The effects of selenium biofortification on mercury bioavailability and toxicity in the lettuce-slug food chain

The effects of selenium biofortification on mercury bioavailability and toxicity in the lettuce-slug food chain

Food and Chemical Toxicology xxx (xxxx) xxxx Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.c...

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Food and Chemical Toxicology xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

The effects of selenium biofortification on mercury bioavailability and toxicity in the lettuce-slug food chain Anja Kavčiča, Bojan Budičb, Katarina Vogel-Mikuša,c,∗ a

Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia National Institute of Chemistry, Hajdrihova 19, SI-1000, Ljubljana, Slovenia c Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia b

ABSTRACT

The effects of foliar Se biofortification (Se+) of the lettuce on the transfer and toxicity of Hg from soil contaminated with HgCl2 (H) and soil collected near the former Hg smelter in Idrija (I), to terrestrial food chain are explored, with Spanish slug as a primary consumer. Foliar application of Se significantly increased Se content in the lettuce, with no detected toxic effects. Mercury exerted toxic effects on plants, decreasing plant biomass, photochemical efficiency of the photosystem II (Fv/Fm) and the total chlorophyll content. Selenium biofortification (Se+ test group) had no effect on Hg bioaccumulation in plants. In slugs, different responses were observed in H and I groups; the I/Se+ subgroup was the most strongly affected by Hg toxicity, exhibiting lower biomass, feeding and growth rate and a higher hepatopancreas/ muscle Hg translocation, pointing to a higher Hg mobility in comparison to H group. Selenium increased Hg bioavailability for slugs, but with opposite physiological responses: alleviating stress in H/Se+ and inducing it in I/Se+ group, indicating different mechanisms of Hg-Se interactions in the food chain under HgCl2 and Idrija soil exposures that can be mainly attributed to different Hg speciation and ligand environment in the soil.

1. Introduction Selenium (Se) is an essential micronutrient for humans and animals, predominantly obtained by consumption of cereals, vegetables, meat and fish (Rayman, 2000). In certain countries Se malnutrition in humans relying mostly on vegetarian diet prevails due to low Se concentration and availability in soils and consequently in crop plants (White and Broadley, 2009). Since direct Se supplementation in diets is less recommendable for poor bioavailability of inorganic Se compounds and possible accidental excess Se intake, agronomic biofortification is considered more advantageous (Hartikainen, 2005). To date, foliar and soil application of Se fertilisers are feasible approaches to increase Se content in the edible parts of crop plants (White and Broadley, 2009) resulting in improved human and animal health (Alfthan et al., 2015). Selenium biofortification was successfully tested and applied in several crops, e.g. wheat (Golob et al., 2018; Riaz et al., 2018), Tartary and hybrid buckwheat (Golob et al., 2018), vegetables (El-Ramady et al., 2014; Germ et al., 2018; Slekovec and Goessler, 2005) and even herbs (Germ et al., 2009), resulting in increased Se contents and improved

nutritional value of the tested plants without significant decrease in crop yields. When added in low concentration (< 20 mg kg−1), Se exerts beneficial effects on plant growth through several mechanisms. Similarly as in humans and animals, Se strengthens the capacity of plants to counteract oxidative stress caused by oxygen radicals produced by internal metabolic or external factors. At proper levels it also delays some of the effects of senescence and may improve utilization of short-wavelength light by plants. In high addition, however, Se is toxic for plants and may trigger oxidative stress and lower crop yields (Hartikainen, 2005; Kolbert et al., 2016). Mercury is a widely dispersed pollutant persisting in the environment in different forms as Hg0, Hg+, Hg2+ and methylmercury (MeHg) (Moreno-Jiménez et al., 2006). Mercury mobility and bioaccessibility is generally low in terrestrial ecosystems, but strongly dependent on Hg speciation and ligand environment in soil. Studies performed in the vicinity of the biggest Hg mines (Almaden, Idrija, Asturias) show that for example in Idrija, Hg is mainly present in the form of HgS (cinnabar) and HgSO4 (Esbrí et al., 2010), while fractionation studies show also the presence of Hg0 (Kocman et al., 2004). The overall Hg mobility as

Abbreviations: C, control potting substrate mixture; H, HgCl2-spiked potting substrate mixture; I, soil collected at Hg contaminated site in Idrija; Se-, without Se; Se +, biofortified with Se; HP, hepatopancreas; M, muscle tissues; MeHg, methylmercury ∗ Corresponding author. Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia. E-mail addresses: [email protected] (A. Kavčič), [email protected] (B. Budič), [email protected] (K. Vogel-Mikuš). https://doi.org/10.1016/j.fct.2019.110939 Received 29 June 2019; Received in revised form 26 October 2019; Accepted 1 November 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Anja Kavčič, Bojan Budič and Katarina Vogel-Mikuš, Food and Chemical Toxicology, https://doi.org/10.1016/j.fct.2019.110939

