United States
Environmental Protection
Agency
Environmental Monitoring
and Support Laboratory
P O Box 15027
Las Vegas NV 89114
EPA 600/7 78 233
December 1978
Research and Development
&EPA
Geothermal Environmental
Impact Assessment:
Procedures for Using
Fauna as Biological
Monitors of Potential
Geothermal Pollutants
Interagency
Energy-Environment
Research
and Development
Program Report
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development. US Environmental
Protection Agency, have been grouped into nine series. These nine broad categories
were established to facilitate further development and application of environmental
technology Elimination of traditional grouping was consciously planned to foster
technology transfer and a maximum interface in related fields The nine series are:
1 Environmental Health Effects Research
2 Environmental Protection Technology
3. Ecological Research
4 Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY—ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the effort
funded under the 17-agency Federal Energy/Environment Research and Development
Program. These studies relate to EPA'S mission to protect the public health and welfare
from adverse effects of pollutants associated with energy systems. The goal of the Pro-
gram is to assure the rapid development of domestic energy supplies in an environ-
mentally-compatible manner by providing the necessary environmental data and
control technology. Investigations include analyses of the transport of energy-related
pollutants and their health and ecological effects; assessments of, and development of,
control technologies for energy systems; and integrated assessments of a wide range
of energy-related environmental issues
This document is available to the public through the National Technical Information
Service, Springfield, Virginia 22161
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EPA-600/7-78-233
December 1978
GEOTHERMAL ENVIRONMENTAL IMPACT ASSESSMENT
Procedures for Using Fauna as Biological Monitors of Potential
Geothermal Pollutants
by
Z. C. Nelson
W. W. Button
A. A. Mullen
W. F. Beckert
G. D. Potter
Monitoring Systems Research and Development Division
Environmental Monitoring and Support Laboratory
Las Vegas, Nevada 89114
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
LAS VEGAS, NEVADA 89114
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring Support
Laboratory-Las Vegas, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
ii
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FOREWORD
Protection of the environment requires effective regulatory actions
which are based on sound technical and scientific information. This infor-
mation must include the quantitative description and linking of P01^'
sources, transport mechanisms, interactions, and resulting effects on man
and his environment. Because of the complexities involved, assessment ot
specific pollutants in the environment requires a total systems approach
which transcends the media of air, water, and land. The Environmental
Monitoring and Support Laboratory-Las Vegas contributes to the formation
and enhancement of a sound integrated monitoring data base through multi-
disciplinary, multimedia programs designed to:
develop and optimize systems and strategies for moni-
toring pollutants and their impact on the environment
demonstrate new monitoring systems and technologies by
applying them to fulfill special monitoring needs of
the Agency's operating programs
This report presents preliminary data on trace element tissue levels,
population characteristics of small mammals and discusses analytical and
field methodology for determining the feasibility of using livestock and
domestic animals as biological monitors of Potential exothermal Plants.
This report is part of an overall program which will combine data on air,
water, soil, flora and fauna in assessing the environmental impact and
design of a monitoring strategy for geothermal resource development. For
further information the reader should contact the Monitoring Systems Exposure
Dose Assessment Branch (MSB) .
George *B. Morgan
Director
Environmental Monitoring and Support Laboratory
Las Vegas
iii
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ABSTRACT
This is the first in a series of reports that covers the feasibility of
utilizing wildlife and domestic animals to design a strategy for assessing the
environmental impact of geothermal resource development. This study is part
of an overall program which will also include data on air, water, soil and
flora.
Animal tissues and animal products were collected in the vicinity of
California and Utah geothermal development sites. These samples are being
assayed for such elements as lead, cadmium, zinc, boron, aluminum and
strontium so as to confirm baseline concentrations in the tissues of area
fauna. Selected samples were analyzed by atomic absorption but most of the
analyses were by optical emission spectrometer, which required freeze-dried,
homogeneously mixed samples. Small mammal population characteristics are
being monitored at Roosevelt Hot Springs, Utah. Those animal species sampled
in the field are being studied under controlled conditions to relate the
ingestion of selected elements to subsequent changes in the elemental concen-
tration of various tissues.
This report presents some preliminary data on trace element concentra-
tions in tissues of wildlife and domestic animals. Concentrations in geother-
mal effluents also were determined. Quality assurance, sample collection,
sample preparation, analytical procedures, and relative abundance of small
mammals are discussed.
iv
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CONTENTS
Foreword ....................... • .....
Abstract ............................. iv
Tables .............................. vi
Introduction ........................... •*•
Trace Element Procedures ..................... 2
Tissue Collection ...................... 2
Laboratory Analysis ..................... 3
Quality Assurance ...................... 3
Census Procedures ......................... 5
Pilot Project ........................... 7
The Study Areas ....................... ?
Trace Element Findings .................... 8
Census Findings ....................... 1?
References ............................ 21
Appendices ............................ 25
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TABLES
Number
1 Outline of Laboratory Studies 6
2 Concentration of Various Elements in Geothermal Fluids. ... 9
3 Elemental Concentrations in Whole Blood 10
A Elemental Concentrations in Carcass, Pelt, and
Gastrointestinal Tracts 11
5 Approximate Distribution of Various Elements in
Kangaroo Rats 13
6 Preliminary Comparisons Between Whole Body Concentrations . . 13
7 Preliminary Data on Elemental Concentrations in Liver
Kidney, and Hair 14
8 Potential Food Preferences for Selected Mammals 18
9 Number of Rabbits/Hares at Roosevelt Hot Springs 19
10 Relative Abundance of Rodents at Roosevelt Hot Springs. ... 19
vi
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INTRODUCTION
The encouraging ecological feature of geothermal energy is that it is
basically a contained system. The effluent rises to a heat exchanger and,
in many cases, would be reinjected down a second well without exit to the
surface atmosphere. Environmental damage therefore should be slight. None-
theless, trace amounts of toxic elements frequently precipitate out of steam
condensates and under some conditions might be transported to surface waters
or dispersed as aerosols. The effect of geothermal development on area
animal life is largely unknown. The delicate nature that comprises a native
environment can be altered by changes in the elemental flow through an
ecosystem, by destruction of habitats, human presence and activity, etc.
(Herman, 1975). To assess any possible impact and to help design a monitoring
strategy for geothermal development, baseline elemental tissue levels are
being collected and population parameters are being monitored.
This report is the first in a series that will lead to the development
of a strategy for monitoring any pollutants to which people might be exposed
as a result of geothermal generation. An EMSL-LV team has been investigating
the use of wildlife and livestock as biologic monitors and presents herewith
a progress report on the first year of research.
The major contribution thus far is the evolution of procedures for
determining levels of trace elements in fauna and wildlife population studies,
with the emphasis on census, food habits and habitat associations.
The field work was done at geothermal project sites in the Imperial
Valley of California and at Roosevelt Hot Springs in Utah. Trace element and
census data appear here in their most preliminary form. Later reports on
faunal monitors will fill in the gaps, with a view to establishing baseline
data, on elements and population, against which later surveys, reflecting the
impact of the power plants, can be compared. The objective is to set the
baselines before industrial development alters the indigenous picture. How-
ever, as this manuscript is written, the fauna team has made significant
progress on quality procedures for trace element analysis and on small mammal
population surveys.
