Distribution and Dispersion of Heavy Metals in the Rock–soil–moss System in Areas Covered by Black Shale in the Southeast of Guizhou Province, China

Black shales are easily exposed duo to human activities such as mining, road construction, and shale gas development, which results in several environmental issues including heavy metals (HMs) pollution, soil erosion and the destruction of vegetation. Moss are widely used to monitor metal pollution in the atmosphere, but few studies on the distribution and dispersion of HMs in the rock – soil – moss system are available. Here, mosses (P. exuosa Harv), growing soils, and corresponding parent rocks were collected from black shale areas. After appropriate pretreatment, samples were analyzed for multiple elements concentration by ICP-AES and ICP-MS. The results show that black shales parent rocks have elevated HMs concentration, and act as a source of multiple metals. Soil signicantly inherit and accumulate heavy metals released from black shale. Signicant positive correlations between HMs in P. exuosa Harv and the growing soils indicate that HMs are mainly originating from geological source rather than atmospheric deposition. Compared with other elements, only the transfer factor (TF) of Cd is greater than 1, the normal functioning of mineral elements (K and Zn) absorption and transportation may contribute to its high tolerance to Cd. Finally, both the BCF and TF for most HMs in P. exuosa Harv are less than 1, indicated that it has a tolerance and exclusion mechanism for these metals. Therefore, the luxuriant and spontaneous growth of P. exuosa Harv could be used as a phytostabilization pioneer plant in the black shale outcrop where vascular plants are rare.


Introduction
Research on black shales have increased in recent years since they are enriched in various harmful elements, including radionuclides, and may have adverse effects on the environment. It has been well documented that black shales are sedimentary rocks containing high concentrations of sulfur and organic carbon, and also host polymetallic deposits which have been mined for Cu, Ni, Zn, Mn, Mo, V, and U (Alloway, 2013; Parviainen and Loukola-Ruskeeniemi, 2019). Black shale is a natural source of heavy metal pollution because weathering, mining, and road construction can result in direct input of HMs to surrounding soils, and then lead to negative effects on soil environmental quality. Particularly, the weathering of HMs-enriched black shales may be one of the most important sources of environmental contamination in the black shales distributed area Peng et al., 2004). The abundance of organic matter and sulphide minerals in black shale make it susceptible to chemical weathering, and produce acid rock drainage after exposed to oxic surface environment (Nordstrom, 2011;Ling et al., 2015;Fru et al., 2016), and subsequently resulted in acidi cation and elevated trace element concentrations in the soils which developed on top of black shales (Cappuyns et al., 2019). Further, the natural weathering processes are enhanced by anthropogenic activities that bring black shale to the surface, and consequently facilitate the release of HMs, transferring into the surrounding soils, water systems, and then translocation occurs to plants and agricultural products cultivated on black shale soils, leading to environmental pollution and human health threats (Liu et al., 2017;Duan et al., 2020). Therefore, it is worth giving more attention to the migration and dispersion of HMs liberated by black shales, as well as the phytoremediation of black shale -associated soils.
It is believed that mosses are primitive terrestrial higher plants and characterized by a simple structure and large surface area as well as no real root systems. Generally, the elements observed in moss depend on deposition and rainfall for nutrients, and subsequently they are used as biomonitors is a wellrecognized technique in studies of atmospheric deposition. Several studies have been conducted on HMs present in mosses associated with gold, zinc, copper and mercury production (Smirnov et al., 2004;Cymerman et al., 2006;Bi et al., 2006;. However, mosses uptake the mineral components partly from the soil substrate, and in uenced by the soil composition. Thus, some authors suggest that mosses also can translocate the metal elements from the soil and conduct them internally (Kłos et al., 2012;Sabovljevć et al., 2020), because they can also perform cushion-like growth, and being more in contact with the soil substrate. Therefore, the pollutants observed in moss samples may originate from geological, biological, and wet and dry deposition sources.
Many moss species play essential roles in water retention and pedogenesis (Jia et al., 2014), they absorb water bearing dissolved mineral elements but also other compounds over their entire surface (Sabovljevć et al., 2020). When mosses appear in soil contamination, the great cation exchange capacity (CEC), absence of a cuticle, and one-cell-thick leaves enable them incapable of avoiding heavy metal adsorption, and as lack stomata, mosses are unable to screen airborne pollutants by closing stomata during stress (Glime, 2007). Hence, moss can e ciently enrich HMs from the polluted environment. Some aquatic bryophytes have been shown to be bioaccumulators of trace metals , and Scopelophila ligulata has been recognized as a Fe-hyperaccumulator because Fe concentration in Scopelophila ligulata was 10-61 times higher than that in normal mosses (Nakajima and Itoh, 2016). Thus, from the perspective of phytoremediation intention, moss species might be considered as a candidate for phytoremediation in heavy metal contaminated areas.
The Lower Cambrian black-rock-series is widely distributed in the Yangtze Platform of South China, and there is regionally developed a typical conformable polymetallic sul de horizon. Guizhou province is one of the typical areas of its development, and it should be emphasized that the eastern Guizhou is an important area for the development of black rock series stratabound metal deposits. In this area, there was a set of black shale rocks between the Dengying Formation and the Liuchapo Formation distributed in Sansui-Shibing-Tianzhu-Majiang region, which was characterized by large-scale vanadium ore and barite ore (Yang et al., 2013;Wang et al., 2016;Wei et al., 2017). In some weathering pro les formed by mining and road construction, few vascular plants can survive in the poor, acidi ed, and arid soil overlying black shales, but some moss species can grow normally and ourish in this challenging environment. However, few information is available on the distribution and migration of HMs, as well as the phytoremediation potential of moss species in the black shale areas.
In this study, the Yacha and Gaoqiao areas underlain by black shales were selected, (I) to investigate the dispersion and distribution characteristics of HMs in rock -soil -moss system; (II) to evaluate the enrichment levels of HMs in soils overlying black shales; (III) to determine whether moss can be used to as a pioneer plant for phytoremediation of HMs in the black shales district.

