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Mercury vapor emissions from the Ingenios in Potosí (Bolivia)
Abstract
Potosí (South West Bolivia) is a well known historical mining site in the world, with mining activity centered at the so-called Cerro Rico. It is an impressive mount formed by rhyolitic rocks affected by intensive hydrothermal alteration, and hosting a complex vein deposit including mainly Ag and Sn minerals. From the start of the mining activity, in the late 16th century, to 1850, the main ore was silver minerals, and from 1850 the silver ores exhausted, and mining activity centered on tin minerals. During the first stage, the silver minerals were treated by amalgamation, using the so-called “método de patio”, which implied the usage of mercury and other compounds as metallurgical agents, and supposed the release of important quantities of mercury to the local environment. This work was carried out at the “ingenios”, milling and mercury processing facilities located next to streams, in order to have the water and mechanical energy needed for the process, and nowadays in ruins. Our results put forward very low mercury vapor concentrations in the region, reaching only occasionally values over 4ng m−3, as well as in the town area, were maximum values reach 31ng m−3 with an average of 5.5ng m−3; detailed surveys at the “Ingenios” demonstrated that in these facilities mercury vapor concentrations were also low, but the excavation of the topsoil causes an important release of the elemental vapor, reaching concentrations over 3000ng m−3. Causes of this low emission of unmodified soil are here interpreted as caused by biological and physicochemical transformation of the metallic mercury accumulated in the soil, to mineral phases such as cinnabar/metacinnabar and/or schuetteite, in reactions mediated by the formation of methylmercury.
... Chiarantini et al. 2016;Hines et al. 2000;Garcia-Ordiales et al. 2017Gray et al. 2014Gray et al. , 2015 is the result of runoff or drainage of mine waste, including the calcines produced during roasting of the ore (Rytuba 2000). Mercury in particular, being a volatile element, can be released to the atmosphere in gaseous elemental mercury (GEM) form from surfaces where Hg can already be present in the substrate as native Hg, as a primary mineral, together with the sulphide (cinnabar) (Higueras et al. 2012;Loredo et al., 2007), or as a by-product of ore processing (Gray et al., 2010;Kotnik et al. 2005). Moreover, the evasion of GEM usually derives from the reduction of Hg 2+ forms to Hg 0 through both abiotic and biotic pathways mainly controlled by solar radiation, and air and soil temperatures (Choi and Holsen 2009;Wang et al. 2005). ...
... A growing number of recent and on-going studies have examined the characteristics of (Cu, Sb, As)-rich minerals from the tetrahedritetennantite group as source of contaminants, the geochemical behaviour of PTEs at such mining sites, and the subsequent environmental issues related to PTE mobility (e.g. Borčinová Radková et al. 2017;Higueras et al. 2012;Majzlan et al. 2018). However, little information is currently available regarding the role of such minerals in terms of the release of PTEs into riverine water and the atmosphere. ...
... Slight increases in GEM concentrations from background levels (up to ~10 ng/m 3 ) were observed near the entrances of the "Finsepol" and "Mulazzani" mine galleries, likely due to the occurrence of Hg in mineral phases similar to that observed by Higueras et al. (2012) in the Potosì mining area (Bolivia), where the occurrence of tetrahedrite was reported. The values of GEM in the air observed in that area were similar to those found in this study at Mt. Avanza, although concentrations from hundreds to thousands of ng/m 3 are frequently reported in sites characterised by notably elevated Hg contents in substrates, such as near larger former Hg mines and cinnabar roasting plants (e.g. ...
The decommissioned fahlore Cu-Sb(-Ag) mine at Mt. Avanza (Carnic Alps, Italy) is a rare example of exploited ore deposits, as the tetrahedrite (Cu6[Cu4(Fe,Zn)2]Sb4S13) is the main ore mineral found. This multi-compartmental geochemical characterisation approach provides one of the first case studies regarding the geochemical behaviour and fate of Hg, Sb, As, Cu, and other elements in solid and water matrices and of Hg in the atmosphere in an environment affected by the mining activity of a fahlore ore deposit. Elevated concentrations of the elements (Cu, Sb, As, Pb, Zn, Hg) associated with both (Zn-Hg)-tetrahedrite and to other minor ore minerals in mine wastes, soils, and stream sediments were observed. Concentrations in waters and stream sediments greatly decreased with increasing distance from the mining area and the Igeo index values testify the highest levels of sediment contamination inside the mine area. Thallium and Ge were associated with the “lithogenic component” and not to sulfosalt/sulphide minerals. Although mine drainage water often slightly exceeded the national regulatory limits for Sb and As, with Sb being more mobile than As, the relatively low dissolved concentrations indicate a moderate stability of the tetrahedrite. The fate of Hg at the investigated fahlore mining district appeared similar to cinnabar mining sites around the world. Weak solubility but the potential evasion of gaseous elemental mercury (GEM) into the atmosphere also appear to be characteristics of Hg in fahlore ores. Although GEM concentrations are such that they do not present a pressing concern, real-time field surveys allowed for the easy identification of Hg sources, proving to be an effective, suitable high-resolution indirect approach for optimising soil sampling surveys and detecting mine wastes and mine adits.
... The Hg T concentrations were higher in urban areas (median: 14.7 μg g −1 , range: 0.177 to 620 μg g −1 ) than rural areas (median: 7.04 μg g −1 , range: 0.068 to 10.6 μg g −1 ). These values are in good agreement with Hg concentrations found in present-day soil near historical Ag mining sites in Potosí of Bolivia (up to 155 μg g −1 ) (Hagan et al., 2011;Higueras et al., 2012), and in the vicinity of former haciendas in Cedral of Mexico (up to 116 μg g −1 in soil and 548 μg g −1 in amalgamation wastes) (Leura Vicencio et al., 2017;Morton-Bermea et al., 2015). The 2-ME extraction method showed the presence Hg I (and Hg II ) in all the 29 samples analyzed (Table S3). ...
... Much higher GEM concentrations were found in the interstitial air at the reprocessed mineral waste and sediment/soil sites within the urban area of Guanajuato (Fig. S2). The maximum GEM value of 44,700 ng m −3 was observed at Plaza Ranas near a former hacienda, which is over an order of magnitude higher than that reported beneath the topsoil of a former patio site at the Cerro Rico mine in Potosí, Bolivia (3000 ng m −3 ) (Higueras et al., 2012). ...
... Similar studies should also be considered for other historical Ag mining regions that employed the patio process. For example, elevated Hg concentrations have been reported in the soil and river sediment in the vicinity of former haciendas in Cedral of Mexico (Leura Vicencio et al., 2017;Morton-Bermea et al., 2015), Potosí of Bolivia (Hagan et al., 2011;Higueras et al., 2012;Hudson-Edwards et al., 2001) and Puno of Peru (Kennedy and Kelloway, 2020). ...
Silver (Ag) production in Hispanic America between the 16th and 19th centuries is thought to be one of the largest sources of anthropogenic mercury (Hg) emissions in history. Recent reviews of the chemistry behind the patio process, which used Hg amalgamation to extract Ag from ore, reveal that a large amount of the Hg may not have been immediately released to the atmosphere; instead, it may have been captured in the form of calomel (Hg2Cl2, in which Hg exists as monovalent HgI) and remained in the local environment. Here we show that Hg used in the patio process centuries ago in the Guanajuato Mining District of Mexico continues to elevate present-day concentrations of gaseous elemental mercury (GEM) throughout the region. In the ground-level air, GEM ranged from 8 to 454 ng m-3, exceeding the Northern Hemispheric average (~1.4 ng m-3) by up to over two orders of magnitude. Much higher concentrations, up to 44,700 ng m-3, were found in the interstitial air of reprocessed mineral waste, sediment, and soil. These highly elevated present-day GEM values are due, at least in part, to the disproportionation of legacy calomel, as supported by the presence of HgI in the reprocessed waste and by the GEM release pattern from calomel disproportionation. Our results imply that the contribution of historical Ag refining to atmospheric Hg emissions must be re-evaluated to account for calomel and its subsequent disproportionation and releases of GEM to the present-day.
... Anomalous Hg emissions, including formation of modern Hg deposits, mark mantle plumes (e.g., Hawaii and Iceland), midocean ridges, volcanic arcs, geothermal fi elds, and active faults (Boström and Fischer, 1969;Eshleman et al., 1971;Aston et al., 1972;Carr et al., 1974Carr et al., , 1975Coderre and Steinthorsson, 1977;Ozerova, 1977;Jin et al., 1989;Stepanov, 1997;Stoffers et al., 1999;Nriagu and Becker, 2003;Rychagov et al., 2009;Kalinchuk and Astakhov, 2014). Processes such as fuel combustion (mainly coal) and artisanal gold mining are the most signifi cant anthropogenic sources of Hg emissions into atmosphere (Higueras et al., 2012;Dalziel and Tordon, 2014;Gworek et al., 2017;Mashyanov et al., 2017;UN Environment, 2019). Elemental mercury (Hg 0 ) vapour comprises more than 95% of the atmospheric Hg species (Sprovieri et al., 2016). ...
... Soil temperature, moisture, redox potential, permeability, and organic matter are important factors that infl uence Hg vapour concentration in soil gas (McCarthy, 1972;Chengliang et al., 1989;Schuster, 1991;Zhang and Lindberg, 1999;Choi and Holsen, 2009;Gu et al., 2011;Yasutake et al., 2011;Higueras et al., 2012;Moore and Castro, 2012;Xie et al., 2019;Esbríl et al., 2020). Chengliang et al. (1989) suggest that Hg in overburden (up to 40 m thick) over the blind Fankou Pb-Zn deposit (>200 m below the surface) in Guangdong (China) occurs mainly as free vapour and adsorbed onto soil colloids such as clays and Fe-Mn oxides. ...
... Such a strong Hg emission from the tailings suggests that Hg amalgamation might have been used to recover gold from crushed ore at the Bentley mine. Mercury amalgamation was extensively used by the mining industry between the mid-1800s and early 1900s (e.g., Higueras et al., 2012;Dalziel and Tordon, 2014). A few weak Hg anomalies (1.7-3.6 ng·m -3 ) also occur on a ridge immediately south of Bentley occurrence. ...
Volatile geogenic components, such as CO2, He, Rn, and Hg, form haloes in soil gas and near-surface air directly above mineral deposits. This contrasts with lithochemical, hydrochemical, and biochemical dispersion haloes that can be laterally displaced or obscured by transported overburden. Mercury vapour surveys have been used in geochemical exploration, because Hg occurs in most types of endogenic ore deposits and is highly mobile. In this study, we measured Hg vapour in air 1-50 cm above ground at 15 sites on Vancouver Island. To evaluate the effectiveness of the method across a range of settings, these sites include different types of known mineralized zones, barren rocks, and faults, both buried and exposed. The direct and continuous analysis via a portable RA-915M mercury analyzer reveals Hg vapour concentrations ranging from 0.5 to 54.4 ng·m-3. The highest Hg concentration was observed above tailings at the Bentley Au occurrence, possibly due to the amalgamation technique used for fine gold extraction between late 1800s and early 1900s. Prominent Hg vapour haloes mark shear-hosted Cu-Ag-Au sulphides at Mount Skirt (13.4x background Hg), epithermal Au-Ag-Cu at Mount Washington (8.9x background Hg), and sediment-covered polymetallic volcanogenic massive sulphide at the Lara-Coronation occurrence (4.2 to 6.6x background Hg). Basalt-hosted Cu-Ag-Au sulphide zones at the Sunro past producer are marked by weak Hg vapour anomalies relative to local background. Faults, including the Leech River fault, which was active in the Quaternary, are also marked by weak Hg vapour anomalies. The study confirms that, although the Hg level is influenced by weather, the real-time Hg vapour measurement of near-surface air can instantly delineate mineralized zones and fault structures that are buried under overburden 10s of m thick. In contrast to soil gas sampling, this simple and rapid technique can be applied to mineral exploration and geological mapping under overburden above any type of surface, including outcrops, talus, bogs, water bodies, snow, and permafrost.
... Another aspect to be considered is the length of time that has passed since the strong Hg inputs in the historic roasting area. Although it was assumed that other than atmospheric emissions, large amounts of Hg 0 were lost in the environment either via spillage or in roasted residues directly discharged into the soil [52], it is possible that this species escaped into the atmosphere or was oxidised and then bound to OM, a process that can quickly occur in oxidised soil layers [107,108]. Moreover, oxidised Hg bound to the more soluble fraction of the OM may have leached to deeper soil layers [43]. ...
... The topsoil from this plot was characterised by a lower Hg and OM content Another aspect to be considered is the length of time that has passed since the strong Hg inputs in the historic roasting area. Although it was assumed that other than atmospheric emissions, large amounts of Hg 0 were lost in the environment either via spillage or in roasted residues directly discharged into the soil [52], it is possible that this species escaped into the atmosphere or was oxidised and then bound to OM, a process that can quickly occur in oxidised soil layers [107,108]. Moreover, oxidised Hg bound to the more soluble fraction of the OM may have leached to deeper soil layers [43]. ...
Considerable amounts of gaseous elemental mercury (Hg⁰) can be released into the atmosphere from Hg-enriched substrates, such as those from former mining areas, posing a potential environmental threat. In this work, Hg⁰ fluxes at the soil–air interface under natural vegetation covers were measured in various locations within the Idrija Hg mining area (Slovenia) and its surroundings. Sites were selected in order to compare Hg⁰ fluxes from both forest soils heavily impacted by historical ore roasting and urban soils characterised by a different degree of Hg enrichment due to the natural occurrence of Hg in rocks or recent mining and roasting processes. Replicate measurements at each site were conducted using a non-steady state flux chamber coupled with a real-time Hg⁰ analyser (Lumex RA-915M). Moreover, topsoil samples (0–2 cm) were analysed for Hg total concentration and speciation. Cinnabar was the predominant Hg form in almost all the sites. Despite Hg⁰ being undetectable in soils using thermo-desorption, substantial emissions were observed (70.7–701.8 ng m⁻² h⁻¹). Urban soils in a naturally enriched area showed on average the highest Hg⁰ fluxes, whereas relatively low emissions were found at the historical roasting site, which is currently forested, despite the significantly high total Hg content in soils (up to 219.0 and 10,400 mg kg⁻¹, respectively). Overall, our findings confirm that shading by trees or litter may effectively limit the amount of Hg⁰ released into the atmosphere even from extremely enriched soils, thus acting as a natural mitigation.
... Low TGM concentrations are normal in areas that are heavily polluted with Hg (but with long-term undisturbed soils) such as mining areas (e.g. Potosí [40,41] and Venezuela [30]). The causes of these low emissions have been interpreted as being due to oxidation of metallic Hg droplets present in the soil [30] and to the formation of stable, non-emitting Hg compounds in the soil, such as metacinnabar (isometric, black HgS) [40] and calomel (Hg 2 Cl 2 ) [41]. ...
... Potosí [40,41] and Venezuela [30]). The causes of these low emissions have been interpreted as being due to oxidation of metallic Hg droplets present in the soil [30] and to the formation of stable, non-emitting Hg compounds in the soil, such as metacinnabar (isometric, black HgS) [40] and calomel (Hg 2 Cl 2 ) [41]. The surroundings of the Usagre Hg mine correspond to pastures with a very low level of agricultural activity, so any of these mechanisms could explain the low TGM emissions from these relatively undisturbed soils. ...
Usagre (Badajoz, SW Spain) mercury mine was active for an undetermined time until its closure in 1971. The ore exploited in this mine was cinnabar (red HgS), and metallurgy was also performed locally by means of primitive furnaces of the Bustamante type. Since the closure of the mine, reclamation measures have not been carried out at the site, and actual passives include a mine shaft, an accessible descending gallery and a mine dump adjacent to the descending gallery. In the work described here, data from two soil geochemistry surveys were combined and analysed. The surveys were separated by a period of 32 years. Measurements of total gaseous mercury (TGM) in the underground mine and its surroundings were also considered. The soil geochemistry included mercury, lead, zinc and copper. The results indicate that soil pollution is mostly related to the trace mineralisations on the surface and they can therefore be interpreted as natural geochemical anomalies. TGM concentrations are extremely high inside the mine but are of very low concern outside the mine.
... But just 10 cm depth below the surface, we observed GEM concentration in the soil interstitial air that were frequently in the range of 10 2 -10 4 ng m − 3 , similar to those measured near fumaroles and other thermal features (Table S7). At some locations, GEM concentrations were several hundred-to several-thousand-times higher than at the surface; the highest of these (on the order of 10 5 ng m − 3 ) represent extreme Hg enrichment for a natural system, and are more similar to soil GEM measured at mining waste sites that involved the use of Hg (e.g., Higueras et al., 2012;Loria et al., 2022). In other cases, typically in areas outside of the more active geothermal features, subsurface GEM was <10 ng m − 3 , within the range of values reported for background soils at similar depths (e.g., Gray et al., 2015;Sigler and Lee, 2006). ...
