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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 I I 77-24,683 PARSONS, James Eugene, 1939- MERCURY TRANSLOCATION AS MEDIATED BY METABIOSIS.
The Ohio State University, Ph.D., 1977 Microbiology
Xerox University Microfilms, Ann Arbor, Michigan 48106
© 1977
JAMES EUGENE PARSONS
ALL RIGHTS RESERVED MERCURY TRANSLOCATION AS MEDIATED BY METABIOSIS
DISSERTATION
Presented in Partial Fulfillm ent of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
James Eugene Parsons, B.S., M.S.
*****
The Ohio State University
1977
Reading Committee: Approved By
J .I. Frea R.M. P fister M.S. Rheins
Adviser Department of Microbiology 11 '"Twinkle, twinkle, little bat? How I wonder what you're at? Up above the world you f ly , Like a teatray in the sky.
Twinkle, twinkle
To the Mad Hatter (90), who through his quixotic actions and incoherent poetry (see above) gave us an accurate portrayal of mercury poisoning. Had he desired, no doubt, he could have conjured up via phyllomancy of his beloved tea ( Thea sinensis) an early glimpse of a sleeping giant, viz. : environmental mercury pollution. ACKNOWLEDGMENTS
In the autumn of 1972 Dr. Robert M. P fister accepted a unique challenge when he agreed to take a "non-traditional" student em barking on the road to opsimathy. The transition and educational process necessary to convert an "old timer" into a neanthropic scien tist progressed quite s a tis fa c to rily , and the thought processes required to transform such an individual into a person who "thinks young" has occurred gradually and progressively. In addition to your faith and friendship, Dr. Pfister, I thank you for your guidance, encouragement, support, and most of a l l , for the oppor tunity to expand my experiences in understanding the relationships that exist between microbial structure and function.
Realizing that I am but one of the multitude of graduate students ministered to by the Department of Microbiology's
Graduate Committee, I wish to thank its members for allowing me to create and pursue an individualized, unique doctoral program, i.e . in my opinion the perfect amalgam of administration, teaching and research.
In addition, I wish to express my deep gratitude to the State of Ohio, The United States Government and to each individual
iv taxpayer of this great country for providing such an educational
opportunity. To my fellow graduate students, this advice — a fonte puro pura defluit aqua — drink freely of it.
For their individual encouragement and inspiration as out
standing teachers, I thank three (3) high school teachers: Ms.
Nora Keville, Ms. Mary Belle Linnell, Mr. George G. Maxfield;
three (3) university professors: Dr. James I. Frea, Dr. Wolfram
Kretschmar, Dr. Melvin S. Rheins, and one (1) university adminis
tra to r: Dr. Arliss L. Roaden.
The patience and interest my family has demonstrated is appreciated. For accepting my routine neglect as a necessary sacrifice from which I hope all will eventually benefit, I am also grateful.
To that cadre of friends that has made l i f e in Columbus an experience in living and not merely existing, a special thanks goes.
And last, but certainly not least, to Patricia S. Camana who through her rendering of the Mad Hatter has v iv id ly demonstrated to us all just what the ancient Roman knew all along — aliena optimum frui insania.
V VITA
November 10, 1939 ...... Born - Lima, Ohio
1 9 6 1 ...... B.S., The Ohio State University, Columbus, Ohio
1963 ...... M.S., The Ohio State University, Columbus, Ohio
1964-196 5...... Teaching Assistant, De partment of Microbiology, The University of Nebraska, Lincoln, Nebraska
1965-196 7 ...... National Aeronautics and Space Administration Trainee, Department of Microbiology, The Univer s ity of Nebraska, Lincoln, Nebraska
1967-196 8 ...... Chief Bacteriologist, State of Nebraska, Depart ment of Health, Division of Laboratories, Lincoln, Nebraska
1968-197 1 ...... Instructor of Medical Microbiology, Department of Medical Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
1972-1973 ...... Teaching Associate, De partment of Microbiology, The Ohio State University, Columbus, Ohio
vi 1973-197 4 ...... Trainee in University Admin is tra tio n , Graduate School, The Ohio State University, Columbus, Ohio
1974-197 5 ...... Teaching Associate, Depart ment of Microbiology, The Ohio State University, Columbus, Ohio
1975-197 7 ...... Director, George Wells Knight International House and Center, The Ohio State U niversity, Columbus, Ohio
1977 ...... Administrative Assistant, Department of Microbiology, The Ohio State University, Columbus, Ohio
PUBLICATIONS
Schmitt, J .A ., R.A. Zabransky, A.S. Janidlo, and J.E. Parsons. 1962. Experimental maduromycosis in the laboratory mouse. Mycopath. Mycol. App. 18:164-168.
Stamm, J.M ., W.E. Engelhard, and J.E. Parsons. 1969. Micro biological study of water-softener resins. Appl. Microbiol. 18:376-386.
FIELDS OF STUDY
Major Field: Microbial Cytology
Studies in Electron Microscopy. Professor Robert M. P fister
Studies in Morphology, Fine Structure and Cell Function. Professor Robert M. P fis te r Studies in Bacterial Physiology and Genetics. Professors James I . Frea and James C. Copeland
Studies in Microbial Parasitism. Professor Melvin S. Rheins
Studies in Clinical Parasitology. Professor Wolfram Kretschmar
Studies in Clinical Virology. Professor Roberta J. White
viii TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS...... iv
VITA...... vi
LIST OF TABLES...... x iii
LIST OF FIGURES...... xiv
INTRODUCTION...... 1
LITERATURE REVIEW ...... 7
Mercury and its compounds ...... 7
Metallic mercury ...... 7 Inorganic mercury compounds ...... 9 Complex ions of mercury ...... 9 Organic mercury compounds ...... 10
Agricultural use of mercury ...... 11 Industrial use of mercury ...... 12 Mercury in medicine ...... 13 Sources of mercury in the environment...... 14 Mercury transformation in the biosphere ...... 19 Conversion between inorganic forms ...... 22
Hg2 + - H g S i 2 2
HgS+ ------► Hg2 + ...... 22
Hg2 + ------> Hgo ...... 2 2
Hg° Hg2 + ...... 23
Conversions between organic and inorganic forms ...... 23
CH3 0CH2 CH2 Hg+ ► Hg2 + ...... 23
ix TABLE OF CONTENTS (Continued)
Page
CH3 Hg+, C2 H5 Hg+, C6 H5 Hg+ Hg2+ and/or Hg°. . . 24
Hg° and/or Hg2+ >- CH3 Hg+ and/or CH3 HgCH3 ...... 25
Conversions between organic forms ...... 27
C6 H5 Hg+------► CH3 Hg+~ ------* CH3 HgCH3 ...... 27
C6 H5 Hg+; z = ? C6 H5 HgC6 H5 ...... 28
CH3 Hg+ ------► CH3 HgCH3 ...... 28
CH3 HgCH3------► CH3 Hg+ ...... 28
Other Conversions in nature ...... 29
(C2h5>4 Pb + Hg2+ ► (C2 H5) 3Pb + C2 H5 Hg+ ..... 29
Interaction between mercury and organisms ...... 29 Detection of mercury and its compounds ...... 31
MATERIALS AND METHODS...... 34
Sample collection ...... 34 Sample processing ...... 34 Total mercury determination ...... 37 "Mercury-free diluent" ...... 40 Aquarium (model la k e ) ...... 40 General layout ...... 40
Aeration ...... 40 F iltra tio n ...... 49 Illumination ...... 49 Temperature ...... 49 W a te r ...... 50
Sediment b e d ...... 50
Aquarium gravel ...... 50 Aquarium pebbles ...... 51 Olentangy River mud ...... 51 Potting soil...... 51 Sand ...... 52
x TABLE OF CONTENTS (Continued)
Page
Ecosystem ...... 52 Regulated mercury s p ill ...... 52 Sam pling ...... 52
Attached planktonic biomass ...... 52 F ilte r entrapped detritus ...... 53 Goldfish...... 53 Sediment cores ...... 53 Snails ...... 54 W a te r ...... 54
Volume measurements ...... 54 Weight measurements ...... 54 Weight vs. volume measurements ...... 55 Scanning electron microscopy ...... 55
Solid specimens ...... 55 Liquid specimens ...... 56
X-ray microanalysis ...... 56
RESULTS...... 58
DISCUSSION...... 89
SUMMARY...... 101
EPILOG...... 103
APPENDICES...... 104
Appendix A Calculation of the potential necessary for oxidation of metallic mercury into divalent m ercury ...... 104
Appendix B Interaction of mercury with biological systems. . . 107
Appendix C Ancillary data for water samples collected from the western basin of Lake E rie ...... 126
Appendix D Data obtained through x-ray microprobe analysis . . 129
xi TABLE OF CONTENTS (Continued)
Page
Appendix E Mathematical expression fo r mercury translocation in model lake bed sediments ...... 136
LITERATURE CITED ...... 141
x ii LIST OF TABLES
Table No. Title Page
1 Sample collection information ...... 60
2 Mercury determination of fractional components - 1 62
3 Mercury determination of fractional components - II ...... 64
4 Mercury level of model components ...... 67
5 Interaction of mercury with biological systems . . 108
6 Physical factors ...... 127
7 Chemical factors ...... 128
8 SAMPLE IDENTITY: Filamentous alga with epiphytic bacteria in s -itu...... 130
9 SAMPLE IDENTITY: Fusiform diatom, in situ...... 131
10 SAMPLE IDENTITY: Pseudomonad isolated from model and cultivated in Hg-containing medium ...... 132
11 SAMPLE IDENTITY: Staphylococcus isolated from model and cultivated in Hg-containing medium . . . 133
12 SAMPLE IDENTITY: Pseudomonad isolated from model and cultivated in Hg-free medium ...... 134
13 SAMPLE IDENTITY: Staphylococcus isolated from model and cultivated in Hg-free medium ...... 135
xi* ai i • LIST OF FIGURES
Figure No. Title Page
1 Mercury transformation in the biosphere ...... 21
2 Flow diagram of procedure u tiliz e d during experimentation ...... 36
3 Schematic of apparatus employed to measure to tal mercury via the cold vapor atomic adsorption spectrophotometric technique ...... 39
4 Calibration curve for a typical mercury standard, absorbance a t 2537 A ...... 42
5 Functional elements of the model lake ...... 44
6 Cross-sectional diagram of s tra tifie d model lake, bed sediments showing depth and composition of each l a y e r ...... 46
7 Illustration of aquarium floor plan ...... 48
8 Kinetics of mercury translocation through model lake bed sediments ...... 69
9 P article mediated mercury m obility in the water column...... 71
10 Mercury accumulation in model lake gastropods. . . 74
11 Mercury accumulation in the model lake fish ( Carrassius auratus) population ...... 76
12 The effect of particle volume on its associated mercury content ...... 79
13 The effect of particle weight on its associated mercury content ...... 81
xiv LIST OF FIGURES (Continued)
Figure No. Title Page
14 P article volume versus particle weight for a given mercury b u rd e n ...... 83
15 Scanning electron micrograph of filamentous alga (FA) with an "Auswuehs" of epiphytic bacteria (B) ...... 85
16A Scanning electron micrograph of a pseudomonad isolated from the model lake benthos ...... 8 8
16B Scanning electron micrograph of a staphylococcus isolated from the model lake benthos ...... 8 8
17 Arhythmic feedback cycle fo r mercury conversion proposed fo r the model employed herein ...... 94
18 Mathematical expression of the interrelationship seen between Time (T) and Distance (D) involved in the translocation of mercury through model lake sediment beds ...... 140
xv INTRODUCTION
Man's environmental manipulations have continued in greater
or lesser degrees for numerous chiliads, but only recently has an
increased awareness of the repercussions of these manipulations
surfaced in the popular consciousness. F irs t man encountered the
inorganic pollutants such as mercury, lead, cadmium, beryllium and asbestos; then the advent of Pandora's panacea, viz., the organic compounds—fa r too numerous to mention herein.
In the 1960's we sparred with the biocides—organophosphates, organochlorines, and herbicides. Grace a Rachel Carson (58), the world became aware of the grim realities resulting from mass indiscriminate spraying.
Again in the 1970's we encounter yet further examples of man's foibles. F irs t in 1973, through a human error no doubt, a toxic f ir e retardant known chemically as polybrominated biphenyl
(PBB) was added to livestock feed. Spontaneous abortions, breeding problems, and/or death of dairy c a ttle focused attention on this problem. Man is now monitoring with horror the movement of the aforementioned chemical through the food chain and the resultant health problems in Homo sapiens. Earlier this year (1977) we began to detect significant amounts of pentachlorophenol (PCP or
1 penta) in milk. PCP, itself a widely used wood preservative
(acquired by cows which rub up against, lick or breathe fumes from wood in feeders and barns), does not appear to be a cause for alarm; dioxins, a by-product of penta manufacture and contaminant, however, are. The la tte r , among man's most toxic poisons, has already made an in it ia l appearance in affected cow's m ilk. At this time it is too early to predict the effects that this, the latest of man's traumas to the environment will have on the food chain or even man himself.
Traditionally speaking, pollution, like prostitution, is any departure from purity. In the environment, however, it has come to mean departure from a normal rather than a pure state; otherwise we would have to say that a given environment is polluted by the organisms, both micro- and macro-, contained therein. If man is to be victorious over his violations of our virginal environment, he must profit from his past indiscretions. Armed with such foreknowledge, we should be able to avoid disaster, or at least defend ourselves against some of its worst consequences. So le t it be with mercury as a pollutant.
Environmental mercury contamination, whether by elemental, inorganic or organometallic compounds, has been recognized recently as a serious water quality problem in many areas of the world. In the past, the general consensus had been that trace metals entering rivers, lakes, or the ocean were rapidly removed from the water phase by eith er chemical or physical interactions with suspended solids and underlying sediments (61). Furthermore,
i t was assumed that once contained in the sediments i t remained in a relatively inert, biologically unavailable form. Recent studies, however, have shown that the aforementioned is not the case; trace metals are readily translocated through the water column (305), mercury its e lf being concentrated via the food chain (72, 97, 133,
145, 180, 182, 210, 281, 351, 352, 402), and subsequently reach as well as accumulate in man (15, 33-36, 59, 65-67, 87, 107, 108,
125-127, 130, 131, 138, 153, 195, 196, 217, 218, 240, 242, 260,
266, 267, 326, 330-332, 336, 343, 377). Although the interest in mercury as a pollutant is re la tiv e ly new to the western hemisphere, this has not been the case in other areas of the world. Japan and
Sweden have had to cope with the problem for years.