Animal tissues and products have been collected at both the California
and Utah sites and are undergoing assay in the effort to establish baseline
concentrations of elements, such as lead', cadmium, zinc, boron, aluminum,
arsenic and strontium. Small mammal population is being monitored at the
Utah site.
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TRACE ELEMENT PROCEDURES
TISSUE COLLECTION
^mp\in8 has been conducted at Imperial Valley on a quarterly
l* C°^leCti°nS consist of Primarily cattle hair and blood with a
nonev LmnT V P°? I ^V^*' Resident waterfowl, fish and occasional
cattle art™ M^ ^SVTn '°llected °n ™ intermittent basis. Since new
Tonlllr f "°nt^Ually b*ln8 Delivered to Imperial Valley the traditional
i v ?f el*ne *an>Plin8 ha* included cattle recently delivered to the
efrtab yeeT P°Ultry SampleS W6re included in the collection
effort because the birds were resident animals. It is expected that dietary
with T ?H SeenUm' flU°rine' mang— -d mercury ould be correted
'
The California collections have also included samples from a locally
nesting water bird, the American Coot (FuUca americana) . Samples of muscle
±!Ti; f' ^' ^ Spleen' kidney' lung' claws> and f«thers have '
been taken from a few birds. Coots are omnivorous and much of their food is
obtained from, or near, the water where aquatic vegetation, aquatic insects
crustaceans, and small fish contribute to their diet. However, flocks of- '
facility6 °CCaSl0nally Si8hted on cultivated fields near a geothermal test
Collections at Roosevelt Hot Springs, Utah have been made on a seasonal
basis to acquire wildlife samples and twice yearly for domestic animal samples.
Samples of hair/wool and blood from sheep and cattle were made in the spring
and fall, seasons that coincided with the roundup and movement of animals
between winter and summer ranges. Rodents were collected in museum special
snap traps, and roadside kills were taken in the case of rabbit collections.
Tissues or organs taken from each animal have included bone, hair, lung liver
kidney and in some cases blood. '
Cattle and sheep samples were sequentially numbered during each collection
period and identified with the date, livestock owner and location of the
feedlot or grazing allotment area. The location, approximate size and total
grazing time for both summer and winter ranges were obtained for Utah live-
stock and, when possible, the geographic origin and time of arrival was
acquired for cattle in the California feedlots. Poultry samples were also
numbered and identified as to owner, location and collection date. Rodents
and lagoraorphs were identified (species), weighed, sexed and, during the
more recent trapping periods, selected body measurements and the reproductive
condition were recorded. During the respective collection efforts, hair or
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wool was clipped from the animal's side and sealed in polyethylene bags.
Blood samples were collected by jugular venipuncture in heparinized syringes
or in heparinized vacutainer tubes and wildlife carcasses were sealed in
polyethylene bags. All samples were transported back to Las Vegas in ice-
filled cooler chests.
Muscle and fat were scraped from the bone samples and the hair samples
were cleaned by the method of Clarke and Wilson (1974). Surface contamina-
tion was removed by washing the hair in separate solutions of soap, acetone
and ethylenediaminetetraacetic acid (EDTA): Wet weights were recorded for
all sample types and the collected material was either freeze-dried (blood,
egg white and yolk, and some rabbit tissues) or oven-dried (rodent tissue,
all hair and bone samples) at 60°C. Dry weights were recorded and the'
individual samples transferred to acid washed polyethylene vials and stored
under refrigerated conditions. The dried samples were subsequently ground
with a cryogenic mill or manually mixed with a Teflon spatula or an agate
mortar and pestle. All laboratory ware, mostly polyethylene, was washed with
dilute nitric acid and followed by a deionized water wash. Stainless steel,
polyethylene and Teflon spatulas, surgical instruments, etc. were cleaned in
a similar fashion. Detailed procedures on sample preparation are described
in Appendix A and B.
LABORATORY ANALYSIS AND QUALITY ASSURANCE
Flame and flameless atomic absorption spectrophotometry, optical emission
spectroscopy and x-ray spectrometry have been used at EMSL-LV for various
projects involving elemental analysis. Additional techniques include neutron
activation, spark source mass spectrometry, pulse polargraphic methods and
anodic stripping voltammetry. A summary comparison of these methods has been
presented by Lisk (1974). In the current study, DC arc optical emission
spectroscopy and flame atomic absorption spectrophotometry were the analytical
tools utilized.
Optical emission spectroscopy has a simultaneous multi-element capability.
The technique is precise, rapid, and adaptable for solid or liquid samples.
The sample is vaporized, usually via electrical discharge, and a fraction of
the atoms are excited to unstable energy levels. When the atoms revert to
their stable states, they emit the absorbed energy as characteristic optical
spectra. A spectrograph disperses the emitted radiation and individual
detectors record the intensities of the spectral lines of interest. These
intensities are related to the concentrations of the elements in the sample.
In our case, 26 elements may be detected simultaneously through standard DC
arc optical emission techniques. The relative standard deviation of this
analytical procedure has been reported at between 3 and 15 percent for finely
ground material with acceptable accuracy above 1 ug/g for most elements
(Alexander et al., 1975).
When solid samples are analyzed in this way, homogeneity is a prerequi-
site since non-homogeneous elemental distribution results in interpretation
errors that cannot be completely eliminated by replicate analyses. An effi-
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cient sample-mixing process is therefore essential, and for our determinations
a cryogenic Tekmar mill (stainless steel chamber and carbide cutting blades)
served well. Liquid standards for optical emission spectroscopy are relative-
ly easy to prepare and use. However, acceptable synthetic standards are very
difficult to prepare from undigested ground material with a uniform elemental
distribution. Dried tissue standards of known elemental concentrations were
therefore acquired from the National Bureau of Standards, and both subsamples
and field-collected material have been routinely analyzed. Furthermore, a
collection of eggs, blood, and liver have been freeze-dried and stored for
repeated assays. The Laboratory of Nuclear Medicine and Radiation Biology,
UCLA conducted three separate analyses on all samples and standards-
Atomic absorption spectrophotometry is a fast, sensitive, and precise
analytical technique; however, it does not have a simultaneous multi-element
capability. A hollow-cathode lamp (one for each element to be analyzed) emits
light of the characteristic frequencies of the element of interest. This
light passes through a flame (acetylene or hydrogen plus air or nitrous oxide)
into which the sample solution is introduced by a nebulizer and the sample
atom, excited by the flame, absorbs part of the radiation emitted by the
hollow-cathode lamp. The resulting weakened light beam goes through a high
resolution spectrometer. The isolated line, characteristic for the element
under investigation, is directed to a photomultiplier tube where the light is
transformed to an amplified electric signal. About 70 metallic elements can
be determined by atomic absorption spectrophotometry.
The samples to be analyzed must be in solution form, meaning tissue
samples must be digested. This handling increases the possibility of contami-
nation and partial loss of sample constituents. Samples undergo wet oxidation
prior to atomic absorption assays. The vast majority of wet oxidation proce-
dures use some combination of four reagents: sulfuric acid, nitric acid,
perchloric acid and hydrogen peroxide. We chose nitric acid since it has
been widespread in its usage. The initial nitric acid digestions conducted
for this study were time consuming and therefore, a commercial microwave oven
has been adapted (Abu - Samra et al., 1975) as a faster alternative. Oven
adaptations included an internal Pyrex liner, exhaust openings at the rear
of the oven and an attached scrubber and exhaust fan to control the acid
fumes. Details of the procedure are given in Appendix I.
