Lecture No. 17. Biomethylation
This throws a whole lot of our assumptions into a cocked hat. Any Eh-pH diagram you look at tells you exactly nothing about methylation.
The Equilibrium Partitioning (EP) approach assumes equilibrium chemistry. Biomethylation is an independent set of processes intimately tied to life processes. And life is itself a major departure from equilibrium.
Background
Since the 1950’s it has been known that some elements can be methylated, and that at least some of the methyl adducts are very toxic. For example, monomethylmercury, CH3Hg+, was responsible for much of the mercury poisoning at Minimata Bay, Japan. Originally it was believed that the methyl compounds of mercury and some other metals were all produced by industry. This was debunked in 1979, when Jensen and Jernelšv discovered natural methylation of mercury. We now know that natural methyl adducts of As, Se, Hg, Sn, Pb, Bi, Sb, and other elements are made by bacteria and higher organisms.
One might assume that the conditions most conducive to methylation would be anoxic-methanic. Like many other things that seem reasonable about methylation, that isn’t true. Different organisms make methyl adducts under conditions ranging from anoxic-methanic to oxic. Each organism has its own reason for methylating a metal or metalloid. I will go through the major methylated elements one by one and look into the mechanisms, the conditions, and the toxicities.
I. Mercury
Mercury has several important inorganic forms:
There are also two methylated forms: (CH3)Hg+ and (CH3)2Hg. The former is a cation that forms a number of salts, such as monomethylmercuric chloride. The latter is a gas at room temperature. Dimethylmercury plays (along with the metal) a major part in the global mercury geochemical cycle.
Background sediment mercury concentration is normally on the order of 10–200 m g/kg. Concentrations found in Minimata Bay in recent decades are around 2000 mg/kg (0.2%). Layers of sediment in Lake Windesmere, England were analyzed. The sediment layers were isotope-dated, and a gradual increase in mercury in recent sediment was found.
AD 520-1260 |
150 ppb Hg |
AD 1400-1800 |
300 ppb |
AD 1870-1895 |
600 ppb |
AD 1915-1950 |
1000 ppb |
Chemical Processes within Sediments
Mercury is strongly sorbed to sediments. (Coatings are important.) It complexes readily to form chelates with organics. These complexes are stable down to about pH 5. Under anoxic-sulfidic conditions, cinnabar forms. However, many sulfate-reducing bacteria also methylate mercury; sulfate reducers are the most important mercury methylators in nature. The biochemical reason appears to be to protect themselves from mercury poisoning. Metal cations move only slowly through bacterial membranes. Mercuric ion (Hg2+) is very toxic. Both of the methyl forms pass through the membrane much more readily than the inorganic form. The process appears, then, to be a way for bacteria to excrete mercury.
Other species are also known to methylate mercury, including aerobic bacteria and fungi. Vitamin B-12 and similar cobalt-containing compounds appear to be the primary methylating agents for mercury. However, there are a few other routes to methylation of Hg2+.
There are also abiotic methylation routes. One is transmethylation, in which a methyl group is transferred from one metal atom to another. Lead, arsenic, and tin readily lose their methyl groups to mercury. For example,
Sn(CH3)3+ + Hg2+ ¨ Sn(CH3)22+ + Hg(CH3)+
Certain organic molecules can also donate methyl groups to mercury. Acetate ion is one example:
CH3COO– + Hg2+ ¨ CO2 + Hg(CH3)+
Fulvic acids have been reported to methylate mercury abiotically.
Biomethylation of mercury is reportedly increased by lower pH. Mercury methylation is increased in waters affected by acid rain. However, since most acid rain is rich in sulfate, we may be seeing an effect of increased sulfate reducing bacterial activity in the sediments.
The bioavailability of monomethylmercury is strongly controlled by hardness; high Ca2+ concentrations in water retard uptake by fish.
Case Study: Canadian Reservoirs
Beginning in the 1960’s several Canadian provinces went on a major dam-building spree. QuŽbec, Ontario, Newfoundland, and Manitoba built a number of large hydroelectric dams that created new lakes in their northern areas. Hydro QuŽbec’s experience was typical.
