Environmental Protection Technology Series
SAMPLING FOR ORGANIC CHEMICALS AND
MICROORGANISMS IN THE SUBSURFACE
Robert S. Kerr Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Ada, Oklahoma 74820
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental 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 ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-77-176
August 1977
SAMPLING FOR ORGANIC CHEMICALS
AND MICROORGANISMS IN THE
SUBSURFACE
by
William J. Dunlap, James F. McNabb,
Marion R. Scalf, and Roger L. Cosby
Ground Water Research Branch
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ADA, OKLAHOMA 74820
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DISCLAIMER
This report has been reviewed by the Robert S. Kerr Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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FOREWORD
The Environmental Protection Agency was established to coordinate
administration of the major Federal programs designed to protect the
quality of our environment.
An important part of the Agency's effort involves the search for
information about environmental problems, management techniques, and new
technologies through which optimum use of the Nation's land and water
resources can be assured and the threat pollution poses to the welfare
of the American people can be minimized.
EPA's Office of Research and Development conducts this search through
a nationwide network of research facilities.
As one of these facilities, the Robert S. Kerr Environmental Research
Laboratory is responsible for the management of programs to: (a) investi-
gate the nature, transport, fate, and management of pollutants in ground
water; (b) develop and demonstrate methods for treating wastewaters with
soil and other natural systems; (c) develop and demonstrate pollution con-
trol technologies for irrigation return flows; (d) develop and demonstrate
pollution control technologies for animal production wastes; (e) develop
and demonstrate technologies to prevent, control or abate pollution from
the petroleum refining and petrochemical industries; and (f) develop and
demonstrate technologies to manage pollution resulting from combinations
of industrial wastewaters or industrial/municipal wastewaters.
This report contributes to that knowledge which is essential in order
for EPA to establish and enforce pollution control standards which are
reasonable, cost effective, and provide adequate environmental protection
for the American public.
William C. Galegar
Di rector
iii
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ABSTRACT
Analyses of low levels of organic chemicals and microorganisms in subsurface
waters and solids are required for realistic assessment of current and potential
pollution of ground water, but are particularly difficult to accomplish because
of problems in sampling often remote and relatively inaccessible subsurface en-
vironments. This report presents procedures currently utilized by the Ground
Water Research Branch of the Environmental Protection Agency for sampling for
organic pollutants and microorganisms in ground waters and subsurface earth
solids.
Technology is described for construction of wells capable of providing repre-
sentative, uncontaminated samples of ground water in compact alluvial formations
at relatively shallow depths and for obtaining cores of subsurface earth solids
suitable for organic and microbial analyses in similar circumstances. Methods
for acquisition of grab samples of ground water suitable for total organic and
microbial analyses and for analyses of volatile organics are presented. Contin-
uous sampling of organics in ground waters lying within approximately 7.5 m
(25 ft) of the surface by sampling units utilizing selected absorbents is
described, including details of adsorbent columns, configuration of and housings
for sampling systems, and sample handling. Procedures for handling and process-
ing of core materials to produce samples amenable to analytical methods for
organics and microorganisms are also presented.
The procedures described provide a basic capability for sampling for organic
pollutants and microorganisms in relatively shallow subsurface environments, and
have potential application in many investigations pertaining to ground-water
pollution. Additional research is needed, however, to further evaluate, improve,
and extend their capabilities.
This report covers a period from July 1975 to January 1977, and work was
completed as of May 1977.
IV
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CONTENTS
Foreword iii
Abstract iv
Figures yi
Acknowledgment vii
1. INTRODUCTION 1
2. CONCLUSIONS 2
3. RECOMMENDATIONS 3
4. SAMPLING OF GROUND WATER 4
WELLS FOR SAMPLING 4
SAMPLING PROCEDURES 5
Preliminary Operations 5
Acquisition of Grab Samples 5
Continuous Sampling 7
5. SAMPLING OF SUBSURFACE SOLIDS 19
ACQUISITION OF CORES 20
HANDLING AND PROCESSING OF CORE MATERIALS 20
6. REFERENCES 26
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FIGURES
Number Page
1 System for acquisition of grab samples of ground water 6
2 Teflon bailer for ground-water sampling 8
3 Ground-water sampling system 10
4 Sampling system housing 11
5 Complete ground-water sampling unit 12
6 Resin adsorption column 14
7 Carbon adsorption column 15
8 Sampling unit and receivers installed at field site 17
9 Core extruding device 21
10 Obtaining a subsample for microbial analysis from a
parent core 23
11 Typical location of microbial and organic samples in
a parent core 24
VI
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ACKNOWLEDGMENT
The assistance of Montie H. Fraser in the design and construction of the
core-sampling equipment described in this report is gratefully acknowledged.
vii
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SECTION 1
INTRODUCTION
The quality of ground water is being increasingly threatened by the con-
tinuing and expanding release of both chemical and biological pollutants into
the earth's crust as the result of various human activities, particularly those
involving disposal of wastes to the land. In order to assess the potential
impact of such activities on ground water and, hence, to provide a rational
basis for strategies to protect the quality of this valuable resource, the
behavior of pollutants in the subsurface and the processes governing this
behavior must be elucidated. This requires the capability for detecting and
measuring low levels of chemicals and microorganisms in both ground waters and
subsurface earth solids. A major problem in development of such analytical
capability is the necessity for obtaining uncontaminated, truly representative
samples from often remote and relatively inaccessible subsurface environments.