Because the material to be analyzed is in solution, the preparation of
standards presents few problems. From EMSL-Cincinnati, standard quality
control solutions have been obtained which contain 15 elements at yg/1
concentrations and which are suitable for dilution to the required concen-
trations. In our work duplicate determinations are usually conducted and
the linearity, which is assurred by instrument components, of the calibration
curve (absorbance versus concentration) is checked and recorded daily. A
blank or solvent is analyzed between each set of 10 samples or standards to
verify baseline stability. These control solutions do not contain sample
residues, but do contain the same digestion reagent residues present in the
regular samples. A few samples were spiked at different concentrations after
sample digestion so that an analytical recovery estimate could be made. At
present atomic absorption analyses are in initial stages and only preliminary
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unevaluated data for arsenic have been obtained. Future analyses will include
other elements and use of standards from EMSL-Cincinnati. The effect of
storage time, container type and grinding technique is being determined with
spiked and unspiked samples. Nonetheless, when sample preparation and analysis
time and overall reagent costs are considered, emission spectroscopy appears
preferable for most routine assays.
Two additional studies have been initiated as part of this program. Those
animal species sampled in the field will be studied under controlled conditions
in an attempt to relate the ingestion of selected elements to subsequent
changes in the elemental concentration of various tissues. Pre- and post-
exposure tissue concentrations will be emphasized. Rodents, rabbits, chickens,
sheep and beef cattle are major representative species in the field sampling
program and have been the main candidate animals for this experimental series.
During 1977 rodents and rabbits, initiating the series, were administered
either oral doses of concentrated geothermal effluents or, in some cases,
single elements of particular interest. The rodents had been collected in
the field, but domestic rabbits were substituted for field lagomorphs because
jackrabbits are difficult to capture and use under laboratory conditions. The
animals were serially sacrificed, for tissue collection, following acute or
chronic exposures, as summarized in Table 1. The results, when completed,
will help determine which tissues and/or bodily divisions might best reflect
environmental exposure. Because of interrelated work, currently in progress
through a grant managed by EMSL-LV, as well as numerous publications on some
of the elements, these laboratory studies have not been extensive. However,
the experiments have been an essential part of the quality assurance effort
inasmuch as the laboratory-generated samples have been analyzed by the same
techniques as those used for the field-collected tissues.
CENSUS PROCEDURES
The relative abundance of lagomorphs was determined through nighttime
observations, and all rabbits and hares visible from the road by spotlight
were recorded. The data are presented as actual counts per kilometer
travelled.
Live traps, baited with rolled oats and placed along census lines, were
used to collect rodents for identification and determination of their relative
abundance at Roosevelt Hot Springs. Two parallel lines were laid out 50
meters apart and 240 meters long each containing 25 Sherman traps at 10-meter
intervals. Two sets of parallel census lines were set at each location
yielding 100 traps. One pair of such lines was 200 meters west of a primary
drilling site and another was about four miles south. Trapping went on for
two days and three nights and, following trap inspection, each captured animal
was identified, sexed, examined for reproductive condition, marked by toe
clipping and released. Results on relative abundance were based on the number
of recaptures and presented as the expected population (Lincoln, 1930; Cockrum,
1962). A similar trapping program is to begin shortly in the Imperial Valley,
California.
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TABLE 1. OUTLINE OF LABORATORY STUDIES CONDUCTED TO DETERMINE ELEMENTAL CONCENTRATIONS
IN TISSUES OF SMALL MAMMALS AT ROOSEVELT HOT SPRINGS
No. of Orally
Dosed Dosing
Animal Animals Material
Desert woodrat
Desert woodrat
Domestic rabbit
9 As203
9 CdCl2
12 As203
ge*
Cumulative
No. of Dose per
Doses Animal
3 600 pg
3 600 yg
3 3.33 mg
1.33 ml
No. of Number and Types of Samples
Control
Animals Blood Liver Kidney Bone
8 17 17 17 17
8 17 17 17 17
8 20 20 20 20
Unwashed
Hair
9
9
20
Gastrointestinal
Pelt Carcass Tract
Desert woodrat
Ord's kangaroo
rat
Laboratory rat
9 CdCl2
6 As203
ge*
6 As203
ge*
3 1.20 mg
10 1.50 mg
2.5 ml
10 1.50 mg
2.5 ml
7 16 16 16
A 10 10 10
7 13 13 13
*In these experiments the arsenic dose was added to geothermal effluent, and the amount of arsenic
spike as well as the volume of effluent administered per animal is shown.
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PILOT PROJECT
STUDY AREAS
Imperial Valley encompasses about 1,700 square miles including 500,000
acres of irrigated farm land where crops are produced throughout the year.
Major vegetable crops include lettuce, onions, melons, tomatoes, and carrots,
while alfalfa, wheat, sugar beets, and cotton are among the prominent field
crops. The valley is also a major cattle-feeding area. Many of the beef
animals are transported great distances to the Imperial Valley feedlots from
Texas, Mississippi or Louisiana. Cattle arrive weighing about 400 pounds and
are processed initially in "calf lots" before transfer to a main lot or leased
pasture plots. One complicating factor in feedlot sampling is that these
animals may graze periodically in several places over the valley. East of
the agricultural fields is a sandy, desert creosote bush (JLcccvea tridentata)
community which serves as the wildlife study plot near East Mesa.
In Imperial Valley, geothermal development is more advanced than at
Roosevelt Hot Springs. Drilling has occurred at several locations including,
Heber, Brawley, East Mesa, Westmoreland and a test facility has been constructed
at Niland which is investigating the use of concentrated brines.
Roosevelt Hot Springs is in southwest Utah on land managed largely for
grazing cattle and sheep, although on irrigated acreage, at considerable
distances from the geothermal resource activity, alfalfa, corn, potatoes and
cereal crops are produced. There are also some dairy associations in the
Minersville ar^ea. Water availability for range animals is a problem and some
grazing allotments must have additional water delivered.
The 40-square-mile geothermal development site ranges in elevation from
about 5200 to 7700 feet northeast of Milford on the western side of the Mineral
Mountain Range. These mountains, extending in a north-south direction, not
only are the dominant geographic feature but also have an obvious effect on
the watershed and climatic conditions within the study area. Sloping foothills
west of the mountains extend out toward the relatively flat Escalante Desert.
The geothermal development at Roosevelt Hot Springs is in the exploratory
stage. Some test wells have been drilled but no power plants have been
constructed.
There are four basic vegetative communities within the Utah geothermal
study area (personal communication K. Brown). Dominant plant species for
the two major communities are the black sage (Artemesia nova) and the pinyon-
juniper association (Pinus edulis and Juniperus osteosperma). Relatively
deep sandy soil along a large wash supports big sagebrush (Artemeaia tridentata)
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and big rabbit brush (Chrysotharmus nauseosus) communities. In addition to
these four major plant groupings, there is a small restricted greasewood
community (SaPcobatus vermimlatus) within the study area. The overall area
is basically semiarid with an annual precipitation of approximately eight
inches and with large annual and daily variations in temperature. However,
localized thunderstorms can be quite severe, and the annual precipitation
generally increases with increasing elevation.
At both study areas, the wells that have been drilled are being tested
prior to completion of commercial plant facilities.
