J. Carl Barrett,1 Harri Vainio,2 David Peakall,3 and Bernard D. Goldstein4
Introduction
There are both commonalities and differences between the sections on human and nonhuman organisms in this report on susceptibility. Many of the techniques are the same, but whereas for human health the main consideration is the protection of susceptible individuals, in environmental terms we are more concerned with susceptible species and communities. Even this concern is double edged, as there are species for which human efforts to control have led to the formation of highly resistant forms. Studies of the mechanisms whereby this resistance has occurred have given much valuable information on the differences between susceptible and resistant populations.
While this section of the joint report follows the sequence of the human health sections, namely, biomarkers of exposure, effect, and susceptibility, emphasis is given to the last category as in general the same biomarkers of exposure and effect are used in human and nonhuman biota. Examples of how these biomarkers are used in the environmental context will be given.
Here we use the term biomarker as a biological change that is a measure of exposure and sometimes of toxic effect caused by environmental chemical(s) at the level of the individual or below. Changes occurring above the level of the individual are referred to as biological indicators.
Figure 2. The role of susceptibility in individuals, biota, populations, and ecosystems.
The discussion here on susceptibility covers a broad canvas. There is first a discussion of biomarkers of susceptibility at the cellular and molecular level, followed by those at the individual level, both long and short term. Then we move up the organizational scale discussing biological indicators of sensitivity at the population, community structure, and ecosystem levels. We conclude with two case examples of individual chemicals or chemical classes to illustrate these points. Finally, we note that humans are part of ecosystems, and the biomarkers of ecosystem susceptibility can also indicate and predict effects of chemicals on humans. The best biological indicators will both warn humans of potential danger and alert us to severe ecosystem damage (Figure 2).
Biomarkers of Exposure and Effect
We regard biomarkers of exposure and effect as essentially a continuum (Figure 2). Whether a biomarker can be used as an indicator of effect as well as of exposure often depends on the depth of our knowledge of a particular case. Thus, we have not made any effort at making this rather artificial separation.
Biomarkers and higher level biological indicators are important tools in ecotoxicology. Their application to studies of chemical effects on nonhuman biota are well documented. However, as this does not fall within the bounds of this discussion, the readers' attention is drawn to a number of authoritative texts (165-167).
Among the hierarchy of biomarkers and biological indicators, representing pollutant-induced changes in biota at all levels of organization (from the molecular to ecosystem), lie the means to identify, classify, and ultimately quantify, the susceptibility of individuals and populations to the detrimental effects of chemical exposure. At the levels of molecule, organelle, and cell, there are a number of changes we may use in our studies of susceptibilities. In the case of DNA adducts, for instance, we can measure the quantity and rate of adduct formation in one individual and compare it to others in the same population. Interspecies variations could also be measured. Similarly, there are a number of important cellular regulation and defense proteins that may be quantified for comparative purposes.
Detoxification proteins such as the mixed-function oxidases (MFO) and the cytochrome P450 enzymes may be measured, as well as the multidrug/multixenobiotic resistance proteins. The levels of such molecules in individuals may give some important clues to the susceptibility of an organism to toxic insult.
Efficient DNA repair has been shown to be an important determinant of chemical and radiation-mediated damage in cells. We may therefore consider the cellular levels of the various repair enzymes as indices of overall susceptibility. Other important molecules are the proteins involved in cell regulation mechanisms. Proteins such as the oncoproteins may be useful, along with the functional analogues of proteins such as p53, coded by the tumor-suppressing gene.
Other indicators of susceptibility at the subcellular level include changes in organelle structure and function, chromosomal aberrations, and the formation of micronuclei to genotoxic agents, as well as the functional integrity of lysosomes. These cellular/molecular indicators of susceptibility may be considered in higher animals and plants to be only components of the overall picture of organism response. Thus they may be used in conjunction with other higher level markers. However, for a large number of single-celled organisms (and simple organisms) they may play a more important role.
