Control of fish diseases
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Laboratory Animal Care↗
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Cockroach-proof containers for dry animal feeds
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Laboratory Animal Care↗
Effects of housing density and cage floor space on C57BL/6J mice
The Guide for the Care and Use of Laboratory Animals (the Guide) is widely accepted as the housing standard by most Institutional Animal Care and Use Committees. The recommendations are based on best professional judgment rather than experimental data. Current efforts are directed toward replacing these guidelines with data-driven, species-appropriate standards. Our studies were undertaken to determine the optimum housing density for C57BL/6J mice, the most commonly used inbred mouse strain. Four-week-old mice were housed for 8 weeks at four densities (the recommended ???12 in2 [ca. 77.4 cm 2]/mouse down to 5.6 in2 [ca. 36.1 cm2]/mouse) in three cage types with various amounts of floor space. Housing density did not affect a variety of physiologic parameters but did affect certain micro-environmental parameters, although these remained within accepted ranges. A second study was undertaken housing C57BL/6J mice with as little as 3.2 in2/mouse (ca. 20.6 cm2). The major effect was elevated ammonia concentrations that exceeded limits acceptable in the workplace at increased housing densities; however, the nasal passages and eyeballs of the mice remained microscopically normal. On the basis of these results, we conclude that C57BL/6J mice as large as 29 g may be housed with 5.6 in2 of floor space per mouse. This area is approximately half the floor space recommended in the Guide. The role of the Guide is to ensure that laboratory animals are well treated and housed in a species-appropriate manner. Our data suggest that current policies could be altered in order to provide the optimal habitation conditions matched to this species' social needs. Copyright 2004 by the American Association for Laboratory Animal Science.
Comparative Medicine↗
Outside the box: Working with wildlife in biocontainment
Research with captive wildlife in Animal Biosafety Level 2 (ABSL2) and 3 (ABSL3) facilities is becoming increasingly necessary as emerging and re-emerging diseases involving wildlife have increasing impacts on human, animal, and environmental health. Utilizing wildlife species in a research facility often requires outside the box thinking with specialized knowledge, practices, facilities, and equipment. The USGS National Wildlife Health Center (NWHC) houses an ABSL3 facility dedicated to understanding wildlife diseases and developing tools to mitigate their impacts on animal and human health. This review presents considerations for utilizing captive wildlife for infectious disease studies, including, husbandry, animal welfare, veterinary care, and biosafety. Examples are drawn from primary literature review and collective 40-year experience of the NWHC. Working with wildlife in ABSL2 and ABSL3 facilities differs from laboratory animals in that typical laboratory housing systems, husbandry practices, and biosafety practices are not designed for work with wildlife. This requires thoughtful adaptation of standard equipment and practices, invention of customized solutions and development of appropriate enrichment plans using the natural history of the species and the microbiological characteristics of introduced and native pathogens. Ultimately, this task requires critical risk assessment, understanding of the physical and psychological needs of diverse species, creativity, innovation, and flexibility. Finally, continual reassessment and improvement are imperative in this constantly changing specialty area of infectious disease and environmental hazard research.
ILAR Journal↗
Rapid diagnosis of avian influenza virus in wild birds: Use of a portable rRT-PCR and freeze-dried reagents in the field
Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal. Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene. The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.
