2008strum

2008strum
EXPOSURE OF MIGRATORY SHOREBIRDS TO ORGANOPHOSPHORUS AND
CARBMATE PESTICIDES AT MIGRATORY STOPOVER AND NON-BREEDING SITES
IN THE WESTERN HEMISPHERE
by
KHARA M. STRUM
B.A., Ohio Wesleyan University, 2001
A THESIS
submitted in partial fulfillment of the requirements for the degree
MASTER OF SCIENCE
Division of Biology
College of Arts and Sciences
KANSAS STATE UNIVERSITY
Manhattan, Kansas
2008
Approved by:
Major Professor
Dr. Brett K. Sandercock
Abstract
Monitoring programs indicate that numerous shorebird populations are subject to ongoing declines. The U.S. Shorebird Conservation Plan lists twenty-seven shorebird species as
species of high concern and seven as highly imperiled, including the Buff-breasted Sandpiper
(Tryngites subruficollis). One hypothesis for ongoing population declines is exposure to toxic
chemicals and pollutants. The purpose of this project was to characterize plasma cholinesterases
(ChEs) of migratory shorebirds and address potential exposure to organophosphorus (OP) and
carbamate (CB) pesticides. Consumption or contact with these pesticides can cause mortality
and a variety of sub-lethal effects. Buff-breasted Sandpipers and other upland shorebirds are
particularly likely to encounter agrochemicals due to their habitat use at the non-breeding
grounds. I sampled migratory shorebirds over three seasons, during north- and southbound
migration in 2006 and 2007 in Texas, Kansas, and Nebraska and during the non-breeding season
in 2007 in Argentina, Uruguay, and Paraguay. I collected blood samples and footwashings from
reference sites, where OP and CB pesticides were not used, and agricultural sites, where these
two insecticides were recommended for control of crop pests. I assessed several variables
known to affect plasma ChE activity including body size, date of capture, time of capture,
condition, sex, and region. Small-bodied species had higher levels of ChE activity in plasma
than large-bodied species. Plasma ChE activities varied with date of capture in 3 of 5 species
sampled in North America. Sex differences were significant in 1 of 4 species tested. Plasma
acetylcholinesterase (AChE) activity was higher among White-rumped Sandpipers sampled in
North America but there was no difference between regions among Buff-breasted Sandpipers.
Time of capture and individual condition did not affect plasma ChE activity. Estimates of
exposure to ChE inhibitors were addressed in five species. Plasma AChE and
butyrylcholinesterase (BChE) activities of Buff-breasted Sandpipers were lower at agricultural
sites in South America but BChE activity was higher at agricultural sites in North America.
There were no differences between sites in four other species tested. A meta-analysis across all
species indicated that in 4 of 6 comparisons habitat type had a negative effect on AChE activity
consistent with exposure to ChE inhibitors but there was a regional positive effect of agricultural
habitat on BChE activity in North America. Comparison of body mass between sites suggested
that use of habitats with potential pesticide application did not affect mass gain. Project results
suggest that 1 of 5 shorebird species tested was exposed to ChE-inhibiting pesticides at the nonbreeding grounds and future monitoring is necessary to assess potential effects at the population
level. This study highlights the importance of complete sampling and addressing variability in
plasma ChEs before making estimates of exposure to OP and CB pesticides. It provides the first
estimates of migratory shorebird exposure to OP and CB pesticides, a potential conservation
issue. Future research should include continued monitoring of Buff-breasted Sandpiper ChE
levels and habitat use. Other sources of anthropogenic declines such as habitat loss and illegal
hunting should be investigated for species that did not show evidence of exposure.
Table of Contents
List of Figures ................................................................................................................................ vi
List of Tables ................................................................................................................................ vii
Acknowledgements........................................................................................................................ ix
CHAPTER 1 - Introduction ............................................................................................................ 1
References................................................................................................................................... 3
CHAPTER 2 - Plasma cholinesterases for monitoring pesticide exposure in Nearctic-Neotropical
migratory shorebirds ....................................................................................................................... 4
Abstract....................................................................................................................................... 4
Introduction................................................................................................................................. 5
Methods ...................................................................................................................................... 7
Shorebird Capture ............................................................................................................... 7
Sample Collection and Preparation..................................................................................... 8
Laboratory Analysis............................................................................................................ 9
Statistical Analysis.............................................................................................................. 9
Results....................................................................................................................................... 10
Discussion................................................................................................................................. 12
References................................................................................................................................. 16
CHAPTER 3 - Exposure of migratory shorebirds to cholinesterase-inhibiting contaminants in the
Western Hemisphere..................................................................................................................... 23
Abstract..................................................................................................................................... 23
Introduction............................................................................................................................... 24
Methods .................................................................................................................................... 27
Shorebird Capture ............................................................................................................. 27
Sample Collection and Preparation................................................................................... 29
ChE Activity ..................................................................................................................... 30
Reactivation Assays .......................................................................................................... 31
Chemical Residue Analyses.............................................................................................. 32
Statistical Analyses ........................................................................................................... 33
iv
Results....................................................................................................................................... 34
Spatial Variation in Plasma ChE Activity ........................................................................ 35
Spatial Variation in Body Mass ........................................................................................ 37
Discussion................................................................................................................................. 38
Spatial Variation in Plasma ChE Activity ........................................................................ 39
Spatial Variation in Body Mass ........................................................................................ 41
Plasma and Brain ChE Activity ........................................................................................ 41
Conclusions....................................................................................................................... 42
References................................................................................................................................. 44
CHAPTER 4 - Conclusions .......................................................................................................... 56
Appendix A - Laboratory controls and inter-lab variation ........................................................... 59
Appendix B - Characterization of shorebird plasma cholinesterases for optimal inhibitor and
substrate concentrations ................................................................................................................ 60
Appendix C - Plasma cholinesterase activity of additional species.............................................. 63
Appendix D - Analyses of variation in plasma cholinesterases of migratory shorebirds using
additional samples......................................................................................................................... 64
v
List of Figures
Figure 2-1 Log10-log10 graph showing the relationship between mean body mass and ChE
activity in 9 species of shorebirds captured during spring migration in the Great Plains
region of the United States. Sample sizes are inside the uppermost x-axis and error bars
represent ± SE. Pectoral Sandpiper (Calidris melanotos) is the higher of the two log10(ChE)
values where log10(body mass) = 1.81.................................................................................. 20
Figure 3-1 Pesticide use effects on AChE (above) and BChE (below) of five shorebird species
captured in North and South America. Mean (units/mL plasma) and standard error (SE) of
ChE activity from individuals sampled from reference sites (R; closed circles) and pesticide
use sites (P; open circles). Site of capture, region (NA = North America and SA = South
America), and species are located below the lower x-axis. Sample sizes are located above
the lower x-axis. An asterisk (*) denotes a significant difference between means using a
two-tailed t-test at an α-level ≤ 0.05..................................................................................... 50
Figure 3-2 Influence of region on AChE (above) and BChE (below) in two shorebird species.
Region of capture (NA = North America, SA = South America) and species are located
below the lower x-axis. Sample sizes are located above the lower x-axis. An asterisk (*)
denotes a significant difference between regional means using a two-tailed t-test at an αlevel ≤ 0.05............................................................................................................................ 51
Figure B-1 Iso-OMPA titration curves of 7 species of migratory shorebirds............................... 61
Figure B-2 Substrate (AThCh) affinity curve of 7 species of migratory shorebirds. ................... 62
vi
List of Tables
Table 2-1 Descriptive statistics of ChE activity (units/mL plasma) for 9 shorebird species
sampled during northbound migration in the Great Plains region of the United States
including sample size of individuals (n)†, mean, standard deviation (SD), minimum (min)
and maximum (max) values.................................................................................................. 21
Table 2-2 Trends in plasma ChE activity of 5 species of shorebirds as a function of date of
capture, time of capture and an index of body condition using log10 transformed ChE
activity. After sequential Bonferroni correction for number of tests, test statistics were
considered significant at an α-level of 0.05 if P < 0.002...................................................... 22
Table 3-1 Common names and Latin names of study species with sample size (n) of individuals
captured in each region. ........................................................................................................ 52
Table 3-2 Descriptive statistics of ChE activity (units/mL plasma) by site and effect size of
habitat use for 7 species of shorebirds captured in South America. Statistics include sample
size (n), mean, standard deviation (SD), minimum (min), and maximum (max). ChE
activity was compared between sites using a two-tailed t-test and a measure of effect size,
Hedges' d. Comparisons were not conducted if n ≤ 5.......................................................... 53
Table 3-3 Descriptive statistics of ChE activity (units/mL plasma) by site and effect size of
habitat use for 9 species of shorebirds captured in North America. Statistics include sample
size (n), mean, standard deviation (SD), minimum (min), and maximum (max). ChE
activity was compared between sites using a two-tailed t-test and a measure of effect size,
Hedges' d. Comparisons were not conducted if n ≤ 5.......................................................... 54
Table 3-4 Mean body mass (g) of 13 species of shorebirds by site and region. Body mass means
that share the same letter superscript are not significantly different within a species using a
two-tailed t-test at an α-level ≤ 0.05. Superscripts A and B are comparisons within regions
(within a row) and superscripts Y and Z are comparisons between sites (within a column).
Comparisons were not conducted if n ≤ 5............................................................................. 55
Table C-1 Plasma ChE activity (units/mL plasma) of 9 shorebird species sampled at migratory
stopover and non-breeding sites with insufficient data for statistical analyses. ................... 63
vii
Table D-1 Differences in plasma ChE activity by sex in 2 species of shorebirds sampled at
migratory stopover sites in North America. Results were considered significant at a α-level
of 0.05 using a two-tailed t-test............................................................................................. 65
Table D-2 Diurnal variation in plasma ChEs of 7 species of shorebirds sampled at reference sites
during migration in North America and at non-breeding sites in South America. Results
were considered significant at an α-level of 0.05 if P < 0.006 after sequential Bonferroni
correction for the number of tests. ........................................................................................ 66
viii
Acknowledgements
I am indebted to many people who provided support and advice as I followed migratory
shorebirds throughout the Western Hemisphere in pursuit of my Master’s degree. My family,
friends, advisors, fellow graduate students, and committee members deserve recognition for their
unrelenting encouragement.
Funding was provided by two grants from the U.S. Fish and Wildlife Service Neotropical
Migratory Bird Conservation Act (NMBCA) and the Migratory Bird Management (MBM)
programs. Additional funding was provided by the Division of Biology, the Graduate Student
Council and the Terry C. Johnson Center for Basic Cancer Research at Kansas State University,
the Society for Environmental Toxicology and Chemistry, The Institute for Environmental and
Human Health at Texas Tech University, the Department of Chemistry at Southern Illinois
University at Edwardsville and Instituto Nacional de Tecnologia Agropecuaria, PPR
Biodiversidad-AERN2 and Project AERN 2622.
I am thankful to Dr. Brett K. Sandercock and Dr. Michael J. Hooper for serving as my
advisors, providing helpful criticism, and sharing their experiences. I also thank Dr. Samantha
Wisely and Dr. David Rintoul for contributing their time and thoughts by serving as members of
my committee. I thank past and current members of the Avian Ecology Lab who provided
insightful comments during my tenure as a graduate student and my fellow graduate students for
passing along their wisdom, especially Page Klug.
Wherever I found myself, I had an amazing team of field technicians and enthusiastic
volunteers. In North America, field assistance was provided by Ashley Casey, Tara Conkling,
ix
Samantha Franks, Kyle Gerstner, Kate Goodenough, Karl Kosciuch, Tara Whitty and the USDA
Wildlife Services Office of Manhattan, Kansas. In South America, Laura Addy, Natalia Bossel,
Noelia Calamari, Julieta Decarre, Graciela Escudero, Andrea Goijman, Benito Haase, Benito
Jaubert, Leandro Macchi, Laura Solari, and Romina Suarez aided with field sampling.
I was fortunate to encounter many gracious landowners, land managers, and biologists
who granted me access to their land. In North America, logistical support was provided by the
staff of Anahuac National Wildlife Refuge, the staff of Quivira National Wildlife Refuge, Mark
Robbins at the University of Kansas, Nebraska Game and Parks Commission, University of
Nebraska at Omaha, and private turf grass farms near Crosby, TX, Colwich, KS, and Lawrence,
KS. International collaborators in South America who provided field sites and logistical support
include the staff of Guyra Paraguay, Robert Owens, Instituto Nacional de Tecnologia
Agropecuaria de Argentina, Reserva Provincial Laguna Mar Chiquita y Bañados Rio Dulce,
Cordoba, Argentina, Hector Caimarys of Guardaparque del Uruguay, and Direccion Nacional de
Recursos Acuaticos del Uruguay.
