U.S. Dept Commerce/NOAA/NMFS/NWFSC/PublicationsNOAA Technical Memorandum NMFS-NWFSC-36       Fish Injuryin the Hylebos Waterwayof Commencement       Bay,Washington
Tracy K. Collier, Lyndal L. Johnson,Mark S. Myers,       Carla M. Stehr, Margaret M. Krahn, and John E. Stein

March 1998

U.S. DEPARTMENT OF       COMMERCEWilliam M. Daley, Secretary

National Oceanic and Atmospheric       AdministrationD. James Baker, Administrator

National Marine Fisheries       ServiceRolland A. Schmitten, Assistant Administrator       for Fisheries 
This item should be cited as follows:
Collier, T.K., L.L. Johnson, M.S. Myers, C.M. Stehr, M.M. Krahn, and J.E. Stein. 1998. Fish injury in the Hylebos Waterway in Commencement Bay, Washington. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-36, 576 p. 

Reference in this document to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA.

Paper copies of this report may be available for a fee from:National Technical Information Service5285 Port Royal RoadSpringfield, VA 221611-800-553-6847orders@ntis.fedworld.gov
CONTENTS
List of Figures

List of Tables 

List of Appendices 

Executive Summary 

Section 1. Juvenile Salmon Injury Study 
Introduction   
Methods   
Sample collection and analysis Statistical methods 
Results   
Chemical contaminant exposure     Liver: chlorinated hydrocarbons Bile: metabolites of aromatic       hydrocarbonsStomach contents: aromatic compounds and chlorinated       hydrocarbons 
Biological indicators of contaminant exposure.     Cytochrome P4501A in liver DNA adducts in liver 

Discussion   of major findings 

Section 2. Flatfish Toxicopathic Injury Study 
Introduction   
Methods   
Sample collection and analysis Statistical methods Inclusion of     additional samples 
Results   
Chemical contaminant exposure     Stomach contents: aromatic compounds and chlorinated hydrocarbons       Bile: metabolites of aromatic hydrocarbons Liver: chlorinated       hydrocarbons 
Biological indicators of contaminant exposure     Cytochrome P4501A in liver DNA adducts in liver Toxicopathic       lesions       
Kidney lesions Liver lesions 
Chemical risk factors for     liver lesion occurrence     Rock sole English sole       
Influence of PAH exposure Influence of PCB exposure         Influence of DDT exposure 
Comparisons with historical data         Lesion prevalences Biliary FACs DNA adducts in liver CYP1A       activity in liver 

Discussion   of major findings 

Section 3. Flatfish Reproductive Injury Study 
Introduction   
Methods   
Sample collection and analysis Statistical methods 
Results   
Chemical contaminant exposure     Aromatic and chlorinated hydrocarbons in stomach contents       Chlorinated hydrocarbons in liver and metabolites of aromatic       hydrocarbons in bile 
Biological indicators of contaminant     exposure     DNA adducts in liver
Toxicopathic liver lesions Condition     factor, growth, and hepatosomatic index Reproductive dysfunction     Ovarian development Ovarian lesions Gonadosomatic index       17§-estradiol concentrations.
Chemical risk factors     Gonadal development Ovarian lesions GSI and plasma estradiol       concentrations

Discussion   of major findings 

Citations
List of Appendices
The following appendices are available only in PDF format - click here to download the Adobe Acrobat reader: Adobe Acrobat

Appendix A - Sampling and Analysis Plan for Hylebos Waterway Fish Injury Studies (42 p. kb)

Appendix B - List of Individual Organic Analytes Measured in Chemical Analyses of Fish Liver Tssue and Stomach Contents for Hylebos Waterway Fish Injury Studies (4 p. kb)

