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Anomalies observed in the Sexual Development
of White Sucker Fish in Boulder Creek

Summary composed by Jim Waterman - BASIN

Intersex and other forms of reproductive disruption in feral white sucker (Catostomus commersoni) downstream of wastewater treatment plant effluent in Boulder, Colorado

The following page is a summary of research conducted by University Colorado Department of Integrative Physiology research personnel:

Alan M. Vajda, Elena M. Lopez, Tammy A. Maldonado, John D. Woodling, and David O. Norris. Department of Integrative Physiology, University of Colorado, Boulder

This summary was composed by Jim Waterman of the BASIN project with the generous cooperation of the above researchers. Please contact BASIN with any comments or corrections.


Feral white suckers (Catostomus commersoni) were collected on Boulder Creek downstream from wastewater treatment plant (WWTP) effluent and from reference sites. This effluent is known to contain endocrine-active compounds including alkylphenols, bisphenol A, and reproductive steroids. Gonadal intersex and other forms of reproductive disruption were found in white suckers collected downstream of WWTP effluent but not at reference sites. The male to female ratio was skewed toward females at the downstream site.

Abnormalities in gonadal morphology, including smaller ovaries, less developed oocytes, and asynchronous follicular development were noted in female white suckers downstream of the WWTP. Elevated plasma vitellogenin was also reported in downstream juvenile white suckers. The reproductive potential of native fishes may be compromised in stream reaches of western states where large volumes of treated wastewater are discharged into relatively small-sized streams.

White Sucker

A long-lived species, the white sucker (Catostomus commersoni) was selected as the target species. White suckers tolerate a wide variety of conditions including river stretches greatly enriched from domestic sewage treatment plant effluents. White sucker have been collected at sites immediately downstream of WWTP effluents in Colorado. Propst (1982) often found the white sucker to be the most common fish at such locations, although numbers were lower than at similar unpolluted sites. Spring sampling dates coincide with spawning in white sucker while fall sampling dates coincide with reduced stream flows and with male and female gametogenesis.

Selection of the white sucker

The White Sucker is widely distributed throughout North America and is common in Front Range streams. A healthy White Sucker population has been observed in Boulder Creek and this species is commonly observed in the vicinity of WWTP outflows. As the research sought to examine effects of anthropogenic impacts, comparison to a reference site upstream of the City of Boulder WWTP effluent discharge was desired. The white sucker is one of the few species found in Boulder Creek both in the warm water stream segments east of the city and the cold water segments flowing through Boulder Canyon, west of the city.

Additional information on the White Sucker

Study Sites

Boulder Creek was sampled above and below the effluent of the City of Boulder wastewater treatment plant (WWTP). Boulder Creek upstream of the Boulder WWTP flows through the city of Boulder and receives all storm water runoff from the municipal area. Pristine comparison sites were not available within the distributional range of the target species. Upstream reference sites were downstream of city of Boulder storm water runoff and upstream of all WWTP effluent. ( Map of water quality monitoring site on Boulder Creek )

Materials and Methods

Fish were collected using electro-fishing equipment with a pulsed DC current in spring and fall of 2003 and 2004. Fishes and sample tissue were handled in the same manner in the spring and fall. Fishes were anesthetized with MS222, weighed to the nearest 1 gram and measured for total length to the nearest mm. Gonads were removed, weighed to the nearest 0.1 gram and preserved in 10% NBF. Relative size and color were noted and any abnormal development described. Small portions of the head, middle, and caudal sections of the gonad were embedded in paraffin for histological analysis. Sections (10mm) were stained using hematoxylin and eosin (Presnell and Schreibman 1997). Ovaries were examined under a light microscope to determine the sex and reproductive stage of each fish. The reproductive stage of the ovaries was determined using a five-stage classification system modified from Goodbred et al. (1997). Plasma vitellogenin was assayed with an anticarp ELISA kit (BioSense) following the manufacturer’s protocols.


  1. Sex ratio skewed towards females downstream of WWTP effluent.
  2. Asynchronous follicular development in downstream females.
  3. Intersex gonads, composed primarily of ovarian tissue, were found in fishes only downstream of WWTP effluent.
  4. Elevated vitellogenin in plasma of juvenile fish downstream of WWTP effluent.
  5. Reduced gonadosomatic index in downstream females.

CU researchers sampled the white sucker populations at reference points and downstream of WWTP outfalls. Approximately equal distributions of male and female were expected and such distributions were found at the reference site. However gender distributions downstream of the WWTP effluent discharge were found to be significantly inconsistent with expectations; with better then 90% female.

While migration of males away from the WWTP discharge area has not been eliminated as a contributing factor, examination of gonadal tissue and vitellogenin proteins support theories that gender distributions are a result of abnormal reproductive tissue development. The few male individuals found at these sites showed significant signs of abnormal development of reproductive tissue

Sexual Differentiation

Typically sexual differentiation occurs in white suckers during the 2nd year of life. Gender development is normally determined by genetics; however in some species sexual differentiation can be influenced by environmental factors, particularly "via exogenous hormonal influences at certain stages of development" (Environmental Biology of Fishes Malcolm Jobling, 1995, pg 297).

Woodling et al. identified skewed sex ratios downstream of WWTP effluent in Boulder, Denver, and Colorado Springs in 2001 (BCWI Forum presentation February 25, 2004.) Further sampling of Boulder Creek in 2002, 2003, and 2004 found the effect of effluent on sex ratio and the occurrence of intersex to be size specific.

Serum Vitellogenin Levels

As female fish approach sexual maturity (several months before spawning) the liver begins to produce vitellogenin, a lipophosphoprotein-calcium complex which is released into the bloodstream and transported to the ovaries. This compound forms the basis of future egg yolk protein and stimulates the development of ovarian follicles. While the initiation of vitellogenin production is believed to be tied to increasing photoperiod it is also marked by increased serum estradiol, suggesting environmental hormones may contribute to elevated vitellogenin levels in downstream juveniles.

Follicular Tissue Development

During the normal development of follicular tissues 2 distinct stages (possibly simultaneous) are observed, one prior to and a second following onset of vitellogeneous. CU researchers found a much wider variation in follicular development in individuals downstream of the WWTP effluent discharge.

A characterization of follicular development in ovaries from size-matched (>200mm) females shows that downstream fish possess significantly more simultaneous ovarian follicular stages than reference fish (P<0.01). All upstream fish examined possess only two follicular stages, one pre-vitellogenic and one post-vitellogenic. Downstream fish possess between one and four simultaneous follicular stages.


While the duration of this study did not address long term population impacts no white sucker population impacts have been observed. It is possible that migration of viable males into the WWTP reach may maintain relatively strong populations however the documented disruption of the normal development of reproductive tissue would be expected to impact the the reproductive success of individuals and conceivable will eventually impact overall populations.

Future Directions

To further characterize the extent of reproductive disruption in the fishes of Colorado's front range rivers CU researchers are continuing to survey the 3 rivers and plan to incorporate laboratory exposure experiments to determine the role of effluent in generating gonadal phenotype.
  1. Determine whether gonadal phenotype in white suckers is influenced by exogenous estrogens.
  2. Determine whether the intersex condition and follicular asynchrony downstream of WWTP effluent is inducible by laboratory exposure to effluent.
  3. Determine whether disruption of follicular synchrony is accompanied by other modifications of life-history phenotype among downstream fish.
  4. Determine whether reproductive disruption in fishes can be detected using non-lethal means.

Literature Cited

Acknowledgments CU Research Funding: US EPA Region, 8 NPDES WQCA #CP988934-01 In-kind support from the Colorado Division of Wildlife

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