... Even though the patio process was phased out centuries ago, soil Hg concentrations in these regions remain highly elevated to the present day. [18][19][20][21] In a recent study in one of such regions, the Guanajuato Mining District (GMD) of Mexico, Loria et al. 22 discovered the presence of exceptionally high concentrations of gaseous element mercury (GEM) in the interstitial air of soil and sediment at or close to the historical mining sites, contributing to highly elevated GEM in the ambient air throughout the entire urban area. They attributed such high GEM concentrations to the disproportionation of calomel (Hg 2 Cl 2 ) formed during the patio process and the volatilization of any remaining liquid mercury that was trapped in present-day soil. ...
In mercury-contaminated soils, mercury sulfides (HgS) occur as nanoparticles in mineral surface coatings. The coatings are composed of ferrihydrite and its replacement by goethite results in the release of the HgS nanoparticles.
... Although concentrations of Hg emitted from mine-contaminated soil and mine waste material were elevated, persistent wind from the south-southwest mountain site (San Joaquin) disperses Hg in the air within a few meters of the ground surface. Other studies have observed increased Hg emissions from soil in mined areas over periods from several hours to days (Higueras et al. 2012). Factor 2 it shows strong correlations for NO − 3 , Na + and Ca 2+ . ...
We measured and compared mercury (Hg) and other ions in rainwater collected in San Joaquin (mining zone) and Juriquilla (urban area), central Mexico, from 2009 to 2012. A total of 274 rainwater samples were collected and analyzed for pH, electrical conductivity, [Formula: see text] Cl-, [Formula: see text] Na+, K+, Ca2+, Mg2+ and Hg. Mercury concentrations in rainwater varied from 24.21 to 248.89 (x-bar = 86.97 ± 10.77) µg L- 1 in San Joaquin (mining zone) and 11.26 to 176.91 (x-bar = 81.51 ± 10.24) µg L- 1 in Juriquilla (urban area). Rainwater sample were collected over periods 1-3 days, depending upon precipitation frequency. Significant correlations (p < 0.05) were found between [Formula: see text] Cl-, [Formula: see text] Na+, K+, Ca2+, Mg2+ and Hg at the San Joaquin site. Significant correlations were obtained between [Formula: see text] Na+, K+, Ca2+, Mg2+ and Hg at the Juriquilla site. In order to determine if there were significant differences among each measured parameter in rainwater collected in San Joaquin and Juriquilla, Kruskal-Wallis test was applied to data. We emphasized that the distribution and concentrations of Hg and the studied ions in rainwater samples were affected by atmospheric dust and local meteorological conditions of wind-speed and direction.
... Hagan et al. (2011) found that total mercury concentrations in ambient soils increased with proximity to the former site of the ribera, the canal that ran through the center of Potosí and supported the amalgamation activities. Another study reported that while present-day ambient mercury vapor concentrations along the ribera in Potosí were low, excavation of topsoil near former mill sites caused the release of elemental mercury vapor in concentrations over 3000 ng/m 3 , which is an order of magnitude greater than the U.S. EPA reference concentration for the inhalation of elemental mercury (300 ng/m 3 ) (Higueras et al., 2012;U.S. EPA, 1995a). While there have been a number of studies related to mercury, studies of other mining-related pollutants (e.g., lead, arsenic, zinc) in Potosí are lacking. ...
Potosí, Bolivia, is the site of centuries of historic and present-day mining of the Cerro Rico, a mountain known for its rich polymetallic deposits, and was the site of large-scale Colonial era silver refining operations. In this study, the concentrations of several metal and metalloid elements were quantified in adobe brick, dirt floor, and surface dust samples from 49 houses in Potosí. Median concentrations of total mercury (Hg), lead (Pb), and arsenic (As) were significantly greater than concentrations measured in Sucre, Bolivia, a non-mining town, and exceeded US-based soil screening levels. Adobe brick samples were further analyzed for bioaccessible concentrations of trace elements using a simulated gastric fluid (GF) extraction. Median GF extractable concentrations of Hg, As, and Pb were 0.085, 13.9, and 32.2% of the total element concentration, respectively. Total and GF extractable concentrations of Hg, As, and Pb were used to estimate exposure and potential health risks to children following incidental ingestion of adobe brick particles. Risks were assessed using a range of potential ingestion rates (50-1000mg/day). Overall, the results of the risk assessment show that the majority of households sampled contained concentrations of bioaccessible Pb and As, but not Hg, that represent a potential health risk. Even at the lowest ingestion rate considered, the majority of households exceeded the risk threshold for Pb, indicating that the concentrations of this metal are of particular concern. To our knowledge, this is the first study to quantify key trace elements in building materials in adobe brick houses and the results indicate that these houses are a potential source of exposure to metals and metalloids in South American mining communities. Additional studies are needed to fully characterize personal exposure and to understand potential adverse health outcomes within the community.
... This is due to the numerous factors involved in the soil to atmosphere transfer process, which is influenced, as mentioned above, by soil Hg speciation, meteorological conditions and time. García-Sánchez et al. [97], Higueras et al. [98] and Guerrero [99] found evidence that Hg contained in soils that remained undisturbed for long periods of time led to changes in mercury speciation and this may reduce the capacity for the release of mercury into the atmosphere in gaseous form. ...
Spain has been the main mercury producer worldwide, with mines or mining districts scattered across its geography. In particular, two main areas show generally higher contents of this element in the soils, namely, Asturias (or the Cantabrian Zone in geological terms) and the Almadén area in the Southern Central Iberian Zone. In this review six different aspects are considered: (1) distribution of total concentrations, (2) mercury mobility and availability, (3) soil to plant transfer, (4) mercury transfer to animal biota, (5) soil to atmosphere transfer and (6) possibility of remediation for sites polluted by mercury. The conclusions drawn from the available results highlight significant differences in contents, mobility and transfer processes depending on the different types of mercury pollution and different climatic conditions. A general background level for Spanish soils can be established at 20 μg kg À1 , but very different ranges can be found in different areas according to the volumetric importance of each source and depending on other local factors. Mercury mining appears to be the most important source of soil pollution, and studies on the possible mobility and transfer to other environmental compartments demonstrate the highest levels at which mercury affects the population living in the proximity of such sources.
... These 2 m Hg air data suggest that although concentrations of Hg emitted from mine waste in mined areas were elevated, persistent wind in southwest Texas disperses Hg in air within a few meters of the ground surface. Other studies have observed decreasing Hg emissions from soil in mined areas over periods of hours to days (García-Sánchez et al. 2006;Higueras et al. 2012), but long-term Hg emissions from soil was not evaluated in this study. soil gas in mined areas are similar to, or higher than, Hg measured in other areas mined for Hg, where concentrations of Hg have been reported to vary from 100 to 6,000 ng/m 3 (Ferrara et al. 1991(Ferrara et al. , 1998b. ...
Samples of soil, water, mine waste leachates, soil gas, and air were collected from areas mined for mercury (Hg) and baseline sites in the Big Bend area, Texas, to evaluate potential Hg contamination in the region. Soil samples collected within 300 m of an inactive Hg mine contained elevated Hg concentrations (3.8–11 µg/g), which were considerably higher than Hg in soil collected from baseline sites (0.03–0.05 µg/g) distal (as much as 24 km) from mines. Only three soil samples collected within 300 m of the mine exceeded the probable effect concentration for Hg of 1.06 µg/g, above which harmful effects are likely to be observed in sediment-dwelling organisms. Concentrations of Hg in mine water runoff (7.9–14 ng/L) were generally higher than those found in springs and wells (0.05–3.1 ng/L), baseline streams (1.1–9.7 ng/L), and sources of drinking water (0.63–9.1 ng/L) collected in the Big Bend region. Concentrations of Hg in all water samples collected in this study were considerably below the 2,000 ng/L drinking water Hg guideline and the 770 ng/L guideline recommended by the U.S. Environmental Protection Agency (USEPA) to protect aquatic wildlife from chronic effects of Hg. Concentrations of Hg in water leachates obtained from leaching of mine wastes varied widely from <0.001 to 760 µg of Hg in leachate/g of sample leached, but only one leachate exceeded the USEPA Hg industrial soil screening level of 31 µg/g. Concentrations of Hg in soil gas collected at mined sites (690–82,000 ng/m3) were highly elevated compared to soil gas collected from baseline sites (1.2–77 ng/m3). However, air collected from mined areas at a height of 2 m above the ground surface contained concentrations of Hg (4.9–64 ng/m3) that were considerably lower than Hg in soil gas from the mined areas. Although concentrations of Hg emitted from mine-contaminated soils and mine wastes were elevated, persistent wind in southwest Texas disperses Hg in the air within a few meters of the ground surface.
... The instrument of choice for the surveys was the widely used LUMEX RA-915, which has proved to be very reliable for GEM studies, having been used in a large number of surveys worldwide; for example, a few studies (among many) conducted during the last decade include those of Palinkas et al. (1990), Sholupov et al. (2004, Higueras et al. (2005Higueras et al. ( , 2006Higueras et al. ( , 2012Higueras et al. ( , 2013, Kotnik et al. (2005), Kim et al. (2006), García-Sánchez et al. (2006a, b), Aiuppa et al. (2007), Almeida et al. (2008), Llanos et al. (2010), Fu et al. (2011), Martínez-Coronado et al. (2011), Kocman et al. (2011), Yasutake et al. (2011 and Vaselli et al. (2013). The LUMEX RA-915 series analyzers for continuous GEM measurements (models RA-915? and 915M) are portable multifunctional atomic absorption spectrometers, with Zeeman background correction, which eliminates the effect of interfering impurities (Sholupov and Ganeyev 1995). ...
Mercury is transported globally in the atmosphere mostly in gaseous elemental form (GEM, [Formula: see text]), but still few worldwide studies taking into account different and contrasted environmental settings are available in a single publication. This work presents and discusses data from Argentina, Bolivia, Bosnia and Herzegovina, Brazil, Chile, China, Croatia, Finland, Italy, Russia, South Africa, Spain, Slovenia and Venezuela. We classified the information in four groups: (1) mining districts where this contaminant poses or has posed a risk for human populations and/or ecosystems; (2) cities, where the concentration of atmospheric mercury could be higher than normal due to the burning of fossil fuels and industrial activities; (3) areas with natural emissions from volcanoes; and (4) pristine areas where no anthropogenic influence was apparent. All the surveys were performed using portable LUMEX RA-915 series atomic absorption spectrometers. The results for cities fall within a low GEM concentration range that rarely exceeds 30 ng m(-3), that is, 6.6 times lower than the restrictive ATSDR threshold (200 ng m(-3)) for chronic exposure to this pollutant. We also observed this behavior in the former mercury mining districts, where few data were above 200 ng m(-3). We noted that high concentrations of GEM are localized phenomena that fade away in short distances. However, this does not imply that they do not pose a risk for those working in close proximity to the source. This is the case of the artisanal gold miners that heat the Au-Hg amalgam to vaporize mercury. In this respect, while GEM can be truly regarded as a hazard, because of possible physical-chemical transformations into other species, it is only under these localized conditions, implying exposure to high GEM concentrations, which it becomes a direct risk for humans.
... The mining history of the Almadén district (Hernández et al., 1999) began some 2000 years ago, when Romans started to use cinnabar as a vermilion red pigment, but it was not until the Arabs invaded this realm that the locality acquired the etymological roots of its present name: Al-maaden (meaning " the mine " ). However, the 'modern' history of Almadén and mercury begins in 1555, when the Spaniard Bartolomé de Medina (1497–1585) discovered the use of mercury in silver processing (Higueras et al., 2012). Thus, from the middle of the 16th Century Almadén had a major strategic importance for the colonization of America, becoming one of the largest early mining and metallurgical centers in Europe. ...
The giant Almadén mercury deposit (Spain) is hosted by the Lower Silurian Criadero Quartzite; in turn this ore-bearing rock unit is cross-cut by the so-called Frailesca unit, a diatreme body of basaltic composition. The geochemical characteristics of the Silurian to Devonian Almadén District volcanic units indicate that these rocks originated from an enriched, evolving mantle source that ultimately yielded basanites–nephelinites to rhyolites, through olivine-basalts, pyroxene-basalts, trachybasalts, trachytes, very scarce rhyolites, and quartz-diabases. The Silurian intraplate alkaline volcanism developed in submarine conditions which triggered widespread hydrothermal activity resulting in Hg ore formation and pervasive alteration to carbonates. The δ18O, δ13C, and δ34S isotopic signatures for carbonates and pyrite suggest different sources for carbon and sulfur, including magmatic and organic for the former and magmatic and sea water for the latter. The most important and efficient natural source of mercury on Earth is by far the volcanic activity, which liberates mercury via quiescent degassing and catastrophic (Plinian) events when eruptions can overwhelm the atmospheric budget of Hg. Thus, we suggest that CO2 degasification and coeval distillation of mercury from the volcanic rocks fed the huge hydrothermal system that led to massive deposition of mercury at Almadén. Build up of Hg0gas in magmatic chambers during waning rifting in the Late Ordovician, followed by renewed volcanism in the Early Silurian, would have resulted in massive degasification of the accumulated mercury. Part of this mercury went into the Criadero Quartzite leading to formation of the huge Almadén deposit and others (e.g., El Entredicho) along the same stratigraphic level. Progressive depletion of the deep seated magmatic Hg stock would have resulted in a drastic reduction in ore deposit size after the Lower Silurian when smaller deposits formed (e.g., Las Cuevas).
... Aquatic biota has the capability to bioconcentrate methyl-Hg up to 10 4 to 10 7 times (Stein et al., 1996). It is also important to consider that time and soil inactivity may lead drastic reduction of Hg emissions (Higueras et al., 2012). The Total Gaseous Mercury (TGM) is represented by the sum of anthropogenic and natural Hg emissions and related to elemental gaseous mercury (GEM) and gaseous oxidized mercury (GOM), while the solid component is named particulate bounded mercury (PBM) (e.g. ...
Para fortalecer el presente estudio de línea base se recomienda seguir las siguientes acciones: b Realizar un barrido de información nacional sobre el Mercurio y sus compuestos, es decir, consolidar el conocimiento e información sobre usos, emisiones y liberaciones, niveles ambientales y opciones de prevención y control. Esta base de datos conlleva un gran esfuerzo por parte de las instituciones involucradas en el tema, pero al mismo tiempo es una herramienta clave de una estrategia de gestión ambiental para el Mercurio, en base a la que se podrá generar nuevos instrumentos legales, técnicos y administrativos para mejorar la situación actual. b Organizar reuniones, foros, debates y charlas de discusión con diferentes sectores (academia, organizaciones sociales, entidades públicas nacionales y sub nacionales) sobre la problemática del Mercurio, donde se identifique el riesgo asociado a la contaminación del Mercurio en el mundo y en Bolivia, y de esta manera generar una conciencia colectiva sobre la necesidad de reducir su uso. b Sistematizar la recopilación de información a través de entrevistas y la creación de formularios adecuados y con respaldo legal para acceder a la información de las entidades públicas y privadas, orientados al tipo de institución. b Focalizar instituciones estratégicas de amplia concentración de información, dedicando más tiempo en la sistematización de la información y modernización de la información dentro de estas instituciones. b Establecer y promover el uso de un protocolo nacional estandarizado para realizar estudios sobre Mercurio en diferentes compartimentos ambientales y biológicos en función de los métodos de colecta, análisis de laboratorios y contenidos de mínimos de los reportes. La determinación de los niveles de Mercurio puede servir para identificar zonas de exposición a la contaminación actual, pero también puede contribuir a predecir potenciales zonas de exposición y contaminación. Gracias a la línea base aquí presentada, se ha identificado dos principales fuentes de exposición antropogénica a la contaminación por Mercurio en Bolivia: 1) el Mercurio atmosférico proveniente de áreas donde se efectúa la tradicional actividad artesanal de recuperación de Oro y 2) el Mercurio liberado en el medio ambiente y particularmente en los medios acuáticos cercanos a industrias y centros mineros. Por medio del presente inventario, se identificó que existen muy pocos estudios sobre el Mercurio atmosférico en las áreas cercanas a centros mineros y grandes industrias
Almost no environmental data on mercury distribution and speciation in soils have been published so far for the Almadén mining district (central Spain), despite its huge size and historic importance. The mercury distribution in soils of the district reveals the existence of high and extremely high mercury values (up to f9000 ppm Hg). The Hg-thermodesorption curves for soils from a decommissioned metallurgical precinct (Almadenejos) and a phytoremediation site show that mercury occurs in the forms of cinnabar and as mercury bound to organic matter. The TEM-EDX study of the highly contaminated anthrosols from Almadenejos (samples with Hg >5000 ppm) shows the existence of cinnabar particles adsorbed to the surface of chlorite grains. Given the generally pyrite-poor character of the ores, and the presence of carbonates in the host rocks, cinnabar solubilization is limited, which in turn mitigates environmental hazards in the district. The only by-product of cinnabar leaching in the mineral
dumps is schuetteite. Preliminary results on local plants (Asparagus acutifolius, Dittrichia graveolens, Marrubium vulgare) show that mercury gets incorporated to roots, stems and leaves, with values of up to about 300 ppm Hg.
