AECT_acid mine water_pdf

Behavioral, Clinical, and Pathological Characterization
of Acid Metalliferous Water Toxicity in Mallards
John P. Isanhart • Hongmei Wu • Karamjeet Pandher •
Russell K. MacRae • Stephen B. Cox •
Michael J. Hooper
Received: 12 October 2010 / Accepted: 17 February 2011 / Published online: 19 March 2011
Ó Springer Science+Business Media, LLC (outside the USA) 2011
Abstract From September to November 2000, United
States Fish and Wildlife Service biologists investigated
incidents involving 221 bird deaths at 3 mine sites located
in New Mexico and Arizona. These bird deaths primar-
ily involved passerine and waterfowl species and were
assumed to be linked to consumption of acid metalliferous
water (AMW). Because all of the carcasses were found in
or near pregnant leach solution ponds, tailings ponds, and
associated lakes or storm water retention basins, an acute-
toxicity study was undertaken using a synthetic AMW
(SAMW) formulation based on the contaminant profile of a
representative pond believed to be responsible for avian
mortalities. An acute oral-toxicity trial was performed with
a mixed-sex group of mallards (Anas platyrhynchos). After
a 24-h pretreatment food and water fast, gorge drinking
was evident in both SAMW treatment and control groups,
with water consumption rates greatest during the initial
drinking periods. Seven of nine treated mallards were kil-
led in extremis within 12 h after the initiation of dose.
Total lethal doses of SAMW ranged from 69.8 to
270.1 mL/kg (mean ± SE 127.9 ± 27.1). Lethal doses of
SAMW were consumed in as few as 20 to 40 min after first
exposure. Clinical signs of SAMW toxicity included
increased serum uric acid, aspartate aminotransferase,
creatine kinase, potassium, and P levels. PCV values of
SAMW-treated birds were also increased compared with
control mallards. Histopathological lesions were observed
in the esophagus, proventriculus, ventriculus, and duode-
num of SAMW-treated mallards, with the most distinctive
being erosion and ulceration of the kaolin of the ven-
triculus, ventricular hemorrhage and/or congestion, and
duodenal hemorrhage. Clinical, pathological, and tissue-
residue results from this study are consistent with literature
documenting acute metal toxicosis, especially copper (Cu),
in avian species and provide useful diagnostic profiles for
AMW toxicity or mortality events. Blood and kidney Cu
concentrations were 23- and 6-fold greater, respectively, in
SAMW mortalities compared with controls, whereas Cu
concentrations in liver were not nearly as increased, sug-
gesting that blood and kidney concentrations may be more
useful than liver concentrations for diagnosing Cu toxicosis
in wild birds. Based on these findings and other reports of
AMW toxicity events in wild birds, we conclude that
AMW bodies pose a significant hazard to wildlife that
come in contact with them.
Availability of clean water sources is critical to the daily
survival of most wild bird species. Migratory species are
dependent on sufficiently regular water sources as they
J. P. Isanhart (&)
U.S. Fish and Wildlife Service, Salt Lake City,
UT 84119, USA
e-mail: john_isanhart@fws.gov
H. Wu
School of Public Health, Wenzhou Medical College,
Wenzhou 325035, People’s Republic of China
K. Pandher
Pfizer, Inc, Groton, CT 06340, USA
R. K. MacRae
U.S. Fish and Wildlife Service, Spokane Valley,
WA 99206, USA
S. B. Cox
The Institute of Environmental and Human Health,
Texas Tech University, Lubbock, TX 79409, USA
M. J. Hooper
U.S. Geological Survey, Columbia, MO 65201, USA
123
Arch Environ Contam Toxicol (2011) 61:653–667
DOI 10.1007/s00244-011-9657-z

migrate to wintering or breeding grounds. Water avail-
ability in the western United States is particularly important
to birds because its scarcity makes it a critical com-
modity. The occurrence of contaminated water sources in
arid or semi-arid areas poses an important threat to local
and migratory birds because their need for water can
often preclude their ability to choose between a variety
of sources (Read 1999). There is a history of mine
water–associated toxicant effects in birds inhabiting the
western United States dating back to waterfowl poison-
ings in lead mine–contaminated rivers and wetlands in
the 1920s (Phillips and Lincoln 1930; Chupp and Dalke
1964).
Acid metalliferous water (AMW) results from (1) the
oxidation and leaching of metals and acid from disturbed
mining sites and tailings piles or (2) the intentional acid
leaching of metals from processed rock to form pregnant
leach solutions from which metals are removed using
electrorefining techniques. Incident data suggest that
waterfowl and passerines are the species most frequently
killed from drinking AMW (Stratus Consulting Inc. 2003;
Stubblefield et al. 1997). There are relatively few data
available on this topic primarily addressing the avian tox-
icity of cyanide-rich water from mining sites (Henny et al.
1994) and acidified water bodies (Foster 1999; Read and
Pickering 1999; Read 1999). Poisoning of birds that con-
sume toxic tailings waters was of particular concern in arid
Australia, with approximately 1000 birds dying annually in
gold mine tailings dams (Minerals Council of Australia
1996; Read 1999). Examples of non-cyanide-associated
bird poisonings in the United States include the deaths of
342 snow geese from AMW at the Berkeley Pit, Butte, MT
(Haglar Bailly Consulting 1996; Stubblefield et al. 1997)
and Canada geese at a petroleum refinery fly ash pond in
Delaware (Rattner et al. 2006). Such anthropogenic land-
scape modifications pose compounding problems for
nomadic or migratory species that are in search of food,
water, and/or resting sites.
Birds require water for the maintenance of cellular
homeostasis, tissue integrity, food digestion, waste excre-
tion, hygiene, and a wide variety of biochemical reactions
(Koutsos et al. 2001). Although some birds are able to
obtain all of the water they require through a combination
of succulent food, insects, and metabolic water, most birds
require drinking water as their primary water source. Cel-
lular dehydration, extracellular dehydration, and osmoreg-
ulatory hormones, primarily angiotensin II, are the primary
physiological stimuli that induce thirst and subsequently
stimulate drinking (Goldstein and Skadhauge 2000).
Dehydrated birds often drink substantially more water than
required, surpassing that consumed by their nondehydrated
counterparts, to restore intracellular and extracellular water
homeostasis, (Takei et al. 1988). Dehydrated migratory
and/or nomadic birds have been observed gorge drinking
at stopover sites to obtain required water resources in
relatively short time periods to regain positive water
balance (Biebach 1990; Klaassen 2004; M. Woodin,
USGS, personal communication). In addition, drinking
rates may increase with increasing osmolarity of the
drinking water (Goldstein and Skadhauge 2000). Migra-
tory birds that use mine-associated metalliferous waters
for stopover sites may be at increased risk of injury or
death as a result of gorge-drinking behavior, a physio-
logical response of dehydrated migrants presented with
high-osmolarity water.
From September to November 2000, United States Fish
and Wildlife Service (USFWS) biologists investigated a
series of incidents involving 221 bird deaths at 3 associated
mine sites located in southwestern New Mexico and
southeastern Arizona (Stratus Consulting, Inc. 2003).
