PREDICTING CONTAMINANT BODY BURDENS FROM EGGS AND EVIDENCE OF MATERNAL TRANSFER IN ALLIGATOR MISSISSIPPIENSIS

RH Rauschenberger, MS Sepúlveda, JJ Wiebe,
DS Ruessler, CM Wieser, and TS Gross

 USGS-Florida Integrated Science Center
and University of Florida, Gainesville, FL.

Presented at the Society for Environmental Toxicology and Chemistry
23rd Annual Meeting, Salt Lake City, UT, November 16 - 20, 2002.

ABSTRACT

Noninvasive methods are useful for monitoring contaminant exposure since they are applicable to endangered species, and allow repeated monitoring of individuals. The present study's objective was to evaluate the use of eggs as a predictor of maternal body burdens in adult alligators.  Adult female alligators (n = 7) and their eggs were collected from Lake Apopka (n = 4) and Lake Griffin (n = 3) in central Florida during June 2001.  Yolk from one or two eggs from each clutch along with maternal tissues (blood, bile, fat, liver, and muscle) were collected, analyzed for lipids, and screened for 30 organochlorine pesticides (OCPs) including DDT and DDT analogs, cyclodienes, mirex and toxaphene.  Mean cumulative OCP burdens for yolks from Lake Apopka and Lake Griffin were 15,108 and 616 ppb wet weight, respectively.  Mean cumulative OC tissue burdens (ppb wet weight) for Lake Apopka females were 55 (blood), 900 (bile), 1,501 (muscle), 2,134 (liver), and 44,650 (fat). Mean cumulative OC tissue burdens (ppb wet weight) for Lake Griffin females were 31 (blood), 87 (bile), 208 (muscle), 153 (liver), and 2,689 (fat).  For all females, the tissue with the greatest number of linear correlations with yolk was fat in which 14 of 15 (93%) chemicals that were detected showed a significant correlation (α = 0.05), followed by liver (11/13, 85%), muscle (7/13, 54%), bile (6/12, 50%), and blood (1/9, 11%) with R-square values ranging from 0.67-0.99. Thus, yolk burdens are predictive of maternal burdens for certain tissues and strongly suggest maternal transfer of selected organochlorines in the American alligator. (Funding was provided by the NIEHS Superfund Basic Research Program Grant #P42-07375.)

INTRODUCTION

Organochlorine pesticides (OCPs) were widely applied across the United States (US) from the early 1940s-1960s in an effort to control malarial mosquitoes and crop-destroying insects. However, in the last few decades almost all OCPs have been removed from US markets due to their well-documented effects on avian eggshell quality, their persistence in the environment, and their ability to biomagnify.  One taxonomic group that appears to be experiencing effects due to OCP exposure is the  American alligator (Alligator mississippiensis). Within a number of sites along the Ocklawaha River in central Florida, alligator populations have experienced increased embryonic mortality and reduced clutch success. Coincidentally, these population level changes have only been observed in lakes with elevated levels of OCPs. The relationship between OCP burdens in maternal tissues, OCP burdens in yolk, and embryo mortality in alligators is not completely understood.  In addition, improved methods are needed for monitoring OCP burdens in adult alligator populations.  

With these challenges in mind, the goals of the present study were to:

1. Determine if OCPs are maternally transferred in alligators.
2. Ascertain the practicality of using egg yolk burdens to predict the OCP burdens in maternal tissues.

METHODS AND MATERIALS

Site descriptions
• Lake Apopka and Lake Griffin (figure 1) were selected as collection sites because previous studies by our laboratory indicated that alligator populations were experiencing increased embryonic mortality and that yolks taken from both sites contained elevated levels of OCPs. However, Lake Apopka egg yolk burdens are in some instances an order of magnitude higher than those from Lake Griffin.  In 2000, for example, p',p-DDE burdens in egg yolk from Lake Apopka averaged 5,105 ppb ( ± 7,225), while p',p-DDE burdens in egg yolk from Lake Griffin averaged 589 ppb (± 895) (T.S. Gross, unpublished data).
Egg collections
• Nests were located via helicopter and airboat surveys. Eggs were placed in plastic bus pans cushioned with nest substrate.  The top of each egg was marked to identify/maintain orientation.  Nesting substrate was placed in between layers of eggs for cushioning.  The top layer of eggs was covered with nesting substrate for insulation.  Clutches were subsequently transported to incubators located at the Florida Fish and Wildlife Conservation Coop. Research Unit (FWCC) in Gainesville, FL
Adult female collections
• After eggs were removed, the nest was recovered and snare-traps were set perpendicular to the direction of the tail –drag.  Traps were checked in the evening and early the next morning.  Trapped females were secured by binding the jaws and limbs to allow for safe transit from the trap site to the USGS-Florida Integrated Science Center (FISC).  At the FISC, animals were weighed, measured, and sacrificed.  A full necropsy was performed and the following tissues were collected and stored in a – 80 C freezer:
• Blood
• Liver
• Fat
• Muscle
• Bile
• Clutches corresponding to each female were transported from the FWCC to the FISC.  Yolk samples were collected and placed with the corresponding maternal tissues. A total of 3 adult females and their clutches were collected from Lake Apopka in addition to 4 adult females and their clutches collected from Lake Griffin.
Chemical analysis
• Lipids:
• For tissues with high lipid content (i.e., fat, yolk), total lipid content was determined by weighing a portion of the tissue sample and then placing the portion into a hexane-acetonitrile mixture for lipid extraction. The solvent is then separated from the lipid and the lipid content is determined gravimetrically. For tissues with low lipid content (i. e. muscle), lipid content was determined by GC-MS.
• Organochlorines:
• Tissue samples were homogenized, weighed, and extracted into ethyl acetate. Sample clean-up includes use of C18 and NH2 SPE (solid phase extraction) cartridges prior to analysis by GC-MS.
Statistical analysis
• Linear regression (SAS, inc.) was used to analyze the relationships between egg yolk-OCP concentrations and tissue concentrations and to formulate model equations. Scatter plots and regression lines between tissue and yolk concentrations were created using Sigma Plot.

