SCOPE 53 - Methods to Assess the Effects of Chemicals On Ecosystems

Once the objectives and relevant scales of an environmental risk assessment have been determined (see Chapter 2), the data collection plan must be designed in a manner that will help establish linkages between contamination and observed adverse effects. Many tools are available to assist the environmental manager in assessing environmental condition. These include chemical analyses, toxicity tests, field surveys and assessments, and special studies such as biomarkers and simulated ecosystem studies (i.e., mesocosms or microcosms). Any one of these approaches may provide the information needed to assess environmental conditions; however, the use of several of these approaches in combination may increase the level of confidence in the assessment. This chapter presents an overview of the approaches that may be used to assess the ecological conditions of both aquatic and terrestrial ecosystems. 

3.1 METHODS SELECTION

After the study objectives have been determined and a design is in place, measurement endpoints or indicators must be selected. Indicators provide data about the assessment endpoints determined to be most relevant to the study objectives. Methods to measure indicators can be grouped into several categories: field-ecological surveys of the contaminated site; chemical analyses of samples from the site; and toxicity tests of site media (water, soil, sediments) either in the laboratory or on site (in situ). Special studies such as bioaccumulation tests and simulated ecosystem studies (e.g., microcosms or mesocosms) may also be used. 

Field surveys can evaluate changes in populations or communities of plants or animals that may be the result of contamination. Preliminary field studies conducted before an entire study is undertaken are recommended, as they may provide information about variability within the study area, thereby helping to define the study design. They may also provide general information on contamination and impacts to biota. Such information helps guide the selection of sampling sites, ecological receptors, and sampling methodologies. Chemical analyses can help determine the extent and quantity of contaminants at the site, while toxicity testing may provide a direct indication of the effects of contamination on site biota. Models can be used to predict the probable fate and transport of chemical contaminants, and, thereby, help determine the most useful plant and animal species for toxicity testing and other special studies. Models and other approaches may also be used to link measured responses in receptors of concern (toxicity test data) to population-level responses (field surveys), and to obtain information on the spatial extent and chemical components of contamination (chemical analyses). The use of each type of test and the careful integration of the data generated help to establish linkages between site impacts and adverse effects observed in biota. 

3.2 OVERVIEW OF METHODS

Methods applicable to the evaluation of chemical effects in both terrestrial and aquatic systems are discussed in the following sections:

3.2.1 CHEMICAL ANALYSES OF SAMPLES

Chemical analyses are an integral component of any chemical effects study. Once preliminary studies have identified the sites to be sampled, chemical analyses of samples (soil and sediment, water, air, or biota) can be conducted to determine concentrations of various contaminants. In some cases, these data may be compared with those collected from an uncontaminated reference site. Chemical analyses are the first step in establishing linkages between adverse effects observed in biota and specific contaminant levels. Information about the total and bioavailable elemental concentrations, the concentrations of organic and inorganic ionic species, and the concentration of molecular organic species added to the environment by various human activities (technological, agricultural, recreational) must be collected. 

Significant advances in analytical chemistry over the past 20 years have provided scientists with numerous methods to characterize environmental contamination. These methods enable researchers to perform rapid and accurate multielemental analyses at a reasonable cost. Similarly, modern organic analytical methods such as gas chromatography (GC), mass spectrometry (MS), high performance liquid chromatography (HPLC), and various combinations of these technologies have enabled chemists to detect very low levels (as low as parts per billion or trillion) of organic environmental contaminants routinely. Some more commonly used analytical methods are presented in Table 3.1. 

The level of complexity of chemical analysis varies with the medium being sampled. Water is perhaps the simplest medium to analyse for chemical contaminants. Even though the concentrations of contaminants in a water sample may be low, the sample can be concentrated before analysis to improve detection and quantification. Analysis of soil and sediment samples may add another level of complexity, along with the difficulty of assuring homogeneity of the sample. 

Analysis of biological tissue (plant or animal) for chemical residues is frequently the most complicated and most expensive task because of the complexity of the tissue matrix. The information obtained from a residue study, however, may be very valuable in ascertaining whether contaminants are bioavailable to biota. Residue or bioaccumulation studies are discussed in greater detail below.

Table 3.1. Major analytical approaches to detect common environmental chemical stressors

If contaminants of interest are likely to be dispersed through the air, they should be monitored. Airborne contaminants include volatile organic compounds, gaseous compounds, and particulates that adhere to airborne particles. However, in many settings including some industrial settings, airborne contaminants are typically present only in low concentrations, thus requiring that large volumes of air be sampled over long periods of time. 

Proper procedures to collect, handle, and store samples are also critical to obtain useful analytical data. Samples must be collected with the appropriate containers, and stored correctly to prevent contamination or loss of the contaminant by leaching or binding to the container and to ensure that metabolic processes have been halted.

3.2.2 TOXICITY TESTS

Toxicity tests provide a direct measure of the bioavailability of toxicants, and, when combined with chemical analyses and field surveys, can help establish linkages between site contamination and adverse ecological effects. Toxicity tests evaluate acute, subchronic, and chronic exposures and measure biological endpoints such as mortality, reproductive performance, growth, and behavioural changes. Also by using toxicity tests, the relative toxicity of a mixture of chemicals can be assessed by taking into account synergistic or antagonistic interactions among chemicals.  

The major disadvantage of traditional laboratory-based toxicity tests is the difficulty in extrapolating effects observed in the laboratory to those observed in the wild. In situ  (literally, "in position") toxicity assessments can help to address this problem. Numerous toxicity tests can be adapted for field use to evaluate the exposure of test organisms in site media, under "normal" conditions encountered in the field. Although in situ toxicity assessments are not as standardized as are laboratory toxicity tests, they are increasingly prominent in the ecological assessment process. 