The most notable examples of environmental contamination with mercury occurred in Japan between 1953 and 1970. In Minamata
(168-170, 219, 353-356), between 1953 and 1961, 121 fishermen or a member of a fisherman's family were stricken with a mysterious illness characterized by cerebellar ataxia, constriction of visual fields, and dysarthria. Of these 121 cases, a total of 46 deaths resulted. Additional cases of mercury induced poisoning, termed
"Minamata Disease," were seen in the coastal town of Niigata and in the riverside villages along the Agano River (165, 374, 375) between 1965 and 1970. Six persons died and another forty-one were irreversibly poisoned. In both incidents, the syndrome appeared mainly among fishermen and their families, and also among other people who fished frequently and/or liked to eat locally
caught aquatic produce. C h aracteristically, the patients in the
Minamata area of Kyushu Island as well as in Niigata Prefecture
had eaten copious amounts of fish and/or shellfish from contaminated
waters.
Even though no deaths were reported in Sweden, the mercury
pollution problem became apparent after seed-eating bird populations
began to decrease drastically (243). This conclusion resulted from
a study of museum specimens (31) which showed that mercury levels
in bird feathers were nearly constant from 1840 to 1940, but in
creased twenty-fold between 1940 and 1965, coincident with the
introduction of alkylmercury compounds as antifungal seed dressings
(31, 42, 185, 360, 361).
Although mercury and its compounds have long been known to be
toxic, only recently has the genetic basis (294, 295, 297) for
said toxicity been established. Utilizing the root cells of
AVliwn cepa organic mercury compounds have been shown to give rise
to c-mitosis and polyploidy (296) in such a mitotic system, whereas
a meiotic system consisting of Drosophila melanogastev, exhibited
unequal recovery of reciprocal gametes due to preferential segrega
tion at firs t meiosis as well as the altered segregation of non-
disjunctional chromosomes in inversion heterozygotes (298). The
la tte r presumably is caused by the changed meiotic synapsis and/or
a changed kinetic activity of the centromeres. I t has not been generally recognized that hazards could arise
from the disposal of mercurials into aquatic biosphere nor was i t
recognized that mercury could undergo a myriad of biochemical
transformations (177, 179, 200, 246). Recent studies (99, 175,
383) indicate that many common inorganic and organic mercury com
pounds which are discharged by industry into public waters, settle
in bottom muds and are converted into alkylmercury compounds, i.e.
mono- and dimethylmercury. Even though both inorganic and organic
compounds enter natural waters, mono- and d i-a lk y l forms of
mercury present the greatest threat to a ll food chains due to
their mobility in water and their solubility in membrane lipids.
Mercury present in fish as well as other aquatic organisms is
almost en tire ly in the methylmercury form.
In order to overcome environmental problems caused by mercury
i t is essential to understand the fate of mercury in aquatic eco
systems. Several interesting questions have been posed by these
observations, and therefore the objectives of this research were:
(a) to study the dynamics of inorganic % organic mercury trans
formations in situ, in a model lacustrine environment; (b) to
follow the fate of mercury, so mobilized through the various
trophic levels of a typical food chain; (e) to elucidate the role
of microparticulates in the active and/or passive transport of mercury; (d) to study cell-mercury interactions; and (e) to
evaluate the hypothesis that a mercury cycle, as such, does indeed exist in nature. Utilizing recent advances made in the microbiologist's armamentarium of techniques, the following
study was initiated. LITERATURE REVIEW
Mercury and Its Compounds
M etallic Mercury
Mercury, also called quicksilver, is a noble metal (128) with the unique characteristic of being a silverw hite liquid at ambient temperatures. This element (symbol, Hg; at. wt., 200.61; at no.,
80; sp. gr., 13.595; melt, pt., -38.87°C; boil. pt. 356.9°C) is soluble only in oxidizing solutions and readily forms alloys called amalgams with some metals, namely: gold, silver, platinum, uranium, copper, lead, sodium and potassium. In addition to occurring naturally as a mixture of isotopes, mercury has a uniform volume expansion over its entire liquid range. This latter property in conjunction with its high surface tension, and inability to wet and/or cling to glass surfaces, makes it extremely useful in the manufacture of measuring devices.
In dry a ir , m etallic mercury is not oxidized, however, after prolonged standing in moist a ir the metal's surface becomes coated with a thin layer of oxide. The metal does not dissolve in a ir- free hydrochloric acid or dilute sulfuric acid, whereas oxidizing acids (nitric, concentrated sulfuric, and aqua regia) when present
7 1n excess w ill yield the corresponding mercury salts. With
chlorine and other halogens, mercury reacts quite read ily.
Many mercury salts exhibit catalytic effects, e.g. :
a. mercuric chloride loaded on activated charcoal for the production of vinyl chloride by the following reaction:
HgClo CH =. CHt + HC1 CH2 = CHC1
(acetylene) (vinyl chloride)
b. mercuric sulfate utilized in the formation of acetaldehyde:
HgS04 CH = CH+ + H20 CH3 CHO
(acetylene) (acetaldehyde)
Mercury is generally classed as a chalcophilic element, that
is, one that tends to concentrate in sulfides. There are two mercuric sulfides, HgS, that have the same composition but different
c ry s ta llin e forms. A few ores (193, 293) contain free mercury, but most of the metal is found as cinnabar, the red sulfide HgS. Arch-
eologists have found it used as a pigment in the ruins of ancient
Egypt, Babylon and at Mohenjo Daro in Pakistan. In the halcyon days
of ancient Rome, much powdered cinnabar was sent there from the
Sierra Morena region of Spain - the mine at Alamden is s till pro
ducing today - for direct use as a cosmetic. Cinnabar is s t ill
employed as a pigment (verm ilion), but its use as a cosmetic has 9 declined due to health and legal considerations. The black sulfide, metacinnabarite, is only occasionally found in mineral deposits.
Inorganic Mercury Compounds
Mercury forms compounds in which i t may have a valence number of eith er 1+ (mercurous) or 2+ (mercuric). The mercurous ion is peculiar in that it is associated into the double ion (Hg 2 ) > not only in solution, but in the solid state of its compounds as well.
Conductivity of mercurous compounds in solutions indicate that they are bivalent electrolytes and x-ray analysis of mercurous crystals confirm that the Hg atoms invariably occur in pairs.
Most mercury compounds are v o la tile , usually decomposing to mercury. A few, notably the halides, sublime without decomposition.
Mercuric compounds are readily reduced to the mercurous state and further to the metal. Conversely, some mercurous compounds (not ably the oxide, and sulfide) are unstable and undergo spontaneous decomposition to the corresponding mercuric compound and free mercury. Mercuric compounds (excepting the halides) hydrolyze readily in aqueous solution, and in most cases are soluble only in the presence of excess acid. Most mercury compounds show a strong tendency to form complexes.
Complex Ions of Mercury
Mercury complexes are f a ir ly numerous. Complexes in which the coordination number is four and the metal is bonded to carbon ( 1 n the cyanides), nitrogen, sulfur or the halogens are among the most important.
The halide complexes exist in two forms, [Hg {Hal)^\ and
Hg (NH3 ) 2 {Hal) 2, where M can be cadmium, potassium or zinc, and the Hal moiety - bromide, chloride, iodide or cyanide.
Mercury has long been known to have a stronger a ffin ity for sulfur than fo r oxygen. Mercaptans, or sulfhydryl groups, as they are more commonly called, have an exceedingly high a ffin ity for mercurials. Almost every toxic action of mercurials is to some extent automatically attributed to an interaction with these ligands to form a complex known as a mercaptide.
In biological systems, sulfhydryl groups are found in a few d iffu s ib le low molecular weight substances such as cysteine, reduced glutathione, CoA, lipoate, and thioglycolate, but the pre dominant sulfhydryl constituents are the proteins. The primary targets fo r interaction and fo r consequent toxicological effects of the mercurials are therefore the proteins. For this reason, it is not surprising that these interactions (1, 19, 29, 37, 166,
308, 309, 316, 346, 348, 362) are especially popular agents for study.
Organic Mercury Compounds
Organic mercury compounds, having the general structure i?-Hg-J, consist of an organic radical such as alkyl, alkoxyalkyl or aryl substituted for R. The X moiety, an anion originating 11 from organic or inorganic substances with a dissociable hydrogen ion {e.g., acids, amides, phenols, th io ls , eta.), is bonded similarly to a salt. Their solubility in water and organic solvents depends on the nature of R and X as does their volatility. For example, the s o lu b ility of phenyl mercury salts in water is lower than that of alkylmercury salts with the same anion; the latter, as a result, are more suitable for water-based fungicidal formula tions.
In general, the short-chain alkylmercury derivatives have a greater v o la tility than phenyl and methoxyethyl mercury compounds.
This difference can, in effect, approach parity by combining an anion such as dicyandiamide with the alkyl radical.
Both aryl and alkyl salts are easily reduced to their diaryl or dialkyl counterparts, and through a series of reactions, elemental mercury. The stability of this group of mercurials varies considerably.
Agricultural Use of Mercury
For more than h a lf a century, mercury fungicides have been utilized for treatment of seeds (20, 94, 233, 303, 306, 307, 387).
In view of the large scale of use, their safety record is notably good, except for the alkylmercury compounds. High levels of such products in the fish (9, 14, 32, 53, 120, 151, 186, 263, 394-396,
398) of industrialized regions, agricultural products (42, 243,
293, 360, 395, 397, 398) of the terrestrial food chain, as well 12
as severe accidents in which hundreds of people in developing
countries have died due to the consumption of cereals (24)
treated by methylmercury or to the consumption of animals (293)
which have consumed such cereals, have recently focused attention
on mercury fungicides. As a re s u lt, the agricultural usage of
alkylmercury compounds has been discouraged. Even though there is much less risk involved with alkoxyalkyl and arylmercury compounds,
their use is still under scrutiny.
Industrial Use of Mercury
The physical properties of mercury are uniquely suited to the demands of the electronic industry (193, 239). This element and/or its derivatives are used to manufacture fluorescent, germicidal, and high-intensity lamps; silent switches; and alkaline batteries.
Several commercial chemical processes also use mercury and its compounds on a large scale. The best known of these is in mercury- cell chlorine-alkali production (315). In this process, chlorine and caustic soda are produced by the following reactions:
Electrolysis NaCl * ClgHanode)
Electrolysis NaCl + NaFta (mercury cathode) SC
H20 NaH9* +• NaOH + #Hg + hHgj- 13
Mercury, as previously stated, is also employed as a catalyst in
several other industrial processes.
There are also other uses of mercurials such as in the amalgamation industry, pharmaceuticals th at produce diuretics
(50, 95, 123, 129, 132, 135, 152, 229, 231, 261, 262, 393) and antiseptics (109), the cosmetic industry, and in the manufacturing of paints.
Mercury in Medicine
In the closing years of the 15th century, descriptions of a new disease began to appear in the lite ra tu re of the day. The f ir s t mention of such a disease is found in an edict issued by the
Diet of Worms on October 7, 1495. In said document, this disease was referred to as "bosen Blattem" (the evil pox); today i t is known as syphylis. As early as 1497 mercury (379) was being advocated by at least two physicians, Johannes Widmann and Corradino
Gilino for syphilotherapy and in 1498 the firs t major book on syphilis was written by Francisco Lopez de Villalobos. In its text, he recognized not only the venereal mode of transmission, described the skin manifestations and later complications of the syndrome, but also deduced the idea of treatment with mercury from a study of the old Arabic literature.
Up to the early years of the 20th century, mercury (192) was the only effective drug available for the treatment of this scourge.
Given per os, in unction, parenterally or via inhalation, its toxic 14
effects on skin, mucous membranes and/or the intestines often out weighed its therapeutic value. In any case, its effects were
suppressive rather than curative.
In recent years, with the advent of modern chemotherapy,
this mode of therapy has been supplanted.
Historically, some of the therapeutic uses (109) of elemental mercury, its inorganic salts, and/or organomercurials are as follows: antiseptic ointments, cathartics, diuretics, germicides, spirochaeticides and in the preparation of dental amalgam. Except for topical use, dental amalgams, and as a disinfectant, mercury compounds are seldom, i f at a l l , employed in modern therapy.
Sources of Mercury in the Environment
Prior to the turn of the century (31, 257, 293, 363), mercury release into the environment was largely accounted for as the result of natural actions, viz. via', (a) the weathering of crustal rocks (7, 57, 105, 124, 143, 147, 188, 289, 345, 359, 372) and (b) vulcanism (400), and/or vaporized metal carried into the atmosphere
(7, 244) from deposits. The form in which mercury appears in rock is not entirely clear; however, it is probably reduced to the metallic form at magmatic temperatures, volatilized, and eventually combined with residual sulfur to form the sulfide, cinnabar (124).
In weathering reactions, these sulfides may be oxidized to the metal (Hg°) or to the soluble mercuric ion (Hg2+).
Natural surface waters (10, 76, 81, 96, 104, 110, 118, 119,
148, 150, 159, 174, 213, 241, 378, 391) contain tolerably small 15 concentrations of mercury except in areas draining mercury deposits.
In d u s tria l, a g ric u ltu ra l, s c ie n tific , and medical uses of mercury and mercury compounds introduce additional quantities of the metal into surface waters. Regardless of its source, the dissipation of mercury in solution appears to be through d ilu tio n , as well as by absorption and/or adsorption on suspended particulate matter and bottom sediments (8, 16, 43, 60, 68, 69, 178, 208, 211, 212, 216,
237, 273, 278, 314, 363, 364).
