by Brendan John Galloway BSc (Honours) UNBSJ, 1997

by Brendan John Galloway BSc (Honours) UNBSJ, 1997
Evaluating the Suitability of Fish Species for Environmental Monitoring Programs.
by
Brendan John Galloway
BSc (Honours) UNBSJ, 1997
MSc UNBSJ, 2000
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
Doctor of Philosophy
In the Graduate Academic Unit of Biology
Supervisor:
Co-Supervisor:
Kelly Munkittrick, PhD, Dept. of Biology, UNBSJ
R. Allen Curry, PhD, Dept. of Biology, UNBF
Examining Board:
Deborah MacLatchy, PhD, Dept. of Biology, UNBSJ
Tillman Benfey, PhD, Dept. of Biology, UNBF
Rob Moir, PhD, Dept. of Social Sciences, UNBSJ
External Examiner: Douglas Holdway, PhD, School of Science, University of
Ontario Institute of Technology
This thesis is accepted by the
Dean of Graduate Studies
THE UNIVERSITY OF NEW BRUNSWICK
March, 2005
© Brendan J. Galloway, 2005
ABSTRACT
Research was conducted to assess the suitability of different fish species
as sentinels for monitoring the effects of multiple stressors on fish populations in
the upper Saint John River and to further evaluate the use of small-bodied fish
for environmental monitoring programs. The effluent discharge areas were
associated with nutrient enrichment, and slimy sculpin showed as much as a
50% increase in condition factor downstream of the effluents, as well as
increases in growth and liver size. Stable isotope data indicated slimy sculpin did
not move between sites, and while the sculpin were capable of demonstrating the
increases in nutrients downstream of the effluents, white sucker showed few
differences between sites. A follow-up study re-examined those two species, as
well as blacknose dace and yellow perch in the same area, and found that
neither species showed as dramatic a response as the sculpin. The differences
in species responses may be related to differences in life history characteristics,
or mobility, or exposure. Results clearly demonstrated the ability of slimy sculpin
to reflect local conditions, but there may be a trade-off between sensitivity and
relevance, depending on the questions being addressed. Slimy sculpin can be a
useful sentinel species for monitoring large rivers that receive multiple industrial
and municipal effluents being discharged in close proximity.
Blacknose dace showed increased variability in gonad size as spawning
time approached, reducing the power and ability to detect differences between
sites. A detailed study examined the biology of four multiple spawning, small-
ii
bodied species (i.e., blacknose dace, northern redbelly dace, golden shiner,
mummichog) to determine the influence of sampling time on data variability. This
information was useful to identify the most suitable time of the year to focus
sampling efforts for these fish species, and determine whether there are
additional challenges to using these species as sentinels.
This PhD project showed that the sentinel fish species selected for a
particular environmental monitoring program will be depend on the a priori study
objectives. The project provided sampling and data interpretation guidance for
the use of small-bodied, multiple-spawning fish species in future environmental
monitoring programs.
iii
ACKNOWLEDGMENTS
Returning to graduate school after completing my M.Sc. seemed like a
crazy thought to me 4 years ago - so crazy that I did it! It’s amazing how plans
can change when great opportunities present themselves. It’s been a long road
(but, fun!!) and I can finally extended my gratitude to all the people who have
helped me along the way.
First and foremost I would like to thank Dr. Kelly Munkittrick for providing
me the opportunity to conduct research towards my Ph.D. degree. Kelly has
been supportive in all aspects of my research and always provided enthusiasm
and optimism (and a Keith’s) when things seemed like they were heading for the
crapper. I greatly appreciate all of the opportunities and advice that Kelly has
provided over the years and I know I’m a better scientist because of it. Thanks
for singing Neil Diamond tunes with me during SETAC karaoke nights too! I
would also like to thank the rest of the Munkittrick clan (Patty, Sara, Jessica) for
allowing me to crash on the couch when I was travelling between Saint John and
Fredericton.
Thanks to Dr. R. Allen Curry for agreeing to be my co-supervisor. I am
sure it was with some uncertainty that he accepted this role, but my thesis work
has greatly benefited from his involvement. I would also like to thank Dr. Simon
Courtenay for being part of my supervisory committee and for his valuable
contributions throughout my studies.
Big thanks to Mr. Craig Wood for his assistance in securing industry
funding for my Industrial Post-Graduate NSERC Scholarship. Additional thanks
iv
to Craig for his encouragement, advice and friendship through my studies – and
providing the fine single malt scotch after TAP meetings too.
Thanks to Sue Dunn and all the folks at NexFor/Fraser Papers
(Edmundston Operations) for their financial assistance, support, and their
willingness to participate in these studies.
My research was all field work and I wish to thank all those people who
unknowing volunteered for “Brendan’s Biology Boot-Camp”. We had some long
days lugging heavy equipment through some rough terrain in bad weather (there
were some nice days too!). Your enthusiasm and hard work made the things
easier, fun, and each trip a success. In no specific order I thank: Kelly
Munkittrick, Steve Currie, Kirk Roach, Michelle Gray, Sandra Brasfield, Mark
Gautreau, Genevieve Vallieres, Jenn Peddle, Nicole Duke, Chris Cronin, Tim
Rees, Megan Findlay, Coral Cargill, Lisa Peters, my Bhutanese buddy Karma
Tenzin, and Andrew “weasel” Halford.
My research was funded through a number of research grants awarded to
Dr. Kelly Munkittrick, including; Toxic Substances Research Initiative (TSRI),
NSERC Discovery Grant, Sir James Dunn Wildlife Research Centre Fund, New
Brunswick Wildlife Council Trust Fund, NWRI, Environment Canada (Atlantic
Region), New Brunswick Innovation Fund, and the Canadian Water Network.
Additional thanks for extensive in-kind support from NexFor, Noranda
Technology Centre, Fraser Papers, New Brunswick Department of Environment,
New Brunswick Department of Natural Resources, Barry Mower and the Maine
v
Department of Environmental Protection, DFO, and the Maine Chapter of the
Nature Conservancy.
I would also like to thank everyone in my family for all the encouragement
and support they have given me throughout my studies – even though they’re still
not sure what I do! Finally, and most important, my biggest thanks goes to my
wife, Tiffanny – for all her love, support, patience and laughter throughout this
journey. Thanks for taking time to help me in the field and lab when I needed
help the most. It’s been a long journey - we can finally move on and seek out
new adventures together.
vi
TABLE OF CONTENTS
ABSTRACT ......................................................................................................... ii
ACKNOWLEDGMENTS ..................................................................................... iv
TABLE OF CONTENTS .................................................................................... vii
LIST OF TABLES ................................................................................................x
LIST OF FIGURES ............................................................................................ xii
CHAPTER 1.
GENERAL INTRODUCTION.................................................. 15
1.1
Environmental Effects Monitoring Program.............................. 15
1.2
Cumulative Effects Assessment .............................................. 17
1.3
Fish Species Selection ............................................................ 20
1.4
Research Hypothesis, Thesis Objectives and Outline ............. 22
1.5
References .............................................................................. 27
CHAPTER 2.
Examination of the Responses of Slimy Sculpin (Cottus
cognatus) and White Sucker (Catostomus commersoni) Collected on the Saint
John River Downstream of Pulp mill, Paper mill, and Sewage Discharges1. .. 33
2.1
Abstract ................................................................................... 33
2.2
Introduction .............................................................................. 34
2.3
Materials and Methods ............................................................ 37
2.3.1
Study area and mill characteristics ............................................ 37
2.3.2
Fish collections .......................................................................... 38
2.3.3
Statistical analyses .................................................................... 42
2.4
2.4.1
Results..................................................................................... 42
October 1999............................................................................. 42
vii
2.4.2
December 1999 ......................................................................... 44
2.4.3
Edmundston Pulp Mill - Fall 2000 Slimy sculpin ........................ 46
2.5
Discussion ............................................................................... 47
2.5.1
White sucker.............................................................................. 49
2.5.2
Ecological significance .............................................................. 51
2.6
Conclusions ............................................................................. 53
2.7
Acknowledgements.................................................................. 54
2.8
References .............................................................................. 70
CHAPTER 3.
Identifying a suitable fish species for monitoring a large river
receiving effluents from a pulp and paper mill, municipal sewage wastewater
facilities, and agricultural runoff2. .................................................................... 75
3.1
Abstract ................................................................................... 75
3.2
Introduction .............................................................................. 76
3.3
Materials and Methods ............................................................ 78
3.3.1
Study area and mill characteristics ............................................ 78
3.3.2
Fish collections .......................................................................... 79
3.3.3
Data Analyses ........................................................................... 81
3.4
Results..................................................................................... 82
3.5
Discussion ............................................................................... 85
3.6
Conclusions ............................................................................. 89
3.7
Acknowledgements.................................................................. 91
3.8
References ............................................................................ 100
viii
CHAPTER 4.
Influence of seasonal changes in relative liver size, condition,
relative gonad size, and variability in ovarian development of multiple spawning
freshwater fish for use in environmental monitoring programs3. ................... 103
4.1
Abstract ................................................................................. 103
4.2
Introduction ............................................................................ 104
4.3
Materials and Methods .......................................................... 107
4.3.1
4.4
Data Analyses ......................................................................... 108
Results................................................................................... 109
4.4.1
Blacknose dace ....................................................................... 109
4.4.2
Golden shiner .......................................................................... 112
4.4.3
Northern Redbelly Dace .......................................................... 113
4.4.4
Mummichog ............................................................................. 114
4.5
Discussion ............................................................................. 116
4.5.1
Reproductive Development ..................................................... 116
4.5.2
Liversomatic Index and Condition Factor ................................ 119
4.5.3
Data Variability, Sample Sizes, and Power ............................. 121
4.6
Conclusions ........................................................................... 125
4.7
Acknowledgements................................................................ 126
4.8
References ............................................................................ 126
CHAPTER 5.
GENERAL DISCUSSION..................................................... 149
5.1
Conclusions ........................................................................... 171
5.2
References ............................................................................ 173
ix
LIST OF TABLES
Table 2.2. Means ± SE (n) of various parameters of adult male and female slimy
sculpin (Cottus cognatus) collected downstream of a pulp mill (DS
Pulp), paper mill (DS Paper), and municipal sewage (DS Mad) and
from reference sites located at Clair, St. Hilaire, and immediately
upstream of the mill (US Pulp). Within a row, differences (p < 0.05)
among sites are denoted by different uppercase letters. .................57
Table 2.3. Means ± SE (n) of various parameters of adult male and female white
sucker (Catostomus commersoni) collected in October 1999. Within a
row, differences (p < 0.05) among sites are denoted by different
uppercase letters. ............................................................................60
Table 2.4. Regression estimates for adult male slimy sculpin (Cottus cognatus)
condition factor and adult female slimy sculpin length-at-age..........61
Table 2.5. Comparative summary of slimy sculpin (Cottus cognatus) and white
sucker (Catostomus commersoni) collected downstream of the
sulphite pulp mill relative to fish collected from St. Hilaire (reference
site), Saint John River, October 1999, New Brunswick, Canada
(modified from Gibbons et al. 1998)a. .............................................62
Table 3.1. Means ± SE (n) of various parameters measured in adult male and
female slimy sculpin (Cottus cognatus) and adult male and female
blacknose dace (Rhinichthys atratulus) collected downstream of a
sulphite pulp mill (D/S Pulp), upstream of the pulp mill diffuser (U/S
Pulp), and downstream of municipal sewage inputs (D/S Mad), and
from reference sites located downstream of the St. Francis River
(Capone) and at St. Hilaire. Within a row, differences (p ≤ 0.05)
among sites are denoted by different uppercase letters. (*Significant
interaction within ANCOVA model; N/A = data not available). .........92
Table 3.2. Regression estimates for adult male slimy sculpin length-at-age
(Cottus cognatus) and adult male blacknose dace (Rhinichthys
atratulus) gonad development. ........................................................95
Table 3.3. Means ± SE (n) of various parameters measured in adult yellow perch
(Perca flavescens) and white sucker (Catostomus commersoni)
collected on the Saint John River, New Brunswick, Canada. Within a
row, differences (p ≤ 0.05) among sites are denoted by different
uppercase letters. ............................................................................96
Table 3.4. A comparison of species responses to the combined effluents at
Edmundston (sewage, pulp mill effluent) and a comparison of fish
responses upstream and downstream of the pulp mill discharge. ...98
Table 4.1. Microscopic characteristics for the determination of oocyte
developmental stages (modified from Blazer 2002).......................132
x
Table 4.2. Means ± SE (n) and minimum and maximum (in parentheses) values
for length, weight, condition factor (k), liversomatic index (LSI), and
gonadosomatic index (GSI) in adult female and male blacknose dace
(Rhinichthys atratulus), golden shiner, mummichog (Fundulus
heteroclitus), and northern redbelly dace (Phoxinus eos) collected
from various sites in southern New Brunswick, Canada. Within a
column, differences (p < 0.05) among sampling dates are denoted by
different superscript uppercase letters. (*Significant interaction within
ANCOVA model)............................................................................133
Table 4.3. Log10 regression estimates for adult female and male blacknose dace
(Rhinichthys atratulus) condition and gonad size and condition factor
for adult female mummichog (Fundulus heteroclitus) gonad size
collected from sites in southern New Brunswick in 2003 and 2004.139
Table 4.4. Relationship of log10 ovary weight to log10 adjusted body weight in
female fish on various dates in 2003 and 2004..............................141
Table 4.5. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for female northern redbelly dace Phoxinus
eos and blacknose dace Rhinichthys atratulus. .............................142
Table 4.6. Comparison of the relationship of log10 ovary weight to log10 body
weight between all female blacknose dace (Rhinichthys atratulus) with
2 year old fish and fish weighing 2 – 4 g 3 year old collected from
Milkish Brook on May 20, 2003......................................................143
Table 5.1. Percent differences in condition factor (k), liversomatic index (LSI),
and gonadosomatic index (GSI) for slimy sculpin, white sucker, yellow
perch, and blacknose dace collected at various times from 1999-2003
(“% Difference” is for municipal sewage and pulp mill effluent
exposed fish relative to reference fish at St. Hilaire). (-) data not
available; NS – not significantly different. ......................................177
xi
LIST OF FIGURES
Figure 2.1. Map of the study area showing the relative location of reference and
exposure fish collection sites (not to scale)......................................63
Figure 2.2. Relative liver size (liversomatic index [LSI]; % of body weight) of
adult male (black bars) and female (white bars) white sucker collected
downstream of the Edmundston pulp mill (DS Pulp) and at reference
sites located upstream of the pulp mill (US Pulp), St. Hilaire, Ogilvie
Lake (males only), and First Lake during the Fall, October 1999.
Refer to Figure 2.1 map for location. ...............................................64
Figure 2.3. Stable-isotope ratios of carbon and nitrogen in adult male (bold
dashed lines) and female (solid lines) slimy sculpin collected from
reference sites at Clair, St. Hilaire, and upstream of the Madawaska
River (US Mad) and from downstream of the Edmundston pulp mill
(DS Pulp) and Madawaska paper mill (DS Paper), October 1999.
Values are expressed as deviations (δ) from standards. Refer to
Figure 2.1 map for location. .............................................................65
Figure 2.4. Summary of "ecologically relevant" changes in condition factor in
female slimy sculpin collected during the Fall 2000 fish survey of the
Saint John River. Fish were collected from various sites, including:
Moody Bridge (MB), Priestly Brook (PB), a site downstream of the St.
Francis River (Capone), sites located upstream (US Nad) and
downstream (DS Nad) of a poultry processing plant, upstream of the
international bridge at Clair, downstream of Baker Brook (DS Baker),
St. Hilaire, upstream of the Madawaska River (US Mad), downstream
of the Madawaska River (DS Mad), upstream of the Iroquois River
(US Iroq), Iroquois River (IR), upstream of the pulp mill diffuser (US
Pulp), downstream of the pulp mill diffuser (DS Pulp), and the Little
Forks (LF). Cross-hatched bars represent reference sculpin collected
from sites on the Saint John River. Black bars represent sculpin from
sites exposed to either poultry processing waste effluent (DS Nad),
municipal sewage wastewater (DS Mad), and pulp mill effluent (DS
Pulp). White bars represent sculpin collected from tributaries. Refer
to Figure 2.1 map for location. .........................................................66
Figure 2.5. Summary of "ecologically relevant" changes in liver size female slimy
sculpin collected during the Fall 2000 fish survey of the Saint John
River. Solid and stippled horizontal lines represent data (i.e., mean ±
25%, mean ± 2SD, respectively) collected at St. Hilaire. Values that
fall between the horizontal lines are considered “normal” for the SJR
at the time of sampling. Cross-hatched bars represent reference
sculpin collected from sites on the Saint John River. Black bars
represent sculpin from sites exposed to either poultry processing
waste effluent (DS Nad), municipal sewage wastewater (DS Mad),
and pulp mill effluent (DS Pulp). White bars represent sculpin
collected from tributaries. Asterisk (*) indicates possible upstream
source of contamination. Refer to Figure 2.1 map for location........68
xii
Figure 3.1. The study area near Edmundston, NB. River flow is from left to right
on the map.......................................................................................99
Figure 4.1. Mean monthly ambient temperature for Milkish Brook during the
study period. Upper bars depict monthly high temperatures and lower
bars depict low temperatures.........................................................144
Figure 4.2. Percentage of each oocyte occurrence (except stage 1, primary
oocytes) in female blacknose dace Rhinichthys atratulus caught in
Milkish Brook. Dark grey bars represent stage 2 oocytes; light grey
bars represent stage 3 oocytes; white bars represent stage 4 oocytes;
bars with a diamond-grid represent stage 5 oocytes; bars with
horizontal-grid represent stage 6 oocytes. .....................................145
Figure 4.3. Percentage of each oocyte occurrence (except stage 1, primary
oocytes) in female golden shiner Notemigonus crysoleucas caught in
Little Chamcook Lake. Dark grey bars represent stage 2 oocytes;
light grey bars represent stage 3 oocytes; white bars represent stage
4 oocytes. ......................................................................................146
Figure 4.4. Percentage of each oocyte occurrence (except stage 1, primary
oocytes) in female northern redbelly dace Phoxinus eos caught in
beaver pond in Keswick River. Dark grey bars represent stage 2
oocytes; light grey bars represent stage 3 oocytes; white bars
represent stage 4 oocytes; bars with a diamond-grid represent stage
5 oocytes; bars with a horizontal-grid represent stage 6 oocytes. .147
Figure 4.5. Percentage of each oocyte occurrence (except stage 1, primary
oocytes) in female mummichog Fundulus heteroclitus caught in a salt
marsh located adjacent to Taylor's Island. Dark grey bars represent
stage 2 oocytes; light grey bars represent stage 3 oocytes; white bars
represent stage 4 oocytes; bars with a diamond-grid represent stage
5 oocytes; bars with a horizontal-grid represent stage 6 oocytes. .148
Figure 5.1. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for all adult female blacknose dace collected
on May 20 (dashed lines) and June 11 (solid lines), 2003. The solid
curved line numbered “1” represents α = 0.05 and β = 0.05; the solid
curved line numbered “2” represents α = 0.10 and β = 0.10; and the
solid curved line numbered “3 represents α = 0.05 and β = 0.20...179
Figure 5.2. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for 2 year old female blacknose dace
collected on May 20 (dashed lines) and June 11 (solid lines), 2003.
The solid curved line numbered “1” represents α = 0.05 and β = 0.05;
the solid curved line numbered “2” represents α = 0.10 and β = 0.10;
and the solid curved line numbered “3 represents α = 0.05 and β =
0.20................................................................................................180
xiii
Figure 5.3. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for 2 – 4 g female blacknose dace collected
on May 20 (dashed lines) and June 11 (solid lines), 2003. The solid
curved line numbered “1” represents α = 0.05 and β = 0.05; the solid
curved line numbered “2” represents α = 0.10 and β = 0.10; and the
solid curved line numbered “3 represents α = 0.05 and β = 0.20...181
Figure 5.4. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for all female northern redbelly dace collected
on May 27 (dashed lines) and June 22 (solid lines), 2003. The solid
curved line numbered “1” represents α = 0.05 and β = 0.05; the solid
curved line numbered “2” represents α = 0.10 and β = 0.10; and the
solid curved line numbered “3 represents α = 0.05 and β = 0.20...182
Figure 5.5. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for 2 year old female northern redbelly dace
collected on May 27 (dashed lines) and June 22 (solid lines), 2003.
The solid curved line numbered “1” represents α = 0.05 and β = 0.05;
the solid curved line numbered “2” represents α = 0.10 and β = 0.10;
and the solid curved line numbered “3 represents α = 0.05 and β =
0.20................................................................................................183
Figure 5.6. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for 2 – 4 g female northern redbelly dace
collected on May 27 (dashed lines) and June 22 (solid lines), 2003.
The solid curved line numbered “1” represents α = 0.05 and β = 0.05;
the solid curved line numbered “2” represents α = 0.10 and β = 0.10;
and the solid curved line numbered “3 represents α = 0.05 and β =
0.20................................................................................................184
xiv
CHAPTER 1.
1.1
GENERAL INTRODUCTION
Environmental Effects Monitoring Program
In Canada, the pulp and paper industry is one of the largest dischargers of
wastewater effluent and has been identified as having potential to disrupt the
growth and reproductive performance of fish [Environment Canada 1997]. In the
early 1990’s, a new regulatory package was developed that included
requirements for pulp and paper mills in Canada to conduct a cyclical
Environmental Effects Monitoring (EEM) program. The objectives of the EEM
program are to assess whether receiving environment changes are associated
with pulp and paper mill effluent exposure to fish, fish habitat, and use of the
fisheries resource. The first two cycles of EEM monitoring identified a variety of
responses in fish populations near pulp mills [Munkittrick et al. 2002]. At some
Canadian sites, exposure of fish to pulp mill effluent has been associated with
delayed sexual maturity, decreased gonad size, increased liver size, and
decreased secondary sex characteristics [Munkittrick et al. 1991; McMaster et al.
1991]. A recent review of the adult fish population survey data from the
Canadian Pulp and Paper Mill EEM program showed that the predominant
response patterns observed in fish populations across Canada were a decrease
in gonad weight and increases in liver weight, condition factor, and weight-at-age
[Environment Canada 2003]. Together, these responses are indicative of
metabolic disruption or endocrine disruption in association with a nutrient
enrichment effect [Environment Canada 2003]. Some of the responses observed
15
in Canada have also been documented in fish exposed to pulp mill effluent in
Scandinavia [Sandstrom et al. 1988; Sandstrom 1994; Tana and Lehtinen 1996;
Sandstrom et al. 2003] and the United States [Adams et al. 1992].
Since the late 1980’s, the Canadian pulp and paper industry has made
significant financial contributions to improving effluent treatment which has
reduced toxicity, nutrient enrichment, and biochemical oxygen demand
[Munkittrick et al. 2000]. Today, the effects of pulp mill effluent on fish
populations are sub-lethal and often fall within the range of natural variability that
can be associated with natural stressors (e.g., temperature) that are not directly
related to anthropogenic inputs [Munkittrick et al. 2000]. As such, separating the
effects of pulp mill effluent exposure on fish health from the effects associated
with natural stressors can be difficult. Other wastewater effluents, such as
municipal sewage, are often discharged in close proximity to pulp and paper mill
discharges. It has been well-documented that wastewater from municipal
sewage facilities contains compounds that can alter the reproductive system of
fish [Jobling and Sumpter 1993; Jobling et al. 1996; Harries et al. 1996; Allen et
al. 1999]. The acute impacts of pulp and paper mill effluents in the past were
gross to the point that the potential impacts of other wastewater discharges (e.g.,
sewage) on the health of aquatic organisms were not recognized. Today,
distinguishing between the effects of sewage and/or other anthropogenic
stressors and pulp and paper mill effluents on fish growth, reproduction, and
survival can be difficult. Attributing responsibility for the existing impacts within a
river responding to multiple stressors to a specific industry is problematic. In
16
Edmundston, New Brunswick, both municipal sewage wastewater and
agricultural inputs (i.e., animal manure runoff) are located upstream of the pulp
mill effluent diffuser. A high priority for research is associated with the need to be
able to separate the relative contributions of multiple discharges and assess the
potential cumulative effects of these discharges at the watershed scale. In order
to address this issue, the objectives of Chapters 2 and 3 were to compare the
whole-organism responses of small-bodied fish (i.e., slimy sculpin, Cottus
cognatus, and blacknose dace, Rhinichthys atratulus) and large-bodied fish (i.e.,
white sucker, Catostomus commersoni, and yellow perch, Perca flavescens)
along a downstream gradient in a section of the Saint John River receiving pulp
and paper mill effluents, municipal sewage, and agricultural runoff (i.e., animal
manure). The goal of the fish collections was to identify a suitable fish species
that could be used to assess the relative contribution of individual anthropogenic
stressors in a section of river receiving multiple stressors.
1.2
Cumulative Effects Assessment
In 1995, the Canadian Environmental Assessment Act (CEAA) required
development proponents to include a cumulative effects assessment (CEA) as
part of future environmental impacts assessments (EIAs). Changes to the CEAA
were based on the fact that long-term environmental impacts occur as a result of
the combined effects of multiple anthropogenic stressors [Hegmann et al. 1999].
Historically, environmental assessments have been conducted without clearly
identifying cumulative effects [Munkittrick et al. 2000]. Instead, traditional
approaches to environmental assessments have focused on stressor-based
17
predictive methods, which concentrate on single stressors (e.g., a single
chemical within a discharge or the discharge of a single effluent) [Munkittrick et
al. 2000]. Specifically, stressor-based CEAs have been conducted as desktop
exercises focused on documenting existing conditions and attempting to identify
potential future stressors, and identifying valued ecosystem components (VECs)
[Munkittrick et al. 2000]. Stressor-based CEAs use the assumed pathways of
impacts to make predictions about the future and propose possible mitigation
strategies [Munkittrick et al. 2000]. It is important to recognize that stressorbased risk assessments and EIAs have successfully helped to reduce the gross
environmental impacts of the past; the main problem with these approaches is
that relatively little site-specific data are used and the predictions made during
pre-development assessments are often not validated with follow up monitoring
during operational and post-operational phases [Munkittrick et al. 2000].
Although proponents have been required to include a CEA within the scope of
the EIA since 1995, there are currently no widely accepted, scientificallydefensible methods for analyzing and evaluating cumulative environmental
impacts [Munkittrick et al. 2000].
Munkittrick et al. [2000] proposed an effects-based cumulative effects
assessment (CEA) program to determine the cumulative impacts of multiple
aquatic stressors on fish populations. The effects-based approach involves
examining factors associated with the status of fish populations in developed and
undeveloped reaches of a large river basin. This approach uses an iterative
assessment program to define the integrated responses of fish to existing
18
conditions and to determine the factors currently limiting fish performance in the
system. The main objectives of an effects-based assessment of cumulative
effects are to document the baseline conditions of fish performance (e.g., growth,
condition, and gonad development), examine yearly variability, and examine
trends in responses over time in order to make some assessment of the existing
“accumulated environmental state” (i.e., the existing state of the system as it has
integrated all existing stressors, both natural and anthropogenic). Performance
is not limited when fish exposed to wastewater effluents exhibit no differences in
age, growth (e.g., weight-at-age), and reproduction when compared to upstream
reference fish. However, if fish showed changes in any of the performance
parameters noted above then one can conclude that limiting or enhancing factors
are present and will need to be confirmed and investigated in more detail through
follow-up studies. In contrast, stressor-based CEA’s do not consider that the
system may already be stressed by natural factors (e.g., resource competition,
predator-prey interactions) and/or other human activities (e.g., fishing pressure)
and only focus on areas where stakeholders perceive a potential stressor exists
(e.g., pulp mill, hydroelectric dam, mining operation). Under the stressor-based
approach, erroneous conclusions regarding potential impacts associated with an
identified stressor (e.g., pulp and paper mill) are likely because other factors
(e.g., natural food limitation or other anthropogenic stressors) which may limited
or enhance the system were not considered. The identification of factors that are
limiting (or enhancing) the existing performance is critical to subsequent
predictive attempts, the design of potential mitigation strategies and the design of
19
future monitoring strategies.
Overall, the main difference between the stressor-based and effects-driven
approaches to CEA’s are the kinds of data required [Munkittrick et al. 2000].
Effects-driven CEA’s are committed to baseline monitoring, adaptive
management, and post-operational monitoring that are not part of stressor-based
assessments [Munkittrick et al. 2000].
One of the key components of many environmental monitoring programs,
including the effects-driven CEA, is the selection of an appropriate sentinel fish
species for monitoring the potential biological impacts of wastewater effluents on
the receiving environment. It is critical that the selected sentinel species offer the
greatest potential for defining stressor influences within the system being studied
[Munkittrick et al. 2000]. Species are selected primarily according to abundance,
exposure, and the ability to measure growth, reproduction and age [Munkittrick et
al. 2000].
1.3
Fish Species Selection
In Canada, large-bodied fish species have commonly been used as
sentinels for monitoring the potential impacts of wastewater effluents (e.g., pulp
and paper mill effluent) on freshwater environments, and include: white sucker
spp. [McMaster et al. 1991; Hodson et al. 1992; Gagnon et al. 1994; Munkittrick
et al. 1994], lake whitefish, Coregonus clupeaformis [Munkittrick et al. 1992],
longnose sucker, Catostomus catostomus [Swanson et al. 1994], and mountain
whitefish, Prosopium williamsoni [Swanson et al. 1994]. Response to pulp mill
effluent exposure have been successfully measured in large-bodied fish, but
20
primarily in situations where fish movement between reference and exposure
sites was impeded by man-made barriers [Hodson et al. 1992; Munkittrick et al.