Conducting parallel toxicity tests in the laboratory and in the field may provide stronger inference of linkages between toxicity and contaminant exposure and reduce the problems associated with laboratory-to-field extrapolations of toxicity data. If the same effects are observed in both types of tests, a stronger case can be made for a causal relationship between the two. 

Toxicity tests may be used in both aquatic and terrestrial habitats; however, procedures for aquatic toxicity testing are more developed than those for terrestrial toxicity testing. Traditional toxicity tests involve a single species, and measure endpoints such as mortality, growth, and reproduction. Numerous types of organisms are used in toxicity testing, including vertebrates (rodents, fish, and birds), invertebrates (plankton, amphipods, and insects), microbes, and plants (aquatic and terrestrial; vascular and non-vascular).  Environmental matrices that can be tested include water, sediment, and soil. Test exposures may be either acute, subchronic, or chronic. Aquatic toxicity tests for acute effects typically require about four days  (representing a small portion of the lifetime of the organism); tests for chronic and subchronic effects range from 7-30 days (representing a much larger portion of the lifetime of the organism). Terrestrial toxicity tests range from hours to days for acute effects to weeks and months for chronic and subchronic effects. Examples of specific aquatic and terrestrial toxicity tests are found below. 

3.2.3 FIELD ASSESSMENTS

Field surveys of terrestrial and aquatic habitats can complement chemical analyses and toxicity testing, and may decrease the uncertainty in the assessment process by providing direct measures of impacts on site biota. Field surveys of contaminated sites provide information about the extent and patterns of contamination, and may help identify sites to sample for chemical analysis. Surveys can indicate the presence of sensitive plant and animal species that may be affected by contamination and also help identify potential species for further study (i.e., toxicity testing). Field surveys also provide information about the effects of contaminants on the structure and function of populations and communities at a site when data from a contaminated site are compared with  data from carefully selected reference sites. 

Community-level field studies generally provide the most information about the biological integrity of a system, while permitting examination of individual taxa within the community. Communities may be assessed from either a structural or functional perspective. An assessment of community structure defines biotic characteristics (e.g., abundance, diversity, and species composition) at a specific point in time, whereas an assessment of community function measures the rate of biological processes (e.g., species colonization rates and nutrient cycling) of the ecosystem.

The use of community-level studies in environmental monitoring is normally performed from a structural perspective, because structural studies take less time, are technically less complex, and facilitate comparisons with data from other kinds of studies. Community-level studies, however, are frequently difficult to interpret, and the causal links with contaminant exposure may not be readily apparent. Contamination often is not the only factor influencing community structure. Natural environmental factors such as temperature, moisture, pH, nutrient availability, and predator-prey relationships also affect community structure. Examples of specific field assessment methods for the aquatic and terrestrial environments are presented below. 

3.2.4 SPECIAL ANALYSES

Special analyses such as bioaccumulation studies, biomarkers, or simulated ecosystem studies provide valuable information for an ecological assessment. Although these kinds of studies are not typically used in  ecological risk assessments, they can be valuable when combined with information from other parts of the assessment process. Examples of some special analyses include bioaccumulation testing of dredged material for ocean disposal or mesocosm studies for pesticide registration.  

3.2.4.1 Bioaccumulation studies

Bioaccumulation studies evaluate the net accumulation of a chemical in an organism through consumption of food or water containing the chemical. Bioaccumulation occurs when the rate at which an organism ingests a chemical exceeds the rate at which it excretes the substance. Bioaccumulation tests measure the actual uptake of a contaminant by organisms, and are especially useful when the contaminants at a site have high bioconcentration factors, such as PCBs or dioxins. Bioaccumulation studies may involve residue studies (i.e., measuring chemical residues in the tissues of organisms from the site) or a controlled study such as measuring residues in organisms exposed to a contaminated medium for a specific length of time, (i.e., a laboratory toxicity test or in situ bioconcentration study). 

Bioaccumulation studies can provide direct measurements of contaminant bioavailability, whereas chemical analyses of soil and sediment cannot. They can also be used to evaluate potential human health risks when used to analyze flora or fauna that may be consumed by humans. Such studies can be difficult to interpret, however, because body burdens of a chemical residue frequently are not directly correlated with adverse effects. Other challenges encountered in bioaccumulation studies include the natural variability between individuals and within a population, interaction between contaminants, and biotransformation within the organism. 

3.2.4.2 Biomarkers

A biomarker is a biological measure of an organism's response to a contaminant. Biomarkers are measurements of biological tissues, fluids, or cells that can be used to determine if an organism has been exposed to a contaminant or if a contaminant has caused biological changes. Biomarkers have been used for many years to assess human exposure and effects but only recently have they been applied to ecological assessments. 

Biomarkers used to assess aquatic or terrestrial organisms generally evaluate physiological, histological, or biochemical parameters. They may provide information not available from residue studies, chemical analyses, toxicity tests, or ecological community studies. By measuring an actual exposure or biological response to a contaminant, biomarkers integrate the temporal exposures of the organism and the multiple pathways of exposure. Also, because most biomarkers provide an estimate of exposure or alterations at organismal or suborganismal levels, the responses observed are recorded after a compound has been metabolized or transformed within the organism, thereby providing a more realistic measure of exposure or changes. 

Presently, few biomarker protocols are widely accepted for use with aquatic or terrestrial organisms, partly because clear causal relationships have not been established for many classes of contaminants. Furthermore, many responses to biomarkers may be produced by environmental influences such as seasonality; and, as with any biological population, intra-specific variability is always a confounding factor. Further discussion of the use of biomarkers in aquatic and terrestrial environments is provided below.

