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Title:
Trace metals and stable isotopes as tracers of life history and trophic connections in estuarine-dependent fish from Tampa Bay, Florida
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English
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Boehme-Terrana, Linae Marie
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University of South Florida
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Tampa, Fla.
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Subjects / Keywords:
Copper
Zinc
Carbon
Nitrogen
Sand seatrout
Tidewater mojarra
Striped striped
Estuary
Bay anchovy
Dissertations, Academic -- Marine Science -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: Florida's estuaries support a wide range of species yet little is known about tracemetal cycling among members of this important ecosystem. To examine the flow of trace metals through the Tampa Bay estuary, four fish species representing different trophic levels were analyzed for copper (Cu), zinc (Zn) and stable isotopes of carbon (C) and nitrogen (N). Species selected were the striped mullet (Mugil cephalus), tidewater mojarra (Eucinostomus harengulus), bay anchovy (Anchoa mitchilli), and sand seatrout (Cynoscion arenarius). Juvenile fish were collected from the Alafia, Hillsborough, Palm, and Little Manatee Rivers. Adults were collected from Tampa Bay. Combinations of trace metal and stable isotope analyses were used to evaluate geographic variability in trace metal concentrations among locations in Tampa Bay and to shed light on trophic pathways that lead to trace metal accumulation.In juvenile mullet, significant trends were found between Zn concentrations, stable isotope ratios, and standard length. Animals of the smallest size classes carry greater concentrations of zinc in their tissues and have distinct stable isotope ratios that reflect their recent life history as offshore planktivorous larvae. Interestingly, the ratio of Zn:Cu concentrations was highly conserved. While species-specific differences were observed, relatively small Zn:Cu variations suggest a possible bioregulatory mechanism that maintains an optimal Zn:Cu ratio even in the presence of elevated absolute metal concentrations. Stable isotope ratios proved to be an effective tracer of ontogenetic changes in fish diet and habitat. Carbon and nitrogen stable isotope analyses revealed that trophic relations between species are established very early in an organism's life history. The bay anchovy, a major prey item of the sand seatrout, has delta¹⁵N values very similar to this predator.Although trophic linkages between trace metals and stable isotopes proved difficult to interpret, the relation between zinc concentrations and delta¹³C values suggested that trace metal concentrations are highest in animals that utilize food webs based on terrestrial carbon.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
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Includes bibliographical references.
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by Linae Marie Boehme-Terrana.
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Document formatted into pages; contains 102 pages.
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Includes vita.

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oclc - 263148471
usfldc doi - E14-SFE0002241
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Trace Metals and Stable Isotopes as Tracers of Life History and Trophic Connections in Estuarine-Dependent Fish from Tampa Bay, Florida by Linae Marie Boehme-Terrana A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy College of Marine Science University of South Florida Major Professor: Robert H. Byrne, Ph.D. David J. Hollander, Ph.D. Mark E. Luther, Ph.D. Richard E. Matheson, Ph.D. Johan Schijf, Ph.D. Edward S. VanVleet, Ph.D. Date of Approval: November 15th, 2007 Keywords: copper, zinc, carbon, nitrogen, sand seat rout, tidewater mojarra, striped mullet, estuary, bay anchovy Copyright 2007, Linae Marie Boehme-Terrana

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Acknowledgements I would like to acknowledge the efforts of my commi ttee: David Hollander, Mark Luther, Ed Matheson, Johan Schijf, and Ted VanVleet. Their advice and expertise greatly enhanced my research. I offer special thanks to my major professor, Bob Byrne. His guidance made me a better scientist. He wanted me t o earn my doctoral degree as badly as I wanted it. Thank you, Bob, for your determina tion. I would like to acknowledge Ernst Peebles for his help with statistics and his never ending knowledge of estuarine dependent fish. Thank you to Molly McLaughlin and the United States Geological Survey in St. Petersburg for donating time and lab space during method development. The LLOYDA Foundation provided financial support in the final stages of my dissertation research. No doctoral candidate compl etes their degree without the support of friends and family. Thank you to my parents, Ma rk and Gretchen Boehme, for their love, emotional support, and encouragement during s ome tough days. Thank you to my sister and brother, Jen and Joel, who would go to t he ends of the earth to help. Thank you to my friends. Without your steadfast support, I w ould not have survived the dissertation process. And finally, I would like to thank my hus band, Joseph Peter Terrana. He put his career on hold to allow me to pursue a Ph.D. J oe provided an unending source of unconditional love, humor, and stress relief throug hout this adventure.

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Note to Reader Note to Reader: The original of this document cont ains color that is necessary for the understanding of the data. The original dissertati on is on file with the USF library in Tampa, Florida.

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i Table of Contents List of Tables iii List of Figures iv Abstract vi 1. Introduction: An Overview of the Tampa Bay Estu ary 1 1.1 Tampa Bay 1 1.2 Estuarine-Dependent Fish 3 1.3 Trace Metals 6 1.4 Stable Isotopes 9 1.5 Objectives 13 2. Variability of Zn and Cu Concentrations among Es tuarine-Dependent Fish from Tampa Bay, Florida 14 2.1 Abstract 14 2.2 Introduction 15 2.3 Methods 18 2.4 Results 20 2.5 Discussion 24

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ii 3. Relationships between Stable Isotopes and Trace Metals in Estuarine-Dependent Fish from Four Tidal Rivers 38 3.1 Abstract 38 3.2 Introduction 39 3.3 Methods 41 3.4 Results 43 3.5 Discussion 45 Summary 61 References 63 Appendices 70 Appendix A All Sample Data for Zn, Cu, d15N and d13C 71 Appendix B Sample Metadata 75 Appendix C Additional Cu & Zn Figures 81 Appendix D Additional Stable Isotope Figures 92 Appendix E Additional Trace Metal/Stable Isotope Figures 96 About the Author End Page

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iii List of Tables Table 1 Mean zinc and copper concentrations, m g/g wet weight one standard error 28 Table 2 Log ([Zn]/[Cu]) the standard deviation 29 Table 3 Mean d13C and d15N 60 Table 4 Additional Sample Data 71 Table 5 Sample Metadata 75

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iv List of Figures Figure 1 Map of sampling locations 30 Figure 2a Zinc in fish from the Tampa Bay estuary 3 1 Figure 2b Copper in fish from the Tampa Bay estuary 31 Figure 3 Log of zinc concentrations for each specie s at each location 32 Figure 4 Log of trace metal concentrations plotted by species for Tampa Bay 33 Figure 5 Log of trace metal concentrations plotted by species for each river 34 Figure 6a Log [Zn] vs. inverse standard length 35 Figure 6b Log [Cu] vs. inverse standard length 35 Figure 7 Log ([Zn]/[Cu]) vs. inverse standard lengt h 36 Figure 8 Log ([Zn]/[Cu]) by species 37 Figure 9 Stable isotope ratios in the bay anchovy 4 9 Figure 10 Carbon stable isotope ratios for each spe cies at each location 50 Figure 11 Nitrogen stable isotope ratios for each s pecies at each location 51 Figure 12 Multidimensional scaling plot showing rel ationship among species 52 Figure 13 Multidimensional scaling plot including z inc concentrations 53 Figure 14 Multidimensional scaling plot including c opper concentrations 53 Figure 15 Multidimensional scaling plot including s tandard length 54 Figure 16 Multidimensional scaling plot including n itrogen stable isotopes 54 Figure 17 Multidimensional scaling plot including c arbon stable isotopes 55 Figure 18 Multidimensional scaling plot including s easonal data 55 Figure 19 Seasonal trends in carbon and nitrogen st able isotopes in the Alafia 56 Figure 20 Seasonal trends in carbon and nitrogen st able isotopes in Tampa Bay 56 Figure 21 Log [Zn] plotted against carbon and nitro gen for striped mullet 57 Figure 22 Log [Zn] plotted against carbon and nitro gen for sand seatrout 57 Figure 23 Stable isotope ratios in estuarine-depend ent fish from Tampa Bay 58

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v Figure 24 Stable isotope ratios in estuarine-depend ent fish from river sites 59 Figure 25 Striped Mullet: Log [Cu] vs. Standard Le ngth 81 Figure 26 Tidewater Mojarra: Log [Cu] vs. Standard Length 82 Figure 27 Sand Seatrout: Log [Cu] vs. Standard Leng th 83 Figure 28 Bay Anchovy: Log [Cu] vs. Standard Length 84 Figure 29 Sand Seatrout: Log [Zn] vs. Log [Cu] 85 Figure 30 Bay Anchovy: Log [Zn] vs. Log [Cu] 86 Figure 31 Striped Mullet: Log [Zn] vs. Log [Cu] 87 Figure 32 Tidewater Mojarra: Log [Zn] vs. Log [Cu ] 88 Figure 33 Log [Zn] vs. Standard Length all specie s, high detail 89 Figure 34 Log [Zn] vs. Log [Cu] by location, all species, expanded axis 90 Figure 35 Cu ( m g/g) and Zn ( m g/g) vs. Standard Length 91 Figure 36 d15N vs. d13C, separated by species 92 Figure 37 d15N vs. d13C, separated by location 93 Figure 38 d15N and. d13C vs. Standard Length 94 Figure 39 d15N vs. d13C including seasonal data 95 Figure 40 Log [Cu] vs. d13C 96 Figure 41 Log [Zn] vs. d13C 97 Figure 42 Log [Cu] vs. d15N 98 Figure 43 Log [Zn/Cu] vs. d13C 99 Figure 44 Log [Zn] vs. d15N 100 Figure 45 Log [Zn/Cu] vs. d15N 101

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vi Trace Metals and Stable Isotopes as Tracers of Life History and Trophic Connections in Estuarine-Dependent Fish from Tampa Bay, Florida Linae Marie Boehme-Terrana ABSTRACT Florida’s estuaries support a wide range of species yet little is known about trace metal cycling among members of this important ecosy stem. To examine the flow of trace metals through the Tampa Bay estuary, four fi sh species representing different trophic levels were analyzed for copper (Cu), zinc (Zn) and stable isotopes of carbon (C) and nitrogen (N). Species selected were the str iped mullet ( Mugil cephalus ), tidewater mojarra ( Eucinostomus harengulus ), bay anchovy ( Anchoa mitchilli ), and sand seatrout ( Cynoscion arenarius ). Juvenile fish were collected from the Alafia, Hillsborough, Palm, and Little Manatee Rivers. Adu lts were collected from Tampa Bay. Combinations of trace metal and stable isotope analyses were used to evaluate geographic variability in trace metal concentration s among locations in Tampa Bay and to shed light on trophic pathways that lead to trace metal accumulation. In juvenile mullet, significant trends were found betw een Zn concentrations, stable isotope ratios, and standard length. Animals of the smallest size classes carry greater concentrations of zinc in their tissues and have di stinct stable isotope ratios that reflect their recent life history as offshore plank tivorous larvae. Interestingly, the ratio of Zn:Cu concentrations was highly conserved. Whil e species-specific differences were observed, relatively small Zn:Cu variations su ggest a possible bioregulatory mechanism that maintains an optimal Zn:Cu ratio eve n in the presence of elevated absolute metal concentrations. Stable isotope rati os proved to be an effective tracer of ontogenetic changes in fish diet and habitat. C arbon and nitrogen stable isotope analyses revealed that trophic relations between sp ecies are established very early in an organism’s life history. The bay anchovy, a maj or prey item of the sand seatrout,

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vii has d15N values very similar to this predator. Although trophic linkages between trace metals and stable isotopes proved difficult t o interpret, the relation between zinc concentrations and d13C values suggested that trace metal concentrations are highest in animals that utilize food webs based on terrestr ial carbon.

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1 Chapter 1: An Overview of the Tampa Bay Estuary Tampa Bay Tampa Bay is the largest open water estuary in Flor ida and the 9th largest shipping port in the nation. The estuary is a relatively well-mixed drowned river basin whose circulation is dominated by deep shipping channels dredged in t he bay (Weisberg & Zheng 2006). Four major rivers provide the majority of fresh wat er: the Hillsborough, Alafia, Little Manatee, and Manatee Rivers (Wolfe and Drew 1990). The total watershed draining into the bay is approximately 6,583 km2. Each river drains its own unique watershed with varying levels of urban development, industry, and agriculture. The Tampa Bay region has undergone extensive development and large popul ation growth. Between 1991 and 2002, the rate of development increased threefold ( Xian & Crane 2005). Pollution introduced through rainwater runoff, powe r plant emissions, trash incinerators, and poorly treated waste water all increase with in creased urban development (Environmental Protection Agency 2005). Possible c ontaminants include nutrients, trace metals, and organic compounds including pesticides and polycyclic aromatic hydrocarbons. In addition to urban development, se veral industries are prominent in the Tampa Bay watershed. Florida’s agriculture industr y produces a large percentage of the winter fruits and vegetables consumed in the United States. Herbicides used to maintain crops contain organic compounds as well as trace me tals that can be toxic to life downstream (O'Shea et al. 1984). Phosphate mining also impacts the rivers flowing into the bay. The phosphate industry is one of the oldes t industries in Florida, accounting for 75% of the nation’s phosphate (Florida Phosphate Co uncil 2002). Phosphate mining mobilizes several toxic elements found in associati on with phosphate deposits. The metals found in highest concentration include titan ium, aluminum, magnesium, iron,

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2 sodium, antimony, arsenic, and tungsten; however, l ower concentrations of other metals are also present (O'Meara et al. 1996). The boatin g industry and associated marinas are additional sources of pollutants. Marinas are a sou rce of lead, copper, cadmium, barium and chromium from boat paint in addition to mercury from lamps and bilge pumps (Florida Department of Environmental Protection 200 3). In 1990, Tampa Bay was designated a part of the Nat ional Estuary Program. Inclusion in the NEP initiated a series of research projects tha t characterized bay habitats, identified sources of anthropogenic pollution, and evaluated s ediment quality. Trace metal research in Tampa Bay has mostly focused on sediments (Long et al. 1991, Alexander et al. 1993, Grabe 1997, Santschi et al. 2001, MacDonald et al. 2004). Sediments from Hillsborough Bay, part of northeast Tampa Bay, contain the highe st contaminant concentrations due to a high percentage of silt derived from nearby urban and industrial areas (Long et al. 1991, MacDonald et al. 2004). Elevated contaminant concen trations are often found in association with fine grain sediment due to higher concentrations of organic matter associated with these sediments as well as a high c harge to mass ratio for fine particles (Eby 2004). Hillsborough Bay receives direct runof f from the city of Tampa as well as water from the Hillsborough, Palm, and Alafia River s. The Hillsborough and Palm Rivers are highly modified river systems, with water contr ol structures restricting natural freshwater flow (Wolfe & Drew 1990). Freshwater fl ow in the rivers is regulated to meet the water needs of surrounding urban areas. These rivers are also used extensively by recreational boaters and marinas and receive signif icant urban runoff. The Alafia River has been modified by the dredging of a deep channel but is considered the least impacted of the three rivers. South of Hillsborough Bay, th e Little Manatee River also contributes water to Tampa Bay. It is less developed than othe r rivers and has been used as a reference site for pollution studies. Its sediment s are characterized by larger grain size and fewer contaminants (Grabe 1997). While trace m etals in sediments are often viewed as metals that have been removed from the food web, recent work indicates benthic infauna can resuspend buried trace metals (Klerks e t al. 2006), making them available in the water colum. Other work indicates that benthic epifauna and benthic microalgae

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3 influence estuarine food webs in Tampa Bay (Holland er & Peebles 2004), and brings into question the impacts that historic sedimentary poll ution could have on current ecosystems. Research on the impact of contaminants on biologica l organisms in the bay spans a wide range of taxa. Compared to sea stars in the Gulf o f Mexico or near the mouth of the bay, sea stars living in Tampa Bay have higher concentra tions of zinc, cadmium, silver and nickel (Lawrence et al. 1993). Work on other inver tebrates in Tampa Bay found elevated contaminant concentrations in oysters from Hillsbor ough Bay and in Bayboro Harbor (Fisher et al. 2000). Research on hard shell clams found high levels of zinc after the organisms were transferred to Bayboro Harbor from a n uncontaminated reference site in the bay (Nasci et al. 1999). Work on organic pollut ants suggests that animals in Tampa Bay have elevated contamination levels compared to fish from other Florida estuaries (Gelsleichter et al. 2005), and that geographic dif ferences in sediment concentrations of organic pollutants are reflected in the tissues of fish (McCain et al. 1996). Estuarine-Dependent Fish Estuaries are semi-enclosed coastal areas where fre shwater and saltwater mix. There are many types of estuaries, each with its own water so urces, shoreline, and watershed characteristics. These productive ecosystems suppo rt an extremely diverse assemblage of flora and fauna. The mixing of salt and fresh water creates a range of aquatic and marine environments which give rise to species adapted to exploit available niches. Estuarinedependent fish are an example of such species. Man y of Florida’s popular game fish, like red drum and snook, are estuarine-dependent species Estuaries serve as a nursery for larval and juvenile estuarine-dependent fish while adult fish spend their lives in the ocean or lower estuary, often spawning offshore. Juvenil e nursery habitats are of special importance. Juvenile fish that lack appropriate hab itat or prey may not survive to adulthood.

