Elemental Levels Analyzed by PIXE in Florida Alligators
J.C. Kuharik, I.I. Kravchenko, F.E. Dunnam, H.A. Van Rinsvelt
Department of Physics, P.O.Box 118440, University of Florida, Gainesville FL 32611
J.P. Ross
Florida Museum of Natural History, P.O.Box 117800, University of Florida, Gainesville, FL 32611
Abstract. Unusual and alarming mortality of alligators (Alligator mississippiensis) has been reported from Lake Griffin,
Florida, where almost 400 dead alligators have been observed since 1997. In addition, the hatch rate for alligator eggs
around Lake Griffin fell below 10% and remains low (30-45%) while the normal hatch rate is typically 80%. Standard
diagnostic methods have been ineffective in determining the cause of the phenomenon. Many possibilities have been
considered including pollutants, nutrition, and toxic algae. Particle Induced X-ray Emission (PIXE) analysis is highly
suitable for investigating concentrations of a wide range of elements in animal tissue. Liver, kidney and spinal cord
tissues from healthy and sick alligators have been analyzed by PIXE for elemental content. Initial results showed
positive correlation between certain elements and neural impairment and morbidity of alligators in Lake Griffin, but
have failed to prove significant.
this study was identification of potentially toxic metals
(lead, mercury, arsenic, tin, cadmium) and results
indicated unremarkable background levels of all
metals tested [2]. The study presented here is an
attempt to use the more sensitive method of PIXE
analysis to identify or eliminate the possibility of a
correlation, whether cause or symptom, between the
alligator mortality and elements within the range of
elements available to PIXE analysis.
INTRODUCTION
An alarming mortality of American alligators
(Alligator mississippiensis) has been reported on Lake
Griffin, Florida. The phenomenon was first observed
in the spring of 1997 when representatives of the
Florida Fish and Wildlife Conservation Commission
conducting population surveys “observed usual
numbers of dead subadult and adult American
alligators”[1]. Symptoms of the disease include
lethargy and unresponsiveness to approach by
humans[1]. The cause remains unknown and a
continuing investigation has yet to yield definitive
results. This study of metals in alligator tissue was
undertaken as part of the larger investigation.
EXPERIMENTAL
Samples were prepared by the following method.
Large pieces (5-12g) of alligator spinal cord, liver and
kidney tissue were first frozen in liquid nitrogen, and
then cut into smaller pieces (approximately ¼”
diameter) using a zirconium ceramic blade and teflon
coated tweezers. The material of the blade and the
teflon coating were utilized to avoid contamination of
the sample from elements found in stainless steel. The
small chunks of tissue were then placed in a freeze
A preliminary investigation of metals in 1998-1999
was conducted by analysis of liver and fat samples
from 8 Lake Griffin and 8 Lake Woodruff alligators by
Inductively Coupled Plasma Argon Emission
Spectroscopy (ICPAES) by School of Veterinary
Medicine University of Pennsylvania. Emphasis of
CP680, Application of Accelerators in Research and Industry: 17th Int'l. Conference, edited by J. L. Duggan and I. L. Morgan
© 2003 American Institute of Physics 0-7354-0149-7/03/$20.00
414
dryer overnight to remove moisture. After moisture
removal, the samples were digested using concentrated
nitric acid (99.999% contaminant free). The digestion
process was performed in a teflon egg placed in a
stainless steel bomb and heated in an oven at a
temperature of 85ºC for a period of 15-20 hours. The
samples were doped with an internal standard of Y2O3
in 5% nitric acid solution to establish levels of yttrium
near 1,000 ppm.
located inside the vacuum chamber, in the horizontal
plane, and making an angle of 135° with the incident
beam direction. The solid angle of the detector was
defined by means of a high purity aluminum
collimator and data were obtained with a “funny”
filter: a 660µm thick Mylar absorber with an axial
aperture of 1% area inserted between the sample and
the detector.
A Kevex 4525P amplifier/pulse
processor was used in conjunction with the detector.
The resolution obtained with the detector-pulse
processor combination was 180eV for the Mn Kα line.
The x-ray spectra were analyzed using the software
Pixfit [3]. A typical PIXE spectrum is shown in figure
1.
Kimfoltm and Isoporetm 0.1µm pore membrane
filters (10µm thick) were used as the backing for the
PIXE targets. The backing materials were chosen
because of their low signature of elements within the
range of inquiry. PIXE analysis of blank targets
revealed only trace amounts of Zn contaminant in the
Kimfoltm and trace amounts of S, Ca, and Fe in the
filter membrane.
