Extremely Elevated Total Mercury and Methylmercury in Forage Plants in a World Large-scale Abandoned Hg Mining Site: A Potential Risk of Exposure to Grazing Animals

Ninety-ve wild forage plants (belonging to 22 species of 18 families) and their corresponding rhizosphere soil samples were collected from wastelands of a world large-scale abandoned Hg mining region for total Hg (THg) and methylmercury (MeHg) analysis. The forage plant communities on the wastelands were dominated by the Asteraceae, Crassulaceae and Polygonaceae families. The THg and MeHg concentrations in the forage plants varied widely and were in the range of 0.10 to 13 mg/kg and 0.19 to 23 μg/kg, respectively. Shoots of Aster ageratoides showed the highest average THg concentration of 12±1.1 mg/kg, while those of Aster subulatus had the highest average MeHg concentrations of 7.4±6.1 μg/kg. Both the THg and MeHg concentrations in the aboveground plant parts exhibited positive correlations with the THg (r=0.70, P<0.01) and MeHg (r=0.68, P<0.01) concentrations in the roots but these were not correlated with the THg and MeHg concentrations in their rhizosphere soils. The species A. ageratoides, A. subulatus, and S. brachyotus showed strong accumulation of Hg and are of concern for herbivorous/omnivorous wildlife and feeding livestock. Taking the provisional tolerable weekly intake (PTWI) values for IHg recommended by the JECFA (2010) for human dietary exposure of 4 ng/g into account, grazing on 1.0 kg of forage (dry weight) by a 65 kg animal would mean that the daily intake of IHg was between 190-13200 μg, which reaches 3-5 order of magnitude higher than the permitted limit, suggesting a potential risk of exposure.


Introduction
Mercury (Hg) is a global pollutant and Hg exposure can increase the risk of cardiovascular disease and have neurological effects on humans, even at low concentrations (Driscoll et al., 2013;Peng et al., 2015). Mining and retorting of cinnabar ores are major sources of metal Hg, which is also one of the major sources of anthropogenic Hg to the environment . The areas impacted by historic Hg mining continue to be threatened by the heavy Hg pollution caused by the abandoned mine-waste calcines (ignited residues). These are enriched with water-soluble secondary Hg compounds, such as meta cinnabar, polymorphic sul de Hg, sulphate Hg, chloride Hg, etc. Those large amounts of water-soluble Hg in e uent discharges from mine-waste calcines can be readily transformed into the more toxic methylmercury (MeHg) under suboxic conditions (Qiu et al., 2005;Lin et al., 2010). MeHg is considered the most harmful form of Hg due to its high lipophilicity (Agency for Toxic Substances and Disease Registry, 2013), so the transformation, bioaccumulation, and biomagni cation of MeHg is of the greatest concern.
China has rich cinnabar deposits, and it ranks third in the world for its total reserves. Of these mines, the Wanshan Hg mine was once known as the "Mercury Capital", and it was the largest elemental Hg production center in China. Activities are exploiting this area in China date back to the Qin Dynasty (220 B.C.). These activities have resulted in severe environmental Hg contamination and generated signi cant quantities of wastelands. THg concentrations as high as 4,400 mg/kg have been found in mine-waste calcines of the Wanshan Hg mine, as high as 790 mg/kg Hg in soils from paddies, and 10,000 ng/L Hg in surface water (Horvat et al., 2003;Qiu et al., 2005). The highest concentrations of Hg in the soils from the abandoned Hg mining region were approximately 2-3 orders of magnitude higher than the 'probable effect concentration' of 1.06 mg/kg Hg, above which harmful effects on organisms are likely Plants provide the basic food energy for animals occupying the areas of former Hg mining. The consumption of the extremely Hgcontaminated forage-plants that may cause Hg accumulation and biomagni cation in primary consumers, which could then enter terrestrial food chains. Mercury, as a long-term hazard in vegetated wastes, may have critical impacts on wildlife dependent on the region (Madejón et al., 2012; Basri et al., 2020). Our recent investigations have shown that MeHg has accumulated in the herbivorous wildlife in the Wanshan Hg mining region to levels that could cause health effects (Abeysinghe et al., 2017;. To better understand the potential risks of THg and MeHg exposure to herbivorous wildlife as well as livestock, the characterization of THg and MeHg in wild plants (forages) is an urgent necessity. It is important to know if forage-plants in contaminated regions are of concern for herbivores.
In the present study, dominant wild forage plants growing on wastelands in the Wanshan Hg mining district, Southwest China were investigated. The objectives were to (1) obtain basic information on the forage plants present and their THg and MeHg levels, (2) elucidate the transfer e ciency of THg and MeHg from the soil to the forage species and factors in uencing this, and (3) clarify which species are of the greatest concern to herbivorous animals and assess the potential risk of exposure.

