Title: Introduction Text: Amphibian species have experienced worldwide declines in populations in recent years. Several factors are involved including habitat destruction, exotic species introduction, and the contamination of surface water and sediments in wetland habitats. Because of their sensitivity to environmental conditions, amphibians can be used as indicator species in the assessment of the ecological health of a wetland. The purpose of this tool is to highlight a new laboratory toxicity test developed by NAVFAC that can be used to evaluate potential risks to amphibians from contaminants in wetland sediments. This test should help the Navy to negotiate more realistic sediment cleanup goals that are based on actual measurements of ecotoxicity and to avoid costly and unnecessary wetland alteration. Visual Description: Picture of a wetland and books about amphibians.

Title: Biology of Amphibians Text: Amphibian taxonomy includes the Kingdom Animalia, Phylum Chordata, Sub-Phylum Vertebrata, and Class Amphibian. There are over 190 species of amphibians in North America from two of the three major amphibian groups including: Salamanders of the order Caudata, which range in length from 6 inches to over 3 feet. All are either strictly aquatic or at least semi-aquatic. Frogs and toads of the order Anura, which are four-legged tailless amphibians. They are usually found in moist or aquatic habitats for at least a portion of their life history. Visual Description: Pictures of different amphibians including frogs, toads, and salamanders.

Title: Amphibian Physiology Text: All amphibians are ectothermic and possess moist permeable skin for oxygen exchange. Visual Description: Graphic of a frog which zooms in on its skin with arrows showing oxygen going in and out.

Title: Ectothermic Text: Amphibians cannot physiologically elevate their body temperature and must do so by regulating their activity type and duration. However, they are able to physiologically lower their body temperature when necessary through evaporative cooling. As a result, amphibians may be both diurnally and nocturnally active as they modify their temporal behavior in order to regulate body temperature.

Title: Moist Permeable Skin Text: Amphibians primarily conduct gaseous exchange through the skin. The skin is thin, highly permeable, and in part breathes for the organisms. Chemical transport occurs readily across the skin. Some amphibians retain gills throughout their lifetime, some are lungless and breathe air through their skin, and others develop lungs for air breathing as adults. These differences and other physiological traits such as glandular/mucus excretions vary the amount of liquid exchange across the skin. This permeability maintains the organisms balance in nature, but also creates the potential for bioaccumulation and intensifies the risk of contaminant exposure for amphibians.

Title: Amphibian Breeding Ecology (1 of 2) Text: An understanding of amphibian breeding behavior is critical to understanding their role as indicator species. Most species of amphibians have a complex, biphasic life cycle. Breeding behavior within subpopulations is generally synchronized in onset and duration due to environmental and seasonal regulation. As a result, entire amphibian populations are potentially at risk from contaminant exposure in breeding areas. For most species, reproduction generally occurs via external fertilization. Visual Description: Picture of an amphibian's eggs.

Title: Amphibian Breeding Ecology (2 of 2) Text: The successful development of amphibian eggs and larvae is hindered by several natural and anthropogenic factors. Natural predation, parasites, and disease can impact the development of amphibian eggs and larvae. However, it has been shown that polychlorinated biphenyls (PCBs), pesticides, acid rain, and ultraviolet-B (UV-B) radiation also have a direct adverse impact on amphibian development. Furthermore, anthropogenic activities may also indirectly affect amphibian development by altering the natural flora surrounding or within water bodies in turn increasing exposure to UV-B radiation, altering the pH, lowering dissolved oxygen levels, and varying food availability. Visual Description: Figure of plankton, fish, frogs, eggs, and a heron being affected by the contaminated sediment in a wetland.

Title: Biphasic Life Cycle Text: Anurans exhibit a biphasic (two-phase) life cycle consisting of aquatic egg and larval stages followed by metamorphosis into a terrestrial juvenile. The life history begins as the tadpole emerges from the egg, breathing oxygen through its gills. Most amphibians change into terrestrial adults. The adult breaths through lungs and the moist outer layer of skin. Every spring, amphibians emerge from hibernation to breed in wetlands. They typically migrate in and out of the aquatic environment to breed. The biphasic lifestyle of amphibians makes them especially vulnerable to impacted air, water, or land.

