Food and Chemical Toxicology xxx (xxxx) xxxx

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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
b
National Institute of Chemistry, Hajdrihova 19, SI-1000, Ljubljana, Slovenia
c
Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia

ABSTRACT

The effects of foliar Se biofortification (Se+) of the lettuce on the transfer and toxicity of Hg from soil con-
taminated 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 biofortifi-
cation (Se+ test group) had no effect on Hg bioaccumulation in plants. In slugs, different responses were ob-
served 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 physio-
logical 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 nutritional value of the tested plants without significant decrease in

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 hu-
mans relying mostly on vegetarian diet prevails due to low Se con-
centration 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
crop yields. When added in low concentration (< 20 mg kg−1), Se ex-
erts 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 pro-
duced 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 environ-
ment 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: anja.kavcic@bf.uni-lj.si (A. Kavčič), bojan.budic@ki.si (B. Budič), katarina.vogelmikus@bf.uni-lj.si (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
A. Kavčič, et al. Food and Chemical Toxicology xxx (xxxx) xxxx

measured by extraction with 0.5M HCl is, however, very low (up to study, e.g. elements, nanoparticles or other chemicals are directly ap-
0.2 mg L−1) (Esbrí et al., 2010), as confirmed also by a low total Hg plied to the plant surface, excluding chemical modification by plant
bioconcentration factor (the ratio between plant and soil Hg con- metabolic pathways (Valant et al., 2012), our study undertakes a more
centration) ranging from 0.4 to 6% (Gnamuš et al., 2000). On the other realistic approach, similar as in Kavčič et al., (2019), where wild
hand, in Almaden and Asturias, Hg mobility is much higher and asso- growing mushrooms accumulating Hg and Se were fed to the Spanish
ciated with the presence of HgCl2 and Hg2+ (Esbrí et al., 2010). Mer- slugs to evaluate Hg/Se bioavailability and toxicity. A linear relation-
cury bioavailability in the soil is also strongly connected to the amount ship was observed between Hg and Se concentration in food and slug
of MeHg, the most mobile and toxic Hg species in biological systems hepatopancreas, while the transport and mobility from HP to the
(Kim and Zoh, 2012). In Idrija it was shown that MeHg formation is muscle tissues depended on Hg ligand environment in food and further
related to decomposition of organic matter, with higher amounts (up to in HP. Complexation of Hg with Se in food or HP restricted Hg accu-
5% of the total Hg) found in organic-matter rich soils (Gnamuš, 2002; mulation in muscle tissues.
Gnamuš et al., 2000; Tomiyasu et al., 2017). The total bioaccessibility In the present study, the plants were grown under Hg exposures and
(gastric and intestinal) of Hg in different vegetables collected from biofortified with Se for six weeks, to absorb and metabolize both ele-
farmland in close proximity to compact fluorescent lamp factories ments before being fed to the primary consumers under controlled
(Zhejiang Province, PR China), where Hg can be found mainly in HgCl2 conditions. The advantage of this system in evaluating metal bioavail-
form, ranged from 34.4% for Chinese mustard greens - xuelihong to ability/toxicity from soil to the food chain over chemical extraction
54.6% for lettuce (Shao et al., 2012), contributing to elevated daily procedures and use of single organisms (plants or invertebrates) is that
uptake of Hg in population consuming the contaminated vegetables. it considers metabolic processes that may alter Hg and Se speciation
Since Hg is toxic already at low concentration and it is difficult to avoid and ligand environment in the primary producer (plant), con-
consumption of vegetables and crops produced in Hg polluted en- sequentially affecting Hg bioavailability and physiological response of
vironments, alternative ways need to be found to decrease Hg exposure the consumer (slug). Based on previous studies (Wang et al., 2016;
through ingestion and to alleviate Hg toxicity. Zidar et al., 2016) the working hypothesis was a protective role of Se
According to research on mammals, Se may protect against stress against Hg-induced stress in lettuce and further in slugs.
induced by Hg and other metals (Boening, 2000; Byrne et al., 1995;
Spiller, 2018) through improved antioxidant activity (Zwolak and
