Effects of acidification on mercury methylation, demethylation, and volatilization in sediments from an acid-susceptible lake

The effect of experimental acidification on mercury methylation, demethylation, and volatilization was examined in surficial sediment samples from a weakly buffered northern Wisconsin lake. All mercury transformations were measured with radioisotopic tracers. Acidification of sediment pH with H2SO4, HCl, or HNO3 significantly decreased 203Hg(II) methylation. Acidification of pH 6.1 (ambient) sediments to pH 4.5 with either H2SO4 or HCl inhibited methylation by over 65%. The decreased methylation was due to the increased hydrogen ion concentration because methylation was not affected by concentrations of Na2SO4 or NaCl equimolar to the amount of acid added. Inhibition of methylation was observed even after prolonged acidification of sediments to pH 5.0 for up to 74 days. Acidification of sediments to pH 5.5, 4.5, and 3.5 with HNO3 resulted in a near complete inhibition of methylation at each pH. Similarly, the addition of equimolar amounts of NaNO3 resulted in a near complete inhibition of methylation, indicating that the inhibition was due to the nitrate ion rather than to the acidity. Demethylation of methyl mercury was not affected by pHs between 8.0 and 4.4, but sharply decreased below pH 4.4. Volatilization of 203Hg(II) from surface sediments was less than 2% of methylation activity and was not significantly different from that in killed sediments. This study indicated that acidification of sediments inhibits mercury methylation and that the observed increase in the mercury burdens in fish from low pH lakes is not due to increased production of methylmercury in sediments.     

Effects of acidification on mercury methylation, demethylation, and volatilization in sediments from an acid-susceptible lake.

The effect of experimental acidification on mercury methylation, demethylation, and volatilization was examined in surficial sediment samples from a weakly buffered northern Wisconsin lake. All mercury transformations were measured with radioisotopic tracers. Acidification of sediment pH with H2SO4, HCl, or HNO3 significantly decreased 203Hg(II) methylation. Acidification of pH 6.1 (ambient) sediments to pH 4.5 with either H2SO4 or HCl inhibited methylation by over 65%. The decreased methylation was due to the increased hydrogen ion concentration because methylation was not affected by concentrations of Na2SO4 or NaCl equimolar to the amount of acid added. Inhibition of methylation was observed even after prolonged acidification of sediments to pH 5.0 for up to 74 days. Acidification of sediments to pH 5.5, 4.5, and 3.5 with HNO3 resulted in a near complete inhibition of methylation at each pH. Similarly, the addition of equimolar amounts of NaNO3 resulted in a near complete inhibition of methylation, indicating that the inhibition was due to the nitrate ion rather than to the acidity. Demethylation of methyl mercury was not affected by pHs between 8.0 and 4.4, but sharply decreased below pH 4.4. Volatilization of 203Hg(II) from surface sediments was less than 2% of methylation activity and was not significantly different from that in killed sediments. This study indicated that acidification of sediments inhibits mercury methylation and that the observed increase in the mercury burdens in fish from low pH lakes is not due to increased production of methylmercury in sediments.     

Fate and distribution of toxic heavy metals in some marine organisms from the eastern Adriatic coast

The paper reports on levels of heavy metals in some organisms from the eastern Adriatic coast and polluted areas near a big urban centre (Split area). Surprisingly high Hg and MeHg concentrations were found in sea bream (Pagellus erythrinus) and striped mullet (Mullus barbatus) from the south-eastern part of the Adriatic. The reason for the high levels are probably geochemical anomalies. Studies of total and organic mercury content in bivalves have also shown that the gills and hepatopancreas had high levels of accumulated mercury. Our results have shown that fish contain significantly higher concentrations of MeHg than mussel. High concentrations of these elements in fish may be a consequence of the methylation process in the visceral organs. Bacterial activity in the intestinal contents is most likely to be responsible for this methylation (Rudd et al., 1980).     

Transformation of mercuric chloride and methylmercury by the rumen microflora

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Transformation of mercuric chloride and methylmercury by the rumen microflora.

