vvEPA
United States
Environmental Protection
Agency
The Enforcement Division
Region V
Chicago IL
EPA-600/9-80-026
May 1980
Research and Development
Proceedings of the
Seminar on Biological
Monitoring and Its Use
in the NPDES Permit
Program
Held at the
Conrad Hilton Hotel
Chicago IL
October 2, 1979
Do not remove. This document
should be retained in the EPA
Region 5 Library Collection.
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U S Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1 Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4 Environmental Monitoring
5 Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9 Miscellaneous Reports
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/9-80-026
May 1980
PROCEEDINGS OF THE SEMINAR
ON BIOLOGICAL MONITORING
AND ITS USE IN THE NPDES
PERMIT "PROGRAM
Chicago, Illinois
October 2, 1979
This seminar was conducted
in cooperation with
The Enforcement Division, Region V
U.S. Environmental Protection Agency
Chicago, Illinois
CENTER FOR ENVIRONMENTAL RESEARCH INFORMATION
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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PREFACE
From the earliest federal water pollution control legislation, the
concept of protection of human health and the environment has been important
in the development of regulations to control waste discharges into the waters
of the United States. The Clean Water Act of 1977 reemphasized this concept
by requiring as a paramount concern the control of toxicant discharges which
may cause damage to human health or the environment. The U.S. EPA and the
state and other pollution control agencies are responding by increasing their
efforts to work with industries and municipalities to identify, evaluate, and
control the discharge of toxicants. Such endeavors during the past few years
have resulted in the installation of industrial and municipal treatment
systems providing major control of conventional pollutants and, directly or
coincidentally, some toxic substances as well.
Nationally, U.S. EPA is developing effluent guidelines and water
quality criteria to control the discharge of a select number of toxicants of
national concern. In addition, U.S. EPA's regional offices and the states
are now working to implement programs to address facilities which may be of
specific local or regional concern because of their particular products,
processes, raw materials, etc., or to address the unique assemblage of
industries in a given river or lake basin.
Two of the tools being utilized to evaluate the potential for toxicant
discharge are biomonitoring and industrial process evaluations. A limited
number of industries having the potential for the discharge of toxicants have
been or will be required to conduct special testing, monitoring, and evalua-
tions utilizing these tools. The information obtained from these assess-
ments, along with national guidelines, is being utilized in the NPDES permit
reissuance process to evaluate the need for additional toxicant controls.
Although many industries, the states, and the U.S. EPA have done some
biomonitoring, the need for increased use of biomonitoring as a tool to
control toxicants has become increasingly evident. Therefore, this seminar
was held to clarify the methods and uses of biomonitoring and its application
to setting limits in NPDES permits. Presentations were made concerning
biological monitoring tests which make use of freshwater and marine bio-
logical systems - phytoplanktons, zooplanktons, macroinvertebrates, fish,
bacteria, etc. - in tests for toxicity in process and waste discharges. The
range of tests include static and flow-through bioassays, including tests for
bioaccumulation, Ames tests which use bacteria to test for mutagenicity, and
some rapid assessment methods such as the fish cough response test.
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TABLE OF CONTENTS
Page
Preface ii
Welcoming Remarks
John McGuire 1
Biomonitoring of Effluents in Perspective
Donald I. Mount 3
The NPDES Permit Policy as It Relates to Biomonitoring
Sandra Gardehring 8
Static Test Using Fish
Ronald Preston 20
Static Test Using Algae
William Miller'. 25
Flow-Through Test Using Fish
William Peltier 31
Bioconcentration Tests for Effluents
Gilman Veith 40
Sediment Bioassay
Max Anderson
Bayliss Prater 47
Tentative Guidelines for Flow-Through Early Life Stage
Toxicity Tests with Fathead Minnows for Use in the U.S.
EPA, OTS-ORD Round Robin Test
Donald I. Mount 62
Effluent Guidelines Limitations and
Lethal Units
Kenneth J. Macek 69
Applicability of the Ames Test in Biomonitoring
Larry Claxton 80
Rapid Assessment Methods (Fish Cough Response, etc.)
Robert Drummond 95
Test Organism Acquisition and Culturing in the Lab
Charles Steiner 97
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WELCOMING REMARKS
John McGuire
Regional Administrator
U.S. EPA, Region V
Chicago, Illinois
I would like to welcome all of you to the seminar on Biological
Monitoring and its use in the NPDES Permit Program. Your participation is,
of course, the key to the seminar. The turnout - some 400 people - suggests
that this seminar is needed and that many of you are aware of the importance
of biomonitoring in developing limitations for toxic substances in NPDES
permits.
Although many industries, the states, and the U.S. EPA have done some
biomonitoring, the need for increased use of biomonitoring as a tool to
control toxicants has become increasingly evident. We thought it would be
useful, therefore, to hold a seminar to clarify the methods and uses of
biomonitoring and its application to setting limits in NPDES permits.
Notices of the seminar were sent to those industries in Region V which we
believed would benefit most from biomonitoring or which might be required
to do so.
While the concept of protecting aquatic life was paramount in the
earlier Federal Water Pollution Control Legislation, more recent legislation,
the Clean Water Act, has emphasized the control of toxic substances with
emphasis on the aquatic environment and human health. We in EPA, the states,
and other pollution control agencies have responded by increasing our efforts
to work with industries and municipalities to surface, evaluate and, where
necessary, control the discharge of toxicants. Industries and municipalities
throughout the country have installed treatment systems which have provided
major control of the more common pollutants and coincidentally some toxic and
persistent chemicals.