The headwaters of the Río Pilcomayo drain the Cerro Rico de Potosí precious metal-polymetallic tin deposits of southern Bolivia. Mining of these deposits has taken place for around 500 years, leading to severe contamination of the Pilcomayo's waters and sediments for at least 200 km downstream. Communities living downstream of the mines and processing mills rely on the river water for irrigation, washing and occasionally, cooking and drinking, although most communities take their drinking water from springs located in the mountains above their village. This investigation focuses on arsenic exposure in people living in riverside communities up to 150 km downstream of the source. Sampling took place in April–May 2003 (dry season) and was repeated in January–March 2004 (wet season) in five communities: El Molino, Tasapampa, Tuero Chico, Sotomayor and Cota. Cota was the control in 2003 and again in 2004; a nearby city, Sucre, and several locations in the UK were also used as controls in 2004. Drinking, irrigation and river waters, hair and urine samples were collected in each community, digested where appropriate and analysed for As using ICP-MS. Arsenic concentrations in drinking waters ranged 0.2–112 g l−1, irrigation water 0.6–329 g l−1, river waters 0.9–12,800 g l−1, hair 37–2110 g kg−1 and urine 11–891 g l−1. All but one drinking water sample was found to contain As below the World Health Organization recommended guideline of 10 g l−1, although a number of irrigation and river water concentrations were above Canadian and Bolivian guidelines. Many As concentrations in the hair and urine samples from this study exceeded published values for non-occupationally exposed subjects. Analysis of mean concentration values for all media types showed that there were no statistically significant differences between the control locations and the communities exposed to known As contamination, suggesting that the source of As may not be mining-related. Arsenic concentration appears to increase as a function of age in hair samples from males and females older than 30 years. Male volunteers over the age of 35 showed increasing urine-As concentrations as a function of age, whereas the opposite was true for the females.
Detailed Spanish records of mercury use and silver production during the colonial period in Potosí, Bolivia were evaluated to estimate atmospheric emissions of mercury from silver smelting. Mercury was used in the silver production process in Potosí and nearly 32,000 metric tons of mercury were released to the environment. AERMOD was used in combination with the estimated emissions to approximate historical air concentrations of mercury from colonial mining operations during 1715, a year of relatively low silver production. Source characteristics were selected from archival documents, colonial maps and images of silver smelters in Potosí and a base case of input parameters was selected. Input parameters were varied to understand the sensitivity of the model to each parameter. Modeled maximum 1-h concentrations were most sensitive to stack height and diameter, whereas an index of community exposure was relatively insensitive to uncertainty in input parameters. Modeled 1-h and long-term concentrations were compared to inhalation reference values for elemental mercury vapor. Estimated 1-h maximum concentrations within 500 m of the silver smelters consistently exceeded present-day occupational inhalation reference values. Additionally, the entire community was estimated to have been exposed to levels of mercury vapor that exceed present-day acute inhalation reference values for the general public. Estimated long-term maximum concentrations of mercury were predicted to substantially exceed the EPA Reference Concentration for areas within 600 m of the silver smelters. A concentration gradient predicted by AERMOD was used to select soil sampling locations along transects in Potosí. Total mercury in soils ranged from 0.105 to 155 mg kg−1, among the highest levels reported for surface soils in the scientific literature. The correlation between estimated air concentrations and measured soil concentrations will guide future research to determine the extent to which the current community of Potosí and vicinity is at risk of adverse health effects from historical mercury contamination.
Silver, Ag, Sn, and Zn ores has been intensively mined and processed at Cerro Rico de Potosí, Bolivia since 1545. Acid mine drainage and processing plant effluent are prime sources of water contamination in the headwaters of the economically and ecologically vital, yet highly impacted, Rio Pilcomayo watershed. Streams receiving drainage from the slopes of Cerro Rico and surrounding landscapes were sampled during the dry (July-August 2006) and wet (March 2007) seasons of one water-year. In-stream waters contain total metals concentrations of up to 16 mg/L As, 4.9 mg/L Cd, 0.97 mg/L Co, 1100 mg/L Fe, 110 mg/L Mn, 4.1 mg/L Pb, and 1500 mg/L Zn with pH and specific conductivity ranging from 2.8-9.5 and 160-5070 μS/cm, respectively. Many of the studied water bodies are more degraded than class "D" of the Bolivian receiving water body criteria, rendering them unfit for domestic or agricultural use. However, some of these waters are currently being used for irrigation and livestock watering. The data indicate that historic and current mining activities have transformed these key natural resources into potential human and environmental health hazards.
Almost no environmental data on mercury distribution and speciation in soils have been published so far for the Almadén mining district (central Spain), despite its huge size and historic importance. The mercury distribution in soils of the district reveals the existence of high and extremely high mercury values (up to ∼9000 ppm Hg). The Hg-thermodesorption curves for soils from a decommissioned metallurgical precinct (Almadenejos) and a phytoremediation site show that mercury occurs in the forms of cinnabar and as mercury bound to organic matter. The TEM-EDX study of the highly contaminated anthrosols from Almadenejos (samples with Hg >5000 ppm) shows the existence of cinnabar particles adsorbed to the surface of chlorite grains. Given the generally pyrite-poor character of the ores, and the presence of carbonates in the host rocks, cinnabar solubilization is limited, which in turn mitigates environmental hazards in the district. The only by-product of cinnabar leaching in the mineral dumps is schuetteite (Hg3SO4O2). Preliminary results on local plants (Asparagus acutifolius, Dittrichia graveolens, Marrubium vulgare) show that mercury gets incorporated to roots, stems and leaves, with values of up to about 300 ppm Hg.
This paper reports the novel use of Zeeman atomic absorption spectrometry using high frequency modulated light polarization (ZAAS-HFM), its theoretical basis and experimental validation. Due to the high frequency modulation of the analytical and reference signals, the temporal background correction error is reduced below 10−5 absorbance units. In addition, the use of ZAAS-HFM enables the operator to increase the apparatus transmittance and therefore to reduce the detection limits and to broaden the dynamic range of the analytical curves.
The mobility, bioavailability and toxicity of mercury in the environment strongly depend on the chemical species in which it is present in soil, sediments, water or air. In mining districts, differences in mobility and bioavailability of mercury mainly arise from the different type of mineralization and ore processing. In this work, synchrotron-based X-ray absorption near-edge spectroscopy (XANES) has been taken advantage of to study the speciation of mercury in geological samples from three of the largest European mercury mining districts: Almadén (Spain), Idria (Slovenia) and Asturias (Spain). XANES has been complemented with a single extraction protocol for the determination of Hg mobility. Ore, calcines, dump material, soil, sediment and suspended particles from the three sites have been considered in the study. In the three sites, rather insoluble sulfide compounds (cinnabar and metacinnabar) were found to predominate. Minor amounts of more soluble mercury compounds (chlorides and sulfates) were also identified in some samples. Single extraction procedures have put forward a strong dependence of the mobility with the concentration of chlorides and sulfates. Differences in efficiency of roasting furnaces from the three sites have been found.
The history of pre-Columbian metallurgy in South America is incomplete because looting of metal artifacts has been pervasive. Here, we reconstruct a millennium of metallurgical activity in southern Bolivia using the stratigraphy of metals associated with smelting (Pb, Sb, Bi, Ag, Sn) from lake sediments deposited near the major silver deposit of Cerro Rico de Potosí. Pronounced metal enrichment events coincide with the terminal stages of Tiwanaku culture (1000 to 1200 A.D.) and Inca through early Colonial times (1400 to 1650 A.D.). The earliest of these events suggests that Cerro Rico ores were actively smelted at a large scale in the Late Intermediate Period, providing evidence for a major pre-Incan silver industry.
Methylmercury (MeHg) is a highly toxic form of mercury that bioaccumulates in aquatic food chains. However, methods to reliably identify sites of MeHg formation or to quantify MeHg production require the use of isotopic tracers, which limits their use. In this paper, a method is presented to quantify the methylation of mercury by a methyltransferase pathway. This methyltransferase pathway is one of the biochemical pathways responsible for biological mercury methylation. Protein is extracted front environmental samples, and mercury methyltransferase (HgMT) activity of soil extracts is calculated by assessing increases in methyltransferase activity induced by Hg addition. In enzyme extracts from pure cultures or soil sampled,, HgMT activity correlated with net MeHg production and Hg consumption, suggesting that HgMT activity can be used to estimate MeHg production in field samples. Over the course of a three-month period in a freshwater wetland, HgMT activity correlated with not MeHg concentrations (r(2) = 0.55; p < 0.057). Furthermore, HgMT activity predicted (r(2) = 0,80; p < 0.01) gross MeHg formation in freshwater wetlands as well as in laboratory microcosms calculated using previously published rate constants. Our results show that a methyltransferase assay, in combination with demethylation estimates, accurately predicts MeHg formation under field and laboratory conditions. This assay does not require the use of mercury added to field samples to estimate activity but rather estimates the biological activity present in the soil by quantifying the amount of enzyme present in the soil. Such an assay is well suited for use in field surveillance programs assessing MeHg formation in a variety of environments.
New high precision 40Ar/39Ar dating of sanidine and biotite from two rhyolitic domes and an ignimbrite, combined with existing fission-track data and
a hydrothermal sericite age, suggests that the world-class Ag deposit at Cerro Rico was emplaced during a protracted period
of magma-related hydrothermal activity beginning at 13.77 ± 0.03 Ma and continuing for at least 0.2 m.y. This may have been
sustained by a large single injection or repeated injections of fractionated Ag-enriched magma into a high-level magma chamber.
K-Ar dating of alunite indicates that supergene oxidation had begun by about 13.5 Ma, soon after dome emplacement, and progressed
semicontinuously for at least 7.5 m.y. This oxidation, while not leading to significant enrichment, has significantly enhanced
the economic viability of the disseminated part of the orebody.
The Cerco de Almadenejos (CDA) is an old metallurgical site located in the province of Ciudad Real (Spain) that operated between 1794 and 1861. The metallurgical precinct was built for the roasting of the Almadén and Almadenejos cinnabar ore to extract Hg metal. A previous pilot geochemical study of soils at the CDA had already shown extremely high concentrations of Hg. To analyze the extent and intensity of contamination, we planned and executed a geochemical survey to cover the CDA and the surrounding areas. The survey covered soils, air, and plants. The planning involved the design of two sampling grids in order to obtain a comprehensive picture of potential environmental hazards in the area: 1) a detailed sampling grid centred on the metallurgical precinct (n = 16 samples; area = 3.6 × 104 m2); and 2) a less detailed sampling grid planned to determine the extension of contamination beyond the metallurgical site (n = 35 samples; area = 1.2 × 106 m2). After variogram modelization of geochemical data, the kriging plots showed that contamination, even if centred at the precinct, extends beyond the site, with Hg concentrations of up to 2200 times those of uncontaminated soils (world baseline). The detailed study of the soils from the precinct shows an extremely high mean concentration of 4220 μg Hg g− 1 (4.2 × 105 times baseline concentration). In turn, these highly polluted soils induce strong emissions of Hg(g), with concentrations of up to 970 ng Hg m− 3. The study of the edible wild asparagus Asparagus acutifolius shows extremely high concentrations of mercury in roots (0.6–443 μg g− 1) and stems (0.3–140 μg g− 1). The data indicate that the study area constitute a hot spot of contamination and is a potential health/environmental hazard for the inhabitants of Almadenejos, livestock, and wild life, that requires immediate action via remediation procedures.
An in situ experiment was established at two environmentally different river sites (one each on the Wisconsin and Fox rivers) to evaluate the extent of methylmercury (MeHg) production in and Hg loss from sediments treated with mercuric chloride (HgCl 2 ) or phenylmercuric acetate (PhHgAc). Bulk sediment was collected from each river site, treated with approximately 1, 10, or 100 ppm Hg (oven‐dry basis) as HgCl 2 or PhHgAc, and returned to the river site with untreated controls for 2, 4, or 12 weeks of equilibration with the aquatic environment. After each sample retrieval, the sediments were analyzed for total Hg using an aqua regia digest and flameless atomic absorption spectrophotometry and for MeHg by gas chromatography using a modified extraction procedure which is described.
Results suggest that Hg losses from stationary sediments are minimal and that sediment transport is probably the major source of Hg movement in a river system. In both sediments, more MeHg was produced from PhHgAc than from a similar concentration of HgCl 2 , and MeHg concentration increased as the Hg treatment with either compound was increased. The MeHg concentrations found in the Wisconsin River sediments were substantially higher than in the Fox River sediments with a similar Hg treatment. The large differences in MeHg production between these two sediments may be partially attributed to their chemical dissimilarities. The Wisconsin River sediment is acidic and contains more organic materials compared to the Fox River sediment which is more alkaline and contains larger amounts of sulfide sulfur.
Glass and fluid inclusions in quartz of rhyolites, porphyries, and granites from mining areas in Japan were investigated. Rhyolites contain various types of glass inclusions and high density liquid inclusions but they are devoid of highly saline polyphase inclusions. Glass inclusions in rhyolites are generally transparent, indicating rapid cooling. Some of them contain dendritic crystals or small euhedral crystals. Porphyries considered to be genetically related to mineralization generally contain highly saline inclusions besides many liquid inclusions. Porphyries also contain many glass inclusions, but in general they are completely or partially devitrified. Size effect on devitrification is remarkable. Larger glass inclusions are easily devitrified, while smaller inclusions occasionally remain transparent. In most granitic rocks highly saline inclusions are not found, but high density liquid inclusions are common. Small granitic stocks related to mineralization, however, are rich in polyphase inclusions. Since fluid inclusions in igneous rocks are generally very small, the ordinary heating-stage and freezing-stage microscopic methods are generally inapplicable. However, microscopic observation of these fluid inclusions, especially of polyphase inclusions, gives valuable information on the possible change in ore-forming fluids during their migration from the center of mineralization. It is inferred that highly saline fluids represented by poly-phase inclusions would have been released from silicate melts in late magmatic stages, and that dilution of these highly saline fluids would be an important factor controlling the ore deposition.
Analysis of mercury-contaminated soil from the flood plain of East Fork Poplar Creek (EFPC) in Oak Ridge, TN, using a scanning electron microscope (SEM) with energy- and wavelength X-ray dispersive spectroscopy (EDS/WDS) and a transmission electron microscope (TEM) with select area electron diffraction (SAED) revealed the presence of submicron, crystalline mercuric sulfide (HgS) in the form of metacinnabar. The HgS formed in place after the deposition and burial of mercury-contaminated soils. A reaction path model developed to describe the geochemical evolution of the soil redox conditions during flooding predicted that the resultant pe and pH of the soil would be within the stability range of HgS. The reaction of mercury with other metal sulfides or sulfhydryl groups in the soil may have also contributed to the formation of HgS. The formation of HgS is significant to the remediation efforts at EFPC because the toxicity, leachability, and volatility of mercury in soils is dependent on the solid phase speciation. Because the local hydrogeochemical conditions are not unique, the forma tion of HgS at this site has implications to other environments as well.
This work reports data of atmospheric mercury for northern Chile. The study was centered in the Coquimbo region, a realm rich in mineral deposits. Some of the mining districts have historic importance and have been exploited almost continuously since the Spanish colonial time (16–18th century). Two of these districts are particularly relevant: (1) Andacollo, initially exploited for gold, and then for copper and gold; and (2) Punitaqui, initially exploited for mercury, and then for copper and gold. The continuous mercury measurement procedures carried out during this survey, have proved to be an excellent tool to detect Hg signatures associated with the mining industrial activities. The combination of cumulative log-probability graphs and atmospheric mercury concentration profiles, allows clear differentiation between areas subjected to agriculture (2–3 ngHg m−3), from those in which mining and metal concentration activities take place (>10 ngHg m−3, most data well beyond this figure). Gold recovery involving milling and amalgamation appear as the most contaminant source of mercury, and yield concentrations in the order of 104–105 ngHg m−3 (Andacollo). Second in importance are the vein mercury deposits of Punitaqui, with concentrations above 100 ngHg m−3, whereas the flotation tailings of the district yield concentrations near to 100 ngHg m−3. The large and modern open pit operations of Andacollo (Carmen: Cu; Dayton: Au) do not show high concentrations of atmospheric mercury.