Chemical-induced changes in many types of cells, tissues and organs can also be seen as markers of susceptibility. A high level of cellular dysfunction results in distinct morphological and biochemical consequences. An example is apoptosis or programmed cell death. The function of a cell may be so impaired, or damage to its DNA so great, that a "suicide" sequence is initiated, resulting in degeneration and death. Such a process may be measured histologically, and the levels of apoptosis measured in a target tissue or organ. Cellular dysfunction may also be measured in a biochemical manner, and tracing increases and/or decreases in tissue specific molecules such as hormones and enzymes may be a key indicator of individual susceptibility.
Finally, physiological parameters are potentially important markers, as they relate directly to fitness and consequently have higher level effects. Also included are homeostatic processes such as thermo- and osmoregulation, alteration to respiration, and changes in cardiac function.
DNA Adducts
32P-postlabeling has been used for a number of environmental studies. Some of the most detailed are those carried out in Puget Sound in the State of Washington. Varanasi et al. (168) used the 1-butanol adduct enhancement method to measure the level of DNA adducts in English sole (Parophyrs vetulus) that were exposed to high concentrations of sediment-associated chemical contaminants and exhibited elevated levels of hepatic neoplasms. The level of DNA adducts in contaminated sites averaged from 17 to 26 nmol/mol nucleotides compared to <0.2 from a control site. The finding that the levels of aromatic hydrocarbons in the sediments of Puget Sound were positively correlated with prevalence of hepatic neoplasms and related lesions in English sole were compared to those found for the starry flounder (Platichthys stellatus) (169). These workers found that the starry flounder had a lower prevalence of hepatic neoplasms and studies suggested that biochemical differences in the metabolism of carcinogenic PAHs can explain the lower susceptibility of the flounder to chemical-induced hepatocarcinogenesis.
The HPLC/fluorescence spectrophotometry approach has been used to show the presence of B[a]P adducts in a population of beluga whale (Delphinapterus leucas) in the St. Lawrence River, whereas no such adducts were found in the brains of whales from the Arctic population (170). The finding of B[a]P adducts in the brains of belugas in the St. Lawrence can be correlated to the high incidence of tumors (cause of death in 18% of the 61 whales post mortemed) in this population.
The formation of DNA adducts has been considered a key step in the initiation of carcinogenesis. However, the presence of DNA adducts in aquatic invertebrates that rarely or never develop neoplasms may obviously be linked to genotoxic end points other than tumorigenesis. Based on good correlations between the induction of DNA adducts and gene mutations, it was hypothesized that, in natural species, a variety of manifestations of the mutational event may actually prove much more biologically and ecologically important than the induction of neoplasia (171).
Strand breakage of DNA has also been used to study the effects of environmental pollutants. Although strand breakage can lead to cell death, in many cases it is repaired before serious damage occurs. Strand breakage as measured by the alkaline unwinding assay is one of a suite of biomarkers used to study the effects of pollution on the liver DNA of bluegill sunfish (Lepomis macrochirus) in rivers of the eastern United States (172). The basic prerequisite for the successful use of DNA adduct and DNA strand breakage in environmental risk assessment studies, however, is the recognition of the fact that lower invertebrates may be incompetent in forming DNA adducts from some highly prevalent pollutants such as PAHs (173).
Some indicators of chemical exposure and effects methods are shown in Table 2.
Biological Indicators of Susceptibility at the Individual Level
The phenomenon of resistance is a striking example of differential susceptibility of organisms to environmental chemicals. There are a number of mechanisms whereby organisms can resist the action of toxic chemicals. These include decreased uptake, increased efflux, increased rate of metabolism of the toxicant, sequestration of the toxin, and increase or repair of target sites within the cell. In some cases more than one mechanism is involved and changes made against one chemical can protect against another; hence, the phenomenon of cross-resistance.
The factors that influence the onset of resistance fall into three broad categories: genetic, biological, and operational. In the first category is the frequency and dominance of the resistant gene. Dominance is important; if the gene is normally recessive because it has disadvantages, such as lower reproductive capacity, the greater the genetic disadvantage, the slower the resistant strain takes over. The shorter the generation turnover and the greater the number of offspring per generation, the more rapidly resistance can occur. For pesticide resistance, the operational factors include the actual pesticide used and the mode and area of application. It is in the area of disease resistance in insects that we have the greatest control (174).