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measured by extraction with 0.5M HCl is, however, very low (up to 0.2 mg L−1) (Esbrí et al., 2010), as confirmed also by a low total Hg bioconcentration factor (the ratio between plant and soil Hg concentration) ranging from 0.4 to 6% (Gnamuš et al., 2000). On the other hand, in Almaden and Asturias, Hg mobility is much higher and associated with the presence of HgCl2 and Hg2+ (Esbrí et al., 2010). Mercury bioavailability in the soil is also strongly connected to the amount of MeHg, the most mobile and toxic Hg species in biological systems (Kim and Zoh, 2012). In Idrija it was shown that MeHg formation is related to decomposition of organic matter, with higher amounts (up to 5% of the total Hg) found in organic-matter rich soils (Gnamuš, 2002; Gnamuš et al., 2000; Tomiyasu et al., 2017). The total bioaccessibility (gastric and intestinal) of Hg in different vegetables collected from farmland in close proximity to compact fluorescent lamp factories (Zhejiang Province, PR China), where Hg can be found mainly in HgCl2 form, ranged from 34.4% for Chinese mustard greens - xuelihong to 54.6% for lettuce (Shao et al., 2012), contributing to elevated daily uptake of Hg in population consuming the contaminated vegetables. Since Hg is toxic already at low concentration and it is difficult to avoid consumption of vegetables and crops produced in Hg polluted environments, alternative ways need to be found to decrease Hg exposure through ingestion and to alleviate Hg toxicity. According to research on mammals, Se may protect against stress induced by Hg and other metals (Boening, 2000; Byrne et al., 1995; Spiller, 2018) through improved antioxidant activity (Zwolak and Zaporowska, 2012) and through binding Hg into insoluble HgSe complexes (Thangavel et al., 1999; Wang et al., 2013; Zhang et al., 2014; Zhao et al., 2004). In plants Hg-Se interactions have been studied by several authors (Tang et al., 2017; Wang et al., 2016; Xu et al., 2019; Zhang et al., 2012; Zhao et al., 2014). Wang et al. (2016) have shown that Se added into the soil inhibits the uptake of MeHg as well as inorganic Hg into rice grains, while foliar Se application has no effect on MeHg plant uptake, concluding that MeHg-Se interactions in soil rather than within the plant might be the key process triggering the decrease in grain Hg levels under Se amendment. Lettuce (Lactuca sativa L.), an annual plant of the Asteraceae family, is widely consumed around the world, and its production exceeded 26 million tons in 2017 (FAO, 2019). Since lettuce is an important part of our everyday diet, Se biofortification of lettuce could help to fight Se malnutrition in Se deficient areas (Smoleń et al., 2014). The lettuce is also known to accumulate relatively large amounts of metals, especially Cd (Baldantoni et al., 2015), As, Ni, Pb, Zn (Boshoff et al., 2015) and also Hg (Miklavčič et al., 2013), thus presenting an important source of hazardous metal dietary intake. In order to quantify the risk of hazardous metal consumption by higher organisms it is necessary to study behaviour of hazardous metals in the food chain and track physiological responses of the test organisms (Boshoff et al., 2015). Our study therefore aimed to explore the effects of foliar Se-biofortification of the lettuce plants, on the transfer and toxicity of Hg from soil contaminated with HgCl2 and soil collected at Hg polluted site in Idrija (the past second largest Hg mine in the world) to terrestrial food chain, with Spanish slugs as primary consumers. Like other invertebrates, slugs accumulate noxious elements and chemicals in their digestive gland (hepatopancreas, HP), occupying the role of appropriate sentinels of environmental pollution (Boshoff et al., 2015; Kavčič et al., 2019; Pauget et al., 2013). In addition, the elements that are mobile within the organisms can be transported and accumulated in the muscle tissues; therefore the muscle to hepatopancreas element concentration ratio can be taken as a measure of element within-organism mobility. Although Spanish slug is listed among the 100 worst invasive species in Europe, it is an important part of food webs, common in diet of various birds and mammals. In addition, experiments with slugs pose no ethical issues, are simple to perform and provide reliable and highly reproducible results (Pauget et al., 2013). While in several metal bioavailability and toxicity studies involving test organisms like terrestrial isopods or snails, the substances under

study, e.g. elements, nanoparticles or other chemicals are directly applied to the plant surface, excluding chemical modification by plant metabolic pathways (Valant et al., 2012), our study undertakes a more realistic approach, similar as in Kavčič et al., (2019), where wild growing mushrooms accumulating Hg and Se were fed to the Spanish slugs to evaluate Hg/Se bioavailability and toxicity. A linear relationship was observed between Hg and Se concentration in food and slug hepatopancreas, while the transport and mobility from HP to the muscle tissues depended on Hg ligand environment in food and further in HP. Complexation of Hg with Se in food or HP restricted Hg accumulation in muscle tissues. In the present study, the plants were grown under Hg exposures and biofortified with Se for six weeks, to absorb and metabolize both elements before being fed to the primary consumers under controlled conditions. The advantage of this system in evaluating metal bioavailability/toxicity from soil to the food chain over chemical extraction procedures and use of single organisms (plants or invertebrates) is that it considers metabolic processes that may alter Hg and Se speciation and ligand environment in the primary producer (plant), consequentially affecting Hg bioavailability and physiological response of the consumer (slug). Based on previous studies (Wang et al., 2016; Zidar et al., 2016) the working hypothesis was a protective role of Se against Hg-induced stress in lettuce and further in slugs. 2. Materials and methods 2.1. Experimental design The experimental set-up consisted of six groups of lettuce plants: two groups were grown in the control potting substrate mixture without and with added Se (C/Se-, C/Se+) and two in the potting substrate mixture spiked with 100 mg kg−1 of HgCl2 (added to the substrate in solution), without and with added Se (H/Se-, H/Se+). The remaining two groups were grown on the soil collected at Hg-contaminated site in Idrija near the chimney of the former Hg smelter (46°00′33.7"N 14°02′02.9"E), (Tomiyasu et al., 2017), again with and without added Se (I/Se-, I/Se+). The basis for the C and H groups was prepared as a mixture of rendzina soil collected near Ljubljana and a standard potting substrate (Cvetal, Agroruše) (2:1 w/w) to match as closely as possible the composition of the soil collected in Idrija. Soil properties are listed in Table 1. Selenium levels were below 0.01 mg kg−1 in potting substrate mixture and soil of all exposure groups. Eight weeks old lettuce (Lactuca sativa L. cv. Exquise) seedlings were Table 1 The properties of the potting substrate mixture and soil used in the experiments with lettuce (EC-conductivity, WHC-water holding capacity). C, control potting substrate mixture; H, HgCl2 spiked potting substrate mixture; I, soil collected in Idrija. Values are averages ± SE, n = 5. C Hg [mg kg−1] pH Soil type Organic matter [%] WHC [%] EC [μS cm-1] Si [mg kg−1] P [mg kg−1] S [mg kg−1] Cl [mg kg−1] K [mg kg−1] Ca [mg kg−1] Mn [mg kg−1] Fe [mg kg−1] Zn [mg kg−1] Rb [mg kg−1] Sr [mg kg−1]

2

H

< 0.001 99.0 ± 1.40 5.6 ± 0.2 6.2 ± 0.2 Rendzina, silty loam 10 ± 2 54 ± 5 240 ± 30 29800 ± 1500 2500 ± 170 380 ± 10 2100 ± 160 7220 ± 200 7880 ± 950 235 ± 1.5 10800 ± 1300 79.4 ± 1.4 55.4 ± 5.2 68.6 ± 8.9

I 101.0 ± 2.00 6.0 ± 0.2 Rendzina, silty loam 9 ± 0.4 43 ± 4 470 ± 50 59100 ± 2700 2330 ± 200 492 ± 47.6 1900 ± 110 9720 ± 290 9900 ± 290 413 ± 49.3 19500 ± 1970 55.2 ± 3.8 80.4 ± 1.5 72.0 ± 2.1