Appendix C - Case Narratives for Hylebos Water Fish Injury Studies 
Juvenile   salmon injury, Biliary FACs
 (7 p. kb)
Juvenile   salmon injury, Cytochrome P4501A
 (10 p. kb) 
Juvenile   salmon injury, DNA Adducts
 (2 p. kb)   
Juvenile   salmon injury, Semivolatile Organics
 (33 p. kb) 
Toxicopathic   Conditions in Flatfish, Biliary FACs
 (5 p. kb) 
Toxicopathic   Conditions in Flatfish, Cytochrome P4501A
 (8 p. kb) 
Toxicopathic   Conditions in Flatfish, DNA Adducts
 (2 p. kb) 
Toxicopathic   Conditions in Flatfish, Semivolatile Organics
 (33 p. kb) Toxicopathic Conditions in Flatfish and Reproductive   Toxicology in Flatfish, Liver, and Kidney Histopathology   
Part     1 (43 p. kb)Part     2 (42 p. kb)
Reproductive   Toxicology in Flatfish, Biliary FACs
 (13 p. kb) 
Reproductive   Toxicology in Flatfish, DNA Adducts
 (4 p. kb) 
Reproductive   Toxicology in Flatfish, Rapid PCB Analysis of Livers
 (24 p.   kb) 
Reproductive   Toxicology in Flatfish, Semivolatile Organics
 (28 p. kb) 
Reproductive   Toxicology in Flatfish, Ovarian Histopathology
 (14 p. kb) 
Reproductive   Toxicology in Flatfish, Somatic Indices
 (11 p. kb) 
Reproductive   Toxicology in Flatfish, Reproductive Steroid RIAs-Plasma 17-b Estradiol
   (36 p. kb) 
Appendix D - Sediment Chemistry Data (59 p. kb) 
Appendix E - Commencement Bay Damage Assessment Quality Assurance Plan (35 p. kb)

List of Abbreviations (Included with Appendix E) 
LIST OF FIGURES
SECTION 1. JUVENILE SALMON INJURY STUDY

Figure 1.1 Sampling site locations for the Juvenile Salmon Injury Study

Figure 1.2 Hexachlorobenzene and hexachlorobutadiene in liver of juvenile salmon

Figure 1.3 PCBs in liver of juvenile salmon

Figure 1.4 DDTs, chlordanes, and dieldrin in liver of juvenile salmon

Figure 1.5 Heptachlor, lindane, and aldrin in liver of juvenile salmon

Figure 1.6 Fluorescent aromatic compounds in bile of juvenile salmon

Figure 1.7 Aromatic hydrocarbons in stomach contents of juvenile salmon

Figure 1.8 Hexachlorobenzene and hexachlorobutadiene in stomach contents of juvenile salmon

Figure 1.9 PCBs in stomach contents of juvenile salmon

Figure 1.10 DDTs, chlordanes, and dieldrin in stomach contents of juvenile salmon

Figure 1.11 Heptachlor, lindane, and aldrin in stomach contents of juvenile salmon

Figure 1.12 Cytochrome P4501A activity and DNA adducts in liver of juvenile salmon

Figure 1.13 Comparisons of Hylebos Waterway juvenile salmon data with historical data from Commencement Bay and the Duwamish Waterway 

SECTION 2. FLATFISH TOXICOPATHIC INJURY STUDY

Figure 2.1 Sampling site locations for the Flatfish Toxicopathic Injury Study

Figure 2.2 Aromatic hydrocarbons in English sole and rock sole stomach contents

Figure 2.3 Polychlorinated biphenyls in English sole and rock sole stomach contents

Figure 2.4 Hexachlorobenzene and hexachlorobutadiene in stomach contents of English sole and rock sole

Figure 2.5 DDTs, chlordanes, and dieldrin English sole and rock sole stomach contents

Figure 2.6 Heptachlor, lindane, and aldrin in English sole and rock sole stomach contents

Figure 2.7 Fluorescent aromatic compounds in bile of English sole and rock sole

Figure 2.8 PCBs in English sole and rock sole liver.

Figure 2.9 Hexachlorobenzene and hexachlorobutadiene in English sole and rock sole liver

Figure 2.10 DDTs, chlordanes, and dieldrin in English sole and rock sole liver.

Figure 2.11 Heptachlor, lindane, and aldrin in English sole and rock sole liver

Figure 2.12 Cytochrome P4501A and DNA adducts in English sole and rock sole liver

Figure 2.13 Liver lesions in English sole and rock sole sampled during the Flatfish Toxicopathic Injury Study.

