EPA-600/3-77-037
April 1977
Ecological Research Series
A CONTROLLED BIOASSAY SYSTEM FOR
MEASURING TOXICITY OF HEAVY
METALS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-77-037
April 1977
A CONTROLLED BIOASSAY SYSTEM FOR
MEASURING TOXICITY OF HEAVY METALS
By
K. H. Mancy and H. E. Allen
Department of Environmental and Industrial Health
School of Public Health
The University of Michigan
Ann Arbor, Michigan, 48109
Contract Number 14-12-591
Project Officer
Gary E. Glass
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor
does mention of trade names or commercial products constitute endorsement
or recommendation for use.
ii
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FOREWORD
Our nation's freshwaters are vital for all animals and plants, yet our
diverse uses of water for recreation, food, energy, transportation, and
industry physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in water.
The Environmental Research Laboratory in Duluth, Minnesota develops methods,
conducts laboratory and field studies, and extrapolates research findings
—to determine how physical and chemical pollution affects
aquatic life
—to assess the effects of ecosystems on pollutants
—to predict effects of pollutants on large lakes through
use of models
—to measure bioaccumulation of pollutants in aquatic
organisms that are consumed by other animals, including
man
This report defines the important parameters that must be measured or
controlled when heavy metals are introduced to the aquatic environment for
the purpose of determining their impact on that system. The toxic response
of an organism is a function of the chemical forms of metals and their
availability to the organism. System parameters which regulate the responses
must be studied for a complete understanding of the bioassay system.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
111
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ABSTRACT
Biological availability of metal micronutrients and metal toxicity are
believed to be dependent on metal oxidation state, complexation, and
solubility, as well as the physicochemical characteristics of the aqueous
phase. Basic design criteria for fish bioassays which are capable of
elucidating the dependency of toxicity on the type and concentration of
various copper species were developed utilizing equilibrium chemical con-
cepts and appropriate analytical techniques.
In order to maintain a desired copper species in the bioassay medium,
synthetic waters were used under well defined physical and chemical conditions.
These solutions were synthesized in accordance with equilibrium models, which
define the distribution of various copper species as a function of the solution's
physical and chemical characteristics. An experimental system was developed
which permitted large volumes of the bioassay waters to be maintained at the
desired chemical equilibria for the duration of the experiment.
Monitoring of the bioassay system included measurements of (a) pH, (b) temper-
ature, (c) flow, (d) specific conductance, (e) calcium, (f) total alkalinity,
(g) dissolved oxygen, and (h) copper species. Novel analytical procedures
were applied for the measurement and the differentiation of copper species.
This included the use of anodic stripping voltammetry and potentiometric mem-
bran electrodes.
The bioassay experimental system was tested by performing toxicity tests at
pH 5.7, 7.0, and 8.1. Results demonstrated the feasibility of conducting
bioassay experiments under conditions of chemical equilibria.
This report was submitted in fulfillment of Contract Number 14-12-591, by The
University of Michigan, under the sponsorship of the Environmental Protection
Agency.
iv
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CONTENTS
Foreword ill
Abstract ±v
List of Figures vi
List of Tables viii
Acknowledgments ix
I Conclusions 1
II Recommendations 2
III Introduction 3
IV Aqueous Chemistry of Copper 14
V Analytical Chemistry of Aqueous Copper 32
VI Development of the Bioassay System 58
VII Design of Bioassay Experiment 69
References 88
Appendices
A Copper Equilibria Computer Program Output 96
B pH and Alkalinity Computer Program Output 103
v
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FIGURES
No. Page
1 Forms of Occurrence of Metal Species in Natural Waters 6
2 Activity of Cupric Ion in Equilibrium with (a) CuO(s);
(b) Cu2(OH)2C03 at Bicarbonate Ion Concentration of
10~3 M; and (c) Cu3^0H)2(C03)2 at Bicarbonate Ion
Concentration of 10~3 M 25
3 Equilibrium Diagram for Copper 26
4 Stability Field Diagram for Tenorite and Malachite as a
Function of pH and Total Carbonate Concentration at
Various Copper Concentrations 29
5 Analytical Scheme for Anodic Stripping Voltammetric
Determination of Metals in Natural Waters 44
6 Anodic Stripping Voltammetric Characterization of
Metallic Species 46
7 Cell and Electrode Arrangement for Anodic Stripping
Voltammetry 48
8 The Effect of Calcium and pH on the Variation of the
Peak Current During the Titration of 5 yg/ml Humic
Acid with Copper 50
9 The Effect of Calcium and pH on the Variation of the
Peak Current During the Titration of 5 ug/ml Huraic
Acid with Cadmium 51
5 +
10 Graphical Representation of Cu^ -Carbonate Ion
Titrations 55
11 Physicochemical and Biological Interrelationships in
Bioassay Experiments 59
vi
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No. Page
12 Partial Pressure of Carbon Dioxide and Amount of
Sodium Bicarbonate (Moles/Liter) Required to Maintain
Total Carbonate = 10~2 Molar or Total Alkalinity =
10 2 Equivalents/Liter 67
13 Partial Pressure of Carbon Dioxide and Amount of
Sodium Bicarbonate (Moles/Liter) Required to Maintain
Total Carbonate = 10~3 Molar or Total Alkalinity =
10 3 Equivalents/Liter 68
14 Change of ASV Signal for Zn2+, Cu2+, and Pb2+ with Time 71
15 Schematic Diagram of Hatchery 73
16 Schematic Diagram of Fish-Conditioning Area 74
17 Schematic Diagram of Bioassay Area 80
18 Schematic Diagram of Bioassay System 81
19 Bioassay Exposure Chamber 82
vii
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TABLES
No. Page
1 Distribution of Soluble Copper as a Function of
pH and Total Carbonate 30
2 Sensitivities of Instrumental Methods for Monitoring
Metals in Water 34
3 Summary of Trace Metal-Humic Acid-Calcium Interactions
as Observed During Titrations with Metal Ions 52
4 Equilibrium and Stability Constants 57
5 Random Sequence for Adding Fish to the Chambers 77
6 Ra'.idom Order for Placing Fish in Bioassay Chambers 78
7 Summary of Bioassay Physicochemical Data 83
8 Summary of Fish Survival Data and Copper Measurements 84
viii
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ACKNOWLEDGMENTS
The authors wish to acknowledge the valuable contributions of the
students and staff of the Environmental Chemistry Program, whose active
participation was essential for the completion of this study. Special
thanks are due to J. Black, D. Minicucci, R. Roberts, T. O'Shea. D.
Johnson, B. Gulp, J. Twork, and R. Lasheen.
In addition, we wish to acknowledge the contributions of Dr. M. Bender,
Dr. W. Matson, and Dr. P. Colby during the initial phase of this study.
The authors also wish to acknowledge the continued interest and helpful
suggestions of the Project Officer, Dr. Gary E. Glass.
ix
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SECTION I
CONCLUSIONS
It is imperative that rigorous physicochemical controls are applied in
heavy metal bioassay experiments, e.g., copper toxicity to fish, in
order to elucidate the dependency of toxicity on the type and concen-
tration of given metal species. Equilibrium chemical concepts can be
used to prescribe appropriate controls which must be verified by appro-
priate analytical techniques.
Novel analytical procedures for the differentiation of copper species
were applied utilizing anodic stripping voltammetry and potentiometric
membrane electrodes. These procedures are also applicable for trace
metal characterization in natural and waste waters.
Anodic stripping titration techniques were used to assess the competi-
tive interaction between trace metals (e.g., Cu, Pb, and Cd) and alka-
line earth metals (e.g. , Ca and Mg) for organic complex formation. Our
findings showed that copper exhibits higher tendency for organic com-
plex formation in comparison to other trace metals. Copper affinity
for organic matter is not affected by high levels of calcium in con-
trast to other trace metals, e.g., Cd. Understanding this type of metal
interaction provides an insight into metal transformations in the
aquatic environment and their toxicity effects.
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SECTION II
RECOMMENDATIONS
Although water quality standards give tolerance limits in terms of total
metal concentrations, the toxicity of different metal species should be
ascertained. This information is essential for the development of
toxicity models including physical, chemical, and biological interac-
tions. These toxicity models may be able to predict the toxic effect
of certain metal species on a given aquatic organism under specified
environmental conditions.
Generally, current metal bioassay practices do not take into account
metal oxidation states, complexation, and solubility. It is therefore
recommended that metal bioassay experiments be conducted under well-
defined physicochemical characteristics of the bioassay medium.
It is recommended that adequate analytical measurements be made so that
meaningful bioassay experiments can be conducted. Such measurements
are required to insure that the added toxicant is present in the bio-
assay medium at the specified level, and to define the physical and
chemical characteristics of the medium.
In view of the increasing awareness of the complexity of chemical trans-
formations in the aquatic environment, it is recommended that efforts
be made to develop reliable analytical systems capable of metal species
characterization. Such analytical tools are essential for the under-
standing and prediction of toxicity to aquatic biota.
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SECTION III
INTRODUCTION
Fish bioassays are perhaps the oldest known procedures for determining
water "safety" and for detecting harmful toxicants. The use of fish
to indicate the presence of polluting materials is similar to the 19th
century practice of using canaries to monitor the presence of toxic
gases in coal mines. In Europe the toxicity of waste effluents is
commonly monitored by placing a known number of fish in a net or screen
box in the effluent stream or the receiving water and observing their
survival at daily intervals. Consequently, fish bioassay tests can be
considered as nonspecific analytical procedures that use fish as in
situ biological sensors.
At the present time water quality criteria and standards are based
largely on data from bioassay and toxicity tests with fish and other
organisms. In fact, acute and chronic toxicity experiments are being
increasingly used to establish guidelines for "safe" levels of toxicants
and other physical and chemical characteristics of the aquatic environ-
ment. Because of the complexity of environmental factors of most nat-
ural aquatic systems, laboratory bioassay experiments, which permit the
maintenance of controlled conditions, are frequently preferred over
field experiments to study the effect of a given toxicant. Neverthe-
less, the ultimate proof of results of laboratory bioassay experiments
must come from studies on natural aquatic communities.
In this report we describe procedures for conducting controlled labor-
atory bioassays. The main thrust of these studies has been the provi-
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sion of rigorous physical and chemical controls of the bioassay experi-
ment. Only under these conditions can the biological response be quan-
tified and understood. The unique aspect of the work was the attempt
to determine the toxicity of different forms of the toxic element,
rather than the effect of total concentration per se. Thus, much
effort was devoted to understanding the aqueous chemistry of copper and
the development of analytical procedures for characterizing the various
metal species. We hope that the reported studies will offer a fresh
outlook on the toxicity of heavy metals to fishes and will aid in
improving the design of bioassay studies.
The role of heavy metals in water quality and their pollutional effects
on lakes, estuaries, and marine environments have received national
attention. Metals such as copper, cadmium, zinc, and cobalt, frequently
found in natural waters in the parts-per-billion range, may serve as
essential micronutrients for enzymatic transformations in trace quanti-
ties and may exert toxic effects on the aquatic biota at higher concen-
trations. Lead and mercury, in particular, are toxic at certain con-
centrations and are considered to be direct signs of man-made pollution.
The effect of trace metals on the quality of natural waters is dependent
on the distribution dynamics of these metals in the aquatic environment
and the types of interactions with the aquatic habitat. Meaningful
investigations should be based on the characterization of the types and
transport dynamics of metals in solution, whether they are adsorbed on
suspended particulate matter,bound by or released from bottom sediments.
The physicochemical characteristics of the aqueous phase and the avail-
ability of organic ligands of natural or pollutional origin greatly
influence the distribution and effect of trace metals on aquatic biota.
The establishment of tolerance limits and water quality standards has
been based on the assumption that the availability of metal micronutri-
ents and the toxicity of metals to biological systems are solely depen-
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dent on metal concentration. This assumption is not valid, since the
availability of metal micronutrients to biological systems and metal
toxicity effects are highly dependent on the degree and type of metal
complex formation. The biological response to trace metals in aquatic
environments is greatly influenced by the presence of metal-binding
ligands of natural or pollutional origin and the form of the metal
species, that is, whether they are in solution or in the form of col-
loidal particulates.
Metals in aqueous environments are conceived to exist in a variety of
forms. To differentiate simply between soluble and insoluble metals is
not easy. Workers in the field frequently adopt an operational defini-
tion of "soluble" and "insoluble" metals by using 0.45y or O.ly membrane
filters.
Metal species in aqueous systems can be categorized as follows:
I. Soluble species
1 - Free ions
2 - Metal complexes with organic or inorganic ligands
II. Insoluble particulates
1 - Colloidal particulates of metal complexes or
aggregates of hydrous metal oxides
2 - Metal complexes adsorbed on suspended particulates
An illustration of size distribution and the variety of species in the
soluble, colloidal, and particulate range is shown in Figure 1, reported
by Stumm and Bilinski . For example, soluble metals can exist as
(a) inorganic complexes and ion pairs, with the ligands such as
OH~, 02~, HC03~, COg"", Cl~, HPO^~, P0^~, S0^~, HS~, S2~, etc.; (b)
organic complexes with amino acids, polysaccharides, amino sugars,
fatty acids, organophosphorus compounds, porphyrin, etc.; and (c) poly-
nuclear complexes.
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< Membrane liltrable ^
l:rcc
metal
ions
Inorganic ion
pairs; inor-
ganic complexes
Diameter range: «
Organic com-
plexes ,
chelates
Metal species
complex bound
to high molec-
ular org. m£»t .
Metal species in
the form of
highly dispersed
colloids
Metal species
sorbed on
colloids
Examples :
Cu.aq
Fe.aq3*
Pb.aq3+
C»2(OH)2+
Pb(CO.)*'
CuCO-
AgSll
CdCl*
CaOH*
Zn(OH)3
Mc-SR
Mc-OOCR
cn,-c=o
M/ 2 \
,Cu
°\ /">
0— C — CH2
Me-lipids
Me— humic— acid
polymers
"lakes"
"Gelbstoffe"
Me polysac-
charides
PeOOU
Fc(OH)3
Mn(N) oxides
Mn 0 0
Na Mn 0
A- S
2
Mcx(OH)y,
MeCOj, MeS
etc . on clays ,
FeOOH or on
Mn(N) oxides
A«2S3fI2~
Figure 1. Forms of occurrence of metal species in natural waters
(1)
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Metal organic interactions have been mentioned repeatedly in recent
literature to explain anomalous metal distribution that cannot be ac-
counted for by known thermodynamic data »»»»»» _ Certain
organic constituents have been postulated to complex with certain
metals, and as a result the availability or toxicity of certain metals
to biological systems is altered. Needless to say, some of these
deductions have been substantiated by the fact that a great many metal
species in natural waters are nonextractable with suitable reagents,
nonseparable by ion exchangers, and nondialysable.
The literature presents diverse opinions on the subject of aquatic metal
(9)
distribution and organic-metal interactions. Corcoran and Alexander
provided indirect evidence for the existence of trace metal organic
complexes in sea water by employing perchloric acid digestion. More
direct evidence was provided by dialysis and neutron activation analy-
sis experiments performed by Slowey and Hood and Slowey and
Jeffrey . More recently, Williams has demonstrated the exis-
tence of organic copper complexes in sea water, and evidence for the
existence of organic complexes with other trace metals, particularly
(13)
iron, has been reported by Koshy and Ganguly and Koshy, Desai, and
Ganguly . Duursma and Sevenhuysen , on the other hand, found that
trace metal-organic interactions are rather insignificant. Recent
radio tracer studies indicate that certain organic compounds (leucine)
exhibit no chelation with trace metals at the levels normally present
in the sea . The existence of trace metal-organic complexes in
fresh water has been suggested by the anodic stripping studies of
Barsdate and Matson , Matson and Roe , and Allen, Matson, and
Mancy . Barsdate provided further evidence through dialysis
studies.
The biological importance of trace metals chelated by organic substances
has also received attention. DeKock showed that iron became readily
available to plant cells and stimulated growth in the presence of humic
-------
acids. This evidence of the stimulatory effect of chelated iron-humic
(21)
acid substances was later used by Prakash and Rashid to partially
explain the increase in growth of marine dinoflagellates in the pre-
(22)
sence of humic acids. Johnston " found that the addition of a mix-
ture of iron, manganese, zinc, copper, and cobalt chelated with EDTA
at pH 7-8 induced abundant growth of unialgal Skeletonema costaturo in
most sea waters enriched with phosphate, nitrate, and silicate. Jones
(23)
found that Escherichia coli does not grow in sea water enriched
with glucose, ammonium, and phosphate ions unless the water is treated
with chelating agents. The inhibitory effect was attributed to the
toxicity of heavy metal ions present in the water. Enhancement of
biological growth in the presence of chelating agents, such as EDTA,
(24)
has been further substantiated by Barber and Ryther , who found that
the presence of chelators increased phytoplankton growth in the Crom-
well Current. Recently, the effect of nitrilotriacetic acid, a strong
chelator, on the growth of estuarine phytoplankton has been reported by
(25)
Erickson, Maloney, and Gentile
Although the literature suggests that some aquatic organisms can selec-
tively concentrate certain metal ions by secreting certain chelating
agents, there is no direct evidence in support of the predominance of
such trace organometallics in the aquatic environment. It is not quite
understood how organic matter in trace quantities can selectively
interact with individual heavy metals, e.g. Cu and Zn, in the presence
(26)
of a large excess of alkaline earth metals, e.g. Ca and Mg . The
question takes on added significance in view of the toxicity studies
(27) (28) (29)
of Cairns and Scheier , Wurtz , Pickering and Henderson , and
Mount . These workers found that the toxicity of zinc and copper
to bluegills, freshwater mollusks, and minnows was less in hard water
than in soft water. Although the mechanism of this inhibition is not
known, chemical interactions of the alkaline earths and trace organics
cannot be overlooked.
-------
In summary, considerable evidence indicates that trace metals in natu-
ral waters are, to a certain extent, present as complex species associ-
ated with trace organic materials. Further evidence is available to
relate these complex organometallics to biological activity and the
toxic effects of trace metals in natural media. However, little in-
formation is available on the dynamics of trace metals in natural
aquatic media and on the distribution and characterization of the
organometallics associated with these trace metals. Furthermore, the
role of alkaline earth ions in the complexation of these trace metals
is presently only a matter of speculation.
TOXICITY OF METALS TO FISH
(31)
Chen discussed in his review how metals act in several ways as
poisons to fish. The metals enter the organism in either of two ways
— through the gills while respiring or through the mouth with swallow-
ing, The entrance of ionic poisons into the gills may be aided by the
excretion of external enzymes to facilitate the active transport of
ions against a concentration gradient. Then, the gill epithelium is
destroyed by the action of certain heavy metals. Therefore, the fish
dies of asphyxiation.
Toxic metals may travel to different parts of the body via the blood-
stream. The poisons may react with cell protoplasm of various tissues
or organs. Insofar as copper toxicity is concerned, a lack of agree-
ment exists concerning the actual "safe level" concentration in water
(32)
Copper concentrations varying from 0.1 to 1.0 mg/1 have been
found by various investigators to be toxic. On the other hand, con-
centrations of 0.015 to 3.0 mg/1 were reported as toxic, particularly
in soft water, to many kinds of fish,
EFFECTS OF CHELATORS ON TOXICITY
Various investigators seem to disagree on the influence of chelation
on metal toxicity to fish. For example, a study of zinc toxicity to
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(33)
fish by Lloyd indicated that suspended zinc sulfate complexes
decreased the survival time of rainbow trout in concentrated solutions.
(34)
Fitzgerald showed that citric acid chelation reduced toxicity of
(35)
copper to minnows 500-fold. Grande in his investigations with
copper in several Norwegian lakes showed that one lake heavily colored
with humic colloids was less toxic to salmonid fishes than the non-
colored lakes, even though both lakes contained the same copper concen-
trations. This phenomenon was described as a masking effect by the
organics on the metal. Masking effect refers to the organics' ability
to reduce or "hide" the toxicity of copper to fish. This apparently
depends upon the type of metal complex present in the water system
which in turn determines the metal's toxicity to organisms.
( 36}
Nielsen and Wium-Andersen in their investigations of copper toxi-
city to plankton found that various organic substances also mask the
effects of metals on algae. The organics appear to be polypeptides
(2)
and other similar substances. Duursma and Sevenhuysen determined
that other organics (phenols, petroleum, and carbon disulfide) present
in the natural system reduced the amount of free copper in sea water.
It is apparent from these investigations that organic chelators defin-
itely affect free copper concentrations in the aqueous system. Whether
the chelators enhance or detract from metal toxicity is still uncertain.