The degree of resistance can be large. For example there are houseflies for which the LD50 for DDT is 3000 times normal, the main mechanism that defends the organism being increased levels of a dehydrochlorinase (175). Resistance among vertebrates is not so evident, although it has been reported in several species of fish, and the case of warfarin resistance in rats is well known.
Resistance can cause considerable changes in the community structure of invertebrates. Man's attempts to control the pests that affect cotton in northeastern Mexico and the southern United States is a good example of the problems that occur with resistance and the alterations of community structure caused by differential susceptibility of organisms to environmental chemicals (176). During the period 1945 to the mid-1950s there was almost complete control of the main pests--the boll weevil and the cotton fleahopper--by OC pesticides and there was a spectacular increase in cotton production. In the mid-1950s it was necessary to switch to OP compounds which increased the costs but cotton was still profitable. Former minor pests, the bollworm and tobacco budworm were becoming major pests. By the mid- to late-1960s all the pests were highly resistant to all insecticides and cotton production dropped markedly. The introduction of integrated pest management was needed to solve this problem.
Individual Susceptibility. Susceptibility of individuals to chemical stress, or their sensitivity to chemicals, is hard to define. Pragmatically, susceptibility may be defined as a lack of resistance, or tolerance, to chemical stress. Such a definition enables the recognition of molecular, biochemical, or physiological mechanisms that constitute a biological defense mechanism against chemical pollution. These include mechanisms dependent upon the activity or levels of mixed-function oxidases, N-acetyltransferases, glutathione S-transferases, glutathione, glucuronidation and sulfation, heat-shock proteins, and metallothioneins. The basic characteristic of these biologic defense/detoxication mechanisms is their inducibility. The induced state of these enzymes, often expressed in thousands of percent increase over natural state, serve as useful biomarkers of exposure/effect. Their activity and inducibility directly influence the outcome of exposure to toxic chemicals (induction of DNA adducts, induction of single strand breaks in the DNA, induction of chromosomal aberrations, modulation of fitness and the frequency of diseases, lethality, extinction of species). In resistant organisms both the natural level and more importantly the inducibility of one or more of these mechanisms is higher than in susceptible individuals. Generally, better response of detoxication/defense mechanisms is inversely proportional to susceptibility. Thus, biomarkers of exposure may be used as short-term indicators of biological susceptibility of individuals to chemicals: low response in these biomarkers will denote a high level of susceptibility. For example, lower inducibility of 7-ethoxyresorufin O-deethylase activity in carp will denote their higher susceptibility to toxic effects of pollutants.
Using the same pragmatic definition of susceptibility as a lack of resistance, one can utilize a recently discovered multixenobiotic resistance mechanism (MXR) as a long-term biomarker of susceptibility of individuals to chemicals. The MXR represents a general biologic first-line defense mechanism, it is taxonomically broadly distributed, and its expression is species dependent. However, there are considerable variations in its expression on an interindividual basis (177). The low expression of activity of P-glycoprotein, a dominant feature in the MXR mechanism (178), in, for example, the snail Monodonta turbinata specimens living in a pristine marine environment, indicates high susceptibility for accumulation of xenobiotics and, consequently, higher likelihood of effects (179). In contrast, specimens of snails living at polluted sites, expressing higher activity of P-glycoprotein due to its induction, are less susceptible to toxic effects of xenobiotics due to a lower level of its accumulation. Thus, the level of expression of MXR may be a good long-term indicator of susceptibility to chemicals. Similarly, species with high levels of MXR (P-glycoprotein) expression, such as mussels (Mytilus galloprovincialis, Mytilus edulis, Crasostera gigas) (180), clam (Corbicula fluminea) (181), marine snail (M. turbinata) (179), marine worm (Urechis caupo) (182), or four marine sponges (183), in contrast to a fresh water mussel (Anodonta cygnea) and a fresh water snail (Paludina vivipara) (179), are not susceptible to chemical pollution. Thus, the low, or nihil, expression of MXR in A. cygnea and P. vivipara are biomarkers of their long-term susceptibility.