Scientists, especially environmental scientists, often encounter trace level concentrations that are typically reported as less than a certain limit of detection, L. Type I left-censored data arises when certain low values lying below L are ignored or unknown as they cannot be measured accurately. In many environmental quality assurance and quality control (QA/QC), and groundwater monitoring applications of the United States Environmental Protection Agency (USEPA), values smaller than L are not required to be reported. However, practitioners still need to obtain reliable estimates of the population mean μ, and the standard deviation (S.D.) σ. The problem gets complex when a small number of high concentrations are observed with a substantial number of concentrations below the detection limit. The high-outlying values contaminate the underlying censored sample, leading to distorted estimates of μ and σ. The USEPA, through the National Exposure Research Laboratory-Las Vegas (NERL-LV), under the Office of Research and Development (ORD), has research interests in developing statistically rigorous robust estimation procedures for contaminated left-censored data sets. Robust estimation procedures based upon a proposed (PROP) influence function are shown to result in reliable estimates of population parameters of mean and S.D. using contaminated left-censored samples. It is also observed that the robust estimates thus obtained with or without the outliers are in close agreement with the corresponding classical estimates after the removal of outliers. Several classical and robust methods for the estimation of μ and σ using left-censored (truncated) data sets with potential outliers have been reviewed and evaluated.
We present data from an early reconnaissance survey (stream sediments, soil, and water Hg chemistry; plants and water crustaceans Hg intake) of the Almadén district (central Spain), that was carried out to establish the potential environmental hazards derived from the anomalous mercury concentrations measured in this realm. The Almadén mercury district (∼300 km2) can be regarded as the largest geochemical anomaly of mercury on Earth. The district includes a series of mercury mineral deposits, having in common a simple mineralogy (dominant cinnabar: HgS, and minor pyrite: FeS2). The ore deposits have been mined for more than 2000 years, and the main mine of the district (Almadén), has been active from Roman times to present day with almost no interruptions. The mercury distribution in soils of the district reveals the existence of high, and extremely high mercury values (up to 8889 μg g− 1), whereas concentrations in stream sediments and waters reach exceptional values of up to 16,000 μg g− 1 and 11,200 ng l− 1 respectively. On the other hand, very high concentrations of methylmercury (MeHg) have been detected in calcines (up to 3100 ng g− 1), sediments (0.32–82 ng g− 1), and waters (0.040–30 ng l− 1). Mercury gets incorporated to edible river crustaceans and plants. The red swamp crayfish Procambarus clarkii, has Hg concentrations of up to 9060 ng g− 1 (muscle) and 26,150 ng g− 1 (hepatopancreas). Regarding plants, the local wild asparagus (Asparagus acutifolius) yields values of up to 298 μg g− 1 Hg. Mercury also escapes to the atmosphere, and mineral deposits, together with metallurgical activities, generate strong anomalies of atmospheric Hg. The most important concentrations relate to the emissions from the Almadén metallurgical roaster, in the order of 14,000 ng Hg m− 3. Additionally, large open pit operations also contribute to the district atmospheric pool of mercury, with high concentrations above 1000 ng Hg m− 3. Thus, no system (rocks, soils, sediments, waters, atmosphere, biota) in the Almadén district is free from strong Hg contamination.
The rocks comprising the Kari Kari massif southeast of the city of Potosi, Bolivia, consist entirely of welded ignimbrites. It is argued that the massif constitutes the resurgent centre of a 20-m.y.-old resurgent caldera. Plutonic rocks are exposed in the south, but volcanic rocks of the caldera rim are exposed in the north, and indicate a shallower erosion level there. The volcanic rocks consist of a coarse moat deposit, consisting of angular fragments of basement material and juvenile clasts, overlain by an extensive garnet-bearing ignimbrite. A plant-fossil-bearing lacustrine deposit was laid down in a lake within the caldera. The Cerro Rico stock, noted for its silver-tin mineralisation, may be a late intrusion along the caldera ring fractures.
The speciation of Hg is a critical determinant of its mobility, reactivity, and potential bioavailability in mine-impacted regions. Furthermore, Hg speciation in these complex natural systems is influenced by a number of physical, geological, and anthropogenic variables. In order to investigate the degree to which several of these variables may affect Hg speciation, extended X-ray absorption fine structure (EXAFS) spectroscopy was used to determine the Hg phases and relative proportions of these phases present in Hg-bearing wastes from selected mine-impacted regions in California and Nevada. The geological origin of Hg ore has a significant effect on Hg speciation in mine wastes. Specifically, samples collected from hot-spring Hg deposits were found to contain soluble Hg-chloride phases, while such phases were largely absent in samples from silica-carbonate Hg deposits; in both deposit types, however, Hg-sulfides in the form of cinnabar (HgS, hex.) and metacinnabar (HgS, cub.) dominate. Calcined wastes in which Hg ore was crushed and roasted in excess of 600 °C, contain high proportions of metacinnabar while the main Hg-containing phase in unroasted waste rock samples from the same mines is cinnabar. The calcining process is thought to promote the reconstructive phase transformation of cinnabar to metacinnabar, which typically occurs at 345 °C. The total Hg concentration in calcines is strongly correlated with particle size, with increases of nearly an order of magnitude in total Hg concentration between the 500–2000 μm and <45 μm size fractions (e.g., from 97–810 mg/kg Hg in calcines from the Sulphur Bank Mine, CA). The proportion of Hg-sulfides present also increased by 8–18% as particle size decreased over the same size range. This finding suggests that insoluble yet soft Hg-sulfides are subject to preferential mechanical weathering and become enriched in the fine-grained fraction, while soluble Hg phases are leached out more readily as particle size decreases. The speciation of Hg in mine wastes is similar to that in distributed sediments located downstream from the same waste piles, indicating that the transport of Hg from mine waste piles does not significantly impact Hg speciation. Hg LIII-EXAFS analysis of samples from Au mining regions, where elemental Hg(0) was introduced to aid in the Au recovery process, identified the presence of Hg-sulfides and schuetteite (Hg3O2SO4), which may have formed as a result of long-term Hg(0) burial in reducing high-sulfide sediments.
The development of the mercury (Hg) amalgamation process in the mid-sixteenth century triggered the onset of large-scale Hg mining in both the Old and New Worlds. However, ancient Hg emissions associated with amalgamation and earlier mining efforts remain poorly constrained. Using a geochemical time-series generated from lake sediments near Cerro Rico de Potosí, once the world's largest silver deposit, we demonstrate that pre-Colonial smelting of Andean silver ores generated substantial Hg emissions as early as the twelfth century. Peak sediment Hg concentrations and fluxes are associated with smelting and exceed background values by approximately 20-fold and 22-fold, respectively. The sediment inventory of this early Hg pollution more than doubles that associated with extensive amalgamation following Spanish control of the mine (1574-1900 AD). Global measurements of [Hg] from economic ores sampled world-wide indicate that the phenomenon of Hg enrichment in non-ferrous ores is widespread. The results presented here imply that indigenous smelting constitutes a previously unrecognized source of early Hg pollution, given naturally elevated [Hg] in economic silver deposits.
In this paper we present the results of the formation of black HgS (metacinnabar) from liquid mercury and elemental sulfur using the mechanical energy provided by a ball mill in different conditions. Metacinnabar formation was observed even after short milling times (15 min) and unreacted liquid mercury was no longer detected after 60 min of milling. The reaction mechanism was monitored with a scanning electron microscope. The impact and friction forces of milling on the Hg and S mixture resulted in the formation of metacinnabar by reducing the size of mercury drops, giving rise to microspheres, and lowering the surface tension to allow sulfur grains to become adhered at the reaction interface. After 60 min of milling, the metacinnabar formation reaction was observed to be more than 99.99% complete, yielding a Toxicity Characteristic Leaching Procedure value of 3.1 microg/L Hg. The reaction product thus complies with the limits of the most stringent Universal Treatment Standard requirements, which allow a maximum TCLP concentration of 25 microg/L.
-rl
METAT IONS IN
BIOTOGICAT SYSTEMS
Edited by Astrid Sigel
Helmut Sigel "oa
Inslitule o, ,norgcfnic Cùemistry University of Bosel
CH-4056 Bdsel, Siyitze ond
VOLUME 34
Mercury and Its Effects on Environmént and Biologry
MnRcpu Dprxen, INc. NEw YoRK . Baspl . HoNc Korlc
ISBN: 0-8247-9828-Z
îhis book is printed on acid-free paper.
COPYRIGHT @ 1997 by MARCEL DEKKER, INC. ALL RIGITTS RTSERVED
11"itr5s1rÀis book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy- ing, microfflming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
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8
Microbial Tbansformation of Mercury Species and Their Importance
in the Biogeochemical Cycle of Mercury
Franco Bald.i
Department of Environmental Biology, University of Siena, Via P.A. Mattioli, 4, I-53100 Siena, Italy
1. INTRODUCTION 214
2. MICROBIAL RESISTANCE TO INORGANIC MERCURY 2t5
2.1. Mercury Resistance in Prokaryotic Cells 2t5
2.2. Mercury Tolerance in Eukaryotic Cells 221
2.3. Role of Mercury-resistant Microbes in the
Environment 223
3. BIOMETTryLATION OF MERCURY 226
3.1. Biosynthesis of Monomethylmercury 228
3.2. Mercury Methylation in the Environment 230
3.3. Dimethylmercury Biosynthesis: The Final Product of
Mercury Methylation 234
4. THE ROLE OF METACINNABAR. THE MOST STABLE MERCURY SPECIES 247
5. NEW VIEW OF THE BIOGEOCHEMICAL CYCLE OF MERCURY
244
213
214 BALDI
ABBREVIéffIONS 247 REFERENCES 248
1. INTRODUCTION
Mercury (Hg) occurs in nature as ionic and elemental mercury. Natural sources include the weathering of cinnabar (HgS) deposits and volcanic and geothermal emissions. Man-made sources include the exploitation of geothermal fields for power generation, combustion of fossil fuels, mining, chloralkali plants, and other minor industrial activities. Syn- thetic organomercurials have been banned for some decades, but more than9OVo oftotal Hgin muscle tissue oftop marine predators, such as in tuna and in seabirds, is monomethylmercury (MMHg), the most toxic species of Hg. MMHg crosses cell membranes by passive transport, and its long half-time in biological tissues leads to high concentrations at the top of the food chain. Minimal increases in the MMHg content of autotrophic organisms produces an unexpectedly large accumulation of Hg (biomagnification) in carnivores.
The transformation of inorganic Hg to MMHg in the environment is commonly accepted to be the work of microbes. Hg transformation by microbes is the result of adaptation to Hg toxicity. The most common detoxifying mechanism is the enz5rmatic reduction of Hgz+ to Hg(O) and confers high resistance to inorganic mercury salts. This narrow- spectrum of Hg resistance is harbored in plasmids in the inducible mer operon. The catalytic protein mercuric reductase is codified by t}:re merA gene. Less common but very important are the broad-spectrum Hg- resistant bacteria, which cleave the C-Hg bond of organomercurials by means of the enzyme organomercurial lyase, codified by the merB gene. Other proteins are involved in the control, binding, and transport of Hg. Other adaptive strategies have been developed to tolerate high Hg concentrations by production of extra- and intracellular polymers and./ or reactive molecules. Under anaerobic conditions, Hg toxicity is dras- tically reduced by organic and inorganic sulfides such as HrS. The latter reacts with Hgz+ and MMHg to produce the stable HgS and the volatile dimethylmercury (DMHg), respectively.
However, MMHg is also produced under these conditions and it is
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 215
still unclear, where, when, and what produces MMHg in the environ- ment. After 30 years of research, the mechanism of accumulation of high percentages of MMHg at the top of the food chain is still a matter of controversy.
The recent development of specific methods has made it possible to determine Hg species at femtomole levels. This has led to the detection of DMHg in the subthermocline of the Pacific and Atlantic Oceans and in the Mediterranean Sea, mangrove sediments, soil, and axenic cul- tures of anaerobic bacteria. It is also produced by the chemical reaction of methylcobalamin and inorganic mercury compounds. These occur- rences suggest that DMHg is of microbial origin and that MMHg is an intermediate product of a detoxifying mechanism, which produces the harmless DMHg. Unfortunately, the double methylation (dimethyla- tion) of Hg is slower than the accumulation of MMHg by cells, because MMHg crosses cell membranes at a high rate and inhibits metabolism more than does ionic mercury. To date, the absence of DMHg in the freshwater lakes of the northern hemisphere suggests that in these ecosystems MMHg is mostly of abiotic origin owing to the high concen- trations ofhumic acid and temperatures farbelow optimal for microbial activity for most of the year. In this chapter, it is sustained that the cycle of Hg is dominated by microbial activity and that bacteria convert toxic forms of Hg to harmless ones to detoxify themselves and the surround- ing environment.
2. MICROBIAL RESISTANCE TO INORGANIC MERCURY
2.1. Mercury Resistance in Prokaryotic Cells
Mercury existed in the environment long before the origin of life, but together with other elements with similar atomic numbers (Pt, Au, Tl, Pb, and Bi) it was not part of the primeval metabolism because of the low concentrations of these elements in the Earth's crust. The loga- rithm (Logro) of relative abundance of Hg is 6.5 orders of magnitude less (-2.5\ than that of the reference element Si = 4 [1]. During the Archean era, when conditions were anoxic on a globale scale, Hg was mainly complexed to sulfides. It only became available to microbes once photo-
216 BALDI
synthesis had evolved massive O, concentrations into the atmosphere. Volatile Hg(O) emitted by intense volcanic activity reacted with sulfides and also with O, and halogens, becoming soluble and toxic. WiLh2IVo of O, in the atmosphere and 2.6 t lgzt g of Cl in the oceans [2], Hg is one of the most toxic elements on Earth today.
Since the origin of life, aerobic bacteria had to adapt to Hg- enriched environments and developed different detoxifying strategies. To date, the most common Hg detoxification mechanism is common in gram-positive and gram-negative bacteria. These microbes transform inorganic Hg to volatile Hg(O) by means of the enzyme mercuric reduc- tase. Hg resistance was discovered by Moore [3] in a Staphylococcus aureus strain at the Torbay Hospital, Exeter, UK. Hg-resistant bacteria were found to grow on peptone agar amended with high concentrations of HgClr. Inorganic mercury resistance was associated with other resis- tance factors (antibiotics) [4] harbored in transferable plasmids. These plasmids conferred resistance to Hg and other metals (Ni and Co) [5]. A separate locus of Hg resistance, among other metal resistances, was observed in Escherichia coli and Salmonella 16l. The same year, Tono- mura et al. [7] isolated the first strain resistant to the organomercurial phenylmercury from contaminated soil. The plasmid-borne origin of broad-spectrum Hg resistance was identified several years later in E. coli and Pseudomonas a.eruginoso [8,9].
After several years ofresearch, the genes involved in narrow and broad-spectrum resistance in mer operorrs were sequenced in gram- positive and gram-negative bacteria [10]. The two products of merA
and merB genes were mercuric reductase and organomercurial lyase, respectively. Today, their molecular weight, kinetic parameters, amino acids, and DNA sequences are well known t10-211. The synthesis of the catabolic genes merA and merB is regulated by merR, which codes for regulatory proteins. Other genes, merP and merT, synthesize pro- teins for the binding and transport, respectively, of Hg species into the cell. A supplementary gene, merC, codes for a further Hg transport protein (MerC), which is embedded in the inner membrane with MerT. A rnerD with some regulatory activity has been found sporadically in some operons. The mer operons are commonly sited on transposons of the "TnJ family". The element Tn501 t22-251originated from the p aeruginosa plasmid and Tn21 element from plasmid R100 (originally from Shigella) wittr substantial homology (>707o) to TnS01 126-291.
'ì
.:
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 217
Genetic information on the mer operoî has been reviewed several times t30-331 and is also discussed in detail in this volume.
Other types of nonenzymatic Hg resistance in bacteria have not been reviewed so often because of varied and sometimes contradictory results. An attempt to clarify the various nonspecific Hg detoxification mechanisms necessitates a few definitions, for example, the terms 119 resistance and llg tolerance are often considered to be synonymous. Here they are defined as follows: Hg resistance is when there is a specific enzyme response to Hg coded by genes for the synthesis of an inducible enzyme, which maintains homeostatic Hg concentrations at subtoxic levels in the cytoplasm. The inhibition pathway has kinetics of the Michaelis-Menten type. Hg tolerance is when there is a nonhomeo- static and nonspecific mechanism towards Hg. Hg tolerance manifests with a gradual inhibitory effect by Hg and other metals. The inhibition pathway depends on the concentrations of reacting compounds such as polymers, which sequester metals, or molecules such as organic and inorganic sulfides, which convert or react with Hg species. These poly- mers and sulfides are produced irrespective of the presence of Hg and other metals. These two terms have a meaning if they are referred to the term Hg sensitivity in bacteria, which are deficient of homeostatic mechanisms and sequestering or reacting agents (better explained in the legend of Fig. L).
