March 2008
River Health ReportOrganic Chemicals in the North Fork & their Relationship to Recent Fish Kills & the Intersex Traits of Smallmouth Bass
Summary
The
Friends of the North Fork of the Shenandoah River has identified 59
organic chemicals in the North Fork, some of which are likely
contributors to the intersex found in smallmouth bass and may be
causing immune suppression that is a contributing factor in recent fish
kills. These findings are a critical step in the investigation of the
role of trace organics in the deterioration of fish health in the
Shenandoah River.
Background
In the spring of 2007, the
Friends of the North Fork of the Shenandoah River (Friends) deployed
trace organic chemical samplers at two locations in the North Fork; one
near Woodstock and one near Mount Jackson. Samples were collected with
two devices, one that collects fat-soluble organic chemicals that would
accumulate in fish tissues and one that collects organic chemicals that
are water soluble and mimics the respiratory exposure of fish. Both
devices are described in more detail in Attachment 1. Samplers were
deployed between March 10th and June 9th. After the samplers were
removed from the river, they were sent to the U. S. Geological Survey
for analysis.
This project was carried out in support of the
Shenandoah River Fish Kill Task Force. Over the last four years, the
North Fork has had unexplained major kills of smallmouth bass, sunfish
and suckers. John Holmes, the chair of the Friends’ Science Committee,
serves on the Task Force that is investigating these kills.
This
project was identified by the Task Force as a high priority for several
reasons. Tissue studies of dead and dying fish indicate that their
immune systems had been compromised. Manmade organic chemicals in the
river can interfere with the effectiveness of their immune systems. In
addition, researchers have found that nearly all male smallmouth bass
in the North Fork exhibit intersex traits (the presence of immature
eggs in their testes). This condition has been linked to certain
manmade organic chemicals that disrupt the normal functions of the
endocrine system, which regulates hormones. With the presence of
manmade organic chemicals implicated in both the fish kills and the
intersex phenomenon, we set out to measure the level of trace organics
in the river during the spring and early summer when the kills have
occurred. Our hypothesis was that there were organic contaminants
present in the river that could contribute to autoimmune deficiency and
endocrine disruption. There was a significant fish kill in progress
when this sampling was done.
Organic Chemicals in the River
Our
study identified 59 specific organic chemicals in the river, including
herbicides, insecticides, pharmaceuticals, PCBs, and other manmade
compounds. The following chemicals were identified in one or more of
the samples:

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Here is an overview of the types of chemicals that we found and where they come from:
Herbicides
– Most of the herbicides we identified are ones used with crops common
to the Shenandoah Valley and are likely entering the river in
stormwater runoff. The highest concentration was for Atrazine, which is
used to kill weeds in fields of corn, soybeans and other crops. Other
herbicides we found include Simazine, Prometon and Trifuralin.
Insecticides
– Several banned insecticides were found in the river. Examples include
DDT, Chlordane and Endrin. They may be present as residuals of earlier
use, or as chemical by-products of similar chemicals that are still
legal. We also found current insecticides. Chlorpyrifos was found. One
of its uses is to control cutworms and other insects in corn. Another
is Permethrin, which has many uses, including the control of insects in
farm crops and the control of mites and lice on chickens.
Pharmaceuticals
– A few common drugs were identified. Most samples contained
Venlafaxine, an anti-depressant marketed under the name Effexor. The
narcotic pain killer, Codeine, was also identified. These drugs are
probably coming into the river through sewage treatment plants and are
the result of the use of these drugs by persons in the Valley.
Polychlorinated
Biphenyls (PCBs) – These are stable (persistent in the environment)
chemicals that have been banned. Their presence is likely associated
with past use.
Polycyclic Aromatic Hydrocarbons (PAHs) – These
compounds generally come from the combustion of fuels or the use of
products derived from coal or petroleum.
Hormones – The samples were
screened for four hormones. One synthetic hormone used in oral
contraceptives was identified. Like the pharmaceuticals, this hormone
is likely coming from sewage and is not fully removed by our sewage
treatment plants.
Other Chemicals – We found other chemical
indicators of human activity. We had samples containing the bug
repellant DEET, caffeine, and two flame retardants,
tri(dichloroisopropyl) phosphate and tri(2-chloroethyl) phosphate. DEET
may come from persons being on or in the river.
