| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Forestry & Natural Resources and School of Civil Engineering, 195 Marsteller St., Purdue University, West Lafayette, Indiana 47907, USA
2 University of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology & Department of Clinical Studies, New Bolton Center, 382 West St. Rd., Kennett Square, Pennsylvania 19348, USA
3 USGS-BRD Florida Integrated Science Center, Center for Aquatic Resource Studies, 7920 NW 71st St., Gainesville, Florida 32653, USA
4 U. S. Fish & Wildlife Service, North Florida Field Office, 6620 Southpoint Dr. South, Suite 310, Jacksonville, Florida 32216, USA
5 Corresponding author (email: mssepulv{at}purdue.edu)
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
|---|
In the last decade, several reports have linked OCPs to endocrine-disrupting effects and population declines in American alligators (Alligator mississippiensis) inhabiting northcentral Florida lakes. Reported endocrine-disrupting effects in alligators from Lake Apopka have included altered secondary sex characteristics, endocrine status, and sex differentiation in hatchlings and juveniles (Gross et al., 1994; Guillette et al., 1994 , 1995, 1999a, 1999b). More recently, increased alligator mortalities have also been described from another Florida lake, Lake Griffin. Since 1997, over 300 adult alligators from this lake have been found either dead or lethargic and unresponsive (Schoeb et al., 2002). Although the ultimate cause(s) for these mortalities has not been determined, detailed clinical and pathological examinations have indicated that the nervous system was primarily affected.
To understand better why populations of American alligators are declining at these sites, a series of field and laboratory studies have been conducted over the last decade by this and other research groups. Field studies have shown significant exposure of alligators to OCPs, especially in animals inhabiting reflooded muck-farm environments (Rauschenberger et al., 2004; Sepúlveda et al., 2004). The most notable finding so far has been the observation that these populations produce embryos that die before or soon after hatching (Woodward et al., 1993; Masson, 1995; Giroux, 1998; Rauschenberger et al., 2004). Thus, our overall working hypothesis is that one or more OCPs found at these sites are causing decreased early-life-stage survivorship in animals, and that this decline is the likely cause for the low recruitment observed at these sites. To date, however, a clear relationship among OCPs and early-life-stage mortality has not been established. Furthermore, it is not clear if effects on offspring quality and survival are the result of direct exposure of early-life stages to OCPs accumulated in the yolk through maternal transfer and/or to impairment of the parent’s ability to produce quality eggs and sperm due to OCPs or to other environmental factors.
The main objective of this study was to determine the ultimate cause of mortality of American alligator late-stage embryos and hatchlings through the conduction of detailed pathological examinations, and to evaluate better the role of OCPs in these mortalities.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
|---|
As part of a long-term health-monitoring program of American alligator populations by our laboratory, egg clutches were collected from several northcentral Florida lakes over the course of two nesting seasons (June/July 2000 and 2001). Lakes Apopka (28°37'N, 81°37'W), Griffin (28°50'N, 81°51'W), and Lochloosa (29°27'N, 82°10'W) and the Emeralda Marsh Conservation Area (28°57'N, 81°48'W), a reclaimed/flooded agricultural marsh east of Lake Griffin, were selected as collection sites. Prior studies by our laboratory (conducted between 1999 and 2002) have indicated vastly different levels of OCP exposure and early-life-stage mortality across these sites. Lake Lochloosa was selected as the reference site because of its low level of OCPs (overall mean for total OCPs in yolk of 231± 30 µg/kg) and high clutch viability, defined as the number of live hatchlings/number of eggs in the clutch (71± 26%) (Rauschenberger et al., 2004). Lake Griffin was selected as an intermediate OCP-exposure site because yolk concentrations average 4,414± 617 µg/kg, and Lake Apopka and Emeralda were selected as high OCP-exposure sites because yolk concentrations average 15,911± 1,786 µg/kg and 15,238± 1,787 µg/kg, respectively (Rauschenberger et al., 2004). In addition, clutch success on Lakes Apopka and Griffin and on Emeralda are below those observed at the reference Lochloosa site (51± 31%, 44± 33%, and 52± 35%, respectively) (Rauschenberger et al., 2004).
Nests were located by aerial survey (helicopter) and/or from the ground (airboat). Prior to their collection, eggs were marked on the top surface with a pencil. Because alligator embryos attach to the top surface of the egg very early during development, marking them prevents from positioning the embryo on the bottom surface of the egg and causing asphyxiation. Complete clutches were transported to the laboratory in plastic pans (48 x 36 x 17 cm) filled with the original nest substrate material and covered with lids that had holes for ventilation. Upon arrival (<8 hr after collection), eggs were candled, and, if viable, placed with the pencil markings facing up in the same plastic tubs. Original nest material was removed from each pan and replaced with sphagnum moss as substrate. Clutches were incubated in an artificial incubation building (7 x 4 m) at a temperature and relative humidity of 30–33 C and 88–92%, respectively. This intermediate incubation temperature will normally result in a 1 : 1 male : female sex ratio (Ferguson and Joanen, 1982).
Eggs were classified as nonviable if they were cracked, lacked a noticeable band around the equatorial center of the egg (also referred to as an unbanded egg), or were banded but contained a dead embryo. The latter eggs are easily distinguishable from live-banded eggs because, when candled, blood vessels from the chrorioallontoid membrane are usually absent and contents appear dark due to a decline in light transmittance. In addition, one viable egg was randomly selected from each clutch, opened, and the embryo collected to determine the age of the clutch. Yolks from these eggs were also collected for later toxicologic analyses (see below).
Monitoring of embryo and hatchling survival
During the course of the ~65-day incubation period, clutches were monitored for embryo survival by candling eggs approximately every 2 wk. Although many embryos died earlier during development, data from these animals are not presented here because it is the subject of a separate publication. Rather, this study focused on determining cause of death in full-term embryos and hatchlings. Full-term or late-stage dead embryos (days 56–65 of age) were those produced from eggs that failed to hatch within a 2-day period after the last egg of a particular clutch had hatched. Animals that died in the process of hatching were also included in this group. In most cases, these were hatchlings that had begun cracking the eggshell and had their heads partially or totally out of the egg. These late-stage embryos were fully necropsied as described below.