Research area
The research areas are located in Sansui County (26°47´-27°04´N, 108°32´-109°04´E) southeast of Guizhou Province, China (Fig. 1). They are typical black rock series vanadium mine that contained a variety of associated HMs (Ag, Ba, Co, Cu, Ga, Mo, Ni, and Pb), the concentrations of vanadium pentoxide (V 2 O 5 ) in rock samples range from 0.07-1.28%, with an average of 0.40%, and part of the rock samples reach the industrial grade (Deng, 2014). The exposed stratum in the study area are mainly of the Niutitang Formation of the Lower Cambrian, and the lithology including black shale, mudstone, and siliceous rock. The average annual temperature is about 15 ℃ and an average annual precipitation is ~ 1147 mm, and the altitude ranges from 450 m to 1470 m. Moreover, the study areas have subtropical climate which is controlled by east Asian monsoon, and this warm and moist climate conditions are favorable for black shales weathering.

Sample collection and pretreatment
In August 2019, we collected a total of 39 samples of mosses, growing soil, and corresponding parent rocks from 13 different sampling sites in the two study areas (Fig. 1), and the information of sampling sites was provided by global positioning system (GPS). The sampling procedure of mosses in this study was referred to wang et al. (2015). Brie y, ve 10 × 10 cm 2 quadrats were established in each site, and then a nal sample (moss, soil, and rock, respectively) was composed of 5 subsamples were carefully packed into polyethylene bags to avoid cross-contamination. In particular, the entire surface layer of the mosses cover in each quadrat was removed down to the growing soil using a sampling knife, which was washed between samplings. After collection, samples were delivered to the laboratory to determine the species and the corresponding statistics.
Species of mosses were identi ed with classical morphological identi cation techniques, using an anatomical lens (HWG-1) and a microscope (XSZ-107), and referred to the atlas"Moss Flora of China".
Mosses samples were classi ed into 7 species in 5 families. Among all the mosses, Pohlia exuosa Harv was luxuriant and spontaneous growth throughout the study areas, accounting for approximately 70% of the total (Fig. 1). Therefore, the P. exuosa Harv was selected as the main moss species to be studied in this study.
It was found that the biomass of mosses growing at some sites was relatively low (the total length of the moss was less than 1cm), and they were not easy to separate the rhizoid and shoot. Thus, four moss samples in each study area, which grew normally and the length of the moss was greater than 3cm, were selected to investigate the distribution of HMs in different mosses tissues (rhizoids and shoots).
All the mosses samples were washed three times with deionized water (18.2 M Ω·cm, 25 ℃) until dust and foreign substance were removed, and each sample was rinsed with the deionized water after being cleaned for 15 min by the ultrasonic cleaner (power ratio: 100%; frequency: 25 kHz) in clean water. And all the samples were dried in a thermostatic air-blower-driven dryer at 45℃ until constant weight. Then, these mosses samples were ground by the portable high speed universal grinder (50 g, AQ-180 E-X, Nail Machinery Ltd., Ningbo, Zhejiang Province, China). Subsequently, the mosses samples were sieved through an 80-mesh sieve (0.18 mm) and homogenized in the laboratory. In addition, rock and soil samples were disaggregated, sieved to − 10 mesh (2 mm), quartered and pulverized in a porcelain mortar to − 200 mesh (0.074 mm), rehomogenized, and repackaged in polyethylene bags for further analysis.