Excellent methods are available for measurement of a functional state of MXR (184), as well as for measurement of concentration of inhibitors of MXR, the chemosensitizers, in environmental samples (185).
Several species or groups can be good indicator species. Very good bioindicators are the water insects, especially species and groups of species of the order Ephemerophera. Ecological niches and needs of their larvae are well known. By feeding on detritus they accumulate in their bodies xenobiotics that can be reliably identified (186).
Behavioral Mechanisms. Individuals can clearly be exposed to chemicals in situations in which they cannot escape or move away from the exposure: then they must rely on biochemical detoxifying methods to avoid effects. Sessile organisms that make up most species in the aquatic world are unable to move away from exposure. However, many organisms, particularly vertebrates, have a number of behavioral and life-history strategies that enable them to avoid exposure to chemicals. These mechanisms make them less vulnerable or less susceptible to the effects of these chemicals.
The most obvious mechanism to avoid chemicals is to move away from exposure; and many animals do this by simply flying, swimming, or walking away. This mechanism functions in many species when they are exposed to high doses of chemicals such as occurs during an oil spill. Birds, many marine mammals, and fish will swim or fly from advancing oil when it is possible to do so (187). However, other fish and some marine mammals will not do so until it is too late to avoid exposure. Most exposures to chemicals, however, occur at such low doses that animals do not, or cannot avoid them.
The impairment of locomotion or abnormal behavior of animals is being used to monitor pollutants. Fish have been used commonly--monitoring the frequency of coughing (188,189), loss of rheotaxis, or disturbance of schooling. These methods are linked with automatic water sampling systems to serve pollutant analysis. Direct measurement of respiration through blood vessels in fish was devised to monitor contaminants (190). Monitoring of mussels' activity is also practical since mussels respond to hazardous chemicals by closing their bulbs. Abnormal web spinning by caddisfly larvae or failure to make nests by other aquatic insects are also useful methods to monitor pollutants. Feeding activity can be another biomarker. A short-cut test with Daphnia using toxicant-induced inhibition of feeding is being studied.
For showing the changes caused by long-term exposure, it is most important to observe changes in the populations of individual species. Changes in populations that are useful include the changes in population dynamics, fertility and fecundity, physiological resistance to external factors, behavior, levels of parasitism or pathogen infestation, and changes in the structure of communities of social insects (186).
Life History Parameters. In addition to behavior, some life history strategies allow animals to avoid exposure, thus rendering them less susceptible to exposure and subsequent effects. Many species migrate during part of the year, removing them from low-level chronic exposure; other species hibernate beneath the ground where they also reduce exposure and thus their vulnerability.
The two life history strategies that render animals most susceptible to the effects of chemicals are a sessile life style and a long life span. Species that are relatively long lived, such as most vertebrates and trees, are more susceptible to chronic exposure to chemicals than those that have a short life span. This factor functions with chronic, low-level exposure rather than acute, toxic levels of chemicals. Species that have a life history strategy that involves short life spans have a reduced susceptibility to chemicals because the potential for bioaccumulation is less and their reproductive potential is higher. Chemicals such as dioxins, PCBs, DDTs, and mercury, which bioaccumulate in internal tissues and are thus available for remobilization into the bloodstream, can have severe effects on organisms that live a long time.
Of the invertebrate bioindicators, insects are the most valuable because of their quantities and diversity, and because of our sufficient knowledge of their taxonomy, morphology, physiology, and ontogeny. Their living areas may be rather limited, their life cycles short, and their reproductive potential enormous, with great variations that can be regulating. These properties are useful for bioindication. The diversity of insect communities is distinct and cumulation of substances is specific; it is not difficult to observe changes in population dynamics and in individual development because the life cycles are short. The reproductive system of insects is very sensitive to biologically active substances (proliferation of follicular cells, change in structure and function of nutritive cells, etc.) This provides the opportunity to use the reproductive system of insects as a model for testing the effects of toxic substances in the environment.