3.2.4.3 Microcosm and mesocosm studies

Microcosm and mesocosm tests are useful intermediates between bioassays and ecosystem experiments. They provide controlled experimental conditions in the laboratory or the field to study changes at any level (population or community or ecosystem) of a chemical or other stressor. Microcosm studies are generally small, contain a few species, and are conducted indoors, whereas mesocosm tests are relatively large, contain most or all the species from an ecosystem, and are usually conducted in outdoor settings. Given the expense and effort required to establish and maintain mesocosms, microcosms are often used when only one or a few species are required for a test. 

Microcosm studies offer several advantages over mesocosm studies and field surveys. Multispecies microcosm studies provide greater ecological "realism" than single-species tests or basic multispecies tests. Microcosm tests are more space-efficient than mesocosm or field studies, and are easier to maintain under uniform conditions essential to replicate and standardize experimental procedures. Furthermore, the chemical, biological, and physical effects of a substance can be determined in one test system, rather than several. Unlike in field tests, microcosms eliminate the chance of contaminating the natural environment. Most importantly, microcosms enable researchers to observe the integrated effects of contaminants on community and ecosystem functions and pathways. 

Data obtained in microcosm studies must be used with caution, however, due to the limitations of such studies. Most limitations result from the fact that a microcosm is an intentionally simplified representation of an ecosystem. Specific limitations include (1) extrapolating observations to the broader environment; (2) the absence of selected components of an ecosystem (i.e., lack of atmospheric deposition); and (3) the use of small population sizes, which may lead to chance extinctions. 

Mesocosm tests are simulated field studies conducted in controlled environments such as artificial ponds or streams, large outdoor tanks, or littoral enclosures in a natural water body. Mesocosm tests also may be conducted in terrestrial habitats to evaluate the effects of chemical contaminants on vegetation. Mesocosm tests employ fully functional ecosystems to predict ecological effects or environmental fate processes and, as such, may be the best possible method to obtain information on how a stressor reacts to actual environmental conditions. A disadvantage of mesocosm studies is the difficulty in discriminating between effects caused by the stressor of interest and the natural variability of the ecosystem or community in question. Mesocosm studies are quite expensive and time consuming; consequently, their usefulness in the ecological risk assessment process may be limited.

3.3 ASSESSMENT OF CHEMICAL IMPACTS TO AQUATIC ECOSYSTEMS

A summary of the methods currently available to measure, diagnose, and quantify the effects of chemicals on aquatic (freshwater, estuarine, and marine) systems is provided below. 

3.3.1 TOXICITY TESTS 

Aquatic toxicity tests have been used extensively for hazard assessment for over 20 years. Many standardized methods have been developed, and published by the US Environmental Protection Agency (EPA), the US Army Corp of Engineers (COE), the Organization of Economic Cooperation and Development (OECD), and the American Society for Testing and Materials (ASTM). Tables 3.2, 3.3, 3.4, 3.5, 3.6 and 3.7 list commonly used protocols for acute and chronic toxicity tests for use with fish and invertebrates from marine and freshwater environments. 

Table 3.2 Some  freshwater acute toxicity tests

The most frequently used laboratory toxicity tests relate the concentration of the chemical in water to the time of death or some other observed manifestation in the test organism. Organisms used in aqueous aquatic toxicity tests include many species of fish, invertebrates, and algae. Commonly used fish species include: fathead minnow (Pimephales promelas) in freshwater systems, and the sheepshead minnow (Cyprinodon variegatus) or silverside (Menidia sp.) in marine and estuarine systems. Commonly used invertebrates include Daphnia sp. or Ceriodaphnia dubia in freshwater and the mysid shrimp (Mysidopsis bahia) in marine systems. Amphipods, such as Hyalella azteca in freshwater and Ampelisca abdita in saltwater, are frequently used to assess sediment toxicity.

Table 3.3 Some estuarine and marine acute toxicity tests (USEPA, 1991b)

Table 3.4 Some freshwater chronic toxicity tests (USEPA, 1989)

The duration of typical toxicity tests with aqueous samples ranges from four days for acute effects with an endpoint of mortality to 7 to 30 days for chronic and subchronic effects on survival, growth, or reproduction. Life-cycle tests are also needed, but are not frequently used, because the duration is long and the cost is high. Test durations to assess sediment toxicity typically range from 10 days or less for acute effects to 30 days for chronic effects. Test endpoints may include survival, reproduction, or emergence.

3.3.2 FIELD ASSESSMENTS

Field assessments of aquatic habitats may include surveys of all populations in the aquatic community including microbes, periphyton, plankton, macroinvertebrates, fish, and macrophytes. Community-level studies provide the most information about the biological integrity of an aquatic system, and allow an evaluation of individual species within the community. Either community structure or function may be assessed; however, structural evaluations are conducted more often, because they take less time, are less complicated, and produce easily interpreted data. A major consideration when assessing community structure is the possible effect by normally variable environmental factors such as salinity, temperature, or shading, as well as by contamination.

Table 3.5 Some estuarine and marine chronic toxicity tests

Any combination of taxonomic groups (algae, invertebrates, or vertebrates) and level of biological organization (individual, population, community, or ecosystem) can be used to assess the health of an aquatic system. Benthic invertebrates are used often in aquatic community studies, because as a group they integrate the effects of present and past conditions; they are generally abundant, relatively immobile, and have relatively long life cycles; and as a group, their ecological relationships are well studied. In addition, sampling procedures are well developed, and a single sampling often collects a considerable number of species from a wide range of phyla. Examples of community structure and function parameters that are used to assess aquatic communities are provided in Table 3.8. Specific procedures to assess the structural or functional parameters of aquatic ecosystems can be found in most aquatic ecology methods manuals such as that of Wetzel and Likens (1990), of EPA (1989), or of ASTM E1383 (1993).

