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

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 © 1999 by CRC Press LLC 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 © 1999 by CRC Press LLC 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 LD 50 , LC 50 , EC 50 , and IC 50 . The definitions are relatively straightforward. LD 50 — The dose that causes mortality in 50% of the organisms tested estimated by graphical or computational means. LC 50 — The concentration that causes mortality in 50% of the organisms tested estimated by graphical or computational means. EC 50 — 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. IC 50 — 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 IC 50 . Table 3.1 Toxicity Data for Compound 1 Dose 0.5 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Compound 1 Cumulative toxicity 0.0 2.0 7.0 23.0 78.0 92.0 97.0 100.0 100.0 Percent additional deaths at each concentration 0.0 2.0 5.0 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). © 1999 by CRC Press LLC 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 LC 50 s. 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 LC 50 s, 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 3.2 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). © 1999 by CRC Press LLC structure activity relationships. Another weakness of the LC 50 , EC 50 , and IC 50 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 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 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Compound 2 Cumulative toxicity 1.0 3.0 6.0 11.0 21.0 36.0 86.0 96.0 100.0 Percent additional deaths at each concentration 1.0 2.0 3.0 5.0 10.0 15.0 50.0 10.0 4.0 Compound 3 Cumulative toxicity 0.0 5.0 15.0 30.0 70.0 85.0 95.0 100.0 100.0 Percent additional deaths at each concentration 0.0 5.0 10.0 15.0 40.0 15.0 10.0 5.0 0.0 © 1999 by CRC Press LLC and suggests that a “Lethal Body Burden” or some other measurement be used to reflect tissue concentrations. These ideas are discussed in a later chapter. Often other terminology is used to describe the concentrations that have a minimal or nonexistent effect. Those that are currently common are NOEC, NOEL, NOAEC, NOAEL, LOEC, LOEL, MTC, and MATC. NOEC — No observed effects concentration determined by graphical or statistical methods. NOEL — No observed effects level determined by graphical or statistical methods. This parameter is reported as a dose. NOAEC — No observed adverse effects concentration determined by graphical or statistical methods. The effect is usually chosen for its impact upon the species tested. NOAEL — No observed adverse effects level determined by graphical or statistical methods. LOEC — Lowest observed effects concentration determined by graphical or statistical methods. LOEL — Lowest observed effects level determined by graphical or statistical methods. MTC — Minimum threshold concentration determined by graphical or statistical methods. MATC — Maximum allowable toxicant concentration determined by graphical or statistical methods. Figure 3.4 Comparison of dose-response curves-1. One of the primary goals of toxicity testing is the comparison or ranking of toxicity. The cumulative plots comparing compound 1 and compound 2 demonstrate the distinct nature of the two different toxicity curves. © 1999 by CRC Press LLC These concentrations and doses usually refer to the concentration or dose that does not produce a statistically significant effect. The ability to determine accurately a threshold level or no effect level is dependent upon a number of criteria including: Sample size and replication. Number of endpoints observed. Number of dosages or concentration. The ability to measure the endpoints. Intrinsic variability of the endpoints within the experimental population. Statistical methodology. Given the difficulty of determining these endpoints, great caution should be taken when using these parameters. An implicit assumption of these endpoints is that there is a threshold concentration or dose. That is, the organism, through compensatory mechanisms or the inherent mode of the toxicity of the chemical, can buffer the effects of the toxicant at certain levels of intoxication (Figure 3.8). In some cases biological effects occur at succeeding lower concentrations until the chemical is removed from the environment. There is much debate about which model of dose vs. effects is more accurate and useful. Figure 3.5 Comparison of dose-response curves-2. Plotting the dose-response curve dem- onstrates that