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Table 2 The lettuce traits separately for the roots, shoots and whole plant where applicable: fresh root and shoot weight (FW), dry root and shoot weight (DW), photochemical efficiency of the photosystem II (Fv/Fm), the total chlorophyll concentration (TChl), Hg, Se, Ca and K root and shoot concentrations and contents, Hg bio-concentration factor (BCF). Values are averages ± SE, n = 5. Different letters next to the values represent statistically significant differences (Duncan's post-hoc test, p < 0.05). C, control potting substrate mixture; H, HgCl2 spiked potting substrate mixture; I, soil collected in Idrija; Se- without Se; Se+ with Se. < LOD – below detection limit, nd-non-determined. Parameter\ Exposure

organ

C/Se-

C/Se+

H/Se-

H/Se+

I/Se-

I/Se+

FW [g]

root shoot root shoot plant shoot shoot root shoot plant root shoot plant root shoot root shoot root shoot root shoot root shoot

8.67 ± 0.89 ab 27.18 ± 3.16 a 1.48 ± 0.16 a 3.08 ± 0.41a 4.56 ± 0.52 a 0.83 ± 0.002 a 6.42 ± 0.95 a < LOD < LOD < LOD nd nd nd nd nd < LOD d 0.18 ± 0.04 c < LOD 0.51 ± 0.06 b 4520 ± 1120 b 7510 ± 1100 b 5890 ± 1800 12550 ± 3140

8.94 ± 0.92 a 19.63 ± 2.83 abc 1.40 ± 0.14 a 2.78 ± 0.47 ab 4.18 ± 0.58 a 0.83 ± 0.003 a 5.30 ± 0.74 ab < LOD < LOD < LOD nd nd nd nd nd 0.98 ± 0.04 b 9.57 ± 0.35 b 1.37 ± 0.16 a 26.8 ± 4.54 a 3800 ± 1150 b 13520 ± 4400 b 7220 ± 2300 13680 ± 6100

3.45 ± 0.74 d 19.67 ± 3.60 abc 0.41 ± 0.13 c 1.51 ± 0.38 b 1.91 ± 0.51 b 0.814 ± 0.008 ab 6.73 ± 1.88 a 19.8 ± 4.56 a 1.12 ± 0.28 b 4.65 ± 0.80 ab 6.27 ± 1.45 a 1.42 ± 0.21 b 7.69 ± 1.38 a 0.200 ± 0.046 a 0.011 ± 0.003 b 0.03 ± 0.03 d 0.66 ± 0.15 c 0.02 ± 0.02 c 0.79 ± 0.12b 14980 ± 6380 a 18500 ± 3200 a 17570 ± 4400 23700 ± 7800

6.37 ± 0.27 bc 23.04 ± 2.37 ab 0.81 ± 0.10 b 2.18 ± 0.32 ab 3.00 ± 0.40 ab 0.826 ± 0.002 a 5.85 ± 1.19 ab 8.67 ± 1.60 b 1.20 ± 0.11 b 3.21 ± 0.46 a 6.96 ± 1.25 a 2.71 ± 0.56 b 9.67 ± 1.71 ab 0.088 ± 0.016 b 0.012 ± 0.001 b 0.86 ± 0.12 c 17.08 ± 3.28 a 0.72 ± 0.15 b 39.4 ± 10.8 a 8880 ± 3230 ab 15630 ± 1100 ab 15770 ± 1400 24400 ± 2900

4.54 ± 0.89 cd 12.20 ± 3.07 c 0.75 ± 0.16 b 1.72 ± 0.47 b 2.48 ± 0.62 b 0.816 ± 0.008 b 2.61 ± 0.58 b 0.80 ± 0.27 c 3.33 ± 1.03 a 2.42 ± 0.62 ab 0.52 ± 0.15 b 5.02 ± 2.10 a 5.53 ± 2.06 b 0.008 ± 0.003 c 0.033 ± 0.01 a 0.11 ± 0.05 d 0.13 ± 0.03 c 0.11 ± 0.06 c 0.25 ± 0.10 b 2950 ± 300 b 8300 ± 1100 b 7650 ± 1900 18070 ± 1400

4.76 ± 0.99 cd 15.08 ± 2.98 bc 0.65 ± 0.13 b 1.74 ± 0.41 b 2.39 ± 0.52 b 0.805 ± 0.012 b 4.52 ± 1.18 ab 1.78 ± 0.25 bc 1.90 ± 0.26 ab 1.87 ± 0.22 b 1.08 ± 0.17 b 3.18 ± 0.91 ab 4.25 ± 0.94 b 0.018 ± 0.002 bc 0.019 ± 0.003 ab 1.54 ± 0.33 a 15.14 ± 0.75 a 1.11 ± 0.43 ab 25.9 ± 5.65 a 4050 ± 170 b 8190 ± 2400 b 17380 ± 7900 21230 ± 6400

DW [g] Fv/Fm TChl [mg/g DW] Hg [μg g−1 DW] Hg [μg organ−1] Hg BCF Se [μg g−1 DW] Se [μg organ−1] Ca [μg g−1 DW] K [μg g−1 DW]

obtained from a gardening company (Vrtnarstvo Škofic, Kranj) and planted in pots (5 per exposure) filled with 0.4 kg of pre-prepared control potting substrate mixture (C); HgCl2 spiked potting substrate mixture (H) and soil collected in Idrija. The substrate around the plant was covered with a black plastic bag to prevent Hg volatilisation. Lettuce was grown in growth chambers for six weeks with a 16/8h day/ night photoperiod, cool white fluorescent illumination of 550 μmol m−2 s−1, a constant temperature of 20 °C, and 50% humidity. The lettuce was regularly watered with tap water and once per week with half-strength Hoagland's nutrient solution (50 ml/pot) (Hoagland and Arnon, 1938) to maintain adequate mineral nutrition. After three weeks of growth, the lettuce in Se+ groups was foliarly sprayed twice per week with Se solution (5 μM potassium selenate K2SeO4) with a total amount of 25 ± 5 μg Se per plant. The slug experiment was performed according to Kavčič et al., (2019). Spanish slugs (Arion vulgaris L.) were collected in the natural environment near Ljubljana and placed individually into plastic containers (5 per exposure), filled with a mixture of gypsum and charcoal (20:1), covered with a filter paper, maintaining moisture in the container at 80–100%. Pellets (~100 mg) prepared from plants of experimental groups (C/Se-, C/Se+, H/Se-, H/Se+, I/Se- and I/Se+) were offered to the slugs in small petri dishes to prevent soaking and degradation. Fresh pellets were supplied daily and the amount of consumed feed per 24h was recorded. The animals were fed for a period of 14 days, while kept under the following conditions: temperature 19–20 °C, 50–55% humidity and 16/8 day/night photoperiod. The plastic containers were cleaned every day under tap and distilled water. At the end of the feeding period the slugs were placed in clean containers and let fasten for 24 h before dissection. Digestive glands (hepatopancreas - HP) and muscle tissues (M) were stored for analysis.

