EPA-600/4-74 01
October 1974
Environmental Monitoring Series
Proceedings of Seminar on Methodology
for Monitoring the Marine Environment
Office of Research and Development
U.S. Environmental Protection Agency
Washington. D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditdoaal grouping
consciously planned to foster technology transfer and
a maximum interface in related fields. The five series
are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the Environmental
Monitoring series. This series describes research
conducted to develop new or improved methods and
instrumentation for the identification and quantification
of environmental pollutants at the lowest conceivably
significant concentrations. It also includes studies to
determine the ambient concentrations of pollutants in the
environment and/or the variance of pollutants as a function
of time or meteorological factors.
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EPA-600/4-74-004
October 1974
PROCEEDINGS
OF
SEMINAR ON METHODOLOGY
FOR
MONITORING THE MARINE ENVIRONMENT
SEATTLE WASHINGTON
OCTOBER 1973
Program Element No. 1HA326
ROAP/Task - PEMP/2
SPONSORED BY
OFFICE OF MONITORING SYSTEMS
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office
Washington, D.C. 20402 - Price $4.90
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EPA REVIEW NOTICE
This report has been reviewed by the Environmental
Protection Agency and approved for publication
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or
recommendation for use.
ii
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EDITORIAL BOARD
Editor
S. Sidney Verner
Editorial Staff
ORD-Headquarters
Francis T. Brezenski
Victor J. Cabelli, Ph. D.
Thomas W. Duke, Ph. D.
Leonard J. Guarraia, Ph.D.
Andrew J. McErlean, Ph.D.
Patrick L. Parker, Ph.D.
Walter F. Rittall
T. Allen Wastier
Barbara J. Wygal
EPA, Region II
NERC-Cincinnati
-Narragansett
NERC-Corvallis
-Gulf Breeze
OAWP-Headquarters
OEGC-Headquarters
Univ. of Texas
Marine Sc. Inst.
NERC-Corvallis
OAWP-Headquarters
OAWP-Headquarters
ill
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SEMINAR
STEERING COMMITTEE
Donald J. Baumgartner, Ph.D.
Francis T. Brezenski
Victor J. Cabelli, Ph.D.
Thomas W. Duke, Ph.D.
Leonard J. Guarraia, Ph. D.
Robert McManus
Patrick L. Parker, Ph.D.
S. Sidney Verner
T. Allen Wastier
NERC-Corvallis
EPA, Region II
NERC-Cincinnati
-Narragansett
NERC-Corvallis
Gulf Breeze
OAWP-Headquarters
OEGC-Headquarters
Univ. of Texas
Marine Sc. Inst.
ORD-Headquarters
OAWP-Headquarters
iv
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FORWORD
The Seminar on Methodology for Monitoring the Marine
Environment was organized to assist the Environmental
Protection Agency in establishing a basis for common
methodological techniques in marine and estuarine water
quality management. The meetings focused on procedures
for sampling and analyzing coastal waters involving the
physical-chemical, biological, and microbiological disciplines.
The Seinar had a three fold purpose: to serve as a forum for
presenting state-of-the-art technology in environmental quality
monitoring of saline and brackish waters; to present recent
developments in sampling and analysis of these waters; and to
clarify and identify problem areas and desirable research goals.
The Seminar was open to anyone with an interest in
techniques for monitoring the environmental quality of coastal
regions. The meeting was necessarily of limited scope and
additional seminars may be useful in the future. The Office of
Monitoring Systems welcomes comments on these proceedings as
well as suggestions for future activities.
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TABLE OF CONTENTS
Foreword [[[ v
Preface [[[ viii
Introduction and Theme
Basic Framework and Field Applications
Mathematical Modeling as a Framework
for Coastal Monitoring ............................. 6
Problems in Measuring Turbidity as a
Water Quality Parameter ............................ 23
Surface Slick Sampling and Analysis .................... 55
A Syfitems Approach to Marine Pollution Monitoring ...... 72
Monitoring for Pollutants
Methods and Problems in Analysis of
Pesticides in the Estuarine Environment ............ 108
Biological Problems in Estuarine Monitoring ............ 126
Determination of Metals in Sea Water ................... •"
Summary of Recent Studies on Biological
Effects of Crude Oils and Oil-Dispersant
Mixtures to the Red Sea (Israel Macrof auna ......... 156
Absorption of Orthophosphates on Borosilicate
and "Citrate of Magnesia Bottles" Polyethylene
and Polyvinyl Surfaces in a Distilled Water
and Seawater Matrix ................................ 180
Development of a Standard Marine Algal Assay
Procedure of Nutrient Assessment ................... 194
Monitoring Seawater for Radionuclides .................. 231
Methods for Monitoring Radioactivity in
Aquatic Biota ...................................... 242
The Properties and Composition of Sludges .............. 259
Making Artemia Sludge Bioassay More
Ecologically Relevant .............................. 275
Monitoring Dredge Spoils ............................... 302
Comparison of Species Diversity and Faunal
Homogeneity Indices as Criteria of Change
in Biological Communities .......................... 316
Microbiological Aspects
Sampling Methods for Microbiological Analyses .......... 334
Microbiological Methods for Monitoring
Marine Waters for Possible Health Effects .......... 359
A Survey of Methods for Monitoring Ecologically
Important Microorganisms in the Marine
Environment ........................................ 384
Monitoring Requirements
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Index of Authorx 411
Appendix A
Agenda A-l
Appendix B
List of Attendees B-l
vii
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PREFACE
This document contains the proceedings of the "Seminar
on Methodology for Monitoring the Maring Environment" 'held in
October 1973. The Seminar and these proceedings are representative
of a continuing effort by the Office of Monitoring Systems to high-
light issues and alleviate, through inter and extra Agency-wide
cooperative efforts, problems of monitoring environmental quality
requiring technical or administrative support.
This Seminar was organized to strengthen the Agency's coastal
monitoring program and was divided into three principle segments,
viz., basic framework and field applications, monitoring for
pollutants which concerned physical-chemical and biological
techniques for investigating eight classes of pollutants; and
microbiological aspects for assessing the quality of marine and
estuarine waters. The theme of the meeting was set by
Dr. Stanley M. Greenfield, Assistant Administrator for Research
and Development. A final paper by Dr. Donald Squires of the
New York State Sea Grant Program addressed the problem of
relating monitoring systems to the users of environmental data.
viii
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INTRODUCTION A1JD THEJ.IL
Stanley M. Greenfield *
I would like to add my words of welcome to those of the
Chairman in wishing you a fruitful and productive meeting.
We have organized this seminar to bring together those
concerned with monitoring the quality of marine and estuarine
waters; to engage in a free exchange of ideas; to learn firsthand
about progress in the development of methodology; and hopefully
to come away with a better understanding of current technology
for coastal and near coastal regions. Perhaps of equal
importance are the bonds we can develop between the academic
community and EPA.
Monitoring of the marine environment is an integral part of
environmental management and the control of pollution, since the
Administrator of the Environmental Protection Agency is thus
provided with information for decision-making relating to the
state of the environment, trends, compliance with standards, etc.
Environmental monitoring data is also essential for assessing and
evaluating control strategies and abatement measures as well as
status and trends in the quality of the marine environment. The
assessment of environmental quality is vitally important in
determining national environmental policy and in keeping the
public informed regarding the condition of the environment.
I should take a moment to explain what I mean by
environmental monitoring. What is meant is the systematic and
continuing observation of environmental quality parameters
including physical-chemical, biological and microbiological
techniques for the purpose of providing a sound data base for
EPA's criteria development, regulatory and operating programs.
The scope of a monitoring program consists of: (a) the
development of sensor devices, instrument systems and analytical
procedures; (b) sample collection and analysis; and (c) data and
information storage and retrieval for the detection and
measurement of pollutants and the assessment of environmental
quality.
*Formerly Assistant Administrator for Research and Development,
U. S. Environmental Protection Agency.
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I need not remind this audience that EPA is basically a
regulatory Agency whose primary function is enforcement of
environmental quality standards in both air and water established
by statute. The monitoring requirements placed on EPA and the
States necessitate a sophisticated sensor network extending
through all the coastal regions. However, such a network must be
v/ell planned and coordinated. The Office of Monitoring Systems
under Will Foster's direction is responsible for developing an
overall monitoring strategy for the Agency. Some of the
important considerations in the development of this strategy are:
. technical, administrative and logistical support
for development of monitoring methodologies;
. development of environmental baselines in order to
identify environmental quality trends;
. timely incorporation of newly developed monitoring
techniques into Agency monitoring networks;
. identification of short-term and long-term monitoring
requi rements;
. standards for methodology in monitoring applications;
. utilization of state-of-the-art sensors, techniques
and procedures;
. a data link between sensors and data storage systems.
These functions spell out the Office of Research and
Development's interest and involvement in monitoring
environmental quality and form the basis for relationships with
other agencies and institutions.
However, all the activities I have enumerated rest on the
premise of a vigorous and competent technological program. In
fact, I would venture to say that only through a reliable,
efficient and timely monitoring program based on the most
advanced concepts in technology v/ill EPA be able to meet the
challenge of restoring and maintaining a viable marine
environment for present and future generations.
Consequently, this meeting was called to further the
development of marine monitoring techniques and methodology and
the subsequent standardization of methodology which is not yet
available and requires a concerted effort by all concerned. We
all recognize the pitfalls and difficulties of the conventional,
i.e., manual techniques for analyzing and evaluating
environmental quality in sea and brackish waters. Unfortunately,
traditional time-honored analytical methodologies all too often
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have not been suited to marine waters and organisms. As an
example, not too long ago the National Science Foundation
conducted an experiment in which lead was dissolved in sea water
to a concentration of 15 ppb; 10 aliquots were distributed to
various university and marine institute laboratories for
analysis; the results that came back reported concentrations of
50-1500 ppb; subsequent repeats of this experiment have brought
results which vary by only an order of magnitude!
Mien we consider the importance of coastal and near coastal
regions to our health and well-being, e.g., as a source of
recreation and food, it is apparent that we must bring the level
of technology of marine monitoring methodology at least 'on a par
with other monitoring methodologies.
While seeking to improve existing technology, it is also
important that we look to the future in our continuing efforts to
advance the state-of-the-art. Historically, the routine
monitoring of environmental pollutants in marine waters has
involved analysis of selected samples collected "in the field".
Such sampling techniques are not only time consuming, but more
important, they have limited utility considering the vast
geographic areas and pollution sources that must be sampled for
truly effective environmental 'monitoring- Clearly, for the
future, the routine monitoring of marine and estuarine waters
will most effectively be conducted through automated devices
operating in the in situ or remote sensing mode and functioning
in real-time.
Needless to say, an operational network of such sensing
instruments, to be widely deployed and effective, must contain
elements which are relatively inexpensive, reliable, sensitive,
accurate, pollutant-specific and easily maintained and
calibrated. Ultimately, the acceptance of advanced monitoring
systems rests on the intrinsic performance of the sensors and
instruments and the degree to which they satisfy the
aforementioned requirements.
Although many advancements in sensor technology may be
expected in the future, improved methodology will always remain a
goal and sensor development will continue to be of great concern
to us. This is particularly so in areas of electro-optical
instrumentation involving correlation or matched filter
techniques and derivative spectrometry, and the class of
electro-chemical probes using ion selective electrodes where
important developments are occurring in sensor technology.
In looking to the future, it is essential that the evolution
of. sensors be closely followed and supported by EPA becadse ever
newer generations of sensing instruments will radically affect
techniques for monitoring the quality of the marine environment.
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Assembled at this meeting are some of the leading exponents
and proponents of marine environmental science to discuss
problems which are fundamental to an effective monitoring
program. I am confident that the results obtained here will
constitute an important contribution to EPA's future monitoring
strategy in the marine environment.
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INTRODUCTORY REMARKS TO SESSION A
BASIC FRAMEWORK AND FIELD APPLICATIONS
MODERATOR: DONALD J. BAUMGARTNER*
This session is entitled Basic Framework and Field
Applications. This is not an entirely satisfactory
descriptive title, but was the closest I could think of
which attempted to describe some of the monitoring problems
we would consider this morning. In initial planning of this
symposium, several titles such as, "Physical",
"Hydrodynamic", "Hydrologic" were considered as descriptive
of the topics to be considered, of currents, salinity, and
temperature for several reasons. First, these systems are
rather well developed and are continually being improved by
the Navy, the National Oceanographic and Atmospheric Agency,
by Oceanographic Institutions, and by the manufacturers of
oceanographic eguipment. These systems which are routinely
employed in oceanographic institutions are well described by
the technical literature of the manufacturers and by reports
in ocanographic journals, The second reason is that the
state-of-the-art of measuring pollutants in coastal waters
is not only much more in need of development but is
certainly more the province of the Environmental Protection
Agency and should receive considerably more attention in
symposiums such as this. Two of the papers in this session
will deal with pollutant measurements having some physical
properties, particulates associated with turbidity, and
surface films.
Taking the work physical in another sense, that of a
physical science, a paper on the application of mathematical
modeling as a fundamental consideration in the design and
operation of a monitoring system will be presented.
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MATHEMATICAL MODELING AS A FRAMEWORK FOR
COASTAL MONITORING
R. J. Callaway *
INTRODUCTION
Several definitions are required as a prelude to this paper.
The first few are taken from Webster's seventh New
Collegiate Dictionary.
mathematics —the science of numbers and their
operations, interrelations,
combinations, generalizations...
model —a system of postulates, data, and
inferences presented as a mathematical
description of an entity or state of
affairs.
monitor —to watch, observe or check, esp. for a
special purpose.
—to keep track of, regulate, or control.
surveillance --close watch kept over a person or group
(as by a detective) .
From these we can suggest that environmental mathematical
modeling, as a monitoring and/or surveillance tool, would be
an attempt to describe, predict, and generalize on pollution
caused transformations and the regulation or control of such
events as guided by or inferred from model output.
MODELS, MODELING AND MODELERS
Several classes of models exist, among them:
models which may be the framework of an eventual working
model! analytical models which usually result from
analytical solutions of simplified (tractable) systems;
numerical models of complex (analytically intractable)
systems; hydraulic models which are scaled down, usually
space distorted, images of real entities or environments;
Environmental Protection Agency,
Pacific Northwest Environmental Research Laboratory,
CorvaHis, Oregon
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niodels which use electronic components as analogues
of physical systems and mathematical operations. The main
emphasis here is on analytical and numerical models.
It has only recently been recognized that environmental
problems occur at the systems level of complexity (12). It
is usually the biologist who has the last say as to how
detrimental a particular situation is or may become. Since
the biologist is rarely a systems engineer, he will do well
to consult with the physicist (or engineer) on certain
problems thus avoiding Cohen's (4) rule: "Physics-envy is
the curse of biology." Likewise, the physicist working on
theoretical biological models should harken to Boorman's (2)
advice: "Physicists who wish to do useful biology should
not try it on their own."
MONITORING SCHEMES RELATED TO THE TYPE OF WASTE INPUT
Figure 1 is a schematic of mathematical formulations of
various waste inputs and the response of a system to the
input. Here, W is waste load concentration, t is time, is
frequency and s is system response. Following Thomann (11) ,
assume a box or reservoir model in which all constituents of
interest are completely mixed. The inputs shown in Figure 1
are discussed from the point of view of the mass balance
equation with constant system parameters:
1) V *p + QC + KVC = W (t)
where
V = volume of system (box), L3
C = concentration of waste, ML-3
t = time, T
Q = advection through box, L3T~>
K = reaction
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Input
Impulse Function
«t
Example
Barge Release
Response
w
t
Step Function
«t
o-
Ocean Outfall
«t
o t
Fourier Series wf
0
Estuarine Outfall
o t
N
Gain
Input Frequency
Phase
GO-
Sum of Impulses
•t
0-
Tanker Oil Spill
n
o t
Random
w
t
0-
Turbulent Interactions
Filter
"2TT/QO
icot
-T T
FIG. 1. Schematic input-output relationships
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In order to monitor impulse response situation which might
be approximated by a barge release of wastes in the open
sea, as hoc procedures will most likely have to be employed.
These will relate to the necessity of following the release
throughout the water column and into the sediment for later
monitoring of the effect on benthic communities, etc. Of
course, in real situations the identity of the point source
cannot be maintained for long; to describe and predict the
spread adequately, equations have been developed which
include more complex advective and diffusive terms but which
still utilize the initial impulse function.
Constant Tilngut
Equation 1 can be easily solved for the step function input
as:
3) c = QV (1 " exp c ~(~~ * K)t])
Equation 3 makes some aspects of the monitoring problem
become clear: as t becomes large the concentration in the
system approaches steady-state i.e., as:
o, C and z- •* 0.
Q+KV dt
Concentration at steady- state is directly proportional to
the waste input and inversely proportional to the system
parameters. Near the source (V-0) , or for a conservative
substance (K = O) , the steady-state concentration is W/Q.
If a given system can be considered as a single well-mixed
box with uni- directional flow then a single fixed instrument
placed anywhere in V could adequately monitor events with
respect to its equilibrium time. In reality, of course, the
actual placement of the instrument will be a function of
several considerations such as distance from the source,
ease of servicing, etc.
Periodic Input
For periodic inputs, the simplest is sinusoidal:
W(t) = W * WoSin(wt*
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C = W + Wo SIN (fa)t+d>)
k
and is seen to be frequency dependent and oscillating about
W. The non-steady periodic output response can be shown to
be (Thomann, op. cit.):
Cp = WoAm(w)
Where
_ i/v
k/V
The system parameters K, Q, V and the input frequency, w,
determine the amplitude and phase characteristics of this
particular system. In general, for any given input which
can be approximated reasonably well by a Fourier series the
total response of the well-mixed system will consist of a
mean response plus periodic responses for each series term.
To evaluate the results, Bode or Nyquist plots are usually
required. For the results above, however, it can be seen
that an increase in input frequency results in an asymptotic
decrease in A to zero; 6(W) approaches */2 asymptotically.
The equilibirium response of the system is
C = C * C
P
where
c = w/k
Knowledge of the input frequency would be required for
determining instrument response times if the input frequency
is high, i. e.f the instrument response time and sampling
frequency at a given station would need to be such that
effluent peaks are not filtered out. In order to relate the
instrument reading to the source the phase lag due to
frequency shift needs to be related to the distance from the
source. For simple flow-through, constant Q systems this is
an easy task; however, for complex tidally varying systems
it is a difficult problem requiring time-series analyses.
For routine purposes this analysis will not usually be
feasible or necessary. Where it is necessary, design
criteria for the sampling period need to be met. These
criteria relate to the period of the expected dominant
frequencies, usually tidal, in the water body.
ArbitrarY_Input
A stationary tanker leaking oil at different rates with time
can be considered as an arbitrary input case. If the rate
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of discharge varies about some mean in an aperiodic fashion,
special mathematical techniques need to be employed because
techniques associated with periodic phenomena are
inapplicable.
An arbitrary input, u(t) can be thought of an consisting of
n unit impulses each with its own amplitude, a , occur ing at
time t :
The response to each impulse is R (t-t ) , a weighting
function which is determined by the system parameters (Q, V,
K) . This function weights all previous inputs up to the
time of observation with the most recent inputs having the
greatest weight. Formally,
X (t) = i(t )R(t-t.) dt,
'
As time t may be expressed as distance from the source then
t = y/v, where y is distance from the source. Here, v = Q/A
- flow through a uniform cross- sectional area and the
convolution integral can be thought of in terms of a steady-
state velocity with the weighting function having less
weight with distance from the source.
Stochastic Input
It may be required to monitor an aperiodic or stochastic
input or a continuous output record such as, dissolved
oxygen in an estuary. Certain periodicities in the D. O.
record will be obvious and related to tidal periods and
diurnal and semi-diurnal fluctuations, however, the effluent
discharged from an outfall may have a stochastic rate of
discharge. Thus, it is implied that two mechanisms may
account for a stochastic output: stochastic waste input
and/or physical and biological processes in the environment.
The analysis and synthesis of these events, i.e., the
analysis of the contribution of the various processes of
waste in order to synthesize the output record, can be
accomplished to some degree, although a complete
interpretation will rarely be attained.
It is unlikely that anything less than a continous or
closely spaced record of the parameter under consideration
will lend itself to the computations required for analysis
of a stochastic record. If it is known that it will be
necessary to monitor and analyze a stochastic event then
special instrumentation and field procedures will have to be
developed with a clear understanding of what is to be
sampled and when. An excellent source of information on the
time— series analysis required is given in Bendat and Pier sol
(1) . Not of small importance is an understanding of what
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the payoff will be and a determination of whether the payoff
justifies the field investigation and subsequent analysis.
MONITORING IN RELATION TO A WORLD MODEL
Forrester's work on world dynamics (5) and a subsequent
study by Meadows et al., (7) provide startling conclusions
as to the fate of the world if pollution is left unchecked.
The above works are certainly open to criticism from a
number of points of view, (e.g., technological advances in
pollution control are omitted) but it is assumed here that
the results of the models are valid in order to emphasize
the role of monitoring on a world scale. Further, it is
suggested that similar smaller scale models may be in order.
Forrester's world model is based on the assumption that the
world can be described in terms of population, capital
investment, pollution, agriculture and natural resources and
that these variables can be described by sets of input data.
Salerno (9) found the model very sensitive to input
assumptions with the result that: either the world is indeed
sensitive to small changes in the input descriptors; or the
model is basically sound but requires more accurate input
data; or the model has serious flaws. The latter view is
echoed by Shubik (10) who considers Forrester's work as M. .
. superficially attractive but nonetheless dangerously
wrong... "
The model of Meadows et al., (op. cit.) is based on
Forrester's work. The authors suggest that w...it is the
only formal model in existence that is truly global in
scope, that has a time horizon longer than thirty years, and
that includes important variables such as population, food
production, and pollution, not as independent entities, but
as dynamically interacting elements, as they are in the.real
world." Four examples of the model output are given in
Figure 2. Results are given from 1900 - 2100; the captions
are self explanatory. It can be seen that the onset of
"pollution" can occur quite rapidly given the assumptions
and constraints of the model. The fact that complex
feedback-feedforward mechanisms exist is clear from the
examples. Figure 2 shows that while initial regulation and
control of pollution and other factors provide for a stable
population, there is an inevitable decline in industrial
growth, resources and food production as pollution
accumulates.
OCEAN TIME SCALES
Geochemists frequently use reservoir or box models of entire
oceans to investigate oceanic circulation, chemical
processes within the ocean and chemical exchange at oceanic
boundaries (6). As there is interchange between estuaries
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VYGfUD .V.OPn1. V/!TH "UNLIMITED"
n:scr;"CcS A:.:D roiurriON CONTROLS
WORLD MODEL WITH "UNLIMITED-
RESOURCES, POLLUTION CONTROLS, AND
INCREASED AGRlCULTtRAL PRODUCTIVITY
7":
A further technological improvement is added 1o the world model
in 1975 to avoid the resource depletion and pollution problems
of previous mocfe( runs. Here we cissume that pollution generation
per unit of mc'ushicl one/ agriculture! output can be reduced fo
one-fourth of »fs J97Q vclt/e. Resource policies are the some as
those in figure 37. These changes allow popu/ofion and industry
to grow unit! the limit of arable land is reached. Food per capita
declines, ana' industrial growth is also slowed as capital is
ted fo food production.
WORLD MODEL WITH "UNLIMITED"
RESOURCES, POLlUTiON CONTROLS, AND "PERFECT"
BIRTH CONTROL
--- T-t^Mtpj
instead of an increase in food oroducfion, on increase in birth
confrol effectiveness is tested as a policy fo overt the food profa-
lem. Since the birth control is voluntary and does not involve any
rolue changes, population continues to grow, but more slowly
than if did in figure 39. Nevertheless, the food crisis is post-
poned for only a decade or two.
Figure 2. World Model Examples
(Meadows et al., 1972)
To ovoid the food crisis of ffe previous model run, overage (and
yield is doubled in 1975 i.i addition to (he pollution and re-
source policies of previous figures. The combination of these three
policies removes so many constraints to growth that population
and industry reach very higf. levels. A/though each unit of indus-
trial production generates much less pollution, total production
rises enough to create a pollution crisis that brings an end fo
growth.
WORLD ^\CD2L \v;r:-: "u;:u::.iTED"
RESOURCES, FOLLUTiON CONTROLS, !MC:;n/,:rj
AGRICULTURAL PRODUCTIVITY, AND "rEP.FECT"
BIRTH CONTROL
-13-
four simultaneous technological pojicies are ir.irodaccd in the
world model in an attempt to avoid Ilie growih-ancf-ccllapje
behavior of previous runs. Resources arc ful/y exploited, anil 75
percent of ,'hose used ore recycled. Pollution cjL-nerafron is re-
duced to ons-fci-rtri of i;s !970 value, la^c.1 yields nrc &'o.;!.,'-.>d,
ond effective method-, of birth conirol ere- mocfo ava,'!:ib:.> to !hc
world popu/.ition. The result is a Vemporory och:ovtmenf of o
constant popuiolion with n wor/j ovoroj-.- i.icomp pc-r co-j.'la
''10' fca^es nearly the pipsent US level. Finally, though, m'u'us-
fria/ growth is halted, and fh« death role riit-j aj resources are
depleted, pollution occu.-)iu!ates, and food production decline.;.
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and coastal waters and coastal and oceanic waters, so may
these interchanges be viewed as local, regional and global
environments requiring equivalently scaled solutions.
Exchange of gases may occur at the air-sea interface and
through reaction within the water column. In the case of
particulates or nutrient balances, changes may occur at the
water-sediment interface within the water column (box) . If
exchange rates are known, then useful information can be
obtained as to the time needed to approach steady-state from
given initial conditions. Ocean estimates of geochemical
processes can then be made. The time frame of these
processes is on the order of decades or centuries. For this
simple model advection between water masses is not allowed;
additional boxes would be needed with advection specified as
an exchange rate.
Within a "global" box two or more regions are inferred.
These may be thought of as individual boxes or connecting
boxes as space or time dependent states approaching
equilibirum by inclusion of additional physical processes or
more detailed constituent mechanisms. Here the time scale
is on the order of a day to a month.
The regional problem can itself be decomposed into a local
situation where individual water parcels are traced. This
decomposition may be sufficiently described by rate
equations (for chemical parameters) . The time scale here
may be on the order of seconds to hours to days.
Of course, one can decompose the local problem even further
or synthesize the one ocean model into a global model.
The system way of looking at things emphasizes that the
processes involved in a given case require consideration of
space and time scales.
As an example of the time involved to approach steady-state
Figure 3 is presented. Here the time required to achieve 95
percent of the concentration difference between a new and
initial steady-state oxygen profile is C-C = =0.95 (C
Ct,0). The solution is given in terms of tSe physical *'***
processes of advection and diffusion versus time in years
for a given consumption rate and is a solution of the
equation:
where
C = concentration
K = vertical eddy diffusivity
U = vertical velocity
~l) = oxygen consumption rate
-14-
-------
0*
Ul
Figure 2. Time tr> steady-state for oxygen concentration
.'. -- 1 km
at
(From Lerman, 1971)
-15-
-------
Figure 3 shows the solution in terms of velocity and
diffusion versus time. For instance, for replenishment by
diffusion only (U=0), and K»=0.2 cm* sec~1, about 4000 years
are required to reach steady-state. Even for more realistic
values large amounts of time were still required to recover
from an incident or approach a new level.
The above reasoning suggest that for some processes global
monitoring is not feasible. Clearly, there are exceptions—
radioactive contamination of ocean waters at depth is an
example. The exceptions are usually related to non-point
source inputs such as atmospheric fallout over wide areas.
We should not expect, however, to routinely detect isolated
point source contributions to global or oceanic pollution
unless they are of extremely high input rates and high level
concentrations.
Regional Time Scales
The question of what is the least sampling frequency
required to monitor a trend or transient cannot be easily
answered for a variety of reasons: the rate processes may
vary (for nonconservative substances) depending on the type
of reaction, depth, water mass, input source and strength,
etc. Possibly the only truly satisfactory method would be
near-continuous sampling coupled'with an averaging or
filtering scheme to detect shifts in water quality not
obvious on a continuous data display.
As an example of the variation of rate processes to be
expected. Table 1 is presented. There is illustrated a wide
spread of observed and calculated oxygen consumption values
(0.0027 - 5.8 ml liter-11 year~M) for differing
environmental conditions (fjords to abyssal waters).
Advection and diffusion markedly affect the replenishment
rate of the resource; the large values observed in fjords
are due to the absence of flushing for long periods of time.
The progressively deteriorating oxygen resource and increase
of phosphate levels in the Baltic below the halocline is
shown in Figure 4. Data from 1900 to 1967 show considerable
scatter but detectable trends on the order of about 0.1 ml/1
0 per year during 1957-1967 and 0.3 mg/1 PO^-P per year.
It is appreciated that there is more to the interpretation
of these figures than meets the eye but the example is
obvious.
LOCAL TIME SCALES
Examples of local (order of kilometers) problems are familar
to everyone and will not be discussed here except to suggest
the following. While scaled down changes (from global to
local) are easier to detect from one level to another lower
-16-
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TABLE 1 Oxygen Consumption In Various Basins
Basin
0 Consumption
(ml liter-' yr-1)
Remarks
Methods
Reference
Santa Barbara
S. California
i
— i
^s
i
6. of Maine
N. Atlantic
Puget Sound
Tofino Inlet
Baltic Sea
1.9 ± 0.3
0.9 ± 0.3
1.8
0.6
0.05
0.36
0.21
5.8
4.1
0.1
May -June 1970, below
500 meters
June-July 1970
200 meters
400 meters
700 meters
Stagnant pool
200 meters
Average of several
locations, fiord basins
Fiord
Landsort Deep (459 m)
Direct measurements
Advection-dif fusion
model
Measured
Adv.-diff. model
Measured
Measured
Measured
Sholkovitz & Gieskes
Sverdrup & Fleming,
Redfield (in Rossby,
Riley, 1951
, 1971
1941
1936)
Barnes & Col lias, 1958
Coote, 1964
Fonselius, 1969
(Estimated from 1957-
1967 Data)
Central Pacific
.0027-.0053
3000 meters
Adv.-diff. model
Munk, 1966
-------
ou
to-
"e •
IW
o
to-
o-
ts
— •-— -
^~~--» 30-
•' "Xx s
\ ^20-
\ 1
'•\ '
]**>
r* A*
* .•
» • ^**
^<^
* •"' ^ • -
: , ^^: ' '
1/ff" '
jS.
00 10 20 30 40 50 60 70 0-Ljj IS54 55 56 57 58 5 ' ' r" ! '' ' ' ^"'
Y e ° r s Y e oM M " 6S M W M M ?<
Dissolved oxygen conlcnl below the haloclinc during the period
1902-1967
Mean values of phosphate in deep water layers (100. 200, 300
and 400 »/) in the Central Baltic Sea during the years 1938 and
1954-1970
Figure 4. Baltic 0?-PO, Relationships
(From Fonselius, 1972)
-18-
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one, there is a corresponding increase in the sampling
frequency and spacing of samples to effect these
observations. A few oxygen observations observed over a
year will exhibit trends on the regional problem on a Baltic
scale but weekly (or shorter) sampling may be required for
estuarine and nearshore monitoring. These broad statements
can be related to a spectrum of time scales necessary to
achieve optiiral monitoring operations. They will in all
likelihood closely resemble the spectrum of driving forces
inherent in any given environment—estuarine to oceanic.
-19-
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SUMMARY AND CONCLUSION
Depending on the goals of the investigator, pollution
problems can be viewed from space scales ranging from
microcosmic to world. Time scales span the range from
seconds to centuries. A monitoring program should seek to
minimize equipment requirements, sampling frequency and the
number of sampling locations. This will be determined by
the type of waste input and by the physical, chemical and
biological processes affecting the concentration and
distribution of the pollutant. For a single parameter,
instrumentation requirements, sample frequency and location
may be optimized—if two or more pollutants are to be
monitored it is not necessarily true that a single sampling
philosophy will satisfy both needs. It is suggested that
modeling may be a valuable adjunct to a monitoring program
in the design stage and as a concomitant activity.
NOTE ADDED:
Boyle (3) noted that there was a typographical error in
the Meadows {op. cit.) world model computer program. He
stated that the error was responsible for the pollution
crisis of the model and concluded that "affluence without
restrictive social policies is apparently attainable."
Meadows and Meadows (8) reported that the error did indeed
exist but had only a small quantitative effect on the
published results. The error was not responsible for the
pollution crisis mode; its removal does not stabilize the
model system and the conclusions are unaffected by the
numerical change.
-20-
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REFERENCES
1, Barnes, C. A., and E. E. Collias. 1958. Some
considerations of oxygen utilization rates in the
Puget Sound. J. Mar. Res. 17:68-80.
2. Bendat, J. s. and A. G. Piersol. 1966. Measurement and
analysis of random data. John Wiley and Sons, Inc.,
New York. 390 pp.
3. Boorman, S. A. 1972. Science 176(4038).
4. Boyle, T. J. 1973. Nature 245, 127.
5. Cohen, J. 1971. Science 172: (675).
6. Coote, A. R. 1964. Physical and chemical study of
Tofino Inlet, Vancouver, B. C. M. Sc. Thesis, U.
Brit. Col. Vancouver, 74p.
7. Fonselius, S. H. 1969. Hydrography of the Baltic Deep
Basin III. Fish. Bd. Sweden, Ser Hydrogr. 23. 97pp.
8. Forrester, J. W. 1971. World dynamics. Wright-Allen,
^ Cambridge.
9. Harrison, N. L., O. L. Loucks, J. W. Mitchell, D. F.
Parkhurst, C. R. Tracy, D. G. Watts, V. J.
Yannacone, Jr. 1970. Science 170(503).
10. Keeling, C. D., and B. Bolin. 1968. The simultaneous
use of chemical tracers in oceanic studies. II. A
three-reservoir model of the North and South Pacific
Oceans. Tellus XX<1):17-54.
11. Lerman, A. 1971. Time to chemical steady-states in
lakes and oceans. Adv. in Chem. series. No. 106.
Nonequilibrium systems in natural water chemistry.
pps. 30-76.
12. Meadows, D. H,, D. L. Meadows, J. Randers and w. W.
Behrens III. 1972. The limits to growth. New
American Library. 207 pps.
13. Meadows, D. H. and D. L. Meadows. 1974. Nature 247,
97.
14. Munk, W. H. 1966. Abyssal recipes. Deep-Sea Res.
13:707-730.
-21-
-------
15. Riley, G. A. 1951. Oxygen, phosphate and nitrate in
the Atlantic Ocean. Bull. Bingham ocean. Collect.
1J:126pp.
16. Rossby, C. G. 1936. Dynamics of steady ocean currents
in the light of experimental fluid mechanics. Pap.
Phys. Ocean Meteor 5{l):U3p.
17. Salerno, J. 1973. Sensitivity in the world dynamics
model. Nature: 244:488-492.
18. Sholkovitz, E. R.f and J. M. Gieskes. 1971. A
physical-chemical study of the flushing of the Santa
Barbara Basin. Limn, and Ocean. XVI(3):479-489.
19. Shubik, M. 1972. Science, 174(4013).
20. Sverdrup, H. U., and R. H. Fleming. 1941. Waters off
the coast of Southern California, March to July
1937. Bull. Scripps Inst. Ocean. 4:261-378.
21. Thomann, R. V. 1972. Systems Analysis and Water
Quality Management. Env. Res. and Applic., Inc.
N.Y. 286 pp.
22.. Walsh, J. J. 1972. Science 176(4038).
-22-
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PROBLEMS IN MEASURING
TURBIDITY AS A WATER QUALITY PARAMETER
R. W. Austin*
1.0 INTRODUCTION
With the accelerated concern for the protection of our environment, in-
cluding the quality of our lakes, streams, and coastal waters, we should assure
ourselves that we are making maximum use of the methods available for monitor-
ing the quality of our water resources. This does not necessarily mean a per-
petuation of methods and standards generated for and borrowed from other fields.
There is merit at this point in time in stepping back and taking a critical
look at the state of the art of optical measurements as they are currently ap-
plied to water quality and the potential which optical measurements have for
providing additional useful information.
The presently popular optical measurement for the purpose of determining
water quality is turbidity. We should ask ourselves, why turbidity? What
does it imply as to water quality? We have come to associate clarity of water
with purity and in some respects, correctly so. In large bodies of water, as
for example, lakes and oceans, we permit the adjective "blue" as an added de-
scriptor of pure water. The implication here is that departure from these
conditions means the presence of either dissolved material causing absorption
or suspended material causing, primarily, scattering. By their presence these
materials cause the water to lose the clear "water-white," or in large quanti-
ties, characteristic blue appearance we tend to associate with water in its
purest state. Thus, green Water may be due to the absorption of blue light by
dissolved material such as yellow substance, or "gelbstoff," attributed to
decomposition products found in the biologically productive areas of the oceans.
Or the green may be due to reflectance of the chlorophyll-bearing phytoplankton
found in these areas. In coastal or estuarine regions, where there may be
*Visibility Laboratory
Scrlpps Institution of Oceanography
University of California, San Diego
San Diego, California 92152
—23—
-------
significant quantities of terrigenous material suspended in the water, the
lack of clarity may be due mostly to the scattering of light by the suspended
silts or clays. It should be remembered, however, that these materials absorb
light as well as scatter, and they do this selectively, that is, they are
colored, in general, and their color varies with the type of material involved.
Moreover, in estuarine waters there is frequently material in solution that
absorbs light and thereby affects the appearance of the water.
Thus, the appearance of water is essentially determined by the nature of
the suspended and dissolved material which it carries, for these materials con-
tribute to the physical processes of scattering and absorption, both of which
are involved in the subjective matter of the appearance of a hydrosol.
However, we should not be concerned solely with the subjective appearance
of the water, although that may be a matter of distinct importance under some
circumstances. Rather, we should seek to use the optical properties of the
water as indicators of its purity or the degree of contamination by undesired
material. Hopefully, by the physical measurement of some optical property or
properties, we can obtain an indication of the type and amount of certain for-
eign materials present in a sample of water--if not on an absolute basis, at
least on a relative scale.
We generally accept this lack of clarity in water as turbidity in the
subjective sense. However, we do not all seem to accept the same physical defi-
nition for turbidity. Workers in colloidal chemistry, for example, carefully
define turbidity as identical with the scattering coefficient alone, intention-
ally excluding the absorption effects from the definition (Kirk Othmer, 1969).
Hodkinson, on the other hand, writing in Aerosol Science (Davies, 1966), defines
turbidity as the product of the extinction coefficient and the path over which
it is measured. Van de Hulst (1957) uses turbidity interchangeably with his
extinction coefficient which is the sum of the absorption and scattering coef-
ficients.
In the current literature of the water treatment and water quality field
there also seems to be some ambiguity. In the Water and Water Pollution Hand-
book, Volume 4, turbidity is equated to the scattering coefficient alone. Yet
in Standard Methods for the Examinations of Water and Wastewater, 13th edition,
-24-
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the statement is made, "Turbidity should be clearly understood to be an expres-
sion of the optical property of a sample which causes light to be scattered
and absorbed rather than transmitted in straight lines through the sample."
Thus, we find in all these definitions recognition that turbidity is re-
lated to scattering, but we do not find agreement on the necessity to include
the effects of absorption when measuring or defining turbidity. We will see
that significant information may be neglected by excluding absorption. We will
also describe some measurement techniques used in optical oceanography that
might be usefully applied to coastal pollution monitoring.
2.0 VARIABILITY OF OPTICAL PARAMETERS
Figure 1 shows an Apollo IX photograph of the eastern United States coast-
line from about the Norfolk/Cape Henry area at the extreme north to Cape Lookout
at the south, an airline distance of 142 nautical miles. Cape Hatteras is about
in the center. At various points along the islands that form the barrier be-
tween Albermarle and Pamlico Sounds and the ocean, we see breaks or inlets with
sediment-laden, turbid water flowing outward and along the coast. We can intuit
something about the direction and relative concentration of the sediments from
the reflectance pattern which the photograph presents. We should note, however,
that the spatial variability points up the complexity of monitoring water qual-
ity from fixed stations. We see here only a two-dimensional representation at
an instant in time. We know also that there are major vertical variations of
equal or greater magnitude and that the whole three-dimensional pattern will
change with tide, storms, rainfall, season, etc.
There are several points that should be emphasized. First, the reflec-
tance of the water is a result of the combined effects of scattering and absorp-
tion: if there is no scattering, the water would appear black regardless of
the absorption; if there is no absorption, scattering due to small particles or
molecules would result in the blue reflectance of clean water, and scattering
due to particles much larger than the wavelength of light would result in an
essentially white-appearing water. It is only when both scattering and absorp-
tion are present that the water can have the full gamut of appearances.
-25-
-------
-
Figure 1. Apollo IX Photography of Eastern United States Coastline
-26-
-------
A second point is that the coastal and estuarine waters are highly variable
both spatially and temporally. This is a fact of nature that affects all types
of monitoring of these waters, not just optical sensing. It may require a dis-
tinct cliange in approach for those who wish to apply the techniques common to
water and waste water examination where a single measurement of a well-mixed
medium may suffice.
A third point, with which most who have tried to perform observations from
the deck of a surface vessel will agree, is that the degree and extent of the
spatial variability is seldom apparent to the observer at the ocean surface.
Thus, the selection of the number and location of sampling stations can be
greatly aided if an aerial reconnaissance of the general area can be performed.
3.0 DISCUSSION
3.1 Transmittance
Let us forget about the word "turbidity" for a moment and review some of
the fundamentals of light transmission through water to see how we might use
the concepts.
As shown in Fig. 2, imagine a beam of essentially parallel light with a
Figure 2
radiance, N_ at x = 0, passing through a medium containing both scattering and
absorption. Radiance will be lost as the beam passes along a path of length £
in tiie medium. The radiance, N«, remaining at £ can be shown to be related to
the input radiance, N , by the expression
N- Ne"(a+ s) (1)
-27-
-------
where a and s are the absorption and scattering coefficients, respectively,
for the medium. The transmittance , T^, for the path £ is simply
_
" e '
It is this transmittance that is measured by a properly designed transmissometer.
We see that it is dependent on both absorption and scattering and further that
we can find the magnitude of the sum of these two coefficients by taking the
natural logarithm of Eq. (2). Thus,
a + s = T An T (3)
* *£
Note that the dimension of these coefficients is reciprocal length. For con-
venience, the sum of the two coefficients is given the specific designation of
volume attenuation coefficient, for which we will use the symbol a. Thus,
a = a + s. (4)
It should be recognized that in general, a, s, and therefore a are functions
of wavelength.
Figure 3 shows a particular mechanization of the transmissometer concept
which .was described by Petzold and Austin (1968) . It utilizes a 1-meter water
path length obtained by folding the instrument at its midpoint by means of a
prism. A 19-millimeter diameter projector beam falls within a slightly larger
sensitive volume of the receiver whether the instrument is in air or in water.
Therefore, the transmissometer may be calibrated against the air path whose
transmittance is assumed to be 100 percent for 1 meter. A correction is intro-
duced, however, to accommodate for the decrease in Fresnel reflectance losses
at the four wetted surfaces when they are immersed. Thus, for this instrument
the sensitivity of the receiver can be adjusted until the indicated air trans-
mittance is 85.5 percent. With this calibration procedure an instrument em-
bodying the proper optical design concepts vrill read the true transmittance of
the v/ater path.
-28-
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VO
o
8
10
g
H«
m
ro
§
o>
rt
n>
o
•o
rt
H-
n
(B
CO
o
o>
rt
H-
O
PORRO PRISM
A
OBJECTIVE LENS
& APERTURE
FIELD STOP
FIELD LENS
LAMP
LIGHT PIPE
SILICON
DETECTOR
WATER PATH
OBJECTIVE LENS
& APERTURE
SHUTTER
-------
In this instrument provision has been made for a portion of the lamp out-
put to be directed to the detector without passing through the water path.
This can be done remotely on command by the operator at any time. Thus, if a
record of this reference reading is made immediately after calibration, the in-
strument sensitivity can be maintained at the calibration level by adjusting
the receiver gain to obtain a repetition of the reference reading.
In any transmissometer design the projector and receiver beams must have
finite angular dimensions. As a consequence, there is a finite amount of flux
included in the measurement which has undergone scattering. Conceptually, the
flux measured by the receiver should be only that which has traversed the path
without being absorbed or scattered. Therefore, the scattered component which
ls_ included in the measurement represents an error. By careful attention to the
design of the optical system this error may be reduced to a point where it is
small compared to the temporal and spatial fluctuations encountered in most
field measurements. The error present, hoxvever, will depend upon the relative
amounts of scattering and absorption of the water and upon the manner in which
the scattering function varies with angle. This particular instrument has been
checked against other transmissometers of good design and against other methods
for determining the attenuation coefficient for a large variety of water condi-
tions. The agreement bettveen the various determinations has been excellent,
that is, within the experimental error, for most cases.
It is important in any optical measurement to be specific about the wave-
length of the radiation used. This is particularly true in transmissometer
measurements because of the marked change in the absorption coefficient with
wavelength. The spectral passband of the instrument can be restricted very
simply by the use of interference filters, by glass or gelatin absorption fil-
ters, or through the use of spectrally monochromatic sources such as lasers.
The usual method for general-purpose instruments such as the one shown here
is to use a combination of one or more gelatin and glass filters to obtain a
moderately narrow spectral passband. In this instrument a filter wheel is
provided to allow the investigator to select the wavelength. In addition, an
infrared blocking filter is used because the high infrared sensitivity of the
-30-
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silicon detector, the large output from the incandescent lamp in the near IR,
and the IR leakage of most dyed gelatin filters combine to give an unwanted
IR response when the instrument is calibrated in air.
;
Figure 4 shows an instrument system based on the concepts just described.
The transmissometer unit is shown at the bottom. The cylindrical enclosure at
the left contains the lamp, the detector, the projector and receiver optics, a
pressure transducer for measuring instrument depth, a thermistor probe for
water temperature measurement, and the necessary electronics. The porro prism
for folding the beam is at the right. The underwater unit is connected to the
surface by a multiple conductor cable. The deck control unit on the right pro-
vides the necessary power for operation of the lamp and electronics in the
underwater unit, plus providing the various control and signal conditioning
functions required for operation. The digital panel meter can display water
transmittance, temperature, instrument depth, or lamp current. Signals are
also provided to a two-pen, x-y recorder shown at left, on which water trans-
mittance and temperature may be recorded simultaneously against depth.
Other versions of this instrument have been built with provisions for
changing the spectral filter by remote command from the surface, for optional
display and recording of attenuation coefficient in lieu of the transmittance,
and for a printer connected to the digital displays to obtain a listing of the
observations.
Figure 5 is an example of the type of record obtained with an instrument
of the type just described. This curve sheet presents profiles of transmit-
tance and temperature as a function of depth taken from an area a few miles
south of the tip of Santa Catalina Island off the California coast. The
region is near the convergence of currents flowing down the east and west sides
of the island and is known to fishery biologists as an area of high biological
activity. We found the chlorophyll level to be moderately high for the region,
and the water color was a very definite green. The temperature profile shows
a number of small inversions near the surface and one at 23. meters probably due
to the mixing of the east and west slope water. Below 37 meters the temperature
uasconstant, indicating a mixed uniform water. The transmittance profile shows
-31-
-------
u>
to
B
ID
*•
o-
o a
n 3
a a
rt (D
O H
rt
O (T>
i-h i-l
n H
3* i-l
ID pi
3
CO CO
n 3
M H-
H- en
•a to
T3 O
01 il
M rt
3 (T)
CO l-(
rt
H- to
rt ><
C CO
ft rt
H- (D
O ft)
I-h <
.D
- J r- '
o o
O
so cr
n v;
w
"3 rt
•< n
•
•
Q
-------
Fig. 5. Transmissometer Profile. Southern California Coastal Waters.
VISIBILITY LABORATORY
UNIVERSITY OF CALIFORNIA
0 J>
DEPTH (METERS!
Fig. 6. Transmissometer Profile. Northern Gulf of California.
-33-
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1.0
o
o
o
o
I—
:D
LLJ
o
.5
.10
.05
BLUE-GREEN (VIS LAB)
HULBURT
CHESAPEAKE BAY
HULBURT DISTILLED WATER
CLARKE AND JAMES (1938)
DISTILLED WATER
400
500
600
680
WAVELENGTH (NANOMETERS)
Fibure 7. Volume Attenuation Coefficient As A Function of Wavelength
For Various Waters
-34-
-------
the same features and others as well. The lew transmittance water (40 percent
per meter) near the surface represents a moderately turbid condition, probably
a resulc of a large phytoplankton population with its associated high chloro-
phyll level and green color. At about 12 meters the transmittance started a
steady increase which was interrupted by a tongue of turbid water intruding
between 23 and 33 meters with a sharp minimum at 27 meters. The same feature
can be seen in the temperature profile, and we might indulge in the conjecture
that the combination was due to a wedge of the warmer, more turbid surface
water. By 37 meters the transmittance was moderately high and uniform below
this depth.
A second profile--this one taken in the northern part of the Gulf of
California near the mouth of the Colorado River--is presented in Fig. 6. It
sho\\rs a different structure with water of intermediate turbidity overlying
heavily silted turbid water at the bottom. The noise-like trace in the 10 to
20 meter region indicates the presence of large particulates, probably zooplank-
ton passing through the light beam. Both of these profiles were taken at a
wavelength of 530 nanometers.
In the vicinity of Santa Catalina Island, measurements were made at five
wavelengths on each station. The spectral changes in the attenuation coeffi-
cient computed from the transmittances in the upper 5 meters of water for three
of these stations are shown in Fig. 7. The \v*aters varied from a very turbid
green productive water shown in the top curve, to the more usual blue-green
coastal water in the middle curve, to the blue almost oceanic water in the
middle of the graph. These may be fairly typical spectral curves for water
that is neither polluted nor silt-bearing. However, we lack adequate data of
this type to make definitive statements about the spectral attenuation coeffi-
cient signatures of various types of water. The spectral transmissometer,
capable of rapidly shifting ivavelengths, is just.now becoming available as a
practical field instrument. The answers it can provide may prove to be very
helpful in monitoring coastal waters. It is, in fact, a kind of submersible
spectrophotometer capable of providing multidimensional spectral data in situ.
For reference, a curve of the attenuation coefficient for a sample of
Chesapeake Bay water reported by Hulburt (1945) is shown near the top of the
-35-
-------
JE
a
O
UJ
O
CJ
UJ
O
BLUE-GREEN VIS LAB
HULBURT
CHESAPEAKE BAY
HULBURT DISTILLED WATER
CLARKE AND JAMES (1938)
DISTILLED WATER
400
500
600
680
WAVELENGTH (NANOMETERS)
Fig. 7. Volume Attenuation Coefficient As a Function
of Wavelength for Various Waters.
-36-
-------
graph. These measurements were performed in the laboratory a day after obtain-
ing the sample. The difference in shape may be attributed in part to the
greater spectral resolution used in the laboratory measurement and perhaps due
in part to changes in the sample after removal from the bay. There is also the
i
distinct possibility that the steeper upxvard slope for the shorter wavelengths
is due to the greater concentrations of dissolved organic decomposition prod-
ucts found in the bay than were present in the coastal water. The rise in the
curve between 580 and 600 nanometers is an absorption band of water found in
most measurements if the spectral resolution and sensitivity are adequate.
The lower curve attributed to Hulburt is based on carefully distilled
water and represents one of several attempts by various investigators to achieve
a measure of the attenuation coefficient for pure \\rater. The approximate lower
limit of attenuation that has been'measured in the spectral region of maximum
transparency, attributed to Clarke and James (1939), is shown at the very bot-
tom of the figure. Recent measurements at the Visibility Laboratory using
laser light in highly filtered fresh water have given results close to the
lower curve.
3.2 SCATTERING
A common measurement technique today in water quality monitoring is neph-
elometry. As usually practiced, the scattering from a sample is measured at
a single angle or small range of angles. Unfortunately, the common practice
for the calibration of these instruments is to use an arbitrary scale devised
originally for a particular form of extinction photometer known as the candle
turbidimeter, or more specifically, the Jackson candle turbidimeter. The units
are called "parts per million turbidity, silica scale," or simply, Jackson
Turbidity Units (JTU's). The calibration resides in the form of a table of
distances in centimeters versus turbidity units. Table I, taken from Standard
Methods, is this calibration. The distances are the depths of the turbid water
Jackson found were required for the image of the flame of a standard candle to
be lost in the glow field caused by the flame's forward scattered light when
-37-
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Table 1
GRADUATION OF CANDLE TURBIDIMETERt
Light Path*
cm
2.3
2.6
2.9
3.2
3.5
3.8
4.1
4.5
4.9
5.5
5.6
5.8
5.9
6.1
6.3
6.4
6.6
6.8
7.0
7.3
7.5
7.8
8.1
8.4
8.7
9.1
9.5
9.9
10.3
10.8
Turbidity
Units
1000
900
800
700
650
600
550
500
450
400
390
380
370
360
350
340
330
320
310
300
290
280
270
260
250
240
230
220
210
200
Light Path*
cm
11.4
12.0
12.7
13.5
14.4
15.4
16.6
18.0
19.6
21.5
22,6
23.8
25.1
26.5
28.1
29.8
31.8
34.1
36.7
39.8
43.5
48.1
54.0
61.8
72.9
Turbidity
Units
190
180
170
160
150
140
130
120
110
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
* Measured from inside bottom of glass tube.
t Standard Methods for the Examination of Water
and Waste Water. Thirteenth Edition.
-38-
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viewed through the particular silica suspension he used. Needless to say, it
has been difficult to reproduce his results with other suspensions, so that
the calibration standard today is actually the table shown here. If one has an
instrument of the design Jackson prescribed, the turbidity for any arbitrary
suspension may be found in JTU's by determining the extinction distance and re-
ferring to the table. The numbers refer only to the equivalent parts per
million of Jackson's silica suspensions and not to any standard physical unit.
The particular silica suspension used originally is probably of no interest to
present-day problems. But the unit or its derivatives persist.
In 1926 Kingsbury and Clarke developed a suspension called Formazin which
could be prepared in accordance with their formulation with a reproducibility
of ±1 percent of the candle turbidimeter reading. Formazin is presently used
as a calibration agent for various types of turbidity measuring instruments,
and the current trend is to call the'unit the Formazin Turbidity Unit or FTU.
Nevertheless, it should be recognized that the standard still goes back to the
candle turbidimeter using the calibration given in the table for parts per
million silica and not, for example, parts per million Formazin.
It is a well-recognized fact that instruments of different optical design
when calibrated against the same turbidity standard may yield widely disparate
answers if used to measure a sample containing different turbidity-generating
material. The basic problem is that the physical principles invoked in the ex-
tinction of the light source are not the same as those used in the single angle
scattering measurement.
This is not to say that useful information cannot be obtained from scatter-
ing measurements or nephelometry. Properly designed instruments calibrated in
well-defined and accepted physical units can be built and may provide useful
data for water quality monitoring. The techniques would be in concert with
those used in optical oceanography, and the data could be compared between in-
vestigators using different instrumentation and with the existing and growing
body of oceanographic data.
The basic scattering measurement may be conceptualized with the aid of
the following development. See Fig. 8. Imagine an irradiance, H , incident
-39-
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H
A*
£. i —
i
— k
N
^
1_
dA
Figure 8
on a small volume, dV, \vhich, for the moment, we will imagine has a projected
area, dA, normal to the direction of propagation, and a length d& along the
direction of propagation. If the volume contains a scattering medium and is
viewed at some distance great compared to its dimensions, it will have an in-
tensity, dl(9), proportional to the incident irradiance and the volume. The
constant of proportionality is called the volume scattering function or VSF,
here denoted 0(0). Thus,
dl(9) = 0(9) • H • dV
or
a (9)
= 1 . dl(9)
(5)
(6)
We can rearrange the terms and obtain
0(9) -^ •
dl(0)
dfl
(7)
We see that HQdA is the power incident on the volume and dl(9) • dfi is the
power leaving the volume in a sol id angle dfl. If the dimensions are kept suitably
small, we may write
0(9) =
0
(8)
-40-
-------
which is the fraction of the power scattered from a beam of light into a small
solid, n, in the direction 9, per unit path length. The dimensions of a(Q) are
reciprocal length times reciprocal solid angle (usually meter"1 x sleradians"1).
If we now integrate the VSF over all angles around the volume, we obtain
the volume scattering coefficient, s, which then represents the total fraction
of the power removed from a beam of light by scattering in any direction, per
unit path length. Thus,
a(0) sin 8 d0 . (9)
Figure 9 shows an optical schematic of an instrument described by Petzold
(1972) which measures the VSF in situ at angles from 10 to 170 degrees. The
projector and receiver have rectangular beams which intersect at the center of
rotation of the projector. The volume of intersection, intersection angle, pro-
jector power, and receiver angular field are all known. The output of the re-
ceiver, therefore, can be converted into a(0) in absolute physical units.
In Fig. 10, six VSF curves obtained with this instrument are shown. The
two additional points shown at very small angles were obtained with another in-
strument capable of measurements at 0.085 to 0.34 degrees from the direction of
propagation. The vertical scale covers eight log cycles and, as a consequence,
some' of the subtleties of the scattering curves tend to be suppressed.
The integration of these volume scattering functions has been performed,
and the values of the resulting scattering coefficients are given in the upper
right of the figure along with measured volume attenuation coefficients for the
same stations. The latter were obtained with the transmissometer described
previously. In general, the VSF's have much the same shape in the forward di-
rection from, say, 90 to 10 degrees. We find changes in the location of the
scattering minimum shifting from 95 degrees for the clearest water shown here
to 150 degrees for the turbid water in San Diego Harbor. In general, the mag-
nitude and shape of a(0) show the greatest variability in the backward direction.
-41-
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2
Prism and
Window
v«
.
0.1 mm Height of 1">3 5 mm
Projector Beam
I
, 1_
| -T— •
l 24.18 mm Height of
\ Receiver Field of View
\
Center) ine of Rotation (Projector)
and Location of Image of Receiver
Field Stop in Water /
/
Aperture and Location of Projector
Field Stop Image in Water
Field Len
\
\
\
l
K
i
i 1
Ar
/ ;
( \
\
\
i
i
!
«— ^<
i
f
1
1
i
Lamp, 100W, Quartz j 1
Iodine Cycle *~
PROJECTOR
J |
1
1
1
1 .
1
1
\
— N.
L_
|
— - ^"* f- Objective
' Lens
1
'
'
('
1
/ Field Stop
~>^
\
1 Exit Pupil and
>f Photocathode
A L
1 1
Photomultiplier Tube
RECEIVER
Sample Volume
3.5 mm Width of
Projector Beam
1
7.57 mm Width of Receiver
Field of View
Figure 9. General Angle Scattering Meter Optical System
-42-
-------
6, ANGLE (DEGREES)
Figure 10. Volume Scattering Function of Sea Water For Various Locations
-43-
-------
However, because the relative magnitude of a(0) in the backward direction is
very much smaller than in the forward direction, the affect of these changes
on the total scattering coefficient, s, is negligible except for the clearest
waters. For example, the rear hemisphere of the scattering function in water
of intermediate to high turbidity contributes typically between 1.2 and 2.5
percent to the total s. The bottom curve shown in Fig. 10 for the moderately-
clear water in the Tongue of the Ocean has 4.5 percent of its scattering to
the rear. Measurements on highly filtered water have shown values as high as
15 percent, and for perfectly clear \vater with only molecular scattering, the
value has a theoretical limit of 50 percent.
Another indicator of the relative importance of the VSF's forward lobe is
the angle required to obtain 50 percent of the total scattering, which may be
called the median scattering angle. Computations performed on available data
from a variety of natural and filtered waters show that this angle varies from
about 5 degrees for very turbid water down to about 2 degrees for intermediate
water with s = 0.27 m"1. It then increases again to larger angles as the water
gets clearer. In the clearest water for which we have data, the median scat-
tering angle is 19 degrees. Thus, for most coastal water the major part of
the scattering coefficient results from the very narrow, 2 to 5 degrees, for-
ward lobe of the VSF.
A corollary of this observation about the VSF is that if the object of
the measurement is to obtain a number relatable to the scattering coefficient,
then the measurement performed by the nephelometer should be at an angle close
to the forward direction. Figure 11 shows the affect of the selection of this
angle on the predictability of the total scattering coefficient. The figure
shows curves for the variability of the normalized volume scattering function,
a(wj, for a range of scattering coefficients of over 100 to 1. As the normal-
ized VSF is simply a(0)/s, it follows that if it is invariant with s, then
c(0) is proportional to s. As \ve see, the current practice of measuring a(8)
at 45, 90, or 135 degrees results in a large variability in the 0(0), whereas
the curve for a(5u) shows a much smaller dependence on the scattering coefficient.
-44-
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b
u.~
to
O
LLJ
.5
.10
.05
.01
.005
.001
I
.005 .01
0 = 5°
0(6) =
0(6)
I
.05 .10
.5
SCATTERING COEFFICIENT, s (nT1)
10°
45°
90°
135°
Figure 11. Normalized Volume Scattering Function
-------
Therefore, it is advisable to measure a(5°)--or even a(10°) if the design of
field-serviceable instruments would be easier and less expensive at 10 degrees--
in order to obtain the best estimate of the scattering coefficient, s.
Whereas these curves were computed from data obtained by the Visibility
Laboratory, other investigators have obtained the same result. Morel (1973)
has made extensive Mie scattering computations for various particle-size dis-
tributions and refractive indices, and he finds that the optimum angle is from
4 to 6 degrees. These scattering data which have been attributed to the Visi-
bility Laboratory have been obtained at a wavelength of about 520 nanometers.
The wavelength for the total scattering coefficient depends on the particle
sizes involved. For pure water with only molecular scattering, the total
scattering coefficient will vary inversely as the 4th power of the wavelength,
and for a polydisperse system having the range of particles found in the ocean,
the coefficient will vary as A"1 to A"2.
3.3 THE ROLES OF SCATTERING COEFFICIENT AND VOLUME ATTENUATION COEFFICIENT
We have developed the concept of beam transmittance as a means of deter-
mining the sum of the absorption and scattering coefficients. We have called
this sum the volume attenuation coefficient, a. We have discussed the measure-
ment of the volume scattering function, 0(0), and the potential which exists
for deducing the total scattering coefficient, s, from such a measurement per-
formed at an appropriately small angle. Theoretically, all values of a and s
could exist in the various proportions necessary to make up the range of a
found in natural waters. In actual fact, the relative amounts of scattering
and absorption found in oceanic and coastal waters are somewhat restricted as
we see on the alpha-scattering diagram presented in Fig. 12. Data from all
measurements where simultaneous a and s were obtained have been plotted here
on a scale of log s versus log a. The parallel diagonal lines represent con-
tours of constant s/a ratio. The curves represent contours of constant absorp-
tion. The sprinkle of points indicates that for the oceanic and coastal waters
represented here the scattering accounts for the major part of the change in
attenuation coefficient as we go from clear water shown at the left of the
-46-
-------
UJ
O
o
.1
VOLUME ATTENUATION COEFFICIENT, a (nT1)
Figure 12. Plot of Scattering Coefficient Data Against Voluem Attenuation
Coefficient Measured Contemporaneously For a Variety of Waters.
All data obtained at a wavenlength of 520 nanometers.
-47-
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diagram to the most turbid water at the right. The absorption does change
somewhat, however, as a increases. These clianges should not be considered in-
significant. It is an artifact of this type of plot that the absorption scale
is greatly compressed in the region of the large s/a values that occur when the
attenuation is large. These data were obtained for a wavelength of 520 nanome-
ters. The picture would differ somewhat for other wavelengths where the absorp-
tion coefficient has a greater or lesser affect particularly for the clearer
waters.