3.3.3 SPECIAL ANALYSES

3.3.3.1 Bioaccumulation studies

Chemicals may enter organisms via food and sediment uptake or by uptake from the water across external membranes and gills. The three basic processes by which contaminants accumulate in aquatic species are bioconcentration, bioaccumulation, and biomagnification. Herein, bioaccumulation is used to represent all three pathways of contaminant uptake.

A first-level bioaccumulation study is used to determine residues in the biota. This study is uncontrolled (i.e., previous contaminant exposure is unknown); however, the study can provide background information, indicating whether the chemicals of interest can be taken up by biota at the site. Its findings are useful in the planning future studies.

A more involved approach to study bioaccumulation is to conduct in situ studies such as stream cage studies. These studies typically involve fish or benthic macroinvertebrates enclosed by a cage either attached to the substrate, suspended in the pelagic zone, or floating. These tests have the advantage of providing more "real-world" conditions; that is, contaminant accumulation proceeds at its normal rate (i.e., impacted by biotransformation and other fate processes) under normal temperature, light, and other exposure parameters. However, the advantages to this type of study are also some of its disadvantages. Many variables cannot be controlled (e.g., pollution slugs, extremely high tides or flows, temperature, light, and food availability). These make the test a more reliable estimator of the real world, but also add additional covariates that in turn make the data more difficult to interpret. Also the potential for escape of test organisms or a predator somehow entering the test enclosure is present. The logistics and costs of these studies also may be quite high.

Controlled environments such as mesocosms, microcosms, and artificial streams also offer a more "real-world" environment, although not to the same degree as in situ studies. They have many of the same advantages and disadvantages as in situ studies. The degree of similarity of controlled ecosystem studies to "real-world" conditions is dependent on the scale of the study (from laboratory flask on up to farm ponds) and its design.

The simplest bioaccumulation study is a laboratory study that tests one species at a time in a defined medium. The simplest of these tests are bioconcentration tests with fish or invertebrates. These tests are typically conducted with single chemicals or well-defined mixtures; however, they may also be modified for contaminated site media such as water or sediment. For instance, the US EPA may require a fish or oyster bioconcentration test for registration of a new chemical under the Toxic Substances Control Act (TSCA) (USEPA, 1992b) or in the pesticide registration process under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (USEPA, 1993b). Data obtained from these tests are used to develop a bioconcentration factor (a unitless value that indicates the degree to which a chemical concentrates in tissue with respect to the concentration in the surrounding medium [water]). The basic premise behind these tests is to expose the test organisms to a known chemical concentration until a steady state is attained (when uptake and depuration are equal) or for a designated time period (ASTM, 1988).

Table 3.6 Some freshwater sediment toxicity tests (ASTM E1383, 1993)

Testing of dredged material is another application to assess the bioaccumulation potential of a substance. These methods have been developed by the U.S. Army Corp of Engineers (1991) and the US EPA to assess the environmental impacts of dredged material dumped into ocean waters (USEPA, 1991a). Methods have also been recently developed to determine the environmental impact of dredged materials dumped into inland and near-coastal waters (USEPA, 1993a). These tests are also applicable to bioaccumulation testing of sediments. In general, these methods consist of exposing an organism (generally a benthic macroinvertebrate) to a quantity of sediment for a designated time (generally 10 days for metals and 28 days for organics or organometallics). Steady state bioaccumulation can then be determined by taking samples at designated intervals from the test chambers (USEPA, 1991b, 1993a).

Table 3.7 some marine and esturine sediment toxicity tests (ASTM E1383, 1993)

3.3.3.2 Biomarkers

Biomarkers are biochemical, physiological, or cellular responses to a stressor. They may provide an indication of exposure to a stressor or of its consequences. They may be used in both field and laboratory settings; and, because the stress is sublethal, they are environmental monitors that can provide early warnings of environmental undesired consequences. Biomarkers such as metallothionein (metal binding proteins) or induction of the cytochrome P₄₅₀ system (mixed function oxidases) have been used as research tools for several years to assess aquatic habitats. However, biomarkers have been employed only recently as environmental monitors.

The measurement of tissue residues (via bioaccumulation by biota) is the most direct biomarker of exposure. However, because some chemicals do not bioaccumulate, this measurement does not take into account toxic effects on the organism, excretion, or biotransformation. Also, the observed effects may not have biological significance. Most other .'typical " biomarkers are indirect measures of exposure or stress that describe either a biological response or exposure that is of biological or toxicological significance. Examples of some commonly used biomarkers in the assessment of aquatic habitats are provided in Table 3.9.

Table 3.8. Community parameters assessed by aquatic field assessment methods (USEPA, 1989)

Biomarkers can provide an added level of detail to an assessment of the aquatic environment, and can more closely infer a causal relationship, thereby decreasing the uncertainty in the conclusions. However, many of these biomarkers are still in the research stage, and, thus, must be used cautiously; a baseline uncontaminated site for comparison may also be required.

3.3.3.3 Controlled ecosystem studies

Controlled ecosystem studies have been used for many years to assess the impacts to aquatic environments, although the focus has been primarily on single chemical impacts. For instance, the USEPA Office of Pesticide Programs previously required simulated field testing (i.e., mesocosm studies) for many pesticides in the registration process. Simulated environment studies of aquatic environments include, in order of size and complexity, microcosms, mesocosms, artificial streams and ponds, and limnocorrals. As size is increased, the more ecosystem components can be added, and a closer replication of a natural system is attained. However, the more complex the model, the more covariants are obtained, and hence the greater the difficulty in interpreting the data. Also, as the complexity of the model increases, the cost and amount of effort involved increases. The ability to replicate systems also becomes increasingly more complex as one moves up the scale in size. However, even with the potential problems involved with the use of the large systems, the information obtained from them can be invaluable in the hazard and risk assessment processes.