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4 In recent years, the definition of “estuarine-depen dent” has been revisited, in view of the large number of species associated with this term. “Estuarine-dependent” as used in this manuscript refers to species whose life cycle would fail if estuarine nursery areas were not available (Able 2005). The diverse strategies of estuarine-dependent fish can be highlighted by focusing on a few species. The bay anchovy, Anchoa mitchilli (Family: Engraulidae) is found across a wide range of latitu des, from Maine to the Gulf of Mexico. Although it is found in the northern reaches of its range only during warm water months (Vouglitois et al. 1987), bay anchovies are present year round in Tampa Bay. As a prey item in the diets of many larger fish (Peebles & Ho pkins 1993), bay anchovy serve as an important link in the trophic system by connecting secondary plankton production to the larger fish that prey upon the anchovy. The diet o f small bay anchovy (<30mm) is dominated by holoplankton. Larger anchovy prey on epifauna, particularly epibenthic crustaceans (Peebles et al. 1996). However, calanoi d copepods maintain a high level of relative importance compared to other dietary items Spawning takes place over most of the year in Tampa Bay (March-October) with a notabl e break during the winter months (Luo & Musick 1991, Peebles et al. 1996). Reproduct ion occurs near the mouths of rivers in areas of high copepod abundance. Bay anchovy gr ow very quickly; some fish can reproduce at approximately 3 months of age (Luo & M usick 1991). Their short generation time makes them an excellent bioindicato r for current estuarine conditions. Another important forage fish is the tidewater moja rra, Eucinostomus harengulus, (Family: Gerreidae). Rather than relying on plankto n, tidewater mojarra feed on benthic polychaetes. Even at lengths as small as 30mm, ove r 80% of their diet is made up of polychaetes. This dietary dominance of polychaetes extends to all size classes (Kerschner et al. 1985, Peebles & Hopkins 1993). T he taxonomy of the tidewater mojarra was under debate during the 1980s, making i nformation on its lifecycle difficult to trace in the literature (Matheson 1983). An ear ly publication on fish from Tampa Bay (Springer & Woodburn 1960) indicated that tidewater mojarra (as E. argenteus ) were found throughout the Tampa Bay estuary, especially in areas of low salinity. Larger sizes of tidewater mojarra move to the coastal areas outs ide the bay or deeper channels where

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5 spawning may occur. Some work suggests that tidewa ter mojarra may also utilize deep channel areas in the bay for spawning (Matheson, pe rs.comm. 2007). The life cycle of striped mullet, Mugil cephalu s (Family: Muglidae), typifies estuarine dependence. Adult mullet spawn in the open Gulf of Mexico over the continental shelf (Arnold & Thompson 1958). Newly spawned mullet are planktonic and feed on plankton in the offshore habitat as they move into their est uarine nursery habitat. Very small juveniles (15-32mm) move into the bay and associate d rivers during the winter months (Pattillo 1997, Nordlie 2000). As they settle in t he rivers, juvenile mullet are deposit feeders, scooping sediment off the bottom (Eggold & Motta 1992). Some studies indicate that mullet feed on general detritus and f ilamentous algae. However, recent research involving the use of stable isotope ratios indicate that benthic microalgae found in the sediments are the primary food source (Holla nder & Peebles 2004). Adult fish are tolerant of a range of salinities, and are often fo und in the shallow parts of the bay (Pattillo 1997). Sand seatrout, Cynoscion arenarius (Family: Sciaenidae), differ from the other specie s in that they are piscivores. Adults spawn in the nears hore regions of the Gulf of Mexico (Pattillo 1997), with larva arriving in their nurse ry habitat at approximately 10mm standard length (Peebles 1996). As is true for mos t estuarine-dependent fish, the larvae and small juveniles initially utilize calanoid cope pods as prey. As they grow, sand seatrout include epifauna in their diet and begin t o feed on juvenile fish. At standard length around 30 mm, fish become the main prey. My sids and amphipods remain part of their diet but have a lower relative importance in larger sand seatrout (Peebles & Hopkins 1993). As adults, sand seatrout can be found near the mouths of rivers, with larger size classes gradually moving into the open bay. Unlike the closely related spotted seatrout ( Cynoscion nebulosus ), sand seatrout are found over muddy bottoms and n ear deep channels. From spring to late summer, adult sand se atrout move to the mouth of the bay and coastal Gulf of Mexico for spawning.

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6 Trace metals Trace metals have diverse properties and are found in aqueous systems over a wide range of concentrations. Despite the wide range of eleme nts available for biological reactions, a few select elements are prominent in organisms fr om single celled bacteria to desert reptiles. Elements that have been incorporated int o biological systems were abundant under the anoxic aquatic conditions that existed du ring the evolution of the first microbes. These elements are still utilized today, although o xygenated conditions have reduced their availability in aquatic systems (Frausto da S ilva & Williams 1991). Many essential elements are now considered growth limiting factors The metals under investigation in this study, zinc (Zn) and copper (Cu), are both tra nsition metals. The +II oxidation state is common to both and Cu can also be founding the +I o xidation state. The concentrations of free Zn and Cu in seawater are low because of st rong interactions with organic compounds (Coale & Bruland 1988, Bruland 1989). In open ocean water, free ion concentrations average approximately 6 nmol/kg for total Zn and 4 nmol/kg for total Cu (Pilson 1998). According to Turner (1981), only 9% of Cu in seawater exists as free ion (pH=8.2, 25C). Free zinc is more abundant with 46% existing as Zn2+. In freshwater, Zn and Cu have higher concentrations. In freshwater a t pH 6, 98% of Zn and 93% of Cu exist as free ions. The mixing conditions within a n estuary make the concentrations of these elements even more variable as these metals c omplex easily with particles in the water. Metals found in the marine environment can be deriv ed from natural sources or introduced by human activity (Fitzgerald et al. 199 8). Anthropogenic pollution has impacted the environment for centuries. Evidence f rom Greenland ice cores shows evidence of lead pollution dating back to metal min ing by the Roman Empire (Nriagu 1996). Today, major anthropogenic sources of metal s include the burning of fossil fuels, trash incineration, mining, urban wastewater runoff and sewage (Seigel 2002). The impact of anthropogenic pollution can extend beyond the source. Industry and agriculture along rivers can impact estuaries far d ownstream, and airborne emissions

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7 from many industries have been implicated in the pr esence of lead and mercury across the globe (Nriagu 1996). Before metals can be incorporated into tissues, the y must be assimilated from the environment. Possible modes of transport include in halation (Rawson et al. 1995), absorption across the skin (Hostynek & Maibach 2006 ), or ingestion. Certain metals can be passed from female organisms to developing offsp ring whether they cross the placental barrier, in the case of mammals, or are p assed on during vitellogenesis in fish. The extent of metal absorption through skin is beli eved to be small compared to other routes (Goyer et al. 1995). Studies indicate that ingestion of prey or milk (in the case of mammals) serves as the primary route for metal upta ke (Goyer et al. 1995). Water consumption or transfer across the gill membranes c an be an important contamination route in fish. However, it appears to be less signi ficant than incorporation from food (Handy 1996, Bury et al. 2003). For animals that br eathe air, evidence suggests that airborne metals can accumulate on lung tissue (Augi er et al. 1993), but the effects of this accumulation route appear localized and remain a su bject of research (Rawson et al. 1995). Trace metals have a wide range of biological functi ons. Metals such as Zn and Fe are key components of biological proteins. Because met als regulate biochemical processes, elevated concentrations of essential metals (Taylor et al. 1996) or the interference of nonessential metals in biochemical reactions (Planello et al. 2007) can have adverse effects on organisms. It has been suggested that animals h ave developed mechanisms to control potentially hazardous metals (Law 1996). One regul atory system involves metallothioneins (MT), metal-binding proteins that regulate the flow of essential metals for biochemical reactions. Metallothioneins are a group of low molecular weight proteins that bind metal through the thiol group of cysteine residues. This group of proteins regulates both essential trace metals and potential ly toxic metals. Metallothioneins are often associated with regulation of Zn availability The highest concentrations of MT occur in the liver and kidney. Not surprisingly, s ignificant correlations have been found

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8 between metallothionein levels and the concentratio ns of Cd and Zn in liver tissue, a known storage site for trace metals. In the presen ce of high metal concentrations, MT bind metals such as Cd, making them unavailable for further reactions (Law 1996). MT has an affinity for mercury as well as Cd (Goyer et al. 1995) but mercury has high affinities for several proteins, unlike Cd, which m akes it less likely to be bound solely by MT. The relationship between metal accumulation and amb ient metal concentrations is complex, involving relationships between uptake mec hanisms and chemical/physical speciation. Arnot and Gobas (2006) defined bioaccu mulation as the “process in which a chemical substance is absorbed in an organism by al l routes of exposure as occurs in the natural environment, i.e., dietary and ambient envi ronment sources.” Animals absorb metals over the course of their lives. Environment al concentrations of metals may remain below the levels mandated by regulatory agencies ye t still create health problems during the lifespan of animals living within that system ( Nendza et al. 1997). Due to complex synergistic effects among pollutants (Das et al. 20 03), the impacts of ambient metal concentrations in the environment, whether attribut able to anthropogenic or natural sources, is likely to remain under investigation fo r some time. Metal accumulation depends on both an animal’s abil ity to absorb metals and an ability to excrete them. There can be major taxonomic differe nces in these metabolic capabilities, especially in the case of invertebrates (Luoma & Ra inbow 2005). For fish species, absorption and excretion rates are similar even in juvenile fish (Zhang & Wang 2007). The bioavailability of elements in prey items also influences metal accumulation (Ni et al. 2000, Zhang & Wang 2006). This may explain the conflicting results found in the current literature on bioaccumulation and biomagnif ication (Luoma & Rainbow 2005). Biomagnification appears to be relatively rare in t race metals (Wang 2002). Trace metals have been used to assess a variety of ecosystem issues. Information on trace metal concentrations has been used to evaluat e the nutritional value of fish and the

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9 benefits of alternative diets (Halver 1989, Lovell 1998). In environmental monitoring applications, trace metals are used to evaluate the influence of local waste water treatment as well as power plant remediation on tra ce metal accumulation in fish (Kirby et al. 2001b). Research on trace metals in Tampa B ay fish populations has primarily focused on mercury concentrations in recreationally important fish (Adams & Onorato 2005). Stable Isotopes Stable isotope analyses have been used for years in the fields of geology and oceanography and have become an integral part of ec osystem studies (Peterson & Fry 1987, Thompson et al. 2005). Arthur et al. (1983) o ffered a succinct explanation of the principals behind the use of stable isotopes in sci entific investigation. Stable isotope analyses are based on the mass difference between i sotopes of the same element. This difference in mass translates into isotopic fractio nation during physical or chemical processes. In addition to fractionation due to phy sical changes like evaporation, certain isotopes are taken up preferentially during biologi cal processes. Peterson and Fry (1987) were among the first to explain how stable isotope information could be applied to the fields of ecology and biology. Carbon, nitrogen, a nd sulfur are the most commonly used elements in isotopic ecosystem studies because thei r light atomic masses makes their fractionations much greater than for heavier elemen ts. Carbon and nitrogen, in particular, participate in many biological reactions, making th em ideal elements for studies of a variety of biological processes. Carbon is an impo rtant element for studies of primary productivity and the origin of carbon for an ecosys tem. Plants take up proportionally more 12C isotope than 13C during photosynthesis (Peterson & Fry 1987). The actual amount of 12C taken up is a function of the photosynthetic path way used by the plant and the amount of 12C in the carbon pool available to the plant. The C3 photosynthetic pathway is so named because carbon dioxide, CO2, from the environment is initially incorporated into a molecule with three carbon atom s. The C-3 pathway relies on the

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10 RuBisCO enzyme to uptake CO2. The C-4 pathway of photosynthesis utilizes the P EP Carboxylase enzyme for the uptake of CO2, incorporating CO2 into a molecule with four carbon atoms. PEP Carboxylase delivers CO2 to RuBisCO. C-4 photosynthetic plants are adapted to conditions of elevated temperature a nd intense sunlight (Lehninger et al. 1993). They include tropical plants like marsh gra ss, Spartina and sugar cane. Plants that use C-3 type photosynthesis pathways preferent ially incorporate 12C relative to plants that use C-4 pathways (Lajitha & Michener 1994), wh ich results in a fractionation between the atmosphere and plant biomass of approxi mately 21‰ for C-3 plants and 7‰ for C-4 plants. Nitrogen is used in ecological stu dies as an indicator of trophic level. The lighter isotope is preferentially excreted in u rine (Peterson & Fry 1987, Sponheimer et al. 2003). This loss of the lighter isotope in c onsumers results in a 3‰ increase in d15N at higher levels of a food chain (Peterson & Fry 19 87). Stable isotope investigations have greatly enhanced food web research. Investigations using stable isotopes offer an advantage over stoma ch content analyses because stable isotope ratios reflect an integrated signal. The st able isotope ratio reflects the prey tissues that are assimilated by the predator while stomach content analyses provide a snapshot of prey identity. Stomach content analyses are biased towards prey with hard, digestionresistant parts. For example, squid beaks remain i n the stomach of a predator much longer than the softer parts of a myctophid fish. R ather than providing a fingerprint of the exact prey species ingested, stable isotope analysi s indicates trophic level (Herzka 2005), sources of carbon, and the importance of benthic co mmunities in feeding processes (Thompson et al. 2005). Isotopic ratios in animal tissues are similar to the C and S isotopic composition of the animal’s diet (Peterson & Fry 1987), with isotopic fractionations on the order of 1‰ per trophic level for carbon. Sulfur isotopes are fractionated when they are taken up by photosynthet ic organisms (Peterson et al. 1986), however, further trophic shifts are very small. Ra ther than using sulfur to detect trophic shifts, sulfur stable isotopes are valuable for det ermining the influence of benthic organic matter and organisms in food webs. Marine sulfate has a d34S value near 20‰. In contrast, d34S values from sediments where sulfate reduction occ urs have isotopic values

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11 near -10‰ (Peterson et al. 1986). Because d13C values change little with trophic level, d13C values are used as indicators of carbon sources. Plants, fish, and benthic species sampled from the terrestrially influenced Suwannee River have d13C values between -31.7‰ and -27.7‰ while those sampled in the Gulf o f Mexico range between -20.2‰ and -16.1‰ (Gu et al. 2001). While these values in dicate that a broad range of isotopic values can be found along Florida’s coast, they fol low the trends reported for carbon from terrestrial versus marine sources. The 10 ‰ di fference between terrestrial and ocean-derived carbon sources overshadows the 1‰ iso topic change seen in carbon with increasing trophic level, especially in studies of estuarine systems. The nitrogen enrichment factor between trophic levels is ~3‰ per trophic level. These nitrogen isotope values can increase by 10-15 ‰ across entire food w ebs, indicating 3-5 trophic transfers (Peterson & Fry 1987). Stable isotopes can be used to trace larval settlin g and life cycle provided that certain sympathetic features exist in an ecosystem. Juvenil e fish must undergo a diet shift when they become adults and they must move to areas that are isotopically distinct. In most cases, movement from a marine habitat to an estuari ne habitat represents a decrease in d13C that is easily monitored in fish tissues. However nitrogen can also trace movements if there are varying nitrogen sources in the estuar y (Hansson et al. 1997). Recent discussions surrounding the definition of estuarine -dependent fish (Able 2005) could be greatly clarified through the use of stable isotope analyses. For example, due to the high turnover that accompanies the addition of new tissu e during growth, the recent habitat of juvenile fish will be reflected in their tissue. La rval fish experience full isotopic turnover in 5-8 days (Herzka & Holt 2000). Juvenile fish exp erience complete turnover on the order of weeks (Herzka 2005). Therefore, regardles s of sampling location for juvenile fish, their recent habitat utilization will be refl ected in the stable isotope ratios of their tissues. This was demonstrated by Weinstein et al. (2000) who noted that bay anchovies sampled on the continental shelf retained the carbo n isotopic ratio associated with nearby estuarine cordgrass. Clearly, isotopic ratios coul d provide valuable information in studies of habitat use by larval and juvenile fish (Herzka 2005).