the concentrations that cause mortality in 50% of the population are distinctly different. However, the slopes of the two curves appear to be the same. In many cases this may indicate that the compounds may interact similarly at the molecular level. © 1999 by CRC Press LLC STANDARD METHODS Over the years a variety of test methods have been standardized. These protocols are available from the American Society for Testing and Materials (ASTM), the Organization for Economic Cooperation and Development (OECD), the National Toxicology Program (NTP), and are available as United States Environmental Pro- tection Agency publications, the Federal Register, and often from the researchers that developed the standard methodology. Advantages of Standard Methods There are distinct advantages to the use of a standard method or guideline in the evaluation of the toxicity of chemicals or mixtures, such as: Uniformity and comparability of test results. Allows replication of the result by other laboratories. Provides criteria as to the suitability of the test data for decisionmaking. Logistics are simplified, little or no developmental work. Data can be compiled with that of other laboratories for use when large data sets are required. Examples are quantitative structure activity research and risk assessment. Figure 3.6 Comparison of dose-response curves-3. Cumulative toxicity plots for compounds 1 and 3. Notice that the plots intersect at roughly 50% mortality. © 1999 by CRC Press LLC The method establishes a defined baseline from which modifications can be made to answer specific research questions. Over the years numerous protocols have been published. Usually, a standard method or guide has the following format for the conduct of a toxicity test using the ASTM methods and guides as an example: The scope of the method or guide is identified. Reference documents, terminology specific to the standards organization, a sum- mary, and the utility of the methodology are listed and discussed. Hazards and recommended safeguards are now routinely listed. Apparatuses to be used are listed and specified. In aquatic toxicity tests, the specifi- cations of the dilution water are given a separate listing reflecting its importance. Specifications for the material undergoing testing are provided. Test organisms are listed along with criteria for health, size, and sources. Experimental procedure is detailed. This listing includes overall design, physical and chemical conditions of the test chambers or other containers, range of concentrations, and measurements to be made. Analytical methodologies for making the measurements during the experiment are often given a separate listing. Acceptability criteria are listed by which to judge the reliability of the toxicity test. Methods for the calculation of results are listed. Often several methods of deter- mining the EC 50 , LD 50 or NOEL are referenced. Specifications are listed for the documentation of the results. Appendixes are often added to provide specifics for particular species of strains of animals and the alterations to the basic protocol to accommodate these organisms. Figure 3.7 Comparison of dose-response curves-4. Although the mid-points of the curves for compounds 1 and 3 are the same, compound 3 is more toxic at low concentrations more typical of exposure in the environment. © 1999 by CRC Press LLC Disadvantages of Standard Methods Standard methods do have a disadvantage. The methods are generally designed to answer very specific questions that are commonly presented. As in the case of acute and chronic toxicity tests, the question is the ranking of the toxicity of a chemical in comparison to other compounds. When the questions are more detailed or the compound has unusual properties, deviations from the standard method should Figure 3.8 Threshold concentration. There are two prevailing ideas on the toxicity of com- pounds at low concentrations. Often it is presumed that a compound has a toxic effect as long as any amount of the compound is available to the organism (A). Only at zero concentration will the effect disappear. The other prevailing idea is that a threshold dose exists below which the compound is present but no effects can be discerned (B). There is a great deal of debate about which model is accurate. © 1999 by CRC Press LLC [...]