Ecotoxicology of Morelet's crocodile in Belize

Over the last two decades, population declines and reproductive impairment have been observed in American alligators (Alligator mississppiensis) inhabiting Lake Apopka, a highly contaminated lake in Florida, USA. Juvenile alligators from the lake have exhibited altered sex-steroid hormone concentrations, abnormal gonadal morphology, and reduced phallus size compared to alligators from a reference lake. No direct cause-effect relationship has been established between these reproductive and endocrine anomalies and environmental contaminants, but results of laboratory and field investigations suggest the potential for contaminant-induced endocrine disruption at various levels of organization in these animals. Although various environmental contaminants considered to be endocrine disrupters have been found in eggs and tissues of crocodilians worldwide, no studies have yet investigated endpoints of endocrine disruption in wild crocodilians outside of Florida. The primary objective of this study was to address this data gap by examining ecotoxicological endpoints in another crocodilian species living in habitats contaminated with endocrine-disrupting chemicals (EDCs), and where appropriate, compare results from this study with those observed for alligators in Florida.

During a pilot study in 1995, multiple organochlorine (OC) pesticides considered to be EDCs were found in eggs of Morelet's crocodiles (Crocodylus moreletii) from three localities in northern Belize. Based on these findings and previous data from Florida showing egg contamination, population declines, and reproductive abnormalities in alligators exposed to many of the same chemicals, a multi-year study was initiated to examine various endpoints of contaminant exposure and response in Morelet's crocodiles living on contaminated and reference sites in northern Belize. Gold Button Lagoon, a man-made lagoon from which contaminated crocodile eggs were collected in 1995, was selected as the contaminated site for this study, while New River Watershed, a more remote site with fewer anthropogenic inputs than Gold Button Lagoon, was selected as the reference site.

Three primary endpoints of endocrine disruption were evaluated in this study. First, vitellogenin induction was examined as an endpoint of exposure to exogenous estrogens or estrogen-mimicking contaminants. Vitellogenin is an egg-yolk precursor protein expressed in all oviparous and ovoviviparous vertebrates. Male and juvenile females normally have no detectable concentration of vitellogenin in their blood but can produce it following stimulation by an exogenous estrogen, such as an EDC. Thus, the presence of vitellogenin in the blood of male or juvenile female crocodiles can serve as an indicator of exposure to an estrogen-mimicking EDC. Of 358 males and juvenile females sampled in this study, no vitellogenin induction was observed, suggesting these animals were likely not exposed to estrogenic xenobiotics. However, many of the animals sampled were later found to contain OC pesticides in their caudal scutes, confirming they had in fact been exposed to OCs (and EDCs). These data suggest the lack of a vitellogenic response should not necessarily be interpreted as an indication that no exposure or other contaminant-induced biological response has occurred.

Second, plasma sex-steroid hormone concentrations were examined as an endpoint of response to EDC exposure in crocodiles from the two study sites. The selection of this endpoint was based on numerous studies reporting altered concentrations of estradiol-17P (E2) and testosterone (T) in alligators from Lake Apopka and other contaminated lakes in Florida. In the present study, few inter-site differences in plasma hormone concentrations were noted. No significant differences in plasma E2 concentrations were detected between sites. However, juvenile males and females from the contaminated site exhibited significantly reduced plasma T concentrations compared to juvenile males and females from the reference site, respectively. This finding was consistent with results from previous studies on alligators in Florida. No other inter-site differences in hormone concentrations were observed. Relationships between body size and hormone concentrations were variable and showed no clear pattern.

Third, male phallus size was examined as a second endpoint of response to EDC exposure in crocodiles from the two study sites. Concurrent with reductions in plasma T concentrations, male alligators from Lake Apopka and other contaminated lakes in Florida have exhibited smaller phallus size compared to animals from a reference lake. Researchers speculate that abnormal hormone concentrations during early life stages may affect anatomical structures dependent on these hormones for proper growth and development (i.e., genitalia). p,p '-DDE, a known anti-androgen, has been detected in alligator eggs and serum from Lake Apopka and was also detected in eggs and scutes of Morelet's crocodiles from the two Belize study sites. Thus, in the present study, male crocodile phallus size and plasma T concentrations were examined as endpoints of response to p,p '-DDE exposure as well as exposure to other contaminants. No differences in mean phallus size were observed between sites, whereas mean plasma T concentrations in juveniles from Gold Button Lagoon were significantly reduced compared with those from New River Watershed.

It was discovered late in the study that New River Watershed exhibited a contaminant profile similar to that observed at Gold Button Lagoon, with multiple OCs detected at similar concentrations in sediments, crocodile eggs, and crocodile caudal scutes at both sites. With the lack of a suitable reference site, it is thus unclear if steroid hormone concentrations and male phallus size observed in this study are within the normal range exhibited by Morelet's crocodiles living in non-contaminated habitats or if they are altered in some way (e.g., increased, reduced). In addition, it is also unclear if inter-site differences in plasma T are the result of exposure to EDCs, natural variation, one or more undetermined factors (e.g., stress), or a combination of these factors.

In general, the results of this study indicate few or no effects of EDC exposure on Morelet's crocodiles inhabiting contaminated wetlands in northern Belize. However, multiple uncertainties encountered in this study make inter-site and inter-study (crocodile to alligator) comparisons difficult and some results equivocal. Thus, the potential effects of EDCs and other contaminants on crocodiles inhabiting these sites should not be assumed to be negligible. Long-term studies are essential to adequately assess the effects of EDCs on crocodilian populations, as these animals are long-lived and many contaminant-induced effects are organizational in nature, occurring during embryonic development but not appearing until later in life.