30 min ramp to 180 °C, 30 min hold at 180 °C, 30 min cooling, 1600 W) of 0.03–0.1g aliquots in 65% nitric acid (Sigma Aldrich). After digestion, the samples were cooled in water bath to prevent evaporation of Hg. Digests were stabilised by HCl and diluted with ultrapure water (Debeljak et al., 2013). Element concentrations in soil and calcium (Ca) and potassium (K) concentration in plants were measured by X-ray fluorescence spectrometry (Nečemer et al., 2008). For XRF analysis a PeduzoT02 (IJS, Slovenia) system equipped with Rh anode and silicon drift diode detector (Amptek) was used. ICP-MS and XRF analyses were validated using standard reference materials (1573a Tomato leaves, NIST and ERMCE464 Tuna fish, Sigma-Aldrich). 2.2.2. Soil analyses Soil samples were dried at room temperature until constant weight. Dried soil was homogenised using mortar and pestle and sieved through a 1 mm sieve. In Table 1, the values of organic matter and pH (Öhlinger et al., 1996), electrochemical conductivity (WTW Multi 340i with TetraCon 325), water capacity (Noggle and Wynd, 1941) and the total element concentrations measured by XRF are given. 2.2.3. Plant analyses At the end of the experiment the photochemical efficiency of the photosystem II in dark adapted state (Fv/Fm) was determined as plant stress indicator (Krause, 1991) by Os-500 modulated fluorometer (OptiSciences), and the shoot height was measured. The plants were harvested, washed with tap and distilled water and separated into roots and shoots. The separate organs were weighed, frozen in liquid nitrogen and freeze-dried for three days (0.001 mbar, −95 °C, ScanVac, LaboGene, Allerød). Dry roots and shoots were pulverized by a mortar and pestle with addition of liquid nitrogen for ICP-MS and XRF analysis. Photosynthetic pigment concentration (the total chlorophylls) were determined in the pulverized lettuce leaves (Lichtenthaler and Buschmann, 2005; Monni et al., 2001). Some of the pulverized plant material was pressed into pellets for slug feed. Since Hg accumulation in the shoots was very low, the six feed mixtures were prepared from roots and shoots in w/w ratio 1:1, with Hg and Se concentrations in the feed presented in Table 3.

2.2. Soil, plant and animal analyses 2.2.1. Element analyses The total Hg and Se concentrations were determined in the plant and animal material by ICP-MS (Agilent 7500ce, Agilent Technologies, Palo Alto, CA) after microwave assisted digestion (MarsXpress, CEM, 3

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Table 3 Mercury and Se concentrations in feed, slug's mortality, feeding rate, growth rate and traits of slug's tissues (hepatopancreas, HP and muscle tissue, M): fresh weight (FW), dry weight (DW), Hg concentration and content, M/HP concentration ratio, Hg bio-concentration factor (BCF), level of lipid peroxidation (MDA), and Se concentration and content. The values are averages ± SE, n = 5. Different letters next to the values represent statistically significant differences (Duncan's post-hoc test, p < 0.05). C, control potting substrate mixture; H, HgCl2 spiked potting substrate mixture; I, soil collected in Idrija; Se-, without Se; Se+, with Se. < LOD – below detection limit, Nd-non-determined. Slug traits\ Exposure Hg in feed [μg g−1 DW] Se in feed [μg g−1 DW] mortality (sum = 60) Feeding rate [mg food/g slug/day] Growth rate [% of FW/day] FW [g] DW [g] Hg [μg g−1 DW] Hg [g organ−1] M/HP conc. ratio Hg BCF MDA [nmol g−1 FW] Se [μg g−1 DW] Se [μg organ−1]

organ

HP M HP M HP M HP M HP M HP M HP M HP M

C/Se-

C/Se+

H/Se-

H/Se+

I/Se-

I/Se+

< 0.001 0.09 ± 0.02 b 1 (2.5%) 7.61 ± v0.39 abc −0.15 ± 0.26 1.17 ± 0.10 a 4.24 ± 0.28a 0.344 ± 0.037 a 0.855 ± 0.071 a < LOD < LOD nd nd nd nd nd 7.52 ± 1.59 d 3.63 ± 0.78 ab 0.14 ± 0.04 d 0.12 ± 0.02 c 0.05 ± 0.02 c 0.11 ± 0.02 d

< 0.001 5.27 ± 0.17 a 1 (2.5%) 9.35 ± 0.36 a 0.05 ± 0.19 1.00 ± 0.09 ab 3.49 ± 0.25 ab 0.250 ± 0.032 ab 0.690 ± 0.058 a < LOD < LOD nd nd nd nd nd 15.69 ± 2.92 c 3.58 ± 0.67 ab 1.21 ± 0.11 b 0.49 ± 0.05 b 0.29 ± 0.03 b 0.33 ± 0.03 b

10.4 ± 2.31 a 0.34 ± 0.07 b 1 (2.5%) 8.64 ± 0.63 ab 0.14 ± 0.18 1.03 ± 0.08 ab 3.38 ± 0.42 ab 0.266 ± 0.026 ab 0.792 ± 0.074 a 1.37 ± 0.069 a 0.027 ± 0.018 bc 0.365 ± 0.038 a 0.020 ± 0.014 ab 0.018 ± 0.011 c 0.131 ± 0.007 b 0.003 ± 0.002 c 7.45 ± 0.48 d 5.58 ± 2.11 a 0.28 ± 0.08 cd 0.13 ± 0.04 c 0.07 ± 0.02 c 0.07 ± 0.03 d

4.94 ± 0.85 b 8.97 ± 1.65 a 0 7.87 ± 0.24 ab 0.05 ± 0.12 1.07 ± 0.06 ab 3.79 ± 0.17 ab 0.285 ± 0.017 ab 0.760 ± 0.034 a 0.951 ± 0.039 b 0.007 ± 0.001 c 0.269 ± 0.016 b 0.005 ± 0.001 b 0.008 ± 0.001 c 0.193 ± 0.008 a 0.001 ± 0.0002 c 6.07 ± 1.53 d 1.94 ± 0.51 b 0.81 ± 0.04 bc 0.33 ± 0.03 b 0.21 ± 0.03 b 0.25 ± 0.02 bc

2.07 ± 0.51 b 0.12 ± 0.03 b 0 7.21 ± 0.8 bc −0.77 ± 1.08 0.76 ± 0.08 b 2.75 ± 0.75 b 0.190 ± 0.016 b 0.358 ± 0.030 b 0.431 ± 0.050 c 0.090 ± 0.027 ab 0.081 ± 0.005 c 0.033 ± 0.012 ab 0.209 ± 0.06 b 0.208 ± 0.024 a 0.044 ± 0.013 b 17.28 ± 1.66 b 2.35 ± 0.45 ab 0.72 ± 0.12 bcd 0.54 ± 0.01 b 0.13 ± 0.01 c 0.19 ± 0.02 cd