These incidents involved a variety of passerine and
waterfowl species, as well as heron, shorebird, and hum-
mingbird mortalities, including a total of at least 24 species
in 10 families. The bird deaths were assumed to be linked
to consumption and/or use of AMW because all of the
carcasses were found near pregnant leach solution ponds,
tailings ponds, and associated lakes or storm water reten-
tion basins (Stratus Consulting, Inc. 2003). Other highly
decomposed bird remains were observed on or near metal-
contaminated waters but were not collected and included in
the total count. Many of the carcasses (approximately 40%)
were found near a 280-acre uncovered tailings pond con-
taining increased concentrations of copper (Cu), zinc (Zn),
aluminum (Al), magnesium (Mg), cadmium (Cd), manga-
nese (Mn), cobalt (Co), and iron (Fe) in standing water
(Table 1). The pH of the pond water was 2.0 at the time of
sampling. The ionic strength was 1.11, calculated as I = ‘
RCi zi
2
, where I = ionic strength, Ci = the molar concen-
tration of ith ion present in the solution, and zi = its charge
(Debye and Hu¨ckel 1923).
The primary goal in this study was to develop an
understanding of how avian drinking behavior can influ-
ence AMW toxicity events and to characterize the likely
behavioral, clinical, and pathological signs of acid metal-
enriched water toxicosis that occur in birds exposed to
contaminated water from the New Mexico and Arizona
mine sites. Because the mine sites of concern were in a pre-
litigation phase, bulk water samples for toxicity studies
were unavailable. To provide a realistic test material for
our studies, we formulated a synthetic acid metalliferous
water (SAMW) based on the components of the tailings
pond where the preponderance of bird carcasses were
discovered in 2000. A second goal was to perform this
assessment under an acute-exposure scenario in water and
food-fasted mallards facing water balance stress similar to
wild avian migrants.
654 Arch Environ Contam Toxicol (2011) 61:653–667
123

Methods
SAMW Preparation and Analysis
Fifty gallons of SAMW were prepared to approximate the
water chemistry of a mine tailings pond where the majority
of known bird mortalities occurred (Stratus Consulting,
Inc. 2003; Table 1), matching site water in terms of both
cation and anion concentrations. The specific gravity of the
SAMW was 1.05. Metals and acid were added as chloride
(Cl), nitrate, fluoride, or sulfate salts to simulate site water
ionic content. Reagents used in SAMW production inclu-
ded Al potassium (K) sulfate (Mallinckrodt Analytical,
Hazelwood, MO), arsenic atomic absorption standard
(Fisher Chemical, Pittsburgh, PA), calcium (Ca) fluoride
(Fisher), CdCl2 (Fisher), CoCl2 (Fisher), chromium (VI)
trioxide (Fisher), CuCl2 (Sigma-Aldrich, St. Louis, MO),
Fe sulfate (Fisher), Mg carbonate hydroxide (Fisher), Mn
sulfate (Fisher), sodium (Na) sulfate (Fisher), nickel sulfate
(Fisher), selenium atomic absorption standard (Fisher),
vanadium pentoxide atomic absorption standard (Fisher),
Zn sulfate (Fisher), and concentrated trace metal–grade
nitric acid (Fisher).
Reagent-grade chemicals, deionized water, and trace
metal–grade nitric acid were added to a 55-gallon poly-
ethylene drum and mixed using a reciprocating pump and
electric mixer until dissolved. Any remaining undissolved
reagents were removed with a GE Smart Water Filtration
system that housed a 15-lm sediment filter. Nitric acid was
used to adjust the pH to 2.0, and pH was confirmed each
day before animal dosing. Concentrations of 15 elements in
the solution were confirmed using flame and furnace
atomic absorption spectroscopy (AAS) and inductively
coupled plasma atomic emission spectroscopy. Method
detection limits (MDLs) for metal combinations in water
were calculated according to United States Environmental
Protection Agency test methods (United States Environ-
mental Protection agency [USEPA] 1994; 40 CFR part
136, Appendix B).
Study Design
All activities involving live animals were carried out under
a Texas Tech University Institutional Animal Care and Use
protocol and in consultation with the university veterinar-
ian. Twenty-four 18- to 20-week-old mallards of equally
mixed sex were commercially obtained and transported
to the Texas Tech University Animal Care Resources
Center. Birds were banded and individually maintained in
0.232-m3
stainless steel cages at 20°C, 40% to 70% rela-
tive humidity, and a 12 h:12 h light-to-dark photoperiod.
Ducks had ad libitum access to feed (Mazuri waterfowl
maintenance diet in pellet form; PMI Nutritional, LLC,
Brentwood, MO), grit, and drinking water. All mallards
were allowed a minimum of 10 days to acclimate to
indoor, caged conditions before initiation of testing, with
their body weight being near or exceeding that recorded on
receipt into the animal facility.
A water-consumption measurement system consisted of
multiple components located both outside and inside the
cage. Drinking-water reservoirs were made from a 1-L
Table 1 Concentrations of
metals from mine-associated
AMW, SAMW dosing solution,
and control drinking water
analyzed by flame and furnace
AAS and inductively coupled
plasma atomic emission
spectroscopy
a
Data from Stratus Consulting,
Inc. (2003). AMW samples
collected from mine site on
September 12, 2000. Nominal
Cl, fluoride, nitrate, and sulfate
concentrations in SAMW were
6552, 379, 5362, and
31,100 mg/L, respectively
Elements Data from USFWS AMW
investigation
(mg/L at pH 2)a
Measured concentrations
from SAMW
(mg/L at pH 2)
Control
drinking
water (mg/L)
MDL (mg/L)
Cu 5840 5943 6.6 0.003
Al 3436 3718 3.5 0.1
Zn 2010 2071 2.3 0.001
Mg 1680 1596 1.8 0.003
Fe 1350 1351 1.2 0.017
Mn 738 746 0.5 0.5
Ca 400 493 0.1 0.1
Cd 21.9 22.2 0.5 0.5
Co 21.7 21.8 0.5 0.5
Na 12.4 17.3 0.1 0.001
Ni 10 10.8 0.5 0.5
Cr 4.2 4.8 0.02 0.02
Se 0.534 0.639 0.01 0.01
V 0.385 0.352 0.01 0.01
As 0.250 0.344 0.01 0.01
Arch Environ Contam Toxicol (2011) 61:653–667 655
123

plastic water bottle with an attached rubber stopper,
straight tubing connector, approximately 8 inches of tub-
ing, ratchet clamp, and a quick-disconnect connector. The
water reservoir was connected to a standard avian drink
cup with a spring-loaded lever (GQF Manufacturing,
Savannah, GA). The majority of spillage drained into
waste-collection devices consisting of an inverted top-half
of a 1-gallon polyethylene jug and funnel connected by
tubing to a 2-L plastic water-bottle waste reservoir. Small
amounts of spillage were also collected using Al pans
located below the immediate drinking area inside the cage.
Evaporative water loss was assumed to be negligible
compared with the use and waste measures made for
consumption determinations. Water consumption was
measured by weighing water in source and waste-water
reservoirs. Water consumed was defined as the difference
between (1) the mass of water loss from the source reser-
voir between the start and end of defined time periods and
(2) the total waste water recovered at the end of the period
from (a) the waste water/spillage reservoir under the
drinking cup; (b) the waste pan immediately under the
drinking area inside the cage; and (c) any remaining water
in the drinking cup.
Total metal doses, calculated for each SAMW-treated
bird as the product of water volume consumed and metal
concentration, were determined for 15 elements in the
SAMW dosing solution.
A preliminary study with six mallards was performed to
develop an understanding of SAMW laboratory drinking
dynamics, behavioral reactions to the dosing solution, time
to death, humane end points for killing the birds, and
pathology findings after a 24-h period of dehydration and
fasting. Based on those findings we adjusted the study
design appropriately. Dosing of each mallard was stag-
gered at 2-min intervals to allow for changing water-bottle
reservoirs and waste-collection bottles at collection time
points. SAMW consumption was adjusted for the density
of the dosing solution and control consumption was based
on 1 g/mL for clean drinking water. Animals were
observed continuously, and behavioral and water con-
sumption data were collected throughout the exposure
period.
