U.S. Dept Commerce/NOAA/NMFS/NWFSC/PublicationsNOAA Technical Memorandum NMFS-NWFSC-4National Benthic Surveillance Project: Northeast CoastFish Histopathology and Relationships Between Lesions and Chemical       Contaminants (1987-89)Lyndal L. Johnson, Carla M. Stehr, O. Paul Olson, Mark S. Myers, Susan       M. Pierce, Bruce B. McCain, and Usha VaranasiNational Marine       Fisheries ServiceNorthwest Fisheries Science CenterEnvironmental       Conservation Division2725 Montlake Blvd. E.Seattle WA 98112       
December 1992
U.S. DEPARTMENT OF COMMERCEBarbara Hackman       Franklin, Secretary       
National Oceanic and Atmospheric       AdministrationJohn A. Knauss, Administrator

National Marine Fisheries ServiceWilliam W. Fox,       Jr. Assistant Administrator for Fisheries 
CONTRIBUTING SCIENTIFIC STAFF 
Carol AirutDonald BrownJohn BuzitisKen CarrascoTracy CollierSin Lam ChanWilliam GronlundJennifer HagenCraig HaleyTom HomLeslie KubinMargaret KrahnTom LeeDan LomaxPaul PleshaCasey RiceHerbert SanbornKaren TilburyCatherine WigrenMary Jean WillisGina Ylitalo
CONTENTS- EXECUTIVE   SUMMARY 
- PREFACE 
- INTRODUCTION 
- MATERIALS   AND METHODS    - Field     Sampling  
  - Laboratory     Analyses 
  - Fish     Age Determination  
  - Histopathological     Analyses 
  - Chemical     Analyses of Sediments, Stomach Contents, Liver Tissue, and Bile  
  - Stomach     Contents Taxonomy  
  - Statistical     Analyses  
  - Heterogeneity     in Lesion Prevalence  
  - Logistic     Regression and Risk Factor Analysis 
- RESULTS       - Chemical     Characterization of Sampling Sites  
  - Sediment     Contaminant Levels at Sampling Sites  
  - Patterns     of Occurrence of Contaminants Within Sediment 
  - Contaminant     Levels in Sediments, Stomach Contents, and Tissues 
  - Stomach     Contents Taxonomy  
  - Hepatic     Lesions  
  - Hepatic     Lesion Prevalences and Intersite Comparisons  
  - Risk     Factors Associated with Hepatic Lesion Occurrence in Individual Fish 
  - Relationships     Between Chemical Contaminants and Hepatic Lesion Prevalences  
  - Renal     Lesions  
  - Renal     Lesion Prevalences  
  - Risk     Factors Associated with Renal Lesion Occurrence in Individual Fish  
  - Relationships     Between Chemical Contaminants and Renal Lesion Prevalences  
  - Fin     Erosion  
  - Fin     Erosion Prevalences 
  - Risk     Factors Associated with Fin Erosion Occurrence in Individual Fish  
  - Relationships     Between Chemical Contaminants and Fin Erosion Prevalences 
- DISCUSSION       - Patterns     of Lesion Occurrence  
  - Toxicopathic     Response to Contaminant Exposure in Winter Flounder 
  - Biological     Risk Factors Associated With Lesion Occurrence  
  - Age      
  - Sampling     Season  
  - Gender      
  - Relationships     Between Chemical Contaminants and Idiopathic Lesions  
  - Hydropic     Vacuolation  
  - Nonspecific     Hepatocellular Necrosis  
  - Neoplasms     and FCA  
  - Proliferative     Lesions  
  - Spongiosis     Hepatis  
  - Megalocytic     Hepatosis/Nuclear Pleomorphism 
  - Renal     Lesions  
  - Fin     Erosion  
  - PCBs     and Idiopathic Disease in Winter Flounder 
- CONCLUSIONS 
- ACKNOWLEDGMENTS 
- CITATIONS    
- TABLES    
- FIGURES 
- APPENDIX     EXECUTIVE SUMMARY      This report presents and interprets the results of pathology studies   conducted on winter flounder (Pleuronectes americanus) between 1987   and 1989 in conjunction with NOAA's National Status and Trends (NS&T)   Program. In these studies, a variety of potentially contaminant-associated   disease conditions were monitored in winter flounder collected from 22   Northeast Coast sites, and the relationship between disease occurrence and   levels of organic chemical contaminants in sediment, stomach contents, and   tissues was examined. Sampling was conducted primarily in spring of 1988 and   1989 (Cycles 5 and 6) as part of the National Benthic Surveillance Project   (NBSP) of the NS&T Program, but to provide a more comprehensive view of   pathological conditions in winter flounder from the Northeast Coast, data on   winter flounder collected in Long Island Sound during 1987 and in Boston   Harbor and adjacent embayments during the winter of 1988 were also included in   this memorandum. In the three studies combined, a total of more than 1,500   fish were examined.  