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12 In addition to providing information about diet and habitat shifts, stable isotopes provide a means to establish relative trophic relationships between species. Das et al. (2000) used stable isotopes and trace metals to address multisp ecies aggregations seen in the eastern tropical Pacific. This study examined the three co mmonly caught species in the tuna fishery: common dolphins, spinner dolphins, and alb acore tuna. Although all three are caught in the same fishing nets, the higher d15N values of tuna compared to the cetacean species indicated that tuna feed at a higher trophi c level than the dolphins. Furthermore, a combination of trace metal and stable isotope ana lyses isolated two subgroups within the tuna (Das et al. 2000), indicating the potentia l for combined trace metal and stable isotope analyses. Stable isotope studies have the ability to greatly enhance our understanding of trace metal pathways. They serve as natural tracers of the tro phic levels and of carbon sources of food webs. Given the increasing urban development of the Tampa Bay region, it is important to understand the concentrations of trace metals in animals that serve as food for both humans and fish. Geographic variability o f contaminants has been found in other Florida estuaries, especially in association with strong pollution sources such as sewage outflows or industrial runoff (Lewis et al. 2002). In addition to information on geographic variability of trace metal concentration s in the estuary, information is needed on the impacts that trace metals have on animals fr om different trophic levels, and on possible trends among different species. Elucidati on of the food web interactions responsible for metal accumulation is an important next step in trace metal research. Prey items serve as the source of trace metals and stable isotopes and link organisms to their food web. When combined in the same study, t race metal and stable isotope analyses could provide valuable information regardi ng the trophic pathways that lead to trace metal bioaccumulation.

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13 Objectives The goal of this research project was to evaluate t he influence of different factors on trace metal accumulation in estuarine-dependent fish. Th ese factors include: Location Species Diet Age Trophic level and interactions Life history changes Interactions between trace metal concentrations and stable isotopes

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14 Chapter 2: Variability of Zn and Cu Concentrations among Estuarine-Dependent Fish from Tampa Bay, Florida Abstract To better understand the geographic variability of zinc (Zn) and copper (Cu) among fish populations in Tampa Bay, Florida, fish were collec ted from four tributary rivers and the bay itself. Estuarine-dependent fish representing different trophic levels were selected for analysis: striped mullet ( Mugil cephalus ), tidewater mojarra ( Eucinostomus harengulus ), bay anchovy ( Anchoa mitchilli ), and sand seatrout ( Cynoscion arenarius ). Muscle tissue was digested using hot block digestio n followed by analysis with Inductively Coupled Plasma Mass Spectrometry (ICP-M S). For most species, adult fish in Tampa Bay carry lower concentrations of trace me tals than river-dwelling juvenile fish. Differences among the rivers were less prono unced. Fish from the Alafia River had the lowest Zn and Cu concentrations while fish from the Hillsborough River consistently had the highest trace metal concentrations. Differ ences in Zn and Cu accumulation were also found among fish species. Bay anchovy had the highest trace metal concentrations of all species, followed by the tidewater mojarra a nd sand seatrout.

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15 Introduction Tampa Bay is the largest open-water estuary in Flor ida and the ninth largest shipping port in the United States. Like many coastal areas, the region has experienced a rapid increase in urban development and a subsequent increase in a nthropogenic pollution. Any increase in pollution is a matter of concern to managers of the bay’s flora and fauna. Increased pollution also impacts human populations because fi sh from Tampa Bay are caught for commercial and recreational purposes. Elevated tra ce metal concentrations could thus pose a threat to public health. Because urban deve lopment and pollution sources vary around the estuary, it is important to understand t he distribution of contaminants and their influence on the organisms living there. Research on anthropogenic pollution in Tampa Bay ha s principally focused on sediments. Results from such work showed that one section of T ampa Bay (Hillsborough Bay and associated rivers) consistently had the highest tra ce metal and organic contaminant concentrations of any area in the bay. This is lik ely due to a higher percentage of silt in this part of the bay as well as Hillsborough Bay’s close proximity to extensive urban and industrial areas (Long et al. 1991, Alexander et al 1993, Grabe 1997, Santschi et al. 2001, MacDonald et al. 2004). Trace metal research on Tampa Bay fish populations has been relatively limited, focusing on methyl-mercury concentrations in recreationally important fish (Adams & Onorato 2005). Some resea rchers have investigated the impact of trace metals in the sediments on other organisms in the bay. Research on sea stars found that those living in Tampa Bay have higher co ncentrations of trace elements than those in other Florida estuaries (Lawrence et al. 1 993). Work on oysters and clams found that those from Tampa Bay areas with elevated sedim ent trace metal concentrations have higher trace metal concentrations in their tissues than those from less contaminated sites (Nasci et al. 1999, Fisher et al. 2000). Spatial variability of trace metal concentrations i n fish has been found in a variety of habitats with strong gradients in land development (Sanger et al. 1999). Research on

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16 striped mullet, Mugil cephalu s (Family: Muglidae), collected in an Australian es tuary found higher trace metal concentrations in mullet g athered near power plant ash discharge than in mullet from undeveloped sections of the estuary (Kirby et al. 2001b). Similarly, work in the Irish Sea found elevated tra ce metals in otoliths from fish collected near sewage discharges (Geffen et al. 2003). Spati al variability in metal concentrations was also found within Florida. Fish caught in Flori da Bay show geographic variation in mercury (Hg) concentrations (Evans & Crumley 2005). Florida Bay fish also have different Hg concentrations than fish living in the Indian River Lagoon (Strom & Graves 2001). Given the importance of Tampa Bay fish to the econo my, and the ever increasing urban development, information is required on geographic variability of trace metals in this system. This project selected four estuarine-depen dent fish species for trace metal analysis, where each species represents a specific trophic group. The bay anchovy, Anchoa mitchilli (Family: Engraulidae), is a plankton feeder, formi ng the apex of a complex planktonic food web. The sand seatrout, Cynoscion arenarius (Family: Sciaenidae), is a piscivore and has a significant i mpact on bay anchovy biomass (Pattillo 1997). The tidewater mojarra, Eucinostomus harengulus (Family: Gerreidae), feeds on benthic infauna (Kerschner et al. 1985). The stripe d mullet, Mugil cephalus (Family: Mugilidae), is an herbivorous grazer as well as a d eposit feeder, taking in organic matter from the sediment in addition to filamentous algae and benthic diatoms (Pattillo 1997). Sampling locations were chosen to span the range of urban development in the Tampa Bay estuary. Sediments from the open areas of Tampa Bay have large grains and are relatively uncontaminated compared to sites near ri vers, marinas, or harbors (Wolfe & Drew 1990, Santschi et al. 2001). The Hillsborough and Palm Rivers are highly modified river systems, with water control structures restri cting natural freshwater flow (Wolfe & Drew 1990). The lower portions of these rivers are used extensively by recreational boaters, contain numerous marinas, and receive sign ificant urban runoff. The Alafia River is relatively close to the Hillsborough and P alm Rivers but its basin is more

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17 influenced by agricultural areas and the phosphate mining industry. The Little Manatee River is less developed than other rivers. Its sedi ments are characterized by larger grain size with fewer contaminants (Grabe 1997). While th e Little Manatee River has been used as a reference site for pollution studies, the site is not considered pristine.

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18 Methods More than 600 fish were collected from five locatio ns: Tampa Bay, the Hillsborough River, Palm River, Alafia River, and Little Manatee River (Figure 1). The Florida Fish and Wildlife Research Institute collected the fish during monthly monitoring surveys. Fish were stored on ice in polyethylene bags in the field until they could be transferred to a -10 C laboratory freezer. Tissues were processed in a class-100 clean air lab oratory according to protocols established by the U.S. Environmental Protection Ag ency (Environmental Protection Agency 2000). Nitrile gloves were used to handle t he fish. Each fish was measured to obtain its standard length. The standard length re presents the length of the fish measured from the tip of the snout (with the mouth closed) t o the base of the caudal fin. Fish were scaled followed by the removal of the upper layer o f skin. Muscle tissue was then collected. Tissue excision with a stainless steel s calpel was performed on a cutting board covered with aluminum foil. Tissues were stored fr ozen in polyethylene bags until digestion. Digestion was achieved following the hot block dige stion method from the Florida Department of Environmental Protection (Method MT-0 60, available from: http://www.dep.state.fl.us/labs/cgi-bin/sop/chemsop .asp). Tissues were weighed into acid-cleaned Teflon digestion tubes. Trace metal g rade HNO3, HCl (Fisher Scientific, Pittsburg, PA) and 30% H2O2 (J.T. Baker, Phillipsburg, NJ) were added while sa mples were heated in a Westco (Danbury, CT) digestion hot block. Samples were run in duplicate or triplicate depending on the amount of tissue available. In the case of small juvenile fish, individual tissues were pooled to ac cumulate sufficient sample mass. Sample masses ranged from 0.1-1 grams. Each digest ion included three procedural blanks. The digestion method was validated using the certif ied reference material DORM-2

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19 (dogfish muscle; National Research Council Canada). Recoveries for Cu and Zn averaged 105% and 115% respectively. Although recov eries are higher than the certified values, they are within the range of recoveries rep orted elsewhere (Al-Yousuf et al. 2000, Asuga et al. 2006). Sample digestates were diluted to 50 mL using trace metal clean water (Milli-Q water) produced with a Millipore (Be dford, MA) purification system. Resulting solutions were analyzed with an Agilent T echnologies 4500 series 200 inductively coupled plasma mass spectrometer (ICP-M S). Measurements were calibrated with an external calibration line using Zn and Cu s ingle-element ICP standards from SPEX CertiPrep (Metuchen, NJ). Cu-63 and Zn-66 wer e used to measure total copper and zinc in each sample as recommended by the instr ument manufacturer. Results were blank-subtracted and are presented on a wet weight basis. The method detection limit (Cu-63: 0.46 m g/l, Zn-66: 1.2 m g/l) was calculated following the procedures of Cle sceri et al. (1998). Data were not normally distributed. However, normal ity was attained using a logtransformation. The log-normal data were analyzed using a combination of linear regression and analysis of co-variance (ANCOVA) wit h the significance level set at a =0.05 (Neave & Worthington 1988, Zar 1996, Weinstei n et al. 2000, Myers & Well 2003, Viana et al. 2005).

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20 Results Table 1 lists mean concentrations of Zn and Cu for each species and location. Not all species were available from all locations. In part icular, sand seatrout were unavailable from the Little Manatee River, and tidewater mojarr a were not available from the Palm and Hillsborough Rivers. Based on their standard l ength, fish collected in Tampa Bay were all adult animals. Zinc concentrations were h ighest in the bay anchovy and tidewater mojarra. The lowest trace metal concentr ations were found in the striped mullet from Tampa Bay. Figures 2a and 2b show zinc and copper concentratio ns observed in muscle tissues of bay anchovy, sand seatrout, striped mullet and tidewate r mojarra. Zinc concentrations range over more than an order of magnitude, and it is evi dent that, for both Zn and Cu, tissue concentrations are strongly related to specimen sta ndard length. This observation mandated an analysis of concentration vs. length an d the analysis of co-variance in the statistical treatment of data. Geographic trends in trace metal concentrations in the Tampa Bay estuary range from higher concentrations in river-dwelling juvenile fi sh to lower concentrations in adult fish from the bay (Figure 3). Tidewater mojarra present the only exception to the dominant trend of higher trace metal concentrations in river fish (Table 1). Tidewater mojarra from the Alafia River have slightly lower Zn concentrati ons than adult mojarra collected in Tampa Bay. However, Cu concentrations do not show s ignificant differences between locations (Appendix C). Compared to other species in this study, tidewater mojarra have a very narrow range of Zn and Cu concentrations acr oss the different sampling locations. In all species except the tidewater mojarra, fish f rom the Hillsborough and Little Manatee Rivers have distinctly higher Zn concentrations tha n those from the bay. Because changes due to growth could contribute to t he differences between fish from Tampa Bay and the rivers, fish samples from the riv er sites were statistically analyzed

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21 without the adult samples. Copper concentrations s how no differences among the rivers. However, significant differences are found for Zn. The general trends that exist between the bay and the rivers persist even when only juven ile fish are considered. The overall trend indicates that concentrations of Zn are highe st in fish from the Hillsborough and Little Manatee Rivers. Palm River fish have interm ediate concentrations. Fish from the Alafia River generally have lower zinc concentratio ns than fish from the Little Manatee and Hillsborough Rivers. These results support the broader analyses which found higher trace metal concentrations in rivers compared to Ta mpa Bay. The general trend in trace metal concentrations is: Hillsborough River Littl e Manatee River Palm River > Alafia River > Tampa Bay. Data were analyzed to investigate interspecific tre nds in trace metal concentrations. Adult fish in Tampa Bay show very distinct, species -specific trends in Zn and Cu accumulation. Bay anchovy and tidewater mojarra hav e higher zinc concentrations than sand seatrout or striped mullet (Figure 4). Simila r trends are seen in Cu concentrations. There is a strong correlation between log Zn and lo g Cu concentrations in adult fish from Tampa Bay (R2=0.7521). The most prominent feature of the graph is the species-specific nature of the concentrations. Mullet have the lowes t Zn and Cu concentrations, with increasing concentrations, respectively, in sand se atrout, tidewater mojarra and bay anchovy. Other sites show species-specific relationships bet ween Zn and Cu, although the order of the species is different (Figure 5). The Little Ma natee River shows a pattern similar to that found in Tampa Bay. For the Hillsborough and Palm Rivers, striped mullet, not bay anchovy, has the highest Zn and Cu concentrations. Correlations between the two metals have R2 values ranging from 0.2109 in the Alafia River to 0.9038 in the Palm River. These linear slopes suggest there maybe be an optim al ratio between Zn and Cu in muscle tissue that is maintained even as concentrat ions increase.

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22 In order to investigate these changes further, quan titative depictions of metal concentrations were obtained using a logarithmic re lationship between metal concentration, MT, and standard length, l : log MT = AM + BM l-1 (1) and it is noted that equation (1) can be equivalent ly written, in exponential form, as MT = a 10B/ with a = 10A. The coefficients obtained in fits of the Figure 6 a,b data using equation (1) are given, respectively, as AZn = 0.571 0.03 and BZn = 19.6 +/1.06 (2) ACu = -0.672 0.03 and BCu = 16.7 +/-1.2 (3) Inspection of Figure 6 shows that as the inverse st andard length varies between approximately 0.003 and 0.05, Zn and Cu concentrati ons vary, on average, by a factor of ten. Furthermore, the slopes (BM) for Zn and Cu given in equations 2 and 3 are quit e similar. This implies that Zn/Cu concentration rati os for the data shown in Figure 3 will be relatively constant. Figure 7 shows Zn/Cu concentration ratios plotted a gainst inverse standard length. The best fit regression of these data is given as log (ZnT/CuT) = (1.253 0.0245) + (2.69 1.04) l -1 (4) The best fit result shown in Figure 7 and equation (4) indicates that, in contrast to the factor of ten variations for absolute concentration s of the trace metals, the Zn/Cu concentration ratio changes by only ~ 32% over the f ull range of standard lengths measured in this study. In absolute terms, the inte rcepts and slopes given in equation (4) translate to a range of Zn/Cu ratios between 18.5 a nd 24.4 as standard length ranges over

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23 approximately one order of magnitude. In view of th e relative constancy in the pooled log (ZnT/CuT) data shown in Figure 7, it is interesting to exam ine these data separated into the trends observed (Figure 8) for each of the spec ies examined in this work. Of the four regressions shown in Figure 8, only a s ingle species (striped mullet) exhibits a positive slope for log (ZnT/CuT) vs. standard length. Even in this case, however, the positive slope is attributable to the influence of seven unusually small mullet with an inverse standard length near 0.04. A regression for the remaining twenty seven mullet has a negative slope (log (ZnT/CuT) vs. standard length) as is the case for the remai ning species. Examination of Figure 8 suggests that diff erences among species for both ZnT/CuT ratios and standard lengths significantly contribu tes to the positive overall slope of the pooled data in Figure 7. Bay anchovies have a ZnT/CuT ratio on the order of 23.6 and relatively large inverse standard lengths. In c onjunction with a relatively large number of striped mullet that have small inverse st andard lengths (0.003 to 0.02) and relatively low ZnT/CuT ratios (ZnT/CuT ~ 12.5), the pooled data shown in Figure 7 have a weak positive slope. The comparisons shown in Figure 8 indicate that the re are small but distinct differences between the ZnT/CuT ratios of individual species. In turn, this implie s that ZnT/CuT ratios are actively regulated for each species (i.e., rela tively small variations are observed in a single species of fish collected in different locat ions). Table 2 provides a summary of ZnT/CuT ratios for each species of fish without considerin g the relatively weak influence of standard length on metal concentration ratios.