Hatchling survival was also monitored for up to 4 wk of age. Two days posthatch, live hatchlings were weighed and measured, tagged, and transferred to indoor rectangular (137 cm x 76 cm x 61 cm) or circular (122 cm x 76 cm) holding tanks. Animals were housed by clutch in groups of 10, thus available floor space was ~0.1 m2 per hatchling. Tanks were filled with clean well water to a depth of ~10 cm, provided with a heating lamp set up for a light cycle of 12 hr light:12 hr dark, and kept clean of debris by rinsing them with well water every other day. As tanks were maintained on a slope, animals had access to both dry and wet environments. Ambient and water temperatures ranged from 28 to 35 C. Tanks were checked daily for the presence of dead hatchlings, which were measured and necropsied as explained below. Animals were not fed during the first 2 wk posthatch because they were expected to use their yolk reserves during this time. At 2 wk of age, hatchlings were fed a pelleted diet (AquaXcelTM4710, Burris Mill & Feed, Inc., Franklinton, Louisiana, USA) every other day ad libitum. At the end of this monitoring period, survivors were returned to their original nest location using geographical positioning system (GPS) information gathered at the time of egg collection. Overall, clutch production was calculated as the total number of hatchlings that survived 1 mo posthatch over the total number of viable eggs set for incubation times 100.
Animal measurements and necropsies
Prior to necropsy, dead late-stage embryos and hatchlings were weighed to the nearest 0.1 g, and head length (HL; base of the skull to tip of the nose), total length (TL; tip of the nose to tip of the tail), and snout-vent-length (SVL; proximal end of vent to tip of snout) were measured to the nearest 0.1 mm with digital calipers. Necropsies were thorough and consisted of measuring several organ weights as well as collecting tissues for histopathologic and microbiologic analyses. Liver, heart, kidneys, lungs, yolk, and fat body were excised from all animals and weighed to the nearest 0.01 g. In crocodilians, the fat body is a morphologically distinct structure located in the coelomic cavity, immediately posterior to the liver. Because body weights differed significantly both within and across clutches, organ weights were corrected by differences in body weight through the calculation of organosomatic indexes. These indices are commonly employed in aquaculture studies that have examined effects of different environmental stressors on the health of aquatic organisms (e.g. Shoemaker et al., 2003; Sang and Fotedar, 2004). Organosomatic indexes were calculated by dividing the weight of the organ by the weight of the animal and multiplying the resulting number by 100. Tissues routinely collected for histopathologic analyses included brain, spinal cord, trachea, lungs, heart, esophagus, stomach, small and large intestines, pancreas, liver, spleen, kidneys, thyroid, thymus, adrenal glands, and tail muscle. In some instances, blood and tissue swabs were also collected for microbiologic analyses. These procedures are explained in more detail below. Because animals were hatched under controlled captive conditions, it was considered unlikely that they would have been parasitized, and thus routine parasite screens were not conducted. In addition, no signs of parasitism were observed during the necropsies.
Histopathology, histochemistry, and indirect immunohistochemistry
Histopathologic analyses were conducted at the Departments of Pathobiology and Clinical Studies, New Bolton Center, University of Pennsylvania. Formalin-fixed (10% buffered formalin) and trimmed tissues were embedded in paraffin, sectioned at 5 µm, mounted on glass slides, air dried, and stained with Mayer’s hematoxylin and eosin (H&E). Selected tissues with lesions were also examined with Gram stain, for better bacterial characterization and with Giemsa stain for Mycoplasma-like organisms and Chlamydiaceae elementary-body identification. Positive controls included the trachea of chickens infected with Mycoplasma gallisepticum and the liver of a bird infected with Chlamydophila psittaci. Selected tissue sections with visible bacteria and inflammation where stained with indirect immunohistochemistry to detect Chlamydophila spp. and Listeria monocytogenes, using primary polyclonal antibodies against specific epitopes of these bacteria. Positive controls included avian tissues infected with Chlamydophila spp. and sheep brain infected with L. monocytogenes. Negative controls included specific pathogen-free tissues and the omission of the primary antibody.
Microbiology
Samples for aerobic/anaerobic bacterial and fungal cultures were collected at necropsy (heart, lung, liver, intestines, and cloaca) from a subset of the animals using CulturetteSwabs® (Becton-Dickinson, Franklin Lakes, New Jersey, USA). Prior to the exposure of organs, the skin was scrubbed with cotton soaked in 70% ethanol. The skin was then cut with a sterile blade, and using flamed scissors and forceps, organs of interest were exposed and a swab sample collected. In addition, blood samples were collected antemortem from the occipital sinuses of sick and dying hatchlings using a sterile 1-ml tuberculin syringe fitted with a 27-gauge 1/2-inch needle after vigorous scrubbing of the skin with cotton soaked in 70% ethanol.
Cultures were performed at the University of Florida’s College of Veterinary Medicine Microbiology laboratory. All samples were plated to the following media: Columbia 5% sheep blood, MacConkey, Columbia CNA, Mycobiotic and Emmons Sab Dex agar. Cultures were incubated at 37 C and examined at either 24 and 48 hr (aerobic) or 48 hr (anaerobic) postinoculation. Anaerobic cultures were conducted using a Bio-BagTM Type A (Becton Dickinson). Blood samples were incubated at 37 C for 30 days and plated to the above media at 24 hr, 4 days, and 21 days postinoculation. Bacterial colonies were identified by Gram staining, morphology, and/or commercial biochemical testing (API®, bio-Meriéux, Hazelwood, Missouri, USA; and RapIDÖANA II Panel®, Remel, Lenexa, Kansas, USA). Fungal cultures were held for 30 days at 32 C and isolates were identified using scotch-tape preparations.
