Introduction to ENVIRONMENTAL TOXICOLOGY Impacts of Chemicals Upon Ecological Systems - CHAPTER 4

CHAPTER 4 Survey and Review of Typical Toxicity Test Methods The importance of understanding the test procedures that are crucial to environ-mental toxicology cannot be underestimated. The requirements of the tests dictate the design of the laboratory, logistics, and the required personnel. In every interpre-tation of an EC50 or an NOEL there should be a clear understanding of the test method used to obtain that estimate. The understanding should include the strengths and weaknesses of the test method and the vagaries of the test organism or organisms. Quite often it is the standard method that is modified by a researcher to answer more specific questions about the effects of xenobiotics. These standard tests form the basis of much of what we know about relative chemical toxicity in a laboratory setting. Table 4.1 lists a number of toxicity tests currently available from a variety of standard sources. This table is not inclusive since there are more specialized tests for specific location or situations. Many more methods exist, some of which are derivatives of basic toxicity tests. More important than memorization of each test procedure is a good understanding of the general thrust of the various toxicity tests, methods of data analysis, and experimental design. A more complete listing of toxicity tests and references for the methods are presented in Appendix A. The following survey starts with single species toxicity tests and concludes with field studies. These summaries are based on the standard methods published by the American Society for Testing and Materials, the U.S. Environmental Protection Agency, and other published sources. Many of these methods are listed in the reference section for this chapter. The survey is broken up into single species and multispecies tests. Although Chapter 3 discussed to some length the various types of toxicity (acute, chronic, partial life cycle, etc.), it is in many ways logical to list them into organismal and ecosystem type tests. That organizational scheme is what is done here. Since it is difficult to include every toxicity test in a volume of this size, representative tests have been chosen for summary. Inclusion here does not imply an endorsement by the authors, but these tests serve as examples of the kinds of toxicity tests used to evaluate environmental hazards. Table 4.1 Partial List of ASTM Standard Methods for Toxicity Evaluation or Testing Biodegradation By a Shake-Flask Die-Away Method Conducting a 90-Day Oral Toxicity Study in Rats Conducting a Subchronic Inhalation Toxicity Study in Rats Conducting Aqueous Direct Photolysis Tests Determining the Anaerobic Biodegradation Potential of Organic Chemicals Determining a Sorption Constant (Koc) for an Organic Chemical in Soil and Sediments Inhibition of Respiration in Microbial Cultures in the Activated Sludge Process Algal Growth Potential Testing with Selenastrum capricornutum Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Mollusks Conducting Reproductive Studies with Avian Species Conducting Subacute Dietary Toxicity Tests with Avian Species Evaluating Environmental Fate Models of Chemicals Measurement of Chlorophyll Content of Algae in Surface Waters Standardized Aquatic Microcosm: Freshwater Using Brine Shrimp Nauplii as Food for Test Animals in Aquatic Toxicology Using Octanol-Water Partition Coefficient to Estimate Median Lethal Concentrations for Fish Due to Narcosis Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes Conducting Acute Toxicity Tests on Aqueous Effluents with Fishes, Macroinvertebrates, and Amphibians Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians Conducting Early Life-Stage Toxicity Tests with Fishes Conducting Life-Cycle Toxicity Tests with Saltwater Mysids Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna Conducting Sediment Toxicity Tests with Freshwater Invertebrates Conducting 10-Day Static Sediment Toxicity Tests with Marine and Estuarine Amphipods Conducting Static 96-h Toxicity Tests with Microalgae Conducting Static Acute Aquatic Toxicity Screening Tests with the Mosquito, Wyeomyia smithii (Coquillett) Conducting Static Acute Toxicity Tests Starting with Embryos of Four Species of Saltwater Bivalve Mollusks Conducting Static Toxicity Tests with the Lemma gibba G3 Conducting a Terrestrial Soil-Core Microcosm Test Conducting Three-Brood, Renewal Toxicity Tests With Ceriodaphnia dubia Hazard of a Material to Aquatic Organisms and Their Uses Assessing the Performance of the