Endocrine disruption in Smallmouth Bass (Micropterus
dolomieu) of the Ohio River Basin
Christopher Barry, Steven Foster
Methods
Fig. 1 – Setup for working up
samples in the field.
Smallmouth Bass
Sample
Collection
Fig. 2 - Electrofishing from
boat.
Sectioning &
Staining
◊ Fish were caught by electrofishing either from
boat for non-wadable streams or using
backpack electrofishing units for wadable
streams.
◊ Fish were either euthanized with Finquel MS222 (99.5% pure tricaine methanesulfonate ) or
sacrificed by severing the spinal cord.
◊ Fish were dissected to determine sex and
male gonads excised in the field.
◊ Testes were stored in formalin for
transportation to the lab.
Fig. 3 – Taking fish mass
data in the field.
Paraffin Embedding
Abstract
◊ tissue wash: 2 x 5 min. in 0.2M sodium
phosphate buffer, pH 7.4.
◊ dehydration series of ethanols:
50% -> 70% -> 90% -> 100% -> 100%
(10 min. ea.)
◊ clearing: 2 x 20 min. in Citrisolv© xylene
substitute.
◊ infiltration: 2 x 1 hr incubation in hot paraffin
◊ embedding: move to fresh hot paraffin and
allow to cool.
Endocrine disruption in Smallmouth Bass (Micropterus dolomieu) of the Ohio
River Basin
Christopher Barry, Steven Foster
The regular occurrence of endocrine disrupting chemicals in US surface waters, as well as their
observed effects in the native wildlife has only been described in the literature in the last 15 years. While
the full range of their effects and the mechanisms through which they act are still under investigation, one
remarkable endpoint for the detection of endocrine disruption in males is the so called intersex condition,
or presence of oocytes in the testes of normally heterosexual fish, also known as ovotestis. In this study,
we both present data on the occurrence of the intersex condition in smallmouth bass of the Ohio River
basin, as well as investigate a method for the quantification of the severity of intersex. Bass were
sampled in 2004 and 2005 from the Kanawha and Muskingum river basins, both tributaries of the Ohio
River, as well as the Back Fork of the Elk, Top Gauley, and West Fork of the Greenbrier River, 3 relatively
undisturbed reference sites in West Virginia. Both boat-mounted and backpack electrofishing equipment
were used for sampling, depending on the size of the river or stream. Fish were sexed via visual
inspection of the gonads upon dissection and the testes of males were excised in the field for histological
processing. Testes were formalin fixed, paraffin embedded, and stained with hematoxylin and eosin
(H&E). Using a method established in European Flounder (Platichthys flesus) by Bateman et al.(Env Tox
& Chem, 2004), the extent of the intersex condition was first quantified using the ovotestis severity index,
or OSI. The proportion of gonadal tissue comprised of oocytes was then quantified using digital image
analysis with ImageJ software. Examination via application of the OSI has revealed cases of intersex in
as many as 100% of fish sampled at one site. All other sites sampled, with the exception of one, showed
some fraction of smallmouth with intersex. Even the reference sites showed some occurrence of
intersex, though in significantly lower numbers. The investigation of the suitability of digital image
analysis using ImageJ shows a correlation between severity analysis by the OSI and severity analysis by
area calculations in ImageJ. Overall, the occurrence of this intersex condition is much more prevalent
than should be expected and must be taken as an alarming warning sign of needed revision of current
water quality management policies.
Conclusion
◊ sectioning: 10µm with an International
Equipment Co. rotary microtome.
◊ transfer sections to 42° C water bath
◊ lift sections from water bath with slide
◊ re-hydration series:
(for compatibility with aqueous stains)
100% -> 100% -> 90% -> 70% -> 50%
(1 min. ea.)
◊ hematoxylin staining: 1 min
◊ rinse:
10 min. in running tap water
dip in deionized water
◊ eosin staining:
- equilibrate 2 min. in 75% ethanol
- stain 2 min. in working strength
eosin solution
◊ final rinse: dip in deionized water
The occurrence of intersex fish at all sites sampled in 2005 was a
surprising finding and raises the question of whether or not intersex occurs
naturally at low levels. However, the findings of higher occurrence of
intersex at impacted sites was to be expected and shows a correlation
between water quality and intersex. The intersex analysis using ImageJ
provides an alternative method for quantifying the severity of ovotestis.
While not necessarily faster than the OSI method, this approach allows for
increased resolution, or a larger range of possible severity values.
The successful demonstration of the histological analysis method at
Marshall paves the way for further collaboration between state and federal
agencies for the continued study of the distribution of intersex condition in
West Virginia fish. This includes not only fish sampled specifically for the
study, but also the potential to work with archived museum specimens.
This information could be very useful in the search for a true reference site.
Can a significant number of samples from a population of fish be found to
demonstrate that a particular site had no occurrences of intersex? Or are
there always at least a small number of fish with this condition?
Mounting
◊ dehydration series:
(for compatibility with non-aqueous medium)
50% -> 70% -> 90% -> 100% -> Citrisolv ©
1 min. ea.
-> 5 min.
◊ mounting: + ~200 µL Fisher Permount ©
+ coverslip
Fig 7 – Backpack electrofishing
unit
gonad
Fig. 4 – Location of fish gonads relative to
other internal anatomy.
Fig. 8 – Intersex severity analysis in ImageJ
References
Individuals’ OSI scores
% Males with Intersex
% Males
with intersex
1) Bateman, K.S., Stentiford, G.D., Feist, S.W. A ranking system for the evaluation of intersex condition in European
flounder (Platichthys flesus). Environmental Toxicology and Chemistry 23:12. 2004 p.2831-2836.