SOURCES OF CHELATING AND SEQUESTERING AGENTS
The major chelating and sequestering agents found in the aquatic envi-
ronment include plant products, microbial metabolites, humic compounds,
lipid soluble fractions, detergents, and sewage effluent. These sources
of complexing agents are discussed below.
(37)
In a review by Pauli concern was given to the exogenic cycle of
matter and the manner of occurrence and distribution of minerals in the
outer surface of the earth. The most effective method of transporting
10
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elements from the lithosphere to the biosphere, including the fresh
water cycle, was reported to involve the biosynthesis of chelating and
sequestering agents. These agents were classified as plant products,
microbial metabolites, or humic compounds, Chelating agents produced
by organisms include lichen acids such as lakmus and orcein, which are
plant-derived polyphenols with two or more aromatic rings.
Humic compounds are derived from decomposition of organic residues
through biological activity and heterogenous catalysis. The humic
compounds are collectors of copper, zinc, nickel, cadmium, lead, man-
ganese, and cobalt. These chelates formed from humic substances and
trapped metals become available for use by various organisms in the
terrestrial and fresh water biocycle,
Slowey and Jeffrey experimented primarily with copper complexes
found in sea water. The largest amounts of extractable copper were
found at depths where sinking, decaying organisms were most likely to
release organic compounds into sea water. Rupture of the phytoplankton
and zooplankton releases some of the organic complexing compounds to
the water. Although the nature of these complexes is not resolved,
some evidence indicates that the copper complex is associated with the
phospholipid, amino lipid, or porphyrin fraction of these lipids. Al-
though these studies concern sea water, fresh waters probably contain
similar complexing organics.
(12)
Williams demonstrated that organically associated copper may be
complexed with phospholipids, carotenoids, or other lipid-soluble frac-
tions. However, it is not clear whether the copper is actually a mo-
lecular complex with an organic ligand or whether it is associated with
organic colloids by bonding within an organic matrix.
Detergents are obviously a significant component of domestic sewage.
(38)
According to Hynes , raw sewage contains about 12 ppm of active
detergent. A significant proportion of detergents remains undegraded
and eventually gets into water bodies. Detergent builders are strong
complexing agents which must be considered in studies of ligands in
natural waters.
11
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EFFECT OF ALKALINE EARTH METALS ON METAL TOXICITY
A first observation of the metallic content of natural water reveals a
predominance of alkalies and alkaline earth metals. Stumm and Morgan
(26}
were therefore prompted to pose the following questions: (1) How
can trace concentrations of organic matter specifically interact with
individual metals? and (2) Does not the presence of Ca2 and Mg2 at
concentrations many orders of magnitude larger than potential organic
complex-forming species blur any complex-forming tendency of the or-
ganic functional groups? These pertinent questions cannot be answered
from present available information on trace metals in the aquatic
environment.
The effect of high concentrations of alkaline earth metals on transi-
tion or heavy metal-organic complex formation can be illustrated by
considering the effect of Ca2 and Mg2 on EDTA complex formation with
Fe(III). The formation of Fe(III)-EDTA complexes is given as
Fe3+ + Y4~ = FeY~; log K = 25.1 (1)
The effect of Ca2 can be expressed as
KFeY
CaY2 + Fe3+ = FeY + Ca2+; log — - =14.4 (2)
where log K Y = 10.7.
Based on the above relationships, FeY cannot be formed to any signi-
ficant extent if the ratio of [Ca2 ] to [Fe3 ] is sufficiently high.
This effect is of course dependent on the relative stability of the
formed complexes. For example, in the case of L = citrate4" the
equilibrium
CaHl" + Fe3+ = FeHL -1- Ca2"1" (3)
is characterized by an equilibrium constant of log (1L., m /KC ) = 7.4.
This indicates that the competitive effect of Ca2+ upon Fe3"1" complexing
by citrate is much smaller than that encountered with EDTA.
12
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Numerous investigators have studied the effect of hardness by measuring
the toxicity of metal solutions with increasing hardness, and several
theories have been advanced to account for their results. Early
workers attributed the decreased toxicity of solutions at high hard-
ness to complexation and precipitation of the metal ions from the
solution. However, with the advent of continuous-flow bioassay tech-
niques, it has been found that some complexation may actually increase
the toxicity of metal solutions . Mount's study showed that at any
given hardness level, as pH increased, the solutions became more toxic.
He attributed this effect to the zinc complexes formed at higher pH
levels.
One of the major difficulties in interpreting the effect of hardness
on heavy metal toxicity stems from the inability to determine whether
the observed effects are a result of calcium-heavy metals competitive
interactions or are due to physiological effects that resulted from
0+ •) +
the presence of Ca or Mg^ , or both. For example, the effect of
hardness could be explained in terms of purely aqueous chemical inter-
actions. Under a given set of conditions, certain heavy metals can
9 +
exist as soluble organic complex species. At relatively high Ca^
concentrations, as a result of metal-ligand exchange reactions, these
heavy metals may be transformed into inorganic metal complexes that
may precipitate as hydroxy compounds at high pH. This effect will be
more pronounced at higher levels of "hardness" and of course is largely
dependent on the pH. Consequently, if the toxicity effects were pri-
marily due to the organic-metal complex, the presence of high levels
of calcium will result in a decrease of its toxicity.
On the other hand, the presence of high levels of Ca2 and Mg2 has
been thought to cause certain physiological changes in fishes that may
result in a decrease in the toxic effects of heavy metals. This was
(33)
shown by Lloyd , who found that preconditioning of fish in hard
water will significantly decrease the animal's susceptibility to toxic
metals.
13
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SECTION IV
AQUEOUS CHEMISTRY OF COPPER
The presence of copper in water is ecologically significant because of
its extreme toxicity to fish and other aquatic organisms. Workers
have studied the toxicity of copper, but none have investigated the
relationship between the speciation of copper and its toxicity. A
comprehensive study of the toxicity of copper is required to provide
the information necessary for evaluating its environmental impact.
SOURCES
Most of the copper entering the aqueous environment is due to the
activities of man. Natural weathering of minerals and the resulting
runoff, which contains copper, contribute an almost insignificant
fraction of the total copper input. The copper washed out of the
atmosphere by precipitation is significant. Lazrus, Lorange, and
(39)
Lodge found an average of 21 Mg/1 Cu in precipitation in the
United States. Kramer found an average of 32 yg/1 total copper
and 22 yg/1 soluble copper in precipitation collected at 36 stations
in northern Ontario. Although the copper reaching bodies of water by
precipitation is a natural occurrence, a large fraction of the copper
in the atmosphere is not of natural origin.
Large amounts of copper enter the natural water system from mining
operations and metallurgic processes. The waste water from copper-
bearing acid mine drainage is usually stored in tailings ponds after
neutralizing the acidity. However, the treated water in the tailings
(41)
pond may contain concentrations of copper as high as 50 ug/1 . The
14
-------
drainage from agina mines may have pH values as Low as 2,5. The acid-
ity of the drainage water has been attributed to the metabolic activity
of certain bacteria, which enhance the oxidation rate of sulfide parti-
(42)
cles by a factor of about 2,000 at the optimum temperature . Pro-
(43)
kovich found concentrations of copper in water near old mines to be
as high as 1,000 mg/1.
Treatment of water with copper sulfate for control of algal growth has
(44 45 46)
been practiced since about 1900 ' ' . In some cases lakes have
been treated with as much as 1.5 million pounds of copper sulfate over
(47)
a period of several decades
FORMATION OF COMPLEXES
The results from many studies indicate that copper in both fresh water
and sea water is held in solution by complexation with naturally
(18}
occurring organic ligands. Allen et al. added 13 yg/1 of copper to
water from the Rouge River. Results of analysis using anodic stripping
voltammetry indicated a logarithmic decrease in peak current with time.
The results were interpreted as a first-order complexation reaction of
copper and excess organic ligands in the river water.
Stability constants for copper hydroxide, oxide (tenorite), and basic
carbonates (azurite, Cu3(OH)2(C03)2; malachite, Cu2(OH)2C03) indicate
that the equilibrium concentration of copper in water with pH greater
than 7 would be very low. In some waters, however, the amount of cop-
per apparently in solution exceeds theoretical limits by several orders
of magnitude. This anomaly has also been explained as a result of
complexation of copper with organic substances called yellow acids,
humic acids, and fulvic acids. Up to 50% of the copper in sea water
can be extracted with chloroform indicating that much of the copper
(12)
in sea water is in some organic complex or sol. Williams found
that 5 to 28% of the copper in sea water was present as complexes with
stability constants greater than 1018.
1.5
-------
Studies concerned with the chemistry of copper in soil have also indi-
cated that copper forms complexes with naturally occurring ligands.
If solutions of copper are stirred with ion exchange resins in both
the presence and absence of humic and fulvic acid the resin will take
up less copper when humic or fulvic acid is present
PRECIPITATION
In the absence of organic ligands , copper is very insoluble at the pH
values found in most natural bodies of water (pH > 7) . When soluble
•
salts of copper (Cu(NC>3)2, CuCl2 , CuSO^) are mixed with natural water
the cupric ion precipitates as copper hydroxide (Cu(OH)2) and will
evolve to tenorite (CuO) or to a basic carbonate, either malachite
(Cu2(OH)2C03) or azurite (Cu3(OH2) (C03)2) , depending on the carbonate
and copper concentration.
COPPER IN FRESH WATER AND SEA WATER
Allen et al. , using anodic stripping voltammetry , found copper con-
centrations of 19 to 108 yg/1 in Rouge River water, 1.8 to 6.8 yg/1 in
Lake Erie, and 0.4 to 19 yg/1 in Lake Michigan. Samples were acidified
before analysis to release copper from organic and inorganic complexes.
Analyses without prior acidification gave much lower results indicating
that most of the copper present was either in complexed or particulate
forms.
(49)
The copper cycle in three Connecticut lakes was described by Riley
In all of the lakes studied both total and particulate (> 0.6 y) copper
concentrations reached a sharp maximum by November. The minimum con-
centrations in all defined forms of copper occurred from February to
September.
Copper in sea water exists primarily in nonionic forms, and its verti-
cal distribution is sharply defined . A peak concentration of over
20 yg/1 occurs near the base of the euphotic zone. The concentration
decreases with depth into the aphotic zone to about 10 yg/1, after
which it stays nearly constant with increasing depth.
16.
-------
SEDIMENTS
In many lakes the concentration of copper in sediments is quite high.
The sediments have served as a sink for the copper that has been added
to lakes for algal control. Detailed profiles of copper in the sedi-
(47)
ment of several lakes are reported by Sanchez-Vazquez . In all
cases the copper concentration was at a maximum in the top layers of
sediment, indicating that most of the copper is from the activities of
man.
WATER QUALITY CRITERIA
The limit set for copper according to U.S. Public Health Service has
been changed twice. The original standard, set in 1925, was 0.2 mg/1.
In 1942 the limit was raised to 3 mg/1, and in 1962 it was lowered to
the present standard of 1 mg/1. The toxicity of copper to human beings
is low, but the present standard was set because of the poor taste
that copper imparts to water. However, because copper is highly toxic
to many organisms the threshold concentration of copper is reported by
(32)
McKee and Wolf to be 0.1 mg/1 in water used for irrigation. For
fish and other aquatic life the desirable concentration limit is 0.02
mg/1 in soft water and 0.05 mg/1 in sea water.
EQUILIBRIUM MODEL OF Cu2+-H20-C0? SYSTEMS
Of particular interest to the study of copper toxicity is the develop-
ment of an equilibrium model describing the homogeneous as well as the
heterogeneous equilibrium distribution of copper between the insoluble
and soluble hydroxy and carbonate complexes. The equilibrium relation-
ships for prediction of the composition of an aquatic system are most
easily solved by numerical digital computer techniques.
2+
A Cu -H20-C02 equilibrium system has been studied in our laboratory
in an attempt to illustrate the chemical relationships that must be
considered in the interpretation of copper toxicity data. However,
great care should be exercised in extending this equilibrium approach
17
-------
to the prediction of metallic species present in natural waters.
Although it is possible to measure the concentrations of inorganic
complexing agents such as hydroxide, carbonate, bicarbonate, sulfate,
chloride, and ammonia and to incorporate these into equilibrium calcu-
lations, prediction of the effect of the organic constituents is not
possible at the present time,
9 +
In the ternary system Cu^ -H20-C02 we are concerned with
(a) the solid phase
CuO, N (tenorite)
(s)
CuC03*Cu(OH)2, x (malachite)
\s/
(CuC03)2'Cu(OH)2, N (azurite)
\s)
and with (b) the aqueous phase
H+, OH~, C02(H2C03C) , HC03~, C032~, Cu2+, CuOH+,
Cu2(OH)22 , Cu(OH)3~, Cu(OK)h2~, CuC03, and
Cu(C03)22~
18
-------
The following equilibria relationships occur in the copper-carbonate
(26)
system ;
Log K
CuO,, + 2 H+ = Cu2+ + H20 7.65 (4)
(tenorite)
Cu2(OH)2C03(g) + 4 H+ = 2 Cu2+ + 3 H20 + C02 , 14.16
(malachite)
or
or
Cu2(OH)2C03 + 3 H+ = 2 Cu2+ + 2 H20 + HC03~ 6.34 (5)
\s /
Cu3(OH)2(C03)2, , + 6 H+ = 3 Cu2+ + 4 H20 + 2 C02, .21.24
\s^ \&)
(azurite)
Cu3(OH)2(C03)2, . + 4 H+ = 3 Cu+ + 2 H20 + 2 HC03" 5.60 (6)
yS /
Cu2+ + H20 = CuOH+ + H+ - 8 (7)
2 Cu2+ + 2 H20 = Cu2(OH)2+ + 2 H+ -10,95 (8)
Cu2+ + 2 C0g~ = Cu(C03)2~ + 2 H+ 10.01 (9)
Cu2 + CO2" = CuC03 6.77 (10)
Cu2+ + 3 H20 = Cu(OH)3~ + 3 H+ -26.3 (11)
Cu2+ + 4 H20 = Cu(OH)J~ + 4 H+ -39.4 (12)
H20 + C02 = H2C03 -1.5 (13)
H2C03 = H+ + HC03" - 6.37 (14)
19
-------
HC03~ = H+ + C0^~ -10.25 (15)
C02 + H20 = HC03~ + H+ - 7.82 (16)
From relationships 13, 14, and 15 it is possible to solve for [HC03~]
and [C03~] in terms of [H+] and [H2C03]:
[HC03 ] = (17)
[H2C03]
[HC03~]
CT [HT]'
[H']2
The expression for total carbonate is:
[Total Carbonate] = CT = [H2C03] + [HC03~] + [C03~]
or
[H2C03] ki [H2C03]
C = [H2C03] + + (19)
•L r TT~ 1 r TT ' T ?
This allows us to define the a values as:
[H2C03] [H+]2
— (20)
20
-------
[C032~]
&2 = ~"~ Z J "~ (22)
CL [H+]2
This permits the calculation of [H2C03] , [HC03 ], and [C032 ] if we know
the pH and the total carbonate concentration.
In the Cu-H20-C02 system there are two distinct equilibrium situations
at any particular pH and carbonate concentration. The first occurs when
the [Cu ] is not large enough to cause precipitation, and the second
when the [Cu ] is large enough to cause the precipitation of tenorite,
malachite, or azurite. These two situations will be discussed separ-
ately.
Case I; No Solid Species Formed
Expressions for each of the soluble species can be obtained be rearrang-
ing equilibria relationships 7, 8, 9, 10, 11, and 12:
[Cu2+](10~8) (l(T10-95)[Cu2+]2
[CuOH ] = , [Cu2(OH)i ] = —
[H ] [H]2
[Cu(C03)2~] = (1010'01)[C02~][Cu2+], [CuC03] = (106-77)[CO§"][Cu2+]
(10~26-3)[Cu2+]
v
[Cu(OH)3] = - v - , [Cu(OH)J~] =
[H ]3
An expression for total copper concentration is:
[Total Copper] = [CuOH~l"] + [Cu(C03)2.~]+[CuC03] + [Cu(OH) 3] + [Cu(OH)2.~]+[Cu2+]
21
-------
This can also be written as;
[Cu2
[Total Copper] - -- v -- + (101 ° -01) [Cu^~] [Cu2+]
(10~26«3)[Cu2+] (10"39-l*)[Cu2"f]
+(106-77)[C02i-][Cu2+] + T- + r +[Cu2+] (23)
[H+]3 [HY
2+
These expressions neglect the dimeric Cu2(OH)2 , which would be signi-
ficant only at much higher concentrations than those present in natural
waters.
We can now define 6 as 3 = [Cu2 ]/[Total Copper], which is equal to:
o o
(24)
ao~8) do"26-3)
— — +(io10-01)[co2i~]2+(io6'77)[co2i]+ — — --
+ "1"
This is a significant equation because it means that the cupric ion
2+
concentration, Cu , can be calculated by knowing the pH, carbonate
concentration, and the total copper concentration as follows:
[Cu2+] = 3 [Total Copper]
where 6 = [Cu2 ]/ [Total Copper] as defined above.
o
If we define Denom as,
Denom = (10~8)/[H+] + (1010« 01 ) [CO2,']2 + (106 -77) [CO2;']
+
(10"26'3 )/[H]3 + (10"39-1+)/[H]t* + 1 (25)
then similar equations can be constructed that can be solved for the
concentration of each of the copper species.
22
-------
The resulting £> values are:
B = [Cu2 ]/[Total Copper] = l/(Denom) (26)
o
BI = [CuOH+]/[Total Copper] = (10~8)/[H+](Denom) (27)
32 = [CuOH3"]/[Total Copper] = (10~26'3)/[H+]3(Denom) (28)
B3 = [Cu(OH)5~]/[Total Copper] = (ICf39^)/[E+]k(Denom) (29)
B4 = [CuC03]/[Total Copper] = (106'77)[C0§~]/(Denom) (30)
B5 = [Cu(C03)2]/[Total Copper] = (1010-01) [CC)|~]2/(Denom) (31)
From these equations:
[Cu2+] = B [Total Copper] (32)
o
[CuOH+] = 3i[Total Copper] (33)
[Cu(OH)3~] = 62[Total Copper] (34)
[Cu(OH)fp] = 63[Total Copper] (35)
[CuC03] = 6^[Total Copper] (36)
[Cu(C03)2~] = B5[Total Copper] (37)
Case II; Solid Species Present
In the presence of a solid copper species, whether tenorite, malachite,
or azurite, the cupric ion concentration at equilibrium will be depen-
dent on the pH, total carbonate, and solid species present.
23
-------
Figure 2 shows the concentration of cupric ion in equilibrium with
tenorite, malachite, and azurite in a bicarbonate concentration of 10~3
M. At any particular pH and bicarbonate level, the precipitate that
dictates the lowest cupric ion concentration will be the predominant
solid species. Tenorite will be the predominant form at pH values
greater than 6 and a bicarbonate concentration of 10 3 M (Figure 3).
Once the solid species has been designated, the cupric ion concentration
will be dictated by that particular solid species:
Tenorite: [Cu2+] = (107-65)[H+]2 (38)
or
+ (107.Q8)[H+]2
Malachite: [Cu2 ] = j- (3^
or
+ (107-08)[H+]2
Azurite: [Cu2 ] = ~r- (40)
[P ]2 3
C02
The concentrations of the soluble species are then easily calculated:
, (10"8)[Cu2+] (10~26-3)[Cu2+]
[CuOH+] = r , [Cu(OH)3"] »
[H'] [H']3
(10'10-95)[Cu2+]2
[Cu(OH)2."] = + > [Cu2(OH)i ]
~
[CuC03] = (106'77)[Cu2+][COp, [Cu(C03)i~] = (1010-01)[Cu2+][C02~]2
24
-------
0
OJ
6
6
g
- 8
i
a
u f
o 10
rH
12
0
o
o
10 12 14
pH
Figure 2. Activity of cupric ion in equilibrium with
(a) CuO(s); (b) Cu2(OH)zC03 at bicarbonate
ion concentration of 10~3 M; and (c) Cu,(OH)?(C03)2
at bicarbonate ion concentration of lO""^ M.
25
-------
7 U
LU
Q.
0.
CS
o
CJ
9
10
12
13
0
10
12
2463
pH
Figure 3. Equilibriun diagram for copper. Total copper concentration equals 10~6 M
and total carbonate concentration equals 10~2 M.
-------
Construction of the Equilibrium Model
A mathematical model can now be constructed that will yield the concen-
tration of each copper species when the total copper concentration,
total carbonate concentration, and pH are known.
This model is constructed by the following steps.
? +
1) Assume no solid species are present and calculate Cu as
explained in case I,
2) Assume a solid species is present and calculate Cu2 as shown
in case II.