Taxa Variation. In the environmental field we have to look not only at susceptible populations of any one species but also at interspecies variations that can cause a differential effect. In this section we examine a number of biomarkers that vary widely in their sensitivity from one group of animals to another.
MIXED-FUNCTION OXIDASES. MFOs are a major component of the defenses of organisms against toxic chemicals in their environment. Originally evolved to handle naturally occurring toxic compounds, they now play an important role in the detoxification of man-made chemicals. Nebert et al. (191) consider that the ancestral cytochrome gene is probably two thousand million years old. The major divergence occurred eight hundred to one thousand million years ago when animals began using plants as food and self-defense mechanisms against toxins in plants evolved. Later, additional families of cytochromes evolved in response to the necessity to metabolize combustion products.
There is some variation between taxa: in general terms, the relative activity of the MFO enzyme epoxide hydrase follows phylogenetic lines with mammals > birds and amphibia > fish, with little overlap between the groupings. Levels in invertebrates are much lower. Glucuronyltransferase activities were much higher in mammals than fish with virtually no overlap. Aldrin epoxidase activity follows the same trend, but in this case there is considerable overlap. A linear log relationship was found between relative activity and body weight for mammals, with man being the outlier having activity lower than would be expected by body weight.
Fish-eating birds show a high correlation coefficient for the regression of activity against body weight, with the values for relative activity being approximately an order of magnitude lower than for mammals of comparable weight. Other species of birds tend to have values intermediate between fish-eating birds and mammals, but the correlation of activity with body weight is considerably weaker. Low values for fish-eating birds are considered to be due to the fact that until xenobiotics contaminated aquatic food chains, the birds had less need of these defense mechanisms. Regrettably no data appear to be available on fish-eating mammals.
The low level of activity in fish has been considered to be due to the fact that until historical times this group of organisms has found the excretory route across the gills effective for removing most xenobiotics. It is clear that this mechanism is quite inadequate to deal with highly liposoluble organochlorine molecules that have been released into the environment by humans in the last few decades.
The relationship between feeding habits and MFO activity has also been demonstrated in insects. A detailed review of the data for aldrin epoxidase has been made by Brattsten (192). He found that aldrin epoxidase activity is lowest in monophagous species, considerably higher in oligophagous, and some 10-fold higher in the most polyphagous species.
The comparison of Phase I and Phase II hepatic transformation in quail and trout to that of a number of mammalian species commonly used in toxicity testing has been made by Gregus et al. (193). They found that the overall metabolism of xenobiotics could vary several hundredfold between species.
TOXICITY OF DIOXINS. The toxicity of dioxins to various mammalian species varies greatly. The most sensitive species known is the guinea pig; the LD50 for the male is 0.6 µg/kg and the female 2.1 µg/kg, the value for the male rat is 22 µg/kg, for the female rat 45 µg/kg and for the rabbit (mixed sex) 115 µg/kg, whereas the least sensitive species is the hamster (1200 µg/kg) (194).
EGGSHELL THINNING. The phenomenon of eggshell thinning, although confined to only one order (Aves) does illustrate some interesting points in susceptibility. Eggshell thinning is caused by DDE and the sensitivity varies greatly. A diet of only a few parts per million will cause 20% thinning (the degree of thinning that causes eggshell breakage and thus reproductive failure) in raptorial birds and some species of fish-eating birds such as the pelican and cormorant. Other species such as gull, terns, and ducks are only moderately sensitive with diets of 40 to 50 ppm being needed to cause biologically significant eggshell thinning. Still other species, such as quail and chicken are completely insensitive and it is impossible to achieve more than a few percent thinning even at the highest dosage that can be used without mortality. One important point from this variation is that the common test species are insensitive and thus even if the measurement of eggshell thinning had been included in test protocols for new pesticides this phenomenon would not then have been recognized. Another point is that the species exposed to the greatest amount of DDE, due to bioaccumulation, are those that are the most sensitive. DDE-induced eggshell thinning caused the decline of many species of raptorial birds, such as the peregrine falcon (Falco peregrinus) and european sparrow hawk (Accipter nisus) over wide areas of the northern hemisphere. These declines have been reversed in areas where bans on DDT have been imposed.