RESULTS and DISCUSSION

Maternal tissue burdens and yolk burdens

• Mean total OCP burdens for yolks from Lake Apopka and Lake Griffin were 15,108 and 616 ppb wet weight, respectively.
• Mean cumulative OCP tissue burdens (ppb wet weight) for Lake Apopka females were 55 (blood), 900 (bile), 1,501 (muscle), 2,134 (liver), and 44,650 (fat).
• Mean cumulative OCP tissue burdens (ppb wet weight) for Lake Griffin females were 31 (blood), 87 (bile), 208 (muscle), 153 (liver), and 2,689 (fat).
• Thus, it is apparent that concentrations of OCPs in tissues (wet weight) varied widely between different  individuals, among tissues of the same individual, and across sites.
• For all females, the tissue with the greatest number of linear correlations with yolk was fat in which 14 of 15 (93%) chemicals that were detected showed a significant correlation (α = 0.05), followed by liver (11/13, 85%), muscle (7/13, 54%), bile (6/12, 50%), and blood (1/9, 11%).
• R-square values for the above correlations ranged from 0.67-0.99 indicating the strong linear relationship between maternal tissue burdens and yolk burdens.
• The ratio of tissue-OCP burdens:yolk-OCP burdens varied according to the type of tissue and the specific chemical.
• After normalizing for lipid content, fat/yolk OCP (total) ratios were very close to 1 for all animals.
• Furthermore, lipid normalization did not increase the number of significant linear correlations between fat and yolk OCP burdens.
• Muscle/yolk OCP (total) ratios exceeded 14 after lipid normalization-which increased the number of significant linear correlations.
• This suggests that although fat tissue contained the highest concentrations of OCPs on a wet weight basis, muscle contained the highest concentrations after lipid normalization (i.e., on a percent-lipid basis).
• Liver was the only tissue in which lipid normalization of OCP burdens resulted in fewer significant linear correlations.
• Lipid normalization of yolk OCP burdens did not affect the number of significant correlations for any of the tissues.

CONCLUSIONS

• The concentrations of OCPs in maternal tissues are closely correlated with the level of lipid content and the extent to which those lipids are mobilized.
• Although OCPs are most likely evenly disbursed throughout an alligator's lipid stores, muscle lipids appear to store higher concentrations than fat.
• The higher OCP burden in muscle lipids, compared to fat and yolk, is due to the fact that lipid stores in muscle are seldom mobilized during yolk production, allowing OCPs to accumulate in these lipids throughout the life of the animal (50 + years for an alligator).
• Furthermore, the mobilization of fat lipids during yolk production and the subsequent restoration of fat stores following nesting season explains the similarity between fat burdens and yolk burdens.
• Egg yolks can be used as a noninvasive method for monitoring the levels of selected lipophilic, chlorinated pesticides in adult female alligators.
• Eggs require less effort and expertise to obtain when compared to capturing adult alligators (i.e., eggs are collected in daylight, while alligators are captured at night).
• Using egg yolks to predict maternal body burdens is more accurate and precise than using blood samples with respect to organochlorine pesticides (OCPs).

PRELIMINARY RESULTS FROM RELATED EXPERIMENTS

In addition to the field study described above, our laboratory is conducting a mesocosm experiment examining maternal transfer of OCPs using a captive breeding population of adult alligators.  Briefly, 24 adult animals were divided into 8 pens (1 male:2 females).  Odd numbered pens were designated treatment pens and even numbered pens were controls. Treated animals were dosed with a mixture of OCPs via one time IP and IM injections in Oct. 2001 and, in March 2002, treatment groups received oral doses of an OCP mixture. Preliminary results indicate that OCP burdens (similar to those observed in wild eggs) can be elicited in eggs from a captive breeding population. This underscores maternal transfer of OCPs as the predominant route of embryonic exposure. This study is the first of its kind to examine how maternally mediated, endogenous OCP exposure affects egg and embryo quality in American alligators.

ACKNOWLEDGEMENTS

This research was supported by NIEHS-SBRP Grant #P42-07375 to T.S. Gross.  The authors would like to thank A. Woodward, N. Kernaghan, J. Basto, A. Harvey, J. Muller, 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.