Sampling problems are particularly acute in analysis of organic pollutants and
microorganisms, which are of major concern because of suspected or known ad-
verse effects on health and the role of microorganisms in processes controlling
pollutant behavior and fate. Introduction of contaminants or any loss of sample
components in sampling for these parameters is likely to produce an inordinate
impact on analytical results because of the low levels usually being determined
and the effects of interferences on analytical procedures.
During the course of investigations concerning the movement and fate of
pollutants in ground water, the Ground Water Research Branch of EPA has developed
and utilized several procedures for sampling organic pollutants and microorganisms
in subsurface environments. These procedures are limited in scope, being gener-
ally applicable only to ground water and earth solids lying within about 7.5 m
(25 ft) of the surface. They represent a first effort, and undoubtedly can and
will be greatly improved in future work. However, it is believed that these pro-
cedures are sufficiently useful to be of interest to others who may be involved
in sampling organic pollutants and microorganisms in the subsurface, either for
research or monitoring purposes. Therefore, the details of the methods and equip-
ment currently utilized by the Ground Water Research Branch for sampling organic
chemicals and microorganisms in ground water and subsurface earth solids are
presented in this report.
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SECTION 2
CONCLUSIONS
The procedures presented in this report provide a basic capability for
sampling for organic pollutants and microorganisms in relatively shallow sub-
surface environments. The techniques and equipment described herein for well
construction and for acquisition of samples from properly constructed wells are
satisfactory for sampling ground water for organics and microorganisms at depths
down to approximately 7.5 m (25 ft) in compact alluvial formations. The coring
and core-processing methods presented may be utilized to sample subsurface earth
solids for organics and microorganisms at slightly greater depths in similar geo-
logical situations. In their present state of development these procedures have
potential application in many situations of possible ground-water pollution, but
additional work is needed to further define, improve, and extend their capabilities.
In particular, further evaluation of the capabilities of the adsorbents employed
for continuous sampling for organics in ground water is needed as a basis for
devising optimum sampling systems, and techniques for constructing sampling wells
and obtaining uncontaminated cores in situations where the use of drilling fluids
appears necessary and for sampling ground water in deeper formations need to be
further explored and perfected.
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SECTION 3
RECOMMENDATIONS
1. The procedures presented In this report, or similar methods based on anal-
ogous principles of sample validity and integrity, should be utilized whenever
possible in ground-water investigations to sample for organic pollutants and
microorganisms in subsurface environments.
2. Additional work should be done to more thoroughly evaluate, improve, and
extend the capability of the procedures described herein for sampling for organic
pollutants and microorganisms in subsurface environments. In particular, the
following needs should be addressed.
a. The qualitative and quantitative efficiency of various adsorbents
for recovering organic compounds from aqueous media should be
thoroughly evaluated to provide an improved basis for selection
of optimum adsorption systems for sampling organics in ground water.
b. A non-adsorbing, non-contaminating submersible pump capable of fitting
into small-diameter well casings should be developed to permit contin-
uous sampling of organics in ground water at depths in excess of
approximately 7.5 m (25 ft).
c. Technology for constructing wells satisfactory for sampling ground
water for organics and microorganisms and for obtaining uncontaminated
cores in loose, non-compacted formations, at depths greater than 12-15 m
(40-50 ft), and in other situations where use of drilling fluids appears
necessary, should be perfected.
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SECTION 4
SAMPLING OF GROUND WATER
WELLS FOR SAMPLING
When possible, wells utilized to sample ground water for organic pollutants
and microorganisms are drilled and completed using technology which minimizes
potential contamination of the aquifer during drilling operations and maximizes
the probability that truly representative ground-water samples can be obtained.
Existing wells are usually of questionable value for this purpose for a number
of reasons, of which the following are principal.
1. Drilling fluids used in ordinary construction of wells for general
use are likely to significantly alter the subsurface chemical and
microbiological environment in the vicinity of such wells.
2. Water from aquifers other than that of interest may be present in
many wells due to their depth, zones of casing, and method of
completion.
3. Surface contamination of wells frequently occurs because of poor
completion practices.
4. Casing materials and pumping equipment may contribute pollutants
to the sampled water or adsorb constituents from it.
Currently, drilling of wells for sampling is accomplished by use of an
auger, thus avoiding the use of drilling fluids and the contamination problems
associated with the use of these materials. This technique is quite effective
for drilling in reasonably compact alluvial materials at relatively shallow
depths. Techniques for drilling satisfactory sampling wells at depths greater
than 12-15 m (40-50 ft), in very loose formations, and all other situations
where the use of drilling fluids appears necessary, remain to be developed.
In the most recent work of the Ground Water Research Branch, 3.8 cm (1.5 in.)
I.D. x 4.45 cm (1.75 in.) O.D. Teflon tubing has been used to case that portion of
sampling wells extending from a few feet above the water table to the bottom of
the borehole. Hence, only this very inert casing material is in contact with
ground water in such wells. Because of the high cost of Teflon tubing, 3.8 cm
(1.5 in.) I.D. galvanized pipe, coupled to the Teflon with a threaded galvanized
coupling, is used to case the upper part of the borehole that lies within the
unsaturated zone and is not directly in contact with the ground water.