Journal of Visualized Experiments↗
Effects of antenna length and material on output power and detection of miniature radio transmitters
The optimal antenna of transmitters used in small aquatic animals is often a compromise between efficient radio wave propagation and effects on animal behavior. Radio transmission efficiency generally increases with diameter and length of the conductor, but increased antenna length or weight can adversely affect animal behavior. We evaluated the effects of changing antenna length and material on the subsequent tag output power, reception, and detection of tagged fish. In a laboratory, we compared the relative signal strengths in water of 150 MHz transmitters over a range of antenna lengths (from 6 to 30 cm) and materials (one weighing about half of the other). The peak relative signal strengths were at 20 and 22 cm, which are approximately one wavelength underwater at the test frequency. The peak relative signal strengths at these lengths were approximately 50% greater than those of 30 cm antennas, a length commonly used in fisheries research. Few significant differences were present in distances for the operator to hear or the telemetry receiver to decode transmitters from a boat-mounted receiving system based on antenna length, but the percent of tagged fish detected passing a hydroelectric dam fitted with an array of receiving systems was significantly greater at the antenna length with peak output power in laboratory tests. This study indicates careful choice of antenna length and material of small transmitters can be used to reduce weight and possible antenna effects on animal behavior, to maximize tag output power and detection, or to balance these factors based on the needs of the application. ?? 2007 Springer Science+Business Media B.V.
Conference Paper↗
Review of concepts useful for maintaining quality of male reproductive field samples for laboratory study
Investigations into cellular and molecular characteristics of male gametes obtained from fish in natural ecosystems require careful sample handling and shipping in order to minimize artifacts. Maintaining sample integrity engenders confident assessments of ecosystem health, whereby animal condition is often reflected by gamete biomarkers - indicators that respond in measurable ways to changes. A number of our investigations have addressed the hypothesis that biomarkers from fish along a pollution gradient are reflective of site location. Species biology and the selected biological endpoints direct choice of parameters such as: temperature, buffer osmolality, time in transit, fixation, cryoprotectants, protease inhibition, and antibiotic inclusion in extender. This paper will highlight case studies, and outline parameters and thoughts on approaches for use by field and laboratory researchers.
Conference Paper↗
Perspectives on animal welfare legislation and study considerations for field-oriented studies of raptors in the United States
Concern for the welfare of animals used in research and teaching has increased over the last 50 yr. Animal welfare legislation has resulted in guidelines for the use of animals in research, but the guidelines can be problematic because they focus on animals used in laboratory and agriculture research. Raptor biologists can be constrained by guidelines, restrictions, and oversight that were not intended for field research methods or wild animals in the wild or captivity. Field researchers can be further hampered by not understanding animal welfare legislation, who is subject to oversight, or that oversight is often provided by a committee consisting primarily of scientists who work with laboratory animals. Raptor researchers in particular may experience difficulty obtaining approval due to use of various species-specific trapping and handling methods. We provide a brief review of animal welfare legislation and describe the basic components and responsibilities of an Institutional Animal Care and Use Committee (IACUC) in the United States. We identify topics in raptor research that are especially problematic to obtaining IACUC approval, and we provide insight on how to address these issues. Finally, we suggest that all raptor researchers, regardless of legal requirements, abide by the spirit of the animal welfare principles. Failure to do so may bring about further regulatory and permitting restrictions. ?? 2010 The Raptor Research Foundation, Inc.
Journal of Raptor Research↗
The humane capture, handling, and disposition of migratory birds
The purpose of this document is to provide guidance on the humane capture, handling, and care of migratory birds trapped for any purpose. It is intended for wildlife managers, regulatory personnel, and individuals that handle or authorize handling of live-captured migratory birds. The U.S. Fish and Wildlife Service – Migratory Bird Program (USFWS), the U.S. Geological Survey Bird Banding Laboratory (BBL), and U.S. Department of Agriculture Animal Plant Health Inspection Service Wildlife Services (WS) collaboratively prepared this document.