Title: External Fertilization Text: Females deposit eggs at or near the water surface, where they are subsequently fertilized by the males. The eggs may be laid in mass, chains, small clumps, or singly attached to aquatic vegetation depending on the species. The purpose of depositing the eggs near the surface is to either warm the eggs by solar radiation for early spring breeders or to expose them to oxygen for mid-summer breeders, when eutrophication is most likely to occur.

Title: Eutrophication Text: Eutrophication is the process by which a body of water becomes enriched in dissolved nutrients that stimulate the growth of aquatic plant life. This typically results in the depletion of dissolved oxygen.

Title: Metamorphosis Text: Metamorphosis represents a critical stage in the amphibian life cycle and is accompanied by numerous complex physiological and anatomical changes. Completion of metamorphosis in larval anurans is characterized by the re-absorption of the tail. The juvenile then physically resembles the adult form. However, some amphibians directly develop from the embryonic stage into adult form without metamorphosis. Others remain aquatic for the duration of their life. More Info Visual Description: Graphic of the metamorphosis of a tadpole into a frog.

Title: Metamorphosis Text: Chemicals in the wetland environment can impair the successful metamorphosis of the amphibian larvae to adult form. Metamorphosis has the potential to mobilize stored energy reserves, along with contaminants that have accumulated over time within these reserves. However, the full extent of the impact that contaminants have on the metamorphic process is relatively unknown.

Title: Habitat Use Text: Amphibians can be found in a variety of habitats in and around wetlands throughout their complex life cycle. Freshwater wetlands serve as an important transition zone between terrestrial uplands and surface water bodies and generally act as a sink for many chemical contaminants. Most amphibians begin their early life in a submerged aquatic environment. During these critical early stages of life, they may be exposed to contaminants present in the sediment and surface water of the wetland. Some amphibian species gradually metamorphose into air breathing adults. The adult amphibian’s habitat ranges from terrestrial to aquatic ecosystems. During adulthood, they may be exposed to contaminants present in the atmosphere, sediment, soil, surface water, and vegetation in their habitat. Visual Description: Pictures of amphibians (frogs and toads) in aquatic and terrestrial habitats.

Title: Amphibian Trophic Status (1 of 3) Text: Amphibians are an important factor in the trophic balance of wetlands serving as both predator and prey to a variety of organisms. Larval stages and tadpoles are large consumers of algae, periphyton, and plankton. Juvenile and adult amphibians are carnivorous and primarily feed on insects, worms, terrestrial and aquatic invertebrates. Some larger amphibian species may also eat small rodents, birds, snakes, or other amphibians. Visual Description: Pictures of amphibian food sources.

Title: Trophic Text: Involving the feeding habits or food relationship of different organisms in a food chain.

Title: Amphibian Trophic Status (2 of 3) Text: Amphibians of all life-stages are a major component of the diet for many predatory vertebrates. The major vertebrate predators of amphibians include mammals such as raccoons, and opossums, birds such as herons and raptors, fish, and some snake species. Adult invertebrates such as crayfish and other arthropods also consume the eggs and larvae stages of many amphibian species. Visual Description: Pictures of amphibian predators including a raccoon, heron, fish, crayfish, and a snake.

Title: Amphibian Trophic Status (3 of 3) Text: The introduction of contaminants into the environment has the potential to disrupt the trophic balance by interfering with the health of prey or predator populations. As an intermediary link in the food web, amphibians can concentrate or bioaccumulate contaminants and transfer them up the food chain to their predators. This magnified risk to top predators not only threatens the health of that single population, but also poses a risk to community diversity. In addition, contaminants concentrated in amphibian tissues may be passed on to their offspring. These contaminants may reduce the likelihood of proper development and directly or indirectly affect the viability of the offspring to survive and successfully reproduce. Visual Description: Figure of a worm, frog, heron, and fox in a food chain.

Title: Navy Assessments and Cleanups Text: The environmental restoration of Navy sites typically involves the determination of cleanup goals through a risk assessment process. Often, it is necessary to determine acceptable contaminant concentrations or cleanup goals that will be protective of nearby ecologically sensitive areas. Ecological risk-based cleanup goals are typically developed using methodologies outlined in Department of Defense (DoD) and other guidance documents. The development of risk-based cleanup goals allows a quantification of the trade-off between costly and potentially environmentally destructive remediation and leaving residual contamination in place. This trade-off is critical in wetland environments because considerable attention has been paid to wetland losses in our nation and remediation near wetland areas is typically environmentally destructive and costly.

