Plasma cholinesterases for monitoring pesticide exposure in Nearctic–Neotropical migratory shorebirds

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 ChE values are a necessary component of monitoring exposure. During northbound migration in 2006, we sampled four upland and five wetland shore-bird 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. Acetylcholinesterase (AChE), the enzyme whose inhibition leads to poisoning symptoms, showed less inter-specific variation than butyrylcholinesterase (BChE). Plasma ChE activities varied with date of capture in three of five species. Sex differences were significant in one of two species tested. Our baseline ChE values for migratory shorebirds provide a framework for future ecotoxicological studies of Nearctic-Neotropical migrant shorebirds.

Full text

641
ORNITOLOGIA NEOTROPICAL 19 (Suppl.): 641–651, 2008
© The Neotropical Ornithological Society
PLASMA CHOLINESTERASES FOR MONITORING PESTICIDE
EXPOSURE IN NEARCTIC-NEOTROPICAL MIGRATORY
SHOREBIRDS
Khara M. Strum
1
, Matilde Alfaro
2
, Ben Haase
3
, Michael J. Hooper
4
, Kevin A. Johnson
5
,
Richard B. Lanctot
6
,
Arne J. Lesterhuis
7
, Leticia López
7
, Angela C. Matz
8
, Cristina
Morales
7
, Benjamin Paulson
5
, Brett K. Sandercock
1
, Julian Torres-Dowdall
9
, & María
Elena Zaccagnini
10
1
Division of Biology, Kansas State University, 116 Ackert Hall, Manhattan, Kansas 66506,
USA. E-mail: kmstrum@ksu.edu
2
Sección Oceanografía, Facultad de Ciencias, Universidad de la República, Iguá 4225,
Montevideo, Uruguay.
3
Fundación Ecuatoriana para el Estudio de Mamíferos Marinos (FEMM) P.O. Box 09-01-
11905, Guayaquil, Ecuador.
4
The Institute of Environmental and Human Health, Texas Tech University, 1207 Gilbert
Avenue, Lubbock, Texas 79416, USA.
5
Department of Chemistry, Southern Illinois University at Edwardsville, Edwardsville, Illinois
62026, USA.
6
USFWS, Migratory Bird Management, 1011 East Tudor Road, Anchorage, Alaska 99503,
USA.
7
Guyra Paraguay, Cnel. Franco 381, Asunción, Paraguay.
8
USFWS, Environmental Contaminants Program, 101-12th
Avenue, Fairbanks, Alaska
99701, USA.
9
Department of Biology, Colorado State University, Fort Collins, Colorado 80523, USA.
10
Instituto de Recursos Biológicos, INTA-CIRN, Los Reseros y Las Cabañas, s/n, 1712
Castelar, Buenos Aires, Argentina.
Resumen. – Colinesterasas en el plasma para monitorear la exposición a pesticidas en aves playe-
ras migratorias. – Los plaguicidas organofosfatados (OP) y carbamatados (CB) son productos agroquí-
micos de uso común en el hemisferio occidental. Estos plaguicidas han causado mortalidad en aves
migratorias y producido efectos fisiológicos adversos en pruebas realizadas con especies de aves cautivas.
Los chorlos y playeros migratorios utilizan una variedad de hábitats cuando pasan el invierno en la zona
templada de Sudamérica y durante su migración a través de las Grandes Llanuras de los Estados Unidos.
Los hábitats con alto riesgo de exposición incluyen arrozales y campos de cultivo de césped en los que se
utilizan productos agroquímicos. La colinesterasa (ChE) es un indicador biológico específico para monito-
rear la exposición a OP y CB y se puede medir usando simples procedimientos de laboratorio. La actividad
de ChE en el plasma es útil como método no letal de monitorear la exposición aviar a los plaguicidas OP y
CB. Muchas variables pueden afectar la actividad enzimática y no siempre es posible realizar ensayos de
reactivación; por lo tanto, los valores de referencia de ChE son un componente necesario del monitoreo de