Embayments sampled included Salem Harbor, Boston Harbor, Massachusetts Bay,   New Bedford Harbor, and Buzzards Bay in Massachusetts; Narragansett Bay in   Rhode Island; Niantic Bay in Connecticut; several sites within Long Island   Sound including New Haven, Norwalk, Bridgeport, and Rocky Point in Connecticut   and Lloyd Point in New York; and Raritan Bay and Great Bay in New Jersey. The   levels and types of chemical contaminants present in sediments from sampling   sites within these embayments differed substantially. Among the most heavily   contaminated sites were Mystic River and Quincy Bay in Boston Harbor, and   Gravesend, West Reach, and East Reach sites in Raritan Bay. Sediments from   these sites had elevated levels of all or most of the contaminants measured in   this study, including aromatic hydrocarbons (AHs), polychlorinated biphenyls   (PCBs), DDTs (DDT and its derivatives), and chlordanes. The New Bedford Harbor   sampling site had very high concentrations of PCBs in sediments, but   relatively low concentrations of other classes of contaminants. Lowest   concentrations of nearly all classes of contaminants in sediment were found at   the Plymouth Entrance site in Massachusetts Bay and at the Rocky Point site in   Long Island Sound. The Rocky Point site served as a reference site for   analyses of intersite differences in lesion prevalences. Detailed information   on concentrations of contaminants in sediments from these sampling sites will   be reported in a forthcoming Technical Memorandum.  Winter flounder were examined for necrotic, sclerotic, and proliferative   lesions in the kidney, fin erosion, and a number of pathological conditions in   the liver, including:    - neoplastic lesions (e.g., hepatocellular and cholangiocellular     carcinomas, liver cell adenomas and cholangiomas);     
  - putatively preneoplastic lesions (i.e., foci of cellular alteration     (FCA));     
  - several unique, presumably degenerative conditions (hydropic     vacuolation, nuclear pleomorphism and megalocytic hepatosis, and spongiosis     hepatis) which have been shown to be associated with toxicant exposure in     previous studies with winter flounder or other fish species ;     
  - nonneoplastic proliferative lesions such as hepatocellular or biliary     regeneration or hyperplasia; and     
  - a variety of nonspecific hepatocellular or biliary necrotic or     degenerative conditions. Some of these lesions, such as liver   neoplasms, FCA, and biliary or hepatocellular degenerative and regenerative   lesions, are well established as histopathologic biomarkers of contaminant   stress in fish and show strong evidence of a contaminant-associated etiology   on the basis of previous field or laboratory studies with winter flounder or   other fish species. Other lesions, such as necrotic and proliferative   conditions in the kidney and fin erosion, have shown some promise as   bioindicators of chemical pollution but require additional testing.    Prevalences of the lesion types described above were determined in winter   flounder from each of the Northeast Coast sampling sites. In addition,   logistic regression was used to examine the effects of selected biological and   contaminant-associated risk factors (e.g., age, gender, site and season of   capture, and levels of chemical contaminants in sediments and fish) on lesion   occurrence. Logistic regression is a multivariate statistical method which is   similar to stepwise multiple regression, but is uniquely suited to binomial or   proportional data such as lesion presence or absence or lesion prevalence.   This technique is frequently applied in epidemiological and epizootiological   studies and has a distinct advantage over simple intersite comparison of   lesion prevalences because it allows biological factors such as age and gender   to be taken into account when evaluating relationships between disease and   site of capture or other parameters associated with contaminant exposure.  Relationships between lesion prevalences and the following major classes of   chemical compounds were examined: high molecular weight AHs containing 4-6   benzene rings (HAHs), low molecular weight AHs containing 1-3 benzene rings   (LAHs), total AHs (LAHs + HAHs), PCBs, DDTs, and chlordanes. These classes of   chemical compounds were chosen as potential risk factors because 1) they are   found at high concentrations in sediments from a number of Northeast Coast   sites; 2) they are generally present in estuaries located near metropolitan   areas throughout the United States and thus represent an index of urban   pollution; and 3) they are generally recognized as toxicants in fish or   mammals. Moreover, concentrations of these contaminants in sediments were   highly correlated with levels of the same compounds or their derivatives in   stomach contents, liver, and bile of winter flounder, indicating that these   compounds were being taken up and accumulated or metabolized by the fish.   