FINAL REPORT
TC-3991-04
INTR0DIWI0N
Prepared by:
TETRA TECH, INC.
P/epared for:
l|.S. ENVIRONMENTA^^OTECTION AGENCY
Region <10 - Office of ihiget Sound
Seattle, WA
and
U.S. Army Corps ;of Engineers
Seattle District
Seattle; WA
March, 1986
TETRA TECH, INC.
11820 Northup Way
Bellevue,'WA 98005
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CONTENTS
Page
PURPOSE 1
SCOPE 2
APPROACH 4
FORMAT 5
CAVEATS 6
11
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FIGURES
Number
Participants of the variable-ranking workshop held on
31 May, 1985
ACKNOWLEDGEMENTS
This chapter was prepared by Tetra Tech, Inc., under the direction
of Dr. Scott Becker, for the U.S. Environmental Protection Agency in partial
fulfillment of Contract No. 68-03-1977. Dr. Thomas Ginn of Tetra Tech was
the Program Manager. Mr. John Underwood and Dr. John Armstrong of U.S. EPA
were the Project Officers.
111
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Introduction
Purpose
March 1986
PURPOSE
Environmental variables in Puget Sound are measured by a wide variety
of organizations, including government agencies, universities, and private
institutions. However, comparisons of results of different studies frequently
are limited because different methods are used to measure the same vari-
able(s). The ability to compare data among different studies is highly
desirable for developing a comprehensive management strategy for the Sound.
This document (i.e., notebook) presents recommended protocols for
measuring selected environmental variables in Puget Sound. The objective is
to encourage most investigators conducting studies such as monitoring programs,
baseline surveys, and intensive investigations to use equivalent methods
whenever possible. If this objective is achieved, most data from future
sampling programs should be comparable among studies. It is recognized that
alternative methods exist for many of the variables considered in this
document and that those methods may produce data of equal or better quality
than do the recommended methods. However, the criterion that data should be
comparable limited the range of methods recommended in this document. It is
also recognized that future research or other circumstances may require
modification or replacement of one or more of the recommended methods. The
loose-leaf format of this document was selected specifically to allow such
changes to be made.
The recommendations in this document pertain primarily to the method-
ological specifications required to measure the selected environmental
variables. Recommendations for study design and data analysis generally
were not included because those considerations vary widely depending upon
the objectives of individual studies. As mentioned previously, the goal of
this document is to ensure that comparable data are generated by different
studies. This does not necessarily require that all studies have the same
initial design, nor that all data are analyzed in the same manner after
being generated. It is recommended, however, that sample collection and
analysis specifications of study designs be similar enough to ensure that
comparable data are produced whenever possible.
As an action separate from the preparation of this document, several of
the recommended protocols will be required for use in government regulatory
permit programs. For example, the Puget Sound Dredged Disposal Analysis
(PSSDA) intends to specify several of the recommended protocols as requirements
when conducting dredged material regulatory testing and disposal site assess-
ments. Use of such standardized procedures is essential for making comparisons
to regulatory standards and reference conditions.
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Introduction
Scope
March 1986
SCOPE
A meeting was convened by U.S. EPA on 31 May, 1985 (Table 1) to determine
the priority of variables for protocol development or documentation. Variables
were ranked on the basis of three major criteria:
1. The frequency with which each variable has been measured in a
variety of studies (e.g., monitoring programs, baseline
surveys, intensive investigations) throughout Puget Sound
2. The importance of each variable for making decisions related
to environmental problems in the Sound
3. The degree to which a variety of methods has been used to
measure each variable.
Using the criteria listed above, eight groups of variables were identified
as having the highest priority for protocol development or documentation.
They include:
• Station positioning considerations
• Conventional sediment variables
• Concentrations of organic compounds in sediment and tissue
• Concentrations of metals in sediment and tissue
• Benthic infaunal variables
t Sediment bioassays
t Pathological conditions in fish livers
• Microbiological indicators.
Recommended protocols for all of these variables are presented in later
sections of this document. In addition to these eight groups of variables,
a number of others were considered appropriate for protocol development.
The loose-leaf format of this document will allow additional protocols to be
included in the future.
In addition to the recommended protocols for each group of variables, a
section on general quality assurance/quality control (QA/QC) procedures is
included in this document. That section identifies the major QA/QC concerns
that should be addressed when collecting and analyzing environmental samples
from Puget Sound.
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TABLE 1. PARTICIPANTS OF THE
VARIABLE-RANKING WORKSHOP HELD
ON 31 MAY, 1985
Name
Organization
John Armstrong
Bob Barrick
Scott Becker3
Joe Blazevich
Don Brown
Joe Cummins
Ray Dalseg
Bob Dexter
Burt Hamner
Ed Long
Dave Mitchell
Paillette Murphy
Jeff Osborn
Jerry Stober
Rich Tomlinson
Jack Word
U.S. EPA
Tetra Tech, Inc.
Tetra Tech, Inc.
U.S. EPA
NOAA
U.S. EPA
Metro
EVS Consultants
U.S. COE
NOAA
Metro
NOAA
Parametrix, Inc.
Univ. of Wash.
Metro
Evans Hamilton, Inc.
a Moderator.
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Introduction
Approach
March 1986
APPROACH
The recommended protocols for each group of variables were developed by
convening a workshop comprised of representatives from most organizations
that routinely measure or use the variables of concern in Puget Sound. The
objective of each workshop was to evaluate various methods and, if possible,
agree upon which methods should be used in the future. Consideration was
given to providing data that will be comparable with the historical database.
Prior to each workshop, the methods used historically in Puget Sound were
evaluated and specific items requiring standardization were identified.
Additional considerations for developing the various recommended protocols
included data quality needs, cost, and availability of equipment and expertise.
Each workshop focused on defining acceptable methods and determining
which of those methods would provide comparable data. If several acceptable
methods did not provide comparable data, the workshop participants were
asked to select only one for future use. As expected, a full consensus
rarely was achieved. However, in many cases, the majority of participants
clearly favored a single method. In other instances, the participants were
relatively evenly divided between recommending two or more methods.
After each workshop, draft protocols were developed. As much as possible,
recommendations of each protocol were based on the majority viewpoint of the
workshop participants. In some cases, a single recommendation could not be
given for a particular specification because no agreement was reached at the
workshop. In such instances, various specifications used by different Puget
Sound investigators were simply described.
Draft protocols were mailed to all workshop participants and other
interested parties for written review. Following this review, comments made
by several reviewers were incorporated into the protocols. Most major
comments made by single reviewers were resolved with each respective reviewer.
After all written reviews were addressed, protocols were finalized and
included as a chapter of this document.
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Introduction
Format
March 1986
FORMAT
Each protocol in this document is designed to stand alone. However, in
many studies several related variables are measured simultaneously. In such
cases, the protocol for one variable may require some modification to be
consistent with that of a second variable. For example, collection of
sediment subsamples for analysis of conventional variables (e.g., particle
size, total volatile solids, total organic carbon) normally does not require
collection equipment to be washed with special solvents. However, if sediment
subsamples also will be collected from the same sample for analysis of
organic compounds, collection equipment for the conventional subsample must
be washed with the same solvent specified for collection of the organics
subsample to avoid contaminating the latter sediment. For studies considering
multiple variables, it is therefore recommended that the protocols for all
relevant variables be reviewed carefully before sampling begins, to ensure
that all appropriate modifications are made.
The formats for most protocols are similar to facilitate use of the
entire document. The following major sections are presented for most protocols:
• Use and limitations - describes what a variable measures and
major limitations to the use of the variable
• Field procedures - describes container type, special cleaning
procedures, collection techniques, sample quantity, preservation
technique, storage conditions, and maximum holding time
• Laboratory procedures - describes analytical procedures (or
provides citations), laboratory equipment, sources of error,
and QA/QC specifications
• Data reporting requirements - describes the kinds of data
that the analytical laboratory should report and the units in
which the data should be reported.
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Introduction
Caveats
March 1986
CAVEATS
Several notes of caution require emphasis before the protocols in this
document are presented. First, these protocols were developed solely to
promote the collection of comparable data in Puget Sound. A variety of
other methods may exist that produce data of equal or better quality than
the recommended protocols. However, the criterion that data should be
comparable limited the range of methods recommended in this document.
A second caveat is that rarely was a full consensus reached with respect
to any aspect of any protocol. Therefore, it should not be construed that
all individuals, agencies, and institutions that participated in this effort
agreed with all of the final products. The recommended protocols are simply
a best effort to represent the majority viewpoints of the many individuals
from diverse backgrounds that attended the workshops or commented on the
draft protocols.
A third caveat is that this document is intended to be dynamic. The
loose-leaf format was selected specifically for this reason. Modifications
or additions to the protocols can therefore be made in the future if needs
or viewpoints change, or if methodological refinements or improvements are made.
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GENQIALQA/QC
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FINAL REPORT
TC-3991-04 Puget Sound Estuary Program
GENERAL QA/QC CONSIDERATIONS
FOR COLLECTING ENVIRONMENTAL
SAMPLES IN PUGET SOUND
Prepared by:
TETRA TECH, INC.
Prepared for:
U.S. ENVIRONMENTAL PROTECTION AGENCY
Region 10 - Office of Puget Sound
Seattle, WA
March, 1986
TETRA TECH, INC.
11820 Northup Way
Bellevue, WA 98005
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CONTENTS
Page
LIST OF FIGURES iii
LIST OF TABLES iv
ACKNOWLEDGEMENTS v
INTRODUCTION 1
SAMPLING PREPARATION 2
SAMPLING PROCEDURES 5
HEALTH AND SAFETY 5
STATION LOCATION 5
SAMPLE HANDLING 6
FIELD PROCEDURES 6
SAMPLE SHIPMENT 7
LABORATORY PROCEDURES 11
SHIPBOARD LABORATORY ANALYSES 12
REFERENCES 14
11
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FIGURES
Number Page
1 An example of a chain-of-custody record 8
2 Examples of a sample analysis request form and a custody
seal 9
TABLES
Number Page
1 Examples of problems frequently encountered during offshore
surveys and recommended solutions to each problem 4
ACKNOWLEDGMENTS
This chapter was prepared originally by Tetra Tech, Inc., for the
Marine Operations Division, Office of Marine and Estuarine Protection,
U.S. EPA, Washington, DC as part of U.S. EPA Contract No. 68-01-6938, Allison
J. Duryee, Project Officer. It was prepared under the direction of Dr. Thomas
Ginn (Program Manager) and Dr.Scott Becker (Project Manager) of Tetra Tech.
Ill
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General QA/QC
Introduction
March 1986
INTRODUCTION
Quality assurance/quality control (QA/QC) procedures are necessary to
ensure that environmental data achieve an acceptable level of quality and
that the level of quality attained is documented adequately. Detailed QA/QC
procedures for the environmental variables considered in this document are
specified in each respective recommended protocol. This section describes
many of the more general QA/QC procedures that should be considered when
collecting and analyzing environmental samples. These procedures are termed
general because they pertain to samples collected for most kinds of environ-
mental variables. They include procedures that should be followed when
samples are collected in the field, shipped to laboratories, and stored or
distributed within laboratories.
No workshop was held to evaluate the general QA/QC procedures for use
in Puget Sound. However, they have been reviewed for use in U.S. EPA's
301(h) marine monitoring programs by U.S. EPA's Office of Research and
Development (Narragansett, RI and Newport, OR), as well as by representatives
from U.S. EPA Regions IX and X.
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General QA/QC
Sampling Preparation
March 1986
SAMPLING PREPARATION
The chief scientist or a designee should thoroughly review the field
survey plan (including QA/QC criteria) before each cruise. The plan should
be checked for completeness and clarity of objectives. A complete plan
should contain the following major elements:
t Identification of scientific party and the responsibilities
of each member
• Statement and prioritization of study objectives
• Description of survey area, including background information
and station locations
t Identification of variables to be measured and corresponding
required containers and preservatives
t Identification of all sample splits or performance samples to
be submitted with the survey samples
• Brief description of sampling methods, including station
positioning technique, sampling devices, replication, and any
special considerations
• Detailed cruise schedule, including time, date, and location
of embarkation and debarkation
• Storage and shipping procedures
• Identification of onshore laboratories to which samples
should be shipped after cruise completion
• Survey vessel requirements (e.g., size, laboratory needs,
sample storage needs)
0 Location and availability of an alternate survey vessel
• All special equipment needed for the survey (e.g., camera,
nets, communication devices).
Study objectives and their prioritization should be understood by all
members of the scientific party. This will ensure that if modifications of
the survey plan become necessary in the field, their impact on the overall
goals of the cruise can be evaluated adequately. After the sampling plan
has been reviewed, contingency plans should be outlined. These plans should
include potential problems and their solutions. Possible solutions to some
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General QA/QC
Sampling Preparation
March 1986
problems frequently encountered during offshore surveys are listed in Table
1. Development of contingency plans can be greatly assisted by reviewing
cruise summary reports and consulting with chief scientists of previous cruises.
The captain of the survey vessel should be provided with a copy of the
survey plan to ensure that it is consistent with the equipment and capa-
bilities of the vessel. Modifications to the ship or cruise plan may be
required.
To ensure that all required sampling equipment and supplies are available
at the time of sampling, an equipment checklist should be constructed.
Spare parts and backup supplies should be included in the inventory.
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TABLE 1. EXAMPLES OF PROBLEMS FREQUENTLY ENCOUNTERED DURING
OFFSHORE SURVEYS AND RECOMMENDED SOLUTIONS TO EACH PROBLEM
Problem
Recommended Solution(s)
Sampling equipment falls or 1s lost overboard
Sampling efficiency reduced by poor weather
Sampling efficiency reduced because of seasickness
Sampling delayed because vessel is Inoperative
A sample 1s partially lost or slightly contam-
inated
Bottom sampler will not penetrate to a sufficient
depth after repeated casts
One or more water bottles fail to trip on
a particular cast
- Have necessary tools onboard to make repairs
• Maintain spare parts Inventory for major
equipment
- Have back-up equipment onboard
- Have SCUBA equipment and divers onboard
for retrieval
- Know where nearest tools, back-up equipment.
or divers are located on shore
- Extend cruise length
- Reschedule cruise
- Ensure scientific crew is large enough
to compensate for reduced personnel
- See solutions to second problem
- Reschedule cruise
- Extend cruise length once vessel Is repaired
- Charter an alternate vessel
- Discard and take another sample
Add weight to the sampler
Move a short distance before taking next
sample
Discard all samples from that cast and
take a new cast
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General QA/QC
Sampling Procedures
March 1986
SAMPLING PROCEDURES
HEALTH AND SAFETY
Guarding the health and safety of the sampling team not only is mandatory
in itself, but also ensures that such concerns do not interfere with the
collection of quality data. It is recommended that all members of the
sampling team wear hard hats and Coast Guard-approved personal flotation
devices when working on deck. Whenever water sampling, grab sampling, and
trawling are being conducted, the danger exists that the block, winch, or
hydrowire will fail or that the water sampler, grab, or trawl assembly
(especially the otter boards) will swing out of control. Any one of these
events could knock someone unconscious (or worse), and possibly overboard.
When sampling in areas that are contaminated by toxic materials, special
equipment (e.g., detectors, respirators) may be required by federal or state
regulations. The use of plastic safety glasses when working with sediments
is a good practice. Sediment particles introduced to the eye can cause
abrasion, infection, or chemical contamination.
Perhaps the most common ailment when sampling is "mal de mer" (i.e.,
seasickness). Although much humor has evolved at the expense of the seasick,
sample quality can be seriously compromised by impaired deck hands. It is
therefore recommended that members of the sampling team who are prone to
seasickness take appropriate medication (e.g., dramamine, scopolamine) prior
to the cruise.
STATION LOCATION
Accurate navigation is essential to ensuring that stations can be
plotted and reoccupied with a high degree of certainty. Although a variety
of navigation and/or position fixing systems are available currently, factors
such as price, availability, and accuracy vary considerably among them. The
station positioning system selected for a given survey should be able to
meet all study design requirements for accuracy and should, at a minimum,
provide a high degree of precision (i.e., repeatablemeasurements). Positioning
systems that are precise but lack a high degree of accuracy may be used
after actual station locations are determined by accurate, independent means
(i.e., "ground-truthed"). For bottom-related samples, all positioning
systems should be used in conjunction with a fathometer to ensure that
sampling occurs at the proper water depth (allowing for tidal stage and any
fathometer corrections). The protocols for station positioning presented in
a later chapter of this notebook should be consulted for details related to
locating stations.
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General QA/QC
Sample Handling
March 1986
SAMPLE HANDLING
After sample collection, proper sample handling ensures that changes in
the constituents of interest are minimized and guards against errors when
shipping and analyzing samples. All stages of sample handling should be
documented adequately. Documentation helps ensure that all sample handling
requirements are carried out and serves as proof that handling was conducted
properly if questions arise later.
FIELD PROCEDURES
It is important throughout any sampling and analysis program to maintain
integrity of the sample from the time of collection to the point of data
reporting. Proper chain-of-custody procedures allow the possession and
handling of samples to be traced from collection to final disposition.
Documents needed to maintain proper chain-of-custody include:
• Field logbook — All pertinent information on field activities
and sampling efforts should be recorded in a bound logbook.
The field supervisor should be responsible for ensuring that
sufficient detail is recorded in the logbook. The logbook
should enable someone else to completely reconstruct the
field activity without relying on the memory of the field
crew. All entires should be made in indelible ink, with each
page signed and dated by the author, and a line drawn through
the remainder of any page. All corrections should consist of
permanent line-out deletions that are initialed. At a minimum,
entries in a logbook should include:
Date and time of starting work
Names of field supervisor and team members
Purpose of proposed sampling effort
Description of sampling site, including information on
any photographs that may be taken
Location of sampling site
Details of actual sampling effort, particularly deviations
from standard operating procedures
Field observations
Field measurements made (e.g., pH, temperature, flow)
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General QA/QC
Sample Handling
March 1986
Field laboratory analytical results
Sample identification
Type and number of sample bottles collected
Sample handling, packaging, labeling, and shipping
information (including destination).
Chain-of-custody procedures should be maintained with the
field logbook. While being used in the field, the logbook
should remain with the field team at all times. Upon completion
of the sampling effort, the logbook should be kept in a
secure area.
• Sample labels — Sample labels must be waterproof and must be
securely fastened to the outside and/or placed inside each
sample container (depending on the kind of sample) to prevent
misidentification of samples. Labels must contain at least
the sample number, preservation technique, date and time of
collection, location of collection, and signature of the
collector. Labels should be marked with indelible ink.
Abbreviated labels may also be placed on the cap of each jar
to facilitate sample identification.
• Chain-of-custody records — A chain-of-custody record (Figure 1)
must accompany every sample. Each person who has custody of
the sample must sign the form and ensure that the samples are
not left unattended unless secured properly.
• Custody seals — Custody seals (Figure 2) are used to detect
unauthorized tampering with the samples. Sampling personnel
should attach seals to all shipping containers sent to the
laboratory by common carrier. Gummed paper seals or custody
tape should be used so that the seal must be broken when the
container holding the samples is opened.
For further information regarding proper chain-of-custody procedures, consult
the policies and procedures manual for the National Enforcement Investigations
Center (NEIC; U.S. EPA 1978).
SAMPLE SHIPMENT
All preseved samples should be shipped immediately after completion of
sampling. This minimizes the number of people handling samples, and protects
sample quality and security. Guidance for shipping hazardous materials can
be found in U.S. Department of Transportation (1984). As samples are prepared
for shipping, the following should be kept in mind:
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8
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U.S. ENVIRONMENTAL PROTECTION AGENCY
CLP Sample Management Office
P.O. Box 818 - Alexandria, Virginia 22313
Phone: 703/JJ7-2K90 - FTS/JJ7-2490
SAS Number
SPECIAL ANALYTICAL SERVICE
PACKING LIST
Sampling Office:
Sampling Contact:
(name]
(phone)
Sampling DateUh
Date Shipped:
Site Name/Code;
Ship To:
Attn:
For Lab Use Only
Date Samples Rec'd:
Received By:
Sample
Numbers
Sample Description
Analysts, Matrix, Concentration
Sample Condition on
Receipt at Lab
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EPA CUSTODY SEAL
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Figure 2. Examples of a sample analysis request form (above)
and a custody seal (below).
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General QA/QC
Sample Handling
March 1986
• Shipping containers should be in good shape and capable of
withstanding rough treatment during shipping.
• Samples should be packed tightly:
Dividers must separate all glass containers
Empty space within shipping boxes should be filled so that
jars are held securely.
• All containers must be leak-proof. If a container is not
leak-proof by design, the interior should be lined with two
heavy-duty plastic bags and the tops of bags should be tied
once samples are inside. Adequate absorbent material should
be placed in the container in a quantity sufficient to absorb
all of the liquid.
• All samples should be accompanied by a sample analysis request.
Variables to be analyzed by the laboratory, and total number
and kind of samples shipped for analysis should be listed on
the request sheet. An example sample analysis request form
is illustrated in Figure 2. The laboratory should acknowledge
receipt of shipment by signing and dating the form, and
returning a copy to the designated QA coordinator.
• A chain-of-custody record for each shipping container should
be filled out completely and signed.
• The original chain-of-custody record and analysis request
should be protected from damage and placed inside the shipping
box. A copy of each should be retained by the shipping
party.
• The custody seal should be attached so that the shipping box
cannot be opened without breaking the seal.
• For shipping containers carrying liquid samples:
A "This End Up" label should be attached to each side to
ensure that jars are transported in an upright position.
A "Fragile-Glass" label should be attached to the top of
box to minimize agitation of samples.
• Shipping containers should be sent by a carrier that will
provide a delivery receipt. This will confirm that the
contract laboratory received the samples and serve as a
backup to the chain-of-custody record.
10
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General QA/QC
Sample Handling
March 1986
• All shipping charges should be prepaid by the sender to avoid
confusion and possible rejection of package by the contract
laboratory.
LABORATORY PROCEDURES
At the laboratory, one person should be designated custodian of all
incoming samples. An alternate should also be designated to serve in the
custodian's absence. The custodian should oversee the following activities:
• Reception of samples
a Maintenance of chain-of-custody records
• Maintenance of sample tracking logs
• Distribution of samples for laboratory analyses
• Sending of samples to outside laboratories
• Supervision of labeling, log keeping, data reduction, and
data transcription
• Storage and security of all samples, data, and documents.
Upon reception of samples, a designated laboratory person should fill
out the chain-of-custody record, indicating time and date of reception,
number of samples, and condition of samples. All irregularities indicating
that sample security or quality may have been jeopardized (e.g., evidence of
tampering, loose lids, cracked jars) should be noted on the sample analysis
request form and returned to the client-designated QA coordinator. In
addition, a designated laboratory person should initiate and maintain the
sample -tracking log that will follow each sample through all stages of
laboratory processing and analysis.
Minimum information required in a sample tracking log includes the
following:
• Sample identification number
• Location and condition of storage
• Date and time of each removal of and return to storage
• Signature of person removing and returning the sample
• Reason for removal from storage
• Final disposition of sample.
11
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General QA/QC
Sample Handling
March 1986
All logbooks, labels, data sheets, tracking logs, and custody records
should have proper identification numbers and be accurately filled out. All
information should be written in ink. Corrections should be made by drawing
a line through the error and entering the correct information. Corrections
should be initialed and dated. Accuracy of all data reductions and tran-
scriptions should be verified at least twice. All samples and documents
should be properly stored within the laboratory until the client authorizes
their removal. Security and confidentiality of all stored material should
be maintained at all times. Before releasing analytical results, all infor-
mation on sample tags, data sheets, tracking logs, and custody records
should be cross-checked to ensure that data pertaining to each sample are
consistent throughout the record.
Originals of the following documents should be sent to the client:
• Chain-of-custody records
• Sample tracking logs
• Data report sheets
• Quality control records.
Copies of all forms should be retained by the laboratory in case originals
are lost in transit.
SHIPBOARD LABORATORY ANALYSES
Depending upon the size and capabilities of the survey vessel, many
environmental variables can be analyzed on board. In general, the laboratory
procedures described for the protocols in this document are applicable to
both shipboard and land-based laboratories. This consistency is important
to ensuring that analytical results will be comparable regardless of which
kind of laboratory generates them.
Although most laboratory procedures are similar between shipboard and
land-based laboratories, a number of additional factors must be considered
when analyzing samples at sea. These factors relate primarily to the remoteness
of the shipboard laboratory from land-based support and the'movement and
limited space of the survey vessel. The major considerations are as follows:
• The design of the laboratory should be efficient, with convenient
equipment locations and adequate storage space.
• The vessel should be equipped with an uninterruptible power
supply that is adequate for operation of all scientific
instruments.
12
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General QA/QC
Sample Handling
March 1986
The laboratory should be well ventilated to remove any toxic
vapors created by chemicals.
The temperature of the laboratory should be controlled,
especially if variations in ambient temperature can influence
particular analyses.
Adequate lighting is necessary, especially for analyses
requiring color discrimination (e.g., titration endpoints).
The laboratory should have adequate water purification apparatus
or be capable of storing water purified on shore.
For storing many kinds of samples, adequate refrigeration and
freezing capabilities are desirable.
The laboratory should never be used as a general passageway
or lounge.
The laboratory should be off limits to unauthorized personnel.
Adequate safety and first aid equipment should be on board,
preferably including an overhead quick-pull safety shower.
Extreme care must be taken when handling samples (for quality
purposes) and hazardous reagents (for safety purposes), as
vessel movement can sometimes be unpredictable.
Backup supplies and instruments should be on board so that
sampling can continue if a piece of equipment is broken or
will not operate properly. A continuously updated inventory
tracking system is useful for maintaining backup equipment.
All equipment should be properly secured to compensate for
predictable and unpredictable vessel movements. Specially
designed racks are useful for this purpose.
Instruments should be checked and calibrated before sailing,
so that problems requiring land-based assistance can be
solved quickly.
Whenever possible, plastic containers should be used instead
of glass because plastic is less susceptible to breakage.
Instruments having digital displays are preferred over those
using needles.
Pre-printed data sheets should be used to ensure that all
required information is recorded.
13
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General QA/QC
References
March 1986
REFERENCES
U.S. Department of Transportation. 1984. Hazardous materials regulations.
U.S. DOT, Washington, DC. Federal Register Vol. 49, Chapter 1, Subchapter
C. pp. 52-792.
U.S. Environmental Protection Agency. 1978 (revised 1983). NEIC policies
and procedures. EPA-330/9-78-001-R. National Enforcement Investigations
Center, Denver, CO.
14
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STATION POSmONING
-------
FINAL REPORT Pygf|J?ounff Estuary Program
TC-3090-05
RECOMMENDED PROTOCOLS
FOR STATION POSITIONING
IN PUGET SOUND
Prepared by:
TETRA TECH, INC.
Prepared for:
RESOURCE PLANNING ASSOCIATES
for:
PUGET SOUND DREDGED DISPOSAL
ANALYSIS (PSDDA)
Monitored by:
U.S. Army Corps of Engineers
Seattle District
Seattle, WA
August, 1986
TETRA TECH, INC.
11820 Northup Way
Bellevue, WA 98005
-------
CONTENTS
Page
LIST OF FIGURES iv
LIST OF TABLES vi
ACKNOWLEDGMENTS vi i
INTRODUCTION 1
POSITIONING METHOD SELECTION PROCEDURE 3
DEVELOPMENT OF SAMPLING PROGRAM 3
Physical Conditions at the Study Site 5
Equipment and Analyses 5
Station Separation 6
Reoccupation 6
Program-Imposed Constraints 7
DEFINITION OF POSITIONING REQUIREMENTS 7
REVIEW AND SELECTION OF POSITIONING METHOD 10
IMPLEMENTATION OF POSITIONING METHOD 11
RECORDKEEPING REQUIREMENTS 12
FIELD RECORDS 12
Initial Survey Description 12
Day Log Entries 12
Station Log Entries 13
REPORTING REQUIREMENTS 14
RECOMMENDED POSITIONING ACCURACIES 15
CLASSIFICATION OF SAMPLING 15
RECOMMENDED ACCURACY 15
RECOMMENDED POSITIONING METHODS 17
REFERENCES 22
ii
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APPENDICES
APPENDIX A: SITE-RELATED POSITIONING LIMITATIONS A-l
APPENDIX B: POSITION ERROR ANALYSIS B-l
APPENDIX C: POSITIONING METHODS AND CONSIDERATIONS FOR SAMPLING IN
PUGET SOUND C-l
OPTICAL POSITIONING TECHNIQUES C-l
ELECTRONIC POSITIONING TECHNIQUES . C-14
RANGE-AZIMUTH SYSTEMS C-43
REFERENCES C-49
APPENDIX D: EVALUATION OF POSITIONING METHODS D-l
POSITIONING PROCEDURES IN USE IN PUGET SOUND D-l
CANDIDATE SYSTEM OVERVIEW D-4
SCREENING CRITERIA D-6
m
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FIGURES
Number Page
1 Positioning method selection procedure 4
2 Components of error in probable area of sample collection 9
3 Comparison of approximate accuracy ranges of some positioning
methods 20
APPENDIX A
A-l Regions of Loran-C signal interference in Puget Sound A-3
APPENDIX B
B-l Line of position measurements for two shore stations
depicting LOP uncertainty (a), and associated error
indicator (b) B-2
B-2 Angle-of-cut effects on fix accuracy B-4
B-3 Illustration of radial error B-7
B-4 Components of error in probable area of sample collection B-10
B-5 Effects of wire angle on the probable (P=0.68) area of
sample collection B-ll
B-6 Effects of navigational accuracy on the probable (P=0.68)
area of sample collection B-13
APPENDIX C
C-l Station positioning by theodolite intersection C-4
C-2 Station fix using position circles C-8
C-3 Three-arm protractor for sextant resections C-9
C-4 Shore target locations to avoid the danger circle C-ll
C-5 Operating modes for radio navigation systems C-19
C-6 Seasonal variations in Loran-C signals at Neah Bay,
Washington 5990 C-32
IV
-------
C-7 Regions of Loran-C signal interference in Puget Sound C-35
C-8 Range-azimuth positioning system area of coverage C-44
APPENDIX D
D-l Approximate accuracy versus distance of some positioning
methods D-8
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TABLES
Number Page
1 Contributors to the station positioning protocols 2
2 Classification of common types of sampling 16
3 Summary of acceptable positioning methods 18
APPENDIX B
B-l Probability versus R/o for elliptical bivariate distributions
with two equal standard deviations B-5
B-2 Circular error probabilities as a function of measurement
standard deviation ratios and error circle radius B-8
APPENDIX C
C-l Summary of vessel positioning methods C-2
C-2 Summary of vernier transit and scale-reading theodolite
characteristics C-6
C-3 Summary of micrometer and digitized theodolite characteristics C-7
C-4 Marine sextant characteristics C-13
C-5 Variable Range Radar (VRR) system characteristics C-15
C-6 Electronic positioning system categories C-17
C-7 Electronic distance measuring instruments C-21
C-8 Total station characteristics C-23
C-9 Short-range positioning system characteristics C-25
C-10 Medium-range positioning system characteristics C-29
C-ll Long-range positioning system characteristics C-30
C-12 Range-azimuth positioning system characteristics C-45
APPENDIX D
D-l Interviewed governmental and private groups that conduct
sampling in Puget Sound D-2
vi
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0-2 Characteristics of positioning methods used for sampling in
Puget Sound D-3
D-3 Evaluation of navigation methods for station positioning D-7
D-4 Typical equipment rental costs (1985 dollars) D-13
ACKNOWLEDGEMENTS
This chapter was prepared by Tetra Tech, Inc., under direction of
Mr. Jeff Stern, for Resource Planning Associates (RPA) for the Puget Sound
Dredged Disposal Analysis (PSDDA), monitored by the Seattle District, U.S. Army
Corps of Engineers (COE) in partial fulfillment of Contract No. DACW67-85-D-
0029. Dr. David Kendall of the U.S. COE was the Technical Coordinator,
and Mr. Gary Bigham of Tetra Tech was the Program Manager. Dr. William
Muellenhoff of Tetra Tech coauthored the appendix on positioning methods
and helped resolve many of the technical issues that arose as the protocols
were being prepared.
VI1
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Station Positioning
Introduction
August 1986
INTRODUCTION
This chapter presents recommended procedures for station positioning
during environmental sampling in Puget Sound. Specifically, the objectives
of this chapter are to:
• Recommend methods for selecting station positioning methods
• Recommend positioning accuracies and associated methods
for specific kinds of sampling.
The recommendations in this chapter are based on the results of a
workshop and written reviews by representatives of most agencies, institutions,
and private firms that fund or conduct environmental research in Puget
Sound (Table 1). At the workshop, it was agreed that no single positioning
method would be adequate for all sampling scenarios. Instead, the group
recommended development of procedures to select appropriate positioning
methods for specific sampling objectives and to ensure consistent implementation
of those methods. A standardized selection procedure was recommended that:
• Accommodates different sampling objectives and site-specific
conditions
• Does not limit the positioning methods that can provide
the accuracy needed or specified
• Allows investigators to use familiar or available systems
• Allows the funding agencies to specify levels of accuracy
appropriate for sampling objectives or database entry
• Incorporates positioning accuracy as a component of sampling
study design regardless of scientific approach.
The description of the selection procedure begins with a review of
interrelationships between sampling program design and positioning require-
ments. Guidance for defining project-specific requirements is provided.
The procedures and equipment that can provide the required accuracies are
identified and an evaluation procedure for comparing candidate positioning
methods is presented. Requirements to standardize the implementation of
positioning methods and reporting of station locations are also presented.
Finally, accuracy levels and corresponding positioning methods are recommended
for three categories of sampling activities (areal samples, point samples,
and special studies).
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TABLE 1. CONTRIBUTORS TO THE STATION POSITIONING PROTOCOLS
Name Organization
John Armstrong* U.S. EPA
Gary Bighama»B Tetra Tech, Inc.
John Oermodya Raven Systems
Ben Huntley3 Sea-Lease, Inc.
David Jamison3 WDNR
Karl Kassebaum U.S. EPA
David Kendall3 U.S. COE
Gary Mausetha Nortec
Gary Minton3 Resource Planning Assoc.
Bill Muellenhoff3 Tetra Tech, Inc.
Bob Parker U.S. COE
Tony Petrillo3 Nortec
Keith Phillips3 U.S. COE
Anthony Roth3 Cooper Consultants
Debra Simecek-Beatty3 NOAA
Jeff Sterna»b Tetra Tech, Inc.
Steve Til ley3 WDNR
Bruce Titus3 URS Engineers
Frank Urabeck U.S. COE
Pete Wilkinson3 Battelle Northwest
3 Attended the workshop held on January 27, 1986.
b Workshop moderators.
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Station Positioning
Method Selection
August 1986
POSITIONING METHOD SELECTION PROCEDURE
A procedure for identifying thfe positioning accuracy appropriate for
a specific sampling objective and the corresponding positioning methods
is presented in this section. The procedure involves identifying specific
factors of the sampling program that affect the accuracy and feasibility
of positioning methods. Next, the individual stations of the sampling
program are used to determine the specific positioning requirements. Available
systems are then evaluated based on positioning requirements and site-specific
constraints to select an appropriate positioning method. These steps are
followed by approval and procurement of the selected system, assembly and
installation of shore stations (if needed), and equipment calibration and
field-testing. The procedure is presented graphically in Figure 1.
DEVELOPMENT OF SAMPLING PROGRAM
Study design and location are important factors in the evaluation
of positioning methods for environmental sampling vessels. Study design
affects required positioning accuracy and location affects feasibility
and accuracy of some positioning methods. Specific design and location
factors include physical conditions and topography of the study site, proposed
equipment and analyses, minimum station separation, station reoccupation,
and program-imposed constraints. All of these factors need to be considered
prior to sampling to ensure that positioning methods do not compromise
quality or interpretation of the data. The design factor most sensitive
to station location (e.g., requiring the highest positioning accuracy)
will initially define the positioning requirements. This initial estimate
should be re-evaluated during the study design to determine whether positioning
limitations will require changes in the sampling program.
The type of positioning accuracy that is needed to meet the specific
sampling objectives of each study should be identified during the evaluation
of effects of design and location factors on positioning requirements.
Absolute or predictable accuracy refers to a method's ability to correctly
define a position by latitude and longitude (Bowditch 1984). Repeatable
or relative accuracy measures a method's ability to return the user to
the same position time after time. The difference between these two accuracies
can be significant. Some of the study design or location factors may affect
one type of accuracy but not the other. Examples are presented under each
subheading below. It is important to identify which accuracy is of concern
for positioning during a specific sampling program before finishing the
following evaluation of effects on study design.
-------
PURCHASE
OR LEASE
SYSTEM
/FUNDING /
AUTHORIZATION /
Figure 1. Positioning method selection procedure.
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Station Positioning
Method Selection
August 1986
Physical Conditions at the Study Site
The ability of a positioning method to achieve its highest projected
accuracy depends, in part, on site-specific conditions. The accuracy of
a position fix from any two points increases as the angle between the lines-
of-position (LOP) on which the vessel must lie approaches 90 degrees. The
area of probable location that can be resolved by a navigation method (the
radial error) varies among locations because of changes in the relative location
of the fix points and the vessel. The number of objects or targets with known
locations that can be used for a position fix depends on the methods used
and, in some cases, on the surrounding terrain. An acceptable fix target
for one method may not be an acceptable target for another method. A preferred
method may not be usable or sufficiently accurate at all locations. For
example, Loran-C cannot be used in some parts of Puget Sound (see Figure A-l,
Appendix A) and the accuracy of visual sighting methods decreases with
distance from shore. Thus, the location (or the combination of locations)
of the study is a principal determinant in the usefulness of a specified
positioning method. Effects of location on the accuracy and applicability
of various positioning methods are described in Appendix A for optical,
radar range, and short-range and long-range electronic positioning methods,
which are commonly used in Puget Sound.
Weather, currents, and other physical factors may also reduce the
achievable accuracy of a positioning method. For example, the relative
drift of the sampling equipment away from the boat under strong currents
or winds can increase with depth. Resulting positioning errors in sample
location (as opposed to boat location) may exceed acceptable limits for
the study if effects of site location on positioning accuracy are not considered
during design of the sampling program.
Equipment and Analyses
The vessel available for the sampling program may affect the study
design by limiting the types of positioning equipment that can be deployed
or set up on board. If a positioning system already exists on the vessel,
it should be evaluated to determine whether its accuracy is adequate for
the sampling program.
Different levels of accuracy are required for different kinds of sampling.
Water column sampling generally does not require a precisely known station
location because the water column is relatively homogeneous compared with
sediments. Trawling transects do not require high positioning accuracies
because the sampled area is large and because the precise location of the
net at any specified moment is uncertain. Accuracy is much more important
for sampling conducted with equipment that penetrates or rests on the bottom
(e.g., cores, grabs). Heavier equipment will usually reduce wire angles
(see discussion under "Define Positioning Requirements," below) and the
area in which the sampler was probably located. For equipment left at
one location for a period of time (e.g., current meters, sediment traps),
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Station Positioning
Method Selection
August 1986
repeatable or relative accuracy to return to the same position is important
for retrieval. Absolute or exact geodetic positions may be less critical.
Sampling of point sources generally requires both high absolute accuracy
for exact location of sources and high relative accuracy for proper definition
of the spatial distribution of sediment pollutant concentrations.
The chemical or statistical analyses to which the collected samples
are subjected should also be considered in determining the required navigational
accuracy. If a gradient of environmental effects is suspected, but the
analytical technique cannot measure small differences in the value of a
specified variable, sampling stations can be located farther apart and
a relatively less accurate positioning method can be used. However, within-
station variability may be more difficult to discern using a less accurate
positioning method. For variables with a patchy distribution, the patch
size could be smaller than the area defined by the repeatable accuracy
of the positioning method, resulting in replicates sampled across community
or physical boundaries. These conditions may not be noticed in the field
and could prevent correct interpretation of the data. Statistical comparisons
with replicate samples (e.g., "synoptic" data, field replicates, time-series
samples) from heterogeneous stations will deserve special attention. The
effects of navigational positioning accuracy and the associated probable
sampling area (area from which samples could have been collected) on statistical
comparisons of data should be considered in the study design.
Station Separation
When sampling stations occur in a grid, along a gradient, or in highly
heterogeneous areas, it is especially important to maintain the statistical
separation between them. Minimum separation should be defined by the diameter
of the probable sampling area. Spatial resolution of station locations
(i.e., probable sampling area) is limited by positioning method accuracy,
depth, and wire angle. The proposed positioning method can be evaluated
to determine whether statistical separation for the proposed station config-
uration will be achieved. Probable sampling area is determined by calculating
the positioning error at a 95 percent probability level and adding the
effect of wire angle. Procedures for determining the spatial resolution
of a single station and adequate station separation are presented later
in this section under "Define Positioning Requirements."
Reoccupation
Station reoccupation (e.g., for replicates or time series samples)
requires accurate positioning techniques to minimize the area sampled.
The variance (s2) of the measured variables (for a specified number of
replicates) usually increases with the area sampled (Holme and Mclntyre
1984). The increase in variance can mask trends or gradients that may
actually exist. With less accurate positioning methods, more replicates
are needed to properly characterize the region and replicate samples are
more likely to have been collected from different patches within the probable
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Station Positioning
Method Selection
August 1986
area sampled. Because repeatable accuracy is typically one order of magnitude
greater than absolute accuracy for a specified navigation method, repeatable
accuracy should be used to determine the probable area sampled at a reoccupied
station. (If the exact geodetic position is important, a highly accurate
system can be used for initial occupation and a less accurate method can
be used for reoccupation. The repeatable accuracy of the less accurate
method may approach the absolute accuracy of the more accurate method.)
Program-Imposed Constraints
Various program-specific constraints may affect the decision on positioning
methods. Sampling time limitations may preclude more logistically demanding
positioning methods. The proximity of the study and reference areas to
one another may require the shore stations to be moved. Operator (e.g.,
investigator, vessel captain) experience with the proposed positioning
system can affect performance. An experienced operator is more likely
to arrange transponders (electronic systems) or landmarks (range or bearing
methods) for complete coverage of the study area and is more likely to
identify potential problem areas prior to the start of the survey. Contractual
obligations may limit changes to the study design, establish maximum expend-
itures for station positioning, or require a minimum achievable accuracy
for the survey. Unless the sampling design demands higher accuracy, funding
agency specifications for positioning requirements should be observed.
DEFINITION OF POSITIONING REQUIREMENTS
Once a study design is drafted, project-specific positioning requirements
can be established. Such requirements are influenced primarily by:
• Characteristics of the survey area (e.g., physical, geo-
graphic)
• Minimum required station separation
• Maximum acceptable area from which the replicate samples
can be collected.
Physical and geographic characteristics of the survey area affect
the applicability of various positioning methods and their achievable
accuracies. For example, as noted earlier, signal interferences in some
parts of Puget Sound may preclude the use of Loran-C as a positioning method.
Accuracy of other methods may be insufficient because objects by which
to establish the position fix (shore stations or targets) are inaccurately
mapped or unavailable. Map accuracy and the ability to locate on a map
the shore stations or objects from which fixes were taken are the largest
potential sources of positioning error. No matter how accurate the positioning
methods are, the locations of sampling stations will always be relative
to the location of fix points on the map used. If the map is not accurate
for locations of landmarks, the station positions determined from those
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Station Positioning
Method Selection
August 1986
landmarks can only be relocated adequately with the same methods. The
exact or geodetic location determined for those stations will not be correct
and other parties that try to relocate the stations using different positioning
methods may sample a different location. Consequently, the most accurate,
up-to-date map of appropriate scale should be used. Recent aerial photographs
that include the survey area can be used to verify map accuracy. The latest
editions of maps from the U.S. Geological Survey and the National Ocean
Survey are generally of sufficient detail and accuracy for positioning
purposes. "Blue line" maps produced by the U.S. Army Corps of Engineers
are made from aerial photographs and should be used if available for the
study area because of their higher accuracy.
Each station location should be plotted and numbered on the map.
Type of sampling, water depth, estimated time on station, and frequency
of occupation should be noted. Biological trawl paths and navigational
hazards should also be indicated. The availability of known targets for
visual or range fixes within acceptable range should be determined for
each station. Candidate locations for shore stations for each positioning
method should be examined. The selected locations should provide coverage
of the entire sampling area. Estimates of position errors should be based
on anticipated LOP or angle errors expected at each station. Limiting
factors within the survey area and at individual sites should be summarized,
based on an inspection of each candidate shore station site. Line-of-site
obstructions, traffic frequency, competing transmitters, air-water boundary
irregularities, accessibility, and security should be evaluated. These
evaluations are expected to provide a basis for judging the feasibility
of a specified positioning method.
Each station location is actually an area from which a sample could
have been collected. The size of this area is determined by the error
inherent in the positioning method (i.e., radial error), the vessel offset
(i.e., distance from the hydrowire to the location on the vessel from which
the station position was determined), and the wire angle (Figure 2). If
one assumes a zero wire angle on the sample gear, the positioning method
error will define the area from which the sample could have been collected.
Any slight wire angle will increase the size of the area as a function
of depth. The probable sampling area can be calculated for each specified
level of probability, depth, wire angle, and accuracy, as explained in
Appendix B, "Position Error Analysis." Probable sampling area should be
calculated for the closest spaced and deepest stations at the 95 percent
probability level [the sum of Equation 3 (p=0.095) and Equation 7 (wire
angle effects) in Appendix B]. This sample area can then be used to determine
the minimum spacing between stations that will guarantee statistical separation
of stations. For studies where station overlap is not a problem, the probable
sampling area can be calculated at a lower probability (using Equation
2 [p=0.68] in place of Equation 3). For example, assuming station overlap
is not a problem (using the sum of Equation 2 and Equation 7), a vessel
using a positioning system with an absolute accuracy of +2 m (+6.6 ft)
in 20 m (66 ft) of water could collect a bottom sample from anywhere within
8
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VESSEL
OFFSET
WIRE
ANGLE
RADIAL ERROR (RADIUS
OF PROBABLE LOCATION)
RADIUS OF PROBABLE SAMPLE COLLECTION AREA
Figure 2.
Components of error in probable area of sample
collection.
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Station Positioning
Method Selection
August 1986
a 66 m2 (710 ft2) probable sampling area. In 100 m (3Z8 ft) of water,
the probable sampling area would increase to 419 m2 (4,508 ft2).
Study objectives may require a small probable sampling area even if
station separation is not a critical design feature. For example, a description
of seasonal variation in benthic populations from a highly heterogeneous
area of Puget Sound would require small probable sampling areas. The size
of the probable sampling area should be determined by the scientific investi-
gators on a case-by-case basis. Where station location and depth are fixed
and the highest level of probability is assumed for the calculations, method
accuracy is the only remaining variable.
Generally, it is sufficient to calculate probable sampling areas at
three levels of accuracy: +2, 20, and 100 m (+6.6, 66, and 328 ft) to
determine the accuracy required for the survey. Both absolute and repeatable
accuracies of positioning methods can be divided into these groupings,
as will be demonstrated in the last section of this document, "Recommended
Positioning Accuracies." Each positioning method will provide accuracies
that could fall anywhere within a certain range depending on site-specific
conditions. The +2, 20, and 100 m (+6.6, 66, and 328 ft) accuracy levels
are generally representative of the highest accuracies achievable under
ideal conditions within the ranges of the various positioning methods.
Candidate positioning methods can be evaluated by accuracy limitations
to identify the most appropriate method. However, state agency or con-
tractually required accuracies should never be exceeded. Having established
accuracy requirements and survey area characteristics, the planner can
then proceed with a detailed review of available systems.
REVIEW AND SELECTION OF POSITIONING METHOD
Positioning methods and associated equipment are described in detail
in Appendix C. Additional information can be found in Ingham (1975), Umbach
(1976), Maloney (1978), Bowditch (1984), and Tetra Tech (1986). Since
any of these methods may be appropriate for a specific study, all should
be considered initially. The investigator can determine if a particular
method will meet calculated accuracy requirements at the survey location
in question. Methods that do not meet accuracy requirements or are inappro-
priate can be eliminated from consideration. The remaining methods should
be examined to identify the most appropriate method for the specific study.
A detailed evaluation of various positioning methods is presented in Appendix D.
Each evaluation procedure must be carefully tailored to the user's
needs and to the specific sampling or monitoring objectives. Factors such
as accuracy, range, cost (procurement or rental), portability, flexibility
(i.e., range of conditions under which the system can operate), security,
user recommendations, training, and service availability are basic to such
an evaluation, but are not all-inclusive. The screening of positioning
methods in Appendix D should be supplemented by project-specific considera-
tions. Other important factors to consider are visibility restrictions
10
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Station Positioning
Method Selection
August 1986
if night or foul weather work is expected, existing equipment on the vessel
to be used, and the rental availability. If purchase of a system is warranted,
additional factors include compatibility with existing equipment, ability
to accommodate future system expansion, and availability of ancillary items
(e.g., data logger, plotter, tracking monitor, or computer). Potential
use of the system for other types of projects may also be relevant. However,
with the expected rapid progress in satellite navigation systems, it may
be desirable to procure a less costly, more labor-intensive system now
and wait 2-5 yr for a satellite system. At a minimum, the positioning
method adopted should provide the accuracy to meet funding agency requirements
and to maintain station separation.
IMPLEMENTATION OF POSITIONING METHOD
Once a positioning method that is adequate for the specific sampling
objective has been selected, the proper setup, calibration, and operational
procedures must be followed to achieve projected accuracies. If the appropriate
equipment is already on board the vessel or the positioning task is hired
out, the responsible party of the cruise should be sure that at least one
member of the field crew is familiar with the positioning method. If the
scientific team is supplying the equipment, appropriate training or experienced
personnel should be provided to ensure proper equipment operation and documen-
tation of positioning data. A backup method should be available on short
notice to avoid loss of ship time if the primary method fails. To ensure
that station locations are accurately occupied regardless of method and
that adequate documentation is available for other parties, record-keeping
requirements should be established, as described in the following section.
11
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Station Positioning
Recordkeeping
August 1986
RECORDKEEPING REQUIREMENTS
FIELD RECORDS
Adequate information to ensure consistent positioning and to allow
reoccupation of stations for replicate sample collection or time-series
monitoring should be kept in a field logbook. More detailed descriptions
facilitate problem-solving and return to the station by the original crew
as well as other parties. Required entries into the logbook for initial
survey description, day log entries, and station log entries are addressed
below.
Initial Survey Description
The positioning method and equipment used should be detailed in the
field logbook. All changes or modifications to standard methods and equipment
should be noted . Names of persons who set up and operate the equipment
should be listed in case questions arise. Locations of on-board equipment
and the reference point on the vessel where the position is actually estimated
(e.g., antennae, sighting position) should be noted. Distance from the
vessel reference point to the hydrowire should be estimated to quantify
the vessel offset component of positioning error. The type of map that
was used for positioning and its identification number or scale should
also be recorded.
Shore station locations should be provided in detail sufficient to
allow another party to occupy the same shore station. Shore stations should
be located on a monumented point, a surveyed structure, or tied into the
local horizontal control at two established benchmarks by surveys performed
at the Third-Order Class I level or higher (Kissam 1981; Umbach 1976).
Existing monumented points can be located from National Ocean Survey Triangu-
lation Diagrams. Shore station descriptions should include the monumented
point reference number, location relative to local surroundings, and access
restrictions. A complete copy of the surveying notes should be included
in the field logbook.
Day Log Entries
Day log entries should record all variations from methods, equipment,
or personnel detailed in the initial survey description. All problems
or irregularities should also be recorded along with the location, time,
and operator. Any weather or physical conditions that might affect achievable
accuracy should be noted. These records help to explain problems that
may not be apparent in the field and can provide information with which
12
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Station Positioning
Recordkeeping
August 1986
to recalculate erroneous station positions. Relocation of any shore station
should be documented in the same detail as for the initial survey description.
All calibration data should be recorded in the day log. All manufacturer-
recommended calibration procedures and frequencies should be observed.
In addition:
• Electronic distance measuring systems (e.g., microwaves)
should be calibrated over water between two monumented points
at the start and end of a survey. The calibration should
be performed at the same range expected for most stations.
Calibration should be checked each day by recording the
readout at the same location (single station systems) or
while crossing the baseline between two shore stations (multiple
station systems).
0 Calibration of Variable Range Radar (VRR) and Loran-C should
be checked each day by recording the readout at the same
location. Two VRR ranges from the same two targets should
be recorded.
t The optical components of shore-based stations should be
calibrated at the start and end of a survey by recording
the angle between the two baselines that run from the station
location to two other monumented points. This orientation
check should be repeated whenever the station is bumped
or moved.
Station Log Entries
Each station location should be recorded in the coordinates or readings
of the method used for positioning in sufficient detail to allow parties
not present at the survey to reoccupy the station in the future. The position-
ing information should be recorded at the time of sample collection (vs. equip-
ment deployment) and for every reoccupation of the station, even during
consecutive replicate sampling. Trawl station positioning data should
be recorded at the beginning and end of every trawl and at each significant
change of direction. Supplemental positioning information that would define
the station location or help subsequent relocation (e.g., anchored, tied
to northwest corner of pier, buoy) should be recorded. If photographs
are used for a posteriori plotting of stations, the roll and frame numbers
should be recorded. Depth, time (tidal height), ship heading, and wire
angle estimation should also be recorded for each occupation of a station.
Conditions at each station should be recorded and checked against
the range of operating conditions known to produce optimal accuracy for
the specified method. Examples of these specific requirements are presented
below for common positioning methods:
13
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Station Positioning
Recordkeeping
August 1986
t VRR - Three fixes from permanent objects should be recorded
for each station. Fixes should all be from targets 0.16 to
6.4 km (0.1 to 4 mi) away and all from the same range scale
on the VRR. Station positions should be supplemented with
Loran-C coodinates, depth, and all visual line-of-site fixes
available.
• Loran-C - The range of Loran-C coordinates for the survey
area should be recorded. This helps the operator notice
signal interference problems and signal jumps (10-skip)
that may otherwise result in recording faulty coordinates
for a station position. Both day and night coordinates
should be recorded from one location inside the study area
to check for a 0.1 usec day/night variation. Any fluctuations
at the 0.1 usec level while on station should be recorded,
including the reading that appeared on the display the majority
of the time.
• Microwave systems - Crossing angles of LOPs should be between
35 and 150 degrees for all stations. Readout should be
noted two to three times before being recorded to eliminate
multipath errors.
REPORTING REQUIREMENTS
Sampling data reports supplied to other parties should include rationale
for selecting the positioning method and recordkeeping standards observed
during data collection. Any specific problems (e.g., wind, currents, waves,
visibility, electronic interferences) that resulted in positioning problems
and those stations affected should be identified. Estimates of the accuracy
achieved during the survey should be included. This information will help
the reader to determine the quality and consistency of the sampling.
Station locations should be reported in State Plane coordinates (specify
north or south zone) or by latitude and longitude (to the nearest second).
Coordinates need not be reported for each replicate collected; a single
set of coordinates for the station is sufficient. Depth corrected to mean
lower low water (MLLW) should be supplied for each station. Coordinates
for the start and end of each trawled section should be provided.
14
-------
Station Positioning
Recommended Accuracy
August 1986
RECOMMENDED POSITIONING ACCURACIES
Accuracy requirements for different types of environmental sampling
requirements are examined in this section. Sampling that requires highly
accurate positioning methods to help ensure quality and consistency in
collected samples and in data interpretation and analysis is described. The
corresponding acceptable positioning methods are identified. This process
is presented for two specific sampling objectives identified by the Puget
Sound Dredged Disposal Analysis (PSDDA).
CLASSIFICATION OF SAMPLING
The sampled medium and collection method can be used to classify sampling
activities. The collected sample will be representative of either a point
location or a larger area. Examples of point and areal samples are provided
in Table 2. For purposes of identifying accuracy requirements for sampling
activities, samples collected from a homogeneous medium (e.g., water column)
are considered representative of a larger area and thus similar to an areal
sample. A third classification of sampling activities which could include
either point or areal samples is also considered in Table 2. This third
group (referred to in this document as special studies) consists of studies
usually used to identify induced effects on the environment. Characterization
of natural variability will be important for special studies and point
samples collected from heterogeneous environments. Accuracy of the positioning
method (i.e., error associated with station location, or spatial resolution
of the location from which the sample could have been collected) will affect
the ability to identify the difference between natural variability and
induced effects. Other sampling will not require this level of accuracy.
Therefore, levels of positioning accuracy can be recommended for the three
classifications of sampling that are identified from Table 2 (special studies,
point sampling at the sediment boundary, and areal sampling including any
samples from the water column).
RECOMMENDED ACCURACY
Accuracy levels of the methods detailed in Appendix C and evaluated in
Appendix D can be categorized as +2, 20, or 100 m (+6.6, 66, and 328 ft)
for the reasons discussed earlier in this document. The highest accuracy
level [+2 m (+6.6 ft)] is recommended for sampling performed under special
studies in Table 2, point sampling studies in Table 2, and studies designed
to build the developing database for the Puget Sound Estuary Program.
The highly accurate station locations possible using +2-m (+6.6-ft) accuracy
will allow more closely spaced stations and better detection of trends or
gradients that could otherwise be masked by variance in replicate samples
collected using less accurate positioning methods. In addition, uniformity
15
-------
TABLE 2. CLASSIFICATION OF COMMON TYPES OF SAMPLING
Point Sampling
Water Column3
Current measurements
Water samples
Sediment traps
Sediment Boundary
Sediment physical/chemical
Bioassay sediments
Interstitial waters
Benthic infauna
Bivalve coring
Areal Sampling
Water Column
Plankton tows
Pelagic fish trawls
Sediment Boundary
Demersal fish trawls
Crab pots or trawls
Bivalve dredging
Special Studies'*
Point source investigations
Time series sampling
Synoptic collections
Spatial gradients (including surface microlayer, dye studies)
Station grids
Dredging site sediment characterizations
a Homogeneous nature of water column makes all water column sampling equivalent
to areal sampling.
b Studies in this classification may contain any of the sampling types
listed above but are considered separately because of additional accuracy
requirements.
16
-------
Station Positioning
Recommended Accuracy
August 1986
of positioning accuracy across these studies is expected to improve compara-
bility of data and is one of the factors that affects overall data quality.
However, if the location or variable sampled does not require the
highest accuracy levels, a lower accuracy level [+20 m (+66 ft)] may be
adequate. For example, results from sampling the relatively homogeneous
benthos of deeper waters would probably not be adversely affected if the
replicate grabs were collected from a larger area. If +20-m (+66-ft) accuracy
is sufficient to address the objective in question, and the funding agency
cannot justify additional funds for the use of higher accuracy in order
to conform to the database requirement, the lower accuracy may be appropriate.
In many cases, point sampling studies could be adequately performed at
the lower level of accuracy. Such exceptions should be evaluated on a
case-by-case basis.
The +20 m (+66 ft) accuracy level should be adequate to collect areal
samples. The more homogenous nature of the medium and larger area sampled
make higher accuracies unnecessary. The exception would be water samples
collected near point sources where location is important in determining
dilution characteristics. The lowest accuracy level [+100 m (+328 ft)]
generally is not recommended.
RECOMMENDED POSITIONING METHODS
Positioning methods that can achieve the two recommended accuracy
levels are presented in Table 3. Approximate ranges of both absolute and
repeatable accuracies for some of the methods are compared in Figure 3.
Methods with +2 to 10 m (+6.6 to 33 ft) accuracy are acceptable for the
higher level of accuracy. Those with +20 to 40 m (+66 to 132 ft) accuracy
are acceptable for the lower level of accuracy. In addition, some of the
advantages and disadvantages for these positioning methods are presented
in Table 3. Absent from Table 3 are some of the optical positioning methods
discussed in Appendix C. While these methods do not offer the accuracy
necessary for most sampling farther from shore, they can be highly accurate
in areas within 0.5 km (1,640 ft) of shore. Along the waterfront of urban
areas and in waterways where other methods may be unsuitable because of
interference or minimum range requirements, optical positioning techniques
may be preferable. Optical range finders and visual line-of-sites can
provide accurate positioning if detailed notes are taken. Positions may
also be determined by taking three photographs to allow for a posteriori
triangulation from aerial maps or "blue lines." Any positioning by optical
methods is improved if multiple methods (e.g., photographs) are employed.
The lead scientific investigator should decide whether the limiting
factor in study design is absolute or repeatable accuracy, what effects
the location will have on positioning capability, and determine which one
of the candidate methods is appropriate for the specific sampling program.
The following determinations of appropriate accuracy levels for specific
17
-------
TABLE 3. SUMMARY OF ACCEPTABLE POSITIONING METHODS
Category
Higher Accuracy
Group
Theodolite
Representative Achievable
Equipment Accuracy
Table C-2 10-30 sec
Table C-3 * 1 m and up
Maximum
Range Cost Advantages
<5 km $1,000-$4,000 Traditional
method.
Inexpensive.
High accuracy.
Successfully
applied.
Restricted areas.
Disadvantages
Line-of-sight.
Two manned shore
stations.
Simultaneous
measurements.
Limits on inter-
section angles.
Area coverage;
station movement.
EON I
Table C-7
1.5-3.0 cm
3 km without $3.500-$15,000
multiple prisms
oo
Total Stations
Table C-8
5-7 cm
<5 km $8,000-$30,000
Microwave
Navigation
Systems
Range-Azimuth
Systems
Satellite Systems
Table C-9
1-3 m
25 km $40,000-$90,000
Table C-12 0.01° and 0.5 m
Table C-ll
1-10 m
<5 km $65,000-$100,000
(optical)
30 km
(elec)
none $150,000-$300,000
(initial units)
under $10.000 later
Extremely accur-
ate. Usable for
other surveying
projects. Cost.
Compact, port-
able, rugged.
Single onshore
station. Other
uses. Minimum
logistics.
No visiblity
restrictions.
Multiple users.
Highly accurate.
Radio line-of-
sight.
High accuracy.
Single station.
Circular coverage.
High accuracy.
Minimum logis-
tics. Use in
restricted/con-
gested areas.
Future cost. No
shore stations.
Motion and
directionality of
reflectors. Line-of-
sight. Visibility,
unless microwave.
Two shore stations.
Ground wave reflec-
tion.
Reflector movement
and directionality.
Prism costs. Line-
of-sight. Optical
or infrared range
limitations.
Cost. Multiple
onshore stations.
Logistics. Security.
Signal reflective
nu11s.
Single user. Cost.
Line-of-sight.
Signal reflective
nulls.
Current coverage.
Initial develop-
ment cost.
-------
TABLE 3. (Continued)
Category
Lower Accuracy
Group
Sextant3
Representative
Equipment
Table C-4
Achievable
Accuracy
+ 10 sec
+ 2 m and up
Maximum
Range
<5 km
Cost Advantages
$1 ,000-$2,000 Rapid. Easy to
implement. Corn-
Disadvantages
Simultaneous
measurement of two
Loran-C
Table C-ll
15 m and
up
none
$1.000-J4,000
Variable Range
Radar
Table C-5
0.5°
25 in
16-72 km
(4.000-$10,000
iron equipment.
Low cost. No
shore party.
High accuracy
closer to shore.
No visibility or
range restric-
tions. No ad-
ditional person-
nel. Low cost,
existing equip-
ment.
No visibility
restrictions.
No additional
personnel. Low
cost, existing
equipment.
a Accuracies greater than +20 m are not common farther than 1 km from shore under
normal operating conditions and is therefore considered in the lower accuracy group.
angles. Target
visibiIities,
location, mainte-
nance. Line-of-
sight. Best in
calm conditions.
Limits on accep-
table angles.
Interference in
some areas. Used
only for reposi-
tioning except in
Iimited areas,
need to locate
station initially
with another
system.
Line-of-sight.
Re Iies on map
accuracies of
targets. Ac-
curacy decreases
with range scale.
-------
ro
o
SEXTANT
OPEN WATERS
NEAR SHORE
VARIABLE RANGE RADAR
LORAN-C MAPPED AREAS
VRR, VISUALS, LORAN-C
OPEN WATERS
NEAR SHORE
MICROWAVE SYSTEMS
SATELLITE SYSTEMS
RANGE-ANGLE SYSTEMS2
REPEATABLE
ACCURACY
20 40 60 80 100
APPROXIMATE ACCURACY RANGE (±m)
a INCLUDES TOTAL STATIONS AND RANGE-AZIMUTH SYSTEMS
120
Figure 3. Comparison of approximate accuracy ranges of some positioning methods.
-------
Station Positioning
Recommended Accuracy
August 1986
sampling objectives are presented as examples of the process. Sampling
studies conducted for PSDDA will consist of two basic objectives:
• To characterize the sediments to be dredged
• To monitor disposal-related impacts and trends at dredged
material disposal sites.
Both objectives are included under special studies. The higher accuracy
is recommended for sediment characterization because exact locations of
the areas to be dredged must be defined for later removal of only the specified
sediments. Dredged areas are usually close to shore. Most of the higher
accuracy methods in Table 3 will meet these requirements close to land.
High repeatable accuracy is required for site monitoring to ensure
that observed trends are temporal and not spatial. Most of these sites
will be over 2 km from shore. Range restrictions may preclude some of
the higher repeatable accuracy systems (see Appendix D; Figure D-l) at
selected sites. Absolute position accuracy may not be important, although
this determination will have to be made for each disposal site and for
individual resources of concern. If absolute positioning is needed the
higher accuracy methods are recommended.
21
-------
Station Positioning
References
August 1986
REFERENCES
Bowditch, N. 1984. American practical navigator. An epitome of navigation.
Defense Mapping Agency Hydrographic/Topographic Center, Washington, DC.
Holme, N.A., and A.D. Mclntyre (eds). 1984. Methods for the study of
marine benthos. Blackwell Scientific Publications. Oxford, U.K. 387
pp.
Ingham, A.E. 1975. Sea surveying. John Wiley and Sons, New York, NY.
306 pp.
Kissom, P. 1981. Surveying for civil engineers. McGraw-Hill Book Co.,
New York, NY.
Maloney, E.S. 1978. Duttons navigation and piloting. Naval Institute
Press, Annapolis, MD. 910 pp.
Tetra Tech. 1986. Evaluation of coastal survey positioning methods for
nearshore marine and estuarine waters. U.S. EPA Office of Marine and Estuarine
Protection, Washington, DC. Tetra Tech, Inc., Bellevue, WA 107 pp.
Umbach, M.J. 1976. Hydrographic manual, fourth edition. U.S. Department
of Commerce, NOAA, Rockville, MD. 414 pp.
22
-------
APPENDIX A
SITE-RELATED POSITIONING LIMITATIONS
-------
Station Positioning
Appendix A - Site-Related Limitations
August 1986
APPENDIX A
SITE-RELATED POSITIONING LIMITATIONS
Location has many effects on the accuracy and applicability of various
positioning methods. Such effects are described below for optical, radar
range, and short-range and long-range electronic positioning methods.
These methods are described because they are commonly used in Puget Sound.
Optical positioning methods rely on the visual resolution of objects
with a known position. Built structures provide more accurate fixes than
land features because sharply defined objects provide better resolution.
The ability to resolve an object decreases with distance. Within 5 km
(3.1 mi) of the shoreline and in more developed areas, accuracy of optical
methods can be comparable to that of electronic methods (Umbach 1976).
However, optical methods are more dependent upon proper operation and target
choice than are other methods. Urban embayments and waterways are therefore
better suited for optical positioning than are regions in central Puget
Sound or areas along less populated, featureless shorelines. The abundance
of accurately located channel markers throughout Puget Sound provides good
sightings in otherwise featureless areas. On featureless shorelines, a
line tangent to the shoreline is used as a line-of-position, with considerable
reduction in accuracy. Use of optical methods is restricted to daylight
hours of good visibility.
Positioning by multiple ranges measured from a variable range radar
system requires fixes on known positions, but eliminates visibility restric-
tions. However, because the radar signal is shadowed beyond the first
object it strikes, the choice of targets can be limited. A second limitation
is possible misidentification of reflection sources in developed areas.
Positions based on misidentified reflection sources are inaccurately located,
but can be reoccupied if the same perceived reflection source is subsequently
used. A third target should be used to crosscheck position determined
from two other targets. All three fixes should be on the same radar range
scale.
Reflections depend on target position and alignment. The most accurate
radar range fixes are based on reflections from objects 0.16-6.4 km (0.1-4 mi)
distant (Crawford, P., personal communication). At stations farther from
shore, adequate targets may not be available. Reflections closer than
0.16 km (0.1 mi) may be erroneous and should not be used. Sloped headlands
and tidal flats are not usable as targets.
Short-range electronic positioning systems (e.g., microwave) involve
at least two shore stations (transponders) and an on-board transmitter.
A-l
-------
Station Positioning
Appendix A - Site-Related Limitations
August 1986
A set of stations is required for each study area. Successful reception
of electromagnetic signals is the critical feature for effective operation
of electronic positioning systems. Signals from the transponders or shore
stations should be received at an angle of 30 to 150 degrees; 90 degrees
is optimal. Signal reception is dependent upon electronic "line-of-sight"
and may be blocked by tight quarters (e.g., waterways, rivers, and shorelines)
and heavy vessel traffic. Such problems are alleviated by newer, more
expensive systems that accommodate over a dozen transponders. With proper
transponder locations, microwave systems can be used to position a vessel
any distance from shore in Puget Sound except closer than 100 m (328 ft)
to one of the transponder locations.
Accuracy of microwave systems depends upon placement of the remote
transponders or shore stations. For example, remote stations not located
on a monumented point will increase error of the vessel position fix.
However, access restrictions, benchmark locations, and line-of-site considera-
tions limit the available transponder locations and achievable angles.
Permanent shore station locations are further limited by availability of
power sources and site security.
Repeatable accuracy of microwave systems, while not affected by transponder
location errors, is dependent upon the 1ine-of-position angle. Certain
combinations of transponder and vessel locations may result in signal cancel-
lation (range holes) and failure to obtain a fix. Occurrence of range
holes varies by location, is impossible to predict, and may force relocation
of a shore station. In developed areas, reflections from metal objects
or buildings may compound the problem or cause jumps in the received signal.
Long-range electronic positioning systems operate on permanent transmitting
stations and user-carried receivers. The only receivable long-range system
in Puget Sound is Loran-C. However, because land masses distort signal
propagation, Loran-C charts are of unknown accuracy in inland waters such
as Puget Sound. In addition, unidentified electronic sources interfere
with Loran-C reception in some areas and prevents its use much of the time
(Figure A-l). However, Loran-C is accurate for repositioning at locations
where readings were recorded in Loran-C on original occupation. Some areas
may exhibit a day/night signal variation of about 0.1 usec (Eaton C., personal
communication) and should be checked when sampling at night.
Recent Loran-C maps based on comparison of Loran-C coordinates with
those from other methods at the same stations support positioning to a
resolution of 0.1 usec of the Loran-C signal [about 37 m (120 ft)] for
limited areas in the vicinity of Elliott Bay (Sturgill, D., personal communi-
cation). Accurate maps for other areas are not available. Distortion
can be defined for areas outside Elliott Bay only by taking readings at
benchmarks around the shoreline. Accuracy is less reliable with distance
from the benchmarks. Therefore, while Loran-C is usable around Elliott
Bay for initial positioning, it can be used in other areas only as a reposi-
tioning tool and will require defining Loran-C station coordinates.
A-2
-------
LARGE AREA REGULARLY AFFECTED
BY POSSIBLY A SINGLE POWERFUL
SOURCE
OCCASIONAL SHORT-TERM INTER-
FERENCE IN ELLIOTT BAY
SEOLA BEACH TO PT. WILLIAMS
POSSIBLE AIRPORT SOURCE
10
NAUTICAL MILES
KILOMETERS
20
«
NOTE: MOST OF THE SOUTH SOUND
ALSO EXHIBITS INTERFERENCE FROM
A SINGLE SOURCE NEAR TACOMA
Figure A-l. Regions of LORAN-C signal interference in Puget
Sound.
A-3
-------
Station Positioning
Appendix A - Site-Related Imitations
August 1986
APPENDIX A
REFERENCES
Crawford, P. 15 November 1985. Personal Communication (phone by Jeff
H. Stern) University of Washington Research Vessel Clifford A. Barnes.
Seattle, WA.
Eaton, C. 9 December 1985. Personal Communication (phone by Jeff H. Stern).
Kittiwake Research Vessel. Seattle, WA.
Sturgill, D. 18 November 1985. Personal Communication (phone by Jeff
H. Stern). Municipality of Metropolitan Seattle, Seattle, WA.
Umbach, M.J. 1976. Hydrographic manual, fourth edition. U.S. Department
of Commerce, NOAA, Rockville, MD. 414 pp.
A-4
-------
APPENDIX B
POSITION ERROR ANALYSIS
-------
Station Positioning
Appendix B - Error Analysis
August 1986
APPENDIX B
POSITION ERROR ANALYSIS
Errors have been traditionally classified in three categories: gross,
systematic, and random (Mikhail and Gracie 1981). Gross errors are mistakes
due to observer carelessness and must be detected and eliminated from positional
measurements. Systematic errors occur due to a deterministic system, in
this case the sextant, range finder, or electronic positioning equipment.
Such errors can be expressed by functional relationships and can therefore
be corrected for in the final measurement. Random errors reflect variations
in measurements that remain after eliminating gross errors and allowing
for systematic errors. As their name implies, such observational errors
are random and have no known functional relationship. Random errors, the
subject of the following discussion, are dealt with on a statistical basis
using probability models.
All positional fixes are in error to some extent. This is true even
if the transmitting stations have been very accurately located and bias
or systematic errors in position coordinates have been eliminated. Random
errors will still cause a variable displacement of position lines about
their computed positions. Each method used to establish a position has
inherent random errors that determine the overall accuracy of a fix. Absolute
or predictable accuracy refers to a method's ability to correctly define
a position by latitude and longitude (Bowditch 1984). Repeatable or relative
accuracy measures a method's ability to return the user to the same position
time after time. The difference between these two accuracies can be signifi-
cant. Therefore, it is important to identify which accuracy is of concern
for positioning during a specific sampling program before determining the
error associated with that accuracy.
Positional accuracy is usually stated in terms of the probability
of being within a certain distance of a desired geographical location.
The line-of-position (LOP) can therefore be considered a strip whose width
is a function of the LOP uncertainty, which can be described by its standard
deviation (o) (Figure B-la). Because a fix involves at least two LOPs,
each having an uncertainty, the LOP crossing boundary forms a parallelogram
(Figure B-lb). The parallelogram area is dependent on the size of the
standard deviations of the two LOPs and the angle at which they cross.
The parallelogram area is given by:
A = (2oi)(2o2) esc* = 4oio2 esc* (D
where 01 and 02 are the LOP measurement standard deviations. Because the
cosecant function becomes large for angles less than 30 degrees and more
B-l
-------
.MEASURED LINE OF
'POSITION NO 2
MEASUREMENT
VALUE SPREADS
MEASURED LINE OF
POSITION No 1
SHORE STA. 1
BASELINE
SHORE STA. 2
a.
ERROR
PARALLELOGRAM
b.
CIRCULAR PROBABLE
ERROR
SOURCE: MODIFIED FROM BOUDITCH 1984
Figure B-l. Line of position measurements for two shore
stacions depicting LOP uncertainly (a), and
associated error indicator (b).
B-2
-------
Station Positioning
Appendix B - Error Analysis
August 1986
than 150 degrees, so too does the size of the error parallelogram (Figure B-2).
Thus, to reduce positional uncertainties, shore station locations should
be located such that very large or very small cut angles do not occur within
the survey area.
Probability theory indicates a 68.27 percent chance of being within
+1.0o of the mean of a set of normally distributed measurements. However,
the probability that the measured position is within the parallelogram
is less. For two sets of measurements, the joint or product probability
is (0.6827)2 x 100 or 46.6 percent. A probable parallelogram is defined
as one for which the probability of a measurement being within its boundary
is 50 percent. This occurs at distances slightly larger than +1.0o (i.e.,
+1.05o). Using +2.0o results in a parallelogram within which it is 91
percent probable that the positional location occurs. The principal drawback
is that the error parallelogram is not a curve of equal probability. An
equal probability contour from a two-LOP fix is an ellipse centered on
the LOP intersections. As with the parallelogram, the area and configuration
of the error ellipse are dependent on the crossing angle and standard deviations
in each set of measurements. The ellipse in which 50 percent of the expected
measurements lie is referred to as the probable ellipse. Techniques for
determining the orientation and the magnitudes of an error ellipse for
various probability levels are presented by Mikhail and Gracie (1981).
It is more common to establish the probability that a position is
located within a circle of a particular radius than to define an error
ellipse. The resultant circular probable error (CPE or CEP) is the radius
of a circle within which 50 percent of the fixes should be located. Bowditch
(1984) provides two methods for determining the sizes of error circles
at a desired probability level, given the standard deviation in each LOP
measurement and the associated pattern crossing angle. The probability
that the measured position is within a circle of a selected radius can
also be determined.
When the standard deviations of two sets of measurements are equal
and the cut angle is 90 degrees, the error figure becomes a circular normal
distribution. Table B-l can be used to determine the probability that
location is within a circle of given radius from the measured position.
At a radial distance (R) from the true position equal to lo (i.e., the
ratio of R/o=l), there is a 39.9 percent chance of being located within
the circle (Table B-l). A 50 percent probability occurs for a circle with
a l.Zo radius (R/o=1.2), and a 90 percent probability occurs for a circle
with a 2.2a radius. These probabilities change as the cut angle varies
from 90 degrees. For example, if the cut angle is 40 degrees, a radius
greater than 3.4o is needed to achieve a 90 percent probability that the
position lies within the circle boundary. Table B-l can also be used to
determine if positioning error is small enough to separate the error circles
of two closely spaced positions. The probability of being located within
a circle can be calculated (at any cut angle) for an error circle radius
B-3
-------
AREA OF
UNCERTAINTY
CD
i
/ 30°
ANGLE OF
/ CUT
90°
ANGLE OF
CUT
POSITION,
LINES
Figure B-2. Angle-of-cuc effects CM. fix accuracy.
-------
TABLE B-l. PROBABILITY VERSUS R/o FOR ELLIPTICAL BIVARIATE
DISTRIBUTIONS WITH TWO EQUAL STANDARD DEVIATIONS
II"
10*
II"
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7
-------
Station Positioning
Appendix B - Error Analysis
August 1986
of one half the distance between the two positions to determine if position
overlap may occur statistically.
Positional accuracy is often quoted using radial error, rms error,
or d , all of which have the same meaning. As with CEP, the error figure
is ar(?ftcle having a radius equal to:
rms = (ox2 + oy2) (2)
where ox and oy are the lo error components along the major and minor axes
of the probability ellipse (Figure B-3). This is often referred to as
the Id or lo fix accuracy. Similarly, a 2o fix is given by:
[(2ox2) + (2oy2)]1/2 (3)
Given the LOP standard deviations oj and 02, the standard deviations along
the basic ellipse axes can be determined by:
oK2 = (1/2 sin2$) { o j2 + o22 + [(c^ + o22)2 - 4(sin0) 0]a2) (4)
and
o2 = (1/2
The probability of being within a Id circle varies from 63 to 68 percent
depending on the ellipticity of the^ error ellipse. For a 2dpms circle,
the probability varies from 95 to 98 percent.
It is common to describe two-dimensional error distributions by two
separate one-dimensional standard deviations along each error axis (ci
and o2). Error circles about a measured position can be constructed for
different LOP standard deviations and cut angles. This can be done by
first calculating ox and oy (the standard deviations along the major and
minor axes of the error ellipse) knowing the crossing angle and the standard
deviations in each measurement (o\ and o2). Table B-2 is then used to
provide probabilities as a function of c, the ratio of the smaller to larger
of ox and cy, and K as the ratio of the error circle radius to the larger
of ox or cv. For example, for a 50° angle of cut, a °i = 15 m (49 ft)
and c2 = 20 m (66 ft), the probability of location within a circle of 30 m
(98 ft) is:
(Ol)2 = 225 m2
(o2)2 = 400 m2
sin2* = 0.5868
Substituting into equations 4 and 5, ox is 29.9 m (98.1 ft) and oy is 13.1 m
(43.0 ft).
B-6
-------
SOURCE: BOWDITCH 1984
Figure B-3. Illustracion of radial error.
B-7
-------
- B-2. CUCJLAR ERROR PROBABILITIES AS A FUNCTION OF MEASUREMENT
STANDARD DEVIATION RATIOS AND ERROR CIRCLE RADIUS
\
•X
0. 1
0.2
0 3
0 4
as
0 6
0 7
0 8
0 9
1 0
,
i
3
5
6
7
8
9
2.0
2. 1
2.3
2.3
2 4
2 5
2.6
2 7
2.8
2 9
3 0
3*3
3 3
3 4
3 5
3 6
3 7
3 8
3 9
4 0
4. 1
4 2
4 3
4. 4
4 5
4.6
4 7
4 S
4 9
j 0
5 1
5 2
i 3
5 4
3 5
J '
3 3
5 9
4 0
0 0
0796557
1585194
2358228
J 108435
3829249
4514938
5160727
5762892
6313797
58208S5
7286679
769S607
3063990
8384867
3663856
S9040U
9108691
9281394
9425669
9544997
9442712
9721931
9785518
9836049
9875807
9906776
9930661
9940897
9962684
9973002
99S0648
9986257
9990332
9993261
9995347
9998818
3997344
999&J53
9999038
9999367
9999587
9999733
9999'2*
9999*92
9999933
9999958
9999974
9*999*4
9999990
9999994
9999998
9999999
' 9999999
1 0000000
1
a i
0443987
1339783
2211804
3010228
3755E84
4457708
5115048
5725957
6288721
6802325
7266597
76X2215
3050848
.0374049
9655127
8897008
9103102
9274964
9422182
9542272
9640598
9720304
9784275
9835108
9875100
9908249
9930271
994S612
9962477
9972853
99S0542
9986182
9940279
9993225
9995323
9996801
9997332
9993545
9999033
9999363
9999585
9999732
9994828
9999891
9999931
9999957
9999974
i 99999*4
9999990
9999994
9999997
9999998
9999999
9999999
1 0000000
1
0 2
0242119
0884533
1739300
2635181
34S1790
• 25S60S
4960683
5604457
6191354
G723586
7202682
7630305
8008554
8140018
9627728
8875060
9085619
9263I2S
9411299
9533775
9434011
9715237
9780408
9832180
9872900
9904612
9929062
9947727
9961934
9972391
9980212
9985949
99901 16
9993112
9995245
9996748
9997747
9998522
9999018
9999353
9999578
9999727
9999826
9999889
9999931
9999957
9999973
9999984
9999(90
9999994
9999997
9999998
9999499
9999999
1 0000000
0.3
0164176
0128396
1318281
2139084
3003001
3846374
4633258
3349387
5993140
6568242
7079681
7532175
7929968
8277048
8577362
8834914
9053766
9237989
9391586
9518415
9622127
9706109
9773450
9820918
9868953
9901674
9926894
9946141
4940684
9971564
0979622
99D5533
9912909
9993105
9996653
•1997733
9"93478
9V9S089
9999334
9999566
4499720
9999821
9999386
9999929
9999954
9949973
1 9999983
9999990
9999994
9999996
9999998
9999999
9999990
1 0000000
i
1
0 «
0123875
0432413
1039193
1743045
2532951
3357384
4170862
4941882
3651564
6291249
6859367
7359558
7793550
9169851
8493071
3768444
9001746
9197275
9359855
9493815
9403170
9691397
9762419
9819594
9862720
9897043
9923483
9943649
9953S79
9970266
4979699
9944480
99)9368
9492513
9994088
9998505
9097633
99934 1 2
9490945
9W9305
99*9547
9999707
9494813
9999881
9999925
9999954
4999971
9999983
9999990
9999994
> 9909996
9999998
9999999
1 9999999
I 0000000
1
0 5
0099377 '
0340193 i
083153.1
1451308
21528SO
2914682
3099305
4474207
3211998
3900953
6524489
7079973
7567265
7989288
8350816
8657559
8913536
9130680
9308615
9454546
9573205
01.60345
4745239
9805703
9853112
9889934
991*260
9439842
9936126
9468294
9977298
9403392
40..H677
9992113
9994559
9996281
9997432
9990111
9999873
9999261
9999519
9999409
9949301
9449074
9999921
9999951
9994970
99999S2
4999989
999U993
9999998
9999998
9999999
9999909
'1 0000000
0 6
0082940
0327123
0719102
1237982
1857448
2548177
3230302
4025G28
4759375
5461319
6116314
4714269
7249673
7720881
SI29287
8478393
8773116
9019110
9222177
9388418
9322991
9631017
9710934
9794661
9837569
987S327
9909944
9933821
99M798
3963205
9471109
9982356
4947407
9991376
9994053
9995418
9997251
999S157
994X774
9999195
9999475
9909661
4999733
•>
-------
Station Positioning
Appendix B - Error Analysis
August 1986
Therefore:
c = 13.1/29.9 = 0.44
and K = 30/29.9 = 1.003
Entering Table B-2 for values K = 1.0 and c = 0.4 gives a probability of
62 percent.
Equipment manufacturers quote accuracy probabilities using various
terminology. Most commonly, CEP and d values are provided. For equal
standard deviations (ox/ay = D, the d * is 1.2 times the CEP. Thus,
quoting the CEP would be more advantageous to a manufacturer than quoting
a d value. However, when system accuracy is given as 100 m (328 ft)
at fife5 2o level, two clarifications are needed: 1) whether absolute or
repeatable accuracy is being quoted, and 2) whether measures linear or
circular error. If the error is circular, the number describes an 86 percent
probability level, whereas if a 2d is referred to, then the probability
of the positioning being within the crrcle is between 95 and 98 percent,
depending on error ellipse axis lengths. Therefore, unless the basis of
an accuracy figure is very explicitly stated, it may be necessary to clarify
its meaning. Only then can the accuracy performance of competitive systems
be compared.
The above discussion is concerned only with the accuracy of the positioning
method. However, the drift of sampling equipment away from the vessel
(which is usually manifested as a wire angle) will affect the size of the
area from which the sample may have been collected. The distance the equipment
may drift is, in effect, added to the radial error of the position fix.
The increase in the probable area above that inherent in the positioning
method is demonstrated in Figure B-4. This larger area of probable sample
location is the total error associated with the location of a sampling
station, and should be considered by the user in determining if the achievable
accuracy is adequate for the purpose of the study.
The increase in the probable sampling location will be a function
of both the wire angle and the depth of the station. The increase of the
sampling area with depth at various wire angles is exhibited in Figure B-5.
The wire angle or equipment drift may not always be noticed or avoidable.
A 5 degree wire angle is not uncommon in shipboard sampling and anything
less than 2 to 3 degrees may not be noticeable. Wire angle may not be
avoidable and the worst-case that may have to be accepted during a study
should be used in determining the probable sampling area. The probable
sampling areas in Figure B-5 have been calculated by adding wire angle
errors to the radial error (calculated from Equation 2) for +2-m (+6.6-ft)
accuracy positioning methods.
The accuracy of the positioning system chosen also will affect the
probable area of sample collection. The less accurate the navigation method,
B-9
-------
VESSEL
OFFSET
WIRE
ANGLE
RADIAL ERROR (RADIUS
OF PROBABLE LOCATION)
RADIUS OF PROBABLE SAMPLE COLLECTION AREA
Figure B-4. Components of error in probable area of sample
collection.
B-10
-------
2500-1
2000-
1500-
r»
oc
1000-
500-
300-
200-
100-
0
NOTE. BASED ON ± 2m ACCURACY AT A
90 DEGREE FIX CALCULATING THE
1 dm, AT 68% PROBABILITY
I
100
I
200
\
300
I
360
DEPTH (m)
Figure B-5. Effects of wire angle on the probable (P = 0.68)
area of sample collection.
B-ll
-------
Station Positioning
Appendix B - Error Analysis
August 1986
the greater the resultant area potentially sampled. The effect of depth
on the probable sampling area as a function of the accuracy of the positioning
method is presented in Figure B-6. The 5 degree wire angle increases the
probable sampling area significantly at depths below 100 m (328 ft).
To ensure that the preferred positioning method will meet all study
goals, the error associated with sample collection at specific stations
should be estimated. The achievable accuracy can be determined by the radial
error utilizing the crossing angle and standard deviations of the LOPs
expected over the entire study area. Either Equation 2 or 3 can be used
to calculate radial errors at specific probabilities. Tables B-l or B-2
can be used to determine probabilities associated with a specific radial
error. The wire angle error component is then added to the radial error.
The distance added to the radial error (see Figure B-4) can be calculated by:
d = z(tane) (6)
where: z = the station depth
9 = the wire angle.
A 5 degree angle may be unavoidable and should always be used to calculate
the wire angle error component. Substituting into Equation 6 gives:
d = 0.09(z) (7)
This distance, added to the radial error, will determine the probable area
of sample collection for any station.
B-12
-------
30.000 -,
90.000 -i
20.000 -
80.000 —
111
cc
±20m
03
i
10,000-
DC
70.000 —
60.000
1100m
NOTE BASED ON A 5 DEGREE WIRE ANGLE
AND 1 d,m> CALCULATED FROM A
90 DEGREE FIX (68% PROBABILITY)
400
100
I
200
I
300
I
400
DEPTH (m)
Figure B-6. Effects of navigational accuracy on the probable (P = 0.68) area of sample
collection.
-------
Station Positioning
Appendix B - Error Analysis
August 1986
APPENDIX B
REFERENCES
Bowditch, N. 1984. American practical navigator. An epitome of navigation.