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In some earlier work, 5 cm (2 in.) PVC pipe was utilized for casing of
sampling wells. This material is relatively inexpensive and easy to use, but
it is less desirable as a casing material than the Teflon tubing-galvanized pipe
combination for two principal reasons. First, organic constituents of ground
water may be adsorbed on the PVC casing. Second, there is evidence that PVC
casing may contribute low levels of organic contaminants to the samples, such
as phthalic acid esters used as plasticizers in PVC manufacture and solvents
from cements used to join lengths of PVC tubing.
Casing materials are carefully cleaned by washing with soap and water,
thorough water rinsing, solvent rinsing, and, finally, rinsing with organic-
free water prior to installation in the borehole.
Wells are gravel-packed with clean, washed pea gravel from the bottom of
the hole to just below the Teflon-galvanized coupling. The casing is cemented
from this point to the surface where a concrete pad approximately 61 x 61 cm
(24 x 24 in.) is installed to accommodate sampling equipment and to aid in
sealing out surface waters. A screw-type cap on the casing prevents contami-
nation when the well is not in use. The well is completed by thorough pumping,
usually with a high-capacity hand diaphragm pump, and is allowed to stabilize
for at least 10 days prior to sampling.
SAMPLING PROCEDURES
Preliminary Operations
Just prior to actual sampling of ground water, the well from which the
samples are to be obtained is thoroughly pumped to insure that all "stale" water
standing in the well-bore is removed and replaced by fresh formation water that
is truly representative of the water in the surrounding aquifer. This is usually
accomplished by means of a "Masterflex" 7015 peristaltic pump powered by a 7545
Variable Speed Drive or 7570 Portable Sampling Drive (Cole-Parmer Instrument Co.,
Chicago, IL). Ground water is pumped from the water table to the inlet tubing of
the pump through "Chemfluor" 6 mm O.D. Teflon tubing, supplied in 366 cm (12 ft)
lengths by Chemplast, Inc., Wayne, NJ. The required length of tubing is fabri-
cated by joining appropriate tubing sections with "Taper-Tite" 6 mm I.D. Teflon
connector assemblies or 6 mm "Chemfluor" Teflon unions, also supplied by Chem-
plast, Inc. The tubing is thoroughly cleaned and sterilized prior to use, and
is very carefully inserted into the well to minimize introduction, of contaminants.
Pumping rates in excess of 500 ml/min are ordinarily used, and a volume of water
equivalent to at least 10 times the volume of water originally standing in the
well casing is removed.
Acquisition of Grab Samples
Grab samples of ground water for direct microbial analysis, biomass deter-
mination by adenosine triphosphate (ATP) analysis, organic carbon determination,
and any other desired analyses for which such samples are suitable, are obtained
by utilizing the system shown in Figure 1. Ground water is drawn up from the
water table through a sterile 6 mm O.D. Teflon tube into a sterile, calibrated
1 liter Erlenmeyer flask by means of a "Masterflex" 7015 peristaltic pump located
on the outlet or downstream side of the sampling flask; hence, the sampled water
contacts only sterile glass and Teflon during the sampling operation.
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TEFLON CONNECTORS
6 MM 1.0. /
TEFLON TUBING
6 MM O.D.
GLASS TUBING
6 MM O.D.
WELL CASING
TYGON
TUBING
OUTLET
PERISTALTIC
PUMP
ERLENMEYER
Figure 1. System for acquisition of grab samples of ground water.
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Samples for direct microbiological analysis are immediately transferred
aseptically to appropriate sample containers, usually 500 ml dilution bottles,
using a propane torch for flaming of glassware. Sample bottles are topped off
to exclude air, and are packed in ice for shipment to the laboratory for analysis.
Samples to be analyzed for ATP as a measure of total biomass (1) are immed-
iately filtered at the sampling point through 0.45 p Mi Hi pore filters, using
sterile Millipore glass filtering equipment with a Gast vacuum pump. The filters
are transferred to foil-sealed, sterile 150 ml beakers and immediately frozen on
dry ice for return to the laboratory.
Samples for organic carbon analysis are transferred from the sampling flask
to thoroughly-cleaned 40 ml vials equipped with Teflon-lined screw caps. They
are quick-frozen on dry ice for shipment and storage until analyzed. Care is
exercised to insure that adequate space remains in the vials for expansion of
the sample upon freezing.
Samples of ground water to be analyzed for highly volatile organics by the
Bellar volatile organic analysis (VOA) method (2) are obtained by means of a
Teflon bailer. The previously-described system ordinarily used for obtaining
grab samples is not suitable for VOA samples because of possible stripping of
highly volatile constituents from the sample under the reduced pressures occurring
in this system.
The Teflon bailer, shown in Figure 2, is either 46 or 91 cm (18 or 36 in.)
in length. It is constructed of 2.5 cm (1 in.) I.D. x 3.8 cm (lh in.) O.D.
Teflon extruded heavy wall tubing plugged at the bottom end with a short length
of 2.5 cm O.D. Teflon extruded rod. Water enters the bailer when it is lowered
into the well through an 0.8 cm (5/16 in.) hole drilled through the end plug and
is prevented from draining out by a 1.9 cm (3/4 in.) diameter glass marble which
fits into a conical seat machined into the top of the plug. The plug fits tightly
inside of the tube comprising the body of the bailer; hence, no adhesives which
might contribute contaminants to sampled water are required to hold it in place.
A cable made by weaving together several strands of guage #24 nickel wire is
used to raise and lower the bailer.
