RESPONSES OF EASTERN MOSQUITOFISH
TO PAPERMILL EFFLUENT EXPOSURE

JJ Noggle¹, DS Ruessler², MS Sepulveda¹, SE Holm^3, TS Gross^1,2

¹University of Florida, Gainesville, FL
²USGS, Florida Integrated Science Center, Gainesville, FL
³Georgia-Pacific Corp., Atlanta, GA

Presented at the 22nd annual meeting of the Society of Environmental Toxicology
and Chemistry, Baltimore, MD, November 11-15, 2001.

ABSTRACT

Past reports have suggested masculinization (elongated anal fins in females) of mosquitofish exposed to papermill effluents.  Following a preliminary collection of mosquitofish from a papermill effluent-dominated stream in spring 2000, an expanded survey was conducted during fall 2000.  Objectives of this larger study included: verification of site effects on anal fin elongation and hormones; assessment of the influence of size/age; comparison of fall and spring collections; and examination of potential seasonality.  Eastern mosquitofish were electroshocked at five sites in and around Rice Creek, which receives effluent from the Georgia-Pacific mill in Palatka, FL. Gender was identified and anal fin and body lengths measured.  A subset of fish was frozen for hormone analyses. Results for females support spring findings: anal fin elongation was present at all sites, with a trend of decreasing elongation with increasing distance from effluent discharge. Division of females into 5 mm size classes did not reveal any influence of size/age on anal fin elongation.  Female hormones for the fall also mirrored spring analyses, with significantly depressed 17β-estradiol and increased testosterone at effluent-dominated sites. In contrast, males responded significantly in the fall compared to a lack of response in the spring.  Males collected in the fall had slightly longer gonopodia at downstream versus reference sites.  They also exhibited some hormonal variation among sites.  Since females responded similarly between seasons yet males did not, a year-long seasonality study is appropriate. These results indicate effects of papermill effluent on mosquitofish secondary sex characteristics, yet effects on reproductive function or success are unknown and also need to be explored.
 

INTRODUCTION

• Masculinized female mosquitofish have been reported in the Fenholloway River (Bortone & Drysdale 1981, Jenkins et al 2001, Orlando et al 2001, Parks et al 2001), Elevenmile Creek (Howell et al 1980, Bortone et al 1989), and in Rice Creek (Bortone 1999) – all streams receiving papermill effluent discharge.
• masculinization: elongation of the female anal fin, resembling the gonopodium of a maturing male (figure 1).
• Seasonality of anal fin elongation in effluent-exposed females was indicated by Cody & Bortone (1997) in Elevenmile Creek.
• The most recent studies of this phenomenon are focused on identifying the bioactive compound, speculated to be an androgen (Raloff 2000, Jenkins et al 2001, Orlando et al 2001, Parks et al 2001).
• Effects on reproductive function are also important to characterize, such as influence of papermill effluent on hormone levels.
• This study was an expansion of a preliminary study on mosquitofish from Rice Creek in the spring of 2000.
   

OBJECTIVES

• To verify site effects on anal fin elongation and hormones in female mosquitofish collected from Rice Creek in spring 2000.
   
• To assess the influence of size/age on anal fin elongation.
   
• To compare fall & spring 2000 mosquitofish collections from Rice Creek, examining any potential seasonality.
   

MATERIALS & METHODS

• Location:
• Rice Creek, tributary of St. Johns River, Palatka, FL, USA.
• receives papermill effluent from Georgia-Pacific pulp & paper mill.
• mill has 2 bleaching lines (bleaching sequence CEHD & C₉₀d₁₀EopHDp) and 1 unbleached line.
• mill products: paper towels, toilet paper, kraft bags.
• during collections, bleaching lines mainly used elemental chlorine, but chlorine substitution began spring 2001 to comply with EPA cluster rules.
• Effluent treatment: aeration and biological treatment through retention ponds (40 days of biodegradation).
• Fall 2000 survey sites: Etonia, SR 100, aerator 1 (AR1), aerator 2 (AR2), lower Rice Creek (LRC) – see figure 2.
• Spring 2000 sites (figure 2) included SR17 bridge (17B) & Rice Creek mouth (RCM), comprising the LRC site of the fall survey.
• spring survey did not include Etonia.
   
• Collection:
• backpack electroshocking and dip nets near vegetated banks.
   
• Measurements:
• gender identification, standard length (fall only), total body length, body weight, total anal fin length, gonopodium (males) or elongation (females), see figure 1 for anal fin measurements.
• Index of anal fin elongation (elongation / total anal fin) calculated to account for body size.
   
• Size classes:
• for the fall collection, females divided into 5 mm size classes to determine if any size/age class accounts for significant portion of females with elongations.
   
• Hormone analysis:
• subset of fish frozen for whole body RIA of 17-beta estradiol & testosterone.
   
• Statistics:
• One-way ANOVA and Tukey test for multiple comparisons to detect significant differences within each sex across field sites and treatments (p<0.05).
• significant differences within each season marked by an asterisk on the graphs.

DISCUSSION

• Lengths and weights only varied for males in the fall, without a site-dependent trend relative to effluent outfall:
• harsher conditions in fall may explain these differences.
• overall these variables are not influenced by effluent exposure.
   
• In females collected in the fall, size/age classes did not distinguish any influence of age or maturation state on length of elongation (Figure 4).
   
• Index of anal fin elongation in females was similar between seasons, exhibiting a significant site-dependent trend relative to effluent outfall (Figure 5).
• elongations were slightly longer in the spring than fall, but not significantly.
   
• In Figure 6, depressed 17-beta estradiol and increased testosterone at effluent-dominated sites was similar for females between seasons.
   
• Males were not influenced by site for anal fins or hormones in the spring (Figures 7 & 8).
   
• However, in the fall, male gonopodia were longer at downstream sites (Figure 7), and 17β-estradiol was significantly high at AR1, with testosterone highest at SR100 (Figure 8).
   
• Season appears to affect males more than females – a year-long seasonality study would help answer questions suggested by this study:
• Do hormones vary seasonally, especially for males, regardless of effluent exposure?
• If female hormone levels are affected by effluent exposure, is reproductive function and success of mosquitofish impaired?
• Will process changes at the Palatka mill, started spring 2001, reverse these trends?

ACKNOWLEDGEMENTS

Georgia-Pacific Corporation, Atlanta, GA, provided funding for this project.