Biological Indicators of Susceptibility to Chemicals at the Population Level
Biological indicators for assessing susceptibility at the population level measure parameters that are a consequence of low activity of mechanisms of resistance detected by molecular and physiological biomarkers. For example, the induction of DNA adducts and DNA strand breakages found in individuals by molecular biomarkers predicted the increase in mutational events in population. Consequently there is a higher susceptibility of populations to diseases, especially viral, bacterial, and parasitic, as the consequence of impairments in the immunosystem. These populations also are more highly susceptible to decreased fitness of population, to impairments in reproduction, to sickness and increased lethality. All these indicators of susceptibility of populations to chemical stress will predict the expected consequences on the level of population such as alterations in adaption, survival and succession. On the ecological level, the most severe effect--extinction of a species--can occur.
Several examples illustrate the use of implications of susceptibility at the population level. In salt marsh ecosystems of North America, salt marsh grass (Spartina alterniflora) is the primary producer; susceptibility of this species to specific chemicals, which results in lowered reproductive output, would have a greater effect on community and ecosystem structure than would susceptibility of other, less important plant species (195). S. alterniflora is susceptible to PAHs from oil pollution; the sensitive vegetative propagules can be killed and the species may take years to recover. Similarly, differences in susceptibility to chemicals among species of algae in salt marsh communities would also have a critical effect on community and ecosystem structure since algae productivity is critical to the overall productivity of the animal communities in the marsh.
Equally important to remember when using cellular and molecular biomarkers as measures of the susceptibility of communities and ecosystems to chemicals, is that changes in molecular and cellular organization must have effects at the population level to be important for community and ecosystem structure.
In many cases, chemical, physical, and biological stressors occur together; and the communities and ecosystems must contend with them at once. Since communities and ecosystems are assemblages of organisms, and thereby are populations that make up the system and are responsible for the stability and repair of that system, the relative susceptibility of that system depends on the susceptibility of individuals and populations within the system.
Biological Indicators of Susceptibility to Chemicals at the Community Level
Within any community or ecosystem the species are not necessarily of equal importance with respect to the maintenance of stability. That is, some species have a keystone role (196). Keystone species include species that provide key nutrients or energy at the lower trophic levels, or species that regulate competition and predation at the higher trophic levels. Where molecular and cellular biomarkers are used to indicate potential susceptibility differences among individuals or species, care must be given to select indicator species that have a key or pivotal role in community structure (197). Chemical disruptions to species that are essential for either the structure or the function of the system will have a greater effect on the susceptibility of the ecosystem than those that are less critical.
Several measures of the structure and function of communities and ecosystems (Table 3) can be used to measure change (167,197-201). The degree of change in ecosystem structure and function that occurs is a measure of susceptibility. That is, if a community or ecosystem undergoes few changes in either structure or function in response to a chemical, biological, or physical perturbation, then it is relatively resistant or can be said to be less susceptible to the effects of a given chemical or class of chemicals.
The methods to evaluate community and ecosystem change are often costly and time consuming, but they can sometimes be used with less technology and equipment. That is, in places or situations where extensive molecular and cellular laboratory facilities are not available, the community and ecosystem measures outlined in Table 3 can be effectively used to determine differences in susceptibility. On the other hand, where such laboratory facilities exist, some of these techniques can be used effectively as surrogates to determine whether individual reproductive potential is compromised, thereby affecting population levels, and by extension, community structure. Table 3 also gives an indication of the difficulties of each methodology, as well as its usefulness for evaluation of the effects of chemicals.
One of the most detailed studies at the community structure level was made in Canada to determine the effects caused by acidification in an experimental lake situation (202). In this study the pH of a poorly buffered lake in northwestern Ontario was reduced from pH 6.8 to pH 5.0 over 8 years. Fish populations started to collapse due to lack of reproduction when the pH reached 5.9, and there were marked changes in the phytoplankton composition although primary production was not decreased. At pH 5.6 thick mats of filamentous algae appeared, which persisted throughout the study. When the stable value of pH 5.0 was reached the species composition of phytoplankton was completely different although primary production remained high; no fish reproduction was occurring; crayfish, leeches, and mayflies were absent; but there was a considerable increase in chlorophyll and no changes in nutrient concentration were observed.