The definitive study was performed on 3 separate days
during a 1-week period, with three control and three
treatment ducks tested per day. Each day, three control
mallards received control drinking water, and three
treatment mallards received SAMW ad libitum in the
morning after a 24-h period of fasting and dehydration.
Food was withheld during the dosing period for both
control and treatment groups. Body mass measurements
were collected for each mallard before the initiation of the
period of dehydration and fasting, at the initiation of
dosing, and at the time of killing or natural death. Body
mass was measured with an electronic balance to the
nearest 0.1 g.
Killing of Animals and Sample Collection
Humane end points for killing were developed with the
Texas Tech University veterinarian. Birds were deter-
mined to be in moribund condition (in extremis) by visual
signs of wing droop, immobility, lack of response to
touch/visual/auditory stimuli, and/or inability to hold head
erect. All treatment ducks were observed until they were
in extremis, weighed, and killed by way of carbon dioxide
asphyxiation. A control bird was killed as close as pos-
sible to the time each treatment bird died. Due to
decreased blood volume and blood pressure in treatment
ducks, we collected approximately five mL blood imme-
diately postmortem using cardiac puncture. Whole-blood
aliquots were placed in microhematocrit tubes for deter-
mination of packed cell volumes (PCVs), placed in serum
separator tubes for serum clinical chemistry, and frozen
for analytical determination of metal concentrations.
Whole blood in serum separator tubes was allowed to clot
at room temperature for 30 min; serum was centrifuged at
6,000 rpm (45089g) for 10 min; and then serum was
frozen at -80°C until analyzed. Serum samples from
SAMW mortalities were diluted with Milli-Q water (18.0
mega-ohm; Millipore, Billerica, MA) to achieve volumes
necessary for clinical chemistry analyses (Hitachi 911
Analyzer; Texas Veterinary Medical Diagnostic Labora-
tory, Amarillo, TX). Additionally, 12 serum samples from
a reference population of nonbreeding, evenly mixed-sex
adult mallards in a non-water-fasted condition were col-
lected and shipped to the diagnostic laboratory for serum
clinical chemistry analyses. Clinical chemistry end points
included total serum protein (TSP), albumin, globulin, Ca,
phosphorus (P), glucose, creatine phosphokinase (CK),
aspartate aminotransferase (AST), uric acid (UA), cho-
lesterol (Chol), alkaline phosphatase (ALP), Na, K, and
chloride (Cl). A series of 8 control samples, analyzed both
diluted and undiluted, demonstrated that, after correction
for dilution, analyte values of the diluted samples ranged
from 96.6% to 108% of the undiluted controls. AST was
the single outlier at 123%. All values were reported
uncorrected.
Grossly observable lesions were documented, and tis-
sues were collected for both metal-residue and histopa-
thological analyses for all birds. Bile was collected from
gall bladders and frozen at -20°C before metal analyses.
Sections of right testis or ovary, right kidney, salt gland,
spleen, liver, pancreas, heart, brain, trachea, right lung,
tongue, esophagus, proventriculus, ventriculus, duodenum,
jejunum, ileum, ceca, and large intestine were fixed in 10%
123

buffered formalin until processed at the Colorado State
University Veterinary Diagnostic Laboratory.
Histopathology
Tissue samples were embedded in paraffin, and 5-lm
sections were histologically analyzed according to routine
hematoxylin-and-eosin staining (Luna 1968). When dic-
tated by histopathologic findings, specific tissue sections
were also stained (VonKassa and Rhodanine methods,
respectively) for Ca and Cu. Primary histopathologic
analysis was performed blindly without knowledge of
treatment. After analysis, observations in treated and con-
trol groups that were indistinguishable both qualitatively
and quantitatively were considered to be background
lesions and deemed unrelated to treatment.
Tissue Metal Analyses
Approximately 0.4 g thawed tissue or fluid samples (liver,
kidney, blood, or bile) were weighed in a 50-mL Teflon
beaker. Samples were digested with trace metal–grade
18 M nitric acid and 30% hydrogen peroxide. Digestion
solutions were volumetrically diluted to 20 mL with Milli-
Q water, transferred to 50-mL plastic centrifuge tubes, and
stored at 4°C until analysis. Samples containing residual
coagulated lipid were centrifuged at 3500 rpm (19179g)
for 10 min or filtered (Whatman no. 1 filter paper).
Cu, Zn, Mg, Mn, and Fe were analyzed using flame
AAS with a deuterium background correction. Values were
reported on a wet-weight (ww) basis. Calibration standards
for these metals were prepared in 3% nitric acid. Spike
returns for all four tissues and fluids were within ±10% of
total. Percent recoveries ±SE for Cu, Zn, Fe, and Mn in a
standard reference material (DOLT-2; National Research
Council Canada) were 96.9 ± 4.1 (n = 3), 94.8 ± 1.4
(n = 3), 83.1 ± 2.2 (n = 3), and 80.4 ± 0.8 (n = 3),
respectively. Mean recoveries of check standards
throughout analyses for all elements and tissues were
±10%. Data were not corrected for percent recoveries of
spikes or reference material. Biological fluid MDLs for Cu,
Zn, Mg, Fe, and Mn were 0.18, 0.10, 2.05, 4.18, and
0.24 lg/g, respectively. Tissue MDLs for Cu, Zn, Mg, Fe,
and Mn were 0.88, 0.64, 5.44, 3.68, and 0.74 lg/g,
respectively. Where metals data were listed as less than ()
a specific value, tissue concentrations were lower than the
lowest calibration standard, or lower than the MDL,
whichever was the greater value. Of the quantified tissue-
metals data, no tissue–metal combination from either
treatment group contained nondetectable levels except for
bile Zn in all groups and Mn in SAMW mortalities. For the
calculation of mean bile Mn concentration for the SAMW
mortalities, one half the MDL (0.12 ug/g ww) was used for
individuals (n = 3 of 7) containing levels lower than the
MDL
Statistical Methods
Measures of central tendency were expressed as the
means ± SEs unless noted otherwise. All data analyzed
using parametric methods were tested for normality and
homogeneity of variances using Komolgorov–Smirnoff
normality and Levene’s tests, respectively. When a non-
Gaussian distribution, heterogeneous variances, or an
unbalanced design was observed, nonparametric tests were
chosen for subsequent analysis. Alternatively, data were
transformed, retested to meet the assumptions of para-
metric methods, and reanalyzed using parametric statis-
tics. A one-way repeated measures analysis of variance
(ANOVA) or Friedman repeated measures ANOVA on
ranks was used to analyze for a treatment and time-related
effect on water consumption for the first three 20-min and
the first three 1-h drinking periods. Differences were fur-
ther analyzed using Tukey pairwise multiple comparison
test to determine differences among and within treatment
groups during water-consumption time periods. ANOVA
on ranks, followed by Dunn’s post hoc test, was used to
test for differences in PCV among reference, control,
and SAMW mallards. Differences between control and
SAMW-treatment group tissue-metal concentrations, as
well as body-mass dynamics, were analyzed using Student
t tests. Student t tests were used to test for differences in
serum clinical chemistry end points between controls and a
reference population of mallards. Clinical chemistry data
from SAMW mallards were not analyzed with statistical
methods due to small serum sample numbers. For these
reasons, clinical chemistry data from five SAMW-treat-
ment mallard serum samples (three mortalities and two
survivors) were compared with lower and upper reference
intervals from the reference population of mallards
(dehydration effect excluded) and with control mallards
(dehydration effect included). Upper and lower ends of the
reference intervals for each serum chemistry end point
were calculated as ±2 SDs of the mean (Burtis et al. 2005).