Study area
The Wanshan Hg mining district, on the eastern edge of the Yun-Gui Plateau in southwestern China (E: 109°07'-109°24'; N: 27°24'-27°3 8'), is the largest industrial metallic Hg production center in China. This region has a typical karst landscape with an average elevation of 850 m. The annual average temperature is 13.4°C, and the mean annual precipitation is 1,400 mm/year. Cinnabar is the main ore mineral associated with metacinnabar, natural metallic Hg, tiemannite, sphalerite, pyrite, and stibnite. The average Hg grade of the ore deposits is higher than 0.25%.
Extensive Hg mining and retorting occurred for 630 years and ceased in 2004. Approximately 125.8 million tonnes of mine-waste calcines were introduced into the environment between the early 1950s and the late 1990s (Qiu et al., 2005). The large historic Hg mining adits of Lengfengdong and Meizixi are headwaters of the rivers Xiaxi and Aozhai, the major aquatic systems in the Wanshan mining district. Large mine-waste calcine piles were placed adjacent to the corresponding adits and generated a signi cant amount of Hg-contaminated wastelands. In the present study, the wastelands generated by the calcine piles from Lengfengdong (LFD), Chongjiao (CJ), and Meizixi (MZX) were selected for investigation ( Fig. 1; Table S1).

Sampling and preparation
Ninety-ve samples of dominant forage plants belonging to 22 species of 18 families, which are favored by grazing animals, were collected from the wastelands. We preferentially sampled herbaceous plants rather than woody species. All of the plants were identi ed to the species level based on descriptions in the Flora of China ( ora.huh.harvard.edu/china/mss/welcome.htm).
During sampling, dominant forage samples were randomly taken from the wastelands, within a sampling grid of 5×5 m. For each sample, three or more similarly sized individual plants of the same species were collected to ensure adequate amounts of tissue for analysis. Plant samples were dug out of the ground with a shovel and separated in situ into aboveground parts (shoots) and roots. In the laboratory, the plants were washed thoroughly with tap water and then with deionized water (DW) three times. Afterwards, the plants were frozen in a freezer and then placed in a vacuum freeze drier (-50°C) for drying. The dry plants were ground and sieved to ne powders using an analytical mill (IKA-A11 basic, IKA, Germany) and nylon sieve (mesh size of 0.18 mm). During processing, the lab equipment was rinsed three times with ethanol cleansing to control cross-contamination among the samples. The ne powder samples were stored in hermetic bags for analysis.
Corresponding rhizosphere soils were simultaneously collected with the plants. Approximately 0.5 kg of rhizosphere soil from the roots of each individual plant was shaken onto a piece of paper, and then the total 1.5 kg of soil collected from the 3 individual plant roots mentioned above was mixed as the nal composite sample. The soils were stored in double polyethylene plastic bags to prevent any cross-contamination. After collection, all of the soil samples were air-dried in the laboratory, thoroughly mixed, and subsequently ground to ne powders using an agate mortar and nylon sieve (mesh size of 0.075 mm). A cleansing process similar to the plant samples preparation was applied to control cross-contamination among the samples.