Table 3.9. Examples of biomarkers (adapted from Huggett et al., 1992; McCarthy and Shugart, 1990; and USEPA, 1989)

The simplest controlled ecosystem study is a microcosm. These studies can range from flask size to aquarium size, and are generally conducted in a laboratory. They range from single species to multispecies systems; but, because they are one step up from a single-species standard laboratory bioassay, their resemblance to the real world is limited. However, recent research has increased the level of complexity in aquatic microcosms.

The standardized aquatic microcosm (SAM), developed by Frieda Taub and associates at the University of Washington, is a gnotobiotic (all organisms in the system are known), mixed flask culture system. This type of system differs from many other microcosms in that a specific ecosystem is not being replicated; rather, general ecosystem processes and functions are being tested. The idea behind the development of this test is to derive a "white rat" model that can be used by investigators worldwide, and can yield easily reproducible results that can be compared and then used in the hazard assessment process (Giesy and Allred, 1985).

To have a standardized aquatic microcosm, the test must be site independent and reproducible, and the water, organisms, and facilities must be available to all (Taub, 1985). This test uses distilled water to which a growth medium and laboratory cultured organisms are added (Taub, 1985). The organisms are easily distinguishable at x40 magnification, have short lifespans, and have been previously used as toxicity testing organisms (Taub, 1985). The test uses fixed numbers of 10 algal species, cladocerans, amphipods, ostracods, protozoans, and rotifers as well as bacteria used as food for the algae and other microorganisms introduced with the zooplankton cultures (Taub and Crow, 1980). The logic is that by initiating microcosms with ample nutrients and small numbers of organisms, the spring-summer behaviour of a temperate aquatic community is being simulated (Taub, 1985). Re-inoculation of organisms is also employed to prevent random extinctions and to mimic immigration (Taub et al., 1986). The test is conducted in gallon jars (24 jars with four treatment groups of six replicates each) under a standard light and temperature regime (Taub, 1985).

The test is run for 56 days after a seven-day assimilation period (Taub, 1985). The biology of the system has been developed to the point that all untreated containers show nitrate depletion, followed by algal increase that is terminated by a Daphnid population increase and an eventual crash of the Daphnid populations (Taub, 1985). Population dynamics as well as ecosystem level variables are monitored. However, measurements of community metabolism such as dissolved oxygen and chlorophyll-A are less variable than the organism counts (Crow and Taub, 1979). Inter- and intra-laboratory reproducibility, relying primarily on copper compounds, has been high (Giesy and Allred, 1985; Taub, 1985; Taub et al., 1986).

The use of SAM as a screening microcosm, not simulating any specific  cosystem, has been supported by other investigators (Haque, 1980). However, it also has been criticized, because of its cost (Shannon et al., 1986) and the fact that artificial communities used in a SAM may not be representative of "natural, coadapted species assemblages" and thus may not be reliable for studies of ecosystem-level properties (Hammons, 1981). 

Mesocosm or simulated field studies are large multispecies controlled ecosystem studies. They may include large pond systems, large outdoor tanks, artificial streams, and enclosures in the pelagic or littoral zone of an aquatic system. Mesocosms are functioning ecosystems, albeit on a smaller scale, and, therefore, provide a measure of both direct and indirect toxicity. However, the disadvantages of mesocosm tests are high logistics to cost, the ability to replicate, and, due to the high numbers of endpoints, difficulty in interpretation of findings (Graney et al., 1994). Mesocosm tests have been used quite extensively in the four-tier FIFRA hazard evaluation process for pesticides.

Tier-4 testing consists of mesocosm (simulated field studies) and actual field studies, and is required on a case-by-case basis depending on the exposure potential and toxicological hazard of the pesticide (Bascietto, 1990; USEPA, 1982). Guidance criteria for registrants on conducting mesocosm testing of pesticides are available (Touart, 1988); however, no standardized methods for mesocosm testing exist, due to the complexity of mesocosms and the specific requirements of each testing situation.

3.4 METHODS TO ASSESS CHEMICAL IMPACTS ON TERRESTRIAL ENVIRONMENTS

Chemically contaminated terrestrial habitats are evaluated in much the same manner as aquatic habitats. After definition of the study objectives, three types of data must be obtained and integrated: those from chemical analyses of samples, toxicity tests (both in the laboratory and in the field), and field or ecological surveys of biota. In addition, incorporation of biomarker studies or controlled ecosystem studies may be appropriate to help diagnose the causes of observed effects on terrestrial habitats and to establish the causal relationships linking contamination to observed changes. The special challenges of studying ecosystems that experience climatic extremes are discussed in later chapters of this book. The most commonly used toxicity tests, field methods, and special analytical techniques for assessing chemical impacts in terrestrial habitats are discussed in this section. Relevant features of the most frequently used toxicity tests are presented in Table 3.10 (tests using vertebrate and invertebrate animal species and soil microbes) and Table 3.11 (tests using vegetation).

3.4.1 TOXICITY TESTS

Terrestrial toxicity tests may be conducted with vertebrates and invertebrates, vegetation, or soil microbes in both laboratory and field situations. Generally, the tests measure toxicity by directly exposing test biota to media samples collected from the site, or indirectly by exposure to eluates (water that has been filtered through soil or sediment samples to remove water-soluble constituents) or leachates prepared from site samples. Most often these tests evaluate acute toxicity on the population level; however, some tests using higher animals evaluate the effects of chronic toxicity on individuals. Many methods evaluate the condition of soils as an indication of chemical toxicity; because soils provide the essential foundation of terrestrial ecosystems, the integrity of the soil can provide an indication of the integrity of the entire ecosystem. References that discuss the methods in greater detail and standardized protocols, where available, are presented in Tables 3.10 and 3.11.