This work would not have been possible without collaboration of Julian Torres-Dowdall
and Matilde Alfaro, two immensely positive and supportive colleagues who guided me through
South America and with whom I look forward to collaborating in the future. I am also indebted
to María Elena Zaccagnini for providing a base of operations for my research expedition and
tackling logistical issues long after my departure from South America.
I am forever grateful to my family for their patience, love, and continued support as I
worked to reach my goal.
x
CHAPTER 1 - Introduction
Nearctic-Neotropical migratory shorebirds represent a taxonomic group with diverse
migration strategies and natural histories (Page and Gill 1994). In the Western Hemisphere,
long-distance migrants breed on the arctic tundra and spend the austral summer in Central and
South America while some short-distance migrants do not venture outside of North America.
Migration along the coast is characterized by large aggregations of shorebirds using a few
important stop-over sites where they exploit abundant, seasonal resources. Shorebird migration
in the interior of North America follows a broad-front pattern and shorebirds are more dispersed
on the landscape to exploit variable resource availability (Skagen and Knopf 1993).
The conspicuous nature of shorebird migration and reliability of arrival time caused many
species to suffer population declines after exploitation for food and sport in the early 19th century
(Forbush 1916, McIlhenny 1943). Although hunting of most species of shorebirds is now illegal,
many species continue to experience population declines. According to the U.S. Shorebird
Conservation Plan (Brown et al. 2001), approximately 45% of migratory shorebird species are
declining and recent analyses of survey data continue to indicate similar trends (Morrison et al.
2006, Bart et al. 2007).
Several factors have been implicated in on-going shorebird population declines.
Anthropogenic factors include exposure to chemicals and pollutants, habitat degradation and
destruction, and continued illegal hunting (Senner and Howe 1984, Zöckler et al. 2003). The
goal of my thesis was to evaluate migratory shorebird exposure to one group of environmental
contaminants, organophosphorus and carbamate pesticides using plasma cholinesterase (ChE)
activity. Specifically, I aimed to 1) assess variation in plasma ChEs of migratory shorebirds
1
during migration and at non-breeding sites, 2) provide reference values of plasma ChEs from
North and South America for this and future ecotoxicological studies, and 3) evaluate exposure
of migratory shorebirds to organophosphorus and carbamate pesticides during migration and at
the non-breeding grounds. The results are applicable to current topics in shorebird conservation.
This thesis is organized into four core chapters. Here, I introduce the study. In chapter
two, I evaluate intrinsic and extrinsic variability of plasma ChEs of migratory shorebirds
captured during migration in North America. I provide reference values of plasma ChEs for
migratory shorebirds sampled during northbound migration in North America. In chapter three, I
provide reference values of plasma ChE activity for migratory shorebirds at non-breeding sites in
South America. I evaluate shorebird exposure to ChE-inhibiting pesticides by comparing ChE
activity in samples collected from agricultural habitats versus samples collected from reference
sites throughout the Western Hemisphere. The last chapter, chapter four, is an overview of
findings and major conclusions of my study.
2
References
Bart, J., S. Brown, B. A. Harrington, and R. I. G. Morrison. 2007. Survey trends of North
American shorebirds: population declines or shifting distributions? Journal of Avian
Biology 38:73-82.
Brown, S. C., B. Hickey, B. Harrington, and R. Gill, eds. 2001. The U.S. Shorebird Conservation
Plan, 2nd ed. Manomet Center for Conservation Sciences, Manomet, Massachusetts.
Forbush, E. H. 1916. A history of the game birds, wild-fowl and shorebirds of Massachusetts and
adjacent states. Massachusetts State Board of Agriculture, Boston, Massachusetts.
McIlhenny, E. A. 1943. Major changes in the bird life of Southern Louisiana during sixty years.
Auk 60:541-549.
Morrison, R. I. G., B. J. McCaffery, R. E. Gill, S. K. Skagen, S. L. Jones, G. W. Page, C. L.
Gratto-Trevor, and B. A. Andres. 2006. Population estimates of North American
shorebirds, 2006. Wader Study Group Bulletin 111:67-85.
Page, G. W., and R. E. Gill, Jr. 1994. Shorebirds in Western North America: Late 1800's to late
1900's. Studies in Avian Biology 15:147-160.
Senner, S. E., and M. A. Howe. 1984. Conservation of Nearctic shorebirds. Pages 379-421 in
Behavior of Marine Animals: Shorebirds, vol. 5. (J. Burger and B. L. Olla Eds.) Plenum
Press, New York, New York.
Skagen, S. K., and F. L. Knopf. 1993. Toward conservation of midcontinental shorebird
migrations. Conservation Biology 7:533-541.
Zöckler, C., S. Delany, and W. Hagemeijer. 2003. Wader populations are declining - how will
we elucidate the reasons? Wader Study Group Bulletin 100:202-211.
3
CHAPTER 2 - Plasma cholinesterases for monitoring pesticide
exposure in Nearctic-Neotropical migratory shorebirds*
Khara M. Strum, Matilde Alfaro, Ben Haase, Michael J. Hooper, Kevin A. Johnson,
Richard B. Lanctot, Arne J. Lesterhuis, Leticia López, Angela C. Matz, Cristina Morales,
Benjamin Paulson, Brett K. Sandercock, Julian Torres-Dowdall, and María Elena Zaccagnini
Abstract
Organophosphorus (OP) and carbamate (CB) pesticides are commonly used
agrochemicals throughout the Western Hemisphere. These pesticides have caused mortalities in
migratory birds and adverse physiological effects in trials with captive birds. Migratory
shorebirds use a variety of habitats during the austral summer in temperate South America and
during migration through the Great Plains of the United States. Habitats where risk of exposure
is high include rice fields and turf grass farms where agrochemicals are used. Cholinesterase
(ChE) is a specific biomarker for monitoring OP and CB exposure and can be measured using
standard laboratory procedures. Plasma ChE activity is useful as a non-lethal means of
monitoring avian exposure to OP and CB pesticides. Many variables can affect enzyme activity
and reactivation assays are not always possible, thus reference values of ChE activity are a
necessary component of monitoring exposure. During northbound migration in 2006, we
sampled four upland and five wetland shorebird species at four pesticide-free sites in North
America, characterizing and measuring plasma ChEs in all shorebird species. Small-bodied
species had higher levels of ChE activity in plasma than large-bodied species.
*
Published as Strum et al. 2008. Ornitología Neotropical 19 (Suppl.): 641-651.
4
Acetylcholinesterase (AChE), the enzyme whose inhibition leads to symptoms of poisoning,
showed less inter-specific variation than butyrylcholinesterase (BChE). Plasma ChE activities
varied with date of capture in 3 of 5 species. Sex differences in BChE were significant in 1 of 2
species tested. Our baseline ChE values for migratory shorebirds provide a framework for future
ecotoxicological studies of Nearctic-Neotropical migrant shorebirds.
Introduction
Organophosphates (OPs) and carbamates (CBs) averaged 68% of the active ingredients in
insecticides used in the United States from 1980 through 2001 (Kiely et al. 2004). The use of
OPs and CBs increased in the 1970’s after organochlorine pesticides (e.g., DDT) were banned
due to health and environmental hazards (e.g., Henny and Bennett 1990). OPs and CBs provide
an alternative to the environmental persistence and bioaccumulation of organochlorines (Blus
2003). In spite of their limited persistence in the environment, many of these chemicals are
highly toxic to avian species and incidental kills of migratory birds are well documented (Basili
and Temple 1995, Goldstein et al. 1999a). Mass mortality incidents have resulted in public
awareness campaigns that emphasized the toxicity of OP and CB pesticides, and in some
countries, lead to laws against the use and manufacture of some of these pesticides (Hooper et al.
1999, Hooper et al. 2003).
Although many highly toxic OPs and CBs are prohibited or highly regulated in the
Americas (Anonymous 2004, USEPA 2007), instances of mortalities and high level exposures
have been reported recently (Pain et al. 2004, Wobeser et al. 2004, Renfrew et al. 2006).
Furthermore, less toxic OPs and CBs continue to be used in agriculture throughout North and
South America. For example, chemicals that inhibit cholinesterase (ChE) are part of the rice
cultivation industry in Uruguay and Argentina (Garamma et al. in Blanco et al. 2006a, MEZ
5
pers. com.). In the United States, OPs and CBs are recommended for pest control on a variety of
crops including rice and turf grass (Fagerness et al. 2001, Merchant 2005, Way and Cockrell
2007).
As part of their annual journey between breeding and non-breeding ranges, migratory
shorebirds cross international boundaries in search of available stop-over habitat. With the loss
of natural wetlands and grasslands (Knopf 1994, Skagen 2006), shorebirds are forced to use
human-altered habitats. Rice fields and turf grass farms provide important alternative wintering
and migratory stopover habitats for shorebirds (Twedt et al. 1998, Corder 2005, Blanco et al.
2006b, Robbins 2007), but also represent potential exposure to ChE-inhibiting chemicals
(Flickinger et al. 1986).
Although ChE activity has traditionally been measured by destructive sampling of brain
tissue, bird populations can be effectively monitored for OP and CB exposure using non-lethal
methods by measuring ChE activities in blood plasma (Hooper et al. 1989, Thompson 1991).
Acetylcholinesterase (AChE), an important enzyme in the central and peripheral nervous
systems, is responsible for the hydrolysis of the neurotransmitter acetylcholine (ACh), at the
nerve–nerve or nerve–effector interface. Without hydrolysis, ACh accumulates in the synapse,
disrupting neurotransmission, impairing behavior and physiology, and eventually leading to
death (Grue et al. 1997, Goldstein et al. 1999a). Plasma ChE activity can demonstrate exposure
levels consistent with intoxication and death in subsets of a population (Hooper et al. 1989,
Goldstein et al. 1999a), as well as a lack of exposure (Goldstein et al. 1999b).
Comparison of ChE activity from field samples to reference values can be used alone or
in conjunction with reactivation assays. Poisoning by OPs and CBs produces similar
physiological effects but reactivation assays allow for differentiation between these two types of
6
poisonings. Reactivation assays also address potential concerns associated with inter-species or
inter-individual ChE variation (Grue 1982, Hill 1989, Fossi et al. 1996). However, reference
values of ChE activity are especially important if reactivation assays cannot be used because
sample volumes are too small or because OP aging results in chemically stable OP–enzyme
bonds (Wilson et al. 1992).
Here, we present reference values of plasma ChE activity for apparently healthy, freeliving individuals of nine shorebird species that use upland and wetland habitats. To describe
ChE activity within and among shorebird species, we tested five factors that are known to affect
ChE activity in other birds: interspecific variation with regard to body mass and intraspecific
variation with regard to sex, body condition and date and time of capture. Our estimates of
plasma ChE activity are among the first values published for shorebirds and will be useful as
reference values in future toxicological studies of Nearctic-Neotropical migratory shorebirds.
Methods
Shorebird Capture
Shorebird capture occurred in three states (Texas, Kansas, and Nebraska) in the United
States, and three countries (Paraguay, Argentina, and Uruguay) in South America from April
through December 2006. Data on body mass data were pooled from all capture sites. The subset
of data used for baseline plasma ChE analysis included individuals captured between 22 April
and 1 June 2006 during northbound migration in the United States at protected wetlands and
grasslands. Northbound migration capture sites included Anahuac National Wildlife Refuge,
Chambers County, TX (29°34’N, 94°32’W), Quivira National Wildlife Refuge, Stafford County,
KS (38°08’N, 98°29’W), Konza Prairie Biological Station, Riley County, KS (39°04’N,
96°33’W) and Kissinger Wildlife Management Area, Clay County, NE (40°26’N, 98°06’W). In
7
2006, rice production at Anahuac National Wildlife Refuge was strictly organic, and there were
restrictions on pesticide application around Quivira National Wildlife Refuge boundaries
(M. Whitbeck pers. com., USEPA 2006). Konza Prairie and Kissinger Wildlife Management
Area are natural preserves that were also pesticide free (E. Horne and R. Souerdyke pers. com.).
Shorebirds were live-captured using mist nets, night-lighting, and drop nets, under applicable
state and federal research permits.
Sample Collection and Preparation
Mass of live-captured birds was measured using a Pesola spring scale (± 1.0 g). Wing
length was measured with a wing rule (± 0.5 mm). Total head, culmen and tarsus length were
measured using vernier calipers (± 0.1 mm). All birds were fitted with a USFWS metal band
with a unique number. When possible, shorebirds were sexed in the field according to Prater et
al. (1977). Upland Sandpipers (Bartramia longicauda) were sexed using molecular markers
based on the CHD gene (Baker et al. 1999, A. E. Casey unpubl. data).