Nationally, EPA is developing effluent limitations and water quality
criteria to control the discharge of a select number of toxicants of possible
national concern. Region V, along with the states, is now working to imple-
ment a program to address those other toxicants which may be of local or
regional concern because of unique concentrations of industries. Biomoni-
toring and industrial process evaluations are some of the tools being util-
ized to evaluate the potential for toxicant discharge. A limited number of
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industries are or will be required to conduct special testing, monitoring and
evaluations utilizing these tools. This information along with the national
guidelines will be utilized in the NPDES permit reissuance process in evalua-
ting the need for additional toxic substances control.
In working with industries to date, a number of common questions have
been raised. The purpose of this workshop is, in part, to answer these
questions and to let you know some of our thinking concerning biomonitoring,
process evaluation, and the need to place greater emphasis on the control of
toxic substances.
I am particularly concerned that efforts to control persistent sub-
stances be given an extremely high priority in the Great Lakes Basin. I need
not recount here the list of these substances which have been found thus far.
We appear to be finding others, also. As U.S. Cochairman of the Great Lakes
Water Quality Board I am charged with the responsibility for implementing the
Water Quality Agreement between the United States and Canada.
That agreement calls for increased attention to and vigilance in the
control of toxic and persistent substances in the Great Lakes Basin. While
we have made and continue to make progress in that area we are obligated to
increase our efforts and to ultimately control these dischargers. The fact
that the Great Lakes also suffer from atmospheric input of many contaminants
only increases the need to reduce or eliminate those dischargers which are
most controllable. While there is an absolute need for heavy, emphasis on the
Great Lakes, we are also aware that significant efforts are needed in our
other major basins such as the Ohio River Basin. To a lesser extent, efforts
are also needed in the Mississippi River Basin.
Biomonitoring represents only one tool for achieving our goals. As
Dr. Mount will discuss later, biomonitoring has not traditionally been used
for measuring persistence. Perhaps it is time we break with tradition and as
appropriate, use all of our tools - biomonitoring, process evaluation and
others - more fully.
Again, I want to welcome you here today to this Biomonitoring Seminar.
I would like now to turn the program over to one of EPA's most distinguished
scientists, Dr. Donald Mount.
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BIOMONITORING OF EFFLUENTS IN PERSPECTIVE
Donald I. Mount
U.S. EPA, Environmental Research Laboratory
Duluth, Minnesota
I'm not exactly sure of the backgrounds of most of you, and I don't
know how much you know about biomonitoring - maybe you know a lot more than I
do. Biomonitoring is a term that means many things to many people and it is
misunderstood because of that. As John said, the thrust of biomonitoring
really relates principally to the protection of organisms, including man, and
I'd like to use a definition or a concept of biomonitoring in a very restric-
tive way this morning as I make my comments so that you'll know what I'm
talking about.
There are many kinds of biomonitoring, including those in which we do
field studies to assess the health of a stream or a lake in relation to an
outfall. But I think the upstream-downstream studies of the fifties are
probably not useful in most heavily industrialized or urbanized locations
simply because there are so many outfalls in close proximity that one cannot
really tell the effects of one outfall from another. And so, although it has
a place in terms of receiving water quality, I don't really see that as being
especially pertinent to the problems that I think we are here to talk about
today, which are problems related to the toxic characteristics of particular
effluents. So, in my comments, I'm going to be talking more about laboratory
or out-of-stream monitoring as opposed to in-stream monitoring, although I
certainly don't want to rule out the use of cages in waste streams and
similar techniques which might be considered in-place monitoring.
I made a statement in the meeting in Washington on the first of August
which I thought was fairly obvious, but I find, upon discussion with other
people since then, it was not really understood. There is really only one
way to determine the toxicity of anything - and that's with an organism. I
don't believe there is any other way to decide what toxicity is or how toxic
something may be without the use of organisms. I think by definition it's
only a response that organisms give to an exposure. There is no electronic
instrument that I know of that measures toxicity directly, although certainly
measurements can be correlated in many instances with toxicity measures.
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An important implication is that there.is no true reference standard
for toxicity. There is no way to know precisely the exact toxicity of some
particular chemical - and I'm talking now about pure chemicals as well as
effluents. This means that the approach to toxicity measurements must be
different than when you measure the amount of mercury in a sample and know
precisely what you have in your standard. How do you know which toxicity
measurement is right and which one is wrong? They are really almost relative
to each other. And maybe everyone is exactly correct for the conditions
under which it was done - but nonetheless you will get different answers.
So, it is inconceivable to me that any discharger who is concerned about the
impact of his effluent on receiving water or any regulatory agency who is
concerned about the control of what we have rather sloppily been calling
"toxics", wouldn't use organisms to determine toxicity because there is no
other way to measure it. If we would use organisms, we would eliminate the
problems of relationships between the response of organisms and the chemical
measurements made with electronic measurements.
The most important reason we have effluent control or pollution con-
trol, be it air, water or whatever, is for the protection of biological
organisms somewhere, including ourselves, of course. There are a few uses of
water which may not be directly biologically related, but for the most part,
they are. It's obvious then that the role of organisms to measure toxicity of
effluents is certainly appropriate and far overdue. If you go back in the
literature - Tarzwell, Doudoroff, and others - were calling for biomonitoring
in the late forties and early fifties. Only just now are we becoming aware
of the value that it has.