... challenges in environmental toxicology is the ability to translate the toxicity tests performed under controlled conditions in the laboratory or test site to the structure and function of real ecosystems This inability to translate the generally reproducible and repeatable laboratory data to effects upon the systems that environmental toxicology tries to protect is often called the lab -to- field dilemma... toxicology Environ Toxicol Chem 15: 59 7-6 03 Molander, S and H Blanck 1992 Detection of pollution-induced community tolerance (PICT) in marine periphyton communities established under diuron exposure Aquat Toxicol 22: 12 9-1 44 © 1999 by CRC Press LLC Moore, D.R.J and P-Y Caux 1997 Estimating low toxic effects Environ Toxicol Chem 16: 76 4-8 01 Rand, G.M and S.R Petrocelli 1985 Introduction In Aquatic Toxicology. .. 111 1-1 116 Landis, W.G., R.A Matthews, A.J Markiewicz, N.A Shough, and G.B Matthews 19 93 Multivariate analyses of the impacts of the turbine fuel jet-a using a microcosm toxicity test J Environ Sci 2: 11 3- 1 30 Landis, W.G., R.A Matthews, A.J Markiewicz, and G.B Matthews 19 93 Multivariate analysis of the impacts of the turbine fuel JP-4 in a microcosm toxicity test with implications for the evaluation of. .. 12.28 (3. 88–4.12) (4.01–4.21) (5 .35 –10 .36 ) (8.04–16.57) 3. 80 3. 99 5 .37 8.00 (3. 59–4.02) (3. 90–4.10) (1.46–10.91) (5.61–11.42) 3. 80 3. 99 5 .37 8.00 (3. 58–4.02) (3. 90–4.10) (1.46–10.91) (5.61–11.42) there is the assumption that the dose-response curve has been correctly linearized As with the other methods, a partial kill is required to establish a confidence interval Comparison of Calculations of Several... large number of toxicity tests that have been developed in environmental toxicology because of the large variety of species and ecosystems that have been investigated However, it is possible to classify the tests using the length of the experiments relative to the life span of the organism and the complexity of the biological community Figure 3. 9 provides a summary of this classification Acute toxicity... predictors of ecological risk Environ Manage 12: 51 5-5 23 Kersting, K 1988 Normalized ecosystem strain in microecosystems using different sets of state variables Verh Int Verein Limnol 23: 164 1-1 646 Kersting, K., and R van Wungaarden 1992 Effects of Chlorpyifos on a microecosystem Environ Toxicol Chem 11: 36 5 -3 72 Landis, W.G., R.A Matthews, and G.B Matthews 1997 The design and analysis of multispecies toxicity... classification of toxicity tests in environmental toxicology? 10 Describe a microcosm and a mesocosm test 11 Describe the lab -to- field dilemma 12 What differences are there between a static and a static-renewal toxicity test? 13 What are the advantages and disadvantages of the recirculating methodology of toxicity testing? 14 Name and describe the best technical method for toxicity testing 15 What is whole-body... the majority of toxicity tests in environmental toxicology are conducted with organisms of unknown origin or field collection Indeed, the cultures often originated from collections and the genetic relationships to the organisms used by other laboratories is poorly known 4 The relative sensitivities to various classes of toxicants of the test species should be known relative to the endpoints to be measured... Materials, Philadelphia, PA, pp 30 3- 3 07 Brooks, D.R., J Collier, B.A Mauer, J.D.H Smith, and E.O Wiley 1989 Entropy and information in evolving biological systems Biol Philos 4: 40 7-4 32 Cairns, J.C., Jr 1986 The myth of the most sensitive species Bioscience 36 : 67 0-6 72 Caux, P-Y and D.R.J Moore 1997 A spreadsheet program for estimating lowtoxic effects Environ Toxicol Chem 16: 80 2-8 06 Chapman, P.M., R.S Caldwell... (gills, stomata), and digestive system Occasionally a toxicant is injected into an aquatic organism, but that is not usually the case in toxicity tests to screen for effects Whole-body exposures are less common when dealing with terrestrial species Often an amount of xenobiotic is injected into the musculature (intramuscular), peritoneum (intraperitoneal), or into a vein (intravenous) on a weight of toxicant . repeatable laboratory data to effects upon the systems that environmental toxi- cology tries to protect is often called the lab -to- field dilemma. Comparisons of laboratory data to field results. respect to: SAS default SAS log 10 ASTM 1 (4.0) Dose 4.00 (3. 88–4.12) 3. 80 (3. 59–4.02) 3. 80 (3. 58–4.02) Log 10 dose 4.11 (4.01–4.21) 3. 99 (3. 90–4.10) 3. 99 (3. 90–4.10) 2 (8.0) Dose 8.02 (5 .35 –10 .36 ). Figure 3. 7 Comparison of dose-response curves-4. Although the mid-points of the curves for compounds 1 and 3 are the same, compound 3 is more toxic at low concentrations more typical of exposure