Defense Mapping Agency Hydrographic/Topographic Center, Washington, DC.
Mikhail, E.M., and G. Gracie. 1981. Analysis and adjustment of survey
measurements. Van Nostrand Reinhold Co., New York, NY. 340 pp.
B-14
-------
APPENDIX C
POSITIONING METHODS AND CONSIDERATIONS
FOR SAMPLING IN PUGET SOUND
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
APPENDIX C
POSITIONING METHODS AND CONSIDERATIONS FOR
SAMPLING IN PUGET SOUND
The various navigational positioning techniques are listed in Table C-l.
This information provides quick-reference review of the performance character-
istics and costs of methods representative of each class. These methods
are grouped by maximum range to facilitate user comparison for the appropriate
sampling sites within Puget Sound. Optical methods are presented first,
followed by electronic systems.
OPTICAL POSITIONING TECHNIQUES
Methods Available
The traditional optical positioning method involves observations of
two horizontal sextant angles between three fixed shore targets, plotted
as a graphical resection using a three-arm protractor or station pointer.
Other optical positioning methods are available (Table C-l), but only this
and the theodolite intersection method are practical in more open waters
[150 m (492 ft) to 5 km (3.1 mi) from shore]. Because the remaining optical
methods cited apply only to river or harbor surveys, at extremely short
ranges, or in very calm water, they are not useful over the range of expected
conditions of sampling in Puget Sound and are only briefly discussed below.
When sampling stations are located relatively close to shore in industrial
areas, they can be identified by relatively inexpensive methods. Use of
a graduated tape or wire is impractical except in the case of very short
distances offshore. Intersecting ranges are used when a number of established
landmarks permit easy selection of multiple ranges that intersect at the
desired sampling point, and accuracy is not critical. Range line and uniform
speed or angle between the line and an onshore target are more applicable
to hydrographic surveys than discrete sampling station locations. Vertical
angle ranging requires a graduated shore line target known as a subtense
bar. A sextant is set so the individual marks on the bar subtend a given
angle at desired distances from shore. This method is limited to extremely
confined areas, relatively short distances, and calm waters. The angle
and stadia method was used historically in very calm waters, but can be
used at longer ranges. This method requires vertical positioning of the
stadia (or rod) on the vessel and careful measurement of angles to the
stadia hairs, enabling a calculation of distance from the observer to the
rod. A position fix is made by measuring the azimuth from a baseline of
known orientation.
C-l
-------
TABLE C-l. SUMMARY OF VESSEL POSITIONING METHODS
Close Range-Direct iup to 5 km (3.1 rrnj]
Horizontal Sextant Angle Resection
Theodolite Intersection from Shore
Subtense Ranging by Vertical Angle
Intersecting Ranges
Range Line and Angle from Shore or Vessel
Angles from Shore and Vessel
Angles and Stadia or Distance from Shore
Range Line and Uniform Speed
Distance Line Ranging
Close Range-Indirect [up to 5 km (3.1 mi)j
Laser
Infrared Electromagnetic Distance
Short Range [up to 40 km (25 mi)J
Variable Range Radar
Microwave Electronic Positioning
(300 MHz-300 GHz)
Medium Range [100-300 km (62-186 mijj
Medium and High Frequency Electronic Positioning
(300 KHz-300 MHz)
Long Range [to 2000 km (1243 mi)j
Low Frequency Electronic Positioning
(30-300 KHz)
Global Positioning
Very Low Frequency, Satellite, Astronomical Observations
(3-30 KHz)
C-2
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
An optical range finder may be used to establish the predetermined
distances to permanent targets. An optical range finder is used by simply
focusing a split-image on the target, enabling the slant distances to the
target to be read off the instrument scale. When combined with a careful
compass reading, this distance reading allows positioning of the sampling
vessel. A survey of accuracies claimed for commercially available instruments
suggests that +3 m (9.8 ft) accuracy can be achieved within approximately
100 m (328 ft) of the sighted targets. The Lietz Model 1200, for example,
provides an accuracy of +1 m (3.3 ft) at 100 m (328 ft). Beyond this distance,
instrumental errors increase rapidly. For the instrument cited, a +9 m
(29.5 ft) accuracy is quoted at 300 m (984 ft). The suggested U.S. list
prices of optical range finders vary from $35 to $120 (Folk, L., personal
communication). Accuracies are affected by the availability of targets
of known position and compass readings. Both of these errors are hard
to quantify, making this method unpredictable and of limited usefulness.
Visual lines have sometimes been used to establish a station position.
This method requires that a minimum of two objects are in alignment, enabling
the vessel to be placed on a common axis extending to the vessel's position.
Simultaneous sighting on a second set of at least two objects places the
vessel at the intersection of the two common axes. The accuracy of each
visual line is highly dependent on the quality of the visual range (e.g.,
alignment), the distance from the alignment objects to the vessel, and
the angle between each range. Also, the number of visual fixes used affects
the magnitude of the positional error. Although this technique is frequently
used for positioning single sampling stations in bays and harbors or otherwise
where a number of convenient alignable targets can be selected, the method
has limitations for large scale monitoring programs. Even when a desired
sampling station has been accurately located using one of the previously
discussed methods, and at that location at least three aligned target-pairs
are visible, it not likely that sufficient alignment target-pairs will
be present for all other desired locations. Also, the unpredictability
of positional error each time a station is reoccupied detracts from the
value of this method.
Because the techniques described here are inexpensive to implement
(as are use of the sextant resection of theodolite intersection methods),
they are attractive to small studies. However, use of more sophisticated
and less labor-dependent techniques may be achievable at moderate costs
by renting or leasing, rather than buying such equipment. All such options
should be considered, provided the recommended absolute positional accuracies
can be achieved by the technique selected.
Theodolite Intersection
Position of the sampling vessel can be established by two onshore
observers who simultaneously measure the angle between a reference object
or shore traverse and the vessel (Figure C-l). A rod or other aiming point
is normally erected on the vessel. Radios, flags, or lights signal the
C-3
-------
Figure C-l. Station positioning by theodolite intersection,
C-4
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
moment at which angle measurements should be made. A theodolite with an
accuracy of +15 sec for single angle measurement, intercept angles near
45°, and a range of 5 km (3.1 mi), should yield a position error less than
+1 m (3.3 ft) (Ingham 1975). Characteristics of theodolites used for such
measurements are summarized in Tables C-2 and C-3.
Although accuracy of this method appears high, its use in open waters
has several distinct disadvantages. Complex arrangements usually are needed
to ensure that angles are measured by two onshore observers at the same
instant of the desired fix. Though not a problem when the vessel remains
on station for a long period of time, it becomes a problem when the vessel
is trawling. Lines from the two theodolites should intersect at nearly
right angles. As indicated in the error analysis discussion, weak position
fixes or corresponding large positional areas of uncertainty result when
the angles measured are less than 30° or more than 150°. To avoid such
extreme angles, onshore observers may have to move frequently, which can
delay a predetermined sampling schedule. Each of the onshore observer
stations must be surveyed to maintain accuracy. Finally, as with all optical
methods, target movement and path interferences (e.g., fog, heavy rain,
or heat waves) can confound the measurements. In spite of these disadvan-
tages, the procedure offers relatively high accuracies at low capital cost
(although labor cost can rapidly add up) and has been successfully applied
in small-scale coastal surveys during favorable weather. It also is advan-
tageous in very restrictive areas or in harbors. It is therefore considered
a candidate method for very limited monitoring programs or surveys.
Sextant Angle Resection
An offshore position can be fixed by measuring the two horizontal
angles between 1ines-of-sight to three identifiable targets with known
positions. When a vessel is underway, the sextant angles must be measured
simultaneously by two observers. The measurement of the first angle between
the center and one outside target allows determination of a circle of position
(COP) on which the sampling vessel must lie (Figure C-2). For example,
when the measured angle is 52°, the first circle of position is plotted
by subtracting this angle from 90° and drawing lines seaward from the siting
targets at the resultant angle of 38° from the baseline. These lines cross
at the center of the COP, which can then be drawn with a compass. This
procedure is repeated using the center and remaining targets, resulting
in the plot of a second COP. In the example, the second angle is 67°,
requiring a plot of radius lines at 23° from the baseline. The intersection
of the two position circles marks the vessel's location.
Position fixes are normally plotted with a station pointer or a three-
arm protractor (Figure C-3). The two measured angles are set by moving
and locking the two outer arms of the protractor, which then is moved over
a nautical chart until the three arms align with the preplotted locations
of the shore targets. The vessel's position is recorded at the center
of the protractor. Because this procedure can be implemented in 10-15
C-5
-------
TABLE C-2. SUMMARY OF VERNIER TRANSIT AND SCALE-READING THEODOLITE CHARACTERISTICS
COMPANY
Benchmark
Berger
Kern
leltz
Nikon
Pentax
Teledyne
Topcon
Warren -Knight
White
Mild
Zelss
MODEL
JENA 020
Bronze 65/45
Aston 67
Project 100
ST-1/6
ST-8/9
Kl-S/ST
BT20/10C
115
TSZOA/S6
NT-2S MK III
Schneider 700/400
GT-4B/6B
TH-60S/60E
OP 107/100-A.ZO
6-15
400U
AG-30B
TL-605E
10-2220/3200
TR-300
TR-303/303PM
T-307AT/309T
T-16
T-05
Th-42/43
VERNIER MICROMETER
OR SCALE DIVISION
20"
20V11
20"
r
1V20"
20"
30"
20"
r
r
r
20V11
20"
r
20"
20"
r
30"
r
2ovr
r
20"
r/20-
20"
20"
POSSIBLE
ESTIMATION
10"
20"/30"
20"
30"
30-/20"
10"
6"
10"
30"
20"/6"
0.2'
6-/20"
6"
20"
20"
15"
20"
10" /30"
6"/10"
12"
10"
10"
U.S. SUGGESTED
LIST PRICE*
S2495
S2125/3850
$1835
$1100
$699/1499
$1699/1899
$3895/3995
$1695/1995
$795
$2495/3695
$3195
$1235/650
$1695/1895
$2500/1895
$1295/1185
$1350
$650
$1595
$2100
$2695/1295
S749
$1879/1995
$2695/2350
$1895
$3950
$3950
•January 1985
C-6
-------
TABLE C-3. SUMMARY OF MICROMETER AND DIGITIZED THEODOLITE CHARACTERISTICS
o
I
COMPANY
Benchmark
Kern
Lletz
Nikon
Pentax
David White
Tope on
Wild
ZeUs
MODEL
JENA 010A
JENA 015B
DKM2-A/T
IKH3/DKM3-A
DT-20E
TM6/10E/20H
TM-1A
HT-1/5A
NT-4D/30/2D
TH-20D/10D/06D
TH-01N
TH 10-20/10
T.308AT/208AT
TH 10-1
OT-20
TL-20DE/10QE/6DE
T-O/T-1
T-2/T-3
T-2000
Th-2
ITh-2
VERNIER MICROMETER
OR SCALE DIVISION
1"
6"
1"
0.5"
20" Accuracy
6V10-/20"
1"
zovi"
6"/10"/20"
20V10V6"
r
20V10"
20-/W
r
LCD Readout 20*
20V10V6-
20"/6"
1V0.2"
0.5'
1"
0.6"
POSSIBLE
ESTIMATION
o.i-
1"
0.1"
0.1"
10" Display
2-/5VIO"
0.1"
6V0.25"
l"/2"/«"
5"/2"/r
0.5"
2"/l"
10"/5"
0.1"
10"/5"/3'
20"/3"
o.r/o.1-
0.1"
-
U.S. SUGGESTED
LIST PRICE*
14295
S3495
15890
SI 2295
S2995
13995/3695/3295
t5995
SI 895/5950
$3695/3595/3095
12850/3500/3690
14900
J2850/3500
12995/3495
(5500
S269S
t 2859/3500/3690
$3995
$5995/1199$
$11995
$5950
116600
January 1985
-------
o
I
00
Figure C-2. Station fix using position circles.
-------
VERNIER
MOVABLE
SIDE
ARMS
INDEX ERROR
ADJUSTMENT
SCREWS
SETTING
SCREW
Figure C-3. Three-arm protraccor for sextant resections,
C-9
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
sec with some experience, it commonly is used in hydrographic surveys where
moving vessel positions are needed. To minimize the parallax error, the
two sextant operators should stand as close as possible when making the
measurements. Sextant angles can routinely be measured to approximately
1 min of arc or better, depending upon the instrument quality and operator
ability. Within 5 km (3.1 mi) of shore stations and at acceptable COP
crossing angles, the resulting accuracy in position is 1 part in 2,500,
or about +2 m (6.6 ft) (Ingham 1975).
Sextant angle resection has been the most widely used positioning
technique in coastal surveying. This is due to its relatively high accuracy,
ease of implementation, and nominal cost of the sextants and the three-
arm protractor. Also, no shore party is required. The procedure does
have some limiting factors, however. Range is ultimately limited by visibility
and by the sizes, elevation, and placement of the shore targets. Also,
it is imperative to follow procedures in locating targets to avoid indeterminate
or weak fixes. For example, a fix cannot be obtained when the vessel and
all three shore targets lie on a common circle (called the danger circle).
This can be avoided by aligning the shore targets along a straight line,
which causes the radius of the danger circle to become infinite. Another
recommended practice to assure strong fixes is to place targets so that
intercepted angles fall between 30° and 140° (ideally between 45° and 60°),
thereby maintaining large position circle cut angles. Shore targets also
may be placed on a curve convex to the observer, with the middle target
nearest the sampling vessel (Figure C-4). Alternately, targets may lie
on a curve concave to the vessel, provided that the anticipated positions
are within the triangle formed by them, they are virtually equidistance
from the vessel, the observed angles are not less than 60°, or the vessel
is well outside the circumscribing circle (Clark 1951; Davis et al. 1966).
At the limits of visibility, sextant angle fixes are likely to be
weak because the angles are small. In such cases, large positional errors
can be caused by small errors in the angles themselves (Umbach 1976).
This problem can be partially offset by using a telescope mounted on the
sextant. Sextants must be in perfect adjustment and angles must be read
with extreme care. Also, the sextant must be of superior quality so that
the angles may be read to the necessary precision. When working at locations
near shore, the sum of the two angles can approach 180°, with one angle
often very large and the other very small. Under such conditions, care
must be taken to mark the two angles simultaneously if the vessel is moving,
or to make several measurements if the vessel is on station because the
angles rapidly changed with slight vessel movements.
Split-fixes (no common center object) may be taken when a three-point
fix is not possible. The vessel position is at the intersection of two
angle loci. A fix is considered strong when the intersection angle is
greater than 45°. While shoreline tangents can be taken when no other
objects are available these fixes are inaccurate in most cases. Split
fixes and shoreline tangents should be used only when absolutely necessary.
C-10
-------
DANGER
CIRCLE
Adapted from Ingham 1975
Figure C-4. Shore target locations to avoid the danger circle.
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
They are inefficient due to the required recording procedures and plotting
time, and they cannot generally be entered into automatic data processing
and plotting systems.
Sextants are classified as vernier or micrometer drum types, the latter
preferred by most users. A well-constructed metal marine sextant is capable
of measuring angles with an instrument error not exceeding 10 sec or 0.1
min of arc (Bowditch 1984). However, as indicated earlier, positional
error will be highly dependent on operator ibility. For accurate work,
a sextant having an arc radius of 162 mm (6.4 in) or more should be selected.
Characteristics of representative sextants are presented in Table C-4.
Sextants must be adjusted prior to the start of the survey and verified
at the conclusion or at least once a week, whichever is more frequent.
Any index correction should be recorded in the survey log. Procedures
for sextant adjustment are provided by Umbach (1976) and Bowditch (1984).
Considering the achievable accuracy when the double horizontal sextant
angle method is properly implemented, this procedure offers an inexpensive
candidate positioning method for limited surveys. However, the method
may be ineffective during poor visibility or periods of heavy weather when
monitoring or seasonal survey work may nonetheless be required. Also important
is the need to construct, survey, and maintain a sufficient number of properly
located shore targets to provide unambiguous positioning. Overall, the
method has merit when the cost of an electronic positioning system cannot
be justified.
Variable Range Radar
While technically not an optical positioning technique, obtaining
ranges from a variable range radar (VRR) consists of two optical estimations
by the user (target location and bearing of radar reflection) and will
be addressed herein. A position can be fixed by measuring the distances
to three targets on the radar screen that can be accurately identified
on a map. A third fix will reduce the chance of error and increase accuracy.
A variable range marker measures the distance to the object (as identified
by its radar reflection). This distance then is drawn with a compass as
a line of position (LOP) on the nautical chart. The intersection of the
three LOPs marks the vessel's position.
Most commercial vessels are equipped with radar for safety and navigation.
Coastal vessel radar usually have ranges from 26 to 116 km (16 to 72 mi).
A variable range marker (VRM), whether built-in or added onto the existing
on-board radar, removes a large portion of operator error in estimating
distances. For positioning at accuracies less than 300 m (984 ft), a VRM
almost always is needed. Range accuracies with the VRM are usually 1 to
2 percent of the range scale, or +25 m (82 ft) at 0.5 km (0.3 mi), the
smallest scale. Accuracy decreases as the range scale is increased. Bearing
C-12
-------
TABLE C-4. MARINE SEXTANT CHARACTERISTICS
SPECIFICATIONS
Arc Range
Instrument
Accuracy
Vernier
Scale
Arc Radius (on)
Telescope
Brightness
Index Mirror (on)
Horizon Mirror Ota. (on)
Illumination
Frew Mtl.
Arc Mtl.
Case
U.S. Suggested
C. PLATH
NAVSTAR
CLASSIC
-5 to «I2S°
< ilO"
0.2'
162
4X40
6X30
57X41
57
Arc A Drum
Brass/Al loy
Brass/Alloy
Mahogany
t!270
NAVSTAR
PROFESSIONAL
-5 to .125°
< 410"
0.2'
162
4X40
57X30
57X30
Yes
Makrolon
Al. Alloy
Plastic
J965
TAMAVA
SPICA
-S to «I25°
< MO"
0.2'
162
««0(7°)0
7X35(6.5°)
100/25
57X42
57
Arc 1 Drum
Aluminum
Bronze
Plastic
S919
TAMAVA
JUPITER
(UNIVISION)
-5 to M25°
<*«•
0.2'
162
4X40(7°»
7X35(6.5°)
100/25
57X42
57
Arc ft Drum
Aluminum
Bronze
Plastic
S71S
TAMAYA
VENUS
-5 to *125°
< 18"
0.2'
138
3X26
75
45X30
45
No
Aluminum
Bronze
Hood
SSBS
UEEMSft
PLATH
-5 to <
< ±10"
0.2'
162
4X40
6X30
57X41
57
.125"
Arc A Drum
Brass
Brass
Mahogany
11674
List Price (1/B5)
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
accuracy is usually less than 1°. Characteristics of representative VRRs
are presented in Table C-5. Generally, analog systems have better resolution
than digital systems, but are not as versatile.
Each position fix will rely on the resolution and identification of
the radar reflection. Resolution of the target position will change with
the range scale. Reflections will depend upon target position and alignment.
The location of the reflection is not always easy to identify. Placement
of the cursor away from the actual reflection surface will introduce error
into the fix. Estimation of the radar target location in relation to the
chart's mapped structures is important. Misidentification of a reflection
source will result in plotting a position at the wrong coordinate, but
will not affect repeatable accuracy if the same target is used. Certain
regions, such as sloped headlands and tidal flats, give inaccurate reflection
because it is impossible to relate the reflection to a map location. The
most accurate radar range fixes are obtained from solid reflections between
0.16 and 6.4 km (0.1 and 4 mi). This keeps the range scale low, resulting
in accuracies greater than +40 m (131 ft) and avoids erroneous readings
caused from very close reflections.
Positioning vessels with VRR should provide sufficient increases in
accuracy over standard radar for most sampling purposes. Repeatable accuracies
can be within +20 m (66 ft). Position fixes from VRR are still available
in almost any type of weather. Most vessels already are equipped with
VRR and other radar can add variable range markers for $1,000 to $2,000.
The newer digital systems, priced in the $5,000 to $10,000 range, offers
multilevel processing for better target pickup and provides map plotting
ability directly on the screen.
ELECTRONIC POSITIONING TECHNIQUES
Electronic positioning methods use the transmission of electromagnetic
(EM) waves from two or more shore stations and a vessel transmitter to
define a vessel's location. The systems are based on the ability to predict
variations in EM wave travel velocity as a function of travel path. Position
is determined by measuring differences in signal arrival times (range-range
mode) or by comparing the phases of received signals to that of the transmitted
signal (hyperbolic ranging).
At their respective maximum ranges, electronic positioning methods
have higher accuracies than visual methods. Measurements can be obtained
regardless of weather and visibility conditions, and operating ranges are
typically much greater than for optical methods. Range can be extended
to 50-100 km (31-62 mi) simply by elevating antennae until signal attenuation
becomes a limiting factor. Shore stations need not be attended, minimizing
personnel requirements. Positional readouts are available as distances or
coordinates, rather than wavelengths or time delays, and deck units are
C-14
-------
TABLE C-5. VARIABLE RANGE RADAR (VRR) SYSTEM CHARACTERISTICS
r>
i
Systems
DECCA
Racal DECCA Marine
Redmond. UA
(206) 885-4713
Furuno U.S.A.
San Francisco, CA
(41S) 873-9393
KODEN/SI-TEX
Norwell, NA
(617) 871-6223
Raytheon Marine
Seattle, UA
(206) 285-6843
Model
R0170 VRM/VP3
R0170 BT
FR-360 MKII
FR-810
FR-1011
T-100
3604
3610
Type
Digital
Digital
Analog
Digital
Analog
Digital
Analog
Analog
Variable
Range Markers
1 (add-on)
1
add-on
2
1
1
1
1
Range
(k«)
77
77
58
116
77
26
58
116
Nominal Accuracy
Range (m) Angle
M
+30 at 0.4 km
+25 at 0.5 km
+36 at 0.5 km
725 at 0.5 km
+22 at 0.5 km
.
-
+0.50
+0.50
<+lo
<+lo
<+lo
+0.50
+10
+10
U.S.
Suggested
List Pricea
$3.990
16.000
$4.400
without VRM
$7.400
$8.300
$2.200
$4.900
$5.400
December, 1985.
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
usually compatible with data processing and automatic plotting equipment.
Position information is continuous, enabling maintenance of a desired location
by dynamic positioning or by traversing along a predetermined path.
The short-range systems of primary importance in survey and monitoring
programs are compact, lightweight, durable, easily calibrated, and relatively
stable. Disadvantages of electronic systems can include cost, particularly
for smaller program requirements, the inconvenience of orienting shore
and on-board units, vandalism of shore stations, and unknown signal propagation
effects (although this should not be a problem over the relatively short
distances to survey points in Puget Sound). As discussed later, costs
can be minimized by sharing the expense among multiple users, by leasing
equipment during survey periods, or by contracting for survey personnel
and equipment.
System Classifications
Electronic positioning systems often are classified by range capability,
which depends largely upon propagation characteristics of the operating
signal. Band width and signal power also influence range capability.
Electronic positioning methods are herein categorized as short-range, medium-
range, and long-range systems. Although short-range systems are emphasized,
other categories also are examined because systems such as Loran-C are
frequently used in parts of Puget Sound. Satellite navigation systems
also are presented because their prices are declining and capabilities
(i.e., coverage and repetitive access) are expected to increase. Categories
of electronic positioning systems, including operating frequencies, wavelengths,
ranges, and representative commercially available equipment, are listed
in Table C-6.
Short-range [0-40 km (0-25 mi)] microwave systems are portable and
best suited for use in the range-range mode. Medium-range systems [to
150 km (93 mi)] also are transportable, although components usually are
bigger and heavier. They are effective in either the range-range or hyperbolic
mode. Long-range [to 2,000 km (1,243 mi)] and global systems transmit
from permanently installed, widely dispersed shore stations or satellites
for multiuser operation.
Comparative Absolute Accuracies
Although it is difficult to specify the positional accuracies achievable
by instruments in each category, some generalizations can be made. Whereas
the optical methods discussed can provide accuracies of +2 m (6.6 ft) for
ranges up to 5 km (3.1 mi) offshore, short-range electronic positioning
systems may provide accuracies of +1-3 m (3.3-9.8 ft) for ranges up to
40 km (25 mi) from shore stations. Comparable medium-range system accuracies
are +5.0 m (16 ft) up to 150 km (93 mi). Long-range systems typically
have accuracies of +50-100 m (164-328 ft) within 350 km (217 mi) of shore
stations, and more than 100 m (328 ft) at greater ranges.
C-16
-------
TABLE C-6. ELECTRONIC POSITIONING SYSTEM CATEGORIES
Category
Representative
Range Systems
Very long range >2,000 km
Very low frequency
Satellite
Long range 0-2,000 km
Low frequency
Medium range 0-150 km
Medium-high frequency
Short range
Radar 0-100 km
Microwave 0-40 km
OMEGA
TRANSIT (NAVSAT)
GEOSTAR
NAVSTAR GPS
SERIES
AERO SERVICE GPS
LORAN-C
VIEUNAV
LAMBDA
SYLEDIS
RAYDIST TRAK IV
HYPER-FIX
ARGO OM-54
HYOROTRAC
DECCA
FURNO U.S.A.
KODEN/SI-TEX
RAYTHEON MARINE
TRISPONGER
MINIRANGER
MICRO-FIX
HYDROFLEX
AUTOTAPE
AZTRAC
POLARFIX
ARTEMIS
C-17
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
Operating Modes
There are two principal radio navigation system operating modes:
the two-range (or range-range) mode and the hyperbolic mode. Some systems
incorporate the advantages of each in a combination mode. These three
modes are presented in Figure C-5. In. the range-range mode, position fixing
is accomplished by measuring the extremely small time intervals required
for EM signals to travel from an on-board master transmitter to one or
more onshore slave stations, and back (Figure C-5a). For a known propagation
velocity, the time interval is converted to a distance (range) from the
slave, defining a single circle of position on which the vessel may lie.
The intersection of two or more such circles (based on signal returns from
two or more slave stations) results in a position fix. This operating
mode usually is restricted to a single user, although single side-band
techniques have been employed to allow multiuser operation (Ingham 1975).
Lane width (distance between points of zero signal phase) remains constant
regardless of distance from the system baseline. The lane width resolution
does not decrease at increasing ranges from the baseline, as is the case
in the hyperbolic positioning mode.
In the hyperbolic mode, the on-board receiver detects the phase difference
of signals arriving from multiple shore-based transmitters. Lines of constant
phase between master and slave transmitters form a hyperbolic pattern of
position lines (Figure C-5b). By measuring the phase difference between
arriving signals, the vessel can be located along one of the position hyper-
bolas. Adding a second master-slave transmitter pair superimposes another
hyperbolic pattern, resulting in a grid network with pattern crossings
(Figure C-5c). Measurement of the signal phase difference from the second
transmitter pair allows vessel positioning on the second pattern, and therefore
"unambiguous" location at the applicable grid crossing point. Phase differences
usually are resolved to 1/100 of the lane width. Resolution of the hyperbolic
system matches that of a range-range system only along the master-slave
transmitter baseline. Because of lane widening for increasing range from
the baseline, the system resolution decreases with vessel distance from
the master-slave transmitter baseline. As indicated earlier, the angle-
of-cut of arriving signals also affects the magnitude of position error.
In hyperbolic systems, extending the baseline length improves cut-angles
of the arriving signals and decreases lane spreading (Ingham 1975).
Short-Range Systems
Electronic Distance-Measuring Instruments—
A position fix is obtainable with two electronic distance-measuring
instruments (EDMI), or one EDMI with an angle measurement by theodolite
or sextant. Distance-measuring instruments that are either electro-optical
(e.g., laser) or electronic (e.g., microwave) are discussed herein. Electro-
optical and microwave distance-measuring devices are extensively used in
C-18
-------
n
i
RANGE-RANGE
GRID CONFIGURATION
B.
HYPERBOLIC PATTERN
LANE
WIDTH
C. HYPERBOLIC
GRID CONFIGURATION
Figure C-5. Operating modes for radio navigation systems
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
land-based surveying. The EDMI master generates a carrier signal which
is directed toward a reflector (in the case of light beams) or a repeater
(for microwaves). The light or microwave beam is modulated at two or three
different frequencies, usually under the control of a precision quartz
crystal oscillator. A phase comparison of the incoming and outgoing beams
enables accurate distance determinations.
The EDMI is relatively new in the surveying field. The geodimeter
of the early 1950s, which used a modulated light beam, was replaced in
the late 1950s by the Tellurometer, which used a modulated microwave signal.
This improvement increased the range and allowed operation in moderate
rain, fog, and darkness. Newer EDMIs have shorter ranges, but due to the
use of solid state electronics, are much more compact, less power-intensive,
and easier to read. The newest EDMIs use highly coherent laser light,
have longer ranges, require even less power, are portable, and are easy
to operate. The so-called "total station" consists of a theodolite for
measuring angles and an EDMI for measuring distances, with outputs recorded
on magnetic or paper tape for subsequent analyses. Under favorable conditions,
EDMI range capabilities are 1.6 km (1 mi) for light-based systems, 80 km
(50 mi) for laser systems, and 150 km (93 mi) for microwave systems.
Properly adjusted and calibrated, an EDMI has few sources of error.
Ground wave reflection can cause error when measurements are made over
water because reflected signals result in faulty distances due to the longer
path lengths. The swing, or cyclic manner, in which reflections are recorded
must be correctly interpreted. At very close range, EDMI accuracy is limited
by a constant of uncertainty. Beyond 0.5-1.0 km (0.3-0.6 mi), accuracies
of 1 part in 25,000 are easily achieved. If meteorological conditions
over the signal path are sufficiently well-known, accuracies of 1 part
in 100,000 can be achieved (Moffitt and Bouchard 1982). Characteristics
of representative short- and long-range electro-optical and microwave distance
measuring devices are presented in Table C-7. As indicated, instruments
with a range of 25 km (16 mi) can be used to measure distances to within
5 cm (2 in), while shorter-distance devices [to 5 km (3.1 mi)] are accurate
to 1.5-3.0 cm (0.6-1.2 In)].
It is apparent, therefore, that accuracies achievable with electronic
distance measurement devices are more than adequate to meet the positioning
requirements for surveys and monitoring programs. In fact, it is the angle-
measuring devices used with EDMIs that limit accuracy, not the EDMI itself.
Probably the major disadvantage of EDMIs is the continual motion and resultant
misalignment of the reflector (in electro-optical systems). Use of microwave
patterns eases directivity requirements.
Total Stations—
An electronic tachymeter, commonly referred to as a total station,
is an instrument for determining the distance, bearing, and elevation of
a distant object. In coastal surveys, it is a shore station instrument
C-20
-------
TABLE C-7. ELECTRONIC DISTANCE MEASURING INSTRUMENTS
COMPANY
Benchmark
Orlando. FL
(305)281-5000
Ceodineter. Inc.
Nova to. CA
(415)677.1256
Kern Instruments
Brewtler. NY
(914)279-5095
Keuffel 1 Esser. Co.
Morrlstown. NJ
(201)285-5000
The Heti Company
Overland Park. KS
(913)492*4900
« Electronics. Inc.
Littleton. CO
(303)795-20<0
Nikon. Inc.
Garden City. NY
(516)222-0200
Pentai. Corp.
Englewood, Co
(303)733-1101
Teludlst. Inc.
Mastic Beach. NY
(516)399-5843
Tope on Instrument Corp.
Paramus, NJ
(201)261-9450
U114 Heerbrugg
Farmingdale. NY
(516)293-7400
MODEL
Surveyor II 1-1
Ceodlmeter 14-A
Geodineter 112/122
Geodlmeter 220
OH 503
Ranger V-A
(HeNe Later)
PulteRanger
RED 2A/2L
MK-I1I, MX- Ml VS
NO 31
PK-81
Tellenat CMW20
(microwave)
DM- S3
Cltatlon-450
01*41 Dlstonat
01-20 Omomat
RANGE (m)
SINGLE PRISM
TRIPLE PRISM
MAX|MUM( prisms)
1600
3000
3500(6)
6000
8000
15000
2500
3600
6000(8/16)
1600
2400
3200(8)
2000
3500
4500(7)
8000
16000
25000
1000
3000
2000/3800
2800/5000
/7000(9)
1600
3000
4000
1900
3200
1400
2000
25000
2000
2500
2900(9)
1600
2300
4000(11)
2500
3500
7000(11)
6000
7000
14000(11)
ACCURACY
(MSE)
i(5 nm • 5 ppm)
1(5 mm • 3 ppm)
*(5 m * 3 ppm)
«(5 nn * 3 ppm)
2(3 m « 2 ppm)
2(5 mm « 2 Ppm)
2(30 W » 150 ppm)
2(5 nm * 5 ppm)
2(5 mm « 2 ppm)
2(5 mm • 5 ppm)
*(5 mm • 5 ppm)
2(5 mm •» 3 ppm)
1(5 mm * 5 ppm)
2(5 mm * 5 ppm)
1(5 nm * 5 ppm)
1(3 mm « 1 ppm)
U.S. SUGGESTED
LIST PRICE
S3.49S
SI 1.300
S6. 250(112}
110.950(122)
SB. 850
S8.995
S20.561
17.500
S4.69S(?A)
$5,950(111)
S7.9SO(II1-VS)
S5.885
S4.790
$16,500
$5.390
$3,995
18,995
$14.995
C-21
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
used to sight the survey vessel reflectors, enabling positional information
to be recorded onshore for subsequent communication to the vessel operator.
In a manual station, the same telescope optics (co-axial) are used to measure
both distance and angles. They basically are theodolites with built-in
EDMI units. With such manually operated units, slope reduction of distances
is done by optically reading the vertical angle and keying it into a built-
in or hand-held calculator (McDonnell, Jr. 1983). A semiautomatic total
station contains a vertical angle sensor for automatic slope reduction
of distances, while horizontal angles are optically read. With an automatic
station, both horizontal and vertical angles are electronically read for
use with slope distances in a data collector or internal computer. A theodolite
with a mount-on EDMI usually is not classified as a total station. An
exception is the modular total station, where the design objective is flexi-
bility of future additional equipment. Such units usually are designed
around an electronic (digitized) theodolite such as the Kern El. Many
total stations are designed to make full use of hand-held calculators (e.g.,
the HP-41CV) for data storage, computations, access to control registers,
testing, calibration, and orthogonal offset determinations. Most manufacturers
offer optional data collectors that serve as electronic supplements to
field books. This permits a convenient interface with a computer and remote
transmission of data using an acoustic modem.
Characteristics of representative total stations are presented in
Table C-8. As indicated, range is dependent upon the number of prisms
available for signal reflection. Such prisms are directional (i.e., must
be pointed towards the shore station), as opposed to omnidirectional prism
arrangements used for range-azimuth navigation systems. Directional prisms
cost approximately $300 for a pair and $500 for a set of three. Although
systems requiring 9 to 11 prisms are expensive, they allow measurements
regardless of vessel orientation. Some manufacturers report displaying
angles or accuracies to 1 sec or less. However, experienced surveyors
know that this level of accuracy requires careful or repeated pointings
at good targets.
For surveys and monitoring programs, single station capability is
attractive. Setup and calibration efforts are minimized, and the logistics
of station movement are much simpler than with a multi-station net. A
total station can be used on other projects when not in use for periodic
monitoring. The $8,000 to $30,000 price range is competitive with the
microwave positioning systems ($40,000 to $100,000), and achievable accuracies
in both range and angle are more than adequate. Instrument capabilities
and costs are reported in the free, bimonthly journal Point of Beginning
[P.O.B. Publishing Company, Wayne, MI (313-729-8400)].
Microwave Navigation Systems-
Short-range electronic positioning systems generally operate at microwave
frequencies that limit the system ranges to "radio 1ine-of-sight." Typically,
such systems are effective between 25 and 100 km (16 and 62 mi) offshore,
C-22
-------
TABLE C-8. TOTAL STATION CHARACTERISTICS
COMPANY
Carl Zeiss, Inc.
Thornwood, NY
(914)848-1600
MODEL TYPE RANGE*
(km)
L M H
RMS3 Send -Auto - 1.5?
- 2.0b
- 3.0e -
Elta 3 Automatic 1.6
PRISMS
1
3
9
3
ACCURACY
RANGE ANGLE
+5-10 mm + Zppm +2"
±10 mn + Zppm +2"
u.s. S'jciiri:
LIST PRICE
58,260
S18.7Z5
Geodlmeter, Inc.
Nova to. CA
(415)883-2367
Kern Instruments
Brewster, NY
(914)279-5095
'et:
•Hand Park. KS
,13)492-4900
MK electronics
Littleton. CO
(303)795-2060
Nikon Instruments
Garden City, NJ
(516)222-0200
Pentax
Englwood, CO
(303)773-1101
Topcon
Paramus, NJ
(201)261-9450
Wild Heerbrug
Farmlngdale, NY
(516(293-7400
Elta 3
Elta 46R
140
E1/DM503
E2/DM503
SMO-3
SOM-3ER
SET-10
MK-III
MK-IV
MK-HYDRO
NTD-4
NTD-1
PX-10D
PX-060
GTS-2B
ET-1
T2000+
DI4L or
DI5
T2000+
DI20
Automatic
Automatic
Automatic
Automatic
Manual
Semi -Auto
Automatic
Automatic
Manual
Automatic
Manual
Manual
Manual
Automatic
Automatic
Automatic
-
.
-
-
1.2
1.8
2.4
3.6
1.5
2.0
2.4
.
.
-
_
.
-
m
.
-
-
m
.
.
-
.
•
-
.
.
-
1.2
1.5
1.7
1.8
2.0
2.3
2.6
2.7
1.6
2.5
3.0
2.0
2.2
3.0
3.8
4.8
2.5
3.5
4.5
0.8
1.4
-
3.0
4.0
3 or 4
1.2
1.8
1.2
1.8
1.4
1.7
1.4
1.7
1.4
2.0
2.6
1.4
2.0
2.6
2.5
3.5
4.5
5.5
6.0
7.0
8.0
9.0
.
.
-
-
3.0
4.0
5.5
6.0
3.0
4.5
5.5
1.2
1.8
2.5+
_
.
-
1.6
2.3
1.6
2.3
m
.
.
-
1.7
2.4
3.0
1.7
2.4
3.0
3.5
5.0
6.0
7.0
9.0
11.0
13.0
14.0
3
6
18
3
1
3
6
8
1
3
7
1
3
9
3
3
3
1
3
1
3
1
3
1
3
1
3
9
1
3
9
1
3
7
11
1
3
7
11
±10 mm
±10 mm
l(5mni «
1(3 mm
1(3 mm
1(5 mm
1(5 mm
+ 2ppm
+ 2ppm
• 5 ppm)
* 2 ppm)
* 2 ppffl)
* 5 ppm)
» 2 ppm)
±2"
±3"
12"
12"
10.5"
10"
5"
6"
digital
direct
Stationary
1(20 mm + 5 ppm)
Moving
1(5 mm
1(5 mm
* 5 ppm)
+ 5 ppm)
!(5ran + 5 ppm)
±(5 mm
1(5 mm
1(5 mm
1(5 mm
1(3 m
l(3mn <
+ 5 ppm)
+ 5 ppm)
+ 5 ppm)
* 5 ppm)
+ 2 ppm)
" 1 ppm)
6"
3"
I"
ID-
S'1
2"
6"
1"
6"
2"
Q.51
digital
S18.7Z5
$12,320
$19.950
$19,175
$22.375
$8,800
$10,800
$11.000
$12,950
$14,950
$16.950
$8,595
subdivision
digital
direct
estimation
digital
$15.985
$8,450
$8,765
estimation
i
0.5"
$7,990
$14,250
521,000
$25,000
$30,000
* Atmospheric visibility: a. low - hazy, 5km
b. medium • clear, 15km
C-23
c. high • very clear, 30km
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
depending on antenna heights and power outputs. Position measurements
are indirect (i.e., by timing the travel of multiple pulsed signals from
a master to two or more remote stations and back; alternately, phase differences
between arriving signals can be measured). Available systems operate in
the range-range mode, the hyperbolic mode, or both. The position fix is
defined by the intersection point of two position circles or hyperbolic
constant-phase lines. Because microwave systems have nominal accuracies
of +1-3 m (+3.3-9.8 ft) from very short ranges to 25-40 km (16-25 mi),
they provide adequate positioning capability for survey work and monitoring
programs. Potential limiting factors include problems with shore station
security, and signal interference in industrial areas or in the vicinity
of radar-intensive military installations. Characteristics of representative
microwave navigation systems are summarized in Table C-9.
Trisponder—The Del Norte Trisponder is an X-band (8,800-9,500 MHz)
positioning system composed of a digital distance-measuring unit (DDMU),
a master station (usually on the vessel), and two remote stations located
at known geographic positions. Each station is a combined transmitter
and receiver. The master station antenna is omnidirectional and each remote
station has a directional antenna. Distances to remote stations are observed
on the DDMU using the range-range mode. A time-sharing feature allows
up to eight users.
The manufacturer quotes a typical range accuracy of +1 m (+3.3 ft),
with an instrument resolution of 0.1 m (0.3 ft). In The Hydrographic Manual.
Umbach (1976) cites a range error for this system of +3 m (+10 ft) with
good field conditions based on the Trisponder Basic Operation Manual published
in 1974. Also cited were tests conducted by the National Ocean Survey
(date unknown), which indicated that temporal electronic drift may cause
measurement variations. Recal ibration is suggested over a measured base
line after every 200 h of operation. However, the tests were conducted
on a Model 202 Trisponder Surveyor System for which the manufacturer claimed
a resolution of 3 m (10 ft) and a positional accuracy of +3 m (+10 ft),
after field calibration.
The currently available microwave system operates with Model 217/218E
transponders and Model 520 or 542 DDMUs. The Model 261 transponder, usable
as a master or remote, has a 5-km (3.1-mi) line-of-sight range. The Model
217/218E transponders operate up to 80 km (50 mi) from shore due to higher
output power. The transponders are designed for use with either of the DDMU
models. The Model 520 will collect four ranges and display two. The Model
542 interrogates four remotes, outputs four sets of data, and also provides
a positioning guidance capability. The cost of a complete system, including
a Model 520 DDMU, a master and two remote 217E/218E transponders, and antenna,
is $40,000 (Buchanan, C., personal communication). For the additional guidance
capability of the Model 542 DDMU, the system cost is $44,500. A less expensive
"black box" version of the DDMU (Model 562) is available for use with an
existing shipboard computer system, allowing more than eight users. Cost
of the Model 562 and other required components is $39,500.
C-24
-------
TABLE C-9. SHORT-RANGE POSITIONING SYSTEM CHARACTERISTICS
r>
i
SYSTEM
TRIPSONDER (520/540 ODMU)
DEL NORTE Technology. Inc
Euie*>. TX
(817)267-3541
FALCON 484 Mini-Ranger
Motorola. Inc.
Tempe. AZ
(602)897-4376
MICRO-FIX
Racal OCCCA Survey. Inc.
Canarlllo. CA
(805)987.8080
HVDROFLEX
Teludist. Inc.
Mastic Beach. NY
(516)399-5843
AUTOTAPE ON-40A/OH-43
Cubic Western Data
San Oiego. CA
(619)268-3100
RANGE FREQUENCY
(kai) ("HO
5 9320-9500(261)
80 9329-9500(21 7E)
80 B800-9000(2I8E)
37 5410-5600
80 54JO
100 3000
ISO 2900-3100
NOMINAL USER
ABSOLUTE CAPABILITY
ACCURACY
1> up to 8
(•ore
optional)
i? up to 20
±1 up to 16
*l .5 single
pTos 3x10 cull i user
di stance (•) option
^0.5 single
~1 : 100 ,000 range
ANIENNA
Vessel Shore
Station
320°?
360°H
2S°¥
360°H „
20°,30°V
360°H
18HV
3CO°H
IOBV
87°/18ql?M
66/50°»
7fljM
15 V
900H
I8°¥.60BH
30°-I20°H
IO°V
COST
140.00C
139,300
$43.000
S63.500
190.000
•January 1985
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
Falcon 484 Mini-Ranger—The Motorola Falcon 484 is a C-band (5,410-5,600
MHz) microwave ranging and positioning system that operates in the two-
range mode from 100 m (328 ft) to 40 km (25 mi). The system consists of
a vessel receiver-transmitter assembly with an ommidirectional antenna,
a range console, and shore-based radar transponders with directional antennae.
Pulsed radar from the survey vessel transmitter interrogates radar transponder
reference stations located at geographically known points. Elapsed time
between transmitted interrogations and the reply from each transponder
is used as the basis for range determinations. Two ranges are used for
trilateral positioning. If three or four ranges are available, range residuals,
sum of squared residuals, and error circle radius data are output for the
least squares position solution. The manufacturer claims a range accuracy
of +2 m (+6.6 ft). Up to 20 users can operate in the same area.
The current cost of a basic Falcon 484 system, including a range processor
control display, receiver/transmitter with ommidirectional antenna, two
reference stations with directional antenna, miscellaneous cables, and
manuals is $39,300 (Jolly, J., personal communication). In an effort to
eliminate one of the two major problems with most microwave positioning
systems (i.e., magneton failures and need to service beacons), Motorola
currently is developing a solid-state beacon using a gun diode. The associated
increase in long-term reliability also will result in decreased range.
However, range capability should remain adequate for positioning needs
in Puget Sound. Costs of the modified transmitters were not available
at time of publication.
Micro-Fix—The Racal Survey Micro-Fix is a range-range microwave posi-
tioning system. With 1ine-of-sight to shore-based transmitters, it is
capable of operating up to 80 km (50 mi) offshore. The system normally
operates at 5,480 MHz, with options at 5,520 and 5,560 MHz. The master
station can interrogate up to eight remote stations (from a possible 32),
with each remote transmitter/receiver unit (T/R) preset to recognize its
own distinctive station code. Up to four separate station groups can be
deployed in the same area without interstation interference. Multiuser
capability allows a maximum of 16 users for each deployed chain. Master
and remote units are interchangeable.
The basic system consists of a master station with a Control Measurement
Unit (CMU), a T/R unit, and two tripod-mounted remote T/R stations. The
vessel's master station interrogates the remote stations sequentially,
triggering reply pulses received by the master T/R and processed by the
CMU to display the corrected ranges. The CMU capabilities include automatic
and continuous self-calibration, track guidance, plotter drive, x-y conversion
(full spheroid and multi-range solution), and slant range correction.
Nominal accuracy is stated by the manufacturer to be +1 m (+3.3 ft). The
cost of a basic Micro-Fix system is $43,000 (Harris, E., personal communica-
tion), including training.
C-26
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
The manufacturer is developing the capability to use the system in
a hyperbolic mode, a combination range-range/hyperbolic mode, or a range/azimuth
mode, but this capability is not yet available. The system also uses circular
polarization techniques to avoid reflective (water surface) signal cancellation
nulls, thereby eliminating the need for a second antenna on the vessel. This
has been accomplished by an antenna design that prevents signal entrance
from reflective angles.
Hydrof 1 ex—Hydrof 1 ex is a short-range to medium-range microwave navigation
'system designed for survey applications where a high degree of accuracy
is required in fixing or tracking a moving vessel. The system operates
at frequencies of 2,920 to 3,300 MHz and has a range of 100 m to 100 km
(328 ft to 62 mi). It consists of a master unit controlled by an HP85
computer with a customized software package, an omnidirectional antenna,
and connecting cables. Each remote unit consists of a transceiver and
either a large range or fan-beam antenna for mounting on a customer-provided
tripod. Accuracy is claimed to be 1 m (3.3 ft) +3xlO~6D, where D is the
distance in meters. The system can be operated in either a two- or three-
range mode. Although single-user is the normal operating mode, a multiuser
option is available. The cost of a master and two remote stations is $63,500
(Baker, W., personal communication).
Autotape DM-40A/DM-43—The Cubic Western Autotape is an S-band range-
range microwave positioning system that operates at ranges of up to 150 km
(93 mi). System components include a shipboard interrogator and range
responders at each fixed onshore station. The OM-43 is capable of working
with three geographic sites. Range information is computed by comparing
the phase shift of the modulated signal transmitted between the interrogator
and responder phase unit to an interrogator reference signal. An Automatic
Position Computing System (APCS) is available for steering information,
real time analog plot of the vessel's track, and magnetic data recording.
The system does not accommodate multiple users. The manufacturer claims
a range accuracy of 0.5 m plus 1:100,000 times the range distance. This
error is due to internal random errors, systematic errors, temperature
variation bias errors, signal strength, component aging, and initial calibration
errors. External errors are said to far exceed internal noise, systematic,
and bias errors. Index of refraction error, which can approach 5 m (16.4 ft)
at 100 km (62 mi), is usually small enough at short ranges to be ignored.
Multipath rms errors (dependent on orientation of reflective objects near
and behind the interrogator omnidirectional antenna) have been observed
from 0 to 3 m (0 to 9.8 ft). The manufacturer states that internal averaging
plus external data smoothing will reduce the effect on Autotape to a small
fraction of a meter, provided the antenna is moving. For this reason,
the system is best suited to applications where the interrogator is on
a moving vessel.
The cost of a basic Autotape system including an interrogator, two range
responders, and associated antennae is $90,000. The same system with the DM-43
and three shore stations is $124,000 (Hempel, C., personal communication).
C-27
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
Medium-Range Systems
Systems in this category typically operate in the medium- to high-
frequency bands (i.e., 1.5-400 MHz), achieving greater ranges using EM
waves that propagate around the earth's surface. Positional accuracies
of medium-range systems vary from a few meters near the base line to tens
of meters at the system's range limits (Ingham 1975). Medium-range systems
must be used with caution in inland waters due to the severe landmass attenu-
ation and water-land interface effects. Such effects usually are manifested
as large calibration variations within a limited survey area. Characteristics
of representative medium-range electronic position fixing systems are summarized
in Table C-10. Because of the limited availability, high capital costs,
and logistical problems posed by the large-sized land stations, these systems
are not discussed further herein. Other systems offer similar accuracies
at reduced costs and are less time intensive for station setup. Various
semi-permanent systems in Table C-10 are discussed in detail in Tetra Tech
(1986).
Long-Range Systems
Long-range and global navigation systems generally operate at low
(30-300 kHz) or very low (less than 30 kHz) frequencies. As in the case
of medium-range systems, EM waves at such frequencies travel for very long
distances, typically limited by transmitter power. Onshore station chains
usually are permanent, for use with an appropriate vessel receiver and
published hyperbolic lattice charts (Ingham 1975). Achievable accuracies
typically are much lower than those of shorter-range systems because long-
range systems are designed for general navigation rather than accurate
positioning. Satellite navigation systems, which operate at much higher
frequencies, afford global coverage at much higher accuracies. Characteristics
of selected long-range navigation systems are presented in Table C-ll.
Loran-C—
Loran, an acronym for long-range navigation, is a pulsed low-frequency
electronic navigation system that operates at 90-110 kHz in the hyperbolic
mode. Loran-C receivers match cycles to measure time differences between
arriving master (M) and secondary (W, X, Y, Z) signals, which are pulse-
and phase-coded to enable source identification (Panshin 1979). The usec
arrival time differences are displayed and can be plotted on a special
Loran-C latticed chart as lines-of-position. Fully automatic Loran-C receivers
simultaneously process signals from two master-secondary station pairs,
displaying LOP information for tracking.
Range capability varies because Loran-C stations radiate peak powers
of 250 kw-2 I*W. Due to the use of low frequencies and large baseline distances
[i.e., 1,850 km (1,150 mi) or more], Loran-C can provide positional information
of reasonable accuracy out to 2,225 km (1,380 mi) with sky waves (Maloney
C-28
-------
TABLE C-10. MEDIUM-RANGE POSITIONING SYSTEM CHARACTERISTICS
SYSTEMS
SYLEDIS
Sercel , Inc.
Houston, TX
(713)492-6688
RAYDIST
Hastings-Raydist
Hampton, VA
<-» (804)723-6531
IN)
*° HYPERFIX
Racal DECCA survey
Camarillo, CA
(805)987-8080
ARGO DM-54
Cubic Western Data
San Diego, CA
(619)268-3100
HYDROTRAC
Odom Offshore Surveys
Baton Rouge, LA
(504)769-3051
RANGE
(km)
300
276 night
740 day
700 day
250 night
740 day
370 night
460 day
230 night
FREQUENCY
(MHz)
420-450
406-434
1.5-2.5
1.6-3.4
1.6-2.0
1.6-4.0
NOMINAL
ABSOLUTE
ACCURACY
±1 LOS
±3 2X LOS
±3
5-10
0.01 lane
display
resolution
4-5
0.05 lane
display
resolution
2-40
0.01 lane
display
resolution
USER ANTENNA
CAPABILITY VESSEL SHORE
Up to 4 Not Provided
(Range-range)
Unlimited
(Hyperbolic)
4/net Omnidirectional
(Range-range)
Unlimited
(Hyperbolic)
(Range-range) Omnidirectional
Unlimited
(Hyperbolic)
Up to 12 Omnidirectional
(Range-range)
Unlimited
(Hyperbolic)
Single Omnidirectional
(Range-range)
Unlimited
(Hyperbolic)
COST*
$ 51,350
$102,200
$113,000
$188,000
$100,000
*January 1985
-------
TABLE C-ll. LONG-RANGE POSITIONING SYSTEM CHARACTERISTICS
SYSTEM
LORAN-C
Multiple Receiver
Manufacturers
LAMBDA
DECCA Survey, Ltd.
England
ONEGA
Multiple Receiver
Manufacturers
VIEUNAV
Navigation Sciences
Bethesda, MO
(301)952-5225
TRANSIT
Multiple Receiver
Manufacturers
GEOSTAR
Geostar Corporation
Princeton. ID
(609)452-1130
NAVSTAR/GPS
Multtple Receiver
Manufacturers
SCRIES
IS! AC. Inc.
Pasadena. CA
(818)793-6130
ACROSERVICE
Houston. TX
(713)784-5800
RANGE
(k»)
2500 day
I8SO Night
400-800
Global
Vre
Coastal
Areas
Global
U.S. Land I
Coastal Areas
Global
Global
Global
FREQUENCY
(KH()
90-110
100-200
10-14
90-110
150 ft 400 MHi
5117-7075
1618-2492
1575 MHi
1228 MHi
1575 MHz
1228 MHi
1575 HHJ
1228 MHi
NOMINAL
ABSOLU1E
ACCURACY
(•»)
185-460
15-90 repeat able
137 day
730 night
1800 day
3700 night
110
90. 1 pass/1 freq.
37-46.1 pass/2 freq.
3-S.Multiple pass
and 2 freq.
1-7
40.C/A.CEP
8-9. P. CEP
< 1 B dlff.
Mode
< 1 n
(1/2 hour)
USER
CAPABILITY
Unlimited
Single 2- range
Unlimited -hyperbolic
Unlimited
Multiple
Unllnlted
Unlimited
P-Ntlitary
C/A Conwrctal
Foreign
Unlimited
Multiple
COST
$1.000-2,000
Call Agent
$4.000-10.000
$40.000 and
$2,000 annual fee
$2.500-10,000 basic
$30.000-45.000 elaborate
Transceiver $450 and
link Rental $10-30/m>.
(Estimated)
$10.000-140.000 Initial
$1.000 eventually
$287.000
$235.000
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
1978). Range achievable at a particular station is dependent upon transmitter
power, receiver sensitivity, noise or interference levels, and signal path
losses (Canadian Coast Guard 1981).
At best, the absolute accuracy of Loran-C in normal operating mode
over short distances using the ground wave varies from 185 to 460 m (0.11 to
0.29 mi), whereas repeatable accuracy varies from 15 to 90 m (49 to 295
ft) depending on the vessel's location within a given coverage area (Dungan
1979; U.S. Coast Guard 1974). Achieving the short-range accuracies cited
above requires proper installation, maintenance, and operation of high-
quality equipment (Canadian Coast Guard 1981). Higher accuracies can be
obtained by operating in a differential mode (i.e., with a supplemental
receiver at a surveyed site onshore which transmits corrections or offsets
to the survey vessel). Available equipment varies from simple receivers
and indicators to fully automated receivers with self-tracking capabilities
that can interface with a vessel's computer. The cost of a Loran-C receiver,
excluding the antenna, varies from less than $1,000 to $2,500-plus.
Although Loran-C frequently is used in Puget Sound for sampling and
monitoring, this application has potential problems. Of particular concern
when operating in areas such as Puget Sound are time and spatial variations
in the Loran-C signals, and signal interferences that prevent operating
in desired survey areas. Inland location [up to 160 km (100 mi)] of Loran-C
chains requires overland signal transmission. This results in phase shifts
that are difficult to predict. Such shifts can cause an erroneous position
location fix. There are also anomalies associated with land-water interfaces
and large structures such as bridges and tall buildings. Crossing-a ^s
also can widely vary from one geographic area to another. In some ca^s,
lines of position are almost parallel, making an accurate fix very difficult.
Noise and interference (e.g., from engines and other electronic equipment)
also can be disruptive, but most Loran-C receivers are equipped with factory-set
or tunable notch filters to minimize such problems.
In an effort to improve navigational capability using the Loran-C
system, the U.S. Coast Guard completed a one-time survey of the east and
west U.S. coasts in which Loran-C positions were compared with those from
a calibrated microwave system. Corrections were obtained for the Defense
Mapping Agency nautical charts, whose LOPs were based on theoretical trans-
mission over water paths (Ryan, R., personal communication). These corrections
do not include seasonal or diurnal signal effects. The land transmission-
path effect (known as the additional secondary phase factor) currently
is under evaluation. The results of a recently completed multi-year West
Coast Stability Study, which extended from San Diego to Vancouver Island,
indicate that the repeatability of Loran-C in Puget Sound is significantly
better than other parts of the country. The annual variation in signals
were on the order of 0.2 usec (Figure C-6). The corresponding 95 percent
confidence ellipses and 2 drms positional repeatabilities at Neah Bay are
+40 m (131 ft). As the System Area Monitor at Whidbey Island is approached,
seasonal stabilities should improve, resulting in improved positional repeat-
C-31
-------
r-i
i
(A>
.3
.2
1
00
-.1
-2
-3
31
Mean
Signs
' • . I
Neah Bay
Yankee
Neah Bay
Zulu
m^i****^**^''*^ 1^11**!^
.4
3
2
1
00
-1
61
28798 863
.041
121
151 181
211 241 271 301
331
361
Julian Day 4
1984
31
Mean
Signs
61
41893.826
.059
121 151 181 211
Julian Day 4
1984
241 271 301 331
361
Figure C-6. Seasonal variations in Loran-C signals at Neah Bay, Washington 5990.
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
August 1986
ability. Based on the study and the propagation model developed, the Coast
Guard can estimate the error ellipse and 2 drms error circles for most
locations within Puget Sound (Slagle, D., personal communication).
Due to differences in the path conditions, measured time differences
(i.e., difference in arrival times of two simultaneously transmitted signals)
often are different than theoretically predicted. In fact, the deviation
varies depending upon the receiver location within the reception area.
So-called spatial variation or grid warpage can be removed by applying
corrections based on measurements at a nearby site that has been accurately
surveyed. Such a procedure was followed by the U.S. Coast Guard during
an extensive survey of Puget Sound's major ship traffic lanes. The purpose
was to establish accurately located way points to which the vessel can
be navigated. Following prescribed turning instructions at a given way
point, the vessel proceeds to the next way point, and repeats the procedure
until the destination is approached. Given an absolute accuracy requirement
of +40 m (131 ft), the Coast Guard conducted simultaneous measurements
at thousands of locations within the Sound with a Motorola Mini Ranger
and a Loran-C receiver. As a result, time differences at geodetically
known way points have been published and data for many interim track points
have been archived. Thus, for these points, a spatial correction can be
made. To maximize the absolute and repeatable accuracy at a given way
point requires input of an additional correction factor to compensate for
the daily and seasonal signal variations earlier addressed.
For purposes of locating sampling stations, it is possible to utilize
the way point data to develop a correction for other nearby locations based
on interpolation of data from the nearest four way point stations (Gazely,
L., personal communication). The accuracy to which the desired time differences
can be calculated and therefore the site located is dependent on distance
from the way points or track points to the desired location.
Metro completed a similar study with fixes from a microwave system
to plot the corresponding Loran-C coordinates within Elliott Bay and surrounding
areas. Absolute accuracies within this mapped region are increased to
40-100 m (131-328 ft) with repeatable accuracies substantially better (Sturgill,
D., personal communication). Metro and other groups update this Loran-C
chart periodically, with a new version expected during the first half of
1986. Because repeatable accuracies of Loran are acceptable for the monitoring
objectives, calibration of Loran-C positions is a reasonable alternative
for long-term monitoring projects by reducing costs incurred with renting
microwave systems (to a single expense). If the study site is within or
near the calibrated Loran-C stations set up by Metro or the Coast Guard,
then interpolation from the nearest stations may be adequate for accuracy
requirements.
Both temporal and spatial variances from a predicted navigation system
grid can be substantially reduced or eliminated by operating in a differential
mode (U.S. Department of Defense and U.S. Department of Navigation 1984).
C-33
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Station Positioning
Appendix C - Positioning Methods
August 1986
A facility (in this case a calibrated Loran-C receiver) may be located
at a fixed point within an area of interest. Loran-C signals are observed
in real time and compared with signals predicted for the known position.
The differences between the observed signal and predicted signal is transmitted
to users as a "differential correction" to upgrade the precision and performance
of the user's receiver processor. For Loran-C, the serving radius for
correction transmission may be up to 320 km (200 mi). The U.S. Coast Guard
studied the ability of the differential Loran-C to meet the 8-20 m (26-66
ft) accuracy requirement of U.S. harbors and harbor approaches. For the
Seattle area, the Coast Guard Double Range Difference MOD 2 Model predicts
that a 20 m (66 ft) 2 drms absolute position accuracy is feasible when
operating in a differential mode (Doughty and May 1985). Tests using Least
Squared Error, Alpha-Beta Filter, and Linear Regression Model approaches
to predict differential Loran-C time difference offsets indicate that a
consistent accuracy within 10 m (33 ft) is not an unreasonable goal for
the particular differential Loran-C system examined on the Thames River
(Bruckner 1985).
Radio frequency (RF) interference was evident during the West Coast
study, both nearby and within the Loran-C band of 90-110 kHz (Blizard and
Slagle 1985). RF interference was noticeably observed at the Tacoma Washington
Harbor Monitor Site, and directly affected the Coast Guard's data acquisition
and collection. During the 16 months of operation at this site, a significant
amount of the data was considered of poor quality, attributable to the
intermittent U.S. Navy transmissions at 76.3 kHz. Apparently the strength
of the signal is so strong that it caused the 100 kHz tuned Loran-C coupler
to oscillate. Conversations with area users indicated that many other
types of receivers also experienced similar problems, thereby limiting
the use of Loran-C in the Tacoma area. The extent to which the problem
is experienced further away from Tacoma will be receiver-dependent. The
U.S. Coast Guard found that use of notch filters in the Hood Canal/Bremerton
area eliminated the problem. Identifing the limit of the interference
area would require transits away from the source area while attempting
to notch out the interference, a task not included in the Coast Guard study.
Other areas with signal interference include relatively small intermittent
sources in Elliott Bay and at Seola Beach near Sea-Tac Airport (Figure C-7).
A large region of northern Puget Sound often is affected apparently because
of another U.S. Navy transmitter. This source precludes reception from
most sets north of Everett (in an area with normally good Loran reception).
Two additional sources of interference found in the West Coast study
were transmitter switching and chain control effects. The AN/FPN-44A tube
transmitters in the U.S. West Coast Chain (9940) located at Fallen, Nevada
(Master); Middeltown, California (X-Ray); and Searchlight, Nevada (Yankee)
are switched every 14 days for routine maintenance, resulting in a signal
offset for approximately 28 days. T,he resultant 40 nanosecond error/offset
C-34
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LARGE AREA REGULARLY AFFECTED
BY POSSIBLY A SINGLE POWERFUL
SOURCE
OCCASIONAL SHORT-TERM INTER-
FERENCE IN ELLIOTT BAY
SEOLA BEACH TO PT. WILLIAMS
POSSIBLE AIRPORT SOURCE
NOTE: MOST OF THE SOUTH SOUND
ALSO EXHIBITS INTERFERENCE FROM
A SINGLE SOURCE NEAR TACOMA
Figure C-7. Regions of LORAN-C signal interference in Puget Sound.
C-35
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Station Positioning
Appendix C - Positioning Methods
August 1986
would most likely be detected and applied in the time difference to the
latitude/longitude conversion algorithm. The Canadian West Coast Chain
(5990) would most likely exhibit similar offsets.
Also apparent in the study was the effect of moving an Alpha-1 site
receiver that maintains the Control Standard Time Differences. When the
9940 Whiskey A-l Monitor was moved in December 1984, a positional grid
shift of 300 nanoseconds occurred. For a typical gradient of 305 m (1,000 ft)
per usec, this is equivalent to a 91.4 m (300 ft) change in position.
Thus, applying Loran-C in a repeatable mode, the user would have found
his "past" position moved by more than 91.4 m (300 ft). In fact, depending
on crossing angle, the difference in positions could even be more. Thus,
users in the repeatable mode must monitor and compensate any changes in
the U.S. West Coast or Canadian Chain (whichever is used) when they occur.
In summary, the accuracies of Loran-C vary depending upon the location.
Prior knowledge of the area reception is required for an adequate determination
of its useability. Achievable accuracies have to be acceptable to the
sampling/monitoring objective for the system to be considered. However,
because of its relatively low cost, ease of installation/ operation, and
reliability in known areas over the range of conditions, Loran-C is a candidate
system to be considered.
Viewnav—
To improve upon the positioning accuracy of standard Loran-C receivers,
Navigation Sciences has developed Viewnav, an interactive computer system
that uses differential Loran-C to position a vessel with a claimed repeatable
accuracy of 4.6 m (15 ft). Absolute accuracy of the system is on the order
of +10 m (+32.8 ft) at the 90 percent confidence level, and +5 m (+16.4
ft) at the 20 percent confidence level (Newcomber, K., personal communication).
Loran-C offsets are obtained by interrogating onshore monitors established
by the company. In addition, a land-based microwave system is used to
calibrate a vessel's initial position or track. The system is particularly
effective in ports and harbor areas where large buildings or the land-water
interface may alter Loran-C readings.
A distinctive feature of the system is an electronic display of the
survey area based on digitized nautical charts. As the vessel moves, its
display position moves relative to depth contours and land boundaries.
Other waterborne radar images in the area also are indicated.
A full system costs approximately $40,000 with a supplementary annual
service fee of $2,000 for chart corrections and equipment maintenance.
The base price includes a mainframe Ai-M16 computer with 512KB of main
memory, a flexible disk drive with 1MB capacity, and a Winchester hard
disk of 10MB capacity. The basic system provides 5MB of chart storage,
C-36
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Station Positioning
Appendix C - Positioning Methods
August 1986
which equates to approximately 650 charts depending on scale selected.
Additional charts can be stored on floppy disks. The manufacturer expects
charts and Loran monitors throughout U.S. coastal areas by 1986.
The system could provide improved accuracies over Loran-C, especially
in areas of Puget Sound that have not been mapped for Loran. The shore
stations require setup time, as does the initial position calibration,
making this system more attractive to long-term monitoring programs. Loran-C
reception limitations again cause limited coverage of the Sound.
Lambda and OMEGA—
These two long-range systems are offshore positioning systems that
do not provide adequate accuracies and/or coverage for station positioning
within Puget Sound. Interested parties are referred to Tetra Tech (1986)
for more information on these systems.
Transit (Navsat)—
The U.S. Navy Navigation Satellite System (originally Project Transit)
consists of a group of satellites in 106-min circular polar orbits at altitudes
of approximately 1,411 km (877 mi). The system also includes ground tracking
stations, a computing center, an injection station, U.S. Naval Observatory
time signals, and vessel receivers and computers. Positional measurements
are based on the Doppler frequency shift that occurs when the relative
distance between the satellite transmitter and vessel receiver changes
(i.e., frequency increase upon closure and frequency decrease upon separation).
Provided the satellite orbits are accurately known, it is possible to locate
the receiver. The nature of the Doppler shift depends upon the exact location
of the receiver relative to the satellite path (Maloney 1978). The system
operates at frequencies of 150 and 400 MHz so that ionspheric corrections
can be made through signal comparison techniques. The vessel's position
is determined based on "known" orbital positions during satellite passage
and measured frequency shifts.
As originally designed, at least one satellite would be within line-
of-site every 35 to 100 min. However, at U.S. East and West Coast latitudes,
the acceptable fix window is approximately every 90 min (Driscoll, C.,
personal communication). This is caused, in part, by the requirement that
a satellite's maximum altitude be between 15° and 75° before a fix is considered
valid. Another problem occurs when two satellites being tracked have approxi-
mately the same closest approach, whereupon it becomes difficult to know
which one is monitored. Typically, each satellite provides four fixes
a day on two successive orbits spaced by 12 h. Because one satellite currently
is inoperative and another has weak batteries, it may take longer (e.g.,
several hours at the equator) to gain a valid fix (Booda 1984).
A static position fix with Transit using single-channel equipment
can be made with an accuracy of approximately 90 m (295 ft). Dual-channel
C-37
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Station Positioning
Appendix C - Positioning Methods
August 1986
receivers improve single-pass accuracy to 37-46 m (121-151 ft) (Hoeber
1981; Maloney 1978). With multiple passes, an rms accuracy of 3-5 m (9.8-
16.4 ft) is claimed by some equipment manufacturers. Using translocation
(i.e., simultaneous measurements at two fixed locations, one of which is
accurately known), estimated positional measurement accuracies are in the
range of 0.5-1.5 m (1.6-4.9 ft) (Oriscoll, C., personal communication;
Moyer, C., personal communication)% Magnavox has developed a satellite
surveying program (MAGNET) that is reportedly capable of simultaneous location
of up to 10 sites with a point-to-point accuracy of 30 cm (1 ft) or better.
However, this system is designed for coordinated geodetic studies over
wide areas using 3 to 10 MX 1505 receivers and recording data from 10 or
more satellite passes (Anonymous 1981).
Transit receiver costs range from $2,500 to $10,000 for basic single
frequency units (Murphy, W., personal communication). More elaborate multiple-
channel systems, sometimes in combination with OMEGA, range in cost from
$30,000 to $52,000 (Jolly, J., personal communication; Driscoll, C., personal
communication).
Use of the Transit system generally is not appropriate for sampling
and monitoring programs because of the inaccuracy of the system for fixes
from either a moving vessel or one occupying stations for a relatively
short time. A fix must be based on a single pass. With satellite passes
at 1-h or 2-h intervals, multiple-pass data acquisition is impractical.
Therefore, only the best single-pass accuracy of 37-46 m (121-151 ft) can
be achieved. Since translocation is designed for two fixed stations rather
than one on a moving sampling vessel, and since MAGNET applies to land
surveying only, neither method will provide a significant increase in accuracy
during the limited time the vessel will be at any one station. However,
the largest disadvantage of the system still is the time constraint between
fixes, which does not make it appropriate for surveys using expensive ship
time.
GEOSTAR—
GEOSTAR is a pulse radio transmission system. Just approved by the
FCC, it will provide satellite information for positions within the continental
U.S. and its coastal waters by 1987. Three geosynchronous satellites (and
a fourth as backup) will orbit the earth at 37,000 km (22,991 mi) at 70°,
100°, and 130° W longitude. System components include transceivers, satellites,
and computers at a ground center. The links between the ground station
and the satellites will operate at 5,117-5,183 MHz and 6,533 MHz, while
user-satellite links will be at 1,618 and 2,492 MHz (Whalen 1984). Should
a satellite fail, the backup would be moved into a proper orbit by telemetry
command from the ground computer facility.
The user will send a command through the transceiver, which relays
the message through the satellites to a central computer at the ground
center, reportedly in less than 1 sec. The signal-arrival times from each
C-38
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Station Positioning
Appendix C - Positioning Methods
August 1986
satellite are used by the ground computer to calculate the position of
the specially coded transceiver. The information is then transmitted back
to the satellites and relayed back to the transceiver in a similar amount
of time.
GEOSTAR will enable a typical single-shot positioning error of +2-7 m
(6-23 ft), according to the developer. When needed, accuracies down to
+ 1 m (3.3 ft) reportedly can be achieved with two-way interaction, signal
analysis, and averaging. Users at a known elevation (e.g., sea level)
will have greater accuracy due to much smaller geometrical dilution of
precision where only two (rather than three) coordinates are required.
Continuous operations in a differential mode also should enable correction
inputs for such errors as ionospheric delays, satellite position drift,
and drifts in satellite electronic delays.
System designers estimate that when operable the cost of a basic hand-
held transceiver with a typewriter keyboard and LCD display will be less
than $1,000. A monthly service charge in the range of $10 to $30 also
is anticipated (Howarth, C., personal communication). At publication,
FCC had just approved GEOSTAR Corporation's application for use of the
requested frequencies. Candidate users are urged to confirm FCC approval,
verify the latest satellite/ground station operating schedule, and obtain
further information on transceiver availability.
Navstar GPS—
The Navstar Global Positioning System (GPS) is a second-generation
satellite navigation system currently under development by the U.S. Department
of Defense. Its purpose is to provide precise, continuous, worldwide,
all-weather, three-dimensional navigation for land, sea, and air applications.
Under current plans, 18 satellites will be launched into three co-planar
orbits 120° apart to provide continuous transmission of time, three-dimensional
position, and velocity messages to system users. The GPS satellites transmit
at 1,227.6 MHz and 1,575.4 MHz to permit the measurement and correction
of ionospheric refraction error. Five developmental satellites currently
are in orbit, providing approximately 4 h of coverage twice daily, separated
by a 12-h period. Continuous two-dimensional positioning information should
be available with 12 satellites by February, 1988, and three-dimensional
coverage is projected for late 1988 or early 1989 (DeGroot, L., personal
communication; Stansell 1984). The system consists of the satellites in
12-h, 20,200-km (12,552-mi) orbits, a U.S. master control station, several
monitoring stations, and small, lightweight, relatively inexpensive receivers.
Signals received from any four Navstar satellites are demodulated, time-
correlated, and processed to obtain precise position information.
Two levels of positioning accuracy are achievable with the Navstar
GPS system. The lower level is obtained from the Standard Position Service
(SPS) using the coarse acquisition or "C/A code." When the system becomes
fully operational, navigational accuracy from these signals should be approxi-
C-39
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Station Positioning
Appendix C - Positioning Methods
August 1986
mately 100 m (328 ft) two-dimensional rms, or a circular probable error
(CEP) of 40 m (131 ft) (Montgomery, B., personal communication). More
accuracy can be achieved using the Precise Positioning Service (PPS) or
"P-code" (i.e., 8-9 m two-dimensional CEP). Additional positioning accuracy
can be achieved by operating in a differential mode, in which receivers
on a vessel and at a known onshore location simultaneously receive the
satellite signals. The onshore receiver is calibrated. Bias corrections
based on signals received at the fixed station are transmitted to the mobile
receiver. These area-specific corrections yield more accurate positional
determinations. With differential GPS, a two-dimensional position should
be definable within a range of 2-5 m (6.6-16.4 ft) (Montgomery 1984; Stansell
1984).
Due to the present lack of full-time coverage, both SPS and PPS are
available to military and civilian users. However, the government intends
to encrypt the P-codes, allowing use only by the military and other authorized
users [e.g., National Ocean Industries Association (NOIA) members]. NOIA
has proposed that all P-code receivers be owned and operated by a single
service company that would provide both the equipment and personnel when
national security conditions are met and no other reasonable navigational
alternatives are available (Stansell 1984). Under U.S. Department of Defense
policy, access to PPS is available to civilian users operating in the "national
interest" (Anonymous 1985). Equipment, cryptography, and support services
will be provided under contract to the user. However, it would appear that
general users in coastal areas will be limited to C/A code equipment. It
may therefore be necessary to operate in the differential mode in order to
achieve the positional accuracies needed for survey and monitoring requirements.
Because the GPS system is in a developmental stage, cost estimates
for the equipment are difficult to make. Several major equipment manufacturers
are in the process of designing receivers with varying capabilities, and
a limited number of models now are available. Some manufacturers envision
that receivers with 100-m (328-ft) accuracy will cost less than $500 when
mass produced (e.g., for automobiles). At the other extreme, for $140,000
Texas Instruments sells the TI4100 Navstar Navigator, said to be capable
of slow dynamic positioning within a few meters, speed within tenths of
a knot, and time to the usec (Montgomery 1984; St. Pierre, R., personal
communication). Motorola anticipates that the initial cost of two stations
needed to operate in the differential mode will be in the $100,000-range
(Sheard, S., personal communication). Magnavox has a five-channel T-Set
GPS Navigator, with real time differential GPS operation as a planned option.
A two-unit system, excluding communications link, costs approximately $100,000
(Driscoll, C., personal communication). Rockwell International sells a
prototype C/A code receiver for $17,500 and anticipates that GPS receivers
will cost less than $10,000 by 1988 (DeGroot, L., personal communication).
Tracer expects initial models to sell for less than $10,000, falling to
around $1,000 in 3-5 yr (Murphy, W., personal communication).
C-40
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Station Positioning
Appendix C - Positioning Methods
August 1986
Using the C/A code in a differential mode, the Navstar GPS positioning
system should provide accuracies within +3 m (+9.8 ft). Errors of 1 m
(3 ft) at a range of ZOO km (12.4 mi) from the reference station are predicted
for daytime operation. Smaller errors are expected at night (Kalafus 1985).
However, testing with the actual satellite signals modified to reduce achievable
accuracy will be required to confirm this capability. The two required
stations may be expensive at first, but competition and the large potential
market are expected to drive the price much lower.
SERIES—
The SERIES positioning and navigation system (Satellite Emission Range
Inferred Earth Surveying) has evolved from a technique originally developed
to measure movements in the earth's crust for tectonic studies and earthquake
predictions. The system enables use of signals from existing Navstar GPS
satellites with no knowledge of either the P or C/A codes by simultaneous
pseudoranging to multiple satellites in a differential mode. Accuracies
on the order of 2 m (6.6 ft) CEP or 5 m (16 ft) two-dimensional rms are
said to be achievable for vessels in a slow dynamic mode (0-10 kn) (ISTAC
1984). This is accomplished by comparing Navstar signal arrivals at one
receiver to the same signals received at another shore-based geodetic reference
mark. By explicit differencing of the frequency and phase measurements
observed from each satellite, it is apparently possible to eliminate the
effects of Navstar signal variations that secure the satellites from an
unauthorized user in an autonomous, single-receiver, real time mode. SERIES
receivers are codeless spectral compressors that simultaneously receive
and incorporate spread-spectra modulations of several Navstar satellites
and then extract the frequency and phase of each satellite in view. Data
are processed by a combination of Doppler positioning and phase ranging.
System components include an unattended, fixed reference station,
a marine vessel station, and a reference link (via satellite or terrestrial
radio) to transfer the reference station signal to the marine vessel for
real time positioning. The reference station includes an SERIES GPS receiver
(MPS-1), a rubidium frequency reference, and a customer-supplied radio
transmitter (200 Hz analog signal). The marine station consists of the
GPS receiver with an integrated Navstar C/A-code-correlating subsystem
for broadcast satellite ephemerides, a rubidium frequency reference, a
microprocessor for real time position computations, and a customer-supplied
receiver for fixed reference station signal reception. A standardized
RS-432 interface is provided for communication of the position information
to the vessel's navigation system.
The initial MPS-1 units are estimated to cost $205,000 for a ship
system and $82,000 for the fixed base system required for differential
mode operation (Whitcomb, J., personal communication). The system will
probably be available on a lease or service-contract basis, as well.
C-41
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Station Positioning
Appendix C - Positioning Methods
August 1986
When operational, the SERIES system may offer a high-accuracy alternative
to the Precise Positioning Service of Navstar GPS or to differential mode
operation with the C/A code. Although the projected cost for an initial
unit is high (to recover research and development expenditures), cost should
decrease with further production. Potential users should confirm price,
verify accuracies achievable from a mobile marine vessel, and identify
specific data-link components that need-to be supplied by the user.
Aero Service GPS—
The Aero Service Division of Western Geophysical Company of America
is developing a codeless satellite navigation system for marine positioning.
The system will enable real time navigation in the slow dynamic mode to
less than 5 m (16.4 ft). For vessels in the semi-static mode (on station)
for 30 min, the system will reportedly yield a position fix accuracy of
less than 1 m (3.3 ft) (Mateker, E., personal communication). The system
will require one unit onshore at a fixed position, and one unit on the
survey vessel. Continuous real time communication between the two systems
may or may not be necessary, depending on steps by the U.S. Department
of Defense to degrade certain elements of the GPS satellite signals. Prototype
testing was scheduled for September, 1985, with production systems planned
for early 1986.
This future system is worthy of note because it will probably be based
on methods now used for land surveying in the MACROMETER Interferometric
Survey System. The MACROMETER is designed for very precise positioning
in a fixed-point (static) mode, using GPS signals but no codes. Company
tracking stations (Phoenix, Arizona and Woburn, Massachusetts) will follow
up to six satellites simultaneously. The MACROMETER is capable of providing
position in terms of latitude, longitude, and ellipsoidal height to within
a few parts per million of the distance from a reference point. In the
continental U.S., 1-3 h of observation will yield first-order (1:100,000)
accuracy in all three coordinates using the GPS satellites. Data for a
second-order or third-order survey point may be collected in less than
1 h (e.g., as little as 15 min of observation).
The MACROMETER surveying system includes two or more field units and
an office data processor. Each V-1000 field unit includes an antenna,
receiver, tape drive, 30 m of cable, time receiver, power package, battery
chargers, generator, and DC/AC inverter. The P-1000 Office Data Processor
computer subsystem includes hard and floppy disk drives, video terminal,
tape drive, printer, modem, and all necessary software. A 1,000 series
two-unit surveying package is currently priced at $235,000. The system
can be rented for $5,000 per V-1000 unit per month, with a 2-mo minimum
on each unit.
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Station Positioning
Appendix C - Positioning Methods
August 1986
RANGE-AZIMUTH SYSTEMS
A number of hybrid positioning systems combine positional data from
various sources to obtain fixes. Such systems usually involve the intersection
of a visual 1 ine-of-position with an electronic line-of-position (Umbach
1976). Visual data may be in the form of sextant angles or theodolite
azimuths. Electronic positional data 'are normally obtained from a microwave
system.
Of particular interest for survey and monitoring programs are dynamic
positioning systems that require only a single shore station and that use
the simultaneous measurement of angle from a known direction and range
to the survey vessel. This range-azimuth method has the advantage of circular
coverage around the shore station (Figure C-8). A single station minimizes
logistical requirements and geometric limitations. Line-of-position inter-
sections are the ideal 90° everywhere within the coverage area. Growth
in the error ellipse is due only to distance from the shore station because
of its independence of the absolute azimuth angle. Accuracy improves as
range decreases, even fairly close to the shore station, and only one unob-
structed line-of-sight is needed. However, such systems allow only a single
user.
Characteristics of three representative systems are summarized in
Table C-12. Two have fully automatic shore stations, requiring attendance
only during setup and alignment. The third requires an onshore operator
at all times. For multiple-day surveys, an automatic station eliminates
the tedium of continuously tracking the survey vessel. The systems offer
much greater flexibility than range-range or hyperbolic systems with multiple
onshore stations. Where positioning requirements extend into ports, estuaries,
or up rivers, the single-station systems offer distinct advantages in covering
restricted or congested survey areas and in establishing an unobstructed
signal path. Each system is distinctive in either its operating medium
(optical, microwave, laser) and/or procedure (i.e., manual or automatic
tracking). The added costs of these systems over standard microwave systems
may be justified where the system also can be used for onshore work during
nonsurvey periods.
AZTRAC
The ODOM Offshore Survey AZTRAC is a semi automated optical angle-measuring
and transmitting system which can be used in conjunction with an independent
distance-measuring system to position a vessel. The AZTRAC system consists
of a modified Wild T16 theodolite, an onshore transmitter, and a vessel
receiver The theodolite has an infinite tangent drive and provides information
in a digital format. Typically, the separate distance-measuring system
consists of a microwave master receiver on the vessel and a remote unit
transmitter located at the theodolite.
C-43
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Figure C-8. Range-azimuth positioning system area of coverage.
C-44
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TABLE C-12. RANGE-AZIMUTH POSITIONING SYSTEM CHARACTERISTICS
SYSTEMS
AZTRAC
Odom Offshore Surveys
Baton Rouge, Louisiana
(504)769-3051
POLARFIX
Krupp Atlas-Elektronik
Webster, Texas
(713)338-6631
ARTEMIS
Andrews Hydrographies
FREQUENCY RANGE
Optical Visible
and
microwave
(typical)
Laser 5000 m
(904 ran)
UHF Telemetry
(406-470 MHz)
9.2-9.3 GHz 10-1400 m or
200-30,000 m
NOMINAL ACCURACY
AZIMUTH RANGE POSITION
0.01 Both depend on accuracy of
ranging system used
0.01° i0.1m±0.1m/km ±0.1m±0.2m/km
0.03° 0.5 m short Not stated
1.5 m long
COST
$22,500 plus
ranging
system cost
cost
$100,000
$70,000 to
$75,000
Houston, Texas
(713)558-2236
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Station Positioning
Appendix C - Positioning Methods
August 1986
For a survey, the AZTRAC theodolite and transmitter are set up at
a known position and the theodolite is zeroed on a known azimuth line of
backsight. The theodolite operator sights and tracks the survey vessel's
ranging antenna or transponder. As the vessel moves, the microwave ranging
system continuously measures distance between the shore station and the
vessel. The tracking motion produces pulses that are decoded by the AZTRAC
transmitter and displayed to the theodolite operator as the angle to the
vessel from the reference azimuth line. The angle also is converted to
BCD serial format and is used to activate the transmitter, which sends
the information to the survey vessel. The AZTRAC receiver converts the
angle information to parallel format and displays it for manual recording.
It simultaneously outputs the angle as serial data for automated recording
or processing by any available on-board computer or plotting system.
AZTRAC is designed to operate with most microwave ranging systems.
Two AZTRAC units working in an azimuth-azimuth mode can provide positioning
in survey areas where reflections from metal structures or electrical noise
from radar and other transmitters limit use of microwave ranging. The
Wild T16 theodolite has a 30X magnification, 27-m (89-ft) field of view
at 1,000 m (3,280 ft), and an angular resolution of 0.01° (36 arc sec); at
a distance of 5 km (3.1 mi), this corresponds to an absolute arc length
error of 0.9 m (3.0 ft).
The National Ocean Survey recently examined range-azimuth positioning
of a vessel moving at a nominal speed of 6 kn (11.1 km/h) at ranges of
up to 3,000 m (9,842 ft). Instruments included a Wild T2 theodolite, from
which angles were manually recorded onshore, and the AZTRAC, whose angles
were recorded on the vessel. Pointing errors (68 percent probability)
of these two instruments were found to be approximately 1.3 m (4.3 ft),
independent of range when standard deviations of right and left movement
data were pooled (Waltz 1984).
The theodolite was sited on a white Del Nprte Trisponder transponder
on the moving vessel. Visibility was good during the 2-day survey (off
Monterey, CA), with calm mornings giving way to afternoon winds of 15 kn
and 0.6-0.9 m (2-3 ft) seas. Range could have been extended much farther
under such conditions, particularly if color had been added to the vessel
target (Waltz, D.A., personal communication). U.S. Army Corps of Engineer
users confirmed the effectiveness and reliability of the AZTRAC system
in conjunction with several different microwave ranging systems (Ard, R.,
personal communication). The system also has proven effective in port
and harbor surveys when it was impossible to achieve optimal shore station
geometry for range-range or hyperbolic operation.
The current cost of the AZTRAC alone is $22,500 (Apsey, B., personal
communication). A system consisting of the AZTRAC, a Motorola Falcon 484
with one reference station (approximately $32,000), and an interface unit
($10,000) would cost $64,500. Thus, provided the required range is achievable
C-46
-------
Station Positioning
Appendix C - Positioning Methods
August 1986
under anticipated visibility conditions, the additional versatility of
the AZTRAC can be realized for $25,000 above the $39,300 cost of a two-
station Falcon 484 range-range microwave system.
POLARFIX
POLARFIX is a dynamic range-azimuth positioning system by Krupp Atlas-
Elektronik. The system uses a scanning (30° horizontal) pulsed laser beam
from a single, fixed, onshore tracking station to follow the survey vessel
(up to 10°/sec) and to transmit range and angle information via telemetry
link. The system incorporates a fully automated onshore tracking station,
which requires no attendance beyond initial station setup and azimuth refer-
encing. The shore station can locate the mast-mounted prism reflectors,
follow the vessel, and, if necessary, relocate the vessel by performing
a routine search pattern based on a record of tracking history. The shore
tracking station consists of a laser-sensing head mounted on a conventional
survey tripod, linked by cable to an integrated control unit that houses
data control, transmission, and telemetry transceiver equipment. An integral
control unit (including a display and a second telemetry receiver), keyboard
terminal, printer, telemetry antenna, and prism reflector assembly are
onboard the vessel.
Under clear operating conditions, a 3-km (1.9-mi) range using a Class I
laser or a 5-km (3.1-mi) range using a Class Ilia laser may be selected.
In foggy weather, range is said to be 1.5 times visible range, due to use
of the pulsed infrared laser. Maximum range achievable varies with the
prism assembly used to reflect the tracking station's laser beam. Single-,
dual-, and triple-ring omnidirectional prism assemblies can be stacked
on the vessel antenna. For average weather conditions, a two-prism assembly
(each with five reflectors) gives approximately 3.5 km (2.2 mi) of range.