3.4.1.1 Invertebrate toxicity tests

Tests using invertebrates (earthworms and insects)

Due to their essential functions in ecosystems, soil invertebrates are useful targets to assess the ecological effects of chemicals. Invertebrate tests measure acute toxicity at the species level (earthworm tests) and the population and community levels (soil insect tests). Primary endpoints are survival, growth (measured as biomass), reproductive success, and behavioural changes. The tests can be used in most habitats, are applicable to a wide range of contaminants, can be conducted fairly rapidly, and offer a range of cost options. Table 3.10 provides more information on these types of tests.

Tests using soil microbes

Soil bacteria and fungi have critical roles in the cycling of carbon, nitrogen, sulphur, and phosphorus, and make substrates available in forms that higher plants and animals can utilize. Because of their unique role in stable ecosystems, the physiological functioning of soil microbes can be very effective in studying the effects of chemicals on terrestrial ecosystems. Toxicity tests using soil microbes measure changes in microbial metabolism, respiration, and nitrogen cycling of soil bacteria and fungi after exposure to contaminants. Short-term microbial toxicity tests are technically simple, rapid, and relatively inexpensive procedures; standard protocols for some tests are available commercially. Table 3.10 provides more information on these tests.

3.4.1.2 Vertebrate toxicity tests

Vertebrate toxicity tests evaluate acute, subacute, and chronic toxicity of chemicals by describing survival and growth (amphibian tests), reproductive success (amphibian, small mammal, and avian tests), and body burdens (small mammal and avian tests) of the test species. All media and most chemicals can be tested by these tests that generally have longer exposure periods than the invertebrate tests previously discussed. The endpoints are easily understood, and are relevant to economically important higher animals. Feeding studies (small mammal and avian toxicity tests) are especially useful to determine the potential uptake of contaminants into food webs and potential human exposure route (if animals tested are representative of a possible human food source). Cellular level tests are available that provide an indication of chemical effects on immune function and genetic material of test species. These tests, while technically complex, provide information about the potential hazards to humans. Standard protocols, many adapted from veterinary medicine, exist for most tests.

Table 3.10. Vertebrate, invertebrate, and microbial test methods to assess the toxicity to terrestrial ecosystems

Table 3.11. Vegetation toxicity test methods to assess chemical impacts to terrestrial ecosystems

3.4.1.3 Vegetation toxicity tests

The vegetation toxicity tests discussed herein are used to test chemicals on crop species.  Using eluates, the tests evaluate acute and subchronic toxicity both directly and indirectly with variable exposure periods (5 to 90 days), and they can be used in most habitats.  The primary endpoints are survival (seed germination test), growth (seedling growth test and root elongation test), reproduction success (vascular plant toxicity tests), and photosynthesis rates (chlorophyll fluorescence assay). The tests are applicable at several levels of organization, and can be applied in both the laboratory and the field to test all types of chemicals. Standard protocols have been adapted from agricultural science that are relatively inexpensive and simple to perform. Seeds of standard test species (e.g., Lactuca sativa, lettuce) are available from commercial sources; seeds of site specific plants can also be tested. These tests offer a wide range of cost options, with some being fairly inexpensive (seed germination and root elongation tests) and others requiring expensive test equipment (photosynthesis inhibition test). Growth conditions must be carefully monitored during these tests, because nutrient limitations can complicate interpretation of toxicity effects.

These tests evaluate chemical effects on all stages of plant development; however, different stages of plant growth exhibit varying sensitivity to chemical insults. For example, seeds are the least sensitive stage of a plant's growth; if seed germination reveals significant toxicity, the environmental consequences are probably severe. Unlike seeds, young roots and seedlings are relatively sensitive to chemical insult, making tests that evaluate those stages especially sensitive assays and particularly effective at demonstrating the effects of low contaminant concentrations and chronic toxicity. Table 3.11 provides detailed information about these tests.

3.4.1.4 In situ toxicity tests

Certain toxicity tests described above have been adapted for use in the field to evaluate in situ toxicity conditions. The most frequently used tests measure earthworm survival, amphibian viability, and seed germination. On-site tests conducted in parallel with laboratory analyses offer certain advantages. In situ toxicity tests can address methodological biases often associated with laboratory tests and the uncertainties associated with extrapolation from laboratory to field and from standard test species to site-specific species. The on-site version of the tests may often reduce cost and workload by eliminating collection, shipping, handling, and disposal of waste, thus permitting the collection and measurement of greater numbers of samples. However, when conducting on-site tests, the environmental conditions at the site, including fluctuation in temperature and moisture conditions, must be considered. To do so may require rejection of many data sets, and also careful planning to be present during optimal conditions. For more detailed descriptions of methods see: earthworm survival test (Callahan et al., 1991; Marquenie et al., 1987; USEPA, 1992); amphibian test (Linder et al., 1991; USEPA, 1992); and seed germination test (USDA 1985; Gorsuch et al., 1990; Linder et al., 1990; USEPA, 1989, 1992; USFDA, 1987b).

3.4.2 FIELD ASSESSMENTS

Field surveys are useful to evaluate the effects of chemicals on terrestrial ecosystems (USEPA, 1989). Field assessments discussed here include remote sensing methods, direct observation or long-term monitoring of permanent plots, field surveys of populations and communities, and adaptations of toxicity tests to field applications. Changes in an ecosystem can be monitored and evaluated at the same site over a long period of time. This technique is especially useful in forest studies when the individuals under study are long lived and the area is less disturbed. Examples of field assessments used to evaluate effects of contaminants are discussed below.