2. Materials and methods
Zaporowska, 2012) and through binding Hg into insoluble HgSe com-
plexes (Thangavel et al., 1999; Wang et al., 2013; Zhang et al., 2014;
2.1. Experimental design
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;
The experimental set-up consisted of six groups of lettuce plants:
Zhang et al., 2012; Zhao et al., 2014). Wang et al. (2016) have shown
two groups were grown in the control potting substrate mixture without
that Se added into the soil inhibits the uptake of MeHg as well as in-
and with added Se (C/Se-, C/Se+) and two in the potting substrate
organic Hg into rice grains, while foliar Se application has no effect on
mixture spiked with 100 mg kg−1 of HgCl2 (added to the substrate in
MeHg plant uptake, concluding that MeHg-Se interactions in soil rather
solution), without and with added Se (H/Se-, H/Se+). The remaining
than within the plant might be the key process triggering the decrease
two groups were grown on the soil collected at Hg-contaminated site in
in grain Hg levels under Se amendment.
Idrija near the chimney of the former Hg smelter (46°00′33.7"N
Lettuce (Lactuca sativa L.), an annual plant of the Asteraceae family,
14°02′02.9"E), (Tomiyasu et al., 2017), again with and without added
is widely consumed around the world, and its production exceeded 26
Se (I/Se-, I/Se+). The basis for the C and H groups was prepared as a
million tons in 2017 (FAO, 2019). Since lettuce is an important part of
mixture of rendzina soil collected near Ljubljana and a standard potting
our everyday diet, Se biofortification of lettuce could help to fight Se
substrate (Cvetal, Agroruše) (2:1 w/w) to match as closely as possible
malnutrition in Se deficient areas (Smoleń et al., 2014). The lettuce is
the composition of the soil collected in Idrija. Soil properties are listed
also known to accumulate relatively large amounts of metals, especially
in Table 1. Selenium levels were below 0.01 mg kg−1 in potting sub-
Cd (Baldantoni et al., 2015), As, Ni, Pb, Zn (Boshoff et al., 2015) and
strate mixture and soil of all exposure groups.
also Hg (Miklavčič et al., 2013), thus presenting an important source of
Eight weeks old lettuce (Lactuca sativa L. cv. Exquise) seedlings were
hazardous metal dietary intake. In order to quantify the risk of ha-
zardous metal consumption by higher organisms it is necessary to study
Table 1
behaviour of hazardous metals in the food chain and track physiological
The properties of the potting substrate mixture and soil used in the experiments
responses of the test organisms (Boshoff et al., 2015). with lettuce (EC-conductivity, WHC-water holding capacity). C, control potting
Our study therefore aimed to explore the effects of foliar Se-bio- substrate mixture; H, HgCl2 spiked potting substrate mixture; I, soil collected in
fortification of the lettuce plants, on the transfer and toxicity of Hg from Idrija. Values are averages ± SE, n = 5.
soil contaminated with HgCl2 and soil collected at Hg polluted site in
C H I
Idrija (the past second largest Hg mine in the world) to terrestrial food
chain, with Spanish slugs as primary consumers. Like other in- Hg [mg kg−1] < 0.001 99.0 ± 1.40 101.0 ± 2.00
vertebrates, slugs accumulate noxious elements and chemicals in their pH 5.6 ± 0.2 6.2 ± 0.2 6.0 ± 0.2
digestive gland (hepatopancreas, HP), occupying the role of appropriate Soil type Rendzina, silty loam Rendzina, silty loam
Organic matter [%] 10 ± 2 9 ± 0.4
sentinels of environmental pollution (Boshoff et al., 2015; Kavčič et al.,
WHC [%] 54 ± 5 43 ± 4
2019; Pauget et al., 2013). In addition, the elements that are mobile EC [μS cm-1] 240 ± 30 470 ± 50
within the organisms can be transported and accumulated in the muscle Si [mg kg−1] 29800 ± 1500 59100 ± 2700
tissues; therefore the muscle to hepatopancreas element concentration P [mg kg−1] 2500 ± 170 2330 ± 200
S [mg kg−1] 380 ± 10 492 ± 47.6
ratio can be taken as a measure of element within-organism mobility.
Cl [mg kg−1] 2100 ± 160 1900 ± 110
Although Spanish slug is listed among the 100 worst invasive species in K [mg kg−1] 7220 ± 200 9720 ± 290
Europe, it is an important part of food webs, common in diet of various Ca [mg kg−1] 7880 ± 950 9900 ± 290
birds and mammals. In addition, experiments with slugs pose no ethical Mn [mg kg−1] 235 ± 1.5 413 ± 49.3
issues, are simple to perform and provide reliable and highly re- Fe [mg kg−1] 10800 ± 1300 19500 ± 1970
Zn [mg kg−1] 79.4 ± 1.4 55.2 ± 3.8
producible results (Pauget et al., 2013).
Rb [mg kg−1] 55.4 ± 5.2 80.4 ± 1.5
While in several metal bioavailability and toxicity studies involving Sr [mg kg−1] 68.6 ± 8.9 72.0 ± 2.1
test organisms like terrestrial isopods or snails, the substances under