Linear regression analysis was used to assess the rela-
tionship between dose and tissue- and fluid-metal concen-
trations in SAMW mortalities. Linear regression analysis
was also used to assess the relationship between total
SAMW consumption or PCV and time to death as well as
the relationship between 20-, 40-, 60-, 120-, and 180-min
SAMW consumption times and time to death. All statistical
analyses were performed with SigmaStat software (version
3.1; Systat Software, San Jose, CA). Results of statistical
tests were considered to be significant at p 0.05.
123

Results
Seven of nine SAMW treatment mallards died or were
killed in extremis from 1.63 to 11.0 h after initiation of
dosing. Two SAMW treatment mallards, one of each sex,
survived exposure, presumably due to decreased con-
sumption of SAMW. The two surviving mallards were
killed and necropsied at 27 and 33 h after dosing. No
control mallards died before being killed during or at the
termination of the study.
Water Consumption Totals and Rates
Water consumption of water-fasted control and SAMW
mallards was substantially greater than that of reference
mallards in a non-water fasted condition. Volumes of water
consumed were significantly different among all three
groups for each of the first three 20-min drinking intervals,
with water-fasted controls consuming approximately twice
the mean volume of water consumed by SAMW mallards
at each time point (Fig. 1).
SAMW mallards drank significantly more water during
the first 20-min period compared with the second and third
20-min drinking periods, and water consumption for con-
trols was significantly greater during the first than the third
20-min drinking period. Water consumption volumes
decreased in the subsequent two 20-min periods for both
control and SAMW mallards.
Water consumption totals for the first 3 h were com-
pared by totaling the initial three 20-min intervals into a
single hour value and comparing it with the second- and
third-hour data. Mean water consumption volumes from
reference mallards in a non-water fasted condition were
B12 mL/kg for each of the first 3 hours and significantly
lower than those of water-fasted control mallards. Mean
SAMW consumption volume was significantly greater
(approximately 10-fold) than reference mallards for the
first hour but not for the second and third hours. For both
control and SAMW mallards, water consumption was
greatest during the first hour and decreased substantially in
the subsequent 2 h. Control totals were different from
SAMW totals for all 3 h of the study. Within water-fasted
controls, first-hour consumption totals were significantly
greater and double those of the second and third hours.
Treatment totals for the first hour, more than twice the
second hour and nearly four times the third hour totals,
were significantly greater than the second- and third-hour
values. Water-consumption data after 3 h of the dosing
study are not presented due to diminishing sample sizes,
deteriorating condition of treatment mallards, and unwill-
ingness of mallards to continue to drink the dosing
solution.
Total SAMW doses ranged from 69.8 to 270.1 mL/kg
(mean ± SE 127.9 ± 27.1) for the seven mortalities.
SAMW doses for the two surviving SAMW mallards were
25.6 and 40.0 mL/kg, respectively (Table 2). There were
no significant relationships between time to death and total
SAMW dose or any of the other water-consumption
intervals.
Signs of Toxicity
Common signs of toxicity among SAMW-treated mallards,
in general order of occurrence, included lateral head
shaking, nasal discharge or oral mucus production, exten-
sive swallowing, ataxia, signs of central nervous system
(CNS) depression, increased breathing rate with shallow
breaths, and death (Table 3). Additional, less common
signs of toxicity included regurgitation, subtle head and/or
body shivering, coughing, and sneezing. Fecal material
from SAMW-treated mallards was usually viscous, dark
green, and lacking visible signs of white urates, whereas
control water-fasted mallards defecated clear and watery
feces. None of these signs were observed in any of the
nonfasted control mallards. In most cases, vigorous lateral
head shaking occurred after the first initial drinks. Oral
mucus production was more common than nasal discharge.
Mucus was usually clear and colorless; however, there
were some instances of blue-green nasal discharge and/or
Fig. 1 Absolute water consumption (mL/kg) during the first three
20-min intervals (A) and first three 1-h intervals (B) of different
mallard treatment groups. Treatments included non-water fasted
mallards (reference) and mallards receiving ad libitum access to
control drinking water (control) or SAMW after a 24-h period of
water deprivation. N = 9 for all bars except SAMW mallards during
the last 1-h time period, where N = 7. Error bars are presented as
SEs. Bars within time steps with different letters differ significantly
from each other (p 0.05). Bars within treatment group with
different numbers differ significantly (p 0.05)
123

oral mucus. Exaggerated swallowing behavior in the
absence of drinking was suggestive of throat irritation and
mucus production. Mallards that consumed enough SAMW
to cause death showed all signs of toxicity through ataxia,
and six of seven treatment mortalities showed signs of CNS
depression. SAMW survivors did not show neurologic signs.
Signs of CNS depression included reoccurring bouts of head
dropping lasting 10 to 15 s and followed by recovery, lack of
response to auditory/visual/touch stimuli, additional head
droop and wing droop, immobility, and/or closed eyelids.
Percent body-mass losses for control and treatment
mallards after a 24-h dehydration period before SAMW
exposure were 6.02% ± 0.43% and 6.66% ± 0.62%,
respectively. The period from initiation of dosing to death
resulted in a mean percent body-mass increase of
0.68% ± 0.38% for controls and a further loss of
6.15 ± 0.73% for treatment mallards. Overall mean per-
cent body-mass loss of control and SAMW treatment
mallards from a hydrated condition to death was
5.38% ± 0.48% and 12.5% ± 0.77%, respectively. Treat-
ment period and overall study duration body-mass losses
were significantly greater in SAMW-treated birds.
Clinical Chemistry and PCV
Serum samples collected from SAMW treatment mallards
were mildly to moderately hemolyzed, whereas control
serum samples were not. Serum Ca, P, glucose, CK, AST,
UA, Na, and K levels from fasted and dehydrated mallards
were all significantly increased compared with the refer-
ence population (Table 4). The most notable differences
between reference and control mallards were observed in
glucose, CK, AST, Na, and K levels, with at least five of
eight individuals in the control group having values greater
than the upper value of the reference interval. Three of five
SAMW-treatment mallards had serum TSP, albumin, and
chloride levels lower then the lower reference interval
value. Five of five SAMW-treatment mallards had serum P,
AST, UA, and K levels greater than the upper reference
interval value, whereas four of five treatment mallards had
CK levels greater than the upper reference interval value.
Treatment-mallard mortalities had decreased mean
serum levels of Ca, glucose, Na, and Cl compared with
control and treatment survivor mallards. Mean glucose
levels were nearly 10-fold lower in treatment mortalities
compared with survivor and control mallards, indicating
severe hypoglycemia in mallards that died from SAMW
consumption. Mean Na and Cl levels were only slightly
lower (20%) in treatment mortalities compared with both
controls and treatment survivors.
Increased mean serum levels of P, ALP, CK, AST, UA,
and K were observed in treatment mortalities compared
with both control and treatment-surviving mallards.