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24 Discussion Within the Tampa Bay estuary, consistently elevated concentrations of Zn and Cu were found in fish from the Hillsborough River. This are a is highly impacted by human activities and modifications, including a dam locat ed in the upper part of the river. Relations between human development and trace metal concentrations in fish, including Zn and Cu, have been found elsewhere. Studies on st riped mullet in Australia found that fish collected near power plants or other anthropog enically-impacted areas had higher levels of trace metals in muscle tissue than fish f rom undeveloped portions of the estuary (Kirby et al. 2001b). Direct comparison between Zn and Cu concentrations in the tissues of fish found in the Tampa Bay estuary and tissue c oncentrations in fish from other estuaries is difficult because only bay anchovy and striped mullet have a widespread distribution. In the event that comparisons with co nspecifics are not possible, comparisons can be made for members of the same fam ily. When necessary in such comparisons, trace metal concentrations were conver ted from dry weight to wet weight by dividing by five as recommended in the literatur e (Ache et al. 2000, Rodriguez-Sierra & Jimenez 2002). Striped mullet in Tampa Bay have similar Zn and Cu concentrations to those found in the Lake Macquarie estuary (Kirby et al. 2001a) but much lower concentrations than those found in striped mullet f rom the Camlik Lagoon in Turkey (Dural et al. 2006). The sites in Australia and Tu rkey are both subject to industrial influences. However, regulations had been enacted a t the Australian site that reduced power plant emissions. Mullet ( Mugil platanus ) from Uruguay had elevated Zn and Cu concentrations while striped weakfish ( Cynoscion guatucupa ) from the same study (Viana et al. 2005) had lower concentrations than t hose found in adult sand seatrout from Tampa Bay. Work in Puerto Rico (Rodriguez-Sierra & Jimenez 2002) showed that mojarra species had lower Cu and Zn concentration t han did tidewater mojarra in the Tampa Bay estuary. Work on anchovies ( Engraulis mordax ) in California indicates that Cu concentrations are lower in anchovies from Tampa Bay (Sydeman & Jarman 1998). Anchovies ( Engraulis encrosicholus ) in Turkey exhibited Zn concentrations similar to those measured in the anchovies of Tampa Bay, but h igher Zn concentrations were found

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25 in other fish (Guner et al. 1998). The rivers around Tampa Bay serve as nursery habita ts for juvenile fish. It has been suggested that geographic trends in trace metal con centrations are not due to differences between Tampa Bay and the rivers; rather they repre sent age related differences between juvenile fish from the rivers and adult fish found in Tampa Bay. Fish undergo many physiological and behavioral changes as they grow. However, the juvenile fish included in this study have complete digestive tracks and or gan systems, making it unlikely that these differences are due to physiological changes. Trace metal assimilation efficiency of juvenile fish is comparable to adult fish (Reinfeld er & Fisher 1994), which suggests that changes in prey items influence trace metals in the ir tissues. Ontogenetic diet shifts occur in many species of fish as increasing size al lows access to a wider variety of prey. Dietary shifts also occur as older fish leave the t idal river habitat for the main estuary where different prey species would be available. L ittle is known about the geographic variability of dissolved copper and zinc concentrat ions in Tampa Bay but work in other estuaries indicates that trace metal concentrations in the water column decrease as one moves away from the river source (Breuer et al. 199 9). Changes in dissolved metal concentrations could influence trace metal availabi lity to food webs via the trace metal content of plankton in the water column. A prominent feature in the Zn-Cu data set (Appendix A) is a species-specific trend in Zn and Cu accumulation (Figure 4). It has been noted that concentrations of Zn are generally higher than those for Cu in muscle tissue (Al-Yousuf et al. 2000, Asuga et al. 2006). However, species-specific trends have not be en reported. Trace metal assimilation efficiencies are similar for fish regardless of spe cies or trophic level (Wang 2002) making it unlikely that observed differences are due to me tabolic processes. Female fish can transfer trace metals to eggs. Research on sturgeon indicates that Zn and Cu concentrations in sturgeon eggs are approximately 1 1 mg/kg and 1.5 mg/kg respectively (Gessner et al. 2002). These concentrations are mu ch lower than the concentrations reported here, making maternal transfer an unlikely source of trace metals for juvenile

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26 fish. The main route for trace metal accumulation is diet (Bury et al. 2003). The influence of diet on trace metal accumulation indic ates that species-specific differences in trace metal concentrations are related to the diffe rent feeding habits of the fish. Bay anchovies feed on plankton but also ingest items fr om the epibenthic region (Peebles et al. 1996). Biomagnification of Zn has been documen ted in plankton-based food webs (Stemberger & Chen 1998). Additional work found el evated Zn in plankton feeders compared to animals from other trophic levels (Barw ick & Maher 2003), suggesting that elevated Zn concentrations in bay anchovies are rel ated to their link with the planktonic food web. The diet of the sandseatrout includes a substantial amount of bay anchovies but also includes other fish and crustacean species More generalized diets can reduce biomagnification of trace metals, possibly explaini ng the lower trace metal concentrations for sand seatrout (Wang 2002). The two remaining species feed on benthic infauna or algae. Striped mullet feed on b enthic algae and organic deposits in the sediments. Benthic diatoms are exposed to trace met als in the sediment. However, diatoms represent the first step in a bioaccumulati on pathway, implying that fish feeding directly on diatoms would not contain as much zinc as fish feeding at higher levels in the food web. The low trophic level utilized by the str iped mullet could explain their relatively low zinc concentrations. While tidewater mojarra also feed in the benthos, they prey on polychaetes, a predatory invertebrate that feeds on other animals and deposits in the sediment (Kerschner et al. 1985). This places the tidewater mojarra one or two trophic levels above the mullet. Polychaetes are a lso known to concentrate zinc in their tissues (Rainbow et al. 2006), providing a source o f elevated Zn to the tidewater mojarra diet. Elevated Zn has been found in other fish spe cies that feed on invertebrates (Papagiannis et al. 2004). This could explain why t idewater mojarra have elevated Zn and Cu concentrations, second only to the bay anchovy. Trace metal concentrations in the sediment are loos ely correlated with geographic trends in trace metal concentrations found in fish from th e Tampa Bay estuary (Long et al. 1991, Grabe 1997, MacDonald et al. 2004). The Hillsboroug h, Palm, and Alafia Rivers drain into Hillsborough Bay, located in northeast Tampa B ay. Sediments from Hillsborough

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27 Bay contain the highest concentrations of contamina nts due to a greater percentage of fine-grain sediment derived from nearby urban and i ndustrial areas (Long et al. 1991, MacDonald et al. 2004). Fish from the Hillsborough River generally had the highest Zn and Cu concentrations. McCain et al. (1996) found e levated concentrations of organic contaminant concentrations in fish and sediment fro m the Hillsborough and Palm Rivers compared to locations in the open bay. Metal enrichment in coastal sediments is significan t throughout Florida and is likely to continue as human development expands (Alexander et al. 1993). While Zn and Cu are toxic only at high concentrations, they co-vary wit h other trace elements like selenium (Kirby et al. 2001b), making them possible proxies of other inorganic contaminants. The loose association between historic concentrations o f trace metals in the sediments and Zn and Cu concentrations measured in fish from Tampa B ay raises the question of whether fish are impacted by decades-old contaminants in th e sediment. Recent work in Tampa Bay indicates that bioturbation by benthic shrimp r esults in a flux of metals to the sediment surface (Klerks et al. 2006). This indica tes that previously contaminated sediments can impact the current ecosystem. Diet i s the main pathway of metals into fish, and many of the fish in this study have links to benthic food webs. Future work should extend to analyses that examine the mechanis ms of contaminant uptake from sediments, focusing on all members of the food web. The addition of stable isotope data to these analyses, especially sulfur, could greatly expand our understanding of trace metal cycling in the food web.

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28 Table 1. Mean zinc and copper concentrations, m g/g wet weight one standard error. Bay anchovy Anchoa mitchilli Sand seatrout Cynoscion arenarius Tidewater mojarra Eucinostomus harengulus Striped mullet Mugil cephalus Zn (g/g) Cu (g/g) n Zn (g/g) Cu (g/g) n Zn (g/g) Cu (g/g) n Zn (g/g) Cu (g/g) n Tampa Bay 11.0 0.5 0.53 0.02 12 4.1 0.2 0.21 0.01 13 8.9 0.4 0.34 0.01 16 2.57 0.09 0.18 0.02 7 Hillsborough River 20 3 0.98 0.08 7 12.8 0.5 0.6 0.2 5 0 46.3 (43.4-49.1) 3.02 (2.66-3.37) 2* Palm River 18 3 0.69 0.19 4 7.1 (7.0-7.2) 0.36 (0.31-0.41) 2* 0 24 9 1.2 0.4 3 Alafia River 15 1 0.57 0.07 14 8.2 0.5 0.6 0.1 18 7.75 0.30 0.30 0.02 14 10 2 0.54 0.07 6 Little Manatee River 25 3 0.83 0.12 10 0 9 1 0.45 0.08 5 13 3 0.9 0.3 16 *Mean and range are presented in place of standard error due to low sample size

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29 Table 2. Average Log ([Zn]/[Cu]) the standard de viation Bay anchovy Anchoa mitchilli Sand seatrout Cynoscion arenarius Tidewater mojarra Eucinostomus harengulus Striped mullet Mugil cephalus Log ([Zn]/[Cu]) n Log ([Zn]/[Cu]) n Log ([Zn]/[Cu]) n Log ([Zn]/[Cu]) n Tampa Bay 1.320.07 12 1.290.09 13 1.420.10 16 1.150.10 7 Hillsborough River 1.300.07 7 1.420.22 5 0 1.19 (0.43 0.53) 2* Palm River 1.450.09 4 1.30 (-0.51 -0.39) 2* 0 1.20.1 3 Alafia River 1.40 0.19 14 1.250.09 18 1.370.09 14 1.100.13 6 Little Manatee River 1.490.08 10 0 1.310.05 5 1 0.06 16 *Mean and range are presented in place of standard error due to low sample size

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30 Figure 1. Map of sampling locations. Fish were ha rvested from Tampa Bay as well as the 1) Hillsborough, 2) Palm. 3) Alafia and 4) Litt le Manatee Rivers.

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31 Figure 2a. Zinc in fish from the Tampa Bay estuary Zn in all fishStandard Length (mm) 050100150200250300350 Zn ( m g/g) 0 10 20 30 40 50 60 Tampa Bay River Fish Figure 2b. Copper in fish from the Tampa Bay estua ry. Copper in all fishStandard Length (mm) 050100150200250300350 Cu ( m g/g) 0 1 2 3 4 Rivers Tampa Bay

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32 Figure 3. Log of the zinc concentration for each sp ecies at each sampling location Sand Sea TroutStandard Length (mm) 050100150200250300 Log [Zn] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Striped MulletStandard Length (mm) 050100150200250300350 Log [Zn] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Alafia Hillsborough Little Manatee Palm Tampa Bay Tidewater MojarraStandard Length (mm) 050100150200250300 Log [Zn] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Bay AnchovyStandard Length (mm) 050100150200250300 Log [Zn] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

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33 Figure 4. Log of the trace metal concentration plot ted by species for Tampa Bay. Tampa BayLog [Cu] -1.0-0.8-0.6-0.4-0.20.0 Log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet R2 = 0.7521 y= 1.3859 +1.1290X

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34 Figure 5. Log of trace metal concentrations plotted by species for each river. Alafia RiverLog [Cu] -0.8-0.6-0.4-0.20.00.20.40.6 Log [Zn] 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet Hillsborough RiverLog [Cu] -0.8-0.6-0.4-0.20.00.20.40.6 Log [Zn] 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Little Manatee RiverLog [Cu] -0.8-0.6-0.4-0.20.00.20.40.6 Log [Zn] 0.8 1.0 1.2 1.4 1.6 1.8 Palm RiverLog [Cu] -0.8-0.6-0.4-0.20.00.20.40.6 Log [Zn] 0.6 0.8 1.0 1.2 1.4 1.6 1.8

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35 Figure 6a. Log [Zn] vs inverse standard length. Log Zn vs invSLInverse Standard Length (mm-1) 0.000.010.020.030.040.050.06 Log Zn ( m g/g) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet R2 = 0.6926 y = 19.5902x + 0.5708 Figure 6b. Log [Cu] vs inverse standard length. Log Cu vs Inverse Standard LengthInverse Standard Length (mm-1) 0.000.010.020.030.040.050.06 Log Cu -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet y = 16.7x -0.672R2 = 0.5640

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36 Figure 7. Zn/Cu Tampa Bay. Log ([Zn]/[Cu]) vs Inverse Standard Length all dataInverse Standard Length (mm-1) 0.000.010.020.030.040.050.06 Log ([Zn]/[Cu]) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet R 2 = 0.04225 y = 2.6945x + 1.2528

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37 Figure 8. Log [Zn]/Log [Cu] for each species Log ([Zn]/[Cu]) vs Inverse Standard Length Sand Sea Trout Inverse Standard Length (mm-1) 0.000.010.020.030.04 Log ([Zn]/[Cu]) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Log ([Zn]/[Cu]) vs Inverse Standard Length Striped Mullet, all mulletInverse Standard Length (mm-1) 0.000.010.020.030.040.05 Log ([Zn]/[Cu]) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Alafia Hillsborough Little Manatee River Palm Tampa Bay Log ([Zn]/[Cu]) vs Inverse Standard Length Tidewater MojarraInverse Standard Length (mm-1) 0.0000.0050.0100.0150.020 Log ([Zn]/[Cu]) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Log ([Zn]/[Cu]) vs Inverse Standard Length Bay AnchovyInverse Standard Length (mm-1) 0.000.010.020.030.040.05 Log ([Zn]/[Cu]) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 R2 = 0.01464 y = -1.3654x +1.3085 R2 = 0.089997 y = -6.5468x + 1.4820 R2 = 0.1935 y = 4.0961x +1.0735 R2 = 0.004557 y = -1.1610x + 1.4247

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38 Chapter 3: Stable Isotopes as Tracers of Life Hist ory and Trophic Connections Abstract Estuarine-dependent fish employ a complex life cycl e that utilizes abundant estuarine production as well as marine habitats in the open e stuary and coastal waters. While estuarine-dependent fish share many life history ch aracteristics, each species has its own unique adaptations. To investigate differences among species, estuarinedependent fish in Tampa Bay were analyzed for stabl e isotope ratios and trace metal concentrations. Natural variations in stable isotop e ratios enable their use as tracers. Stable isotopes can be used to document diet shifts in juvenile fish and the movement of juvenile fish to their adult habitat. In Tampa Bay, both nitrogen and carbon stable isotopes provide information regarding the movement of fish within the estuary. Elevated d15N values in the river habitat utilized by juvenile fish make it possible to trace the life-history-related movement of estuarin e-dependent fish in Tampa Bay. Combined stable isotope and trace metal observation s show that, for copper and zinc, the impact of terrestrial influences can override t rophic bioaccumulation or biomagnification.