Certain nonspecific mechanisms of Hg detoxification reported in the past are dubious, because Hg(O) volatilization analysis was not always performed to check for mercuric reductase. In defining other nonspecific detoxifying mechanisms, it is important to know whether enzymatic transformation of Hg is occurring.
A type of Hg tolerance was reported in Staphylococcus aureus strains M3, SC, and SD isolated from humans and in strain K1015 from a culture collection (Giessen, Germany). The mechanism of tolerance was based on extracellular binding of Hg by polysaccharide polymer com- pounds, which were not decomposed in the presence of Hg2+ by extra- cellular lytic enzymes [34]. All the strains were initially sensitive to a minimum inhibitory concentration of 2 Fg'ml,-t HgClr. After serial transfers, strains adapted to grow up to 20 Fg'ml,-t of HgClr. Hg tolerance was associated with a complete loss of degrading enzymes, such as lipases and phospholipases, and reduced production of a-hemo- lysin, B-hemolysin, and protein A. The enzymatic activities decreased as Hg tolerance increased.
218
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Resìstance
80
8oo v40
FIG. 1. Diagram showing the differences between Hg resistance, Hg tolerance, and Hg sensitivity in bacteria. The latter is the reference used to define the other two. A Hg-sensitive strain is by definition inhibited by the minimal concentrations of Hg. Hg resistance is due to a detoxifying mechanism specific for Hg and inhibition is due to saturation of mercuric reductase. Hg toìerance is nonspecific for Hg and is based on sequestration or reactive compounds which are produced irrespective of metal concentrations.
Another type of Hg tolerance was reported, for example, in a strain of Pseudomonas oleouorans isolated from activated sludge. This strain was tolerant up to 350 pg.ml-l of Hg2+, 100 Fg.ml,-t of Cdz+,40 pg'mL 1 of Cr6+, and 1000 FB.ml,-t of Cu2+. Tfansmission electron microscope measurements revealed cells with many electron-dense granules in the cyboplasm. Chemical analysis showed that about 80Zo of the Hgtaken up was in the cytoplasm and the rest in the cell envelope fraction. In the insoluble cytoplasmic fraction, Hg was found in polysac- charide polyphosphates and lipopolysaccharide fractions [BS]. This in- tracellular metal sequestering mechanism was also found in the cyano- bacterium Plectonemq boryanum, which produces polyphosphate bodies that sequester significant amounts of various metals. When exposed to 100 pg.ml--l of Hg2+ as HgClr, these autotrophic cells accumulated significant Hg concentrations in the polyphosphate bodies and little in
Sensit ivity
Tólerance
o lo f*-"",X"nr.u,1o.r,r',i8o
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 219
the cytoplasm [36]. Other subcellular structures such as extra- and intracellular membrane compartmentalization of Hg in intrathylacoid whorls can be regarded as a further detoxifying mechanism conferring additional Hg tolerance to P boryanum [37,38].
The synthesis of metal-binding compounds may also be a mecha- nism of Hg detoxification. A metal-binding metallothionein protein was isolated among prokaryotes in Synechococcus sp. [39]. This type of protein is characterized as a group of low molecular weight molecules with a high metal and cysteine content f40,411. They were first found in the equine renal cortex [421. The metallothionein-like proteins have been found more in eukaryotic than prokaryotic cells [39,43,44]' T}:,e precise role of these proteins is unknown, but they are certainly in- volved in heavy metal detoxification [38,45]. In Synechococcus strains PCC 630 and PCC 7942,the metallothionein was found to be a product of gene smtA 146,471.
A further Hg-detoxifying mechanism, based on nonenzymatic re- duction of inorganic Hg, was reported for the cyanobacterítm Synecho- cocctts bacillaris. A strain that volatilízedHg2+ to Hg(O) at a rate of 6.6 pmol.pg-t. Chlo d-1, which is about 30 times that carried out by micro- algae [481. The mechanism of Hg volatilization was not enzymatic and is still unknown; however, in phytoplankton, the smaller cyanobac- teria were found to be the main Hg reducers. It would be worthwhile to study this nonspecific Hg transformation in detail. Hg volatilization was very low compared with the enz;rmatic efficiency of the gram- positive and gram-negative aerobic heterotrophic bacteria, but it may be important in relation to the environmental impact of Hg mobiliza- tion in the hydrosphere, since cyanobacteria can reach concentrations of >108 cell'L-r [48].
In anaerobic organisms, Hg biotransformation is not well under- stood and other mechanisms of Hg tolerance have been hypothesized [49,50] . Variable tolerance to inorganic and organic mercury com- pounds was found in the strictly anaerobic bacteria Bacteroides rumi- nicola and in Clostridiunt perfringens, which was isolated from clinical settings and sewage [511. Inorganic mercury resistance in these isolates was neither inducible nor plasmid mediated.
Under laboratory conditions, Hg tolerance often manifests in an- aerobes, because the strains are gTown in the presence ofhigh concen- trations of cysteine, a reducing agent. The addition of 1mM of cysteine
220 BALDI
chloride to the medium increases the Hg tolerance 17-fold [51]. Anaer- obic species such as Bacteroides succinogenes, B. fibrosoluens, and Megasphera elsdenii showed Hg tolerances to 100 Fg . ml,-t Hgz+ and to 326 pg'mL-rHgz+, respectively, because they were cultured in media rich in cysteine or cysteine plus rumen fluid from cattle [52]. A 40Vo reduction by Hg volatilization was found during 20-min incubation with 2o3}Ig2+ added into the media in the absence of sulfydryl groups with induced and uninduced strains of Enterobacter cloacae, B. ruminicola, and,E. coli [5]). However, Hg volatilization was not rapid or complete, as it is for the enzymatic Hg resistance.
The strictly anaerobic Clostridium cochlearium strain T-2 was found to be tolerant to 800 Fg.mL 1HgCl, in a medium containing 0.0l%o cysteine [53]. This strain was also demonstrated to be able to degrade MMHg by the virtue of a plasmid which did not harbor mer operon but had the capability to produce HrS. This compound has been demonstrated to have a detoxifying activity on MMHg, which is trans- formed to DMHg and B-HgS 1541. Desulfouibrio desulfuricans tolerated 100 times more MMHg than the broad-spectrum Hg-resistant Pseudo- monas putida strain FB-1, which can degrade 1 pg of MMHg en- zymatically to CHn and Hg(0) in t h [551. Organic sulfides, therefore, transform or bind Hg and significantly reduce its toxicity in anaerobic environments. This means that Hg is harmless and stable in anoxic compartments, and microbes may not need any enzymatic and./or energy-dependent mechanism to reduce Hg toxicity.
Another mechanism of Hg tolerance, the oxidative demethylation of MMHg under anoxic conditions, was recently reported [56,57]. La- beled raCHrHgI spiked into two strains of Desulfouibrio was converted to laCHn from the methyl group of MMHg and laCHn and 1aCO, were formed by a methylotrophic methanogenic culture grown on trimethyl- amine. Tlaces of CHn were also observed during the conversion of MMHg (reductive dimethylation) to DMHg in axenic cultures of D. desulfuricans [58]. Oxidative demethylation of MMHg was also identi- fied in sediments of the Carson River, Nevada. Inhibition of sulfate reduction by additions of molybdate resulted in a significantly de- pressed demethylation of the oxidation/reduction ratio. Addition of sul- fates to sediment slurries stimulated the production of laCO, from [1aC]MMHg, whereas additions of 2-bromo-ethane-sulfonic acid (BESA), an inhibitor of methanogenesis, blocked raCHa production [52].
Ì
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 221
2.2. Mercury Tolerance in Eukaryotic Cells
The enzymatic Hg resistance conferred by the flavoenzyme mercury reductase has not yet been found in eukaryotic cells. The mer operon was successfully transferred to a recipient strain of Saccharomyces cereuisiae, but the efficiency of the detoxifnng mechanism was reduced by one order of magnitude with respect to the donor E. coli S-17-1 t591. The modified yeast-synthesized Hg reductase was five times more resi- stant than the Hg-sensitive S. cereuisiae strain Afl22. The reduced efficiency of Hg volatilization in eukaryotic cells is probably due to a slower passive diffusion of Hg(O) than in the small prokaryotic cells. This experiment probably suggests why the detoxifying enzymatic mechanism of prokaryotic cells did not develop in eukaryotic cells.
Natural Hg volatilization as Hg(0) has, however, been reported in the yeasts Cryptococcus sp., Candida albicans, and Saccharomyces cereuisiae [60,61]. The Cryptococcus sp. strain exposed to high concen- trations of HgCl, reduced Hg2+ significantly in the supernatant, but on the other hand, the yeast cells accumulated up to 90 pg' g-1 of total Hg when exposed to 180 pg'ml,-l HgClr. Electron micrographs of un- stained cells showed Hg associated with the cell wall and intracellular vacuoles. Elemental analysis did not provide any information about the Hg species complexed to the wall, but the fact remains that this Hg is retained by the cells and does not diffuse away as does Hg(O).
Hg volatilization has also been reported in the green algae Chla' mydomonas sp. and Chlorella pyrenoidosa 162,631. The latter vol- atilized Hg(0) in 8 days, after a lag phase of 4 days, by means of low molecular weight (smaller than 1200 Dalton) intracellular reducing factor t631. Hg volatilization has even been found in phyboplankton growing in defined media, natural seawater, and freshwater [48]. Ma- rine eukaryotic phytoplankton (>3 pm) reduced Hg'* to Hg(0), but this conversion was much lower than in cyanobacteria, which were the predominant autotrophic Hg reducers. The mechanism of the slow Hg volatilization (0.5 pmol'pg-l Chlo 6-t) which occurs in the diatom Thalassiosira weissflogii and in the green algae Dunaliella tertiolecta
and P au lou a lutheri is unknown . ln T. w e i s sflogll, this nonspecifi c metal reduction is probably due to agents which are also able to reduce Cu2+ to Cu+ at a higher rate [64].
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4-5
è0
èo
-l e4
h 6)?
,-^
Mercury chloride (pglml-)
FIG. 2. Accumulation of total Hg in algal cells exposed for 3 h to different concentrations of HgClr: dead cells of Scenedesmzs sp. (r) and in Scenedesmus acutus (o) and in photosynthesizing cells of Scenedesrnus sp. (n) and Scene- desmus acutus (o). In the latter two, the low accumulation was related to a higher rate of photosynthesis.
A different mechanism of Hg tolerance was recently found in green microalgae isolated from polluted areas in Ttrscany, Italy. Hg tolerance ín Scenedesmus sp. was related to lower Hg uptake in the photo- synthesis and respiratory modes of the Hg-tolerant strain than in the Hg-sensitive Scenedesmus dcutus (Fig. 2). In dead cells, Hg accumula- tion was similar for both strains t651. An energetic mechanism was hypothesized to be associated with the photosynthetic activity of the Hg-tolerant strain. In Scenedesmus sp., high O, evolution rates and a high intra- and extracellular pH may also be responsible for the chemi- cal conversion of spiked soluble HgClo to the less soluble mercury
rì
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 223
hydroxide [Hg(OH)r] in the medium, which became highly aerobic and alkaline (pH = 10 in 3 h) [66,67]. So the reduced Hg uptake by the Hg- tolerant Scenedesmus sp. additionally was due to the a possible co- precipitation of Hg(OH)2 with colloidal particles associated with the other mineral hydroxides of the medium. In other experiments, Scene- desmus obliquus has been observed to form colloids offerric hydroxides, phosphates, and carbonates and to coprecipitate ionic Cu and Cd [68,69]. Moreover, it was hypothesized that HS(OH)z formed in the colloidal fraction associated with photosynthetic pigment distribution in the seawater column [70].
2.3. Role of Mercury-resistant Microbes in the Environment
Since the 1970s, several reports have been published on narrow- and broad-spectrum Hg-resistant bacteria isolated from different fresh- water [71] and marine environments [72,73]. All isolates carried out the same detoxifyrng reactions with the two enzymes, mercuric reductase and organomercurial lyase. Broad-spectrum Hg-resistant bacteria are less common than those of the narrow spectrum. The latter were found in the estuary of Minamat aBay , Japan [74] . Thousands of strains were subsequently isolated from sediments and were characterized in terms of eight different pathways of inorganic and organic compounds, as reported in Table 1 t751.
In the Mediterranean area, the first isolation of Hg-resistant bac- teria was near pyrite and cinnabar mines in T\rscany, Italy [76]. Out of 37 aerobic heterotrophic species isolated, only six volatilized Hg2* to Hg(0). A chemolitotrophic T. ferroxidans strain SW9K-1was isolated from pyrite mines t77l.It showed a constitutional Hg resistance similar to strain BA-4 isolated from a coal mine settling pond in Wyoming [78]. Thirty-six Hg-resistant strains of 106 heterotrophic bacteria were later isolated from the Fiora River, which drains an area of cinnabar deposits in southern T\rscany. Seven strains were classified as broad- spectrum Hg-resistant and degraded MMHg [79]. In all37 strains, Hg resistance was due to the volatilization of Hg2+ to Hg(0), and it was always inducible.
The inducibility of Hg resistance is an evident adaptation to Hg
TABLE 1
Volatilization Patterns and Frequency of Mercury Compound-volatilizing Strains from Minamata Bay and Control Stations
Pattern of volatilizationu l2345678strains
Total
Hgz+VVVVVVVV
Methyl-Hg
Ethyl-Hg
Thimerosal FluoresceineHgacetate NV Phenyl-Hg acetate NV lnr
NV NV
NV NV
NV NV NV l\n/ lln/
NV NV V V V V NV NV V V V V V NV V VVVVV NV V NV NV NV V NV NV V NV NV NV V V
V V
p-Chloro Hg benzoate
NostrainsMinamataBay 10 16 I 11 3 1 6 19 75 (n = 1428)
Control(n=3176, 18 16 4
12 value 0.28 5.34b 8.83" 10.6c 0.20 0.07 13.3c 41.8" 42.5"
uV, Volatilization; NV, no volatilization.
bp < 0.05.
? < 0.01.
Source: Reproduced with permission from [75].
5 I 2 0 0 54
ru r\) è
(D
t- 0
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 225
stress. Mercury ions alter microbial community structure and function [80]. The mechanism of adaptation in situ was studied in detail by Barkay [81] in different freshwater, salt marsh and marine environ- ments. If water samples were preexposed to HgCl, (250 p"g. L-1), the Hg residence time was shorter than in freshwater. In environments rich in humic acid (salt marsh), Hg binding to organic material slowed down Hg volatilization and its residence time. Hg volatilization has been correlated with its bioavailability for bacteria. If Hg is complexed by chlorine in seawater, the reduction of Hg(II) to Hg(0) is faster than in
freshwater samples, where inorganic Hg occurs mainly as hydroxides and is therefore less available [31,81]. When Hg is added, the population of Hg-resistant bacteria increases in percentage, but it decreases in biodiversity [81].
Similar experiments were carried out with two to four orders of magnitude lower concentrations of HgCl, (from 0.02 to 2 pg'l-t) spiked in a controlled freshwater system. In the water column, the unexposed microbial community increased suddenly from 0.02 to 2lVo in a 12-h experiment and then gradually decreased over a week to almost pris- tine values. During sampling, there was an instant decrease in the Hg- sensitive population in24h on the one hand and, on the other hand, an increase from one to seven different Hg-resistant species in week-long experiments [82]. It is not clear whether the Hg-resistant species were uninduced but already present in the controlled system or ifthey be- came Hg-resistant by horizontal gene transfer from the frrst Hg- resistant isolate. This aspect of Hg resistance mobility in a natural microbial population was studied in "in situ mating" experiments with epilithic bacteria isolated from a river in southern Wales using a natu- ral plasmid pQMl harboring narro\M-spectrum Hg resistance [83]. A highly signifrcant linear relationship was found between the loga- rithmic transfer frequency of the plasmid in relation to water tempera- ture changes from 6 to 21"C. The transfer frequency increased by 10-fold for every 2.6oC increase.
The determination of mer genes in Tn21 was also used to deter- mine the role of this transposon in the adaptation of microbial commu- nities to ionic mercury [84]. Cross hybridization with Tn2l was found ín50Vo of Hg-resistant bacteria strains in two freshwater communities, but only in l27o of strains represented in two saline communities. It is a common finding that Hg-resistant strains from different environ-
226 BALDI
ments do not hybridize with the same genetic probe [85,86]. However, it is always possible to determine a positive correlation between metal resistance and Hg contaminations as measured by using the plate count method [87] and/or hybridization with genetic probes [83,84]. Some- times no relationship between metal content and the relative percent- age of resistant microbes is found, because below the minimum metal threshold concentration there is no response. Because Hg resistance is inducible, the detoxifying mechanism is switched off at very low Hg concentrations.