Concentrations of organic contaminants in all compartments were correlated   with disease prevalences to evaluate the consistency of observed   relationships; however, contaminant levels in liver and bile were considered   to have the greatest relevance as toxicologic risk factors because they   correspond most closely to actual uptake of toxicants by animals.     Significant Findings       - Overall, hepatic lesions in winter flounder proved to be extremely     reliable histological markers of chemical contaminant exposure. Hydropic     vacuolation and nonspecific necrotic lesions showed particular utility as     indicators of anthropogenic stress. Both lesions occurred at high     prevalences (25 to 50%) in winter flounder from heavily contaminated sites,     and showed a clear gradient in prevalence from heavily to minimally     contaminated sites. In addition, they were closely correlated with levels of     several classes of organic contaminants in fish liver and bile, sediments,     and stomach contents.     
  - Hepatic neoplasms, foci of cellular alteration, and nonneoplastic     proliferative lesions such as biliary hyperplasia were also found in winter     flounder from several heavily contaminated sites, but these lesions were not     highly correlated with chemical contaminant levels in liver, bile, stomach     contents, or sediments. The lack of correlation can be partly attributed to     the relatively low prevalences (2 to 10%) of neoplastic, preneoplastic, and     proliferative lesions in winter flounder sampled in this study. Also, there     is a long latency period for the development of neoplasms and related     lesions. Consequently, even if contaminant exposure is a critical factor in     their etiology, they may not be highly correlated with contaminant levels in     sediment at the sampling site, or with levels of contaminants in the animals     at the time of collection. This is especially likely to be the case in     younger animals, or in a relatively mobile species such as winter flounder.     The problem is compounded by the fact that for certain key environmental     carcinogens such as AHs, current methodologies estimate only short-term, not     chronic exposure. There is substantial evidence from both field and     laboratory studies that neoplastic and preneoplastic lesions in fish are     associated with exposure to environmental toxicants. However, because of     their relatively low prevalence in many marine species and their long     latency period, they may be less suitable as biomarkers of anthropogenic     stress in field monitoring programs than earlier-occurring and more     prevalent toxicopathic lesions.     
  - The compounds most frequently identified as risk factors for     pathological conditions in winter flounder were AHs, DDTs, and chlordanes.     In addition to these chemical contaminants, several biological factors,     particularly age and sampling season, had a significant influence on disease     prevalence. However, because AHs, DDTs, and chlordanes generally occurred     together in sediments, and animals were exposed to them simultaneously, it     was difficult to evaluate the relative importance of these toxicants in the     development of pollution-associated disease in winter flounder.     
  - PCBs did not emerge as significant risk factors for any pathological     condition observed in winter flounder. Not only did PCB levels in sediment,     stomach contents, and liver fail to correlate with lesion prevalences, but     perhaps more importantly, fish sampled from one site, New Bedford Harbor,     that had high levels of PCBs in sediments but relatively low levels of other     contaminants including AHs, had low prevalences of all pathological     conditions monitored in this study. These data, however, do not exclude PCBs     from a role as secondary etiologic agents for neoplastic lesions or     regenerative and degenerative conditions in winter flounder.     
  - Prevalences of renal lesions were not highly correlated with levels of     organic contaminants in sediments, stomach contents, bile or liver of winter     flounder. Of the three classes of lesions monitored in this study     (proliferative, necrotic, and sclerotic), only necrotic lesions showed any     evidence of a relationship to contaminant exposure.     
  - Fin erosion prevalences were relatively high (e.g., 25 to 30%) at     certain heavily contaminated sites especially within Boston Harbor, and were     significantly correlated with contaminant concentrations in sediments and     stomach contents, and with biliary FAC concentrations. However, because     biotic factors such as fungal or bacterial pathogens as well as xenobiotic     contaminants may be associated with the development of fin erosion, it is     not as specific a biomarker as other toxicopathic lesions.     