Trace metals exist in a myriad of different forms within the sediment-water system. Part may stay in the interstitial water as free (or complexed) ions, adsorbed on solids, or attached to surficial ligands on particulates. Some may incorporate with in soluble organic or inorganic matters of authigenic solids. Others may imbed in the crystalline matrix of the sediment. The current awareness of mercury as a pollutant of aquatic systems has aroused much interest in the exchange of metallic moieties between the sediment-water interface. This so called benthic boundary layer is considered to be the site of important interactions which greatly affect the behavior of trace metals in the overlying water.
Various mechanisms of metal m obilization have been proposed, for example, diffusion (292), desorption (305), dissolution (49,
163), redox reaction (347), complex formation (69, 101, 234, 264,
305, 325), biological effects (70, 164), or physical disturbance.
Although lim ited to areas with a winter snow problem, a unique form of mercury desorption, as a result of s a lt pollution, 16
1s being encountered. With the increased usage of sodium chloride—calcium chloride as a highway deicing agent, excessive amounts of chloride (C l- ) ion are encountered in waterways. Due to its strong affinity for mercury, release from polluted bed sediments occurs. In addition to being a serious contaminant itself, road salt in natural waters can acerbate contamination by mercury and undoubtedly by other toxic heavy metals.
With the advent of civilization, human activities have had a profound impact on the release of mercury and/or its compounds into the environment. For centuries, man's contribution was mainly limited to its release through the atmosphere (13, 173, 198) from fossil fuels (88, 184, 197), lig n ite and/or petroleum (22).
Mercury in the atmosphere ultim ately reaches the earth either by dry fallout or by precipitation where it is captured by the soil.
Rainfall-induced erosion and leaching return it, in part, to these same streams or other waters.
As industrialization developed, smelting processes for other metals, the ores of which contain mercury, added to our contribu tion. Gradually, as man developed sophisticated needs, numerous and varied uses for mercurials were found. While some are conservative of the metal, others allow leakage, and s till others deliberately introduce mercury compounds into the environment.
Among those promoting leakage, the use of the flow mercury cathode cell in the c h io r-a lk a li industry is the greatest offender (n). 17
Recognition that m etallic mercury through its oligodynamic
action could function as an insecticide (227) opened the door for the use of mercurials in the agriculture industry. Use of mercury compounds in the production of fungicides, which are employed as seed dressings, foliage sprays, and for garden-lawn applications, as well as slimicides in the pulp-paper industry
(46) are among the major deliberately introduced sources of this element in the environment.
Due to'their indiscriminate discharge into nature, man often finds himself bearing the brunt of his accidental exposure to mercury and/or its compounds (357). Although probably not major contributors to environmental pollution, effluents from hospitals, dental fa c ilitie s (276), chemical laboratories, and homes should not be ignored.
Several of mankind's most heinous encounters with mercury poisoning have been as a direct result of the interjection of anthropogenic mercury into his food chain. The firs t indication that methylmercury may present a threat to public health came from the epidemics of Minamata Bay (168-170, 219, 353-356) on the
Shiranuhi Sea and Niigata (165, 374, 375), Japan. In both instances the etiology was traced to the release of methylmercury compounds and/or their precursors from plastic industries in which inorganic mercury compounds were used as catalysts. In these instances, the transfer of mercury from the factory effluent to those a fflic te d was mediated by edible aquatic animals. Consumption 18
of pork products (155, 293) from hogs fed on treated seed wheat
and homemade bread (24) prepared from similar grain (i.e.-treated
with methyl- or ethylmercury fungicides) were responsible for out
breaks in Iraq , Guatemala, Pakistan and New Mexico. Since these
tragic occurrences of mercury poisoning, considerable attention
has been directed towards an elim ination and understanding of the
occurrence of these complexes in the environment.
Almost simultaneously, the Swedes (31, 185, 243) demonstrated
that mercury to x ic ity was the cause of widespread m ortality f ir s t
in seed-eating birds, followed by the predators. The appearance
of this phenomenon correlates closely with the introduction of
mercurial fungicides, such as methylmercury dicyandiamide, as
seed dressings in their agrarian programs. Decisive action did
not follow immediately. The event that finally triggered Sweden's
sweeping assault on mercury pollution was the impounding of a
carload of imported Swedish eggs by the health authorities in
Vienna. Following the appearance of mercury in eggs and other
Swedish foodstuffs (32, 42, 186, 243, 360, 394-398), Sweden
embarked on one of the most far reaching and effective environmental
cleanup programs to date.
More recently, detection of abnormally high concentrations of mercury in fish caught in Lake St. C la ir and Lake Erie brought the
problem of mercury contamination of natural water systems to public
attention in North America (11, 23, 41, 74, 92, 134, 248, 321, 376,
401). In retrospect, i t appears that the North American continent
is no exception to trends seen elsewhere in the world—considerable
amounts of mercury and/or its compounds have also been released
into the environment, and of that, most has found its way into
natural water systems. This has been documented in the United
States (12, 64, 84, 230, 407) and is undoubtedly true in other countri es.
Mercury Transformation in the Biosphere (Figure 1)
Generally speaking, one finds mercury being discharged into nature in one of the following forms: (a) as m etallic mercury,
Hg°; (b) as inorganic divalent mercury, Hg2+; (a) as phenylmercury,
C6H5Hg+; (d) as methylmercury, CH 3 Hg+; (e) as ethylmercury, C 2 H5 Hg+ or (f) as methoxyethylmercury, CH 3 0 CH2 CH2 Hg+.
To understand the ecological effects on the d iffere n t kinds of discharges and the risk factors involved, the transforming reactions between the d iffe re n t compounds of mercury in nature are of central significance. The consequences of these transforming reactions are p a rtic u la rly obvious when i t concerns the deposits of mercury in the bed sediments of waterways, which can be mobilized through conversion to other, more biologically active forms. These deposits are prim arily made up of phenylmercury found in fib e r banks, downstream from pulp-paper m ills , and inorganic mercury, either metallic or divalent with its high affinity for organic mud. Figure 1. Mercury transformation in the biosphere. • Inorganic Compounds □ ■ Organic Compounds
i- _i ©
[>0 22
Conversion Between Inorganic Forms
Hg° Hg2+—------> HgS*
Hg2+— - > HgS+ (176)
I f sulfide ions (S^_) are present mercuric su lfid e, with an
extremely low aquatic solubility, is formed. Under anaerobic
conditions, such as those prevailing in bottom muds, mercuric
sulfide seems to be stable.
HgST— > Hg2* (99, 176)
Normally, under aerobic conditions, mercuric sulfide is slowly
• oxidized to sulfite (SC^") and/or sulfate (SO^- ). The conversion
of to divalent ions—complexed to other compounds--prepares
mercury for further conversion. Direct methylation of mercuric
sulfide has also been reported, however, at a much slower rate.
H g 2 + - — , Hgo (4} 5 is 205, 247, 369, 380, 414)
Reduction of divalent mercury may occur in nature under re
ducing conditions where the redox potential is in favor of this
transformation. Bacteria of the genus Pseudomonas have also been
shown as a method of detoxification, to carry out this conversion.
The extent to which this conversion occurs naturally, and its 23
significance for the turnover of mercury in the environment is not
known.
Hg° ► Hg2+ (162, 177)
Conversion of m etallic mercury to divalent mercury ions can and has been shown to occur under conditions present at the bottoms of lakes and rivers. This is an oxidation and, as such, dependent upon the redox-potential in the medium. The potential necessary for this reaction to occur can be calculated from the following formula:
E = 850 + 30 log a where the coefficient a is a measure of the binding strength of the complexes between divalent mercury and the available complexing agents. For mercury complexes with organic mud, the coefficient a 21 has been calculated to be > 10 . This means that oxidation of m etallic mercury to inorganic divalent mercury w ill take place in an aquatic environment [see Appendix A] i f organic substances and oxygen are present.
Conversions Between Organic and Inorganic Forms
CH30CH2CH2Hg+ ------* Hg2+ (176)
In an acid medium, a ll alkoxyalkylmercury compounds decompose in a manner sim ilar to that seen with methoxyethylmercury below: 24
CH30CH2CH2Hg+ + H+ * CH30H + CH2 = CH2f + Hg2+.
CHoHg , C,H,-Hg , C6H5Hg+ *• Hg2+ and/or Hg° (40, 100, 114, — ------— ------115, 191, 339- 342, 366, 367, 369, 404, 414)
A ll known organometallic compounds can be degraded into inor ganic mercury either biologically, chemically and/or physically
(by UV light). The stability of the organomercurials, however, is very different. In general, it can be said that the short-chain alkylmercury compounds are the most stable, and that stability increases as the carbon chain decreases.
Evidence suggests that microbial decomposition of organomer- p. p+ curials involves cleavage of the C-Hg bond, reduction of Hg to
Hg°, and liberation of the corresponding alkanes (114). For example, a member of the genus Pseudomonas isolated from the soil and grown in a medium containing phenylmercuric acetate (PMA) appeared to bind PMA to the cell surface prior to being reduced to metallic mercury (368). It was shown that a reduced nicotinamide adenine dinucleotide (NADH) generating system and a sulfhydryl com pound were required to form Hg°. Thus, the common in tra c e llu la r reductant NADH may be responsible fo r mercury metabolism in micro organisms. Sim ilarly, C2H 5 Hg+, C5 H5 Hg+, and CH3Hg+ were degraded to Hg° and ethane, benzene, and methane, respectively (114).
Relatedly, Spangler et al (340) isolated 207 bacterial cultures from fish and sediments taken from Lake St. C la ir. Thirty 25 cultures were capable of aerobic demethyl ation with twenty-two and twenty-one of the above thirty being facultative anaerobes and anaerobes, respectively. These authors further confirmed that the degradation of methylmercury was a reductive demethyla- tion reaction resulting in the formation of methane and inorganic
(Hg° or Hg2+) mercury.
Hg° and/or Hg2+ ------► CH3Hg+ and/or CHoHgCHo (99, 139, 181, ------“ 183, 225, 304, 339, 383)
The idea that mercury could be biologically methylated was f ir s t proposed by Japanese engineers to explain "Minamata Disease".
However, during the investigation, they found that it was possible to conclude the formation of methylmercury had occurred within the factory. Subsequently, Swedish researchers gave new impetus to the study of the diagenetic process of biological methylation. In the bottom sediments from aquaria and/or natural bodies of water, the net result of the process could be mono- or dimethylmercury, and the rate of biological methylation of mercury was found to be well correlated with general microbiological activity in the sediment.
The mechanism of methylation was later the subject for many studies. Among the earlier reports was that of Woods and his co-workers (409) who showed nonenzymatic methylation of mercury by c e ll-fre e extracts of a methanogenic bacterium. Methylcobalamin served as the donor of methyl groups in the presence of ATP and a mild reductant. Several years later it was found that mercury 26
could also be methylated in a neutral water solution by a purely abiotic reaction (38, 167). Again the methyl donor was methyl- cobalamin; the reaction progressed both aerobically and anaerobical ly . Another rather unique mechanism of in vivo methylation was studied in aerobic cultures of Neurospora arassa (224). Through a series of experiments, the methylation of mercury was termed an
"incorrect" synthesis of methionine. A more complete picture of the mechanisms involved in the methylation of mercury under both aerobic and anaerobic conditions has been provided by Wood et ai
(410) and Dunlap (91).
The a b ility to methyl ate mercury does not seem to be restricted to one or a few types of microorganisms, but seems to be a wide spread process. Thus the mercury methylation a c tiv ity in the environment is enhanced by the same conditions as the general microbiological activity. This also means that the highest methyla- tion rate in the aquatic environment occurs in the uppermost part of the organic sediment and on suspended organic particulates. In natural waters, when oxygen is exhausted, hydrogen sulfide starts to be formed and the divalent mercury is bound as mercuric sulfide.
In this form mercury is generally not available for methylation under anaerobic conditions, and even under aerobic conditions the methylation rate is low. Investigations (409) have shown that Hg2+, whether discharged in itially in this state or chemically oxidized from m etallic mercury, is in fact methylated in waters and natural 27
sediments by bacteria under anaerobic conditions, be it enzymatically
as with the methanogenic bacteria or non-enzymatic via the transfer
of methyl groups from Co3+ to Hg2+ in biological systems. Fagerstrom and Jernelov (99) reported that methylation also occurred in the top
layer of sediments i f they were continuously oxygenated. Further more, a ll microorganisms capable of synthesizing alkyl B-12 type compounds are capable of CH 3 Hg+ synthesis (167, 407). From experi mental data i t appears that a ll forms of mercury may be converted directly or indirectly to either mono- or dimethylmercury. An alkaline pH favors a higher proportion of CHgHgC^ as related to
CH3 Hg+ because the former is rapidly degraded to the la tte r in an acidic environment. Additionally, it has been found (225) that mercury methylation rates are influenced by a number of environ mental and biological parameters, such as: (a) temperature; (b) pH; (a) organic concentration expressed as organic sediment index i.e ., the product of the percent organic carbon and organic nitrogen in a given sample; (d) microbial activity; and (e) con centration of available mercury.
In addition to the aforementioned, microorganisms residing in the intestine and in the slime on fish have been shown to methyl ate mercury; however, the fish itself does not seem to do so (282).
Conversions Between Organic Forms
C6H5H9+ ------*■ ch3h9+ <____ — CH3HgCH3 (177) 28
The conversion in bottom sediment of phenylmercury to mono-
and dimethylmercury has been studied and shown to occur. The
p o s s ib ility that the formation of both end products proceeds along more than one synthetic pathway exists.
C6H5Hg+ > C6H5HgC6H5 (250, 368)
Phenylmercuric acetate, an organomercurial that has been widely used as a fungicide and slimicide, was found to be metabolized quickly by soil and aquatic microorganisms. One of the major meta bolic products was id en tified to be diphenylmercury. Subsequently this reaction has been shown to be reversible.
CH3Hg+ ------► CH3HgCH3 (177)
Dimethylmercury has been shown to be formed from monomethyl - mercury in decomposing fish and from inorganic mercury in sediments.
Here the formation of dimethylmercury from inorganic mercury is regarded as a two-step process with monomethylmercury as an in te r mediate product.
