aect_acid mine water_pdf

Behavioral, Clinical, and Pathological Characterizationof 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

� 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: [email protected]

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 Huckel 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

SO4a 3978 ± 842 1020 1244

Cla 838 ± 177 215 262

NO3a 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 parenthesesa 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 perioda 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 analysisb Survived exposure to SAMW. These birds were killed either 27 or 33 h after exposurec n = 7 for SAMW-exposed mallards (mortalities only); n = 9 for control mallardsd 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 valuesa 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.

References

Adams AW, West JL, Kars AJ (1969) Some effects on turkeys of

nitrate in the drinking water. Poult Sci 48:1222–1229

Adams AW, Cunningham FE, Monger LL (1975) Some effects on

layers of sodium sulfate and magnesium sulfate in their drinking

water. Poult Sci 54:707–714

Bartholomew GA, Cade TJ (1963) The water economy of land birds.

Auk 80:504–539

Bast C (1991) Toxicity summary for sulfate. Prepared for Oak Ridge

Reservation Environmental Restoration Program. Available at:

http://rais.ornl.gov/tox/profiles/sulfate_f_V1.html. Accessed 7

July 2008

Beyer WN, Audet DJ, Morton A, Campbell JK, LeCaptain L (1998)

Lead exposure of waterfowl ingesting Coeur d’Alene River

Basin sediments. J Environ Qual 27:1533–1538

Biebach H (1990) Strategies of trans-Sahara migrants. In: Gwinner E

(ed) Bird Migration. Spring-Verlag, Berlin, Germany,

pp 352–367

Blem CR (1976) Patterns of lipid storage and utilization in birds.