3) If the value for Cu2 obtained in step (1) is larger than Cu2
calculated in step (.2) , then precipitation will occur and all
calculations should proceed as described in case II.
If the value for Cu2 obtained in step 1 is smaller than Cu2 deter-
mined in step 2, then no precipitation occurs and the concentrations of
soluble species should be computed as described in case I.
This procedure may be repeated with different pH values and carbonate
concentrations to determine the variation in copper species under dif-
ferent chemical conditions. This variation of parameters is a repeti-
tive process, which has been readily accomplished with the use of a
computer, Sample computer program output is shown in Appendix A.
A particularly useful method of presenting these data is to plot the pH
vs. the log of the concentration of the different copper species, hold-
ing the total copper and carbonate concentrations constant. An example
of this procedure is shown in Figure 3. With a total copper concentra-
tion of 10 6 M and a total carbonate concentration of 10 2 M, cupric
ion predominates from pH 0 to 6, CuCOs from pH 6 to 8.3, and tenorite
from pH 8.3 to 12.5.
In the system described the fraction of the metal present as a given
species will be independent of the total metal concentration at any pH
and carbonate value as long as the total metal concentration does not
become so great as to permit dimeric metallic complexes and providing
27
-------
that no solid species are present. If metal precipitates form, in-
creasing the total metal concentration will not change the concentra-
tion of metal in solution or the species of the metals. Rather, only
the quantity of solid will increase. The pH region over which the
solid is stable is dependent on the total amount of metal in the system
(Figure 4). The range of stability increases as the concentration in-
creases. Since the dominant form of the metal at pH values less than
that at which tenorite forms is soluble copper carbonate, it should be
expected that the carbonate concentration in addition to the pH would
influence the lower limit of tenorite stability. This is shown in
Figure 4 by the curved portion of the lower stability boundary. The
effect of carbonate is more pronounced as the total copper decreases.
At high copper and carbonate concentrations malachite becomes a stable
species, but it will transform to tenorite by decreasing either the
total copper or carbonate concentration. Again it should be mentioned
that equilibrium calculations must be used with caution. The equilib-
rium constants used in the calculations are generally obtained at
higher metal concentrations and ionic strength than are present in the
natural environment. Often, predicted stable solid species are not
found under experimental conditions. For example, although tenorite is
the predicted stable solid, we did not find it to form within 1 week in
laboratory systems. Rather, solid copper hydroxide Cu(OH)2 was present.
With the same model the predominance of any given copper species as a
function of pH and carbonate content can be calculated. This is illus-
trated in Table 1 for a total copper concentration of 1 x 10 6 M.
Such equilibrium calculations are considered useful for toxicity studies.
They can be used to predict the form of copper under defined experi-
mental conditions. This is particularly relevant since available ana-
lytical techniques have not been refined enough to distinguish between
metal complexes in solution.
28
-------
2 —
3
8
bo
o
7
T
TENORITE
COPPER
CONC,
(MOLAR)
10
-5
10
-4
10
-6
10
-7
0 5
7
9
pH
11
13
Figure 4. Stability field diagram for tenorite and
malachite as a function of pH and total
carbonate concentration at various copper
concentrations.
29
-------
Table 1. DISTRIBUTION OF SOLUBLE COPPER AS A FUNCTION OF pH AND TOTAL CARBONATE
OJ
o
Moles/liter
PH
5.5
7.0
8.0
5.5
7.0
8.0
5.5
7.0
8.0
5.5
7.0
8.0
5.5
7.0
8.0
[co3lt
IxlO"5
IxlO"5
IxlO"5
5xlO~5
5xlO~5
5xlO~5
1x10'"
IxlO"1*
1x10""
5x10-"
SxlO"1*
SxlO'1*
1x1 0~3
lxlO~3
IxlO"3
Soluble Cu3
io"6
10-6.3
10-7.99
10~6
10-6.26
10-7.81
10~6
10"5-22
10-7.66
10~6
10"6
ID-MS
10~6
10~6
10~6.86
Cu2+
100
89
44
100
81
29
100
74
20
100
43
6
98
28
3
CuOH+
-------
Table 1. (Continued)
Moles/liter Soluble copper (%)
PH
5.5
7.0
8.0
5.5
7.0
8.0
[co3]t
5xlO~3
5xlO~3
5xlO~3
IxlO"2
IxlO"2
IxlO"2
Soluble Cua Cu2+ CuOH+ Cu(OH)3 Ci
10~6 93 <1 <1
10~6 7 7 <1
10~6-17 <1 1 <1
10~6 89 <1 <1
10~6 4 <1 <1
10"6 <1 <1 <1
2 2 —
i(OHH CuCOa Cu(COs)2
cl 6 <1
cl 91 <1
cl 95 <1
cl 11 <1
cl 95 <1
a 91 8
a Total copper = lO"6'00 M
-------
SECTION V
ANALYTICAL CHEMISTRY
OF AQUEOUS COPPER
Discussions in this section include a critical review of existing
methods for trace copper measurement, with emphasis on their applica-
bility and limitations. More detailed discussions are presented on
electroanalytical techniques, specifically anodic stripping voltammetry
and potentiometric membrane electrodes. These techniques have been
primarily used in this study in conjunction with the bioassay experi-
ments .
GENERAL REQUIREMENTS FOR TRACE METAL ANALYSIS
The principles and procedures of analysis for trace metals, including
copper, in the aqueous environment have been described by Hume ,
Mancy , Morrison , and others. The qualities most often noted
for an ideal method of trace metal analysis are detection limits, sen-
sitivity, selectivity, the ability to distinguish free from complexed
metal, and general applicability for use with natural samples. The
analytical method should have low detection limits, sufficient for
direct determination of the trace metal without pretreatment or precon-
centration steps. Copper, for instance, occurs at levels of 10 7 to
10 8 M in natural waters, and an appropriate method should be able to
reach this level of detection with reliability. Selectivity implies
the capability to distinguish a signal that is due strictly to the
trace metal in question and not to the presence of any interfering ion
that may be present in the test solution.
32
-------
Ideally speaking, the analytical procedure should also be capable of
defining the metal species under investigation. Lack of information
on the speciation of trace metals has been a main obstacle to the
elucidation of their role in natural waters. Information on the type
of species present (e.g., type of complexes and their stability and
rates of complex formation) is a prerequisite to a better understanding
of the distribution and function of trace metals in aquatic environ-
ments.
Copper does not exist in simple ionic form in natural waters. The work
of Corcoran and Alexander , Slowey and Hood , and Foster and
(54)
Morris has led them to believe that as much as 50 percent of the
copper present in sea water is in the form of organic complexes.
/ o / \ ^ o o \
Later work by Barber and Ryther and Johnston has shown that the
organic complexes of copper can be correlated to the biological activ-
ity in sea water.
The applicability of a method sums up a variety of factors of practical
consideration. The reliability, reproducibility, and precision of the
method in question all fall under the heading of applicability. Other
fundamental considerations are (1) the portability and ruggedness of
equipment for field operations, (2) the time required for a single
analysis, (3) the simplicity of the operation, and finally (4) economy,
space, and additional equipment requirements.
Available Analytical Methods for Trace Copper Analysis
The analyst can select among a number of analytical procedures with
different performance characteristics ~ . Various trace analysis
techniques and their general sensitivity ranges are shown in Table 2.
The following discussion will center on the applicability of some of
the more common techniques for copper and includes spectrophotometric,
radiochemical, and electroanalytical methods of trace copper determina-
tion.
33
-------
Table 2, SENSITIVITIES OF INSTRUMENTAL METHODS
FOR MONITORING METALS IN WATER3
Method
Polarography
Molecular Absorption
Atomic Absorption (Flame)
Atomic Emission
Square Wave Polarography
Sweep Voltammetry
Emission Spectroscopy (Arc)
X-Ray Fluorescence
Pulse Polarography
Molecular Fluorescence
Spark Source Mass Spectrometry
Anodic Stripping
Neutron Activation
Atomic Absorption (Flameless)
Chemiluminescence
Emission Spectroscopy (Plasma)
Sensitivity
100
100
100
100
10
10
10
10
1
1
1
0.1
0.1
0.1
0.1
0.1
» PPb (yg/1)
- 1,000
- 1,000
- 1,000
- 1,000
100
100
100
- 100
10
10
10
10
10
10
10
10
For any method the sensitivity will vary according to the
particular metal.
34
-------
Optical Techniques — The optical techniques most commonly used in
trace copper determination include atomic absorption spectrophotometry
and colorimetry. In recent years atomic absorption has become one of
the most popular methods of analysis for copper in natural and waste
waters because of its selectivity, speed, and relative simplicity of
analysis. The technique requires as little as a 5- to 10-ml sample to
perform analysis, and sensitivities in the region of microgram of cop-
per per liter have been achieved with normal flame absorption techniques
Non-flame techniques can improve on this sensitivity, but are
more costly and are severely lacking in precision. The main limita-
tions of both flame and flameless atomic absorption techniques are:
(a) metal species cannot be determined; (b) the technique is strictly
a laboratory procedure and cannot be used in the field; and (c) cost
per analysis is high in comparison to electroanalytical procedures.
Colorimetry, in contrast to atomic absorption, is simple and inexpen-
sive. The American Public Health Association et al. prescribe
three colorimetric methods for use in copper determination, and these
methods are capable of achieving detection limits near 20 ppb depending
on the experimental conditions and the presence of interfering ions.
1. The cuprethol method achieves its maximum sensitivity with
100-ml nessler tubes for visual comparison, but mercurous,
nickel, silver, bismuth, and cobalt ions can act as signifi-
cant interferences.
2. The bathocuproine method is just as sensitive as the cupre-
thol method, and no ions interfere significantly when in
concentrations below the parts-per-million range.
3. The most specific reagent for copper, neocuproine, is most
commonly used for analysis of aqueous copper.
35
-------
The main limitation with coloriraetric procedures is lack of selectivity
and the effect of interfering ions. In addition, colorimetric proce-
dures require a number of preparatory steps as well as a separation or
concentration step. Colorimetric procedures are also incapable of
metal speciation since complex forms of copper may interact slowly or
not at all with the color-developing compound.
Radiochemical Techniques — Neutron activation analysis is a very sen-
sitive analytical procedure with detection limits in the parts-per-
( 58}
trillion (ppt) range . Hence, no preconcentration step is necessary.
Nevertheless the matrix effect can be quite significant and may result
in supressing the signals. Beyond matrix effects, the most serious
limitations of activation analysis are its inability to distinguish
species of trace metals and the elaborate, sophisticated, and expensive
equipment required for operation.
Miscellaneous Techniques -— Several techniques that are not easily
categorized have been used in recent years for trace analysis. Mass
spectroscopy is a very sensitive technique for trace copper analysis,
but extensive sample preparation is required and the overall method is
(59)
time consuming and expensive and requires skilled analysts
Electron spin resonance has also been used for the analysis of copper
(60)
Electroanalytical Techniques — Among the various electroanalytical
techniques particularly applicable for the analysis of copper in
aqueous samples are (a) potentiometric membrane electrodes, (b) conven-
tional and pulse polarography, and (c) anodic stripping voltammetry.
These techniques vary in their limit of detection from 10~5 M for con-
ventional polarography to 10 10 M for anodic stripping voltammetry.
Perhaps the unique advantage of electroanalytical techniques lies in
their ability to distinguish between free and complexed metallic species,
Additional advantages of these methods are their capability of in situ
3-6
-------
measurement, their high selectivity, and their easy adaptability for
field operations. Furthermore, electroanalytical measurement of metals
in aqueous samples is considerably less expensive than atomic absorption
spectrophotometry. The comparison is based on capital cost of equipment
and cost per analysis including manpower requirements.
The oldest of the above-mentioned techniques is conventional polaro-
graphy, with a detection limit for copper of about 10 5 M, a range of
practical operation of 10 2 to 10 5 M, and a precision of 1 - 3 percent.
If preconcentration steps are used, the sensitivity can be lowered to
about 10~7 M.
Cathode-ray polarography or oscillographic polarography has been used
for analysis of natural water and wastewaters with a detection limit of
10 7 M ' . This technique involves the use of a cathode-ray oscil-
loscope to measure the current-potential curves of applied (saw-tooth)
rapid potential sweeps during the lifetime of a single mercury drop.
Multiple techniques are also applicable. The peak current in the re-
sulting polarogram is related to the concentration of the electroactive
species for a reversible reaction. Oscillographic polarography has the
advantages of (1) relatively high sensitivity, (2) high resolution, and
(3) rapidity of analysis. Copper can be determined at the 50 ppb level
((."})
in natural waters by this technique
The basic polarographic techniques are further modified in pulse and
differential pulse polarography, both of which may be used to increase
the sensitivity and minimize the effects of interferences . Pulse
polarography has the advantage of extending the sensitivity of deter-
mination to approximately 10 8 M. The pulse technique is based on the
application of short potential pulses of 50 msec on either a constant
or a gradually increasing background voltage. Following application of
the pulse, current measurements are usually done after the spike of
charging current has decayed. The limiting current in pulse polaro-
graphy is larger than in classical polarography. In derivative pulse
polarography, the voltage pulse is superimposed upon a slowly changing
37
-------
potential (about 1 mV per sec) and the difference in current between
successive drops is recorded versus the potential.
Differential pulse polarographic techniques are based on measuring the
current twice during the lifetime of each mercury drop and recording
the difference . Current peak-shaped curves are obtained where the
peak height is proportional to the metal concentration. This technique
is 100 - 1,000 times more sensitive than classical polarography, and 10
times more sensitive than nondifferential pulse techniques.
Anodic stripping voltammetry (ASV), sometimes referred to as inverse
polarography, is a simple yet extremely useful electroanalytical tech-
nique for trace metal analysis. It offers a detection limit of as low
as 10 8 to 10 1° M for copper, which is well within the range of natur-
ally occurring levels. Moreover, ASV, along with other electrochemical
methods, has the ability to distinguish free from complexed metal. The
method is highly specific for copper and, even at very low concentra-
tions, the precision is near 5 percent. The equipment required is
relatively inexpensive, readily transported, and has been used in the
field.
The application of ASV to environmental studies has until recently
received little mention in the literature. Matson and coworkers have
been the forerunners of bringing attention to this useful technique.
Their work involves studies on the Great Lakes ' , arctic lakes ,
and sewage effluents . Evidence for the existence of trace metal-
organic complexes is clearly presented in these works; however, the
techniques utilized for the characterization of these complexes general-
ly involved only acid digestion and acid titration procedures. The only
other available study on the application of ASV to the characterization
r t o\
of trace metal complexation is that of Zirino and Healy , Their
study of the analysis of zinc in sea water by ASV included the observa-
tion that the peak current associated with zinc decreased as the pH
increased. This phenomenon was explained on the basis of the increased
formation of ZnCC>3 or Zn(OH>2 with increasing pH.
38
-------
Other environmental studies with ASV have been primarily concerned with
the quantitative determination of trace metals in aquatic systems.
Peterson, Brant, and Mancy recently used the composite mercury-
graphite electrode (CMGE) in the study of the -distribution of copper
and lead in a municipal water system. This study included seasonal,
daily, and hourly variations in the concentrations of these metals at
various points along the distribution route of municipal water. Varia-
tion in the concentrations of the metals was related to residence times
in the pipes as well as variations in the original source of the water
and in the rate of consumption. Anodic stripping voltammetry was chosen
as the primary analytical technique for the characterization of copper
in the bioassay study. In the following section a detailed discussion
of the experimental procedures and methods of metal speciation is
presented.
ANODIC STRIPPING VOLTAMMETRY
Introduction
Anodic stripping voltammetry (ASV) is a combination of (a) a concentra-
tion step (pre-electrolysis, cathodic deposition, or plating) in which
the metal ion (or ions) of interest is reduced by controlled potential
electrolysis, either by deposition as a metal onto a solid microelec-
trode or by formation of an amalgam with the mercury of a mercury drop
or mercury film electrode and (b) a following step consisting of an
anodic dissolution process (reverse electrolysis or stripping) in which
the metal is oxidized and returned to the solution. As with other elec-
trochemical techniques ASV is a nondestructive method of analysis.
The concentration step is carried out for a definite time under repro-
ducible conditions such that the concentration onto the electrode is
quantitative (total plating) or such that a reproducible fraction of the
desired component is deposited from solution (partial plating). By
controlling the potential during this process, more easily electrolyzed
constituents may be separated. The concentration step is thus performed
39
-------
by applying to the electrodes a potential which is cathodic of the
polarographic halfwave potential by some 0.3 to 0.4 volts (where the
current has its limiting value) and maintaining this potential for a
given period of time under reproducible conditions of stirring, type
and area of microelectrode, and composition of medium.
After a short "rest period" to allow the solution to become quiescent,
the stripping process is initiated. The stripping step is performed by
a voltammetric scanning procedure, which produces a response proportion-
al to the amount of material deposited. The resulting stripping "vol-
tammogram" produces peaks whose heights are generally proportional to
the concentrations of the corresponding electroactive metal ions and
whose potentials are a qualitative indication of the nature of the
species present in solution. On this basis, the important character-
istics of the peak are its height (peak current, i , in microamperes),
its width at h± (w, , in volts or millivolts), and its potential (E ,
p -2 p
in volts). These characteristics are affected by the type of micro-
electrode used and by the rate of voltage change (sweep rate, v, in
mV/sec) used in the stripping process.
Anodic stripping voltammetry, which is also known as linear potential
sweep stripping chronoamperometry, stripping analysis, anodic amalgam
voltammetry, and inverse voltammetry, has been successfully applied to
the analysis of Ag, Au, Ba, Bi, Cd, Ga, Ge, Hg, In, K, Mn, Ni, Pb, Pt,
Sb, Sn, Tl, and Zn , in addition to the analysis of Cu.
40
-------
Theory
In the plating step, metal deposition follows a first-order rate
expression
Q = Q (1 - e~kt)
vt vo '
where Q = number of coulombs equivalent to metal deposited at
time t (coulombs)
Q = number of coulombs equivalent to total amount of metal
in test solution (coulombs)
t = time of electrolysis (seconds)
k = rate constant defined by the expression (sec )
k = DA/V6 (41)
where D = diffusion constant of the electroactive species in solution
(cm /sec)
2
A = test electrode surface area (cm )
3
V = volume of the solution (cm )
6 = effective thickness of the diffusion layer (cm)
It is essential to conduct the plating step at conditions of constant
stirring, temperature, cell geometry, and supporting electrolyte.
The stripping step is normally conducted in a quiescent solution. The
voltammetric technique normally employed during the stripping phase is
potential sweep chronoamperometry (application of an anodic linear
potential sweep); however, other voltammetric procedures may also be
applied. Of those which have been attempted, square wave polarography
, radio frequency techniques , differential polarography ,
(74)
and pulse polarography have proven successful.
41
-------
The working electrode employed in this study was the thin-film mercury
graphite electrode . The current-potential equations for the an
stripping curves with mercury thin-film electrodes were derived and
verified b
equations:
graphite electrode . The current-potential equations for the anodic
srcur;
verified by Roe and Ton! . Their derivations result in the following
i = nFA!C° v/e (42)
EP • Eo + ¥
where n = number of electrons in the electrode reaction
F = Faraday
A = surface area of the electrode
1 = thickness of the mercury film
C = concentration of reduced metal in the electrode
R
v = rate of potential change
= nF/RT
6 = thickness of the unstirred layer of solution adjacent
to the electrode
Dn = diffusion coefficient of the oxidized species in solution
These equations predict that i is a linear function of v, and E is a
P P
linear function of log v. The equations have been experimentally veri-
fied and have been found to be valid at low sweep rates. Large values
of v can cause departures from the equations because of a breakdown in
the assumptions that (a) 5 is a constant from one sweep rate to the next
and (b) the diffusion rate of deposited metal out of the mercury film is
extremely rapid compared to the sweep rate. The effect may be signifi-
cant for i , but is only slight for E .
P P
Regardless of which type of electrode is used in ASV, the analytical
results obtained from the stripping curve are always dependent in some
way on the sweep rate and on some property of the geometry of the elec-
42
-------
trode. These factors profoundly influence the peak characteristics,
namely, the peak current, the peak potential, and the peak width.
I)iagnostic Criteria
Trace metals in the aquatic environment are frequently found in the
parts-per-billion range or lower (10~"8 - 10~10 M) . With most analytical
procedures such low levels necessitate a preconcentration step before
the actual analysis. Anodic stripping voltamraetry with the composite
mercury-graphite electrode (CMGE), however, has a great enough sensitiv-
ity to permit direct determinations of metal ions at these concentration
levels. In addition, it is a nondestructive process which permits
repeated determinations on the same sample. Therefore, ASV is a tech-
nique highly suited for environmental samples.