Now, let me insert one other word of caution. None of us are pro-
posing that biomonitoring is a replacement for chemical measurements - that's
just clearly not the case. Both are needed - they are both tools that tell
us different things about the effluent. And in some cases chemical analysis
may be far better than biomonitoring for what we want to know. But I think
that if you will stop for a moment you will realize that the kinds of chemi-
cals that we are now focusing our attention upon are not BOD and suspended
solids but complex organic chemicals. To measure those chemicals is a real
job. If you think that the cost of purchasing the electronic equipment to
make these measurements is bad, even though it may be $1/2 million in some
instances, the cost of buying competent chemists who can operate that equip-
ment and who know what it's telling them is an even greater cost. The
capital cost of the equiment is small compared to the manpower to operate an
installation like that. And so, we certainly do need to have cheaper and
more effective methods to use as routine tools and save those costly analyses
for places where we really need them. So, if we talk in terms, as some
speakers later today may, of $450 or $500 for a biomonitoring test, that cost
pales when compared to the alternate costs if we have to go the analytical
route. I remind you again that we are not now talking about measuring BOD,
which is a cheap thing to do. We are talking about much more difficult
measurements for a great many more chemicals. That's not to say there won't
be surrogate measures to which we can relate toxicity as we learn more about
it, but at the present state-of-the-art, that doesn't seem to be possible.
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Some of the advantages of biomonitoring, as opposed to only chemical
monitoring, are that it probably more closely approximates the receiving
water conditions than nearly anything we can do, and it does consider the
interactions that may occur between the components of a waste stream. Again,
for the most part, I'm talking about complex wastes that are made up of many
things and vary in quantities. This is the difference between working with
industrial wastes and classical toxicology. I'm not considering in these
comments biomonitoring for human health. There is another line of defense
which makes that concern a little less important in that few of us drink
untreated water. The protection of the organisms does not have that second
line of defense. So our discussion more commonly, and maybe rightly, is
focused on aquatic organisms rather than on the human health aspect even
though I think we all agree that the health aspect is more important.
While there may be additive or synergistic properties in wastes there
are also antagonistic properties or subtractive properties, whatever you want
to call them. From this standpoint we get at the problem of mixtures very
effectively - probably as effectively as any way we can approach the question
at the present time, because the state of toxicology in the aquatic area, and
I think also in the mammalian area, is not really advanced far enough yet to
know how to deal with interactions. You can measure the interaction of one
mixture but there's no way to relate that information to another mixture. I
suggest as proof of this, you ask your physician what he knows about the
interaction of commonly used drugs in your body and you'll find out that
the state-of-the-art is embryonic.
A third distinct advantage is that we are measuring the target re-
sponse and not an index of it. When you measure COD, TOC, or mercury in a
waste, you are not measuring the same condition as exists in the receiving
water. You come much closer in a biomonitoring test.
Let me digress to comment on how animals behave in pickle jars. There
may be differences between the way animals respond in glass bottles and the
way they respond in the stream, but the alternative is to do it all in the
stream. If any of you have done much in the way of in-stream work, you know
the tremendous cost that's involved and the tremendous frustration you often
feel. It may grate against your scientific credibility to think about
extrapolating responses of animals in bottles to real-life situations. But
there is a two-edged sword which I'm afraid industry, in particular, is not
aware of. If we can't extrapolate with effluents, then when registering a
new chemical under TOSCA, I don't see where EPA has any other choice but to
say that you can't extrapolate those either. That means testing lake trout,
shovel-head catfish, and elephants. My point is, either we extrapolate or we
grind to a halt country-wide. I don't know what position the agency's going
to take - I don't make policy in the agency - but if they ask me, I'm going
to say, if we can't extrapolate on effluent biomonitoring, then we can't
extrapolate on the registration of new chemicals either under FIFRA or under
TOSCA. There's going to be some error, but we all work with error daily.
More often than not, the difference in response of animals in the bottle is
not because they're responding differently than in the stream; we are just
not aware of all the conditions to which they are exposed. If you look at
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the costs of biomonitoring and compare them to the probable costs of chemical
analysis, biomonitoring is an efficient use of resources.
There are obviously disadvantages. The world's problems are not going
to be solved by a simple 96-hour or 24-hour bioassay. Some disadvantages are
that there will be physical-chemical changes in the properties of the wastes
after they are released into the receiving water. If we run tests with the
whole effluent, even though we may use receiving water as a diluent, we are
not going to measure all of the same chemical and physical interactions that
go on in a stream. But that is equally true of BOD measurements. It's a
difficult and rather subjective art, I think you'll all have to agree, to get
from a BOD test to the DO in a stream. And we have a very comparable problem
when we work with whole effluents. But nevertheless, you can certainly gain
a good first cut at what may be of concern in your waste by use of biomoni-
toring.
It's difficult to measure chronic effects, whether they be chronic
toxicity, carcinogenicity, or bioconcentration, without going to rather
extensive and longer-term tests. At the present time, we simply don't have
any overnight test that looks all that promising to get at the chronic
effects. So, you cannot expect to go from a simple short effluent test to
a no-effect concentration or a safe concentration in the receiving water, and
have any credibility. We are not trying to go from an effluent to a safe
receiving water concentration with a simple lethality test. It's just
nonsense to think you can do that as yet.
As John mentioned earlier in his comments, most of our biomonitoring
to date has not attempted to measure persistence. If there is one property
about chemicals on which we ought to focus, a property which we ought to
eliminate above others, it is persistence in the environment. If you think
of previous headlines in the newspapers of the last ten years and identify
the chemicals named - methyl-mercury, DDT, Dieldrin, PCB's, Mi rex, Kepone -
they all have that property in common - they are environmentally persistent.
If I were making new chemicals or concerned about the effects of my discharge
on receiving water, I would clearly want to know what the persistence of
this material is. If we did not have persistent chemicals in discharges, all
of our toxicity or environmental contamination problems would be local prob-
lems - they would not be ubiquitous like we have had in the past. We are
not as adept at effectively measuring persistence with simple tests. I think
we are much better at measuring toxicity, but measuring persistence is
doable.
