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 EC 50 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. © 1999 by CRC Press LLC 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 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 (K oc ) 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 © 1999 by CRC Press LLC 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 CaCO 3 ) is recommended for tests with D. pulex, and moderately hard water (80 to 100 mg/l as CaCO 3 ) 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 © 1999 by CRC Press LLC 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 4.2 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 D. magna D. magna Age of test organisms ≤ 24-h old ≤ 24-h old Number of organisms per chamber 10 10 (minimum) Experimental design Test vessel type and size 100 ml beakers 250 ml Test solution volume 80 ml 200 ml Number of replicates per sample 2 (minimum) 3 (minimum) Feeding regime Various combinations of trout chow, yeast, alfalfa, green algae, and diatoms given in excess Do not feed Test duration 21 days 48 h Physical and chemical parameters Water temperature (°C) 20°C 20 ± 2°C Light quality Ambient laboratory levels Ambient laboratory levels Light intensity Up to 600 lux 540 to 1080 lux Photoperiod 16 h light and 8 h dark (with 15- to 30-min transition) 16 h light and 8 h dark pH range 7.0–8.6 7.0–8.6 DO concentration 40–100% 60–100% Aeration Not necessary None Endpoint Survival, growth, and reproduction Immobilization © 1999 by CRC Press LLC 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 Table 4.3 Summary for Conducting Three-Brood, Renewal Toxicity Tests with Ceriodaphnia dubia Test type Static renewal/chronic Organisms Ceriodaphnia dubia Age of test organisms <12 h old Experimental design Test vessel type and size 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 Number of replicates 10 Total number of organisms At least 10 Number of organisms per chamber 1 Feeding regime 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 Test duration 7 days Physical and chemical parameters Temperature 25° ± 1°C Test solution pH Not specified DO concentration 40–100% Endpoint Reproduction © 1999 by CRC Press LLC Other test organisms can be used if necessary for a particular toxicity assessment or research. The methodology is very adaptable. Depending upon the test organism, between 2 × 10 4 and 5 × 10 4 cells are used to inoculate the test vessel and the concentration of cells is determined daily. Cell counts are made daily by using a hemocytometer or an electronic particle counter such as the Coulter Counter. Chlorophyll a can be measured spectrophotometrically or fluorometrically. The fluorometric determinations are more accurate at low con- centrations of test organism. Other measurements that have been used include DNA content, ATP charge, and 14 C assimilation. If only standing biomass is the endpoint to be measured then only cell concen- tration at the end of the exposure period has to be determined. However, measure- ments such as area under the curve and growth rate are important variables in determining the ecological impacts of a toxicant. These valuable endpoints require Table 4.4 Summary of Test Conditions for Conducting Static 96-H Toxicity Tests with Microalgae Test type Static Organisms Fresh water species: Selenastrum capricornutum, Scenedesmus subspicatus, Chlorella vulgaris, Microcystis aeruginosa Anabaena flos-aquae, Navicula pelliculosa; Salt water species: Skeletonema costatum, Thalassiosira pseudonana, and Dunaliella tertiolecta Number of organisms per chamber (±10%) Selenastrum capricornutum and other freshwater green algae 2 × 10 4 cells/ml Navicula pelliculosa 2 × 10 4 cells/ml Microcystis aeruginosa 5 × 10 4 cells/ml Anabaena flos-aquae 2 × 10 4 cells/ml Saltwater Species 2 × 10 4 cells/ml Experimental design Test vessel type and size Sterile Erlenmeyer flasks of borosilicate glass, any size Test solution volume Not to exceed 50% of the flask volume for tests conducted on a shaker, and not more than 20% of the flask volume for tests not conducted on a shaker Number of replicate chambers per sample 2 or more Test duration 96 h Physical and chemical parameters Water temperature 24 ± 2°C for freshwater green and blue-green alga 20 ± 2°C for Navicula pelliculosa and other saltwater alga Light quality Continuous “cool-white” fluorescent Light intensity Should not vary by more than ±15% 60 µE m –2 /s –1 (4300 lm/m 2 ) for freshwater diatoms and green algae 30 µE m –2 /s –1 (2150 lm/m 2 ) for freshwater blue-green algae 82–90 µE m –2 /s –1 (5900 to 6500 lm/m 2 ) for Thalassiosira 60 µE m –2 /s –1 (4300 lm/m 2 ) for Skeletonema Photoperiod 14 h light/10 h dark for Skeletonema Test solution pH 7.5 ± 0.1 for freshwater 8.0 ± 0.1 for saltwater Endpoint Biomass, cell number, area underneath the growth curve, chlorophyll content © 1999 by CRC Press LLC measurements of cell density each day for the duration of the toxicity test. Other measurements to ensure the replicability of the data include