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)

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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

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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

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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

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 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

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       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

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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

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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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 Lewis,  J.  C.   1970.   Wildlife  Census Methods:  A Resume.   J. Wildl. Dis.
 6:356-364.

Lincoln, F. C.  1930.   Calculating Waterfowl Abundance  on  the  Basis of Banding
Returns.  U.S. Dept. Agric. Cir. 118:1-4.


                                     22

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 Lisk, D. J.  1974.  Recent Developments in the Analysis of Toxic Elements
 Science 184 : 1137-1141.

 Lloyd, E.   1967.  A Comparison of the Metabolism of Calcium and Strontium in
 Rabbit and Man.  pp. 167-174.  In Strontium Metabolism.  (J. M. A.  Lenihan,   '
 J. F. Loutit, and J. H. Martin, eds.).  Academic Press, London.

 Miller, W. J.  1969.  Absorption, Tissue Distribution, Endogeneous  Excretion
 and Horaeostatic Control of Zinc in Ruminants.  Am.  J. Clin. Nutr.  22:1323-1331,

 Miller, W. J.  1975.  New Concepts and Developments in Metabolism and Homeo-
 stasis of  Inorganic Elements in Dairy Cattle.  J.  Dairy Sci. 58:1549-1560.

 Morgareidge,  K.  1963.  Metabolism of Two Forms of  Dietary Arsenic  by the
 Rat.   Agric.  Food Chem. 11:377-378.

 Nelson, Z. C.,  K. S. Moor,  and W. G.  Bradley.  1975.   Utilization of Food,
 Space and  Time  by Rodents of a Juniper-Pinyon Community of Southern Nevada.
 Amer. Soc. Mammal.  (Proceedings Abstracts).

 O'Farrell, M. J., D. W. Kaufman,  and  D.  W.  Lundahl.  1977.   Use of  Live
 Trapping With the Assessment Line Method for Density  Estimation.  J. Mammal
 58:575-582.

 Peoples, S. A.   1964.   Arsenic Toxicity in  Cattle.  Ann.  N.Y.  Acad.  Sci.
 111:644-649.

 Peoples, S. A.   1975.   Review of  Arsenical  Pesticides,  pp. 1-12.   In
 Arsenical  Pesticides (E.  A.  Woolson,  ed.).   American  Chemical  Society,
 Washington.                                                          *

 Schieler   L.  1976.  Geothermal Effluents,  Their Toxicity and  Prioritization.
      i          Pr°C' FirSt Worksh°P Sampling Geothermal Effluents.   EPA-600/9-
Shapiro, H. A.  1967.  Arsenic Content of Human Hair  and  Nails;   its
tation.  J. Forensic Med. 14:65-71.

Smith, H. D., C. P. Jorgensen, and H. D. Tolley.   1972.   Estimation of  Small
Mammal Using Recapture Methods:  Partitioning of  Estimator Variables.   Acta.
Theriol. 17:57-66.

Smith, M. H. , R. Blessing, J. G. Chelton, J. B. Gentry, F. B. Golley, and
J. T. McGinnis.  1971.  Determining  Density for  Small Mammal Populations
Using A Grid and Assessment Lines.  Acta.. Theriol. 16:105-125.

Smith, M. H., R. H. Gardner, J. B. Gentry, D. W.  Kaufman, and M.  J. O'Farrell.
1975.  Density Estimations of Small Mammal Populations,   pp. 25-53.  In Small
Mammals:  Their Productivity and Population Dynamics  (F.  B. Golley,
K. Petrusewicz, and L. Ryszkowski, eds.).  Cambridge  Univ. Press, London.
                                     23

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Tipton  I. H., and M. J. Cook.  1963.  Trace Elements in Human Tissue.  Part
II.  Adult Subjects From the United States.  Health Phys. 9:103-145.

Tipton, I. H., and P. L. Stewart, and P. G. Martin.  1966.  Trace Elements  in
Diets and Excreta.  Health Phys. 12:1683-1689.

Underwood, E. J.  1977.  Trace Elements in Human and Animal Nutrition.
Academic Press, New York.  545 pp.

Williams,  0.   1959.   Food Habits of the Deer Mouse.  J. Mammal. 40:415-419.
                                    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.
                         27

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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.
                                 28

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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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