There are special considerations in using effluent tests. They cannot
be done only once. Effluents are variable from day to day as all who have
worked with effluents know. Such toxicity measurements are only as good as
the representativeness of the sample. If the sample is not typical of the
waste, then neither is the toxicity, and you cannot say much about the
impact on the receiving water. Biomonitoring tests must be cheap enough,
simple enough, and short enough, so that they can be done on enough samples
to characterize the waste. What I have said is equally true of chemical
measurements as well. Measuring the TOC in a waste once a month is not
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telling us much for many wastes. There is no sense in making the toxicity
measurement any better than the representativeness of the sample used.
One message I hope to leave with you from this discussion this morning
is that it's time we consider toxicity as a property of wastes which may need
to be reduced and which we should measure. This does not necessarily mean
that effluents must have zero toxicity. Toxicity is relative and we are
concerned about those where the exposure that will occur in the receiving
water is harmful.
DISCUSSION
Question: I know very little about the testing that you are speaking
of - this biological monitoring - but if we do have to put this
into industry, in what form do you think it will be? Will this
be a fish pond, or are there any different types of scientists or
chemists that it involves? What does it mean? What will we have
to resolve - the topics here at issue?
Answer: In general terms without getting into specifics of tests which we
are going to do later today, it's clear that tests must be
simple, doable by something other than the PhD, and they must
have a reasonable cost. I think if we don't achieve those things
in the effluent tests, we have achieved very little. I think
Sandy and perhaps some of the other speakers may be in a better
position to know what form that takes. We've been talking in
terms of tests that can be done offsite in most instances.
Question: Would EPA consider it good enough to hire a free-lance com-
pany out to do the biomonitoring? In Syracuse there is a company
that just runs bioassays, and we send a lot of our samples
there.
Answer: Again, I can't speak for the agency, but those of us in the
agency who have been talking about these problems I think all
recognize the essential roles which consulting firms or consul-
ting laboratories must play in this program. I think whether a
discharger goes to a consultant or does it himself will depend in
large part on the size of his operation, the kind of staff that
he has, and the particular location. But I don't think any of us
at this point are ruling out the use of consulting firms.
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THE NPDES PERMIT POLICY AS IT RELATES TO BIOMONITORING
Sandra Gardebring
Director, Enforcement Division
U. S. EPA, Region V
Chicago, Illinois
Good morning. I would like to add my personal welcome to that of John
McGuire, the Regional Administrator, and to thank you for your enthusiastic
response to our seminar on biomonitoring. The fact that so many of you are
here underscores the timeliness of this topic and the value of biomonitoring
as a tool in achieving clean water.
What are we really talking about when we say biomonitoring? As used
in this conference, biomonitoring, or biological monitoring tests are studies
which make use of freshwater or marine biological systems - phytoplankton,
zooplankton, macroinvertebrates, fish - and in some cases bacteria - to test
for toxicity in process and waste discharges. The range of tests includes
static and flow-through bioassays, Ames tests, and some rapid assessment
methods such as fish cough response. Depending on which test is used, the
data generated can indicate acute or chronic toxicity, carcinogenicity,
rnutagenicity, or can suggest potentially problematic bioaccumulation of
organic chemicals, trace metals, or other toxicants.
However, it was the passage of the 1972 Act, and its requirement of a
nationally uniform water discharge permitting program with technology based
standards, which finally focused federal and state resources on water pollu-
tion clean up. In this region, we issued about 7,800 industrial permits
with BPT or water-quality-based requirements, addressing a wide range
of water pollutants. Emphasis was often on the more common pollutants such
as suspended solids, BOD, and pH, along with more persistent toxicants such
as heavy rnetals and PCBs. The long process of treatment design and instal-
lation was started, and although some dischargers had to be "persuaded"
through federal and state enforcement actions, overall, our efforts at
achieving compliance in this region have been quite successful. Some 75
percent of industrial dischargers- in our six states now meet the limitations
of their NPDES permits, and the remainder of the major permittees, almost
without exception, are the subject of state or federal enofrcement actions
aimed at bringing about their compliance.
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During this same period, several other things were going on which
brought us to where we are today. First, the instrument makers and our
friends in the laboratories combined to bring us tools and techniques which
allow the discovery and quantification of water pollutants at lower and lower
levels - parts per million, parts per billion, parts per trillion. At the
same time, we weren't very good at telling whether the presence of such
pollutants - even so-called "exotics" - at these very low levels was signifi-
cant or not. The "safe" levels in ambient air or water just weren't known
for many pollutants.
Secondly, both the public and those of us whose business is pollution
control became more and more aware of the real risks associated with the
unregulated flow of these chemically complex materials into our environment.
PBBs, PCBs, Mi rex, Si 1 vex, Dioxin, Kepone - terms known heretofore primarily
to white-coated men in chemistry labs - became, almost literally, household
words. The removal of solids and the increase in dissolved oxygen in our
waterways was a truly remarkable and wonderful effort, but it clearly was not
enough when chemically complicated materials with unknown biological effects
continued to be discharged.
The state and federal environmental regulatory agencies began to
respond under existing laws; a process which was accelerated under the teYms
of a consent decree agreed to by EPA and the Natural Resources Defense
Council in 1976. That agreement, which ended an NRDC lawsuit related to
toxic effluents, required EPA to test for the presence of 129 specific
toxicants in waste water discharges from 34 industrial categories and to
control any of these priority pollutants found at harmful levels via toxic
pollutant effluent guidelines regulations. Certain of the technology based
limitations and water quality criteria needed to carry out the terms of the
decree have begun to appear this year in the Federal Register.
However, we simply cannot afford to wait for or depend solely upon the
effort to develop categorical limitations for the 129 pollutants to solve our
toxic pollutant problems.