After the reservoirs filled with water, mercury levels in fish rose dramatically. The local Indians, who depended on a diet high in fish, began showing symptoms of mercury poisoning. Here are a few examples of contaminated fish:
| Fish | Reservoir | Pre-reservoir Hg concentration | Year 2 | Year 4 |
| Whitefish | La Grande 2 | 0.09-0.17 ppm | 0.26 ppm | 0.51 ppm |
| Suckers | Opinacca | 0.13-0.19 | — | 0.40 |
| Pike | Caniapiscan | 0.52-0.60 | — | 1.24 |
| Pike | La Grande 2 | 0.6 | 1.28 | 2.05 |
The Canadian mercury limit in edible fish is 0.5 ppm.
What happened?
There was no industrial source and there was no increase in any outside mercury source, natural or man-made.
It turns out that the earth’s atmosphere acts as part of a giant still. Elements that can exist in the gas phase tend to evaporate from tropical and subtropical regions and condense out at high northern and southern latitudes. Ever since the end of the last ice age, northern Canada has been a mercury sink. The soil and the plants contain elevated mercury levels.
When the lakes were filled, a bunch of the mercury went into solution within a few years. At depth the lakes had anoxic-sulfidic conditions because of the abundant organic matter on the flooded ground. The conditions were ideal for mercury methylation.
Monomethylmercury is very bioavailable. The fish had high monomethylmercury levels in their muscle tissues. Pike, being near the top of the food chain, had very high mercury concentrations. People, being even higher on the chain, got mercury poisoning.
It was necessary in many cases to empty and refill the reservoirs. The mercury on the ground was not infinite, and after most of it went into solution and was sent downstream to Hudson Bay, the refilled lakes returned to safe mercury levels. After one generation, so did the fish. But, for a lot of local people, the damage was done.
In most freshwater and marine systems, methylmercury makes up no more than 1% of the total mercury. However, environments have been found where up to 5% of the mercury is methylated.
II. Arsenic
There are several naturally-occurring forms of inorganic arsenic:
Arsenic can be methylated by bacteria, algae, fungi, vascular plants, and animals. There are two series of methylated arsenic compounds.
The methylated arsenic (V) compounds include
These As (V) compounds are produced by algae, cyanobacteria, arthropods, fish, mammals, and other organisms. They are much less toxic than arsenate; each added methyl group drops the toxicity by a factor of about 10. Since a lethal dose of arsenic in the form of arsenate salts is about 6 g, a lethal dose of arsenic in the form of trimethylarsine oxide is about 6 kg. The monomethyl and dimethyl forms are produced by many aerobic organisms as a way of detoxifying arsenic. Arsenate can be eliminated through the kidneys, but it does a lot of damage going through. The methylated arsenates can also be eliminated through the kidneys, but with much less tissue damage.
In aquatic environments, algae and cyanobacteria ("blue-green algae") methylate arsenate to monomethyl and some dimethyl As (V), some of which is excreted and some of which is retained. The plankton (e.g., shrimp) consume the algae and produce a higher percentage of dimethyl As (V). The animals higher on the food chain consume the plankton and methylate the arsenic further, producing some trimethylarsine oxide.
In most streams, less than 1% of the arsenic is methylated. In lakes, particularly eutrophic ones, over 50% of the arsenic may be methylated. There is definitely a seasonal variation in the amount of methylation that seems to be related to variations in the water’s microbial ecology as temperatures change.
A large number of marine animals go further, incorporating methylated arsenic into arsenoribosides, arsenolipids, and other complex organic compounds. Ocean water is 1–2 ppb arsenic, but the methylation and further incorporation of arsenic into complex molecules does not appear to be just a matter of detoxifying it. There appears to be a metabolic reason why these animals produce these compounds. An average lobster dinner contains about 30 mg of arsenic, but the arsenic is in a form that is not bioavailable to humans. (By comparison, 100 mg of As in the form of As2O3 will kill most people.)
There is also a series of methylated As (III) compounds:
These compounds are extremely toxic. Arsine itself is highly toxic, and it gains toxicity with each added methyl group. Dimethylarsine is produced by methanogenic bacteria and other anaerobes. Some fungi such as molds of the Scopulariopsis genus make trimethylarsine.
This brings us to an interesting case study:
Schweinfurter Green and Gosio Gas
In 1778 the first copper arsenate dye was described in a paper by Carl Wilhelm Scheele. A number of similar pigments were formulated during the next few decades. Around 1814 the Wilhelm Sattler Dye and White Lead Company in Schweinfurt began making a mixed copper acetate-arsenate salt that had markedly superior properties. It gave a beautiful green color to paint, cloth, candy (!) and paper. Unlike most pigments in use then, it did not turn grey or dull on exposure to sulfides in the air.