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

The accumulation of inorganic mercury from sea water by the plaice, Pleuronectes platessa L

The accumulation of inorganic mercury from sea water by plaice eggs, larvae, and adult fish has been studied using ²⁰³HgCl₂ as a tracer. The isotope was rapidly accumulated and the levels of accumulation have been related to stable element concentrations. High concentration factors were attained by many organs, but the distribution of the ²⁰³Hg was markedly different from that of the stable element. Whereas the largest fraction of the body burden of mercury is contained by muscle, only a slow rate of accumulation into this tissue was observed. In addition, the mercury in fish muscle is of the methyl form: no evidence for the methylation of the tracer was obtained.     

Constitutive synthesis of a transport function encoded by the Thiobacillus ferrooxidans merC gene cloned in Escherichia coli.

Mercuric reductase activity determined by the Thiobacillus ferrooxidans merA gene (cloned and expressed constitutively in Escherichia coli) was measured by volatilization of 203Hg2+. (The absence of a merR regulatory gene in the cloned Thiobacillus mer determinant provides a basis for the constitutive synthesis of this system.) In the absence of the Thiobacillus merC transport gene, the mercury volatilization activity was cryptic and was not seen with whole cells but only with sonication-disrupted cells. The Thiobacillus merC transport function was compared with transport via the merT-merP system of plasmid pDU1358. Both systems, cloned and expressed in E. coli, governed enhanced uptake of 203Hg2+ in a temperature- and concentration-dependent fashion. Uptake via MerT-MerP was greater and conferred greater hypersensitivity to Hg2+ than did uptake with MerC. Mercury uptake was inhibited by N-ethylmaleimide but not by EDTA. Ag+ salts inhibited mercury uptake by the MerT-MerP system but did not inhibit uptake via MerC. Radioactive mercury accumulated by the MerT-MerP and by the MerC systems was exchangeable with nonradioactive Hg2+.     

Methods for Measuring Specific Rates of Mercury Methylation and Degradation and Their Use in Determining Factors Controlling Net Rates of Mercury Methylation

A method was developed to estimate specific rates of demethylation of methyl mercury in aquatic samples by measuring the volatile ¹⁴C end products of ¹⁴CH₃HgI demethylation. This method was used in conjunction with a ²⁰³Hg²⁺ radiochemical method which determines specific rates of mercury methylation. Together, these methods enabled us to examine some factors controlling the net rate of mercury methylation. The methodologies were field tested, using lake sediment samples from a recently flooded reservoir in the Southern Indian Lake system which had developed a mercury contamination problem in fish. Ratios of the specific rates of methylation/demethylation were calculated. The highest ratios of methylation/demethylation were calculated. The highest ratios of methylation/demethylation occurred in the flooded shorelines of Southern Indian Lake. These results provide an explanation for the observed increases in the methyl mercury concentrations in fish after flooding.     

Methylation and demethylation of mercury under controlled redox, pH and salinity conditions.

In estuarine sediments, the microbially mediated processes of methylation, demethylation, and volatilization determine the state and overall toxicity of mercury pollutants. The effects of redox potential (Eh) and salinity on the above microbial processes were investigated in reactors constructed to allow for continuous monitoring and adjustment of the pH (6.8) and Eh of freshly collected estuarine sediments. For measurements of methylation and demethylation activity, sediment slurries adjusted to appropriate salinity were spiked with HgCl2 or CH3HgCl, respectively, and were incubated in the reactors. Methylmercury was measured by gas chromatography. Volatilized elemental mercury (Hg0) was trapped and determined by cold vapor atomic absorption spectrometry. Volatilization of Hg0 and CH3HgCH3 were found to be minimal. Methylation of Hg2+ was favored at Eh-220 mV as compared to +110 mV. At -220 mV, high salinity (2.5%) inhibited methylation, and low salinity (0.4%) favored it. At +110 mV, the salinity effect was less pronounced. Demethylation of CH3HgCl was favored at +110 mV regardless of the salinity level. Low redox potential under low salinity conditions inhibited demethylation, but high salinity reversed this inhibition. These findings are helpful for interpreting and predicting the behavior of mercury pollutants in estuarine sediments.     

