domain: sde chim

text:
Zoyne Pedrero Zayas

Pedrero Zayas
email: zoyne.pedrerozayas@univ-pau.fr

CHAPTER 8 Mercury

Keywords: certified reference materials, diet, fish, food, GC, HPLC, ICP-MS, isotopic dilution analysis, isotopic fractionation, MC-ICP-MS, methylmercury, mercury, mercury species, non-chromatographic methods, rice, speciation

Mercury (Hg) pollution is an acknowledged major environmental problem. Considering its extreme toxicity, Hg has recently been included in the top ten list of chemicals of major public health concern according to the World Health Organization. Once released into the environment, it is transformed in aquatic ecosystems by microorganisms into the neurotoxic methylmercury. The hazardous effect is then biomagnified through the trophic/food chain. Diet is considered the main exposure pathway of Hg in humans. Therefore, safety values have been established by food safety authorities in order to protect consumers. Seafood, followed by rice, is the primary source of Hg in the human diet. A variety of analytical methodologies is available for the analysis of Hg and its species in food. This chapter presents recent advances in the determination of Hg in foodstuffs. Special attention is given to innovative Hg (species) extraction and preconcentration systems assisted by nanoparticles. Non-chromatographic approaches, as an alternative to classical chromatographic approaches used for speciation are detailed. The potential and limitations of Hg isotopic analysis in food is also discussed.

INTRODUCTION

Mercury (Hg) pollution is considered a major environmental and public health concern. Because of its toxicity, Hg has been recently included in the top ten hazardous chemicals by the World Health Organization (WHO). Pregnant women and children in early life are considered the most vulnerable population to Hg harmfulness. Toxic effects can be lethal and include affections of the nervous, digestive and immune systems, and on lungs and kidneys.

Taking into account the capability of Hg to bind thiols [START_REF] Harris | The chemical form of mercury in fish[END_REF], its interaction with essential proteins and enzymes leading to their dysfunction seems to be the origin of such toxicity.

Despite occupational exposure (i.e., miners, dentists) -dental amalgams being undisputable sources of Hg -diet appears as the main exposure pathway of Hg in humans. In general, seafood consumption is recognized as the most common pathway of Hg human exposure. It is especially troubling considering the recent and important increase of Hg in oceanic waters [START_REF] Lamborg | A global ocean inventory of anthropogenic mercury based on water column measurements[END_REF]. Anthropogenic activities, such as mining and coal burning are responsible for the increased Hg levels in the atmosphere and in oceanic surfaces [START_REF] Lavoie | Mercury transport and human exposure from global marine fisheries[END_REF]. Microorganisms in aquatic ecosystems play a crucial role since they biotransform inorganic Hg (iHg) into methylmercury (MeHg which is present in its free form as CH3Hg + ). The latter exhibits high levels of toxicity and it is easily bioaccumulated through the food chain resulting in serious social and health effects.

Considering Hg toxicity, food safety authorities set the maximal acceptable levels for Hg in food. The established Hg values in foodstuffs depend on their nature. For food supplements, the maximum level is as high as 0.10 mg kg -1 in the final product. In the case of fishery products comprising crustaceans and muscle meat of fish (except predatory ones), it is fixed at 0.5 mg kg -1 wet weight and for predatory fish species as bonito, eel, marlin, sharks and tuna, among others, it is 1 mg kg -1 wet weight (COMMISSION REGULATION (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs). These regulations are effective for fresh and processed fish.

A tolerable weekly intake (TWI) has been set as a safe consumption threshold in order to avoid Hg exposure risks and is regularly reevaluated. The European Food Safety Authority set in 2012 a new MeHg TWI at 1.3 µg kg -1 bodyweight, lower than that established by the Joint Food and Agriculture Organization (FAO)/WHO Expert Committee on Food Additives of 1.6 µg kg -1 bodyweight. However, due to seafood consumption, a significant part of the global population, mainly from developing countries, is exposed to higher Hg levels than the thresholds established by food safety authorities [START_REF] Lavoie | Mercury transport and human exposure from global marine fisheries[END_REF].

Taking into account that diet is the principal source of exposure to Hg, its toxicity strongly related to its speciation, and Hg species are toxic at low concentrations, the analytical chemistry community is continuously seeking for advances in foodstuffs analysis methods. Currently, the main goals of the new and trendy analytical approaches are the development of sensitive, cost-effective and green methods for the determination of Hg and its species. Isotopic fractionation analysis also appears as a fresh strategy for the identification and discrimination of Hg sources in food products, adding another dimension to total Hg quantification and speciation. In this chapter, current and promising approaches for Hg analysis in food are described.

FOOD MATRICES WHERE Hg IS OFTEN DETERMINED

According to a recent report of FAO (2016), fisheries and aquaculture are very important sources of food, nutrition, income and livelihood for hundreds of millions of people around the world. Driven by rising domestic income, consumers in emerging economies (where consumption was previously based on locally available products) are experiencing a diversification of the types of available fish through an increase in fishery imports. The significant growth in fish consumption has enhanced people's diets around the world through diversified and nutritious food. Fish consumption represents in many countries the dominant source of proteins. Therefore, the high seafood consumption could lead to significant risks due to MeHg ingestion.

Since seafood is considered a major contributor of Hg through diet, the quantification of Hg and its species in such products grabbed the attention of the analytical chemistry community. As a consequence, most of the Hg speciation studies in foodstuffs correspond to the analysis of fish and other seafood. The new methodologies developed for Hg speciation in fish and seafood are presented all along the text.

Rice is a dominant global crop, recognized to be one of the most important sources of Hg in human diet. Microbial Hg methylation is considered the main source of this organomercurial species in paddy soils. The traditional rice culture practice involves several flooding processes, which lead to anaerobic conditions facilitating iHg methylation by sulfate reducing bacteria [START_REF] Meng | Distribution patterns of inorganic mercury and methylmercury in tissues of rice (Oryza sativa L.) plants and possible bioaccumulation pathways[END_REF]. In addition, the use of iodomethane as fumigant, enhances Hg methylation in soil under sunlight, increasing MeHg exposure from rice [START_REF] Yin | Fumigant methyl iodide can methylate inorganic mercury species in natural waters[END_REF]. After soil uptake by the plant, Hg is transported to the edible part [START_REF] Feng | Mercury pollution in Guizhou, Southwestern China -An overview[END_REF][START_REF] Horvat | Total mercury, methylmercury and selenium in mercury polluted areas in the province Guizhou, China[END_REF]. It constitutes a potential risk in Hg polluted areas like Hg-contaminated mining regions, where Hg values reach up to 500 ng g - 1 [START_REF] Horvat | Total mercury, methylmercury and selenium in mercury polluted areas in the province Guizhou, China[END_REF]. In such regions, the most important MeHg exposure source is not fish, but rice consumption [START_REF] Feng | Human exposure to methylmercury through rice intake in mercury mining areas, Guizhou province, China[END_REF]. MeHg intake through rice ingestion has been reflected on the levels of MeHg in hair of inhabitants of such areas [START_REF] Horvat | Total mercury, methylmercury and selenium in mercury polluted areas in the province Guizhou, China[END_REF][START_REF] Li | Mercury Isotope Signatures of Methylmercury in Rice Samples from the Wanshan Mercury Mining Area, China: Environmental Implications[END_REF].

Rice seeds consist of a hull and a nutritious bran coat layer surrounding the endosperm and inner embryo. Brown rice is the result of removing the hull (inedible) and can be consumed in this state. Further processing yields to "white" or "polished" rice. The distribution of Hg species varies according to the fraction of the rain grains. Mostly, iHg is located in hull and bran, while MeHg is found in edible white rice. Rice processing leads to a release of up to 78% of iHg, which is contrasting with MeHg that remains almost untouched [START_REF] Meng | Localization and Speciation of Mercury in Brown Rice with Implications for Pan-Asian Public Health[END_REF]. The analysis of commercial rice and baby-food rice from European markets (2 samples of each type) reveals a higher proportion of MeHg in babyfood. It is attributed to the processing practice that includes rice washing and cooking. It is suggested that iHg could be lost during these steps, leading to higher MeHg percentage [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF].

Hg speciation in rice is a challenge principally taking into consideration the difficult extraction from the starch matrix of Hg species without provoking their degradation/interconversion and the lack of rice CRMs (Certified Reference Materials) for MeHg. In absence of certified MeHg values in rice matrices, other biological CRMs are used for method validation despite the remarkable matrix mismatching [START_REF] Brombach | Methylmercury varies more than one order of magnitude in commercial European rice[END_REF][START_REF] Jiménez-Moreno | Fast method for the simultaneous determination of monomethylmercury and inorganic mercury in rice and aquatic plants[END_REF]. Species specific isotope dilution is definitely an elegant solution for the validation of analytical methods in rice. The addition of the isotopically enriched species allows tracking potential species degradations/transformations and losses during the analytical procedure [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF][START_REF] Feng | Investigation of Hg uptake and transport between paddy soil and rice seeds combining Hg isotopic composition and speciation[END_REF].

Alkaline and acid digestion including several subsequent extractions and backextractions steps are commonly used [START_REF] Jiménez-Moreno | Fast method for the simultaneous determination of monomethylmercury and inorganic mercury in rice and aquatic plants[END_REF]. Two fast and sensitive methodologies based on both, acid and basic microwave (MW) extraction of Hg species followed by their derivatization (ethylation) and analysis by gas chromatography (GC) coupled to Atomic Fluorescence Spectrometry (AFS) via pyrolysis, were developed. Basic digestion uses 25% tetramethylammonium hydroxide while 6N HNO3 is chosen for acid treatment. Compared to traditional methods for Hg speciation in this matrix, it implies a noticeable time reduction (it is less time consuming) (from sample preparation to analysis). The sum of Hg(II) (being present in its free form as Hg 2+ ) and MeHg in the CRMs was similar for acid and basic digestions. However, it should be taken into account that adequate blanks are only obtained for acid MW digestion, being more suitable for samples with low Hg contents. Limits of detection (LODs) were in the range of ng g -1 (0.1-0.7 ng g -1 ) [START_REF] Jiménez-Moreno | Fast method for the simultaneous determination of monomethylmercury and inorganic mercury in rice and aquatic plants[END_REF].

Considering the economic rises on developing countries, there is an urgent need for developing inexpensive and simple but accurate and sensitive Hg speciation analysis methods [START_REF] Lin | Ultrasensitive Speciation Analysis of Mercury in Rice by Headspace Solid Phase Microextraction Using Porous Carbons and Gas Chromatography-Dielectric Barrier Discharge Optical Emission Spectrometry[END_REF]. Inspired by this urgency, a fresh ultrasensitive Hg speciation method by headspace solid-phase microextraction (HS-SPME) using porous carbons (PCs) and GC-Dielectric Barrier Discharge (DBD) Optical Emission Spectrometry (OES) was developed.

The applicability of the method was demonstrated by the analysis of 12 rice samples from both, unpolluted and abandoned Hg mining districts in China. The protocol for Hg species extraction from rice comprised the addition of 10.0 mL of 25% (w/v) potassium hydroxide-methanol to 1 g of grounded samples and the mixture was maintained in a water bath at 65 °C during 4 h. The resulting digest was diluted to 25 mL with methanol. Hg(II), ethylmercury (EtHg, CH3CH2Hg + ) and MeHg were derivatized with NaBPH4 to their volatile species and preconcentrated by HS-SPME assisted by PCs. In-house prepared PCs SPME presented superior qualities than commercial SPME considering the simultaneous and outstanding extraction efficiency for the three Hg species that was maintained even after 200 extraction cycles. The comparison of Hg species quantification by the new GC-DBD-OES method and conventional high-performance liquid chromatography (HPLC)inductively coupled plasma mass spectrometry (ICP-MS) showed evidence of a complete matching of the results. The sum of the Hg(II) and MeHg coincided with the total Hg concentration determined by ICP-MS after MW assisted acid digestion. LODs were 0.5, 0.75, and 1.0 μg kg -1 for Hg(II), MeHg, and EtHg, respectively, with a recovery between 90 and 105% [START_REF] Lin | Ultrasensitive Speciation Analysis of Mercury in Rice by Headspace Solid Phase Microextraction Using Porous Carbons and Gas Chromatography-Dielectric Barrier Discharge Optical Emission Spectrometry[END_REF]. DBD-OES benefits from low power consumption, simple set up, high electron temperature and weak background radiation. Considering the small size and compactness of DBD-OES, it can be directly connected to the GC capillary column by a T tube conveniently introducing Ar discharge gas. Direct coupling without using transport tubes prevents loss of Hg and memory effect. This represents a pioneer application of GC-DBD-OES on elemental speciation, used before for volatile carbon containing compounds and halohydrocarbons [START_REF] Lin | Ultrasensitive Speciation Analysis of Mercury in Rice by Headspace Solid Phase Microextraction Using Porous Carbons and Gas Chromatography-Dielectric Barrier Discharge Optical Emission Spectrometry[END_REF]. Despite the PCs HS-SPME-GC-DBD-OES method was developed and applied on rice samples, its accuracy validation using fish protein (DORM-4) and lobster hepatopancreas (TORT-3) CRMs reveals the potential of this approach on the analyses of complex matrices as seafood and other foodstuffs. The potential coupling of DBD-OES with miniaturized GC should be promising in the field of Hg speciation due to its compactness, and low power and gas consumption [START_REF] Lin | Ultrasensitive Speciation Analysis of Mercury in Rice by Headspace Solid Phase Microextraction Using Porous Carbons and Gas Chromatography-Dielectric Barrier Discharge Optical Emission Spectrometry[END_REF].

Rice is one of the scarce food matrices where the analysis of Hg is not restricted to the quantification of its most common species, Hg(II) and MeHg. In view of the public health concern associated to the consumption of this staple product, several studies focus on the understanding of Hg accumulation pathways by different approaches as molecular speciation [START_REF] Meng | Localization and Speciation of Mercury in Brown Rice with Implications for Pan-Asian Public Health[END_REF][START_REF] Feng | Investigation of Hg uptake and transport between paddy soil and rice seeds combining Hg isotopic composition and speciation[END_REF], imaging [START_REF] Meng | Localization and Speciation of Mercury in Brown Rice with Implications for Pan-Asian Public Health[END_REF], and isotopic analysis [START_REF] Li | Mercury Isotope Signatures of Methylmercury in Rice Samples from the Wanshan Mercury Mining Area, China: Environmental Implications[END_REF][START_REF] Feng | Investigation of Hg uptake and transport between paddy soil and rice seeds combining Hg isotopic composition and speciation[END_REF].

Hg imaging analyses in food are not common and one of the rare examples corresponds to Hg characterization in rice. Synchrotron radiation microscopic Xray fluorescence (SR-μXRF) provides the Hg map in rice grains, as well as for other elements as Ca, Cd, Se, Cu, Fe, Mn, Zn and K. The principal Hg location is the surface of brown rice grains, corresponding to the pericarp and aleurone layer. The elements K, Ca, Fe, Mn and Cd are co-localized at the surface of the rice grain. It is attributed to the absorption of atmospheric iHg [START_REF] Meng | Localization and Speciation of Mercury in Brown Rice with Implications for Pan-Asian Public Health[END_REF]. Speciation by X-ray absorption near-edge spectroscopy (XANES) is limited to bran samples due to their higher Hg content. Results indicate that Hg(II) and MeHg are both binding cysteine (Cys). Although the structure of the Cys-rich bioligands binding the species are not identified, it has been suggested that Hg(II) could be associated to phytochelatins and MeHg to proteins. These differences on speciation could justify the higher mobility of MeHg-Cys, actively transported to the endosperm during seed ripening [START_REF] Meng | Localization and Speciation of Mercury in Brown Rice with Implications for Pan-Asian Public Health[END_REF].