Because of our
interest in the intersex phenomenon, extracts from the samplers were
evaluated for the presence of chemicals that imitate estrogen. That
testing showed a high level of endocrine interference for the full
mixture of organics in our samplers. The complete USGS report
presenting the test methods and the laboratory results can be found in
Attachment 2.
It is important to qualify our results in
several ways. First, the concentrations that we found are averages for
periods of 40-50 days. Peak concentrations would have been much higher.
Second, this report only includes the chemicals that are in the river
water; organics are also likely to be present in the river sediment,
which we did not sample. Third, while the USGS analyzed our samples for
several hundred chemicals, there are many other manmade organics that
could be in the river but were not evaluated. Fourth, we only collected
samples in the spring and early summer of one year. Concentrations may
be different in other parts of the year or in other years. Lastly, we
are only reporting on chemicals that were present at levels above the
method detection limits (MDL) of the instruments used by the
laboratory. Other chemicals were likely present at levels below the
sensitivities of those instruments.
Implications for the Intersex Phenomenon and the Fish Kills
Researchers
believe that the intersex found in smallmouth bass is related to
chemicals that are in the river as a result of human activities in the
area. (For more information on the presence of intersex in the
Shenandoah River, see the related report below which is Attachment 3.)
Journal of Aquatic Animal Health
Article: pp. 242–253 | Abstract | PDF (708K)
Intersex (Testicular Oocytes) in Smallmouth Bass from the Potomac River and Selected Nearby Drainages
V. S. Blazer*,a, L. R. Iwanowicza, D. D. Iwanowicza, D. R. Smithb, J. A. Youngb, J. D. Hedrickc, S. W. Fosterd, and S. J. Reesere
U.S. Geological Survey, National Fish Health Research Laboratory, 11649 Leetown Road, Kearneysville, West Virginia 25430, USA
U.S. Geological Survey, Aquatic Ecology Branch, 11649 Leetown Road, Kearneysville, West Virginia 25430, USA
West Virginia Division of Natural Resources, 1 Depot Street, Romney, West Virginia 26757, USA
U.S. Army Corps of Engineers, Huntington District, Post Office Box 9, Apple Grove, West Virginia 25502, USA
Virginia Department of Game and Inland Fisheries, 517 Lee Highway, Verona, Virginia 24482, USA
Abstract.—Intersex,
or the presence of characteristics of both sexes, in fishes that are
normally gonochoristic has been used as an indicator of exposure to
estrogenic compounds. In 2003, during health assessments conducted in
response to kills and a high prevalence of skin lesions observed in
smallmouth bass Micropterus dolomieu
in the South Branch of the Potomac River, the presence of immature
oocytes within testes was noted. To evaluate this condition, a severity
index (0–4) was developed based on the distribution of oocytes within
the testes. Using gonad samples collected from 2003 to 2005, the number
of histologic sections needed to accurately detect the condition in
mature smallmouth bass was statistically evaluated. The reliability of
detection depended on the severity index and the number of sections
examined. Examining five transverse sections taken along the length of
the gonad resulted in a greater than 90% probability of detecting
testicular oocytes when the severity index exceeded 0.5. Using the
severity index we compared smallmouth bass collected at selected sites
within the South Branch during three seasons in 2004. Seasonal
differences in severity and prevalence were observed. The highest
prevalence and severity were consistently noted during the
prespawn–spawning season, when compared with the postspawn season. In
2005, smallmouth bass were collected at selected out-of-basin sites in
West Virginia where fish kills and external skin lesions have not been
reported, as well as at sites in the Shenandoah River, Virginia (part
of the Potomac drainage), where kills and lesions occurred in
2004–2005. The prevalence of testicular oocytes is discussed in terms
of human population and agricultural intensity.