Case Studies
Lead. Lead is an example of how biomarkers can be used at different levels of organization. Concentrations of lead in tissues such as blood, liver, and kidney are measures of exposure. Analysis of levels of lead and other heavy metals, however, is costly and requires technical knowledge and sophisticated equipment. However, 
-aminolevulinic acid dehydratase (-ALAD) activity can also be measured in blood as a biomarker of exposure and effect (203). It is a cheaper and easier method of analysis than of lead itself.
Additionally, the effects of lead can be measured with a number of neurobehavioral assays, since one of the primary nonlethal effects of lead is on cognitive, psychomotor, and other neural processes in both human (204) and nonhuman biota (205,206). Lead can cause mortality directly, or can cause lowered reproductive success through depression of clutch and egg size, mortality of embryos, depression of growth, and disruption of reproductive behavior in a variety of species, especially on birds, the work on which has been key (206). There are differences in susceptibility among birds: seabirds such as gulls and terns are more susceptible than chickens, ducks, and passerines (207-209).
Lead shows the classic trophic level relationships with respect to vulnerability: species that are higher on the food chain have higher levels of exposure than those that are lower on the food chain (206). However, susceptibility will also vary with such factors as developmental maturity at hatching, at least in birds. That is, lead disrupts parental recognition, making those species dependent on recognition (i.e., precocial birds) more susceptible to the effects of lead than species that do not depend on this recognition (210). Humans are at a relatively high trophic level and clearly show effects of lead. These effects, especially the neurological and behavioral ones, are particularly apparent during development, and persist for many years in humans (204).
Lead can also have disruptive effects at the population and community levels. The abnormalities caused by lead can reduce survival and lower reproduction success, resulting in lowered population levels for impacted populations (206). The effects in humans have the potential to severely alter social structure and behavior of populations that are severely exposed (204). As early as the 1950s, Bellrose demonstrated that lead poisoning could depress population levels by differentially affecting those wounded by hunters, and the response was dose related (211). Similarly, birds nesting along roadways are heavily exposed to lead from gasoline, while those in more remote areas are not. Animals near smelters are also heavily exposed through the aquatic food chain. By differentially affecting different species, the species composition of communities can be affected by lead, leading ultimately to changes in ecosystem structure. Because organisms higher on the food chain are more susceptible, this end of the system would be more affected.
While the mechanisms whereby lead affects hemoglobin formation (e.g., inhibition of -ALAD) are well known, the mechanisms disrupting neural systems, development, and behavior are not well established. These phenomena have been quantified, without clarifying the mechanisms. Phenomena include differences in cell growth and neuronal projections.
PCBs and Dioxins. The mechanism of action of PCBs, polychlorinated dibenzofurans (PCDFs), and polychlorinated dibenozo-p-dioxin (PCDDs) is considered to proceed via initial binding to a high affinity, low capacity cytosolic receptor protein. The identification of the Ah receptor (212) with stereospecific, high affinity binding to 2,3,7,8-TCDD was a key finding in bringing molecular biology into the realm of toxicology. Examining the toxicological and receptor binding data, Poland and Knutson (213) concluded that it was likely that these compounds exert their toxicity through the cytosol receptor.
The ability of specific PCBs, PCDFs, and PCDDs to induce the P450I system is greatly influenced by the degree of chlorination and the chlorine substitution pattern. The most toxic PCBs are those that are unsubstituted in the ortho positions, i.e., 3,3´,4,4´- tetrachlorobiphenyl (TCB), 3,3´,4,4´,5-pentachlorobiphenyl, and 3,3´,4,4´,5,5´-hexachlorobiphenyl, which allows the molecule to assume a co-planar configuration. There is a close relationship between aryl hydrocarbon hydroxylase (AHH) induction and body weight loss, and AHH induction and thymic atrophy, although the interactions of enzyme induction, receptor binding, and toxicological manifestations are very complex and our knowledge is far from complete.
