Effects of acidification on the mobility of metals and metalloids: an overview.

The exchange rates for metals and metalloids between sediments, soils, water and aquatic biota are discussed in terms of normal and acidified ecosystems. Where it is possible, the pathways for a number of toxic elements are presented with special emphasis on the impact of acidification on changing chemical speciation, and on the potential toxicity of such acid-generated chemical species. Concerns for the impact of acidification on the stability of ecosystems, the safety of drinking water, bioaccumulation in fish and in plants are addressed.


Introduction Evolution Aspects of Metal Ion Transport
Before examining some of the processes involved in element uptake by living cells, let us consider some of the selection principles involved in the chemistry of life. To accomplish this end several fundamental questions must be asked: * Which elements are essential for the growth and cell division of microorganisms, plants, and animals? * Why were these elements selected in the evolution of microorganisms ot almost 4 x 109 years ago? * What is the role of the geosphere in determining the uptake of essential elements? * What is the role of the biosphere in the selection of these elements? Obviously the uptake of elements and their utilization by living cells depends on chemical and physical properties of each element. Of the 92 elements in the periodic table, 30 have been found to be required for microbial life, although not all of these 30 elements are necessary for the growth and cell division of every microbial species. Therefore, in addition to the bulk elements, carbon, nitrogen, hydrogen and oxygen, 26 other elements are required in intermediate to trace amounts. An overabundance of any of these elements can cause an intracellular toxic buildup which often results in death. It is therefore of great significance to understand the impact of large-scale environmental perturbations on the availability of both essential and nonessential elements. The reason for the selection of these 30 elements in the evolution of microorganisms appears to have been determined by two parameters: the abundance of elements in the earth's crust and the solubility of these *Gray Freshwater Biological Institute, University of Minnesota, Navarre, MN 55392. elements in water under strictly anaerobic conditions. Table 1 presents a list of elements in order of their crustal abundances. Of the top 26 elements, 22 are found to be essential for life in higher organisms. Nonessential elements are generally of low abundance in the earth's crust and therefore should not be effective in competing with essential elements in cells through their specific transport systems.
The effect of solubility properties of elements under strictly anaerobic conditions is best illustrated by the biological utilization of iron and the rejection of aluminum. Under anaerobic conditions iron will be present in complexes of lower oxidation sate, e.g., Fe(II) salts which would be water-soluble and available for transport into primitive anaerobic bacteria. However, the solubilization of abundant elements such as Al and Pb requires low pH, and so such elements are available to living cells entering the aqueous environment acidified below pH 6.0 (1).

Essentiality Versus Toxicity of Metal Ions
Williams (2,3) has documented those parameters which lead to the uptake of either too little or too much of individual essential elements for the sustenance of cell growth and cell division. For example, the failure of a cell to transport and utilize sufficient essential metal could arise as follows: (1) low availability; (2) excessive competition from other metal ions with similar chemical properties, e.g., Co(II) and Ni(II, Ca(II) and Cd(II), P043and AsO43, Li' and Na+, etc.; (3) inadequate synthesis of carrier molecules by the cell; (4) excessive excretion of metal ions by the cell; (5) failure of the energy-driven uptake systems.