pH, temperature, and light intensity. Whenever possible, toxicant concentration should also be taken at the beginning and end of the test. Errors in measurement, degradation, or volatilization can produce a concentration different from that of the expected or nominal concentration. Good microbiological sterilization technique is required to ensure a minimum of cross-contamination with other algae and to prevent the introduction of bacteria. The degradation of the toxicant by introduced bacteria can alter the apparent toxicity, even to the point of eliminating the test compound from the media. Another interesting aspect of this test is the enhancement of algal growth often found at low concentrations of toxicant. The spontaneous hydrolysis or other break- down of the test compound may provide nutrients as well as nutrients contained in effluents. It is crucial that the data be appropriately plotted and analyzed. Acute Toxicity Tests with Aquatic Vertebrates and Macroinvertebrates As with the daphnid toxicity tests, toxicity tests using a variety of fish species, amphibians, and macroinvertebrates have long been the standbys of aquatic toxicity evaluations. Table 4.5 summarizes the species and methods used in these tests. One of the major problems in conducting these toxicity tests is the reliable supply of healthy test organisms. Many of the fish species used to stock ponds and lakes are available through hatcheries. Specialist suppliers also exist for the species that are routinely used for toxicity evaluations. In some cases it is required that wild organisms are collected and acclimated to the laboratory environment before con- ducting the toxicity test. Animals collected from the wild have some advantages and some drawbacks. The major advantage is that if the organism is collected locally the sensitivity demonstrated in the toxicity test is representative of that particular native population. Care must be taken, however, to not unduly stress the collected organisms or the resultant stress may cause an overestimate of the toxicity of the compound being examined. The major difficulty of using organisms collected from wild populations is the variation among populations in sensitivity to the toxicant or to the laboratory culture collections. With mobile organisms it may be difficult to consistently collect organisms from the same breeding population. Also, the act of collecting the organisms may seriously deplete their numbers, especially in areas near the testing facility. Care should be taken not to deplete local populations. Another solution is to maintain a habitat adjacent to the facility as a source of the test organisms under the control and regulation of the testing laboratory. Another difficulty in conducting a broad series of toxicity tests is the assurance of adequate water quality and volume for a variety of species. For testing freshwater species the solution is often the investment in a well system with the water filtered and sterilized. Occasionally the testing facility may be adjacent to a body of water that can supply a consistent and uncontaminated source of water for the culture of the test organisms and also act as a source of dilution water. Laboratories on the Great Lakes or marine laboratories often have access to large volumes of relatively clean water. The least desirable but often the only option available is the use of distilled tap water for culture and dilution. At the least, the tap water should be © 1999 by CRC Press LLC Table 4.5 Summary for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians Test type Static, renewal, flow-through Organisms Fresh water Vertebrates Frog ( Rana sp.), toad ( Bufo sp.), coho salmon ( Oncorhynchus kisutch ), rainbow trout ( Oncorhynchus mykiss ), brook trout ( Salvelinus fontinalis ), goldfish ( Carassius auratus ), fathead minnow ( Pimephales promelas ), channel catfish ( Ictalarus punctatus ), bluegill ( Lepomis macrochirus ), green sunfish ( Lepomis cyanellus ) Invertebrates Daphnids ( Daphnia magna, D. pulex, D. pulicaria ), amphipods ( Gammarus lacustris, G. fasciatus,G. pseudolimnaeus ), crayfish ( Orconectes sp. , Combarus sp. , Procambarus sp., Pacifastacus leniusculus ), stoneflies ( Pteronarcys sp.), mayflies ( Baetis sp. , Ephemerella sp., Hexagenia limbata, H. bilineata ), midges ( Chironomus sp.), snails ( Physa integra, P. heterostropha, Amnicola limosa) , planaria ( Dugesia tigrina ) Saltwater Vertebrates Sheepshead minnow ( Cyprinodon variegatus ), mummichog ( Fundulus heteroclitus ), longnose killifish ( Fundulus similis ), silverside ( Menidia sp.), threespine stickleback ( Gasterosteus aculeatus ), pinfish ( Lagodon rhomboides ), spot ( Leiostomus xanthurus ), shiner perch ( Cymatogaster aggregata ), tidepool sculpin ( Oligocottus maculosus ), sanddab ( Citharichthys stigmaeus ), flounder ( Paralichthys dentatus, P. lethostigma ), starry flounder ( Platichthys stellatus ), English sole ( Parophrys vetulus ), herring ( Clupea harengus ) Invertebrates Copepods ( Acartia clausi, Acartia tonsa ), shrimp ( Penaeus