2
A. Kavčič, et al. Food and Chemical Toxicology xxx (xxxx) xxxx

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-con-
centration 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 8.67 ± 0.89 ab 8.94 ± 0.92 a 3.45 ± 0.74 d 6.37 ± 0.27 bc 4.54 ± 0.89 cd 4.76 ± 0.99 cd
shoot 27.18 ± 3.16 a 19.63 ± 2.83 abc 19.67 ± 3.60 abc 23.04 ± 2.37 ab 12.20 ± 3.07 c 15.08 ± 2.98 bc
DW [g] root 1.48 ± 0.16 a 1.40 ± 0.14 a 0.41 ± 0.13 c 0.81 ± 0.10 b 0.75 ± 0.16 b 0.65 ± 0.13 b
shoot 3.08 ± 0.41a 2.78 ± 0.47 ab 1.51 ± 0.38 b 2.18 ± 0.32 ab 1.72 ± 0.47 b 1.74 ± 0.41 b
plant 4.56 ± 0.52 a 4.18 ± 0.58 a 1.91 ± 0.51 b 3.00 ± 0.40 ab 2.48 ± 0.62 b 2.39 ± 0.52 b
Fv/Fm shoot 0.83 ± 0.002 a 0.83 ± 0.003 a 0.814 ± 0.008 ab 0.826 ± 0.002 a 0.816 ± 0.008 b 0.805 ± 0.012 b
TChl [mg/g DW] shoot 6.42 ± 0.95 a 5.30 ± 0.74 ab 6.73 ± 1.88 a 5.85 ± 1.19 ab 2.61 ± 0.58 b 4.52 ± 1.18 ab
Hg [μg g−1 DW] root < LOD < LOD 19.8 ± 4.56 a 8.67 ± 1.60 b 0.80 ± 0.27 c 1.78 ± 0.25 bc
shoot < LOD < LOD 1.12 ± 0.28 b 1.20 ± 0.11 b 3.33 ± 1.03 a 1.90 ± 0.26 ab
plant < LOD < LOD 4.65 ± 0.80 ab 3.21 ± 0.46 a 2.42 ± 0.62 ab 1.87 ± 0.22 b
Hg [μg organ−1] root nd nd 6.27 ± 1.45 a 6.96 ± 1.25 a 0.52 ± 0.15 b 1.08 ± 0.17 b
shoot nd nd 1.42 ± 0.21 b 2.71 ± 0.56 b 5.02 ± 2.10 a 3.18 ± 0.91 ab
plant nd nd 7.69 ± 1.38 a 9.67 ± 1.71 ab 5.53 ± 2.06 b 4.25 ± 0.94 b
Hg BCF root nd nd 0.200 ± 0.046 a 0.088 ± 0.016 b 0.008 ± 0.003 c 0.018 ± 0.002 bc
shoot nd nd 0.011 ± 0.003 b 0.012 ± 0.001 b 0.033 ± 0.01 a 0.019 ± 0.003 ab
Se [μg g−1 DW] root < LOD d 0.98 ± 0.04 b 0.03 ± 0.03 d 0.86 ± 0.12 c 0.11 ± 0.05 d 1.54 ± 0.33 a
shoot 0.18 ± 0.04 c 9.57 ± 0.35 b 0.66 ± 0.15 c 17.08 ± 3.28 a 0.13 ± 0.03 c 15.14 ± 0.75 a
Se [μg organ−1] root < LOD 1.37 ± 0.16 a 0.02 ± 0.02 c 0.72 ± 0.15 b 0.11 ± 0.06 c 1.11 ± 0.43 ab
shoot 0.51 ± 0.06 b 26.8 ± 4.54 a 0.79 ± 0.12b 39.4 ± 10.8 a 0.25 ± 0.10 b 25.9 ± 5.65 a
Ca [μg g−1 DW] root 4520 ± 1120 b 3800 ± 1150 b 14980 ± 6380 a 8880 ± 3230 ab 2950 ± 300 b 4050 ± 170 b
shoot 7510 ± 1100 b 13520 ± 4400 b 18500 ± 3200 a 15630 ± 1100 ab 8300 ± 1100 b 8190 ± 2400 b
K [μg g−1 DW] root 5890 ± 1800 7220 ± 2300 17570 ± 4400 15770 ± 1400 7650 ± 1900 17380 ± 7900
shoot 12550 ± 3140 13680 ± 6100 23700 ± 7800 24400 ± 2900 18070 ± 1400 21230 ± 6400