642
STRUM ET AL.
la exposición. Durante la migración hacia el norte del 2006, tomamos muestras de cuatro especies de aves
playeras de altiplanicie y cinco especies de aves playeras de humedales en tres estados de Norteamérica,
caracterizando y midiendo los niveles de ChE en el plasma en todas las especies. Las especies de cuerpo
pequeño tienen niveles más altos de actividad de ChE en el plasma que las especies de cuerpo grande. La
acetilcolinesterasa (AChE), la enzima cuya inhibición lleva a los efectos de envenenamiento, muestra
menos variación entre las especies que la butirilocolinesterasa (BChE). La actividad de ChE en el plasma
mostró variación según la fecha de captura en tres de cinco especies. Diferencias por sexo fueron significa-
tivas en una de las dos especies testeadas. Nuestra investigación presenta valores referenciales de colineste-
rasa para aves playeras migratorias y provee el marco para futuros estudios ecotoxicológicos en especies de
aves playeras Neotropicales y Neárticas.
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 dur-
ing 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 agro-
chemicals 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 ChE values are a necessary component of moni-
toring exposure. During northbound migration in 2006, we sampled four upland and five wetland shore-
bird 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 spe-
cies. Acetylcholinesterase (AChE), the enzyme whose inhibition leads to poisoning symptoms, showed less
inter-specific variation than butyrylcholinesterase (BChE). Plasma ChE activities varied with date of cap-
ture in three of five species. Sex differences were significant in one of two species tested. Our baseline
ChE values for migratory shorebirds provide a framework for future ecotoxicological studies of Nearctic-
Neotropical migrant shorebirds. Accepted 20 December 2007.
Key words: Carbamate, cholinesterase, ecotoxicology, organophosphate, waders.
INTRODUCTION
Organophosphates (OPs) and carbamates
(CBs) averaged 68% of insecticide active
ingredients 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 haz-
ards (e.g., Henny & Bennett 1990). OPs and
CBs provide an alternative to the environ-
mental persistence and bioaccumulation of
organo-chlorines (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 & Temple 1995,
Goldstein et al. 1999a). Mass mortality inci-
dents have resulted in public awareness cam-
paigns 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 expo-
sures have been reported recently (Pain et al.
2004, Wobeser et al. 2004). 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 culti-

643
SHOREBIRD PLASMA CHOLINESTERASES
vation industry in Uruguay and Argentina
(Garamma et al. fide Blanco et al. 2006, MEZ
pers. comm.). 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 &
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 into human-altered habitats. Rice fields
and turf grass farms provide important alter-
native wintering and migratory stopover habi-
tats for shorebirds (Twedt et al. 1998, Corder
2005, Blanco et al. 2006, 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 effec-
tively 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 impor-
tant enzyme in the central and peripheral ner-
vous systems, is responsible for the hydrolysis
of the neurotransmitter acetylcholine (ACh),
at the nerve–nerve or nerve–effector inter-
face. Without hydrolysis, ACh accumulates in
the synapse, disrupting neurotransmission,
impairing behavior and physiology, and even-
tually leading to death (Grue et al. 1997, Gold-
stein et al. 1999a). Plasma ChE activity can
demonstrate exposure levels consistent with
intoxication and death in subsets of a popula-
tion (Hooper et al. 1989, Goldstein et al.
1999a), as well as a lack of exposure (Gold-
stein 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 poisonings. Reactivation assays also
address potential concerns associated with
inter-species or inter-individual ChE variation
(Grue 1982, Hill 1989, Fossi et al. 1996). How-
ever, reference values of ChE activity are
especially important if reactivation assays can-
not be used because sample volumes are too
small or because OP aging results in chemi-
cally stable OP–enzyme bonds (Wilson et al.
1992).
Here, we present reference values of
plasma ChE activity for apparently healthy,
free-living individuals of nine shorebird spe-
cies 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 (Par-
aguay, Argentina, and Uruguay) in South
America from April through December 2006.
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. Data from
individuals captured in South America were
not included in plasma ChE analyses but con-
tributed to mean mass calculations. North-
bound migration capture sites included