Wallpaper was just becoming popular in Europe at that time. Schweinfurter Green became very widely used in wallpapers all over Europe. The paper, then as now, was stuck to walls with a starch-based paste.
Northern Europe has a cool, damp climate, even in summer. This climate is ideal for the growth of mildew, and that laid the groundwork for a widespread health problem.
Green wallpaper became very popular in Germany, France, and other countries. Besides Schweinfurter Green there were a number of green copper-arsenic dyes made by competing companies: Paris Green, Bremer Green, Kaiser Green, etc. People began to notice that, in bedrooms with green wallpaper, all the bedbugs died. (This was seen as a major benefit and led to increased sales of the paper.) However, two other phenomena became widespread: (1) a garlic odor was often present in rooms with such wallpaper, and (2) many people who slept in rooms with green wallpaper got sick and died.
In 1838 the Prussian government forbade the use of poisonous substances as dyes for wallpaper. The law was not strictly enforced, and many other jurisdictions in Germany and elsewhere in Europe went on allowing their use. And no one knew what was causing the deaths.
In 1897 an Italian chemist, B. Gosio, established that the starch in the paste and sizing was being consumed by a common mold called Penicillium brevicaulum, which today is called Scopulariopsis brevicaula. The mold was taking up arsenic from the dye and was excreting it as a gas. The gas had a strong garlic odor and was very toxic. Gosio knew the gas was an organoarsenic compound but was unable to identify it exactly. The poisonous vapors from green wallpaper became known as "Gosio gas."
In 1945 Frederick Challenger identified the gas as trimethylarsine. We now know that the fungus converts arsenate to trimethylarsine oxide through a complex series of biochemical reactions. If it stopped there, there would be no problem. However, as a last step the oxide is reduced to trimethylarsine and the gas is excreted. This gas is what killed all those people.
III. Selenium
Selenium is methylated by plants, animals, and microorganisms to dimethylselenide (CH3SeCH3) and dimethyldiselenide (CH3Se2CH3), as well as to dimethylselenone ((Ch3)2SeO2) and several less common species. The methylselenide and methylselenone compounds are volatile and the first two (at least) are important in the global selenium geochemical cycle. Marsh plants like cattails and bulrushes methylate a good deal of selenium in areas with high selenium content. Even more selenium is methylated under drier, more oxic conditions by various fungi and bacteria. Selenium methylation is partly a detoxification route. Formation of dimethylselenide (which is not very toxic) is a way to eliminate hydrogen selenide (which is extremely toxic).
Selenium in Closed Drainage Basins in the West
In most Western States there are deposits of Cretaceous-age shale. This shale is high in selenium, and weathering of the shale releases selenium into groundwater and surface waters. This is normally not a problem, since the selenium levels are low enough to be beneficial rather than toxic. In areas where the water is used for irrigation, however, if the runoff from irrigated fields is allowed to drain into closed basins, evaporation does its work, and the selenium concentration in water and soil increases. Eventually the selenium reaches toxic levels. Plants take up selenium and animals eat the plants and get poisoned. The best-known selenium disaster was at Kesterson Reservoir in California. The Coast Ranges contain a lot of Cretaceous shale and the drainage from those mountains contains significant dissolved selenium. Before the advent of large-scale irrigation there was no problem, but during the 20th Century much of the San Joaqu’n Valley has been irrigated. A drainage system for the irrigated fields was planned. The San Luis Drain was to carry runoff from the fields and from the drain tiles under the fields to San Francisco Bay. This drain was blocked when people downstream realized how much pesticide residue and fertilizer would get dumped into the Sacramento-San Joaqu’n delta and the bay; it would have had severe impacts on the local fishery, which was already under stress from the State Water project and from urban runoff. The San Luis Drain never got built, and the water dead-ended at Kesterson Reservoir near Los Ba–os. Kesterson was also a Federal game refuge that attracted many ducks and geese migrating along the Pacific Flyway. The reservoir functioned mainly as an evaporation basin. Evapoconcentration caused the selenium levels in the soil to build up to toxic levels, the plants took up excessive selenium, and birds started getting poisoned. Drainage to the reservoir was cut off, and it was allowed to dry up. A very expensive cleanup was carried out that involved hauling soil to a hazardous waste dump.