Toxicology
One viable egg was randomly collected from each clutch, opened, and yolk contents stored in glass amber vials at –20 C until analyzed for OCPs as described in Rauschenberger et al. (2004). Previous work done in our laboratory has shown that OCP concentrations in alligator yolk does not differ across eggs within a particular clutch (Gross, unpubl. data). Thus, yolk OCP concentrations in a randomly selected egg are representative of the whole clutch. Briefly, yolks were first homogenized and a 2–5-g sample extracted into ethyl acetate. Samples were purified using C18 and NH2 SPE (solid-phase extraction) cartridges, and pesticide concentration determined using a gas chromatograph (Hewlett Packard HP-6890, Wilmington, Delaware, USA) and a mass spectrometer (HP 5973) in electron impact mode according to U.S. Environment Protection Agency (EPA) method 8270C (USEPA, 1990). Identification of all analytes and quantitation for toxaphene was conducted in full-scan mode, where all ions are monitored. To improve sensitivity, selected ion monitoring (SIM) was used for the quantitation of all other analytes, except kepone. Samples were analyzed at least three times. For quantitation, a five-point standard curve was prepared for each analyte (R2
0.99). Fresh curves were analyzed with each set of 20 samples. Each standard and sample was fortified to contain a deuterated internal standard, 5 µl of US-108 (120 µg/ml; Ultra Scientific), added just before analysis. All samples also contained a surrogate, 2 µg/ml of tetrachloroxylene (Ultra Scientific) added after homogenization. Duplicate quality-control samples were prepared and analyzed with every 20 samples (typically at a level of 1.00 or 2.50 µg/ml of 
-BHC, heptachlor, aldrin, dieldrin, endrin, and p, p'-DDT) with an acceptable recovery ranging of 70–30%. Repeated analyses were conducted as allowed by matrix interferences and sample availability. Pesticide concentrations in this study are presented as nonlipid normalized wet-weight values.
Statistical analyses
Statistical analyses were performed using SAS 9 software (SAS, 2002). Two-way analyses of covariance (ANCOVA) (PROC GLM) were used to test the effects of site and animal age (full-term embryo vs. hatchling) or animal condition (live vs. dead) on the dependent variables (body measurements and organosomatic indices). Clutch identification was used as a covariate in these analyses. Total OCPs were compared across sites using a one-way analysis of variance (ANOVA). The ANOVAs were followed by pairwise comparisons using Tukey’s multiple comparison tests. The frequency of abnormalities was compared across sites and levels of OCP contamination using chi-square tests (PROC FREQ). Data are reported as mean± SEM unless otherwise indicated. Statistically significant differences were declared at P
0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
|---|
). Late developmental and post-hatch mortality of necropsied alligators differed across sites and was highest in Lake Griffin (121 animals from 12 clutches, 24%), followed by Emeralda (56 animals from four clutches, 9%), Lake Apopka (56 animals from seven clutches, 7%), and Lochloosa (three animals from two clutches, 1%).
|
). Early through midincubation embryo mortality (up to day ~57 of incubation) was included for this calculation, although, as already discussed, these embryos were not further examined as part of this study. As expected, clutch production followed an opposite trend to that of embryo and hatchling mortality (necropsied animals only) and was lowest in Griffin and Emeralda (33± 10% and 35± 20%, respectively), intermediate in Apopka (57± 10%), and highest in Lochloosa (78± 18%) (Table 1).
The developmental stage at which these mortalities occurred also differed across sites, with dead animals being composed of mostly full-term embryos (60%) in the case of Lake Griffin or hatchlings (79–88%) in the case of Lake Apopka and Emeralda. All three dead animals from Lochloosa were late-stage embryos (Table 1).
Comparisons of body measurements and organosomatic indices between late-stage embryos and hatchlings within and across sites are summarized in Table 2. Regardless of site, there was an ~7% increase in HL, SVL, and TL between late-stage embryos and hatchlings (from 34 to 36, 11 to 12, and 22 to 24 cm, respectively). In terms of organosomatic indices, some did not change with age (fat body and kidney, 0.35–0.49 and 1.0–1.1%, respectively), whereas others decreased (yolk somatic index, overall decrease of 51%, from 18% to 8.7%) or increased (liver: 13%, 2.8–3.2%; lung: 43%, 0.8–1.4%; and heart: 25%, 0.6–0.8%). Changes in body weight and organosomatic indices between both age classes, however, were not consistent across sites. For instance, declines in body weight between late-stage embryos and hatchlings were only observed in Griffin animals (decline of 15%, from 48 to 41 g). Similarly, declines in yolk somatic index were most pronounced in Emeralda (65%), and Griffin (49%) animals compared to Apopka (24%) (Table 2). A similar but opposite trend was observed with lung and heart somatic indices, with overall increases of 45 and 26% and of 43 and 25% for Griffin and Emeralda, respectively, compared with an increase of only 15 and 7% for the same indices in Apopka alligators.
|
. Lochloosa animals were not included in these analyses because of small sample size. From this comparison, differences in body measurements across sites were most evident in hatchlings compared with late-stage embryos. Indeed, the size of late-stage embryos did not differ among the three sites studied (overall means of 48 g for body weight and 34, 11, and 23 cm for HL, SVL, and TL, respectively), whereas hatchlings from Lake Apopka were significantly heavier when compared with hatchlings from Lake Griffin and Emeralda (50 vs. 42 g). Although Apopka hatchlings were also larger (HL: 37 vs. 36 cm; TL: 25 vs. 24 cm), these 1-cm differences might not be biologically significant. The SVL in hatchlings did not differ across sites (overall mean of 12 cm). There was an opposite trend in organosomatic indices across sites between late-stage embryos and hatchlings. For instance, liver, kidney, and heart somatic indices did not differ in late-stage embryos from the different sites (overall means of 2.8, 1.0, and 0.6%, respectively), but were increased in hatchlings from Griffin compared with Apopka and Emeralda for liver and kidney somatic indices (3.4 vs. 3% and 1.3 vs. 1%) and from Griffin and Emeralda compared with Apopka for heart somatic index (0.8 vs. 0.6%). In contrast, fat somatic index differed across sites only in late-stage embryos (overall means of 0.8, 0.3, and 0.2% for Apopka, Griffin, and Emeralda, respectively) with hatchlings having an overall mean of 0.5% regardless of site. A similar trend was observed for lung somatic index (overall means of 1.1% for Apopka and of 0.8% for Griffin and Emeralda late-stage embryos, with no differences across sites for hatchlings [1.4%]). Finally, yolk somatic index was increased significantly in late-stage embryos from Emeralda (24%) compared with same-age alligators from Lakes Griffin (18%) and Apopka (11%) and did not differ across sites in hatchlings (overall mean of 8.7%) (Table 2).