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39 Introduction Estuarine-dependent fish utilize a unique life hist ory strategy. Adult fish live in the open areas of the estuary and nearby coastal regions. S pawning generally occurs in the lower estuary, along the coast or offshore. Larval estuar ine-dependent fish migrate back to the estuary before settling in a nursery habitat (Able 2005). Fish settlement refers to the arrival of larval or juvenile fish to a substrate-b ased habitat from the pelagic environment where they were spawned. Carbon and nitrogen stabl e isotopes can be used to trace larval settlement and life history strategies of es tuarine-dependent fish (Herzka & Holt 2000) because they provide detail about the timing of habitat shifts and dietary changes (Herzka 2005). Due to instrument limitations for t his study, only carbon and nitrogen will be considered here. Carbon can be used as a g eographic tracer. Primary producers in rivers carry a carbon isotopic signature that is close to the d13C of soil organic matter, -26‰ (Peterson & Fry 1987). In the marine environm ent, dissolution of CO2 into surface waters results in a small fractionation resulting i n dissolved d13CO2 of ~1‰ (Fry 2006). The subsequent uptake of CO2 by phytoplankton during photosynthesis produces fu rther fractionation whereby d13C ranges from -19 to -24‰ (Fry 2006). In the Tamp a Bay estuary, coastal phytoplankton range from -19 to -2 2‰ (Hollander & Peebles 2004). Observations of d13C can differentiate between a food source influence d by terrestrial carbon and one that involves an estuarine or marine habitat. Nitrogen isotopes are generally used to determine an animal’s trophic pos ition within the food web. Because fish have higher d15N than their prey, an increase of approximately 3‰ per trophic level, it is possible to establish relative trophic positi ons. Comparison between locations must take into account the nitrogen and carbon isotopic values at the base of the food web. When comparing isotopic values between locations, i t is important to know the isotopic baseline for each site. The d15N or d13C baseline refers to the isotopic ratio of the primary producer that serves as the base of a local food web (Post 2002). Isotopic baselines, especially d15N, can be influenced by seasonal change and anthrop ogenic inputs from agricultural runoff or wastewater treat ment. It is important to monitor the d15N baseline for each location before making decision s regarding relative trophic

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40 position between locations (Hansson et al. 1997). The Tampa Bay estuary is surrounded by strong gradi ents of urban and agricultural development. The northeastern portion of the bay i s highly developed and impacted by anthropogenic pollution from urban runoff and an ac tive commercial shipping port. The southeastern edge of the bay is rural with large dr ainage fields for septic tanks as well as agricultural areas. Both urban development and agr icultural land use serve as possible sources of pollution in the bay. Pollution sources can include nitrogen enrichment from pasture runoff or water treatment plants, agricultu ral pesticides, power plant emissions, and trace metal pollution from marinas. With a ran ge of anthropogenic sources found around the bay, it is informative to combine invest igations of trace metal concentrations in estuarine-dependent fish with stable isotope inv estigations. The combination can provide insight into the geographic variability of trace metals and stable isotopes and elucidate links between trace metal concentrations and local food webs While estuarine-dependent fish share a variety of l ife history characteristics, they span a wide range of sizes, feeding strategies, and prey t ypes. In order to include a spectrum of size, trophic level, and feeding strategies, four s pecies were selected for geographic and trophic analyses: striped mullet ( Mugil cephalus ), tidewater mojarra ( Eucinostomus harengulus ), bay anchovy ( Anchoa mitchilli ), and sand seatrout ( Cynoscion arenarius ). Sampling these species from the tidal rivers and th e bay itself, allows examination of geographic trends in stable isotopes, trophic relat ions of each species, and links between trace metal accumulation and local food webs.

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41 Methods Fish were collected by the Florida Fish and Wildlif e Research Institute during monthly monitoring surveys in Tampa Bay and associated tida l rivers. Samples were stored on ice until they could be transferred to a freezer. Tissu e collection took place in a class-100 clean air laboratory according to protocols establi shed by the U.S. Environmental Protection Agency (2000). Nitrile gloves were used to handle the fish. Tissue collection for trace metals was performed with a stainless ste el scalpel on a cutting board covered with aluminum foil. Fish were scaled followed by t he removal of the upper layer of skin. Muscle tissue was then collected. While more than 6 00 individual fish were collected, juvenile fish samples were pooled to obtain suffici ent tissue for analysis. Samples for trace metal analyses were stored frozen in polyethy lene bags until digestion. Samples for stable isotope analyses were stored frozen in alumi num foil until they could be prepared for analyses. Samples were prepared for trace metal analyses usin g the hot block digestion method from the Florida Department of Environmental Protec tion (Method MT-060-1.3) as discussed in Chapter 2. Once digested, samples wer e analyzed with an Agilent Technologies 4500 series 200 inductively coupled pl asma mass spectrometer (ICP-MS). Samples for stable isotope analyses were dried at 5 0C for 24 hours. Dried tissue was ground into a fine powder using a mortar and pestle Duplicate portions of each sample where weighed in tin cups on a microbalance. Sampl es were combusted using a Carlo Erba 2500 Series I elemental analyzer. Carbon and nitrogen isotopic ratios were measured using a continuous-flow outlet system on a Finnigan MAT Delta Plus XL stable isotope mass spectrometer. All C and N isot opic analyses were run in duplicate, and the spectrometer was calibrated using one of tw o standard reference materials (SRM) from the National Institute of Standards and Techno logy (NIST): SRM 1577b, bovine liver, or SRM 1570a, spinach leaves. These standar ds are not certified for isotopic composition, but their isotopic composition was est ablished in the laboratory using repeated analyses of the material to provide isotop ic guidelines. Carbon isotope ratios

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42 measured in samples were standardized to the isotop ic ratios of Pee Dee Belemnite, a carbonate from the Cretaceous Pee Dee formation loc ated in South Carolina, USA. PDB is the accepted zero point standard for carbon isot ope abundances. Atmospheric nitrogen was used as the zero point standard for N isotopes. Carbon and nitrogen isotopic values are expressed in d notation according to the following equation:G d X = [(Rsample/Rstandard – 1)] 1000G(1)G where X is 13C or 15N and R is the value of 13C/12C or 15N/14N respectively. The Kolmogorov-Smirnov normality test indicated tha t stable isotope data were normally distributed. Trace metal data were log-normal in d istribution. Stable isotope data were analyzed using ANOVA with Tukey’s Test for multiple comparisons with the significance level set at a =0.05. Combined trace metal and stable isotope da ta were analyzed using non-metric multidimensional scaling (MDS) procedures found in the software package PRIMER (Plymouth Routines in Multi variate Ecological Research) (Clarke et al. 2006).

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43 Results Bay anchovy adults and juveniles have the most terr estrially influenced carbon of all species in this study (Figure 9). Carbon isotopic v alues for bay anchovies range from -28 to -20‰, indicating that adult bay anchovies remain in areas of terrestrial influence through out their life. By comparison, other speci es show mean d13C carbon values close to marine phytoplankton as adults and terrestrially derived carbon as juveniles (Figure 10, Table 3). Carbon isotopic values of juvenile sand s eatrout range from -22.51‰ in the Hillsborough River to -20.37‰ in the Palm River, wh ile adult sand seatrout have mean d13C values of -18.45‰. A similar trend is seen in ti dewater mojarra, where juvenile d13C values range from -23.13 to -21.41‰, and adults h ave enriched values with an average of -16.32‰. Nitrogen isotopes indicate a d istinct trophic separation (Figure 11, Table 3). Bay anchovy and sand seatrout have d15N ranging from 12-15‰ ,while values for striped mullet and tidewater mojarra range from 9-12‰ (Figure 11, Table 3). With the established life history of these species, and the relative trophic position of each species determined through stable isotope analyses, non-metric multidimensional scaling (MDS) was applied to the full data set. Zinc and c opper concentrations, d13C and d15N and standard length were used as variables. MDS ana lysis allows all variables in a data set to be related to each other. Results of MDS ar e evaluated using 2-dimensional plots of a multidimensional relationship. The accuracy o f the conversion to 2 dimensions is indicated by the stress value on each plot. The rel ative position of points on the graph indicates relatedness. Points that are close toget her share similarities with respect to the variables under consideration. Points on the oppos ite sides of the graph are most different. Variables of interest are then overlaye d on the plot in order to see trends related to the arrangement of data points. The MDS plot indicates that the most distinct group is composed of mullet and anchovies (lower right quadrant, Figure 12). Addition of Zn a nd Cu concentrations to the plot (Figure 13 & 14) shows that the distinct anchovy an d mullet on the right side of the

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44 figure also have elevated trace metal concentration s. Standard length data reveal that the fish with elevated trace metal concentrations are a mong the smallest fish in the study (Figure 15). Surprisingly, overlaying d15N onto the plot shows no clear trends (Figure 16). The d13C overlay (Figure 17) shows few trends except a pos sible slight influence of terrestrial carbon influence on the distinctive fis h. Introduction of seasonal trends to the plot reveals that the small distinctive fish were c aught exclusively during the dry season (Figure 18). To further investigate the relationship between d13C and trace metal concentrations, values for these variables were plotted using conve ntional 2-dimensional plots. Plots of d15N against season revealed elevated d15N in sand seatrout and bay anchovy from the Alafia River in the dry season (Figure 19). The exi stence of seasonal trends in the bay (Figure 20) was weak or inconclusive. Attempts to correlate seasonal change with trace metal concentrations revealed no significant trends With the exception of striped mullet, plots of d15N against the logarithm of Zn and Cu concentrations (Figure 21) did not reveal significa nt trends between nitrogen ratios and trace metal concentrations. Comparisons between d13C and trace metal concentrations proved to be much more informative. A general trend of decreasing trace metal concentrations with increasing d13C can be seen in Figures 21 & 22. Sand seatrout and striped mullet undergo large growth and habitat shi fts during their life. This shift is not seen in the bay anchovy which remains connected to a habitat influenced by terrestrial carbon throughout its life cycle.

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45 Discussion Fish in Tampa Bay have distinct interspecific troph ic relationships that are defined by stable isotope ratios. Trophic relationships found in adult fish (Figure 23) are similar to those found in juvenile fish from the rivers (Figur e 24). The bay anchovy and sand seatrout, with relatively elevated d15N, are almost a full trophic level higher than the striped mullet or tidewater mojarra. The nitrogen d ata for mullet and mojarra are scattered and statistical tests do not separate the m into different groups. This isotopic separation reflects the feeding habits of these fou r species. Tidewater mojarra and striped mullet are both benthic feeders, while bay anchovie s rely on plankton and seatrout are primarily piscivorous. Surprisingly, the bay ancho vy has d15N values which are similar to one of its predators, the sand seatrout. The rel ative trophic positions of the species investigated in this study are established very ear ly in the organisms’ life histories. Statistical analyses of stable isotope ratios in ju venile and adult fish are similar. Even in the nursery rivers, bay anchovy and sand seatrout h ave higher d15N than tidewater mojarra and striped mullet. The four species are a lso distinguishable by their carbon isotopic values. Bay anchovy have d13C values that are strongly influenced by terrestria l sources. Carbon isotope values of sand seatrout, striped mullet and tidewater mojarra are almost 4 ‰ higher than those of the bay anchovy. Th is reflects the use of marine habitats by sand seatrout, striped mullet and tidewater moja rra. Adult bay anchovies maintain their association with rivers as adults, which are often areas of high copepod abundance (Peebles et al. 199 6). While bay anchovies are an important prey item for sand seatrout, the complex planktonic food web that is utilized by bay anchovies has many trophic steps, creating enri ched d15N values. As juvenile bay anchovies leave the rivers, a decrease in d15N is observed in adults, consistent with fish moving away from the elevated riverine d15N (Figure 9). In contrast, the smallest sand seatrout have d13C values influenced by terrestrial carbon (Figure 1 0), with fish greater than 100 mm shifting to a marine carbon source. Th e known life history of sand seatrout is inconsistent with the trophic information reveal ed by nitrogen isotopes. In sand

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46 seatrout, a clear trend of increasing d15N values with increasing length is observed for lengths less than 150 mm (Figure 11). As juvenile sand seatrout grow, they consume larger and larger prey, increasing their trophic le vel. Once fish attain an adult length near 150mm, there is a gradual decrease in the d15N of the fish. Although this indicates that adult sand seatrout switch to prey of a lower troph ic level, diet studies do not support that interpretation (Peebles 1996). It has been reporte d that d15N in Tampa Bay is much lower than values found in the rivers (Hollander & Peeble s 2004). Therefore fish moving into the bay will utilize a food web based on lower d15N values than those in the river, explaining the lower d15N values in adult fish. Shifts in d15N values also correspond to habitat changes in the tidewater mojarra and stripe d mullet (Figure 11). This suggests that elevated d15N values in the rivers allow stable nitrogen isotop es to be used as a tracer for the life history of estuarine-dependent fish. Striped mullet life history can also be traced usin g stable isotope data. A distinctive feature in the striped mullet d15N graph is the obvious cluster of very small mullet near a d15N of 8‰. This is well below the 12‰ value seen in the other juveniles. These small mullet are querimana-stage juveniles. Querimana ref ers to mullet that have entered the estuary but not yet settled into a nursery river. B ecause mullet spawn over the continental shelf, young mullet entering the estuary retain the nitrogen signature associated with an offshore nitrogen source. This interpretation is s upported by d13C values which indicate a marine carbon source for their diet (Figure 10). J uvenile fish grow quickly and lose their marine signature as they add biomass during growth (Herzka & Holt 2000). This explains the decrease in d13C values for juvenile mullet of the next size class Seasonal changes have a strong influence on the riv er and bay environments. Florida’s seasons principally consist of dry and wet seasons. The wet season spans the months of June-September (Schmidt & Luther 2002). Juvenile fi sh from the Alafia River show seasonal trends in their stable isotope ratio. San d seatrout and bay anchovy have enriched d15N values during the dry season (Figure 12). Some e vidence of enriched nitrogen during the dry season was found in the Lit tle Manatee River; however,

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47 insufficient sample numbers prevent further analyse s among other rivers. Seasonal trends were not as prominent in the bay (Figure 13). In fa ct, data from the tidewater mojarra suggest that fish in Tampa Bay have depleted nitrog en during the dry season, in contrast to the trend in the Alafia River. The strong season al signal seen in the Alafia River raises the question of whether similar changes would be ex pected in the other rivers and what impacts might be observed for trace metal concentra tions. Given that terrestrial d13C values are associated with elevated zinc concentrat ions, changes in trace metal concentrations could be expected during the wet sea son when increased runoff from precipitation brings an influx of terrestrially der ived carbon into the rivers. There is a clear seasonal trend in the Alafia isotope data, ho wever, no clear trends emerged between seasonality and trace element concentrations. Othe r variables that change with season such as standard length make making seasonal impact s difficult to interpret. A large body of research has attempted to link trac e element concentrations to trophic level. For some metals, like mercury, the link is relatively clear (Strom & Graves 2001, Evans & Crumley 2005). However, no link between tro phic level and trace metal concentrations was observed in this study. It is p ossible that elevated d15N values in the rivers masked trophic relations. Elevated d15N in the rivers make it appear that juvenile sand seatrout are feeding on a higher trophic level than adults. However, even among samples from the same river and season, it was not possible to establish a relationship between d15N and trace element concentrations. While trends were expected between trace metal accu mulation and d15N, only d13C proved to be predictive for metal accumulation. Fi sh with the highest Zn concentrations in the study are bay anchovy, and d13C values for these fish indicate they rely on terrestrial based food webs throughout their life. In contrast, striped mullet and sand seatrout markedly change from a dependence on terre strial carbon as juveniles to marine carbon as adults. As d13C isotopic values move towards values representing coastal phytoplankton, a decrease in zinc concentrations is seen in fish tissues (Figures 21 & 22). The lack of correlation between stable isotopes and trace metal concentrations is

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48 puzzling. Both trace metal accumulation and stable isotope ratios are based on diet. Diet is the primary pathway for trace metal accumulation and the isotopic composition of an animal follows a very predictable fractionation tha t is based on the isotopic composition of the animal’s diet. Sulfur isotopes would provid e additional information about the influence of the sediments on the trace metal conce ntrations. However, instrumentation for sulfur isotopic analyses were not available in this study. Stable isotopes are effective tracers of the life h istory of estuarine-dependent fish. This study indicated that distinct shifts in diet and ha bitat are closely linked to stable isotope ratios. Movements are most distinct for the sand s eatrout and striped mullet because their life history involves distinct changes in diet and habitat. The most complete account of life history was seen in the striped mullet because samples included animals that had not yet settled. Even with a species like the bay anch ovy, which remain mostly in the bay, smaller shifts are seen as fish move away from rive rs. Changes in nitrogen isotopic values have been seen in fish that live in a gradie nt that includes a source of elevated d15N such as a water treatment plant (Schlacher et al. 2005). There have also been indications that migratory juvenile fish may have i ntermediate nitrogen ratios if they live in an environment with geographic variations in the d15N baseline (Hansson et al. 1997). This observation is extended in the current work th rough the use of many size classes and species. Future work should include sulfur isotopic ratios in order to evaluate the influence of sedimentary contamination on trace met al concentrations in fish.