Am Zool 16:67–684

Burtis CA, Ashwood ER, Bruns DE (2005) Tietz Textbook of Clinical

Chemistry and Molecular Diagnostics, 4th edn. Elsevier/Saun-

ders, Philadelphia, PA

Calder WA (1981) Scaling of physiological processes in homeother-

mic animals. Annu Rev Physiol 43:301–322

Carmi N, Pinshow B, Porter WP, Jaeger J (1992) Water and energy

limitations on flight duration in small migrating birds. Auk

109(2):268–276

Chupp NR, Dalke PD (1964) Waterfowl mortality in the Coeur

D’Alene River Valley, Idaho. J Wildl Manag 25:692–703

Custer CM, Custer TW, Hill EF (2007) Mercury exposure and effects

on cavity-nesting birds from the Carson River, Nevada. Arch

Environ Contam Toxicol 52(1):129–136

Cutler PL, Morrison ML (1998) Habitat use by small vertebrates at

two water developments in southwestern Arizona. Southwest Nat

43:155–162

Daniels JI (1988) Evaluation of military field-water quality. Volume

4. Health criteria and recommendation for standards, Part 1.

Chemicals and properties of military concern associated with

natural and anthropogenic sources. AD UCRL-21008 Volume 4,

Part 1

Dawson WR (1982) Evaporative losses of water by birds. Comp

Biochem Physiol A 71:495–509


123

http://rais.ornl.gov/tox/profiles/sulfate_f_V1.html

Debye P, Huckel E (1923) The theory of electrolytes. I. Lowering of

freezing point and related phenomena. Physikalische Zeitschrift

24:185–206

Di Giulio RT, Scanlon PF (1984a) Heavy metals in tissues of

waterfowl from the Chesapeake Bay, USA. Environ Pollut

(Series A) 35:29–48

Di Giulio RT, Scanlon PF (1984b) Sublethal effects of cadmium

ingestion on mallard ducks. Arch Environ Contam Toxicol

13:765–771

Extension Toxicology Network (1996) Copper sulfate. Pesticide

Information Project of Cooperative Extension Offices of Cornell

University, Oregon State University, the University of Idaho,

and the University of California at Davis and the Institute for

Environmental Toxicology, Michigan State University. Avail-

able at: http://extoxnet.orst.edu/pips/coppersu.htm. Accessed 4

Dec 2006

Foster SD (1999) The biological and physiological effects of excess

copper in juvenile mallards (Anas platyrhynchos): an investiga-

tion of the toxicity of acid mine drainage in waterfowl. Master’s

thesis, Colorado State University, Fort Collins, CO

Gasaway WC, Buss IO (1972) Zinc toxicity in the mallard. J Wildl

Manag 36:1107–1117

Gerstenberger SL (2004) Mercury concentrations in migratory

waterfowl harvested from southern Nevada wildlife management

areas, USA. Environ Toxicol 19(1):35–44

Goldstein DL, Skadhauge E (2000) Renal and extrarenal regulation of

body fluid composition. In: Whittow GC (ed) Sturkie’s Avian

Physiology. Academic, London, England, pp 265–298

Gustin MS, Taylor GR Jr, Leonard TL (1994) High levels of mercury

contamination in multiple media of the Carson River drainage

basin of Nevada: Implications for risk assessment. Environ

Health Perspect 102(9):772–778

Haglar Bailly Consulting, Inc (1996) Supplemental injury assessment

report: Clark Fork River NPL Site NRDA, lethal injuries to snow

geese, Berkeley Pit, Butte, MT. Prepared for Montana Natural

Resource Damage Assessment Litigation Program

Henderson BM, Winterfield RW (1975) Acute copper toxicosis in the

Canada goose. Avian Dis 19:385–387

Henny CJ, Hallock RJ, Hill EF (1994) Cyanide and migratory birds at

gold mines in Nevada, USA. Ecotoxicology 3:45–58

Henny CJ, Blus LJ, Hoffman DJ, Sileo L, Audet DJ, Snyder MR

(2000) Field evaluation of lead effects on Canada geese and

mallards in the Coeur d’Alene River Basin, Idaho. Arch Environ

Contam Toxicol 39:97–112

Henny CJ, Hill EF, Hoffman DJ, Spalding MG, Grove RA (2002)

Nineteenth century mercury: hazard to wading birds and

cormorants of the Carson River, Nevada. Ecotoxicology

11:213–231

Jensen LS, Dunn PA, Dobson KN (1991) Induction of oral lesions in

broiler chicks by supplementing the diet with copper. Avian Dis

35:969–973

Kienholz EW (1968) Effects of salts in drinking water of turkeys. Ann

Nutr Health 23:3–25

Klaassen M (1996) Metabolic constraints on long-distance migration

in birds. J Exp Biol 199:57–64

Klaassen M (2004) May dehydration risk govern long-distance

migratory behavior? J Avian Biol 35:4–6

Kobayashi Y, Shimada A, Umemura T, Nagai T (1992) An outbreak

of copper poisoning in mute swans (Cygnus olor). J Vet Med Sci

54(2):229–233

Koutsos EA, Matson KD, Klasing KC (2001) Nutrition of birds in the

order Psittaciformes: a review. J Avian Med Surg 14:257–275

Krista LM, Carlson CW, Olson OE (1961) Some effects of saline

waters on chicks, laying hens, poults, and ducklings. Poult Sci

40:938–944

Leberg PL, Spengler TJ, Barrow WC Jr (1996) Lipid and water

depletion in migrating passerines following passage over the

Gulf of Mexico. Oecologia 106:1–7

Luna LG (1968) Manual of Histological Staining Methods of the

Armed Forces Institute of Pathology, 3rd edn. McGraw-Hill,

New York, NY

Lynn JC, Chambers CL, Rosenstock SR (2006) Use of wildlife water

developments by birds in southwest Arizona during migration.