Differences in body measurements between live (2 days posthatch) and dead hatchlings (late-stage embryos) are presented in Figure 1. From this comparison, there was a significant decline in body weights in dead hatchlings from Griffin (a 23% decline, from 52 to 40 g) and Emeralda (a 16% decline, from 51 to 43 g) compared with their live counterparts (P<0.0001, F = 32, df = 5). There were no differences in body weights between dead and live Apopka hatchlings (52 and 50 g, respectively). Head length was decreased in dead hatchlings regardless of site (P<0.0001, F = 19, df = 5), although this decline was most evident again in Griffin and Emeralda (a 2.5% decline, from 36.5 to 35.6 cm, and a 5% decline, from 37.4 to 35.6 cm, respectively) compared with Apopka animals (a 1.6% decline, from 37.1 to 36.5 cm). There were no differences in TL or SVL between dead and live hatchlings.
|
). The most prevalent condition was the presence of edema (80 animals affected, which corresponds to an overall prevalence of 34%), which was either localized internally around the heart and lungs (32% of the edema cases) or abdominal cavity (ascites, 9%), and/or externally affecting limbs (30%) and jaw and neck (29%). Internal edema was evidenced as a layer of white opaque fibrinous material surrounding the lungs and heart. The second most prevalent abnormality was hyperemic or congested organs (73 animals, 29% prevalence), with 80% of cases having hyperemia of the yolk sac (43%) and lungs (37%), and the remaining 20% having hyperemia of the kidneys (8%), gastrointestinal tract (7%), and liver (5%). Severely emaciated alligators were observed at a prevalence of 14%, with over half of the cases diagnosed in Griffin animals. Gross deformities were observed at a very low prevalence (eight animals, 3% prevalence) and consisted of vertebral (scoliosis) and tail (kinked) abnormalities at 31% prevalence each; the presence of extra digits (23%); and conjoined twins (15%). Supernumerary digits consisted of up to three extra digits/limb, and most cases were animals that had abnormal forelimbs (i.e., curved, edematous, or abnormal size). Conjoined twins were attached to each other through the yolk sac and were late-stage embryos that died before hatching. Approximately 9% of the dead animals examined were also diagnosed with other abnormalities, which included oral pin-point, white granulomas (47%); gout, evidenced as the presence of enlarged kidneys and whitish cream prominent tubular contents (33%); liver and lung granular foci of 1–2 mm in diameter (8 and 4%, respectively); and heart petechiae (8%).
|
). Over half of the histopathologic changes were lung lesions, mostly heterophilic pneumonias (30%), considered of bacterial origin because of microscopic observation of bacterial organisms, with or without the presence of edema (16%) and atelectasis (9%). Other lesions included the presence of hepatocellular lipidosis, or fatty liver (12%); presence of bacteria in the blood or septicemia (8%); and generalized inflammations of the gastrointestinal tract (gastritis and enteritis, 7%) and kidneys (nephritis, 6%). Most cases of fatty livers were diagnosed during necropsy and were characterized by the presence of yellow tan and friable livers. Other categories of lesions (overall prevalence of 12%) included three cases of tracheitis; two cases of epicarditis and thymus heterophilic infiltration; and one case each of steatitis, leptomeningitis, splenitis, hepatitis, and mineralization of renal tubules. The bacteria observed were all Gram-negative coccobacilli. There were no significant lesions suggesting the presence of Chlamydiaceae or viral infection. In particular, no elementary bodies and no viral inclusion bodies were identified. The number of animals examined for microbiologic endpoints was limited to 14 animals belonging to six clutches. Of 10 blood samples analyzed, eight were positive for one or more of the following bacterial organisms: Aeromonas hydrophila, Serratia marcescens, Bacillus sp., Bacteroides sp., Clostridium sp., Staphylococcus sp., and a Gram-negative rod. Corynebacterium sp. was isolated from one of four lung samples. From this same clutch, this organism plus Clostridium sp. was also isolated from the liver. One animal from Lake Griffin was examined for Salmonella and the results were negative. No fungal organisms were isolated in this study.
There were significant differences in total yolk OCP concentrations across sites. Yolks from Emeralda contained approximately 25 and 2 times the OCPs compared with Griffin and Apopka (31,763± 3,777 µg/kg compared with 1,242± 481 µg/kg and 13,240± 5,046 µg/kg, respectively) (P<0.0001, F = 28, df = 2). Regardless of site, over 95% of the OCP mixture was composed of DDT and related metabolites (mostly p,p'-DDE) and of cyclodiene pesticides (mainly dieldrin and chlordanes). To determine the relationship between exposure to high concentrations of pesticides and the occurrence of different gross and histologic abnormalities, animals were divided into three groups based on degree of contamination: low (100–900 µg/kg, n = 11 clutches), medium (1,500–8,500 µg/kg, n = 8 clutches), and high (26,000–52,000 µg/kg, n = 5 clutches) OCP body burdens. The result of this analysis is shown in Figure 3. Except for a slight increase in the prevalence of histologic lesions with degree of OCP contamination, there was no other significant association between high pesticide burdens and presence of abnormalities.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
|---|
In this study, late embryonic and hatchling mortality differed across sites and was highest in Lake Griffin (24%), compared with Emeralda (9%), Apopka (7%), and Lochloosa (1%). Lochloosa and Apopka clutches were of high (78%) and intermediate (57%) quality, respectively, in terms of percentage of hatchlings that survived 1 mo posthatch. Although clutches from Emeralda experienced only about a third of the late-stage embryo and hatchling mortality compared with Griffin, clutch productivity was about the same for both sites (34%). This was due to a higher rate of embryo deaths in the Emeralda site during the first weeks of incubation. A decreased survival in early life stages of American alligators from polluted sites is consistent with previous reports (Masson, 1995; Giroux, 1998; Sepúlveda et al., 2004), although viability values in clutches from Lake Apopka in the present study were higher than reported in the early 1990s by Woodward et al. (1993). Causes for low clutch viability could be related to either external and/or internal factors, such as flooding and desiccation of eggs; altered eggshell, yolk, or albumen quality; infertility; and/or increased zygote and embryonic rates.
