ORGANOCHLORINE PESTICIDES AND EMBRYONIC
MORTALITY IN ALLIGATORS AND LARGEMOUTH BASS:
INVESTIGATING CAUSAL RELATIONSHIPS AND MECHANISMS

R. Heath Rauschenberger, Maria S. Sepúlveda, Jon J. Wiebe,
Janet E. Buckland, D. Shane Ruessler, and Timothy S. Gross

Department of Physiology, College of Veterinary Medicine, University of Florida
 and U.S. Geological Survey, Florida Integrated Science Center

Presented at the Superfund Basic Research Program Annual Conference,
Tucson, AZ, November 3-6, 2002.

Abstract

Our laboratory has been using field and laboratory studies to evaluate the incidence of developmental mortality in largemouth bass (Micropterus salmoides) and the American alligator (Alligator mississippiensis) as a function of environmental exposure to organochlorine pesticides (OCPs).  The sampling areas in Central Florida have included several reference and contaminated lakes, as well as restored wetland areas that were formerly agricultural properties.  The predominant contaminants from these sites are DDT and derivatives, toxaphene, dieldrin, and chlordane. In largemouth bass, OCP exposure does not decrease spawning or hatch rates. However, our studies suggest OCP exposure increases fry mortality at the swim-up stage and causes alterations in endocrine function (reduction of sex steroids and vitellogenin) in adults.  In alligators, field studies indicate hatch rates are reduced in contaminated sites when compared to reference sites.  Similarly, reduced hatch rates have been observed in eggs of a captive, breeding population of adult alligators that received oral doses of an ecorelevant mixture of OCPs. Low hatch rates are due to increased neonatal mortality during the first 35 days of development with some mortality occurring just prior to hatching.  Analyses of alligator eggs and embryos have not indicated developmental anomalies or disease as the cause for this mortality, but rather an overall retardation of growth in clutches from high-OCP sites.  In conclusion, our field and laboratory studies suggest that reproductive alterations are occurring in two vertebrate classes exposed to OCPs and that similar mechanisms may be involved due to both classes experiencing embryonic mortality rather than infertility.  

Specific Aim 1:

Conduct field studies to determine the relative contributions of fertilization failure, losses during in ovo development, and decreased perinatal viability to decreased reproductive success in alligators and largemouth bass at impacted sites, and to determine which organochlorinated pesticides (OCP) are most closely related with adverse effects at each life stage.

Specific Aim 2:

Conduct mesocosm and laboratory treatment studies to determine the direct and multi generational effects of muck farm environments on reproduction, endocrine function, and sexual differentiation in largemouth bass, and to determine which OCP or combinations are most closely associated with adverse effects.

Specific Aim 3:

To establish the causal role and the life stage affected for selected OCPs or combinations of OCPs using controlled experimental treatment studies in alligators.

Largemouth Bass Studies

Field Study

Objective

Compare reproductive success of OCP-naturally exposed bass (Eustis Farm) with controls (Lowry-Brown Farm)

Methodology

• In early February 2001, 36 bass (1:1 sex ratio) from each site were stocked in two 0.1 acre ponds
• Endpoints Measured:
• Reproductive biomarkers: sex hormones in both sexes & gonad histology in females
• Spawning success: number nests built and fry produced to swim-up stage was monitored until mid-March

Site Description

• Lowry-Brown Farm (Low Exposure Site):
• Approx. 350 acres
• Flooded in 1992 and stocked with largemouth bass in 1994
• Bass have reproduced at or near predicted levels
• Site was pasture and palmetto for 7 years prior to flooding
• Total [OCP]* in fillet: 15 ppb
   
• Eustis Farm (High Exposure Site): 
• Approx. 1100 acres
• Flooded in 1992 and stocked with largemouth bass in 1994
• Little to no reproductive success for largemouth bass
• Site was farmed for 25 years prior to flooding
• Total [OCP] in fillet: 376 ppb

Evidence of Developmental Mortality in Bass

Spawning Results:

• Lowry bass produced 13 nests (possibly 14) and the number of fry produced to swim-up stage averaged 4,786 (900 – 9,916)
   
• Eustis bass produced 11 nests, however no fry were produced to swim-up stage from these fish

Conclusions

• Sex steroids were decreased in bass from the OCP-exposed muck farm as compared to the reference
• Although bass from both sites were capable of spawning, fry produced by OCP-exposed fish were unable to reach the swim-up stage
• Developmental abnormalities observed in 3-day old fry included alterations to the head, vertebral column, and yolk sac
• These effects may be due to direct toxicity and/or production of "poor" quality eggs/embryos

           Pond (0.1 acre) with spawning mats ....    