Mercury translocation in and evaporation from soil. I. soil lysimeter experiments with 203Hg‐radiolabeled compounds

Due to a considerable increase of anthropogenic mercury emissions, the mercury load of many soils has risen significantly, for instance in northern Europe. Understanding the fate of mercury in soils is a prerequisite for assessing the effects of ecotoxicological concern. This paper presents a method for obtaining qualitative and quantitative information about mercury translocation in and evaporation from soil. Soil lysimeters were treated with ²⁰³Hg‐labeled HgCl₂ and CH₃HgCl and irrigated with artificial rain. It was demonstrated that the leaching of Hg can be detected by measuring the relative y‐activity throughout the soil profile by means of Na(TI)I detectors. Furthermore, the set‐up was designed to allow detection of Hg volatilization from soil by using traps of iodized charcoal, followed by a potassium peroxodisulfate solution and measuring the γ‐activity. The amount of radioactive Hg in soil leachate was measured by a Na(Tl)I well‐type detector after upconcentration. The determination of monomethyl ²⁰³Hg was been performed by extraction procedures that isolate the methyl mercury compounds. The amount of ²⁰³Hg retained in the soil profile and the real depth of leaching were determined by stratifying the soil profile at the end of the experiment and measuring the y‐activity. With control of all pathways of Hg, the experimental design allows performance of a mass balance analysis.     

Factors influencing changes in mercury concentrations in lake water and yellow perch (<Emphasis Type="Italic">Perca flavescens</Emphasis>) in Adirondack lakes

Over the past 20+ years, fish with elevated concentrations of mercury (Hg) have been observed in remote lake districts, including the Adirondack region of New York. Across eastern North America studies have also reported a negative correlation between fish Hg concentration and lake pH. Recent controls in emissions of sulfur dioxide (SO₂) have resulted in some improvement in the acid–base status of acid-impacted surface waters including Adirondack lakes. In addition, there has been an apparent decrease in atmospheric Hg deposition. A synoptic survey of 25 lakes in the Adirondacks was conducted in 1992–1993 to analyze spatial patterns of Hg in the water column and yellow perch (Perca flavescens). The same cluster of 25 lakes was resurveyed in 2005–2006 to evaluate if changes in lake concentrations of Hg species or fish Hg have occurred. We observed a varied response of changes in water chemistry and fish Hg concentrations. In twelve of the resurveyed lakes the yellow perch had lower Hg concentrations, six lakes had yellow perch with higher Hg concentrations, and in seven lakes yellow perch Hg concentrations did not change significantly (α = 0.05). Four variables appear to influence the change in yellow perch Hg concentrations in the Adirondacks: watershed area, elevation, change in pH, and change in fish body condition. We hypothesize that as the acidity in lakes is attenuated, the lakes may become more productive and/or water quality conditions less stressful to fish leading to increasing fish body condition. As fish body condition improves, fish exhibit “growth dilution” of tissue contaminants leading to lower fish Hg concentrations.     

Interactions between tellurium and mercury in murine lung and other organs after metallic mercury inhalation: a comparison with selenium