This can be extended to approximately 5.0 km (3.1 mi) with the addition
of more assemblies. Although a 5 km (3.1 mi) distance from shore will
cover most of the Sound, this does add limitations to range in some areas.
Range accuracy is reported as 0.1 m +0.1 m/km (0.3 ft +0.5 ft/mi)
of measured range. Azimuth accuracy is said to be 0.01° or better. The
resulting positional accuracy at 1, 3, and 5 km (0.6, 1.9, and 3.1 mi)
is approximately 0.3, 0.6, and 1.0 m (1, 2, 3.2 ft), respectively. The
positional algorithm given is +0.1 m +0.2 m/km (+0.3 ft +1.1 ft/mi). Current
cost of the system is $100,000 (Guillory, J., personal communication).
ARTEMIS
The ARTEMIS by the Christiaan Huygenslabortorium (Holland) is a distance-
bearing type of microwave positioning system capable of measurements at
ranges of 10 m (32.8 ft) to 30 km (18.6 mi), and angles from 0° to 360°
from a single fixed shore station. Accuracies at the two-sigma or 95 percent
level are given as +1.5 m (4.9 ft) distance, and +0.03° azimuth, equivalent
to +0.5 m/km (2.6 ft/mi).
C-47
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Station Positioning
Appendix C - Positioning Methods
August 1986
Angle measurements are based on automatic tracking antennas on the
vessel and at the shore station. Once locked, the two antennae move always
pointing towards each other. A maximum combined tracking speed of 3°/sec
is allowable to achieve the specified angle error. The direction of the
fixed station antenna is accurately measured with a precision shaft coder,
which is mechanically coupled to the ma-in shaft of the antenna. Measured
angle data are transferred to the mobile station via the established continuous
microwave channel. The same microwave link is used to measure distance
by controlled interruption of the microwave signal. Both angle and distance
usually are displayed on the Mobile Control Data Unit, although readout
at the shore station also is feasible. The microwave link also is used
for voice communication between the two stations without disturbing the
data being transmitted.
The vessel's positioning equipment consists of the Mobile Control
Data Unit (MCDU), a Mobile Antenna Unit (MAU), the antenna, cables, a telephone
handset, and a speaker. The shore station consists of a Fix Antenna Unit
(FAU), Fix Control Data Unit (FCDU), an antenna, cables, a telephone handset,
a speaker, and a telescope for initial directional alignment. The shore
station requires attendance only for setup, referencing, and periodic battery
checks. The bearing is electronically referenced to a geodetic grid by
siting the unit in a known reference direction and manually adjusting the
observed direction readout to the correct value.
Limiting factors (common to all microwave systems) include radio line-
of-site conditions (obstacle-free for optimum performance) and multipath
interference due to reflections from the sea surface. The latter can be
reduced by proper adjustment of antenna heights. Where survey area traffic
is heavy, signal interruptions could unlock the two tracking antennae.
Manual relocking by a shore station attendant or automatic relocking using
autosearch, an option available at added cost, would then be necessary.
Although signals generally are unaffected by rain and fog, there is some
range reduction during heavy rains or snowfalls.
The present system costs $70,000-$75,000, depending on the options
selected (Coupe, C., personal communication). The system may be rented
from Andrews Hydrographies, Inc. (Houston, Texas) for approximately $850
per day.
C-48
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Station Positioning
Appendix C - Positioning Methods
August 1986
APPENDIX C
REFERENCES
Anonymous. 1981. Satellite program surveys 10 sites simultaneously.
Sea Technology. 22(5):41.
Anonymous. 1985. Precision code unavailable to civil sector GPS users.
Sea Technology. 26(1):77.
Apsey, B. 10 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Odom Offshore Surveys, Inc., Baton Rouge, LA.
Ard, R. 18 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). U.S. Army Corps of Engineers San Francisco District,
San Francisco, CA.
Baker, W.F. 11 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Teludist, Inc., Mastic Beach, NY.
Blizard, M.M., and O.C. Slagle. 1985. LORAN-C West Coast stability study.
Technical paper presented at the Fourteenth Annual Wild Goose Association
Convention, October 23, 1985, Santa Barbara, CA.
Booda, L.L. 1984. Civil use of Navstar GPS, a matter of debate. Sea
Technology. 25(3):17-18.
Bowditch, N. 1984. American practical navigator. An epitome of navigation.
Defense Mapping Agency Hydrographic Center.
Bruckner, D.C. 1985. Differential Loran-C: estimator improvement and
local system implementation. Technical paper presented at the Fourteenth
Annual Wild Goose Association Convention, October 23, 1985, Santa Barbara,
CA.
Buchanan, C. 31 December 1984. Personal Communication (phone by Dr. William
P. Muellenhoff). Del Norte Technology, Inc., Euless, TX.
Canadian Coast Guard. 1981. A primer on Loran-C. TP-2659. Aids and
Waterways, Canadian Coast Guard, Department of Transportation, Ottawa,
Ontario, Canada. 29 pp.
Clark, D. 1951. Plane and geodetic surveying for engineers. Constable
and Company, Ltd., London, UK. 636 pp.
C-49
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Station Positioning
Appendix C - Positioning Methods
August 1986
Coupe, C.J. 23 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Andrews Hydrographic Services Inc., Houston, TX.
Davis, R.E., F.S. Foote, and J.W. Kelly. 1966. Surveying theory and practice.
McGraw-Hill Book Co., New York, NY. 1096 pp.
DeGroot, L. 15 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Rockwell International, Cedar Rapids, IA.
Doughty, R.A., and W.K. May. 1985. Differential Loran-C...where do we
really need it. Technical paper presented at the Fourteenth Annual Wild
Goose Association Convention, October 23, 1985, Santa Barbara, CA.
Driscoll, C. 15 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Magnavox, Torrance, CA.
Dungan, R.G. 1979. How to get the most out of Loran-C. SG 54. Extension
Marine Advisory Program, Oregon State University, Corvallis, OR. 12 pp.
Folk, L. 21 March 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Kuker-Rankin, Inc., Seattle, WA.
Gazley, L. 17 December 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). U.S. Coast Guard Office of Navigation, Radionavigation
Division, Washington, DC.
Guillory, J. 28 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Krupp Atlas-Elektronik, Webster, TX.
Harris, E. 3 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Del Norte Technology Inc., Camarillo, CA.
Hempel , C. 10 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Cubic Western Data, San Diego, CA.
Hoeber, J.L. 1981. An update on worldwide navigation systems - the present
and the year 2000. Sea Technology. 22(3):10-13.
Howarth, C. 16 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Geostar Corp., Princeton, NJ.
Ingham, A.E. 1975. Sea surveying. John Wiley and Sons, New York, NY.
306 pp.
ISTAC. 1984. Marine Positioning Sensor. MTS-1. Technical Data Sheet
ISTAC-SERIES. ISTAC, Inc., Pasadena, CA. p. 47.
Kalafus, R.M. 1985. Differential GPS standards. Satellite navigation
system is a promising new tool of high accuracy. Sea Technology 26:52-54.
C-50
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Station Positioning
Appendix C - Positioning Methods
August 1986
Jolly, J. 31 December 1984. Personal Communication (phone by Dr. William
P. Muellenhoff). Jon B. Jolly, Inc., Seattle, WA.
Jolly, J. 15 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Jon B. Jolly, Inc., Seattle, WA.
Maloney, E.S. 1978. Duttons navigation and piloting. Naval Institute
Press, Annapolis, MD. 910 pp.
Mateker, E.J., Jr. 17 January 1985. Personal Communication (phone by
Dr. William P. Muellenhoff). Litton Aero Service, Houston, TX.
McDonnell, Jr. 1983. Total station survey. Point of Beginning. 8:16-30.
Moffitt, F.H., and H. Bouchard. 1982. Surveying. Harper and Row Publishing,
New York, NY. 834 pp.
Montgomery, B.O. 1984. Navstar GPS - A giant step for navigation and
positioning. Sea Technology. 25(3):22-23.
Montgomery, B. 15 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Texas Instruments, Lewisville, TX.
Moyer, C. 15 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Motorola, Inc., Tempe, AZ.
Murphy, W. 15 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Tracer, Inc., Austin, TX.
Newcomer, K.E. 21 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Navigation Sciences, Inc., Bethesda, MD.
Panshin, D.A. 1979. What you should know about Loran-C receivers. SG 50.
Extension Marine Advisory Program, Oregon State University, Corvallis,
OR. 8 pp.
Ryan, R. 11 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). USCG Pacific Area, Government Island, Alameda, CA.
Sheard, S. 15 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). Motorola Inc., Tempe, AZ.
Slagle, 0. 31 October 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). U.S. Coast Guard Research and Development Center, Avery
Point, Groton, CT.
C-51
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Station Positioning
Appendix C - Positioning Methods
August 1986
Stansell, Jr., T.A. 1984. GPS marine user equipment. Magnavox Technical
Paper MX-TM-3381-84. Presented at the Global Civil Satellite Navigation
Systems Conference, The Royal Institute of Navigation, London, UK.
St. Pierre, R. 15 January 1985. Personal Communication (phone by Dr. William
Muellenhoff). Texas Instruments, Lewisville, TX.
Sturgill, D. 18 November 1985. Personal Communication (phone by Jeff
H. Stern). Municipality of Metropolitan Seattle, Seattle, WA.
Tetra Tech. 1986. Evaluation of survey positioning methods for nearshore
marine and estuarine waters. U.S. EPA Office of Marine and Estuarine Pro-
tection, Washington, DC. Tetra Tech, Inc., Bellevue, WA. 107 pp.
Umbach, M.J. 1976. Hydrographic manual, fourth edition. U.S. Department
of Commerce, NOAA, Rockville, MD. 414 pp.
U.S. Coast Guard. 1974. Loran-C user handbook. CG-462. U.S. Coast Guard,
Washington, DC. 25 pp.
U.S. Department of Defense and U.S. Department of Transportation. 1984.
Federal radionavigation plan. DOD-4650.4, DOT-TSC-RSPA-84-8. Washington, DC.
Waltz, D.A. 1984. An error analysis of range-azimuth positioning, pp. 93-
101. In: Proc. of National Ocean Service Hydrographic Conference. April
25-27, Rockville, MD.
Waltz, D.A. 18 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). National Ocean Service, Norfolk, VA.
Whalen, W.L. 1984. Geostar positioning system using satellite technology.
Sea Technology. 25(3):31-34.
Whitcomb, J.H. 14 January 1985. Personal Communication (phone by Dr. William
P. Muellenhoff). ISTAC, Inc., Pasadena, CA.
C-52
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APPENDIX D
EVALUATION OF POSITIONING METHODS
-------
Station Positioning
Appendix D - Methods Evaluation
August 1986
APPENDIX D
EVALUATION OF POSITIONING METHODS
Methods to position at stations in Puget Sound for collection of environ-
mental samples are addressed in this appendix. After an overview of each
method is presented, the available positioning methods are evaluated and
comparative information is presented to facilitate selection of a method
appropriate to meet sampling objectives. Positioning methods and associated
equipment are described in detail in Appendix C.
POSITIONING PROCEDURES IN USE IN PUGET SOUND
Equipment, procedures, advantages, and limitations of positioning
methods used in Puget Sound were identified from interviews with various
governmental and private groups that conduct environmental sampling within
Puget Sound (Table D-l). These methods, their accuracies as expressed
by the field personnel who work with them, and the limiting restrictions
are summarized in Table D-2. The majority of the sampling done in Puget
Sound in recent years has been accomplished with the following five positioning
methods:
• Sextant angle resection
• Variable range radar (VRR)
• Loran-C
• Combinations of VRR, visual fixes, and Loran-C
• Microwave systems.
Many smaller research vessels in Puget Sound are equipped with VRR
and positioning by this method is usually used when VRR fixes are considered
adequate for station location definition. However, if higher accuracies
are required, VRR fixes are supplemented by visual line-of-site fixes,
bottom depth readout, and Loran-C. Loran-C cannot initially be used to
locate a station in most areas of Puget Sound. Loran-C coordinates are
recorded, however, to help improve repositioning accuracy. Overall, the
combination of methods should increase accuracies closer to shore (visual
fixes) and with steeper bottom slopes (fathometer).
Of the other methods practiced in Puget Sound, sextant angles are
used the least. Because of visibility effects, it is difficult to estimate
the accuracy of this method, which can change considerably between stations.
D-l
-------
TABLE D-l. INTERVIEWED GOVERNMENTAL AND PRIVATE GROUPS
THAT CONDUCT SAMPLING IN PUGET SOUND
GOVERNMENTAL AGENCIES AND INSTITUTIONS
Federal
National Marine Fisheries Service (NOAA)
National Ocean Survey (NOAA)
Pacific Marine Environmental Laboratory (NOAA)
U.S. Army Corps of Engineers
U.S. Environmental Protection Agency
State
Department of Ecology
Department of Natural Resources
Department of Social and Health Services
University of Washington, College of Ocean and Fisheries Sciences
Local
Municipality of Metropolitan Seattle (Metro)
PRIVATE FIRMS
Battelle Northwest Laboratories
Cooper Consultants, Inc.
Evans Hamilton, Inc.
EVS Consultants
Northern Technical Services, Inc.
Parametrix, Inc.
URS Engineers
RESEARCH VESSELS
Clifford A. Barnes (UW)
DNR III (WDNR)
Kittiwake (Private)
Liberty (Metro)
McArthur (NOAA)
Harold K. Streeter (NOAA)
D-2
-------
TABLE D-2. CHARACTERISTICS OF POSITIONING METHODS
USED FOR SAMPLING IN PUGET SOUNDa
o
co
Method
Sextant Angle
Resection
Variable Range
Radar (VRR)
Loran-C
VRR Visual Fixes,
Loran-C
Microwave Systems
Areas
Open Areas
Nearshore
All
Limited
Open Areas
Nearshore
All
User Reported Accuracy Ranges (+m)°
Absolute Repeatable Restrictions Use Among Local Groups
100
10
70
40
30
10
2.5
- 200
- 20
- 120
- 100
- 100
- 40
- 10
<100 Visibility, landmarks
Similar <2 - 3 km from shore
20 - 50 Increases with distance,
featureless topography
10 - 30 Only in Loran-C
mapped areas
20 Repeatability drops in areas
with no Loran reception
Similar <3-4 km from shore, Loran not as
critical to repeatability
1-3 Transponder locations
Low
Low
Medium
Medium
High
Medium
High
a As identified through user interviews.
b Reported accuracy ranges are consistent with manufacturer claims with
the exception that sextant accuracies only achieve highest accuracies near-
shore.
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Station Positioning
Appendix D - Methods Evaluation
August 1986
Therefore, sextants are used mainly during region characterization studies
when location is not critical. When strict absolute accuracy is needed,
microwave systems are frequently used. In areas that have been mapped
for Loran-C range lines, absolute accuracies as good as or better than
VRR can be achieved with Loran-C. As more stations are occupied in these
mapped areas, comparisons of Loran-C signals with other fixes help define
areas of signal warpage from land ar building interactions, increasing
absolute accuracy. These systems and their methods are discussed in detail
in Appendix C.
CANDIDATE SYSTEM OVERVIEW
Methods other than those commonly used in Puget Sound for station
positioning are available. Characteristics, major advantages, and major
disadvantages of each method listed below are summarized in this section.
Detailed descriptions are presented1in Appendix C.
0 Multiple horizontal angles
Theodolite
Sextant angle resection
• Multiple electronic ranges
Variable range radar
Distance-measuring instruments
Microwave systems
Loran-C
Satellite ranging
t Range and angle
Theodolite and EDMI
Total station
Range-azimuth positioning systems.
Multiple Horizontal Angles
Theodolites have the necessary angular acuracies at the anticipated
maximum ranges. They are commonly used as surveying instruments and cost
$2,000 (30-sec accuarcy) to $4,000 (10-sec accuracy). At least two theodolites,
two operators, a siting target on the vessel, and a three-way communications
link to coordinate fixes are required (see Appendix C, Figure C-l). Theodolites
can be used only during daylight hours of good visibility.
Sextant angle resection techniques offer adequate angular accuracy
( + 10 sec) and sextants costs $1,000-$5,000. A three-arm protractor is
required for plotting positions. Two oprators should take simultaneous
fixes on moving vessels. Because the operators are on board, a separate
D-4
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Station Positioning
Appendix D - Methods Evaluation
August 1986
communication link is not necessary and they can also serve as crew. However,
the method requires highly visible shore targets and is therefore useful
only during daylight hours of good visibility. In addition, it is difficult
for even an experienced operator to shoot an accurate fix from a moving
platform in adverse weather.
Multiple Electronic Ranges
Positioning with multiple electronic VRR ranges will be less accurate
over anticipated distances than electronic positioning methods. Equipment
costs range from $4,000 to $10,000. Weather and visibility rarely limit
use and extra personnel are not needed to help navigate.
Positioning with Electronic Distance-Measuring Instruments (EDMI)
offers adequate accuracy but marginal range beyond 3 km (1.9 mi) without
multiple prisms. EDMI systems cost $8,000-$15,000 apiece for long-range
units and approach $40,000 for systems with prisms. EDMIs require two
staffed stations, a three-way communications link to coordinate fixes,
and multiple prism assemblies.
Several microwave navigation systems with sufficient accuracy and
adequate range are available for $40,000-$90,000. These systems comprise
two shore stations and an on-board transmitter. With an additional shore
station, the hyperbolic mode can provide multiple user capability. Limitations
include geometry of shore stations; vessel position in the coverage area
(i.e., crossing angle limitations); and possible interferences from line-of-site
obstructions, sea-surface reflective nulls, and land-sea boundaries.
Positioning from Loran-C ranges offers acceptable repeatable accuracy
for relocating at station locations for most sampling objectives. Receivers
cost $1,000-$4,000 and do not require additional personnel. Limitations
include interference in some areas of Puget Sound and the need to initially
locate stations with another method.
Transit satellite-based methods currently do not offer sufficient
accuracy except with multiple passes, which are impracticable when a sampling
station is only briefly occupied. In the future, required accuracies will
be achievable using Navstar GPS satellite-based techniques ($10,000-$40,000
for first units; $1,000 for subsequent production models). Independent
geosynchronous satellite networks, such as GEOSTAR, may become available
at a system interrogator cost of $450 plus a monthly fee. This method
is in the early planning stages and recently received FCC approval. Satellite
methods do not require additional personnel.
Range and Angle
A theodolite and EDMI could be paired with a communications link for
approximately $10,000-$12,000. Total stations developed for this purpose
range in cost from $9,000 for a manual station to $15,000-$25,000 for a
D-5
-------
Station Positioning
Appendix D - Methods Evaluation
August 1986
fully automated station. Optical and infrared range limitations exist, and
the optical components can be operated only during daylight hours of good
visibility. The range-azimuth positioning methods examined (see Appendix C)
provide sufficient positional accuracy with a single station, and cost
between $65,000 for a manual tracking and $70,000-$100,000 for fully automated
tracking.
SCREENING CRITERIA
Candidate systems were evaluated for accuracy, flexibility (i.e.,
range of conditions under which the system can operate, including use for
other purposes), portability, reliability, servicing requirements, availability,
cost, and convenience. Results of the evaluation are presented in Table D-3.
Methods are presented across the top of this table in order of increasing
range capability. Methods that could be eliminated from further consideration
for some sampling objectives are marked by an asterisk. Limitations that
could preclude further consideration included inability to operate at night
or in poor visibility (e.g., optical methods) or availability of comparable
methods at lower cost with fewer logistical problems (e.g., medium-range
systems). The investigator has to determine the project-specific considerations
that could be restrictive enough to preclude use of some positioning methods
and to determine if the accuracies required for the study can be achieved.
For purposes of this evaluation, medium-range sytems were dropped from
consideration and optical methods were considered only for nearshore sampling.
Remaining positioning methods were reevaluated for range capability, accuracy,
availability, capital and operating costs, and merits of use. This information
is summarized below.
Range Considerations
Most of the systems noted for restrictive limitations have inadequate
range capabilities for visibility and/or distance reasons. At sampling
sites farther from land in Puget Sound, VRR will have to be used at a larger
scale and a lower accuracy. Sextants and (to a lesser extent) systems
that use theodolites may not permit adequate resolution of targets. Lower
resolution results in reduced accuracy. The rest of the remaining systems
listed in Table D-3 have adequate ranges to cover most areas of Puget Sound
(Figure D-l).
Accuracy
Various methods can meet repeatable accuracies of +20 m (+66 ft).
VRR can consistently position within +20 m (+66 ft) in areas close to shore
with supplement from visual fixes and farther from land in areas with good
Loran-C reception. Loran-C alone can reposition within +20 m (+66 ft)
in the Elliott Bay area that has recently been mapped with a Loran signal
grid. Also, under good conditions, sextants can easily reposition within
this distance. Near shore, sextants may approach the accuracy of microwave
systems under perfect conditions. Microwave systems, range-azimuth systems,
D-6
-------
TABLE D-3. EVALUATION OF NAVIGATION METHODS FOR STATION POSITIONING
Sextant
Angle
Resection Theodolite
Accuracy
Absolute
Repea table
Flexibil ity
Portabil ity
Reliabil ity
Servicing
Cal ibration
Maintenance
Availabil ity
Equ i pmen t
Service
Rental
Cost
Convenience
M-L
H-M
M-L*
H
H
L
H
H
M-L
H
H
L
H-M
H
L*
M
H-M
L
M
H
M
H
H
L
Variable
Range
Radar
(VRR)
L
M
H
L
H-M
H
H
H
H
L
H-M
H
VRR,
Visuals,
Loran-C
M-L
H-M
H
H-L
H-M
H-M
H
H
H
H-L
H-M
H
Total
Stations
H-M
H
M-L*
M
H-M
M-L
M
n
M-L
M-L
M-L
L
Microwave
Systems
H
H
H-M
H
H-M
L
H
H
M
H
L
L
Range- Medium-
Azimuth Range
Systems Systems Loran-C
H-N
H
M-L*
H
H-M
L
M
M-L
M-L
L
L*
L
H-M
M
M-L
L
H-M
H-M
H-M
L*
M-L
L
L*
L*
M
M
H-L
H
H
H-M
H
H
H-M
H
H
H
GEUbTAK GPi
H h-11
H H-M
H H
H-M H-L
? M
H H
H H
? M
* M
? '
H-L M-L
H H-L
H = High ranking (adequate, above average, inexpensive, infrequent).
M = Medium ranking (marginal, average, intermediate).
L = Low ranking (not adequate, below average, expensive, frequent).
• - Significant enough limitation to preclude use as a positioning method
under some conditions.
' = Not enough information available to evaluate.
-------
oo
3
o
<
2
Q-
LU
a:
100 —
120
MICROWAVE SYSTEMS
AND SOME RANGE-ANGLE
SYSTEMS
PUGET SOUND
MAXIMUM RANGE
10 20
DISTANCE FROM SHORE (TARGETS) (km)
Figure D-l. Approximate accuracy versus distance of some positioning methods.
-------
Station Positioning
Appendix D - Methods Evaluation
August 1986
total stations, and theodolites can reposition a vessel within +2 m (+6.6 ft).
The accuracies of the latter three methods will deteriorate with the visi-
bility. The GPS satellite method accuracies, which vary with satellite
code access, are between those of microwave systems and the repeatable
accuracy of Loran-C and VRR. GEOSTAR will be almost as accurate as the
microwave systems.
The only systems that are capable of obtaining absolute accuracies
greater than +20 m (+66 ft) in most locations are the electronic ranging
systems (microwave-based or satellite). Near shore, optical methods and
VRR can usually reach these absolute accuracies but are dependent on the
available targets and calm, clear conditions.
Availability
The satellite systems will not be usable for an undetermined period
of time. GPS does not yet have enough satellites in orbit to give consistent
fixes without long time delays. GEOSTAR will not be operational for a
few years. The other candidate systems are available.
Capital and Operating Costs
GPS capability is expected to cost from $10,000 to $40,000. Cost
of later models is expected to drop to $1,000. Proposed GEOSTAR interrogators
are expected to cost $450 plus a monthly use fee. VRR models range from
$4,000 to $10,000. Loran-C units cost $1,000-$4,000. Little or no operating
cost is associated with these methods and the vessel captain can operate
them. Microwave systems typically cost $40,000-$95,000, not including
high operating expenses. Range-azimuth systems range from $64,500 to $100,000
and offer greater flexibility for other work and reduced logistical problems.
Remaining systems (total stations for $9,000 manual, $15,000-$25,000 automatic;
EDMI and theodolites for $10,000-$12,000; and pairs of sextants or theodolites
for $4,000-$8,000) are all significantly lower in initial cost. However,
these are typically much more labor-intensive and logistically complicated,
and can have high operating costs.
Short-term rental or long-term leasing of equipment should be considered
as an alternative to purchase of a positioning system. A short-term rental
limits cost to only the actual surveying periods and avoids maintenance
responsibilities. These arrangements are also attractive during periods
of rapid navigation system development (e.g., satellite systems) if such
developments indicate that adequate systems will be available in the near
future at lower capital costs.
The monthly rental cost of equipment is often set at 10 percent of
the suggested retail cost. However, short-term rates for many items are
categorized by the number of rental days (e.g., 1-10, 11-90 and 91 plus).
Rent/purchase options are available with a set amount of the rental fee
(e.g., 50-80 percent) applied to the purchase price when the option is
D-9
-------
Station Positioning
Appendix D - Methods Evaluation
August 1986
exercised. Typical rental costs (subject to change) for items of interest
are shown in Table D-4. Evaluation of the cost-effectiveness of applicable
systems should include rent/lease options as well as procurement. If the
user has little experience in a particular system, it may also be appro-
priate to include the cost of one or more equipment technicians or operators
(up to $300 per day plus expenses) to provide reliable navigation data
for each survey.
Merits of Use
When repeatable positioning is the primary concern, both Loran-C and
VRR methods have adequate accuracies for most sampling objectives. A combi-
nation of VRR, visual fixes, Loran-C, and recording station depth will
improve repeatable accuracy. Both Loran-C and VRR are routinely carried
on research vessels, limiting logistical requirements. The combined method
also offers redundancy that minimizes the effect on positioning capability
if one of the systems malfunctions.
Sextant fixes are affected by distance, visibility, and rough water.
The variability in positioning accuracy across the range of conditions
that can be expected make this method questionable for consistent operations
across all stations. In good conditions, sextant fixes can improve accuracies
over those obtainable for VRR or Loran-C. However, if the investigator
is trying to locate specific stations, targets and angles have to be pre-
determined. Sextants are also valuable as a backup positioning method
when the primary system malfunctions.
The shore-based optical methods are not as affected by rough water.
Logistical requirements include location of the shore stations and one
or two shore station operators. Range-azimuth and total stations offer
the advantages of a single shore station and a built-in communications
link, which will limit logistical requirements. Two stations have to be
staffed and a three-way communications link has to be provided for theodolite
positioning. These systems can achieve accuracies of approximately +2 m
(+6.6 ft), but accuracies can deteriorate with visibility and distance
to shore for the optical components of these systems.
Microwave systems offer high accuracies [+2 m (+6.6 ft)], but at a
high cost. Microwave systems are not limited by visibility restrictions
or ranges in Puget Sound. The additional logistical requirements of microwave
positioning add complexity to the study design and implementation. When
available (approximately 1988), consistent satellite fixes are expected
to offer the increased accuracies of microwave sytems with the simplicity
of Loran-C.
The radius of the probable sampling area could be reduced by <50 m
(165 ft) by using a +2-m absolute accuracy system rather than a +20-m absolute
accuracy system. In deeper waters (e.g., increased sampling area but more
homogeneous benthos), the added accuracy would only be advantageous if
D-10
-------
TABLE 0-4. TYPICAL EQUIPMENT RENTAL COSTS (1985 DOLLARS)
Category
Theodolites
EOMIs
Total Stations
1"
6-10"
20"
1.0-2.0 km
2.0-6.0 km
>6.0
Automatic
Manual
1-10 Days
25-35
22-25
15-18
25-30
25-65
40-50
75-110
40-50
Rental Period
11-90 Days 91+ Days Monthly
18-21
13-15
10-13
15-19
18-39
25-30
45-70
25-45
12-15
9-12
7-10
10-13
15-20
16-22
30-60
16-20
Hydrographic Systems
Trisponder 202 175 2,750
Cubic Auto Tape
(3 range) 200 105 90
Motorola Mini
Ranger II
(including 2 trans-
ponders) 200 105 90 3,750
(additional trans-
ponders) 36 18 12
D-ll
-------
Station Positioning
Appendix 0 - Methods Evaluation
August 1986
station grid size or absolute positions were critical. In shallower, near
shore waters, the differences in achievable accuracy decrease as resolution
of positioning targets improves. However, spatial heterogeneity also
increases. The investigator will have to determine if the increase in
probable sampling area [now less than 30 m (98 ft) in radius] that results
from using less accurate systems will compromise quality of the collected
data. If stations were initially located using a highly accurate positioning
method (e.g., microwave system) and position coordinates were simultaneously
defined in coordinates of a lower accuracy method (e.g., Loran-C, VRR,
visual fixes), the station could be reoccupied using the less accurate
method, without significant loss in absolute accuracy.
D-12
-------
SEDIMENT CONVENTIONAL*
-------
FINAL REPORT --_,..
TC-3991-04 Puget Sound Estuary Program
RECOMMENDED PROTOCOLS FOR MEASURING
CONVENTIONAL SEDIMENT VARIABLES
IN PUGET SOUND
Prepared by:
TETRA TECH, INC.
Prepared for:
U.S. ENVIRONMENTAL PROTECTION AGENCY
Region 10 - Office of Puget Sound
Seattle, WA
March, 1986
TETRA TECH, INC.
11820 Northup Way
Bellevue, WA 98005
-------
CONTENTS
Page
LIST OF TABLES v
INTRODUCTION 1
COLLECTION OF SURFICIAL SEDIMENTS FOR PHYSICAL AND CHEMICAL
VARIABLES 4
INTRODUCTION 4
DESIGN OF SAMPLER 4
PENETRATION DEPTH 5
OPERATION OF SAMPLER 5
SAMPLE ACCEPTABILITY CRITERIA 6
SAMPLE COLLECTION 6
PARTICLE SIZE 9
USE AND LIMITATIONS 9
FIELD PROCEDURES 9
Collection 9
Processing 10
LABORATORY PROCEDURES 10
Analytical Procedures 10
QA/QC Procedures 15
DATA REPORTING REQUIREMENTS 16
TOTAL SOLIDS I7
USE AND LIMITATIONS 17
FIELD PROCEDURES 17
Collection 17
Processing 17
LABORATORY PROCEDURES 17
Analytical Procedures 17
QA/QC Procedures I8
DATA REPORTING REQUIREMENTS 19
ii
-------
TOTAL VOLATILE SOLIDS (TVS) 20
USE AND LIMITATIONS 20
FIELD PROCEDURES 20
Collection 20
Processing 20
LABORATORY PROCEDURES 20
Analytical Procedures 20
QA/QC Procedures 21
DATA REPORTING REQUIREMENTS 22
TOTAL ORGANIC CARBON (TOC) 23
USE' AND LIMITATIONS 23
FIELD PROCEDURES 23
Collection 23
Processing 23
LABORATORY PROCEDURES 23
Analytical Procedures 23
QA/QC Procedures 25
DATA REPORTING REQUIREMENTS 25
OIL AND GREASE (FREON EXTRACTABLE) 27
USE AND LIMITATIONS 27
FIELD PROCEDURES 27
Collection 27
Processing 27
LABORATORY PROCEDURES 28
Analytical Procedures 28
QA/QC Procedures 30
DATA REPORTING REQUIREMENTS 31
TOTAL SULFIDES 32
USE AND LIMITATIONS 32
FIELD PROCEDURES 32
Collection 32
Processing 32
-------
LABORATORY PROCEDURES 32
Analytical Procedures 32
QA/QC Procedures 35
DATA REPORTING REQUIREMENTS 36
TOTAL NITROGEN 37
BIOCHEMICAL OXYGEN DEMAND (BOD) 38
USE AND LIMITATIONS 38
FIELD PROCEDURES 38
Collection 38
Processing 38
LABORATORY PROCEDURES 38
Analytical Procedures 38
QA/QC Procedures 41
DATA REPORTING REQUIREMENTS 41
CHEMICAL OXYGEN DEMAND (COD) 42
USE AND LIMITATIONS 42
FIELD PROCEDURES 42
Collection 42
Processing 42
LABORATORY PROCEDURES 43
Analytical Procedures 43
QA/QC Procedures 45
DATA REPORTING REQUIREMENTS 45
REFERENCES 46
iv
-------
LIST OF TABLES
Number Page
1 Contributors to the sediment conventional protocols 2
2 Recommended sample sizes, containers, preservation,
techniques, and holding times for sediment conventional
variables 3
3 Withdrawal times for pipet analysis as a function
of particle size and water temperature 14
ACKNOWLEDGEMENTS
This chapter was prepared by Tetra Tech, Inc., under the direction of
Dr. Scott Becker, for the U.S. Environmental Protection Agency in partial
fulfillment of Contract No. 68-03-1977. Dr. Thomas Ginn of Tetra Tech was
the Program Manager. Mr. John Underwood and Dr. John Armstrong of U.S. EPA
were the Project Officers. Ms. Julia Wilcox of Tetra Tech helped resolve
many of the technical issues that arose as the protocols were being
prepared.
-------
Conventional Sediment Variables
Introduction
March 1986
INTRODUCTION
Recommended methods for measuring the following conventional sediment
variables in Puget Sound are presented in this chapter:
Particle size
Total solids
Total volatile solids
Total organic carbon
Oil and grease
Total sulfides
Total nitrogen
Biochemical oxygen demand
Chemical oxygen demand.
Each method is based on the results of a workshop and written reviews by
representatives from most organizations that fund or conduct environmental
research in Puget Sound (Table 1). The purpose of developing these
recommended protocols is to encourage all Puget Sound investigators
conducting monitoring programs, baseline surveys, and intensive invest-
igations to use standardized methods whenever possible. If this goal is
achieved, most data collected in Puget Sound should be directly comparable
and thereby capable of being integrated into a sound-wide database. Such a
database is necessary for developing and maintaining a comprehensive water
quality management program for Puget Sound.
Each recommended protocol describes the use and limitations of the
respective variable; the field collection and processing methods; and the
laboratory analytical, QA/QC, and data reporting procedures. Each
recommended analytical procedure was modified from Plumb (1981). The
general collection and holding recommendations for each variable are
presented in Table 2.
Although the following protocols are recommended for most studies
conducted in Puget Sound, departures from these methods may be necessary to
meet the special requirements of individual projects. If such departures
are made, however, the funding agency or investigator should be aware that
the resulting data may not be comparable with most other data of that kind.
In some instances, data collected using different methods may be compared if
the methods are intercalibrated adequately.
-------
TABLE 1. CONTRIBUTORS TO THE SEDIMENT CONVENTIONAL PROTOCOLS
Name
John Armstronga
Bob Barricka
Scott Becker*»b
Gordon Bilyard*
Chuck Boatman*
Eric Crecelius3
Joe Cummins
Bob Dexter3
George Ditsworth
John Downing9
Bruce Duncan
Mark Fugiel*
Arnold Gahler
Carolyn Gangmark
Roy Jones
Dave Kendall
Shawn Moore*
Gary Mauseth*
Mike Nelson*
Ahmad Nevissi*
Wally Triol*
Frank Urabeck
Steve Vincent*
Fred Weinmann*
Julia Wilcoxa
Carolyn Wilson
Jack Word*
Organization
U.S. EPA
Tetra Tech, Inc.
Tetra Tech, Inc.
Tetra Tech, Inc.
URS Engineers
Battelle Northwest
U.S. EPA
EVS Consultants
U.S. EPA
Nortec
U.S. EPA
Am Test, Inc.
U.S. EPA
U.S. EPA
U.S. EPA
U.S. COE
Am Test, Inc.
Nortec
Laucks Testing Labs
Univ. of Washington
Parametrix, Inc.
U.S. COE
Weyerhauser Company
U.S. COE
Tetra Tech, Inc.
U.S. EPA
Evans-Hamilton, Inc.
Attended the workshop held on June 28, 1985.
Workshop moderator.
-------
TABLE 2. RECOMMENDED SAMPLE SIZES, CONTAINERS, PRESERVATION TECHNIQUES,
AND HOLDING TIMES FOR SEDIMENT CONVENTIONAL VARIABLES
Minimum
Sample
Variable Size (g)*
Particle size
Total solids
Total volatile
solids
Total organic
carbon
Oil and grease
Total sul fides
Total nitrogen
Biochemical
oxygen demand
Chemical oxgyen
demand
100-15QC
50
50
25
100
50
25
50
50
Contained
P,G
P.G
P,G
P,G
G only
P,G
P,G
P,G
P.G
Preservation
Cool, 4° C
Freeze
Freeze
Freeze
Cool, 4° C, HC1;
Freeze
Cool, 40 C,
IN zinc acetate
Freeze
Cool, 40 C
Cool, 40 C
Maximum
Holding
Time
6 mod
6 mod
6 mod
6 mod
28 daysd
6 mod
7 daysd
6 mod
7 dayse
7 days6
a Recommended field sample sizes for one laboratory analysis. If additional
laboratory analyses are required (e.g., replicates), the field sample size
should be adjusted accordingly.
b p = polyethylene, G = glass.
c Sandier sediments require larger sample sizes than do muddier sediments.
^ This is a suggested holding time. No U.S. EPA criteria exist for the
preservation of this variable.
e This holding time is recommended by Plumb (1981).
-------
Conventional Sediment Variables
Surficial Sediment Collection
March 1986
COLLECTION OF SURFICIAL SEDIMENTS
FOR PHYSICAL AND CHEMICAL VARIABLES
INTRODUCTION
This section describes the protocols required to collect an acceptable
subtidal surficial sediment sample for subsequent measurement of physical
and chemical variables. This subject has generally been neglected in the
past and sampling crews have been given relatively wide latitude in deciding
how to collect samples. However, because sample collection procedures
influence the results of all subsequent laboratory and data analyses, it is
critical that samples be collected using acceptable and standardized
techniques.
DESIGN OF SAMPLER
In Puget Sound, the most common sampling device for subtidal surficial
sediments is the modified van Veen bottom grab. However, a variety of
coring devices is also used. The primary criterion for an adequate sampler
is that it consistently collect undisturbed samples to the required depth
below the sediment surface without contaminating the samples. An additional
criterion is that the sampler can be handled properly on board the survey
vessel. An otherwise acceptable sampler may yield inadequate sediment
samples if it is too large, heavy, or awkward to be handled properly.
Collection of undisturbed sediment requires that the sampler:
• Create a minimal bow wake when descending
• Form a leakproof seal when the sediment sample is taken
• Prevent winnowing and excessive sample disturbance when
ascending
• Allow easy access to the sample surface.
Most modified van Veen grabs have open upper faces that are fitted with
rubber flaps. Upon descent, the flaps are forced open to minimize the bow
wake, whereas upon ascent, the flaps are forced closed to prevent sample
winnowing. Some box corers have solid flaps that are clipped open upon
descent and snap shut after the corer is triggered. Although most samplers
seal adequately when purchased, the wear and tear of repeated field use
eventually reduces this sealing ability. A sampler should therefore be
monitored constantly for sample leakage. If unacceptable leakage occurs,
the sampler should be repaired or replaced. If a sampler is to be borrowed
or leased for a project, its sealing ability should be confirmed prior to
sampling. Also, it is prudent to have a backup sampler on board the survey
vessel in case the primary sampler begins leaking during a cruise.
-------
Conventional Sediment Variables
Surficial Sediment Collection
March 1986
The required penetration depth below the sediment surface is a function
of the desired sample depth (see Penetration Depth). Generally, it is
better to penetrate below the desired sample depth to minimize sample
disturbance when the sampling device closes. Penetration depth of most
sampling devices varies with sediment character, and generally is greatest
in fine sediments and least in coarse sediments. Sampling devices generally
rely upon either gravity or a piston mechanism to penetrate the sediment.
In both cases, penetration depth can be modified by adding or subtracting
weight from the samplers. Thus, it is optimal to use a sampler that has a
means of weight adjustment. If a sampler cannot consistently achieve the
desired penetration depth, an alternate device should be used.
Once the sampler is secured on board the survey vessel, it is essential
that the surface of the sample be made accessible without disturbing the
sample. Generally, samplers have hinged flaps on their upper face for this
purpose. The opem'ng(s) in the upper face of the sampler should be large
enough to allow easy subsampling of the sediment surface. If an opening is
too small, the sample may be disturbed as the scientific crew member
struggles to take a subsample.
PENETRATION DEPTH
For characterizing surficial sediments in Puget Sound, it is
recommended that the upper 2 cm of the sediment column be evaluated. When
collecting the upper 2 cm of sediment, it is recommended that a minimum
penetration depth of 4-5 cm be achieved for each acceptable sample.
Although the 2-cm specification is arbitrary, it will ensure that:
• Relatively recent sediments are sampled
• Adequate volumes of sediments can be obtained readily for
laboratory analyses
• Data from different studies can be compared validly.
Sampling depths other than 2 cm may be appropriate for specific purposes.
For example, the upper 1 cm of sediment may be required to determine the age
of the most recently deposited sediments. By contrast, a sample depth much
greater than 2 cm may be required to evaluate the vertical profile of
sediment characteristics or to determine depth-averaged characteristics
prior to dredging. If a sampling depth other than 2 cm is used, comparisons
with data from 2-cm deep samples may be questionable.
OPERATION OF SAMPLER
The sampling device should be attached to the hydrowire using a ball-
bearing swivel. The swivel will minimize the twisting forces on the sampler
during deployment and ensure that proper contact is made with the bottom.
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Conventional Sediment Variables
Surficial Sediment Collection
March 1986
For safety, the hydrowire, swivel, and all shackles should have a load
capacity at least three times greater than the weight of a full sampler.
The sampler should be lowered through the water column at a controlled
speed of approximately 1 ft/sec. Under no circumstances should the sampler
be allowed to "free fall" to the bottom, as this may result in premature
triggering, an excessive bow wake, or improper orientation upon contact with
the bottom. The sampler should contact the bottom gently and only its
weight or piston mechanism should be used to force it into the sediment.
After the sediment sample is taken, the sampler should be raised slowly
off the bottom and then retrieved at a controlled speed of approximately 1
ft/sec. Before the sampler breaks the water surface, the survey vessel
should head into the waves (if present) to minimize vessel rolling. This
maneuver will minimize swinging of the sampler after it breaks the water
surface. If excessive swinging occurs or if the sampler strikes the vessel
during retrieval, extra attention should be paid to evaluating sample
disturbance when judging sample acceptability.
The sampler should be secured immediately after it is brought on board
the survey vessel. If the sampler tips or slides around before being
secured, extra attention should be paid to evaluating sample disturbance.
SAMPLE ACCEPTABILITY CRITERIA
After the sampler is secured on deck, the sediment sample should be
inspected carefully before being accepted. The following acceptability
criteria should be satisfied:
• The sampler is not over-filled with sample so that the
sediment surface is pressed against the top of the sampler
• Overlying water is present (indicates minimal leakage)
• The overlying water is not excessively turbid (indicates
minimal sample disturbance)
• The sediment surface is relatively flat (indicates minimal
disturbance or winnowing)
t The desired penetration depth is achieved (i.e., 4-5 cm for a
2-cm deep surficial sample).
If a sample does not meet all criteria, it should be rejected.
SAMPLE COLLECTION
After a sample is judged acceptable, the following observations should
be noted on the field log sheet:
• Station location
• Depth
-------
Conventional Sediment Variables
Surficial Sediment Collection
March 1986
• Gross characteristics of the surficial sediment
Texture
Color
Biological structures (e.g., shells, tubes, macrophytes)
Presence of debris (e.g., wood chips, wood fibers, human
artifacts)
Presence of oily sheen
Odor (e.g., hydrogen sulfide, oil, creosote)
• Gross characteristics of the vertical profile
Changes in sediment characteristics
Presence and depth of redox potential discontinuity
(rpd) layer
• Penetration depth
• Comments related to sample quality
Leakage
Winnowing
Disturbance.
Before subsamples of the surficial sediments are taken, the overlying
water must be removed. The preferred method of removing this water is by
slowly siphoning it off near one side of the sampler. Methods such as
decanting the water or slightly cracking the grab to let the water run out
are not recommended, as they may result in unacceptable disturbance or loss
of fine-grained surficial sediment and organic matter.
Once the overlying water has been removed, the surficial sediment can
be subsampled. It is recommended that subsamples be taken using a flat
scoop shaped like a coal shovel. The shoulders of the scoop should be 2 cm
high. This device will allow a relatively large subsample to be taken
accurately to a depth of 2 cm. Coring devices are not recommended because
generally they collect small amounts of surficial sediment and therefore
require repeated extractions to obtain a sufficient volume of material for
analysis of conventional sediment variables. A curved scoop is not
recommended because it does not sample a uniform depth. Because accurate
and consistent subsampling requires practice, it is advisable that an
experienced person perform this task.
When subsampling surficial sediments, unrepresentative material should
be removed in the field under the supervision of the chief scientist and
noted on the field log sheet. The criteria used to determine representa-
tiveness should be determined prior to sampling.
Finally, if samples are to be analyzed for trace metals or priority
pollutant organic compounds, sample contamination during collection must be
avoided. All sampling equipment (i.e., siphon hoses, scoops, containers)
should be made of noncontaminating material and should be cleaned appro-
priately before use. Samples should not be touched with ungloved fingers.
In addition, potential airborne contamination (e.g., stack gases, cigarette
smoke) should be avoided. Detailed guidance for preventing sample
-------
Conventional Sediment Variables
Surficial Sediment Collection
March 1986
contamination is given in the protocols for metals and organic compounds in
other chapters of this notebook.
8
-------
Conventional Sediment Variables
Particle Size
March 1986
PARTICLE SIZE
USE AND LIMITATIONS
Particle size is used to characterize the physical characteristics of
sediments. Because particle size influences both chemical and biological
variables, it can be used to normalize chemical concentrations according to
sediment characteristics and to account for some of the variability found in
biological assemblages. Particle size is also an important variable for
marine engineering purposes. In addition to Plumb (1981), a variety of
other references discuss the uses and measurement of particle size (e.g.,
Krumbein and Petti John 1938; Folk 1968; Buchanan 1984).
Particle size can be characterized in a wide range of detail. The
grossest divisions that generally are considered useful for characterizing
particle size distributions are percentages of gravel, sand, silt, and
clay. However, each of these size fractions can be subdivided further so
that additional characteristics of the size distribution (e.g., mean
diameter, skewness, kurtosis) can be determined.
Particle size determinations can either include or exclude organic
material. If organic material is removed prior to analysis, the "true"
(i.e., primarily inorganic) particle size distribution is determined. If
organic material is included in the analysis, the "apparent11 (i.e., organic
plus inorganic) particle size distribution is determined. Because true and
apparent distributions may differ, detailed comparisons between samples
analyzed by these different methods are questionable. It is therefore
desirable that all samples within each study (at a minimum) and among
different studies (if possible) be analyzed using only one of these two
methods.
FIELD PROCEDURES
Collection
Samples can be collected in glass or plastic containers. A minimum
sample size of 100-150 g is recommended. If unrepresentative material is to
be removed from the sample, it should be removed in the field under the
supervision of the chief scientist and noted on the field log sheet.
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Conventional Sediment Variables
Particle Size
March 1986
Processing
Samples should be stored at 4° C, and can be held for up to 6 mo before
analysis. Samples must not be frozen or dried prior to analysis, as either
process may change the particle size distribution.
LABORATORY PROCEDURES
Analytical Procedures
• Equipment
Sieve shaker
Ro-Tap or equivalent
Drying oven
Constant temperature bath
Analytical balance
0.1 mg accuracy
Desiccator
Clock
With second hand
Standard sieves
Appropriate mesh sizes
Sieve pan and top
Sieve brush
Funnel
1-L graduated cylinders
50-mL beakers
20-mL pipets
Water pique or squirt bottle
Glossy paper
Dispersant
1 percent sodium hexametaphosphate = 1 percent commercially
available Calgon
Distilled water.
0 Sample preparation
-Allow samples to warm to room temperature.
Homogenize each sample mechanically, incorporating any overlying
water.
Remove a representative aliquot (approximately 25 g) and analyze
for total solids content. This information can be used to
estimate the dry weight of the aliquot used for particle size
analysis. The efficiency of the entire analysis can then be
evaluated by adding the dry weights of all sample fractions and
comparing this sum with the estimated dry weight of the original
aliquot.
Remove a second representative aliquot for wet sieving. The
aliquot can range from 20 g for muddy sediments to 100 g for sandy
sediments. The critical factor for sample size determination is
10
-------
Conventional Sediment Variables
Particle Size
March 1986
the weight of fine-grained material that will be used for the
pipet analysis. Ideally the total dry weight of fine-grained
material in the 1-L graduated cylinder should equal approximately
15 g. However, total weights between 5 and 25 g are considered
acceptable. Total weights outside this range are not considered
acceptable and it is recommended that aliquot size be modified to
bring the amount of fine-grained material into the acceptable
range.
Weigh the wet sample to the nearest 0.01 g.
Organics oxidation - this step removes organic material from the
sample.It is optional and depends upon the objectives of each study.
Place the sediment sample in a large beaker (>_2 L).
Add 20 ml of 10 percent hydrogen peroxide solution and mix.
Let the sample stand until frothing stops.
Once frothing stops, add an additional 10 ml of hydrogen peroxide
solution.
Continue adding 10-mL portions of hydrogen peroxide solution until
no frothing occurs on addition.
Boil the sample to remove any excess hydrogen peroxide.
Be careful that material is not lost from the beaker during
frothing and boiling.
Wet-sieving - this step separates the sample into size fractions
greater than 62.5 urn (i.e., sand and gravel) and less than 62.5 urn
(i.e., silt and clay)
Place the 62.5-um (4 phi) sieve in a funnel, with a 1-L graduated
cylinder underneath. Moisten the sieve using a light spray of
distilled water.
Place the sample in a beaker, add 20-30 mL of distilled water, and
stir to suspend fine-grained material.
Pour the sample into the sieve and thoroughly rinse the beaker and
stirrer with distilled water.
Wash the sediment on the sieve with distilled water using a water
pique or squirt bottle having low water pressure. Aggregates can
be gently broken using a rubber policeman.
Continue wet sieving until only clear water passes through the
sieve. Try to ensure that the rinsate does not exceed
approximately 950 mL. This can generally be accomplished by
sieving a sample quantity that is not too large and by efficient
use of the rinse water. Both of these techniques may require
experimentation before routine wet sieving is started.
Gravel-sand fraction - this fraction is subdivided further by
mechanically dry sieving it through a graded series of screens.
Wash the coarse fraction into a preweighed 50-mL beaker using
distilled water. Rinse the sieve thoroughly.
11
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Conventional Sediment Variables
Particle Size
March 1986
Dry the coarse fraction to constant weight at 90 +_ 2° C. The
drying temperature is less than 100° C to prevent boiling and
potential loss of sample.
Cool the sample to room temperature in a desiccator.
Weigh the cooled sample to the nearest 0.1 mg.
Set up a nest of sieves that will divide the coarse fraction into
the desired number of subfractions. Set up the sieves in a graded
series of mesh sizes, with the coarsest mesh on top and the finest
mesh on the bottom. The bottom sieve always should have a mesh
size of 62.5 urn (4 phi). Place a solid pan on the bottom of the
stack and a lid on top of the stack. At a minimum, the coarse
fraction should be separated into gravel and sand fractions, using
a sieve with a mesh size of 2 mm (-1 phi).
Add the sample to the uppermost sieve. Complete transfer can be
ensured by using a sieve brush to remove any material adhering to
the beaker. The sieve brush can also be used to gently break up
aggregated sediment.
Shake mechanically for exactly 15 min using the Ro-Tap (or equiva-
lent). A shaker having an automatic timer is preferable.
After shaking, empty the contents of each sieve onto a glossy
piece of paper (e.g., wax paper). To empty a sieve, invert it and
tap it on the table several times while ensuring that all edges
hit the table at the same time. If the sieve is not tapped
evenly, the meshes may be distorted. After tapping the sieve,
ensure complete removal of the sample by brushing the back of the
screen. After brushing the back of the screen, turn the sieve
over and brush out any particles adhering to the sides of the
sieve or the inside of the screen.
Add the fraction that passed through the bottom sieve (e.g., 4
phi) and was retained by the solid pan to the silt-clay fraction
of that sample.
Weigh each remaining size fraction to the nearest 0.1 mg.
Sum the weights of all size fractions and compare the result with
the initial weight of the coarse fraction. Losses and inac-
curacies should be less than 5 percent of the initial weight.
Losses and inaccuracies tend to increase with increasing number of
fractions.
Large amounts of organically derived fragments (e.g., wood debris,
grass, shells) or any unusual material in any size fraction should
be noted on the laboratory log sheet.
Silt-clay fraction - this fraction is subdivided further using a pipet
technique that depends upon the differential settling rates of
different particles. Because additions to this fraction may be made
after mechanical sieving of the gravel-sand fraction (see above), it is
recommended that the silt-clay analysis for each sample not be
conducted until the gravel-sand analysis has been completed.
Add 10 mL of the dispersant to 990 mL of distilled water.
Determine the weight of dispersant in a 20-mL aliquot of this
12
-------
Conventional Sediment Variables
Particle Size
March 1986
mixture by pipeting a 20-mL aliquot into each of five tared
beakers, drying the samples to constant weight at 90 +_ 2 C,
cooling the samples in a desiccator, weighing the cooled samples,
and calculating the mean weight of dispersant in the five
samples. This weight multiplied by the number of 10-mL additions
of dispersant to each sample will be subtracted from the weight of
each sediment fraction at the end of the pipet analysis.
Add 10 ml of the dispersant to each sample suspension in the 1-L
graduated cylinders.
Mix each suspension by either stoppering and inverting the
cylinder or by using the up and down motion of a perforated disc
plunger.
Allow the mixed suspension to stand for 2-3 h and check for signs
of flocculation. Flocculation can be recognized by a curdling and
rapid settling of lumps of particles or by the presence of a thick
soupy layer on the bottom of the cylinder passing abruptly into
clear water above.
If flocculation occurs, add dispersant in 10-mL increments until
no noticeable flocculation occurs. Record the volume of
dispersant added.
When ready to conduct the pipet analysis, bring the sample volume
to 1 L by adding distilled water, mix the suspension thoroughly,
and place the cylinder in a constant-temperature water bath. If
the volume is greater or less than 1 L, the factor for converting
the weight of the sediment in each 20-mL aliquot to that in the
total volume must be modified accordingly.
After 20 sec, withdraw a 20-mL aliquot from a depth of 20 cm below
the surface of the suspension using a pipet. The pipet should be
marked for the specified sampling depths and should be inserted
vertically into the settling cylinder when the aliquot is taken.
A suction bulb may be used on the open end of the pipet to
facilitate sampling. It is critical that the suspension be
disturbed as little as possible when pipet aliquots are taken.
Transfer the 20-mL aliquot to a preweighed 50-mL beaker. Rinse
the pipet into the beaker using 20 mL of distilled water.
Withdraw 20-mL aliquots at a depth of 10 cm below the surface of
the suspension at the appropriate time(s) listed in Table 3. A
formula for calculating withdrawal times is given by Folk (1968)
and Buchanan (1984). If a withdrawal is missed, the suspension
can be stirred again and the missed withdrawal can be taken at the
appropriate time after settling begins. It is not necessary to
withdraw the initial 20-mL aliquot when this corrective action is
conducted.
Transfer these additional 20-mL aliquots to 50-mL preweighed
beakers, each time rinsing the pipet into the respective beaker
using 20 mL of distilled water
Dry all aliquots to constant weight at 90 + 2° C. A drying temp-
erature less than 100° C is used to prevenT boiling and potential
loss of sample.
13
-------
TABLE 3. WITHDRAWAL TIMES FOR PIPET ANALYSIS AS A FUNCTION
OF PARTICLE SIZE AND WATER TEMPERATURE3 ••
Diameter
Finer
than
(phi)
4.0
5.0
6.0
7.0
8.QC
9.0
10.0
Diameter
Finer Withdrawal
than Depth
(urn) (cm) 18° C
62.5
31.2
15.6
7.8
3.9
1.95
0.98
20
10
10
10
10
10
10
20s
2mOs
8mOs
31m59s
2h8m
8h32m
34h6m
Elapsed Time for Withdrawal of Sample in
Hours (h) , Minutes (m), and Seconds (s)
19° C 20° C 21° C 22° C 23° C 24° C
20s
Im57s
7m48s
31ml Is
2h5m
8hl8m
33hl6m
20s
Im54s
7m36s
30m26s
2h2m
8h6m
32h28m
20s
Im51s
7m25s
29m41s
Ih59m
7h56m
31h40m
20s
Im49s
7ml 5s
28m59s
Ih56m
7h44m
3 Oh 56m
20s
Im46s
7m5s
28ml8s
Ih53m
7h32m
30hl2m
20s
Im44s
6m55s
27m39s
Ih51m
7h22m
29h30m
a Modified from Plumb (1981).
b It is critical that temperature be held constant during the pipet analysis.
c Breakpoint between silt and clay.
-------
Conventional Sediment Variables
Particle Size
March 1986
Cool dried samples to room temperature in a desiccator.
Weigh cooled samples to the nearest 0.1 mg.
• Calculations
- The total weight of a phi-size interval in the 1-L graduated
cylinder is determined as follows:
Phi weight (g dry weight) = 50[(A-C)-(B-C)]
Where:
A = weight (g) of residue in a 20-ml aliquot for a
given phi-size boundary
B = weight (g) residue in a 20-mL aliquot for the next
larger phi-size boundary
C = mean weight (g) of dispersant in a 20-mL aliquot.
QA/QC Procedures
It is critical that each sample be homogenized thoroughly in the
laboratory before a subsample is taken for analysis. Laboratory
homogenization should be conducted even if samples were homogenized in the
field.
After dry-sieving a sample, all material must be removed from the
sieve. This can be accomplished by tapping the rim of the sieve evenly on a
hard surface and by brushing the screen.
The total amount of fine-grained material used for pipet analysis
should be 5-25 g. If more material is used, particles may interfere with
each other during settling and the possibility of flocculation may be
enhanced. If less material is used, the experimental error in weighing
becomes large relative to the sample size.
Before pipet extractions can be made, the sample must be homogenized
thoroughly within the settling cylinder. Once the pipet analysis begins,
the settling cylinders must not be disturbed, as this will alter particle
settling velocities. Care must be taken to disturb the sample as little as
possible when pipet extractions are made.
After a pipet extract has been transferred to a drying beaker, any
sample adhering to the inside of the pipet must be removed. This can be
accomplished by drawing 20 mL of distilled water into the pipet and adding
this rinse water to the drying beaker.
Dried samples should be cooled in a desiccator and held there until
they are weighed. If a desiccator is not used, the sediment will accumulate
ambient moisture and the sample weight will be overestimated. A color-
indicating desiccant is recommended so that spent desiccant can be detected
15
-------
Conventional Sediment Variables
Particle Size
March 1986
easily. Also, the seal on the desiccator should be checked periodically,
and, if necessary, the ground glass rims should be greased or the "0" rings
should be replaced.
It is recommended that triplicate analyses be conducted on one of every
20 samples, or on one sample per batch if less than 20 samples are
analyzed. It is also recommended that the analytical balance, drying oven,
and temperature bath be inspected daily and calibrated at least once per
week.
DATA REPORTING REQUIREMENTS
The weight of each sediment fraction should be reported to the nearest
0.0001 g dry weight. The laboratory should report the results of all
samples analyzed (including QA replicates) and should note any problems that
may have influenced data quality.
16
-------
Conventional Sediment Variables
Total Solids
March 1986
TOTAL SOLIDS
USE AND LIMITATIONS
Total solids are the organic and inorganic materials remaining after a
sample has been dried completely. This variable is commonly used to convert
sediment concentrations of substances from a wet-weight to a dry-weight
basis. It typically is measured in conjunction with other variables.
Total solids values are operationally defined, because results depend
on drying temperatures. For example, temperature-dependent weight losses
occur from volatilization of organic matter, mechanically occluded water,
water of crystallization, and gases from heat-induced chemical
decomposition. By contrast, weight gains may result from oxidation
processes. To provide data that are comparable among different studies, it
is therefore critical that drying temperatures be standardized.
FIELD PROCEDURES
Collection
Samples can be collected in glass or plastic containers. Samples can
be collected in the same containers as samples for other variables, if total
solids is to be measured in conjunction with those variables. A minimum
sample size of 50 g is recommended. If unrepresentative material is to be
removed from the sample, it should be removed in the field under the
supervision of the chief scientist and noted on the field log sheet.
Processing
Samples should be stored frozen and can be held for up to 6 mo under
that condition.
LABORATORY PROCEDURES
Analytical Procedures
• Equipment
-Muffle furnace
550° C capacity
Drying oven
Desiccator
17
-------
Conventional Sediment Variables
Total Solids
March 1986
Analytical balance
0.01 g accuracy
100-mL evaporating dishes
Porcelain, platinum, or Vycor.
• Equipment preparation
-Ignite clean evaporating dishes at 550 +_ 10° C for 1 h in a muffle
furnace to remove any remaining organic material.
Cool ignited dishes to room temperature in a desiccator.
Weigh each cooled dish to the nearest 0.01 g and store in the
desiccator.
• Sample preparation
-Allow frozen sediment samples to warm to room temperature
Homogenize each sample mechanically, incorporating any overlying
water.
Transfer a representative subsample (approximately 25 g) to a
preweighed evaporation dish.
Weigh the undried sample to the nearest 0.01 g.
• Analytical procedures
- Dry the sample to constant weight at 103 ^ 2° C.
Cool the dried sample to room temperature in a desiccator.
Weigh the cooled sample to the nearest 0.01 g.
• Calculations
-Total solids content is determined as follows:
Percent sol Ids -
Where:
A = weight (g) of dish and dry sample residue
B = weight (g) of dish
C = weight (g) of dish and wet sample.
QA/QC Procedures
It is critical that each sample be thoroughly homogenized in the
laboratory before a subsample is taken for analysis. Laboratory
homogenization should be conducted even if samples were homogenized in the
field.
Evaporating dishes must be ignited at 550° C before being used for
total solids analysis. This step ensures that dishes are free from organic
contaminants.
Dried samples should be cooled in a desiccator and held there until
they are weighed. If a desiccator is not used, the sediment will accumulate
18
-------
Conventional Sediment Variables
Total Solids
March 1986
ambient moisture and the sample weight will be overestimated. A color-
indicating desiccant is recommended so that spent desiccant can be detected
easily. Also, the seal on the desiccator should be checked periodically
and, if necessary, the ground glass rims should be greased or the "0" rings
should be replaced.
It is recommended that triplicate analyses be conducted on one of every
20 samples or on one sample per batch if less than 20 samples are analyzed.
It is also recommended that the analytical balance and drying oven be
inspected daily and calibrated at least once per week.
DATA REPORTING REQUIREMENTS
Total solids should be reported as a percentage of the wet weight of
the sample to the nearest 0.1 unit. The laboratory should report the
results of all samples analyzed (including QA replicates) and should note
any problems that may have influenced sample quality.
19
-------
Conventional Sediment Variables
Total Volatile Solids (TVS)
March 1986
TOTAL VOLATILE SOLIDS (TVS)
USE AND LIMITATIONS
Total volatile solids represent the fraction of total solids that are
lost on ignition at a higher temperature than that used to determine total
solids. Total volatile solids is used as a crude estimate of the amount of
organic matter in the total solids.
Total volatile solids is operationally defined by the ignition temper-
ature. Total volatile solids content does not always represent the organic
content of a sample because some organic material may be lost at the drying
temperature and some inorganic material (e.g., carbonates, chlorides) may be
lost at the ignition temperature. Because of the temperature dependence of
total volatile solids, valid interstudy comparisons require the use of
standardized drying and ignition temperatures.
FIELD PROCEDURES
Collection
Samples can be collected in glass or plastic containers. A minimum
sample size of 50 g is recommended. If unrepresentative material is to be
removed from the sample, it should be removed in the field under the
supervision of the chief scientist and noted on the field log sheet.
Processing
Samples should be stored frozen and can be held for up to 6 mo under
that condition.
LABORATORY PROCEDURES
Analytical Procedures
• Equipment
-Muffle furnace
550° C capacity
Drying oven
Desiccator
Analytical balance
0.01 g accuracy
100-mL evaporating dishes
Porcelain, platinum, or Vycor.
20
-------
Conventional Sediment Variables
Total Volatile Solids (TVS)
March 1986
• Equipment preparation
- Ignite clean evaporating dishes at 550 +_ 10° C for 1 h in a muffle
furnace to remove any remaining organic material.
Cool ignited dishes to room temperature in a desiccator.
Weigh each cooled dish to the nearest 0.01 g and store in the
desiccator.
t Sample preparation
- Allow frozen sediment samples to warm to room temperature.
Homogenize each sample mechanically, incorporating any overlying
water.
Transfer a representative subsample (approximately 25 g) to a
preweighed evaporating dish.
• Analytical procedures
- Dry the sample to constant weight at 103 +_ 2° C.
Cool the dried sample to room temperature in a desiccator.
Weigh the cooled sample to the nearest 0.01 g.
Ignite the sample at 550 + 10° C to constant weight. Make sure
that the samples do not flare up when placed in the oven, as
sediment may be lost from the crucibles. If sample flashing is a
problem, it is recommended that the muffle furnace be cooler than
550° C when samples are placed inside, and that the temperature
gradually be increased to 550° C.
Weigh each cooled sample to the nearest 0.01 g.
• Calculations
: TVS content is determined as follows:
Perc(int TVS . i
Where:
A = weight (g) of dish and dry sample residue
B = weight (g) of evaporation dish
C = weight (g) of dish and ignition residue.
QA/QC Procedures
It is critical that each sample be thoroughly homogenized in the
laboratory before a subsample is taken for analysis. Laboratory
homogenization should be conducted even if samples were homogenized in the
field.
Evaporating dishes (or crucibles) must be ignited at 550° C before
being used for total volatile solids analysis. This step ensures that the
dishes are free from volatile contaminants.
21
-------
Conventional Sediment Variables
Total Volatile Solids (TVS)
March 1986
Dried and combusted samples should be cooled in a desiccator and held
there until they are weighed. If a desiccator is not used, the sediment
will accumulate ambient moisture and the sample weight will be
overestimated. A color-indicating desiccant is recommended so that spent
desiccant can be detected easily. Also, the seal on the desiccator should
be checked periodically and, if necessary, the ground glass rims should be
greased or the "0" rings should be replaced.
It is recommended that triplicate analyses be conducted on one of every
20 samples or on one sample per batch if less than 20 samples are analyzed.
It is also recommended that the analytical balance, drying oven, and muffle
furnace be inspected daily and calibrated at least once per week.
DATA REPORTING REQUIREMENTS
Total volatile solids should be reported as a percentage of the dry
weight of the uncombusted sample to the nearest 0.1 unit. The laboratory
should report the results of all samples analyzed (including QA replicates)
and should note any problems that may have influenced data quality.
22
-------
Conventional Sediment Variables
Total Organic Carbon (TOC)
March 1986
TOTAL ORGANIC CARBON (TOC)
USE AND LIMITATIONS
Total organic carbon is a measure of the total amount of nonvolatile,
volatile, partially volatile, and particulate organic compounds in a
sample. Total organic carbon is independent of the oxidation state of the
organic compounds and is not a measure of the organically bound and
inorganic elements that can contribute to the biochemical and chemical
oxygen demand tests.
Because inorganic carbon (e.g., carbonates, bicarbonates, free C02)
will interfere with total organic carbon determinations, samples should be
treated to remove inorganic carbon before being analyzed.
FIELD PROCEDURES
Collection
Samples can be collected in glass or plastic containers. A minimum
sample size of 25 g is recommended. If unrepresentative material is to be
removed from the sample, it should be removed in the field under the
supervision of the chief scientist and noted on the field log sheet.
Processing
Samples should be stored frozen and can be held for up to 6 mo under
that condition. Excessive temperatures should not be used to thaw samples.
LABORATORY PROCEDURES
Analytical Procedures
• Equipment
-Induction furnace
e.g., Leco WR-12, Dohrmann DC-50, Coleman CH analyzer,
Perk in Elmer 240 elemental analyzer, Carlo-Erba 1106
Analytical balance
0.1 mg accuracy
Desiccator
Combustion boats
10 percent hydrochloric acid (HC1)
Cupric oxide fines (or equivalent material)
Benzoic acid or other carbon source as a standard.
23
-------
Conventional Sediment Variables
Total Organic Carbon (TOC)
March 1986
• Equipment preparation
-Clean combustion boats by placing them in the induction furnace at
950° C. After being cleaned, combustion boats should not be
touched with bare hands.
Cool boats to room temperature in a desiccator.
Weigh each boat to the nearest 0.1 mg.
§ Sample preparation
•Allow frozen samples to warm to room temperature.
Homogenize each sample mechanically, incorporating any overlying
water.
Transfer a representative aliquot (5-10 g) to a clean container.
• Analytical procedures
- Dry samples to constant weight at 70 +_ 2° C. The drying
temperature is relatively low to minimize loss of volatile organic
compounds.
Cool dried samples to room temperature in a desiccator.
Grind sample using a mortar and pestle to break up aggregates.
Transfer a representative aliquot (0.2-0.5 g) to a clean,
preweighed combustion boat.
Determine sample weight to the nearest 0.1 mg.
Add several drops of HC1 to the dried sample to remove car-
bonates. Wait until the effervescing is completed and add more
acid. Continue this process until the incremental addition of
acid causes no further effervescence. Do not add too much acid at
one time as this may cause loss of sample due to frothing.
Exposure of small samples (i.e., 1-10 mg) having less than 50
percent carbonate to an HC1 atmosphere for 24-48 h has been shown
to be an effective means of removing carbonates (Hedges and Stern
1984). If this method is used for sample sizes greater than 10
mg, its effectiveness should be demonstrated by the user.
Dry the HCl-treated sample to constant weight at 70 ^2° C.
Cool to room temperature in a desiccator.
Add previously ashed cupric oxide fines or equivalent material
(e.g., alumina oxide) to the sample in the combustion boat.
Combust the sample in an induction furnace at a minimum
temperature of 950 ^10° C.
t Calculations
-If an ascarite-filled tube is used to capture COg, the carbon
content of the sample can be calculated as follows:
Percent carbon . A(Q.27|9)(100)
24
-------
Conventional Sediment Variables
Total Organic Carbon (TOC)
March 1986
Where:
A = the weight (g) of C02 determined by weighing the
ascarite tube before and after combustion
B = dry weight (g) of the unacidified sample in the
combustion boat
0.2729 = the ratio of the molecular weight of carbon to the
molecular weight of carbon dioxide
A silica gel trap should be placed before the ascarite tube to
catch any moisture driven off during sample combustion.
Additional silica gel should be placed at the exit end of the
ascarite tube to trap any water that might be formed by reaction
of the trapped C02 with the NaOH in the ascarite.
If an elemental analyzer is used, the amount of CQ.2 will be
measured by a thermal conductivity detector. The instrument
should be calibrated daily using an empty boat blank as the zero
point and at least two standards. Standards should bracket the
expected range of carbon concentrations in the samples.
Q.A/QC Procedures
It is critical that each sample be thoroughly homogenized in the
laboratory before a subsample is taken for analysis. Laboratory
homogenization should be conducted even if samples were homogenized in the
field.
Dried samples should be cooled in a desiccator and held there until
they are weighed. If a desiccator is not used, the sediment will accumulate
ambient moisture and the sample weight will be overestimated. A color-
indicating desiccant is recommended so that spent desiccant can be detected
easily. Also, the seal on the desiccator should be checked periodically
and, if necessary, the ground glass rims should be greased or the "0" rings
should be replaced.
It is recommended that triplicate analyses be conducted on one of every
20 samples, or on one sample per batch if less than 20 samples are
analyzed. A method blank should be analyzed at the same frequency as the
triplicate analyses. The analytical balance should be inspected daily and
calibrated at least once per week. The carbon analyzer should be calibrated
daily with freshly prepared standards. A standard reference material should
be analyzed at least once for each major survey.
DATA REPORTING REQUIREMENTS
Total organic carbon should be reported as a percentage of the dry
weight of the unacidified sample to the nearest 0.1 unit. The laboratory
should report the results of all samples (including QA replicates, method
25
-------
Conventional Sediment Variables
Total Organic Carbon (TOC)
March 1986
blanks, and standard reference measurements) and should note any problems
that may have influenced sample quality. The laboratory should also provide
a summary of the calibration procedure and results (e.g., range covered,
regression equation, coefficient of determination).
26
-------
Conventional Sediment Variables
Oil and Grease (Freon-Extractable)
March 1986
OIL AND GREASE (FREON-EXTRACTABLE)
USE AND LIMITATIONS
Oil and grease tests measure all material recovered as a substance
soluble in a nonpolar solvent (e.g., Freon) under acidic conditions. Oil
and grease includes such compounds as hydrocarbons, vegetable oils, animal
fats, waxes, soaps, greases, and related industrial compounds.
In addition to oil and grease, the solvent may dissolve other kinds of
substances, such as sulfur compounds, organic dyes, and chlorophyll. Oil
and grease is therefore operationally defined by the kind of solvent and
analytical methods used. Standardized procedures are essential for valid
interstudy comparisons. Because oil and grease values obtained using the
gravimetric technique sometimes do not correlate with values obtained using
the IR technique, it is recommended that a single technique be used within
each study (at a minimum) and among different studies (if possible).
FIELD PROCEDURES
Collection
Samples should be collected only in glass containers having TFE-lined
lids. Although aluminum-lined lids can be used, seawater eventually will
corrode the aluminum. Before being used, containers and lids should first
be washed with a warm aqueous detergent mixture and then, in sequence,
thoroughly rinsed with hot tap water, rinsed at least twice with distilled
water, rinsed once with Freon (i.e., l,l,2-trichloro-l,2,2-trifluoroethane,
and dried at 105 +_ 2° C for 30 min. A minimum sample size of 100 g is
recommended. If unrepresentative material is to be removed from the sample,
it should be removed in the field under the supervision of the chief
scientist and noted on the field log sheet.
Processing
If samples cannot be analyzed within 24 h, they can be preserved with
approximately 1 mL of concentrated hydrochloric acid (HC1) per 80 g of
sample. Acid-preserved samples should be stored at 4° C, and can be held
for up to 28 days in that condition. Although U.S. EPA has not established
a recommeded maximum holding time for oil and grease in sediments, 28 days
is consistent with the recommended holding time for acid-preserved water
samples. Samples can also be preserved by freezing at -20° C, and can be
held under that condition for up to 6 mo. Samples must be kept field-moist
during storage because they may lose apparent oil and grease as a result of
drying.
27
-------
Conventional Sediment Variables
Oil and Grease (Freon-Extractable)
March 1986
LABORATORY PROCEDURES
Analytical Procedures
• Equipment
-Infrared spectrophotometer
IR technique only
Analytical balance
Gravimetric technique only, 0.1 mg accuracy
Extraction apparatus, Soxhlet
Vacuum pump or other source of vacuum
Extraction thimble, paper
Concentrated hydrochloric acid (HC1) or concentrated sulfuric acid
(H2S04)
Magnesium sulfate monohydrate
Prepare MgSO^HgO by drying a thin layer of MgS04«7H20
overnight in an oven at 103° C
Freon (l,l,2-trichloro-l,2,2,-trifluoroethane), boiling point 47°
C
The solvent should leave no measurable residue- on evapor-
ation; redistill if necessary
Grease-free cotton
Extract nonabsorbent cotton using Freon
Oil reference standard
If the identity of oil and grease in a sample is unknown, a
mixture of 15.0 ml n-hexadecane, 15.0 ml isooctane, and 10.0
ml chlorobenzene should be used as the standard. This is the
same reference oil used for water samples in U.S. EPA Method
413.2 (U.S. EPA 1983). If the identity of oil and grease is
known, the standard can be comprised of the same substance as
that in the sample.
• Sample preparation
•Allow samples to warm to room temperature.
Homogenize each sample mechanically, incorporating any overlying
water.
Remove a representative aliquot (approximately 25 g) and analyze
it for total solids content.
Remove a representative aliquot (approximately 20 g) and weigh it
to the nearest 0.1 mg.
Transfer the weighed aliquot to a 150-mL beaker for oil and grease
analysis.
• Oil and grease extraction
- Acidify the sample to pH = 2 using concentrated HC1 or con-
centrated H2S04.
Add 25 g MgSO^HgO to the acidified sediment sample. Stir to make
a uniformly smooth paste that is spread on the beaker wall. Allow
to stand 15-30 min until solidified.
28
-------
Conventional Sediment Variables
Oil and Grease (Freon-Extractable)
March 1986
Following solidification, remove the solids and grind in a
porcelain mortar. The use of a desiccated, uniformly ground
sample improves the efficiency of the extraction process.
Add the ground sample to a paper extraction thimble. The beaker
and mortar should be wiped with a small piece of filter paper that
has been soaked in Freon. Add the filter paper to the paper
thimble.
Fill the thimble with glass wool or small glass beads. Extract
the prepared sample using Freon in a Soxhlet apparatus at a rate
of 20 cycles/h for 4 h. Stir at least twice during extraction to
prevent channeling. If the final extract is turbid, filter the
sample through grease-free cotton into a clean flask. Rinse the
initial sample container and the cotton with Freon and add the
washing to the filtered sample.
Oil and grease concentration of the extract can be determined
using either the infrared spectrophotometry or the gravimetric
method.
Infrared spectrophotometry method
-Quantitatively transfer the sediment extract to a convenient size
volumetric flask and dilute to volume with Freon.
Prepare calibration standards using the reference oil.
Transfer required amounts of the reference material into 100-mL
volumetric flasks using microliter pipettes. Dilute to volume
with Freon.
The most appropriate pathlength for the quartz cells to be used in
the spectrophotometric determination is determined by the expected
sample concentration. The following information is presented as a
guide for selecting cell length:
Pathlength. cm Expected Range, mg
T~~^ 4 - 40
5 0.5-8
10 0.1 - 4
Based on observed ranges of oil and grease in sediments, it may be
necessary to dilute the sample extracts to the working ranges
indicated above.
Scan the standards and samples from 3,200 to 2,700 cm'1 using a
recording infrared spectrophotometer. Freon should be used in the
reference beam of a dual beam instrument or to zero a single beam
instrument. The absorbance of the 2930-cm"1 peak should be used
to construct a standard curve.
Prepare a standard curve by plotting measured absorbance vs. oil
and grease concentration of the standards. Compare the absorbance
of the Freon extract to the standard curve to determine the oil
and grease concentration.
Calculate oil and grease concentration as follows:
Concentration (mg/kg dry weight) =
29
-------
Conventional Sediment Variables
Oil and Grease (Freon-Extractable)
March 1986
Where:
X = concentration of oil and grease in the Freon
extract, mg/L
V = volume of Freon extract, L
g = wet weight of sediment extracted, g
% S = percent total solids in the sediment sample
(expressed as a decimal fraction).
• Gravimetric method
•Quantitatively transfer the sediment extract to a tared distilling
flask. Rinse the extract container with Freon and add to the
distilling flask.
Distill the Freon from the extraction flasks using a water bath at
70° C.
After the solvent has been evaporated, place the flask on a warm
steam bath for 15 min and draw air through the flask by means of
an applied vacuum for the final 1 min.
Cool the sample in a desiccator for 30 min.
Weigh the cooled sample to the nearest 0.1 mg.
Calculate oil and grease concentration as follows:
Concentration (mg/kg dry weight) = (A
Where:
A = weight (mg) of tared flask and oil and grease residue
B = weight (mg) of tared flask
g = wet weight (g) of sediment extracted
%S = percent total solids in the sediment sample (expressed
as a decimal fraction)
1000 = factor for converting mg/g to mg/kg.
QA/QC Procedures
Because the results of an oil and grease analysis are extremely
sensitive to the methods used, comparable results can be obtained only by
strict adherence to all methodological details.
It is critical that each sample be thoroughly homogenized in the
laboratory before a subsample is taken for analysis. Laboratory
homogenization should be conducted even if samples were homogenized in the
field.
It is recommended that triplicate analyses be conducted on one of every
20 samples, or on one sample per batch if less than 20 samples are
analyzed. Also, a method blank should be analyzed for each batch of samples
extracted.
30
-------
Conventional Sediment Variables
Oil and Grease (Freon-Extractable)
March 1986
DATA REPORTING REQUIREMENTS
Oil and grease concentrations should be reported as mg/kg dry weight to
no more than three significant figures. The laboratory should report the
results of all samples (including QA replicates and method blanks) and
should note any problems that may have influenced sample quality. The
laboratory should also provide a description of the calibration procedures
and standards used to determine oil and grease concentrations by infrared
spectrophotometry.
31
-------
Conventional Sediment Variables
Total Sulfides
March 1986
TOTAL SULFIDES
USE AND LIMITATIONS
Total sulfldes represent the amount of acid-soluble H2S, HS", and S2~
in a sample. Sulfides are measured because they may be toxic and because
they may create unaesthetic conditions. This method cannot be used if a
measure of only water-soluble sulfides is desired. A measure of water-
soluble sulfides might be desired if an estimate of biologically available
sulfides is needed.
Sulfides are difficult to sample because some may be lost through
volatilization and/or gas stripping and some may be lost through oxidation
by dissolved oxygen.
FIELD PROCEDURES
Collection
Samples can be collected in glass or plastic containers. A minimum
sample size of 50 g is recommended. If unrepresentative material is to be
removed from the sample, it should be removed in the field under the
supervision of the chief scientist and noted on the field log sheet.
Processing
Samples for total sulfides should be preserved in the field by adding 2
N zinc acetate solution (approximately 5 mL per 30 g of sediment) and
swirling the mixture. Samples should be stored in the dark at 4° C and
analyzed as soon as possible. It is critical that air contact with samples
be minimized and samples be kept moist, to minimize oxidation. Although
U.S. EPA has not established a recommended maximum holding time for sulfides
in sediments, a maximum holding time of 7 days would be consistent with the
holding time recommended for sulfldes in preserved water samples.
LABORATORY PROCEDURES
Analytical Procedures
• Equipment
-Distillation apparatus, all glass
For large samples, a suitable assembly consists of a 1-L
pyrex distilling flask with Graham condenser as used for the
analysis of phenols. A section of glass tubing should be
32
-------
Conventional Sediment Variables
Total Sulfides
March 1986
connected to the tip of the condenser so that it reaches the
bottom of the collection tube.
Distillate collection tubes
Short-form Messier tubes, graduated at 50 and 100 ml.
Spectrophotometer
For use at 650 nm and providing a light path of 1 in or
greater.
Nitrogen, water-pumped
Zinc acetate, 2 N
Dissolve 2215 g of ln(Cz^Qz)Z'2^ZQ in distilled water and
dilute to 1 L.
Zinc acetate, 0.2 N
To 100 ml of 2~^Zn(C2H302)2'2H20 add several drops of acetic
acid and dilute to 1 L.
Sulfuric acid solution, 1:1
Carefully add 500 ml of concentrated H2S04 to 500 ml of
distilled water in a 1-L flask. Mix continuously and cool
under running water while combining reagents. Cool solution
before using.
Dilute sulfuric acid solution, approximately 0.1 N^
Dilute 5 mL of 1:1 ^$04 to 1 L with distilled water.
Stock amine solution
Dissolve 2.7 g of N, N-dimethyl-p-phenylenediamine sulfate
and dilute to 100 ml with 1:1 H2S04 solution. This solution
is stable for approximately 1 wk.
Working amine solution
Dilute 2 ml of stock amine solution to 100 ml with 1:1 H2S04
solution. Prepare fresh daily.
Ferric chloride solution
Dissolve 100 g of FeCl3«6H20 in hot distilled water and
dilute to 100 ml. Cool before use.
Standard potassium biniodate solution, 0.025 N^
Accurately weigh out 0.8124 g KH(I03)2 and dissolve in
distilled water. Dilute to 1 L.
Standard sodium thiosulfate titrant, 0.025 N_
Dissolve 6.205 g Na2$203'5H20 in distilled water and dilute
to 1 L. Preserve with 5 ml chloroform. Standardize against
standard potassium biniodate using starch as an indicator.
Potassium iodide solution
Dissolve 5 g of KI in distilled water and dilute to 100 ml.
Treated hydrochloric acid
Place one or two strips of aluminum in a small beaker of
concentrated HC1. Following the subsequent reaction, the
acid is poured off and is ready to use.
Oxygen-free dilution water
Pass nitrogen gas through a sufficient quantity of distilled
water for dilution requirements. A minimum of 10 min is
required to displace oxygen in the water.
Sodium sulfide, reagent, crystal.
33
-------
Conventional Sediment Variables
Total Sulfides
March 1986
t Standards preparation
:Prepare 0.01 N_ sulfide solution as follows: weigh approximately
1.2 g of large crystal Na2S-9H20. Wash the crystals several times
with distilled water. Discard the washings and add the washed
crystals to 975 ml of nitrogen-saturated distilled water. Dilute
to 1 L.
Pipet 20 ml of stock sulfide solution into 100 ml of oxygen-free
water. Add 5 ml of KI solution, 20 ml of 0.025 N KH(I03)2
solution, and 10 ml of 0.1 N_ H2S04. Titrate with 0.025~^ Na2S203
solution using starch as an endpoint indicator. Carry a blank
through the procedure and calculate the amount of reacted iodine
from the difference between the blank and standard titrations.
Because 1 ml of 0.025 N_ KH (103)2 is equivalent to 0.400 mg of
sulfide ion, calculate the sulfide concentration in the stock
solution.
Calculate the volume of stock solution that contains 0.2 mg
sulfide and add this amount to 900 ml of oxygen-free water.
Dilute to 1 L. This is the working standard containing 2 ug
S/mL. Sulfide solutions are extremely unstable and must be
prepared fresh and used immediately. Stability is increased by
using nitrogen-saturated water for dilution.
Prepare a standard curve by dilution of the working sulfide
solution.- Pipet 20 ml 0.2 N_ Zn(C2H302)2 into a series of 50-mL
Nessler tubes. Add the required amounts of sulfide solution to
each Nessler tube, taking care to pipet the solution below the
Zn(C2H302)2 level. Dilute to 50 ml with oxygen-free water.
Equilibrate the temperature of the standards to 23-25° C using a
water bath while the colorimetric reagents are added. Add 2 ml
dilute amine-sulfuric acid solution to the standard, mix, and add
0.25 ml (5 drops) FeCl3 solution. Mix the solution and allow 10
min for color development. Measure the absorbance at 650 nm.
t Sample preparation
:Allow samples to warm to room temperature.
Homogenize each sample mechanically, incorporating any overlying
water.
Remove a representative aliquot (approximately 25 g) and analyze
for total solids content.
Remove a representative aliquot for total sulfides analysis. The
aliquot should not contain more than 50 ug of sulfide.
Weigh the aliquot to the nearest 0.1 mg.
• Distillation
-Set up the distillation apparatus. The transfer tube from the
condenser should reach the bottom of the distillated collection
tube. The condenser should be attached so that it can be easily
moved up or down when diluting the distillate or adding reagents.
Pipet 20 ml of 0.2 N^ Zn(C2H302)2 into a 100-mL Nessler tube and
lower the condenser so that the transfer tubing reaches below the
34
-------
Conventional Sediment Variables
Total Sulfides
March 1986
level of the liquid. Attach a distilling flask and pass nitrogen
gas through the system for at least 10 min.
Transfer the sample aliquot to the distillation flask. Bubble
nitrogen gas through the sample to remove any oxygen dissolved in
the sample. A small amount of sulfide may be driven over by the
gas, so be sure that the only exit is through the zinc acetate
solution in the collecting tube.
Discontinue nitrogen evolution and add rapidly several boiling
stones, two drops of methyl orange indicator, and enough treated
HC1 to change the color from orange to red. Stopper as quickly as
possible and heat slowly. The slower the heating rate, the
greater the contact time between the evolved H2S and Zn(C2H302)2
and the less chance of sulfide loss.
Distill the solution until approximately 20 ml of distillate has
been collected (roughly 5-8 min after the solution commences to
boil). Turn off heat and remove the stopper in the distillation
flask to keep the distillate from being sucked back up the
condenser. Raise the transfer tube above the 50-mL mark on the
collection container and dilute the solution to 50 ml.
Place the distillates in a water bath at 23-25° c. Add 2 mL
dilute amine solution and mix. Add 0.25 mL (5 drops) FeCl3
solution and mix. Allow 10 min for color development and measure
sample absorbance at 650 nm.
t Calculations
•Prepare a standard curve by plotting absorbance of the standards
vs. sulfide concentration. Ensure that the standards cover the
range of concentrations expected in the samples. Determine the
sulfide concentration of the sample distillate by comparing sample
absorbance with the standard curve.
Calculate total sulfides concentration as follows:
Concentration (mg/kg dry weight) =
Where:
C = sulfide concentration in distillate, mg/L
0.05 = sample volume of distillate, L (as written)
g = wet weight of sediment aliquot, g
%S = percent solids of sediment as a decimal fraction.
QA/QC Procedures
It is critical that each sample be thoroughly homogenized in the
laboratory before a subsample is taken for analysis. Laboratory
homogenization should be conducted even if samples were homogenized in the
field.