  - The toxicopathic response (e.g., hydropic vacuolation of hepatocellular     and biliary cells and biliary hyperplasia, and a predominance of     cholangiocellular neoplasms over hepatocellular neoplasms) typically     exhibited by winter flounder sampled from urban sites is quite different     from patterns of lesion occurrence associated with the histogenesis of     neoplasia in mammals and fish species such as medaka, rainbow trout, and     English sole. The types of lesions found in winter flounder in contaminated     areas have also been noted in other bottomfish (e.g., starry flounder     (Platichthys stellatus) and white croaker (Genyonemus     lineatus) ) at contaminated sites on the West Coast sampled in the     NBSP. These differences between starry flounder, white croaker, and winter     flounder and species such as English sole suggest that toxicopathic     response, and possibly histogenesis of neoplasia, may follow more than one     pathway in teleost fish. In these studies we were not able to   evaluate the effects of a number of potential toxicological risk factors such   as certain metals, especially biologically-active forms of metals including   organometals, dioxins, and coplanar PCBs, because appropriate cost-effective   methodologies amenable for large-scale biomonitoring are not currently   available. Development of such methodologies and their incorporation into the   NBSP could bring about significant advances in our understanding of the   toxicopathic effects of such contaminants and their importance in the   development of pollution-associated disease. Our ability to identify   relationships between environmental toxicants and disease conditions in   benthic fish would be further enhanced by the utilization of indicators (e.g.,   xenobiotic-DNA adducts) that measure chronic exposure to labile toxicants,   such as AHs, and by determination of concentrations of selected contaminants,   including CHs, in individual fish. Such data may be used to develop predictive   dose-response models linking contaminant levels in individuals with the risk   of disease.    Although biomonitoring studies such as those reported here could benefit   greatly by inclusion of a broader suite of contaminants and biological   endpoints, the data gathered thus far provide significant indications that   certain environmental contaminants are potential risk factors for pathological   conditions observed in winter flounder, a benthic fish used as an indicator   species for the assessment of environmental quality on the Northeast Coast.   This correlational evidence, while being circumstantial, provides valuable   insights into the potential involvement of certain groups of contaminants in   disease initiation and progression, and will serve as a basis for the   formulation of definitive cause-and-effect studies with potential etiologic   agents present in urban environments.       PREFACE   The National Benthic Surveillance Project (NBSP) was initiated in 1984 as a   component of the National Oceanic and Atmospheric Administration's (NOAA's)   National Status and Trends (NS&T) Program, which was designed to assess   the status of, as well as long-term changes in, the environmental quality of   the Nation's coastal and estuarine waters. The NBSP is a cooperative effort   between the National Marine Fisheries Service and the Coastal Monitoring and   Bioeffects Division of the Office of Ocean Resources Conservation and   Assessment of NOAA's National Ocean Service. The project's specific objectives   are to measure concentrations of chemical contaminants in sediment and in   bottom-dwelling fish species at selected sites in urban and nonurban   embayments, to determine the prevalences of pathological conditions and other   bioindicators of contaminant exposure in these same fish species, and to   evaluate temporal trends in the above-mentioned parameters. One hundred sites   in embayments along the Atlantic, Gulf and Pacific coasts, including Alaska,   have been sampled since 1984, with each annual sampling referred to as a   "cycle" (e.g., 1984 = Cycle 1). Prior to Cycle 5, the Northeast Fisheries   Center was responsible for Northeast Coast NBSP sampling, and results of these   studies are presented in Zdanowicz et al. (1986) and NOAA (1987, 1988).  This Technical Memorandum presents and interprets the results of   histopathological studies conducted on winter flounder (Pleuronectes   americanus formerly Pseudopleuronectes americanus) sampled from   Northeast Coast sites between 1987 and 1989 as part of the NS&T Program.   Pathological conditions described include a variety of idiopathic liver,   kidney, and fin lesions. Some of these lesions, such as liver neoplasms and   related lesions, are well established as histopathologic biomarkers of   contaminant stress, while others have shown some promise as indicators but   require additional testing. Prevalences of these lesions in winter flounder   sampled from sites with different degrees of chemical contamination are   compared. In addition, lesion prevalences are correlated with chemical   exposure data to better understand the relationship between environmental   contaminants and fish disease. This memorandum is not meant to comprehensively   review the marine pollution literature; however, pertinent references are   included in discussions of the most significant findings.  