The effluent being extracted during these activities contains many
chemical elements. The main mineral constituents are the bicarbonates,
carbonates, sulfates and dichlorides of sodium, potassium, magnesium and
calcium. The proportions of these minerals vary with the location and depth
of the wells, where the water also contains varying amounts of less common
elements, some of them at levels that cause environmental concern. Table 2
presents data on the concentration of various elements in geothermal brine
for Roosevelt Hot Springs and Imperial Valley sites.
Chemical elements tend to circulate through characteristic pathways
within an ecosystem and travel between the abiotic component and the living
organisms. The ecosystem, as a functional unit, has evolved in such a way
as to remain viable and stable within the context of these cycles. However,
human activities greatly accelerate the movement of many elements, and the
cycles can become overloaded or irregular, changes that are obviously important
for biologically essential elements but less apparently critical, but of
concern, for non-essential elements if there is an immediate or long-term
detrimental effect.
TRACE ELEMENT FINDINGS
Some of the 1977 collection-year tissue assays have been completed for
cattle blood and for selected wildlife species. Elemental concentrations in
the whole blood of Imperial Valley cattle are shown in Table 3. Concentrations
in the carcass, washed pelt, unwashed gastrointestinal tract and whole body
(extrapolated) are shown in Table 4 for kangaroo rats captured at Roosevelt
Hot Springs. Percentages of the total body amounts found in each of these
divisions are shown in Table 5 and some comparisons in dry weight concentration
between kangaroo rats and laboratory rats appear in Table 6. The elemental
concentrations in liver, kidney and hair tissue from Great Basin pocket mice
collected at Roosevelt Hot Springs are shown in Table 7. All analytical data
are considered preliminary and will be compared with the results from ongoing
assays, future collections, analysis and evaluation of quality assurance data.
The information will be summarized ultimately as baseline concentrations
for the sentinel animals. It should be pointed out, however, that, while
blood is relatively easy to collect, it is affected by a variety of homeostatic
mechanisms, and elemental concentrations in it may tend to remain constant.
This homeostasis permits the body's internal environment to remain constant
8
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TABLE 2. CONCENTRATIONS OF VARIOUS ELEMENTS IN GEOTHERMAL FLUIDS
AT ROOSEVELT HOT SPRINGS AND IMPERIAL VALLEY
Element
Aluminum
Arsenic
Barium
Boron
Chromium
Cobalt
Copper
Iron
Lead
Silicon
Strontium
Tin
Zinc
Roosevelt
Hot Springs
(Ug/ml)*
0.32
-
trace***
-
trace
trace
trace
7.2
0.5
150.0
0.8
trace
0.1
Niland
California
(Mg./g)**
-
-
250.0
390.0
-
-
-
2000.0
80.0
-
440.0
-
500.0
Imperial Valley, Calif.
"worst case composite"
(UR/*)**
-
15.0
570.0
-
1.80
0.40
10.0
4200.0
400.0
-
740.0
0.65
970.0
* In-house analysis of one site by optical emission
** From Schieler, 1976, compiled from several sources
*** "trace" is less than 0.1 yg/ral
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Table 3. ELEMENTAL CONCENTRATIONS IN WHOLE BLOOD OF IMPERIAL VALLEY
CATTLE DURING MAY 1977 (Mean values expressed in yg/g
wet weight; standard deviation and ranges are also shown).
13 Adult 20 New-
Element Feedlot Animals Arrival Calves
„. 3.82 ± 0.9 3.92 ± 0.5
Zinc
(2.64 - 6.25) (2.74 - 5.18)
1.16 ± 0.2 1.04 ± 0.3
Copper
(0.80 - 1.46) (0.68 - 1.92)
457.90 ± 75.1 345.16 ± 46.8
Iron
(312.62 - 558.64) (237.96 - 421.11)
An . 1.20 ± 0.2 1.06 ± 0.5
Aluminum
(0.93 - 1.52) (0.74 - 3.08)
0.52 ± 0.1 0.46 ± 0.1
Boron
(0.33 - 0.68) (0.21 - 0.65)
c... 7.81 ± 1.1 7.04 ± 1.6
Silicon
(6.06 - 9.33) (4.92 - 12.08)
C4. ... 0.21 ± 0.0 0.25 ± 0.0
Strontium
(0.18 - 0.28) (0.18 - 0.32)
n . 0.70 ± 0.1 0.64 ± 0.2
Barium
(0.56 - 0.92) (0.31 - 1.06)
r , . 0.74 ± 0.1* 0.58**
Cadmium
(0.67 - 0.82)
* Values based on three animals, others <3.0 wg/g dry weight
** Value for one animal, others <3.0 ug/g dry weight
Concentrations for lead were <1.0 pg/g dry weight
10
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Table 4. ELEMENTAL CONCENTRATIONS IN CARCASS, PELT, AND GASTROINTESTINAL TRACT FROM
KANGAROO RATS (Dipodomys ordii) COLLECTED AT ROOSEVELT HOT SPRINGS
DURING MAY 1977 (Mean values expressed in ug/g wet weight;
standard deviations and ranges are also shown).
Element
Carcass
Pelt
Unwashed
G.I. Tract
Extrapolated
Whole Body
Zinc
Copper
Iron
Aluminum
Silica
Strontium
Barium
42.61 ± 11.6
(28.86 - 38.36)
2.47 ± 0.3
(2.07 - 3.00)
76.42 ± 9.81
(60.57 - 96.93)
5.15 ± 1.5
(3.59 - 8.17)
11.47 ± 3.0
(6.83 - 14.63)
12.80 ± 4.9
(4.79 - 19.76)
4.26 ± 0/8
(2.74 - 5.43)
10.18 ± 2.4
(7.63 - 14.73)
3.99 ± 0.6
(3.05 - 4.85)
136.65 ± 36.1
(60.89 - 184.01)
103.03 ± 36.1
(43.63 - 163.52)
202.79 ± 75.8
(76.80 - 332.29)
3.31 ± 2.6
(1.24 - 10.19)
1.94 ± 0.9
(0.73 - 4.21)
42.48 ± 13.5
(25.77 - 68.28)
5.31 ± 1.4
(3.29 - 7.41)
116.12 ± 54.3
(60.00 - 184.12)
32.72 ± 24.4
(7.05 - 65.00)
254.44 ± 231.5
(77.48 - 874.76)
2.32 ± 1.1
(0.93 - 3.89)
3.75 ± 1.4
(2.31 - 7.15)
35.96 ± 8.3
(26.94 - 45.34)
3.05 ± 0.4
(2.41 - 3.84)
91.75 ± 13.8
(75.91 - 118.86)
27.17 ± 9.3
(16.49 - 43.92)
71.06 ± 28.8
(47.72 - 132.81)
9.98 ± 3.4
(4.18 - 15.07)
3.75 ± 0.7
(2.56 - 4.64)
-------
Cont. Table 4. ELEMENTAL CONCENTRATIONS IN CARCASS, PELT, AND GASTROINTESTINAL TRACT FROM
KANGAROO RATS (Dipodomys ordii) COLLECTED AT ROOSEVELT HOT SPRINGS
DURING MAY 1977 (Mean values expressed in Pg/g wet weight;
standard deviations and ranges are also shown).