1.84 ± 0.20 b 8.34 ± 0.47 a 0 5.91 ± 0.86 c −0.65 ± 0.19 0.83 ± 0.10 ab 3.54 ± 0.69 ab 0.214 ± 0.025 b 0.424 ± 0.067 b 0.427 ± 0.061 c 0.147 ± 0.052 a 0.089 ± 0.004 c 0.063 ± 0.022 a 0.359 ± 0.117 a 0.232 ± 0.033 a 0.08 ± 0.028 a 23.71 ± 0.63 a 3.05 ± 0.14 ab 6.9 ± 1.05 a 2.38 ± 0.47 a 1.43 ± 0.08 a 0.95 ± 0.07 a

2.2.4. Animal analyses Animal mortality was monitored on a daily basis and animal feeding rate was determined as the amount of the feed eaten per day (Table 3). At the end of the feeding experiment the slugs were dissected. Fresh HP and M biomass was determined. Aliquots of 80 mg, to be extracted with 2 mL of 80% ethanol (Hodges et al., 1999), were taken for malondialdehyde (MDA) analysis as a measurement of lipid peroxidation and membrane damage, and the rest of the material was frozen in liquid nitrogen and freeze-dried as described above. After microwave assisted digestion, as described above, Hg and Se in HP and M were determined by ICP-MS.

multiplied by 400 g of soil per pot), Splant is Hg content in the plant organs (Hg concertation in the organ [μg g−1 DW ] multiplied by organ biomass), Sfeed is Hg content [μg] in consumed feed (Hg concentration in a mixture of lettuce roots and shoots [μg g−1 DW ] multiplied by the amount of consumed feed [g]) and Sslug is Hg content [μg] in the slug HP or M [μg] (Hg concentration [μg g−1 DW ] in HP or M multiplied by dry biomass of HP or M [g]). 2.2.6. Statistical analysis In a one-way ANOVA test the effects of different exposures were compared followed by Duncan's post hoc test (Statistica Statsoft 7.0). Differences at p < 0.05 were considered significant. Two way clustering analysis based on Euclidian distances with a heat chart of “z transformed” parameters measured in the lettuce and the slugs was performed in the “R” project for statistical computing (i386 3.4.3) (Singh et al., 2014).

2.2.5. Bioaccumulation and bioavailability Metal bioaccumulation in an organism or tissue can be quantified by bioconcentration factor (BCF) (Table 3), calculated as the ratio between the metal concentration in the organism and its environment (soil or feed) (de Vries et al., 2007):

BCFplant [%] = BCFslug [%] =

Cplant *100 Csoil

3. Results

(Eq. 1)

Cslug *100 Cfeed

3.1. Plants

(Eq. 2)

A reduction in plant biomass production is one the most reliable indicators of plant stress. In our experiment a lower root and shoot dry biomass was observed in Hg exposed plants in comparison to the controls (Table 2). Selenium biofortification had no effect on the root and shoot biomass production of the C/Se+ and I/Se+ plants. There was, however, improved root biomass production in H/Se+ plants (Table 2). Exposure to HgCl2 induced bolting of the lettuce plants as evident from the increased plant heights in the H/Se- and H/Se+ exposures (Fig. 1a), while in the remaining exposures the plants retained rosettes till the end of experiment (Fig. 1a and b). Selenium biofortification had no effect on the plant height and no other visual toxicity symptoms such as chlorosis or necrosis were observed. Foliar spraying of the lettuce leaves with Se (~25 μg per plant) resulted in significantly increased Se concentration in the shoots (Table 2). Selenium was also transported from the shoots to the roots, significantly increasing the root concentration of Se+ plants. Selenium concentration in the shoots seemingly increased with Hg exposure.

−1

where Csoil is Hg concentration [μg g DW] in the soil, Cplant is Hg concentration [μg g−1 DW] in the plant (roots or shoots), Cfeed is Hg concentration [μg g−1 DW] in the slugs feed and Cslug is Hg concentration [μg g−1 DW] in the slug (HP or M). BCF could be an inadequate indicator of metal uptake since it does not take into account the growth of an organism (concentration or dilution of pollutant) and the feeding rate of the animal. For this reason, bioaccumulation index (BI) (Fig. 4) is calculated according to the equation Eq. (3) for plants and Eq. (4) for slugs:

BIplant [%] =

BIslug [%] =

Splant *100 Ssoil

(Eq. 3)

Sslug *100 Sfeed

(Eq. 4) −1

where Ssoil is Hg content [μg] in soil (Hg soil concentration [μg g

DW] 4

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Fig. 2. a) The relationship between Hg concentration in the shoots and photochemical efficiency of the photosystem II (Fv/Fm) and the total chlorophyll contents (TChl) and b) the relationship between Hg and Ca concentrations in the roots. Values are averages ± SE, n = 5.

Interestingly, the highest Hg levels in the shoots were measured in I/Seand I/Se+ plants (Table 2). Selenium biofortification had no effect on shoot Hg concentration or content and neither on the whole-plant Hg level (Table 2). The photochemical efficiency of the photosystem II (Fv/Fm ratio) and the total chlorophyll (TChl) content decreased with increasing Hg concentrations in the lettuce shoots (Table 2, Fig. 2a). The lowest values of both were found in I/Se- and I/Se+ plants with the highest Hg shoot levels (Fig. 2a, Table 2). Selenium biofortification had no effect on the TChl and Fv/Fm values. The levels of macronutrients K and Ca were also measured in the roots and shoots as indicators of the membrane stability (van Doorn and Woltering, 2008). There were no effects of Hg exposure or Se biofortification on K level (Table 2), while Ca concentration increased with increasing Hg concentration in roots (Fig. 2b). Selenium biofortification decreased Ca accumulation in roots of H/Se+ plants, in line with lower root Hg concentration (Table 2).

Fig. 1. a) Plant's height and b) visual appearance of the lettuce at different exposures: C, non-contaminated substrate; H, artificially HgCl2 spiked substrate; I, soil collected at Hg contaminated site in Idrija; Se-, without Se; Se+, biofortified with Se. The values are averages ± SE, n = 5; different letters above the columns represent statistically significant differences (Duncan's posthoc test, p < 0.05).

