1010 POLLUTION EFFECTS ON FISH INTRODUCTION During the past twenty-five years, man has subjected the earth to an ever increasing variety of chemical substances never before encountered by living organisms. Many of these foreign substances, which originated from industrial, agricultural and municipal sources, are seriously threaten- ing the natural environmental conditions established over geologic time by evolutionary processes. Organisms, includ- ing man, are finding it more and more difficult to escape the consequences of exposure to these compounds, many of which are known to interfere with life sustaining biochemi- cal and reproductive mechanisms. Little is known, however, concerning the effects the vast majority of these pollutants will have on plants and animals that inhabit this planet. The aquatic environment is the ultimate destination of most of these contaminants as a result of rainfall and runoff from the lands. The establishment of water quality requirements for the protection of fish life must, therefore, become an item of first priority. The task is complicated, however, by the fact that dif- ferent species of fish as well as different developmental states of the same or different species exhibit wide variations in their sensitivity or tolerance to foreign materials. In addition, the basic biochemical and physiological information needed to evaluate the harmful effects foreign substances have on organ- isms is severely lacking for fish and other aquatic organisms. It is the purpose of this chapter to briefly discuss the current state of knowledge in regard to the response of fishes to environmental contaminants. Because of the serious lack of information in the area of fish toxicology, † it is not pos- sible, nor is it within the scope of this discussion, to pre sent even an outline of the toxic effects of the many foreign sub- stances which fish encounter. Instead, representative com- pounds have been selected to illustrate the principles that are believed to play a major role in determining how fish respond to environmental contaminants. Unfortunately, these principles are based almost entirely on research per- formed over the last several decades dealing with the toxi- cologic responses of mammalian species, including man, to drugs and foreign compounds. Until equivalent research is performed on fishes, we will be forced to rely on these mammalian principles when attempting to evaluate the effects foreign compounds have on fish species. MECHANISMS OF TOXICITY Foreign substances can injure organisms through a vari- ety of mechanism but these can generally be grouped into two categories: specific and non-specific injury. Chemicals that produce non-specific injury usually do so at the site of exposure, such as skin, respiratory membranes, oral mucosa, and intestinal mucosa. Frequently, such injury is the result of the caustic or corrosive nature of the chemical with the cellular responses being directly related to the concentration of toxicant within the cell or tissue involved. Agents pro- ducing this type of injury are commonly referred to as “pri- mary irritants” because they induce local, minor to severe inflammatory responses and occasionally extensive necrosis (death) of cells. Excellent examples of these chemicals are acids, bases and aldehydes, all of which are strong irritants to mucous and gill membranes. Other compounds, unlike the non-specific action of the primary irritants, can have a high degree of specificity and act at low dose levels at certain receptor sites to produce pathological change in specific cells with subsequent altera- tion of the function of organ systems. The degree of injury is dependent on the efficiency of the repair mechanisms for the tissue involved. If a cell is not irreversibly damaged, func- tional and structural characteristics may return to normal. Cells that are permanently injured at usually replaced by fibrous (scar) tissue. Fibrosis can seriously impair the func- tional capability of the tissue or organ involved. Examples of substances that exhibit specific toxicologi- cal actions are those that produce mutations by interfering with the genetic machinery of germ cells. Mutation of a dominant gene may express itself immediately and produce fatal abnormalities (teratogenic effects) or early fetal death. Other mutations may not express themselves for several gen- erations and then suddenly appear creating serious abnor- malities for that individual and its offspring. Chemicals may be carcinogenic (e.g. diethylnitrosamine and aflatoxin) and initiate the growth of malignant tumors in fish and mammals (Stanton, 1965; Ashley et al. , 1964). Many toxicants directly or indirectly affect reproductive