Element
Carcass
Pelt
Unwashed
G.I. Tract
Extrapolated
Whole Body
Lead
Cadmium
Magnesium
Manganese
Boron
***
648.66 ± 89.2
(475.59 - 775.05)
0.58 ± 0.2
(0.37 - 0.94)
****
1.19 ± 0.9
(0.51 - 2.53)
*
112.28 ± 40.0
(75.31 - 217.68)
1.86 ± 0.7
(1.06 - 3.27)
2.55 ± 4.2
(0.15 - 14.40)
***
778.15 ± 225.3
(500.44 - 1142.97)
29.20 + 10.3
(18.65 - 47.89)
1.13 ± 0.4
(0.62 - 2.00)
0.24 ± 0.2**
(0.10 - 0.54)
***
543.72 ± 71.4
(399.39 - 640.13)
3.37 ± 1.1
(1.73 - 4.57)
0.605 ± 0.8
(0.10 - 2.90)
* Undetectable
** Values for four animals
*** Concentration <3.0 yg/g dry weight
**** Concentration <2.0 yg/g dry weight
-------
Table 5. APPROXIMATE DISTRIBUTION OF VARIOUS ELEMENTS IN KANGAROO RATS
CAPTURED AT ROOSEVELT HOT SPRINGS DURING 1977 (Amounts
expressed as percentages of the whole body total
amounts in carcass, pelt and gastrointestinal tract)
Element
Aluminum
Silicon
Barium
Iron
Zinc
Boron
Carcass
14
12
81
60
83
-
Pelt
76
60
10
29
6
70
Unwashed
G.I. Tract
10
28
9
11
11
30
Table 6. PRELIMINARY COMPARISONS BETWEEN THE WHOLE BODY ELEMENTAL
CONCENTRATIONS FOR KANGAROO RATS AND LABORATORY RATS (Mean
values expressed in yg/g of dry weight ± two standard errors)
Animal Zinc Aluminum Strontium Boron
Kangaroo 112.65 ± 20.9 84.42 ± 19.5 31.44 ± 7.9 11.65 ± 1.6
Rats
Laboratory 8fi fi ± fi>5 69>8g ± fi 2 4>gA ± Q g 3>Q7 ± Q<4
Rats
13
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Table 7. PRELIMINARY DATA ON ELEMENTAL CONCENTRATIONS IN LIVER, KIDNEY
AND HAIR FROM GREAT BASIN POCKET MICE (Perognathus parvus) COLLECTED
AT ROOSEVELT HOT SPRINGS DURING 1977 (Mean values expressed in ug/g
wet weight for liver and kidney and in ug/g dry weight for hair;
standard deviations and ranges are also shown).
Element
Liver
Kidney
Hair
Zinc
Copper
Boron
Aluminum
Strontium
Lead
Cadmium
17.27 ± 4.9
(8.89 - 23. 64)
4.83 ± 0.8
(3.41 - 5.45)
6.26 ± 4.0
(0.09 - 12.73)
22.46 ± 22.9
(8.51 - 56.62)
0.61 ± 0.2
(0.31 - 0.84)
**
9.75 ± 4.8 67.36 ± 58.7
(4.66 - 19.91) (16.15 - 162.50)
5.44 ± 2.1 19.84 ± 4.0
(2.04 - 9.41) (15.20 - 25.10)
9.36 ± 5.9 21.46 ± 10.2
(2.03 - 25.63) (10.10 - 33.20)
12.12 ± 12.5 852.00 ± 584.6
(1.56 - 39.45) (237.50-1700.00)
0.47 ± 0.3 13.14 ± 9.9
(0.17 - 1.30) (2.00 - 28.30)
* 2.93 ± 0.7
(2.20 - 3.60)
** **
* Undetectable
** Values <3.0 vg/g dry weight
14
-------
at all times. For example, during periods of shortages of needed requirements,
a combination of factors such as increased assimilation, restricted excretion
and/or release of material from storage sites allows the blood and the tissues
to maintain normal physiological levels. Opposite phenomena occur during
periods of excess intake: less assimilation, increased excretion and shunting
of material to storage sites.
Homeostatic control routes by which ruminants adapt to varying intakes
of calcium, magnesium, sodium, potassium, chlorine, iron, zinc, copper, iodine,
cobalt, manganese, molybdenum, selenium, fluorine, nickel and cadmium have
been summarized by Miller, 1975. These control routes include changes in
the percentage of elemental uptake from the gastrointestinal tract, in the
amount of an element excreted and in the elemental deposition in certain
tissues. In the case of hair, elemental components have been sequestered
from the metabolic processes and therefore are not part of a fluctuating
elemental pool. Furthermore, the keratinous (fibrous protein) outer structure
of each hair is resistant to chemical alteration. Results from hair analysis
will be used to establish baseline concentrations and, in the case of rodent
hair, to determine whether the elemental concentrations in hair reflect
elemental concentration in the body organs. Hair has been reported to reflect
environmental exposure and bodily stores (Hammer et al., 1971; Corridan, 1974;
Hopp, 1976), although others (Huckabee et al., 1972) have not supported this
correlation. It is hoped that further analyses will determine if blood, hair
and other organs are feasible as indicators of environmental exposure.
An essential part of evaluating elemental tissue levels is an understanding
of what is known about selected elements of interests (i.e., absorption,
distribution, retention etc.). At present the elements of major interest for
this study are aluminum, arsenic, boron, cadmium, lead, strontium and zinc.
Aluminum has not been shown to be an essential element although Underwood
(1977) speculated that it may be involved in cellular energy metabolism. It
is poorly absorbed, so that a two-fold increase in oral exposure does not
significantly affect retention (Underwood, 1977), and it is excreted mainly
in the feces. In humans the highest levels are found in the lung and skin,
and it is completely turned over after 30 days (Tipton and Cook, 1963; Tipton
et al., 1966). Wild rats absorbed 38% of ingested aluminum (Kaufman et al.,
1976). When high doses of aluminum are given to rats (200 mg/kg), excretion
via urine increased and body retention was mainly in the liver, testes and
bone (Underwood, 1977).
Arsenic, a non-essential, toxic element is widely distributed in the
body (Underwood, 1977). Body retention varies based on chemical form. Rats
retained 11.4% of naturally occurring arsenic, 14.8% of As03 and 18.9% of
As205 (Coulson et al., 1935; Morgareidge, 1963). Hair and nails are select
places for arsenic determinations since they serve as major deposition sites
(26% and 15%, respectively, of total body burden), but they also act as
excretory routes and may not reflect body stores (Peoples et al., 1975);
however, they do accumulate the element over long periods of exposure (Shapiro,
1967). Other storage sites include liver, heart, skin and spleen (Peoples,
15
-------
1964, 1975). Absorbed arsenic is excreted at the rate of 74.6%, mainly via
urine (Coulson et al., 1935).
Boron is an essential element for higher plants but not for animals
(Underwood, 1977). Boron is found throughout the human body in levels between
0.5 f.o 1.5 Mg/g dry weight with highest levels in bones. Ingestions of large
amounts of boron (boric acid) reportedly leads to increased levels in the
brain (Underwood, 1977). In humans, it was found that over 30 days boron was
more than completely turned over (Tipton et al., 1966). In normal adults the
major sites of deposition were found to be the air passages, brain and liver
(Tipton and Cook, 1963).
