EFFECT OF IN VIVO EXPOSURE TO p,p-DDE AND DIELDRIN ON SEASONAL GONADAL DEVELOPMENT AND STEROIDOGENESIS IN FEMALE LARGEMOUTH BASS

Muller, J.K.¹, Arnold, B.S.², Monck E.K.¹, Borgert, C.J.^1,3, and Gross, T.S.^1,2

 ¹College of Veterinary Medicine, University of Florida, Gainesville, FL.
²USGS - Florida Integrated Science Center, Gainesville, FL.
 ³Applied Pharmacology and Toxicology, Inc. Alachua, FL.

Presented at the The Society of Environmental Toxicology and Chemistry (SETAC) meeting in November 2002.

Abstract

Previous work from this laboratory has indicated that persistent organochlorine pesticides have a wide range of effects on steroidogenesis and reproductive function in fish. The current study investigated potential reproductive effects of two pesticides, p,p-DDE and Dieldrin, on female largemouth bass (Micropterus salmoides floridanus). Sixty-day slow matrix release pellets were inserted (IP) to produce specific internal doses of these chemicals. Twenty-five fish were placed into each of eight treatment groups: sham, placebo (matrix only pellet), 2.5mg DDE, 5.0mg DDE, 10.0mg DDE, 0.25mg Dieldrin, 0.5mg Dieldrin, and 1.0mg Dieldrin.  These doses were chosen to create body burdens similar to those reported for largemouth bass in sites where effects were reported. Exposure to each pesticide began before the onset of the reproductive season (January) with the full dose being released prior to spawning (March).  After sixty days, each fish was bled and plasma collected for circulating hormone analysis.  Fish were sacrificed, gonad and liver collected for contaminant analysis and the determination of GSI and LSI. Gonads were placed in explant culture for analysis of steroidogenesis in vitro.  Preliminary contaminant analysis indicates that pellets did not release a consistent dose in each category. Analyses were based upon actual body burden in the liver and gonad. Results show a decrease in estradiol production but no change in 11-ketotestosterone levels in bass treated at the high dose of p,p-DDE and Dieldrin,a change in hormone levels that may be associated with altered reproductive success. No change in GSI or LSI was observed for Dieldrin or p,p-DDE, regardless of dose.  Altered steroid synthesis following exposure of largemouth bass to organochlorine pesticides may be associated with non-synchrony of spawning and reduced reproductive success.
 

Introduction

Organochlorine pesticides (OCPs) had broad usages throughout the United States to control termites, mosquitoes and other pests in the mid-1900s. The use of OCPs was banned in the U.S. in the 1970s, but due to the persistence of these chemicals and their ability to biomagnify, they remain in the environment Many agricultural endeavors along the Ocklawaha river system in Florida (Fig. 1) applied OCPs. Sites that show high OCP contamination are associated with abnormal reproductive function in alligators and fish (Gross, 1994; Marburger, 1999).  Two of the OCPs found in highest concentration at these sites are p,p-DDE and Dieldrin (Marburger, 2002). These pesticides were selected for development of a single chemical dose response relationship to reproductive function in largemouth bass (Micropterus salmoides floridanus). Largemouth bass were chosen as the experimental model because of their top-predator status in the aquatic food chain, which allows for intense biomagnification of lipophilic compounds, such as organochlorine pesticides.

Objectives

• Induce body burdens of two organochlorine pesticides in female largemouth bass throughout reproductive season.
• Assess changes in reproductive capacity post-exposure.
   

