Using Chorioallantoic Membranes for Non-Lethal Assessment of

Chapter 20

Downloaded by STANFORD UNIV GREEN LIBR on September 17, 2012 | http://pubs.acs.org Publication Date: November 1, 2000 | doi: 10.1021/bk-2001-0771.ch020

Using Chorioallantoic Membranes for Non-Lethal Assessment of Exposure and Effect in Oviparous Wildlife 1

George P. Cobb, Donald M . Norman , Pattie D. Houlis, and Tim A . Bargar 2

The Institute of Environmental and Human Health, Texas Tech University and The Texas Tech University Health Sciences Center, Lubbock, TX 79416

Assessing chemical exposure during ecotoxicology studies presents unique analytical problems. Sacrificing enough organisms to obtain sufficient numbers of tissue samples for reliable exposure assessment may impact community dynamics. Also, lethal sampling is not appropriate when evaluating threatened or endangered wildlife. Chorioallantoic membranes (CAMs) may be used as a surrogate tissue for evaluation of chlorinated hydrocarbon exposure in oviparous species. Residues in CAMs are highly correlated with residues in eggs,(for total PCBs ,r =0.592 to 0.782; p= 0.02 to 0.0001). Residues in CAMs also correlate with monooxygenase activity in hatchling and maternal liver. As chlorination within a given class of compounds increases, more of the compound is retained in the egg yolk and is ultimately resorbed into the hatchling. Interestingly, lower chlorinated PCBs are preferentially transferred from maternal reserves to eggs. The C A M technique has been validated with chicken eggs in laboratory studies. Contaminant exposure in heron, alligator and sea turtle populations have been successfully monitored using this approach in field studies. 2

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Current address: Pacific NW Pesticide Consulting Group, 2112 NW 199 Street, Shoreline, WA 98117. Current address: National Ocean Service, National Océanographie and Atmospheric Administration, Charleston, SC 29412.

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© 2001 American Chemical Society In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Introduction Assessment of wildlife exposure to chlorinated organic contaminants has historically used residue analysis [1-8]. There is also a significant move toward biomarkers of exposure and effect [9-14]. While providing important information, most residue and biomarker approaches are lethal to the organisms being studied. Lethal sampling can have deleterious effects on wildlife populations and removes individual organisms from further study. Such population alterations bias any reproductive, growth, prédation or habitat use endpoints that may be desired. Lethal sampling techniques are not possible with threatened or endangered species. These obstacles can be prevented by collecting tissue samples non-lethally and by drawing strong correlations between chemical concentrations and biomarker response [12-14].

Nonlethal Monitoring of Eggs Thefirstnonlethal tissue sampling from eggs utilized eggshell membranes from peregrine falcon (Falco peregrinus) eggs. Eggshell membranes are located between egg contents and the egg shell. These membranes were used to estimate chlorinated hydrocarbon content in museum specimens [15] andfreshlyhatched eggs [16]. In a modification of this approach, Norman utilized chorioallantoic membranes (CAMs) of great blue heron (Ardea herodias fannini) eggs from the Puget Sound Ecosystem [17]. CAMs are highly vascular membranes that are formed just before organogenesis. CAMs perform gas exchange, nutrient transport from the yolk, and excretory containment functions [18]. The CAM remains in the egg after hatching or is loosely attached to the hatchling's abdomen. The nonessential nature of the post hatching C A M allows noninvasive tissue collection for subsequent chemical analysis [19].

Study Species Great blue herons {Ardea herodias) are accepted as good indicators of contaminant presence in estuarine ecosystems [20,21]. The heron's diet and resident nature contribute heavily to its utility as an indicator species. Based on these characteristics, three heron colonies in The Puget Sound ecosystem monitored for Chlorinated hydrocarbon uptake [17,19,22]. Effects of chlorinated pesticide exposure on American alligators {Alligator mississippiensis), has been evaluated [23,24], but alligator exposure to PCBs is poorly defined [25-27]. To better characterize this exposure, Bear Island (BI) and Yawkey Wildlife Center (YWC), were monitored as reference and contaminated areas, respectively. BI is in the ACE Basin, a National Estuarine Research Reserve, at the confluence of the Ashepoo, Combahee, and Edisto Rivers. YWC is located on the shore of Winyah Bay, one hundred and fifty miles northeast of BI. These two study areas are situated in the tidal areas of two distinctly separate watersheds. From

In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


277 1989 through 1992, South Carolina Department of Health and Environmental Control advised against fish and shellfish consumption from Winyah Bay due to chlorinated hydrocarbon contamination [28,29]. Alligators consume primarily aquatic prey which allows accumulation of chlorinated hydrocarbons. The different historical PCB concentrations at the two study sites allowed comparison of PCB uptake by alligators. Data concerning contaminants in sea turtles are rare. Most research concerning PCB effects on reptiles has utilized more readily available turtle species [30,31]. Mean PCB concentrations have been determined in snapping turtles {Chelydra serpentina) from the Hudson River and from Lake Ontario, Canada [31,32]. Little data exist concerning PCBs in loggerhead sea turtles {Caretta caretta) [33,34]. We have found no studies correlating PCB concentrations with adverse effects in sea turtles. Utilization of CAMs as tissues for chemical analysis would allow residue and biological effect data to be collected for sea turtles and other threatened species.

Laboratory Studies Even though field research in our laboratory has demonstrated that PCB and DDE concentrations in CAMs correlate to concentrations in whole eggs [19] and that CAMs collected from hatched eggs can be used to assess contaminant uptake by oviparous wildlife near hazardous waste sites [22,27,35], the utility of the C A M technique has been questioned. The basis for this question is a study in which concordance analysis showed partitioning of chlorinated organic compounds between C A M and egg tissue did not follow passive diffusion models [36]. This finding implicates active transports in Chlorinated hydrocarbon movement among egg tissues. Therefore, laboratory dosing studies were conducted to better define the effect of chlorination and other structural characteristics on contaminant distribution between C A M and egg tissue. The following text describes the toxicological foundation and field applicability of C A M monitoring.

METHODS Sample Collection and Handling Three wildlife species were evaluated during field studies: great blue herons, American alligators, and loggerhead sea turtles (18,19,24,35). All eggs were collected with the supervision of State or Federal wildlife officials. Heron eggs were collected by climbing trees and retrieving eggs directlyfromnests. Heron CAMs were retrieved directlyfromthe ground below colonies. Twenty-one alligator eggs were collected from YWC, and twenty eggs were collected from BI. Three eggs were collectedfrom13 of 14 nests. Eggs were collected at least 48 hours following hatching of siblings from a given nest. Twenty one unviable loggerhead sea turtle eggs were collected from the Hatchling Headstart Program at the Cape Romain Island National Wildlife Refuge. Eggs were kept on ice in the field and placed in glass containers for storage at

Using Chorioallantoic Membranes for Non-Lethal Assessment of

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