Final Report↗
Guidelines for use of fishes in research
The 2004 and 2014 Guidelines were developed to provide a structure that advances appropriate attention toward valid experimental designs and procedures with aquatic animals while ensuring humane treatment of the experimental subjects. At a practical level, the Guidelines are intended to provide general recommendations on field and laboratory endeavors, such as sampling, holding, and handling fishes; to offer information on administrative matters, including regulations and permits; and to address typical ethical concerns, such as perceptions of pain or discomfort experienced by experimental subjects. These Guidelines must be recognized as guidelines . They are not intended to provide detailed instructions but rather to alert investigators to a broad array of topics and concerns to consider prior to initiating study. At a comprehensive level, the principles upon which these Guidelines are based are broadly applicable, and many of the described practices and approaches can be adapted to situations involving other aquatic animal species and conditions. Understanding the differences between fishes and other vertebrates, especially mammals, is critically important to conducting scientifically sound research with fishes. Disparities in life histories and mortality rates in fishes versus other vertebrates are critical in designing sustainable sampling levels in fish populations. The UFR Committee points out that (1) compared to mammalian populations, adult populations of many fish species persist despite very high natural mortality rates in juvenile stages by virtue of the fact that most species lay thousands or tens of thousands of eggs; (2) because of these mortality patterns, research on fishes, especially field research or research on early life stages, can involve, and often requires, much larger numbers of research subjects than does research on mammals; and (3) the animal handling and husbandry requirements for fishes are fundamentally different from those for mammals and other vertebrates, in general. Policies, regulations, and recommendations developed for research on mammals, birds, reptiles, or even amphibians are frequently inappropriate for research with fishes. The Guidelines also address some of the ethical concerns that motivate guidelines used for research with other vertebrates, while being mindful of the unique physiology and general nature of fishes. The Guidelines were developed for general use by investigators within the United States; therefore, the roles, responsibilities, and informational needs of Institutional Animal Care and Use Committees (IACUCs) were given specific attention. All United States institutions that use vertebrate animals for research, teaching, research training, and biological testing are required to create an IACUC to oversee and evaluate all aspects of the institution’s animal care and use program. Investigators from other nations who read this document may disregard specific references to U.S. state and federal laws and regulations, as their institutional infrastructure and processes may differ from those of an internal committee such as IACUCs. The principles described herein, however, are applicable to research on fishes regardless of geographic location. Investigators in other nations may benefit by modifying any of the specific provisions pertaining to the United States, thereby adopting guidelines consistent with the laws and regulations of their own government. The UFR Committee urges that the Guidelines be endorsed and adopted (adapted, where necessary) by those state and federal authorities with regulatory responsibilities for fishes, offices with federal oversight (e.g., National Institutes of Health, Office of Laboratory Animal Welfare; http://grants.nih.gov/grants/olaw/olaw.htm ) as well as by universities and other institutions and authorities using fishes and aquatic animals within their research and teaching programs.
Report↗
Bureau of Sport Fisheries and Wildlife Pesticide-Wildlife Review: 1959
Research findings of the Bureau of Sport Fisheries and Wildlife, State agencies and independent research workers in Ala., Ark., Fla., Ga., La., Mass., Mich., Mont., N. Dak., Tex., and Wis. are summarized in this report together with recommendations for reducing damage from pest control operations. Major topics discussed are: Scope of Pesticide-Wildlife Problem; Effects on Wildlife-General; Laboratory Studies and Toxicology; Direct and Indirect Effects of Pesticides on Wildlife; Recent Pesticide Legislation; Value of Wildlife; and Recommendations for Safeguarding Wildlife Values during Pest Control. To avoid undue hazards to wildlife, applications must not exceed the toxicity equivalent of the following concentrations of DDT to the respective forms of wildlife: 0.1 pounds of DDT/acre for crustaceans; 0.2 for fish; 1.0 for amphibians; 2.0 for reptiles and birds; and 5.0 for most mammals. Other suggestions are: 1) Chemical treatment should be used only when entomological research has proved it to be necessary; 2) Before pesticides are used, the effects on different kinds of animals and on animals living in different habitats should be known and carefully considered; 3) Only minimum quantities of chemicals necessary to achieve adequate control of pests should be applied; 4) Pesticides should not be applied to areas that are any larger than is necessary and the chemicals that are used should be the ones whose effects are no more long-lasting than necessary; 5) Whenever possible, chemicals should be applied at the seasons of the year when wildlife damage will be least; 6) Conscientious effort should be made to be sure that pesticides are applied at no more than the intended rates and that no areas receive double doses. Alternates to chemical control are suggested. Among these are biological control, modified agricultural practices, destruction of insect wintering quarters, and the manipulation of water levels.