Additionally, ASV also offers the unique opportunity to study trace
metal complexation at the environmentally significant levels. This is
based on the following diagnostic procedures for the differentiation of
free and complexed metals and for the characterization of metal com-
plexes: (a) acid digestions; (b) variations of ASV parameters, namely,
sweep rate, plating time, and plating potential; (c) titrations of
natural water samples with acid, metals which cannot be determined by
ASV, metals which can be determined by ASV, and titrations of the trace
metals in water samples with organic ligands. Total metal content can
be determined by digesting the sample before ASV analysis, and filtra-
tion separates dissolved from particulate fractions on the basis of
particle size. The analytical scheme is illustrated in Figure 5.
In a discussion of the types of complex systems that might be encouraged
in bioassay waters, there are two broad classifications with several
subclassifications depending on the types of ligands present:
Case I. Excess trace metal (M)
a. (M) and (MLL)
b. (M) and (MNL)
c. (M) and (MLL) and (MNL)
43
-------
SAMPLE
digestion
total
digestion
filtration
particulate
dissolved
addition of materials
variation of ASV parameters
H
electroactive metals
non-electroactive metals
ligands
sweep rate
plating time
stripping medium
plating potential
mixing
Figure 5. Analytical scheme for anodic stripping voltammetric
determination of metals in natural waters,
44
-------
Case II. Excess ligand, (LL) or (NL)
a, (LL) and (MLL) and (MNL)
b. (LL) and (NL) and (MNL) where the nonlabile
complex (MNL) is the stronger complex
c. (NL) and (MLL) and (MNL)
d. (LL) and (NL) and (MNL) where the labile
complex (MLL) is the stronger complex
In this classification M stands for the metal, LL is a labile ligand
which forms complexes whose rate of dissociation is faster than the rate
of plating, and NL is a nonlabile ligand which forms complexes whose
rate of dissociation is slower than the rate of plating. Note that the
definition of labile and nonlabile ligands is an operational definition
based on the dissociation kinetics of their respective complexes. This
should be differentiated from the definition of weak and strong com-
plexes which have therraodynamic significance based on stability con-
stants. Note also that the above classification assumes a water sample
containing a mixture of available ligands.
Based on the above limitations of the terms labile and nonlabile, it
can be concluded that i measurements can differentiate between free
P
metals and labile metal complexes on one hand and nonlabile metal com-
plexes on the other hand. A further differentiation between labile and
nonlabile metal complexes is possible from data obtained during metal-
organic ligand titrations. E measurement can generally distinguish
between free metals and complexed metals, both labile and nonlabile.
The stability constants can easily by calculated from E measurements.
Furthermore, the technique is suitable for assessing rates of metal
complexation. This is illustrated in Figure 6.
Results from the titrations of metal by the addition of organic ligands
provide evidence for metal-organic complexation; however, this technique,
in most cases, cannot be used to characterize the type of metal-organic
interaction. On the other hand, metal-organic titration in which the
metal is the titrant is a useful diagnostic criterion for the character-
ization of both labile and nonlabile ligand-metal interactions
45
-------
i Measurements-
•FREE METAL'
COMPLEXED METAL-
-LABILE METAL COMPLEXES'
•E Measurements
P
E and i
P P
Measurements
L—NONLABILE METAL COMPLEXES-
Metal Titrant
Titrations
Figure 6. Anodic stripping voltammetric characterization of metallic species.
-------
Experimental
Heath polarography system, Model EUW-401, (Heath Company, Benton Har-
bor, Michigan) and a chopper stabilizer, Model EUA-19-4, were used in
this work. A polarographic module interface unit, Model 1014, (Envi-
ronmental Sciences Associates, Inc., Newtonville, Massachusetts) in
conjunction with a Heath operational amplifier, Model EUW-19A, was used
to provide the necessary voltammetric deposition and stripping modes
for the simultaneous operation of four cells. Voltammograms were re-
corded on a Honeywell Electronik 194 strip chart recorder (Honeywell,
Inc., Fort Washington, Pennsylvania).
Measurements were performed in 100-ml fused quartz beakers, and the
electrodes were inserted into the cells through rubber stoppers. The
cell and electrode arrangement is shown in Figure 7.
The reference and counter electrodes were both separated from the solu-
tion by 4-mm-diameter Vycor plugs. These plugs were attached to the
electrode proper (composed of 4-inch lengths of 4-mm-diameter Pyrex
tubing) by means of sleeves of Teflon tubing.
The counter electrode was filled with supporting electrolyte. Electri-
cal contact was made by means of a platinum wire inserted in the tube.
Contact to the solution in the cell was made through the porous Vycor
plug. A saturated mercury-mercurous sulfate reference electrode was
(78)
used and constructed as described by Lingane . The test electrode
was a composite mercury-graphite electrode (CMGE). The electrode sur-
2
face area was 2.2 cm . Detailed discussions on the preparation and
properties of the CMGE are found elsewhere ' .
A nitrogen-carbon dioxide gas mixture was used for deaeration of the
test solution. The pH was controlled by using appropriate partial
pressures of carbon dioxide.
47
-------
Test Electrode (CMGE)
Counter Electrode (Pt)
Rubber
Stopper
Vycor Plugs
and
Mixture
\
V
\
/
1
-
0
o
\
/
V-
n
'/
/
/
/
/
/
/
/
/
/
/
/
L
°0^
, < Reference Electrode
/ (Hg/Hg2S04)
^ — Teflon Gas Purger
/
O GS
7
100 ml Quartz
Beaker
Figure 7. Cell and electrode arrangement for anodic
stripping voltammetry.
48
-------
STUDY OF COMPETITIVE METAL INTERACTIONS
Water samples containing 5 ppm humic acid were titrated with copper in
the presence and absence of 40 ppm calcium. The titration curves shown
in Figure 8 illustrate the competitive interaction between copper and
calcium for humic acid ligands . The lack of peak current signal at
low Cu concentrations was believed to be a result of the formation of
strong nonlabile complexes. At high pH values the capacity for non-
labile metal complex formation is greater than at low pH values. After
the nonlabile ligand has been saturated, further addition of copper to
the test solution is accompanied by an increase in peak current signals.
The presence of calcium exhibits little or no effect on copper-humic
acid interactions. Thus, copper competes favorably with calcium for
the formation of a nonlabile complex with humic acid.
For comparative purposes Figure 9 shows the same titration curves with
cadmium. These titration curves are clearly linear, indicating predom-
inantly cadmium-humic acid labile interactions. Furthermore, the pre-
sence of calcium causes a release of cadmium from its complexes and
allows for a more rapid interaction between the trace metal and the
test electrode. This is evidenced by the increased peak currents ob-
tained in the presence of calcium.
Examples shown in Figures 8 and 9 represent two different heavy metal-
organic interactions. Copper forms relatively stable, nonlabile com-
plexes with humic acid, but cadmium seems to form more labile com-
plexes. Calcium competes favorably with cadmium, and not with copper
for complex formation with humic acid. Additionally, humic acid
represents a mixture of labile and nonlabile ligand characteristics.
Because in the case of cadmium, the addition of calcium causes an in-
crease in peak currents, we believe that the cadmium-humic acid inter-
action is not exclusively labile. A summary of metal-humic acid-calcium
interactions for a number of trace metals is given in Table 3.
49
-------
•H
pH=8.5
0
pH-7.1
10 12 14
Q
(Cu) x 10 (moles/liter)
Figure 8. The effect of calcium and pH on the variation of
the peak current during the titration of 5 wg/ml
humic acid with copper.
O Calcium absent
O Calcium present
50
-------
20
0
40
20
40
1
.H^ 20
0
pH=8.5
pH=7.1
pH=6.2
0 2 4 6 8 10
(Cd) x 10 (moles/liter)
Figure 9. The effect of calcium and pH on the variation of
the peak current during the titration of 5 pg/ml
humic acid with cadmium.
O Calcium absent
D Calcium present
51
-------
Table 3.
SUMMARY OF TRACE METAL-HUMIC ACID-CALCIUM INTERACTIONS
AS OBSERVED DURING TITRATIONS WITH METAL IONS
Metal
Nature of Interactions
Bismuth
Cadmium
Copper
Indium
Lead
Thallium
Nonlabile interactions becoming
increasingly labile with increasing
pH; successful calcium competition
for labile ligands
Labile interactions; successful
calcium competition
Strong nonlabile interactions
increasing with increasing pH;
successful calcium competition
for labile ligands
Nonlabile interactions; successful
calcium competition for labile
ligands
Increasing lability with increasing
pH; successful calcium competition
increasing with increasing pH
Labile interactions; successful
calcium competition
52
-------
POTENTIOMETRIC MEMBRANE ELECTRODES
An Orion cupric ion selective electrode was used in this study to mea-
2+
sure Cu concentrations. Absolute potentiometric measurements followed
a Nernstian relationship. The detection limit for this electrode system
was 10 ' M. Pertinent information on metal speciation can be achieved
from potentiometric titrations.
The formation of the soluble complex species CuC03 has been studied by
/o/-)\ (81 } ( 82^
Scaifev , Silmanv , and Stiff ^ . This complex is found in
natural waters at pH 6.5 to 8.0; its concentration is governed by the
concentration of carbonate.
o+ -
The equilibrium constants for the reaction between Cu and HC03 were
( 82}
studied. A modified procedure from that described by Stiff was
used. This included rigorous control of pH and total carbonate. Based
/go\
upon the techniques reported by Stiff , but in which both copper
activity and pH were monitored , we have determined the equilibrium
constant for the reaction of cupric and bicarbonate ions to form CuC03.
Three concentrations of sodium bicarbonate (1.0 x 10 3, 2 x 10 3, and
5 x 10~3 M) and three concentrations of copper (1.58 x 10~6 , 7.9 x 10~6 ,
and 1.58 x 10~5 M) were used.
It was assumed that the following two equilibrium relationships govern
the copper-bicarbonate system:
Cu2+ + HC03~ + CuHC03+, K = [CuHC03+] /[Cu2+] [HC03~] (44)
Cu2+ + HC03~ t CuC03 + H+, K = [CuC03] [H+] /[Cu2+] [HC03~] (45)
53
-------
Under these conditions, the total copper [Cu ] is approximated by the
following equation:
[Cu ] = [Cu2+] + [CuC03] + [CuHC03+], or
[CuHC03+] + [CuC03] = [Cut] - [Cu2+], (46)
which by substitution gives,
([Cutl - [Cu2+])/[Cu2+][HC03~] - K! + K2/[H+]. (47)
The values of ([Cu ] - [Cu2+])/[Cu2 ][HCO ~] when plotted against the
+
corresponding values of I/[H ] would be linear if equations (45) and
(46) govern this system. The slope of the line will be equal to K2,
and the intercept of the ([Cu^] - [Cu2+])/[Cu2+][HC03~] axis will be
equal to KJ.
The results obtained with different bicarbonate solutions are shown in
Figure 10. The graphs have the following features: (a) The intercepts
on the ([Cu ] - [Cu2 ])/[Cu2 ][HC03~] axis are all very close to the
t +
origin showing that K} is close to zero and that CuHC03 is a virtually
o
non-existent species. (b) Below a pH value of 8.0 in 5.0 x 10 M
bicarbonate, pH 7.6 in 2.0 x 10 3 M bicarbonate, and pH 7.3 in 1.0 x
10~3 M bicarbonate equations (44) and (45) do represent the system and
2+
since KJ. is nearly zero [Cut] - [Cu ] = [CuC03]. (c) At pH values
greater than those indicated the plots are nonlinear, indicating the
formation of a complex or complexes in addition to CuC03. The values
of K were computed from the slopes of the linear portions of the
graphs. The values of K£ vary with varying total ionic strength. The
thermodynamic equilibrium constant K2 ' was calculated from the equation:
(aCuC03) (V)/(aCu2+) (a-)' (48)
54
-------
80
70
60
50
40
30
"A"
1 .0 x ID"3 M
[HCO§]
o
20
10
0
0 40 80 120
1/[H+] x IQ6
80
70
60
50
40
30
20
10
5.0 x 10~3 M
[HCO-]
1 I
50 100 150 200 250
1/[H+] x 106
20 40 60 80 100
1/[H+] x 106
Figure 10. Graphical representation of Cu2+ - carbonate ion titrations.
-------
The value of (a ) is 1.0 since CuC03 is an uncharged species, and
3 (83)
the other activity coefficients were taken from Kielland and Stiff
(82)
The stability constant of the complex species CuC03 can be evaluated
from the following equations:
Cu2+ + HC03~ £ CuC03 + H+ (49)
and C032~ + H+ + HC03 , K3' = [aHC03~]/[aC032~][aH+
(50)
a
where denotes activity. In summing equations (49) and (50),
Cu2+ + C032~ J CuC03
-a
CuC03]
a o+ a
[aCu2+][aC03
or pKit' = pK2' + pK3'. (52)
The equilibrium and stability constants for this reaction at different
bicarbonate ion concentrations are given in Table 4.
By using a value of -10.3 for pK3' as reported in the literature ,
the pKit values determined from our experimental data were found to be "
-7.3. We realize that pKV determined from our results is higher than
(82) (81)
those reported by Stiff and by Silman . Nevertheless, our re-
sults were quite reproducible as indicated in Table 4.
56
-------
Table 4, EQUILIBRIUM AND STABILITY CONSTANTS
Bicarbonate
concentration pK.2* PK.1+'
[M]
1.0 x 10~3 2.94 -7.3
2.0 x 10~3 2.96 -7.3
5.0 x 10~3 3.00 -7.3
57
-------
SECTION VI
DEVELOPMENT OF THE BIOASSAY SYSTEM
A vast amount of literature on metal toxicity is based on bioassay
experiments. Many of the available data resulted from studies with
limited objectives, e.g., the determination of the effect of waste
discharge on a receiving water. The remaining data are for the most
part ambiguous and difficult to interpret. In fact, most bioassay
data, if not all, have resulted from studies based on limited or no
consideration of the underlying chemistry. Regrettably, these data
are the only information available on which water quality criteria
can be based. At present, there is a pressing need for a better data
base — one which is founded upon scientifically conceived and rigor-
ously defined bioassay studies.
The chief task in this study was the design of a bioassay system in
which physicochemical and biological interrelationships were accounted
for and in which independent variables could be rigorously controlled.
The main interrelationships that need to be considered in the study of
metal toxicity are illustrated in Figure 11 in terms of (1) physico-
chemical characteristics of bioassay water, (2) concentration of metal
species, (3) metal uptake by test organism, and (4) biological response.
In this schematic representation the central factor is the physicochemi-
cal characteristics of the bioassay water. These properties will dic-
tate the concentration of the toxic metal species in the bioassay water
(arrow 1-2) and influence metal uptake by the test organism (arrow 1-3)
and the biological response (arrow 1-4). The concentration of toxic
58
-------
1. Physicochemical character-
istics of Bioassay Water
>
2-1
\
1-2
\
/
2. Concentration of
Toxic Metal Species
2-3
\
3. Metal Up
Test Or
/
4-3
A
\
/
take by
ganism
3-4
\
s
4. Biological Response
< l~2
\
{ 1-4
^
Figure 11. Physicocheraical and biological interrelationships
in bioassay experiments .
59
-------
metal species will in turn influence the physicochemical characteristics
of the bioassay water (arrow 2-1) through adsorption, complexation, ion
exchange, and other reactions of the metal species. Metal uptake by the
test organism is largely dependent on the concentration of the different
species of the toxic metal (arrow 2-3). The biological response of the
test organism is determined by the rate and magnitude of the metal up-
take (arrow 3-4). In turn the physiological status of the test organism
as indicated by the biological response will significantly influence
metal uptake (arrow 4-3).
Bioassays have been conducted in one of two ways: (1) in static systems,
usually to establish the minimum dilution of an industrial waste in
which fish or other aquatic organisms survive, or (2) in dynamic systems,
either in the laboratory or in the field, to determine the toxicity of
a given material. In neither approach, especially where metals are con-
cerned, has the investigator been able to effectively control the chemi-
cal and physical variables to permit his results to be applied to other
situations. It is virtually impossible to control all the variables in
a laboratory bioassay. Nevertheless, field experiments are not the com-
plete answer. Although the field test indicates the toxicity of the
metal under consideration in the environment, the investigator cannot
use this method to understand the role of each of the important physico-
chemical variables in producing the observed effect. The main physico-
chemical variables that need to be controlled are hydrodynamics, temper-
ature, ionic strength, light, dissolved oxygen, pH, alkalinity, and the
chemical composition of the bioassay medium. Some of these variables,
such as temperature and light, are usually considered by most investi-
gators, but others are rarely taken into account.
This section does not review the physical and chemical variables affect-
ing toxicity. A critical review of some of these factors has been pub-
. . . , . , (85)
lished elsewhere
60
-------
CHEMICAL SPECIES OF METAL TOXICANTS
Few bioassays have been conducted so that the results can be inter-
preted in terras of the toxicity of specific forms of the metal. No
studies have been conducted that controlled pH, alkalinity, and hardness
independently. Mount investigated the effect of pH and total hard-
ness on the acute toxicity of zinc to fish. Hardness was controlled at
values of 50, 100, and 200 mg/1 CaC03 by mixing limestone spring water
and deionized water. The pH was controlled by the addition of strong
acid or base. Addition of the strong acid or base will, besides con-
( 7 f\~\
trolling the pH, change the alkalinity of the system . For example,
at a hardness of 100 mg/1 the alkalinities were 16, 56, and 102 mg/1
CaCOs at pH 6, 7, and 8, respectively. The change in the alkalinity of
the system is due to the rapid establishment of chemical equilibrium
between the solution and the atmosphere.
Most investigators consider alkalinity and hardness to be related since,
in nature, waters of high alkalinity tend also to be high in hardness.
These two properties are, however, basically not related since alkalin-
ity refers to the system's acid-neutralizing capacity and hardness re-
fers to its divalent metal concentration or its capacity to complex with
EDTA. A solution of calcium chloride will have a high level of hardness
and a low level of alkalinity, whereas a sodium carbonate solution will
have a low hardness and a high alkalinity.
The concentrations of carbonate, bicarbonate, and carbon dioxide in an
aquatic system are of importance primarily because of their roles in pH
control, complexation of metals, and control of metal solubility. Below
( 86}
is presented a simple method, developed by Roberts and Allen , for
the control of total carbonate or total alkalinity and pH in the bioas-
say experiment. This technique is applicable to natural as well as
synthetic waters.
The formation of significant quantities of metal carbonate or hydroxy
complexes will affect the chemical relationships presented. The amount
of base and the partial pressure of carbon dioxide required to establish
61
-------
a desired set of conditions can be predicted if the appropriate equilibr-
rium constants in the equations presented are included.
EQUILIBRIUM RELATIONSHIPS IN THE CARBONIC ACID SYSTEM
Equilibrium Relationships of Total Alkalinity and pH
The total alkalinity of a sample is defined as its acid-neutralizing
/ f\ /• \
capacity . This can be expressed as:
Total Alkalinity = 2[C032"] + [HC03~] + [OH~] - [H+] (53)
where the brackets indicate concentrations, and the alkalinity is ex-
pressed in equivalents per liter. For a sample exposed to the atmos-
phere the concentrations of carbonate and bicarbonate ions are related
to the partial pressure of carbon dioxide (P ) by the following
CO2
relationships:
[co32 ]
and
Hf7-82p
c°2
[HC03 ] = - - - (55)
+
By substituting these relationships in equation (54) the following
equation results:
10-17. 85p lO"7-82?
C02 C02
[Total Alkalinity] = - -r-; - + - T
[H }2 [H+]
+
1 (56)
62
-------
In this expression for total alkalinity the pH and partial pressure are
the variables, but this expression will always equal zero unless a base
is added to the solution. This can best be shown by an example.
In a solution of sodium bicarbonate, sodium carbonate, or sodium hydrox-
ide, the following anion-cation balance will hold:
[Na+] + [H+] = 2[C032~] + [HC03~] + [OH~] (57)
Rearrangement gives :
[Total Alkalinity] = [Na+] = 2[C032~] + [HC03~] + [OH~] - [H+] (58)
The total alkalinitv is equal to the amount of base in solution and
will equal zero if no base is present.
We can now substitute the sodium ion concentration for total alkalinity
in equation (57), which on rearrangement yields an expression for the
partial pressure of carbon dioxide as a function of the total alkalinity
(or sodium ion concentration) and the pH:
10-17.85 + 10-7.82[H+]
This equation is important because it shows that the [H ] can be con-
trolled by the partial pressure of carbon dioxide and the amount of base
added to the solution. Both of these variables can be controlled by the
investigator.