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49 Figure 9. Stable isotope ratios in the bay anchovy from all locations. Bay Anchovyd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Alafia Hillsborough Little Manatee Palm Tampa Bay

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50 Figure 10. Carbon stable isotopes for each species at each sampling location. Sand Sea Trout all locationsStandard Length (mm) 050100150200250300 d13C -28 -26 -24 -22 -20 -18 -16 -14 -12 Bay Anchovy all locationsStandard Length (mm) 152025303540455055 d13C -28 -26 -24 -22 -20 -18 -16 -14 -12 Alafia Hillsborough Little Manatee Palm Tampa Bay Tidewater Mojarra all locationsStandard Length (mm) 405060708090100110 d13C -28 -26 -24 -22 -20 -18 -16 -14 -12 Striped Mullet all locationsStandard Length (mm) 050100150200250300350 d13C -28 -26 -24 -22 -20 -18 -16 -14 -12 d13C and Standard Length

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51 Figure 11. Nitrogen stable isotopes for each specie s at each sampling location. Sand Sea Trout all locationsStandard Length (mm) 050100150200250300 d15N 6 8 10 12 14 16 Tidewater Mojarra all locationsStandard Length (mm) 405060708090100110 d15N 6 8 10 12 14 16 Bay Anchovy all locationsStandard length (mm) 152025303540455055 d15N 6 8 10 12 14 16 Alafia Hillsborough Little Manatee Palm Tampa Bay Striped Mullet all locationsStandard Length (mm) 050100150200250300350 d15N 6 8 10 12 14 16 d dd d15N and Standard Length

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52 Figure 12. Multidimensional plot showing the relat ion among species. NormaliseResemblance: D1 Euclidean distance A A A A A A A A A A A A C C C C C C C C C C C C C C C C E E E E E E E E E E E E E E M M M M M M M M M M M M M M M M A A A A A C C C C C M M A A A A A A A A A A E E E E E M M M M M M A A A A C C M M M A A A A A A A A A A C C C C C C C C C C C C E E E E E E E E E E E E E E M M M M M M M2D Stress: 0.01

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53 Figure 13. Multidimensional plot including zinc co ncentrations ( m g/g). Figure 14. Multidimensional plot including copper concentrations ( m g/g).

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54 Figure 15. Multidimensional plot including standar d length (mm). Figure 16. Multidimensional plot including nitrogen isotopes. NormaliseResemblance: D1 Euclidean distance d15N 6 9 12 15 A A A A A A A A A A A A C C C C C C C C C C C C C C C C E E E E E E EE E E E E E E M M M M M M M M M M M M M M M M A A A A A C C C C C M M A A A A A A A A A A E E E E E M M M M M M A A A A C C M M M A A A A A A A A A A CC C C C C C C C CC C E E E E E E E E E EE E E E M M M M M M M2D Stress: 0.01 NormaliseResemblance: D1 Euclidean distance St Lengths 40 160 280 400 A A A A A A A A A A A A C C C C C C C C C C C C C C C C E E E E E E E E E E E E E E M M M M M M M M M M M M M M M M A A A A A C C C C C M M A A AA A A A A A A E E E E E M M M M M M A A A A C C M M M A A A A A A A A A A C CC C C CC C C CC C E E E E E E E E E EE E E E M M M M MMM 2D Stress: 0.01

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55 Figure 17. Multidimensional plot including carbon isotopes. Figure 18. Multidimensional plot including seasona l data. NormaliseResemblance: D1 Euclidean distance d13C -28 -22 -16 -10 A A A A A A A A A A A A C C C C C C C C C C C C C C C C E E E E E E E E E E E E E E M M M M M M M M M M M M M M M M A A A A A C C C C C M M A A AA A A A A A A E E E E E M M M M M M A A A A C C M M M A A A A A A A A A A C CC C C CC C C CC C E E E E E E E E E EE E E E M M M M MMM 2D Stress: 0.01 NormaliseResemblance: D1 Euclidean distance season D W A A A A A A A A A A A A C C C C C C C C C C C C C C C C E E E E E E E E E E E E E E M M M M M M M M M M M M M M M M A A A A A C C C C C M M A A A A A A A A A A E E E E E M M M M M M A A A A C C M M M A A A A A A A A A A C C C C C C C C C C C C E E E E E E E E E E E E E E M M M M M M M2D Stress: 0.01

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56 Figure 19. Seasonal trends in carbon and nitrogen stable isotopes in the Alafia River. Red indicates the dry season. Blue indicates the w et season. Bay Anchovyd13C -28-26-24-22-20-18-16 d15N 10 12 14 16 May 05 Jul 05 Oct 05 Jan 06 Unk late 05-06 Sand Sea Troutd13C -28-26-24-22-20-18-16 d15N 9 10 11 12 13 14 15 16 Jan 06 May 05 Aug 06 Sep 06 Oct 05 Alafia River Figure 20. Seasonal trends in carbon and nitrogen s table isotopes in Tampa Bay. Red indicates the dry season. Blue indicates the wet s eason. Sand Sea Troutd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Feb 06 Aug 06 Tidewater Mojarrad13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Jun 05 Oct 05 Tampa Bay

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57 Figure 21. Log [Zn] plotted against carbon and nitr ogen isotopic ratios for striped mullet. d13C -28-26-24-22-20-18-16-14-12 log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Striped Mullet querimana excludedd15N 6810121416 log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Figure 22. Log [Zn] plotted against carbon and nitr ogen isotopic ratios for sand seatrout. Sand Sea Trout all datad15N 6810121416 Log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 d13C -28-26-24-22-20-18-16-14-12 Log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

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58 Figure 23. Stable isotope ratios in estuarine-depe ndent fish species from Tampa Bay. Tampa Bay all speciesd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet

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59 Figure 24. Stable isotope ratios in estuarine-depe ndent fish species from river sites. Alafia River all speciesd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet Hillsborough River all speciesd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Little Manatee River all speciesd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Palm River all speciesd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16

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60 Table 3. Mean d13C and d15N standard error. Bay anchovy Anchoa mitchilli Sand seatrout Cynoscion arenarius Tidewater mojarra Eucinostomus harengulus Striped mullet Mugil cephalus d13C d15N n d13C d15N n d13C d15N n d13C d15N n Tampa Bay -23.2 1.2 12.00 0.58 10 -18.45 0.21 12.77 0.19 12 -16.32 0.67 10.17 0.44 14 -18.0 1.1 9.26 0.40 7 Hillsborough River -23.08 0.51 13.52 0.31 5 -22.5 1.0 12.15 0.84 5 -19.45 0.16 8.79 0.02 2 Palm River -21.02 0.51 14.96 0.26 4 -20.37 0.69 14.0 1.3 2 -17.2 1.0 9.91 0.87 3 Alafia River -21.23 0.25 13.56 0.19 13 -20.95 0.35 12.72 0.37 17 -21.41 0.81 11.42 0.43 14 -21.23 0.53 10.55 0.26 16 Little Manatee River -24.16 0.66 13.33 0.14 10 -23.13 0.90 12.62 0.35 5 -21.16 0.53 10.69 0.73 6 *Mean and range are presented in place of standard error due to low sample size

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61 Summary Differences in tissue trace metal concentrations we re found between locations. Animals living in the bay exhibited lower trace metal conce ntrations than animals living in the rivers. Slight differences in trace metal concentr ations were noted between river sites, with the Hillsborough River emerging as a site with elevated zinc. Species-specific trends were found in trace metal accumulation, possibly du e to diet. Age may be a factor in trace metal accumulation as younger animals living in the rivers have higher trace metal concentrations than adults. This could be due to o ntogenetic changes in feeding, possibly a higher feeding rate, or geographic differences in ambient zinc and copper concentrations at different locations. The higher concentrations in juvenile fish are not maintained in adult animals. The ratio of Zn:Cu i n fish tissues provides intriguing insight into trace metal concentrations in these sp ecies. The ratio is highly conserved between species, even when absolute concentrations vary over an order of magnitude. Trophic relationships that exist in adulthood are e stablished early in the life of the fish. Life history changes are distinct in estuarine-depe ndent fish and can be traced using stable isotopes. Observations of trace metal conce ntrations and stable isotopes indicated that increased influence of terrestrial carbon is r elated to elevated trace metal concentrations. It was surprising to see similar t rophic levels between the bay anchovy and sand seatrout given the well established predat or-prey relationship between these species. Mini-mullet (querimana stage) carry disti nct trace metal and isotopic signatures that reflect their recent life history as offshore larva. With the exception of nitrogen isotopes, many facto rs influence the accumulation of trace metals in estuarine dependent fish. Geographic dif ferences in trace metal concentrations

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62 impact Zn and Cu accumulation in fish across the ba y. However, terrestrial influence and its impact on the diet of each fish are the best pr edictors of trace metal concentrations in tissues. Trace metals and stable isotopes are useful for stu dy of estuarine-dependent fish. Future work should include more extensive sampling of juve nile fish to investigate exactly when changes occur. The integration of trace metal conc entrations with growth models could provide insight into the shift from elevated metal concentration in young fish to lower concentrations in adults. Future work should also include more adult species from additional locations to expand baseline data on tra ce metals within the bay.

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70 Appendices

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71 Appendix A. Additional Sample Data Table 4: All Sample Data for Copper, Zinc, d15N and d13C Sample ID Cu 63 Cu 65 Zn 66 Zn 68 d dd d 15 N d dd d 13 C A505A01 0.395 0.2756 8.5334 8.3696 14.025692 21.49558 A505A02 0.4034 0.3029 10.032 9.9063 13.822692 21.64958 A505A03 0.4296 0.3347 9.4073 9.1385 14.064692 22.81208 A507A01 1.403 1.3961 13.401 13.081 12.23 21.98 A510A03 0.6121 0.5931 9.6772 9.2272 13.56 21.12 A507A02 0.4729 0.4387 16.033 15.952 A510A01 0.5431 0.3447 11.786 11.586 13.7825 19.84562 A510A02 0.5612 0.3562 11.896 11.779 13.6695 20.19362 A5UUA01 0.3682 0.3024 18.07 17.965 13.1895 21.06162 A5UUA02 0.5183 0.4319 16.013 15.859 13.51 21.56962 A5UUA03 0.4069 0.3408 21.113 20.843 13.5525 21.88812 A601A01 0.6925 0.5754 19.093 18.981 14.400692 20.31183 A601A02 0.6741 0.5557 18.72 18.678 14.172692 20.06433 A601A03 0.6912 0 .5786 19.333 19.301 C506A01 0.4212 0.3574 8.9874 9.0195 C510A04 0.5149 0.5124 8.6364 8.6542 13.86 21.06 C510A05 0.3179 0.2578 5.7842 5.6884 14.23 19.85 C505A01 0.4216 0.3777 7.2433 6.9117 13.786192 21.75008 C505A02 0.4832 0.3953 7.7279 7.4936 1 3.538192 21.15958 C601A01 0.1946 0.1363 3.9798 3.9238 15.492692 19.68358 C510A02 0.6814 0.5301 11.863 11.811 13.67525 20.79491 C510A03 0.461 0.3392 8.6976 8.6587 13.50675 21.31941 C609A02 0.5248 0.5248 9.4756 9.4295 10.454077 22.90677 C609A05 0.4 339 0.4339 7.1736 7.1935 11.64825 19.65191 C609A06 0.6635 0.6635 7.059 7.1678 10.63575 22.19491 C609A07 12.75375 21.73791 C609A04 0.6149 0.5332 8.2914 8.2339 11.82975 22.25241 C608A01 0.2919 0.2383 7.755 7.5279 11.97925 18.81191 C608A02 0 .3167 0.2506 7.136 6.9296 12.14075 18.79641 C609A01 0.8333 0.8333 11.14 10.656 10.58325 23.50541 C609A03 0.4857 0.4857 9.3973 9.1111 11.47775 21.49341 C510A01 0.592 0.4773 10.988 10.923 14.58 19.105 E506A01 0.4589 0.4566 7.3362 7.1981 11.73 25.49 E506A02 0.3916 0.3897 7.131 7.139 12.101 25.96612 E506A03 0.4079 0.364 8.0576 8.5896 12.0015 25.76212 E506A04 0.4799 0.4093 10.042 9.8999 12.098 26.70362 E608A09 0.2263 0.1839 6.3522 6.4295 11.71725 19.70191 E608A10 0.2012 0.1637 4.9823 4.6006 11. 43225 19.33541 E608A05 0.338 0.2591 8.2863 8.2991 11.290577 19.57927 E608A06 0.282 0.2109 8.037 7.9491 10.913577 19.16027 E608A07 0.4273 0.3644 7.4397 7.4028 11.092077 19.31077 E608A08 0.278 0.2177 7.7635 7.6231 11.374077 19.38177 E608A01 0.2958 0.2389 8.0979 7.9243 10.99925 19.71491 E608A02 0.2696 0.2076 8.1337 7.9269 10.96025 21.04691

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72 Appendix A. (Continued) Sample ID Cu 63 Cu 65 Zn 66 Zn 68 d dd d 15 N d dd d 13 C E608A03 0.3024 0.2253 8.3922 8.2447 11.228577 19.35077 E608A04 0.3018 0.2235 7.7091 7 .4988 10.986077 19.23077 M601A01 1.3489 1.2141 31.505 31.897 8.4141923 17.91783 M601A02 1.396 1.2613 32.587 32.708 8.0956923 17.65933 M606A01 0.498 0.4593 4.7052 4.7367 11.462346 22.86062 M606A02 0.4631 0.4154 5.6236 5.7365 10.574346 20.39162 M60 6A03 0.4874 0.443 5.8309 5.8482 11.063346 20.61412 M606A04 0.4919 0.4457 5.1951 5.2395 11.598846 21.60812 M606A05 0.4607 0.4394 5.2904 5.3412 10.972346 18.27212 M606A06 0.5597 0.5597 5.7551 5.6887 11.159346 21.45462 M606A07 0.522 0.5017 6.0052 6.09 42 10.855346 21.31712 M606A08 0.5367 0.5156 7.0788 7.1269 10.840846 21.11512 M606A09 0.494 0.4542 6.6246 6.7107 11.508346 21.60712 M606A10 0.5538 0.534 6.6869 6.7687 10.622346 20.26612 M604A01 0.8506 0.813 11.972 12.016 10.948577 23.47677 M604A02 0.8556 0.8178 9.4549 9.5546 10.778577 23.96427 M604A03 0.6407 0.613 9.3978 9.4303 10.511077 25.50327 M608A01 0.6753 0.6477 4.9902 4.8603 9.3800769 21.60927 A506C01 0.7107 0.5906 17.354 17.386 8.7199167 21.47392 A507C03 0.5858 0.4952 12.466 12.327 10.797192 20.71558 A507C01 0.4692 0.3636 15.902 15.714 12.347 21.71462 A507C02 0.4842 0.3885 13.894 13.754 12.192 22.00412 C602G01 0.238 0.2137 3.6268 3.5931 12.967308 18.92525 C602G02 0.1807 0.1551 3.7827 3.7263 12.625808 17.73625 C602G03 0.1753 0.1469 4.3217 4.2922 12.432808 18.21125 C602G04 0.2356 0.2024 3.6586 3.5902 12.530808 17.42775 C602G05 0.2292 0.192 4.5067 4.4453 12.648308 19.08025 C602G06 0.2216 0.195 3.6651 3.597 11.403808 17.24675 C602G07 0.2469 0.2195 3.9752 3.9285 12.217808 18.33025 C602G08 0.2336 0.1994 3.7112 3.6583 12.519808 19.60725 C602G09 0.1708 0.1468 4.3461 4.2748 13.400808 19.26325 A510H01 1.1087 1.1033 25.628 25.659 14.02 21.94 A510H02 0.6578 0.6061 11.4 11.437 14.140917 21.85892 A510H03 0.949 0.9444 15.2 44 15.215 13.369417 23.23642 A512H01 1.0273 0.9129 20.507 20.299 A512H02 1.282 0.9757 30.042 30.853 12.399577 24.38777 A512H03 0.8304 0.6312 15.3 15.259 A512H04 1.0333 0.833 25 25.567 13.673917 23.95192 C507H01 1.243 1.237 13.497 13.404 14.58 19.1 C510H01 0.3187 0.2414 11.204 11.124 13.63 21.12662 C510H02 0.3981 0.3961 11.741 11.638 11.177 23.84312 C510H03 0.3646 0.3628 13.526 13.393 11.334 23.65912 C510H04 0.4603 0.4581 13.8 13.705 10.051 24.73312 M601H01 2.6646 2.503 43.363 43.553 8. 7756923 19.28133 M601H02 3.3699 3.1949 49.141 49.783 8.8066923 19.60983 A508L01 0.7052 0.6815 14.952 14.785 12.719692 24.13758 A505L01 0.8644 0.7588 25.98 25.982 12.586192 26.76883