35
-------
Conventional Sediment Variables
Total Sulfides
March 1986
It is recommended that triplicate analyses be conducted on one of every
20 samples, or on one sample per batch if less than 20 samples are
analyzed. Fresh standards should be used to calculate a calibration curve
for each batch of samples. The analytical balance should be inspected daily
and calibrated at least once per week.
DATA REPORTING REQUIREMENTS
Total sulfides should be reported as mg/kg of sediment dry weight to
the nearest 0.1 unit. The laboratory should report the results of all
samples (including QA replicates) and should note any problems that may have
influenced sample quality. The laboratory should also describe the
calibration curve used to determine total sulfide concentrations.
36
-------
Conventional Sediment Variables
Total Nitrogen
March 1986
TOTAL NITROGEN
If the elemental analyzer used to measure total organic carbon can also
measure total nitrogen, it is recommended that the latter variable be
measured simultaneously with TOC. Total nitrogen values in sediments
generally are used to compare carbon-to-nitrogen ratios. A separate total
nitrogen analysis using a technique other than the elemental analyzer method
is not considered equivalent for calculating carbon-to-nitrogen ratios.
37
-------
Conventional Sediment Variables
Biochemical Oxygen Demand
March 1986
BIOCHEMICAL OXYGEN DEMAND (BOD)
USE AND LIMITATIONS
Biochemical oxygen demand is a measure of the dissolved oxygen consumed
by microbial organisms while assimilating and oxidizing the organic matter
in a sample. This test is used to estimate the amount of organic matter
that is available to organisms, in contrast to other tests used to estimate
the total amount of organic matter (e.g., total volatile solids, total
organic carbon, chemical oxygen demand).
In addition to oxygen used for degrading organic matter, biochemical
oxygen demand may also include oxygen used to oxidize inorganic material
(e.g., sulfide, ferrous iron) and reduced forms of nitrogen.
The method described by Plumb (1981) involves the use of freshwater
bacteria as the seed and buffered distilled water as the dilution water.
An alternative procedure for marine sediments is to use marine bacteria as
the seed and filtered, oxygenated seawater as the dilution water. If
seawater is used, it does not need to be buffered.
FIELD PROCEDURES
Collection
Samples can be collected in glass or plastic containers. A minimum
sample size of 50 g is recommended. If unrepresentative material is to be
removed from the sample, it should be removed in the field under the
supervision of the chief scientist and noted on the field log sheet.
Processing
Samples should be stored at 4° C, and can be held for up to 7 days
under that condition. Samples should be kept field-moist and air contact
should be prevented to minimize oxidation.
LABORATORY PROCEDURES
Analytical Procedures
t Equipment
-Incubator
Thermostatically controlled at 20° +_ \o c.
All light should be excluded to prevent the photosynthetic
production of dissolved oxygen by algae in the sample.
38
-------
Conventional Sediment Variables
Biochemical Oxygen Demand
March 1986
Incubation bottles
300-mL capacity, with ground glass stoppers.
Phosphate buffer solution
Dissolve the following in distilled water: 8.5 g potassium
dihydrogen phosphate, K^POy, 21.75 g dipotassium hydrogen
phosphate, ^HPtty; 33.4 g disodium hydrogen phosphate hepta-
hydrate, N32HP04-7H20; and 1.7 g ammonium chloride, NH/iCl.
Dilute to 1 L. The pH of this buffer should be 7.2 without
further adjustment. If dilution water is to be stored in the
incubator, the phosphate buffer should be added just prior to
using the dilution water.
Magnesium sulfate solution
Dissolve 22.5 g MgS04«7H20 in distilled water and dilute to 1
L.
Calcium chloride solution
Dissolve 27.5 g anhydrous CaCl2 in distilled water and dilute
to 1 L.
Ferric chloride solution
Dissolve 0.25 g FeCl3«6H20 in distilled water and dilute to 1
L.
Dilution water
Store distilled water in cotton-plugged bottles for a
sufficient length of time to become saturated with dissolved
oxygen. The water should be aerated by shaking a partially
filled bottle or using a supply of clean compressed air. The
distilled water used should be as near as possible to 20° C
and of high purity. Place the desired volume of distilled
water in a suitable bottle and add 1 ml each of phosphate
buffer, magnesium sulfate, calcium chloride, and ferric
chloride for each liter of water.
Seeding material
Satisfactory seed may sometimes be obtained by using the
supernatant liquor from domestic sewage that has been stored
at 20° C for 24-36 h. Use the seed that has been found by
practical experience to be the most satisfactory for the
particular material under study. Only past experience can
determine the amount of seed to be added per liter but the
amount should give an oxygen depletion of approximately 2
mg/L. The amount of seed required may vary with the source
of the seed. Seeded dilution water should be used the same
day it is prepared.
Standards preparation
- Prepare a stock BOD standard solution by dissolving 0.150 g
reagent grade glucose and 0.150 g reagent grade glutamic acid in 1
L of distilled water. The solids should be dried for 1 h at
103° C prior to weighing.
39
-------
Conventional Sediment Variables
Biochemical Oxygen Demand
March 1986
• Sample preparation
-Allow samples to warm to room temperature.
Homogenize each sample mechanically, incorporating any overlying
water.
Remove a representative aliquot (approximately 25 g) and analyze
for total solids content.
Remove a representative aliquot (approximately 5 g) and weigh it
to the nearest 0.1 mg.
Transfer the weighed aliquot to a BOD bottle for analysis.
• Analytical procedures
-Fill each BOD bottle with dilution water and place the samples in
the incubator. Ensure that air bubbles are not trapped in the BOD
bottles. Prepare a blank consisting of dilution water in a
separate BOD bottle. Make sure that there is a water seal in the
neck of each sample bottle and blank when placed in the
incubator. Replenish the water seals on all bottles each morning.
Determine the initial dissolved oxygen concentration of each
sample and blank using the Winkler titration method or a dissolved
oxygen probe. This can best be accomplished by directly measuring
the dissolved oxygen concentration in the dilution water. This
method is recommended because sediment may cause a rapid
consumption of oxygen, making it difficult to obtain a stable
initial dissolved oxygen reading. If a probe is used for oxygen
measurement, the same sample can be used for immediate dissolved
oxygen demand and biochemical oxygen demand.
Incubate samples and blanks for 5 days at 20 +_ 1° C. Determine
residual dissolved oxygen concentrations in the incubated samples
using the analytical method of choice. The most reliable BOD
determinations will occur in those samples with a residual
dissolved oxygen concentration of at least 2 mg/L and a dissolved
oxygen depletion of at least 2 mg/L.
It is recommended that the dilution water be incubated as a check
on its quality. To do this, fill two BOD bottles with unseeded
dilution water. Stopper one bottle, fill the water seal, and
place in the incubator at 20 +_ 1° C for 5 days. Analyze the
second sample to determine initial dissolved oxygen
concentration. Following the 5-day period, determine dissolved
oxygen in the incubated sample. The oxygen depletion should not
be more than 0.2 mg/L and preferably not more than 0.1 mg/L. If
these values are exceeded, the quality of the dilution water or
the treatment of samples (e.g., filling of water seals) should be
considered suspect.
Prepare a working BOD standard solution by diluting 20 mL of the
stock solution to 1 L with seeded dilution water. Fill three BOD
bottles and incubate at 20 + 1° C for 5 days. The resulting BOD
of these samples should Fe 218 *_ 11 mg/L. Any appreciable
deviation from these expected results may raise questions on the
quality of the dilution water, the viability or suitability of the
seed material, or the analytical technique.
40
-------
Conventional Sediment Variables
Biochemical Oxygen Demand
March 1986
• Calculations
-Sediment BOD is calculated as follows:
BOD (mg/kg dry weight) =
Where:
0 = dissolved oxygen concentration at time zero, mg/L
F = dissolved oxygen concentration after 5 days, mg/L
b = volume of BOD bottle, ml
g = wet weight of sediment sample used, g
%S = percent solids in sediment sample (expressed as a
decimal fraction.
QA/QC Procedures
It is critical that each sample be thoroughly homogenized in the
laboratory before a subsample is taken for analysis. Laboratory
homogenization should be conducted even if samples were homogenized in the
field.
It is recommended that triplicate analyses be conducted on one of every
20 samples, or on one sample per batch if less than 20 samples are
analyzed. A dilution water blank and glucose-glutamic acid standard should
be analyzed at the same frequency as the triplicate analyses.
DATA REPORTING REQUIREMENTS
Biochemical oxygen demand should be reported as mg/kg dry weight, to
the nearest 0.1 unit. The laboratory should report the results of all
samples analyzed, including QA replicates, seeded dilution water blanks,
unseeded dilution water blanks, and glucose-glutamic acid standards. The
laboratory should also note any problems that may have influenced sample
quality.
41
-------
Conventional Sediment Variables
Chemical Oxygen Demand
March 1986
CHEMICAL OXYGEN DEMAND (COD)
USE AND LIMITATIONS
Chemical oxygen demand is a measure of the oxygen equivalent of the
organic matter content of a sample that is susceptible to oxidation by a
strong chemical oxidant at elevated temperature and reduced pH. The test
was devised as an alternative to the biochemical oxygen demand test for
estimating organic matter. For samples from a specific source, chemical
oxygen demand can be related empirically to biochemical oxygen demand, total
organic carbon, or total volatile solids and then used for monitoring after
a relationship has been established.
Major limitations of the chemical oxygen demand test are that it is not
specific for organic matter and that correlations with other measures of
organic carbon are not always found. Inorganic substances such as Fe^+,
MnZ+t and S2" can increase the consumption of oxidizing agent during the
test. Plumb (1981) recommends that chemical oxygen demand not be equated
with organic matter in sediments.
FIELD PROCEDURES
Collection
Samples can be collected in glass or plastic containers. A minimum
sample size of 50 g is recommended. If unrepresentative material is to be
removed from the sample, it should be removed in the field under the
supervision of the chief scientist and noted on the field log sheet.
Processing
Samples should be stored at 4° C and can be held under that condition
for 7 days. Samples must be kept field-moist and free from air contact
during storage to prevent air oxidation.
42
-------
Conventional Sediment Variables
Chemical Oxygen Demand
March 1986
LABORATORY PROCEDURES
Analytical Procedures
• Equipment
•Reflux apparatus
Consisting of 250- or 500-mL Erlenmeyer flasks with ground
glass 24/40 neck1 and 300-mm jacket Liebig, West, or
equivalent condensers2 with 24/40 ground glass joint.
Hot plate
Having sufficient power to produce 1.4 W/cmz (9 W/inz) of
heating surface, or equivalent, to ensure adequate refluxing
of the sample.
Standard potassium dichromate solution, 0.250 H_
Dissolve 12.259 g KgCrgOy primary standard grade, previously
dried at 103° C for 2 h, in distilled water and dilute to 1
L. The addition of 0.12 g/L sulphamic acid will eliminate
interference due to nitrites in the sample at concentrations
up to 6 mg/L.
Sulfuric acid reagent
Concentrated H2S04 containing 22 g silver sulfate, Ag2S04,
per 9-lb bottle. Allow 1 or 2 days for dissolution.
Standard ferrous ammonium sulfate titrant, 0.25 N_
Dissolve 98 g Fe(NH4)2 (SO/^'SHgO in distilled water.
Carefully add 20 ml concentrated HgSO^ cool, and dilute to 1
L. This solution must be standardized against I^C^Oy
daily. To standardize the ferrous ammonium sulfate, dilute
10 ml standard potassium dichromate solution to approximately
100 mL. Carefully add 30 ml concentrated H2S04 and allow to
cool. Titrate with ferrous ammonium titrant, using 2-3 drops
of ferroin indicator.
(ml K?Cr?07)(0.25)
Normality = 1—LJ.
[ml Fe(NH4)2(S04)2]
Ferroin indicator
Dissolve 1.485 g 1,10-phenantroline monohydrate and 0.695 g
ferrous sulfate, FeS04«7H20, in water and dilute to 100 ml.
Alternatively, a commercially prepared indicator can be
purchased.
Silver sulfate
Ag2s°4i reagent powder.
Mercuric sulfate
HgS04, analytical-grade crystals.
iCorning 5000 or equivalent.
2Corm'ng 2360, 91548, or equivalent.
43
-------
Conventional Sediment Variables
Chemical Oxygen Demand
March 1986
• Sample preparation
-Allow samples to warm to room temperature.
Homogenize each sample mechanically, Incorporating any overlying
water.
Remove a representative aliquot (approximately 25 g) and analyze
for total solids content.
Remove a representative aliquot (approximately 2 g) and weigh it
to the nearest 0.1 mg.
Transfer the weighed aliquot to a reflux flask for COD analysis.
Wash the sediment into the flask with a minimum amount of
distilled water (i.e., <25 ml).
t Analytical procedures
-Place several boiling stones or glass beads and 1.0 g HgS04 in the
reflux flask with the sample.
Add 25 ml 0.25 ^ <2CR207 to the flask and mix thoroughly.
Slowly, and with constant mixing, add 75 ml of sulfuric
acid-silver sulfate solution. Ensure that the mixture is well
mixed to avoid localized superheating.
Attach the sample flask to a condenser and reflux for 2 h. If the
added dichromate dissipates during reflux, either 1) repeat, using
a smaller sample size, or 2) carefully add additional 0.25 N_
K2C"r2°7 through the condenser. Be sure to record any added
dichromate.
Allow the sample to cool and rinse the condenser with 40-50 ml
distilled water.
Add an additional 50 ml of distilled water to the sample and allow
to cool to room temperature.
Add 3-5 drops of ferroin indicator and titrate with 0.25 N^
Fe(NH4)2(504)2 to a sharp color change (blue-green to
reddish-brown).
For a blank, reflux 25 ml of distilled water, 25 ml of 0.25 N_
K2Cr2°7» * Q HgS04, several glass beads or boiling stones, and 75
ml of sulfuric acid-silver sulfate solution for 2 h. Cool, add
3-5 drops of ferrion indicator, treat as a sample and titrate with
0.25 N_Fe(NH4)2(S04)2.
• Calculations
-Sediment COO is determined as follows:
COD (mg/kg dry weight) = 'A'
Where:
A = volume of 0.25 N Fe(NH4)2(S04)2 for blank titration, mL
B = volume of 0.25 FT Fe(NH4J2(S04)2 for sample titration, ml
N = normality of (TT25 N^ Fe(^4)2(504)2 used for titration,
eq/L
8,000 = equivalent weight of oxygen, mg/eq
44
-------
Conventional Sediment Variables
Chemical Oxygen Demand
March 1986
g = wet weight of sample, g
%S = percent solids in sediment sample (expressed as a decimal
fraction.
QA/QC Procedures
It is critical that each sample be thoroughly homogenized in the
laboratory before a subsample is taken for analysis. Laboratory
homogenization should be conducted even if samples were homogenized in the
field.
It is recommended that triplicate analyses be conducted on one of every
20 samples, or on one sample per batch if less than 20 samples are
analyzed.
DATA REPORTING REQUIREMENTS
Chemical oxygen demand should be reported as mg/kg of sediment dry
weight to the nearest 0.1 unit. The laboratory should report the results of
all samples analyzed (including QA replicates and method blanks) and should
note any problems that may have influenced data quality. The laboratory
should also report the results of the standard test.
45
-------
Conventional Sediment Variables
References
March 1986
REFERENCES
Buchanan, J.B. 1984. Sediment analysis, pp. 41-65. In: Methods for
the Study of Marine Benthos. N.A. Holme and A.D. Mclntyre (eds). Blackwell
Scientific Publications, Boston, MA.
Folk, R.L. 1968. Petrology of sedimentary rocks. Hemphill Publishing
Co., Austin, TX. 172 pp.
Hedges, J.I., and J.H. Stern. 1984. Carbon and nitrogen determinations
of carbonate-containing solids. Limnol. Oceanogr. 29:657-663.
Krumbein, W.C. and F.J. PettiJohn. 1938. Manual of sedimentary petrography.
Appleton-Century Crafts, NY. 549 pp.
Plumb, R.H. 1981. Procedures for handling and chemical analysis of sediment
and water samples. Technical Report EPA/CE-81-1. U.S. Army Corps of Engineers,
Vicksburg, MS.
U.S. Environmental Protection Agency. 1983. Methods for chemical analysis
of water and wastes. EPA 600/4-79-020 (Revised 1983). Environmental Monitoring
and Support Laboratory, Cincinnati, OH.
46
-------
ORGANIC COMPOUNDS
-------
METALS
-------
FINAL REPORT Pi/qef Sound Estuary Program
TC-3090-04
RECOMMENDED PROTOCOLS
FOR MEASURING METALS
IN PUGET SOUND WATER, SEDIMENT,
AND TISSUE SAMPLES
Prepared by:
TETRA TECH, INC.
Prepared for:
RESOURCE PLANNING ASSOCIATES
for:
PUGET SOUND DREDGED DISPOSAL
ANALYSIS (PSDDA)
Monitored by:
U.S. Army Corps of Engineers
Seattle District
Seattle, WA
August, 1986
TETRA TECH, INC.
11820 Northup Way
Bellevue, WA 98005
-------
CONTENTS
Page
LIST OF TABLES iv
ACKNOWLEDGMENTS iv
INTRODUCTION 1
GENERAL 1
PRECAUTIONS AND LIMITATIONS 1
SAMPLE COLLECTION 4
PRECOLLECTION 4
SOURCES OF CONTAMINATION 4
WATER COLUMN SAMPLES 6
PARTICULATE SAMPLES 7
SURFICIAL SEDIMENT SAMPLES 10
TISSUE SAMPLES 13
ANALYTICAL METHODS 16
SAMPLE PREPARATION 16
INSTRUMENTAL METHODS 18
RECOMMENDED INSTRUMENTAL METHODS 18
METALS SPEC IAT ION 19
QUALITY ASSURANCE/QUALITY CONTROL 20
QA/QC MEASURES INITIATED BY THE ANALYTICAL LABORATORY 20
QA/QC MEASURES INITIATED IN THE FIELD 21
CORRECTIVE ACTIONS 23
DATA REPORTING 24
DATA REPORT PACKAGE 24
BACKUP DOCUMENTATION 24
REFERENCES 25
APPENDICES
A: SELECTION OF METALS FOR PROTOCOL DEVELOPMENT A-l
B: EXCERPTS FROM EXHIBITS B, D, E, AND G (U.S. EPA 1985) B-l
C: ELUTRIATE AND FRACTIONATION METHODS (PLUMB 1981) C-l
11
-------
D: TOTAL ACID DIGESTION METHOD FOR SEDIMENT (RANTALA AND
LORING 1975) D-l
E: HN03/HC104 DIGESTION METHOD FOR TISSUE (TETRA TECH 1986a) E-l
F: APDC/MIBK EXTRACTION METHOD FOR SALT WATER (GREENBERG ET AL.
1985) F-l
G: DFAA INSTRUMENTAL AND SPECTROPHOTOMETRIC METHODS (U.S. EPA
1979a) G-l
111
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TABLES
Number Page
1 Contributors to the metals protocols 2
2 Sample preservation and storage parameters 5
ACKNOWLEDGMENTS
This chapter was prepared by Tetra Tech, Inc., under the direction
of Mr. Robert Barrick, for Resource Planning Associates (RPA) for the Puget
Sound Dredged Disposal Analysis (PSDDA) monitored by the Seattle District,
U.S. Army Corps of Engineers (COE) in partial fulfillment of Contract
No. DACW67-85-D-0029. Dr. David Kendall of U.S. COE was the Technical
Coordinator. The primary author of this chapter was Dr. Charles Lytle
of Tetra Tech, Inc.
1v
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Metals
Introduction
August 1986
INTRODUCTION
GENERAL
This chapter presents recommended protocols for measuring metals in
water, sediment, and tissue samples from Puget Sound. The list of metals
considered and their limits of detection (LODs) are summarized in Appendix A.
The main objectives of this report are to:
• Elucidate acceptable method options for each metal meeting
the minimum LODs specified for water, sediment, and biological
samples from Puget Sound
• Prioritize all method options for each metal and make recom-
mendations on the best method for each appropriate matrix.
The recommendations presented in this chapter are based on the results
of a workshop and detailed reviews by representatives from most organizations
that fund or conduct environmental research in Puget Sound (Table 1).
The purpose of developing these recommended protocols is to encourage all
Puget Sound investigators conducting monitoring programs, baseline surveys,
and intensive investigations to use standardized methods whenever possible.
If this goal is achieved, most data collected in Puget Sound should be
directly comparable and thereby capable of being integrated into a sound-wide
database. Such a database is necessary for developing and maintaining
a comprehensive water quality management program for Puget Sound.
Although the following protocols are recommended for most studies
conducted in Puget Sound, departures from these methods may be necessary
to meet the special requirements of individual projects. If such departures
are made, however, the funding agency or investigator should be aware that
the resulting data may not be comparable with most other data of that kind.
In some instances, data collected using different methods may be compared
if the methods are intercalibrated adequately.
PRECAUTIONS AND LIMITATIONS
Although well-established procedures for proper sample handling and
analysis are essential for collecting data of high quality, standard protocols
do not exist for all the required methods. Approved methods exist for
the analysis of metals in water (e.g., U.S. EPA 1979a, ASTM 1983), but
only interim procedures have been proposed for sediment and tissue samples
(U.S. EPA 1977, Plumb 1981). New considerations may need to be addressed
and additional guidelines provided to maximize the quality and efficiency
of the procedures.
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TABLE 1. CONTRIBUTORS TO THE METALS PROTOCOLS
Roy Arakia
Bob Barricka«b
Robert Clark3
Eric Crecelius3
Ray Dalsega
Bob Dexter3
Mark Fugiel3
Michael Higgins3
Roger Kadeg3
David Kendall3
Mary Beth Lanza3
Chuck Lytle3
Pamela Navaja3
Matt Schultz3
Mike Shelton
Jeff Stern3
Steve Twiss3
Washington Dept. of Ecology
Tetra Tech
NOAA/NMFS
Battelle Northwest
Metro
EVS Consultants
Am Test
Analytical Technologies
Envirosphere
U.S. COE
Laucks Testing Labs
Tetra Tech
Washington DSHS
Envirosphere
Weyerhaeuser Co.
Tetra Tech
Washington Dept. of Ecology
3 Attended the workshop held on January 17, 1986.
b Workshop moderator.
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Metals
Introduction
August 1986
The instrumental methods proposed are prone to chemical and matrix
interferences that can either suppress or enhance the analyte signal (Skoog
1985). Special attention should be given when they are likely to be present
as noted in the individual protocols. If any such interferences are suspected,
it is extremely important that their effect be determined and corrective
action taken.
Because of the low limits of detection proposed for many environmental
analyses, clean field and laboratory procedures are especially important.
Sample contamination can occur during any stage of collection, handling,
storage, or analysis. Potential contaminant sources must be known and
steps taken to minimize or eliminate them (Murphy 1976).
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Metals
Sample Collection
August 1986
SAMPLE COLLECTION
PRECOLLECTION
Container Materials
The best containers for the collection and/or storage of trace metal
samples are made of quartz or tetrafluoroethylene (TFE). Because these
containers are expensive, the preferred container material is either poly-
propylene or linear polyethylene, with a polyethylene cap. Borosilicate
glass may be used, but lids must not have aluminum or cardboard liners
(Greenberg et al. 1985).
For tissue samples, scalpels should be made of corrosion-resistant
stainless steel, while tweezers and cutting surfaces should be plastic
or TFE (Tetra Tech 1986a). Stainless steel homogenizer blades should be
replaced with tantalum.
The recommended container materials, sample sizes, preservation techniques,
and storage lifetimes for all the metals of concern in water, sediment,
and tissue are summarized in Table 2.
Cleaning Methods
Prior to use, containers and any glass or plastic parts associated
with the sampling equipment should be thoroughly cleaned with a detergent
solution, rinsed with tap water, soaked 24 h at 70° C in an acid solution
of 1:1 HN03 or 1:1 HC1, and then rinsed with metal-free water. Do not
use chromic acid for any cleaning purpose. For stainless steel parts,
omit the acid soaking step (Greenberg et al. 1985).
SOURCES OF CONTAMINATION
In the field, sources of contamination include sampling gear, lubricants
and oils, engine exhaust, airborne dust, and ice used for cooling samples.
During sample handling and analysis, contamination sources include exposure
to airborne dust, insufficiently clean sample containers, contact with
inappropriate materials (e.g., rubber o-rings, aluminum-lined caps, steel
containers), contaminated reagents, and carryover in testing instruments
due to insufficient cleaning or flushing between samples.
Exposure to airborne dust can be minimized by using capped containers
and by keeping physical sample handling to a minimum. Use of recommended
container materials and cleaning methods should minimize contamination
from storage containers. Reagents should be ACS Reagent Grade or better
and should never be returned to their stock containers once removed. Carry-
over can be avoided by carefully following instrument manufacturers' instruc-
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TABLE 2. SAMPLE PRESERVATION AND STORAGE PARAMETERS
Sample
Analyte
Total or dissolved
metals (except Hg)a
Total or
dissolved Hga
Particulate Metals
All metals
Elutriate Studye
Fractionation Studye
All metals (except Hg)
Hg
Matrix
H20
H20
H20
Sediment
H20
Sediment
Sediment
Tissue
Tissue
Containerb
P.G.TFE9
G.TFE
P.G
P,G
P|G
P,G
P,G
P,G
Size
1 L
250 ml
1 gal
50 gc
12 L
3 L
1 L
5 gc.h
0.2 gc,h
Storage
Preservative
HN03 PH <2
HN03 pH <2
i
Freeze
4° C
4° C
Freezef
Freezef
Lifetime
6 mo
28 days
i
6 mod
--
—
6 mod
28 daysd
a U.S. EPA 1982.
b P = linear polyethylene, G = borosilicate glass, TFE = tetrafluoroethylene.
c Wet weight.
d Suggested. No EPA criteria exist. Hg holding time 28 days.
e Plumb 1981. Storage time "as short as possible," analyses to be completed
"within 1 wk of sample collection."
f Post-dissection.
g If aliquot for Hg taken from this 1 L sample, cannot use linear polyethylene.
h Weight is a minimum for one sample. Studies using specific organs may require more.
i Samples should be filtered as soon as possible and always within 24 h. Workshop attendees
recommend that filtering be done shipboard rather than in lab on shore.
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Metals
Sample Collection
August 1986
tions. Sources of contamination in the analytical laboratory and on shipboard
have been thoroughly reviewed by Mart (1979a,b).
WATER COLUMN SAMPLES
Water column samples frequently are collected using water bottles.
Water bottle samplers are relatively simple devices that generally consist
of some type of cylindrical tube with stoppers at each end and a closing
device that is activated by a messenger or an electrical signal. The most
commonly used samplers of this description are the Kemmerer, Van Dorn,
Niskin, and Nansen samplers. Each device samples a discrete parcel of
water at any designated depth. Frequently, multiple water samplers are
fixed on a rosette frame so that several depths can be sampled during one
cast or replicate samples can be taken at the same depth.
Prior to deployment, the stoppers of water bottle samplers are cocked
open on the sampling vessel. At this step, it is critical that the interior
of the sampler and stoppers remain free from contamination. All members
of the sampling team should therefore avoid touching the insides of the
sampler and stoppers, and all samplers and stoppers must be rinsed thoroughly.
After cocking, the sampler is lowered to a designated depth. The
sampler must be open at both ends so that water is not trapped within the
device as it is being lowered through the water column. Once the sampler
reaches the desired depth, it should be allowed to equilibrate with ambient
conditions for 2-3 min before being closed.
After equilibration, the closing device can be activated by messenger
or electrical signal, and the sampler can be retrieved. It is recommended
that at least two samplers be used simultaneously for each depth. A second
sampler will provide a backup to the primary sampler in case the latter
device misfires or won't trigger. This will eliminate the need for an
additional cast. A second sampler will also supplement the primary sampler
if the volume collected by the latter device is too small for all required
subsampling and rinsing. To ensure that all subsamples at a particular
depth are collected from the same water parcel, it is essential that they
all be taken from a single cast. Multiple casts using a single water sampler
will not meet this objective. Sample water must therefore be used conserva-
tively after collection.
Once the water sampler is brought on board the sampling vessel, the
stoppers should be checked immediately for complete seals. If a stopper
is not properly sealed, water from the sampled depth may have leaked out
upon retrieval and been replaced by water from shallower depths. Because
this kind of contamination can bias results, the entire water sample should
be rejected.
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Metals
Sample Collection
August 1986
PARTICULATE SAMPLES
This section provides guidance on the collection of the particulate
fraction of water samples from in-plant process streams, effluents, and
receiving waters. All devices capable of separating particulate material
from water samples may contribute contaminants to the sample taken for
analysis. The level of this "blank" contamination must be determined in
advance and reduced when possible.
There are several techniques for obtaining the particulate fraction
of water samples, including different filtration procedures, settling/centrifu-
gation, centrifugation, and continuous-flow centrifugation. The latter
technique may not be appropriate for metals because the equipment required
contains a number of metallic parts. A backflush-filtration technique
has also been described in an intercomparison study (Horowitz 1986), but
is not appropriate for chemical analyses because of selective losses of
the filtered material prior to analysis.
Filtration may be prefereable to other techniques for many analyses
because it requires less expensive equipment (i.e., a high speed centrifuge
is not required), and provides a sample that is suitable for direct chemical
analysis (i.e., a residue on a filter that can be extracted or digested).
The amount of particulate material that may be collected on a filter (e.g.,
typically <1 g) may limit this technique for low-level analyses, including
metals. Centrifugation techniques can yield comparable results and may
be used to collect much larger amounts of particulate material than by
filtration (e.g., several grams), but require careful and complete transfer
of the sample from the centrifuge prior to analysis.
Horowitz (1986) describes a centrifugation technique (fixed angle-
head, or swing-bucket rotor; not a continuous-flow system) for metals analysis
that he recommends when large-scale studies are undertaken (e.g., 2,000-
3,000 samplers per year). Bates et al. (1983) describe a system using
continuous-flow centrifugation (Son/all Model SS-3 or RC-5 high-speed centri-
fuge) for collection of particulate material for organic analyses (specifically
hydrocarbons). One interpretation of the results of this study that may
have implications for metals analyses was the possibility that continuous-
flow centrifugation may not recover fine-grained particles in the l-2um
range as efficiently as filtration. Although both techniques recovered
between 70 and 90 percent of the particles in this size range, these very
small particles may have a higher loading of metals than larger particles.
Sample Compositing and Preservation
Ideally, samples of in-plant process streams or effluents should be
collected as a flow-proportionate composite of at least 5 days of mechanical
interval sampling to yield a time-integrated measurement. If flow-proportion-
ated samples cannot be collected (i.e., aliquots collected in proportion
to the changing flow rate of the stream), instantaneous grab samples can
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Metals
Sample Collection
August 1986
be substituted, but should still be composited over several days collection
whenever possible. It is usually unreasonable to composite receiving water
samples over such periods of time in a routine sampling program, and it
may be unreasonable to store composited process stream or effluent samples
over several days when total volumes greater than approximately 20 L (i.e.,
5 gal) are required.
Samples for metals analysis should be stored under refrigeration in
1-gal polyethylene cubitainers. Until use, the cubitainers should be stored
(collapsed) in plastic bags. The cubitainers should be carefully rinsed
with several aliquots of the sample prior to filling to remove any dust
particles. An acid pre-wash step is not considered necessary for the
cubitainers.
Filtration of the entire sample should be conducted at one time, and
this process may take from one to several hours to complete. A potential
preservation technique is addition of mercuric chloride (for organic analyses
only), or a 2 percent sodium azide solution. The latter preservative has
worked well with sediment trap samples collected by NOAA, but requires
access to a fume hood for sample processing which is a distinct limitation.
Suspended solids samples cannot be preserved with 100 percent effectiveness
(Greenberg et al. 1985). Hence, preserved sample bottles should be stored
under refrigeration (2°C) prior to filtration to minimize decomposition
of solids. No samples should be held for longer than 7 days.
Sample Volumes
A 4-L composited sample (i.e., 1-gal) is recommended for the analysis
of metals in particulate samples. The volume required for analysis is
a function of the concentration of particulate material in the stream and
the concentration of metals in the particulate material. Horowitz (1986)
recommends the collection of 100 mg of particulate material for analysis
of metals, but this quantity cannot always be obtained with a 4-L sample.
Larger sample volumes (e.g., 20-L) can be processed but are not considered
necessary for most purposes. Smaller sample sizes (e.g., 5-30 mg) have
been successfully analyzed for most metals using graphite furnace and ICP
techniques and a final dilution volume of 10 mL digestate (Tetra Tech 1985).
Sample Processing
Preparation of Filters-
Polycarbonate membrane filters (e.g., Nuclepore) of 0.4-um pore size
are recommended for filtration of water samples for metals analyses. The
142-mm diameter filters are required to filter the recommended minimum
of approximately 4-L of sample. The membrane filters do not require any
pretreatment prior to weighing, and can be taken directly from the manu-
facturer's container. Each filter must be carefully weighed to the nearest
0.1 mg prior to filtering. Unlike glass fiber filters, the amount of residue
8
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Metals
Sample Collection
August 1986
collected on membrane filters can be accurately weighed. The dry weight
of the filtered material is used directly to determine the concentration
of metals associated with the particulate material.
Because the 142-mm filters are so light, care must be taken to prevent
wrinkling when placing the filter in the filter holder. Air currents can
easily disrupt the placement of the filter, and once wet the filters must
be carefully moved to their final position on the holder to prevent tearing.
A TFE filter holder is recommended, but a plastic filter holder may also
be used.
Filtration of Samples
After the tared filter has been placed into the filter holder, a plastic
squeeze bottle should be used to completely rinse the filter with 10 percent
high purity HC1 followed by deionized, distilled water until neutral pH
has been established. A water aspirator can be used to provide suction
for the rinsing. The acid rinse will leach any dust particles that may
have adhered to the filters (such particles can contribute zinc and potentially
other contaminants to the analysis).
Once the filter has been properly rinsed, the filter holder should
be reassembled and a siphon tube attached between the filter holder and
the cubitainer holding the water sample (place the cubitainer above the
filter holder to provide gravity-feed). The cubitainer should be vigorously
shaken prior to the filtration to dislodge particles that may have adhered
to the sides. Using the water aspirator, siphon the entire water sample
through the filter holder. After the water has passed through the filter,
disconnect the filter holder from the aspirator while the aspirator is
still running.
Carefully open the filter holder and, using plastic forceps, fold
the filter in thirds (like a crepe) to protect the filtered material.
Finally, with two pairs of forceps gently hold down one end of the filter
while folding the filter in half from the opposite end. Place the folded
filter in a polycarbonate petri dish. Seal the petri dishes, label,and
store frozen until the metals analysis can be started. Prior to digestion,
the filters should be dried at no more than 60°C and weighed to determine
total sample weight (the original weight of the clean filter must be supplied
to the laboratory). Filters dried at higher temperatures may suffer losses
of the more volatile metals.
At least one filter blank should be prepared by going through the
entire filtration setup procedure (i.e., including the acid-rinse step)
in the absence of a water sample. Fold and place the filter blank in a
polycarbonate petri dish as with other samples. At least one filter blank
should consist of an untreated folded membrane filter taken directly from
the manufacturer's box (to check efficiency of the rinse step).
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Metals
Sample Collection
August 1986
SURFICIAL SEDIMENT SAMPLES
This section describes the protocols required to collect an acceptable
subtidal surficial sediment sample for subsequent measurement of physical
and chemical variables. This subject has generally been neglected in the
past and sampling crews have been given relatively wide latitude in deciding
how to collect samples. However, because sample collection procedures
influence the results of all subsequent laboratory and data analyses, it
is critical that samples be collected using acceptable and standardized
techniques.
Design of Sampler
In Puget Sound, the most common sampling device for subtidal surficial
sediments is the modified van Veen bottom grab. However, a variety of
coring devices is also used. The primary criterion for an adequate sampler
is that it consistently collect undisturbed samples to the required depth
below the sediment surface without contaminating the samples. An additional
criterion is that the sampler can be handled properly on board the survey
vessel. An otherwise acceptable sampler may yield inadequate sediment
samples if it is too large, heavy, or awkward to be handled properly.
Collection of undisturbed sediment requires that the sampler:
• Create a minimal bow wake when descending
• Form a leakproof seal when the sediment sample is taken
a Prevent winnowing and excessive sample disturbance when
ascending
• Allow easy access to the sample surface.
Most modified van Veen grabs have open upper faces that are fitted with
rubber flaps. Upon descent, the flaps are forced open to minimize the
bow wake, whereas upon ascent, the flaps are forced closed to prevent sample
winnowing. Some box corers have solid flaps that are clipped open upon
descent and snap shut after the corer is triggered. Although most samplers
seal adequately when purchased, the wear and tear of repeated field use
eventually reduces this sealing ability. A sampler should therefore be
monitored constantly for sample leakage. If unacceptable leakage occurs,
the sampler should be repaired or replaced. If a sampler is to be borrowed
or leased for a project, its sealing ability should be confirmed prior
to sampling. Also, it is prudent to have a backup sampler on board the
survey vessel in case the primary sampler begins leaking during a cruise.
The required penetration depth below the sediment surface is a function
of the desired sample depth (see Penetration Depth). Generally, it is
better to penetrate below the desired sample depth to minimize sample
disturbance when the sampling device closes. Penetration depth of most
sampling devices varies with sediment character, and generally is greatest
in fine sediments and least in coarse sediments. Sampling devices generally
rely upon either gravity or a piston mechanism to penetrate the sediment.
10
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Metals
Sample Collection
August 1986
In both cases, penetration depth can be modified by adding or subtracting
weight from the samplers. Thus, it is optimal to use a sampler that has
a means of weight adjustment. If a sampler cannot consistently achieve
the desired penetration depth, an alternate device should be used.
Once the sampler is secured on board the survey vessel, it is essential
that the surface of the sample be made accessible without disturbing the
sample. Generally, samplers have hinged flaps on their upper face for
this purpose. The opening(s) in the upper face of the sampler should be
large enough to allow easy subsampling of the sediment surface. If an
opening is too small, the sample may be disturbed as the scientific crew
member struggles to take a subsample.
Penetration Depth
For characterizing surficial sediments in Puget Sound, it is recommended
that the upper 2 cm of the sediment column be evaluated. When collecting
the upper 2 cm of sediment, it is recommended that a minimum penetration
depth of 4-5 cm be achieved for each acceptable sample.
Although the 2-cm specification is arbitrary, it will ensure that:
• Relatively recent sediments are sampled
• Adequate volumes of sediments can be obtained readily for
laboratory analyses
• Data from different studies can be compared validly.
Sampling depths other than 2 cm may be appropriate for specific purposes.
For example, the upper 1 cm of sediment may be required to determine the
age of the most recently deposited sediments. By contrast, a sample depth
much greater than 2 cm may be required to evaluate the vertical profile
of sediment characteristics or to determine depth-averaged characteristics
prior to dredging. If a sampling depth other than 2 cm is used, comparisons
with data from 2-cm deep samples may be questionable.
Operation of Sampler
The sampling device should be attached to the hydrowire using a ball-
bearing swivel. The swivel will minimize the twisting forces on the sampler
during deployment and ensure that proper contact is made with the bottom.
For safety, the hydrowire, swivel, and all shackles should have a load
capacity at least three times greater than the weight of a full sampler.
The sampler should be lowered through the water column at a controlled
speed of approximately 1 ft/sec. Under no circumstances should the sampler
be allowed to "free fall" to the bottom, as this may result in premature
triggering, an excessive bow wake, or improper orientation upon contact
with the bottom. The sampler should contact the bottom gently and only
its weight or piston mechanism should be used to force it into the sediment.
11
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Metals
Sample Collection
August 1986
After the sediment sample is taken, the sampler should be raised slowly
off the bottom and then retrieved at a controlled speed of approximately
1 ft/sec. Before the sampler breaks the water surface, the survey vessel
should head into the waves (if present) to minimize vessel rolling. This
maneuver will minimize swinging of the sampler after it breaks the water
surface. If excessive swinging occurs or if the sampler strikes the vessel
during retrieval, extra attention should be paid to evaluating sample
disturbance when judging sample acceptability.
The sampler should be secured immediately after it is brought on board
the survey vessel. If the sampler tips or slides around before being secured,
extra attention should be paid to evaluating sample disturbance.
Sample Acceptability Criteria
After the sampler is secured on deck, the sediment sample should be
inspected carefully before being accepted. The following acceptability
criteria should be satisfied:
• The sampler is not over-filled with sample so that the sediment
surface is pressed against the top of the sampler
0 Overlying water is present (indicates minimal leakage)
• The overlying water is not excessively turbid (indicates
minimal sample disturbance)
• The sediment surface is relatively flat (indicates minimal
disturbance or winnowing)
• The desired penetration depth is achieved (i.e., 4-5 cm
for a 2-cm deep surficial sample).
If a sample does not meet all criteria, it should be rejected.
Sample Collection
After a sample is judged acceptable, the following observations should
be noted on the field log sheet:
0 Station location
0 Depth
0 Gross characteristics of the surficial sediment
Texture
Color
Biological structures (e.g., shells, tubes, macrophytes)
Presence of debris (e.g., wood chips, wood fibers,
human artifacts)
Presence of oily sheen
Odor (e.g., hydrogen sulfide, oil, creosote)
0 Gross characteristics of the vertical profile
Changes in sediment characteristics
Presence and depth of redox potential discontinuity
(rpd) layer
12
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Metals
Sample Collection
August 1986
• Penetration depth
0 Comments related to sample quality
Leakage
Winnowing
Disturbance.
Before subsamples of the surficial sediments are taken, the overlying
water must be removed. The preferred method of removing this water is
by slowly siphoning it off near one side of the sampler. Methods such
as decanting the water or slightly cracking the grab to let the water run
out are not recommended, as they may result in unacceptable disturbance
or loss of fine-grained surficial sediment and organic matter.
Once the overlying water has been removed, the surficial sediment
can be subsampled. It is recommended that subsamples be taken using a
flat scoop shaped like a coal shovel. The shoulders of the scoop should
be 2 cm high. This device will allow a relatively large subsample to be
taken accurately to a depth of 2 cm. Coring devices are not recommended
because generally they collect small amounts of surficial sediment and
therefore require repeated extractions to obtain a sufficient volume of
material for analysis of conventional sediment variables. A curved scoop
is not recommended because it does not sample a uniform depth. Because
accurate and consistent subsampling requires practice, it is advisable
that an experienced person perform this task.
When subsampling surficial sediments, unrepresentative material should
be removed in the field under the supervision of the chief scientist and
noted on the field log sheet. The criteria used to determine representa-
tiveness should be determined prior to sampling.
Finally, if samples are to be analyzed for trace metals or priority
pollutant organic compounds, sample contamination during collection must
be avoided. All sampling equipment (i.e., siphon hoses, scoops, containers)
should be made of noncontaminating material and should be cleaned appropriately
before use. Samples should not be touched with ungloved fingers. In addition,
potential airborne contamination (e.g., stack gases, cigarette smoke) should
be avoided. Detailed guidance for preventing sample contamination is given
in a previous section of this chapter.
TISSUE SAMPLES
Collection
The major difficulty in trace metal analyses of tissue samples is
controlling contamination of the sample after collection. In the field,
sources of contamination include sampling gear, grease from winches or
cables, engine exhaust, dust, or ice used for cooling. Care must be taken
during handling to avoid these and any other possible sources of contamination.
For example, during sampling the ship should be positioned such that the
engine exhausts do not fall on deck. To avoid contamination from melting
13
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Metals
Sample Col lection
August 1986
ice, the samples should be wrapped in aluminum foil and placed in watertight
plastic bags. The outer skin of the fish or shell of the shellfish acts
as protection against metals contamination from the aluminum foil.
Sample resection and any subsampling of the organisms should be carried
out in a controlled environment (e.g., dust-free room). In most cases,
this requires that the organisms be transported on ice to a laboratory,
rather than being resected on board the sampling vessel. It is recommended
that whole organisms not be frozen prior to resection if analyses will
be conducted only on selected tissues, because freezing may cause internal
organs to rupture and contaminate other tissue. If organisms are eviscerated
on board the survey vessel, the remaining tissue (e.g., muscle) may be
wrapped as described above and frozen.
Resection is best performed under "clean room" conditions. The "clean
room" should have positive pressure and filtered air. The "clean room"
should also be entirely metal-free and isolated from all samples high in
contaminants (e.g., hazardous waste). At a minimum, care should be taken
to avoid contamination from dust, instruments, and all materials that may
contact the samples. The best equipment to use for trace metal analyses
is made of quartz, TFE, polypropylene, or polyethylene. Stainless steel
that is resistant to corrosion may be used if necessary. Corrosion-resistant
stainless steel is not magnetic, and thus can be distinguished from other
stainless steels with a magnet. Stainless steel scalpels have been found
not to contaminate mussel samples (Stephenson et al. 1979). However, low
concentrations of heavy metals in other biological tissues (e.g., fish
muscle) may be contaminated significantly by any exposure to stainless
steel. Quartz utensils are ideal but expensive. To control contamination
when resecting tissue, separate sets of utensils should be used for removing
outer tissue and for removing tissue for analysis. For bench liners and
bottles, borosilicate glass would be preferred over plastic if trace organic
analyses are to be performed on the same sample.
Resection should be conducted by or under the supervision of a competent
biologist. For fish samples, special care must be taken to avoid contaminating
target tissues (especially muscle) with slime and/or adhering sediment
from the fish exterior (skin) during resection. The incision "troughs"
are subject to such contamination. Thus, they should not be included in
the sample. In the case of muscle, a "core" of tissue is taken from within
the area boarded by the incision troughs, without contacting them. Unless
specifically sought as a sample, the dark muscle tissue that may exist
in the vicinity of the lateral line should not be mixed with the light
muscle tissue that consitutes the rest of the muscle tissue mass.
Prior to use, utensils and bottles should be thoroughly cleaned with
a detergent solution, rinsed with tap water, soaked in acid, and then rinsed
with metal-free water. For quartz, TFE, or glass containers, use 1+1 HN03,
1+1 HC1, or aqua regia (3 parts cone HC1 + 1 part cone HN03) for soaking.
For plastic material, use 1+1 HNQ.3 or 1+1 HC1. Reliable soaking conditions
are 24 h at 70° C (Greenburg et al. 1985). Do not use chromic acid for
14
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Metals
Sample Collection
August 1986
cleaning any materials. Acids used should be at least reagent grade.
For metal parts, clean as stated for glass or plastic, except omit the
acid soak step. If trace organic analyses are to be performed on the same
samples, final rinsing with methylene chloride is acceptable.
Sample size requirements can vary with tissue type (e.g., liver or
muscle) and detection limit requirements. In general, a minimum sample
size of 6 g (wet weight) is required for the analysis of all priority pollutant
metals. To allow for duplicates, spikes, and required reanalysis, a sample
size of 50 g (wet weight) is recommended. Samples can be stored in glass,
TFE, or high-strength polyethylene jars.
Processing
Samples should be frozen after resection and kept at -20° C. Although
specific holding times have not been recommended by U.S. EPA, a maximum
holding time of 6 mo (except for mercury samples, which should be held
a maximum of 28 days) would be consistent with that for water samples.
When a sample is thawed, the associated liquid should be maintained
as a part of the sample. This liquid will contain lipid material. To
avoid loss of moisture from the sample, partially thawed samples should
be homogenized. Homogenizers used to grind the tissue should have tantalum
or titanium parts rather than stainless steel parts. Stainless steel blades
used during homogenization have been found to be a source of nickel and
chromium contamination.
15
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Metals
Analytical Methods
August 1986
ANALYTICAL METHODS
SAMPLE PREPARATION
Water: Total Metals
Freshwater samples to be analyzed by graphite furnace atomic absorption
spectroscopy (GFAA) should be prepared using an HN03/H202 digest. Analysis
by inductively-coupled plasma emission spectroscopy (ICP) or direct flame
atomic absorption spectroscopy (DFAA) requires an HN03/HC1 digest. Samples
to be analyzed for mercury by cold vapor atomic absorption spectroscopy
(CVAA) must have a separate, 100-mL aliquot digested using potassium per-
manganate and potassium persulfate. All three procedures are detailed
in Exhibit D of the Contract Laboratory Program (U.S. EPA 1985), which
is attached to this report as Appendix B.
Because digestion creates excessive salt concentrations that interfere
with instrumental methods, saltwater samples must be extracted. Extraction
with ammonium pyrrolidine dithiocarbamate/methyl isobutylketone (APDC/MIBK)
is recommended and is detailed in Appendix F (Greenburg et al. 1985).
Water; Dissolved Metals
According to convention, "dissolved" substances are those contained
in the filtrate passing a 0.45 urn filter. Samples to be analyzed for dissolved
metals must be filtered prior to preservation and subsequent digestion.
However, filters are a significant source of trace element contamination.
Membrane filters (e.g., polycarbonate, fluorocarbon) are significantly
cleaner than paper (Murphy 1976).
It is recommended that 0.45 urn polycarbonate filters without grid
marks be used for the filtration of water samples and that they be cleaned
by drawing through 500 mL 1:1 HN03 followed by 1 L metal-free water (to
neutral pH). Prior to use, filters must be stored in a tightly-closed
container to avoid contamination from airborne dust.
The filtrate should be acidified to pH<2 using 1:1 HN03. If a precipitate
appears upon acidification, the sample must be digested as for total metals.
Water; Particulate Metals
Particulate substances are operationally defined as those retained
on a 0.45 urn filter. Polycarbonate filters should be used and cleaned
as detailed in the previous section. After cleaning, the filters should
be dried to constant weight at 60 C. A detailed protocol for shipboard
filtration of large volumes of seawater using commercially-available equipment
is given by Mart (1979b). A completely submersible, self-contained filtration
apparatus, capable of filtering up to 1,000 L at a flow rate of 50-200 mL/min,
16
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Metals
Analytical Methods
August 1986
is commercially available from Seastar Instruments Ltd., Sidney, British
Columbia, Canada. After sample filtration, the filters should be dried
again to constant weight at 60 C. Total sample weight is then calculated
by difference.
After weighing, the entire filter plus sample is digested as for sediment
samples as detailed in Appendix D.
Sediment; Bulk Analysis
The two most common digestion methods employed for sediment samples
are total and strong acid. Total digestion is the most severe and employs
hydrofluoric acid to aid in the breakdown of silicate matrices and/or perchloric
acid to aid in the decomposition of organic matter. The use of a total
acid digest has been recently endorsed by the NOAA/NBS Quality Assurance
Workshop (December 5-6, 1985).
Strong acid digestion employs nitric acid, some ratio of nitric and
hydrochloric acids, or nitric acid and hydrogen peroxide. Strong acid
digestions are not as efficient as total acid methods for the solubilization
of certain metals (e.g., aluminum, chromium) that are tightly matrix-bound,
but they do not require special labware or hoods. The efficiencies of
various strong acid digestion methods have been reviewed by Plumb (1981).
Although Plumb concludes that the literature for strong acid digests supports
the use of HN03:HC1, the U.S. Environmental Protection Agency Contract
Laboratory Program (CLP) requires the use of HNC^rHgOz (U.S. EPA 1985).
The detailed procedure for the latter method is given in Exhibit D of the
CLP, which is attached to this report as Appendix B.
A hydrofluoric acid/aqua regia total acid digestion was agreed upon
at the January 17, 1986, PSDDA/PSEP workshop. The detailed protocol is
attached to this report as Appendix D (Rantala and Loring 1975).
Sediment: Elutriate and Fractionation Studies
The elutriate test is used to estimate the types and amounts of substances
likely to be released to the water column from sediments during dredging
operations. Fractionation studies estimate the distribution and mobility
of substances occurring in sediments. Detailed procedures for these two
tests are given by Plumb (1981) and are attached to this report as Appendix C.
The filtered samples from the elutriate test are treated as "dissolved
metals" samples discussed above. The filtered samples from the six different
"phases" of the fractionation procedure should be analyzed directly.
Tissue
Workshop participants agreed upon a nitric acid/perchloric acid digestion
for tissue samples. Perchloric acid is especially useful for the dissolution
of fats. The detailed procedure for this method is attached to this report
as Appendix E (Tetra Tech 1986a). Equivalent methods may be used (e.g.,
17
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Metals
Analytical Methods
August 1986
U.S. EPA 1977) if accuracy and precision can be demonstrated to the level
specified in the Quality Assurance/Quality Control section of this report.
INSTRUMENTAL METHODS
The instrumental methods currently available for the analysis of trace
metals include electrochemical (e.g., differential pulse polarography),
spectrophotometric (e.g., silver diethyldithiocarbamate), atomic absorption
spectrophotometric, atomic emission spectrophotometric, x-ray fluorescence,
and neutron activation.
While any analytical method might be used as long as the documented
instrument or method detection limits meet the required LODs specified
(Appendix A), certain instrumental techniques have drawbacks and are not
recommended. For example, both electrochemical and spectrophotometric
techniques are time-consuming and are not amenable to the analysis of large
numbers of samples. Neutron activation requires the use of expensive nuclear
equipment that is not widely distributed. Certain specialized techniques
designed for the analysis of particular metal species (e.g., organotins)
also require the use of research-grade instrumentation often extensively
modified from commercially available equipment (e.g., Donard and Weber 1985).
RECOMMENDED INSTRUMENTAL METHODS
The LODs recommended in Appendix A were arrived at by matching U.S. EPA
water quality criteria (for water analyses) and the lowest concentrations
expected in Puget Sound sediments and marine tissues against the detection
limits possible for each matrix using agency-approved instrumental methods.
For mercury, the only instrumental method adequate for these requirements
for all matrices is cold vapor atomic absorption (CVAA). For tissue, graphite
furnace atomic absorption (GFAA) is required for all the other priority
pollutant metals of concern.
For sediment, inductively-coupled plasma emission spectroscopy (ICP)
methods are adequate for copper, nickel, and zinc. All other priority
pollutant metals of concern in sediment require GFAA to achieve the LODs.
For water, ICP methods are adequate for cadmium and zinc. All other
priority pollutant metals of concern in water require GFAA to achieve the
LODs.
Atomic absorption analysis via hydride generation (HYDAA) can lower
the detection limits for antimony, arsenic, and selenium by a factor of
10-100 (Skoog 1985). This technique may be substituted for GFAA for samples
in which low concentrations of these three elements are expected, or it
may be used routinely. The U.S. EPA-approved method for arsenic by hydride
generation (U.S. EPA 1979a) is included in Appendix G and can be adapted
for the analysis of selenium and antimony. Hydride generation may be coupled
to ICP instead of AA for certain elements (e.g., selenium and antimony).
18
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Metals
Analytical Methods
August 1986
However, this method may be used only if accuracy and precision can be
demonstrated to the levels specified in the Quality Assurance/Quality Control
section of this report.
The U.S. EPA approved methods for GFAA and ICP analysis for all the
metals of concern except mercury and for the CVAA analysis of mercury are
detailed in Exhibit D of Appendix B (U.S. EPA 1985). It must be emphasized
that GFAA requires duplicate injections and matrix spikes. If spike recovery
is not within specific limits as detailed in the CLP (Exhibit E of Appendix B),
the method of standard additions (MSA) may be required. The monitoring
of GFAA spike recoveries is critical for all low level samples and is especially
critical for the analysis of lead.
The use of matrix modifiers has become widespread in the GFAA analysis
of freshwater (Manning and Slavin 1983), saltwater (Slavin et al. 1984,
Manning and Slavin 1978), and biological samples (Hinderberger et al. 1981).
No agency-approved protocol was found. However, matrix modifiers may be
used, especially for lead, arsenic, selenium, and antimony, as long as
accuracy and precision can be demonstrated to the levels specified in the
Quality Assurance/Quality Control section of this report.
Although X-ray fluorescence (XFS) may achieve the LCDs listed in
Appendix A, no agency-approved method was found. XFS may be used if accuracy
and precision can be demonstrated to the levels specified in the Quality
Assurance/Quality Control section of this report.
Other agency-approved instrumental techniques with detection limits
higher than those specified in Appendix A (e.g., direct flame atomic absorption)
may be used only if the sample concentration exceeds twice the documented
detection limit of the method. These U.S. EPA-approved methods are detailed
in Appendix G (U.S. EPA 1979a).
METALS SPECIATION
Because the techniques for the analysis of arsenic and organotin species
are not widely used, are not routine, and have inadequate QA/QC documentation,
no protocols were recommended at the January PSDDA/PSEP workshop. Although
not yet tested, it was suggested that methyl mercury may possibly be extracted
by vacuum distillation (e.g., Hiatt and Jones 1984) and then analyzed by
gas chromatography/mass spectrometry.
19
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Metals
QA/QC
August 1986
QUALITY ASSURANCE/QUALITY CONTROL
QA/QC MEASURES INITIATED BY THE ANALYTICAL LABORATORY
Standard laboratory practices for cleanliness as applied to glassware,
reagents, solvents, gases, and instruments must be followed. For additional
guidelines not covered in this report, see Sections 4 and 5 of Handbook
for Analytical Quality Control in Water and Wastewater Laboratories (U.S. EPA
1979b).
Instrumental QA/QC Checks
Instrumental QA/QC checks necessary for all the U.S. EPA-approved
methods discussed in the previous section include:
• Calibration blank
• Initial calibration and calibration verification
• Continuing calibration verification
• U.S. EPA Standard Reference Samples
• ICP interference check sample analysis (for ICP only).
Guidelines for instrumental calibration are given in U.S. EPA Methods
for Chemical Analysis of Water and Wastes (U.S. EPA 1979a). In general,
all instruments must be calibrated daily and each time the instrument is
set up. Calibration procedures will follow the procedures specified for
each analysis in the U.S. EPA protocols. In addition (as specified for
the CLP, Appendix B), after an instrument system has been calibrated, the
accuracy of the initial calibration shall be verified and documented for
every analyte by the analysis of U.S. EPA Quality Control Solutions. Where
a certified solution of an analyte is not available from U.S. EPA or any
source, analyses shall be conducted on an independent standard at a concen-
tration other than that used for calibration, but within the calibration
range (e.g., tin is not currently present in U.S. EPA Quality Control Samples).
When measurements for the certified components exceed the control limits,
the analysis must be terminated, the problem corrected, the instrument
recalibrated, and the calibration reverified.
For ICP and AA analyses, all work shall be performed using continuing
calibration as outlined in the CLP Statement of Work with regard to Exhibit E,
Sections 1 and 2 (see Appendix B). Frequency is 10 percent or every 2 h
during an analysis run, whichever is more frequent.
20
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Metals
QA/QC
August 1986
Method QA/QC Checks
Contract laboratories should perform the quality control checks listed
below:
0 Preparation blank
• Spiked sample analysis
t Replicate sample analysis
0 GFAA method of standard addition (if necessary)
0 Laboratory control sample analysis (Standard Reference Materials).
Details on the use and application of these checks along with control
limits, reporting requirements, and corrective actions are discussed in
Exhibit E of Appendix B (U.S. EPA 1985). The frequencies of application
of these checks are 5 percent or one per batch, whichever is more frequent.
The control limits are +20 percent relative percent difference for duplicates,
75-125 percent recovery for spikes, and 80-120 percent recovery for the
analysis of standard reference materials (SRMs). For the purpose of QA/QC,
the contract required detection limits (CRDLs) shall be those listed in
Table 6 of Appendix A. For batches of five samples or less, the minimum
QC checks should be a preparation blank and the analysis of an SRM. If
an analyte is not in the SRM, a matrix spike must be analyzed for that
particular analyte.
In general for small batches (i.e., analytical duplicates > matrix spikes. If
several small batches of the same matrix are analyzed sequentially (i.e.,
for several small projects), an SRM can be analyzed at a frequency of 5 percent
overall, with at least one sample duplicate analyzed per individual batch.
If any QA/QC check does not meet the established criteria, the laboratory
QA officer should notify the project QA coordinator and the methods should
be adjusted or, if necessary, data qualifications and reasons for noncompliance
to criteria should be submitted with the analytical data report.
QA/QC MEASURES INITIATED IN THE FIELD
In addition to the QA/QC checks listed above, the following four checks
may be initiated at the time of sample collection (Plumb 1981). These
checks may not replace any of the QA/QC measures outlined above but may
be included as part of the overall QA/QC program.
21
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Metals
QA/QC
August 1986
Transfer (Preservation) Blanks
Reagents can become contaminated after a period of use in the field.
The transfer blank will shown this and any contamination introduced during
shipping.
A sample container is filled with distilled water to the same volume
as that for samples and preserved as if it were a normal water or sediment
sample. This blank is then sent to the laboratory for analysis.
Cross-Contamination Blanks
Field equipment can exhibit carryover from one sample to the next
if not thoroughly cleaned between sampling. The cross-contamination blank
is designed to verify the absence of carryover.
Decontaminated sample-handling equipment (spatulas, augers, core barrels)
is wiped with a clean lab tissue, which is then placed in a scalable container.
Alternatively, the equipment is rinsed with distilled/deionized water,
and the water is collected and preserved as if it were a normal water sample.
Blind Replicate Samples
A collected sample is homogenized and split in the field into at least
three identical aliquots, and each aliquot is treated and identified as
a separate sample. The replicates are sent blind to the laboratory. The
mean, standard deviation, and relative percent standard deviation are calculated
by the project QA coordinator.
In addition, a collected sample may be split in the field into two
aliquots, and one aliquot sent for analysis to a different or "reference"
laboratory. The relative percent difference is calculated by the project
QA coordinator. If project constraints require the use of more than one
laboratory, their comparability must be established using certified reference
materials.
Blind Standard Reference Materials
A standard reference material is placed in a sample container at the
time of sample collection and sent blind to the laboratory. The percent
recovery is calculated by the project QA coordinator.
It is recommended that the same control limits be established as for
the laboratory QA/QC checks. The project QA coordinator must inform the
laboratory if the control limits are exceeded and corrective actions must
be taken.
22
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Metals
QA/QC
August 1986
CORRECTIVE ACTIONS
If the concentration of the field or laboratory blank is greater than
the required detection limit, all steps in the sample handling should be
reviewed. Many trace metal contamination problems are due to airborne
dust. Keeping containers closed, and rinsing all handling equipment immediately
before use minimizes dust problems. In the field, mercury-filled thermometers
are a potential source of severe mercury contamination. In the laboratory,
samples for mercury analysis should be isolated from items such as polarographs
or COD reagents.
Poor replication may be caused by inadequate mixing of the sample
before taking aliquots, inconsistent contamination, inconsistent digestion
procedures, or instrumentation problems. Instrumentation problems may
be isolated by recalibration and calibration blank analysis. Inconsistent
digestion may be due to analyte loss; see below for corrective action.
Also, hotplates may not hold a constant temperature across their surfaces.
This can be remedied by changing the position of digestion vessels at regular
intervals during heating.
Poor performance on the analysis of the Standard Reference Material
(SRM) or poor spike recovery may be caused for the same reasons as poor
replication. However, if replicate results are acceptable, poor SRM performance
or poor spike recovery may be caused by loss of analyte during digestion.
To check for analyte loss during digestion and for low recovery due to
interferences during analysis, spike the sample after digestion and compare
the analysis to the predigestion spike. If the results are different,
the digestion technique should be adjusted. If the results are the same,
dilute the sample by at least a factor of five and reanalyze. If spike
recovery is still poor, standard additions, matrix modifiers, or another
method is required.
23
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Metals
Data Reporting
August 1986
DATA REPORTING
Concentrations of elements from sediment samples should be reported
on a dry weight basis. Those from tissue samples should be reported on
a wet weight basis along with the percent moisture content (wet/dry ratio)
of the tissue. If the tissue sample is too small to do both a metals analysis
and a moisture determination, omit the latter.
DATA REPORT PACKAGE
The data report package for analyses of each sample should include:
• Tabulated results in units as specified for each matrix
in the analytical protocols (Appendices B, D, E, and G),
validated and signed in original by the laboratory manager
0 Any data qualifications and explanation for any variance
from the analytical protocols
• Results for all of the QA/QC checks initiated by the labora-
tory
• Tabulation of instrument and method detection limits.
BACKUP DOCUMENTATION
All contract laboratories are required to submit results that are
supported by sufficient backup data and quality assurance results to enable
independent QA reviewers to conclusively determine the quality of the data.
The laboratories should be able to supply legible photocopies of original
data sheets with sufficient information to unequivocally identify:
t Calibration results
• Calibration and preparation blanks
0 Samples and dilutions
• Duplicates and spikes
• Any anomalies in instrument performance or unusual instrumental
adjustments.
24
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Metals
References
August 1986
REFERENCES
American Society for Testing and Materials. 1983. Annual book of ASTM
standards. Water and Environmental Technology. Vol. 11.01. ASTM, Phila-
delphia, PA. 752 pp.
Bates, T.S., S.E. Hamilton, and J.D. Cline. 1983. Collection of suspended
particulate matter for hydrocarbon analyses: Continuous flow centrifugation
vs. filtration. Estuarine Coast. Shelf Sci. 16:107.
Donard, O.F.X., and J.H. Weber. 1985. Behavior of methyltin compounds
under simulated estuarine conditions. Environ. Sci. Technol. 19:1104-1110.
Greenberg, A.E., R. Trussel, and L.S. Clersceri (eds). 1985. Standard
methods for examination of waste and wastewater. American Public Health
Association, Washington, DC. 1268 pp.
Hiatt, M.H., and T.L. Jones. 1984. Isolation of purgeable organics from
solid matrices by vacuum distillation. U.S. Environmental Protection Agency,
Las Vegas Laboratory, Las Vegas, NV.
Hinderberger, E.J., M.L. Kaiser, and S.R. Koirtyohann. 1981. Furnace
atomic absorption analysis of biological samples using the L'Vov platform
and matrix modification. Atomic Spec. 2:1-7.
Horowitz, A.J. 1986. Comparison of methods for the concentration of suspended
sediment in river water for subsequent chemical analysis. Environ. Sci. &
Technol. 20:155-160.
Manning, D.C., and W. Slavin. 1978. Determinatin of lead in a chloride
matrix with the graphite furance. Anal. Chem. 50:1234-1238.
Manning, D.C., and W. Slavin. 1983. The determination of trace elements
in natural waters using the stabilized temperature platform furnace. Applied
Spec. 37:1-11.
Mart, L. 1979a. Prevention of contamination and other accuracy risks
in voltamnetric trace metal analysis of natural waters. Part I: Preparatory
steps, filtration, and storage of water samples. Fresenius Z. Anal. Chem.
296:350-357.
Mart, L. 1979b. Prevention of contamination and other accuracy risks
in volammetric trace metal analysis of natural waters. Part II: Collection
of surface water samples. Fresenius Z. Anal. Chem. 299:97-102.
25
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Metals
References
August 1986
Murphy, T.J. 1976. The role of the analytical blank in accurate trace
analysis, pp. 551-539. In: Accuracy in Trace Analysis: Sampling, Sample
Handling, and Analysis. National Bureau of Standards Special Publication
422. NBS, Washington, DC.
Plumb, R.H., Jr. 1981. Procedure for handling and chemical analysis of
sediment and water samples. Technical Report EPA/CE-81-1. U.S. EPA and
Corps of Engineers, U.S. Army Engineers Waterways Experiment Station, Vicksburg,
MS.
Rantala, R.T.T., and D.H. Loring. 1975. Multi-element analysis of silicate
rocks and marine sediments by atomic absorption spectrophotometry. Atomic
Abs. Newsletter. 14(5):117-120.
Skoog, D.A. 1985. Principles of instrumental analysis. Third edition.
Saunders, Philadelphia, PA. pp. 270-277, 282-284, 303-304.
Slavin, W., G.R. Carnrick, and D.C. Manning. 1984. Chloride interferences
in graphite furnace atomic absorption spectrometry. Anal. Chem. 56:163-168.
Stephenson, M.D., M. Martin, S.E. Lange, A.R. Flegal, and J.H. Martin.
1979. California mussel watch 1977-1978. Volume II: Trace metals concen-
trations in the California mussel, Mytilus californianus. SWRCB Water
Quality Monitoring Report No. 79-22. Sacramento, CA. 110 pp.
Tetra Tech. 1985. Commencement Bay nearshore/tideflats remedial investiga-
tion. Final Report (Volume I). Prepared for Washington Department of
Ecology and U.S. EPA. Tetra Tech, Inc., Bellevue, WA.
Tetra Tech. 1986a. Bioaccumulation monitoring guidance: 4. analytical
methods for EPA priority pollutants and 301(h) pesticides in tissues from
estuarine and marine organisms. Final report prepared for the U.S. EPA
by Tetra Tech, Inc., Bellevue, WA.
Tetra Tech. 1986b. Protocols for collection of particulate samples
organic and inorganic analysis. Tetra Tech, Inc., Bellevue, WA.
for
U.S. Environmental Protection Agency. 1977 (revised October, 1980). Interim
methods for the sampling and analysis of priority pollutants in sediments and
fish tissue. Environmental Monitoring and Support Laboratory, Cincinnati, OH.
U.S. Environmental Protection Agency. 1978 (revised 1983). NEIC Policies
and procedures. EPA-330/9-78-001-R. National Enforcement Investigations
Center, Denver, CO.
U.S. Environmental Protection Agency. 1979a (revised March, 1983). Methods
for chemical analysis of water and wastes. EPA-600/4-79-020. Environmental
Monitoring and Support Laboratory, Cincinnati, OH.
26
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Metals
References
August 1986
U.S. Environmental Protection Agency. 1979b. Handbook for analytical
quality control in water and wastewater laboratories. EPA-600/4-79-019.
National Environmental Research Center, Cincinnati, OH.
U.S. Environmental Protection Agency. 1982. Handbook for sampling and
sample preservation of water and wastewater. EPA-600-82-029. Environmental
Monitoring and Support Laboratory, Cincinnati, OH. 402 pp.
U.S. Environmental Protection Agency. 1984. Guidelines establishing test
procedures for the analysis of pollutants. U.S. EPA, Washington, DC.
Federal Register, Vol. 49, No. 209, pp. 43234-43436.
U.S. Environmental Protection Agency. 1985. Contract laboratory program
statement of work (SOW), inorganic analysis, multi-media, multi-concentration.
SOW No. 785. U.S. EPA, Washington, DC.
27
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APPENDIX A
FINAL REPORT
TASK A-l
SELECTION OF METALS FOR PROTOCOL DEVELOPMENT
-------
CONTENTS
Page
LIST OF TABLES A-1ii
TASK A-l: SELECTION OF METALS FOR PROTOCOL DEVELOPMENT A-l
INTRODUCTION A"1
EVALUATION OF U.S. EPA PRIORITY POLLUTANT METALS A-2
METALS SPECIATION A~5
EVALUATION OF NON-PRIORITY POLLUTANT METALS A-5
RECOMMENDED LIMITS OF DETECTION A-6
SUMMARY A'14
REFERENCES A~15
A-li
-------
TABLES
Number Page
1 Metals of concern as indicated by Puget Sound field studies A-3
2 Limits of detection for water, sediment, and tissue
matrices by instrument A-9
3 U.S. EPA Water Quality Criteria A-10
4 Summary of metal concentrations in sediments from Puget Sound
reference areas A-ll
5 Minimum and maximum metal detection limits reported for
tissue samples A-12
6 Recommended limits of detection for water, sediment,
and tissue matrices A-13
A-iii
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TASK A-l
SELECTION OF METALS FOR PROTOCOL DEVELOPMENT
INTRODUCTION
Recent studies have documented high concentrations of a number of
U.S. EPA priority pollutant metals and metalloids in the sediments of
industrialized embayments in Puget Sound relative to reference areas.
To ensure the acceptability of dredging and dredged material disposal,
federal and state agencies must be able to accurately and routinely analyze
for these substances in Mater, sediment, and biological samples. Because
of the number of agencies involved and different program objectives, a
standardized protocol is necessary to ensure comparability of results.
It is the objective of Task A to develop this protocol.
A consensus approach involving federal, state, and local institutions
will be used to develop both standardized chemical tests and standardized
test interpretation guidelines for use by all Puget Sound regulatory agencies
concerned with dredged materials. Protocols developed for each metal (or
group of metals that can be analyzed simultaneously) will include guidance
for field sampling, analytical procedures, and quality control requirements.
When possible, existing procedures are to be recommended, and modified
as appropriate. Draft protocols will be reviewed by a workshop of experts
to reach a consensus on the recommended requirements. After incorporation
of the workshop comments, a final protocol report will be drafted for final
review.
The purpose of Task A-l is to:
• Recommend appropriate metals and metalloids for which analytical
protocols will be proposed under the Puget Sound Dredged
Disposal Analysis (PSDDA) program
A-l
-------
• Recommend limits of detection (LOD) for the analysis of
each element in water, sediment, and tissue samples.
The thirteen U.S. EPA priority pollutant metals include three elements
(antimony, arsenic, and selenium) that are classified as metalloids, which
are elements that do not strictly occur as metals in the environment.
Following U.S. EPA convention and for ease of discussion, all of these
elements will be referred to as metals.
EVALUATION OF U.S. EPA PRIORITY POLLUTANT METALS
Protocols were requested by PSDDA for all metals either documented
or likely to be of concern in Puget Sound. In addition, justification
was required for the exclusion of any U.S. EPA priority pollutant metal.
Reports for four recent field investigations in Puget Sound were reviewed
during the selection process (Malins et al. 1980, 1982; Galvin et al. 1984;
Battelle 1985; Tetra Tech 1985a). Historical data from previous investigations
summarized by Dexter et al. (1981) and recommendations for metals of concern
proposed in several summary reports of contamination in Puget Sound were
also reviewed (Konasewich et al. 1982; Jones and Stokes 1983; Quinlan et
al. 1985).
The thirteen U.S. EPA priority pollutant metals and data reports providing
evidence of their potential concern in Puget Sound are listed in Table 1.
Criteria typically used to determine if a metal is of concern include high
toxicity (measured in laboratory studies), high concentration relative
to sediments from reference areas located away from industrialized areas
of Puget Sound, and widespread distribution in Puget Sound. While all U.S. EPA
priority pollutant metals are considered toxic, metals identified to be
of concern have been found in high concentration only in limited areas
of the Sound (e.g., antimony, arsenic, cadmium, copper, lead, mercury,
and zinc at the Fourmile Rock disposal site relative to the rest of central
Puget Sound; Galvin et al. 1984).
A-2
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TABLE 1. METALS OF CONCERN AS INDICATED BY PUGET SOUND FIELD STUDIES
U.S. EPA
Priority Dexter Mai ins Galvin
Pollutant et. al . et. al . Battelle et. al.
Metal 1981* 1982 1985 1984
Antimony (Sb) x x
Arsenic (As) x x x x
Beryllium (Be)
Cadmium (Cd) x x
Chromium (Cr)
Copper (Cu) x x x x
Lead (Pb) x x x x
Mercury (Hg) x x x x
Nickel (Ni)
Selenium (Se) x
Silver (Ag) x x
Thallium (Tl)
Zinc (Zn) x x x x
Tetra
Tech
1985a
x
x
X
X
X
X
X
X
X
Frequency
of Concern
(Out of 5)
3
5
0
3
0
5
5
5
1
1
3
0
5
* Secondary source (summarizes pre-1980 historical data).
A-3
-------
Two areas of consensus are indicated by Table 1. First, three metals
(beryllium, chromium, and thallium) were not identified by any report as
of concern. Although potentially toxic, these metals are not present in
Puget Sound sediments in concentrations that exceed natural levels. These
three metals are not recommended for protocol development. Second, five
metals (arsenic, copper, lead, mercury, and zinc) were identified in all
reports as of concern because of their high concentration in urban embayments.
A potential association with adverse biological effects was also noted
in some reports. These five metals are recommended for protocol development.
Three additional metals (silver, cadmium, and antimony) were found
in elevated concentration by a majority of the investigators. Of these
metals, silver was found in high concentrations at relatively few sites
(Malins et al. 1982; Battelle 1985; Tetra Tech 1985a). Silver is extremely
toxic even at low concentrations, it is a potentially useful tracer of
sewage effluent, and historically its concentration in Puget Sound sediments
has increased faster than that of other metal pollutants (Crecelius, E.,
22 October 1985, personal communication). Because of these factors, silver
has been identified as a metal of concern. Cadmium is also highly toxic
and is recommended for protocol development. Although antimony is less
toxic, its high concentration in selected areas (e.g., sediments contaminated
with copper smelter slag) warrants its inclusion.
Selenium was reported in high concentration in only one investigation
(Malins et al. 1982). Several summary reports subsequently used these
data to justify including selenium as a metal of concern (Konasewich et
al. 1982; Jones and Stokes 1983; Quinlan et al. 1985). The instrumental
technique used to measure selenium in the investigation (ion-coupled plasma
emission spectroscopy) is subject to substantial spectral interferences
(Parson et al. 1980). All other investigations using alternative techniques
(e.g., graphite furnace atomic adsorption) have reported uniformly low
or undetected concentrations of selenium. Therefore, selenium is not recom-
mended for protocol development.
In Commencement Bay, nickel was found in somewhat elevated concentrations
at a few sites exhibiting biological effects (Tetra Tech 1985a). Although
A-4
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the available data suggest that nickel primarily derives from natural sources,
there are insufficient data to dismiss nickel as a potential metal of concern
in those few areas. Therefore, nickel is recommended for protocol development.
METALS SPECIAT ION
The toxicity of metals is highly dependent upon their chemical form
(speciation). For example, inorganic arsenic in the +3 oxidation state
is much more toxic than in the +5 oxidation state. Inorganic arsenic is
toxic if ingested by humans, yet organic arsenic compounds are relatively
nontoxic to mammals. In contrast, mercury salts are relatively nontoxic
because of low solubilities, but methylated mercury compounds are highly
toxic. Hence, quantitative determinations of different metal species could
seriously affect interpretation of dredged material test results.
Recent studies (e.g., Crecelius et al. 1984; U.S. EPA 1980) have shown
that arsenic and mercury exist predominantly as methylated compounds in
marine biota. Dominance by one chemical form reduces the need for quantitative
speciation studies as routine measurements. However, in some cases (e.g.,
health risk assessment) a precise determination of metals species in different
bioaccumulation studies may be necessary.
In the water column, total concentrations of arsenic and mercury are
low because of dilution, and in sediments, matrix effects confound speciation
studies. Speciation techniques in general are difficult, time consuming,
and nonroutine (Crecelius et al. 1984). Because of the apparent small
incremental gain in knowledge from speciation analyses in biota for all
but a few applications, routine analyses do not appear to be cost effective.
Development of procedures for speciation analyses to be used in special
circumstances will require a consensus from a workshop of experts, to be
held as part of Task A.
EVALUATION OF NON-PRIORITY POLLUTANT METALS
None of the reports reviewed included a metal of concern that was
not already on the U.S. EPA priority pollutant list. However, selected
A-5
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non-priority pollutant metals could be of use in studies of dredged material.
Their value in determining sediment quality values is being investigated
as part of a separate task for PSDDA and the Puget Sound Estuarine Program.
Chemical data can be normalized to account for physical and physicochemical
differences among samples. These differences may mask trends in chemical
concentrations that are useful in interpreting environmental data. Metals
from pollution sources tend to be selectively enriched within fine-grained
sediment fractions having a high content of hydrous manganese or iron oxides.
The incorporation of metals in these oxides may also affect their toxicity.
Therefore, normalization of metal concentrations to the iron or manganese
content of sediments may be useful for determining sediment quality values.
It may also help account for the dilution of contaminated sediments by
materials from unrelated sources during transport and deposition.
Manganese and iron are recommended for inclusion in the metals protocol.
RECOMMENDED LIMITS OF DETECTION
Environmental analytical chemists have not universally agreed upon
a convention for determining and reporting the lower detection limits of
analytical procedures. Furthermore, the basis for detection limits reported
in the literature is rarely given. Values reported as lower detection
limits are commonly based on instrumental sensitivity, levels of blank
contamination, and/or matrix interferences and have various levels of statis-
tical significance. The American Chemical Society's Committee on Environmental
Improvement (CEI) defined the following types of detection limits in an
effort to standardize the reporting procedures of environmental laboratories
(Keith et al. 1983):
• Instrument Detection Limit (IDL) — the smallest signal
above background noise that an instrument can detect reliably.
• Limit of Detection (LOD) — the lowest concentration level
that can be determined to be statistically different from
A-6
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the blank. The recommended value for LOD is 3o, where o
is the standard deviation of the blank in replicate analyses.
• Limit of Quantitation (LOQ) — the level above which quantitative
results may be obtained with a specified degree of confidence.
The recommended value for LOQ is 10o, where o is the standard
deviation of blanks in replicate analyses.
• Method Detection Limit (MDL) — the minimum concentration
of a substance that can be identified, measured, and reported
with 99 percent confidence that the analyte concentration
is greater than zero. The MDL is determined from seven
replicate analyses of a sample of a given matrix containing
the analyte (Glaser et al. 1981).
The CEI recommended that results below 3o should be reported as "not detected"
(ND) and that the detection limit (or LOD) be given in parentheses. In
addition, if the results are near the detection limit (3o to 10a, which
is the "region of less-certain quantitation"), the results should be reported
as detections with the limit of detection given in parentheses.
The CEI definitions are useful for establishing a conceptual framework
for detection limits, but are somewhat limited in a practical sense. The
IDL does not address possible blank contaminants or matrix interferences
and is not a good standard for complex environmental matrices, such as
tissues. The LOD and LOQ account for blank contamination, but not for
matrix complexity and interferences. The high 10o level specified for
LOQ helps to preclude false positive findings, but may also necessitate
the rejection of valid data. The MDL is the only operationally defined
detection limit and provides a high statistical confidence level but, like
the LOQ, may be too stringent and necessitate the rejection of valid data.
The detection limits recommended in this report are not strictly based
on the CEI definitions. Instead, they are considered to be typically attainable
values based on the best professional judgment and experience of analytical
chemists who considered the instrumental sensitivity of affordable equipment,
A-7
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common problems with blank contamination and matrix interferences, and
reasonable levels of laboratory analytical effort. The recommended values
are not absolute, as analytical procedures and laboratory precision can
affect attainable detection levels. The detection limits recommended herein
fall between the IDL and MDL as defined by the CEI.
The LOD for metals selected for protocol development must be specified
low enough so that data from the analysis of a particular matrix (i.e.,
water, sediment, or biota) will be comparable and of acceptable quality.
LOD reported for the recommended metals are summarized in Table 2 for each
matrix type analyzed by a variety of instrumental techniques: direct flame
AA (DFAA), graphite furnace AA (GFAA), cold vapor AA (CVAA), and ion-coupled
plasma emission spectroscopy (ICP).
Several factors affect achievable detection limits regardless of the
analytical procedure. These include the available sample size, presence
of interfering substances, range of pollutants to be analyzed, and level
of pollutant introduced as a contaminant during sampling or analysis.
LOD given in Table 2 are provided as examples of typical attainable levels.
These limits are not intrinsically constant for a particular method, but
also depend on the precision attainable by an individual laboratory. Each
laboratory must evaluate its own precision and thus establish its own detection
limits. Large variations from the LOD in Table 2 may occur depending on
the analytical instrumentation and technique and on individual sample charac-
teristics.
Recommendations of LOD must be based, in part, on the analytical require-
ments to quantify concentrations found in relatively "clean" reference
areas, and to approach water criteria levels. Table 3 lists the U.S. EPA
water quality criteria. The range of concentrations of the recommended
metals in sediments from Puget Sound reference areas is shown in Table 4.
The range of detection limits reported for these metals in marine tissue
samples is shown in Table 5. Recommended LOD are summarized in Table 6.
The recommended LOD for sediments and tissues were selected by considering
attainable LOD for different instruments (Table 2), U.S. EPA water quality
criteria (Table 3), and reported environmental levels (Tables 4 and 5).
A-8
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TABLE 2. LIMITS OF DETECTION FOR WATER, SEDIMENT,
AND TISSUE MATRICES BY INSTRUMENT
Antimony
Arsenic
Cadmium
Copper
Iron
Lead
Mercury
Manganese
Nickel
Silver
Zinc
(Sb)
(As)
(Cd)
(Cu)
(Fe)
(Pb)
(Hg)
(Mn)
(Ni)
(Ag)
(Zn)
ICP
0.032
0.053
0.004
0.006
NAd
0.042
0
NA
0.015
0.002
0.007
Water3
DFAA
0
0
0
0
.2
—
.005
.02
NA
.1
GFAA
0.
0.
0.
0.
0.
003
001
0001
001
NA
001
.0002 (CVAA)
NA
0.04
0
0
.01
.005
0.
0.
0.
NA
001
0002
00005
Sedimentb
ICP GFAA
3.2
—
4.0
0.6
0.7
4.2
0.01
2.0
1.5
0.7
0.2
0.1
0.1
0.1
0.1
—
0.1
(CVAA)
—
0.1
0.1
0.2
ICP
10
3
0.4
0.6
NA
4
0
NA
1
0.7
0.2
Tissue0
DFAA GFAA
—
—
0.1
0.1
NA
1.0
0.02
0.02
0.01
0.01
NA
0.03
.01 (CVAA)
NA
0.5
0.1
0.1
NA
0.02
0.01
0.2
a DFAA and GFAA data from U.S. EPA 1979; ICP data from U.S. EPA 1984.
Values are mg/L.
b ICP data from Tetra Tech 1984; GFAA and CVAA data are detection limits
that can be reasonably attained by various laboratories. Under strict
conditions these limits can be lowered (e.g., Battelle 1985). Values are
mg/kg dry weight for 5 g (wet) sediment in a 100 mL digest.
c Tetra Tech 1985b. Values are ug/g wet weight for 5 g tissue in a 50 mL
digest.
d NA = Not applicable; iron and manganese used as natural tracers for sediments
only.
A-9
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TABLE 3. U.S. EPA WATER QUALITY CRITERIAa (ug/L)
Metal
Antimony
Arsenic
Cadmium
Copper
Lead
Mercury
Nickel
Silver
Zinc
EPA UQC
—
36
9.3
2.9
5.6
0.025
7.1
2.3
58
a U.S. EPA 1980a and U.S. EPA 1985.
A-10
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TABLE 4. SUMMARY OF METAL CONCENTRATIONS IN SEDIMENTS
FROM PUGET SOUND REFERENCE AREAS
Range
(mg/kg dry wt)
Antimony
Arsenic
Cadmium
Copper
Lead
Mercury
Nickel
Silver
Zinc
U O.lb
1.9 -
0.1 -
5 -
U 0.1
0.01 -
4 -
0.02 -
15 -
- 1.7
17
1.9
74
- 24
0.28
47
3.3
100
Mean
(mg/kg dry wt)
0.32C - 0.38d
7.2
0.67
32
9.8C - 9.8d
0.08
28
1.2
62
Detection
Frequency
12/32
34/34
24/24
28/28
21/28
38/38
26/26
26/26
26/26
Reference
Sites*
1,2,3,4,7,8,9
1,2,3,4,7,8,9
1,2,3,4,6,9
1,2,3,4,5,6,9
1,2,3,4,5,6,9
1-9
1,2,3,4,5,9
1,2.3,4,5,9
1,2,3,4,5,9
a Reference sites: 1. Carr Inlet 4. Case Inlet 7. Nisqually Delta
2. Samish Bay 5. Port Madison 8. Hood Canal
3. Dabob Bay 6. Port Susan 9. Sequim Bay
b U: Undetected at the limit of detection shown.
c Mean calculated using 0.00 for undetected values.
d Mean calculated using the reported detection limit for undetected values.
References:
(Site 1) Tetra Tech 1985a; Crecelius et al. (1975).
(Site 2) Battelle (1985).
(Site 3) Battelle (1985).
(Site 4) Crecelius et al. (1975); Mai ins et al. (1980).
(Site 5) Mai ins et al. (1980).
(Site 6) Mai ins et al. (1981).
(Site 7) Crecelius et al. (1975).
(Site 8) Crecelius et al. (1975).
(Site 9) Battelle (1985).
A-ll
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TABLE 5. MINIMUM AND MAXIMUM METAL DETECTION LIMITS REPORTED
FOR TISSUE SAMPLES (FROM TETRA TECH 1985b)
Element
Antimony
Arsenic
Cadmium
Copper
Lead
Mercury
Nickel
Silver
Zinc
Detection Limit
(ug/g-wet weight)
Minimum Maximum
0.01 1.0
Always detected
(minimum = 0.72)
0.001 0.75
Always detected
(minimum = 0.052)
0.030 1.6
0.0004 0.09
0.019 1.0
0.001 0.27
Always detected
(minimum = 1.42)
A-12
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TABLE 6. RECOMMENDED LIMITS OF DETECTION
FOR WATER, SEDIMENT, AND TISSUE MATRICES
Antimony
Arsenic
Cadmium
Copper
Iron
Lead
Mercury
Manganese
Nickel
Silver
Zinc
Water9
3
1
0.1
1
NAd
1
0.26
NA
1
0.2
1
Sedimentb
0.1
0.1
0.1
0.1
0.7
0.1
0.01
2.0
0.1
0.1
0.2
Tissue0
0.02
0.02
0.01
0.01
NA
0.03
0.01
NA
0.02
0.01
0.2
a ug/L.
D mg/kg dry weight.
c ug/g wet weight.
d NA = Not applicable; iron and manganese
used as natural tracers for sediments only.
e This detection limit can be lowered to
0.02 ug/L by using a special, gold-amalgamation-
cold-vapor technique (Bloom and Crecelius
1983). However, accuracy and precision must
be demonstrated to the levels specified in
the QA/QC section of the main body of this
report.
A-13
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Recommended LOD for water are for graphite furnace analysis, except for
cold vapor atomic adsorption for mercury.