Breast milk is a food matrix particularly interesting that allows the Hg exposure estimation of lactating women and newborns, who constitutes an extremely sensitive population. MeHg is able to cross the mammary gland, being present in the colostrum and breast milk [START_REF] Sundberg | Placental and lactational transfer of mercury from rats exposed to methylmercury in their diet: Speciation of mercury in the offspring[END_REF]. Hg content in breast milk is strongly linked to maternal exposure. Mothers' diet and their dental amalgams are the main sources of Hg in breast milk [START_REF] Palkovicova | Maternal amalgam dental fillings as the source of mercury exposure in developing fetus and newborn[END_REF][START_REF] Richardson | Mercury exposure and risks from dental amalgam in the US population, post-2000[END_REF][START_REF] Vieira | Total and methyl-mercury in hair and milk of mothers living in the city of Porto Velho and in villages along the Rio Madeira, Amazon, Brazil[END_REF]. The percentage of MeHg varies in a wide range according to the diet, where highest values correspond to fish-consuming populations [START_REF] Vieira | Total and methyl-mercury in hair and milk of mothers living in the city of Porto Velho and in villages along the Rio Madeira, Amazon, Brazil[END_REF][START_REF] Rebelo | Mercury in breast milk from women in the Federal District, Brazil and dietary risk assessment for breastfed infants[END_REF].

Most of the analytical methods for Hg quantification in breast milk involved acid digestions and relatively long sample treatments [START_REF] Iwai-Shimada | Methylmercury in the breast milk of Japanese mothers and lactational exposure of their infants[END_REF][START_REF] Miklavčič | Biomarkers of low-level mercury exposure through fish consumption in pregnant and lactating Slovenian women[END_REF]. The characterization of Hg content and speciation in large sets of samples (i.e., breast milk banks, epidemiological studies) claims for sensitive, cost-effective and validated analytical approaches. Hg quantification by advanced mercury analyzer demonstrated to be a good alternative to expensive ICP-MS analysis, with a simple sample treatment based on freeze-drying [START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF].

GC coupled to different detection systems like cold vapor (CV)-AFS [START_REF] Vieira | Total and methyl-mercury in hair and milk of mothers living in the city of Porto Velho and in villages along the Rio Madeira, Amazon, Brazil[END_REF][START_REF] Rebelo | Mercury in breast milk from women in the Federal District, Brazil and dietary risk assessment for breastfed infants[END_REF][START_REF] Miklavčič | Biomarkers of low-level mercury exposure through fish consumption in pregnant and lactating Slovenian women[END_REF] and electron capture detection [START_REF] Iwai-Shimada | Methylmercury in the breast milk of Japanese mothers and lactational exposure of their infants[END_REF] has been proposed for the separation of Hg species in breast milk after their derivatization into volatile forms. ICP-MS has recently been hyphenated to chromatographic techniques for Hg speciation in breast milk in order to exploit the advantages of isotopic dilution (ID) analysis, which enhance accuracy by the correction of potential losses and/or species transformation during the analytical procedure [START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF].

MeHg speciation methods in breastmilk based on both, GC-isotopic dilution (ID)-ICP-MS and HPLC-ID-ICP-MS, exhibit similar quantification limits (~1 µg kg -1 ) and accuracy (94-97%). Although GC is more efficient considering the time required for species separation, this method involves a tedious derivatization step. In contrast, sample treatment for HPLC is simpler, limited to a preconcentration by freeze-drying and dissolution of the dried breastmilk on a minimum volume of water [START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF]. Taking into account the similarity on the analytical characteristics, both approaches can be used for the analysis of this complex matrix. HPLC-ID-ICP-MS was chosen in a recent French epidemiological study of 180 breast milk samples. Hg and MeHg values reach up to 16.9 μg Hg kg -1 and 0.43 μg Hg kg -1 wet weight, respectively [START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF].

In addition to quantification of Hg and its species, a metalloproteomic study has also been carried on breast milk. Quantification of Hg in the breast milk proteome, obtained using two-dimensional electrophoresis after protein precipitation with acetone, indicates that Hg is principally associated to proteins with molecular masses in the range from 14 to 26 kDa [START_REF] Cerbino | Metalloproteomics Approach to Analyze Mercury in Breast Milk and Hair Samples of Lactating Women in Communities of the Amazon Basin, Brazil[END_REF].

Nuts in general are an important source of essential minerals, proteins and unsaturated fat. They are consumed worldwide and often recommended to be included in diet in order to reduce risks of coronary diseases. They are comprised in the list of foodstuffs analyzed in national dietary studies since they became habitually consumed [START_REF] Millour | Sb and Al levels in foodstuffs from the 2nd French total diet study[END_REF]. Hg content in such products can be higher when grown in contaminated regions. The content of Hg is attributed to the soil-plant interaction since nuts are isolated by the shell, therefore excluding the potential Hg atmospheric contribution. The concentration in nut peel is up to 10 times higher than in the enclosed nuts, endorsed to the shield role of peel, preventing the transfer of toxic elements as Hg and Cd to nuts [START_REF] Rodushkin | Levels of inorganic constituents in raw nuts and seeds on the Swedish market[END_REF]. Acid digestion by using HNO3 or HNO3/HF mixture of nuts samples in a MW system is largely used for the quantification of Hg, as part of a multi-elemental analysis, by ICP-MS [START_REF] Millour | Sb and Al levels in foodstuffs from the 2nd French total diet study[END_REF][START_REF] Rodushkin | Levels of inorganic constituents in raw nuts and seeds on the Swedish market[END_REF]. The fat fraction in edible nuts varies between 25 to 60% and does not contain Hg [START_REF] Silva | Determination of total mercury in nuts at ultratrace level[END_REF]. Defatting strategies are often included in the sample treatment protocol. For ICP-MS analysis, a defatting step consisting of vigorously shaking 20 g of grounded sample with 100 mL of a (2:1, v/v) chloroform-methanol solution, repeated three times, is included before acid digestion on a MW system [START_REF] Kannamkumarath | Studies of various elements of nutritional and toxicological interest with different molecular weight fractions in Brazil nuts[END_REF].

Direct mercury analyzer (DMA) appears as an alternative for Hg quantification by a cost-effective method. Despite one of the main advantages of this technique is the non-requirement of sample pretreatment, in the case of nuts, the presence of fat causes interferences during the pyrolysis processes due to the production of high volumes of fumes, conducing to erroneous Hg measurements. Defatting is guaranteed by vigorous shaking during 10 min of 3 g of samples with 15 mL of (2:1, v/v) chloroform-methanol solution. The LOD for the analysis of 100 mg sample by DMA is 0.08 µg kg -1 (LOQ: 0.3 µg kg -1 and RSD 2-14%) [START_REF] Silva | Determination of total mercury in nuts at ultratrace level[END_REF].

Hg concentration in diverse types of nuts from different geographical origin is commonly lower than 5 µg kg -1 [START_REF] Millour | Sb and Al levels in foodstuffs from the 2nd French total diet study[END_REF][START_REF] Rodushkin | Levels of inorganic constituents in raw nuts and seeds on the Swedish market[END_REF][START_REF] Silva | Determination of total mercury in nuts at ultratrace level[END_REF]. In Brazil nuts, Hg reaches up to 8000 µg kg -1 . It is principally associated (72%) to the NaOH extractable fraction, mainly binding biomolecules between 0.36 and 14.4 kDa [START_REF] Kannamkumarath | Studies of various elements of nutritional and toxicological interest with different molecular weight fractions in Brazil nuts[END_REF].

Honey's benefits as antioxidant and anti-inflammatory raise the interest in its consumption. Honey is worldwide largely consumed due to its exceptional nutritional value. The increase on popularity of such natural product is also accompanied by authenticity and geographical origin concerns. The safety quality of honey depends, among others, on the level of some toxic elements as Hg. Honey, considered a well-known environmental indicator, is able to accumulate metals and metalloids. Hg is often included in a wide list comprising other mineral and trace elements analyzed in honey in order to determine the quality and safety of honey, but also for the assessment of biological and geographical origin by chemometric methods. It is therefore usually analyzed by multi-elemental analytical techniques like ICP-MS after nitric acid MW digestion [START_REF] Millour | Sb and Al levels in foodstuffs from the 2nd French total diet study[END_REF][START_REF] Pohl | Recent achievements in element analysis of bee honeys by atomic and mass spectrometry methods[END_REF][START_REF] Quinto | Characterization, chemometric evaluation, and human health-related aspects of essential and toxic elements in Italian honey samples by inductively coupled plasma mass spectrometry[END_REF][START_REF] Stocker | Trace and mineral elements in royal jelly and homeostatic effects[END_REF]. The DMA alternative avoids any sample treatment and a complete analysis of Hg in five min [START_REF] Vieira | Development and comparison of two analytical methods to quantify the mercury content in honey[END_REF]. According to the Hg values reported in honey, an ordinary consumption of this product does not seem to represent a risk for human health.

ADVANCES ON THE DETERMINATION OF TOTAL Hg IN FOOD

Hg usually belongs to a long list of toxic elements to be analyzed in food products in order to evaluate the dietary exposure of a population to food contaminants. ICP-MS is therefore the most popular technique chosen by food control laboratories/institutions due to its multi-elemental character and its sensitivity. Hg is commonly quantified, after MW assisted digestion, in a wide type of matrices including cereals, eggs (and eggs products), baby food, drinks, snacks, ice creams, spices, meat, sweeteners, honey, and confectionery [START_REF] Millour | Sb and Al levels in foodstuffs from the 2nd French total diet study[END_REF][START_REF] Pohl | Recent achievements in element analysis of bee honeys by atomic and mass spectrometry methods[END_REF][START_REF] Pandelova | Zn contents in baby foods from the EU market: Comparison of assessed infant intakes with the present safety limits for minerals and trace elements[END_REF]. In spite of the undeniable capacity of this technique for Hg determination in food, the analytical community actively works on the development of innovative approaches dealing with specific situations like the treatment of great numbers of samples, and environmentally friendly and miniaturized solutions, among others. Representative advances on the analysis of Hg in food are presented hereafter.

Advanced or direct mercury analyzers (AMA or DMA) are techniques for Hg quantification in food that can be used for solid and liquid samples. One of the main advantages of direct mercury analyzer compared with the primary techniques used for Hg analysis in food -atomic absorption spectrometry (AAS), AFS, inductively coupled plasma atomic emission spectrometry (ICP-AES) and mass spectrometry (ICP-MS)-is the possibility of analyzing Hg at ultra-trace levels without any sample pre-treatment. Short analysis time, easy use, and cost effectiveness associated are some of the main advantages of the method. DMA is based on the thermal decomposition of the sample followed by Hg amalgamation by gold system subsequent atomic absorption detection. It has successfully been applied to diverse food matrices like honey, nuts, infant food, seafood and animal edible tissues as equine muscle, bovine kidney and swine kidney, and poultry muscle [START_REF] Ribeiro | Development and validation of a method for the determination of Hg in animal tissues (equine muscle, bovine kidney and swine kidney, and poultry muscle) by direct mercury analysis (DMA)[END_REF].

DMA is the technique chosen by European countries authorities to carry out national diet studies on infant and toddlers, resulting on the analysis of more than 370 infant food samples [START_REF] Guérin | Mercury in foods from the first French total diet study on infants and toddlers[END_REF][START_REF] Martins | Total mercury in infant food, occurrence and exposure assessment in Portugal[END_REF]. Accurate dietary exposure assessment claims for the detection and quantification limits as low as possible. The most recent DMA method, developed and validated for (infant) food analysis, exhibits detection and quantification limits of 0.3 µg kg -1 fresh weight and 0.6 µg kg -1 , respectively [START_REF] Guérin | Mercury in foods from the first French total diet study on infants and toddlers[END_REF].

Although DMA is principally associated to solid samples, it has been validated for the quantification of Hg in breast milk. The comparison of ICP-MS and DMA in this food matrix shows that quantification limits for both analytical approaches are in the range between 2 and 5 µg Hg kg -1 , tending to be lower by DMA. Considering the similar sensitivity, Hg quantification by direct analysis represents a better option due to the superior sample throughput. ICP-MS analysis implies a previous digestion of the sample, in this case based on mineralization with aqua regia on a heating block during 3 h. In contrast, DMA does not require any sample treatment. Breast milk samples are exclusively freeze-dried before analysis and Hg is quantified by the standard addition method [START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF].

Unquestionably, DMA has an enormous potential for Hg analysis in food. Nevertheless, the main limitation of this technique is its exclusive success on total Hg concentration, not extended to speciation. A few recent examples suggest the possibility of using thermal release for speciation in solid samples [START_REF] Rezende | Quantification and speciation of mercury in streams and rivers sediment samples from Paracatu, MG, Brazil, using a direct mercury analyzer®[END_REF], not yet widely extended. The recent combination of a thiourea derivative polymer with DMA leads to the screening of Hg(II) and MeHg in food, specifically in fish [START_REF] Zarco-Fernández | Development of a thiourea derivative polymer combined to a Direct Mercury Analyser for screening and pre-concentration of mercury species[END_REF].

Electro thermal vaporization (ETV) appears as a direct sampling technique with a versatile formation and high sample introduction efficiency. It has been coupled to diverse detection systems as AAS, AFS, ICP-OES/MS. The gas phase enrichment method guarantees an optimum interference separation by trapping the analyte. A gold amalgamator is frequently used for selective Hg trapping, separating the element from other pyrolysis gases and concentrating it before thermal induced release into the detector. In a similar way, a tungsten coil is used for the separation of Cd from the matrix interferences [START_REF] Wang | Direct determination of trace mercury and cadmium in food by sequential electrothermal vaporization atomic fluorescence spectrometry using tungsten and gold coil traps[END_REF].

Gold and tungsten coil traps were integrated for the first time into one ETV-AAS system for the quantification of two toxic elements usually simultaneously tracked in foodstuffs. Considering the noticeable differences on vaporization temperature between Hg and Cd, a sequential vaporization of these elements in food matrices was obtained by using an improved on-line ashing furnace. The whole analytical procedure is carried out within 10 min, including sample pre-treatment, with a LOD of 0.7 pg for Hg (and 0.5 pg for Cd). Mushrooms, green tea, corn, milk and spinach are some of the food samples already analyzed using this method. The coupling to miniaturized detection AAS or AFS systems will promote the application of this easy, green and digestion free method for in situ monitoring of Hg (and Cd) in food [START_REF] Wang | Direct determination of trace mercury and cadmium in food by sequential electrothermal vaporization atomic fluorescence spectrometry using tungsten and gold coil traps[END_REF].

Electrochemistry represents a fast, simple, and sensitive alternative, suitable for Hg routine analysis. In addition to the low cost, compared with other analytical techniques (ICP-MS, CVAAS, CVAFS), it is portable and appropriate for in situ determination. In general terms, Hg is preconcentrated on the electrode followed by stripping, mainly by anodic stripping voltammetry (ASV). Electrochemical techniques for Hg detection are a hot topic. They are characterized by a high preconcentration capacity, achieving low LODs. Most of the recent advances on this subject correspond to the development of electrode surfaces allowing the preconcentration of lowest amounts of Hg. DNA-based assays and nanostructured electrodes are the most used methodologies in the last years [START_REF] Martín-Yerga | Recent advances in the electrochemical detection of mercury[END_REF].