644
STRUM ET AL.
Anahuac National Wildlife Refuge, Chambers
County, TX (29
o
34’N, 94
o
32’W), Quivira
National Wildlife Refuge, Stafford County,
KS (38
o
08’N, 98
o
29’W), Konza Prairie
Biological Station, Riley County, KS (39
o
04’N, 96
o
33’W), and Kissinger Wildlife
Management Area, Clay County, NE
(40
o
26’N, 98
o
06’W). In 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 mea-
sured with a wing rule (± 0.5 mm). Total
head, culmen and tarsus length were mea-
sured using vernier calipers (± 0.1 mm). All
birds were fitted with a USFWS metal band
with a unique number. When possible, shore-
birds were sexed in the field according to
Prater et al. (1977). Upland Sandpipers (Bartra-
mia longicauda) were sexed using molecular
markers based on the CHD gene (Baker et al.
1999, A. E. Casey unpubl.).
Blood was collected using a 27-gauge nee-
dle 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 –20ºC for less than
one month and transferred to –80ºC until lab-
oratory analysis could be conducted. All sam-
ples were assayed within one year of
collection.
Laboratory analysis. Samples were thawed
immediately before ChE activity determina-
tion. As a first step, six plasma samples from
each species were pooled for characterization
of optimal enzyme dilution and reagent
(acetylthiocholine-iodide [AThCh] and tetra-
isopropyl pyrophosphoramide [iso-OMPA])
concentrations. ChE activity was determined
using the method of Ellman et al. (1961) as
modified by Gard & Hooper (1993) for use in
a 96-well spectrophotometric plate reader
(Molecular Devices, Palo Alto, CA) with
Softmax Pro software (Molecular Devices,
Palo Alto, CA). Final volume of each assay
was 250 µL and contained the following com-
ponents: 0.05 M final concentration (FC) of
Trizma buffer (pH 8.0), 3.23x10
–4
M FC of
5,5-dithio[bis-2-nitrobenzoic acid] (DTNB),
diluted enzyme sample, and 1.00x10
–3
M FC
of AThCh. To separate butyrylcholinesterase
(BChE) from AChE, samples were incubated
with the BChE-specific inhibitor iso-OMPA
at FCs between 1.0x10
–4
M and 1.0x10
–5
M
according to the characterization of each spe-
cies. 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
o
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 absor-
bance units per min to µmoles AThCh hydro-
lyzed per min (units) per milliliter 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 activ-

645
SHOREBIRD PLASMA CHOLINESTERASES
ities fell within ± 3 SD of the mean except for
two TChE and BChE values for the Least
Sandpiper (Calidris minutilla) which were over
4.5 times the mean for this species. These two
outliers were removed from subsequent analy-
sis. All data presented are in raw form but sta-
tistical results are based on log
10
-transformed
data to correct for allometric scaling. General
FIG. 1. Log
10
-log
10
plot showing the relationship between mean body mass and plasma ChE activity in
nine species of shorebirds captured during migration in the Great Plains 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 log
10
(ChE) values where log
10
(body mass) = 1.81.

646
STRUM ET AL.
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 6-h blocks each accord-
ing to the following criteria: 1 = 0–05:59 h, 2
= 06:00–11:59 h, 3 = 12:00–17:59 h, and 4 =
18:00–23:59 h. 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 condi-
tion. PCA analyses were based on four mor-
phological measurements, total head, culmen,
wing, and tarsus, and were calculated sepa-
rately 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 posi-
tive in seven of nine species; in the remaining
two species one eigenvector was negative (K.
M. Strum unpubl.). 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 analy-
sis from 138 individuals of nine species. All
samples were used in analysis of AChE activ-
ity and after removing two outliers from
Least Sandpiper BChE and TChE activity,
136 samples were used. We calculated average
body mass for these nine species from cap-
tures of 511 individuals at migratory and non-
breeding sites throughout the Western Hemi-
sphere. Our study species included: Amer-
ican Golden-Plover (Pluvialis dominica), Kill-
deer (Charadrius vociferus), Upland Sandpiper,
Buff-breasted Sandpiper (Tryngites subruficollis),
Pectoral Sandpiper (Calidris melanotos), White-
TABLE 1: Descriptive statistics of ChE activity (µmol AThCh hydrolysed/min per mL plasma) for nine
shorebird species sampled during northbound migration in the Great Plains of the United States including
sample size of individuals (n)
†
, mean, standard deviation (SD), minimum (min) and maximum (max)
values.
Species n TChE AChE BChE
Mean SD Min Max Mean SD Min Max Mean SD Min Max
American Golden-Plover
Killdeer
Upland Sandpiper
Buff-breasted Sandpiper
Pectoral Sandpiper
White-rumped Sandpiper
Stilt Sandpiper
Least Sandpiper
Semipalmated Sandpiper
2
5
25
21
7
34
5
20
19
1.20
1.56
1.81
2.22
3.22
3.29
3.82
3.25
6.38
0.01
0.55
0.70
0.43
0.80
0.80
0.67
0.94
2.95
1.20
0.91
0.69
1.71
1.53
1.95
2.89
1.20
1.28
1.21
2.42
3.29
3.22
4.01
5.05
4.72
4.67
10.78
0.37
0.41
0.24
0.40
0.48
0.72
0.52
0.45
0.46
0.00
0.16
0.14
0.19
0.12
0.38
0.16
0.13
0.22
0.37
0.21
0.02
0.16
0.32
0.30
0.37
0.27
0.07
0.37
0.59
0.65
0.89
0.66
2.31
0.69
0.81
0.89
0.83
1.15
1.57
1.83
2.74
2.56
3.31
2.80
5.92
0.01
0.45
0.67
0.31
0.72
0.69
0.60
0.89
2.85
0.83
0.69
0.51
1.43
1.21
1.53
2.45
0.81
1.21
0.84
1.89
3.11
2.56
3.35
3.99
4.03
4.16
10.11
†
Total sample size. Estimates of TChE and BChE exclude two outliers from Least Sandpipers.