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 con-
tainers (5 per exposure), filled with a mixture of gypsum and charcoal
(20:1), covered with a filter paper, maintaining moisture in the con-
tainer at 80–100%. Pellets (~100 mg) prepared from plants of experi-
mental 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 de-
gradation. Fresh pellets were supplied daily and the amount of con-
sumed 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 con-
tainers and let fasten for 24 h before dissection. Digestive glands (he-
patopancreas - HP) and muscle tissues (M) were stored for analysis.
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,
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 diges-
tion, 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 (Opti-
Sciences), and the shoot height was measured. The plants were har-
vested, 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, Labo-
Gene, 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.

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A. Kavčič, et al. Food and Chemical Toxicology xxx (xxxx) xxxx

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 organ C/Se- C/Se+ H/Se- H/Se+ I/Se- I/Se+

Hg in feed [μg g−1 DW] < 0.001 < 0.001 10.4 ± 2.31 a 4.94 ± 0.85 b 2.07 ± 0.51 b 1.84 ± 0.20 b
Se in feed [μg g−1 DW] 0.09 ± 0.02 b 5.27 ± 0.17 a 0.34 ± 0.07 b 8.97 ± 1.65 a 0.12 ± 0.03 b 8.34 ± 0.47 a
mortality (sum = 60) 1 (2.5%) 1 (2.5%) 1 (2.5%) 0 0 0
Feeding rate [mg food/g slug/day] 7.61 ± v0.39 abc 9.35 ± 0.36 a 8.64 ± 0.63 ab 7.87 ± 0.24 ab 7.21 ± 0.8 bc 5.91 ± 0.86 c
Growth rate [% of FW/day] −0.15 ± 0.26 0.05 ± 0.19 0.14 ± 0.18 0.05 ± 0.12 −0.77 ± 1.08 −0.65 ± 0.19
FW [g] HP 1.17 ± 0.10 a 1.00 ± 0.09 ab 1.03 ± 0.08 ab 1.07 ± 0.06 ab 0.76 ± 0.08 b 0.83 ± 0.10 ab
M 4.24 ± 0.28a 3.49 ± 0.25 ab 3.38 ± 0.42 ab 3.79 ± 0.17 ab 2.75 ± 0.75 b 3.54 ± 0.69 ab
DW [g] HP 0.344 ± 0.037 a 0.250 ± 0.032 ab 0.266 ± 0.026 ab 0.285 ± 0.017 ab 0.190 ± 0.016 b 0.214 ± 0.025 b
M 0.855 ± 0.071 a 0.690 ± 0.058 a 0.792 ± 0.074 a 0.760 ± 0.034 a 0.358 ± 0.030 b 0.424 ± 0.067 b
Hg [μg g−1 DW] HP < LOD < LOD 1.37 ± 0.069 a 0.951 ± 0.039 b 0.431 ± 0.050 c 0.427 ± 0.061 c
M < LOD < LOD 0.027 ± 0.018 bc 0.007 ± 0.001 c 0.090 ± 0.027 ab 0.147 ± 0.052 a