mechanism, gonadal growth and development, spawning behavior, and fry survival by specific toxicological actions. This can frequently result in a depletion or extinction of a susceptible species. † Toxicology is the scientific discipline that deals with the quantita- tion of injurious effects on living systems resulting from chemi- cal and physical agents that bring about alterations in cell or organ structure and function. C016_009_r03.indd 1010C016_009_r03.indd 1010 11/18/2005 1:12:29 PM11/18/2005 1:12:29 P © 2006 by Taylor & Francis Group, LLC POLLUTION EFFECTS ON FISH 1011 FACTORS THAT INFLUENCE TOXICITY Several biological, physical and chemical factors play a part in determining the ultimate toxicological consequences of a foreign compound on an organism. To produce injurious effects, a toxicant must achieve an adequate concentration at its sites of action. The concentration attained at these sites in the animal obviously depends on the amount of foreign compound present in the animal’s environment. Equally important, however, are the extent and rate of the toxicant’s absorption, distribution, binding or localization in tissues, inactivation, and excretion. These factors are depicted in Figure 1 (Goodman and Gilman, 1970). The following sections discuss each of these aspects that play the major role in the response of organisms to foreign compounds. Absorption of the Toxicant Generally speaking, a compound with specific toxicologi- cal actions must first be absorbed and distributed (biological translocation) in an organism before it can reach its specific site of action and exert its toxic effect. The ease with which foreign substances are absorbed, therefore, is a significant factor in determining the toxicity of foreign compounds. Absorption of substances by fish occurs through the skin, oral mucosa, intestinal mucosa, and gills. Because it is nec- essary that gill surfaces be exposed to large volumes of water for the maintenance of adequate blood levels of CO 2 and O 2 , this organ is an especially vulnerable site for the absorp- tion of foreign materials. Fish are, therefore, exceptionally susceptible to toxicants that readily cross the gill epithe- lium. In addition, fish acquire many foreign substances from their diet by absorption across the gastrointestinal mucosa. It should be mentioned, however, that studies are severely lacking about this and other mechanisms of translocation of foreign substances in fishes. The biochemical and physicalchemical properties of a compound determine both its ability to cross biological membranes and its distribution within an organism. In gen- eral, the non-ionized, non-polar forms of compounds are significantly soluble in fat, i.e. lipid soluble, and are there- fore readily transported across the lipidal components which characterize animal-cell membranes (Whittaker, 1968). The chemical structure of a foreign compound also determines its ability to react with biological molecules as well as its susceptibility to biotransformation (metabolism) by organ- isms. The ability of an animal to metabolize foreign com- pounds is important to that organism because the products (metabolites) formed usually are less toxic (but occasionally greater) than the parent compound. A more detailed discus- sion of biotransformation mechanisms appears later in this chapter. Distribution of the Toxicant Once the toxicant is absorbed and has entered an organism’s blood or lymphatic system, it is readily transported and dis- tributed to sites of action, centers of metabolic breakdown or detoxication, storage, and excretion. Most toxicants are transported reversibly bound to blood proteins with only a small portion existing in the free or unbound state. Compounds must usually exist in this unbound state to react with biological molecules (receptors) and interfere with biochemical mechanisms. Therefore, the total amount of plasma protein available for the binding and transport of toxic substances plays an important role in the toxicological consequences of these compounds (Petermann, 1961). It is interesting to note that fish, when compared to mammals, have a distinct disadvantage in this regard since Locus of Action Receptor Free Toxicant Bound Toxicant Plasma Bound Toxicant Metabolites Excretion Absorption Free Toxicant Tissue Depots Bound Free Biotransformation FIGURE 1 C016_009_r03.indd 1011C016_009_r03.indd 1011 11/18/2005 1:12:30 PM11/18/2005 1:12:30 P © 2006 by Taylor & Francis Group, LLC 1012 POLLUTION EFFECTS ON FISH they have much less plasma protein. Compounds that have a high tendency to bind to plasma proteins may compete or displace one another from binding sites when they exist together in the blood. This may be