Blood was collected using a 27-gauge needle and heparinized capillary tubes (70 µL)
from the brachial vein of the wing. Total blood collected per bird ranged between two to six
capillary tubes (140 – 420 µL) and was <1% of the bird’s body mass (Gaunt et al. 1999). Blood
samples were transferred to 0.5 mL screw cap cryovials, stored on wet ice in the field, and
centrifuged within 8 hours to separate plasma from red blood cells. Plasma samples were stored
at -20°C for less than one month and transferred to -80°C until laboratory analysis could be
conducted. All samples were assayed within one year of collection.
8
Laboratory Analysis
Samples were thawed immediately before ChE activity determination. As a first step, six
plasma samples from each species were pooled for characterization of optimal enzyme dilution
and reagent (acetylthiocholine-iodide [AThCh] and tetraisopropyl pyrophosphoramide [isoOMPA]) concentrations (see Appendix B). ChE activity was determined using the method of
Ellman et al. (1961) as modified by Gard and Hooper (1993) for use in a 96-well
spectrophotometric plate reader (Molecular Devices, Palo Alto, CA, USA) with Softmax Pro
software (Molecular Devices). Final volume of each assay was 250 µL and contained the
following components in 0.05 M Trizma buffer (pH 8.0): 3.23 x 10-4 M final concentration (FC)
of 5,5-dithio[bis-2-nitrobenzoic acid], diluted enzyme sample, and 1.00 x 10-3 M FC of AThCh.
To separate butyrylcholinesterase (BChE) from AChE, samples were incubated with the
BChE-specific inhibitor iso-OMPA at FCs between 1.0 x 10-4 M and 1.0 x 10-5 M according to
the characterization of each species. BChE was calculated as the difference between total
cholinesterase (TChE) and AChE activity in the presence of iso-OMPA. All samples were run in
triplicate at 25°C with the spectrophotometer set in kinetic mode. Absorbance was measured at
412 nm at 15 s intervals for 180 s with 0 s lag time. ChE activities were converted from
absorbance units per min to µmoles AThCh hydrolyzed per min (units) per mL of plasma using
an extinction coefficient of 13,600 (cm x M)-1.
Statistical Analysis
All statistical analyses were conducted using procedures of SAS (ver 9.1, SAS Institute,
Cary, NC, USA). All ChE activity fell within ±3 SD of the mean except for two TChE and
BChE values for Least Sandpiper which were over 4.5 times the mean for this species. These
two outliers were removed from subsequent analysis. All data presented are in raw form but
9
statistical results are based on log10-transformed data to correct for allometric scaling. General
linear models (Proc GLM) were used to determine the relationship of plasma ChEs among
species using a single factor fixed effects ANOVA. Regression models (Proc REG) were
calculated for plasma ChEs and time of capture, date of capture, and body condition for species
with ≥ 15 samples. For those species where sex could be reliably determined, sex differences in
plasma ChEs were compared using a Student’s t-test (Proc TTEST). Time of capture was
divided into four time periods of six-hour blocks each according to the following criteria:
1 = 0h – 05:59h, 2 = 06:00h – 11:59h, 3 = 12:00h – 17:59h, and 4 = 18:00h – 23:59h.
A multivariate index of body condition was computed by regressing the mass of each individual
at capture on PC1 from principal components analysis (PCA), using the residuals as an index of
body condition. PCA analyses were based on four morphological measurements, total head,
culmen, wing, and tarsus, and were calculated separately for each species. PC1 explained
between 34% and 66% of the variation in the four morphometrics. PC1 was an index of body
size because all eigenvectors were positive in seven of nine species; in the remaining two species
one eigenvector was negative (K. M. Strum unpubl. data). Average mass for each species was
calculated using a larger dataset of captured birds that included the subset used in ChE analysis.
All tests were two-tailed and considered significant at an α−level = 0.05 after Bonferroni
correction for the number of tests (Rice 1989).
Results
During northbound migration, we captured 174 individuals from 16 shorebird species,
and obtained sufficient plasma for ChE analysis from 138 individuals of 9 species. All samples
were used in analysis of AChE activity and after removing the two outliers of Least Sandpiper
TChE and BChE activity, 136 samples were used. We calculated average body mass for these
10
nine species from captures of 511 individuals at migratory and non-breeding sites throughout the
Western Hemisphere. Our study species included: American Golden-Plover (Pluvialis
dominica), Killdeer (Charadrius vociferus), Upland Sandpiper, Buff-breasted Sandpiper
(Tryngites subruficollis), Pectoral Sandpiper (Calidris melanotos), White-rumped Sandpiper
(C. fuscicollis), Stilt Sandpiper (C. himantopus), Least Sandpiper (C. minutilla), and
Semipalmated Sandpiper (C. pusilla).
TChE and BChE were highly correlated (r2 = 0.983, P < 0.001, n = 136). TChE and
AChE were also significantly correlated (r2 = 0.533, P < 0.001, n = 136) though less variation in
TChE could be explained by AChE. Some results are reported for BChE and AChE only. TChE
values for comparisons to other studies can be obtained by combining our AChE and BChE
values provided that substrate, substrate concentration, and assay temperature are identical.
Plasma BChE activity varied negatively with body size (F8,127 = 20.3, P < 0.001) as did AChE
(F8,129 = 11.0, P < 0.001; Fig. 2-1). Mean AChE ranged from 0.24 units/mL (± 0.14 SD, n = 25)
in Upland Sandpipers to 0.72 (± 0.38 SD, n = 34) in White-rumped Sandpipers, whereas mean
BChE ranged from 0.83 (± 0.01 SD, n = 2) in American Golden-Plovers to 5.92 (± 2.85 SD,
n = 19) in Semipalmated Sandpipers (Table 2-1). Values for Least Sandpiper outliers were
BChE: 15.68 and 19.66, TChE: 16.04 and 20.17. Both of these individuals were females and had
longer than average wing chord (≥ 100 mm).
Sex differences in plasma ChEs were evaluated in two species, Semipalmated and
Upland Sandpipers. Mean BChE was lower in male Upland Sandpipers (1.28 ± 0.54 SD, n = 13)
than females (1.89 ± 0.67 SD, n = 12; t23 = 2.60, P = 0.016). However, mean AChE was not
significantly different between male (0.20 ± 0.07 SD, n = 13) and female (0.28 ± 0.19 SD,
11
n = 12) Upland Sandpipers (t14.9 = 0.50, P = 0.615 [unequal variance]). Similarly, mean plasma
ChEs did not differ between male (AChE: 0.55 ± 0.25 SD, n = 7; BChE: 5.70 ± 2.95 SD, n = 7)
and female Semipalmated Sandpipers (AChE: 0.41 ± 0.19 SD, n = 12; BChE: 6.04 ± 2.92 SD,
n = 12, AChE: t17 = -1.02, P = 0.323; BChE: t17 = -0.06, P = 0.956).
In four species, the relationship between plasma ChEs and date of capture, time of
capture and body condition were analyzed (Table 2-2). Three species showed trends in ChE
activity as a function of capture date. Levels of BChE activity increased throughout the capture
period in Upland Sandpipers (r2 = 0.276, F1,23 = 8.8, P = 0.007) and Least Sandpipers
(r2 = 0.298, F1,16 = 6.79, P = 0.019). AChE activity increased throughout the capture period in
White-rumped Sandpipers (r2 = 0.117, F1,32 = 4.24, P = 0.048). Trends were marginally
significant in these three species after Bonferroni corrections for the number of tests (Rice 1989).
Other plasma ChE components did not vary with capture period in any of these species. There
was no significant relationship between time of capture or body condition for any species tested.
Discussion
Interspecific variation in plasma BChE activity decreased with increasing shorebird mass
similar to results from a study of plasma ChEs in European raptors (Roy et al. 2005). Massspecific metabolic demands decrease as shorebird body size increases (Kvist and Lindström
2001), which may be a partial explanation for the inverse relationship between shorebird plasma
ChE activity and body mass. Based on the high correlation between TChE and BChE, most of
the variation in shorebird TChE can be attributed to BChE activity. BChE has been shown to
successfully buffer AChE inhibition from some OP chemicals (Leopold 1996, Parker and
Goldstein 2000). Birds lack A-esterases which hydrolyze OP and CB pesticides (Aldridge 1953)
12
and higher levels of BChE activity may provide some protection against poisoning and
information about exposure.
Inclusion of all ChE activity results is important when presenting baseline ChE values,
however extreme outliers may influence statistical tests. For this reason, we removed two
outliers from our dataset before analysis. The causes of extreme BChE activity were unknown
but the two individuals with outlier values could have had liver damage or unusual levels of fat
metabolism during migration (Rattner and Fairbrother 1991, Valle et al. 2006).
Due to the interspecific variation in ChE activity with regard to body size, our data can be
used to estimate normal plasma ChE activity levels of species without reference values for field
sample comparison. While there is no substitute for species-specific reference values, patterns of
mass-specific variation in plasma ChE activity provide an initial framework for assessing
exposure in other shorebird species.
We found sex differences in mean plasma BChE in one species, the Upland Sandpiper.
At the time of capture, females were heavier than males (female mean mass = 166 g, n = 12;
male mean mass = 136 g, n = 13) and would be expected to have lower plasma ChE activities
based on the interspecific results of this study. However, females of Upland Sandpiper had
higher mean BChE activity than males. Our results may be related to breeding condition because
Upland Sandpipers evaluated in this study had recently arrived on the breeding grounds and
many females were at an egg-laying stage (B. K. Sandercock unpubl. data). An increase in
plasma ChEs during egg-laying has been reported in other avian species (Rattner and Fairbrother
1991). Samples of Upland Sandpiper during the non-breeding season as well as samples from
males and females of other shorebird species on the breeding grounds are needed to further
investigate this idea.
13
The condition of individual shorebirds was not related to plasma ChE activity in our
study. This is an important result since the physiological stress of migration can result in interindividual variation in body condition depending on the time since arrival at a stopover site and
the distance traveled prior to capture. Individuals in better condition presumably have more fat
and muscle translating into larger relative mass (Schulte-Hostedde et al. 2005), unrelated to ChE
activities. However, birds that died from anti-ChE exposure had lower fat and muscle scores due
to reduced food intake after poisoning (Grue 1982). In our study, body condition was not used
as an indicator of chemical exposure as it might be in another study.
Increases in plasma ChEs were marginally significant in three species throughout the
capture season. In each case, the percnt of variation explained in plasma ChE activity was fairly
low (r2 < 0.3). Seasonal variation in mean plasma ChEs has been detected in other migratory
birds and has been attributed to changes in diet (Goldstein et al. 1999b). In shorebirds, variation
in plasma ChEs during the capture season could be due to changes in diet or to changes in
physiological condition caused by changes in organ size during migration (Piersma and Gill
1998). Further investigation of the relationship between ChEs and date will be conducted using
data from individuals sampled in South America. With larger datasets from additional sites,
seasonal patterns in ChE activity may be more apparent.
The new data presented here provide a starting point for understanding variation in
plasma ChEs in Nearctic-Neotropical migratory shorebirds. Future analyses should be
conducted with samples collected at non-breeding sites in South America as well as the breeding
grounds. The relationship of plasma ChEs to environmental covariates should be further
explored to provide a more complete picture of shorebird plasma ChEs throughout the annual
cycle. Data on ChE activity could then be used to assess shorebird exposure to ChE-inhibiting
14
pesticides at any time of year. Once exposure is determined, efforts could be focused on affected
species to evaluate if the level of exposure poses a population threat. If so, efforts could begin
on developing regulations for OP and CB pesticides through partnerships with local and
international governments. If future studies demonstrate that shorebird exposure to ChEinhibitors is limited, this information will be used to redirect research efforts into other possible
causes of shorebird population declines.
15
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19
Figure 2-1 Log10-log10 graph showing the relationship between mean body mass and ChE
activity in 9 species of shorebirds captured during spring migration in the Great Plains
region of the United States. Sample sizes are inside the uppermost x-axis and error bars
represent ± SE. Pectoral Sandpiper (Calidris melanotos) is the higher of the two log10(ChE)
values where log10(body mass) = 1.81.