Received: April 21, 2007; Accepted: September 15, 2007; Published Online: December 31, 2007
DOI: 10.1577/H07-031.1
Journal of Aquatic Animal Health 2007;19:242–253
* Corresponding author: vblazer@usgs.gov
The most
likely mechanism for this is exposure to chemicals that disrupt the
endocrine system of the fish. The endocrine system releases hormones
that control the reproductive system. An endocrine disruptor is a
chemical that interferes with that function of the endocrine system and
changes the growth, development or reproduction of the animal. More
than half of the chemicals we found are known or suspected endocrine
disruptors. In addition, the screening test for estrogen imitators
clearly shows that the mixture of organics in the river can have that
effect. Therefore, we feel that we have identified organic chemicals as
likely contributors to the level of intersex in smallmouth bass in the
river.
Finally, there is the question of whether the organics
we found in the river are the cause of the fish kills. Because Virginia
does not have water quality standards for all of these chemicals, there
is no regulatory trigger against which to compare these results. None
of the fish kill research done to date suggests that the kills are the
result of a direct toxic effect of a single chemical. Instead, there is
a widely held view that the immune systems of the fish in the North
Fork are compromised and, as a result, the fish are more susceptible to
disease when the kills occur. The chemicals we found in the river are
likely to compromise the immune systems of the fish. Certain of the
organics in the river are known to disrupt immune systems. For example,
one researcher has found that Atrazine causes immune suppression and
endocrine disruption in frogs when they are exposed to levels of
Atrazine lower than the ones we found (Hayes et. al, 2002 and Hayes et
al, 2006). The scientific literature has references to immune system
interference for a number of the other chemicals we have identified,
including Chlordane, Hexachlorobenzene, Simazine, Venlafaxine,
Permethrin, Chlorpyrifos, and PCBs. Further, the “soup” of organics in
the river may have a cumulative effect on fish. That is, the level of
any one compound may cause no harm but the collective mixture can cause
harm, as a result of chronic exposure and synergistic effects. We are
not able to say definitively that the presence of these chemicals is
causing the fish kills. However, we are able to say that their presence
is evidence of one potential cause of, or contributor to, the kills.
(For a more thorough review of endocrine disruption and immune system responses to the organic chemicals, see Attachment 4)
Implications for Human Health
Human
health considerations are outside the scope of this study. However, any
potential risk that these chemicals pose may extend to the drinking
water supplies in the Valley. Recent studies have shown that trace
organics of the kind we have found are going through water treatment
systems and are present in public drinking water supplies for many US
cities (Donn et. al, 2008). The towns of Woodstock, Strasburg and
Winchester all get their raw water from the North Fork down river from
our sampling locations. As far as we know, the public water systems in
the Valley have not been tested for trace organics at the levels we
studied.
Next Steps We are continuing our investigation of trace organics in the river. We are taking the following steps:
- We
are sharing our test results with other fish kill researchers and we
will work through the Fish Kill Task Force to compare our results with
the results of other water, sediment and fish sampling in the
Shenandoah River.
- We will present these results to the State
Department of Health and the State Department of Environmental Quality
and ask that they determine if these chemicals pose any risk to the
citizens of the Valley. We will specifically request testing of
drinking water supplies to determine if these same types of chemicals
are present in our drinking water and what risk that may pose.
- We
will support further studies of the fish kills and the intersex of the
smallmouth bass. We will ask our local, state and federal
representatives to support funding of those studies.
- We will work to
have the sources of water pollution in the Valley reduced so that we
can restore the health and safety of the North Fork. We do not need to
await the final determination of the causes of either the fish kills or
the intersex to take steps to reduce the levels of organics in the
river. There are separate major initiatives to reduce the levels of
nitrates and phosphates in the river and restore parts of the river
that are impaired by high levels of bacteria and sediment. Steps to
achieve those goals will also reduce the flow of organics into the
river.
Acknowledgments
The Friends have many people to
thank for the success of this project. The laboratory analysis of the
samples was a complex, time-consuming, and expensive task. A grant from
the Virginia Environmental Endowment and a matching grant from an
anonymous foundation allowed us to pay for that laboratory work. The
laboratory work itself was carried out by the USGS Columbia
Environmental Research Center. Dr. David Alvarez and the entire USGS
team helped us greatly with the design of the study and the
interpretation of the results. John Holmes, the head of the Friends’
Science Committee, served as the Project Manager. Mary Gessner, Bud
Griswold, and Jeff Kelble assisted with the deployment and retrieval of
the samplers. They were joined by Bob Luce in the preparation of this
report.