SUMMARY
U.S. EPA priority pollutant metals recommended for inclusion in protocol
development include antimony, arsenic, cadmium, copper, lead, mercury,
nickel, silver, and zinc. U.S. EPA priority pollutant metals excluded
include beryllium, chromium, selenium, and thallium. Speciation protocols
may be useful for selected bioaccumulation studies (i.e., arsenic and mercury),
but are not recommended for routine monitoring, or for analysis of waters
or sediments. Iron and manganese are recommended for inclusion in the
protocol specification because of their potential use as geochemical tracers
and as potentially important factors in the generation of sediment quality
values.
The list of metals recommended for the development of sediment quality
values (SQV) in Puget Sound (Tetra Tech 1985c) includes all 13 priority
pollutant metals. These were presented only as potential candidates for
metals of concern. The purpose of the SQV report in this regard was to
provide guidance for selecting contaminants of concern rather than to actually
select the contaminants. Thus, the SQV list contains some metals specifically
excluded in this report.
Limits of detection are recommended for the U.S. EPA priority pollutant
metals after a consideration of attainable detection limits for each matrix
type and expected levels in the environment (sediments and tissues). Based
on this comparison, maximum detection limits (often meaning lower cost)
are recommended that would still be sufficient for characterizing each
matrix in a dredged materials testing program. Detection limits for speciation
analyses (e.g., inorganic arsenic or methylmercury in tissues) are roughly
similar to those attainable for the total metal analysis.
A-14
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REFERENCES
Battelle Northwest. 1985. Detailed chemical and biological analyses of
selected sediments from Puget Sound. Volume 1. Prepared for U.S. Environmental
Protection Agency, Region X. Battelle Marine Research Laboratory, Sequim, WA.
300 pp.
Bloom, N.S., and E.A. Crecelius. 1983. Determination of mercury in seawater
at sub-nanogram per liter levels. Marine Chem. 14:49-59.
Crecelius, E. 22 October 1985. Personal Communication (phone by Dr. Charles
R. Lytle). Battelle Marine Research Laboratory, Sequim, WA.
Crecelius, E.A., and C.W. Apts. 1984. Concentration and speciation of
arsenic in flatfish and crabs collected from Commencement Bay. Tacoma-Pierce
County Health Department, Tacoma, WA. 14 pp.
Dexter, R.N., D.E. Anderson, E.A. Quinlan, L.S. Goldstein, R.M. Strickland,
S.P. Pavlov, J.R. Clayton, Jr. R.M. Kocan, and M. Landolt. 1981. A summary
of knowledge of Puget Sound related to chemical contaminants. NOAA Technical
Memorandum OMPA-13. National Oceanic and Atmospheric Administration, Boulder,
CO. 435 pp.
Galvin, D.V., G.P. Romberg, D.R. Houck, and J.H. Lesniak. 1984. Toxicant
pretreatment planning study. Summary Report. Municipality of Metropolitan
Seattle, Seattle, WA. 202 pp.
Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde. 1981.
Trace analyses for wastewaters. Environ. Sci. Technol. 15:1426-1435.
Jones & Stokes. 1983. Water quality management program for Puget Sound.
Prepared for U.S. Environmental Protection Agency, Region X. Jones & Stokes
Associates, Inc. Sacramento, CA.
Keith, L.J., W. Crummet, J. Deegan, Jr., R.A. Libby, J.K. Taylor, G. Weatler.
1983. Principles of environmental analysis. Anal. Chem. 55:2210-2218.
Konasewich, D.E., P.M. Chapman, E. Gerencher, G. Vigers and N. Treloar.
1982. Effects, pathways, processes, and transformation of Puget sound
contaminants of concern. NOAA Technical Memorandum OMPA-20. National
Oceanic and Atmospheric Administration, Boulder, CO. 357 pp.
Malins, D.C., B.B. McCain, D.W. Brown, A.K. Sparks, and H.O. Hodgins.
1980. Chemical contaminants and biological abnormalities in central and
southern Puget Sound. NOAA Technical Memorandum OMPA-2. National Oceanic
and Atmospheric Administration, Boulder, CO. 295 pp.
A-15
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Malins, D.C., S.L. Chan, B.B. McCain, D.W. Brown, A.K. Sparks, and H.O.
Hodgins. 1981. Puget Sound pollution and its effects on marine biota.
Progress report to OMPA for the period May 1 to September 30, 1980. National
Marine Fisheries Service, Seattle, WA.
Malins, D.C., B.B. McCain, D.W. Brown, A.K. Sparks, and H.O. Hodgins.
1982. Chemical contaminants and abnormalities in fish and invertebrates
from Puget sound. NOAA Technical Memorandum OMPA-19. National Oceanic
and Atmospheric Administration, Boulder, CO. 168 pp.
Parson, M.L., A. Forster, and D. Anderson. 1980. Atlas of spectral inter-
ferences in ICP spectroscopy. Plenum Press, New York, NY. 644 pp.
Quinlan, E.A., P.M. Chapman, R.N. Dexter, D.E. Konasewich, C.C. Ebbesmeyer,
G.A. Erickson, B.R. Kowalski, and T.A. Silver. 1985. Toxic chemicals
and biological effects in Puget Sound: states and scenarios for the future.
Draft. NOAA Technical Memorandum. National Oceanic and Atmospheric Admini-
stration, Boulder, CO. 334 pp.
Tetra Tech. 1984. Laboratory analytical protocol for the Anaconda smelter
RI/FS. Tetra Tech, Bellevue, WA.
Tetra Tech. 1985a. Commencement Bay nearshore/tideflats remedial investi-
gation. Volume 1. Prepared for Washington State Department of Ecology
and U.S. Environmental Protection Agency, Region X. Tetra Tech, Inc.,
Bellevue, WA.
Tetra Tech. 1985b. Bioaccumulation monitoring guidance: 3. Recommended
analytical detection limits. Prepared for U.S. Environmental Protection
Agency, Washington, DC. Tetra Tech, Inc., Bellevue, WA.
Tetra Tech. 1985c. Task 3. Evaluation of approaches for the development
of sediment quality values for Puget Sound. Draft report prepared for
Resource Planning Associates for the U.S. Army Corps of Engineers by Tetra
Tech, Bellevue, WA.
U.S. Environmental Protection Agency. 1979. Methods for chemical analysis
of water and wastes. U.S. Environmental Protection Agency Environmental
Monitoring and Support Laboratory, Cincinnati, OH.
U.S. Environmental Protection Agency. 1980a. Water quality criteria.
U.S. Environmental Protection Agency, Washington, DC. Federal Register,
Vol. 46, No. 156, Part IV. pp. 79318-79379.
U.S. Environmental Protection Agency. 1980b. Ambient water quality criterion
for mercury. U.S. Environmental Protection Agency, Washington, DC.
U.S. Environmental Protection Agency. 1984. Guidelines establishing test
procedures for the analysis of pollutants under the clean water act; final
rule and interim final rule and proposed rule. U.S. Environmental Protection
Agency, Washington, DC. Federal Register, Vol. 49, No. 209, Part VII.
pp. 199-204.
A-16
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U.S. Environmental Protection Agency. 1985. Water quality criteria.
U.S. Environmental Protection Agency, Washington, DC. Federal Register,
Vol. 50, No. 146, Part II. pp. 30784-30796.
A-17
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APPENDIX B
EXCERPTS FROM
EXHIBIT B: REPORTING REQUIREMENTS AND DELIVERABLES
EXHIBIT D: ANALYTICAL METHODS
EXHIBIT E: QUALITY ASSURANCE/QUALITY CONTROL
EXHIBIT G: CHAIN-OF-CUSTODY AND DOCUMENT CONTROL PROCEDURES
(U.S. EPA 1985)
-------
CONTRACT LABORATORY PROGRAM
STATEMENT OF WORK
(SOW)
INORGANIC ANALYSIS
Multi-Media
Mulci-Concentracion
SOW No. 785
July 1985
Based on: Caucus Inorganics Prococol
B-l
-------
EXHIBIT B,
REPORTING REQUIREMENTS AND DELIVERABLES
B-2
-------
REPORTING REQUIREMENTS AND DELIVERABLES
The contractor shall provide reports and other deliverables specified hereunder.
Specific reports are described below. The Contract Reporting Schedule which
follows the report descriptions gives delivery schedule and report distribution
Information, Including addresses for report designees. All' reports to multiple
addresses must be sent out concurrently by whatever method necessary to meet the
contract required time limits. All reports must also be submitted in a legible
fora or resubmission will be required at no additional cost to the Agency.
Report Description
A* Weekly Progress Reports - Tabulation of samples received, date of
receipt, and a tabulation of problems encountered.
B. Sample Traffic Report (Original Lab Copy for Return to SMO) - Copy of
SHO Sample Traffic Report with lab receipt information and original
Contractor signature.
C. Sample Data Package - Data report package for analyses of each
sample (including all required QA/QC-Exhibit E) must be complete
before submission and shall Include:
1) Copies of completed SMO Sample Traffic Reports with receipt of
information completed for all samples reported in data package.
2} The cover page for the inorganic analysis data package (Exhibit
B) including general comments, Statement of Work (SOU) Number,
QC Report 0, sample EPA cross reference numbers, footnotes used
in the data package, and the statement on use of ICP background
and interelement corrections for the samples. The SOV number
defines the Statement of Work used to obtain the reported values.
3) Tabulated results in ug/L for aqueous samples or mg/kg for solid
samples (identification and quantity) of specified chemical
constituents (Exhibit C) by the specified analyses (Exhibit 0),
validated and signed in original signature by the Laboratory
Manager, and reported on Form I.* The results for solid samples
will be reported on a dry weight basis. Percent solids are not
required on aqueous samples* If the value or the result is greacer
than or equal to the Instrument Detection Limit (IDL), corrected for
dilutions, as determined in Exhibit E, report the value and Indicate
the analytical method used for the metals* Use P_ for ICP, A_ for
flame AA and J_ for furnace AA. All dilutions not* required by the
contract and Tffectlng the IDL, must be noted on an element by ele-
ment basis on Form I. If the value is less than the Contract Re-
quired Detection Limits (CRDL) in Exhibit C, put the value in
brackets, e.g. lib). If the element was analyzed for but not de-
tected, report the instrument detection limit value with a "IT
*In che event the Laboratory Manager cannot validate all data reported for
each sample, he/she will provide a detailed description of the problems
associated with the sample.
B-3
-------
(e.g.. LOU). Use an "E" as the footnote Co indicate an estimated
value or value noc reported due to the presence of interference,
and an explanatory note oust be included on the cover page. If
the duplicate sample analysis is not within the control limits,
flag it with an asterisk (*}. Use "S" as a footnote to indicate
a value determined by Method of Standard Additions (MSA). If the
correlation coefficient (r) for method of standard additions is
less than 0.995, flag the value with a '+' sign. If duplicate
injection precision criteria specified for Furnace AA analysis in
Exhibit E cannot be net, flag the data with an "M". If the spike
sample recovery is not within control limits, flag the data with
the letter N. Report results to two significant figures for values
from 0 to 100 and three significant figures for results greater
than 100, with the exception of Mercury (see Mercury Methods -
Exhibit D). For rounding rules, follow the EPA Handbook of
Analytical Quality Control in Water and Wastewater Laboratories
(EPA-600/4-79-019).
Under the comments section on Form I, provide a brief physical
description of the sample using the following guidelines:
A. Water samples - Coloration and clarity
B. Solid samples - Coloration, texture and artifacts
Recommended Descriptive Terms
Coloration; Red, blue, yellow, green, orange, violet, white,
colorless, brown, grey, black
Clarity: Clear, cloudy, opaque .
Texture; Fine (powdery), Medium (sand), Coarse (large crystals
or rocks)
Also note any significant changes that occur during sample prepara-
tion (i.e. coloration shifts, emulsion formation).
4) Analytical results for samples and spikes, duplicates, standards,
ICP Interference Check Samples, reagent blanks, laboratory control
samples, Instrument detection limits and holding times on-QA Forms
II, III, IV, V, VI, VII, VIII, IX and X. Multiple forms require
identification (i.e. Form IIA, Form IIB etc.) Summarize each full
method of standard addition performed on Form VIII.
5) Legible photocopy of raw data (sequential measurement readout
record) clearly labeled with sufficient information to unequivocally
Identify:
a) calibration standards (including prep date).
b) calibration blanks and preparation blanks.
B-4
-------
c) initial and continuing calibration verification standards and
Interference check samples.
d) diluted and undiluted samples (by EPA number) and all weights,
dilutions and volumes used to obtain the reported values. If
the volumes, weights and dilutions are consistent for all sam-
ples in a given Case, a general statement outlining these
parameters is sufficient.
e) duplicates.
f) spikes (indicating standard solutions used, final spike concen-
trations, volumes involved)*
g) any instrument adjustments, data corrections or other apparent
anomalies on the measurement record including all data voided
or data not used to obtain reported values*
h) all information for furnace analysis contained in Fora VIII
(Exhibit E), clearly identified on the raw data including
sample #, initial single spike data, Z recovery, full MSA data,
MSA correlation coefficient, slope and intercept of linear fit,
and final sample concentration (standard addition concentra-
tion) .
6) Copies of digestion logs for the 1C?, flameless AA and Eg prepar-
ations and of the distillation log for cyanide. These logs must
Include: 1) date, 2) sample weights and volumes, 3) sufficient
information to unequivocably identify which QC samples (i.e. LCS,
preparation blank) correspond to each batch digested, 4) comments
describing any significant sample changes or reactions which occur
during preparation, 5) indication of pH <2 or >12, as applicable.
7) The order of raw data in the data package shall be: 1) ICF, 2)
Flame AA, 3) Furnace AA, 4) Hg, 5) CN, 6) Digestion and Distilla-
tion logs, 7) Percent Solids. All raw data must include intensities
(1C?) and absorbances (AA) unless instrument direct readout is in
concentration units.
8) For each reported value, the data package must contain all raw data
from the instrument used to obtain that value and the QA/QC values
reported. All instruments must provide a hard copy of the instru-
ment readout (i.e. stripcharts, printer tapes, etc.). A photocopy
of the instrument readout must be included in the raw data package.
Information shall include a key to abbreviations, with response units
stated, and a cross reference to EPA sample numbers.
D. Results of Intercoaparison/Perfomance Evaluaclon(PE) Sample Analyses
Tabulation of analytical results for Interconparlson/PE Sample analyses
include all requirements specified in C above.
B-5
-------
E. Complete Case File Purge (formerly termed the "Document Control and Chain-of-
Custody Package")
The Complete Case File Purge package must include all laboratory records
received or generated for a specific case that have not been previously
submitted to EPA as deliverable. These items include but are not limited
to: sample tags, custody records, sample tracking records, analysts logbook
pages, bench1 sheets, instrument readout records, computer printouts, raw
data summaries, instrument logbook pages (including instrument conditions),
correspondence, and the document inventory.
Shipment of each Complete Case File Purge package by first class mail,
overnight carrier, priority mail or equivalent is acceptable. Custody
seals, which are provided by EPA, must be placed on shipping containers and
a document inventory and transmittal letter included. The laboratory is
not required to maintain any documents for a case after shipment; however,
it is recommended that you maintain a copy of the document inventory and
transmittal letter.
F. Quarterly Verification of Instrument Parameters
The Contractor muse perform and report quarterly verification of instrument
detection limits by methods specified in Exhibit E and report type and
model 0 for each instrument used on this contract. For the 1CP instrument-
ation and methods, the Contractor must also report quarterly: linearity
range verification, interelement correction factors, wavelengths used and
integration times. This information is reported using Forms XI, XII and
XI11. Submissions of Quarterly Verification of Instrument Parameters oust
include the raw data used to determine those values reported.
G. Results of Solid Laboratory Control Samples (LCS)
Tabulation of analytical results and QC for the solid LCS sample analysis,
as specified in Exhibit E.
B-6
-------
CONTRACT REPORTING SCHEDULE
Report
A. Weekly Progress
Report
B. Sample Traffic
Re pore
C. Sample Data
Package
D* Results of Incer-
comparison Study/
FE Sample Analysis
E. Compilation of
Complete Case File
Purge Package
F. Complete Case
File Purge
Package
C. Quarterly
Verification
of Instrument
Parameters
H. Results of Solid
LCS Analysis
Mo.
1 Copies
1
1
3
1
I
1
2
2
Delivery
Schedule*
Weekly
7 days f roo
receipt of
sample
35 days from
receipt of
sample
35 days from
receipt of
sample
7 days after
data submission
180 days after
data submission
or 7 days from
request by PO
or SMO
Quarterly:
15th day of
January, April,
July, October
Monthly: 15th
of every month
SMO( 1 )
X
X
X
X
f*
A
ENSL/
1 LV(2>
X
X
X
X
1 Region/
J Clienc(3)
X
ICEAT
UEIC(4)
X
•Schedule designated in calendar days.
B-7
-------
REPORT DISTRIBUTION ADDRESSEES;
(1) USEPA
Contract Laboratory Program
Sample Management Office (SMO)
P.O. Box 318
Alexandria, VA 22313
(2) USEPA
Environmental Monitoring
Systems Laboratory (EI1SL/LV)
P.O. Box 15027
Us Vegas. NV 8911*
AITN: Data Audit Staff
For overnight delivery service,
use street address:
300 H. Lee Street
Alexandria, VA 22314
(3) USEPA REGIONS:
For overnight delivery service,
use street address:
944 E. Harmon, Exec. Ccr. Ha. 226
Us Vegas, NV 49109
ATTN: Data Audit Scaff
(4)
The CLP Sample Management Office will provide the contractor with the list
of addresses for distribution to the ten EPA Regions. SMO will provide
the Contractor with updated Regional address/name lists as necessary
throughout the period of. the contract*
USEPA
HE1C Contract Evidence Audit Team
12600 West Coifax Ave., Suite C310
La lie wood, CO 802 IS
B-3
-------
U.S. EPA Cone race Laboratory Program
Sample Management Office
P.O. Box 818 - Alexandria, VA 22313
703/557-2490 FTS: 8-557-2490 Date
COVER PACE
INORGANIC ANALYSES DATA PACKAGE
Ub Name Case No.
SOW No. Q«C. Report No.
Ub Receipt Date
Sample Numbers
EPA No. Lab ID No. EPA No. Lab ID No.
Comments:
ICP interelement and background corrections applied? Yes No .
Lf yes, corrections applied before _____ or after generation of raw data.
Footnotes:
MR - Net required by contract at this time
Form I:
Value - If the result is a value greater than or equal to the instrument
detecion limit but less than the contract-required detection limit,
report the value in brackets (i.e., [10]). Indicate the analytical
method used-with V (for ICP), A (for Flame AA) or. F (for Furnace AA).
U - Indicates element was analyzed for but not detected. Report with che
instrument detection limit value (e.g., 10U).
E - Indicates a value estimated or not reported due to the presence of
interference. Explanatory note included on cover page.
s - Indicates value determined by Method of Standard Addition.
U - Indicate? spike sample recovery is not within control limits.
* - Indicaces duplicate analysis Is not within control limits.
+ - Indicates the correlation coefficient for method of standard addition is
less than 0.995
M - Indicates duplicate Injection results exceeded control limits.
Indicate method used: P for ICP; A for Flame AA and F for Furnace.
B-9
-------
Form I
U.S. EPA Contract Laboratory Program
Sample Management Office
P.O. Box 818 - Alexandria, VA 22313
703/557-2490 FTS: 8-557-2490
EPA Sample No.
Date
LAB NAME
SOW NO.
LAB SAMPLE ID. NO.
INORGANIC ANALYSIS DATA SHEET
CASE NO.
QC REPORT NO.
Elements Identified and Measured
Concentration: Low
Matrix: Water
Soil
Medium
Sludge Other
ug/L or mg/kg dry weight (Circle One)
1* Aluminum 13. Magnesium
2. Antimony
3. Arsenic
4. Barium
5. Beryllium
6. Cadmium
7. Calcium
8. Chromium
9. Cobalt
10. Copper
11. Iron
12. Lead
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Manganese
Mercury
Nickel
Potassium
Selenium
Silver
Sodium
Thallium
Vanadium
Zinc
Precent Solids (Z)
Cyanide
Footnotes:
For reporting results to EPA, standard result qualifiers are used
as defined on Cover Page. Additional flags or footnotes explaining
results are encouraged. Definition of such flags oust be explicit.
and contained on Cover Page, however.
Comments:
Lab Manager
B-10
-------
Fora II
Q. C. Report No.
LAB NAME
DATE
INITIAL AND CONTINUING CALIBRATION VERIFICATION3
CASE NO.
SOW NO.
UNITS
Compound Initial Calib. 1 Continuing Calibration2
Metals:
5. Beryllium
Cobalt
12. Lead
16. ' Nickel
19. Silver
20. Sodium
21. Thallium
22. Vanadium
23. Zinc
Other:
inide
True Value
.
m
Found
•
•
ZR
•
•
True Value
•
Found
ZR
Found
•
ZR
Method4
1 Initial Calibration Source
2 Continuing Calibration Source_
3 Control Limits: Mercury and Tin 80-120; Other Metals 90-110; Cyanide 85-115
4 Indicate Analytical Method Used: P - ICP; A - Flame AA; F - Furnace AA
-------
LAB NAME
DATE
Fora III
Q. C. Report No.
BLANKS
CASE NO,
UNITS
Matrix
Compound
Metals:
1 • Aluminum
2. Antimony
3, Arsenic
A. Barium
5. Beryllium
6. Cadmium
7. Calcium
8. Chromium
9. Cobalt
10* Copper
11. Iron
12. Lead
13. Magnesium
14. Manganese
15. Mercury
16. Nickel
17. Potassium
18. Selenium
19. Silver
20 • Sodium
21. Thallium
22. Vanadium
23. Zinc
Other:
'
Cyanide
Initial
Calibration
Blank Value
*
•
Continuing Calibrate
Blank Value
1
i
2 3
m
4
•
•
Preparation Blank
Matrix: Matrix:
1 2
•
•
B-12
-------
LAB NAME
DATE
Fora IV
Q. C. Report No.
ZCP INTERFERENCE CHECK SAMPLE
CASE NO.
Check Sample I.D.
Cheek Sample Source
Units
Compound
Metals:
1. Aluminum
2. Antimony
3. Arsenic
A. Barium
5. Beryllium
6. Cadmium
7. Calcium
8. Chromium
. Cobalt
10* Copper
11* Iron
12. Lead
13. Magnesium
14. Manganese
15. Mercury
16. Nickel
17. Potassium
18. Selenium
19. Silver
20. Sodium
21. Thallium
22. Vanadium
23. Zinc
Other:
Control Limit si
Mean
Std. Dev.
True2
•
.
Initial
Observed
ZR
Final
Observed
ZR
* Mean value based on n •
True value of EPA ICP Interference Check Sample or contractor standard.
B-13
-------
Fora V
Q. C. Report No.
SPIKE SAMPLE RECOVER*
LAB NAME
DATE
CASE NO.
EPA Sample No.
Lab Sample ID No.
Units
Matrix
Compound
Metals:
1 . 'Aluminum
2t Antimony
3* Arsenic
4. Barium • •
5* Beryllium
6 . Cadai un
7 . Calcium
8. Chromium
9. Cobalt
10. Copper
12. Lead
13. Magnesium
14. Manganese
15. Mercury
16. Nickel
17 i Potassium
18. Selenium
19. Silver
20. Sodium
23. Zinc
Other:
Control Limit
ZR
75-125
»
•
_ __ m
•
•
m
•
-
•
•
H
N
-
•
•
«•
*•
-
-
-
W
-
M
Spiked Samole
Result (SSR)
•
Sample
Result (SR)
Spiked
Added (SA)
•
•
•
ZRl
1 ZR " USSR - SR)/SA] x 100
"N"- cue of control
"MR*- Not required
Comments:
B-L4
-------
LAB NAME
DATE
Fora VI
C. Report No.
DUPLICATES
CASE NO.
EPA Sample No.
Lab Sample ID No.
Units
Matrix
Compound
Metals:
1 . Aluminum
2. Antimony
3. Arsenic
4. Barium
5. Beryllium
6. Cadmium
7. Calcium
8. Chromium
9. Cobalt
J. Copper
11. Iron
12. Lead
13. Magnesium
14. Manganese
15. Mercury
16. Nickel
17. Potassium
18. Selenium
1*». Silver '
20. Sodium
21. Thallium
22. Vanadium
23. Zinc
Other:
ranide
Control Limit*
•
.
•
•
Sample(S)
B
Dupllcate(D)
.
RPD2
* Out of Concrol
1 To be added at a later date.
2 RPD • [|S - D|/((S + D)/2)] x 100
NC - Non calculable RPD due to value(s) less than CRDL
B-15
-------
Form VII
Q.C. Report No.
LAB NAME
INSTRUMENT DETECTION LIMITS AND
LABORATORY CONTROL SAMPLE
CASE NO.
DATE
LCS NO.
Compound
Metals:
1. Aluminum
2. Antimony
3* Arsenic
4. Barium
5. Beryllium
b« Cadmium
7. Calcium
8. Chromium
9. Cobalt
10. Copper
11. Iron
12. Lead
13. Magnesium
14. ilanganese
15. Mercury
16. Nickel
17. Pocassiua
18. Selenium
19. Silver
20. Sodium
21. ThalliuD
22. Vanadium
23. Zinc
Other:
Cyanide
Required Dececcion
Limits (CRDL)-ug/!
200
60
10
200
5
5
sooo
10
so
25
100
5
5000
15
0.2
40
5000
5
10
5000
10
50
20
10
Instrument Detection
Limits (IDL)-ug/!
ICP/AA Furnace
ID# ID*
•
-
NR
f.'R
Lab Control Sample
ug/L mg/kg
(circle one)
True Found ZR
•
NR - Noc required
B-16
-------
Fora VIII
Q.C. Report No. __
STANDARD ADDITION RESULTS
LAB NAME
DATE
CASE NO.
UNITS
EPA
Sample 9
Element
•
—
•
—
—
0 ADD
ABS.
1 ADD
CON.
ABS1
2 ADD
CON.
ABS.1
3 ADD
CON.
ABS.1
FINAL
CON. 2
r*
•
1 CON is the concentration added.. ABS. is the instrument readout in absorbance or
concentration.
2 Concentration as determined by MSA
*"r" is the correlation coefficient.
+ - correlation coefficient is outside of control window of 0.995.
B-17
-------
Form IX
Q. C. Report No.
ZCP SERIAL DILUTIONS
LAB NAME
CASE NO.
EPA Sample No.
DATE
Lab Sample ID No.
Units
Matrix
Compound
Metals:
1 . Aluminum
Initial Sample
Concentration I)
Serial Dilution1
Result (S)
Z Difference2
2. Antimony
3* Arsenic
4. Barium
5. Beryllium
6» Cad^yp
7. Calcium
8 • Chromium
9. Cobalt
10* Copoer
11. Iron
12. Lead
13. Magnesium
14. Manganese
15. Nickel
16. Potassium
17. Selenium
18. Silver
19. Sodium
20. Thallium
21. Vanadium
22. Zinc
Other:
|
1 Diluted sample concentration corrected for 1:4 dilution (see Exhibit D)
2 Percent Difference • I1 " sl x 100
I
NR - Not Required, initial sample concentration less than 10 times IDL
NA - Not Applicable, analyte not determined by ICP
B-18
-------
Form X
QC Report No.
HOLDING TIMES
LAB NAME
DATE
CASE NO.
EPA
Sample No*
Matrix
Date
Received
•
Mercury
Prep Date
Mercury
Holding Time
(Days)
CN Prep
Date
.
CN
Holding Time
(Days)
.
B-19
-------
Fora XI (Quarterly)
INSTRUMENT DETECTION LIMITS
LAB NAME
DATE
ICP/Flame AA (Circle One) Model Number
Furnace AA Number
Element
1 • Aluminum
2. Antimony
3. Arsenic
4. Barium
5. Beryllium
6. Cadmium
7. Calcium
8. Chromium
9. Cobalt
10. Copper
11. Iron
12. Lead
Wavelength
(nm) J
CXDL
(ug/L)
200
60
10
200
5
5
5000
10
50
25
10U
5
IDL
(ug/L)
Element
13. Magnesium
14. Manganese
15. Mercury
16. Nickel
17. Potassium
18. Selenium
19. Silver
20. Sodium
21. Thallium
22. Vanadium
23. Zinc
Wavelength
-------
Fora XIZ (Quarterly)
ICP InterelemcQt Correction Factors
LABORATORY,
DATE
ZCP Model Number
Analyte
1. Antimony
7. Cobalt
8. Copcer
9. Lead
10* Manganese
12. Nickel
IS. Silver
IS. Vanadium
19. Zinc
Analyte
Wavelength
(na)
•
Intereleaent Correction Factors
for
Al
Ca
•
. Fe
Mg
COMMENTS:
B-21
Manager
-------
Fora XII (Quarterly) (cont'd)
ICP Ineerelemenc Correction Factors
LABORATORY^
DATE
ICP Model Number
Analyte
1. Antimony
4. Beryllium
5. Cadmium
6. Chromium
7. Cobalt
8. Copper
9. Lead
12. Nickel
15. Silver
19. Zinc
Analyte
Wavelength
(nm)
•
•
Interelement Correction Factors .
for
•
•
COMMENTS:
Lab Manager
B-22
-------
Fora XIII (Quarterly)
ICP Linear Ranges
LAB NAME
DATE
ICP Model Number
Analyte
1. Aluminum
2. Antimony
3. Arsenic
4. Barium
5. Beryllium
6. Cadmium
7. Calcium
8. Chromium
9. Cobalt
10. Copoer
11. Iron
12. Lead
Integration
Time
(Seconds)
Concen-
tration
(us/L)
Aaalyte
13. Magnesium
14. Manganese
15. Mercury
16. Nickel
17. Potassium
IS. Selenium
19. Silver
20. Sodium
21. Thallium
22. Vanadium
23. Zinc
Integration
Time
(Seconds)
Concen-
tration
(ui/L)
Footnotes: • Indicate elements not analyzed by ICP with the notation "NA",
COMMENTS:
Lab Manager
B-23
-------
EXHIBIT D
ANALYTICAL METHODS
B-24
-------
Analytical Methods
Any analytical method specified in Exhibit D may be utilized as long as the
documented instrument or method detection limits meet the contract required
detection levels (Exhibit C). Analytical methods with higher detection limits
may be used only if the.sample concentration exceeds twice the documented
detection limit of the instrument or method. When an analyte concentration
exceeds the calibrated or linear range, reanalysis of the prepared sample is
required after appropriate dilution. All samples must initially be run un-
diluted (i.e. final product of sample preparation procedures). Both diluted
and undiluted sample measurements must be contained in the raw data.
Labware must be acid cleaned according to EPA's manual "Methods for Chemical
Analysis of Water and Wastes' (2) or as equivalent procedure. Samples must be
opened and digested in a hood. Stock solutions for standards may be purchased
or made up as specified in the analytical methods. Deionized water (Type II)
is equivalent to distilled water. All sample dilutions should be made with
acidified water to maintain constant add strength. Background corrections are
required for flame AA measurements below 350 na and all furnace AA measurements.
Before water sample preparation is initiated, check the pH of all water samples
and note in the sample preparation log if it is less than 2 for metals and
greater than 12 for cyanide.
NOTE: Reference numbers appear in parentheses throughout this section.
See the last page of this exhibit for reference list.
1. Furnace AA/IC?/Flame AA - Water
Furnace Atomic Absorption
A. Sample Preparation (Furnace Digestion Procedure for Waters)
Shake sample and transfer 100 mL of well-mixed sample to a 250 mL
beaker, add 1 mL of (1+1) HN03 and 2 mL 30Z H202. Cover with watch
glass or similar cover, heat for 2 hours at 95*C or until the volume
is reduced to- between -25 and 50 mL (make certain samples do not boil).
Cool sample and filter (see Mote 1) to remove insoluble material and
bring back to 100 mL with deionized distilled water. The sample is
now ready for analysis.
Concentrations so determined shall be reported as "total".
If Sb is to be determined by furnace, use the dlgestates prepared for
ICP/AA analysis.
B. Analytical Procedures
See Attachment 1 (3).i
Each furnace analysis requires at least 2 burns, except when performing
full method of standard additions (MSA).
B-25
-------
Inductively Coupled Plasma (ICP) and Plane Atomic Absorption (AA)
A. Sample Preparation (ICP/Flame AA Digestion Procedure for Water)
Shake sample and transfer 100 mL of:a veil mixed sample to a beaker.
Add 2 mL of (1+1) HN03 and 10'mL of (1+1) HC1 to the sample. Cover with
watch glass or similar cover and heat on a steam bath or hot plate until
the volume has been reduced to between 25 and 50 mL making certain the
sample does not boil. After this treatment, cool sample and filter to
remove insoluble material that could clog the nebulizer. (See Note 1.)
Adjust the volume to 100 mL with deionized distilled water. The sample
is now ready for analysis.
Concentrations so determined shall be reported as "total."
NOTE 1: In place of filtering, the sample after dilution and mixing
may be centrifuged or allowed to settle by gravity overnight
to remove insoluble material.
B. Analytical Procedures
ICP - See Attachment 3.
Flame AA - See Attachment 4.
2. Furnace AA/ICP/Flame AA - Sediment, Sludges and Soils
A. Sample Preparation - Acid Digestion of Sediments, Sludges and Soils
See Attachment 1.
B. Analytical Procedures
Furnace AA - See Attachment 2.
Each furnace analysis requires at least 2 burns, except for full MSA.
ICP - See Attachment 3.
Flame AA - See Attachment A.
3. Mercury in Water
See Attachment 5 (manual) and 5A (automated).
4. Mercury in Sediment
See Attachment 6.
5. Cyanide in Water
See Attachment 7.
6. Cyanide in Sediment
See Attachment 8.
7. Determination of Percent Solids
See Attachment 9.
B-26
-------
ATTACHMENT 1
SAMPLE PREPARATION OF SEDIMENTS, SLUDGES AND SOILS
1. Scope and Application
i.i This method is an acid digestion procedure used Co prepare sediments,
sludges, and soil samples for analysis by flame or furnace atomic
absorption spectroscopy (AAS) or by inductively coupled argon plasma
spectroscopy (1C?). Samples prepared by this method may be analyzed
by AAS or ICP for the following metals:
Aluminum Chromium Potassium
Antimony Cobalt Selenium
Arsenic Copper Silver
Barium Iron Sodium •
Beryllium Lead Thallium
Cadmium Magnesium Vanadium
Calcium Manganese Zinc
Nickel
2. Summary of Method
NOTE: A separate digestion procedure is required for furnace AA and ICP
analysis.
2.1 A representative 1 g (wet weight) sample is digested in nitric acid and
hydrogen peroxide. The dlgestate is then refluxed with either nitric
acid or hydrochloric acid. Hydrochloric acid is used as the final
reflux acid for the furnace AA analysis of Sb, the flame AA or ICP
analysis of Al, Sb, Ba, Be, Ca, Cd, Cr, Co, Cu, Fe, Pb, Mg, Mn,
Nit K, Ag, Na, Tl, V and Zn. Nitric acid is employed as the final
reflux add for the furnace AA analysis of Aa, Be, Cd, Cr, Co, Cu, Fe,
Pb, Mn, Ni, Se, Ag, Tl, V, and Zn. A separate sample shall be dried
for a total solids determination (Exhibit D, Attachment 9).
3. Apparatus and Materials
3.1 250 ml beaker or other appropriate vessel.
3.2 Watch glasses
3.3 Thermometer that covers range of 0° to 200*C
3.4 Whatman No. 42 filter paper or equivalent
4. Reagents
4.1 ASTM Type II water (ASTM D1193): Hater must be monitored.
4.2 Concentrated Nitric Acid (sp. gr. 1.41)
B-27
-------
4.3 Concentrated Hydrochloric Acid (sp. gr. 1.19)
4.4 Hydrogen Peroxide (30%)
5. Sample Preservation, and Handling
5.1 Non-aqueous samples muse be. refrigerated upon receipt uncil analysis.
6. Procedure
6.1 Mix Che sample thoroughly to achieve homogeniety. For each digestion
procedure, weigh (to the nearest O.Olgns) a 1.0 to 1.5 gm portion of
sample and transfer to a beaker.
6.2 Add 10 ml of 1:1 nitric acid (HNC^), mix the slurry, and cover with
a watch glass. Heat the sample to 95 °C and reflux for 10 minutes
without boiling, Allow the sample to cool, add 5 ml of concentrated
HN03, replace the watch glass, and reflux for 30 minutes. Do not
allow the volume to be reduced to less than 5 ml while maintaining
a covering of solution over the bottom of the beaker.
6.3 After the second reflux step has been completed and the sample has
cooled, add 2 ml of Type II water and 3 ml of 30Z hydrogen peroxide
(H202>* Return the beaker to the hot plate for warming to start the
peroxide reaction. Care must be taken to ensure that losses do not
occur due to excessively vigorous effervescence. Heat until effer-
vescence subsides, and cool the beaker*
6.4 Continue to add 30Z H202 in 1 ml aliquot s with warming until the
effervescence is minimal or until the general sample appearance is
unchanged. (NOTE: Do not add more than a total of 10 ml 302 H202. )
6.5 If the sample is being prepared for the furnace AA analysis of Sb,
the flame AA or 1CP analysis of Al, Sb. Ba, Be, Ca, Cd, Cr, Co, Cu,
Fe, Pb, Mg, Hn, Hi, K, Ag, Ha, Tl, V, and Zn, add 5 ml of 1:1 HC1
and 10 ml of Type II water, return the covered beaker to the hot
plate, and heat for an additional 10 minutes. After cooling, filter
through Whatman No. 42 filter paper (or equivalent) and dilute to
100 ml with Type II water (or centrifuge the sample - see Note 1).
The diluted sample has an approximate acid concentration of 2.5*
(v/v) HC1 and 5Z (v/v) HN03. Dilute the dlgestate 1:1 (200 ml final
volume) with the delonized water. The sample is now ready for
analysis.
6.6 L£ the sample is being prepared for the furnace analysis of As, Be,
Cd, Cr, Co, Cu, Fe, Pb, Hn. Ni, Se, Ag, Tl, V, and 2n, continue heat
ing the acid-peroxide digestate until the volume has been reduced to
approximately 2 ml, add 10 ml of Type II water, and warm the mixture
After cooling, filter through Whatman No. 42 filter paper (or equi-
valent - see Note 1) and dilute to 100 ml with Type 11 water (or
centrifuge the sample). The diluted digestate solution contains
B-28
-------
approximately 22 (v/v) HN03. Diluce the digestace 1:1 (200 ml final
volume) with deionized water. For analysis, withdraw aliquocs of
appropriate volume, and add any required reagent or matrix modifier.
The sample is now ready for analysis.
7. Calculations
7.1 A separate determination of percent solids oust be performed
(Exhibit D, Attachment 9).
7.2 The concentrations determined in the digest are to be reported
on the basis of the dry weight of the sample.
Concentration (dry wt.) (mg/kg) . C x V
wHTs
where C • Concentration (mg/L)
V • Final volume in liters after sample preparation
W » Weight in kg of wet sample
S - Z Solids/100
R£F: Modification of Method 3050 from. SW-846, Test Methods for Evaluating
Solid Waste, EPA Office of Solid Waste and Emergency Response, July 1982.
8. Bibliography
1. Modification (by committee) of Method 3050, SW-846, 2nd ed., Tesc
Methods for Evaluating Solid Waste. EPA Office of Solid Waste and
Emergency Response, July 1982.
B-29
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ATTACHMENT 2
ANTIMONY
Method 204.2 CLP-M (Atomic Absorption, furnace technique)
Optimum Concentration Range: 20-300 ug/1
Approximate Detection Limit: 3 ug/1
Preparation of Standard Solution
1. Stock solution: Carefully weigh 2.7426 g of antimony potassium
tartrate (analytical reagent grade) and dissolve in delonlzed
distilled water. Dilute to 1 liter with deionized water.
1 mL - 1 mg Sb (1000 og/L).
2. Prepare dilutions of the stock solution to be used as calibration
standards at the time of analysis. These solutions are also to be
used for "standard additions".
3. The calibration standards must be prepared using the same type of
acid and at the same concentration as will result in the sample to
be analyzed after sample preparation.
Instrument Parameters (General)
1. Drying Time and Temp: 30.sec-125'C.
2. Ashing Time and Temp: 30 sec-800°C.
3. Atomizing Time and Temp: 10 sec-2700'C.
4. Purge Gas Atmosphere: Argon
5. Wavelength: 217.6 no
6. Other operating parameters should be set as specified by the
particular'instrument manufacturer.
Notes
1. The above concentration values and instrument conditions are for a
Perkin-Elmer HGA-2100, based on the use of a 20 ul Injection, contin-
uous flow purge gas and non-pyrolycic graphite and are to be used as
guidelines only. Smaller size furnace devices or those employing
faster rates of atomlzatlon can be operated using lower atomizaclon
temperatures for shorter time periods than the above recommended
.-settings.
2. The use of background correction is required.
3. Nitrogen may also be used as the purge gas.
4. If chloride concentration presents a matrix problem or causes a loss
previous to atomlzation, add an excess 5 mg of ammonium nitrace Co
the furnace and ash using a ramp accessory or with incremental seeps
until the recommended ashing temperature is reached.
B-30
-------
5. For every sample anal/zed, verification is necessary Co determine
that method of standard addition is not required (see Exhibit E).
6. If method of standard addition is required follow the procedure
given in Exhibit E.
B-31
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ARSENIC
Method 200.2 CLF-M (Atomic Absorption, furnace technique)
Optimum Concentration Range: 5-100 ug/1
Approximate Detection Limit: 1 ug/1
Preparation of Standard Solution
1. Stock solution: Dissolve 1.32U g of arsenic trioxide, As203
(analytical reagent grade) in 100 ml of delonlzed distilled water
containing 4 g NaOH. Acidify the solution with 20 ml cone. UNO3
and dilute to 1 liter. 1 ml • 1 mg As (1000 mg/1).
2. Nickel Nitrate Solution, 52: Dissolve 24.780 g of ACS reagent grade
Ni(N03)2*6H20 in delonlzed distilled water and make up to 100 ml.
3. Nickel Nitrate Solution, 12: Dilute 20 ml of the 52 nickel nitrate
to 100 ml with deionized distilled water.
4. Working Arsenic Solution: Prepare dilutions of the stock solution to
be used as calibration standards at the time of analysis. Withdraw
appropriate allquots of the stock solution, add 1 ml of cone. HN03,
2 ml of 302 &202 and 2 ml of the 52 nickel nitrate solution. Dilute
to 100 ml with deionized distilled water.
Sample Preparation
1. Add 100 ul of the 52 nickel nitrate solution to 5 ml of the digested
sample. The sample*is now ready for injection into the furnace.
Instrument Parameters (General)
1. Drying Time and Temp: 30 sec-125°C.
2. Ashing Time and Temp: 30 sec-1100°C.
3. Atomizing Time and Temp: 10 sec-2700aC.
4. Purge Gas Atmosphere: Argon
5. Wavelength: 193.7 nm
6. Other operating parameters should be set as specified by the
particular Instrument manufacturer.
Notes
1. The above concentration values and Instrument conditions are for a
Perkln-Elmer HGA-2100, based on the use of a 20 ul injection, purge
gas interrupt and non-pyrolytic graphite. Smaller size furnace
devices or those employing faster rates of atomlzation can be operated
using lower atomlzation temperatures for shorter time periods than the
above recommended settings.
2. The use of background correction is required.
3. For every sample analyzed, verification is necessary to determine
that method of standard addition is not required (see Exhibit E).
4. If method of standard addition is required, follow the procedure
given in Exhibit E).
5. The use of the Electrodeless Discharge Lamps (EDL) for the light
source is recommended. B-32
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CADMIUM
Method 213.2 CLP-M (Atomic Absorption, furnace technique)
Optimum Concentration Range: 0.5-10 ug/1
Approximate Detection Limit: 0.1 ug/1
Preparation of Standard Solution
1. Stock solution: Carefully weigh 2.282g of cadmium sulfate, 3 Cd
8 B20 (analytical reagent grade) and dissolve in delonized distilled
water. Make up to 1 liter with delonized distilled water. 1 mL »
1 mg Cd (1000 mg/L).
2. Ammonium Phosphate solution (40Z): Dissolve 40 grams of ammonium
phosphate, (NH^ZHPO^ (analytical reagent grade) in delonized distilled
water and dilute to 100 ml.
3. Prepare dilutions of stock cadmium solution to be used as calibration
standards at the time of analysis. To each 100 ml of standard and
sample alike add 2.0 ml of the ammonium phosphate solution. The
calibration standards must be prepared using the same type of acid and
at the same concentration as will result in the sample to be analyzed
after sample preparation.
Instrument Parameters (General)
1. Drying Time and Temp: 30 sec-125'C.
2. Ashing Time and Temp: 30 sec-500'C.
3. Atomizing Time and Temp: 10 sec-1900°C.
4. Purge Gas Atmosphere: Argon
5. Wavelength: 228.8 nm
6. The operating parameters should be set as specified by the particular
instrument manufacturer.
Notes
1. The above concentration values and instrument conditions are for a
Perkln-Elmer HGA-2100, based on the use of a 20 ul injection, contin-
uous flow purge ga's and non-pyrolytic graphite and are to be used as
guidelines only. Smaller size furnace devices or those employing
faster rates of atomlzation can be operated using lower atomization
temperatures for shorter time periods than the above recommended
settings.
2. The use of background correction is required.
3. Contamination from the work area is critical in cadmium analysis.
Use pipet tips which are free of cadmium.
4. For every sample analyzed, verification is necessary to determine
that method of standard addition is not required (see Exhibit E).
S. If method of standard addition is required, follow the procedure given
in Exhibit E. B-33
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LEAD
Method 239.2 CLP-M (Atomic Absorption, furnace technique)
Optimum Concentration Range: 5-100 ug/1
Approximate Detection'Limit: 1 ug/1
Preparation of Standard Solution
1. Stock solution: Carefully weigh 1.599 g of lead nitrate, Pb (N03)2
(analytical reagent grade), and dissolve in deionlzed distilled water.
When solution is complete, acidify with 10 mL redistilled HN<>3 and
dilute to 1 liter with deionized distilled water. 1 mL • 1 mg Pb
(lOOOmg/L).
2. Lanthanum Nitrate solution: Dissolve 58.64 g of ACS reagent grade
La2<)3 la 100 ml cone. HN(>3 and dilute to 1000 ml with deionized
distilled water. 1 ml - 50 mg La.
3. Working Lead solution: Prepare dilutions of stock lead solution to be
used as calibration standards at the time of analysis. The calibration
standards must be prepared using the same type of acid and at the same
concentration as will result in the sample to be analyzed after sample
preparation. To each 100 ml of diluted standard add 10 ml of the
lanthanum nitrate solution.
Sample Preparation
1. To each 100 ml of prepared sample solution add 10 ml of the lanthanum
nitrate solution.
Instrument Parameters (General)
1. Drying Time and Temp: 30 sec-125'C.
2. Ashing Time and Temp: 30 sec-500'C.
3. Atomizing Time and Temp: 10 sec-2700'C.
4. Purge Gas Atmosphere: Argon
5. Wavelength: 283.3 nm
6. Other operating parameters should be set as specified by the particular
instrument manufacturer.
Notes
1. The above concentration values and instrument conditions are for a
Perkin-Elmer HGA-2100, based on the use of a 20 ul Injection, contin-
uous flow purge gas and non-pyrolytic graphite and are to be used as
guidelines only. Smaller size furnace devices or those employing
faster rates of atomizatlon can be operated using lower acomizacion
temperatures for shorter time periods than the above recommended .
settings*
B-34
-------
Method 239.2 CLP-M (cone.)
2. The use of background correction is required.
3. Greater sensitivity can be achieved using the 217.0 na line, but
the optimum concentration range is reduced. The use of a lead
electrodeless discharge lamp at this lower wavelength has been
found to be advantageous. Also a lower atomization temperature
(24UO'C) may be preferred.
4. To suppress sulfate interference (up to 1500 ppm) lanthanum is
added as the nitrate to both samples and calibration standards.
(Atomic Absorption Newsletter Vol. K5, No. 3, p. 71, May-June 1976).
5. Since glassware contamination is a severe problem in lead analysis,
all glassware*should .be cleaned immediately prior to use, and once
cleaned, should not be open to the atmosphere except when necessary.
6. For every sample analyzed, verification is necessary to determine
that method of standard addition is not required (see Exhibit E).
7. If method of standard addition is required, follow the procedure
given in Exhibit E.
B-35
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SILVER
Method 272.2 CLP-M (Atomic Absorption, furnace technique)
Optimum Concentration Range: 1-25 ug/1
Approximate Detection Limit: 0.2 ug/1
Preparation of Standard Solution
1. Stock solution: Dissolve 1.575 g of AgN03 (analytical reagent
grade) in deionized distilled water. Add 10 mL of concentrated
and make up to 1 liter. 1 mL » 1 mg Ag (1000 mg/L).
2. Prepare dilutions of Che stock solution to be used as calibration
standards at the time of analysis. These solutions are also to be
used for "standard additions".
3. The calibration standards must be prepared using the same type of
acid and at the same concentration as will result in the sample to
be analyzed after sample preparation.
Instrument Parameters (General)
1. Drying Time and Temp: 30 sec-125'C.
2. Ashing Time and Temp: 30 sec-400*C.
3. Atomizing Time and Temp: 10 sec-2700'C.
4. Purge Gas Atmosphere: Argon
5. Wavelength: 328.1 nm
6. Other operating parameters should be set as specified by the
particular instrument manufacturer.
Notes
1. The above concentration values and instrument conditions are for
a Perkin-Elmer HCA-2100, based on the use of a 20 ul injection,
continuous flow purge gas and non-pyrolytic graphite and are to
be used as guidelines only. Smaller size furnace devices or those
employing faster rates of atomization can be operated using lover
atomization temperatures for shorter time periods than the above
recommended settings.
2. The use of background correction is required.
3. The use of hallde acids should be avoided.
4. If absorption to container walls or formation of AgCl is suspected,
see NOTE 3 under the Direct Aspiration Method (Exhibit D, Attachment 4).
5. For every sample analyzed, verification is necessary to determine
that method of standard addition is not required (see Exhibit E).
6. If method of standard addition is required, follow the procedure
given in Exhibit E.
B-36
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ATTACHMENT 3
Method 200.7 CLP-M*
INDUCTIVELY COUPLtD PLASMA-ATOMIC EMISSION SPECTROMETRIC METHOD
FOR TRACE ELEMENT ANALYSIS OF WATER AND WASTES
1. Scope and Application
1.1 Dissolved elements are determined in filtered and acidified samples.
Appropriate steps must be taken in all analyses to ensure that
potential interferences are taken into account. This is especially
true when dissolved solids exceed 150U mg/L. (See 5.)
1.2 Total elements are determined after appropriate digestion procedures
are performed. Since digestion, techniques increase the dissolved
solids content of the samples, appropriate steps must be taken to
correct for potential interference effects. (See 5.)
1.3* Table 1 lists elements along with recommended wavelengths and typical
estimated instrumental detection limits using .conventional pneumatic
nebulizatlon. Actual working detected limits are sample dependent
and as the sample matrix varies, these concentrations may also vary.
In time, other elements maybe added as more information becomes
available and as required.
1.4 Because of the differences between various makes and models of
satisfactory instruments, no detailed instrumental operating
instructions can be provided. Instead, the analyst is referred
to the Instructions provided by the manufacturer of the particular
instrument.
2. Summary of Method
2.1 The method describes a technique for the simultaneous or sequential
multielement determination of trace elements in solution. The basis
of the method is the measurement of atomic emission by an optical
spectroscopic technique. Samples are nebulized and the aerosol
that is produced is transported to the plasma torch where excitation
occurs. Characteristic atomic-line emission spectra are produced by
a radio-frequency inductively coupled plasma (ICP). The spectra are
dispersed by a grating spectrometer and the intensities of the line
are monitored by photomultiplier tubes. The photocurrents from Che
photomultiplier tubes are.processed and controlled by a computer
system. A background correction technique is required co compensate
for variable background contribution to the determination of trace
elements. Background must be measured adjacent to analyte lines on
*CL?-M Modified for the Contract Laboratory Program
B-37
-------
Method 200.7 CLP-M (cont.)
samples during analysis. The position selected for the background
intensity measurement, on either or both sides of the analytical
line, will be determined by the complexity of the spectrum adjacent
to the analyte line. The position used'must be free of spectral
interference and reflect the same change in background intensity as
occurs at the analyte wavelength measured. Background correction is
not required in cases of line broadening where a background correc-
tion measurement would actually degrade the analytical result. The
possibility of additional interferences named In S.I (and tests for
their presence as described In 5.2) should also be recognized and
appropriate corrections made.
3. Definitions
3.1 Dissolved — Those elements which-will pass through a 0.45 urn
membrane filter.
3.2 Suspended — Those elements which are retained by a 0.45 urn
membrane filter.
3.3 Total — The concentration determined on an unfiltered sample
following vigorous digestion.
3.4 Instrumental detection limits — See Exhibit E, pages 2-4.
3.5 Sensitivity — The slope of the analytical curve, i.e. functional
relationship between emission intensity and concentration.
3.6 Instrument cheek standard — A multielement standard of known
concentrations prepared by the analyst to monitor and verify
Instrument performance on a dally basis. (See 7.6.1.)
3.7 Interference cheek sample — A solution containing both interfering
and analyte elements of known concentration that can be used co
verify background and interelement correction factors. (See 7.6.2.)
3.0 Quality control sample — A solution obtained from an oucslde source
having known concentration values to be used to verify the calibra-
tion standards. (See 7.6.3.)
3.9 Calibration standards — A series of known standard solutions used
by the analyst for calibration of the instrument (i.e., preparation
of the analytical curve). (See 7.4.)
3.10 Linear dynamic range — The concentration range over which the
analytical curve remains linear as determined in Exhibit E.
3.11 Reagent blank — A volume of deionlzed, distilled water containing
the same acid matrix as the calibration standards carried through
the entire analycical scheme. (See 7.5.2.)
B-38
-------
Method 200.7 CLP-M (cone.)
3.12 Calibration blank — A volume of deionized, distilled water acidified
vlth HN03 and UC1. (See 7.5.1.)
3.13 Method of standard addition — The standard addition technique
Involves the use of the unknown and the unknown-^plus-a-known amount
of standard by adding known amounts of standard to one or more
allquots of the processed sample solution.
4. Safety
4.1 The toxlcity or carcinogenicity of each reagent used In this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. The laboratory
is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified
in this method. A reference file of material handling data sheets
should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are
available and have been identified (11.7, 11.8 and 11.9) for the
information of the analyst.
5. Interferences
5.1 Several types of interference effects may contribute to in-
accuracies in the determination of trace elements. They can be
summarized as follows:
5.1.1 Spectral interferences can be categorized as 1) overlap of
a spectral line from another element; 2) unresolved overlap
of molecular band spectra; 3) background contribution from
continuous or recombination -phenomena; and 4) background
contribution from stray light from the line emission of high
concentration elements. The first of these effects can be
compensated by utilizing a computer correction of the raw
data, requiring the monitoring and measurement of the inter-
fering element. The second effect may require selection of
an alternate wavelength. The third and fourth effects can
usually be compensated by a background correction adjacent
to the analyte line. In addition, users of simultaneous
multi-element instrumentation must assume the responsibility
of verifying the absence of spectral interference from an
element that could occur in a sample but for which there is
no channel in the instrument array. Listed in Table 2 are
some Interference effects for the recommended wavelengths
given in Table 1. The data in Table 2 are intended for use
only as a rudimentary guide for' the indication of potential
spectral interferences. For this purpose, linear relations
between concentration and intensity for the analytes and the
interferents can be assumed. The interference information,
which was collected at the Ames Laboratory1, is expressed as
'Ames Laboratory, USUOE, Iowa State University, Ames Iowa 50011
B-39
-------
Method 200.7 CLP-M (cone.)
analyte concentration equivalents (i.e. false analyte concen-
trations) arising from 100 mg/L of the interferenc element.
The suggested use of this information is 'as follows: Assume
that arsenic (at 193.696 mn) is to be determined in a sample
containing approximately 10 mg/L of of aluminum. According
to Table 2, 100 mg/L of aluminum would yield a false signal
for arsenic equivalent to approximately 1.3 mg/L. Therefore,
10 mg/L of aluminum would result in a false signal for arsenic
equivalent to approximately 0.13 mg/L. The reader is cautioned
that other analytical systems may exhibit somewhat different
levels of interference Chan those shown in Table 2, and that
the Interference effects must be evaluated for each individual
system. Only those interferents listed were investigated and
the blank spaces in Table 2 indicate that measurable inter-
ferences were not observed from the interferent concentrations
listed in Table 3. Generally, interferences were discernible
if they produced peaks or background shifts corresponding to
2-5Z of the peaks generated by the analyte concentrations also
listed in Table 3.
At present, information on the listed silver and potassium
wavelengths are not available but it has been reported that
second order energy from the magnesium 383.231 nm wavelength
Interferes with the listed potassium line at 766.491 an.
5.1.2 Physical interferences are generally considered to be effects
associated with the sample nebulization and transport proc-
esses. Such properties as change in viscosity and surface
tension can cause significant inaccuracies especially in
samples which may contain high dissolved solids and/or acid
concentrations. The use of a peristaltic pump may lessen
these Interferences. If these types of interferences are
operative, they must be reduced by dilution of the sample
and/ or utilization of standard addition techniques. Another
problem which can occur from high dissolved solids is sale
buildup at the tip of the nebulizer. This affects aerosol
flow race causing instrumental drift. Vetting Che argon
prior to nebulization, the use of a tip washer, or sample
dilution have been used to control this problem. Also, it
has been reported that better control of the argon flow race
improves instrument performance. This is accomplished with
the use of mass flow controllers.
5.1.3 Chemical interferences are characterized by molecular conr-
pound formation, lonlzation effects and solute vaporization
effects, formally these effects are not pronounced with the
ICP technique, however, if observed they can be minimized by
careful selection of operating conditions (that is, incldenc
power, observation position, and so forth), by buffering of
the sample, by matrix matching, and by standard addition
procedures. These types of interferences can be highly
dependent on matrix type and the specific analyce element.
B-40
-------
Method 200.7 CLP-M (cone.)
5.2 For each group of samples of a similar matrix type and concentration
(i.e., low, medium) for each Case of samples, or for each 20 samples
received, whichever is more frequent, the following tests must be
performed prior to reporting concentration data for analyte elements.
5.2.1 Serial dilution— If the analyte concentration is suffi-
ciently high (minimally a factor of 10 above the instrument
detection limit after dilution), an analysis of a 1:4 dilution
must agree within 10 percent of the original determination.
Serial dilution results must be reported on QC Report Form IX.
Samples identified as Field Blanks cannot be used for serial
dilution analysis.
If the dilution analysis is not within 10Z, a chemical or
physical interference effect should be suspected, and the
data oust be flagged with an T.
6. Apparatus
6.1 Inductively Coupled Plasma-Atomic Emission Spectrometer.
6.1.1 Computer controlled atomic emission spectrometer with back-
ground correction.
6.1.2 Radiofrequency generator.
6.1.3 Argon gas supply, welding grade or better.
6.2 Operating conditions — Because of the differences between various
makes and models of satisfactory instruments, no detailed operating
instructions can be provided. Instead, .the analyst should follow the
instructions provided by the manufacturer of the particular instrument.
Sensitivity, Instrumental detection limit, precision, linear dynamic
range, and interference effects oust be investigated and established
for each individual analyte line on that particular Instrument. All
measurements must be within the instrument linear range where correc-
tion factors are valid. It is the responsibility of the analyst to
verify that the instrument configuration and operating conditions
used satisfy the analytical requirements and to maintain quality con-
trol data confirming instrument performance and analytical results.
7. Reagents and standards
7.1 Acids used in the preparation of standards and for sample processing
must be ultra-high purity grade or equivalent. Redistilled acids
are acceptable.
7.1.1 Acetic acid, cone, (sp gr 1.06).
7.1.2 Hydrochloric acid, cone, (sp gr 1.19).
7.1.3 Hydrochloric acid. (1+1): Add 500 mL cone. HC1 (sp gr 1.19)
to 400 mL delonlzed, distilled water and dilute Co L liter.
' B-41
-------
Method 200.7 CLP-M (cone.)
7.1.4 Nitric acid, cone, (sp gr 1.41).
7.1.5 Nitric acid. (1+1): Add 500 mL cone. HN03 (sp gr'1.41) to
400 mL deionized, distilled water and dilute to 1 liter.
7.2 Deionized, distilled water; Prepare by passing distilled water
through a mixed bed of cation and anion exchange resins. Use
deionized, distilled water for the preparation of all reagents,
calibration standards and as dilution water. The purity of this
water must be equivalent to ASTM Type II reagent water of Specifi-
cation D 1193 (14.t>).
7.3 Standard stock solutions may be purchased or prepared from ultra
high purity grade chemicals or metals. All salts must be dried for
1 h at 105° unless- otherwise specified.
(CAUTION: Many metal salts are extremely toxic and may be fatal if
swallowed. Wash hands thoroughly after handling.) Typical stock
solution preparation procedures follow:
7.3.1 Aluminum solution, stock. 1 mL • 100 ug Al: Dissolved 0.100
g of aluminum metal in an acid mixture of 4 mL of (1+1) HC1
and 1 mL of cone. HN03 in a beaker. Warm gently to effect
solution. When solution is complete, transfer quantitatively
to a liter flask, add an additional 10 mL of (1+1) HC1 and
dilute to 1000 mL with deionized, distilled water.
7.3.2 Antimony solution stock, 1 oL - 100 ug Sb: Dissolve 0.2669
g K(SbO)C4h406 in deionized distilled water, add 10 mL (1+1)
ECl and dilute to 1000 mL with deionized, distilled water.
7.3.3 Arsenic solution, stock. 1 mL • 100 ug As: Dissolve 0.1320
g of As203 in 100 mL of deionized, distilled water containing
0.4 g NaOH. Acidify the solution with 2 mL cone. HNC>3 and
dilute to 1,000 mL with deionized, distilled water.
7.3.4 Barium solution, stock. 1 mL - 100 ug Ba: Dissolve 0.1516 g
Bad2 (dried at 250°C for 2 hrs)-in 10 mL deionized, distilled
water with 1 mL (1+1) UC1. Add 10.0 mL (1+1) HC1 and dilute
to 1,000 mL with deionized, distilled water.
7.3.5 Beryllium solution, stock, 1 mL • 100 ug Be: Do not dry.
Dissolve 1.966 g BeSO4'4H20, in deionized, distilled water,
add 10.0 mL cone. UN03 and dilute to 1,000 mL with deionized,
distilled water.
7.3.6 Boron solution, stock, 1 mL • 100 ug B: Do not dry. Dissolve
0.571b g anhydrous (13803 in deionized, distilled water and
dilute to 1,000 nL. Use a reagent meeting ACS specifications
keep the bottle tightly stoppered and store in a desiccator
to prevent the entrance of atmospheric moisture.
B-42
-------
Method 200.7 CLP-M (cone.)
7.3.7 Cadmium solution, stock. 1 mL - 100 ug Cd: Dissolve 0.1142
g CdO in a minimum amount of (1+1) HliC^. Heat to increase
rate of dissolution. Add 10.0 mL cone. HN03 and dilute to
1,000 mL with deionized, distilled water.
7.3.8 Calcium solution, stock. 1 mL - 100 ug Ca: Suspend 0.2498 g
CaO>3 dried at IbO'C for 1 h before weighing in deionized,
distilled water and dissolve cautiously with a minimum amount
of (1+1) HNC>3. Add 10.0 mL cone. HN(>3 and dilute to 1,000 mL
with deionized, distilled water.
7.3.9 Chromium solution, stock. 1 mL • 100 ug Cr: Dissolve 0.1923
g of Cr03 in deionized, distilled water. When solution is
complete acidify with 10 mL cone. H1K>3 and dilute to 1,000 mL
with deionized, distilled water.
7.3.10 Cobalt solution stock. 1 mL • 10 ug Co: Dissolve 0.1000 g
of cobalt metal in a minimum amount of (1+1) HN03. Add 10.0
mL (1+1) UC1 and dilute to 1,000 mL with deionized, distilled
water.
7.3.11 Copper solution, stock. 1 mL • 100 ug Cu: Dissolve 0.1252
g CuO in a minimum amount of (1+1) UM03* Add 10.0 mL cone.
HN03 and dilute to 1,000 mL with deionized. distilled water.
7.3.12 Iron solution, stock, 1 mL • 100 ug Fe: Dissolve 0.1430 g
Fe2<>3 in a warm mixture of 20 mL (1+1) UC1 and 2 mL of cone.
HN03* Cool, add an additional 5 mL of cone. EN03 and dilute
to 1,000 mL with deionized, distilled water.
7.3.13 Lead solution, stock. 1 mL - 100 ug Pb: Dissolve 0.1599 g
Pb(N03)2 in a minimum amount of (1+1) HN03. Add 10.0 mL of
cone. HN03 and dilute to 1,000 mL with deionized, distilled
water.
7.3.14 Magnesium solution, stock, 1 mL - 100 ug Mg: Dissolve 0.1658
g MgO in a minimum amount of (1+1) HM^. Add 10.0 mL cone.
HN03 and dilute to 1,000 mL with deionized, distilled water.
7.3.15 Manganese solution, stock. 1 mL - 100 ug Mn: Dissolve O.lOOO
g of manganese metal in the acid mixture, 10 nL cone. HC1
and 1 mL epnc. UNO3, and dilute to 1,000 mL with deionized,
distilled water.
7.3.16 Molybdenum solution, stock, 1 mL • 100 ug Mo: Dissolve
0.2043 g (NH4)2Mo04 in deionized, distilled water and dilute
to 1,000 mL.
B-43
-------
Method 200.7 CLP-M (cone.)
7.3.17 Nickel solution, stock. 1 mL - 100 ug Ni: Dissolve 0.1000 g
of nickel metal in 10 nL hot cone* HN03, cool and dilute co
1,000 nL with deionized, distilled water.
7.3.1S Potassium solution, stock. 1 nL - 100 ug K: Dissolve 0.1907
g KC1, dried at 110'C, in deionized, distilled water. Dilute
to 1,000 mL.
7.3.19 Selenium solution, stock. 1 mL • 100 ug Se: Do not dry.
Dissolve 0.1727 g H2Se°3 (actual assay 94.6Z) in deionized,
distilled water and dilute co 1,000 mL.
7.3.20 Silica solution, stock, 1 mL • 100 ug S102: Do not dry.
Dissolve 0.4730 g Na2Si03'9H20 in deionized, distilled water.
Add 10.0 mL cone. BNOj and dilute to 1,000 mL with deionized,
distilled water.
7.3.21 Silver solution, stock. 1 mL - 100 ug Ag: Dissolve 0.1575 g
AgN(>3 in 100 mL of deionized, distilled water and 1U mL cone.
HN<>3. Dilute to 1,000 mL with deionized, distilled water.
7.3.22 Sodium solution, stock. 1 mL « 100 ug Na: Dissolve 0.2542 g
MaCl in deionized, distilled water. Add 10.0 mL cone. HN03
and dilute to 1,000 mL .with deionized, distilled water.
7.3.23 Thallium solution, stock. 1 mL - 100 ug Tl: Dissolve 0.1303
g T1N03 in deionized, distilled water. Add 10.0 mL cone.
HNOj and dilute to 1,000 mL with deionized, distilled water.
7.3.24 Vanadium solution, stock. 1 mL - 100 ug V: Dissolve 0.2297
NH4V03 in a minimum amount of cone. HN03* Heat to Increase
rate of dissolution. Add 10.0 mL cone. HN03 and dilute to
1,000 mL with deionized, distilled water.
7.3.25 Zinc solution, stock. 1 mL - 100 ug Zn: Dissolve 0.1245 g
ZnO in a minimum amount of dilute HM03. Add 10.0 mL cone.
HM03 and dilute to 1,000 mL with deionized, distilled water.
7.4 Mixed calibration standard solutions — Prepare mixed calibration
standard solutions by combining appropriate volumes of the stock
solutions in volumetric flasks. (See 7.4.1 thru 7.4.5.) Add 2
mL of (1+1) UN03 and 1U mL of (1+1) HC1 and dilute to 100 mL with
deionized. distilled water. (See Notes 1 and 6.) Prior to pre-
paring the mixed standards, each stock solution should be analyzed
separately to determine possible spectral interference or the
presence of Impurities. Care should be taken when preparing che
mixed standards that the elements are compatible and stable.
Transfer the mixed standard solutions to a FEP fluorocarbon or
unused polyethylene bottle for storage. Fresh mixed scandards
should be prepared as needed with the realization that concencraclon
B-44
-------
Method 20U.7 CLP-M (cone.)
can change on aging. Calibration standards must be initially
verified using a quality control sample and monitored weekly for
stability (see 7.6.3). Although not specifically required, some
typical calibration standard combinations follow when using those
specific wavelengths listed in Table 1.
7.4.1 Mixed standard solution I — Manganese, beryllium, cadmium,
lead, and zinc.
7.4.2 Mixed standard solution II — Barium, copper, iron, vanadium,
and cobalt.
7.4.3 Mixed standard solution III — Molybdenum, silica,
arsenic, and selenium.
7.4.4 Mixed standard solution IV — Calcium, sodium, potassium,
aluminum, chromium and nickel.
7.4.5 Mixed standard solution V — Antimony, boron, magnesium,
silver, and thallium.
NOTE 1: If the addition of silver to the recommended
acid pombination results in an initial precipitation
add 15 mL of deionized distilled water and warn the
flask until the solution clears. .Cool and dilute to
100 mL with deionized,' distilled water. For this
acid combination the silver concentration should be
limited to 2 mg/L. Silver under these conditions, is
stable in a tap water matrix for 30 days. Higher
concentrations of silver require additional HC1.
7.5 Two types of blanks are required for the analysis. The calibration
blank (3.13) is used in establishing the analytical curve while the
reagent blank (preparation blank, 3.12) is .used to correct for
possible contamination resulting from varying amounts of the acids
used in the sample processing.
7.5.1 The calibration blank is prepared by diluting 2 mL of
UMU3 and 10 mL of (1*1) HC1 to 100 mL with deionized,
distilled water. (See Note 6.) Prepare a sufficient
quantity to be used to flush the system -between standards
and samples.
7.5.2 The reagent blank (or preparation blank - See Exhibit E)
must contain all the reagents and in the same volumes as
used in the processing of the samples. The reagent blank
must be carried through the complete procedure and contain
the same acid concentration in the final solution as the
sample solution used for analysis.
B-45
-------
Method 2CU.7 CLP-M (cone.)
7.6 In addition the calibration standards, an instrument check standard
(3.6), an interference check sample (3.7) and a quality control
sample (3.8) are also required for Che analyses.
7.6.1 The instrument check standard for continuing calibration
verification is prepared by the analyst by combining com-
patible elements at a concentration equivalent to the mid-
point of their respective calibration curves. (See 10.1.3.)
7.0.2 The interference check sample is prepared by the analyst,
or obtained from EPA if available (Exhibit E).
7.6.3 The quality control sample for the initial calibration
verification should be prepared in the same acid matrix
as the calibration standards and in accordance with the
Instructions provided by the supplier. EPA will either
supply a quality control sample or information where one
of equal quality can be procured. (See 10.1.1.)
8. Procedure
8.1 Set up Instrument with proper operating parameters established in
Section 6.2. The instrument must be allowed to- become thermally
stable before beginning. This usually requires at least 30 min.
of operation prior to calibration.
8.2 Initiate appropriate operating configuration of computer.
8.3 Profile and calibrate Instrument according to instrument manufac-
turer's recommended procedures, using mixed calibration standard
solutions such as those described in Section 7.4. Flush the system
with the calibration blank (7.5.1) between each standard. (See
NOTE 7.) (Use the average intensity of multiple exposures for both
standardization and sample analysis to reduce random error.)
NOTE 7: For boron concentrations greater than 500 ug/L extended
flush times of 1 to 2 minutes may be required.
8.4 Begin the sample run flushing the system with the calibration blank
solution (7.5.1) betveen each sample. (See NOTE 7.) Analyze the
instrument check standard (7.6.1) and the calibration blank (7.5.1)
each 10 samples.
9. Calculation
9.1 Reagent blanks (preparation blanks) should be treated as specified
in Exhibit E.
9.2 If dilutions were performed, the appropriate factor muse be applied
to sample values.
9.3 Data oust be reported in ug/L.
B-46
-------
Method 200.7 CLP-M (cone.)
10. Quality Control (Instrumental)
10.1 Check the instrument standardization by analyzing appropriate
quality control check standards as follows:
10.1.1 A quality control sample (7.6.3) must be used daily for the
initial calibration verification (See Exhibit E). A fresh
dilution of this sample shall be analyzed every week there-
after to monitor their stability. If the results are not
within *10Z of the true value listed for the control sample,
prepare a new calibration standard and recalibrate the
instrument. If this does not correct the problem, prepare a
new stock standard and a new calibration standard and repeat
the calibration.
10.1.2 Analyze the calibration blank (7.5.1) at a frequency of 10Z.
The result should be within ± contract required detection
levels (Exhibit C). If the result is not within the control
level, terminate the analysis, correct the problem and
recalibrate the instrument (See Exhibit E).
10.1.3 For continuing calibration verification, analyze an appro-
priate instrument check standard (7.6.1) containing the
elements of interest at a frequency of 10Z. This check
standard is used to determine instrument drift. If agree-
ment is not within ±102 of the expected values, the analysis
is out of control. The analysis must be terminated, the
problem corrected, the instrument recalibrated, and the
preceding 10 samples reanalyzed (See Exhibit E).
10.1.4 To verify interelement and background correction factors
analyze the ICP interference check sample (7.6.2) at the
beginning, and end of the sample run or a nimimum of twice
per 8 hour work shift whichever is more frequent. The check
sample must be analyzed initially at least 5 times repeti-
tively to establish a mean value and standard deviation.
Results must fall within the established control limits. If
not, terminate the analysis, correct the problem, recalibrate
the instrument, and reanalyze the samples (See Exhibit E).
11. Bibliography
1. Winge, R. K., V.J. Peterson, and V.A. Tassel, "Inductively Coupled
Plasma-Atomic Emission Spectroscopy Prominent Lines," EPA-600/4-79-017.
2. Winefordner, J.D., "Trace Analysis: Spectroacopic Methods for Elements,
Chemical Analysis, Vol. 46, pp. 41-'42.
B-47
-------
Method 200.7 CLP-M (cont.)
3. Handbook for Analytical Quality Control in Water and Wastevater
Laboratories, EPA-600/4-79-019.
4. Garbarino, J.R. and Taylor, H.E., "An Inductively-Coupled Plasma
Atomic Emission Spectrometric Method for Routine Water Quality
Testing," Applied Spectroscopy .3p_i No< 3(1979).
5. "Methods for Chemical Analysis of Water and Wastes," EPA-600/4-79-020.
6. Annual Book of ASTM Standards, Part 31.
7. "Carcinogens - Working With Carcinogens," Department'of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
8. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976),
.
9. "Safety in Academic Chemistry Laboratories, American Chemical Society
Publications, Committee on Chemical Safety, 3rd Edition, 1979.
10. "Inductively Coupled Plasma-Atomic Emission. Spectrometric Method of
Trace Elements Analysis of Water and Waste", Method 200.7 modified
by CLP Inorganic Data/Protocol Review Committee; original method by
Theodore D. Martin, EMSL/Cinclnnati.
B-48
-------
Method 200.7 CLP-M (cone.)
Element
TABLE 1 - RECOMMENDED WAVELENGTHS<2) AND ESTIMATED
INSTRUMENTAL DETECTION LIMITS
Wavelength,
Estimated Detection
Limit, ug/L<2)
Aluminum
Antimony
Arsenic
Barium
Beryllium
Boron
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Molybdenum
Nickel
Potassium
Selenium
Silica (Si02)
Silver
Sodium
Thallium
Vanadium
Zinc
308.215
206.833
193.696
455.403
313.042
249.773
226.502
317.933
267.716
228.616
324.754
259.940
220.353
279.079
257.610
202.030
231.604
766.491
196.026
288.158
328.068
588.995
190.864
292.402
213.856
45
32
53
2
0.3
5
4
10
7
7
6
7
42
30
2
8
15
seeO)
75
58
7
29
40
8
2
The wavelengths listed are recommended because of their sensitivity and
overall acceptance. Other wavelength may be substituted if they can
provide the needed sensitivity and are treated with the same corrective
techniques for spectral interference. (See 5.1.1). The use of alternate
wavelengths must be 'reported (in nm) with the sample data.
The estimated instrumental detection limits as shown are taken from
"Inductively Coupled Plasma-Atomic Emission Spectroscopy-Promlnent Lines,
EPA-600/4-79-017. They are given as a guide for an instrumental Hole.
The actual method detection limits are sample dependent and may vary as
the sample matrix varies.
highly dependent on operating conditions and plasma position.
B-49
-------
TABLE 2. EXAMPLE OP ANALYTE CONCENTRATION EQUIVALENTS (ng/L) ARISING FROM
INTEKPEKENTS AT THE 100 ng/L LEVEL
Analyte
Aluminum
Antimony
Araenlc
Barium
Beryllium
Boron
Cadmium
„ Calcium
CO
' Chromium
0
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Molybdenum
Nickel
Selenium
Silicon
Sodium
Thallium
Vanadium
Zinc
Wavelength,
nm
308.215
206.833
193.696
455.403
313.042
249.773
226.502
317.933
267.716
228.616
324.754
259.940
220.353
279.079
257.610
202.030
231.604
196.026
288.158
•588.995
190.864
292.402
2IJ.H56
Al Ca
w a
0.47
1.3
__ __
—
0.04
__ —
— —
— —
__ — .
—
__ __
0.17
0.02
0.005 —
0.05
— —
0.23
__ __
—
0.30
__ _ _
— — — —
Interferent
Cr Cu Pe Mg Hn Nl
— 0.21
2.9 — 0.08 — —
0.44
— __ __ — — — —
— — — — —
_ n 11 __ — — —
__ _— U.Jfc
— — 0.03 — — 0.02
0.08 — 0.01 0.01 0.04 —
— o.003 — 0.04
0.03 — 0.005 — — 0.03
0.003 — — —
0.12
__ — — — — —
0.11 — 0.13 — 0.25 —
0.01 — 0.002 0.002
0.03 —
— — — — — — — — — — — —
0.09
0.07
— — — —
— — ~~ •"— ~*~ ~~ ~~
0.05 -- 0.005
— 0.14 -- -- — 0.29
Ti
__
.25
~~
—
0.04
~
—
0.03
—
0.15
0.05
^^
~
0.07
^~»
—
— —
— —
—
0.08
— —
0.02
™~
V
1.4
0.45
1.1 •
—
0.05
""
—
0.03
0.04
—
0.02
""
—
0.12
^^
—
—
~
0.01
—
^"™
—
™~
-------
Method 200.7 CLP-M (cone)
TABLE 3. INTERFERENT AND ANALYTE ELEMENTAL CONCENTRATIONS USED
FOR INTERFERENCE MEASUREMENTS IN TABLE 2 (EXHIBIT 0)
Analytes
Al
As
B
Ba
Be
Ca
Cd
Co
Cr
Cu
Fe
Mg
Mn
Mo'
Na
Nl
Pb
Sb
Se
Si
Tl
V
Zn
(mg/L)
10
10
10
1
1
1
10
1
1
1
1
1
1
10
10
10
10'
10
10
1
10
1
10
Interferents
Al
Ca
Cr
Cu
Fe
Mg
Mn
Ni
Tl
V
(mg/L)
1000
1000
200
200
1000
1000
200
200
200
200
B-51
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ATTACHMENT 5
MERCURY
Method 245.1 CLF-M* (Manual Cold Vapor Technique)
1. Scope and Application
1.1 In addition to inorganic forms of mercury, organic mercurials
may also be present. These organo-mercury compounds will not
respond to the cold vapor atomic absorption technique unless they
are first broken down and converted to mercuric ions. Potassium
permanganate oxidizes many of these compounds, but recent studies
have shown that a number of organic mercurials, including phenyl
mercuric acetate and methyl mercuric chloride, are only partially
oxidized by this reagent. Potassium, persulfate has been found to
give approximately 100Z recovery when used as the oxldant with
these compounds. Therefore, a persulfate oxidation step following
the addition of the permanganate has been included to insure thac
organo-mercury compounds, if present, will be oxidized to the
mercuric ion before measurement. A heat step is required for
methyl mercuric chloride when present in or spiked to a natural
system. For distilled water the heat step is not necessary.
1.2 The range of the method may be varied through instrument and/or
recorder expansion. Using a 100 ml sample, a detection limit of
0.2 ug Hg/1 can be achieved (see Appendix 11.2).
2. Summary of Method
2.1 The flameless AA procedure is a physical method based on the
absorption of radiation at 253.7 nm by mercury vapor. Organic
mercury compounds are oxidized and the mercury is reduced to the
elemental state and aerated from solution in a closed system.
The mercury vapor passes through a cell positioned in the light
path of an atomic absorption spectrophotomecer. Absorbance (peak
height) la measured as a function of mercury concentration and
recorded in the usual manner.
*CLP-M modified for the Contract Laboratory Program
B-52
-------
Method 245.1 CLF-H (cone.)
3. Sample Handling and Preservation
3.1 Until more conclusive data are obtained, samples should be
preserved by acidification with nitric acid to a pH of 2 or
lover Immediately at the time of collection.
4. Interference
4.1 Possible lnterferen.ce from sulflde Is eliminated by the addition
of potassium permanganate. Concentrations as high as 20 mg/1 of
sulflde as sodium sulflde do not Interfere with the recovery of
added inorganic mercury from distilled water.
4.2 Copper has also been reported to Interfere; however, copper
concentrations as high as 10 mg/1 had no effect on recovery of
mercury from spiked samples.
4.3 Sea waters, brines and industrial effluents high in chlorides
require additional permanganate (as much as 25 ml). During the
oxidation step, chlorides are converted to free chlorine which
will also absorb radiation of 253 nm. Care must be taken to
assure that free chlorine is absent before the mercury is reduced
and swept into the cell.- This may be accomplished by using an
excess of hydroxylamlne sulfate reagent (25 ml). In addition,
the dead air space in the BOD bottle must be purged before the
addition of stannous sulfate. Both inorganic and organic mercury
spikes have been quantitatively recovered from the sea water using
this technique.
4.4 Interference from certain volatile organic materials which will
absorb at this wavelength is also possible. A preliminary run
without reagents should determine if this type of interference is
present (see Appendix 11.1).
5. Apparatus
5.1 Atomic Absorption Spectrophotometer: (See Note 1) Any atomic
absorption unit having an open sample presentation area in which
to mount the absorption cell is suitable. Instrument settings
recommended by the particular manufacturer should be followed.
NOTE !:• Instruments designed specifically for the measurement
of mercury using the cold vapor technique are commercially available
and may be substituted for the atomic absorption spectrophotometer.
B-53
-------
Method 245.1 CLP-M (cone.)
5.2 Mercury Hollow Cathode Lamp: Vestinghouse WL-22847, argon filled,
or equivalent.
5.3 Recorder: Any multi-range variable speed recorder that is compatible
'with the UV detection system is suitable.
5.4 Absorption Cell: Standard spectrophotometer cells 10 cm long, having
quartz end windows may be used. Suitable cells may be constructed
from plexiglass tubing, 1" O.D. Z 4-1/2". The ends are ground per-
pendicular to the longitudinal axis and quartz windows (1" diameter
Z 1/16" thickness) are cemented in place. The cell is strapped to
a burner for support and aligned in the light beam by use of two 2"
by 2" cards. One Inch diameter holes are cut in the middle of each
card; Che cards are than placed over each end of the cell. The cell
is then positioned and adjusted vertically and horizontally to find
the maximum transmlttance.
5.5 Air Pump: Any peristaltic pump capable of delivering 1 liter of air
per minute may be used. A Masterflex pump with electronic speed
control has been found to be satisfactory.
5.6- Flowmeter: Capable of measuring an air flow of 1 liter per minute.
5.7 Aeration Tubing: A straight glass fit having a coarse porosity.
Tygon tubing is used for passage of the mercury vapor from the
sample bottle to the absorption cell and return.
5.8 Drying Tube: 6" Z 3/4" diameter tube containing 20 g of magnesium
perchlorate (see Note 2). The apparatus is assembled as shown in
Figure 1.
NOTE 2: In place of the magnesium perchlorate drying tube, a small
reading lamp with 60V bulb may be used to prevent condensation of
moisture Inside the cell. The lamp is positioned to shine on the
absorption cell maintaining the air temperature In the cell about
10°C above ambient.
6. Reagents
6.1 Sulfuric Acid, Cone: Reagent grade.
6.1.1 Sulfuric acid, 0.5 N: Dilute 14.0 ml of cone, sulfuric acid
to 1.0 liter.
6.2 Nitric Acid, Cone: Reagent grade of low mercury content (see Note
3). NOTE 3: If a high reagent blank is obtained, it may be neces-
sary eo distill the nitric acid.
B-54
-------
Method 245.1 CLP-H (cone.)
6.3
6.4
6.5
6.6
6.7
Scannous Sulfate: Add 25 g stannous sulface to 250 ml of 0.5
N sulfuric acid. Th'is mixture is a suspension and should be
stirred continuously during use. (Stannous chloride may be used
la place of stannous sulfate.)
Sodium Chloride-Hyroxylamine Sulfate Solution: Dissolve 12 g of
sodium chloride and 12 g of hydroxylamine sulfate in distilled
water and dilute to 100 ml. (Hydroxylamine hydrochloride may be
used in place of hydroxylamine sulface.)
Potassium Permanganate: 5Z solution, w/v. Dissolve 5 g of
potassium permanganate in 100 ml of distilled water.
Potassium Persulfate: 5Z solution, w/v.
persulfate In 100 ml of distilled wace.r.
Dissolve 5 g of potassium
Stock Mercury Solution: Dissolve 0.1354 g of mercuric chloride in
75 ml of distilled water. Add 10 ml of cone, nitric acid and
adjust the volume to 100.0 ml. 1 ml • 1 mg Hg.
+ 'BUBBLER
SAMPLE SOLUTION
IN BOO BOTTLE
ABSORPTION
CELL
SCRUBBER
CONTAINING
A MERCURY
ABSORBING
MEDIA
Figure 1. Apparatus for Flaneless Mercury Determination.
3-55
-------
Method 245.1 CLP-M (cone.)
6.8 Working Mercury Solution: Make successive dilutions of the stock
mercury solution to obtain a working standard containing 0.1 ug per
ml. This working standard and the dilutions of the stock mercury
solution should be prepared fresh daily. Acidity of the working
standard should be maintained at 0.15Z nitric acid. This acid
should be added to the flask as needed before the addition of the
aliquot.
7. Calibration
7.1 Transfer 0, 0.5, 1.0, 5.0 and 10.0 ml aliquots of the working
mercury solution containing 0 to 1.0 ug of mercury to a series of
300 ml BOO bottles. Add enough distilled water to each bottle to
make a total volume of 100 ml. Mix thoroughly and add 5 ml of cone.
sulfuric acid (6.1) and 2.5 ml of concr nitric acid (6.2) to each
bottle. -Add 15 ml of KMn<>4 (6.5) solution to each beetle and allow
to stand at least 15 minutes. Add 8 ml of potassium persulfate (6.6)
to each bottle and heat for 2 hours in a water bath maintained at
95"C. Cool and add 6 ml of sodium chloride-hydroxylamine sulfate
solution (6.4) to reduce the excess permanganate. When the solution
has been decolorized wait'30 seconds, add 5 ml of the stannous
sulfate solution (6.3) and immediately attach the bottle to the
aeration apparatus forming a closed system* At this point the
sample is allowed to stand quietly without manual agitation. The
circulating pump, which has previously been adjusted to a rate of
1 liter per minute, is allowed to run continuously (see Note A).
The absorbance will increase and reach maximum within 30 seconds.
As soon as the recorder pen levels off, approximately 1 minute,
open the bypass valve and continue"the aeration until the absorbance
returns to its minimum value (see Note 5). Close the bypass valve,
remove the stopper and frit from the BOD bottle and continue the
aeration. Proceed with the standards and construct a standard
curve by plotting peak height versus micrograms of mercury.
NOTE 4: An open system where the mercury vapor is passed through
the absorption cell only once nay be used instead of the closed
system.
NOTE 5: Because of the toxic nature of mercury vapor precaution
must be taken to avoid its Inhalation. Therefore, a bypass has
been included in the system to either vent the mercury vapor into
an exhaust hood or pass the vapor through some absorbing media,
such as:
a) equal voliimes of 0.1 M KMn04, and 10Z ^SOt
b) 0.252 iodine in a 32 a KI solution
A specially treated charcoal that will adsorb mercury vapor is also
available from Barnebey and Cheney, E. 8th Ave. and N. Cassidy St.,
Columbus, Ohio 43219, Cat #580-13 or 0580-22.
B-56
-------
Method 245.1 CLF-H (cone.)
8. Procedure
S.I Transfer 100 ml, or an aliquot diluted to 100 ml, containing not
more than 1.0 ug of mercury, to a 300 ml BOD bottle. Add 5 oil of
eulfuric acid (6.1) and 2.5 ml of cone, nitric acid (6.2) mixing
after each addlton. Add 15 ml of potassium permanganate solution
(6.5) to each sample bottle (see Note 6). For sewage samples
additional permanganate may be required. Shake and add additional
portions of potassium permanganate solution, if necessary, until the
purple color persists for at least 15 minutes. Add 8 ml of potassium
persulfate (6.6) to each bottle and heat for 2 hours in a water bath
.at 95'C.