DNA-based electrodes exploit the strong bound between Hg(II) and thymine (T) DNA bases, allowing the preconcentration of Hg. DNA-based electrodes exhibit extremely low LODs, but they are less stable and more time-consuming than nanostructured electrodes. In general, the longer analysis time and the stability difficulties of DNA-based assays hamper their application in complex samples [START_REF] Martín-Yerga | Recent advances in the electrochemical detection of mercury[END_REF].

Electrodes modified with lead nanoparticles with thiol-functionalized polysiloxilane [START_REF] Tyszczuk-Rotko | Thiol-functionalized polysiloxanes modified by lead nanoparticles: Synthesis, characterization and application for determination of trace concentrations of mercury(II)[END_REF], nickel nanoparticles with carbon composites [START_REF] Veerakumar | Nickel Nanoparticle-Decorated Porous Carbons for Highly Active Catalytic Reduction of Organic Dyes and Sensitive Detection of Hg(II) Ions[END_REF], among a long list, have been used for Hg determination. The high affinity of gold towards Hg justifies the larger application of gold nanoparticles (AuNPs) on electrode surface for Hg determination [START_REF] Martín-Yerga | Recent advances in the electrochemical detection of mercury[END_REF]. In general, nanostructured electrode surfaces with high electroactive areas and excellent surface properties guarantee a sensitive detection in a short time. Nanoporous materials appear as a prominent approach, characterized by shorter analysis times and relatively simpler procedures. Screenprinted electrodes are expected to be used for the development of future commercial Hg sensors, principally due to their low-cost, disposable and miniaturized properties [START_REF] Martín-Yerga | Recent advances in the electrochemical detection of mercury[END_REF].

Most of the applications of electrochemical detection correspond to Hg analysis in water so far [START_REF] Martín-Yerga | Recent advances in the electrochemical detection of mercury[END_REF][START_REF] Suherman | Recent developments in inorganic Hg 2+ detection by voltammetry[END_REF]. An ultrasensitive and reusable electrochemical biosensor for the determination of mercury ions (Hg(II)) in water in the range of 10 ng L -1 to 1 µg L -1 has been developed based on thymine modified gold nanoparticles/reduced graphene oxide nanocomposites [START_REF] Wang | Functionalized gold nanoparticles/reduced graphene oxide nanocomposites for ultrasensitive electrochemical sensing of mercury ions based on thymine-mercurythymine structure[END_REF]. The development of new assays and the analytical improvement of electrochemical methods should open new perspectives for the analysis of more matrices, like biological and food samples. Some examples are already present in the literature, describing successful Hg characterization in urine and blood [START_REF] Martín-Yerga | Recent advances in the electrochemical detection of mercury[END_REF][START_REF] Suherman | Recent developments in inorganic Hg 2+ detection by voltammetry[END_REF]. Indium-tin oxide surfaces have been used to develop nanoporous gold electrodes for the detection of Hg. They have been primarily used in water but also in milk analysis achieving a detection limit of 0.03 µg L -1 [START_REF] Lin | Electrochemical detection of Hg(II) ions based on nanoporous gold nanoparticles modified indium tin oxide electrode[END_REF].

The application of electrochemical Hg detection in food is incipient. A variety of food samples comprising cockles [START_REF] Isa | Amperometric determination of nanomolar mercury(II) by layered double nanocomposite of zinc/aluminium hydroxide-3(4methoxyphenyl)propionate modified single-walled carbon nanotube paste electrode[END_REF][START_REF] Chaiyo | Highly sensitive determination of mercury using copper enhancer by diamond electrode coupled with sequential injection-anodic stripping voltammetry[END_REF], seaweed [START_REF] Chaiyo | Highly sensitive determination of mercury using copper enhancer by diamond electrode coupled with sequential injection-anodic stripping voltammetry[END_REF], milk [START_REF] Lin | Electrochemical detection of Hg(II) ions based on nanoporous gold nanoparticles modified indium tin oxide electrode[END_REF], breast milk [START_REF] Farahi | Effect of KNO3 to remove silver interferences in the determination of mercury(II): Application in milk and breast milk samples[END_REF] and vegetable origin samples like red chili and tomato [START_REF] Isa | Amperometric determination of nanomolar mercury(II) by layered double nanocomposite of zinc/aluminium hydroxide-3(4methoxyphenyl)propionate modified single-walled carbon nanotube paste electrode[END_REF] has been successfully analyzed. However, the existing studies on foodstuffs focus mainly in fish samples [START_REF] Isa | Amperometric determination of nanomolar mercury(II) by layered double nanocomposite of zinc/aluminium hydroxide-3(4methoxyphenyl)propionate modified single-walled carbon nanotube paste electrode[END_REF][START_REF] Afkhami | Construction of a modified carbon paste electrode for the highly selective simultaneous electrochemical determination of trace amounts of mercury(II) and cadmium(II)[END_REF][START_REF] Giacomino | Anodic stripping voltammetry with gold electrodes as an alternative method for the routine determination of mercury in fish. Comparison with spectroscopic approaches[END_REF][START_REF] Guerreiro | Characterization of an electrochemical mercury sensor using alternating current, cyclic, square wave and differential pulse voltammetry[END_REF]. Under optimal conditions, anodic stripping voltammetry gold nanoparticle-modified glassy carbon electrode achieves a detection limit of 0.001 µg L -1 with a short deposition time of 2 min in fish. The analytical performance of the electrochemical approach is comparable to conventional DMA analysis [START_REF] Giacomino | Anodic stripping voltammetry with gold electrodes as an alternative method for the routine determination of mercury in fish. Comparison with spectroscopic approaches[END_REF] Compared to DMA, the main drawback of electrochemical techniques is the need of longer analysis time and a wet sample digestion which implies the generation of waste solution with possible loss of analyte by vaporization [START_REF] Giacomino | Anodic stripping voltammetry with gold electrodes as an alternative method for the routine determination of mercury in fish. Comparison with spectroscopic approaches[END_REF].

Sample Treatment for Total Hg Determination Assisted by Solid-Phase Extraction

Effective isolation of the analyte and preconcentration are common sample treatment requirements for trace element analysis. In general terms, solid-phase extraction (SPE) is the technique that conquered the preconcentration of Hg from real samples. Low reagent consumption, simplicity and minimum cost are their main advantages. Assisted by the tremendous progress in nanomaterials, there are noticeable advances on SPE for Hg determination in food.

Dispersed solid-phase extraction represents an alternative to classical SPE. It consists in the direct addition of the solid phase to the agitated sample solution during an optimized time. Therefore, no special equipment is required for sample treatment. The enriched solid phase is mechanically recovered (i.e., centrifugation, filtration) and the analyte is therefore eluted with an adequate solvent for further analysis. This SPE alternative is robust, fast, low cost and simply operated. Matrix solid-phase dispersion (MSPD) is particularly interesting in sample preparation of solid, semisolid and highly viscous samples. The sample is blended with a solid support in a mortar with a pestle. The solid support plays an abrasive role leading to the disruption of sample architecture. Sample components are consequently dispersed on the surface of the support particles. The solid homogeneous mixture is commonly packaged into a cartridge for analyte elution with a suitable solvent [START_REF] Duarte | Assessment of modified matrix solid-phase dispersion as sample preparation for the determination of CH3Hg + and Hg 2+ in fish[END_REF].

The association of matrix solid phase dispersion assisted by multiwall carbon nanotubes (MWCNTs-MSPD) with single drop solute on electrode glow discharge induced (SD-SEGD) chemical vapor generation constitutes an important advancement on Hg quantification in micro amount samples. The MWCNTs-MSPD consists in blending 1 mg of the solid sample with 0.5 mg of MWCNTs for 5 min using an agate pestle. The homogeneous solid mixture is placed in the MSPD column and Hg species retained are extracted by 100 μL of eluent containing 0.5% L-Cys and 4% HCOOH. Hg species are efficiently converted to Hg 0 by SD-SEGD-CVG and further transported to AFS for detection. The accuracy of the method was validated by the analysis of several biological CRMs and it was applied to fish samples [START_REF] Chen | Single-Drop Solution Electrode Discharge-Induced Cold Vapor Generation Coupling to Matrix Solid-Phase Dispersion: A Robust Approach for Sensitive Quantification of Total Mercury Distribution in Fish[END_REF]. LOD improved 100 fold with this robust SD-SEGD-CVG-AFS method, in comparison to conventional CVG-AFS where the micro amount of sample is significantly diluted by the carrier solution. In addition, memory effect, typically associated to CVG or pneumatic nebulization ICP-MS is not observed. The absence of memory effect is attributed to several factors as: the low sample volume (20µL), the inhibition of adsorption in connecting tubes by the presence of L-Cys and the minimized transport tube length [START_REF] Chen | Single-Drop Solution Electrode Discharge-Induced Cold Vapor Generation Coupling to Matrix Solid-Phase Dispersion: A Robust Approach for Sensitive Quantification of Total Mercury Distribution in Fish[END_REF]. The combination of MWCNTs-MSPD and SD-SEGD-CVG-AFS is an absolutely promising approach for Hg quantification in foodstuffs. It could be particularly interesting for Hg analysis in rare and/or luxurious food products such as caviar, saffron and truffles, where minimal amounts of samples are available for analysis.

Magnetic SPE (MSPE) is a new kind of solid-phase extraction based on the combination of a non-magnetic adsorbent with a magnetic material [START_REF] Yavuz | Magnetic dispersive solid phase extraction with graphene/ZnFe2O4 nanocomposite adsorbent for the sensitive determination of mercury in water and fish samples by cold vapor atomic absorption Microchem[END_REF]. Magnetic nanoparticles (MNPs) have a high surface area and sorption capacity. Iron, nickel, cobalt and their oxides constitute the core of most of the MNPs. The surface of the MNPs is frequently covered by molecular imprinted polymers, silica or graphene, in order to avoid nanoparticles aggregation [START_REF] Karadjova | Nanomaterials for elemental speciation[END_REF]. Once the sorption of the analyte is complete on the magnetic sorbent, they are separated by an external magnetic field. Therefore, any additional step of physical separation (i.e., centrifugation, filtration) is required. The analyte is eluted by magnetic separation of the regenerated solvent, resulting in a simple and fast methodology [START_REF] Karadjova | Nanomaterials for elemental speciation[END_REF]. Considering the multiple advantages of MSPE, there is an unquestionable trend on its application to Hg (and other heavy metal ions). So far, it has been particularly used for water analysis, but several examples of Hg analysis in food show the analytical potential of the method (Table 1) [START_REF] Hemmati | Magnetic nanoparticle based solid-phase extraction of heavy metal ions: A review on recent advances[END_REF].

Fe3O4 nanoparticles are excellent supports for SPE due to their magnetic properties, making easy the separation from the solution by using a magnet. They exhibit high thermal and chemical stability and can be modified to develop selective sorbents [START_REF] Najafi | A novel magnetic ion imprinted nano-polymer for selective separation and determination of low levels of mercury(II) ions in fish samples[END_REF]. However, they tend to form aggregates and are susceptible to oxidation. Surface decoration with specific coating avoids these drawbacks and potentially increases their selectivity even in complex matrices. Several examples show its potential for the analysis of Hg in food [START_REF] Najafi | A novel magnetic ion imprinted nano-polymer for selective separation and determination of low levels of mercury(II) ions in fish samples[END_REF][START_REF] Seidi | Magnetic dispersive solid phase extraction based on polythiophene modified magnetic graphene oxide for mercury determination in seafood followed by flow-injection cold vapor atomic absorption spectrometry[END_REF][START_REF] Tadjarodi | A magnetic nanocomposite prepared from chelator-modified magnetite (Fe3O4) and HKUST-1 (MOF-199) for separation and preconcentration of mercury(II)[END_REF][START_REF] Zhu | Speciation of mercury in water and fish samples by HPLC-ICP-MS after magnetic solid phase extraction[END_REF].

Magnetized polymers, including both ion imprinted and non-imprinted polymers have been used to determine Hg(II) in food [START_REF] Najafi | A novel magnetic ion imprinted nano-polymer for selective separation and determination of low levels of mercury(II) ions in fish samples[END_REF][START_REF] Abolhasani | Determination of Hg(II) ions in sea food samples after extraction and preconcentration by novel Fe3O4@SiO2@polythiophene magnetic nanocomposite[END_REF]. Uptake time, sample pH, and nanosorbent amount influence the sorption procedure. Type and concentration of the eluent, volume and time of elution are the main parameters to be considered for elution optimization.

Silica is one of the most ideal shells, since it enhances mechanical and chemical stability in extreme acid/basic media. Polymeric materials have been used to modify the surface of such MNPs used on the extraction and preconcentration of Hg(II). They have been applied to the determination of Hg in complex food matrices [START_REF] Abolhasani | Determination of Hg(II) ions in sea food samples after extraction and preconcentration by novel Fe3O4@SiO2@polythiophene magnetic nanocomposite[END_REF]. The surface of Fe3O4@SiO2 NPs was modified by polythiophene using a self-assembly of thiophene onto Fe3O4 NPs. The sorbent, resulting on the modification of Fe3O4@SiO2 presents a sorption capacity of 59 mg g -1 . Quantification by CV-AAS of the eluted Hg(II) provides a detection limit of 0.02 ng mL -1 . The method was validated by the good correlation between the Hg certified and experimentally obtained values in fish CRMs, DOLT 4 and DORM 2. The magnetic nanosorbent was therefore used for the quantification of Hg in shrimp, fish and canned tuna [START_REF] Abolhasani | Determination of Hg(II) ions in sea food samples after extraction and preconcentration by novel Fe3O4@SiO2@polythiophene magnetic nanocomposite[END_REF].

Ion imprinted polymers (IIPs) appear as an advanced generation of polymeric materials. The presence of specific binding sites from the metal ion/ligand guarantees an exceptional selectivity. IIPs particles are grafted on various substrates in order to increase active surface area and favor the extraction efficiency. IIPs are obtained by several polymerization methods as precipitation, suspension and bulk polymerization [START_REF] Hemmati | Magnetic nanoparticle based solid-phase extraction of heavy metal ions: A review on recent advances[END_REF]. Surface imprinting of MNPs is a modern and promising alternative for Hg analysis that combines a magnetic core with a highly selective surface (IIP) [START_REF] Najafi | A novel magnetic ion imprinted nano-polymer for selective separation and determination of low levels of mercury(II) ions in fish samples[END_REF].

A novel IIP based on N-(pyridin-2-ylmethyl)ethenamine (V-Pic) was grafted on Fe3O4 NPs. It was used with success for the quantification of Hg in fish. Hg was extracted from the sample solution (pH adjusted at 8) by 2 min contact with 0.001 g of the sorbent. After isolation of the MNPs containing Hg, by the action of a magnet, Hg ions were eluted with 5 mL solution of 0.1 mol L -1 of HCl and 0.1 mol L -1 of EDTA. Finally, the eluent was analyzed by ICP-OES. The procedure exhibited excellent analytical performance, with a relative standard deviation (RSD) of 1.5% and a 0.03 ng mL -1 detection limit [START_REF] Najafi | A novel magnetic ion imprinted nano-polymer for selective separation and determination of low levels of mercury(II) ions in fish samples[END_REF].