647
SHOREBIRD PLASMA CHOLINESTERASES
rumped Sandpiper (Calidris fuscicollis), Stilt
Sandpiper (Calidris himantopus), Least Sand-
piper, and Semipalmated Sandpiper (Calidris
pusilla).
TChE and BChE were highly correlated
(r
2
= 0.984, P < 0.001, n = 136). TChE and
AChE were also significantly correlated (r
2
=
0.533, P < 0.001, n = 136) though less varia-
tion in TChE could be explained by AChE.
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 sub-
strate, substrate concentration, and assay tem-
perature are identical. Plasma BChE activity
varied negatively with body size (F
8,127
= 20.3,
P < 0.001) as did AChE (F
8,129
= 11.0, P <
0.001, Fig. 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
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 eval-
uated 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, t
23
= 2.60, P = 0.016). However, mean AChE
was not significantly different between male
(0.20 ± 0.07 SD, n = 13) and female Upland
Sandpipers (0.28 ± 0.19 SD, n = 12, t
14.9
=
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 Semipal-
mated Sandpipers (AChE: 0.41 ± 0.19 SD, n
= 12; BChE: 6.04 ± 2.92 SD, n = 12, AChE:
t
17
= –1.02, P = 0.324, BChE: t
17
= –0.06, P =
0.956).
In four species, the relationship between
plasma ChEs and date of capture, time of cap-
ture and body condition were analyzed. 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 (r
2
= 0.276, F
1,23
= 8.8, P = 0.007) and Least Sandpipers (r
2
=
TABLE 2: Trends in plasma ChE’s of five shorebird species as a function of date of capture, time of cap-
ture and an index of body condition using log
10
transformed ChE activity. After sequential Bonferroni cor-
rection for number of tests, test statistics were considered significant at an α−level of 0.05 if P < 0.002.
Species ChE type Date of capture Time of capture Index of body
condition
df FP ≤ df FP ≤ df FP ≤
Upland Sandpiper
Buff-breasted Sandpiper
White-rumped Sandpiper
Least Sandpiper
Semipalmated Sandpiper
log(AChE)
log(BChE)
log(AChE)
log(BChE)
log(AChE)
log(BChE)
log(AChE)
log(BChE)
log(AChE)
log(BChE)
1,23
1,23
1,19
1,19
1,32
1,32
1,18
1,16
1,17
1,17
0.0
8.8
0.0
2.2
4.2
2.5
0.1
6.8
0.1
3.1
0.956
0.007
0.891
0.152
0.048
0.123
0.817
0.019
0.736
0.097
1,23
1,23
1,19
1,19
1,32
1,32
1,16
1,14
1,17
1,17
0.9
0.0
0.3
0.8
0.1
0.2
0.6
0.0
0.3
0.1
0.359
0.836
0.858
0.373
0.750
0.653
0.452
0.886
0.615
0.715
1,23
1,23
1,19
1,19
1,32
1,32
1,18
1,16
1,16
1,16
1.8
0.2
0.2
0.4
0.0
0.3
0.5
0.0
0.0
0.7
0.193
0.640
0.692
0.550
0.876
0.599
0.498
0.977
0.926
0.419