Title: Navy Wetlands are a Prime Habitat for Amphibians Text: Wetland habitats often form the majority of open space at Naval facilities. This phenomenon is illustrated at the Naval Air Station South Weymouth in Massachusetts, where palustrine wetlands comprise approximately 40% of the 1,400 acre facility. Visual Description: Figure of the continental united states. Massachusetts advances with South Weymouth NAS identified.

Title: Palustrine Wetland Text: Palustrine comes from the Latin word "palus" or marsh. Wetlands within this category includes all nontidal wetlands dominated by trees, shrubs, persistent emergents, emergent mosses or lichens, and all such wetlands that occur in tidal areas where salinity due to ocean-derived salts is below 0.5%. It also includes some wetlands lacking such vegetation, but with all of the following four characteristics: area less than 20 acres active wave-formed or bedrock shoreline features lacking water depth in the deepest part of basin less than 2 meters at low water salinity due to ocean derived salts less than 0.5%

Title: Amphibians as Ecological Indicators in Wetlands Text: Amphibians are considered to be an indicator species for the ecological health of a given habitat. Although many species exhibit a response to environmental stressors, the combination of their biphasic life cycle and unique physiology make amphibians an excellent gauge of ecosystem health. Visual Description: Picture of a wetland.

Title: Appropriate Use of Ecological Endpoints Text: Amphibians play a key ecological role in most wetlands. However, because of the limited availability of chronic ecotoxicity data for amphibians, environmentally acceptable endpoints for environmental restoration are often based on data from more sensitive aquatic species. These sensitive species include fathead minnows and daphnids. These aquatic species have been widely studied, but may not even inhabit the wetland environment at a given site. The inappropriate use of these aquatic species may complicate ecological risk-based management decisions at Navy sites. As a result of using overly sensitive aquatic species that are not representative of wetlands habitats to make risk-based decisions, the Navy runs the risk of remediating wetlands when no remediation is required. This overestimate of risk could also result in potentially disruptive wetland alterations. Conversely, at some sites, the tests with nonwetland aquatic species that do not have similar life stages could lead to the conclusion that no unacceptable risks exist at the site. However, early life stage amphibians at the site may be still be at risk.

Title: Endpoint Text: An endpoint is a measurable characteristic of a population, species, or group that is affected by an environmental stressor or contaminant. Examples include mortality, reproductive impairment, growth impairment, behavioral changes, and other effects. Refer to Section 2.3 of this link for more information on ecological endpoints.

Title: Amphibian State of the Science Text: NAVFAC's research into this topic began with a literature review of both surface water and sediment toxicity screening levels, with a focus on the following list of 11 contaminants: Cadmium Chromium Copper Lead Mercury Nickel Zinc PCBs 4,4-DDT PAHs Ordnance/explosive compounds

Title: Main Amphibian Toxicology Databases Text: Two recently published compilations of amphibian ecotoxicity data were the main sources utilized to establish the state of the science on amphibian ecotoxicity studies (these studies are summarized on the next page): Ecotoxicology of Amphibians and Reptiles This resource, published by the Society of Environmental Toxicology and Chemistry (SETAC), provides summaries of several studies that have been conducted with amphibians exposed to a variety of contaminants. RATL Database Database of Reptile and Amphibian Toxicology Literature (RATL). This resource, published by the Canadian Wildlife Service as a Technical Report, contains numerous data extracted from primary literature for reptiles and amphibians. Visual Description: Screenshots of amphibian toxicology databases.

Title: Database References Text: SETAC Sparling, D.W. Ecotoxicology of Organic Contaminants to Amphibians. In: Ecotoxicology of Amphibians and Reptiles. Eds. D.W. Sparling, G. Linder and C.A. Bishop. SETAC Technical Publication RATL Pauli, B.D., J.A. Perrault, and S.L. Money, 2000. RATL: A Database of Reptile and Amphibian Toxicology Literature. National Wildlife Research Centre 2000, Canadian Wildlife Service, Environmental Conservation Branch. Technical Report Series Number 357.

