Christopher Barry, Steven Foster
 
M.U. Confocal Facility
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Fig. 1 Setup for working up samples in the field

         
     
     
Fig. 2 Electrofishing from boat
   
     
   
     

Abstract:

...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.

    
               
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Fig. 4 Location of fish gonads relative to other internal anatomy
   
 
Fig. 3 Taking fish mass data in the field
     
   
     
   
 

Introduction:

...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.

   
               
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Fig. 5 Occurrence of intersex at each site
    
                   
     
   
      Methods:            
                   
     

 

      

   
Sample Collection
Sectioning and 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 MS-222 (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

◊ 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

                   
                   
                   
 

 

      

 

    
Paraffin Embedding
Mounting
       
       
◊ 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.		 ◊ 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. 6 Individuals’ OSI scores
    
                   
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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.

   
     
Fig 7 Backpack electrofishing unit
  
       
     
   
                   
 
   
     
Fig. 8 Intersex severity analysis in ImageJ
    
                 
 
                   
     
         
     
Fig. 9 10x image of ovotestis
   
Fig. 10 40x image of ovotestis
   
     
   
     

Conclusion:

... 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?

    
     
   
                   
                   
                   
     

References

      
Acknowledgements
    
       
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.

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.

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.

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.		   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		  
     
     
                   
 


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