648
STRUM ET AL.
0.298, F
1,16
= 6.79, P = 0.019), whereas levels
of AChE increased throughout the capture
period in White-rumped Sandpipers (r
2
=
0.117, F
1,32
= 4.24, P = 0.048). Trends were
marginally significant in all three species after
Bonferroni corrections for the number of
tests (Rice 1989). Other components of
plasma ChE did not vary with capture period
in any of these species (Table 2). There was
no significant relationship between time of
capture or body condition for any species
tested (Table 2).
DISCUSSION
Interspecific variation in plasma BChE activ-
ity decreased with increasing shorebird mass
similar to results from a study of plasma
ChEs in European raptors (Roy et al. 2005).
Mass-specific metabolic demands decrease as
shorebird body size increases (Kvist & Lind-
ström 2001), which may be a partial explana-
tion 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 chem-
icals (Leopold 1996, Parker & Goldstein
2000). Birds lack A-esterases which hydrolyze
OP and CB pesticides (Aldridge 1953) and
higher levels of BChE activity may provide
some protection against poisoning and infor-
mation about exposure.
Inclusion of all ChE activity results is
important when presenting baseline ChE val-
ues, 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 lipid metabolism during migration
(Rattner & Fairbrother 1991, Valle et al. 2006).
Due to interspecific variation in ChE
activity with regard to body size, our data can
be used to estimate normal plasma ChEs of
species without reference values for field
sample comparison. While there is no substi-
tute for species-specific reference values, pat-
terns 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, female Upland Sandpip-
ers had higher mean BChE activity than
males. Our results may be related to breeding
condition because Upland Sandpipers evalu-
ated in this study had recently arrived on the
breeding grounds and many females were
at an egg-laying stage (B. K. Sandercock
unpubl.). An increase in plasma ChEs during
egg-laying has been reported in other avian
species (Rattner & Fairbrother 1991). Sam-
ples of Upland Sandpipers during the non-
breeding season as well as samples from
males and females of other shorebird species
on the breeding grounds are needed to fur-
ther investigate this idea.
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
inter-individual variation in body condition
depending on the time since arrival at a stop-
over site and the distance traveled prior to
capture. Individuals in better condition pre-
sumably have more fat and muscle translating
into larger relative mass (Schulte-Hostedde et
al. 2005), unrelated to ChE activities. How-
ever, birds that died from anti-ChE exposure
had lower fat and muscle scores due to
reduced food intake after poisoning (Grue

649
SHOREBIRD PLASMA CHOLINESTERASES
1982). In our study, body condition was not
used as an indicator of chemical exposure but
it might be in another study.
Increases in plasma ChEs were marginally
significant in three species throughout the
capture period. In each case, the variation
explained in plasma BChE was fairly low (r
2
<
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, varia-
tion in plasma ChEs during the capture sea-
son could be due to changes in diet or to
changes in physiological condition caused by
changes in organ size during migration
(Piersma & 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 pat-
terns 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 shore-
birds. 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 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 regula-
tions for OP and CB pesticides through part-
nerships with local and international
governments. If future studies demonstrate
that shorebird exposure to ChE-inhibitors is
limited, this information will be used to redi-
rect research efforts into other possible causes
of shorebird population declines.
ACKNOWLEDGMENTS
For access to field sites and logistical support,
we thank Matt Whitbeck and the staff of
Anahuac National Wildlife Refuge; Dave Hil-
ley and Jim Sellers and the staff of Quivira
National Wildlife Refuge; Joel Jorgensen from
Nebraska Game and Parks Commission and
John McCarty and LaReesa Wolfenbarger at
the University of Nebraska at Omaha. Ashley
Casey, Tara Conkling, Kyle Gerstner, Kate
Goodenough, Karl Kosciuch and USDA
Wildlife Services Office of Manhattan, Kan-
sas provided field assistance in North Amer-
ica. Laura Addy, Natalia Bossel, Noelia
Calamari, Julieta Decarre, Andrea Goijman,
Benito Jaubert, Leandro Macchi, Laura Solari,
and Romina Suarez aided with field sampling
in South America. Chuck Otte from Kansas
State University Extension Office provided
assistance with pesticide use information.
Stephanie Jones and Suzanne Fellows of the
U.S. Fish and Wildlife Service provided logis-
tical support for the project. Funding for field
work and lab analyses was provided by grants
from the Neotropical Migratory Bird Conser-
vation Act (NMBCA) and Migratory Bird
Management (MBM) programs of the U.S.
Fish and Wildlife Service.
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