3.4 THE RELATIONSHIP BETWEEN JTU AND a
We can attempt to relate the turbidity unit scale to the volume attenua-
tion coefficient, a, by the following empirical method. Duntley (1971) states
that, in his experiments with an underwater light source in lake water, the
envelope of the lamp disappears in the glow field generated by the forward
scattering from the light at about 15 attenuation lengths, i.e., r = 15/a,
where r is the distance through the water from lamp to observer at the extinc-
tion of the lamp image. Cousteau (private communication) has reported similar
findings in the Mediterranean Sea. Duntley repeated his test in a tank with
artificial scattering agents and found that the source disappeared at 16 at-
tenuation lengths, possibly due to the angular scattering properties of the
particular scattering agent used. In any event, we have no evidence that the
threshold distance is likely to depart appreciably from the 15-attenuation
length value in natural water of moderate to good clarity.
We will now apply that value to the much more turbid water for which the
candle turbidimeter was designed and use the calibration values for the tur-
bidimeter given earlier. If the JTU or concentration of silicates is propor-
tional to the attenuation coefficient, then we might deduce from Duntley's
findings that
(JTU)k = a = ii
where k is the coefficient of porportionality and r is the threshold or extinc-
tion distance.
-48-
-------
Note that this is a large extrapolation from the type of water in which
the observations were made, however. The clearest water for which the candle
turbidimeter is calibrated has an extinction distance of 72.9 centimeters (or
0.729 meter), and the corresponding attenuation coefficient would be a =
20.6 m"1 or an attenuation length of I/a- 0.05 meters! Such waters are seldom
encountered except in very dirty bays and harbors or in silt-laden rivers. Con-
ventional transmissometers with a path length of 1.0 meter are, for practical
purposes, incapable of measuring a signal through such water. An instrument
with a 10-centimeter path (0.1 meter) could, however, make a reasonable as-
sessment of the transmittance of such water and even of water having a somewhat
higher attenuation coefficient.
Figure 13 shows the calibration for the candle turbidimeter, i.e., tur-
bidity units plotted against threshold distance in centimeters. The plot has
been made on a log-log scale as the range of the variables extends over ap-
proximately 1.5 log cycles. On such a plot an inverse relationship betxveen
JTU and r would be a straight line with a slope of minus one. Such a line is
shown (dotted) for the relationship: JTU = 2000/r (where r is in centimeters).
It can be seen that the turbidimeter calibration curve has a greater slope than
-1 and, in fact, appears to have a change in slope at about 30 centimeters.
Thus, the simple inverse relationship does not hold precisely, although the
reason does not appear obvious unless it is caused by multiple scattering. It
may be that the straight line inverse relationship is a satisfactory approxima-
tion to the true extinction distance versus "parts per million silica," con-
sidering the errors and precision of the procedure.
If we use the straight line approximation shown in Fig. 13 and measure
the distances in meters vice centimeters in order to have a in the more con-
ventional units of I/meter, then we find that
a = 0.75 JTU (m'1).
We can debate whether the constant should be 1.0 or 0.5, but it is more likely
to depend upon the nature of the scattering properties of the particular
-49-
-------
1000
500
5. 200
CO
h-
z
100
00
CC.
O
50
20
10
JTU = 2000/r
JTU vs. r From Table I
10
20
50
100
THRESHOLD DISTANCE, r(cm)
Figure 13.' Plot of Jackson Turbidity Units, JTU, Against Threshold or Extinction
Distance, r, in Centimeters. Solid line from Table I. Dotted line
is a plot of JTU = 2000/r.
-50-
-------
suspended material and the amount of absorbing material present than upon the
niceties of any theoretical development. It is unlikely, in fact, that a
single constant relating JTU and a can be applied to various suspensions apt
to be encountered in the use of the candle turbidimeter.
To perpetuate the use of the JTU or FTU in measurement problems of coastal
and offshore monitoring would seem, however, to be a path of dubious wisdom.
The numbers so generated relate only to other data obtained with instruments
of identical design and have little or no physical significance. On the other
hand, instruments are available which are capable of measuring the optical
variables encountered in marine waters and of providing data in units which are
accepted and understood by workers in the field of optical oceanography through-
out the world.
4.0 SUMMARY
Water turbidity, although due primarily to the presence of suspended mate-
rial with its concomitant scattering, is also affected by absorption. The mea-
surement of turbidity by image extinction methods such as the candle turbidi-
meter recognizes the effects of scattering (particularly forward scattering)
and absorption. Most of the presently used nephelometric techniques for as-
sessing turbidity in water quality work ignore the effect of absorption and
make a turbidity determination proportional to the volume scattering function
at some large angle (or range of angles) from the direction of propagation. In
addition, these instruments are calibrated in units (JTU's or FTU's) that have
little physical significance and can only be correlated to measurements made
with instruments of identical optical design that have been calibrated against
the same standard suspension.
Techniques and instrumentation exist for determining the volume attenua-
tion coefficient, a, in widely accepted absolute physical units by using trans-
missometers of proper optical design and water path length. This attenuation
coefficient is probably closely related to the extinction distances found by the
candle turbidimeter but not necessarily to the JTU or FTU determinations obtained
-51-
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by nephelometry. These latter determinations ignore the effects of absorption
and are usually made at large angles where the correlation between volume
scattering function, a(0), and total scattering coefficient, s, is poor, as
shown in Fig. 11. If scattering measurements are made at around 5 degrees
from the forward direction, however, the correlation between a(9) and s is
better and nephelometry can provide a good estimate of s. If a sufficiently
large body of data such as that shown in Fig. 12 can be obtained for the range
of variables of interest, it might lead to an empirical path,from a(0) to s
to a. If this is found to be the case, a useful technique may be developed
whereby small angle scattering measurements can be related to the total attenu-
ation coefficient of the medium. For some situations scattering measurement
may be preferred as when waters having a large range of turbidities must be
measured with a single instrument.
For all such optical measurements, the instruments should be calibrated
in accepted, absolute physical units and careful attention given to the spectral
range of the radiation involved. Both absorption and scattering are wavelength
dependent and the results of the mesurements and the conclusions drawn may de-
< - -v-*..*
pend upon the spectral response of the instruments used. Furthermore, a poten-
tially significant characteristic for the study of water quality is neglected
if the spectral dependence of these coefficients is not utilized.
-52-
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BIBLIOGRAPHY
Beyer, G. L., "Turbidimetry and Nepehlometry," in Kirk-Othmer, Ed., Encyclo-
pedia of Chemical Technology (Interscience Publishers, New York, 1969),
2nd ed., Vol. 20, pp. 738-748.
Clarke, G. L. and H. R. James, "Laboratory Analysis of the Selective Absorption
of Light by Sea Water," J. Opt. Soc. Am. 29_ (February 1939), pp. 43-55.
Duntley, S. Q., "Underwater Lighting by Submerged Lasers and Incandescent
Sources," University of California, San Diego, Scripps Institution of
Oceanography, Visibility Laboratory, SIO Ref. 71-1 (June 1971).
Hodkinson, J. R., "The Optical Measurement of Aerosols," in C. N. Davies, Ed.,
Aerosol Science (Academic Press, Inc., New York, 1966), pp. 287-357.
Hulburt, E. 0., "Optics of Distilled and Natural Water," J. Opt. Soc. Am. 35_
(November 1945), pp. 698-705.
Morel, Andre", "Diffusion de la Lumiere par les Eaux de Her. Resultats Experi-
ment aux et Approche Theorique," in AGARD Lecture Series No. 61_on Optics
of the Sea (Interface and In-Water Transmission and Imaging), (Technical
Editing and Reproduction Ltd., London, August 1973), pp. 3.1-1 - 3.1-76.
Petzold, T. J. and R. W. Austin, "An Underwater Transmissometer for Ocean Sur-
vey Work," University of California, San Diego, Scripps Institution of
Oceanography, Visibility Laboratory, SIO Ref. 68-9 (April 1968).
Petzold, T. J., "Volume Scattering Functions for Selected Ocean Waters," Univer-
sity of California, San Diego, Scripps Institution of Oceanography, Visi-
bility Laboratory, SIO Ref. 72-78 (October 1972).
-53-
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BIBLIOGRAPHY (Cent.)
Standard Methods for the Examination of Water and Wastewater, 13th ed., M. J.
Taras, A. E. Greenberg, R. D. Hoak and M. C. Rand, Eds., American Public
Health Assn., New York, Chap. 100, Sec. 163, 163A, -163B, and 163C, pp.
349-356.''
Van de Hulst, H. C., Light Scattering by Small Particles (John Wiley and Sons,
Inc., New York, 1957).
Water and Water Pollution Handbook, Vol. 4, Leonard L. Ciaccio, Ed. (Marcel
Dekker, Inc., New York, 1973), Chap. 27, pp. 1431-1686.
-54-
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SURFACE SLICKS AND FILMS -
A NEED FOR CONTROL
Walter F. Rittal]*
INTRODUCTION
Surface slicks and films, as natural phenomena, have been under
investigation for many years. LaFond (1969) has defined sea
surface slicks as "...streaks or patches of relatively calm water
adjacent to rippled water." This definition was adequate for his
purposes but too simplistic as a definition of a potential pollutant.
As research has progressed from the mechanisms of formation and
movement to their physical-chemical composition, a series of results
have been published that forewarn of potentially dangerous
concentrating effects at the air-water interface.
PURPOSE
The purpose of this document is one of emphasis--designed to
draw attention to the significance of surface accumulations.
Discussions will center on one specific non-natural input area,
namely that resulting from municipal waste discharges via outfalls.
The regulations governing such discharges will be briefly examined
to ascertain their ability to ensure adequate control and finally
the discussions will turn to current research efforts.
The areas to be discussed will include work that has attempted
to assess the significance of the problem. Methods applicable to
field sampling, analysis of constituents and quantitative control
will be put forth as candidate methods for the standardization.
THE PROBLEM
Garrett (1965) found that such surface layers are rich in fatty
esters, free fatty acids, fatty alcohols and hydrocarbons - a
* Pacific Northwest tnvironmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon
-55-
-------
finding that has been demonstrated repeatedly by subsequent
investigators. Harvey (1966) in studies both in California and
Hawaii found the number of algae and protozoa in a defined
surface slick to be some four times the concentration at a depth
of ten centimeters. This concentration factor of 4 was recently
demonstrated again by Selleck (1973).
Bacteria important to the public health have been found in these
surface layers, and as was demonstrated by Amies, (1956) films in
swimming pool isolate the bacteria from the chlorine of the water
below. Coliform concentrations as high as 1000 organisms per
100 ml have been reported for surface slicks near ocean outfalls.
These counts however have been shown to be directly related to
floating sewage type particles contained in the slick or film
layer.
The health hazard may not be strictly aquatic. Randall and
Ledbetter (1955) demonstrated that enormous quantities of nuclei
containing enteric and respiratory pathogens can be transferred
to the atmosphere from the surface films of activated sludge ponds.
This mechanism was recently used to explain the high incidence
of respiratory disease in Mexico in an area downwind of an
existing sewage treatment facility. Woodcock (1955) showed that
bursting air bubbles were possible mechanisms for this transfer
and from the works of others it has been shown that from 0.3 to
0.7 microgram of film material accompanies each microgram of sea
salt released to the atmosphere.
It has also been demonstrated by Riseborough (1968) that the
microorganisms that dominate this film layer concentrate pesticides;
a bioaccumulatory effect, the first of many such transfers possible
through the food chain. As research continues the complexity of
the problem becomes more obvious. Duce and Quinn (1972) have
-56-
-------
recently reported findings that reveal the surface layer to be
an effective concentrator of trace metals, with estuarine
concentrating factors* going as high as 50.
These factors help to show the general scope of the problem,
however, we have completely avoided the more complex areas of
carcinogenic and synergistic effects that may occur in this light
available zone. These concerns are real ones and they must be
considered in any monitoring program designed to be responsive
to the total system. It is quite common to see emphasis in
monitoring placed on the bulk water column and on one of the two
interfaces that exist - namely the sediment-water interface
with little or no recognition of the importance of the air-water
interface.
* The concentrating factor is defined as the ratio of the surface
layer concentration to the concentration at a specified depth
in the water column (often 10 cm).
-57-
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REGULATIONS
Under existing federal law each state is allowed to establish its
own water quality standards. The New Jersey, Alabama and California
laws will be discussed here.
NEW JERSEY
New Jersey designed its own regulations to comply with the federal ,
guidelines and adopted the following regulation for the control
of surface floatable solids, oil, grease, color, turbidity and
sett!cable solids: "None noticeable in the water or deposited
along the shore or on the aquatic substrate in quantities
detrimental to the natural biota. None which would render the
waste unsuitable for the designated uses."
ALABAMA
Alabama's regulations, apparently as a result of noncompliance,
were set by the federal government and contain only one specific
reference to floating materials. This reference is contained
in a definition of a minimum secondary treatment facility -
"...a facility which at design flow is capable of removing
substantially all floating and settleable solids..." These
standards reflect an attempt to prevent the discharge from
being readily apparent and it seems quite obvious that in these
regulations no specific quantitative control was established.
CALIFORNIA
California has maintained its reputation for unconventional
action and met the problem head on. Regulations were established
-58-
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that limit the hexane extractable content to levels felt to
preclude surface contamination. The standard specifies that the
concentration of grease and oil (hexane extractables) on the waters
o
surface shall not exceed 10 mg/m more than 50 percent of the
2
time, nor 20 mg/m more than 10 percent of the time. To cover
the area of floating particulates - levels were also established
that preclude concentrations of particles of wastewater origin
P
above 1.0 mg/m (dry wt) more than 50 percent of the time and 1.5
2
mg/m more than 10 percent of the time.
There is no doubt about the need for more quantitative regulations -
there is, however, serious doubts as to the effectiveness of
standards based on slick or film concentrations alone. The
objective here should not be only the prevention of visible
slicks, but rather the prevention of adverse concentration of any
material deemed harmful to one or more segments of the total
ecosystem. California has taken the first step by establishing
specific limits, providing a mechanism for additions and modifications
as our level of knowledge and experience grows.
-59
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OCEAN OUTFALLS
Municipal waste discharges via outfalls are not the only source
of slick and film materials but they are inputs that have been
studied. A stationary surface slick has been found to exist
above the enormous Hyperion outfall off Los Angeles. This slick,
figure 1, was visible during most sampling trips in 1971 - 1973 -
trips which have coincided with the existence of strong density
stratification and a submerged waste plume. This stationary
slick was relatable to the waste discharge because: it assumes
the same Y configuration characteristic of the diffuser; it
contained a high number of floating parti oilates identifiable
as being of sewage origin; and because it contained high levels
of coliform bacteria.
How does one assess the significance of these findings? In a 1971
report to the Southern California Coastal Water Research Project
it
(SCCWRP), Dr. Selleck, the principal investigator suggested that
coliform organism die off did not occur at the rate of 90 percent
in 6.5 hours as conventional techniques would indicate, but
based on the inclusion of surface layer concentrations would be
closer to 35 percent in 6 hours. The bacteria in the slick were
associated with floating particulate material and apparently
protected from normal lethal effects. These findings also suggest
that the diluting and dispersive nature of outfall diffuser systems
may not be nearly as efficient as the gross numbers would indicate
when applied only to constituents with densities less than that
of the ambient fluid.
Dilution calculations are meaningless in attempting to predict
surface concentrations of surface film materials or particulates.
-60-
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81/2
Tow Line
Screen Sampler
500/Lipore Size
Canvas
• *•"'.' ' ' * ^^ *' AJT v " ' ' V^'' >v •' *»•* '" '* "' .-*.* * __ ft. \ tt ' • •*.'•-.** .
•..:•••.-.:. ".;."^~~T'-fe.— .:• •;•.•;• •: " -••.;:•. Profile View:;-/. :.. .•-.:'•'
Figure 2 TRAWL NET SAMPLER
-------
WASTEWATER EFFLUENTS
Procedures are now being tested to try to relate the field results
obtained by the above methods to the character of the effluent itself.
-62-
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SUMMARY AND RECOMMENDATIONS
Experimental and field data available in the literature have
shown that surface concentrations of films, slicks and particulates
are important factors in effective pollution control. Ocean
outfalls have been shown to be possible sources and existing
water quality regulations have been questioned as to their
control effectiveness.
Nonuniformity in sample collection and analyses was identified
as one of the more critical areas needing attention. A need that
should go hand-in-hand with the establishment of quantitative
regulations. Candidate methods for the standardization of field
collection procedures were presented for: surface slicks;
surface bacteria; micro and floating particulates.
Two recommendations are in order. First, EPA, in compliance
with current legislation, is about to publish regulations for
Coastal Water Quality. They will appear for comment - in the
Federal Register on October 18, 1973. Specific quantitative limits
on surface concentrations are lacking and interested parties
are urged to respond. Secondly, EPA's Quality Assurance Division,
responsible for the standardization of procedures used in the
monitoring and control of pollution, needs input from all
concerned parties. Your participation will help to effectively
select, test and finally standardize the more appropriate procedure.
-63-
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SCALE 1:50,000
N
I
Ch
.Y • '•'
.MARINA
EL REY
APPARENT SLICK
MOVEMENT
I,000'/HR.
DROGUE RECOVERY
0.33 KNOT SURFACE
DRIFT OVER OUTFALL
Figure 1 SAMPLING STATIONS 1 & 2, SANTA MONICA BAY—APRIL 7, 1971
-------
These materials are not prevented from accumulating on the air-water
interface by such intermediate phenomena. They are affected, in
terms of areal concentration, by the gross size of the diffuser,
the size of the waste plume, its height of rise and of course
the physical-environmental parameters descriptive of the water
body itself i.e., the water motion, depth, density and characteristic
diffusive nature. This argument applies equally well to the
bacterial distributions found associated with floating particulates.
It is unlikely that the long term accumulative nature of a
continuous discharge could ever be realized in open coastal waters
because of high rates of degradation and bioutilization. Physical
factors such as wind and waves also contribute here acting to
break up and disperse the material when certain limits are exceeded.
The dispersed materials, however, do not vanish, and when the winds
and waves subside they may become part of another surface slick far
removed from any point source and hence be interpreted as a
completely natural slick.
Surface slicks and films have been shown to be partially composed
of long chain fatty acids and hydrocarbons. Farrington and Quinn
(1970) investigated the concentration of these materials in
treated wastewater effluents and have estimated that somewhere
between 28,000 and 140,000 metric tons were discharged to coastal
waters during 1970; an input that equals or betters reported
accidental oil spills during the same period.
These calculations.don't have to be precise to warn us of a
potential danger. We are very cognizant of the acute effects
of large oil spills and we know that there are chronic effects
as well. The point here is simple - municipal waste discharges
contain essentially the same level of hydrocarbons and must
-65-
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also carry with them some chronic effects. Little is known about
the ability of coastal ecosystems to degrade these materials and
as has been indicated simple dilution is not enough.
It would be wrong to conclude that all ocean outfalls produce
stationary surface slicks since we don't know how differing
degrees of treatment nor discharge method influences the net
effect. It is reasonable, on the other hand, to conclude that
based on field data the currently applied aesthetic-visible-
res trictions have not been totally effective in the true sense
of environmental protection.
-66-
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SAMPLING METHODOLOGY
It is difficult to assess the true significance of surface slicks
and films because of the current nonuniformity in the procedures
used to collect and analyze such materials. EPA has been sponsoring
research designed to fill this need through the development of basic
procedures for film collection, surface bacteria collection and
the collection of representative near surface samples of particulates.
Analytical methods are being explored that have application to coastal
monitoring schemes.
Preliminary research findings, backed by two years of effort have
found the following procedures simple to apply and effective in
areas both with and without visible surface films.
SURFACE SLICKS AND FILMS
The collection of the surface slick samples is accomplished
with the use of a flexible screen made of glass cloth.
Laboratory tests showed that such screens collected salt water
by capillary action to a depth of 0.15 mm. The size of the cloth
used in field applications is 0.1 m square.
The actual sampling sequence is as follows: The glass cloth is
solvent washed in the laboratory to minimize the background
concentration of the specific constituent to be analyzed for
(in this case normally a hexane extractable one), and placed
in a glass container with a teflon lid. The cloth is placed
on the sea surface using a special device designed to spread
the cloth and prevent contamination. The cloth is allowed to
wet completely at which time it is retreived using glass rods
and returned to its solvent rinsed glass container and sealed.
-67-
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There is one undesirable effect from this procedure and that is
the loss of water as the cloth is folded and reinserted into the
container. This loss may be corrected for, but the significance
of this correction is as yet unknown.
BACTERIAL SAMPLING
Nylon netting of coarse weave was used for this purpose. Laboratory
studies indicated good bacterial release when the sample was
washed with a sterile buffered solution. The netting used had
a two filament knit with 0.75 to 1.0 mm openings and was found
to collect a layer averaging about 0.1 mm at 22°C. In reducing
the data the area sampled was calculated by dividing the volume
of water by 0.1 mm rather than assuming that the full 0.1 m
square was the true area. It is noted that the pretreatment
given the cloths did cause some shrinkage. Samples were washed
with 150 ml sterile buffer, capped shaken (at least 50 times)
and aliquots withdrawn, filtered immediately and incubated for
later counting.
MICRO PARTICULATES
A 0.55 sq m nylon net was used for this purpose. The procedure
for all surface layer collections was essentially the same.
This sample is refrigerated and preserved with formalin until
it can be examined.
FLOATING PARTICULATES
A trawl net was used to collect floating particulates from the
ocean's surface. The sampler, see Figure 2, was towed at 1.5
knots. The water entering the device is forced over a submerged
airfoil and then is forced through a 500 y removable nylon screen.
The area sampled averages 87 cm wide and 3.0 cm in thickness.
-68-
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ACKNOWLEDGEMENTS
The methodology and procedures discussed in this paper were
developed as part of an EPA Research Grant (800373) to the
University of California, Berkeley. The principal investigator
was Dr. R. E. Selleck. Others working under his direction
included Ralph Carter and Lloyd Bracewell, doctoral candidates
at the University.
-69-
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REFERENCES
Amies C. R. (1956). Surface films on Swimming Pools. Pub.
Health India, 4: pp 31-35.
Anon. (1971). Rules and Regulations Establishing Surface
Water Quality Criteria New Jersey Dept. of Environmental
Protection. Richard J. Sullivan, Commissioner - June 30, 1971.
Anon. (1972). Interstate Waters of the State of Alabama - Federal
Register, Vol. 37, No. 49 (40 CFR 120) March 11, 1972.
Anon. (1972). Water Quality Control Plan - Ocean Waters of
California - State of California Water Resources Control
Board - Adopted July 6, 1972.
Anon. (1972). Surface Phenomena Study Sanitary Engineering
Research Laboratory Report No. 72-9. University of
California, Berkeley, June 1972.
Duce, R. A., et. al. (1972). Enrichment of Heavy Metals and
Organic Compounds in the Surface Micro layer and Narragansett
Bay. Science Vol. 176, p. 161, 1972.
Farrington, J. W. and J. G. Quinn. (1973). Petroleum Hydrocarbons
and Fatty Acids in Waste Water Effluents. Journal WPCF, Vol.
45, No. 4, April 1973, pp. 704-712.
Garrett, W. D. (1965). Collection of Slick Forming Materials
From the Sea Surface. Limnol. Oceanog. 10:602-605.
Harvey G. W. (1966). Microlayer Collection From the Sea Surface.
A New Method and Initial Results. Limnol. Oceanog. 11: 608-613.
-70-
-------
LaFond, E. C., and K. LaFond. (1969). Perspectives of Slicks,
Streaks, and Internal Wave Studies. Bulletin of the Japanese
Society of Fisheries Oceanography, November 1969, p. 49-57.
Randall, C. W. and J. 0. Ledbetter. (1966). Bacterial Air
Pollution From Activated Sludge Units. Amer. Ind. Hyg.
Assoc. J. 27:pp 506-519.
Riseborough, R. W. (1968). Pesticides: Transatlantic Movements
in the Northeast Trades. Science 159: pp 1233-35.
Selleck, R. E. and R. Carter. (1973). Evaluation of Floatables
of Wastewater Origin in the Vicinity of Marine Outfalls.
Proceedings 3rd Annual Technical Conference Estuaries of
the Pacific Northwest, Oregon State University Report,
March 1973.
Selleck, R. E. and R. Carter. (1971). Progress Report - EPA Grant
16070 FKO - The Significance and Control of Waste Water Floatables
in Coastal Waters.
Selleck, R. E. and L. Bracewell. (1973). Progress Reports -
EPA Grant R800373 - The Significance and Control of
Wastewater Floatables in Coastal Waters.
Woodcock, A. H. (1955). Bursting Bubbles and Air Pollution.
Sewage and Industrial Wastes. Vol. 27, No. 10, pp 1189-1192.
-71-
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A SYSTEMS APPROACH TO MARINE POLLUTION MONITORING
S. P. Pavlou*, T. E. Whitledge, J. c. Kelley, and J. J. Walsh
INTRODUCTION
Perturbation of ecosystem {or ecological) stability by
the introduction of man-made chemical pollutants in the
biosphere is a problem of international concern.
To date, the detection, identification, and measurement
of biologically active pollutants in the marine environment
has been accomplished in some detail (trace metals,
synthetic organics, radionuclides). End effects due to
biological amplification (extinction of certain species and
potential toxification problems for man) have also been
investigated with great concern. However, despite the
steadily increasing sophistication in experimentation and
the introduction of new analytical techniques in the field
of pollution research, useful and predictive information
concerning the response of biological systems to man-made
perturbation is often empirical, descriptive, and generally
obtained by scattered investigations and non-rigorous crash
programs.
Marine pollution monitoring is a complex problem and
requires the utilization of multidisciplinary scientific
knowledge and a well organized field program for
understanding the transfer of pollutants and interactive
processes taking place throughout a given marine ecosystem,
as well as predicting and controlling their consequences.
This type of research has been initiated only recently.
We are currently engaged in international research
involving evaluation of the physical, chemical, and
biological dynamics of marine ecosystems within various
ocean upwelling regions and assessment of biological
response of marine ecosystems subjected to man-made
perturbations. For the second case, while primary emphasis
has been placed on eutrophic systems |Puget Sound, Southern
California Bight), preliminary work has been initiated on
oligotrophic systems (Eastern Mediterranean) to compare the
impact of nutrient and toxic compound addition on these two
distinctly different ecosystems.
*Department of Oceanography, University of Washington
-72-
-------
This type of multidisciplinary approach has been adopted
to serve the following priorities: developing a
sophisticated understanding of marine ecosystems under man-
made perturbations; acquiring a predictive capacity for
short and long term response of the primary productivity and
fish population within a specific coastal area subjected to
high pollution inputs; and assisting governmental agencies
in establishing effective water quality standards.
The overall framework and a summary of the new
methodology as is currently employed during our field
investigations, is described below.
OVERALL FRAMEWORK
A schematic diagram showing the important processes
characterizing a polluted marine ecosystem is given in
Figure 1. Upon introduction of a chemical in the marine
environment, the degree of interaction with the biological
components of the system is a critical factor in assessing
the overall impact to the ecosystem. Assuming that the
physical parameters of the system (advection, diffusion,
turbulent mixing and boundary conditions) are defined, the
response to the perturbation depends on a number of
important physiochemical factors: (1) the chemical
structure of the microcomponent, (2) the reactivity with the
medium (alteration of the equilibrium condition of the
system* s reservoir), (3) the reactivity with various
constituents of the medium {chemical speciation) , <**) the
transport process to the biological site of interest, (5)
the specific interaction on the cell wall, (6) the
inhibition of enzymatic processes within the cell reflected
by the alteration of the growth characteristics of the
organism, and {7) the recycling and regeneration process.
These considerations have some important connotations to
the monitoring methodology, as well as the construction of a
simulation model of a polluted area. Quantitative
definition of the above physiochemical factors and their
utilization in the theoretical framework are necessary for
making sound predictions about the resiliency of the system
under a given pollution load. Therefore, a rigorous
monitoring program should include a capability for obtaining
data on the chemical inhibition of various biological rate
parameters {photosythesis, nutrient uptake, enzymatic
activity, grazing, etc.) characterizing the primary trophic
levels of the ecosystem under investigation.
la order to satisfy these priorities, a capacity has
been established for carrying out controlled experiments on
-73-
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ATMOSPHERE
RUN-OFF
DOMESTIC
SEWAGE
INDUSTRIAL
WASTE
-J
^
EXCHANGES
ACROSS THE
SYSTEM
BACTERIA ON
DETRITUS AMD
SEDIMENTS
Figure 1. Block Diagram of Important Processes Characterizing a Polluted
Marine Ecosystem
-------
the components of the system, describing the spatial
relationships of these components, integrating the
components into a general system model, predicting the
behavior of the system, and testing the predictions in the
field. These aspects have been approached and developed
simultaneously to accomplish the proper interrelations.
Together with an intense physical oceanographic program, the
chemical and biological studies include the following
capabilities:
1) An experimental laboratory in which the specific
effects of particular limiting nutrients and chemical
inhibitors on a selected species of phytoplankton can be
studied under controlled conditions.
2) A shipboard data acquisition/reduction (DA/DR) and
presentation facility (PF), heavily graphics oriented. A
block diagram of the DA/DR system is shown in Figure 2.
This facility is essentially a bank of sensors (including
chemical AutoAnalyzers ) and a computer capable of
acquiring, editing, and analyzing data from the sensor array
and presenting this data graphically as it is acquired. A
data storage facility is also necessary. The DA/PF has been
developed and is currently in operation in our laboratories.
3) Experimentation on nutrient, pollutant and energy
fluxes through the primary trophic levels of the food chain,
carried out in the field with naturally occurring organisms
and ambient water.
<») A computer simulation model of a polluted marine
ecosystem. This model is currently being constructed and
will be used to integrate the results of the field
measurements, the field and laboratory experiments, and
other related information, in order to predict the behavior
of the total system under specific pollution stresses. The
model identifies important diagnostic features of the
ecosystem which can be verified by the other three
activities of the program. Utilization of the data
acquisition system allows us to interpret the behavior of
the system under study and assess the time dependence of its
state.
OPTIMUM ANALYSIS SCHEME
The actual analysis of the area under investigation is
carried out as shown in Figure 3.
1) Using the data acquisition system, continuous
records of the surface distributions of the important
variables (temperature, salinity, the nutrients.
-75-
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Integrated Sampling and DA/DR System
i
-0
UNDERWAY MODE
SENSOR BANK
T, S, hv,
Vw, N|, Chi,
Nekton,
Zooplankton
Autoanalyzer
DATA
STORAGE
DATA ACQUISITION
DATA REDUCTION
Maps, Statistical
Analysis (regression, correlation
and factor analysis)
Model Interaction
DATA OUTPUT
AND DISPLAY
BATCH MODE
EXPERIMENTAL
Results
Routine data
MODELS
Figure 2. Integrated Sampling and DA/DR System
-------
OPTIMUM ANALYSIS SCHEME (CYCLE)
I
FIELD DEFINITION AND SURFACE DISTRIBUTION OF X
X| « T,S,N,,02,Chlor, OTHER IMPORTANT VARIABLES
(REMOTE SENSING, UNDERWAY MAPPING)
PHYSICAL MEASUREMENTS
CURRENT
rw»
hv
ISOLATION OF DYNAMIC
REGION
(SPATIAL GRADIENTS OF X
AND BIOMASS)
ROUTINE DATA COLLECTION
PC, PN, PSI, Chlor
SEDIMENT ANALYSIS
SUSPENDED PARTICULATE
PHYTOPLANKTON
ZOOPLANKTON
DOM DOC
POLLUTANT FRACTIONATION
EXPERIMENTATION
lkC AND Nj UPTAKE (PHYTOPLANKTON
CHELATION AND TOXICITY
ENZYMATIG ESTIMATES OF RATE PROCESSES
GRAZING
REMINERALIZATION
MODELLING
DESCRIPTION OF THE STANDING CROPS AND RATES OF THE SYSTEM
MODIFICATION OF THE FOOD QUALITY (C/N VARIATION)
SPECIES SUCCESSION
SEDIMENT WATER QUALITY CHANGES
SENSITIVITY ANALYSIS OF SAMPLING PLAN FOR VALIDATION STUDIES
Figure 3. Optimum Analysis Scheme of a Polluted Marine Ecosystem
-77-
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chlorophyll, etc.) are measured in the area of interest.
From this information surface maps of the distributions are
produced and hydrographic stations are occupied to define
the overall structure in the region. When the most dynamic
part of the area has been identified, detailed attention is
focused upon it. This phase usually requires 1 to 3 days,
depending on the size of the sampling grid.
2) Another continuous record of the surface variables
is recorded while the ship steams over a fine grid in the
area. Surface contour maps are produced and displayed
continuously as the data is acquired and edited (4-10
hours).
3) On the basis of the surface distributions, sites are
chosen for experimentation. These sites may be selected
because they display certain features such as different
limiting nutrient structures, well developed phytoplankton
blooms, freshly upwelled water, abrupt gradients of specific
pollutants, or for being in the dispersive field of a sewage
outfall (4-20 days).
4) When the first set of experiments has begun, the
surface distributions are again mapped and new experiments
initiated.
5) AS soon as the experimental data are available, they
are coupled with the distribution data and simulation
experiments are initiated. The results of the model
prediction are compared with the incoming data and the
comparison is evaluated by the scientists in the field.
INTEGRATED SAMPLING AND DATA ACQUISITION/DATA REDUCTION SYSTEM
Interactive Real Time Information System (IRIS)
The various components of the shipboard real time DA/DR
system are shown in Figure 4. They include the hull mounted
pumping system (HUMPS), the towed underway pumping system
(TUPS), salinity-temperature-depth sensors (STD), the
hydrographic sampling bottles, radiation and meterological
sensors, as well as other auxiliary instrumentation for
various data gathering. The AutoAnalyzer and fluorometer
receive the samples either continuously or in a batch mode
(Figure 2). IRIS collects the information on the peripheral
processor through analog to digital conversion and sends it
to the central processor for program manipulation and output
display. A generalized schematic diagram of IRIS is shown
in Figure 5,
-78-
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HUMPS
TUPS
STD
' 1
SURFACE
PUMPING
SYSTEM
SURFACE
TiA
SENSORS
I ;_
TUPS
PUMP
TUPS
C,T,D
CONTROL!^
I |
PLESSEY
S,T,D
ROSETTE
HYDROGRAPHIC
BOTTLES
6-0 ROSETTE
12 30LNISKINS
AUTOANALYZER
CHEMISTRY
SAUNOMETER
H~~l I"*"* Hi
r
USER TERMINALS
L
Figure 4. Schematic Flow Diagram for Biological Sampling
-79-
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INDUSTRY COMPATIBLE
MAGTAPE (TU-IO)
I
CO
o
INTERACTIVE
GRAPHICS
(GT-40)
CTD
INTERACTIVE
GRAPHICS
(GT-40)
CTD
LINEPRINTER/
GRAPHICS
(GOULD 4800)
OPERATORS
CONSOLE
(LA 30)
cro
CALCOMP 563
O
DIGITAL I/O
(DRIIA/B)
A/D
CONVERTER
(AD01)
INTERPROCESSOR LINK
ODOD
OO GO
8K WORDS
—I MEMORY
CPU
(11/45)
SWAPPING USER DATA AND
DISK PROGRAM MAGTAPE
(RSII) (TU56)
USER DATA AND
PROGRAM MAGTAPE
(TU56)
OPERATORS
CONSOLE
(LA30)
CARTRIDGE
DISK
(3-RK05)
Figure 5. A General Schematic Diagram of the IRIS Computer System
-------
Data stored in the IRIS system is available for output
in several forms, depending on the type of data and the
user«s preference. Together with the standard tabular
output, vertical distribution of station data (depth
profiles of specific variables), vertical distribution of
data from several stations (vertical contour plots), two-
dimensional surfaces or subsurface maps of station data
(surface contour plots) and two-dimensional underway maps
can be constructed. A capability for three-dimensional
mapping will also be available in the future. All of these
forms of output may be displayed on an oscilloscope console
or obtained as hard copy on a printer. Examples of typical
data output are shown in Figures 15 and 26, with a
discussion of the observations following in the Shipboard
Experimentation section.
Towed Underway Pumping §YJ&§S OSES)
The "traditional" sampling procedure, as shown in Figure
6, involves continuous underway surface mapping to determine
the horizontal distribution of temperature, salinity,
nutrient and flourescence. This operation is conducted at
night. By compiling this information with the sampling data
obtained during the day, the vertical distribution of these
properties is defined. To alleviate this time consuming
process, we have designed a towed underway pumping system
(TUPS) which has a three-dimensional mapping capability. A
typical sampling cycle with TUPS is shown in Figure 7.
The ship steams the normal zig-zag cruise track while an
active depressor (in our case, a model 3000 Batfish, Hermes
Electronics, Ltd.), oscillates vertically in a sinusoidal
fashion from the surface to a maximum depth of 100 meters.
The depressor was chosen to provide a variable depth
capability without having excessive cable wear and to
minimize the strength and weight requirements of the storage
and retrieval reel. The Batfish contains a hydraulic servo-
control mechanism, a Guildline CTD and a Deming water pump.
The system can be towed at 10 knots over a depth range of 0-
110 meters while pumping water up to deck at 10 gal/min
under a pressure of 10 Ibs/in . The pumping hoses are made
of polyurethane and are free from synthetic organics or
trace metal contaminants. The CTD in the Batfish is
accurate to + .00 0/00 in salinity units and + ,02°C.
The whole system consists of a winch (used only for
storage, deployment and retrieval), a faired cable
containing the pumping hoses, electrical conductors and a
coaxial cable (Figure 8), the Batfish, and the emergency
recovery system. The Batfish is towed with an approximate 3
-81-
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CONTLNJJOUS SURFACE ANAL.YS»S
TRADITIONAL
Figure 6. BAMPUIUG DESIGN
-------
I
CD
U)
I
TOWED UIMQERWAV
SYSTEIVI CTUPS1
-------
I
00
3/8" 9X19 AMOAL
TORQUE BALANCED
WIRE ROPE WITH -
.68"0.0. .80*1.0.
POLYURETHANE HOSE
1.02" 0.0. .95"l.D.
POLYURETHANE HOSE
10 CONDUCTOR NO. 18
CABLE WITH
POLYURETHANE JACKET
POLYETHYLENE JACKET
(14,800 Ib. BREAKING
POLYURETHANE
FAIRED JACKET
COAXIAL CABLE WITH
POLYURETHANE JACKET
7 CONDUCTOR NO. 12
CONTROL CABLE WITH
POLYURETHANE JACKET
Figure 8. Specifications of the Tups Faired Cable
-------
to 1 scope, as shown in Figure 9. The overall deck
configuration of TUPS is shown in Figure 10 and a block
diagram of the power control components is displayed in
Figure 11.
PHYSICAL MEASUREMENTS
The physical description of a polluted marine region and
the definition of the physical processes governing the
distribution of discharged material are important factors in
establishing a successful monitoring program.
Data on the circulation patterns of the study area are
necessary for obtaining information on the residence times
of discharged microconstitutents, describing the flushing
characteristics of the area, and correlating pollutant
accumulation with the physical state of the system.
We usually obtain information on the circulation
features of the area by deploying drogues for monitoring the
water movements. The typical drogue consists of a parachute
or cross (acting as a sea anchor) attached to a float
surrounding a metal pole manifold equipped with a light and
radar reflector. The ship follows the drogue and positions
are noted every 1 or 2 hours. After a few tidal cycles, the
gross circulation pattern can be deduced. Anchored current
meters are also deployed occasionally to measure speed and
direction of currents at a fixed location. Progressive
vector diagrams from the current meter data are then
constructed for interpretation.
CHEMICAL AND BIOLOGICAL DATA COLLECTION
Numerous samples for both chemical and biological
analyses are collected routinely at the sampling stations.
Samples collected from 5-liter water sampling bottles are
used for salinity, oxygen, nitrate, nitrite, ammonium,
silicate, phosphate, and trace metal analyses. Temperature
data are obtained with reversing thermometers.
Phytoplankton samples collected with twelve 30-liter water
samplers, in a rosette configuration, are used for
Phytoplankton standing stock data and isotopic tracer
experiments. Biomass is determined by routinely measuring
chlorophyll, particulate carbon, particulate nitrogen,
particulate silica, respiratory electron transport activity
(ETS) and total amount of particulate matter in the form of
a size-frequency specturm. Zooplaakton and sediments are
also collected for trace organic analysis. Sampling
methodology for plankton includes standard net techniques as
well as special devices such as the Large volume Filter
-85-
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I
00
100 m
Figure 9. Towing Configuration of the Batfish
-------
TENSION METER
WEAK LINK
i
00
POWER COMPONENTS
WATER FOR
SENSOR ARRAYS
Figure 10. The Overall Deck Configuration of Tups
-------
115 V
BATFISH
POWER
SUPPLY
BATFISH
SERVO
CONTROL
BOX
r •
1
1
1
SERVO
.. OATdCMJ
CTD
w 1 \J
HYDRAULIC
Bl 1MB
220V
or
440 V
WINCH
CONTROL
BOX
L_ 1
WINCH
Figure 11. Block Diagram of the TUPS Power Control Components
-88-
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(LVF) and the Protected Closed Net (PCN) for phytoplankton
and zooplankton collections respectively, as used in our
trace organics program. Schematic diagrams of these devices
are shown in Figures 12 and 13.
SHIPBOARD EXPERIMENTATION
Assessment of the biological impact of marine outfalls
on the primary productivity of the area has been
accomplished by following photosynthetic and respiratory
activity as well as changes in phytoplankton nutrient
uptake. Radioactive tracers and stable isotopes have been
commonly used to monitor these rate processes. Shipboard
toxicity studies include growing of natural phytoplankton
populations in the ambient water and monitoring changes in
»*C, **N-uptake and respiration rates; observing the
approach of laboratory cultured phytoplankton and
zooplankton to toxicity threshold behavior under the actual
trace compound load of the system; pulsing of natural
populations with higher trace compound concentration in
reference to the ambient levels and monitoring their growth
response and their adaptability to the perturbation.
Carbon fixation is measured by the traditional »*C
method with shipboard analysis carried out by a gas flow
geiger counter or a scintillation counter. Nitrogen and
silica utilization is determined by nutrient uptake
experiments with *SN and 2»si tracers. The isotopes are
analyzed on a shipboard mass spectrometer. Incubation
experiments are performed onboard ship using standard
procedures associated with the simulated |.n situ methods.
Trace organic (chlorinated hydrocarbons) accumulation in
phytoplankton, suspended particulate matter, zooplankton and
sediments are measured by shipboard gas chroraatography and
confirmed by mass spectrometry. Anodic stripping voltaraetry
has been used occasionally onboard ship for some trace metal
determination in seawater (e.g., Zn, Cu, Pb).
SOME PRELIMINARY RESULTS
The data presented below can be used as an example of
the inherent advantages in adopting a systems and real time
approach to marine pollution monitoring. The area
investigated is the coastal waters of the Southern
California Bight, off the White's Point Outfall in Los
Angeles (OUTFALL I, II cruise series, 1972-73, R/V T.G.
Thompson) . A general map of the region, together with
station locations during the OUTFALL-II studies is shown in
Figure 14.
-89-
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INTAKE
LINE
PRIME LINE
OUT
SCAVENGE
LINE
BALLAST
Figure 12. SCHEMATIC DIAGRAM OF THE HV FILTER
-------
PC RELEASE "VINE
RETRIEVAL LINE
WEIGHT ( 10 KG )
COD END xSKIRT
Figure 13. Configuration of the PCN
(a) Landing stage
(b) Towing stage
-------
45'
25'
118° 20'
LOS ANGELES
PALOS VERDES
HILLS
(VICENTE •. .£,•& • •'.•'
l-.jj.. .. ......ai**&i-. * :
IO4.I06A
I03.IO6B
I05,I06\*9' '
%i
x 90
• •
99.I06E
15'
SAN PEDRO
BAY
.98
89
,100,106 F
,ot,
106 D
97,106 G
45'
33!
40
2,1060
96.I06H
,94,106 J
I
^95,1061
I
OUTFALL
251
II8»20'
15'
Figure 1A. Station Locations During OUTFALL-11 Cruise
-92-
-------
The major objectives of these investigations were to
evaluate the extent of the submerged sewage field, to assess
the degree to which the dispersive field might influence the
biological productivity of the area, and to determine
whether the sewage field is affected by upwelling.
Following a brief aerial survey {2-4 hrs) for locating
distinct surface water and bionass structures in terms of
temperature and chlorophyll gradients, a preliminary field
analysis was carried out with all underway surfaces sensors
in operation.
Typical grid maps for temperature, nutrients and
chlorophyll fluorescence are shown in Figures 15 through 21.
Although temperature, phosphate, nitrate, nitrite and
siliate show very little variation in the vicinity of the
outfall, chlorophyll fluorescence and ammonium concentration
display distinct maxima. These surface maps are produced
immediately after completing the grid so that the output
data may be used in planning the location of hydrographic
and productivity stations in the most dynamic region of the
area.
The submerged sewage field was located by utilizing
salinity-temperature-depth |STD) sensor array data similar
to the profile shown in Figure 22. The observed temperature
and salinity values were used to calculate the density,
denoted as where = (density-1) x 1000 on the plot.
Station 093, located near Long Point, shows a characteristic
profile of low salinity water at about 7 meters,
corresponding to the thermocline depth and reflecting the
position of the submerged field. Other STD station data
also confirm the general presence of the sewage field in the
thermocline region.
Vertical contour plots of nutrients for a longshore
transect are shown in Figures 23 to 26. The distribution
characteristics correlate well with the STD data. However,
nitrate and silicate show reduced sensitivity in locating
the sewage field when compared to ammonium or phosphate.
Ammonium is a good tracer for the discharge effluent because
of low natural background and high concentrations in the
discharged effluent.
The concentration of ammonium is so large in the sewage
field that it may still be apparent after other discharged
substances have been reduced to background values. Ammonium
concentrations of five times the normal, or twice the
maximum oceanic values have been observed in areas where no
-93-
-------
15.64, . • . ,5
• • * V
L
: /' -"4'
•._' . »l^Vii5.i2
• 15.30 . •(5.08 . .A.
I II 1 I I I I I I I I I I I I I I I I II I I I | | I I I I I | |
TEMPERATURE - GRID 28, 7 MAY 73. R = 5
Figure 15. CONTOUR INTERVAL = O.2OO
-------
I
<&
(Jl
. yp g ;
• mf 31.85V
I I I I I I I I I I I I I I I I I I I I I I I | I
CHL-GRID28, 7 MAY 73. R=5
Figure 16. CONTOUR |NTERVAL = 8.000
-------
I
VD
0.70
I I I I I I I I I I I I I I I I I 1 I I I I I I I I I I I I I I | |
NH4 - GRID 28, 7 MAY 73. R=5
Figure 17. CONTOUR INTERVAL = O.*OO
-------
-J
I
-091 • •
I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I |
Figure 18.
P04 - GRID 28, 7 MAY 73. R = 5
CONTOUR INTERVAL = 0.400
-------
03
I
/ • v/.v/w - /*.
°°f • oo60
r>—<•/{
7 . • V^
I I I I I I I I I I I I I I I I I I I I I I I I
Figure 19.
NO3-6RID 28, 7 MAY 73. R = 5
CONTOUR INTERVAL » O.O2O
-------
O.27
• •
•
0.20 . . .
• • • v
• 0.27 *
. •* • •
• 0.44, '0.40 . • ,
O.IO» °'43
. *0.49« *0.55 0 *
0.54
*0.22
I I I I I I I I I I I I I I I I I I I I I I I I I I | || || | | |
Figure2o. N02-GRID 28, 7 MAY 73. R = 5
CONTOUR INTERVAL = 0.400
-------
o
o
I
3.66 .
. ,3J5. ••,•••*•
9
3.41
• 3.55 .
.• 3.29 . *4.07 •
• • • • 4.21
•v63.-"-. •'
/136,3 3.41 . .3.03
^^ ^P
i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i
Figure 21.
SIO4 - GRID 28, 7 MM 73. R = 5
CONTOUR INTERVAL » I.6OO
-------
54.00
32.5O
10.0 •
20.0 •
30.0 ••
40*0 • •
50.0 - -
STD PLOT
TT07B 093
E4.50
1
ESrOO
1
ES.SO
—I—
26.00
S6.SO
87. 00
0.00 2.50 S.OO 7.50 10.00 12.56 15.00 17.50
H 1 1 1 1 I 1 1—
33.00
33.50
34.00
34.50
+0
eo.oo
+T
3S.OO
60.0
FIGURE 22. LATITUDE 33-43.4
U3NGJTLOE llfl-23'0
T = TEMPERATUre - C
S = SrtLINITT - 0/00
0 = SIQMA-T
-101-
0ATE 0 MAY 1973
BEGIN 500 END SOO
-------
• 1.22 •0.41 *0.44 •0.46 «0.62
5__ »l.22 •0.67 »0.46 •2.51 •0.59
15
20
25__
30 H-
35
40
45_|_
50
• 4.46
I I I I I I I I I
Figure 23.
93-92-91-90-89 AMMONIUM 0-50 METERS
CONTOUR INTERVAL = 20.000
-102-
-------
• 2.46
• 18.56
• 0.10
• 2.97
• 0.47
• 19.11
I I I I I I I I I I I I I
Figure 24. 93-92~91"9Q-89 NITRATE 0-50 METERS
CONTOUR INTERVAL « 4.000
-103-
-------
• 10.69
• 10.77
• 709
• 17.52
• 4.49
5.06
• 4.96
• 5.08
• 5.60
• 7.92
• 6.29
• 6.52
• 9.71 \-^Je.84
• 14.47 «2.98
• 20.35 • 17.94
I I I I I I I I
Figure 25.
93-92-91-90-89 SILICATE 0-50 METERS
CONTOUR INTERVAL = 4.000
-104-
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0.59
•0.54
• 0.67
•0.55
I I I I I I I
Figure 26.
93-92-91-90-89 PHOSPHATE 0-52 METERS
CONTOUR INTERVAL « O.800
-105-
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discontinuities in the temperature, salinity, silicate or
phosphate fields were found.
Data collected on OUTFALL-1I showed upwelling to be an
important process involved in the performance of a sewage
outfall. Usually the outfalls are designed to keep the
sewage field in a subsurface layer; however, complications
arise when upwelling brings subsurface water to the surface.
Figure 27 shows a vertical contour plot of ammonium on an
offshore transect. As the sewage field approaches the coast
the ammonium isopleths are displaced towards the surface.
Coincident with the high ammonium, lower water temperatures
were also noted at a shallow depth on the inshore stations.
The increased concentration of ammonium in the euphotic zone
correlated well with the large dinoflagellate bloom observed
in the area.
SUMMARY
From the above considerations, a potentially effective
marine pollution monitoring program should satisfy the
following criteria?
1. Utilization of a multidisciplinary approach.
2. Implementation of a rigorous and sophisticated field
program involving methodology for real time data
acquisition and reduction.
3. Understanding the transport, interactive processes,
and biological consequences of the ecosystem under
investigation.
4. Development of a predictive capacity (simulation)
and field validation of the theoretical framework
for evaluating short and long term responses.
5. Establishment of control capability and effective
water quality standards (input control) as
determined by the systems investigation results.
-106-
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058
060
To.09
FIGURE 27,
-107-
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METHODS AND PROBLEMS IN ANALYSIS OF PESTICIDES
IN THE ESTUARINE ENVIRONEMENT
A. J. Wilson, Jr. and J. Forester
Gulf Breeze Environmental Research Laboratory,
Gulf Breeze, Florida
The presence of pesticides in the marine environment has
been well documented. Cox (4) reported DDT concentrations
in sea water along the Pacific Coast to range from 0.0023
parts per billion (microgram/liter) off Oregon and
Washington to 0.0056 parts per billion off Southern
California. Residues of DDT and dieldrin were detected in
livers of fishes by Duke and Wilson (6) from the
Northeastern Pacific Ocean and in gray whales by Wolman and
Wilson (16) . Other documentation of chlorinated
hydrocarbons in the marine environment is presented by
Goldberg et al. (7).
The Gulf Breeze Environmental Research Laboratory at
Gulf Breeze, Florida, an associate laboratory of the
National Environmental Research Center, Corvallis, Oregon,
has been conducting research on the effects of pesticides in
the marine environment since 1958. Since that time the
laboratory has analyzed over twenty thousand samples for
these pollutants in water, sediment, oysters, crabs, fish,
birds and mammals. From 1965 until 1972 this facility
analyzed over eight thousand samples for the National
Pesticide Monitoring Program as reported by Butler (3).
This report describes analytical methods employed by this
Program, some recent studies in water analysis, and the need
for adequate analytical quality control in marine
monitoring.
NATIONAL PESTICIDE MONITORING PROGRAM
From studies at Gulf Breeze, bivalve mollusks appeared
to be suitable animals to use as indicators of estuarine
pollution. Adult bivalve mollusks are sessile, permitting
repeated sampling of the same population. In addition,
experiments by Butler (2) indicated that bivalve mollusks
readily accumulate chlorinated hydrocarbons, consequently,
oysters, mussels and clams were the primary indicator
organisms in this program.
-108-
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Molluscs were collected at about 30-day intervals at 183
estuarine sites in 15 coastal states. Approximately 15
individuals were taken from each station by other agencies,
prepared in their laboratories, and shipped to Gulf Breeze
for analyses. The rationale for all analyses being
conducted at Gulf Breeze was based on the premise that these
methods of analyses would be consistent from station to
station and month to month. This would eliminate variation
in methodology and permit a more reliable interpretation of
seasonal and geographic trends. In addition, it was not
economically feasible at the outset to equip several
satellite laboratories and conduct a suitable
interlaboratory quality control program.
Prior to the start of the program several preservatives
were evaluated to find a method that would allow shipment of
samples without dry ice. when samples were dehydrated by
mixing them with a 9: 1 mixture of anhydrous sodium sulfate
and Quso, a micro fine silica, they could be held at room
temperature for up to 15 days without loss or degradation of
tche chlorinated hydrocarbon. This procedure allowed
shipment of samples in aluminum foil by surface mail from
the collecting laboratory to the Gulf Breeze Laboratory.
Analytical Procedures
Molluscs were analyzed for aldrin, chlordane, o, p* and
p,p* isomers of DDT and its metabolites, dieldrin, endrin
heptachlor, heptachlor epoxide, lindane, methoxychlor,
mirex, and toxaphene.
Sample_Pregaration
The tissues of 15 individuals were shucked into a
one-pint Mason jar and thoroughtly homogenized with an
Osterized blender. Approximately 30 g of the homogenate was
added to a second Mason jar and blended with a 9:1 mixture
of sodium sulfate and Quso. By alternately chilling and
blending, a free-flowing powder was obtained. The blended
sample was wrapped in aluminum foil and shipped to Gulf
Breeze. Upon receipt of the sample, it was weighed and
extracted in a Soxhlet apparatus for 4 hours with petroleum
ether.
-109-
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TABLE 1
PERCENTAGE RECOVERY OF PESTICIDES
FROM FORTIFIED OYSTER SAMPLES
PESTICIDE
DDE
ODD
DDT
ACTUAL (PPM)
0.033
0.033
0.34
0.34
0.067
0.067
0.70
0.70
0.10
0.10
1.0
1.0
FOUND (PPM)
0.026
0.026
0.30
0.29
0.061
0.064
0.67
0.63
0.087
0.094
0.95
0.92
% RECOVERY
79
79
88
85
91
96
96
90
87
94
95
92
I
H
H
O
I
-------
The extracts were then purified by concentrating and
transferring the extract to 250 ml separatory funnels. The
extracts were diluted to 25 ml with petroleum ether and
partitioned with two 50 ml portions of acetonitrile
previously saturated with petroleum ether. The acetonitrile
was evaporated to dryness and the residue eluted from a
Florisil column, Mills et aJL. (11). In this technique,
inceasing proportions of ethyl ether to petroleum ether were
used to elute fractions containing increasingly polar
insecticides.
Quantitation and Qualitatj.on
The extracts were analyzed with Varlan Aerograph
electron capture gas chromatographs. Extracts were injected
into at least two 180 cm x 2 mm (ID) columns of different
liquid phases. The following columns have been used:
DC-200, QF-1, EGS, OV-101, mixed DC-200/QF-1, and mixed
OV-101/OV-17, Liquid phases and gas chromatographic
parameters were adjusted so that p,p* DDT would elute in
approximately 12 minutes. The lower limit of detection for
a 30 g mollusc sample was 0.010 parts per million
(milligrams/per kilogram). Residues were reported on a wet
weight basis without adjustment for recovery rates. Thin
layer chromatography and MpM values after Bowman and Beroza
(1) were used for additional confirmation of compound
identity.
Extraction efficiencies were determined by re-extracting
samples for longer periods of time and with different
solvent systems. Recovery rates were determined by
fortification of samples with known levels of pesticides.
Table 1 shows typical recovery rates cf DDT and its
metabolites from fortified oyster samples. The values were
adjusted to account for naturally-occurring DDT residues.
SEA WATER ANALYSIS
Prior to 1971, the Gulf Breeze Laboratory belonged to
the Bureau of Commerical Fisheries, United States Department
of Interior. During those early days, there was little
known regarding the effects and kinetics of pesticides in
the marine environment, consequently, in addition to
scientific publications, the laboratory frequently published
-111-
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quarterly and annual progress reports containing provisional
or preliminary data.
In 1968 the author submitted the following report for
inclusion in a Progress Report of the Bureau of commercial
Fisheries (15).
STABILITY OF PESTICIDES IN
SEA WATER
We began preliminary studies to determine
the stability of pesticides in sea water. Three
p.p.b. of aldrin, p,p'-DDT, malathion, and
parathion in aceton were added separately to
four clear glass, one-gallon bottles containing
sea water (salinity 29.8 p.p.t.; pH 8.1), one
chemical per bottle. After an initial sample
of the water was analyzed, the bottles were
sealed and completely immersed in an outdoor
flowing sea-water tank. Table 5 shows the con-
centration of the chemical at the indicated time
interval.
Although we used natural sea water in these
preliminary experiments, the tests will be
repeated with sterile artificial sea water so
that the relative stability of the pesticides can
be evaluated under standardized experimental
conditions.
Because these studies showed a rapid loss of DDT in sea
water, the report received a great deal of attention. As
stated, these were preliminary studies but, unfortunately,
amny readers carried the data beyond the scope of the
experiment. Obviously, additional studies were required to
account for the rapid decline of these pesticides before any
conclusions could be made.
Since these studies were conducted, several
investigators have reported on the transport of pesticides
in marine waters. Cox (4) reported that adsorption of DDT
is implicated in the uptake mechanism for algal cells. His
experiments also indicate that particules less than 1-2>c
diameter carry most of the DDT residues in whole sea water.
Working in the laboratory with six species of marine algae,
Rice and Sikka (13) found that all species concentrated DDT
to levels many times higher than the original concentration
of the medium. Transformation of DDT and cyclodience
-112-
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Table 5.—Stability of pesticides in natural sea water
(salinity 29.8 p.p.t.; pH 8.1)
I
H
H
U)
I
Pesticide
p.p'-DDT
p, p f -DDE*. .
p, p f -DDD*. .
Aldrin**
Dieldrin*. .
Malathion. . . .
Parathion. . . .
Days after start of experiment
0 6
P.p.b. P.p.b.
2.9 .75
.096
2.6 .58
.74
3.0 <0.2
2.9 1.9
17
P.p.b.
1.0
.95
.081
.096
1.0
<0.2
1.25
24
P.p.b.
.27
.065
.041
<0.01
1.0
1.0
31
P.p.b.
.18
.034
.038
<0.01
.75
.71
38
P.p.b.
.16
.037
.037
<0.01
.56
.37
^Metabolites of parent compound.
**From the seventeenth day onward, 2 unidentified peaks ap-
peared on the chromatographic charts after aldrin had eluted.
-------
insecticide took place in surface films, plankton, and algae
but not in water from the open ocean according to Patil et
al. (12).
Recently, experiments have been conducted to determine
the cause of loss of DDT in the 1968 studies at Gulf Breeze.
The experiments were repeated under similar conditions with
the exception that duplicate samples were analyzed. In 1968
the salinity was 29.8 ppt (parts per thousand) and the
incubation temperature averaged 29 C; in 1973, the salinity
was 24.0 ppt and the incubation temperature averaged 12 C.
Figure 1 shows the percentage recovery of DDT (including DDE
and DDD) during the two experiments. The results are
similar except for the 17 day 1968 analysis.
Experiments were then performed to determine if DDT was
adsorbed to the walls of the test containers. Additional
experiments were designed to determine if DDT was converted
to DDA, a water soluble metabolite of DDT, which would not
have been detected by the method of analyses used in the
initial study. These experiments showed that less than 1%
of the DDT was adsorbed to the walls of the glass bottles
andd furthermore that there was no conversion to DDA.
Since petroleum ether was the solvent used for
extracting the DDT from sea water, the following studies
were initiated to evaluate the extraction efficienceies of
other solvent systems. Duplicate one-gallon bottles of
clear glass, containing 3.5 liters of sea water or distilled
water, were fortified with 10.5 jug of p,p* DDT in 350 uf. of
acetone to yield a concentration 3.0 ppb. Duplicate 500 ml
samples were taken from each bottle and extracted with one
of the following solvents: three 50 ml portions of
petroleum ether, two 50 ml portions of 15% ethyl ether in
hexane followed by one 50 ml portion of hexane, or three 50
ml portions of methylene chloride. All solvents were dried
with sodium sulfate, concentrated to an appropriate volume
and analyzed by electron capture gas chromatography. Just
prior to extraction, all samples were fortified with o,p*DDE
to evaluate the integrity of the analyses. The recovery
rate of o,p«DDE in all tests was greater than 89%,
indicating no significant loss during analyses.
After initial sampling, the bottles were sealed and
incubated at 20 C under controlled light conditions (12
hours light, 12 hours dark). Duplicate samples of 500 ml
were extracted at various time intervals.
Tables 2-5 show the average percentage recovery of
p,p*DDT extracted from duplicate sea water or distilled
-114-
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100
(Jl
I
0
PERCENTAGE RECOVERY OF DDT AND
METABOLITES IN 1968-1973 EXPERIMENTS
Figure 1. Percentage Recovery of DDT and Metabolites in 1968 and 1973 Experiments
-------
TABLE 2
PERCENTAGE RECOVERY OF P,Pf DDT FROM SEA WATER
BY DIFFERENT EXTRACTION SOLVENTS
EXTRACTION SOLVENT
DAY
PETROLEUM
ETHER
93
67
15% ETHYL ETHER
IN HEXANE
93
66
METHYLENE
CHLORIDE
93
76
-------
TABLE 3
PERCENTAGE RECOVERY OF P,Pf DDT FROM SEA WATER
BY PETROLEUM ETHER AND METHYLENE CHLORIDE
DAY
PETROLEUM ETHER
90
67
METHYLENE CHLORIDE
95
85
-------
TABLE 4
PERCENTAGE RECOVERY OF P,P' DDT FROM SEA WATER AND
DISTILLED WATER BY PETROLEUM ETHER AND METHYLENE CHLORIDE
SEA WATER
DISTILLED WATER
DAY
0
14
PETROLEUM
ETHER
90
58
46
METHYLENE
CHLORIDE
94
78
68
PETROLEUM
ETHER
90
90
94
METHYLENE
CHLORIDE
91
91
92
-------
TABLE 5
PERCENTAGE RECOVERY OF P,PfDDT FROM SEA WATER
INCUBATED UNDER DIFFERENT LIGHT AND TEMPERATURE CONDITIONS
DAT
12 HOUR LIGHT AND
12 HOUR DARK AT 20 C
DARK AT 5 C
87
88
69
81
68
86
-------
water samples up to 14 days after initiation of the
experiment. p,p*DDE was the only metabolite measured, and
since it never exceeded 216 of the parent compound it is not
included in the percentage recoveries. The sea water was
collected adjacent to the Gulf Breeze Laboratory in Santa
Rosa Sound and the salinity ranged from 16 ppt to 21 ppt.