In addition to this publication, a separate Technical Memorandum is in   preparation to provide detailed information on contaminant levels in winter   flounder tissues and in sediments at the Northeast Coast sampling sites (Brown   et al. in prep.). While data on sediment and tissue contaminant concentrations   will be utilized in this pathology memorandum, the chemistry report should be   consulted for more comprehensive information on tissue and sediment chemistry   for Northeast Coast NBSP studies conducted during 1988 and 1989.     INTRODUCTION   The National Benthic Surveillance Project (NBSP), a component of the   National Oceanic and Atmospheric Administration's (NOAA's) Status and Trends   (NS&T) Program, is designed to assess the status of the Nation's coastal   and estuarine waters and monitor long-term changes in marine environmental   quality. A key component of the NBSP is the utilization of histopathologic   alterations in fish tissues as indicators of the effects of exposure to   environmental contaminants on marine organisms. These histopathologic   biomarkers are pathological conditions that either morphologically resemble   lesions induced by the experimental exposure of mammals or fish to toxicants,   or show evidence of a contaminant-associated etiology in field studies because   they occur either exclusively or at significantly increased prevalences in   fish from contaminated sites.  Probably the most commonly used histopathologic biomarkers are neoplastic   and preneoplastic liver lesions, although recent studies indicate that certain   associated degenerative and regenerative conditions may be equally or even   more useful as biomarkers of anthropogenic stress (Myers et al. in press,   Varanasi et al. in press). Liver cancer and related lesions have been reported   in several species of bottomfish (e.g., English sole (Pleuronectes   vetulus, formerly Parophrys vetulus) (McCain et al. 1977; Malins   et al. 1984, 1985; Myers et al. 1987), white croaker (Genyonemus   lineatus) (Malins et al. 1987; Myers et al. 1991, 1992); Atlantic tomcod   (Microgadus tomcod) (Smith et al. 1979; Cormier 1986)) from   chemically contaminated coastal areas. Associations between concentrations of   chemical contaminants in sediments and fish tissues and certain of these   lesions have been observed in field surveys (Malins et al. 1984, 1985; McCain   et. al. 1988; Vogelbein et al. 1990), and have been further substantiated by   statistical analyses and epizootiological models (Rhodes et al. 1987, Myers et   al, 1990, 1991, 1992; Landahl et al. 1990). Moreover, in some species,   including English sole, cause-and-effect relationships between selected   contaminants and hepatic lesions similar to those observed in field-sampled   animals have been demonstrated in long-term laboratory studies (Varanasi et   al. 1987, Schiewe et al. 1991). Other pathological conditions, including fin   erosion and renal lesions, have also shown some promise as bioindicators of   contaminant exposure in fish (Dethlefsen 1980, Cross 1985, Wellings et al.   1976, Sherwood and Mearns 1977, Cross et al. 1985, Sindermann 1990,   Reimschuessel et al. 1990, Hawkins et al. 1989, McCain et al. 1992), but   require additional testing in both the field and the laboratory to confirm   their chemical etiology.  Previous field studies suggest that liver lesions and other disease   conditions in winter flounder (Pleuronectes americanus formerly   Pseudopleuronectes americanus) may be reliable bioindicators of   environmental degradation on the Northeast Coast. The occurrence of neoplastic   and non-neoplastic liver disease in fish from contaminated areas, especially   from sites within Boston Harbor, is well-documented, both in earlier NBSP   sampling conducted by the Northeast Fisheries Center (Zdanowicz et al. 1986,   NOAA 1987, 1988) and in studies by other investigators (Murchelano and Wolke   1985, 1991; Moore 1991, Gronlund et al. 1991). Elevated prevalences of fin   erosion have also been reported in flounder from contaminated sites along the   Northeast Coast (Williams 1981, Ziskowski and Murchelano 1975). At present,   however, there is relatively little information available on how levels of   specific contaminants in sediments and winter flounder tissues are related to   disease occurrence, or on the effects of biological factors such as age and   gender on disease prevalence in this species.  