Circular↗
Antibiotic, pharmaceutical, and wastewater-compound data for Michigan, 1998-2005
Beginning in the late 1990's, the U.S. Geological Survey began to develop analytical methods to detect, at concentrations less than 1 microgram per liter (ug/L), emerging water contaminants such as pharmaceuticals, personal-care chemicals, and a variety of other chemicals associated with various human and animal sources. During 1998-2005, the U.S. Geological Survey analyzed the following Michigan water samples: 41 samples for antibiotic compounds, 28 samples for pharmaceutical compounds, 46 unfiltered samples for wastewater compounds (dissolved and suspended compounds), and 113 filtered samples for wastewater compounds (dissolved constituents only). The purpose of this report is to summarize the status of emerging contaminants in Michigan waters based on data from several different project-specific sample-collection efforts in Michigan during an 8-year period. During the course of the 8-year sampling effort, antibiotics were determined at 20 surface-water sites and 2 groundwater sites, pharmaceuticals were determined at 11 surface-water sites, wastewater compounds in unfiltered water were determined at 31 surface-water sites, and wastewater compounds in filtered water were determined at 40 surface-water and 4 groundwater sites. Some sites were visited only once, but others were visited multiple times. A variety of quality-assurance samples also were collected. This report describes the analytical methods used, describes the variations in analytical methods and reporting levels during the 8-year period, and summarizes all data using current (2009) reporting criteria. Very few chemicals were detected at concentrations greater than current laboratory reporting levels, which currently vary from a low of 0.005 ug/L for some antibiotics to 5 ug/L for some wastewater compounds. Nevertheless, 10 of 51 chemicals in the antibiotics analysis, 9 of 14 chemicals in the pharmaceuticals analysis, 34 of 67 chemicals in the unfiltered-wastewater analysis, and 56 of 62 chemicals in the filtered-wastewater analysis were detected. Antibiotics were detected at 7 of 20 tested surface-water sites, but none were detected in 2 groundwater samples. Pharmaceuticals were detected at 7 of 11 surface-water sites. Wastewater compounds were detected at 25 of 31 sites for which unfiltered water samples were analyzed and at least once at all 40 surface-water sites and all 4 groundwater sites for which filtered water samples were analyzed. Overall, the chemicals detected most frequently in Michigan waters were similar to those reported frequently in other studies nationwide. Patterns of chemical detections were site specific and appear to be related to local sources, overall land use, and hydrologic conditions at the time of sampling. Field-blank results provide important information for the design of future sampling programs in Michigan and demonstrate the need for careful field-study design. Field-replicate results indicated substantial confidence regarding the presence or absence of the many chemicals tested. Overall, data reported herein indicate that a wide array of antibiotic, pharmaceutical, and organic wastewater compounds occur in Michigan waters. Patterns of occurrence, with respect to hydrologic, land use, and source variables, generally appear to be similar for Michigan as for other sampled waters across the United States. The data reported herein can serve as a basis for future studies in Michigan.