Bailers are sterilized by autoclaving before use to minimize introduction of
contaminants into the wells being sampled. Ground-water samples obtained are very
carefully poured from the bailer into clean serum bottles of appropriate size
(usually 125 ml), with caution being exercised to avoid turbulence which might
result in loss of volatile organics and/or excessive oxygenation of the samples.
The serum bottles are topped-off to avoid including gas spaces in the samples and
are tightly closed with Teflon-lined septums held in place by aluminum crimp-on
seals (Precision Sampling Corporation, Baton Rouge, LA). The sealed VOA samples
are packed in ice and returned to the laboratory for analysis at the earliest
convenient time.
Continuous Sampling
Sampling of ground water for organic pollutants other than those amenable to
the VOA procedure is accomplished by continuous sampling procedures utilizing
selected adsorbents to concentrate and recover the organic constituents. These
procedures are based on work described by: Junk et al. (3); Breidenbach et al. (4);
Buelow, Carswell, and Symons (5); and, Endres and Herman (6).
7
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NICKEL WIRE
CABLE
l-l/4"o.D.xl" I.D. TEFLON
EXTRUDED TUBING,
18 TO 36"LONG
s-^ ils~- 3/4" DIAMETER
[~V ' GLASS MARBLE
DIAMETER TEFLON
EXTRUDED ROD
5/16 DIAMETER
HOLE
Figure 2. Teflon bailer for ground-water sampling,
8
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A special ground-water sampling system is used in which ground water is
pumped directly from the water table through two columns of adsorbent in series
by means of a "Masterflex" 7014 peristaltic pump (powered by a 7545-10 Variable
Speed Drive) located on the outlet, or downstream, side of the columns (Figure 3).
Teflon tubing (6 mm O.D.) conveys the ground water from the zone of saturation to
the inlet of the first column. Columns, which vary in configuration depending on
the absorbent for which they are designed, are constructed of glass or glass and
Teflon. Connecting tubing is 6 mm O.D. glass, and components are joined together
by 6 mm I.D. "Taper-Tite" Teflon connectors. These components are thoroughly
cleaned to eliminate organic contaminants prior to assembly of the system. Hence,
ground water being sampled contacts only clean glass or Teflon (excepting the
adsorbents) until it has passed through both adsorbent columns. Loss of solutes
by adsorption on sampling equipment and introduction of organic contaminants
during the sampling operation are, therefore, virtually precluded.
Sampling systems are installed in specially constructed housings to form
self-contained sampling units which are easily transported and set up over wells
in the field and which afford protection to the sampling system components against
damage by weather, small animals, and accidental breakage. A detailed drawing of
a sampling system housing is presented in Figure 4.
Overall dimensions of the housing are approximately 61 cm (24 in.) high,
58 cm (23 in.) wide, and 49 cm (19.5 in.) deep. Access to the interior is gained
both through a door in the front side and through the top, which is hinged to ,
open to the rear. A handle on the top facilitates transport of the unit. Space
is provided for installation of a "Masterflex" Variable Speed Drive, fitted with
as many as three peristaltic pump heads, on the floor at the rear of the housing,
and for mounting the control unit for the pump drive on the rear wall. Vent holes
in the back and left side walls permit dissipation of heat from the pump drive
motor. Two female electrical receptacles are provided inside the housing, mounted
in a standard electrical box on the side interior wall. A male motor plug base
mounted in the side wall behind this box serves as a receptacle for connecting
the housing to an external 110 V electrical source through a suitable extension
cord.
Holes of approximately 8 mm (0.3 in.) diameter drilled in removable sections
of the housing floor provide passage for the Teflon lines of the sampling systems
from the housing into the casing of a well being sampled. The removable floor
sections facilitate manipulation of the tubing during installation of a sampling
unit over a well and yet permit effective closure of the housing when the unit is
in place.
A 12 mm (0.5 in.) aluminum rod, mounted on the side walls of the housing by
"Flexaframe" foot plates (Fisher Scientific Co., Pittsburgh, PA), extends across
the interior to serve as the support upon which adsorbent columns are installed.
Standard laboratory extension clamps and "Flexaframe" 90° connectors are used for
column installation. As many as three pairs of columns can be accommodated by a
housing; hence, three different sampling systems may be employed simultaneously
for sampling ground water from a single well.
A complete ground-water sampling unit, consisting of a housing containing
two complete sampling systems, is shown in Figure 5.
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GLASS TUBING
6 MM 0.0.
TEFLON
CONNECTOR
6MM I.D.
TO WASTE
RECEIVER
PERISTALTIC
PUMP
-TEFLON TUBING
6MM 0.0.
WELL
CASING
Figure 3. Ground-water sampling system.
10
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Figure 4. Sampling system housing.
11
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Figure 5. Complete ground-water sampling unit.
12
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Adsorbent columns currently being used are prepared from macroreticular
resin (XAD-2, Rohm and Haas Co., Philadelphia, PA), activated carbon (Filtrasorb
200, Calgon Corporation, Pittsburgh, PA), or polyamide (Polyamide Woelm, ICN
Pharmaceuticals, Inc., Cleveland, OH).
