Effects of Paper Mill Effluents on Secondary Sex Characteristics in Mosquitofish

JJ Noggle¹, DS Ruessler², MS Sepulveda¹, SE Holm³, TS Gross^1,2

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

Presented at the 40th annual meeting of the Society of Toxicology
in San Fransisco, CA, March 25-29, 2001.

ABSTRACT

Studies have reported masculinization of female mosquitofish exposed to paper mill effluents.  Masculinization has been measured as an elongation of the anal fin into a gonopodium-like structure. However, these studies have not adequately considered age, maturation stage, nor dose-dependence to enable the use of this response as a screening and testing protocol.  Mosquitofish are sexually dimorphic and exhibit maturation-dependent secondary sex characteristics: adult males have an elongated anal fin/gonopodium, whereas females exhibit a gender specific anal/gravid spot and urogenital papilla. The current study evaluated the relationship between paper mill effluent exposure and secondary sex characteristics in mosquitofish under controlled and natural exposures. Eastern mosquitofish (Gambusia holbrooki) were exposed to 20,40, and 100% effluent concentrations for 60 days. Adult fish were collected at multiple locations in Rice Creek, an effluent-dominated site from Georgia-Pacific's Palatka, FL, operation. Detailed morphological measurements were recorded: body size, anal fin length, gonopodium/elongation, and urogenital papilla. In the field study, anal fin elongation in females was detected at all sites (including the reference) and a significant trend of decreasing elongation with increasing distance downstream from the effluent outfall was apparent. However, anal fin lengths in these females were not gonopodium-like in size or structure, although associated with altered urogenital papilla. In addition, naturally exposed females had decreased estradiol and increased testosterone at sites nearest to the effluent outfall. Male mosquitofish showed no significant influence of effluent exposure on gonopodial morphology or endocrine status.  Laboratory exposures did not result in masculinization of female mosquitofish.  These results do not support the utilization of mosquitofish as a screening and testing model for paper mill effluent exposure without additional development and validation.
 

INTRODUCTION

Over twenty years ago, researchers discovered female mosquitofish living in paper mill effluent-dominated streams that resembled males (Howell et al, 1980; Bortone & Drysdale, 1981). Normally mosquitofish are sexually dimorphic with maturation-dependent secondary sexual characteristics: males having a projection of the anal fin called the gonopodium, and females having an anal/gravid spot and a urogenital papilla. Paper mill effluent-exposed females had developed an elongation of the anal fin into a gonopodium-like structure (Figure 2), and the phenomenon was termed masculinization. This masculinization was inducible in most laboratories by bacterially degraded phytosterols (Denton et al., 1985; Hunsinger et al., 1988; Howell & Denton, 1989), plant compounds found in paper mill effluent.  Although components of paper mill effluent have induced masculinization, past studies have not exposed these fish to whole effluent in a controlled setting.  Preliminary evidence of masculinization has also been documented in the field (Bortone & Cody 1999). The few studies of reproductive effects such as brood size are preliminary and ambiguous (Rosa-Molinar & Williams, 1984; Krotzer, 1990), so the impact this phenomenon might have on reproduction and population status is questionable.  The influence of age, maturation stage, and dose-dependence have not been adequately examined either, especially when considering this species as a biomonitoring tool.  The current project paired tank and field experiments to address some of these lesser-studied aspects in mosquitofish masculinization.
 

OBJECTIVES

Ø To confirm the presence of abnormal female mosquitofish in paper mill effluent-dominated sites.

Ø To examine the effect of paper mill effluent on secondary sex characteristics and hormone levels in mosquitofish.

Ø To study any dose-dependent response of secondary sex characters to paper mill effluent in mosquitofish.

Ø To evaluate the response of mosquitofish exposed to whole effluent dilutions in a controlled setting.
 

MATERIALS & METHODS

Paper Mill of Interest

Georgia-Pacific Corporation (GP) operates a pulp and paper mill in Palatka, FL.  This mill has 2 bleaching lines (bleaching sequence CEHD & C₉₀d₁₀EopHDp) and 1 unbleached line, producing paper towels, toilet paper, and kraft bags.  The bleaching lines mainly use elemental chlorine, but process changes to comply with EPA cluster rules will go into effect in 2001 at which time chlorine substitution will begin. Effluent is treated by aeration and biological treatment through retention ponds (40 days of biodegradation), then discharged into Rice Creek, a tributary of the St. Johns River.

Field Collection

Eastern mosquitofish, Gambusia holbrooki,  were collected by dip net along vegetated banks at six sites in Rice Creek and St. Johns River at varying distances from the effluent outfall (Figure 1) in the spring of 2000. Several morphological parameters were measured: total body length and weight, urogenital papilla (females), total anal fin, anal fin, gonopodium (males), and elongation (females). Figure 2 defines the fin parameters, which were measured using a dissecting scope and ocular micrometer. Indices of anal fin elongation were calculated using total anal fin and total body length to help control for any influence size had on elongation or gonopodium. In addition, an index of female urogenital papilla width was calculated for similar reasons.  Finally, a subset of fish from each site were analyzed for whole body hormone concentrations (17β-estradiol and testosterone).

Preliminary Tank Exposures

A unique tank system has been established on site at the GP Palatka mill for whole effluent exposure of aquatic species (Figure 3). As a pilot study, some tanks were stocked with approximately one thousand Eastern mosquitofish each and exposure lasted 10 weeks during spring 2000.  Fish were fed flakes once or twice a week.  Upon termination of exposure, surviving mosquitofish were preserved and morphological parameters were measured equivalent to those described for the field collection. Indices of elongation were likewise calculated.

Statistical Analyses

One-way ANOVA and Tukey test for multiple comparisons were used to detect significant differences within each sex across field sites and treatments (p<0.05).  Significant differences are marked by an asterisk on the graphs.

CONCLUSIONS

Ø Gender-specific influence of paper mill effluent on mosquitofish secondary sex characteristics and hormones.

Ø Both studies showed no significant influence of paper mill effluent on males for any response parameters.

Ø Females appeared to be affected by effluent in the field collection but not so clearly in the preliminary tank exposures.

Ø Field collection results imply an androgenic response in female mosquitofish exposed to paper mill effluent. Assuming effluent concentration directly related to distance from effluent outfall, a dose-dependent response can be also be concluded.

Ø However, such a conclusion cannot be made for female mosquitofish exposed to whole effluent in tanks.

Ø This lack of response to paper mill effluent could be caused by two different factors:

• the confounding factor of small sample size (fish survival in the tanks was very low, probably due to high fish density), or
   
• an absence of the factor(s) exerting the androgenic response in females found in the field collection.

Ø Continued and expanded study of this field/tank dichotomy is currently under way, including: increased sample sizes for both field and tank experiments; more appropriate reference field sites; better fish survival in tanks; analysis of fish within and among size/age classes based on standard length; and hormone analysis for tank-exposed fish.