3.2. Animals In total, three slugs out of 60 died during the 2 weeks of exposure (Table 3). The highest dry and fresh weight of hepatopancreas (HP) and muscle tissues (M) was recorded in the C/Se-slugs and the lowest in I/Seand I/Se+ slugs. These results are in line with the slug growth rate, which was also the lowest in I/Se- and I/Se+ slugs. Feeding rate was the highest in C/Se+ slugs and the lowest in I/Se+ slugs (Table 3). Selenium biofortification increased Se concentration in slugs HP and M (Table 3), with preferential storage of Se in HP. The highest Se concentrations were measured in I/Se+ slugs due to the lower slug biomass compared to C and H slugs.

When the shoot biomass was considered, it became apparent that this effect should mainly be attributed to the decrease in shoot biomass of Hg exposed plants (Table 2). The level of Hg in the controls was < 0.001 μg g−1. In Hg exposed plants, Hg mainly accumulated in the roots (Table 2). In roots of H/Se + plants, significantly lower Hg concentration was measured than in H/Se-plants (Table 2), but no effect of Se biofortification showed in root Hg contents (Table 2), due to the higher root biomass of H/Se+ plants. 5

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Fig. 4. Bioavailability of Hg for the roots, shoots and whole plants, and slugs HP and M calculated as Hg bioaccumulation index (BI) at different exposures: H, artificially HgCl2 spiked substrate; I, soil collected at Hg contaminated site in Idrija; Se-, without Se; Se+, biofortified with Se. The values are averages ± se, n = 5; different letters above the columns represent statistically significant differences (Duncan's post-hoc test, p < 0.05). Hg BI was not calculated for C/ Se- and C/Se+ exposures.

observed in I/Se- and I/Se+ slugs. Selenium biofortification decreased the Hg concentration in M of H/Se+ slugs, but not in I/Se+ slugs. In H slugs a significantly lower HP/M Hg concentration ratio was observed (Table 3) than in I slugs. The ratio significantly increased in I/ Se+ slugs, pointing to a higher transfer rate of Hg from HP to M in the presence of Se. The MDA level measured as indicator of the lipid peroxidation and membrane damage in HP was the highest in I/Se- and I/Se+ slugs (Table 3), in spite of the lowest Hg concentrations. MDA level increased in C/See + and I/Se+ exposures in comparison C/Se- and I/Se-. The MDA levels measured in M were the highest in H/Se- slugs, and the lowest in H/Se+ slugs. No correlation was observed between MDA and HP Hg concentrations, but MDA correlated rather with HP Se concentration (Fig. 3b).

Fig. 3. a) Two dimensional hierarchical clustering based on Euclidian distanced of z-transformed averages of plant traits at different exposures: C, non-contaminated substrate; H, artificially HgCl2 spiked substrate; I, soil collected at Hg contaminated site in Idrija; Se, biofortified with Se. Sh, shoots; R, roots; P, plant; H, height; FW, fresh weight; DW, dry weight; TChl, the total chlorophylls. b) Two dimensional hierarchical clustering based on Euclidian distanced of ztransformed averages of slug traits at different exposures: C, non-contaminated substrate; H, artificially HgCl2 spiked substrate; I, soil collected at Hg contaminated site in Idrija; Se, biofortified with Se. H, hepatopancreas; M, muscle tissue; FW, fresh weight; DW, dry weight; GR, growth rate; FR, feeding rate; MDA, malondialdehyde levels.

3.3. Mercury bioavailability for plants and slugs Mercury bioaccumulation determined as BCF significantly decreased in H/Se+ roots in comparison to H/Se-, while there was no difference between I/Se- and I/Se+ groups, although the BCFs were lower than for H plants (Table 2). No effect of Se biofortification was seen for the shoot BCF, although lower levels were calculated for H plants. The slug HP BCFs increased in H/Se+ group in comparison to H/ Se-, while no effect of Se biofortification was observed in I group (Table 3). The highest M BCF was, however, calculated for I/Se+ slugs, with significantly lower values found in I/Se- and H slugs. Overall mercury bioavailability for plants as assessed by

Mercury accumulated mainly in slug HP, with an order of magnitude lower level in M (Table 3). The highest Hg concentration was found in HP of H/Se- slugs and significantly lower in H/Se+ slugs followed by I/Se- and I/Se+ slugs, reflecting Hg concentration in the feed. Interestingly, for muscle tissues the highest Hg concentration was 6

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bioaccumulation index BI (Eq. (3)) was very low (Fig. 4a). The values for H/Se- and H/Se+ roots were higher than those of I/Se- and I/Se + plants, while the corresponding values for the shoots were more or less comparable. Selenium biofortification did not affect Hg bioavailability at the root, shoot or at the whole plant level. For the slugs significantly higher HP BI were found in exposures with added Se (Fig. 4b). For M BI, Se showed no effect in H exposures, but it significantly increased Hg bioavailability in I/Se+ exposure.

within-plant mobility may be governed by Hg speciation and ligand environment in soil. According to the studies performed in Idrija region, soil from Idrija may also contain MeHg, adding to the already complex Hg uptake and translocation mechanisms in the plants grown in Idrija soil. The sampling site of the Idrija soil, is located near the chimney of the former Hg smelter (Tomiyasu et al., 2017), containing significant amounts of MeHg (up to 200 μg kg−1), easily produced from atmospherically deposited Hg (Tomiyasu et al., 2017, 2012). In comparison to inorganic Hg forms, MeHg is highly mobile and toxic and may be easily transported from the roots to the leaves and further to the seeds (Feng et al., 2016; Wang et al., 2016). Vegetables sampled in Idrija (e.g. chicory) contained up to 12.7 mg kg−1 of the total Hg with up to 0.6 mg kg−1 (5%) of MeHg (Miklavčič et al., 2013), additionally confirming the presence of MeHg in soil (Kocman et al., 2004). Two-way clustering analysis based on Euclidian distances (Fig. 3a) showed that physiological response of lettuce depended mainly on the Hg exposures, while Se addition induced only minor effects. The controls clustered separately, while more similar responses were observed in Hg exposed plants characterized by lower biomass production, Fv/ Fm and TChl concentrations. Although Hg concentration in the lettuce edible parts may be tolerable for consumption, HgCl2 induces bolting as indicated by increased plant height and the changed plant morphology in H plants, while the plants in other groups retained rosette growth form till the end of experiment. When early bolting occurs, the main characteristics of leaf vegetable performance include a premature differentiation of flower buds, reduction in the number of leaves, textural decline, development of a bitter flavour, and the inability to form a compact leaf ball - the traits that are highly undesirable from the consumer point of view (Hao et al., 2018). It was reported that Hg vapour as well as other forms of Hg induce early senescence through increase in ethylene synthesis (Speitel and Siegel, 1975) closely related to premature bolting. In addition, Hg accumulated in the shoots negatively affected the TChl levels and photochemical efficiency of the photosystem II (Chen and Yang, 2012): the lowest levels found in I plants indicate that Hg may be incorporated into the photosynthetically active leaf tissues and not only deposited/bound on the leaf surface. Increase of Hg uptake was also accompanied by increased Ca level in plants. The trait was already observed in maize grown in HgCl2 contaminated substrate, where Ca level increased in rhizodermis, the main Hg storage site in the roots (Debeljak et al., 2018). Calcium is involved in signalling during programmed cell death (O'Brien and Ferguson, 1997), therefore its concentration increases in tissues affected by Hg stress (Debeljak et al., 2018).