100.00%
2.5
90.00%
80.00%
2)
Katherine E. Liney, Susan Jobling, Jan A. Shears, Peter Simpson, and Charles R. Tyler. Assessing the Sensitivity
of Different Life Stages for Sexual Disruption in Roach (Rutilus rutilus) Exposed to Effluents from Wastewater Treatment
Works. Environmental Health Perspectives 113:10. 2005 p.1299-1307.
2
60.00%
OSI
50.00%
1
30.00%
0.5
20.00%
10.00%
Individuals
Fig. 5 - Occurrence of intersex at each
site
Fig. 6 - Individuals’ OSI scores
Results
With the exception of one site on the Kanawha River, all sites sampled were found to have at least one fish
with intersex, including reference sites. However, the number of cases, and severity of intersex were
significantly higher at impacted sites. (see figs. 5 & 6)
The microscopic histological analysis was successfully demonstrated at Marshall using the fish from East
Lynn. Furthermore, the intersex analysis via area calculations in Image J yielded very similar results to
intersex severity analysis using the OSI.
MR3 Tuscarawas 7
MR3 Tuscarawas 4
MR3 Tuscarawas 1
MR2 Tuscarawas 9
MR2 Tuscarawas 2
MR1 Walhonding 17
MR1 Walhonding 7
MR1 Walhonding 4
WV3 West Fork of Greenbrier 30
WV3 West Fork of Greenbrier 28
WV3 West Fork of Greenbrier 26
WV3 West Fork of Greenbrier 23
WV3 West Fork of Greenbrier 21
WV3 West Fork of Greenbrier 17
WV3 West Fork of Greenbrier 15
WV3 West Fork of Greenbrier 13
WV2 Back Fork of Elk 19
WV2 Back Fork of Elk 12
WV2 Back Fork of Elk 9
WV2 Back Fork of Elk 6
WV3 West Fork of Greenbrier 7
Site
WV2 Back Fork of Elk 3
WV1 Gauley 11
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1
While endocrine disrupting chemicals (EDCs) in aquatic environments have come under
increasing scrutiny during the last decade, their mechanisms of action and effects are still largely
unknown. Therefore, even with more information on the presence and distribution of these
chemicals, it is difficult to predict how they will impact both human and wildlife communities. For this
reason, this study investigates the occurrence of an endpoint of endocrine disruption in smallmouth
bass, the intersex condition, or ovotestis. While this does not reveal any information on exactly
which EDCs are causing the endocrine disruption, it proves that the EDCs responsible are present
in sufficient concentrations to impact wildlife.
EDCs consist of a very large range of chemicals that interact with the hormonal regulation
systems of many animals. They can be human and veterinary pharmaceuticals, plasticizers,
pesticides, metals, and many other organic chemicals that exhibit reactions at very low
concentrations, especially with long term exposure. In this study, smallmouth bass were chosen as
the target species because of their widespread geographic distribution and ability to thrive in water
ranging from the small, wadable, relatively unimpacted reference streams to rivers as large as the
Ohio. In this study, the Muskingum and Kanawha drainages were chosen as representatives of
significantly impacted sites, while the Top Gauley, Back fork of the Elk, and West Fork of the
Greenbrier represent the smallest, relatively unimpacted sites which were still large enough to
support smallmouth populations. Fish from these sites were collected through collaboration with the
US ACE and then processed for histological analysis by the USGS. In addition, fish from East Lynn
Lake were collected in order to prove the histological method and analysis at Marshall.
3)
Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., Buxton, H.T. Pharmaceuticals,
Hormones, and Other Organic Wastewater Contaminants in U.S. Streams, 1999-2000: A National Reconnaissance.
Environmental Science and Technology 36. 2002 p.1202-1211.
40.00%
0.00%
Introduction
1.5
WV1 Gauley 3
% Males with intersex
70.00%
4)
Johnson, M.J. Hall, L.C. The estrogenicity of selected herbicides and adjuvants endocrine disruption capabilities
of surflan and oryzalin. A report prepared for the division of environmental analysis, California Dept. of Transportation
Interagency Agreement Nos. 43A0014 AND 43A0073
5)
Peck, M., Gibson, R.W., Kortenkamp, A., Hill, E.M. Sediments are major sinks of steroidal estrogens in two united
kingdom rivers. Environmental Toxicology and Chemistry 23:04. 2003 p. 945-952.
6)
Schmitt, C.J., V.S. Blazer, G.M. Dethloff, D.E. Tillitt, T.S. Gross, W.L. Bryant Jr., L.R. DeWeese, S.B. Smith, R.W.
Goede, T.M. Bartish, and T.J. Kubiak. Biomonitoring of Environmental Status and Trends (BEST) Program: Field
Procedures for Assessing the Exposure of Fish to Environmental Contaminants. U.S. Geological Survey, Biological
Resources Division, Columbia, (MO): Information and Technology Report USGS/BRD-1999-0007. iv+35pp. +
appendices. Columbia, MO. 1999.
Acknowledgements
Steven Foster, limnologist – USACE, Huntington District - funding, equipment, expertise
West Virginia Dept. of Environmental Protection – Watershed Assessment Section - equipment, manpower
US Geological Survey – Fish Health Branch of Leetown Science Center - Dr. Vickie Blazer’s lab –
- expertise, lab facilities, consultation
David Neff – help with microscopy and histology topics
Dr. Somerville – thesis advisor - conception of topic, funding, consultation
NASA Research Grant – for partial funding