Organomercurial resistance is another known type of Hg resis- tance, but there have been few papers on organomercurial adaptation and not much is known about the distribution of merB (the gene coding for organomercurial lyase) in the environment. Significant Hg resis- tance has been found in bacterial communities isolated from New Hope Pond and Reality Lake (Tennessee) contaminated by Hg(0) t881. MMHg resistance seemed to be slightly higher in the pond communities than in those of control water. However, the response of the microbial commu- nities was feeble because of the very low Hg concentrations. When 10 pg.L-lMMHg was added, the MMHg resistance of the microbial com- munity of the pond water increased significantly. Conversely, in a con- trolled freshwater system, there was no microbial response to MMHg additions and no broad-spectrum Hg-resistant bacteria were isolated from the water column after a week of exposure to 2 p"g' L-l of this organometal t821.
3. BIOMETHYLATION OF MERCURY
In more than 30 years of environmental studies on the biogeochemical cycle of Hg, there are still many unsolved questions on MMHg bio- synthesis. It is not clear whether MMHg synthesis is an enzymatic process or just a chemical reaction with methylcobalamin or whether the vitamin B, producers are the only candidates for methylating Hg. We do not know if bacteria first produce dimethylmercury (DMHg) which is instantly degraded to MMHg or whether DMHg is the final product of methylation. DMHg may possibly be a mechanism of Hg detoxifrcation. The importance of environmental parameters in Hg
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 227
methylation is unclear as are the relative weights of abiotic and biolog- ical methylation. To answer all these questions and others, a key point is to determine MMHg and DMHg and other related Hg species specifi- cally and accurately.
Recent methodologies based on derivatization of inorganic and organic Hg to volatile compounds by reaction with reduced boron com- pounds and their determination at picomole levels by gas chromatogra- phy in line with atomic fluorescence spectrophometry [89,90], atomic absorption spectrophotometry [91], or Fourier transform infrared spec- troscopy [92,93] brought the presence of DMHg in the environment back to the attention ofresearchers. So until the 1990s, except in spo- radic papers, the only organic Hg species reported in the environment was MMHg.
In the past, MMHg concentrations in sediments, microbial cul- tures, and other matrices were overestimated owing to analytical arte- facts related to the low reliability of methods for extracting MMHg from complex matrices such as sediments, microbial cultures, and others [94]. For example, the additions of natural thiols, coenz5rme M, methanethiol, dimethylsulfide, and halomethane to bacterial cultures caused an increase of solvent-extractable Hg, but it was not MMHg [93]. To understand the role of microorganisms in MMHg synthesis from several decades of studies, it is important to consider not only the different analytical techniques but also the different protocols, which may include additions of vitamin B, or similar congener. Bacteria were often "forced" to produce MMHg by adding methyl donors, so that it was often impossible to distinguish biological activity from chemical reactions.
From a historical point of view, MMHg and DMHg of natural origin were first determined by gas chromatography-mass spectrome- try (GC-MS) analysis in aquarium sludge and in rotten fish spiked with HgClr. Both organomercurials were found after 4 and 7 weeks of incu- bation under anaerobic conditions, suggesting that Hg2+ alkylation may have a bacterial origin and that anaerobic conditions could effect this reaction [95]. MMHg and DMHg were also determined by thin layer chromatography (TLC) in cell-free extract of a methanogenic strain, MOH, isolated from symbiotic mixed cultures in a canal mud from Delft, Holland [96]. In the organomercurial synthesis test of Hgz+, the cofactor methylcobalamin (CH.-Co(III)-5,6-dimethyl benzimid-
228
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azolyl-cobamide) was added to cell-free extract, which is a good sub- strate for methanogenesis. Inorganic Hg inhibited methane production, reduced vitamin Bu to Brr., and produced the two alkylated forms, MMHg and DMHg. This rapid conversion suggested that methyl group transfer from co3+ to Hg2+ could be nonenzymatic (trans-alkylation). A few years later, Imura et al. [97] demonstrated that Hgz+ could be methylated chemically by methylcobalamin in a few hours, at BZ.c in the dark, under mild reducing conditions, and in the absence of cell extract. DMHg and MMHg were detected with silica gel thin layer in a three-solvent system. It was suggested that the initial product of this reaction was DMHg for equimolar quantities of Hgclr. DMHg was then
converted to MMHg by further action of HgClr, as follows [92]: 2CHs-Bu + HgCl, ___> (CHr)rHg
(CHr)rHg + HgCl, ---> 2CHrHgCl
since the demonstration that inorganic Hg was chemically meth- ylated to MMHg and DMHg, it was generally accepted to convert DMHg to MMHg for routine analysis by gas chromatography equipped with electron capture detector (GC-ECD) [gg,99]. This derivatizatìon proce_ dure was welcomed because of the high volatility of DMHg and to avoid its possible decomposition in the GC column. so the tw-o organomer- curials were treated with inorganic acids and DMHg conrreried quan-
titatively to MMHg. This analytical simplification arù became popular among chemists, engineers, biologists, and other scientists interested in Hg pollution; however, the cycle of Hg was lacking information on Hg mobilization and reduction in Hg toxicity.
3.1. Biosynthesis of Monomethylmercury
From the 1970s, studies of the transformation of inorganic Hg to MMHg flourished in many environmentar laboratories. Many ref,orts dealt with MMHg synthesis in pure cultures t100_1041 and it seemed that both prokaryotes and eukaryotes could methylate Hgz+. This synthesis was increased by the addition of vitamin B, to the curture íedium.
vitamin Brr, in its methylated form, was therefore considered the main substrate for MMHg synthesis. This growth factor is known to be pro_ duced copiously by aerobic and anaerobic microbes: pseudomonos d.eni-
(1)
e)
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 229
trificans [105,106], Rhodospirillum rubrum [I07], Salmonella typhi- murium [108], Escft.erichia coli ft091, Propionibacterium shermanii ILIO), Eubacterium limosum [111], methanogens [112-115], acetogenic bacteria [116], clostridia [49,117] and sulfate-reducing bacteria [118].
In one of the first studies under laboratory conditions, MMHg production was determined in cultures of gram-positive and gram- negative species (Pseudomonas fluorescens, Mycobacteriurn phlei, Ba- cillus megaterium, E. coli, andAerobacter aerogenes) and fungal species (Aspergillus niger, Scopulariopsis breuicaulis) 1702). MMHg was deter- mined by GC-ECD in experiments with and without vitamin Brr. Small amounts of MMHg were formed by all bacteria on addition of vitamin B',. All three fungal species methylated Hg. When Dl-methionine was added to the medium, the fungi were more sensitive to HgClr. No significant increase in MMHg concentrations was detected inA. niger in the presence of 10 Fg.ml,-t HgClr.
Alternative Hg methylation with vitamin B, were tested. For example, Hg methylation was studied in Ne urospord crassa on the basis of methionine production [119], since Hg tolerance was associated with the methionine synthesis loci in a Staphylococcus strain [120]. In N. crasscr., methionine was found to increase toxicity in the presence of HgCl, but not MMHg production. The fungus could tolerate MMHg toxicity in the presence of other methyl donors like choline and betaine. MMHg increases were found in cultures by adding amino acids such as Dl-homocysteine and Dl-homoserine. In these experiments, MMHg was determined only by the GC-ECD technique. No other specific ana- lytical methods were used. The production of MMHg during methionine synthesis has not yet been confirmed in other biological systems. Since this experiment, many investigators have unsuccessfully used DL- homocysteine in pure cultures to improve MMHg synthesis through the S-adenosyl-methionine (SAM) pathway. SAM is another important
methyl donor with potential methylation for Hg2+; however, Hg meth- ylation did not take place with this compound in a yeast strain of Saccharomyces cereuisiae, a good SAM producer [121] . In some bacterial species, we cannot exclude the indirect involvement of other methyl donors in the enhancement of Hg methylation in certain environments.
For more than 15 years, it was generally accepted that the meth- ylation of Hg was performed by prokaryotes in aerobic and anaerobic environments. Until compeau and Bartha [122] demonstrated that
230 BALDI
MMHg was synthesized in anoxic estuarine sediments (-220mV), sulfate-reducingbacteria were suspected to be the main bacterial group involved in MMHg biosynthesis in situ [123], although it has been proved for years that the presence of sulfides inhibited this process in soil, sediment, and bacterial cultures 1122,124-1261.
The principal experiment that demonstrated the effrciency of sul- fate reducers in Hg methylation was performed with slurries of salt marsh sediments incubated in an anaerobic chamber [123] by spiking 75 pg'ml,-r of HgCl, into the system. Experiments for inhibition or enhancement of MMHg synthesis were carried out by adding different organic sources: pyruvate, acetate, and lactate, which stimulated three- fold more MMHg synthesis than glucose. The addition of BESA, a specific inhibitor of methanogenesis, significantly increased MMHg synthesis in 10 days. Conversely, additions of molybdate, a specific inhibitor of methanogenesis and sulfate-reduction, suppressed MMHg synthesis (Fig. 3). This experiment showed that sulfate reducers were the predominant, if not exclusive, Hg methylators in this environment. Potential Hg methylation was fully expressed only when sulfate was limiting and carbon sources were available at low salinity. However, one strain that methylated Hg better at 0.5 M than at 0.2 M of chloride was isolated from a high-salinity enrichment culture [127].
MMHg synthesis by D. desulfuricans, isolated from salt marsh sediments [123], was studied by following the biosynthesis of the amino acids serine and glycine with radiolabeled l3-laClsodium pyruvate 11281. A frnal methylene group transported by N5,N10-methylene- tetrahydrofolate (THF) was converted to a methyl group and was then transferred to vitamin B,, [118], which finally methylated Hg2*. The role of cobalamins in MMHg synthesis was again confirmed to be funda- mental, and they are still the only compounds known to transfer the carbanion (CH;) to Hg2+ [129-1311.
3.2. Mercury Methylation in the Environment
One of the first determinations of MMHg in the environment was performed at the time of the trial between Japanese civil authorities and the Chisso factory at Minamata 11321. The dispute centered on whether the MMHg in the bay was of industrial origin or naturally
r-
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 231
0246810
Timc (daYs)
FIG. 3. Synthesis of MMHg in anoxic estuarine sediment slurry spiked with Hgz* at 75 p,g. g-t (dw) (o) and the effects on the process of 30 mM BESA (.) and 20 mM NarMoOa (t), specific inhibitors of methanogenesis, and sulfate reduction, respeitively. Error bars represent the range ofvalues in duplicate samples and are omitted when smaller than the symbols. (Reproduced with permission from [123].)
biosynthesized by microbes. The defense held that it has been demon- strated that in Sweden, bacteria could methylate Hg [95]. When high concentrations of MMHg were found in the industrial effiuent in 1971 t1331, the authorities closed the industrial plant. It was estimated that this industry dumped about 4.1tons of MMHg out of 82 tons of total Hg through the Hyakken outlet from 1932 to 1971 [132,134].
After subsequent incidents as occurred in Iraq [135-137]' MMHg was banned everywhere, and today its concentration in the environ- ment is generally of natural origin. It was identified in estuarine sedi- ments from the Mississippi Delta [99]. Hg methylation was also studied in situ in estuarine sediments from San Francisco Bay [138]' High con- centrations of HgCl, were spiked in natural shallow and deep sedi- ments, and MMHg was detected after 0, 18, and 28 days. This experi-
232
BALDI
ment led to the hypothesis that MMHg was biosynthesized in sedi- ments but also degraded by bacteria (demethylation). The totai concen- tration of MMHg in the sediments would therefore be the resultant between the methylation/demethylation rate. This difference between the Hg methylation/demethylation process in the oxic/anoxic zone was well demonstrated in further reports [88,139-143]. The inducible growth of aerobic bacteria which degrade MMHg is likely to occur in aerobic sediments, when the concentration of this organomercurial reaches the threshold value for induction of organomercurial lyase synthesis in broad-spectrum Hg-resistant bacteria. In general, it has been estimated that an average of LÙvo of plasmids confer resistance to organic mercury in aerobic bacteria populations [81]. However, this conventional percentage can be nil or higher than 10 depending on the
bioavailable MMHg in the ecosystem [82,88].
Most of the studies on Hg methylation in situ have been performed
in the northern hemisphere where MMHg is unexplainably high in fish [144-1471in the absence of local contamination. The low concentrations of Hg are probably from emissions of fossil fuel plants and subsequent condensation in cold areas ofnorthern Europe, northern united states, and canada [148-150]. since fish living in lakes of these regions contain high concentrations of MMHg, there is much interest in the origin of
MMHg in these environments.
Mercury methylation is generally studied in situ in the water
column, sediments, and surface aggregates by radiochemical tech- niques. For example, high production of MMHg by sediment-floc in non-mercury-contaminated lakes was about 10 times faster than in Hg- contaminated lakes [151]. This confirms that the absence of the non- induced broad-spectrum Hg-resistant bacteria in pristine areas leads to the formation of more MMHg than in Hg-polluted areas where MMHg degradation is induced.
Microbial Hg methylation in the environment does not depend only on redox potential but also on several other parameters such as pH, temperature, and nutrients. The importance of pH on MMHg for- mation seems restricted mainly to freshwater systems adjacent to in- dustrialized areas in North America and northern Europe where most of the water basins have a low pH owing to acid depositions and high concentrations of humic substances. An increase in Hg methylation in the water column was observed, for example, in the Canadian Shield
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 233
lakes when the water was acidic [142]. MMHg formation in the fresh- water environment is stimulated at the water-sediment interface in lakes at low pH and possibly also in the aerobic water column. Lowering the pH at the aerobic/anaerobic sedimenVwater interface resulted in a two- to threefold increase in the rate of MMHg production [142,152]. The effect of pH on Hg methylation may differ between lakes with aerobic waters; for instance, MMHg production was greater in the epilimnetic water of Little Rock Lake (Wisconsin) at pH 6.1than in an artificiallly acidified aerobic basin at pH 4.7 [142]. However, sometimes the immobilization of Hg by sulfides, which was probably the reason for the decreased rates of Hg methylation in some acidified subsurface sediments, cannot be excluded [153-155].
An additional phenomenon correlated with low pH was the release of MMHg from the sediment surface. The behavior of dissolved organic carbon (DOC), which binds Hg2+, DaY be an additional factor that contributes to Hg methylation in the water column of acidified lakes. One of the effects of lake acidification on DOC is its sedimentation as DOC- aluminum complexes from the water column [156-158]. The precipita- tion of DOC and thus the loss of Hg-binding sites might also explain the stimulation of free Hg methylation in the water column. Thus, acidifica- tion reduces the metal binding efficiency of DOC and stimulates water column methylation of more available inorganic mercury.
The influence of temperature on MMHg biosynthesis was studied in sediment and water of the upper Wisconsin River [139]. It was found that the top 4 cm of sediment exhibited the greatest Hg methyla- tion activity, especially in a site significantly enriched with total Hg, whereas in the water column, methylation was almost undetectable' Mercury methylation was higher at an optimum temperature of 35'C under laboratory conditions, and it was stimulated by addition of pro- tein hydrolysates and anoxic conditions [139]. The low temperatures in the northern hemisphere do not favor microbial activity or Hg methyla- tion. This suggested that MMHg can be of abiotic origin; at least in the coldest periods of the year when the temperature is far from the optimal microbial growth. Studies of Hg methylation by psychrophilic bacteria have not yet been performed. So the main abiotic sources of MMHg in these ecosytems are probably humic substances [].59,1601 and/or rains
[161-163].
With regard to the influence of nutrient additions in controlling
234 BALDI
methylation of Hg, it has been found in situ that additions of tryptic-soy broth to sediments increased Hg methylation [151]. In natural systems, nutrients for bacteria are from periodic resuspension of the sediment and plankton debris. In the most polluted sites in the Wisconsin lakes, the highest Hg methylation yield is reached late in summer because of the high organic matter content when the river is low [139]. The high concentration of DOC in lakes often coincides with an increase in MMHg, mainly in the particulate organic matter [143]. The addition of different nutrients, especially pyruvate, to salt marsh sediments in- creased Hg methylation at least threefold t1231.
All these parameters-redox potential, pH, temperature, enzy- matic demethylation, and nutrients-play an important role in MMHg mass balance in an ecosystem. In one lake, low pH may be important in stimulating Hg methylation, whereas in another, the decomposition of newly flood-borne organic material or more anaerobic conditions are more important ft42). The biological origin of MMHg is difficult to differentiate from abiotic formation, especially in cold freshwater envi- ronments. The demethylation of MMHg to Hg(0) and CH, is of biolog- ical origin and occurs mainly at the oxic/anoxic interface, at mild tem- peratures, and with a good supply of nutrients. However, in other habitats, the double methylation of Hg (dimethylation) to DMHg seems to prevail over monomethylation.
3.3. Dimethylmercury Biosynthesis: The Final Product of Mercury Methylation
For 30 years, there was little interest in DMHg [95-97,122,164] for lack of a routine analytical method for its determination and because this species was considered a mere by-product of MMHg formation. It was recently demonstrated [131] that methylcobalamin, the only compound permitting methylation of inorganic Hg, produced DMHg as the final product and not MMHg as it was previously suggested [97,98,126].
