Introduction to ENVIRONMENTAL TOXICOLOGY Impacts of Chemicals Upon Ecological Systems - CHAPTER 3

CHAPTER 3 An Introduction to Toxicity Testing Toxicity is the property or properties of a material that produces a harmful effect upon a biological system. A toxicant is the material that produces this biological effect. The majority of the chemicals discussed in this text are of man-made or anthropogenic origin. This is not to deny that extremely toxic materials are produced by biological systems, venom, botulinum endotoxin, and some of the fungal afla-toxins are extremely potent materials. However, compounds that are derived from natural sources are produced in low amounts. Anthropogenically derived compounds can be produced in the millions of pounds per year. Materials introduced into the environment come from two basic types of sources. Point discharges are derived from such sources as sewage discharges, waste streams from industrial sources, hazardous waste disposal sites, and accidental spills. Point discharges are generally easy to characterize as to the types of materials released, rates of release, and total amounts. In contrast, nonpoint discharges are those mate-rials released from agricultural run-offs, contaminated soils and aquatic sediments, atmospheric deposition, and urban run-off from such sources as parking lots and residential areas. Nonpoint discharges are much more difficult to characterize. In most situations, discharges from nonpoint sources are complex mixtures, amounts of toxicants are difficult to characterize, and rates and the timing of discharges are as difficult to predict as the rain. One of the most difficult aspects of nonpoint discharges is that the components can vary in their toxicological characteristics. Many classes of compounds can exhibit environmental toxicity. One of the most commonly discussed and researched are the pesticides. Pesticide can refer to any compound that exhibits toxicity to an undesirable organism. Since the biochemistry and physiology of all organisms are linked by the stochastic processes of evolution, a compound toxic to a Norway rat is likely to be toxic to other small mammals. Industrial chemicals also are a major concern because of the large amounts trans-ported and used. Metals from mining operations, manufacturing, and as contaminants in lubricants also are released into the environment. Crude oil and the petroleum products derived from the oil are a significant source of environmental toxicity because of their persistence and common usage in an industrialized society. Many of these compounds, especially metal salts and petroleum, can be found in normally uncontaminated environments. In many cases, metals such as copper and zinc are essential nutrients. However, it is not just the presence of a compound that poses a toxicological threat, but the relationships between its dose to an organism and its biological effects that determine what environmental concentrations are harmful. Any chemical material can exhibit harmful effects when the amount introduced to an organism is high enough. Simple exposure to a chemical also does not mean that a harmful effect will result. Of critical importance is the dose, or actual amount of material that enters an organism, that determines the biological ramifications. At low doses no apparent harmful effects occur. In fact, many toxicity evaluations result in increased growth of the organisms at low doses. Higher doses may result in mortality. The relationship between dose and the biological effect is the dose-response relationship. In some instances, no effects can be observed until a certain threshold concentration is reached. In environmental toxicology, environmental con-centration is often used as a substitute for knowing the actual amount or dose of a chemical entering an organism. Care must be taken to realize that dose may be only indirectly related to environmental concentration. The surface-to-volume ratio, shape, characteristics of the organisms external covering, and respiratory systems can all dramatically affect the rates of a chemical’s absorption from the environment. Since it is common usage, concentration will be the variable from which mortality will be derived, but with the understanding that concentration and dose are not always directly proportional or comparable from species to species. THE DOSE-RESPONSE CURVE The graph describing the response of an enzyme, organism, population, or biological community to a range of concentrations of a xenobiotic is the dose-response curve. Enzyme inhibition, DNA damage, death, behavioral changes, and other responses can be described using this relationship. Table 3.1 presents the data for a typical response over concentration or dose for a particular xenobiotic. At each concentration the percentage or actual number of organisms responding or the magnitude of effects is plotted (Figure 3.1). The dis-tribution that results resembles a sigmoid curve. The origin of this distribution is straightforward. If only the additional mortalities seen at each concentration are plotted, the distribution that results is that of a normal distribution or a bell-shaped curve (Figure 3.2). This distribution is not surprising. Responses or traits from organisms that are controlled by numerous sets of genes follow bell-shaped curves. Length, coat color, and fecundity are examples of multigenic traits whose distribution results in a normal distribution. The distribution of mortality vs. concentration or dose is drawn so that the cumulative mortality is plotted at each concentration. At each