Selenium is known to form complexes with heavy metals in the blood and thus increase the retention time of the metals in several organs, especially in the reticulo-endothelial system. Selenium may similarly cause retention of mercury in the lung after metallic mercury (Hg0) inhalation. This study, comparing the effects of tellurium with those of selenium (both in group '6b' of the periodical system), showed that Te(IV) was as effective as Se(IV) and Se(VI) (all given in a dose of 10 mumol/kg body wt.) in retaining inhaled 203Hg0 (1.5 mumol/kg body wt.) in the lung (presumably 203Hg2+ after oxidation). Te(VI) had to be given in a dose of 100 mumol/kg body wt to produce the same effect. As in the lung, also in other organs tellurium caused a dose-dependent increase in mercury retention. At a dose level of 10 mumol Te(IV) per kg body wt. the mercury retention ratios (treated/control) were 140 for the lung and 8.6 for the whole body. The corresponding figures for Te(VI) (10, 30 and 100 mumol/kg body wt.) were 10, 73 and 120 and 3.7, 3.9 and 4.3, respectively. Retention of i.v. injected 203HgCl2 was increased by pre-administration of tellurium, again in a dose-dependent manner and Te(IV) being 3-10 times more effective than Te(VI). The kidney and the spleen were the dominant organs, as is the case after Se pretreatment. Anions of other elements, arsenite, arsenate, chromate, molybdate and wolframate (30 mumol/kg body wt.), did not affect the retention of 203Hg in lung or any other organ, or in the whole body after inhalation of 203Hg0. It is suggested that Te(IV) may easily be reduced to Te2- (in analogy with selenium) which may complex with Hg2+. The liability for Te(VI) to be reduced to Te2- appears to be approx. 10 times lower.     

Determination of mercury and organomercurial resistance in obligate anaerobic bacteria

A methodology for determining the minimum inhibitory concentration of inorganic and organomercurial compounds for obligate anaerobic bacteria is described. A wide variation in the susceptibility of anaerobic clinical and sewage isolates was observed. Isolates of Bacteroides ruminicola and Clostridium perfringens resistant to mercury were examined for their plasmid content and ability to demonstrate inducible resistance. None of the resistant anaerobes contained any plasmids, while resistant facultative isolates from the same source contained several plasmids. In 24 h, resistant strains of clostridia and Bacteroides volatilized 20 and 43% of the 203Hg2+ added to cultures, while Escherichia coli R100 and a sewage isolate of Enterobacter cloacae volatilized 63 and 27%, respectively, of the added 203Hg2+. Attempts to induce mercury resistance in the aerobic isolates were successful, but no induction was seen in the anaerobes. Thus, mercury resistance in these anaerobic isolates was neither inducible nor plasmid mediated.     

Laboratory Study of Chemical Speciation of Mercury in Lake Sediment and Water under Aerobic and Anaerobic Conditions

Chemical speciation and partitioning of radiolabeled HgCl₂ were studied in model aquatic systems consisting of undisturbed eutrophic lake sediment and water in plastic cylinders. The cylinders were either gradually made anaerobic by a gentle flow of N₂-CO₂ or kept aerobic by air flow. The proportion of methylated ²⁰³Hg was significantly higher, in both water and sediment, in the anaerobic systems than in the aerobic systems. The composition and total concentration of fatty acids originating from bacterial phospholipids, as well as the concentration of vitamin B₁₂, including related cobalamins, were similar in sediments from the anaerobic and aerobic systems. Bacterial cell numbers were, on average, 3.6 times higher in the anaerobic water columns than in the aerobic ones. Volatilization of ²⁰³Hg occurred in all systems except in an autoclaved control and was of similar magnitudes in the anaerobic and aerobic systems. Incorporation of ²⁰³Hg into the sediment was significantly faster in the aerobic systems than in the anaerobic systems. These results suggest that episodes of anoxia in bottom waters and sediment cause an increase in net mercury methylation and, hence, an increase in bioavailable mercury.     

Formation of Methyl Mercury by Bacteria

Twenty-three Hg2+-resistant cultures were isolated from sediment of the Savannah River in Georgia; of these, 14 were gram-negative short rods belonging to the genera Escherichia and Enterobacter, six were gram-positive cocci (three Staphylococcus sp. and three Streptococcus sp.) and three were Bacillus sp. All the Escherichia, Enterobacter, and the Bacillus strain were more resistant to Hg2+ than the strains of staphylococci and streptococci. Adaptation using serial dilutions and concentration gradient agar plate techniques showed that it was possible to select a Hg2+-resistant strain from a parent culture identified as Enterobacter aerogenes. This culture resisted 1,200 mug of Hg2+ per ml of medium and produced methyl mercury from HgCl2, but was unable to convert Hg2+ to volatile elemental mercury (Hg0). Under constant aeration (i.e., submerged culture), slightly more methyl mercury was formed than in the absence of aeration. Production of methyl mercury was cyclic in nature and slightly decreased if DL-homocysteine was present in media, but increased with methylcobalamine. It is concluded that the bacterial production of methyl mercury may be a means of resistance and detoxification against mercurials in which inorganic Hg2+ is converted to organic form and secreted into the environment.     