Given that there are nearly 50,000 chemical compounds in commercial
usage; given that more than 12,000 of them are suspected toxicants, 1,500
possible carcinogens, and many others considered capable of injuring human
health and aquatic biota; and given that we know little or nothing about the
synergistic effects of these compounds at even low levels, we must begin
addressing the specific problems in our states, whether or not they are
related to the 129 consent decree toxicants.
It is in this effort that biomonitoring, when combined with process
evaluation and chemical analysis, can be a potent tool both for discovering
and for limiting the discharge of toxic substances, and that is how we intend
to use it.
Let me provide a few more details about our approach.
EPA authority for requiring biomonitoring stems from Section 308 of
the Clean Water Act. The states, in order to receive NPDES authority, are
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required to have similar authority. Our review indicates that all of the
states in Region V do have similar authority and can require biomonitoring
and process evaluation either as part of a permit or as a separate indepen-
dent information-gathering requirement.
Perhaps some of the most important questions, particularly to indus-
try, are: Who are the target industries? Who should monitor? And beyond
that, how will the results be interpreted and used? As I have noted pre-
viously, biomonitoring is only a tool and as such it must be applied with
full knowledge of its limitations. It can be relatively expensive. The
results are sometimes less than clear and the tests often must be repeated.
On the other hand, biomonitoring is useful in telling us the response
of an organism to a complex effluent. The interaction - simple additive
effects and synergisms - of chemicals in an effluent and the ultimate effects
upon aquatic life are difficult to judge from available literature and
criteria. Many, if not most of the bioassays used to develop criteria are
run with one, perhaps two, or rarely three substances interacting. Informa-
tion on substances which bioaccumulate is even more limited and various
biomonitoring tests can be used to derive data to address these toxicant
effects.
I am sure that during the seminar you will explore many more of the
limitations and benefits of biomonitoring. I have highlighted only a few of
those just to indicate our own awareness of the need for reasonable applica-
tion of biomonitoring and all other tools.
Returning to those questions I posed previously: Who will be required
to biomonitor? How will they be selected?
Let me outline generally our thinking. Since most of the state and
federal interest is focused on major dischargers, those lists provide a
natural place to start. There are between 500 and 600 industries on the
major list in Region V. Those industries which might well be required to
biomonitor include chemical, pharmaceutical, pesticide, plastics, iron and
steel, rubber, chlor-alkali, paper, and other similar chemical-type facili-
ties.
In part, the level of treatment may also play a part in our decision.
Certainly a process stream receiving treatment with carbon filtration will be
looked upon differently than one receiving more conventional treatment. An
industry discharging only noncontact cooling waters with total process
stream recycle or deep-well injection may also be viewed differently.
Furthermore, I want to make it clear that we do not intend to ignore
those dichargers which have been previously classified as minor. We believe
that there are more than a few of those dichargers which may contain signifi-
cant contaminants and require closer scrutiny than they have received in the
past. Again, a careful evaluation of the facility, the process involved,
present treatment and the likelihood of a toxicant being in the discharge
will be necessary before biomonitoring is required.
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Finally, discharges to the Great Lakes or its tributaries will receive
emphasis for finding and eliminating or severely limiting those substances
which bioaccumulate and may pose a long term hazard to other aquatic life or
higher animals including man. Our agreement with the Canadians, and the
general tendency of bioaccumulative substances to be particularly troublesome
in the Great Lakes, forces us to place more emphasis in that area. However,
our emphasis on the Great Lakes will not be to the exclusion of other major
drainages such as the Ohio and Mississippi Rivers and their tributaries.
It should be obvious that the need for biomonitoring will largely
be determined on a case-by-case basis. Once the determination is made that
biomonitoring is needed, then the specific test or series of tests must
also be determined. Much will depend upon the nature of the contaminants
or suspected contaminants. In some cases, "failure" in one test may lead
to another. In a few cases where data presently exists, long term bioaccu-
mulation may be required directly. We estimate at present that 15 to 20
industries may need to conduct long term bioaccumulation tests. It is
also estimated that 150 to 200 industries will require other forms of bio-
monitoring.
How then will the results of these tests be applied? How will permit
limitations be developed from test results? Let me again outline our general
approach. At the outset, I should note that state water quality standards
generally require that nothing be toxic within the mixing zone. Some states
specify that the concentration of a substance within the mixing zone shall
not exceed the 96-hour TLM for that substance. We interpret these require-
ments to mean that the discharge at the end of the pipe must not be toxic.
In addition, state water quality standards require that outside the mixing
zone concentrations of substances must not exceed 1/10 of the 96-hour TLM.
These standards will be used to develop permit limits for appropriate toxi-
cants. In some cases the toxicant will not cause a problem at the discharge
or exceed 1/10 of the 96-hour limit at the edge of the mixing zone, but it
may bioaccumulate in aquatic organisms or be a known carcinogen, mutagen,
or teratogen. For some of these toxicants we will have the information
necessary to develop appropriate control levels for protecting aquatic
organisms and human health. For others, very little will be known and we
will have to work with the states and the industry to develop appropriate
control levels based on data derived from biomonitoring, process evaluations,
and toxicity reduction plans.
At this point I would like to address those of you in the audience who
represent industry. Much of what I have said so far has been addressed more
to the state people - engineers, biologists, lawyers - those often intimately
involved in the NPDES permit program. In some cases the states are ahead of
us in the biomonitoring area. In other states little if any biomonitoring
has occurred. In addition to exchanging information with our states, part of
the purpose of this seminar is to spread ideas and information and to let you
know what we are thinking about. For those of you who may be most affected
by what we are saying and proposing here, we believe it is to your advantage
not to wait. We believe it is to your advantage to seize the initiative
before the states or EPA knock at your door.