Metal organic frameworks (MOF) have also been used to modify Fe3O4 NPs, for the extraction of several toxic metal ions [START_REF] Hemmati | Magnetic nanoparticle based solid-phase extraction of heavy metal ions: A review on recent advances[END_REF]. MOFs have highly porous-crystalline structures based on various coordinative connections among metal ions and organic linkers, resulting on materials of outstanding features such as diverse geometrical configurations, tunable pore size, large surface area, and mechanical/thermal stability. The shapes and sizes of the porous determine their selectivity, which justify the application on SPE of such nanocomposites, relatively quickly prepared [START_REF] Hemmati | Magnetic nanoparticle based solid-phase extraction of heavy metal ions: A review on recent advances[END_REF].

The combination of magnetic SPE and MOF lead to superior high-yield extraction in complex matrices. Magnetic MOFs are easily dispersed and magnetically isolated from the extraction medium. Fe3O4 NPs have been modified with 4-(5)imidazoledithiocarboxylic acid and then reacted with trimesic acid and Cu(II) acetate to form the metal-organic framework capable of extracting Hg(II). This material has been employed for the separation and preconcentration of Hg(II) in fish and canned tuna samples. It has a great sorption capacity (254 mg g -1 ) and is reusable during up to 12 sorption/elution cycles, without decreasing extraction efficiency. Hg(II) is eluted with a 1.1 mol L -l thiourea solution and subsequently analyzed by CV AAS. The detection limit and the relative standard deviation of the method are 10 ng L -1 and <8.3%, respectively [START_REF] Tadjarodi | A magnetic nanocomposite prepared from chelator-modified magnetite (Fe3O4) and HKUST-1 (MOF-199) for separation and preconcentration of mercury(II)[END_REF].

Magnetic carbon nanomaterials exhibit exclusive physicochemical properties, exceptional thermal/chemical stability and large sorption capacity [START_REF] Hemmati | Magnetic nanoparticle based solid-phase extraction of heavy metal ions: A review on recent advances[END_REF]. The versatile structure of graphene and graphene-based materials explains the exceptional adsorption of metal ions (i.e., Hg(II)), among others [START_REF] Yavuz | Magnetic dispersive solid phase extraction with graphene/ZnFe2O4 nanocomposite adsorbent for the sensitive determination of mercury in water and fish samples by cold vapor atomic absorption Microchem[END_REF][START_REF] Seidi | Magnetic dispersive solid phase extraction based on polythiophene modified magnetic graphene oxide for mercury determination in seafood followed by flow-injection cold vapor atomic absorption spectrometry[END_REF]. The most recent example of magnetized carbonaceous materials corresponds to the incorporation of ZnFe2O4 nanoparticles on graphene sheets. The integration of this mixed oxide, ZnFe2O4, prevents agglomeration of graphene sheets and improves their stability and adsorption capacity. This material, with a preconcentration factor of 30 and a high reusability (50 cycles) was used for the fast extraction of Hg(II) from water and fish samples. Hg was quantified by CV-AAS exhibiting a detection limit of 10 ng L -1 and a precision (RSD) of 2.7% [START_REF] Yavuz | Magnetic dispersive solid phase extraction with graphene/ZnFe2O4 nanocomposite adsorbent for the sensitive determination of mercury in water and fish samples by cold vapor atomic absorption Microchem[END_REF].

The application of MSPE on column minimizes reagent volume consumption and sample handling in comparison to MSPE in batch. However, there is a limited number of applications so far, using MSPE on column. Hg isolation from food of animal and vegetable origin (i.e., fish, cabbage, potatoes, green pepper, etc.) was achieved by an on column method with a new biosorbent based on the use of fungi Coprinus micaceus and γ-Fe2O3. Biomagnetic-sorbents, that match with low cost and green procedures, have been used in MSPE. Bacteria, yeast, fungi, and algae are some of the biomaterials employed. In general terms, structure stability is one of the main limitations of this approach. These materials are not widely used for Hg(II) extraction up to now. The fungal biomass immobilized with γ-Fe2O3 NPs with a sorption capacity of 26.2 mg g -1 was incorporated to a polyethylene column (1x10 cm size). The sample solution, resulting on the digestion of the mentioned foodstuffs, was passed through the MSPE column by using a peristaltic pump. Hg(II) ions eluted with 5 mL of 1 mol L -1 HCl and were analyzed by ICP-OES. The method, validated with water and fish muscle CRMs presents excellent LODs [START_REF] Özdemir | Magnetic solid phase extractions of Co(II) and Hg(II) by using magnetized C. micaceus from water and food samples[END_REF].

The experience related with the use of on-line SPE for Hg detection is scarce, but based on the examples corresponding to other elements some challenges have been identified. The main limitations are the high backpressure on the SPE column and the feasibility of reconditioning/exchange after degradation [START_REF] Hemmati | Magnetic nanoparticle based solid-phase extraction of heavy metal ions: A review on recent advances[END_REF]. In general, backpressure difficulties are due to decreased column permeability with NPs. Off-line Hg detection, performed by a large choice of analytical techniques (Table 1), is commonly used after MSPE. However, the handling could lead to sample contamination and there is also a risk of non-quantitative recovery of the analyte due to losing aliquots of the sorbent. The on-line hyphenation of SPE, is expected to allow automatization and consequently accuracy and precision. The on-line coupling minimizes the use of solvents and the efficient factor is favored.

The immobilization of the magnetic SPE material onto the inner walls of a knotted reactor placed in the injection valve of the flow injection manifold allows the development of an on-line detection system [START_REF] Vereda Alonso | Development of an on-line solid phase extraction method based on new functionalized magnetic nanoparticles. Use in the determination of mercury in biological and sea-water samples[END_REF]. The flow injection magnetic solidphase microextraction coupled to CV-ETAAS was used for Hg determination in seafood (fish, mussels). The proposed configuration does not present backpressure problems and represents a promising solution in routine analysis. MNPs were coated with silica, which is recognized by its unique features of ordered pore network, high surface area and versatility regarding further functionalization. The resulting silica coated MNPs were modified with 1,5-bis(di-2-pyridyl)methylene thiocarbohydrazide. The Hg(II) adsorption capacity of the material was 5.22 mg g -1 . This green analytical approach is rapid, selective, easy to use and the analytical performances (LOD: 7.8 ng L -1 ; RSD: 1.7%) are comparable to similar methods for Hg determination [START_REF] Vereda Alonso | Development of an on-line solid phase extraction method based on new functionalized magnetic nanoparticles. Use in the determination of mercury in biological and sea-water samples[END_REF].

Despite the irrefutable popularity of MSPE for the analysis of Hg(II) in food and environmental samples, its use in Hg speciation is limited to a few applications in food analysis [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF][START_REF] Zhu | Speciation of mercury in water and fish samples by HPLC-ICP-MS after magnetic solid phase extraction[END_REF][START_REF] Londonio | Online solid phase extraction-HPLC-ICP-MS system for mercury and methylmercury preconcentration using functionalised carbon nanotubes for their determination in dietary supplements[END_REF][START_REF] López-García | Determination of ultratraces of mercury species using separation with magnetic core-modified silver nanoparticles and electrothermal atomic absorption spectrometry[END_REF].

RECENT ADVANCES IN SPECIATION ANALYSIS OF MERCURY IN FOOD

Hg speciation in food is principally restricted to the quantification of two species: Hg(II) and MeHg, since food safety authorities exclusively regulate the content of such species. In general terms, the most common analytical methods for Hg speciation consist in the hyphenation of a highly effective separation technique and a sensitive elemental detection (i.e., ICP-MS, ICP-OES, AAS, AFS). The most popular separation techniques are unequivocally GC and HPLC. ICP-MS detection is favored by the possibility of multi-elemental analysis, high sensitivity, wide linear range, and isotopic information. There is a clear upward trend in analytical chemistry towards the development of green and environmentally friendly methods. Hg analysis in food is also concerned by this purpose. The progress on nanomaterials undeniably assists with the development of miniaturized, sensitive and green approaches for the speciation of Hg in foodstuffs. Several non-chromatographic methods have recently been developed for Hg speciation in food (Fig. 1). GC is one of the traditional separative techniques used for Hg speciation. The hyphenation of GC-ICP-MS is recognized as a powerful tool for Hg speciation. The detection by ICP-MS allows isotopic dilution analysis enhancing accuracy and precision on Hg species determination [START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF][START_REF] Clémens | Mercury speciation in seafood using isotope dilution analysis: A review[END_REF]. A recent scheme for ultrasensitive Hg speciation (Hg(II), MeHg, and EtHg) analysis in rice proposes the hyphenation of GC to DBD-OES. Compactness and cost-effectiveness are some of the main advantages of this approach thanks to the small size, low power, and low gas consumption of DBD-OES [START_REF] Lin | Ultrasensitive Speciation Analysis of Mercury in Rice by Headspace Solid Phase Microextraction Using Porous Carbons and Gas Chromatography-Dielectric Barrier Discharge Optical Emission Spectrometry[END_REF].

In general terms, GC is characterized for shorter separation times and higher resolution in comparison to HPLC. Nevertheless, contrary to HPLC separation, GC requires the derivatization of the Hg species present in food to volatilize them. This crucial step of sample preparation processes is long, tedious and strongly influenced by the sample matrix. For these reasons, HPLC is often chosen for Hg speciation in food [START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF].

Hg Speciation in Food by Using High Pressure Liquid Chromatography

HPLC is widely used for Hg speciation in food. The main advances in this regard are largely assisted by nanomaterials, from species extraction [START_REF] Chen | Single-Drop Solution Electrode Discharge-Induced Cold Vapor Generation Coupling to Matrix Solid-Phase Dispersion: A Robust Approach for Sensitive Quantification of Total Mercury Distribution in Fish[END_REF][START_REF] Londonio | Online solid phase extraction-HPLC-ICP-MS system for mercury and methylmercury preconcentration using functionalised carbon nanotubes for their determination in dietary supplements[END_REF][START_REF] Deng | Online solid sampling platform using multi-wall carbon nanotube assisted matrix solid phase dispersion for mercury speciation in fish by HPLC-ICP-MS[END_REF] to interfaces on the detection system [START_REF] De Quadros | Mercury speciation by high-performance liquid chromatography atomic fluorescence spectrometry using an integrated microwave/UV interface. Optimization of a single step procedure for the simultaneous photo-oxidation of mercury species and photo-generation of Hg 0[END_REF][START_REF] Li | Determination and speciation of Hg using HPLC-AFS by atomization of this metal on a UV/nano-ZrO2/HCOOH photocatalytic reduction unit[END_REF][START_REF] Yin | Vapour generation at a UV/TiO2 photocatalysis reaction device for determination and speciation of mercury by AFS and HPLC-AFS[END_REF].

Chromatographic Separation Assisted by Solid-Phase Extraction

The key role of SPE on recent methods for Hg analysis in food is not limited to total Hg quantification approaches, but extended to Hg speciation studies. Dispersed solid-phase extraction has a recognized potential on the extraction of elemental species despite being principally applied in the isolation of organic compounds. In comparison to conventional species extraction methods like sonication, heating treatments or MW radiation, MSPD is carried out at room temperature and atmospheric pressure reducing the risks of species instability/degradation. Additionally, this quantitative and fast extraction approach can be considered environmentally friendly due to the low waste generated and the use of reagents with low toxicity. A few but promising analytical methods exploit the advantages of this sample treatment approach for Hg speciation [START_REF] Duarte | Assessment of modified matrix solid-phase dispersion as sample preparation for the determination of CH3Hg + and Hg 2+ in fish[END_REF][START_REF] Deng | Online solid sampling platform using multi-wall carbon nanotube assisted matrix solid phase dispersion for mercury speciation in fish by HPLC-ICP-MS[END_REF].

The first Hg speciation study using MSPD, achieved in 2013, consisted in blending for 3 min 0.2 g of fish sample with 0.5 g of SiO2, acting as solid support. Hg species were quantitatively extracted with 5 mL of 0.5 mol L -1 NaCl and 4.2 mol L -1 HCl solution with 1 min of stirring time. After derivatization with sodium tetraphenylborate, separation and determination of Hg(II) and MeHg were performed by GC coupled to mass spectrometry (MS). Considering the matrix effect, quantification was carried out by matrix-matched calibration. After method validation with CRMs, Hg speciation was carried out in tuna, shark and guitar fish. The relative standard deviation was lower than 9.5% and LODs for MeHg and Hg(II) were 0.06 and 0.12 μg g -1 , respectively [START_REF] Duarte | Assessment of modified matrix solid-phase dispersion as sample preparation for the determination of CH3Hg + and Hg 2+ in fish[END_REF].

The hyphenation of MSPD with HPLC represents an evolution with respect to GC speciation considering the non-requirement of derivatization steps. The coupling of these techniques constitutes the basis for a simple on-line solid sampling platform for Hg speciation in fish. MSPD step was improved by the use of multiwall carbon nanotubes (MWCNTs) considered an excellent solid support for Hg species extraction [START_REF] Chen | Single-Drop Solution Electrode Discharge-Induced Cold Vapor Generation Coupling to Matrix Solid-Phase Dispersion: A Robust Approach for Sensitive Quantification of Total Mercury Distribution in Fish[END_REF][START_REF] Londonio | Online solid phase extraction-HPLC-ICP-MS system for mercury and methylmercury preconcentration using functionalised carbon nanotubes for their determination in dietary supplements[END_REF][START_REF] Deng | Online solid sampling platform using multi-wall carbon nanotube assisted matrix solid phase dispersion for mercury speciation in fish by HPLC-ICP-MS[END_REF]. This carbonaceous nanomaterial is characterized by a large surface area and outstanding mechanical properties. 4 mg of MWCNTs and 1 mg of fish were blended for MSPD, using a solution containing 2% (v/v) HCl and 1.5% (m/v) L-Cys as eluent. Extracted Hg species were directed to the HPLC-ICP-MS system and separated by reversed-phase liquid chromatography. The LODs were 9.9 ng g -1 for Hg(II) and 8.4 ng g -1 for MeHg, whereas the LOQs were 21.5 ng g -1 for Hg(II) and 18.3 ng g -1 for MeHg (RSD <10% for Hg(II) and <12.5% for MeHg) [START_REF] Deng | Online solid sampling platform using multi-wall carbon nanotube assisted matrix solid phase dispersion for mercury speciation in fish by HPLC-ICP-MS[END_REF]. The on-line speciation method assisted by MWCNTs is superior to previous MSPD approaches from several points of view. It involves high sample throughput, a low sample (1 mg) and solid support (4 mg) consumption without any derivatization of the extracted species minimizing the contamination risk [START_REF] Deng | Online solid sampling platform using multi-wall carbon nanotube assisted matrix solid phase dispersion for mercury speciation in fish by HPLC-ICP-MS[END_REF].

Functionalization of MWCNTs is a simple process that increases their selectivity. MWCNTs are decorated with poly-L-methionine, a sulfur-based substrate, on a novel on-line SPE-HPLC-ICP-MS system for Hg(II) and MeHg preconcentration from fish muscle and fish oil-based dietary supplements [START_REF] Londonio | Online solid phase extraction-HPLC-ICP-MS system for mercury and methylmercury preconcentration using functionalised carbon nanotubes for their determination in dietary supplements[END_REF]. The preconcentration microcolumn contained a mixture of the functionalized MWCNTs with inert particles of a low density polyethylene wax (Epolene) (1:10) to avoid aggregation and back pressure. The stable sorbent has a life time of at least 575 cycles with an enrichment factor up to 190. Hg(II) and MeHg eluted from the preconcentration microcolumn were separated by HPLC on a C18 column in approximately 10 min using a ternary mixture of 0.5% formic acid, 0.2% 2-mercaptoethanol and 20% methanol as mobile phase [START_REF] Londonio | Online solid phase extraction-HPLC-ICP-MS system for mercury and methylmercury preconcentration using functionalised carbon nanotubes for their determination in dietary supplements[END_REF]. This approach is very attractive considering the LODs (15-17 ng L -1 ) and the high sample throughput (5 samples h -1 ), using minimum amount of substrate without derivatization of the mercurial species [START_REF] Londonio | Online solid phase extraction-HPLC-ICP-MS system for mercury and methylmercury preconcentration using functionalised carbon nanotubes for their determination in dietary supplements[END_REF].