CH3HgCH3 ------»■ CH3Hg+ (176)
At low pH (< 5.6) dimethylmercury breaks down to monomethyl mercury. Exposure of CH3HgCH3 to UV lig h t yields m etallic mercury with the monomethyl-derivative serving as an intermediate. 29
Other Conversions in Nature
(C2H5 ) 4 Pb + Hg2+ ------► (c2h5>3 Pb + C2H5Hg+
Transalkylation of divalent inorganic mercury with tetraethyl
lead, from gasoline additives, yields monoethylmercury as one of
its byproducts.
Interaction Between Mercury and Organisms
In the current literature numerous accounts of the inter actions between mercury and organisms at a ll levels are related; the most current of these citations are presented in tabular form
in Appendix B.
Certain general observations, however, can be made in relation to aquatic organisms. Microbial conversions of inorganic reserves to organomercury compounds concurrently demonstrate increased s o lu b ility in the overlying water, thus improving elemental m obility within the suspending matrix, in addition to increasing their solu b ility in the lipid components of biologically active membranes.
Whether the aquatic protists can extract methylated mercury com pounds from water in preference to the assimilation of inorganic compounds directly from the surrounding medium or not, they are able to concentrate (382) the mercury within themselves to levels considerably higher than those prevailing in th eir environment.
Further up the food chain, the mercury concentration in organisms 30
(214) increases either by absorption directly from the milieu, with
food sources from lower trophic levels (133), or by a combination
of both means. Should these organisms, by they macro- or micro- in
nature, remain in the same locale u n til th e ir demise, the mercury
in their cells and/or tissues can be returned to nature in several
ways, namely: (a) evaporation as mono- or dimethylmercury through
decomposition, (b) return to the sediment pool via reductive
demethylation, or (a) volatilization of methylated mercury compounds
in sediments and/or soils.
What, then, is the ultimate fate of the mercury contaminated
aquatic environment? Although a modicum of mercury is no doubt
removed from a given locale through vaporization and movement of macroflora and macrofauna, m obility of the biomass and colloidal
particulates, be it voluntary or not, is likely to remove more mercury. Thus, i f a mercury source is depleted, a body of water
theo retically could be expected to cleanse it s e lf of its mercury
burden. In fa c t, however, the likelihood of this occurring has recently been demonstrated to be low by experiments showing the rate with which microbial methylation in bottom sediments brings about the mobilization of bound mercury (177), the presence of a depot of readily available mercury within the sediment bed, and the replenishment of said depot by reductive demethylation. 31
Detection of Mercury and its Compounds
There are three methods commonly used for the determination of mercury in biological samples: CaJ colorimetric determination with dithizone (116), (b) neutron activation analysis (73, 82, 93, 102,
137, 255, 290, 311, 392), and (a) atomic absorption spectrophotometry
(3, 146, 202, 275). Numerous new techniques (18, 63, 79, 117, 144,
154, 160, 187, 220, 235, 236, 337, 338, 358, 371), many of which employ thin layer and gas chromatography, have been introduced in hopes of not only increasing sensitivity but also differentiating between organic and inorganic forms of mercury. Several papers (62,
75, 149, 228, 319, 370) recently have also pointed out sources for error in our standard methods.
Colorimetric analysis of mercury is the traditional method, and it is s till used occasionally today because of its low cost, simplicity and sensitivity (0.01 ppm). However, the use of this method has declined since the advent of atomic absorption spectro photometry.
Determination of trace amounts of mercury can also be accom plished by neutron activation analysis. The concentration of mercury in the sample is determined by irrad iatin g the samples and then detecting the gamma radiation from the resulting radioactive species with a Ge(Li) detector. The primary advantage of neutron activation analysis is its extreme s e n s itiv ity . As l i t t l e as 0.5 ppb of mercury can be measured. However, the disadvantages are that i t 32
1s a time consuming procedure, it is expensive, and it has to be
run by skilled and experienced personnel. Therefore, for the
present, its complexity and cost lim it its use.
By fa r, the most popular and common method of determining
trace amounts of mercury in biological samples is by employing
atomic absorption spectrophotometry. The concepts relating to atomic absorption are based on the premise that i f a solution containing a metallic species is aspirated into a flame, an atomic vapor of the metal w ill be formed. Some of these m etallic atoms w ill be excited to higher energy levels, but an overwhelmingly larger percentage w ill remain in the ground state. These ground- state atoms of a particular element will absorb light radiation, from a source other than the flame, in th e ir own specific resonance wavelength, i.e., the wavelength of light emitted when an atom returns from its lowest excited state to the ground state. Thus, i f lig h t of this wavelength is passed through a flame containing atoms of the element, part of that light will be absorbed and the absorption will be proportional to the density of the atoms in the flame.
When the flame is used to produce the atomic vapor this method is termed flame atomic absorption spectrophotometry.
Another procedure has been developed for mercury wherein the atomic vapor is produced from mercury in solution by reduction of
Hg^+ to Hg° followed by aeration of the solution. This does not 33
involve the use of a flame and is termed flame!ess or cold vapor atomic absorption spectrophotometry. In using this procedure, as l i t t l e as 0.2 ppb of mercury can be measured in water samples.
The detection portion of an analysis takes less than a minute to perform, but additional time is needed for sample prepara tio n . However, this is usually less time consuming than both the dithizone method and the neutron activation method. Atomic absorp tion spectrophotometry is more sensitive and also more expensive than the dithizone method. It is not as expensive as neutron activation analysis, nor are highly qu alified personnel needed to perform the analysis; but, neutron activation is more sensitive than atomic absorption spectrophotometry in the analysis of biological samples. At the present time, flameless atomic absorp tion spectrophotometry is the preferred method for total mercury analysis, and it is the method of choice for this study. MATERIALS AND METHODS
Sample Collection
Between September, 1967 and June, 1969, 5 gallon water samples were collected by boat at various stations in the western basin of
Lake E rie. Each sample was collected using a hand pump from midway between the water surface and the lake bottom (usually 15 to 20 feet in the western basin). All samples were stored in chemically cleaned glassware at 4°C until transportation, processing and/or testing could be accomplished. Many of the 360 samples analyzed were collected prior to and during the major mercury scare in the lake.
Sample Processing (Figure 2)
Upon reaching the laboratory, 100 ml aliquots of each sample were removed fo r analysis (see Appendix C_). The remainder of each
5 gallon sample was processed by continuous-flow high-speed centrifugation (Sorvall RC-2B equipped with a Szent-Gorgi continuous- flow attachment) at 4°C with a 45 ml per minute flow rate and a gravitational force of 27,000. This permitted the removal of particles down to 0.5 pm. The supernatant was then passed through
34 • Figure 2. Flow diagram of procedure u tiliz e d during
experimentation.
35 Centrifugation 27,000 x g - flow rate 45 ml/min. Pellet I Supernatant I |(D
Centrifugation 27,000 xg-flow rate llml/min. Sucrose Gradient Centrifugation 1,500 x g-l hour
1 Pellet II Supernatant n (j2)
Sucrose Gradient Centrifugation 1,500 x g -l hour
— @>
(# ) « Fraction Number 36
.Sample; *5 gallon' UJ
Centrifugation 27,000 x g - flow rate 45 ml/min. Pellet I Supernatant 1 |(D
Centrifugation 27,000 xg-flow rate llml/min. Sucrose Gradient Centrifugation 1,500 x g-l hour
Pellet H Supernatant II (*2) (D>- Sucrose Gradient Centrifugation 1,500 x g -l hour
— 0
(#) « Fraction Number the centrifuge at 27,000 x g with a flow rate of 11 ml/min to
affect the removal of colloids down to 0.1 pm. Solid residue
from these fractionations were subsequently placed on top of
gradients constructed of sucrose with a linear density of 1.0765
(19% wt/vol) to 1.2241 (49% wt/vol) and centrifuged at 1,500 x g
for 1 hour (221-223). Bands were collected by using a Beckman
tube-cutting device and the particulates contained therein were
either dialysed (utilizing Union Carbide dialysis tubing) against
or washed in "mercury-free diluent" (see below) by high-speed
centrifugation (27,000 x g for 30 min.).
Total Mercury Determination
Utilizing the technique of Hatch and Ott (146) in conjunction
with the apparatus described below (Figure 3) the total mercury
content of each fraction was determined by flame!ess or cold vapor
atomic absorption spectrophotometry. Aliquots of each specimen
under study were transferred to 250 ml round-bottomed flasks. To
the contents of each flask were added 25 ml 18 N sulfuric acid,
10 ml 7 N nitric acid, and enough "mercury-free diluent" to make
100 ml. Treating each reaction flask individually, 20 ml of a
sodium chloride-hydroxylamine sulfate solution (60 ml of a 25%, wt/vol, hydroxylamine sulfate and 50 ml of a 30%, wt/vol, sodium
chloride solution diluted to 500 ml with "mercury-free diluent")
followed by 10 ml of a 10% wt/vol stannous sulfate solution in Figure 3. Schematic of apparatus employed to measure total
mercury via the cold vapor atomic adsorption
spectrophotometric technique.
38 © Perkin Elmer 40 3 Atomic Absorption Spectrophotometer
Hollow Cathode Lamp Digital Readout
Absorption Cell
Air Intake
Exhaust Hood
Varistalic Drying Pump Agent Reaction Flask
CO 1 0 40
0.5 N sulfuric acid was added. Immediately the reaction vessel
was attached to the aeration apparatus forming a closed system.
The mercury vapor thus produced was analyzed fo r its absorption at o 2537 A in a quartz-windowed cell. Absorbance values displayed on
the digital readout were recorded for 4 min at 30 sec intervals.
These readings were averaged, reduced by that of the reagent
control and utilized for calculating the total mercury content of
a given sample by comparison with curves prepared from known
standards, e.g.: Figure 4.
"Mercury-Free Diluent11
All water utilized to make reagents as well as dilutions was
triple distilled, filtered via 0.45 ym membrane filtration (M in i
pore) and steam sterilized (121°C, 15 psi).
Aquarium (Model Lake)
In an attempt to study a regulated mercury spill in a con trolled environment, a model lake was created in a 20 gallon aquarium (Figures 5 -7).
General Layout (Figure 5)
Aeration
Laboratory a ir , a fte r passing through either the
fritted glass sparger or the filtration unit (packed
to a depth of 10 cm with Finny F ilte r Floss - Finney Figure 4. Calibration curve for a typical mercury standard, o absorbance at 2537 A.
41 M W at Absorbance
Total Mercury Ifiq) ZP Figure 5. Functional elements of the model lake.
43 ©
Air Pump
^•Affluent
Suction-— =6=;
® ©
©______Top View
4S. Figure 6 Cross-sectional diagram of stratified model lake,
bed sediments showing depth and composition of
each layer.
45 46
© Depth of Layer Composition of Layer
to 2 7 .0 cm Water Column
2 .0 cm Aquarium Pebbles
1.5 cm Aquarium Gravel
0 cm Sand
0 .5 cm Potting Soil 0 .5 cm Olentangy River Mud 0 .5 cm Potting Soil
1.0 cm Sand Figure 7. Illustration of aquarium floor plan.
47 Aquarium Floor Plon
8cm
77.5 cm
Site of Regulated Mercury Fish Feeding Station (Hg°) Spill □ Sampling Site for Sediment Cores 49
Product, Inc.; 602 Main Street; Cincinnati, Ohio
45202) was adjusted to yield a to ta l flow rate of
500 ml/min.
F iltra tio n
F ilte r Floss, moistened with "mercury-free diluent" was packed to a depth of 10 cm and replaced every 7 days.
F ilte r entrapped detritus was dislodged from expended
floss by gentle washing with "mercury-free diluent."
Materials thusly collected were dried at 50°C for 24
hours, weighed, resuspended by vortex action in 65.0 ml of the same diluent and analyzed fo r to ta l mercury content.
Illumination
Light was provided by means of a plant stimulating fluorescent bulb (Sylvania-Enhance) mounted in the aquarium cover. The distance from the bulb surface to the water interface was 6 cm. At weekly intervals, the lamp surface and both sides of its portal were cleaned with commercial window cleaner (Windex) to remove accumulated film s.
Temperature
The aquarium and its contents were allowed to equilibrate to ambient laboratory temperatures and 50
kept within that range, i.e ., fluctuating between
20°C and 25°C.
Water
A ll water u tilize d within the model was double
distilled, filtered (via 0.45 ym Mi H i pore) to remove
suspended particulates, and autoclaved (121°C, 15 psi)
to eliminate unwanted protists. The affluent to effluent
flow rate (Figure 5) was adjusted to 2.0 ml/min.
Sediment Bed (Figure 6)
The sediment bed of the model lake was constructed in the following manner, beginning at the bottom and progressing to the topmost, layers of sand (1.0 cm), potting soil (0.5 cm), Olentangy
River mud (0.5 cm), potting soil (0.5 cm), sand (1.0 cm), Aquarium
Gravel (1.5 cm), and Aquarium Pebbles (2.0 cm) were s tra tifie d .
Aquarium Gravel
Pure natural white Aquarium Gravel (Noah's Ark
Pet Center; 1603 West Lane Avenue, Columbus, Ohio
43221) with an average diameter of 2 mm was u tiliz e d .
Prior to usage, the gravel was washed three times in
"mercury-free diluent" and dried at 80°C. Aquarium Pebbles
Following a triple rinse with "mercury-free diluent" and drying at 80°C, Black Decorative Aquarium Pebbles
(Melody Brand Products; Maud, Ohio) having a mean diameter of 0.5 cm were employed as the top layer.
Olentangy River Mud
Mud collected immediately after the firs t Spring thaw (April 5, 1976) was obtained along the bank of the Olentangy River approximately 200 yards south of the Drake Union (The Ohio State University - Columbus
Campus). This layer served as our inoculum in that it contained in addition to the mercury methylating microbes found in most sediments (177); water mites of the genus T y rrellia\ several genera of gastropods, i . e . , Campeloma and Helisoma', and copious amounts of oligochaete worms, viz., Tubifex spp.
Potting Soil
Stim-U-Plant Potting Soil (Stim-U-Plant Labora tories, Inc.; Columbus, Ohio 43216) was employed throughout this study. 52
Sand
Pure s ilic a sand, 20-30 mesh (850-600 ym) was
thrice washed in "mercury-free diluent" and dried
at 80°C for use.