A specific analytical approach was proposed to measure alkyl-Hg forms simultaneously by purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy (PT-GC-FTIR) [1311. The determination was performed in sealed vessels to avoid losses due to
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 235
the rapid volatilization of DMHg. The new technique provided impor- tant new information:
1. DMHg was the final organic Hg species in the reaction be- tween methylcobalamin and ionic Hg. MMHgwas formed first and then transformed to DMHg.
2. The mono- and dimethylation of Hg depended on the tempera- ture according to the Arrhenius equations:
143.5
ln(MMHg.) = 6.161 - T (3)
R2 = 0.997
ln(DMHg.) = 2.758 -
R2 = 0.944
7r.34
(4)
where MMHg, is the methylation rate and DMHg" is the double meth- ylation rate of Hg and the temperature, I in 'C.
3. The second methylation step for MMHg, either formed in solu- tion or spiked into the system, was as fast as the first methylation step. The alkylation rate of CHrHgCl to give DMHgwas calculated at 20 and 37'C and is expressed by the following equation:
ln (DMHg.) = 6.431 - t46.9 (ol T
4. The reaction of methylcobalamin with different inorganic Hg salts (vitamin: Hg ratio 10:1) showed that for Hg salts with a higher ionic component of the Hg bond, such as in HgSOn, DMHg formed more rapidly than with halogenated salts (HgCl, and HgIr) that have a higher covalent component.
5. The high volatility of DMHg necessitated a suitable sampling for gaseous compounds [1311. During analysis, 25Vo of DMHg was lost during sample transfer from the reaction vial to the purging vessel of the PT-GC-FTIR apparatus.
The formation of DMHg was demonstrated in axenic cultures of D. desulfuricans strain LS, which is known to synthetize both vitamin B, and MMHg, after additions of Hg2+, during fermentation [118]. This strain also formed DMHg from added MMHg but by a different path-
236 BALDI
way. Experiments carried out with the sulfate-reducer strain LS dem- onstrated the disappearance of MMHg from the cultures with HrS production from the dissimulative reduction of sulfates. However, it was previously observed that chemical additions of HrS made MMHg volatile [165] and led to the formation of white crystals which were identified as dimethylmercury sulfide (DMHgS; i.e., CHrHg-S-HgCHr) [166]. In time, the crystals turned from white to black; in this secondary reaction, the volatile DMHg and solid metacinnabar (B-HgS) formed.
Under laboratory conditions, active cells ofD. desulfuricans strain LS cultured in Postgate medium with sulfate immediately produced DMHgS from MMHg additions (100 pg'ml,-r). An authentic standard was not used to identify it, but the chemical species identification was based on the mass spectra from molecular fragmentation (mass/charge ntio [m I z)). The fragments were: m / z = 47 for [CHs - S] +, m / z = 202 for [Hgi *, m / z = 217 for [CHrHg] +, m / z = 249 for [CHr-Hg- S]+, m / z = 264 for [CH, - Hg- S - CH3l +, and, m / z = 464 for [CH, - Hg- S - Hg- CHrl +. The mass/charge ratios of various peaks identifred this species as DMHgS (Fig. a). DMHgS was determined by GC-MS after chloroform extraction from culture suspensions [54]. The formation of DMHg from MMHg and then from DMHgS was monitored for 15 days of incubation of strain LS. During this period, B-HgS was also formed, precipitating in the cultures. Tbansformation of MMHg to DMHg and F-HgS occurs as follows:
2MMHg + H2S -+ [DMHgSl -+ DMHg + B-HgS (6)
During reaction (6), traces of CHn formed, probably by further degradation of DMHg due to the weakly acid conditions (pH = 6.2) in the bacterial culture [54].
Degradation of MMHg by HrS from sulfate-reducing bacteria was also reproducible in sediments. MMHg spiked into anoxic marine sedi- ments was also transformed into DMHg, although the intermediate form, DMHgS, was not found. A method to extract DMHgS species from complex matrices still has to be developed. As in axenic cultures of D. desulfuricans, MMHg disappeared from anaerobic sediments with the evolution of DMHg. This transformation was of microbial origin, be- cause DMHg was not significantly produced after sediments were treated with molybdate, a strong inhibitor of sulfate-reducing bacteria Í58,1221. The transformation of MMHg to DMHg was followed in 8-day experiments in the same anoxic sediments spiked with 10 pg of CH,HgCl.
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 237 Abundanc!
4000
0
10 12 14 Time (min)
MaslCharge
FIG. 4. Mass spectrum of dimethylmercury sulfide (DMHgS) extracted with chloroform from a culture of D. desulfuricans strain LS. The relative retention time (top) and the abundance of molecular fragmentation (bottom) are shown. (Reproduced with permission from [54].)
The disappearance of MMHg was complementary to the formation of DMHg in the sediments (Fig. 5).
In the light ofthe biotransformation of MMHgby HrS, the workby Pan-Hu et al. [53] suggests that C. cochlearium strain T-2P probably has a similar mechanism of detoxification' In this strain, MMHg- degradation activity is harbored in a conjugative plasmid together with HrS production. The strain T-2P was thought to split the C-Hg bond of MMHg; however, no loss of volatile Hg from the medium was found, because it was presumed that the volatile inorganic Hg reacted with HrS to produce HgS [167J. Conversely, the C. cochlearium-cured strain T-2C lost both the ability to degrade MMHg and to produce HrS, and it became sensitive to mercurials, acquiring the capacity to produce MMHg from HgCl, in the absence of vitamin B, or otherwise. Hence,
238
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fo 40O
02468
Time (days)
FIG. 5. Kinetic studies in anoxic sediment samples from Pialasse di Ravenna, polluted many ypars ago with MMHg from an acetaldehyde factory similar to lhut of Mi.rumata Bay, demonstrate that still today, sediment incubated for 9 days at 28"C and spiked with 10 pg of CHrHgCl converts this to DMHg (A), whereas MMHg (A) disappears. (Reproduced with permission from [58].)
there is a further affinity between C. cochlearium T-2C and D' de' sulfuricans strain LS. When the latter is grown in a fermentative mode, it also produces MMHg by the vitamin B12 pathway [118]. These sim- ilarities between c. cochlearium and D. desulfuricans stggest that
Clostrid.ium probably also transforms MMHg to DMHg by HrS evolu- tion. It will be interesting to determine the MMHg tolerance in other bacteria which produce HrS by secondary metabolism.
The dimethylation of inorganic Hg to DMHg is one of the best mechanisms of Hg tolerance, since DMHg is more volatile than Hg(O) formed by the flavoenzyme mercuric reductase. DMHg forms outside the cell in concomitance with HrS evolution. Unlike MMHg, it did not accumulate in D. desulfuricans cells [54] or in the cells of the broad- spectrum Hg-resistant Pseudomonas putida strain FB1 when the wild
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 239
strain FBl and the cured strain FB6 were exposed for 3 h to 5 pg'ml,-r of DMHg (Fig. 6A). Conversely, MMHg (1 pg'ml,-l) rapidly accumu- lated in the cured strain FB6. The wild strain FBl seemed unable to degrade DMHg in vivo, but degraded MMHg to Hg(0) (Fig. 6B). The nondegradability of DMHg in vivo may have been due to the fact that this Hg compound did not interact with the cells because of its high volatility. Experiments to demonstrate DMHg degradation by cell-free extracts are necessary.
During this decade, the interest in DMHg distribution in the environment has coincided with the development of new specific and sensitive routine determination methods. DMHg was first found at femtomolar levels (max = 555 -l- 154 fM, n-3) in subthermocline equa- torials waters (>200 m) of the Pacific Ocean, and it was inversely correlated with dissolved o, [168J. Then DMHg was also found ranging from 10 fM to 0.31 pM in deep waters of the North Atlantic ocean between Greenland and Norway and around Iceland. The highest DMHg concentrations were found in the deepest waters at most sta- tions [70]. The presence of this Hg species in deep ocean waters could be the result of an in situ production or advective transport from other zones; modeling calculation favor the former hypothesis [70]. The pro- duction of sulfide by sulfate-reducing bacteria is probably the only
cause known so far of DMHg formation in the ocean depths and also in more oxygenated waters but where sulfides still occur t1691. DMHg formation can therefore occur under suboxic/oxic conditions as sug- gested by laboratory studies [95,96,123]. In the Mediterranean Sea (Alboran Sea and Strait of Gibraltar), DMHg has also been detected in oxygenated subthermocline waters, where oxygen concentrations were above 150 rrM [170].
DMHg formation in sediments was first studied in a mangrove ecosystem at sepetiba Bay, Brazil [171]. These sediments were contami- nated by man-made effluents containing high concentrations of sn and Hg. High concentrations (211 ng' g-1 to 233 ng'g-1 dw) of DMHg were found in sediment cores at a depth of 10 cm, with a minimum value at 30- to 40-cm depth. These concentrations were several orders of magni- tude higher than in ocean waters. High concentrations of humic sub- stances presumably stabilize and sequester DMHg in mangrove sedi- ments tÚll;high concentrations of sulfate and an optimal temperature range for microbial growth would also have facilitated DMHg formation
240
BALDI
l (x)
^80
:o 60 è
5. 40 è!
0
B
o0<
0
60 90 r20 150 180 Time (min)
Ì:lu oo
A+ì-.--
'I'inrc (nrin)
FIG. 6. (A) Accumulation of organomercurials in a wild strain FBI (o, o) of broad-spectrum Hg-resistant Pseudomonas putida and the respective cured strain FB6 (o, l) after exposure for 3 h at 28"C to 1 pg.mL-1 CHrHgCl (o, o), and 5 pg'mL-1 of (CHs)zHg (r, .). Total Hg was determined by atomic absorp- tion spectrometry (AAS) by previous mineralization and atomization of Hg in the samples. (B) Volatilization as Hg(O) from cultures of a wild strain FB1 (o, o) of broad-spectrum Hg-resistant Pseudomonas putid.a and the respective cured strain FB6 (D, r) after exposure for 70 min at room temperature to 1pg.ml,-t CHrHgCl (o, o), and 5 pg'ml-t of (CHr)rHg (r, r). Hg(0) concentrations were calculated on the basis of the relative pressure at a given temperature with respect to elemental Hg(0) standard injected into the atomization cell of AAS.
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 241
in this environment. This process was of course attributed to the reac- tion with free sulfides but also to a mechanism of trans-alkylation due to high concentrations of methylated tin forms (mono-, di-, and tri- methyltin). Monomethylation of inorganic Hg by methyltin was known [172], but double methylation (dimethylation) of Hg, although likely, has yet to be proven.
The formation of DMHg from MMHg was recently reported in salt marsh sediments from Marina di Ravenna, Italy [58], and floodplain soil of the Elbe River, Germany, containing low concentrations of MMHg. In this experiment, Hg(0) was also determined in the degassed soil samples together with DMHg [173]. These two volatile Hg species were easily purged from soil with Nr. A total of 1.4 ng'g-1 (dw) of DMHg was released from the soil suspensions. Sulfide additions to the soil rapidly produced new DMHg even at sulfide additions as low as 1.5 mg'g-1 from which no DMHg evolution was detectable by chemical reaction [174]. In the future, DMHg will be determined in many other environments, and its importance in the cycle of Hg will probably be reconsidered.
The only ecosystems in which DMHg has been analyzed but not detected so far are the freshwater environments of acid lakes in North America and northern Europe. MMHg is the only organomercurial found in such ecosystems. This is probably explained by (1) acid condi- tions which favor MMHg stability [70,96], (2) low concentration of sulfate as electron acceptor for sulfate reducing bacteria, and (3) the mainly abiotic origin of MMHg in these lakes.
4. THE ROLE OF METACINNABAR. THE MOST STABLE MERCURY SPECIES
Mercury sulfide (HgS) occurs in nature as red-hexagonal (a-form, cin- nabar) or black cubic crystals (B-form, metacinnabar). The latter are mainly produced by microbial activity with evolution of HrS with Hg compounds. HgS is very stable and is conserved naturally in the Hg reserve pool of the Earth, where its turnover is in terms of millions of years. Natural events such as new volcanic and geothermal activities, metamorphogenesis, and the mining of cinnabar deposits, free Hg from sulfides so that it enters the Hg exchangeable pool.
242
BALDI
HgS is stable in most reducing environments. The extreme insolu- bility of HgS may be increased under very reducing conditions by con- version of Hg ions to Hg(O) or by the formation of the more stable HgSS- ion at high pH Í67 ,175,1761. The release of Hg from HgS may occur via a complex mechanism depending on a positive redox potential, low pH, and high concentrations of Cl- t671.
The reaction of inorganic Hg2+ with HrS is immediate and is the main cause of Hg loss from the water column under anoxic conditions [100,101,122,124-t261. HgS should precipitate immediately because of its high density (d = 7.67), but under laboratory conditions in the presence of sulfate-reducing bacteria, the reaction between Hgz+ and HrS takes place in a floating aggregate formed by a polysaccharide *àt"i" with embedded bacteria and newly formed microcrystals of HgS
[177]. Heavier crystals later leave the polysaccharide envelope under the effect of gravity. under the light microscope, the microbial aggre- gate has areas of different density. The high density to light is due to crystals and the lower density to bacteria.
The environmental meaning of this laboratory experiment is that the biological reaction between free inorganic Hg and free sulfides occurs in a naturally controlled system (exudated polysaccharide floc) produced by microbes in which sulfides from anaerobic respiration are retained in this microhabitat t1781. The reaction is more efficient than in aqueous solutions from which Hrs degasses at a higher rate. In the controlled system ofpolysaccharide aggregate, this reaction takes place with less gas diffusion and Hgs precipitation becomes possible even in suboxic anÙor oxic conditions.
The polysaccharide floc exuded by sulfate-reducing bacteria pro- vides an optimal crystallization site owing to hyperconcentrations of Hg2*, H*, and 52- which may be complexed by active sites of polysac- charides [179]. The formation of Hgs crystals by sulfide activity is produced both from inorganic Hg but also from MMHg as already reported t5al. MMHg is first precipitated as DMHgS, but in a second- ary step, when DMHg degasses, F-HgS is the most stable form of Hg as shown by x-ray spectra with relative intensity peaks at 26"35',30"55"
43"75', and 51"80' (x-ray diffractometry analysis) of dry cell pellets. The latter was obtained by centrifuging D. desulfuricans cultures spiked with 100 pg' ml,-l of Hg in two separated bottles (cap' 1L) spiked with HgCl, and CHrHgCl additions after 15 days of incubation at28C (Fig. 7).
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 243
51" 80' 43" 75' 30" 55' 26" 35',
FIG. 7. Spectra of p-HgS (metacinnabar) obtained by x-ray diffractometry with the highest peak of the relative intensity (1007o) at 26'35' theta and the minor ones. Spectrum A is B-HgS (from MMHg transformation by 1 liter of a D. desulfuricans strain LS culture spiked with 100 [rB.ml.-t of CHrHgCl and incubated at 28"C for 15 days) ofthe solid acid-insoluble residue after centrifu- gation of cell suspension (10.7 mg.L-t). Spectrum B is B-HgS (from a culture of D. desulfuricons strain LS spiked with HgCl, [100 p.g.mL 1] incubated at 28"C for 15 days) ofthe solid acid-insoluble residue after centrifugation ofcell suspen- sion (97.4 mg.L-t).
Although HgS is the most stable species of Hg, it is reported that traces of bioavailable Hgz+ can be removed from this sulfide into sedi- ments where they are accumulated by fish t1801. A Hg-resistant Thio- bacillus fenoxidans strain has been found to produce Hg(O) enzymati- cally from red cinnabar mixed with pyrite (1:10 w:w), but it could not grow chemolithotrophically on this sulfide as enerry source t781. A Hg- sensitive T. ferroxidazs strain could not grow on pyrite mixed with red cinnabar. This suggests that probably there was a concentration offree inorganic Hg on the crystals; otherwise the toxicity of cinnabar and Hg(O) degassing cannot be explained [781. When metal sulfides, an anaerobic product, are exposed to air, they may react with oxygen to produce Hg oxides and,/or sulfates on the surface. For example, we cannot exclude that some types of HgS are more reactive than others, like reactive pyrite (FeSo) [181], called also "fireballs", which can be set
244 BALDI
on fire because of rapid natural oxidation [182]. Halogens such as Cl- can facilitate the removal of Hg from HgS in marine systems [67].