concentration the total numbers of organisms that have died by that concentration are plotted. The presen-tation in Figure 3.1 is usually referred to as a dose-response curve. Data are plotted as continuous and a sigmoid curve usually results (Figure 3.3). Two parameters of this curve are used to describe it: (1) the concentration or dose that results in 50% of the measured effect and (2) the slope of the linear part of the curve that passes Table 3.1 Toxicity Data for Compound 1 Dose Compound 1 Cumulative toxicity Percent additional deaths at each concentration 0.5 1.0 2.0 0.0 2.0 7.0 0.0 2.0 5.0 3.0 4.0 5.0 6.0 7.0 8.0 23.0 78.0 92.0 97.0 100.0 100.0 15.0 55.0 15.0 5.0 3.0 0.0 Note: All of the toxicity data are given as a percentage of the total organisms at a particular treatment group. For example, if 7 out of 100 organisms died or expressed other endpoints at a concentration of 2 mg/kg, then the percentage responding would be 7%. Figure 3.1 Plot of cumulative mortality vs. environmental concentration or dose.The data are plotted as cumulative number of dead by each dose using the data presented in Table 3.1. The x-axis is in units of weight to volume (concentration) or weight of toxicant per unit weight of animal (dose). through the midpoint. Both parameters are necessary to describe accurately the rela-tionship between chemical concentration and effect. The midpoint is commonly referred to as a LD50, LC50, EC50, and IC50. The definitions are relatively straightforward. LD50 — The dose that causes mortality in 50% of the organisms tested estimated by graphical or computational means. LC50 — The concentration that causes mortality in 50% of the organisms tested estimated by graphical or computational means. EC50 — The concentration that has an effect on 50% of the organisms tested estimated by graphical or computational means. Often this parameter is used for effects that are not death. IC50 — Inhibitory concentration that reduces the normal response of an organism by 50% estimated by graphical or computational means. Growth rates of algae, bac-teria, and other organisms are often measured as an IC50. Figure 3.2 Plot of mortality vs. environmental concentration or dose. Not surprisingly, the distribution that results is that of a normal distribution or a bell-shaped curve.This distribution is not surprising. Responses or traits from organisms that are controlled by numerous sets of genes follow bell-shaped curves. Length, coat color, and fecundity are examples of multigenic traits whose distribution result in a bell-shaped curve.The x-axis is in units of weight to volume (concentration) or weight of toxicant per unit weight of animal (dose). One of the primary reasons for conducting any type of toxicity test is to rank chemicals as to their toxicity. Table 3.2 provides data on toxicity for two different compounds. It is readily apparent that the midpoint for compound 2 will likely be higher than that of compound 1. A plot of the cumulative toxicity (Figure 3.4) confirms that the concentration that causes mortality to half of the population for compound 2 is higher than compound 1. Linear plots of the data points are super-imposed upon the curve (Figure 3.5) confirming that the midpoints are different. Notice, however, that the slopes of the lines are similar. In most cases the toxicity of a compound is usually reported using only the midpoint reported in a mass per unit mass (mg/kg) or volume (mg/l). This practice is misleading and can lead to a misunderstanding or the true hazard of a compound to a particular xenobiotic. Figure 3.6 provides an example of two compounds with the same LC50s. Plotting the cumulative toxicity and superimposing the linear graph the concurrence of the points is confirmed (Figure 3.7). However, the slopes of the lines are different with compound 3 having twice the toxicity of compound 1 at a concentration of 2. At low concentrations, those that are often found in the environ-ment, compound 3 has the greater effect. Conversely, compounds may have different LC50s, but the slopes may be the same. Similar slopes may imply a similar mode of action. In addition, toxicity is not generated by the unit mass of xenobiotic but by the molecule. Molar concentra-tions or dosages provide a more accurate assessment of the toxicity of a particular compound. This relationship will be explored further in our discussion of quantitative Figure 3.3 The sigmoid dose-response curve. Converted from the discontinuous bar graph of Figure 3.2 to a line graph. If mortality is a continuous function of the toxicant, the result is the typical sigmoid dose-response curve. The x-axis is in units of weight to volume (concentration) or weight of toxicant per unit weight of animal (dose). Table 3.2 Toxicity Data for Compounds 2 and 3 Dose 0.5 Compound 2 Cumulative toxicity 1.0 Percent additional 1.0 deaths at each concentration Compound 3 Cumulative toxicity 0.0 Percent additional 0.0 deaths at each concentration 1.0 2.0 3.0 6.0 2.0 3.0 5.0 15.0 5.0 10.0 3.0 4.0 5.0 6.0 7.0 8.0 11.0 21.0 36.0 86.0 96.0 100.0 5.0 10.0 15.0 50.0 10.0 4.0 30.0 70.0 85.0 95.0 100.0 100.0 15.0 40.0 15.0 10.0 5.0 0.0 structure activity relationships. Another weakness of the LC50, EC50, and IC50 is that they reflect the environmental concentration of the toxicant over the specified time of the test. Compounds that move into tissues slowly may have a lower toxicity in a 96-h test simply because the concentration in the tissue has not reached toxic levels within the specified testing time. L. McCarty has written extensively on this topic ... - tailieumienphi.vn