Ecosystem
One week following the establishment of an equilibrium in
the aquarium, 36 goldfish (Carrassius auratus) averaging 4 g in
weight, were introduced into the ecosystem. Fish were fed Long-
liv e Shrimp-el-etts Pelleted Fish Food (The Hartz Mountain Corp.;
Harrison, New Jersey 02029) daily (1 pellet/fish) at the feeding
station (Figure 7). The ecosystem was then allowed to re
equilibrate for a one-month period.
Regulated Mercury S pill
Following the removal of base lin e sediment cores, 1 gram of metallic mercury (Hg°) was introduced, at the appropriate site
(Figure 7), into the mud layer via a pyrex standpipe. Said glass
tube was gently removed by a twisting action to re-stratify the
bed sediment.
Sampling
Attached Planktonic Biomass
At 7 day intervals, gelatinous materials attached
to the inner glass surface of the aquarium were removed using a single-edge razor blade (Gem). After drying
at 50°C for 24 hours and being weighed, specimens were
resuspended using a Vortex Mixer in 65 ml of "mercury-
free diluent" and analyzed for total mercury content.
F ilte r Entrapped Detritus
See previous section entitled "Filtration."
Goldfish
At the requisite time intervals, individual fish
were sacrificed by placing them in liquid nitrogen
(-196°C), dried at 50°C for 48 hours, weighed and
suspended in 65 ml of "mercury-free diluent." Total mercury content was determined following digestion of
the entire specimen with 25 ml 18 N sulfuric acid and
10 ml 7 N nitric acid. A 48 hour digestion at ambient
temperature was employed.
Sediment Cores
U tilizin g a truncated 25 ml p ip ette, sediment
cores were taken weekly from pre-selected sites
(Figure 7). Following drying at 50°C for 24 hours,
samples were mechanically pulverized, weighed, and analyzed for total mercury content utilizing the
technique designed by Hatch and Ott (146) for rock
samples. 54
Snails
Gastropods were processed in the same fashion as
goldfish (see previous section), with one exception;
the digestion period at ambient temperatures was
shortened to 24 hours.
Water
S ixty-five ml aliquots were processed in a manner
identical to those samples removed from the western
basin of Lake Erie.
Volume Measurements
Water suspended particulates were measured following vortex mixing by placing 10.0 ml in a 12 ml graduated (in 0.1 ml sub
divisions) conical centrifuge tube and centrifuging (Sorvall GLC-1)
in a swinging-bucket head at 1000 rpm for 10 min. When measurements were completed, the sediment was resuspended and the en tire 10.0 ml specimen was diluted to a fin al volume of 65 ml with "mercury-free diluent" and analyzed for total mercury content.
Weight Measurements
Removal of suspended particulates was accomplished by c e n tri fugation at 1000 rpm for 10 min. Following drying at 50°C for 24 hours each specimen was weighed. Aliquots were resuspended in 65 ml of "mercury-free diluent" and analyzed for mercury.- 55
Weight vs. Volume Measurements
Volume Measurements were made according to the aforementioned protocol; upon completion each sample was resuspended, dried at
50°C for 24 hours, and weighed.
Scanning Electron Microscopy
Solid Specimens
Colonies of bacteria isolated from the model lake
were cultivated on Trypticase Soy Agar (BioQuest,
Cockeysville, Maryland 21030) fo r 24 hours at 25°C,
fixed for 1 hour with 6% (vol/vol) glutaraldehyde, and
excised with a cork borer. Each cylinder so obtained
was dehydrated by passage through a series of graded
ethyl alcohol solutions (30%, 50%, 70%, 80%, 90%, 95%,
100% for 10 minutes each), placed in 100% amyl acetate
as an intermediate fluid (i.e . - one that is miscible with
both water and the transitional fluid [CO^]), and critical
point dried (Samdri; PVT-3 C ritic a l Point Drying Apparatus;
Biodynamics Research Corp., Rockville, Maryland 20852).
Dry samples were attached to aluminum "pin-type" specimen
mounts (Structure Probe, Inc.; West Chester, Pennsylvania
19380) with conducting paint and stored in a dessicator
un til sputter coated in a Hummer I I I DC Sputter Coater o (Technics; Alexander, Virginia 22310) with 200 A of gold. 56
Each specimen was examined in a Hitachi S-500
Scanning Electron Microscope (H itachi, Ltd.; Tokyo,
Japan) operating at 20 KV, and specimen images were
recorded on Poloroid Land Film Type 55/Positive-
Negative. Micrographs were made from the original
negatives directly on Ektamatic SCF paper.
Liquid Specimens
Two m illilite r s of the liquid specimens suspended
in 6% glutaraldehyde fixative and two 3.05 mm diameter
punched copper grids (0 Mesh) were placed in premoistened
Union Carbide dialysis tubing. Upon completion of dehy
dration and critical point drying (see above) the tubing
was Cut, grids with adhering specimens removed, and the
la tte r mounted with conducting paint on aluminum "pin-
type" specimen stubs. At this point, liquid specimens
were treated in a manner identical to solid ones.
X-Ray Microanalysis
Liquid specimens consisting of either model lake samples or
Trypticase Soy Broth (BioQuest; Cockeysville, Maryland 21030)
cultures of isolates from said aquarium were washed three times
in "mercury-free diluent" to remove unwanted elements from the
suspending matrix. Following the final centrifugation at 15,000 g, a heavy suspension of each specimen was made in "mercury-free 57
diluent" and 0.5 ml was placed on pure carbon "pin-type" specimen mounts (Structure Probe, Inc.; W estchester, Pennsylvania 19380). o Each specimen was dried in vaeuo, sputter coated with 50 A of
carbon and analyzed using x-ray excitation with an energy dispersive
spectrometer. The electron probe microanalysis was conducted in a
Hitachi S-500 Scanning Electron Microscope with an ORTEC 6230 S i(L i)
X-ray Detector and a Multi-Channel (1024) Analyzer (Oak Ridge,
Tennessee 37830) under the auspices of Dr. Thomas N. Taylor, De partment of Botany, The Ohio State University.
In order to determine the fate of soluble mercury compounds found in the model lake's water column or their effect on microbial isolates, the latter were grown in Trypticase Soy Broth (25°C, 24 hrs) made with aquarium water and filte r sterilized (0.45 ym;
M illip o re ). Sample processing and examination were as previously mentioned. RESULTS
In it ia lly the work conducted for this project was concerned with the presence of mercury in the western basin of Lake Erie
(Table 1). Previous work has demonstrated: that mercury has been deposited into this area of the lake; that the aqueous environment of this region contains a large number of particulates (232, 287,
288), both inorganic and organic; and that there are microorganisms capable of methylating the mercury pool present in the sediment.
However, l i t t l e information was available concerning which, i f any, of the micro-components of the water column play a sig n ifican t role in mercury translocation. Utilizing the protocol delineated in
Figure 2 in conjunction with standard curves, mercury levels were determined for water samples and component fractions from pre determined sites in the western basin of Lake Erie (Tables 2 and 3).
The results of these analyses indicated that mercury is: (a) present in varying amounts and locales of the western basin, (b) consistently present in higher measurable levels in areas of the lake away from the stronger lake currents, i.e., in bays and/or harbors, (a) readily detectable for long periods of time, (d) particle associated, and
Ce) present in amounts directly related to particle density.
58 Table 1. Sample collection information.
59 60
TABLE 1
Sample Date Number Location* Collected
16 B 9-09-67
39 A 6-10-68
42 B 6-17-68
43 B 6-21-68
51 B 7-02-68
69 B 8-02-68
91 A 8-26-68
105 D 10-15-68
106 A 10-22-68
107 C 10-29-68
115 B 5-16-69
122 B 6-26-69
A = Rattlesnake Island Area
B = Middle Island Area
C = Put-In-Bay and G ibraltar Island Harbor Area
D = Sandusky Bay Area Table 2. Mercury determination of fractional components - I.
61 TABLE 2
Sample Number
39______91______106______105 Total Mercury in Parts Per B illio n
X 34 51 63 564
2 22 33 42 372
3 10 16 19 181
4 1 1 1 12
5 1 2 2 16
6 1 2 2 19
7 1 2 2 23
8 2 3 3 37
9 2 4 4 46
10 4 7 8 79
11 9 12 19 131
12 8 14 16 167
13 1 0 2 11
14 0 1 1 6
15 0 1 1 9
16 0 1 1 13
17 0 1 1 14
18 1 1 2 17
19 1 1 2 20
20 2 2 3 35
21 3 5 5 51 Table 3. Mercury determination of fractional components - I I .
63 64
TABLE 3
______Sample Number ______16 42 43 51 69 115 122 Total Mercury in Parts Per B illio n
1 56 139 200 260 383 398 452
2 42 98 172 182 290 347 388
3 11 39 21 74 91 41 57
4 1 3 3 3 15 16 15
5 2 4 3 5 18 17 16
6 2 7 6 6 20 19 18
7 4 9 14 8 22 21 26
8 4 11 19 11 32 31 33
9 5 14 24 17 36 36 41
10 8 19 39 49 49 73 84
11 15 31 58 81 97 127 147
12 8 28 13 63 79 32 46
13 2 9 5 9 11 4 10
14 0 1 0 3 0 0 1
15 0 1 0 4 3 0 1
16 0 2 0 5 4 1 1
17 0 2 0 6 7 1 1
18 1 3 1 6 9 2 2
19 1 4 2 8 12 3 4
20 3 7 3 13 17 6 8
21 3 8 7 17 26 19 27 65
In an attempt to observe the kinetics of mercury transloca
tion throughout the bed sediment and its overlying water column as well as to study the entry of mercury into the food chain and its concentration via movement from lower to higher trophic levels, a laboratory model (Figures 5 and 7) of a lake was developed. After the bottom sediment was stratified (Figure 6), the flo ra and fauna added, and lake currents simulated, a 1 month period was allowed for an equilibrium to be established in the ecosystem. Once baseline data was obtained for a ll model components
(Table 4) a regulated mercury (Hg°) spill was introduced into the test system. U tiliz in g the techniques previously described, total mercury levels of the various components were monitored over a 10- month period.
As can be seen from the data in Figure 8, mercury is f ir s t detectable in sediment cores from Site #1 (Figure 7) 2 weeks post-introduction and shortly thereafter (+4 weeks) at the other
Sites. Equilibrium is reached throughout the entire sediment a fte r 13 weeks. Upon closer examination, i t appeared that mercury moved outward from its in itia l s ite in an in fin ite series of concentric circles and as the distance from said source increased, the time required for mercury to reach any subsequent sampling
Sites (Figure 7) decreased.
A fter traversing the entire sediment bed, mercury next appears in the overlying water column (Figure 9) and eventually reaches an equilibrium — 0.6 of that found in the sediment. Several weeks Table 4. Mercury level of model components.
66 TABLE 4
Total Mercury Sampl e in yg per gram
Aquarium Gravel 0.025
Aquarium Pebbles 0.041
Fish Food Pellets 0.005
Olentangy River Mud 0.013
Potting Soil 0.005
Sand 0.008
Test Sediment Core 0.027
Tubifex spp. 0.016
Water 0.000 Figure 8. Kinetics of mercury translocation through model
lake bed sediments.
68 fJLg, Hg 0.02 04 .0 0 6 .0 0 0.10 08 .0 0 0.12 0.14 Weeks ie*4 * Site ie*3 * Site 2 Site# I site
20 VO cn Figure 9. P article mediated mercury m obility in the water
column.
70 fJLg /g , Hg 04 .0 0 8 .0 0 06 .0 0 002 IO . O 0* ak Water Tank itr Entrapped Filter tahd Planktonic Attached Biomass Detritus
Weeks JL-JI © 20 72 following the detection of mercury in tank water, we in itially detected the accumulation of a planktonic biomass attached to glass surfaces. Subsequent examination of this material showed that i t contained mercury (Figure 9) and its mode of accumulation suggested that i t was derived from the surrounding water matrix.
Approximately 4 weeks after the firs t appearance of an attached film on glass surfaces, both phytoplankton and zooplankton (con taining mercury) were visually detected in tank waters. Mechanical concentration of said suspended particulates occurred via f i l t r a tion (Figure 9).
The gastropod components of our ecosystem thrived and produced numerous offspring throughout the time span of our experiment.
After an in itial lag period, both species concentrated mercury 1000 fold in relatio n to th e ir surrounding milieu (Figure 10). Mercury accumulation in both species closely corroborates what is known concerning th e ir feeding habits (C.B. Stein, Personal Communication), viz.'. mercury appears in Helisoma tvivolvis only after it appears in the periphyton film attached to glass, known to be a source of nutriment and in Campeloma deeisa subsequent to the appearance of a floe of decomposing organic matter deposited between the 2 2 nc* and
2 4 th Week on the underlying rocks. The latter snail is character is tic a lly found burrowing through soft mud and feeds on decomposing organic material present in or on it.
A similar case can be made for the model's ichthyic constituents
(Figure 11); once mercury accumulation and concentration (1000 x) is Figure 10. Mercury accumulation in model lake gastropods.
73 /X g /g , Hg 140 100 120 0 8 0 6 0 4 20 2 6 8 2 4 2 0 2 16 12 8 4 -«=»■- oplm decisa Compelomo eioa trivolvis Helisoma
Weeks 623 4 0 4 -p* Figure 11. Mercury accumulation in the model lake fish
(Carrassius auratus) population.
75 Carassius auratus
28 32 36 40
CT»•x i 77
in itia te d i t proceeds at a rate and to levels unaccountable for
by externally provided foodstuffs (Table 4).
As mentioned previously, data obtained from the fractionated
water samples collected in situ clearly suggested (Tables 2 and 3)
that the mercury load of a given sample is present in amounts
directly related to particle density. To test this hypothesis,
suspended elements (mostly organic plankton) of the model's eco
system were scrutinized. A linear relationship between both
packed biomass volume (Figure 12) and dry particulate weight
(Figure 13) in relatio n to total mercury content was observed.
Finally by plotting the volume of suspended particulates against
th e ir dry weight (Figure 14) we note that the mercury load
carried, either actively or passively, via organic particles is
surface associated.