Materials and Methods

• Female largemouth bass were housed in 4 replicate concrete runs with a flow through aquatic system.
• Exposure was attained by insertion of a 60-day slow release matrix pellet (Innovative Research of America, Inc.) intraperitoneally along with a pit tag:
• DDE pellets contained: 2.5, 5.0, or 10.0 mg p,p-DDE
• Dieldrin pellets contained: 0.25, 0.5, 1.0 mg Dieldrin
• Placebo: matrix only pellet
• Sham:  insertion of pit tag only
• Exposure period ran from January to March which covers most of the vitellogenic phase of the reproductive period.
• On day 60 fish were weighed , measured, bled, organs collected and weighed.
• Blood plasma was collected by centrifugation at 4°C at 3,000rpm for 15 minutes.
• Circulating sex steroid hormones – 17β-estradiol and 11-ketotestosterone were determined by RIA.
• After the 60-day exposure period, the following were calculated:
• GSI (gonad weight/body weight *100),
• LSI (liver weight/body weight *100) and
• K, condition factor, (weight/length³ *100,000)
• Contaminant analysis on gonadal tissue from 5 fish per treatment was done by GC/MS.
• Sections of gonadal tissue were taken for histological determination of reproductive stage.
• Statistical analysis was done by regression of chemical concentration against all parameters (SAS).

Implanting a Largemouth Bass

Results

Upon receipt of contaminant analysis, it was realized that the pellets did not release a consistent dose in each treatment level and some fish had exposure to both p,p-DDE and Dieldrin.  All analysis was based upon actual chemical load in the gonadal tissue.

Discussion

Single chemical exposure of female largemouth bass (Micropterus salmoides floridanus) was employed to develop a dose response relationship between organochlorine pesticide exposure and reproductive function.  Despite the exposure method releasing inconsistent doses, GC/MS analysis of gonadal pesticide burdens provided a continuous quantitative measurement of exposure level for each fish. Induced body burdens were targeted to mimic those of largemouth bass in sites in the Ocklawaha River system where reproductive dysfunction has been observed.   Gonadal DDT derivatives and Dieldrin exposures in these areas  range from 1300 to 4200 ppb and 70 to 130 ppb, respectively (Marburger, 2002).  Achieved gonadal concentrations in this study were within or slightly higher than the target range for p,p-DDE and extremely above the target range for Dieldrin.  However, significant decreases in circulating estradiol were found only for high levels of  p,p-DDE and Dieldrin exposure.  This indicates that Dieldrin alone is not a significant endocrine modulator at concentrations found in the wild, whereas p,p-DDE may be. Lastly, the OCP burdens achieved in this study did not show any dose response relationship to health status or relative organ size of largemouth bass, indicating only a cellular/tissue level effect of high doses of p,p-DDE and Dieldrin when present as single chemicals.
 

Conclusions

• Because there was a decrease in circulating 17β-estradiol levels and not 11-ketotestosterone, one possible mechanism of toxicity for p,p-DDE and Dieldrin is a blockage of the sex steroid synthesis pathway at the conversion of androstenedione or testosterone to estradiol by aromatase.
• Other studies on wild bass populations show a decrease in not only estradiol, but 11-ketotestosterone as well. This is suggestive of a mixture effect that varies from the effects of a single chemical exposure.
• Further research is needed to test OCP mixtures that mimic exposures of wild populations in contaminated areas.
   

References

Gross, T.S., L.J. Guillette, Jr., H.F. Percival, G.R. Masson, J.M. Matter, and A.R. Woodward, 1994.  Contaminant induced reproductive anomalies in Florida alligators.  Comparative Pathology Bulletin 4:2-8.

Marburger J.E., W.E. Johnson, T.S. Gross, D.R. Douglas, and J. Di, 2002.   Residual organochlorine pesticides in soils and fish from wetland restoration areas in central Florida. Wetlands (in press).

Marburger J.E., W.E. Johnson, D.R. Douglas, and T.S. Gross, 1999.  Pesticide contamination of fish and sediments in the Emeralda Marsh Conservation Area: Relevance to fisheries establishment in flooded muck farms.  Technical Memorandum No. 31 to St. Johns River Water Management District.
 

Acknowledgements

This research was funded by the American Chemical Council. Contaminant analysis was provided by the Analytical Toxicology Core Laboratory, Center for Human and Environmental Toxicology, University of Florida. Histology provided by Histology Tech Services, Gainesville, FL.  Special thanks to Marisol Sepulveda, Carla Wieser, Shane Ruessler, Jon Wiebe, Nikki Kernaghan, Jessica Grosso, and Janet Buckland.