Sample analyses
The soil pH was measured in a 1:2.5 (soil: water w/v) mixture by a glass electrode pH meter (PHS-3E, Shanghai, China). OM was measured by the classic low-temperature external heat potassium dichromate oxidation-colorimetric method (Cao et al., 2020). In addition, all samples were sent to an accredited laboratory (ALS Minerals-ALS Chemex Co. Ltd. Guangzhou, China) to determine the concentrations of major and trace elements. The analysis procedure for the rosk and soil samples was as follows Zhang et al., 2020).
Each sample was digested in two methods. Firstly, 0.25 g of sample was accurately weighted and digested by a concentrated acid mixture (a ratio of 1 : 2.5 : 2 : 2.5 for the HClO 4 : HNO 3 : HF : HCl) in an oven at ~ 190℃for 48 h, cooled to room temperature, heated on a preheated hot plate (150℃) to get rid of excess acid until crystalline solid was formed, and diluted to a steady volume (12.5 mL) with 2% hydrochloric acid. The nal solution was then analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES, America, Varian VISTA) and inductively coupled plasma mass spectrometry (ICP-MS, America, Agilent 7700x). The other sample (0.50 g) was dissolved in aqua regia (1:3, v/v: HNO 3 and HCl), digested slowly at ~ 190℃for 48 h in a graphite heating block, and then placed and heated on a preheated hot plate (150℃) under a fume hood until white fumes appeared and crystalline solid was formed. Afterwards, the resulting solution was diluted to volume with deionized water, mixed and analyzed by ICP-AES, followed by ICP-MS for the remaining suite of elements. According to the actual characteristics of the sample, the digestion effect and interelement spectral interferences, the comprehensive value was the nal test result. As for the moss samples, 1.0 g moss powder was accurately weighted and added with 5 mL HNO 3 (ultra-pure grade) into a Te on digestion vessel and digested ~ 8 h slowly at room temperature, and then heated for ~ 3 h on a preheated hot plate (150°C) under the fume hood. The remaining solid crystal was dissolved and transferred into a volumetric ask after cooling; the nal volume was precisely adjusted to 25 mL using 2% hydrochloric acid. And then mixed thoroughly respectively and analyzed by ICP-MS.

Pollution assessment of HMs in soil substrates
The geo-accumulation index (I geo ) was rst proposed by Muller, which has been widely used to assess the degree of HMs pollution in soil and sediment (Muller, 1969). Rahn presented a useful way to determine whether a particular element was found in greater abundance than what might be expected from crustal sources, introducing the concept of "enrichment factors" (Rahn, 1971). In this study, I geo and EF are applied to determine the level of HMs contamination and the degree of HMs enrichment in soil, respectively. They are calculated using the following equations: (1)

The bioconcentration factor (BCF)
BCF was an important quantitative indicator of plant contamination and has commonly been used for estimating metal transfer from soil to plants. It represented the ratio of the element concentration in the plants to that in the soil (Fayiga et al., 2004). In this study, it is used to evaluate the accumulation capacity of HMs in moss and calculate through the following equation: BCF is the bioconcentration factor of the HMs in mosses, C plant is the metal element concentration in the moss, and C soil is the elemental concentrations in the growing soil of the mosses.