3.4.2.1 Remote sensing methods

Remote sensing may be used in several ways to assess vegetation of chemically contaminated sites. The main sources of radiometric data are the Landsat Multi Spectral Scanner (MSS) in the US, the Thematic Mapper (TM) in the US, and the Systeme Probatoire d'Observation de la Terre (SPOT) in France (USEPA, 1989; Koeln et al., 1994). Resolution for the three types of data are: MSS: 80 metres; TM: 30 metres; and SPOT: 20 metres. For improved resolution, infrared and conventional photography from fixed-wing aerial aircraft may be supplemented. Remotely sensed data offer the following advantages: relatively unlimited accessibility; safe, non-intrusive assessment and monitoring; and the existence of historical data (MSS since 1972; TM since 1982; SPOT since 1984), the opportunity to assess large-scale seasonal and annual vegetation patterns (USEPA, 1989). Data derived from remote sensing methods can be used to map vegetation boundaries, estimate photosynthesis rates and drought stress, detect the effects of natural pests epidemics, and assess forest decline due to air pollutants. Advantages of remote sensing methods are discussed in greater detail in the contributed chapters.

3.4.2.2 Direct observation or ground truthing of remotely sensed data

A primary use of field survey methods is "ground truthing" or verification of remotely sensed data by direct observation in the field to determine the vegetation types and habitats present. A semi-quantitative method like the Relevé method, which has been used for many years worldwide, is usually sufficient to develop a description and patterning of the plant species present (Braun-Blanquet, 1932). This method will provide information sufficient to plan additional studies of plant and animal species to evaluate the influence of chemicals on site biota.

3.4.2.3 Long-term monitoring of permanent assessment plots

Long-term monitoring of permanently marked plots is often useful to follow changes in a terrestrial ecosystem (Clarke, 1986; Bonham, 1989; USEPA, 1989). This technique has been used extensively in forest habitats by national resource management agencies such as the US Forest Service. Permanent forest plots evaluate parameters indicative of overall forest condition and habitat suitability, (e.g., vegetation demographics, soil surface conditions, primary productivity, and nutrient cycling). Such long-term monitoring may be costly. The data quality is very dependent on the selection of the representative sample location. Detailed descriptions of the various types of permanent assessment procedures are presented later in this volume.

3.4.2.4 Population surveys

Vegetation surveys¾community structure and floristics studies

These methods provide quantitative or qualitative information to help establish the extent and magnitude of chemical impacts on vegetation. Plant community parameters (e.g., species density and percent cover) of existing site plant communities or of defined test plant communities grown in a representative mesocosm in test soil are used to identify effects of chemicals. Endpoints are species abundance, species dominance, community structure, age-class or size-class distributions, and species distribution patterns. Vegetation sampling data provide information that can be linked with site history and toxicological information to establish causation. Vegetation sampling methods have been used widely in basic and applied plant ecology for many years. Many field sampling techniques exist, ranging from pseudo-quantitative to quantitative methods relying on defined-area plots (quadrants) or various plotless sampling including lines, points, or variable radius methods. Detailed descriptions of these methods are presented by Kapustka et al. (1989), Pfleeger (1991), USEPA (1992), Weinstein and Laurence (1989), and Weinstein et al. (1991).

Animal population surveys

Surveys of animal populations present on a site, when compared to references sites, provide information about the impact of contamination on demographic parameters and ecological diversity (USEPA, 1989; Bookout, 1994). To accurately estimate if vertebrate and invertebrate populations have been adversely affected by site contamination, a census or estimation of population numbers of resident species, age and sex ratios, reproduction rates and rearing success, and survival and mortality rates must be conducted (Johnson, 1994). An understanding of the life cycles and behaviour of animal species expected to be found at the site, as determined by studying a reference site, helps to guide the sampling plan.

Two major approaches to estimate animal populations are direct counts and indirect methods of observing the animals present (Johnson, 1994). Direct counts of representative sample sites are more cost-effective than conducting a complete census. The most common direct methods are sampling along transects (Anderson et al., 1976) and within quadrants, recording direct observations by field teams, or sampling with capture traps. Selection of sampling times and capture method is determined by evaluating reference sites as well as documentation in the literature. Field teams must be experienced in animal handling and identification. If captured animals are to be used in long-term studies or toxicity tests, the individuals must be handled and marked in a manner that does not cause injury or death.

When direct observational methods are impossible, indirect evidence of animal life must be utilized. The sampling design and statistical interpretations are the same as for direct observation or capture data. Commonly used types of indirect signs include: dens, burrows, or nests; tracks, faeces, calls; and counts of carcasses. The use of indirect signs alone is problematic, but can be used cautiously for interpretation (Davis and Winstead, 1980).

3.4.3 SPECIAL ANALYSES

Tests that will provide information unavailable from chemical analyses, toxicity tests, or field assessments of the chemically contaminated site are often desirable. Demonstrating that a chemical is present in elevated levels at the site or that the chemical is toxic to biota is at times insufficient, but the chemicals must be shown to be incorporated into the food chain. Residue studies can provide this kind of information, and give indications of possible routes of exposure to humans through their food sources. Soil microcosm studies provide a picture of the overall impact on soil ecosystem functioning that is important to evaluate chemical impacts on primary productivity of plant and animal resources of value to human society. Biomarkers may also provide an indication of actual contaminant exposure or effects at the organismal level. Biomarkers and soil microcosm studies used to evaluate contaminant stress in terrestrial organisms are described below.

3.4.3.1 Biomarkers

Biomarkers are increasingly incorporated into assessments of aquatic and terrestrial ecosystems. General discussions of the utilization of biomarkers can be found in McCarthy and Shugart (1990) and Huggett et al. (1992).