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73 Appendix A. (Continued) Sample ID Cu 63 Cu 65 Zn 66 Zn 68 d dd d 15 N d dd d 13 C A505L02 0.5994 0.5068 18.307 18.169 13.160192 25.52483 A505L04 0.5051 0.4312 17.177 17.155 13.303692 25.14183 A505L05 0.5014 0.4367 17.849 17.728 13.511692 25.96883 A505L06 0.4777 0.4264 18.732 18.699 13.568692 26.02433 A505L03 0.5963 0. 4931 20.94 20.879 14.094692 20.75533 A601L01 1.1897 0.7268 34.863 34.858 13.496417 23.55042 A601L02 1.3267 1.0103 34.615 34.485 13.513917 21.73192 A601L03 1.5273 1.3523 45.508 45.591 13.315917 21.96792 E508L02 0.45 0.3945 8.564 8.5999 11.79 25.1 E508L01 0.7529 0.6433 14.251 14.4 11.785 25.105 E604L01 0.3608 0.3196 7.489 7.7168 13.267417 21.09642 E604L02 0.3635 0.3201 7.3052 7.4258 13.427917 21.04592 E604L03 0.3324 0.2728 8.2676 8.4404 12.828417 23.28342 M508L05 0.5552 0.4749 9.5831 9.6142 11.39 22.63 M508L01 0.8072 0.7072 14.286 14.207 11.196192 22.31283 M508L02 0.5668 0.4873 9.9458 9.9286 11.681 21.921 M508L03 0.4896 0.4157 8.511 8.4424 11.60 20.48 M508L04 0.5557 0.4831 7.5471 7.441 11.246667 19.66722 M601L01 2.4827 2.0484 29.457 29.558 7.0436923 19.92283 A509P01 1.2672 1.261 26.543 26.355 14.23 22.49 A601P01 0.5061 0.3898 15.773 15.572 15.4405 20.94162 A601P02 0.5123 0.3696 16.779 16.565 15.0715 20.37912 A601P03 0.459 0.3121 13.16 13.163 15.0965 20.27812 C507P01 0.4088 0.3264 7.2126 7.2037 12.68 21.06 C601P01 0.3075 0.1906 6.9758 6.9044 15.305692 19.67358 M507P01 0.492 0.3895 5.7751 5.748 11.63 15.16 M601P01 1.6544 1.4146 33.583 33.776 9.3096923 18.30833 M601P02 1.5738 1.2484 31.171 31.382 8.7831923 18.09483 C5 11T01 0.2782 0.2396 4.9327 4.9804 A510T01 0.4973 0.3764 6.8694 6.8792 A606T01 0.5231 0.4924 9.1293 9.182 13.334846 23.88212 A606T02 0.4445 0.4148 10.08 10.141 13.076346 23.11412 A606T03 0.4763 0.4444 10.124 10.213 6.5013462 13.16512 A606T04 0.4 156 0.3993 10.423 10.553 12.618846 26.06712 A606T05 0.5016 0.4686 11.709 11.857 12.866346 24.57612 A606T06 0.455 0.4086 10.749 10.797 13.414346 21.93362 A608T01 0.594 0.5354 12.478 12.317 12.620077 25.00477 A608T02 0.6094 0.5471 12.104 11.922 11.54 1577 25.19927 A608T03 0.5779 0.5173 12.428 12.302 11.701577 24.80077 A608T04 0.6238 0.5719 13.36 13.219 11.896577 A608T05 0.6479 0.5898 12.89 12.76 12.43775 24.14241 C608T01 0.1482 0.1381 3.0019 3.0028 13.28375 18.59741 C608T02 0.184 0.1437 4.873 9 4.6775 13.76375 18.47541 C608T03 0.2051 0.1957 4.4776 4.3206 13.44175 18.44641 E506T01 0.2548 0.2095 9.1633 9.3407 11.015917 16.93092 E506T04 0.2926 0.2527 9.3102 9.381 11.068417 17.18342 E506T05 0.3064 0.2548 11.248 11.392 10.99 16.43

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74 Appendix A. (Continued) Sample ID Cu 63 Cu 65 Zn 66 Zn 68 d dd d 15 N d dd d 13 C E506T06 0.3282 0.2754 9.9053 10.088 11 16.38 E506T07 0.3219 0.2731 12.071 12.323 10.986917 16.90392 E506T02 0.3511 0.2886 8.5794 8.5713 10.908417 19.09942 E506T03 0.3967 0.3303 6.9956 6.9596 14.489417 22.97392 E506T09 0.2981 0.2534 8.081 7.9616 E510T01 0.3563 0.3041 7.5707 7.5767 8.7216923 14.15308 E510T02 0.3455 0.2819 9.1057 9.0804 8.6321923 13.60308 E510T03 0.339 0.2699 9.3131 9.312 8.9166923 16.11208 E510T04 0.3769 0.3122 8.5669 8.487 8.5056923 13.30758 E510T05 0.3408 0.293 8 8.4199 8.3622 9.2086923 16.49708 E510T06 0.3713 0.3204 8.4922 8.44 8.9471923 14.83258 E510T07 0.388 0.328 8.3999 8.3763 E510T08 0.3114 0.2669 6.742 6.7141 8.9846923 14.12558 M506T01 0.1889 0.188 2.7011 2.6365 7.47 16.8 M506T02 0.2257 0.2246 2. 4939 2.3865 9.1861667 17.11022 M506T03 0.2488 0.2476 2.4836 2.4141 9.3996667 15.61022 M506T04 0.1577 0.1569 2.1373 2.0879 8.8596667 16.58622 M506T05 0.132 0.1313 2.5186 2.4922 10.959667 20.26522 M506T06 0.1586 0.1578 2.8122 2.7743 9.9473077 15.579 75 M506T07 0.1758 0.175 2.8217 2.7616 8.9768077 23.78275

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75 Appendix B: Sample Metadata Table 5. Sample Metadata Sample ID Genus Species Location FIM ID Mean Standard Length (mm) Individual Lengths (mm) A505A01 Anchoa mitchilli Alafia River 49.3 49 51 48 A505A02 Anchoa mitchilli Alafia River 45.8 47 50 44 42 A505A03 Anchoa mitchilli Alafia River 40.8 42 44 40 37 A505L01 Anchoa mitchilli Little Manatee River TBM200505 26.3 26 26 24 25 26 30 27 29 26 24 A505L02 Anchoa mitchilli Little Manatee River TBM200505 26.5 28 25 25 26 28 27 26 25 27 28 A505L03 Anchoa mitchilli Little Manatee River TBM200505 26.7 24 28 27 27 26 30 24 28 26 27 A505L04 Anchoa mitchilli Little Manatee River TBM200505 27.2 25 24 28 26 25 30 33 30 25 26 A505L05 Anchoa mitchilli Little Manatee River TBM200505 26.4 27 28 27 29 24 26 25 27 25 26 A505L06 Anchoa mitchilli Little Manatee River TBM200505 27 29 28 26 25 25 28 27 30 27 25 A507A01 Anchoa mitchilli Alafia River TBM2005074306 20.5 18 19 20 23 25 19 21 19 21 20 A507A02 Anchoa mitchilli Alafia River TBM2005074307 30.3 32 33 30 37 34 26 31 29 26 25 A508L01 Anchoa mitchilli Little Manatee River TBM2005080913 27.5 20 27 26 24 30 32 30 31 21 34 A509P01 Anchoa mitchilli Palm River TBM2005094404 23.9 25 27 23 25 24 22 23 25 23 22 A510A01 Anchoa mitchilli Alafia River TBM2005104703 44.8 46 44 47 42 A510A02 Anchoa mitchilli Alafia River TBM2005104703 46 48 47 45 44 A510A03 Anchoa mitchilli Alafia River TBM2005104601 25.5 31 22 25 28 27 24 22 22 25 29 A510H01 Anchoa mitchilli Hillsborough River TBM2005104104 24.8 26 24 23 26 24 24 26 24 25 26 A510H02 Anchoa mitchilli Hillsborough River TBM2005104104 25.4 25 26 26 24 24 25 24 27 26 27 A510H03 Anchoa mitchilli Hillsborough River TBM2005104104 30.7 31 30 31 A510T01 Anchoa mitchilli Tampa Bay TBM2005104702 44.3 43 45 45 A512H01 Anchoa mitchilli Hillsborough River TBM2005124402 23 22 24 25 22 23 22 23 24 22 A512H02 Anchoa mitchilli Hillsborough River TBM2005124402 24.1 23 24 27 25 22 26 22 24 24

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76 Appendix B: (Continued) A512H03 Anchoa mitchilli Hillsborough River TBM2005124402 27 25 26 25 24 28 26 24 32 33 A512H04 Anchoa mitchilli Hillsborough River TBM2005124402 32 39 32 A5UUA01 Anchoa mitchilli Alafia River 45 47 46 42 A5UUA02 Anchoa mitchilli Alafia River 46.7 43 53 44 A5UUA03 Anchoa mitchilli Alafia River 43.5 45 42 44 43 A601A01 Anchoa mitchilli Alafia River TBM2006013101 30.3 30 31 30 31 29 30 32 30 31 29 A601A02 Anchoa mitchilli Alafia River TBM2006013101 28.9 26 27 28 26 31 32 25 30 33 31 A601A03 Anchoa mitchilli Alafia River TBM2006013101 28.5 33 26 31 30 26 29 29 26 28 27 A601L01 Anchoa mitchilli Little Manatee River TBM2006010213 24 27 24 22 24 22 21 27 25 25 23 A601L02 Anchoa mitchilli Little Manatee River TBM2006010213 24.1 26 24 24 25 23 22 23 25 25 24 A601L03 Anchoa mitchilli Little Manatee River TBM2006010213 24.8 25 26 24 23 25 25 25 25 A601P01 Anchoa mitchilli Palm River TBM2006014601 30.2 31 25 32 33 31 27 29 30 32 32 A601P02 Anchoa mitchilli Palm River TBM2006014601 29.8 29 35 30 29 31 30 30 29 28 27 A601P03 Anchoa mitchilli Palm River TBM2006014601 37.3 41 40 32 39 38 36 41 34 35 37 A606T01 Anchoa mitchilli Tampa Bay TBM2006062802 38 37 39 38 A606T02 Anchoa mitchilli Tampa Bay TBM2006062802 39.25 40 39 40 38 A606T03 Anchoa mitchilli Tampa Bay TBM2006062802 37.25 39 36 37 37 A606T04 Anchoa mitchilli Tampa Bay TBM2006062802 36.7 37 37 36 A606T05 Anchoa mitchilli Tampa Bay TBM2006062802 37.7 40 38 35 A606T06 Anchoa mitchilli Tampa Bay TBM2006062802 44.7 46 46 42 A608T01 Anchoa mitchilli Tampa Bay TBM2006080209 41 42 41 40 A608T02 Anchoa mitchilli Tampa Bay TBM2006080209 44 43 44 45 A608T03 Anchoa mitchilli Tampa Bay TBM2006080209 42 42 41 43 A608T04 Anchoa mitchilli Tampa Bay TBM2006080209 41.7 41 39 45 A608T05 Anchoa mitchilli Tampa Bay TBM2006080209 48.5 49 48 C505A01 Cynoscion arenarius Alafia River TBM20050531 32.7 33 36 35 33 34 25 C505A02 Cynoscion arenarius Alafia River TBM20050531 31.8 32 31 27 29 33 39 C506A01 Cynoscion arenarius Alafia River TBM2005064308 41.3 25 30 69

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77 Appendix B. (Continued) C507H01 Cynoscion arenarius Hillsborough River TBM2005074708 29.75 27 33 31 28 C507P01 Cynoscion arenarius Palm River TBM2005074509 56 37 75 C510A01 Cynoscion arenarius Alafia River TBM2005104706 200 C510A02 Cynoscion arenarius Alafia River TBM2005104609 62.5 62 63 C510A03 Cynoscion arenarius Alafia River TBM2005104609 85 C510A04 Cynoscion arenarius Alafia River TBM2005104609 61 62 60 C510A05 Cynoscion arenarius Alafia River TBM2005104609 29.8 27 33 31 28 C510H01 Cynoscion arenarius Hillsborough River TBM2005104104 48 45 51 C510H02 Cynoscion arenarius Hillsborough River TBM2005104104 40.7 45 38 39 C510H03 Cynoscion arenarius Hillsborough River TBM2005104104 39.7 41 36 42 C510H04 Cynoscion arenarius Hillsborough River TBM2005104104 30.5 27 30 31 33 32 30 C511T01 Cynoscion arenarius Tampa Bay TBM2005114302 150 C601A01 Cynoscion arenarius Alafia River TBM2006014504 105 C601P01 Cynoscion arenarius Palm River TBM2006014601 190 C602G01 Cynoscion arenarius Gulf of Mexico Coast 240 C602G02 Cynoscion arenarius Gulf of Mexico Coast 280 C602G03 Cynoscion arenarius Gulf of Mexico Coast 275 C602G04 Cynoscion arenarius Gulf of Mexico Coast 220 C602G05 Cynoscion arenarius Gulf of Mexico Coast 200 C602G06 Cynoscion arenarius Gulf of Mexico Coast 250 C602G07 Cynoscion arenarius Gulf of Mexico Coast 225 C602G08 Cynoscion arenarius Gulf of Mexico Coast 210 C602G09 Cynoscion arenarius Gulf of Mexico Coast 220 C608A01 Cynoscion arenarius Alafia River TBM2006084602 59.3 65 57 56 C608A02 Cynoscion arenarius Alafia River TBM2006084602 51 50 52 51 C608T01 Cynoscion arenarius Tampa Bay TBM2006040404 170 C608T02 Cynoscion arenarius Tampa Bay TBM2006040404 179 C608T03 Cynoscion arenarius Tampa Bay TBM2006040404 184

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78 Appendix B: (Continued) C609A01 Cynoscion arenarius Alafia River TBM2006093111 35.7 35 36 36 C609A02 Cynoscion arenarius Alafia River TBM2006093111 35.7 37 35 35 C609A03 Cynoscion arenarius Alafia River TBM2006093111 39 39 40 38 C609A04 Cynoscion arenarius Alafia River TBM2006093111 44 47 42 43 C609A05 Cynoscion arenarius Alafia River TBM2006093112 42 42 42 C609A06 Cynoscion arenarius Alafia River TBM2006093112 33.5 33 34 C609A07 Cynoscion arenarius Alafia River TBM2006094507 67 E506A01 Eucinostomus harengulus Alafia River TBM2005064301 54.7 54 53 57 E506A02 Eucinostomus harengulus Alafia River TBM2005064301 62.3 64 62 61 E506A03 Eucinostomus harengulus Alafia River TBM2005064301 59.3 61 58 59 E506A04 Eucinostomus harengulus Alafia River TBM2005064302 59.5 61 58 E506T01 Eucinostomus harengulus Tampa Bay TBM2005064301 92 E506T02 Eucinostomus harengulus Tampa Bay TBM2005064301 84 E506T03 Eucinostomus harengulus Tampa Bay TBM2005064301 88 E506T04 Eucinostomus harengulus Tampa Bay TBM2005064301 94 E506T05 Eucinostomus harengulus Tampa Bay TBM2005064301 92 E506T06 Eucinostomus harengulus Tampa Bay TBM2005064301 90 E506T07 Eucinostomus harengulus Tampa Bay TBM2005064301 93 E506T09 Eucinostomus harengulus Tampa Bay TBM2005064301 98 E508L01 Eucinostomus harengulus Little Manatee River TBM2005080907 49 51 47 E508L02 Eucinostomus harengulus Little Manatee River TBM2005080911 58 62 54 E510T01 Eucinostomus harengulus Tampa Bay TBM2005100605 100 E510T02 Eucinostomus harengulus Tampa Bay TBM2005100605 103 E510T03 Eucinostomus harengulus Tampa Bay TBM2005100605 101 E510T04 Eucinostomus harengulus Tampa Bay TBM2005100605 102 E510T05 Eucinostomus harengulus Tampa Bay TBM2005100605 106 E510T06 Eucinostomus harengulus Tampa Bay TBM2005100605 101 E510T07 Eucinostomus harengulus Tampa Bay TBM2005100605 98