A computer program for the computation of the P required to establish
(86)
equilibrium at any pH and total alkalinity is presented. in
Appendix B. Results for 10 l , 10 2 , and 10~3 eq/1 for the pH range
4.0 to 12.0 are presented in Appendix B. The results computed and
the titles given them in the computer output are total carbonate concen-
63
-------
tration (TC03), the logarithm of the total carbonate concentration
(LOG TC03), the logarithm of the partial pressure of carbon dioxide
(LOG PC02), and the logarithms of the molar concentrations of C032~,
HC03~, and H2C03 (LOG C03, LOG HC03, and LOG H2C03, respectively).
Equilibrium Relationships of Total Carbonate and pH — The total car-
bonate, [C032~] + [HC03~] + [H2C03], is also controlled by the partial
pressure of carbon dioxide and the pH according to the following rela-
tionship:
10-18.15P 10"7-82P
CO2 CO2
[Total Carbonate] = + + lO"1
[H+]2 [H+j co2 (60)
where [H2C03] = lO"1-47? (61)
Solving equation (60) for the partial pressure of carbon dioxide,
[Total Carbonate][H*]2
(62)
an expression is obtained that can be used to calculate the necessary
partial pressure of carbon dioxide for any desired pH and total carbon-
ate concentration. The concentration of sodium ion necessary to estab-
lish the desired conditions is given by equation (60) , which can be
rewritten
(10-17.85+10-7.82[H+])p [IlV-lO'1"-00
co2
[Na ] = - v --- T - (63)
[H ]2 [H+]
Addition of NaHC03, Na2C03 , or NaOH is necessary so that the total car-
bonate and pH may be independently varied.
64
-------
Calculation Procedures for Synthetic Waters —
Case I: Total Alkalinity and pH Control.
The procedure for controlling total alkalinity and pH in solu-
tions made from distilled or deionized water is very simple:
1) Decide what total alkalinity is desired and add that
molar concentration of NaHC03, NaOH, or half as much
Na2C03 to the solution.
2) Mix carbon dioxide with air or a nitrogen-oxygen mixture
and bubble the final mixture through the solution,
varying the amount of carbon dioxide until the desired
pH is obtained and maintained in equilibrium.
The pH may be varied simply by adjustment of the amount of car-
bon dioxide in the mixture, and the alkalinity will remain
constant at any pH.
Case II: Total Carbonate and pH Control.
The control of total carbonate and pH involves the following
calculation:
1) Calculate the amount of NaHCOa, NaOH, or NaaCOs neces-
sary for the desired total carbonate and pH from the
following equation which can be derived from equations
62 and 63.
[Total Carbonate] (10"17«85+10~7«82[H+]) [H+]2-10"llt •°°
2) After the addition of the sodium ion, the pH is adjusted
with a mixture of carbon dioxide and air or a nitrogen-
oxygen mixture as described in the total alkalinity
case. Once the desired pH has been obtained, the total
carbonate concentration should also be at the desired
level. These calculations and additions of base must be
done for each pH and total carbonate concentration.
65
-------
Figures 12 and 13 show the partial pressures of carbon dioxide and the
amounts of base necessary to maintain total alkalinities (eq/1) or total
_ O _ O
carbonate concentrations (moles/1) or 10 and 10 . There is a minimum
pH that can be maintained for any designated total carbonate concentra-
tion and a maximum pH that can be obtained for any desired alkalinity.
The maximum pH decreases by one unit as the alkalinity decreases ten-
fold. In Figures 12 and 13 the maximum pH decreases from 12 to 11 as
— O — O
the total alkalinity decreases from 10 to 10 eq/1. The minimum pH
that can be maintained in the case of total carbonate control is not
directly proportional to the total carbonate concentration. As the
_2 —3
total carbonate decreases from 10 to 10 M the minimum pH increases
from 4.0 to 4.7.
Limitations — Although it is theoretically possible to achieve any pH
and total alkalinity or total carbonate by this procedure, practical
constraints limit its usable range. At low pH's and high total alka-
linity or total carbonate, high concentrations of free carbon dioxide
may be lethal to fish. At high pH's the high ionic strength present in
the control of total carbonate may prove deleterious; also it may be
difficult in practice to control the low carbon dioxide pressure re-
quired to maintain a high pH. These problems should not develop in the
control of bioassay chemical conditions because extreme values of pH
and total alkalinity or total carbonate would not be desired.
Generally, the predominant factor in controlling pH and alkalinity is
the carbonate system. However, certain bioassay conditions may signi-
ficantly alter the total alkalinity, total carbonate, and pH levels.
For example, high hardness, alkalinity, and pH may cause carbonate pre-
cipitation and reduce the total alkalinity. High concentrations of
metal toxicants such as lead may also result in the formation of metal
hydroxides, which would lower the alkalinity. These conditions can be
recognized ff the pH obtained is significantly lower than the predicted
value.
66
-------
PH
Figure 12. Partial pressure of carbon dioxide and amount
of sodiun bicarbonate (moles/liter) required
to maintain total carbonate = 10~2 molar (solid
line) or total alkalinity = 10~2 equivalents/
liter (dashed line). Total carbonate cannot be
below pH 4.0, and total alkalinity cannot be
above pH 12.0.
67
-------
12
1/4
Figure 13. Partial pressure of carbon dioxide and moles/liter
sodium bicarbonate required to maintain total
carbonate -10 molar (solid line) or total
alkalinity = 10 3 equivalents/liter (dashed line).
Total carbonate cannot be controlled below pH 4.7
and total alkalinity cannot be controlled above
pH 11.0.
68
-------
SECTION VII
DESIGN OF BIOASSAY EXPERIMENT
As stated earlier, it is essential that the chemical factors affecting
copper toxicity to fishes be controlled. Although numerous reports
deal with the toxicity of copper, none includes an account of copper
species, e.g., free copper ions, inorganic and organic copper complexes,
and colloidal dispersions of copper species.
In our bioassay test facility the following variables were controlled:
(1) ionic strength; (2) inorganic ligands: hydroxide and carbonate;
(3) calcium; and (4) copper species. The following variables were mea-
sured during the testing of the bioassay facilities: (1) toxicity as
measured by standard bioassay analysis techniques; (2) concentration of
copper species including free, labile, nonlabile, particulate, and
total; and (3) characteristics of the bioassay water including pH, cal-
cium, carbonate, dissolved oxygen, and turbidity. The system design
insures that the bioassay water is of the desired chemical composition
and that it does not change during the course of the bioassay. This
stable condition is accomplished by using a synthetic bioassay water
that is allowed to reach chemical equilibrium before the beginning of
the bioassay, by using continuous flow bioassays, and by measuring the
chemical composition of the bioassay water throughout the test. Special
precautions must be taken to insure that water samples do not change
between collection and analysis.
Since the attainment of chemical equilibrium is often a slow process,
the bioassay water must be prepared and allowed to reach equilibrium
before exposing the test organism to it. An example of the slow at-
69
-------
tainment of equilibrium is illustrated in Figure 14. Lead, copper, and
zinc nitrates were added at an initial concentration of 2.5 x 10 6 M
in an aqueous solution of 10 3 eq/1 total alkalinity and pH 9. Pulse
polarographic measurement of copper, lead, and zinc as a function of
/ OJ \
time indicates a slow process of metal transformation . Consider-
able time is needed to assure the attainment of equilibrium. Conse-
quently, the common practice of adding the toxic agent immediately
before exposure of the test organism should be viewed with skepticism.
Needless to say, analyses conducted to verify the existence of a given
metal species at steady-state conditions may not be valid since mea-
surements are invariably made much later than the moment of exposure.
This discrepancy limits our ability to extrapolate from data obtained
uch ;
(89)
/• QO\
with "diluters," such as those described by Mount and Warner and
Esve.lt and Conners
Flow bioassays were deemed necessary to insure that chemical conditions
did not change during the bioassay. In addition to the usually cited
deficiencies of static bioassays (loss of toxicant through biological
uptake or adsorption and the build-up of toxic metabolic waste pro-
ducts), waste products released by the test organism may react with
copper resulting in changing the chemical species of the toxicant
present.
HATCHERY AND BIOASSAY SYSTEM
Fish Hatchery
A fish hatchery was established in which the necessary test organisms
(juvenile guppies, Lebistes retieulatus) were raised and conditioned to
the required environmental conditions before their use in a bioassay.
Over 600 adult guppies were maintained in two 70-gal. aquaria. Aquatic
plants covered the surface to provide cover for the young. Illumina-
tion was maintained 18 hours a day, and newborn fish were removed daily
and transferred to 5-gal. aquaria.
70
-------
CK5
c
3 n
ET
IN3 (U
+ o
H-
rr O
00
D
r-o
3
Q-
100
50
20 —
10 —
5 —
2 —
10
pH = 9
-3
alkalinity =10 eq/1—
20 30 40
TIME (Minutes)
50
60
-------
The juvenile fish to be used in the bioassay were approximately 10 days
to 2 weeks old. Guppies in this age group are no more susceptible to
copper poisoning and stress than older fish. This factor was demon-
strated during static bioassays in which two groups of fish of differ-
ent ages were subjected to identical copper concentrations. In all
tests the older fish died at the same rate as the younger fish. Because
it is easier to maintain a large number of younger fish, the 10-day to
2-week-old guppies were used as experimental organisms.
Ann Arbor tap water was used for the hatchery water supply. It was
dechlorinated by passage through a charcoal filter, and the pH was main-
tained in the 7.1 - 7.4 range. The conditioned water was held in a
250-gal. aluminum tank; the water in the 70-gal. aquaria was continu-
ously exchanged. The water supply system and hatchery are shown sche-
matically in Figure 15. Water level in the 250-gal. reservoir was
maintained by a float valve. The pH was measured, and the output from
the pH meter was used as the input for a controller with a built-in
recorder, enabling the record to be checked for pH deviations outside
the acceptable range. The controller was used to activate two peristal-
tic pumps to dispense acid and base. Although this system is capable of
maintaining a narrower pH span, the limits were set to provide a pH
within a tolerable range without requiring the pumps to be activated
excessively. At times only a single pump delivering acid was utilized;
the high pH (greater than pH 10) of the Ann Arbor water, as compared to
the desired pH, eliminated the need for addition of base.
Fish Conditioning
The fish-conditioning system, shown in Figure 16, included a 250-gal.
equilibrium tank that contained water of the required chemical compo-
sition. A heater and thermoregulator on a styrofoam float guided by
plastic rods maintained the conditioning water at 25° C. Water from
the equilibrium tank flowed through a series of 5-gal. aquaria con-
taining the fish to be conditioned for a bioassay. The aquaria were
equipped with heaters and constant-level siphons.
72
-------
1
2
3
4
5
6
7
Charcoal Filter
Float Valve
pH Electrode
Heater-Thermostat
Acid and Base Lines
Air Stone
Water Outlet
Ann Arbor Tap
Water Inlet
8 Light Fixture
9 Constant Head Siphon
10 Air Stone
11 Heater
12 Aquarium Filter
13 Rustrak RCpH Recorder-Contl.
14 Ministatic Acid and Base Pumps
To Acid &
Base Bottles
pH Control Center
11
10
Equilibrium Tank
70 Gallon Aquarium
Figure 15. Schematic diagram of hatchery.
-------
CONDITIONING WATER
LINE
AQUARIUM WITH
HEATER AND
CONSTANT HEAD
SIPHON
INDICATING LIGHT
FLOAT WITH HEATER
AND THERMOSTAT
5-GALLON AQUARIA
CONDITIONING EQUILIBRIUM TANK
Figure 16. Schematic diagram of fish-conditioning area.
-------
The fish were acclimated for 10 days. Two days before the beginning of
a bioassay, the conditioned fish were transferred to their appropriate
bioassay chambers. The conditioning water was then diverted to these
chambers. This procedure eliminated stress and death of fish due to
damage or weakening by transfer from the conditioning aquaria to the
bioassay chambers.
Moassay Water
The test solutions were prepared several days in advance of their use
to permit the solutions to reach chemical equilibrium and to allow time
for chemical analyses of the water before beginning a bioassay. The
required chemicals were added to charcoal-filtered, deionized water
contained in 250-gal. fiberglass-covered plywood tanks. Sodium bicar-
bonate was added to the water to control the pH. Then, carbon dioxide
and air were mixed in a 3 x 1 x 1-foot baffled fiberglass-covered ply-
wood mixing box. The gas mixture was bubbled through the test water;
large aquarium stanes were used for gas dispersion. The partial pres-
sure of carbon dioxide was adjusted until the desired pH of the water
was obtained. The theoretical considerations of the control of pH in
this manner have been discussed previously.
To control hardness calcium sulfate was added to the equilibrium tanks.
Magnesium sulfate, sodium chloride, and potassium sulfate, all neces-
sary ions for proper physiological function of the fish, were also
added to the water. Sodium sulfate was added to obtain constant ionic
strength for the experiments. The concentration of each of the chemi-
cals was: [Ca2+] = 2 x lO"4 M; [C032~] = 1 x 10~3 M; [Cl~] = 30 ppm;
+ 2+
[K ] = 4 ppm; and [Mg ] = 4 ppm.
Randomization and Exposure of Fish for Bioassays
In the bioassay, the fish must be assigned to the bioassay chambers in
a random manner since the fish removed from the aquarium may be the
weakest. Randomization prevents placing all the weakest fish in a sin-
gle chamber. A computer program was written that gives a random
75
-------
sequence for assigning fish to bioassay chambers. Fish were placed in
1-quart plastic containers until each container had one fish, then a
second fish was added to each chamber. This process was continued
until the required number of fish had been added to each container.
The fish in each container were then transferred to the identically
designated bioassay chamber. Plastic containers were used because they
could be clustered in a small area around the aquarium. This arrange-
ment minimized the time required to accomplish the randomization and
also minimized the time that a fish had to be kept in the net.
The computer program (Table 5) provides the random sequence for adding
the fish to the chambers. The variables L and M in the program are,
respectively, the number of bioassay chambers to be used in the experi-
ment and the total number of fish to be placed in each chamber. The
computer output for 12 bioassay chambers and 20 fish per chamber has
been included (Table 6). This computer program has as its essential
part the subroutine RANDU which generates the random numbers. This
subroutine produces the same results as the subroutine of the same name
available in IBM's Scientific Subroutine Package/System 360. The sub-
routine has been rewritten for use on the IBM 1130 computer and was
supplied by Professor L.S. Whitlock, Department of Biostatistics, School
of Public Health, The University of Michigan.
After all the fish were randomized to their respective chambers, they
were allowed to acclimatize to a 30 ml/min flow of conditioning water
for approximately 48 hours. Then the actual test-water solutions con-
taining copper flowed into the chambers at about 30 ml/min. The bioas-
say continued for 96 hours. At 8-hour intervals the chambers were
checked to measure various characteristics including pH, temperature,
resistivity, flow rate, and dissolved oxygen. Also, at these times
dead fish were removed from the chambers. For experimental purposes
lack of pectoral fin movement indicated death. The time and the day
of each fish death were recorded on data sheets throughout the 96-hour
experiment.
76
-------
Table 5.
RANDOM SEQUENCE FOR ADDING FISH TO THE CHAMBERS
DIMENSION J(14)
WRITE(1,200)
200 FORMAT(' GIVE ME 2 ODD RANDOM NUMBERS IN (14,IX,14)FORMAT.'/
1' BOTH NUMBERS SHOULD BE LARGER THAN 4000')
READ(6,300)1X1,1X2
300 FORMAT(14,IX,14)
1X1=1X1/2*2+1
1X2=1X2/2*2+1
WRITE(5,400)
400 FORMAT(1H1,8X,'RANDOM ORDER FOR PLACING FISH IN BIOASSAY CHAMBERS'
HIT FISH NUMBER 1A IB 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B
2 7A 7B'/1X,68('*'))
L=12
M=20
DO 60 K=1,M
DO 5 1=1,L
5 J(I)=-99
NC=0
40 CALL RANDU(IX1,IX2,X)
DO 20 1=1,L
IF(X*L-I)10,20,20
10 IF(J(I))30,40,40
30 J(I)=NC+1
NC=NC+1
IF (NC-L)40,60,60
20 CONTINUE
60 WRITE(5,100)K,(J(I),I=1,L)
100 FORMAT(1HO,6X,I3,3X,UI4)
CALL EXIT
END
77
-------
Table 6.
RANDOM ORDER FOR PLACING FISH IN BIOASSAY CHAMBERS
FISH NUMBER 1A IB 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B 7A 7B
********************************************************************
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
4
12
2
2
2
10
6
12
11
11
11
3
11
8
9
5
6
8
3
2
8
8
5
9
1
3
1
3
12
1
2
12
10
1
1
11
1
2
2
3
9
4
4
11
9
8
8
7
1
6
3
5
6
12
12
3
3
4
12
7
10
2
12
3
3
7
10
1
8
8
10
1
8
2
7
2
4
12
1
1
2
1
1
5
5
6
5
4
3
4
5
4
7
7
6
4
5
5
11
9
12
6
8
7
10
11
7
6
2
7
8
9
3
10
8
12
10
1
4
8
5
11
10
12
11
5
4
9
4
3
9
8
9
6
11
8
2
6
5
12
3
7
3
10
6
9
2
11
5
5
1
2
1
4
5
10
8
7
7
10
7
5
6
6
8
12
3
8
10
12
6
10
5
5
3
9
12
9
9
4
11
9
11
4
4
1
12
2
9
9
12
6
4
11
10
6
9
3
8
5
1
10
9
1
12
2
9
5
6
10
7
11
2
3
4
7
11
11
10
6
6
3
7
8
7
4
11
10
7
2
4
7
12
9
2
1
7
10
6
11
78
-------
Exposure Facility
The experimental facility is shown in Figures 17 and 18. The test water
flowed from the equilibrium tank to the bioassay chambers through 25-
foot coils of stainless steel tubing contained ir, three large water
baths connected by siphons and a pump. Medical-grade silicone tubing
was used for connections. The bioassay chambers were contained in the
water bath to permit better temperature control and to ease the disposal
of effluent water. Excess water in the bath was removed by a standpipe.
The temperature of both the bath and the water contained in the exposure
chambers could be regulated with a half-degree precision.
The exposure chambers (Figure 19) were fabricated from glass and sili-
cone sealant. These chambers had a volume of approximately 1 liter, and
the flow of water through the chambers was baffled by the placement of a
plastic screen across each end. The flow pattern through the chamber,
as determined by dye tracing, was even.
TOXICITY EXPERIMENTS
Three bioassay experiments were conducted for the sole purpose of asses-
sing the validity of the newly developed concepts and techniques of
chemical control. The time required to conduct one bioassay was between
2 and 3 weeks. Physicochemical data for pH, temperature, electrical
resistance, hardness, alkalinity, and total carbonate are presented in
Table 7. Alkalinity was determined by potentiometric titration, and
total carbon was determined by total carbon analysis.
Data for bioassays at pH 8.10, 7.00, and 5.67 (bioassays A, B, and C,
respectively) are presented in Table 8. Percentage survival was deter-
mined at 48 hours; in most cases the mortality at 96 hours' exposure
was so high that the data were not usable. In bioassay A all fish died
at the highest copper concentration. In addition, percentage survival
was lower at 130 ppb total copper than at 278 ppb. In bioassays B and
C there were no anomalies in the data.
79
-------
DEMINERALIZED WATER LINE
00
o
BIOASSAY
EQUILIBRIUM TANKS
WALK V
PUMP OUTLET
WATER BATH TANKS
Figure 17. Schematic diagram of bioassay area.
-------
CONDITIONING
WATER LINE
V
ON-OFF
VALVE
EQUILIBRIUM TANK
WATER BATH
TANK
Figure 18. Schematic diagram of bioassay system.
81
-------
MATERIALS; DOUBLE STRENGTH GLASS
PLASTIC SCREENING
BONDED WITH SILICONS RUBBER SEALER
^
4
2~
\
f
" 8 '""
3
16
— i
31
15 4 J
16
2
\
r5
G16
i
U. .„. r1
rL
h r^
5
3
|DIAX
— .
J
3
\
j
5
8
^
Figure 19. Bioassay exposure chamber.