Title: Data Limitations Text: The primary objective of NAVFAC's research project was to develop a standardized approach or test method for assessing the risk to amphibians from contaminated sediments. The existing test methods and literature related to amphibians focused primarily on the impacts associated with exposure to contaminated surface water and not contaminated sediment. In addition, a variety of species, endpoints, and testing durations have been used to evaluate effects. In general, there is a lack of consistent information regarding the potential toxicological impacts to amphibians from contaminated sediments or hydric soils in wetlands. Click here for references for the main sources utilized for surface water toxicity study information. Visual Description: Table summarizing surface water toxicity studies for cadmium, chromium, copper, lead, mercury, nickel, zinc, DDT, PAHs, and PCBs.

Title: Comparison of Amphibian Toxicology Data to AWQC Text: Amphibian thresholds are generally much higher than Ambient Water Quality Criteria (AWQC); however, it is important to recognize that this evaluation considered only lethal effects data. It is possible that the results would differ markedly for sub-lethal effects data, or if exposure duration and life stage data were considered. Click here for information on the calculated values included in the table. It is apparent that there is insufficient data available to develop numerical surface water and sediment quality screening values for amphibians for the contaminants evaluated. Few bulk sediment or interstitial water studies with amphibians were found in the literature. Visual Description: Table comparing surface water screening benchmarks to calculated centiles for cadmium, copper, mercury, zinc, and DDT.

Title: AWQC Text: Few nationally accepted values are currently available for conducting screening level Ecological Risk Assessments (ERAs). Exceptions are the EPA (1986) AWQC, which identify chronic and acute water concentrations considered to be protective of freshwater and marine aquatic biota. These values may be used directly as screening ecotoxicity values (SEVs) for calculating aquatic biota hazard quotients (HQs): the ratio of the exposure estimate to an effects concentration considered to represent a “safe” environmental concentration or dose.

Title: Calculation of 10th and 50th centile Text: These five contaminants were selected for further evaluation of lethal effects data because they had the most complete data sets available in the scientific literature review. In order to establish preliminary effects concentrations for these chemicals in water, the 10th and 50th centile of the toxicity distribution were calculated from the results of existing amphibian studies. The observed lethal endpoints from all amphibian species and measured effects were incorporated into the dataset for the calculations.

Title: Toxicity Testing Protocol Development Text: During Phase II of NAVFAC's study, a series of laboratory tests were conducted to identify the following parameters necessary for the development of a short-term chronic exposure test: The most appropriate and available test species, The most sensitive age of the test organisms, The most appropriate test length, The most appropriate test system, and The most sensitive test endpoints The studies were conducted in two phases including: Test Development and Test Refinement. Results of the Test Refinement Phase were used to define the test specifications. Visual Description: Pictures of different laboratory tests. It includes photos of sediment used in study, beakers on shaker tables, and other lab equipment.

Title: Test System Overview Text: More detail can be obtained by clicking on highlighted cells in the table. In summary, young tadpoles are placed in beakers containing sediment and overlying water. The overlying water in each beaker is replaced continuously via a flow-through delivery system. The beakers are placed in a water bath or environmental chamber that is held constant; water chemistry is measured on the appropriate days. When the tadpoles reach stage 25 (all external evidence of gills is gone), they are fed on a daily basis. At the end of the test (10 days), final overlying water chemistry samples are collected and measured. All living organisms are counted and removed for sublethal (width and body length) measurements. Sediment and/or water can be collected for chemical analysis, if necessary. Visual Description: Table summarizing the system used to test an amphibian species.

Title: Y0817 Study Results: Test Species Text: The amphibians tested were the anuran species Rana pipiens and Bufo americanus. Tadpoles of Rana pipiens can be obtained easily from about early November through late March. Between late March and mid-May field collected tadpoles of Rana and Bufo can be obtained and do well in the laboratory. Bufo may be more sensitive to copper than Rana, but less sensitive to chloride salts and commercial deicers.