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79 Appendix B: (Continued) E510T08 Eucinostomus harengulus Tampa Bay TBM2005100605 101 E604L01 Eucinostomus harengulus Little Manatee River TBM2006040404 51 50 52 E604L02 Eucinostomus harengulus Little Manatee River TBM2006040404 44.7 44 46 44 E604L03 Eucinostomus harengulus Little Manatee River TBM2006040404 51 55 47 E608A01 Eucinostomus harengulus Alafia River TBM2006084603 63.5 62 65 E608A02 Eucinostomus harengulus Alafia River TBM2006084603 56 58 54 E608A03 Eucinostomus harengulus Alafia River TBM2006084602 47.3 49 48 45 47 E608A04 Eucinostomus harengulus Alafia River TBM2006084602 46.3 47 46 46 E608A05 Eucinostomus harengulus Alafia River TBM2006084602 50.3 50 51 50 E608A06 Eucinostomus harengulus Alafia River TBM2006084602 54.8 55 55 53 56 E608A07 Eucinostomus harengulus Alafia River TBM2006084602 56.7 57 56 57 E608A08 Eucinostomus harengulus Alafia River TBM2006084602 60 60 59 61 E608A09 Eucinostomus harengulus Alafia River TBM2006084602 59.7 62 58 59 E608A10 Eucinostomus harengulus Alafia River TBM2006084602 68 71 65 M506T01 Mugil cephalus Tampa Bay TBM2005061202 279 M506T02 Mugil cephalus Tampa Bay TBM2005061202 305 M506T03 Mugil cephalus Tampa Bay TBM2005061202 257 M506T04 Mugil cephalus Tampa Bay TBM2005061202 246 M506T05 Mugil cephalus Tampa Bay TBM2005061202 260 M506T06 Mugil cephalus Tampa Bay TBM2005061202 210 M506T07 Mugil cephalus Tampa Bay TBM2005061202 275 M507P01 Mugil cephalus Palm River TBM2005074502 59 60 58 M508L01 Mugil cephalus Little Manatee River TBM2005080907 53 52 56 51 M508L02 Mugil cephalus Little Manatee River TBM2005080907 72.5 71 74 M508L03 Mugil cephalus Little Manatee River TBM2005080907 98 M508L04 Mugil cephalus Little Manatee River TBM2005080907 102 M508L05 Mugil cephalus Little Manatee River TBM2005080907 60.7 61 60 61 M601A01 Mugil cephalus Alafia River TBM2006013104 25.3 27 27 26 24 24 22 28 26 26 27 26 26 21 24 26

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80 Appendix B: (Continued) M601A02 Mugil cephalus Alafia River TBM2006013104 25.5 22 28 27 25 24 22 29 26 20 27 25 28 27 26 27 M601H01 Mugil cephalus Hillsborough River TBM2006014201 24.8 25 25 27 25 23 24 26 25 23 26 22 23 26 27 M601H02 Mugil cephalus Hillsborough River TBM2006014201 25.7 26 25 23 27 24 27 26 24 26 27 26 26 27 26 M601L01 Mugil cephalus Little Manatee River TBM2005080907 24 23 22 26 M601P01 Mugil cephalus Palm River TBM2006014605 25.9 26 23 24 24 26 24 29 27 23 29 27 28 27 26 25 M601P02 Mugil cephalus Palm River TBM2006014605 26 25 26 26 27 26 24 29 22 28 28 25 25 24 25 30 M604A01 Mugil cephalus Alafia River TBM2006044303 51.8 49 52 50 56 M604A02 Mugil cephalus Alafia River TBM2006044303 57.8 60 57 58 56 M604A03 Mugil cephalus Alafia River TBM2006044303 69 65 73 M606A01 Mugil cephalus Alafia River TBM2006063105 88 M606A02 Mugil cephalus Alafia River TBM2006063105 89 M606A03 Mugil cephalus Alafia River TBM2006063105 84 M606A04 Mugil cephalus Alafia River TBM2006063105 80 M606A05 Mugil cephalus Alafia River TBM2006063105 76 M606A06 Mugil cephalus Alafia River TBM2006063105 70 M606A07 Mugil cephalus Alafia River TBM2006063105 72 M606A08 Mugil cephalus Alafia River TBM2006063105 72 M606A09 Mugil cephalus Alafia River TBM2006063105 74 M606A10 Mugil cephalus Alafia River TBM2006063105 75 M608A01 Mugil cephalus Alafia River TBM2006084606 95

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81 Appendix C: Additional Trace Metal Figures Figure 25. Striped Mullet: Log [Cu] vs. Standard Le ngth Striped MulletStandard Length (mm) 050100150200250300350 Log [Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6

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82 Appendix C: (Continued) Figure 26. Tidewater Mojarra: Log [Cu] vs. Standard Length Tidewater MojarraStandard Legnth (mm) 405060708090100110 Log [Cu] -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0

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83 Appendix C: (Continued) Figure 27. Sand Seatrout: Log [Cu] vs. Standard Len gth Sand Sea TroutStandard Length (mm) 050100150200250300 Log [Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2

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84 Appendix C: (Continued) Figure 28. Bay Anchovy: Log [Cu] vs. Standard Lengt h Bay AnchovyStandard Length (mm) 152025303540455055 Log [Cu] -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3

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85 Appendix C: (Continued) Figure 29. Sand Seatrout: Log [Zn] vs. Log [Cu] Log [Zn] vs Log [Cu] Sand Sea TroutLog [Cu] -0.8-0.6-0.4-0.20.00.2 Log [Zn] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

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86 Appendix C: (Continued) Figure 30. Bay Anchovy: Log [Zn] vs. Log [Cu] Log [Zn] vs Log [Cu] Bay AnchovyLog [Cu] -0.8-0.6-0.4-0.20.00.2 Log [Zn] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

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87 Appendix C: (Continued) Figure 31. Striped Mullet: Log [Zn] vs. Log [Cu] Log [Zn] vs Log [Cu] Striped MulletLog [Cu] -0.8-0.6-0.4-0.20.00.20.40.6 Log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Alafia Hillsborough Little Manatee Palm Tampa Bay

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88 Appendix C: (Continued) Figure 32. Tidewater Mojarra: Log [Zn] vs. Log [Cu ] Log [Zn] vs Log [Cu] Tidewater MojarraLog [Cu] -0.8-0.6-0.4-0.20.00.2 Log [Zn] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

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89 Appendix C: (Continued) Figure 33. Log [Zn] vs. Standard Length all specie s, high detail Tidewater MojarraStandard Length (mm) 406080100 Log [Zn] 0.6 0.7 0.8 0.9 1.0 1.1 1.2 Sand Sea TroutStandard Length (mm) 050100150200250300 Log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 Bay AnchovyStandard Length (mm) 20304050 Log [Zn] 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Striped MulletStandard Length (mm) 0100200300 Log [Zn] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Alafia Hillsborough Little Manatee Palm Tampa Bay

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90 Appendix C: (Continued) Figure 34. Log [Zn] vs. Log [Cu] by location, all species, expanded axis Hillsborough RiverLog [Cu] -0.6-0.4-0.20.00.20.40.6 Log [Zn] 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Little Manatee RiverLog [Cu] -0.6-0.4-0.20.00.20.40.6 Log [Zn] 0.8 1.0 1.2 1.4 1.6 1.8 Palm RiverLog [Cu] -0.6-0.4-0.20.00.20.4 Log [Zn] 0.6 0.8 1.0 1.2 1.4 1.6 Alafia RiverLog [Cu] -0.8-0.6-0.4-0.20.00.2 Log [Zn] 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet

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91 Appendix C: (Continued) Figure 35. Cu ( m g/g) and Zn ( m g/g) vs. Standard Length Copper in all fishStandard Length (mm) 050100150200250300350 Cu ( m g/g) 0 1 2 3 4 Rivers Tampa Bay Standard Length (mm) 050100150200250300350 Zn ( m g/g) 0 10 20 30 40 50 60 Tampa Bay River Fish

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92 Appendix D: Additional Stable Isotope Figures Figure 36. d15N vs. d13C, separated by species Striped Mullet Isotopes6 7 8 9 10 11 12 13 14 15 16 -28-26-24-22-20-18-16-14 d dd d13C‰d dd d15N‰ Alafia Hillsborough Little Manatee Palm Tampa Bay Tidewater Mojarra Isotopes6 7 8 9 10 11 12 13 14 15 16 -28-26-24-22-20-18-16-14 d dd d13C‰d dd d15N‰ Alafia Little Manatee Tampa Bay Sand Sea Trout Isotopes6 8 10 12 14 16 -28-26-24-22-20-18-16-14 d dd d13C‰d dd d15N‰ Alafia Hillsborough Palm Tampa Bay Bay Anchovy Isotopes6 8 10 12 14 16 -28-26-24-22-20-18-16-14 d dd d13C‰d dd d15N‰ Alafia Hillsborough Little Manatee Palm Tampa Bay

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93 Appendix D: (Continued) Figure 37. d15N vs. d13C, separated by location Palm River6 7 8 9 10 11 12 13 14 15 16 -28-26-24-22-20-18-16-14 d dd d13C‰d dd d15N‰ Bay Anchovy Sand Sea Trout Striped Mullet Hillsborough River6 7 8 9 10 11 12 13 14 15 16 -28-26-24-22-20-18-16-14 d dd d13C‰d dd d15N‰ Bay Anchovy Sand Sea Trout Striped Mullet Alafia River6 7 8 9 10 11 12 13 14 15 16 -28-26-24-22-20-18-16-14 d dd d13C‰d dd d15N‰ Bay Anchovy Sand Sea Trout Tidewater Mojarra Striped Mullet Little Manatee River6 7 8 9 10 11 12 13 14 15 -28-26-24-22-20-18-16-14 d dd d13C‰d dd d15N‰ Bay Anchovy Tidewater Mojarra Striped Mullet

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94 Appendix D (Continued) Figure 38. d15N and. d13C vs. Standard Length d13C and Standard LengthStandard Length 050100150200250300350 d13C -28 -26 -24 -22 -20 -18 -16 -14 -12 d15N and Standard LengthStandard Length (mm) 050100150200250300350 d15N 6 8 10 12 14 16

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95 Appendix D (Continued) Figure 39. d15N vs. d13C including seasonal data Bay Anchovyd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Sand Sea Troutd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Dry Season Wet Season Tidewater Mojarrad13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Striped Mulletd13C -28-26-24-22-20-18-16-14-12 d15N 6 8 10 12 14 16 Tampa Bay

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96 Appendix E: Trace Metal and Stable Isotope Figures Figure 40. Log [Cu] vs. d13C Striped Mullet all sitesd13C -28-26-24-22-20-18-16-14-12 log [Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Tidewater Mojarra all sitesd13C -28-26-24-22-20-18-16-14-12 log [Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Sand Sea Trout all sitesd13C -28-26-24-22-20-18-16-14-12 log[Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Bay Anchovy all sitesd13C -28-26-24-22-20-18-16-14-12 log[Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Alafia Hillsborough Little Manatee Palm Tampa Bay

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97 Appendix E (Continued) Figure 41. Log [Zn] vs. d13C Bay Anchovy all sitesd13C -28-26-24-22-20-18-16-14-12 Log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Alafia Hillsborough Little Manatee Palm Tampa Bay Sand Sea Trout all sitesd13C -26-24-22-20-18-16-14-12 Log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Tidewater Mojarra all sitesd13C -28-26-24-22-20-18-16-14-12 log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Striped Mullet all sitesd13C -28-26-24-22-20-18-16-14-12 Log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

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98 Appendix E (Continued) Figure 42. Log [Cu] vs. d15N Bay Anchovy all sitesd15N 6810121416 log [Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Alafia Hillsborough Little Manatee Palm Tampa Bay Sand Sea Trout all sitesd15N 6810121416 log [Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Tidewater Mojarral all sitesd15N 6810121416 log[Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Striped Mullet all sitesd15N 6810121416 log [Cu] -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6

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99 Appendix E (Continued) Figure 43. Log [Zn/Cu] vs. d13C Sand Sea Trout all sitesd13C -28-26-24-22-20-18-16-14-12 Log ([Zn]/[Cu]) 0.8 1.0 1.2 1.4 1.6 1.8 Tidewater Mojarra all sitesd13C -28-26-24-22-20-18-16-14-12 Log ([Zn]/[Cu]) 0.8 1.0 1.2 1.4 1.6 1.8 Bay Anchovy all sitesd13C -28-26-24-22-20-18-16-14-12 Log ([Zn]/[Cu]) 0.8 1.0 1.2 1.4 1.6 1.8 Alafia Hillsborough Little Manatee Palm Tampa Bay Striped Mullet all sitesd13C -28-26-24-22-20-18-16-14-12 Log ([Zn]/[Cu]) 0.8 1.0 1.2 1.4 1.6 1.8

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100 Appendix E (Continued) Figure 44. Log [Zn] vs. d15N Striped Mullet all sitesd15N 6810121416 log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Tidewater Mojarra all sitesd15N 6810121416 log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Sand Sea Trout all sitesd15N 6810121416 log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Bay Anchovy all sitesd15N 6810121416 log [Zn] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Alafia Hillsborough Little Manatee Palm Tampa Bay

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101 Appendix E (Continued) Figure 45. Log [Zn/Cu] vs. d15N Bay Anchovy all sites d15N 6810121416 Log ([Zn]/[Cu]) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Alafia Hillsborough Little Manatee Palm Tampa Bay Sand Sea Trout all sitesd15N 6810121416 Log ([Zn]/[Cu]) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Tidewater Mojarra all sitesd15N 6810121416 Log ([Zn]/[Cu]) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Striped Mullet all sitesd15N 6810121416 Log ([Zn]/[Cu]) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

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102 Linae Marie Boehme-Terrana was raised in Florida wh ere she attended St. Petersburg High School’s International Baccalaureate program. Growing up in a state surrounded by water, marine science quickly capture d her imagination. She pursued her interest in marine science by attending the Uni versity of Miami from 1991-1994. She majored in Biology with minors in Chemistry, Ps ychology, and Marine Science, graduating with general honors in 3.5 years. Follo wing her undergraduate work she gained practical experience in marine science throu gh seasonal employment as an aerial observer in the Florida Department of Enviro nmental Protection’s right whale monitoring program. For the next few years she wor ked as a research assistant within the University of South Florida’s College of Marine Science. In 1996, she was admitted to the USF master’s degree program followe d by a transfer to the doctoral program in 1998. During her time at the college, sh e was awarded a National Science Foundation GK-12 OCEANS Fellowship which gave her t he opportunity to bring marine science into local elementary school classro oms.


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Boehme-Terrana, Linae Marie.
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Trace metals and stable isotopes as tracers of life history and trophic connections in estuarine-dependent fish from Tampa Bay, Florida
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by Linae Marie Boehme-Terrana.
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[Tampa, Fla.] :
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2007.
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ABSTRACT: Florida's estuaries support a wide range of species yet little is known about tracemetal cycling among members of this important ecosystem. To examine the flow of trace metals through the Tampa Bay estuary, four fish species representing different trophic levels were analyzed for copper (Cu), zinc (Zn) and stable isotopes of carbon (C) and nitrogen (N). Species selected were the striped mullet (Mugil cephalus), tidewater mojarra (Eucinostomus harengulus), bay anchovy (Anchoa mitchilli), and sand seatrout (Cynoscion arenarius). Juvenile fish were collected from the Alafia, Hillsborough, Palm, and Little Manatee Rivers. Adults were collected from Tampa Bay. Combinations of trace metal and stable isotope analyses were used to evaluate geographic variability in trace metal concentrations among locations in Tampa Bay and to shed light on trophic pathways that lead to trace metal accumulation.In juvenile mullet, significant trends were found between Zn concentrations, stable isotope ratios, and standard length. Animals of the smallest size classes carry greater concentrations of zinc in their tissues and have distinct stable isotope ratios that reflect their recent life history as offshore planktivorous larvae. Interestingly, the ratio of Zn:Cu concentrations was highly conserved. While species-specific differences were observed, relatively small Zn:Cu variations suggest a possible bioregulatory mechanism that maintains an optimal Zn:Cu ratio even in the presence of elevated absolute metal concentrations. Stable isotope ratios proved to be an effective tracer of ontogenetic changes in fish diet and habitat. Carbon and nitrogen stable isotope analyses revealed that trophic relations between species are established very early in an organism's life history. The bay anchovy, a major prey item of the sand seatrout, has deltaN values very similar to this predator.Although trophic linkages between trace metals and stable isotopes proved difficult to interpret, the relation between zinc concentrations and deltaC values suggested that trace metal concentrations are highest in animals that utilize food webs based on terrestrial carbon.
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Dissertation (Ph.D.)--University of South Florida, 2007.
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Text (Electronic dissertation) in PDF format.
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Adviser: Robert H. Byrne, Ph.D.
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Copper.
Zinc.
Carbon.
Nitrogen.
Sand seatrout.
Tidewater mojarra.
Striped striped.
Estuary.
Bay anchovy.
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Dissertations, Academic
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x Marine Science
Doctoral.
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