Michigan↗
Guidelines for use of fishes in research: Revised and expanded
The Guidelines for the Use of Fishes in Research (2014; 2014 Guidelines ), now available through the American fisheries Society (AFS) website and in print from the AFS bookstore, is a resource to aid researchers and regulatory authorities regarding responsible, scientifically valid research on fish and aquatic wildlife. The Guidelines for the Use of Fishes in Field Research (American Society of Ichthyologists and Herpetologists [ASIH] et al. 1987, 1988) emphasized field research and was followed by the 2004 Guidelines including laboratory research topics. Each version of the Guidelines has been jointly endorsed and/or published by the ASIH, the American Institute of Fishery Research Biologists (AIFRB), and AFS--each focusing on the scientific understanding, global conservation, and sustainability of aquatic animals, fisheries, and ecosystems. Changes with time necessitate revisions to make the Guidelines consistent with contemporary practices and scientific literature so to remain relevant as a technical resource. This document provides not only general principles relevant for field and laboratory research endeavors but includes specific requirements for researchers working within the United States and outside of the country. Within the scope of their expertise, the 2014 Uses of Fishers in Research (UFR) Committee members updated and revised sections, resulting in a 90-page 2014 Guidelines having undergone through peer review. As before, topical areas were addressed (see Table of Contents on page 416). Expanded coverage was provided on U.S. and international agencies and programs relevant to research with fishes. The Surgical Procedures and the Marking and Tagging section received special focus by a UFR Subcommittee. Feeds and Feeding and the Administration of Drugs, Biologics and Other Chemicals are just some of the newly added topics. The 2014 Guidelines is user-friendly by way of hyperlinks to external Internet sites, intradocument sections, and tables of acronyms with corresponding terms, low regulatory priority drugs, and Office of International des Epizooties notifiable disease agents. Again, the Institutional Animal Care and Use Committee (IACUC) role is explained, expectations for research are provided, and a brief checklist for IACUC readiness is included. Overall, the ,i>2014 Guidelines is the taxon-specific resource for our professional societies and is a principal document for standards on the care and use of fish and aquatic vertebrates in research.
Fisheries↗
Assessment of general health of fishes collected at selected sites in the Great Lakes Basin In 2012
During the past decade, there has been a substantive increase in the detection of “emerging contaminants”, defined as a new substance, chemical, or metabolite in the environment; or a legacy substance with a newly expanded distribution, altered release, or a newly recognized effect (such as endocrine disruption). Emerging contaminants include substances such as biogenic hormones (human and animal), brominated flame retardants, pharmaceuticals, personal care products, plasticizers, current use pesticides, detergents, and nanoparticles. These contaminants are frequently not regulated or inadequately regulated by state or Federal water quality programs. Information about the toxicity of these substances to fish and wildlife resources is generally limited, compared to more highly regulated contaminants, and some classes have been shown to cause affects (for example feminization of male fish, immunomodulation) that are not evaluated via traditional toxicity testing protocols. As a result, these compounds may pose a substantial, but currently poorly documented threat to aquatic ecosystems. Failure to identify and understand the impacts of these emerging contaminants on fish and wildlife resources may result in deleterious impacts to Great Lakes resources that can result in adverse ecological, economic and recreational consequences. The U. S. Fish and Wildlife Service received funding through the Great Lakes Restoration Initiative (GLRI) for an Early Warning Program to detect and identify emerging contaminants and to evaluate the effects of these contaminants on fish and wildlife. The U.S. Geological Survey (WV Cooperative Fish and Wildlife Research Unit and National Fish Health Research Laboratory, Leetown Science Center) developed and implemented a biological effects monitoring protocol to assist in this program. Fish collections and measurements of biomarkers of exposure in Fall 2010 and Spring 2011 occurred at individual sites within select Areas of Concern (AOCs). They provided an assessment of the utility of the suite of biomarkers and also identified sites for more in-depth analyses. Selected areas are characterized as areas with known emerging contaminants, sensitive or listed species, areas downstream from municipal wastewater discharges or receiving waters for industrial facilities, and/or areas susceptible to agricultural or urban contamination, or harbors or ports. The results of the 2010- 2011 studies were summarized in Blazer et al. 2014 a, b, c; Braham et al. in review and Blazer et al. in review.