NOTE 6: The same amount of KMn04 added to the samples should be
present in standards and blanks.
Cool and add 6 ml of sodium chloride-hydroxylamine sulfate (6.4) to
reduce the excess permanganate (see Note 7). After a delay of at
least 30 seconds add 5 ml of stannous sulfate (6.3) and immediately
attach the bottle to the aeration apparatus. Continue as described
under Calibration.
NOTE 7: Add reductant in 6 mL increments until KMn<>4 is completely
reduced.
9. Calculation
9.1 Determine the peak height of the unknown from the chart and read
the mercury value from the standard curve.
9.2 Calculate the mercury concentration in the sample by the formula:
ug Hg in 1,000
ug Hg/1 • aliquot X
volume of aliquot in ml
9.3 Report mercury concentrations as follows: Below 0.2 ug/1, 0.2U;
between 0.2 and 10 ug/1, one decimal; above 10 ug/1, whole numbers.
10. Appendix
10.1 While the possibility of absorption from certain organic substances
actually being present in the sample does exist, EMSU has noc encoun-
tered such samples. This is mentioned only to caution the analyse
of the possibility. A simple correction that may be used is as
follows: If an interference has been found to be present (4.4), the
sample should- be analyzed boch by using the regular procedure and
again under oxidizing conditions only, that is without the reducing
reagents. The true mercury value can then be obtained by subtracting
the two values.
B-57
-------
10.2 If additional sensitivity is required, a 200 ml sample wich
recorder expansion may be used provided the instrument does not
produce undue noise. Using a Coleman MAS-50 with a drying tube
if magnesium perchlorate and a variable recorder, 2 mv was set
to read full scale. With these conditions, and distilled water
solutions of mercuric chloride at concentrations of 0.15, 0.10,
0.05 and 0.025 ug/1 the standard deviations were +0.027, +0.0006,
+0.01 and +0.004. Percent recoveries at these levels were 107,
83, 84 and~~96Z, respectively.
10.3 Directions for the disposal of mercury-containing wastes are
given in ASTM Standards, Part 31, "Water", p. 349, Method D3223
(1976).
Bibliography
1. Kopp, J. F., Longbottom, M. C. and Lobring, L. B. "Cold Vapor
Method for Determining Mercury", AWWA, vol. 64, p. 20, Jan. 1972.
2. Annual Book of ASTM Standards, Part 31, "Water", Standard D3223-73,
p. 343 (1976).
3. Standard Methods for the Examination of Water and Wastewater 14th
Edition, p. 136 (1975).
B-58
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ATTACHMENT 5A
MERCURY
Method 245.2 CLP-M* (Automated Cold Vapor Technique)
1. Scope and Application
1.1 The working range is 0.2 to 20.0 ug Hg/1.
2. Summary of Method
2.1 The flaneless AA procedure is a physical method based on the
absorption of radiation at 253.7 nm by mercury vapor. The mercury
is reduced to the elemental state and aerated from solution. The
mercury vapor passes through a cell*positioned in the light path of
an atomic absorption spectrophotometer. Absorbance'Cpeak height) is
measured as a function of mercury concentration and recorded in the
usual manne r.
2.2 In addition to inorganic forms of mercury, organic mercurials may
also be present. These organo-mercury compounds will not respond
to the flameless atomic absorption technique unless they are firsc
broken down and converted to mercuric 'ions. Potassium permanganate
oxidizes many of these compounds, but recent studies have shown that
a number of organic mercurials, including phenyl mercuric acetate
and methyl mercuric chloride, are only partially oxidized by this
reagent. Potassium persulfate has been found to give approximately
100Z recovery when used as the oxidant with these compounds. There-
fore, an automated persulfate oxidation step following the automated
addition of the permanganate has been included to insure that organo-
mercury compounds, if present, will be oxidized to the mercuric ion
before measurement.
3. Sample Handling and Preservation
3.1 Until more conclusive data are obtained, samples should be preserved
by acidification with nitric acid to a pH of 2 or lower immediately
at the time of collection("•) (see Exhibit F).
* CLP-M Modified for the contract Laboratory Program
B-59
-------
Method 245.2 CLP-M (cont.)
4. Interference (see NOTE 1)
4.1 Some sea waters and waste-waters high in chlorides have shown a posi-
tive interference, probably due Co the formation of free chlorine.
4.2 Interference from certain volatile organic materials which will •
absorb at this wavelength is also possible. A preliminary run under
oxidizing conditions, without stannous sulfate, would determine if
this type of interference is present.
4.3 Formation of a heavy precipitate, in some wastewaters and effluents,
has been reported upon addition of concentrated sulfuric acid. If
this is encountered, the problem sample cannot be analyzed by this
method.
4.4 Samples containing solids must be blended and then mixed while being
sampled if total mercury values are to be reported.
NOTE 1: All the above interferences can be overcome by use of the
Manual Mercury method.
5* Apparatus
5.1 Technicon Auto Analyzer consisting of.:
5,1.1 Sampler II with provision for sample mixing.
5.1.2 Manifold.
5.1.3 Proportioning Pump II or III.
5.1.4 High temperature heating bath with two distillation coils
(Technicon Part #116-0163) in series.
5.2 Vapor-liquid separator (Figure 1).
5.3 ' Absorption cell, 100 mm long, 10 mm diameter with quartz windows.
5.4 Atomic Absorption Spectrophotometer (see Note 2): Any atomic
absorption unit having an open sample presentation area in which
to mount the absorption cell is suitable. Instrument settings
recommended by the particular manufacturer should be followed.
NOTE 2: Instruments designed specifically for the measurement of
mercury using the cold vapor technique are commercially
available and may be substituted for the atomic absorption
Spectrophotometer.
5.5 Mercury Hollow Cathode Lamp: Westinghouse VL-22847, argon filled,
or equivalent.
3-60
-------
Method 245.2 CLP-M (cone.)
5.6 Recorder: Any multi-range variable speed recorder that is compat-
ible with the UV detection system is suitable.
5.7 Source of cooling water for jacketed mixing coil and connector A-7.
5.8 Heat lamp: A small reading lamp with 60V bulb may be used to
prevent condensation of moisture inside the cell. The lamp is
positioned to shine on the absorption cell maintaining the air
temperature in the cell about 10'C above ambient.
6. Reagents
6.1 Sulfuric Acid, Cone: Reagent grade
6.1.1 Sulfuric acid, 2 N: Dilute 56 ml of cone, sulfuric acid to
1 liter with distilled water.
6.1.2 Sulfuric acid, 10Z: Dilute 100 ml cone, sulfuric acid to
1 liter with distilled water.
6.2 Nitric acid, Cone: Reagent grade of low mercury content.
6.2.1 Nitric Acid, 0.5Z Wash Solution: Dilute 5 ml of concentrated
nitric acid to I liter with distilled water.'
6.3 Stannous Sulfate: Add-50 g stanhous sulfate to 500 ml of 2 N
sulfuric aeid £6.1.1). This mixture is a suspension and should be
stirred continuously during use.
MOTE 3: Stannous chloride may be used in place of stannous sulfate.
6.4 Sodium Chloride-Hydroxylamlne Sulfate Solution: Dissolve 30 g of
sodium chloride and 30 g of hydroxylamine sulfate in distilled
water to 1 liter.
NOTE 4: Hydroxylamine hydrochloride may be used in place of
hydroxylamine sulfate.
6.5 Potassium Permanganate: 0.5Z solution, w/v. Dissolve 5 g of
potassium permanganate in 1 liter of distilled water.
6.6 Potassium Permanganate, 0.1 N: Dissolve 3.16 g of potassium
permanganate in distilled water and dilute to 1 liter.
6.7 Potassium Persulfate1: 0.5Z solution, w/v. Dissolve 5 g potassium
persulfate in 1 liter of distilled water.'
6.H Stock Mercury Solution: Dissolve 0.1354 g of mercuric chloride in
75 ml of distilled water. Add 10 ml of cone, nitric acid and
adjust the volume to 100.0 ml. . 1.0 ml • 1.0 mg Hg.
B-61
-------
Method 245.2 CLP-M (cone.)
6.9 Working Mercury Solution: Make successive dilutions of the stock
mercury solution (6.8) to obtain a working standard containing 0.1
ug per ml. This working standard and the dilutions of the stock
mercury solution should be prepared fresh daily. Acidity of the
working standard should be maintained at 0.15Z nitric acid. This
acid should be added to the flask as needed before the addition of
the aliquot. From this solution prepare standards containing 0.2,
0.5, 1.0, 2.0, 5.0, 10.0, 15.0 and 20.0 ug Hg/1.
6.10 Air Scrubber Solution: Mix equal volumes of 0.1 N potassium
permanganate (6.6) and 10Z sulfuric acid (6.1.2).
7. Procedure
7.1 Set up manifold as shown in Figure 2.
7.2 Feeding all the reagents through the system with acid wash solution
(6.2.1) through the sample line, adjust heating bath to 105°C.
7.3 Turn on atomic absorption spectrophotometer, adjust instrument
settings as recommended by the manufacturer, align absorption' cell
in light path for maximum transmlttance and place heat lamp directly
over absorption cell.
7.4 Arrange working mercury standards from 0.2 to 20.0 ug Hg/1 in sampler
and start sampling. Complete loading of sample tray with unknown
samples.
7.5 Prepare standard curve by plotting peak height of processed standards
against concentration values. Determine concentration of samples by
comparing sample peak height with standard curve.
NOTE 5: Because of the toxic nature of mercury vapor, precaution
must be taken to avoid its Inhalation. Venting the mercury vapor
into an exhaust hood or passing the vapor through some absorbing
media such as:
a) equal volumes of 0.1 N KMn04(6.6) and 10Z H2S04 (6.1.2).
b) 0.25Z iodine in a 32 KI solution, is recommended.
A specially treated charcoal that will adsorb mercury vapor is also
availale from Barnebey and Cheney, E. 8th Ave. and North Cassidy St.,
Columbus, Ohio 43219, Cat, #580-13 or #580-22.
7.6 After the analysis if complete put all lines except the H^SOt line
in distilled water to wash out system. After flushing, was out the
H2S04 line. Also flush the coils in the high temperature heating bach
by pumping stannous sulfate (6.3) through the sample lines followed
by distilled water. This will prevent build-up of oxides of manganese.
B-62
-------
Method 245.2 CLP-M (cont.)
Bibliography
1. Wallace R. A., Fulkerson, W., Shults, W. D., and Lyon, W. S., "Mercury
in the Environment-The Human Element", Oak Ridge National Laboratory,
ORNL/NSF-EP-1 p. 31, (January, 1971).
2. Hatch, W. R. and Ott, W. L., "Determination of Sub-Microgram Quantities
of Mercury by Atomic Absorption Specrophotometry". Anal. Chem. 40, 2085
(1968).
3. Brandenberger, H. and Bader, H., "The Determination of Nanogram Levels
of Mercury in Solution by a Flameless Atomic Absorption Technique",
Atomic Absorption Newsletter £, 101 (1967).
4. Brandenberger, h. and Bader, H., "The Determination of Mercury by
Flameless Atomic Absorption II, A Static Vapor Method", Atomic
Absorption Newsletter J.,53 (1968).
5. Goulden, P. D. and Afghan, B. K. "An Automated Method for Determining
Mercury in Water", Technlcon, adv. in Auto. Analy. .2, p. 317 (1970).
6. "Interim Methods for the Sampling and Analysis of Priority Pollutants in
Sediments and Fish Tissue," USEPA, Environmental Monitoring and Support
Laboratory, Cincinnatin, Ohio, august 1977,- revised Occtober 1980.
7. Op. clt. (#1), Methods 245.1 or 245.2.
B-63
-------
Method 245.2 CLP-M (cone.)
AIR AND
SOLUTION
M
AIR
OUT
7/23 T
0.7 cm 10
0.4 em ID
14 cm
SOLUTION
OUT
Figure 1. Vapor liquid separator.
B-64
-------
AA
SPECTROPNOTOMETER
Dl
MflflftflOO
0000
lUnn Ilia
Long
251. 7nn
20/hr
RECORDER
V«nt
Waste
€:
S-3
09
I
5
HEATING BATH
I PSI
i;
r.
l/.in
3.90
2.00 |'DO
3
CO MM03 Uash
g-
p.
3.90
SFi
^.90
3.90
JJ2.
3.90 co
i.20
1
Air
AIR
SCRUBBER
10Z SnS0
4
3Z NaCl
4
J.50
1.20
K?S]0|
2.76
(Con)
1.90
l.l'l
:?•
Sanplc
n
o
a
2JDO
Air
1.30
.51 KHn06
Proportioning Pump III
Figure 2. Mercury .Manifold AA-1.
* Acid Flem iCRUBBEft
** Teflon
••* Claa«
-------
ATTACHMENT 6
MERCURY (in Sediments)
Method 245.5 CLP-M* (Manual Cold Vapor Technique)
1. Scope and Application
1.1 This procedure measures total mercury (organic and inorganic) in
soils, sediments, bottom deposits and sludge type materials.
1.2 The range ofrthe method is 0.2 to 5 ug/g. The range may be extended
above or below the normal range by increasing or decreasing sample
size or through instrument and recorder control.
2. Summary of Method'
2.1 A weighed portion of the sample is acid digested for 2 minutes at
95'C, followed by oxidation with potassium permanganate and potassium
persulfate. Mercury in the digested sample is then measured by the
conventional cold vapor technique.
2.2 An alternate, digestion involving the use of an autoclave is describe
in (a.2).
3. Sample Handling and Preservation
3.1 Because of the extreme sensitivity of the analytical procedure and
the omnipresence of mercury, care- must be taken to avoid extraneous
contamination. Sampling devices and sample containers should be
.ascertained to be free of mercury; the sample should not be exposed
to any condition in the laboratory that may result in contact or
air-borne mercury contamination.
3.2 Refrigerate solid sanples upon receipt.
3.3 The sample should be analyzed without drying. A separate I solids
determination is required. (Exhibit D, Attachment 9).
4. Interferences
4.1 The same types of interferences that may occur in water samples are
also possible with sediments, i.e., .sulfides, high copper, high
chlorides, etc.
*CLP-M modified for the Contract Laboratory Program
B-66
-------
Method 245.5 CLP-M (cone.)
4.2 Volatile materials which absorb at 253.7 nm will cause a positive
interference. In order to remove any interfering volatile materials,
purge the dead air space in the BOD bottle before the addition of
stannous sulfate.
4.3 Sample containing high concentrations of oxidizable organic materials,
as evidenced by high chemical oxygen demand values, may not be com-
pletely oxidized by this procedure. When this occurs, the recovery
of organic mercury will be low. The problem can be eliminated by
reducing the weight of the original sample or by Increasing the
amount of potassium persulfate (and consequently stannous chloride)
used in the digestion.
5. Apparatus
5.1 Atomic Absorption Speetrophotometer (see Note 1): Any atomic absorp-
tion unit having an open sample presentation area In which to mount
the absorption cell is suitable. Instrument settings recommended by
the particular manufacturer should be followed.
NOTE 1: Instruments designed specifically for the measurement of
mercury using the cold vapor technique are commercially available
and may be substituted for the atomic absorption spectrophotometer.
5.2 Mercury Hollow Cathode Lamp: Westlnghouse WL-22847, argon filled,
or equivalent.
5.3 'Recorder: Any multi-range variable speed recorder that is compatible
with the UV detection system is suitable.
5.4 Absorption Cell: Standard spectrophotometer cells 10 cm long, having
quartz end windows may be used. Suitable cells many be constructed
from pexlglass tubing, 1" O.D. X 4-1/2". The ends are ground perpen-
dicular to the longitudinal axis -and quartz windows U" diameter X
1/16" thickness) are cemented in place. Gas inlet and outlet ports
(also of plexiglass but 1/4" 0.0.) are attached approximately 1/2"
from each end. The cell is strapped to a burner for support and
aligned in the light beam to give the maximum transmittance.
NOTE 2: Two 2" X 2" cards with one inch diameter holes may be
placed over each end of the cell to assist in positioning the cell
for maximum transmittance.
5.5 Air Pump: Any peristaltic pump capable of delivering 1 liter or air
per minute may be used. A Masterflex pump with electronic speed
control has been found to be satlsfatory. (Regulated compressed air
can be used in an open one-pass system'.}
5.6 Flowmeter: Capable of measuring an air flow of 1 liter per minute.
B-67
-------
Method 245.5 CLP-M (cont.)
5.7 Aeration Tubing: Tygon tubing is used for passage of the mercury
vapor from the sample bottle to the absorption cell and return.
Straight glass tubing terminating in a coarse porous frit is used
for sparging air into the sample*
5.8 Drying Tube: 6" X 3/4" diameter tube containing 20 g of magnesium
perchlorate (see Note 3). The apparatus is assembled as shown in
the accompanying diagram.
NOTE 3: In place of the magnesium perchlorate during tube, a small
reading lamp with 60V bulb may be used to prevent condensation of
moisture inside the cell. The lamp Is positioned to shine on the
absorption cell maintaining the air temperature in the cell about
1U*C above ambient.
6. Reagents
6.1 Sulfuric acid, cone.: Reagent grade of low mercury content.
6.2 Nitric acid, cone.: Reagent grade of low mercury content.
6.3 Stannous Sulfate: Add 25 g stanaous sulfate to 250 ml of 0.5 N
•ulfuric acid (6.2). This mixture is a suspension and should be
stirred continuously during use.
6.4 Sodium Chloride-Hydroxylamine Sulfate Solution: Dissolve 12 g of
sodium chloride and 12 g of hydroxylamine sulfate in distilled water
and dilute to 100 ml.
NOTE 4: A 102 solution of stannous chloride may be substituted
for (6.3) and hydroxylamine hydrochloride may be used in place of
hydroxylamine sulfate In (6.4).
6.5 Potassium Permanganate: 52 solution, v/v. Dissolve 5 g of potassium
permanganate in 100 ml of distilled water.
6.6 Potassium Persulfate: 52 solution, w/v. Dissolve 5 g of pocassium
persulfate In 100 ml of distilled water.
6.7 Stock Mercury Solution: Dissolve 0.1354 g of mercuric chloride in
75 ml of distilled water. Add ml of cone, nitric acid and adjust
the volume to 100.0 ml. 1.0 - 1.0 mg Kg.
6.8 Working Mercury Solution: Make successive dilutions of the stock
mercury solution (6.7) to obtain a working standard containing 0.1
ug/ml. This working* standard and the dilution of the stock mercury
solutions should be prepared fresh dally. Acidity of the working
standard should be maintained at 0.152 nitric acid. This acid
should be added to the flask as needed before the addition of che
aliquot.
B-68
-------
Method 245.5 CLP-M (coat.)
7. Calibration
7.1 Transfer 0, 0.5, 1.0, 5.0 and 10 ml aliquots of Che working mercury
solutions (6.8) containing 0 to 1.0 ug of mercury to a series of 300
ml BOD bottles. Add enough distilled water to each bottle to make a
total volume of 10 ml. Add 5 ml of cone. ^SOfc (6.1) and 2.5 ml of
cone. H1K>3 (6.2) and heat 2 minutes in a water bath at 95°C. Allow
the sample to cool and add 50 ml distilled water, 15 ml of KMn04
solution (6.5) and 8 ml of potassium persulfate solution (6.6) to
each bottle and return to the water bath for 30 minutes. Cool and
add 6 ml of sodium chloride-hydroxylamine sulfate solution (6.4) to
reduce the excess permanganate. Add 50 ml of distilled water.
Treating each bottle individually, add 5 ml of stannous sulfate
solution (6.3) and Immediately attach the bottle to the aeration
apparatus. At this point the sample is allowed-Co stand quietly
without manual agitation. The circulating pump, which has previously
been adjusted to a rate of 1 liter per minute, is allowed to run
continuously. The absorbance, as exhibited either on the spectro-
photometer or the recorder, will Increase and reach maximum within
30 seconds. As soon as the recorder pen levels off, approximately 1
minute, open the bypass valve and continue Che aeration until the
abscrbance returns to its minimum value (see Note 5). Close the
bypass valve, remove the fritted tubing from the BOD bottle and
continue the aeration. Proceed with the standards and construct a
standard curve by plotting peak height versus micrograms of mercury.
NOTE 5: Because of the toxic nature of mercury vapor precaution
must be taken to avoid its inhalation. Therefore, a bypass has been
included in the system to either vent Che mercury vapor into an
exhaust hood or pass the vapor through some absorbing media, such as:
a) equal volumes of 0.1 N KMn04 and 10Z H2S04
b) 0.25Z iodine in a 3Z KI solution
A specially treated charcoal that will absorb mercury vapor is also
available from Barnebey and Cheney, E. 8th Avenue and N. Cassidy
Sreet, Columbus, Ohio 43219
b. Procedure
8.1 Weigh a representative 0.2 g portion of wet sample and place in the
bottom of a BOD bottle. Add 5 mL of sulfurlc acid (6.1) and 2.5 mL
of concentrated nitric acid (6.2) mixing after each addition. Heat
two minutes in a water bath at 95*C. Cool, add 50 ml distilled
water, 15 mL potassium permanganate solution (6.5) and 8 mL of
potassium persulfate solution (6.6) to each sample bottle. Mix
thoroughly and place in the water bath for 3U minutes at 95°C. Cool
and add 6 mL of sodium chloride-hydroxylamine sulfate (6.4) to reduce
the excess permanganate. Add 55 mL of distilled water. Treating
each bottle individually, add 5 mL of stannous sulfate (6.3) and
immediately attach the bottle to the aeration apparatus. Continue
as described under (7.1)..
8-69
-------
Method 245.5 CLP-M (cent.)
8.2 An alternate digestion procedure employing an autoclave may also be
used. In this method 5 ml of cone* ^SQ^ and 2 ml of cone. HN03
are added Co the 0.2 g of sample. 5 ml of saturated KMnO^ solution
and 8 ml of potassium persulfate solution are added and the bottle is
covered with a piece of aluminum foil. The sample is autoclaved at
121*C and 15 Ibs. for 15 minutes. Cool, make up to a volume of 100
ml with distilled water and add 6 ml of sodium chloride-hydroxylamine
sulfate solution (6.4) to reduce the excess permanganate. Purge the
dead air space and continue as described under (7.1).
9. Calculations
9.1 Measure the peak height of the unknown from the chart and read the
mercury value from the standard curve.
9.2 Calculate the mercury concentration in the sample by the formula:
ug Hg in the aliquot
ug Bg/g - wt of the aliquot in gms (based upon dry wt of the sample)
9.3 Report mercury concencrations as follows: Below 0.1 ug/gm, 0.1U;
between 0.1 and 1 ug/gm, to the nearest 0.01 ug; between 1 and 10
ug/gm, to nearest 0.1 ug; above 10 ug/gm, to nearest ug.
Bibliography
1. Bishop, J. N., "Mercury in Sediments'", Ontario Water Resources
Conm., Toronto, Ontario, Canada, 1971.
2. Salma, M., private communication, EPA Cal/Nev. Basin Office,
Almeda, California.
3. "Interim Methods for the Sampling and Analysis of Priority Pollutants
in Sediments and Fish Tissue," USEPA Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio, August 1977, Revised October
1980.
4. Op. eit. («), Methods 245.1 or 245.2.
B-70
-------
EZHIBIT E
QUALITY ASSURANCE/QUALITY CONTROL REQUIREMENTS
B-7I
-------
GENERAL QA/QC CONSIDERATIONS
Standard laboratory practices for laboratory cleanliness as applied
to glassware and apparatus oust be adhered to. Laboratory practices with
regard to reagents, solvents, and gases should also be adhered to. For
additional guidelines regarding these general laboratory procedures, please
see Sections 4 and 5 of the Handbook for Analytical Quality Control in
Water and Wastevater Laboratories EPA-600/4-79-019, USEPA Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio, March 1979.
B-72
-------
QUALITY ASSURANCE REQUIREMENTS
The quality assurance/quality control (QA/QC.) procedures defined below must
be used by the Contractor when performing the methods specified in Exhibit
D. When additional QA/QC procedures are specified in the methods in Exhibit
D, the Contractor must also follow these procedures. The cost of performing
all QA/QC procedures specified in this Statement of Work is included in the
price of performing the bid lot.
The purpose of this document is to provide a uniform set of procedures for
the analysis of inorganic constituents of samples, documentation of methods
and their performance during a survey, and verification of the sample data
generated. The program will also assist laboratory personnel in recalling
and defending their actions under cross examination if required to present
court testimony in enforcement ease litigation.
The prime function of the QA/QC program outlined here is the definition of
procedures for the evaluation and documentation of sampling and analytical
methodologies and the reduction and reporting of data. The objective is to
provide a uniform basis for sample collection and handling, instrument and
methods maintenance, performance evaluation, and analytical data gathering
and reporting. Although it is impossible to address all analytical situations
In one document, the approach taken here is to define minimum requirements
for all major steps relevant to any inorganic analysis. In many instances
where methodologies are available, specific quality control procedures are
Incorporated into the method documentation. Ideally, samples involved in
enforcement actions are analyzed only after the methods have met the minimum
performance and documentation requirements described in this document.
The Contractor must participate In the Laboratory Audit and Intercomparlson
Study Program run by EPA, EMSL-Las Vegas. The Contractor can expect to
analyze two samples per three-month contract period for this program.
The Contractor must perform and report to SMO and EMSL/LV on Form XI quarterly
verification of instrument detection limits (IDL) by the method specified in
Exhibit E, by type and model for each instrument used on this contract. All
the IDLs must meet the CXDLs specified in Exhibit C. For ICP methods, the
Contractor must also report linearity range verification, all interelement
correction factors, wavelengths used and integration times on a quarterly
basis on Forms XII and XIII.
For QA/QC procedures, two different types of "samples" are specified. A
"sample received" is the field or PE sample each of which has an EPA number.
An "analytical sample" is each "analysis" performed (i.e. each cup, tube or
container in autosamplers, etc),. A "frequency of 102" means once every 10
analytical samples.
The Contractor must report all QC data in the exact format shown in Forms
II - XIII (See Exhibit B).
B-73
-------
This section outlines the minimum QA/QC operations necessary to satisfy the
analytical requirements of the contract. The following (JA/QC operations must
be performed as stated in this exhibit:
1. Initial Calibration and Calibration Verification
2. Continuing Calibration Verification
3. Preparation Blank Analysis
4. Interference Check Sample Analysis
5. 1C? Serial Dilution Analysis
6. Matrix Spike Analysis
7. Duplicate Sample Analysis
8. Furnace AA QC Analysis (Method of Standard Additions may be required
under certain conditions).
9. Laboratory Quality Control Sample Analysis
1. Initial Calibration and Calibration Verification
Guidelines for instrumental calibration are given in EPA 600/4-79-020
and/or Exhibit D. Instruments must be calibrated daily and each time
the instrument is set up*
Instrument Calibration
For atomic absorption systems, calibration standards are prepared by
diluting the stock mecal solutions at the time of analysis. Low calibra-
tion standards must be prepared fresh each time an analysis is to be made
and discarded after use. Prepare'a blank and at least three calibration
standards in graduated amounts in the appropriate range* One atomic
absorption calibration standard must be at the CSDL except for mercury.
The calibration standards must be prepared using the same type of acid- or
combination of acids and at the same concentration as will result in the
samples following sample preparation. Beginning with the blank and working
toward the highest standard, aspirate or Inject the solutions and record
the readings. Calibration standards for furnace- procedures should be
prepared as described in the individual methods for that metal.
For cyanide and mercury, follow the calibration procedures outlined in
Exhibit D. One cyanide calibration standard must be- at the CSDL. For 1C?
•y8cams, calibrate the instrument according to instrument manufacturer's
recommended procedures.
Initial Calibration Verification Standards
After the ICP, AA and cyanide systems have been calibrated, the accuracy
of the initial calibration shall be verified and documented for every
analyce by the analysis of EPA Initial Calibration Verification Solution.
8-74
-------
The initial calibration verification standard must be run at each wave-
length used for analysis.
If the Initial Calibration Verification Solution is not available from
EPA, or where a certified solution of an analyte is not available from any
source, analyses shall be conducted on an independent standard at a concen-
tration other than that used for calibration, but within the calibration
range* An independent standard is defined as a standard composed of the
analytes from a different source than those used in the standards for the
initial calibration. For ICP, the Initial Calibration Verification Solu-
tion must be run at each wavelength used in the analysis of the sample.
When measurements exceed the control limits of Table 1, the analysis must
be terminated, the problem corrected, the instrument recalibrated, and the
calibration reverified.
The values for the Initial and subsequent continuing calibration veriflea-
ions shall be recorded on Form II (see Exhibit B) for ICP, AA, and cyanide
analyses, as indicated.
Instrument Detection Limit Determination
Before any field samples are analyzed under this contract, the•instrumental
detection limits (in ug/L) must be determined within 30 days of the start
of the analyses and at least quarterly (every 3 months), and must meet the
levels specified in Exhibit C. The instrumental detection limits (in ug/L)
•hall be determined by multiplying by 3, the average of the standard devia-.
tions obtained on three nonconsecutive days from the analysis of a standard
solution (each analyte in reagent water) at a concentration 3-5 times IDL,
with 7 consecutive measurements per day*
For each Case, IDLs must be reported on QC Report Fora VII. If multiple
AA Instruments are used for the analysis of an element within a Case, the
highest IDL for the AAs must be reported for that Case. Use the same
reporting procedure for multiple ICPs.
Linear Range Analysis
To verify linearity near the CRDL for ICP analysis, the contractor must
analyze an ICP standard at two times the CRDL at the beginning and end of
each sample analysis run, or a minimum of twice per 8 hour working shift,
whichever Is more frequent. This standard must be run for all elements
analyzed by ICP except Al, Be,- Ca, Fe, Mg, Ha and K.
For ICP analysis, a linear range verification check standard must be
analyzed and reported quarterly for each element on Form XII. The standard
must be analyzed during a routine analytical run performed under this con-
tract. The analytically determined concentration of this standard must be
wiehln +_ 52 of the true value. This concentration is the upper llmlc of
the ICP~iinear range beyond which results cannot be reported under this
contract.
B-75
-------
Calibration Blank
A calibration blank is analyzed each time Che instrument is calibrated, at
the beginning and the end of the run, and at a frequency of 10Z during the
run. The results for the calibration blank solution shall be recorded on
Fora III for ICP, AA and cyanide analyses, as indicated. Blanks are to be
reported down to the instrument detection limit. If this blank result is
greater than the CRDL (Exhibit C), terminate analysis, correct the problem
and recalibrate.
2. Continuing Calibration Verification
To assure calibration accuracy during each analysis run, one of the follow
ing standards is to be used for continuing calibration verification and must
be analyzed for each analyte at a frequency of 10Z or every 2 hours during
an analysis run, whichever is more frequent. The standard must also be
analyzed for each analyte at the beginning of the run and after the last
analytical sample. The analyte concentrations in the continuing calibration
standard must be one'of the following solutions at or near the mid-range
levels of the calibration curve:
1. EPA Solutions
2. NBS SUM 1643a
3. A contractor-prepared standard solution
TABLE 1. INITIAL AND CONTINUING CALIBRATION VERIFICATION
CONTROL LIMITS FOR INORGANIC ANALYSES
Z of True Value (EPA Set)
Analytical Method Inorganic Species Low Limit High Limit
ICP/AA
Cold Vapor AA
Other
Metals
Mercury
Cyanide
90
80
85
110
120
115
The same continuing calibration standard must be used throughout the analysis
runs for a case of samples received.
If the deviation of the continuing calibration verification is greater than
the Control Limits specified in Table 1, the instrument must be recalibrated
and the preceding 10 samples reanalyzed for the analyses affected. Information
regarding the continuing verification of calibration shall be recorded on Fora
II (see Exhibit B) for ICP, AA and cyanide as indicated.
B-76
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3. Preparation Blank Analysis
At least one preparation blank (or reagent blank), consisting of deionized
distilled water processed through each sample preparation procedure (i.e.
water, solids) performed for each Case, must be prepared and analyzed with
every 20 samples received, or with each batch* of samples digested whichever
is more frequent (see Exhibit D). The first 20 samples of a Case are to be
assigned to preparation blank one,'and the second 20 samples to preparation
blank two, etc. (see Form III). Each data package must contain'the results of
all the preparation blank analyses associated with the samples in that Case.
This blank is to be reported for each Case and used in all analyses to
ascertain whether sample concentrations reflect contamination in the
following manner:
1. If the concentration of the blank is less than or equal to the con-
tract required detection level (Exhibit C), no correction of sample
results is performed.
2. If the concentration of the blank is above the contract required de-
tection level: For any group of samples associated with a particular
blank, the concentration of the sample with the least concentrated
analyte oust be 10X the blank concentration, or all samples associated
with the blank and less than 10 times the blank concentration must be
redigested and reanalyzed, with the exception of an Identified aqueous-
soil field blank. The sample value is not to be corrected for the
blank value.
The values for the preparation blank shall be recorded in ug/L on Fora
III (see Exhibit B) for ICP, AA, and cyanide analyses, as indicated.
4. ICP Interference Cheek Sample Analysis
To verify inter-element and background correction factors the Contractor
oust analyze and report the results for an 1C? Interference Check Sample at
the beginning and end of each sample analysis run (or a minimum of twice per
8 hour working shift, whichever is most frequent). The ICP Interference
Cheek Sample must be obtained from EPA (EMSL/LV) if available. Results for
the check sample analysis during the analytical runs must fall within Che
control Halt of ±202 of the true value for the analytes included in the
Interference Check Sample. If not, terminate the analysis, correct the
problem, recalibrate, reverlfy the calibration, and reanalyze the samples.
If the ICP Interference Check Sample is not available from EPA, an independ-
ent ICP Cheek Sample must be prepared with interferent and analyte concen-
trations at the levels specified in Table 2 (Exhibit E). The mean value and
standard deviation must be 'established by initially analyzing the check sam-
ples at lease 5 times repetitively for each parameter on Form IV. Resulcs
must fall within the control limit of +20Z of the established mean value.
Results of all Interference Check Sample analyses for all ICP parameters
must be recorded on Form IV (see Exhibit B).
*A group of samples prepared ac the same time.
B-77
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5. 1C? Serial Dilution Analysis
Prior to reporting concentation data for the analyte elements, the Con-
tractor must analyze and report the results of Che ICP Serial Dilution
Analysis. The ICP Serial Dilution Analysis must be performed on each
group of samples of a similar matrix type (i.e. water, soil) and concen-
tration (i.e. low, medium) for each Case of samples, or for each 20 samples
received, whichever is more frequent. Samples, identified as field blanks
cannot be used for serial dilution analysis. If the analyte concentration
Is sufficiently high (minimally a factor of 10 above the instrumental
detection limit after dilution), an analysis of a 1:4 dilution must agree
within 10 percent of the original determination. If the dilution analysis
Is not within 10Z, a chemical or physical Interference effect should be
suspected, and the data must be flagged with an "E." Serial dilution
results must be reported on QC Report Form IX.
6. Spiked Sample Analysis
The spiked sample analysis is designed to provide information about the
effect of che sample matrix on the digestion and measurement methodology.
The spike is added before the digestion and prior to any distillation
steps (I.e., CH~). At lease one spiked sample analysis must be performed
on each group of samples of a similar matrix type (i.e. water, soil) and
concentration (i.e. low, medium) for each Case of samples or for each 20
samples received, whichever is more frequent.* Samples identified as
field blanks cannot be used for spiked sample analysis. The analyte spike
must be added in Che amount given in Table 3 (Exhibit E) for each element
analyzed. If. two analytical methods are used to. obtain the reported
values for the same element for a Case of samples (i.e. 1C?, GFAA), spike
samples must be run by each method used. If the spike recovery is not
within the limits of 75-125Z, the data of all samples received associated
with that spiked sample must be flagged with the letter "N". An excep-
tion to this rule is granted in situations where the sample concentration
exceeds the spike concentration by a factor of four or more. In such a
case, the spike recovery should not be. considered and the data shall be
reported unflagged even if the percent recovery does not meet the 75-125Z
recovery criteria. In the instance where there is more than one spiked
sample per matrix per Case, if one spike sample recovery is not within
contract criteria, flag all the samples of the same matrix in the Case.
Individual component percenc recoveries (ZR) are calculated as follows:
ZRecovery - (SSR-SR) x 100
SA
Where SSR - Spiked Sample Result
SR • Sample Result
SA - Spike Added
When sample concentration is less than the instrument detection limit, use
SR • 0 for purposes of calculating ZRecovery. The spiked sample results
must be reported on Form V for ICP, AA and cyanide analyses, as indicated.
*EPA may require additional spike sample analysis, upon request, for which the
contractor will be paid.
B-78
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TABLE 2. 1NTERFERENT AND ANALITE ELEMENTAL CONCENTRATIONS USED FOR 1C?
INTERFERENCE CHECK SAMPLE
Analytes
Ba
Be
Cd
Co
Cr
Cu
Mn
Ni
Fb
V
Zn
(mg/L)
0.5
0.5
1.0
0.5
0.5
0.5
0.5
1.0
1.0
0.5
1.0
Interferents
AI
Ca
Fe
Hg
(mg/L)
500
500
200
500
TABLE 3. SPIRING LEVELS1 FOR SPIKED SAMPLE ANALYSIS
Element
Aluminum
Antimony
Arsenic
Barium
Beryllium
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Mercury
Nickel
Potassium
Selenium
Silver
Sodium
Thallium
Vanadium
Zinc
Cyanide
For ICP/AA
(ug/L)
Water
2,000
500
2,000
50
50
*
200
500
250
1,000
500
*
200
400
*
50
*
500
200
Sediment-1
*
500
2,000
50
50
*
200
500
250
*
500
*
500
500
*
50
*
500
500
For
Water
100
20
5
20
10
50
Furnace AA
(ug/L)
Sediment1
.
100
40
5
50
10
50
Other
(ug/L)
1
100
NOTE: Elements without spike levels and not designated with an asterisk, should
be spiked at appropriate levels t
levels shown indicate concentrations in the final dlgestate of the spiked
sample (200 mL FV)
No spike required.
B-79
-------
Duplicate Sample Analysis
Ac least one duplicate sample must be analyzed from each group of samples
of a similar matrix type (i.e. water, soil) and concentration (i.e. low,
medium) for each Case of samples or for each 20 samples received, whichever
is more frequent.* Samples identified as field blanks cannot be used for
duplicate sample analysis. If two analytical methods are used to obtain the
reported values for the same element for a Case of samples (i.e. ICP, GFAA),
duplicate samples must be run by each method used. The relative percent
differences'*' (RPD) for each component are calculated as follows:
RPD - " , 100
Where RPD • Relative Percent Difference
D} • First Sample Value
D£ • Second Sample Value (duplicate)
The results of the duplicate sample analysis must be reported on Form VI.
(See, Exhibit B). A control limit of ± 20Z for RPD shall be used for sample
values greater than 5 times the contract required detection level (CRDL).
A control limit of + the CRDL shall be used for sample values less than
5 times the CRDL (Exhibit C), and this control limit (+CRDL) should be
entered in the "Control Limit" column on Form VI. If one result is above
the 5 x CRDL level and the other is below, use the + CRDL criteria. If
either sample value is less than the CRDL, the RPD is not calculated and
is indicated as "NC" on Fora VI.
If the duplicate 'sample results are outside the control limits, flag all
the data for samples received associated with that duplicate sample with
an "*' on Forms I and V. In the instance where there is more than one
duplicate sample per Case, if one duplicate result is not within contract
criteria, flag all the samples of the same matrix in the Case. The percent
difference data will be used by EPA to evaluate the long-term precision of
the methods for each parameter. Specific control limits for each element
will be added to Form VI at a later date based on these precision results.
8. Furnace Atomic Absorption (AA) QC Analysis
Because of the nature of the Furnace AA technique, the special procedures
summarized in Figure 1 will be required for quantltation. (These procedures
are not meant to replace those included in Exhibit D of this document, but
will supplement the guidance provided therein.)
*EPA may require additional duplicates be analyzed, on request, for
which the contractor will be paid.
^Relative percent difference is equivalent to relative range of duplicates (RR).
B-80
-------
Figure 1.
FURNACE ATOMIC ABSORPTION ANALYSIS SCHEME
PREPARE AND ANALYZE SAMPLE
AND ONE SPIKE (2 X C.R.D.L.)
(double injections required)
ANALYSIS WITHIN CALIBRATION RANGE
NO
DILUTE SAMPLE
YES
RECOVERY OF SPIKE >40%
YES
if NO, repeac only once
if StillwI BTJIC DATA UTTH AN "E"
NO
SAMPLE ABSORBANCE >50Z of SPIKE ABSORBANCE*
NO
YES
SPIKE RECOVERY <85Z OR >115Z
NO
1
YES
QUANTITATE BY MSA WITH 3 SPIKES
AT 50, 100 AND 150Z OF SAMPLE
ABSORBANCE
(only single injections required)
CORRELATION COEFFICIENT >0.995
if NO,
repeac only once
YES
FLAG DATA WITH "a
if still NO
REPORT SAMPLE AS
-------
1. All furnace analyses, except during Full Methods of Standard Addition
(MSA), will require duplicate injections for which the average absorb-
anee or "concentration" will be reported. All analyses must fall
within the calibration range. The raw data package oust contain
both absorbance or "concentration" values, the average value and che
relative standard deviation (RSD) or'coefficient of variation (CV).
For concentrations greater than CRDL,' che duplicate injection readings
oust agree within 202 RSD or CV, or che sample must be rerun once.
If the readings are still out, flag the value with an "M" on Form I.
2. All furnace analyses for each sample will require at least a single
analytical spike to determine if the MSA will be required for quanci-
tation. Analytical spikes are not required on the predlgest spike
samples. The spike* will be required to be at a concentration (in
the sample) twice the contract required detection limit (CRDL). The
percent (ZR) of the spike, calculated by the sane formula as Spiked
Sample analyses (Exhibit E), will then determine how the sample will
be quantitated as follows:
a) If the spike recovery is less than 40Z, the saaple must be diluted
and rerun with another spike. Dilute che sample by a factor of 5
to 10 and rerun. This seep oust only be performed once. If after
the dilution the spike recovery is still <40Z, report data and
flag with an "E" to indicate interference problems.
b) If the spike recovery is-greater than 40Z and the sample absorbance
or concentration is <50Z of the spike4", report the sample as less
than the CRDL or less than the CRDL times the dilution factor if
the sample was diluted.
c) If the sample absorbance or concentration is >50Z of the spike*
and the spike recovery is between 85Z and 115Z, the sample should
be quantitated directly from the calibration curve.
d) If the sample absorbance or concentration is >50Z of the spike*
and the spike recovery is less than 85Z or greater than 115Z, the
sample must be quantitated by MSA.
3. The following procedures will be incorporated into MSA analyses.
a) Data from MSA calculations must be within the linear range as
determined by the calibration curve generated at the beginning of
the analytical run.
•Spikes are post dlgesc spikes (to be prepared prior to analysis by adding a
known quantity of the analyte to an aliquot of the digested sample. The
unspiked sample aliquot must be compensated for any volume change in che spike
samples by addition of deionized water to the unspiked sample aliquot.
"''"Spike" is defined as (absorbance or concentration of spike sample) minus
(absorbance or concentration of the saaple).
-------
b) The sample and three spikes oust be analyzed consecutively for
MSA quantisation (the "initial" spike run data is specifically
excluded frotn use la the MSA quantication). Only single injec-
tions are required for MSA quantitation.
c) Spikes* should be prepared such that:
• Spike 1 ia approximately 502 of the sample absorbance.
- Spike 2 is approximately 100Z of the sample absorbance.
- Spike 3 is approximately 1502 of the sample absorbance.
d) The data for each MSA analysis should be clearly identified in
the raw data documentation along with the slope, intercept and
correlation coefficient (r) for the least squares fit of the data
and Che results reported on Form VIII. Reported values obtained
by MSA are flagged on the data sheet (Fora I) with the letter "V".
e) If the correlation coefficient (r) for a particular analysis is
less than 0.995 the MSA analyses oust be repeated once. If the
correlation coefficient is still <0.995, the results on Form I
oust be flagged with "+".
9. .Laboratory Control Sample Analysis
Laboratory Control Sample (LCS) - Aqueous and solid laboratory quality
control samples must be analyzed for each analyte using the saae sample
preparation and analytical methods employed for the EPA samples received.
The aqueous LCS solution must be obtained from EPA (if unavailable, the
EPA Initial Calibration Verification solution may be used).' The aqueous
LCS must be prepared and analyzed with the samples for each of the pro-
cedures applied to each case of samples received. One aqueous LCS muse
be analyzed for every 20 samples received, or for each batch* of samples
digested whichever is more frequent (see Exhibit D). Each data package
must contain the results of all the LCS analyses associated with the
samples on that Case. For cyanide, the distilled mid-range calibration
standard may be used as the aqueous LCS (see Section 8.3.2.1, Exhibit D).
An aqueous LCS is not required for mercury analysis.
*Spikes are post digest spikes to be prepared prior to analysis by adding
a known quantity of the analyse to an aliquot of the digested sample. The
unspiked sample aliquot must be compensated for any volume change in the
spiked samples by addition of deionlzed water to the unspiked sample aliquot,
+A group of samples prepared at the same time.
.8-83
-------
All aqueous LCS results will be reported on Form VII in terms of true
concentration and percent recovery (ZR) as calculated by:
ZR • (Observed/True) x 100
where "observed" is the measured concentration. If the Z recovery far
the aqueous LCS falls outside the control limits of 80Z - 120%, the
analyses must be terminated, the problems corrected and the previous
samples associated with that LCS re-analyzed (ie., previous 19 samples
or the batch of samples from the case).
Once a month, a solid LCS, available from EMSL-LV oust be prepared and
analyzed using each of the procedures applied to the solid samples received.
If this EPA solid LCS is unavailable, other EPA Quality Assurance Check
samples or other certified materials may be used.
The monthly results of the solid LCS samples should be reported on a
duplicate Form VII and submitted monthly to EMSL/Las Vegas and SMO on
the 15th of every month.
If the percent recovery for the solid LCS sample is outside the control
limits established by EPA, no further sample analyses may be done until
the analytical problems are solved, and satisfactory LCS results are
obtained.
5-84
-------
EXHIBIT G
CHAIN-OF-CUSTODY AND DOCUMENT
CONTROL PROCEDURES
B-85
-------
NOTES
The contractor shall not deviate from the procedures described herein without
Che prior written approval of the Contracting Officer: Provided, that the
Contracting Officer nay ratify in writing such deviation and such ratification
shall constitute the approval required herein.
B-86
-------
Specificaclons for Chain-of-Cuscody and
Document Control Procedures
The contractor oust have written standard operating procedures (SOP) for
receipt of samples, maintenance of custody, tracking the analysis of samples
and assembly of completed data and must follow these SOPs. These procedures
are necessary to ensure that analytical data collected under this contract
are acceptable for use in EPA enforcement case preparations and litigation.
The Contractor's SOPs shall provide mechanisms and documentation to meet each
of the following specifications and shall be used by EPA for the basis for
laboratory evidence audits.
1. The contractor shall have a designated sample custodian responsible for
receipt of the samples.
2. The contractor shall have written SOPs for receiving and logging in of
the samples. The procedures shall include documentation of the sample
condition, maintenance of custody and sample security and documentation
of verification of sample tag information against custody records.
3. The contractor shall have written SOPs for maintenance of the security
of the sample after log in and shall demonstrate security of the sample
storage and laboratory areas.
4. The contractor shall have written SOPs for tracking the work performed on
any particular sample. The tracking system shall include standard data
logging formats, logbook entry procedures and a means of controlling log-
book pages, computer printouts, chart tracing and other written or printed
documents relevant to the samples. Logbooks, printed forms or other writ-
ten documentation must be available to describe the work performed in each
of the following stages of analysis:
o
o Sample receipt
o Sample preparation
o Standard preparation/tracking
o ICP analysis
o Flame, flameless and cold vapor AA analysis
o Non-metal Inorganics analysis
o Data reduction
o Uata reporting
5. The contractor shall have written SOPs for organization and assembly
of all documents relating to each tPA case. Documents shall be filed
on a case specific basis. The procedures must ensure that all documents
including logbook pages, sample tracking records, measurement readout
records,, computer printouts, raw data summaries, correspondence and any
other written documents having reference to the case, are compiled in
one location for submission to EPA in the Complete Case File Purge package
(formerly termed the "Document Control and Chain-of-Custody Package.")
The Case File Purge package must be compiled within 7 days of submission
. of che sample data package.The system must include a document numbering
and Inventory procedure.
B-87
-------
6. The Complete Case File Purge package includes but is not limited to:
sample tags, custody records, sample cracking records, analyses log-
book pages, bench sheets, measurement readout records, extraction and
analysis chronicles, computer printouts, raw data summaries, instrument
logbook pages, correspondence, and the document inventory*
B-88,
-------
Hazardous Waste Disposal Sice
Contract Analytical Support
Chain-of-Custody and Document Control Procedures
Sample Control
A sample is physical evidence collected from a facility or from the environment.
An essential part of this investigations effort is the control of the evidence
gathered. To accomplish this, the following chain-of-custody and document •
control procedures have been established.
Sample Identification
Each sample bottle shall be labeled with a tag containing the sample number and
sample, description to identify the contents of the bottle. Additionally, the
sample number shall be marked on the outside of any special packaging container
to facilitate identification. Typical sample tags are shown in Figure 1.
Chain—of-Custody Procedures
Because of the nature of the data being collected., the possession of samples
must be traceable from the time the samples are collected until they are intro-
duced as evidence in legal proceedings. To maintain and document sample custody,
the chain-of-custody procedures described here are followed.
A sample is under custody if:
1. It is in your actual possession, or
2. It is in your view, after being in your physical possession, or
3. It was in your possession and then you locked or sealed it up to prevent
tampering, or
4. It is in a secure area.
To assure custody of samples during transport and shipping, each sample within
a packing container is recorded on a chain-of-custody record shown in Figure 2.
Each sample number Is recorded and the number of containers shipped is recorded
on the sheets. Also record the other information regarding the project, sam-
ples (or shipper if returning empty bottles), method of shipment and remember
to sign and date the sheet. The original custody sheet is then placed inside
the package (protected from damage) and the package sealed. Sample containers,
shipping boxes, coolers or other packages may be sealed by using the seal shown
in Figure 3. The seal must be placed so the container cannot be opened without
breaking the'seal.
B-89
-------
Upon receipt of samples in custody, Inspect the package and note any damage co
the sealing tape or custody seals. Note on the custody record or other log-
book that the seals or locks were intact upon receipt if no tampering or damage
appears to have occurred. Open the package and verify that each item listed on
the sheet is present and correctly identified. If all data and samples are cor-
rect, sign and date the "received by Laboratory by" box. In the event errors
are noted, record the discrepancies in the remarks column (initial and date each
comment) then sign the chain-of-custody record. Report discrepancies to the
'Sample Management Office for remedies.
Laboratory Document Control
The goal of the Document Control Program is to assure that all documents for
a specified case (group of samples) will be accounted for when the project is
completed. The program Includes a document numbering and inventory procedure
for preparation of the specified documentation packages for each case.
Logbooks
All observations and results recorded by the laboratory but not on preprinted
data sheets are entered Into permanent laboratory logbooks. Data recorded are
referenced with the case number, date and analyst's signature at the top of the
page. Data from only one case are recorded per page. When all the data from a
case is compiled, copies of all logbook entries must be included in the documen-
tation package.
Instrument logs are also limited to one case per page with the ease number
recorded at the top of each page. Copies of these logs must also be included
in the final documentation package.
Corrections to Docuaentation
All documentation in logbooks and other documents shall be in ink. If an
error Is made in a logbook assigned to one individual, that person should make
corrections simply by crossing a line through the error and entering the correct
information. Changes made subsequently are dated and initialed. Corrections
made to other daca records or non-personal logbooks are made by crossing a
single line through the error, entering the correct information and initialing
and* dating the correction.
Consistency of -Documentation
Before releasing analytical results, the laboratory assembles and cross checks
the information on sample tags, custody records, lab bench sheets, personal and
instrument logs andother relevant data to ensure that data pertaining to each
particular sample or case Is consistent throughout the record.
-------
Doeuaene Numbering and Inventory Procedure
In order Co provide docuoenc accouncabilicy of che completed analysis records,
each Item in a case is inventoried and assigned a serialized number and an
identifier associating it TO the case and Region.
Case # - Region - Serialized number
For example - 75-2-0240
All documents relevant to each case including: logbook pages, bench sheets,
instrument readings, chare recordings, custody records, etc., are inventoried.
Each data generator (analyst) is responsible for ensuring that all documents
generated are placed in the file for inventory and returned to EPA. Figure 4
is an example of a document inventory*
Confidential Information
Any samples or information received with a request of confidentiality is handled
as "confidential." A separate, locked file is maintained and segregated from
other nonconfidential information. Data generated from confidential samples is
treated as "confidential." Upon receipt of confidential information, the DCO*
logs these documents into a Confidential Inventory Log. The information is
then made available to authorized personnel, but only after it has been signed
out to that person by the DCO. The documents shall be returned to the locked
file at the conclusion of each working day. Confidential information may not
be reproduced except upon approval by and under the supervision of the DCO.
Any reproduction must be kept to an absolute minimum. The DCO will enter all
copies into the document control system and apply the same requirements as
the original. In addition, this Information may not be destroyed except upon
approval by and under the supervision of the EPA program manager and EPA con-
tracting officer. The DCO shall remove and retain the cover page of any con-
fidential information disposed of for one year and shall keep a record of che
destruction in the Confidential Inventory Log.
*DCO is a document concrol officer assigned by che contractor to maintain
control of confidential information.
-------
tf-l'f'J
yoo?
*
MI:
*!•* I Hi'
I
.§
!
i
v>EPA
UNfTED STATES ENVIRONME^AL PROTECTION AGENCY
Figure 1. Example sample tag.
B-92
-------
r*K'mONMINtA|
O" ** !•' I nliM
AGf NCV
CHAIN OF CUSTOOV Rf COHO
oc
c
00 3
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r.
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o:
n
o
rncij NO f
ntuiri MAMr
SAI''PII"S '
««• no
- -
„.„
-•
IIMI
•_
-•
q...n,u.,l.,db, ,».,-.,...
1
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—
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4
-•
-
--
—
-
D«lr / Tim*
D.i./rm,
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"•»--•••» -V
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Rtc*iv*d by ISfpMiM**! t
Rrr* iv»d by 1 S*p*iwr«l
R»c*i**«l >n* ItltQtftinty hf
/////// ««»»M
•
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•
Hd.nqu.iKtdbv IS^M.^ O.It f.B- RK.IMd b* n.*«~~>
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UiMiki
-------
"IV3S AQOJLSnO
CUSTODY SEAL
L - -
Date -
Signature
Figure 3. Example custody seal made of perforated paper stock.
-------
1. Scudy plans or project plans.
2. Saaple traffic records, weekly reports.
3. Custody records, sample tags, sample loop.
4. Laboratory logbooks, personal logbooks, instrument logbooks (or
appropriate copies of logbook pages).
5. Laboratory data (sorted by sample), calibration and quality
control data results.
6. Data summaries and reports.
7. All other documents, forms or records referencing the samples.
Figure 4. Example Document Inventory Format for each case*.
B-95
-------
APPENDIX C
ELUTRIATE AND FRACTIONATION METHODS
(PLUMB 1981)
-------
Elutriate test
The elutriate test is a simplified simulation of the
dredging and disposal process wherein predetermined amounts of
dredging site water and sediment are mixed together to approximate
a dredged material slurry.2'20 The elutriate in the supernatant
resulting from the vigorous 30-min shaking of one part sediment from
the dredging site with four parts water (vol/vol) collected from
the dredging site followed by a 1-hr settling time and appropriate
centrifugation and O.U5 v filtration. Thus, it will be necessary to
collect both water and sediment samples to perform the elutriate test.
When evaluating a dredging operation, the sediment should be collected
at the dredging site and the water should be collected at the dredging
and the disposal site. To evaluate a fill material activity, samples
should be collected from the source of the fill material and the water
should be collected from the disposal site.
Water sample collection. Collection should be made with an
appropriate noncontaminating water sampling device. Either discrete
samplers such as Kemmerer or Van Dorn samplers or continuous collectors
such as submersible pumps may be used. The volume of water required
will depend on the number of analyses to be performed. For each sample
to be subjected to elutriate testing, it is suggested that a minimum of
It £ be collected at the disposal site and 8 i be collected at the
dredging site to evaluate a dredging operation and/or 12 i be collected
at the disposal site to evaluate a fill material disposal operation.
This will provide li i of water for analyses and sufficient water to
prepare triplicate 3-& elutriates. (Each elutriate should yield
2.0 to 2.2 i of standard elutriate for analysis.) If the samples are
to be analyzed for trace organics or a large number of constituents,
a proportionately larger initial sample should be collected.
Samples must be stored in glass containers if trace
organic analyses are to be performed. Generally, either plastic or
C-l
-------
glass containers may be used for other parameters. The samples should
be maintained at U°C until analyzed but never frozen. The storage
period should be as short as possible to minimize changes in the
characteristics of the water. Disposal site water should be analyzed
or split and preserved immediately. The remainder of the water should
be used in the elutriate test, which should be processed within 1 week
of collection.
Sediment sample collection. Samples should be taken from
the fill or the dredging site with a grab or a corer. Approximately 3&
of sediment or fill material would provide sufficient sample to prepare
triplicate 3-i elutriates. Again, if the resultant standard elutriates
are to be analyzed for trace organics or a large number of constituents,
a proportionately larger initial sample should be collected.
Samples may be stored in plastic bags, jars, or glass
containers. However, if trace organic analyses are to be performed,
glass containers with teflon-lined lids are required. A special
precaution that must be taken with sediment samples is to ensure
that the containers are completely filled with sample and that air
bubbles are not trapped in the container. This step is necessary to
minimize sample oxidation that could influence elutriate test
results.2'22
The samples should be stored immediately at U°C. They must
not be frozen or dried prior to use. The storage period should be as
short as possible to minimize changes in the characteristics of the
sediment. It is recommended that samples be processed within 1 week
of collection.
Apparatus. The following apparatus are required to perform
the elutriate test. Prior to use, all glassware, filtration equipment,
and filters should be washed with 5 to 10 percent (or stronger)
hydrochloric acid (HCl) and then rinsed thoroughly with deionized
water. The necessary apparatus include:
a. Acid-rinsed plastic bottles for collection of water
samples.
b. Plastic Jars or bags ("Whirl-Pak," plastic freezer
container's, etc.) for collecting dredged or fill
material samples.
C-2
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c_. Laboratory shaker capable of shaking 2-i flasks at
approximately 100 excursions/minute. Box type or
wrist-action shakers are acceptable.
d_. Several 1-i graduated cylinders.
e_. Large (15 cm) povder funnels.
£. Several 2-1, large-mouth graduated Erlenmeyer flasks.
£. Vacuum or pressure filtration equipment, including
vacuum pump or compressed air source, and an appropriate
filter holder capable of accomodating U7-, 105-, or
155-m-diameter filters.
h. Membrane filters with a O.U5-V pore-size diameter.
~" The filters should be soaked in 5 M HC1 for at least
2 hr prior to use.
i_. Centrifuge capable of handling six 1- or 0.5-A centri-
fuge bottles at 3000 to 5000 rpm. International Model
K or Sorval Super Speed are acceptable models.
J_. Wide-mouth, 1-gal capacity glass Jars with teflon-
lined screw-top lids for use as sample containers
when samples are to be analyzed for trace organics.
(It may be necessary to purchase Jars and teflon
sheets separately; in this case, the teflon lid
liners may be prepared by the laboratory personnel.)
Test procedure. The stepwise test procedure is given below:
a. Subsample a minimum volume of 1 I each of dredging site
and disposal site water. If it is known in advance
that a large number of measurements are to be performed,
the size of each subsample should be increased to meet
the anticipated needs.
b. Filter an appropriate portion of the disposal site
~ water through an acid-soaked 0.1*5-y pore-size membrane
filter that has been prerinsed with approximately
100 ml of disposal site water. The filtrate from the
rinsing procedure should be discarded.
£. Analyze the filtered disposal site sample as soon as
possible. If necessary, the samples may be stored at
U°C after splitting and the appropriate preservatives
have been added (Table 2-U). Filtered water samples
may also be frozen with no apparent destruction of
sample integrity.
d_. Repeat steps a., b, and c_ with dredging site water.
This step is omitted with a fill material sample.
e. Subsample approximately 1 iof sediment from the well-
~~ mixed original sample. Mix the sediments and
unfiltered dredging site water in a volumetric
sediment-to-water ratio of 1:U at room temperature
(22 +_ 2°C). This is best done by the method of
C-3
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volumetric displacement.23 One hundred mililiters of
unfiltered dredging site water is placed into a
graduated Erlenmeyer flask. The sediment subsample is
then carefully added via a powder funnel to obtain a
total volume of 300 ml. (A 200-ml volume of sediment
will now be in the flask.) The flask is then filled
to the 1000-ml mark with unfiltered dredging site
water, which produces a slurry with a final ratio of
one volume sediment to four volumes water.
This method should provide 700 to 800 ml of water for
analysis. If the analyses to be run require a larger volume of water,
the initial volumes used to prepare the elutriate slurry may be
proportionately increased as long as the solid-to-liquid ratio remains
constant (e.g. mix UOO ml sediment and 1600 ml unfiltered dredging
site water). Alternately, several 1-i sediment/dredging site water
slurries may be prepared as outlined above and the filtrates combined
to provide sufficient water for analysis. The procedure continues as
follows:
£. (1) Cap the flask tightly with a noncontaminating
stopper and shake vigorously on an automatic
shaker at about 100 excursions per minute for
30 min. A polyfilffl-covered rubber stopper is
acceptable for minimum contamination.
(2) During the mixing step given above, the
oxygen demand of the dredged material may cause
the dissolved oxygen concentration in the elutriate
to be reduced to zero. This change can alter
the release of chemical contaminants from dredged
material to the disposal site water and reduce
the reproducibility of the elutriate test. l
If it is known that anoxic conditions (zero
dissolved oxygen) will not occur at the disposal
site or if reproducibility of the elutriate test
is a potential problem, the mixing may be
accomplished by using a compressed air-mixing*
procedure instead of the mechanical mixing
described in Step f. (l). After preparation of
the elutriate slurry, an air-diffuser tube is
inserted almost to the bottom of the flask.
Compressed air should be passed through a
deionized water trap and then through the
diffuser tube and the slurry. The flow rate
should be adjusted to agitate the mixture
* This procedure can cause the loss of highlj volatile chemical con-
stituents. If volatile materials are of concern, compressed air
mixing should not be used.
C-4
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vigorously for 30 min. In addition, the flasks
should be stirred manually at 10-min intervals
to ensure complete mixing.
£. After 30 min of shaking or mixing with air,
allow the suspension to settle for 1 hr.
li. After settling, carefully decant the supernatant
into appropriate centrifuge bottles and then
centrifuge. The time and revolutions per minute
during centrifugation should be selected to reduce
the suspended solids concentration substantially
and, therefore, shorten the final filtration
process. After centrifugation, vacuum or pressure
filter approximately 100 ml of sample through a
O.U5-U membrane filter and discard the filtrate.
Filter the remainder of the sample to give a
clear final solution (the standard elutriate) and
store at U°C in a clean, noncontaminating container
in the dark. The filtration process is intended
for use when the standard elutriate is to be analyzed
for conventional chemical contaminants. When the
elutriate is to be analyzed for organic contaminants
and PCB's, filtration should not be used since
organic concentrations can be reduced by sorption.
Centrifugation should be used to remove particulate
matter when the standard elutriate is to be
analyzed for specific organics.
i_. Analyze the standard elutriate as soon as possible.
If necessary, the samples may be stored at U°C
after splitting and the appropriate preservatives
have been added.
j_. Prepare and analyze the elutriate in triplicate.
The average of the three replicates should be
reported as the concentration of the standard
elutriate.
Sediment fractionation
Chemical constituents associated with sediments may be
distributed in many chemical forms. The purpose of a fractionation
procedure is to better define this distribution. This objective is
achieved by leaching a sample with a series of successively harsher
extraction agents. Reagents used in the procedure to be described
below consist of interstitial water, ammonium acetate, hydroxylamine,
hydrogen peroxide, citrate-dithionate, and hydrofluoric acid-nitric
acid.
The premise of the fractionation procedure is that a
specific geochemical phase is defined by a specific chemical
C-5
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extraction agent. Thus, the ammonium acetate extract is referred to as
the exchangeable phase, and the citrate-dithionate extract is referred
to as the moderately reducible phase. These relationships have not been
rigorously demonstrated and, therefore, the fractions are only opera-
tionally defined.
The use of fractionation results at this time appears to be
limited to supporting other studies. That is, results have greater
value in sediment research studies than in the regulatory decision-
making process. Limited results with sediment fractionation data
suggest a higher correlation vith the more labile sediment phases
(interstitial water, exchangeable phase) and elutriate test results11
and long-term water quality changes.l°
A major limitation of the fractionation procedure is that
previous experience is limited to heavy metals and nutrients. A broad
spectrum analysis of the individual fractions has been limited by small
sample size, particularly the interstitial water fraction.
Sample collection. Samples should be collected with a grab
or a corer. Because the distribution of sediment-associated chemicals
can be altered by processes such as drying and oxidation, samples should
be kept wet and exposure to the atmosphere should be minimized. Samples
collected with a grab or dredge must be quickly transferred to a con-
tainer and air bubbles must be excluded from the container. Corer
samples should be sealed in the core liner and returned to the labora-
tory in an upright position. Previous studies have shown that a 15-cm
section from a 7.5-cm-diamcter core can provide sufficient material to
perform fractionation studies for eight metals and four nutrients.11
The samples should be stored immediately at U°C. They
must not be frozen or dried prior to use. The storage period should
be as short as possible to minimize changes in the distribution of
chemical constituents in the sediments. It is recommended that samples
be processed within 1 week of collection.
Apparatus. The following apparatus is required to perform
the elemental partitioning procedure. Prior to use, all glassware,
filtration equipment, and filters should be washed with 5 to 10 percent
HC1 and then rinsed thoroughly with deionized water. This list of
C-6
-------
equipment includes:
a. Plastic Jars or bags ("Whirl-Pak," plastic freezer
~ containers, etc.) for collecting grab or dredge
samples or polyethylene liners for the collection of
core samples.
b_. Glove box or disposable glove bag.
£. Polarographic oxygen analyzer or alternate method to
confirm the absence of oxygen in glove bag.
£. Utensils for splitting cores and handling samples in the
glove bag.
e_. 250-ml and 500-ml polycarbonate centrifuge bottles.
f_. Refrigerated centrifuge.
£. Vacuum filtration apparatus.
h. 150-ml and 120-ml polyethylene storage bottles.
i_. Blender or porcelain mortar and pestle.
j_. Top-loading balance.
k. Weighing dishes.
1^ Digestion block or hot plate.
m. Teflon beakers.
la. 50-ml volumetric flasks.
Test procedure. A stepwise sediment fractionation procedure
is given in the following paragraphs.
To begin the procedure, prepare a glove box or disposable
glove bag. Flush the system with nitrogen gas and maintain a positive
pressure nitrogen atmosphere. Oxygen-free conditions in the glove bag
or box should be verified with a polarographic oxygen analyzer prior
to sample processing. Initial sample handling and all steps in the
interstitial water and ammonium acetate extractions should be conducted
under a nitrogen atmosphere.
Acid wash all hardware to be used in the extractions in
6 N HCL and thoroughly rinse with distilled water to minimize sample
contamination during processing.
The initial separation removes the interstitial water phase.
This is accomplished by first placing the sealed sediment sample in the
glove bag. After reestablishing the nitrogen atmosphere, extrude the
sediment core from its liner into a flat plastic container. If the
core is to be sectioned vertically, 15-cm sections of a 7.5-cm-diaraeter
C-7
-------
core have been shown to provide sufficient material for the sequential
fractionation procedure. Each core section should be split into halves
vith one half (approximately 300 cc) being used for the interstitial
vater testing and the remaining half used for all other analyses. Place
the half section for the interstitial water analysis in an oxygen-free,
polycarbonate 500-ml centrifuge bottle in the glove bag and seal.
Centrifuge the sample in a refrigerated centrifuge (U°C) at 900
revolutions per minute (M.3,000 * g) for 5 min. This should be
sufficient to recover UO percent of the total sediment water. After
centrifugalion, return the sample to the glove bag and vacuum filter
the interstitial water through a O.U5-V pore-size membrane filter.
Transfer the filtered sample to an acid-washed polyethylene bottle and
acidify to pH 1 with HC1 for preservation.
If the sediment sample is not to be sectioned vertically,
decant excess water and blend the core or dredge sample. Place
approximately 300 cc of the blended sample in an oxygen-free, poly-
carbonate 500-ml centrifuge bottle and seal. Centrifuge this sample
for 5 min at 900 revolutions per minute (I3j000 * g) in a refrigerated
centrifuge (U°C) and then filter through a O.U5-U pore-size membrane
filter under a nitrogen atmosphere. The filtered sample may be
analyzed immediately or split for preservation and storage.*
The exchangeable phase is determined on the unused half of
the wet sediment sample that was blended for interstitial water
analysis. Blend the wet sediment with an electrically driven poly-
ethylene stirrer contained in the glove bag. Remove a subsample of
the homogenized sediment sample (blended core section, core, or grab
sample) for percent solids determination.
Weigh a second subsample (approximately 20 g dry weight
of each homogenized sediment section into an oxygen-free, tarred,
250-ml centrifuge tube containing 100 ml deoxygenated 1 N^ ammonium
acetate, producing a suspension with an approximate solid-to-liquid
ratio of 1:5. Adjust the pH of the acetate solution to the pH of the
surface sediments. Seal the samples and then place on a wrist-action
shaker for 1 hr. Centrifuge the samples at 6000 revolutions per minute
for 5 min and return them to the glove bag for further processing under
C-8
-------
the nitrogen atmosphere. Filter the sample through O.U5-V pore-size
membrane filters, retaining both the filtrate and the solid residue.
The filtrate may be analyzed immediately or split and
preserved for specific constituents, as discussed for water samples.
This extract will also include the interstitial water components
since a fresh blended sediment sample was used. Therefore* measured
concentrations should be reduced to compensate for the interstitial
water. This can be accomplished as:
Wt exchangeable material = (Vol ext) (Cone ext) -
(Wt Sample) (l-< Solids) , .
(density water) uw<;
where
(Vol ext) = volume of ammonium acetate extract
(Cone ext) = analytical concentration in ammonium acetate extract
(Vt Sample) = wet weight of sample for ammonium acetate extraction
% Solids = percent solids in sample
(density water) = density of water at temperature of sample
(IWC) = interstitial water concentration of sample
The easily reducible phase is performed with the solid
residue from the exchangeable phase determination. This step and all
subsequent steps in the fractionation procedure can be conducted outside
the glove bag. Add 50 ml 1?2 sparged distilled-deionized water to the
centrifuge tube containing the solid residue from the 1 N. ammonium
acetate extraction. Agitate the sample with a stainless steel spatula
or a glass-stirring rod to ensure good washing efficiency. Centrifuge
the suspension of 6000 revolutions per minute and discard the liquid
phase. A portion of the solid residue will have to be set aside at
this point for a redetermination of percent solids.
Blend the remaining sediment residue and transfer a 2-g
(dry weight equivalent) subsample to a 250-ml Erlenmeyer flask. Add
100 ml of 0.1 M hydroxylamine hydrochloride-0.01 M nitric acid solution.
The resultant suspension will have a solid-to-extractant ratio of
approximately 1:50. Seal the sample and place the suspension on a
wrist-action shaker (or equivalent) for 30 min. Centrifuge the sample
C-9
-------
at 6000 revolutions per minute for 5 min. Decant and filter the liquid
phase through 0.1»5-U pore-size membrane filters. The filtrate may be
treated as a water sample and analyzed immediately or split and preserved
for specific constituents.
The solid residue is used in the organic and sulfide phase
extraction. Wash the residue from the easily reducible phase vith 50 ml
distilled water. After agitating the suspension, centrifuge the sample
at 6000 revolutions per minute for 5 min and discard the supernate.
*
Subsample the residue for a percent solids determination so the organic
and sulfide results can be expressed on a dry weight basis. Add 50 ml
of 30 percent hydrogen peroxide to the washed residue and adjust the pH
to 2.5 with HC1. (The purpose of the pH adjustment is to prevent any
released metals from precipitating.) Digest the sample at 95°C for
6 to 8 hr. Add 100 ml of 1 If ammonium acetate buffered at pH 2.5 to
the digestate and shake for 1 hr. Centrifuge the sample at 6000 revo-
lutions per minute for 5 min and filter the sample through 0.1* 5-w
pore-size membrane filters. Treat the filtrate as a water sample
and analyze immediately or split and preserve as required. Retain
the solid residue.
The next fraction in the 'sequence is the moderately
reducible phase. Wash the organic and sulfide phase solid residue
with 50 ml of distilled water; centrifuge as described earlier; and
discard the supernate. Redetermine percent solids on a subsample
of the residue. Add 100 ml of a citrate-dithionate solution
(16 g sodium citrate + 1.67 g sodium dithionate/100 ml distilled
water) and mechanically shake the suspension for 17 hr. Centrifuge
the sample at 6000 revolutions per minute for 5 min and filter the
supernate through a O.b5-V pore-size membrane filter. Analyze the
filtered sample for moderately reducible constituents and retain
the solid residue for further treatment.
The sample for residual phase digestion is obtained by
washing the moderately reducible phase residue with 50 ml of distilled
water; centrifuging at 6000 revolutions per minute for 5 min; and
discarding the supernate. Dry the residue at 105°C and transfer
C-10
-------
a 0.5-g dry weight subsample to a teflon beaker. Add 15 ml hydro-
fluoric acid and 10 ml concentrated nitric acid; cover the beaker;
and digest at 175°C. After evaporation to near dryness, add 8 ml
fuming nitric acid stepwise in 2-ml increments. Continue evaporation
to near dryness. Add 6 N. HC1 to dissolve the residue, heating if
necessary. Quantitatively transfer the solution to a 50-ml volumetric
flask and dilute to volume. Analyze the sample immediately or preserve
subsamples for specific constituents.
A schematic flov diagram for the fractionation procedure
is presented in Figure 2-2. When this procedure is used, a built-in
quality control check is to total each of the operationally defined
phases and compare to a total digest of the sample. The data should
be considered suspect if they differ by more than 5 to 10 percent.
Bulk analysis
A bulk analysis provides a measure of the total concen-
tration of a specific constituent in the sample being analyzed. This
is accomplished by subjecting a sample to strong oxidation, acid
digestion, or organic solvent extraction. The procedure is similar
to that used for the residual phase digestion in the elemental
partitioning procedure discussed earlier. Total sediment concentrations
can be used to compare different sites and to identify major point
sources. However, because of the harshness of the extraction pro-
cedure, information on chemical distribution and/or potential
environmental impact is lost.
Sample collection. Samples for total analyses may be
collected with a dredge or grab sampler or a core sampler. Approxi-
mately 1 to 2 I of sediment or fill material should be taken from the
proposed project site and placed in plastic Jars or containers. If
trace organic constituents are to be determined, the sample should be
stored in a glass container or a second sample of approximately the
same size should be collected and stored separately in glass containers.
Samples should be stored at U°C.
Upon reaching the laboratory, sediment samples may be
stored wet, air dried, or frozen. The selection between these
preservation techniques should be'based primarily on the specific
C-ll
-------
I
t-»
ro
NTERSTITIAL WATER
PHASE
SEDIMENT SAMPLE I
(Preserve
Anaerobic
Integrity)
Section and
Centrifuge
•^RESIDUE
upernatant
(Filter)
Acidify
(pH 1-2)
SOLUTION
ANALYZE AS
INTERSTITIAL
WATER PHASE
EXCHANGEABLE PHASE
EXTRACT with
IN NHtOAc
-Centrifuge
(Preserve
Anaerobic
Integrity)
Wast
Supernatant
(Filter)
Acidify
SOLUTION
ANALYZE AS
EXCHANGEABLE
PHASE
EASILY REDUCIBLE
PHASE
EXTRACT wi th
NH2OH-HC1
Centrifuge
Supernatant
" (Filter)
SOLUTION
ANALYZE AS
EASILY
REDUCIBLE
PHASE
ORGANIC +
SULFIDE PHASE
DIGEST with
H202
(pH 2.5)
at 95°C
Extract as
Exchange-]
able
(pH 2.5)
•RESIDUE
Supernatant
(Filter)
SOLUTION
ANALYZE AS
ORGANIC +
SULFIDE
PHASE
MODERATELY REDUCIBLE
PHASE
EXTRACT with Na2S20H
RESIDUAL PHASE
DIGEST at 95°C
fh HF +
, +
ming)
s
entrifuge
Wash
IESIDUE
Supernatant
I (Filter)
SOLUTION
ANALYZE AS
MODERATELY
REDUCIBLE
PHASE
(Centrifuge)
I
SOLUTION
ANALYZE AS
RESIDUAL
PHASE
Figure 2-2. Elemental partitioning for sediment characterization
-------
parameter to be determined and, secondarily, on personal choice.
Several parameters such as pH, redox, total solids, and volatile
solids must be run on wet samples. Other parameters may change due
to oxidation (chlorine demand, BOD, COD, SOD, sulfides), volatili-
zation (phenolics, volatile solids), or chemical instability (carbamates,
herbicides). Samples to be analyzed for these parameters should be
processed as soon as possible using subsamples of the original vet
sample. Samples to be analyzed for particle size (dispersed), total
organic carbon (TOC), metals (except possibly mercury), chlorinated
hydrocarbon pesticides, and PCB's may be stored wet, dried, or frozen.
Apparatus. The specific equipment necessary will vary
depending on the chemical constituent(s) to be analyzed in the total
sediment digest or the total sediment organic extract. Specific
needs and cleanup procedures are discussed with each parameter in
Section 3.
Test procedure. The following stepwise procedure is
recommended for the processing of sediment or fill material samples
to be analyzed for total or bulk content:
a_. Decant any overlying water that may have been collected
with the dredge or corer.
b_. Blend the dredge, core, or sectioned core sample.
£. Transfer an aliquot of the homogenized sample to a
tarred weighing dish and weigh. Dry the sample at
105°C to a constant weight. This information will
allow calculation of percent solids in the sample
and to report subsequent bulk analysis results on a
milligram-per-kilogram dry weight basis. The dried
sample from the percent solids determination may be
subjected to further chemical analysis for those
parameters not affected by the drying process.
If volatile solids are to be determined, record the
weight of the crucible and the dried sample used in
the percent solids determination. Place the sample
in an electric muffle furnace and ignite the sample
at 600°C for 60 min. Remove the sample from the
furnace, allow to cool, and desiccate for 30 min
prior to weighing. Report the weight lost on
ignition as percent volatile solids.
d_. Transfer a second subsample of the blended sample to
a suitable container for pH and oxidation-reduction
(redox) potential determinations. The sample size
C-13
-------
should be sufficient to allow the electrodes to be
inserted to a depth of U to 6 cm. Allow sufficient
time for the electrode responses to stabilize and
record the respective pH and redox values.
e_. Set aside subsamples of the wet, blended sample for the
analysis of time-dependent or unstable chemical
constituents. Parameters in this category include
biological oxygen demand, chemical oxygen demand,
sediment oxygen demand, chlorine demand, herbicides and
carbamates, phenolics, sulfides, nitrogen, phosphorus,
and oil and grease. Thus, as many as 12 subsamples
(if all listed analyses are to be performed) will be
required. Suggested sample sizes for each aliquot are
presented in Figure 2-3. These analyses should be
initiated as soon as possible to minimize the effects
of sample alteration due to handling and storage.
f_. Set aside separate subsamples for the analysis of
particle size, carbon, metals, and chlorinated
hydrocarbons. However, because of the increased
stability of these constituents (relative to those
in Step £, above), the aliquots may be taken from the
initial wet, blended sample, or a sample that has been
dried or frozen for storage. Required subsample sizes
are presented in Figure 2-3.
Individuals performing bulk analysis of sediment samples
should be aware of the fact that analytical results may be affected by
sample handling and storage procedures. The following special caveats
are maintained here because of the importance of this fact and again
with the appropriate analytical procedure in Section 3:
a_. It is preferable to determine Eh and pH values in the
field as soon as the sample is collected since there is
no way to stablize these parameters. If this is not
possible, these parameters should be determined as
soon as possible in the laboratory using a wet sample.
Sample handling should be-kept to a minimum to avoid
sample dehydration or sample oxidation.
b_. Percent solids and specific gravity also must be
determined on a sample of original moisture content.
The sample should be handled in such a manner to
minimize water loss and sample dehydration.
c_. Cation exchange capacity can be influenced by sample
drying. Therefore, it is recommended that this
parameter be determined on original moisture content
samples.
d_. Chlorine demand, biological oxygen demand, chemical
oxygen demand, and sediment oxygen demand are all
C-14
-------
Dredge or Grab
Sample
Mineralogical
Compos i t i on
10-25 9
Total
Organic
Ca rbon
&
Total
Inorganic
Carbon
1-2 q
Heavy Metals
1-10 g
PCB's &
Pesticides
10-100 q
Time-dependent Parameters
Process Immediately
Particle Size
25-50 q
Whole
Core
1 BLEND SAMPLE
^ Dry Sample L
^ freeze Sample L
*"" for storage ™
Note: All sa
a dry
Section
Core
^
^ ^
Dry Sample pH
105 *C Eh
% Solids 50-100 q
5-20 q
600 "C
Volatile Solids
mple sizes given on
weight basis.
— »
_k
— fr
,__*
W
— •+
_fc
— •*
— 1
— 1
-H
—4
Cation Exchange Capacity
5-10 q
Chlorine Demand
1-2 g
Biochemical Oxygen Demand
0.5-5.0 q
Chemical Oxygen Demand
0.5-5.0 q
Sediment Oxygen Demand
10-100 q
Herbicides & Carbamates
10-100 q
(Phenolics
10-50 a
{Sul fides
1-5 a
{ Spec! fie Gravity
250 q
\ Nitrogen forms
t 0.5-10 q
\ Phosphorus forms
T 0.5-10 q
J Oi 1 6 Grease
5-50 g
Figure 2-3. Sediment sample splitting for bulk analysis
-------
measures of the reducing capacity of the sample being
analyzed. Since sediments are frequently reduced and
contain elevated concentrations of ferrous iron,
manganous manganese, and sulfide that can be oxidized
by atmospheric oxygen, these parameters should be run
on vet samples.
e_. Herbicides and carbamates are chemically unstable with
relatively short half-lives. Immediate extraction of
the original sample with methylene chloride reduces
the possibility of chemically or biologically catalyzed
decomposition and increases herbicide and carbamate
stability.
£. Phenolic compounds may be lost by volatilization during
storage. Therefore, samples to be analyzed for phenols
should not be dried and storage time should be minimized.
If immediate analysis is not possible, storage by
freezing may be acceptable. Subsequent sample thawing
should be accomplished at low temperature to reduce
phenol loss by volatilization.
£. Sulfides in the sample may be lost by volatilization
and oxidation. It is recommended that sample contact
with atmospheric oxygen be minimized between sample
collection and analysis to reduce this effect. This
can best be accomplished by excluding air bubbles from
sample containers and minimizing sample storage time.
h. Some forms of nitrogen that are expected to occur in
sediments (nitrites) are unstable in the presence of
oxygen and can be lost on sample drying. In addition,
sample composition may be altered by the uptake or
loss of volatile ammonia. Therefore, sample processing
should begin as soon as possible using a sample of
original moisture content.
i^ The distribution of phosphorus forms may be altered by
~ changes in other sample constituents. For example,
the oxidation of iron in a sample may precipitate
soluble phosphate. Therefore, if soluble phosphate
is to be determined, wet samples should be processed
as soon as possible. If total phosphate is the only
parameter of concern, analysis can be conducted on a
wet, dried, or frozen sample.
j_. The oil and grease content of samples may be reduced
due to volatilization. Consequently, sample drying
prior to analysis is not recommended.
k_. The selection of a storage technique for samples to be
analyzed for particle size depends on the method of
analysis. If apparent particle size is to be run,
a wet sample should be used. However, if dispersed
particle size is to be run, a wet, dried, or frozen
sample may be used.
C-16
-------
1^ Most of the heavy metals are stable and samples
scheduled for analysis can be stored in a vet, dried,
or frozen state. The selection of a storage method
can affect the distribution of a metal among various
forms, but the total concentration should be unaffected.
Tvo possible exceptions are mercury and selenium, which
can be lost by volatilization. This particularly is
true if the samples are dried above 60°C.
m. Chlorinated hydrocarbon pesticides and PCB's are stable
and probably unaffected by the method of sample storage.
Improved stability can be achieved by immediate
extraction of the original sample with an organic
solvent and is suggested, but not essential.
11. The analysis of a sediment or fill material sample for
specific constituents will require a sample digestion
or sample extraction technique. Since the selection of
a digestion solution or solvent is dependent on the
analysis to be performed, this information is presented
with the specific analytical techniques.
Dredged material may be subjected to several types of
testing. This section has provided guidance for conducting elutriate
testing, elemental partitioning, and bulk analysis of sedimentary
samples. Since each of these procedures measures a different property
of the sample, different storage requirements are required for samples
to be subjected to each testing procedure. Therefore, this section
has also provided detailed guidance for the handling of sedimentary
samples from the time of collection until the time of analysis.
C-17
-------
References
1. Environmental Protection Agency. "Draft Interim Analytical
Methods Manual for the Ocean Disposal Permit Program."
EPA; Washington, D.C. (January 197M-
2. Environmental Effects Laboratory. "Ecological Evaluation of
Proposed Discharge of Dredged or Fill Material into Navigable
Waters. Interim Guidance for Implementation of Section UoMb)(l)
of Public Law 92-500 (Federal Water Pollution Control Act
Amendments of 1972)." Miscellaneous Paper D-76-17. U. S.
Army Engineer Waterways Experiment Station, CE; Vicksburg,
Mississippi (1976).
3. Environmental Protection Agency. "NPDES Compliance Sampling
Manual." Enforcement Division, Office of Water Enforcement,
Compliance Branch, EPA; Washington, D.C. 139 P. (1977).
U. American Public Health Association. Standard Methods for the
Examination of Water and Wasjtewater Including Bottom Sediments
and Sludges. lUth Edition. American Public Health Association,
New York, New York. 1193 p. (1975).
5. Sly, P. G. "Bottom Sediment Sampling." Proc. 12th Conf. Gr.
Lakes Res. pp. 883-898 (1969).
6. Howmiller, R. P. "A Comparison of the Effectiveness of Ekman
and Ponar Grabs." Trans. Amer. Fish. Soc. 100:560-561* (1971).
7. Wigley, R. L. "Comparative Efficiencies of Van Veen and Smith-
Mclntyre Grab Samplers as Revealed by Motion Pictures." Ecology
1*8:168-169 (1967).
8. Hudson, P. L. "Quantitative Sampling with Three Benthic Dredges."
Trans. Amer. Fish. Soc. 99:603-607 (1970).
9. Christie, N. D. "Relationship Between Sediment Texture, Species
Richness, and Volume of Sediment Sampled by a Grab." Marine
Biology 30:89-96 (1975).
10. Brannon, J. M., Plumb Jr., R. H., and Smith, I. "Long Term Release
of Contaminants from Dredged Material." Technical Report D-78-l*9.
U. S. Army Engineer Waterways Experiment Station, CE, Vickstmrg,
Mississippi. 66 p. (1978).
11. Brannon, J. M., Engler, R. M., Rose, J. R., Hunt, P. G., and
Smith, I. "Selective Analytical Partitioning of Sediments to
Evaluate Potential Mobility of Chemical Constituents During
Dredging and Disposal Operations." Technical Report D-76-7.
U. S. Army Engineer Waterways Experiment Station, CE; Vicksburg,
Mississippi. 90 p. (1976).
12. Jenne, E. A., and Luoma, S. N. "Forms of Trace Elements in Soils,
Sediments, and Associated Waters: An Overview of Their Determi-
nation and Biological Availability." U. S. Geological Survey;
Menlo Park, California. Presentation at Biological Implications
C-18
-------
of Metals in the Environment. 15th Life Sciences Symposium;
Hanford, Connecticut. 71* P- (September 29 - October 1, 1975).
13. Chen, K. Y., Gupta, S. K., Sycip, A. Z., Lu, J. C. S., Knesevic, M.,
and Choi, W. W. "Research Study on the Effect of Dispersion
Settling, and Resediznentation on Migration of Chemical Constituents
During Open Water Disposal of Dredged Materials." Contract Report
D-76-1. U. S. Army Engineer Waterways Experiment Station, CE;
Vicksburg, Mississippi. 221 p. (1976).
Ik. Lee, G. F. "Chemical Aspects of Bioassay Techniques for
Establishing Water Quality Criteria." Water Res. 7:1525-15'*6
(1973).
15. Environmental Protection Agency. "Methods for Chemical Analysis
of Water and Wastes." National Environmental Research Center,
EPA; Cincinnati, Ohio. EPA-625/6-7U-003. 298 p. (197*0.
16. Sherma, J. "Manual of Analytical Quality Control for Pesticides
and Related Compounds in Human and Environmental Samples."
Contract 68-02-1727. EPA Health Effects Research Laboratory;
Research Triangle Park, North Carolina. EPA-600/1-76-017.