Those of you interested in learning more about the
Friends and supporting our efforts should go to our about us page.
AttachmentsOrganic Chemicals Passive Samplers – SPMD and POCIS
Investigation
of Organic Chemicals Potentially Responsible for Mortality and Intersex
in Fish of the North Fork of the Shenandoah River, USGS Report 2008-1093
Intersex
(Testicular Oocytes) in Smallmouth Bass from the Potomac River and
Selected Nearby Drainages, Journal of Aquatic Animal Health 19:242-253,
2007
Evidence of Endocrine Disruptors and Immune System
Suppressors in the Results of the 2007 Trace Organics Monitoring
Project on the North Fork of the Shenandoah River
References
Donn, Jeff, Mendoza, Martha, and Justin Pritchard, “AP Probe Finds Drugs in Drinking Water”, Associated Press, March, 2008.
Hayes, Tyrone B, Atif Collins, Melissa Lee, Magdelena Mendoza, Nigel Noriega,
A.
Ali Stuart, and Aaron Vonk. “Hermaphroditic Demasculinized Frogs After
Exposure to the Herbicide Atrazine at Low Ecologically Relevant Doses”,
Proceedings of the National Academy of Sciences of the United States of
America. Vol 99, No. 8. 5476-5480, 2002.
Hayes, Tyrone B., Paola
Case, Sarah Chui, Duc Chung, Cathryn Haeffele, Kelly Haston, Melissa
Lee, Vien Phoung Mai, Youssra Marjuoa, John Parker and Mable Tsui,
“Pesticide Mixtures, Endocrine Disruption, and Amphibian Declines: Are
We Understanding the Impact?” Environmental Health Perspectives. April,
2006. No. 114. 40 – 50.
Attachment 1 Organic Chemicals Passive Samplers – SPMD and POCISIn
this study, the Friends of the North Fork deployed two types of
samplers: Semipermeable Membrane Device (SPMD) and Polar Organic
Chemical Integrative Sampler (POCIS). The two work together to collect
the trace organics in the water to which fish are exposed. The SPMD is
designed to sample the non-polar (hydrophobic), fat-soluble organic
chemicals in water. The POCIS is designed to sample the polar,
water-soluble organic chemicals in water.
Both the SPMD and
the POCIS are integrative samplers that provide an average
concentration of chemicals over the time period that the samplers are
in the river. In our case, they were deployed for 42 to 50 days. Both
are also known as passive samplers, meaning that they have no
mechanical or moving parts and do not require a power source or
maintenance while they are in the river.
The SPMD consists of
lipid oil inside a membrane. Chemicals pass through the membrane and
remain in the oil. The SPMD is housed in a protective plastic container
while it is in the river. The SPMD mimics the bioaccumulation of
organic contaminants in the fatty tissues of fish.
The POCIS
consists of a solid sorbent inside a membrane that allows water and
dissolved chemicals to pass through to the sorbent where the chemicals
are trapped. The POCIS is housed in a protective stainless steel cage
while it is in the river. The POCIS mimics the respiratory system of
the fish.
Both are state-of-the-art samplers created by the United States Geological Service for studies of the type we have done.
For more information on these sampling devices, go to: http://biology.usgs.gov/contaminant/passive_samplers.html
Attachment 2Investigation of Organic Chemicals Potentially Responsible for Mortality and Intersex in Fish Prepared in cooperation with the Friends of the North Fork of the Shenandoah River 
Journal of Aquatic Animal Health
Article: pp. 242–253 | Full Text | PDF (708K)
Intersex (Testicular Oocytes) in Smallmouth Bass from the Potomac River and Selected Nearby Drainages
Attachment 4
Evidence
of Endocrine Disruptors and Immune System Suppressors in the Results of
the 2007 Trace Organics Monitoring Project on the North Fork of the
Shenandoah River
This document provides an overview of the
endocrine system, the way in which certain chemicals can disrupt that
system and the evidence for such chemicals being found in the samples
collected by the Friends of the North Fork in 2007. This document
should be read in conjunction with the report from the Friends entitled
“River Health Report: Organic Chemicals in the North Fork and their
Relationship to Recent Fish Kills and the Intersex Traits of Smallmouth
Bass” and the laboratory report from USGS entitled “Investigation of
Organic Chemicals Potentially Responsible for Mortality and Intersex in
Fish of the North Fork of the Shenandoah River.”