0.0
20
19
34
5
7 21
5
2 25
-0.2
-0.4
log(AChE) = -0.311log(Mass) + 0.155
r ² = 0.388
-0.8
1.0
log(BChE) = -0.702log(Mass) + 1.562
r ² = 0.652
0.8
0.6
0.4
0.2
0.0
-0.2
1.0
log(TChE) = -0.633log(Mass) + 1.527
r ² = 0.695
0.8
0.6
0.4
0.2
0.0
1.4
1.6
1.8
2.0
2.2
20
American Golden-Plover
Upland Sandpiper
Killdeer
Pectoral Sandpiper
Buff-breasted Sandpiper
Stilt Sandpiper
Log10(Mean Shorebird Mass)
White-rumped Sandpiper
Semipalmated Sandpiper
-0.2
Least Sandpiper
Log10(Plasma Cholinesterase Activity)
-0.6
Table 2-1 Descriptive statistics of ChE activity (units/mL plasma) for 9 shorebird species
sampled during northbound migration in the Great Plains region of the United States
including sample size of individuals (n)†, mean, standard deviation (SD), minimum (min)
and maximum (max) values.
Species
American Golden-Plover
Killdeer
Upland Sandpiper
Buff-breasted Sandpiper
Pectoral Sandpiper
White-rumped Sandpiper
Stilt Sandpiper
Least Sandpiper
Semipalmated Sandpiper
n
2
5
25
21
7
34
5
20
19
mean
1.20
1.56
1.81
2.18
3.22
3.23
3.82
3.37
6.38
TChE
SD min
0.01 1.20
0.55 0.91
0.70 0.69
0.38 1.71
0.80 1.53
0.80 1.95
0.67 2.89
0.81 1.92
2.95 1.28
max
1.21
2.42
3.29
3.11
4.01
4.75
4.72
4.67
10.78
mean
0.37
0.41
0.24
0.35
0.48
0.72
0.52
0.45
0.46
†
AChE
SD min
0.00 0.37
0.16 0.21
0.14 0.02
0.11 0.16
0.12 0.32
0.38 0.30
0.16 0.37
0.10 0.32
0.22 0.07
Total sample size. Estimates of TChE and BChE exclude two outliers of Least Sandpiper.
21
max
0.37
0.59
0.65
0.65
0.66
2.31
0.69
0.60
0.89
BChE
mean SD min
0.83 0.01 0.83
1.15 0.45 0.69
1.57 0.67 0.51
1.79 0.27 1.43
2.74 0.72 1.21
2.56 0.69 1.53
3.31 0.60 2.45
2.92 0.76 1.33
5.92 2.85 1.21
max
0.84
1.89
3.11
2.24
3.35
3.99
4.03
4.16
10.11
Table 2-2 Trends in plasma ChE activity of 5 species of shorebirds as a function of date of
capture, time of capture and an index of body condition using log10 transformed ChE
activity. After sequential Bonferroni correction for number of tests, test statistics were
considered significant at an α-level of 0.05 if P < 0.002.
Date of Capture
df
F
P≤
1,23 0.0 0.956
1,23 8.8 0.007
Time of Capture
df
F
P≤
1,23 0.9 0.359
1,23 0.0 0.836
Index of Body
Condition
df
F
P≤
1,23 1.8 0.193
1,23 0.2 0.640
Buff-breasted Sandpiper log(AChE)
log(BChE)
1,19
1,19
0.0
2.2
0.891
0.152
1,19
1,19
0.3
0.8
0.858
0.373
1,19
1,19
0.2
0.4
0.692
0.550
White-rumped Sandpiper log(AChE)
log(BChE)
1,32
1,32
4.2
2.5
0.048
0.123
1,32
1,32
0.1
0.2
0.750
0.653
1,32
1,32
0.0
0.3
0.876
0.599
Least Sandpiper
log(AChE)
log(BChE)
1,18
1,16
0.1
6.8
0.820
0.019
1,16
1,14
0.6
0.0
0.452
0.886
1,18
1,16
0.5
0.0
0.498
0.977
Semipalmated Sandpiper log(AChE)
log(BChE)
1,17
1,17
0.1
3.1
0.736
0.097
1,17
1,17
0.3
0.1
0.615
0.715
1,16
1,16
0.0
0.7
0.926
0.419
Species
Upland Sandpiper
ChE Type
log(AChE)
log(BChE)
22
CHAPTER 3 - Exposure of migratory shorebirds to cholinesteraseinhibiting contaminants in the Western Hemisphere
Khara M. Strum, Michael J. Hooper, Kevin A. Johnson, Richard B. Lanctot,
Brett K. Sandercock, and María Elena Zaccagnini
Abstract
Migratory shorebirds traverse long distances during their annual movements and access
to quality habitats is critical to completion of a successful migration. Many natural habitats have
been degraded or destroyed and shorebirds increasingly utilize agricultural habitats. Use of
cultivated fields and similar habitats may increase risk of exposure to cholinesterase (ChE)inhibiting pesticides. To evaluate exposure of migratory shorebirds to organophosphorus (OP)
and carbamate (CB) pesticides, we sampled birds at stopover sites during spring and fall
migration in North America and at non-breeding sites in South America. Birds were sampled
from reference sites with no known OP or CB applications and pesticide use sites where
agrochemicals were recommended for control of crop pests. Blood samples and footwashes
were collected from live-captured individuals. Plasma acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) activity levels of Buff-breasted Sandpipers were lower at pesticide
use sites in South America but BChE activity was higher at pesticide use sites in North America.
There were no differences between reference sites and pesticide use sites in four other species
tested. Regional differences in plasma ChE activities from reference sites were tested in two
species. Plasma AChE activity was higher among White-rumped Sandpipers sampled in North
America but there was no difference between regions for Buff-breasted Sandpipers. A metaanalysis across all species indicated 4 of 6 effect sizes were negative for AChE but there was an
23
overall positive effect of pesticide use sites on BChE activity in North America. Comparisons of
body mass between sites and regions suggest that habitat use did not affect maintenance of body
mass. Our study provides the first prospective estimates of shorebird exposure to ChE-inhibiting
pesticides. Overall, our results suggest that 1 of 5 shorebird species tested was exposed to
ChE-inhibiting pesticides at the non-breeding grounds and continued monitoring is necessary.
We highlight the importance of complete sampling and addressing variability in plasma ChEs as
part of evaluating potential exposure to organophosphorus and carbamate pesticides.
Introduction
Ongoing declines in populations of migratory shorebirds have spurred research to
investigate possible environmental factors driving population dynamics range-wide. Exposure to
environmental contaminants, habitat loss and degradation, disturbance, and illegal hunting have
been suggested as anthropogenic factors that may limit shorebird populations (Senner and Howe
1984, Page and Gill 1994, Zöckler et al. 2003). Previous research on shorebird exposure to
environmental contaminants has focused on heavy metals, organochlorines, and polychlorinated
biphenyls (White et al. 1980, White et al. 1983, Custer and Myers 1990, Custer and Mitchell
1991, Burger et al. 1993, Hui 1998, Hui et al. 2001, McFarland et al. 2002). Few studies have
investigated exposure of migratory shorebirds to pesticides that act by cholinesterase (ChE)
inhibition (Mitchell and White 1982, Fair et al. 1995, Iko et al. 2003). These chemicals are
potent insecticides used to control agricultural pests throughout North and South America. For
example, over 49 million kg of insecticides were applied to protect crops in the United States in
2001. Of these, 70% were ChE-inhibiting organophosphorus (OP) and carbamate (CB)
pesticides (Kiely et al. 2004). These two types of pesticides replaced organochlorines due to
their relatively rapid degradation and lack of bioaccumulation. Nevertheless, OPs and CBs are
24
highly toxic over the short-term and may pose a risk to non-target, avian species. Migratory
birds have been poisoned while exploiting resources in agricultural habitats in South America
(Basili and Temple 1995, Goldstein et al. 1999a). In addition to mortality, sub-lethal exposure to
OPs and CBs can cause a suite of physiological impairments including loss of migratory
orientation and decreased flight speed (Vyas et al. 1995, Grue et al. 1997, Brasel et al. 2005).
The Great Plains region of the United States is an important flyway for migratory
shorebirds that breed at arctic or north temperate latitudes and winter in Southern South America
(Skagen et al. 1999). The landscape of the Great Plains flyway is dominated by agriculture.
Similarly, non-breeding habitats in South America have largely been converted into agricultural
and rangeland areas (Wetmore 1927, Soriano et al. 1992, Isacch and Martínez 2003, Blanco et al.
2006). Thus, evaluation of potential exposure to ChE-inhibiting pesticides is important to
conservation efforts of migratory shorebirds.
Life history traits and migratory pathways of shorebirds may determine risk of pesticide
exposure for different species. Nearctic-Neotropical shorebird migrants can be categorized into
two groups based on their habitat requirements: upland and wetland. Upland shorebirds prefer
drier habitats comprised of low vegetation and include American Golden-Plover (Pluvialis
dominica), Upland Sandpiper (Bartramia longicauda), and Buff-breasted Sandpiper (Tryngites
subruficollis; Myers and Myers 1979, Isacch and Martínez 2003). These three species regularly
use agricultural habitats at stopover and wintering sites where they may come into direct contact
with OPs and CBs. Upland shorebirds consume a variety of invertebrates, including agricultural
pests, whose population numbers increase during shorebird migration (Houston and Bowen
2001, Nagoshi and Meagher 2004, Isacch et al. 2005). Wetland species, such as Least Sandpiper
(Calidris minutilla), Pectoral Sandpiper (C. melanotos), and White-rumped Sandpiper
25
(C. fuscicollis) require habitats with standing water, and frequently use rice fields and other
agricultural areas where use of ChE inhibitors may be widespread (Hands et al. 1991, Skagen
and Knopf 1993, Twedt et al. 1998, Skagen et al. 2005, Blanco et al. 2006). In fact, small
numbers of shorebirds have been found dead in rice fields after the application of carbofuran, a
potent anti-ChE (Flickinger et al. 1980; 1986, Littrell 1998).
ChE activity in brain or plasma is often measured as an indication of exposure to OP and
CB pesticides (Soler-Rodriguez et al. 1998). Measurement of brain ChE activity is useful for
diagnosing OP or CB poisoning post-mortem but requires euthanizing the bird. On the other
hand, plasma ChE activity is sensitive to low dose exposure to OPs and CBs and can be used to
monitor sub-lethal exposure among live-captured individuals (Hooper et al. 1989, Thompson
1991, Wilson et al. 1991, Soler-Rodriguez et al. 1998). There are two main types of ChE in
avian plasma. Acetylcholinesterase (AChE) is responsible for hydrolysis of the neurotransmitter
acetylcholine and maintains proper functioning of the central and peripheral nervous systems.
The exact function of butyrylcholinesterase (BChE) is unknown although it has been shown to
successfully buffer AChE inhibition from some OP compounds (Leopold 1996, Parker and
Goldstein 2000).
Both enzymes can be used to monitor exposure to OP and CB pesticides by comparison
of field samples to reference values or by reactivation of inhibited enzyme (Hooper et al. 1989,
Strum et al. 2008). Plasma ChEs can vary on spatial and temporal scales (Goldstein et al. 1999b)
but reference values rarely control for these factors. Similarly, reactivation attempts can be
hampered by dealkylation and spontaneous reactivation of inhibited enzymes (Wilson et al.
1992). Evaluating OP and CB exposure in migrant birds must account for sources of variability
in reference values that can interfere with transcontinental ChE monitoring efforts.
26
The purpose of our study was to evaluate exposure of migratory shorebirds to ChEinhibiting pesticides. We sampled shorebirds at stopover sites in North America and at their
non-breeding grounds in South America. In previous work, we reported reference values of
plasma ChE activity for nine species of migratory shorebirds sampled at stopover sites in the
Great Plains and addressed variability of plasma ChEs (Strum et al. 2008). Here, we provide the
first values for non-breeding shorebirds in South America. Moreover, we compared plasma ChE
activity and body mass between sites with no OP and CB use versus sites where ChE-inhibitors
were recommended for pest control. We calculated an effect size of habitat type on ChE activity
across all species. We also tested for variation in plasma ChEs by region to assess the need for
reference values at different temporal and spatial scales. If shorebirds were exposed to
pesticides, we predicted plasma ChE activity and mean body mass would be lower in samples
from sites with insecticide use (Grue et al. 1997, Goldstein et al. 1999b). We predicted a change
in ChE by region if dietary changes were occurring between the breeding and non-breeding
grounds (Goldstein et al. 1999b). Our study represents the first large-scale, prospective
evaluation of anti-ChE pesticide exposure to address a potential conservation threat to migratory
shorebirds throughout their range (Hooper et al. 2003).
Methods
Shorebird Capture
We captured shorebirds in three states (Texas, Kansas and Nebraska) in the United States
from April to August 2006 and March to June 2007, and three countries (Argentina, Paraguay,
and Uruguay) in South America from September to December 2007. We concentrated our
capture efforts at natural sites with no known pesticide use (reference sites) and agricultural sites
27
where OPs and CBs were recommended for pest control according to national or state crop
guidelines (pesticide use sites).