Each target was prepared by
dropping 100µl of the sample solution onto the
backing and then dried in a vacuum chamber. Each
selection of digested sample tissue was analyzed 4 or
more times to obtain mean average values with an
estimated error of 15%.
Livers and kidneys from 16 alligators were tested
using PIXE analysis. Initially, spinal cord tissue from
4 alligators was also examined but showed very low
concentrations of only a few elements in the range of
inquiry and for this reason was not investigated
further. The 16 alligators involved in the liver/kidney
study were taken from two different lakes. Five
healthy and seven unhealthy were taken from Florida’s
Lake Griffin. Four healthy alligators from Florida’s
Lake Woodruff, which is considered to be a healthy
ecosystem, served as a control group. Each alligator
was assigned a numerical impairment value using a
subjective evaluation of behavior in the field. A value
of 0-1 indicates normal avoidance behavior, 2-4
indicates increasing levels of impairment and 5
indicates completely moribund. The alligators selected
for this study all had an impairment value of 1, 3, or 4.
The samples were bombarded with a proton beam
of 2.5 MeV generated using a 5 SDH Peletron NEC
tandem accelerator. Before striking the samples the
beam passed through a gold foil to homogenize the
intensity distribution. Total beam intensity was kept
below 35nA to avoid overheating and charge build up
on the sample. The characteristic x-rays were detected
with a 30mm2x3mm thick Kevek Si(Li) detector
FIGURE 1. Sample PIXE spectrum of a liver from an unhealthy alligator.
415
FIGURE 2. Mean average (+/- 15%) elemental concentrations given in ppm for selected elements. Dot shaped data points
represent individual alligators and triangles represent impairment groups.
C u K id n e y
C u L iv e r
1 2 .0
5 0 .0
4 5 .0
1 0 .0
4 0 .0
3 5 .0
ppm Cu
ppm Cu
8 .0
6 .0
4 .0
3 0 .0
2 5 .0
2 0 .0
1 5 .0
1 0 .0
2 .0
5 .0
0 .0
0 .0
0
1
2
3
4
0
5
1
2
3
4
5
Im p a ir m e n t
Im p a ir m e n t
F e K id n e y
F e L iv e r
1 2 0 0 .0
2 0 0 0 0 .0
1 8 0 0 0 .0
1 0 0 0 .0
1 6 0 0 0 .0
1 4 0 0 0 .0
ppm Fe
ppm Fe
8 0 0 .0
6 0 0 .0
4 0 0 .0
1 2 0 0 0 .0
1 0 0 0 0 .0
8 0 0 0 .0
6 0 0 0 .0
2 0 0 .0
4 0 0 0 .0
0 .0
0
1
2
3
4
2 0 0 0 .0
5
0 .0
Im p a ir m e n t
0
1
2
4
5
R b L iv e r
4 5 .0
4 0 .0
3 5 .0
3 5 .0
3 0 .0
3 0 .0
2 5 .0
2 5 .0
ppm Rb
ppm Rb
3
Im p a irm e n t
R b K id n e y
2 0 .0
1 5 .0
1 0 .0
2 0 .0
1 5 .0
1 0 .0
5 .0
5 .0
0 .0
0
1
2
3
4
0 .0
5
0
Im p a irm e n t
1
2
S e K id n e y
4
5
4
5
4
5
S e L iv e r
3 0 .0
1 4 .0
2 5 .0
1 2 .0
2 0 .0
1 0 .0
ppm Se
ppm Se
3
Im p a ir m e n t
1 5 .0
1 0 .0
8 .0
6 .0
4 .0
5 .0
2 .0
0 .0
0
1
2
3
4
5
0 .0
0
Im p a ir m e n t
1
2
3
Im p a ir m e n t
Z n K id n e y
Z n L iv e r
1 0 0 .0
9 0 .0
1 2 0 .0
8 0 .0
1 0 0 .0
6 0 .0
ppm Zn
ppm Zn
7 0 .0
5 0 .0
4 0 .0
8 0 .0
6 0 .0
4 0 .0
3 0 .0
2 0 .0
2 0 .0
1 0 .0
0 .0
0 .0
0
1
2
3
4
0
5
1
2
3
I m p a ir m e n t
Im p a ir m e n t
416
The study is the first to apply PIXE techniques to
the study of alligators.
We have successfully
developed techniques that allow application of PIXE
to a variety of organic samples (liver, kidney, and
spinal cord). The PIXE method is demonstrated to
provide reasonably precise estimates of metals from
the same sample using very small amounts of tissue.
The levels of metals detectable are an order of
magnitude smaller than by other standard techniques.