The XAD-2 resin is purified by sequential solvent extraction with methanol,
acetonitrile, and ethyl ether in 2200 ml modified Soxhlet extractors, and stored
under methanol until used for column preparation (3). Filtrasorb 200 activated
carbon is boiled in a 5% solution of hydrochloric acid in organic-free water for
3 hr, washed thoroughly with organic-free water to remove all chlorides, dried
at 150° C for several hours in a clean oven, and stored in glass-stoppered jars
until used. Polyamide Woelm (hereafter called PAW) is suspended in distilled
water about 2 hr prior to column preparation and washed after packing by passing
successively through each column the following solvents: 100 ml water; 100 ml
2-propanol:water (1:1); 200 ml 2-propanol; 100 ml 2-propanol:water (1:1); and,
200 ml water.
XAD-2 and PAW are packed from methanol and water slurries, respectively, to
produce 9 x 130 mm adsorbent beds in glass columns fabricated from 12 mm O.D.
borosilicate glass tubing. These columns, shown in Figure 6, are equipped with
a 3-way Teflon stopcock at each end to permit retention of fluid in the adsorb-
ent during transport and bypassing of the sample stream during sampling. The
two column sections are held together by a "Taper-Tite" Teflon connector assembly
which permits easy disassembly for packing and elution. The columns are plugged
at both ends with solvent-extracted glass wool, with the short, or inlet, sections
being completely filled with this material to serve as a filter protecting the
adsorbent from particulate matter which might be present in the sample stream.
During packing and elution the columns are inverted from the normal sampling
position, with the short (sample-inlet) end replaced by a reservoir. After
packing and assembly, the XAD and PAW columns are kept sealed and never allowed
to become dry.
Carbon adsorption columns are prepared by placing 70 g of dry, purified
Filtrasorb 200 in a 30 mm I.D. borosilicate glass column (Figure 7), thus pro-
ducing a carbon bed approximately 230 mm in length. The columns are equipped
with 50/30 ball and socket joints to permit disassembly for packing and removal
of the carbon, which is retained in place during sampling by solvent-washed
glass wool plugs. Except during actual use, the columns are kept tightly sealed
by inserting 6 mm O.D. Teflon plugs into 6 mm I.D. "Taper-Tite" Teflon connectors
attached to the column inlets and outlets.
A system comprised of an XAD-2 column and an activated carbon column arranged
such that the sampled water first contacts the resin column is currently used for
sampling ground water for organic pollutants which are readily amenable to gas
chromatography. A system employing two polyamide columns in series is used to
sample for pollutants of relatively greater molecular weight and polarity,
including particularly those substances capable of forming hydrogen bonds.
For actual sampling, suitable systems are assembled and installed in housings
at the laboratory. Organic-free water is pumped through XAD and PAW columns to
clear them of solvents, and the systems are sealed by closing appropriate stop-
cocks and installing "Taper-Tite" connectors containing glass or Teflon plugs on
the ends of tubing. The complete sampling units are then transported to the field
13
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STOPCOCK
3-WAY TEFLON
GLASS WOOL
PLUG
TEFLON
CONNECTOR
12 MM I.D.
-TEFLON TUBING
6 MM I.D.
Figure 6. Resin adsorption column.
14
-------
30 MM
GLASS WOOL
PLUG
230
MM
§ 50/30 JOINT
Figure 7. Carbon adsorption column.
15
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and set in place over the casing heads of the wells to be sampled, with the Teflon
lines from the sampling systems extending down the well-bores into the ground water.
As mentioned previously, the wells are pre-pumped just prior to set-up of sampling
units to insure the presence of fresh formation water in the casings when sampling
is begun. To preclude the entry of contaminants into the wells, the casing heads
are kept tightly covered during the sampling period. This is accomplished by
closing them with plastic or metal caps or plugs containing holes just large
enough to accommodate the Teflon sampling lines, or by sealing the spaces between
the sampling lines and casing walls with heavy aluminum foil.
Sampling is conducted by continuously pumping ground water through the sam-
pling systems for periods approximating seven days at flow rates usually ranging
from 10 to 25 ml/min. Flow rates of 15 to 20 ml/min are considered optimum, but
are often difficult to maintain because of changing resistances of the columns
to fluid flow, principally as a result of accumulation of particulate matter in
the glass wool plugs holding the adsorbents in place.
Total volumes of ground water sampled ordinarily range from 100-250 1, and
are determined by collecting and measuring the water leaving the sampling systems.
For this purpose, water from the outlets of the peristaltic pumps is conveyed by
Tygon tubing through the back walls of the sampling system housings into cali-
brated waste receivers consisting of 32 gal. molded polyethylene trash cans which
have been calibrated in liters.
In Figure 8 a sampling unit and waste receivers are shown in place at a field
site. As this photograph shows, both unit and receivers are anchored in place by
means of wooden stakes and wire cable as a precaution against high winds.
Upon completion of sampling, the adsorbent columns are sealed and the sampling
units are immediately returned to the laboratory for disassembly and elution or
extraction of the adsorbents. XAD-2 and PAW columns are sealed while completely
filled with sample water, while carbon columns are drained of excess liquid
before sealing.
For elution, XAD-2 and PAW columns are inverted and the short inlet sections
containing glass wool plugs on which considerable particulate matter may have
collected are replaced by 300 ml reservoirs. XAD-2 columns are drained of ex-
cess water and eluted with 20 ml of acetone followed by 80 ml of chloroform, as
suggested by Webb (7). The combined eluates are dried with anhydrous sodium
sulfate and reduced to the desired volume in Kuderna-Danish evaporators. PAW
columns are eluted with 30 ml of 2-propanol, and the eluates are carefully
reduced in volume in rotary evaporators operated at about 100 mm pressure and
temperatures not exceeding 25° C.
