Fish Kill Investigation 
 
 
March 2008
 
 
River Health Report
 
Organic 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:  
  
 
 
 
 
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 at Attachment 3.)  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:
  1. 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.
  2. 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.
  3. 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.
  4. 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 website at  http://www.fnfsr.org  
 
Attachments
  1. 1.Organic Chemicals Passive Samplers – SPMD and POCIS
  2. 2.Investigation of Organic Chemicals Potentially Responsible for Mortality and Intersex in Fish of the North Fork of the Shenandoah River, USGS Report 2008-1093
  3. 3.Intersex (Testicular Oocytes) in Smallmouth Bass from the Potomac River and Selected Nearby Drainages, Journal of Aquatic Animal Health 19:242-253, 2007  
  4. 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
 
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 POCIS
In 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 2
Investigation 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
 
                                                          
 
 
Attachment 3
 
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.