Cadmium is a non-essential toxic element, which lacks an effective
homeostatic control. It has a reported half-life of 33 years in humans
(Underwood, 1977). Intestinal absorption varies slightly between species,
but most studies indicate assimilation of 2%-10% of a given dose (Friberg
et al., 1971; Fleischer et al., 1974), with an average of 6% (Fassett, 1975).
This is in contrast to 40% reported for respiratory retention (Fassett, 1975).
Retention of cadmium has been estimated at 3%-8% over a 50-year period for
humans (Friberg et al., 1971) and as low as 1% over 1 year for rats (Fleischer
et al., 1974). Two-thirds of cadmium absorbed is found in the liver and
kidney (Underwood, 1977), and some is deposited in hair. Excretion is mainly
via the feces while urinary excretion accounts for only about 1% of absorbed
dose (Friberg et al., 1971).
Lead, another non-essential, toxic element is absorbed at different rates
based on speciation and age: sheep and rabbits 1.3% of oral dose (Baxter,
1950), adult rats 4.7% (Forbes and Reina, 1972), and man 5%-10% (Underwood,
1977). In humans 90% is retained in the skeleton, but high lead intake raises
levels in the liver, kidney and hair. Lead is excreted slowly via bile and
feces. In cattle the excretion is 91%-97% in the feces and 1.3%-2.4% in the
urine (Underwood, 1977).
Strontium absorption varies with animal species: wild rats 33% of digested
ash free food (Kaufman et al., 1976), while rabbits absorbed 13.5% of oral dose
(Lloyd, 1967). The average absorption rate in animals is 5%-25% (Underwood,
1977), and 99% of retained strontium is found in the bone (Underwood, 1977).
The strontium deposited in adult soft tissues is concentrated in the intestinal
tract, aorta and larynx (Tipton and Cook, 1963). Absorbed strontium is excreted
mainly via urine.
Zinc is an essential trace element in animals as part of many metallo-
enzymes. Its assimilation and excretion is homeostatically controlled. The
uptake of zinc varies from 34% of digested ash free food in wild rodents to
30% of dose in cattle (Miller, 1969; Kaufman, 1976). In cattle, 40% of orally
administered 65Zn was retained after 10 days (Miller, 1969). The highest
levels of zinc are found in the hair, bone, skin, liver and kidney (Miller,
1969; Underwood, 1977). Zinc is excreted (70%) via the feces (Underwood, 1977).
16
-------
CENSUS FINDINGS
Monitoring the population parameters of small mammals has been added to
this study to include some indication of ecosystem stability. Characteristics
to be studied include food preferences, habitat association, species compo-
sition, and abundance. Rodents were selected for primary emphasis in the
census due to their expected abundance, ease of collection and importance in
the food web (food source for snakes, hawks, owls, coyotes, foxes, etc.) as
well as their own particular food preferences (Table 8).. Related studies are
in progress on the vegetative characteristics at Roosevelt Hot Springs. Most
studies involving censusing (estimate of abundance) utilize removal, non-
removal or non-trapping (e.g. direct observation) techniques. Non-removal
trapping, involving a mark and release procedure, was selected to accomplish
the study objectives (Lincoln, 1930; Hayne, 1949). This approach assumes
that every marked animal will become randomly re-distributed in the population
and will have the same probability of future capture as an unmarked (not
previously captured) animal. The method further assumes that, during an
individual trapping effort, dramatic changes (death, migration, immigration,
etc.) are not occurring.
Some changes in the animal population probably will be noted that are
unrelated to the geothermal operation. Even if the species within a community
remain relatively stable, their absolute numbers frequently will fluctuate.
Cyclic fluctuations can result from seasonal reproductive patterns that
themselves are not identical from year to year. Furthermore, long-term fluc-
tuations will be virtually impossible to predict during this study period,
and no effort is currently being made to estimate potential changes in the
occurrence or transmission of pathogens, changes in parasitic infestation or
potential hazards to long-term reproductive success.
Relative abundance determinations for lagomorphs have indicated a popu-
lation decrease from June to July (Table 9). The majority of animals sighted
were blacktailed jackrabbits (Lepus californicus). Unless a relationship
between roadside populations and the general population can be established,
these data can be used only to reflect fluctuations from one census period
to another. Estimating rabbit and hare populations by the roadside count
technique has been a common census method (Lewis, 1970), although livetrapping
(Edwards and Eberhardt, 1967) and pellet plots to establish indirect counts
(Adams, 1959) have been employed under various conditions.
Both rodent census sites at Roosevelt Hot Springs were located along
the margins of pinyon-juniper communities that, to some extent, represented
vegetative transition zones. Table 10 presents relative abundance data for
rodents associated with sagebrush and sagebrush-juniper communities as well
as a composite value for animals captured in both plant groupings.
The numerical decrease noted for July might suggest a seasonal trend
but these limited observations do not allow for a definitive statement. Col-
lections made using the census line approach will provide the relative infor-
mation but not a density estimate (number of animal per unit area). To
determine density, the effective trapping area must be known, and this area
17
-------
Table 8. POTENTIAL FOOD PREFERENCES OF SELECTED MAMMALS
POSSIBLY OCCURRING AT ROOSEVELT HOT SPRINGS
Animals
Percentage of-Total Diet
Seeds Vegetation Insects Vertebrates
Blacktailed Jackrabbit
Lepus califomicus
Antelope Ground Squirrel
Ammospermophilus leucwrus
Desert Woodrat
Neotoma lepida
Merriam's Kangaroo Rat
Dipodomys merriami
Long-tailed Pocket Mouse
Perognathus formosus
Little Pocket Mouse
Perognathus longimembris
Deer Mouse
Peromysous maniculatus
Brush Mouse
Peromysaus boylii.
Pinyon Mouse
Peromysaua truei
Pinyon Mouse
Peromysaus
1.0
45.5
1.0
82.0
82.9
85.6
70.0
82.2
56.1
56.1
99.0
32.0
98.8
16.0
16.1
12.9
9.0
11.2
25.9
25.9
11.5
trace
2.0
1.2
1.3
21.0
6.5
17.4
17.4
7.5
Values, based on the yearly average of stomach contents,
were taken from reports on Nevada, Wyoming or Arizona
mammals (Williams, 1959; Hayden, 1966; Bradley, 1968;
Bradley and Mauer, 1971; Nelson et al., 1975).
18
-------
Table 9.
NUMBER OF RABBITS/HARES COUNTED AT ROOSEVELT HOT SPRINGS
DURING JUNE AND JULY 1977
Month
June
July
Census
Area
Test Area
Distance
Control
Test Area
Distance
Control
Number of animals
per km travelled
2.03
0.92
1.40
Table 10. RELATIVE ABUNDANCE OF RODENTS AT ROOSEVELT HOT SPRINGS
DURING JUNE AND JULY 1977
Month
June
July
Collection
Area
Test Area
Distance
Control
Test Area
Distance
Control
Vegetative
Association
Sagebrush
Sagebrush/ Juniper
Total
Sagebrush
Sagebrush/ Juniper
Total
Sagebrush
Sagebrush/ Juniper
Total
Sagebrush
Sagebrush/ Juniper
Total
Relative*
Abundance
17.50
24.00
39.00
16.70
23.40
40.30
12.00
10.50
21.67
8.75
24.00
32.45
* Expressed as P = SM/R, where P = total population,
S = number of unmarked and marked animals captured
on the last day, M = total number of animals marked
and released, R = number of recaptures occurring on
last day (Lincoln, 1930).
