Table Of ContentEFFECTS OF PAPER MILL EFFLUENTS ON THE HEALTH AND
REPRODUCTIVE SUCCESS OF LARGEMOUTH BASS (MICROPTERUS
SALMOIDES): FIELD AND LABORATORY STUDIES
By
MARIA SOLEDAD SEPULVEDA
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIALFULFILLMENT
OFTHE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2000
ACKNOWLEDGMENTS
My
deepest thanks to Dr. Timothy Gross for having accepted me as his graduate
student. He introduced me to the field of fish ecotoxicology and endocrine disruption,
and offered me the unique opportunity to work with an interdisciplinary research team at
USGS-BRD
the Florida Caribbean Science Center Ecotoxicology Laboratory
(Gainesville, FL). Ecotoxicology staffmembers and friends Shane Ruessler, Carla
Wieser, Jon Wiebe, Nikki Kernaghan, Kelly McDonald, and Vincent and Lisa Centonze
assisted in innumerable tasks ranging from the treatment, collection and processing of
fish; analysis ofplasma samples for reproductive hormones; to the conduction of
spawning studies. Lyn Day and Meri Nantz helped in the purchase ofequipment and in
the day-to-day process oftrying to get things done. My most sincere appreciation to all of
them. I will always remember my days at the "ecotoxicology lab"!
I would also like to thank my othercommittee members: Drs. Evan Gallagher and
Steve Roberts (Center for Environmental and Human Toxicology, College ofVeterinary
Medicine, UF, Gainesville, FL); Dr. Trenton Schoeb (Department of Pathobiology,
College ofVeterinary Medicine, UF, Gainesville, FL), and Dr. Nancy Denslow
(Department ofBiochemistry and Molecular Biology, College ofMedicine, UF,
Gainesville, FL). Their comments and suggestions greatly improved the quality ofmy
research and writing. Also, Dr. Schoeb's help in the interpretation ofhistology slides and
11
EROD
Dr. Gallagher's assistance in the conduction of and other liver enzyme assays are
greatly acknowledged.
This dissertation would not have been possible without the support ofthe
Georgia-Pacific Corporation, Atlanta, GA. through a two-year grant awarded to Dr.
Timothy Gross. My deepest thanks to Stewart Holm, from Georgia-Pacific, for his help
in designing and overseeing this project. Also thanks to Myra Carpenter (Georgia-
Pacific, Palatka Operation, FL) who was responsible for the building and maintenance of
the treatment tank system in Palatka.
I also thank Karen Sheehy (Center for Environmental and Human Toxicology,
UF, Gainesville, FL) for her assistance in the EROD analyses, as well as Kevin Kroll and
Marjorie Chow (Center for Biotechnology, UF, Gainesville, FL) for conducting the 1998
vitellogenin analyses. I am specially thankful to Kevin, who kindly spent time training
me in the "art" ofconducting ELISAs for the detection ofvitellogenin in bass.
I would like to thank Bill Johnson and other staff from the Florida Game and
FreshwaterFish Commission (Fisheries Research Laboratory, Eustis, FL) for providing
boats and personnel for the collection of largemouth bass from the St. Johns River and for
aging the fish using otolith analyses. John Higman (St. Johns Water Management
District) provided invaluable information on fish and sediment chemical data from sites
along the lower St. Johns River. Jay Harrison and Galin Jones (Department ofStatistics,
UF, Gainesville, FL) assisted in the statistical analyses.
Finally, with special recognition and love, I thank my husband, Hugo Ochoa, my
daughter Natalia Ochoa, and my mother Pura Luque, for their invaluable collaboration,
111
support, and most of all for their patience. I would not have been able to finish this
degree without them.
iv
1
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS
ii
ABSTRACT
viii
CHAPTERS
INTRODUCTION AND BACKGROUND
1 1
Introduction 1
General Aims ofEcotoxicological Studies 2
Biomarkers ofExposure and Effects 2
Levels ofBiological Organization 3
Effects ofEndocrine Disrupting Chemicals in Wildlife 4
The Pulp and Paper Industry 5
Introduction 5
Pulp and Paper Manufacturing Process Sequence 5
Pollution Outputs 9
Wastewater Treatment Technology 10
The Pulp and Paper Cluster Rules 1
Georgia-Pacific's Paper Mill Plant in Palatka, Florida 12
General Description 12
Ongoing Improvements 13
Sublethal Physiological Effects ofPulp and Paper Mill Effluents on Fish 13
General Health Effects 14
Liver Health Effects 17
Reproductive Health Effects 22
The Largemouth Bass {Micropterus salmoides) 27
General Description 27
Geographic Distribution 28
Habitat and Range 28
Growth and Feeding Habits 29
Reproduction 30
Significance ofthis Work 33
Organization ofDissertation 34
COMPARISON OF REPRODUCTIVE PARAMETERS FROM FLORIDA
2
LARGEMOUTH BASS {MICROPTERUS SALMOIDES FLORIDANUS)
SAMPLED FROM REFERENCE AND CONTAMINATED SITES IN THE
ST. JOHNS RIVER AND TRIBUTARIES 36
Introduction 36
Materials and Methods 39
Sampling Sites and Fish Collection 39
Chemical Analysis from Fish Tissues 40
Bleeding, Necropsies, and Age Determination 41
Reproductive Endpoints 41
LiverEROD Activity 46
Statistical Analyses 47
Results 48
Chemical Analysis from Sediments and Fish Tissues 48
Physiological and Reproductive Endpoints 48
Discussion 53
3 IN VIVO ASSESSMENT ON THE REPRODUCTIVE EFFECTS OF
PAPER MILLEFFLUENTS ON LARGEMOUTH BASS 91
Introduction 91
Materials and Methods 92
Animals and Holding Facility 92
Effluent Characteristics 93
Exposure Conditions 94
Reproductive Endpoints 94
Statistical Analyses 95
Results 96
Females 96
Males 98
Both Sexes 99
Discussion 100
4 IMPACT OF PAPER MILLEFFLUENTS ON LARGEMOUTH BASS
HEALTH: FIELD AND LABORATORY STUDIES 128
Introduction 128