Title: Test Species Text: Northern Leopard Frogs, Rana pipiens, are common in North America. They are found from the Atlantic Coast to eastern California, Oregon, and Washington; from northern Canada to as far south as southern Mexico, although they are not found in the Southeastern United States. More Info The American toad, Bufo americanus, is located in the midwest to eastern region of North America. They range east of the Rocky Mountains to the Atlantic Coast, and from mid Canada to Mexico. More Info Visual Description: Picture of the Northern Leopard Frog and the American Toad.

Title: Rana Pipiens Text: Rana pipiens (Northern Leopard Frog) is a small- to medium- sized frog, with a total body length of 5 to 9 cm. Body coloration is green to light brown. Yellow-outlined, oval, black spots cover the back. It also has two lightly colored lines on ridges that run the length of the back for oxygen exchange. Adult Rana pipiens overwinter in the mud at the bottom of lakes and ponds and emerge in the spring when air temperature reaches approximately 10 degrees Celsius. The breeding season runs from March through May, depending upon the latitude within the animal’s range. A female lays up to 6,000 eggs that form a large floating mass. The eggs hatch in about two weeks. Tadpoles are omnivores, feeding on algae, plants, and dead organisms, including other tadpoles. Tadpoles complete the metamorphosis to adults in 10 to 13 weeks, but this is somewhat dependent upon temperature and availability of food. Rana pipiens involve 46 stages from fertilized egg to air-breathing adult. Eggs hatch approximately at stage 20, which occurs approximately six days after fertilization. Stage 25 can be identified by the complete loss of external gills. From stage 25 until adulthood, stage is generally identified by limb bud development and in later stages, reabsorption of the tail and mouth size.

Title: Bufo Americanus Text: The Bufo americanus toad is very thick, rough, and darkly spotted. Each spot has one or two prominent "warts." These warts can be colored red and yellow. This toad's skin color is normally a shade of brown but changes color depending upon the temperature, humidity and the amount of stress the toad is experiencing. The bellies are a white or yellow color. The sexes can be distinguished in three ways. Males have dark colored throats, of black or brown, and females have white throats. Females are larger than males and are over all a lighter color. All toads have defensive chemicals in their skin. Each female can produce 2,000 to 20,000 eggs. Breeding occurs in the months of March or April, but may extend into July. It is usually triggered by warming temperatures and longer days. The eggs are black on top and white on the bottom (counter-shaded), and embedded in long strings of clear sticky gel. Female toads prefer ponds without fish to lay their eggs in. The larvae (tadpoles) are dark, almost black, with smooth skin, round bodies and a somewhat rounded tail. Newly-metamorphosed toadlets are similar to the adult toad: mottled brown with dark spots and bumps. They are cryptically colored, and are active mainly at night, making it harder for predators to find them. Most adult toads have short legs and round bodies. There are four toes on the front legs. Five toes are connected together by a webbing on their hind legs. The American toad typically hunts at night, and is most active in humid and wet conditions. Most prey is captured with their wide sticky tongues. They also may use their front legs in order to eat larger food. They grasp their food and push it into their mouths. An American toad can eat one hundred insects in a night.

Title: Y0817 Study Results: Test Organism Age Text: Younger organisms are generally more sensitive to toxicants than older organisms. For sediment tests, organisms should not be older than about 72 hours at test initiation.

Title: Y0817 Study Results: Test Duration Text: Conducting test for longer periods of time does not result in substantially lower statistical endpoints (e.g., No Observable Effects Concentrations (NOECs)).