2000]. Using large-bodied fish for environmental monitoring in large, open rivers
has become a concern for a number of reasons, including: many large-bodied
fish species are capable of extensive movement beyond effluent exposure areas
[Swanson et al. 1994], insufficient numbers of fish captured to properly assess
potential impacts of pulp mill effluent exposure [Hodson et al. 1992], and
exploitation of commercially important species (e.g., lake whitefish) may obscure
or confound impacts associated with environmental stressors such as pulp mill
effluent [Gibbons et al. 1998a].
Recent evidence has shown that small, non-migratory fish species can be
used as alternative sentinel species in large, open water systems where mobility
may be an issue [Gibbons et al. 1998a; Munkittrick et al. 2000]. Many smallbodied fish species, such as cottids and cyprinids, are less mobile relative to
large-bodied species, possess a smaller home range, and exhibit territorial
behaviour [Van Vliet 1964; Hill and Grossman 1987; Minns 1995]. Together,
these characteristics increase the probability that the measured physiological,
biochemical and whole-organism responses in small-bodied fish species will
reflect the local environmental conditions where they were caught [Gibbons et al.
1998a]. In addition, small-bodied fish species are more abundant than largebodied fish species, which facilitates sampling; they are short-lived and therefore
exhibit alterations in growth and reproduction quicker than longer-lived, large-
21
bodied species; and they are not subjected to sport or commercial fishing
pressures [Gibbons et al. 1998a]. The main disadvantage of using small-bodied
species is the lack of understanding of life-history characteristics [Munkittrick et
al. 2000]. To address this issue, seasonal changes in liver size, condition factor,
gonad size, and variability in female gonad development were monitored in four
multiple spawning, small-bodied fish species (see Chapter 4).
Few studies have compared the whole-organism responses of small-bodied
and large-bodied fish exposed to anthropogenic stressors such as pulp mill
effluent. Gibbons et al. [1998b] compared the responses of trout-perch
(Percopsis omiscomaycus) and white sucker downstream of a pulp mill on the
Kapuskasing River in Northern Ontario. Results from this study showed smallbodied fish (i.e., trout-perch) and large-bodied fish (i.e., white sucker) exposed to
pulp mill effluent exhibited different whole-organism responses, but the
underlying mechanisms (i.e., size-specific mortality and/or recruitment problems)
responsible for the responses were similar. It is evident that small-bodied and
large-bodied fish in some rivers can exhibit differences in whole-organism
responses. However, due to a lack of comparative studies, it not known whether
small-bodied and large-bodied fish in other rivers will show differences in wholeorganism responses.
1.4
Research Hypothesis, Thesis Objectives and Outline
My research hypothesis was to determine whether fish can be used to
assess the relative contribution of individual anthropogenic stressors to the
existing environmental conditions in a river exposed to multiple anthropogenic
22
stressors. More specifically, the objectives of my thesis were to compare the
whole-organism responses of small-bodied fish (i.e., slimy sculpin and blacknose
dace) and large-bodied fish (i.e., white sucker and yellow perch) along a
downstream gradient in a river exposed to pulp and paper mill effluents,
municipal sewage wastewater, and agricultural runoff (i.e., manure); identify the
fish species that is best suited to assess the relative contribution of individual
anthropogenic stressors in a river exposed to multiple anthropogenic stressors;
determine which life history characteristics most influenced the ability of a fish
species to exhibit the measured whole organism responses; and provide
sampling guidance for the use of multiple spawning, small-bodied fish species for
use in environmental monitoring programs.
The objective of the first chapter (Chapter 2) was to compare the wholeorganism responses of white sucker and slimy sculpin (Cottus cognatus)
exposed to multiple wastewater effluents in the Saint John River near
Edmundston. The null (Ho) hypotheses were:
Ho1: There are no differences in the mean age, energy use (i.e., length-at-age,
gonad size), or energy stores (i.e., liver size, condition factor) of slimy sculpin
and white sucker exposed to pulp mill effluent, paper mill effluent, municipal
sewage wastewater, and manure relative to upstream reference fish.
Ho2: There are no differences in the stable carbon (13C/12C) and nitrogen
(15N/14N) isotope signatures of slimy sculpin exposed to pulp mill effluent, paper
mill effluent, municipal sewage wastewater, and agricultural runoff (i.e., manure)
23
relative to upstream reference fish.
The Saint John River near Edmundston is a complex receiving
environment. The river receives effluent from a pulp mill, a paper mill, three
treated sewage discharges, and other untreated sewage releases enter the river
over a 10-km reach. In addition, two tributaries carry manure into the system.
Results from previous work on the Moose River system in Ontario showed largebodied fish and small-bodied fish exposed to pulp mill effluent could exhibit
differences in whole-organism responses [Munkittrick et al. 2000]. Because
there was a lack of comparative studies, it was unknown whether different fish
species would exhibit different whole organism responses in a river that is more
industrialized (i.e., Saint John River) relative to the Moose River system - could
fish be used to discriminate individual wastewater effluents in a relatively small
section of the Saint John River receiving multiple wastewater effluents? The
results from the research on white sucker and slimy sculpin collected from the
Saint John River near Edmundston are presented in Chapter 2, “Examination of
the responses of slimy sculpin (Cottus cognatus) and white sucker (Catostomus
commersoni) collected on the Saint John River downstream of pulp mill, paper
mill, and sewage discharges”.
After identifying differences in the whole-organism responses of white
sucker and slimy sculpin collected from the upper Saint John River near
Edmundston, I wanted to test the hypothesis that body size, rather than other
inter-specific differences, was responsible for the differences in the whole
24
organism responses of slimy sculpin and white sucker documented in Chapter 2.
Would white sucker and yellow perch show similar responses? Would the whole
organism responses of slimy sculpin and blacknose dace be similar? Would the
whole organism responses of white sucker and slimy sculpin be consistent
between years? The null (Ho) hypotheses were:
Ho1: There are no differences in the mean age, energy use (i.e., length-at-age,
gonad size), energy stores (i.e., liver size, condition factor), or stable carbon and
nitrogen isotope signatures of slimy sculpin and blacknose dace exposed to pulp
mill effluent, municipal sewage wastewater, and manure relative to upstream
reference fish.
Ho2: There are no differences in the mean age, energy use (i.e., length-at-age,
gonad size), energy stores (i.e., liver size, condition factor), or stable carbon and
nitrogen isotope signatures of white sucker and yellow perch exposed to pulp mill
effluent, municipal sewage wastewater, and manure relative to upstream
reference fish.
Ho3: There are no differences in the stable carbon (13C/12C) and nitrogen
(15N/14N) isotope signatures of small-bodied fish and large bodied fish.
Ho4: There are no inter-annual differences in the whole-organism responses of
slimy sculpin and white sucker.
In addition, it was also important to further contribute to the scientific
understanding of the suitability of small-bodied fish species for environmental
monitoring programs. Results from this work are presented in chapter 3,
“Identifying a suitable fish species for monitoring a large river receiving effluents
25
from a pulp and paper mill, municipal sewage wastewater facilities, and
agricultural runoff”.
One of the main challenges of incorporating small-bodied fish species in
environmental monitoring programs is a lack of basic biological information.
Blacknose dace (Chapter 3) showed increased variability in the reproductive
endpoints and the data suggested sampling was conducted at a time of the year
when gonad development was highly variable. There was a need to investigate
this further in order to develop better guidance on how to address reproductive
investment in multiple-spawning fish species. To address this issue, blacknose
dace (Rhinichthys atratulus), golden shiner (Notemigonus crysoleucas), northern
redbelly dace (Phoxinus eos), and the estuarine mummichog (Fundulus
heteroclitus) were collected at various times during the pre-spawning, spawning,
and post-spawning seasons. The objectives of this work were to examine the
annual reproductive cycles of blacknose dace, golden shiner, northern redbelly
dace, and the estuarine mummichog; examine seasonal variability in gonad
development by monitoring changes in the coefficient of determination values
(i.e., r2 values) for regressions of ovary weight and body weight; increase our
understanding of natural source(s) of variability in gonad development; identify
suitable techniques to minimize data variability; and reduce sample size
requirements for detecting a critical effect sizes. The null hypothesis (Ho) was:
Ho: There are no differences in the variability of female ovarian development
during the pre-spawning, spawning, and post-spawning seasons for blacknose
dace, golden shiner, northern redbelly dace, and the estuarine mummichog.
26
Results of this work are presented in chapter 4, “Influence of seasonal
changes on the suitability of multiple spawning freshwater fish species for
examining reproductive impacts of stress”.
The final chapter (Chapter 5) provides a discussion and summary of the
significance of the whole-organism response patterns of slimy sculpin, blacknose
dace, white sucker, and yellow perch exposed to multiple anthropogenic
stressors in a section of the Saint John River near Edmundston, New Brunswick.
The suitability of the slimy sculpin for monitoring sections of rivers receiving
multiple wastewater effluents discharges is discussed. Sampling guidance for
use of multiple spawning, small-bodied fish species in environmental monitoring
programs is also considered. Suggestions for future research needs and
conclusions from the research are also provided.
For this thesis, length-at-age was used to estimate growth; fecundity (i.e.,
total number of eggs per female) and relative gonad size were used to estimate
reproduction; and condition factor and relative liver size were used to estimate
energy storage.
1.5
References
Adams, M.S., Crumby, W.D., Greeley, M.S., Shugart, L.R., Saylor, C. 1992.
Responses of fish populations and communities to pulp mill effluents: a
holistic assessment. Ecotox Environ Safe 24: 347-360.
Allen, Y., Scott, A.P., Matthiessen, P., Haworth, S., Thain, J.E., and Fiest, S.
1999. Survey of estrogenic activity in United Kingdom estuarine and
27
coastal waters and its effects on gonadal development of the flounder
Platichthys flesus. Environ Toxicol Chem 18: 1791-1800.
Environment Canada. 1997. Environment Canada’s national strategy for
addressing endocrine disrupting substances in the environment.
http://www.ec.gc.ca/eds/strat_e.htm.
Environment Canada. 2003. National assessment of pulp and paper
environmental effects monitoring data: a report synopsis. National Water
Research Institute, Burlington, Ontario. NWRI Scientific Assessment
Report Series No. 2. 28p.
Gagnon, M.M., Dodson, J.J., Hodson, P.V., Van Der Kraak, G., and Carey, J.H.
1994. Seasonal effects of bleached-kraft mill effluent on reproductive
parameters of white sucker (Catostomus commersoni) populations of the
St. Maurice River, Quebec, Canada. Can J Aquat Sci 51: 337-347.
Gibbons, W.N., Munkittrick, K.R., and Taylor, W.D. 1998a. Monitoring aquatic
environments receiving industrial effluents using small fish species 1:
Response of spoonhead sculpin (Cottus ricei) downstream of a bleachedkraft pulp mill. Environ Toxicol Chem 17: 2227-2237.
Gibbons, W.N., Munkittrick, K.R., and Taylor, W.D. 1998b. Monitoring aquatic
environments receiving industrial effluents using small fish species 2:
Comparison between responses of trout-perch (Percopsis omiscomaycus)
and white sucker (Catostomus commersoni) downstream of a pulp mill.
Environ Toxicol Chem 17: 2238-2245.
28
Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, P., Routledge,
E.J., Rycroft, R., Sumpter, J.P., and Tylor, T. 1996. A survey of
estrogenic activity in United Kingdom inland waters. Environ Toxicol
Chem 15: 1993-2002.
Hill, J., and Grossman, G.D. 1987. Home range estimates for three North
American stream fishes. Copeia 1987: 376-380.
Hodson, P.V., McWhirter, M., Ralph, K., Gray, B., Thivierge, D., Carey, J.H., Van
Der Kraak, G., Whittle, D.M., and Levesque, M. 1992. Effects of
bleached kraft mill effluent on fish in the St. Maurice River, Quebec.
Environ Toxicol Chem 17: 2227-2237.
Jobling, S., and Sumpter, J.P. 1993. Detergent components in sewage effluent
are weakly oestrogenic to fish : An in vitro study using rainbow trout
(Oncorhynchus mykiss) hepatocytes. Aquat Toxicol 27 : 361-372.
Jobling, S., Sheahan, D.A., Osborne, J.A., Matthiessen, P., and Sumpter, J.P.
1996. Inhibition of testicular growth in rainbow trout (Oncorhynchus
mykiss) exposed to estrogenic alkylphenolic chemicals. Environ Toxicol
Chem 15: 194-202.
Minns, C.K. 1995. Allometry of home range size in lake and river fishes. Can J
Fish Aquat Sci 52: 1499-1508.
McMaster ME, Van Der Kraak GJ, Portt CB, Munkittrick KR, Sibley PK, Smith IR,
Dixon DG. 1991. Changes in hepatic mixed-function oxygenase (MFO)
activity, plasma steroid levels and age at maturity of a white sucker
29
(Catostomus commersoni) population exposed to bleached kraft pulp mill
effluent. Aquat Toxicol 21: 199-218.
McMaster, M.R., Munkittrick, K.R., Jardine, J.J., Robinson, R.D., and Van Der
Kraak, G. 1995. Protocol for measuring in vitro steroid production by fish
gonadal tissue. Can Tech Rep Fish Aquat Sci 1961: 78.
Munkittrick, K.R., Portt, C.B., Van Der Kraak, G., Smith, I.R., Rokosh, D.A. 1991.
Impact of bleached kraft mill effluent on population characteristics, liver
MFO activity, and serum steroid levels of a Lake Superior white sucker
(Catostomus commersoni) population. Can J Fish Aquat Sci 48:13711380.
Munkittrick, K.R. 1992. A review and evaluation of study design considerations
for site-specifically assessing the health of fish populations. J Aquat
Ecosys Health 1: 283-293.
Munkittrick, K.R., Van Der Kraak, G., McMaster, M.E., Portt, C.B. 1992.
Response of hepatic mixed function oxygenase (MFO) activity and plasma
sex steroids to secondary treatment of bleached kraft pulp mill effluent and
mill shutdown. Environ Toxicol Chem 11: 1427-1439.
Munkittrick, K.R., Van Der Kraak, G., McMaster, M.E., Portt, C.B., van den
Heuvel, M.R., and Servos, M.R. 1994. Survey of receiving-water
environmental impacts associated with discharges from pulp mills. 2.
Gonad size, liver size, hepatic EROD activity and plasma sex steroid
levels in white sucker. Environ Toxicol Chem 13: 1089-1101.
30
Munkittrick, K.R., McMaster, M.E., Van Der Kraak, G., Portt, C., Gibbons, W.N.,
Farwell, A., and Gray, M. 2000. Development of methods for effectsdriven cumulative effects assessment using fish populations: Moose River
Project. SETAC Press, Pensacola, FL, USA.
Munkittrick, K.R., McGeachy, S.A., McMaster, M.E., Courtenay, S.C. 2002.
Review of cycle 2 freshwater fish studies from the pulp and paper
Environmental Effects Monitoring program. Water Quality Res J Can 37:
49-77.
Sandstrom, O., Neuman, E., and Karas, P. 1988. Effects of a bleached pulp mill
effluent on growth and gonad function in Baltic coastal fish. Water Sci
Tech 20: 107-118.
Sandstrom, O. 1994. Incomplete recovery in a coastal fish community exposed
to effluent from a modernised Swedish bleached kraft mill. Can J Fish
Aquat Sci 51: 2195-2205.
Sandstrom, O., Forlin, L., Grahn, O., Lander, L., Larsson, A., and Lindesjoo, E.
2003. Assessments of the environmental impact of Swedish pulp and
paper mill effluents at the beginning of the next century. In Stuthridge,
T.R., van den Heuval, M.R., Marvin, N.A., Slade, A.H., and Gifford, J.,
eds, Environmental Impacts of Pulp and Paper Waste Streams. SETAC,
Pensacola, FL, USA (CD) pp 499-504.
Swanson, S.W., Schryer, R., Shelast, R., Kloepper-Sams, P.J., Owens, J.W.
1994. Exposure of fish to biologically treated bleached-kraft mill effluent.
31
3. Fish habitat and population assessment. Environ Toxicol Chem 13:
1497-1507.
Tana, J., and Lehtinen, K. 1996. The aquatic environmental impact of pulping
and bleaching operations – an overview. The Finnish Environment,
Environmental Protection, Helsinki, Finland.
Van Vliet, W.H. 1964. An ecological study of Cottus cognatus (Richardson,
1836) in Northern Saskatchewan. MA Thesis, Department of Biology,
University of Saskatchewan. 155p.
Wassenar, L.I. and Culp, J.M. 1996. The use of stable isotope analyses to
identify pulp mill effluent signatures in riverine food webs. In Servos,
M.R., Munkittrick, K.R., Carey, J.H., and Van Der Kraak, G., eds,
Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie
Press, Delray Beach, FL, USA. pp 413-424.
32
CHAPTER 2.
Examination of the Responses of Slimy Sculpin (Cottus
cognatus) and White Sucker (Catostomus commersoni) Collected on
the Saint John River Downstream of Pulp mill, Paper mill, and
Sewage Discharges1.
1
Published: Galloway, B.J., Munkittrick, K.R., Currie, S., Gray, M.A., Curry, R.A.,
and Wood, C. 2003. Examination of the responses of slimy sculpin (Cottus
cognatus) and white sucker (Catostomus commersoni) collected on the Saint
John River downstream of pulp mill, paper mill, and sewage discharges.
Environmental Toxicology and Chemistry 22: 2898-2907.
2.1
Abstract
As part of a larger survey on cumulative effects within the Saint John River
basin, a fish survey was conducted near Edmundston, New Brunswick, in the fall
of 1999 using slimy sculpin (Cottus cognatus) and white sucker (Catostomus
commersoni). The discharge environment receives effluent from a pulp mill, a
paper mill, three sewage discharges, and tributaries receiving agricultural runoff.
Sculpin collected downstream of the sewage discharges and pulp mill effluent
had greater growth, condition, and liver size than reference fish, but no significant
differences in gonad size. Stable isotope data indicated slimy sculpin did not
move across the river to an area exposed to paper mill effluent or to sites
upstream of the pulp mill effluent diffuser. Female sculpin collected downstream
of the paper mill showed no significant differences in length, body weight, age,
condition factor, liver size, and gonad size compared to fish from reference sites.
33
Female white sucker collected downstream of the pulp mill did not differ
significantly in any measured parameter compared to reference fish. Liver sizes
of white sucker from the Saint John River were outside of the range considered
to be indicative of uncontaminated riverine sites. In 2000, sculpin collected
downstream from a poultry-processing facility had larger livers and lower
condition factors, suggesting that the site is contaminated. We found no
significant differences in sculpin length, weight, condition (except for males), and
liver size in sculpin collected downstream from the pulp mill in October 2001.
The responses of slimy sculpin and white sucker differed and may be related to
differences in life history characteristics. Results from this study indicate the
slimy sculpin is a suitable fish species for monitoring rivers that receive multiple
industrial and municipal effluents.
2.2
Introduction
Over the last seven years, we have been developing an effects-driven
cumulative effects assessment (CEA) framework for the Moose River basin in
Northern Ontario [Munkittrick et al. 2000; Munkittrick and McMaster 2000]. This
evaluation is an iterative approach: in it, the population-level responses to
aquatic stressors are used to direct diagnostic studies to identify factors limiting
performance of fish in the system. Population response patterns of fish to aquatic
stressors have been developed during previous work on metal mining effluent
[Munkittrick and Dixon 1989a; Munkittrick and Dixon 1989b], pulp mill effluents
[Munkittrick et al. 1991], interactions of hydroelectric and pulp mill discharges
[Munkittrick et al. 2000] and sewage and pulp mill effluents [Frank et al. 1998]. To
34
continue the development of this CEA framework, several questions were
highlighted by the Moose River studies, including (1) would the approach be as
successful in a system with more complicated waste inputs? and (2) how do
potential contributions from non-point sources affect interpretation?
The Saint John River basin was selected as the study site for a number of
reasons. Among these are the facts that it has more development than the
Moose River basin, and a larger population base. There are hydroelectric
developments and pulp mill discharges within the system (similar to the Moose
River basin study site). There is also considerable agricultural development
along the system, plus multiple sewage discharges, and other industrial
developments (e.g., poultry-processing facility). The estuarine area near the
mouth of the Saint John River is complicated by multiple discharges, and offers
the potential for future expansion of the work into estuarine and marine areas.
Various monitoring and research programs have been implemented on the Saint
John River, but few linkages exist between them.
Canadian pulp and paper mills are required to conduct a cyclical
Environmental Effects Monitoring (EEM) program to determine if environmental
effects are evident when the mill discharges effluents that are in compliance with
effluent guidelines. The monitoring program includes requirements for
monitoring effluents, benthic invertebrate communities and fish populations; the
fish program has recently been reviewed [Munkittrick et al. 2002]. The first cycle
of EEM for pulp and paper mills reported in 1996; cycle 2 reported April 2000. As
part of the 1996 Cycle I EEM report for the Fraser Inc., Edmundston pulp mill
35
(Edmundston, New Brunswick, Canada), the adult fish survey identified a number
of responses in fish downstream of the mill discharge [BAR Environmental Inc.
1996]. Yellow perch (Perca flavescens) collected downstream of the pulp mill
diffuser had lower growth compared to fish from upstream reference sites.
Gonads in male yellow perch were also smaller and females were less fecund.
White sucker (Catostomus commersoni) below the mill had greater growth rates
and gonad size, but lower indices of liver size.
The Saint John River near Edmundston receives effluent from a pulp mill,
a paper mill, and three treated sewage discharges, and other untreated sewage
releases enter the river over a 10-km reach. In addition, two tributaries carry
agricultural runoff into the system. The objectives of this study were to develop a
database to identify the background fish performance levels of the upper Saint
John River basin, examine whether the differences in fish reported in 1996 were
still present in 1999, and examine the selection of reference sites. Specifically,
we hypothesized that large-bodied and small-bodied fish from the upper Saint
John River would show differences in performance, and that small-bodied fish
would be the most suitable sentinel species for monitoring anthropogenic impacts
in the upper Saint John River.
Fraser Papers Inc. (Edmundston pulp mill, Edmundston, New Brunswick,
Canada), Nexfor (Noranda Technology Centre, Pointe-Claire, Quebec, Canada),
and Environment Canada (National Water Research Institute, Saskatoon,
Saskatchewan, Canada) participated in a variety of studies as part of Fraser
Papers Inc. commitment to EEM studies for cycle 2. These on-site projects
36
included a 90-d flow-through fathead minnow (Pimephales promelas) bioassay
[Parrott et al. 2000], a flow-through microcosm invertebrate exposure [Culp et al.
2003], and the fish collections (present study). Additional studies are
documenting the chemicals in pulp mill effluent that can be accumulated by fish
[Hewitt et al. 2003].
Based on results from the 1999 fish survey, the objectives of the fish
survey conducted in 2000 were to locate potential sources of contamination on
the Saint John River upstream of our reference site in Clair, and to examine
annual variability at sampling sites located on the mainstem of the Saint John
River near Edmundston, N.B.
2.3
Materials and Methods
2.3.1 Study area and mill characteristics
The Saint John River originates in headwater lakes in northern Maine
(USA) and travels almost 700 km to the mouth, located at the city of Saint John,
at the Bay of Fundy. Most of the drainage area (approximately 51%) is located in
New Brunswick; while the remaining area is located in the state of Maine (36%)
and the province of Quebec (13%) [BAR Environmental Inc. 1994]. Near
Edmundston, the Saint John River forms part of the international border between
Canada and the State of Maine. The flow of the Saint John River near
Edmundston is unregulated and undergoes considerable annual fluctuations; the
dominant substrate in the river is gravel [BAR Environmental Inc. 1994]. There
are no barriers to fish movement on the mainstem of the Saint John River near
37
Edmundston. However, a dam located near the mouth of the Madawaska River,
upstream of the mill discharge, restricts the movement of fish into the tributary.
The sulphite pulp mill (Edmundston, New Brunswick, Canada) and paper
mill (Madawaska, Maine, USA) are located on the north and south sides of the
Saint John River, respectively (Figure 2.1). The pulp mill produces
approximately 550 air-dried metric tonnes (ADMT) of sulphite pulp per day, 350
ADMT of ground wood pulp per day, 120 ADMT of boxboard per day, and 100
tonnes of de-inked product per day [BAR Environmental Inc. 1994]. Most of the
pulp produced at the Edmundston mill is shipped across the Saint John River to
the paper mill, which produces uncoated groundwood paper and coated fine
paper [BAR Environmental Inc. 1994]. The pulp mill discharges approximately
75,500 m3 of secondary-treated effluent per day from an aerated lagoon facility
(retention time ~7 days) into the north side of the Saint John River, about 4 km
downstream from the mill. The paper mill discharges primary-treated effluent into
the south side of the Saint John River, about 4 km upstream of the pulp mill
effluent diffuser.
2.3.2 Fish collections
White sucker (Catostomus commersoni) and slimy sculpin (Cottus
cognatus) were consistently captured in our field collections in the upper Saint
John River. In the fall of 1999, several large back-to-back rainfall events led to
very high water levels and prevented collection of yellow perch near the effluent
outfall.
38
We attempted to capture 20 adult fish of each sex at each site. Reference
fish were collected from sites located ~35 km above the pulp mill outfall near
Clair; at St. Hilaire (~16 km upstream), and at a site immediately upstream of the
Edmundston pulp mill diffuser (Figure 2.1). Fish were also collected from sites
located downstream of the pulp mill, paper mill, and municipal sewage
discharges. White sucker were collected in areas with deep pools and moderate
flowing water between October 21 and 29, 1999, using a gasoline generatorpowered electrofishing unit (Smith-Root GPP 7.5, Smith-Root Inc., Vancouver,
WA, USA) mounted in an inflatable raft boat. White sucker were collected from
St. Hilaire, at a site ~0.5 -1.0 km upstream of the pulp mill effluent outfall and a
site downstream (~0.5 -2.0 km) of the Edmundston pulp mill diffuser. White
sucker were also collected from reference sites: Ogilvie Lake (1999) and First
Lake (2000) (Table 2.1). White sucker are benthic feeders that have been
extensively used in other monitoring programs [Munkittrick et al. 2000;
Munkittrick et al. 2002; Environment Canada 1997; Munkittrick et al. 1998].
Slimy sculpin were collected between October 1-16 and December 14-16,
1999 using a battery-powered backpack electrofisher (Smith-Root Model 12B,
Smith-Root Inc., Vancouver, WA, USA). Sculpin were collected with dip nets
where rock/rubble substrates could be found in fast-moving water (runs and
riffles) approximately 0.5-1.5 m deep. Attempts to obtain sculpin from other
downstream sites were unsuccessful due to unsuitable habitat.
Slimy sculpin are widely distributed throughout shallower portions of the
Saint John River basin [Scott and Crossman 1998]. Slimy sculpin feed primarily
39
on aquatic invertebrates; they can reach a maximum length of ~13 cm and have
a life span of 5-6 years [Van Vliet 1964]. Sculpin spawn in spring and can reach
sexual maturity at age one, but body size appears to be the determinant of
maturity [Van Vliet 1964].
Fish were rendered unconscious by concussion, followed by spinal
severance. We then measured length (i.e., total length for sculpin, and forklength for white sucker), body weight, liver weight, and gonad weight. White
sucker were aged by counting annuli on clean, dried opercula under a dissecting
microscope. The annuli counts were verified by two readers. Slimy sculpin were
aged by counting annuli on sagittal otoliths according to the methods of MacKay
et al. [1990] and Gibbons et al. [1998a]. Briefly, sagittal otoliths were removed
surgically from each fish and placed in propylene glycol for ≥ 24 h before being
inspected under a dissecting microscope. If annuli were difficult to count, the
sagittal otoliths were mounted on slides and ground thinner using 400 grit sand
paper until the annuli became visible.
White muscle samples were taken from male and female sculpin for stable
isotope analysis. These analyses were used to determine residency patterns of
slimy sculpin collected at each site during the fall 1999 study on the Saint John
River. Approximately 0.5 g of white muscle tissue was removed from each fish
and placed in drying oven at 65 °C for 48 h. The dried tissue samples were
individually ground into a fine powder with a mortar and pestle, and were stored
in clean glass scintillation vials before isotopic analysis. Stable-isotope ratios
(e.g., 13C/12C, 15N/14N) are expressed as delta (δ) values and are measures of a
40
parts-per-thousand (‰) difference between the sample isotope ratio and that of
an international standard (carbon – Pee Dee Belemnite; nitrogen – atmospheric
nitrogen) [Peterson and Fry 1987]. Delta is expressed in terms of the numerator
(i.e., heavier stable isotope; e.g., 13C). Samples that have more negative delta
values contain less 13C or 15N relative to samples with higher delta values and
are termed depleted for an isotope. Samples that have higher delta values are
considered enriched.
Relative abundance of slimy sculpin collected during December 14-16,
1999, was estimated using catch-per-unit-effort (CPUE). In 2000, slimy sculpin
were collected between August 22 and September 13 from the same sites
described for the 1999 fish survey; these fish were sampled for whole-body
characteristics as outlined above. Additional sites were sampled during the 2000
fish survey (Table 2.1). In 2001, slimy sculpin were collected during August 2029 from a site downstream of the pulp mill effluent discharge point, and from
reference sites immediately upstream of the pulp mill diffuser and St. Hilaire
(Figure 2.1). Age data for male and female slimy sculpin collected in 2000 and
2001 was not available.
It is important to note that, in a previous fish survey, exposure to pulp mill
effluent was confirmed by the presence of 12-chlorodehydroabietic and 14chlorodehydroabietic resin acids in the bile of yellow perch and white sucker
captured downstream of the pulp mill effluent diffuser (same site used in the
present survey) [BAR Environmental Inc. 1996]. For the present study, stable-
41
isotope ratios of carbon (13C/12C) and nitrogen (15N/14N) measured in sculpin were
used to assess site fidelity and infer exposure.