Chinese Hamster Ovary Cell/Hypoxanthine Guanine Phosphoribosyl Transferase Gene Mutation Assay SINGLE SPECIES TOXICITY TESTS Daphnia 48-H Acute Toxicity Test This test along with the fish 96-h acute toxicity test is one of the standbys in aquatic toxicology. Daphnia magna and D. pulex are the common test species. D. magna require a relatively hard water for their culture. D. magna are large, commonly available, and easy to culture. D. pulex are not quite as large as D. magna and tolerate softer water. It is recommended that the test organisms be derived from adults, three generations after introduction into the specific laboratory media. Water quality is a major factor in the performance of any laboratory aquatic toxicity test. Care must be taken to eliminate other sources of mortality, such as chlorine of chlorinated organics, heavy metal contamination, and contamination by organics in the groundwater or reservoir supply. In some labs with access to high-grade tap or well water, only a minor purification system is required. However, in many cases a further filtration and distillation step may be required. Soft dilution water (40 to 48 mg/l as CaCO3) is recommended for tests with D. pulex, and moderately hard water (80 to 100 mg/l as CaCO3) is recommended for tests with D. magna. A dilution water is considered acceptable if Daphnia spp. show adequate survival and reproduction when cultured in the water. Sodium pentachlorophenate (NaPCP) is the reference toxicant that has been suggested for toxicity tests using daphnids. The use of a reference toxicant is important in confirming the health of the daphnia and the quality of the water and test methodology. In general, 10 neonates that are less than 24 h old are placed in 125 ml beakers containing 100 ml of test solution with five concentrations and a negative control. The tests are usually run in triplicate. Death is difficult to observe so immobility of the daphnia is used as the endpoint. An organism in considered immobile (nonmotile) if it does not resume swimming after prodding with a pipet or glass rod. Measure-ments are made at 24-h intervals. No feeding occurs during the course of this toxicity test. The daphnia 48-h toxicity test is a useful screen for the toxicity of single compounds, mixtures, or effluents. In some cases the daphnid toxicity test has been used to evaluate the potential pathology or other potential problems with genetically engineered organisms. The advantages of the daphnid toxicity test are short time frame, small amounts of hazardous waste are generated, and the test is inexpensive. Often daphnids are more sensitive than vertebrates to a variety of toxicants. The disadvantages include the time-consuming maintenance of test stocks and the sen-sitivity of the organisms to water quality. The chronic or partial life cycle toxicity test with D. magna is an attempt to look at growth and reproductive success of the test organisms. This test is contrasted to its acute counterpart in Table 4.2. The test follows a set of daphnia through the production of three broods with generally a measurement of growth (length or mass) of the original organisms along with the numbers of offspring derived from each animal. One of the most controversial aspects of this test has been the food source during the study. A number of mixtures have been tried with interesting results. A mixture of trout chow and algae has been demonstrated to provided excellent growth, but there are concerns about the consistency of the ingredients. Many laboratories use a combination of algae, Ankistrodesmus convolutus, A. falcatus, Chlamydomonas reinhardii, and Selenastrum capricornutum as the food source. This toxicity test is usually run as a static renewal but some researchers have used a continuous flow set up with a proportional diluter. Handling the organisms during the transfer to new media is a potential problem for inexperienced technicians. Occasionally it is difficult to set up concentrations for the test if the median values for the chronic endpoints are close to the values for a toxicant that induce mortality over the duration of the experiment. Loss of replicates can occur if the mortality rates are high enough. Use of the dose-response curve of the acute data Table 4.2 Comparison of the D. magna 48-h Acute Toxicity Test with the Common D. magna Chronic Toxicity or Partial Life Cycle Test Test type Chronic (partial life cycle) Acute 48 h Organisms Age of test organisms Number of organisms per chamber Experimental design Test vessel type and size Test solution volume Number of