Hatchlings from Apopka and Lochloosa not only survived at an increased rate during the first month of age compared with Griffin and Emeralda animals, but were also significantly larger at the time of hatching. This difference in weight was true for both dead and live hatchlings. In addition, there were significant site differences in organ size and energy allocation and utilization (Lochloosa animals were not included in this analysis because of small sample size). An increased rate in yolk utilization during embryonic development in Apopka animals could explain their larger size at the time of hatching. Indeed, yolk somatic indices were almost doubled in late-stage embryos from Griffin and Emeralda (21%) compared with same-age alligators from Lake Apopka (11%). In contrast, animals from Griffin and Emeralda hatched with a larger yolk sac that declined in size at a much higher rate during the first month posthatch (49% to 65% decline) compared with Apopka alligators (24% decline). In addition, Apopka alligators hatched with about four times the amount of body fat reserves compared with Griffin and Emeralda animals. Thus, increased survival during the first weeks posthatch in Apopka animals could be explained by a larger body size coupled with an increase in body fat reserves at the time of hatching.
An increased body size in hatchlings from Lake Apopka is consistent with previous reports. Richey (2001) also reported significant differences in body size at hatching, with lowest values in Griffin and Emeralda and highest in Apopka animals. Milnes et al. (2001) examined hatchlings from three lakes in northcentral Florida (Woodruff, Orange, and Apopka) and hypothesized that lake effects resulting from several environmental (such as food and resource availability, nutrient levels, and contaminant levels) and demographic (such as age) factors, and not genetic differences, affect hatchling size through influences on the maternal contribution to the embryonic environment. A difference in survival and hatchling size across sites in the present study supports this lake-effect hypothesis and the idea of a differential preovulatory parental investment in alligators inhabiting different lakes.
Recent studies in different species of reptiles have shown that both body size and amount of yolk reserves at the time of hatching are important predictors of early survival, and that these two traits usually are negatively correlated to each other (Congdon et al., 1999). Increased hatchling survival has been linked to larger yolk reserves at the time of hatching because of a delay in time to starvation during the transition to an exogenous feeding regime (Congdon and Gibbons, 1985, 1989). In addition, because yolk is an important source of immunoglobulins in egg-laying vertebrates (Picchietti et al., 2001), a more complete utilization of these reserves during embryonic development should allow the newly hatched animals to better deal with pathogens, increasing the chances for survival. Larger yolk reserves at the time of hatching, however, imply that a decreased conversion of egg material for embryo growth and development has taken place during incubation, with the resulting production of smaller hatchlings (Congdon et al., 1999). Results from the present study support the bigger-is-better hypothesis because larger hatchlings survived for longer periods, despite their smaller yolk sac at the time of hatching. Because larger hatchlings also had proportionally larger fat bodies, this additional source of energy should also be considered as an important predictor of early survival in reptiles.
Growth retardation in dead embryos and hatchlings
Results from this study indicate general growth impairment for sick and dying hatchlings compared with live counterparts. Differences in size between live and dead hatchlings were more pronounced in animals from Griffin and Emeralda compared with Apopka. Indeed, over half of the cases of severe emaciation were diagnosed in Griffin animals. Causes for decreased growth are unknown at this time, but could be related to alterations in the nutritional composition of eggs in animals inhabiting polluted sites, as discussed in more detail later.
Gross pathology, histopathology, and microbiology
In this study, the most prevalent pathologic changes among dead animals were the presence of edema (overall prevalence of 34%), hyperemic organs (29%), inflammation (30%), and gross deformities (3%). The cause for the generalized edema could be related to the presence of bacterial infections because abnormal retention of fluids has been described in several species of crocodilians with septicemia. For instance, swelling of limbs and abdomen was reported in Indian crocodiles (Gavialis gangeticus) infected with Clostridium sp. and Planococcus sp. (Misra et al., 1993). Hydropericardium, ascites, and lung edema have also been reported from Nile crocodiles (Crocodylus niloticus) infected with Chlamydia sp. (Huchzermeyer et al., 1994), and American alligators infected with Mycoplasma alligatoris suffered from periocular, facial, neck, and limb edema (Clippinger et al., 2000). Generalized edema could also result from exposure to organochlorine contaminants, as discussed in more detail later.
Histologic lesions were prevalent, occurring in close to a third of the animals examined. However, percent occurrence of lesions differed across sites and was approximately half as prevalent in Griffin compared with Apopka and Emeralda animals. Over half of the histopathologic cases were lung lesions, mostly heterophilic pneumonias of bacterial origin with or without the presence of edema and atelectasis. Other histopathologic findings included the presence of liver lipidosis or fatty liver; septicemia; gastritis and enteritis; and nephritis.
In the present study, several opportunistic bacterial organisms were isolated, including Aeromonas hydrophila, Serratia marcescens, Bacillus sp., Bacteroides sp., and Staphylococcus sp. from blood, Corynebacterium sp. from lung, and Clostridium sp. from blood and liver. These are all recognized pathogens in reptiles and other vertebrates. No fungal organisms were isolated or morphologically identified in this study. Bacterial septicemias, with particular involvement of certain organs, are a frequent cause of illness and mortality in crocodilian hatchlings. In most cases, Gram-negative bacteria are involved, including Aeromonas hydrophila, Salmonella spp., and less often Clostridium septicum, Edwarsiella tarda, Escherichia coli, Klebsiella sp., Pasteurella multocida, Providencia rettgeri, Pseudomonas spp., and Staphylococcus sp. (Ladds and Sims, 1990; Huchzermeyer, 1991; Buenviaje et al., 1994; Ladds et al., 1996; Camus and Hawke, 2002). In Florida, both A. hydrophila and A. shigelloides have been implicated as the cause of mortality of free-ranging American alligators (Shotts et al., 1972). However, A. hydrophila has also been isolated from apparently healthy American alligators (Gorden et al., 1979).
Because the number of samples analyzed for the presence of bacteria and fungi was limited, some pathogenic agents might have been missed during this study. Nevertheless, most cases of pneumonia and tissue inflammation probably had bacterial involvement because of the microscopic observation of heterophilic infiltration and bacteria in the affected organs. Whether bacteria were the primary cause of the problem or the result of an opportunistic invasion and later infection remains unknown at this time. In at least three cases, pneumonia was probably triggered after inhalation of yolk contents (inhalation pneumonia) by full-term embryos. These cases were diagnosed histologically by the presence of a proteinaceous yolk-like material in the airways of affected animals. There were no lesions or inclusions suggestive of accumulation of viral and/or viral-induced cell proteins to indicate the presence of a productive viral infection. Sources of bacteria in the present study are unknown at this time but may be related to opportunistic organisms present in the environment and/or endogenous intestinal bacteria that were able to cross the mucosal barrier and cause disease (Huchzermeyer, 2002).
