An Evaluation of Environmental Contaminants and Developmental Toxicity for the American Alligator
 in Central Florida

Maria Sepúlveda, Jon Wiebe, Alfred Harvey, James Basto,
Shane Ruessler, Elaine Roldan, and Timothy Gross

Department of Physiological Sciences, College of Veterinary Medicine,
University of Florida, Gainesville, FL, 32611

USGS - Florida Integrated Science Center, Gainesville, FL 32653.

Presented at the Society of Toxicology, 40th Annual Meeting, San Francisco, California.  March 25 – 29, 2001.

Abstract

         Previous studies have reported decreased egg viabilitiy/hatch rates and increased neonatal mortalities for American alligators (Alligator mississippiensis) from Lake Apopka, Florida. The cause(s) for these developmental effects are unclear, but it is possible that they are related to exposure to environmental contaminants. The present study evaluated alligator clutch, egg, and hatchling parameters in relation to exposure to organochlorine pesticides (OCP) in different Lakes from central Florida. Alligator egg clutches (24 - 35 per site) were collected from lakes Apopka, Griffin, and from the reclaimed/flooded agricultural properties adjacent to Lake Griffin (Emeralda Marsh). Orange Lake served as the reference site for this study. Clutches were monitored for fecundity, number of unbanded eggs, hatch rates, and embryonic mortality. Non-viable eggs were opened to estimate the stage of embryonic loss, and neonatal mortality classified as either pre-hatch (early: 1 – 35 days; late: 36 – hatching) or post-hatch. Several morphological parameters were measured from a subset of eggs and hatchlings.  In addition, [OCP] were measured in over 100 yolks representing different stages of development and viability conditions (live, unbanded, and dead).  Yolk [OCP] were ranked in the order: Apopka > Emeralda > Griffin > Orange, and the primary pesticides observed were: DDE and DDD, toxaphene, cyclodiene pesticides (mainly dieldrin), chlordanes, mirex, and methoxychlor.  Fecundity was increased and hatch rates were reduced in Apopka, Emeralda, and Griffin clutches when compared to Orange Lake clutches.  Low hatch rates in these sites were due to increased neonatal mortality, mainly during the first 35 days of embryonic development. Lower hatch rates, however, were not related to increased yolk [OCP], although there was a positive relationship between [DDE] and percentage of unbanded eggs. Live eggs had similar [OCP] when compared to non-viable eggs (unbanded and early embryonic mortality).  In addition, higher [OCP] in yolk did not seem to result in eggshell thinning nor in the production of smaller hatchlings. Although almost nothing is known about [OCP] in alligator eggs and their effects, [OCP] in this study fell below those known to induce reproductive alterations in different fish and avian species.  Environmental contaminants, however, may still be playing a role in the observed decreased egg viabilities by affecting normal reproductive function (vitellogenesis) in females. In addition, it remains unknown whether these pesticide mixtures might be acting in more than just an additive manner, exerting direct toxicity to the developing embryos.

Objectives

The overall objective of this project was to:

v     Conduct field epidemiologic studies to evaluate embryonic development and mortality of the American alligator as a function of parental exposure to organochlorine pesticides (OCP) in Central Florida.

Materials and Methods

Study Design:
         Between June and July 2000, alligator egg clutches (24 to 35 per site, total of 111 clutches) were collected from lakes Apopka, Griffin, and from the reclaimed/flooded agricultural properties adjacent to lakes Griffin (Emeralda Marsh).  Orange Lake served as the reference site for these studies (Fig. 1). From previous studies, it was known that OCP concentrations in alligator tissues are highest in animals from Apopka and Emeralda, with intermediate and low concentrations in alligators from Lakes Griffin and Orange, respectively. In the laboratory, eggs were candled to determine whether they were fertile or "banded", and if so, nested in moist sphagnum moss and incubated at 30.5^oC and 99% humidity in an artificial incubation building.  One egg/clutch was opened to determine stage of development.  The following parameters were recorded from all clutches: fecundity (clutch size); percentage of "unbanded" eggs (which probably result from very early embryonic mortality, [(number of unbanded eggs)/(fecundity) x 100]; percent hatchability [(number of eggs that hatched)/(fecundity) x 100], and neonatal mortality. Eggs were opened when diagnosed as non-viable to estimate the stage of embryonic loss, and neonatal mortality was classified as either pre-hatch (early: 1-35 days of development; late: 36 - hatching), or post-hatch.  Eggshell thickness and hatchling parameters (body weights, total length, snout-vent length, and head lengths) were also measured in a subset of eggs/hatchlings.  A total of 103 yolks from the three most impacted sites (Apopka, Emeralda, and Griffin) representing different stages of egg development and mortality, were analyzed for OCP concentrations via GC-MS.

Statistical Analyses:
         Analysis of variance (ANOVA) followed by a Tukey's test was used to evaluate differences on clutch parameters across sites. Since the concentration of some OCP in yolk was affected by age of development ( Fig. 2 ), differences in contaminant levels across sites were determined using analysis of covariance (ANCOVA). Linear regressions of clutch, egg, and hatchling parameters and yolk OCP concentrations were also performed.  Statistical significance was declared at p < 0.05.

Conclusions

v   Yolk [OCP] were ranked in the order: Apopka > Emeralda > Griffin > Orange, and the primary pesticides observed were: DDE and DDD, toxaphene, cyclodiene pesticides (mainly dieldrin), chlordanes, mirex, and methoxychlor (Fig. 2).
v   Fecundity was increased and hatch rates were reduced in Apopka, Emeralda, and Griffin clutches when compared to Orange Lake clutches.  Low hatch rates in these sites were due to increased neonatal mortality (Fig. 3).
v Lower hatch rates, however, were not related to increased yolk [OCP], although there was a positive relationship between [DDE] and percentage of unbanded eggs (Table 1).
v Live eggs had similar [OCP] when compared to non-viable eggs (unbanded and early embryonic mortality) (Fig. 4).
v Higher [OCP] in yolk did not seem to result in eggshell thinning nor in the production of smaller hatchlings (Table 1).
v [OCP] in this study fell below those known to induce reproductive alterations in different fish and avian species ^(1,2).
v Environmental contaminants, however, may still be playing a role in the observed decreased egg viabilities by affecting normal reproductive function (vitellogenesis) in females.  In addition, it remains unknown whether these pesticide mixtures might be acting in more than just an additive manner, exerting direct toxicity to the  developing embryos.

References

1. Jarvinen A.W., and G.T. Ankley.  1999. Linkage of effects to tissue residues: development of a comprehensive database for aquatic organisms exposed to inorganic and organic chemicals. Pensacola FL: SETAC.  364 pp.
2. Beyer, W.N., G.H. Heinz, and A.W. Redmon-Norwood.  1996. Environmental contaminants in wildlife: interpreting tissue concentrations.  Pensacola FL: SETAC.  494 pp.

Acknowledgements

v NIEHS, Superfund Project: "Chlorinated pesticides and developmental mortality in wildlife".
v University of Florida, Department of Physiological Sciences, College of Veterinary Medicine.
v USGS - Florida Integrated Science Center.
v Florida Fish and Wildlife Conservation Commission.