the mechanism of danger- ous toxic interactions because plasma protein binding sites become so saturated that a greater percentage of unbound toxicant exists in the blood than would normally be present with only one toxicant. When the degree of plasma binding is high and the rate of release is low, plasma proteins can act as a storage depot for the bound substances. Storage depots also frequently result from a particular affinity a toxicant may possess for certain organs or tissues. Examples of this are the chlorinated hydrocarbon pesticides which are stores in body fat and heavy metals such as copper and mercury which are stored in the liver and kidney of fish (Life, 1969). Many foreign compounds are capable of producing a specific effect, that is, are selectivity toxic, on a specific bio- logical system or systems. These systems are said to be the site of locus of action of the chemical. The site may be con- fined to one anatomic location within the animal, or may be diffusely located throughout the animal. Two fundamental types of mechanisms are responsible for the selective action of chemicals on cells or cellular mechanisms. The first type is a result of factors that increase the con- centration of the toxicant at specific cell or tissue sites. This is accomplished in the organisms by mechanisms of selec- tive translocation and biotransformation. One good example is the renal tubular (kidney cell) injury produced in fish exposed to copper. Because copper is excreted by the kidney it accumulates at tubular cells. As the concentration rises injurious levels are reached and these cells are damaged or destroyed (Life, 1969). A second mechanism in the selective toxicity of chemicals on cells involves the presence of specific targets or receptor systems in exposed cells. In this case, the concentration of the toxicant is the same for all cells, but only certain cells are affected. This is due to the specificity of action of the toxi- cant on receptors that are normally occupied by endogenous hormonal or neurohormonal substances. Organophosphorus compounds such as parathion and malathion are good exam- ples of selectively toxic agents that act in this way. These cholinesterase inhibitors act to inhibit the enzyme respon- sible for hydrolysis of the neurotransmitter, acetylcholine. In this example cholinesterase is considered to be the receptor. Prevention of the hydrolysis of acetylcholine results in the continuous stimulation of post-synaptic sites throughout the central and peripheral nervous systems and rapid death due to respiratory paralysis is the usual outcome. Biotransformation and Excretion of the Toxicant The duration and intensity of injurious action of many for- eign compounds are largely determined by the degree and speed at which an organism can eliminate these compounds (Conney, 1967). The kidneys of both marine and fresh water fishes have been shown to share with mammalian kidneys the primary role of ridding an animal of potentially toxic compounds (Forster, 1961, 1967). The excretion of substances by the kidney is largely determined by lipid solubility characteristics of the com- pounds as they enter renal tubules. Molecules with a high degree of lipid solubility are readily re-absorbed from the renal tubule through lipoidal membranes back into the cir- culating blood and consequently are not excreted. It is only through certain specific biochemical transformations of these foreign compounds by the organism itself that lipid solubili- ties are altered and tubular excretion is successful (Brodie and Erdos, 1962). These reactions or transformations can be classified as oxidations, reductions, hydrolyses and synthe- ses (conjugation). Most animals, including fish, transform (metabolize) foreign compounds in two successive phases, the first phase consisting of a variety of oxidations, reductions, and hydro- lyses and the second phase of a limited number of synthe- ses or conjugations (Williams, 1967). Phase I reactions can result in: 1) The inactivation of a toxic compound; 2) The conversion of an initially inactive compound into a toxic compound; and 3) The conversion of a toxic compound into another toxic compound. The second phase of biotransformation, consisting of synthetic reactions, most often results in the conversion of toxic compounds into inactive excretory products. This con- cept of the metabolism of foreign compounds can be repre- sented as in Figure 2 (Williams, 1967). Biochemical reactions of both phases of metabolism are catalyzed by enzymes located in various organ systems, and it is from the study of the qualitative and quantitative variations in these enzymes that an evaluation of detoxifying capacities can be made for