This report presents the results of pathological studies conducted on   winter flounder collected from Northeast Coast sites between 1987 and 1989 as   part of the NS&T Program. The major objectives of the study were 1) to   determine the prevalences of fin erosion and selected hepatic and renal   lesions in winter flounder from the Northeast Coast sampling sites; 2) to   examine the effects of biological variables such as age, gender, and sampling   season on lesion occurrence; and 3) to assess the relationship between levels   of chemical contaminants in sediments and fish and lesion prevalences.  Sampling was primarily conducted during Cycles 5 and 6 (1988 and 1989) of   the Northeast Coast NBSP, but to provide a more comprehensive view of   pathological conditions in winter flounder from the Northeast Coast, data from   winter flounder collected in Long Island Sound in 1987 (Gronlund et al. 1991)   and in Boston Harbor and adjacent embayments during the winters of 1988 and   1989 (Johnson et al. 1992, in press) are also included in this report.   Histopathological examination of tissues and chemical analyses were carried   out in our laboratory in a uniform fashion for all three studies, so all data   included here are comparable.  Lesion types monitored in winter flounder included fin erosion; necrotic,   sclerotic, and proliferative lesions in the kidney; and a number of suspected   toxicopathic liver lesions such as neoplasms, putatively preneoplastic foci of   cellular alteration, and nonneoplastic proliferative and degenerative   conditions. A variety of other pathological conditions, such as parasitic   infections, were noted in flounder examined in this study, but they will not   be described in detail in this report as there is little evidence that they   have a chemical etiology (Hinton et al. 1992).  The following classes of chemical contaminants were included in the   analysis as potential risk factors : high molecular weight aromatic   hydrocarbons (AHs) containing 4-5 benzene rings (HAHs), low molecular weight   AHs containing 2-3 benzene rings (LAHs), total AHs (LAHs + HAHs),   polychlorinated biphenyls (PCBs), DDT and its derivatives, DDD and DDE (DDTs),   and chlordanes. These classes of chemical compounds were chosen because 1)   they were present at high concentrations in sediments from a number of   Northeast Coast urban sites; 2) they are among the most prevalent contaminants   in estuaries adjacent to metropolitan areas throughout the United States, and   thus represent an index of urban pollution (Varanasi et al. 1988, 1989a); and   3) they are generally recognized as toxicants in fish or mammals (Klaassen et   al. 1986).  Concentrations of these organic contaminants or their metabolites were   measured in several compartments (i.e., sediments, stomach contents, liver,   and bile) to give a comprehensive picture of exposure and relationships   between contaminant levels in all three compartments and idiopathic disease.   However, contaminant levels in liver and bile were considered to have the   greatest relevance as toxicologic risk factors, because they are most   representative of the actual uptake of toxic compounds by fish. It should be   noted that other types of contaminants, such as dioxins or organometals, may   also play a role in the development of disease in winter flounder and other   fish species. However, because methodologies for measuring concentrations of   these compounds in fish and sediments that are cost-effective and appropriate   for a large-scale monitoring program are not currently available, it was not   possible to include them in the NBSP at this time.  Trace metal analyses of sediments, liver tissue, and stomach contents were   conducted by the Southeast Fisheries Science Center, and data on metal   concentrations will not be presented here. Analyses of metals from previous   NBSP samplings on both the West Coast (Varanasi et al. 1988, 1989a) and the   Northeast Coast (NOAA 1988) indicated that there was little relationship   between tissue concentrations of metals and concentrations of metals in   sediment in any of the fish species examined. These findings suggest that   metals may not be bioavailable to fish, or that their uptake and accumulation   are governed by complex processes that make tissue concentrations of metals a   poor indicator of environmental exposure. Consequently, no attempt was made to   correlate metal concentrations with fish disease.  Logistic regression (Breslow and Day 1980, Schlesselman 1982) was used to   assess the influence of biological and contaminant-associated risk factors on   lesion occurrence. The logistic function is considered a basic model for   dose-response relationships and logistic regression analysis is frequently   applied in epidemiological and epizootiological studies. This technique is   similar to commonly-used stepwise multiple regression procedures, but is   uniquely suited to binomial or proportional data (e.g., lesion prevalence or   lesion presence or absence). Results of these analyses and their relationship   to other studies of pollution-associated disease in winter flounder will be   discussed below.  Table   of Contents