MNPs are suitable adsorbents for micro MSPE of different Hg species depending on the reagent used for functionalization [START_REF] López-García | Determination of ultratraces of mercury species using separation with magnetic core-modified silver nanoparticles and electrothermal atomic absorption spectrometry[END_REF]. Magnetic core-modified silver nanoparticles (Fe3O4@Ag) functionalized with the sodium salt of 2mercaptoethane-sulphonate adsorb Hg(II) selectively. However, a functionalization of the NPs with L-Cys (Fe3O4@Ag@Cys) leads to the retention of the inorganic species, but also monomethyl mercury, dimethyl mercury, ethyl mercury, phenyl mercury and diphenyl mercury. Once eluted with KI, total Hg was measured by ETAAS using silver nitrate and potassium permanganate solutions for chemical modification. Organomercurial species are desorbed in two fractions (MeHg + dimethylmercury (Me2Hg, (CH3)2Hg) + EtHg and phenylmercury (PhHg, C6H5Hg + ) + diphenylmercury (Ph2Hg, (C6H5)2Hg)). Therefore, this method cannot be considered a complete Hg speciation tool, but an easy screening of the organic and inorganic fractions. It was applied to the analysis of edible oils and the low amount of sorbent required (3 mg) facilitated the miniaturization of the procedure. Inorganic and organic Hg levels in vegetal edible oils are lower than the LOD (0.01 µg L -1 ), contrasting with fish oils where the concentration ranged between 0.08 and 0.45 µg kg -1 [START_REF] López-García | Determination of ultratraces of mercury species using separation with magnetic core-modified silver nanoparticles and electrothermal atomic absorption spectrometry[END_REF].

An advanced method, by using the same magnetic-core NPs, enabled precise quantification of organomercurial species. Fe3O4@SiO2@γmercaptopropyltrimethoxysilane MNPs were used for Hg speciation in water and fish samples. After ultrasound-based extraction of Hg species from fish muscle, the MNPs efficiently retained Hg(II), MeHg and PhHg. A solution of 0.1 mol L -1 HNO3 and 4% (m/v) thiourea was used for analytes desorption. After magnet isolation of the sorbent, Hg species were quantified by HPLC-ICP-MS. The proposed method is characterized by outstanding preconcentration factors (around 175 enhancement factor), fast separation (8 min) and high sensitivity (LOD: 0.49-0.74 ng L -1 ) [START_REF] Zhu | Speciation of mercury in water and fish samples by HPLC-ICP-MS after magnetic solid phase extraction[END_REF].

A robust, cost-effective, and sensitive method was developed for MeHg speciation in rice and rice products without derivatization. It consists of a simple sample preparation followed by on-line SPE preconcentration coupled to HPLC-CV-AFS [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF]. Rice and baby-food rice samples (0.3 g) were digested with 3 mL THAMH (25% (m/v)) for 20 min at 55 °C and 20 min at 60 °C and 1600 W in open vessels. After addition of 2 mL HCl, the mixture was heated again in the MW for 20 min at 55 °C and 20 min at 60 °C. Approximately 60% of the filtered supernatant was diluted up to 50 mL, of which 35 mL were loaded on the SPE column (2.1 x 3 mm) at a speed of 5 mL min -1 . A commercial thiol/thiourea silica material was used as sorbent for SPE. Hg species were eluted with 1.5 mmol L -1 ammonium pyrrolidine dithiocarbamate in 75% (v/v) methanol and separated on-line by a C8 HPLC column. Post column addition of bromine as oxidant and UV irradiation assured oxidation of MeHg to Hg(II). Conventional reduction with acidic tin (II) chloride was used to convert Hg(II) to Hg 0 . The sample throughput of the SPE preconcentration coupled to HPLC-CV-AFS method is four per hour [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF].

Contrary to fish Hg (speciation) analysis that benefits from a wide range of CRMs for method validation, there are no CRMs for MeHg in rice. Therefore, an elegant solution was cross-validation against standard addition and species specific isotope dilution (SSID)-GC-ICP-MS, which showed no significant differences versus the external calibration with SPE-HPLC-CV-AFS [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF][START_REF] Brombach | Methylmercury varies more than one order of magnitude in commercial European rice[END_REF]. Isotopically enriched 199 Hg(II) spike was added to the sample in order to track abiotic MeHg formation in this organic sample. The isotopic standard was enriched in isotope 199 at 98% and was spiked in excess (20-folds) in order to clearly see any alteration on the natural Hg isotopic ratio, attributed to abiotic MeHg formation. The natural isotopic ratio of MeHg remained unalterable, which guaranteed the absence of artificial MeHg formation during the whole analytical procedure-from digestion until determination. Therefore, the MeHg found in rice corresponded to the occurrence of this organomercurial species in food products [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF].

The LOD for MeHg by this simple and reliable approach is 0.4 ng L -1 . In the analyzed rice grains and baby-food rice samples from local European shops, the concentration varied between 1.56 and 2.69 µg kg -1 with a RSD ranging between 5.2 and 16.7%. It is a cost effective method for Hg speciation based on the detection by CV-AFS in comparison with GC/HPLC-ICP-MS approaches. Therefore, it is attractive for routine food control laboratories [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF].

Trends in the Choice of Stationary and Mobile Phases in HPLC for Hg Analysis

HPLC is largely used for Hg speciation analysis, mainly using reversed-phase (RP) chromatographic mode (Table 2). Mobile phases in RP may contain high salt levels and considerable amount of organic solvents. It has negative consequences on the operations of some detectors, such as nebulizer obstruction and interface deposition of carbon and salt in ICP-MS. Species separation by HPLC implies long separation times reducing analytical efficiency and throughput [START_REF] Cheng | Ion-pairing reversed-phase chromatography coupled to inductively coupled plasma mass spectrometry as a tool to determine mercurial species in freshwater fish[END_REF]. The large volumes of mobile phase waste, rich on toxic organic compounds, place this technique at a great distance of green chemistry. Hg speciation by ion-pairing-RP-ICP-MS using aqueous mobile phases containing minimal quantities of salts, promises to solve the previously mentioned problems.

Two advanced ion-pairing HPLC methods were developed based on positively and negatively charged ion-pairing reagents (tetrabutylammonium hydroxide -TBAH and sodium dodecylbenzene sulfonate -SDBS), where sodium 3-mercapto-1propylsulfonate (MPS) and L-Cys were included respectively in the mobile phases to transform mercury species into negative and positive Hg-complexes for good resolution. The addition of phenylalanine (Phe) enhanced the analytical efficiency and reduced waste production through reducing the baseline separation time in combination with short C18 guard columns (5 µm, 12.5 mm × 2.1 mm i.d.) [START_REF] Cheng | Ion-pairing reversed-phase chromatography coupled to inductively coupled plasma mass spectrometry as a tool to determine mercurial species in freshwater fish[END_REF]. Sample preparation consisted of ultrasonication (15 min at 40 °C) of 0.5 g of fish with two successive additions of 10 mL of a solution containing 5 mol L -1 HCl and 2.0 mmol L -1 Cys. After centrifugation and 0.45 µm filtration, 5 µL of the extract were injected. The analysis of five freshwater fish samples evidenced that this green and efficient method could be exploited for (food) routine analysis laboratories [START_REF] Cheng | Ion-pairing reversed-phase chromatography coupled to inductively coupled plasma mass spectrometry as a tool to determine mercurial species in freshwater fish[END_REF]. Hg(II), MeHg, EtHg and PhHg were baseline separated by two consecutive C18 guard columns with optimized mobile phases (2.0 mmol L -1 SDBS + 2.0 mmol L -1 Cys + 1.0 mmol L -1 Phe (pH 3.0) and 4.0 mmol L -1 TBAH + 2.0 mmol L -1 MPS + 2.0 mmol L -1 Phe (pH 6.0)). The LODs were 0.015, 0.014, 0.028 and 0.042 µg L -1 for Hg(II), MeHg, EtHg and PhHg, respectively. The analysis of fish tissues CRMs demonstrated a good recovery of Hg species (91-106%) and the accuracy of the method [START_REF] Cheng | Ion-pairing reversed-phase chromatography coupled to inductively coupled plasma mass spectrometry as a tool to determine mercurial species in freshwater fish[END_REF].

Rice consumption is one of the most important sources of exposure to arsenic (As). Therefore, speciation methods enabling the simultaneous speciation of As and Hg in this food product are particularly interesting. Ion-pairing reversed-phase liquid chromatography coupled to ICP-MS was used for the simultaneous speciation of four As species as well as Hg(II), EtHg and MeHg. Taking into account the compromise of best extraction for Hg (60% of total Hg) and As species, 1% HNO3 MW assisted extraction was chosen. The species were separated by ion pairing-RP liquid chromatography within 20 min (including 10 min of re-equilibration) by using tetrabutylammonium hydroxide (TBAH) as ion-pairing reagent [START_REF] Fang | Simultaneous determination of arsenic and mercury species in rice by ion-pairing reversed phase chromatography with inductively coupled plasma mass spectrometry[END_REF]. Hg species were quantified by external calibration. The LODs of the simultaneous As/Hg speciation method were 0.04, 0.01 and 0.03 µg L -1 for Hg(II), EtHg, and MeHg, respectively [START_REF] Fang | Simultaneous determination of arsenic and mercury species in rice by ion-pairing reversed phase chromatography with inductively coupled plasma mass spectrometry[END_REF]. The main advantage of this approach is the simultaneous speciation of three Hg and four As species in a single extraction and chromatographic run without any requirement of organic solvents in the mobile phase.

Ionic liquids (ILs) have demonstrated to be a fresh alternative to conventional ionpairing agents, such as tetraalkylammonium salts, for Hg speciation by RP-HPLC-CV-AFS. MeHg, EtHg, and Hg(II) were separated using a C18 column and a gradient developed by mixing methanol and a solution composed of 0.4% (v/v) 1octyl-3-methylimidazolium chloride [C8mim]Cl, 100 mmol L -1 NaCl and 20 mmol L -1 buffer citric acid/citrate (pH 2.0) within 12 min. LODs were 0.05, 0.06 and 0.11 µg L -1 for MeHg, EtHg and Hg(II), respectively, which are comparable to other methods [START_REF] Castro Grijalba | Usefulness of ionic liquids as mobile phase modifiers in HPLC-CV-AFS for mercury speciation analysis in food[END_REF]. The method was applied to a variety of samples, including fresh seafood, canned fish, yeast and garlic. Hg species were extracted from food samples (300 mg) by sonication during 10 min with 5 mL of a 5 mol L -1 solution. 5-fold dilution for seafood samples and 10-fold for yeast and garlic was proposed to avoid possible matrix effect. The extraction yield varied according to the matrix, from 51% for yeast up to 87% for seafood. Hg species were separated by HPLC and organic species were photo-oxidized on-line in a UV digestion unit by using a 0.75% (w/v) K2S2O8 solution [START_REF] Castro Grijalba | Usefulness of ionic liquids as mobile phase modifiers in HPLC-CV-AFS for mercury speciation analysis in food[END_REF]. In the analyzed foodstuffs, Hg levels were in a range between 0.3 and 0.7 µg g -1 and Hg species distribution varied according to the food sample. MeHg was the major species in seafood while Hg(II) dominated in yeast and garlic [START_REF] Castro Grijalba | Usefulness of ionic liquids as mobile phase modifiers in HPLC-CV-AFS for mercury speciation analysis in food[END_REF].

For HPLC analysis, the development of new stationary phases based on nanomaterials opens new perspectives on Hg speciation. A recent example is the successful use of graphene as stationary phase in HPLC. Graphene is a new class of carbon-based nanomaterial, commonly obtained by graphite oxidation and subsequent reduction processes; it does not contain metal impurities. This nanomaterial has outstanding sorptive properties and therefore a great trace metal preconcentration capacity [START_REF] Bendicho | Nanoparticle-assisted chemical speciation of trace elements, TrAC[END_REF]. Both surfaces of the planar sheet are accessible for sorption, resulting in a huge specific surface area. Graphene oxide is oxidized graphene, keeping the singular structures and properties of graphene.

In general terms, the application of graphene to trace metal speciation is rare so far. The unique adsorption properties of graphene and graphene oxide have been exploited for the first time on the development of HPLC stationary phase for Hg speciation. Hg species (inorganic and organic ones) complexes with 2-thiosalicylic acid were attracted by graphene oxide due to π electron stacking. The coupling of the graphene oxide based column to ICP-MS provided LODs of 0.016, 0.027, 0.032 and 0.068 µg L -1 , for Hg(II), MeHg, EtHg and PhHg, respectively [START_REF] Cheng | Graphene oxide as a stationary phase for speciation of inorganic and organic species of mercury, arsenic and selenium using HPLC with ICP-MS detection[END_REF]. Graphene and graphene oxide based columns (4.6 mm i.d. and 5 cm long) did not show different results regarding retention time and peak resolution. The separation of inorganic and organic mercury species was achieved in 12 min, by using 2 mmol L -1 TSA and 5 mmol L -1 phenylalanine solution at pH 8 with a flow rate of 1.0 mL min -1 [START_REF] Cheng | Graphene oxide as a stationary phase for speciation of inorganic and organic species of mercury, arsenic and selenium using HPLC with ICP-MS detection[END_REF].

The comparison with a commercial C8 column (5µm, 4.6 mm i.d. and 5 cm long) demonstrated the superior analytical performance of the graphene based developed column [START_REF] Cheng | Graphene oxide as a stationary phase for speciation of inorganic and organic species of mercury, arsenic and selenium using HPLC with ICP-MS detection[END_REF]. The GO@SiO2 based column, successfully used for Hg speciation in fish, showed its feasibility for routine Hg speciation analysis by HPLC-ICP-MS. The success of the graphene oxide based column on the retention of Hg species evidenced the potential of this carbon-based nanomaterial as a powerful adsorbent in preconcentration procedures [START_REF] Cheng | Graphene oxide as a stationary phase for speciation of inorganic and organic species of mercury, arsenic and selenium using HPLC with ICP-MS detection[END_REF].

Vapor Generation of Hg Species Assisted by Nanomaterials

A considerable number of applications of nanomaterials on Hg speciation corresponds to their use in efficient interfaces between liquid chromatography and the detection by CV-AFS (Table 3). Photochemical vapor generation is an advanced approach that can also be successfully hyphenated to chromatographic and flow injection on one side and atomic detection methods, as AFS, on the other side. It consists of the generation of reducing radicals from low molecular weight organic compounds under UV irradiation. Laser and microwave irradiation are also effective on the generation of free radicals with similar analytical purposes [START_REF] Wu | Microwave-enhanced cold vapor generation for speciation analysis of mercury by atomic fluorescence spectrometry[END_REF][START_REF] Wu | Applications of chemical vapor generation in non-tetrahydroborate media to analytical atomic spectrometry[END_REF].