replicates per sample Feeding regime Test duration Physical and chemical parameters Water temperature (°C) Light quality Light intensity Photoperiod pH range DO concentration Aeration Endpoint D. magna £24-h old 10 100 ml beakers 80 ml 2 (minimum) Various combinations of trout chow, yeast, alfalfa, green algae, and diatoms given in excess 21 days 20°C Ambient laboratory levels Up to 600 lux 16 h light and 8 h dark (with 15- to 30-min transition) 7.0–8.6 40–100% Not necessary Survival, growth, and reproduction D. magna £24-h old 10 (minimum) 250 ml 200 ml 3 (minimum) Do not feed 48 h 20 ± 2°C Ambient laboratory levels 540 to 1080 lux 16 h light and 8 h dark 7.0–8.6 60–100% None Immobilization should help in identifying useful boundary conditions for the higher concentrations of xenobiotic. Closely related to the D. magna partial life cycle toxicity test is the three-brood renewal toxicity test with Ceriodaphnia dubia (Table 4.3). The test was developed in an attempt to shorten the amount of time, amount of toxicant, and the cost of performing chronic-type toxicity tests. This methodology has proven useful in a variety of roles, especially in the testing of effluents. One of the drawbacks and advantages of the method is the small size of the test organism. Adult C. dubia are about the same size as first instar D. magna. Handling the first instars and even the adults often takes a dissecting microscope and a steady hand. Conversely, the small size enables the researcher to conduct the test in a minimum of space and the rapid reproduction rate makes the method one of the shortest life cycle type tests. As with the D. magna tests, one of the problems has been in the successful formulation of a food to ensure the health and reproducible reproduction of the C. dubia during the course of the toxicity tests. A combination of trout chow, yeast, rye grass powder, and algae have been used. Nonetheless, the C. dubia three-brood toxicity test has been proven to be useful and replicable. Clonal variability in sensitivity to toxicants does exist. Soares, Baird, and Calow (1992) examined the relative sensitivities of nine clones of D. magna of sodium bromide and 3,4-dichloroaniline (3,4-DCA) using chronic NOECs and LOECs as endpoints. All tests were conducted in the same laboratory. There was as much as a 15 fold difference in the LOECs in the tests using sodium bromide. A two- to Table 4.3 Summary for Conducting Three-Brood, Renewal Toxicity Tests with Ceriodaphnia dubia Test type Organisms Age of test organisms Experimental design Test vessel type and size Number of replicates Total number of organisms Number of organisms per chamber Feeding regime Test duration Physical and chemical parameters Temperature Test solution pH DO concentration Endpoint Static renewal/chronic Ceriodaphnia dubia <12 h old Test has been conducted with 30 ml beaker with 15 ml of test solution; can use any container made of glass, Type 316 stainless steel, or fluorocarbon plastic if a) each C. dubia is in a separate chamber or compartment and b) each chamber can maintain adequate DO levels for the organism; chambers should be covered with glass, stainless steel, nylon, or fluorocarbon plastic covers or Shimatsu closures 10 At least 10 1 Various combinations of trout chow, yeast, rye grass powder, and algae have been used; types of algae include: Ankistrodesmus convolutus, A. falcatus, Chlamydomonas reinhardii, and Selenastrum capricornutum 7 days 25° ± 1°C Not specified 40–100% Reproduction four-fold difference in LOECs was observed for the 3,4-DCA toxicity tests. Interclonal variation was therefore substantial. These results demonstrated the importance of iden-tifying the specific clone used in toxicity testing when attempting to compare results. Algal 96-H Growth Toxicity Test The purpose of this toxicity test is to examine the toxicity of materials to a variety of freshwater and marine algae and it is summarized in Table 4.4. In aquatic systems algae are generally responsible for a large percentage of the primary pro-duction. Impacts upon the unicellular photosynthetic organisms could have long-lasting impacts to the community. Numerous test organisms have been used in this toxicity test, but those currently recommended by the ASTM guidelines are Freshwater: Green Algae: Selenastrum capricornutum, Scenedesmus subspicatus, Chlorella vulgaris Blue-green algae (bacteria): Microcystus aeruginosa, Anabena flos-aquae Diatom: Navicula pelliculosa Saltwater: Diatom: Skeltonema costatum, Thalassiosira pseudonana Flagellate: Dunaliella tertiolecta ... - tailieumienphi.vn