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Looking early at your facility gives you all the options, including
one of the most important elements - time. With no regulatory onus you are
free to investigate and experiment. You are able to select the most cost-
effective approach and time with due regard to plant operation and least
interference with union rules, worker scheduling, and the other constraints
which generally translate to time and money. Nothing is less cost-effective
and more disruptive than when the regulatory agency imposes a tight schedule
on an industry because of concerns generated by a process totally outside of
your control. You also should utilize biomonitoring in evaluating which
treatment scheme you would install to control conventional or other toxic
substances so that you can obtain the greatest incidental control of toxi-
cants.
For instance, we are just beginning a program to collect samples from
selected river basins for a broad organic scan of fish flesh to detect sub-
stances which may have bioaccumulated. These scans tell us whether or not
something of concern has been or is being discharged. Where something is
found, for example, hexachlorobenzene (HCP), chlordane, PCBs or any of
dozens of other compounds, we will then begin to look for probable sources.
Knowing the nature of the various industries in a basin we can usually
pinpoint likely sources or a probable source. This will lead to inquiries
to the industry such as a 308 letter requiring process evaluation, biomoni-
toring or additional chemical sampling. Those of you who receive such
inquiries may not have a problem but it's pretty certain that your company
will feel some anxiety and there will be time constraints for your response.
In such an instance, you can no longer plan the most optimum and least
costly path - you're under the gun. To avoid that situation you need to
begin now to do some of these things on your own. What are your processes?
What chemicals go into them? What by-products are produced? What are the
trace contaminants? If one process seems to pose a problem, should you have
a bioassay run or check for bioaccumulation? What process modifications can
you make to reduce or eliminate the problem as opposed to end-of-pipe treat-
ment? We are not asking you to do everything at once. We are asking that
you establish priorities, putting a high priority on toxic substances,
particularly those which are persistent, which bioaccumulate or which rep-
resent a human health hazard. By taking the initiative you will be able to
establish priorities and work within the many other constraints. In the
meantime, we are encouraging the states to initiate fish flesh monitoring,
biomonitoring, process evaluation, and other tools of the pollution control
business.
If any of these lead to your industry you will be ahead of the game
and probably save money if you've already done some of these things on your
own schedule. It should be obvious that the direction we have outlined is
for a long term - we do not expect biomonitoring or process evaluation to
suddenly appear in all NPDES permits. However, as these permits are reissued
this year and in 1980-81 we will be stressing these areas and they will begin
to appear more often in permits.
In summary, the tools we are discussing today are not new, but they
will receive increased use in the future particularly as they enable us to
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identify and control toxic substances. We are hopeful that this seminar will
provide greater understanding of where we are-headed: increased cooperation
between state and federal agencies and industry; greater interplay between
biologists, engineers, and administrators of agencies and companies; and
finally greater understanding and use of biomonitoring as a tool. These in
turn, are, of course, only steps along the way to our ultimate goal: fish-
able and swimmable waters in all parts of this country.
DISCUSSION
Question: I am wondering if biomonitoring will be acceptable to the 129
priority pollutants.
Answer #1: If one runs bioassays on an effluent, recognizing that an efflu-
(Mount) ent is a complex mixture, the 129 priority pollutant assessments
will never be satisfied. I don't think I could really answer
that question for certain, but as much as I understand the
consent decree, I would think that biomonitoring would not be
acceptable to the 129 priority pollutants. It might be useful,
but it seems to me that the consent decree requires certain
levels of treatment; I don't think that toxicity is one of the
parameters by which that treatment is devised.
Answer #2: I think that answer is generally correct. I would not want
(Garde- to say, however, that biomonitoring will not be part of the pro-
bring) cess that we will use when dealing with those particular parame-
ters. We are all kind of feeling our way along on the consent
decree pollutants, and I think there will be some use of biomoni-
toring in that process. However, it won't be solely a toxicity-
based approach.
Question: Along those lines, in our experience, we have found that we
are exceeding a level of toxicity for priority pollutants, but in
our biomonitoring program there is no toxicity. What can you do
about the inconsistency there?
Answer: I don't think that it's an inconsistency. I mentioned this morn-
(Mount) ing that there will be antagonisms in the effluents as well as
the opposite, which is what we always hear about - the synergis-
tic properties. I think that one ought to try to separate
biomonitoring from the specific" consent decree program because
they arose out of different contexts even though they are gener-
ally both aimed at controlling toxicity. We have all felt, for a
long time, that when you put mixtures together different things
happen than when you keep them separate, which is why I think
biomonitoring effluents is so valuable. It gives us a better
handle on the real world impact in those situations.
Question: What if we do scan the river basins and collect data by bio-
monitoring on our effluents although these are not on your
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consent decrees. When it comes down to the actual permit process
and legal proceedings, all of the data which we collect before
the process begins becomes evidence both in our favor and in many
cases against us - am I correct?
Answer: I think that's generally correct. All I can suggest to you, sir,
(Garde- is that your alternative is simply to wait, as I indicated, until
bring) you get a 308 letter. The 308 letter will have a time schedule
in it, but if you think you have a problem with some contaminant
or other toxic material that you can identify in your effluent,
you're better off to begin now. I wouldn't suggest that you
charge out and hire the consulting firm this lady mentioned
earlier, but I would suggest that you might want to come in and
sit down with the state officials or people from my staff and
talk about the kinds of things that should be looked at.
Question: What do you anticipate your reaction will be, from an enforcement
point of view, if a toxicant which wasn't listed in the per-
mittee's application or control in the permit shows up through the
biomonitoring?