5. NEW VIEW OF THE BIOGEOCHEMICAL CYCLE OF MERCURY
The role of microrganisms in the cycle of Hg is known as outlined above, but it is still underestimated. A large part of the Hg cycle is undoubtedly dominated by microbial activity in aquatic and terrestrial environ- ments. Here we have described the capacity of microorganisms to adapt to Hg pollution and to transform toxic Hg species to harmless ones. Microbes control Hg toxicity by maintaining homeostatic subtoxic levels not only in the cell cytoplasm but also in the surrounding environ- ment, which sooner or later leads to detoxification of the polluted site. Understanding of the Hg cycle in some historical polluted areas by industry is possible in the light of microbial reactions under anaerobic conditions.
For example, why did the Minamata disease develop only in Japan and not in other parts of the world, such as Italy, where similar indus- tries were located? The Minamata disease was diagnosed by Dr. Hoso- kawa (Director of Minamata Factory Hospital) in May 1956. The first case in the Minamata district appeared in 1953. In 1959, the disease was recognized to be of epidemic proportions among fishermen and their families [132]. The epidemic had its origin in 1932, when the Shin- Nihon Chisso Hyno Co. began to use HgCl, and HgSOn as a catalyst to produce acetaldehyde and vinyl chloride [133] and to dump its effiuent in the bay. The factory was not closed down until 1971. Further alkyl Hg contamination was found at Niigata, where a similar factory of the Showa Denko Co. was discharging MMHg into the Agano River. Many more cases of the Minamata disease were added to the previous list. In 1988, 1750 cases of Minamata sSmdrome were certified of 12 336 persons who applied for certification [134]. Officially, 750 people died of this environmental disease [183].
At that time, several factories in other parts of the world were producing acetaldehyde from acetylene using inorganic Hg as catalyst. At least two factories using the same process were known in Europe: the
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 245
ANIC factory at Marina di Ravenna, Italy, and the Huls factory at Marl, Germany. Not many people are aware that a similar ecological disaster could also have occurred in Europe. The Italian case was studied com- paratively by Professor Ui from Tokyo University, who studied also the epidemic at Niigata. Fortunately, no clinical cases were observed in Italian fishermen, who were eating 3-4 kg of fish per week, at Marina di Ravenna and "the rumor of suicide cats was not heard" [132] . This was because the Italian fishermen did not eat the local fish, except eels, because it smelled of oil from nearby raffineries that discharged wastes in the same area as the acetaldehyde factory. This area is characterized by a semiclosed marsh area consisting of branched channels of shallow water and anoxic sediments, which can reach a temperature as high as 34"C at 8 a.m.
If a comparison between the geography of Minamata Bay and the Pialasse (salt marsh) at Marina di Ravenna is made [132,184]' it emerges that in Japan, MMHg was discharged in the bay where fisher- men caught their food, whereas in Italy, the industry discharged into an anoxic marsh, with very limited exchange with the open sea [185]. Today at Marina di Ravenna, the concentrations of total Hg are still enormously high, although industrial activity stopped several years ago. In the first 10 cm of sediments of the most polluted site, there is an average level of 400 pg'g-t (dw). This concentration is by far higher than in Minamata Bay, where sediments with concentrations above 25
pg. g I (dw) have now been removed, transported elsewhere, and mixed
with soil [134,186].
At Pialasse di Ravenna, a concentration of 1000 pg'g-1 of total Hg
was reached at a depth of 80 cm in a sediment core [185]. However, no "Minamata" cases have been observed so far. The explanation for this discrepancy between Japan and Italy is that the toxic MMHg was rapidly converted to the harmless species DMHg and HgS in the anoxic marsh. This was demonstrated, as reported above (Sec. 3.3), by adding MMHg to this polluted sediment (see Fig. 5). This sediment sample contained 0.1 pg'g-r (0.016Vo) of MMHg versus 600 pg'g-t of total Hg
[58]. The environmental conditions of Pialasse favor sulfate-reducing bacteria which produce HrS, because the sediments and waters are anoxic and the temperature is warm enough to transform the toxic MMHg to DMHg and HgS rapidly. So the Minamata disease did not break out at Marina di Ravenna because MMHg was converted to
246
BALDI
(CH3)2Hg
,;;i i:,r,Hg 'i ' ,,,i *g Àlr*iun.;oiii
FIG. 8. The biochemical cycle of Hg. Under aerobic conditions, inorganic Hg can (1) be reduced by Hg-resistant bacteria to the volatile Hg(0) if the Hg concentration exceeds the threshold for Hg induction of t}:'e mer operon; (2) accumulate in the food chain; (3) be complexed to dissolved organic matter (DOC); (4) be adsorbed on particulate organic matter (POC) in the water column in the oxic/anoxic zone; (5) be transformed to methylmercury (MMHg) and its concentrations are the result of microbial methylation/demethylation pro- cesses. (6) MMHg can be accumulated faster than Hg2+ itr the food chain or sequestered by POC and DOC.-Under anoxic conditions in the presence of HrS from microbial dissimulative reduction of sulfate: (7)Hgz* precipitates as solid metacinnabar (p-HgS), (8) MMHg is converted to the instable dimethylmercury sulfide (DMHgS, CH,Hg-S-HgCH3); (9) this species transforms into volatile dimethylmercury (DMHg) and solid p-HgS; (10) DMHg degrades under mild acid conditions to CHn and (11) some free Hg2+ may be volatilized to Hg(O) by nonspecific reducing agents of microbial origin.
AIR
MICROBES AND THE BIOGEOCHEMICAL CYCLE OF Hg 247
harmless species. Of course, the fact of local oil-smelly fish also pro- tected the population from a catastrophe. Eels contained at that time relative low concentrations of MMHg (from 0.1to 6.37 pg.g-r, fw) in comparison with other predators caught at Minamata Bay t1321. If MMHg would have been discharged by the ANIC factory directly into the sea, as in the case of the Chisso factory at Minamata, severe con- tamination of the open costal waters sediments and biota could be expected. In the 1970s, fish caught offshore in the shallow waters ofthe Adriatic Sea showed low total Hg concentrations in muscle tissues ranging from 0.05 to 0.3 pg'g-1 (fw) for anchovies (Engraulis en- crasiculus) and sardines (Sardina pilchardus). These concentrations were lower than those of the same species caught from the T\'rrhenian Sea, which was polluted by Hg from the weathering of a cinnabar mine tailing and slightly higher than those of fish from the Atlantic Ocean [187]. The aquatic marsh system of Pialasse di Ravenna, therefore, functioned as a barrier for MMHg and as a natural bioremediation site. This interpretation is based on the chemical instability of MMHg under anoxic conditions and subsequent reduction of toxicity. The cycle of Hg can be outlined as in Fig. 8. MMHg reacts with HrS to form the instable species DMHgS, which degrades to the volatile DMHg and the solid HgS. Under mild acidic conditions, DMHg can be further degraded to CH., and some inorganic Hg can be reduced to Hg(0). The only residue is HgS. These transformations probably occurred at the "Pialasse di Ravenna" but not in Minamata Bay.
ABBREVIATIONS
AAS atomic absorption spectrometry BESA 2-bromo-ethane sulfonic acid Chlo chlorophyll o
DMHg dimethylmercury
DMHgS dimethylmercury sulfide DOC dissolved organic carbon dw dry weight
fM femtomolar (= 10-15 M) fi^/ fresh weight
248
BALDI
GC-ECD gas chromatography with electron capture detector GC-MS gas chromatography-mass spectrometry
MMHg monomethylmercury
pM picomolar (= 19-tz 14;
POC particulate organic matter
PT-GC-FTIR purge-and-trap gas chromatography in line with
Fourier transform infrared spectroscopy SAM S-adenosylmethionine
THF tetrahydrofolate
TLC thin layer chromatography
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Trace element definition and functions, and inputs into soils from the most important anthropogenic sources, related and not related to agricultural practices, of general and local or incidental concern, are discussed in the first part of this review. Trace element inputs include those from commercial fertilizers, liming materials and agrochemicals, sewage sludges and other wastes used as soil amendments, irrigation waters, and atmospheric depositions from urban, industrial, and other sources. In the second part of the review, the most important ascertained effects of soil trace elements on human health are presented. The possible relations found between some specific soil trace elements, such as Cd, Se, As and others, and cancer incidence and mortality, and diffusion of other important human diseases are reviewed. Brief conclusions and recommendations conclude this review.
A pilot-scale model was constructed to determine if a wetland treatment system (WTS) could effectively remove low-level mercury from an outfall located at the Department of Energy's Savannah River Site. Site-specific hydrosoil was planted with giant bulrush, Scirpus californicus, and surface amended with gypsum (CaSO4) prior to investigating the biogeochemical dynamics of sediment-based sulfur and mercury speciation. On average, the pilot WTS decreased total mercury concentrations in the outfall stream by 50%. Transformation of mercury to a more "bioavailable" species, methylmercury, was also observed in the wetland treatment system. Methylmercury formation in the wetland was ascertained with respect to sediment biogeochemistry and S. californicus influences. Differences in sulfate-reduction rates (SRRs) were observed between mesocosms that received additional decomposing Scirpus matter and mesocosms that were permitted growth of the submerged macrophyte, Potamogeton pusillus. Relative abundance measurements of sulfate-reducing bacteria (SRB) as characterized using oligonucleotide probes were also noticeably different between the two mesocosms. A positive correlation between increased sulfide, dissolved total mercury, and dissolved methylmercury concentrations was also observed in porewater. The data suggest that soluble mercury-sulfide complexes were formed and contributed, in part, to a slight increase in mercury solubility. Observed increases in methylmercury concentration also suggest that soluble mercury-sulfide complexes represent a significant source of mercury that is "available" for methylation. Finally, a volunteer macrophyte, Potamogeton pusillus, is implicated as having contributed additional suspended particulate matter in surface water that subsequently reduced the pool of dissolved mercury while also providing an environment suitable for demethylation.
Methylmercury (MeHg) is a highly toxic form of mercury that bioaccumulates in aquatic food chains. However, methods to reliably identify sites of MeHg formation or to quantify MeHg production require the use of isotopic tracers, which limits their use. In this paper, a method is presented to quantify the methylation of mercury by a methyltransferase pathway. This methyltransferase pathway is one of the biochemical pathways responsible for biological mercury methylation. Protein is extracted from environmental samples, and mercury methyltransferase (HgMT) activity of soil extracts is calculated by assessing increases in methyltransferase activity induced by Hg addition. In enzyme extracts from pure cultures or soil samples, HgMT activity correlated with net MeHg production and Hg consumption, suggesting that HgMT activity can be used to estimate MeHg production in field samples. Over the course of a three-month period in a freshwater wetland, HgMT activity correlated with net MeHg concentrations (r2 = 0.55; p < 0.057). Furthermore, HgMT activity predicted (r2 = 0.80; p < 0.01) gross MeHg formation in freshwater wetlands as well as in laboratory microcosms calculated using previously published rate constants. Our results show that a methyltransferase assay, in combination with demethylation estimates, accurately predicts MeHg formation under field and laboratory conditions. This assay does not require the use of mercury added to field samples to estimate activity but rather estimates the biological activity present in the soil by quantifying the amount of enzyme present in the soil. Such an assay is well suited for use in field surveillance programs assessing MeHg formation in a variety of environments.
Speciation of Hg and conversion to methyl-Hg were evaluated in mine wastes, sediments, and water collected from the Almadén District, Spain, the world's largest Hg producing region. Our data for methyl-Hg, a neurotoxin hazardous to humans, are the first reported for sediment and water from the Almadén area. Concentrations of Hg and methyl-Hg in mine waste, sediment, and water from Almadén are among the highestfound at Hg mines worldwide. Mine wastes from Almadén contain highly elevated Hg concentrations, ranging from 160 to 34,000 microg/g, and methyl-Hg varies from <0.20 to 3100 ng/g. Isotopic tracer methods indicate that mine wastes at one site (Almadenejos) exhibit unusually high rates of Hg-methylation, which correspond with mine wastes containing the highest methyl-Hg concentrations. Streamwater collected near the Almadén mine is also contaminated, containing Hg as high as 13,000 ng/L and methyl-Hg as high as 30 ng/L; corresponding stream sediments contain Hg concentrations as high as 2300 microg/g and methyl-Hg concentrations as high as 82 ng/g. Several streamwaters contain Hg concentrations in excess of the 1000 ng/L World Health Organization (WHO) drinking water standard. Methyl-Hg formation and degradation was rapid in mines wastes and stream sediments demonstrating the dynamic nature of Hg cycling. These data indicate substantial downstream transport of Hg from the Almadén mine and significant conversion to methyl-Hg in the surface environment.
Metallurgic calcines with very high mercury and methylmercury content from the Almadén mining district were analyzed by synchrotron-based microprobe techniques. Information about mercury speciation was obtained by micro-EXAFS (microscopic extended X-ray absorption fine structure) spectroscopy, whereas elemental associations were evaluated by micro-XRF (microscopic X-ray fluorescence analysis) mapping. Complementary characterization methodologies, including X-ray diffraction (XRD), inductively coupled plasma-optical spectroscopy (ICP-OES), as well as a sequential extraction scheme (SES), were used to predict the potential availability of mercury. Analysis of total metal content revealed extremely high concentrations of mercury and iron (between 7 and 35 and 65-70 g kg(-1), respectively) and high zinc concentrations (2.2-2.5 g kg(-1)), whereas other metals such as copper, nickel, and lead were found at low concentration levels (30-300 mg kg(-1)). Micro-EXAFS results indicate that cinnabar (HgS(red)) is one of the main species within the studied mercury-rich particles (5-89% of total mercury content), together with more soluble mercury compounds such as Hg3(SO4)02 (schuetteite) and HgO (5-55% of total mercury content). Additionally, element-specific micro-XRF maps of selected mercury-rich particles in the studied samples revealed an evident correlation among Hg-Pb-Ni (and S), indicating a possible geochemical linkage of these elements. Correlations were also found among Fe-Mn and Hg, which have been attributed to sorption of mercury onto oxyhydroxides of Fe and Mn. This finding was supported by results from a sequential extraction scheme, where a significant
The Río Pilcomayo heads on the Cerro Rico de Potosí precious metal-polymetallic tin deposits of Southern Bolivia. Mining of the Potosí deposits began in 1545 and has led to the severe contamination of the Pilcomayo's water and sediments for at least 200 km downstream of the mines. This investigation addresses the potential human health affects of metal and As contamination on four communities located along the upper Río Pilcomayo by examining the potential significance of human exposure pathways associated with soils, crops and water (including river, irrigation and drinking water supplies). The most significantly contaminated agricultural soils occur upstream at Mondragón where Cd, Pb and Zn concentrations exceed recommended guideline values for agricultural use. Further downstream the degree of contamination decreases, and metal concentrations are below Dutch, German and Canadian guideline values. Metal and As concentrations in agricultural products from the four communities were generally below existing guidelines for heavy metal content in commercially-sold vegetables. Thus, the consumption of contaminated produce does not appear to represent a significant exposure pathway. A possible exception is Pb in carrots, lettuce and beetroots from Sotomayor and Tuero Chico; 37% and 55% of the samples, respectively, exceeded recommended guidelines. Most communities obtain drinking water from sources other than the Río Pilcomayo. In general, dissolved concentrations of metals and As in drinking water from the four studied communities are below the WHO guideline values with the exception of Sb, which was high at Tasapampa. The inadvertent ingestion of contaminated water from irrigation canals and the Río Pilcomayo represents a potential exposure pathway, but its significance is thought to be minimal. Given the degree of soil contamination in the area, perhaps the most significant exposure pathway is the ingestion of contaminated soil particles, particularly particles attached to, and consumed with vegetables. The risks associated with this pathway can be reduced by thoroughly washing or peeling the vegetables prior to consumption. Other exposure pathways that are currently under investigation include the consumption of contaminated meat from livestock and poultry, which drink polluted waters and the ingestion of contaminated wind-blown dust.
Diagnóstico Previo—Aporte al Plan de Desarrollo Municipal de Potosí Honorable Alcaldía Municipal Potosí
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Potosí 2007–2011. Honorable Alcaldía Municipal Potosí, Programa BOL/AIDCO/
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Graphical Arrays of Chemical-Specific Health Effect Reference Values for Inhalation Exposures
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U.S. EPA, 2009. Graphical Arrays of Chemical-Specific Health Effect Reference Values for
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2010. XANES speciation on mercury on three mining districts—Almadén, Asturias
(Spain), Idria (Slovenia). Journal of Synchrotron Radiation 17, 179–186.
Archival education in Spain
- Cortés Alonso
Cortés Alonso, V., 1988. Archival education in Spain. American Archivist 51, 330-335.
Diagnóstico Previo-Aporte al Plan de Desarrollo Municipal de Potosí
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Potosí 2007-2011. Honorable Alcaldía Municipal Potosí, Programa BOL/AIDCO/
2002/0467 APEMIN II. http://www.apemin.eu/PDM/PDMPotosi.pdf.
A Legacy of Nearly 500 Years of Mining in Potosí
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Potosí, Bolivia: Stream Water Quality. In: Barnhisel, R.I. (Ed.), Published by ASMR,
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