Further evidence to corroborate this hypothesis was gleaned
from energy dispersive x-ray analysis in the scanning electron microscope (313) of model lake particulates, in situ, and micro
bial isolates from therein [ see Appendix d\. In sampling d irectly
from the aquarium, q u alitative detection of mercury was seen with
some particulates, such as a filamentous alga, but not with
others, e.g. the fusiform diatom. Close scrutiny of electron micrographs revealed the presence of an epiphytic film of bacteria
on the mercury containing alga (Figure 15).
As microorganisms are known to play an important role in the
transformation and mobilization of mercury, the epiphytic bacterium Figure 12. The effect of particle volume on its associated
mercury content.
78 0.14
0.12
0.10
0.08 o> I
J 0.06
0.04
0.02
O' Figure 13. The effect of particle weight on its associated
mercury content.
80 0.14
0.12
0.10
9 0.08
=*. 0.06
0.04
002
0.2 0.4 0.6 0.8 2.0
00 Figure 14 P article volume versus particle weight for a given
mercury burden.
82 83
in
in
•n-r cvj E
c m
in
m
CM CM Figure 15. Scanning electron micrograph of filamentous alga
(FA) with an "Auswuohs" of epiphytic bacteria (B).
84 10 MM 86 was isolated, id e n tifie d as a pseudomonad (Figure 16A), and sub sequently shown to contain mercury [ see Appendix d]. Other bacteria, including a Gram positive staphylococcus (Figure 16B)
[Appendix z5], have been isolated from the model lake environment and also demonstrate the a b ility to retain mercury. Figure 16A. Scanning electron micrograph of a pseudomonad
isolated from the model lake benthos.
Figure 16B. Scanning electron micrograph of a staphylococcus
isolated from the model lake benthos.
87 5 MM DISCUSSION
From the experiments evaluating the role played by particu
lates in lacustrine mercury movement (Tables 1, 2, and 3), one
read ily notes that the mercury content of the western basin varies
with the location from which the specimen was taken as well as
the date. As to be expected, areas of the lake with diminished water flow, viz.'. the bays and/or the harbors have a tendency to
show elevated levels of mercury, whether this is due to entrapment of locally solubilized deposits, accumulation from external
sources, or by means of comparison, depletion of the mercury borne particulates in less quiescent areas by rapid surface movement is not discernable. Data contained herein is in concert with the observations of Kovacik and Walters (211) who showed, through th e ir work with sediment cores taken in the western basin, that Rattlesnake
Island lie s w ithin an area showing only background values of mercury whereas Middle Island is in a province rich in surface mercury pollution. The la tte r is due, no doubt in part, to contaminated waters from Lake St. C la ir entering via the Detroit River and traveling long-shoreward along the northern most or Canadian shore.
Through differential centrifugation it was shown that the majority of the mercury burden of a given water column lies within
8 9 the organic component (planktonic elements > 0.5 pm) while the
remaining amounts are associated with colloidal inorganics {e.g.
clay, 0.5-0.1 pm). No matter in which of the two distinct com
ponents i t is found, mercury has unequivocally been shown to be
associated with suspended particulates; the amount on any one
given particle being directly correlated to its density. The mechanisms involved in this so called "particle adsorption" can
in part be explained by the affinity of mercury for the sulfhydryl
group which can bind i t to suspended organic matter, both liv in g ,
like plankton, or non-living, like peat and humus. No doubt, the
affinity for lipids of elemental mercury dissolved in water and
the predilection of mono- and dimethylmercury for these very same membrane components, relative to their solubility in water, fa c ili
tates th e ir adsorption by aquatic organisms. Other than Krauskopf' observations (213) that microcrystalline iron oxides and mont- morillonite clay absorbed mercuric ion from water, little is known concerning the adsorption of mercury on inorganic substrates, their ion-exchange properties, or differential adsorption for the numerous inorganic species in solution and/or suspension. The probability, however, that microbes may colonize particulates - be they inorganic (71, 252-254, 403, 405, 406, 416) or organic (48) - or that through microbial metabolism a zoogloeal mass encases inorganic particles converting them to "pseudo-organic particulates should not be discounted. One example of such a relationship is 91 found herein (Figure 15), viz., that which the limnologist terms as an "Auswuohs" - the development of epiphytes on higher plants.
There are, unfortunately, few experimental analyses of the process
involved in attachment (44, 251); the well-defined chemical composition of resins, however, provides a reasonable model system for the study of such surface phenomenon (1, 344, 417).
Regardless of whether mercury is transported actively or passively (140), the initial contact with such an intermediary particle is surface-associated (280, 368); this is analogous to mechanisms reported for other ions (226, 310). X-ray analysis, with the beam voltages employed and their resultant information depth, presented herein [ see Appendix d\ confirm these observations.
Baseline mercury determinations (Table 4) of model lake components revealed our choice of inoculum, Olentangy River mud, to be low—0.013 yg/g—in mercury content. The validity of this information was confirmed by ascertaining the level of mercury accumulation in Tubifex species (Table 4) from their environment.
Concentration was shown to be by a factor of 1.23, well within the range (1.20±0.26) described by Jernelov (177).
After the introduction of mercury into the equilibrated ecosystem, one f ir s t detects the appearance of mercury migrating through the bed sediments (Figure 8 ). The initial time lag seen between the metal's introduction and its detection can be accounted fo r i f one considers the possible mechanisms involved in benthonic mediated mercury translocation. A priori evidence 92 suggests that a major factor in such mechanisms is the d iffe re n tia l s o lu b ility exhibited by the various mercury compounds. Although absorption by organisms may be facilitated by the affinity for lipids of elemental mercury dissolved in water, as already noted, it is not likely that this is an important factor since mercury occurs predominately in the mercuric form in oxygenated water where aquatic organisms must liv e . On the other hand, however, methylmercury compounds are more soluble in lipids than are mercuric ion or m etallic mercury in solution; they are also about
100 times more soluble in lipid s than in water (166). This allows methylmercury compounds to penetrate more readily than the inorganic forms of mercury into c e lls , and as a consequence increases the mobility. The key to this theory resides in the conversion of metallic (Hg°) mercury to methylated derivatives and its associated increase in solubility. By combining known facts concerning mercury methyl ation with the de facto data contained herein, we theorize that the following sequence of events (Figure 17) has occurred:
A. Upon exposure to the m etallic mercury, microorganisms
from the heterogenous population of the benthos are
selected that are tolerant of both inorganic and
organic mercury compounds, and capable of producing
CHgHg+ from eith er Hg° or Hg^+ . Figure 17. Arhythmic feedback cycle for mercury conversion
proposed for the model employed herein.
93 Gastropods
Plankton
Methylation
|2 +
,2+1
( h^s)
lO -P* 95
B. An increase in numbers of the previously selected
microbes occurred with the concurrent establishment
of the necessary enzymatic machinery to bring about
methylation.
C. Microbially mediated methylation of the inorganic
mercury in solution occurred in the top layer of
the continuously oxygenated sediment (99).
D. Biologically initiated autocatalytic mobilization
of methylated mercury occurs from the rapidly
advancing front of multiplying microbes.
E. In the lower layers of the sediment bed and/or areas
where microbial metabolism has depleted the oxygen
supply, microorganisms are selected (40) that while
being r e tr a c tile to both forms of mercury are n + capable of producing Hgu from either CHgHg or CH^HgCH^.
F. The increase in numbers of such microbial populations
(step E) is paralleled with a like increase in their
metabolic processes.
G. Reductive demethyl ation of methylmercury to methane
and inorganic mercury was promoted by the myriad of
bacteria thus stimulated (114, 340, 341, 367). i By coupling the concomitant methylation of inorganic mercury
and demethylation of methylmercury with the selection and growth
(or m otility) of specialized microbial populations, one can 96 readily visualize the cascade of events necessary to in itia te and bring about the translocation of mercury. Biocenological reactions such as the one proposed herein are fa r from unique in nature; in fact, due to the innate disposition of this particular pratityasamutpada, it can most accurately be termed an arhythmic feedback cycle (85, 388). Delays encountered between mercury's introduction into a test system such as ours and its detection, are most likely due to either of the following mechanisms, or a combination thereof: (a) sociological adaptation (413) i.e., a shift in species composition or species abundance in a community in relatio n to the environmental conditions; the process of adapta tion ending in the adapted or climax association which is in equilibrium with a certain combination of ecological factors actually dominating the environment for a s u fficien t period of time and/or (b) transfer of the extrachromosomal plasmid (141,
256, 268, 270, 327, 389) mediating metal resistance among the heterogeneous population via cotransduction in Gram positive staphylococci (206, 207, 258, 269, 271, 302, 349) and/or conjugation in the Gram negative bacilli (203, 204, 238, 334, 349,
350).
Alteration of microbial populations via the transfer of plasmids fo r mercury resistance can now be shown to have an environmental impact as well as possible commercial value (317, 385, 386).
Following the accidental spillage of crude o il into a mercury contaminated environment, partitioning of mercury occurs in the 97 overlying layer. Even though concentration of mercury in the oil
is often 4,000 times higher than in the sediment and 300,000 times higher than the water milieu, mercury-resistant populations are
known to degrade the o il in toto. Insight into such permutations mediated by microorganisms within our environment should allow mankind the luxury of not being left floundering both in oil and a morass of ignorance.
Let us now fo r a moment consider our original mercury source— a 1 g sphere of m etallic mercury. In lig h t of our theory, what is its fate? With each cycle of the previously mentioned series of events the surface area of inorganic mercury increases; subsequent to and in conjunction with this change in availability of mercury we have an ever-increasing population of actively metabolizing microorganisms available to translocate said element, thus requiring less time. Examination of our data con cerning the dynamics of mercury in bed sediments allows us to predict its course mathematically using the following formula
[see Appendix e] : T = A ( l- e ~ ^ ) . Caution should, however, be employed in applying this relationship elsewhere.
Upon the establishment of a mercury equilibrium in the s tra tifie d , model lake sediments, mercury begins to appear in the overlying water column. Microscopic examination of the water matrix showed that the appearance of mercury correlated d ire c tly with the migration of the planktonic biomass composed of phytoplankton, protists, and zooplankton into the overlying layer. As these 98 particulates migrate through the fluid medium, they have a pre dilection for attachment to solid surfaces, mainly the aquarium glass. Once the periplankton film encases all exposed surfaces
(approximately 4 weeks after its in itial appearance), macroscopic examination of tank waters reveals an increasing population of mercury containing particulates readily removed by filtr a tio n
(Figure 9).
At this stage of the experimentation (Figure 10), mercury begins to appear in the brown, ramshorn-shaped gastropod, viz.:
Helisoma trivolvis. This flat-coiled snail is characteristically a browsing species which feeds on the algal component of the peri- plankton film with its own attached peri phytic, methyl ating bac teria. Aquarium enthusiasts employ this species for this very reason, to keep the glass clear of the algal film which otherwise obscures the view of fis h or other aquarium animals (C.B. Stein,
Personal Communication).
Between the 22nd and 24th weeks, a dense floe of organic, decomposing detritus appeared on the surface of the underlying stratum. Shortly thereafter, mercury was detected in ovate-conical, green-pigmented--
With the advent of unattached, organic micro-particulates in
the water column, one notices the simultaneous accumulation of
mercury in the resident goldfish ( Carassius auratus). This obser
vation suggested two possible mechanisms for the mercury uptake
seen, i.e ., (a) mercury containing elements in suspension supple
mented the diet of these fish, and/or (b) inorganic mercury in
solution was methylated and adsorbed by the fish directly from
water via bacteria growing on their slimy bodies. The linear
increase of mercury and its concentration in goldfish tissue
(Figure 11), may actually continue for years, rather than weeks,
as had been reported with other species of fish (21). From our
experiments we can only hypothesize the fate of mercury as it climbs one trophic level of the food chain and encounters Homo sapiens.
In an attempt to ascertain how a mercury burden is trans located via these water borne, organic particles, our attention was focused on th e ir physical attrib u tes, namely: volume and weight. Data contained herein (Figures 12-14) shows that while mercury increases with the weight of such particulates; a more profound relationship (increase), however, is demonstrated with their volume. Upon examination of a given sample for these two parameters, i t was observed that the weight of a given microbial population reached a plateau whereas the volume of the very same cells contained therein continued to increase. This clearly suggested that mercury translocation mediated via the particulates 100 under study was a surface related phenomenon. X-ray microanalysis v e rifie d the presence of particulates having a surface-associated mercury burden among the constituents of the biomass. Indeed, this is not a surprising observation since numerous other activities seen in protists are largely due to their high surface to volume ra tio .
In summary, mercury probably moves through the environment in a number of important ways, e.g. : (a) the translocation through the bottom sediments as described herein and which may be considered as a biologically autocatalytic process, (b) the translocation on micro- and macro-particulates in the water column in a relationship probably bearing upon surface to volume ratio, and/or (a) the mobilization of mercury in association with the motility of the benthonic macrofauna, such as oligochaete worms, gastropods and fis h . SUMMARY
1. The mercury burden found in water columns of Lake Erie's
western basin is particle associated; whether inorganic or
organic in nature, the load carried by individual particles
is directly related to their density.
2. Through the use of a model lake, i t has been theorized that
mercury translocation within the underlying bed sediments is
cyclical in nature, i.e. the concomitant methylation of in
organic mercury in aerobic conditions and demethyl ation of
methylmercury compounds where the oxygen level is low or
depleted, and the result of microbial initiation in the
benthos.
3. Translocation of mercury in the model lake bed sediments
was expressed mathematically.
4. Observations on the time interval required to translocate
mercury by biocenosis are suggestive of either sociological
adaptation or plasmid mediated resistance.
5. Mercury translocation mediated via organic particulates in
the model's water column--be it active or passive--was a
101 surface related phenomenon largely due to their high
surface to volume ratio.
X-ray microanalysis verifie d the presence of particulates
having a surface-associated mercury burden among the constituents of the model's biomass.
Mercury concentration was observed as the element moved from lower to higher levels of a typical food chain
(procaryotes »- eucaryotes >• gastropods and/or fis h ). EPILOG
. .T o the wide world and
all her fading sweets ..."