Great Lakes↗
A standard operating procedure for the surgical implantation of transmitters in juvenile salmonids
Biotelemetry is a useful tool to monitor the movements of animals and is widely applied in fisheries research. Radio or acoustic technology can be used, depending on the study design and the environmental conditions in the study area. A broad definition of telemetry also includes the use of Passive Integrated Transponder (PIT) tags, either separately or with a radio or acoustic transmitter. To use telemetry, fish must be equipped with a transmitter. Although there are several attachment procedures available, surgical implantation of transmitters in the abdominal cavity is recognized as the best technique for long-term telemetry studies in general (Stasko and Pincock, 1977; Winter, 1996; Jepsen, 2003), and specifically for juvenile salmonids, Oncorhynchus spp. (Adams and others, 1998a, 1998b; Martinelli and others, 1998; Hall and others, 2009). Studies that use telemetry assume that the processes by which the animals are captured, handled, and tagged, as well as the act of carrying the transmitter, will have minimal effect on their behavior and performance. This assumption, commonly stated as a lack of transmitter effects, must be valid if telemetry studies are to describe accurately the movements and behavior of an entire population of interest, rather than the subset of that population that carries transmitters. This document describes a standard operating procedure (SOP) for surgical implantation of radio or acoustic transmitters in juvenile salmonids. The procedures were developed from a broad base of published information, laboratory experiments, and practical experience in tagging thousands of fish for numerous studies of juvenile salmon movements near Columbia River and Snake River hydroelectric dams. Staff from the Western Fisheries Research Center's Columbia River Research Laboratory (CRRL) frequently have used telemetry studies to evaluate new structures or operations at hydroelectric dams in the Columbia River Basin, and these evaluations typically require large numbers of tagged fish. For example, a study conducted at the dams on the Columbia River and funded by the U.S. Army Corps of Engineers required tagging and monitoring of 40,000 juvenile salmon during a 3-month migration period (Counihan and others, 2006a, 2006b; Perry and others, 2006). To meet the demands of such a large study, the authors and CRRL staff refined the SOP to increase efficiency in the tagging process while maintaining high standards of fish care. The SOP has been used in laboratory and field settings for more than 15 years, and consistently has produced low mortality rates (<1 percent) and transmitter loss rates (<0.01 percent) in the 24-36 hours after tagging. In addition to describing the detailed surgical procedures required for transmitter implantation, this document provides guidance on fish collection, handling and holding, and the release of tagged fish. Although often overlooked, or at least underemphasized, these processes can have a large impact on the outcome of the tagging procedure. Stress associated with the individual steps in handling and tagging can be cumulative and lethal (Maule and others, 1988; Wedemeyer and others, 1990; Portz and others, 2006), so the goal is to provide the best possible fish care at every step in order to manage the overall effect on study fish.
Open-File Report↗
Handbook of acute toxicity of chemicals to fish and aquatic invertebrates : summaries of toxicity tests conducted at Columbia National Fisheries Research Laboratory, 1965-78
Acute toxicity is a major subject of research at Columbia National Fisheries Research Laboratory for evaluating the impact of toxic chemicals on fishery resources. The Laboratory has played a leading role in developing research technology for toxicity testing and data interpretation. In 1965-78, more than 400 chemicals were tested against a variety of invertebrates and fish species representative of both cold- and warm-water climates. The use of acute toxicity tests for assessing the potential hazard of chemical contaminants to aquatic organisms is well documented (Boyd 1957; Henderson et al. 1960; Sanders and Cope 1966; Macek and McAllister 1970). Static acute toxicity tests provide rapid and (within limits) reproducible concentration-response curves for estimating toxic effects of chemicals on aquatic organisms. These tests provide a database for determining relative toxicity of a large number of chemicals to a variety of species and for estimating acute effects of chemical spills on natural aquatic systems; they also assist in determining priority and design of additional toxicity studies. Acute toxicity tests usually provide estimates of the exposure concentration causing 50% mortality (LC50) to test organisms during a specified period of time. For certain invertebrates, the effective concentration is based on immobilization, or some other identifiable endpoint, rather than on