Excessive element uptake can occur through the reversal of the above five factors. It is important to recognize that living cells are not at equilibrium with their external environment and therefore a kinetic approach Table 1. Crustal abundance of elements in the earth's surface, and a list of some of the elements required to support life.a"b 0, Si, Al,* Fe, Na, Ca, Mg, K, H, Mn, P, S, C, V, Cl, Cr, Zn, Ni, Cu, Co, N, Pb,* Sn,* Br, Be,* As, F, Mo, W, Tl,* I, Sb,* Cd,* Se All the rest are less than 0.1 ,ug/g. a Elements followed by an asterisk have no known biological function.
'Range of concentration 0 = 46.6% to Se which is 0.1 ,ug/g. to metal ion transport, binding, toxicity, and resistance to toxicity is much more meaningful than a thermodynamic approach. This biochemical basis for toxicity is complicated by the great variety of reactions at the molecular and cellular levels even in closely related organisms and tissues. Several strategies for resistance to intoxication have been identified (4). Williams (3) has pointed out that metal ion interactions in biology can be divided into three classes: ions in fast exchange with biological ligands; ions in intermediary exchange with biological ligands; ions in slow exchange with biological ligands.
Examples of those elements in fast exchange include the alkali metals Na+ and K+, the alkali earth metals Ca2" and Mg2, and of course, H+. Those which can sometimes be in intermediary exchange are Fe2+ and Mn2, . Examples ofthose in slow exchange are generally in the active sites of metalloenzymes, e.g., Fe" , Zn2+, Ni2 , Cu2+. Metal-to-metal interactions and covalency predominate in the slow-exchange metals and this provides the basis for stability. However, competition for those metal ions in fast exchange is often severe.
As outlined in the previous section, living cells have membranes which cause the initial barrier for metal ion uptake. In prokaryotes the external cell membrane represents the only barrier, but in eukaryotes there are many membranous organelles which can partition metal ions by a variety of mechanisms. A comparative study of metal ion resistance between blue-green algae (prokaryotes) and green algae (eukaryotes) demonstrates how important membranes are to metal ion uptake and toxicity (4). The green algae are much more resistant to high concentrations of toxic metal ions such as Cu2+ and Ni2+ than are the blue-green algae (5). The external cell membrane of prokaryotes carefully selects those ions which are in fast exchange, e.g., the rejection of Na+ and Ca2+ and selection of K+ and Mg + (6). In eukaryotes spatial partitioning of metals occurs, even for those in slow exchange, because metal-binding macromolecules can be partitioned in different organelles and in tissues of different cell lines, e.g., metallothionen binding Zn2+, Cd2, Cu2+, and Hg2e in the kidney cortex. Once the cell-buffering capacity for essential metal ions is exceeded toxicity becomes evident. Toxic effects become evident at much lower concentrations for nonessential metals. In the natural environment nonessential metals seldom reach concentrations in excess of 1 ug/g (i.e., 1 ppm) ( Table 1). Examples of circumstances in which natural higher metal concentrations occur are active volcanic regions, such as deep-sea vents, hot springs, and volcanic lakes. However, the combustion of fossil fuels by advanced industrial society has greatly contributed to acidification of natural waters which has a profound effect on the availability of both essential and non-essential metals and metalloids. In that which follows, I shall attempt to summarize the potential human health effects of acid rain.