W. Shakespeare (324)
103 APPENDIX A
CALCULATION OF THE POTENTIAL NECESSARY FOR OXIDATION
OF METALLIC MERCURY INTO DIVALENT MERCURY
104 APPENDIX A
An important qu ality about divalent inorganic mercury is its affinity for organic mud. This binding to organic mud is extremely strong, with an a-coefficient (i.e.--a-coefficient is a measure of 21 the binding strength of a complex) greater than 1 0 in comparison to other complexes. As reported by Jernelov (177), Werner sug gested that the potential necessary for the oxidation of
o * HgO >- Hg ions could, under certain conditions, be written as follows:
fHq2* Total! E = 850 + 30 log a
Utilizing a theoretical value of 2 ppm for [Hg2+ TotalJ which r 21 is s 1 0 ~ 3 M and an a-coefficient of 1 0 , the potential necessary to oxidize m etallic mercury to divalent mercury ions is:
[io-51 E = 850 + 30 log 10
E = < + 80 mv
It is, therefore, readily apparent that this potential (80 mv) favors the oxidation of mercury fa r more than the original EQ (850 mv).
105 106
APPENDIX A (Continued)
^ 2^ The complex formation forces the reaction Hg° h Hg + p i 2e" to the rig h t by reducing [Hg J and, hence, altering the oxida- 2+ tion potential to an obtainable level in sediments. If the [Hg
Total] is lowered by any means, the potential necessary for the oxidation is, of course, further decreased. APPENDIX B
Table 5. Interaction of mercury with biological systems.
107 TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Abvamis bvarna bream 394, 395
Accipiter gentilis goshawk 384
Acinetobactev spp. bacterium 386
Actitis macularia spotted sandpiper 92
Aldrovandia macrochir halosaur 26
Alectovis graeca chukar 335
Allium cepa onion 294-296, 298
Alnus crispa alder 323
Aloe arborescens aloe 156
Alosa pseudoharengus alewife 161, 199
Axribloplites vupestris rock bass 161, 199
Amia calva bowfi n 199
- - - amoeba 328
Ammotretis spp. flounder 299
Anas acuta pintail duck 17
Anas discovs blue-winged teal 17
Anas platyrhynchos mallard 17, 92, 245
Anquilla anquilla eel 45
Anquilla vostvata American eel 161, 415
Anquilla vulgaris eel 171, 395
108 109
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Antarctophthirus callorhini sucking lic e 190
Antimora rostrata morid 26, 77
Aplodinotus grunniens sheepshead, 199, 320 freshwater drum
Ardea herodias great blue heron 92, 158
Argyropleecus spp. hatchet fish 402
Arthrobacter spp. bacterium 385
Arvipis trutta Australian salmon 299
Avtemia spp. brine shrimp 328
Asclepias tuberosa butterfly weed 156
Asellus aquaticus hogslater 186
Asio otus long-eared owl 31
Aspergillus clavatus mold 328
Aspergillus niger mold 19, 383
Aspergillus spp. mold 303, 342
Astaaus fluviatilis freshwater crayfish 161
Aythya affinis lesser scaup 92
Azolla spp. plant 328
Bacillus megaterium bacterium 162, 303,
Bacillus spp. bacterium 139, 342
Bacillus subtilis bacterium 162, 247,
barley seed 328 110
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Bathylogus spp. bathypelagic fish 402
Bathysaurus agassizi benthopelagic fish 26
Betula nana dwarf birch 323
Be tula papyrifera subsp. humilis white birch 323
Botaurus lentiginosus American bittern 92
Bougainvillaea spp. bougainvillea 328
Branta canadensis Canada goose 92
Brevoortia tyrannus menhaden 161
Bruchid quadrimaculatus weevil 227
Bubo bubo eagle owl 31
Bursavia spp. protozoa 328
Buteo buteo buzzard 31
Callovhinus ursinus Northern fu r seal 6 , 157, 190, 384
Callorynchus milii elephant shark 299
Cambavus spp. crayfish 92
Cancer magister crab 279
Cancer productus crab 279
Carassius auratus goldfish 161, 199
Carcinus maena shore crab 171
Carpiodes cyprinus q u ill back 199 Ill
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
CasmevodLus albus American egret 92, 158
Catostomus oommersoni white (yellow) sucker 320
Catostomus spp. sucker 199
Ceratophyllum demevsim coontail 1 0 0
Cevatopteris spp. plant 328
Chaetoceros costatum marine diatom 1 2 2
ChaVinwea brevibarbis macrourid 26
Chalinura earapina macrourid 26
Charadrius ahloropus common gallinule 92
- - - Chinese radish 328
Chironomus spp. chironomid larvae 333
Chlcmydomonas veinhavdi alga 28
Chlccrnydornonas spp. alga 272, 328
Chlidonias nigev black tern 92
Chlovella pyvenoidosa alga 25, 189, 249
Chlorella spp. alga 272
Chryptoohironornus spp. chironomid larvae 333
Civous ayaneus hen harrier 31
Citvobactev spp. bacterium 162, 385
Cladophova spp. alga 328
Clostvidiwn cochleavium bacterium 340, 341
Clostvidiwn sticklandii bacterium 91, 249 112
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Clostridium thermoacet-ieum bacterium 91, 249
Clupea harengus Atlantic herring 92, 161
Clupea sprattus sprat 83
Clupeidae herring 92
Codhliobotus miyabeanus brown spot of rice 303
Coelotanypus spp. chironomid larvae 333
Codiaeum spp. croton 156
Coleus spp. coleus 328
Coregonus albuta whitefish 384, 395
Coregonus artedii tu llib e e 320
Coregonus elupeaformis lake whitefish 199, 320
Coregonus lavaretus whitefish 395
Coregus artedii cisco 199
corn plant and seed 328
Corydalus spp. hellgrammite 92
coturnix quail 245
Crangon orangon shrimp 83
Crassostvea gigas Pacific oyster 279, 322
Crassostrea wirginioa Eastern oyster 80, 209, 318
Crassuta spp. plant 328
Cryptoaoocus spp. yeast 51
cucumber plant and seed 328
Cyolothone Spp. fish 402 113
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Cyprinidae mi nnow 92
Cyprinus carpio carp 199, 259
Daphnia magna copepod 39
Detichon urbica house martin 361
Dendrobivm spp. orchid 328
Diaphus theta myctophid 201
Drosophila melanogaster f r u it fly 294, 295 297, 298
Etodea canadensis el odea 100
Erriberiza calandra corn bunting 31
Engrautis japonica anchovy 351, 352
Engrautis mordax anchovy 161, 2 0 1
Enterobacter aerogenes bacterium 139, 383
Entevobaoter spp. bacterium 139, 380, 385
Erol-ia alpina dunlin 92
Escherichia coli bacterium 162, 203-205, 247, 303, 334, 348-350, 362, 368, 383
Escherichia spp. bacterium 139
Esox lucius Northern pike 98, 161, 171, 186, 199, 320, 384, 394, 395
Esox masquinongy muskellunge 199 114
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Esox nigev chain pickerel 161, 415
Euoophia spp. mysid 402
Euthynnus pelanris skipjack tuna 402
Falao peregrinus peregrine 31, 361
Flavobaeterium spp. bacterium 385
FontinaZis spp. moss 186
Fusavivm nivale snow mold 233, 303
Fvsavivm spp. seedling blight mold 303
Gadus aeglefinus haddock 394
Gadus morhua cod 83, 142, 161, 394, 395
GaZeovhinus australis school shark 299
GaZZinuZa chlovopus common gallin ule 92
Galtus galtus domestic fowl 360
grey partridges 245
Haii-aetus alh-ic%Vla white-tailed (sea) eagle 31, 384
HaZiohoevus grypus grey seal 161
Hadera helix English ivy 156
Helianthus debilis sunflower 156
Heliosoma eampanulata snail 100 115
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Helminthosporiwn avenae le a f spot of oats 303
Helminthosporium gramineum leaf stripe of barley 233, 303
Helminthosporium sigmoideum stem rot of rice 303
Helminthosporium spp. le a f spot of maize 303
Helminthosporium teres net blotch of barley 303
Helobdella spp. leech 186
Hiodon alosoides goldeye 320
Hiodon tergisus mooneye 199
Holosteum umbellatum jagged chickweed 323
Homarus vulgaris lobster 161
Hydropsyche pelluciduta caddisfly 186
Ictalurus punctatus channel catfish 199
Ictalurus spp. bullhead 199
Ictiobus spp. buffalo 199
Irisine spp. plant 328
Isoperla spp. stonefly 186
Juniperus virginiana red cedar 323
Klebsiella aerogenes bacterium 247 TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Lagopus lagopus willow grouse 31, 361
Lampanyctus ritteri myctophid 2 0 1
Larus delawarensis ring-billed gull 92
Latridopsis forsteri trumpeter 299
Lebistes reticulata guppies 1 0 0 , 215
Ledum palustre subsp. decumbens Labrador tea 323
Leionura atun snoek 299
Lerrma spp. plant 328
Lepomis gibbosus pumpkinseed 199
Lepomis macrochirus bluegill 56
lettuce seed 328
Leucothrix mucor bacterium 386
Ligustrum spp. privet 156
Lota lota burbot 199, 320
Lota vulgaris eel pout 394, 395
Lucioperca sandra pike-perch 394, 395
Lumbricidae earthworm 92
Lupinus luteus lupine 303
Lutra lutra o tter 384
Lycopersicon esculentum tomato 156
Macrocystis pyrifera macroalga 2 0 1
Mareca spp. widgeon 17 % 117
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
- - - marigold seed 328
Mentha crispa mi nt 156
Mentha piperita peppermint 156
Mentha spicata spearmint 25
MerZangus merZangus whiting 83
Me thanobaeteriwn omeZianskii bacterium 27, 52, 249, 408, 409, 411
Mioropterus doZomieui smallmouth bass 199
Mieropterus saZmonoides largemouth bass 199
Morone americana white perch 415
Morone saxatiZis striped bass 77
MotaeiZZa aZba white wagtail 361
Moxostoma spp. redhorse 199
MugiZ cephaZus sea mullet 299
MusteZa vison mink 384
MusteZus antarotious gummy shark 299
Mya arenaria clam 279
Mycobacterium phZei bacterium 383
MytiZus eduZis mussel 8 6 , 279
MytiZus gaZZoprovinciaZis mussel 8 6
Nansenia spp. fish 402
Navodon spp. leatherjackets 299
NemadactyZus maaropterus perch 299 118
TABLE 5
SCIENTIFIC NAME , COMMON NAME REFERENCE
Neomys fodiens water shrew 361
Nephrolepis exalta Boston fern 156
Neurospora orassa mold 224
Noeardia astevoides actinomycete 386
Nocavdia covall'ina actinomycete 386
NoQardia otitidis-oaviarwi actinomycete 386
Notemigonus ovysoleuaas golden shiner 92
Notropis atherinoides Lake Emerald shiner 92
Notropis spp. shiner 199
Nyoticopax nyet'Caorax black-crowned night heron 92, 158
Nymphea alba water l i l y 186
Ommastrophes bartamii. squid 402
Oncovhyndhus kisutoh coho salmon 199
Oncorhynchus nevka sockeye salmon 312
onion seeds 328
Oniscus spp. land isopod 328
Ophiophalis spp. brittle star 402
Opuntia spp. prickly pear cactus 328
Oxalis covnioulata oxalis 156
Padhysandra terminalis pach.ysandra 156 TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Pagophilus groenlandicus harp seal 161
Palaemonetes vulgaris grass shrimp 300
Pandion haliaetus osprey 384
Parus coeruleus blue t i t 361
Parus mac or great tit 361
Pelargonium spp. geranium 156
Peniaillium roqueforti mold 303, 368
Penioillium spp. mold 303, 342
Perea flaveseens yellow perch 92, 199, 320, 415
Perea fluviatilis perch 384, 394, 395
Perdix perdix Hungarian partridge 31, 361, 390
Phaeodaetyturn tricornutum phytoplankton 201, 272
Phasianus colchieus pheasant 2, 31, 47, 53, 136, 194, 245, 265, 335, 361, 384
Philodlna spp. invertebrate 328
Phoma betae black leg of sugarbeet 233
Physieulus barbatus cod 299
_ _ _ pinto bean plant 328
Pirieularia oryzea rice blast 303
Pisea mariana black spruce 323
Pisum sativum pea 303
- — — planaria 328 120
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Platyoephalus bassensis sand flathead 89, 299
Pleuroneotes flesus flounder 171
Pleuroneotes platessa plaice 83, 142, 283, 285, 286, 395
Podieeps oris talus great-created grebe 384
Podilymbus podiceps pied-billed grebe 92
Podooarpus spp. plant 328
Pomatomus saltatrix bluefish 77
Pomoxis Spp. crappie 199
Proechinophthirus fluctus sucking lic e 190
Proteus spp. bacterium 247
Prunus persioa peach 156
Pseudolabrus spp. parrot fish 299
Pseudomonas aeruginosa bacterium 37, 162, 238 303, 349, 368 386
Pseudomonas fluoresaens bacterium 25, 162, 383
Pseudomonas pyocyanea bacterium 247
Pseudomonas spp. bacterium 114, 115, 303 318, 366-368, 385, 386
Pusa hispida saimensis ring seal 161, 171, 190 384
Pyrenophora avenae mold 307, 308
Pythium ultimwn mold 19 TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Querous alba white oak 323
Querous lyrata over-up oak 323
Querous Spp. oak 156
Querous stellata post oak 323
Rasa clavata thornback ray 284
Rana catesbeiana bullfroq 92
Rana pipiens leopard frog 92
Rhizobium leguminosarum root nodule bacterium 386
Rhizobium melitoti root nodule bacterium 386
Rhizobium solani root nodule bacterium 19
Rhizobium spp. root nodule bacterium 303
Rhodopseudomonas capsulata bacterium 172
Rhus copallina winged sumac 323
Rhus glabra smooth sumac 323
Rooous ohrysops white bass 199, 412
Rooous mississippiensis yellow bass 199
- - - rose 156
- - - rye seed 328
Saocharotmyces cerevisiae baker's yeast 280, 329, 383 I
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Salmo gairdneri rainbow trout 121, 199, 274 282, 312
Salmo ocla sea trout 399
Salmo salar A tlantic salmon 161, 399, 415
Salmo salvelinus char 395
Salmo trutta brown trout 199
Salmonella spp. bacterium 334
Salmonella typhimurium bacterium 247
Salmonella typhosa bacterium 362
Salvelinus fontinalis brook trout 161, 199, 415
Salvelinus namaycush lake trout 21, 161, 199, 320, 389
Scarabaeidae beetles 92
Sclerotinia borealis snow mold 303
Scopulariopsis brevicaulis mold 383
Selenastrum capricornutum alga 103
Semotilus spp. dace 92
Seriola quinqueradiata yellow tail 351, 352
Seriolella maculata . mackerel 299
Sewanus s err anus sea perch 171
Serrivomer sector snipe eel 402
Sialis spp. ald e rfly 186
Sillaginodes punctatus spotted whiting 299
Sorex minutus pygmy shrew 361 I
123
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Spiraea beauverdiana spi raea 323
Spiraea salicifolia spiraea 156
Spirodela spp. plant 328
Squalus acanthius spiny dogfish 106
Squalus spp. dog shark 299
Staphylococcus aureus bacterium 30, 206, 247, 258, 271, 302 303, 348, 349 362, 368
Staphylococcus spp. bacterium 139
Stenobrachius leucopsaris myctophid 2 0 1
Stentor spp. protozoan 328
Sterna hirundo common tern 92
Stizostedion canadense sauger 199, 320
Stizostedion vitreum vitreum walleye 199, 320
Streptococcus faecalis bacterium 247
Streptococcus spp. bacterium 139
Strix aluco tawny owl 31
Sus scrofa pig 291, 373
Synedra ulna diatom 1 1 1 , 1 1 2
Synedra ulna var. danica diatom 113
Tarletonbeania crenularis myctophid 2 0 1
Tetrahymena pyriformis protozoan 365 TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Thamnophis sirtalis gardersnake 92
Thunnus atatunga tuna 77
Thunnus albaaares yellowfin tuna 77, 277
Thunnus albacora tuna 77
Thunnus thynnus tuna 77
Titletia caries wheat bunt 233
Titletio foetida wheat bunt 303
Titletia tritici wheat bunt 303
Trachrus japonicus jack mackerel 351
Turbatrix spp. invertebrate 328
Turdus merula blackbird 361
turnip seed 328
Typhula spp. snow mold 303
Uca pugitator fid d le r crab 381
Urocystis occulata stripe smut of rye 303
Ustilago avenae loose smut of oats 233, 303
Ustilago hordei covered smut in barley 233, 303
Vanellus vaneltus lapwing 361
Vibrio spp. bacterium 385, 386 125
TABLE 5
SCIENTIFIC NAME COMMON NAME REFERENCE
Vioia faba broad bean 156
Volvox spp. alga 328
Xvphias gladius swordfish 301
Zalophus californianus California sea lion 55 0
APPENDIX C
ANCILLARY DATA FOR WATER SAMPLES COLLECTED FROM
THE WESTERN BASIN OF LAKE ERIE
Table 6 . Physical factors.