an organism (Williams, 1967). Phase I reactions are carried out by enzymes of normal metabolic routes and by enzymes which occur in the smooth endoplasmic reticulum of liver cells. When these cells are ruptured in the laboratory by homogenization the endoplas- mic reticulum undergoes fragmentation. High-speed cen- trifugation separates these fragments from the remaining cell constituents. These fragments are referred to as micro- somes and it is the microsomal enzymes that are involved in the metabolism of many drugs and foreign compounds. Microsomal enzymes do not generally act on lipid-insoluble compounds but rather convert lipid-soluble materials by oxi- dative and reductive processes to less lipid-soluble metabo- lites, which are more polar substances and, therefore, readily Phase I Drug Activation or inactivation Oxidation reduction and/or Hydrolysis products Phase II Inactivation Synthetic o r conjugation products FIGURE 2 C016_009_r03.indd 1012C016_009_r03.indd 1012 11/18/2005 1:12:30 PM11/18/2005 1:12:30 P © 2006 by Taylor & Francis Group, LLC POLLUTION EFFECTS ON FISH 1013 excretable by the kidney. Without these biotransformations, the effects of some foreign substances on organisms would last for months (Brodie et al., 1965). Excretion of these lipid- insoluble metabolites can be achieved by active secretion at the tubules of the kidney or by passive transport across the glomerular membrane into the renal tubule (Forster, 1961, 1967). Since the metabolite has been transformed to a less lipid-soluble derivative, it will not diffuse back into the plasma for recirculation subsequent to passing through the glomerulus into the tubule. The microsomal enzymes were, until recently, thought to be concerned only with the metabolism of compounds which are normally regarded as foreign to the body. Conney (1967) has recently shown that steroid hormones and other normal body constituents are also substrates of the drug- metabolizing enzymes in liver microsomes and he suggests that this enzyme system may play a significant role in their regulation and physiological action. These enzyme systems are thought to operate by a mixed function oxidase mecha- nism whereby NADPH reduces a component in microsomes which reacts with molecular oxygen to form an “active oxygen” intermediate. The “active oxygen” is then trans- ferred to the drug or toxicant (Gillette, 1963). Key enzymes in the overall reaction are NADP-cytochrome c reductase, the flavin enzyme involved in the oxidation of NADP, cyto- chrome P-450, and NADPH cyctochrome P-450 reductase, which acts to reduce oxidized cytochrome P-450 (Gillette and Sasame, 1966). Many foreign compounds can alter these key enzymes and enhance or impair the ability of liver microsomal enzymes to metabolize other foreign compounds and ste- roids (Conney, 1967). Halogenated hydrocarbon insecti- cides have long been known to be potent stimulators of mammalian drug-metabolizing enzymes (Hart and Fouts, 1963; Hart, Shultice and Fouts, 1963). Buhler (1966) selectively induced drug-metabolizing enzymes in rain- bow trout by exposing these animals to DDT or phenyl- butazone. Organophosphate insecticides are unlike the halogenated hydrocarbons in that they inhibit, rather than stimulate, the metabolism of drugs and steroids by liver microsomes, when given chronically to rats (Rosenberg and Coon, 1958; Welch et al., 1967). Some heavy metals (Fe ϩϩ , Cu ϩϩ , Zn ϩϩ and Co ϩϩ ) have also been shown to be inhibitory to drug metabolism in mice and rats (Peters and Fouts, 1970). Alterations in microsomal enzyme metabolizing capacities can substantially alter an animal’s response to foreign compounds as well as its ability to hydroxylate steroid hormones and other normal body con- stituents (Conney, 1967). Altered steroid metabolism can directly affect the animal’s ability to cope with environ- mental stresses as well as seriously impair reproductive mechanisms. Other than Buhler’s (1966) work on rainbow trout, little is known about the effects foreign compounds have on the microsomal drug metabolizing capacities of fishes. In addition, the current state of knowledge dealing with metabolism of foreign compounds in fishes is, at best, scanty (Adamson, 1967). THE TOXICITY OF COMPLEX EFFLUENTS Most industrial effluents contain mixtures of two or more substances. These complex effluents present special prob- lems in evaluating their toxicities to fish and other organisms. In some cases two agents with similar pharmacologic actions can produce a response that is equal to the summation of the effects of the individual agents or greater than the summa- tion of the independent effects of the two agents. The latter response is called “potentiation” and