Metals and Metalloids of Concern
Mercury Recent analyses on oil shale show that primitive organisms were actively synthesizing organometals and organometalloids. Therefore biomethylation must have given certain microorganisms selective advantages for the elimination of heavy metals such as mercury and tin, and for metalloids such as arsenic and selenium. The synthesis of less polar organometallic compounds from polar inorganic ions has certain advantages for cellular elimination by diffusion-controlled processes (7,8). The microbial synthesis of organometallic compounds from inorganic precursors is well understood in both the terrestrial environment and in the sea. Mechanisms for B12-dependent synthesis of metal-alkyls have been discovered for the metals Hg, Pb, Tl, Pd, Pt, Au, Sn, Cr and for the metalloids As and Se (8)(9)(10)(11)(12)(13)(14). Pathways for the synthesis of organoarsenic compounds have also been show to occur by a mechanism involving Sadenosyl-methionine as the methylating coenzyme (14).
The ecological significance of B12-dependent biomethylation is best illustrated by B12-dependent and independent strains of Clostridium cochlearium. The B12dependent strain is capable of methylating Hg(II) salts to CH3Hg(II), whereas the B12-dependent is incapable of catalyzing this reaction. Both strains transport Hg(II) into cells at the same rate, but the independent strain is inhibited by at least 40-fold lower concentration of Hg(II) than the dependent strain. This result clearly demonstrate that Cl. cochlearium uses biomethylation as a mechanism for detoxification giving the organism a clear advantage in mercury-contaminated systems.
After methylmercury is released from the microbial system, it enters food chains as a consequence of its rapid diffusion rate. In the estuarine environment, the reduction of sulfate by Desulfovibrio species to produce hydrogen sulfide is quite important in reducing CH3Hg(II) concentrations by S2--catalyzed disproportionation to volatile (CH3)2Hg and insoluble HgS. It should be pointed out here that there is overwhelming evidence to support the notion that membrane trasport of methylmercury is diffusion-controlled. Fluorescence techniques and high-resolution NMR show that diffusion is the key to CH3Hg(II) accumulation (7). Also, a field study of the uptake of CH3Hg(II) by tuna fish in the Mediterranean fits perfectly the diffusion model for biota in tuna fish food chains (16). An updated view of the mercury cycle is presented in Figure 1. At this point let us discuss some of the individual reactions of this mercury cycle in more detail especially since we have an appreciable understanding of the molecular biology and biochemistry of the equilibria (17): CH3Hg-= Hg2e =Hge (1) Microorganisms have been isolated which catalyze the reactions described above for both the forward reaction to Hg0 and the back reaction to CH3Hg'.
The enzymes which carry out the forward reactions are coded by DNA ofbacterial plasmids and transposons and not by normal chromosal genes. Therefore it is not too surprising that mercuric and organomercurial strains of bacteria have been isolated from a variety of ecosystems such as soil, water, and marine sediments (14)(15)(16)(17)(18)(19). The enzymology of methylmercury hydrolysis and mercuric ion reduction is now understood in some detail (18)(19)(20)(21). In fact, the sequence of the active site of mercuric ion reductase is now determined and has been found to be identical to that of glutathione reductase (22). Clearly, the reduction of Hg2e to Hg0, which is volatile, represents a very effective detoxification mechanism. Much less is known about the reverse reaction, i.e., the oxidation of Hg0 to Hg2e. However, an enzyme which is critical to the oxygen cycle (catalase) will carry out this reaction. Microbial methylation of mercuric ion is also widespread (23,24). Biomethylation has been shown to occur in sediments and in human feces (25). The important role played by sulfide in the biological cycle for mercury is presented in Figure 1. Hydrogen sulfide is extremely effective at volatilization and precipation of mercury through disproportionation chemistry in the aqueous environment. The same is true for the volatilization and precipation of lead compounds. This chemistry is important to the mobilization ofmetals < from the aquatic environment into the atmosphere. Such reactions occur in polluted lakes, rivers, coastal zones, estuaries, and salt marshes where Desulfovibrio z species have access to sulfate in anaerobic ecosystems zs (Fig. 1). The disproportionation of organometals by H2S 3 is outlined in Eqs. (2) and (3). 2CH3Hg+ + H2S -* (CH3)2Hg + HgS 2(CH3)3Pb+ + H2S -* (CH3)4Pb + (CH3)2PbS (2) ( Once in the atmosphere volatile organmetals, such as dimethylmercury, are unstable because metal-carbon bonds are susceptible to homolytic cleavage by light (Fig. 1). The impact of acidification on the concentrations of intermediates in the mercury cycle is most interesting, and has been tested experimentally in both Swedish and Canadian Lakes (26,27).
At low pH the rate of synthesis of methylmercury by acid tolerant microorganisms is lower than with the more diverse populations of bacteria which grow in sediments under ambient conditions. However, any dimethylmercury which is produced by base-catalyzed disproportionation is converted to monomethylmercury by acid hydrolysis [Eq. (4)]: (CH3)2Hg + H+ -* CH3Hg+ + CH4 t (4) This reaction can increase that fraction of methylmercury which is available to the food chain and lead to increased concentrations of methylmercury in predatory fish such as northern pike. Such a reaction has been found to be consistent in both Swedish and Canadian Lakes which are undergoing acidification (26,27).
The increase in concentrations of methylmercury in predatory fish is also apparently due to decreasing populations of fish in lakes which are undergoing acidification (i.e. the bioaccumulation rate for methylmercury is extremely rapid, but the predator population is small; therefore fewer fish must accumulate more methylmercury).
It is clear from Swedish studies that most of the input of mercury into freshwater lakes occurs by the exchange ofmercury from soils in regions where acid precipitation is severe (26). Extensive studies in Scandinavia show that the concentration of mercury in topsoils has increased about fivefold since the beginning of the industrial revolution. In the U.S., mercury additions to the terrestrial environment are more recent. However a report published by the National Academy of Sciences (28) estimates that over 3000 tons of mercury is smelted from coal in the generation of electricity in the U.S., and this appears to be a major uncontrolled input.