3.4.3.2 Soil microcosm studies

Soil microcosm studies evaluate ecological effects and environmental fate and transport of solid and liquid contaminants by measuring adverse effects on growth and reproduction of native vegetation or crop plants. The tests also measure the uptake and cycling of nutrients in the terrestrial ecosystem. Possible endpoints include: primary productivity; bioaccumulation and translocation of contaminants in plant tissue; and nutrients lost in leachates. Originally designed to test grassland or agricultural soils, the tests have been widely used in numerous systems, and are standardized through ASTM E1197 (1992). Tests can be readily adapted to site-specific conditions, and have been used to evaluate complex chemical wastes, hazardous wastes, and agricultural chemicals. Because of the ease of collecting leachate, these tests are especially suitable to monitor nutrient losses in leachates. Regrettably, the duration of these tests is relatively long (6-8 weeks), and few commercial laboratories currently conduct them. More detailed descriptions of such methods are found in USEPA (1992b) and Van Voris et al. (1985).

3.5 DIAGNOSIS OF ENVIRONMENTAL PROBLEMS AND ESTABLISHMENT OF CAUSATION

This chapter has presented some of the varied tools that enable scientists to define the condition of chemically contaminated sites and to establish linkages between site contamination and observed adverse effects. These tools include chemical analyses, toxicity tests, field surveys, special studies such as biomarkers and simulated ecosystem studies, and mathematical modelling to evaluate assumptions (Figure 3.1). Each tool provides specific kinds of data, which, if used alone, are certainly useful; however, the greatest contribution of each tool is in providing a unique portion of an entire environmental puzzle. Chemical analyses of site samples show the extent and quantity of contamination. Data from toxicity tests (laboratory and in situ) demonstrate that contaminants are capable of causing damage. Field observations indicate the extent and patterns of contamination and provide supporting evidence that site populations have been altered structurally and functionally.

Once ecotoxicity has been measured using the tools described above, the next step is to demonstrate that the contaminants caused the damage. In most cases, the extent and nature of the toxic effects (especially in a retrospective risk assessment) do not unequivocally demonstrate which agent was responsible for the injury. Correlations between a substance and a form of damage may be obvious, but causation is most difficult to prove. As with other fields of toxicology, the scientist and decision maker may need to rely on statistical associations, because causal relationships can be established only indirectly.

Figure 3.1 Types of data used in an ecological risk assessment

Scientists have used the criteria proposed by bacteriologist, Robert Koch, in the early 1900s when medical science was first struggling to understand cholera, typhoid fever, and tuberculosis, as a way to establish the weight of evidence for causation. These criteria can be adapted as guidelines to establish whether a chemical is causing (or has caused) the observed alterations; they are summarized as follows (as restated by Evans, 1976):

1. The effect is more pronounced in exposed biota.
2. The exposure is more frequent among biota exhibiting the effect.
3. The incidence of the effect is higher in exposed populations.
4. The appearance of the effect should follow exposure.
5. The responses should follow exposure along a gradient from mild to severe.
6. Exposure triggers a measured response, with a higher probability after exposure, and should not be seen in unexposed biota.
7. Experimental reproduction of the effect occurs more frequently in exposed biota.
8. Elimination of the suspected agent decreases the incidence of the effect.

If these criteria are met by the totality of the data, then causation can be claimed with a relatively high degree of certainty. The environmental manager must be aware of the many limitations inherent in ecological data when making management decisions. However, if the appropriate steps are followed (i.e., clear statement of objectives, sound study design, selection of appropriate assessment methodologies, careful data interpretation, and QA/QC integrated into the entire study process), the data will be sufficiently sound and comprehensive to make confident estimates of risk on which to base environmental decisions.

3.6 RECOMMENDATIONS

3.6.1 RECOMMENDATIONS FOR ENHANCING ASSESSMENTS OF AQUATIC AND TERRESTRIAL ECOSYSTEMS

1. Further research should be conducted to characterize pristine aquatic ecosystems so that deviations from the natural situation resulting from chemical contamination can be reliably quantified.

2. Monitoring programs should be implemented to estimate baseline data and to gain increased understanding of the natural variability within ecosystems. Longterm monitoring programs should be established as soon as possible to detect adverse effects of chemicals on ecosystems.

3. Additional research needs to be conducted using communities or organisms in micro- and meso-cosms to establish dose-response relationships at levels above that of the single species. Often unclear is how change at one level of a community affects organisms at other levels in aquatic ecosystems. Therefore, mechanistic links must be established among effects at different levels of biological organization. Effects at the global level, in particular, deserve further attention.

4. Synergistic and antagonistic effects among chemicals need to be better understood in aquatic ecosystems.

5. Research should continue to identify useful biomarkers in nontemperate biomes, especially in polar and tropical regions. Uncertainties resulting from extrapolation of data generated in temperate biomes make these tests less useful than in other regions. What holds for a particular concentration of a pollutant in one ecosystem may not necessarily apply to all ecosystems due to differences in local conditions.

6. Accidental releases of chemicals should be viewed as an opportunity to increase understanding of the nature and severity of adverse effects and the efficacy of current monitoring techniques for chemical contaminants.

7. Recovery from accidental releases and chronic exposures, and the potential to manipulate the natural recovery process, should be studied. The robustness of ecosystems should be studied to increase understanding of the ability of a system to withstand chemical exposure without irreversible damage.

8. The importance of timeframes and time scales needs greater attention. The timeframe in which results are needed should be considered when selecting assessment and monitoring techniques. In addition, the nature of a chemical release and dispersal (i.e., patterns and rates; acute and chronic toxicity) should be considered when selecting methods.

9. Alternative methods should be developed that will more easily allow the translation of ecological characterizations into value or benefit characterizations.

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