Answer: I think it's a little hard to answer that question in the
(Garde- abstract. The purpose of the program that we have outlined here
bring) is to identify dischargers that have toxic materials present in
their effluents. Recognizing the limitations that are suggested
through the use of this word, I think we all generally know what
we're talking about. If we identify the presence of a discharge
that has that kind of material through biomonitoring, we'll
ultimately be looking to some kind of permit limitation. It may
be a negotiated limitation; it may be one based on a legal
process that you're painfully familiar with, but we will, I
suspect, in at least some of those cases, be looking to permit
limitations based on the presence of that material.
Question: What happens to these laboratory tests - are these data sub-
ject to subpoena?
Answer: I presume that they are. I know no reason why they would not be,
(Garde- although, as I said, I am more of an administrator than a lawyer.
bring) I can't imagine that they would not be subject to subpoena unless
they are subject to some kind of confidentiality claim through
one of the provisions of the Act.
Question: What if there is a natural toxicant in the waters coming into our
plants and that through our waste treatment systems it's not
removed - are we responsible for that?
Answer #1: Well, I think it's a legal question, not a biological one. How-
(Mount) ever, I think that there should not, in general, be any problem
in measuring the increased toxicity, if there is any which your
effluent has as a result of natural toxicants, by appropriate
biomonitoring tests.
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Answer #2: There is a specific answer to that question. I know that the Act
(Garde- deals with that very specifically within the regulations and
bring) unfortunately it has escaped me. I will try to get someone to
get the answer for you. (See statement hy Glen Pratt, p. 44 --
Ed.)-
Answer #3: I don't really see that the problem is any different, in princi-
(Mount) pie, from other situations where particular chemicals or sub-
stances, especially metals, are more toxic in one type of receiv-
ing water than in another. I think it's the same situation as
when there are properties of receiving waters which are not good
for the organisms and therefore affect the tests done in those
waters.
Answer #4: I would just like to comment on an earlier question asked by this
(Garde- gentleman referring to whether or not biomonitoring would replace
bring) screening for the 129. It is our intent, in the June 14 draft-
collated permit application performance regulations, not to use
biomonitoring as a screen for the 129 in some cases. We do,
however, encourage that in certain situations the permit writer
include biomonitoring as part of the recorded departments in
developing the permit limitations. I just wanted to make that
clear and that the NRDC consent decree is not necessarily the
driving force behind the indigenous biomonitoring program. It
was just something that got us started, and now that my office in
Washington is in the process of posing the consolidated permit
application form regulations as well as what we call the biomoni-
toring policy protocol, biomonitoring can be used on a case-by-
case basis in addition to greater chemical test condition.
Question: What is the status of application factors these days? The labor-
atory fish production indices and all these other things that
your lab has been involved with. Is it basically a 96-hour
median tolerance limit or is an artificial number chosen in order
to give safe dosages? Is there any flexibility in this proce-
dure? What's the present-day status? Is it going to be 1/10,
1/100, or what?
Answer: Well, I'm not sure, unless you have an hour, that I can answer
(Mount) that question. I think it's fair to say that if you do not have
long-term toxicity data on an effluent or on a chemical, that is,
all you have is some estimate of legality from a short test,
there isn't any better way to predict the no-effect level than
with the application factor. Now having said that, I should also
say that there is a lot of error in doing it this way. As a
result, there will probably be some error in any prediction.
Short of having data, I think it's about the only tool that I
know of right now. Based on comments that we received on the
guidelines for the consent decree criteria documents, we are now
thinking along those lines mainly because one of the early
comments that came back consistently was that you cannot general-
ize among chemicals. We agree with this. We are accepting that
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this is the situation, and we are faced with this question:
You have chemical X and you have only 96-hour lethal data on
it. What is the estimated no-effect level? We believe that
the application factor is eminently better than guessing. I want
to quibble with some of your terminology. It is not a safety
factor; I think I heard you mention the word safe or something
like that, but maybe I'm misquoting you. In any event, the
application factor, as it is being proposed, is an attempt to get
from the lethal level to the no-effect level, and I think that's
different from a safety factor. We do know that one can put
chemicals in categories in terms of the magnitude and the appli-
cation factor, for example, some are on the order of 1/10 or
less, some are on the order of 1/1000 or more, and others fall in
between. So there are some generalized differences that you can
see among chemicals, but when you try to decide whether the
application factor is 1/12 or 1/11 or 1/15, you find that the
data are simply not good enough to allow you to do that kind of
precise work. How much of that is due to experimental error,
problems of techniques and variations between laboratories, and
how much is real? We simply don't know.
Question: We've heard that we're going to have to run chemical analyses for
specific toxicants, and, based on your comments, if we're always
within limits we should never see any negative response of the
biotest for that parameter. Isn't the biotest kind of redundant
on that?
Answer: The question was, if one is within the chemical limits as mea-
(Mount) sured by analytical chemistry, one would not expect to see
toxicity in a biomonitoring test, and the answer to that is no.
I think you will expect to see it from time to time, and we do.
The reasons are, first of all, that the limitations are clearly
not on all chemicals in any consent decree. And the second
point is that we have not yet come to grips in the consent decree
v/ork or in any other work - all of these standards deal really
with the problem of nature.
Question: Let's just assume that you are operating a waste treatment plant
for industry. You have biomonitoring set up, and you determine
that your effluent is toxic; you don't necessarily know what
to attribute it to. What's your next step to correct the prob-
lem, since that's what we're interested in doing?
Answer: I think chemical analysis is the next step, along with more toxi-
(Mount) city measurements, and I think the way in which most people go
about it, if there are multiple streams making up the effluent
streams, is to go back up the line and see where the toxicity
is coming from - where most of it is coming from. I think it is
far cheaper to locate the source with biomonitoring and make the
identity with chemical analysis, than to try to do it with
chemicals all the way. If you go the analytical route, you may
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measure 475 chemicals before you find the one that's causing the
problem. But if toxicity is your concern then it seems obvious
that toxicity ought to be the endpoint in the measurements that
you make. Ultimately, of course, you'll have to identify it
analytically.