82
-------
Table 7. SUMMARY OF BIOASSAY PHYSICOCHEMICAL DATA
00
U)
Bioassay A Bioassay B
Variable
X n s X n s
pH 8.10 67 0.12 7.00 66 0.06
Temperature
(°C) 24.8 56 0.2 24.7 49 0.3
Electrical Resistance
(ohms) 3230 48 78 3200 18 51
Hardness
(meq/1) 0.404 6 0.010 0.284 6 0.032
Alkalinity
(meq/1) 0.975 6 0.012 0.789 6 0.013
Total Carbonate:
(mM) Measured 0.95 6 0.035 0.87 6 0.038
(mM) Calculated
from Alkalinity 0.98 0.97
Bioassay C
X3 n s
5.67 120 0.33
24.9 38 0.2
4007 84 129
0.412 6 0.012
0.130 6 0.002
0.95 6 0.036
0.75
Arithmetic average
-------
TABLE 8. SUMMARY OF FISH SURVIVAL DATA AND COPPER MEASUREMENTS3
Total
Statistic Copper
Bioassay A X
(pH 8.10) N
s
X
N
s
X
N
s
X
N
s
X
N
s
X
N
s
<1
1
— — —
42
4
3
70
4
6
130
4
11
278
4
46
343
4
42
Soluble
Copper
— —
__ —
26
2
8
32
2
2
56
2
4
78
2
23
154
2
19
Partic-
ulate
Copper
— .__
— "— —
32
2
9
42
2
5
77
2
4
131
2
16
229
2
3
Free &
Labile Percentage
Copper Survival
—
6
2
2
10
2
1
22
2
1
39
2
1
51
2
1
100.0
92.5
42.5
7.5
15.5
0.0
. . . continued
Copper values are given in yg Cu/1.
84
-------
Table 8. (Continued)
SUMMARY OF FISH SURVIVAL DATA AND COPPER MEASUREMENT5
Statistic
Bioassay B X
(pH 7.00) N
s
X
N
s
X
N
s
X
N
s
X
N
s
X~
N
s
Total
Copper
1
39
3
12
62
3
3
155
3
18
254
3
49
340
3
46
Soluble
Copper
__^
27
2
9
40
2
8
104
2
40
185
2
74
244
2
12
Partic-
ulate
Copper
—
16
2
7
26
2
2
54
2
10
68
2
34
95
2
18
Free &
Labile
Copper
10
2
4
15
2
0
71
2
38
96
2
56
109
2
58
Percentage
Survival
97.0
92.5
76.5
50.0
33.0
13.0
. . .continued
values are given in yg Cu/1.
85
-------
Table 8. (Continued)
SUMMARY OF FISH SURVIVAL DATA AND COPPER MEASUREMENT3
Statistic
Bioassay C X
N
s
X
N
s
X
N
s
X
N
s
X
N
s
X
N
s
Total
Copper
<1
1
39
4
5
70
4
8
171
3
7
284
4
14
450
4
26
Parti- Free &
Soluble ulate Labile Percentage
Copper Copper Copper Survival
100.0
97.0
79.5
63.5
46.5
28.0
— — —
Copper values are given in Mg Cu/1,
86
-------
In bioassays A and B total, soluble, particulate, free, and labile
copper were measured. However, only total copper was determined in
bioassay C because at pH 5.67 total copper was equal to soluble copper,
hence particulate copper was absent. In addition, total copper at this
pH is equal to free and labile copper.
The performance characteristics of the bioassay experiments substantiate
the validity of the newly applied techniques of chemical controls.
Results of bioassays reported in Table 8 need further validation due
to lack of replications and should not be used as fish toxicity data
per se.
87
-------
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95
-------
APPENDIX A
AQUEOUS COPPER EQUILIBRIA AT CONSTANT TOTAL CARBONATE
AND VARIABLE pH VALUES
96
-------
0.010000
COPPIIU o.oooooi
PH
0.9
9.10
0.20
0.30
0.40
0.50
0.60
3.70
O.tO
O.OQ
I. CO
1.10
1.20
1.30
1.40
1.50
1.60
1.70
l.»3
1.90
2.00
2.10
2.20
2.30
2.*0
2.50
2.60
2.70
?.RO
2.90
3.00
3.10
3.20
3.31
3.'»0
3.50
3.60
3.70
3. "0
3.90
4.03
4.1 3
4.70
4.30
4.40
*.5T
*.'>0
4.70
«. °.o
4.90
CU
6.00
6.00
6.00
6. CO
6.00
6.00
6.00
6.00
6.00
6.00
6.03
6.00
••.03
6.00
6.30
fe.OO
6.00
6.00
6.0'J
6.00
6.00
6.00
<• .00
6.01)
6.10
6.00
6.00
6.00
6.00
6.00
A. 00
6.00
6.00
6. TO
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6. 00
6.00
6.00
6.00
6.00
6.00
CUOH
14.00
13.90
13.80
13.70
13.60
13.50
13.40
13.30
13.20
13.10
13.00
12.90
12. 33
12.70
12. AJ
12.50
12.40
:-'.30
12.23
12.10
12.03
u. 93
11.10
11.70
11.60
11.50
1 1.40
11.10
11.20
11.10
11 .00
10.90
10. "0
10.7'J
10.60
10. 10
10.40
10. 30
10.^3
in. 10
10.03
9.93
9.<*0
9.70
9.60
9.50
°.40
0.30
9.20
9.10
CUOH1
14.30
14.00
14.00
14. 30
14.00
14.30
14.00
14.00
14.30
14.00
- 14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14. JO
14.00
14.00
14.00
14.00
14.00
1 4. JO
14.00
14.00
14.30
14.00
14.00
14.00
14.00
1 4.00
14.00
14.00
14.00
14.00
I4.';o
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
CUOM4
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
'4.00
14.00
14.00
14.00
14.00
14.00
14.00
1't. 00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
|4. 00
14. 00
14.00
14. on
14.00
14.00
I'-.GO
14.00
1'. .00
14.00
14.00
14.00
14. 00
14.00
I4.no
14.00
14.00
ruco3
14.00
14.00
I'^.OO
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
i3.«3
13.63
13.41
13.23
13.03
12. <)3
12.63
1?.43
1?.23
1?.03
11. H3
11.63
11.43
11.23
11.03
10. »3
10.63
10.43
10.21
10.03
°. S3
9.63
9.43
9.23
9.04
B.F.4
S.64
8.44
«.25
6.05
C(J(C03)2
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.0'J
14.0')
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14. OO
14.00
14.00
!<•. 00
14.00
14.00
14.00
1 4.00
14.00
I '..00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
I4.no
14. 'JO
14.00
14.00
14.00
I4.no
14.00
14.00
14.00
14.00
14.00
14.00
14.00
13.63
CU2IOHI2
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
I '..00
14.00
14. OC
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14. PO
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
SOL. CU
6. 00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.30
6.00
6.00
6.00
6.00
6.00
6.00
6.00
ft. 00
6.00
6.00
6.00
6.00
6.00
t.OO
6.00
6.00
6.00
6.00
6 . JO
6.30
6.00
6.00
6.00
6.00
6.30
6.00
6.30
6.30
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
CU PUFC.
14.00
14.00
14.00
14.00
14.00
14.03
14.30
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.03
14.00
14.00
14.03
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.03
14.00
14.00
14.00
14.00
14.00
14.00
14.00
U.OO
14.00
SPECIES
NO PRFC.
NO PHEC.
NO PREC.
NO P3FC.
NO P9EC.
NO PHEC.
NO PREC.
NO "HEC.
NO PUPC.
NO PREC.
NO PREC.
NO P1EC.
NO °RFC.
NO P<*EC.
NO PREC.
NO PREC.
NO P1FC.
NO »REC.
NO PRFC.
NO P9FC.
NO PREC.
NO PREC.
NO PR?C.
NO OREC.
NO PRFC.
NO PREC.
NO PREC.
NO PR^C.
NO DRFC.
NO PREC.
NO PR^C.
NO PREC.
NO PREC.
NO PREt.
NO PRFC.
NO PREC.
NO PREC.
NO PRFC.
NO PRrC .
NO PREC.
NO PREC.
NO PRFC.
NO PREC.
NO PREC.
NO PR*C.
NO PREC.
NO PREC.
NO PR EC.
NO PREC.
NO PREC.
-------
en
PH
5.00
5.10
5.20
5.30
5.40
5.50
5.63
5.70
5.81
5. O1
6. no
6.10
"•.21
6.30
6.40
6.50
6.60
6.70
&.80
6.00
7.00
T.10
7.20
7.30
7.40
7.? 0
7.60
7.70
7.«0
7."0
1.00
?. 10
1.20
".30
1.40
8. 50
P. 60
J.70
1.10
1.00
9.00
9.10
9.20
9.30
9.41
9.50
9.60
9.70
9.90
9.90
CO
6.01
<-.01
6.02
6.02
6.94
6.05
6. J1
6.11
6.16
6. ?2
6. 10
6.39
6.4"
6.'0
6.71
6.13
6.~5
7.07
7.19
7.30
7.41
7.52
7.64
7.74
7.15
7 . •"">
6.17
1.17
" .21
1.19
1 .50
R.61
1 . 75
0.95
9.15
•3.35
0.55
9.75
9.T>
10.15
10.35
11.15
10.75
10.05
11.15
11 .35
11.55
11.75
11.95
12.15
CUOH
9.01
i.-n
8.82
1. 72
P.ft4
1. 5r
9.43
R.41
«. 3'.
R. 1?
(J. 30
•:, . 20
o.2V
0.10
P. 31
1.33
B. 3^
R. 37
8.31
R.40
8.41
1.42
R.44
R.44
1.45
1 . 't6
?.47
1.47
R.4H
R. VO
P. 50
R.51
». 5r)
P.")1!
1.75
8.15
fl.95
9.05
0.15
9.25
0 . 3 '•
0.45
c. 55
9.65
9.75
9.15
9. 95
10.35
10.15
10.25
CU1H3
14.00
14.00
14.00
1 4.00
1 * . 0 1
14.00
14.00
14.00
I'-.OO
14.00
14.00
14.00
14.00
1 4. 00
13.81
13.63
13.41:
13.27
13. OB
12. -50
12.71
12.52
12.54
12.14
11.95
11.76
11.57
11.37
11. in
10.99
1C. MO
10.61
10.45
10.35
10.25
10.15
10.05
9.95
9.15
9.75
9.65
'> . 5 5
".45
9.33
9.25
9.15
0.05
8.95
8.85
8.75
CUOH4
14.00
14.00
14.00
14.00
14. OO
14.00
1<». 00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14. JO
14.00
14.00
14.00
14.00
14.00
1 '«. 01
14. OC
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
I '..00
14.00
;4.oo
14.00
13. 55
13.75
13.^5
13.35
13. li
12.05
17.75
12.55
12.35
12.15
11.99
CDC 03
7.16
7.67
7.48
7.30
7.12
6.15
6.7Q
6.64
6. 51
6.40
6.31
6.23
ft. 17
ft. 13
6. 10
6.07
6. Of
6.04
6.03
6.03
6.02
6.02
6.02
ft. 02
6.02
6.0?
6.02
6.02
6.03
6.03
6.04
6.05
6.09
ft. 19
6.21
'..39
6.49
6.59
6.69
6.PO
6. "0
7.01
7.11
7.22
7.33
7.44
7.56
7.68
7. BO
7.93
C U I CO i 1 2
13. ?4
1 2 . f 6
12.47
1?. 10
11.73
11 .37
11.03
10. 70
10.39
10. 11
9.84
9. f. 1
9.39
Q. 19
9.01
p.es
6.69
1. 55
l.'l
R. 2°
R. 16
P. 04
7.93
7.f'2
7.71
7. f I
7. SO
7.40
7.30
7.21
7. 11
7.02
6.96
6.96
6.96
6.96
6.V6
6.^-7
6.07
6. '"8
6.")
7. no
7.01
7.03
7.05
7.07
7.10
7.14
7.18
7.23
Ci)2inH|2
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
1 4.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
12.05
12.25
12.45
12.65
12. «5
13.05
13.25
13.45
13.65
13.15
14.00
14.00
14.00
14. OO
14.00
14.00
14.00
14.00
SOL. CU
6.00
6.00
6.00
ft. 00
6.00
6.00
6.00
ft. 00
6.00
fr.UO
6.00
6.00
6.00
6.00
6.00
6.00
6.10
6*00
6.00
6.00
6.00
6.00
6.00
6.10
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.03
6.12
6.20
6.2fl
6. 36
6.44
6.51
6.58
6.64
6.7(1
6.76
6.81
6.16
6.91
6.97
7.02
7.08
7.14
CU PRFC.
14.03
14.00
14.00
14.00
14.01
14.00
14.10
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.10
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
7.15
6.62
6.43
6.32
6.25
6.?0
6.16
6.1 3
6.11
6. 10
6.01
6.07
6.06
6.06
6.05
6.04
6.04
6.03
SPF.C IFS
NO PREC.
NO PREC.
NO °RFC.
NO PRFC.
NO PREC.
NO PREC.
NO PREC.
NO PREC.
NO PRFC.
NO PRCC.
NO PREC.
NO PREC.
NO PRFC.
NO PREC.
NO PRFC.
NO PRFC.
NO PP?C.
NO PRFC.
NO PREC.
NO PREC.
NO PRFC.
NO PRFC.
NO PRFC.
NO PREC.
NO PREC.
NO PREC.
NO PRFC.
NO PR=C.
NO PRFC.
NO PREC.
NO PREC.
NO PRFC.
TE^ORITE
T^'JORl TF
TFNORITE
TENOR1TE
TENORITE
TFNORITE
TENORITE
TENORITE
TFNORITE
7FNORITF
TENOR1TE
TENORITE
TENORITF
TENORITE
TENORITE
TFNORITF
TENORITE
TENO«m
-------
PH
11.00
10. 11
10.20
10.13
10.40
10. SO
10.60
10.70
10. PO
10.90
11.03
11.10
11.20
11.30
11.40
11.50
11. 40
11.70
11.80
11.93
12.00
12.10
12.20
12.10
12.40
12.53
12.60
12.70
12. «1
12.90
13.00
11.10
13.20
13. 3J
11.40
1.1. 50
13.60
13.70
13.AO
13.90
14.00
CU
12.35
12.55
12. 75
12.95
13. 15
13.35
13.55
13.75
13.95
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.10
14.00
14.00
14.00
14.00
14.11
14.00
14.00
14.30
14.00
14.00
14.00
CUOH
10. J5
10.45
10. 55
10.65
10.75
10.85
10.95
11.05
11.15
11.25
11.35
11.45
11.55
11.65
11.75
11.85
11.95
12.05
12.15
12.27
12.35
12.45
12.55
12.65
12.75
12. 85
13.02
13.25
1 3.48
13. Tl
13.95
14.00
14. 90
14.00
14.00
14.00
14.00
14.30
14.00
14.00
14.00
CUOH3
R. 65
fi.55
P . '*K
E.35
B.75
".15
P. 05
7.95
7.35
7.75
7.65
7.55
7.45
7.35
7.25
7.15
7.05
6.05
6. 85
6. 75
6.65
6.55
6.45
6. 35
6.25
6.15
6. 12
6.15
6. 11
6.21
6.25
6.30
*>. 35
6.41
6.48
6.55
6.67
6.70
6.78
6.86
6.95
CUOH4
11.75
11.55
11.35
11.15
10.95
10.75
10.55
10.35
10.15
9.95
o. 75
9.55
0.35
9. 15
fl.95
6. 75
P. 55
8.35
8.15
7.S5
7.75
7.55
7.35
7.15
6.05
6.75
6.62
6.55
f .4"
6.41
6.35
6.30
6.25
6.21
6.18
6.15
6.12
6.10
6.08
6.06
6.09
CI/C03
8.06
8.19
».33
8.48
".63
8.79
9.<36
9.13
9.30
9.48
0.^6
9. =4
10.03
10. 72
10.41
10.61
10.80
11.00
11.19
11.39
11. '-9
11.79
11.09
12.18
1?.3S
1 ? . 58
1?. 15
13.18
11.51
1 3 . B4
14.00
14.00
14.no
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
CU(C03)2
7.21
7.37
7.45
7.54
7.65
7.77
7.P9
?.C3
8.11
8.34
8.50
8.67
8.84
9.02
".21
9.39
0.58
9.78
c.97
10. 16
10.36
10.56
10. 75
10.05
11.15
11.35
11.61
11.94
12.27
12.61
12.95
13.29
13.65
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
CU2(nHI2
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
SOL. CU
7.21
7.28
7.36
7.44
7.52
7.59
7.64
7.67
7.67
7.64
7.50
7.51
7.43
7.13
7.24
7.14
7.04
6.93
6.83
6.72
6.62
6.51
6.40
6.29
6.17
6.05
6.00
6.00
6.00
6.30
6.00
6 . ,10
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
CU PRE
*• 03
6.02
6.02
6.02
6.01
6.01
6.01
6.01
6.01
6.01
6.01
6.01
6.02
6.02
6.03
6.03
6.04
6.05
6.07
6.09
6.12
6.16
6.22
6.32
6.49
6.94
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14. OO
14.30
14.00
14.00
14.00
14.00
SPEC IPS
TFNORITE
TENORITE
TENORITE
TFNORITF
TFNORIT?
TENORITE
TENORITE
TENORITE
TENORITE
TENQRITF
TENOR ITE
TENORITE
TENORITE
TFNORITF
TFNnaiT*
TENORITF
TENORITE
TENORITE
TENORITE
TENORITE
TENOTITE
NO "RFC.
NO P»FC.
NO PREC.
NO PRSC.
NO PRFC.
NO "REC.
NO P*EC.
NO PREC.
NO PRFC.
NO PRFC.
NO PREC.
NO PREC.
NO PR«=C.
NO PREC.
NO PREC.
-------
o.oonoo
COPPER= 0.000001
o
o
PH
0.0
9.10
9.20
0.30
0.43
0.10
0.60
0.73
0.*}
0.90
.00
.10
.21
.31
.*0
.50
l.AO
1.70
I. HI
I. 93
3.00
2.10
2.2 1
z.-n
2.40
2.50
2. AT
2. TO
2.»0
Z.93
3.00
3.10
3.70
3.30
3.40
*.50
3.*0
3.70
3. fa
3.90
4.00
4.10
4.20
4.30
4.40
4.50
4.60
4.70
4.80
4.10
cu
6.00
6.00
6.00
IS. 00
IS. 00
6.00
ft. 00
•>. oo
s.oo
ft. 00
ft. 00
ft. 00
ft .00
ft. 03
*. 30
6.00
ft.OJ
ft . m
ft. 00
ft. CO
ft. 00
ft. 00
ft. 10
f..?1
ft. 00
ft. 00
6.00
ft. 00
A. 00
ft. 00
ft. 00
6.00
ft. 00
ft .00
6.0J
ft. 00
6.00
6.00
ft .01
ft. 00
6.00
ft. 0-1
ft. 00
ft. 03
6.00
ft. 00
6.00
6.00
6.00
6.00
CD'IH
14.03
13.90
13.33
13.7Q
13.60
13.50
1 3.40
13. 3D
13.20
13. 10
13. QT
12.90
U.SO
12.70
12.63
12. SO
I2.'«0
1 '. > 3
1 7 . 1 0
12. 10
12.00
1 1 .90
1 l.flO
11.70
11 .SO
1 1 .-JO
11 .'.0
11.. 40
11. '0
11.10
11.00
1C. 90
10.30
10.70
10.60
13. SO
10.40
10.30
10.20
10.10
10.00
9.9Q
9.40
9.70
9.60
9.50
9.40
9.30
9.20
9,10
CUOH3
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.T.O
14.00
14.00
I'-.on
1 4.00
14.00
14.00
14.00
I 4.00
14.00
1 '..00
I '..00
14.00
I '..00
14.00
1 4.00
14.00
14.00
1 4.00
14.00
14.00
14.00
14.00
1 4.00
14.00
14.00
14.00
14.00
14.00
1 4.00
14.00
14.00
14.00
14.00
I'.OO
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14,00
CHr
-------
PM
5.00
5.10
5.23
5.30
5.43
?.53
5.60
5.70
5.«0
5.93
6.00
6.10
5.?0
6.30
"..*3
6.53
A. 60
6.73
A. 80
S.90
7.00
7.10
7.20
7.30
7.40
7. 53
7.61
7.73
7.13
7.10
9.00
8.10
«.?0
1.33
8.40
8.50
K.60
<».70
«.«3
1.90
9.03
9.n
9.20
0.30
9.43
9.53
«.60
9.70
9. A3
9.90
CU
6.03
5.00
6.03
C.OO
6. no
6.01
6.01
6.01
6.02
6.03
6.04
6.06
6.39
6.1?
r>.!6
6.21
6.26
6.32
6.39
'..47
6. 55
6.ft4
6.75
ft. 9S
7.15
7.35
7.55
7.75
7.?5
8.16
«. 15
«. 55
•?. 71
M.9S
". 15
".35
'•(.•i-i
9. 75
T.ns
10.15
10.35
I').S5
10. T>
10.95
11.15
11. 35
11.55
11.75
11.95
12.15
CUOH
9.0'3
8.?0
8.80
8.70
8.63
8.51
fl.41
0.31
8.7?
ff.13
r.T.