Table 2 shows that immediately after the sea water (21
ppt) was fortified with 3.0 ppb of DDT all solvent systems
removed 93% of the DDT. After six days of incubation this
level of recovery was not observed with any of the solvents
tested. However, methylene chloride was more efficient than
petroleum ether or 15X ethyl in hexane. Part of this
experiment was repeated with sea water (16 ppt) and
incubated for 4 days with similar results (Table 3) .
An experiment was performed with sea water (21 ppt) and
distilled water using petroleum ether and methylene
chloride. Table 4 shows that immediately after
fortification, recoveries were greater than 90S for water
and solvents.
After 14 days, similar recoveries were observed only in
distilled water. In sea water however, there was 49S and
28% reduction in recovery with petroleum ether and methylene
chloride respectively. Since distilled water is devoid of
particulate matter, this study suggests that DDT may be
absorbed or adsorbed to plankton or particulate matter in
sea water and the sorbed material was not removed resulting
in low recoveries of DDT. This would explain the initially
high extraction efficiency of DDT followed by the decline in
recovery as DDT was associated with the particulate phase.
Since methylene chloride was the most polar solvent used, it
would have a greater affinity for removing the sorbed DDT.
In another test, duplicate bottles containing sea water
(20 ppt) and DDT were incubated under controlled lighting
conditions at 20 C and another set incubated at 5 C without
light. Both were extracted with methylene chloride at
various time intervals. Table 5 shows low recovery at 14
days under controlled lighting condition. However, those
samples incubated at 5 C in total darkness did not show a
significant decrease in recovery rate. Since the metabolic
activity of plankton was probably inhibited under these
temperatures and lighting conditions, these results suggest
that DDT may be absorbed rather than adsorbed by plankton.
However, Rice and Sikka (13) comparing the uptake of DDT by
-120-
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living and dead algae found that cells accumulated equal
amounts of the pesticide.
Interaction of pesticides between water and particulate
matter are complex. Not only do light and temperature
appear to alter equilibria, but other physical and chemical
factors have effects. Evaluating liquid-liquid extraction
techniques of herbicides from riwer water, Suffet (14)
observed that the isopropyl ester of 2,4-D was adsorbed to
particulate matter in river water and that the amount
changed by alteration of the pH of the water. Huang and
Liao (10) found that adsorption of DDT to clays was rapid
but the amount differed with the type of clay. A mixed
culture of algae consisting mainly of Vaucfaenia had a
greater adsorption for DDT than bentonite according to Hill
and Mccarty (9). Cox (5) reported that in natural marine
populations virtually all of the DDT available for uptake
was incorporated onto phytoplankton, but this may only
account for 10X of the DDT residues recoverable from whole
sea water.
These experiments support the work of other
investigators in that DDT and other pesticides are extremely
hydrophobic and can easily be adsorbed or absorbed by
suspended matter from liquid solutions. The 1968
experiments at Gulf Breeze supports the concept that
physical or chemical transformations of pesticides altered
the extraction efficiences of the solvent and prevented
complete recovery of the compounds. Obviously, additional
work needs to be done to account for all of the chemical
added to the test system.
It is difficult to relate laboratory findings directly
to these of the estuary or open oceans. However, the
laboratory data illustrate clearly some problems that could
be encountered in monitoring sea water for pesticide
pollution. The conventional analyses of water samples by
liquid-liquid extraction techniques may provide invalid data
if suspended matter is not considered. Standardized methods
are needed to analyze the water column and suspended
material separately.
Recently, a synthetic resin, Amberlite XAD-2 was
evaluated as an adsorption medium for chlorinated
hydrocarbons dissolved in sea water by Harvey (8). This
technique utilizes large volumes of sea water and therefore
permits greater sensitivity in analysis. In addition, the
method eliminates the problems encountered in transport of
large samples of water. Pollutants could be adsorbed on the
resin at the sampling site and shipped to the appropriate
-121-
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analytical laboratory for desorption. Since tee resin only
removes the dissolved portion, additional samplings would be
required to determine the levels absorbed or adsorbed to
particulate matter.
ANALYTICAL QUALITY CONTROL
All pesticide residue laboratories should maintain an
adequate analytical quality control program. The program
should include both an intra-laboratory performance
evaluation of personnel and methodology and an
inter-laboratory sample exchange program. These programs
are time consuming but are essential to the generation of
valid analytical data.
Table 1 shows the recovery rates of oyster samples
fortified with known concentrations of pesticides.
Fortification techniques provide data only on the recovery
efficiency of the total analytical procedure and not on the
extraction step. Field residues may be subject to physical
and chemical transformation and therefore raay not be in the
same physical or chemical state as the fortified sample.
Table 2 illustrates the errors that can result from
fortified samples. Extraction of water samples immediately
after fortification yields relatively high recovery
efficiency with all solvent systems in sea water. Analyses
several days later show the relative inefficienceis of the
solvents systems used in sea water. Regardless of the
analytical method used or the substrate being extracted,
recovery data must be obtained on the extraction efficiency
and the total analytical procedure.
There are several other factors in residue laboratories
which, if ignored, may lead to inaccurate data. To name a
few: (a) all glassware must be clean and free of residues;
(b) the purity of all reagents used during analyses must be
determined; (c) the accuracy of analytical standards must be
maintained; (d) the condition of all components of the gas
chromatograph must be optimized; and (e) laboratory
personnel should be thoroughly trained.
An area that needs further study is the use of internal
standards in marine pesticide monitoring. Currently, the
Gulf Breeze Laboratory is no longer affiliated with a large
monitoring program. Almost all samples submitted for
analysis are of known identity. Most of these samples are
fortified with an internal standard, just prior to analysis.
The standard is usually a compound which behaves in an
analytically similar way to the compound of interest. This
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technique is valuable in assessing the validity of the
analysis. This same technique could also be applied to
marine monitoring samples if an appropriate compound could
be found that would not interfere with the monitored
pesticides. It would be extremely valuable in laboratories
with large sample volumes where close supervision of
laboratory operations is not possible.
The analysis of marine samples for chlorinated
hydrocarbon pesticides is at times complicated by the
presence of polychlorinated biphenyl compounds (PCB). These
compounds are industrial pollutants and are produced in the
United states under the trade name Aroclor. They have
chromatograph retention times similar to the organochlorine
pesticides and therefore complicate the analysis when both
are present in a sample, several techniques have been
described for the separation of PCB from organochlorine
pesticides. A review of these methods were presented by
Zitko and Choi (17). These techniques are time consuming
and, in general, semiquantitative, In addition,
differential absorption or metabolism of the Aroclor isomers
in marine biota prevent accurate analysis of the PCB's. In
view of these facts and the large number of samples that
could result from a global monitoring program, this area of
analysis required further study and/or standardization.
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REFERENCES
1. Bowman, M. C. and M. Beroza, 1965. Extraction p-Values
of Pesticides and Related compounds in Six Binary
Solvent Systems. J. Asspc. Agr. Chem. 48: 943-952.
2. Butler, P. A., 1966. Pesticides in the Marine
Environment. J. Ajajjl. Ecol. 3 (Suppl): 253-259.
3. Butler, P. A., 1973. Organochlorine Residues in
Estuarine Molluscs 1965-1972 National Pesticide
Monitoring Program. jPestJys. Monit. J. 6: 238-362.
4. Cox, J. L., 1971. DDT Residues in Seawater and
Particulate Matter in the California current System.
y- §- Ii§h. Wildl. Serv. Fish. Bull. 69: 443-450.
5. Cox, J. L., 1972. DDT Residues in Marine Plankton.
B§§i
-------
11. Mills, P. A.f J. F. Oaley and E. A. Gaither, 1963.
Rapid Method for Chlorinated Pesticide Residue in
Non-Fatty Foods. J. Assoc. Agr. Chem. 46: 186-191.
12. Patil, K. C. , F. Matsumura and G. M. Boush, 1972.
Metabolic Transformation of DDT, Dieldrin, Aldrin
and Endrin by Marine Microorganisms. Environ. Sci.
Techno^. 62 629-632.
13. Rice, C. P. and H. Sikka, 1973. Uptake and Metabolism
of DDT by Six Species of Marine Algae. J. Agr. food
Chem. 21: 148-152.
14. Suffet, I. H., 1973. The p-Value Approach to
Quantitative Liquid-Liquid Extraction of Pesticides
and Herbicides from Water. 3. Liquid-Liquid
Extraction of Phenoxy Acid Herbicides from Water.
J. Agr. Food Chem. 21: 591-598.
15. Wilson, A. J., J. Forester and J. Knight, 1970.
Chemical Assays. 1969 Prog. Rep., Center for
Estuarine and Menhaden Research, Gulf Breeze, Fla.
0. S. Fish Wildl. Serv. CJ.rc. 335: 18-20.
16. Wolman, A. A. and A. J. Wilson, 1970. Occurrence of
Pesticides in Whales. Pestic. Monit. J. 4:8-10.
17. Zitko, V. and P. Choi, 1971. PCS and Other Industrial
Halogenated Hydrocarbons in the Environment. Fish.
Res. Board Can. Technical Report No. 272, Biological
Station, St. Andrews, N. B.
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BIOLOGICAL PROBLEMS IN ESTUARINE MONITORING
P. A. Butler *
INTRODUCTION
Awareness of the extent of persistent organochlorine
pollution both in the physical environment and the biota has
made apparent the need for continuing surveillance programs
to assess the problems. During the period 1965-72, samples
collected in the National Estuaries Monitoring Program, as
well as in other studies, revealed some of the difficulties
involved in the interpretation of residue data. Bivalve
molluscs are efficient bioassay tools for identifying the
ebb and flow of pollutants in surrounding waters, and the
monthly monitoring of molluscan populations made obvious the
dynamic nature of organochlorine pollution in the estuary.
The image of polluted versus unpolluted estuaries was soon
modified by monitoring data that indicated instead the
movement of relatively discrete masses of clean and polluted
water through the estuary. Organochlorine residues in
molluscs fluctuated from month to month in response to the
sometimes transitory nature of the pollution and contrasted
sharply at times with residues observed in other elements of
the associated biota.
To gain increased understanding of the significance of
residue levels, sample collections in problem areas were
intensified in variety, frequency, and size; and additional
work was undertaken under laboratory conditions. The
experimental program demonstrated that the uptake and
retention of persistent residues varied unpredictably with
the environmental element sampled. It became clear that
surveillance or monitoring systems had to be carefully
designed if they were to provide answers to specific program
objectives.
*Gulf Breeze Envivonmental Research Laboratory,
Gulf Breeze, Florida
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A primary concern is the extent of environmental
degradation - with the implied intention of reversing or at
least halting harmful pollution trends. But individual
agency goals are much more specific. For expedience and
because of monetary restrictions, programs are usually
designed to clarify rather narrowly defined sectors of the
whole pollution picture. Often, the foremost question
concerns only the existence of a human health problem, and
because of some environmental pollution problems may go
uninvestigated. The idea that any changes harmful to the
environment will eventually affect man is still not
generally accepted.
The basic needs for environmental surveillance require
programs designed to provide current data that will be
adequate to identify later deleterious changes. Monitoring
data should indicate existing problems as well as their
extent in time and space. To provide information of this
type, protocols must take into account the influence of
various physiological and ecological factors affecting the
sample selected for study. Some of the anomalous residues
data acquired so far are understandable, but reasons for
others are less certain. The following discussion of
factors affecting persistent organochlorine residues
indicates some of the options available in selecting the
most informative sample types.
FIELD AND LABORATORY OBSERVATIONS
Residue differentials resulting from kind of species
monitored;
The necessity for utilizating different species to
monitor pesticides in different coastal areas prompted the
conduct of laboratory experiments to determine the relative
sensitivity of molluscan species selected for their
diversity in salinity tolerance. A number of controlled
experiments have shown the relative uniformity of DDT
residue formation in the eastern oyster (Crassostrea
Y-irainica) under varying estuarine conditions of salinity
and temperature. observations of fcur other molluscan
species exposed simultaneously to a mixture of common
organochlorine pesticides indicated considerable variation
in the relative rates at which residues were acquired, then
lost when clean water was restored to them (Table 1) (3) .
Studies were directed toward evaluating residue flushing
rates in the hard clam (Mercenaria mercenaria) because of
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Bioassay animal
Magnification in
Body after 5
days exposure
Percentage loss
after 7 days
in clean water
Soft clam {Mya arenaria)
Eastern oyster (Crassostrea
yirginica)
Marsh clam (Ranqia cuneata)
Asiatic clam (Corbicula f luminea)
Hard clam (Mercenaria mercenaria)
3000
1200
700
600
500
7u
50
50
30
75
Table 1. Average biomagnification and depuration rates of a
of seven common chlorinated pesticides by molluscs exposed
in a flowing seawater system.
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the prevalence of this species in the New England area and
its observed poor performance in storing DDT residues. The
biological half-life of pesticide residues in molluscs under
the same conditions is of importance in determining the
movement of pollutants in the estuary. Clams and oysters
with DDT residues of about UO ppm were placed in aquaria
with uncontarainated flowing seawater. The clams flushed out
50 percent of their DDT residue within five days. At the
end of 15 days less than 2% of the original DDT burden
remained. In contrast, the oysters still contained 50% of
the residue at the end of 15 days and significant amounts of
DDT were present after a month. Clearly, clams would have
to be sampled more frequently than oysters to determine
pollution inputs.
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Uptake and depuration rates in molluscs under
laboratory conditions must be accepted as relative figures
and extrapolated to field conditions with caution. In one
study, for example, oysters exposed to mercury accumulated
residues of about 28 ppm (8). Relatively small declines in
residues took place in the following 160 days of depuration.
The authors concluded from the data that mercury residues
acquired by oysters in nature might require years to decline
to acceptable levels. Their experimental conditions were
described as a 'natural system1 but the oysters were
supplied only 2 i clean water/animal per hour. This volume
of water should be compared to their well-known utilization
of up to 30 Si per hour even under experimental conditions
(11) . It is probable that in this experiment, the mercury
was continuously recycled and the oysters never had
opportunity to adequately flush their tissues.
The mercury exposure experiment is perhaps comparable to
a situation monitored in Mecox Bay, Long Island (6). The
water of this bay are typically isolated from the ocean by
sand bars, and only periodically do winter storms cut the
bars and permit flushing (10). In the period 1966-72, the
oysters in this bay were continuously contaminated with DDT
and at a maximum level higher than that observed in any of
the other 16 estuarine stations monitored in the New York
despite apparently low use records of DDT in the area. In
my opinion, these oysters were continuously contaminated
with recycled DDT because of the inadequate supply of clean
water.
Species differentials in pesticide uptake are apparent
in populations monitored in Conscience Bay, New York (6).
During the first half of 1968, monthly mussel samples
(MY-tilus ®<|ujyL§) from this bay contained about 50 ppb of DDT
and its metabolites. In the latter half of the year and
until April 1969, hard clam was substituted because it was
easier to collect. During this period no DDT residues were
detected. In April and thereafter, the mussel was again
utilized and DDT residues were found to be once more 50 ppb
or higher. In this case, the convenience of collecting hard
clam samples 'determined' the presence or absence of DDT
pollution in the bay.
Similar discrepancies were observed in simultaneous
collections of spot (Leiostomus xanthurus) and oysters in a
south Carolina estuary in 1968. Residues of DDT occurred in
all monthly samples of the fish and levels fluctuated
between 100 and 300 ppb although the fish sampled were of
uniform size. Oyster samples showed DDT pollution was
present only during the first six months of the year. If
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DDT pollution was present after that, it was not stored by
the oyster at levels chemically detectable. Numerous other
studies have demonstrated the retention and gradual increase
of organochlorine residues in fish, at least until their
first spawning period. Consequently, it is not possible to
determine from periodic fish analyses the seasonal
occurrence of DDT pollution in an estuary.
Unexpected variations occur in the sensitivity or
selectivity of the biota in the same ecosystem to
organochlorine pollution even though the species monitored
are presumed to occupy similar tropic levels. These
differences are well-illustrated in a study of monitoring
methods conducted in Virginia (12) . Sample analyses
demonstrated the existence of residues of three different
polychlorinated biphenyl compounds {Aroclors 1242, 1254,
and 1260) in different types of sairples. All samples were
large enough to minimize individual variation. In March,
Aroclor 1242 was detected only in anchovies (Anchoa sp.);
Aroclor 1254 was present in silversides (Menidia sp.),
oysters and two series of plankton tows. A third series of
plankton samples contained only Aroclor 1260. Three months
later, Aroclor 1242 was present in both anchovies and
silversides while Aroclor 1254 was no longer found in
silversides but was still present in oysters and plankton.
Aroclor 1260 was not detected. These variations in residue
accumulations in a short period in cne river system are not
easily explained {Table 2).
March June
Aroclor 1242 Fish A I/ Fish A
Fish S
Aroclor 1254 Fish S 2/
Oysters Oysters
Plankton Plankton
Sediment Sediment
Aroclor 1260 Plankton
I/ Anchovies
2/ Silversides
Table 2, Estuarine samples containing residues of PCB's at two
sampling periods
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Past dependence on fortuitous samples (found dead,
happened to be caught in a net, etc.) to assess pollution
levels has resulted in an uneven if not confused picture of
general environmental contamination. Eroad community
studies frequently show persistent organochlorine residues
differing by an order of magnitude in species of similar
habit (9, 20). Consequently, valid assessment of persistent
residues in any one species of a community requires not only
an understanding of its position in the trophic structure
but also its variability as compared to similar species.
Residue differentials resulting from age of individuals
monitorgd.
In fish and other vertebrates, the localization of
organochlorines is highly lipid tissues and their
persistence, at least in part, for long periods is well-
documented. For example, DDT tends to accumulate gradually
in pinfish (Lagodon rhomboides) and an approximately tenfold
increase from 0-to-l-year fish has been documented (14).
There is an approximate doubling of DDT residues in lake
trout in the Great Lakes each year up to age 10 (17).
Accumulation of dieldrin residues in these fish are
proportional to but age occurs at lower magnitude.
Approximate doubling of mercury levels during the first few
years has been reported in salmon (Salmo salar) from rivers
in Sweden (18) .
In contrast, organochlorine residues in molluscs do not
persist from year to year in the absence of pollution
despite their affinity for lipid tissues. Residue levels
fluctuate widely depending on the input of pollutants to the
estuarine system and not on the age of the mollusc. Residue
levels can be correlated frequently with pesticide usage in
the drainage system when oysters of similar size are
monitored. However, oysters of different size do react
differentially to similar pollutant levels and, in general,
residues per gram of tissue are higher in small oysters than
in oysters of significantly larger size. Although the data
are not at hand, I assume that this difference is a function
of the larger amount of water circulated (filtered) per gram
of tissue in small oysters compared to large ones. This
higher biological magnification of residues in small oysters
might be disasterous since, with a given amount of
contaminated tissue consumed, predators would ingest much
more DDT from small as opposed to large oysters.
This demonstrated increase in persistent residues with
age complicates interpretation of data in many instances
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where the age of individuals making up the sample are
unavailable and difficult to ascertain.
Residue differentials resulting from variations among
individuals.
For obvious reasons, uniform populations or aggregates
of individuals are selected as often as possible as
indicators of environmental degradation. But both
laboratory and field studies demonstrate that there can be
much individual variation in levels of organochlorine
residues. DDT residue levels in a presumably uniform sample
of yearling pinfish in a Florida estuary ranged from 13.7
ppm to 0.5 ppm (1«»). The standard error in these data was
more than 23OX of the arithmetic mean. The average in two
of the fish was 13.2 ppm while the average residue in the
other eight fish was only 1.5 ppm DDT. Not all groups
analyzed have shown this diversity but it must be
anticipated in data interpretations. Diversity in residue
levels of molluscan populations is less extreme and may be
illustrated by one group of mature oysters exposed to DDT
under controlled conditions in flowing seawater for 96
hours. In the 10 individuals, total DDT residues ranged
from about 4 to 25 ppm with a mean value of 11.6 ppm. In
this case, the standard deviation was only about 50% of the
mean.
Much larger samples must be collected to obtain more
uniform data, but this is a costly and time-consuming
process. We undertook in 1971 as exploratory project to
examine the merits of larger sample size (12). Oysters, two
species of fish, plankton and sediment cores were collected
concurrently at two stations in two Virginia rivers at a
two-month interval. Ten samples of 15 oysters and 10
samples of 25 fish were collected at each station. In
general, PCB«s were the principal residues found and the
spread of values was reasonably small. Standard errors, for
example, were about 15 to 30% of the arithmetic means. Data
on sediment cores were less uniform even though samples were
collected in a restricted area. In one series, for example,
only 3 of 10 cores had measurable residues of PCB. This
means that had only replicate cores been taken there would
have been about a 50-50 chance that one of the cores would
have contained a residue. These data are significant in
light of the fact that sometimes only a single sediment core
is collected to assess pollution in an entire estuary.
Numerous studies have shown that the magnitude of
persistent residue levels in sediments is usually inversely
proportional to grain size. In consequence, care must be
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exercised in selecting representative sample sites, as well
as in analyzing an adequate number of samples.
Despite the mathematical pleasure in achieving
uniformity in sampling results, it must be emphasized that
averaging data, either in the electric blender or the
calculator, may lead to serious management errors. Animals,
in general, are not responsive to average pollution levels,
they survive and flourish as a result of environmental
extremes. A single high incidence of endrin in the
environment can be satisfactorily averaged away on paper,
but at the time of its occurrence all of the endemic animals
may have been killed.
There is a further important consideration in assessing
the importance of organochlorine residues in aquatic biota.
It is axiomatic that, with a given level of environmental
pollution, the sensitive species and the sensitive
individuals of more tolerant species will be affected first.
In one experiment, pinfish were fed a diet contaminated with
about 4 ppm of DDT (4). At the end of the 14th day, the ten
surviving fish were sacrificed and found to have average
residues of about 4 ppm. The 25 fish dying during the 2-
week period averaged about 0.6 ppm of DDT, less than 1/6 as
much as the resistant individuals, obviously, the magnitude
of residues occurring in apparently healthy populations is
not necessarily an indication of tolerable pollution levels.
Residue differentials showing seasonal variations.
Monitoring programs and laboratory studies in which
periodic samples have been collected at sufficiently short
intervals may show clearly defined seasonal variations in
residue levels. Such cyclic changes in the presence of
relatively constant pollution loading are indicative of
basis physiological changes in the monitored species. For
example, studies of speckled seatrout in Texas showed a 75%
decrease in gonad DDT residues in mature fish in the late
fall (7). Lowe reported a more than 50% decline in DDT and
a 70* decline in toxaphene residues in oysters continuously
exposed to these pollutants (15). Oysters exposed
simultaneouly to PCB, dieldrin and DDT in the laboratory
lost from 45 to 8Q% of the residues within a short period
(16). Whole body residues of mercury in oysters are also
reported to decline seasonally in a manner similar to the
organochlorine compounds (8).
In each case, the abrupt declines have been clearly
identified with the normal spawning period of the animal in
question. That there should be a significant percentage
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loss of such residues on a seasonal basis is entirely
predictable in view of the localization, for example, of DDT
oyster gametes (1).
Seasonal declines in levels of organochlorine residues
in oyster are also clearly associated with fluctuating
levels in the input of pollutants into the aquatic system.
In southwest Florida, the former agricultural use of DDT was
intensified just prior to the harvest of sweet corn and
sugar cane. Residue levels of DDT in oysters collected
monthly in an associated river basin reflected this
management practice; peak DDT residues in oysters were as
much as lOOOx higher than minimal residue levels in 1967-68
(6).
Organochlorine residue patterns in estuarine biota
resulting from agricultural and industrial practices may be
masked by the effects of marked changes in river discharge
in the drainage basin because of rainfall variations.
SS§i^S§ §ifl§£«Qtials affected bv. body region monitored.
Regardless of the mode of entry of organochlorine into
living tissues, there is a partitioning and partial
immobilization of these compounds in fatty tissues because
of their lipophilic nature. Such segregation is clearly
demonstrated in fish in which adipose tissues are highly
localized. In coho salmon, for example, DDT residues in a
mid-body fillet range from an average of about 65 ppm in
fatty tissues to less than 6 ppm in the muscle. The whole
body DDT residue of similar fish was about 12 ppm (17) .
As discussed above, residue levels increase with age in
fish. In aquatic mammals the localization of residues in
older individuals may be even more striking. In a dead
porpoise (Tursiogs truncatus) found near Pensacola, Florida,
total DDT residues ranged from about 1.5 ppm in blood, 7 ppm
in the muscle, 9 ppm in the brain to 33 ppm in the liver,
and more than 500 ppm in the blubber (13) . Even in oysters,
where total body fat is only about H%f there is a marked
localization of DDT residues in the digestive gland, and,
seasonally, in the gonad which contains more fat than other
organs.
In general monitoring or surveillance programs, the
localization of persistent residues in particular body
regions has little importance from the point of view of
either human health or resource protection so long as the
monitored species is small enough to be analyzed on a whole-
body basis. Residues large enough to warrant further
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investigation will show up in such analyses regardless of
their location in the body.
It is quite another matter, however, if as in market-
basket surveillance programs, only the products, e.g.,
lobster and shrimp tails or tuna muscle, are examined. It
is conceivable then the edible portions would contain
negligible organochlorine residues while the discarded body
parts could contain amounts detrimental to the productivity
of the animal itself or to other animals preying on it in
nature.
SUMMARY
Successful monitoring of the estuarine environment for
persistent organochlorine pollutants is dependent in large
measure on the collection of appropriately biased samples.
Statistically randomized sample collections are
unsatisfactory for the simple reason that pollution patterns
are not random. The transport of persistent residues are
dependent on a large number of biological factors which in
turn are modified by physical and chemical parameters of the
environment.
Monitoring programs have several basic functions and
requirements. First, they should record existing residues
of persistent pollutants that occur at significant trophic
levels in aquatic ecosystems. Sample collection protocols,
as well as analytical procedures, must be sufficiently
standardized to ensure the comparability of data, not only
from one area to another but also from year to year. It is
essential that monitoring programs collect comparable data
for sufficiently long periods of time so that pollution
trends can be identified. Finally, it should be stressed
that monitoring data must be transmitted on a timely basis
to action agencies. Agencies mandated to identify and
regulate pollution sources, agencies with resource
protection responsibilities, and agencies concerned with
human welfare must have clearly established communications
channels with environmental monitoring programs.
ACKNOWLEDGEMENTS
I wish to thank Alfred J. Wilson and Jerrold Forester of
the Gulf Breeze Environmental Research Laboratory and Roy
Schutzmann of the Pesticides Monitoring Laboratory of the
Environmental Protection Agency for many of the chemical
analyses reported.
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LITERATURE CITED
1. Butler, Philip A., 1966. The problems of pesticides in
estuaries. Trans. Amer. Fish. Soc. Spec. Publ.
3:110-115.
2. Butler, Philip A., 1966. Pesticides in the marine
environment. J. Appl. Ecol. 3 (Suppl):253-259.
3. Butler, Philip A., 1968. Pesticide residues in
estuarine mollusks. in Proc. Natl. Symp. Estuarine
Pollut., p. 107-121, Stanford University, Stanford,
Calif., 1967.
4. Butler, Philip A., 1969. The significance of DDT
residues in estuarine fauna, in Chemical Fallout,
p. 205-220. C. C. Thomas, Springfield, 111.
5. Butler, Philip A., 1969. Monitoring pesticide
pollution. BioScience, 19 (10):889-91.
6. Butler, Philip A., 1973. Organochlorine residues in
estuarine molluscs, 1965-1972 - National Pesticide
Monitoring Program. Pestic. Monit. J., 6(4):238-
362.
7. Butler, Philip A., Ray Childress, and Alfred J. Wilson,
Jr. 1970. The association of DDT residues with
losses in marine productivity. In M. Ruivo
(editor), Marine Pollution and Sea Life, p. 262-266.
Fishing News (Books) Ltd. London.
8. Cunningham, P. A. and M. R. Tripp, 1973. Accumulation
and depuration of mercury in the American oyster
CJTassostrea virginica. Mar. Biol. 20:14-19.
9. Duke, T. W., J. I. Lowe and A. J. Wilson, Jr., 1970. A
polychlorinated biphenyl (Aroclor 1254) in the
water, sediment, and biota cf Escambia Bay, Florida.
Bull. Environ, contam. Toxicol. 5(2):171-180.
10. Foehrenbach, Jack, 1970. N. Y. State Dept. of
Environmental Conservation, personal communication.
11. Galtsoff, Paul S., 1964. The American oyster,
Crassostrea virainica Gmelin. U. S. Fish Wildl.
Serv. BullT 64:480 p.
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12. Gillespie, J. R., Jr., 1972. Estuarine Monitoring
Program - sample Evaluation Project. NMFS Contract
No. N-042-37-72(N). (unpublished)
13. Gulf Breeze Environmental Research LAboratory, Gulf
Breeze, Florida, unpublished data.
1U. Hansen, David J. and Alfred J. Wilson, Jr., 1970.
Significance of DDT residues from the estuary near
Pensacola, Fla. Pestic. Monit. J. 4(2):51-56.
15. Lowe, Jack I., Paul D. Wilson, Alan J. Rick, and Alfred
J. Wilson, Jr., 1971. Chronic exposure of oysters
to DDT, toxaphene and parathion. Proc. Natl.
Shellfish Assoc., 61:71-79.
16. Parrish, Patrick R., 1973. Chronic effects of three
toxic organics on the American oyster, Crassostrea
yirgjnica. Proc. Natl. Shellfish Assoc. Meeting.
June 1973~. Abstract.
17. Reinert, Robert E., 1969. Insecticides and the Great
Lakes. Limnos Magazine, Great Lakes Foundation,
2(3) :<*-9.
18. Reinert, Robert E., 1970. Pesticide Concentrations in
Great Lakes Fish. Pestic. Monit. J. 3 (<») :233-2«»0.
19. Westoo, G., 1973. Methylmercury as percentage of total
mercury in flesh and viscera of salmon and seatrout
of various ages. Science 181:567-568.
20. Woodwell, George M., Charles F. Wurster, Jr. and Peter
A. Isaacson, 1967. DDT residues in an east coast
estuary. Science, 156:821-824.
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DETERMINATION OF METALS IN SEA WATER
D. H. Kampbell
Surveillance and Analysis Division, EPA Region II,
Edison, New Jersey
1. INTRODUCTION
Pollution by heavy metals in the marine environment can
occur from waste dumping, direct wastewater discharge, and
from flow of polluted rivers. The precipitating action by
sea water salts on waste colloidal material should sediment
significant portions of the metals. The sheer volume of sea
water will greatly dilute the remaining portion.
Nevertheless, constant dumping or discharge of metals in
fixed regions over a long period of time can only be
detrimental.
Natural sea water is a complex mixture of virtually
every chemical element. It differs from fresh water by
having a much higher dissolved salt content. The salt
matrix causes interference problems with nearly all
conventional fresh water metal analytical methods. The
normal composition of individual metals in marine waters is
less than 20 g/1 (ppb). Localized pollution can increase
the level many fold. The analyst must give much effort and
attention to get consistent and reliable results for such
low concentrations.
2. METALLIC FORMS
The chemical forms of metals in sea water can be divided
into six fractions {Table I). They are inorganic
particulate, organic particulate, particle adsorbed,
inorganic complexed, organic complexed, and ionic.
The complexed and ionic can be classified as dissolved.
A method of analysis may detect all or only part of the
forms present. The fraction that responds will be dependent
on sample pretreatment and the analytical technique used.
For example, filtration should separate particulates from
dissolved fractions, Ion exchange resins may perform a
complete metal separation by retaining all fractions either
-139-
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TABLE I
METALLIC FORMS IN SEAWATER
Example
Inorganic Particulate
Organic Particulate
Particle Adsorbed
Inorganic Complexed
Organic Complexed
Ionic
Me S
Me In Plant Life
Me* On Colloidal Material
MeCl*-
Me-humates
Me*
-140-
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by chelation or particle entrapment. Chemical forms of
metals in sea water are variables that must be considered
when interpreting the analytical measurement.
3. SAMPLE PREPARATION
Great care is required during collection and preparation
of samples for them to be representative of a specific
marine locality. Metals loss or contamination in sample
containers, by chemical changes and by impure reagents, are
potential problems that need be controlled.
Most trace metal analytical methods are not capable of
detecting metals at the extremely low concentrations and in
the presence of salt matrix interferences characteristic of
sea water. Much of the matrix can be removed and the metals
enriched by chemical separation. Tested ways of separating
metals from sea water are precipitation, volatilization,
solvent extraction, ion exchange, and electrodeposition
(Table II).
Precipitation or crystallization is a classical method
of separation. A prepared reagent can be added to a sample
to collect the metals of interest. Generally, recoveries
are good, but separations tend to be nonspecific and time
consuming.
Volatilization has wide applicability in analytical
chemistry, The volatile separations of arsenic, selenium,
and mercury from their parent matrix are some common
applications. Evaporation of sea water to concentrate
metals is for all practical means useless because of the
large amount of salt residue obtained.
Solvent extraction has been widely used to separate a
single metal or a group of metals from water. Two popular
metal extractants are dithizone chelation in chloroform and
ammonium pyrolidine dicarbamate chelation (APDC) in
methylisobutyl ketone (MIBK). Solvent extraction is
relatively simple and rapid, but seldom does complete
extraction of metals occur.
Electrolysis has been used to separate metal ions from
sea water by deposition onto an electrode. Deposition
behavior is influenced by many variables and must be closely
controlled. The anlytical method of anodic stripping
polarography is an application of the technique.
-141-
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TABLE II
TECHNIQUES FOR CHEMICAL SEPARATION
Precipitation
Volatilization
Solvent Extraction
Ion Exchange
Electrodeposition
-142-
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4. ANALYTICAL APPROACH
Refined instrumentation has proliferated the methods for
trace metal analysis (Table III).
Atomic absorption is widely used for metals analysis.
The instruments are durable and will detect some 40
elements. Detection is quite specific. Sensitivity is
frequently near or lower than 1 ppm. Flameless atomic
absorption is even more sensitive, but high background
attenuation caused by sea water makes a precise direct
analysis difficult.
Gas chromatography has been used to analyze metals as
specific organocomplexes. The technique should have parts
per billion detection ranges with an electroncapture
detector. Sample preparation would require solvent
extraction after formation of specific metal-chelates.
Anodic stripping voltammetry is an analytical
application of electrochemistry. Metallic concentrations in
water as low as 10-*° molar are detectable with 100 ml
samples. Measurements must be carefully controlled to be
reproducible and accurate. Anodic stripping voltammetry has
been applied to shipboard analyses of Cd, Cu, Pb, and Zn.
The instrument responds to free metal ions or complexes that
break down during analysis. Selective ion probes may
eventually provide a continuous monitoring system for heavy
metals. Ion electrodes are available for detecting Cu, Pb,
Ag, and Cd, but their application to sea water is limited.
Metals have long been determined by emission
spectroscopy. It is very specific and can assay a sample
for amny elements, simultaneously. Preliminary metal
separation from sea water is required to attain detectable
amounts and to reduce serious matrix interferences.
Spark source mass spectrometry could furnish a rapid and
sensitive method of metals analysis, if matrix interferences
from sea water are overcome. Costs for instruments,
maintenance, and personnel make this a very expensive
method.
Neutron activation analysis has been applied to sea
water. Detection limits are within the range of a normal
metals content of sea water. Multielement determinations
can be done. Interferences from radioactive sodium would
require chemical separation or long storage periods. The
-143-
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TABLE III
PHYSICAL TECHNIQUES FOR MTALS ANALYSIS
Atomic Absorption Spectrosoopy
Chromatography
Electrocherai stry
Emission Spectroscopy
Mass Spectrcwetry
Radiochemistry
Spectrophotometzy
X-Ray Spectroscopy
-144-
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equipment is very expensive. The analysis techniques
require highly trained laboratory personnel.
Spectrophotometry methods are capable fo providing
reliable analyses at the ppm and below level for many
metals. Spectrophotometers are durable and inexpensive
instruments. Many physical and chemical factors influence
precision and accuracy.
Multispectrometer X-ray fluorescence instruments are
available that will detect up to 20 elements in a solid
sample, simultaneously. Solid state detectors have improved
detection limits, but not enough for the low metals level in
dried sea water salts. Sea water sediments could be rapidly
analyzed by x-ray fluorescence.
5. APPLICATIONS
The arsenic level in sea water has been reported to be
3 jig/1 (30) . A popular method of arsenic analysis has been
by atomic absorption {13} with a closed system arsine
generator. The detection limit of 5 jag/1 can be lowered
slightly by concentrating the sample before analysis. This
can be done by water loss during a heated-digestion step.
Differential pulse polarography has been used as an
analytical technique for arsenic (15). Other inorganic ions
may interfere, but the method is rapid and will detect
arsenic in the pg/1 range. Portmann and Riley (19) have
described a way to separate and concentrate arsenic from sea
water by co-crystallization with thionalide. The
concentrate was reacted to form a molybdate blue complex for
spectrophotometric measurement. They reported an arsenic
recovery of 98%. The analysis required three days to
complete.
The amount of beryllium in the open sea has been
reported to be about 0.0002 jag/1 (14). Ion exchange was
used to concentrate the beryllium to a level that could be
detected by emission spectroscopy. The beryllium detection
limit by conventional atomic absorption is about H orders of
magnitude higher than that normally found in the sea. A
very large sample would be needed to obtain a detectable
concentrate, solvent extraction of chelated beryllium
followed by detection with gas chroraography has possible
-145-
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applications for sea water analysis. Ross and Sievers (25)
have established a detection limit of 4 x 10~»*g Be with
electron-capture detection of trifluoroacetylacetone
chelated beryllium.
Cadmium
The quantity of cadmium usually present in sea water is
near 0.1 ;ug/1 (23). Both neutron activation analysis and
anodic stripping voltammetry have sufficient sensitivity for
direct measurements at such a low concentration. Anodic
stripping voltammetry is more easily available and suitable
for water chemists. A method using anodic stripping
voltammetry for the determination of Cd, Cu, Pb, and Zn in
sea water has been reported by Rojahn <2t). His technique
was rapid, sensitive, and the reproducibility was very good.
Atomic absorption is an often used way to determine cadmium
in high salt systems after ion exchange concentration and
separation from the parent matrix. Although ion exchange
separation is time consuming, it is simple and with care,
precision can be within 515. The method for cadmium analysis
used in our laboratory is similar to that reported by Windora
and Smith (32), except we use Dowex A-1 resin. A more rapid
determination of cadmium at higher levels can be made by
solvent extraction (10).
Chromium
The level of chromium in typical sea water is about O.t
jig/1 (8) . concentration by one order of magnitude will
place the chromium level within the detection limit of
conventional atomic absorption. Solvent extraction by
APDC/MIBK will accomplish the task after Cr3* is oxidized
because only Cr6* chelates with the APDC (16), The
concentrate is easily within the detection range of analysis
by atomic absorption equipped with a flameless graphite
furnace device (18). Chau, Sim, and Wong (7) used a
technique that lowered the analytical range for chromium by
atomic absorption to 0.2 »g/1 with a reproducibility of
_*0. 06 jig/1. They extracted chromium acetylacetonate into
MIBK after co-precipitation of chromium with Fe (OH) from
liter samples of sea water.
Cobalt
The cobalt content of sea water is about 0.1 jug/1 (30).
Cobalt has been satisfactorily determined by atomic
-146-
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absorption after separation and concentration from sea water
by either ion-exchange (32) or solvent extraction (12, 18).
Cobalt as low as 0.001 ftg/I can normally by measured in
neutron activated sea salts after a two-month storage
period.
Copper
The copper content of sea water is about 1 jug/1 (30).
Copper has been analyzed by atomic absorption after
concentration by solvent extraction (28) or ion exchange
(32). Riley and Taylor (21) report that copper can be
retained quantitatively from filtered sea water by
Chelex-100 and eluted completely with 2N nitric acid.
Kuwata, Hisatomi, and Haseqawa (10) successfully extracted
copper from sea water with sodium diethyldithiocarbamate
into MIBK. Rojahn (24) has demonstrated that anodic
stripping voltammetry works veil for direct analysis of
copper in sea water.
Iron
The amount of iron normally present in sea water is near
10 Jug/1 (30) . Numerous colorimetric methods are available
for determination of soluble iron in sea water. The
phenanthroline color is commonly used. A procedure has been
described by Strickland and Parsons (29). The method is
practically free of interferences from normal sea water
constituents. Segar and Gonzales (26) have demonstrated
that iron can be determined directly by atomic absorption
with a flameless graphite furnace device. Iron can also be
analyzed by flame atomic absorption after concentration by
solvent extraction (28).
-147-
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Lead
Lead in sea water is normally found at a concentration
of 0.03 jag/1 <30). A good anodic stripping voltammetry
technique for shipboard analysis for lead has been described
by Zirino and Healy (33). Rojahn (24) reported good
reproducibility for lead analysis in sea water by a similar
polarographic technique. Lead can be concentrated from sea
water for analysis by atomic absorption using ion exchange
chelation (21). A rather large sample would be needed to
obtain sufficient lead for flame atomic absorption analysis.
Manganese
The manganese concentration in sea water is near 2 >ig/l
(30). An atomic absorption analysis method with manganese
concentration by ion exchange has been reported by Riley and
Taylor (23). Their accuracy and precision was quite good.
A very sensitive malachite green spectrophotometrie method
for direct manganese analysis has been outlined by
Strickland and Parsons (29).
Mercury
The normal mercury content of sea water is 0,03 xig/1
(30). A simple technique of concentrating mercury from two
to 10 liters sea water into 20 ml of scrubbing solution has
been described by Topping and Pirie (31). Their technique
permits analysis by flaraeless atomic absorption of samples
containing as low as 0*002 *tgHg/l. A dithizone-chloroform
extraction technique similar to that described by Chau and
Saitoh (6) is used in our laboratory. Mercury levels in
samples as low as 0.05 ug/1 have been detected using a
conventional flameless atomic absorption unit. Several
commercial mercury vapor analyzers are available that have
detection limits below that normally found in sea water.
Chemical separation of mercury from a sea water aliquot can
be achieved by selective deposition onto cadmium sulfide
pads or glass disks impregnated with gold. The collectors
can be heated to volatilize mercury for measurement.
Anderson, Evans, Murphy, and white (1) could detect as low
as 0.001 »g Hg by this technique. A sensitive direct
measurement device has been described by Braman (2). His
detector was a rubber membrane probe permeable to reduced
mercury and helium carrier gas. The gas stream passes
through a dc discharge cell from which the mercury emission
intensity is recorded. Hater samples containing from 0.01
-148-
-------
to 0.16 jag/1 were analyzed without any apparent matrix
interferences.
Molybdenum
The molybdenum content of sea water is about 10 jag/1
(30) . A technique for ion exchange concentration has been
described by Riley and Taylor (22) . The molybdenum
concentrate was measured spectrophotometrically. Molybdenum
has been separated from sea water with the chelates chitosan
and diethylaminoethylcellulose by Muzzarelli and Rocchetti
(17). They analyzed the concentrate by f lameless atomic
absorption. A molybdenum collector of thorium hydroxide was
used by Kim and Zeitlin (9). Final determination was done
spectrophotometrically with the Mo-thiocyanate complex.
They reported a molybdenum recovery near 100X.
Nickel
The nickel content of sea water is about 2 pg/1 (30) .
Many of the chemical separation techniques of ion exchange
(21) and solvent extraction (3) for cadmium are also
applicable to nickel. Rampon and cuvelier (20) reported
that nickel in sea water can be determined at 1 ug/1 levels
by atomic absorption after extraction with dimethylgloximate
in chloroform.
Selenium
The selenium level in sea water is about 0. H jig/1 (30).
Selenium, like arsenic, can be determined by atomic
absorption with the closed system generator. The sample
level need be at least 2 jug/1. A spectrophotometric method
using diaminobenzidine has been reported by chau and Riley
(5) for selenium analysis of sea water. They concentrated
selenium by co-precipitation with iron hydroxide. A very
sensitive and rapid technique has been devised by Shimoishi
(27) using an electron-capture detector on a gas
chromatograph. As low as 0.02 jag/I selenium was detected.
Silver
The silver concentration in sea water is about 0.05 jig/1
(30) . The very high sensitivity for silver by f lameless
atomic absorption makes it an attractive choice for
analysis. Solvent extraction of Ag-dithizonate could
-149-
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probably be used to separate silver from sea water. A
spectrophotometric method for silver after co-
crystallization with tiiionalide has been described by Lai
and Weiss (11). The crystal concentrates were digested in
acid. Estimation of silver was made from the red-violet
color reaction with rhodanine.
Vanadium
The vanadium content of sea water is about 2 ug/1 (30).
Spectrophotometric determination of vanadium with
diaminobenzidine after concentration from sea water by ion
exchange has been described by Piley and Taylor (22) . They
obtained a complete recovery of added vanadium using
Chelex-100 resin. Although the precision was relatively
poor, Segar and Gonzalez (26) have shown that direct
analysis by flameless atomic absorption can be achieved.
Normal sea water contains about 10 jug/1 of zinc (30).
Techniques of anodic stripping polarography and atomic
absorption spectroscopy have been used successfully in
numerous laboratories to measure zinc in sea water. Burre11
and wood (4) used a tantalum sample boat accessory with
atomic absorption to directly determine zinc in 0,25 ml sea
water samples. They reported a detection limit of 0.0002 Mg
Zn. A zinc extraction procedure by Nix and Goodwin (18) for
lake water should be applicable to sea water. Chemical
separation of zinc by Chelex-100 resin from sea water has
been satisfactorily done by Windom and Smith (32). Atomic
absorption was their choice for the final analysis. Zirino
and Healy (33) described an anodic stripping voltammetry
method used for shipboard analysis of zinc. They provided
evidence that pH dependent metal complexes occur in sea
water. Their lowest detectable level of zinc was 0.2 Aig/1.
Table IV lists the concentration of metals often found
in sea water and the methods that have been successfully
used for the analysis of the particular metals.
-150-
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TAJL.E_IV
APPLICATIONS
Element
Arsenic
Beryllium
Cadmium
Chromium
Cobalt
Copper
Iron
Lead
Manganese
Normal Amount
In Seawater
wg/1
Method
Reference
3.
0.0002
0.1
0.<4
0.1
1.
10.
0.03
2.
Atomic Absorption
Differential Pulse Polarography
Co-crystallization - Spectrophotometry
Ion Exchange - Emission Spectroscopy
Gas Chromography
Anodic stripping Polarography
Ion Exchange - Atomic Absorption
Solvent Extraction - Atomic Absorption
Flameless Atomic Absorption
Ion Exchange - Atomic Absorption
Solvent Extraction - Atomic Absorption
Ion Exchange - Atomic Absorption
Solvent Extraction - Atomic Absorption
Anodic stripping Polarography
Flameless Atomic Absorption
Spectrophotometry
Solvent Extraction - Atomic Absorption
Anodic Stripping Polarography
Ion Exchange - Atomic Absorption
Ion Exchange - Atomic Absorption
Manning (1971)
Myers 6 Osteryoung (1973)
Portmann & Riley (1964)
Merrill, et. al. (1960)
Ross 6 Sievers (1972)
Rojahn (1972)
Windom 6 Smith (1972)
Chau, et. al. (1968)
Nix 6 Goodwin (1970)
Windom & Smith (1972)
Lee 6 Burrell (1972)
Riley 6 Taylor (1968)
Kuwata, et. al. (1971)
Rojahn (1972)
Segar & Gonzalez (1972)
Strickland 6 Parsons 1968
Spencer 6 Brewer (1969)
Zirino 6 Healy (1972)
Riley 6 Taylor (1968)
Riley 6 Taylor (1968)
i
H
Ui
M
I
-------
Element
Normal Amount
In Seawater
Jig/I
Method
Reference
Mercury
0.03
Molybdenum
Nickel
Selenium
Silver
Vanadium
10.
2.
0.4
0.05
2.
Zinc
10.
Flameless Atomic Absorption
Cold Vapor Atomic Absorption
Solvent Extraction - Cold Vapor AA
Membrane Probe
Coprecipitation - Spectrophotometry
Ion Exchange - Spectrophotometry
Solvent Extraction - Atomic Absorption
Solvent Extraction - Atomic Absorption
Coprecipitation - Spectrophotometry
Gas Chromatography
Cocrystallization - Spectrophotometry
Ion Exchange - Spectrophotometry
Flameless Atomic Absorption
Flameless Atomic Absorption
Ion Exchange - Atomic Absorption
Anodic Stripping Polarography
Anderson, et. al. (1971)
Topping £ Pirie (1972)
Chau & Saitoh (1970)
Brauman (1971)
Kim & Zeitlin (1970)
Riley & Taylor (1968)
Brooks, et. al. (1967)
Rampon & Cuvelier (1972)
Chau & Riley (1965)
Shimoishi (1973)
Lai 6 Weiss (1962)
Riley 6 Taylor (1968)
Segar & Gonzalez (1972)
Burrell 8 Wood (1969)
Windom & Smith (1972)
Zirino 8 Healy (1972)
-------
6. SUMMARY
Analytical methods are described to determine As, Be,
Cd, Cr, Co, Cu, Fe, Pb, Mn, Hg, Mo, Ni, Se, Ag, V, and Zn in
sea water.
Analysis of all metals can be accomplished with various
degrees of convenience by atomic absorption. Direct
analysis by flameless atomic absorption has many desirable
attributes, but utilization cannot be maximized until the
interferring action of sea water components is overcome.
Anodic stripping voltammetry is becoming increasingly useful
for sea water analysis. Direct measurements for Cd, Cu, pb,
and Zn have been made. Major ions in sea water do not
interfere. Neutron activation analysis and spark source
mass spectrometry have been utilized as analytical
techniques. Gas chromatography has possible applications
for sea water metal analysis.
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REFERENCES
1. Anderson, D., J. Evans, J. Murphy, and W. White. Anal.
Chem. 43:1511 (1971).
2. Braman, R. Anal. Che. U3:1462 (1971).
3. Brooks, R., B. Presley, and I. Kaplan. Talanta 14r809
(1967).
4. Burrell, D. and G. Wood, Analytical Chin. Act 48s45
(1969) .
5. Chau, Y. and J. Riley. Anal. Chim. Acta 33:36 (1965).
6. Chau, Y. and H. Saitoh. Envir. Sci. 6 Techn. 4:839
(1970).
7. Chau, Y., S. Sim, and Y. Wong. Anal. Chim. Act
43:13(1968).
8. Chuecas, L. and J. Riley. Anal. Chim. Act 35:240
(1966) .
9. Kim, Y. and H. Zeitlin. Anal. Chim. Acta 51:516 (1970).
10. Kuwata, K., K. Hisatomi, and T. Haseqawa. Atom. Abs.
Newsletter 10:111 (1971).
11, Lai, M. and H. Weiss. Anal. Chem. 34:1012 (1962).
12. Lee, M. and D. Burrell. Anal. Chim. Acta 62:153
(1972).
13. Manning, D. Atom. Abs. Newsletter 10:123 (1971).
14. Merrill, J., E. Lyden, M. Honda, and J. Arnold.
Geochim. Cosmochim. Acta 18:108 (1960).
15. Myers, D. and J. Osteryoung. Anal. Chem. 45:267
(1973).
16. Midgett, M. and M, Fishman. Atom. Abs. Newsletter
6:128 {1967).
17. Muzzarelli, R. and R. Rocchetti. Anal. Chim. Acta
64:371 (1973).
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18. Nix, J. and T. Goodwin. Atom. Abs. Newsletter 9:119
(1970).
19. Portmann, J. and J. Riley. Anal. Chim. Acta 31:509
(1964).
20. Rampon, H. and R. Cuvelier. Anal. Chim. Acta 60:226
(1972).
21. Riley, J. and D. Taylor. Anal. Chim. Acta 40:479
(1968).
22. Riley, J. and D. Taylor. Anal. Chim. Acta 41:175
(1968).
23. Riley, J. and D. Taylor. Deep Sea Research 15:629
(1968).
24. Rojahn, T. Anal. Chim. Acta 62:438 (1972).
25. Ross, W. and R. Sievers. Envir. Sci. € Techn. 6:155
(1972).
26. Segar, D. and J. Gonzalez. Anal. Chim. Acta 58:7
(1972).
27. Shimoishi, Y. Anal. Chira. Acta 64:465 (1973).
28. Spencer, D. and P. Brewer. Geochim. cosmochim. Acta
33:325 (1969).
29. Strickland, J. and T. Parsons. A Practical Handbook of
Seawater Analysis, Bull. 167, Fisheries Res. Board
of Canada (1968).
30. Thompson, G. SPEX Speaker Vol. XVI - No. 2, 2 (1971).
31. Topping, G. and J. Pirie. Anal. Chim. Acta 62:200
(1972).
32. Windom, H. and R. Smith. Deep Sea Research 19:727
(1972).
33. Zirino, A. and M. Healy. Envir. Sci. 6 Techn. 6:243
(1972).
-155-
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RECENT STUDIES ON BIOLOGICAL EFFECTS OF CRUDE OILS AND
OIL-DISPERSANT MIXTURES TO RED SEA MACROFAUNA
R. Eisler
National Marine Water Quality Laboratory,
Narragansett, Rhode Island 02882
G. W. Kissill
Israel Oceanographic and Limnological Research Institute
Elat, Israel
Y. Cohen
Zoology Department, Hebrew University
Jerusalem, Israel
INTRODUCTION
With the closure of the Suez Canal in 1967, the deep-
water harbor at Elat, Israel, became increasingly important
as an oil terminal (Figure 1). In the 12 months ending in
July 1973, more than 25 million tons of crude oil were
unloaded, most of which was transported by supertankers in
the 200,000-250,000 ton class. From Elat, oil is
transported via the Elat-Ashkelon pipeline and is eventually
processed at European or Israeli refineries. Spills are
common at Elat; a majority of them were estimated at less
than 1,000 liters, but at least two greater than 10,000
liters were observed within the past year. Oil spill
countermeasures are extensive and include mechanical
containment devices around vessels unloading, use of straw
and other oil-absorbing materials in the event of spillage,
cessation of unloading operations with the advent of
southerly winds in order to protect the luxury bathing
beaches to the north, and heavy financial penalties to
polluters. The use of chemical oil dispersants are
prohibited at Elat in order to minimize damage to the biota;
however, this is not rigidly enforced.
Because virtually nothing was known regarding the
influence of crude oil or chemical oil counteractants on
local coral reef ecosystems, the Hebrew University in 1972
initiated a continuing series of investigations in this
subject area. This account summarizes progress during the
first year of laboratory studies and is extracted wholly
from the findings of Cohen («), Eisler (5, 6, 7), and Eisler
and Kissil (8). Specifically, we report on acute toxicity
to representative species of marine macrofauna of two common
grades of crude oils (which together comprise more than 80*
of all crude oil unloaded at Elat), a chemical oil
-156-
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MEDITERRANEAN
SEA
TURKEY
\AGHA JARI
PERSIAN
GULF/
ARABIAN
SEA
Figure 1. Geographic Loaction of Eilat -157-
-------
dispersant used extensively in Northern Israel, and mixtures
of oil and dispersant at realistic application levels.
Bioassays were conducted under static as well as continuous
flow conditions. Depth-toxicity interactions were evaluated
using deep (2.0 m) tanks. Sublethal and latent effects of
crudes and oil-dispersant mixtures on physiology,
metabolism, and behavior were investigated as were short-
term degradation and bioaccumulation of oil.
TEST TOXICANTS
Three materials were evaluated: Iranian crude oil from
the Agha-Jari field located at the head of the Persian Gulf
(Figure 1), Sinai crude oil from the vicinity of Abu Rodes
situated on the Gulf of Suez (Figure 1), and a chemical oil
dispersant used extensively on the Mediterranean Coast of
Israel. Oil samples were kindly provided by the
Elat-Ashkelon Pipeline Company and were taken from tankers
unloading at Elat in September, 1972. These were stored in
tightly sealed 25 liter containers until use. Iranian
crude, which comprises about 60% of the total unloaded at
Elat, when compared to Sinai crude (20% of total) is low is
sulfur, asphaltenes, carbon residues, and especially
viscosity (Table 1). The Sinai crude transported by tankers
is a mixture of 20% offshore field and 80% onshore field
(Table 1). Dispersant samples were supplied by Chemotas
Ltd. of Tel Aviv. Chemotas stated that the dispersant
consisted of 1556 surfactant and 85% solvent. Composition of
the surfactant fraction, according to Chemotas spokesman,
is: 70S ethylene oxide; 15X linear alcohols in the C11-C15
range; 10% organic impurities, mostly amines; and 536 water.
The solvent fraction consits of about 65% aromatics and 3556
high boiling aliphatics.
ACUTE TOXITY
Static Jar Tests
Methods: static bioassays were conducted in wide mouth
glass jars containing 3 liters of aerated, unfiltered
seawater. Each test was conducted at a salinity of 41 o/oo,
a temperature of 22*1°C, dissolved oxygen greater than **.Q
mg/1, and pH of 8.1+0.1 unit; these conditions approximated
those of nearby waters as described by Oren (11). The test
animals were adults from locally abundant groups of
coelenterates (1 species), molluscs (4 species), crustaceans
(2 species) , echinoderms (1 species), and juvenile teleosts
(2 species) . Species used were: Heteroxenia fuscescens, an
-158-
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Field Location
Properties
Spec. grav.
at 60°F.
API
Kinematic viscositj
at 50°F
at 100°F
Pour point
Sulfur, wt %
Asphaltenes, wt %
Carbon residue, wt%
(1)
Agha Jari field,
Persian Gulf,
offshore.
0.847
35.6
14.4
5.66
+10
1.35
0.60
3.7
(2)
Belayim field,
Slani,
onshore
0.922
31.8
796.0
93.0
+6
3.29
7.7
10.2
(2)
Belayim field,
Sinai,
offshore
0.875
30.2
134.0
13.0
+6
1.8
1.54
4.8
(1) from Fallah et al (1972)
(2) data supplied by Israel Institute of Petroleum. Sample tested for toxicity was
mixture of 80% onshore field, 20% offshore.
Table |. Some properties of crude oils used in studies on toxicity of
oils to Red Sea macrofauna.
-159-
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octocoral; Prupa granulata. a gastropod drill: Nerita
forskali, a littoral gastropod; Mytilus variabilis, a
mussel; Acanthopleura haddoni. a chiton; Calcinus latens, a
hermit crab; Palaemon pacifica, a shrimp; Echinometra
mathei, a sea urchin; Parupeneus barberinus, goatfish; and
siganus riyulatus, rabbitfish.
Assay organisms were added to the jars about 30 minutes
before the test substances. Agitation of the media-toxicant
solution as recommend by LaRoche, et al (10) was not
essential according to results of preliminary studies that
we conducted. Details of methodology and procedures are
listed in LaRoche, et al (10) as modified by Eisler (5).
Results: Concentrations of all compounds tested that
were fatal to 50% of the individuals in 168 hours (LC-50
(168 hr.) are presented in Table 2, From these data we
concluded that both grades of crude oil are relatively
innocuous when compared to the dispersant; that toxicity of
oil-dispersant mixtures reflect the biocidal properties of
the dispersant alone; and that fishes and crustaceans are
among the most sensitive groups tested, similar results
were reported by earthy and Arthur (3) and LaRoche, et al
(10).
In another series of studies with juvenile rabbitfish,
we investigated the effect of tiroe in the aerated assay
medium on toxicity in high concentrations of Iranian crude
oil, Sinai crude oil, and dispersant. For the
concentrations tested each compound behaved differently
during a maximum period of 168 hours in the assay medium
before living organisms were added. For example, Iranian
crude oil became progressively less toxic with time, and the
dispersant lost all of its biocidal properties within 2
hours (Table 3). But Sinai crude became increasingly more
toxic with time (Table 3). To account for these phenomena
it would appear that the more volatile fractions of Iranian
crude, as well as dispersant, must contain most of the
acutely lethal components; for Sinai crude, the non-volatile
components would be the most toxic. Additional bioassays
with rabbitfish confirmed that the surfactant fraction of
the dispersant, i^e.. the ethylene oxide-containing fraction,
contained virtually all of the toxic properties. Also, the
lower boiling components of Iranian crude and the highest
boiling fractions of Sinai crude were indeed the most toxic
(Table 4).
-160-
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Animal
Coelenterates
octocoral*2/
Molluscs
drill
snail
mussel
chiton
Crustaceans
hermit crab
shrimp
Echinoderms
urchin
Teleosts
goatfishl/
rabbitfish
Iranian
crude
oil
12
30+
17
30+
30+
21
2
30+
6
0.8
Sinai
crude
oil
30+
30+
30+
30+
30+
30+
16
30+
16
15
Dispersant
.018
.064
.045
.018
.013
.006
.006
.006
.006
.010
Oil-
dispersant
mixtures ^
.110+
. .. . -.
.152
_____
--- —
_
.096
.061.
.150
I/ Values are in ml/1 test compound added eo jars at start,
2/96 hour values
3/ 48 hour values
Table 2. Concentrations of two crude oils, a chemical dispersant, and
oil-dispersant mixture (10 parts oil - 1 part dispersant v/v)
fatal to 50% of selected species of Red Sea macrofauna in 168
hours.
-161-
-------
Fish added to
medium - hours after
test substance
168
120
72
24
2
1
0
Iranian crude
(1.9 ml/1)
12
18
44
63
—
—
75
Sinai crude
(22 ml/1)
75
75
63
50
—
—
25
Dispersant
(0.020 ml/1)
0
0
0
0
0
25
75
Controls
Table 3. Effect of time in aerated medium on toxicity of high con-
centrations of two crude oils and one dispersant to juve-
nile rabbitfish. Values represent percent dead at 168
hours (n « 6 to 8 fish/time interval).
-162-
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Boiling fraction Type oil % dead-in 96 h
Low, <145°C Iranian 100
Intermediate, 145-210"C Iranian 50
High, >210°C Iranian 0
Controls 0
Low, <80'C Sinai 0
Intermediate, 80-115°C Sinai 0
High, >115°C Sinai 50
Controls 0
Table 4. Toxicity of three boiling fractions of two crude oils to
•Juvenile rabbitfish. Values equal percent dead at 96
hours during exposure to 3.3 ml/1 under static conditions.
-163-
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Continuous Flow Tank Tests
Methods: Assay tanks (Figure 2) were 2.0 meters high,
1.0 meter in diameter, with a window 60 cm wide by 125 cm
high. Each tank was divided into two compartments by
perforated partitions placed 0.2, 1.0, and 1.8 m from the
surface. Construction of tank and partitions was
fiberglass. Onfiltered seawater was introduced in a spray
pattern via perforated pipes at the rate of about 5
liters/minute from a constant head tank. This flow is
equivalent to a water turnover rate of about 4,8 daily. All
incoming water passed through the oil film; but data on
degree of mixing and turbulence are lacking. Waste water
exited via standpipes situated below the bottom partition
then traversed oil traps and sand filters before discharge
into the Gulf of Elat. Seawater composition was as
described in the previous section. A more detailed account
of methods and procedures is presented in Eisler and Kissil
(8) and Eisler (7) .
Assay animals were introduced 1-2 days prior to the
toxicants. Because of space limitations only a limited
number of comparatively high concentrations were tested.
The highest level of oils added to each tank was 10 ml/1;
this is equivalent to 15 liters added at the surface. In
terms of liters per m2 surface area, a concentration of 10
ml/1 for the tank tests equaled 19.10; for the jar tests
this was 1.33.
Results: A comparison of acute toxicity values to Red
Sea macrofauna derived from static jar tests and from
continuous flow tank tests is shown in Table 5. In the 20
instances of paired observations for which comparisons are
possible, toxicity values derived from tank tests were
higher, i-ej.. test compounds were less toxic in 11 of the 20
cases and lower in only one instance (crustaceans vs
dispersants); in the remaining 8 cases there was
insufficient data to reach a conclusion (Table 5). It is
probable that the lower mortality observed in tanks when
compared to jars is associated with lower biomass/1 medium
as well as metabolite and toxicant removal by the constant
flushing action.