The translocation factor (TF)
TF was de ned as the ratio of element concentrations in the aerial parts to those of the roots (Zhang et al., 2012;Zou et al., 2012). In this study, it is calculated to assess the capability of mosses to accumulate the metal element, absorbed by rhizoid, into the shoot. It is calculated as follows: Where TF is the translocation factor of the metal element in moss, and C ngt is the metal element contents in the shoots, and C rhi is the metal element contents in the rhizoids.

QA/QC
Quality assurance and quality control for the HMs analyses were validated using duplicates, method blanks, and standard reference materials (SRM). Standard reference materials (SpL03 representing plant samples, GBM908-10 representing soil samples, and MRGeo08 representing rock samples) were analyzed. The method blank was lower than the detection limits in all samples, and their HMs recoveries were 90-110%. The relative standard deviations of the HMs in the soil substrate and mosses samples for the duplicate analysis were both < 10%.

HMs concentrations in black shales
Ranges and mean concentrations of HMs in black shales of the Yacha and the Gaoqiao areas are shown in Table 1 Table 2

The results of soil heavy metals pollution evaluation
As shown in Fig Cd > Mo > Pb > As. Likewise, Cd exhibits the relatively high variation coe cient of all elements (163.1% in YC and 84.1% in GQ, respectively), this is very consistent with the rock and soil samples. In addtion, the average concentration of Cu was signi cantly higher than that of other HMs (P < 0.05), and part of moss samples have a Cu content greater than 1000 mg kg − 1 , which is probably due to the high copper content in the corresponding soil (Table 2). However, the average concentration of Cu (847.4 mg kg − 1 in YC, 688.6 mg kg − 1 in GQ, respectively) in all moss samples is lower than 1000 mg kg − 1 , indicating that P. exuosa Harv is not a hyperaccumulator for the investigated elements. Table 4 Heavy metal concentrations of P. exuosa Harv (mg kg − 1  (1) Standard deviation; (2) VC (Variation Coe cient (%)) = standard deviation*100/mean.

Bioconcentration factors and transfer factors of heavy meals in P. exuosa Harv
A plant's ability to accumulate metals from soils can be estimated using the BCF, according to A plant's ability to translocate metals from roots to shoots is measured using the TF, which is de ned as the ratio of metal concentration in the shoots to the roots. In this study, P. exuosa Harv is not an accumulator of most HMs in shoots, and their transfer to its shoots parts from its rhizoids is restricted.
As shown in Table 5, the content of all the investigated metals in shoots of P. exuosa Harv are lower than rhizoids except for Cd, and the average TFs in all the sites of Cd, As, Pb, Cu, Cr, Ni, and Mo are 1.49, 0.29, 0.48, 0.56, 0.28, 0.90, and 0.59, respectively, ( Fig. 3B). Similarly, Cd exhibit the maximum TF value compared to other metal elements, which is consistent with the BCF of Cd. Table 5 Range and mean of heavy metal concentrations in P. exuosa Harv tissues (mg kg − 1 ).  . In this study, the concentration of typical mineral elements in P. exuosa Harv tissues (rhizoids and shoots) are also given in Table 5. The average content of Mg and P in rhizoids are higher than that in shoots, whereas the average content of K and Zn in rhizoids are lower than that in shoots in both study areas. As shown in Fig. 4, the average TF in all the sites of Mg, P, K, and Zn are 0.92, 0.64, 2.35, and 1.04, respectively. Interestingly, the TFs of K and Zn are great than 1, and signi cantly higher than that of Mg, this trend is well in agreement with the found TF of Cd, imply that the translocation of Cd in shoots may have a synergistic effect with K and Zn. Table 5 Ranges and means of mineral element concentrations in P. exuosa Harv tissues (mg kg − 1  4. Discussion