AFS is a convenient method for Hg determination characterized by its sensitivity, selectivity, and relatively low cost instrumentation. The sensitivity, and consequently the LODs, are strongly influenced by the sample introduction efficiency. Vapor/hydride generation is largely used, guaranteeing matrix separation and an efficient analyte transport. Vapor/hydride generation by using tetrahydroborate is the most frequent approach for Hg determination [START_REF] De Quadros | Mercury speciation by high-performance liquid chromatography atomic fluorescence spectrometry using an integrated microwave/UV interface. Optimization of a single step procedure for the simultaneous photo-oxidation of mercury species and photo-generation of Hg 0[END_REF]. The chemical vapor generation method based on the use of tetrahydroborate suffers of interferences that degrade the sensitivity and reproducibility of the method, and the large amount of hydrogen generated can dramatically affect the plasma/flame source. The photocatalytic properties of TiO2 are exploited on the development of an efficient interface for Hg speciation by HPLC-CV-AFS [START_REF] Yin | Vapour generation at a UV/TiO2 photocatalysis reaction device for determination and speciation of mercury by AFS and HPLC-AFS[END_REF]. The method allows direct vapor generation of Hg species on nano TiO2 under UV irradiation in the presence of a mixture of formic acid and sodium formate, acting as a hole scavenger. Compared to the most frequently used system KBH4/NaOH-HCl, the direct photocatalysis vapor generation on nano TiO2 clearly exhibited better analytical performance [START_REF] Yin | Vapour generation at a UV/TiO2 photocatalysis reaction device for determination and speciation of mercury by AFS and HPLC-AFS[END_REF]. The method was applied on the investigation of Hg species (Hg(II), MeHg, EtHg and PhHg) in fish, oysters and others seafood, with a LOD between 10 and 70 ng L -1 , according to the mercurial species. The principle of on-line photocatalyst-assisted vapor generation using nano TiO2, has also successfully been used in the hyphenation of HPLC with ICP-MS [START_REF] Chen | Determination of methylmercury and inorganic mercury by coupling short-column ion chromatographic separation, on-line photocatalyst-assisted vapor generation, and inductively coupled plasma mass spectrometry[END_REF].

Nanosized TiO2 has also been included in the integrated MW/UV interface for Hg species determination by HPLC-AFS. Hg(II), EtHg, MeHg were converted to Hg 0 by the combined action of UV and MW in presence of formic acid and Cys in the mobile phase. This method allows the replacement of the commonly used tetrahydroborate system by an environmentally friendly alternative for Hg speciation with LODs between 0.15 and 0.35 µg L -1 in fish [START_REF] De Quadros | Mercury speciation by high-performance liquid chromatography atomic fluorescence spectrometry using an integrated microwave/UV interface. Optimization of a single step procedure for the simultaneous photo-oxidation of mercury species and photo-generation of Hg 0[END_REF].

The use of ZrO2-NPs improves the efficiency of the photocatalytic system, compared to nanosized TiO2 [START_REF] Karadjova | Nanomaterials for elemental speciation[END_REF]. A UV/nano-ZrO2/HCOOH photocatalytic reduction unit was developed as an on-line interface between HPLC and AFS for Hg speciation. Hg species were transformed into atomic Hg on nanosized ZrO2 upon UV irradiation. The sensitive novel system reached LODs of 13, 16 and 24 ng L -1 for MeHg, EtHg and Hg(II), respectively, and was successfully applied to the analysis of fish and other seafood samples [START_REF] Li | Determination and speciation of Hg using HPLC-AFS by atomization of this metal on a UV/nano-ZrO2/HCOOH photocatalytic reduction unit[END_REF].

Advanced oxidation using Fe3O4 MNPs constitutes another example of how nanoparticles assist Hg speciation. Hydride generation/cold vapor inactive species were transformed in active ones by efficient on-line post-column advanced oxidation using Fe3O4 MNPs without MW/UV irradiation. The LODs of this simple, efficient and green approach were 0.7, 0.8, 0.9 and 1.1 µg L -1 , for Hg(II), EtHg, PhHg and MeHg, respectively. Peak broadening was reduced due to the very short length of Fe3O4 MNPs column (2.5 cm). This approach, which was applied to Hg speciation in fish, does not require the complex experimental set up of conventional oxidation techniques and eliminates the use of toxic oxidation reagents [START_REF] Ai | Advanced oxidation using Fe3O4 magnetic nanoparticles and its application in mercury speciation analysis by high performance liquid chromatography-cold vapor generation atomic fluorescence spectrometry[END_REF].

Hg Speciation in Food by Non-Chromatographic Methods

Chromatographic methods are absolutely well established on Hg speciation in food. However, the high cost of the instrumentation and analysis as well as the complex sample pre-treatment used in these conventional methods motivated the development of non-chromatographic approaches. Non-chromatographic methods are based on the individual chemical and physical properties of the different species. Low solvent consumption, promptness, and preconcentration are some of the advantages of such methods. The functionalization of nanomaterials opens new perspectives on the development and application of non-chromatographic approaches. Some of these recent and promising methods are presented hereafter.

Electrochemical vapor generation (ECVG) is an important sample introduction

technique based on the reaction between H• derived from the cathode of the electrolytic cell and the analyte. In comparison to photochemical vapor generation (PVG), ECVG is more efficient and environmentally friendly considering that it only requires a simple electrolytic cell and a power supply [START_REF] Zhang | Speciation of inorganic-and methyl-mercury in biological matrixes by electrochemical vapor generation from an l -cysteine modified graphite electrode with atomic fluorescence spectrometry detection[END_REF]. ECVG has been coupled to spectroscopic techniques as AAS and AFS for Hg quantification in a wide type of samples. However, its application to speciation is relatively new [START_REF] Shi | Highly efficient electrocatalytic vapor generation of methylmercury based on the gold particles deposited glassy carbon electrode: A typical application for sensitive mercury speciation analysis in fish samples[END_REF].

The first example corresponds to the development of a L-Cys modified graphite cathode, which efficiently converts Hg(II) and MeHg to Hg vapor [START_REF] Zhang | Speciation of inorganic-and methyl-mercury in biological matrixes by electrochemical vapor generation from an l -cysteine modified graphite electrode with atomic fluorescence spectrometry detection[END_REF]. Taking into account that at low current mode (0.2 A) exclusively Hg(II) is efficiently converted to Hg vapor and at high current mode (2.2 A), both MeHg and Hg(II) can be reduced efficiently, a sensitive speciation method was established relying on the current control. The novel non-chromatographic speciation method was validated by the analysis of several CRMs and applied on Hg speciation in seafood. The superiority of ECVG, compared to conventional CVG was demonstrated since it avoids sodium tetraethylborate and oxidizing agents consumption, minimizing costs and potential environmental pollution risks [START_REF] Zhang | Speciation of inorganic-and methyl-mercury in biological matrixes by electrochemical vapor generation from an l -cysteine modified graphite electrode with atomic fluorescence spectrometry detection[END_REF].

An innovator compact method based on flow injection photochemical vapor generation (PVG) coupled with miniaturized solution cathode glow discharge atomic emission spectroscopy (SCGD-AES) was developed for the determination of Hg and its species [START_REF] Mo | Flow Injection Photochemical Vapor Generation Coupled with Miniaturized Solution-Cathode Glow Discharge Atomic Emission Spectrometry for Determination and Speciation Analysis of Mercury[END_REF]. This novel method combines the small size, independence of gases and low power consumption of SCGD-AES with the simplicity, miniaturization feasibility and green analytical characteristic of PVG. The SCGD was generated between a miniature hollow titanium tube and a solution emerging from a glass capillary. An automated stopped-flow operation allowed increasing the UV irradiation time on-line. Hg vapor, generated by PVG, was transported to the SCGD for excitation, and finally the emission signals were recorded by a miniaturized spectrograph. Inorganic and organic Hg species (MeHg and EtHg) exhibited dissimilar responses at different wavelengths and powers from the UV lamp. Therefore, Hg speciation was possible by the variation of wavelengths and powers from the UV lamp and irradiation times. Inorganic and organic species were converted to Hg 0 for the determination of total Hg with 8 W/254 nm UV lamp and 60 s irradiation time; while only Hg(II) can be reduced to Hg 0 and determined selectively with 4 W/365 nm UV lamp and 20 s irradiation time. Consequently, organic mercury concentration was calculated by the difference on total Hg and inorganic Hg concentrations. Hg(II), less toxic than organic species, was successfully used as a primary standard for the quantification of Hg considering the similar sensitivity at 8 W/254 nm. This pioneer PVG-SCGD-AES approach results on a 365-fold improvement in Hg(II) LOD (0.2 µg L -1 ) compared to SCGD-AES. The method was validated for water and biological samples including fish and could be extrapolated to other elements [START_REF] Mo | Flow Injection Photochemical Vapor Generation Coupled with Miniaturized Solution-Cathode Glow Discharge Atomic Emission Spectrometry for Determination and Speciation Analysis of Mercury[END_REF]. Nickel demonstrated to be an optimum enhancement reagent for the PVG efficiency of Hg(II) and MeHg. Under optimized conditions, the addition of nickel (10 mg L -1 ) increased the signal intensity of Hg(II) and MeHg around 1.5 times compared to that without nickel ion. Separative techniques like GC and HPLC could be potentially combined to PVG-SCGD-AES, opening new possibilities for Hg speciation analysis [START_REF] Mo | Flow Injection Photochemical Vapor Generation Coupled with Miniaturized Solution-Cathode Glow Discharge Atomic Emission Spectrometry for Determination and Speciation Analysis of Mercury[END_REF].

Double liquid-liquid extractions and/or selective derivatization are commonly used for Hg speciation by non-chromatographic methods [START_REF] Covaci | Methylmercury determination in seafood by photochemical vapor generation capacitively coupled plasma microtorch optical emission spectrometry[END_REF]. A recent example is the introduction of methyl isobutyl ketone on the sample treatment of seafood as degassing agent leaving Hg into the water phase. MeHg was extracted from an aliquot of the aqueous phase with HBr, CuCl2 and toluene, and back-extracted into a L-Cys-sodium acetate solution. This method was proposed for quantification of both total Hg and MeHg in two consecutive steps using a single Hg commercial standard solution by heating vaporization AAS. Despite most analytical studies focused on fish muscle, and gonads are also consumed. The analysis revealed remarkable differences between the MeHg fraction in sea urchins and squids glands [START_REF] Yoshimoto | Simple analysis of total mercury and methylmercury in seafood using heating vaporization atomic absorption spectrometry[END_REF]. This protocol, validated by the analysis of several CRMs, is much simpler and cost-effective in comparison with chromatographic techniques [START_REF] Yoshimoto | Simple analysis of total mercury and methylmercury in seafood using heating vaporization atomic absorption spectrometry[END_REF].

A non-chromatographic Hg speciation method based on double liquid-liquid extraction and measurement by flow injection UV photochemical vapor generation capacitively coupled plasma microtorch optical emission spectrometry (UVPVG-μCCP-OES) was developed for Hg determination in fish and others seafood [START_REF] Covaci | Methylmercury determination in seafood by photochemical vapor generation capacitively coupled plasma microtorch optical emission spectrometry[END_REF].

In brief, Hg species extracted from 0.2 g of lyophilized fish and seafood samples were confined in 10 mL hydrobromic acid (47%), followed by MeHg extraction in toluene and back-extraction in 1% L-Cys solution. MeHg was quantified by UV-PVG-μCCP-OES and external calibration against Hg(II) standards in formic acid. LOD and LOQ (2 µg kg -1 and 6 µg kg -1 , respectively) were improved in comparison to thermal decomposition atomic absorption spectrometry (TDAAS) reference method and other methods used for Hg speciation in seafood [START_REF] Covaci | Methylmercury determination in seafood by photochemical vapor generation capacitively coupled plasma microtorch optical emission spectrometry[END_REF]. Hg(II) was determined by the difference between total Hg and MeHg concentrations [START_REF] Covaci | Mercury speciation in seafood using non-chromatographic chemical vapor generation capacitively coupled plasma microtorch optical emission spectrometry method -Evaluation of methylmercury exposure[END_REF]. This new miniaturized approach involves a low power/Ar consumption plasma microtorch (100 mL min -1 ) and a low resolution microspectrometer. The developed method is simple and safe concerning sample preparation through elimination of conventional, harmful reductants and attractive for using unsophisticated instrumentation [START_REF] Covaci | Mercury speciation in seafood using non-chromatographic chemical vapor generation capacitively coupled plasma microtorch optical emission spectrometry method -Evaluation of methylmercury exposure[END_REF].

A modification of this method, based on ultrasound assisted extraction of Hg(II) species with formic acid go further on its green concept. In that case the use of toluene is no longer required, formic acid being the unique reagent. Concentration of MeHg was determined by subtraction of Hg(II) amount from total Hg. The eco-scale non-chromatographic method, with LODs and LOQs of 4.8 and 14.4 µg kg -1 respectively, was applied on Hg speciation in fish [START_REF] Covaci | A highly sensitive eco-scale method for mercury determination in water and food using photochemical vapor generation and miniaturized instrumentation for capacitively coupled plasma microtorch optical emission spectrometry[END_REF].

Non-Chromatographic Speciation Assisted by SPE

Amberlite XAD-series resins, easy derivatized and highly stable in a wide pH range, are on the top of sorbents used for speciation. Aminated Amberlite XAD-4 resin was proposed as a solid-phase extractant for Hg speciation in seafood (fish and mussel). Hg(II) and MeHg were sequentially eluted from the aminated XAD-4 resin column coupled to flow injection cold vapor generation AAS. 400 mg of AAXAD-4 were placed into a SPE cartridge column (3 mL volume, 8 mm i.d., 65 mm length) and sample solution passed through the column at a flow rate of 1.5 mL min -1 . Hg(II) and MeHg eluted consecutively with 10 mL of 0.1% (w/v) thiourea in 3% (v/v) HCl and 10 mL of 6.0 mol L -1 HCl solution at 1.5 mL min -1 flow rate, respectively. Hg(II) was directly quantified by FI-CVG-AAS, while MeHg was previously oxidized before analysis with 20 µL of 1% (m/v) KMNO4. The enhancement factor for Hg(II) and MeHg was 11.7 and 17.2, respectively, and the column did not show significant decay in the Hg species extraction efficiency up to 60 cycles. This method (LOD: 0.148 and 0.157 µg L -1 for Hg(II) and MeHg, respectively) allowed semiautomatic quantification of Hg species in seafood [START_REF] Çaylak | Use of an aminated Amberlite XAD-4 column coupled to flow injection cold vapour generation atomic absorption spectrometry for mercury speciation in water and fish tissue samples[END_REF].

IIPs also assist Hg speciation in food [START_REF] Zarco-Fernández | Development of a thiourea derivative polymer combined to a Direct Mercury Analyser for screening and pre-concentration of mercury species[END_REF][START_REF] Dakova | Non-chromatographic mercury speciation and determination in wine by new core-shell ion-imprinted sorbents[END_REF][START_REF] Zarco-Fernández | A new specific polymeric material for mercury speciation: Application to environmental and food samples[END_REF]. For example, one of the few Hg speciation studies in wine consists of the high selectivity and preconcentration towards Hg(II) of a non-chromatographic method by using a core-shell ionimprinted sorbent. Airborne particulate matter deposition and the grapevine intake from the soil/groundwater system explained the presence of Hg, and other toxic elements in wine [START_REF] Dakova | Non-chromatographic mercury speciation and determination in wine by new core-shell ion-imprinted sorbents[END_REF]. Considering the low Hg content, usually at sub µg L -1 , the speciation required preconcentration steps.