For all species and all compounds assayed in the large
tanks, survival was the same in both compartments in 40 out
of 55 tests (Table 6). In the 15 instances where
differential survival was observed between compartments, it
was the lower compartment (1.0 to 1.8 m from surface) that
exhibited higher survival in 14 out of 15 instances (Table
6). It appears that a depth protective effect exists and
-164-
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I.,I
2 Constant head and continuous flow tanks system
used in oil and dispersant toxicity bioassays.
-------
Iranian Crude Oil
Jar
loelenterates^/ 1
toll uses2/ 3 to 10
Irustaceans3/ 1 to 3
:chinoderms*/ 30+
releosts5/ 0.3 to
1
Tank
10+
10+
10+
10+
10+
Sinai Crude Oil
Jar
30+
10 to 30
3 to 10
30+
3 to 10
Tank
10*-
10+
10+
10+
10+
Chemical Dispersant
Jar
0.01Q
0.003 to
0.020
0.003
0.003
0.003
Tank
0.030
0.010 to
0.100
0.001
0.003
Q.010
Oil -Dispersant Mixtures6/
Jar
0.110+
0.110
0.011 to
0.033
0.033
0.033 to
0.110
Tank
0.110+
0.110+
co.no
:0.110
;0.110+
1) Organism assayed for both jar and tank tests was Heteroxehia fuscescens. an octocoral.
2) For Jar tests: Oruoa qranulata (gastropod), Nerfta forskali (gastropod). Mytilus varlabilus (mussel), Acanthopleura haddonl (chiton):
tests: Pinctada fucada (pearl oyster), Trochus.;dep.tatJLS (gastropod). Circe crocea (clam).
3) Jar tests; naTaemon paciflca (shrimp*, Ca1c1pus~1atens (hermit crab); tank tests: Panulirus pencHiatus (lobster).
4) Jar tests: Echinometra mathei (short-spined sea urchin); tank tests: Diadema setosum (long-splned sea urchin).
5) Jar tests: Siganus rfvulatus (rabbitfish). Parupeneus barberinus (goatfish); tank test; S. nvulatus.
6) Mixtures contain 91% oil, 9% dispersant v/v.
tank
Table 5.. Comparison of crude oil and oil-dispersant toxidty values to Red Sea fauna derived from static jar tests and from continuous flow tank
tests. Values are in ml/1 toxicant added to medium at start allowing at least 90% survival in 168 hours.
-------
Total
number
tests
No. tests with
differential
survival
between
upper and
lower
No. tests with
survival higher
in
upper lower
(0.2-1.0 m) (1.0-1.8 m)
compartments compartment compartment
Dispersant
Iranian crude oil
Sinai crude oil
Oil-dispersant mixtures
Controls
19
8
7
14
7
8
2
1
4
0
0
0
0
1
0
8
2
1
3
0
Total 55 15 1 14
Table 6. Tank tests: Effect of distance from surface on survival of
selected species of Red Sea raacrofauna. Exposure was for
168 hours to various concentrations of chemical dispersant,
crude oils, and oil-dispersant mixtures.
-167-
-------
that this observation merits further elucidation in future
toxicity testing of oils and oil counteractants.
SUBLETHAL EFFECTS
Iranian .Crude
After immersion for 168 hours under static assay
conditions, several species exhibited abnormal behavior
patterns when compared to controls. Mussels, for example,
did not adhere to the walls of the test jar or to other
mussels by byssal thread attachment, were slow to close
valves in response to mechanical stimuli, and secreted
copious quantities of mucous. About 6036 of the mussels
immersed in 30 ml/1 were afflicted, 40% at 3 ml/1, 10% in 1
ml/1 and none at lower concentrations tested. Snail
(Nerita) and drill (Drup.a) distress syndromes were
characterized by extreme sluggishness in closure of
operculum following tactile stimulation, inability to
complete closure of operculum, failure to adhere to sides or
bottoms of jar, and excessive mucous secretion. For snails,
80% showed this pattern at 10 ml/1, 40% at 3 ml/1, and none
at lower levels of Iranian crude. Drills were more
resistant with only 10% in stress at 30 ml/1 and none at
lower levels. Rabbitfish exhibited a lowering in blood
hematocrit values and a pronounced enlargement of the liver
(Table 7); these results were statistically significant at
the 0,01 level. However, all rabbitfish survived at least
six months posttreatment in flowing seawater aquaria with
apparently normal appetite and growth; unfortunately, data
are lacking on posttreatment hematocrits and SLI values.
In flowing water tank tests, octocorals were affected
during immersion in 10 ml/1 Iranian crude to the extent of
reduction in tentacular pulsation rate. Reduction was most
pronounced (100% inhibition in 96 hr) among those held
nearest (0.2 m) the surface and least pronounced (40%
inhibition in 96 hr) among groups held furthest (1.8 m) from
the surface; intermediate values were observed at
intermediate depths.
Sinai Crude
Mussels and snails that survived immersion for 168 hours
in high concentrations under static conditions showed
distress, as described previously. More than 90% of mussels
held in 30 ml/I were abnormal at 168 hr; this dropped to 60%
-168-
-------
Treatment Hematocrit SLI
Iranian crude
0.30 ml/1 -7 +55
1,00 mlII -26 -1-105
Sinai crude
10.0 ml/1
Dispersant
0.001 ml /I
0.003 ml/1
0,010 ml/1
-16
-10
+2
-18
+158
+2
+5
+21
^Control values were 47.18 for hematocrit and 1.155 for somatoliver
index.
Table 7. Effect of two crude oils and a dispersant on blood hematocrit
and somatoliver index (liver wt/body wt X 100) of juvenile
rabbitfish, Siganus rivulatus surviving exposure for 168 hours.
Values are percent deviation from controls. '
-169-
-------
at 10 ml/I and zero at 3 ml/1 and lower. An identical
pattern was observed for snails.
Rabbitfish subjected to 10 ml/1 Sinai crude in jars
showed a lowering of blood heraatocrits and marked increases
in liver size (Table 7). As was true for Iranian crude,
there were no deaths during a posttreatment observation
period of six months.
Dispersant
In jar tests, stress was observed in 30% of mussels held
at 0.02 ml/1, with all animals outwardly normal at lower
concentrations. There is a trend towards lower blood
hematocrits and increasing SLI among rabbitfish (Table 7),
but these differences were not statistically significant at
the 0.01 level.
OilrDispersant Mixtures
Snails exhibited distress after immersion in oil-
dispersant mixtures for 168 hr. More than 80% appeared
abnormal in a mixture of 0.1 ml/1 Iranian crude plus 0.01
ml/1 dispersant, the lowest concentration tested. At higher
sublethal levels tested, 100% manifested the stress syndrome
described earlier.
LATENT EFFECTS (MOLLUSCS)
This section summarizes effects of high sublethal
concentrations of Iranian crude oil and dispersant on
posttreatment predation rate of the gastropod, Drup.a
granulata, on the mussel, MYtilus variabilis and on number
of egg cases deposited by Drupa.
Oil
Groups of mussels and drills were exposed separately for
168 hr to 10 ml/1 of Iranian crude in static jar assays.
Afterwards, mussels (n = 65) and drills (n = 15) were placed
in flowing seawater aquaria until 95% or more of the mussels
were consumed by the drills. Four combinations were
possible using oil-exposed drills (D+), oil-exposed mussels
(M+), control drills (D~), and control mussels (M~) : namely,
D+M+, D+M-, D~M+, and D~M~ (controls). In 28 days almost
-170-
-------
all of the mussels in the D~M- group had been consumed
compared to 1/3 this level in the D+M+ group, and 1/2 this
level when only one species, either predator or prey, was
similarly exposed (Figure 3) . We speculate that at least
two mechanisms may be operable to account for this
phenomenon: (a) whereby oil taken in by mussels acted as a
drill repellant; (b) another involving inhibition of the
drill's radular apparatus or digestive enzymes; the effects
are apparently cumulative. The effect of different time-
concentration combinations other than 168 hr-10 ml/1 oil on
posttreatment predation rate is unknown and appears to
require additional research.
The average number of egg cases deposited by individual
over the 28 day period ranged between 4.2 and 14.2
(Figure 4) . Fecundity of Drupa seems to be a direct
function of mussel consumption (Figure 3) j however, more
research is needed to establish this pattern conclusively.
Dispersant
Immersion of Druga and Mytilus in 0.003 ml/1 for 168 hr
did not affect posttreatraent predation rate over a period of
32 days. But fecundity of untreated drills preying on
untreated mussels was 7 times higher than groups where both
had been initially subjected to dispersant and 3 to 5 times
higher than groups where only one species had been subjected
to dispersant. We concluded that high sublethal levels of
the dispersant tested interferes with egg case deposition by
Drupa during the first month posttreatment.
DEGRADATION
Gas chromatogaras of crude oil from the Agha Jari field
indicate that the most prominent peaks are those of normal
paraffins, extending from at least decane (C10H 22) to penta-
triacontane (CoeHo?) with the majority in the q2 -c^
range. Details of the analytical procedure are presented by
Cohen (1973). Gas chromatograms of weathered oil samples
taken from the large tanks after 96 hr exposure at 23°c
suggest the loss of low boiling compounds in the CIQ "C15
range (Figure 5). This observation lends support to tf
findings of Blumer et al (2) that evaporation and
dissolution are the initial degradation processes.
-171-
-------
Figure 3. Destruction of Mussels U.S. Time
8 12 16 20 24
TIME, in days post-treatment
-------
-j
CO
8
12 16 20 24
TIME, in days post-treatment
Figure 4. Deposition of Egg Cases U.S. Time
28
-------
o
a
w>
a:
w
a
K
o
u
u
» C"
IRANIAN CRUDE OIL
NON-WEATHERED
WEATHERED FOR 96 HOURS IN CONTINUOUS
FLOW TANK SYSTEM
Fig* 5* Chromatograms of Iranian crude oil before and after weathering
for 96 hours la continuous flow tank system.
-------
BIQACCUMULATIQN
Colonies of the octocoral, Heteroxenia fuseescens,
exposed to 10 ml/1 crude oil for 96 hours showed marked
uptake in the Cn-C15 range and also in the region above
C 23 (Figure 6) . Extreme care was taken that colonies were
not in actual contact with oil film. Colonies subjected to
only 3 ml/1 oil for 96 hours showed little uptake in the
C -^-C 15 range, but the hydrocarbon distribution above c,,
remained in close agreement with that of the higher
application level. It is possible that the reduced C..-C.,.
fraction in tissues may be due to evaporative loss of those
fractions prior to incorporation.
The amount of oil taken up by the most contaminated
corals, i.e. those exposed to 10 ml/1 oil for 96 hr was less
than 1% of the total hydrocarbon content of control corals.
We conclude that corals, although comparatively resistant to
crude oil under simulated natural conditions, are capable of
incorporating oil from the surrounding medium into its
tissues. Bioaccumulation and long-term retention of fuel
oil components by shellfish is documented by Blumer (1).
However, the metabolic implications of Blumer*s observations
and our own findings are imperfectly understood and appear
to merit further investigation.
RECOMMENDATIONS
On the basis of studies summarized herein as well as
available personnel and equipment, we suggest a broadening
of the oil research program at the Elat Marine Biological
Laboratory during the coming year in the areas of bioassay,
chemical residues, behavior, and field monitoring studies.
Specifically, we recommend the following:
Bioassay
Laboratory testing of chemical oil dispersants and other
oil counter actants, both alone and in combination with
target oils at realistic application levels, should continue
using representative species of Red Sea biota. Bioassays
are to be conducted under conditions of continous flow in
containers of sufficient height (2.0 m is suggested) to
account for possible depth protective effects. Test species
would include various life history stages of hermatypic and
non-hermatypic corals, perciform teleosts, bivalve molluscs,
decapod crustaceans, echinoid echinoderms, and conceivably
-175-
-------
60
100
150
200
Temp.:(«C)
300
I
15
I
35
T i me ( min.)
I
45
I
55
65
Fig. 6* 'Gas chromotograms of Heteroxenia fuscescens before and after
exposure to 10 ml/1 Iranian crude oil for 96 hours.
-------
other groups. Availability of toxicants in the media to
assay organisms should be ascertained using appropriate
chemical procedures. Research thrust in this problem area
would be on long-term biocidal properties of test compounds,
as modified by physiochemical factors and dosing rates.
Chemical Res idues
Rate and extent of bioaccuraulation of crude oils, oil
counteractants, and mixtures should be determined under
simulated field conditions. This would include data on
accumulation from the medium and through marine food chains
using gas chromatography and other techniques. Research
emphasis should be on hydrocarbon residues from flesh of
commercially important species, especially teleosts, but
also crustaceans and bivalve molluscs.
Behavior Studies
Impact of sublethal concentrations of crude oils and
oil-counteractant mixtures to biota, with special reference
to inshore fauna, should be evaluated during exposure and
afterwards, we suggest that priority be given to type
studies of attractance-repellence, stamina and predator
avoidance, feeding behavior, long-term effects on fecundity,
and disease resistance.
Fieldstudies
We recommend that a live well monitoring capability be
established near oil-unloading terminals, and other
potential point sources. At this time adult lobsters appear
promising as suitable and sensitive indicators. Also,
dispersant compounds proven under laboratory condition's to
be low in toxicity, comparatively degradable, and efficient
oil counteractants should be promptly field tested on
typical coral reef ecosystems utilizing suitable containment
materials. For all studies, we recommend a chemical residue
analytical capability.
ACKNOWLEDGEMENTS
We are especially obligated to Prof. F. Dov For,
Director of the Marine Biological Laboratory for
encouragement and financial support. We acknowledge the
efforts of staff members of the MBL and the Israel
-177-
-------
Limnological and Oceanographic Research Institute for
contributing generously of their time in collection of assay
organisms and maintenance of test facilities. The technical
assistance of personnel from the Israel Institute of
Petroleum, The Elat-Ashkelon Pipeline Company, the
municipality of Elat, and Chemotas, Ltd. was appreciated.
Finally, the editorial assistance of various EPA scientists,
especially Drs. Jan Prager, Peter Rogerson, and
Eric Schneider is acknowledged.
-178-
-------
REFERENCES
1. Blumer, M., 1972. Oil Contamination and the Living
Resources of the Sea. In Ruivo, M. (ed.) Marine
Pollution and Sea Life, Fishery Trading News (Books)
Ltd., London: 476-481.
2. Blumer, M., M. Erhardt, and J. H. Jones, 1973. The
Environmental Fate of Stranded Oil. Deep Sea Res.
20 (3): 233-261.
3. earthy, J. D. and D. R. Arthur (eds), 1968. The
Biological Effects of oil Pollution on Littoral
Communities. Suppl. to Vol. 2, Field Studies
Council, London: 198 pp.
4. Cohen, Y., 1973. Effects of cCrude oil on the Red Sea
Alcyonarian Heteroxenia fuscescens. M. S. Thesis,
Hebrew University of Jerusalem, Dept. zoology,
Jerusalem, Israel.
5. Eisler, R., 1974 a. Acute Toxicities of Crude Oils and
Oil-Dispersant Mixtures to Red Sea Fishes and
Invertebrates. Israel J. Zool., in gress.
6. Eisler, R., 1974 b. Latent Effects of Iranian Crude Oil
and an Oil-Dispersant to Red Sea Molluscs. Israel
J. Zool., in press.
7. Eisler, R., 1974. Static and Continous Flow Bioassay of
Crude Oil and Oil-Dispersant Mixtures to Red Sea
Macrofauna. Marine Biology, in preparation.
8. Eisler, R. and G. Wm, Kissil, 1974. Toxicity and
Sublethal Effects of Crude oils and Oil-Dispersant
Mixtures to Juvenile Rabbitfish, Signaus Rivulatus.
Trans. Amer. Fish. Soc., in review.
9. Fallah, A., A. Babakhshan, M. Shahab, and A. Manoosi,
1972. Correlated Data of Iranian Crude oils. J.
Inst. Petrol. 58 (560): 75-82.
10. LaRoche, G., R. Eisler, and C. M. Tarzwell, 1970.
Bioassay Procedures for Oil and oil-Dispersant
Toxicity Evaluation. J. Water Poll. Contr. Feder.
42: 1982-1989.
11. Oren, O. H., 1962. A Note on the Hydrography of the
Gulf of Eylath. Contrib. Knowl. Red Sea, Sea Fish.
Res. Sta., Haifa, Israel, Bull. 30, (21): 2-14.
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ADSORPTION OF ORTHOPHOSPHATE ON BOROSILICATE AND "CITRATE OF MAGNESIA
BOTTLES" POLYETHYLENE AND POLYVINYL SURFACES IN A DISTILLED WATER AND
SEAWATER MATRIX
D. F. Krawczyk and M. W. Allen *
The chemical analysis of nutrients in marine and
estuarine waters has been going on since before the turn of
the century. A glance at the oceans where upwelling
liberates an abundance of nutrients will provide the
realization that the oceans are an important source of food
supply. The task of analyzing marine and estuarine waters
accurately and precisely is the responsibility of the
oceanographic chemist who has historically accomplished his
analytical work on site. Those tests that could be
conducted on shipboard were performed immediately. Thus, an
oceanographic vessel would carry burets, pipettes, and
meters that the chemist needed to perform analyses. If for
some reason an analysis could not be performed immediately,
then the techniques of preservation were freezing and cold
storage until analysis could be completed.
Today, the oceanographic vessel can be equipped with
probes and automated analytical equipment allowing precise
analysis to be accomplished on site (1,2). However, insight
to some of the problems that may be encountered on
oceanographic vessels can be gleaned from a passage from
Scientj.f j.c American's special "Oceans" issue.
Being an oceanographer is not quite the same as being a
professional sailor. Oceanographers have the best of
two worlds—both the sea and the land. Yet many of
them, like many sailors, find it extraordinarily
satisfying to be far from the nearest coast on one of
the small, oily and uncomfortable ships of their trade,
even in the midst of a vicious storm, let alone on one
of those wonderful days in the Tropics when the sea and
air are smiling and calm.
I think the chief reason is that on shipboard both the
past and future disappear. Little can be done to remedy
the mistakes of yesterday; no planning for tomorrow can
reckon with the unpredictability of ships and the sea.
To live in the present is the essence of being a seaman.
* Pacific Northwest Environmental Research Laboratory
-180-
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The work of an oceanographer, however, is inextricably
related to time. To understand the present ocean he
mast reconstruct the present ocean, he must reconstruct
his history, and to test and use his understanding he
needs to be able to predict—both what he will find by
new observations and future events in the sea. (3).
The relevance of the above comments to the present study
lies mainly in the framework and magnitude of "time" it
reveals for the meaningful interpretation of marine samples.
A mechanical breakdown of equipment aboard ship would
require back-up systems for storage of samples until
analysis was possible, in facing the problem of storage
consideration must be given to the potential problem of
adsorption, especially in the case of orthophosphate. Heron
(H), Hassenteufeul (5), and Ryden (6) have all reported an
adsorption of phosphate on glass, polyethylene,
polypropylene, and polycarbonate. The problem presented a
variety of situations which were attributed to adsorption of
phosphate onto surfaces. The problem of adsorption, it
should be noted, will affect the accuracy of the analysis
but not necessarily the precision.
EXPERIMENTAL
This study concerns itself with the measurement of
orthophosphate in distilled water and seawater after contact
with borosilicate glass, "citycate of magnesia bottle", thin
wall polyethylene Cubitainer®, commercial grade wall
polyethylene and polyvinyl chloride bottles as sample
holding containers.
The newly purchased, first-time borosilicate glass and
soda lime glass (citrate) bottles, were washed in phosphate-
free detergent, rinsed with deionized water, rinsed with
double distilled water and dried. The unused polyethylene
containers and the PVC bottles were used without any
pretreatment. Five replicates of sample were analyzed. A
second set of five replicates received 40 mg/1 of mercuric
chloride. To a third set of five replicates orthophosphate
was added. The addition was made with a microliter pipette
from a concentrated orthophosphate solution. A fourth set
of five replicates received an addition of mercuric chloride
to provide a 10 mg/1 concentration and an incremental
addition of orthophosphate again spiked via microliter
pipette. An analysis of waters used is presented in Table
1.
© registered trademark
-181-
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TABLE 1. Characteristics of Water
Parameter
pH
Conductivity
Total Inorganic Carbon
Total Organic Carbon
Kjeldahl Nitrogen
Nitrate Nitrogen
Nitrite Nitrogen
Total Phosphate Phosphorus
Orthophosphate Phosphorus
Total Chromium
Total Cobalt
Total Copper
Total Iron
Total Lead
Total Manganese
Total Zinc
*Salinity in parts per thousand
Concentration
Units
mhos*
mg/1
mg/1
mg/1
ug/i
ug/l
ug/l
ug/l
ug/l
ug/l
ug/i
ug/l
ug/i
ug/i
ug/l
Distilled
Water
6.8
0.6
<1
< .3
< .01
<1
<1
<1
<1
<1
<1
1
20
<5
<1
1
Seawater
7.6
33.3*
26
1.2
.2
200
2
35
35
<5
< 20
15
380
<25
46
7
-182-
-------
The rationale in using off-the-shelf bottles was an
attempt to model a shipboard situation where a mechanical
breakdown would force the use of alternative systems. An
assumption is made that time would not permit a thorough
preparation for use of sample bottles.
The waters were analyzed for orthophosphate phosphorus
using the automated pbosphomolybdate procedure. Details of
the analysis are available (7,8,9). A salt correction was
made where analysis of marine waters was conducted (10). A
single analysis was performed on each bottle. The data for
orthophosphate analysis of distilled water and seawater is
presented in Tables 2-8.
The results presented in Table 2 on borosilicate glass
when compared to data of Table 1 indicate that phosphate or
some constituent providing a positive phosphate level is
being leached from glassware very quickly. Some indication
of release of phosphate was also evidenced in the "Citrate
of Magnesia Bottle" but the data do not appear as convincing
as in the case of the borosilicate glass. The examination
of these data after the seventh day required analysis in
triplicate on a selected number of bottles on the eighth
day. The results of the triplicate analysis are presented
in Table 4. These data indicate that there were some
problems in the data reported on the sixth day. Thus, the
analytical quality control (AQC) aspects of presented data
would allow for deletions of the data from the sixth day.
It also lends credence to the assumption that the numbers
obtained from the borosilicate glass portions of the study
are meaningful.
The consistency of the data in Table 5, with the
exception of the data portion after six days which was
rejected based on AQC, provides still more evidence that the
values in Table 2 have some meaning. A second group of
borosilicate bottles was washed with phosphate-free
detergent, rinsed, dried, rerinsed with double distilled
water and dried. The data for this second set are presented
in Table 6. Triplicate analyses on a selected number of
bottles were conducted after the second day. The purpose of
the analyses after 2 days is to provide validity for the use
of a single analysis per bottle concept. The second rinsing
with distilled water seemed to have provided some
improvement in leaching of some component providing a
positive orthophosphate level. A hydrochloric acid wash
followed by a phosphate-free detergent wash followed by a
distilled water rinse followed by drying provided
orthophosphate analyses that were similar to blanks and were
less than 1/ag/l.
Two types of polyethylene bottles were used in this ^
study. A bottle identified as a polyethylene cubitainer w
of one liter size (produced from hot melted polyethylene
-183-
-------
TABLE 2. Borosilicate Glass - 500 ml size
Distilled Water Matrix
Bottle
Number
Treatment
I
I-1
00
I
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
None
None
None
None
None
HgCl, *Added
HgCl« *Added
HgCl, *Added
HgCl, *Added
HgClJ *Added
Spike **
Spike **
Spike **
Spike **
Spike **
Spike + HgCU Added
Spike + HgCl- Added
Spike + HgClp Added
Spike + HgClp Added
Spike + HgCl« Added
Zero time
ug/1
Orthophosphate
Phosphorus
1
1
8
3
3
2
12
3
15
8
17
12
6
10
9
7
13
11
9
14
After 2 days
Contact yg/1
Orthophosphate
Phosphorus
3
4
14
2
1
2
2
3
4
10
9
8
7
6
9
13
8
9
8
6
After 6 days
Contact yg/1
Orthophosphate
Phosphorus
5
2
1
8
4
21
12
4
5
8
4
6
9
10
9
9
8
9
8
14
After 7 days
Contact jag/1
Orthophosphate
Phosphorus
4
3
17
9
3
3
2
3
4
3
4
14
9
9
10
10
11
8
9
15
*HgCl/> = 40 mg/1 in sample
**SpiKe =4 yg/1 P
-------
TABLE 3. "Citrate of Magnesia Bottle"
Distilled Water Matrix
HL
00
(Jt
Bottle
Number
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Treatment
Zero time
yg/1
Orthophosphate
Phosphorus
None
None
None
None
None
HgCl2
HgCl|
HgCl,
HgCl*
HgClp
Spike
Spike
Spike
Spike
Spike
Spike
Spike
Spike
Spike
Spike
Added*
Added*
Added*
Added*
Added*
Added**
Added**
Added**
Added**
Added**
+ HgCl9
+ HgCl,
+ HgCl-
+ HgCl,
+ HgClg
Added
Added
Added
Added
Added
3
6
3
2
3
1
0
0
0
2
^»
-
-
-
-
-
-
-
-
-
After 2 days
Contact yg/1
Orthophosphate
Phosphorus
1
1
1
1
1
1
1
1
1
1
7
7
7
6
14
5
6
6
6
6
After 6 days
Contact yg/1
Orthophosphate
Phosphorus
14
9
10
1
1
1
1
1
1
1
1
1
1
5
6
5
5
12
6
6
After 7 days
Contact yg/1
Orthophosphate
Phosphorus
12
2
2
1
1
1
2
2
2
2
2
6
15
11
9
7
8
8
8
7
*HgCl« = 40 mg/1 in sample
**Spifce = 4 yg/1
-------
TABLE 4. Triplicate Analysis on Selected Bottles
After the Eighth Day of Contact in a
Distilled Water Matrix
Eighth Day Replicate Analysis
iag/1 Orthophosphate
Seventh First Second Third
Bottle
Type
Borosilicate Glass
Borosilicate Glass
Borosilicate Glass
Borosilicate Glass
**CMB Glass
**CMB Glass
**CMB-Glass
**CMB Glass
Polyethylene
Polyethylene
Polyethylene
Polyethylene
Polyethylene
Polyethylene
Polyethylene
Polyethylene
PVC
PVC
PVC
PVC
Bottle
Number
3
6
11
16
21
28
31
38 p
Cubitainer*
CubitainerR
CubitainerE
Cubitainer
Molded Bottle
Molded Bottle
Molded Bottle
Molded Bottle
Molded Bottle
Molded Bottle
Molded Bottle
Molded Bottle
Treatment
None
HgCl9
Spike*
Spike *+ HgCl9
None L
HgCl2
Spike*
Spike** HgCl9
None *
HgCl?
SpikS*
Spike*+ HgCU
None
Hgci2
Spike*
Spike*+ HgCl2
None
HgCl9
Spike*
Spike*+ HgCl2
Sevei
Da:
»
17
3
4
10
12
2
8
2
1
4
1
6
7
2
1
5
5
Ib
1
10
8
1
1
5
4
1
1
7
7
1
1
5
6
1
1
4
5
15
1
10
8
1
1
5
4
1
1
5
7
1
1
5
5
1
1
3
3
14
2
11
9
2
1
4
5
1
1
5
7
4
4
1
1
2
2
*Spike level = added 4 yg/1 of orthophosphate
** CMB = "Citrate of Magnesia Bottle"
CubitainerR Registered trademark of Hedwin Corp.
(a) Data from seventh day is for comparative purposes
-186-
-------
TABLE 5. Plastic Bottles
Distilled Water Matrix
Bottle
Type
Polyethylene*
Polyethylene*
Polyethylene*
Polyethylene
Polyethylene
,L Polyethylene
oo Polyethylene
i Polyethylene
PVC
PVC
PVC
PVC
b
>bj
b)
b)
a
a
a
a
Treatment
None
HgCl,
Zero Time
Mean of
Five Replicates
P9/1
Orthophosphate
Phosphorus
;**
Spike*** HgCl2
None
HgCl,
Spike **
Spike*** HgCl2
None
HgCl 2
Spikl**
Spike*** HgCl2
6
5
1
1
3
3
3
2
After 2 Days
Mean of
Five Replicates
yg/1
Orthophosphate
Phosphorus
2
1
5
7
6
6
1
1
5
5
After 6 days
Mean of
Five Replicates
yg/1
Orthophosphate
Phosphorus
6
6
6
1
3
3
3
1
3
4
5
*Polyethylene one liter Cubitainer
**Spike Level = added 4 yg/1 of Orthophosphate
(a) Polyethylene molded bottle
(b) Polyvinylchloride molded bottle.
- registered trademark Hedwin Corp.
After 7 Days
Mean of
Five Replicates
yg/1
Orthophosphate
Phosphorus
1
1
7
7
1
2
6
7
2
1
5
7
-------
TABLE 6. Borosilicate Glass Special Rinse
Distilled Water Matrix
Bottle
Number
101
102
103
104
105
106
107
108
i 109
rollO
112
113
114
115
116
117
118
119
120
Treatment
None
None
None
None
None
HgCl9
HgCi:
HgC12
Spiki
Spike*
Spike*
Spike*
Spike*
HgC12
HgCl*
HgCl|
HgCl*
**
Spike*
Spike*
Spike*
Spike*
Spike*
Zero Time
yg/1
Orthophosphate
Phosphorus
2
1
9
2
1
4
4
2
6
6
5
8
7
6
6
4
7
7
1
1
After 1 Day
yg/1
Orthophosphate
Phosphorus
4
3
3
3
7
5
4
4
5
4
7
8
5
5
5
9
5
8
7
After 2 Days
yg/1
Orthophosphate
Phosphorus
After 2 Days
yg/1
Orthophosphate
Phosphorus
1
After 2 Days
yg/1
Orthophosphate
Phosphorus
1
*Spike level = added 4 yg/1 of Orthophosphate.
-------
sheets with fused seams) was one of the polyethylene bottles
used. A molded polyethylene bottle of 250 ml volume was the
second polyethylene bottle used. A clear polyvinyl chloride
widemouthed, molded bottle of one liter size was the sample
container noted as polyvinyl chloride bottle.
The data in Tables 4 and 5 indicate little change in
orthophosphate in a distilled water matrix during the seven-
day period.
The next experiment dealt with potential losses of
orthophosphate as a function of time in a seawater matrix.
The seawater used in this study was taken from a well-aged
composite held in a tank filled to 20 percent from a
January, 1973 collection and 80 percent from an April, 1973
collection at Depoe Bay, Oregon. As in the first study,
five replicates in four groups (natural water, water * HgCla
, water * spike, water + spike t HgCla) were prepared with
the .single difference being the concentration of the
orthophosphate after 2 days and after 16 days. The results
of the analyses are present in Table 7. The observed
differences between the data from the second to sixteenth
day are presented in Table 8. It should be pointed out in
Table 7 that the spike level was added to a distilled water
matrix and used as a control. The borosilicate glass
bottles used for control were previously acid washed, rinsed
with distilled water and dried. A second set of acid washed
borosilicate glass bottles was used for the seawater matrix
experiment. The data for the second set is also shown in
Table 7 with analysis being conducted only on the sixteenth
day.
DISCUSSION
The sample bottles were kept at laboratory room
temperature in laboratory light and handled in the same
manner as any sample collected for analysis. No special
shaking was provided for the bottles.
The data in Tables 2, 4, and 6 indicate the borosilicate
bottles must be cleaned with hydrochloric acid and rinsed
well with distilled water before use as collection bottles.
When the quality of water sample approaches that of
distilled water, a significant error in release of materials
from borosilicate bottles may occur. The interesting point
in Table 5 is the use of off-the-shelf
-------
TABLE 7. Seawater Matrix
r
H
O
Bottle
Type
Borosllicate Glass
"CMB"
Polyethylene
Cubitainer K
Polyethylene
Molded Bottles
PVC
Molded Bottles
Dist. H20 in
Borosilfcate
Borosilicate Glass
Acid Washed
Treatment
None
HgCl9
Spike
HgCl2
HgCU
Spike
HgCl2
HgCl,,
Spike
HgCU
(a)
+ Spike(a)
(a)
-i- Spike
(a)
+ Spike
(a)
(a)
Spike" (a)
HgCl2 + Spike(a)
None
HgCl,
Spike (a)
HgCl2 + Spike (a)
None
Spike (a)
None
HgCl2
Spike (a)
HgCl2 + Spike (a)
After 2 Days
Contact Mean of
Five Replicates
P9/1
Orthophosphate
Phosphorus
33.6
39.4
59.6
66.2
34.4
34.2
56.4
58.0
34.1
34.9
59.1
59.2*
32.6
34.5
61.6
62.6
32.8
33.7
61.3
58.9
1.0
20.4
"CMB" Citrate of Magnesia Bottle
(a) Spike level 25.1 ug/1 ± 3.1 yg/1 (n=20)
* N=4 R
Cubitainer registered trademark of Hedwin Corp.
Standard
Deviation
4.6
4.8
3.6
3.5
.5
.8
1.4
1.4
.2
.3
.6
1.0*
.9
.5
1.2
1.1
.4
.4
.5
3.9
2.8
After 16 Days
Contact Mean of
Five Replicates
yg/i
Orthophosphate
Phosphorus
44.0
50.
63,
70.
36.0
37.8
57.
59.
.4
.2
.4
.3
.4
3.4
30.7
19.6
.6
.4*
,5
59.
1
38,
29.8
64.8
35.4
38.0*
61
61
.4
.3
1.0
20.8
38,
35.
68,
60.4
Standard
Deviation
4.0
6.8
1.1
2.9
.71
.1
.6
.4
2.3
4.3
4.3
2.1
.6*
.4
1.7
1.1
.9
.1*
.7
3.3
1.8
2.4
.5
3.0
.9
-------
TABLE 8. Magnitude of Change Noted Between 2nd & 16th Days
Seawater Matrix
Container
Borosilicate Glass
"CMB" Glass
Polyethylene Cubitainer
Polyethylene Molded Bottle
Polyvinyl Chloride Molded Bottle
Treatment
None
HgCl,
Spikfe (a)
HgCl2 + Spike (a)
None
HgCl-
Spike (a)
HgCl9 + Spike (a)
None''
HgCl?
Spikl (a)
HgCl2 + Spike (a)
None
HgCl,
Spiki (a)
HgCl2 + Spike (a)
None
HgCl2
Spike (a)
HgCl2 t Spike (a)
Change From
Day 2 - Day 16
•09/1
+10.4
+11.0
+ 3.6
+ 4.2
1
6
3.6
.9
+ 1.4
-30.7
- 4.2
-39.5
.4
-31.2
+ 4.0
-31.8
+ 2.4
+ 2.6
+ 4.3
.1
2.4
Pooled
Standard
Deviation
yg/1
4.3
5.9
2.7
3.2
.6
.6
1.1
1.0
*
3.0
*
1.7
*
.5
*
1.1
.7
.2
.6
3.6
"CMB" Citrate of Magnesia Bottle
(a) Spike level 25.1 yg/1 ± 3.1 yg/l (n=20)
* Pooled standard deviation not computed because of substantial change in data from
second to sixteenth day.
Cubitainer* Registered trandmark of Hedwin Corp.
-191-
-------
days both in untreated seawater and seawater receiving
incremental additions of orthophosphate phosphorus. No
significant changes were observed in polyethylene bottles
that contained 40 mg/1 of mercuric chloride either in the
seawater or orthophosphate-spiked seawater, No attempt was
made to identify the cause for loss. The data in Table 8
point out an orthophosphate phosphorus loss level of 40-7 to
39.5 ug/1. Although the surface area in a cubitainer©was
four times the surface area in a molded polyethylene bottle,
these differences appear to be a constant, or at least
within experimental error.
Two theories can be advanced: (a) biological uptake
with inhibition in glass or polyvinyl chloride bottles, or
(b) preferential adsorption of mercuric chloride upon
adsorbing sites available on the polyethylene surface which
would inhibit adsorption of orthophosphate ions. Of the two
theories the biological uptake theory appears less likely
because of the loss of such a significant level of
orthophosphate.
It is interesting to note that little change was
observed in the polyvinyl chloride bottles in orthophosphate
concentrations both in unpreserved and preserved samples.
An effective AQC program permitted the rejection of
questionable data.
SUMMARY
Adsorption or loss of orthophosphate in glass bottles
does not seem to be a problem at low orthophosphate
concentration in a distilled water matrix up through seven
days. However, glass bottles must have proper acid
treatment to be satisfactory containers for low phosphate
water samples because of leaching of a constituent that
provides a positive phosphate reaction.
In seawater, losses of orthophosphate phosphorus were
noted after a sixteen day period in polyethylene bottles.
If mercuric chloride is added to seawater, polyethylene
bottles can be used for holding samples for orthophosphate
analysis without any pretreatment (e.g. acid washing,
washing and rinsing). Polyvinyl chloride bottles can be
used as seawater sample storage containers as received from
the supplier without any pretreatment.
Borosilicate glass bottles must be acid washed and
thoroughly rinsed before holding samples for orthophosphate
analysis.
-192-
-------
REFERENCES
1. Grasshoff, K. 1970. A simultaneous multiple channel
system for nutrient analysis in seawater with analog
and digital data record in advances in automated
analysis. Vol. I. Industrial Analysis Technicon
International Congress 1969. pp. 133-143. Mechiad
Inc. White Plains, N. Y.
2. Coote, A. R. , Duedall, I. ». and Hiltz, R. S. 1972.
Automatic analysis at sea in advances in automated
analysis. Vol. II. industrial Analysis Technicon
International Congress, 1970. pp. 3<47-351. Futura
Publishing Co. Mt. Kisco, N. Y.
3. Revelle, R. The Ocean. Scientific American. Vol. 221
(3) 55. September, 1969.
4. Heron, J. 1962. "Determination of Phosphate in Water
after storage in Polyethylene." Limnology and
Oceanography. 7:316-321.
5. Hassenteufel, W., Jagitsch, R., and Koczy, F. F. 1963.
"Impregnation of glass surface against sorption of
phosphate traces." Lignoj.ogy and Oceanography.
8:152-156.
6. Ryden, J. C., Syers, J. K., and Harris, R, F. 1972.
"Sorption of Inorganic Phosphates by Laboratory
Ware. Implications in Environmental Phosphorus
Techniques," Analyst 97:903-908.
7. Murphy, J., and Ruby, J. 1962. "A modified single
solution method for determination of phosphate in
natural water.1* Anal Chim. Acta. 27:31-36.
8. Methods for Chemical Analysis of Water and Wastes, 1971.
Environmental Protection Agency, pp. 246-257.
NERC-AQCL, Cincinnati, Ohio.
9. Johnson, D. L. 1971. "Simultaneous determination of
arsenate and phosphate in natural waters."
Environmental Science and Technology. 5:411-414.
10. Atlas, E. L., Hager, S. W., Gordon, L. I., Park, P. K.
1971. A practical manual for use of Technicon
Autoanalyzer * in seawater nutrient analysis revised
Technical Report 215. pp. 24-25. Dept. of
Oceanography School of Science Oregon State
University.
-193-
-------
DEVELOPMENT OF A STANDARD MARINE ALGAL ASSAY
PROCEDURE FOR NUTRIENT ASSESSMENT
D. T. Specht and W. E. Miller*
INTRODUCTION
The need for a rapid, accurate means to assess water
quality in marine and estuarine situations is no less urgent
than for freshwater systems. Estuaries are particularly
valuable and sensitive resources (1,2,), for which no
standardized primary producer bioassay for water quality
exists. Bioassays have been developed for very specific
purposes, but most are narrow in design and require highly
specialized personnel to operate and evaluate (3, 4, 5, 6,
7, 8, 10, 11, 12,
Inasmuch as estuaries are the "nurseries11 of virtually
all commercially important shellfish, fish and their food
organisms, it becomes vitally important to be able to
accurately assess their capabilities for primary
productivity and to determine and predict the impact of the
addition of various nutrients and wastewaters upon that
productivity (15, 5, 16, 17, 11, 13, 18, 23, 19, 20, 21, 22,
42).
For reasons of simplicity and consistency, the protocol
for the marine algal assay follows closely that of the
freshwater algal assay — "Algal Assay Procedure: Bottle
Test" (39) published in August 1971. This is particularly
true of the physical facilities required, incubation
conditions and methodology.
ESTABLISHMENT OF CRITERIA FOR BIOASSAY ORGANISM
Unlike freshwater systems, estuaries in particular pose
the problem of interpretation of varying salinities on the
effect of nutrient concentration biomass production. While
no algal species was thought to be unaffected by varying
salinity, the most ideal candidate would be one that
responded in a linear and predictable manner to such
changes. Accordingly, this criterion was judged to be of
overriding importance. A species was desired that would
*Pacific Northwest Environmental Research Laboratory,
Corvallis, Oregon
-194-
-------
also be genetically stable, reproduce asexually, and would
not adhere to the walls of the flasks or tend to clump.
Other major problem areas to be considered were the
array of factors that govern the consistency of growth
response such as light intensity, temperature, influence of
pH, relative size of inoculum as well as selection and
modification of a standard stock medium in which the
intracellular nutrient carryover in the inoculum would be
minimal.
A significant consideration is the ease of evaluation or
measurement of the algal biomass produced. For purposes of
consistency, all results would be converted to equivalent
dry weight, as compared with standard gravimetric methods.
(39) .
SELECTION OF CANDIDATE TEST SPECIES
Considering the established criteria, candidate algal
species were selected on several bases looking for three
distinct classes of algae. For biostimulation, one should
consider pollution tolerant algae, such as the green algae
Punaliella tertiojlecta, and Nannochloris atoraus. For
toxicity or inhibition considerations, one should look
toward typical crustacean or bivalve food organisms such as
Isochrysis lai^SEi/ Cyclotella menenghiana, and
Thalassiosira pseudonana. For situations in which nitrogen
is limiting, a nitrogen fixing blue-green alga should be
considered. Some species to be examined are Coocochloris
elabens and Tr ichodesmium sp. {24) .
These algae represent three taxonomic classes: the
green algae, blue-green algae, and diatoms. Thus far, most
of the work has centered around the green flagellate,
Dunaliella^ tertiolecta Butcher {DUN clone), kindly provided
by Dr.~John Ryther, Woods Hole Oceanographic Institution.
This organism has shown the most linear response for every
parameter examined (25). An additional advantage of
PJ!S§!i§!i
-------
DEVELOPMENT OF STANDARD MEDIA RESPONSE CORVES
The initial work with Dunaliella was performed using a
modified Burkholder's Artificial Sea Water (ASW) (21). This
is one of several potentially amenable media which will
support algal growth at relatively low nutrient levels (28,
29), Table 1.
A- SalinitY Tolerance and Salt Intercast-Ion
Although it was assumed that all algae would be affected
to some degree by the salinity of the media, it was
desirable to find the alga with the most linear response and
tolerance to manipulation of salinity, primarily between 5 %o
and 35%,. In order to establish linearity of response,
levels of salinity chosen were 5, 8, 12, 16, 20, 24, 30 and
35/* , Figure 1. The initial tests were made with rather
high levels of nutrients; but later, nutrient response
trials showed essentially the same linearity at much lower
levels. The data thus produced provided the basis for a
three-dimensional plot, such as Figure 2, which portrays
response "surfaces" according to salinity-nutrient
interactions. These responses appear to be predictable,
within limits, Figure 2.
-196-
-------
TABLE 1
MARINE ALGAL ASSAY PROCEDURE SUMMARY: Eutrophication and Lake
Restoration Branch, PNERL,
NERCC, EPA
*• TEST ORGANISM; Puna1 Jell a tertiolecta Butcher (DUN clone)
available from the Eutrophication and Lake Restoration Branch,
PNERL, EPA. 200 SW 35th St., Corvallis, OR 97330; or WHOI.
II. ENVIRONMENTAL CONDITIONS: In general, follow exactly the
ALGAL ASSAY PROCEDURE BOTTLE TEST (NERP, EPA, August, 1971)
with the following exceptions:
TEMPERATURE: 18 - 20°C
SAMPLE SIZE: 100 ml in a 500 ml Erlenmyer flask
INOCULUM: Inoculate with 1 ml washed (w/ 20 %>« ASW
less N & P, by serial centrifugation) 5-8
day old culture at a concentration of 10,000
cells/ml (to give a final concentration in
the flask of 100 cells/ml, equivalent to
approximately 0.03 mg dry wt/1).
111• BASAL MEDIUM: Modified Burkholder's Artificial Seawater (ASW)
with NAAM levels of the following nutrients; N, P, Fe, and NaEDTA.
Use Analytical Reagent or Reagent Grade chemicals, and double
glass distilled water.
Compound grams/I grams/41
NaCl 23.48 93.92
Na2S04 3.92 15.68
NaHC03 0.19 0.76
KC1 0.66 2.64
KBr 0.096 0.384
H3B03 0.026 0.104
MgCl2»6H20 10.61 42.44
SrCl2'6H20 0.04 0.16
CaCl2-2H20 1.469 5.876
H20 to 1,000 ml 4,000 ml
filter through prewashed 0.45 ym membrane filter.
-197-
-------
FOR DILUTION TO VARIOUS SALINITIES: (4 liter batches)
Salinity, °/00
35
30
24
20
16
12
8
5
ASW stock, 1
4.0
3.428
2.742
2.285
1.828
1.371
0.914
0.571
H20, 1 (glass distilled)
0.0
0.571
1.257
1.714
2.171
2.628
3.085
3.428
For any given final salinity, mix well, adding the following
NAAM levels of nutrients:
NaN03
KHP0
4
NaEDTA
102 mg/41 batch (4.2 mg N/l)
4.176 mg/41 batch (0.186 mg P/l)
1200 yg/41 batch (300 yg/1 )
*NAAM trace metal mix (minus FeCl3)
filter through .45 ym membrane filter,
add AFTER filtration, sterilized FeCl3, 384 yg/41 batch (33.05 yg Fe/1).
Add the following: 0.0928 g H3B03; 0.208 g MnCl2'4H20; 0.016 g
ZnCl2; 0.714 mg CoCl2'6H20; 0.0107 mg CuCl2'2H20; 3.63 mg N
2H20; make up to 500 ml, adding 1 ml of this concentrate to
each liter of media.
-198-
-------
I
H
VO
CD
Dunaliella tertiolecta
Burkholder's ASW + NAAM nutrients
Fig. 1
SALINITY, %o
© first run
A second run
slope 5.521
r=.942, 14° freedom
35
-------
Fig. 2 Growth of Dunaliella at various salinities and
phosphorus concentrations.
-------
B- Temperature and Light.
Several trials were made at 24+ 2°C, the standard
temperature used in the freshwater Algal Assay Procedure.
However, responses were both irregular and too rapid for
accurate assessment; and replication was particularly poor.
A temperature of 18°c was found to yield satisfactory
results in these same terms (25).
Light levels of 400 ft-c * 10 percent {1300yuw/cm2; 4300
lux) proved satisfactory for Dunaliella, and no reason was
seen to change this from the freshwater Algal Assay
Procedure.* *
The work so far with Tfaalassipsira has been done at
20*2°C, with no shaking, and 550 ft-c ± 10 percent |1850
w/cm2; 5920 lux according to data provided by D. P. Larsen
(30) in his work with this species. Good replication has
been obtained under these conditions.
** The energy level output of a bank of six 48 inch "cool
white" fluorescent lamps
-------
C. Biomass Determination_
Several methods of growth measurement, indirect or
direct, may be employed. Direct cell counting, using a
haemocytometer with a microscope, is relatively accurate at
high population levels, but very slow. Gravimetrically
determined dry weights are likewise time consuming and may
be at variance with calculated dry weights if substantial
bacterial growth occurs in samples, or substantial amounts
of ferric-magnesium hydroxide forms in the sample. Also,
accurate dry weights are difficult to obtain at low algal
biomass concentrations.
In the development of this test, electronic particle
counters were used, with frequent cross checks by
gravimetric dry weight determinations and haemocytometer
counts. Cell counts at either 1:10 or 1:100 dilution in
filtered isotonic saline electrolyte were made, recording
mean cell volumes (MCV) from an accompanying mean cell
volume computer. Since Punali,glla is substantially larger
than Selenastrum capricornuturn,aperture current and
amplification settings used for DunajLielj.a are generally
one-haIf that used to count Selenastrum in the freshwater
assay procedure. Generally, the electronic particle counter
was found to be the most feasible method of dealing with
large numbers of samples, but could not be economically
justified for casual or infrequent assays.
D- Nutrient Response
The initial assumption was that estuarine waters are
nitrogen limited for algal growth (11, 21, 5, 31, 32, 33).
One recent sampling survey of the Yaquina estuary showed
that, while water from the lower bay was predictably
nitrogen limited for algal growth, water from the upper bay
and its tributaries was shown to be phosphorus limited for
algal growth. The marine algal assay, using Dunaliella, can
serve to define this boundary, which can change not only
with the season but with each tide and fluctuations of
tributary inflow.
With respect to this situation, the response curves
developed for Dunaliella show that the alga will respond to
concentrations at least as low as 2.5 ug P/l, 10 ug ammonia
N/l, and 50 ug nitrate N/l in defined medium.
The curves were developed from additions (spikes)
ranging from 2.5 to 50 ug/1 in five steps for phosphorus.
Figure 3; 10 to 1,000 ug in five steps for nitrate nitrogen,
Figure 4 and ammonia nitrogen. Figures 5, 6. All assays
-201-
-------
5%o ASW
+N A AM nutrients
- phosphorus
O + 0 mg P/l
-OO25mg P/t
.005 mg P/l "
+ .01 mg P/l
.025mg P/l "
+ .05mg P/l
lO'rr-r-r
10* r
I6%o ASW
+NAAM nutrients
- phosphorus
O + 0 mg P/l
O +.0025 mg P/l
a t.OOSmg P/l ~
A + .oi mg P/l
O + .O25mgP/l ~
V +.05mg P/l -
MA 022373
8
12
16
2O 24
DAYS
Growth response of Dunoliello terliolecta
DAYS
Growth response of Dunollella tertiolecta
to3
xlO'
0>
3
I
10"
10
,-2
20%o ASW
+NAAM nutrients
- phosphorus
O + 0 mg P/l
O +.0025mg P/l
a +.005mgP/l ~
A + .01 mg P/l "
O + .025mg P/l
V 4.05mg P/l -
MA 022373
10s
or
a
10'
8
12
18
20
24
10
.-2
35%o ASW
+NAAM nutrients .
- phosphorus
O +0 mg P/'
©•KO025mgP/'
B t.OOSmg P/l ":
A + .01 mg m
O+.O25mgP/l
V4.O5mgP/l -
MA03O973R
I
I
0 4
8
12
16
20 24
DAYS
Growth response of Dunoliella tertiolecta
DAYS
Growth response of Dunoliella terliolecto
rig. 3
-202-
-------
10* I- i i i I i i i
5%0 ASW
+ NAAM nutrients
- nitrogen
low NO 5 -N
O + 0 mg N/l
0 + .0lmg N/t
+.05mg N/l
A +.lOmg N/l
O + .50 mg N/l
V + 1.0 mg N/l
MA 03O973
I03
£10°
o
10
8
DAYS
Growth response of Dunoliella tertiolecta
10
.-2
I
I6%o ASW
+ NAAM nutrients
- nitrogen
low NOg -N
O + 0 mg N/l
O + .0lmg N/l
a + .05 mg N/l
A+.IOmg N/l
O f .50 mg N/l
V + 1.0 mg N/l
MA O30973
I
I
O 4
8
12
16
2O 24
DAYS
Growth response of Dunaliella tertiolecta
20%0 ASW
+ NAAM nutrients
- nitrogen
O t O mg N/l
O +.0lmg N/!
-r.05mg N/l
A-t-.IOmg N/l
.50mg N/l
V + I.Omg N/l
MA 030973
I , . . I . .
I03
IOZ
K
O
10
12
16
20 24
,,DAYS
Growth response of Dunoliello tertiolecto
Fig. 4
10
-203-
.-2
35%o ASW
+ NAAM nutrients
- nitrogen
low NO 3 -N
O 10 mg N/l
O+.OImg N/l
o -r.05mg N/l
A-i-.lOmg N/l
O-r.50mg N/l
V + I.Omg N/l
MA 030973
. , I . .,].,.
12
16
2O 24
DAYS
Growth response of Punai'ieila tertiolecta
-------
to3
v.10'
o>
2
I
1^
>
10
10'
5%oASW _
+NAAM nutrients .
-nitrogen
lowNH*-N
O + 0 mg N/l
G + .01 mg N/l
D -f .05mg N/l
A + . I mg N/l
O + .5mg N/l
V + I.Omg N/l
MA 032373
I03
10*
10
8
12
16
20
24
I I I I I I I
I6%«ASW
+NA AM nutrients .
-nitrogen
lowNH*-N
O + 0 mg N/l
G 4.0lmg N/l .
a + .05 mg N/l
A •*•. I mg N/t
O-t-.Smg N/l
V +• l.0mg N/l
MA 032373
I , , , I . , . I , , ,
8
12
16
20 24
DAYS
Growth response of Dunaliella tertiolecta
DAYS
Growth response of Dunoliella tertiolecta
I03rr
20%oASW _:
+NAAM nutrients -
-nitrogen
lowNH*-N
O + 0 mg N/l
G + .01 mg N/l _
a + .05mg N/l :
A +. I mg N/l -
O -r .5mg N/l
V + I.Omg N/l "
a
o
10'
0 4
• •
DAYS
Growth response of Dunaliella tertiolecta
12
35%oASW
+NAAM nutrients
-nitrogen
lowNH^-N
O + O mg N/l
O +X>lmgN/l
Q + .05 mg N/l
A+.l mg N/l
O + .5mg N/l
V- I.OmgN/1
MA 032373
J.
16
20 24
DAYS
Growth response of Dunoliella tertiolecta
Fig. 5
-204-
-------
o>
>l
v.
Q
ion
o
o
1C?
Calculated Regression Line
Slope Cor.Coef fr) 16° Freedom
O 5%o
O I6%o
a 20%o
35%o
A
66.7
78.1
770
73.2
.998
.995
.993
.985
MA 032373
NHj-N ,mg/l
1:0
r.5
Fig. 6 Growth response of Dunaliella tertiolectatdavl4
-------
were run in triplicate along with controls.
Nitrilotriacetic acid (NTA) was examined as a possible
nitrogen source (34, 35, 36, 11), or for possible growth
depressing or enhancing effects by chelation in actual
estuarine field samples. There was no significant yield
difference between control and spikes ranging from 50 to
1,000 mg/1 NTA nitrogen {expressed as nitrogen, 7.3 percent
N by weight) , Table 1.
Although ammonia nitrogen was more stimulatory than
nitrate nitrogen in our ASW (37) , there appeared to be a
growth rate difference but no yield difference in spiked
field samples. Figures 14, 15, 17 and 18.
In Burkholder»s ASW <5 %o to 35%o salinity Dunaliella
produces an average of 1.076 mg dry weight per ^ig of
phosphorus, 0.0318 mg dry weight per jig of nitrate nitrogen,
and 0.0796 mg dry weight per jag of ammonia nitrogen, (Table
2).
One outstanding characteristic of the use of Dunaliella
was the consistency of data replication. An example of this
is the result of the investigation of NTA as a nitrogen
source in natural water samples. NTA appeared to have
little, if any, effect on the growth of puna^iel^a • either
stimulatory or inhibitory. In the experiment, the control
and four levels of NTA (+0.05 to 1.0 mg/1 as N) with three
replicates each gave a total of fifteen replicates for each
of six natural waters (all Oregon coastal estuaries) . In
all cases, a t-test (13 degrees of freedom) showed no
significant difference between control and spikes, and no
correlation was shown between an increase in NTA and dry
weight produced. The normalized standard deviation for the
entire run of 90 flasks was less than + 15 percent.
F . standard Inoculum Level
In order to avoid problems of nutrient carry-over in a
healthy inoculum, the size of the inoculum was reduced from
an initial 1,000 cells per ml (0.3 mg/1 dry weight) to 100
cells per ml (0.03 mg/1 dry weight). Although there was a
detectable lag in growth of about two days, growth rate was
unaffected and the final yield was virtually identical at
day 10 or 12, Figure 7. The inoculum was dispensed in a 1
ml volume.
-206-
-------
Table 2. BIOMASS PRODUCED PER UNIT OF
NUTRIENT BY DUNALIELLA AT DAY 14 IN
DEFINED
MEDIA
mg dry weigh t/yg of nutrient
Salinity P
5 °/oo 0.557
±0.158
16 °/oo 0.930
±0.240
20 °/oo 1.170
±0.164
35 °/oo 1.129
±0.232
Average 1 .076
N03~ - N
0.0096
±0.0014
0.0308
±0 .0394
0.0331
±0.0379
0.0315
±0.0361
0.0318
NH4+ - N P:N03- - N P:NH4+ - N
0.0747 1:58 1:7.4
±0.0075
0.0844 1:30.2 1:11.0
±0.0058
0.0766 1:35.3 1:15.3
±0.005
0.0765 1:41.1 1:13.5
±0.0193
0.0796 1:41.1 1:13.5
-207-
-------
10s L I i i | i i i | I i I | i I i | i i I I ' ' ' :
IOZ r
5%o ASW+ Full NAAM nutrients
inoculum strength cells/nil
o 100/ml
250/ml
CD lOOO/ml
I , , , I , , , I , , , , ,
0 4 8 12 16 20 24
DAYS
Growth response of Punch el la tertiolecta
20%o ASW+Full NAAM nutrients
inoculum strength cells/ml
o 100/ml
25O/ml
O I000/ml
IA 022373
I , , , I , , , I , , , I . , .
12 16 20 24
DAYS
Growth response of Dunoliella tertiolecta
16 %o ASW+Full NAAM nutrients
inoculum strength cells/ml
o lOO/ml
250/ml
a 1000/ml
4 8 12 16 20 24
DAYS
Growth response of Dunaliello tertiolecta
I03 L I I I | I I I
35%o ASW+ Full NAAM nutrients
inoculum strength cells/ml
© 100/ml
250ml
o 1000/ml
10
.-2
04 8 12 16 20 24
Growth response of Dunaliella tertiolectq
Fig 7
-208-
-------
FIELD TESTS
The majority of field work has been done with water from
the Yaquina Estuary, Newport, Oregon, Figure 8. It is
relatively near, and has been studied extensively by other
EPA and Oregon State University personnel. Some samples
were also obtained from several sites in Puget sound and
assayed in cooperation with the Washington State Department
of Ecology.
In general, several observations may be made: (a) the
marine algal assay indicated whether nitrogen or phosphorus
constituted a growth-limiting nutrient in estuarine waters;
(b) with a somewhat extended incubation period for bioassay,
the test organism could adjust to the lower salinities of
freshwater tributaries (although a lower limit of 3.5/0*
salinity has been noted for Dunaliella (25) j to indicate
whether nitrogen or phosphorus was algal growth limiting;
and (c) the bioassay could show the point of changeover in
the estuary from phosphorus to nitrogen limitation.
Because of the likelihood of growth limitation by light
or elements other than nitrogen or phosphorus (iron,
manganese, or, in the case of diatoms, silicon) this
bioassay may not predict the full potential of a water to
produce algal growth, However, it does seem reasonable that
the indications of nutrient limitation given by the assay
should be accurate in a relative sense (38). It should be
kept in mind all samples were membrane filtered and stored
at 4°C until assayed.
The ability of Dunaliella to respond to low level
nitrogen spikes to a natural water sample can be seen in
Figure 9, which illustrates the changeover from nitrogen to
phosphorus limitation in a sample from the upper Yaquiaa
estuary. In this case, in addition to control, nitrate
nitrogen was added at the levels of 0.02, 0.1, 0.2, 0.5, and
1.0 mg/1 as nitrogen (0.001 to 0.05 mg P/l, and N + P
combinations were also added). The growth response started
to decline just below 0.5 rag/1 total nitrogen, plateauing at
about 23 mg dry wt/1 with the 1.0 rag N/l spike. However, if
0.05 mgP/1 was added in addition to the 1.0 mgN/1 spike,
approximately 36 mg dry wt/1 was attained, If all points on
the plot are considered, except the 1.0 mg N/l set, a line
of very good fit can be plotted (r = .986, t = 26.7, 19
degrees of freedom, significant at the 0.1 percent level)
showing that about 97 percent of the change in dry weight
can be attributed to a change in the level of nitrogen
present. If one considers only nitrogen, the plateau of 23
mg dry weight/1 defined by the 0.5 and 1.0 rag N/l spikes
-209-
-------
Charlie's Dock
(16.0 miles) ...
Toledo Public
Boat Landing
(70 miles)
Yaquina River
Bridge
(21.5 miles)
Elk City
Boat Dock
19.5 miles)
MILL CREEK
Elk Creek
Bridge
(21.5 miles]
MARINE ALGAL ASSAY FIELD SAMPLING SITES
YAQUINA ESTUARY, NEWPORT, OREGON
Fig. 8
-------
35-
N 30-
a
•o
25H
£ 20-
o»
I
Q
0)
"5
o
15-
2 10-
5-
0
'(+.05 mg
P/l )
(no P
spike)
Toledo Public Boot Launching Site
Toledo, Oregon-Yaquina Estuary 25% Salinity
Slope 30783
r* 386<0.l%
t* 26.7I5
-------
Table 3. CHEMICAL PARAMETERS OF YAQUINA ESTUARY
AND TRIBUTARY SURFACE GRAB SAMPLES, 28 June 1973,
AT OR NEAR HIGH WATER, ON INCOMING TIDE
K)
I
Sampling Site
Parameter
NH4+ -N- mg/1
,M03" -N- mg/1
Tot. sol. P mg/1
Ortho-P mg/1
Sulfate, mg/1
Sol. Fe, yg/1
Sol . Mn, yg/1
Salinity, /oo
Conductivity, ymho
Tot. Sol. C, mg/1
Sol . reactive Si, mg/1
OS U Small
Boat Dock
0.036
0.120
0.035
0.03
3100
240
20
30.76
49,200
24.5
.68
Toledo Public
Boat Landing
0.018
0.129
0.015
0.013
896
100
27
14.14
23,200
15.8
2.2
Burpee
0.076
0.416
0.015
0.011
256
100
34
2.882
5,800
9
4.6
Charlie's Dock
0.06
0.430
0.015
0.019
10
140
11
<2.84
208
5.4
4.8
Elk City
0.038
0.498
0.015
0.009
3
140
13
<2.84
69
4.7
5.25
Elk River,
Upstream
0.025
0.315
0.015
0.011
<2.5
160
10
<2.84
67
4.8
5.4
Yaquina River,
Upstream
<0.001
0.560
0.01
0.008
4
140
9
<2.84
65
4.5
5.5
-------
demonstrates the advent of phosphorus limitation in this
sample. The multiple levels of spikes also show that, when
nitrogen is limiting, growth responses are linear with
respect to the spike level until some other nutrient or
physical factor becomes limiting.
Control dry weights for a series of bioassays by the
Washington State Department of Ecology on Puget Sound
samples collected by EPA Region X personnel are shown in
figure 10. A plot of day 10 dry weights against total
nitrogen accounted for 83 percent of the variation in dry
weight, indicating that nitrogen was probably the limiting
nutrient for algal growth.
Phosphorus was found to be limiting in a sample taken on
November 1, 1972 at the Burpee sampling site one day after
the onset of heavy winter rains. Figures 11, 12. Nitrogen,
however, appeared to be limiting downstream at the Toledo
and OSU dock stations. After 6 weeks of sample storage, the
status of the Toledo sample as probably nitrogen limiting
was confirmed and the phosphorus limitation in the Burpee
sample was reaffirmed. This indicated that the apparent
phosphorus limitation was real and not a condition related
to decreased salinity. Instead it was probably caused by
the increased nitrogen content of the rainwater runoff.