Soil
For THg determination, approximately 0.1-0.2 g (accurate to 0.0001 g) soil samples were weighed and placed into plastic tubes. Then, 5 mL DW and 5 mL fresh aqua regia (HCl : HNO 3 = 3 : 1, v/v) were added. The samples were rested for 5 min, and then, 1 mL BrCl was added for water bath digestion at 95°C for 3 h. The digestate was left for 24 h. Following this, 400 µL NH 2 OH·HCl was added to remove the free halogens, and the samples were brought to a xed volume of 50 mL with DW. Approximately 5 mL of the digestate was taken for Hg analysis, similar to the methods used for the plants.
For MeHg determination, approximately 0.3-0.4 g (accurate to 0.0001) soil samples were weighed and placed into 50 mL plastic centrifuge tubes. Next, 1 mL of 2 mol/L CuSO 4 and 4 mL concentrated HNO 3 :H 2 O = 1:3 (v/v) were added. Then, 5 mL ultra-pure CH 2 Cl 2 was added and shaken for 30 min to extract the MeHg into the solvent. Afterwards, the CH 2 Cl 2 solvent phase was collected in a 50 mL Te on bottle. Approximately 30 mL of DW was added and then the MeHg was back-extracted into the new water phase. The extract was brought to a xed volume of 50 mL with DW (Liang et al., 1996). Approximately 5 mL aliquots were taken for MeHg GC-CVAFS analysis, similar to the procedure used for the plants.
For the soil pH measurements, approximately 10 g soil samples were weighed and placed into plastic vials. Next, 25 mL of DW without CO 2 was added, mixed for 2 min, and left to settle for 30 min (Lu, 2000). The soil pH was determined using a pH meter (PHS- For soil organic matter (OM) determination, approximately 0.5-1.0 g of soil was weighed and placed into colorimetric tubes.
Concentrated sulfuric acid was added and then OM measurement followed a water bath-potassium dichromate volumetric method (Lu, 2000).

Quality Assurance/Quality Control
Quality assurance/quality control (QA/QC) measures employed consisted of the use of a standard working curve, blanks, sample duplicates, matrix spikes, and the certi ed reference materials of lichen (BCR-482), lobster hepatopancreas (TORT-2), Chinese yellowred soil (GBW07405), and estuarine sediment (ERM-CC580), as further described below and in the Supplementary Material (Table S2).
For THg, the method was validated using the reference materials BCR-482 and GBW07405. An average total Hg concentration of 0.475 ± 0.02 mg/kg (n = 5) was obtained for the lichen standard BCR-482, which was within the range of the certi ed value of 0.48 ± 0.02 mg/kg. For the soil, GBW07405 was used, and the measured concentration of 0.32 ± 0.02 mg/kg (n = 5) was within acceptable range of the certi ed value of 0.29 ± 0.04 mg/kg.
For MeHg, the obtained value of 75.0 ± 3.1 µg/kg (n = 5) met the certi ed value of 75.5 ± 3.7 µg/kg for the ERMCC-580 soil standard. In addition, the obtained value of 155 ± 25 µg/kg (n = 5) met the certi ed value of 152 ± 13 µg/kg for the TORT-2 plant standard. The recovery of THg and MeHg in the solid samples was in the range of 95-109%, and 87-108%, respectively.

Calculations of BCFs and TFs of IHg and MeHg
In the present study, the IHg and MeHg BCFs were de ned as the ratios of their concentration in the plant roots to that in the soil ( Figure S1). A positive correlation was detected between the shoots and roots (r 2 = 0.48, P < 0.0001; Fig. 3a). ageratoides recorded the highest THg concentrations both in the shoots and roots, which may be of great concern for herbivorous/omnivorous grazing animals.