2.3.3 Statistical analyses
Analysis of variance (ANOVA) was used to test for differences in mean
length, weight, and age of fish between sites; Tukey’s post hoc comparisons
were used to examine individual site differences when more than two sites were
compared. Analysis of covariance (ANCOVA) was used to assess site
differences in length-at-age, and the relationships between weight and length
(condition factor), liver size, and gonad size. Except for length-at-age and
condition, adjusted body weight (total wt – organ wt) was used as a covariate in
the ANCOVA model. Data were log10 transformed where appropriate before
performing ANOVA and ANCOVA, and sexes were analyzed separately. All data
analyses were done using SYSTAT® (SPSS, SYSTAT, Chicago, IL, USA)
statistical software. Gonad weights and liver weights were calculated as percent
adjusted body weight for summary purposes.
2.4
2.4.1
Results
October 1999
Male and female slimy sculpin and white sucker collected downstream of
the pulp mill showed few differences in age, when compared to fish from
reference sites (Tables 2.2, 2.3). Male sculpin collected downstream of the pulp
mill were significantly longer, heavier, had larger livers and higher condition
factors and had greater length-at-age (data not shown) relative to reference fish
42
from St. Hilaire and Clair (Tables 2.2, 2.4). Female sculpin from pulp mill
exposed and reference sites did not differ in length (Table 2.2), but exposed
female sculpin were heavier, had higher condition factors, greater length-at-age
and larger livers (livers and body weight higher relative to one reference site
only) (Tables 2.2, 2.4). No significant differences were found for male and female
gonad weights for either sculpin or white sucker collected from the Saint John
River. Both male and female white sucker downstream of the pulp mill showed
no significant differences in fork length, body weight, condition (males only), liver
size or length-at-age (data not shown) compared to reference sites. Male and
female white sucker collected at St. Hilaire and upstream and downstream of the
pulp mill had larger livers than fish collected from First Lake and Ogilvie Lake
(reference sites; see Figure 2.2). Male white sucker from First Lake and Ogilvie
Lake exhibited a LSI similar to reference suckers collected in northern Ontario
during the Moose River studies (Missinaibi, 0.85 ± 0.03 [Munkittrick et al. 2000]).
We found no significant differences in male and female sculpin length,
body weight, age, and gonad size for fish collected downstream of paper mill
effluent versus fish from Clair and St. Hilaire (Table 2.2). Female sculpin also
showed no differences in condition factor or liver size. However, male sculpin
from effluent-exposed sites had greater length-at-age compared to fish from Clair
and St. Hilaire, and larger livers relative to fish from St. Hilaire.
Male sculpin exposed to sewage effluent were longer, heavier, had higher
condition factors and had larger livers than fish from reference sites (Table 2.2).
Female sculpin collected near the sewage outfall were longer, heavier, older and
43
had greater length-at-age compared to reference fish, but did not differ in liver
size (Table 2.2). Female gonad development in exposed fish was reduced
relative to fish at Clair, but not St. Hilaire (Table 2.2).
The trends in stable isotope signatures for male and female sculpin
collected at each site were similar. Male and female sculpin downstream of the
Edmundston pulp mill and Madawaska paper mill and upstream of the
Madawaska River were enriched in 13C compared to fish collected at Clair and
St. Hilaire (Figure 2.3). Male and female sculpin downstream of the Edmundston
pulp mill were less 15N-depleted than fish collected downstream of the
Madawaska paper mill and upstream reference sites (Figure 2.3).
2.4.2 December 1999
Due to small gonad sizes in October, sculpin were collected again in
December when gonad development had progressed [prespawning female
sculpin had a gonadosomatic index (GSI) of 35.9% ± 2.8; Gray unpublished data]
and liver sizes were larger. We also added another sampling site, ~ 0.4 km
upstream of the pulp mill diffuser. Similar to October, male sculpin downstream
of the pulp mill were longer, heavier, and younger than fish at St. Hilaire (Table
2.2). These fish also had greater length-at-age (data not shown), but did not
differ significantly in gonad size. Female sculpin collected from within the effluent
plume were of similar age to reference fish (p=0.054), but were longer, heavier,
had larger livers, greater gonad size and higher condition and length-at-age,
compared to fish at St. Hilaire.
44
Approximately 85 sculpin were captured per h of electrofishing at the pulp
mill effluent exposed site, and 43 sculpin were captured per h of electrofishing at
the reference site (immediately upstream of the pulp mill diffuser). But only about
27 sculpin were caught per h of electrofishing at the reference site in St. Hilaire
(data not shown).
Results from the 1999 fish survey showed that white sucker collected from
various sites on the Saint John River exhibited few differences in liver size, but
white sucker from the Saint John River sites had larger livers compared to white
sucker from lake sites in Northern New Brunswick and reference rivers in
Northern Ontario. Slimy sculpin collected downstream of the Madawaska River
and pulp mill discharge had larger livers and faster growth (as evident from
otoliths analyses) compared to fish from upstream reference sites. Stable
isotope analysis suggested that the sculpin reflect the environmental conditions
at each sampling site; thus, they seem to have a relatively small home range.
The relatively small differences among sites, for white sucker, suggest either that
(1) white sucker are mobile, and thus do not reflect local environmental
conditions, or (2) there may be additional upstream inputs of pollutants. The
larger livers (without a concomitant increase in condition factor) of sculpin
collected at Clair, relative to Saint Hilaire reference sites, suggested that
upstream areas should be evaluated for potential contamination. This was done
in the fall of 2000.
45
2.4.3 Edmundston Pulp Mill - Fall 2000 Slimy sculpin
Female sculpin from sites where pulp mill effluent was present, in dilute
form, showed no difference in length and body weight relative to fish from
reference sites. However, they did have greater condition factors (Table 2.2;
Figure 2.4). Female sculpin from downstream of the pulp mill diffuser had a LSI
that was reduced relative to fish at Clair, but not relative to fish collected
immediately upstream of the pulp mill (p=0.06) or from St. Hilaire (p=0.08) (Table
2.2; Figure 2.5).
Sculpin were collected 15 km above the Clair collection site, immediately
downstream of a poultry processing operation in Saint-Francois-de-Madawaska,
NB. These fish had larger liver sizes (Figure 2.5) compared to fish from any of
the other sites sampled; condition factor was not different from sites immediately
upstream or downstream (Figure 2.4). Condition factor was lower in fish
downstream of major tributaries at the St. Francis River (Capone), Baker Brook,
and the Iroquois River, but not the Madawaska, which receives municipal
sewage (Figure 2.5).
Health-related parameters for sculpin from the Saint John River showed
significant differences within and between years (Table 2.2). Differences in the
magnitude of the parameters measured in fish between 1999 and 2000/2001
may have been related to inconsistencies in the sampling times; seasonal
changes in liver size, and possible changes in exposure would be expected due
to seasonal changes as well.
46
2.5
Discussion
The general response of slimy sculpin downstream of the sulphite pulp mill
suggests an overall increase in energy storage and utilization [Munkittrick et al.
2000; Gibbons and Munkittrick 1994]. Male sculpin exposed to pulp mill effluent
were longer, heavier, and in better condition than males from reference sites.
Female sculpin collected downstream of the pulp mill diffuser were heavier than
reference fish collected at St. Hilaire. Increases in body size have been
documented in fish exposed to pulp mill effluent [Gibbons et al. 1998a;
Munkittrick et al. 1994; Gibbons et al. 1998b], and may result from increased
water temperatures and/or indirect food web effects related to increased nutrient
concentrations in the exposure area [Gibbons et al. 1998b]. Fish collected near
some other pulp mills have shown delayed maturity, reduced body size, reduced
gonad size, and increased liver size [McMaster et al. 1991; Munkittrick et al.
1991; Munkittrick et al. 1992]. Although reproductive investment was difficult to
estimate, more sculpin were captured per h of electrofishing (as reflected by
CPUE) downstream of the pulp mill suggesting increased productivity.
The whole-organism parameters measured in slimy sculpin collected
downstream of the Edmundston pulp mill diffuser were not due to effluent toxicity.
A fathead minnow test was conducted during the same time period as our fish
survey [Parrott et al. 2000]. Results from the fathead minnow test showed that
when fish were exposed to 10% final effluent, an environmentally relevant
concentration survival was not affected, fish exhibited increased growth (length
and weight), and liver size was unaffected [Parrott et al. 2000]. The increased
47
energy storage and utilization observed in slimy sculpin collected downstream of
the pulp mill diffuser appeared to be the result of a nutrient enrichment effect
associated with the mill effluent. Benthic invertebrates contained in an artificial
mesocosm and exposed to Edmundston pulp mill effluent had a higher level of
emergence, compared to controls and N and P concentrations were greater
downstream of the mill, suggesting increased production due to enrichment [Culp
et al. 2003]. An enrichment effect also was noted in a previous environmental
assessment of the Edmundston pulp mill, which showed an increase in benthos
abundance and benthos richness downstream of the pulp mill diffuser [BAR
Environmental Inc. 1996]. Nutrient enrichment effects on benthic invertebrates
have been observed in other Canadian studies monitoring the potential impacts
of pulp mill effluent on the receiving environment. Lowell et al. [1996], for
example, reported that pulp mill effluent exposure resulted in significant
increases in growth and molting frequency of mayflies.
Establishing the residency patterns and home ranges of slimy sculpin is
important. The stable isotope data suggest that sculpin collected in 1999 from
the SJR near Edmundston had a small home range and the measured wholeorganism responses reflected local conditions. The δ13C and δ15N values of
sculpin collected downstream of the pulp mill suggest these sites were
isotopically enriched relative to reference sites in Clair and St. Hilaire (see Figure
2.3). Pulp mill effluent has been shown to be isotopically distinct from
background levels within a river, and may be a useful pulp mill effluent tracer
[Wassenar and Culp 1996]. In fact, δ13C values of sculpin downstream of the mill
48
closely resemble the carbon signature of terrestrial plants [Peterson and Fry
1987], indicating the terrestrial source of the carbon enrichment downstream of
the mill.
In 1999, water discharge was 7351 CFS during the fish survey
(http://waterdata.usgs.gov/me/nwis/uv/?site_no=01014000) and female sculpin
collected immediately upstream of the pulp mill did not differ much from fish
exposed to pulp mill effluent. However, during the fish survey in 2000, water
discharge was 1551 CFS and female sculpin collected upstream of the pulp mill
had greater LSI and lower condition factor scores, relative to fish that were
exposed to pulp mill effluent. Low water levels during the 2000 fish survey would
have resulted in sculpin being exposed to higher concentrations of pulp mill
effluent, compared to fish collected in 1999. If effluent from the Fraser
Edmundston pulp mill had a negative impact on fish performance in the receiving
environment, we would have expected to see fish with increased liversomatic
index (LSI) and a concomitant decrease in condition. However, this was not the
case. Understanding the potential influence of municipal sewage and non-point
sources of pollution on fish growth and reproductive performance in the upper
SJR is important and warrants further investigation.
2.5.1 White sucker
Male and female white sucker demonstrated different responses to the
effluent in terms of overall body characteristics when compared to slimy sculpin.
This was surprising, because both species were collected from the same sites
49
and should have had similar exposure regimes (see Table 2.5). Liver size did
not appear to be affected in white sucker collected on the Saint John River.
However, white sucker liver size was greater, relative to white sucker collected
from two pristine New Brunswick lakes (see Figure 2.2). The LSI of white sucker
from the two New Brunswick reference lakes were similar to LSI values for white
sucker collected from reference sites in Northern Ontario, and white suckers from
the Saint John River had livers similar in size to those of white sucker collected
downstream of a sulphite pulp mill in Kapuskasing, Northern Ontario [Munkittrick
et al. 2000]. The increase in white sucker liver size might be reflective of the
“normal” situation for the Saint John River, or could be due to one or more
unidentified upstream source(s) of contamination. Alternatively, the condition is
associated with the mobility of white sucker in this river system. Additional
investigation is needed to resolve among these possibilities. Presently, it is
evident that the elevated LSI of white sucker in the SJR near Edmundston is not
reflective of a “normal” situation. A potential source of contamination was
identified upstream of Clair during the 2000 fish survey, but, it is not yet clear
how this source limits or enhances fish performance in the upper SJR. The
upper SJR near Edmundston has no natural or man-made barriers to fish
movement; therefore, it is likely that the responses of white sucker reflect
movement of the species throughout the river. If so, measured differences
between reference fish and fish captured in the exposure area may not be
attributed to a particular source of contamination.
50
2.5.2 Ecological significance
Identifying ecologically relevant changes in fish organ size, growth rates, or
energy storage for the SJR will be important for understanding the potential of
future stressors to impact local fish populations [Munkittrick et al. 2000]. The
largest difference observed for LSI occurred in female sculpin in 1999; mean LSI
values went from 1.14 ± 0.13 at St. Hilaire to 2.47 ± 0.33 downstream of the pulp
mill, a 117% increase (see Table 2.2). The largest difference in condition factor
seen occurred in female sculpin during the fall of 2000; mean condition factor
went from 1.03 ± 0.02 at St. Hilaire to 1.18 ± 0.05 downstream of the pulp mill, a
15% increase (see Table 2.2). The Canadian EEM program for pulp mill
effluents recently completed the second cycle of monitoring [Munkittrick et al.
2002]. Fish comparisons are available in this data set for 65 pulp mill locations.
The 5% of sites that have the largest increase in liver size were above a 70%
increase, and the 5% of sites that had the greatest decrease in liver size were
greater than 31% decreases [Munkittrick et al. 2002]. For condition factor, the
5% of sites that demonstrated the largest increases in condition were greater
than 17% increase, and the 5% with the greatest decreases were more than a
5% decrease. Clearly, the differences seen in some years downstream of the
city of Edmundston are large, relative to changes seen at other sites.
No guidelines have yet been developed for interpreting the ecological
relevance of changes in organ size and condition. It is widely accepted that
individual or suborganismal changes that result in population or community level
changes are ecologically relevant. However, it can be very difficult to quantify
51
changes at population and community levels when sites are not far apart and
there are no barriers to fish movement between sites. The EEM program
recommends being able to detect a difference in parameters of >25% (10% of
condition factor) [Ribey et al. 2002]. The EEM program is working towards
developing critical effect sizes to determine when changes are considered
serious.
Other suggested means for determining if changes have ecological relevance
are based on the magnitude of differences that are observed. For example,
ecological relevance may be inferred if changes are outside the range seen at
reference sites, or if differences are greater than two standard deviations from
the average for fish from reference sites [Munkittrick et al. 2000]. The long-term
average for condition factor at St. Hilaire was 0.93 ± 0.11 for female sculpin. The
range of “normal” levels, based on 2 SD would be 0.71 to 1.15, and averages
greater than this were found in some collections. From this, we can conclude
that the differences downstream of the sewage and pulp mill discharges are
large, relative to what has been recorded at other sites, and can be outside of
what would be considered “normal” for this species in New Brunswick.
Overall, the results for slimy sculpin suggest that fish collected downstream of
municipal sewage and pulp mill effluent live in a nutrient-rich environment that
promotes growth. The site differences in sculpin energy storage and utilization
observed may also have been related to differences in water temperature, but
this is not known. At this point, the interim conclusions regarding the factors that
may limit or enhance fish performance in the SJR should be viewed as
52
hypotheses and will need to be confirmed through follow-up studies. The healthrelated parameters measured in slimy sculpin that were not ecologically relevant
still need to be considered when evaluating the potential risks associated with
existing and future industrial developments that may impact fish performance
[Munkittrick et al. 2000].
2.6
Conclusions
Slimy sculpin downstream of the sewage discharges and pulp mill effluent
had greater growth, condition, and liver size, but no significant differences in
gonad size relative to reference fish. The results for white sucker were not
consistent with slimy sculpin, suggesting white sucker may not be a suitable
environmental monitoring species for this portion of the SJR. The responses of
slimy sculpin and white sucker in the present study were not consistent with
results from the cycle 1 EEM. The stable isotope signatures of slimy sculpin
exposed to pulp mill and paper mill effluent were different, which confirmed
previous plume delineation studies that demonstrated the paper mill and pulp mill
effluent were not mixing within the study area. The differences observed in the
whole-organism response of sculpin and suckers may be related to fish mobility.
Slimy sculpin whole-organism responses reflect site conditions and are a suitable
sentinel species for an effects-driven cumulative effects assessment of the Saint
John River. Together, results from the fish survey (present study), fathead
minnow (Pimephales promelas) flow-through bioassay [Parrott et al. 2000], and
the microcosm flow-through invertebrate exposure [Culp et al. 2003] suggest that
53
the increased energy storage and utilization of organisms exposed to pulp mill
effluent is related to increased nutrients.
2.7
Acknowledgements
This project is receiving funding from TSRI (Project 205), NBCFWRU,
NexFor and Fraser Papers Inc., Sir James Dunn Wildlife Research Centre Fund,
New Brunswick Wildlife Council Trust Fund, NWRI, Environment Canada
(Atlantic Region), Saint John River ACAP. We also acknowledge extensive inkind support from NexFor, Noranda Technology Centre, Fraser Papers, NBDOE,
NBDNRE, Maine DEP, DFO, and the Maine Chapter of the Nature Conservancy.
We acknowledge Stable Isotopes in Nature Lab at UNB. Student support from
NSERC, UNB, NB Female Mentorship Program, NB Student Employment
Program. KRM receives support from a NSERC Discovery Grant, and from the
Canada Research Chairs program.
54
Table 2.1. Location and description of Saint John River sites sampled for fish in 1999-2001. All sites are located in
Canada, except for Moody Bridge and Priestly Brook, which are located in Maine, USA.
Site ID
Site
a
number
Year sampled
Site location
46°57.394’ N
66°54.769’ W
47°30.567’ N
68°15.072’ W
47°35.480’ N
68°07.462’ W
47°21.337’ N
68°16.235’ W
47°21.272’ N
68°16.358’ W
47°21.465’ N
68°16.541’ W
47°21.683’ N
68°16.558’ W
47°21.594’ N
68°16.762’ W
47°21.612’ N
68°19.532’ W
47°21.505’ N
68°19.578’ W
47°17.243’ N
68°22.862’ W
47°18.038’ N
68°42.605’ W
47°14.866’ N
68°36.317’ W
47°14.483’ N
68°42.605’ W
47°12.865’ N
68°48.956’ W
Ogilvie L
18
1999
First L
17
1999
LF
16
2000
DS Pulp
15
1999-2001
DS Paper
14
1999
US Pulp
13
1999-2001
IR
12
2000
US Iroq
11
2000
DS Mad
10
1999-2000
US Mad
9
1999-2000
St. Hilaire
8
1999-2001
DS Baker
7
2000
Clair
6
1999-2000
DS Nad
5
2000
US Nad
4
2000
Approximate
distance from pulp
mill outfall (km)
110 km northeast
Reference lake located in northern New Brunswick.
31 km north
Reference lake located on the Green River.
21 km northeast
0.6 km upstream
Little Forks (LF) Branch of Green River, a tributary of the
Green River.
Site located on the SJR downstream of the Fraser Inc.
Edmundston pulp mill effluent diffuser.
Site located on the SJR ~ 4400 m downstream of the Fraser
Inc. Madawaska paper mill effluent diffuser.
Site located on the SJR immediately upstream of the pulp
mill diffuser and downstream of the Iroquois River.
Iroquois River (IR). A tributary of the SJR.
0.7 km upstream
Site located on the SJR upstream of the Iroquois River.
4 km upstream
Site located on the SJR downstream of the Madawaska
River
Site located on the SJR upstream of the Madawaska River.
1.3 km downstream
0.2 km across SJR
0.4 km upstream
4.7 km upstream
16 km upstream
Description
32 upstream
Site near St. Hilaire located upstream of the city of
Edmundston.
Site located downstream of Baker Brook.
39 km upstream
Site located near the town of Clair, New Brunswick.
44 km upstream
Site located on the SJR downstream of poultry processing
facility.
Site located upstream of poultry processing facility.
44 km upstream
55
Year sampled
Site location
3
2000
PB
2
2000
MB
1
2000
47°11.196’ N
68°52.912’ W
46°49.533’ N
68°32.700’ W
46°37.733’ N
69°46.800’ W
Site ID
Site
a
number
Capone
a
Approximate
distance from pulp
mill outfall (km)
52 km upstream
Description
101 km upstream
Priestly Brook (PB). Reference site located on the SJR in
the State of Maine.
Moody Bridge (MB). Reference site located on the SJR in
the State of Maine.
123 km upstream
Site located downstream of the St. Francis River.
Site numbers correspond to sites shown in Fig. 1. DS=Downstream; US=Upstream.
56
Table 2.2. Means ± SE (n) of various parameters of adult male and female slimy sculpin (Cottus cognatus) collected
downstream of a pulp mill (DS Pulp), paper mill (DS Paper), and municipal sewage (DS Mad) and from reference
sites located at Clair, St. Hilaire, and immediately upstream of the mill (US Pulp). Within a row, differences (p <
0.05) among sites are denoted by different uppercase letters.
Sex,
Date
Male
Oct
1999
Parameter
DS Mad
65.3 ± 1.2 (22)A
68.2 ± 1.4 (47)AB
73.7 ± 1.5 (20)BC
Weight (g)
2.38 ± 0.13 (22)A
2.98 ± 0.19 (47)A
4.19 ± 0.24 (20)BC
Age (y)
2.3 ± 0.2 (18)A
2.3 ± 0.2 (34)A
2.0 ± 0.0 (11)A
0.84 ± 0.01 (22)*
0.89 ± 0.01 (43)*
1.03 ± 0.02 (20)*
0.96 ± 0.05 (22)AB
0.83 ± 0.07 (43)A
1.09 ± 0.08 (20)BC
1.16 ± 0.07 (22)A
1.32 ± 0.09 (42)A
1.22 ± 0.11 (20)A
K
LSI
b
GSIc
Length (mm)
Weight (g)
Age (y)
K
LSI
GSI
Sept
2000
St. Hilaire
Length (mm)
a
Dec
1999
Clair
-
72.5 ± 1.0 (18)A
3.62 ± 0.13 (18)A
2.9 ± 0.2 (18)A
0.95 ± 0.02 (18)A
1.20 ± 0.07 (18)A
1.32 ± 0.08 (18)A
Length (mm)
82.6 ± 1.1(18)B
73.4 ± 3.5 (9)A
Weight (g)
6.04 ± 0.24(18)B
4.58 ± 0.65 (9)A
K
1.07 ± 0.02(18)A
1.10 ± 0.02 (9)A
LSI
2.08 ± 0.15(18)B
1.60 ± 0.15 (9)AB
-
US Pulp
DS Pulp
DS Paper
-
77.3 ± 2.0 (11)C
69.4 ± 3.4 (17)ABC
4.65 ± 0.29 (11)C
3.46 ± 0.53 (17)AB
2.1 ± 0.2 (11)A
2.1 ± 0.3 (17)A
1.00 ± 0.02(11)*
0.92 ± 0.02(17)*
1.52 ± 0.12(11)C
1.23 ± 0.13(17)BC
1.30 ± 0.08(11)A
1.42 ± 0.14(17)A
-
81.9 ± 2.1 (20)B
78.3 ± 3.1 (10)AB
5.38 ± 0.78 (10)AB
1.06 ± 0.03 (10)A
1.38 ± 0.13 (10)B
6.04 ± 0.38 (20)B
2.4 ± 0.2 (20)B
1.08 ± 0.02 (20)B
1.44 ± 0.06 (19)A
1.41 ± 0.08 (20)A
-
57
Sex,
Date
Parameter
GSI
Aug
2001
Length (mm)
Weight (g)
K
LSI
GSI
Clair
0.49 ± 0.05(18)B
-
St. Hilaire
DS Mad
DS Paper
-
-
-
0.72 ± 0.06 (10)B
71.6 ± 1.9 (21)A
-
73.6 ± 1.5 (19)A
73.7 ± 1.7 (25)A
4.34 ± 0.28 (19)A
4.45 ± 0.37 (25)A
1.06 ± 0.02 (19)B
1.07 ± 0.02 (25)B
1.61 ± 0.09 (19)B
2.10 ± 0.11 (25)A
3.89 ± 0.38 (21)A
1.01 ± 0.01 (21)A
1.85 ± 0.10 (21)A
-
Length (mm)
62.4 ± 1.8 (21)A
57.9 ± 1.5 (17)A
75.1 ± 2.2 (19)B
Oct
1999
Weight (g)
2.09 ± 0.17 (21)AC
1.65 ± 0.13 (17)A
4.35 ± 0.42 (19)B
Age (y)
1.5 ± 0.2 (20)A
1.4 ± 0.2 (12)A
2.6 ± 0.2 (14)B
K
0.83 ± 0.02(21)A
0.83 ± 0.02(13)A
0.97 ± 0.02 (19)BC
LSI
1.61 ± 0.08(21)AC
1.14 ± 0.13(13) B
1.40 ± 0.12 (19)AB
GSI
1.38 ± 0.10(21)A
1.10 ± 0.11(13)AB
1.20 ± 0.12 (20)B
Sept
2000
DS Pulp
0.49 ± 0.06 (9)AB
Female
Dec
1999
US Pulp
-
-
-
-
63.4 ± 2.5 (13)A
63.3 ± 2.2 (12)A
2.60 ± 0.31 (13)C
2.28 ± 0.23 (12)AC
1.5 ± 0.3 (13)A
1.5 ± 0.2 (12)A
0.96 ± 0.03(13)C
0.85 ± 0.02(12)AC
2.47 ± 0.33(13)C
1.56 ± 0.14(12)ABC
1.35 ± 0.23(13)AB
1.30 ± 0.12(12)A
Length (mm)
-
64.6 ± 1.6 (17)A
-
71.7 ± 3.0 (9)B
70.7 ± 1.8 (18)B
-
Weight (g)
-
2.43 ± 0.18 (17)A
-
3.67 ± 0.41 (9)B
3.52 ± 0.26 (18)B
-
Age (y)
-
2.6 ± 0.2 (16)A
-
2.6 ± 0.2 (9)A
2.6 ± 0.2 (18)A
-
K
-
0.88 ± 0.02 (17)A
-
0.98 ± 0.04 (9)B
0.98 ± 0.02 (18)B
-
LSI
-
2.94 ± 0.09 (17)A
-
3.17 ± 0.11 (9)AB
3.34 ± 0.09 (18)B
-
GSI
-
3.79 ± 0.20 (17)A
-
5.44 ± 0.25 (9)B
4.52 ± 0.26 (18)AB
-
Length (mm)
79.2 ± 2.4 (10)A
66.1 ± 2.2 (15)B
-
74.1 ± 2.1 (16)A
70.4 ± 1.2 (5)AB
-
Weight (g)
5.46 ± 0.47 (10)A
3.13 ± 0.35 (15)B
-
4.47 ± 0.39 (16)A
4.10 ± 0.07 (5)AB
-
K
1.08 ± 0.03 (10)AB
1.03 ± 0.02 (15)B
-
1.05 ± 0.02 (16)B
1.18 ± 0.05 (5)A
-
LSI
2.93 ± 0.19 (10)B
2.33 ± 0.19 (15)AB
-
2.30 ± 0.12 (16)AB
1.61 ± 0.20 (4)A
-
58
Sex,
Date
Parameter
GSI
Aug
2001
Clair
0.69 ± 0.03 (10)A
St. Hilaire
DS Mad
US Pulp
DS Pulp
DS Paper
0.79 ± 0.06 (10)AB
-
1.08 ± 0.08 (13)B
0.78 ± 0.08 (5)AB
-
Length (mm)
-
64.8 ± 1.9 (18)A
-
72.5 ± 1.9 (19)B
60.5 ± 1.5 (11)A
-
Weight (g)
-
2.75 ± 0.26 (18)A
-
4.12 ± 0.34 (19)B
2.30 ± 0.18 (11)A
-
K
-
0.97 ± 0.02 (18)A
-
1.04 ± 0.02 (19)B
1.02 ± 0.02 (11)AB
-
LSI
-
2.48 ± 0.17 (18)A
-
2.03 ± 0.16 (19)A
2.39 ± 0.30 (11)A
-
GSI
-
-
-
-
-
-
a
K = 100000*(body weight/(total length3)); bLSI = 100*(liver weight/(body weight – liver weight)); cGSI = 100*(gonad
weight/(body weight – gonad weight)); *Significant interaction term within ANCOVA model; (-) Data not available.
59
Table 2.3. Means ± SE (n) of various parameters of adult male and female white
sucker (Catostomus commersoni) collected in October 1999. Within a
row, differences (p < 0.05) among sites are denoted by different
uppercase letters.
Sex
Male
Parameter
Fork length (cm)
Body weight (g)
Age (y)
Ka
LSIb
GSIc
Reference sites
St. Hilaire
Upstream Pulp Mill
37.0 ± 0.5(20)A
39.6 ± 0.4(20)B
676 ± 26(20)A
793 ± 23(20)B
5.7 ± 0.3(18)A
5.9 ± 0.2(20)A
1.33 ± 0.02(20)A
1.28 ± 0.02(20)A
1.36 ± 0.06(20)A
1.28 ± 0.03(20)A
4.10 ± 0.19(20)A
4.59 ± 0.25(20)A
Study site
Downstream Pulp Mill
38.4 ± 0.5(20)AB
736 ± 29(20)AB
5.8 ± 0.2(19)A
1.29± 0.02(20)A
1.38 ± 0.07(20)A
4.91 ± 0.18(20)A
Female
Fork length (cm)
Body weight (g)
Age (y)
K
LSI
GSI
Fecundity, no. eggs
42.4 ± 0.3(20)A
1025 ± 27(20)A
7.4 ± 0.4(20)A
1.33 ± 0.01(20)B
1.83 ± 0.06(20)A
6.90 ± 0.35(20)A
22960 ± 1103(20)A
41.9 ± 0.5(21)A
943 ± 33(21)AB
6.2 ± 0.3(20)A
1.28 ± 0.02(21)A
1.73 ± 0.04(21)A
6.36 ± 0.30(21)A
22336 ± 1359(20)A
41.5 ± 0.6(20)A
913 ± 34(20)B
6.2 ± 0.3(19)A
1.28 ± 0.02(20)A
1.67 ± 0.05(20)A
6.00 ± 0.26(20)A
20724 ± 1096(20)A
a
K = 100000*(body weight/(fork length3)); bLSI = 100*(liver weight/(body weight –
liver weight)); cGSI = 100*(gonad weight/(body weight – gonad weight)).