Laboratory Study

Objective

Evaluate, under controlled conditions, the effects of two of the most common OCPs (DDE and Dieldrin) on reproductive success of largemouth bass

Methodology

• Between December and January 2002, 700 adult bass were implanted with slow-releasing pellets (Innovative Research Inc.) that contained the following chemicals:
• Controls, DDE (2.5, 5.0, 10 ppm), Dieldrin (0.25, 0.5, 1.0 ppm)
• Bass were implanted with either 60-day-releasing pellets or 90-day-releasing pellets
   
• Endpoints Measured:
• Growth and survival
• Reproductive biomarkers: sex hormones, vitellogenin, gonad histology
• Reproductive success:
• Number of nests produced
• Egg Size
• Survival at swim-up stage
• Fry growth
• Frequency of deformities
• Concentration of DDE and Dieldrin were measured in gonads/eggs (approx. 10 females/treatment)

Results

Percent Mats With Eggs

DDE and Dieldrin Concentrations Elicited in Eggs via Implanted Pellets (* no. of females)

Conclusions- Bass Studies

• OCP burdens can be elicited in eggs via the implantation of time-release OCP pellets in adult female bass.
• The effects observed in the field study are possibly the result of exposure to complex mixtures since exposure to two OCPs did not elicit same effects as field study.
• The high variability in clutch parameters in the pellet study suggests future studies should incorporate larger samples.

Alligator Studies

Embryo Morphometric, Histopathology, and Hatching Studies

• Hatching Study
• Monitor hatch rates and egg viability across reference and contaminated sites
   
• Morphometric Study
• Eggs are opened at specific develop. stages and embryos are measured (Mass, Head Length, Total Length, Eye Length, Distance b/t Limbs)
   
• Histopathology Study
• Embryos from Morph. Study are used to evaluate: Organogenesis, presence of lesions and lesion types and affected organs
   
• Unbanded egg / Blastodisk Study
• Prior studies by our lab (Beverly Arnold and Dave Rotstein) indicate the presence of paternal DNA contribution to blastodisks obtained from unbanded eggs indicating very early embryonic mortality
• Obj. of blastodisk study is to confirm through histological examination whether unbanded eggs are unfertilized eggs or are the result of early embryonic mortality
   
• Procedure
• Collected 10 – 25 clutches per site (total 4 sites)
• Stage of Development is determined
• 2 egg yolks per clutch are collected for OCP analysis
• Each clutch is split randomly for hatching and developmental studies (Morphometric Study + Histo. Study)
   

• Maternal Body Burden/Maternal Transfer Study
• Examine the relationship between maternal exposure (body burdens) and embryonic mortality
   
•  Determining the use of egg yolk [OCP] to predict maternal tissue [OCP]
   
•  Examination of organ-somatic indices as one method of determining if female is affected by OCP exposure

CAPTIVE ADULT ALLIGATOR STUDY

• Methodology for Captive Alligator Study
• 24 adult alligators were distributed across 8 pens (1 male:2 females)
• Four breeding groups were not treated with OCPs (control group)
• Four breeding groups received OCP treatment via dietary exposure (treated group)
• Eggs were collected after oviposition
• Two eggs were sampled to determine OCP concentrations
• Remaining eggs were artificially incubated to determine clutch viability (# live hatchlings / # eggs incubated)

CONCLUSIONS – Alligator Studies

Hatching and Contaminant Survey Studies

• Indicate the continued trend of decreased hatch rates observed in clutches obtained from OCP contaminated sites
   
• Indicate the continued trend of elevated OCP concentrations in maternal tissues and egg yolks obtained from OCP contaminated sites
   
• Indicate that PCB and PAH concentrations are very similar across all sites (OCP-clean & OCP contaminated)

Morphometric Study

• Indicates gross deformities occur but, do not appear to be the leading cause of embryo death
   
• Indicates that growth retardation is occurring in clutches with low hatch rates and in those exposed to OCPs

Maternal Transfer Study

• Indicates that OCPs are maternally transferred to embryo and that maternal tissue burdens can be estimated from yolk burdens via linear regression analyses

Captive Adult Alligator Study

• Indicates that OCP concentrations similar to those found in wild eggs from contaminated sites can be reproduced in captive eggs by exposure of captive, adult female alligators to OCPs.
   
• Indicates that poor hatch rates similar to those observed in wild clutches from contaminated field sites can be reproduced in captive clutches of captive, adult female alligators exposed to OCPs.
   
• Underscores maternal transfer as the route of embryonic OCP exposure [instead of OCPs from external media passing through the eggshell to the embryo].
   
• First study to induce endogenous embryonic OCP exposure (via maternal transfer) in a crocodilian model (previous studies have used topical eggshell painting or egg injection techniques).

ACKNOWLEDGMENTS

This research was supported by NIEHS-SBRP grant # P42-07375 to T.S. Gross. The authors would like to thank C. Wieser, T. Smith, J. Grosso, A. Woodward, N. Kernaghan, J. Basto, A. Harvey, J. Muller, J. Noggle, and P. Wilkinson for help in collecting alligators and eggs.  We thank D. Gross for conducting the necropsies. Analytical chemistry was conducted at the Analytical Toxicology Core Laboratory, Center for Environmental and Human Toxicology, University of Florida. Alligators and eggs were collected under permit from the Florida Fish & Wildlife Conservation Commission. All laboratory work was performed in full compliance with guidelines set forth by the University of Florida Institutional Animal Care and Use Committee and the USGS-FISC Animal Care and Use Committee.