Figure 2.14 Liver lesions in rock sole sampled during the Flatfish Reproductive Injury Study

Figure 2.15 Liver lesions in rock sole sampled during the combined Flatfish Toxicopathic and Reproductive Injury Studies

Figure 2.16 Liver lesions in English sole from the combined Flatfish Injury Studies

Figure 2.17 Liver lesions in English sole and rock sole from the combined Flatfish Injury Studies, compared to historical data

Figure 2.18 Fluorescent aromatic compounds in bile of English sole and rock sole from the Flatfish Toxicopathic Injury Study, compared to historical data

Figure 2.19 DNA adducts in English sole and rock sole from the Flatfish Toxicopathic Injury Study, compared to historical data

Figure 2.20 Cytochrome P4501A in liver of English sole and rock sole from the Flatfish Toxicopathic Injury Study, compared to historical data

SECTION 3. FLATFISH REPRODUCTIVE INJURY STUDY

Figure 3.1 Sampling sites for the Flatfish Reproductive Injury Study

Figure 3.2 Contaminants in female English sole stomach contents

Figure 3.3 Hexachlorobenzene in female English sole liver

Figure 3.4 Fluorescent aromatic compound concentrations in bile of female English sole

Figure 3.5 DDTs in female English sole liver

Figure 3.6 Summed PCB congeners in female English sole liver

Figure 3.7 TCDD toxic equivalents in female English sole liver

Figure 3.8 DNA adducts in female English sole liver

Figure 3.9 Liver lesions in female English sole.

Figure 3.10 Mean size at age for female English sole.

Figure 3.11 Length-weight relationships in female English sole.

Figure 3.12 Condition factor in subadult and adult female English sole.

Figure 3.13 Hepatosomatic index in female English sole

Figure 3.14 Percentages of adult female English sole with developing yolked eggs

Figure 3.15 Percentages of female English sole with developing yolked eggs by age class

Figure 3.16 Estimated probability of ovarian development with age in English sole

Figure 3.17 Ovarian lesion prevalences in female English sole.

Figure 3.18 Gonadosomatic index in adult female English sole.

Figure 3.19 Plasma 17ß-estradiol concentrations in adult female English sole.

Figure 3.20 Liver HCB as a risk factor for precocious sexual maturation in subadult female English sole

Figure 3.21 Fluorescent aromatic compounds (FACs) in bile as a risk factor for inhibited gonad development in adult female English sole

Figure 3.22 Projected egg and larval production by age in female English sole. 

LIST OF TABLES

SECTION 1. JUVENILE SALMON INJURY STUDY

Table 1.1 Sampling sites and dates for the 1994 Juvenile Salmon Injury Study

Table 1.2 Numbers of composite samples collected for 1994 Juvenile Salmon Injury Study 

SECTION 2. FLATFISH TOXICOPATHIC INJURY STUDY 

Table 2.1 Site of capture and age as risk factors for liver lesions in rock sole, Flatfish Toxicopathic Injury Study

Table 2.2 Site of capture and age as risk factors for liver lesions in English sole, Flatfish Toxicopathic Injury Study

Table 2.3 Site of capture and age as risk factors for liver lesions in English sole, combined studies

Table 2.4 Biliary FACs as risk factors for liver lesions in English sole, combined studies

Table 2.5 Chemical risk factors for liver lesions in English sole, combined studies

Table 2.6 Hepatic DNA adducts as risk factors for liver lesions in female English sole, Flatfish Reproductive Injury Study

Table 2.7 Hepatic DNA adducts as risk factors for liver lesions in English sole, combined studies

Table 2.8 Chlorinated hydrocarbons (measured by HPLC/PDA) as risk factors for liver lesions in female English sole on an individual basis, Flatfish Reproductive Injury Study

Table 2.9 Chlorinated hydrocarbons (measured by HPLC/PDA) as risk factors for liver lesions in female English sole, based on mean concentrations and prevalence data, Flatfish Reproductive Injury Study 
SECTION 3. FLATFISH REPRODUCTIVE INJURY STUDY 
Table 3.1 PCB congeners in female English sole liver