The Hg(II) ion-imprinted sorbents were synthesized via copolymerization using MAA as a functional monomer, as a crosslinking agent in the presence of Hgcomplexes with 1-pyrrolidinedithiocarboxylic acid, as chelating agent. Silica gel acted as supporting material, chemically modified with 3-(trimethoxysilyl)propyl methacrylate. The sorbent exhibited a high specificity for the retention of Hg(II) without interferences of the organic matrix and benefits from a fast kinetics of sorption and desorption processes. Hg speciation in white and red wines (20-50 mL) was carried out in a column solid-phase extraction scheme using 100 mg of sorbent. Hg(II) was quantified in column eluates by CV-AAS and MeHg concentration was determined by the difference between total Hg and Hg(II) concentrations [START_REF] Dakova | Non-chromatographic mercury speciation and determination in wine by new core-shell ion-imprinted sorbents[END_REF]. The LOD for Hg(II) (0.02 µg L -1 ), was comparable to those obtained by GC-ICP-MS in wine without requirement of derivatization steps. This sorbent exhibited a great chemical and mechanical stability, unalterable after more than 50 cycles of sorption/desorption [START_REF] Dakova | Non-chromatographic mercury speciation and determination in wine by new core-shell ion-imprinted sorbents[END_REF].

The combination of a thiourea derivative polymer with DMA was proposed for screening and pre-concentration of Hg species. The method was based on the use of a new selective polymer, synthesized by using 2-(methacryloylamino) ethyl 2methyl acrylate as cross-linker, 1-phenyl-3-(-3-vinyl phenyl) thiourea as monomer and azobisisobutyronitrile as initiator. Hg species were extracted from fish by ultrasonication and loaded on the highly selective polymer. The speciation strategy consisted of the specific release of MeHg at 120 °C, followed by the standard DMA temperature up to 600 °C for the quantification of Hg(II). On the first heating step (at 60 °C), MeHg was recovered up to 50%, being the rest of the organomercurial species, measured in conjunction with Hg(II) after application of higher temperatures [START_REF] Zarco-Fernández | Development of a thiourea derivative polymer combined to a Direct Mercury Analyser for screening and pre-concentration of mercury species[END_REF]. The method was therefore considered for rapid screening, and not for precise quantification of Hg species. This innovative approach provides a fast MeHg screening in food with simple handling and sample pretreatment.

A more advanced approach was based on the use of a new polymeric material that allowed, for the first time, the retention of both species, Hg(II) and MeHg. After sequential elution, Hg(II) and MeHg were quantified by CV-AFS. The polymer was synthesized by using vinyl derivative of 8-hydroxiquinoline as monomer, and 2-(methacryloylamino) ethyl 2-methyl acrylate as co-monomer in presence of azobisisobutyronitrile, as initiator. At least 20 cycles of Hg species sorption/desorption can be carried out, with a reproducibility of 5% [START_REF] Zarco-Fernández | A new specific polymeric material for mercury speciation: Application to environmental and food samples[END_REF]. The analytical procedure, with a quantification limit of 0.015 mg kg -1 , was validated with a tuna CRM (BCR 464) and applied to commercial fish. Hg species extraction from lyophilized fish samples was achieved with 5 mol L -1 HCl under ultrasonication. Highly acidic fish extracts were diluted before loading on the polymer cartridge in order not to disturb the optimum sorption loading, at neutral pH. 1 mL of the fish extract was loaded on a conventional glass solid-phase extraction cartridge. Hg(II) was eluted by the addition of 3 mL of 2 mol L -1 HCl in methanol for Hg (II), followed by the MeHg elution with 3 mL of NaClO 20% in 1 mol L -1 HCl. The proposed method involves minimum handling and could be exploited for Hg speciation in routine laboratories [START_REF] Zarco-Fernández | A new specific polymeric material for mercury speciation: Application to environmental and food samples[END_REF]. This rapid, low cost, and simple Hg speciation approach also reduces considerably the reagents waste compared with conventional speciation approaches.

Non-Chromatographic Methods Assisted by Nanomaterials

An innovative recent technology proposes the use of gold particles deposited glassy carbon electrode (Au/GCE) as cathode for ECVG combined with AFS for Hg speciation. The Au/GCE exhibited outstanding catalytic property for electrochemical conversion of MeHg to gaseous mercury. Highly consistent or distinct difference between the AFS signals of MeHg and Hg(II) could be achieved by control of ECVG electrolytic parameters. The method, based on the divergent electrochemical reactivity of Hg(II) and MeHg showed high sensitivity (LOD 0.44 µg kg -1 ) and good repeatability (<6%, RSD) in fish analysis [START_REF] Shi | Highly efficient electrocatalytic vapor generation of methylmercury based on the gold particles deposited glassy carbon electrode: A typical application for sensitive mercury speciation analysis in fish samples[END_REF]. This sensitive, green and simple technique has an enormous potential in food routine laboratories.

A visual detection method of MeHg and EtHg in fish samples has been developed based on DNA-templated alloy Ag-Au NPs. The speciation approach relies on the ability of DNA sequences containing T to combine with organomercurial species. The different and tunable binding force of DNA sequence to Hg species by modifying the number and locations of T bases explains the application of T-rich DNA on Hg speciation [START_REF] Chen | Specifically and Visually Detect Methyl-Mercury and Ethyl-Mercury in Fish Sample Based on DNA-Templated Alloy Ag-Au Nanoparticles[END_REF].

Two T-rich aptamers were designed: HT5 for exclusive recognition of MeHg and HT7 that recognizes MeHg and EtHg. Therefore, quantification of EtHg was carried out by subtraction of MeHg obtained with HT5 from the total MeHg and EtHg concentration obtained with HT7. In presence of Ag + and Au + , the aptamers preferentially bind the organomercurial species and consequently the formation of alloy Ag-Au NPs after reduction producing the color change of the solution (from colorless to purple). The presence of Hg(II) and other metallic ions, even at higher concentration than MeHg and EtHg did not interfere the detection of these organic species. It demonstrates the excellent recognition specificity of the designed aptamers [START_REF] Chen | Specifically and Visually Detect Methyl-Mercury and Ethyl-Mercury in Fish Sample Based on DNA-Templated Alloy Ag-Au Nanoparticles[END_REF].

This method allows bare eye detection of the organomercurial species at 1.0 mg kg - 1 . LODs are around 10 times lower by using UV-Vis spectrometer as detector. The method was applied to the detection and quantification of organomercurial species in fish, extracted by MW acid digestion with a recovery of 101-109% (RSD <8%). The instrument-free visual discrimination and detection of MeHg and EtHg constitutes a simple, rapid, and cost effective alternative for Hg speciation in food [START_REF] Chen | Specifically and Visually Detect Methyl-Mercury and Ethyl-Mercury in Fish Sample Based on DNA-Templated Alloy Ag-Au Nanoparticles[END_REF].

Carbon dots (CDs), a new alternative to quantum dots, are fluorescent nanoparticles with excellent optical probes (highly photo stables, strong fluorescence and tunable color emission). They are attractive also considering that in contrast to quantum dots, their simple synthesis, using eco-friendly carbon sources (i.e., glucose, sucrose) does not involve precursors containing toxic elements, becoming a green alternative [START_REF] Bendicho | Nanoparticle-assisted chemical speciation of trace elements, TrAC[END_REF][START_REF] Costas-Mora | In situ building of a nanoprobe based on fluorescent carbon dots for methylmercury detection[END_REF].

The applications of CDs in elemental speciation are still scarce but promising. A fast and simple fluorescent assay has been recently developed for MeHg detection using D-fructose as carbon source. Ultrasonication was used for simultaneous CDs fabrication and selective recognition of the target analyte. The assay was accomplished within 1 min, exhibiting a LOD of 5.9 nmol L -1 of MeHg (RSD 2.2%). Hg species were extracted from marine animal tissues CRMs (0.4 g) by ultrasonication during 4 min at 12% of amplitude in 5 mol L -1 HCl solution. MeHg was quantified by the fluorescent assay after filtration (0.45 µm) and clean up (C18 cartridges) of the fish extracts. MeHg recognition was attributed to the hydrophobicity of this species and its capability to facilitate a non-radiative electron/hole recombination [START_REF] Costas-Mora | In situ building of a nanoprobe based on fluorescent carbon dots for methylmercury detection[END_REF]. The validation of this environmentally friendly method with water and marine animal tissues CRMs guarantees its successful application in (sea)food and beverages.

ADVANCES AND TRENDS IN THE USE OF Hg STABLE ISOTOPES

The use of stable isotope tracers is a great support for developing analytical methods for Hg speciation. The addition of isotopically enriched species, usually 199 Hg(II) and 201 MeHg, allows monitoring and correction of losses and/or species transformation from the sample preparation steps. Isotopic dilution analysis is a powerful tool in analytical quality assurance and quality control. Its application in Hg analysis in food is extended to a wide variety of samples as seafood [START_REF] Clémens | Mercury speciation in seafood using isotope dilution analysis: A review[END_REF][START_REF] Clémens | Mercury speciation analysis in seafood by species-specific isotope dilution: Method validation and occurrence data[END_REF], rice [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF][START_REF] Feng | Investigation of Hg uptake and transport between paddy soil and rice seeds combining Hg isotopic composition and speciation[END_REF] and breast milk [START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF].

Species specific isotopic dilution is a smart solution to the quantification of Hg species in absence of CRMs. In complex rice and breast milk samples, it proves the validation of speciation methods based on both GC and HPLC hyphenated to ICP-MS [START_REF] Brombach | Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation[END_REF][START_REF] Feng | Investigation of Hg uptake and transport between paddy soil and rice seeds combining Hg isotopic composition and speciation[END_REF][START_REF] Vacchina | Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk[END_REF].

The potential of Hg stable isotopes in food is not restricted to isotopic dilution analysis. Hg isotopic fractionation-the natural isotopic abundance variation-can be also exploited in dietary studies.

Hg is recognized as a "model element" for isotopic fractionation studies. It has several stable isotopes that exhibit mass-dependent and mass-independent fractionation [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF], which, combined, provide precious information on sources discrimination, reactivity and fate of Hg in a biogeochemical context. The improvement of analytical methods and the development of multiple collector inductively coupled mass spectrometry (MC-ICP-MS) has drastically expanded the research on non-traditional stable isotope deviated from traditional geological applications.

The great relevance of Hg isotopic signature on a large variety of subjects such as the elucidation of metabolic pathways [START_REF] Feng | Investigation of Hg uptake and transport between paddy soil and rice seeds combining Hg isotopic composition and speciation[END_REF][START_REF] Feng | Specific pathways of dietary methylmercury and inorganic mercury determined by mercury speciation and isotopic composition in zebrafish (Danio rerio)[END_REF][START_REF] Kritee | Microbial stable isotope fractionation of mercury: A synthesis of present understanding and future directions[END_REF][START_REF] Kwon | Absence of fractionation of mercury isotopes during trophic transfer of methylmercury to freshwater fish in captivity[END_REF][START_REF] Yin | Stable Mercury Isotope Variation in Rice Plants (Oryza sativa L.) from the Wanshan Mercury Mining District, SW China[END_REF], tracking of pollution sources [START_REF] Perrot | Tracing sources and bioaccumulation of mercury in fish of Lake Baikal -Angara River using Hg isotopic composition[END_REF][START_REF] Perrot | Higher mass-independent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF], discrimination of human exposure pathways [START_REF] Laffont | Hg speciation and stable isotope signatures in human hair as a tracer for dietary and occupational exposure to mercury[END_REF][START_REF] Rothenberg | Stable Mercury Isotopes in Polished Rice (Oryza sativa L.) and Hair from Rice Consumers[END_REF][START_REF] Sherman | New insight into biomarkers of human mercury exposure using naturally occurring mercury stable isotopes[END_REF][START_REF] Li | Assessing sources of human methylmercury exposure using stable mercury isotopes[END_REF], and biogeochemical processes [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF][START_REF] Masbou | Hg Stable Isotope Time Trend in Ringed Seals Registers Decreasing Sea Ice Cover in the Alaskan Arctic[END_REF][START_REF] Point | Methylmercury photodegradation influenced by seaice cover in Arctic marine ecosystems[END_REF][START_REF] Renedo | Seabird Tissues As Efficient Biomonitoring Tools for Hg Isotopic Investigations: Implications of Using Blood and Feathers from Chicks and Adults[END_REF][START_REF] Renedo | Identification of sources and bioaccumulation pathways of MeHg in subantarctic penguins: A stable isotopic investigation[END_REF] has been demonstrated. The potential of the Hg isotopic pattern can be extended to food products.

Hg isotopic composition has been determined in rice and fish. Hg isotopic in fish is an excellent tool for tracking Hg (pollution) sources in aquatic ecosystems. Moreover, the isotopic pattern of Hg in this foodstuff is extremely useful for the determination of Hg human exposure. In general, an enrichment of heavier isotopes of approximately 2‰ (δ 202 Hg) is observed in hair as compared to the levels of these isotopes in consumed fish. Additionally, the preservation of mass independent fractionation signatures in human's hair and urine allows the discernment between occupational exposure (gold mining, dental professionals) and fish ingestion [START_REF] Laffont | Hg speciation and stable isotope signatures in human hair as a tracer for dietary and occupational exposure to mercury[END_REF][START_REF] Sherman | New insight into biomarkers of human mercury exposure using naturally occurring mercury stable isotopes[END_REF]. The clear differences between Oceanic and coastal fish Hg isotopic patterns provide the hints to successfully identify Oceanic and mixed seafood consumers by the analysis of their hair [START_REF] Li | Assessing sources of human methylmercury exposure using stable mercury isotopes[END_REF]. More recently, the characterization of Hg stable isotopes enabled the discernment of different populations of European seabass, Dicentrarchus labrax, what can be extrapolated to high resolution dietary discrimination in a local scale [START_REF] Cransveld | Mercury Stable Isotopes Discriminate Different Populations of European Seabass and Trace Potential Hg Sources around Europe[END_REF].

In the case of rice, it has provided, among others, crucial information about sources of Hg contamination. The fraction of atmospheric Hg in the plants follows the trend: leaf > stem > seeds > roots [START_REF] Yin | Stable Mercury Isotope Variation in Rice Plants (Oryza sativa L.) from the Wanshan Mercury Mining District, SW China[END_REF]. The combination of Hg speciation and isotopic composition revealed the dissimilar uptake and translocation pathways of Hg species from paddy soil to the edible part. The fact that seeds, containing higher MeHg proportion, exhibit significantly heavier Hg isotopes is understood as a facilitated MeHg uptake and transport to the seed in comparison to inorganic Hg species [START_REF] Feng | Investigation of Hg uptake and transport between paddy soil and rice seeds combining Hg isotopic composition and speciation[END_REF].

In general, most of Hg isotopic studies are so far restricted to elemental isotopic pattern determination. The major drawbacks of Hg isotopic analysis are: 1) the high cost of the multicollector instrument, being exclusive of few laboratories; 2) the reduced number of CRM for the validation of Hg elemental isotopic characterization and the absolute lack of such materials regarding the isotopic composition of Hg species and 3) the elevated concentrations usually required for a precise analysis.