Unnumbered (1976).
17. Environmental Protection Agency. "Handbook for Analytical
Quality Control in Water and Wastewater Laboratories."
Environmental Monitoring and Support Laboratory, Environmental
Research Center; Cincinnati, Ohio. Draft Report (1978).
18. Delfino, J. J. "Quality Assurance in Water and Wastewater
Analysis Laboratories." Water and Sewage Works 12U(7):79-8U
(1977).
19. Anon. "Federal Environmental Monitoring: Will the Bubble Burst?"
QA Report. Environmental Science and Technology 12:126U-1269
(1978).
20. Keeley, J. W., and Engler, R. M. "Discussion of Regulatory
Criteria for Ocean Disposal of Dredged Materials: Elutriate
Test Rationale and Implementation Guidelines." Miscellaneous
Paper D-7U-1U. U. S. Army Engineer Waterways Experiment
Station, CE; Vicksburg, Mississippi (l97M.
21. Lee, G. F., Piwoni, M. D., Lopez, J. M., Mariani, G. M.,
Richardson, J. S., Homer, D. H., and Saleh, F. "Research Study
for the Development of Dredged Material Disposal Criteria."
Technical Report D-75-1U. U. S. Army Engineer Waterways
Experiment Station, CE; Vicksburg, Mississippi (1975).
22. Plumb, R. H., Jr. "A Bioassay Dilution Technique to Assess the
Significance of Dredged Material Disposal." Miscellaneous Paper
D-76-6. U. S. Army Engineer Waterways Experiment Station, CE;
Vicksburg, Mississippi. 16 p. (1976).
23. Neff, J. W., Foster, R. S., and Slowey, J. F. "Availability of
Sediment-Adsorbed Heavy Metals to Benthos with Particular Emphasis
on Deposit Feeding Infauna." Technical Report D-78-U2. U. S.
C-19
-------
Army Engineer Waterways Experiment Station, CE; Vicksburg,
Mississippi. 286 p. (1978).
Lee, G. F. and Plumb, R. H. Jr. "Literature Review on Research
Study for the Development of Dredged Material Disposal Criteria."
Contract Report D-7U-1. U. S. Army Engineer Waterways Experiment
Station, CE; Vicksburg, Mississippi. ll*5 p. (1971*).
C-20
-------
APPENDIX D
TOTAL ACID DIGESTION METHOD FOR SEDIMENT
(RANTALA AND LOR ING 1975)
-------
REAGENTS
1. Concentrated hydrofluoric, hydrochloric, and nitric acids
[ultrex grade (Baker) or equivalent]
2. 2.5 percent boric acid - dissolve 50 g [ultrex grade (Baker)
or equivalent] boric acid in 2 L distilled-deionized water.
3. 30 percent hydrogen peroxide [ultrex grade (Baker) or equivalent],
PROCEDURE
1. If mercury is to be analyzed, a separate, 0.4-g (wet) aliquot
is digested and analyzed by the cold vapor AA technique
detailed in Appendix B. (Note that the sample weight must
be doubled to achieve the 0.01-mg/kg dry weight detection
limit specified in Table 6 of Appendix A.)
2. Dry a representative portion of sediment overnight at 60 C.
Grind to 100 mesh.
3. Weigh 200-mg dried sample into a decomposition vessel.
(Vessels of various sizes may be obtained from: (a) Savillex
Corp., 5325 Hwy. 101, Minnetonka, MN, USA 55343; (b) H.K. Morri-
son and Sons Ltd., Mount Uniacke, N.S., Canada; (c) Parr
Instrument Company, 211-53rd St., Moline, IL, USA 61265;
(d) Uniseal Decomposition Vessels Ltd., P.O. Box 9463, 31 094
Haifa, Israel - distributed in the USA by Columbia Organic
Chemicals Co., P.O. Box 1045, Camden, SC 29020.) Add 0.5 mL
cone. HN03.
4. In a fume hood, warm on a hot plate at low setting and open
to the atmosphere until the brown N02 fumes are absent.
To aid digestion, a few drops of 30 percent H202 may be
added.
D-l
-------
5. Cool to room temperature. Add 0.75 mL cone. HC1 and 3.0 ml
cone. HF. Seal the digestion vessel.
6. Heat in an oven for 2 h at the digestion temperature recommended
by the manufacturer (105°C - 130°C). (Note: a longer digestion
time may be necessary for some sediments if visible particulates
are present after the addition of boric acid. Some fluorides,
such as CaF2 are insoluble in the digestion acids and appear
as a greyish sludge. These will dissolve upon addition
of boric acid. Also, flakes of black, undigested carbon-
aceous matter may also be present. These will not dissolve
upon boric acid addition, but will not interfere with the
analysis.)
7. Remove vessel from oven and cool to room temperature. Add
20 mL of 2.5 percent boric acid. Reseal vessel and return
to oven at recommended temperature for 1 h.
8. Remove vessel from oven and cool to room temperature. Quanti-
tatively transfer contents to a 25 ml polyethylene volumetric
flask and bring to the mark with distilled-deionized water.
9. Digestate may be analyzed directly using the recommended
atomic spectroscopic instrumental techniques.
D-2
-------
MULTI-ELEMENT ANALYSIS OF SILICATE ROCKS AND MARINE
SEDIMENTS BY ATOMIC ABSORPTION SPECTROPHOTOMETRY
R. T. T. Remain and D. H. Loring
Department of ike Environment
Fisheries and Marine Service
Marine Ecology Laboratory
Bedford Institute of Oceanography
Dartmouth, Nova Scotia, Canada
ABSTRACT
A rapid method it described for the de-
tenninition of 21 dementi in silicate
tocki and marine sediments by flame
atomic •bwrpiion. The method often
good accuracy and precision in major,
minor, and trace element determinations
aad it very suitable for marine f eochea-
ical studies. Precision and accuracy bare
been evaluated by analysing seven
TJ.S.G.S. rock standards.
RESUMC
On decril une meihode rapide pour la
determination de 21 elements dans des
roches silicates el de* sediments marins,
par absorption atomique de flsmme.
La methode donne une bonne exactitude
et une bonne precision pour la determi-
nation d'elementi majeurs, mineurs el
traces « esl Ires sdapfee a des eludes
de feochinic marine. L'fvaluation de la
precision et de I'eianitude • M faite
par 1'analyse de sept standards de rocbe
uses
ZUSAMMENFASSUNG
Es wird cine rascne Meihode fur Benin-
muttf vn 21 Qeaemea in Silikaiiestei-
nan and Meeres-Sedimenten mitielt
Fknunen-AtomsbsorptioD beacbrieben
Die Methode bietet fur die Bestimmunf
der Haupt.-Neben.-vnd Spurenelemente
cine jute Riehtifkeii und Primion und
tigntl aicn susieieichBet fur leochem-
Ische Siudien in Bereich des Meeres
Praasion und Richliikeit wurden durcb
Analyse von 7 USCS Cesieins-Sid. be
slimmt.
INTRODUCTION
Recent trends in the field of atomic absorption spectre*-
copy have been toward flameless alemisation. Severe
matrix problems are encountered, however, in samples such
as silicate rock* and marine sediments. Remedies such as
selective volatilization and the method of standard addi-
tions have failed to eliminate these problems in complex
solutions (1).
Flame alomisation, for which matrix interferences can
generally be controlled satisfactorily, is one of the most
suitable methods for (he analysis of silicate rocks and
marine sediment*.
The method described here consists of an acid decom-
position in a Teflon bomb followed by flame atomic
absorption for the delerminstion of 21 elements in silicate
rocks and marine sediments. It is more comprehensive in
terms of number of elements than other reported schemes
based on HF decomposition (23,4).
For the assessment of relative accuracy and the precision
of this method, U.S.G.S. rock standards granite G>2 and
diabase W-l have been analysed for 21 major, minor, and
trace elements: Al, Ca, Fe, K, Mg, Mn. Na, Si, Ti, Ba, Be,
Co. Cr, Cu. Li, Ni, Pb, Rb, Sr. V and Zn.
In addition, U.S.C.S. rock standards andesite AGV-1,
basalt BCR-1, dunile DTS-1, pranodiorile GSP-1 and peri-
dotite PCC-1 were analysed for 12 trace elements.
For some elements, notably Ba, Be, G, Pb, Rb and V.
which were determined by this method, no atomic absorp-
tion values were reported by Flanagan in his compilation
of the rock standard data (5).
PROCEDURE
Sample Preparation
Table 1 shows the analytical scheme for the major and
trace elements.
For the major and some minor elements, a 0.1-g sample
is decomposed with HF and aqua regia in a Teflon vessel",
(6) and initially diluted to 100 ml. From this solution Mn.
Si, and also Ni for DTS-1 and PCC-1 are determined
directly.
Solid KC1 is added to separate aliquots to give a potas-
sium concentration of 1500 ppm, ana these are anal) zed
forBa,Sr,andTi.
For the determinations of AX Ca, Fe, Mg, and Na. a
further 20X dilution is made to give the desired dilution
factor of 2X10* for these elements in the KC1 (1500 pj.m)
matrix. A similar dilution is also made for the delrrinina-
tion of K by substituting NaCl as the diluent
For trace element determinations, a separate 1-p sample
TABIEI
Sample Preparation Diagram for Silicate Rock Analyses
0.1-a, sample -+ Teflon vessel decomposition
-»1000X dilution
-»1000X dilution in 1500 ppm K
-»2000X dilution in 1500 ppm K
-»2000X dilution in 1500 ppm Na
1-g sample —» Teflon vessel decomposition—» 100X dilution
Mn,Si
Ba. Sr, Ti
Al, Ca. Fe, Mg, Na
K
Be, Co, Cr. Cu, li,
Ni,Pb.Rb,V,Zn
from H. X. Monijcn ind Sons Ltd. Mount llniicke,
H«nU Co., N. S., Cmnids.
ATOMIC ABSORPTION NEWSUHER
Vol. 14. No. 5, September-October 1975
D-3
-------
I- r|rmmpo«rrl in ihr Trflon tr»»c! and ililulrrl In 100 ml
Frutn lni« ."ululion Br, Co. Cr. Cu. Li. Ni. Pli. Rb. V. and
Zn are determined direct!).
Decomposition Proctdur*
A finely ground sample of 0.1 ft or 1 g is weighed ac-
curately in the Teflon vessel and welled 1 ml of aqua regia.
Six ml of concentrated HF are added ilowl), and the cover
it placed on liphlly. The vessel is immersed in a water bath
<90°C-100°C) for'l hr Care should be taken that the vessels
remain immersed during the entire heating period. Follow-
ing the heating, the vessels should immediate!) be removed
from the water bath and placed in cold water. The water
level should not reach the threaded cover. These precau-
tions are necessary to avoid sucking water into the vessels
by the vacuum created during cooling. After cooling, the
contents are washed into a 100-ml polypropylene volu-
metric flask containing 5.6 g H9BO, that has been shaken
with approximately 20 ml of H90. The flask is shaken to
complete the dissolution and made up to volume. The
aolutions are stored in polypropylene bottles.
Sample solutions thus prepared are extremely stable. In
more concentrated trace element solutions, a gelatinous
precipitate of borosilicates will form due to the high con-
centration of silicon. This has no adverse effect on trace
element determinations (6), and the removal of the bulk
of silicates is desirable.
Sf ondoref Preparation
Single-element standards for major and minor elements
can be used. For the sake of convenience, however, a com-
bined slock standard solution was prepared. This, as well
as other standards, was prepared from 1000-pprn standards.
The 1000-ppm standards were prepared either in the
laboratory or obtained commercially. No specific details
are given here for preparing these solutions. Such informa-
tion is readily available (7).
The exceptions are the Si, Ti, and Cr standards which
are described below. A combined slock standard solution
of 300 ug/ml Al and 100 ug/ml Ca. Fe, K, Mg, Mn, and
Na was prepared. This stock standard was diluted 25, 50,
75. 100. 200. and 400 times for working standards. All
working standards were prepared in 1500 ppm K except
standards for K which were prepared in 1500 ppm Na.
The decomposition blank contained 1 ml aqua regia. 6 ml
concentrated HF. and 5.6 g H3B03/100inl.
A 1000-ppm Si standard was prepared from a silica rod
by the same decomposition procedure as described for the
samples. Working standards of 200-400 ppm Si were pre-
pared in • decomposition blank solution.
A 1000-ppm Si siandard was prepared from a silica rod
bj the »ame decomposition procedure as described for the
sample*. Working standards of 200-400 ppm Si were pre-
pared in a decomposition blank solution.
The 1000-ppm Ti siandard was prepared from TiOj by
the same decomposition procedure described for the sam-
ples. Working standards of 2-10 ppm Ti were prepared in
the decomposition blank with 1500 ppm K.
Single-element standards were prepared for Ba. Be, Cr,
Rb. Sr. and V. A combined Mock siandard solution of
50 tig -'ml each was prepared for Co, Cu. Li, Ni. Pb, and Zn.
V orkinp standards of 0.1-2.0 ppm for these elements were
prepared in the decomposition blank and are stable for
»r\eral months.
Working ftandards of 0.1-2.0 pfim Ba were prepared in
llir decomposition blank with 1500 ppm K.
In preparing IV Mamiards it i* aHv^aMr to u*r rom
mernalh atailaMr stork «tjnr)arrl •><>lulion<> >inrr bcr\l
Hum dust i» potential!) loxic M orkinp Mandsrd* of 5-10
ppb Be were prepared in ihr flrcompo*il>un blank
A 1000-ppm Cr Manriard wa* prepared from K.-CrjO;
Working standard* of 0 1-1.5 ppm Cr *ere prepared in the
decomposition blank The best semilixil) for Cr is obtained
in the air-acetylene flame, but it is first necessary to reduce
Cr** to Cr14 in the solution because the standards give a
low absorption if the Cr is not reduced. This reduction was
carried out b) adding 2 ml perchloric acid and 1 ml 307r
H-O. to 20 ml of 1000 ppm Cr**, followed by healing and
dilution to 1000 ml to obtain a 20-ppm Cr** siandard (8).
This standard waa diluted with the decomposition blank
solution for working standards. Reduction of Cr** to Cr**
is not necessary for Cr determination in a nitrous oxide-
acetylene flame, but this method is less sensitive.
Working standards of 0.2-3.0 ppm Rb were prepared in
the decomposition blank with sufficient K added to match
the sample concentration. The absorption of Rb is sup-
Eressed by the decomposition blank solution and enhanced
) K. Enhancement by K b progressive to about 600 ppm
K, above which suppression by K occurs. Thus, the addition
of excessive amounts of K results in a vastly suppressed
absorption signal and cannot be recommended. Enhance-
ment by Na is small. When 200 ppm of Na and K were
added to a 1-ppm Rb standard in tie decomposition blank.
the combined enhancement was about 2% more than in
200 ppm K only. Since the determination is highly depend-
ent on K concentration, it was first necessary to determine
the K content of the samples and match it to the standards.
Since the absorption of Rb was observed to be linear at
least to 3 ppm Rb for a particular K concentration, several
standard curves could be drawn by using just one standard
in each case. Where large numbers of samples are analyxed.
it is not practical to match the sample concentration of K
in each standard, but the Rb concentration can be inter-
polated from other standard curves. It remains to be in-
vestigated whether another alkali metal could be used in
large amounts to compensate for interferences without
causing suppression of Rb absorption.
Working standards of 0.1-1.0 ppm Sr were prepared in
the decomposition blank with 1500 ppm K.
Working standards of 0.3-5.0 ppm V were prepared in
the decomposition blank with 100 ppm Al and 500 ppm K.
Both Al and K enhance the vanadium absorption. No
further enhancement was observed above 100 ppm Al and
500 ppm K.
APPARATUS
A Perkin-Elmer Model 303 atomic absorption speclro-
photometer equipped with a Deuterium Background Cor-
rector and a 10-mV full-scale deflection recorder was used
for the determination of all elements except Pb which was
determined using a Perkin-Elmer Model 306 atomic ab-
sorption spectropholomeler. Scale expansion was 10X or
less for all determinations. Light sources for K, Na, and
Rb were Osram arc-discharge lamps. A Perkin-Elmer EDL
was the light source for Pb. The Be light source was a
Westinghouse hollow cathode lamp, and the remaining
elements were determined with Perkin-Elmer Inlensitron*'
single-element hollow cathode lamps. Acetylene was thr
fuel for all elements. Air was used as the oxidant, except
for Al. Ca, .Mg, Si. Ti. Ba. Be, Sr, and V for which nitrous
oxide was emplo)ed.
0-4
ATOMIC ABSORPTION NEWSLETTER
Vol. 14, No. 5. September-October 1975
-------
INTERFERENCES
Matrix mterfrrrnrr* on major and minor element* are
reduced b\ dilution in formp the concenlration to the linear
portion of the ar»*orhance curve, loniution is controlled b>
additions of 1500 ppm K or Na to standard." and samples.
Chemical suppression by elements such as silicon and
aluminum on calcium and magnesium absorption was
avoided b\ the use of a nitrous oxide-acel)lene flame. This
eliminated the need for lanthanum which is essential in an
air-acetylene flame. No interferences were observed for
Mn and Si in a fluobonc-boric acid matrix.
lonization in Ba and Sr determinations Has controlled
by adding 1SOO ppm K to samples and standards. Stan-
dards for Cr, Rb, and V require special attention as dis-
cussed under standard preparation. Molecular absorption
is corrected by the Deuterium Background Corrector in
Co, Ni, and Pb determinations. No interferences were ob-
served for Be, Cu, Li, and Zn in a fluoboric-boric acid
matrix.
RESULTS AND DISCUSSION
Table 11 indicates the relative accuracy of the major and
minor element data for W-l and C-2 by the comparison of
our values to those compiled by Flanagan (9). The preci-
sion is expressed as a coefficient of variation. To obtain
these data, 6 separate portions of W-l and G-2 were
analysed.
Table III indicates the relative accuracy of the trace
element determinations for teven U.S.G.S. standard rocks
by comparison of our values to those compiled by Flanagan.
The precision is expressed as a coefficient of variation
(Table IV). These data are based on 3 separate portions
TABLE II
Major Elements in U.S.G.S. Standard Rocks (%)
Al
Co
Fe
K
M9
Mn
Na
Si
Ti
W-l
G-2
W-l
G-2
W-l
G-2
W-l
G-2
W-l
G-2
W-l
G-2
W-l
G-2
W-l
G-2
W-l
G-2
A
7.94
8.15
7.83
1.39
7.76
1.85
0.53
374
3.99
0.46
0.132
0.026
1.59
3.02
24.6
32.3
0.64
0.30
B
7.68
8.11
7.83
MO
7.96
1.90
0.56
3.62
3.86
0.44
0.127
0.025
1.60
2.94
24.6
31.5
0.67
0.31
Coeff.
1.5
2.7
0.7
1.9
0.4
5.0
4.7
1.2
0.1
4.1
1.1
4A
1.2
0.8
2.6
0.8
1.5
2.6
A Flanagan (8)
B This work
nf thr rock -ample
Clo*e a^rn-nirnt with Flanagan'* \alur- ran 1«- nolc.l Im
most clement* Zinc talues for DTS-1 ind PCC-1 arr )<•«
Tin* could be due to incomplete decomposition n( rhru
milrv An appreciable amount of chromite which rmilil not
br dcroinjio-rd entirel) is contained in DTS-1 and PCC-1 .
therefore, it was not possible to determine Cr accurnirU
in thene samples.
Nickel values for ACV-1 and W-l are comparatively
low. An attempt was made to determine Ni without back-
ground correction. The positive error that resulted, how-
ever, was considerably larger than the negative error w ith
background correction. It has been shown that very large
positive or negative error* are possible in the determina-
tion of Pb, Zn, Ni, and Co in geological materials which
depend upon the concentration of the trace elements and
major elements in the sample solution (101. The dilution
factor of 100X and the fluoboric-boric acid matrix used in
this method minimize these errors.
The precision for major and minor elements is ^ 55
and for trace elements generally < 10%.
For most major elements the relative accuracy is com-
parable and precision improved over that reported b>
Buckley and Cranston. However, for trace elements present
in low concentrations, both the relative accuracy and pre-
cision are significantly improved over the values reported
by the above authors. No complete comparison can be
made at this time with other scheme; due to the lack of
published statistical data on all the elements we hate
studied by this method.
The method described here is ideal for major geochemi-
cal studies on marine sediments (11) and is now used in
this laboratory for routine analyses. Although the organic
matter is not completely oxidized during the decomposi-
tion, the residue is generally <3#, and its elemental
contribution is neligible. The method is rapid as a larpe
number of inexpensive Teflon vessels can be used simul-
taneously and the dilutions carried out with commercial
sampler-diluters.
CONCLUSION
The method described for multi-element analyse.- of
silicate rocks and marine sediments by flame atomic ab-
TABLE IV
Precision (Coefficient of VoriotionJ for the
Determination of Trace Elements in
* U.S.G.S. Standard Rockt(% |
Bo
Be
Co
Cr
Cu
Li
Ni
Pb
Rb
Sr
V
Zn
AGV-1
2.0
2.8
14
5.6
4.2
2.6
—
4.3
2.3
2.2
1.2
1.3
BOM
5.7
2.2
7.3
3.9
6.7
2.5
—
15
1.2
2.9
2.4
1.6
W-l
3.2
4.0
2.4
0.5
3.7
2.2
4.5
—
4.1
5.1
0.7
0.7
ors-t
—
_
0.9
_
4.2
6.4
—
—
—
—
—
1.5
G-2
1.1
1.7
7.9
6.1
4.3
0.8
—
11
2.1
1.6
6.0
4.1
GSP-1
1.4
1.9
12
1.9
4.9
1.5
—
8.6
0.5
3.5
4.3
10
PCC-I
_
_
09
_
5.6
10
—
—
_
—
7.5
18
ATOMIC ABSORPTION NEWSLETTER
Vol. 14. No. 5. September-October 1975
D-5
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TABLE 111
Trace Elements in U.S G S. Standard Rockt I pom)
la (a)
(b)
»• (e)
(b)
Pb{o)
(b)
Rb(o)
(b)
Sr (a)
(b)
V (a)
(b)
Zn(a)
(b)
a Flanagan (8)
b Thitwork
AGV-J
1208
1140
3
2.6
14
18
12
9
40
59
12
12
18.5
12
35
40
67
67
657
698
125
128
84
90
ICft-7
675
630
1.7
2.1
38
36
18
11
18
15
13
14
16
<12
16
16
47
49
330
363
399
446
120
129
W-1
160
156
0.8
0.9
47
41
114
116
110
122
14
14
76
66
8
<«
21
21
190
194
264
285
86
86
075- r
2.4
<5
_
<0.5
133
128
—
not analyzed
7
7
2
2.7
2269
2420
14
19
_
not analyzed
0.35
<2
10
<15
45
32
G-2
1870
1970
2.6
2.9
5.5
7
7
7
12
12
37
34
5
<12
31
34
168
166
479
457
35
42
85
87
GSM
1300
1230
13
1.7
6
9
12
12
33
31
32
30
12.5
<12
51
59
254
248
233
245
53
62
98
105
PCC-I
1.2
<5
—
.
B. F. J. Jolinmn. T. C Woodi*. Jr. and J. M. Cumminc», Jr.. Au
Absorption Ne»»lcH- II. 118 (1972).
9. F. J. Flintpjo, Ceaeblm. Cotmotb'm. Aeu J7,1189 (1973).
10. C. J. S. Covett and R. E. Vhiiebecd. J. Ceeehem. E«pl t. 121
11973).
II. D. H. Urine tnd D. J. C. Noia. Bulletin 182, Fiskeriw Resetrcb
Board of Canida.
D-6
ATOMIC ABSORPTION NEWSLETTER
Vol. H, No. S, September-October 1975
-------
APPENDIX E
HN03/HC104 DIGESTION METHOD FOR TISSUE
(TETRA TECH 1986a)
-------
11.0 PROCEDURE
11.1 Homogenize samples prior to analysis to ensure that a representative
aliquot is taken. Any grinder or homogenizer that has been found to be
free of contamination may be used. Samples should be ground wet to avoid
losses of volatile elements (e.g., Hg, Se) during drying. The liquid associated
with a sample after thawing should be retained as part of the sample.
11.2 Transfer the sample paste to a container suitable for storage. If
not immediately analyzed, the samples should be frozen (-20° C) until required.
Containers should be tightly sealed to prevent moisture loss or gain during
storage.
11.3 Dry Weight Determination - if sample size permits and dry-weight
concentrations are required, dry weight determinations may be performed
as follows: transfer an aliquot of approximately 3 g (weighed to the nearest
0.1 g) to a pre-weighed dish. Allow the sample to dry in an oven at 105° C
overnight, and determine the solid residue weight to the nearest 0.1 g.
The percent total solids is calculated as:
TS = [dry residue wt (g)]/[west sample wt (g)]
Dry-weight determinations should not be made at the cost of having insufficient
sample for metals analysis. Significant decreases in the size of samples
used for extraction will decrease attainable detection limits.
11.4 Accurately weigh representative aliquots of homogenized tissue to
the nearest 0.1 mg. If sample size permits, approximately 5 g is required
to maintain optimum detection limits. Transfer the weighed tissue to a
pre-cleaned 125-mL Erlenmeyer flask equipped with an all-glass reflux cap.
Analyze a sufficient number of reagent blanks, sample duplicates, analyte
spikes, and certified reference materials concurrently.
E-l
-------
11.5 Add 10.0 ml of concentrated nitric acid (ACS grade or better), replace
cap and swirl. Allow flask to stand at room temperature for about 15 h
in a dust-free ventilated environment. Periodically swirl the contents
to help solubilize the tissue.
11.6 After 15 h, gently heat the flask to approximately 100° C - hold
at this temperature for 1 h. Gradually increase the temperature in 50° C
increments to a maximum of 250° C. Continue digesting until all tissue
has been solubilized. This usually takes about 4 h. Do not rush the initial
digestion as losses of volatile elements will likely occur. Once digestion
is complete, cool flasks to room temperature and add 4.0 ml of perchloric
acid.
CAUTION: Perchloric acid is a strong oxidizing agent. The analyst
must be fully aware of the precautions associated with
its use. This procedure (i.e., use of perchloric acid
at sub-fuming temperatures) has been safety performerd
without a perchloric hod, but a perchloric hod is strongly
recommended nonetheless. Laboratories that do not
carefully monitor perchloric acid digestions will be
endangered without perchloric hoods. Safety precautions
and background information pertaining to perchloric
acid can be found in Schilt (1979; Perchloric Acid
and Perchlorates. G. Frederick Smith Chemical Company,
Columbus, OH.).
11.7 Return flasks to the hotplate which has been cooled to about 200° C.
Continue heating for 1 h, then increase plate temperature to 300° C. Hold
at this temperture until all traces of nitric acid fumes have disappeared
and the solutions have become clear. Do not overheat flasks or allow perchloric
fumes (dense white) to appear. If perchloric fumes appear, reduce heat
immediately. Remove the exracts when clear and cool them to room temperature.
E-2
-------
11.8 When the digestion is complete, rinse the caps into the flasks and
transfer the extract to a pre-cleaned 100-mL volumetric flask. Rinse the
Erlenmeyer flask three times with DDW and combine with the extract previously
added to the volumetric flask. Adjust the volume with DDW and transfer
to a precleaned plastic bottle.
NOTE: Some elements are not as stable as others in solution and
therefore should be analyzed first. Stability can be determined by daily
analysis of the extracts. However, the following can be used as a guideline:
Sb, Pb, Hg, Se, and Ag - analyze within 1 day
As and Cd - analyze within 2 days
Cr, Cu, Ni and Zn - analyze within 1 wk
Be and Tl - to be determined.
E-3
-------
APPENDIX F
APDC/MIBK EXTRACTION METHOD FOR SALT WATER
(GREENBURG ET AL. 1985)
-------
303 B. Determination of Low Concentrations of Cadmium,
Chromium, Cobalt, Copper, Iron, Lead, Manganese, Nickel,
Silver, and Zinc by Chelation with Ammonium Pyrrolidine
Dithiocarbamate (APDC) and Extraction into Methyl Isobutyl
Ketone (MIBK)
1. Apparatus
a. Atomic absorption spectrometer and as-
sociated equipment: See Section 303.2.
b. Burner head, conventional. Consult
manufacturer's operating manual for sug-
gested burner head.
2. Reagents
a. Air See 303A.20.
b. Acetylene: See 303A.24.
c. Metal-free water: See 303A.2c.
d. Methyl uobutyl ketone (MIBK). re-
agent grade. For trace analysis, purify
MIBK by redistillation or by sub-boiling
distillation.
e. Ammonium pyrrolidine dithiocarba-
mate (APDC) solution: Dissolve 4 g APDC
in 100 mL water. If necessary, purify
APDC with an equal volume of MIBK.
Shake 30 s in a separatory funnel, let sep-
arate, and withdraw lower portion. Discard
MIBK layer.
/ Nitric acid. HNO,, cone, ultrapure.
g. Standard metal solutions: See 303A.2/.
h. Potassium permanganate solution,
KMnO,, 5% aqueous.
i. Sodium sulfate. Na,SO4, anhydrous.
/ Water-saturated MIBK: Mix one part
F-l
-------
ATOMIC ABSORPTION SPECTROMETRY
161
purified MIBK with one pan water in a
separatory funnel. Shake 30 s and let sep-
arate. Discard aqueous layer. Save MIBK
layer.
k. Sodium azide solution: Dissolve 0.1 g
NaN, in water and dilute to 100 mL.
3. Procedure
a. Instrument operation: See Section
303A.3a. After final adjusting of burner
position, aspirate water-saturated MIBK
into flame and gradually reduce fuel flow
until flame is similar to that before aspi-
ration of solvent.
b. Standardization: Select at least three
standard metal solutions (prepared as in
303A.2/) to bracket expected sample metal
concentration and to be, after extraction,
in the optimum concentration range of the
instrument. Adjust 100 mL of each stand-
ard and 100 mL of a metal-free water blank
to pH 3 by adding \N HNO, or \N NaOH.
For individual element extraction, use the
following pH ranges to obtain optimum
extraction efficiency:
Element
pH Range for Optimum
Extraction
Ag 2-5 (complex unstable)
Cd 1-6
Co 2-10
Cr 3-9
Cu 0.1-8
Fe 2-5
Mn 2-4 (complex unstable)
Ni 2-4
Pb 0.1-6
Zn 2-6
NOTE For Ag and Pb extraction the op-
timum pH value is 2.3 ± 0.2. The Mn
complex deteriorates rapidly, resulting in
decreased instrument response. Unless Mn
can be analyzed immediately after extrac-
tion, use another analytical procedure.
Transfer each standard solution and
blank to individual 200-mL volumetric
flasks, add 1 mL APDC solution, and
shake to mix. Add 10 mL MIBK and shake
vigorously for 30 s. (The maximum volume
ratio of sample to MIBK is 40.) Let con-
tents of each flask separate into aqueous
and organic layers, then carefully add
water down the side of each flask to bring
the organic layer into the neck and acces-
sible to the aspirating tube.
Aspirate organic extracts directly into
the flame (zeroing instrument on a water-
saturated MIBK blank) and record ab-
sorbance. With some instruments it may
be necessary to convert percent absorption
to absorbance by using a table generally
provided by the manufacturer.
Prepare a calibration curve by plotting
on linear graph paper absorbances of ex-
tracted standards against their concentra-
tions before extraction.
c. Analysis of samples: Rinse atomizer by
aspirating water-saturated MIBK. Aspi-
rate organic extracts treated as above di-
rectly into the flame and record
absorbances.
With the above extraction procedure
only hexavalent chromium is measured. To
determine total chromium, oxidize triva-
lent chromium to hexavalent chromium by
bringing sample to a boil and adding suf-
ficient KMnO. solution dropwise to give a
persistent pink color while the solution is
boiled for 10 min. Destroy excess KMnO.
by adding 1 to 2 drops NaN, solution to
the boiling solution, allowing 2 min for the
reaction to proceed. If pink color persists,
add 1 to 2 more drops NaN, solution and
wait 2 min. Heat an additional 5 mm. Cool,
extract with MIBK, and aspirate.
During extraction, if an emulsion forms
at the water-MIBK interface, add anhy-
drous Na2SO, to obtain a homogeneous or-
ganic phase.
Determine recovery of silver whenever
matrices change.
162
METALS (300)
To avoid problems associated with in-
stability of extracted metal complexes, de-
termine metals immediately after extrac-
tion.
4. Calculations
Calculate the concentration of each
metal ion in micrograms per liter by re-
ferring to the appropriate calibration curve.
F-2
-------
APPENDIX G
DFAA INSTRUMENTAL AND SPECTROPHOTOMETRIC METHODS
(U.S. EPA 19793)
-------
ANTIMONY
Method 204.1 (Atomic absorption, direct aspiration)
STORE! NO. Total 01097
Dissolved 01095
Suspended 01096
Optimum Concentration Range: 1-40 mg/1 using a wavelength of 217.6 nm
Sensitivity: 0.5 mg/1
Detection Limit: 0.2 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 2.7426 g of antimony potassium tartrate (analytical
reagent grade) and dissolve in deionized distilled water. Dilute to 1 liter with deionized
distilled water. 1 ml = 1 mgSb (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the time of
analysis. The calibration standards should be prepared using the same type of acid and at
the same concentration as will result in the sample to be analyzed either directly or after
processing.
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 through 4.1.4 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory.
Instrumental Parameters (General)
1. Antimony hollow cathode lamp
2. Wavelength: 217.6 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Fuel lean
Analysis Procedure
1. For analysis procedure and calculation, see "Direct Aspiration", part 9.1 of the Atomic
Absorption Methods section of this manual.
Approved for NPDES
Issued 1974
Editorial revision 1978
G-l
-------
Interferences
1. In the presence of lead (1000 mg/1), a spectral interference may occur at the 217.6 nm
resonance line. In this case the 231.1 nm antimony line should be used.
2. Increasing acid concentrations decrease antimony absorption. To avoid this effect, the
acid concentration in the samples and in the standards should be matched.
Notes
1. Data to be entered into STORET must be reported as ug/I.
2. For concentrations of antimony below 0.35 mg/1, the furnace procedure (Method 204.2)
is recommended.
Precision and Accuracy
1. In a single laboratory (EMSL), using a mixed industrial-domestic waste effluent at
concentrations of 5.0 and IS mg Sb/1, the standard deviations were ±0.08 and ±0.1,
respectively. Recoveries at these levels were 96% and 97%, respectively.
G-2
-------
ARSENIC
Method 206.3 (Atomic Absorption—gaseous hydride)
STORET NO. Total 01002
Dissolved 01000
Suspended 01001
1. Scope and Application
1.1 The gaseous hydride method determines inorganic arsenic when present in
concentrations at or above 2 ug/1. The method is applicable to drinking water and most
fresh and saline waters in the absence of high concentrations of chromium, cobalt,
copper, mercury, molybdenum, nickel and silver.
2. Summary of Method
2.1 Arsenic in the sample is first reduced to the trivalcnt form using SnClj and converted to
arsine, AsH3, using zinc metal. The gaseous hydride is swept into an argon-hydrogen
flame of an atomic absorption spectrophotometer. The working range of the mehtod is
2-20 ug/1. The 193.7 nm wavelength line is used.
3. Comments
3.1 In analyzing drinking water and most surface and ground waters, interferences are rarely
encountered. Industrial waste samples should be spiked with a known amount of arsenic
to establish adequate recovery.
3.2 Organic forms of arsenic must be converted to inorganic compounds and organic matter
must be oxidized before beginning the analysis. The oxidation procedure given in
Method 206.S (Standard Methods, 14th Edition, Method 404B, p. 285, Procedure 4.a)
has been found suitable.
3.3 For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
3.4 For quality control requirements and optional recommendations for use in drinking
water analyses, see part lOof the Atomic Absorption Methods section of this manual.
3.5 Data to be entered into STORET must be reported as ug/1.
4. Precision and Accuracy
4.1 Ten replicate solutions of o-arsenilic acid at the 5,10 and 20 ug/1 level were analyzed by
a single laboratory. Standard deviations were ±0.3, ±0.9 and ±1.1 with recoveries of 94,
93 and 85%, respectively. (Caldwell, J. S., Lishka, R. J., and McFarren, E. F.,
"Evaluation of a Low Cost Arsenic and Selenium Determination at Microgram per Liter
Levels", JAWWA., vol 65, p 731, Nov., 1973.)
Approved for NPDES and SDWA
Issued 1974
G-3
-------
5. References
3.1. Except for the perchloric acid step, the procedure to be used for this determination is
found in: Standard Methods for the Examination of Water and Wastewater, 14th
Edition, p!59, Method 301 A(VII),( 1975)
G-4
-------
ARSENIC
Method 206.4 (Spectrophotometric-SDDC)
STORE! NO. 01002
Inorganic, Dissolved 00095
Inorganic, Total 00997
Inorganic, Suspended 00996
1. Scope and Application
1.1 The silver diethyldithiocarbamate method determines inorganic arsenic when present in
concentrations at or above 10 ug/1. The method is applicable to drinking water and most
fresh and saline waters in the absence of. high concentrations of chromium, cobalt,
copper, mercury, molybdenum, nickel, and silver. Domestic and industrial wastes may
also be analyzed after digestion (see 3.3).
1.2 Difficulties may be encountered with certain industrial waste materials containing
volatile substances. High sulfur content of wastes may exceed removal capacity of the
lead acetate scrubber.
2. Summary of Method
2.1 Arsenic in the sample is reduced to arsine, AsH3, in acid solution in a hydrogen
generator. The arsine is passed through a scrubber to remove sulfide and is absorbed in a
solution of silver diethyldithiocarbamate dissolved in pyridine. The red complex thus
formed is measured in a spectrophotometer at 535 nm.
3. Comments
3.1 In analyzing drinking water and most surface and ground waters, interferences are rarely
encountered. Industrial waste samples should be spiked with a known amount of arsenic
to establish adequate recovery.
3.2 It is essential that the system be airtight during evolution of the arsine, to avoid losses.
3.3 If concentration of the sample and/or oxidation of any organic matter is required, refer
to Method 206.5. [Standard Methods. 14th Edition, Method 404B, p. 284, Procedure 4.a
(1975)]. For sample handling and preservation, see part 4.1 of the Atomic Absorption
Methods section of this manual.
3.3.1 Since nitric acid gives a negative interference in this test, use sulfuric acid as a
preservative if only inorganic arsenic is being measured.
3.4 1-Ephedrine in chloroform has been found to be a suitable solvent for silver
diethyldithiocarbamate if the analyst finds the odor of pyridine objectionable [Anal.
Chem. 45,1786(1973)].
3.5 For quality control requirements and optional recommendations for use in drinking
water analyses, see part 10 of the Atomic Absorption Methods section of this manual.
Approved for NPDES and SDWA
Issued 1971
Editorial revision 1974
G-5
-------
4. Precision and Accuracy
4.1 In a round-robin study reported by Standard Methods a synthetic unknown sample
containing 40 ug/1, as As, with other metals was analyzed in 46 laboratories. Relative
standard deviation was ±13.8% and relative error was 0%.
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 14th Edition, p. 283,
Method 404A( 197 5).
G-6
-------
ARSENIC
Method 206.5 (Sample Digestion Prior to Total Arsenic
Analysis by Silver Diethyldithiocarbamate or Hydride
Procedures)
1. Scope and Application:
1.1 Both the silver diethyldithiocarbamate spectrophotometric method and the AA hydride
procedure measure inorganic arsenic. Therefore, if either of these procedures are being
employed for the purpose of measuring total arsenic (inorganic plus organic), all
organically bound arsenic must first be converted to an inorganic form prior to the
analytical determination. This may be accomplished with H]S04-HNOj.
2. Procedure
2.1 To a suitable sample containing from 2 to 30 ug of arsenic, add 7 ml (1 +1) H2SO4 and 5
ml cone HNO3. Evaporate the sample to SO3 fumes. Caution: If the sample chars, stop
the digestion immediately, cool and add additional cone HNO3. Continue digestion
adding additional cone HNO, as necessary.
2.2 If the sample remains colorless, or straw-yellow during evolution of SO3 fumes, the
digestion is complete.
2.3 Cool the digested sample, add about 25 ml distilled water, and again evaporate to SO}
fumes to expel oxides of nitrogen.
2.4 The sample is now ready for analysis using either the hydride or spectrophotometric
procedure.
3. Interferences
3.1 All traces of nitric acid must be removed before either the spectrophotometric or the
hydride procedures are applied. Oxides of nitrogen should be expelled by taking the
sample to fumes of SO3.
4. Notes
4.1 The digestion step may be carried out in a flask on a hot-plate or in a Kjeldahl apparatus.
This digestion step may also be used, in effect, to concentrate the sample, inasmuch as
any size volume may be processed.
Bibliography
1. Standard Methods for the Examination of Water and Wastewater, p285, method 404B, step 4a,
14th Edition (1975).
Approved for NPDES and SDWA
Issued 1978
G-7
-------
CADMIUM
Method 213.1 (Atomic Absorption, direct aspiration)
STORE! NO. Total 01027
Dissolved 01025
Suspended 01026
Optimum Concentration Range: 0.05-2 mg/1 using a wavelength of 228.8 nm
Sensitivity: 0.025 mg/1
Detection Limit: 0.005 ing/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 2.282 g of cadmium sulfate (3CdSO«»8H,O, analytical
reagent grade) and dissolve in deionized distilled water. 1 ml = 1 mg Cd (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the time of
analysis. The calibration standards should be prepared using the same type of acid and at
the same concentration as will result in the sample to be analyzed either directly or after
processing.
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 through 4.1.4 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory.
Instrumental Parameters (General)
1. Cadmium hollow cathode lamp
2. Wavelength: 228.8 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Analysis Procedure
1. For analysis procedure and calculation, see "Direct Aspiration", part 9.1 of the Atomic
Absorption Methods section of this manual.
Approved for NPDES and SDWA
Issued 1971
Editorial revision 1974
G-8
-------
Notes
1. For levels of cadmium below 20 ug/1, either the Special Extraction Procedure given in
Part 9.2 of the Atomic Absorption methods section as the furnace technique, Method
213.2 is recommended.
2. Data to be entered into STORET must be reported as,ug/l.
3. For quality control requirements and optional recommendations for use in drinking
water analyses, see part 10 of the Atomic Absorption Methods section of this manual.
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was conducted by
the Quality Assurance and Laboratory Evaluation Branch of EMSL. Six synthetic
concentrates containing varying levels of aluminum, cadmium, chromium, copper, iron,
manganese, lead and zinc were added to natural water samples. The statistical results for
cadmium were as follows:
Standard
Number True Values Mean Value Deviation Accuracy as
of Labs ug/liter ug/Iiter ug/liter % Bias
74 71 70 21 -2.2
73 78 74 18 -5.7
63 14 16.8 11.0 19.8
68 18 18.3 10.3 1.9
55 1.4 3.3 5.0 135
51 2.8 2.9 2.8 4.7
G-9
-------
COPPER
Method 220.1 (Atomic Absorption, direct aspiration)
STORET NO. Total 01042
Dissolved 01040
Suspended 01041
Optimum Concentration Range: 0.2-5 mg/1 using a wavelength of 324.7 nm
Sensitivity: 0.1 mg/1
Detection Limit: 0.02 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.00 g of electrolyte copper (analytical reagent grade).
Dissolve in 5 ml redistilled HNO, and make up to 1 liter with deionized distilled water.
Final concentration is 1 mg Cu per ml (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the time of
analysis. The calibration standards should be prepared using the same type of acid and at
the same concentration as will result in the sample to be analyzed either directly or after
processing.
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 thru 4.1.4 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory.
Instrumental Parameters (General)
1. Copper hollow cathode lamp
2. Wavelength: 324.7 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Analysis Procedure
1. For analysis procedure and calculation, see "Direct Aspiration", part 9.1 of the Atomic
Absorption Methods section of this manual.
Approved for NPDES
Issued 1971
Editorial revision 1974 and 1978
G-10
-------
Notes
1. For levels of copper below SO i/g/1, either the Special Extraction Procedure, given in part
9.2 of the Atomic Absorption Methods section or the furnace technique. Method 220.2,
is recommended.
2. Numerous absorption lines are available for the determination of copper. By selecting a
suitable absorption wavelength, copper samples may be analyzed over a very wide range
of concentration. The following lines may be used:
327.4 nm Relative Sensitivity 2
216.S nm Relative Sensitivity 7
222.S nm Relative Sensitivity 20
3. Data to be entered into STORET must be reported as ug/1.
4. The 2,9-dimethyl-1, 10-phenanthroline colorimetric method may also be used (Standard
Methods, 14th Edition, p. 196).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was conducted by
the Quality Assurance and Laboratory Evaluation Branch of EMSL. Six synthetic
concentrates containing varying levels of aluminum, cadmium, chromium, copper, iron,
manganese, lead and zinc were added to natural water samples. The statistical results for
copper were as follows:
Standard
Number True Values Mean Value Deviation Accuracy as
of Labs ug/liter ug/liter tig/liter % Bias
91 302 305 56 0.9
92 332 324 56 -2.4
86 60 64 23 7.0
84 75 76 22 1.3
66 7.5 9.7 6.1 29.7
66 12.0 13.9 9.7 15.5
G-ll
-------
IRON
Method 236.1 (Atomic Absorption, direct aspiration)
STORE! NO. Total 01045
Dissolved 01046
Suspended 01044
Optimum Concentration Range: 0.3-5 mg/1 using a wavelength of 248.3 nm
Sensitivity: 0.12mg/l
Detection Limit: 0.03 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.000 g of pure iron wire (analytical reagent grade) and
dissolve in 5 ml redistilled HNO3, wanning if necessary. When solution is complete make
up to 1 liter with deionized distilled water. 1 ml = 1 mg Fe (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the time of
analysis. The calibration standards should be prepared using the same type of acid and at
the same concentration as will result in the sample to be analyzed either directly or after
processing.
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 thru 4.1.4 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory.
Instrumental Parameters (General)
1. Iron hollow cathode lamp
2. Wavelength: 248.3 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Analysis Procedure
1. For analysis procedure and calculation, see "Direct Aspiration", part 9.1 of the Atomic
Absorption Methods section of this manual.
Approved for NPDES
Issued 1971
Editorial revision 1974 and 1978
G-12
-------
Notes
1. The following lines may also be used:
248.8 nm Relative Sensitivity 2
271.9 nm Relative Sensitivity 4
302.1 nm Relative Sensitivity 5
252.1 nm Relative Sensitivity 6
372.0 nm Relative Sensitivity 10
2. Data to be reported into STORET must be reported as ug/1.
3. The 1,10-phenanthroline colorimetric method may also be used (Standard Methods,
14th Edition, p. 208).
4. For concentrations of iron below 0.05 mg/1, either the Special Extraction Procedure
given in part 9.2 of the Atomic Absorption Methods section or the furnace procedure,
Method 236.2, is recommended.
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was conducted by
the Quality Assurance and Laboratory Evaluation Branch of EMSL. Six synthetic
concentrates containing varying levels of aluminum, cadmium, chromium, copper, iron,
manganese, lead and zinc were added to natural water samples. The statistical results for
iron were as follows:
Standard
Number True Values Mean Value Deviation Accuracy as
Of Labs ug/liter ug/liter tig/liter % Bias
82 840 855 173 1.8
85 700 680 178 -2.8
78 350 348 131 -0.5
79 438 435 183 -0.7
57 24 58 69 141
54 10 48 69 382
G-13
-------
LEAD
Method 239.1 (Atomic Absorption, direct aspiration)
STORET NO. Total 01051
Dissolved 01049
Suspended 01050
Optimum Concentration Range: 1-20 mg/1 using a wavelength of 283.3 nm
Sensitivity: 0.5 mg/1
Detection Limit: 0.1 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.S99 g of. lead nitrate, Pb(NO3)2 (analytical reagent
grade), and dissolve in deionized distilled water. When solution is complete acidify with
10 ml redistilled HNO3 and dilute to 1 liter with deionized distilled water. 1 ml = 1 mg
Pb( 1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the time of
analysis. The calibration standards should be prepared using the same type of acid and at
the same concentration as will result in the sample to be analyzed either directly or after
processing.
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 thru 4.1.4 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory.
Instrumental Parameters (General)
1. Lead hollow cathode lamp
2. Wavelength: 283.3 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Analysis Procedure
1. For analysis procedure and calculation, see "Direct Aspiration", part 9.1 of the Atomic
Absorption Methods section of this manual.
Approved for NPDES and SDWA
Issued 1971
Editorial revision 1974 and 1978
G-14'
-------
Notes
1. The analysis of this metal is exceptionally sensitive to turbulence and absorption bands in
the flame. Therefore, some care should be taken to position the light beam in the most
stable, center portion of the flame. To do this, first adjust the burner to maximize the
absorbance reading with a lead standard. Then, aspirate a water blank and make minute
adjustments in the burner alignment to minimize the signal.
2. For levels of lead below 200 i/g/1, either the Special Extraction Procedure given in part
9.2 of the Atomic Absorption Methods section or the furnace technique, Method 239.2,
is recommended.
3. The following lines may also be used:
217.0 nm Relative Sensitivity 0.4
261.4 nm Relative Sensitivity 10
4. For quality control requirements and optional recommendations for use in drinking
water analyses, see part 10 of the Atomic Absorption Methods section of this manual.
5. Data to be entered into STORET must be reported as ug/1.
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was conducted by
the Quality Assurance and Laboratory Evaluation Branch of EMSL. Six synthetic
concentrates containing varying levels of aluminum, cadmium, chromium, copper, iron,
manganese, lead and zinc were added to natural water samples. The statistical results for
lead were as follows:
Standard
Number True Values Mean Value Deviation Accuracy as
of Labs iig/liter ug/liter iig/liter % Bias
74 367 377 128 2.9
74 334 340 111 1.8
64 101 101 46 -0.2
64 84 85 40 1.1
61 37 41 25 9.6
60 25 31 22 25.7
G-15
-------
MANGANESE
Method 243.1 (Atomic Absorption, direct aspiration)
STORET NO. Total 01055
Dissolved 01056
Suspended 01054
Optimum Concentration Range: 0.1-3 mg/1 using a wavelength of 279.5 nm
Sensitivity: 0.05 mg/1
Detection Limit: 0.01 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.000 g of manganese metal (analytical reagent grade)
and dissolve in 10 ml of redistilled HNO3. When solution is complete, dilute to 1 liter
with 1%(V/V)HC1.1ml = 1 mgMn (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the time of
analysis. The calibration standards should be prepared using the same type of acid and at
the same concentration as will result in the sample to be analyzed either directly or after
processing.
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 thru 4.1.4 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory.
Instrumental Parameters (General)
1. Manganese hollow cathode lamp
2. Wavelength: 279.5 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Analysis Procedure
1. For analysis procedure and calculation, see "Direct Aspiration", part 9.1 of the Atomic
Absorption Methods section of this manual.
Approved for NPDES
Issued 1971
Editorial revision 1974 and 1978
G-16
-------
Notes
1. For levels of manganese below 25 ug/1, either the furnace procedure. Method 243.2, or
the Special Extraction Procedure given in part 9.2 of the Atomic Absorption Methods
section is recommended. The extraction is carried out at a pH of 4.5 to 5. The manganese
chelate is very unstable and the analysis must be made without delay to prevent its re-
solution in the aqueous phase.
2. The following line may also be used:
403.1 nm Relative Sensitivity 10.
3. Data to be entered into STORET must be reported as ug/1.
4. The persulfate colorimetric method may also be used (Standard Methods, 14th Edition,
p225).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was conducted by
the Quality Assurance and Laboratory Evaluation Branch of EMSL. Six synthetic
concentrates containing varying levels of aluminum, cadmium, chromium, copper, iron,
manganese, lead and zinc were added to natural water samples. The statistical results for
manganese were as follows:
Standard
Number True Values Mean Value Deviation Accuracy as
of Labs ug/liter ug/liter ug/liter % Bias
77 426 432 70 1.5
78 469 474 97 1.2
71 84 86 26 2.1
70 106 104 31 -2.1
55 II 21 27 93
55 17 21 20 22
G-17
-------
NICKEL
Method 249.1 (Atomic Absorption, direct aspiration)
STORE! NO. Total 01067
Dissolved 01065
Suspended 01066
Optimum Concentration Range: 0.3-5 mg/1 using a wavelength of 232.0 nm
Sensitivity: 0.15 mg/1
Detection Limit: 0.04 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 4.953 g of nickel nitrate, Ni(NO3)2»6H2O (analytical reagent
grade) in deionized distilled water. Add 10 ml of cone, nitric acid and dilute to 1 liter
with deionized distilled water. 1 ml = 1 mg Ni (1000 mg/1).
2. Prepare dilutions of the stock nickel solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type of
acid and at the same concentration as will result in the sample to be analyzed either
directly or after processing.
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 thru 4.1.4 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory.
Instrumental Parameters (General)
1. Nickel hollow cathode lamp
2. Wavelength: 232.0 nm
3, Fuel: Acetylene
4. Oxidant: Air
5. Type of Flame: Oxidizing
Analysis Procedure
1. For analysis procedure and calculation, see "Direct Aspiration", part 9.1 of the Atomic
Absorption Methods section of this manual.
Approved for NPDES
Issued 1974
Editorial revision 1978
G-18
-------
Interferences
1. The 352.4 nm wavelength is less susceptible to spectral interference and may be used.
The calibration curve is more linear at this wavelength; however, there is some loss of
sensitivity.
Notes
1. For levels of nickel below 100 ug/1, either the Special Extraction Procedure, given in
part 9.2 of the Atomic Absorption Methods section or the furnace technique, Method
249.2, is recommended.
2. Data to be entered into STORET must be reported as ug/1.
3. The heptoxime method may also be used (Standard Methods, 14th Edition, p 232).
Precision and Accuracy
1. In a single laboratory (EMSL), using a mixed industrial-domestic waste effluent at
concentrations of 0.20,1.0 and 5.0 mgNi/1, the standard deviations were ±0.011, ±0.02
and ±0.04, respectively. Recoveries at these levels were 100%, 97% and 93%,
respectively.
G-19
-------
SILVER
Method 272.1 (Atomic Absorption, direct aspiration)
STORE! NO. Total 01077
Dissolved 01075
Suspended 01076
Optimum Concentration Range: 0.1-4 mg/1 using a wavelength of 328.1 nm
Sensitivity: 0.06 mg/1
Detection Limit: 0.01 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 1.575 g of AgttO3 (analytical reagent grade) in deionized
distilled water, add 10 ml cone. HNO3 and make up to 1 liter. 1 ml = 1 mg Ag (1000
mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the time of
analysis. The calibration standards should be prepared using nitric acid and at the same
concentration as will result in the sample to be analyzed either directly or after
processing.
3. Iodine Solution, 1 N: Dissolve 20 grams of potassium iodide, KI (analytical reagent
grade) in 50 ml of deionized distilled water, add 12.7 grams of iodine, I2 (analytical
reagent grade) and dilute to 100 ml. Store in a brown bottle.
4. Cyanogen Iodide (CNI) Solution: To 50 ml of deionized distilled water add 4.0 ml cone.
NH4OH, 6.5 grams KCN, and 5.0 ml of 1.0 N I2 solution. Mix and dilute to 100 ml with
deionized distilled water. Fresh solution should be prepared every two weeks."'
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 thru 4.1.3 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory;
however, the residue must be taken up in dilute nitric acid rather than hydrochloric to
prevent precipitation of AgCl.
Approved for NPDES and SOW A
Issued 1971
Editorial revision 1974
Technical revision 1978
G-20
-------
Instrumental Parameters (General)
1. Silver hollow cathode lamp
2. Wavelength: 328.1 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Analysis Procedure
1. For the analysis procedure and the calculation, see "Direct Aspiration", part 9.1 of the
Atomic Absorption Methods section of this manual.
Motet
I. For levels of silver below 30 tig/1, either the Special Extraction Procedure, given in part
9.2 of the Atomic Absorption Methods section or the furnace procedure, Method 272.2,
is recommended.
2. Silver nitrate standards are light sensitive. Dilutions of the stock should be discarded
after use as concentrations below 10 mg/1 are not stable over long periods of time.
3. If absorption to container walls or the formation of AgCl is suspected, make the sample
basic using cone. NH4OH and add 1 ml of (CNI) solution per 100 ml of sample. Mix the
sample and allow to stand for 1 hour before proceeding with the analysis.1"
4. The 338.2 nm wavelength may also be used. This has a relative sensitivity of 2.
5. Data to be entered into STORET must be reported as ug/1.
Precision and Accuracy
1. In a round-robin study reported by Standard Methods, a synthetic sample containing SO
ug Ag/1 was analyzed by SO laboratories with a reported standard deviation of ±8.8 and
a relative error 10.6%.
References •
1. "The Use of Cyanogen Iodide (CNI) as a Stabilizing Agent for Silver in Photographic
Processing Effluent Sample", Owerbach, Daniel, Photographic Technology Division,
Eastman Kodak Company, Rochester, N.Y. 146SO.
2. Standard Methods for Examination of Water and Wastewater, 14th Edition, p. 148,
Method 301A.
G-21
-------
ZINC
Method 289.1 (Atomic Absorption, direct aspiration)
STORE! NO. Total 01092
Dissolved 01090
Suspended 01091
Optimum Concentration Range: 0.05-1 mg/1 using a wavelength of 213.9 nm
Sensitivity: 0.02 mg/1
Detection Limit: 0.005 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.00 g of zinc metal (analytical reagent grade) and
dissolve cautiously in 10 ml HNO3. When solution is complete make up to 1 liter with
deionized distilled water. 1 ml = 1 mg Zn (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the time of
analysis. The calibration standards should be prepared using the same type of acid and at
the same concentration as will result in the sample to be analyzed either directly or after
processing.
Sample Preservation
1. For sample handling and preservation, see part 4.1 of the Atomic Absorption Methods
section of this manual.
Sample Preparation
1. The procedures for preparation of the sample as given in parts 4.1.1 thru 4.1.4 of the
Atomic Absorption Methods section of this manual have been found to be satisfactory.
Instrumental Parameters
1. Zinc hollow cathode lamp
2. Wavelength: 213.9 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Analysis Procedure
1. For the analysis procedure and the calculation, see "direct aspiration" part 9.1 of the
Atomic Absorption Methods section of this manual.
Approved for NPDES
Issued 1971
Editorial revision 1974
G-22
-------
Notes
1. High levels of silicon may interfere.
2. The air-acetylene flame absorbs about 25% of the energy at the 213.9 nm line.
3. The sensitivity may be increased by the use of low-temperature flames.
4. Some sample container cap liners can be a source of zinc contamination. To circumvent
or avoid this problem, the use of polypropylene caps is recommended.
5. The dithizone colorimetric method may also be used (Standard Methods, 14th Edition, p
26S).
6. For concentrations of zinc below 0.01 mg/1, either the Special Extraction Procedure
given in part 9.2 of the Atomic Absorption Methods section or the furnace procedure,
Method 289.2, is recommended.
7. Data to entered into Storet must be reported as ug/1.
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was conducted by
the Quality Assurance and Laboratory Evaluation Branch of EMSL. Six synthetic
concentrates containing varying levels of aluminum, cadmium, chromium, copper, iron,
manganese, lead and zinc were added to natural water samples. The statistical results for
zinc were as follows:
Standard
Number True Values Mean Value Deviation Accuracy as
of Labs ug/liter ug/liter tig/liter % Bias
86 281 284 97 1.2
89 310 308 114 -07
82 56 62 28 11.3
81 70 75 28 6.6
62 7 22 26 206
61 11 17 18 56.6
G-23
-------
BENTH1C JNFAUHA
-------
SEDIMENT BiOASSAYS
-------
FINAL REPORT
TC-3991-04 PugetSound Estuary Program
RECOMMENDED PROTOCOLS FOR
CONDUCTING LABORATORY BIOASSAYS
ON PUGET SOUND SEDIMENTS
Prepared by:
TETRA TECH, INC.
and
E.V.S. CONSULTANTS, INC.
Prepared for:
U.S. ENVIRONMENTAL PROTECTION AGENCY
Region 10 - Office of Puget Sound
Seattle, WA
May, 1986
TETRA TECH, INC.
11820 Northup Way
Bellevue, WA 98005
-------
CONTENTS
LIST OF TABLES v
ACKNOWLEDGEMENTS v
INTRODUCTION l
BIOASSAY SELECTION 4
FIELD COLLECTION OF SUBTIDAL SURFICIAL SEDIMENTS 11
DESIGN OF SAMPLER U
PENETRATION DEPTH 12
OPERATION OF SAMPLER 13
SAMPLE ACCEPTABILITY CRITERIA 13
SAMPLE COLLECTION 14
SAMPLE HOMOGENIZATION 15
CONCURRENT COLLECTION OF SEDIMENT CHEMISTRY AND BIOASSAY SAMPLES 15
GENERAL QUALITY ASSURANCE/QUALITY CONTROL GUIDELINES 16
NEGATIVE CONTROLS 16
POSITIVE CONTROLS 16
TEST ORGANISMS 16
REFERENCE TEST SAMPLES 16
BLIND TESTING 17
MAINTENANCE/MEASUREMENT OF WATER QUALITY 17
STANDARD LABORATORY PROCEDURES 17
AMPHIPOD SEDIMENT BIOASSAY 18
USE AND LIMITATIONS 18
11
-------
FIELD PROCEDURES 19
Collection 19
Processing 19
LABORATORY PROCEDURES 19
Test Animals 20
Control Sediment 21
Test Sediment 21
Bioassay Seawater 22
Facilities and Equipment 22
Bioassay Procedure 23
Experimental Design 25
DATA REPORTING REQUIREMENTS 26
BIVALVE LARVAE SEDIMENT BIOASSAY 27
USE AND LIMITATIONS 27
FIELD PROCEDURES 28
Collection 28
Processing 28
LABORATORY PROCEDURES 28
Bioassay Species 29
Bioassay Sediment 29
Bioassay Seawater 29
Facilities and Equipment 30
Bioassay Procedure 30
Controls 32
DATA REPORTING REQUIREMENTS 32
ANAPHASE ABERRATION SEDIMENT BIOASSAY 33
USE AND LIMITATIONS 33
FIELD PROCEDURES 33
Collection 33
Processing 33
LABORATORY PROCEDURES 34
Cell Cultures 34
Sediment Extraction 34
Culture Conditions 36
Bioassay Procedure 37
Controls 38
111
-------
DATA REPORTING REQUIREMENTS 38
MICROTOX SEDIMENT BIOASSAY (ORGANIC EXTRACT) 40
USE AND LIMITATIONS 40
FIELD PROCEDURES 41
Collection 41
Processing 41
LABORATORY PROCEDURES 41
Facilities and Equipment 41
Sediment Extraction 41
Bioassay Procedure 43
Controls 44
DATA REPORTING REQUIREMENTS 44
MICROTOX SEDIMENT BIOASSAY (SALINE EXTRACT) 45
USE AND LIMITATIONS 45
FIELD PROCEDURES 45
Collection 45
Processing 45
LABORATORY PROCEDURES 46
Preparation of Sediment Extract 46
Bioassay Procedure 46
Controls 47
DATA REPORTING REQUIREMENTS 48
OTHER PROMISING TECHNIQUES 49
REFERENCES 51
1v
-------
TABLES
Number Page
1 Contributors to the sediment bioassay protocols 2
2 Summary of laboratory bioassay tests with marine sediments 5
3 Applicability and relevance of sensitive laboratory
sediment bioassay tests used in more than one study 8
4 Categorization of selected sediment bioassays by type of
test and type of effect 10
ACKNOWLEDGEMENTS
This chapter was prepared by Tetra Tech, Inc., under the direction
of Dr. Scott Becker, for the U.S. Environmental Protection Agency in partial
fulfillment of Contract No. 68-03-1977. Dr. Thomas Sinn of Tetra Tech
was the Program Manager. Mr. John Underwood and Dr. John Armstrong of
U.S. EPA were the Project Officers. The principal author of this chapter
was Dr. Peter Chapman of E.V.S. Consultants, Inc.
-------
Laboratory Sediment Bioassays
Introduction
May 1986
INTRODUCTION
This document presents recommended methods for conducting the following
laboratory sediment bioassays in Puget Sound:
• Amphipod bioassay
• Bivalve larvae bioassay
• Anaphase aberration bioassay
• Microtox bioassay
Organic extraction
Saline extraction.
Each recommended method is based on the results of a workshop and written
reviews by representatives from most organizations that fund or conduct
environmental studies in Puget Sound (Table 1). The purpose of developing
these recommended protocols is to encourage all Puget Sound investigators
conducting monitoring programs, baseline surveys, and intensive investigations
to use standardized methods whenever possible. If this goal is achieved,
most data collected in the Sound will be directly comparable and thereby
capable of being integrated into a sound-wide database. Such a database
is necessary for developing and maintaining a comprehensive water quality
management program for Puget Sound.
The sediment bioassays considered in this document are the tests used
most frequently by a variety of Puget Sound investigators. However, other
bioassays have been used or are currently being developed for use in Puget
Sound. Several of these additional tests are described in the final section
of this document (see Other Promising Techniques).
Each recommended protocol describes the use and limitations of the
respective variable; the field collection and processing methods; and the
laboratory analytical, quality assurance/quality control (QA/QC), and data
reporting procedures. In developing each protocol, it was recognized that
the field of sediment bioassays is relatively new and is expanding rapidly.
The loose-leaf format of this document will allow modification of the recom-
mended protocols, if necessary, and inclusion of additional protocols.
Although the following protocols are recommended for most studies
conducted in Puget Sound, departures from these methods may be necessary
to meet the special requirements of individual projects. If such departures
are made, however, the funding agency or investigator should be aware that
the resulting data may not be comparable with most other data of that kind.
1
-------
TABLE 1. CONTRIBUTORS TO THE SEDIMENT BIOASSAY PROTOCOLS
John Armstrong3
Richard Batiuk
Scott Becker3
Alice Benedict3
Jim Buckley3
Rick Cardwell3
Gary Chapman .
Peter Chapman3'0
Joe Cummins3
Paul Dinnel
Bruce Duncan3
Jim Fisher3
Burt Hamner3
Dave Jamison3
Dave Kendall3
Dick Kocan3
Mary Lou Mills3
Phillip Oshida
Diane Robbins
Pat Romberg3
Brian Ross
Norman Rubinstein
Mike Schiewe3
Margaret Stinson3
Jerry Stober3
John Strand3
Rick Swartz
Dave Terpening3
Jim Thornton3
Milton Tunzi
Don Weitkamp3
Ron Westley3
Les Williams3
Jack Word3
U.S. EPA
U.S. EPA
Tetra Tech
NOAA
Metro
Envirosphere Co.
U.S. EPA
EVS Consultants
U.S. EPA
Univ. of Washington
U.S. EPA
Weyerhaeuser Co.
U.S. COE
Wash. Dept. of Natural Resources
COE
of Washington
Dept. of Fisheries
U.S.
Univ
Wash
U.S. EPA
Invert Aid
Metro
U.S. EPA
U.S. EPA
NOAA
Wash. Dept. of Ecology
Univ. of Washington
Battelle Northwest
U.S. EPA
U.S. EPA
Wash. Dept. of Ecology
U.S. EPA
Parametrix
Wash. Dept. of Fisheries
Tetra Tech
Evans-Hamilton
Attended the workshop held on October 22, 1985.
Workshop moderator.
-------
Laboratory Sediment Bioassays
Introduction
May 1986
In some instances, data collected using different methods may be compared
if the methods are intercalibrated adequately.
Before protocols for specific bioassays are described, sections are
presented on 1) the criteria used to identify the tests considered in this
document, 2) protocols for field collection of surficial test sediments,
and 3) general QA/QC procedures that apply to all sediment bioassays.
-------
Laboratory Sediment Bioassays
Introduction
May 1986
BIOASSAY SELECTION
A large number of sediment bioassays have been developed and used
in recent years (Table 2). Many of these tests have been used to evaluate
the toxicity of Puget Sound sediments. Eight tests (Table 3) were selected
for further consideration based on the following criteria:
• Sensitivity - each test has detected biological effects
in a variety of sediments
• Usage - each test has been used in more than one study in
Puget Sound.
Of the eight sediment bioassays selected for consideration, four were
identified as suitable for general application in Puget Sound for reasons
outlined in Table 3. These four tests are categorized in Table 4 in terms
of kind of test and kind of effect measured. The other tests identified
in Table 3, together with some of the additonal tests mentioned in Table 2,
are considered as other promising techniques at the end of this chapter.
-------
TABLE 2. SUMMARY OF LABORATORY BIOASSAY TESTS
WITH MARINE SEDIMENTS
Taxon
Investigators
ACUTE LETHAL BIOASSAYS
Phytoplankton:
two species test
Annelid worms:
* Monopylephorus cuticulatus
* Glycinde picta
Crustaceans:
(Copepods)
* Acartia tonsa
* Tigriopus sp.
(Amphipods)
* Eogammarus confervicolus
* Eohaustorius washingtonianus
* Grandifoxus grandis
T* Rhepoxynius abronius
(Cumaceans)
several species
(Shrimp)
* Palaemonetes pugio
Wurster (1982)
Chapman et al. (198Za)
Swartz et al. (1979)
Shuba et al. (1978)
Shuba et al. (1978)
Chapman et al. (1982a)
Ott et al. (in prep.)
Pierson et al. (1983)
Swartz et al . (1979; 1981;
1982; 1985a,b); Ott et al . (in
prep.); Chapman et al. (1982a,b;
1984); Chapman and Fink (1983);
Chapman and Barlow (1984);
Malins et al. (1985); Tetra
Tech (1985); Battelle (1986)
Swartz et al. (1979)
Shuba et al. (1978)
-------
TABLE 2. (Continued)
Bivalve Molluscs:
* Macoma inquinata
* Protothaca staminea
* Rangia cuneata
Mya arenaria
Fish:
*
Gasterosteus aculeatus
(threespine stickleback)
Oncorhynchus kisutch
(coho salmon)
Oncorhynchus tshawytscha
(Chinook salmon)
Fundulus heteroclitus
(mummichog)
Leiostomus xanthurus
(spot)
SUBLETHAL BIOASSAYS
Respiration rate of the
oligochaete worm,
Monopylephorus cuticulatus
Oyster larvae. Crassostrea gigas,
partial life-cycle bioassay
Surf smelt, Hypomesys pretiosus
pretiosus, partial life-cycle
Bioassay
Full life-cycle bioassay with
the polychaete worm,
Capitella capitata
Swartz et al. (1979)
Swartz et al. (1979)
Shuba et al. (1978)
Tsai et al. (1979)
Chapman et al. (1982a)
Legore and DesVoigne (1973)
Pierson et al. (1983)
Tsai et al. (1979);
Hargis et al. (1984)
Tsai et al. (1979);
Hargis et al. (1984)
Chapman (in press); Chapman
et al. (1982a,b; 1984; 1985);
Chapman and Fink (1983)
Schink et al . (1974); Cummins
et al . (1976); Chapman and
Morgan (1983); Chapman et
al . (1983; 1985); Pierson
et al. (1983); Mai ins et al .
(1985); Tetra Tech (1985);
Battelle (1986)
Chapman et al. (1983); Malins
et al. (1985)
Chapman et al. (1983);
Chapman and Fink (1984)
-------
TABLE 2. (Continued)
T*
Histopathological measures of
toxicity to English sole,
Parophrys vetulus, with 3 mo.
sediment exposure
Phytotoxicity to marsh plant
Cyperus esculentus
Sea urchin fertilization assay
with Strongylocentrotus
purpuratus
Full life-cycle bioassays with
nematodes Chromadorina germanica
and Diploeaimella punicea
Partial life-cycle bioassays
with the copepod Tigriopus
californicus
CYTOTOXIC/GENOTOXIC BIOASSAYS
T* Mitotic abnormalities (anaphase
aberrations) and cell prolifera-
tion in vitro
MICROBIAL ACTIVITY BIOASSAYS
T* Microtox, Photobacterium
phosphoreum, bacterial
luminescence
McCain et al. (1982)
Lee et al. (1982)
Ross et al. (1984)
Malins et al. (1985)
Tietjen and Lee (1984)
Misitano (1983) ; Mai ins et
al. (1985)
Chapman et al. (1982a,b; 1983;
1984; 1985); Landolt and Kocan
(1984a,b)
Williams et al. (in press);
Schiewe et al. (1985); Malins
et al. (1985)
* Indicates used in Puget Sound.
T Sensitive method used in more than one study.
-------
TABLE 3. APPLICABILITY AND RELEVANCE OF SENSITIVE LABORATORY
SEDIMENT BIOASSAY TESTS USED IN MORE THAN ONE STUDY
Bioassay
Type'
Acceptable for
Immediate Use
in Puget Sound?
Comment
Amphipod 10-day test
(Rhepoxynius abronius)
L.S
Yes
Oligochaete respiration
rate measurements
(Monopylephorus cuticulatus)
No
Bivalve larvae 48-h test
(Crassostrea gigas,
Mytilus edulis
L,S
Yes
gigas.
Echinoderm sperm test
L.S
No
Surf smelt partial life-
cycle test (Hypomesus
pretiosus pretiosus)
L.S.C
No
Copepod partial life-cycle L,S,C
test (Tigriopus californicus)
No
Simple test, reproducible,
of ecological relevance,
applicability verified
in numerous studies,
extensive usage to date
in Puget Sound
Provides useful information,
but requires specialized
equipment and expertise
not generally available
(Chapman et al. 1985)
Simple test, indicator
of toxicity, relevance
and applicability verified
in several independent
studies, extensive usage
to date in Puget Sound
Simple test, can be conducted
year-round by using different
echinoderm species, but
interfering factors cannot
yet be eliminated (Ross
et al. 1984; Malins et
al. 1985; Dinnel and
Stober 1985)
Smelt eggs not available
year-round, results confounded
by excessive natural
var iab i1ity (Chapman
et al. 1983, 1985)
Simple test, can be conducted
year-round, but procedure
has not been fully validated
(D. Misitano, NMFS, pers.
ccmm.; P. Chapman, E.V.S.
Consultants, unpub. data)
8
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TABLE 3. (Continued)
In vitro anaphase aberration
tests with rainbow trout
gonad cells (Salmo gairdneri)
Yes
Microtox, bacterial
luminescence bioassay
(Photobacterium phosphoreum)
Yes
One of the few methods
to measure possible genotoxic/
cytotoxic effects, moderate
usage to date in Puget
Sound, requires a higher
level of expertise than
other tests but can be
conducted by more than
one laboratory in Puget
Sound
One of the few methods
to measure possible effects
on bacteria, shows good
promise, moderate usage
to date in Puget Sound
a L = Lethal; S = Sublethal; C = Chronic; G = Genotoxic.
-------
TABLE 4. CATEGORIZATION OF SELECTED SEDIMENT BIOASSAYS
BY KIND OF TEST AND KIND OF EFFECT
Kind of Effect3
Kind of Test Lethal Sublethal Genotoxic
Solid and liquid phase:
Rhepoxynius abronius X X
Bivalve larvae X X
Extracts:
Microtox X
Anaphase aberrations X
a None of these tests measures chronic effects (i.e., defined as longer
term than 10 days, involving partial life-cycle testing).
10
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Laboratory Sediment Bioassays
Sediment Collection
May 1936
FIELD COLLECTION OF SUBTIDAL SURFICIAL SEDIMENTS
This section describes the protocols required to collect an acceptable
subtidal surficial sediment sample for subsequent analysis by a laboratory
sediment bioassay. This subject has generally been neglected in the past
and sampling crews have been given relatively wide latitude in deciding
how to collect samples. However, because sample collection procedures
influence the results of all subsequent laboratory and data analyses, it
is critical that samples be collected using acceptable and standardized
techniques.
DESIGN OF SAMPLER
In Puget Sound, the most common sampling device for subtidal surficial
sediments is the modified van Veen bottom grab. However, a variety of
coring devices (e.g., box corer, Kasten corer) are also used. The primary
criterion for an adequate sampler is that it consistently collect undisturbed
samples to the required depth below the sediment surface without contaminating
the samples. An additional criterion is that the sampler can be handled
properly on board the survey vessel. An otherwise acceptable sampler may
yield inadequate sediment samples if it is too large, heavy, or awkward
to be handled properly.
Collection of undisturbed sediment requires that the sampler:
• Create a minimal bow wake when descending
• Form a leakproof seal when the sediment sample is taken
• Prevent winnowing and excessive sample disturbance when
ascending
• Allow easy access to the sample surface.
Most modified van Veen grabs have open upper faces that are fitted with
rubber flaps. Upon descent, the flaps are forced open to minimize the
bow wake, whereas upon ascent, the flaps are forced closed to prevent sample
winnowing. Some box corers have solid flaps that are clipped open upon
descent, and snap shut after the corer is triggered. Although most samplers
seal adequately when purchased, the wear and tear of repeated field use
eventually reduces this sealing ability. A sampler should therefore oe
monitored constantly for sample leakage. If unacceptable leakage occurs,
the sampler should be repaired or replaced. If a sampler is to be borrowed
or leased for a project, its sealing ability should be confirmed prior
11
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Laboratory Sediment Bioassays
Sediment Collection
May 1986
to sampling. Also, it is prudent to have a backup sampler on board the
survey vessel if the primary sampler begins leaking during a cruise.
The required penetration depth below the sediment surface is a function
of the desired sample depth (see Penetration Depth). Generally, it is
better to penetrate below the desired sample depth to minimize sample
disturbance when the sampling device closes. Penetration depth of most
sampling devices varies with sediment character; it is greatest in fine
sediments and least in coarse sediments. Sampling devices generally rely
upon either gravity or a piston mechanism to penetrate the sediment. In
both cases, penetration depth can be modified by adding or subtracting
weight from the samplers. Thus, it is optimal to use a sampler that has
a means of weight adjustment. If a sampler cannot consistently achieve
the desired penetration depth, an alternate device should be used.
Once the sampler is secured on board the survey vessel, it is essential
that the surface of the sample be made accessible without disturbing the
sample. Generally, samplers have hinged flaps on their upper face for
this purpose. The openings in the upper face of the sampler should be
large enough to allow easy subsampling of the sediment surface. If an
opening is too small, the sample may be disturbed as the field member struggles
to take a subsample.
PENETRATION DEPTH
For characterizing the toxicity of surficial sediments in Puget Sound,
it is recommended that the upper 2 cm of sediment be evaluated. When collecting
the upper 2 cm of sediment, it is recommended that a minimum penetration
depth of 4-5 cm be achieved for each acceptable sample.
Although the 2-cm specification is arbitrary, it will ensure that:
• Relatively recent sediments are sampled
• Adequate volumes of sediment can be obtained readily for
laboratory analyses
• Data from different studies (historical or ongoing) can
be compared validly.
Sampling depths other than 2 cm may be appropriate for specific purposes.
For example, when toxicity determinations are made for marine dredge disposal
permitting, sediments collected from depths as great as several meters
may be tested. It should be remembered, however, that if a sampling depth
other than 2 cm is used, comparisons with data from 2-cm deep samples may
be questionable.
12
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Laboratory Sediment Bioassays
Sediment Collection
May 1986
OPERATION OF SAMPLER
The sampling device should be attached to the hydrowire using a ball-
bearing swivel. The swivel will minimize the twisting forces on the sampler
during deployment and ensure that proper contact is made with the bottom.
For safety, the hydrowire, swivel, and all shackles should have a load
capacity at least three times greater than the weight of a full sampler.
The sampler should be lowered through the water column at a controlled
speed of approximately 1 ft per sec. Under no circumstances should the sampler
be allowed to "free fall" to the bottom, as this may result in premature
triggering, an excessive bow wake, or improper orientation upon contact
with the bottom. The sampler should contact the bottom gently, and only
its weight or piston mechanism should be used to force it into the sediment.
After the sediment sample is taken, the sampler should be raised slowly
off the bottom and then retrieved at a controlled speed of approximately
1 ft per sec. Before the sampler breaks the water surface, the survey
vessel should head into the waves (if present) to minimize vessel rolling.
This maneuver will minimize swinging of the sampler after it breaks the
water surface. If excessive swinging occurs or if the sampler strikes
the vessel during retrieval, extra attention should be paid to evaluating
sample disturbance when judging sample acceptability.
The sampler should be secured immediately after it is brought on board
the survey vessel. If the sampler tips or slides around before being secured,
extra attention should be paid to evaluating sample disturbance.
SAMPLE ACCEPTABILITY CRITERIA
After the sampler is secured on deck, the sediment sample should be
inspected carefully before being accepted. The following acceptability
criteria should be satisfied:
• The sampler is not overfilled with sample so that the sediment
surface is pressed against the top of the sampler
• ' Overlying water is present (indicates minimal leakage)
• The overlying water is not excessively turbid (indicates
minimal sample disturbance)
• The sediment surface is relatively flat (indicates minimal
disturbance or winnowing)
t The desired penetration depth is achieved (i.e., 4-5 cm
for a 2-cm deep surficial sample).
If a sample does not meet any one of these criteria, it should be rejected.
13
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Laboratory Sediment Bioassays
Sediment Collection
May 1986
SAMPLE COLLECTION
After a sample is judged acceptable, the following observations should
be noted on the field log sheet:
• Station location
• Depth
• Gross characteristics of the surficial sediment
Texture
Color
Biological structures (e.g., shells, tubes, macrophytes)
Presence of debris (e.g., wood chips, wood fibers, human
artifacts)
Presence of oily sheen
Odor (e.g., hydrogen sulfide, oil, creosote)
• Gross characteristics of the vertical profile
Changes in sediment characteristics
Presence and depth of redox potential discontinuity
(rpd) layer
• Penetration depth
t Comments relative to sample quality
Leakage
Winnowing
Disturbance.
Before subsamples of the surficial sediments are taken, the overlying
water must be removed. The preferred method of removing this water is
by slowly siphoning it off near one side of the sampler. Methods such
as decanting the water or slightly cracking the grab to let the water run
out are not recommended, as they may result in unacceptable disturbance
or loss of fine-grained surficial sediment and organic matter.
Once the overlying water has been removed, the surficial sediment
can be subsampled. It is recommended that subsamples be taken using a
flat scoop shaped like a coal shovel. The shoulders of the scoop should
be 2 cm high. This device will allow a relatively large subsample to be
taken accurately to a depth of 2 cm. Coring devices are not recommended
because generally they collect small amounts of surficial sediment, and
therefore require repeated extractions to obtain a sufficient volume of
material for analysis of conventional sediment variables. A curved scoop
14
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Laboratory Sediment Bioassays
Sediment Collection
May 1986
is not recommended because it does not sample a uniform depth. Because
accurate and consistent subsampling requires practice, it is advisable
that an experienced person perform this task.
Finally, sample contamination during collection must be avoided.
All sampling equipment (e.g., scoops, containers) should be made of non-
contaminating material, and should be cleaned appropriately before use.
It is recommended that all objects coming in contact with the sample be
made of glass, stainless steel, or TFE (i.e., tetrafluoroethylene; e.g.,
Teflon). To avoid contamination, all sampling equipment should be washed
thoroughly with pesticide-grade methylene chloride prior to initial use
and between use for each sample.
SAMPLE HOMOGENIZATION
Both compositing of samples for chemistry and bioassay testing, and
subsampling bioassay samples for different types of testing require thorough
mixing (homogenization) of the initial sample. This is particularly important
when the contents of several samples are required to provide sufficient
material for testing.
Compositing and homogenization can be accomplished by transferring
sediment to a solvent-rinsed glass or stainless steel bowl and thoroughly
homogenizing by stirring with a stainless steel spoon and spatula until
textural and color homogeneity are achieved. The bowl and all utensils
should be solvent-rinsed between composites, and kept covered with aluminum
foil to prevent airborne or other contamination.
CONCURRENT COLLECTION OF SEDIMENT CHEMISTRY AND BIOASSAY SAMPLES
If sediment chemistry samples are being collected along with sediment
bioassay samples they should be collected from the same sample. Specifically,
composites of several samples or of individual samples should be combined
and mixed before aliquots are removed for sediment chemistry and sediment
bioassay determinations. Sample homogenization and collection of bioassay
aliquots should be conducted so that chemical aliquots are not contaminated
in the process. The protocols for collecting aliquots for analyses of
organic compounds and metals should be consulted in the appropriate chapters
of this notebook to ensure that collection procedures for bioassay aliquots
are compatible with those recommended for chemical aliquots.
15
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Laboratory Sediment Bioassays
General QA/QC
May 1986
GENERAL QUALITY ASSURANCE/QUALITY CONTROL GUIDELINES
The following general QA/QC procedures apply to all sediment bioassays.
QA/QC procedures specific to each bioassay for which protocols are detailed,
and that differ from the following generic guidelines, are incorporated
into individual bioassay protocols.
NEGATIVE CONTROLS
All bioassays must be conducted using well-established negative (clean)
controls. For every test series with a particular organism, one bioassay
test chamber or series of chambers must contain clean, inert material plus
diluent seawater. The complete bioassay series must be repeated if more
than 10 percent of the control animals die or show evidence of sublethal
effects.
POSITIVE CONTROLS
All bioassays should be conducted using well-established positive
(toxic) controls. These controls involve the use of reference toxicants.
Reference toxicants are used to provide insight into mortalities or increased
sensitivity that may occur as a result of disease, changes in tolerance/sensi-
tivity, and loading density. Reference toxicants can also provide insight
into nonlethal effects that occur due to acclimation, insensitivity, or stress
tolerance developed in handling and bioassay. Accordingly, concurrent bioassays
using a reference toxicant should be implemented for each test series.
TEST ORGANISMS
Only healthy organisms of similar size and life history stage should
be used in bioassays. Taxonomic identifications of bioassay organisms
must be confirmed by a qualified taxonomist.
REFERENCE TEST SAMPLES
Control sediments generally are those from which test animals were
collected. As such, physical and chemical sediment characteristics (e.g.,
grain size, organic content) may be very different from those of the test
sediments. Where this is the case, one or more reference sediments should
be added to the test series. Reference sediments should be collected from
an area documented to be free from chemical contamination and should represent
the range of important physical and chemical variables found in the test
sediments. Data derived from such a sample, if in fact it is totally free
of contamination, can be used to partition toxicant effects from unrelated
effects such as those of sediment grain size.
16
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Laboratory Sediment Bioassays
General QA/QC
May 1986
BLIND TESTING
All treatment and bioassay containers should be randomized and testing
should be conducted without laboratory personnel knowing sample identities.
Replicates of each treatment should be assigned a code number during testing
and randomized in the test sequence.
MAINTENANCE/MEASUREMENT OF WATER QUALITY
Bioassays involving exposure of organisms in aqueous media require
that the media be uncontaminated and that proper water quality conditions
be maintained to ensure the survival of the organisms, and to ensure that
undue stress is not exerted on the organisms unrelated to the test sediments.
At a minimum, the following variables must be measured at the beginning
and termination of testing: salinity, dissolved oxygen, pH, and temperature.
STANDARD LABORATORY PROCEDURES
Standard laboratory procedures must be followed in all testing. These
include proper documentation, proper cleaning, avoidance of contamination,
and maintenance of appropriate test conditions. All unusual observations
or deviations from established procedures must be recorded and reported.
17
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Laboratory Sediment Bioassays
Amphi pod Bioassay
May 1986
AMPHIPOD SEDIMENT BIOASSAY
USE AND LIMITATIONS
The amphipod (Rhepoxynius abronius) sediment bioassay is used to charac-
terize the toxicity of marine sediments. This bioassay may be used alone
as a screening tool in broad-scale sediment surveys, in combination with
sediment chemistry and in situ biological indices, and in laboratory experi-
ments addressing a variety of sediment and water quality manipulations.
The amphipod bioassay is not appropriate for sediments that have an
interstitial salinity of less than 15 ppt. In addition, the following
constraints apply:
• An interstitial salinity of 25 ppt is necessary to ensure
that there are no salinity effects. If interstitial salinities
are between 15 and 24 ppt, they must be adjusted upward.
• Grain size may have an effect on the animals at extremes
of fine and coarse material. If the clay content of the
test sediments exceeds 50 percent or the gravel content
exceeds 35 percent, controls for the effects of particle
size distribution are recommended.
• As in all bioassays using natural populations, there is
a possibility that relative sensitivity of the amphipods
will vary with season or other factors. Accordingly, a
positive control is recommended. This should comprise a
96-h LC50 measurement with a reference toxicant (e.g., CdCl2
or NaPCP) conducted in the absence of sediment.
t Identification of R. abronius must be confirmed by a qualified
taxonomist prior to initiation of the bioassay, and represen-
tative specimens should be preserved and archived for future
reference.
• Predators generally are not a problem in the Dioassay, but
potential problems can be avoided by observation and reasonable
care.
18
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Laboratory Sediment Bioassays
Amphi pod Bioassay
May 1986
FIELD PROCEDURES
Collection
Test Animals--
Amphipods are obtained using benthic grabs (e.g., van Veen, Smith-
Mclntyre) or small dredges. If a dredge is used, a short haul (10 m) will
minimize potential damage to the animals during collection. Approximately
one third more animals than are required for the bioassay are collected.
Surface and bottom seawater salinity and temperature are measured at the
collection site. Sediment temperature is recorded from the first and last
dredge sample. It is recommended that bioassays be conducted within 10 days
of amphipod collection.
Sediment--
Both control and test sediment should be collected in solvent-cleaned
glass jars having TFE-lined lids. Each jar should be filled completely
to exclude air. A minimum sediment sample size of 0.25 L for each bioassay
beaker is recommended for both kinds of sediment.