Figure 13 shows the results of assays on water samples
collected at the same site on August 1972, It can be seen
from control flasks that growth correlates very well with
total N but not with salinity. On June 28, 1973 samples
were taken in the Yaguina estuary. Figure 14, from OSU dock,
Elk City, and approximately 2 miles further up each of the
two tributaries, Elk River and Yaquina River. Salinities
ranged from 30.8%o (49,300 Amho conductivity) at the OSD
boat dock station to less than 2.8%o (65 .«.mho) in the
Yaquina River station [See Table 3 for chemical analysis of
sample (40) ].
After physiological adjustment, Dunaliella grew in these
essentially freshwater situations and gave a statistically
significant indication of the growth limiting nutrient,
Figures 14, 15. In this case, it is evident from the curves
that nitrogen was limiting at the OSO dock station, Figure
14. At the Toledo station {14 %c salinity) the situation is
somewhat indistinct; however a t-test shows that the growth
in the sample spiked with phosphorus is significantly
greater at the 5 percent level than that in the control or
the nitrate and ammonia spiked samples. These tests
indicate phosphorus limitation.
-213-
-------
I03 L I I I I I I I I I I I I I I I I I
10
A.-Mud Bay
B.-DabobBay
C.-Oyster Boy
D.-Oakland Bay
E-S.HoodCanol
(POTLATCH)
Filtered-No Spikes
1
1
Fig 10
4 8 12 16 20 24
DAYS
Algal Assay of Puget Sound Sites
Spring 1973
-214-
-------
10'
A+1.ON
0+.O5P+1.0N
* siqnificantly different from control
Growth response of Dunaliello tertiolecta
Yoquina Bay, OSU Dock, II/I/72 33.8%o salinity
1O2
10'
o Control
o *O5 P
A+1.0 N
GH-O5P+1ONJ
* significantly different from control
0
Ddays
1O
14
Growth response of Dunaliello tertiolecta
Yaquina Bay, OSU Dock, II/I/72 33.8%0 salinity
(sample stored 6 weeks)
A +1.0 N
0+05P+1ON
* significarrtly different from control
days
Growth response of Dunaliella tertiolecto
Yaquina Bay,Toledo, 11/1/72 24%osalinity
1O2-
cn
101
£
Q
1CP
©Control
<•> +O5 P
A +1.0 N
0+D5P+1DN
* significantly different from control
0
udoys
10
14
Growth response of Dunaliella tertiolecta
Yaquina Bay,Toledo, II/I/72 24%o salinity
(sample stored 6 weeks
-215-
-------
icy
D)
Q
10°
0
o Control
o +.O5 P
A+1.O N
0*Q5P+1QN.
significantly different from control
3
6da
ys
10
14
Growth response of Dunoliella tertiolecta
YaquinaBay, Burpee, I I/I/72,!7%o salinity
o Control
o +.05 P
ION
0*.05P+1DN_
significantly different from control
3days
Growth response of Dunoliella tertiolecta
YaquinaBay, Burpee, II/I/72 I7%»salinity
(sample stored 6 weeks)
Fig. 12
10 15 20 25 30 35
Yaquina Bay,8 August I972
& = xlO~2mg./l total N
o - %, Salinity
Growth response of Dunaliella tertiolecto
Fig. 13
-216-
-------
CONTROLS
O Yoquina.OSU Dock
O Toledo
& Burpee
& Charlie'sDock
O Elk City
V Elk River
> Yaquina River
MA 070373
10*
4 8 12 16 20 24
DAYS
Growth response of Puna 11 el la tertiolecta
io3 fc, . . i , . i
IOZ
3
I
r-
10'
lO"2
e significantly different from
control (O) and N spikes (O,A)
at day 8 and 10at the5%level.
I
•Yaquina Boy, Toledo, 14.1%.
O Control
O +0.05mg P/l
El +l.0mg NO] -N A
& -fl-Omg NHJ -N /I
O +0.05ma P/l + l.0mg NOj -N /I
V +0.05mg p/i +I.OmgNH*-N /I
^cignif icanf ly different from control
MA 071373
I . . . I , . , I , , . I , ,
16
20
24
4 8 12
DAYS
Growth response of Dunaliella tertiolecta
I
t-
*
I/.0
IO
10"
10*
T~r
YaquinoBay.OSUDock 308%.
O Control
O +0.05 mg P/l
B +l-0ma N05 -N /I
A +l.0mg NHJ -N /I
O *0.05mgP/l tLOmgNOt -N /I
* +O05mg P/( 4-UOmg NHJ-N /I
•significantly different from control
MA07I373
I
12
16
20 24
DAYS
Growth response of Dunoliello tertiolecto
IW . i i i j i i i i i i i i i i i ^^^T i | i i T »
IOZ r
Burpee, 2.9%. salinity
O Control
O +0.05mg P/!
d +1.0 mg NO] -N /I
A +l.0mg NHJ -N /I
0 +0.05mg P/l •fl.OmgNOj -N /I
» +0.05nig P/l *l.0mgNHJ-N l\
*8J9nlficontly different from control
MA 070313
I , . . I , , . I . . . I ,
16
20 24
4 8 12
DAYS
Growth response of Dunaliella tertiolecta
Fig. 14
-217-
-------
10 i i i i i
Charlie's Dock, <2B%.
O Control
0 +0.05 mg P/l
+1.0 mg N0§ -N /I
+l.0mg NHJ -N /I
O +0.05mgP/1 tLOmgNOj -N /I
+ 0.05mg P/I + 1.0 ing NH* -N /I
*signif icontly different from control
IO
12
16
20 24
DAYS
Growth response of Dunaliella tertiolecta
IOZ r
Elk River,
O Control
+O.O5ntg P/l
B -H.Omg NOJ -N XI
tl.Omg NH^ -K /I
O +0.05mgP/l +IX>mgNO" -N /I
+QO5mg P/I
*siflnif(coBtly diff0ront from control
MA 070373
12
16
20 24
DAYS
Growth response of Dunoliello tertiolecto
ElkCity,<2.8%.
O Control
© +0.05mg P/I
B +l.0mg N03 -H
+|.0mg NHJ -N t\
+O.OSmgP/l +IOmgN03 -V A
+0.05mg P/I +U)mgNH*-N /I
•significantly different from control
MA 070373
I , . , I , , . I i i i I i i
12 16 20 24
DAYS
Growth response of Dunoliella tertiolecto
l03F-r-T-r
Yoquina River, Up»treom,<2.8%«
O Control
© +0.05 mg P/l
B *l.0mg NOl -N-A
A •fl.Omg NHj -N-/I
O +OX»5mgP/l *\OmgWT3 -N /•
v +a05mg P/l 4-|jOmgNH*-N /)
*«igni?icantly diff arent from control
MA07I373
I ... I ... I ... I ...
8
12
16
20 24
DAYS
Growth response of DunoUello tertiolecto
Fig 15
-218-
-------
MARINE ALGAL ASSAY FIELD SAMPLING SITES
SOUTHERN OREGON COASTAL ESTUARIES
Fig. 16
-------
Q
10"
10"
,-1
0
Coos Bay
at North Bend, Or.
O Control
O *Q05mg.P/L
e »l.0mg. NOj -N-/L
& *1.0mg. NH? -N-/L
O *O05mg.P/L»1.0mg.NO3-N-/L
? 40.05mg.P/L*1.0mg.NH;-N-/L '
I , i , I . . ,
2O 24
4 8 12 16
DAYS
Growth response of Dunaliella
tertblecta
I
O
10
ri
10"
?£.
(5
Coos Bay at
Horsefall Road Bridge.
G Control
O »OO5imP/L
& »1.Omg. NHI -N-/L i
O .0.05mg.P/L-1.0mg.N03-N-/L
^ «O.05rng.P/L«1.Omg.NH4-N-/L n
| , . , | , . , I i . , I , , . I , .
4 8 12
16
20 24
DAYS
Growth response of Dunaliella
tertblecta
Siuslaw River
at Florence, Or
O Control
O «QD5mg.RL
O •1.0mg. NOo -N-/U
A «1.0mg. NHI -N-/L
O »005mg.PC»1.0rr«.NCft-N-/L
»0.05mg.P/L*1.0mg.NH|-N-/L
i—I—i I. i—i—i—l
12
DAYS
Growth response of Dunaliella
tertblecta
103
10'
(-
T.
10"
102
Umpqua River
at Reedsport.Or
O Control
O «OD5mg.Ri.
1.0mg.
-N-/L
ft"'4-"&"'£"'&"&"&
_ iu DAYS
Growth response of Dunaliella
tertblecta
Fig. 17
-220-
-------
Alsea River
at Waldport, Or.
O Control
O •QOSmg.fi'L
e *l.omg. NCK -N-/L
A *1.0mg. NrC-N-/L
-— PC."
*OO5mg.RL*1.0rng.NH4-N-/L
. I . , , I , . . I . . ,
8
12 16;
Growth response of DunalJglla
tertioledta
icr
10'
]E?10
2O 24
10"
>
1O"
I ' i M
Yaquina River
at Newport, Or
O control
O *Q05mg.fi|L
Q *1.0mg. NO§ -N-/L
A «1.0mg. NHt -N-/L
O *OD5rtig.RL«1.Qmg.NC5-M-fl.
I
I
I
O 4 8 12^ 16 ^O
DAYS
Growth response of Dunaliella
tertiolecta
24
IO'FTHT-T-
10'
I
I-
10°
10
10*'
O Coos Bay at North Bend
O Coos Bay at Horsefall Rd. Br
Q Umpqua River at Reedsport
& Sustaw River at Florence
0 Alsea River at Waldport
-------
Table 4. CHEMICAL PARAMETERS OF 6 OREGON
ESTUARY SAMPLES AFTER MEMBRANE FILTRATION
Surface Grab Samples, 25 July 1973,
on Incoming Tide at or near High Water
Sampling Site
Parameter
UH4+ -N- mg/1
N03~ -N- mg/1
Total P mg/1
Ortho-P mg/1
to Sulfate mg/1
N)
*? Sol. Fe, yg/1
Sol. Mn, yg/1
Salinity, °/oo
Conductivity, ymho
Tot. Sol. C, mg/1
Sol . reactive Si , mg/1
Coos Bay at
North Bend
0.145
0.044
0.03
0.028
3200
240
40
31.18
47,500
25
0.4
Coos Bay at Umpqua River
Horsefall Rd. Bridge at Reedsport
0.118
0.019
0.035
0.028
3300
240
40
31.14
47,500
26
0.4
0.027
0.003
0.01
<0.001
1500
80
20
11.62
19,500
13
2.5
Si us law River
at Florence
0.059
0.009
0.01
0.01
2500
120
20
21.97
35,000
18
0.72
Alsea River
at Waldport
0.103
0.082
0.035
0.036
3400
240
30
33.09
50,400
25
0.84
Yaquina River
at Newport
0.100
0.11
0.035
0.03
3700
240
30
32.88
50,300
25
0.72
-------
i
ro
to
LO
I03
10'
10'
10*
Abert Lake.Ore.
O +.05P + I.ON
A -f |.0mg/l N
O -t .05mg/I P
O Control
Filtered only
Conductivity :50,000umho _
I
I
I
8
12
DAYS
16
20
24
Fig.19 Growth response of DunaiiellQ tertiolecta
in alkaline fresh water
I03 L I I I I I I I I I I I I I I I I '"' I
10s
10'
I
H
10"
10"
Abert Lake.Ore
+ .05P+I.ON
I.Omg/l N
O+ .05mg/l P
O Control
Autoclaved and
filtered
Conductivity^
20
4 8 12 16
DAYS
Growth response of DunQliello tertiolecta
in alkaline freshwater
24
-------
At the Burpee station, Figure 14, (2.97** salinity, 5800
phosphorus was shown to be the limiting growth factor. Sample spiked
with phosphorus grew significantly more than control or those spiked
with nitrogen.
Samples from the remainder of the stations, all essentially freshwater
(208 -65**mho conductivity), showed marked lags in growth patterns.
All of these stations responded significantly to spikes containing
phosphorus but not nitrogen, which were not significantly different
than control. It should be noted that replication was not as good in
low salinity or freshwater as in higher salinities with Dunaliella, but
more experimentation should yield a workable test protocol to
establish acceptable replicability.
The next set of samples was taken on July 25, 1973, from five southern
Oregon coastal estuaries, from Coos Bay on the south to Yaquina Bay
on the north (Figure 16). These estuaries, sampled on the incoming tide
at or near high water, were nitrogen limited with the exception of the
Umpqua River at Reedsport. This sample, 11.6 °loo salinity, responded
in a significant manner to spikes containing phosphorus, but not to
nitrogen [Figures 17, 18; See Table 4 for chemical analysis of
samples (40)].
A further application of the marine assay using D. tertiolecta is
exemplified by results obtained on water samples from Albert Lake,
a highly alkaline (50,000^ mho) drainage sink in inland
southeastern Oregon. Attempts to assay this water with Selenastrum
capricornutum using the freshwater Algal Assay Procedure failed when
the inoculum was killed by the high alkalinity (salinity). However,
from Figure 19 we can see that after an initial adjustment lag,
Dunaliella shows nitrogen to be the growth limiting nutrient.
-224-
-------
VI. SUMMARY "
The effort to design and evaluate a marine version of the
Algal Assay Procedure: Bottle Test has resulted in the
establishment of the suitability of Dunaliella tertiolecta
Butcher (DUN clone) as a highly versatile and consistent
bioassay organism for nutrient assessment in marine, estuarine,
and some freshwater situations.
-225-
-------
REFERENCES
1. Ketchum, Bostwick H. 1971. Population, Natural
Resources, and Biological Effects of Pollution of
Estuaries and coastal Maters. In: Man»s Impact on
Terrestrial and Oceanic Ecosystems. William H.
Mathews, Frederick E. Smith, and Edward D. Goldberg,
(Eds.)- MIT Press, Cambridge, Mass. pp. 59-79.
2. Inman, Douglas L., and Birchard M. Brush. 1973. The
Coastal challenge. Science 181s20-31.
3. Atkins, w. R. G. 1923. The Phosphate Content of Fresh
and Salt Waters in its Relationship to the Growth of
Algal Plankton. J. Mar. Biol. Assoc. 13:119.
4. Smayda, T. J. 1970. Growth Potential Bioassay of Mater
Masses Using Diatom Cultures: Phosphorescent Bay
(Puerto Rico) and Caribbean Waters. Helgol. Wiss.
Meeresunters. 20:172-194.
5. Goldman, Joel C., Kenneth R. Tenore, and Helen I.
Stanley. 1973. Inorganic Nitrogen Removal from
Wastewater: Effect on Phytoplankton Growth in
Coastal Marine Waters. Science 180:955-956.
6. Skulberg, Olav. 1964. Algal Problems Related to the
Eutrophication of European Water Supplies, and a
Bioassay Method to Assess Fertilizing Influences of
Pollution on Inland Waters. In: Algae and Man.
Ed. D. F. Jackson. Plenum Press, N. Y. pp. 262-
299.
7. Glass, Gary E. 1973. Bioassay Techniques and
Environmental Chemistry. Ann Arbor Science Publ.,
Inc. Ann Arbor, Mich.
8. Dierberg, Forrest E. 1972. Development of an Algal
Assay for Estuarine Water Quality. MS Thesis,
University of North Carolina, Dept. of Environmental
Sciences and Engineering.
9. McPhee, Craig. 1961. Bioassay of Algal Production in
Chemically Altered Waters. Limnol. and Oceanogr.
6:416-422.
-226-
-------
10. Tarzwell, C. M. 1971. Bioassays to Determine Allowable
Waste Concentrations in the Aquatic Environemnt.
In: H. A, Cole (Organizer). A Discussion on
Biological Effects of Pollution in the Sea. Proc.
Roy. Soc. Lond. B 177 <1048):279-285.
11. Mitchell, Dee. 1973. Algal Bioassays for Estimating
the Effect of Added Materials Upon the Planktonic
Algae in Surface Waters. In: Bioassay Techniques
and Environmental Chemistry, 6. E. Glass (Ed.).
Ann Arbor Sci. Publ., Inc., Ann Arbor, Mich. pp.
153-158.
12. Tallqvist, Torsten. 1973. Algal Assay Procedure
{Bottle Test) at the Norwegian Institute for Water
Research. In: Algal Assays in Water Pollution
Research: Proceedings from a Nordic Symposium,
Oslow, 25-26 October, 1972. Nordforsk. Secretariat
of Environmental Sciences, Helsinki. Publ. * 1973-2.
pp. 5-17.
13. Tallqvist, Torsten. 1973. (b) Use of Algal Assay for
Investigating a Brackish Water Area. In: Algal
Assays in Water Pollution Research: Proceedings
from a Nordic Symposium, Oslow, Sciences, Helsinki.
Publ. t!973-2. pp. 11L-123.
14. Fitzgerald, G. P. 1972. Bioassay Analysis of Nutrient
Availability, In: Nutrients in Natural Waters,
Herbert E. Allen and J. R. Kramer (Eds.). Wiley
Interscience, N. Y. pp. 147-170.
15. Edmondson, W. T.* and Y. H. Edmondson. 1917.
Measurements of Production in Fertilized Salt Water.
Jour. Mar. Res. 6:228-215.
16. Thomas, William H. 1971. Effects of Nutrients on Growth
of Algae: Tropical Oceanic Phytoplankton. Final
Report to National Science Foundation. IMN
Reference 71-9. Scripps Inst. Oceanogr.
17. Ryther, John H., and William M. Dunstan. 1971.
Nitrogen, Phosphorus, and Eutrophication in the
Coastal Marine Environment. Science 171:1008-1013.
-227-
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18. O«Sullivan, A. J. 1971. Ecological Effects of Sewage
Discharge in in the Marine Environment. In: H. A.
Cole (Organizer), A Discussion on Biological Effects
of Pollution in the Sea. Proc. Roy. Soc. Lond. B.
177(1048):331-351.
19. Waite, Thomas and and Ralph Mitchell. 1972. The Effect
of Nutrient Fertilization on the Benthie Algae O£va
lactuca.. Zeit. Botanica Marina 15(3) s 151-156.
20. Preston, A., and P. C. Wood. 1971. Monitoring the
Marine Environment. In: H. A. Cole (Organizer), A
discussion on Biological Effects of Pollution in the
Sea. Proc. Roy. Soc. Long. B 177(1048):451-462.
21. Ryther, J. H., and R. R. L. Guillard. 1959. Enrichment
Experiments as a Means of Studying Nutrients
Limiting to Phtoplankton Production. Deep Sea Res.
6:65-69.
21. Ryther, John H. 1954. The Ecology of Plankton Blooms in
Biol. Bull. 106(2):198-209.
23. Apollonia, s. 1973. Glaciers and Nutrients in Arctic
Seas. Science 180:491-493.
24. Taylor, Barrie P., Chun C. Lee, and John S. Bunt. 1973.
Nitrogen-fixation Associated with the Marine Blue-
green Alga, Trichodesmium, as measured by the
Acetylene-reduction Technique. Archiv. Mikrobiol.
88:205-212.
25. McLachlan, J. 1960. The Culture of Dunaliella
tertiolecta Butcher - A Euryhaline Organism. Can.
J. Microbiol. 6:367-379.
26. Provasoli, L. 1963. Organic Regulation of
Phytoplankton Fertility. In: M. N. Hill (Ed.), The
Sea. Vol. II. Wiley, N. Y. pp. 165-219.
27. Burkholder, P. 1963. Some Nutritional Relationships
Among Microbes of the Sea Sediments and Waters. Ins
Symposium on Marine Microbiology, Ed., C. H.
Oppenheimer. pp. 133-150. Thcmas, Springfield.
28. Kester, Dana R., Iver W. Duedall, Donald N. Connors, and
Ricardo M. Pytkowicz. 1967. Preparation of
Artificial Seawater. Limnol. and Oceanogr.
12(1) :176-179.
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29. Lyman, J., and R. H. Fleming. 1940. Composition of
Seawater. J. Mar. Res., 3:13*4-146.
30. Larsen, D. P. 1973. Personal communication.
31. Welch, Eugene B. 1968. Phytoplankton and Related
Water-quality Conditions in an Enriched Estuary. J.
Water Poll. Contr. Fed. 40(10):1711«1727.
32. Eppley, R. W., A. F. Carlucci, O. Holm-Hansen, D.
Kiefer, J. J. McCarthy, and P. M. Williams. 1971,
Evidence for Eutrophication in the Sea Near Southern
California Coastal Sewage Outfalls, July, 1970.
California Oceanic Fisheries Investigations Reports.
UCSD 10P20-90.
33. Thomas, William H. 1970. Effect of Ammonium and
Nitrate concentration on Chlorophyll Increases in
Natural Tropical Pacific Phytoplankton Populations.
Limnol. and Oceanogr, 15(3) :386-394.
34. Erickson, S, J., T. E. Maloney, and J. H. Gentile.
1970. Effect of Nitrilotriacetic Acid on the Growth
and Metabolism of Estuarine Pfeytoplankton. J. Water
Poll. Contr. Fed. 42(8) Pt. 2:E329-R335.
35. Rudd, J. W. M., B. E. Townsend, and R. D. Hamilton.
1973. Discharge of Nitrilotriacetate (NTA) from Two
Sewage Treatment Facilities Located in a Mid-
continential Climate, J. Fish, Res. Bsd. Can.
30(7):1026-1030.
36. Sturm, R. N., and A. G. Payne. 1973, Environmental
Testing of Trisodiura Nitrolotriacetate: Bioassays
for Aquatic Safety and Algal Stimulation, Ins
Bioassay Techniques and Environmental Chemistry. G.
E. Glass (Ed.). Ann Arbor Science Publ., Inc. Ann
Arbor, Mich.
37. Paasche. S. 1971. Effect of Ammonia and Nitrate on
Growth, Photosyntheses, and Ribulosediphosphate
Carboxylase Content Of Dunaliella tertiolecta.
Physiol. Plant. 25:294-299.
38. Maloney, T. E., William E, Miller, and Tamotsu
Shiroyama. 1972. Algal Responses to Nutrient
Additions in Natural Waters. I. Laboratory Assays.
In: Nutrients and Eutrophication. G. E. Likens
(Ed.). Special Symposia, Volume I, 1972. pp. 134-
140. Am. Soc. Limn, and Oceanogr., Inc.
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39. National Eutrophication Research Program. 1971. Algal
Assay Procedure Bottle Test. Environmental
Protection Agency, O. s. Gowt. Printing Off s
1972-795-146/1. Region X.
40. Anonymous. 1971. Methods for Chemical Analysis of
Water and and Wastes. Environmental Protection
Agency. U. S. Govt. Printing Office.
41. Tyler, John E. 1973, Applied Radiometry. Oceanogr.
Mar. Biol. Ann. Rev. Vol. 11:11-15.
42. Ketchum, Bostwick H. 1972. The Water's Edge: Critical
Problems of the Coastal Zone. MIT Press, Cambridge,
Mass. pp. 157-160.
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MONITORING SEAWATER FOR RADIONUCLIDES
B. Kahn and D. M. Montgomery *
Introduction
Radioactivity has been monitored in the environment to
assess radiation exposure and protect public health for
approximately thirty years. The first source of radiation
pollution was atomic energy research laboratories, then
fallout from testing nuclear weapons was added, and most
recently effluents from nuclear power stations and
associated facilities have become a primary concern. The
world-wide fallout from nuclear test explosions in the
atmosphere stimulated extensive measurements and research to
describe the movement of radionuclides in the environment,
including the marine ecosystem. Some of the attempts to
interpret marine radioactivity data led to programs for
studying transport phenomena with artificial and natural
radionuclides as tracers.
Measurements of radionuclides in seawater by many
nations are thoroughly documented. The state of the art and
current trends are indicated by proceedings of a symposium
sponsored by the International Atomic Energy Agency (IAEA)
at Seattle in 1972 (1), and in a recent comprehensive review
by the NAS-NRC Panel on Radioactivity in the Marine
Environment (2), Monitoring methods and problems are
summarized in two IAEA publications on marine radioactivity
studies (3, 1); the more recent one (3) has probably the
best available discussion of problems and possible
solutions.
The basis for monitoring radioactivity is the
consideration of critical radionuclides, pathways, and
populations (5) , where "critical" is defined as most
important in leading to radiation exposure. This approach
is necessary because there are too many radionuclides,
available pathways, and potentially exposed persons to
Radiochemistry and Nuclear Engineering Facility,
Office of Radiation Programs and National
Environmental Research center, US EPA,
Cincinnati, Ohio
-231-
-------
consider all with equal effort. Computational models of
radiation exposure may be developed to identify and quantify
the critical components, with research undertaken to
evaluate uncertain vectors and measure dispersion and
transfer factors. A primary purpose of a monitoring program
is to test compliance with legal limits; monitoring data may
also be used to confirm computational models, provide
numerical factors applicable to the specific locale and
check source terms.
The main considerations that shape programs for
monitoring radioactivity in the marine environment are:
1. numerous radionuclides have to be measured;
2. radionuclides in seawater are at extremely low
concentrations—levels of the order of 0.1 to 1
picocurie (pCi) per liter, that correspond in the
case of »osr, for example, to approximately 10-»7 M;
3. some radionuclides take several chemical or physical
forms unlike those of indigenous stable nuclides;
and
4. potential radiological hazards usually arise from
concentration of radionuclides in biota exposed to
seawater.
Analytical Procedures
Radiochemical analysis of seawater usually combines
radionuclide accumulation, chemical separation, and nuclear
radiation detection. Whether all three are needed depends
on the nuclear and chemical characteristics of the
radionuclide, its concentration relative to the required
measurement sensitivity, and possible interferences. For
example, radionuclides that emit relatively penetrating
gamma rays and are measured only to assure compliance with
radiation protection standards (6) may not have to be
concentrated or separated chemically.
Distinctly different approaches are followed if the
analysis requires a few milliliters, a few liters, or
hundreds to thousands of liters. In the first case, the
sample is treated like other liquids (7, 8) by
radioanalytical procedures such as the ones compiled by the
NA3-NRC Radiochemistry Subcommittee (9). In the second
case, these methods are adapted, with some modifications, to
concentrate the radionuclide from the initially larger
volumes of water. In the third case, very large volumes of
seawater usually are passed through collectors that have
been found effective for one or several radionuclides of
interest and the accumulated radionuclides are then
-232-
-------
analyzed. As an alternative to these analyses, the
radionuclide concentration may be measured in situ by
suspending a radiation detector over or within the water, or
exposing a measured fraction of the water to the detector.
Concentration of radionuclides from water has been
accomplished by coprecipitation, ion exchange, absorption or
solvent extraction. Many of the processes for
simultaneously concentrating a variety of radionuclides are
not applicable for seawater, however, because of its high
salt content. A complete review of all concentration
methods used for determining radionuclides from seawater
would be prohibitively long, but some of the concentration
techniques employed for the more radiologically important or
frequently analyzed radionuclides are listed in Table 1.
Concentration by coprecipitation with Fe(OH)- or absorption
on MnO2 has been most widely used. Among the radionuclides
included, 9°Sr and 137Cs have been been of primary concern
because of their accumulation in seawater from global
fallout. Most procedures for *°Sr involve precipitation of
strontium carbonate or oxalate. Cesium-137 is generally
concentrated by absorption on a variety of inorganic ion
exchangers.
Although the concentration methods listed in Table 1
have been used for volumes up to 1000 liters, their
application for sample volumes in excess of several hundred
liters is limited by need for special equipment or
facilities for handling large volumes. A sampler developed
by Silker et al. (32) for concentrating radionuclides from
seawater eliminated this limitation. The sampler combines
filtration of particulate radionuclides and absorption of
dissolved radionuclides on an aluminum oxide bed in an
integral unit. Where several thousand liters must be
processed, seawater is pumped through the sampler at a flow
rate of 40 liters/minute. The main disadvantage of the
system is the low retention efficiency for many
radionuclides on the aluminum oxide bed, For monitoring
purposes, the collection efficiency for the radionuclides of
interest must be determined.
Recent studies (33) at a nuclear power station that
discharges liquid wastes into seawater agree with earlier
observations (1) that effluent radionuclides are not
necessarily in the most commonly encountered chemical state.
Concentration techniques, therefore, had to be tested with
samples spiked with radioactive waste solutions being
discharged. Filtration and ion exchange were employed for
quantitatively recovering s*Mn, ««Co, *°Co, »=»*cs a«d l37Cs
from 400 liters of seawater, Seawater was pumped through
-233-
-------
I
to
Table 1
Concentration Methods for Hadionuclides in Seawater
Radionuclide
Volume Analyzed
(liters)
Insol.
Hydroxides*
s«Co,
•»Sr, »°Sr
Concentration Method
1-10
20
100-200
5-50
50-200
1-50
Extraction into isoamyl alcohol
or n-butanol
10-1000 Coprecipitation with Fe {OHK
Absorption on MnO
Ion exchange on chelating resin
Precipitation of Sr as carbonate
or oxalate
Precipitation as Zr~(PO,), and
Nb20 5 H20
Coprecipitation with Cos
Absorption on inorganic ion
exchangers AMP* or metal
ferrocyanides
Coprecipitation with BiPO, or
Fe(OH)3
References
10,11
11-15
16
17-22
23
24
25-28
29-31
, »**Ce
«• AMP: ammonium phophomolybdate
-------
0.45-u membrane filters in cartridges at a flow rate of 15
liters/minute to collect particulate radionuclides. The
filtrate was then passed through Chelex-100 chelating ion
exchange resin to concentrate 5*Mn, 5»Co, and *<>Co, and
through ammonium hexacyanocobalt ferrate (NCFC) on silica
gel for »3*Cs and »3*cs retention. A disadvantage was the
relatively slow (12 liters/hour) flow rate necessitated by
the slow exchange rate of the chelating resin.
Selection of the appropriate radiation measurement
instrument also contributes to detection sensitivity. For
radionuclides that emit gamma rays, the Ge(Li) detector with
multichannel analyzer is now usually preferred. The high
detector resolution permits simultaneous measurement of
numerous photon-emitting radionuclides with precise
identification by gamma-ray energy. Moreover, it analyzes
the sample nondestructively and can process volumes of
several liters. Instruments for radionuclides that emit
only alpha particles, beta particles, or weak x-rays have
lower limits of detection, but, with few exceptions,
chemical separation is needed before measurement.
In §itu measurements of beta particles have been
obtained with Geiger-Mueller counters, and of gamma rays,
with Nal(Ti) detectors, either directly in the water or in
accumulation media such as ion-exchange resins. Such
systems are more efficient than sampling programs in
providing immediate and continuous measurements of
radioactivity in water, but the counting efficiency is
uncertain, contamination is an ever-present possibility, and
the detection sensitivity is usually not as high.
Problems
Difficulties in sampling the marine environment,
analyzing samples, and determining radioactivity values are
discussed in many of the cited references, particularly
reference (3) . Acquaintance with this body of knowledge
will prevent wasted effort and the dissemination of
misinformation. Recommended solutions, however, may only
apply to specific sources, samples, and situations. After
selecting the monitoring methods that appear to be most
suitable, they must be thoroughly tested. The following
categorizing of a source-oriented monitoring program is
attempted to separate some of these problems for more
convenient individual consideration:
1. SamBlingJTechnigue. If the sampling program is to yield
accurate"resuits of average radionuclide concentration or
total amount, sampling locations and sampling frequencies
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(both horizontal and vertical) must be carefully selected.
The sampling program must then be continually evaluated for
the influence of differences in sampling locations and times
with the best sampling and analysis techniques (see below).
As in other monitoring programs, care should be used to
insure the integrity of the sample.
2. Sample storage. Radionuclides in a stored water sample
may change their chemical or physical form, deposit
irreversibly on container surfaces and suspended material,
or be lost to the air. Tests may show that the radionuclide
or another constituent of the sample should be separated
immediately after collection, that a particular container
material is suitably inert, or that losses will be prevented
by adding acid, base, stable isotopic carrier, or complexing
agent. Freezing the sample, lining the container with
another material, or analyzing container as well as sample
may solve the problem. Although these difficulties may
arise with any substance, they are particularly serious for
radionuclides at their usual extremely low concentration.
Tests must be performed with actual samples, not with
radioactive tracers that may be in different chemical forms.
It can be anticipated that a process suitable for one
radionuclide will not necessarily apply to some others.
3« Radionuclide identification. It is tempting to identify
the radioactive constituents of a sample by analyzing a
convenient volume with a simple procedure such as gamma-ray
spectrometry. However, some radiologically significant
radionuclides may be below the detection limit or emit no
gamma rays and are overlooked by this procedure. All
potentially significant radionuclides should be identified
at the source before dilution, when detection is easier, and
then measured for confirmation in seawater with appropriate
volumes and radiation detectors.
4, Radignuclide.concentration and purification. The most
effective approach, generally, is to test initially those
methods that have been used successfully in similar
programs, particularly those evaluated by several
laboratories. Even such procedures, however, must be tested
with the samples of interest. A different chemical form of
trie radionuclide in the sample and the presence of
interfering substances may invalidate a method that was
effective in other situations or had been tested with
routinely available radioactive tracers. Hence, the
chemical state of the radionuclide must either be
determined, or sufficiently altered by the analytical
procedure to permit effective treatment for collection,
purification, and preparation for measurement. A high yield
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in the usual radiochemical procedure of carrier or tracer
addition and recovery then provides a useful index of method
reliability with regard to radionuclide collection.
5. Radionuclide^measurement. Accuracy of results is
assured by quality control of the following factors: (a)
absence of consistent errors in radiochemical analysis
demonstrated by analyzing "known" samples; (b) magnitude of
random errors quantified by replicate analyses; (c) level of
radioactive contaminants measured by "blank" analyses; (d)
calibration of radiation detector for counting efficiency of
specific radionuclides or as function of radiation energy
with radioactivity standards that are from or traceable to
NBS; (e) normal functioning of radiation detection
instrument checked by counting performance standards and
"background11, and (f) identification of radionuclides
confirmed by observing decay characteristics such as type
and energy of radiation, half life, and genetic
relationships. The effectiveness of such programs is
enhanced by separating the responsibility for analyses from
that for quality control results and participating in
intercomparisons with other laboratories, such as the IAEA
program (1).
6« Data conf irmation. Atypical samples, unknown losses,
radioactive contaminants in the environment or the
laboratory, and calculational errors are common causes of
misinformation in the literature. Most questionable values
would be promptly recognized as such by comparison with
results for similar samples in the same or other programs,
and concentration values in related samples, such as biota
or sediment.
7. Data reportingand evaluation. As a minimum
requirement, the uncertainty in the results and limits of
detection must be reported, and the significance of results
discussed in terms of the source, "local background" values,
and radiation exposure by critical pathways. Information on
other exposure pathways and comparisons with similar
situations elsewhere are also useful. Data reports that are
undefined with regard to accuracy and radiation impact are
at best incomplete and at worst the cause of unending
problems.
Summary and Conclusions
An acceptable radiological monitoring program can be
prepared on the basis of extensive experience with both
routine programs and research concerning radionuclide
transport in the marine environment. Many difficulties must
be considered: those common to most marine monitoring
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programs; those associated with the chemical state of the
major radionuclides in effluents (including their extremely
low concentration); and those related to measuring numerous
radionuclides at very low levels. Participation in the
program by appropriate specialists is a necessity, and use
of only an oceanographer or a radiochemist when both are
needed usually leads to initially unrecognized difficulties.
The following activities can be specifically recommended
to EPA for assuring an adequate radiological monitoring
program:
1. Participation with organizations such as the IAEA to
appoint expert groups for designating reference methods for
monitoring.
2. A testing program for reference methods to confirm their
applicability and observe their limitations.
3. Development of transport models for point sources.
tt. Preparation of quality control guides for radiochemical
analysis.
5. Support for, and participation in, laboratory
intercomparisons of seawater samples with radionuclides of
interest.
6. Support for availability of appropriate NBS
radioactivity standards in useful forms.
Many of these activities are components of existing programs
in EPA, but some might well be expanded.
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1 • Radioactive Contamination of the Marine Environment,
International Atomic Energy Agency, Vienna, 1973.
2. Radioactivity in the Marine Environment, National
Academy of Science, Washington, D.~C., 1971.
3« Reference Methods for Marine Radioactivity studies.
International Atomic Energy Agency, Vienna, 1971.
*• Methods of Surveying and Monitoring Marine
Radioactivity, Safety Series No. 11, International
Atomic Energy Agency, Vienna, 1965.
5. Committee 4 of the International Commission on
Radiological Protection, Principles of Environmental
Monitoring Related to the Handling of Radioactive
Materials, ICRP Publication 7, Pergamon Press, New
York, 1965.
6. USAEC, "Standards for Protection Against Radiation",
Title 10, Code of Federal Regulations, Part 20, U.S.
Govt. Printing Office, Washington, D. C. 1965.
7. Kahn, B., "Determination of Radioactive Nuclides in
Water", in Water and Water Pollution Handbook, L. L.
Ciaccio, ed,, Marcel Dekker, Inc., New York, 1973,
1357.
8. "Radioactive Nuclides in Water", in Manual on Water,
American Society for Testing and Materials,
Philadelphia, 1969, 287.
9. Subcommittee on Radiochemistry, "Radiochemistry of
Cadmium", USAEC Rept. NAS-NS-3001, 1969, to
"Radiochemistry of Plutonium", USAEC Rept. NAS-NS-
3058, 1965.
10. Flynn, W. W., Meehan, W. R., Anal. Chem. Acta. 63
(1973) 483-8.
11. Chakravarti, D., Lewis, G. B., Palumbo, R. F., Seymour,
A. H., Nature (London) 203 (1964) 571-5.
12. Cutshal, N., Johnson, V., Osterberg, C., Science 152
(1966) 202-3.
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13. Mccurdy, D. E., Russo, J. J., Quarterly Progress Report
(April 25-July 15, 1972) New Jersey Dept. of
Environmental Protection, Trenton (1972).
14. Slowey, J. F., Hayes, D., Divon, B., Hood, D. W.,
Symposium on Marine Geochemistry, USAEC Report NYO-
3450-1 (1965) 109-29.
15. Yamagata, N. G., Iwashima, K., Nature (London) 200.
(1963) 157.
16. Lai, M. G., Goya, H. A., USAEC Rept. USNRDL TR-67-11.
17. Miyake, Y., Saruhashi, K. , Katsuragi, Y., Pap.
Meteorol. Geophys., Tokyo 11 (1960) 188-90.
18. Rocco, G. G., Broecker, W. S., J. Geophys, Res. 68
(1963) 4501-12.
19. Sugihara, T. T., James, H. I., Troianello, E. J. Bowen,
V. T., Anal. Chem. 31 (1959) 44-9.
20. Higano, R., Shiozaki, M., Marine Res. Lab. Hydrogr.
Office Japan 1 (1960) 137-45.
21. Azahazha, E. G., "Methods for the Determination of
Strontium-90 in Sea Water", V. I. Baranov and L. M.
Khitrov, eds., Akad. Nauk SSR, Oceanographic
Commission (1964) 177-84 (Israel Program Scientific
Translation 1966). (Available from U.S. Dept. of
Commerce, Springfield, Va.,)
22. Sutton, D. C. , Kelly, J. J., USAEC Rept. HASL-196
(1968) .
23. Hampson, B. L., Analyst 88 (1963) 529-33.
24. Yamagata, N., Iwashima, K., "Cobalt Sulphides, an
Effective Collector for Radioruthenium Complexes in
Seawater", in Rapid Methods f_or Measuring
Radioactivity in the Environment, IAEA, Vienna,
1971, 85-90,
25. Folsom, T. R., Saruhashi, K,, J. Radiat. Res. 4 (1963)
39-53.
26. Boni, A. L., Anal. Chem 3j8 (1966) 89-92.
27. Prout, W. E., Russell, E. R., Groh, H. J., J. Inorg.
Nucl. Chem. 27 (1965) 473-9.
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28. Kourim, V., Rais, J., Million, B., J. Inorg. Nucl.
Chem. 26 (1964) 1111-5.
29. Filial, K. C., Smith, R. C., Folsom, T. R., Nature
(London) 203 (1964) 568-77.
30. Talvitie, N. A., Anal. Chem. 43 (1971) 1827-30.
31. Sakanoue, M., Nakaura, M., Imai, T., " Deter mi nation of
Plutonium in Environmental Samples", in Rapid
Methods for Measuring Radioactivity in the ~
Environment, IAEA, Vienna, 1971, 171-81.
32. Silker, W. B., Perkins, R. w., Rieck, H. G., Ocean
Engrg. 2 (1971) 49-55.
33. Montgomery, D. M., Krieger, H. L., Kahn, B., "Monitoring
Low Level Radioactive Aqueous Discharges from a
Nuclear Power Station in a Seawater Environment" in
Surveillance around Nuclear Installations,
International Atomic Energy Agency, Vienna, to be
published.
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METHODS FOR MONITORING RADIOACTIVITY IN AQUATIC BIOTA
V. A. Nelson, W. R. Schell and A. H. Seymour*
I. INTRODUCTION
The general scope of this paper will be limited to a
discussion of monitoring for radioactivity in aquatic biota,
and the specific purpose will be: to review pertinent
literature concerning monitoring radioactivity in the
aquatic environment; to discuss the critical pathway and
specific activity approaches to sampling; to outline two
monitoring programs conducted by the Laboratory of Radiation
Ecology (LRE); and to describe sample collection, sample
handling, chemical procedures and radiometric equipment used
in monitoring programs now in progress at the Laboratory of
Radiation Ecology.
Literature concerning monitoring radioactivity in the
marine environment is extensive, and only a brief review
will be presented in this report. A principal source of
information on this subject is found in the publications of
the International Atomic Energy Agency (IAEA). The 1970
IAEA report, "Reference Methods for Marine Radioactivity
Studies,*1 details procedures for sample collection, storage
and preparation, describes analytical methods for several
radionuclides, and is a good general purpose reference
manual for monitoring studies. Other general references
include "Methods of Surveying and Monitoring Marine
Radioactivity," (3); the "Manual on Environmental Monitoring
in Normal Operations," (4); "Environmental Contamination by
Radioactive Materials," (S); and the Environmental
Protection Agency (EPA) technical report, "Environmental
Radioactivity Surveillance Guide," (24).
Of the references dealing principally with radiochemical
techniques employed in monitoring, the most complete
practical survey of procedures available is prepared by the
USAEC Health and Safety Laboratory, entitled "HASL
Procedures Manual" (23). This document contains information
on counting procedures, sample treatment, analytical
separations and data reporting. The details of the
procedures have been tested by both HASL and contractor
laboratories. Other useful documents discussing
Laboratory of Radiation Ecology,
College of Fisheries,
University of Washington.
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radiochemical analysis are the 1967 Public Health service
report, "Radioassay Procedures for Environmental Samples",
the 1966 World Health Organization report, "Methods of
Radiochemical Analysis"; and two IAEA reports, "Quick
Methods for Radiochemical Analysis," (6); and "Rapid Methods
for Measuring Radioactivity in the Environment," (8) .
Two National Academy of Sciences publications of
interest are, "Radionuclides in Food," (14) which discusses
sources, levels and does evaluations of both natural and
man-produced radionuclides in food and water, and
"Radioactivity in the Marine Environment," (13) which
provides a summary of what has been learned about the
sources, distribution and concentration of radionuclides in
the marine environmment and their effects upon marine
organisms and man.
II* Monitoring Programs
Sampling and analytical procedures have been developed
in our laboratory, or adapted from the procedures of others,
to determine the concentrations of radionuclides in many
types of environmental samples. (In this regard, the term
"environmental samples" refers to all types of samples—
plants and animals as well as air, water, soil and
sediments.) The procedures have been used for the
collection and analyses of a large variety of samples from
far-ranging geographical areas. The samples have been
obtained in conjunction with the Laboratory's research
programs which include monitoring radionuclides at Amchitka
Island in the Aleutian Islands, studies of the distribution
of radionuclides at the former test sites of Bikini and
Eniwetok Atolls in the Marshall Islands, monitoring of the
marine environment of Washington State to delineate
distribution of radionuclides produced by the Hanford Atomic
Works, conducting a survey of a proposed nuclear power plant
site, and determining levels of natural lead-210 and
polonium-210 in the marine environment.
A. Objectives
A common reason for the initiation of a sampling
program is to gather data that ultimately may be necessary
to assess the human radiation exposure from radioactive
materials released to the environment. Another reason is to
make use of the radionuclides that are present in the
environment as tags or tracers that can provide basic
information about environmental processes, studies that may
be, but are not necessarily, related to the assessment of
human radiation exposure. In some situations the amount or
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type of radionuclides released may not be significant enough
to justify a monitoring program based on either of the above
reasons; however, a monitoring program solely to satisfy
public interest as to the fate of the radionuclides released
to the environment may still be required. In the opinion of
the authors, and others (22, 27), monitoring radioactive
waste disposal operations to provide protection for marine
resource organisms (salmon, crabs, etc.) is not necessary if
ICRP standards for human health are maintained.
B. Approaches
Two of the approaches to a monitoring program
concerned with public health are the critical pathway
approach and the specific activity approach.
The critical pathway approach is a study of what happens
to a radionuclide from the time is is introduced into the
aquatic environment until it reaches man, and is described
in detail in the International Commission on Radiological
Protection (ICRP) Publication No. 7 (9). Briefly, it
suggests that if all of the radionuclides present in a
particular environment and all of the ways by which they can
reach man are investigated, it may be found that only one or
two radionuclides and pathways need to be investigated as
they will be more critical to human health than all of the
other radionuclides and pathways combined.
A schematic outline of the critical pathway approach, as
adapted from Preston, 1969, is shown in Figure 1. The
figure suggests that there are three steps to be followed in
order to determine the maximum permissible amount of
radionuclide(s) to be discharged daily as an environmental
contaminant. First, the concentration of the
radionuclide(s) in the receiving water and in the food web,
and the dietary and living habits of the local population
are established; second, the critical pathways are
identified from the above information; and third, the dose
to the human population is estimated, the dose compared to
the permissible exposure as defined by the ICRP and the
results of the comparison are related to the daily discharge
rate.
Normally, in the first stages of a critical pathway
monitoring program, subsidiary pathways also will be sampled
until enough data are available to confirm or reject
selection of the critical pathways. However, by combining
present knowledge of the behavior of radionuclides in the
aquatic environment with knowledge of local food organisms
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and local human population habits, a reasonable allocation
of sampling effort can be made at an early planning stage.
The concept of the specific activity approach was
presented in NAS-NRC publication 985 (12) and has been
discussed in several recent publications, including Kaye and
Nelson (10), Preston (15), and the NAS RIME Report (13).
This approach provides a method for establishing maximum
permissible concentration values for radionuclides in
seawater (MPCC) and, basically, states that the specific
activity of a radionuclide in seawater should be no greater
than the specific activity for the same radionuclide in tbe
critical organ of man. Specific activity in this context is
defined as microcuries of the radioisotope per gram of
element; for example, uCi of *«Fe per g of Fe. For the
critical organ of man, the specific activity is the ratio of
the maximum permissible burden for the critical organ as
defined by ICRP to the weight of the total element in the
critical organ; for seawater, the specific activity is the
ratio of the MPCC value in uCi per kg to the weight of the
total element per kg {of seawater). If the specific
activity for seawater and for the critical organ of man are
equated, then,
MPCC (uCi/kg) = (specific activity in critical organ)(g of
element per kg seawater)
Another presentation of the specific activity approach
is the schematic outline prepared by Preston (15) and given
in Figure 2. The specific activity in Figure 2 is defined
as the limiting specific activity for seawater on the
hypothesis that if the specific activity for the critical
organ is not exceeded in seawater, then the maximum
permissible burden for the radionuclide in the critical
organ can not be exceeded in man by eating seafoods,
regardless of amount.
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ESTIMATED ACTIVITY LEVEL IN WATER AT EQUILIBRIUM FROM
A KNOWN DISCHARGE FATE
LOCAL DIETARY AND LIVING HABIT INFORMATION
CONCENTRATION FACTORS FOR ORGANISMS OR MATERIALS IN
CRITICAL PATHWAYS
ACTIVITY LEVELS IN CRITICAL PATHWAYS
*
LOCAL DIETARY AND LIVING HABIT INFORMATION
DAILY INTAKE/DAILY EXPOSURE
ICRP MAXIMUM PERMISSIBLE DAILY INTAKE/EXPOSURE
MAXIMUM PERMISSIBLE DAILY DISCHARGE RATE
FIGURE 1. CRITICAL PATHWAY APPROACH TO RADIOACTIVE WASTE
DISPOSAL ASSESSMENT (ADAPTED FROM PRESTON, 1969)
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Poster, Ophel and Preston (2) made a careful comparison
of both the critical pathway and specific activity
approaches for determining allowable concentrations of
radionuclides in seawater. They pointed out that both
approaches require knowledge about the kinds, amounts, and
the physical and chemical forms of the radionuclides
released, about dilution and distribution of the
radionaclides in the receiving water due to mechanical and
natural processes, and about the availability of the
radionaclides to the biota. However, the critical pathway
approach also requires information about the concentration
of radionuclides from seawater by marine organisms and about
the kinds and amounts of seafoods eaten by the local
population, whereas the specific activity approach does not.
These are difficult parameters to establish and limit the
reliability of the critical pathway approach. However, the
specific activity approach provides a very conservative
estimate of the allowable concentration of radionuclides in
seawater because it assumes that all of manfs food comes
from the sea and does not fully compensate for the reduction
in the specific activity value that occurs as the
radionuclide moves from the sea to man. This approach is
not fully applicable for radionuclides that do not have a
stable counterpart in seawater, the critical organ, or both
and is not applicable for radionuclides that have the
gastrointestinal tract as the critical organ.
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ICRP_MAXIMUM_PERMISSIBLE ORGAN JBODYX BURDEN.QF^RADIQNUCLIDE
ORGAN (BODY) CONTENT OF STABLE NUCLIDE
MAXIMUM PERMISSIBLE SPECIFIC ACTIVITY IN SEAWATER
CONCENTRATION OF STABLE ELEMENT IN SEAWATER
MAXIMUM PERMISSIBLE CONCENTRATION OF RADIONUCLIDE IN SEAWATER
EQUILIBRIUM CONCENTRATION FROM UNIT RATE OF DISCHARGE
MAXIMUM PERMISSIBLE RATE OF DISCHARGE
FIGURE 2. SPECIFIC ACTIVITY APPROACH TO AQUEOUS RADIOACTIVE WASTE
DISPOSAL ASSESSMENT (FROM PPESTON, 1969)
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In practice, both the specific activity and the critical
pathway approaches may be used to predict the impact of the
release of radionuclides. The specific activity approach
may be used to provide a first approximation of the
magnitude of the problem but the critical pathway approach
is usually selected as the means of precisely defining the
limits of the problem. Usually, the program begins with a
pre-release general survey of the receiving water and of the
local population to establish the existing levels of
radioactivity in the environment and to provide information
for making predictions about the quantity of radionuclides
to be released. Then, after the release of the
radionuclides begins, the general survey is repeated and is
later followed by a monitoring program that is limited to
sampling only the critical pathways that lead to man.
Indicator organisms (organisms, preferably sessile,
available and abundant, which exhibit high concentration
factors and long biological half-lives for the radionuclides
of interest) whose radionuclide content may be related to
that found in a critical pathway organism may also be
sampled. If the predicted pathways and those to the local
populations are not confirmed by direct measurements after
discharge has begun, an adjustment is made in the release of
the radionuclides.
C. Examples
Monitoring programs to delineate the movement of
radionuclides in the marine environment have been conducted
by personnel of the Laboratory of Radiation Ecology (LRE,
previously known as the Applied Fisheries Laboratory and the
Laboratory of Radiation Biology) since 1916, when monitoring
at the Pacific Test Site, Bikini Atoll, was begun, some of
the programs currently in progress at Bikini and nearby
atolls were noted in the introduction. A brief description
of two of these programs will be given to illustrate typical
monitoring studies.
iQiwetok - in 1946, Bikini and Eniwetok Atolls were
selected by the US Government for the testing of nuclear
devices. The indigenous Marshallese people were removed
from these atolls prior to the commencement of testing,
which lasted from 19U6 to 1958. Recently, Bikini Atoll was
returned to the Trust Territory Government and Eniwetok
Atoll will soon be returned. Before the Eniwetok people
return to their atolls, a comprehensive radiometric survey
of air, water, soil, sediments and biota of the atoll was
required to provide data to assess the radiological hazards
to people returning to Eniwetok to live. The field portion
of this survey was conducted from October, 1972 to February,
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1973. It was a joint effort of the Lawrence Livermore
Laboratory and a number of other laboratories, including
LRE, and was coordinated by the Nevada Operations Office of
the OS Atomic Energy Commission.
Since the level of environmental radioactivity present
at Eniwetok in 1964 and earlier is known from past surveys
(26) , background information was available to assist in the
design of the sampling program. The areas were selected for
sampling because they are potential resettlement sites or
were previous collection sites. In addition, "control11 fish
for the aquatic biota program were obtained from Kwajalein
Atoll, over 300 miles to the southeast of Eniwetok.
There are more than 700 species of fish at Eniwetok but
only a few species of reef, benthic and pelagic fishes were
selected for use in this study. The species selected were
chosen for one or more of the following reasons: they are
commonly eaten by the Marshallese; they are relatively
abundant at most collection sites; they are representative
of a feeding habit; or there is previous relevant
radiometric information about the species. Selected
invertebrates were also collected for analysis.
All samples were analyzed for gamma- emit ting
radionucludes and many for iron- 55 (5SFe) , strontium-90
f'OSr) , and plutonium-239, 240
The results of these analyses and of analyses from the
other portions of the survey, along with information on the
probable living and feeding habits of the returning Eniwetok
people, has provided a basis for calculating dose rates to
the people. Dose rates, following varying degrees of clean-
up of radiological hazards on the atoll, have been
calculated. When both the radiological hazard and the
sociological factors are known, then decisions can be made
about courses of action to reduce the radiation dose to the
returning Eniwetok people.
..., Washington - The second monitoring program I would like
to discuss is a study of the distribution of Hanford-
produced radionuclides in marine organisms of Washington.
The original plutonium production reactors at Hanford, near
Richland, Washington released radionuclides into the
Columbia River from 1944 until January 1971, when the last
of these reactors was shut down permanently. The
distribution of Hanford- produced radionuclides in the marine
environment from the mouth of the river — 550 km from the
Hanford reactors — , along the Washington Coast, in the
Strait of Juan de Fuca, and in Puget Sound has been
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monitored by LRE since 1961, At present, two and a half
years after the last reactor shutdown, some radiomiclides
are still present in water, biota, and sediments, due to
recycling^ of the long half-lived radionuclides. It is
expected that within time the levels of most remaining
radionuclides will be below the limits of detection.
To determine the distribution of Hanford-produced
radionuclides, an extensive preliminary survey was begun in
1961 to establish radionuclide distribution in the marine
environment by species, by tissues, by season, and by areas.
Samples collected from over twenty intertidal locations
along the Washington-Oregon coast between Cape Flattery and
Coos Bay, which are 260 km north and 350 km south of the
river mouth, respectively. As knowledge was acquired as to
radionuclide distribution in the organisms, eight coastal
areas were selected as regular collecting stations for
invertebrates and algae, and four coastal bays, plus puget
Sound, were selected for oyster collection sites. Plankton
and demersal fish were also collected at sites extending 240
km off the coast.
Preliminary results indicated that the distribution of
Hanford-produced radionuclides was best monitored by zinc-65
(*5Zn) distribution; hence, sampling was focused on those
organisms concentrating 65Zn. Although other species were
still collected, the organisms sampled at most stations and
analyzed routinely were mussels, sea anemones, and selected
algae.
This type of sampling program continued from 1961
through 1963, at which time it was felt that the original
objectives of the survey had been met. Results are reported
by Seymour and Lewis (18) .
The program was renewed in 1965, one year after
completion of the original program. It was resumed in order
to observe changes in the levels of radionuclides in marine
organisms associated with the permanent shutdown of the
eight Hanford plutonium-production reactors between 1965 and
1971. This program emphasized collection of coastal
organisms, especially mussels, which were known to
concentrate *5Zn, although other radionuclides were
measured.
All samples were analyzed for gamma-emitting
radionuclides. Results of these analyses indicated that the
amount of *«Zn declined with increasing distance from the
river mouth and time after reactor shutdown. Further
information on this and other corollary studies can be found
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in Seymour and Nelson (19), Seymour (17) , and in annual
reports of the Laboratory of Radiation Ecology to the
Division of Biomedical and Environmental Research, OS Atonic
Energy Commission.
III. Sample Preparation
Field samples are frozen as soon as possible after
collection and then stored until prepared for analyses. To
prepare for gamma-spectrum analyses, the samples are thawed,
dissected if necessary, dried at 95C to a constant weight,
ground to a homogeneous mixture in a food blender and then
packaged in a standard container for gamma counting.
Analyses for alpha or beta-emitting radionuclides requires
chemical separation of the radionuclide from an aliquot of
the dried, ground sample and deposition of the extracted
radionuclide on a planchette for counting. To concentrate a
dried sample for radiochemical separation the sample is
either dry or wet-ashed. Dry-ashing in a muffle furnace to
remove all organic material is easier than wet-ashing but it
is necessary to maintain strict temperature control to
ensure that the more volatile radionuclides are not lost
(11). Wet-ashing is a better technique to get samples into
solution, but it is more time-consuming than dry-ashing
(21). After ashing, the sample can be dissolved in acids
or, if it is still insoluble, a fushion technique with
hydrofluoric acid can be used. For refractory radionuclides
this is most important, since they often are not soluble in
mineral acids.
Once the samples are in solution the various group and
specific element separations can be made. Radio-tracers or
standard chemical techniques are used to determine the
chemical yield. If the concentration of an element is low
in the sample, a carrier for the radionuclide must be used.
It is important to measure the stable element in each sample
in order to determine the amount of carrier needed in the
chemical procedure.
Some of the radionuclides not detected by gamma
spectrometry but commonly determined by alpha or beta
counting at LRE are «spe, »«Sr, 239,339,zvopu, and ^H. A
brief discussion of the methods used for analysis of these
radionuclides will be presented here to indicate those
procedures found most useful for -aquatic biota.
Because of the low concentrations of 90Sr and Pu
expected in marine biota, samples up to 500 g wet weight are
concentrated for analysis. To extract »<>Sr the BASL "Rapid
Technique for «<>sr" is used. This procedure employs solvent
-252-
-------
extraction of yttrium-90 C»oy) which is measured by liquid
scintillation methods. Yttrium carrier is added to measure
the yttrium yield on extraction. If the first extraction of
yttrium is contaminated by rare earths such as europiura-155
or 152, the sample is stored for two weeks and the
extraction repeated. Since the europium is removed by the
first extraction, the second extraction should yield «oy
alone.
The chemical procedure most suitable for plutoniure
analysis requires an initial solvent extraction of the
trans-uranium elements, followed by selective elution of the
Pu, using a method adapted from Buttler (1) . with certain
samples of high salt content, co-precipitation of Pu with
calcium phosphate and ion exchange, using an an ion exchange
resin, was found to be satisfactory (28) . Following
extraction, Pu was plated on platinum discs by
electrodeposition.
The procedure adopted for sspe analyses is the method
described in HASL-300 (23). By this method, iron-55 is
separated by solvent extraction and is plated on copper
discs by electrodepostion.
The LRE monitoring program at Amchitka includes the
measurement of 3H in free water. Tritium incorporated in
the water molecule is the most mobile of all radionuclides
produced in underground nuclear detonations, and hence
tritium in water is a monitor of radionuclide leakage from
the site of the Amchitka detonations. Because of the
difficulty of measuring tritium at low concentrations, the
limits of detection commonly reported in the literature are
200 tritium units (1 TO = 3.2 pCi/liter) . This limit is not
adequuate for a reliable measurement of the present levels
of 3H in rainfall, which ranges from 25-350 TD. Lower
limits of detection can be attained by a electrolytic
enrichment process but this procedure is excessively time-
consuming. For these reasons, the Laboratory has adapted
the procedure of Sauzay and schell (16) which provides a
relatively simple method for analysis of 3H in free water at
limits of detection of about 15 TO. The method, in essence,
is simply the distillation of water from the samples of
temperature differential and vacuum, mixing of the
distillate with Insta-gel, and counting the sample in a
liquid scintillation spectrometer. Biotic samples were
processed in the same way as the water samples except that
the samples were lyophilized rather than heated to prevent
decomposition.
-253-
-------
The equipment used to measure 3H, ssFe, *°Sr, and Pu is
discussed in the following section.
IV. Counting Equipment
Equipment used in the laboratory to measure
radioactivity in aquatic biota samples may be divided into
three basic categories, alpha, beta, and gamma or X-ray
counters.
Gamma counters commonly used at LRE are sodium iodide
(thallium activated) and germanium diode {lithium drifted)
detectors, with an intrinsic Ge detector used for selected
radionuclides such as americium-241 (2*VAra) and europium-155
(tssEu). The Nal detectors have a greater efficiency but
lower energy resolution for gamma-emitting radionuclides
than the Ge(Li) detectors. Hence, the Nal detectors are
used for counting samples that contain only a few
radionuclides, and especially if the energy of their gamma
photons are not similar (e.g., fish muscle containing *°K
and »3*Cs). Besides having better resolution of gamma
energy, a Ge (Li) detector also is capable of efficiently
operating over a wider energy range (50 KeV to 2 MeV) than
is a Nal detector (250 KeV to 2 MeV). Thus, our Ge(Li)
detectors are used for samples containing a mixture of
radionuclides which would be difficult to resolve on a Nal
detector or for samples containing radioeleraents (24*Ara,
etc.) emitting gammas of energies below the efficient
working range of the Nal detector. One requirement of the
Ge(Li) detector system is the need to keep the diode cooled
by liquid nitrogen. To obtain the gamma energy spectrum of
a sample with a mixture of radionuclides, either a system
with a 4Hx 5" Nal crystal detector and a 400-channel
analyzer or a system with a Ge (Li) diode detector and a
Ge(Li) diode detector and 4096-channel analyzer are used.
To obtain the gamma count of a sample with a single
radionuclide or the total gamma count of a sample with a
mixture or radionuclides, a system with a 3*1 x 3* Nal
crystal detector and a single-channel analyzer are used.
The intrinsic Ge detector is used for counting samples
that emit low energy gamma photons or X-rays in the range
from about 6 KeV to 400 KeV. This system has a low
efficiency but excellent resolution and is capable of
differentiating between x-ray energies 300 to 700 eV apart,
depending on the energy of the x-rays being measured. The
diode of this system also must be cooled by liquid nitrogen
but only during operation.
-254-
-------
A gas proportional tube and a 200-channel analyzer make
up a system that is used for iron-55 counting. This system
has an anticoincidence shield which results in a low
background {2 CPM). The electronic components of this
system also are used with an in situ underwater Nal probe
recently used at Eniwetok to measure gamma-emitting
radionuclides in lagoon sediments.
Two types of beta counters are used in our laboratory;
one, a total beta system, the other, a liquid scintillation
system. The latter is used mainly for counting *osr-90Y and
3H, for which this system has a low background and high
efficiency. The total beta system has an anticoincidence
shield that reduces the background to about 1 count per
minute and is about 30X efficient for szp. Beta counts must
be corrected for the counts that are absorbed by the sample
matrix on the counting planchette. A reliable estimate of
this factor, self absorption, may be difficult to obtain.
