COMPARISON OF ANAL FIN MORPHOLOGY
IN FEMALE GAMBUSIA AMONG THREE FLORIDA PULP & PAPER MILLS

JJ Noggle¹, WK Bradley², D Borton², JT Smith³, TS Gross^1,3

¹University of Florida, Gainesville, FL
²National Council for Air and Stream Improvement, Inc. (NCASI), Vanceboro, NC
³USGS/Florida Integrated Science Center, Gainesville, FL

Presented at the 23rd annual meeting of the Society of Environmental Toxicology and Chemistry,
Salt Lake City, UT, November 16-20, 2002.

ABSTRACT

For over 20 years, anal fin elongation in female gambusia has been reported in receiving streams of pulp and paper mills. Studies suggested masculinization of females and development of a gonopodium, the male copulatory organ, and often focused on multiple sites in Florida, including Elevenmile Creek, the Fenholloway River, and Rice Creek. The current study compared anal fin size and structure among these streams, and evaluated potential site-specific and mill process relationships. Approximately 200 fish were collected at reference, upstream, pre-discharge, and downstream sites for anal fin and sex steroid analyses.  Water was analyzed for selected chemicals (resin acids, fatty acids, phytosterols, total organic carbon, condensable tannins, and polyphenolics) and significant differences between mills and sites were noted.  Anal fin elongation in female gambusia was apparent at effluent-dominated sites within the Fenholloway River and Rice Creek, however, anal fin elongation was not observed in Elevenmile Creek. In the Fenholloway River greatest elongation occurred at the downstream site, with a trend of increasing elongation with increasing distance from the pre-discharge site. In contrast, in Rice Creek greatest elongation occurred at the discharge site with a trend of decreasing elongation with increasing distance from the outfall. Maximal anal fin elongation never approached the male gonopodium in length or in terminal differentiation. Indeed the mean 4:6 anal fin ray index for affected females was 1.5 while normal males and females had an index of 2.5 and 1.1 respectively.  Results of hormone analyses indicate female gambusia had increased testosterone at effluent-dominated sites as compared to upstream sites for all streams. These data imply mill effluent may alter testosterone production in female gambusia. Anal fin data suggest an association between elongation and exposure to high concentrations of effluent. Dose-dependence is not consistent among mills, perhaps due to differences such as tree species, end products, dilution rates of effluent, etc. Increasing anal fin elongation down the Fenholloway River supports the hypothesis that biotransformation products of phytosterols induce elongation. Although the Fenholloway River had the highest concentrations of naturally derived compounds (i.e. resin acids, phytosterols, etc.) a relationship between effects and distinct chemical classes or specific chemicals is yet unclear.
 

INTRODUCTION

Ø Over twenty years ago researchers discovered female gambusia that resembled males living in Elevenmile Creek, a paper mill effluent-dominated stream in the panhandle of Florida (Howell et al, 1980).

Ø Also found in Fenholloway River (Bortone & Drysdale 1981, Jenkins et al. 2001, Parks et al. 2001) & Rice Creek (Bortone & Cody 1999), two other Florida streams.

Ø Normally gambusia are sexually dimorphic with maturation-dependent secondary sexual characteristics (figure1):

Ø Adult males have a projection & differentiation of the anal fin called the gonopodium.
Ø Adult females lack a gonopodium (they have a gravid spot and urogenital papilla).

Ø Paper mill effluent-exposed females had developed an elongation of the anal fin resembling a gonopodium-like structure, and the phenomenon was termed "masculinization".

Ø This masculinization was inducible in most laboratories by bacterially degraded phytosterols (Denton et al., 1985; Hunsinger et al., 1988; Howell & Denton, 1989), naturally derived plant compounds found in pulp & paper mill effluent.

Ø Hypothesized phytosterols degraded into androgens such as androstenedione.
Ø Recent research focused on androgen activity & mechanistic responses (Jenkins et al. 2001, Parks et al. 2001, Orlando et al. 2002).

Ø Variation in the masculinization response among different pulp & paper mills has never been directly addressed.

Ø Important to determine if response will be used for screening & testing procedures.
 

MATERIALS & METHODS

Field sites

Ø Three unique systems receiving pulp & paper mill effluent: Rice Creek, Fenholloway River, and Elevenmile Creek (figure 2).
Ø Similar types of sites were chosen for each system (see figure 3), many of which have been historically sampled.
Ø Many differences among mills (figure 4) such as tree species utilized, end products, and treatment of effluent.

Fish Collection

Ø Collected approximately 200 fish per site by backpack electroshocker and/or dip nets (figure 5).
Ø Also depicted in figure 5, fish for hormone analysis were processed on site (20-30 females per site): anesthetized (MS222), body length & weight measured, digitally photographed, and frozen.

Ø Remaining fish counted and preserved in 10% formalin.

Water Analysis

Ø A water sample was taken at each site & water quality (such as pH, conductivity, turbidity) was measured.
Ø Chemical analyses included: resin acids, fatty acids, phytosterols, TOC, condensable tannins, polyphenolics, chlorophenolics, metals, neutral semi-volatiles.

Hormone analysis

Ø Whole body hormone analysis for 17beta-estradiol and testosterone using RIA procedure.

Anal fin morphology

Ø Anal fin rays 4 & 6 were measured from digital pictures using SigmaScan ProⒸ? 5.0 for all fish frozen for hormones.
Ø Anal fins of preserved fish were measured manually (ocular micrometer in dissecting scope) & by computer.
Ø Manual measurements will allow better comparison to historical collections in future studies.

SUMMARY

Ø Highest concentrations of naturally derived effluent components and largest responses (anal fin elongation and testosterone) at Fenholloway River system (figures 6, 7, & 8).

Ø Intermediary values at Rice Creek system.
Ø Lowest values at Elevenmile Creek system.

Ø Positive dose-dependent response of anal fin elongation is supported at Rice Creek system (figure 8).

Ø Opposite effect at Fenholloway River system.

Ø Anal fin elongation and testosterone levels do not correlate (figures 7 & 8).

Ø High testosterone values and low degree of elongation at reference sites for Fenholloway River.
Ø Decreased testosterone and increased elongation with increasing distance from outfall in Fenholloway River.
Ø High testosterone at pre-discharge site for Elevenmile Creek, although lack of significant elongation.
 

CONCLUSIONS

Ø Association between anal fin elongation and exposure to high concentrations of pulp & paper mill effluent.

Ø Threshold of response for anal fin elongation may lie between chemical concentrations in Rice Creek and Elevenmile Creek effluents.
Ø Dose- dependence unclear – may reflect mill differences (figure 4) or environmental variation.

Ø Increase in anal fin elongation down Fenholloway River supports activity of breakdown products and not parent compounds.

Ø Lack of relationship between anal fin elongation and testosterone indicates these responses may occur by separate mechanisms and/or stressors (mill differences described in figure 4, environmental differences).

Ø Variability in hormones among sites without effluent exposure indicates reproductive stage may influence hormone status (currently under investigation).
Ø Relationship between observed effects and distinct chemical classes is yet unclear.
 

ACKNOWLEDGEMENTS

Ø National Council for Air and Stream Improvement, Inc. (NCASI) provided funding for this project.

Ø Special thanks for the cooperation and feedback provided by the pulp & paper mills associated with this study.