Wildl Soc Bull 34(3):592–601

Lynn JC, Rosenstock SR, Chambers CL (2008) Avian use of desert

wildlife water developments as determined by remote videog-

raphy. West N Am Nat 68(1):107–112

MacMillen RE (1962) The minimum water requirements of mourning

doves. Condor 64:165–166

Minerals Council of Australia (1996) Tailings storage facilities at

Australian gold mines. Submission to the Senate Environment,

Recreation, Communications and the Arts References Commit-

tee, Canberra, Australia

Nabhan GP, Holdsworth AR (1999) State of the biome: Uniqueness,

biodiversity, threats and the adequacy of protection in the

Sonoran Bioregion. The Wildlands Project, Tucson, AZ. Cited

in: Lynn JC, Chambers CL, Rosenstock SR (2006) Use of

wildlife water developments by birds in southwest Arizona

during migration. Wildl Soc Bull 34(3):592–601

National Research Council of the National Academies (2005) Mineral

tolerance of animals, 2nd revised ed. National Academy Press,

Washington, DC

O’Brien CA, Waddell RB, Rosenstock SS, Rabe MJ (2006) Wildlife

use of water catchments in southwestern Arizona. Wildl Soc Bull

34(3):582–601

Phillips JC, Lincoln FC (1930) American waterfowl: their present

situation and the outlook for their future. Houghton Mifflin, New

York, NY

Pullar EM (1940a) The toxicity of various copper compounds and

mixtures for domestic birds. Aust Vet J 16(4):147–162

Pullar EM (1940b) The toxicity of various copper compounds and

mixtures for domestic birds. 2. Aust Vet J 16(5):203–213

Puls R (1994) Mineral Levels in Animal Health, 2nd edn. Sherpa

International, Clearbrook, BC

Rattner BA, McKernan MA, Eisenreich KM, Link WA, Olsen GH,

Hoffman DJ et al (2006) Toxicity and hazard of vanadium to

mallard ducks (Anas platyrhynchos) and Canada geese (Brantacanadensis). J Toxicol Environ Health A 69(4):331–351

Read JL (1999) A strategy for minimizing waterfowl deaths on toxic

waterbodies. J Appl Ecol 36:345–350

Read JL, Pickering R (1999) Ecological and toxicological effects of

exposure to an acidic, radioactive tailings storage. Environ

Monit Assess 54:69–85

Rogers DT Jr, Odum EP (1964) Effects of age, sex, and levels of fat

deposition on major body components in some wood warblers.

Auk 81:505–513

Seiler RL, Lico MS, Wiemeyer SN, Evers DC (2004) Mercury in the

Walker River Basin, Nevada and California – sources, distribu-

tion, and potential effects on the ecosystem. U.S. Geological

Survey Scientific Investigations Report 2004-5147

Sheridan TE, Nabhan GP (1978) Who killed the Gila? In: Water in a

thirsty land: Sonoran desert and mountain river history. Pinon

Press, Prescott, AZ, pp 1630. Cited in Lynn JC, Chambers CL,

Rosenstock SR (2006) Use of wildlife water developments by

birds in southwest Arizona during migration. Wildl Soc Bull

34(3):592–601

Sileo L, Beyer WN, Mateo R (2004) Pancreatitis in wild zinc-

poisoned waterfowl. Avian Pathol 32(6):655–660

Smyth M, Coulombe HN (1971) Notes on the use of desert springs by

birds in California. Condor 73(2):240–243


123

http://extoxnet.orst.edu/pips/coppersu.htm

Stratus Consulting, Inc (2003) Preassessment screen for the Chino,

Tyrone, and Morenci Mine Sites, Grant County, New Mexico,

and Morenci, Arizona. Prepared for the United States Fish and

Wildlife Service, Albuquerque, NM

Stubblefield WA, Baroch J, Dressen P, Spraker TR, Getzy DM (1997)

Evaluation of the toxic properties of acid mine drainage water to

snow geese. Abstract No. 009, 18th Annual Meeting Society of

Environmental Toxicology and Chemistry, San Francisco, CA,

November 16–20

Takei Y, Okawara Y, Kobayashi H (1988) Water intake induced by

water deprivation in the quail, Coturnix coturnix japonica.

J Comp Physiol B 158:519–525

Torre-Bueno JR (1978) Evaporative cooling and water balance during

flight in birds. J Exp Biol 75:231–236

United States Environmental Protection Agency (1993) Wild-

life Exposure Factors Handbook. Office of Research and

Development, United States Environmental Protection Agency,

Washington, DC. EPA/600/R-93/187

United States Environmental Protection Agency (1994) Test methods

for evaluating solid waste: Physical/chemical methods. Environ-

mental Protection Agency, Office of Solid Waste and Emergency

Response

Wayne DM, Warwick JJ, Lechler PJ, Gill GA, Lyons WB (1996)

Mercury contamination in the Carson River, Nevada: a

preliminary study of the impact of mining wastes. Water Air

Soil Pollut 92:391–408

Yapp WB (1956) Two physiological considerations in bird migration.

Wilson Bull 68:312–327

Yapp WB (1962) Some physical limitations on migration. Ibis

104:86–89


123

aect_acid mine water_pdf

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