19
-------
is usually based on some multiple of the trap spacing or home ranges of
individual species (Jorgensen and Hayward, 1965; Smith et al., 1975). A
number of trapping techniques are available for this purpose (Smith et al.,
1971; Smith et al., 1972; and O'Farrell et al., 1977). We are currently
utilizing a square grid configuration (a series of parallel lines of traps
spaced 15 m apart). The wooden staked grid patterns have been positioned
at the two Roosevelt Hot Springs collection sites and future population
estimates will be reported as the number of animals per hectare. Trapping
techniques, density and the associated problems in assessing small mammal
populations have been reviewed by many investigators (Smith et al., 1975).
It should be noted that for comparison purposes variables such as the trapping
configuration (trap spacing), trap type and response, bait, duration of the
census, season of collection, climatic conditions, overall habitat character-
istics, as well as the census calculations methods, must all be considered.
Associated with population studies, identifications of several mammalian
wildlife species were made at Roosevelt Hot Springs during 1977 and verified
utilizing Fautin (1946) and Hall and Kelson (1959). The species included:
the blacktailed jackrabbit (Lepus aal-i-fornicus"), Nuttall's cottontail
(Syvilagus nuttalli), Desert cottontail (Syvilagus audubonii), rock squirrel
(Spermophilus variegatus) antelope ground squirrel (Armospermophilus leueurus),
least chipmunk (Eutamias minimus), Great Basin pocket mouse (Perognathus
pawns), Ord's kangaroo rat (Dipodomys ordii), western harvest mouse
(Reithrodontomys megalotis), canyon mouse (Peromysaus arinitus), deer mouse
(Peromysaus maniculatus), brush mouse (Peromysaus boylii), desert woodrat
(Neotoma lepida), western big-eared bat (Plecotus townsendii), coyote (Cariis
latrans), long-tailed weasel (Mustela frenata), Badger (Taxidea taxus) and
mule deer (Odoooileus hemionus).
Preliminary data on species composition, combining both live trap and
snap trap collections, indicate that the Great Basin pocket mice were the
predominant rodent for Roosevelt Hot Springs trapping areas accounting for
69.8% of the captures followed by: deer mice, 14.3%; Ord's kangaroo rats,
11.1%; western harvest mice, 2.4%; desert woodrats, 1.6%; and canyon mice,
0.8%. Numerical relationships between species (i.e., composition and/or
diversity) may be more useful to the program than a population assessment
since changes in species indices are used frequently in pollution ecology
to measure the stress in an ecosystem (Beijer and Jernelov, 1928).
20
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Herman, E. R. 1975. Geothermal Energy. Noyes Data Corp., Park Ridge, New
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Bradley, W. G. 1968. Food Habits of the Antelop Ground Squirrel in Southern
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Bradley, W. G., and R. A. Maues. 1971. Reproduction and Food Habits of
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Trioxide. J. Nutr. 10:255-270.
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Edwards, W. R., and L. Eberhardt. 1967. Estimating Cottontail Abundance
From Livetrapping Data. J. Wildl. Mgmt. 31:87-96.
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I. C. T. Nisbet, and S. Epstein. 1974. Environmental Impact of Cadmium:
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Forbes, G. B., and J. C. Reina. 1972. Effect of Age on Gastrointestinal
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24
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APPENDIX A
SAMPLE PREPARATION FOR TRACE ELEMENT ANALYSIS
• BY ATOMIC ABSORPTION SPECTROSCOPY
A. Laboratory Equipment, Reagents and Personnel.
1. Equipment
a. Sample holding and storage containers (jars, bags,
vials, etc.) should be made of polyethylene or a
similar inert plastic.
b. All other items used in sampling and sample handling
(surgical instruments, spatulas, forceps, tongs, etc.)
should be either Teflon-coated or made of plastic or
stainless steel.
c. Flasks or beakers used during microwave digestion
should be made of Teflon or of a high-grade
borosilicate glass. These containers should be used
for either high- or low-level samples only, depending
on previous use, and be so marked.
d. Containers, flasks and instruments are cleaned prior
to use. Wash in warm, detergent-supplemented water
and rinse with deionized water. Soak for at least
four hours in 1:1 cone. HN03/H20(V/V) or in 1:1:2
HN03/HCL/H20(V/V). Rinse with deionized water and
oven dry at 50-60°C. Cover washed and dried equipment
until needed.
e. Dissecting tables are cleaned with dilute nitric acid
and covered with disposable plastic lined pad. Fresh
pads and gloves as well as fresh (cleaned) sets of
instruments are used for each animal.
2. Reagents
a. All water used must be deionized.
b. Reagent impurity concentrations must be low enough
that they do not interfere with elements of interest.
25
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3. Personnel
a. Persons involved in either washing operations or
in tissue sampling will wear laboratory coats and
sterile surgical gloves.
b. All persons involved in sample digestion must wear
rubber aprons, rubber gloves and safety glasses
(or preferably safety masks).
c. Samples, glassware, instruments, etc., should never
be touched with bare hands. Gloves are always worn
and cleaned tongs or forceps are used. Smoking is
not allowed in the laboratory.
d. All personnel involved in any aspect of sampling,
sample handling, or sample preparation must be
thoroughly familiar with the study procedures
and objectives as well as difficulties associated
with trace element work.
B. Tissue Collection, Preparation and Storage.
1. General Procedures
a. Clean, label, cap and weigh sample containers and
assemble materials needed for dissection.
b. Perform dissection and place isolated tissues in
respectively labeled containers. Cap and weigh
containers recording net tissue wet weight.
c. Place open containers in drying oven (60°C) and
dry to constant weights. Close containers, record
sample dry weight and store containers (with sample)
in freezer.
Note: Freeze drying is the preferred
drying method as potential for contam-
ination is usually reduced at lower
temperatures. Freeze drying is used
whenever equipment is available.
2. Procedures Applicable to Blood, Hair and Egg Samples.
a. The following procedure applies to blood samples.
(1) Samples are collected in sterile
heparinized syringes (or vacutainer
tubes) with sterile needles. Syringes
26
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are emptied into labeled, weighed
polyethylene vials.
(2) Weighed samples are stored in the
freezer.
b. The following procedure applies to hair samples.
(Modified from Clarke and Wilson, 1974).
(1) To remove surface contamination,
swirl hair sample (using forceps)
in a hot detergent solution (10 ml
commercial detergent in 600 ml of
deionized water) and rinse in dis-
tilled water. Wrap sample in a
6 x 12" piece of cheesecloth, fold
the ends over and secure them with
white adhesive tape. Write identi-
fication number on the tape with an
indelible marker.
(2) Slightly agitate the sample in deionized
water and wring it out. Place sample
in a hot detergent solution (10 ml
detergent + 600 ml deionized water),
squeeze it repeatedly with a glass .rod
and wring out.
Repeat step (2).
(3) Rinse sample in deionized water and
wring it out. Place sample in acetone
(300 ml), agitate and squeeze it
repeatedly with a glass rod and then
wring out.
(4) Place sample in 600 ml of a hot,
filtered, saturated solution of
ethylenediaminetetraacetic acid
(EDTA) in deionized water, agitate
and squeeze with a glass rod. After
5 minutes, remove and wring out the
sample, wash sample twice in deionized
water and wring out thoroughly.