The endocrine
system consists of glands and hormones (chemical messengers) that
regulate the growth, metabolism, reproduction, and the functioning of
many organs of an animal. The major hormones that regulate the
reproductive system are estrogens and androgens (e.g., testosterone).
Endocrine
disruptor chemicals (EDCs) are synthetic or natural chemicals that can
interact with estrogen receptors in animal cells, alter the function of
the endocrine system, and adversely affect the animal’s health. EDCs
mimic natural hormones by virtue of their size and molecular structure.
Even at low concentrations they can stimulate, block, or modify
reactions in the endocrine system.
It has been reported
(Thacker, 2005) that there may be as many as 87,000 chemicals that can
act as endocrine disruptors. However, the vast majority of these
chemicals have not yet been adequately tested to determine whether they
are known or merely suspected EDCs. Much more research is needed to
establish mechanisms of disruption, dose/response relationships, and
the effects of mixtures of EDCs (Kavlock et al., 1996; National
Research Council, 1999). Known natural EDCs are found in some plants
(phytoestrogens) or in fungi (mycotoxins) on plants, but in general
these are benign to animals.
Synthetic chemicals that are EDCs
include: dioxins; PCBs; benzo(a)pyrene; phthalates; bisphenol A;
alkylphenols; many insecticides, herbicides, and fungicides; heavy
metals, and synthetically produced hormones identical to natural
hormones, such as those in human oral contraceptives and certain animal
feed additives (National Research Council, 1999; European Commission
for the Environment Research website
http://ec.europa.eu/research/endocrine/background_disruption_en.html
Evidence
that endocrine disruption has occurred in the North Fork comes from the
work of Dr. Vicki Blazer of the U.S. Geological Survey and her
colleagues. They found that all 8 male smallmouth bass they collected
from the North Fork in the fall of 2004 displayed intersex
characteristics. The male fish had precursor egg cells within their
reproductive organs (testicular oocytes); this phenomenon is suggested
as a biological marker for endocrine disruption (Blazer et al., 2007).
Importantly, endocrine disruption has also been shown to compromise or
modulate the immune systems of several fish species (Aaltonen et al.,
1997; Baier-Anderson and Anderson, 2000; Dunier et al., 1995).
Immunomodulation in the North Fork has been suggested by the finding of
bacteria, fungi, and parasites as causes of skin lesions on the fish
(Blazer et al., 2007). Whether such immunomodulation has allowed
normally warded-off diseases to prevail, resulting in fish kills, is a
major topic of investigation by researchers working with the Shenandoah
River Fish Kill Task Force.
We now turn our attention to the
specific chemicals that were found in the river during the 2007
sampling program. As reported in the USGS report, a number of
organochlorine pesticides and PCBs were measured in the samples
collected with the Semi-Permeable Membrane Device (SPMD).
Concentrations greater than the method quantification limit (MQL) were
found for the pesticide trifluralin, the pesticide/fungicide
hexachlorobenzene, the insecticide chlorpyrifos, the now-banned
pesticides trans-chlordane and cis-chlordane, related metabolite
oxychlordane, and related constituent trans-nonachlor. DDT metabolites
o,p’DDE and p,p’DDE were also found, as were heptachlor epoxide (formed
from the banned pesticide heptachlor) and dieldrin (used in termite
control and as a wood preservative). PCBs were detected but were at
concentrations below the MQL. All of these chemicals are known or
suspected EDCs.
Similarly, other agricultural herbicides and
insecticides were found in the Polar Organic Chemical Integrative
Sampler (POCIS) sampler. Concentrations greater than the MQL were found
for atrazine, desethylatrazine (a degradation product of atrazine),
prometron, simazine, and carbaryl. All of these are known or suspected
EDCs
Analyses for three natural hormones and one synthetic
hormone detected only 17 α-ethynylestradiol, the synthetic estrogen
hormone found in birth control pills, above the MDL in the POCIS
sampler at Mt. Jackson deployed in the late spring period.