4. Discussion 4.1. Plants The potting substrate mixture and the soil collected in Idrija were all very low in Se, as reported generally for Slovenia (Žnidarčič, 2011). Foliar spraying of Se significantly increased Se concentration in the shoots as well as roots, indicating Se assimilation and transfer to the roots via phloem (Kápolna et al., 2009), but Se concentration in the roots still remain low (below 1.5 μg g−1 DW). No toxic effects of Se foliar spraying in line with similar low-Se biofortification experiments (El-Ramady et al., 2014; Germ et al., 2018; Slekovec and Goessler, 2005; Zidar et al., 2016) were observed. The lettuce biofortified with Se contained on average 15 μg Se g−1 DW in the edible parts. Considering 83% of water, the amount in FW yielded 1.8 μg Se g−1. Three servings of fresh salad (75 g) prepared from this lettuce per week would therefore fully cover recommended daily uptake of Se (55 μg) (Institute of Medicine, 2015). Mercury levels in edible lettuce parts ranged from 1.1 μg g−1 DW in H and 2.6 μg g−1 DW in I plants. For humans, provisional tolerable weekly intake (PTWI) for inorganic mercury is 4 μg kg−1 body weight (BW) and for MeHg 1.6 μg kg−1 BW (“WHO/JECFA,” 2007), equivalent to 280 and 112 μg, respectively for a 70 kg person per week. In the same approximation as for Se uptake (83% water content, three servings (75g) of salad per week), the weekly intake would yield 30 μg for H and 70 μg for I lettuce, which is well below PTWI, but nevertheless adding a significant amount of Hg to the diet. Significantly higher root Hg concentration was observed in HgCl2 exposed plants, while in the shoots higher Hg level was detected in plants grown in soil from Idrija. In HgCl2 contaminated potting substrate mixture containing approximately 10% of organic matter, Hg2+ dissociated from HgCl2 in water solution was probably bound to organic matter (humic acids) and clay particles (Yu et al., 2004), representing a pool of exchangeable and bioavailable Hg2+ for the root uptake. As evident from Hg localization and speciation studies by laser-ablation ICP-MS and X-ray absorption spectroscopy, in plants cultured in HgCl2 contaminated potting substrate mixture, the majority of Hg2+ is bound to thiol (-SH) ligands of the cell-wall components in the root rhizodermis and cortex (Debeljak et al., 2018, 2013; Kodre et al., 2017). The Hg2+ ions are thus strongly immobilized and prevented from translocation to the upper plant parts, as also confirmed in our study where only a low level of Hg root-to-shoot translocation was detected. In comparison to HgCl2 contaminated potting substrate mixture, Hg speciation and ligand environment in Idrija soil is much more complex. The majority of Hg is present as cinnabar (HgS) (Esbrí et al., 2010; Kocman et al., 2004), that is poorly soluble, with very low level of bioaccumulation and bioavailability for plants, as confirmed by root BCF and BI. The reason for the relatively high shoot Hg accumulation in I plants could be the presence of Hg0 in Idrija soil (Kocman et al., 2004) that can volatilize from soil and condense on the leaf surface, or enter inner leaf tissues through stomata openings (Martínez-Trinidad et al., 2013; Patra and Sharma, 2000). Mercury in the shoots of I plants may therefore originate both from root-to-shoot transport and air-borne Hg, preventing any firm conclusions on the effects of Se biofortification on Hg root-to-shoot translocation and shoot uptake in Se biofortified lettuce. Since a different behaviour is observed in plants grown in HgCl2 contaminated potting substrate mixture, we can conclude that Hg

4.2. Animals The slugs fed with Se biofortified lettuce showed increased Se and MDA levels when compared to the controls, indicating that Se biofortification may induce Se overload and toxicity in invertebrate fauna. In a study performed with Se hyperaccumulating plants (Hanson et al., 2003) it was shown that caterpillars for example avoid eating Se rich plants due to severe toxicity, while snails (Mesodon ferrissi) were not distracted by Se and were proven more resistant to Se toxicity. The slugs fed for two weeks by the lettuce grown at H and I Hg exposures, showed differences according to physiological parameters and Hg uptake. As apparent from the two-way clustering analysis (Fig. 3b), the I slugs clustered separately from the C and H slugs. The I slugs had the lowest HP and M fresh and dry biomass, the lowest feeding rate and negative growth rate, together with the highest level of Hg accumulation in the muscle tissues. In I/Se+ group the highest BI, Se and HP-MDA levels were observed, indicating that Se increased Hg bioavailability and induced additional stress in this group. Reduction of slug's biomass and severe membrane damage as measured by significantly increased HP-MDA levels in I/Se- slugs, resulted in preconcentration of Se in HP of I/Se+ slugs exerting additional toxic 7

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effects, probably through interaction with thiol groups of different enzymes and proteins (Zwolak and Zaporowska, 2012). On the other hand, the H/Se+ slugs grouped together with the C, indicating that Se mitigated stress in comparison with the H/Se-group. In both groups Hg was mainly retained in HP with only low transfer rate to the muscle tissues. Selenium biofortification lowered Hg accumulation in HP of H/Se+ group, as well as in muscle tissues, which was consequently reflected in a significantly lower M-MDA level. Similar decrease in Hg uptake in the presence of Se was also observed in a study where the isopods (Porcellio scaber) were fed by plants grown in HgCl2 spiked soil and foliarly sprayed with Se (Zidar et al., 2016). The decrease of Hg uptake in the presence of Se was not observed in the slugs fed by plants grown in soil from Idrija, similarly as in Porcellio scaber (Zidar et al., 2016). Further studies are needed to decipher the mechanisms behind this behaviour, but it is highly likely that response is governed by Hg speciation, namely the presence of MeHg in soil and further in plant.