The benefits of using PIXE for this type of study are
clear. PIXE allows for simultaneous examination of a
wide range of metals in a tissue sample size as small as
30mg. In the search for a cause to an unknown
disease, the importance of being able to use only a
small amount of tissue for analysis cannot be
understated. By utilizing only a small amount of
tissue, PIXE analysis provides a great deal of
information with only a small cost to the total amount
of material available for study. Thus, sample material
can be reserved for future studies or for other types of
analysis.
DISCUSSION
Initially 6 to 9 duplicate analyses were conducted
on several samples to evaluate if increasing the
number of analyses increased precision of the
estimate.
The coefficient of variation (Std
deviation/mean) did not consistently improve as
additional analyses of the same sample were pooled.
Therefore we analyzed four subsets from each sample
and pooled the results to give a sample mean value.
Results of the study are given in figure 2 as pooled
sample means of element concentration for individual
alligators and for impairment groups. The values
obtained for Cu, Fe and Zn are in the same range as
the University of Pennsylvania ICPAES study
mentioned above. In that study selenium was below
the detectable limit in most samples but fell in the
same range of values 1 – 2.5 ppm wet weight
(calculated assuming water content is around 75%).
One published value for selenium in alligator livers is
also comparable, 0.64 + 0.09 ppm wet weight [5]. We
therefore feel confident that the values obtained in our
current study are accurate. Iron values are highly
variable and we suspect that contamination from steel
equipment (scalpels, knives) during necropsy and also
variation in quantities of blood contained in the tissues
may be a factor.
While this study has failed to demonstrate
significant differences in metal concentrations in
tissues between alligators from different lakes and
different health status due to the high variability
between alligators, we believe this to be a natural
property of these biological samples. We further
believe that the technique outlined above clearly has
potential to provide high-resolution analysis for metals
in biological samples for ecological studies.
Initially, our results taken from half of our total
sample group indicated that alligators from Lake
Griffin appear to have depleted levels of selenium in
their liver and kidney. Alligators from Lake Griffin
had significantly lower levels of selenium than
alligators from Lake Woodruff for both liver (t = 2.9
P<0.05, 23 df) and kidney (t= 4.5,P<0.01 df=67).
Impaired alligators had significantly lower selenium in
the kidney than unimpaired alligators (t= 4.2,P<<0.01,
df=58). However the difference in selenium in the
liver (one tailed t= 1.5, P= 0.06, df =66) failed to be
significant at alpha=0.05. None of the other metals
showed significant differences.
ACKNOWLEDGMENTS
The authors would like to thank Adele Luta and
Ralph Kelly for their assistance in developing the
sample preparation procedure used in this study. Also,
the authors would like to thank the University Scholars
Program at the University of Florida for providing
financial assistance to student researchers.
REFERENCES
We examined this effect in more detail by doubling
our sample size with healthy and sick alligators from
Lake Griffin. This expanded data set has not clarified
the apparent trends in metal levels. Increasing the
sample size has increased the sample variance and the
apparent differences between the lakes and between
healthy and sick alligators are obscured by the
variability of the data. While the general trends are
still apparent in the average values, we are unable to
confirm that these differences are robust with this data
set. Additional fine-grained statistical analysis may
allow us to apportion some of the variance to other
factors (e.g. alligator size, sex).
417
1.
Schoeb, T.R., Heaton-Jones, T.G., Clemmons, R.M.,
Carboneau, D.A., Woodward, A.R., Shelton, D.,
Poppenga, R.H., “Clinical and Necropsy Findings
Associated with Increased Mortality Among American
Alligators of Lake Griffin, Florida,” in Journal of
Wildlife Diseases, 38(2), 2002, Wildlife Disease
Association, pp.320-327.
2.
Ross, J. P. “Effect of toxic algae on alligators and
alligator egg development.” Tech Research Report
Project FL 03 to Water Resources Research Center
USGS- University of Florida 2000
3.
Coldwell R.L., Van Rinsvelt, H.A. “Pixfit-A Special
Analysis Program for PIXE” in Application of
Accelerators in Research and Industry – 1997, edited
by J.L.Dugan and I.L.Morgan, AIP Conference
Proceedings 475, New York: American Institute of
Physics, 1997, pp. 555-558.
4.
Burger, J., M. Gochfeld, Rooney, A. A., Orlando, E. F.
Woodward A. R. & Guillette L. J.. 2000. “Metals and
Metalloids in Tissues of American Alligators in three
Florida lakes” in Arch. Environ. Contam. Toxicol.
2000, 38: pp 501-508.
418