Problems that have to be faced before one can use this approach for wildlife toxicological investigations in the field are extrapolations from species to species and extrapolations from cell culture to the intact animal. Brunstrom and co-workers have carried out studies on avian embryos. Marked differences were found in the sensitivity of the chicken, pheasant, turkey, duck, and gull (214-216). They found that the pheasant was 50 times less sensitive than the chicken, and other species were even less sensitive. This emphasizes the difficulties of interspecies comparison since the chicken and pheasant both belong to the order Phasianidae.
The application of this complex biochemistry to field investigations has been based on expressing the complex mixtures of PCBs, PCDFs, and PCDDs as dioxin equivalents (TCDD-EQ). Based on their affinity for the Ah receptor, the activity of the individual congeners are assigned a value relative to the most active compound (2,3,7,8-TCDD) which is given a value of 1. These toxic equivalent factors are multiplied by their concentration to give TCDD-EQ for each compound. Although even the co-planar PCBs are a good deal less active than 2,3,7,8-TCDD, their concentrations are much higher and thus they contribute more than the dioxins to the total TCDD-EQs. Now it is possible to do the process in reverse and use the degree of enzyme induction to estimate the TCDD-EQs. This bioassay approach is rapid and inexpensive compared to the conventional chemical analysis by gas chromatography-mass spectrometry.
Good correlations have been found between TCDD- EQ of egg contents and both the reproductive success and incidence of deformities in fish-eating birds in the Great Lakes of North America. In terms of sensitivity, it is of note that the cormorant is some 20 times more sensitive than the Caspian tern (Hydroprogne caspia). In the most contaminated areas of the Great Lakes, the productivity success has been linked to population declines.
Ethical Issues and Ecosystem Susceptibility
There are at least three areas where ethical considerations impact methods evaluation for ecological systems: scientist versus all other stakeholder views, government actions and ecosystem vulnerability, and, the conflict between using biomarkers in ecosystems for understanding the ecosystem itself and using ecosystems as indicators for human health [see section on ethics below, and Soskolne (15)]. Increasingly, the responsibilities of scientists toward stakeholders (including scientists) are obscure with respect to how well the methodologies actually measure the phenomena in question. It is unclear, therefore, how soon scientific "findings" should be released or made available, how stringent the criteria for acceptance of an effect must be (given the accuracy of the methods), and how to resolve conflicts between scientific viewpoints that develop as a result of methodology differences.
Certain communities and ecosystems are more susceptible to damage from exposure to chemicals by virtue of their species diversities, unique species assemblages, or presence of endangered species. In these cases, ecologists and ecotoxicologists have a responsibility to make the susceptibility known, and where possible, to affect decisions to reduce the potential for exposure to chemicals.
For example, the National Research Council examined the susceptibilities of offshore communities in the United States to oil pollution. They determined that the coast of Florida was very vulnerable because of the presence of subtropical coral reefs and manatees (both limited in the continental United States). As a result, the U.S. government decided not to allow offshore oil exploration in Florida.
There is a conflict between the use of biomarkers as indicators of ecosystem health for itself, compared to using these biomarkers only as indicators of human exposure. We would argue that both are important uses of biomarkers, and biomarkers should be developed that fit and evaluate the effects of chemicals in both. We have an ethical responsibility to preserve ecosystem integrity on a worldwide basis.
Conclusions and Recommendations
The following steps should be undertaken to protect organisms, species, communities, and ecosystems:
a) development of biomarkers at the cellular and molecular level that cross-cut taxonomic levels, including the vast diversity of invertebrates
b) study of molecules and mechanisms conserved across the animal kingdom; these highly conserved mechanisms may also have implications for higher animals including humans
c) development of biomarkers that integrate across all levels of biological organization because some chemicals may have a greater effect at lower trophic levels while others will be more apparent at higher levels
d) development of biological indicators that identify ecosystems susceptible to chemicals
e) Development of biomarkers that are rapid and inexpensive and thus capable of being widely used.
Last Update: June11, 1997