setiferus, P. duorarum, P. aztecus ), grass shrimp ( Palaemonetes pugio, P. intermedius, P. vulgaris ), sand shrimp ( Crangon septemspinosa ), shrimp ( Pandalus jordani, P. danae ), bay shrimp ( Crangon nigricauda ), mysid ( Mysidopsis bahia, M. bigelowi, M. almyra ), blue crab ( Callinectes sapidus ), shore crab (Hemigrapsus sp., Pachygrapsus sp.), green crab (Carcinus maenas), fiddler crab (Uca sp.), oyster (Crassostrea virginica, C. gigas), polychaete (Capitella capitata) Age and size of test organisms All organisms should be as uniform as possible in age and size. Fish: juvenile; weight between 0.1–5.0 g; total length of longest fish should be no more than twice that of the shortest fish Invertebrates: except for deposition tests with bivalve mollusks and tests with copepods, immature organisms should be used whenever possible Daphnids: less than 24 h old; Amphipods, mayflies, and stone flies: early instar Midges: second or third instar Saltwater mysids: less than 24 h post-release from the brood sac Do not use ovigerous decapod crustaceans or polychaetes with visible developing eggs in coelom Amphibians: use young larvae whenever possible © 1999 by CRC Press LLC Experimental design Test vessel type and size Smallest horizontal dimension should be three times the largest horizontal dimension of the largest organism Depth should be at least three times the height of the largest organism Solution volume At least 150 mm deep for organisms over 0.5 g each and at least 50 mm deep for smaller organisms Feeding regime Feed at least once a day a food that will support normal function Test duration Daphnids and midge larvae: 48 h All other species: 96 h in static tests, at least 96 h in renewal and flow-through test Physical and chemical parameters Water temperature (°C) Fresh water Vertebrates Frog, Rana sp. (22) Toad, Bufo sp. (22) Coho salmon, Oncorhynchus kisutch (12) Rainbow trout, Oncorhynchus mykiss (12) Brook trout, Salvelinus fontinalis (12) Goldfish, Carassius auratus (17, 22) Fathead minnow, Pimephales promelas (25) Channel catfish, Ictalurus punctatus (17, 22) Bluegill, Lepomis macrochirus (17, 22) Green sunfish, Lepomis cyanellus (17, 22) Invertebrates Daphnids, Daphnia magna, D. pulex, D. pulicaria (20) Amphipods, Gammarus lacustris, G. fasciatus, G. pseudolimnaeus (17) Crayfish, Orconectes sp., Combarus sp., Procambarus sp., (17, 22) Pacifastacus leniusculus (17) Stoneflies, Pteronarcys sp. (12) Mayflies, Baetis sp., Ephemerella sp. (17) Mayflies, Hexagenia limbata, H. bilineata (22) Midges, Chironomus sp. (22) Snails, Physa integra, P. heterostropha, Amnicola limosa (22) Planaria, Dugesia tigrina (22) Saltwater Vertebrates Sheepshead minnow, Cyprinodon variegatus (22) Mummichog, Fundulus heteroclitus (22) Longnose killifish, Fundulus similis (22) Silverside, Menidia sp. (22) Threespine stickleback, Gasterosteus aculeatus (17) Pinfish, Lagodon rhomboides (22) Spot, Leiostomus xanthurus (22) Shiner perch, Cymatogaster aggregata (12) Tidepool sculpin, Oligocottus maculosus (12) Sanddab, Citharichthys stigmaeus (12) Flounder, Paralichthys dentatus, lethostigma (22) Starry flounder, Platichthys stellatus (12) Table 4.5 (continued) Summary for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians Test type Static, renewal, flow-through © 1999 by CRC Press LLC doubly distilled and filtered before being used to make culture media. Systems that use distilled water supplied by a central system, filtered through an ion exchange system and then glass distilled have proven reliable. Unfortunately, the necessity of using distilled water cuts down on the volumes available for large-scale, flow-through tests systems. Finally, it is important to constantly monitor the quality of the water source. The choice of deionizing or filtering units is also important. Apparently, some resins do leach out small amounts of materials toxic to fish and invertebrates. A positive control using a toxicant with well-known LC 50 values should give an indication of the suitability of the test solutions. Measurement of variables such as hardness, pH, alkalinity, and, in the case of marine systems, salinity, can prevent disasters or unreliable test results. The fish species used in these tests can be far ranging, although the most popular are the fathead minnow (Pimephales promelas), bluegill (Lepomis macrochirus), the channel catfish (Ictalarus punctatus), and the rainbow trout (Oncorhynchus mykiss). Andromonas fish are usually represented by the Coho salmon (O. kisutch). Marine species used are often the sheepshead minnow (Cyprinodon variegatus), mummi- chog (Fundulus heteroclitus), and silversides (Menidia sp.). English sole, Parophrys vetulus (12) Herring, Clupea harengus (12) Invertebrates Copepods, Acartia clausi (12) Acartia tonsa (22) Shrimp, Penaeus setiferus, P. duorarum, P. aztecus (22) Grass shrimp, Palaemonetes pugio, P. intermedius, P. vulgaris (22) Sand shrimp, Crangon septemspinosa (17) Shrimp, Pandalus jordani, P. danae (12) Bay shrimp, Crangon (17) Mysid, Mysidopsis bahia, M. bigelowi, M. almyra (27) Blue crab, Callinectes sapidus (22) Shore crab, Hemigrapsus sp., Pachygrapsus sp. (12) Green crab, Carcinus maenas (22) Fiddler crab, Uca sp. (22) Oyster, Crassostrea virginica, C. gigas (22) Polychaete, Capitella capitata (22) Light quality Not specified Light intensity Not specified Photoperiod 16 h light/8 h dark with a 15 to 30 min transition period Test solution pH Very soft: 6.4–6.9 Soft: 7.2–7.6 Hard: 7.6–8.0 Very hard: 8.0–8.4 DO concentration 60–100% for static test during first 48 h 40–100% for static test after 48 h 60–100% for renewal and flow-through tests (all times) Endpoint Death, immobilization Table 4.5 (continued) Summary for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians Test type Static, renewal, flow-through © 1999 by CRC Press LLC [...]... Brunswick on the east to Alberta on the west (FF of C, 48 0 to 48 2) Channel Catfish (Ictalurus punctatus) Description: Average length is 14 to 21 in (356 to 533 mm), weight is 2 to 4 lb Color: Individuals less than 12 to 14 in (305 to 356 mm) are pale blue to pale olive with silvery overcast; adults with dorsal surface of head and back and upper side © 1999 by CRC Press LLC steel-blue to gray, lower sides... the laboratory Figure 4. 1 illustrates the course of events over the 64 days of the experiment and Table 4. 13 provides a tabular overview The microcosms are prepared by the introduction of 10 algal, 4 invertebrate and 1 bacterial species into 3 l of sterile defined medium Test containers are 4 l glass jars An autoclaved sediment consisting of 200 g silica sand and 0.5 g of ground chitin are autoclaved... migration of other organisms into the test plot Ecosystems ranging from agroecosystems to wetlands have been examined in this manner Compared to the aquatic multispecies toxicity tests the terrestrial systems have not undergone the same level of standardization This is due to the length of time most of these tests require and the specialized nature of most of the test systems rather than any lack of completeness... the number of animals required in a toxicity test Finally, it is often possible to refine the methodology as to require fewer animals Biochemical and physiological indicators of toxicant stress or indications of mechanisms can help to reduce the number of animals or even the need for such testing Although useful, and forming the backbone of most toxicological research, the single-species toxicity test... mosquito larvae This group is probably the most adapted of all aquatic insects The larvae of this group are often used as an indicator of environmental quality Habitats of immatures range from littoral marine waters to mountain torrents, from mangrove swamps to Arctic bogs, and from clear, deep lakes to heavily polluted waters They can be expected in almost all inland waters Most species are bottom-dwelling,... useful is an atlas of malformations making it easier to score the results of the toxicity test Given the relative ease of performing the toxicity test and the supporting documentation, FETAX has found a rapid acceptance as a teratogenicity screen in environmental toxicology © 1999 by CRC Press LLC Table 4. 12 Listing of Current Multispecies Toxicity Tests Aquatic microcosms Benthic-pelagic microcosm... One species of filamentous green alga c) One species of nitrogen-fixing blue-green alga (bacteria) d) One grazing macroinvertebrate e) One benthic, detrital-feeding macroinvertebrate f) Bacteria and protozoa species 1 l beakers covered with a large petri dish 50 ml of acid washed sand sediment and 900 ml of Taub # 82 medium [20], into which 50 ml of inoculum was introduced 4 5 10 ml of stock community... stored in a cooler at 4 C Many SUMMARY This chapter reviewed a wide variety of toxicity tests, yet only a small fraction of the toxicity tests that are currently performed or that exist These tests cover the entire range of biological organization that can be expected to fit into a laboratory or outdoor contained setting There are a few caveats that must be dealt with when dealing with the topic of toxicity... being 50 to 60°F (10.0 to 15.5°C) (FF of C, 1 84 to 191) Brook Trout (Salvelinus fontinalis) Description: Average length is 10 to 12 in (2 54 to 305 mm); breeding males may develop a hook (or kype) at the front of the lower jaw Color: Back is olive-green to dark brown, at times almost black, sides lighter, becoming silvery white below; light green or cream-colored wavy lines or vermiculations on top of head... classed as to period and mode of exposure Two examples of mammalian tests are summarized in Tables 4. 6 and 4. 7 The small mammal toxicity tests were originally and are still used primarily for the extrapolation of toxicity and hazard to humans The advantage of this developmental process is that a great deal of toxicity data does occur for a variety of compounds, both in their structure and their mode of action . 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. 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. 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