Two alpha counting systems are used at LRE, a total
alpha counter and an alpha diode counter. Using a zinc
sulfide screen as detector, the total alpha counting system
counts about SOX of all alpha particles emitted by a sample
without resolving the energies of the particles. For
samples with a single alpha emitting radionuclide the total
alpha counting system is used and for samples with more than
one alpha emitting radionuclide the alpha spectrometer
system is used. By selection of the proper method and
equipment, it* s possible to measure all types of
radioactivity in environmental samples.
V. conclusions
In conclusion, personnel charged with monitoring a
radioactive waste disposal operation from a nuclear reactor
or other sources can find relevant information in the
published literature as to methods and procedures to be used
to quantitatively and qualitatively identify the
radionuclides present in the environment. These procedures
may have to be altered to fit the particular situation, but
reference to the literature and consultation with
specialists presently working in the field usually can
resolve any difficulties that may arise.
-255-
-------
References
1. Buttler, F. E. 1968. Rapid bioassay methods for
Plutonium, neptunium, and uranium* Health Physics.
Pergamon Press ^5:19-24.
2. Foster, R. F., I. L. Ophel and A. Preston. 1971.
Evaluation of human radiation exposure, p. 210-260.
In Radioactivity in the Marine Environment.
National Academy of Sciences Publ. ISBN 0-309-
01865-X, Washington, D. C.
3. International Atomic Energy Agency. 1965. Methods of
Surveying and Monitoring Marine Radioactivity.
Safety Series No. 11. STI/PUB/86. IAEA. Vienna.
95 pp.
H. International Atomic Energy Agency. 1966. Manual on
Environmental Monitoring in Normal Operation.
Safety Series No. 16, STI/POB/98. Vienna. 70 pp.
5. International Atomic Energy Agency. 1969a.
Environmental Contamination by Radioactive
Materials. Proceedings Series. STI/PUB/226. IAEA,
Vienna. 736 pp.
6. International Atomic Energy Agency. 1969b. Quick
Methods for Radiochemical Analysis. Technical
Report Series No. 95, STI/DOC/10/95. IAEA, Vienna,
62pp.
7. International Atomic Energy Agency. 1970. Reference
Methods for Marine Radioactivity Studies. Technical
Report series No. 118 - STI/DOC/10/118. IAEA,
Vienna. 284 pp.
8. International Atomic Energy Agency. 1971. Rapid
Methods for Measuring Radioactivity in the
Environment. Proceedings Series - STI/PUB/289.
IAEA, Vienna. 967 pp.
9. International Commission on Radiological Protection.
1966. Principles of environmental monitoring
related to the handling of radioactive materials. A
report by Committee 4. ICRP Publication 7, Pergamon
Press, Oxford. 11 p.
-256-
-------
10. Kaye, S. V, and D. J. Nelson. 1968. Analysis of
specific activity concept as related to
environmental concentration of radionuclides. Nuc.
Safety 9:53-58.
11. Nakaiaura, R. , Y. Suzuki, E. Kawachi and T. Oeda. 1972.
The loss of radionuclides in marine organisms during
thermal decomposition. J. Radiation Research
13(3) :
12. National Academy of Sciences - National Research
Council. 1962. Disposal of low-level radioactive
waste into Pacific coastal waters. NAS-NRC Publ.
985. Washington, D. C. 87 pp.
13. National Academy of Sciences. 1971. Radioactivity in
the Marine Environment. NAS Publ. ISBN 0-309-01865-
X, Washington, D. C. 272 p.
14. National Academy of Sciences - National Research
Council. 1973. Radionuclides in foods. NAS Publ.
ISBN 0-309-02113-8, Washington, D, C. 97p.
15. Preston, A. 1969. Aquatic monitoring programs, p. 309-
321 in Environmental contamination by radioactive
materials. Proc. IAEA symposium, 24-28 March 1969,
Vienna,
16. Sauzay, G. and W. R. Schell. 1972. Analysis of low
level tritium concentrations by electrolytic
enrichment and liquid scintillation counting.
International Journal Applied Radiation and
Isotopes, 23; 25-33.
17, Seymour, A. H. 1966. Accumulation and loss of zinc-65
by oysters in a natural environment, p. 605. In
Proc. symposium on Disposal of Radioactive Wastes
into Seas, Oceans, and Surface Waters, 1966, IAEA,
Vienna, Austria.
18. Seymour, A. H. and G. B. Lewis. 1964. Radionuclides of
Columbia River origin in marine organisms,
sediments, and water collected from the coastal and
off-shore waters of Washington and Oregon, 1961-
1963. U.S. Atomic Energy Commission Report OWFL-86.
-257-
-------
19. Seymour, A. H. and V. A. Nelson. 1973a. Biological
half-lives for zinc and mercury in the Pacific
oyster, Crassostrea gigas, p. 849-856. In D. J.
Nelson (edT7, Proc. Third National Symposium on
Radioecology, 10-12 May 1971. Oak Ridge, Tenn.
CONF-710501.
20. Seymour, A. H. and V. A. Nelson. 1973b. Decline of
Zn in marine mussels following the shutdown of
Hanford reactors, p. 277-286. In Proc. Symp. on
Radioactive contamination of the Marine Environment,
10-11 July 1972, Seattle, Washington. Publ. IAEA,
Vienna, Austria.
21. Smith, G. F. 1953. The wet ashing of organic matter
employing hot concentrated perchloric acid, the
liquid fire reaction. Analytics Chimica Acta
8 (5): 397-421.
22. Templeton, W. R. , R. E. Nakatani and E. E. Held. 1971.
Radiation effects, p. 223-239. In Radioactivity in
the Marine Environment. NAS-NRC Publ. ISBN O-309*
01865-X, Washington, D. c.
23. U. s. Atomic Energy Commission, Health and Safety
Laboratory. 1972. HASL Procedures Manual, (ed.)
John Harley. HASL-300, National Technical
Information Service, Springfield, Virginia.
24. U. S. Environmental Protection Agency. 1972.
Environmental Radioactivity Survillance Guide. US
E.P.A. Technical Report ORP/SID 72-2. National
Technical Information Service, Springfield,
Virginia. 25 pp.
25. U. S, Public Health Service. 1967, Radioassay
Procedures for Environmental Samples. Environmental
Health series, Radiological Health. PHS publication
No. 999-RH-27. U.S. Govt, Printing Office,
Washington, D. C.
26. Welander, A. D. et al. 1967. Bikini-Eniwetok studies,
1964: Part II. Radiobiological studies, U. S.
Atomic Energy commission Report UWFL-93.
27. Woodhead, D. S. 1971. The biological effects of
radioactive waste. Proc. Roy. Soc. Lond. B
|77;423-437.
28. World Health Organization. 1966. Methods of
Radiochemical Analysis. WHO. Geneva. 163 pp.
-258-
-------
THE PROPERTIES AND COMPOSITION OF SLUDGES
R. B. Dean and J. E, Smith, Jr. *
Production^an.dTreatment_of Sludges
Sludge is a liquid-solid mixture containing contaminants
removed from waste water by physical, biological, and
chemical treatments. Although sludge contains solids, one
problem of its treatment is the removal of water that is in
close association with waste solids. The major part of the
cost of sludge treatment is directly related to the tons of
water associated with each ton of solids. A typical
digested sludge contains about 20 tons of water for each ton
of solids. A thin, waste-activated sludge from biological
treatment may contain well over 100 tons of water per ton of
solids (Table 1). Dewatering and drying sludge are
expensive operations that can cost as much as $50 per ton of
dry solids produced.
The quantities of typical sludges as they are removed
from clarifier tanks or thickeners are shown in Table 2.
Several types of sludge may be produced at different stages
of a conventional waste treatment system. Figure 1 shows a
typical flow sheet for an activated sludge plant. A
trickling filter plant would use essentially the same flow
sheet, substituting a rock or plastic-filled "filter" for
the aerator. The biological sludge sloughed off a trickling
filter is frequently called humus in England. It is similar
but not identical to the organic humus found in good soils.
Raw primary sludge consists of readily settleable
organic matter, fine sand, and silt. It is highly
putrescible and cannot be stored even for a few hours in
warm weather without some type of stabilization to prevent
odors from decomposition.
Advanced Waste Treatment Research Laboratory,
Cincinnati, Ohio
-259-
-------
TABLE 1
WATER CONTENT OF SLUDGES
Treatment Percent Moisture Tons of Water/Ton Sludge Solids
Primary Sedimentation 95 19
Chem. Precipitation 93 13.3
Trickling Filters
g Humus - Low Rate 93 13.3
i
Humus - High Rate 97 32.4
Activated Sludge 98-99 ~65.6
Well Digested Sludge
Primary Treatment 85-90 ~7.0
Activated Sludge 90-94 ~11.5
-------
TABLE 2
I
tvj
en
TYPICAL QUANTITIES OF SLUDGE PRODUCED
IN WASTEWATER TREATMENT PROCESSES
Treatment
Plain Sedimentation
Trickling Filter Humus
Chem. Precipitation
Activated Sludge
Keefer
(1940)
2,950
745
5,120
19,400
Fair & Imhoff
(1965)
3,530
530
5,100
14,600
Babbitt
(1953)
2,440
750
5,250
18,700
McCa be
Eckenfelder
3,000
700
5,100
19,400
&
(1963)
(Given as gallons/million gallons sewage treated)
-------
RAW
SEWAGE
NJ
CTi
to
I
JtV
,•:••:•*.
,\J
'.;.'
11 U»
« IX 1
SECONDARY
m
EFFLUENT
3)DIGESTED
SLUDGE
Figure 1.
BIOLOGICAL SEWAGE TREATMENT
-------
Waste-activated sludge (WAS) is the product of
biological multiplication of microorganisms feeding on
soluble and suspended organic matter in the presence of
dissolved oxygen. A major part of the microbial sludge is
returned to the aerator; but a fraction, representing net
growth, is wasted. Waste- activated sludge is also
putrescible. It may be treated separately; it may be
combined with primary sludge for further treatment; or it
may sometimes be discharged directly to the influent sewer
to be collected with the primary sludge.
Most of the bacteria in waste- activated sludge are floe-
forming Zooglea, which are related to Pseudomonads. Up to
90 percent of the Zoogleal mass is extracellular" jelly
secreted as bacterial capsules (4) . This gelatinous mass
entraps small particles and organisms in floes that settle
with the bacteria, leaving a clarified, final effluent.
Primary sludge, of course, contains high concentrations
of fecal cbliform bacteria, especially Escherichia co^i. It
also contains lesser quantities of intestinal and
respiratory tract organisms, many of which may be disease-
causing (12) . Cholera, thyphoid, and dysentary are diseases
commonly associated with fecal wastes (7) . Amoeboid cysts,
intestinal worm eggs, and parasitic fungi may be transmitted
by sludge (15) . Sewage treatment removes disease organisms
from the effluent primarily by sedimentation; however, the
organisms may remain in a viable state in the sludge
The high bacterial content of raw sludge causes
putrefaction within a few hours because oxygen is used up
and anaerobic forms take over. Controlled putrefaction is
called anaerobic digestion. If sludge is held for 10 to 30
days in a digester, methane fermentation converts about half
of the organic matter to gases. A mixture of methane and
carbon dioxide is produced that has about one half the
heating value of natural gas and is contaminated with
hydrogen sulfide and other odorous substances. There is
enough energy in the gas to operate normal mechanical
equipment and air compressors in a sewage treatment plant.
As digestion converts the solids to gases, the mass of
sludge to be treated in subsequent operations is
substantially reduced, well-digested sludge does not
putrefy further; it is a dark liquid resembling an emulsion
of crude oil and frequently has a similar odor.
Digestion is usually carried out as a semi- continuous
process (28) . A fraction of the digester contents is
withdrawn periodically, usually every day, and the volume is
made up with raw sludge. The contents of the digester are
-263-
-------
mixed by gas evolution, usually supplemented by mechanical
mixing or recirculation. If 5 percent of the contents of a
digester is replaced each day, the nominal detention time
will be 20 days (100X/5X per day); however, 5 percent of the
removed sludge will have received only one day's exposure to
anaerobic conditions. Digested sludge is frequently
thickened by settling and the supernatant foul liquor is
returned to the plant for further treatment.
Characteristics of digester supernatant, which also
represent the composition of the interstitial water in
sludge are shown in Table 3.
Because anaerobic digestion is easily upset by a variety
of toxic influents, aerobic digestion {or aerobic
stabilization) of sludge is sometimes used, especially by
small treatment plants because it entails lower capital
expenditures. With aerobic digestion in this process, waste
sludges are aerated, as in secondary treatment, until a
large part of the organic solids have been destroyed (18).
There is no generation of fuel gas, and energy is required
to provide aeration. The process is inherently more stable
than anaerobic digestion, and the final product is also free
from further putrefaction.
Although conditions in digesters are unfavorable for the
multiplication of most pathogenic organisms, they are not
lethal, and the principal bactericidal effect appears to be
related to a natural dieoff with time. Kenner et al. (13)
have reported on bacterial concentrations in digested
primary sludge and undigested waste-activated sludge. Table
4 shows representative data. The large differences between
raw waste-activated sludges A and B, which were taken 11
days apart, are not unexpected and probably represent day-
to-day fluctuations, consistent differences between aerobic
and anaerobic destruction of pathogens have not been
reported.
Phosphates are sometimes removed from sewage effluents
by chemical precipitation. Chemicals may be added at any
stage of treatment, including treatment of the final
effluent in a tertiary process. The added chemicals alter
the physical properties of the sludges produced, sometimes
favorably and sometimes quite unfavorably. They increase
the mass of sludge produced and, of course, also increase
its phosphate content. Chemicals that have a significant
influence on the bulk properties of sludges include lime,
alum, and iron salts. Lime treatment adds calcium
carbonate, phosphate, and sometimes magnesium hydroxide to
the sludge. Alum and iron salts add the respective aluminum
and ferric phosphates and the hydroxides. All three
-264-
-------
TABLE 3
PROPERTIES OF DIGESTER SUPERNATANT
(MUNICIPAL WASTEWATER SLUDGE)
Item Standard Rote High Rate
Total susp.
solids (mg/l) 4,000-5,000 10,000-14,000
Total solids
(mg/l) 2,000-3,000 4,000-6,000
BOD (mg/l) 2,000-3,500 6,000-9,000
N> Volatile
solids (mg/l) 650-3,000 2,400-3,800
Alkalinity
(MO)(mg/l) 1,000-2,400 1,900-2,700
H2S(mg/l) 70-90 190-440
HN3~Nitrogen
(mg/l) 240-560 560-620
pH 7.0-7.6 6.4-7.2
After Maliva et al.,1971
-------
hydroxides are bulky and hard to filter (1). The same
chemicals may also be added to sludge, after it has been
separated from the effluent, to condition or stabilize it.
Sludge is sometimes treated with large doses of chlorine
that produce hydrochloric acid and chlorinated compounds and
destroy ammonia and pathogens (9).
Heat treatment of sludge to improve dewaterability is
carried out at temperatures above 160° for about half an
hour. These conditions will completely destroy all living
organisms. If oxygen is supplied together with heat
treatment, some of the organic matter may be oxidized; the
process is then called wet oxidation. All heat treatment
processes increase the concentration of soluble organic
matter and ammonia in the supernatant liquor or "soup."
This soup, although sterile when it is produced, is a rich
nutrient broth that can putrefy if allowed to come into
contact with bacteria from the environment. The dewatered
sludge is, however, resistant to putrefaction (3, 20).
Lagoon or other storage for many months is frequently
depended upon to reduce the numbers of pathogenic organisms,
particularly those that cannot multiply outside the human
body (10, 14). Storage may be necessary, in any case, if
sludge is disposed of only part of the year, and additional
storage lagoons can be built into the system to provide more
protection against transmission of disease. Because sludge
will settle in lagoons to form a mud that may become too
thick to pump, resuspension in water or effluent may be
necessary before the sludge can be removed from the lagoon.
Comggsitign_of_Sfudges
Sludges analyses are normally reported on a dry weight
basis. Only a few substances such as salts and ammonia are
soluble in sludge water; when sludge is dewatered, they will
be lost in proportion to the extent of dewatering. Most
substances in sludge are insoluble or are absorbed on sludge
solids; these will be retained when sludge is dewatered
unless they are present in the suspended-solids fraction.
Many so-called dewatering treatments, especially
centrifugation and gravity thickening, produce a centrate or
filtrate that contains a significant fraction of suspended
solids. The concentrate from a scroll-conveyor, solid-bowl
centrifuge may contain as much as 70 percent of the solids
in the feed (22). A differently designed centrifuge has
achieved 99.9% solids capture with the assistance of
polymers (30). Pressure filters can also produce filtrates
with much less than 1.0% of the feed solids concentration
(8).
-266-
-------
Sewage varies in flow and composition from hour to hour
and day to day reflecting the living patterns of the
population. It also varies from place to place depending
primarily on industrial contributions. The normal operation
of a sludge digester provides the equivalent of a 10-day
moving average of sludge composition, which effectively
damps out hourly and diurnal variations, solids drawn from
a sludge thickener or from an inefficiently stirred digester
will vary significantly in solids content with time of
withdrawal and will reflect variations in solids
concentration within the container. Therefore, a carefully
designed sampling scheme mast be used to obtain a true
representation of any given batch of sludge (16). Sludge
settles in lagoons leaving a surface layer of oil and a
gradient of solids content increasing downward. Since
sludge is a non-Newtonian fluid, there will be little or no
lateral mixing in lagoons, and significant variations from
point to point may be expected in lagoons that have been
filled over a long period of time (29). Analyses of
multiple samples, rather than replicate analysis of a
composite sample, gives confidence in the sampling scheme
and provides an estimate of the variability of the data.
Organic matter in sludge consists of food residues,
bacterial solids, cellulose, humus-like compounds
structurally related to lignin, .hydrocarbons, and
chlorinated hydrocarbons of low volatility, fragments of
plastics and rubber, and miscellaneous synthetics such as
adsorbed detergents and paint latex.
Grease in sludge is defined as the material extractable
by hexane from an acidified sample under rigorously
standardized conditions (26). The grease fraction includes
soluble hydrocarbons, fatty acids, waxes, and chlorinated
hydrocarbons. The potentially floatable fraction of sludge
includes grease and insoluble fragments of rubber and
polyethylene.
Inorganic constituents of sludge, with the exception of
nitrogen compounds and mercury, may be determined after the
organic matter has been oxidized. Dry ashing in a muffle
furnace at 450°-600°C or wet ashing in mixtures of oxidizing
acids may be used. Major inorganic components of sludge may
be similar to those found in soil, oxides, carbonates,
silicates and phosphates of calcium, magnesium, iron, and
aluminum make up the bulk of the inorganic fraction of
sludge (Table 5).
Sludge contains the major plant nutrients nitrogen,
phosphorus, and potassium at levels that are about one-fifth
-267-
-------
TABLE 4
cn
oo
BACTERIA IN SEWAGE SLUDGE
(per 100m!)
Raw Primary
Trickling Filter
Raw WAS - A
Raw WAS
(Thickened) -B
Raw WAS - C
Anaerobic
Digested Primary
Aerobic Digested
WAS
Anaerobic
Digested WAS
Iron Primary
Lime Primary
pH 9.0
Lime Primary
pH 11.5
Fecal coli
(x 106)
11.4
11.5
2.8
20
2.0
0.39
0.66
0.32
32
32
0.014
Salmonella
460
93
74
9,300
2,300
29
150
7.3
460
1,500
<3.0
Pseu domon as
46,000
110,000
1,100
2,000
24,000
34
100,000
1,000
21,000
24,000
<3.0
After Kenner, 1972
-------
TABLE 5
SUMMARY OF MAJOB AND MINOR ELEMENTS IN SLUDGE
Aluminum
Antimony
Arsenic
Barium
Beryll
Boron
Cadmium
Calcium
Chromium
Cobalt
Copper
Gallium
Iron
Lead
Magn
Mang
Mercury
Molybd
Nickel
Phosph
Potass
Silicon
Silver
Sodium
Stront
Sulfur
Tin
Titanium
Vanadium
Zinc
(mg/g
Primary
art Sludge
im 5.1
ly n.a.*
: 1.2
2.2
Ivan 0.0025
0,10
n 0.19
n n.a.
am 2.0
0.22
2.0
n 0.06
16.1
1.0
turn 10.6
3se 0.78
/ 0.005
enum 0.36
0.52
or us 3.8
ium n.a.
n n.a.
0,24
4.0
ium O.13
n.a.
0.95
urn 14.8
urn 2.1
6.9
ium 1.7
dried sludge)
Activated
Sludqe
10.0
n.a.
1,2
1.2
0.0035
0.070
0.35
13.0
4.3
0.002
1.1
0.05
40.5
1.5
7.0
0.31
0.02
0.20
0.38
19.9
4.2
40.0
0.15
4.4
0.16
10.1
0.50
11.8
0.7
3.3
10.0
Digested
Sludge___
17.9
0.9
n.a.
1.4
0.0025
0.046
0.26
33.5
2.3
n.a.
1.6
0.05
30.6
1.9
7.5
0.98
n.a.
0.25
0.38
12.8
2.8
162
0.20
6.2
0.26
12.3
0.60
14.2
5.2
4.0
2.0
*n.a. not available
After Salotto et al., 1971
-269-
-------
l
K)
-«J
O
TABLE 6
MINERAL NUTRIENTS PERCENT
OF DRY SLUDGE SOLIDS
Total N 3.5 - 6.4
Organic N 2.0 - 4.5
p 0.8 - 3.9
P2O5 1.8 - 8.7
K 0.2 - 0.7
K2O 0.24 - O.84
After Peterson, J.R., 1972.
-------
of those found in chemical fertilizers. Table 6 expresses
average analyses, as the elements and in agricultural units
of sludge. Sludge that has been digested and dewatered by
filtration, centrifugation, or by drainage on drying beds
will lose to half of its nitrogen as ammonium salts in the
removed water. Potassium is also lost when digested sludge
is dewatered, Phosphates are present in sludge both as
organic compounds and as precipitates with calcium,
magnesium, aluminum, and iron depending upon the treatment
given. Activated sludge can remove significant quantities
of phosphates either as organic phosphate |21) or as calcium
phosphate (7). Alum, iron, or lime may be used to
precipitate phosphates from wastewater. Digestion will
reduce ferric phosphate to ferrous but most of the phosphate
remains as the insoluble mineral vivianite (27).
Sludge contains almost all of the metal ions that are
discharged to sewers or extracted from plumbing. Heavy
metals occuring in sludge in quantities which are
significant in foods, include zinc, copper, nickel, cadmium,
mercury, and lead. Arsenic, selenium, manganese, and
molybdenum may occasionally be present in significant
quantities from industrial sources.
The composition of sludges varies so much that
meaningful data can only be obtained by applying appropriate
statistical methods. The variance or the spread are as
important as the average or the geometric mean, and
conclusions based on a few spot analyses are likely to be
misleading. Obtaining a representative set of samples is
perhaps even more important than choosing the best method of
analysis. Methods developed for the collection of
unconsolidated sediment samples from lakes and marine waters
can profitably be applied to the sampling of sludge lagoons.
-271-
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REFERENCES
1. Adrian, D. D. , and Smith, J. E., Jr. (1972).
"Dewatering Physical -Chemical Sludges," Proc. Conf.
on Application of New Concepts of Physical-Chemical
Wastewater Treatment, Vanderbilt University, Sept.
18-22, Pergamon Press, Inc. 273-289.
2. Babbitt, H. E. (1953) . "Sewerage and Sewage
Treatment," John Wiley and Sons, Inc. (N.Y. ), 7th
edition.
3. Brooks, R. B. (1970). "Heat Treatment of Sewage
Sludge," water Pollution Control, 92-99 and 221-
231.
4. Dugan, P. R. , and Picrum, H. M. (1972) . "Removal of
Mineral Ions from Water by Microbially Produced
Polymers," Proc. of the 27th Annual Purdue Inc.
waste Conf., May 2-U.
5. Fair, G. M. , and Imhoff, K. (1965). "Sewage Treatment,"
John Wiley and Sons, Inc. (N.Y.), 2nd edition.
6. Ferguson, J. F. , Jenkins, D. , and Eastman, J. (1973).
"Calcium Phosphate Precipitation at Slightly
Alkaline pH Values," J. 'Water Poll. Control Fed.
, 620-631.
7. Frobisher, M. (1965). "Fundamentals of Microbiology",
w. B, Saunders Co. (Philadelphia) , 7th edition.
8. Gerlich, J, W. , and Rockwell, M. D. (1973). "Pressure
Filtration of Wastewater Sludge with Ash Filter
Aid, "Research Report No. EPA-R2-73-231 for the U.S.
EPA by the City of Cedar Rapids, Iowa, under Grant
11060 EZX,
9. Green, J. E. (1972) . "Sludge Oxidation," The American
City., Oct. 1972.
10. Hinesly, T. D, , Braids, O. C., and Molina, J. E.
(1971) . "Agricultural Benefits and Environmental
Changes Resulting from the Use of Digested Sewage
Sludge on Field Crops." An Interim Report on a
Solid Waste Demonstration Project, U.S. EPA SW-30d.
11. Keefer, C. E. (1940). "Sewage Treatment Works,"
McGraw Hill Book Co., Inc. (N.Y.).
-272-
-------
12. Kenner, B. A., Dotson, G. K.f and smith, J. E., Jr.
(1971). "Simultaneous Quantitation of Salmonella
Species and Pseudomonas aeruginosa", U.S. EPA,
National Environmental Research Center, Cincinnati,
Ohio.
13. Kenner, B. A. {1972). In-house report, U.S. EPA,
National Environmental Research Center, Cincinnati,
Ohio, March 31, 1972.
14. Koser, A. (1967). "The Use of Sewage and sewage Sludge
in Agriculture from the Point of View of Veterinary
Hygiene," Schr. Reihe Kuratoriums Kulturbauw. 16,
25-42 (German).
15. Krige, P. R. (1964). "A Survey of the Pathogenic
Organisms and Helminthic Ova in Composts and Sewage
Sludge," J. lost. Sew. Purif., 215-220.
16. Langford, L. L. (1961). "Digester Volume
Requirements," Water and Sewage Works, Reference
Number, Oct. 31, 1961,R-353"~to R-358.
17. Levin, G. V., and Shapiro, J. (1965). "Metabolic
Uptake of Phosphorus by Wastewater Organisms," J.
Water Poll. Control Fed. ,37 <6) ,800-821.
18. Loehr, R. C. (1965). "Aerobic Digestion Factors
Affecting Design,*1 Water and Sewage Works, Ref. No.
112, R-169 to R-180.
19. Maliva, J. F., Jr., and DiFilippo, J. (1971).
"Treatment of Supernatants and Liquids Associated
with Sludge Treatment," Water and Sewage H£dss»
Ref. No. 118, p. R-30.
20. Mayrose, D. T., and Walsh, J. J. (1973). "Heat
Conditioning of Sewage Sludge—Dorr-Oliver's Farrar
System," presented at N.Y. Water Poll. Control
Assn. Meeting, Jan. 1973.
21. McCabe, J., and Eckenfelter, W. W, (1963). "Advanced in
Biological Waste Treatment," Pergamon Press.
22. Parkhurst, J. D., Rodrigue, R. F., Miele, R. P., and
Hayashi, S. T. (1973). "Summary Report: Pilot
Plant Studies on Dewatering Primary Digested
Sludge," EPA-670/2-73-043 August 1973.
-273-
-------
23. Peterson, J. R., Lue-hing, C., and Zenz, D. R. (1972).
"Chemical and Biological Quality of Municipal
Sludge, "Symposium on Recycling Treated Municipal
Waste Water and Sludge Through Forest and
Croplands," Pennsylvania State University,
University Park, Pennsylvania.
24. Salotto, B. V., and Farrell, J. B. <1971).
"Preliminary Report—The Impact of Sludge
Incineration on Air and Land," prepared by Advanced
Waste Treatment Research Laboratory, NERC,
Cincinnati, for EPA Task Force on Sludge
Incineration, July 12, 1971.
25. Schaffer, R. B., Van Hall, C, E,, McDermott, G. N.,
Barth, D., stenger, V. A., Sebesta, S. J., and
Griggs, S. H. (1965). "Application of a Carbon
Analyzer in Waste Treatment." J. Water Poll.
Control Fed. 37, 1545-1566.
26. Standard Methods for the Examination of Water and
Wastewater (1971), Amer. Public Health Association,
New York, N. Y., 13th edition.
27. Thomas, E. A. (1965). "Phosphate Elimination in the
Activated Sludge of Mannedorf and Phosphate Fixation
in Lake and clarifier sludge," Viertellahrsschr.
Naturforsch. Ges. Zurerich 110, 419-34 (Dec.).
28. Transactions of the 15th Annual Conf. on Sanitary
Engineering (1965), Univ. of Kansas Publ., Bull, of
Engineering and Architecture No. 54.
29. Ullrich, A. H. (1967). "Use of Wastewater
Stabilization Ponds in Two Different Systems," J.
Water Poll. Control Fed. .39(6), 965-977. * ~
30. Vermehren, P. (1971). "Centrifuging of Sludge," Sixth
Nordic Symposium About Water Research titled
"Chemical Removal of Nutrients Salts from Effluent,"
23 April 1970. Nordforsk Publication 1971:5.
-274-
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MAKING ARTEMIA SLUDGE BIOASSAY MORE
ECOLOGICALLY RELEVANT
R.J. Nadeau and B.J. Pastalove*
Introduction
The Ocean Dumping Final Regulations and Criteria, under
the Marine Protection Research and Sanctuaries Act of 1972,
requires chemical and physical analysis of the waste
materials as well as a bioassay using an appropriate
sensitive marine species {Federal Register, Oct. 15, 1973) .
Obviously, the most appropriate species would be those
endemic to the area of immediate impact of open ocean waste
disposal, namely the pelagic or open water plankton.
The microcrustacean Artemia salina was selected as the
organism of choice for the bioassay requirements because,
unlike most endemic zooplankton, it can be reared and
maintained under laboratory conditions. Ar££I£i£ is readily
available at most pet stores, easily cultured at room
temperatures, and has been used as a bioassay organism in
the past. Artemia does not occur naturally within the
marine environment, within the United states, it exists as
disjunct allopatric populations in Great Salt Lake, Utah,
and San Francisco Bay, California. In tropical areas it is
often found in hypersaline impoundments (salinas) that are
used for generating commercial grade salt.
Essentially, the utility of the Artemia bioassay lies in
its capacity to rank the toxicity of materials destined for
ocean disposal* The problem with the Artemia bioassay is
that it is being used as a partial measure of the
environmental impact of disposed materials.
The Interim Criteria limits the permissible
concentration of wastes being disposed of in the mixing zone
to 0.01 of the TL of the appropriate sensitive marine
organism (i.e., Artemia). The application factor (0.01) is
derived from recommendations of the National Technical
Advisory Committee (1).
*Surveillance and Analysis Division, Region II, Edison, N,J,
-275-
-------
No bioassay information is available on municipal sewage
sludge using endemic pelagic zooplankton species, except for
the preliminary work performed by the Sandy Hook Marine
Laboratory for the Army corps of Engineers (2) .
A series of experiments were conducted to determine the
relative sensitivities of Artemia and the microcrustaoean
zooplankton species endemic to the municipal sewage sludge
dump site within the New York Bight.
Materials and Methods
Standard Bioassay Method
The bioassay used in this study was an adaptation of the
standard method developed by Tarzwell (3) for determining
oil and oil dispersant toxicity levels of Artginia saJ4f*a-
The standard method can be summarized as follows:
1- A, salina eggs from the San Francisco Bay area are
hatched in a tray and collected by drawing them to a light;
20 Artemia are transferred by means of a pipette to holding
dishes, along with 20 ml of artificial seawater (pH 8.0 -
8.2; salinity 30°/oo) and held for 24 hours.
2. Five concentrations of toxicant, each with five
replicates and a control, plus five concentrations of a
standard toxicant, sodium dodecyl sulfate USP grade (DOS)
are prepared, for a total of 35 dishes.
3. Working from a stock solution, the proper amount of
toxicant needed to produce the answered concentrations in a
total test volume of 100 ml is drawn off and added to
Carolina culture dishes (3.5 x 1.5 in.) along with 80 ml of
artificial seawater; with the addition of 20 Artemia and 20
ml seawater, the total volume equals 100 ml.
4. The dishes are incubated at 20°C for 48 hours, at
which time the numbers of live and dead individuals are
determined for each culture dish and TL values are
calculated by interpolation according to Standard Methods
Standard Versus Modified Bioassay Method
In several instances, either modifications or additions
were made to the standard method proposed by Tarzwell (3) .
They includes
-276-
-------
1. The use of natural seawater instead of standard
artificial seawater to avoid subjecting the delicate
indigenous species to the stresses of salinity and pH
changes; and using the natural seawater on Artemia to
eliminate salinity and pH as variables.
2. The use of an aeration hatching system for Artemia.
3. The use of Artemia larvae (see Figure 1) 5 to 10
hours after hatching versus Tarzwell's use of 24 hour
larvae.
4, The use of 10 individuals in each test dish rather
than 20 to ease the counting, which became tedious because
the Artemia became buried in the highly concentrated sludge.
5. The monitoring of pH, temperature, salinity and
dissolved oxygen.
6. The use of a shaker table aeration system to
compensate for the high oxygen demand of sludge (an air hose
pump system would have been to unwieldly as well as too time
consuming to install).
7. The modification of counting procedures to
accommodate the special problems of working with heavy
sludge concentrations, e.g., allowing the sludge to settle,
using the phototactic response of Artemia, searching with
the light beam, etc.
Modified_B^oassay Method
Experimental Organisms:
Only San Francisco Bay area Artemia saj.ina eggs were
used. They were hatched in a 1-liter funnel with a
continuous attached air supply; the eggs were kept in
constant motion during this period.
Indigenous zooplankton were collected at two sites. The
first was an offshore area approximately 4 to 6 miles east
of the Shrewsbury Rocks, Sandy Hook, contiguous to the
sludge dumping grounds. The second site was a land station
in the Shrewsbury River, a tidal river on the Jersey shore.
Thirty gallons of water were collected during each sampling
period and these periods occurred five times over a 10 week
span. Salinity and temperature readings were also made
during collection.
-277-
-------
•I
Figure I.
Artemia salina nauplii larvae (5 - 10 hours) 3.1 cm = 50y)
630x.
-278-
-------
The organisms were transported to the laboratory in
cooled aerated containers to ensure their viability
(temperatures during transport were 20 + 2°C) and were kept
in the laboratory in 20 gallon tanks (18 * 2°C) with a
constant air supply. Other than for the phytoplankton
already in the tanks, the organisms were not fed.
Age of Organisms:
For Artemia. a period of 2H to 30 hours elapsed between
the time the eggs were first placed in the hatching
apparatus and the beginning of the bioassay.
With the endemic species, age determination was not
possible, although efforts were made to select mature adults
only. Generally, organisms were used 2 days following their
collection, but if a sufficient number of individuals
survived until the following week (as usually happened),
then a second run was made on the same species.
General Procedures:
For both Artemia and the endemic species, either four or
five concentrations of the standard toxicant, DOS were
prepared for a maximum total of 72 dishes.
The Carolina culture dishes used as test containers were
washed in detergent, rinsed with hot water, and oven dried.
Preparation of Test Concentrations:
From the results of preliminary screening tests, a
general range of toxicant (sludge) concentrations to be used
on Arteraia and on the endemic species was chosen. For
Artemia, the range was 103 to 10s ppm (see Figure II) and
for the natural species, 1 to 103 ppm. A sample of
municipal sewage sludge from the Middlesex Regional Sewage
Plant, South Amboy, New Jersey, was kept under refrigeration
(2°C) in a plastic (Nalgene) container and used during the
2-month testing period. This was done to avoid the inherent
variability of using different samples of sludge (5).
Physical and chemical analysis of Middlesex sludge is
included in Table 1.
Approximately 8 liters of seawater (collected during the
sampling periods) were filtered through 0.4u membrane
filters (Millipore) to remove phytoplankton and detritus.
This water was then used in preparing the test
concentrations.
-279-
-------
Figure II.
Heavy concentrations of sewage sludge in Carolina culture
dishes during bioassay.
-280-
-------
PHYSICAL AND CHEMICAL ANALYSIS OF SEWAGE SLUDGE FROM
MIDDLESEX COUNTY SEWAGE TREATMENT PLANT TO BE BARGED TO THE DUMPING GROUNDS
Parameter
PH
COD mg/1
acidity (supernatant)mg/1 (CaCO )
" (diluted) mg/1 (CaCO )
alkalinity (supernatant) mg/1 (CaCO )
" (diluted) mg/1 (CaCO )
total solids mg/1
total volatile solids %
suspended solids mg/1
suspended volatile solids %
phenols mg/1
total phosphate-P mg/1
total sulfide mg/1
total Kjeldahl-N mg/1
ammonia-N mg/1
Sampling Data
1 June «73
5.6
69,000
1,400
1,350
) 3,770
6,400
50,000
67.3
MM
—
—
470
16.0
-
—
6 June *
5.1
87,000
2,110
1.260
3,200
5,870
67,000
69.0
54,300
77.6
15.6
633
27.2
1,400
169.0
to
GO
Heavy Metal Analysis from a Composite of the Above Samples
Plus Two Earlier Samples
Parameter
Arsenic
Beryllium
Cadmium
Chromium
Copper
Lead
Mercury
Nickel
Selenium
Zinc
Vanadium
Concentration fmq/11
0.18
<0.02
6.1
3.8
16.5
11.8
2.6
2.42
0.015
38.5
<0.2
-------
A stock solution of 10s ppra concentration of sludge was
volumetrically prepared: 100 ml of sludge were removed from
the container with a wide-bore pipette and added to 900 ml
of seawater. From this stock solution, various dilutions
were made; and 100 ml of the appropriate concentration were
dispensed in properly labeled test dishes.
Standard toxicant solutions were prepared in a similar
manner. The ranges used were 1 to 10 ppm for Artemia and
0.1 to 3.0 ppm for the endemic species.
Introduction of Test Organisms:
The previously prepared Artemia were counted out with a
small pipette and 10 individuals were transferred to each
sample.
A sample of the zooplankton was removed from the holding
tank and individuals of the most prevalent species were
pipetted into each test dish.
Aeration:
To ensure that oxygen levels in the test dishes remained
greater than 4 ppm, a shaker table apparatus {see Figure
III) was designed, on which,,all the culture dishes could be
placed. The combination of a relatively large surface area
and a constant mixing action from oscillation was found
sufficient to maintain dissolved oxygen concentrations above
4 ppm.
Exposure:
The entire test ran for 48 hours at a constant
temperature of 20*1°C (temperature controlled room) under a
regime of 8 hours of low light and 15 hours of dark. The
shaker table aeration system was operating at all times.
Observations:
Four physical parameters {dissolved oxygen, salinity,
temperature and pH) were monitored at the start of each test
run, after 24 hours, and again after 48 hours at the
conclusion of the test. A dissolved oxygen probe (Yellow
Springs Instrument Co., Model 54 ), an index refractometer
(American Optical Co.), a glass thermometer, and a pH meter
(Beckman, Model 96) were used to monitor the four parameters
respectively.
-282-
-------
. ,.-^^^^^^^^^^^^^^^^^
Figure III.
Shaker table assembly for holding Carolina culture dishes
during bioassay.
-283-
-------
TL 50 values at both 24 hours and 48 hours were
determined by counting only the living organisms in each
test dish.
Counting &rtem.ia samples (see Figure IV) was especially
difficult because of the high concentrations of sludge being
used. The general procedure consisted of a 5- minute
settling period after which the samples were placed on a
black surface (for greater contrast) and a strong bean of
light was held at the side of the dish. Individuals in the
supernatant fluid swam towards the light source where they
could be counted. The procedure became tedious because, at
higher sludge concentrations, most of the Artemia were
buried within the settled solids. To reach the light source
they had to first work their way out into the supernatant
fluid. This process, in some cases, took upwards of 5
minutes. By illuminating the side of the dish for a long
period of time and then carefully searching the top of the
settled material, the sides of the dish, and the mid-depths
with the light beam, most individuals could be counted.
The TL^Q values obtained after 24 hours were not as
accurate as those after 48 hours because it was difficult to
count moving nauplii. At 48 hours, when the run was
terminated, the nauplii were removed as they were counted,
and the accuracy of the count was thus improved.
The nature of the sludge made it impossible to find dead
individuals, much less probe them to detect movement.
Therefore, for the purposes of this test, a positive
phototactic response was the criterion for survival, i.e.,
those individuals that were unable to emerge from the
settled material in response to the light source were
considered dead.
The method used to count Artemia was also used for the
endemic species. There was comparatively little solid
matter in suspension {that range of sludge concentrations
was lower than that for Artemia) , and it was easier to
locate live individuals in the reflection of the light beam.
However, unlike Artemia , which were strongly attracted by
the light source, the endemic zooplankton exhibited only a
weak phototactic response. This necessitated a thorough
search of all areas of the dish (especially the edges) while
trying to keep track of the rapidly moving individuals.
-284-
-------
Figure IV.
Counting Artemia nauplii using a pencil light source,
-285-
-------
Calculations:
5Q values were calculated by plotting on semi-log
paper, the concentrations at which greater than 50 percent
and less than 50 percent survived (y axis) and the percent
survival at these two concentrations ix axis) . A straight
line is drawn between the two points; where the straight
line crosses the 50 percent survival line is the TL^Q •
Where possible, TI^0 »s for 24 and 48 hours were calculated
for Artemia and endemic species.
Timetable:
An entire 48-hour test run, with preparation, took a
total of 5 days. A general timetable is as follows:
Day._l: Field collection of zooplankton sample and
seawater.
Day_2: Prepare new batch of Artemia
Filter collected seawater
Decide on range of sludge concentrations to be
used
Label clean glassware
Prepare concentrations and set up test apparatus
2§Y_3: Place Arteir4.a in test dishes
Collect endemic zooplankton from holding tank
and place in test dishes fl to 4 hours
depending on species abundance)
Take readings of physical parameters of
representative concentrations (i.e.,
control, highest concentration, and median
concentrations)
Dav._4: Make 24-hour survival counts |2 to 4 hours)
Take readings of physical parameters
Day_5: Make 48-hour survival counts (2 to 4 hours)
Take readings of physical parameters
-286-
-------
AddeQdum._to
-------
TABLE 2
MEDIAN TOLERANCE LIMIT (in ppm) FOR 50 PERCENT OF TEST POPULATIONS AFTER
24 HOUR AND 48 HOUR EXPOSURE TO SEWAGE SLUDGE
Date
Temperature
»C
Artend a nauplii
24 hour 48 hour
Indigenous Species
24 hour 48 hour Name
April '73 3.0 all alive all alive
April '73 23.5 all alive 12,000
July '73 20 ± 1.5«C all alive 22,500
Aug. '73 20 ± 1.5°C all alive 31,000
NJ
00
f Aug. '73 20 + 1.5«C *
Aug. '73 20 ± 1.5°C **
Sept. '73 20 ± 1.5*C all alive 15,075
200
100
15.0
49.0
16.0
All alive
Less than
500.0
370.0
all dead
all dead
4.4
32.5
2.0
330.0
500.0
205.0
Centropaqes
hamatus
Centrop.age§
Acartia
tonsa
E§£lSll§DU§
Barvus
Paracalanus
parvus
Unidentified
harpacticoids
Oithona
similis
Paracalanus
p.aryus
* 50* mortality did not occur with 48 hours; 46$ mortality
in 5 x 10* ppm
** 50% mortality did not occur within 48 hours; 34* mortality
in 7 x 10* ppm
-------
TABLE 2
MEDIAN TOLERANCE LIMIT (in ppm) FOR 50 PERCENT OF TEST POPULATIONS AFTER
24 HOUR AND 48 HOUR EXPOSURE TO SEWAGE SLUDGE
Date
April • 73
April «73
Temperature Ar£§.mia nauplii Indigenous Species
°C 24 hour 48 hour 24 hour 48 hour Name
3.0
23.5
all alive all alive 200
all alive 12,000 100
all dead centropages
hamatus
all dead Centre pages
to
00
-------
Table 3 summarizes the sensitivity of each species
compared with that of Artemia as determined from the
experimental runs completed through 48 hours. Those species
that had replicate runs showed a wide range of
sensitivities. In part, this could be attributed to the
increasing TL5Q of Artemia (Table 2).
It is difficult to hypothesize which substances (Table
1) in the sludge are actually responsible for toxicity
without a complex factoral experimental design using pure
constituents in various concentration combinations. This
was neither practical nor necessary for the preliminary
study presented here but should be investigated,
particularly to stimulate advancement of sewage treatment
technology.
Modifying Factors
Te mpe rature:
The temperature-controlled room in which the bioassays
were run, kept thermal changes at a minimum; water
temperatures during ail bioassays were 20 £ 1.5°C (the
recommended temperature regime is 20 * 1°C).
During any one bioassay, there were small increases in
temperature (less than 2 degrees, C) over the 48-hour
period. However, observed fluctuations in temperature such
as these would not significantly affect the TL 50 values (6).
Salinity:
As a result of evaporation, the salinity increased from
the initial to the 48-hour reading (generally salinity was 3
to 7 °/oo greater at the end of the bioassay). Although
this is actually a fairly steep increase, the results should
not have been significantly affected because:
1. The evaporation was slow enough to let all the
organisms adapt to the change.
2- Artemia is certainly resistant to high salinity.
3. Estuarine microcrustaceans can tolerate high
salinity even though they are not usually found in such
waters. Their absence does not result from an inability to
withstand higher salinities, but from their inability to
sucessfully compete with larger oceanic forms.
-290-
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TABLE 3
SENSITIVITY FACTORS RANGE FOR INDIGENOUS SPECIES
(PL Artemia 48 hour/TL Indigenous Species 48 hour)
Name
Paracalanus Earyus (Claus)
Acartia tonsa Dana
Unidentified Harpacticoid
Qithgna similis Claus
Centropages hamatus (Lilljeborg)
Dana
i
to
VO
Sen s itiyity . Fac-tors
73 - 953 (100 - 1000)
35 - 5113 (50 - 5000)
212
30
120
Ultra sensitive (could not
be maintained in laboratory)
-------
4. Those species indigenous to open coastal waters can
tolerate high salinity levels.
Dissolved Oxygen:
The dissolved oxygen levels in all but the very highest
sludge concentrations ( >50,000 ppm) were greater than 4 ppm
(the recommended "critical" level).
Within the controls, DO levels were constant and high (>
6 ppm). Within the toxicant concentrations, DO levels were
lower (which could be related to the high oxygen demand of
sludge) but increased during the bioassay. Critically low
initial dissolved oxygen readings occurred at 5 X 10* ppm
(DO < 3 ppm) , 7 x 10* (DO < 2 ppm) and 10s ppm (DO < 2 ppm),
but by the end of the 48-hour period, levels had increased
to greater than 4 ppm.
Discussion
From our study it is obvious that the endemic
microcrustacean species, when exposed to the sludge
concurrently with the nauplii, are more sensitive to the
Middlesex county municipal sewage sludge than are Artemia
nauplii.
Researchers at the Sandy Hook Marine Laboratory (2)
exposed copepods (species not delineated) to sewage sludge
at concentrations equal to or greater than those expected
under present normal ocean dumping procedures (results are
summarized below).
Concentration Results
IfiBlI Duration time = 72 hours
10,000 95% mortality after 2 hr.
5,000 50% mortality after 2 hr.
3,334
1,000 No mortality
No mortality was observed at 1,000 ppm (2 to 10 times
the TL 50 concentration derived from our studies). As stated
before (see Materials and Methods) , municipal sewage sludge
varies greatly in composition, particularly from plant to
plant. The sludge in the Sandy Hook studies came from the
municipal sewage treatment plant at Bed Bank, New Jersey.
It did not contain any of the industrial waste components
-292-
-------
treated by the Middlesex County Treatment Plant. The
difference in sludge composition could be responsible for
the difference in results.
The copepods and cladocerans, being ecotypes to Artemia
occupy important ecological niches as filter-feeding primary
consumers within the pelagic community and as food for all
the zooplankton-grazing fish species.
Centropages hamatus (Lilljeborg), the calanoid copepod
species used initially to discern a sensitivity difference
between Artemia larvae and endemic microcrustaceans, is a
spring-summer form with wide salinity tolerance (7). It was
the largest copepod used in the study and comprises an
important bulk of the filter-feeder biomass during its peak
abundance. Distribution and abundance studies indicated
Centropages, including a congeneric form C. typicus (see
Figure V)., to be the fourth most abundant copepod genus in
the New York Bight <9 to 14 x 10* individuals per cubic
meter) (2).
Paracalanus paryus (Glaus) (see Figure VI) , the smallest
calanoid used in the studies, ranks second in abundance; it
occurs throughout the year, and is most abundant in the New
York Bight during the fall (20 x 103 individuals per cubic
meter) (2). The animals used for this study were collected
from waters adjacent to the sewage dumping site. Because
Paracalanus survived well under laboratory conditions,
replicate runs on this species, using the initial
collection, were possible when additional offshore
collecting was not feasible. However, Paracalanus showed a
wide range of sensitivity to the sludge, and was most
sensitive in the early tests. It is possible that the
organisms used in the last test were mere tolerant (TL,-0 =
10 times that of the most sensitive test), the more
sensitive individuals having died during the 2 weeks the
population was held in the laboratory.
Acartia tonsa Dana (see Figure VII), a temperate-
tropical calanoid form dominant during the summer in Middle
Atlantic coastal waters, was collected from the Shrewsbury
River, a tidal river on the New Jersey shore. This inshore
collection site provided an abundance of individuals when
offshore collecting trips were not possible. Acartia is
tolerant to estuarine-marine conditions and is abundant
during the summer and fall (7). This species is relatively
sensitive to municipal sewage sludge (TL50 - <».4 at 48
hours) especially when compared with Artemia.
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Figure V.
Centropages typicus Kroyer, a calanoid copepod congeneric
to C. hamatus, seasonally abundant in the New York Bight
(2.2 cm = 50y) 435x.
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Figure VI.
Paracalanus parvus (Glaus), a small calanoid copepod,
seasonally abundant in the New York Bight (3.6 cm 50y)
720x.
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"""••••••l^l
Figure VII.
Acartia tonsa Dana, a calanoid copepod, abundant during the
summer and fall in New York Bight (2.8 cm = 50y) 560x.
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Sithona sindlis (Claus) (see Figure VIII), the only
cyclopoid copepod used in the experiment, was collected
offshore in late August. This genus is the most abundant
and frequently occurring copepod in the New York Bight, It
occurs year round and has peak abundance during July (2) .
Wilson (8) reports that O. similis is littoral and is often
found in tidal pools and salt ponds. Oithona ranks high in
abundance, but probably is an unlikely food source for
zooplankton-eating fish due to its small size (2).
Although not as sensitive as some of the other endemic
species, Oithona must still be considered a very critical
species due to its great abundance and frequency of
occurrence. An impact upon this species could detrimentally
influence the pelagic ecosystem of the New York Bight.
An unidentified harpacticoid species (see Figure IX) was
used for one of the bioassay runs. Although not as
sensitive as some of the other endemic species, it was still
more sensitive to sludge than Artemia. Harpacticoids are
commonly associated with the mud-water interface of the
benthos; but they are also adventitious plankton dwellers,
particularly in the detritus-laden waters of the Middle
Atlantic Coast (9). These forms can become abundant in the
coastal bays and inlets (10). Unfortunately, little is
known about harpacticoids as a group, particularly in the
taxonomic areas, and workable indentification keys are not
available.
A marine cladoceran, Penilia avirostris Dana (see Figure
X), was used during one of the tests. Heavy mortalities
(80X-90% after 2U hr.) occurred within the control group and
invalidated the bioassay. Penilia is also a filter feeder,
but it is more sporadic in occurrence and abundance in the
Middle Atlantic coastal waters than are the endemic copepod
species. Marine cladocerans, like harpacticoid copepods,
are adventitious plankton forms but are sporadically
abundant (7) . In the New York Bight, Penilia and Podon
occasionally dominate the zooplankton during July and August
(2).
Experimental conditions of the bioassay certainly do not
replicate the environmental and ecological variables present
in the natural planktonic ecosystem of the New York Bight,
and it would be presumptuous to extrapolate from these
lethal concentrations for each species to the real world
situation. At best, these tests provide the information
needed to rank the sensitivity of indigenous species to
Middlesex County sewage sludge.
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<
fi
Figure VIII.
Oithona similis Glaus, a cyclopoid copepod, the most abundant
year round microcrustacean in the New York Bight (4.9 cm =
50y) 990x.
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•I
Figure IX.
Unidentified harpacticoid, an adventitious microcrustacean
often found in the plankton of coastal estuaries and
inlets (3.4 cm = 50y) 685x.
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Figure
Pen ilia avirostris Dana
found in large
50y) 410x.
a marine cladoceran occasionally
numbers in the New York Bight. (2.0 cm =
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Conclusion
Seasonally dominate endemic zooplankton species should
be used as the organisms of choice for the ocean disposal
bioassay criteria, especially in light of our knowledge that
these species are much more sensitive than the Artemia
nauplii. At the present time, however, this is not~~feasible
because the availability of these species from the
laboratory stocks is extremely limited in number and kind.
Field collecting could provide the organisms, but it would
be unwieldly, time consuming and expensive for each
laboratory performing the bioassays. Special holding
facilities would be required to maintain the organisms while
the bioassay was being performed,
In lieu of direct replacement of Artemia nauplii with
seasonally dominant zooplankton species, derivations of
sensitivity factors for each of the indigenous should be
determined using as many of the life stages as possible.
These sensitivity factors should be determined in
laboratories that have the expertise and equipment to work
with regionally indigenous species.
Summary
Comparative experiments using recommended bioassay
procedures for Artemia nauplii and endemic microcrustacean
zooplankton have illustrated that endemic species have a
greater sensitivity to municipal sewage sludge than have
Artemia nauplii. It has also shown, based upon these
results, that the allowable concentation {0.01 of the
Artemia 48-hour TL «-0) may be too high to prevent impact upon
the plankton in the mixing zone. In this case, the Criteria
may not be sufficient to prevent damage, both lethal and
sublethal, to the organisms first exposed to the dumped
materials, namely the microcrustacean populations.
To increase the value of using the bioassay criteria for
protecting the marine ecosystem from the impact of disposed
materials, sensitivity factors must be determined for
additional endemic species. These sensitivity factors
coupled with the present application factors, could then be
applied to the toxicity values derived from the Artemia
bioassay to increase the use of these bioassays as a device
for protecting the ecology of our Nation's coastal waters.
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References
1. United States National Technical Advisory Committee
1968. Report of the committee on Water Quality
Criteria. FWPCA, U.S. Dept. of Interior 23« pps.
2. sandy Hook Marine Laboratory. 1972. "The Effects of
Waste Disposal in the New York Bight, Final Report,
Section III: Zooplankton studies."
3. Tarzwell, C. M. 1969. "Standard Methods for the
Determination of Relative Toxicity of Oil
Dispersants and Mixtures of Dispersants and Various
Oils to Aquatic Organisms". In Proc. Joint
Conference on Prevention and Control of Oil Spills.
API and FWPCA, December 15-17, 1969, N.Y. pp. 179-
186.
U. Standard Methods for Examination of Water and
Wastewaters, 1971, 13th Edition, American Public
Health Association, New York.
5. Gross, M. G. 1970. "New York Metropolitan Region - a
Major Sediment Source", Water Resources Research,
6|3) :927-931.
6. Sprague, J. B. 1970. "Measurement of Pollutant
Toxicity to Fish, II. Utilizing and Applying
Bioassay Results", Water Resources Research, 4:3-32.
7. Jeffries, H. P. and W, C. Johnson. 1972. "Distribution
and Abundance of Zooplankton in Coastal and Offshore
Environmental Inventory - Cape Hatteras to Nantucket
Shoals", Marine Publication Series, pps. 1-93,
Section IV.
8. Wilson, C. B. 1932. "The Copepods of the Woods Hole
Region, Massachusetts" Bull. U.S. National Museum.
158. 635 pps.
9. Gosner, K. L. 1971. "Guide to Identification of Marine
and Estuarine Invertebrates". Wiley-Interscience,
New Jersey, 693 pps.
10. Durand, J. B., and R. J. Nadeau. 1972. "Water
Resources Development in the Mullica River Basin.
Part I. Biological Evaluation of the Mullica River
- Great Bay Estuary", New Jersey Water Resources
Research Institute, Rutgers University.
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MONITORING DREDGE SPOILS
L. S. Slotta and K. J. Williamson *
INTRODUCTION
The majority of U.S. waterways depend on dredging to
insure adequate water depths for shipping. Other important
functions of dredging include creation of land areas; mining
of underwater mineral deposits; correction of erosion; and
excavation of sand, gravel, shells and rocks.
Types_ of.Dredges
Dredges can be typically categorized as either hydraulic
or mechanical. Hydraulic dredges mix large volumes of water
with the sediment and the fluidized slurry is pumped away as
a sludge. This type of dredge results in the discharge of
large volumes of water that have become in direct contact
with the dredged sediments. As a result, these waters may
reflect the pollutional nature of the dredge spoil.
The dredged sediments which are termed "spoils" can be
disposed of by several different procedures. For hopper
dredge operations, the spoils are collected in large
sedimentation tanks (hoppers) aboard the dredge. These are
then dumped within the estuary or offshore. For pipeline
dredging operations, the sediment slurry may be pumped to a
nearby diked area which is subsequently filled with the
spoil.
Mechanical dredges directly resemble dry-land excavation
machines and are usually mounted on a fcarge. This type of
dredge is primarily used for projects with rocky deposits
and for limited operations. Such dredges create fewer
environmental concerns since interaction of the sediments
with the water column is minimized. The spoils typically
are barged to a land or water disposal site.
*Civil Engineering Department, Oregon State University
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Scope of U.S. Dredging
Dredging activities remove and redeposit tremendous
guantities of material. In the D.S. in 1972, maintenance
dredging and new dredging projects accounted for the
transfer of over 300 million cu. yds. and 80 million cu.
yds. of dredge spoils, respectively. Total costs of these
projects exceeded $150 million (2) .
A soils characterization of the spoil materials of
navigation channels which are maintenance dredged annually
revealed that:
"By far the largest category (approximately
153,000,000 cu. yds. per year) is that classified
as mixed sand and silt. About half this value is
associated with the coastal areas of the United
States, and the other half the inland rivers.
Approximately 30,000,000 cu. yds. per year of that
category including sand, gravel, and shell is
dredged from the nation's inland waterways, while
the remaining 22,000,000 cu. yds. is dredged fro»
the coastal zone. The ill-defined materials mud,
clay, silt, topsoil and shale account for 80,000,000
cu. yds. per year, all but 8,100,000 cu. yds. of
which are dredged from the eastern one-third of the
United States. Finally, although the group
including organic muck, sludge, peat, and municipal-
industrial wastes accounts for only 1,400,000 cu,
yds. per year, some of the more pressing
environmental problems are associated with this
group. Generally speaking, the materials dredged
and disposed of in inland waterways are sand and
gravel. The moving sand bottoms of many of the
nation's navigable rivers have been a supply of sand
and gravel for construction purposes for years.
Again, generally, in lakes, harbors, and many areas
of the coastal zones where the carrying capacity of
the water is guite low, the dredged materials often
consist of small, light particles such as clays and
silts" (2) .
Environmental concerns in relation to dredging have
risen due to the relatively fragile nature of estuarine
ecosystems and the widespread use of dredging in estuaries.
Particular interest has been generated around spoil disposal
by filling wetlands and dumping in estuarine waters. Only
the latter case and other dredging activities that could
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directly affect water and benthic environments are examined
in this paper.
Many positive environmental impacts have been documented
for dredging in addition to the obvious creation and
maintenance of channels. Improved circulation which results
from the removal of choked inlets can increase production of
shellfish and fish due to the increased availability of
food. Increased circulation also can reduce the impact of
man-made wastes which are frequently discharged into
estuaries. In many cases, dredge spoils are economically
processed to produce sand and gravel for construction.
In contrast to the several positive impacts, many
potential negative environmental impacts have been cited
(Table 1). These impacts result from various physical
alterations such as the change in the underwater topography,
the removal of benthic animals and plants and the discharge
of large quantities of particulate matter into the water
column. In all cases, serious degradation of water quality
and destruction of ecological systems may occur.
Table 1. Potential Negative Environmental impacts
of Dredging of Sediments
Alteration of the
Estuarine Environment
Environmental Impact
Changed Topography
Removal of Benthic Animals
Removal of Benthic Plants
Discharge of Particulate Matter
Alteration of currents. Tides,
Salinity Regimes, and Water
Quality
Significant Animal Kills,
Alteration of Important
Habitats
Alteration of Pelagic and
Benthic Habitats, Increased
Instability of Benthic Deposits
Increased Turbidities and Sed-
imentation Rates, Release of
Soluble Pollutants
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SCOPE
During the past fifteen months, an interdisciplinary
team at Oregon State University, under the sponsorship of
the NSF-RANN program, has been conducting research on the
environmental effects of dredging in estuarine waters. This
paper will report the potential acute and chronic
environmental impacts of dredging examined as part of this
study. A proposal of guidelines to minimize the acute
impacts and the identification of research needs to
effectively monitor dredging projects will also be
presented.
ACUTE ENVIRONMENTAL IMPACTS
The interactions within estuaries are highly complex and
involve geological, hydraulic, biological, chemical, social,
economic and political factors. Presently, the impacts of
dredging are primarily identified as acute changes in the
important system properties of one or several of these
categories. However, dredging also induces many potential
long-term chronic impacts that also must be incorporated
into the decision-making process. To fulfill national goals
of protecting our environments, dredging must be regulated
and both acute and chronic environmental impacts must be
considered. The potential acute and chronic problems and
monitoring procedures to regulate their impacts are
described in the following sections.
Altered C ir_cu j.at ion
Dredging can have a wide influence on estuarine
environments by altering the circulation patterns. Many Of
the biological species are adversely affected by permanent
changes in either salinity or temperature which result from
the circulation changes. St. Amant (20) and Waldo (22) both
reported long term changes in biological production to such
dredge induced alterations. Seme populations such as
herbivore Acartia tonsa (10) are extremely sensitive to
specific circulation patterns which can be significantly
altered by either spoil removal or disposal.
These studies suggest that dredging that could
significantly alter circulation patterns needs to be
regulated and at least monitored. The necessary techniques
of determining circulation patterns by airphoto analysis
have been developed and successfully utilized (4,24).
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Phfsical Remova1 of Organisms
The roost apparent biological impact of dredging pertains
to the removal of benthic organisms in the dredge spoil.
Although this process probably does result in a large kill
of these organisms, the impact does not appear to be,
significant for small scale dredging operations. Harrison,
Lynch and Altschaeff1 (8); Saila, Pratt and Polgar (IH) ; and
Slotta, et al. (19), all measured an immediate decrease in
the infaunal populations after dredging, but a fairly rapid
repopulation did occur.
Burial of Organisms
The ability of animals to withstand the adverse effects
of burial in areas near the dredge site or in the spoil site
depends primarily on their behavior and morphology. Species
such as large polychaetes and bivalves which can burrow have
been shown to survive burial of up to 21 cm of sediments
(15) . However, attached sessile species are probably killed
by burial of any magnitude. Numerous authors (see Saila,
Pratt and Polgar, 1971, for a review) have reported acute
kills from burial of various benthic organisms including
oysters. Slotta, et al. (19) reported that readjustment of
benthic infauna to former abundance levels occurred within
two weeks of spoiling. Thus the impacts in the spoil areas
also do not seem to be significant for small localized
projects.
The rapid recovery rates at both the dredge site and
spoils area have been attributed to a resistant biological
population (19). It has been hypothesized that the
dredging-related activities, such as marine traffic, also
disturb the benthic deposits, at short time intervals, which
tends to ecologically favor organisms resistant to
disruptions.Thus, acute biological impacts of dredging in
areas subjected to dredging-related activities may not be
significant even though large guantities of sediments are
removed and deposited.