60
Table 2.4. Regression estimates for adult male slimy sculpin (Cottus cognatus)
condition factor and adult female slimy sculpin length-at-age.
Date
October
1999
Sex
Parameter
Site type
M
Condition Factor
Reference
Site
location
Clair
M
M
M
Condition Factor
Condition Factor
Condition Factor
Reference
Study
Study
M
Condition Factor
F
F
F
F
F
2
Slope
Intercept
n
p
r
2.81
-4.74
22
<0.0001
0.95
St. Hilaire
DS Mad
DS Pulp
3.21
2.57
2.46
-5.43
-4.18
-3.98
47
20
11
<0.0001
<0.0001
<0.0001
0.96
0.88
0.96
Study
DS Paper
3.11
-5.23
17
<0.0001
0.98
Length-at-age
Length-at-age
Length-at-age
Reference
Reference
Study
Clair
St. Hilaire
DS Mad
0.25
0.21
0.45
1.76
1.75
1.70
20
12
14
<0.0001
<0.0001
<0.0001
0.82
0.89
0.89
Length-at-age
Length-at-age
Study
Study
DS Pulp
DS Paper
0.28
0.21
1.74
1.78
13
12
<0.0001
<0.0001
0.81
0.75
61
Table 2.5. Comparative summary of slimy sculpin (Cottus cognatus) and white
sucker (Catostomus commersoni) collected downstream of the sulphite
pulp mill relative to fish collected from St. Hilaire (reference site), Saint
John River, October 1999, New Brunswick, Canada (modified from
Gibbons et al. 1998)a.
Parameter
Length
Body weight
Condition
Age
Length-at-age
GSI
LSI
a
Slimy sculpin
Male
Female
+
+
+
0
+
0
+
0
+
+
0
+
0
+
White sucker
Male
Female
0
0
0
0
0
0
0
0
0
0
0
0
0
(0) = no change; (+) = significant increase; (-) = significant decrease.
62
16
17
Quebec, Canada
M
ad
rde
r
Site 18,
180 km
Ri
ve
r
ve
Ri
rB
roo
Pulp mill
k
7 St. Hilaire
de
sad
-M
Ri
ve
r
3
ka
as
aw
Co
nc
is
13
15
Paper mill
oi
nc
ra
Fra
12
14
.F
St
St.
9
10
r
ke
r
11
Ba
i ve
nR
s
New Brunswick, Canada
ee
Gr
oi
qu
Iro
o
Pr
ci a
vin
o
lB
aw
as
ka
nn
er
s
4
S
n
oh
tJ
ai n
8
N
Clair
6
5
r
ve
Ri
Maine, USA
Site 1, 123 km
Site 2, 101 km
Pulp mill effluent lagoon
system and outfall
Paper mill effluent lagoon
system and outfall
Municipal sewage lagoon
and outfall
Figure 2.1. Map of the study area showing the relative location of reference and
exposure fish collection sites (not to scale).
63
2.0
1.6
LSI (%)
1.2
0.8
0.4
0.0
First L
Ogilvie L
St. Hilaire
US Pulp
DS Pulp
Figure 2.2. Relative liver size (liversomatic index [LSI]; % of body weight) of
adult male (black bars) and female (white bars) white sucker collected
downstream of the Edmundston pulp mill (DS Pulp) and at reference
sites located upstream of the pulp mill (US Pulp), St. Hilaire, Ogilvie
Lake (males only), and First Lake during the Fall, October 1999. Refer
to Figure 2.1 map for location.
64
12
11.5
11
DS Paper
15
N 0/00
10.5
10
Clair
St. Hilaire
9.5
US Mad
9
8.5
DS Pulp
8
7.5
-28 -27 -27 -26 -26 -25 -25 -24 -24 -23 -23 -22 -22 -21 -21 -20
13
C 0/00
Figure 2.3. Stable-isotope ratios of carbon and nitrogen in adult male (bold
dashed lines) and female (solid lines) slimy sculpin collected from
reference sites at Clair, St. Hilaire, and upstream of the Madawaska
River (US Mad) and from downstream of the Edmundston pulp mill (DS
Pulp) and Madawaska paper mill (DS Paper), October 1999. Values
are expressed as deviations (δ) from standards. Refer to Figure 2.1
map for location.
65
Figure 2.4. Summary of "ecologically relevant" changes in condition factor in
female slimy sculpin collected during the Fall 2000 fish survey of the
Saint John River. Fish were collected from various sites, including:
Moody Bridge (MB), Priestly Brook (PB), a site downstream of the St.
Francis River (Capone), sites located upstream (US Nad) and
downstream (DS Nad) of a poultry processing plant, upstream of the
international bridge at Clair, downstream of Baker Brook (DS Baker), St.
Hilaire, upstream of the Madawaska River (US Mad), downstream of
the Madawaska River (DS Mad), upstream of the Iroquois River (US
Iroq), Iroquois River (IR), upstream of the pulp mill diffuser (US Pulp),
downstream of the pulp mill diffuser (DS Pulp), and the Little Forks (LF).
Cross-hatched bars represent reference sculpin collected from sites on
the Saint John River. Black bars represent sculpin from sites exposed
to either poultry processing waste effluent (DS Nad), municipal sewage
wastewater (DS Mad), and pulp mill effluent (DS Pulp). White bars
represent sculpin collected from tributaries. Refer to Figure 2.1 map for
location.
66
67
LF
Mainstem Saint John River
IR
DS Pulp
US Pulp
US Iroq
DS Mad
US Mad
St.Hilaire
DS Baker
US Clair
DS Nad
US Nad
Capone
PB
MB
Condition Factor (K)
1.30
Tributaries
1.20
1.10
1.00
0.90
0.80
0.70
0.60
Figure 2.5. Summary of "ecologically relevant" changes in liver size female slimy
sculpin collected during the Fall 2000 fish survey of the Saint John
River. Solid and stippled horizontal lines represent data (i.e., mean ±
25%, mean ± 2SD, respectively) collected at St. Hilaire. Values that fall
between the horizontal lines are considered “normal” for the SJR at the
time of sampling. Cross-hatched bars represent reference sculpin
collected from sites on the Saint John River. Black bars represent
sculpin from sites exposed to either poultry processing waste effluent
(DS Nad), municipal sewage wastewater (DS Mad), and pulp mill
effluent (DS Pulp). White bars represent sculpin collected from
tributaries. Asterisk (*) indicates possible upstream source of
contamination. Refer to Figure 2.1 map for location.
68
69
LF
Mainstem Saint John River
IR
DS Pulp
US Pulp
US Iroq
DS Mad
US Mad
St.Hilaire
DS Baker
4.00
US Clair
DS Nad
US Nad
Capone
PB
MB
LSI (%)
4.50
Tributaries
*
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
2.8
References
BAR Environmental Inc.
1994.
Environmental effects monitoring pre-design
historical information for Fraser Inc., Edmundston sulphite pulp mill.
Guelph, ON, Canada.
BAR Environmental Inc. 1996. Environmental effects monitoring for Fraser Inc.
Edmundston sulphite pulp mill final report. Guelph, ON, Canada.
Culp, J.M., Cash, K.J., Glozier, N.E., and Brua, R.B. 2003. Effects of pulp mill
effluent on benthic assemblages in along the Saint John River, Canada.
Environ Toxicol Chem 22: 2916-2925.
Environment
Canada.
1997.
Fish
Survey
Expert
Working
Group:
Recommendations from Cycle 1 review. Ottawa ON: Environment Canada.
EEM/1997/6. 262p.
Frank, M., McMaster, M.E., Munkittrick, K.R., Savoie, M.C., and Wood, C.
Effects of sulphite and bleached kraft pulp and paper mill effluents on
yellow perch and Johnnie darters. 25th Aquatic Toxicity Workshop,
Quebec, QC, Canada, October 18-21, 1998, pp 53.
Gibbons, W.N., and Munkittrick, K.R. 1994. A sentinel monitoring framework for
identifying fish population responses to industrial discharges.
J Aquat
Ecosyst Health 3: 227-237.
Gibbons, W.N., Munkittrick, K.R., and Taylor, W.D. 1998a. Monitoring aquatic
environments receiving industrial effluents using small fish species 1:
Response of spoonhead sculpin (Cottus ricei) downstream of a bleachedkraft pulp mill. Environ Toxicol Chem 17: 2227-2237.
70
Gibbons, W.N., Munkittrick, K.R., McMaster, M.E., and Taylor, W.D.
1998b.
Monitoring aquatic environments receiving industrial effluents using small
fish species 2: Comparison between responses of trout-perch (Percopsis
omiscomaycus) and white sucker (Catostomus commersoni) downstream
of a pulp mill. Environ Toxicol Chem 17: 2238-2245.
Hewitt, L.M., Pryce Hobby, A.C., Parrott, J.L., Marlatt, V., Wood, C., Oakes, K.,
and Van Der Kraak, G.
2003.
Accumulation of ligands for aryl
hydrocarbon and sex steroid receptors in fish exposed to treated effluent
from a bleached sulphite/groundwood pulp mill. Environ Toxicol Chem 22:
2890-2897.
Lowell, R.B., Culp, J.M., Wrona, F.J., and Bothwell, M.L. 1996. Effects of pulp
mill effluents on benthic freshwater invertebrates: food availability and
stimulation of increased growth and development.
In Servos MR,
Munkittrick KR, Carey JH, Van Der Kraak GJ, (eds.), Environmental fate
and effects of pulp and paper mill effluents.
St. Lucie Press, Delray
Beach, FL, USA, pp 525-532.
MacKay, W.C., Ash, G.R., and Norris, H.J. (eds.). 1990. Fish ageing methods
for Alberta. RL&L Environmental Services, Edmonton, AB, Canada.
McMaster, M.E., Van Der Kraak, G., Portt, C.B., Munkittrick, K.R., Sibley, P.K.,
Smith, I.R., and Dixon, D.G. 1991. Changes in hepatic mixed-function
oxygenase (MFO) activity, plasma steroid levels and age at maturity of a
white sucker (Catostomus commersoni) population exposed to bleached
kraft pulp mill effluent. Aquat Toxicol 21: 199-218.
71
Munkittrick, K.R., and Dixon, D.G. 1989a. An holistic approach to ecosystem
health assessment using fish population characteristics.
Hydrobiologia
188/189: 122-135.
Munkittrick, K.R., and Dixon, D.G. 1989b. Use of white sucker populations to
assess the health of aquatic ecosystems exposed to low-level contaminant
stress. Can J Fish Aquat Sci 46: 1455-1462.
Munkittrick, K.R., Portt, C.B., Van Der Kraak, G.J., Smith, I.R., and Rokosh, D.A.
1991. Impact of bleached kraft mill effluent on population characteristics,
liver MFO activity, and serum steroid levels of a Lake Superior white
sucker (Catostomus commersoni) population.
Can J Fish Aquat Sci
48:1371-1380.
Munkittrick, K.R., McMaster, M.E., Portt, C.B., Van Der Kraak, G., Smith, I.R.,
and Dixon, D.G. 1992. Changes in maturity, plasma sex steroid levels,
hepatic mixed function oxygenase activity, and the presence of external
lesions in lake whitefish (Coregonus clupeaformis) exposed to bleached
kraft mill effluent. Can J Fish Aquat Sci 49: 1560-1569.
Munkittrick, K.R., Van Der Kraak, G., McMaster, M.E., Portt, C.B., van den
Heuvel, M.R., and Servos, M.R. 1994.
Survey of receiving-water
environmental impacts associated with discharges from pulp mills.
2.
Gonad size, liver size, hepatic EROD activity, and plasma sex steroid
levels in white sucker. Environ Toxicol Chem 13: 1089-1101.
Munkittrick, K.R., McMaster, M.E., McCarthy, L.H., Servos, M.R., and Van Der
Kraak, G. 1998. An overview of recent studies on the potential of pulp
72
mill effluents to impact reproductive function in fish. J Toxicol Environ
Health Part B 1:101-125.
Munkittrick, K.R., and McMaster, M.E.
2000.
Effects-driven assessment of
multiple stressors using fish populations. In: Ferenc, S.A., and Foran,
J.A., (eds.), Multiple stressors in ecological risk and impact assessment:
Approach to risk estimation. SETAC Press, Pensacola, FL, USA, pp 2765.
Munkittrick, K.R., McMaster, M.E., Van Der Kraak, G., Portt, C., Gibbons, W.N.,
Farwell, A., and Gray, M. 2000. Development of methods for effectsdriven cumulative effects assessment using fish populations: Moose River
Project. SETAC Press, Pensacola, FL, USA.
Munkittrick, K.R., McGeachy, S.A., McMaster, M.E., Courtenay, S.C.
2002.
Overview of cycle 2 freshwater fish studies from the pulp and paper
Environmental Effects Monitoring program. Water Quality Res J Can 37:
49-77.
Parrott, J.L., Wood, C.S., Boutot, P., Blunt, B.R., Baker, M.A., and Dunn, S.
Fathead minnow long-term growth/reproductive tests to assess final
effluent from a bleached sulphite mill.
4th International conference on
environmental impacts of the pulp and paper industry, Helsinki, Finland,
June 12-15, 2000, pp 207-212.
Peterson, B.J., and Fry, B. 1987. Stable isotopes in ecosystem studies. Ann
Rev Ecol Syst 18: 293-320.
73
Ribey, S.C., Munkittrick, K.R., McMaster, M.E., Courtenay, S., Langlois, C.,
Munger, S., Rosaasen, A., and Whitley, G.
2002. Development of a
monitoring design for examining effects in wild fish associated with
discharges from metal mines. Water Quality Res. J. Can. 37: 229-249.
Scott, W.B., and Crossman, E.J. 1998. Freshwater Fishes of Canada. Galt
House, Oakville, ON, Canada.
Van Vliet, W.H. 1964. An ecological study of Cottus cognatus (Richardson,
1836)
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Northern
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MA
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University
of
Saskatchewan, Saskatoon, Saskatchewan, Canada.
Wassenar, L.I., and Culp, J.M. 1996. The use of stable isotope analyses to
identify pulp mill effluent signatures in riverine food webs.
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M.R., Munkittrick, K.R., Carey, J.H., Van Der Kraak, G., (eds),
Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie
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74
CHAPTER 3.
Identifying a suitable fish species for monitoring a large
river receiving effluents from a pulp and paper mill, municipal
sewage wastewater facilities, and agricultural runoff2.
2
Published: Galloway, B.J., Munkittrick, K.R., Curry, R.A., Wood, C., and Dunn,
S. 2004. Identifying a suitable fish species for monitoring a large river receiving
effluents from pulp and paper mill, municipal sewage wastewater facilities, and
agricultural runoff. In Borton, D.L., Hall, T.J., Fisher, R.P., and Thomas, J.F.,
(eds) Fifth International Conference on Fate and Effects of Pulp and Paper Mill
Effluents, June 1-4, Seattle, WA: 169-181.
3.1
Abstract
Identifying the impacts of individual aquatic stressors on fish health in rivers
exposed to multiple stressors is difficult and has become the focus of recent
Canadian studies on environmental effects. There has been a movement over
the past 5 years towards using small-bodied fish species for monitoring, because
of their presumed lower mobility. There have not been many direct comparisons
between the results of monitoring studies using small and large-bodied species.
The objective of this project was to determine whether differences in species
responses exist, and whether they can be explained by differences in life history
characteristics and mobility. This paper will compare the responses of 4 fish
species upstream and downstream of a city in a river receiving wastes from a
pulp mill, paper mill, multiple sewage discharges and agricultural runoff. The
study outlines data gaps associated with ongoing studies.
75
3.2
Introduction
Identifying the potential impacts of individual aquatic stressors on fish
health in rivers exposed to multiple stressors is a difficult task. Over the past few
years there has been increased interest in using small-bodied fish (e.g., Cottids
and Cyprinids) for monitoring the potential impacts of point source [Frank et al.
1998; Gibbons et al. 1998a; Gibbons et al. 1998b; Munkittrick et al. 2000;
Munkittrick et al. 2002] and non-point sources of contaminants [Gray et al. 2002].
In Canada, a recent review of cycle 2 fish studies (1997-2000) for the pulp and
paper Environmental Effects Monitoring (EEM) program noted an increase in the
use of small-bodied fish from cycle 1 (1993-1996) [Munkittrick et al. 2002]. Many
small-bodied fish species are well suited for monitoring complex receiving
environments since they are presumed to be less mobile and more abundant
than large-bodied fish species (e.g., Catostomids). The main disadvantage of
using small-bodied fish species for EEM programs is the lack of basic life-history
information [Munkittrick et al. 2002]. A recent literature survey of the Aquatic
Sciences and Fisheries Abstracts (Cambridge Scientific Abstracts, Bethesda,
MD.), yielded a paucity of detailed reproductive information for some of the smallbodied fish used in the cycle 2 pulp and paper EEM program, including:
blacknose dace (Rhinichthys atratulus), bluntnose minnow (Pimephales notatus),
logperch (Percina caproides), and creek chub (Semotilus corporalis). The lack of
detailed life history information for small-bodied fish can hinder the design and
interpretation of environmental monitoring programs.
76
In 1999, a collaborative research agreement was initiated between Fraser
Papers Inc. (Edmundston pulp mill, Edmundston, New Brunswick, Canada),
Nexfor (Noranda Technology Centre, Pointe-Claire, Quebec, Canada), and
Environment Canada (National Water Research Institute, Saskatoon,
Saskatchewan, Canada) to participate in a variety of studies as part of Fraser
Papers Inc. commitment to Environmental Effects Monitoring (EEM) studies for
cycle 2. The studies including an on-site 90-d flow-through fathead minnow
(Pimephales promelas) bioassay [Parrott et al. 2003], a flow-through microcosm
invertebrate exposure [Culp et al. 2003], an assessment of bioavailable pulp mill
effluent chemicals accumulated by fish during short term exposures [Hewitt et al.
2003], and wild fish collections [Galloway et al. 2003].
The objectives of the present study were to clarify local impacts on wild
fish, expand field collections to include other species to examine potential
species differences in responses, continue to de-couple other confounding
factors at this study area using less mobile small-bodied fish, and contribute to
furthering the scientific understanding of the suitability of small-bodied fish
species for environmental monitoring programs. We compared the responses of
blacknose dace and slimy sculpin (small-bodied fish species) with yellow perch
and white sucker (large-bodied fish species) to determine whether the responses
of small-bodied fish and large-bodied fish would be different.
77
3.3
Materials and Methods
3.3.1 Study area and mill characteristics
The Saint John River originates at Fifth St. John Pond in northern Maine
(USA) and travels ~700 km to the mouth, located at the city of Saint John, at the
Bay of Fundy. Approximately 51% of the drainage area is located in New
Brunswick; while the remaining area is located in the state of Maine (36%) and
the province of Quebec (13%) [11]. Near Edmundston (approximately 450 km
from the river mouth), the Saint John River forms part of the international border
between Canada and the State of Maine and extends for ~120 km. The flow of
the Saint John River near Edmundston is unregulated and undergoes
considerable annual fluctuations; the dominant substrate in the river is gravel
[11]. There are no barriers to fish movement on the mainstem of the Saint John
River near Edmundston. However, a dam located near the mouth of the
Madawaska River, upstream of the mill discharge, restricts the movement of fish
in the tributary.
The bleached-sulphite pulp mill (Edmundston, New Brunswick, Canada)
and paper mill (Madawaska, Maine, USA) are located on the north and south
sides of the Saint John River, respectively (Figure 3.1). The pulp mill produces
daily approximately 550 air-dried metric tonnes (ADMT) of sulphite pulp, 350
ADMT of ground wood pulp, 120 ADMT of boxboard, and 100 tonnes of de-inked
product [BAR Environmental Inc. 1994]. At the time of our survey, the wood
furnish consisted of ~70% fir and ~30% spruce and the bleaching sequence was
(Dc-Ep-DH-): Dc = chlorination stage with 75% chlorine dioxide substitution; Ep =
78
extraction stage with sodium hydroxide reinforced with hydrogen peroxide; DH =
bleaching stage with chlorine dioxide and sodium hypochlorite; (-) = washing
stage. The pulp mill discharges more than 73,000 m3 of secondary-treated
effluent per day from an aerated lagoon facility (retention time ~7 days) into the
north side of the Saint John River, about 4 km downstream from the mill. The
paper mill discharges secondary-treated effluent into the south side of the Saint
John River, about 4 km upstream of the pulp mill effluent diffuser. A plume
delineation study has shown the paper mill effluent and pulp mill effluent do not
mix in our fish collection area [11].
3.3.2 Fish collections
Adult fish of each sex were targeted during each site collection.
Prespawning slimy sculpin (Cottus cognatus) were collected between March 4 to
26, 2002 and prespawning blacknose dace (Rhinichthys atratulus) were collected
between June 11 to 14, 2002 using a backpack electrofisher (Smith-Root Model
12B, Smith-Root Inc., Vancouver, WA, USA). Fish were collected from a site
located below a bleached-sulphite pulp mill (D/S Pulp; lat 47°21.337’N, long
68°16.235’W) and a site located downstream of municipal sewage inputs (D/S
Mad; 47°21.612’W, long 68°19.532’W). Reference fish were collected from a site
immediately upstream of the pulp mill diffuser (U/S Pulp; lat 47°21.465’N,
68°16.541’W), a site ~ 20 km upstream of the mill diffuser (St. Hilaire; lat
47°17.243’N, long 68°22.862’W), and a site located ~ 52 km upstream of the
town of Edmundston, below the St. Francis River (“Capone”; lat 47°11.196’N,
79
long 68°52.912’W). Ice cover in March prohibited the collection of slimy sculpin
from the reference site located at St. Hilaire. Male blacknose dace were not
captured from the site below the St. Francis River or immediately upstream of the
pulp mill effluent diffuser. Adult yellow perch (Perca flavescens) were collected
by angling between September 20-24, 2002 and white sucker (Catostomus
commersoni) were collected using 3” monofilament gillnets between October 1823, 2002 from a site located downstream of the pulp mill and from a reference
site at St. Hilaire.
Fish were rendered unconscious by concussion, followed by spinal
severance, and length, body weight, liver weight, and gonad weights were
recorded. Aging material was collected for determination of survival (i.e., mean
age) and growth (i.e., length-at-age). The age of white sucker and yellow perch
was determined by counting annuli on clean, dried opercula under a dissecting
microscope. Slimy sculpin and blacknose dace were aged by counting annuli on
sagittal otoliths. The fecundity of female slimy sculpin was estimated in the field
by counting the total number of eggs per fish. For female white sucker and
yellow perch, approximately 1 g of ovarian tissue was collected in the field and
placed in a 10 % buffered formalin solution. In the lab, the ovarian tissue was
blotted dry, reweighed, and the total number of eggs was counted. These results
were used to estimate the total number of eggs per fish (total fecundity).
Fecundity estimates for female blacknose dace were not available.
White
muscle samples were obtained for stable isotope analyses and were used to
estimate residency patterns of fish collected at various sites in our survey.
80
3.3.3 Data Analyses
Analysis of variance (ANOVA) was used to test for differences in mean
length, weight, and age of fish between sites; Bonferroni post hoc comparisons
were used to examine individual site differences when more than two sites were
compared. Analysis of covariance (ANCOVA) was used to assess site
differences in length-at-age, and the relationships between weight and length
(condition factor), liver size, and gonad size. Except for length-at-age, condition
and stable isotope ratio analyses, adjusted body weight (total wt – organ wt) was
used as a covariate in the ANCOVA model. All data were log10 transformed
before performing ANOVA and ANCOVA, and sexes were analyzed separately.
All data analyses were done using SYSTAT® (SPSS, SYSTAT, Chicago, IL,
USA) statistical software. For ANCOVA, slopes were consider different when p <
0.01. Gonad weights and liver weights were calculated as percent-adjusted body
weight for summary purposes.
When gonad size of female dace was analyzed, it became evident that
females could be divided into two groups: smaller fish had a GSI less than 8%,
and exhibited large variations in gonad development while larger fish with a GSI
≥ 8% exhibited much less variability. For this study, comparisons for this species
were restricted to larger fish and female dace with a GSI less than 8% were
excluded from the analyses.
White muscle samples were obtained from female fish for stable 13C/12C
and 15N/14N isotope analyses and were used to determine site fidelity of fish
collected at each site. A small piece of white muscle was removed from each
81
fish and placed in drying oven at 65°C for at least 48 h. Dried tissue samples
were individually ground into a fine powder and stored in clean glass scintillation
vials before isotopic analyses. Stable-isotope ratios (e.g., 13C/12C, 15N/14N) are
expressed as delta (δ) values and are measures of a parts-per-thousand (‰)
difference between the sample isotope ratio and that of an international standard
(carbon – Pee Dee Belemnite; nitrogen – atmospheric nitrogen) [12]. Delta is
expressed in terms of the numerator (i.e., heavier stable isotope; e.g., 13C).
Samples that have more negative delta values contain less 13C or 15N relative to
samples with higher delta values and are termed depleted for an isotope.
Samples that have higher delta values are considered enriched.
3.4
Results
Male and female slimy sculpin collected downstream of the sewage and
pulp mill effluent were significantly longer and heavier than fish collected from a
reference site located downstream of the St. Francis River (i.e., Capone) (Table
3.1). In contrast, male and female yellow perch and white sucker exhibited no
site differences in length and body weight (Table 3.3). Female blacknose dace
collected downstream of the pulp mill effluent diffuser and municipal sewage
wastewater were significantly shorter relative to reference fish at Capone, but not
St. Hilaire (Table 3.1). Female dace collected downstream of the sewage inputs
from the Madawaska River (i.e., D/S Mad) showed reduced body weight relative
to fish collected at upstream reference sites (Table 3.1). Male dace exhibited no
significant site differences in length (p=0.19) or weight (p=0.31) (Table 3.1).
82
Female sculpin collected downstream of sewage inputs from the
Madawaska River and pulp mill effluent diffuser were significantly older relative to
fish at Capone (Table 3.1). Male sculpin exposed to pulp mill effluent were the
same age as reference fish from Capone, but sewage-exposed males were older
(Table 3.1). For male and female sculpin, the slopes of the regression lines for
length-at-age at the site located downstream of the Madawaska River, upstream
of the pulp mill, and downstream of the pulp mill were higher than the reference
site at Capone (Table 3.2). Male and female white sucker exhibited no
significant site differences in age (Table 3.3) and length-at-age (data not shown).
Male yellow perch collected downstream of the pulp mill were older relative to
fish at St. Hilaire, but this was not the case for females (Table 3.3). Female dace
collected downstream of the pulp mill were the same age as reference fish at
Capone and St. Hilaire, but males collected downstream of the pulp mill were
younger than fish at St. Hilaire (Table 3.1). Perch and dace exhibited no
significant site differences in length-at-age (data not shown).
Liver size in male sculpin from the pulp mill site and sewage site was
significantly larger compared to fish from Capone, but this was not the case for
fish captured immediately upstream of the pulp mill diffuser (Table 3.1). There
were no significant site differences in female sculpin liver size (Table 3.1). Liver
size in male and female dace exposed to pulp mill effluent was significantly
greater compared to reference fish at Capone (females only) and St. Hilaire
(Table 3.1). Male yellow perch downstream of the pulp mill showed a significant
increase in liver size relative to upstream reference fish, but not females (Table
83
3.3). In contrast, male and female white sucker exposed to pulp mill effluent
exhibited significant decreases in liver size compared to upstream reference fish
(Table 3.3).
The condition factor of male and female sculpin downstream of the pulp
mill, upstream of the pulp mill, and downstream of the sewage was significantly
higher relative to reference fish at Capone. In addition, sculpin collected
downstream of the sewage discharges had significantly higher condition factors
relative to fish exposed to pulp mill effluent (Table 3.1). Male and female dace
exhibited no significant site differences in condition factor (Table 3.2). Male
yellow perch captured downstream of the pulp mill had significantly higher
condition factors compared to reference fish, but this was not the case for female
perch or male and female white sucker (Table 3.3).
There were no significant site differences in gonad size for male and
female slimy sculpin, yellow perch, and white sucker (Table 3.1). Likewise, there
were no significant site differences in fecundity of female sculpin, yellow perch,
and white sucker (Tables 3.1, 3.3). Female dace exposed to pulp mill effluent
and sewage wastewater exhibited no significant differences in ovary size
compared to reference fish at Capone and St. Hilaire (Table 3.1). For male dace,
the slope of the regression lines for gonad development were higher at the sites
located downstream of the Madawaska River and St. Hilaire when compared to
fish exposed to pulp mill effluent (Table 3.2).
Female sculpin captured downstream of the pulp mill effluent diffuser were
significantly more enriched in 13C than reference fish at Capone and fish
84
immediately downstream of sewage, but not fish captured immediately upstream
of the pulp mill diffuser (Table 3.1). Female sculpin collected immediately
downstream of the sewage discharges from the Madawaska River and upstream
of the pulp mill diffuser were significantly more enriched in 15N relative to
reference fish at Capone and fish exposed to pulp mill effluent (Table 3.1).