The low prevalence of deformities observed in the present study (3%) is in agreement with reports from other crocodilian species. For instance, in the Indian gharial (Gavialis gangeticus), deformities were detected in 6% of 1,061 hatchlings examined and 80% of them consisted of defects to the vertebral column (Singh and Bustard, 1982). Similar low prevalences of deformities were reported by Boede and Sogbe (2000) in American (Crocodylus acutus) and Orinoco (C. intermedius) hatchling crocodiles (1–42 days). These authors reported ~5% deformities to the tail, fore limbs, and maxillary bones. Although most researchers agree that these deformities are of congenital origin, some have considered these abnormalities to be due to exposure of embryos to extreme temperature and humidity conditions (Ayarzaguena, 1990; Blanco-Márquez, 1997).
The presence of enlarged, pale, fatty livers was a relatively common finding in the present study. Because in reptiles, accumulation of fat in liver is just another way of allocating energy for times of need (i.e., hibernation and fasting), the mere presence of intrahepatic lipid can be quite normal and not synonymous of a pathologic condition (Divers and Cooper, 2000). Hepatic lipidosis, however, has also been reported in association with infectious diseases such as adenovirus and chlamydiosis (Huchzermeyer et al., 1994; Huchzermeyer, 2002).
Relationship between OCPs and abnormalities
Yolk OCP concentrations reported in this study (7,062± 1,245 µg/kg) are well within ranges considered toxic for many species of egg-laying vertebrates (Jarvinen and Ankley, 1999). However, except for a slight increase in the prevalence of histologic lesions with increased OCP contamination, there was no other significant association between high pesticide burdens and presence of abnormalities.
In American alligators, results have clearly demonstrated reduced egg hatch-ability for eggs collected from OCP-contaminated sites. Nonetheless, OCP concentrations in eggs do not correlate with embryo survival rates, suggesting the potential for OCP-maternal-mediated effects on egg quality and/or the involvement of other environmental factors. For other egg-laying vertebrate species, there are numerous laboratory and field studies linking OCP exposure to early-life-stage mortality. OCPs can induce early-life-stage mortality through three mechanisms: 1) direct toxicity to developing embryos, 2) altered maternal vitellogenesis and oogenesis leading to changes in egg composition, and/or 3) immunosuppression. Direct toxicity of chlorinated organics has been extensively characterized after exposure of early-life stages of fish to aryl hydrocarbon receptor (AhR) agonists (mostly dioxins and polychlorinated biphenyls [PCBs], but also OCPs) (Smith and Cole, 1973; Monod, 1985; Guiney et al., 1997; Henry et al., 1997). In these studies, mortality was associated with yolk sac and pericardial edema, craniofacial alterations, and severe and generalized hemovascular damage. An interesting finding from these studies is that the same effects occurred regardless of exposure route (i.e., maternal transfer, waterborne, or injections), suggesting these AhR agonists exert their effects on early-life stages through nonmaternal-mediated mechanisms. In fish-eating birds, exposure to organochlorine contaminants also induces embryotoxicity, a condition known as Great Lakes embryo mortality, edema, and deformity syndrome (GLEMEDS), and characterized by increased embryo mortality, growth retardation, subcutaneous pericardial and peritoneal edema, and congenital deformities of the bill and limbs (Gilbertson et al., 1991). This syndrome closely resembles chick edema disease observed in chickens after in ovo exposure of hens to dioxins. A second mode of action of OCPs is through an interference with vitellogenesis and oogenesis in adult females, leading to changes in egg composition that are later translated in decreased embryo viability. The types, amounts, and relative proportions of lipids and fatty acids, protein, water, vitamins, and minerals present in eggs have all been identified as crucial for the normal development of embryos. As vitellogenins are the major source of nutrition for the developing embryo and also serve as transporters of hormones and antibodies for all egg-laying vertebrates (Babin, 1992; Picchietti et al., 2001), a decline in circulating levels of these proteins in OCP-exposed females could result in alterations in egg composition and thus in embryo survival. Alterations of fatty-acid profiles in yolk (Pushpanjali et al., 2000; Stanton et al., 2003) and of protein contents in albumin (Fernie et al., 2003) have been reported in avian eggs exposed to several chlorinated organics. A final mechanism by which OCPs could increase mortality in early-life stages is through imunosuppression. In the present study, the presence of mixed bacterial infections of multiple organ systems suggest imunosuppression and enhanced susceptibility to opportunistic pathogens. As already discussed, imunosuppression and increased susceptibility to infectious agents could have been the result of stress related to captive conditions. Indeed, in juvenile American alligators, stress has been shown to alter plasma corticosterone concentrations and white blood-cell parameters (Lance and Elsey, 1999). In the present study, immunosuppression could also have been related to exposure to OCPs. A significant positive relationship between total egg OCPs and the incidence of histological lesions, most of which included inflammation of lungs and other major organs, supports this link.
There are conflicting reports on the effects of OCPs on the immune function of American alligators. Gross (1997) found decreased antibody responses and hypoplastic lymphoid tissue and bone marrow in Apopka hatchlings, and Rooney (1998) found that juvenile Apopka alligators had smaller thymic medullary/cortical ratios and smaller splenic lymphocyte sheaths. Richey (2001), however, exposed alligator eggs to OCPs and found no evidence of immunosuppression as determined by a battery of tests that measured both cellular and humoral immune function.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
|---|
BABIN, P. J. 1992. Research needs for the risk assessment of health and environmental effects of endocrine disrupters: A report of the U.S. EPA-sponsoredworkshop. EnvironmentalHealth and Perspectives 104: 715–740.
BENTON, J., D. DOUGLAS, AND L. PREVATT. 1991. Completion report as required by federal aid in fish restoration: Wallop-Breaux Project F-30-18, Ocklawaha Basin Fisheries Investigations Study XII. Lake Apopka Fisheries Studies. State of Florida Game and Fresh Water Fish Commission, Eustis Fishery Laboratory, Eustis, Florida.
BLANCO-MARQUEZ, P. A. 1997. Enfermedades degenerativas óseas y articulares en caimán del Orinoco, Crocodylus intermedius, caimán de la costa, C. acutus. Presentación de tres casos. In Memorias IV Reunión Regional Grupo Especialista de Cocodrilos de América Latina y el Caribe (GEC/UICN). Tabasco, México, pp. 26–33.