lethality. The application of the LC50 has gained acceptance among toxicologists and is generally the most highly rated test for assessing potential adverse effects of chemical contaminants to aquatic life (Brungs and Mount 1978; American Institute for Biological Sciences 1978 a ). The literature contains numerous papers dealing with the acute toxicity of chemicals to freshwater organisms. However, there is a tremendous need for a concise compendium of toxicity data covering a large variety of chemicals and test species. This Handbook is a compilation of a large volume of acute toxicity data from the Columbia Laboratory and its field laboratories. It presents definitive acute toxicity data on 271 chemicals tested against a variety of freshwater invertebrates and fishes. The chemicals represent all major groups of pesticides, as well as numerous industrial chemicals. This compilation should serve as a useful database for the many agencies and organizations dealing with research and management programs concerned with the impact of chemicals on aquatic resources. The Columbia Laboratory has played a major role in developing currently used standard methodology for static acute toxicity testing. The use of standardized methodology greatly reduces variation in results. The data presented here have been carefully scrutinized to eliminate tests that failed to follow acceptable procedures. Handling of test organisms and procedures for static toxicity tests followed those described by Lennon and Walker (1964) and Macek and McAllister (1970), and conform well with those recommended by Brauhn and Schoettger (1975) and the Committee on Methods for Toxicity Tests with Aquatic Organisms (1975). The species of fish and invertebrates that were tested are listed in phylogenetic order in Tables 1 and 2. Fish were obtained from Federal and State hatcheries as either eggs or fry. Original stocks of invertebrates were collected and cultured from wild populations with no known source of contamination; these populations were replenished regularly. The invertebrates were cultured in the Laboratory by methods similar to those described by Sanders and Cope (1966). Test chemicals usually consisted of technical or analytical grade samples of known purity. Formulations of the chemicals were also tested when available. When purity of test chemicals was known, all calculated concentrations were based on percent active ingredients. Stock solutions were prepared immediately before each test, with commercial grade acetone as the carrier solvent. Occasionally, ethanol or dimethyl-formamide was substituted. Solvent concentrations did not exceed 0.5 mL/L in final dilution water. Test water (dilution water) was reconstituted from deionized water of at least 10 6 ohms resistivity by the addition of appropriate reagent grade chemicals (Marking 1969). Water was buffered to maintain a pH of 7.2 to 7.5, an alkalinity of 30 to 35 mg/L, and a hardness of 40 to 50 mg/L as CaCO 3 . Test water was mixed thoroughly and aerated before transfer into test chambers. Fish were acclimated to dilution water by gradually changing the water in acclimated tanks from 100% well water to 100% reconstituted water over a 1- to 3-day period at the desired testing temperature. Invertebrates were acclimated from well water to dilution water over a 4- to 6-h period. Toxicity tests were conducted under static conditions without aeration, and the organisms were not fed during acclimation or testing. Temperature of test solutions was maintained within ± 1°C of that required for a given test. Toxicity tests with fish were conducted in 18.9-liter (5-gal) wide-mouthed jars containing 15 liters of test solution. Fingerling fish weighing 0.2 to 1.5 g were tested at each concentration. Caution was taken not to exceed 0.8 g of test organisms per liter of solution. Duplicate test chambers were used to accommodate larger fish. Test chambers varied in size for invertebrates, depending on the species used; volume of test solution ranged from 0.25 to 4 liters. At least 10 organisms were exposed to each concentration for all definitive tests. At least six concentrations were used per toxicity test. The tests began upon initial exposure to the toxicant and continued for 96 h. Immobilization tests with invertebrates were conducted for only 48 h. The number of dead or affected organisms in each test chamber were recorded and the dead organisms were removed every 24 h; general observations on the condition of test organisms were also recorded at these times. Toxicity data were analyzed by a statistical method described by Litchfield and Wilcoxon (1949) to determine LC50 (theoretical estimate of the concentration lethal to 50% of the test animals) and 95% confidence intervals. This method is recommended by the American Public Health Association (1971) and by Sprague (1969) for determining median lethal concentrations. The procedure is easily modified for computing a single LC50 when replicate tests are performed.
Resource Publication↗