Female dace collected downstream of the pulp mill were significantly enriched in
13
C relative to reference fish at St. Hilaire, but showed no site differences in 15N
(Table 3.1). Interestingly, female yellow perch showed no significant site
differences in either 13C or 15N (Table 3.3). Female white sucker exposed to pulp
mill effluent were enriched in 13C and depleted in 15N relative to reference fish at
St. Hilaire (Table 3.3).
3.5
Discussion
The general response patterns of slimy sculpin are consistent with our
previous work and suggest an overall increase in energy storage and utilization
via a nutrient enrichment effect [Munkittrick et al. 2000; Gibbons and Munkittrick
1994]. Male and female sculpin exposed to pulp mill effluent were longer,
heavier, and were in better condition and showed increased length-at-age
relative to reference fish. Similar to our results from 1999 [Galloway et al. 2003],
male and female sculpin exposed to pulp mill effluent exhibited no significant site
differences in gonad size relative to reference fish. Slimy sculpin invest most
their energy into gonad development during the winter when the river is icecovered, water temperatures and flow are low, and pulp mill effluent
85
concentrations are elevated. The absence of site differences in March indicates
that pulp mill effluent exposure was not adversely affecting sculpin gonad
development.
A series of fathead minnow life cycle studies were conducted on site at the
time of the fish collections [Parrott et al. 2003], and fathead minnow exposed to
10% effluent in a transportable laboratory showed increased growth (length and
weight), increased condition factor, and increased liver size. The wild fish
responses were not attributable to the effluent. Although sculpin showed
increased body size, liver size and condition relative to the upstream reference
sites, there were no differences relative to a site immediately upstream of the
diffuser, and condition was lower than fish collected downstream of the sewage
discharges in Edmundston.
Sculpin living downstream of the pulp mill had a unique 13C signature
relative to reference fish at Capone and fish downstream of municipal sewage
(i.e., D/S Mad) indicating a carbon-enriched environment. It is important to note
that female sculpin 13C and 15N signatures from the present study were similar to
our 1999 results (data not shown) [Galloway et al. 2003], providing further
evidence that sculpin movements are limited and the integrated response
patterns are site-specific. Sculpin exposed to municipal sewage wastewater (i.e.,
D/S Mad) had a distinct 15N signature relative to fish at Capone and downstream
of the pulp mill effluent diffuser, but not fish captured upstream of the pulp mill. If
sculpin captured downstream of the pulp mill were moving to upstream sites then
15
N signatures should have been similar, but this was not the case. Furthermore,
86
our stable isotope results support other studies that have shown sites receiving
pulp mill effluent and municipal sewage wastewater can have distinct stable
isotope signatures relative to upstream sites and can be used to trace the
geographical extent of specific wastewater effluent streams [Wayland and
Hobson 2001; Wassenar and Culp 1996]. We also know that sculpin on the north
shore of the river do not mix with the sculpin on the south shore [Galloway et al.
2003].
White sucker and blacknose dace also showed different stable isotope
signatures between St. Hilaire and Edmundston, showing that these fish were not
mixing between the reference site and the site downstream of the pulp mill.
Similar to sculpin, blacknose dace showed larger livers (both sexes) downstream
of the pulp mill, relative to the reference site, but these differences were also
apparent upstream at the sewage sites and can not be attributed to the pulp mill.
We can not expect that the larger-bodied species do not mix between the
south and north shores of the river (approximately 50 m at this location). White
sucker showed no differences between the upstream reference site and the pulp
mill site, except for smaller livers downstream of the pulp mill. This species did
not respond to the combined effects of the sewage and mill discharges. Yellow
perch males showed increased liver size and condition factor, but no other
significant differences were present in males or females.
Using changes in mean age, energy expenditure (e.g., Length-at-age,
gonad weight, fecundity), and energy storage (e.g., condition factor, liver size)
between exposed and reference sites it is possible to identify the potential
87
mechanisms that resulted in the measured response patterns of each fish
species [Munkittrick et al. 2000; Gibbons et al. 1994]. For example, sculpin
collected downstream of the pulp mill effluent diffuser showed no differences in
age (males only), but showed increased length-at-age, condition, body size, and
liver size (males only) when compared to reference fish suggesting an increase
in food and/or habitat [Munkittrick et al. 2000; Gibbons et al. 1994]. The sculpin,
dace and perch all responded to better food conditions downstream of the city of
Edmundston (Table 3.4), although the changes can be attributed more to the
sewage inputs than to the input of the pulp mill effluent. Culp et al. [2003]
demonstrated that invertebrate and algal production was increased by the pulp
mill effluent relative to upstream river situations, but the fish apparently did not
detect the increase associated with the mill. Furthermore, Parrott et al. [2003]
demonstrated that the effluent alone was sufficient to further increase growth and
liver size in fathead minnow life cycle studies, although this again was not
translated into a field effect. Liver size and condition did not increase in dace or
sculpin above that seen immediately upstream of the outfall, and stable isotope
levels demonstrated that the sculpin were not moving between sites.
The decrease in liver size of white sucker downstream of the pulp mill
outfall is puzzling; both male and female liver sizes are more than 20% smaller at
the downstream site. The white sucker are probably the species in this study
that best integrates the stressors within the reach of the area, and would mix
between sides of the rivers with some upstream and downstream movement.
Studies conducted further downstream on the Saint John River have
88
demonstrated that the white sucker is not very mobile outside of the spawning
season with a home range outside of the spawning season of 1 to <2 km
[Doherty et al. 2003]. Furthermore, the stable isotopic ratios reported here show
that the downstream and upstream fish were not mixing. White sucker were
remarkably similar between the two sites outside of the reduction in liver sizes.
The liver sizes downstream are identical to those reported previously for white
sucker during the fall at this site [Galloway et al. 2003], and the difference reflects
a change at the reference site. This will be further investigated.
3.6
Conclusions
•
Slimy sculpin downstream of the sewage discharges and pulp mill effluent
had greater growth, condition, and liver size, but no significant site
differences in gonad size relative to reference fish. The results of the
present survey along with previous work at these sites [see Parrott et al.
2003; Culp et al. 2003; Hewitt et al. 2003] support a nutrient enrichment
effect in fish exposed to the combined effluents from the sewage
discharges and the pulp mill. Stable isotope data showed slimy sculpin
have a very limited home range, the whole-organism responses are sitespecific, and as such, slimy sculpin are suitable sentinel species for an
effects-driven cumulative effects assessment of the Saint John River.
•
White sucker and yellow perch showed few site differences in any of the
measured whole-organism parameters and the responses were not
consistent with slimy sculpin or previous work done at these sites,
suggesting white sucker and yellow perch may not be as sensitive as the
89
small-bodied species for picking up changes within a limited reach of the
river.
•
White muscle stable 13C and 15N isotope signatures in white sucker, yellow
perch, and blacknose dace indicated movement is limited. However,
stable 13C and 15N isotope signatures in blacknose dace and yellow perch
did not show as much isotopic separation as white sucker and slimy
sculpin suggesting their prey items may not be incorporating pulp mill
effluent derived-carbon into their diet. Future work will need to be
conducted to understand the influence of pulp mill effluent and municipal
sewage wastewater on the local food web.
•
There is substantial lack of basic biological information for many smallbodied fish (e.g., Cyprinids), which can hinder scientifically sound data
interpretation and recommendations. Many cyprinids are multiple
spawners; Environment Canada’s Environmental Effects Monitoring (EEM)
regulations suggest these fish should be sampled prior to the initial
spawning event when all fish are presumed to be at the same stage of
reproductive development. For the present study, pre-spawning female
blacknose dace exhibited a great deal of variability in gonad development
relative to slimy sculpin. Currently, we are working to provide additional
guidance for sampling and interpreting data for multiple spawning smallbodied used for environmental monitoring programs.
90
3.7
Acknowledgements
This project received funding from the Toxic Substances Research Initiative
(Project 205), NexFor and Fraser Papers, and NBCFWRU. We also
acknowledge extensive in-kind support from NexFor, Noranda Technology
Centre, and Fraser Papers. The invaluable help of K. Roach, A. Halford, K.
Tenzin, and J. McPhee in the field is appreciated. Graduate student support
from NSERC and UNB. KRM receives support from a NSERC Discovery Grant,
the Canadian Water Network National Centres of Excellence and from the
Canada Research Chairs program.
91
Table 3.1. Means ± SE (n) of various parameters measured in adult male and female slimy sculpin (Cottus cognatus) and
adult male and female blacknose dace (Rhinichthys atratulus) collected downstream of a sulphite pulp mill (D/S
Pulp), upstream of the pulp mill diffuser (U/S Pulp), and downstream of municipal sewage inputs (D/S Mad), and
from reference sites located downstream of the St. Francis River (Capone) and at St. Hilaire. Within a row,
differences (p ≤ 0.05) among sites are denoted by different uppercase letters. (*Significant interaction within
ANCOVA model; N/A = data not available).
Sites
Species
Sex
Parameter
Capone
Sculpin
M
Fork length (mm)
65.2 ± 1.0(22)A
Body weight (g)
F
St. Hilaire
D/S Mad
U/S Pulp
D/S Pulp
N/A
92.7 ± 1.4(29)B
85.8 ± 2.1(26)BC
82.9 ± 3.0(24)C
2.85 ± 0.17(22)A
N/A
10.6 ± 0.5(29)B
7.71 ± 0.50(26)C
7.37 ± 0.77(24)C
Age
2.8 ± 0.1(22)A
N/A
3.5 ± 0.1 (29)B
3.3 ± 0.1(26)AB
2.9 ± 0.2 (24)A
Ka
1.01 ± 0.01(22)A
N/A
1.30 ± 0.02(29)C
1.17 ± 0.02(26)B
1.16 ± 0.03(24)B
LSIb
1.84 ± 0.10(22)A
N/A
3.06 ± 0.15(29)BC
2.28 ± 0.11(26)AC
2.68 ± 0.11(23)C
GSIc
1.46 ± 0.05(22)AB
N/A
1.56 ± 0.05(29)A
1.71 ± 0.06(26)B
1.51 ± 0.07(24)AB
Fork length (mm)
56.3 ± 0.09(33)A
N/A
83.9 ± 1.7 (30)B
77.6 ± 1.5(29)B
80.0 ± 2.5 (22)B
92
Sites
Species
Sex
Parameter
Capone
Sculpin
F
Body weight (g)
1.82 ± 0.10(33)A
Age
Dace
M
F
St. Hilaire
D/S Mad
U/S Pulp
D/S Pulp
N/A
7.79 ± 0.45 (30)B
5.42 ± 0.32(29)C
6.27 ± 0.67(22)C
2.4 ± 0.1(33)A
N/A
3.6 ± 0.1 (30)B
2.9 ± 0.1(28)C
3.2 ± 0.2 (22)BC
K
0.99 ± 0.02(33)A
N/A
1.31 ± 0.04 (30)B
1.13 ± 0.02(29)C
1.13 ± 0.02(22)C
LSI
4.25 ± 0.11(33)A
N/A
4.62 ± 0.13 (30)A
4.39 ± 0.09(29)A
4.30 ± 0.16 (21)A
GSI
14.9 ± 0.5(33)A
N/A
15.7 ± 0.8 (30)A
16.9 ± 0.4(29)A
16.0 ± 1.3(22)A
Fecundity, no. eggs
60.9 ± 3.5(33)A
N/A
266 ± 16 (30)A
185 ± 12(29)A
208 ± 21 (22)A
δ13C (‰)
-28.7 ± 0.3 (8)A
N/A
-28.3 ± 0.6 (8)AB
-26.4 ± 0.4 (8)BC
-25.0 ± 0.9 (8)C
δ15N (‰)
8.9 ± 0.1 (7)A
N/A
11.5 ± 0.1 (7)B
11.0 ± 0.1 (7)B
8.4 ± 0.2 (7)C
Fork length (mm)
N/A
57.9 ± 1.0(33)A
57.1 ± 0.9(20)A
N/A
55.3 ± 1.0(18)A
Body weight (g)
N/A
2.00 ± 0.11(33)A
1.92 ± 0.09(20)A
N/A
1.76 ± 0.13(18)A
Age
N/A
2.4 ± 0.1(33)B
2.3 ± 0.1(20)AB
N/A
2.1 ± 0.01(17)A
K
N/A
1.00 ± 0.01(33)A
1.02 ± 0.01(20)A
N/A
1.02 ± 0.02(18)A
LSI
N/A
1.19 ± 0.08(33)A
1.67 ± 0.11(19)B
N/A
2.27 ± 0.18(17)B
GSI
N/A
0.83 ± 0.04(30)*
0.66 ± 0.06(17)*
N/A
1.02 ± 0.10(18)*
63.5 ± 1.3(24)AB
58.5 ± 1.5(14)B
Fork length (mm)
64.0 ± 0.8(20)A
59.7 ± 1.3(12)AB
59.2 ± 1.0(22)B
93
Sites
Species
Sex
Parameter
Capone
St. Hilaire
D/S Mad
U/S Pulp
D/S Pulp
Dace
F
Body weight (g)
2.90 ± 0.11(20)A
2.91 ± 0.19(24)A
2.22 ± 0.19(14)B
2.36 ± 0.18(12)AB
2.33 ± 0.13(22)AB
Age
3.0 ± 0.1(20)A
2.8 ± 0.1(23)AB
2.4 ± 0.1(14)B
2.8 ± 0.1(12)AB
2.6 ± 0.1(22)AB
K
1.10 ± 0.01(20)A
1.10 ± 0.02(24)A
1.08 ± 0.01(14)A
1.09 ± 0.03(12)A
1.10 ± 0.01(22)A
LSI
2.46 ± 0.19(20)A
2.68 ± 0.15(24)AC
3.20 ± 0.27(14)C
3.36 ± 0.22(12)BC
3.36 ± 0.14(22)BC
GSI
14.0 ± 0.8(20)A
18.6 ± 1.2(24)B
14.1 ± 1.2(14)AB
15.8 ± 1.5(12)AB
14.9 ± 0.9(22)AB
δ13C (‰)
N/A
-25.1 ± 0.1 (8)A
N/A
N/A
-24.6 ± 0.2 (8)B
δ15N (‰)
N/A
10.4 ± 0.1 (8)A
N/A
N/A
10.1 ± 0.1 (8)A
94
Table 3.2. Regression estimates for adult male slimy sculpin length-at-age
(Cottus cognatus) and adult male blacknose dace (Rhinichthys
atratulus) gonad development.
Species
Sex
Sculpin
M
F
Dace
M
Parameter
Length-atage
Length-atage
Length-atage
Length-atage
Length-atage
Length-atage
Length-atage
Length-atage
Gonad
weight
Gonad
weight
Gonad
weight
Site
Locations
Capone
Site Types
Slope
Intercept
n
p
r2
Reference
0.16
1.74
22
0.017
0.25
D/S Mad
Study
0.38
1.76
29
<0.0001
0.44
U/S Pulp
Reference
0.51
1.67
26
<0.0001
0.81
D/S Pulp
Study
0.62
1.63
24
<0.0001
0.82
Capone
Reference
0.30
1.64
33
<0.0001
0.70
D/S Mad
Study
0.45
1.68
30
<0.0001
0.80
U/S Pulp
Reference
0.34
1.73
28
<0.0001
0.80
D/S Pulp
Study
0.50
1.65
22
<0.0001
0.81
St. Hilaire
Reference
0.98
-2.09
33
<0.0001
0.54
D/S Mad
Study
2.04
-2.50
20
<0.0001
0.56
D/S Pulp
Study
0.38
-1.88
18
0.28
95
Table 3.3. Means ± SE (n) of various parameters measured in adult yellow perch
(Perca flavescens) and white sucker (Catostomus commersoni)
collected on the Saint John River, New Brunswick, Canada. Within a
row, differences (p ≤ 0.05) among sites are denoted by different
uppercase letters.
Species
Yellow
perch
White
sucker
Yellow
perch
White
sucker
Reference site
Study site
Sex
M
Parameter
Fork length (cm)
St. Hilaire
Downstream Pulp mill
19.1± 1.2(14)A
19.4 ± 0.4(19)A
M
Body weight (g)
Age
Ka
LSIb
GSIc
Fork length (cm)
115 ± 22(14)A
2.8 ± 0.4(14)A
1.42 ± 0.02(14)A
0.86 ± 0.05(14)A
5.52 ± 0.60(14)A
39.6 ± 0.5(13)A
114 ± 8(19)A
3.6 ± 0.3(19)B
1.52 ± 0.03(19)B
1.01 ± 0.04(19)B
7.12 ± 0.69(19)A
39.3 ± 0.4(19)A
F
Body weight (g)
Age
K
LSI
GSI
Fork length (cm)
839 ± 33 (13)A
6.6 ± 0.5(13)A
1.35 ± 0.02(13)A
1.97 ± 0.10 (13)A
5.24 ± 0.21 (13)A
25.2 ± 1.6 (10)A
798 ± 24(19)A
6.1 ± 0.4(19)A
1.31 ± 0.02(19)A
1.50 ± 0.13 (19)B
4.98 ± 0.15(19)A
22.5 ± 0.5 (42)A
243 ± 45 (10)A
2.8 ± 0.1 (38)A
1.34 ± 0.03 (10)A
1.20 ± 0.07 (10)A
2.56 ± 0.17 (10)A
37556 ± 5854 (9)A
169 ± 11 (42)A
2.9 ± 0.4 (10)A
1.41 ± 0.02 (42)A
1.26 ± 0.05 (42)A
2.41 ± 0.11 (42)A
30446 ± 2460 (31)A
F
Body weight (g)
Age
K
LSI
GSI
Fecundity, no. of
eggs
δ13C (‰)
δ15N (‰)
Fork length (cm)
-23.6 ± 0.3(8)A
11.8 ± 0.3(7)A
42.7 ± 0.5(22)A
-23.0 ± 0.2 (7)A
10.3 ± 0.3(7)A
41.6 ± 0.6 (26)A
Body weight (g)
Age
K
LSI
GSI
Fecundity, no. of
eggs
1015 ± 30(22)A
8.0 ± 0.4(17)A
1.30 ± 0.02(22)A
2.30 ± 0.06(22)A
6.95 ± 0.33(22)A
18667 ± 943(22)A
933 ± 35(26)A
7.2 ± 0.4(22)A
1.29 ± 0.02(26)A
1.81 ± 0.06(26)B
6.08 ± 0.32(26)A
16803 ± 1294 (21)A
96
Species
White
sucker
Sex
F
Reference site
Study site
St. Hilaire
Downstream Pulp mill
δ C (‰)
-24.8 ± 0.8(8)A
-22.6 ± 0.3 (8)B
δ15N (‰)
10.0 ± 0.4(8)A
8.4 ± 0.3(8)B
Parameter
13
97
Table 3.4. A comparison of species responses to the combined effluents at
Edmundston (sewage, pulp mill effluent) and a comparison of fish
responses upstream and downstream of the pulp mill discharge.
Species
Sculpin*
Dace
Sex
Male
Reference* versus downstream pulp
mill
Age Body Gonad Liver Condition
Size size
size
↑
0
↑
↑
0
Female
↑
↑
0
0
↑
Male
↓
0
0
↑
0
0
0
↑
0
Female
Perch
Sucker
Male
↑
0
0
↑
↑
Female
0
0
0
0
0
Male
0
0
0
↓
0
Female
0
0
0
↓
0
Upstream pulp mill versus downstream
Age
Liver
size
0
Gonad
size
0
Condition
0
Body
Size
0
0
0
0
0
0
0
0
0
0
0
* Reference site is Capone.
98
aR
iv
er
D/S Mad
iver
ia
inc
v
Pro
lB
er
ord
sR
uoi
Iroq
M
ad
aw
as
k
Quebec, Canada
U/S Pulp
D/S Pulp
Pulp mill
Paper mill
New Brunswick, Canada
St. Hilaire
St.
Fra
nc i
sR
ive
Capone
r
in
Sa
nR
h
o
tJ
Pulp mill effluent
lagoon and diffuser
r
ive
Maine, USA
Paper mill effluent
lagoon and diffuser
Municipal sewage
facilities
Figure 3.1. The study area near Edmundston, NB. River flow is from left to right
on the map.
99
3.8
References
BAR Environmental Inc.
1994.
Environmental effects monitoring pre-design
historical information for Fraser Inc., Edmundston sulphite pulp mill.
Guelph, ON, Canada.
Culp,J.M., Cash, K.J., Glozier, N.E., and Brua, R.B. 2003. Effects of pulp mill
effluent on benthic assemblages in along the Saint John River, Canada.
Environ Toxicol Chem 22: 2916-2925.
Doherty, C.A., Curry, R.A., and Munkittrick, K.R. 2003. Tracking adult white
sucker movements near point source discharges in the Saint John River,
New Brunswick, Canada. Water Quality Res J Can: Submitted.
Frank, M., McMaster, M.E., Munkittrick, K.R., Savoie, M.C., and Wood, C.
Effects of sulphite and bleached kraft pulp and paper mill effluents on
yellow perch and Johnnie darters. 25th Aquatic Toxicity Workshop,
Quebec, QC, Canada, October 18-21, 1998, pp 53.
Galloway, B.J., Munkittrick, K.R., Currie, S., Gray, M.A., Curry, R.A., and Wood,
C. 2003. Examination of the responses of slimy sculpin (Cottus cognatus)
and white sucker (Catostomus commersoni) collected on the Saint John
River downstream of pulp mill, paper mill, and sewage discharges.
Environ Toxicol Chem 22: 2898-2907.
Gibbons, W.N., and Munkittrick, K.R. 1994. A sentinel monitoring framework for
identifying fish population responses to industrial discharges.
J Aquat
Ecosyst Health 3: 227-237.
100
Gibbons, W.N., and Munkittrick, K.R., and Taylor, W.D.
1998a. Monitoring
aquatic environments receiving industrial effluents using small fish species
1: Response of spoonhead sculpin (Cottus ricei) downstream of a
bleached-kraft pulp mill. Environ Toxicol Chem 17: 2227-2237.
Gibbons, W.N., Munkittrick, K.R., McMaster, M.E., and Taylor, W.D.
1998b.
Monitoring aquatic environments receiving industrial effluents using small
fish species 2: Comparison between responses of trout-perch (Percopsis
omiscomaycus) and white sucker (Catostomus commersoni) downstream
of a pulp mill. Environ Toxicol Chem 17: 2238-2245.
Gray, M.A., Curry, R.A., and Munkittrick, K.R.
2002.
Non-lethal sampling
methods for assessing environmental impacts using a small-bodied
sentinel fish species. Water Quality Res J Can 37: 195-211.
Hewitt, L.M., Pryce Hobby, A.C., Parrott, J.L., Marlatt, V., Wood, C., Oakes, K.,
Van Der Kraak, G. 2003.
Accumulation of ligands for aryl hydrocarbon
and sex steroid receptors in fish exposed to treated effluent from a
bleached sulphite/groundwood pulp mill. Environ Toxicol Chem 22: 28902897.
Munkittrick, K.R., McMaster, M.E., Van Der Kraak, G., Portt, C., Gibbons, W.N.,
Farwell, A, and Gray, M. 2000. Development of methods for effectsdriven cumulative effects assessment using fish populations: Moose River
Project. SETAC Press, Pensacola, FL, USA.
Munkittrick, K.R., McGeachy, S.A., McMaster, M.E., Courtenay, S.C.
2002.
Overview of cycle 2 freshwater fish studies from the pulp and paper
101
Environmental Effects Monitoring program. Water Quality Res J Can 37:
49-77.
Parrott, J.L., Wood, C.S., Boutot, P., Blunt, B.R., Baker, M.A., and Dunn, S.
2003.
Fathead minnow long-term growth/reproductive tests to assess
final effluent from a bleached sulphite mill. Environ Toxicol Chem 22:
2908-2915.
Peterson, B.J., and Fry, B. 1987. Stable isotopes in ecosystem studies. Ann
Rev Ecol Syst 18: 293-320.
Scott, W.B., and Crossman, E.J. 1998. Freshwater Fishes of Canada. Galt
House, Oakville, ON, Canada.
Wayland, M., and Hobson, K.A. 2001. Stable carbon, nitrogen, and sulphur
isotope ratios in riparian food webs on rivers receiving sewage and pulpmill effluents. Can. J. Zool. 79: 5-15.
Wassenar, L.I., and Culp, J.M. 1996. The use of stable isotope analyses to
identify pulp mill effluent signatures in riverine food webs.
In Servos,
M.R., Munkittrick, K.R., Carey, J.H., Van Der Kraak, G., (eds),
Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie
Press, Delray Beach, FL, USA, pp 413-424.
102
CHAPTER 4.
Influence of seasonal changes in relative liver size,
condition, relative gonad size, and variability in ovarian development
of multiple spawning freshwater fish for use in environmental
monitoring programs3.
3
Submitted for Publication: Galloway, B.J., and Munkittrick, K.R. 2005. Influence
of seasonal changes in relative liver size, condition, relative gonad size, and
variability in ovarian development of multiple spawning freshwater fish for use in
environmental monitoring programs. Journal of Fish Biology.
4.1
Abstract
The use of forage fish for environmental monitoring programs has
increased over the past few years because they are relatively easy to capture,
many species exhibit site fidelity, and they are usually relatively abundant. The
main disadvantage of using forage fish for environmental monitoring programs is
the lack of basic life-history information, which can hinder study design and data
interpretation. The objectives of this study were to collect basic biological
information to assist in understanding the biology of selected forage fish for use
in environmental monitoring programs. Specifically, we examined seasonal
changes in condition factor, liversomatic index (LSI), gonadosomatic index (GSI),
and ovarian histology in four common cyprinids found in Atlantic Canada:
blacknose dace Rhinichthys atratulus, northern redbelly dace Phoxinus eos,
golden shiner Notemigonus crysoleucas and the mummichog Fundulus
heteroclitus. All four species are batch-spawners that have an extended
103
spawning season (spring-summer). GSI and LSI profiles of female blacknose
dace, mummichog, and golden shiner were similar, decreasing gradually during
the spawning season. GSI and LSI of female northern redbelly dace peaked
twice suggesting oocyte recruitment was continuous throughout the spawning
season. In addition, regressions of ovary weight to adjusted body weight were
more variable in northern redbelly dace relative to the other fish species. For
female blacknose dace, variability in the relationship between ovary weight and
adjusted body weight was lowest during the early prespawning period (r2=0.93)
and highest just before spawning (r2=0.43), and this variability was minimized by
selecting either 2-year-old fish or fish that weighed 2 – 4 g. Minimizing sources
of natural variability in metrics required for environmental monitoring studies is
particularly important since it will increase the probability of detecting impacts
associated with contaminant exposure.
4.2
Introduction
In Canada, the use of large-bodied fish species in many Environmental
Effects Monitoring programs has decreased [Munkittrick et al. 2002] because of
concerns over mobility issues (i.e., fish moving out of the area receiving
wastewater effluents). The use of small-bodied fish for environmental monitoring
programs has increased over the past few years because they are relatively easy
to capture, many species exhibit site fidelity, and they are usually relatively
abundant. Small-bodied fish have also been used in environmental monitoring
programs in the United States [Yeardley, Jr 2000; Fraker et al. 2002; Noggle et
al. 2004] and New Zealand [Richardson 1997]. The main disadvantage of using
104
small-bodied fish for environmental monitoring programs is the lack of basic lifehistory information, which can hinder study design and data interpretation.
There are a number of challenges to using small-bodied species for
environmental assessments, including populations can sometimes show
localized differences in performance because of their reliance on small habitat
patches. Additionally, there is often a paucity of background biological
information on which to base study designs. Furthermore, several small-bodied
species available for use in monitoring programs have multiple spawning periods
during the summer. It becomes difficult to ensure synchronous sampling
between exposed and reference populations that may have relatively small
differences in environmental conditions [LeBlanc et al. 1997]. In an attempt to
avoid the complications of multiple spawning periods and differences in timing
between groups of fish, Environment Canada has provided guidance for
monitoring programs that suggests that the sampling of multiple spawning fish be
conducted in the prespawning period prior to the onset of the first spawn of the
year [Environment Canada, 1997, 2001]. At this time, it was assumed that
development would be synchronized between individuals, and if changes were
not evident prior to the first spawn, the consequences of small changes later in
the season would be less important.
Recently, blacknose dace Rhinichthys atratulus (Hermann) were used in a
monitoring program examining the consequences of multiple wastewater
105
discharges in the upper Saint John River, New Brunswick, Canada [Galloway et
al. 2004]. During these studies it was apparent that the strength of the
relationship between gonad size and body size in this multiple spawning fish
species decreased as the sampling season was approached. This decreased
statistical power and complicated the interpretation of data. The present study
examined four species of multi-spawning, small-bodied cyprinids found in Atlantic
Canada to examine the influence of season and reproductive activity on the
potential power of statistical comparisons and the ability to distinguish site
differences.
Seasonal changes in condition factor, liversomatic index (LSI),
gonadosomatic index (GSI), and ovarian histology in blacknose dace R.
atratulus, northern redbelly dace Phoxinus eos (Cope), golden shiner
Notemigonus crysoleucas (Mitchell), and estuarine mummichog Fundulus
heteroclitus L. were examined. The mummichog is a common minnow found
along the Atlantic coast and been used in environmental monitoring programs
[Leblanc et al. 1997; Couillard and Nellis 1999; Dube and MacLatchy 2000] and
its life history has been well studied [Taylor and DiMichele 1980; Wallace and
Selman 1981; Shimizu 1997]. However, there is a paucity of detailed biological
information (especially for reproduction) for blacknose dace, northern redbelly
dace, and golden shiner in Canada and specifically for the Maritime Provinces.