7.<>6
7.3T
7.92
7.76
7.71
7.66
7.62
7.59
7.57
7. 55
7.54
7.55
7.55
7.75
7.95
7.95
8.05
*. IS
1.25
P. 35
r,.4S
a.ss
H.65
W.75
fi.85
".95
c'.05
". 15
9.25
9.35
".AS
''.Si
9.65
".75
9.85
'».95
10.05
10.15
10.25
CUOH3
I*. 00
14.00
14.00
14.00
I4.no
14.00
14.00
14.00
14.00
14.00
14.03
14.10
M.79
13.52
13.26
13.01
I?. 76
12.52
12.29
12.07
11.85
11.04
11.45
11. J5
11.25
11.15
11. OS
10.95
10.85
10. 7S
10.65
10.55
10.45
10.35
10.25
10. 15
10.05
Q.95
P. 85
9.75
^.65
9.SS
9. '.5
9.35
0.25
". 15
o.05
fl.95
fl.85
8.75
CUOH4
14.00
14.30
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
I4.no
14.00
14.00
14.00
I4.no
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.30
14.00
14.00
14.00
14.00
14.00
14.00
14.00
13. "5
13.75
13.r.5
13.35
13.15
12.95
12.75
12.55
12.35
12.15
11.95
cur. 03
».R5
8.66
B.47
P. 27
8.09
7.90
7.72
7.54
7.37
7.21
7.05
6.°1
6.77
6.65
6. 54
6.45
6.37
6.30
6.24
'..20
*. 16
6.13
6.13
6.22
6.31
6.41
6.50
6.60
t: . t 9
6.79
6. P9
6.99
7.09
7.19
7.29
7.39
7.49
7.59
7.69
7.80
7.90
«. PI
8.11
8.22
fl.33
8. 44
8. 56
8.6*
8.80
8.93
CUICr)3)2
14.00
14.00
14.00
14. CO
13.70
13.33
12. "6
12.60
12.25
11.91
11. 5}
11.28
10.99
10.71
10.46
10.22
10. 01
0.81
°.62
o.46
9.30
9.16
f>.05
9.03
9.01
".00
8.99
R.98
8.97'
8.97
8. 96
P. 96
P. 96
fi.96
8.96
8.96
8.96
fl.97
8.°7
«.98
8.99
9.00
9.01
9.03
9.05
9.07
9.10
9.14
9.18
9.23
Cll2IPH)2
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
1'..00
14.00
14.00
14.00
10.05
10. ?5
10.45
10.65
1U.05
11.05
11.25
11.45
11.65
11. «5
12.05
1?.25
12.45
12.65
12.85
13.05
13. ?5
13.45
13.65
13.85
14.00
14. "0
14.00
14.00
14.00
14.00
14.00
14.00
SOL. CU
6.00
6.00
6.00
6.10
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.02
6.13
6.24
6.35
6.45
6.55
f .66
6.76
6.86
6.16
7.06
7.16
7.26
7.36
7.46
7.56
7.65
7.75
7.85
7.04
8.03
8.12
8.20
8.28
8.34
8.39
8.43
ft. 44
CU PREC.
14.00
14.30
14.00
14.00
14.00
14.03
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
7.26
6.58
6.37
6.26
6.19
6.14
6.11
6.03
6.06
6.05
6.04
6.03
6.02
6.02
6.92
6.01
6.01
6.01
6.01
6.01
6.00
6.00
6.30
6.00
6.00
6.00
6.00
6.00
SPECIES
NO p«*r..
NO PREC.
NO PREC.
NO PREC.
NO PRFC.
NO PR=C.
NO PREC.
NO PRCC.
NO PREC.
NO PRFC.
NO PREC.
NO PRFC.
NO PREC.
NO PREC.
NO PREC.
NO PREC.
NO PREC.
NO PREC.
NO PRFC.
NO PREC.
NO PREC.
NO PRFf.
TFNflRITF
TENORITE
TENQRITE
TEWJRITE
TC^IRITF
TENORITE
TENORITF
TENflRITE
TENTITE
T?NORITF
TFNORITE
TFNORITF
TENORITE
TENORITF
TENORITF
TENORITE
TENHR1TE
TFNPRITF
TF'JORITF
TfN'IRtTF
TENORITF
TEN1RITF
TENORITF
TE>gO
-------
PH
13.01
10.10
in. ?o
10.30
13.40
10.51
lO.ftO
11.70
10. JO
10.00
11 .00
11.10
11.20
11.30
11. *0
11.50
11.61
11 .70
ll.'O
11. Oil
12.00
12.11
12.20
12.30
12.40
12 .50
12.60
12.71
12. "1
1 ?.91
13.00
13.10
13.20
13.30
13.40
13.50
13.61
13. TO
13.80
13. "0
14.00
CU
17. 15
1'.55
12,75
1 7 .qc>
13.15
13.35
1? . 51;
13.75
13. "5
I'-.OO
14.00
14.11
1 4 . 00
14,00
1-..00
14.10
14.01
14.01
1 '•• . 00
1 'i . 0 0
14.00
14.00
14.00
14.00
14.00
14,00
14.10
14.00
14,03
14,10
14.00
14,00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
CUD'<
10.35
11. '-5
10.55
11.55
10.75
10. 95
10.^5
11.0*
11.15
11.75
11. 35
11.45
1 1. 55
1 1 . 65
11.75
11.85
11.15
1?. 15
12.15
17.25
12.35
12.45
12.55
12.65
12.75
12.85
13.02
13.25
1 1.48
1 3.71
13.95
14. CO
14.00
1 4. 10
14.00
14.00
14.00
14.00
14.00
14.00
14.00
CUO'13
8.65
8.^5
F, . 4 5
°. ?"•
Q.?5
°. 15
1.05
7. 5 5
7.85
7.75
7.65
7.55
7.45
7.35
7.25
7.15
7.05
ft. 95
6.85
6. 75
6. 65
6. "5
6.45
6.35
6.25
6.15
6.12
ft. 15
6.18
6.21
6.25
6.30
6.35
6.41
6.48
6.5S
6.62
6.70
6.78
6.86
6.95
CIH)H4
11.75
11 .55
11.35
11.15
1(1. ^
10.75
10.55
10.35
10. 15
9. Q5
9. 75
0.^5
9.35
9. 15
K.05
8.75
0.55
8.35
(!. 15
7.T-,
7.75
7. 55
7.35
7. 15
6.95
ft. 75
ft. 62
6.55
ft. 48
ft. 41
ft. 35
6.30
ft. 25
6.21
6. 18
ft. '15
ft. 12
ft. 10
6.08
6.06
6.05
euros
9.06
•3.10
9.33
9.48
9.63
9.79
9.96
10. 13
10.30
10.^8
10.66
13.^4
11.03
11.22
11.41
11.61
11. °0
12.^0
12.10
12.39
12.59
12.79
12.99
13.18
13.38
13.58
13.35
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
CUICH3)2
9.29
9.37
1.45
9. 54
9.65
9.77
9 . °9
1 0 . P 3
10.13
10. 34
10. 50
1 0 . ft 7
10. B4
11.02
11.21
11. 39
11.5ft
11 .78
11. °7
12.16
12.36
12.56
I?. 75
12. 35
13. 15
13.35
13.61
13.94
1 4 . (; 0
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
CM2 (riH»Z
14.00
14.00
14.00
14.00
14.00
14.00
1 4.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14. CO
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
SOL. C'J
P. 44
8.41
f .36
8.29
8.21
P. 13
8.04
7.94
7.84
7.75
7.65
7.55
7.44
7.34
7.24
7.14
7.04
6.93
6.83
6.72
ft. 62
6.51
6.40
6.29
ft. 17
ft. 05
6.00
ft. 00
6.00
ft. 00
ft. 00
6.00
6.00
6.00
ft. 00
6.00
6.00
6.00
6.00
6.00
6.00
CU I'PFC.
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
ft. 01
6.01
6.01
6.01
6.02
6.02
6.03
6.03
6.04
6.05
6.07
6.03
6.12
6. 1 «>
6.22
6.32
6.49
6.94
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14. OC
14.00
14.00
14.00
14.00
SPFCIFS
TENORITF
TFNORITE
TENORHF
TFNORITE
TENORITE
TFNORITE
TFNORITE
TENHRITE
TFNORITE
TCNORTTE
TENORITE
TFNORITF
TENORITE
TFNORITF
TFNORITE
TFNORITE
TFNORITE
TFNHRITE
TENOR ITE
TF'JP'MTP
TENORITE
TFNORITF
TENORITE
TFNORITE
TENHRIT?
TENORITF
NO PRCC.
NO PRFC.
NO PRFC.
NO PRFC.
NO PREC.
NO PREC.
NO PRFC.
NO PREC.
NO PREC.
NO PRCC.
NO PREC.
NO PREC.
NO PREC.
NO PREC.
NO PREC.
-------
APPENDIX B
COMPUTATION OF EQUILIBRIUM pH AND TOTAL ALKALINITY
AT DIFFERENT PARTIAL PRESSURES OF CARBON DIOXIDE
103
-------
PH» 4.00
EQUILIBRIUM
PIC02>
ATM
***INITIAL***
NA2CC3
MG/L
EOUIV
CAC03
********* **********FINAL*************** *******
H2CC3 HC03 COS
************* PCLAR *************
ALKAL INITV
EU/L
MAXIMUM
CALCIUM
MCLflR
FINAL
IONIC
STRENGTH
MG/L
0.2222E
0.3703E
0.5184E
0.6665E
0.8146E
0.9628E
0.1110E
0.1259E
0.1407E
0.1555E
0.1703E
0.1851E
0.1999E
0.?l*7E
0.2295E
0.2443E
0.2592E
0.2740E
0.28R8E
0.3036E
0.3184E
0.3332E
0.3460E
0.3628E
0.3776E
0.3925E
0.4073E
_JU«221JL
0.4369E
0.4517E
01
01
01
01
01
01
02
02
02
02
02
02
02
02
Of
02
02
02
02
02
02
02
02
02
02
02
02
02
02
02
12.387
24.775
37.162
49.550
61.938
74.325
P6.713
99.100
111.488
123.876
136.263
UB.651
161.039
173.426
185.814
198.201
210.589
222.977
235.364
247.752
260.139
272.527
284.915
297.302
309.690
322.078
334.465
346.853
359.240
371.626
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
0.7530E-
0.1254E
0.1756E
0.2258E
0.2760E
0.3262E
0.3764E
0.4^66E
0.4768E
0.5269E
0.5771E
0.6273E
0.6775fc
0.7277E
0.7779E
0.8281E
0.8783E
0.9284E
0.9786E
0. 1028E
0.1079E
0.1129E
0.1179E
0.1229E
0. 1279E
0.1329E
0.1380E
0.1430E
0.1480E
0.1530E
01
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
01
01
01
01
01
01
01
01
01
01
01
0.2997E-03
0.4995E-03
0.6993E-03
0.8991E-03
0. 1098fc-02
0. 1298E-02
0.14986-02
0.1698E-C2
0. 1998E-02
0.2097E-02
0.2297E-C2
0.2497E-02
0.2697E-C2
0.2897E-C2
0.3096E-02
0.3296E-02
0.3496E-02
0.3696E-02
0.3896E-02
0.4095E-02
0.4295E-02
0.4495E-02
0.4695E-02
0.4895E-02
0.5094E-02
0.5294E-C2
0.5494E-02
0.5694E-02
0.5894E-02
0.6093E-02
0.1502E-09
0.2503E-09
0.3505E-09
0.4506E-09
0.550et-09
0.6509E-09
0.7510E-09
0.85l2b-09
0.9513E-09
0. 1C51E-08
0. 1151E-08
0. 1251E-08
0. 135U-08
0. 1452E-08
0. 1552C-08
0. 1652E-08
0. 175^E-08
0.1R52E-08
0. 1952E-08
0.2052E-08
0.2153E-08
0.225JE-08
0.2353b-08
0.2453E-08
0.2553E-08
0.2653E-08
0.2753E-08,
0.2853E-08 '
0.2954E-08
0.3054E-08
0.1908E-03
0.3996E-03
0.5994E-C3
0.7992E-03
0.9990E-03
0.1198E-02
0. 1398E-02
0.15'^8E-02
0.1798E-02
0. 199RE-02
0.2197E-02
0.2397E-02
0.2Sr;7fc-02
0.27')7E-02
0.2997E-02
0.3196E-02
0.3396E-02
0.35T6E-02
0.3796E-02
0. 3996E-02
0.4105E-02
0.4395E-02
0.45g5E-02
0.4795E-02
0.4995E-02
0.5194E-02
0.5394E-02
0.5594E-02
0.579AE-02
0.5994E-02
0.3335E
0.2C01E
0.1429E
0.1112E
0.9099E
0.7699E
0.6672E
0.58676
0.5268E
0.4766E
0.4351E
0.4003E
0.37C7E
0.3451E
0.3228E
0.3033E
0.2H59E
0.2705E
0.2566E
0.2441E
0.2327E
0.2224E
0.2129E
0.2042E
0.1962E
0.1888E
0.1819E
0.1756E
0.1696E
0.1640E
02
02
02
02
01
01
Cl
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
01
0.2990E-03
0. 49965-03
0.6994E-03
0.8992E-03
0. 1099E-02
0. 1298E-02
0. 1498E-02
0.1MPE-02
0.1898E-02
0.2098E-02
0.2'297E-02
O.P497E-02
0.2697E-02
0.2897E-02
0.3097E-02
0.3296E-02
0.3496E-02
0.3696E-02
0. 3896E-02
0.4096E-02
0.42T5E-02
0.4495E-02
0.4695E-02
0.4895E-02
0.5095E-02
0.5294E-02
0.5494E-02
0.5694E-02
0.5894E-02
0.6094E-02
-------
PH= 5.00
EQUILIBRIUM
PJC02)
ATM
***INITIAL***
NA2C03
MG/L
EBUIV
CAC03
************* ******FINA|_**********************
H2C03 HC03
************* KCLAR
CC3
*************
ALKALINITY
EC/L
MAXIMUM
CALCIUM
MOLAR
FINAL
IONIC
STRENGTH
MG/L
0.1555E 00
0.3036E 00
0.4517E 00
0.5998E 00
0.7*796 00
0.8960E 00
0.1044E 01
0.1192E 01
0.134CE 01
0.1488E 01
0.1636E 01
0.1784E 01
0.1932E 01
0.2080E 01
0.2229E 01
0.23776 01
0.2525fc 01
0.2673E 01
0.2821E 01
0.29C.9E 01
0.31l7fc 01
0.3265E 01
0.3413k 01
0.3562E 01
0.3710E 01
0.3B58E 01
0.4006E 01
0.4154E 01
0.4302E 01
0.4450E 01
12.387
24.775
37.162
49.550
61.938
74.325
66.713
99.100
111.488
123.676
136.263
14H.651
161.039
173.426
Ifl5.814
198.201
210.589
222.977
235.364
247.752
260.139
272.527
284.915
297.302
309.690
322.078
334.465
346.853
359.240
371.628
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
IPO
190
200
210
220
230
240
250
260
270
280
290
300
0.5269E-02
0.1028E-01
0.1530E-01
0.2032E-01
0.2534E-01
0.3036E-01
0.3538E-01
0.4040E-01
0.4541E-01
0.5043E-01
0.5545E-01
0.6047E-01
0.6549E-01
0.7051E-01
0.7553E-01
0.8055E-01
0.8556E-01
0.905flE-01
0.9560E-01
0.1C06E 00
0.1056E 00
0. 1106E 00
0.1i:>6F 00
0.1206E 00
0.1257E 00
0.1307E 00
0.1357E 00
0.1407E 00
0.1457E 00
0.1508E 00
0.2097E-03
0.4095E-C3
0.6093E-C3
O.F091E-03
0.1008E-02
0.1208E-02
0.140BE-C2
0.1608E-C2
0.1B08E-02
0.2007E-C2
0.2207E-C2
0.2407E-02
0.2607E-C2
0.2R07E-02
0.3006E-02
0.3206t-02
0.3406E-02
0.3606E-02
0.3806E-C2
0.4005E-02
0.42C5E-02
0.44C5E-C2
0.4605t-02
0.4805E-C2
0.5004E-02
0.5204E-02
0.5404E-02
0.5604E-02
0.5804E-02
0.6003E-02
0.1051E-08
0.2052E-08
0.3C54E-08
0.405bE-08
0.5056h-08
0.6058E-08
0.7C59E-08
0.8061E-08
0.9062E-08
0.1C06E-07
0.1106E-07
0.1206E-07
0.1306C-07
0. 1406E-07
0.1507E-07
0.1607E-07
0.1707E-07
0. 1807E-07
0.1907E-07
0.2007E-07
0.2107E-07
0.2208E-07
0.2308E-07
0.2408E-07
0.2508E-07
0.2608E-07
0.2708E-07
0.2808E-07
0.2908E-07
0.3009E-07
0.1998E-03
0.3996E-03
o.sgg'.E-os
0.7992E-03
0.99^0E-03
O.H9RE-02
0. 139RE-02
0.1598E-02
0.179RE-02
0.1998E-02
0.2197E-02
0.2397E-02
C.2597t-02
0.2797E-02
0.2997t-02
0.3196E-02
0.3396E-02
0.3596E-02
0.3796E-02
0.3996E-02
0.4195E-02
0.4395E-02
0.4595E-02
0.4795E-02
0.4995E-02
0.5194E-02
0.5394E-02
0.55S4E-02
0.5794E-02
0.5994E-02
0.4766E 01
0.2441E 01
0.1640E 01
0.1235E 01
0.991CE 00
0.8272E 00
0.7C99E 00
0.6217E 00
0.553CE OC
0.49POE 00
0.4529E 00
0.4153E 00
0.3H35E 00
0.3562E 00
0.3325E 00
0.3118E 00
0.2935E 00
0.2772E 00
0.2627E 00
0.24-36E 00
0.2377E OC
0.2269E 00
0.2171E 00
0.2081E 00
0.1998E 00
0.1921E 00
0.1850E 00
0.1784E 00
0.1722E 00
0.1665E 00
0.2098E-03
0.4096E-03
0.6094E-03
0.8092E-03
O.l009fc-02
0.120PE-02
0.1408b-02
0.1608E-02
0.1806E-02
0.200Rt-02
0.2207E-02
0.2407E-02
0.2607E-02
0.2807E-02
0.3Q07E-02
0.3206E-02
0.3406E-02
0.3606E-02
0.3806E-02
0.4006E-02
0.4205E-02
0.4405E-02
0.4605E-02
0.4805E-02
0.5005E-02
0.5204E-02
0.5404t-02
0.5604E-02
0.5804E-02
0.6004fc-02
-------
PH= 6.00
EQUILIBRIUM
PCCC2)
ATM
***INITIAL***
NA2CC3
MG/L
EQUIV
CAC03
MG/L
*******************FINAL*************** *******
H2C03 HC03 C03
************* PCLAR *************
ALKALINITY
EG/L
MAXIMUM
CALCIUM
MOLAR
FINAL
IONIC
STRENGTH
0.1488E-01
0.2969b-01
0.4450E-01
0.5931E-01
0.7412E-01
O.B893E-01
0.1037E 00
0.1185E 00
0.1333E 00
0.1481E 00
0.16Z9E 00
0.1777E 00
0.1926E 00
0.2074E 00
0.2222E 00
0.2370E 00
0.2518E 00
0.2666E 00
0.2814E 00
0.2962E 00
0.3110E 00
0.3258E 00
0.3407E 00
0.3555E 00
0.3703E 00
0.385U 00
0.3999E 00
0.4147E 00
0.4295E 00
0.44436 00
12.387
24.775
37.162
49.550
61.938
74.325
86.713
99. 100
111.486
123.876
136.263
148.651
161.039
173.426
185.814
198.201
210.589
222.977
235.364
247.752
260.139
272.527
284.915
297.302
309.690
322.078
334.465
346.853
359.240
371.628
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
IflO
190
200
210
220
230
240
250
260
270
280
290
300
0.5043E-03
0.1006E-02
0.1507E-02
0.2009E-02
0.2511E-02
0.3013E-02
0.3515E-02
0.4017E-02
0.4518E-02
0.5020E-02
0.5522E-02
0.6024E-02
0.6526E-02
0.7028E-02
0.7529E-02
0.8031E-02
0.853?E-02
0.9035E-02
0.9537E-02
0.1003E-01
0. 1054E-01
O.H04E-01
0. 1154E-01
0.1204E-01
0.1254E-01
0. 1304E-01
0. 1355E-01
0.1405E-01
0.1455E-01
0.1505E-01
0.2007E-03
0.4005E-03
0.6003E-03
0.6001E-03
0.9998E-03
0.1199E-02
0. 1399E-02
0. 1599E-C2
0. 1799E-02
0. 1998E-02
0.2198E-C2
0.2398E-02
0.2598E-02
0.2797E-02
0.2997E-02
0.3197E-02
0. 33976-02
0.3597E-02
0.3796E-02
0.3996E-02
0.4196C-02
0.4396E-02
0.4595E-02
0.4795E-02
0.4995E-02
0.5195E-02
0.5395E-C2
0.i594E-02
0.5794E-02
0.5994E-02
0.1C06E-07
0.2C07E-07
0.3C08E-07
0.4C10E-07
0.5Cllfc-07
0.6C12E-07
0.7013F-07
0.8015E-07
0.9016E-07
0. 1C01E-06
0.1101E-06
0. 1202E-06
0. 1302E-06
0. 1402E-06
0. 1502E-06
0. 1602E-06
0. 170^6-06
0. lPO?b-06
0. 190
-------
PM» 7.00
EQUILIBRIUM
PCCC2)
ATM
***INITIAL***
NA2C03
HG/L
E8UIV
CAC03
************ *******FINA L******* ***************
H2C03 HC03
************* KCLAR
CC3
*************
ALKALINITY
EQ/L
MAXIMUM
CALCIUM
MOLAR
FINAL
IONIC
STRENGTH
MG/L
O.U79E-02
0.2959E-02
0.4438E-02
0.59186-02
0.7398E-02
0.86776-02
0.1035E-01
O.U83E-01
0.13316-01
0.1479E-01
0.16276-01
0.1775E-01
0. 19236-01
0.2071E-01
0.2219E-01
0.2367E-01
0.25156-01
0.26636-01
0.2811E-01
0.2959E-01
0.3107E-01
0.3255E-01
0.3403E-01
0.3551E-01
0.3699E-01
0.3847E-01
0.3995E-01
O.M43E-01
0.4290E-01
0.4O8E-01
12.387
24.775
37.162
49.550
61.938
74.325
86.713
99.100
111.48R
123.876
136.263
148.651
161.039
173.426
1R5.814
198.201
210.589
222.977
235.364
247.752
260.139
272.527
284.915
297.302
309.690
322.078
334.465
346.853
359.2*0
371.628
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
1RO
190
200
210
220
230
240
250
260
270
280
290
300
0.50136-04
0.1002E-03
0.1504E-03
0.2005E-03
0.25066-03
0.3008E-03
0.3509E-03
0.4010E-03
0.4512E-03
0.5013E-03
0.5515E-03
0.6016E-03
0.6517E-03
0.7019E-03
0.7520E-03
0.8021E-03
0.85236-03
0.90246-03
0.95266-03
0. 1002E-02
0. 10526-02
0.1103E-02
0.1153E-02
0.12036-02
0.1253E-02
0.1303E-02
0.1353E-02
0.1403E-02
0.1453E-02
0.1504E-02
0.1996E-03
0.3992E-03
0.5988E-C3
0.7984E-03
0.9980E-03
0.1197E-02
0.1397E-02
0.15966-02
0. 1796E-02
0.19966-02
0.2195E-02
0.2395E-02
0.2594E-02
0.27946-02
0.29946-02
0.3193E-02
0.33936-02
0. 35926-02
0.3792E-02
0.3992E-C2
0.4191E-02
0.4391E-02
0.4590E-C2
0.4790E-02
0.4990t-02
0.5189E-02
0.53B9E-02
0.5588E-02
0.5788E-02
0.5988E-02
0. 1000E-06
0.2000E-06
0.3C01E-06
0.4C01E-06
0.5C01E-06
0.600?E-06
0.7002t-06
0.8002E-06
0.9C03E-06
O.lCOOE-05
0.1100E-05
0. 12006-05
0.1300E-05
0.14006-05
0. 15006-05
0.166E-C2
0.5010E-02
0.4554E-02
0.4175E-02
0.3853E-02
0.3b78E-02
0.3340E-02
0.3131E-02
0.29476-02
0.2783E-02
0.2636E-02
0.2505E-02
0.2385E-02
0.2277E-C2
0.2178E-02
0.20B7E-02
0.2004E-02
0.1926E-02
0.18556-02
0.17896-02
0.1727E-02
0.1670E-02
0.