Table 3.2 Chemical risk factors for reproductive abnormalities in female English sole

Table 3.3 Contaminant exposure vs. gonad size and plasma estradiol concentrations in subadult and adult female English sole

Table 3.4 Contaminant exposure vs. precocious steroid production and gonad growth in subadult female English sole
Contributing Investigators(in alphabetical order)Bernadita F. AnulacionDonald W. BrownTom HomO. Paul OlsonSusan M. PierceWilliam L. ReichertHerbert R. SanbornSean Y. SolGina M. YlitaloContributing scientific staff(in alphabetical order)Nicolaus G. AdamsBrian D. BillJennie L. BoltonDaryle BoydRichard H. BoyerCynthia A. BucherDouglas G. BurrowsJon BuzitisSena CamarataLawrence P. Chicchelly, Jr.Josie EllisBich-Thuy L. EberhartBarbara L. FrenchWilliam D. GronlundBeth H. HornessLarry HufnagleAnna N. KagleyKyle E. KardongKari KoponenCheryl A. KroneTom LeeDaniel P. LomaxRonald W. PearceKaren Peck-MillerPaul D. PleshaCatherine A. SloanFrank C. SommersSylvester SpencerScott StolzMaryjean L. WillisEXECUTIVE SUMMARYBeginning in the mid-1970s, studies by the Environmental Conservation Division of the Northwest Fisheries Science Center had shown that there was a strong relationship between chemical contaminants in sediments, and increased prevalences of liver disease, including cancer, in bottomfish in Puget Sound. In the mid-1980s, these studies were extended to show that bottomfish from contaminated areas also were experiencing a wide range of reproductive impairment, including failure to develop eggs, failure to spawn, lowered egg fertility, and lowered production of normal larvae. Also beginning in the mid-1980s, studies of juvenile salmon in urban waterways of Puget Sound began to show that these fish were accumulating contaminants as they made their passage from freshwater to saltwater, and this contaminant exposure was linked to impaired immune function and reduced growth, which are key elements involved in the early ocean survival of salmon. 
The Hylebos Waterway is one of several waterways included in the Commencement Bay Superfund Site. The Commencement Bay Natural Resource Trustee Council (the Trustees) is concerned that hazardous substances present in the sediments of the Hylebos Waterway have caused and are continuing to cause injury to natural resources. Members of this council include NOAA, the U.S. Fish and Wildlife Service, the Washington State Department of Ecology, the Washington State Department of Natural Resources, the Puyallup Tribe of Indians, and the Muckelshoot Indian Tribe. As the Trustees became aware of the severity of the chemical contamination in the sediments of the Hylebos Waterway, concerns were raised as to whether the flatfish feeding in the Hylebos Waterway or the juvenile salmon migrating through it were suffering injury as a result of their exposure to toxic contaminants. Accordingly, studies to assess fish injury were designed, approved, and funded, and these studies are the subject of this publication.

The overall objective of the Hylebos Waterway fish injury assessment investigation was to document the current status of contaminant exposure and associated injuries in fish indigenous to the Hylebos Waterway, to quantify the extent of these injuries, and to evaluate links between these injuries and chemical contaminants in this waterway. The basic design of the studies was derived from substantial previous effort performed by the National Marine Fisheries Service, documenting several relationships between exposure of fish to chemical contaminants and the occurrence of biological impacts. The three specific objectives were to 1) assess chemical contamination and the potential for associated injuries in juvenile salmonids, 2) determine toxicopathic conditions (e.g., liver disease) in flatfish, and 3) determine contaminant-induced reproductive dysfunction in flatfish. The field collections for these studies were conducted between May of 1994 and April of 1995, analyses of samples were completed by June of 1996, and analysis of data was completed by January of 1997.