Title: Test Protocol Daily Activities Text: Day -1 Place 100 ml of homogenized sediment, including control sediment, in each of the test chambers. Add 175 ml of overlying water to each chamber. Add the water carefully to avoid, as much as possible, suspension of sediment. Do not start flow-through system yet. Day 0 Begin flow-through system. Water flow rate should be slow, so as not to disturb the sediment in the test beakers. Set the rate so that the test chamber volume is replaced two to four times during each 24-hour period. After at least one hour collect overlying water for initial water characterization (dissolved oxygen [DO], temperature, pH, conductivity, hardness, alkalinity, ammonia, and total residual chlorine). If DO in any test chamber is less than 3.0 mg/L, increase the flow rate of the incoming water slightly. This must be done for all test chambers. After one hour, recheck the DO, if it is still low, begin aeration of all test chambers. Set aeration tubes or pipettes (Pasteur pipettes work well) so that the tip is no more than 0.5 cm under the water’s surface. After aerating the test chambers for approximately 30 minutes, recheck the DO to ensure that the level has increased to greater than 3.0 mg/L. If total ammonia concentrations are greater than 5.0 mg/L, a second sample should be collected and retested. If ammonia levels are still high, then the test can proceed but a notation should be made of the high levels. Ammonia concentrations greater than 5.0 mg/L may be high enough to cause adverse effects to the test organisms. Add five tadpoles to each test chamber. At less than or equal to 72 hours in age, all tadpoles should be very close in size; avoid using animals that are noticeably small or large. Also, do not use animals that exhibit unusual behavior or are deformed. To transfer organisms, use a glass pipette and gently place them in the test chambers. Release organisms under the water’s surface. Minimize the amount of water transferred with the organisms. Rinse the pipette with deionized water before obtaining more organisms. After all organisms have been placed in the test chambers, return the chambers to the water bath or environmental chamber. Check DO within one to two hours after the organisms have been added to the chambers. If DO is low (less than 3.0 mg/L) follow the procedures described above for increasing flow or adding aeration. From the remaining batch of tadpoles, select 5 to 10 for possible examination of metamorphic stage. These organisms should be preserved with 70% isopropanol or 10% formalin. If the tissue concentration of specific chemicals is to be measured, additional organisms must be collected for determination of initial concentration. The amount of tissue needed for analysis varies with the specific analyte. Check with the analytical laboratory to determine how much tissue will be needed. Animals for tissue analysis must be frozen unless they are processed and analyzed immediately. Days 1-9 Examine organisms from at least three beakers each day to determine metamorphic stage. At hatch, tadpoles are at stage 20. It takes approximately 4 to 6 days for hatched tadpoles to reach stage 25, when feeding begins. Therefore, if tests are initiated with less than 24 hr-old organisms, feeding will begin about midway through the test. However, if tests are initiated with 72-hour organisms, feeding may begin on day 1 or 2. If organisms are at stage 25, feeding should begin with approximately 4 mg of ground, dry TetraMin per chamber. Adding excess food should be avoided since it can cause a reduction in DO concentrations that may result in mortality. Each chamber should be examined for living organisms each day. If no organisms are seen swimming, then the chamber should be removed and examined carefully. Dead organisms must be removed. The following water characterizations are made: Temperature: continuously in the water bath or environmental chamber and in each treatment (one replicate only) on days 3, 6, and 9. DO: daily in each treatment (one replicate only) and in any chamber where mortality has occurred or where water quality is in question. pH: in each treatment (one replicate only) on days 3, 6, and 9 and in any chamber where mortality has occurred or where water quality is in question. Ammonia: at least twice in each treatment during the course of the study. For example, days 3 and 7. Day 10 Final water characterizations are made: Temperature, DO, pH, conductivity in each test treatment. Hardness and alkalinity may be measured as well. Remove live organisms from each test chamber and transfer them to small beakers (glass or plastic) containing 10 to 20 ml of clean (unchlorinated) water. Tadpoles can easily blend in with some sediment and often move very little, even with prodding. Test chambers should be examined thoroughly to find any live organisms. When pouring out water for chemistry or disposal, pour the water through a net to catch any tadpoles that may have been missed. Live tadpoles must be anesthetized or killed before sublethal measurements can be made. The use of 3-aminobenzoic acid ethyl ester (MS-222) is recommended. To each of the small beakers containing tadpoles, add approximately 1 ml of a stock solution (2 g/liter) of MS-222. If organisms continue to move after several minutes, add a few additional drops of the anesthetic. Tadpoles should not be left in the MS-222 solution for an extended period of time since tadpoles will begin to fall apart. Using a clear metric ruler, measure the maximum body width and body length (snout to base of tail) to the nearest 0.5 mm.

Title: Y0817 Study Results: Control Sediment Text: Tadpoles grow better when exposed to a natural control sediment rather than a formulated sediment.

Title: Y0817 Study Results: Test Temperature Text: Organisms grow adequately and remain healthy when tested at a temperature of 23 degrees Celsius.