strongly dependent on Hg speciation and ligand environment in soil. In case of HgCl2 soil contamination, Hg2+ ions are more available for the plant root uptake, but strongly immobilized in the roots by S-ligands with only low translocation to the shoots. When HgCl2 contaminated plant material is eaten by slugs, Hg is immobilized in the digestive system and hepatopancreas, with low translocation rate to muscles. In this exposure, Se alleviates Hg stress or has no effect on plant performance, but decreases Hg concentration in slug hepatopancreas and muscles. Although Se increases Hg bioavailability for hepatopancreas, toxicity effects are lesser due to strong Hg immobilization (probably as HgSe). On the other hand, in Idrija soil Hg speciation and ligand environment is very complex. The majority of Hg is present as insoluble HgS with traces of Hg0 and MeHg. When MeHg is present in soil, although in very small amounts, this results in increased Hg root to shoot translocation and toxicity expressed as decreased Fv/Fm and photosynthetic pigment content in plants. When such plants are eaten by slugs, higher amount of Hg is transported to muscles, and toxicity effects are exhibited already at very low concentrations. In this case Se addition increases Hg bioavailability and induces additional stress to already stressed slugs. Further studies are however needed to decipher the mechanism behind these responses.

4.3. Mercury bioaccumulation and bioavailability Mercury bioaccumulation and bioavailability for plants and slugs were assessed through bioconcentration factor (BCF) and bioaccumulation index (BI) with the latest considering not only organ Hg concentration, but also the biomass production (plants) and growth and feeding rate (animals). For the plants, lower bioaccumulation and bioavailability from the soil to the roots was confirmed in soil from Idrija when compared to HgCl2 spiked soil, since the majority of Hg in soil is in the form of HgS (Tomiyasu et al., 2012). The positive effect of Se biofortification seen as a BCF decrease in H/Se+ roots was, however, not observed when calculating BI (considering plant biomass), similarly as in the study by Wang et al. (2016), where foliar Se application had no effect on Hg uptake and translocation in rice plants. For the slugs our results show that Hg accumulated in the lettuce plants grown in Idrija soil is more bioavailable and better translocated from HP to the muscle tissues, as indicated by higher BCF, BI and M/HP values. Mercury speciation studies in muscle tissues of invertebrates, fish and mammals show that more than 90% of Hg is present in these tissues in highly bioavailable, mobile and toxic MeHg form (Rua-Ibarz et al., 2016; Sadhu et al., 2015; Wagemann et al., 1998). Higher Hg accumulation levels in plant shoots and muscle tissues of I slugs may therefore indicate the presence of MeHg in soil and further in the food chain in line with other studies performed in Idrija (Gnamuš, 2002; Gnamuš et al., 2000; Tomiyasu et al., 2017, 2012). On the other hand, in the HgCl2 spiked soil the probability of Hg methylation is negligible, which is in line with much lower root-to-shoot or HP-to-M translocation due to strong complexation of Hg2+ with S-ligands (Kodre et al., 2017). Interestingly, Se increased Hg bioavailability for HP from both H/Se + and I/Se+ plants and for M from I/Se+ plants, but with different physiological response observed in H and I groups, implying different mechanisms of Hg-Se interactions in the both Hg exposures. In plants grown in water soluble HgCl2 contaminated soil the presence of Hg2+ is expected, strongly interacting with thiol groups leading to immobilization of Hg in plants roots. Due to low Se concentration in roots Hg-Se interactions are less probable. Small amount of lipophilic and highly toxic MeHg on the other hand could explain high Hg mobility and toxic effects on slugs fed by lettuce grown in Idrija soil. Although MeHg targets S-ligands, organic Se compounds as selenocysteine and selenomethionine play major role in MeHg immobilization due to much higher affinity (Spiller, 2018). Since in plants only small amount of selenate is converted to seleno-organic compounds, and due to highly lipophilic nature, MeHg remains much more mobile in the food chain than Hg2+.

Author contribution AK and KVM designed the study and wrote the paper, AK performed experiments with lettuce and slugs and analyses. BB performed the ICPMS analysis. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The study was financed by the Postgraduate researchers funding ARRS (Young Researcher) and by the Slovenian Research Agency (ARRS) P1-0212 (Plant biology), J7-9418 and J1-8156. We acknowledge technical help of Tanja Murn, who was financed by Josef Stefan Institute, Slovenia. References Alfthan, G., Eurola, M., Ekholm, P., Venäläinen, E.-R., Root, T., Korkalainen, K., Hartikainen, H., Salminen, P., Hietaniemi, V., Aspila, P., Aro, A., Selenium Working Group, 2015. Effects of nationwide addition of selenium to fertilizers on foods, and animal and human health in Finland: from deficiency to optimal selenium status of the population. J. Trace Elem. Med. Biol. 31, 142–147. https://doi.org/10.1016/j. jtemb.2014.04.009. Baldantoni, D., Morra, L., Zaccardelli, M., Alfani, A., 2015. Cadmium accumulation in leaves of leafy vegetables. Ecotoxicol. Environ. Saf. 123, 89–94. https://doi.org/10. 1016/j.ecoenv.2015.05.019. Boening, D.W., 2000. Ecological effects, transport, and fate of mercury: a general review. Chemosphere 40, 1335–1351. https://doi.org/10.1016/S0045-6535(99)00283-0. Boshoff, M., Jordaens, K., Baguet, S., Bervoets, L., 2015. Trace metal transfer in a soil–plant–snail microcosm field experiment and biomarker responses in snails. Ecol. Indicat. 48, 636–648. https://doi.org/10.1016/j.ecolind.2014.08.037. Byrne, A., Škreblin, M., Falnoga, I., Al-Sabti, K., Horvat, M., 1995. Mercury and selenium perspectives from Idrija. Acta Chim. Slov. 42, 175–198. Chen, J., Yang, Z.M., 2012. Mercury toxicity, molecular response and tolerance in higher plants. Biometals 25, 847–857. https://doi.org/10.1007/s10534-012-9560-8. de Vries, W., Römkens, P.F. a M., Schütze, G., 2007. Critical soil concentrations of cadmium, lead, and mercury in view of health effects on humans and animals. Rev. Environ. Contam. Toxicol. 191, 91–130. Debeljak, M., van Elteren, J.T., Špruk, A., Izmer, A., Vanhaecke, F., Vogel-Mikuš, K., 2018. The role of arbuscular mycorrhiza in mercury and mineral nutrient uptake in maize. Chemosphere 212, 1076–1084. https://doi.org/10.1016/j.chemosphere. 2018.08.147. Debeljak, M., van Elteren, J.T., Vogel-Mikuš, K., 2013. Development of a 2D laser ablation inductively coupled plasma mass spectrometry mapping procedure for mercury in maize (Zea mays L.) root cross-sections. Anal. Chim. Acta 787, 155–162. https://doi.

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