Role of Na+ in transport of Hg2+ and induction of the Tn21 mer operon.

The effects of sodium ions on the uptake of Hg2+ and induction of the Tn21 mer operon were studied by using Escherichia coli HMS174 harboring the reporter plasmids pRB28 and pOS14. Plasmid pRB28 carries merRT', and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterless luciferase gene cassette in pUCD615. The bioluminescent response to 1 microM Hg2+ was significantly inhibited in E. coli HMS174(pRB28) in minimal medium supplemented with sodium ions at 10 to 140 mM. After initial acceleration, light emission declined at 50 nM Hg2+ in the presence of Na+. The mer-lux assay with resting cells carrying pRB28 and 203Hg2+ uptake experiments showed increased induction and enhanced mercury uptake, respectively, in media supplemented with sodium ions. The presence of Na+ facilitated maintenance of bioluminescence in resting HMS174(pRB28) cells induced with 50 nM Hg2+. External K+ stimulated bioluminescent response in HMS174(pRB28) and HMS174(pOS14) grown in sodium phosphate minimal medium devoid of potassium ions. Sodium ions appear to facilitate mercury transport. We propose that sodium-coupled transport of mercuric ions can be one of the mechanisms for mercury uptake by E. coli and that the Na+ gradient may energize the transport of Hg2+.     

Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum.

A Chesapeake Bay water isolate of Mycobacterium scrofulaceum containing a 115-megadalton plasmid (pVT1) grew in the presence of 100 microM HgCl2 and converted soluble 203Hg2+ to volatile mercury at a rate of 50 pmol/10(8) cells per min. Cell extracts contained a soluble mercuric reductase whose activity was not dependent on exogenously supplied thiol compounds. The enzyme displayed nearly identical activity when either NADH or NADPH served as the electron donor. A spontaneously cured derivative lacking pVT1 failed to grow in the presence of 100 microM HgCl2 and possessed no detectable mercuric reductase activity.     

Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.     

Mercury-selenium interactions in relation to histochemical staining of mercury in the rat liver

Summary Selenium has been suggested to enhance the histochemical staining of mercury when sections of tissue are subjected to the silver-enhancement method. In the present study, histochemical staining patterns of mercury in tissue sections of rat livers were compared with the actual content of organic and inorganic Hg in the livers, in both the presence and the absence of Se. Rats were injected intravenously with 5g of Hgg^–1 body weight as methyl [²⁰³Hg] mercury chloride (MeHg) or as [²⁰³Hg]mercuric chloride (Hg²⁺). After 2h, half the rats received an additional intraperitoneal injection of 2g of Se g^–1 body weight as sodium [⁷⁵Se]selenite. All the rats were killed 1h later. Homogenized liver samples were prepared for mercury analysis by two different methods: alkaline digestion and ultrasonic disintegration. Quantitative chemical analysis based on benzene extrction of the radioactively labelled Hg compounds showed that the chemical form of mercury, either organic or inorganic, was preserved from its administration to its deposition in the liver. Light and electron microscopy demonstrated that no silver enhancement of Hg occurred when MeHg alone was present in the sections of tissue, whereas MeHg accompanied by Se induced a moderate deposition of silver grains. In contrast, sections containing Hg²⁺ alone yielded some staining, and the addition of Se increased the staining dramatically. The results of the present study show that acute selenite pretreatment is a prerequisite for the histochemical demonstration of methyl mercury, and greatly increases the staining of inorganic mercury when applying the silver-enhancement method.