Title: Y0817 Study Results: Solution Renewal Text: Sediment tests in flow-through chambers are preferable over static-renewal systems because of the buildup of ammonia. Ammonia concentrations in excess of 5 mg/L could cause sublethal effects to anurans.

Title: Y0817 Study Results: Feeding Text: Tadpoles grow better when fed TetraMin or TetraMin mix rather than the frog food available commercially.

Title: Y0817 Study Results: QA/QC Criteria Text: If survival in test treatments is greater than in the control, then it can be concluded that field-collected sediments are not acutely toxic. If mortality is highly variable, survival may be due to variations in water chemistry, variability in organism health, or differences in how chambers were treated. Highly variable water chemistry may indicate the sediment was not sufficiently homogenized or differences in flowrate.

Title: Validation of Study Results Text: The amphibian toxicity testing protocol was validated during Phase III of the NAVFAC study. The validation effort was completed by conducting the testing in accordance with the protocol developed in Phase II of the study. A series of tests were carried out with sediment spiked with cadmium, copper, lead, and zinc. The tests evaluated the amphibian responses and explored the effects of additional test variables such as the metals concentration and total organic carbon content of the sediment.

Title: Test Endpoints and Point Estimates Text: Test endpoints used to make risk management decisions include a statistical analysis of survival, body width, and body length data gathered during laboratory testing. Point estimates are seldom used in sediment tests because there is generally no known concentration gradient of a particular chemical of concern. In addition, sediments may contain multiple toxicants that could act independently or have synergistic, additive, or antagonistic effects. However, point estimates may be calculated; click here for more information. Visual Description: Bar chart of NOEC Cu vs. Biological Endpoints (survival, width, and body length) for different age groups.

Title: Calculating Point Estimates Text: Point estimates could be calculated based upon the percent (weight or volume) of a test sediment mixed with a nontoxic control sediment. If this method is used, then both sediments should have approximately the same moisture fraction so that the percentage estimates are reasonably accurate. Point estimates could also be used if samples are collected along a known concentration gradient for one particular chemical and no other chemicals of concern are present. Finally, if spiked sediment tests are conducted where different treatments of sediment contain variable but known quantities of a particular chemical, then point estimates can be made.

Title: NAVFAC Study Considerations Text: In general, the study results suggest that relative to the toxicity testing endpoints evaluated, amphibian test thresholds were generally substantially higher than AWQC and other literature-derived benchmarks. In addition, it was determined that copper and zinc toxicity was strongly associated with the amount of organic carbon in the tested sediment. High levels of organic carbon tended to bind these metals into the sediment, reducing their concentration in water and decreasing toxicity. Visual Description: Bar chart of Percent Survival vs. Water Dissolved Organic Carbon Concentration for different copper concentrations.

Title: Assessing Amphibians at Your Site Text: The following decision flow diagram has been developed to guide wetland risk management decisions at your site. The diagram begins with the Tier I Screening Ecological Risk Assessment (SERA) and if necessary, through the Tier II Baseline Ecological Risk Assessment (BERA). Click the "Next" button to initiate the Tier I level evaluation for your site. More information about the Navy's ERA process Visual Description: A flow diagram to guide risk management decisions at wetlands.

Title: Tier I: Wetland Habitat Text: Wetlands provide habitat for an incredible array of plants and animals. Over one-third of all threatened and endangered species of animals live in wetlands, and nearly half use wetlands at some time in their lives. Amphibians are a common resident of wetlands, relying on wetlands for breeding, laying their eggs in and around wet areas. During the winter, amphibians may hibernate under leaves or mud until the warmth and ensuing water of spring bring them back out.

Title: Tier I: Exposure Pathways Text: A complete exposure pathway is one in which the chemical can be traced or expected to travel from the source to a receptor that can be affected by the chemical. For Navy guidance on identifying potential exposure pathways visit Section 2.3 of this web site,

Title: Tier I: Benchmarks Text: The Environmental Sciences Division of Oak Ridge National Laboratory developed and compiled a comprehensive set of ecotoxicological screening benchmarks for surface water, sediment, and surface soil applicable to a range of aquatic organisms, soil invertebrates, and terrestrial plants. These benchmarks, or updates performed in collaboration with the Center for Information Studies at the University of Tennessee and the Bechtel Jacobs Corp., are provided as a searchable database. Links to supporting technical reports from which the benchmarks were obtained are also provided.