This is particularly surprising for blacknose dace since it is considered to be one
of the most common minnow species in the Maritime Provinces and may serve
106
as an important food source for economically important trout species [Scott and
Crossman 1998].
Seasonal variability in the ovary weight to body weight relationship (i.e.,
coefficient of determination [r2] values for linear regressions equations) of female
fish was also monitored to: (1) identify the time of the year when the relationship
between ovary weight and adjusted body weight (i.e., total body minus gonad
weight) was most variable (i.e., low r2 values), (2) increase our understanding of
natural source(s) of variability in gonad development, (3) identify suitable
techniques to minimize data variability and (4) reduce sample size requirements
for detecting a critical effect size when designing cost-effective studies in the
future.
4.3
Materials and Methods
Fish were collected from various sites in southern New Brunswick
between May 2003 and April 2004. Blacknose dace were collected using a
backpack electrofisher [Smith-Root Model 12B, Smith-Root Inc., Vancouver, WA,
USA] from Milkish Brook (N 45.37977°, W 66.13306°). Northern redbelly dace
were collected from a beaver pond in the Keswick River watershed (N 46.09523°,
W 66.97490°; females only); golden shiner were collected from Little Chamcook
Lake (N 45.15228°, W 67.107320°); baited minnow traps were used to collect
mummichogs from a salt marsh located adjacent to Taylor's Island in Saint John
(N 45.22953°, W 66.13082°). Captured fish were transferred to the laboratory in
107
coolers filled with ambient water, rendered unconscious by concussion, followed
by spinal severance, and length (± 1 mm), body weight (± 0.01 g), liver (± 0.001
g) weight, and gonad weights (0.001 g) were recorded. Fish were aged by
counting annuli on sagittal otoliths. The annuli counts were verified by two
readers.
Ovaries were fixed in 10% buffered formalin and transferred to 70%
ethanol for storage. Paraplast-embedded tissues were sectioned (4 µm) with a
rotary microtome and stained with Ehrlich's haematoxylin and counterstained
with eosin Y. Ovarian sections were examined with a light microscope to
determine the percent of various oocyte stages present at each sampling time.
One hundred oocytes sectioned through the nucleus were counted to determine
the relative proportion of different developmental stages. The ovarian stage
terminology follows Blazer [2002] (see Table 4.1). Ovarian section images were
obtained from an Olympus™ BX40 microscope with Sony™ Exwave HSD digital
video camera attachment.
4.3.1 Data Analyses
Analysis of variance (ANOVA) was used to test for differences in mean
length, weight, and age of fish between collection dates; Bonferroni post hoc
comparisons were used to compare means. Analysis of covariance (ANCOVA)
was used to assess differences in the relationships between weight and length
108
(condition factor), and body weight and liver size and gonad size. Except for
condition, adjusted body weight (total wt – organ wt) was used as a covariate in
the ANCOVA model. All data were log10 transformed before performing ANOVA
and ANCOVA, and sexes were analyzed separately. All data analyses were
done using SYSTAT® (SPSS, SYSTAT, Chicago, IL, USA) statistical software.
ANCOVA is robust even when the slopes are not equal, so slopes were
considered different when p < 0.01 (Hamilton et al. 1993). Gonad weights and
liver weights were calculated as percent-adjusted body weight for summary
purposes.
4.4
Results
4.4.1 Blacknose dace
Female blacknose dace exhibited few significant differences in fork length
(68.6 ± 0.54 mm, n=141), weight (3.56 ± 0.10 g, n=141) or age (mean 2.6 years,
n=90), between sampling periods (Table 4.2). Male blacknose dace showed no
significant differences in fork length (65.1 ± 0.6 mm, n=100) or body weight (3.02
± 0.09 g, n=100), (Table 4.2). Condition factors (slope of body weight versus
length) were highest for both sexes in May relative to all other sampling times,
except males in August (Table 4.3).
Initial spawning for blacknose dace occurred in early June when mean
water temperatures reached 15.0°C (Figure 4.1). In early May, females showed
a strong relationship between ovarian size and adjusted body weight (body
weight-gonad weight), with an average GSI of 9%, (Table 4.2) and r2=0.97 (see
109
Table 4.4). Ovarian size continued to increase, peaking on May 20 and
remaining high in some fish (>20%) until mid-June. Coefficients of determination
(i.e., r2) values declined to 0.80 in late May and 0.43 in early June (Table 4.4).
Female blacknose dace ovaries in early May contained mostly oocytes in the
cortical alveoli and early vitellogenic stages (Figure 4.2). The appearance of midvitellogenic oocytes increased in late May, and showed a slight reduction by June
11, however, some females still contained mid-vitellogenic and mature oocytes
(Figure 4.2).
The highest GSIs for female blacknose dace were recorded on May 20
(25%) and June 11 (21.2%); the highest male GSIs were recorded on May 4
(1.67%) and May 20 (1.53%) (Table 4.2). By July 28, mean female and male
GSIs were significantly reduced relative to pre-spawning values; ovaries
contained mostly previtellogenic oocytes (Figure 4.2), and the r2 value for the
ovary weight to adjusted body weight relationship in female blacknose dace was
high at 0.76 (Table 4.4).
It has been well documented and generally understood that temperature
plays a critical role in many aspects of the physiology and biochemistry of fishes
and increasing water temperatures and photoperiod are often associated with
increased gonad development and the onset of spawning activity in fishes
inhabiting temperate waters [see Pankhurst and Porter 2003]. In Milkish Brook,
mean monthly water temperatures increased from 10.7°C in May to 15.0°C in
110
June (Figure 4.1), which corresponded to the largest increase in female GSI,
increased presence of vitellogenic oocytes, and increased condition (see Table
4.2). Liver weights did not decrease during this same time period, indicating
energy investment in gonad development from May to June was probably from
active feeding. Generally, increased water temperatures are associated with
increased feeding activity in fish inhabiting temperate waters. Mean water
temperature increased ~2°C from June to reach 17.1°C in July (Figure 4.1); it
was evident that blacknose dace spawning occurred at these temperatures since
male and female GSIs, LSIs, and condition all decreased during this time (Table
4.2). Mean water temperatures remained constant from July to August at ~ 17°C
(Figure 4.1) and female GSIs showed a significant increase during this time
(Table 4.2).
Liver sizes of female and male blacknose dace varied seasonally and
decreased during spawning. Female blacknose dace had mean LSI values of ~
3% in May and were significantly reduced by June 11 with the appearance of
stage 5 oocytes (exogenous vitellogenesis) in the gonads (Table 4.2, Figure 4.2).
By July 28, female LSI values were significantly reduced relative to pre-spawning
values and remained low by August, which corresponded to a decline in the
presence of vitellogenic eggs and appearance of atretic eggs (Table 4.2, Figure
4.2). Male LSIs peaked in early May, remained virtually unchanged by June 11,
and were significantly reduced relative to pre-spawning values by July and
August (Table 4.2).
111
4.4.2 Golden shiner
Female golden shiners were a similar size during the collections (p>0.05);
mean fork length (76.5 ± 0.9 mm, n=140), body weight (4.77 ± 0.21g, n=140),
and age (3.1 ± 0.1 years, n=101). Male shiners were identical in size, with an
average fork length of 76.5 ± 1.1 (n=80) and body weight 4.77 ± 0.26 (n=80).
Female and male golden shiner GSIs peaked in late June and remained elevated
by early July (Table 4.2). Ovaries exhibited an increase in the presence of
mature oocytes on June 22 (Figure 4.3), which corresponded to the lowest
recorded r2 value for the ovary weight to adjusted body weight relationship (Table
4.4). By early August, mean GSIs for female and male golden shiners were
significantly reduced relative to fish collected in late July, however, female fish
with GSIs >10% were still present (Table 4.2). By late August, mean GSIs of
male and female golden shiners were significantly reduced relative to prespawning fish and ovaries contained primary oocytes only (Table 4.2, Figure
4.3). The gonad weight to adjusted body weight relationship of females collected
on August 27 was strong with an r2 value of 0.91 (Table 4.4).
There were no significant differences in female and male condition factor
between sampling times, except males on August 29 (Table 4.2). Except for
April 24, female LSI values were similar between sampling times (Table 4.2).
Male golden shiner showed no significant changes in LSI between sampling
times (Table 4.2).
112
4.4.3 Northern Redbelly Dace
Female northern redbelly dace exhibited few differences in mean fork
length (54.6 ± 0.3 mm, n=354), body weight (1.56 ± 0.03 g, n=354) and age (2 ±
0.06 years, n=154) during the study period (Table 4.2). Gonad development in
female northern redbelly dace showed the most variability among the species
sampled. Mean GSIs in prespawning female northern redbelly dace were low in
April (<5%), increased significantly by May 27 and peaked on June 12 at almost
10% (Table 4.2). Histological sections of female P. eos ovaries confirmed that
the increase in mean GSI from April to June corresponded to an increase in the
percentage of late vitellogenic (stage 4) oocytes present within the ovary (Figure
4.4). During this same time period, r2 values for the ovary weight to adjusted
body weight relationship in female redbelly dace were consistently low (< 0.4,
Table 4.4). By late June, mean GSIs were significantly reduced relative to
earlier samples and the presence of post-ovulatory follicles (POFs) confirmed
spawning had occurred. However, it is important to note that some individual fish
still had GSIs >20% and ovaries contained late stage vitellogenic oocytes and
fully mature oocytes (Table 4.2, Figure 4.4).
The lowest r2 value for the ovary weight to adjusted body weight
relationship in female redbelly dace was recorded in late June (Table 4.4). The
highest female northern redbelly dace GSI values were measured on May 27
(21.5%), June 12 (21.5%), and June 22 (23%) (Table 4.2). After a decline over 4
weeks, mean GSI of females collected in July and August were ~ 2%, were
significantly reduced relative to prespawning fish in April, and remained low to
113
October (Table 4.2). The ovaries of fish collected in August contained
previtellogenic oocytes only (Figure 4.4). Coefficient of determination values for
the ovary weight to adjusted body weight relationship in female redbelly dace
were all >0.5 from July to October (Table 4.4).
The liversomatic index of female northern redbelly dace was variable with
few statistically significant differences throughout the season. LSI peaked at two
periods: in April, afterwards the LSI decreased 28% by June 12, which
corresponded to a 59% increase in GSI; and in late June, corresponding to the
maximum GSI observed (i.e., 23%) (Table 4.2). By July 27, LSI of female
northern redbelly dace was significantly reduced relative to pre-spawning fish
and remained low to October (Table 4.2). Condition of prespawning female
northern redbelly dace was low in April, increased in June and remained high
until late July (Table 4.2). After 4 weeks, females had returned to pre-spawning
condition and remained unchanged by October (Table 4.2).
4.4.4 Mummichog
Female mummichog collected in April and June were shorter and lighter
relative to fish collected on other dates (Table 4.2); males exhibited no significant
differences in fork length (65.7 ± 0.9 mm, n=86) and body weight (3.40 ± 0.15 g,
n=86) during our collections. The mean fork length, body weight, and age of all
female mummichog was 65.3 ± 0.7 mm (n=239), 3.39 ± 0.11 g (n=239), and 2.4
114
± 0.1 years (n=109), respectively. Female mummichogs collected in June had a
mean age of 2.0 ± 0.1 (n=42) and were significantly younger relative to females
collected in May, July, and August with a mean age of 2.5 ± 0.1 (n=69).
Mean GSIs of female mummichog were low (< 5%) in April and early May
(Table 4.2). On May 8, mummichog ovaries consisted of mostly previtellogenic
oocytes and few early vitellogenic oocytes (Table 4.2, Figure 4.5). Variability in
the ovary weight to adjusted body weight relationship for female mummichog was
low in April and May when r2 were 0.93 and 0.85, respectively (Table 4.4).
Female GSIs peaked in late June with the appearance of fully mature oocytes
(Table 4.2, Figure 4.5), which corresponded to increased variability in the ovary
weight to adjusted body weight relationship (Table 4.4). Mean GSIs of female
mummichogs were low in July (Table 4.2), but gonad development was highly
variable relative to other sampling times (Table 4.4). By late August, female
GSIs ranged from ~1-3% (similar to October), ovaries contained mostly primary
oocytes, and variability in the gonad weight to adjusted body weight relationship
was low and approaching pre-spawning conditions (Tables 4.2, 4.4; Figure 4.5).
Mean GSIs for male mummichogs peaked by late June, were significantly
reduced by late July, and remained low to October (Table 4.2).
ANCOVA results showed the slopes for ovary weight versus adjusted
body weight were significantly different (F=4.675; d.f. = 5, 227; P<0.0005, see
Table 4.3). For female mummichog, slopes of the regression for condition were
115
significantly different (ANCOVA; F=5.502; d.f.=2,227;p<0.0005). Thus there
were seasonal differences in the rate at which body weight increased with length
(see Table 4.3). The fattest male mummichogs were collected on July 28 and
October 12 and corresponded to elevated LSIs (Table 4.2).
4.5
Discussion
The objectives of our research on the four multi-spawning, small-bodied
fish species from southern New Brunswick were to document seasonal changes
in gonad development, liver size, and condition (metrics required for many
environmental monitoring programs) during the pre-spawning, spawning, and
post-spawning seasons; attempt to identify and minimize sources of natural
variability in gonad development; and provide guidance to facilitate the use of
multi-spawning, small-bodied fish species for regional, national, and international
environmental monitoring programs.
4.5.1 Reproductive Development
The results of the present study indicate blacknose dace, northern
redbelly dace, and golden shiner, like some other cyprinids, have an extended
spawning season (spring-summer), with multiple spawning events [Tartar 1969;
Delahunty and DeVlaming 1980; Heins and Rabito 1986; DeHaven et al. 1992;
116
Rinchard and Kestemont 1996; Roberts and Grossman 2001]. Mummichog
followed a similar pattern, as has been previously reported [Shimizu 1997].
Releasing small batches of eggs several times during the reproductive season
increases the probability that some offspring will survive despite fluctuating
environmental conditions, predation pressure, and/or resource competition (e.g.,
suitable spawning habitat/food/shelter). Our results also demonstrated that
oocyte development in the four fish species was asynchronous; there were
considerable differences in the patterns of ovarian maturation between northern
redbelly dace and the other fish species; and rates of oocyte development differ
between individuals of a species. Together, histology and regressions of ovary
weight to adjusted body weight were critical for understanding seasonal
variability in ovarian development both within and between species. Except for
northern redbelly dace, all female fish sampled showed the same seasonal
pattern of variability in gonad development (see Table 4.4). For example,
variability in female blacknose dace gonad development was low (r2=0.93) during
the early pre-spawning season when the ovaries contained few vitellogenic
oocytes, increased (r2=0.43) just before the onset of spawning activity when the
ovary contained two batches of maturing oocytes, and gradually the variability
decreased as the post-spawning season approached and the ovaries contained
only primary oocytes.
Histology showed the increases in variability in gonad development
resulted from individual differences in oocyte maturation as fish approached the
117
first spawning event. The exact mechanisms controlling the rate of growth of
maturing oocytes in fish is not completely understood [Tyler and Sumpter 1996].
Studies with rainbow trout [Oncorhynchus mykiss; Tyler et al. 1990], and cod
[Gadus morhua; Kjesbu et al. 1996, Tomkiewicz et al. 2003] have shown there is
considerable variability in individual oocyte growth in maturing ovaries. Thus, it is
likely that the increased variability seen in female blacknose dace, mummichog,
and golden shiner ovaries during the pre-spawning period resulted from
differences in the growth rates of individual oocytes in the early stages of
vitellogenesis that would be released later in the spawning season.
Gonad development in female northern redbelly dace was highly variable
throughout the study relative to the other three species and was highly variable
among individuals. Histological sections of ovarian tissue were invaluable to our
understanding of variability in female northern redbelly dace gonad development.
Specifically, post-ovulatory follicles (POFs) were observed in the ovaries of some
fish that also contained maturing oocytes suggesting at least one batch of
oocytes was recently released. Some female northern redbelly dace had ovaries
containing remnant ripe eggs, atretic eggs, and maturing oocytes indicating the
fish had recently spawned and was preparing to release another batch of eggs.
The histological results for female northern redbelly dace suggest individuals
release small batches of eggs daily throughout an extended spawning season.
The high variability in female northern redbelly dace gonad development is due
to the fact that ovaries contain oocytes in various maturity stages during the
118
entire reproductive season and that oocytes are continuously recruited into
vitellogenesis.
4.5.2 Liversomatic Index and Condition Factor
LSI and condition factor data for the fish species collected in the present
study provided important information about the patterns of energy use and
storage during the pre-spawning period, spawning period, and post-spawning
period. The seasonal changes in LSI were particularly pronounced in female fish
and there were differences between species. Liver energy stores in female
blacknose dace were not fully utilized during the early stages of gonadal
development. Liver weights of female blacknose dace were highest during the
pre-spawning period in May when the largest increase in gonad weight and
condition occurred. If the liver was the main energy source for oocyte
development in female blacknose dace then we should have seen a decline in
LSI from May 4 to May 20, but this was not the case. In fact, female LSI values
remained virtually unchanged in May suggesting most of the energy for early
gonad development was derived from feeding. Female blacknose dace LSIs
declined from May 20 to June 11, which corresponded to a slight decrease in
GSI and presence of mature (stage 5) oocytes within the ovary. Female LSI and
GSI values showed the largest decline from June 11 to July 28. Taken together,
seasonal changes in LSI of female blacknose dace suggests energy invested
into early gonad growth (i.e., first batch of eggs released) is probably derived
119
from active feeding, while most of the liver energy stores are utilized for
maturation of subsequent batches of eggs to be released later in the spawning
season. Future studies will be needed to improve our understanding of this
issue. The pattern of energy use for gonad development in male blacknose dace
was similar, although not as pronounced. Changes in female blacknose dace
condition factor mirrored the changes in GSI.
As in bleak [Alburnus alburnus; Rinchard and Kestemont 2003], LSIs of
northern redbelly dace did not decrease continuously during the spawning
season, but showed two peaks: in April, just before the spawning season and
again in late June. The changes in northern redbelly dace LSI occur because
batches of oocytes are going through vitellogenesis during the entire spawning
season, which was also the case for the bleak [Rinchard and Kestemont 2003].
LSI and condition remained relatively stable during the spawning season in male
and female golden shiner collected from Little Chamcook Lake suggesting liver
energy stores were not seriously depleted and fish were possibly feeding. Again,
this is difficult to confirm without stomach content analysis. Mummichog liver
energy stores declined as gonad weights increased, but it is important to note
that fish condition continuously increased during the pre-spawning period and
remained relatively high during the spawning season indicating fish were feeding
to maintain liver energy stores.
120
It is important to note that the seasonal changes in female blacknose dace
LSI differed from the other multiple spawning fish used in this study suggesting
the pattern of oocyte recruitment and maturation is different between blacknose
dace and the other species.
4.5.3 Data Variability, Sample Sizes, and Power
It is our goal to facilitate the use of multi-spawning, small-bodied fish in
environmental monitoring programs. In order to accomplish this goal, we
identified sources of data variability and selected techniques that could be used
to minimize data variability, which would allow us to determine the sample sizes
required in order to detect a 25% difference (i.e., critical effect size) in gonad size
between a reference and exposure site at a specified power level [see
Munkittrick et al. 2002; Lowell et al. 2003]. In Canada, for example, Federal
government regulations require pulp and paper mills and metal mining operations
to design and implement monitoring programs capable of detecting a 25%
difference in gonad size between an exposed site (i.e., site receiving effluent)
and reference site [Environment Canada 1997].
The number of samples required to detect a difference between
populations depends on the variability, the size of difference you want to detect,
the power required (1-β), and the statistical certainty (α). The batch-spawning
fish investigated in this paper demonstrated changes in variability during the
121
sample season that would dramatically affect the sample sizes required to detect
a standardized difference between populations. For blacknose dace collected in
early May (r2=0.97), the sample size required to detect a difference is much
smaller than during late June when the r2 is < 0.45. Based on all female
blacknose dace data collected on May 20 and June 11, the sample sizes
required to detect a 25% difference in gonad size are shown in Table 4.5. On
May 20 when the standard deviation was about 24% of the mean (i.e., CV=24%);
a sample size of 16 to 24 fish (depending on site) would be required to detect a
25% difference in gonad size when α and β were 0.05 and 0.20, and when both
α and β equalled 0.10. On June 11, the standard deviation for all female
blacknose dace data increased to about 41% of the mean (i.e., CV = 41%), as
such, future studies during this time period would require sample sizes of 47 to
71 (Table 4.5).
It is possible to reduce the variability by restricting the samples to include
fish of a similar age or body size. Sample sizes required to detect a 25%
difference in gonad size for all female blacknose dace collected on May 20 and
June 11 were compared to 2 year old fish and fish weighing 2 – 4 g at three
different levels of power (i.e., α=0.05 and β=0.05; α=0.05 and β=0.20; α=0.10
and β=0.10). The r2 for the regression of ovary weight to adjusted body weight
for all female blacknose dace collected on May 20 was 0.80 and when 2 - 4 g fish
or 2 year old fish were selected the r2 value increased to 0.88 and 0.94,
respectively (Table 4.6). When only 2 – 4 g female blacknose dace were
122
considered, the standard deviation was reduced to 20% of the mean on May 20
and 17 fish per site were required to detect a 25% difference in gonad size when
α=0.05 and β=0.05, but only 11 fish per site were required when α=0.05 and
β=0.20 or when α and β both equalled 0.10 (Table 4.5). When 2 year old female
blacknose dace were selected only, the CV was reduced to about 15% of the
mean on May 20 and less than 10 fish per site were required to detect a 25%
difference in gonad size. Selecting 2 year old fish or fish that ranged in size from
2 – 4 g would reduce some of the variability associated with gonad development
and increase our ability to detect potential reproductive impacts associated with
contaminant exposure if they exist and would also allow us to collect all of the
required reproductive end-points for the EEM program.
However, these techniques were not successful at reducing variability
during all seasons or for all species. For example, the variability in gonad
development in 2 year old female blacknose dace increased sample size
requirements by about 13.5 - 14.9 times in June (i.e., spawning season) relative
to May (i.e., pre-spawning season). Overall, it is evident that choosing a certain
age and/or size class of fish during certain times of the reproductive season will
reduce data variability and sample sizes required to detect critical effect sizes
with a specified level of power, but these techniques will not work for all species
(e.g., northern redbelly dace).
123
The timing of sampling for small-bodied fish (both single and multispawners) is critical to the success of a study and can only be accomplished
when sufficient basic biological information is available. However, the existing
guidance for sampling prior to the first spawn is not always the best timing. As
the proximity of the first spawn approached, the correlation reduced in strength in
most species. This is probably associated with a reduced synchronization of the
second spawning event between individuals. Furthermore, female northern
redbelly dace showed considerable more variability in gonad development during
the entire study relative to the other fish. Using data for female northern redbelly
dace collected on May 27 (highest spring r2 value) and June 22 (lowest r2 value),
we calculated the sample sizes required to detect a 25% difference in gonad size
at the same three power levels noted above. On May 27, the CV for all female
northern redbelly dace was 57% and sample sizes between 81-134 fish per site
would be required to detect of 25% difference in gonad size (Table 4.5). On
June 22, when the CV was 75%, the sample sizes required per site were more
than double that required on May 27 (Table 4.5). Sampling only 2 year old or 2 –
4 g female northern redbelly dace on May 27 and June 22 did not substantially
reduce data variability or sample size requirements as the CV remained around
50% during each sampling time for both groups (Table 4.5).
In some species, selecting a specific age or size class of fish may be
useful to reduce the data variability associated with gonadal development during
the pre-spawning season. Reducing the variability will reduce the sample size
124
required to detect critical effect sizes with a specified level of power. Conversely,
it also apparent that the techniques used to reduce sample variability may not be
as useful during certain periods of the reproductive season and may not be
suitable for all fish species.
4.6
Conclusions
Our results demonstrate blacknose dace, northern redbelly dace, golden
shiner, and mummichog have extended spawning seasons (spring-summer),
which has been reported for some of these species in other locations. However,
there are differences in the onset of spawning, which is probably related to
geographical differences in environmental conditions. The suite of indices used
in this study provided information about the seasonal changes in energy use and
storage, particularly as it relates to gonad growth. Female blacknose dace
appear not to rely on liver energy stores when the maximum increases in gonad
size occur and may rely on energy derived from active feeding. Future studies
will be needed to clarify this issue. Understanding the basic biology of multiple
spawning, small-bodied fish potentially used in environmental monitoring
programs is important for study design and data interpretation. Overall, we know
the strength of the gonad size – adjusted body weight relationship varies during
the season for each species and that some fish species (i.e., northern redbelly
dace) show more seasonal variability in this parameter than others (i.e.,
blacknose dace). Selecting a specific age class or size class of fish will reduce
125
some of the data variability associated with gonadal development and will also
reduce sample size requirements for future studies in some fish species during
certain times of the year, but these techniques may not be suitable for all
species. Multi-spawning, small-bodied fish can be successfully used in
environmental monitoring programs, but additional basic biological research will
be required in order to continue to facilitate their use.
4.7
Acknowledgements
This project received funding from the Canadian Water Network, Toxic
Substance Research Initiative (Project 205), New Brunswick Innovation Fund,
NexFor and Fraser Papers. The invaluable help of K. Tenzin, J. Peddle, G.
Vallieres, and T. Galloway in the field is greatly appreciated. Graduate student
support for BJG from NSERC Industrial-Post Graduate Scholarship Program and
UNB. KRM receives support from a NSERC Discovery Grant, the Canadian
Water Network Networks of Centres of Excellence and from the Canada
Research Chairs Program.
4.8
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131
Table 4.1. Microscopic characteristics for the determination of oocyte
developmental stages (modified from Blazer 2002).
Ovarian stage
Ovarian stage
code
Description of the most advanced
oocytes present within ovary.
Previtellogenic (Primary
Growth)
1
Previtellogenic (Cortical
alveoli)
2
Early vitellogenic
3
Mid Vitellogenic
4
Mature
5
Atretic
6
Post-spawning
7
Includes chromatin nucleolar
oocytes (single nucleolus) and
perinucleolar oocytes (multiple
nucleoli).
Cortical alveoli oocyte with cortical
alveoli beginning to appear in the
periphery of the cytoplasm.
Cortical alveoli present throughout
the cytoplasm but yolk globules
become apparent.
Cortical alveoli are densely packed
into the cell periphery with most of
the cytoplasm containing yolk
globules.
Yolk globules fuse into a
homogenous mass.
Breakdown of the nucleus, vitelline
envelope, and increase in the size
and number of the follicular cells.
Post-ovulatory follicles and
previtellogenic oocytes.
132
Table 4.2. Means ± SE (n) and minimum and maximum (in parentheses) values for length, weight, condition factor (k),
liversomatic index (LSI), and gonadosomatic index (GSI) in adult female and male blacknose dace (Rhinichthys
atratulus), golden shiner, mummichog (Fundulus heteroclitus), and northern redbelly dace (Phoxinus eos)
collected from various sites in southern New Brunswick, Canada. Within a column, differences (p < 0.05) among
sampling dates are denoted by different superscript uppercase letters. (*Significant interaction within ANCOVA
model).