Processing
Test Animals--
Contents of the dredge are gently washed into a container using seawater
of similar temperature and salinity to that at the collection depth. Samples
that show evidence of contamination (e.g., oil sheen) are rejected. Amphipods
will typically bury in the sediment and if necessary can be held in the
containers for several hours (at the bottom temperature) prior to sieving.
It is preferable to minimize the delay from collection to sieving. To
avoid handling stress, each dredge sample is placed in a separate container.
Animals are maintained and transported in clean coolers (with ice as neces-
sary).
Sediment--
Both control and test sediment should be stored at 40 c in the dark.
Holding time should not exceed 14 days.
LABORATORY PROCEDURES
The laboratory procedures are those described by Swartz et al. (1985)
with the following changes incorporated:
t The salinity of the overlying water is increased to 28 ppt
• Reburial is omitted as a response variable
19
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Laboratory Sediment Bioassays
Amphipod Bloassay
May 1986
• A 1.0-mm screen is used to sieve out amphipods prior to
initiation of testing
t Holding time for amphipods is standardized to between 4 and
10 days
• Sediment holding time prior to testing is set at a maximum
of 14 days
• Additional details are provided concerning maintenance and
transportation of amphipods, confirmation of taxonomic identi-
fications, and freeing of amphipods trapped during testing
by water surface tension
• A specific procedure for interstitial salinity adjustment
is recommended
• A positive control is stipulated and data on 96-h LC50 values
for two reference toxicants are provided.
Test Animals
Sieving—
A 1.0-mm sieve is used to remove j?. abronius from sediment. Mature
amphipods (3.0-5.0 mm total length) are used in the sediment bioassay.
Gentle sieving is essential to reduce handling stress. The sieve is placed
in a large tub filled with seawater at ambient collection site bottom salinity
and temperature. The entire contents of each holding container, including
water, are washed through the sieve using seawater pumped at low pressure
through a fan spray nozzle. The sieve can be shaken gently, but the bottom
of the screen must be beneath the water surface at all times. Material
retained on the screen is washed into buckets for sorting. Large pieces
of detritus and obvious predators are discarded. If there is a delay of
more than 1 n before sorting begins, the buckets should contain enough
sieved sediment to allow the amphipods to bury. The buckets must be kept
at collection site temperature. Aeration may be necessary.
Sorting--
An aliquot of detritus or sediment containing amphipods is placed
in a sorting tray. Healthy, active animals are removed with a bulb pipette
(5-mm opening) and placed in 10-cm diameter finger bowls filled with 28 ppt
seawater and a 2-on deep layer of 0.5-mm sieved collection site sediment.
Twenty amphipods are held in each bowl and enough bowls are prepared to
provide at least one third more specimens than are required for the bioassay.
For R. abronius, seawater temperature during sorting must not exceed 18° c.
Filled finger bowls are submerged in holding tanks supplied with flowing
20
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Laboratory Sediment Bioassays
Amphipod Bioassay
May 1986
water or aeration where temperature and salinity approximate bioassay condi-
tions. If temperature and salinity adjustments are necessary, they should
be made gradually. Healthy R. abronius will remain in the finger bowl
sediment and can be retrieved easily when the bioassay is set up. Amphipods
should be acclimated to laboratory conditions for a minimum of 4 days and
a maximum of 10 days before testing.
The identification of amphipods as R. abronius must be confirmed by
a qualified taxonomist. In addition, representative specimens from each
bioassay series should be preserved and archived for future reference.
Control Sediment
j*. aoronius typically inhabits well-sorted, fine sand. Suggestions
for sTeving and settling may have to be adjusted for other sediment types.
Approximately 0.25 L of control sediment is collected for each bioassay
beaker. This sediment is sieved twice: first, to remove the test species
and other macrobenthos and second, to adjust interstitial water salinity.
The entire contents of one or more sediment samples, including water and
suspended particulate matter, are sieved through a 0.5-mm screen without
allowing overflow from the container. After the first sieving, the sediment
is allowed to settle for at least 4 h. Overlying water is then decanted
and the sediment resieved through a 0.5-mm screen into water of the bioassay
salinity (28 ppt for R. abronius). Again, the sediment is allowed to settle
for at least 4 h (preTerably overnight), overlying water is decanted, and
the control sediment is held at 40 c until the bioassay chambers are prepared.
Test Sediment
Approximately 0.25 L of test sediment are necessary for each bioassay
beaker. The natural geochemical properties of test sediment collected
from the field must be within the tolerance limits of the test species.
R. abronius may be adversely affected by salinity stress if the interstitial
water salinity is below 25 ppt. Interstitial salinities below 25 ppt may
therefore require adjustment upwards. This species is tolerant of a broad
range of sediment types. However, controls for the effects of particle
size distribution are recommended if the clay content exceeds 50 percent
or the gravel content exceeds 35 percent.
The j*. abronius test requires a minimum water column salinity regime
of 28 ppt7 When the interstitial (i.e., pore water) salinity is below
25 ppt, it must be raised if this test is to be used. The following procedure
is recommended in such cases. The interstitial salinity of the sediments
is determined (e.g., by refractometer) and the sediments are placed in
the bioassay chambers with overlying water of a salinity calculated to
raise interstitial salinities to a minimum of 25 ppt. The sediments are
then carefully and slowly stirred by hand with a clean glass rod for 1
min, and allowed to settle and equilibrate. The majority (approximately
21
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Laboratory Sediment Bioassays
Amphi pod Bioassay
May 1986
75 percent) of the overlying water is then carefully decanted and the inter-
stitial salinity in each chamber confirmed prior to bioassay initiation.
The decant water is retained, salinity adjusted if necessary, and used
as the overlying water in the bioassay. Sediments are mixed after adding
the decant water. After the bioassays terminate, the interstitial salinities
are reconfirmed.
Bioassay Seawater
Seawater used in the bioassay is maintained at a salinity of 28 +1 ppt
and temperature of 15 +_lo c. If a series of experiments is planned, test
temperature and salinity should be the same throughout the series. The
bioassay seawater must be uncontaminated, which may necessitate collection
of seawater at the amphipod collection site. The quantity of seawater
required is dependent on sieving and holding needs and on the number of
bioassay chambers.
Bioassay seawater is passed through a filter with 0.45-um pore diameter.
If necessary, salinity is reduced by addition of deionized distilled water
or raised by addition of clean oceanic water or reagent grade chemicals (ASTM
Practice for Conducting Acute Toxicity Tests with Fishes, Macro invertebrates,
and Amphibians [E 729]). Seawater is prepared within 2 days of the bioassay
and stored in covered, clean containers at the bioassay temperature.
Facilities and Equipment
The bioassay chamber is a standard 1-L glass beaker (10-cm internal
diameter) covered with an 11.4-on diameter glass watchglass. The beakers
are placed in a shallow water bath or temperature-controlled room with
overhead aeration source. Aeration to each beaker is provided through
a 1-mL glass pipette that extends between the beaker spout and watchglass
to a depth not closer than 2 cm from the sediment surface. Air is bubbled
into the beakers at a rate that does not disturb the sediment. The bioassay
temperature is maintained by either the water bath or room temperature
control.
All glassware is cleaned by washing with laboratory detergent; rinsing
in turn with distilled water, 10 percent nitric acid (HN03) or hydrochloric
acid (HC1), and distilled water. Large plastic containers and plastic
sieves used for preparation and storage of sediment and seawater are precon-
ditioned initially by soaking for 24 h in seawater and rinsed after each
use with clean seawater. They are used only for bioassays and stored in
a clean room. Sieves and containers used to collect and store amphipods,
seawater, and control sediment are kept separate from those used for test
sediment.
22
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Laboratory Sediment Bioassays
Amphipod Bioassay
May 1986
Bioassay Procedure
Approximately 175 ml of test sediment are placed in the bottom of
the 1-L bioassay chamber to create a 2-on deep layer. Beakers are filled
to 950 ml with 28 ppt seawater, covered with a watchglass, and placed in
a 150 c water bath. Constant illumination is provided by overhead lights.
Water in the beakers is aerated without disturbing the sediment surface.
Twenty amphipods are placed in each beaker. The bioassay is terminated
after 10 days of exposure.
The primary response criterion is survival after 10 days exposure
to test or control sediment. The secondary response criterion is daily
emergence of amphipods from toxic sediment.
Initiation--
The day before the bioassay is initiated, each test sediment sample
is mixed within its storage container and an aliquot sufficient to make
a 2-cm deep layer is added to a bioassay beaker. For replicate bioassay
samples, the weight of sediment necessary to make a 2-cm deep layer (approxi-
mately 175 ml) in the first beaker is added to the other replicates. The
same procedure applies to control sediment. Treatments are randomly assigned
to prenumbered bioassay beakers.
The sediment aliquot in the beaker is settled by smoothing with a
spoon, and bubbles are removed by tapping the beaker against the palm of
the hand. A disk (attached to a string for removal) is placed on the sediment
surface. This minimizes sediment disruption as bioassay seawater is added
up to the 750-mL mark on the beakers. This disk is removed and rinsed
in bioassay water between beakers and changed between treatments. The
beakers are covered with watchglasses, put into the 150 c water bath or
temperature-controlled room, and aerated. The beakers are allowed to equili-
brate overnight to bioassay conditions. Normal room lighting is maintained
continuously during the bioassay. If the experimental design requires
monitoring of sediment chemistry (e.g., metals, total volatile solids,
Eh), additional beakers must be set up for this purpose. Monitoring the
quality of seawater overlying the sediment can be accomplished in the bioassay
beakers without disturbing the sediment. Temperature is recorded from
a thermometer maintained in a separate beaker containing control sediment
and bioassay water but no amphipods.
On the day the bioassay is initiated, amphipods are distributed among
all beakers so that each receives 20 individuals. It is usually not logis-
tically possible to distribute amphipods to all beakers at the same time,
so it necessary to select a portion of the beakers (as many as 15) to be
processed together. The exact number of beakers to receive amphipods at
one time is dependent on the size and design of the experiment. At least
one replicate from each treatment, including control sediment, is processed
at a time if possible. Otherwise, selection is random.
23
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Laboratory Sediment Bloassays
Amphi pod Bioassay
May 1986
Amphipods are removed from the holding sediment using a 1.0-mm sieve,
and then transferred to sorting trays. About one third more fingerbowls
are removed from the holding tank at one time than are required for the
number of beakers. This allows selection of active, healthy animals for
the bioassay. Amphipods are removed from the sorting tray and sequentially
distributed among clean 10-cm fingerbowls each containing 150 ml of bioassay
seawater without sediment. The number of amphipods distributed to each
fingerbowl is recounted by transferring them to a separate fingerbowl.
Amphipods are added to the bioassay beakers by placing a black plastic
disk on the seawater surface and gently pouring the entire contents of
the fingerbowl into the beaker. The fingerbowl is washed with bioassay
water to remove adhering amphipods. The seawater level is brought up to
950 mL with bioassay water, and the disk is removed and rinsed between
samples. Amphipods are allowed to bury in the sediment and any that are
floating on the seawater surface are pushed down with the edge of the beaker
cover. After 1 h, amphipods that have not buried are removed and replaced.
Normally, less than 1 percent of the animals will fail to bury in 1 h.
Monitoring--
If samples for chemical analysis are desired, seawater and sediment
samples can be taken from beakers at the initiation of the bioassay. A
small quantity of seawater can be taken from beakers at the initiation
of the bioassay, but chemistry beakers have to be sacrificed to obtain
sediment samples. This is accomplished by siphoning the overlying seawater
without disturbing the sediment surface and then taking appropriate sediment
aliquots for chemical analyses. It is not necessary to add amphipods to
chemistry beakers that are sacrificed at the initiation of the bioassay,
but amphipods are added to those sacrificed later. Certain sediment and
water quality variables (e.g., dissolved oxygen, pH, Eh) can be monitored
by inserting analytical probes into the chemistry beakers.
During the course of the bioassay, certain observations are made daily.
Temperature in the beaker set up for this purpose is monitored. Lighting
and aeration systems are checked. Each beaker is carefully examined but
not disturbed except for the temporary removal of the aeration pipette
and watchglass. Notes are made on sediment appearance and unusual conditions.
The number of amphipods that have emerged from the sediment, either floating
on the water surface or lying on top of the sediment, is counted and recorded.
Amphipods that have emerged are not removed, even if they are dead. These
data are used to document the temporal pattern of emergence. Amphipods
trapped by surface tension at the water surface are gently pushed down
with a clean instrument (e.g., pipet, glass rod, beaker cover).
24
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Laboratory Sediment Bioassays
Amphipod Bloassay
May 1986
Termination--
The bioassay is terminated after 10 days of exposure. After daily
observations are recorded, the contents of the bioassay beakers are sieved
through a 0.5-mm screen. Material retained on the screen is placed in
clean bioasssay water in a sorting tray. The number of live and dead amphipods
is recorded. The sum of these numbers may not always equal 20 because
of death and subsequent decomposition of amphipods. An amphipod is counted
as alive if there is any sign of life (e.g., pleopod twitch observed under
magnification, response to gentle prodding with a clean instrument).
Experimental Design
Logistics--
A typical sediment bioassay involves about 50 to 60 bioassay beakers.
Collection and preparation of animals, sediment, and seawater requires
at least four people for 2 days. Three or four people are required on
the days experiments are initiated and terminated. One person can monitor
the experiment in progress.
Controls--
Five replicates of the amphipod collection site control sediment are
included in all bioassays. These beakers comprise a negative (clean) control
that allows comparisons among experiments and among laboratories of the
validity of the procedures used in individual investigations. At least
90 percent of the amphipods in the control replicates should survive. The
design of field surveys may include a reference sediment involving five
replicate samples from an area believed to be free from sediment contamination.
This provides a site-specific basis for comparison of potentially toxic and
nontoxic conditions. Experiments in which contaminants are added to sediment
may require control replicates to determine effects of solvent addition.
A positive (contaminated) control is also required for all testing.
This involves determining 96-h LC50 values for R. abronius exposed in clean,
filtered seawater without sediment to reference toxicants (following standard
bioassay procedures and under the same general test conditions as the sediment
bioassays). Such data are necessary to determine the relative sensitivity
of the animals (e.g., seasonal difference in sensitivity) for each test
series to ensure comparability of the data. Two commonly used reference
toxicants are reagent-grade CdCl2 and NaPCP. Swartz et al. (1986) determined
a 96-h LC50 of 1.61 mg/L for CdCl2, while Cummins and Gangmark (in preparation)
found that the 96-h LC50 for NaPCP ranged from 0.30 to 0.39 mg/L over a
2-yr period. Either reference toxicant may be used, but the acute lethality
results must be reported along with the sediment bioassay results. Bioassays
to establish an LC50 involve four or five logarithmic concentration series
and a control. At least one treatment should give a partial response below
25
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Laboratory Sediment Bioassays
Amphipod Bioassay
May 1986
the LC50 and one above the LC50. Statistical procedures for the LC50 estimate
are given in APHA (1985).
Response Criteria--
Emergence from sediment and survival are the two response criteria
examined in all bioassays. Emergence data are used to monitor" a behavioral
response of the amphipods during the 10-day exposure. Survival after 10
days of exposure is the primary criterion of toxicity. Each of these response
criteria must be monitored in a "blind" fashion; that is, the observer
must have no knowledge of the treatment of the sediment in the beakers.
This is accomplished through randomization of beaker numbers.
DATA REPORTING REQUIREMENTS
The following data should be reported by all laboratories performing
this bioassay:
• Water quality measurements during testing (i.e., DO, temperature,
salinity, pH).
• Daily emergence for each beaker and the 10-day mean and
standard deviation for each- treatment
• 10-day survival in each beaker and the mean and standard
deviation for each treatment
• Interstitial salinity values of test sediments
• 96-h LC50 values with reference toxicants
• Any problems that may have influenced data quality.
26
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Laboratory Sediment Bioassays
Bivalve Larvae Bioassay
May 1986
BIVALVE LARVAE SEDIMENT BIOASSAY
USE AND LIMITATIONS
The bivalve larvae bioassay technique is described in Standard Methods
(APHA 1985) and by ASTM (1985) as a rapid and reliable indicator of environ-
mental quality. Pacific oysters (Crassostrea gigas) and blue mussels (Mytilus
edulis) are recommended for testing. During the first 48 h of embryonic
development, fertilized oyster and mussel eggs normally develop into free-
swimming, fully shelled larvae (prodissoconch I). Failure of the eggs
to survive or the proportion of larvae developing in an abnormal manner
is used as an indicator of toxicity.
This sediment bioassay can be used to characterize the toxicity of
marine sediments. It may be used alone as a screening tool in broad-scale
sediment surveys, in combination with sediment chemistry and in situ biological
indices, and in laboratory experiments addressing a variety of sediment
and water quality manipulations.
The bivalve larvae bioassay can be used in sediments that have interstitial
salinities less than 1 ppt, as the sediments are mixed and equilibrated with
seawater prior to testing. However, because further testing is required to
determine the validity of using this technique with such low salinity sediments,
this bioassay is not recommended for sediments that have an interstitial
salinity of less than 10 ppt. In addition, the following caveats apply:
• Bivalve larvae such as those of C^. gigas normally are not
associated with the types of sediments that generally are
tested using this method. Hence, this bioassay is only
an indicator of relative toxicity and is not usually of
direct ecological significance.
• Spawning of £. gigas occurs naturally in the Puget Sound
area in summer. The natural spawning period for H. edu 1 is
is late spring to early summer. Both of these bivalves
can be induced to spawn at other times of the year, but
may show decreased viability of gametes. Gamete viability
may also vary depending on the brood stock used. Accordingly,
a positive control is recommmended. This should comprise
a 48-h EC50 measurement with a reference toxicant (either
CdCl£ or NaPCP) conducted in the absence of sediment.
• It is possible that abnormalities induced during testing
may be underestimated due to poor recovery of living larvae
from the sediments. Accordingly, it is recommended that
27
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Laboratory Sediment Bioassays
Bivalve Larvae Bioassay
May 1986
a few sediment samples from each set of bioassays conducted
with this technique be examined to determine whether living
larvae are present in the sediment. The results should
be quantified and reported.
FIELD PROCEDURES
Collection
Both control and test sediment should be collected in solvent-cleaned
glass jars having TFE-lined lids. Each jar should be filled completely
with sediment to exclude air. A minimum sediment sample size of 20 g for
each bioassay chamber is recommended for both kinds of sediment.
Processing
Both control and test sediment should be stored at 40 c in the dark.
Holding time should not exceed 14 days.
LABORATORY PROCEDURES
The following procedures apply equally to larvae of both £. gigas
and M. edulis. and are as described by Chapman and Morgan (1983) with the
following changes incorporated:
• The salinity of test water is adjusted to 28 ppt
• Replication is increased from two to five to al low adequate
statistical comparisons
• Larvae of M. edulis are included in the bioassay protocol
t Sediment resuspension in the bioassay containers is adequately
accomplished by vigorous shaking for 10 sec; there is no
need to rotate the containers for 3 h at 10 rpm
t Twenty grams of sediment is suspended in 1 L of seawater
rather than 15 g in 750 mL
• Sediment holding time prior to testing is set at a maximum
of 14 days
• pH is not adjusted before the bioassay starts and is only
monitored
• A positive control is recommended
t Additional details provided by ASTM (1985) for conditioning
and spawning adults are included.
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Laboratory Sediment Bioassays
Bivalve Larvae Bioassay
May 1986
Bioassay Species
For a given test or series of related tests, adult bivalves (brood
stock) are obtained from the same source, either commercial rearing facilities
(oysters) or a chemically uncontaminated area (mussels). Within 24 h of
collection or purchase, adults should be transported to the test laboratory
and placed into flowing seawater similar in character to that from which
they were taken. Rough handling; extended periods of desiccation; and
abrupt changes in temperature, salinity, or other water quality variables
must be avoided as these may induce premature spawning or render the stock
useless for later controlled spawning or both. Upon receipt, adults should
be cleaned of fouling organisms and detritus and placed in flowing seawater
for conditioning.
Adult bivalves are held at recommended conditioning temperatures to
stimulate final maturation of the gametes. The desired conditioning temperature
(20 +20 c for oysters and 14 +20 C for mussels) should be attained gradually
at an increment not exceeding 2° C per day. Conditioning may extend from
a few days to several weeks depending on the physiological and gametogenic
status of the adults. The length of the conditioning period is determined
empirically by periodic sacrifical examination and spawning of representative
individuals. Adults should be spawned or discarded within 2-3 wk after
attaining acceptable maturity because gamete quality will deteriorate rapidly
with excessive conditioning.
Bioassay Sediment
The bivalve larvae bioassay is conducted with sediment controls in
addition to seawater controls. Control sediment typically consists of
material collected from an area documented to be free of chemical contamination
and of proven nontoxicity to bivalve larvae.
Bioassay Seawater
Seawater used in the bioassay is maintained at a salinity of 28 +1 ppt
and temperature of 20 +10 C. The bioassay seawater must be uncontaminated
and of acceptably low toxicity. The standard biological criterion of accepta-
bility is that the larvae, spawned by adults in the dilution water, must
not incur more than 10 percent abnormal development or 30 percent mortality
during 48 h of exposure to the dilution water (ASTM 1985).
If necessary, salinity of the bioassay water is reduced by addition
of deionized distilled water or raised by addition of clean oceanic water
or reagent grade chemicals (ASTM Practice for Conducting Acute Toxicity
Tests with Fishes, Macroinvertebrates, and Amphibians [E729]). Seawater
is prepared within 2 days of use and is stored in clean, covered containers
at the requisite temperature.
29
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Laboratory Sediment Bioassays
Bivalve Larvae Bioassay
May 1986
Facilities and Equipment
The bioassay chamber is a clean, acid-rinsed, 1-L glass bottle with
a screw-top lid. Bioassays are conducted with the bottles in shallow water
baths, incubators, or temperature-controlled rooms.
If adults are to be conditioned for spawning out-of-season, a continuous
supply of temperature-controlled seawater is needed. Raw seawater can
be used for holding and conditioning, but feeding the adults a natural
or cultivated alga is necessary to deter starvation. The flow rates used
for adult conditioning must be high enough to prevent water quality degradation
and provide as much food as possible to the adults.
Tanks and trays are necessary for holding the adults, and a water
bath, incubator, or temperature-controlled room is necessary during the
bioassay. Adult holding and conditioning tanks should be cleaned at least
weekly to prevent accumulation of organic matter and bacteria. Dead specimens
should be removed immediately and the tanks cleaned. The tanks should
be cleaned with detergent and rinsed with clean seawater, and if microbial
growth is present, rinsed with 200 mg/L of hypochlorite and then seawater.
With enriched waters and elevated conditioning temperatures, more frequent
cleaning may be required. All new test chambers should be washed with
detergent and rinsed in turn with water, pesticide-free acetone, water,
acid, and then water.
Bioassay Procedure
The bioassay procedure can be summarized as follows. Adult bivalves,
conditioned as necessary in the laboratory, are induced to spawn with selected
thermal and biological (i.e., sperm) stimulation. Selected densities of
the resulting embryos are exposed to the test or control sediments for
48 h, during which the embryos normally will develop into prodissoconch I
larvae. Toxicity is measured based on abnormal shell development and larval
death.
The bivalves are spawned by rapidly raising the water temperature
to 5-100 c above the conditioning temperature. Females are additionally
stimulated to spawn by the addition of sperm from a sacrificed or naturally
spawned male.
Spawning is conducted by placing the bivalves in individual, clean
Pyrex dishes containing filtered, UV-treated seawater. Fertilization is
accomplished within 1 h of spawning by combining eggs and sperm in a 1-L
Nalgene beaker. The fertilized eggs are then washed through a 0.25-mm
Nitex screen to remove excess gonadal material and suspended in 2 L of
filtered, UV-treated seawater at incubating temperature. The embryos are
kept suspended by frequent agitation using a perforated plunger and used
in the bioassay within 2 h of fertilization. When microscopic examination
of fertilized eggs reveals the formation of polar bodies, egg density is
30
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Laboratory Sediment Bioassays
Bivalve Larvae Bioassay
May 1986
determined from triplicate counts of the number of eggs in 1.0-mL samples
of a 1:99 dilution of homogeneous egg suspension.
Sediment bioassays are conducted in clean (i.e., rinsed with dilute
acid) 1-L glass bottles. Twenty grams (wet weight) of the appropriate
sediment is added to each bottle and volume is brought up to 1 L with filtered,
UV-treated seawater (28 ppt salinity) to make a final concentration in
all containers of 20 g (wet weight) of sediment per liter of seawater.
All test treatments are replicated five times. The sediment controls contain
20 g/L of clean sediment. In addition, a control series is prepared consisting
of clean seawater without sediment.
The sediments are suspended by vigorous shaking for 10 sec, the embryos
are added, and the suspended sediments are allowed to settle. No additional
agitation is provided. The seawater control is treated similarly.
Within 2 h of fertilization, each container is inoculated with 20,000-
40,000 developing embryos to give a concentration of about 20-40 per mL.
The embryo concentration at 0 h should be confirmed by collecting replicate
10-mL samples from control cultures and preserving them in 5-percent formalin.
The containers are covered and incubated for 48 h at 20 +lo c under a 14-h
light:10-h dark photoperiod. Test vessels are not aerated during the bioassay.
After 48 h, the bioassay can be terminated in one of two ways. In
the first method, the contents of each container are carefully poured through
a 38-um Nitex sieve without disturbing the settled sediment, thereby retaining
and concentrating the surviving larvae (larvae caught in the sediments
are almost always dead). The concentrated larvae are washed into a 100-mL
graduated cylinder, quantitatively transferred to 8-mL screw-cap vials,
and preserved in 5 percent buffered formalin. In the second termination
method, the water and larvae overlying the settled sediment in each container
are carefully poured into a clean 1-L beaker. This water is then stirred,
and a 10-mL aliquot of the well-mixed sample is removed by pi pet and placed
in a 10-mL screw-cap vial. The contents of each vial is preserved in 5
percent buffered formalin.
Preserved samples (equal in volume to those containing 300-500 larvae
in controls) are examined in Sedgewick-Rafter cells under 100X magnification.
Normal and abnormal larvae are enumerated to determine percent survival
and percent abnormality. Percent survival is determined as the number
of larvae surviving in each test container relative to the seawater control.
Larvae that fail to transform to the fully shelled, straight-hinged, 'D1-shaped
prodissoconch I stage are considered abnormal.
Salinity and dissolved oxygen are initially adjusted in each container
to 28 ppt and a minimum of 90 percent saturation, respectively. These
variables and pH are measured in each container at the start and termination
of the bioassay.
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Laboratory Sediment Bioassays
Bivalve Larvae Bioassay
May 1986
Controls
Five replicates of control sediment and five replicates of control
seawater alone are included in all bioassays. These comprise negative
(clean) controls that allow comparison among experiments and among laboratories
of the validity of the procedures used in individual investigations. At
least 70 percent of the larvae must survive the 48-h exposure with seawater
alone, and of these at least 90 percent must show no abnormalities. The
design of field surveys may include a reference sediment from an area believed
to be free from chemical contamination. This provides a basis for comparison
of potentially toxic and nontoxic conditions. Experiments in which contaminants
are added to sediment may require control replicates to determine effects
of solvent addition.
A positive (toxic) control is also required. This involves determining
48-h LC50 and EC50 values for bivalve larvae exposed to reference toxicants
in clean, filtered, UV-treated seawater without sediment (following standard
bioassay procedures and under the same general test conditions as the sediment
bioassays). Such data are necessary to determine the relative sensitivity
of the larvae. Two commonly used reference toxicants are reagent-grade
CdCl2 and NaPCP. Either reference toxicant may be used, but the results
must be reported along with the sediment bioassay results. Bioassays to
establish an LC50 or an EC50 involve four or five logarithmic concentration
series and a control. At least one treatment should give a partial response
below the LC50 and EC50 and one above the LC50 and EC50. Statistical procedures
for the LC50 and EC50 estimates are given in APHA (1985) and ASTM (1985).
DATA REPORTING REQUIREMENTS
The following data should be reported by all laboratories performing
this bioassay:
• Water quality measurements at the beginning and end of testing
(e.g., dissolved oxygen, temperature, salinity, pH)
• Individual replicate and mean and standard deviation data
for larval survival after 48 h
• Individual replicate and mean and standard deviation data
for larval abnormalities after 48 h
• 48-h LC50 and EC50 values with reference toxicants
t Data on larval presence in the sediment
• Any problems that may have influenced data quality.
32
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Laboratory Sediment Bioassays
Anaphase Aberration Bioassay
May 1986
ANAPHASE ABERRATION SEDIMENT BIOASSAY
USE AND LIMITATIONS
This sediment bioassay is used to characterize the genotoxicity of
marine sediments. It may be used alone as a screening tool in broad-scale
sediment surveys, in combination with sediment chemistry and in situ biological
indices, and in laboratory experiments addressing a variety of sediment
and water quality manipulations.
This bioassay can be used with any type of sediment, regardless of
the interstitial salinity or grain size characteristics. However, the
following caveats apply:
• The bioassay depends on a chemical extraction procedure
that is specific for neutral, nonionic organic compounds.
Other classes of contaminants such as metals and highly
acidic and basic organic compounds are not efficiently ex-
tracted. Thus, characterization of sediment toxicity is
directed towards neutral compounds such as aromatic and
chlorinated hydrocarbons.
• "Natural" genotoxicity may occur in marine sediments due
to the decomposition of plant species containing genotoxic
substances that evolved as a means of protecting plants
from parasites and predators. Thus, positive genotoxic
responses may be noted in areas generally regarded as pris-
tine.
FIELD PROCEDURES
Collection
Sediment should be collected in solvent-rinsed glass jars having TFE-
lined lids. Each jar should be filled completely to exclude air. A minimum
sediment sample size of 100 g is recommended for each test.
Processing
Sediment samples should be stored frozen at -200 c within 8 h of collec-
tion. Holding time should not exceed 6 mo.
33
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Laboratory Sediment Bioassays
Anaphase Aberration Bioassay
May 1986
LABORATORY PROCEDURES
As previously mentioned, this technique depends on a chemical extraction
procedure. Extraction procedures recommended are those presently used
by the National Marine Fisheries Service, National Oceanic and Atmospheric
Administration in Puget Sound (MacLeod et al. 1984).
The following procedure is synthesized from Kocan et al. (1982), Chapman
et al. (1982), Kocan and Powell (1985), and Kocan et al. (1985). Accordingly,
this procedure supersedes previous published methodologies.
Cell Cultures
Although any cell type can be used in this test, the rainbow trout
gonad cell (RT6-2) is recommended because it has been used extensively
for this purpose, is readily obtainable, and is easy to cultivate. It
has numerous mi tot ic figures when growing exponentially and the rmtotic
cells are large in comparison to those of other species, thereby making
it easy to count the damaged anaphase cells. This cell type is sensitive
to a wide range of organic chemical compounds (Kocan et al. 1982) as well
as complex mixtures of chemicals that may occur in marine sediments (Kocan
and Powell 1985; Kocan et al. 1985). Other cells used for this purpose
are the bluegil1-sunfish line (BF-2), human foreskin fibroblasts, newt
cells, and several plant cells (in vivo). Generally these cells can be
obtained from any state or federal fish disease diagnostic laboratory,
from investigators at both university and federal laboratories, or from
the American Type Culture Collection (Rockville, MD).
Sediment Extraction
Test materials must consist of substances that are compatible with
the growth of cells in culture. Generally this can be accomplished using
organic extracts of environmental material such as marine sediments.
Extractions should be made using pesticide-grade reagents that have
been tested for toxicity to the cells prior to their use for extraction.
This initial toxicity testing can be done by evaporating a volume of the
reagent equivalent to that which would be used for the actual extraction,
and adding this to the cultures in varying amounts dissolved in the solvent
(e.g., dimethyl sulfoxide, DMSO) to be used for cell exposure. If the
maximum anticipated amount of the solvent blank to be used in the final
test does not significantly affect the cell cultures, the extraction can
proceed without concern about the possible toxic effect of the solvents.
Once the extracts have been made, gravimetric determinations of their absolute
organic content must be made so that comparable organic concentrations from
each site can be used in cell cultures exposed to extracts from different
locations. This is accomplished by first weighing the tube to be used
for extract storage to the nearest 0.001 g, adding the extract in the solvent,
evaporating the solvent with nitrogen, and reweighing the tubes. The difference
34
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Laboratory Sediment Bioassays
Anaphase Aberration Bioassay
May 1986
between the original tube weight and the final tube weight is the weight
of the sample extract. The sample extract is then redissolved in spectral-
grade DMSO so that each sample contains the same weight/volume ratio of
extract (e.g., mg/mL). In this way it is possible to expose each set of
cultures to exactly the same amount of organic extract, thereby making
comparisons possible on an organic content basis. If relative toxicities
from site to site are needed, one can extrapolate back to the original
organic content of each sample and compare sites based on total amounts
of organic compounds present. The following detailed procedures for the
preparation of sediment extracts follow those of Chapman et al. (1982)
and MacLeod et al. (1984).
Sediment samples are frozen and stored until just prior to extraction.
Each sample is then thawed and rehomogenized by thorough stirring. An
aliquot of approximately 20 g wet weight is transferred to a clean, tared
beaker, dried to constant weight (80° C), desiccated, and reweighed to
determine the percent water. A second aliquot (approximately 150 g wet
weight) is transferred to a tared, solvent-cleaned, 315-mL stainless steel
centrifuge bottle with a TFE-lined screw-cap, and weighed. The sample
is then serially extracted with pesticide-grade solvents.
Methanol (50 mL) is added to each centrifuge bottle. The bottle is
tightly capped, shaken vigorously for 2 mm, and centrifuged at 2,000 rpm
for 5 min. The clean solvent is decanted into a 1-L separatory funnel.
The procedure is performed twice more and the methanol extracts are combined
in the separatory funnel, which is then closed and covered with aluminum foil.
One hundred milliliters of a dichloromethane/methanol (2:1 v/v) solution
is added to the centrifuge bottle, the cap is closed tightly, and the bottle
is shaken vigorously for 2 min to ensure complete mixing. The bottle is
then placed in a shaker table overnight (approximately 18 h), following
which the sediment is settled by centrifugation at 2,000 rpm for 5 min
and the solvent decanted into the separatory funnel with the methanol.
A second 100-mL aliquot of the dichloromethane/methanol (2:1) solution
is added, the bottle is shaken vigorously, and the bottle is then placed
on the shaker table for 6 h. The sediments are again settled with centrifu-
gation and the solvents are decanted.
The remaining sample is shaken vigorously for 2 min with approximately
30 mL of dichloromethane. It is then centrifuged and the solvent is decanted
into the separatory funnel. Another 100 mL of dichloromethane is added
to the bottle, the cap is secured, the bottle is shaken vigorously, and
the bottle is placed on the shaker table overnight. The sediments are
again settled with centrifugation and the solvent is decanted into the
separatory funnel. A final 30-mL rinse of dichloromethane is added, the
bottle is shaken vigorously, centrifuged, and decanted. The sediment is
then discarded.
35
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Laboratory Sediment Bioassays
Anaphase Aberration Bioassay
May 1986
Approximately 500 ml of clean, distilled water is added to the combined
solvents in the separatory funnel. The funnel is carefully swirled and
inverted (with frequent venting) for 2 min. The liquid phases are allowed
to separate and the dichloromethane (lower) layer is drained into a 500-mL
separatory funnel. The aqueous layer is re-extracted twice with 20 mL
of dichloromethane and the remainder is discarded. The dichloromethane
fractions are combined in the 50-mL funnel, transferred, with rinsing,
back to the 1-L funnel, and re-extracted with another 500 ml of distilled
water. The dichloromethane is drained into the 500-mL funnel and the aqueous
layer is extracted once more with 20 mL of dichloromethane. The latter
solvent is added to the 500-mL funnel and the aqueous layer is discarded.
The dichloromethane is drained from the 500-mL separatory funnel through
approximately 20 g of combusted and washed anhydrous sodium sulfate that
is held in a 30-mL glass conical centrifuge tube with the tip cut off.
The effluent from this mini-column is discharged into a 500-mL Kuderna-Danish
flask with a 15-mL receiver. When empty, the 500-mL separatory funnel
is rinsed with 20 mL of dichloromethane, which is drained through the sodium
sulfate column into the flask. The column is washed a final time with
10 mL of dichloromethane and drained into the flask.
Boiling chips are added to the Kuderna-Danish flask and a 3-ball Snyder
column is placed on top. The solvent volume is reduced to about 5 mL using
a hot water bath. When cooled, the sides of the flask are rinsed into
the receiver with dichloromethane. The receiver is removed and the contents
are quantitatively transferred to a tared conical centrifuge tube with
a ground glass stopper. The sample is taken almost to dryness using the
hot water bath, and stored wrapped in aluminum foil in a desiccator with
the stopper open slightly until a constant weight is achieved upon reweighing
the tube. This weight is the amount of extractable organic material.
After weighing, the tube is closed and wrapped fully in aluminum foil,
ready for anaphase aberration testing. Extracts are treated with 1 mL
of spectrophotometric-grade DMSO for 24 h with frequent stirring on a vortex
mixer. The DMSO is then removed to a glass vial and used as "stock" solution.
Because all extracted material is not dissolved in the DMSO during testing,
the centrifuge tubes are dried and reweighed to determine the exact amount
used in testing (fraction soluble). Both stock and extract solutions are
stored in the dark under nitrogen until applied to the cell cultures.
Culture Conditions
RTG-2 cells as well as most fish cell lines grow in a variety of com-
mercially available culture media. The Leibovitz L-15 medium was found
to be most consistent in terms of ease of preparation, use without special
buffers or carbon dioxide incubators, and long-term storage capability.
This medium can be obtained from any scientific supply house that carries
cell/tissue culture materials. The medium comes as a dry powder that is
added to distilled water, autoclaved or filter-steril ized, and stored in
36
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Laboratory Sediment Bioassays
Anaphase Aberration Bioassay
May 1986
a refrigerator at 4° C until it is used. Full instructions are available
with the medium when it is purchased. Generally, heat-deactivated fetal
calf serum is added to any culture medium at 10 percent concentration to
ensure that the proper growth factors are present. This serum can also
be purchased in 100-mL to 1-L lots already sterilized from the scientific
suppliers. Before the cells are placed in the medium, pH should be adjusted
to 7.1-7.3 using either sodium bicarbonate or HC1. Some laboratories use
HEPES buffer (either acid or base in a commercially available product)
in place of the bicarbonate or HC1. This culture system differs from that
originally described by Kocan et al. (1982) only because it has been modified
to simplify the laboratory procedures.
Bioassay Procedure
Cells are grown and tested at 180 c on standard, clean microscope
slides or on 1x5 cm coverslips in Leighton tubes, depending on the amount
of test material available. The cells are placed into the culture system
1 day prior to the actual exposure to ensure that they have had a chance
to attach to the glass substrate and begin growing. On the following day
(18-24 h later), the culture medium is removed and the test material is
added. This should consist of normal L-15 medium dissolved in DMSO, to
which the organic extract has been added. The DMSO should be from a pretested
lot to ensure that it is nontoxic to the system and should not exceed 0.5 per-
cent (v/v) of the culture medium (e.g., 5 uL/mL). This can be reduced
to 0.1 percent DMSO if toxicity is a problem or if minimal DMSO is required
for conservation of extract. Exposure time should be 48 h from the time
of addition of the treated medium until fixation. Damaged cells can be
observed for longer periods even after the toxic substance has been removed,
but the maximum response does not increase beyond 48 h.
An initial screening test must be conducted to determine the actual
extract dilutions to be used for this bioassay. Ideally, dilutions tested
for anaphase aberrations comprise both the highest concentration of extract
(mg/L) that permits continued cell proliferation (i.e., nontoxic) and a
second concentration one dilution lower. This method ensures that a sufficient
number of mitotic figures are present to score for chromosome damage.
Based on previous experience in Puget Sound, the following six extract
dilutions should be prepared: 50, 25, 10, 5, 2 and 1 ug/mL. Cells are
first exposed to these concentrations for each sediment extract tested,
and then the concentrations that inhibit mitosis are determined. Although
identical extract dilutions may not be used to test each sediment sample,
all results are normalized to organic content which has been previously
determined.
To determine mitotic effects and anaphase aberrations, the slides
or coverslips containing the cells are removed from the culture medium
and fixed in methanol:acetic acid (3:1). The methanol is absolute and
the acetic acid is glacial (undiluted). Following 15-60 min in the fixative
(no adverse effects occur if they are left longer) the slides are air dried,
37
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Laboratory Sediment Bioassays
Anaphase Aberration Bioassay
May 1986
then placed into 3 percent Gurr's R66 Geimsa stain for 15-30 min. This
stain is made up in Sorensen's buffer (pH 6.8). Optimum staining is determined
empirically by examining the slides with a microscope at various intervals
after placing them in the stain. Problems usually occur because of too
little staining time rather than excessive staining. If excessive staining
does occur, the cells can be destained in Sorensen's buffer. The staining
system selectively stains the condensed chromosomes undergoing mitosis
and has very little effect on the cytoplasm of the cell, which allows good
resolution of the small fragments of chromosome that are not associated
with the main chromosome bundles.
Once the cells have been stained, they are mounted on microscope slides
(if on coverslips) or are covered with coverslips (if grown on slides)
to facilitate microscopic examination and scoring. Slide identification
labels are covered with a piece of tape to prevent observer bias while
scoring. Three replicate slides are made of each exposure concentration
with two concentrations for each sediment extract. Each slide is then
examined at 500X to l.OOOX until a minimum of 100 anaphase cells is observed
and scored. In this way, there will be three replicates per dose with
100 anaphase cells per replicate.
The numbers and percents of normal and abnormal anaphases are recorded.
Cells are scored as abnormal if they contain any of the previously described
chromosomal lesions reported for this test (Nichols et al. 1977; Kocan
et al. 1982; Chapman et al. 1982a; Kocan and Powell 1985; Kocan et al.
1985).
Controls
Controls consist of 1) untreated cultures used as negative controls
to ensure that the culture conditions (e.g., medium, serum) are not toxic
to the cells, 2) a solvent blank to ensure that the residue from the solvents
used during the extraction procedure is not cytotoxic or genotoxic (generally
done prior to actual testing and includes the DMSO that will be used in
the final solution), and 3) a positive control consisting of cultures exposed
to several concentrations of a known genotoxic agent to indicate that the
test system is functioning properly and does indeed respond to genotoxic
substances. One possible positive control is a 0.25-ug/mL concentration
of benzo(a)pyrene. This level of exposure should result in an anaphase
aberration frequency of 50-65 percent (Chapman et al. 1982a).
DATA REPORTING REQUIREMENTS
The following data should be reported by all laboratories performing
this bioassay:
t Initial screening data for the determination of extract
concentrations
38
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Laboratory Sediment Bioassays
Anaphase Aberration Bioassay
May 1986
• Individual replicate and mean and standard deviation data
for numbers of normal and abnormal anaphases observed
• Types of anaphase aberrations observed
• Frequency of anaphase aberrations observed with the positive
control
• Any problems that may have influenced data quality.
39
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Laboratory Sediment Bioassays
Microtox Bioassay - Organic Extract
May 1986
MICROTOX SEDIMENT BIOASSAY (ORGANIC EXTRACT)
USE AND LIMITATIONS
The Microtox bioassay is a rapid, sensitive method of toxicity testing
based on light emission by the luminescent bacterium (Photobacterium phos-
phor eum) in the presence and absence of aqueous toxicants. The emitted
light is a product of the bacterial electron transport system and thus
directly reflects the metabolic state of the cell. Accordingly, decreased
luminescence following exposure to chemical contaminants provides a quanti-
tative measure of toxicity. The assay was developed for use in freshwater
habitats to assess the toxicity of waterborne pollutants (Bulich et al.
1981) and has been adapted for use in the marine environment to assess
toxicity of organic sediment extracts (Schiewe et al. 1985).
This sediment bioassay is used to characterize the toxicity of marine
sediments. It may be used alone as a screening tool in broad-scale sediment
surveys, in combination with sediment chemistry and in situ biological
indices, and in laboratory experiments addressing a variety of sediment
and water quality manipulations.
The Microtox bioassay can be used with any type of sediment, regardless
of interstitial salinity or grain size characteristics. However, the following
caveats apply:
• The bioassay depends on a chemical extraction procedure
that is specific for neutral, nonionic organic compounds.
Other classes of contaminants such as metals and highly
acidic and basic organic compounds are not efficiently ex-
tracted. Thus, characterization of sediment toxicity is
directed towards neutral compounds such as aromatic and
chlorinated hydrocarbons.
• Naturally occurring toxic substances may be present in and
extracted from marine sediments. Hence, relatively high
toxicity occasionally may be noted in areas generally regarded
as free from chemical contamination.
40
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Laboratory Sediment Bioassays
Microtox Bioassay - Organic Extract
May 1986
FIELD PROCEDURES
Collection
Sediment should be collected in solvent-rinsed glass jars having TFE-
lined lids. Each jar should be filled completely to exclude air. A minimum
sediment sample size of 500 g is recommended for each test.
Processing
Sediment samples should be stored frozen at -200 c within 8 h of collec-
tion. Holding time should not exceed 6 mo.
LABORATORY PROCEDURES
Facilities and Equipment
The bioassay is performed using the Beckman Model 2055 Microtox Toxicity
Analyzer System, a temperature-regulated photometer equipped with a photo-
multiplier. Freeze-dried bacteria, reconstitution solution (i.e., organic-free
distilled water), diluent, and other necessary materials can be purchased
from Microbics Corporation, Carlsbad, California. The test procedure is
conceptually quite straightforward. However, the methodology requires
careful attention to detail and requires 1-2 wk to become technically profi-
cient.
Sediment Extraction
As previously mentioned, this technique depends on a chemical extraction
procedure. Extraction procedures recommended are those presently used
by the National Marine Fisheries Service, National Oceanic and Atmospheric
Administration in Puget Sound (MacLeod et al. 1984).
• Thaw sediment, decant and discard excess water. Homogenize
sediment with stainless steel spoon. Discard large pebbles,
shells, seaweed, wood, crabs, etc.
t Weigh out 10 +0.5 g sediment to the nearest 0.01 g and place
in a dichloromethane-rinsed centrifuge bottle
• Set aside approximately 10 g of the homogenized sediment
for the determination of dry weight
t Centrifuge sediment sample for 5 min at 1,000 x g and discard
water
• Add 100 mL spectral-grade dichloromethane and 50 g sodium
sulfate to sediment sample
41
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Laboratory Sediment Bioassays
Microtox Bioassay - Organic Extract
May 1986
Manually shake bottle until contents are loose and free-flowing
and then roll 16 h (overnight) on a tumbler
Centrifuge sediment 5 min at 1,000 x g and save the dichloro-
methane extract
Add another 100 mL dichloromethane to sediment and tumble
for 6 h (during day)
Centrifuge for 5 min at 1,000 x g and save the dichlorcmethane
extract
Repeat extraction a third time with 100 mL dichloromethane
for 16 h (overnight) and collect extract by centrifugation
Combine the three 100-mL portions of the dichloromethane
extract
Add three or four boiling chips to flask containing the
dichloromethane extract and attach to Synder column
Concentrate extract to 10-15 mL in a 600 c water bath and
then transfer it to a concentrator tube
Wash down the flask two times with 3-4 mL dichloromethane
and add washings to the tube
Add a boiling chip to the tube and, using a tube heater,
concentrate the extract to greater than 0.9 but less than
1.0 mL
Adjust volume to 1.0 mL with dichloromethane (at this point
0.1 mL may be removed for 6C/MS analyses)
Add 3 mL of hexane to the remaining 0.9 mL of extract and
concentrate to 2 mL
Remove 100 uL of extract and add to a concentrator tube
containing 3 mL ethanol
Place tube in heater block and concentrate to 2 mL
Adjust volume to 3.0 mL with ethanol (the standard sediment
extract used in Microtox testing)
Extraction blanks are prepared using an identical procedure
but without the sediment.
42
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Laboratory Sediment Bioassays
Microtox Bioassay - Organic Extract
May 1986
Bioassay Procedure
The organic extract approach uses the basic Microtox method described
in the Microtox Operating Manual (Beckman Instruments 1982) and by Bulich
et al. (1981).
• Reconstitute lyophilized bacteria with 1-mL double-distilled,
charcoal-filtered water in a Microtox cuvette and place
in 40 c holding well. Bacterial suspensions are used within
8 h of reconstitution.
• Prepare three or more widely spaced primary dilutions (e.g.,
5.0, 0.5, 0.05 percent extract [v/v]) in double-distilled,
charcoal-filtered water.
• Adjust concentration of each primary dilution to 2 percent
NaCl by adding 0.1 ml 22 percent NaCl per mL of diluted
extract.
• Use these diluted extracts in a range-finding assay to determine
an appropriate primary dilution for the definitive assay
described below. The primary dilution should cause a 65-90 per-
cent decrease in bioluminescence in 15 min. Methods for
the range-finding assay are the same as those for the definitive
assay except usually only three concentrations are tested
without replication.
• For the definitive assay, two-fold serial dilutions (i.e.,
5.0, 2.5, 1.25, and 0.625 percent) of extract are prepared
in 2 percent NaCl. A 2 percent NaCl blank is also prepared
for testing to measure spontaneous decay of light production
which occurs naturally independent of treatment.
0 In each of 10 test cuvettes, a 10 uL aliquot of bacterial
suspension is added to 500 uL of diluent and incubated for
15 min in the incubation wells. This assures temperature
equilibration and stability of bioluminescence.
• After 15 min, initial levels of light emission are measured
in each of the 10 test cuvettes.
• At 30-sec intervals, 500 ml al iquots of each concentration
of extract are added to two of the cuvettes (i.e., two replicates
each of the four extract dilutions and the saline blank).
Timing is critical because bioluminescence gradually decreases
over time.
43
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Laboratory Sediment Bioassays
Microtox Bioassay - Organic Extract
May 1986
Exactly 5 min after addition of the sediment extract, light
emission is measured at 30-sec intervals and in the same
sequence used for extract additions in the preceding step.
Light emission is measured again at 15 min and 30 min.
Immediately after testing each sediment extract, an ethanol-
only control is assayed using the same primary dilution
sequence used in the sediment extract test. The ethanol-only
data are used to adjust the sediment extract data for the
contribution of the solvent vehicle.
Estimates of the 15-min EC50s (i.e., the concentration of
extract causing a 50 percent reduction in bioluminescence)
are obtained using linear regression analyses (see Microtox
Operating Manual). Briefly, the percent inhibition of light
emitted at each test concentration and time point is converted
to a gamma value which is defined as the ratio of light
lost to light remaining. Gamma values are normal i zed for
natural decline in light production measured over time as
described by Bulich et al. (1981) and further adjusted for
the contribution of the ethanol vehicle. The natural log
of gamma is regressed on the natural log of extract concentration
and the EC50 is calculated from the regression equation.
A statistical procedure based on Fieller's theorem (Finney
1964) is used to calculate a 95-percent confidence interval.
Controls
• Ethanol, sodium lauryl sulfate, or other suitable compound
should be used to assess daily bioassay performance and
to determine differences in response among lots of bacteria.
• Bioassay repeatability is evaluated by duplicate testing
of 10 percent of the sediment extracts.
DATA REPORTING REQUIREMENTS
The following data should be reported by all laboratories performing
this bioassay:
• Range-finding assay results
t Raw light emission data for each test series
• 15-min EC50 data and 95-percent confidence intervals for
each test series and for controls
• Any problems that may have influenced data quality.
44
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Laboratory Sediment Bioassays
Microtox Bioassay - Saline Extract
May 1986
MICROTOX BIOASSAY (SALINE EXTRACT)
USE AND LIMITATIONS
The use of saline extracts of sediment for the Microtox bioassay has
been described by Williams et al. (in press). This technique is not presently
recommended for widespread use in Puget Sound as it requires further research
to evaluate test variables and effects of sediment storage and extraction
techniques on the precision of the results. However, the techniques are
well developed and are provided herein for ease of use in possible future
research efforts.
The organic and saline extract approaches both use the basic Microtox
method described in the Microtox Operating Manual (Beckman Instruments
1982) and by Bulich et al. (1981). The major difference is in the preparation
of test samples. Each procedure is specific with regard to the classes
of contaminants that are tested for toxicity and, in general, the results
for each approach can be viewed as complementary.
A limitation of the saline extract Microtox bioassay procedure is
that only the water-soluble fraction of sediment-adsorbed trace metals
and organic pollutants are extracted from the sediments. Thus, contaminants
with extremely low water solubilities (PCBs) will tend to be partitioned
almost exclusively onto sediment particles and are unlikely to occur in
high concentration in the saline extract used in toxicity testing. Another
limitation is the need to establish a correction factor for changes in
bacterial luminescence caused by variation among samples in sediment pore-
water salinity. Although Williams et al. (in press) showed that salinity-
induced changes in luminescence were negligible for sediments taken from
Commencement Bay, other estuarine sediments may have a greater range of
pore-water salinities and may require a salinity correction factor. Finally,
the assay requires further research to demonstrate precision of the extraction
procedure and stability of sediment toxicity during periods of prolonged
sediment storage (i.e., 2 wk).
FIELD PROCEDURES
Collection
Sediment should be collected in solvent-rinsed glass jars having TFE-
lined lids. Each jar should be filled completely to exclude air. A minimum
sediment sample size of 200 g is recommended for each test.
45
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Laboratory Sediment Bioassays
Microtox Bioassay - Saline Extract
May 1986
Processing
Sediment samples should be stored at 40 c in the dark. Holding time
should not exceed 14 days.
LABORATORY PROCEDURES
With slight modification, sample processing follows procedures described
by Williams et al. (in press). Precleaned glass test tubes are filled
with a minimum of 60 g of sediment, sparged with high purity nitrogen gas,
tightly sealed with TFE-lined caps, stored in the dark at 40 C, and assayed
within 14 days following collection.
Preparation of Sediment Extract
• Remove 30 g of sediment from each test tube with a stainless
steel spatula, place in 30-mL glass containers equipped
with a fritted glass cap, and add 10.0 mL of Mirotox diluent
(2.0 percent NaCl w/v in double-distilled, organic-free
water).
• Wash the sediment-diluent slurry for 24 h in the dark at
40 c by gentle agitation (100 rpm) on a rotary shaker table.
• Transfer the sediment slurry to 30-mL Corex tubes and centrifuge
for 15 min at 9,000 rpm (9,770 x g) in a refrigerated (40 C)
centrifuge.
• Draw the supernatant off by pipette, place it in a clean
test tube, cool on ice and use immediately in preparation
of serial dilutions for the Microtox bioassay.
Bioassay Procedure
• Rehydrate a vial of freeze-dried bacteria with 1.0 mL of
reconstitution solution, cover with parafilm, store in a
40 C well on the Microtox analyzer, and use within 5 h of
rehydration.
• Prepare 100, 50, 25, 12.5 and 0 percent serial dilutions
of the sediment supernatant in Microtox diluent. The 0 percent
dilution is a reagent blank needed to measure spontaneous
decay in bacterial luminescence independent of any treatment.
t In each of 10 test cuvettes, add 10 uL of the rehydrated
bacterial suspension to 350 uL of diluent and incubate for
15 min in one of the 150 c wells on the analyzer. This
assures temperature equilibration of the bacterial suspension
and stability of luminescence.
46
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Laboratory Sediment Bioassays
Microtox Bioassay - Saline Extract
May 1986
• After 15 min, measure initial luminescence in each of the
10 test cuvettes.
• At 30-sec intervals, add 500 uL aliquots of each supernatant
dilution to two of the cuvettes (e.g., two replicates each
of the four test dilutions and the saline blank). Timing
is critical because toxicant-induced decrease in luminescence
begins as soon as the sediment supernatant is added to the
bacterial suspension.
• Exactly 15 min after addition of the sediment supernatants,
measure luminescence at 30-sec intervals and in the same
sequence used for supernatant additions in the preceding
step.
t Calculate percent decrease in luminescence relative to the
reagent blank using the formula:
Percent Decrease = [(RI0-It)/(RIo)] x 100
where:
I0 = initial luminescence
It = luminescence at the end of 15 min
R = blank ratio.
The blank ratio is calculated by:
R = Bt/Bo
where:
B0 = initial luminescence of the reagent blank
Bt = luminescence of the reagent blank after 15 min.
Controls
• Clean reference sediments used as negative controls.
t Construction of a calibration curve to determine salinity
induced changes in bacterial luminescence.
• Use of sodium arsenate as a reference toxicant to assess
day-to-day performance of the bioassay and to determine
differences in toxic response among lot numbers of bacteria.
• Verification of a dose-response relationship between bacterial
luminescence and sediment extract concentration.
47
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Laboratory Sediment Bioassays
Microtox Bioassay - Saline Extract
May 1986
DATA REPORTING REQUIREMENTS
The following data should be reported by all laboratories performing
this bioassay:
• Percent decrease in luminescence for each concentration
of supernatant (e.g., saline sediment extract) tested.
• Determination of a significant dose-response relationship
by least-squares regression of percent decrease in luminescence
on the logarithm of sample dilution.
• Determination of EC50 values and 95-percent confidence limits
for the reference toxicant.
48
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Laboratory Sediment Bioassays
Other Promising Techniques
May 1986
OTHER PROMISING TECHNIQUES
This section describes promising sediment bioassay techniques that
may be generally applicable in Puget Sound following further detailed testing
and validation of the methods. The techniques are listed in terms of relative
priority:
1. The echinoderm sperm bioassay has been used in sediment
bioassays using elutriates but may be affected by the physical
presence of particles, masking any chemical toxicity (Ross
et al. 1984; Malins et al. 1985; Dinnel and Stober 1985).
If this test can be successfully adapted to sediment bioassay
testing, it should prove useful. Echinoderms such as sea
urchins and sand dollars may be usable interchangeably,
allowing testing to occur over most of the year. Draft
protocols have been developed by the U.S. EPA (Narragansett
Laboratories) for conducting effluent toxicity tests with
the sea urchin Arbacia punctulata (Nacci et al. in review).
These protocols may be appl icable in large measure to Puget
Sound sediment testing. In addition to the echinoderm sperm
bioassay, the echinoderm embryo bioassay described by Dinnel
and Stober (1985) may also be applicable to sediment testing
(Ross et al. 1984), allowing a wider range of life-cycle
testing for this group of organisms.
2. Extension of the bivalve larvae sediment bioassay technique
described earlier to bivalve species other than the Pacific
oyster and blue mussel should be possible. ASTM (1985)
has standardized procedures for only four species: Pacific
oyster (Crassostrea gigas), blue mussel (Mytilus edulis).
Eastern oyster (Crassostrea virginica), and quanog (Mercenaria
mercenaria). APHA (1985) only discusses larvae tests with
oysters. However, Puget Sound species such as the Olympia
oyster (Ostrea lurida), Macoma balthica, and geoduck (Panopea
generosa) may be useful alternative species for use in the
bivalve larvae sediment bioassay.
3. Three techniques other than the echinoderm sperm test that
were listed in Table 3 but not recommended for widespread
use in Puget Sound are: 1) the oligochaete, Monopylephorus
cuticulatus. respiration rate test; 2) the surf smelt, Hypomesus
pretiosus pretious, partial life-cycle test; and, 3) the
copepod. Tigriopus californicus, partial life-cycle test.
These tests are all sensitive methods, but are not immediately
applicable. The oligochaete respiration test and the copepod
49
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Laboratory Sediment Bioassays
Other Promising Techniques
May 1986
partial life-cycle test are highly promising tests that,
if further developed, may be suitable for inclusion in future
updates of this document. The surf smelt egg to larvae
test may be useful if natural variability can be overcome.
In any case, some type of fish egg development test (e.g.,
herring or cod) would be extremely valuable for future use
in Puget Sound, even though it is recognized that such species
cannot be tested year-round.
4. Life-cycle bioassays with nematodes as mentioned in Table 2,
although not used to date in Puget Sound, may provide a
rapid and effective means of determining chronic toxicity
of sediments.
5. The U.S. EPA (Narragansett Laboratories) is presently developing
a solid phase sediment toxicity test using the infaunal
amphipod Ampelisca abdita (Gentile et al. 1985). This species
apparently is similar to Rhepoxynius abronius with respect
to toxicant sensitivity. However, it is found in fine sediments,
whereas £. abronius is not (Scott, K.J., 31 October 1985,
personal communication). Ampelisca spp. also occur along
the west coast of North America, and this animal may be
a useful species for testing those fine sediments that may
have some measure of physical effect on R. abronius.
6. The U.S. EPA (Narragansett Laboratories) is presently finalizing
testing protocols for complex effluents in marine waters.
Four such tests have been developed: 1) 7-day survival/growth/
reproduction with the mysid, Mysidopsis bahia; 2) larval
fish growth/survival with the sheepshead minnow, Cyprinodon
variegatus; 3) sea urchin (Arbacia punculata) sperm eel 1
tests; and 4) sexual reproduction tests with the alga Champia
parvula. The sea urchin sperm cell test is already noted
in this document as a promising technique. The other three
tests may be useful methods to test the toxicity of sediment
elutriates.
7. The Federal Register (U.S. EPA 1985) recently introduced
formal test guidelines for several types of bioassays under
the Toxic Substances Control Act. Two of these tests are
not listed above: algae and penaeid shrimp acute tests.
These may be useful methods to test the toxicity of sediment
elutriates.
50
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environmental mixtures. IV. M.D. Waters, S.S. Sandhu, J. Lewtas, L. Claxton,
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Kocan, R.M., K.N. Sabo, and M.L. Landolt. 1985. Cytotoxicity/genotoxicity,
the application of cell culture techniques to the measurement of marine
sediment pollution. Aquat. Toxicol. 6:165-177.
Landolt, M.L., and R.M. Kocan. 1984a. Lethal and sublethal effects of
marine sediment extracts on fish cells and chromosomes. Helgol. Meeresunters
38:125-139.
Landolt, M.L., and R.M. Kocan. 1984b. Anaphase aberrations in cultured
fish cells as a bioassay of marine sediments. Mar. Environ. Res. 14:497-498.
Lee, C.R., B.L. Flosom, Jr., and R.M. Engler. 1982. Availability and
plant uptake of heavy metals from contaminated dredged material placed
in flooded and upland disposal environments. Environ. Int. 7:65-72.
Legore, R.S., and D.M. DesVoigne. 1973. Absence of acute effects on threespine
sticklebacks (Gasterosteus aculeatus) and coho salmon (Oncorhynchus kisutch)
exposed to resuspended harbor contaminants. J. Fish. Res. Board Can. 30:124-
1242.
MacLeod, Jr., W.D., D.W. Brown, A.J. Friedman, 0. Maynes, and R.W. Pearce.
1984a. Standard analytical procedures of the NOAA National Analytical Facility,
1984-1985. Extractable toxic organic compounds. NOAA Tech. Memo. NMFS
F/NWC-64. 110 pp.
MacLeod, W.D., D.W. Brown, A.J. Friedman, 0. Maynes, and R.W. Pearce.
1984b. Standard analytical procedures of the NOAA (National Oceanic and
Atmospheric Administration) National Analytical Facility, 1984-1985: extract-
able toxic organic compounds. NOAA-TM-NMFS-F/NWC-64. 102 pp.
Mai ins, D.C., S-L. Chan, U. Varanasi, M.H. Schiewe, J.E. Stein, D.W. Brown,
M.M. Krahn, and B.B. McCain. 1985. Bioavailability and toxicity of sediment-
associated chemical contaminants to marine biota. Unpublished report prepared
by NOAA/NMFS, Seattle, WA. 30 pp.
McCain, B.B., M.S. Myers, U. Varanasi, D.W. Brown, L.D. Rhodes, W.D. Gronlund,
D.G. Elliott, W.A. Palsson, H.O. Hodgins, and D.C. Malins. 1982. Pathology
of two species of flatfish from urban estuaries in Puget Sound. EPA-600/
7-82-001. 100 pp.
Misitano, D. 1983. Effects of contaminated sediments on reproduction
of a marine copepod. pp. 26-28. In: NOAA/NMFS Quarterly Report Oct.-Dec.,
1983.
Nacci, D.E., R. Walsh, and E. Jackim. (In review.) Guidance manual for
conducting sperm cell tests with the sea urchin, Arbacia punctulata. for
use in testing complex effluents. U.S. Environmental Protection Agency,
Environmental Research Laboratory, Narragansett, RI. 16 pp.
53
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Nichols, W.W., R.C. Miller, and C. Bradt. 1977. In vitro anaphase and
metaphase preparations in mutation testing, pp. 225-233. In: Handbook
of Mutagenicity Test Procedures. B.J. Kilbey, M. Legator, W.W. Nichols,
and C. Ramel (eds). Elsevier Scientific Publishing Co., New York, NY.
Ott, F.S., P.O. Plesha, R.D. Bates, C. Smith, and B.B. McCain. (In prep.)
Developing sediment bioassays as an ecological monitoring tool. U.S. Dept. of
Commerce, Natl. Marine Fish. Serv.
Pierson, K.B., B.D. Ross, C.L. Melby, S.D. Brewer, and R.E. Nakatam.
1983. Biological testing of solid phase and suspended phase dredge material
from Commencement Bay, Tacoma, Washington. U.S. Army Corps of Engineers
DACW67-82-C-0038. 59 pp.
Ross, B., P. Dinnel, and Q. Stober. 1984. Marine toxicology, pp. 291-370.
In: Renton Sewage Treatment Plant Project: Duwamish Head. Q. Stober
and K. Chew (eds). Fisheries Research Institute FRI-UW-8417.
Schiewe, M.H., E.G. Hawk, D.I. Actor, and M.M. Krahn. 1985. Use of a
bacterial bioluminescence assay to assess toxicity of contaminated marine
sediments. Can. J. Fish. Aquat. Sci. 42:1244-1248.
Schink, T.D., W.E. Westley, and C.E. Woelke. 1974. Pacific oyster embryo
bioassays of bottom sediments from Washington waters. Washington State
Department of Fisheries. 24 pp.
Scott, K.J. 31 October 1985. Personal Communication (Contact by P.M.
Chapman). U.S. Environmental Protection Agency, Environmental Research
Laboratory, Narragansett, RI.
Shuba, P.J., H.E. Tatum, and J.H. Cornall. 1978. Biological assessment
methods to predict the impact of open water disposal of dredged material.
Dredged Material Research Program, Tech. Rpt. D-78-50. Dept. of Army,
Vicksburg, MS. 77 pp.
Swartz, R.C., W.A. DeBen, and F.A. Cole. 1979. A bioassay for the toxicity
of sediment to marine macrobenthos. J. Water Pollut. Control Fed. 51:944-950.
Swartz, R.C., D.W. Schultz, G.R. Ditsworth, W.A. DeBen, and E.A. Cole.
1981. Sediment toxicity, contamination, and benthic community structure
near ocean disposal sites. Estuaries 4:258.
Swartz, R.C., W.A. DeBen, K.A. Sercu, and J.O. Lamberson. 1982. Sediment
toxicity and the distribution of amphipods in Commencement Bay, Washington,
U.S.A. Mar. Pollut. Bull. 13:359-364.
Swartz, R.C., W.A. DeBen, O.K. Phillips, J.O. Lambserson, and F.A. Cole.
1985a. Phoxocephalid amphipod bioassay for marine sediment toxicity.
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of the Seventh Annual Symposium. ASTM STP 854. R.D. Cardwell, R. Purdy,
and R.C. Banner (eds). American Society for Testing and Materials, Phila-
delphia, PA.
54
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Swartz, R.C., D.W. Schults, G.R. Ditsworth, W.A. DeBen, and F.A. Cole.
1985b. Sediment toxicity, contamination, and macrobenthic communities
near a large sewage outfall, pp. 152-175. In: Validation and predictability
of laboratory methods for assessing the fate and effects of contaminated
aquatic ecosystems. ASTM STP 865. T.P. Boyle (ed.).
Swartz, R.C., G.R. Ditsworth, D.W. Schults, and J.O. Lamberson. 1986.
Sediment toxicity to a marine infaunal amphipod: cadmium and its interaction
with sewage sludge. Mar. Environ. Res. 18:133-153.
Tietjen, J.M., and J.J. Lee. 1984. The use of free-living nemotodes as
a bioassay for estuarine sediments. Mar. Environ. Res. 11:233-252.
Tsai, D.F., J. Welch, K.W. Chang, J. Shaeffer, and L.E. Cronin. 1979.
Bioassay of Baltimore Harbor sediments. Estuaries 2:141-153.
U.S. Environmental Protection Agency. 1985. Toxic Substances Control
Act test guidelines; final rules. U.S. EPA, Washington, DC. Federal Register,
Vol. 50, No. 188, Parts 796, 797, and 798. pp. 39252-39584.
Williams, L.G., P.M. Chapman, and T.C. Ginn. (In press.) A comparative
evaluation of sediment toxicity using bacterial luminescence, oyster embryo,
and amphipod sediment bioassays. Mar. Environ. Res.
Wurster, C.F. 1982. Bioassays for evaluating chemical toxicity to marine
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Project Office. 13 pp.
55
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FISH PATHOLOGY
-------
MICROBIOLOGY
-------
FINAL REPORT
TC-3991-04 Pugef Sound Estuary Program
RECOMMENDED PROTOCOLS FOR
MICROBIOLOGICAL STUDIES
IN PUGET SOUND
Prepared by:
TETRA TECH, INC.
and
E.V.S. CONSULTANTS, INC.
Prepared for:
U.S. ENVIRONMENTAL PROTECTION AGENCY
Region 10 - Office of Puget Sound
Seattle, VVA
November, 1986
TETRA TECH, INC.
11820 Northup Way
Bellevue, WA 98005
-------
CONTENTS
Page
LIST OF TABLES iii
INTRODUCTION 1
MICROBIOLOGICAL MEASUREMENTS CURRENTLY MADE IN PUGET SOUND 3
RECOMMENDATIONS FOR FUTURE STUDIES 5
BACTERIAL INDICATORS 5
PRIMARY PATHOGENS 5
SPECIAL SAMPLING CONSIDERATIONS 6
WATER COLUMN 6
SEDIMENTS 6
TISSUE 7
USES AND LIMITATIONS OF RECOMMENDED BACTERIAL INDICATORS 8
FECAL COLIFORM BACTERIA AND FECAL COLIFORM BACTERIA/E. COLI 8
ENTEROCOCCI 10
CLOSTRIDIUM PERFRINGENS 11
LABORATORY PROCEDURES FOR RECOMMENDED BACTERIAL INDICATORS 12
QUALITY ASSURANCE/QUALITY CONTROL (QA/QC) 14
REFERENCES 15
11
-------
TABLES
Number
1
2
Contributors to the microbiology protocols
Current bacteriological measurements in Puget Sound
3 Recommended bacterial indicators for monitoring in Puget
Sound
4 Recommended laboratory procedures for bacterial indicators
Page
2
4
9
13
ACKNOWLEDGEMENTS
This chapter was prepared by Tetra Tech, Inc., under the direction
of Dr. Scott Becker, for the U.S. Environmental Protection Agency (EPA)
in partial fulfillment of Contract No. 68-03-1977. Dr. Thomas Ginn of
Tetra Tech was the Program Manager. Mr. John Underwood and Dr. John Armstrong
of U.S. EPA were the Project Officers. Mr. Peter Nix of E.V.S. Consultants,
Inc., was the primary author of this chapter.
iii
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Microbiology Protocols
Introduction
October 1986
INTRODUCTION
This chapter presents recommendations for measuring microorganisms
in Puget Sound. The recommendations are based on the results of a workshop
and on written reviews by representatives from most organizations that
fund or conduct microbiological studies in the Sound (Table 1). The purpose
of developing these recommendations is to encourage all Puget Sound investi-
gators conducting monitoring programs, baseline surveys, and intensive
investigations to use standardized methods whenever possible. If this
goal is achieved, most data collected in Puget Sound should be directly
comparable and thereby capable of being integrated into a sound-wide database.
Such a database is necessary for developing and maintaining a comprehensive
water quality management program for Puget Sound.
The initial section of this chapter describes those microorganisms
currently being measured in Puget Sound. In subsequent sections recom-
mended microorganisms for future studies are identified, and special consider-
ations for sampling water, sediment, and tissue are discussed. Finally,
the uses and limitations of the recommended microorganisms are discussed,
and laboratory and quality assurance/quality control (QA/QC) procedures
are recommended.
It is recognized that departures from the general recommendations
made herein may be necessary to meet the special requirements of individual
projects. If such departures are made, however, the funding agency or
investigator should be aware that the resulting data may not be comparable
with most other data of that kind. In some instances, data collected using
different methods may be compared if the methods are intercalibrated adequately.
-------
TABLE 1. CONTRIBUTORS TO THE MICROBIOLOGY PROTOCOLS
Name
Organization
Carlos Abeyta
John Armstrong
Scott Becker
Norma Christopherson
Alfred Dufour
Gary Fraser
Mike Glass
Peggy Hammer
Andrew Heyward
Nancy Jensen
Lawrence Kamahele
Larry Kirchner
Jack Lilja
Steve Martin
Jack Matches
June Nakata
Peter Nix
Mike Schiewe
Jay Vasconcelos
Marleen Wekell
U.S. Food and Drug Adminstration
U.S. Environmental Protection Agency
Tetra Tech, Inc.
Washington Department of Social and
Health Services
U.S. Environmental Protection Agency
Washington Department of Social and
Health Services
Washington Department of Social and
Health Services
U.S. Environmental Protection Agency
Municipality of Metropolitan Seattle
Washington Department of Ecology
Seattle-King County Health Department
Seattle-King County Health Department
Washington Department of Social and
Health Services
U.S. Army Corps of Engineers
University of Washington
Seattle-King County Health Department
E.V.S. Consultants, Inc.
National Oceanic and Atmospheric
Administration
U.S. Environmental Protection Agency
U.S. Food and Drug Administration
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Microbiology Protocols
Current Measurements
October 1986
MICROBIOLOGICAL MEASUREMENTS CURRENTLY MADE
IN PUGET SOUND
A variety of agencies sample for bacteria in the water, sediment,
or biota of Puget Sound. These agencies and the various bacterial indicators
(i.e., of pathogens) and/or primary pathogens they evaluate are listed
in Table 2. Measurements of bacterial indicators that are currently used
include:
t Total counts of coliform bacteria (i.e., total coliform
bacteria)
t Counts of fecal coliform bacteria (i.e., fecal coliform
bacteria)
t Counts of Escherichia col i as a fraction of fecal coliform
bacteria (i.e., fecal coliform bacteria/E. coli)
t Counts of enterococci, a subset of fecal streptococci (i.e.,
enterococci)
• Counts of Clostridium perfringens (i.e., C. perfringens)
0 Counts of heterotrophic bacteria by standard plate count
methods (i.e., standard plate counts).
-------
TABLE 2. CURRENT BACTERIOLOGICAL MEASUREMENTS IN PUGET SOUND*
Agency
Routine Monitoringb
Water Tissue
Special Projects
U.S. FDA
(Seattle)
U.S. EPA
(Region 10)
Seattle-King Co.
Health Dept.
Wash. Oept. of
Ecology
Metro
Total coliform bacteria (I)
Fecal conform bacteria (I)
Fecal streptococci (I,NR)
Total collfonn bacteria (I)
Fecal conform bacteria (I)
Fecal conform bacteria (I)(NR)
Total conform bacteria (I)
Fecal conform bacteria (I)
Fecal conform bacteria (I) Fecal conform bacteria (I)
Vibrio spp. (P)
C. perf
C. perfrlngens (P)
Aeromonas hyd
Yerslnia spp.
Aeromonas hydrophlla (I,P)
>. (P)
Occasional (most work is
1n fresh water)c
C. perfrinqens (I,P)
E. coll (I)
Vibrio parahaemolytlcus (P)
Enterococci (I)
Wash. Dept. of
Social and
Health Services
Fecal conform bacteria (I)
Fecal conform bacteria (I)
Standard plate counts (I)
I - indicator of primary pathogens or specific pollution sources. P - primary pathogen,
NR - not routine.
b No agency in Puget Sound conducts routine monitoring of bacteria in sediments.
c Marine studies have included the fate and survival of fecal colifonm bacteria and heterotrophic
bacteria In relation to dredging operations.
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Microbiology Protocols
Future Studies
October 1986
RECOMMENDATIONS FOR FUTURE STUDIES
BACTERIAL INDICATORS
Reliance on a single bacterial indicator or a suite of indicators
may not be desirable as a general rule because the objectives of different
studies may vary substantially. For example, requirements for water pollution
monitoring differ from those for evaluating the acceptability of shellfish
for human consumption. In general, however, the following bacterial indicators
should be considered for use in most surveys of bacterial indicators in
Puget Sound:
• Fecal coliform bacteria
t Fecal coliform bacteria/E. coli
• Enterococci
t C. perfringens.
These indicators might be used individually or in various combinations
depending upon the specific objectives of each study. The best uses and
the limitations of each indicator are discussed later in this chapter.
PRIMARY PATHOGENS
In some cases, disease outbreaks may require the direct investigation
and identification of primary pathogens because use of an indicator is
inappropriate. For example, outbreaks of acute gastroenteritis may show
no correspondence between the presence of a specific indicator, such as
fecal coli form bacteria and the primary pathogen, such as Yersinia entero-
colitica (Hunger et al. 1980). Direct identification of primary pathogens
may also be appropriate during reconnaissance surveys in areas lacking
historical data. The choice of pathogen should be determined by the nature
of the disease and by seasonal considerations. For example, Vibrio bacteria
generally are found in the warmer months, whereas Yersinia bacteria are
more prevalent during colder periods of the year.
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Microbiology Protocols
Sampling Considerations
October 1986
SPECIAL SAMPLING CONSIDERATIONS
Choice of an optimum matrix for sampling (e.g., water column, sediment,
interstitial water, or tissue) will depend upon the objectives of each
study. For example, public health impacts are most likely through ingestion
of contaminated shellfish or contact with contaminated water. Shellfish
tissues or recreational waters may therefore be the materials most appropriate
for analysis in studies focusing on health risks. The identification of
present point sources of pollution can best be determined by analysis of
water samples, whereas long-term pollution trends may best be described
by sediment analysis.
WATER COLUMN
Water sampling can result in highly variable data because bacteria
are not uniformly distributed throughout the water column (Gameson 1983)
and sample volumes generally are limited to 50-100 mL. One major cause
of spatial heterogeneity is the tendency for bacterial cells to concentrate
in a thin microlayer on the surface of the water. Because bacterial abundances
in the microlayer may exceed abundances in underlying surface water by
several orders of magnitude (Hardy 1982), it is reconmended that the microlayer
and underlying water be sampled separately. However, sampling of the microlayer
requires specialized techniques that have yet to be standardized. Also,
collection and analysis of samples from both the microlayer and underlying
water at each station may be too expensive for many routine monitoring
programs. Thus, if separate samples cannot be collected within the constraints
of a particular program, it is recommended that the microlayer be included
in the sample by using the traditional "scoop" method of surface water
sampling (U.S. EPA 1978). This method involves plunging an open bottle
straight down to a depth of 15-30 cm below the water surface, moving it
horizontal to the surface while tipping it slightly to let trapped air
escape, and removing the bottle in a vertical position. It is recommended
that samples be collected using a wide-mouth (12-15 cm) bottle to facilitate
inclusion of the microlayer. •
SEDIMENTS
Sediments are known to be heterogeneous with respect to types and
numbers of bacteria. In addition, the bacteriological composition of sediments
may have little relationship to public health impacts. For these reasons,
routine monitoring of sediments in Puget Sound presently is not undertaken
by any organization (Table 1), and is not recommended except under special
circumstances.
One special application of sediment monitoring is analysis for C. per-
fringens to trace the distribution of sewage. Because spores of this bacterium
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Microbiology Protocols
Sampling Considerations
October 1986
are associated with fecal pollution and survive over long periods, they
offer the advantage of providing a cumulative record of sewage influence
suitable for long-term monitoring surveys. However, the fact that the
spores are persistent in the environment, and thus accumulate, renders
analysis for C. perfringens in sediments inappropriate as a basis for providing
regulatory guidelines.
TISSUE
Sampling and analysis of shellfish tissue present fewer problems than
sampling and analysis of water and sediment (see APHA 1985a). Shellfish
sampling is very important because the consumption of shellfish as food,
sometimes in the raw state, may present a serious public health hazard.
Shellfish offer several advantages for sampling: they concentrate
bacteria, can be sampled relatively easily, and reflect pollution levels
over relatively long periods in both sediment and water. In Puget Sound
it is recommended that one or several shellfish species of recreational
or commercial importance be sampled routinely at each major harvesting
area. The use of a small number (preferably one) of species as standards
will reduce the variation among stations and sampling periods that results
from interspecific differences in the propensity to concentrate bacteria.
Because the whole organism is eaten, the whole body should be prepared
for analysis.
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Microbiology Protocols
Use and Limitations
October 1986
USES AND LIMITATIONS OF RECOMMENDED BACTERIAL INDICATORS
Recommended bacterial indicators for monitoring in Puget Sound are
presented in Table 3 for each kind of sample matrix.
FECAL COLIFORM BACTERIA AND FECAL COLIFORM BACTERIA/E. COLI
The density of fecal col i form bacteria has commonly been used as an
indicator of fecal pollution and as an indicator of the presence of pathogens.
The widespread use of fecal col i form bacteria as an indicator has the advantage
of providing a basis for comparisons with historical data. However, there
are several distinct, and often substantial, limitations to using fecal
coliform bacteria for these purposes. In addtion, the densities of colifomt
bacteria may not accurately reflect public health risks (Hanes and Fragala
1967). Recent research has indicated that, although many species of non-patho-
genic enteric bacteria (e.g., coliform bacteria) are viable in aquatic
systems, they are not totally recoverable using conventional techniques
(Roszak et al. 1984; Xu et al. 1982). Normal culturing techniques may
seriously underestimate the concentrations of these organisms in the environ-
ment. Because the degree of recoverability may also depend on variable
environmental factors (e.g., nutrient concentrations), it may be difficult
to develop general bacteriological standards that could apply to all areas.
Cabelli et al. (1983) concluded that fecal coliform bacteria were
inferior to enterococci as indicators of the presence of pathogens in marine
recreational waters with respect to evaluating public health risks. Survival
times for coliform bacteria are substantially less than for many pathogens
(Borrego et al. 1983), complicating efforts to correlate counts of fecal
coliform bacteria with the densities of pathogens at any specific time.
Another problem associated with the use of fecal col i form bacteria as indicators
is the fact that they are not specific to mammalian fecal pollution. For
example, the fecal coliform bacterium Klebsiel la is common in pulp mill
effluents.
For all of the above reasons, data on fecal coliform bacteria cannot
in themselves be considered adequate for a thorough assessment of public
health risks. Fecal coliform bacteria continue to be used as an indicator
because other indicators also have deficiencies, and because measurements
of fecal coliform bacteria provide a basis for comparisons with historical
data. The lack of specificity of fecal coliform bacteria to mammalian
fecal pollution prompted the development of a membrane filtration method
for enumerating E. coli (Dufour et al. 1981). Unlike fecal coliform bacteria
as a group, E. coli are specific to mammalian fecal pollution. The enumeration
method incorporates a technique for distinguishing E. coli from other fecal
coliform bacteria. The result is notated as fecal coliform bacteria/ E. coli.
However, with respect specifically to recreational water quality criteria,
8
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TABLE 3. RECOMMENDED BACTERIAL INDICATORS
FOR MONITORING IN PUGET SOUND
Matrix
Objective
Water*
Sediment
Tissue
Recreational use
evaluation (e.g.,
swimming)
Pollution monitoring
and/or water quality
surveys
Shellfish consump-
tion evaluation
Fecal conform
bacteria
Enterococci
Fecal conform
bacteria
L. coll
C^ perfringens
Enterococci
Fecal coliform
bacteria
D
Fecal conform
bacteria
C. perfringens
Fecal conform
bacteria
E. coli
C. perfrinqens
Fecal conform
bacteria
a Fecal conform bacteria are recommended here because current water quality
standards are based on them. If these standards change to include only
enterococci, it may still be useful to measure fecal coliform bacteria
to maintain continuity with historical databases.
b U.S. EPA and National Oceanic and Atmospheric Administration (NOAA) currently
are co-sponsoring research Into the application of the enterococci and
E. coli indicators for shellfish harvesting waters.
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Microbiology Protocols
Use and Limitations
October 1986
U.S. EPA has not recommended the use of E. coli for marine and estuarine
waters.
Laboratory analyses of fecal coliform bacteria are conducted using
one of two most probable number (MPN) techniques (i.e., EC and A-l) or
using a membrane filtration (MF) technique. The statistical reliability
of the MF technique is greater than the MPN procedures (APHA 1985b). However,
factors such as turbidity may reduce counts for the MF technique (Berger
and Argaman 1983). Thus, the results generated by the MF and MPN techniques
may not be directly comparable, and their inconsistent use by Puget Sound
organizations limits comparisons of data gathered by different agencies.
ENTEROCOCCI
Enterococci are streptococcus bacteria indigenous to the intestines
of warm-blooded animals. U.S. EPA recommends their use as indicators of
fecal pollution in recreational waters because of the previously mentioned
limitations of fecal coliform bacteria analysis, and because of the following
characteristics:
• Because the concentration of enterococci has a greater corres-
pondence to the incidence of gastroenteritis than do concen-
trations of E. coli or fecal coliform bacteria, it is a
better indicator of public health risk in recreational marine
waters (Cabelli et al. 1983).
0 Enterococci may die off more slowly in sediments than fecal
coliform bacteria (Van Donsel and Geldreich 1971), and therefore
be better indicators of sediment contamination.
• Because they are tolerant to high salinity, enterococci
are of particular value in analysis of marine waters (Coler
and Litsky 1976).
0 Taxonomic identification of streptococcus bacteria can be
undertaken easily (e.g., API biochemical strips) and, unlike
the situation with fecal coliform bacteria, can reveal the
kinds of mammalian pollution (e.g., humans, livestock).
This advantage arises from the fact that particular kinds
of mammals harbor characteristic species of streptococcus
bacteria (e.g., S. bovis in cattle).
0 Techniques of gene fingerprinting (DMA analyses) have been
undertaken using streptococcus bacteria and can more positively
link bacteria in the environment to identical bacteria found
in source effluents, thereby confirming the source(s) of
contamination. Although not suitable for routine monitoring
surveys, genetic analysis may be useful in certain research
applications.
10
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Microbiology Protocols
Use and Limitations
October 1986
CLOSTRIDIUM PERFRINGENS
C. perfringens is consistently associated with fecal wastes and provides
a usable, state-of-the-art marker for delineating the deposition and/or
movement of sewage participates that is more reliable than the traditional
coliform bacteria indicators (Emerson and Cabelli 1982). C. perfringens
is recommended for water, sediment, and tissue because it is present in
wastewater at concentrations of 10^-104 per 100 mL (Fujioka and Shizumura
1985), and because its resistance to chlorination and environmental factors
closely resembles that of enteric viruses (Bisson and Cabelli 1980).
11
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Mi,c.ro'bio,l qgy Protocol s
' La'bbratfory .Procedures
October 1986
LABORATORY PROCEDURES FOR RECOMMENDED BACTER'lAL INDICATORS
Recommended laboratory procedures for the bacterial indicators listed
in Table 3 are summarized in Table 4.
12
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TABLE 4. RECOMMENDED LABORATORY PROCEDURES FOR BACTERIAL INDICATORS
Test Organisms
Laboratory Procedures
Water
Sediment
Tissue
Fecal collform
bacteria
Fecal coliform
bacteria/E. coll
Enterococci
C. perfringens*
MPN tubes using A-I
broth (APHA 1985b)
(fecal coliform
bacteria/100 mL)
MPN tubes using A-I
broth (APHA 1985b)
(fecal coliform
bacteria/100 mL)
mTEC (DuFour et
1981)
(E. coll/100 n»L)
al
mE (Levin et al. 1975)
(enterococci/100 mL)
MPN tubes using iron
milk (St. John et al.
1982) (C. perfrin-
gens/100 mL)
MPN tubes using iron
milk (St. John et al.
1982) (C. perfrin-
gens/100 g)
MPN tubes using EC
broth (APHA 1985a)
(fecal coliform
bacteria/100 mL)
MPN tubes using iroi
milk (St. John et al
1982) (C. perfrin-
gens/100 mL)
a Two laboratory techniques are available for C. perfringens: mCP by membrane filtration foi
water (Bisson and Cabelli 1979) and sediment (Emerson and Cabelli 1982), and. iron milk tube
using MPN techniques (St. John et al. 1982). The latter method is recommended (pending an
comparative data) because the procedure is simpler and less costly.
13
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Microbiology Protocols
QA/QC
October 1986
QUALITY ASSURANCE/QUALITY CONTROL (QA/QC)
Laboratory and analytical QA/QC procedures are discussed in detail
in U.S. EPA (1978) and APHA (1985b). Special problems exist in microbiological
analyses because analytical standards, known additions, and reference samples
generally are not available. However, a minimum QA/QC program should include:
• Ten percent of the total number of samples analyzed in duplicate
0 Ten percent of the total number of samples split and analyzed
by two or more laboratories
• Sterile distilled water transported to the field, transferred
to a sample bottle, and processed routinely to ensure samples
were not contaminated during collection and transport
• Repeated sampling at one site during varying conditions
(e.g., tides, weather) to evaluate variability in the field.
14
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Microbiology Protocols
References
October 1986
REFERENCES
APHA. 1985a. Laboratory procedures for the examination of seawater and
shellfish. 15th ed. American Public Health Association. 144 pp.
APHA. 1985b. Standard methods for the examination of water and wastewater.
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