MeHg
Forage-plants showed broad ranges of MeHg concentrations in the roots and shoots, ranging from 0.19 to 23 µg/kg and 0.28 to 11 µg/kg, respectively ( Table 2; Fig. 2b). The highest average MeHg concentration was found in the shoots of A. subulatus at 7.4 ± 6.1 µg/kg, followed by S. brachyotus with 3.5 ± 2.5 µg/kg, while the lowest was in B. camperstris with 0.49 ± 0.11 µg/kg. S. brachyotus exhibited the highest average MeHg concentration in its roots with 13 ± 10 µg/kg, followed by A. subulatus at 9.4 ± 8.0 µg/kg, while the lowest was in A. ageratoides at 1.1 ± 0.76 µg/kg. As expected, the root MeHg exhibited a signi cant positive correlation to the MeHg in the shoots (r 2 = 0.46, P < 0.0001; Fig. 3b), suggesting a strong transport of MeHg from the roots to the aboveground parts of the plants. Different species exhibited different capabilities for MeHg bioaccumulation. In addition to A. subulatus, which recorded the highest levels of MeHg concentration (greater than 10 µg/kg on average) in both the shoots and roots, species S. brachyotus, C. edulis, and P. oleracea also showed high MeHg concentrations, particularly in their roots, with a range of 6.

TFs
The TFs for IHg in the plants varied widely, ranging between 0.12 and 13. The lowest TF value differed from the highest by more than In the present study, though soil Hg played an important role in the absorption and enrichment of Hg in the plants, it was found that the rhizosphere soil pH and OM may also play important roles in the process of Hg uptake and transfer in plants. Our results indicate that there is a complex mechanism for Hg uptake, particularly for plants growing in heavily Hg-contaminated sites.

Potential risks for herbivores
The forage plants investigated in the present study are usually consumed by domestic animals such as cows, goats, and poultry. Currently, critical limits for Hg in plants (grass) for grazing animals are not available. Therefore, JECFA PTWI values for human intake were cited in the risk assessment for herbivores (Gramss and Voigt, 2014). Taking the provisional tolerable weekly intake (PTWI) value 4 ng/g for IHg recommended by the JECFA (2010) for human dietary exposure from foods other than sh and shell sh into account, daily ingestion of 0.037 µg IHg by a 65 kg animal is acceptable. Grazing on 1.0 kg of the shoots of the forage-plants (dry weight) would mean that the daily intake of IHg and MeHg was between 190-13200 µg, which reaches 3-5 orders of magnitude higher than the permitted limit. Moreover, grazing animals, such as cows and sheep are highly sensitive to contamination due to the ingestion of soil along with grass intake, hence, the heavily contaminated soil with both THg and MeHg is another point of concern.

Conclusions
The dominant forage plants collected from the wastelands of this world large-scale Hg mine exhibited high concentrations of both THg and MeHg, ranging from 0.085 to 13 mg/kg and 0.059 to 24 µg/kg, respectively. Species A. ageratoides, C. barometz, I. batatas, and P. sikkimensis exhibited THg greater than the high hazard level of 3 mg/kg in their shoots. Moreover, comparable levels of MeHg in A. subulatus, C. edulis, S. brachyotus, and P. oleracea to that found in rice were observed. Among of those species, A. ageratoides, C. barometz, and I. batatas showed high abilities to accumulate and transfer both IHg and MeHg to their shoots, with their TFs being greater than 1.0. The species A. ageratoides, A. subulatus, and S. brachyotus in the present study may pose the highest risks for THg and MeHg exposure to biota due to their strong Hg accumulation abilities and abundant biomass. Because the investigated forage plants were widely consumed by herbivorous/omnivorous wildlife and feeding livestock, the high THg and MeHg concentrations could directly result in Hg accumulation and biomagni cation in the terrestrial food chain. Thus, future studies on the plantherbivorous-carnivorous food chain are urgently needed to clarify the risks from THg, and particularly MeHg, exposure to wildlife and feeding livestock. Declarations