During northbound migration in 2006, four reference sites in North America included
coastal prairie and organic rice fields at Anahuac National Wildlife Refuge, Chambers County,
TX (29°34’N, 94°32’W), salt marshes at Quivira National Wildlife Refuge, Stafford County, KS
(38°08’N, 98°29’W), native grassland at Konza Prairie Biological Station, Riley County, KS
(39°04’N, 96°33’W), and wetlands at Kissinger Wildlife Management Area, Clay County, NE
(40°26’N, 98°06’W). Rice production at Anahuac National Wildlife Refuge was strictly organic
and there were restrictions on application of pesticides around Quivira National Wildlife Refuge
boundaries (M. Whitbeck pers. com., USEPA 2006). Konza Prairie and Kissinger Wildlife
Management Area are natural reserves that were also pesticide free (E. Horne and R. Souerdyke
pers. com.). During southbound migration in 2006 and northbound migration in 2007,
shorebirds were sampled at one reference site, salt marshes of Bolivar Flats Shorebird Sanctuary
(29°78’N, 94°77’W) and three agricultural sites, turf grass farms near Lawrence, KS (38°58’N,
95°14’W), Colwich, KS (37°46’N, 97°32’W), and Crosby, TX (29°54’N, 95°03’W).
Shorebirds were captured during the non-breeding season in 2006 in Southern South
America. Three reference sites included inundated grasslands along the shoreline of Bahía de
Asunción, Paraguay (25°16’S, 57°37’W), salt marshes and shoreline of Laguna Mar Chiquita,
Cordoba, Argentina (30°32’S, 62°17’W), and coastal grasslands of Laguna de Rocha, Rocha
Department, Uruguay (34°40’S, 54°17’W). Bahía de Asunción and Laguna Mar Chiquita are
designated as sites of regional and hemispheric importance for shorebirds by the Western
Hemisphere Shorebird Reserve Network. Bahía de Asunción is centrally located within the city
of Asunción with no adjacent agriculture. Laguna Mar Chiquita and Laguna de Rocha are
28
nationally protected parks, neither of which had OPs or CBs applied to local capture areas during
the field season (H. Caimarys and E. Martin pers. com.). Two agricultural sites sampled in
South America included rice fields and cattle pastures near San Joaquin, Santa Fe Province,
Argentina (30°42’S, 60°52’W) and Coronilla, Rocha Department, Uruguay (33°48’S, 53°39’W).
Shorebirds were live-captured using mist nets, drop nets, and night-lighting under
applicable state, federal, and international research permits. In addition, a small number of
Buff-breasted Sandpipers were collected by shotgun at two sites, turf grass farms near Lawrence
and Colwich, KS. After sampling, all birds were deposited as study skins at the University of
Kansas, Museum of Natural History (Robbins 2007).
Sample Collection and Preparation
To examine body size and condition of shorebirds among sites, seven morphological
measurements were recorded for each individual. Mass of live-captured birds was measured
using a Pesola spring scale (±1 g). Wing length was measured with a wing rule (±0.5 mm).
Total head, culmen and tarsus length were measured using vernier calipers (±0.1 mm). Fat
deposits in the furcula, sides, and abdomen were visually scored on a 9-point scale from zero to
eight, with zero indicating no fat and eight indicating large fat depots (Bairlein 1995). If
possible, shorebirds were sexed in the field according to Prater et al. (1977). Upland Sandpipers
and Buff-breasted Sandpipers were sexed using molecular markers based on the CHD gene
(Baker et al. 1999, A.E. Casey unpubl. data, K. M. Strum unpubl. data.). All birds were fitted
with a USFWS metal band with a unique number.
Field samples for pesticide exposure included blood, brain tissue, and footwashes. Blood
was collected using a 27-gauge needle and heparinized capillary tubes (70 µL) from the brachial
vein of the wing. Total blood collected per bird ranged between two to six capillary tubes
29
(140 to 420 µL) and was <1% of the bird’s body mass (Gaunt et al. 1999). Blood samples were
transferred to 0.5 mL screw cap cryovials, stored on wet ice in the field, and centrifuged within
8 hours to separate plasma from red blood cells. Plasma samples were stored at -20°C for one to
four weeks and transferred to -80°C until laboratory analyses could be conducted. Collected
individuals were wrapped in chemically-clean foil, placed on wet ice in the field and frozen at
-80°C within 8 hours. Brains were excised partially-frozen, placed into 2.0 mL Eppendorf tubes
and immediately refrozen at -80°C. The tarsi and feet of captured and collected individuals were
rinsed with 15 mL pesticide grade 2-propanol collected in an amber glass jar with a Teflon lined
lid via a stainless steel funnel. Footwashes were stored on wet ice in the field and transferred to
-20°C until analyses could be conducted. All samples were analyzed within one year of
collection.
ChE Activity
Laboratory analyses of ChE activity were conducted at The Institute for Environmental
and Human Health (TIEHH) at Texas Tech University and at the Division of Biology at Kansas
State University (KSU). Samples analyzed at TIEHH were read on a SpectraMax plate reader
(Molecular Devices, Palo Alto, CA, USA) with Softmax Pro software (Molecular Devices).
KSU samples were analyzed in a Model 680 microplate reader (Bio-Rad Laboratories, Hercules,
CA, USA) with Microplate Manager software (ver 5.2.1, Bio-Rad Laboratories). Assay
conditions and reagent concentrations were identical between labs to minimize inter-lab
differences. To quantify within and between lab variation, aliquots of horse serum (Invitrogen
Corporation, Carlsbad, CA, USA) of known ChE activity were run on each plate containing
shorebird samples (see Appendix A).
30
Sample preparation differed according to tissue type. Plasma samples were thawed
immediately before ChE activity determination and diluted to a species-specific concentration
with cold 0.5 M, 7.4 pH buffer. Brains were thawed and homogenized using the Tissue Miser
electric homogenization tool (Fisher Scientific, Pittsburgh, PA, USA) at a 1:3 ratio of
tissue:buffer. ChE activity was determined immediately after homogenization on a 60- or 72fold serially diluted sample.
All ChE activity for plasma and brain was determined using the method of Ellman et al.
(1961) as modified by Gard and Hooper (1993) for use on a 96-well plate reader. Final volume
of each assay was 250 µL and contained the following components in 0.05 M Trizma buffer
(pH 8.0): 3.23 x 10-4 M final concentration (FC) of 5,5-dithio[bis-2-nitrobenzoic acid], diluted
enzyme sample, and 1.00 x 10-3 M FC of acetylthiocholine-iodide (AThCh). To separate activity
of BChE from AChE, a portion of each sample was incubated with the BChE-specific inhibitor,
tetraisopropyl pyrophosphoramide (iso-OMPA), at FC 1.0 x 10-4 M. BChE activity was
calculated as the difference between total cholinesterase (TChE) measured without iso-OMPA
and AChE activity in the presence of iso-OMPA. All samples were run in triplicate at 25°C with
the spectrophotometer set in kinetic mode. Absorbance was measured at 412 nm at 15 s intervals
for 180 s with a lag time of 0 s. ChE activities were converted from absorbance units per min to
µmoles AThCh hydrolyzed per min (units) using an extinction coefficient of 13,600 (cm x M)-1
and were expressed as units per mL of plasma or units per g of brain tissue.
Reactivation Assays
Mean plasma ChE activity was lower at pesticide use sites than at reference sites in one
species, the Buff-breasted Sandpiper (see Results). Reactivation assays were conducted to test
for OP and CB inhibition of enzyme activity on samples from this species with an adequate
31
volume after dilution (≥ 1.3 mL). Reactivation of OP-inhibited enzyme required two aliquots of
diluted sample. One aliquot was incubated with 1.00 x 10-4 M FC pyridine-2-aldoxime
methochlorine (2-PAM) at 25°C to induce chemical reactivation. The remaining aliquot
received an equal volume of distilled water and was incubated alongside the sample with 2-PAM
as a negative control. Aliquots were assayed after 0.5 hr. CB-inhibited enzyme reactivation
analysis also required two additional sample aliquots. One aliquot was incubated at 37°C to
induce spontaneous reactivation whereas the other aliquot was maintained on ice as a negative
control. Both aliquots were assayed after one hr.
Chemical Residue Analyses
Footwash samples were screened against reference standards for 13 OPs and 7 CBs at a
detection limit of 0.5 ug/mL or 1 ug. OP residues were analyzed on a HP 6890 gas
chromatograph (GC) equipped with a nitrogen-phosphorous detector (NPD). The column
(J & W Scientific, Rancho Cordova, CA, USA) was a 30-m x 0.25 mm i.d. fused silica capillary
coated with a DB-1701 stationary phase (film thickness of 0.25 µm) and a carrier gas (He) flow
rate of 1.5 mL/min. Injector and detector temperatures were 250°C and 275°C, respectively. CB
residues were analyzed on a HP 1100 Series liquid chromatograph (HPLC) with a Post-Column
Derivatizer (PCX) 5200® (Mountain View, CA, USA) and a fluorescence detector (excitation
and emission wavelengths of 330 nm and 465 nm, respectively). The column (Discovery® C8,
Supelco, St. Louis, MO, USA) was 250 mm x 4.6 mm (5 µm particle size) operated at a flow rate
of 0.8 mL/min. Standard operating conditions were a water:methanol ratio of 88:12 for 2 min,
followed by an increase in methanol to 34:66 over 40 min with hold time of 4 min followed by
an increase to 100% methanol in 0.1 min with a final hold time of 3 min.
32
For both GC and HPLC analyses, an internal standard adjusted for some of the internal
variation of the instrument, while an intermittent standard was run after every 5 injections to
assess variability. If the intermittent standard varied by more than 10%, the sample set was
re-run. A five-point standard curve was constructed from constant volume injections of
calibration standards. Computer-generated peak areas were used to measure sample
concentrations in an external standard method.
Statistical Analyses
Statistical analyses were performed with Program SAS (ver 9.1.3, SAS Institute, Cary,
NC, USA). We performed our meta-analysis with Program MetaWin (ver 2.0, Sinauer
Associates, Sunderland, MA, USA). An important covariate for variation in plasma ChE activity
is interspecific variation in body size and we conducted analyses separately for each species of
shorebird (Roy et al. 2005, Strum et al. 2008).
Mean ChE activity averaged from triplicate readings was used in analyses if the
coefficient of variation was below 10%. Z-tests were used to identify outliers where
observations were ≥ 3SD of the overall mean for each species. Comparisons of mean ChE
activities between regions (North vs. South America) and between reference sites and pesticide
use sites within regions were performed using t-tests (Proc TTEST). Tests for differences
between regions and between reference sites and pesticide use sites were two-tailed. Tests for
differences between regions were calculated using data from reference sites only.
Despite considerable sampling effort, we were unable to capture all species at each
combination of region and site, which precluded use of standard statistical analysis with factorial
models. Thus, to synthesize the effect of habitat (reference vs. pesticide use) on ChE activity
among shorebirds, we calculated individual, regional, and cumulative effect sizes, using
33
Hedges’ d as a measure of effect size (Gurevitch and Hedges 1993). Our meta-analysis
quantified an overall effect of treatment (pesticide use sites) on a response variable (ChE
activity). The magnitude of the effect size (d) is unbounded and can be positive or negative.
According to Cohen (1988), values of d ≤ 0.2 are small, d = 0.5 are moderate, and d ≥ 0.8
indicates a large effect. We used reference sites as the control and negative effect sizes indicate
that individuals captured at pesticide use sites had lower ChE activity than birds sampled at
reference sites.
ChE reactivation was evaluated using a two-tailed t-test (Proc TTEST). To evaluate
reactivation of OP-inhibited enzyme, we compared the post-incubation ChE activity of the
sample incubated with 2-PAM versus the negative control. Similarly, CB inhibition of enzyme
was tested by comparing post-incubation activity of the incubated sample versus the sample kept
on ice. Some sample volumes were insufficient to conduct all reactivation analyses resulting in
unequal sample sizes between assays.
One potential effect of ChE inhibition can be reduced feeding and anorexia (Grue 1982,
Grue et al. 1997). To test for a physiological effect of ChE inhibition, we first examined whether
fat score was correlated with body mass. We then used body mass as an indicator of fitness and
compared mean body mass between regions and treatments (sites) within a region using a two
sample t-test (Proc TTEST). Comparisons between regions were conducted using data from
reference sites only.