Yeast
estrogen screen assays completed by the USGS on both the SPMD and POCIS
extracts show that relative estrogenicities of the mixture of chemicals
from POCIS are many times more than for SPMD at equivalent deployments.
In fact, the SPMD levels were viewed as typical for background levels
while the POCIS levels were particularly high. Seemingly, this
indicates that intersex consequences should be more attributable to
polar chemicals that are collected in the POCIS. However, further
investigation could show this not to be the case. The non-polar
lipophilic chemicals in the SPMD will bioaccumulate to a much greater
extent than will polar hydrophilic POCIS chemicals. Biomagnification of
SPMD chemicals as they move up the food chain to the top predators,
especially smallmouth bass, may be an important factor.
The
number of chemicals sampled by SMPD and POCIS and carefully analyzed in
this North Fork study is indeed impressive. However, because of the
vast number of known or suspected EDCs, it cannot be comprehensive for
EDCs. Some of the known EDCs not analyzed in this study include
bisphenol A (lacquer for lining food cans), nonylphenol and alkylphenol
ethoxylates (surfactants, plasticizers), phthlates (plasticizers),
dioxins, polybrominated diphenyl ethers (flame retardants), and heavy
metals. One chemical that should be looked at closely is arsenic.
Arsenic is a known EDC (Watson and Yager, 2007). The state has observed
arsenic in some fish from the area. One potential source of arsenic in
the river is roxarsone, an arsenic containing compound that is added to
some poultry feed and can end up in poultry litter that is used as a
fertilizer. It is now under study for its environmental effects
(Schreiber et al., 2004).
This trace organics monitoring study
has verified and quantified the presence of a number of endocrine
disrupting chemicals in the North Fork of the Shenandoah River. There
may be more. This study is a vital part of the investigations to
determine the connection between EDCs, intersex condition in fish, fish
kills, and the health of the river.
References
Aaltonen,
T. M., Valtonen, E. T., and E.I. Jokinen, 1997, Immunoreactivity of
roach, Rutilus rutilus, following laboratory exposure to bleached pulp
and paper mill effluents: Ecotoxicology and Environmental Safety, v.
38, p. 266-271.
Blazer, V.S., L.R. Iwanowicz, D.D. Iwanowicz,
D.R. Smith, J.A. Young, J.D. Hedrick, S. W. Foster, and S. J. Reeser,
2007, Intersex (testicular oocytes) in smallmouth bass from the Potomac
River and selected nearby drainages. Journal of Aquatic Animal Health,
v. 19, p. 242-253.
Baier-Anderson, C., and R.S. Anderson, 2000,
Suppression of superoxide production by chlorothalonil in striped bass
(Morone saxatilus) macrophages: the role of cellular sulfhydryls and
oxidative stress: Aquatic Toxicology, v. 50, p. 85-96.
Dunier,
M., C. Veronet, A. K. Siwicki, and V. Verlhac, 1995, Effect of lindane
exposure on rainbow trout (Oncorhynchus mykiss) immunity. IV.
Prevention of nonspecific and specific immunosuppression by dietary
vitamin C (ascorbate-2-polyphosphate): Ecotoxicology and Environmental
Safety, v. 30, p. 259-268.
Kavlock, R. J. et al., 1996, Research
needs for the risk assessment of health and environmental effects of
endocrine disruptors: a report of the U.S. EPA-sponsored workshop:
Environmental Health Perspectives, v. 104, supplement 4, p. 1-26.
National Research Council, 1999, Hormonally active agents in the environment: National Academy Press, Washington D.C., 430 p.
Schreiber,
M., D. Chambers and J. White, 2004, Cycling of organoarsenic compounds
in agricultural watersheds: [abstr. H21c-1031] American Geophysical
Union, Fall Meeting.
Thacker, P.D., 2005, Sensing environmental
estrogens with glowing yeast: Environmental Science & Technology
Online News website:
http://pubs.acs.org/subscribe/journals/esthag-w/2005/jul/tech/pt_yeast.html.
Watson,
W.H., and J.D. Yager, 2007, Arsenic: extension of its endocrine
disruption potential to interference with estrogen receptor-mediated
signaling: Toxicological Sciences, v. 98, no. 1, p. 1-4.
To see how this project began and see photos of the samplers go to
our Trace Organics Page.
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