Although mean K levels of SAMW treatment mortali-
ties were only slightly increased compared with controls,
mean P, ALP, CK, AST, and UA levels ranged from
Table 2 Constituent doses in SAMW mortalities and mean and high
doses for surviving mallards
Constituent Total dose (mg/kg body mass)
Mortalities (n = 7) Survivors (n = 2)
Mean ± SE Mean High
Cu 760.2 ± 160.9 194.9 237.7
Al 475.6 ± 100.6 122.0 148.7
Zn 264.9 ± 56.1 67.9 82.8
Mg 204.1 ± 43.2 52.3 63.8
Fe 172.8 ± 36.6 44.3 54.0
Mn 95.4 ± 20.2 24.5 29.8
Ca 63.1 ± 13.3 16.2 19.7
Cd 2.8 ± 0.60 0.73 0.89
Co 2.8 ± 0.59 0.72 0.87
Na 2.2 ± 0.47 0.57 0.69
Ni 1.4 ± 0.29 0.35 0.43
Cr 0.61 ± 0.13 0.16 0.19
Se 0.08 ± 0.02 0.02 0.03
V 0.04 ± 0.01 0.01 0.014
As 0.04 ± 0.01 0.01 0.014
SO4
a
3978 ± 842 1020 1244
Cla
838 ± 177 215 262
NO3
a
686 ± 145 175.9 214.5
Fla
48.5 ± 10.3 12.4 15.2
a
Total doses based on nominal constituent concentration in SAMW
Table 3 Signs of toxicity and associated means and ranges of times to signs of toxicity among SAMW-exposed mallards
Statistical measure Time (min) to signs of toxicity among SAMW-exposed mallards
Head
shaking
Nasal discharge/
mucus
Exaggerated
swallowing
Ataxia CNS depression/
dazed
Breathing
change
Death
Mean timea
(min) 11 (9) 45 (9) 50 (9) 117 (7) 259 (6) 183 (5) 305 (7)
Range of times (min) 0–52 9–106 10–175 31–252 86–652 86–350 98–661
Data are presented only for birds that demonstrated each specific sign. Number of mallards showing sign is in parentheses
a
Two of nine mallards survived exposure due to decreased SAMW consumption and are not included in the calculation of mean time to death
123

approximately 2 to 15 times greater in treatment mortalities
compared with controls. However, the variability of these
five end points for treatment mortalities was relatively high
and exceeded that of controls, likely due to decreased
serum sample sizes of SAMW-treated mallards.
Mean PCV values from treatment mortalities were
significantly greater than those of controls and the refer-
ence population (p 0.001). Mean PCVs from the two
SAMW-treatment survivors were higher than the upper
level of the reference interval but lower than mean values
from treatment mortalities. There was a significant rela-
tionship between PCV and time to death (p = 0.05;
r2
= 0.57; n = 7), with greater PCVs coinciding with
shorter times to death.
Pathology
Common grossly observable abnormalities in SAMW-
treated mallards included presence of increased clear or
blue-green mucus and associated discoloration of the
mucosa of the esophagus, proventriculus, ventricular
kaolin, and intestine, as well as in the proximal trachea and
nasopharynx. Other abnormalities that were less common,
although more severe, included petechial hemorrhages on
the serosal surface of the duodenum and localized ulcer-
ations of the ventriculus (mostly along the proventricular–
ventricular junction) and the duodenum. Reddening of the
proventriculus and erosion and reddening of the mucosa of
the proximal duodenum were noted in seven SAMW-
treated birds, two of which were survivors. Similar lesions
were absent in control birds.
Histopathologically, mild chronic portal hepatitis and
mild to marked chronic heterophilic tracheitis were
observed both in treated and control mallards and were
considered background findings that were unrelated to
treatment. Minimal to mild splenic lymphoid necrosis was
noted in five SAMW-treated birds and in two control birds.
Lymphoid necrosis is often a manifestation of stress in
Table 4 Serum clinical chemistry values for a reference mallard population and for food and water-restricted control, SAMW mortalities, and
SAMW survivors
Reference population Controls SAMW mortalitiesa
SAMW survivorsb
Mean ± SD Reference
interval
Mean ± SD Relative to
reference values
Mean ± SD Relative to
reference values
Mean Relative to
reference values
N = 12 Lower Upper N = 8 Below Above N = 3 Below Above N = 2 Below Above
TSP (g/dl) 4.39 ± 0.55 3.29 5.49 4.33 ± 0.54 – – 3.78 ± 1.25 1 – 3 2 –
Albumin (g/dl) 2.27 ± 0.16 1.95 2.59 2.34 ± 0.27 1 1 2.14 ± 0.68 1 – 1.56 2 –
Globulin (g/dl) 2.12 ± 0.44 1.24 3 1.96 ± 0.28 – – 1.67 ± 0.58 1 – 1.45 – –
A/G ratio 1.09 ± 0.15 0.79 1.39 1.18 ± 0.1 – – 1.31 ± 0.08 – – 2.52 – 1
Ca (mg/dl) 11.5 ± 0.46 10.6 12.44 12.2 ± 0.92* – 3 10.8 ± 0.51 1 – 12.2 – –
P (mg/dl) 4.58 ± 0.92 2.74 6.42 7.85 ± 2.27* – 4 14.3 ± 5.79 – 3 8.6 – 2
Glucose (mg/dl) 179 ± 16 147 211 258 ± 81* – 5 30 ± 21 3 – 240.6 – 1
ALP (U/l) 96 ± 26 44 148 109 ± 68 2 2 192 ± 96.1 – 1 108 – –
CK (U/l) 295 ± 90 115 475 1186 ± 972* – 6 6579 ± 4797 – 3 902 – 1
AST (U/l) 13 ± 2 9 17 44 ± 15* – 8 393 ± 100 – 3 73 – 2
UA (mg/dl) 3.57 ± 1.2 1.17 5.97 5.29 ± 1.52* – 3 79.7 ± 22.7 – 3 27 – 2
Chol (mg/dl) 308 ± 32 244 372 317 ± 49 1 1 370 ± 162 1 2 261 – –
Na (meq/l) 149 ± 2.48 144.87 154.79 161 ± 9.96* – 6 135 ± 23.5 2 1 166 – 2
K (meq/l) 2.67 ± 0.37 1.93 3.41 7.32 ± 4.11* – 8 10.0 ± 4.64 – 3 4.5 – 2
Na/K ratio 57.2 ± 7.7 41.8 72.6 27.6 ± 11.7* 8 – 15.6 ± 7.1 3 – 82.1 – 1
Cl (meq/l) 104 ± 2 100 108 103 ± 6 2 2 91 ± 6 3 – 107 – 1
PCV 48 ± 2 44 52 50 ± 5c
1 2 75 ± 6c,d
– 7 63 – 2
Serum chemistry results from the reference population were collected from individuals in a non-water fasted state after regular food removal
during overnight lights-out period
a
Serum samples from treatment mallards were diluted either 1:4, 1:3, or 1:9 with 18.0 mega-ohm water. All parameter values were corrected for
dilution factors but not for recoveries. Serum sample quantities from all other treatment mortalities were not sufficient for analysis
b
Survived exposure to SAMW. These birds were killed either 27 or 33 h after exposure
c
n = 7 for SAMW-exposed mallards (mortalities only); n = 9 for control mallards
d
Significantly different than control and reference values (p 0.001; ANOVA on ranks-Dunn’s test)
* p 0.05 compared with reference population
123

animals and is most likely a nonspecific effect. Increased
incidence in treated birds compared with controls is most
likely an indicator of increased stress in the treated birds.
SAMW treatment-related histopathologic lesions were
limited to the esophagus, proventriculus, ventriculus, and
duodenum. The esophagus in four of seven treated birds
exhibited varying degrees of mucous gland ectasia with or
without associated heterophilic inflammation. Some glands
were obliterated by heterophilic inflammation and necro-
sis. Condensed blue discoloration was often noted at the
opening of the esophageal glands on the mucosa. This
condensed material was negative for Ca and Cu by special
stains (VonKassa and Rhodanine methods, respectively).