Hg [g organ−1] HP nd nd 0.365 ± 0.038 a 0.269 ± 0.016 b 0.081 ± 0.005 c 0.089 ± 0.004 c
M nd nd 0.020 ± 0.014 ab 0.005 ± 0.001 b 0.033 ± 0.012 ab 0.063 ± 0.022 a
M/HP conc. ratio nd nd 0.018 ± 0.011 c 0.008 ± 0.001 c 0.209 ± 0.06 b 0.359 ± 0.117 a
Hg BCF HP nd nd 0.131 ± 0.007 b 0.193 ± 0.008 a 0.208 ± 0.024 a 0.232 ± 0.033 a
M nd nd 0.003 ± 0.002 c 0.001 ± 0.0002 c 0.044 ± 0.013 b 0.08 ± 0.028 a
MDA [nmol g−1 FW] HP 7.52 ± 1.59 d 15.69 ± 2.92 c 7.45 ± 0.48 d 6.07 ± 1.53 d 17.28 ± 1.66 b 23.71 ± 0.63 a
M 3.63 ± 0.78 ab 3.58 ± 0.67 ab 5.58 ± 2.11 a 1.94 ± 0.51 b 2.35 ± 0.45 ab 3.05 ± 0.14 ab
Se [μg g−1 DW] HP 0.14 ± 0.04 d 1.21 ± 0.11 b 0.28 ± 0.08 cd 0.81 ± 0.04 bc 0.72 ± 0.12 bcd 6.9 ± 1.05 a
M 0.12 ± 0.02 c 0.49 ± 0.05 b 0.13 ± 0.04 c 0.33 ± 0.03 b 0.54 ± 0.01 b 2.38 ± 0.47 a
Se [μg organ−1] HP 0.05 ± 0.02 c 0.29 ± 0.03 b 0.07 ± 0.02 c 0.21 ± 0.03 b 0.13 ± 0.01 c 1.43 ± 0.08 a
M 0.11 ± 0.02 d 0.33 ± 0.03 b 0.07 ± 0.03 d 0.25 ± 0.02 bc 0.19 ± 0.02 cd 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 mal-
ondialdehyde (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.
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):
Cplant
BCFplant [%] = *100
Csoil
Cslug
BCFslug [%] = *100
Cfeed
where Csoil is Hg concentration [μg g −1
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 con-
centration [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 di-
lution 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:
Splant
BIplant [%] = *100
Ssoil
Sslug
BIslug [%] = *100
Sfeed
−1
where Ssoil is Hg content [μg] in soil (Hg soil concentration [μg g
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 trans-
formed” parameters measured in the lettuce and the slugs was per-
formed in the “R” project for statistical computing (i386 3.4.3) (Singh
et al., 2014).
3. Results
(Eq. 1)
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 con-
trols (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.
(Eq. 3) Foliar spraying of the lettuce leaves with Se (~25 μg per plant) re-
sulted in significantly increased Se concentration in the shoots
(Table 2). Selenium was also transported from the shoots to the roots,
(Eq. 4)
significantly increasing the root concentration of Se+ plants. Selenium
DW] concentration in the shoots seemingly increased with Hg exposure.