BOEDE, E. O., AND E. SOGBE. 2000. Diseases in Orinoco crocodile (Crocodylus intermedius) and American crocodile (Crocodylus acutus) kept in Venezuelan farms. Revista Cientfica FCV-LUZ 10: 328–338.
BUENVIAJE, G. N., P. W. LADDS, AND S. C. MANOLIS. 1994. Disease-husbandry associations in farmed crocodiles in Queensland and the Northern Territory. Australian Veterinary Journal 71: 165–173.[Medline]
CAMUS, A. C., AND J. P. HAWKE. 2002. Providencia rettgeri-associated septicemia and meningoen-cephalitis in juvenile farmed American alligators Alligator mississippiensis. Journal of Aquatic Animal Health 14: 149–153.
CLIPPINGER, T. L., R. A. BENNETT, C. M. JOHNSON, K. A. VLIET, S. L. DEEM, J. OROS, E. R. JACOBSON, I. M. SCHUMACHER, D. R. BROWN, AND M. B. BROWN. 2000. Morbidity and mortality associated with a new mycoplasm species from captive American alligators (Alligator mississippiensis). Journal of Zoological Wildlife and Medicine 31: 303–314.
CONGDON, J. D., AND J. W. GIBBONS. 1985. Egg components and reproductive characteristics of turtles: Relationships to body size. Herpetologica 41: 194–205.
———., AND ———. 1989. Posthatching yolk reserves in hatchling American alligators. Herpetologica 45: 305–309.
———., R. D. NAGLE, A. E. DUNHAM, C. W. BECK, O. M. KINNEY, AND S. R. YEOMANS. 1999. The relationship of body size to survivorship of hatchling snapping turtles (Chelydra serpentina): An evaluation of the "bigger is better" hypothesis. Oecologia 121: 224–235.
DIVERS, S. J., AND J. E. COOPER. 2000. Reptile hepatic lipidosis. Seminars in Avian and Exotic Pet Medicine 9: 153–164.
FERGUSON, M. W., AND T. JOANEN. 1982. Temperature of egg incubation determines sex in Alligator mississippiensis. Nature 296: 850–853.[Medline]
FERNIE, K., J. SMITS, AND G. BORTOLOTTI. 2003. Developmental toxicity of in ovo exposure to polychlorinated biphenyls: I. Immediate and subsequent effects on first-generation nestling American kestrels (Falco sparverius). Environmental Toxicology and Chemistry 22: 554–560.[Medline]
GILBERTSON, M., T. KUBIAK, J. LUDWIG, AND G. FOX. 1991. Great Lakes embryo mortality, edema, and deformities syndrome (GLEMEDS) in colonial fish-eating birds: Similarity to chick-edema disease. Journal of Toxicology and Environmental and Health 33: 455–520.
GIROUX, D. M. 1998. Lake Apopka revisited: A correlation analysis of nesting anomalies and DDT contaminants. Masters Thesis, University of Florida, Gainesville, Florida.
GORDEN, R. W., T. T. HAZEN, G. W. ESCH, AND C. B. FLIERMANS. 1979. Isolation of Aeromonas hydrophila from the American alligator, Alligator mississippiensis. Journal of Wildlife Diseases 15: 239–243.[Abstract]
GROSS, D. A. 1997. Thymus, spleen, and bone marrow hypoplasia and decreased antibody responses in hatchling Lake Apopka alligators. Masters Thesis, University of Florida, Gainesville, Florida.
———., T. S., L. J. GUILLETTE, H. F. PERCIVAL, G. R. MASSON, J. M. MATTER, AND A. R. WOODWARD. 1994. Contaminant-induced reproductive anomalies in Florida alligators. Comparative Pathology Bulletin 26: 2–8.
GUILETTE, L. J., T. S. GROSS, G. R. MASSON, J. M. MATTER, H. F. PERCIVAL, AND A. R. WOODWARD. 1994. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environmental Health & Perspectives 102: 680–688.[Medline]
———., D. A. CRAIN, A. A. Rooney, AND D. B. PICKFORD. 1995. Organization versus activation: The role of endocrine-disrupting contaminants (EDCs) during embryonic development in wildlife. Environmental Health and Perspectives 103: 57–164.
———., J. W. BROCK, A. A. ROONEY, AND A. R. WOODWARD. 1999a. Serum concentrations of various environmental contaminants and their relationship to sex steroid concentration and phallus size in juvenile American alligators. Archives of Environmental Contamination and Toxicology 36: 447–455.[Medline]
———., A. R. WOODWARD, D. A. CRAIN, D. B. PICKFORD, A. A. ROONEY, AND H. F. PERCIVAL. 1999b. Plasma steroid concentrations and male phallus size in juvenile alligators from seven Florida lakes. General and Comparative Endocrinology 116: 356–372.[Medline]
GUINEY, P. D., R. M. SMOLOWITZ, R. E. PETERSON, AND J. J. STEGEMAN. 1997. Correlation of 2,3,7,8-tetrachloro-p-dioxin induction of cytochrome P4501A in vascular endothelium with toxicity in early life stages of lake trout. Toxicology and Applied Pharmacology 143: 256–273.[Medline]
HENRY, T. R., J. M. SPITSBERGEN, M. W. HORNUNG, C. C. ABNET, AND R. E. PETERSON. 1997. Early life stage toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish (Danio rerio). Toxicology and Applied Pharmacology 142: 56–68.[Medline]
HUCHZERMEYER, F. W., G. H. GERDES, C. M. FOGGIN, K. D. A. HUCHZERMEYER, AND L. C. LIMPER. 1994. Hepatitis in farmed hatchling Nile crocodiles (Crocodylus niloticus) due to chlamydial infection. Journal of the South African Veterinary Association 65: 20–22.[Medline]
HUCHZERMEYER, K. D. A. 1991. Treatment and control of an outbreak of salmonellosis in hatchling Nile crocodiles (Crocodylus niloticus). Journal of the South African Veterinary Association 62: 23–25.[Medline]
———. 2002. Diseases of farmed crocodiles and ostriches. Revue Scientifique et Technique de l Office International des Epizooties 21: 265–276.[Medline]
JARVINEN, A., AND G. ANKLEY. 1999. Linkage of effects to tissue residues: Development of a comprehensive database for aquatic organisms exposed to inorganic and organic chemicals., SETAC Press, Pensacola, Florida, 358 pp.