represents the condition whereby one compound is made more toxic in the presence of another compound which alone may produce minimal or no pharmacologic effect. Potentiation poses a special problem for the aquatic environment as well as the terrestrial environ- ment for it is possible that a certain combination of relatively harmless substances may result in an unpredictable high level of toxicity that would seriously threaten the existence of one or many species. Usually, however, the effect of two agents is the summation of responses to each agent. Occasionally, the effect of a toxic substance is reduced on the addition of another substance, a phenomenon referred to as antagonism. In some cases the antagonistic substance may or may not be toxic when present by itself. Synergism (potentiation and summation) and antagonism are poorly understood phenomena and greatly confuse the understanding and prediction of the toxic effects of industrial effluents. The majority of toxicological studies conducted in this and other countries on both mammals and fish deal with the effects of single substances on organisms; only a few stud- ies are currently investigating the responses of organisms to complex mixtures of substances. Synergism and antagonism are worthy of further investigation, for little is known about the basic mechanisms governing these processes. RESISTANCE OF FISH TO TOXICANTS Resistance of animals to chemicals has been known to exist for some time now and has posed serious obstacles in the control of insects and bacteria. While the mechanisms of resistance remain thus far a mystery, we know that they are genetically based. Most susceptible populations of animals have an occasional individual which exhibits resistance and it is this member that provides the genetic material for selec- tion pressures to act upon. Resistance has best been demonstrated with various pesticides in several natural populations of fishes (Ferguson et al., 1964, 1966), but these findings have attracted little attention. Many biologists have, in fact, believed this phe- nomenon to be beneficial to these animals, especially since they are useful to man. Recently, some disturbing evidence has emerged sug- gesting that pesticide-resistant vertebrates pose a major hazard in natural ecosystems and that they may be creating serious toxicological problems for man (Ferguson, 1967). The reason for this is some resistant animals carry massive residues of unaltered pesticides in their tissues and aggra- vate the already serious problem of biological magnification. C016_009_r03.indd 1013C016_009_r03.indd 1013 11/18/2005 1:12:30 PM11/18/2005 1:12:30 P © 2006 by Taylor & Francis Group, LLC 1014 POLLUTION EFFECTS ON FISH Animals whose resistance is due to enhanced abilities to metabolize toxic substances to inactive metabolites do not, however, contribute to biological magnification. Biological magnification results when a foreign sub- stance enters plants and small animals and is then passed rapidly along food chains to larger animals. As this happens the substance becomes more and more concentrated until it reaches dangerous levels in the large predacious fish, many of which are consumed by birds and mammals including man. Clearly, the more resistance a fish has for the particu- lar toxicant in its tissues the greater the likelihood it will be consumed by animals living on the land. Unfortunately, these animals may not have equivalent levels of resistance and may be unable to adequately deal with these toxicants. CURRENT METHODS OF EVALUATING TOXICITY IN FISHES The establishment of water quality standards for concentra- tions of toxic pollutants that will be safe for fish has recently become a major concern of Federal and State Governments in pollution control (Water Quality Criteria, 1968). Efforts in this regard have centered around determining lethal limits of toxicants by establishing a TLm (tolerance limit, median) of various species exposed to toxicants for periods of time up to 96 hr (Sprague, 1969). These short term studies have been valuable in defin- ing the upper limits of toxicity but have not considered the subtle deleterious effects of foreign compounds which may not be evident for weeks, months, or longer (Water Quality Criteria, 1963). These responses to toxicity may manifest themselves in appetite changes, metabolic alterations, dis- orders of the nervous system, reproductive changes, behav- ioral abnormalities, or alteration of vital functions which are not immediately lethal. For this reason, investigations have only recently been conducted to measure toxicity in terms of survival, growth, and reproductive alterations resulting from long-term exposure to sublethal