Its composition was uncertain, but it could have repre-
sented coagulated mucous and/or other precipitate from
SAMW.
Compared with control birds, the proventricular mucosa
of treated birds (9 of 9) was variably eroded, denuded, and
covered by an amorphous layer of mucin and granular
eosinophilic material (interpreted as fibrin). Often along the
eroded epithelium, there was basophilic discoloration of
the connective tissue scaffold, suggesting mineralization.
Additionally, there was marked congestion with or without
heterophilic inflammation in the lamina propria and sub-
mucosa. The proventricular glands were within normal
limits in treated and control animals. A male mallard sur-
vivor consuming the lowest dose of SAMW represented
the most acute morphologic change in the proventricu-
lar mucosa. In this bird, individual or small clusters of
mucosal epithelial cells were degenerative to necrotic
(as indicated by cellular swelling, cytoplasmic eosino-
philia, and pyknosis) and in the process of being sloughed.
In other areas, there was complete loss of mucosal epi-
thelium and the denuded connective tissue exhibited the
basophilic discoloration described previously. In the two
SAMW-treatment survivors, there was an apparent attempt
at re-epithelization of focally extensive areas of the mucosa
as suggested by lining of the mucosa by flattened epithelial
cells compared with columnar cells in the controls.
Changes in the ventriculus were noted in eight of nine
SAMW-treated and one of nine control mallards (Table 5).
The changes in the control mallard included minimal
infiltrate of heterophils in the submucosa. In contrast, the
changes in the treated mallards were markedly more
prominent and included a greater heterophilic response in
the submucosa, with degenerate heterophils extending into
the kaolin layer of some birds. Also, erosion or ulceration
of the kaolin layer with subjacent congestion and hemor-
rhage were noted in most treated mallards (7 of 9). It is
notable that the changes in the ventriculus persisted while
the proventriculus exhibited signs of repair in the SAMW-
treated survivors.
Changes in the small intestine were noted in six of nine
SAMW mallards and included increased mucus and
coagulated protein on the mucosal epithelial surface of the
jejunum, small intestine congestion, and hemorrhaging as
well as one case of coagulative necrosis in the duodenal
lamina propria and denudation of the duodenal tips of villi.
Tissue-Metal Residues
Blood and kidney tissue-metal concentrations from
SAMW-treated mallards were increased compared with
control mallards (Table 6). Mean kidney Cu, Zn, Mg, and
Mn concentrations were significantly greater in SAMW
mortalities compared with controls. Kidney Cu and Mn
concentrations were approximately 6- and 3.5-fold greater
in SAMW mortalities compared with controls. There was
no difference in kidney Fe concentration between SAMW
mortalities and controls. Mean blood Cu, Zn, Mg, and Fe
concentrations from SAMW mortalities were significantly
greater than control mallards as well. Blood Cu levels were
approximately 23 times greater in SAMW mortalities
compared with controls, and mean blood Zn levels from
SAMW mortalities were approximately twice the mean of
controls. There were no significant differences in liver
metal concentrations; however, mean liver Cu concentra-
tions were approximately 50% greater in lethally exposed
mallards than in controls. Bile concentrations of Cu and
Mn in SAMW mortalities were significantly greater than
those from controls; however, there were no differences in
bile Mg and Fe concentrations between the two groups.
A significant relationship was observed between Cu
dose and kidney Cu (p = 0.011, r2
= 0.758) and Cu dose
and blood Cu (p = 0.029, r2
= 0.649) in the mortalities.
There was also a significant relationship between Mn dose
and kidney Mn concentration (p = 0.004, r2
= 0.838).
There were no other significant relationships observed
between metal dose and tissue- or fluid-metal concentra-
tions for any other combinations in the mortalities.
Discussion
The synthetic acid mine tailings pond water was highly
toxic to mallards, with seven of nine mallards dying as
Table 5 Nature of histopathological changes in the ventriculus after
acute SAMW and control water treatments in mallard ducks
Treatment Erosion or
ulceration
of kaolin
Heterophilic
inflammation
Congestion
and
hemorrhage
SAMW treated (all birds) 7/9 7/9 7/9
SAMW-treated survivors 2/2 2/2 2/2
Control 0/1 1/1 0/1
123

quickly as 98 min after first exposure. The average
SAMW consumption rate in the first 20 min was approx-
imately 81 mL/kg/h (based on 27 mL/kg consumed),
which indicates there is high potential for acute mortality
in birds that are willing to drink water with such high
concentrations of toxic metals. Most of the clinical signs
of toxicity, which were suggestive of renal dysfunction
(increased UA, P, and K, and decreased Cl), liver damage
(increased AST), heart or muscle damage (increased CK),
potential biliary obstruction (increased ALP), dehydration
(increased hematocrit), hemolysis, and/or shock are sim-
ilar to previous reports of acute Cu or acid mine water
toxicosis in waterfowl (Henderson and Winterfield
1975; Stubblefield et al. 1997). Time to death from
other reports of Cu or AMW-related waterfowl mortali-
ties (Henderson and Winterfield 1975; Stubblefield et al.
1997) have been similar to our findings, with birds being
found in extremis or dead within 12 to 24 h after first
ad libitum exposure. It is not clear if the significant
correlation between PCV and time to death may be
indicative on the importance of systemic dehydration in
the death of affected mallards or if dehydration is a
reflection of renal dysfunction.
The majority of SAMW metal concentrations were
lower than those considered to be acutely toxic to avian
species (NRC 2005). Although their concentrations would
be considered increased compared with those of more
typical drinking water sources encountered by birds, the
majority are nutritionally essential, homeostatically regu-
lated, and not bioaccumulated to a degree of toxicological
concern and therefore pose less threat of toxicity to
exposed birds. The general mechanisms of toxicity for
SAMW metals include oxidative damage, antagonistic
effects on metabolism of other minerals, and perturbations
in acid–base homeostasis and electrolyte balance (NRC
2005). Complexity of AMW constituents, potential inter-
active effects, and the diversity of specific toxic effects
pose a challenge to understanding the ultimate cause of
death in AMW-exposed birds.
Acute Toxicity of Cu and Other AMW Contaminants
Of the metals in solution, Cu likely played an important
role in the toxicity of this water. Cu doses in this study
ranged from 415 to 1605 mg/kg. The lower limits of Cu
lethality occur at doses of 160 and 240 mg/kg body wt in
Table 6 Mean ± SE concentrations of elements detected in liver, kidney, blood, and bile from SAMW-treatment and control mallards using
flame AAS
Tissue Concentrations of elements (lg/g ww ± SE)
Cu Zn Mg Fe Mn
Liver control 156 ± 47 59.2 ± 2.7 300 ± 6.9 1389 ± 196 5.3 ± 0.3
SAMW (mortality) 210 ± 39.3 57.7 ± 5.4 289 ± 15.0 1122 ± 208 14.6 ± 0.9**
SAMW (survivor) 327 69.7 266 749 9.6
406, 247 75.6, 63.8 290, 243 849, 649 9.4, 9.8
Kidney control 7.5 ± 0.48 21.8 ± 0.67 248 ± 9.8 148 ± 7.9 3.9 ± 0.39
SAMW (mortality) 43.1 ± 4.1*** 28.7 ± 2.0** 298 ± 19.6* 201 ± 18 14.7 ± 1.4***
SAMW (survivor) 20.8 25.0 270 174 8.0
11.9, 29.8 21.7, 28.4 247, 294 183, 164 4.7, 11.3
Blood control 2.0 ± 0.3 5.6 ± 1.1 99.2 ± 6.6 424 ± 6.6 0.24
SAMW (mortality) 45.9 ± 6.5*** 14.6 ± 2.2** 144 ± 6.6*** 632 ± 24.8*** 0.24
SAMW (survivor) 3.4 6.8 123 563 0.24
1.8, 5.0 7.0, 6.7 132, 114 600, 525
Bile control 39.4 ± 5.7 0.10 192 ± 12.7 8.1 ± 2.6 0.24
SAMW (mortality) 70.6 ± 14.0* 0.10 203 ± 25.9 8.7 ± 1.5 34.1 ± 18.8a
SAMW (survivor) 118 0.10 201 10.8 48.1
63, 174 138, 264 8.8, 12.7 8.0, 88.1
N = 9 for each tissue–metal combination in controls; N = 7 in SAMW mortalities; and N = 2 in SAMW survivors. Tissue-metal concentrations
that fell below the lowest calibration standard or MDL, whichever was the greater value, were reported as ‘‘’’ that value. Values for SAMW
survivors are means with individual values
a
Four of seven samples with concentration above MDL; 50% MDL used for three remaining samples
* p 0.05
** p 0.01
*** p 0.001 (significantly different from control)
123

mallards (400 mg CuSO4/kg [Pullar 1940a, b] and 600 mg
CuSO4/kg [EXTOXNET 1996]). All seven of the lethally
treated SAMW mallards, as well as one of the survivors,
consumed more than the lowest lethal dose (Table 2).