-------
PH- B.QO
O
CO
EQUILIBRIUM
PICG2)
ATM
***INITIAL***
NA2C03
MG/L
EBUIV
CAC03
MG/L
***************** **FINAL**********************
H2CC3 HC03 C03
************* KCLAR *************
ALKALINITY
EC/L
MAXIMUM
CALCIUM
POLAR
FINAL
IONIC
STRENGTH
0. 14596-03
0.2925E-03
0.43926-03
0.5858E-03
0.73246-03
O.B791E-03
0.1025E-02
0.1172E-02
0.1319E-02
0.1465E-02
0.1612E-02
0. 175Hfc-02
0.19056-02
0.2052E-02
0.2198E-02
0.2345b-02
0.2492E-02
0.263BE-02
0.278SE-02
0.2932E-02
0.3078E-02
0.3225E-02
0.3372E-0?
0.351BE-02
0.3665E-02
0.3812E-02
0.3958b-02
0.4105E-02
0.4251E-02
0.4398E-02
12.387
24.775
37.162
49.550
61.938
74.325
86.713
99.100
111. 488
123.876
136.263
146.651
161.030
173.426
105.814
196.201
210.589
222.977
235.364
247.752
260.139
272.527
284.915
297.302
309.690
322.078
334.465
346.853
359.240
371.628
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
mo
190
200
210
220
230
240
250
260
270
280
290
300
0.4944E-05
0.9913E-05
0. 1488E-04
0. 19B5E-04
0.2482E-04
0.2978E-04
0. 3475E-04
0. 3972E-04
0.4469E-04
0.4966E-04
0.5463E-04
0.5960E-04
0.6457E-04
0.6954E-04
0.7450E-04
0.7947E-04
0.8444E-04
0.8941E-04
0.9438E-04
0.9935E-04
0. 1043F-03
0.1092E-03
0. 1142E-03
0. 1192E-03
0.1241E-03
0.1291E-03
0.1341E-03
0.1391E-03
0.1440E-03
0.1490E-03
0. 1968E-03
0.3946E-03
0.5924E-C3
0.7902E-03
0.9881E-03
0.1185E-02
0. 13fi3t-C2
0. 1581E-C2
0.1779E-02
0.1977E-02
0.2175E-02
0.2372E-02
0.2570C-C2
0.2769E-02
O.P966E-02
0.3164F-02
0.3361E-02
0.3559E-02
0.3757F-02
0. 3955E-02
0.4153fc-02
0.4350C-C2
0.4548E-C2
0.4746fc-02
0.4944E-C2
0.5142E-02
0.5340E-02
0.5537E-C2
0.5735E-02
0.5933E-02
0.9865E-06
0.1977E-05
0.2969E-05
0.3960E-05
0.4952E-05
0.5943E-05
0.6935E-05
0.7926E-05
0.8916E-05
0.9909fc-05
0. 1090E-04
0.1189E-04
0.128«E-04
0. 1387E-04
0. 1486E-04
0.158^-04
0.1684E-04
0. 1784E-C4
0.1883E-04
0.19R^E-04
0.2C81E-04
0.218CE-04
0.2279E-04
0. 23786-04
0.2478E-04
0.2577E-04
0.2676E-04
0.2775E-04
0. 28746-04
0.29736-04
0.1998E-03
0.3996E-03
0.5994E-03
C.7992E-03
0.9990E-03
0.1198E-02
0.1398E-02
0.1598E-02
0. 179PE-02
0. 1998E-02
0.2197E-02
0.2397E-02
0.2597E-02
0.2797F-02
0.2997E-02
0.3196E-02
0. 33966-02
0.35966-02
0.3796fc-02
0. 3996E-02
0.4195t-02
0.4395E-02
0.4595E-02
0.4795E-02
0.4995E-02
0.5194E-02
0.5394E-02
0.5594E-02
0.5794E-02
0.5994E-02
0.5080E-02
0.2533E-02
0.1687E-02
0.1265E-02
0.1012E-02
0.8432E-03
0.7226E-03
0.6322E-03
0.5619E-03
0.5C57E-03
0.4597E-03
0.4214E-C3
0.3t"50E-03
0.3612E-03
0.3371E-03
0.3160E-03
0.2974E-03
0.2P09E-03
0.2661E-03
0.2528E-03
0.2407E-03
0.2298E-03
0.2198E-03
0.21C6E-03
0.2022E-03
0.1944E-03
0.1872E-03
0.1805E-03
0. 17436-03
0.1685E-03
0.2007E-03
0.4015E-03
0.6023E-03
O.fiOUE-03
0.1003E-02
0. 1204E-02
0.1405E-02
0. 1606E-02
0.1807fc-02
0.2007E-02
0.2208E-02
0.?409E-02
0.2610E-02
0.2811E-02
0.3011E-02
0.3212E-02
0. 34136-02
0.3614E-02
0.3815E-02
0.40156-02
0.42166-02
0.4417E-02
0.4618E-02
0.4819E-02
0.5019E-02
0.52206-02
0.5421E-02
0.56226-02
0.58226-02
0.60236-02
-------
PH» 9.00
EQUILIBRIUM
PIC02)
ATM
***INITIAL***
NA2C03
MG/L
EQUIV
CAC03
******* ************FINAL**********************
H2C03 HC03
************* PCLAR
CC3 ALKALINITY
************* EC/L
MAXIMUM
CALCIUM
POLAR
FINAI.
IONIC
STRENGTH
KG/L
0.1278E-04
0.2625E-04
0.3971E-04
0.5317E-04
0.6663E-04
0.80096-0'.
0.9356fc-04
0.1070E-03
0.1204E-03
0.1339E-03
0.1474E-03
0.1608E-03
0.1743E-03
O.I877E-03
0.2012E-03
0.2147fc-03
0.2281E-03
0.2416E-03
0.2551E-03
P.26B5E-03
0.2820E-03
0.2954E-03
0.3089E-03
0.3224E-03
0.3358E-03
0.3493E-03
0.3627E-03
0.3762E-03
0.3897E-03
0.*031E-03
12.387
24.775
37.162
49.550
61.938
74.325
86.713
99.100
111.488
123.876
136.263
148.651
161.039
173.426
185.814
198.201
210.589
222.977
235.364
247.752
260.139
272.527
284.915
297.302
309.690
322.078
334.465
346.853
359.240
371.628
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
0.4333E-06
0.8894E-06
0.1345E-05
0.1801E-05
0.2257E-05
0.2714E-05
0.3170E-03
0.3626E-05
0.4082E-05
0.4538E-05
0.4994E-05
0.5450E-05
0.5907E-05
0.6363E-05
0.6819E-05
0.7275E-05
0.7731E-05
0.81B7E-05
0.8644E-05
0.9100E-05
0.9b56t-05
0. 1C01E-04
0. 1046E-04
0.1092E-04
0.1138E-04
0.1183E-04
0.1229E-04
0.1274E-04
0.1320E-04
0.1366E-04
0.1725E-03
0.3S4lb-03
0.5357E-03
0.7173E-03
0.8988E-03
0.1080E-02
0. 12626-02
0.1443fc-C2
0.1625E-C2
0.1806E-02
0.1988E-02
0.2170E-02
0.2351E-02
0.2533E-02
0.2714E-02
0.2896E-C2
0.307PE-02
0.3259E-02
0.344U-02
0. 362?E-02
0.3804E-02
0.3986E-C2
0.4167E-02
0.4349E-02
0.4530E-02
0.4712E-02
0.4894C-02
0.5075E-02
0.5257E-02
0.5438E-02
0.8645E-05
0. 1774E-04
0.2684E-04
0.3595E-04
0.4505E-C4
0.5415E-04
0.6325E-04
0.7235E-04
0.8145E-04
0.9C55E-04
0.9966E-04
0.1C87E-03
0.1178E-03
0.1269E-03
0.136UE-03
0.1451E-03
0.1542E-03
0.1fc33E-03
0. 17?4fc-03
0.1815E-03
0.1906E-03
0. 1997E-03
0.2C88E-03
0.2179E-03
0.227CE-03
0.2361E-03
0.2452E-03
0.2543E-03
0.2634E-03
0.2725E-03
0. 1998E-03
0.39T6E-03
0.5994E-03
0.799PE-03
0.9990E-03
C.H98E-02
0.1398E-02
0.1598E-02
0.1798E-02
0.1998E-02
0.2197E-02
0.2397E-02
0.2597E-02
0.27T7E-02
O.P997E-02
0.3196E-C2
0.3396E-02
0.3596E-02
0.3796E-02
0.3996E-02
0.4195E-02
0.4395E-02
0.4595E-02
0.4795E-02
0.4995E-02
0.5194E-02
0.5394E-C2
0.5594E-02
0.5794E-02
0.5994E-02
0.5796E-03
0.2624E-03
0.1866E-03
0.1394E-03
0.1H2E-03
0.9255E-04
0.7923E-04
0.6926E-04
0.6152E-C4
0.5534E-04
0.5028E-04
0.4608E-04
0.4252E-C4
0.3947E-04
0.3683E-04
0.3452E-04
0.3248E-04
0.3067E-04
0.2905E-04
0.2760E-04
0.262RE-04
0.250PE-04
0.2399E-04
0.2299E-04
0.2207E-04
0.2122E-04
0.2043E-04
0.1970E-04
0.19C2E-04
0.1838E-04
0.2084E-03
0.4173E-03
0.6262E-03
0.8351E-03
0.1044E-02
0.1252E-02
0. I'f616-02
0.1670E-02
0.1879E-02
0.2088E-02
0.2297E-02
0.2506E-02
0.2715E-02
0.2924E-02
0.3133E-02
0.3341E-02
0.3550E-02
0.3759E-02
0.3068E-02
O.M77E-02
0.43R6E-02
0.4595E-02
0.4804E-02
0.5013E-02
0.5222E-02
0.5430E-02
0.5639E-02
0.5848E-02
0.6057E-02
0.6266E-02
-------
PH'10.00
EQUIL IBRIUH ***INITIAL*** *************«,*****FINAL**********************
PICC2)
ATM
NA2C03
MG/L
ECUIV
CAC03
H2C03 HC03
************* NCLAR **
C03
***********
ALKALINITY
EQ/L
MAXIMUM
CALCIUM
KOLAR
FINAL
IONIC
STRENGTH
MG/L
0.3694fc-06
0.1109E-05
0.1848E-05
0.258fiE-05
0.3328E-05
0.4067E-05
0.4807E-05
0.5547E-05
0.6286E-0!>
0.7026E-Ot>
0.7766fc-05
0.8506t-05
0.9245E-05
0.9985E-05
0.1072E-04
0.1146E-04
0.122CE-04
0.1294E-04
0.1368E-04
0. 1442E-04
0.1516E-04
0.1590E-04
0.1664E-04
0.1738E-04
0.1812E-04
0.1886E-04
0.1960E-04
0.2034E-04
0.2108E-04
0.2182E-04
12.387
24.775
37.162
49.550
61.938
74.325
86.713
99.100
111.488
123.876
136.263
148.651
161.039
173.426
185.814
198.201
210.589
222.977
235.364
247.752
260.139
272.527
284.915
297.302
309.690
322.078
334.465
346.853
359.2*0
371.628
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
0. 1251E-07
0.3758E-D7
0.6264E-07
0.8771E-07
0. 1127E-06
0.1378E-06
0. 1629E-06
0.1879E-06
0.2130E-06
0.2380E-06
0.2631E-06
0.2882E-06
0.3132E-06
0.3383E-06
0.3634E-06
0.3884E-06
0.41 35E-06
0.4386E-06
0.4636E-06
0.4887C-06
0.5137E-06
0.5388E-06
0.5639E-06
0.5889E-06
0.6140E-06
0.6391E-06
0.6641E-06
0.6892E-06
0.7U3E-06
0.7393E-06
0.4984E-04
0.1496E-03
0.2494E-03
0.3491E-03
0.4489E-03
0.5487E-03
0.6485E-03
0.7483t-C3
0.8480E-03
0.9478E-03
0.1047E-C2
0.1147E-02
0.1247E-02
0.1347E-C2
0.1446E-02
0.1546E-02
0.1646E-02
0. 1746E-02
0.1845E-02
O.C1945E-02
0.2045E-02
0.2145E-02
0.2245fc-02
0.2344E-02
0.2444E-02
0.2544E-02
0.2644E-02
0.2743E-02
0.2843E-02
0.2943E-02
0.2497E-04
0.7498E-04
0.1249E-03
0.175CE-03
0.225CE-03
0.2750E-03
0.325CE-03
0. 3750E-03
0.425CE-03
0.4750E-03
0. 525CE-03
0.5750E-03
0.t250E-03
0.6751E-03
0.7251E-03
0.7751E-03
0.0251E-03
O.P751E-03
0.9251E-03
0.9751E-03
0. 1C25E-02
0.1C75E-02
0.1125E-02
0.1175E-02
0.12256-02
0.1275E-02
0.1325E-02
0.1375E-02
0.1425E-02
0.1475E-02
0.1998E-03
0. 3996E-03
0.5994E-03
0.7992E-03
0.9990E-03
0.1 198E-02
0.1398E-C2
0. 15986-02
0.1798E-02
0. 1998E-02
0.2197E-02
0.2397E-02
0.2597E-02
0.2797E-C2
0.2997E-02
0. 3106E-02
0.3396E-02
0.3516E-02
0.3796E-02
0.3996E-02
0.4195E-C2
0.4395E-02
0.4595E-02
0.4795E-02
0.4995E-02
0.5194E-02
0.5394E-02
0.5594E-02
0.5794E-02
0.5994E-02
0.2006E-03
0.6A83E-04
0.4009E-04
0.2863E-04
0.2227E-04
0. 1FS22E-04
0.1541E-04
0. 1336E-04
0.1179E-04
0.1C54E-04
0.9545E-05
0.8715E-05
0.8017E-05
0.7423E-05
0.6911E-C5
0. 64656-05
0.6074E-05
0.5726E-05
0.5417E-05
0.5139E-05
0.48886-05
0.4661E-05
0.4454E-05
0.4264E-05
0.4090E-05
0.3930E-05
0.3781E-05
0.3644E-05
0.3516E-05
0.3397E-05
0.2247E-03
0.4745E-03
0.7243E-03
0.9742E-03
0.1224E-02
0.1473E-02
0.1723E-02
0.1973E-02
0.2223E-02
0.2473E-02
0.2722E-02
0.2972E-02
0.3222E-02
0.3472E-02
0.372?E-02
0.3971E-02
6.4221E-02
0.4471E-02
0.'. 721E-02
0.4971E-02
0.5220E-02
0.5470E-02
0.5720E-02
0.5970E-02
0.6220E-02
0.6470E-02
0.6719E-02
0.6969E-02
0.72196-02
0. 74696-02
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/3-77-037
2.
3. RECIPIENT'S ACCESSION NO.
TITLE AND SUBTITLE
A CONTROLLED BIOASSAY SYSTEM FOR MEASURING TOXICITY
OF HEAVY METALS
5. REPORT DATE
April 1977 issuing date
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
K. H. Mancy and H. E. Allen
a. PERFORMING ORGANIZATION REPORT NO.
. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Environmental & Industrial Health
School of Public Health
The University of Michigan
Ann Arbor, Michigan 48109
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
Contract No. 14-12-591
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory-Duluth
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final Project Report
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Biological availability of metal micronutrients and metal toxicity are believed to
be dependent on metal oxidation state, complexation, and solubility as well as the
physicochemical characteristics of the aqueous phase. Basic design criteria for
fish bioassays which are capable of elucidating the dependency of toxicity on the
type and concentration of various copper species were developed utilizing equili-
brium chemical concepts and appropriate analytical techniques.
In order to maintain a desired copper species in the bioassay medium, synthetic
waters were used under well-defined physical and chemical conditions. These solu-
tions were synthesized in accordance with equilibrium models, which define the
distribution of various copper species as a function of the solution physical and
chemical characteristics. An experimental system was developed which permitted
large volumes of the bioassay waters to be maintained at the desired chemical
equilibria for the duration of the experiment.
Monitoring of the bioassay system included measurements of (a) pH, (b) temperature,
(c) flow, (d) specific conductance, (e) calcium, (f) total alkalinity, (g) dissolved
oxygen, and (h) copper species. Novel analytical procedures were applied for the
measurement and the differentiation of copper species. This included the use of
anodic stripping voltammetry and potentiometric membrane electrodes.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Experimental Design
Bioassay
Toxicity
Fishes
Chemical Equilibrium
Metal Complexes
06/F
06/T
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
121
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
111
U S GOVFDNMENI PRINTING Of fKt. 1977- "> 5 7 - 0 56 ; 5t>00
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