4
A. Kavčič, et al.
Fig. 1. a) Plant's height and b) visual appearance of the lettuce at different
exposures: C, non-contaminated substrate; H, artificially HgCl2 spiked sub-
strate; 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).
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
Food and Chemical Toxicology xxx (xxxx) xxxx
Fig. 2. a) The relationship between Hg concentration in the shoots and pho-
tochemical 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/Se-
and 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 bio-
fortification 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).
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/Se-
and 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.
A. Kavčič, et al.
Fig. 3. a) Two dimensional hierarchical clustering based on Euclidian distanced
of z-transformed averages of plant traits at different exposures: C, non-con-
taminated 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 z-
transformed averages of slug traits at different exposures: C, non-contaminated
substrate; H, artificially HgCl2 spiked substrate; I, soil collected at Hg con-
taminated 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.
Mercury accumulated mainly in slug HP, with an order of magni-
tude 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
Food and Chemical Toxicology xxx (xxxx) xxxx
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 con-
centration (Fig. 3b).
3.3. Mercury bioavailability for plants and slugs
Mercury bioaccumulation determined as BCF significantly de-
creased 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
A. Kavčič, et al.
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 bioavail-
ability 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.
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 there-
fore 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 sub-
strate 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 rhizo-
dermis and cortex (Debeljak et al., 2018, 2013; Kodre et al., 2017). The
Hg2+ ions are thus strongly immobilized and prevented from translo-
cation 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 spe-
ciation 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 let-
tuce. Since a different behaviour is observed in plants grown in HgCl2
contaminated potting substrate mixture, we can conclude that Hg
7
Food and Chemical Toxicology xxx (xxxx) xxxx
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 sam-
pling 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 de-
posited Hg (Tomiyasu et al., 2017, 2012). In comparison to inorganic
Hg forms, MeHg is highly mobile and toxic and may be easily trans-
ported 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 con-
trols 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 dif-
ferentiation 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 nega-
tively 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 af-
fected by Hg stress (Debeljak et al., 2018).
4.2. Animals
The slugs fed with Se biofortified lettuce showed increased Se and
MDA levels when compared to the controls, indicating that Se bio-
fortification 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 sig-
nificantly increased HP-MDA levels in I/Se- slugs, resulted in pre-
concentration of Se in HP of I/Se+ slugs exerting additional toxic
A. Kavčič, et al.
effects, probably through interaction with thiol groups of different en-
zymes 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 accumula-
tion 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 de-
crease 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 me-
chanisms 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.
4.3. Mercury bioaccumulation and bioavailability
Mercury bioaccumulation and bioavailability for plants and slugs
were assessed through bioconcentration factor (BCF) and bioaccumu-
lation index (BI) with the latest considering not only organ Hg con-
centration, 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 im-
mobilization 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+.
5. Conclusions
Mercury bioavailability and toxicity for the terrestrial food chain is
8
Food and Chemical Toxicology xxx (xxxx) xxxx
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 perfor-
mance, 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 en-
vironment is very complex. The majority of Hg is present as insoluble
HgS with traces of Hg0 and MeHg. When MeHg is present in soil, al-
though in very small amounts, this results in increased Hg root to shoot
translocation and toxicity expressed as decreased Fv/Fm and photo-
synthetic pigment content in plants. When such plants are eaten by
slugs, higher amount of Hg is transported to muscles, and toxicity ef-
fects 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.
Author contribution
AK and KVM designed the study and wrote the paper, AK performed
experiments with lettuce and slugs and analyses. BB performed the ICP-
MS analysis.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence 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 acknowl-
edge technical help of Tanja Murn, who was financed by Josef Stefan
Institute, Slovenia.
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