In addition to clinical signs of metal toxicosis, signs of
metal-induced pathology were observed in SAMW-treated
mallards. Based on the nature of the gross and histopa-
thology findings, it appears that the majority of the
pathology from the SAMW solution is related to the
extremely high concentration of Cu. Characteristic changes
found in acute Cu toxicosis are hemorrhage or necrosis of
the liver and kidney, proventricular and ventricular
necrosis, intestinal hemorrhage, increased liver Cu con-
centrations, and, sometimes, stomatitis (Henderson and
Winterfield 1975; Jensen et al. 1991; NRC 2005; Pullar
1940a, b). Mallards from our studies demonstrated all of
these changes with the exception of consistent liver and
kidney lesions. Decreased exposure duration and sudden
mortality were likely responsible for the lack of stomatitis
and kidney damage in our study.
Liver Cu residue is a commonly used and reliable index
of exposure in cases of acute Cu toxicosis. Reported mean
liver Cu concentrations in wild adult mallard ducks range
from 35 to 585 lg/g dry weight (dw). Although field-col-
lected mallards usually have liver Cu concentrations
100 lg/g dw (Chupp and Dalke 1964; Di Giulio and
Scanlon 1984a), laboratory-control mallards have been
reported as having greater levels in the range of approxi-
mately 300 to 600 lg Cu/g dw (Di Giulio and Scanlon
1984a, b). Such a wide range in liver Cu concentrations is
likely the result of dietary differences between wild and
laboratory-maintained mallards. Canada geese (Branta
canadensis) displaying similar clinical signs and patho-
logical findings as SAMW-treatment mallards from our
study contained 56 to 97 lg Cu/g ww in livers (Henderson
and Winterfield 1975). Wild Canada goose liver Cu con-
centrations typically range from 6 to 30 lg/g ww
(approximately 20 to 100 lg/g dw; Puls 1994), whereas
mute swan liver Cu concentrations range from 120 to
360 lg/g dw (Kobayashi et al. 1992). Liver Cu concen-
trations from lethally exposed mallards in this study ranged
from 81 to 391 lg/g ww or 270 to 1302 lg/g dw (assuming
70% moisture), re-emphasizing the wide range of liver Cu
concentrations that may be associated with Cu-related
mortality incidents. In cases of acute AMW mortality
incidents, Cu accumulation in the blood or kidney would
be a better predictor of acute Cu toxicosis.
Although the SAMW had high concentrations of several
potentially toxic metals, and several metals were at
increased levels in more than one tissue in SAMW-treated
mallards compared with controls, the tissue-residue data do
not reflect acutely toxic levels for any of the metals except
Cu. For instance, mean liver and kidney Zn concentrations
can range from 600 to 1100 and 1000 to 1700 lg/g dw,
respectively, in Zn-poisoned mallards (Gasaway and Buss
1972) and were 280 and 220 ug/g dw, respectively, in a
nonlethally intoxicated mallard (Sileo et al. 2004). Mean
liver and kidney Zn concentrations from SAMW mortali-
ties in our study were 57.7 and 28.7 lg/g ww (92 and
96 lg/g dw), which are similar to liver and kidney Zn
concentrations from our control mallards and other repor-
ted control mallards (Gasaway and Buss 1972). This is
important because Zn concentrations that lead to waterfowl
mortalities and toxicity in the wild, by way of degenerative
pancreatitis (which did not occur in SAMW-dosed mal-
lards), demonstrate increased Zn in liver and kidney as well
as the pancreas, which we did not chemically analyze
(Sileo et al. 2004). These findings do not, however, pre-
clude the potential that interactions between Cu and other
metals may have led to modification in the toxicity of the
SAMW, although these phenomena would need testing
well beyond the scope of this investigation.
Other toxic metals, such as Hg and Pb, were not detected
in field AMW samples or were at concentrations not con-
sidered to be an acute threat to avian wildlife health.
Although the sites of concern in southwestern New Mexico
and southeastern Arizona did not contain AMW with
hazardous concentrations of such metals, other mining-
associated sites in the western United States do contain
increased and potentially hazardous concentrations of other
toxic metals, especially Hg and Pb, in water, soil, sediment,
and biota (Beyer et al. 1998; Gustin et al. 1994; Seiler et al.
2004; Wayne et al. 1996), where bioaccumulation has been
documented in terrestrial (Custer et al. 2007) and aquatic
avian species (Gerstenberger 2004; Henny et al. 2000;
Henny et al. 2002; Seiler et al. 2004). Therefore, other
metals of potential concern not included in our laboratory
AMW study could play a role in the toxicity of mine
wastewater to avian wildlife.
In addition to the metals, the acidic nature of SAMW
and the high sulfate and nitrate content may have con-
tributed to the overall toxicity of the solution. Sixty-seven
percent of ducklings died after a 5-day exposure to drink-
ing water at pH 3.0 without any added metals (Foster
1999). Acid-only solutions may produce age-dependent
toxicity in avian species; however, data are limited, and
further investigation is warranted. Ingestion of high con-
centrations of sulfate salts can also be detrimental to a
bird’s health. It is commonly noted that sulfates can have a
cathartic and laxative effect in exposed organisms (Daniels
1988), with Mg and Na sulfate being more potent laxatives
than Ca sulfate (Daniels 1988). Sulfate ions can induce
laxative effects by causing retention of excess fluid in the
intestinal lumen and increasing motor activity in the small
and large intestine (Bast 1991). More severe sulfate effects
in birds include alteration of acid–base balance (metabolic
123

acidosis) and potentially death (NRC 2005). For example,
100% mortality, visceral gout, and kidney necrosis were
observed in laying hens after 12 continuous days of
receiving 16,000 mg/L total sulfate in the form of Na or
Mg sulfate in drinking water (Adams et al. 1975). The
sulfate concentrations of site AMW and SAMW from our
study were approximately 76,000 mg/L (Russ MacRae,
USFSW, personal communication) and 31,100 mg/L, res-
pectively; therefore, sulfate potentially could have added to
the toxicity of metals despite the absence of kidney lesions.