Title: Tier I: Background Data Text: Background data is site-specific.

Title: BERA Tier II Evaluation Text: If the Tier 1 SERA identifies a number of contaminants that may pose unacceptable risks to ecological resources, then the decision would be to proceed to a Tier 2 Baseline Ecological Risk Assessment (BERA). The Tier 2 BERA consists of a number of steps designed to provide a scientifically based and defensible assessment of exposure and hazard to ecological resources that will support a risk management decision regarding site cleanup. All information gathered and toxicity testing during a Tier II are completed using site specific data. The initial step involves collecting hydric soil samples for additional chemical analyses. Amphibian toxicity testing may also occur at this time. Please click the "Yes" or "No" buttons to evaluate your site. Click on the green question buttons if you would like more information about the question being asked. Visual Description: Figure zooms in on Tier II of the flow decision diagram.

Title: Tier II: PBT's Text: PBT means persistent, bioaccumulative, and toxic. PBT pollutants are toxic chemicals that persist in the environment and bioaccumulate in food chains. They pose considerable risks to human health and ecosystems. The biggest concerns about PBTs are that they transfer rather easily among air, water, and land, and span boundaries of programs, geography, and generations. Click here for further information on PBTs

Title: Tier II: Laboratory Toxicity Testing Text: Standard Operating Procedures developed for conducting laboratory toxicity testing can be found on page 22.

Title: Tier II: Laboratory Toxicity Testing Text: Additional response actions may be required (e.g., additional studies, remedial activities, monitoring)

Title: Toxicity Testing Protocol Text: Laboratory studies are one way of assessing toxicity directly. The purpose of this SOP is to help evaluate possible effects of chemical stressors in sediments and hydric soils on amphibians in natural ecosystems. The test method uses an early life stage of a native North American species, and lethal and sub-lethal toxicity endpoints that are relevant to typical assessment endpoints considered by the Navy in their ecological risk assessments.

Title: QA/QC Criteria Text: Quality Assurance/Quality Control (QA/QC) criteria for the data includes evaluating the data for the following: Survival in all test treatments Variability within a treatment Water chemistry

Title: QA/QC Criteria Text: If survival in test treatments is greater than the control, then it can be concluded that field-collected sediments are not acutely toxic.

Title: QA/QC Criteria Text: If mortality is highly variable and scattered throughout the test, then the test might not be acceptable. Highly variable survival may be due to variations in water chemistry (e.g., elevated ammonia due to excess food in some chambers), variability in organism health, or differences in how chambers were treated (e.g., different amounts of food or flow rates of overlying water).

Title: QA/QC Criteria Text: Highly variable water chemistry may indicate the sediment was not sufficiently homogenized or differences in flow rates.

Title: Summary Text: This training tool is designed to allow the Navy and DoD to develop more environmentally relevant risk assessments. Risk managers can use this information to identify cleanup levels and set remediation goals avoiding costly and unnecessary wetland alteration based on use of inappropriate ecological endpoints. Click here to view the Final Amphibian Ecological Risk Assessment Guidance Manual that presents a standardized risk assessment protocol for evaluating potential risks to amphibians at Navy wetland sites. Visual Description: Picture of a field with a lake.

Title: Links Text: Websites containing information about amphibians USGS Checklist of Amphibian Species and Identification Guide The Frogs of New England Environmental Education for Kids Critter CornerRay Rasmussen's Website: The Northern Leopard Frog All About Frogs Website: The Northern Leopard Frog Nova Scotia Frogs Websites containing amphibian ecotoxicity data Society of Environmental Toxicology and Chemistry (SETAC) Reptile and Amphibian Toxicology Literature (RATL) ”Websites containing ecological risk assessment information” Navy Ecological Risk Assessment Process Ecological Benchmarks developed and compiled by Oak Ridge National Laboratory USEPA PBT Chemical Program

Title: Contact Information Text: For more information about the approach for assessing risks to amphibians, please contact: PRTH_NFESCT2@navy.mil