The contents of activated carbon columns are emptied into clean borosilicate
glass pans, dried, and extracted successively with chloroform and ethanol, using
essentially the methods of Buelow, Carswell, and Symons (5). The carbon chloro-
form and carbon alcohol extracts are reduced in volume as needed for further study.
Columns of each adsorbent, which are prepared exactly as those used for
sampling but which are allowed to contact only organic-free water, are processed
along with the sampling columns to provide blank extracts or eluates.
16
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Figure 8. Sampling unit and receivers installed at field site.
17
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The eluates and extracts produced by the sampling procedures described
above comprise essentially uncontamlnated concentrates of many of the trace
organic constituents of the sampled ground water. They are suitable for analysis
by currently available methods to obtain valuable information concerning the
identity and quantity of organic pollutants in ground water (8).
18
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SECTION 5
SAMPLING OF SUBSURFACE SOLIDS
Subsurface earth solids, as well as ground water, must be sampled and
analyzed for organic pollutants and microorganisms if investigations pertaining
to the potential impact on ground water quality of activities releasing pollut-
ants into the earth's crust are to be effective. There are several principal
reasons for this requirement.
1. Only by analysis of earth solids from the unsaturated zone under-
lying pollutant-releasing activities can those pollutants which
are moving very slowly toward the water table because of sorption
and/or physical impediment be detected and their rates of movement
and degradation measured. Such pollutants, which probably include
a major proportion of organics and microorganisms, are not likely
to be detected in ground water until the activities releasing
them have been in operation for protracted periods. Because of
their potential for long-term pollution of ground water, it is
imperative that the behavior of these pollutants in the subsurface
be established at the earliest practicable time.
2. Analyses of organic pollutants in solid samples from the zone of
saturation are needed for a realistic evaluation of the total
extent and probable longevity of organic pollution in an aquifer.
Such analyses provide a measure of the quantity of pollutants
which are sorbed on aquifer solids and which are in equilibrium
with, and in essence serve as a reservoir for, pollutants in
solution in the adjacent ground water.
3. Microbial populations which may be involved in the biological
alteration of pollutants in subsurface formations are likely to
be in such close association with subsurface solids that they
will not be present in well waters in numbers which are quantita-
tively indicative of their presence in the formations; hence,
analysis of subsurface solids are needed for accurate evaluation
of such populations.
4. Even when the best well construction and ground-water sampling
procedures are used, it is difficult to completely eliminate the
possibility that contaminating surface microbes may be present in
ground-water samples. Solids taken from the interior of cores
carefully obtained from the zone of saturation probably provide
the most authenic samples of aquifer microorganisms that can be
obtained.
19
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Successful sampling of subsurface earth solids for organics and microorga-
nisms requires both acquisition of cores of subsurface solids at desired depths
in a manner minimizing potential contamination and proper handling and processing
of the core material obtained to insure its integrity and produce samples suitable
for determinative analytical procedures.
ACQUISITION OF CORES
In most of the studies conducted thus far by the Ground Water Research
Branch, sampling depths have been shallow enough and strata have been sufficiently
compact to permit use of an auger and dry-tube coring procedure for acquisition of
cores. In this procedure, a hole is opened to the desired sample depth by augering
and a "dry-tube" core sampler consisting of a simple steel tube approximately 46 cm
(18 in.) in length and 7.6 cm (3 in.) in diameter is then placed on the bottom of
the hole and pushed into the undisturbed formation. The core barrel is usually
fitted with a steel drive shoe of slightly smaller inside diameter than that of
the barrel to facilitate removal of the core. The core is retained in the barrel
by wall friction. The augering-coring procedure is repeated sequentially to
obtain a succession of cores until the maximum desired sampling depth is attained.
If needed, the borehole opened in the coring operation is completed as a ground-
water sampling well.
Contamination problems are minimized in the auger and dry-tube coring proced-
ure, principally because no drilling fluid is used. However, the procedure has
been utilized to maximum depths no greater than 25 ft to date and is subject to
definite problems when sampling in relatively loose, poorly compacted formations,
particularly in saturated zones. The use of a "hollow-stem" auger to hold the
hole open during sampling and modification of the sampler to provide better
retention of the core material in the tube offer some hope for extending the
utility of the procedure, but these techniques have not been evaluated in the
field.
For deeper sampling where use of drilling fluids is necessary, limited use
has been made of a piston sampler for collecting organic samples. The piston
sampler employs a sample tube identical to the dry-tube core barrel but drilling
fluid pressure is used as the driving force. The sampler sits on the bottom of
the borehole at the end of the drill stem. A shear pin maintains the sample
tube in an "up" position until a vent plug is dropped down the drill stem to the
sampler. Pressure of the drilling fluid then shears the pin, forcing the tube
through the bottom of the hole into the uncored material below the borehole.
Considerable additional work is needed, however, to develop optimum methods for
obtaining core samples with the piston sampler and to evaluate the efficiency
of this device for sampling of organics and microorganisms. In particular, the
extent of contamination of cores by drilling fluids and methods for avoiding such
contamination need to be further explored.