(5) Dry sample at 60°C, open cheesecloth
and transfer sample to a preweighed,
labeled polyethylene container. Record
net weight of clean hair sample and
store in freezer.
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c. The following procedure applies to egg samples.
(1) Wash intact eggs with a detergent
solution and with deionized water.
(2) Break individual eggs and separate
into yolk, white and shell. Transfer
each fraction to respective preweighed,
labeled polyethylene containers. Record
wet weight and store sealed containers
in the freezer.
Note: Egg yolk may contain a high fat
content and an extraction process must
be employed to remove lipids.
C. Microwave Assisted Digestion of Biological Samples.
Note: Personnel conducting sample digestions
must wear rubber gloves, apron and safety
goggles or preferably safety masks. Accurate
records of digestion steps are essential.
1. Sample Size
a. Sample size for microwave digestion should not
exceed 1 g dry weight or, in the case of whole
blood, should not exceed 5 g wet weight.
b. Samples should be treated with a minimum quantity
of the purest acid available. Recommended nitric
acid additions are 10 ml per 0.5 g of dry tissue
or per 2.5 g of whole blood.
c. Volumetric flask size will be determined by
initial sample size or requirements for replicate
analyses.
2. Reagents
a. Concentrated nitric acid is transferred to a
container (borosilicate) of convenient size and
placed in hood.
b. Acid container labels must indicate preparation
date and container contents. Do not allow dust
to accumulate on the exterior of the container.
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3. Digestion Procedure
a. Transfer approximately 0.5 g of dried tissue to
a preweighed 100 ml Teflon beaker or to a 125 ml
Erlenmeyer flask. Record tissue weight.
b. Add 10 ml cone, nitric acid to sample, swirl and
allow to stand a few minutes (starts digestion).
c. Place beaker (containing sample) in microwave oven
and turn switch to "high." When acid begins to
boil, turn switch to "medium-low." As digestion
progresses, digest color changes from dark to
light; brown fumes are emitted. Digestion time
is not critical, but is approximately one minute
per ml of acid used. As digestion approaches
completion the solution should be clear or straw-
colored and transparent. The digestion should be
stopped when the solution volume is approximately
0.5 ml.
Note: If solution is not light colored
and transparent more acid may need to be
added and the digestion step repeated.
d. Turn switch to "off." When all fumes have been
removed by exhaust system, open oven door, remove
samples with tongs and cover sample beaker and
transfer beaker to the hood for cooling.
e. Add 3-4 ml of deionized water to beaker, swirl and
pour contents into volumetric flask. Rinse beaker
with 2-3 ml of deionized water and add this to
flask. Bring to volume with deionized water.
Note: If precipitate remains in flask,
add HC1 or another suitable solvent
dropwise (not to exceed 5.0% of volume)
until precipitate is dissolved. Water
is then added to volume.
f. Transfer solution from volumetric flask to a poly-
ethylene container for storage. Seal the labeled
container and place it in the freezer.
D. Potential Errors During Sample Preparation and Storage.
1. Low Values
a. Incomplete sample digestion may result in incomplete
solubilization.
29
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b. Overheating of sample during digestion may result
in partial volatilization of certain elements
(e.g., mercury, arsenic).
c. Careless manipulation of samples and sample solutions
may result in spillage.
d. Under certain conditions, elements of interest may
be absorbed to container wall or to precipitates
formed during digestion or storage.
e. During sample storage period, bacterial action may
increase volatility of certain elements (e.g.,
formation of methyl mercury). This can be suppressed
by freezing samples and keeping storage time to a
minimum.
2. High Values
a. Use of less-than-clean laboratory equipment and use
of impure reagents will produce inaccurate values.
b. Samples can also be contaminated by airborne dusts,
mists and fumes.
c. Partial evaporation of water from improperly sealed
digest containers will concentrate the solutes.
30
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APPENDIX B
PREPARATION OF TISSUE SAMPLES FOR OPTICAL
EMISSION SPECTROSCOPY ANALYSIS
Note: All equipment used in these procedures will have been
thoroughly cleaned as was previously described.
1. Large samples (muscle, pelt, carcass, gastrointestinal tract, etc.)
are isolated and dried as described earlier. The dried, frozen
samples are homogenized (usually 2 to 3 minutes) in a Tekmar
analytical grinding mill and part of the sample submitted for
analysis.
2. Small samples (rodent organs) are isolated and dried as described
earlier. Since the dry weight of these small organs is usually
between 0.01 and 0.8 grams, each organ is cut into small pieces
and the entire sample submitted for analysis.
3. Hair samples are washed as described, dried, cut into sections
4 to 5 mm long and submitted for analysis.
4. Blood and eggs (separated into yolk and albumin) are dried, then
manually powdered and mixed with a Teflon rod and submitted for
analysis.
31
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/7-78-233
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
GEOTHERMAL ENVIRONMENTAL IMPACT ASSESSMENT: Procedures
for Using Fauna as Biological Monitors of Potential
Geothermal Pollutants
5. REPORT DATE
December 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Z.. C. Nelson, W. W. Sutton, A. A. Mullen, W. F. Beckert
G. D. Potter
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Monitoring and Support Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, Nevada 89114
10. PROGRAM ELEMENT NO.
1NE827
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency—Las Vegas, NV
Office of Research and Development
Environmental Monitoring and Support Laboratory
Las Vegas, Nevada 89114
13. TYPE OF REPORT AND PERIOD COVERED
Progress Report, 1977
14. SPONSORING AGENCY CODE
EPA/600/07
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This is the first in a series of reports that covers the feasibility of utilizing
wildlife and domestic animals to design a monitoring strategy for assessing the
environmental impact of geothermal resource development. This study is part of an
overall program which will also include data on any water, soil and flora.
Animal tissues and animal products were collected in the vicinity of California
and Utah geothermal development sites. These samples are being analyzed for selected
elements so as to confirm baseline concentrations in tissues of area fauna. Small
mammal populations characteristics are also being monitored at Roosevelt Hot Springs,
Utah. Laboratory studies are being conducted to relate the ingestion of selected
elements to subsequent changes in elemental concentration of various tissues.
This report presents some preliminary data on trace element concentrations in
tissues of wildlife and domestic animals. Concentrations in geothermal effluents also
were determined. Quality assurance, sample collection, relative abundance of small
mammals and, especially, methodology (sample preparational and analytical procedures)
are discussed.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Held/Group
Geothermal Energy
Biological Indicators
Environmental Monitoring
Trace Elements
Terrestrial Ecosystems
Natural Abundance
Environmental Impact
Livestock
Wildlife
loosevelt Hot Springs, Utah
Imperial Valley, Californi;
Relative Abundance
Tissue Preparation
Analytical Procedures
Optical Emission Spectrome
Atomic Absorption
Small Mammals
06, C, F
07, B
10
try
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report I
UNCLASSIFIED
21. NO. OF PAGES
40
20. SECURITY CLASS (Thispage>
UNCLASSIFIED
22. PRICE
A03
EPA Form 2220-1 (Rev. 4-77)
PREVIOUS EDITION IS OBSOLETE
>)• "U.S. GOVERNMENT PRINTING OFFICE:" 1979-684-269/2110 Region No. 9-1
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