Parent rock -soil -moss relationships
As shown in Fig. 5, all the investigated elements have the similar distribution trend in the rock-soil-moss system. That is, the black shales parent rocks have elevated HMs concentrations and act as a source of HMs. It is widely accepted that extreme acidity and sul de minerals were usually related to high solubility of HMs, and therefore increased concentration in the soil (Sabovljevć et al., 2020). Here, we noticed that the soil inherited and enriched metal elements obviously which released from black shale, leading the investigated HMs concentrations in soil are higher than that in the corresponding black shales parent rocks. It was believed that heavy metal elements released from black shales (such as sul des, silicates, and carbonates) may migrate as ions under acidic conditions, and soil OM played a key role in capturing and retaining these metal ions (Pašava et al., 2003;Perkins et al., 2015). In this study, higher OM contents (average 16.73% for YC, average 10.74% for GQ, respectively) determine more metal sorption sites and more metal chelators, and facilitate the increased accumulation and retention of HMs (Shu et al., 2016). Therefore, the released HMs from the weathering of black shale parent materials contribute considerably to their accumulation and pollution in soils.
A hot issue is whether the HMs in mosses mainly originate from atmospheric deposition or from the soil substrate. Some moss species were used to monitor the atmospheric deposition of trace elements (Smirnov et al., 2004;Cymerman et al., 2006). However, the geochemical properties of hosting soils have been shown to have a signi cant effect on the elemental concentrations in plants. We also nd from Fig. 5 that the gradient diffusion of all the investigated HMs decreasing from soil substrate to rhizoids, and from rhizoids to upper shoots (except for Cd) is obviously. This nding is similar to the results of Sabovljevć et al. (2020). Moreover, taking all sites into consideration, signi cant linear relationship correlations (except for Pb) are found between the concentration data of the same elements in P. exuosa Harv and corresponding soil (Fig. 6), con rming that HMs are mostly originating from soils substrate that P. exuosa Harv lives on and less from the atmospheric deposition. Mosses are plants with relatively simple morphology, but unistratose leaves and uniseriate rhizoids provide a large surface area for cation exchange, allowing the free uptake of dust particles and droplets of moisture (Wang et al., 2015). Thus, the metals-containing dust particles derived from the black shale parent materials are absorbed and utilized by the P. exuosa Harv. This is well in agreement with the reported, con rming that the uplifting of the pollutant-containing dusts from the soil or substrate were absorbed and translocated by mosses (Kłos et al., 2012;Sabovljevć et al., 2020). Therefore, as mentioned above, black shale parent rocks act as the major source of heavy metal pollutants, and then cause geogenic pollutants input into soil and P. exuosa Harv in the black shale distributed areas.

Tolerance mechanisms for heavy metals in P. exuosa Harv
A tolerant plant has speci c physiological mechanisms that collectively enable it to function normally even in the presence of high concentrations of HMs. In this study, few plants can survive in soil overlying black shales that is highly acidic and rich in multi-metals, but P. exuosa Harv are capable of growing and ourishing in this challenging environment. More speci cally, P. exuosa Harv grow normally and function well in soil contaminated heavily (up to 486.0 mg kg − 1 Cd and 2220 mg kg − 1 Cu in YC-2 site), suggesting that moss growing on the soil are tolerant of these metals. Thus, P. exuosa Harv exhibit a high tolerance to multiple metals, and might be a promising candidate for remediation in contaminated soils derived from black shales.
In this study, the average BCFs for all the HMs of P. exuosa Harv are lower than 1, indicating that P. exuosa Harv can insulate HMs in contaminated growth soil to reduce their effect on its tissues ( Fig. 2A). Likewise, all the HMs concentrations of the shoots are lower than those in rhizoid (TF < 1) except for Cd (Fig. 2B), indicated that P. exuosa Harv tend to accumulate and retain HMs in their rhizoids. Uniseriate rhizoids of P. exuosa Harv provide a large surface area for cation exchange, allowing the free uptake of dust particles that were derived from the black shale soil. Thus, it is believe that P. exuosa Harv can immobilize and retain HMs through absorption and accumulation by rhizoids, adsorption onto rhizoids, or precipitation within the rhizosphere (Yoon et al., 2006). A similar result was found in the previous study conducted in indigenous zinc smelting area that a tolerant plant (Juncus effusus) can immobilize HMs through absorption and accumulation by root, and caused BCF and TF lower than 1 for most HMs . This might be a protective behavior for the mosses to accumulate HMs in the rhizoids vacuoles and limit their transportation to the upper shoot tissues, and some studies have found similar results (Shanker et al., 2005;Huang and Wang, 2010). Overall, P. exuosa Harv is a plant that is tolerant of high concentration of HMs and restricts transferring these metals from the soil to its rhizoids and from the rhizoids to the shoots. Restriction of upward movement from rhizoids into shoots can be considered as an important tolerance mechanism for P. exuosa Harv.
Plants were de ned as hyperaccumulators when they accumulate more than 100 mg kg − 1 of Cd or more than 1000 mg kg − 1 of As, Cr, Cu and Pb (Baker et al., 1994). The average content of all the investigated metal elements in P. exuosa Harv is not greater than 1000 mg kg − 1 ( In general, mosses have lower biomass compared with vascular plants, which might limit their potential for broad applications. However, the lower biomass might not be a problem, because the main functions of Moss-dominated biological soil crusts including resistance to erosion and increasing soil OM content (Belnap et al., 2004;Lange and Belnap, 2016), which facilitates the accumulation and retention of HMs instead of migrating to other environmental medias (e.g., paddy soil, surface water, and sediment, etc).
Moreover, mosses are less likely to be preyed by animals, thus reducing the risk of HMs into the high trophic level via food chain. Therefore, as mentioned above, we suggest that P. exuosa Harv may be considered as an excellent candidate for phytostabilization in the black shale distributed areas where vascular plants are rare.