Since Kesterson’s closure, farms and irrigation districts in the valley have created their own evaporation basins. After five to ten years those basins have begun to show hazardous selenium levels.
There have been selenium problems in other parts of the West, but none of them have been as severe as Kesterson’s. Benton Lake, north of Great Falls, is another of those game refuge/evaporation basins. Selenium levels have become hazardous in recent years. Yi-Chiang Zhang did his UM doctoral work on the Benton Lake situation. He showed that the selenium levels could be kept low by managing water flow into the lake so that half the basin was dry at any given time. Under dry conditions, the plants and soil biota would methylate the selenium and thus eliminate it in volatile form.
IV. Other Elements
Tin is methylated by a number of plants, including salt-water marsh grasses of the genus Spartina. The tin is re-released when the plants die and decay. Methyltin compounds, which range from CH3Sn3+ to (CH3)4Sn, are fairly toxic.
Lead is methylated by a number of microorganisms. Methyllead compounds, especially tetramethyllead ((CH3)4Pb), are extremely toxic. Tetramethyllead is a gas under ambient conditions. It is easily absorbed through the lungs into the bloodstream, and it crosses the blood-brain barrier very easily. Thus it is much more toxic than other lead compounds. The air around old battery dumps may be hazardous for this reason.
Methyl adducts of several other metalloids (antimony, bismuth, and tellurium) form naturally. They are not usually considered major environmental problems because of the rarity of these elements. However, trimethylstibine, the antimony counterpart of trimethylarsine, is extremely toxic. Methylated tellurium is also quite toxic. Bismuth compounds usually contain some antimony unless they are carefully purified. In the 19th Century, bismuth preparations were widely used for indigestion. The early bismuth products often contained antimony. People taking them produced enough trimethylstibine so that its garlicky odor was noticeable in their breath. The odor was called "bismuth breath."
In one form, cobalt is readily methylated. Cobalamin, or vitamin B-12, consists of a cobalt atom held in a complex organic molecule. The cobalt is usually bound to a cyanide radical (cyanocobalamin), a methyl radical (methylcobalamin), or a water molecule (aquocobalamin). Methyl-B-12 is one of the ways organisms (including humans) transfer methyl groups — to DNA, to other organic molecules, and to metals and metalloids. (The cyanide, by the way, does not make B-12 toxic.)
Determining Geochemical Background
The question often arises, "Is a stream/lake/soil contaminated?" Another question is, "Where did the contamination come from?" These questions are not always easy to answer.
We will examine the case of suspected contamination caused by mining activity. Parts of this approach could be applicable to contamination from other industrial activity (e.g., mercury waste, PCB’s), residential development, etc.
The first thing to address is the natural level of the contaminant of interest. The level of contamination — the enrichment factor — must be known. This may help in establishing the geographical extent of the contamination.
So how do we find the natural level? One approach is to compare the local level to average global values. For example, arsenic is present in the crust at an average abundance of 5 ppm, in average fresh waters at less than 1 ppb, and in seawater at 1-2 ppb. We can then work out a "typical" enrichment or contamination index. This actually works pretty well for nonmetals and synthetic organics. It is not very good for metals and metalloids. For such elements, the characteristics of a site may make it non-average or atypical. For example, the neighborhood of an orebody is normally enriched in the elements present in the ore. Another good example is shaly soil, which is naturally high in selenium.
To try to get around this problem, another very typical approach is to limit the study by the type of sample and geochemical environment. A local baseline is found with the same characteristics of vegetation, geology, climate, and so on. This works very well if an appropriate baseline site can be found. However, such an appropriate baseline site can be hard or impossible to find. Again, this method works well for synthetic organics.
A third approach is to choose a baseline on the basis of biology. An environment with "healthy biota" is found, and it is used as a baseline. The "test site" is compared with the "baseline site."
The problem is that none of these approaches addresses the case of "natural" contaminants or contaminants unrelated to the mining activity that is suspected to cause contamination.
Here is an alternate approach to finding background concentrations in mineralized areas.
To previous lecture: Lecture No 16. Metal Toxicity II
To next lecture: Lecture No 18. Determining Geochemical Background
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