HANDLING AND PROCESSING OF CORE MATERIALS
As soon as a core is obtained, the drive shoe is removed and the sample tube
is placed into a hydraulic extruding device (Figure 9). As the core sample is
forced out of the tube, the first 5 to 8 cm (2-3 in.) are cut off with a sterile
scalpel and discarded, or used for analyses of chemical or physical parameters.
20
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SAMPLE
HYDRAULIC CYLINDER
Figure 9. Core extruding device.
-------
The center of the core is then subsampled to obtain sample material suitable for
microbial analysis by pushing a sterile 1.3 cm (0.5 in.) I.D. stainless steel
tube into the core for about 15 cm (6 in.), as shown in Figure 10. The sub-
sample is extruded with a sterile rod into appropriate containers. By subsampling
the interior of the original core, microbial contamination from the core barrel
and coring operations can be prevented (9).
The type of microbiological sample container which is used is dependent on
the type of analysis to be performed. For culturing of aerobic organisms any
sterile container is suitable if analyses are to be performed within a few hours.
If there is to be a significant delay before the sample is used, care is exer-
cised to keep the sample in a manner that prevents major changes in the microbial
content. Thus, polyethylene bags, which allow the passage of air but not water
vapor, are used as sample containers if aerobic organisms are to be cultured in
order to give the samples access to air and yet keep them from drying. Samples
are maintained if possible at the temperatures at which they are sampled. Other-
wise they are refrigerated.
Since subsurface environments of any depth are usually reducing in nature,
the enumeration and identification of anaerobic microorganisms is essential if
the total microbial composition of the system is to be known. Because many
anaerobic bacteria are known to be extremely sensitive to oxygen, it is important
that samples which will be used in anaerobic culturing procedures be handled in
a manner that minimizes exposure to air. This is accomplished by extruding sub-
samples into sterile glass tubes from which the air is replaced quickly with an
oxygen-free gas. Two methods have been utilized for air removal and replacement.
In one method the sample tube is closed with a cotton plug and placed in an
anaerobic jar from which the oxygen is removed either by catalytic means or by
the use of a vacuum pump-replacement gas system (usually oxygen-free nitrogen).
In the second method, the sterile glass tube containing the subsample is fitted
with a gas-tight rubber septum stopper. A needle is pushed through the septum
and the tube is evacuated with a vacuum pump and filled with a sterile, oxygen-
free gas such as nitrogen. This process of evacuation and gas replacement is
repeated at least three times.
Samples to be analyzed for ATP (Adenosine Triphosphate) content are taken in
the same manner as microbial samples. Subsamples are placed into sterile metal
cans which are closed and immediately placed into liquid nitrogen contained in
an insulated polystyrene box. Frozen samples are returned to the laboratory on
dry ice and stored at -45° C in a low temperature freezer until being analyzed.
After a sub-core for microbial analysis has been removed from the parent core,
a 10 cm (4 in.) length of core material for organic analysis is obtained. This is
achieved in two steps. First, a 5 cm (2 in.) section of core is extruded from
the barrel, carefully detached by means of a clean spatula or scalpel, and dropped
directly into a thoroughly cleaned 14 x 8 x 5 cm (5-11/16 x 3-1/4 x 2 in.) disposable
aluminum baking pan. The procedure is then repeated to obtain a second 5 cm section
of core in the same pan. The pan is covered tightly with clean aluminum foil and
placed in an insulated polystyrene box containing liquid nitrogen to quick-freeze
the sample material. A maximum of two organic samples are usually obtained from
a single 46 cm parent core. Organic and microbial samples are ordinarily taken
from the same location in the core, as shown in Figure 11, to facilitate data
correlation.
22
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Figure 10. Obtaining a subsample for microbial
analysis from a parent core.
23
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E
u
n
O
_ i
O
MICROBIAL SAMPLE
1.3 X 15.2 cm
ORGANIC SAMPLE
7.6 X 10.2 cm
MICROBIAL SAMPLE
1.3 X 15.2 cm
ORGANIC SAMPLE
7.6 X 10.2 cm
Figure 11. Typical location of microbial and
organic samples in a parent core.
24
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The frozen samples of earth solids are returned to the laboratory on dry ice,
stored temporarily at -45° C in a low-temperature freezer, and freeze-dried as soon
as possible in a bulk type freeze drier (Universal Sub-Mobile 15, The Virtis
Company, Inc., Gardiner, NY). Each sample of dried solids is carefully crushed
and mixed to obtain a better degree of homogeneity. These samples are then trans-
ferred to thoroughly cleaned 475 ml (16 oz) wide-mouth jars with Teflon lined caps
and stored at -45° C until subjected to further processing or analysis.
Samples of dried core material are subjected to gross organic analysis, such
as total organic carbon, without further processing. Samples suitable for more
definitive organic analysis, including identification of individual compounds,
are prepared by solvent extraction of the solid samples. This is achieved by
extracting samples consisting of approximately 150 g of dried solids in large
Soxhlet extractors for 48 hr with an azeotropic mixture of 87% chloroform and
13% methanol. Teflon fiber extraction thimbles (43 x 123 mm "Zitex," Chemplast,
Inc., Wayne, NJ) are used. New thimbles are pre-extracted for 72 hr with chloro-
form-methanol (87:13) to remove manufacturing impurities, while used thimbles are
washed in detergent solution in an ultrasonic cleaner, thoroughly rinsed in tap
and distilled water, and extracted overnight with chloroform-methanol prior to
reuse.