Results
We sampled a total of 671 individuals of 14 shorebird species at stopover sites and nonbreeding sites in the Western Hemisphere. Our study species included 11 species of latitudinal
34
migrants and 3 species of Neotropical endemics. Number of individuals of a species captured
ranged from 5 (South American Painted-Snipe) to 122 (White-rumped Sandpiper; Table 3-1).
Of the captured individuals, 452 birds had sufficient sample volumes of plasma for
analyses of ChE activity. Two Least Sandpipers had BChE activity 15 to 20 SD greater than the
mean for this species as reported in Strum et al. (2008) and were omitted from statistical
analyses. In South America, mean AChE (units/mL plasma) ranged from a low of 0.28 (Buffbreasted Sandpiper) to 0.61 (Lesser Yellowlegs) and mean BChE activity ranged from 0.58
(South American Painted-Snipe) to 5.38 (Lesser Yellowlegs; Table 3-2). In North America, we
found that mean AChE ranged from a low of 0.27 (Upland Sandpiper) to 0.73 (White-rumped
Sandpiper) and mean BChE activity ranged from a low of 1.63 (Upland Sandpiper) to 8.34
(Semipalmated Sandpiper; Table 3-3).
Spatial Variation in Plasma ChE Activity
Variation in plasma ChEs by site (reference vs. pesticide use) was apparent in 1 of 5
species tested. Buff-breasted Sandpiper AChE and BChE activity was 36.7% (t21 = -2.26,
P = 0.034) and 31.6% lower (t21 = -2.91, P = 0.008) at pesticide use sites in South America
(Table 3-2, Fig. 3-1). However, Buff-breasted Sandpiper BChE activity was 21.0% higher in
individuals sampled from pesticide use sites in North America (t39 = 3.47, P ≤ 0.001; Table 3-3,
Fig. 3-1). There were no further significant differences in ChE activity as a function of habitatspecific pesticide use in the other shorebird species with sufficient sample numbers for
comparison (Tables 3-2 and 3-3, Fig. 3-1).
AChE and BChE activity from reference sites differed between regions for 1 of 2 species
(Tables 3-2 and 3-3, Fig.3- 2). White-rumped Sandpiper AChE was 19.3% higher in individuals
sampled in North America than South America (t97 = 2.50, P = 0.014). Similarly, mean BChE
35
activity was 26.7% higher for Pectoral Sandpipers sampled in North America but statistical
inferences were not possible due to the small sample size from South America. There was no
significant difference in mean plasma AChE or BChE activity between regions in Buff-breasted
Sandpipers sampled at reference sites.
Mean TChE activity of Buff-breasted Sandpiper brains collected at turf grass farms was
24.3 units/g brain (± 3.1 SD, n = 6; min: 18.5, max: 27.5). Plasma TChE of Buff-breasted
Sandpipers sampled at pesticide use sites in North America was 2.7 units/mL plasma
(± 0.6 SD, n = 20) and represented 10.9% of brain TChE activity.
The effect size of habitat on ChE activity was variable among species and between
regions and overall, effect sizes were non-significant for AChE (d = -0.15, 95% CI: -0.41 to
+0.10) and BChE (d = 0.18, 95% CI: -0.35 to +0.60; Tables 3-2 and 3-3). Species-specific effect
sizes for AChE were negative for 4 of 6 comparisons and ranged from a low of -0.96 in
Buff-breasted Sandpipers to +0.27 in American Golden-Plovers, both sampled in South America.
Effect sizes for BChE were negative for 1 of 6 comparisons and ranged from -1.21 in
Buff-breasted Sandpipers captured in South America to +1.05 in Buff-breasted Sandpipers
captured in North America. There was a significant, positive effect of site on BChE activity in
North America (d = +0.58, 95% CI: +0.20 to +1.05) but not in South America (d = -0.11,
95% CI: -1.21 to +0.19). There were no significant regional effects on AChE activity
(NA: d = -0.20, 95% CI: -0.29 to +0.10; SA: d = -0.11, 95% CI: -0.47 to +0.27). Based on the
Qw statistic for heterogeneity, there was variation in BChE effect sizes among species in South
America (Qw = 6.64, P = 0.04) but not in North America (Qw = 3.52, P = 0.17). There were no
differences in effect sizes among species for AChE in North or South America (NA: Qw = 0.51,
P = 0.78; SA: Qw = 4.93, P = 0.085).
36
Plasma ChE reactivations were attempted in samples collected from Buff-breasted
Sandpipers in South America. Not all samples had adequate volume for reactivation analyses
and 12 samples from reference sites and 5 samples from pesticide use sites were tested for
reactivation of plasma ChE activity. Plasma AChE and BChE of Buff-breasted Sandpipers did
not reactivate using the 2-PAM reactivation method in samples from pesticide use sites (AChE:
t8 = -0.07, P = 0.95; BChE: t8 = 0.02, P = 0.99) or reference sites (AChE: t22 = 0.33, P = 0.74;
BChE: t22 = 0.45, P = 0.66). Likewise, plasma ChEs did not reactivate via spontaneous
reactivation for CB-inhibition from pesticide use sites (AChE: t8 = -0.03, P = 0.97; BChE:
t8 = -0.43, P = 0.68) or reference sites (AChE: t20 = -0.37, P = 0.72; BChE: t20 = -9.26,
P ≤ 0.001). No chemical residues were found in any footwash extraction.
Spatial Variation in Body Mass
Linear regression analyses indicated that fat score was a function of body mass in 7 of 13
species (r2 ≥ 0.4, P ≤ 0.05). Thus, we used only body mass as an indicator of individual
quality. There was sufficient data for regional and site comparisons in 7 species. Two of four
species showed site differences in body mass in South America and all three species tested
showed differences in North America (Table 3-4). In all but one species, mean body mass was
higher at pesticide use sites than at reference sites. In South America, Buff-breasted Sandpipers
were 9.7% heavier (t22 = 2.46, P = 0.023) and White-rumped Sandpipers were 5.7% heavier at
pesticide use sites (t84 = 2.27, P = 0.026) while American Golden-Plovers (t45 = 1.39, P = 0.17)
and Common Plovers (t14 = -0.11, P = 0.91) showed no difference in mean body mass between
sites of capture. In the Northern Hemisphere, Buff-breasted Sandpipers were 9.9% heavier
(t62 = 2.41, P = 0.019), Pectoral Sandpipers were 30.5% heavier (t35 = 4.21, P ≤ 0.001), and
Upland Sandpipers were 17.2% lighter (t65 = 1.91, P = 0.06) at pesticide use sites.
37
Two of three species tested showed regional differences in mean body mass; in all cases,
body mass was greater for individuals sampled in North America (Table 3-4). White-rumped
Sandpipers were 6.6% heavier (t104 = 3.16, P = 0.002) and Buff-breasted Sandpipers were 13.1%
heavier (t35 = 3.49, P ≤ 0.001) in North America. Stilt Sandpiper body mass did not differ
between regions (t16 = 0.11, P = 0.91).
Discussion
We conducted intensive sampling of 14 species of migratory shorebirds across the Western
Hemisphere to evaluate potential exposure to ChE-inhibiting pesticides. We used plasma ChEs
to evaluate exposure to organophosphorus (OP) and carbamate (CB) pesticides because the
sampling procedure was non-lethal and is sensitive to most OPs and CBs (Soler-Rodriguez et al.
1998). Our results demonstrate that of three species with sufficient data for comparisons
between reference sites and pesticide use sites, one Nearctic-Neotropical migratory shorebird,
the Buff-breasted Sandpiper, was likely exposed to ChE-inhibiting pesticides at non-breeding
sites in South America and that two other species, American Golden-Plovers and White-rumped
Sandpipers, were likely not. Of three species captured during migration in North America, no
evidence of exposure was detected. A meta-analysis indicated negative effects of habitat (site)
use on AChE activity in 4 of 6 comparisons and on BChE activity in one species. However, there
was a significant positive effect of habitat use on BChE in North America. No evidence of
negative physiological effects due to ChE-inhibitor exposure or habitat use was detected using
body mass.
38
Spatial Variation in Plasma ChE Activity
Plasma ChEs differed by site (reference vs. pesticide use) only in the Buff-breasted
Sandpiper. In South America, mean plasma AChE and BChE activities of Buff-breasted
Sandpipers were lower at pesticide use sites than reference sites and the magnitude of the
differences was consistent with inhibition by ChE-inhibitors reported in other migratory birds
(Goldstein et al. 1999a). In contrast with this result, BChE activity of Buff-breasted Sandpipers
sampled at pesticide use sites in North America was 21% higher than individuals sampled at
reference sites. This is opposite to the expected pattern if individuals were exposed to ChE
inhibitors but is consistent with differences in total plasma ChE activity found in Mountain
Plovers (Charadrius montanus) at an agricultural site compared to a reference site in North
America (Iko et al. 2003).
We found negative effect sizes on AChE activity in 2 of 3 species in each region ranging
from small (-0.10) to large (-0.96) effect sizes. Conversely, habitat type had a significant
positive effect on plasma BChE activity of shorebirds in North America using Hedges’ d.
Although both cumulative effect sizes were non-significant, the upper confidence interval for the
overall effect on AChE was slightly greater than zero. AChE serves an important role in
neurotransmission and is generally used to measure exposure to OPs and CBs. Given the
number and magnitude of negative species-specific effect sizes on AChE activity, we conclude
that shorebirds are exposed to ChE-inhibiting pesticides on the non-breeding grounds.
We found limited evidence for regional variation in ChE activity. Plasma AChE was
higher in North America than South America for 1 of 2 species (White-rumped Sandpipers).
BChE activity was also higher in North America for Pectoral Sandpipers though the sample size
from South America was small. Regional variation might be expected due to three
39
non-exclusive factors. First, lower AChE activity in South America relative to North America
could be due to changes in diet during movements between hemispheres (Goldstein et al. 1999b).
Second, the amount of subcutaneous fat may partially explain differences in plasma ChE
activity. A significant positive correlation between body fat and serum BChE activity has been
reported in human patients (Randell 2005). Last, exposure to other contaminants not measured
in this study could be affecting plasma ChE activity. Dieter (1974) found that chronic, low-dose
dietary exposure of quail to DDE (a metabolite of the organochlorine DDT), Aroclor 1254
(a polychlorinated biphenyl), and mercuric chloride (a heavy metal) affected plasma TChE
activity.
In cases like the Buff-breasted Sandpiper, where plasma ChE activity is lower at an
agricultural site where pesticides were recommended for use, reactivation of plasma ChEs
in vitro can be helpful in determining exposure to ChE inhibitors. However, we were unable to
successfully reactivate plasma ChEs by two independent methods. Our results are somewhat
inconclusive because some chemicals (i.e., di-methyl OPs) form a stable OP-enzyme complex
through dealkylation and resist chemical reactivation. In contrast, some OPs and CBs readily
undergo spontaneous reactivation (dephosphorylation or decarbamylation) leading to normally
functioning plasma enzymes not long after poisoning, making inhibition difficult to detect
(Wilson et al. 1992, Hunt and Hooper 1993). The rate of dealkylation or ageing is usually
inversely related to the rate of dephosphorylation (Wilson et al. 1992).
Extraction of chemical residues from footwashes can also provide evidence of exposure
to ChE inhibitors. Analysis of footwashings from this study yielded no evidence of dermal
exposure to OPs or CBs. We made efforts to sample shorebirds from areas where OPs and CBs
were recommended for pest control, but the actual spatial and temporal use patterns of these
40
pesticides were unknown. The disparity in Buff-breasted Sandpiper plasma ChE activity
between reference sites and pesticide use sites in South America deserves further study by
residue analyses of prey items collected in the field and assessments of exposure tied to known
anti-ChE applications (Hooper et al. 1989, Wilson et al. 1991).
Spatial Variation in Body Mass
One effect of ChE inhibition is reduced foraging ability and anorexia resulting in
decreased body mass (Grue 1982, Grue et al. 1997). We tested for an effect of exposure to OPs
and CBs by using body mass as an indicator of fitness, and made comparisons between sites and
regions. Although shorebird body mass varied between habitats, our results indicated that
shorebirds were able to maintain and even have higher body mass at pesticide use sites.
Specifically, we were unable to detect an effect of exposure to ChE inhibitors in Buff-breasted
Sandpipers. Regional body mass differences also existed in some species. Individuals sampled
in North America had heavier body masses which could be a result of variation in the migratory
status of these individuals. Shorebirds may maintain and use large fat stores during migration
while they are comparatively lean during stationary periods at non-breeding grounds (Harrington
et al. 1991).