Table 7. Chemical factors.
126 TABLE 6
PHYSICAL FACTORS
NUM LOC DI_ VE IE WT DPT T
16 B 0 0 0 76 71 15 06
39 A 8 05 80 62 15 09
42 B 3 1 0 63 65 30 05
43 B 4 15 6 8 67 15 04
51 B 7 15 76 69 15 05
69 B 4 08 75 74 15 08
91 A 1 2 0 63 76 15 0 2
105 D 5 1 0 75 61 15 03
106 A 5 2 0 61 59 04 1 1
107 C 7 30 45 51 1 0 04
115 B 7 0 2 80 56 15 07
1 2 2 B 6 1 0 76 6 6 15 07
NUM = Sample Number VE = Wind Velocity LOC = Location TE = A ir Temperatu re DI = Wind Direction WT = Water Temperature 1 = North DPT = Depth 2 = Northeast T = Turbidity With Secchi 3 = East Disc 4 = Southeast 5 = South 6 = Southwest 7 = West 8 = Northwest
127 9
TABLE 7
CHEMICAL FACTORS L C 0 NUM LOC pH N03 no2 ALK SO4 CL
16 B 8 . 0 0 .150 .300 .013 1 0 0 19 15
39 A 7.90 .150 4.08 .015 1 0 0 23 23
42 B 8.45 .150 4.99 .008 90 32 25
43 B 8.40 .180 27.5 .049 90 40 25
51 B 7.90 .190 4.98 . 0 2 2 90 31 25
69 B 7.30 . 2 0 0 4.98 .015 90 26 20
91 A 8.30 .310 4.99 .005 90 31 25
105 D 8.60 .300 4.00 . 0 0 0 90 50 26
106 A 8.50 .200 .100 .010 70 2 0 20
107 C 8.45 .300 9.00 .004 90 32 25
115 B 7.30 .180 7.50 . 0 1 2 90 2 2 20
1 2 2 B 8.70 . 1 0 0 4.50 .007 90 19 20
NUM = Sample Number LOC = Location pH = -log [H+] PO4 = Phosphate NOo = Nitrate NO2 = N itrite ALK = Total Alkalinity SO4 = Sulfate CL = Chloride
128 0
APPENDIX D
DATA OBTAINED THROUGH X-RAY MICROPROBE ANALYSIS
Table 8 . SAMPLE IDENTITY: Filamentous alga with epiphytic
bacteria, in situ.
Table 9. SAMPLE IDENTITY: Fusiform diatom, in situ.
Table 10. SAMPLE IDENTITY: Pseudomonad isolated from model and
cultivated in Hg-containing medium.
Table 11. SAMPLE IDENTITY: Staphylococcus isolated from model
and cultivated in Hg-containing
medium.
Table 12. SAMPLE IDENTITY: Pseudomonad isolated from model and
cultivated in Hg-free medium.
Table 13. SAMPLE IDENTITY: Staphylococcus isolated from model and
cultivated in Hg-free medium.
129 0
TABLE 8
SAMPLE IDENTITY: Filamentous alga with epiphytic bacteria, in s itu
DATA COLLECTION TIME (SEC): 400.000
ENERGY INTENSITY RESIDUAL ERROR ELEMENT LINE (KEV) (CPS) (CHI-SQ)
SI Kal 1.740 18.030 49.064 K&l 1.836 0.240 13.933
P Kal 2.013 64.439 12.678 KBl 2.139 1.215 17.292
S Kal 2.307 205.165 24.771 K31 2.464 9.235 17.374
CL Kal 2.622 7.228 2.969 Ka2 2.620 3.615 2.969 K31 2.815 0.567 0.384
FE Kal 6.403 3.300 0.762 Ka2 6.390 1.649 0.762 KBl 7.057 0.681 0.218
HG Lai 9.987 1.240 0.603 La2 9.896 0.124 0.489 LB1 11.821 0.661 1.076 L82 11.992 0.265 1.058 LB3 11.993 0.080 0.966 L34 11.561 0.052 0.158 Lyl 13.828 0.141 0.127 Ly 2 14.160 0.014 0.050 LX 8.720 0.035 0.017 Ma 2.195 20.854 26.442 M3 2.282 10.133 26.082 MY 2.487 0.579 15.833
TOTAL CHI-SQ: 5.825
130 TABLE 9
SAMPLE IDENTITY: Fusiform diatom, in s itu
DATA COLLECTION TIME (SEC): 400.000
ENERGY INTENSITY RESIDUAL ERROR ELEMENT LINE (KEV) (CPS) (CHI-SQ)
SI Kal 1.740 1985.919 686.875 KBl 1.836 26.484 577.390
s Kal 2.307 524.046 161.950 KBl 2.464 23.582 75.035
FE Kal 6.403 1.408 0.530 Ka2 6.390 0.703 0.530 KBl 7.057 0.290 0.091
TOTAL CHI-SQ: 204.520
131 TABLE 10
SAMPLE IDENTITY: Pseudomonad isolated from model and cultivated in Hg-containing medium
DATA COLLECTION TIME (SEC): 400.000
ENERGY INTENSITY RESIDUAL ERROR ELEMENT LINE (KEV) (CPS) (CHI-SQ)
SI Kal 1.740 4.270 1.787 KBl 1.836 0.057 2.399
S Kal 2.307 70.158 130.136 KBl 2.464 3.153 128.961
CR Kal 5.414 0.936 4.079 Ka2 5.405 0.468 4.079 KBl 5.946 0.173 0.328
FE Kal 6.403 0.708 37.839 Ka2 6.390 0.354 37.839 KBl 7.057 0.146 0.513
CU Lai 0.930 1.502 8.103 LB1 0.950 0.301 8.328 LB3 1.023 0.015 7.504 LX 0.811 0.074 876.026
HG Lai 9.987 0.222 30.819 La2 9.896 0.022 2.269 LB1 11.821 0.119 2.415 LB2 11.922 0.048 11.262 LB3 11.993 0.014 17.684 LB4 11.561 0.009 0.066 Lyl 13.828 0.025 0.024 Ly2 14.160 0.003 0.198 LX 8.720 0.006 0.012 Ma 2.195 2.672 80.558 MB 2.282 1.297 129.319 My 2.487 0.074 125.541
TOTAL CHI-SQ: 38.757
132 TABLE 11
SAMPLE IDENTITY: Staphylococcus isolated from model and cultivated in Hg-containing medium
DATA COLLECTION TIME (SEC): 400.000
ENERGY INTENSITY RESIDUAL ERROR ELEMENT LINE (KEV) (CPS) (CHI-SQ)
SI Kal 1.740 17.120 70.330 KBl 1.836 0.228 32.999
S Kal 2.307 423.602 64.663 KBl 2.464 19.060 13.216
FE Kal 6.403 1.063 0.283 Ka2 6.390 0.531 0.283 KBl 7.057 0.219 0.044
HG Lai 9.987 0.957 0.407 La2 9.896 0.095 0.255 LBl 11.821 0.510 0.529 LB2 11.922 0.205 0.709 LB3 11.993 0.062 0.603 LB4 11.561 0.040 0.293 ly i 13.828 0.108 0.077 Ly 2 14.160 0 . 0 1 1 0.134 LX 8.720 0.027 0.063 Ma 2.195 18.979 76.226 MB 2.282 9.220 72.413 My 2.487 0.527 10.914
TOTAL CHI-SQ: 8.903
133 TABLE 12
SAMPLE IDENTITY: Pseudomonad isolated from model and cultivated in Hg-free medium
DATA COLLECTION TIME (SEC): 400.000
ENERGY INTENSITY RESIDUAL ERROR ELEMENT LINE (KEV) (CPS) (CHI-SQ)
NA Kal 1.041 6.600 33.068
MG Kal 1.253 23.369 18.311
AL Kal 1.486 11.699 28.183
P Kal 2.013 423.135 176.700 KBl 2.139 7.975 82.608
S Kal 2.307 53.484 56.127 KBl 2.464 2.407 1.554
CL Kal 2.622 5.241 2.723 Ka2 2.620 2.621 2.723 KBl 2.815 0.411 6.588
K Kal 3.313 187.398 230.122 Ka2 3.310 93.698 230.122 KBl 3.589 28.219 120.856
CA Kal 3.691 7.044 4.595 Ka2 3.687 3.522 5.561 KBl 4.012 1.068 0.520
TOTAL CHI-SQ: 101.321
134 TABLE 13
SAMPLE IDENTITY: Staphylococcus isolated from model and cultivated in Hg-free medium
DATA COLLECTION TIME (SEC): 400.000
ENERGY INTENSITY RESIDUAL ERROR ELEMENT LINE (KEV) (CPS) (CHI-SQ)
NA Kal 1.041 10.683 38.589
MG Kal 1.253 37.000 54.036
P Kal 2.013 400.716 195.990 KBl 2.139 7.552 83.168
S Kal 2.307 60.570 51.926 KBl . 2.464 2.726 2.250
CL Kal 2.622 19.799 3.310 Ka2 2.620 9.902 3.310 KBl 2.815 1.554 2.094
K Kal 3.313 37.336 31.598 Ka2 3.310 18.668 31.598 KBl 3.589 5.622 18.712
CA Kal 3.691 22.585 6.358 Ka2 3.687 11.291 6.333 KBl 4.012 3.426 0.809
TOTAL CHI-SQ: 72.693
135 APPENDIX E
MATHEMATICAL EXPRESSION FOR MERCURY TRANSLOCATION IN
MODEL LAKE BED SEDIMENTS
136 APPENDIX E
MATHEMATICAL EXPRESSION FOR MERCURY TRANSLOCATION IN
MODEL LAKE BED SEDIMENTS
Graphic representation [see Figure 18) of the kinetics of mercury translocation through model lake bed sediments (Figure
8 ), plotting Time (expressed in weeks) on the y-axis and distance
(expressed in centimeters) on the x-axis at any one given mercury
concentration yields the following mathematical expression (78) of the functions:
T = A (l-e"kD)
T = value on the y-axis (Time)
D = value on the x-axis (Distance)
k = constant
A = Asymptote (Figure 18)
Using the aforementioned equation, solving for k:
T = A (l-e“kD)
T = A-Ae~kD
T-A = -Ae"kD
137 APPENDIX E (Continued)
I f ex = y, then log^ y = x, yielding:
logN [TiAj = . kD
Substituting actual data from Figure 8 , viz.: T = 5 weeks and
D = 17 cm with the Asymptote from Figure 18, one determines that:
k (constant) = 0.0408
Now by utilizing our in itia l equation: T = A (l-e "^ ) with experimental findings from our model, we can now determine the course of mercury mobility, -i.e., when (T) mercury w ill arrive at any given point (D) within the sediment bed or vice versa. Figure 18. Mathematical expression of the interrelationship
seen between Time (T) and Distance (D) involved
in the translocation of mercury through model lake
sediment beds.
139 Asymptote
Point from Actual Data Point from Mathematical Extrapolation
20 40 60 90 IOC cm
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