LADDS, P. W., AND L. D. SIMS. 1990. Diseases of young captive crocodiles in Papua New Guinea. Australian Veterinary Journal 67: 323–330.[Medline]
———., J. BRADLEY, AND R. G. HIRST. 1996. Providencia rettgeri meningitis in hatchling saltwater alligators (Crocodylus porosus). Australian Veterinary Journal 74: 397–398.[Medline]
LANCE, V. A., AND R. M. ELSEY. 1999. Hormonal and metabolic responses of juvenile alligators to cold shock. Journal of Experimental Zoology 283: 566–572.
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, USA. Wetlands 22: 705–711.
MASSON, G. R. 1995. Environmental influences on reproductive potential, clutch viability and embryonic mortality of the American alligator in Florida. PhD Dissertation, University of Florida, Gainesville, Florida.
MILNES, M. R., A. R. WOODWARD, AND L. J. GUILLETTE. 2001. Morphological variation in hatchling American alligators (Alligator mississippiensis) from three Florida lakes. Journal of Herpetology 35: 264–271.
MISRA, P. R., D. KUMAR, G. M. PATNAIK, R. P. RAMAN, AND A. SINHA. 1993. Bacterial isolates from apparently healthy and diseased crocodiles (Gavialis gangeticus). Indian Veterinary Journal 70: 375–376.
MONOD, G. 1985. Egg mortality on Lake Geneva charr (Salvelinus alpinus L.) contaminated by PCB and DDT derivatives. Bulletin of Environmental Contamination and Toxicology 35: 531–536.[Medline]
PICCHIETTI, S., G. SCAPIGLIATI, M. FANELLI, E. BARBATO, S. CANESE, L. MASTROLIA, M. MAZZINI, AND L. ABELLI. 2001. Sex-related variations of serum immunoglobulins during reproduction in gilthead sea bream and evidence for a transfer from the female to the eggs. Journal of Fish Biology 59: 1503–1511.
PUSHPANJALI, P. A. K., R. L. PRASAD, AND A. KUMAR. 2000. An avian model for the study of in vivo embryo toxicity of endosulfan influence on lipid pattern of liver tissue. Indian Veterinary Journal 77: 451–453.
RAUSCHENBERGER, R. H, J. J. WIEBE, M. S. SEPULVEDA, J. E. BUCKLAND, AND T. S. GROSS. 2004. Predicting maternal body burdens of organo-chlorine pesticides from eggs and evidence of maternal transfer in Alligator mississippiensis. Environmental Toxicology and Chemistry 23: 2906–2915.[Medline]
RICHEY, L. J. 2001. Effects of endocrine-disrupting contaminants on the immune system of hatchling American alligators. PhD Dissertation, University of Florida, Gainesville, Florida.
ROONEY, A. A. 1998. Variation in the endocrine and immune system of juvenile alligators: Environmental influence on physiology. PhD Dissertation, University of Florida, Gainesville, Florida.
SANG, H. M., AND R. FOTEDAR. 2004. Growth, survival, haemolymph osmolality and organosomatic indices of the western king prawn (Penaeus latisulcatus Kishinouye, 1896) reared at different salinities. Aquaculture 234: 601–614.
SAS INSTITUTE, INC. 2002. SAS Version 9.0. SAS Institute Inc., Cary, NC, USA.
SCHOEB, T. R., T. G. HEATON-JONES, R. M. CLEMMONS, D. A. CARBONNEAU, A. R. WOODWARD, D. SHELTON, AND R. H. POPPENGA. 2002. Clinical and necropsy findings associated with increased mortality among American alligators of Lake Griffin, Florida. Journal of Wildlife Diseases 38: 320–337.[Abstract]
SEPULVEDA, M. S., J. J. WIEBE, D. C. HONEYFIELD, J. P. HINTERKOPF, W. E. JOHNSON, AND T. S. GROSS. 2004. Organochlorine pesticides and thiamine in eggs of largemouth bass and the American alligator, and their relationship with early life-stage mortality. Journal of Wildlife Diseases 40: 782–786.
SHOEMAKER, C. A., P. H. KLESIUS, C. LIM, AND M. YILDIRIM. 2003. Feed deprivation of channel catfish, Ictalurus punctatus (Rafinesque), influences organosomatic indices, chemical composition and susceptibility to Flavobacterium columnare. Journal of Fish Diseases 26: 553–561.[Medline]
SHOTTS, E. B. JR., J. L. GAINES, L. MARTIN, AND A. K. PRESTWOOD. 1972. Aeromonas-induced deaths among fish and reptiles in a eutrophic inland lake. Journal of the American Veterinary Medical Association 161: 603–607.[Medline]
SINGH, L. A. K., AND H. R. BUSTARD. 1982. Congenital defects in the gharial Gavialis gangeticus (Gmelin). British Journal of Herpetology 6: 215–219.
SMITH, R. M., AND C. F. COLE. 1973. Effects of egg concentrations of DDT and dieldrin on development in winter flounder (Pseudopleuronectes americanus). Journal of Fisheries Research Board of Canada 12: 1894–1898.
STANTON, B., J. DEWITT, D. HENSHEL, S. WATKINS, AND B. LASLEY. 2003. Fatty acid metabolism in neonatal chickens (Gallus domesticus) treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3',4,4',5-pentachlorobiphenyl (PCB-126) in ovo. Comparative Biochemistry and Physiology C 136: 73–84.
U.S. ENVIRONMENTAL PROTECTION AGENCY (USEPA). 1990. Determination of semivolatile organic compounds by gas chromatography-mass spectrometry. Test Methods for Evaluating Solid Waste, 2nd Edition. Method 8270B. Test methods for evaluating solid waste (SW-846). Office of Solid Waste, USEPA, Washington, D.C.
WOODWARD, A. R., H. F. PERCIVAL, M. L. JENNINGS, AND C. T. MOORE. 1993. Low clutch viability of American alligators on Lake Apopka. Florida Scientist 56: 52–63.
Received for publication 19 October 2004.
| ||||||||||||||||||||||||||||||||||||||||||||||||