At high doses, nitrates can also be lethal to birds. Fifty
percent of turkey poults died after a 1-week exposure to
3990 mg Na nitrate/L, and 60% died after 21 days of
exposure (Adams et al. 1969). One hundred percent mor-
tality in turkey poults was observed after 5-day treatment
with drinking water nitrate concentrations as low as
5320 mg/L, with signs of toxicity including subnormal
growth, salivation, uncoordination, kidney enlargement,
and tissue lesions similar to those observed in salt toxicosis
cases (Adams et al. 1969). We observed uncoordination in
all mallards consuming a lethal dose of SAMW containing
approximately 5400 mg nitrate/L; however, tissue lesions
in our study were not similar to those observed in avian salt
toxicosis cases, and death occurred in 12 h, which was
not congruous with the findings of Adams et al. (1969).
Based on poultry sulfate (Adams et al. 1975; Kienholz
1968; Krista et al. 1961) and nitrate (Adams et al. 1969)
toxicity literature, it is unlikely that mallards exposed to
SAMW could have died from sulfate or nitrate toxicosis
within the observed times to death.
Avian Water Balance and its Role in Contaminated
Water Toxicity
Dehydration and subsequent thirst were likely the two most
important factors driving SAMW-treated mallards to con-
sume lethal doses of SAMW in our study. These birds, as
well as fasted and dehydrated control mallards, fulfilled
their water needs by consuming relatively large amounts of
water in a short period of time. Similar gorge-drinking
behavior has been observed in birds arriving at water after
migratory flights (Biebach 1990; Klaassen 2004; Marc
Woodin, USGS, personal communication). In this study, a
lethal dose was consumed in as little as 20 to 40 min after
first exposure. This adaptive strategy may be of use for
birds using water sources in arid regions of the United
States. This behavior could also be used by birds con-
suming AMW from ponds, puddles, or streams, such as
those found in the desert southwest where vegetation is
scarce or absent. For example, mildly dehydrated mourning
doves are able to drink approximately 157% of their daily
ad libitum intake and 386% of the minimum daily
requirement in only one or two draughts that last only 1
minute (MacMillen 1962). Species that use such behavioral
adaptations and that are not completely averse to AMW
would be at increased risk to injury from exposure to
acid-contaminated water.
Daily water-consumption volumes and rates are not
known for the majority of avian species and are generally
estimated using allometric equations based on the work of
Bartholomew and Cade (1963) and Calder (1981). These
allometric equations are acquired from documents such as
the USEPA Wildlife Exposure Factors Handbook (1993)
and used in ecological risk assessments. Although estima-
tions of daily water requirements typically suffice for
exposure and effects assessments, such mathematically
derived values may underestimate the potential for AMW-
induced injury in cases where birds ingest water at greater
consumption rates and for shorter durations than allometric
equations would predict. Because birds can consume large
volumes of water quickly, water-consumption measure-
ments more frequently than 24-h intervals are necessary to
provide accurate exposure estimates for birds that use toxic
water bodies, such as AMW.
The importance of water balance to birds during
migration remains unclear. Several studies have suggested
that water balance is an important physiological constraint
on migratory bird species (Carmi et al. 1992; Klaassen
1996; Leberg et al. 1996; Yapp 1956, 1962). Other studies
have shown migratory birds, some of which are generally
considered to maintain water balance without free water
consumption, drinking from water catchments or other
water resources in semi-arid or arid stopover habitats
(Cutler and Morrison 1998; Lynn et al. 2006, 2008;
O’Brien et al. 2006; Smyth and Coulombe 1971). Alter-
nately, other studies conclude that energy demands (i.e., fat
stores and body mass) are the primary limiting factor that
influences migration and that birds can use different
migration strategies to avoid water loss (Blem 1976;
Biebach 1990; Dawson 1982; Rogers and Odum 1964;
Torre-Bueno 1978). Nevertheless, water imbalance
decreases flight efficiency and can force flying migrants
experiencing dehydration to land and rehydrate. When
natural water sources become scarce, when riparian areas
are decreased or altered (such as in the southwestern
United States [Nabhan and Holdsworth 1999; Sheridan and
Nabhan 1978]), or when contaminated water sources have
been created, clean water availability becomes an impor-
tant factor in determining the survival of both migratory
and resident birds.
Forensic Tools for Identifying AMW Toxicity
Wildlife biologists who find birds injured or dead on or
near AMW bodies can take some important actions to
determine whether or not the water is responsible for
123

inducing injury or death. For incapacitated birds, signs of
AMW toxicity may include those listed in this article,
especially lethargy, wing droop, and the inability to stand,
walk, or hold the head erect. Some waterfowl have been
observed swimming in circles and been unable to hold their
head out of the water (unpublished data, Russell MacRae).
Depending on the dose of acid, metals, and other water
constituents, incapacitated birds may or may not be able to
recover from AMW-induced injury. When possible, blood,
and kidney samples should be collected from injured birds
for determination of metals, and feet, legs, and oral cavity
examined for lesions caused by exposure to AMW. For
birds found dead on or near AMW bodies, carcass condi-
tion should be examined and determined whether it will be
sufficient for forensic analysis. For carcasses in fair to good
condition, gross pathological analysis, tissue harvesting,
and formalin fixing of AMW target tissues (esophagus,
proventriculus, ventriculus, and duodenum) should be
performed as soon as possible. Extent and intensity of
lesions in tissues will be dependent on the metals and dose
ingested. Pathologies not present in SAMW-exposed birds
from our study may be present in birds injured on other
AMW bodies; therefore, sections from all tissue types
harvested in this study should be excised from birds in the
field, and eyes and sections of skin should be removed for
determination of ocular and dermal injuries as well. For
carcasses in poor condition, tissue-metal residues will
likely be the only useful diagnostic tool. In addition to
behavioral and pathological signs of toxicity, clinical and
hematological end points, such as UA, K, P, AST, CK, and
PCV, may be of use in avian forensic investigations of
AMW toxicity.
Conclusion
Based on the findings from our study and other reports of
AMW toxicity events in wild birds, we conclude that
AMW bodies pose a significant hazard to wildlife that
come in contact with them. Birds that are not averse to
AMW have a potential increased risk of injury after oral
exposure. Thousands of migrating birds are likely to be
injured every year in the western United States due to
exposure to AMW. Little is known about the potential
population level effects that lethal and sublethal exposures
could have on avian species. We have presented data
concerning the acute toxicity of a SAMW that reflect a
documented exposure scenario with metal concentrations
that can be found in pregnant leach solution ponds, tailings
ponds, and mine site process water-storage ponds associ-
ated with a mining complex in southeastern Arizona and
southwestern New Mexico. Further studies of the broad
range of metal and acid-associated exposure scenarios are
warranted to fully assess the hazards to be found at active
and abandoned mines along avian migratory pathways.
Acknowledgments We thank Melanie Barnes, Gopal Coimbatore,
the Colorado State University Veterinary Pathology Laboratory, and
the Texas Veterinary Medical Diagnostic Laboratory for performing
analytical and diagnostic procedures. George Cobb, Ann Maest,
Michael Fry, Mike Hart, Amber Matthews, Toby McBride, and ani-
mal care assistants also contributed substantially to this research. We
thank Kevin Reynolds, Karen Cathey, Susan Finger, Barnett Rattner,
and Nelson Beyer whose reviews improved earlier versions of this
manuscript. This work was funded by the Department of Interior
Natural Resource Damage Assessment and Restoration Program with
additional support from the U.S. Geological Survey Columbia Envi-
ronmental Research Center and The Institute of Environmental and
Human Health at Texas Tech University. Any use of trade, product, or
firm names is for descriptive purposes only and does not imply
endorsement by the U.S. Government.
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