In uences of Cd on the absorption of mineral elements in P. exuosa Harv
Compared with other heavy metal elements, only Cd has a transfer factor greater than 1, indicating that P.
exuosa Harv growing on the soil contaminated by multiple metals are most e cient in translocating Cd and accumulating signi cant levels of Cd in upper shoots. This may be attributed to ine cient immobilisation of Cd by the rhizoids of P. exuosa Harv, resulting in their e cient translocation and accumulation to shoots, as evidenced by the acidi ed soil (Table 2) and increase the potential for Cd uptake at the lower pH. Thus, P. exuosa Harv growing at the black shale soils exhibited increased Cd levels in their shoots compared to their rhizoids, which may represent a strategy for Cd toxicity reduction, by which P. exuosa Harv take up harmful Cd to non-photosynthetically active organs such as shoots to alleviate Cd toxicity by biomass diffusion. Moreover, it is worth noticing that the nonessential Cd can indirectly affect plant growth by in uencing the absorption of mineral nutrients during this process (Feng et al., 2018). Signi cant correlation between the TFs of metal elements indicated that plants had a synergistic effect on the absorption of these metals (Yoon et al., 2006). As shown in Fig. 7, there are signi cant correlations between the TF of Cd and K, Zn in P. exuosa Harv samples (p < 0.05), this means the P. exuosa Harv, which is effective in translocating Cd, is also effective in translocating K and Zn and vice versa. Indeed, it has been reported the increasing Zn 2+ activities plays an important role in the increasing transfer ratio of Cd from root to shoot (Cai et al., 2019), con rming that rhizoids-to-shoots mineral elements (K and Zn) transport is stimulated by nonessential Cd.
Plants need to prevent damage from non-essential metals and ensure the proper homeostasis of essential elements. Previous studies showed that Zn-Cd interaction was important because of their chemical properties were similar and sharing transporters, and synergistic interactions were observed in . Thus, potential synergistic absorption of Cd on K and Zn in P. exuosa Harv is realizable in this study, con rming that the mechanism underlying the regulation of mineral element absorption might exist in P. exuosa Harv to alleviate Cd toxicity. Overall, the normal functioning of K and Zn absorption and transportation might contribute to its high tolerance to Cd.

Conclusions
The black shale rock samples are highly enriched in HMs and may cause geological pollution for the surrounding environment. Also, the concentration of HMs in black shales are characterized by a high variability for the trace elements, especially Cd, Cr, and Ni, indicating an overall chemical heterogeneity within the black shales parent rocks. Also, black shale acts as a release source of metal elements, leading to signi cant inheritance and enrichment of these metals in the soil. Signi cant correlation of HMs between P. exuosa Harv and growing soil indicating HMs in P. exuosa Harv are primarily originating from growing soils and less from the atmospheric deposition. P. exuosa Harv can naturally grow in and cover the black shale areas where vascular plants are rare, and the BCFs and TFs of all metal elements are not higher than 1 (except for Cd), indicating that moss might be considered as a promising pioneer plant for phytoremediation in the black shale areas.
Our study show that Cd affects the absorption and transport of K and Zn by P. exuosa Harv obviously, and a mechanisms underlying the regulation of K and Zn absorption might exist in P. exuosa Harv to resist Cd toxicity. However, more case studies on the detoxi cation of Cd by the absorption and metabolism of K and Zn in P. exuosa Harv are needed.