Species
Sex
Date
Blacknose
F
May 4, 2003
Dace
F
F
F
F
May 20, 2003
June 11, 2003
July 28, 2003
Aug 19, 2003
Length (mm)
Body Weight (g)
Ka
LSIb
GSIc
69.9±1.9(16)AB
3.84±0.42(16)AB
1.07± 0.03(16)*
3.17± 0.21(16)C
9.07±0.47(16)A
(62.0-93.0)
(2.37-8.96)
(0.88 - 1.29)
(1.34 - 4.39)
(6.28 - 13.3)
70.6±1.1(24)A
4.13±0.23(24)A
1.14± 0.02(24)*
3.33 ± 0.24(24)C
16.5±0.8(24)B
(63.0-78.0)
(2.51-6.18)
(1.00 - 1.35)
(0.42 - 5.11)
(10.3 - 25.0)
70.9±1.0(35)A
4.05±0.18(35)A
1.11± 0.02(35)*
2.09± 0.12(35)A
12.9±0.9 (35)A
(61.0-83.0)
(2.19-6.46)
(0.93 - 1.35)
(0.73 - 3.94)
(2.92 - 21.2)
65.3±1.1(29)B
2.95±0.15(29)B
1.04 ± 0.01(29)*
0.92± 0.05(29)B
2.33±0.14(29)C
(58.0-83.0)
(2.05-6.00)
(0.89 -1.19)
(0.47 - 1.56)
(0.31 - 3.65)
67.0±1.0(37)AB
3.07±0.15(37)B
1.00 ± 0.01(37)*
0.96± 0.05(37)B
3.41±0.11(37)D
(59.0-81.0)
(2.02-5.28)
(0.89 - 1.07)
(0.25 - 1.64)
(1.11-4.56)
133
Species
Sex
Date
Length (mm)
Body Weight (g)
Ka
LSIb
GSIc
Blacknose
M
May 4, 2003
65.0±1.4(24)A
3.05±0.22(24)A
1.05±0.02(24)*
2.16±0.18(24)A
1.00±0.05(24)A
(51.0-74.0)
(1.11-4.94)
(0.84-1.27)
(0.76-3.83)
(0.66-1.67)
68.0±0.97(21)A
3.27±0.15(21)A
1.03±0.01(21)*
1.76±0.10(21)A
1.00±0.04(21)A
(60.0-77.0)
(2.24-4.88)
(0.93-1.13)
(1.05-2.59)
(0.66-1.53)
62.1±1.8(15)A
3.06±0.26(15)A
1.24±0.03(15)*
1.69±0.21(15)A
0.58±0.08(15)B
(51.0-73.0)
(1.61-4.81)
(1.08-1.45)
(0.68-3.23)
(0.06-1.17)
64.7±1.0(22)A
2.88±0.12(22)A
1.05±0.02(22)*
0.94±0.05(22)B
0.30±0.03(22)C
(57.0-72.0)
(1.81-3.87)
(0.93-1.23)
(0.57-1.44)
(0.08-0.73)
64.7±1.9(18)A
2.85±0.25(18)A
1.00±0.01(18)*
0.84±0.08(18)B
0.30±0.05(18)C
(50.0-77.0)
(1.20-4.97)
(0.91-1.09)
(0.38-1.48)
(0.03-0.71)
78.6±1.9(20)AB
5.02±0.45(20)AB
0.99± 0.02(20)A
1.48±0.10(20)AB
7.12±0.84(20)B
(67.0-94.0)
(2.57-9.22)
(0.85 - 1.15)
(0.84-2.36)
(1.11-13.4)
74.0±1.4(41)B
4.35±0.32(41)AB
1.02± 0.01(41)A
1.35±0.06(41)A
5.15±0.51(41)B
(60.0-106)
(2.15-13.9)
(0.88-1.17)
(0.57-2.43)
(1.27-17.2)
82.1±2.6(26)A
6.14±0.69(26)A
1.00± 0.02(26)A
1.62±0.09(26)AB
2.58± 0.42(26)AC
(59.0-107)
(1.80-15.7)
(0.88-1.17)
(0.85-2.53)
(1.05-10.7)
Dace
M
M
M
M
Golden
F
May 20, 2003
June 11, 2003
July 28, 2003
Aug 19, 2003
June 22, 2003
Shiner
F
F
July 9, 2003
Aug 7, 2003
134
Species
Sex
Date
Golden
F
Aug 29, 2003
Shiner
F
Golden
M
April 24, 2004
June 22, 2003
Shiner
M
M
M
Northern
F
July 9, 2003
Aug 7, 2003
Aug 29, 2003
May 27, 2003
redbelly Dace
F
June 12, 2003
Length (mm)
Body Weight (g)
Ka
LSIb
GSIc
77.0±1.7(19)AB
4.68±0.43(19)AB
0.98± 0.02(19)A
1.38±0.06(19)AB
1.58± 0.07(19)A
(64.0-95.0)
(2.51-10.8)
(0.82-1.26)
(0.94-1.96)
(1.01-2.23)
73.6±1.7(34)B
4.15±0.35(34)B
0.97± 0.02(34)A
1.71±0.08(34)B
2.75±0.22(34)C
(55.0-96.0)
(1.55-9.41)
(0.73-1.17)
(0.66-2.95)
(1.06-6.09)
77.5±1.9(20)AB
4.95±0.40(20)A
1.02± 0.02(20)A
1.14± 0.05(20)A
2.37± 0.20(20)*
(65.0-98.0)
(2.71-9.10)
(0.91-1.22)
(0.82-1.57)
(1.09-4.75)
79.1±2.4(21)A
5.57±0.56(21)A
1.05± 0.02(21)A
1.11± 0.05(21)A
1.81± 0.14(21)*
(62.0-102)
(2.21-12.0)
(0.77-1.23)
(0.60-1.45)
(0.74-2.66)
79.3±2.5(16)A
5.37±0.70(16)A
1.00± 0.02(16)AC
1.16± 0.07(16)A
1.12± 0.17(16)*
(66.0-104)
(2.39-13.6)
(0.83-1.21)
(0.54-1.79)
(0.33-2.33)
71.3±1.5(23)B
3.47±0.28(23)B
0.92± 0.01 (23)C
0.98± 0.05(23)A
0.45± 0.03(23)*
(62.0-94.0)
(2.09-8.70)
(0.81-1.05)
(0.47-1.44)
(0.14-0.87)
56.8 ± 00.8(60)B
1.76±0.08(60)B
0.93 ± 0.01(60)C
2.08±0.08(60)AC
9.70 ± 0.71(60)A
(43.0-72.0)
(0.71-3.98)
(0.76 - 1.19)
(0.39 - 3.30)
(0.44 - 21.5)
52.8±0.9(45)A
1.49±0.08(45)ABC
0.97±0.02(45)AD
1.77±0.09(45)A
9.94 ± 0.85(45)A
135
Species
Sex
F
Northern
F
Date
June 22, 2003
July 27, 2003
redbelly Dace
F
F
F
Mummichog
F
F
F
Aug 27, 2003
Oct 12, 2003
April 24, 2004
May 8, 2003
June 20, 2003
July 28, 2003
Length (mm)
Body Weight (g)
Ka
LSIb
GSIc
(44.0-65.0)
(0.58-2.87)
(0.68 - 1.30)
(0.77 - 4.19)
(0.39 - 21.5)
55.5±0.8(72)AB
1.72±0.08(72)AB
0.97±0.01(72)ACD
2.50 0.09(72)BC
6.98±0.62(72)CD
(44.0-75.0)
(0.75-4.13)
(0.83 - 1.18)
(0.78 - 4.49)
(0.44 - 23.0)
54.5±1.2(31)AB
1.59±0.11(31)ABC
0.93±0.02(31)DC
1.71±0.08(31)A
2.07±0.22(31)B
(45.0-66.0)
(0.88-2.79)
(0.80 - 1.13)
(0.53 - 2.70)
(0.74 - 6.25)
53.3±0.8(50)AB
1.36±0.07(50)C
0.87 ± 0.01(50)B
2.04±0.09(50)AC
2.09 ± 0.11(50)B
(45.0-70.0)
(0.85-3.20)
(0.77 - 0.99)
(0.61 - 3.79)
(0.67 - 4.09)
53.4±0.8(38)AB
1.37±0.06(38)AC
0.87 ± 0.01(38)BC
1.94±0.09(38)ABC
3.20±0.22(38)BCD
(42.0-63.0)
(0.62-2.61)
(0.78 - 1.04)
(0.95 - 3.11)
(0.68 - 5.71)
54.6±0.8(50)AB
1.50±0.07(50)ABC
0.89 ± 0.01 (50)B
2.27±0.10 (50)C
4.03±0.28(50)D
(45.0-66.0)
(0.81-2.89)
(0.79 - 1.07)
(0.87-4.12)
(1.00-9.59)
72.9±1.9(21)A
3.89±0.30(21)A
0.95±0.02(21)*
3.61± 0.22(21)A
4.45±0.16(21)*
(57.0-82.0)
(1.66-5.62)
(0.82-1.09)
(1.62-5.28)
(2.89-5.26)
56.7±1.4(43)B
2.37±0.21(43)B
1.21± 0.02(43)*
3.28± 0.17(43)A
23.7± 1.0(43)*
(42.0-83.0)
(0.94-7.03)
(0.96-1.67)
(1.54-6.73)
(8.81-37.6)
69.6±0.83(45)A
4.21±0.16(45)A
1.22± 0.01(45)*
4.03± 0.13(45)AB
1.85± 0.16(45)*
136
Species
Sex
F
F
F
Mummichog
M
M
M
M
M
Date
Aug 28, 2003
Oct 12, 2003
April 24, 2004
May 8, 2003
June 20, 2003
July 28, 2003
Aug 28, 2003
Oct 12, 2003
Length (mm)
Body Weight (g)
Ka
LSIb
GSIc
(59.0-88.0)
(2.47-8.08)
(1.09-1.45)
(2.34-5.34)
(1.15-7.08)
71.3±1.3(40)A
4.39±0.24(40)A
1.16± 0.01(40)*
3.35± 0.12(40)A
1.52± 0.05(40)*
(45.0-90.0)
(1.01-8.72)
(0.98-1.32)
(1.88-4.72)
(1.05-2.47)
66.2±1.5(41)A
3.69±0.30(41)A
1.17± 0.01(41)*
5.32 ± 0.15(41)C
1.84± 0.05(41)*
(54.0-89.0)
(1.73-9.51)
(0.95-1.35)
(2.54-7.42)
(1.20-2.93)
60.0±1.3(49)B
2.27±0.20(49)B
0.95± 0.01(49)*
4.07± 0.14(49)B
3.46 ± 0.10(49)*
(48.0-85.0)
(0.93-7.46)
(0.80-1.22)
(2.02-6.12)
(1.93-5.76)
65.5±2.8(12)A
2.88±0.40(12)A
0.94± 0.03(12)B
2.88± 0.28(12)A
1.53± 0.23(12)B
(46.0-83.0)
(0.75-6.05)
(0.77-1.06)
(1.57-4.95)
(0.36-3.17)
73.1±2.6(8)A
4.09±0.49(8)A
1.02± 0.02(8)B
1.51± 0.19(8)B
2.33± 0.15(8)B
(68.0-90.0)
(3.07-7.09)
(0.97-1.18)
(1.15-2.84)
(1.46-2.95)
63.1±2.7(20)A
3.44±0.43(20)A
1.22± 0.02(20)A
3.59± 0.23(20)A
0.52± 0.07(20)A
(39.0-79.0)
(0.66-6.45)
(1.08-1.34)
(2.08-5.52)
(0.10-1.13)
64.5±1.3(22)A
3.10±0.20(22)A
1.12± 0.01(22)C
3.47± 0.14(22)A
0.39± 0.03(22)A
(52.0-75.0)
(1.37-5.02)
(0.97-1.22)
(2.28-4.85)
(0.15-0.87)
66.5±1.3(24)A
3.67±0.24(24)A
1.20 ±0.02(24)A
5.54± 0.22(24)C
0.63± 0.10(24)A
137
Species
Sex
Date
Length (mm)
Body Weight (g)
Ka
LSIb
GSIc
(55.0-78.0)
(1.75-5.91)
(1.05-1.35)
(3.46-7.83)
(0.13-2.01)
a
K = 100000*(body weight/(total length3)); bLSI = 100*(liver weight/(body weight – liver weight)); cGSI = 100*(gonad
weight/(body weight – gonad weight)).
138
Table 4.3. Log10 regression estimates for adult female and male blacknose dace
(Rhinichthys atratulus) condition and gonad size and condition factor for
adult female mummichog (Fundulus heteroclitus) gonad size collected
from sites in southern New Brunswick in 2003 and 2004.
Species
Blacknose
Dace
Sex
F
M
Slope
3.45
Intercept
-5.80
n
16
p
<0.0005
r2
0.94
May 20, 2003
3.69
-6.22
24
<0.0005
0.96
June 11, 2003
3.04
-5.02
35
<0.0005
0.90
July 28, 2003
2.79
-4.60
29
<0.0005
0.93
Aug 19, 2003
3.02
-5.03
37
<0.0005
0.97
Condition
Factor
Condition
Factor
Condition
Factor
Condition
Factor
Condition
Factor
May 4, 2003
3.71
-6.27
24
<0.0005
0.98
May 20, 2003
3.14
-5.25
21
<0.0005
0.92
June 11, 2003
3.04
-4.99
15
<0.0005
0.95
July 28, 2003
2.56
-4.18
22
<0.0005
0.92
Aug 19, 2003
3.21
-5.39
18
<0.0005
0.99
Parameter
Condition
Factor
Condition
Factor
Condition
Factor
Condition
Factor
Condition
Factor
Collection Date
May 4, 2003
Golden shiner
M
Gonad Size
Gonad Size
Gonad Size
Gonad Size
June 22, 2003
July 9, 2003
Aug 7, 2003
Aug 29, 2003
0.72
0.69
1.80
0.93
-1.46
-1.56
-2.58
-2.34
20
21
16
23
0.013
0.002
<0.0005
0.004
0.30
0.41
0.70
0.34
Mummichog
F
Gonad Size
Gonad Size
Gonad Size
Gonad Size
May 8, 2003
June 20, 2003
July 28, 2003
August 28,
2003
October 12,
2003
April 24, 2004
1.04
0.73
1.33
1.16
-1.38
-0.58
-1.97
-1.92
21
43
45
40
<0.0005
<0.0005
<0.0005
<0.0005
0.86
0.60
0.47
0.89
1.18
-1.83
41
<0.0005
0.94
1.07
-1.49
49
<0.0005
0.87
May 8, 2003
3.35
-5.68
21
<0.0005
0.96
June 20, 2003
2.89
-4.73
43
<0.0005
0.94
July 28, 2003
3.05
-5.01
45
<0.0005
0.95
August 28,
2003
3.25
-5.39
40
<0.0005
0.98
Gonad Size
Gonad Size
F
Condition
Factor
Condition
Factor
Condition
Factor
Condition
Factor
139
Species
Mummichog
Sex
F
Parameter
Condition
Factor
Condition
Factor
Collection Date
October 12,
2003
April 24, 2004
Slope
3.36
Intercept
-5.59
n
41
p
<0.0005
r2
0.98
3.39
-5.72
49
<0.0005
0.98
140
Table 4.4. Relationship of log10 ovary weight to log10 adjusted body weight in
female fish on various dates in 2003 and 2004.
Species
Sex
Sample Date
Regression Equation
p
r2
n
Blacknose
Dace
F
May 4, 2003
Y= 0.15x – 0.20
<0.0005
0.97
16
May 20, 2003
June 11, 2003
July 28, 2003
Y= 0.27x - 0.35
Y= 0.15x - 0.08
Y= 0.04x - 0.05
<0.0005
<0.0005
<0.0005
0.80
0.43
0.76
24
35
29
August 19, 2003
Y= 0.05x - 0.04
<0.0005
0.87
37
June 22, 2003
Y=0.104x – 0.134
<0.0005
0.58
20
July 9, 2003
Y=0.142x – 0.346
<0.0005
0.63
41
Aug 7, 2003
Y=0.041x – 0.087
<0.0005
0.61
34
Aug 29, 2003
Y=0.024x – 0.037
<0.0005
0.91
20
May 27, 2003
Y= 0.146x – 0.073
<0.0005
0.38
60
June 12, 2003
June 22, 2003
Y=0.089x + 0.013
Y=0.062x + 0.011
<0.0005
<0.0005
0.28
0.19
45
72
July 27, 2003
Aug 27, 2003
Y=0.044x - 0.031
Y=0.027x - 0.008
<0.0005
<0.0005
0.56
0.64
31
50
Oct 12, 2003
April 24, 2003
Y=0.056x - 0.031
Y = 0.039x+0.002
<0.0005
<0.0005
0.55
0.27
38
50
May 8, 2003
June 20, 2003
July 28, 2003
Aug 28, 2003
Oct 12, 2003
April 24, 2004
Y=0.048x – 0.011
Y=0.158x + 0.126
Y=0.045x – 0.107
Y=0.016x – 0.003
Y=0.022x-0.012
Y = 0.038x-0.007
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
0.85
0.58
0.39
0.79
0.94
0.93
21
43
45
40
41
49
Golden
Shiner
Northern
Redbelly
Dace
Mummichog
F
F
F
141
Table 4.5. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for female northern redbelly dace Phoxinus eos
and blacknose dace Rhinichthys atratulus.
Sample sizes at specified power
levels
α=0.05,
β=0.05
α=0.05,
β=0.20
α=0.10,
β=0.10
Species
Collection
Date
Group
Coefficient
of Variation
(CV,%)
Northern
Redbelly Dace
May 27
All fish
57
134
81
89
2 year old
2–4g
56
44
126
79
76
48
83
52
June 22
All fish
2 year old
2–4g
75
44
57
237
157
134
143
95
81
156
104
88
May 20
All fish
2 year old
2–4g
24
15
20
24
9
17
15
6
11
16
6
11
June 11
All fish
2 year old
2–4g
41
56
52
71
134
104
43
81
63
47
89
69
Blacknose Dace
142
Table 4.6. Comparison of the relationship of log10 ovary weight to log10 body
weight between all female blacknose dace (Rhinichthys atratulus) with 2
year old fish and fish weighing 2 – 4 g 3 year old collected from Milkish
Brook on May 20, 2003.
Group
Regression Equation
R2
n
All fish
Y= 0.27x - 0.35
0.80
24
2 year old
Y= 0.18x – 0.20
0.94
8
2–4g
Y= 0.23x - 0.34
0.88
12
143
25
Water Temperature (°C)
20
15
10
5
0
May
June
July
Aug
Sept
Oct
Month
Figure 4.1. Mean monthly ambient temperature for Milkish Brook during the
study period. Upper bars depict monthly high temperatures and lower
bars depict low temperatures.
144
40
May 4
May 20
July 28
June 11
Aug 19
35
Percent (%)
30
25
20
15
10
5
0
2
3
4
2
3
4
2
3
4
5
2
6
2
6
Egg stage
Figure 4.2. Percentage of each oocyte occurrence (except stage 1, primary
oocytes) in female blacknose dace Rhinichthys atratulus caught in
Milkish Brook. Dark grey bars represent stage 2 oocytes; light grey
bars represent stage 3 oocytes; white bars represent stage 4 oocytes;
bars with a diamond-grid represent stage 5 oocytes; bars with
horizontal-grid represent stage 6 oocytes.
145
30
June 9
June 22
Aug 29
25
Percent (%)
20
15
10
5
0
2
3
4
2
3
4
2
Egg stage
Figure 4.3. Percentage of each oocyte occurrence (except stage 1, primary
oocytes) in female golden shiner Notemigonus crysoleucas caught in
Little Chamcook Lake. Dark grey bars represent stage 2 oocytes; light
grey bars represent stage 3 oocytes; white bars represent stage 4
oocytes.
146
60
May 27
50
June 12
June 22
Aug 27
Percent (%)
40
30
20
10
0
2
3
4
2
3
4
2
3
4
5
6
2
6
Egg stage
Figure 4.4. Percentage of each oocyte occurrence (except stage 1, primary
oocytes) in female northern redbelly dace Phoxinus eos caught in
beaver pond in Keswick River. Dark grey bars represent stage 2
oocytes; light grey bars represent stage 3 oocytes; white bars represent
stage 4 oocytes; bars with a diamond-grid represent stage 5 oocytes;
bars with a horizontal-grid represent stage 6 oocytes.
147
45
May 8
June 20
July 28
Aug 28
40
35
Percent (%)
30
25
20
15
10
5
0
2
3
4
2
3
4
5
2
6
2
6
Egg stage
Figure 4.5. Percentage of each oocyte occurrence (except stage 1, primary
oocytes) in female mummichog Fundulus heteroclitus caught in a salt
marsh located adjacent to Taylor's Island. Dark grey bars represent
stage 2 oocytes; light grey bars represent stage 3 oocytes; white bars
represent stage 4 oocytes; bars with a diamond-grid represent stage 5
oocytes; bars with a horizontal-grid represent stage 6 oocytes.
148
CHAPTER 5.
GENERAL DISCUSSION
The previous chapters have presented the results of my research designed
to assess the suitability of various fish species for environmental monitoring
programs. Overall, my research hypothesis was to determine whether fish can
be used to assess the relative contribution of individual anthropogenic stressors
to the existing environmental conditions in a river exposed to multiple
anthropogenic stressors.. More specifically, the objectives of my thesis were to
compare the whole-organism responses of small-bodied fish (i.e., slimy sculpin
and blacknose dace) and large-bodied fish (i.e., white sucker and yellow perch)
along a downstream gradient in a river exposed to pulp and paper mill effluents,
municipal sewage wastewater, and agricultural runoff (i.e., manure); identify the
fish species that is best suited to assess the relative contribution of individual
anthropogenic stressors in a river exposed to multiple anthropogenic stressors;
determine which life history characteristics most influenced the ability of a fish
species to exhibit the measured whole organism responses; and provide
sampling guidance for the use of multiple spawning, small-bodied fish species for
use in environmental monitoring programs.
The first part of my thesis focused on comparing the whole-organism
responses of white sucker and slimy sculpin collected downstream of pulp and
paper mill effluents, municipal sewage wastewater, and agricultural inputs on the
Saint John River near the city of Edmundston in northwest New Brunswick.
Results from the study showed that white sucker and slimy sculpin exhibited
differences in whole-organism responses, despite being collected from the same
149
sites. Slimy sculpin collected downstream of the municipal sewage discharges
and pulp mill effluent had greater growth, condition, and liver size and exhibited
no significant differences in gonad size relative to sculpin collected from
upstream reference sites.
In some monitoring situations, there is a lot of concern from stakeholders
that the measured whole-organism responses of fish do not reflect site-specific
environmental conditions. Since the receiving environment near Edmundston is
complex, establishing the residency patterns and home ranges of slimy sculpin
was an important issue to address. The stable carbon (δ13C) and nitrogen (δ15N)
isotope data showed that slimy sculpin collected from the Saint John River near
Edmundston had small home ranges (< 500 m), and the measured whole
organism responses reflected local conditions. In addition, stable carbon (δ13C)
and nitrogen (δ15N) isotope values of sculpin collected downstream of the pulp
mill, paper mill, and municipal sewage showed these sites were isotopically
enriched relative to upstream reference sites located at Clair and St. Hilaire. The
pulp mill and paper mill are located on opposite sides of the Saint John River, in
Canada (New Brunswick) and the United States (Maine), respectively. Sculpin
collected downstream of the paper mill showed no significant differences in
length, body weight, age, condition factor, liver size, and gonad size compared to
fish from upstream reference sites. Interestingly, the sculpin collected
downstream of the paper mill had unique stable isotope signatures relative to
sculpin collected immediately across the river and downstream of the pulp mill
showing sculpin were not moving across the river either. Gray [2003], also
150
showed slimy sculpin exhibit a high degree of spatial and temporal residency
within watercourses of New Brunswick using PIT tags and stable isotopes.
It was also surprising that the relative liver sizes of white sucker collected
from sites located downstream of the pulp mill effluent, upstream of the pulp mill
effluent, and the reference site located at St. Hilaire on the Saint John River did
not appear to be affected. However, with further analyses, white sucker liver size
was greater relative to white sucker collected from First Lake and Ogilvie Lake
(two pristine New Brunswick lakes near Edmundston). The LSI of white sucker
from the two New Brunswick reference lakes were similar to LSI values for white
sucker collected from reference sites in northern Ontario, and white suckers from
the Saint John River had livers similar in size to those of white sucker collected
downstream of a sulfite pulp mill in Kapuskasing, northern Ontario [Munkittrick et
al. 2000]. The increase in white sucker liver size may be reflective of the normal
situation for the Saint John River or it could be due to one or more unidentified
upstream source(s) of contamination (i.e., poultry processing facility). On the
other hand, the white sucker liver sizes could have been associated with the
mobility of the species in this river system. It was apparent that follow-up work
was required to help determine among these possibilities and whether the
responses persisted.
One of the most challenging, controversial, and criticized aspects of
environmental monitoring programs is any attempt to determine the ecological
151
relevance of the measured changes in a particular set of parameters. The initial
fish collections for my thesis were part of a larger effects-driven cumulative
effects assessment (CEA) of the Saint John River basin. The overall goal of the
effects-driven CEA is to help identify both natural factors (e.g., water
temperature, resource competition, etc.) and anthropogenic factors (e.g., pulp
mills, municipal sewage, hydro dams, etc.) that may enhance or limit fish
survival, growth, and reproduction and determine whether the current situation is
sustainable [Munkittrick et al. 2000]. As such, white sucker and slimy sculpin
performance was used as baseline data in an initial attempt to identify
ecologically relevant changes (i.e., magnitude of changes) in fish energy storage
and utilization for the upper Saint John River and would be important for
understanding the potential of future stressors to impact local fish populations. In
addition, the patterns of whole-organism responses could help identify the
potential cause(s) of the measured responses [Gibbons and Munkittrick 1994;
Munkittrick et al. 2000]. Since baseline fish performance data for the upper Saint
John River near Edmundston were not available, fish performance data from the
national pulp and paper EEM program were used to help put into context the
relative magnitude of the differences in fish performance downstream of the
municipal sewage (i.e., DS Mad) and pulp mill (i.e., DS Pulp) relative to fish
collected at reference sites located upstream of Edmundston. Intra- and interannual differences in the magnitude of fish performance responses are
presented in Table 5.1. The magnitude of responses and changes in the
patterns of the measured responses outlined in Table 5.1 provided invaluable
152
information regarding the initial assessment of fish performance in the upper
Saint John River near the city of Edmundston and will be discussed in more
detail in the following paragraphs.
Except for male GSI, sculpin collected downstream of municipal sewage
and pulp mill effluent discharge points in October 1999 exhibited increases in
condition, LSI, and GSI (Table 5.1). During this same time, male sculpin
collected downstream of the sewage and pulp mill effluent exhibited a small
decrease in GSI, but female sculpin exhibited an increase in GSI and this was
more evident for fish collected downstream of the pulp mill effluent (Table 5.1).
Some of the measured responses in sculpin downstream of the municipal
sewage and pulp mill effluent were very large relative to the average national
responses seen in the pulp and paper EEM program [Munkittrick et al. 2002;
Lowell et al. 2004]. The largest difference observed for LSI in female sculpin
collected downstream of the pulp mill occurred in 1999; mean LSI values went
from 1.14 ± 0.13 at St. Hilaire to 2.47 ± 0.33 downstream of the pulp mill, a 117%
increase, and is close to the largest measured responses reviewed in Cycle 2
and 3 of the EEM program [Munkittrick et al. 2002; Lowell et al. 2004]. The
largest measured difference in condition factor of female sculpin downstream of
the pulp mill occurred during the fall of 2000; mean condition factor went from
1.03 ± 0.02 at St. Hilaire to 1.18 ± 0.05 downstream of the pulp mill, a 15%
increase, also a large increase within the context of the EEM program. One of
the most interesting observations was in sculpin collected downstream of
153
municipal sewage wastewater. Except for female GSI, male and female sculpin
collected downstream of the sewage showed very large increases in condition
and liver size relative to reference fish and fish exposed to pulp mill effluent. In
fact, the increases in condition factor for sculpin collected downstream of the
sewage were greater than any of the measured responses outlined in the
national review of the Cycle 2 and Cycle 3 pulp and paper EEM program
[Munkittrick et al. 2002; Lowell et al. 2004].
Consistency in the patterns of the measured whole-organism responses
between species and between sexes is also important to consider. Male and
female sculpin collected downstream of the sewage and pulp mill effluent in
March 2002 (a few weeks before spawning) showed concurrent increases in
condition, LSI, and GSI which were also noted in female sculpin collected
downstream of the pulp mill in December 1999 (Table 5.1). Increases in
condition and LSI with concomitant increase in both male and female GSI have
also been noted in spoonhead sculpin and longnose sucker collected
downstream of a mill in Hinton, Alberta; yellow perch in Cabano, Quebec; and
white sucker in Jonquieres, Quebec and reflects an increase in food availability
downstream of the discharges, consistent with nutrient enrichment [Gibbons et
al. 1998; Munkittrick et al. 2002]. Inconsistent responses in fish parameters
reflecting energy use and storage (i.e., condition, LSI, GSI) can indicate
metabolic disruption. For example, the pattern of responses for male sculpin
collected downstream of sewage and pulp mill effluent in October 1999 has also
154
been noted at another Canadian pulp mill [Munkittrick et al. 1994]. This response
pattern has also been referred to as a “Jackfish Bay” type of response or
metabolic disruption as fish seem to access enough food but show decreases in
gonad size [Gibbons and Munkittrick 1994; Munkittrick et al. 2000]. It is the most
common response pattern observed in the fish population surveys for the
national pulp and paper EEM program [Environment Canada 2003]. Collecting
sculpin in late summer-early fall could lead some investigators to erroneously
conclude that metabolic disruption may be an issue in fish exposed to sewage
and pulp mill effluent in Edmundston.
Slimy sculpin show a different pattern of reproductive development than
many other Canadian spring-spawning species. Gonadal recrudescence does
not initiate in females until very late fall, and continues throughout the winter
period [Gray 2003]. When sculpin were collected closer to the spawning season
(December 1999) and just a few weeks prior to the spawning season (March
2002) the responses of both male and female sculpin were the same (i.e.,
increased condition, LSI, and GSI) and suggested that fish were living in nutrientrich environments that promoted growth. These responses patterns also
suggested that sculpin were not investing energy into reproduction during the late
summer – early fall and collecting fish during these times would provide no
suitable reproductive information.
For future studies with slimy sculpin in the upper Saint John River near the
city of Edmundston, sampling should be done in late fall – spring when gonadal
155
development has sufficiently progressed such that all reproductive parameters
can be measured. It would also be more likely to detect potential reproductive
impacts during a time of the year when the fish is investing increased energy into
reproduction. However, the availability of a spring, prespawning period varies
from year to year, dependent on the pace of snow melt and the timing of the start
of the spring freshet.
Together, data from my initial fish survey and the fathead minnow (P.
promelas) flow-through bioassay [Parrot et al. 2003] and microcosm flow-through
invertebrate exposure [Culp et al. 2003] suggested that the improved energy
storage and utilization of aquatic biota exposed to pulp mill effluent near the city
of Edmundston was related to increased nutrients.
In contrast, male and female white sucker consistently exhibited
decreases (except male LSI and GSI in October 1999) in the measured wholeorganism responses and the magnitude of the differences varied between years
(Table 5.1). Decreases in all three parameters have also been measured in
silver redhorse sucker in Kingsey Falls, Quebec; shorthead redhorse sucker in
Trenton, Ontario; yellow perch in Nackawic, New Brunswick; and rock bass in
Shawinigan, Quebec [Munkittrick et al. 2002]. A decrease in condition, liver size,
and gonad size indicates decreased food availability, but it is not possible to
determine from the initial data which cases have reduced food at the exposure
site, versus those with increased food at the reference site [Munkittrick et al.
156
2002]. Future studies would be required to determine whether these responses
persisted; if the response pattern persisted then an alternative reference site
would be selected and the benthic invertebrate community would be examined in
detail [Munkittrick et al. 2002].