The objective of the Juvenile Salmon Injury Study was to determine to what degree juvenile chum (Oncorhynchus keta) and chinook salmon (Oncorhynchus tshawytscha) captured from the Hylebos Waterway might be exposed to organic contaminants, and to compare the levels of exposure observed to previous studies where such exposures have been linked to biological dysfunction. The results showed that juvenile chum and chinook salmon from the Hylebos Waterway are being exposed to a wide range of chemical contaminants, compared to fish from hatcheries or reference estuaries. These contaminants include high and low molecular weight polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs, including the toxic congeners 105 and 118), hexachlorobutadiene (HCBD), hexachlorobenzene (HCB), DDTs, heptachlor, and several pesticides. Moreover, the concentrations of contaminants in juvenile chinook and chum salmon from the Hylebos Waterway are comparable to levels previously shown to be associated with biological injury in juvenile chinook salmon. For example, contaminant concentrations similar to those measured in tissues, stomach contents, and fluids of juvenile salmon from the Hylebos Waterway are associated with impaired growth, suppression of immune function, and increased mortality following pathogen exposure in chinook salmon collected from another contaminated waterway in Puget Sound, the Duwamish River. To what extent these effects are occurring in juvenile salmon from the Hylebos Waterway, however, was not specifically determined in the investigation. Additional research is underway to further assess injury to these fish, by examining the potential of the toxicants present in the Hylebos Waterway to elicit biological dysfunction such as impaired disease resistance and reduced growth. 

The objectives of the Flatfish Toxicopathic Injury Study, were to 1) assess the extent of chemical contaminant exposure of English sole (Pleuronectes vetulus) and rock sole (Pleuronectes bilineatus) residing in the Hylebos Waterway by examining chemical concentrations, early biochemical responses, and prevalences of liver lesions, and 2) compare liver lesion prevalences and biochemical responses with historical data to determine if conditions have changed since the late 1970s and 1980s. Results showed that English sole and rock sole residing in the Hylebos Waterway are being exposed to several classes of toxic chemical contaminants, including PAHs, PCBs, DDT (and its derivatives), HCB, and HCBD. These exposures were also clearly reflected in elevated levels of DNA adducts and cytochrome P4501A (CYP1A) activities in liver. Higher prevalences of injury in the form of toxicopathic liver lesions were detected in English sole from the Hylebos Waterway, in comparison to the reference site. The chemical risk factors most commonly associated with hepatic lesion occurrence in Hylebos flatfish were exposure to PAHs, PCBs, and DDTs. The frequency of injury in the form of hepatic lesions closely paralleled levels of DNA damage in liver in fish from the study sites. In comparing the results of this investigation with earlier work done in the Hylebos Waterway, it was found that lesion prevalences and biochemical measures of early response to contaminant exposure had not changed appreciably since the late 1970s, and 1980s, respectively. Thus, there has been little or no improvement in the Hylebos Waterway over the past decade or more with respect to this particular fish injury. 

The Flatfish Reproductive Injury Study was undertaken to assess contaminant-associated changes in gonadal development and reproductive steroid concentrations in female English sole. Because of its potential to impact population growth and consequently fisheries productivity, reproductive impairment is potentially one of the most damaging effects of aquatic pollution on marine fish. Accordingly, considerable effort was expended on this portion of the Hylebos Waterway fish injury investigation. Similar to the other portions of the investigation, it was shown that adult female flatfish from the Hylebos Waterway are being exposed to several classes of toxic contaminants, including PAHs, PCBs, pesticides, and HCB. Moreover, this contaminant exposure was associated with several types of injury in female English sole from the Hylebos Waterway. Most notably, 40-50% of juvenile sole in the Hylebos Waterway showed precocious sexual maturation, and 20-30% of adult sole showed inhibited gonadal development. Whereas the inhibition of gonadal development was most closely associated with indicators of exposure to PAHs, the increased prevalence of precocious sexual maturation was associated with increased exposure to both chlorinated hydrocarbons and PAHs. Substantial proportions of fish also exhibited DNA damage and hepatic lesions. The reproductive injury observed in Hylebos fish would presumably reduce the number of eggs and larvae contributed by these animals to the English sole population in Commencement Bay and southern Puget Sound, and the associated decline in recruitment could reduce the overall productivity of the sole population and its resilience to the impacts of other environmental stressors. 

On to Section One