Materials and Methods 129
Field Study 129
Laboratory Study 134
Results 136
Field Study 136
Laboratory Study 137
Discussion 138
EFFECTS OF PAPER MILLEFFLUENTS ON REPRODUCTIVE
5
SUCCESS OF LARGEMOUTH BASS 165
Introduction 165
Materials and Methods 166
In Vivo Experiment 166
Spawning Study 169
Results 173
VI
1
In Vivo Experiment 173
Spawning Study 178
Discussion 180
6 IN VITRO STEROIDOGENESIS BY GONADALTISSUES FROM
FEMALE LARGEMOUTH BASS EXPOSED TO PAPER MILL
EFFLUENTS AND RESIN ACIDS 224
Introduction 224
Materials and Methods 225
Effluent Characteristics 225
In Vivo Exposures 226
In Vitro Gonadal Cultures 227
Statistical Analyses 228
Results 229
Experiment 229
1
Experiment 2 229
Experiment 3 230
Discussion 230
7 GENERAL CONCLUSIONS ECOLOGICAL SIGNIFICANCE, AND
,
FUTURE RESEARCH NEEDS 242
General Conclusions 242
Field Studies 242
In Vivo Studies 243
In Vitro Studies 245
Ecological Significance 245
Future Research Needs 247
Additional Field Studies 248
Mesocosms Studies 248
Evaluate Effects on Other Aquatic Organisms 249
Evaluate Biological Effects ofMill Improvements 249
Mechanistic Studies 250
REFERENCES 248
BIOGRAPHICAL SKETCH 268
VI
Abstract ofDissertation Presented to the Graduate School
ofthe University ofFlorida in Partial Fulfillment ofthe
Requirements for the Degree ofDoctor ofPhilosophy
EFFECTS OF PAPER MILL EFFLUENTS ON THE HEALTH AND REPRODUCTIVE
SUCCESS OF LARGEMOUTH BASS (MICROPTERUS SALMOIDES): FIELD AND
LABORATORY STUDIES
By
Maria S. Sepulveda
August 2000
Chairman: Timothy S. Gross
MajorDepartment: Veterinary Medicine
The effects ofbleached kraft paper mill effluents (BKME) on the health and
reproduction oflargemouth bass (Micropterus salmoides) were examined through field
BKME-
and laboratory studies. During 1996/97 and 1998, bass were collected from both
exposed (located at different distances downstream from the effluents discharged by the
Palatka Paper Mill) and reference streams, and parameters compared across sites.
Although concentrations of sex steroids and vitellogenin and induction of
biotransformation enzymes were altered in bass from exposed streams, there were no
differences on gonad weights, fecundities, and age distributions across sites. Some health
endpoints were altered in bass collected from exposed streams, but these fell within
normal ranges and were probably not associated with detrimental health effects.
Laboratory studies that involved exposures ofbass to different concentrations of
BKME (10, 20, 40, and 80%) for up to 56 days were conducted during the reproductive
Vlll
seasons of 1998 and 1999. In contrast to what was observed in the field, bass exposed to
Palatka's BKME responded with changes at the biochemical-level (decline in sex steroids
and vitellogenin) that were usually translated into tissue/organ-level responses (declines
in gonad weights and retardation of gonad development). The majority ofthese responses
were observed afterexposures to at least 20% BKME concentrations. These changes,
however, did not result in lower fecundities, egg sizes, or hatchabilities. Later
evaluations offry numbers revealed significant negative effects ofeffluent exposure on
survivorship, with a threshold effluent concentration of 10%. This decline was probably
caused by an increased frequency ofdeformities coupled with alterations on growth. It
was hypothesized that these changes could have resulted from alterations in "egg quality"
due to chronic failure ofparental reproductive systems after almost two months of
effluent exposures, and/or to acute embryo toxicity after translocation ofpersistent
organic compounds from the mother to the developing embryo.
Results from in vitro cultures showed significant declines in the production of70-
estradiol by follicles collected from BKME-exposed females. These declines paralleled
changes in plasma 17p-estradiol observed in females during the in vivo studies, and
suggested the direct action ofchemical(s) at the gonad level. There were no dose-
response changes associated with resin acid exposures, which would suggest the action of
chemicals other than resin acids as possible causative agents ofthe reproductive
alterations observed in BKME-exposed largemouth bass.
IX
CHAPTER
1
INTRODUCTION AND BACKGROUND
Introduction
Forover a century, men have used seas, lakes, rivers, and other sources ofwater
as final resting points for many industrial and agricultural contaminants. This chemical
release to the environment has prompted scientists around the world to evaluate the
potential effects ofsuch pollutants on both human and ecosystem health. Since fish play
a fundamental role in aquatic ecosystems, they have been widely used as monitors of
environmental health and quality. Earlier studies on the effects ofenvironmental
contaminants in fish and other wildlife were focused on examining and reporting obvious
and rather catastrophic responses, such as big kills or die-offs. Because stricter
environmental regulations nowadays have overall decreased the toxicity ofchemicals that
are being released to the environment, acute lethality is no longer a likely response in
wild populations inhabiting contaminated areas. Long-term exposure to low
concentrations ofpollutants, however, is still ofmajorconcern because it can seriously
affect the ability of individual animals to grow, survive to adulthood, and reproduce
leading to population number declines and eventually extinction. Measuring these subtle
responses in fish populations in response to environmental contaminants is the focus of
this dissertation.