The analysis is based on a complete digestion of the sample, usually by acid digestion, followed by isotopic determination through continuous CV-MC-ICP-MS. In order to diminish the problems associated with Hg isotopic characterization at ultra-trace levels, an on-line pre-concentration method was developed based on the hyphenation of a commercially available cold vapor generation dual gold amalgamation system to a multi-collector ICP-MS. The automated method supports a rapid throughput and consumes less than 100 mL of sample, representing a reduction of the sample volume of more than two orders of magnitude in comparison to existing off-line pre-concentration strategies. This method proved to be accurate and precise for the determination of total Hg isotopic composition of fish [START_REF] Bérail | Determination of total Hg isotopic composition at ultra-trace levels by on line cold vapor generation and dual gold-amalgamation coupled to MC-ICP-MS[END_REF].

The relatively recent coupling of GC to MC-ICP-MS [START_REF] Epov | Approach to measure isotopic ratios in species using multicollector-ICPMS coupled with chromatography[END_REF] constitutes a remarkable evolution on isotopic analysis since it allows the characterization of both Hg species, named compound-specific stable isotope analysis (CSIA) method. An automated on-line preconcentration system, which allows injections volumes up to 100 folds higher than in a conventional GC-MC-ICP-MS scheme has been introduced in order to be able to analyze samples with low Hg content. Preconcentration and transfer of Hg species (Hg(II) and MeHg) to the column was achieved with a programmed temperature vaporization injector. Hg species were efficiently preconcentrated and fish reference materials with concentrations down to 150 ng g -1 were successfully analyzed.

Another alternative to the isotopic characterization of MeHg is via a selective extraction method for MeHg, followed by continuous CV-MC-ICP-MS [START_REF] Masbou | Application of a selective extraction method for methylmercury compound specific stable isotope analysis (MeHg-CSIA) in biological materials[END_REF]. It has been applied to the analysis of rice samples. Briefly, 0.25-0.5 g were extracted with 5 mL of 30% w/w NaBr in 4 mol L -1 H2SO4, 10 mL of 2.5% w/w CuSO4 and 10 mL of toluene. The mixture was shaked for 1 h at 420 rpm, in order to transfer MeHgBr from the matrix to the toluene phase, followed by a back-extraction with aqueous sodium thiosulfate solution (0.005 mol L -1 ) to convert the MeHgBr into a stable MeHg-thiosulfate complex. Hg isotopic characterization of MeHg was then carried out by CV-MC-ICP-MS after dilution of the MeHg-thiosulfate solution with a 3:1 v/v mixture of HNO3 and HCl. Concerning the stable isotopic composition of Hg(II), it was determined by isotopic mass balance difference between total Hg and MeHg isotopic patterns. The method was validated with a fish CRM and applied to the analysis of rice and human hair samples in order to understand human exposure pathways [START_REF] Li | Mercury Isotope Signatures of Methylmercury in Rice Samples from the Wanshan Mercury Mining Area, China: Environmental Implications[END_REF].

A promising chromatographic methodology has recently been validated for Hg isotope ratio measurement of MeHg in fish tissues at environmentally relevant levels. It is based on the isocratic separation of Hg(II) and MeHg in a reversedphase HPLC column with a mobile phase containing L-Cys as complexing agent.

One of the main advantages of this approach is the non-requirement of derivatization steps. The MeHg fraction isolated from the chromatographic columns was completely oxidized, prior to CV-MC-ICP-MS, due to the challenges driven by the non-reactivity of MeHg with SnCl2 and, interferences caused by the presence of reduced sulfur in the mobile phase [START_REF] Entwisle | Hg isotope ratio measurements of methylmercury in fish tissues using HPLC with off line cold vapour generation MC-ICPMS[END_REF]. Considering that for the moment there are not commercially available CRMs for isotopic composition of Hg species, it is extremely valuable to have the possibility of contrasting the measurements obtained by different methods, such as GC and HPLC coupled to MC-ICP-MS.

CONCLUDING REMARKS

The urgency to develop sensitive and environmentally friendly methodologies promotes the advances in the analysis of Hg in food. Such development is greatly supported by the progress in nanomaterials that assist in the whole procedure comprising Hg (species) extraction, preconcentration, separation and detection. Hg isotopic fractionation emerges as a powerful tool on dietary studies, allowing the discernment among several Hg sources of exposure. In addition, the identification of pollution sources in food is also possible through the Hg isotopic signature. It would be extremely valuable on the development of strategies that alleviate the impact of Hg contamination sources in foodstuffs.

Seafood monopolizes the attention of the analytical community that validated a wide variety of analytical approaches for such matrix. However, the global consumption of rice, considered an important source of Hg, claims for adequate methodologies to this complex samples.

Hg analysis in food is motivated by the human risk associated to dietary exposure. Consumer's guidelines are based on the assumption that MeHg from food is absolutely absorbed after human ingestion. However, it seems to overestimate the real assimilation of MeHg, since the nature of the matrix, fish species, cooking processes and the co-ingestion of certain food components seems to play a relevant role on the potential human assimilation of ingested Hg [START_REF] Afonso | Evaluation of the risk/benefit associated to the consumption of raw and cooked farmed meagre based on the bioaccessibility of selenium, eicosapentaenoic acid and docosahexaenoic acid, total mercury, and methylmercury determined by an in vitro digestion model[END_REF][START_REF] Alves | Oral bioaccessibility of toxic and essential elements in raw and cooked commercial seafood species available in European markets[END_REF][START_REF] Bradley | A review of mercury bioavailability in humans and fish[END_REF][START_REF] Girard | Cooking and co-ingested polyphenols reduce in vitro methylmercury bioaccessibility from fish and may alter exposure in humans[END_REF][START_REF] Jadán Piedra | Reduction of mercury bioaccessibility using dietary strategies[END_REF][START_REF] Ouédraogo | Effects of various cooking methods and food components on bioaccessibility of mercury from fish[END_REF]. For example, cooking practices like, steaming, boiling, grill and frying have been evaluated [START_REF] Afonso | Evaluation of the risk/benefit associated to the consumption of raw and cooked farmed meagre based on the bioaccessibility of selenium, eicosapentaenoic acid and docosahexaenoic acid, total mercury, and methylmercury determined by an in vitro digestion model[END_REF][START_REF] Alves | Oral bioaccessibility of toxic and essential elements in raw and cooked commercial seafood species available in European markets[END_REF][START_REF] Ouédraogo | Effects of various cooking methods and food components on bioaccessibility of mercury from fish[END_REF][START_REF] He | Factors affecting the bioaccessibility of methylmercury in several marine fish species[END_REF], resulting in a reduction of the potential absorption at the intestinal epithelium level up to 90% [START_REF] Alves | Oral bioaccessibility of toxic and essential elements in raw and cooked commercial seafood species available in European markets[END_REF], The co-ingestion of polyphenol-rich beverages like tea and coffee or pure polyphenols are leading to a decrease on Hg bioaccessibility [START_REF] Girard | Cooking and co-ingested polyphenols reduce in vitro methylmercury bioaccessibility from fish and may alter exposure in humans[END_REF][START_REF] Ouédraogo | Effects of various cooking methods and food components on bioaccessibility of mercury from fish[END_REF][START_REF] He | Factors affecting the bioaccessibility of methylmercury in several marine fish species[END_REF].

The existing methods for Hg analysis in food focus on the quantification of Hg and its species based on the consumer guidelines, which set safe consumption thresholds. Nevertheless, some new risk assessment criteria are under development.

A new seafood safety index named Selenium health benefit value (HBVSe) is a good case in point, which is based on the observation that Se is an antagonist of Hg toxicity. It considers both, MeHg and Se concentrations in fish, and shows more reliable accuracy than predictions based exclusively on MeHg [START_REF] Ralston | Selenium Health Benefit Values: Updated Criteria for Mercury Risk Assessments[END_REF]. In summary, the future and perspectives of Hg analysis in food will be absolutely influenced by the trends and evolution of consumption guidelines.

CONFLICT OF INTEREST

None Declared

ACKNOWLEDGEMENT

The author thanks The Agence Nationale de la Recherche (MERSEL Project No. ANR-18-CE34-0004-01) for funding and R. Saz for English editing.

LIST OF ABBREVIATIONS

AAS

ACKNOWLEDGEMENT

The author thanks R. Saz for English editing.

Figure 1 :

 1 Figure 1: Chromatographic and non-chromatographic approaches for Hg speciation in food.

Table 1 :

 1 Application of magnetized nanomaterials for MSPE of Hg in food.

	Magnetic solid-	Analyte	Isolation	Detection	Elution solvent	Contact	Elution	pH	Nano-	Sorption	LOD (ng L -1 )	RSD	Cycles	Matrix	Ref.
	phase					time	time		sorbent	capacity		(%)
						(min)	(min)		amount	(mg g -1 )
									(mg)
	Fe3O4@SiO2@pol	Hg(II)	batch	CV-AFS	3.0 mL, 0.3 mol L -1	6.8	8.4	5.2	26	59	20	< 9.2	-	Shrimp, fish,	[61]
	ythiophene			(off-line)	thiourea in 0.34 mol									canned tuna
					L -1 HCl solution
	Fe3O4@IIP based	Hg(II)	batch	ICP-OES	5 mL solution 0.1	2	10	8	10	147	30	1.47		Fish	[57]
	on N-(pyridin-2-			(off-line)	mol L -1 HCl and 0.1
	ylmethyl)ethenam				mol L -1 EDTA
	ine (V-Pic)
	GO/Fe3O4@	Hg(II)	batch	FI-CV-	2.5 mL HCl 1.7 mol	21	2	6.5	20	1	25	<10	-	Seafood	[58]
	polythiophene			AAS	L -1
				(off-line)
	MNPs	Hg(II)	on	CV-	0.6 mL min -1 ,	2	1.7	5	50	5.22	7.8	1.7	At	Mussel, fish	[63]
	functionalized		column	ETAAS	thiourea 6%,								least
	with 1,5-bis(di-2-			(on-line)	NaBH4 0.002%,								550
	pyridyl)methylen				NaOH 0.5%
	e
	thiocarbohydrazi
	de
	graphene/ZnFe2O	Hg(II)	batch	CV-AAS	5 mL 2.0 mol L -1	3	3	6	100	4.1	10	2.7	50	Fish	[54]
	4			(off-line)	HCl
	Fe3O4@DTIM-	Hg(II)	batch	CV-AAS	3.5 mL 1.1 mol L -1	8	11	6	24	254	10	<8.3	12	Fish, canned	[59]
	HKUST-1(Cu)			(off-line)	thiourea									tuna
	bio-MSPE (C.	Hg(II)	on	ICP-OES	5 mL 1.0 mol L -1	-	1.7	5	100	26.2	40	-	35	Potato,	[62]
	micaceus		column	(off-line)	HCl									cabbage,
	immobilized with													ketchup,
	γ-Fe2O3)													green pepper,
														meat, fish,
														chicken, milk
	Fe3O4@SiO2@γ-	Hg(II),	batch	HPLC-	0.5 mL	15	2	4	5	29.8 (for	0.74 (for Hg(II)),	<10		Fish	[60]
	mercaptopropyltr	MeHg,		ICP-MS	0.1 mol L -1 HNO3					Hg(II)),	0.67 (for MeHg),
	imethoxysilane	PhHg		(off-line)	and 4% (m/v)					98.1 (for	0.49 (for PhHg)
					thiourea					MeHg)
	poly-L-	Hg(II),	on	HPLC-	10% HCl + 20%	-	10	9	3	-	15 (for Hg(II)),	<5	575	Fish oil based	[64]
	methionine-	MeHg	column	ICP-MS	methanol						17 (for MeHg)			dietary
	MWCNTs			(on-line)										supplements

Table 2 :

 2 Some recent examples of Hg speciation in food by methods based on HPLC separation.

	Chromatographic	Column	Mobile phase	Hg	Detection	Time	LOD (ng L -1 )	Samples	Ref.
	method			species	system	(min)
	RP	C8 (4.6 x	1.5 mmol L -1 ammonium	MeHg	CV-AFS	14	0.4 (MeHg)	Rice, baby-	[11]
		150 mm, 5	pyrrolidine dithiocarbamate					food rice
		µm)	in 75% (v/v) methanol
	Ion-pairing-RP	C18 (4.6 x	Gradient: (A) 5 mmol L -1	Hg(II),	ICP-MS	20	10 (EtHg),	Rice, rice	[72]
		150 mm, 5	TBAH, 10 mmol L -1	EtHg,			40 (Hg(II)),	flour
		µm)	NH4H2PO4; (B) 5% (v/v)	MeHg			30 (MeHg)	standard
			methanol, 0.1% (m/v) L-
			Cys, 0.06 mmol L -1
			CH3COONH4 (pH 7.1)
	Ion-pairing-RP	C18 guard	2.0 mmol L -1 SDBS + 2.0	Hg(II),	ICP-MS	3	15 (Hg(II)),	Fish	[71]
		columns (5	mmol L -1 Cys + 1.0 mmol	MeHg,			14 (MeHg),
		µm, 12.5 mm	L -1 Phe (pH 3.0); 4.0 mmol	EtHg,			28 (EtHg),
		x 2.1 mm	L -1 TBAH + 2.0 mmol L -1	PhHg			42 (PhHg)
		i.d.)	MPS + 2.0 mmol L -1 Phe
			(pH 6.0)
	Ion-pairing-RP	C18 (150	Gradient: (A) 100% (v/v)	Hg(II),	CV-AFS	12	11 (Hg(II)),	Fresh	[73]
		mm x 4.6	methanol (B) 0.4% (v/v)	MeHg,			50 (MeHg),	seafood,
		mm)	[C8mim]Cl; pH 2.0; 0.02	EtHg			60 (EtHg)	canned
			mol L -1 citric acid/citrate					fish, yeast,
			buffer; 0.1 mol L -1 NaCl					garlic
	Graphene oxide	GO@SiO2	2 mmol L -1 TSA and 5	Hg(II),	ICP-MS	12	16 (Hg(II)),	Fish	[75]
	column	column (500	mmol L -1 phenylalanine	MeHg,			27 (MeHg),
		mm x 4.6	(pH 8)	EtHg,			32 (EtHg),
		mm)		PhHg			68 (PhHg)

Table 3 :

 3 Some examples of nanomaterials used as efficient interfaces between liquid chromatography and CV-AFS detection for Hg speciation in foodstuffs.

	Hg	Matrix NPs (action)	Analytical	Mobile phase	LOD	Ref.
	species			technique		(µg L -1 )
	Hg(II),	Fish	TiO2	HPLC-AFS 1% methanol, 1%	0.15-0.35	[68]
	EtHg,		(photocatalysis)		formic acid, 5 mmol
	MeHg				L -1 Cys, 0.15 mol L -1
					NaClO4.
	Hg(II),	Fish,	TiO2 (UV/TiO2	HPLC-AFS acetonitrile and water	0.01-0.07	[70]
	EtHg,	oysters	photocatalysis		(65:35)
		and	reaction device)		containing 1.5 mmol
	PhHg	others			L -1 ammonium
		seafood			pyrrolidine
					dithiocarbamate
					(pH 5.5)
	Hg(II),	Fish,	ZrO2	HPLC-AFS 10% HCOOH, pH 3	0.013-	[69]
	EtHg,	oysters	(UV/ZrO2/HCOOH			0.024
	MeHg	and	photocatalytic
		others	reduction unit)
		seafood
	Hg(II),	Fish	Fe3O4 (on-line	HPLC-AFS acetonitrile and water	0.7-1.1	[79]
	MeHg,		advanced oxidation)		(10:90) at pH 6.8
	EtHg,				containing 0.12%
	PhHg				(m/v) L-Cys