Cadmium and Lead
The primary focus for the potential for human exposure to cadmium and lead has been on the collection of drinking water and concomitant acid corrosion of these metals directly into drinking water supplies. This corrosive aspect of drinking water can be solved, and in fact has been solved for lead in Glasgow, by adjusting the pH of that city water supply with lime, before distribution. Similar measures will understandably have to be taken into consideration in acid-susceptible reservoirs and in private cisterns in regions of the USA where acid rain is severe.

Aluminum
Aluminum is the third most abundant element in the earth's crust. The chemistry of this element under both aerobic and anaerobic conditions makes it unavailable for uptake by living organisms unless those organisms grow at low pH. A detailed study of Swedish lakes shows that the solubilization of aluminum salts becomes increasingly important at the pH falls below 6.0 (1). Similar conclusions are reached by Driscoll in his study of aluminum in acid susceptible lakes in North America (29). The toxicity of aluminum to higher organisms, including man, very much depends on the inorganic chemical speciation of its salts. For example, acute chronic toxicity studies in rats show that Al(OH)3 is not toxic at exceedingly high doses, but AlC13 has low LD50 dose (30). The transport and toxicity of aluminum complexes in living organisms including man is not understood, although there appears to be a correlation between Ca2+ transport and Al + transport (30). This is to be expected from the arguments on the exchange rates of elements which I alluded to earlier in this chapter. The connection between aluminum uptake and diseases of the central nervous system has been adequately documented by Wills (31) and Perl (32).
What is missing from this field is a complete understanding of toxic species, and biochemical mode of action of this element. Noninvasive techniques such as in vivo A127 NMR should help in unravelling some of these basic questions. However, it is clear that acid precipitation causes dramatic increases in concentrations of aluminum in freshwater systems. The experience in Guam could be repeated in North America unless we address this most serious situation.
At this point I have no ideas on how to prevent the third most abundant element (in the earth's crust) from being solubilized by acidification without the application of broad scale neutralization procedures.

Arsenic and Selenium
There has been very little discussion of the volatization of the metalloids arsenic and selenium by comparison with sulfur from fossil fuel combustion or from mining and smelting activities. The impact of acidification on the metabolic pathways for arsenic and selenium requires attention primarily because of the differences in reactivity of their oxyanions in acid versus neutral conditions. At acid pH's the standard reduction potential for the SevI/SeIv couple is + 1.15 V, and for the Asv/AsIII couple it is + 0.559 V; under these conditions, methylation of arsenic and selenium species to more volatile and less toxic forms should be slowed down considerably which would be expected to lead to a buildup of the more toxic inorganic anions in water.
However, at pH 7.0 and above the Eo for Sev/SeIv changes to +0.05 V and for Asv/AsLII -0.67 V, this allows efficient methylation by the methylating coenzyme S-adenosylmethionine to produce volatile methylated metabolites (33,34).
This change in reactivity of the oxyanions of arsenic and selenium is likely to be of critical importance to the buildup of concentrations of these elements in surface waters and sediments in the long term. At least this recognition points up the importance of future research efforts in the distribution of these extremely toxic elements.

Effects of Competing Cations and Anions on the Uptake of Toxic Metals and Metalloids
It is clear that inorganic ions compete for transport pathways in living organisms, and that many of the "good ions" such as Mg + and Ca2+ effectively compete with the "bad ions" such as Cd2+ and Al3+ in biological membranes (35). Clearly one remedial option is to ensure that the concentrations of Ca2 + and Mg2 + in waters exceed the concentrations of ions such as Al>+ and Cd2 . This of course depends very much on the nature of the complexing anions in the water. Much more needs to be done on the actual inorganic speciation of toxic metals in acid-susceptible surface waters. For example, while it is recognized that the concentration of aluminum is very high in the drinking water sources in Guam, to my knowledge there is no information on aluminum speciation in this drinking water. This requires the development of more sophisticated analytical procedures. The first phase of any analytical procedure is the development of appropriate separation techniques. Such separation techniques have been developed using gas chromatography, liquid chromatography, or ion chromatography which are specifically designed for inorganic analysis. However, the connection ofthese techniques with mass spectrometry for the structural determination of inorganic species, has been slow in developing even though such techniques are well advanced in the determination of organic structures.

Concerns
It is understood that any chemical species in the environment may move from its source through the physical environment and into biota where it may cause either a beneficial or a toxic effect at the cellular level. The pathways taken by each element involve many chemical species, and the distribution of these species is determined by thermodynamic and kinetic considerations. Those species which are stable thermodynami-cally can often be isolated and quantitated. Ideally the precise chemical structures of each of these stable compounds, or complexes, should be analyzed in that matrix where they exist naturally. Unfortunately, in the stressed system entropy increases and there is a concomitant increase in instability in both the physical and biological context. The acidification of the aqueous environment has already been shown to cause significant stress on ecosystems. Dr. Stokes presented an example of the total colonization of some acid susceptible lakes in Canada with a single species of a filamentous alga. As diversity of populations decrease to only a few acid tolerant species, then the normal balance for the cycling of elements changes appreciably. This has already been shown to be the case for mercury and aluminum, and may well be the case for the metalloids arsenic and selenium. The net effect of this instability is an increase in the concentration of methylmercury in predatory fish and an increase in the concentration of soluble aluminum species in surface waters. The consumption of methylmercury-contaminated fish has already been the cause of major health problems in Japan and more recently in China, and the consumption of drinking water containing high concentrations of aluminum is implicated in the epidemiology of Alzheimer's disease in Guam.
I wish to acknowledge the support of the NIH AM 18101 for support of some of the research discussed in this review.