Overall, the results from my initial study showed slimy sculpin collected
downstream of municipal sewage and pulp mill effluent were living in a nutrientrich environment that facilitated growth and indicate the slimy sculpin is a suitable
fish species for monitoring complex receiving environments. The responses of
slimy sculpin and white sucker differed, and were perhaps in relation to
differences in life history characteristics. In my initial attempt to identify
ecologically significant changes in fish performance for the upper Saint John
River, I showed that the differences downstream of the sewage and pulp mill
discharges were quite large relative to what has been observed other sites in
Canada receiving pulp mill effluent and can be outside of what would be
considered normal for slimy sculpin in New Brunswick.
Although white sucker and slimy sculpin showed different whole-organism
responses, I wanted to determine whether other large-bodied and small-bodied
fish would show similar responses. Would white sucker and yellow perch show
similar responses? Would the responses of slimy sculpin and blacknose dace be
similar? Would the white sucker and slimy sculpin results be consistent between
years? The second objective of my thesis was to deal with these issues (see
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Chapter 3). In this study, I attempted to clarify local impacts on wild fish, expand
fish collections to include other species to examine potential species differences
in responses, continue to de-couple other confounding factors at this study area
using less mobile small-bodied fish, and contribute to furthering the scientific
understanding of the suitability of small-bodied fish species for environmental
monitoring programs. I compared the responses of blacknose dace and slimy
sculpin (small-bodied fish species) with yellow perch and white sucker (largebodied fish species) to determine whether the responses of small-bodied fish and
large-bodied fish would be different.
The general response patterns of slimy sculpin were consistent with my
previous study and provided further evidence of an overall increase in energy
storage and utilization via a nutrient enrichment effect. Male and female sculpin
exposed to pulp mill effluent were longer, heavier, and were in better condition
and showed increased length-at-age relative to reference fish. Similar to my
previous study, male and female sculpin exposed to pulp mill effluent exhibited
no significant site differences in gonad size relative to reference fish. Slimy
sculpin invest most of their energy into gonad development during the winter
when the river is ice-covered, water temperatures and flow are low, and pulp mill
effluent concentrations are elevated. The absence of site differences in March
indicated that pulp mill and sewage effluent exposure did not adversely affect
sculpin gonad development. Similar to sculpin, blacknose dace showed larger
livers (both sexes) downstream of the pulp mill, relative to the reference site, but
158
these differences were also apparent upstream at the sewage sites and could not
be attributed to the pulp mill effluent. Overall, the magnitude of the responses of
male and female blacknose dace collected downstream of the sewage and pulp
mill effluent varied but some were large (e.g., 91 % increase in male LSI
downstream of the pulp mill) (Table 5.1).
Sculpin living downstream of the pulp mill had unique 13C signatures
relative to fish at the reference site located downstream of the St. Francois River
(i.e., “Capone”) and fish downstream of municipal sewage (i.e., D/S Mad)
indicating a carbon-enriched environment. The stable isotope results from this
study were similar to results from my previous study and provided conclusive
evidence that sculpin movements in the Saint John River near the city of
Edmundston were limited and the integrated response patterns were sitespecific. Another interesting finding of this study was that sculpin exposed to
municipal sewage wastewater had a distinct 15N signature relative to fish
collected at a reference site located downstream of the St. Francois River (i.e.,
“Capone”) and downstream of the pulp mill effluent diffuser, but not fish captured
upstream of the pulp mill. If sculpin captured downstream of the pulp mill were
moving to upstream sites then 15N signatures should have been similar, but this
was not the case. In fact, the stable 15N signatures of sculpin collected
immediately downstream of the sewage and at a site located immediately
upstream of the pulp mill effluent diffuser showed that these fish were feeding at
a higher trophic level relative to pulp mill effluent exposed fish and reference fish.
159
The results suggest that nutrient enrichment from the municipal sewage is so
great that sculpin are able to attain a body size that would allow them to feed at a
higher trophic level relative to other sculpin populations in the vicinity.
Furthermore, the stable isotope results support other studies [Wassenar and
Culp 1996; Wayland and Hobson 2001] that have shown sites receiving pulp
mill effluent and municipal sewage wastewater can have distinct stable isotope
signatures relative to upstream sites and can be used to trace the geographical
extent of specific wastewater effluent streams.
White sucker and yellow perch showed few site differences in any of the
measured whole-organism parameters and the responses were not consistent
with slimy sculpin or previous work done at these sites, and suggested white
sucker and yellow perch were not as sensitive as sculpin for picking up changes
within a limited reach of the river. The decreases in white sucker liver size
downstream of the pulp mill outfall were puzzling; male and female LSIs were
reduced at the pulp mill site by 24% and 27%, respectively. I could not exclude
the possibility that the larger-bodied species did not mix between the south and
north shores of the Saint John River (about 50 m at the section near
Edmundston) or other areas immediately upstream or further downstream of the
mill diffuser. The white muscle stable 13C and 15N isotope ratios reported show
that white sucker collected downstream of the pulp mill effluent diffuser were not
mixing with fish collected at the upstream reference site located at St. Hilaire.
Doherty [2003], using radio telemetry, showed white sucker movements in the
160
middle section of the Saint John River were limited outside the spawning season
and tagged fish had a winter home range of < 2.5 km. If white sucker in the
upper portion of the river near Edmundston also moved ≤ 2.5 km then it is likely
that the fish were moving between the north and south sides of the river with the
possibility of some limited upstream and downstream movements. Thus, the
measured sucker responses were indicative of the environmental conditions
within in a much larger section of the river (i.e., potentially a 2.5 km radius from
the capture site) and not those immediately downstream of the pulp mill effluent
diffuser and within about 50 m of the shore, which the sculpin reflected.
Yellow perch males showed increased liver size and condition factor, but
no other significant differences were present in males or females. Except for
female GSI, male and female yellow perch whole-organism responses indicated
a nutrient rich-environment downstream of the pulp mill (Table 5.1); these fish
were collected in a deep pool in a backwater eddy downstream of the diffuser.
Stable 13C and 15N isotope signatures in blacknose dace and yellow perch did
not show as much isotopic separation as white sucker and slimy sculpin
suggesting their prey items may not be incorporating pulp mill effluent derivedcarbon into their diet. Future work will be needed to better understand the
influence of pulp mill effluent and municipal sewage wastewater on the local food
web.
161
Taken together, the results from chapters 2 and 3 suggest white sucker
and yellow perch may not be the most suitable species for monitoring multiple
wastewater effluents discharged in close proximity, such as the situation near the
city of Edmundston. However, as a result of their life history characteristics (e.g.,
trophic position, mobility) white sucker and perch are probably the fish species
that best integrate all of the stressors (both natural and anthropogenic) in this
section of the Saint John River. Ultimately, the best choice of fish species to be
use in a monitoring program will depend on the question(s) that need to be
addressed and the guidance that can be provided. If, for example, a researcher
wants to examine the impacts of multiple effluents discharged in close proximity
within a limited river reach on fish performance where mobility is an issue, then
small-bodied fish (e.g., cottids) are the best candidate species. These species
exhibit high site-fidelity and are able to show changes in whole-organism
responses that reflect localized conditions, within a limited reach of the river.
However, if the geographic scale of a study is expanded and the objective is to
assess the integrated impacts of stressors on fish performance within a
watershed then the use of large-bodied fish species (e.g., Catostomids) would be
more appropriate.
The most challenging aspect of this study was incorporating an additional
small-bodied fish species into the field program. Many small-bodied fish species
are particularly well suited for monitoring complex aquatic receiving environments
since they are presumed to be less mobile, easily captured, and more abundant
162
than large-bodied fish species. However, the main disadvantage of using smallbodied fish species for monitoring programs (e.g., pulp and paper and metal
mining EEM programs) is the lack of basic life-history information. In general,
there is a paucity of detailed reproductive information for many small-bodied fish
that can be used in the pulp and paper and metal mining EEM programs. This is
troublesome; many metal mines in Canada are located in remote locations and
discharge into small headwater streams where many of these fish species live
and will be the only sentinel species available. It will be difficult to design studies
and interpret data, and field costs will be higher using small-bodied fish when
sufficient basic biological information is absent. For this study, I consulted Scott
and Crossman (1998) and other scientific literature via searchable databases
(e.g., Aquatic Sciences and Fisheries Abstracts, Science Citation Index) to try
and determine the most suitable time to collected pre-spawning blacknose dace
in the upper Saint John River near Edmundston. However, none of these
information resources provided any detailed reproductive information for the
region and generically suggested that spawning occurred from May to June when
water temperatures reached about 21°C. Pre-spawning female blacknose dace
were collected from river sites near Edmundston during the first week of June
2002 and showed increased variability in gonad development relative to that
seen in sculpin. Smaller female blacknose dace showed increased variability in
gonad development relative to the larger females. The variability was not due to
differences in sampling time since all fish were collected during the same period.
It was difficult to determine the exact reason why female dace showed this
163
variability in gonad development during the pre-spawning period since there was
no relevant biological information available.
I knew that dace were capable of exhibiting site differences in some of the
measured whole-organism parameters, but I wanted to know why female dace
exhibited increased variability in gonad development. As a result of this
research, a number of interesting follow-up questions were generated, including:
Do blacknose dace spawn once or multiple times during the reproductive
season? Do other small-bodied, multiple spawning fish exhibit the same
variability in gonad development during the pre-spawning period? Is there a
better time of the year to sample these fish in order to reduce variability? These
questions were addressed in chapter 4.
The objectives of chapter 4 were to collect basic biological information for
multiple spawning, small-bodied fish species and provide sampling guidance for
their use in future environmental monitoring programs. When using multiple
spawning fish in monitoring programs, Environment Canada recommends that
sampling should be conducted in the prespawning period prior to the onset of the
first spawn of the year so that potential complications associated with differences
in synchronicity of spawning between groups of fish can be avoided
[Environment Canada, 1997]. At this time, it was assumed that development
would be synchronized between individuals, and if changes were not evident
prior to the first spawn, the consequences of small changes later in the season
164
would be less important. To address the issues associated with using multiplespawning, small-bodied fish in monitoring programs I examined seasonal
changes in condition factor, liversomatic index (LSI), gonadosomatic index (GSI),
and ovarian histology in blacknose dace, northern redbelly dace, golden shiner,
and estuarine mummichog. I was also interested in assessing seasonal changes
in the ovary weight to body weight relationship (i.e., coefficient of determination
[r2] values for linear regressions equations) of female fish so that I could have a
better understanding of seasonal variability in gonad development and whether
different size classes of female fish showed similar seasonal variability. More
specifically, I attempted to identify the time of the year when the relationship
between ovary weight and body weight was most variable (i.e., low r2 values);
increase our understanding of natural source(s) of variability in gonad
development; identify suitable techniques to minimize data variability; and reduce
sample size requirements for detecting a critical effect size when designing costeffective studies in the future.
Data from this study provided a number of interesting findings. Overall, I
found that all the fish species I collected have extended spawning seasons
(spring-summer) and the suite of indices measured provided sufficiently detailed
information about the seasonal change in energy use and storage, particularly as
it relates to gonad growth. Oocyte development in the four fish species was
asynchronous and there were considerable differences in the patterns of ovarian
maturation between northern redbelly dace and the other fish species collected.
165
Oocyte development also differed between individuals of a species. For
blacknose dace, the first spawning occurred in early June when mean water
temperatures reached about 15.0°C (min.-max: 10.3 to 21.6°C). Interestingly,
female blacknose dace appeared not to rely solely on liver energy stores when
the maximum increases in gonad size occurred (i.e., 45% increase from May 4 to
May 20) and may rely on energy derived from active feeding. Future studies will
be needed to clarify this issue.
It was evident that the strength of the gonad size – body weight
relationship varied during the season for each species and that some fish
species showed much more seasonal variability in this parameter relative to
others and suggested that the timing of collections is very important when using
these fish species in monitoring programs. The most striking example of
seasonal variability in ovary development was that of northern redbelly dace.
Gonad development in this species was highly variable throughout the
reproductive season relative to the other species. Histology was an important
tool that helped me better understand the source of this variability. Ovaries of
northern redbelly dace contained oocytes in all developmental stages; as such,
there appeared to be no synchronicity in the timing of spawning events among
individuals, which would account for the high degree of variability observed
throughout the season. During the peak of the northern redbelly spawning
season (i.e., May – June), female GSI values ranged from < 1% to > 20%. It was
evident that seasonal variability in ovary development was higher in some
166
species (i.e., northern redbelly dace) relative to other species (i.e., blacknose
dace) and that timing of sample collections would be very important to take into
consideration.
Due to poor weather conditions and/or poor river conditions, it is
sometimes difficult to collect fish during the most optimal time of the year to
assess potential reproductive impacts associated with a particular anthropogenic
stressor. One of the key objectives of this study was to have a better
understanding of some of the basic biology of the collected fish species, attempt
to facilitate their use in environmental monitoring programs, and reduce sample
size requirements for future studies. In order to do this, I identified sources of
data variability and selected basic techniques that I hoped could be employed to
reduce some of the data variability and determine the sample sizes required in
order to detect a specified critical effect size (i.e., 25%) in gonad size between a
reference and exposure site at a specified power level. I chose a critical effect
size of 25% because the Canadian Federal government regulations require pulp
and paper mills and metal mining operations to design and implement monitoring
programs capable of detecting a 25% difference in gonad size between an
exposed site and reference site [Environment Canada 1997, 2002]. By selecting
a specific age class or size class of fish I was able to reduce some of the data
variability associated with seasonal gonadal development, which helped to
substantially reduce sample size requirements for future studies with some fish
species (i.e., blacknose dace) during certain times of the year, but it was
167
apparent that these techniques were not as suitable for other species (i.e.,
northern redbelly dace). The sample size required to detect a 25% difference in
gonad size in female blacknose dace was much smaller during the pre-spawning
period in May when compared to late June when the r2 was < 0.45. More
specifically, on May 20 the coefficient of variation (CV) was about 24% and a
sample size of 16 to 24 fish was required to detect a 25% difference in gonad
size when α and β were 0.05 and 0.20, and when both α and β equalled 0.10
(Figure 5.1). On June 11, however, the CV for all female blacknose dace data
increased to about 41%, as such, future studies during this time period would
require sample sizes of 47 to 71 (depending on the power level) (Figure 5.1). I
was able to reduce the variability (and sample sizes) by selecting only fish of a
similar age or body size, but this only worked for certain times of the year and not
all species. When 2 year old female blacknose dace were selected only, the CV
was reduced to about 15% on May 20 and less than 10 fish per site were
required to detect a 25% difference in gonad size (Figure 5.2). In contrast,
selecting 2 year female blacknose dace on June 11 actually increased the
variability (CV = 56%) and greater sample sizes were required (Figure 5.2).
Selecting only 2-4 g female blacknose dace on May 20 reduced some of the
variability (CV = 20%), but not as much as selecting only 2 year old fish;
nonetheless, sample sizes were reduced (Figure 5.3). Again, this was not the
case on June 11 when selecting 2-4 g female blacknose dace increased
variability (CV = 52%) and sample sizes requirements (Figure 5.3).
168
Since female northern redbelly dace showed the most seasonal variability
in gonad development I wanted to determine whether these techniques could
substantially reduce the sample size requirements for this species. On May 27,
the CV for all female northern redbelly dace was 57% and sample sizes between
81-134 fish per site were required to detect a 25% difference in gonad size
(Figure 5.4). On June 22, variability in gonad development was higher and even
larger sample sizes were required (Figure 5.4). Unfortunately, using only 2 year
old or 2 – 4 g female northern redbelly dace on May 27 or June 22 did not
substantially reduce data variability. The CV for 2 year old female northern
redbelly dace captured on May 27 was 56% and sample size requirements were
83-126 fish per site (Figure 5.5). The CV for 2-4 g fish was reduced to 44%, but
52-79 individuals per site were still required (Figure 5.6). Regardless of the
technique, large sample sizes were still required on June 22 for 2 year old
(Figure 5.5) and 2 – 4 g (Figure 5.6) female northern redbelly dace. Taken
together, results for female northern redbelly dace suggest that this species is
not a suitable sentinel fish species for environmental monitoring programs, at
least in southern New Brunswick. Correlation coefficients of under 0.4 mean that
sample sizes are very high. Increased seasonal variability in gonad development
would make it too difficult to detect potential reproductive impacts associated with
contaminant exposure. In addition, it has been well documented that northern
redbelly dace readily hybridize with other species, which would also confound
data interpretation.
169
Addressing the female blacknose dace variability issue noted in chapter 3
was important. The variability in gonad development in small female dace noted
in Chapter 3 is likely due to the timing of sampling (variability in female gonad
development increased as the spawning time approached and we sampled
blacknose dace between June 11 to 14; the first week of the spawning season)
and the presence of sexually immature fish and/or first time spawners, which can
hinder data analysis. Young slimy sculpin [Brasfield 2004] and trout-perch
[Gibbons et al. 1998] spawning for the first time also show increased variability in
gonad development leading these authors to conclude that their inclusion would
obscure interpretation of results.
Most importantly, multi-spawning, small-bodied fish can be successfully
used in environmental monitoring programs, but additional basic biological
research may be required in order to continue to facilitate their use. The
Canadian EEM program uses the variability in gonadal size to determine sample
size requirements for sampling. It is important to have high correlations between
gonad weight and body weight to reduce the potential impacts of sampling on the
population. Variability is increased in multiple spawning fish species because:
a)
Younger fish of some species only spawn once, versus multiple
times in older fish, inflating variability. The variation can be
reduced in some species by selecting restricted size ranges or
ages of fish to increase power.
b)
The first spawn of the season in most species is synchronized,
170
probably by spawning temperature. However, the timing of the
second spawn is not, and variability increases as spawning time
approaches because of the size of the asynchronicity of the
second clutch of eggs. Variability can be reduced by sampling
very early in gonadal development.
c)
Some multiple spawning species may have too much variability
(r2< 0.4) throughout the spawning season. This may be due to
asynchronicity of the first spawn, or the potential for some
individuals to not spawn every year. Species with low
correlations should be avoided for reproductive investigations
until we develop a better understanding of the biological basis for
the variability.
5.1
Conclusions
The differences in life-history characteristics of fish species are important
when choosing a sentinel species since these characteristics will influence the
sensitivity and/or tolerance of the species to contaminant exposure. For the
upper Saint John River, large-bodied species (e.g., white sucker) are best suited
for assessing the overall health at a watershed or reach scale since their
responses reflect a relatively wider geographical scale. Small-bodied fish
species (e.g., sculpin) are best suited to assess the relative contribution of
individual effluents in sections of rivers receiving multiple effluents discharged in
171
close proximity. The biggest challenge of using small-bodied fish in
environmental monitoring programs is the paucity of basic biological information
for both single and multiple spawning species. It is also evident from this thesis
that basic biological information can significantly reduce the numbers of fish
required for the EEM program while still collecting all the relevant biological
information.
Future research will need to focus on assessing the impact of sewage and
pulp mill effluent on local food webs. Some of the responses of sculpin exposed
to municipal sewage near Edmundston were larger than any of the responses
associated with pulp mill effluents across Canada and will need to be
investigated in more detail. Detailed studies on the life history of large bodied
fish in the upper Saint John River near Edmundston should be initiated and the
number of collection sites should be expanded in order to better understand and
continue to identify natural and anthropogenic stressors which were causing the
measured responses (or lack thereof in the case of white suckers) observed in
this thesis. Continuing to collect detailed life history information for small-bodied
fish will be particularly important when designing fish studies for the Federal
metal mining EEM program. In Canada, most metal mines are located in remote
areas and discharge into small, headwater streams that have limited fish
populations and fish diversity. Large-bodied fish are not available in many of
these locations and lethal sampling of large numbers of fish may posed a serious
threat to the very local fish community that the EEM is designed to protect.
172
Continued research on developing non lethal techniques and community surveys
and data interpretation guidance for the use of small-bodied fish species would
be particularly beneficial for metal mining studies. Finally, research partnerships
between government, academia, and industry need to continue and expand so
that we can continue to better understand the impacts of complex industrial and
municipal wastewater effluents and agricultural activities on the health of aquatic
biota. These partnerships will not only allow us to continue to identify previously
unknown areas (and contaminants) of concern, but will also facilitate the
development and implementation of socially acceptable and economically viable
mitigation strategies.
5.2
References
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short-lived fishes: what Cottus has taught us. Poster presentation at 31st
Annual Aquatic Toxicity Workshop, October 24-27, Charlottetown, PEI.
Culp,J.M., Cash, K.J., Glozier, N.E., and Brua, R.B. 2003. Effects of pulp mill
effluent on benthic assemblages in along the Saint John River, Canada.
Environ Toxicol Chem 22: 2916-2925.
Doherty, C.A., R.A. Curry and K.R. Munkittrick. 2003. Tracking adult white
sucker movements near point source discharges in the Saint John River,
New Brunswick, Canada. In Borton, D.L., Hall, T.J., Fisher, R.P., &
Thomas, J.F., (eds), Pulp & Paper Mill Effluent Environmental Fate &
Effects, June 1-4, Seattle, WA, pp. 123 - 132.
Environment Canada. 1997. Fish Survey Expert Working Group:
173
Recommendations from Cycle 1 review. Ottawa ON: Environment Canada.
EEM/1997/6. 262p.
Environment Canada. 2002. Metal mining guidance document for aquatic
environmental effects monitoring. EEM/2002. June 2002.
Environment Canada. 2003. National assessment of pulp and paper
environmental effects monitoring data: a report synopsis. National Water
Research Institute, Burlington, ON. NWRI Scientific Assessment Report
Series No. 2.
Gibbons WN and Munkittrick KR. 1994. A sentinel monitoring framework for
identifying fish population responses to industrial discharges. J. Aquat.
Ecosyst. Health 3: 227-237.
Gibbons WN, Munkittrick KR, McMaster ME, Taylor WD. 1998. Monitoring
aquatic environments receiving industrial effluents using small fish species
2: Comparison between responses of trout-perch (Percopsis
omiscomaycus) and white sucker (Catostomus commersoni) downstream
of a pulp mill. Environ Toxicol Chem 17: 2238-2245.
Gray MA. 2003. Assessing non-point source pollution in agricultural regions of
the upper St. John River basin using the slimy sculpin (Cottus cognatus).
PhD thesis, University of New Brunswick. Fredericton, NB, Canada.
Lowell R, Ring B, Pastershank G, Walker S, and Trudel L. 2004. National
assessment of data from the pulp and paper EEM program: cycle 3 and
comparisons to earlier cycles. Pulp and paper EEM detailed information
session, November 25-26, Edmonton, AB, Canada.
174
Munkittrick KR, Van Der Kraak GJ, McMaster ME, Portt CB, van den Heuvel MR,
Servos MR. 1994. Survey of receiving water environmental impacts
associated with discharges from pulp mills. 2. Gonad size, liver size,
hepatic EROD activity, and plasma sex steroid levels in white sucker.
Environ Toxicol Chem 13:1089–1101.
Munkittrick, K.R., McMaster, M.E., Van Der Kraak, G., Portt, C., Gibbons, W.N.,
Farwell, A., and Gray, M. 2000. Development of methods for effectsdriven cumulative effects assessment using fish populations: Moose River
Project. SETAC Press, Pensacola, FL, USA.
Munkittrick KR, McGeachy SA, McMaster ME, Courtenay SC. 2002. Overview
of cycle 2 freshwater fish studies from the pulp and paper Environmental
Effects Monitoring program. Water Quality Res J Can 37: 49-77.
Parrott, J.L., Wood, C.S., Boutot, P., Blunt, B.R., Baker, M.A., and Dunn, S.
2003. Fathead minnow long-term growth/reproductive tests to assess
final effluent from a bleached sulphite mill. Environ Toxicol Chem 22:
2908-2915.
Scott WB, Crossman EJ. 1998. Freshwater Fishes of Canada. Galt House,
Oakville, ON, Canada.
Wassenar LI, Culp JM. 1996. The use of stable isotope analyses to identify pulp
mill effluent signatures in riverine food webs. In Servos MR, Munkittrick
KR, Carey JH, Van Der Kraak GJ, (eds), Environmental Fate and Effects
of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA,
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Wayland M, Hobson KA. 2001. Stable carbon, nitrogen, and sulphur isotope
ratios in riparian food webs on rivers receiving sewage and pulp-mill
effluents. Can. J. Zool. 79: 5-15.
176
Table 5.1. Percent differences in condition factor (k), liversomatic index (LSI),
and gonadosomatic index (GSI) for slimy sculpin, white sucker, yellow
perch, and blacknose dace collected at various times from 1999-2003
(“% Difference” is for municipal sewage and pulp mill effluent exposed
fish relative to reference fish at St. Hilaire). (-) data not available; NS –
not significantly different.
Sex Species
Date
Parameter
M
October
1999
K
Slimy
Sculpin
December
1999
August
2001
March
2002*
F
Slimy
Sculpin
October
1999
December
1999
Municipal
Sewage
16
Pulp mill
Effluent
12
LSI
GSI
K
31
-8 (NS)
-
83
-2 (NS)
14
LSI
GSI
K
-
20 (NS)
7 (NS)
6
LSI
GSI
K
29
LSI
GSI
K
66
7 (NS)
17
LSI
GSI
K
23 (NS)
9 (NS)
-
LSI
GSI
September K
2000
LSI
GSI
August
K
2001
LSI
GSI
14 (NS)
15
46
3 (NS)
16
117 (NS)
23 (NS)
11
-
14
19 (NS)
13
-
-31 (NS)
-1.3 (NS)
5 (NS)
-
-4 (NS)
-
177
March
2002*
M
White
sucker
October
1999
October
2002
F
White
sucker
October
1999
October
2002
M
F
M
F
K
32
14
LSI
GSI
K
9 (NS)
5 (NS)
-
1 (NS)
7 (NS)
-3 (NS)
LSI
GSI
K
-
2 (NS)
20 (NS)
-3 (NS)
LSI
GSI
K
-
-24
-5 (NS)
-4
LSI
GSI
K
LSI
GSI
Yellow
September K
Perch
2002
LSI
GSI
Yellow
September K
Perch
2002
LSI
GSI
Blacknose June 2002 K
Dace
LSI
GSI
Blacknose June 2002 K
Dace
LSI
GSI
-
-5 (NS)
-8 (NS)
-0.8 (NS)
-21
-13 (NS)
7
17
29 (NS)
5 (NS)
2 (NS)
5 (NS)
-6 (NS)
2 (NS)
40
-26
-2 (NS)
91
23
0 (NS)
19 (NS)
-24 (NS)
25
-20 (NS)
178
300
1
250
Number of Fish
200
2
3
150
100
n=71
50
n=47
n=43
n=24
n=16
n=15
0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
CV (%)
Figure 5.1. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for all adult female blacknose dace collected on
May 20 (dashed lines) and June 11 (solid lines), 2003. The solid
curved line numbered “1” represents α = 0.05 and β = 0.05; the solid
curved line numbered “2” represents α = 0.10 and β = 0.10; and the
solid curved line numbered “3 represents α = 0.05 and β = 0.20.
179
300
1
250
Number of Fish
200
2
3
150
n=134
100
n=89
n=81
50
n=6
n=9
0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
CV%
Figure 5.2. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for 2 year old female blacknose dace collected
on May 20 (dashed lines) and June 11 (solid lines), 2003. The solid
curved line numbered “1” represents α = 0.05 and β = 0.05; the solid
curved line numbered “2” represents α = 0.10 and β = 0.10; and the
solid curved line numbered “3 represents α = 0.05 and β = 0.20.
180
300
1
250
Number of Fish
200
2
3
150
n=104
100
n=69
n=63
50
n=17
n=11
0
0
10
20
30
40
50
60
70
80
CV (%)
Figure 5.3. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for 2 – 4 g female blacknose dace collected on
May 20 (dashed lines) and June 11 (solid lines), 2003. The solid
curved line numbered “1” represents α = 0.05 and β = 0.05; the solid
curved line numbered “2” represents α = 0.10 and β = 0.10; and the
solid curved line numbered “3 represents α = 0.05 and β = 0.20.
181
90
300
1
250
n=237
Number of Fish
200
2
3
n=156
150
n=143
n=134
100
n=89
n=81
50
0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
CV (%)
Figure 5.4. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for all female northern redbelly dace collected
on May 27 (dashed lines) and June 22 (solid lines), 2003. The solid
curved line numbered “1” represents α = 0.05 and β = 0.05; the solid
curved line numbered “2” represents α = 0.10 and β = 0.10; and the
solid curved line numbered “3 represents α = 0.05 and β = 0.20.
182
85
300
1
250
Number of Fish
200
2
n=157
3
150
n=126
n=104
n=95
100
n=83
n=76
50
0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
CV (%)
Figure 5.5. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for 2 year old female northern redbelly dace
collected on May 27 (dashed lines) and June 22 (solid lines), 2003.
The solid curved line numbered “1” represents α = 0.05 and β = 0.05;
the solid curved line numbered “2” represents α = 0.10 and β = 0.10;
and the solid curved line numbered “3 represents α = 0.05 and β = 0.20.
183
85
300
1
250
Number of Fish
200
2
3
150
n=134
100
n=88
n=81
n=79
n=52
50
n=48
0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
CV (%)
Figure 5.6. Sample sizes required to detect a 25% difference in gonad size at
different levels of power for 2 – 4 g female northern redbelly dace
collected on May 27 (dashed lines) and June 22 (solid lines), 2003.
The solid curved line numbered “1” represents α = 0.05 and β = 0.05;
the solid curved line numbered “2” represents α = 0.10 and β = 0.10;
and the solid curved line numbered “3 represents α = 0.05 and β = 0.20.
184
80
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