Turbidity and Suspended Sp^ids
The most commonly reported effect of dredging on water
guality is an increase in turbidity and suspended solids.
However, some investigators have concluded that such
increases do not represent a significant impact
(6,11,12,14,21). This conclusion has been reached based on
two premises. First, the increase in turbidity and
suspended solids occurs over localized areas which pelagic
species can probably avoid. Second, periodic high turbidity
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levels are part of the evolutionary experience of estuaries.
Sediments are resuspended by wind, waves and tidal scour and
large sediment loads are carried with the winter fresh water
flows. Schubel (17) has reported a 20-fold increase in
suspended sediment concentrations in Chesapeake Bay caused
by natural occurences. With this evolutionary experience,
many estuarine animals are tolerant to suspended-solids
laden waters. Saila, Polgar and Rogers (15) cited several
examples of tests with fish and lobsters held in water with
several grams/liter of suspended sediments; no significant
mortalities were measured. Thus turbidity related impacts
do not seem to be significant in those cases cited.
Nutrient^Release
Nutrients in the various chemical forms of nitrogen and
phosphorus are commonly released frcm dredge spoils which
results in significant increases in the ambient
concentrations. Cronin, et al. (6) reported increases near
the discharge plume from 50 to 1,000 times ambient total
phosphorus and total nitrogen levels. However, no increase
in phytoplankton was observed. Windom (21) also reported
large releases of nutrients in his studies of five estuaries
on the southeastern coast of the United States. However, in
contrast to Cronin's results, significant algal growth was
reported when dredge spoils were placed in contact with the
receiving waters in closed bottle experiments. Stimulation
of algal growths was also noted from light-dark bottle
experiments at the dredging sites. In most cases, such
factors as the localized nature cf most dredging projects,
the large dispersion in most estuaries and the decrease in
available light from increased turbidity will reduce the
potentiality of serious environmental problems from nutrient
stimulation.
Oxygen Demand
Dredging operations lead to the release of organic
materials and inorganic materials (such as sulfides) which
create an oxygen demand within the overlying waters/ Under
certain conditions, significant reductions of dissolved
oxygen concentrations can result during dredging operations
(3). In addition, dredging operations may expose benthic
deposits of high oxygen demand which previously had been
covered by relatively clean materials. The settlement of
organic material suspended by dredging operations on the
surfaces of benthic systems and can cause an increased
benthic oxygen demand. The reverse can also be true if
dredging operations lead to the removal of polluted
sediments.
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High concentrations of free sulfides within the deposits
and the release of free sulfides to the overlying water and
atmosphere as a direct or indirect result of dredging
operations can be environmentally significant for a number
of reasons. First, the release of free sulfides can
increase the benthic oxygen demand rate and thus lead to a
decline in the aerobic zone of the deposit and a rapid
lowering of the DO concentrations within the overlying
waters. Second, free sulfides, particularly hydrogen
sulfide, are toxic at low concentrations to fish,
crustaceans, polychetes, and a variety of benthic micro-
vertebrates (7,9,18). Actual toxic concentrations reported
in the literature usually represent only initial sulfide
concentrations below 0.075 mg/1 {pH 7.6-8.0) were found to
be significantly harmful to rainbow trout, sucker, and
walleye, particularly to the eggs and fry of these fish (5).
Heavy Metals
The release of heavy metals from polluted sediments as a
result of dredging has been postulated by many authors.
However, in sediments where sulfides are being produced, the
possible chemical transformations from resuspension become
quite complex. Presently it is unknown whether heavy metals
will be released from sulfide bearing sediments.
Ferrous sulfides are common minerals in anaerobic
sediments and are probably responsible for the
characteristic black color. Preliminary studies at OSU have
shown that heavy metals absorb on both Fe(III) oxides and
Fe(II) sulfides. In addition, the heavy metals are readily
co-precipitated in both Fe (III)oxides and Fe(II) sulfides.
From these results, it is hypothesized that heavy metals
will not be released to the water column upon resuspension
and either will be absorbed, co-precipitated and
incorporated within the sulfide-bearing sediments; a similar
hypothesis has been proposed by Windom {2H). The hypothesis
is in agreement with the data reported by Windom (24) and
May (12) in which heavy metals present in the dredge spoils
were not released to the water column.
More research is required to elucidate the important
mechanism occurring in this process. Present data are not
adeguate to establish exact criteria.
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Tpxi c_Organi c s
Important organics in relation to the toxicity of dredge
spoil include the organochloride insecticides, the
organophosphorus insecticides and the polychlorinated
biphenols. The possible adverse effects of spoils
contaminated with these compounds are numerous; however,
direct cause and effect relationships are not well
documented. More research is needed in this area in
relation to monitoring methods and acceptable criteria.
CHRONIC ENVIRONMENTAL IMPACTS
As described in the last section, the acute impacts of
dredging are highly complex and not well-defined. Even less
is known about the extent of chronic or long term
environmental impacts. These chronic impacts include not
only dredging but also such activities as shipping,
industrialization, and urbanization which alter the
environment in complex ways. The measurement of such
chronic impacts requires an understanding of the geological,
hydraulic, biological and chemical factors which control the
interactions in estuaries.
Presently, the impacts of dredging have been primarily
identified as acute changes in important system properties.
Little is known of chronic impacts for two reasons. First,
chronic impacts are not so immediately apparent upon
examination of a problem. An understanding of the system
properties is often required to sort out the chronic problem
from the multitude of other changes. In reference to
dredging, the understanding of important system properties
has been almost non-existent. Second, the detection of
chronic impacts requires long-term research. Several
potential chronic impacts will be briefly discussed in this
section.
Particle Size,Change
A dominant feature of hopper dredging activities is the
resuspensions of bottom sediments. AS a dredge suction head
passes through a dredge site, surface sediments drawn into
the head and pass to the hopper. Seme of the material
around the suction head is disturbed mechanically and thrown
into suspension. Heavier particles settle out after the
disturbance passes, while lighter particles remain in
suspension due to ambient turbulence and may be transported
from the original site by local currents. The material
which passes into the hopper is initially in suspension, but
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the heavier particles settle to the hopper bottom. The
lighter particles remain in suspension and some are returned
to the estuary water column via the hopper overflow.
At the spoil area, the contents of the hopper are
released and settle to the bottom as a slurry. Surface
shear during descent and impact-induced mixing at the bottom
resuspend a portion of the material; again, the fines may be
transported from the spoil site. As a result of repeated
resuspensioning and settling and the subsequent loss of
fines, dredge spoils may contain smaller fractions of fines
than occur at the dredge site.
Specifically, it was observed on the coos Bay hopper
dredge project that a five-fold increase in mean particle
size occurred in the spoils immediately after spoiling and
persisted for two months. The escaping fines probably
contributed to long term siltation in the adjacent shallow
areas.
The dependence of animal populations on a specific
particle size distribution has been clearly identified.
Rhoads and Young (13) reported that suspension feeders and
benthic infauna are largely confined to sandy or firm mud
bottoms. Sanders (16) showed that suspension feeders in
Long Island Sound comprised 80' percent of the organisms on
coarser sediments. Selective and non-selective deposit
feeders were the dominant forms in fine sediments. Thus it
can be concluded that changes in particle size from dredging
operations probably seriously affect the distribution of the
benthic populations.
Reduced Sediment Turnover
Polluted dredge spoils are often deposited behind "water
tight berms" and/or in diked, sacrificial channels, in
either case, a sediment system results in the spoil deposit
area in which the bottom deposits have increased stability
over their previous condition. With this increased
stability the sediments are turned over less frequently and
the build-up of anoxic, sulfide-bearing sediments can
result. In addition, more organics are deposited in this
relatively quiescent region which further encourages the
growth of sulfate-reducing bacteria. The end result in the
spoil disposal area can be s significant reduction in the
biological populations present before spoiling. Similar
results can occur in open estuarine areas when dredging
causes reduced current velocities throughout an estuary.
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Increased Sediment: Turnover
Dredging can increase current velocities through the
removal of inlet barriers, channelization, or the removal of
eel grass. These increased current velocities may
subsequently increase the turnover of the sediments, which,
as the reverse of the previous case, can also adversely
affect the biological communities.
Resistant^BiQloqical Communities
Preliminary studies (19) have suggested that the benthic
infaunal communities may become modified in an estuary which
has repeated dredging. These communities may become adapted
to a more or less continual resuspension of the sediments
and its persistence may actually depend on this turnover.
The turnover in some estuaries may depend more upon the prop
wash of large ships than on the continual maintenance
dredging.
RESEARCH NEEDS
In relation to monitoring of dredging projects the
following research areas should be addressed.
Improve Monitoring RegudLrements
A system needs to be developed in which the required
parameters to be monitored varies with the degree of
pollution. Some easily measured parameters (e.g., volatile
solids) that roughly correlate with pollution potential
should be used to determine both the sampling methods and
the required parameter to be monitored. For low volatile
solids (<2% by dry wt.), little additional monitoring would
be necessary; for high volatile solids (>10X by dry wt.)
many tests both before and during the dredging would be
necessary.
Release of_Heavy Metals and Toxic Hydrocarbons
Research needs to be initiated to determine if heavy
metals can be released from dredge spoils under natural
environmental conditions. Additional studies are required
to elucidate the important transport mechanisms and the
environmental impacts of the chlorinated hydrocarbons which
are known to exist in high concentrations in certain
sediments.
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The exact role of turbidity as a controlling factor is
relatively unknown for estuaries. Work concerning long term
increases from all man-made activities including dredging
and the possible impacts of such increases should be
established.
Benthie Deposits
Man-made activities in estuaries undoubtedly alter the
rate at which benthic deposits are turned over. Natural
causes include tides, currents, freshwater flows and benthic
burrowers. Important man-related causes are dredging, ship
props, and channelization. The interrelationship and
importance of each of these activities needs to be further
examined.
CONCLUSIONS
1. Present monitoring technology is available to measure
many parameters in relation to dredging projects.
2. Criteria are reguired to specify which parameters should
be monitored.
3. More research is necessary to elucidate cause and effect
relationships especially in relation to chronic impacts.
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REFERENCES
1. Berner, R. A. Principles of Chemical Sedimentolggy.
McGraw-Hill Book Co., 1971.
2. Boyd, M. B., Saucier, R. T., Keeley, J. W., Montgomery,
R. L., Brown, R. D., Mathis, D. B., and Guise, C. J.
U.S. Army Corp of Engineers, "Disposal of Dredge
Spoil - Problem Identification and Assessment and
Research Program Development", Technical Report H-
72-8, 1972.
3. Brown, Ci L., and Clark, R. "Observations on Dredging
and Dissolved Oxygen in a Tidal Waterway,"
Water Resources Research * j»:6, 1381, 1968.
4. Burgess, F. J., and James, W. P. "Airphoto Analysis of
Ocean Outfall Dispersion", EPA Program No. 16070
RNS, 1971.
5. Colby, P. I., and Smith, L. L., Jr. "Survival of
walleye Eggs and Fry on Paper Fiber Sludge Deposits
in Rainy River, Minnesota",
Transactions of the American Fisheries Society,
96:279, 1967.
6. Cronin, L. E., et al. "Gross Physical and Biological
Effects of Overboard Spoil Disposal in Upper
Chesapeake Bay", Natural Resources Institute,
University of Maryland, Contribution 397, 1970.
7. Fenchel, T. "The Ecology of Marine Microbenthos. IV.
Structure and Function of the Benthic Ecosystem, Its
Chemical and Physical Factors and the Microfaune
communities with Special Reference to the Ciliated
Protozoa", Ophelia, 6:1, 1969.
8. Harrison, W., Lynch, M. P., and Altschaeffl, A. G.
"Sediments of Lower Chesapeake Bay with Emphasis on
Mass Properties," J. of sed. Petrol.. 3J»:4, 727,
1964.
9. Ivanov, M. V. "Microbiological Processes in the
Formation of Sulfur Deposits", translated from
Russian by S. Nemchonok. Israel Program for
Scientific Translations. Jerusalem, Israel.
10. Johnson, J. K., and Miller, C. E. "The Dynamics of an
Isolated Population of ACartia Tunsa in Yaquina
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Bay, Oregon", 3rd Annual Technical conference on
Estuaries, Corvallis, Oregon, 1973.
11. Hacking, J. G. "Canal Dredging and silting in Louisiana
Bays", University of Texas, Publication of the
Institute of Marine Science. 7:262, 1962.
12. May, E. G. "Environmental Effects of Hydraulic Dredging
in Estuaries," Alabama Marine_jesources Bulletin.
9:1, 1973.
13. Rhoads, p., and Young, D. "The Influence of Deposit-
Feeding Organisms on Sediment Stability and
Community Trophic Structure", Journal of Marine
Research. 28:2, 1970.
11. Saila, S. B., Pratt, S. D., and Polgar, T. T. "Dredge
Spoil Disposal in Rhode Island Sound", Marine
Technical Report No. 2, University of Rhode Island,
1972.
15. Saila, S. B., Pratt, S. D., and Polgar, T. T.
"Providence Harbor Improvement Spoil Disposal site
Evaluation Study, Phase II", University of Rhode
Island, 1972.
16. Sanders, H. L. "Oceanography of Long Island Sound,
1972-1951. The Biology of Marine Bottom
Communities," gingham Oceanography coll. Bull.,
15:315, 1956.
17. schubel, J. "Turbidity Maximum of the Northern
Chesapeake Bay", Science. i§l:1013, 1968.
18. Servizi, J. A., Gordon, R. W., and Martens, D. w.
"Marine Disposal of Sediments from Bellingham Harbor
as Related to Sockeye and Pink Salmon Fishers",
International Pacific Salmon Fisheries commission
Progress Report, No. 23, 1969.
19. Slotta, L. S., Sollitt, C. K., Bella, D. A., Hancock, D.
R., McCauley, J. E. , and Parr, R. A. "Effects of
Hopper Dredging and In-Channel Spoiling (October 1,
1972) in Coos Bay, Oregon", Oregon state University,
1973.
20. St. Amant, L. S. "Oysters, Water Bottoms and Seafood",
Seventh Biennial Report, Louisiana wildlife and
Fisheries commission, 1956-57:71, 1956.
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21. Sullivan, B. "Zooplankton and Dredging: Literature
Review and Suggestions for Research", Department of
Oceanography, Oregon State Oniversity, 1973.
22. Waldo, E. "Louisiana Oyster Story", Seventh Biennial
Report, Louisiana wildlife and Fisheries Commission,
1956-57, 93, 1956.
23. weise, H. G. "Airphoto Analysis of Estuarine
Circulation". M.S. Thesis, Oregon State University,
1973.
2U. windom, H. L. "Processes Responsible for Hater Quality
Changes During Pipeline Dredging in Marine
Environment", WODA V, 1973.
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COMPARISON OF SPECIES DIVERSITY AND FAUNAL HOMOGENEITY
INDICIES AS CRITERIA OF CHANGE IN BIOLOGICAL COMMUNITIES
R. C. Swartz,* W. A. DeBen,* and A. J. McErlean**
•*' INTRODUCTION
The structure of marine communities is frequently used
as an indication of the environmental consequences of
pollution (21). Prevention of major ecosystem alterations
necessitates monitoring programs that can detect subtle
changes before irreversible damage bas occurred. Data
analysis procedures should therefore be sensitive to
relatively minor differences in biological conditions.
Species diversity indices have become very popular criteria
(sometimes the only criterion) of the "stability" or
condition of stressed ecosystems (1, 2, 5, 10, 13, 20, 2U,
25, 26, 27) . Other community characteristics such as
species composition, homogeneity, density, and population
dynamics are often ignored. In this report we will compare
the ability of a variety of structural indices to
discriminate between closely related assemblages of demersal
fishes and macroinvertebrates at four stations in Yaquina
Bay, Oregon.
MATERIALS AND METHODS
Pata_Collection
Demersal fish and epibenthic crustaceans were collected
by bottom trawl at four stations in the Yaquina Bay, Oregon
from March 1967 through Febraury 1968. Two surveys, usually
separated by a two-week period, were made each month. Data
from each station have been pooled on a monthly basis for
analysis. The stations were located from near the Yaquina
Bay entrance (station 1) to Weiser Point, 7.6 km up the Bay
(Table 1). Collections were made with a semi-balloon 5.9 m
(headrope length)) trawl net with a 3.8 cm body mesh and 1.3
cm (stretched size) bag liner. All specimens were
identified to the species level and enumerated.
* Pacific Northwest Environmental Research Laboratory,
Corvallis, Oregon
**Office of Technical Analysis, Environmental Protection Agency,
Washington, D.C.
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DataAnalysis
Density
Because of differences in trawling distances at the four
stations, densities were compared on the basis of catch of
individuals per unit effort. The minimum effort was a 550 m
total monthly trawl distance at station 3. Faunal densities
for stations 1, 2 and H were calculated by multiplying the
observed catch by the ratio of 550 m to the actual monthly
trawl distance at each station.
Table 1. Description of trawl stations.
Station
Number
Location
Distance from entrance Approximate
to jetties at Newport, trawl
Oregon distance
First rock finger
downbay from Hwy.
101 bridge to south
bridge support.
Parallel to Newport
breakwater.
Buoy 15 to opposite
Conquille Point.
Between Yaquina and
Weiser Points.
l.U-1.8 km
2.6-3.U km
5,7-6.0 km
7.0-7.6
400m
750m
275m
560m
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Species diversity
Species diversity is dependent on the number of species
(richness) and the distribution of individuals among the
species (evenness) (16). Shannon's (19) information-
theoretical measure of mean species diversity per individual
(H1) is sensitive to and increases with both species
richness and evenness. The value of H* is proportional to
the uncertainity of identification of an individual selected
at random from a multi-species population (16). H1 was
calculated with the aid of the tables of Lloyd, Zar, and
Karr (1968) according to:
H* *y (N logi() N - J n
where n is the number of individuals of the i— species, N
is the total number of individuals, and S is the total
number of species.
The evenness of each collection was determined a J1 , the
ratio of observed H1 to maximum H1 for observed 8(16).
Maximum H1 occurs when all species are represented by the
same number of individuals. Thus for H1 max:
£ ni 10*io ni=s< |logio !>
and the equation for H1 reduces to:
H* max = Iog10 S
The evenness index J» = H*/H* max = H'/log^ S
Indices of species richness were determined in three
ways:
1. the observed number of species (S) in each monthly
collection
2. the predicted number of species per unit effort
(S ) . Sanders' (18) rarefaction method was used
toRpredict the number of species that would have
been present if the trawling distance had been 550 m
per month at all stations. All n /N were calculated
for the original sample. The percentage that one
individual would represent in the 550 m trawl
(1/N550) was determined from the faunal density
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data. It was assumed that all species for which (r
/N) > U/N550) would be present in the rarefied
sample. In addition, some of the rarer species for
which (ni/N)<(l/N55o> would also be present. Their
contribution to S was determined by
-------
M
J J A
MONTH
N
Fig. I. Seasonal changes in faunal density (catch per 550
m trawl distance) at stations 1-4.
M
Fig. 2. Seasonal changes in species richness (SR) P«r 550
m monthly trawl collection at stations 1-4.
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Species^diversity
Species richness
The spatial-temporal distribution patterns of S(observed
number of species) and s (rarefied number of species per
550 m monthly trawl) were essentially identical. Only the
latter will be presented, s increased during the summer at
all stations and, compared ta density, there were relatively
small differences in species richness between stations (Fig.
2). The maximum S at each station was highest at 3 (21
species) and lowest at 1 (15.1 species), although during two
months (September and October) S R at 1 was higher than at
any other station. There was very little difference in
species richness when data for the entire year were pooled:
SR= 28.8, 28.0, 28.0, and 29.9 for stations 1 through 4,
respectively.
Margalef's (8) richness index, d, was also generally
higher during the summer, but its seasonal pattern was not
as obvious as that of SR (Fig. 3). Since d is a function of
the ratio of richness to density [ (S-l)/lnN], the index
value was often lowest at station 3 where density was
usually 2 to 3 times greater than at the other stations.
The highest d occurred at station 1 for the October
collection which contained 16 species and 67 individuals.
Evenness
There was little evidence for a seasonal cycle in
Pielou*s (16) evenness index (J1) at stations 1 and 2 (Fig.
4). J» at stations 3 and 4 was very low from May through
July and it was consistently lower than J1 at 1 and 2 during
late spring and the entire summer. During the remainder of
the year, there were no clear differences in J1 at the four
stations.
Information-theoretical index of species diversity
Shannon1s (19) index (H1) increased during the warmer
months at stations 1 and 2 {Fig. 5), but the seasonal
pattern was not as obvious as that of species richness. Hl
was lower at stations 3 and 4 than at 1 and 2 from late
spring to early fall. Like J*, H* at 3 and 4 was relatively
low during the months of highest faunal densities (May-
July) .
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1.2
Fig. 3. Seasonal changes in Margalef's (1951) specie*
richness index (d) of sf of ions 1-4.
I-Or
0.8
0.6
0 4
0 2
-------
Faunal homogeneity
Except for September, the fauna 1 affinity index between
monthly samples at stations 1 and 2 (annual mean i S-* t —
= 40.0 * 7.6) was consistently higher than between 1 and'T
(X = 1976 » 6.1) or 1 and 4 (x- = 17.5 * 6.4) (Fig. 6). The
assemblage at station 2, however, was just as closely
related to stations 3 (x = 44.2 £ 8.4) and 4 (35 = 39.3 *
7.4) as 1 (Fig. 7) . Highest fauna 1 homogeneity occurred
between stations 3 and 4 (x - 68.9 * 7.9) (Figs. 8 and 9).
Thus, the analyses of fauna 1 homogeneity indicated the
existence of two distinct assemblages at stations 3-4 and 1,
and an intermediate demersal community at station 2. This
pattern was evident throughout the year.
Spatial-temporal distribution o£ species
The buffalo sculpin was the most abundant species at
station 1, and second most abundant at 2 (Fig. 10) . It
ranked 14th at 3 and 15th at 4. Thus its spatial
distribution accounted for much of the relatively high
affinity between stations 1 and 2 and low affinity between
1-3 and 1-4. Most of the specimens in our collections were
juveniles < 10 cm in total length. They were most abundant
from April through October.
Cancer maaister
The dungeness crab was the most dominant species at
station 2 and more common at 3 and 4 than at 1 (Fig. 11) .
Juvenile crabs enter Yaquina Bay in large numbers during
June and July and most of the population returns to the
ocean in the fall.
Parophrvs yetulus
The English sole was the most numerous species at
stations 3 and 4 (Fig. 12.) . It ranked 3rd and 7th in
abundance for the entire year at stations 2 and 1,
respectively. Only a few specimens in our samples and none
in those of Westrheim (22) from the same area were adults.
Although present throughout the year, P. yetulus was most
common from May through August.
* Sx = standard error
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I00r
STATION I
MAMJJASONDJF
Fig. 6. Fauna I affinity between station I and stations
2, 3, and 4.
100
80
z
u. 60
10
20
STATION 3
4
/\
/
..
I
.o
o"
MAMJJASONDJF
MONTH
Fig. 8. Fauna! affinity between station 3 and stations 1,2,
and 4.
100 r
80
so
40
20
STATION 2
J 1 1
M A M J J A SONDJF
MONTH
Fig. 7. Faunal affinity between station 2 and stations 1,3
and 4. '
Fig. 9. Faunal affinity between station 4 and stations I,
2, and 3.
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zoo
MAMJJA SONDJF
MONTH
Fig. 10. Seasonal distribution of the buffalo sculpin (Enophrys
bison) at stations 1-4.
i 100
O
tc.
Fig. II. Seasonal distribution of the Dungeness crab (Cancer
magister) at stations 1-4.
1400
1200 -
IOOO
800
o
> 600
a
o
£ 4
a
z
200
Paraphrys vetulus
J J A
MONTH
S 0
Fig. 12. Seasonal distribution of the English sole (Porophr
vetulus) at stations 1*4.
500
400
100
•o
Lump«nui saottto
Fig. 13. Seasonal distribution of the Pacific snakeWenny
(Lumpenus sogitto) at stations I-4.
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Lumpenus sagitta
The Pacific snakeblenny was the second most abundant
species at stations 3 and 4, ranked eighth at station 2, and
was not collected at station 1 (Fig. 13) . Like ParoEhryj
vetulus, If- saai^ta was very abundant in the late spring and
early summer. From May to August these two species
represented 7 OX of the individuals collected at stations 3
and 4.
Platichthys stellatus
The starry flounder was common throughout the survey
area. It ranked 4th, 6th, 5th , and 6th in total abundance
at stations 1 through 4, respectively (Fig. 14) . It was
least abundant in the fall, but was present in more monthly
collections (45 of 48) than any other species.
Mature specimens of the viviparous pile perch entered
Yaquina Bay during the spring. Many young-of- the- year were
present at station 4 in August and at lower Bay stations in
September (Fig. 15) . The spatial-temporal distribution of
R. vacca contributed to several deviations from the normal
pattern~of faunal affinity between the stations. The
minimum index value between stations 3 and 4 occurred in
August when R. vacca was abundant at 4 and rare at 3.
Maximum aff inity~"between stations 1-3 and 1-4 was observed
in September when pile perch were common at all stations.
Crago nigricauda
The black-tailed shrimp is of interest because of its
unique seasonal distribution (Fig. 16) . It was the only
species common from late fall to early spring and rare or
absent during the summer.
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1 60
zo
Plotichthy. sHllolus
JASOND J F
Fig. 14. Seasonol distribution of th« storr» flounder IPtotichthye
stellatus) at stations 1-4.
IOO
Raccochilus vacco
MAMJJASOND J F
MONTH
Fig. 15. Seasonal distribution of the pile parch (Raccochilus
yqcco.) at stations I - 4.
200r
rx:
Crago nigricouda
MAMJ J A S 0 N 0 JF
MONTH
Fig. 16. Seasonal distribution of the block-tailed shrimp
(Crago nigncouda) at stations 1-4
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DISCUSSION
Spatial-temporal changes in the structure of the demersal
community of Yaquina Bay are complex. No single index
adequately reflected all of the differences between the four
stations. In fact, the interpretation of some indices may
have been misleading in the absence of other community and
population characteristics.
The density of all individuals was relatively high in
the upper bay (stations 3 and 4) and low near the bay
entrance (stations 1 and 2) . This difference was due
primarily to the very high abundance of just two species
<£i£2£hrv.§ Y§tulus and Lumpenus sagjtta) at stations 3 and
H, and was not characteristic of the rest of the assemblage.
The extreme dominance of P. vetulus and L. sagitta explains
the reduction in J1 at the upper bay stations from May to
August.
Species richness (S,.) was very similar at all stations
but the fauna1 affinity index demonstrated major changes in
either species composition or dominance. Margalef's index,
d, was influenced by both richness and density patterns.
These parameters are best analyzed separately as SR and
catch per unit effort or space.
H1, the most popular diversity index in pollution
studies, showed very little difference between the stations.
Its value is a function of species richness and evenness,
but it did not indicate the strong seasonal cycle of S at
all stations nor the obvious difference in evenness between
stations 1-2 and 3-* from May through August. Species
diversity indices derived from information theory (H* and
J1) are not sensitive to changes in species composition or
the dominance rank of species provided that S and evenness
do not change, obviously H» should not be the sole or even
primary criterion of biological conditions in marine
ecosystems which often exhibit strong spatial-temporal
gradients of species composition.
Neither density nor any of the species diversity indices
evidenced the consistent differences in faunal homogeneity
between the stations. The affinity index demonstrated the
close relationship between the assemblages at stations 3 and
4, the intermediate nature of the fauna at station 2, and
the dissimilarity between the communities at station 1 and
stations'3-4. This simple index can also be used to
document temporal changes in community structure. For
example, at station 3 the affinity index was 79.8% for the
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June and July collections and 15.7% for the June and
December collections. This analysis is sensitive to
evenness, species composition, and the relative abundance of
individual species. It is not a good measure of richness
because rare species enter or leave the community with
little impact on the percent distribution of individuals
between species. It will also not indicate differences in
the density of the total community.
An analysis of the dynamics of individual populations
was an essential part of the description of the demersal
community of Yaquina Bay. The spatial and temporal
distributions of species revealed no discrete community
types with characteristic species composition and dominance.
The concept of gradient analysis in which species are
considered to be distributed independently according to
their own environmental requirements is a more appropriate
basis for description (23). Thus, some of the species (e.g.
Elatichthys stellatus) were common at all of the stations,
while others occurred in maximum abundance at discrete
positions of the gradient: Engphyrys bison at 1 and 2,
Cancer magister at 2, Lumpenus sagitta and Parophrys vetulus
at 3 and U. The dominant organisms showed strong seasonal
-iist i ibution patterns associated with migratory (e.g. Cancer
!liijt-et§l) or reproductive (e.g. Racochilus yacca) cycles.
Many populations were most abundant during the warmer months
(not always the same month), but Crago nigricauda was least
common during the summer. The independent spatial-temporal
distribution patterns of single populations explain many of
the apparently erratic changes in "community"
characteristics. As noted above, the appearance of large
numbers of young-of-the-year Rhacochilus vacca at station 1
in August and their subsequent migration down and out of the
Bay in September resulted in deviations from the otherwise
consistent pattern of faunal homogeneity between stations.
A multitude of faunal density, diversity, richness,
evenness, and affinity indices have been proposed and
compared on the basis of ecological and mathematical theory
and empirical observations of their ability to discriminate
between samples (3, U, 6, 9, 11, 12, 13, 14, 15, 16, 18,
24). Many of these reports attempted to identify the "best"
index. The objective of most marine monitoring programs
conducted by regulatory agencies is to document changes in
biological conditions that can be correlated with water
quality or other pollution parameters. Our study has
demonstrated that many characteristics of the biota should
be examined. No single index is sufficient. We found that
total faunal density, species richness (18), evenness (J»;
16), and an affinity index (17) were helpful in describing
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closely related demersal communities. Differences in the
spatial-temporal distribution of individual species
populations provided the most obvious discrimination between
collections.
ACKNOWLEDGEMENTS
Donald Baumgartner and William Clothier designed the
sampling program. We also thank William Clothier and George
Ditsworth for their participation in the field sampling.
Faith Cole for assistance in data processing, and John Frey
for preparing the figures. Mrs. Grace Boden kindly typed
the manuscript.
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REFERENCES
1. Bechtel, T. J. and B. J. Copeland. 1970. Fish species
diversity indices as indicators of pollution in
Galveston Bay, Texas. Contr. Mar. Sci. 15:103-132.
2. Copeland, B. J. and T. J. Bechtel. 1971. Species
diversity and water quality in Galveston Bay, Texas.
Water, Air, and Soil Pollution 1:89-105.
3. DeBenedictis, P. A. 1973. On the correlations between
certain diversity indices. Am. Nat. 107:295-302.
4. Fager, E. W. 1973. Diversity: a sampling study. Am.
Nat. 106: 293-310.
5. Harkins, R. D. and R. E. Austin. 1973. Reduction and
evaluation of biological data. J. Water Poll.
Control Fed. 45: 1606-1611.
6. Hurlbert, S. H, 1971. The nonconcept of species
diversity: A critique and alternate parameters.
Ecology 52: 577-586.
7. Lloyd, M., J. fi. Zar, and J. R. Karr. 1968. On the
calculation of information - theoretical measure of
diversity. Amer. Midi. Natur. 79:257-272.
8. Margalef, R. 1951. Diversidad de especies en
cominudades naturales. Pro. Ins. Biol. Appl. 9: 5-
27.
9. MeErlean, A. J. and J. A. Mihursky. 1968. Species
diversity species abundance of fish populations: an
examination of various methods. Proc. 22nd Ann.
Conf., Southeastern Assoc. Game Fish Commissioners.
10 p.
10. McErlean, A. J., S. G. o»Connor, J. A. Mihursky, and C.
I. Gibson. 1973. Abundance, diversity and seasonal
patterns of estuarine fish populations. Est. and
Coastal Mar. Sci. 1:19-36.
11. Mclntosh, R. P. 1967. An index of diversity and the
relation of certain concepts to diversity. Ecology
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12. Menhinick, E. F. 196a. A comparison of some species -
individuals diversity indices applied to samples of
field insects. Ecology 45:859-861.
13. Pearson, E. A., P. N. Storrs, and R. E. Selleck. 1967.
Some physical parameters and their significance in
marine waste disposal. In Pollution and Marine
Ecology, T. A. Olson and~F. J. Burgess (eds.).
Interscience Publ., John Wiley & sons, New York. p.
297-315.
14. Pielou, E. C. 1966a. Shannon's formula as a measure of
specific diversity: its use and misuse, Amer. Nat.
100: 463-465.
15. Pielou, E. C, 1966b. The measurement of diversity in
different types of biological collections. J.
Theoret. Biol. 13: 131-144.
16. Pielou, E. C. 1970. An introduction to mathematical
ecology. Wiley-Interscience. New York. 286 p.
17. Sanders, H. L. 1960. Benthic studies in Buzzards Bay.
III. The structure of the soft-bottom community.
Limn. Oceanogr. 5:138-153.
18. Sanders, H, L. 1968. Marine benthic diversity: a
comparative study. Am. Nat. 102: 243-282.
19. Shannon, C. E. 1948. A mathematical theory of
communication. Bell Syst. Tech. J. 27: 379-423,
623-656.
20. Storrs, P. N., E. A. Pearson, H. F. Ludwig, R. Walsh and
E. J. Stann. 1969. Estuarine water quality and
biologic population indices. Proc. 4th Int. Conf.
water Pollut. Res. Prague, pp. 901-910.
21. Swartz, R. C. 1972. Biological criteria of
environmental change in the Chesapeake Bay.
Cheasapeake Sci. 13(Suppl.):S17-S41.
22. Westrheim, S. J. 1955. Size composition growth and
seasonal abundance of juvenile English sole
i£a£2£h£Y.§ yetulus) in Yaquina Bay. Fish Coram. of
Oregon~Res. Briefs 6: 4-9.
23. Whittaker, R. H. 1967. Gradient analysis of
vegetation. Biol. Rev. 49: 207-264.
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24. Wilhm, J. L. 1967. Comparison of some diversity
indices applied to populations of benthic
macroinvertebrates in a stream receiving organic
wastes. J. Water Poll. Control Fed. 39: 1673-1683.
25. Wilhm, J. L. 1970. Range of diversity index in benthic
macroinvertebrate populations. J. Water Poll.
Control Fed. 42: R221-R224.
26. Wilhm, J. L. and T. C. Dorris. 1966. Species diversity
of benthic macroinvertebrates in a stream receiving
domestic and oil refinery effluents. Am. • Midi.
Natur. 76:
27. Wilhm, J. L. and T. C. Dorris. 1968. Biological
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18: 477-481.
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SAMPLING METHODS FOR MICROBIOLOGICAL ANALYSES
R. R. Colweil*
Since the marine environment encompasses the coastal,
off-shore and deep ocean regions of the world oceans,
sampling problems cannot be truly appreciated without some
knowledge of the underwater terrain, i.e., that unique
topography and geography of the marine environment which may
include valleys, plains, peaks, ridges, shelves and sills
submerged to depths from a meter to several miles. The
water overlying this vast ocean territory disguises the
great heterogeneity of the deep ocean. Too often,
investigators are oblivious to the changing seascape as they
move from sampling station to sampling station. The geology
of each region of the world oceans can be vastly different.
For example, sediments may be composed predominately of
sand, silt, gravel or other soil types. Replicability of
samples adds another dimension to the problem. These
factors pose severe problems in obtaining representative
samples preliminary to describing the microbiology of a
given sector of the marine environment.
A sampling regimen undertaken in a monitoring program
will, of necessity, involve ship time, with the
sophistication of the vessel ranging from a small "whaler"
to an ocean-going oceanographic vessel. The work
undertaken, therefore, is further limited by the facilities
of the vessel out of which the investigator must operate. A
monitoring program aboard ship can involve highly
sophisticated procedures or extremely primitive or
simplistic techniques.
In our studies, we have had available a range of
vessels, including the R/V EASTWARD (Fig. 1) for deep ocean
work. The R/V EASTWARD offers laboratory facilities
permitting relatively complicated experiments at sea.
Another vessel, the R/V RIDGELY WARFIELD is employed in our
work in the Chesapeake Bay (Fig. 2) . Laboratory space or\
the R/V WARFIELD is sufficient and well-designed so that
several investigators can operate simultaneously and in
reasonable comfort {Fig. 3).
*Department of Microbiology, University of Maryland
-335-
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Figure 1. The R/V EASTWARD, operated out of Beaufort,
North Carolina, by Duke University with
support from the National Science
Foundation.
-336-
-------
Figure 2.
fron the
-337-
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Figure 3. Laboratory aboard the P/V RIDGELY WARFIELD.
Microbiological analyses can be carried out
with relative ease aboard the ship.
-338-
-------
For shallow water work, however, the size of the craft
is necessarily much smaller, and on-station work thereby is
significantly reduced. The Maryland Department of Natural
Resources and the Environmental Protection Agency, Annapolis
Laboratory, operate small craft suitable for estuarine work
(Figs. 4 and 5) . For very shallow streams and inlets,
motor "whalers" or row-boats are used; in such craft it is
feasible only to collect samples that are then returned to
the laboratory for analysis, if sampling is done relatively
close to the laboratory, little or no problem in delay of
sample processing is encountered, if samples must be
collected at great distances (more than several hours
journey) from the home laboratory, either the samples are
flown back to the laboratory or a mobile field laboratory
unit is required.
The sampling equipment employed in collecting water,
sediment and biota for microbiological analyses is
relatively primitive. Marine microbiology research and
monitoring are seriously hampered by the lack of proper
equipment for sampling and in situ study. There has been a
much too limited effort to improve the technology for
environmental assessment and analysis, clearly, sampling
equipment for microbiological work is a problem area and
deserves attention.
Determination of microbial densities can be accomplished
by direct microscopic or total viable count methods,
measurement of adenosine triphosphate (ATP) , diaminopiraelic
acid (DAP) , muramic acid or deoxyribonucleic acid (DNA) , or
the estimation of the production of heterotrophic bacteria
using C1* uptake, measurement of oxygen consumption,
bacterial photosynthesis, or bacterial chemosynthesis. For
these determinations, water and sediment samples are usually
collected in glass or clear plastic bottles,
Microbiological research places heavy demands on
sampling techniques. The microbiological sampler has to be
sterilizable. This eliminates the use of the Nansen
-------
**»-
Maryland Department of Natural Resources craft
made available for field work.
-340-
-------
*»,„ :,
Figure 5. Research craft operated by the Environmental
Protection Agency, Annapolis Laboratory.
Courtesy of Dr. D. Lear.
-341-
-------
Figure 6a. The Nansen Bottle,
-342-
-------
6b. The van Dorn Bottle.
These sampling bottles, used by biological
and chemical oceanographers, are unsuitable
for microbiological analyses.
-343-
-------
shallow waters, from 1-5 feet., many investigators choose
the expediency of a sterile reagent bottle attached to a
string or rope (Fig. 7). Thus, there is a need for a
standardized, lightweight, hand operated, sterilizable water
sampler which permits taking a sample aseptically, of known
volume, and at a known depth in shallow waters.
Rubber bulbs of approximately 300 ml volume were
introduced as sampling vessels by 2oBell (13) and Sieburth
(10) (Fig. 8). They can be autoclaved and evacuated by
deflation and used to a depth of several hundred meters.
When non-elastic plastic material is used for a sampling
vessel, filling must be operated by an external source of
power. This is realized in the Niskin sampler (9). Upon
triggering by a messenger, two plates open in V-fashion by
spring power, thus inflating a plastic bag. The intake tube
of silicone rubber is cut by a sharp blade and, after
filling of the bag, is closed by a clamp. There are 1.2, 3
liter and 5 liter models of the sampler (Fig. 9).
In the bulb and bag samplers, contamination of the water
sample may result from breaking the glass capillary or from
cutting the rubber tube intake by a non-sterile object.
Furthermore, the samples are taken near the sampling gear,
and the hydro-wire can be heavily contaminated by passing
through the neuston layer. Escherichia coli, contaminating
the surrounding surface water of a research vessel, has been
shown by Jannasch (3) to be carried down to great depths by
the sampling gear. To prevent such contamination, a special
sampling device was constructed and tested by Jannasch and
Maddux (4). This device consists of a plastic syringe
operated by gravity. The sample intake is a tube of
silicone rubber 10 cm long which, before autoclaving, is
covered by a piece of dialysis tubing. In operation, the
sampler swings about 60 cm away from the hydro-wire and a
plunger sucks the sample into the syringe. Experiments
showed that heavy artificial contamination of the sampling
gear with a tracer organism did not result in contamination
of the sample. The sampling vessel contains no air and can
be operated at any depth. However, only 50 ml of water can
be sampled. Thus, the application of such a sampler, in its
present configuration, is limited. There is no reason why a
larger volume sampler could not be designed and developed to
operate on this principle.
Multiple sampling, i.e., many individual samples at a
given depth cannot be obtained at the present time. That
is, samples cannot be replicated except by raising and
lowering a hydrographic wire outfitted with fresh sterile
-344-
-------
Figure 7,
A sterile bottle attached to a light line or
string often is resorted to by
microbiologists sampling in very shallow
waters.
-345-
-------
Figure 8. The J-Z sampler devised by ZoBell and his
associates {ZoBell, 1946) .
-346-
-------
Figure 9. The Niskin sampler. The deep-ocean water
sampler most frequently used used by marine
microbiologists.
-347-
-------
samplers or by hanging samplers one above another, which is
not precisely the same as taking multiple, simultaneous
samples. Development of such a sampler appears
technologically feasible.
There is no sterile sediment sampler available for use
in deep waters. Cores are taken by hand in shallow waters
or by corers on a hydro-wire. Microorganisms from the
surface can be carried down on the outside of the core.
After retrieval, however, the core can be frozen in the core
liner to prevent further contamination of the inner part.
If at all possible, the material should be analyzed
immediately {Fig. 10).
A variety of grab samplers have been developed {Figs.
lla, b, c).
A. sediment sampler designed by Van Donsel and Geldreich
at the EPA Cincinnati laboratory operates in the following
manner. The sampler is closed during delivery to the
bottom. On contact with the bottom surface, it is opened,
taking the sediment sample. A messenger-activated noose
then closes the sediment sampling bag. This sampler shows
great promise for shallow coastal waters and for estuaries.
However, for deeper water, there are problems with opening
and closure of this device. Based on our experience, hand-
operated samplers are not efficient in waters of depths
greater than six feet.
We are presently developing a deep ocean environmental
unit which will permit capture arid retrieval of deep water
samples in a sterile chamber, without alteration of
temperature and pressure. Prototype testing is anticipated
in early 1974.
Collection of biota for microbiological examination
requires dredging or tonging, in the case of molluscs, and
trawling for fish. In sampling crabs, some semblance of
asepsis can be followed if blood, i.e., haemolymph samples,
are required, since sterile hypodermic syringes can be used
to withdraw the haemolymph. For neuston, or for floating
vegetation, samplers have been devised, but none has been
universally accepted. Plankton samples are collected
usually by employing nets towed through the water mass under
stud y.
Microbiological water quality monitoring has been
considered as preeminently an agar plate and colony counting
procedure, following closely the methods used for
enumerating coliforms or other indicator organisms. This
-348-
-------
Figure 10.
A coring device with a removal, autoclavable
core liner. Frequently used to collect
sediment samples for microbiological
analyses.
-349-
-------
Figure 11.
a. "Petite Ponar" sampler. (Wildlife Co.,
Saqinaw, Michigan.)
-350-
-------
II b. Grab Sampler.
-351-
-------
I Ic. Dredge sampler.
-352-
-------
very narrow perspective should be expanded and broadened so
that microbiological monitoring can evolve to encompass such
techniques as: (1) measuring metabolic activity of mixed
microbial populations by »*c uptake of labeled lactose or
sucrose, a potentially valuable way to estimate survival and
function of enterics in a stream or estuary, and
-------
Figure 12. Aseptic removal of haemolymph from a Chesapeake
Bay Blue Crab.
-354-
-------
Figure 13.
Seasonal variation in percent of HgCl - resistant
bacteria in Chesapeake Bay sediment. The
percent of total, viable, aerobic,
heterotrophic bacterial count (TVC) capable
of growth on a solid medium containing 6 ppm
HgCl was determined. Sediment samples from
Chesapeake Bay Stations B-l, 1972 (A) ; B-2,
1972 and 1973 (£,0); and EB-1, 1972 (*,O)
were plated and incubated at 25 C for 1
week. (Nelson and Colwell, 1973.)
-355-
-------
Figure 14. Seasonal variation in petroleum-degrading
bacteria in Chesapeake Bay. (Walker and
Colwell, 1973.) TVC = total viable count of
aerobic, heterotropbic bacteria; PD =
petroleum degrading microorganisms.
-356-
-------
the microfoial ecology of the estuaries and ocean regions to
interpret the data obtained, even when these indices are
correctly measured. Instead, sharp focus ought to be placed
on the bacterial species found in water and sediments and on
those distributional variations which follow sedimentation
patterns and which are correlated with tides, currents and
movements of water masses. Furthermore, the survival and
function of microorganisms, both autochthonous and
allochthonous, in the sediments and suspended sediments
should be studied, for it is in sediments and bottom sludges
that "over-wintering11 or long-term survival of
microorganisms occur and in the suspended sediments that
spatial transfer of microorganisms takes place.
Finally, we should re-shape our microbiological
approaches so that an understanding of microbial "degraders"
and "mineralizers" in the environment is achieved. With
such knowledge, these microorganisms can be put to work as
well as controlled. In situ microbial activities provide
the key to microbial ecology, not the static measurement of
parameters. The latter comprises the "uncertainty
principle** of microbiological sampling, i.e., that which is
measured is immediately the past; the present changes as the
measurement is made, hence the future, i.e., prediction from
the measurement, is uncertain, if not erroneous.
Technologically, improvements in the methods for measuring
indicator organisms and indices of pollution, such as total
coliforms, dissolved oxygen, biological oxygen demand, etc.,
may eventually result in accuracies to the nth decimal
point, but the intrinsic value of these measurements will
remain limited, for the simple reason that an index does not
tell us enough of what we need to know about a dynamic and
ever-shifting environment. Simplistic approaches to
environmental problems are inadequate, at best, and
potentially disastrous. At the present time, improvement in
sampling methods and equipment for obtaining data should be
the major, first-order priority.
ACKNOWLEDGMENT
The author wishes to acknowledge support received from
the Biological Oceanography Program, National Science
Foundation, Grant No. GA-36235, for the field studies and
development of a deep ocean environmental sampler cited in
the manuscript.
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-------
REFERENCES CITED
1. Colwell, R. R. , Lovelace, T. E., Wan, L., Kaneko, T. ,
Staley, T., Chen, P. K., and Tubiash, H.
(1973). VU>rio parahdemolvticus - isolation,
identification, classification and ecology. J.
Milk Food Technol, 36: 202-213.
2. Hobbie, J. E., Holm-Hansen, O., Packard, T. T., Porneroy,
L. R., Sheldon, R. W., Thomas, J. P., and Wiebe,
W. J. (1972) . Distribution and activity of
microorganisms in ocean water. Limnol.
Oceanogr. 17: 544-555.
3. Jannasch, H. W. (1968) , Competitive elimination of
Enterobacteriaceae from seawater. Appl.
Microbiol. 16: 1616-1618.
4. Jannasch, H. W. and Maddux, W. S. (1967). A note on
bacteriological sampling in seawater. J. Mar.
Res. 25: 185-189.
5. Jannasch, H. W, (1972). New approaches to assessment
of microbial activity in polluted waters. Inz
Water Pollution Microbiology. R. Mitchell
(ed.). Wiley, New York.
6. Kaneko, T, and Colwell, R. R. (1973). Ecology of
Vibrio parahaemglyticus in Chesapeake Bay. J.
Bacteriol. 113: 24-32.
7. Nelson, J. D., Jr. and Colwell, R. R. (1973).
Microbial ecology of mercury-resistant bacteria
in Chesapeake Bay. J. Microbial Ecology. In
Press,
8. Nelson, J. D., Jr., Blair, W., Brinckman, F. E.,
Colwell, R. R., and Iverson, W. P. (1973).
Biodegradation of phenylmercuric acetate by
mercury-resistant bacteria. Appl. Microbiol.
26: 312-326.
9. Niskin, S. J. (1962). A water sampler for
microbiological studies. Deep-Sea Res. 9: 501-
503.
10. Sieburth, J. McN. (1963), A simple form of ZoBell
bacteriological sampler for shallow water.
Limnol. Oceanogr. 8: 489-493.
-358-
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11. Sorokin, Y. I. (I960), Bacteriological water bottle.
Bull. Inst, Biol. Reservoirs. Acad. Sci,,
USSR, 6: 53-54.
12. Walker, J. D. and Colwell, R. R. <1973). Mcrobial
degradation of model petroleum at low
temperatures. J. Microbial Ecology, In Press.
13. ZoBell, Cm E. (1946). Marine Microbiology. Chronica
Botanica Co., Waltham, Mass.
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MICROBIOLOGICAL METHODS FOR MONITORING MARINE WATERS
FOR POSSIBLE HEALTH EFFECTS
V.J. Cabelli,* F. T. Brezenski,**, A. P. Dufour* and M. A. Levin*
Standardized, quantitative methods for monitorning the
marine environment for the range of microorganisms which
could cause health effects in man are the exception rather
than the rule. This statement is valid if a standardized
method is defined as one whose accuracy, precision and
sensitivity have been evaluated and found satisfactory with
marine environmental samples collected from a number of
geographically different areas. This situuation derives in
part from limited knowledge as to which health effects
indicator or pathogenic microorganisms should be quantified.
In turn, this stems from a paucity of epidemiological data
relating the incidence of illness to the density of one or
more microbial pathogens or indicators in marine waters, the
underlying sediments and the fauna and flora therein. For
example, most regulatory agencies monitor natural
recreational waters for coliform organisms to determine the
degree of fecal contamination (26); and there is no question
that these waters should be monitored for fecal pollution,
since the potential for transmission of enteric disease does
exist and operational criteria are necessary. Yet, in
Stevenson's (31)epidemiological study on recreational
waters, upper respiratory complaints were more common than
gastrointestinal disturbances. Another example concerns the
requirement for monitoring marine waters for Vibrio
Barahemqlyticus, a major causative agent of food poisoning
in Japan (15,28) and one which has been isolated from
coastal waters and shellfish in the United States (3,14,23).
Although V. parahemolyticus has been clearly established as
the etiological agent of disease outbreaks, there are
insufficient data on its relationship to pollution.
* Northeast Water Supply Research Laboratory,
Narragansett, R I.
** Surveillance and Analysis Division, Region II.
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Thus, with reference to microbiological methodology for
monitoring the marine environment, the more immediate
question concerns the organisms whose enumeration is more
likely to provide information on health hazards to
recreationists, individuals consuming marine fauna and flora
as foods and, possibly seaside populations exposed to
aerosols produced by wave action. The sources of these
pathogenic microorganisms in the marine environment can be
(a) raw and treated fecal wastes from warm-blooded animals,
(b) certain industrial effluents, such as those from food
processing, wood pulp, textile finishing, and certain
chemical plants, and (c) "dumped" materials such as dredged
materials, sludge and garbage. In addition, nutrient
pollution resulting from industrial and municipal wastes and
even thermal pollution could result in the propagation to
hazardous levels of aquatic microorganisms capable of
producing disease in man. Non-halophilie species, such as
Klebsiella sp. Pseudoraonas aeruginQsa and Aeromonas
hydropnilaf must be considered along with the halophile,
Vibrio BarafaemolYticus since estuarine as well as coastal
waters are to be monitored.
SOURCES OF PATHOGENIC MICROORGANISMS
Feca1 Was tes
Traditionally the human pathogens of concern in
untreated and treated fecal wastes are those excreted from
infected individuals or carriers, i.e., enteropathogenic
viruses, Salmonella, Shigella, enteropathogenic Eschericjria
coli, and in some iparts of the world the cholera bacillus.
However, consideration also should be given to certain
"opportunistic" pathogens such as P. aeruginosa (19), A.
hydrophila (17,32), and Klebsiella (13) which, although
infrequently present in small numbers in human and animal
feces, appear to propagate during the course of sewage
treatment and can be discharged in large numbers in
effluents which are insufficiently treated and chlorinated.
P. aeruginosa and A. hydrophila in densities of about 10*
and~10* per ml respectively, were obtained from
unchlorinated effluents at various stages of treatment at
each of nine sewage treatment plants examined. Furthermore,
the distribution of coliform types appears to change through
sewage treatment. Data collected in our laboratory suggest
that, concurrent with the decrease in E, coli levels through
the course of sewage treatment, there is at times an
increase in Enterobacter and Klebsiella densities. Table 1
and Figure l~show the decrease in P. aeruqinQsa and A.
hydrophila densities with the increase in the distance
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-------
Sampling
station
N 1
2
3
4
5
6
7
Mean
P. aeruginosa
24
24
18
27
17
7.7
4.2
recovery/ 100 ml
2 2
Fecal coliforms
1100.
1500.
1400.
490.
600.
63.
47.
Sampling stations shown in Fig. 1.
2
Number of samples: 7, collected between February and December.
Table 1. Pseudomonas and fecal coliform densities in Providence River, R.I,
-362-
-------
N5 (600) \
TU
N6
(63)
x
N7 (47)
J F
M A M J J
month
Figure 1. Seasonal and geographic variations of Aeromonas hydrophila densities
at stations N 1-7 in Providence River and upper NarragansCtt Bay.
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-------
downstream from sanitary pollution sources in the Providence
River, Rhode Island.
Industrial, £f fluents
Klebsiella is both a coliform and an "opportunistic"
pathogen which can cause upper respiratory and urinary tract
infections in man. High densities of this organism have
been recovered from waters receiving the discharges from
pulp mills (6) . The authors have found large numbers of
this organism and P. aerugingsa^ a major cause of "swimmer's
ear", in the effluents from a textile finishing plant and a
chemical plant (Table 2.) Since about 50% of the Klebsiella
isolates we have obtained from industrial effluents would be
defined as fecal coliforms, total and even fecal coliform
densities in the receiving waters could be misinterpreted as
indicating high levels of contamination with the fecal
wastes of warm-blooded animals. However, this does not
preclude the need to measure Klebgiella_densities in waters
receiving such effluents, since this organism is a human
pathogen. Furthermore, it has been observed that the
frequency of mouse pathogenic Klebsiella strains isolated
from textile finishing plant effluents was no less than that
from clinical sources (10) .
At least four aquatic microorganisms, P. aeruginosat A.
K. gneumgniae^ and V. parahemolyticus are
capable of causing disease in man and multiplying in the
aquatic environment. All four of these organisms can be
found in significant numbers in estuarine waters, depending,
of course, upon seasonal and other factors. Additional data
are needed on the relationship of the levels of these
organisms to the proximity of nutrient pollution sources.
Significance of ^Sources
The sources of human pathogens present in the estuarine
environment are summarized in Table 3, With the exception
of the enteropathogenic viruses, Salmonella and Shiqel la
species, the remaining pathogens all have extra-fecal
sources. If epidemiological data do reveal significant
health hazards associated with their presence at given
Densities, these hazards may not be "indicated" by coliforms
alone. Even with reference to those microbial pathogens
which have no extra-fecal source, several investigators,
most recently Dutka (12) , have questioned the validity of
recreational water standards based on coliforms and even
fecal coliforms. Dutka (11) cited several instances in
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Recovery/100 ml in effluents from
n____. _B Textile Chemical
Organisms Finishing Plants
Plants
Total Conforms 5.3 x 107 7.0 x 105
E. coli < 105 1.0 x 105
Klebsiella 2.7 x 107 6.0 x 105
Enterobacter 2.6 x 107 < 105
Citrobacter < io5 < 10
P. aeruginosa 1.3 x IO5 5.8 x IO6
Table 2. Distribution of coliform types in industrial effluents
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Source of the organism
Pathogen Feces Indust. Run-off Aquatic
Entoroviruses •*• - -
ShicjeLla +
A. hydroghj.^a + *
P. aeruginosa «• + + +
¥.• parahemQlyticus - *
C. botulinum - - -f *•
Feces and sewage wastes (30, 5, 24) .
Certain types of industrial effluents, i.e. pulp mills (4).
Run-off from "uncontaminated soils" (24).
Multiplication in the aquatic environment reported (5, 24, 21).
Table 3. Sources of some pathogenic bacteria found in the
marine environment
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which there was no consistent relationship of coliform or
fecal coliform levels to Salmonella densities or in which
salmonellae were isolated from waters with very low coliform
densities.
The question of the consistency of coliform-
enteropathogen relationships is the problem of extra-fecal
sources of the more commonly used indicators of fecal
pollution. These are shown in Table 4. The implication
from this table affirms what several workers in the field
have pointed out, i.e., that E. coli is the most specific
indicator of pollution from the fecal wastes of warm-blooded
animals (12). However, in monitoring marine waters for the
range of pollution sources, it would be more illuminating to
obtain data on the distributions of the "component genera"
of the coliform population. A procedure for obtaining such
data will be described later in this report.
STATUS OF MICROBIAL METHODOLOGY
The status of quantitative, microbiological methods
evaluated for use in monitoring the marine environment is
presented in Table 5. Available methods were categorized
either as a standard Method (SM), i.e. one which appears in
Standard Methods for the Examination of Hater and Wastewater
(2) or in the Bacteriologica1 Analytical Manual published by
the Division of Microbiology, Food and Drug Administration
(1) , as a method which has been designated as tentative
(TSM) in Standard Methods for the Examination of Water and
Wastewater, or as an experimental method which has received
some degree of evaluation with samples collected from the
marine environment. The term "None" is shown for those
instances in which there is no method meeting the above
criteria. Standardized enumerative methods for routine
monitoring may not be required for all the organisms listed
in Table 5. Rather, these organisms should be considered in
determining which microbiological measurements should be
made to obtain the data needed to derive criteria and
standards. The state of the art with regard to methods for
monitoring the marine environment is overstated in Table 5.
Thus, in several instances where there are "Standard
Methods'* for quantitating microbial pathogens or indicators,
such as P. aeruqinosa, total coliforms, fecal coliforms, and
fecal streptococci, the methods have not been sufficiently
evaluated with samples from marine waters. For example, the
method for P. aeruqinosa, as given in the current edition of
"Standard Methods", has been evaluated and found
unsatisfactory for use with marine waters. It is being
replaced with a membrane filter (MF) method (24) .
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Bacterial Indicator
Coliforms
E.coli
Klebsiella
Enterobacter
Citrobacter
Fecal coliforms
Fecal streptococci
source of organism
Indust. Run-off Aquatic
* or -
(*)
(*)
Tndust. - certain types of industrial effluents
Run-off from "uncontaminated soils" (9, 16, 25).
Multiplication in the aquatic environment reported (30, 1, 4, 20)
Portion of coliform population which may multiply.
Table a. Sources other than feces for bacteria used as indicators
of fecal pollution
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Organism
Water
Collection from
Sediment
Fauna
Enters viruses
Salmonella
-V
Shigella
P. aeruginosa
A. hydrophila
Coliforms
E. coli
Klebsiella sp.
Enterobacter
Citrobacter
Fecal coliforms
Fecal streptococci
V. parahemolyticus
C. botulinum
C. perfringens
Exp.
None1
None
TSM1
None1
SM1
None
None1
None
None
SM1
SM1
Exp.1
None
None
None
None
None
SM2
None
None
None
None
SM2
SM2
SM2
SM2
SM2
None
None
None
None
None
SM2
None
None
None
None
SM2
SM2
SM2
SM2
SM2
SM "Standard Methods for the Examination of Water and Wastewater" ( 2) , FDA
method
TSM Tentative "Standard Method".
Exp. Evaluated experimental method.
None No method evaluated with marine samples.
Experimental procedure developed or under development for marinr recreational
waters .
2
FDA method assumes isolation methods can be used in MPN method for quantifi-
cation.
Table 5. Quantitative, microbiological methods evaluated for use in the
monitoring of marine environment
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Unpublished data from several laboratories concerned
with the examination of marine water samples have indicated
that the mEndo and roFC procedures for the enumeration of
total and fecal coliforms, respectively, underestimate these
populations. Samples of the data collected in our
laboratory which support this contention are shown in Table
6, which compares the recovery of total colifornts by the mC
and mEndo procedures and Table 7 which compares mC to the
mEndo and "standard" MPN methods for the recovery of total
coliforms. Tables 8 and 9 provide the same comparisons for
fecal coliform recoveries obtained by the mC, mFC and MPN
procedures. With 47 estuarine water samples collected' from
Raritan Bay, fecal coliform recoveries by the MPN method
were about twice those by the mFC procedure, on the average.
The KF method, which some investigators recommend as
providing the best estimate of fecal streptococci in fresh
water, was found to markedly underestimate the densities of
this group of bacteria in marine waters in the northeast
United States. This can be seem from Table 10 which
compares the densities of fecal streptococci by the KF
method to those by the mSD procedure developed in bur
laboratory (25) .
There remains some question whether the microbial
methodology for use with sediment and fauna! samples as
indicated in Table 5 will, in fact, provide quantitative
estimates of the accuracy and precision required. Most of
these methods are or can be used as multiple tube procedures
which yield most probable number (MPN) estimates, with the
attendant problems of precision or reproducibility,
Furthermore, with faunal, sediment and even turbid water
samples, it is not certain whether single cells or particles
composed of one or more viable organisms are being counted.
ON-GOING METHODS RESEARCH
Studies towards the development of microbiological
methods specifically applicable to monitoring the marine
environment for health effects microorganisms are on-going
at a few laboratories. Those organisms for which methods
are being developed at our own laboratory are indicated in
Table 5 with the superscript "1". The major thrust of our
effort has been to obtain accurate and precise methods for
use in epidemiological-microbiological trials being
conducted to establish criteria for recreational water. It
is beyond the scope of this report to describe all the
methods which have been developed or are undergoing
development. However, some examples are given to illustrate
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Sample
source
Providence
River
Boston
Harbor
Recovery per
mC
2000
5300
1500
120,000
290
100
14
100 ml by
mEndo
1100
3200
550
90,000
140
70
8
Table 6. Comparison of confirmed coliform recoveries from
marine waters by mC and mEndo procedures
Relationship
No. of samples
mC > by 5%
mC < by 5%
< 5% diff.
Comparison
mEndo
33
25
7
1
of recoveries by mC to
MPN
20
14
6
0
Confirmed by picking colonies to lactose brotu.
Table 7. Confirmed coliform recoveries from New York Bay by
the mC procedure as compared to those by the mEndo
and MPN methods
-371-
-------
Sample
source
Recovery per 100 ml by
fecal mC mFC
Providence
River
2200
7700
280
1000
Boston
Harbor
New York
50
2100
6000
1100
1000
Table 8. Comparison of confirmed fecal coliform recoveries from
marine waters by fecal mC and mFC procedures
Relationship
No. of samples
fecal mC > by 5%
fecal mC < by 5%
< 5% diff.
Comparison
mFC
16
12
3
1
of recoveries by mC to
EC MPN
16
8
6
2
Table 9. Confirmed fecal coliform recoveries from New York Bay by
the fecal mC procedure as compared to those by the mFC
and EC MPN methods
-372-
-------
Conf. Rec.
mSD1
404
243
150
47
70
230
153
46
310
10
/100 ml
KF
8
6
6
2
4
17
12
4
26
1
mSD/KF
50.
40
25.
24.
18.
14
13
12
12
11.
Median
Conf. Rec.
mSD
46
7
32
38
14
34
121
25
13
mSD/KF ratio 11.
/100 ml
KF
5
1
5
8
4
13
50
43
61
mSD/KF
9.2
7.