Chloroform-methanol extracts are passed through pre-extracted glass fiber
filters into 500 ml round bottom flasks, using Millipore 47 mm glass filter
holders modified with 24/40 glass joints on the outlets to eliminate possible
contamination from use of rubber stoppers. The filtered extracts are then
reduced in volume by means of rotary evaporators to provide samples suitable
for comparison and identification studies.
25
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SECTION 6
REFERENCES
1. Cheer, Sue, John H. Gentile, and C. S. Hegre. Improved Methods for ATP
Analysis. Anal. Biochem. 66:102-114, 1974.
2. Bellar, T. A., and J. J. Lichtenberg. Determining Volatile Organics at
Microgram-per-Litre Levels by Gas Chromatography. J. Amer. Water Works
Assn. 66:739-744, 1974.
3. Junk, G. A., J. J. Richard, M. D. Grieser, D. Witiak, J. L. Witiak, M. D.
Arguello, R. Vick, H. J. Svec, J. S. Fritz, and G. V. Calder. Use of
Macroreticular Resins in the Analysis of Water for Trace Organic Contami-
nants. J. Chromatogr. 99:745-762, 1974.
4. Breidenbach, A. W., J. J. Lichtenberg, C. F. Henke, D. J. Smith, J. W.
Eichelberger, and H. Stierle. The Identification and Measurement of
Chlorinated Hydrocarbon Pesticides in Surface Waters. U.S. Department
of the Interior, Federal Water Pollution Control Administration,
Washington, D.C. 1966. pp. 5, 6, 8-12, 33, 34.
-.- j -LJ
5. Buelow, R. W., J. K. Carswell, and J. M. Symons. An Improved Method for
Determining Organics by Activated Carbon Adsorption and Solvent Extraction -
,,-r Parts I and II. J. Amer. Water Works Assn. 65:57-72 and 195-199, 1973.
6. Endres, Horst and Helmut Herman. Preparative and Analytical Separation
of Organic Compounds by Means of Chromatography on Polyamide. Angew.
Chem. Int. Ed. 2:254-260, 1963.
7. Webb, R. G. Isolating Organic Water Pollutants: XAD Resins, Urethane
Foams, Solvent Extraction. EPA-660/4-75-003, U.S. Environmental Protection
Agency, Con/all is, Oregon, 1975. pp. 3-8.
8. Webb, R. G., A. W. Garrison, L. H. Keith, and J. M. McGuire. Current
Practice in GC-MS Analysis of Organics in Water. EPA-R2-73-277, U.S.
Environmental Protection Agency, Corvallis, Oregon, 1973. 91 pp.
9. Parkinson, D., T. R. G. Gray, and S. J. Williams. Methods for Studying
the Ecology of Soil Micro-organisms. International Biological Programme
Handbook No. 19, Blackwell Scientific Publications, Oxford, England, 1971.
p. 7.
26
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/2-77-176
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
SAMPLING FOR ORGANIC CHEMICALS AND MICROORGANISMS
IN THE SUBSURFACE
5. REPORT DATE
August 1977
issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
William J. Dunlap, James F. McNabb, Marion R. Scalf,
and Roger L. Cosby
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Robert S. Kerr Environmental Research Laboratory
Office of Research & Development
U.S. Environmental Protection Agency
Ada, Oklahoma 74820
10. PROGRAM ELEMENT NO.
1BA609
11. CONTRACT/GRANT NO.
N/A
12. SPONSORING AGENCY NAME AND ADDRESS
Robert S. Kerr Environmental Research Lab.-Ada, OK
Office of Research & Development
U.S. Environmental Protection Agency
Ada. Oklahoma 74820
13. TYPE OF REPORT AND PERIOD COVERED
In-House 7/75 - 1/77
14. SPONSORING AGENCY CODE
EPA/600/15
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Procedures currently utilized by the Ground Water Research Branch of the Environ-
mental Protection Agency for sampling for organic pollutants and microorganisms in
ground waters and subsurface earth solids are presented. Technology is described for
construction of wells capable of providing representative, uncontaminated samples of
ground water in compact alluvial formations at relatively shallow depths and for obtain
ing cores of subsurface earth solids suitable for organic and microbial analyses in
similar circumstances. Methods for acquisition of grab samples of ground water suitabl
for total organic and microbial analyses and for analyses of volatile organics are pre-
sented. Continuous sampling of organics in ground waters lying within approximately
7.5 m (25 ft) of the surface by sampling units utilizing selected absorbents is describ
including details of adsorbent columns, configuration of and housings for sampling sys-
tems, and sample handling. Procedures for handling and processing of core materials to
produce samples amenable to analytical methods for organics and microorganisms are also
presented.
The procedures described provide a basic capability for sampling for organic
pollutants and microorganisms in relatively shallow subsurface environments, and have
potential application in many investigations pertaining to ground-water pollution.
Additional research is needed, however, to further evaluate, improve, and extend their
capabilities. This report covers a period from July 1975 to January 1977. and work was
d,
^pmpietea as or nay iy//
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Ground Water
Sampling
Organic Compounds
Microorganisms
Subsurface Investigations
Coring
Sampling Wells
Core Acquisition
Organic Sampling
13B
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
35
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
27
U.S. GOVERNMENT PRINTING OFFICE: 1977-757-056/6529 Region No. 5-11
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