levels of pollutants (Water Quality Criteria, 1968). The concept of a “maximum accept- able toxicant concentration (MATC)” has originated from these studies and is defined as the highest continuous con- centration of a toxicant that does not significantly decrease the laboratory fish production index; an index developed by Mount and Stephen (1967) which takes into account sur- vival, growth, reproduction, spawning behavior, viability of eggs, and growth of fry. Because the toxicity of most pollutants varies with water characteristics and fish species, Mount and Stephen (1967) proposed the use of an “application factor,” (calculated by dividing the MATC of the 96-hr TLm value), to determine safe concentrations of toxic pollutants which, when deter- mined for one species of fish in one type of water, may be applicable to other waters and other species. Studies are currently underway to test the practicality of this approach (Mount, 1968; Mount and Stephen, 1969). The “application factor” approach may improve upon present methods of estimating safe concentrations of toxicants. At best, however, this approach requires considerable time for collection and evaluation of data and measures only the end result of a multitude of biochemical, physiological, metabolic, pharmacological and pathological responses to toxicants. Little definitive information is gained concerning the mechanism which produces the gross changes upon which the “application factor” is based. Because of the virtual impossibility of thoroughly assess- ing the individual, cumulative, synergistic and antagonistic effects of the numerous substances continually being intro- duced into our environment, it is imperative that we know the basic metabolic, physiologic and toxicologic responses of fish to compounds representative of broad categories of foreign substances. Only then will we be in a position to predict intelligently the biological effects of toxicants and regulate their concentrations to assure protection of this very important biological resource. If fish toxicologists continue to consider only the effect of a substance on the labora- tory fish production index without understanding causative mechanisms, they will severely limit the amount of informa- tion available for making meaningful decisions so desper- ately needed in water pollution control programs. Investigations of the disposition of foreign compounds in fish will shed valuable information on the evolution of enzymes that metabolize drugs, on drug metabolic pathways and excretion, and on factors affecting the biological half- life of foreign compounds in their lower species as well as higher vertebrates including man (Adamson, 1967). The edi- the collection of fishes with fin erosion or other deformities. A few concentrated on fresh water streams (Reash and Berra, 1989; Sindermann, 1979) whereas a preponderance focused on marine or estuarine environments with and without pol- example. Reash and Berra found that the incidence of fish erosion was significantly greater at polluted stream sites compared to unpolluted sites. REFERENCES Adamson, R.H. (1967) Fed. Proc., 26, 1047. Ashley, L.M., J.E. Halver and G.N. Wogan (1964) Fed. Proc., 23, 105. Brodie, B.B., G.J. Cosmides and D.P. Rall (1965) Science, 148, 1547. Brodie, B.B. and E.G. Erdos (1962) Proceedings of the First International Pharmacological Meeting, 6 , Macmillan, New York. Buhler, D.R. (1966) Fed. Proc., 25, 343. Conney, A.H. (1967) Pharm. Rev., 19, 317. Cross, J.N. (1985) Fish Bull. , 83, 195. Ferguson, D.E. (1967) Trans. Thirty-Second North Am. Wildlife and Natu- ral Resources Conf. Ferguson, D.E., D.D. Culley, W.D. Cotton and R.P. Dodds (1964) Bio Science, 14, 43. Ferguson, D.E., J.L. Ludke and G.G. Murphy (1966) Trans. Am. Fish. Soc., 95, 335. Forster, R.P. (1961) Kidney cells, in The Cell, Ed. by J. Brachet and A.E. Mirshy, 5 , pp. 89–161. Academic Press, New York. Forster, R.P. 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BARDACH University of Michigan C016_009_r03.indd 1015C016_009_r03.indd 1015 11/18/2005 1:12:30 PM11/18/2005 1:12:30 P © 2006 by Taylor & Francis Group, LLC . of oxidations, reductions, and hydro- lyses and the second phase of a limited number of synthe- ses or conjugations (Williams, 1967). Phase I reactions can result in: 1) The inactivation of. Investigations of the disposition of foreign compounds in fish will shed valuable information on the evolution of enzymes that metabolize drugs, on drug metabolic pathways and excretion, and on factors. concentrations to assure protection of this very important biological resource. If fish toxicologists continue to consider only the effect of a substance on the labora- tory fish production