6.4
4.8
3.5
2.6
2.4
.58
.21
1 In excess of 95% of the isolates identified as S_. fecalis, S_. faecium,
S. durans or S. zymogenes.
Table 10. Comparison of "fecal streptococci" densities in New York
City beaches by the KF and mSD procedures
-373-
-------
Organism
Total co li forms
Fecal coliforms
E. coli
Klebsiella
Enterobacter
Citrobacter
P. aeruginosa
Recovery per 100 ml at
Coney Island Rockaways
(19th St.) (67th St.)
2490 58
453 28
403 24
356 12
1678 15
67 2
45 7.6
Table 11. Distribution of coliform types at New York City beaches
Organisms
Total coliforms
Fecal coliforms
E. coli
Klebsiella
Enterobacter
Citrobacter
P . aeruginos a
Recovery/ 100 ml in water at
Site 1 Site 2 Site
2.1 x 104 3.1 x 106 5.6
3.1 x 106 2.8
2.1 x 104 < 105 <
< 103 3.1 x 106 2.8
< 103 < 105 2.8
< 103 < 105 <
35 41
3
x 104
x 104
103
x 104
x 104
103
8
Table 12. Distribution of coliform types at fresh water sites
-374-
-------
the direction of the research and the types of information
which can be obtained thereby.
Coliforms
A membrane filter procedure for the quantification of
coliforms in marine waters has been developed (10). In the
development of the method (mC) certain prerequisites were
considered: (a) the method should permit the examination of
large quantities of water and provide more precise estimates
than can be obtained with MPN procedures; (b) it should
allow the option of measuring total coliforms, fecal
coliforms and component genera of the coliform population;
(c) an oxidase test should be included to exclude Aeromonas
hydrophila, since some strains of this organism are lactose
positive and aerogenic; and (d) except when confirmation is
required, "picking" of colonies for biochemical or
serological identification should be minimized. The
achievement of the last prerequisite has been furthered by
use of in situ substrate tests in which the membrane filter,
following incubation on a selective-differential medium, is
sequentially transferred to one or more substrates
containing appropriate indicator systems. It can be seen
that in the mC procedure, as shown in Figure 2, colonies are
picked for identification. However, encouraging results
have been obtained by following the in situ urease test for
Klebsiella with an in situ oxidase test to eliminate A.
hydrophila and then an i.n situ indole test, such as
suggested by Delaney (8) for differentiating citrobacter and
Enterobacter from E. coli. The type of information which
can be obtained from the mC procedure is shown in Table 11.
The E. coli to Enterobacter ratios indicate that the sources
of pollution reaching the two beaches are different. In
this instance the fecal coliform-total coliform ratios would
have provided the same insights. Such would not be the case
in waters receiving industrial effluents containing large
numbers of Klebsiella. The occurence of water samples in
which Enterobacter, Klebsiella. or E. coli constitute the
major component of the coliform population is shown in Table
12.
legal Streptococci
A flow diagram for the conduct of the mSD method tor
fecal streptococci is shown in Figure 3. In this procedure,
incubation at 41 C on a medium highly selective for Group D,
streptocci (to date, neither Streptoccus salivarius,s.
Sitis, s. e
-------
MC
(count and mark typical colonies)-
in situ urease test
(pick up to 15 colonies)
confirmation in
lactose broth
I
no gas
(discard)
gas
Nutrient agar plate
oxidase test
CTC-
discard
PCTC-
H S
__2
!
Citrate
"1
Klebsie]1a
Citrobacter
Enterobacter
E.coli
•a
b_
e.
d
Presumptive totalcoliforms
Urease negative coliforms (UNC)
Typical, urease positive colonies, rarely do not confirm
Partially Confirmed Total Coliforms = UHC Total'picled + Klebsiella
Confirmed Total Coliforms = UHC °*id nf§».1fc^> POS' + Klebsiella
Total picked
— for calculating coliform densities
Figure 2. Flow diagram for total coiiforms and distribution of coliform types,
Fecal coliform estimate can be obtained by performance of EC test
from lactose positive confirmatory tubes.
-376-
-------
mSD
in situ esculin test
I
(discard)
Fecal streptococci
! (
I
I
I
Confirmation
I
; , 1
Pick colonies to BHI
Bile-Esculin Agar
Growth plus
black ppt.
in medium
Identification
by
Fluorescent Antibody
Oth
ler
(discard)
BHI - Brain Heart Infusion broth.
Figure 3. Flow diagram of mSD procedure for fecal streptococci.
-377-
-------
Aeronomas bydrophila
The objective of eliminating the picking of colonies for
identification has not been achieved with this procedure.
None of many compounds tested were found to completely
inhibit coliforms and other organisms present in marine
waters while allowing quantitative recovery of A.
Incubation of the filters at 37 C, an in situ
oxidase test, and transfer of typical colonies to double
sugar iron agar (containing mannitol as the "major" sugar,
inositol as the "minor" sugar and no Nad) permits the
differentiation of Aeromgnas hvdrophila from coliforms, non-
fermentative pseudomonads and achromobacters, salt requiring
vibrios, Aeromonas salmonicida and A. shigelloides.
NEEDED METHODOLOGY
As evaluated, a reasonably facile and minimally
destructive method is needed for dispersing roulticell
particles into single cell units and separating the
microorganisms from sediment particles to which they adhere.
The resulting microbial suspensions then could be assayed by
conventional plating or MF procedures. In addition, an
assay for the spores of C^ostgidium perfriggens could
provide a sensitive means of monitoring the dispersal and
persistence of sewage sludge and, possibly, dredge spoils
"dumped" into marine waters. Methods are available for the
enumeration of marine sediments, especially if the
aforementioned procedure is developed.
It has been shown by Miyamoto et aj.. (27) with Vibrio
parahemolvticus, by Kauter and associates (22) with
Clostridium botulinum, and in our own laboratory with
Klebsiella (23) , A. hvdrophila (5) , and V. parahemolvticus
that the numbers of environmental isolates which are
"pathogenic", as defined by mouse pathogenicity or
toxigenicity, generally constitute minor portions of the
populations, since some of these organisms are
opportunists, it may be that an "unstressed" mouse is not
the animal of choice to model pathogenicity for
"susceptible" humans. More likely, only a portion of the
environmental .isolates are, in fact, pathogenic; and the
enumerative methods may have to be modified so that they are
both selective and differential for the "pathogenic"
biotypes within these species.
DISCUSSION AND CONCLUSIONS
The state of the art concerning methodology for the
quantification of health effects indicators in the marine
-378-
-------
mA
in situ oxidase test
i
typical, ox +
ADS IA
others
ox neg. or
atypical ox. +
Alk. slant, Acid butt
no. H2S, oxidase pos.
Aeromonas hydrophila
I
I
Confirmation
Dextrose broth with gas tube
I
1
Perm
gas
I
Perm Oxid Neg
no
gas
(dis card)
Perm - acid inside and outside Durham tube.
Oxid - acid outside Durham tube.
Neg - alkaline throughout.
ADSIA - trypticase, 1.5%; yeast extract, 0.3%; polypeptone, 0.5%;
inositol, 1.0%; mannitol, 0.15%; ferric sulfate, 0.02%;
sodium thiosulfate, 0.03%; phenol red, 0.003%; agar, 1.2%.
Sterilized and prepared, dispensed, inoculated as described
for kligler iron agar.
Figure 4. Flow diagram of mA procedure for aermonas.
-379-
-------
environment must be upgraded. The situation stems from
several factors:
1. a paucity of epidemiological data relating health
hazards to individuals associating with the marine
environment to some indicator or indicators of the
water quality.
2. insufficient evaluation of existing methodology when
applied specifically to the enumeration of the
organism in the marine environment.
3. the willingness, in the past, to rely too heavily on
coliforms and even fecal coliforms as the sole
indicators of water quality.
Efforts are being made to correct this situation. A
long-range research program to develop criteria for marine
recreational waters is in progress. Central to this effort
are epidemiological-raicrobiological studies to relate the
incidence of illness among swimmers to some health effects
indicator(s) of water quality at the beaches. A series of
trials to evaluate the epidemiological and available
microbiological methodology have been completed. In
addition, these trials will produce a limited amount of data
- the first such data to become available since Stevenson's
(31) studies - comparing the rates of illness by type and
severity among swimmers and non-swimming controls. The
study was conducted at two beaches that were markedly
different in their pollution levels according to existing
criteria. Methods such as those described herein were used
to enumerate a number of possible microbial indicators of
water quality. However, until a sufficient quantity of such
epidemiological-microbiological data becomes available, we
must rely on existing criteria, standards and monitoring
procedures as being the best available.
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-------
REFERENCES
1. Bacteriological Analytical Manual for Foods, 3 ed.,
1973. FDA, Dept. Health Education Welfare,
Washington, D. c.
2. Standard Methods for Examination of Water and
Wastewater, 13 ed., 1971. American Public Health
Association.
3. fiaross, J., and J. Liston. 1970. Occurrence of Vibrio
parahemolyticus and Related Hemolytic Vibrios in
Marine Environments of Washington State, Appl.
Microbiol. 20:179-186.
4. Bordner, R. H., and B. J. Carroll, seminar on the
Significance of Fecal coliforra in Industrial Wastes.
Natl. Field Inv. Cent. Denver, Col. July 1972.
5. Cabelli, V. J. 1973. Occurrence of Aeromonads in
Recreational Water. American Society for
Microbiology Proceedings, p. 32.
6. Blosser, R. O. 1971. Experience with Indicator
Organism Tests in Determining the Bacteriological
Quality of Pulp and Paper Mill Effluents and Their
Receiving Waters. National council of the Paper
Industry for Air and Stream Improvement, Technical
Bulletin No. 244, pp. 56.
7. Deaner, D. A. and K. D. Kerri. 1969. Regrowth of Fecal
Coliforms, J. Am. Water Works Assoc. 60.:465-468.
8. Delaney, J. E., J. A. McCarthy, R. J. Grasso. 1962.
Measurement of E. coli Type I by the Membrane
Filter. Water and Sewage Works 1JD9-389.
9. Duncan, D. W. and W. E. Razzell. 1972. Klebsiella
Biotypes Among Coliforms Isolated from Forest
Environments and Farm Produce. Appl. Microbiol.
24:933-938.
10. Dufour, A. P., V. J. Cabelli, and M. A. Levin. 1973.
Occurrence of Klebsiella species in Wastes from a
Textile Finishing Plant. Abstracts of the Annual
Meeting, American Society for Microbiology, p. 3.
11. Dutka, B. J., J. B. Bell, P. Collins and J. Popplow.
1969. A Bacteriological Study of the Rainy River,
-381-
-------
Conducted for the Advisory Board on Water Pollution,
Rainy River, and Lake of the Woods, International
Joint Commission. Department of National Health and
Welfare (Canada). Division of Public Health
Engineering, Manuscript Report NO. KR-69-1, pp. 42.
12. Dutka, B. J. 1973. Coliforms are an Inadequate Index
of Water Quality. J. Environmental Health 36:39.
13. Eickhoff, T. C. 1972. Kjebsiella pneumoniae Infection:
A Review with Reference to the Water-Borne
Epidemiologic Significance of K. pneumoniae Present
in the Natural Environment. National Council of the
Paper Industry for Air and Stream Improvement,
Technical Bulletin No. 254, pp. 24.
14. Fishbein, M., I. J. Mehlman and J. Pitcher. 1970.
Isolation of Vibrio parahemplyticus from the
Processed Meat of Chesapeake Bay Blue Crabs. Appl.
Microbiol. 20:176-178.
15. Fujino, T., Y. Ohuno, D. Nakada, A* Aoyama, K. Fukae, T.
Mukae and T. Ueho. 1956. On the Bacteriological
Examination of Shirasu-Food Poisoning. Med. J.
Osaka Univ. 4:299-304.
16. Geldreich, E. E., C. B. Huff, R. H. Bordner, P. W.
Kabler and A. F. Clark. 1962. The Fecal Coli-
aerogenes Flora of Soils from Various Gographical
Areas. J. Appl. Bacteriol. 25:87-93.
17. Gilardi, G. L. 1967. Morphological and Biochemical
Characteristics of Aergmonas punctata (hydrophila,
1igui faciens) Isolated from Human Sources. Appl.
Microbiol. i5:417-421.
18. Harmon, S. M., D. A. Lautter and J. T. Peeler. 1971.
Comparison of Media for the Enumeration of
Clostridium perfrinqens. Appl. Microbiol. 2.1:922-
927.
19. Hoadley, A. W. 1968. On the Significance of
Pseudomonas aerugingsa in Surface Waters. J. New
Eng. W,W.A. 82:99-11.
20. Hulka, S. C., S. R. Keen and E. JM. Davis. 1973.
Sediment Coliform Populations and Post Chlorination
Behavior of Wastewater Bacteria. Water and Sewage
Works, Oct. pp. 79-81.
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21. Kaneko, T. and R. R. Colwell. 1973. Ecology of Vibrio
par ah envoi vticus in Chesapeake Bay. J. Bact. 113:24-
32.
22. Kautter, D. A., S* A. Harmon, R. K. Lynt, Jr. and T.
Lilly, Jr. 1966. Antagonistic Effect on
Clostridium bot ul inure Type E by organisms Resembling
it. Appl. Microbiol. ^1: 616-6 22.
23. Krantz, G. H., R. R. Colwell and E. Lovelace. 1969.
Vibrio par ahemol vticus from the Blue Crab,
Callinectes sapidus in Chesapeake Bay. Science
164:1286-1287.
24. Levin, M. A. and Ca belli, y. J. 1972. Membrane Filter
Technique for Enumeration of Pseudomonas aeruginosa.
Appl. Microbiol. 24:864-870.
25. Levin, M. A., Ca belli, V. J. and Dufour, A. P. 1973.
Quantitation of Fecal Streptococci in the Marine
Environment. American society for Microbiology
Proceedings, p. 37.
26. Mechalas, B. J., Hekiman, X. K. , Schinagi, L. A. and
Dudley, R. H. 1972. Water Quality Criteria Data
Book, Volume 4, W.P.C.R.S., Series 18040 DA20472.
*
27. Miyamoto, Y., T. Kato, Y. O'Bara, S. Akilama, K.
Takizawa and S. Yamai. 1969. Hemolytic
Characteristic of Vibrio parapfaemol vticus; Its
Close Correlation with Human Pathogenicity. J.
Bacteriol. 100:1147-1149.
28. Sakazaki, R. S. , Lawanami and H. Fukumi. 1963. Studies
on the Enter opathogenic. Facultatively Halophilic
Bacterium, vibrio parahemolvticus . 1.
Morphological, Biochemical and serological
Properties and its Taxonomical position. Jap. J.
Med. Sec. and Biol._16 161-1 88.
29. Rosebury, Theodor. 1962. Microorganisms Indigenous to
Man. McGraw-Hill Book company.
30. Shuval, H* I., Judith Cohen and Robert Kolodney. 1973.
Regrowth of coliforms and Fecal coliforms in
Chlorinated wastewater Effluents. Water Res. 7:537-
546.
-383-
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31. Stevenson, A. H. 1953. Studies of Bathing Water
Quality and Health. J. Am. Public Health Assn.
43:259.
32. Von Gravenitz, A. and A. Mensch. 1968. The Genus
Aeromonas in Human Bacteriology. Report of 30 Cases
and Review of the Literature* New Eng. J. Med.
278:245-219.
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A SURVEY OF METHODS FOR MONITORING ECOLOGICALLY IMPORTANT
MICROORGANISMS IN THE MARINE ENVIRONMENT
J. P. Buck*
Monitoring implies keeping track of something that warns
or instructs relative to a specific purpose: i.e., how much
of what is where. The time element, however, compounds the
problem. Continuous monitoring may provide a rate of change
measurement but does it say what is happening? And how
often and for how long is monitoring carried out? some
additional definitive insight is offered by a task group of
the National Academy of Sciences (1971). In speaking to the
question of the effects of pollutants on marine organisms,
and depending on the level of interest (i.e., from cellular
level to community dynamics), a span of minutes to decades
may be appropriate. Thus between 10 minutes and 10 years, a
5 X 10s span in differences in various monitoring
procedures may exist.
A more significant concern may be that of what
constitutes an "ecologically important" microorganism. If
those organisms which have been monitored on the basis of
ability to detect, convenience, or tradition are truly
ecologically important, surely others have been treated as
ecologically unimportant. More likely there are levels of
importance. Many studies have shown the distribution of a
wide variety of microorganisms in marine and estuarine
waters. The question is, what are they doing?
Consider a bacterium in an estuary which supports a
shellfish population; perhaps a species of Flavobacterium,
Achromobacter, or some other common nonindicator or
nonpatfaogenic genus. With no other information, we probably
would not seek to monitor the presence of these bacteria and
could consider them ecologically unimportant. However,
shellfish do accumulate bacteria (e.g., Cabelli and
*Marine sciences Institute,
Marine Research Laboratory
University of Connecticut,
Noank, CT 06340
-385-
-------
Hefferman, 1970) and thus assume an ecological significance
in terms of mollusc nutrition. Further, newer information
indicates that some strains of "common" bacteria can
transform environmentally critical compounds such as PCBs
(Ahmed and Focht, 1973a,b) and chlorinated hydrocarbons
(Leshniowsky et al., 1970; Pfaender and Alexander, 1972;
Sethunathan and Yoshida, 1973). In this context, certain
"unimportant" genera now do assume an ecological importance.
Going one more step, the observation of biomagnification in
Aergbacter aerogenes and Bacillus subtilis is significant
(Johnson and Kennedy, 1973) . Here are two very common,
"low-profile" bacteria in which cellular residue
magnification factors were up to 4,300 fold compared with
water. For subsequent food chain events we now have very
ecologically important microorganisms. On microbiological
grounds, the shellfish may be safe but unless chemical
standards are utilized, high pesticide levels will be
present in marketable products as a result of microbial
activity.
To be sure, much of these data are derived from in vitro
studies and therein lies another problem. What about in
situ activities-what is the level of correlation between the
two or, as above, they are there-what are they doing?
Considerable attention has been directed to potential
ecological catastrophy, e.g., Torrey Canyon, Santa Barabara
oil spill, massive offshore dumping and the like. There is
no doubt that these are major events but should we not be
concerned also, on a day to day basis, with the endemic,
chronic, almost "accepted" addition of materials to the
aquatic environment? Included here are local sources of
effluents, marinas (Mack and D'ltri, 1973), fuel depots,
near-shore dumping (Biggs, 1968), small manufacturing plants
and so forth. For example, some monitoring of particular
areas has shown the presence of nontraditional
microorganisms of possible use as indicators of hydrocarbons
(Turner and Ahearn, 1970) and human sewage (Cook, 1970).
From studies of these sites seeking specific types of
organisms, we may be able to know what to look for in
catastrophies. One point is that, while we cannot predict a
major oil spill, we do know the siting of sewage outfalls,
power plants, refineries, chemical plants, etc. in advance
of construction and can monitor these if we know what to
look for. Moreover, it can be done in an atomosphere of
logic over a long "before" period of time rather than in a
panic following an "after" ecological tragedy such as a
major oil spill. Baseline studies, for as long as possible,
cannot be overemphasized.
-386-
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Any consideration of methods for assessing anything must
presume that the techniques being used (a) do detect the
organisms sought and (b) that the organisms, once detected,
do indicate what we assume they do. There are, of course,
"standard methods" for the examination of water and
wastewater (APHA et al. , 1971; EPA, 1971) and dairy products
(Walter, 1967) and "recommended methods11 for the
consideration of foods (Sharf, 1966) and seawater and
shellfish {APHA, 1970). These are desirable in the sense
that at least data from various samples and areas are
comparable.
The choice of microbiological methods to be used for a
particular circumstance will have to be based on experience.
Obviously no research group can monitor all microorganisms
at all times; options will have to be exercised. The state-
of-the-art at present is such that each study, as an
individual case, should consist of certain basic procedures
and yet allow for experimentation with new methods. We
know, for example, that marine bacteria are essentially
psychrophilic and quite susceptible to relatively small
temperature change (Morita, 1966). In an estuary, these
populations will be mixed with eurythermal types from
freshwater or soil sources. One should also consider the
salinity effect; to what extent are marine bacteria tolerant
of inorganic dilution and how do river or terrestrial
bacteria survive a seawater gradient? To examine adequately
the ecological role of any of these organisms will require a
variety of methods and cultural manipulations and demands
both field and laboratory integration. To be sure, the
standard laboratory plating techniques can be be employed
with a battery of selective and differential media and
temperatures of incubation. But this is rather of an all or
nothing result. There are, however, some interesting
variations on the common plate count theme. Agar "dip-
slides" |Mara, 1972) can be used to approximate total and
coliform counts for routine monitoring. A double medium
technique can be used to study stressed cell injury. One
medium allows growth of uninjured cells only while a second
shows growth of both uninjured and injured cells. Using
lack of colony formation on the former as an index of
unrepaired injury, recovery can be detected on the latter
medium. This technique has been used for studying lethal
and inhibitory effects of sodium chloride on thermally
stressed bacteria (Erwin and Haight, 1973). Surely, wider
application to environmental problems can be imagined.
A more sophisticated supplement to plate counts is
offered by the use of continuous culture or chemostat
techniques whereby substrate level, temperature, salinity,
-387-
-------
pH, Eh, etc. can be managed on a sliding scale to attempt to
simulate field observations on special environmental
additives in question. Within this laboratory scheme,
microscopy, sophisticated biochemical analyses, and other
techniques can be superimposed. Emphasis must be placed on
the consideration of each situation as unique. The exact
choices of monitoring methods and alterations therein are
dependent on geographic location of the site, uses (real or
potential) of the water, existing hydrographic conditions,
and degree of anticipated environmental stress. Because of
this, our degree of acceptance of standard methods for the
examination of ecologically important microorganisms in the
marine environment is questionable.
In any monitoring situation, we look normally at a
particular core of organisms, both micro and macro. Among
the former are the bacteria and phytoplankton. Recent
studies on phytoplankton indicate that, in some areas, the
dominant organisms are the very small one (Malone, 1971;
NOAA, 1972; Reynolds, 1973) and easily missed by traditional
techniques. Considering the indicator potential of
microalgae in general in ecological assessments of thermal
(e.g., Patrick, 1969; Lanza and Cairns, 1972) and chemical
(Carpenter et al., 1972; Brook and Baker, 1971; Hamilton et
al., 1970) additions, we may well be overlooking the major
portion of the plankton biomass.
In our own laboratory, we have studied the effects of
thermal addition (Buck and Rankin, 1972; Foerster et al.,
197ft), treated sewage effluent (Pilcher and Buck, 1973), and
dredge spoils (Bireley, thesis in preparation) on,
respectively, a river, an estuary, and Long Island Sound.
Using the "usual" parameters of total bacterial counts and
numbers of total and fecal coliforms as well as counts of
phytoplankton and certain physiological groups of bacteria
(proteolytic, amylolytic, lipolytic), we have observed no
drastic quantitative or qualitative impacts on the
environment other than very local ones. This is, of course,
pleasing but cannot be regarded necessarily as a true
synopsis of the ecological impact of these additions on the
total ecosystem. We should not extrapolate lack of effect
on a few to many. Some in situ studies of nitrifying
bacteria under thermal enrichment (Pierce and White, 1973;
Schwert and White, 1973) have shown an apparent correlation
between temperature and nitrification. In another study, a
modified BOD test was used to correlate activity of
ammonium-oxidizing bacteria with temperature (Roch and
Kaffka, 1972). Surely the types of bacteria involved in
these and other transformations {Burchard, 1971; Colwell,
1972) are ecologically important yet rarely included in
-388-
-------
routine monitoring studies; usually, a very small inventory
of the total microbial population is considered. Perhaps
representatives of the classical producing, consuming, and
transforming members of the ecosystem deserve attention if
such a cross section could be subject to mutual agreement
and if one takes care in assigning labels , (Bunt, 1968).
The aquatic environment consists of mixed cultures of a
highly competitive nature and biochemical diversity cited
above. The addition of a pollutant causes change in the
external environment and may lead to stimulation or
inhibition of the indigenous populations. The effects may
be obvious, in terms of blooms or mass mortalities, or more
subtle unless carefully monitored. This could well extend
to the study of specific pollutants as chemotactic agents
(Adler et al. , 1973; Chet et al., 1973) and their influence
on biotransformation.
General methods and concepts in organic mineralization
continue to be advanced (Hood, 1970; Poraeroy, 1970) and will
affect changes in our approach to the microbial
transformation of carbon compounds. Of parallel interest
are the rates of uptake and release of ecologically
significant phosphorous-containing molecules (Johannes,
1964; Satotni and Pomeroy, 1965; Abbott, 1969). Similarly,
studies on the rate of transformation of nitrogen compounds
(Goering and Dugdale, 1966; Yoshida and Kimata, 1969) are
necessary. The ability to detect the various types of
nitrogen bacteria involved (i.e., Finstein, 1968; Kawai and
Sugahara, 1971) as well as activity under different
conditions (Carlucci and McNally, 1969) form a mandatory
base for continued work. We are only beginning to detect
competition for such common compounds as urea (Remsen et
ajl., 1972) and ami no acids (Burnison and Morita, 1973). In
addition, the sulfur cycle (Kellogg et al., 1972) represents
a complex series of chemical interactions. Only recently
have certain groups of participant bacteria been studied
closely (Truper et al., .1969; Tuttle and Jannasch, 1972,
1973) and specific econiches continue to be revealed
(Fenchel and Riedl, 1970). some problems in methods for
enumeration have appeared (Mara and Williams, 1970).
Given these few examples 01 classical element cycling
concepts, it must be admitted that our sum knowledge of the
intricacies involved is limited. And we know even less of
the effects of various pollutants on the activities of the
organisms concerned. For basic studies, the enrichment or
selective culture technique (Schlegel and Jannasch, 1967) is
a powerful tool for getting at specific metabolic types of
organisms in the entire biomass. While a very old
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technique, a wide variety of individual methods are
available (Aaronson, 1970; Rodina, 1970). For example, the
use of enrichment culture in silica gel medium has been used
successfully for the detection of petroleumlytic bacteria in
seawater (Seki, 1973) . Modification and sophistication to
an even greater extent should prove valuable in applying
these techniques to particular problems. Rate studies are
emphasized in connection with all of these. An occurrence
is one thing but the temporal aspect is really what we are
after - how fast is something being turned over and to what
extent is it being influenced by an environmental additive?
Mention should be made also of the effects of heavy
metals on microorganisms. Some examples have been shown for
nickel (Cobet et al., 1970), copper (Erickson et al. , 1970),
lead (Tornabene and Edwards, 1972), and mercury (Nelson et
al., 1973). what effect these and other elements have on
ecologically important processes is largely unknown but
these should be worked into monitoring programs if
identified as present as additives. Analytical methods for
heavy metal detection are improving; however, the laboratory
culture of bacteria in ecologically present quantities of
some heavy metals requires very careful manipulation in
terms of inorganic contamination of synthetic media.
The EPA Ecological Research Series of publications are
valuable sources of methodology for assessing pollution
effects on fish and other commercially important species.
While much is known of micrpbial disease in fish (Sinderman,
1966) and shellfish (Sprague, 1971), little information is
available on pollution effects on the etiological agents
although some data have appeared (e.g., Pippy and Hare,
1969) . Relatively "new11 diseases of marine life (Haskin et
al., 1966; Vendros et al., 1971) and even man (Zeligman,
1972; Jolly and SeaburyT 1972) demand revised methodology
for detecting the causative organisms and the effects of
pollutants on them. Organic and inorganic nutrients and
thermal additions would seem to be among the obvious
environmental stimulants to be considered in monitoring
studies connected with the occurrence of specific pathogens.
Here again, refinement of an existing selective culture
seems an appropriate place to start.
Often overlooked is the biochemical diversity of
microorganisms. In most studies, a marine pseudomonad would
not attract special attention yet one such isolate (Buck et
al., 1963) is capable of deaminating with subsequent
production of ammonia and ichthyotoxicity as well as
precipitating calcium carbonate (Greenfield, 1963). These
are all ecologically important activities if occurring in
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situ, close scrutiny may well show such biological
diversity in many bacteria.
Even recognizing quantitative and qualitative variations
(Buck, 1974), numbers of microbes (bacteria, phytoplankton)
will always be recorded and the debate over their
significance will continue. Certainly detection of numbers
is more meaningful when some qualitative data accompany them
and most useful when some notion of activity or indicator
role is clearly assigned. For example, Palmer (1969)
suggest that certain genera and species of microalgae can be
used to rate water samples for organic pollution levels.
Species diversity among intertidal macroalgae shows similar
responses (Borowitzka, 1972).
A lesson from the microbiological space program could be
useful (Merek and Oyama, 1968); i.e., assume little and
apply a broad brush approach at first, then gradually
narrowing. A wide spectrum of nutritional types could be
sought wherein an environmental additive in question may
influence metabolic activity. Also, w
-------
felt in environmental additions. One current interest in
eutrophication involves the role of bacteria in producing CO
for algal respiration rather than a phosphate and/or nitrate
stimulation (Kuentzel, 1969; Lange, 1970). If indeed such a
relationship exists, then appropriate monitoring of
particular environments, in conjunction with laboratory
studies (O'Brien, 1972), will clarify the hypothesis.
We must think too in terms of future use of marine
environments for culture of economically valuable animals
(or plants) or their products (George Washington University,
1971a, b). The oceans do have size and dilution factors in
their favor; however, confinement of small areas for rearing
of single species may yield new problems in the form of
epizootics and epiphytotics. Continuous monitoring of eggs,
larvae, and adults (Johnson et al. , 1971) will be required
for possible recognition of microbial-induced pathological
conditions and other effects (Webber, 1973).
Some years ago, pollution equated with sewage. Today,
of course, environmental additives are many and varied. In
some cases, technology is wanting for critical
identification of certain exotic, compounds and some
obviously persist for long periods and are widespread
(Harvey et al., 1973). we know little of the ultimate fate
of these materials or how long their chemical uniqueness
will remain a mystery to microorganisms. In fact, even
"usual" organic matter is somewhat refractory on the ocean
floor (Jannasch et al., 1972; Jannasch and Wirsen, 1973).
Perhaps more interest should be paid to macroorganisms
in aquatic habitats. Monitoring of fouling (McNulty, 1970;
Ingnatiades and Becacos-Kontos, 1970) and benthic (Reish,
197f; Goodnight, 1973; McDaniel, 1973) organisms have shown
some useful applications (and problems) associated with
their determination in various situations. The American
oyster responds to various levels of PCBs (Lowe et al.,
1972) and heavy metals (Calabrese et al., 1973).
Biomagnification appears again in the examination of fish
bile which may concentrate certain compounds over 1000 fold
from water levels (Lech et al. , 1973) .
Any attempt to detail all available methods for
microbiological monitoring would be hopelessly naive.
Fortunately, we are updated periodically on technology
(e.g.. Water Pollution Control Federation, 1973) and
reminded occasionally of our task (e.g., Tarzwell, 1962;
Wilhm and Dorris, 1968). Overviews of monitoring
instrumentation do appear (Ciaccio, 1971; Ballinger, 1972)
and, recently, a most valuable volume became available on
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methodology in microbial ecology (Rosswall, 1973). General
(e.g., Mackenthun, 1966; McKee, 1967; Ludwig and Storrs,
1970) and specific (Kneiys, 1971; Norris et al. , 1973;
Hlavka, 1973) approaches to particualr areas can be
consulted also.
Certain uses of photography, particualrly infrared, have
great value in water quality monitoring (Merriman, 1970;
Punk and Flaherty, 1972). On the organism level, infrared
color photography has been employed for detection of
unstained microbes in natural habitats {Casida, 1967).
Detection and activity of periphytic bacteria can be
accomplished with modifications of light microscopy {Bott
and Brock, 1970). Development of the scanning electron
microscope has revealed an elegant picture of in situ
associations {Brooks et al., 1972; Gessner et al., 1972).
Fluorescent techniques are applicable to chlorophyll-
containing forms (Wood and Oppenheimer, 1962). Another use
involving the fluorescent antibody technique for
determination of fecal pollution has provided rapid
identification and quantitation of indicator bacteria
(Abshire and Guthrie, 1971). Serological tracing has been
applied also with microscopy (Glantz, 1968).
Approaches to the study of microbial activity in waters
have been reviewed admirably by Fjerdingstad (1971) and
Jannasch (1969, 1972). Included are discussions of biomass
determinations by ATP measurement and assessments of growth
and substrate uptake which include oxygen exchanges and the
production of measureable color reactions using TTC. Some
interesting in situ applications have appeared on growth
rates of bacteria in river water and sediment uptake of
substrate (e.g., Hendricks, 1972; Wood and Chua, 1973).
Autoradiography {Brock and Brock, 1966) may be added as a
method for further exploration {see Rosswall, 1973), Gas
chromatography offers another new tool for certain processes
and environments (Payne, 1973; Francis et al., 1973).
Bioassay of organic compounds {vitamins, etc.) in
natural waters {Belser, 1963) has provided valuable data for
productivity studies and ecological interactions. Algae not
only require growth stimulants {Provasoli, 1963) but also
produce them (Carlucci and Bowes, 1970). A provisional
algal assay procedure {PAAP; Weiss and Helms, 1971) has been
developed to measure the total algal nutrient potential of
natural waters by utilizing the waters as a culture medium
yielding a final algal biomass as a nutrient indicator.
This test has been modified {Cain and Trainor, 1973) to
overcome some inadequacies and now represents a simple.
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fast, reproducible, and field applicable technique. Some
studies have shown the value of marine animals as bioassay
agents for hydrocarbons in water (LaRoche et al., 1970).
Given the ingenuity and pool of scientific talent
available, the above review of existing methodology will
predictably be synthesized into less primitive and more
meaningful monitoring. Sorensen and Moss (1973) describe in
detail some inherent problems in the implementation of the
National Environmental Policy Act. They see the process in
three steps of increasing complexity; (a) impact
identification, (b) prediction of impacts, and (c)
evaluation of a specific impact. In all stages, monitoring
of some sort will be included, as well as in post-approval
activities. The critical point here is that we must not get
tied to untested assessment methods. Constant review and
evaluation will engender change in methods and/or approach
to include introduction of new techniques and the
elimination of others where warranted. In addition, some
legal (McManus, 1973} and philosphical
-------
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MONITORING SYSTEMS AND THEIR USERS: IS THERE A RELATIONSHIP?
(What's It All About, Alfie?)
D. F. Squires *
When I agreed to speak to you this afternoon, I told
your seminar chairman that I was far from qualified as an
authority on environmental monitoring. Perhaps that is why
I am here—late in the session. This speech's title for
print has the usual formal flavor. The real title is,
"What's It All About, Alfie?"
Let me begin by stating that monitoring our environment
in a continued, sustained, coordinated, thoughtful way is
probably one of man's most necessary activities. We live in
an age of environmental awareness. A new crop of voters is
coming of age-those to whom Earth Day was a call to arms to
rectify the past sins of their elders. We must not forget
that to those young students--in high school or younger—the
environmental crisis is very real and has made a strong
impression. When, for us older types under intense social
pressures, our way of life and our standard of living are
threatened by energy problems, it is easy for us to
compromise the environment and to forget conveniently about
those dramatic issues. To a.younger generation, letting up
on pollution abatement procedures may seem a cop out--and
they may not agree. This situation may lead to a new
confrontation, demanding from the scientific community data
of a new kind. The scientific community must be prepared to
understand--and to interpret—what is happening in the
natural world around us.
My current home is on the east coast, in New York State.
Over 30 years ago changes began to occur in the coastal
waters of Long Island—and in the life sustained by those
waters. One of the first indicators of change was an
increase of starfish in Long Island Sound. Those predators,
coupled with a disastrous sequence of storms, resulted in
failure of the oyster industry, change persisted. Some of
the flounder population began to go; other fisheries became
marginal or collapsed. Lobsters, it is true, became more
abundant; and the eel grass, Zostera, recovered from a
ruinous setback by disease, bringing with its regrowth
stronger shellfish populations, change persists. That
change is the catalyst of a new, massive, and probably
rationally unresolvable conflict between recreational and
commercial fishing interests.
* N.Y. State Sea Grant Program
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Now recall the very rough sequence of events I have
sketched. Causation for most is not known. Disease in the
Zostera is documented; but about the rest, we have to
surmise—guess. In the conflict raging bitterly between
sports and commercial interests, facts are notably lacking.
Some attribute diminishing fish populations to long-term
oceanic shifts, others to overfishing. Everybody blames
"pollution." All are probably causative factors of the
changes in population structure. Fingers are pointed,
accusations are leveled, much heat is generated, but very
little light is shed.
Alan Longhurst and his colleagues (1) recently called
for "deliberately mounted and well-sustained Ocean
monitoring operations." They make important points:
1. That "pollution monitoring schemes, in the ocean or
elsewhere, can only succeed if the natural effects
of the changing physical environment are both
understood and monitored continuously and
indefinitely";
2. That "natural fluctuations in animal populations
have already been ascribed incorrectly to the
effects of pollutants*1;
3. That "a serious impact on the environment [ could 3
pass unnoticed through ignorance of natural
population instability."
Longhurst and his colleagues document their stance with
eloquent examples of biological fluctuations in ocean
populations; the fin-fish collapse on the east coast in the
19th century, the complex instabilities of "El Ninos,"
Pacific sardine fluctuations, and others.
Their point is that when we judge man's impact on the
ocean we must avoid the twin pitfalls of;
1. Assuming that the ocean's vastness, its chemical
buffering system, and the complexity of the food web
will render it capable of absorbing insult after
insult;
2. Regarding populations of marine organisms as
inherently stable and thinking of changes in them as
solely the result of man's intervention.
To cry "Eco-catastropheJ" prematurely is to create
another Amchitka. To prophesy the end of the world is
surely the way to lose the attention of one's audience. We
must beware of crying wolf—or, to cite a recent example,
Acanthaster_. The "Crown-of Thorns" was a cause^celebrex
aptly named for its effect on many members of our society.
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Up went the rallying cry, "Save our coral reefs from the
depradations of the starfish!" But were we witnessing a
subtle effect of man's intrusion by insecticides and other
pollutants? Or were we, as now seems the case, witnessing a
population explosion of a heretofore rare species? We lose
credibility with each false clarion call.
Let me turn to another concern which may be new to you.
Technocrats no longer stalk the halls of power in
Washington. Consumerism has arisen in the land. Science
has fallen from the favored position, although its successor
has not been chosen. Our society is skeptical of the
technological fix, of the knowledgeable but distant
technologist with all the answers. We scientists are being
pushed to move in new directions. As demands upon science
change, so must the system for meeting those demands.
William Baker, president of the Bell Telephone Laboratories,
testified to the House Committee on Science and Astronautics
recently. He cited the change in demand from performance
systems to service-oriented economic systems. The hardware
of the 1940*3, 1950*s, and 1960«s does not meet the 1970*s
challenge to science and engineering to solve problems in
the service of man—problems of ecology, universal health
care, public transport, costly food production and
distribution—problems with a huge consumer factor.
You who are engaged in environmental monitoring are
serving new masters: you are answerable to a very broad
constituency. Are you aware of them? Science for science's
sake is no longer enough to pay the way. Are you becoming
alert to the needs and the frailties of the new masters?
Let me pose some questions and then relate a case study.
Do people understand what you are doing when you monitor
the environment—when you model the environment? Do they
understand the limitations? Are you telling the taxpayer
footing the bill all that he should know? Is it really
getting through to him?
I submit that the answer to all is No! To illustrate, I
am going to cite from a recent report by one of New York's
Sea Grant extension agents oa the high water problems on the
Lake Ontario shoreline. I should have said, "I'll share
this report with you" - That is extensionese for, "You
listen while I talk." I will admit at the outset that the
monitoring system and model involved are primitive compared
to the weighty issues you have been discussing, but the
social interactions are real, important, and critical to the
future of monitoring programs.
In the winter of 1972-73— this past winter—there was
weak ice cover on Lake Ontario with few periods of strong,
hard freeze. The usual coastal buffer, a thick ice sheet,
was not present. During January and February coastal
erosion was greater than usual and breakwaters were
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undercut. When high winds on March 18 and 19 brought storm
waves to the southern shores, extensive erosion, wave
overwash and flooding occurred—all exacerbated by the
eroded breakwaters. Lake levels in Lake Ontario then were
at least one and a half feet lower than the maximal levels
reached later, in the summer. By April and May, the
winter's high precipitation in the upper Great Lakes caused
rapidly rising lake levels. The situation reached serious
proportions in the summer. Public conception of these
events is now blurry but was confused even in May. The
flooding resulting from storm waves merges, in their minds,
with flooding from high water levels, despite the fact that
recompense depends in large part upon the distinction
between the two. Memories are short, hindsight is not
always clear, cause and effect often aren*t linked. After
all, most coastal dwellers assume that when they develop
land, erosion will somehow cease.
Now, all these issues have been further clouded by
controversy over control of lake levels and the Corps of
Engineers' "Operation Foresight." Although Operation
Foresight had been announced as a dike system to protect
against flooding, it became an erosion control project.
Slippage due to technical factors, union problems, and lack
of easements from property owners delayed the project from
March until September and did not protect against flooding.
The Corps of Engineers, relatively blameless in this
connection, has been caught by the failure of Operation
Foresight. The entire episode is now viewed by many
citizens as a federal conspiracy to maintain high lake
levels for the benefit of commerce and the electrical
generation industry.
This idea is reinforced by the fact that the Lake Levels
Control Board is chaired by the corps. The Board regulates
lake levels based on examination of three-month
precipitation levels, but the model does not include the
storage capacity of the upper lakes. Only 20 percent of
Lake Ontario's water is derived from directly received
precipitation.
some people now charge that for the Corps of Engineers
to have announced Operation Foresight in December of 1972,
the Corps must have perceived the high water threat as early
as the summer of 1972. Therefore: (a) high levels reached
in the summer of 1973 were quite preventable and (b) were
the result of a federal-commercial conspiracy so that (c)
disaster relief programs are in fact a payoff. Reason has
little sway in such a hotly fired controversy.
The need for "getting through to the audience" is
obvious.
1. Coordinated information directed towards local and
county governments, perhaps by Sea Grant's advisory
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service agents, could have pointed up the increased
potential for storm damage because of ice-free
conditions.
2. Weather data collection and dissemination could be
vastly improved. It is now sent to Cleveland for
processing, and comes back to Rochester as
forecasts. No distribution is made to Civil Defense
or other coordinative agencies.
3. The predictive model for lake levels can be improved
by incorporating data on storage in the upper lakes.
Let me summarize. Public opinion and facts are not
always identical. We all need to work hard—very hard—at
improving public understanding of what we are doing and what
our limitations are. I have tried to make several points
today. You in monitoring systems, we in other branches of
science, need to give a lot of thought to the people we
serve, and to their information needs—their education. A
new generation, more knowledgeable and much more skeptical
than the old, is with us. While a compelling case can be
made for monitoring systems of long duration, we must share
information we already have which can be used in meeting
daily disasters, and at the same time guard against drying
wolf. I'm speaking not of doing a public relations job, but
of making really thoughtful attempts to educate our ultimate
consumer, the public. Our most urgent task is to get
thoroughly in touch with the users of environmental
monitoring systems—and I do not mean the data banks of this
world, another subject, one I will carefully avoid.
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INDEX OF AUTHORS
Austin, T.
Problems In Measuring Turbidity as a Water
Quality Parameter 23
Buck, J. D.
A Survey of Methods for Monitoring Ecologically
Important Microorganisms in the Marine Environment 384
Butler, P. A.
Biological Problems in estuarine Monitoring 126
Cabelli, V. J. and F. T. Brezenski, A. P. Dufour, M. A. Levin
Microbiological Methods for Monitoring Marine
Waters for Possible Health Effects 359
Callaway, R. J.
Mathematical Modeling as a Framework for
Coastal Monitoring 6
Colwell, R. R.
Sampling Methods for Microbiological Analysis 334
Dean, R. B. and J. E. Smith, Jr.
The Properties and Composition of Sludges 259
Eisler, R.
Summary of Recent Studies on Biological Effects of
Crude Oils and Oil-Dispersant Mixtures to the
Red Sea (Israel) Macrofauna 156
Greenfield, S. M.
Introduction and Theme 1
Kahn, B. and D. M. Montgomery
Monitoring Seawater for Radionuclides 231
Kampbell, D. H.
Determination of Metals in Sea Water 139
Krawczyk, D. F. and M. W. Allen
Absorption of Orthophosphates on Borosilicate and
"Citrate of Magnesia Bottles"/ Polyethylene and
Polyvinyl Surfaces in a Distilled Water and
Sea Water Matrix 180
Nadeau, R. and B. J. Pastolove
Making Artemia Sludge Bioassay More
Ecologically Relevant 275
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Nelson, V. A. and W. R. Schell, A. H. Seymour
Methods for Monitoring Radioactivity in
Aquatic Biota 242
Pavlou, S. and T. E. Whitledge, J. C. Kelley, J. J. Walsh
A Systems Approach to Marine Pollution Monitoring 72
Rittall, W. F.
Surface Slick Sampling and Analysis 55
Slotta, L. S. and K. J. Williamson
Monitoring Dredge Spoils 302
Specht, D. T. and W. E. Miller
• Development of a Standard Marine Algal Assay
Procedure for Nutrient Assessment 194
Squires, D. F.
Monitoring Systems and Their Users: is
a Relationship? 406
Swartz, R. C., W. A. DeBen, and A. J. McErlean
Comparison of Species Diversity and Faunal Homogeneity
Indices as Criteria of Change in Biological Communities 316
Wilson, A. J., Jr.
Methods and Problems in Analysis of Pesticides
in the Estuarine Environment 108
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APPENDIX A - AGENDA
SEMINAR ON METHODOLOGY
FOR
MONITORING THE MARINE ENVIRONMENT
Time: October 16 - 18, 1973
Place: Washington Plaza Hotel
Fifth Avenue at Westlake
Seattle, Washington
Chairman: S. Sid Verner
EPA, Office of Research and Development
Tuesday, October 16, 1973
7:45 AM Registration
8:15 AM Introduction and Theme
Dr. Stanley Greenfield
Assistant Administrator for Research and Development
Session A
Basic Framework and Field Applications
Moderator: Don Baumgartner, NERC-Corvallis
8:30 AM Mathematical Modeling as a Framework for Coastal Monitoring
R. J. Callaway, NERC-Corvallis
Turbidity as a Water Quality Parameter
R. Austin, Scripps
Surface Slick Sampling and Analysis
W. Rittall, NERC-Corvallis
An Integral Shipboard Surveillance and Analysis Program
S. Pavlou, U. of Wash.
A-l
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10:30 AM Coffee Break
11:00 AM Discussion
12:00 PM Lunch
Session B
Monitoring for Pollutants
Moderator: Pat Parker, U. Of Texas
1:30 PM Pesticides
Methods and Problems in Analysis of Pesticide in the Estuarine
Environment
A. J. Wilson, Jr., GBERL
Problems in Monitoring Data Interpretation
P. A. Butler, GBERL
3:30 PM Coffee Break
4:00 PM Metals
Methods for Analysis in Sea Water
D. H. Kampbell, EPA-Region II
Metals and Biological Availability in the Marine Environment
D. K. Phelps, NMWQL
6:30 PM No Host Mixer
to
8:00 PM
Wednesday. October 17. 1973
Session B (Continued)
8:00 AM Petroleum Products
Recent Work in Monitoring Petroleum Pollution of the Ocean
M. H. Feldman, NERC-Corvallis
Summary of Recent Studies on Biological Effects of Crude
Oils and Oil-Dispersant Mixtures to the Red Sea (Israel)
Macrofauna
R. Eisler, NMWQL
A-2
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10:00 AM Coffee Break
10:30 AM Thermal Pollution
Problems Using Marine Waters for Industrial Cooling
J. C. Prager, NMWQL
11:30 AM Lunch
1:00 PM Nutrients
Absorption of Orthophosphates on Pyrex, Soft Glass,
Polyetheylene and PVC in Water
D. Krawczyk, NERC-Corvallis
Development of a Standard Marine Algal Assay Procedure for
Nutrient Assessment
D. T. Specht and W. E. Miller, NERC-Corvallis
3:00 PM Coffee Break
3:30 PM Radionuclides
Monitoring for Radionuclides in the Marine Environment
B. Kahn, NERC-Cincinnati
Methods for Monitoring Radioactivity in Aquatic Biota
V. A. Nelson, W. R. Schell, A. H. Seymour, U. of Wash.
5:30 PM Adjourn
ThursdayT October 18, 1973
Session B (Continued)
8:00 AM Sludges and Benthic Material
The Properties and Composition of Sewage Sludges
R. B. Dean, J. E. Smith, Jr., NERC-Cincinnati
Making Bioassays of Marine Ecology Relevant
R. Nadeau, EWQRL
10:00 AM Coffee Break
A-3
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10:30 AM Dredge Spoils
Methods for Monitoring Dredge Spoils
L. S. Slotta, OSU
An Analysis of the Application of Diversity Indices and Other
Measures to the Problem of Quantifying Environmental Change
R. Swartz and A. J. McErlean, OEGC
12:30 PM Lunch
Session C
Microbiological Aspects
Moderator: Len Guarraia, OAWP
1:30 PM Sampling Methods for Microbiological Analyses
R. R. Colwell U. of MD
Microbiological Methods for Monitoring Marine Waters £or
Possible Health Effects
V. J. Cabelli, F. T. Brezenski, A, P. Dufour, M. A. Levin, £?A
Methods for Monitoring the Marine Environment for Ec.> logical ly
Important Marine Organisms
J. D. Buck, U. of CT
3:30 PM Coffee Break
Session D
Monitoring Requirements
4:00 PM Monitoring Systems and Their Users: Is There a Relationship?
D. F- Squires, NY State Sea Grant Program
Adjourn
A-4
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APPENDIX B - LIST OF ATTENDEES
R. W. Austin
952 Amiford Drive
San Diego, CA
Thomas W. Backman
NVC/San Diego
4321 Meritt Blvd.
La Mesa, CA
Dick Bauer
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
Don Baumgartner
Environmental Protection Agency
200 S.W. 35th
Corvallis, OR
Roger Bean
Battelle Company N.W.
P.O. Box 999
Richland, WA
Paul R. Becker
Corps of Engineers
P.O. Box 631
Vicksburg, MI
C. Byron Behrens
Clean Sound Co-op
2406 13th Avenue, S.W.
Seattle, WA
Sam Ben-Yaakov
UCLA
Dept. of Geology
Los Angeles, CA
J. N. Blazevich
Environmental Protection Agency
Region X Lab
1200 6th Avenue
Seattle, WA
Robert L. Booth
Environmental Protection Agency
NERC, AQCL
Cincinnati, OH
Dale E. Brandon
Alyeska Pipeline
2100 Travis Street
Houston, TX
F. Brezenski
Environmental Protection Agency
Rariton Arsenal
Edison, NJ
Herbert Bruce
NOAA
328 W. 10th
Juneau, AK
John Buck
University of Connecticut
15 Osag Lane
Noank, CT
Philip Butler
Environmental Protection Agency
Sabine Island
Gulf Breeze, FL
Victor J. Cabelli
Environmental Protection Agency
8. Ferry Road
Nargansett, RI
John A. Calder
Florida State University
Dept, of Oceanography
Tallahassee, FL
R. J. Galloway
Environmental Protection Agency
PNERL
200 S, 35th Street
Corvallis, OR
B-l
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Robert Campbell
P.O. Box 25827
Bldg. 5-3
Denver Federal Center
Denver, CO
Roy Carpenter
University of Washington
Oceanography Dept.
WB-10
Seattle, WA
T. C. Carver
NMFS-NOAA
Washington, DC
Donald Casey
Environmental Protection Agency
P.O. Box 5035 Riverstation
Rochester, NY
John Christian
Environmental Protection Agency
Water Quality Standards
WSM - Room 807A
Washington, DC
Robert C. Clark, Jr.
NMFS-NOAA
Northwest Fisheries Center
2721 Montlake Blvd. E.
Seattle, WA
John R. Clayton, Jr.
University of Washington
Dept. of Oceanography
Seattle, WA
Ed Coate
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
R. R. Colwell
University of Maryland
Dept. of Microbiology
College Park, MD
Geraldine Cox
Raytheon
P.O. Box 360
Portsmouth, RI
Joseph M. Cummins
Environmental Protection Agency
Region X
P.O. Box 283
Herbert Curl, Jr.
Oregon State University
Corvallis OR
Ray Dalsey
Metro
410 W. Harrison Street
Seattle, WA
Brock W. de Lappe
University of California
P.O. Box 245
Bodeva Bay, CA
Ed De Nike
Dept. of Ecology
P.O. Box 829
Olympia, WA
Robert Dexter
University of Washington
Dept. of Oceanography
Seattle, WA
Ralph Domenowske
Metro
410 W. Harrison Street
Seattle, WA
R. C. Dugdale
University of Washington
Seattle, WA
Thomas W. Duke
Environmental Protection Agency
Gulf Breeze Lab
Gulf Breeze, FL
B-2
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R. Eisler
Environmental Protection Agency
NMWQL
Nargansett, RI
Rex L. Eley
P.O. Box 631
Waterways Eng. Station
Vicksburg, MS
Dale R. Evans
NOAA-NMFS
P.O. Box 1668
Juneau, AK
Jim Everts
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
M. H. Feldman
200 S.W. 35th
Corvallis, OR
Street
Dale Ferrier
Dept. of Ecology
Olympia, WA
James H. Finger
Environmental Protection Agency
College Station Road
Athens, GA
E. V. Fitzpatrick
240 Highland Avenue
Needham Heights, MA
Lee Fortier
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
Wolfgang Fuhs
NYS Dept. of Health
Div. Labs & Research
New Scoll Avenue
Albany, NY
Arnold Gahler
Environmental Protection Agency
Region X
Redmond, WA
C. S. Glam
NSF - IDOE
1800 G Street, NW
Washington, DC
L. Guarraia
Environmental Protection Agency
OAWP
Washington, DC
Robert D. Harp
Environmental Protection Agency
NFIC
Federal Center - Bldg. 53
Denver, CO
Howard Harris
NOAA
2725 Montlake Blvd. E.
Seattle, WA
Michael Healy
National Science Foundation
Washington, DC
Doreen Hill
Environmental Protection Agency
OEGC
805 Blossom Drive
Rockville, MD
Barry Holliday
ODMR
COE - Waterways Experimental Station
P'.O. Box 631
Vicksburg, MS
Kent Hughes
NOAA
Headquarters Bldg. 5 - Room 805
6010 Executive Blvd.
Rockville, MD
B-3
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David Jamison
Dept. of Natural Resources
Olympia, WA
Harlen H. Johnson
CH M/Hill
777 106th Avenue, N.E.
Bellevue, WA
Thomas Jofaes
Environmental Protection Agency
Region V
1600 Patterson
Suite 1100
Dallas, TX
Bernd Kahn
Radiochemistry & Nuclear
Eng. Lab
Cincinnati, OH
Don Kampbell
Environmental Protection Agency
GSA Raritan Depot
Edison, NJ
Neva Karrick
Northwest Fisheries Center
2725 Montlake Blvd. E.
Seattle, WA
Daniel B. Keane
Environmental Protection Agency
Redmond Lab
Redmond, WA
John W. Keeley
P.O. Box 631
Vicksburg, MS
Wesley L. Kinney
Environmental Protection Agency
OAWP
1043 WSME
Washington, DC
J. S. Kittredge
University of Texas
MBI
Galvaston, TX
A. T. Knecht
Atlantic Richfield Co.
400 E Sibley
Harvey, IL
Charles B. Koons
Esso Production Research Co.
P.O. Box 218
Houston, TX
Daniel F. Krawczyk
Environmental Protection Agency
NERC-Corvallis
200 S.W. 35th
Corvallis, OR
Ron Kreizenbeck
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
Katherine Krogslund
University of Washington
Dept. of Oceanography
Seattle, WA
T. L. Ku
University of Southern California
Dept. of Geology
Los Angeles, CA
Jerry Larrance
Northwest Fisheries Center
2725 Montlake Blvd. E.
Seattle, WA
John R. Lariviere
Dept. of Environmental Quality
1234 S.W. Morrison Street
Portland, OR
Jeff Layton
CH M/Hill
777 106th Avenue, NE
Bellevue, WA
Robert E. Lee
Environmental Protection Agency
Research Triangle Park, NC
B-4
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Paul Lefcourt
Environmental Protection Agency
Office of Research & Development
Washington, DC
Joseph Lewis
Water Quality and Non Point
Source Control Division
Washington, DC
Christopher J. Libbey
3-M
3-M Center
St. Paul, MN
Dorothy Lowman
University of Washington
Dept. of Oceanography
Seattle, WA
Edward Lukjanowicz
U.S. Navy
Seattle, WA
Helen McCammon
Environmental Protection Agency
Region I
JFK Federal Bldg.
Boston, MA
Vance McClure
NOAA
P.O. Box 98
Tiburon, CA
Andrew J. McErlean
Environmental Protection Agency
OEGC
Office of Technical Analysis
WSM - Rm. 3211
Washington, DC
Leslie McMillion
P.O. Box 15027
Las Vegas, NV
Nick J. Malueg
Environmental Protection Agency
Region X
15345 N.E. 36th Street
Redmond, WA
Stephen Meith
Dept. of Environmental Quality
8148 S.W. Burtn
Portland, OR
Calvin Menzie
SQ, BSFW
Washington, DC
M. Dwayne Milner
Environmental Protection Agency
6608 Hornwood Drive
Houston, TX
David R. Minard
5 Norma Court
Novato, CA
Don Moore
Environmental Protection Agency
NFIC
Denver, CO
Donald Morris
Naval Civil Engineering Lab
Port Mueneune, CA
Ernst W. Mueller
Arctic Environmental Research Lab
College, AK
Bill Muir
Environmental Protection Agency
WSME - Rm. 945
Washington, DC
Royal Nadeau
Edison Water Quality
Research Lab
Edison, NJ
Francis Nelson
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
Victor A. Nelson
University of Washington
RT. 1, Box 38
Hansville, WA
B-5
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Richard Ogar
Atlantic Richfield Co.
271 W. Kingtut
Lynden, WA
Lawrence Olinger
Environmental Protection Agency
Escambia Bay Recovery Study
Gulf Breeze, FL
H. E. Ohanian
Interstate Electronics
707 E. Vermont Avenue
Anaheim, CA
John Osborn
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
Patrick Parker
University of Texas
Port Aransas, TX
D. Patten
NMFS
Box 21
Mukilteo, WA
S. Pavlou
University of Washington
4416 51st N.E.
Seattle, WA
Donald K. Phelps
Environmental Protection Agency
Room 710
Narragansett, RI
Herbert E. Pintler
Environmental Protection Agency
100 California Street
San Francisco, CA
Stephen V. W. Pope
Environmental Protection Agency
15345 N.E. 36th
Redmond, WA
Jan C. Prager
Environmental Protection Agency
National Marine Water Quality Lab
P.O. Box 277
West Kingston, Rl
Murice 0. Rinkel
SUSIO
830 1st Street, S.
St. Petersburg, FL
Robert Risebrough
142 Vicente
Berkeley, CA
W. Rittall
1215 N.W. Hillcrest Drive
Corvallis, OR
W. Rosenberg
Environmental Protection Agency
RBgion I
Boston, MA
Herb Sanborn
NMFS
2725 Montlake Blvd. E.
Seattle, WA
William Sargent
Boeing Co.
P.O. Box 3707
Seattle, WA
Bill Schmidt
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
A. James Seidl
Fish & Wildlife Service
813 D Street
Anchorage, AK
John Shackleford
Dept. of Social & Health Services
Water Chemistry - Rm. 1509
Smith Tower Building
Seattle, WA
B-6
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T. Shafik
Environmental Protection Agency
Research Triangle Park, NC
David Shaw
University of Alaska
Fairbanks, AK
Charles Simenstad
Fisheries Research Institute
University of Washington
Seattle, WA
June Siva
515 S. Flower Street
Los Angeles, CA
L. S. Slotta
1540 N.W. Dixon
Corvallis, OR
Edmund H. Smith
Pacific Marine Station
University of Pacific
Dilen Beach, CA
J. E. Smith, Jr.
Environmental Protection Agency
AWTRL - NERC
Cincinnati, OH
R. D. Spear
Environmental Protection Agency
Region II
26 Federal Plaza
New York, NY
David T. Specht
Environmental Protection Agency
Environmental Research Lab
200 S.W. 35th Street
Corvallis, OR
Donald Squires
NYS Sea Grant Program
99 Washington Avenue
Albany, NY
William Stang
P.O. Box 25227
Bldg. 5-3
Denver Federal Center
Denver, CO
Thomas Stanley
Environmental Protection Agency
Chief, Control Branch
Washington, DC
Richard Swartz
Environmental Protection Agency
Marine Science Center
Newport, OR
Frieda B. Taub
University of Washington
College of Fisheries
WH-10, Rm. 212
Seattle, WA
William L. Templeton
Battelle Northwest
P.O. Box 999
Richland, WA
Thomas 0. Thatcher
Battelle Northwest
P.O. Box 999
Richland, WA
R. C. Timme
Interstate Electronics Corp.
Environmental Engineering Div.
707 E. Vermont
Anaheim, CA
R. Y. Ting
Battelle Northwest
P.O. Box 999
Richland, WA
J. R. Vandernorst
Battelle Northwest
P.O. Box 999
Richland, WA
Sid Verner
Environmental Protection Agency
Office of Research & Development
Washington, DC
Robert L. Vickery
Environmental Protection Agency
1600 Patterson Street
Suite 1100
Dallas, TX
B-7
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Stuart Wakeham
University of Washington
Oceanography & Chemistry Depts.
Seattle, WA
T. A. Waatler
Rt, 3
P.O. Box 340A
Annapolis, MD
Cornelius Weber
Environmental Protection AGency
Air Quality Control Lab
1014 Broadway
Cincinnati, OH
T. E. Whitledge
University of Washington
Dept. of Oceanography
Seattle» WA
R. E. Wildung
Battelle Northwest
P.O. Box 999
Richland, WA
Kenneth J. Williamson
Oregon State University
Civil Engineering Dept.
Corvallis, OR
A. J. Wilson
Environmental Protection Agency
Sabine Island
Gulf Breeze, FL
Carolyn Wilson
Environmental Protection Agency
Region X
1200 6th Avenue
Seattle, WA
Homer R. Wolfe
Environmental Protection Agency
P.O. Box 73
Wenatchee, WA
H. L. Young
Environmental Protection Agency
Region IX
610 Central Avenue
Alameda, CA
Bob Zeller
Environmental Protection Agency
Region V
1 N. Wacker
Chicago, IL
John Yearsley
Environmental Protection
Region X
1200 6th Avenue
Seattle, WA
Agency
B-8
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/k-jk-OOk
2.
3. RECIPIENT'S ACCESSION-NO.
TITLE AND SUBTITLE
Proceedings of Seminar on Methodology for
Monitoring the Marine Environment
S. REPORT DATE
October
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
S. S. Verner, Editor-in-Chief
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Protection Agency
Office of Monitoring Systems - RD-688
Washington, D.C. 20460
10. PROGRAM ELEMENT NO.
1HA326 PEMP/2
11. CONTRACT/GRANT NO.
2. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
N/A
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This document contains the proceedings of the "Seminar on Methodology
for Monitoring the Marine Environment" held in October 1973. The
Seminar was organized to strengthen the Agency's coastal monitoring
program and focused on physical-chemical biological, and microbiologica
techniques for assessing the quality of marine and estuarine waters.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATl Field/Group
Coastal Monitoring
Estuarine Ecosystems
Marine Pathogens
Marine Pollutants
18. DISTRIBUTION STATEMENT
19. SECURITY CLASS (ThisReport)
21. NO. OF PAGES
20. SECURITY CLASS (This page}
22. PRICE
EPA Form 2220-1 (9-73J
U.S. GOVERNMENT PRINTING OFFICE: 1974—582-415:157
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