Plasma and Brain ChE Activity
Buff-breasted Sandpiper brain TChE activity (24.3 units/g brain ± 3.1 SD, n = 6) was
similar to values published for other shorebird species. Using similar ChE analysis methods,
Mitchell and White (1982) reported 22.8 units/g brain (± 5.6 SD, n = 60) for Western
Sandpipers, 16.2 units/g brain (± 3.4 SD, n = 55) for Long-billed Dowitchers (Limnodromus
scolopaceus), and 19.4 units/g brain (± 4.5 SD, n = 30) for American Avocets (Recurvirostra
americana) sampled at Laguna Madre, Texas during the non-breeding season. Custer and
41
Mitchell (1991) reported values between 11.1 and 17.8 units/g brain (14.7 ± 1.8 SD, n = 55) for
Willets (Catoptrophorus semipalmatus) sampled from the same area.
Conclusions
Our field study is one of the first prospective studies to evaluate exposure to ChEinhibiting pesticides in migratory species. Overall, most species showed little evidence of
exposure. However, our results suggest that Buff-breasted Sandpipers were exposed to
ChE-inhibiting pesticides in South America and pesticide use sites could have potentially
negative effects on plasma ChE activity in other shorebird species. Buff-breasted Sandpiper
samples with depressed ChE levels originated from one field site in Argentina where other
species were not captured in large numbers. We suggest continued cooperation and
collaboration with investigators and universities in Argentina to continue monitoring of
Buff-breasted Sandpiper plasma ChE activity levels at the non-breeding grounds. Further
monitoring could also provide more robust sample sizes for species where we provide baseline
estimates of ChE activity but lacked complete sampling in this study.
The expansion of agriculture into native habitats will continue to pose a conservation
challenge for migratory species. Future research in ecotoxicology of migratory birds requires
continued collaboration with international partners to identify local sites of exposure and the
initiation of new partnerships to expand monitoring efforts to other sites of interest. The
development and use of new pesticide formulations that are less toxic to non-target species and
the coordination of pesticide applications to provide maximum benefit to farmers while posing
little threat to migratory species are also needed. Additionally, studies using stable isotopes and
radio-telemetry can increase our knowledge of habitat use by shorebirds during migration and at
42
non-breeding sites (Fry et al. 1998, Farmer et al. 2004). This knowledge can help better assess
exposure potential and focus future monitoring efforts in high-risk areas.
43
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49
Figure 3-1 Pesticide use effects on AChE (above) and BChE (below) of five shorebird
species captured in North and South America. Mean (units/mL plasma) and standard
error (SE) of ChE activity from individuals sampled from reference sites (R; closed circles)
and pesticide use sites (P; open circles). Site of capture, region (NA = North America and
SA = South America), and species are located below the lower x-axis. Sample sizes are
located above the lower x-axis. An asterisk (*) denotes a significant difference between
means using a two-tailed t-test at an α-level ≤ 0.05.
0.7
AChE Activity ± SE
(units/mL plasma)
0.6
0.5
0.4
*
0.3
0.2
0.1
0.0
3.5
BChE Activity ± SE
(units/mL plasma)
3.0
2.5
*
2.0
*
1.5
1.0
0.5
0.0
64 16
21 20
15
8
R P
R P
R P
SA
SA
NA
White-rumped
Buff-breasted
Sandpiper
Sandpiper
11 21
27 18
38
5
R P
R P
R P
SA
NA
NA
Pectoral
American
Upland
Sandpiper Golden-Plover Sandpiper
50
Figure 3-2 Influence of region on AChE (above) and BChE (below) in two shorebird
species. Region of capture (NA = North America, SA = South America) and species are
located below the lower x-axis. Sample sizes are located above the lower x-axis. An
asterisk (*) denotes a significant difference between regional means using a two-tailed t-test
at an α-level ≤ 0.05.
1.0
AChE Activity ± SE
(units/mL plasma)
0.8
*
0.6
0.4
0.2
0.0
3.0
BChE Activity ± SE
(units/mL plasma)
2.5
2.0
1.5
1.0
0.5
0.0
35
21
64
NA
SA
White-rumped
Sandpiper
15
NA
SA
Buff-breasted
Sandpiper
51
Table 3-1 Common names and Latin names of study species with sample size (n) of
individuals captured in each region.
Common Name
Nearctic-Neotropical Migrants
American Golden-Plover
Lesser Yellowlegs
Upland Sandpiper
Semipalmated Sandpiper
Least Sandpiper
Western Sandpiper
White-rumped Sandpiper
Pectoral Sandpiper
Stilt Sandpiper
Buff-breasted Sandpiper
Common Snipe
Latin Name
Pluvialis dominica
Tringa flavipes
Bartramia longicauda
Calidris pusilla
Calidris minutilla
Calidris mauri
Calidris fuscicollis
Calidris melanotos
Calidris himantopus
Tryngites subruficollis
Gallinago gallinago
South American Endemics
Southern Lapwing
Collared Plover
South American Painted-Snipe
Vanellus chilensis
Charadrius collaris
Nycticryphes semicollaris
52
North America (n) South America (n)
3
3
67
60
65
76
36
38
12
67
3
47
6
86
42
6
24
3
-
6
16
5
n
27
27
6
6
64
64
3
3
15
15
3
3
3
3
Reference
mean SD
min
0.37 0.13 0.18
1.08 0.30 0.51
0.61 0.20 0.32
4.38 1.23 2.99
0.59 0.19 0.20
2.32 0.87 0.28
0.52 0.03 0.48
1.84 0.32 1.50
0.41 0.16 0.23
1.79 0.28 1.07
0.31 0.08 0.22
2.26 0.61 1.60
0.44 0.17 0.28
0.89 0.17 0.72
* Indicates result is significant using a two-tailed t-test at an α-level of 0.05
** Indicates result is significant using a two-tailed t-test at an α-level of 0.01
ChE Type
AChE
BChE
Lesser Yellowlegs
AChE
BChE
White-rumped Sandpiper
AChE
BChE
Pectoral Sandpiper
AChE
BChE
Buff-breasted Sandpiper
AChE
BChE
Common Snipe
AChE
BChE
South American Painted-Snipe AChE
BChE
Species
American Golden-Plover
Comparisons were not conducted if n ≤ 5.
53
max
0.65
1.71
0.89
5.81
1.05
4.72
0.54
2.12
0.73
2.18
0.37
2.82
0.62
1.05
n
18
18
16
16
39
39
8
8
2
2
Pesticide Use
mean SD
min
0.40 0.07 0.26
1.14 0.33 0.70
0.57 0.19 0.24
2.34 0.61 1.42
0.39 0.14 0.12
2.09 0.53 0.92
0.26 0.13 0.13
1.23 0.66 0.40
0.47 0.11 0.39
0.58 0.10 0.51
South America
max
0.53
1.77
0.94
3.37
0.86
3.47
0.49
2.65
0.55
0.65
t
0.66
0.60
-0.24
0.10
-2.26
-2.91
-
P≤
0.51
0.55
0.82
0.92
0.03*
0.01**
-
d
+0.27
+0.19
-0.10
+0.02
-0.96
-1.21
-
maximum (max). ChE activity was compared between sites using a two-tailed t-test and a measure of effect size, Hedges' d.
SD(d)
0.30
0.30
0.28
0.28
0.46
0.47
-
shorebirds captured in South America. Statistics include sample size (n), mean, standard deviation (SD), minimum (min), and
Table 3-2 Descriptive statistics of ChE activity (units/mL plasma) by site and effect size of habitat use for 7 species of
ChE Type
AChE
BChE
AChE
BChE
AChE
BChE
AChE
BChE
AChE
BChE
AChE
BChE
AChE
BChE
AChE
BChE
AChE
BChE
N mean
2 0.57
2 3.68
38 0.27
38 1.63
41 0.50
41 8.34
7 0.48
7 5.16
42 0.47
42 2.61
35 0.73
35 2.56
11 0.51
11 2.82
21 0.40
21 1.83
3 0.35
3 2.06
Reference
SD
min
0.32 0.34
0.85 3.08
0.20 0.01
0.61 0.60
0.19 0.07
4.54 1.21
0.17 0.24
3.59 3.08
0.16 0.27
0.86 0.81
0.37 0.30
0.68 1.53
0.13 0.32
0.67 1.21
0.19 0.16
0.31 1.43
0.06 0.32
0.85 1.39
** Indicates result is significant using a two-tailed t-test at an α-level of 0.01
Common Snipe
Buff-breasted Sandpiper
Pectoral Sandpiper
White-rumped Sandpiper
Least Sandpiper
Western Sandpiper
Semipalmated Sandpiper
Upland Sandpiper
Species
Lesser Yellowlegs
Comparisons were not conducted if n ≤ 5.
54
max
0.79
4.29
1.06
3.11
0.89
20.4
0.76
13.1
0.99
4.16
2.31
3.99
0.67
3.95
0.89
2.56
0.42
3.02
n
5
5
2
2
21
21
20
20
-
Pesticide Use
mean SD
min
0.29 0.20 0.06
1.73 1.41 0.28
0.43 0.15 0.33
1.57 0.03 1.55
0.47 0.13 0.26
3.02 0.74 1.77
0.35 0.18 0.11
2.31 0.56 1.00
-
North America
max
0.59
1.19
0.54
1.59
0.75
4.43
0.99
3.40
-
t
0.28
0.35
-0.70
0.73
-0.80
3.47
-
P≤
0.78
0.73
0.49
0.47
0.43
0.01**
-
d
+0.10
+0.13
-0.30
+0.27
-0.26
+1.05
-
maximum (max). ChE activity was compared between sites using a two-tailed t-test and a measure of effect size, Hedges' d.
SD(d)
0.48
0.48
0.37
0.37
0.32
0.33
-
shorebirds captured in North America. Statistics include sample size (n), mean, standard deviation (SD), minimum (min), and
Table 3-3 Descriptive statistics of ChE activity (units/mL plasma) by site and effect size of habitat use for 9 species of
Table 3-4 Mean body mass (g) of 13 species of shorebirds by site and region. Body mass
means that share the same letter superscript are not significantly different within a species
using a two-tailed t-test at an α-level ≤ 0.05. Superscripts A and B are comparisons within
regions (within a row) and superscripts Y and Z are comparisons between sites (within a
column). Comparisons were not conducted if n ≤ 5.
Common Name
Mass (g)
North America
Site* n Mean SD min
Nearctic-Neotropical Migrants
American Golden-Plover
Lesser Yellowlegs
Upland Sandpiper
Semipalmated Sandpiper
Least Sandpiper
Western Sandpiper
White-rumped Sandpiper
Pectoral Sandpiper
Stilt Sandpiper
Buff-breasted Sandpiper
Common Snipe
South American Endemics
Collared Plover
South American Painted-Snipe
South America
max Site* n Mean
SD min
max
R
R
R
P
R
R
P
R
R
R
P
R
R
P
R
3
3
62
5
60
62
3
76
36
13
25
12
22
45
3
141.0
100.0
149.0Y
131.0Z
29.4
23.1
19.3
26.9
37.7A
58.7Y
77.8Z
54.0A
63.4A,Y
70.6Z
118.0
10.4
19.5
20.6
9.8
5.4
3.6
0.6
2.9
4.5
13.7
13.0
9.8
7.5
12.7
5.2
134.0
80.0
118.0
124.0
20.5
18.0
19.0
22.0
31.0
44.5
53.0
44.0
46.0
42.0
115.0
153.0
119.0
212.0
148.0
42.0
36.0
20.0
39.0
50.0
90.0
92.0
72.0
79.0
101.0
121.0
R
P
R
R
P
R
P
R
R
P
R
28
18
6
70
16
3
39
6
15
9
3
131.0Y 7.5 103.0
135.0Y 9.2 119.0
91.5
24.4 72.0
B,Y
35.2
3.2 25.5
Z
37.3
3.0 33.0
49.6
2.1 48.0
63.2
11.9 35.5
A
53.5
2.1 51.0
B,Y
55.1
5.8 42.0
Z
61.1
5.4 50.5
121.0
5.9 117.0
155.0
149.0
139.0
45.0
45.0
52.0
86.0
56.0
62.0
65.5
128.0
-
-
-
-
-
-
R
P
R
P
5
11
3
2
32.8Y
32.6Y
64.6
63.0
37.0
37.5
69.0
67.0
*R = Reference site, P = Pesticide use site
55
3.6
3.6
3.8
5.7
29.0
26.0
62.0
59.0
CHAPTER 4 - Conclusions
Migratory shorebird populations face on-going global population declines. Effective
conservation of shorebird populations requires an understanding of factors affecting shorebirds
throughout their annual cycle. Shorebirds cover vast geographic expanses during migration and
at the non-breeding grounds. Likewise, population limiting factors can be encountered at any
stage. My thesis addressed one potential cause for migratory shorebird declines, exposure to
environmental contaminants at the non-breeding grounds. Specifically, I estimated exposure to
organophosphorus (OP) and carbamate (CB) pesticides during migration and at non-breeding
sites and tested for an effect of exposure by comparing body mass between habitats. I addressed
variability of the specific biomarker, cholinesterase (ChE), used to determine exposure to these
chemicals. I provide the first comprehensive data available on exposure of migratory shorebirds
to ChE-inhibitors and provide important information regarding conservation of these species.
