in food. EFSA J 7 (10), 1351. EFSA Panel on Contaminants in the Food Chain (CONTAM), 2010. Scientific opinion on lead in food. EFSA Journal 8 (4), 1570. Available at: http://www.efsa.europa. 4. Conclusion eu/en/efsajournal/pub/1570 (accessed 04.12.18). EFSA Panel on Contaminants in the Food Chain (CONTAM), 2012. Scientific opinion The results of the study highlight the importance of investi- on the risk for public health related to the presence of mercury and methyl mercury in food. EFSA Journal 10 (12), 2985. 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