Variability in plasma ChEs is important when estimating exposure to OPs and CBs across
different spatial and temporal scales. I found that shorebird plasma ChEs decreased as body size
increased. Thus, comparisons of enzyme activity must be conducted separately for each species.
Intrinsic factors such as sex and body condition and extrinsic factors such as time of capture and
date of capture can also affect ChE activity. In North America, plasma butyrylcholinesterase
(BChE) increased with date of capture in two species, Upland Sandpipers and Least Sandpipers
and plasma acetylcholinesterase (AChE) increased with capture date in one species,
White-rumped Sandpipers. Sex differences were found in only one species, the Upland
Sandpiper; females had higher BChE than males. Time of capture and body condition did not
affect ChE activity in any species tested in North America. Regionally, White-rumped
56
Sandpiper plasma AChE was higher in North America. My results indicate the importance of
evaluating environmental and biological influences on plasma ChEs in field toxicology studies.
I assessed exposure of migratory shorebirds to ChE inhibitors in five species by
comparing ChE activity of samples collected at reference sites to samples collected from
agricultural areas during the same temporal and spatial framework. I found evidence for
exposure to ChE-inhibitors in South America in one species, the Buff-breasted Sandpiper. In
contrast, plasma ChEs were higher at use sites in North America for the same species. There
were no differences in plasma ChE activity between habitats in any other species tested. Using a
meta-analysis, I determined that agricultural habitats had a negative effect on AChE activity in 4
of 6 comparisons, an indication that sub-lethal exposure may be occurring in other migratory
shorebird species. However, the cumulative effect sizes were non-significant. My results
suggest that some species of migratory shorebirds sampled in this study were exposed to
ChE-inhibiting pesticides at the non-breeding grounds.
The conversion of natural habitats to agricultural land uses will continue to pose a
conservation threat to migratory shorebirds. While shorebirds can exploit available resources in
agricultural habitats, exposure to agrochemicals is a threat to the conservation of at least one
species of migratory shorebird. I recommend continued monitoring of Buff-breasted Sandpipers
and other shorebird species in more diverse agricultural habitats and continued cooperation with
international partners. Future research should include interviews with farmers to increase
knowledge of local pesticide use. Initiation of education campaigns to educate farmers about the
toxicity of OPs and CBs and to devise alternative pest management regimes is needed.
Monitoring should also be expanded to other agricultural regions of the Americas that are
57
important to non-breeding shorebirds. These measures will ensure thorough attention to this
conservation issue.
Although I made preliminary estimates of exposure to OPs and CBs in non-breeding
shorebirds, I did not evaluate behavior of exposed individuals. Thus, I could not make
inferences about the potential behavioral effects of sub-lethal exposure. Exposure-induced
behavioral changes can have important consequences on individual condition and survival.
Additionally, my study addressed exposure of migratory shorebirds to one type of environmental
contaminant, ChE-inhibiting pesticides. Other studies have focused on heavy metals and DDT
metabolites at the non-breeding grounds (e.g., see Chapter 3) but effects of these contaminants
have received little attention at the breeding grounds. Contaminants may have independent or
synergistic negative effects on individuals that could decrease fecundity. Research on the
breeding grounds should incorporate exposure to contaminants as a variable in reproductive and
behavioral studies.
Overall, my data provides insight into one anthropogenic source of shorebird population
declines. Habitat loss and degradation and illegal hunting may also contribute to population
declines. Shorebirds exploit a variety of habitats during migration and at the non-breeding
grounds, however the amount of time spent in and the relative importance of each habitat is
unknown. Using tools such as stable isotopes and radio telemetry we can infer foraging vs.
roosting habitat and estimate exposure risk to ChE inhibitors and other anthropogenic sources of
declines. This will help determine the significance of these risk factors to population declines of
migratory shorebirds.
58
Appendix A - Laboratory controls and inter-lab variation
Horse serum was used as a laboratory standard to compare within and between lab
variation at Kansas State University (KSU) and the Texas Institute for Environmental and
Human Health (TIEHH). There was no difference in mean ± SD standard serum activity
between labs for either AChE (KSU: 19.3 ± 5.9 SD, n = 53; TIEHH: 18.6 ± 7.0 SD, n = 46;
t97 = 0.47, P = 0.64) or BChE (KSU: 221.7 ± 22.2 SD, n = 53; TIEHH: 222.6 ± 10.4 SD, n = 46;
t76 = -0.25, P = 0.80, [unequal variance]) when temperature, substrate, and inhibitor
concentrations were identical 1.0 x 10-4 M). This comparison of standards validates the use of
ChE activity from different labs in combined statistical analyses. Overall, within lab variation
was higher for BChE activity measured at KSU (10.0% CV) than TIEHH (4.7% CV).
59
Appendix B - Characterization of shorebird plasma cholinesterases
for optimal inhibitor and substrate concentrations
As a first step, plasma ChEs were characterized for optimal inhibitor (iso-OMPA) and
substrate (AThCh) concentrations. Iso-OMPA is a BChE-specific inhibitor and is used to
separate AChE and BChE activity. BChE activity is calculated as the difference between total
cholinesterase (TChE) measured without iso-OMPA and AChE activity in the presence of
iso-OMPA. Ideal concentrations of iso-OMPA are sufficient to inhibit all BChE without
inhibiting AChE activity. The optimal concentration of iso-OMPA is found within the plateau
region of a graph depicting %TChE Activity vs. log10[iso-OMPA]. In this study the optimal
concentration of iso-OMPA was similar for all species, 1.0 x 10-4 M (Fig. B-1, arrow).
Similarly, Substrate concentration must be chosen to maximize enzyme activity without
inhibiting sample enzyme. Optimal substrate concentrations are found at the peak of the bellshaped curve of AChE activity vs. log10[AThCh]. Optimal substrate concentration was similar
for all species, 1.0 x 10-4 M (Fig. B-2, arrow).
Species abbreviations in Figures B-1 and B-2 are as follows: AMGP = American
Golden-Plover, UPSA = Upland Sandpiper, SESA = Semipalmated Sandpiper, LESA = Least
Sandpiper, WRSA = White-rumped Sandpiper, PESA = Pectoral Sandpiper, BBSA =
Buff-breasted Sandpiper.
60
Figure B-1 Iso-OMPA titration curves of 7 species of migratory shorebirds.
90
AMGP
UPSA
SESA
LESA
WRSA
PESA
BBSA
80
% TChE ± SD
(units/mL plasma)
70
60
50
40
30
20
10
0
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
log10 [iso-OMPA]
61
-4.0
-3.5
-3.0
-2.5
Figure B-2 Substrate (AThCh) affinity curve of 7 species of migratory shorebirds.
20
18
AChE Activity ± SD
(units/mL plasma)
16
14
12
AMGP
UPSA
SESA
LESA
WRSA
PESA
BBSA
10
8
6
4
2
0
-6.5
-6.0
-5.5
-5.0
-4.5
-4.0
log10 [AThCh]
62
-3.5
-3.0
-2.5
*R = Reference site, P = Pesticide use site
South American Endemics
Southern Lapwing
Vanellus chilensis
Collared Plover
Charadrius collaris
P
R
P
Common Name
Scientific Name
Site*
Nearctic-Neotropical Migrants
Snowy Plover
R
Charadrius alexandrinus
Spotted Sandpiper
R
Actitis macularis
Solitary Sandpiper
R
Tringa solitaria
Baird's Sandpiper
R
Calidris bairdii
Dunlin
R
Calidris alpine
Long-billed Dowitcher Limnodromus scolopaceous
R
Wilson's Phalarope
R
Phalaropus tricolor
sites with insufficient data for statistical analyses.
6
1
5
63
0.229 0.066 0.161 0.317
0.326
0.278 0.083 0.155 0.383
0.391
0.710 0.116 0.547 0.806
0.811
0.439
0.695 0.431 0.390 1.000
1.235
0.725 0.173 0.603 0.848
1
4
1
1
2
1
2
max
mean
n
AChE
SD min
max
1.158 0.561 0.647 2.096
1.183
1.158 0.513 0.647 2.096
1.895
1.494 0.532 2.264 4.000
1.183
3.184
2.631 0.019 2.618 2.645
1.258
6.200 1.776 4.944 7.456
mean
BChE
SD min
Table C-1 Plasma ChE activity (units/mL plasma) of 9 shorebird species sampled at migratory stopover and non-breeding
Chapter 3.
endemics were sampled only at pesticide use sites or had only one sample collected from reference sites and were not included in
Nearctic-Neotropical migratory shorebird species were not included in Chapter 2 because n ≤ 5. Two species of South American
Nine species of shorebirds lacked sufficient data for inclusion in Chapters 2 and 3. Reference values of ChE activity of 7
Appendix C - Plasma cholinesterase activity of additional species
Appendix D - Analyses of variation in plasma cholinesterases of
migratory shorebirds using additional samples
Sex differences and diurnal variation in plasma ChE activity of shorebirds sampled
during north-bound migration in 2006 were reported in Chapter 2. The following analyses were
conducted according to statistical methods described in Chapter 2 but contain additional data
from samples collected during south-bound migration in North America and at non-breeding
sites in South America in 2006.
Plasma BChE was 22.4% higher in female Upland Sandpipers than males (Table D-1).
No other differences between sexes were detected in AChE or BChE. These results are
consistent with previous analyses (see Chapter 2).
There was significant variation in plasma BChE activity in 1 of 7 species tested
(Table D-2). Semipalmated Sandpiper BChE activity (units/mL plasma) was higher in
individuals captured between 06:00 – 11:59h (11.6 ± 4.5 SD, n = 14) than individuals captured
between 12:00 – 17:59h (4.4 ± 1.9 SD, n = 4) or 18:00 – 23:59h (6.9 ± 3.7 SD, n = 22). No other
species showed variation in plasma ChE activity with time of capture.
64
Table D-1 Differences in plasma ChE activity by sex in 2 species of shorebirds sampled at
migratory stopover sites in North America. Results were considered significant at a α-level
of 0.05 using a two-tailed t-test.
Species
Upland Sandpiper†
ChE Type Region
AChE
NA
BChE
‡
Semipalmated Sandpiper AChE
NA
BChE
†
n
16
16
29
29
Female
Mean
0.26
1.87
0.49
8.61
Sexed using molecular markers; ‡Sexed using Prater et al. 1977
*Indicates a significant result at an α-level ≤ 0.05
65
SD
0.19
0.64
0.22
4.85
n
22
22
9
9
Male
Mean
0.27
1.45
0.53
7.02
SD
0.21
0.52
0.21
4.05
t
-0.1
2.18
-0.6
0.89
df
36
36
36
36
P≤
0.925
0.037*
0.580
0.381
Table D-2 Diurnal variation in plasma ChEs of 7 species of shorebirds sampled at reference
sites during migration in North America and at non-breeding sites in South America.
Results were considered significant at an α-level of 0.05 if P < 0.006 after sequential
Bonferroni correction for the number of tests.
Species
American-Golden Plover
Region ChE Type
SA
log(AChE)
log(BChE)
Time of Capture
df
F
P≤
1,25
1.2 0.284
1,25
1.6 0.212
Upland Sandpiper
NA
log(AChE)
log(BChE)
1,36
1,36
4.8 0.035
0.1 0.716
Buff-breasted Sandpiper
NA
log(AChE)
log(BChE)
1,19
1,19
0.0 0.858
0.8 0.373
SA
log(AChE)
log(BChE)
1,12
1,12
2.5 0.139
4.6 0.053
NA
log(AChE)
log(BChE)
1,32
1,32
0.1 0.750
0.2 0.653
SA
log(AChE)
log(BChE)
1,62
1,62
2.8 0.102
0.5 0.485
Pectoral Sandpiper
NA
log(AChE)
log(BChE)
1,9
1,9
0.6 0.466
0.0 0.864
Least Sandpiper
NA
log(AChE)
log(BChE)
1,37
1,37
0.1 0.800
0.2 0.697
Semipalmated Sandpiper
NA
log(AChE)
log(BChE)
1,38
1,38
0.2 0.701
8.5 0.006*
White-rumped Sandpiper
*Indicates a significant result at an α-level ≤ 0.05
66
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