Question: Isn't it possible to get a toxic result and not actually have
a toxic condition? What if there is not enough DO in your efflu-
ent stream, or another condition exists that is not necessarily
favorable to the organisms that you are using?
Answer: I think what you're referring to is the situation where the
(Mount) conditions of the test are different from the conditions of the
receiving water. In that case, I think the test would be consi-
dered improper.
Question: If all of these companies and all of these laboratories that
are government run get into bioassay, and we choose something
like rhododendron roots or something, isn't there going to be a
logistic shortage of test vehicles and equipment? How long will
it be before you have enough supply to meet the demand where you
think this is profitable?
Answer: I don't think it's a problem because I think that the profit
(Mount) motives of those who do the test are such that they will stay
well ahead of the rate at which the regulatory agencies can
implement this. If that is not the case then I think it's
obvious, and common sense dictates, that we have to move at a
pace so that we can keep up with the demand. But, you will be
surprised at how many firms already have this capability, and the
evidence for this is the fact that the price is coming down. Now
it may not be in absolute dollars but it certainly is in real
dollars. There was a time when a static bioassay was $800, now
they're considerably cheaper in a number of places. So I really
think that that's not a big problem. We're not talking about
sophisticated equipment at this stage nor expensive equipment
compared to the modern analytical chemistry lab. I think the
problems in measuring 129 chemicals will be more costly than
this.
Question: You know that the chlorination process causes carcinogens, and
I am wondering if your biomonitoring program will take this
into consideration, maybe change, modify, or ban the chlorination
process?
Answer #1: I know of no specific program addressed particularly to the
(Mount) chlorine problem. I am aware of the controversy in the regula-
tory as well as the scientific community. I suppose that some
of the bioassays that will be run may identify chlorine as a
particular problem and then we would deal with it at the parti-
cular site. I don't know that any of the biomonitoring that we
have been talking about here, which is really intended to be
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fairly site-specific, would go to any sort of national policy or
program change with regard to chlorination. I think that issue
exists in the agency, in state agencies, and in Canada, and is
the subject of a lot of conversation. There has been some change
in the policy and there may indeed be more, but at this point I
know of no particular biomonitoring effort.
Answer #2: I know of a number of places in the country where they have gone
(Garde- to dechlorination in order to avoid the problems of the toxicity
bring) of chlorine itself. Whether chlorine itself is a problem as far
as aquatic toxicity is concerned would depend on how much time
elapses before you measure that toxicity, and that's why the
comments I've made about persistence are particularly important.
But with regard to the problem of forming other chemicals that
may be carcinogens or other such properties, I think it's recog-
nized that there are some that are formed in certain kinds of
situations. It seems to me that it's going to be a negotiation
between the trade-offs, which are what we gain by chlorinating
and what we lose by chlorinating. It is simply a conflict of not
being able to do everything perfectly; there has to be a trade-
off. I think that we may come to recognize, in our society as a
whole and in this field as a whole, that there are worse villians
running around than coliforms.
Question: When we think of other large industrial nations, where do they
stand on the subject of biomonitoring and its application to
regulations? Can you give us some information on that? Are we
the leaders, or are we the followers? Are we in step with the
rest of the world?
Answer #1: I can't give you any information. I can't say anything about the
(Mount) requirements of regulatory agencies in other countries. But I do
know that there is much activity in Europe, for example, in
England and South Africa, in developing biomonitoring tests.
Answer #2: The only place I know of now is over in the United Kingdom on the
(Garde- Thames where they've instituted continuous monitoring have had
bring) systems going for the past about 6-10 years or so - very active.
Question: What is the perceivable time frame as far as post regulations?
Answer: In terms of our biomonitoring requirements, I can't give you any
(Garde- specific number. Certainly as we go into the next round of
bring) permit issuance and as we look toward BAT, we will be starting
down that road. Within Region V, as I said, we are starting to
target particular geographic areas where problems may exist. We
are doing that through the fish flesh analyses and other data
gathering that we have. So, it will be, if not in the imme-
diately forseeable future, within the next year, into the
calendar years 80 and 81, as we go through the next round of
permit issuance. It's coming very soon.
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Question: Do you envision biomonitoring for POTW1s eventually?
Answer #1: I guess Glen (Pratt) should probably answer this question because
(Garde- he's the pretreatment expert, but let me give you my own personal
bring) perspective. I think that that really depends on the extent to
which we are able to put into effect an effective pretreatment
program. We know that there are a tremendous number of toxic
materials that pass through normal POTW systems and get out into
the waterways. I think the way to go about it is to look into
some process evaluations for industries that discharge in the
public systems, and I would say generally for this round at
least, we will be looking principally at industrial dischargers.
For municipal dischargers, our focus will be on getting the
pretreatment program going.
Answer #2: Let me briefly add to that. There might be a few cases where we
(Pratt) would be talking about having the municipality do biomonitoring
on its influent rather than on its effluent. It would be used as
part of a program to go back and control specific industries or
to look for industries that might need control. I know we are
looking, in several cases, at having biomonitoring requirements
for specific industries that would be going into municipal
systems. In general, to emphasize what Sandy said, we are going
to be looking in our normal reissuance process so that when a
given permit comes up for reissue, particularly in the short-term
permits, we're going to be requiring biomonitoring as part of the
permitting process, as well as where we have become aware of
specific problems within a given watershed. So I think it's
going to be through both of those channels. As far as the
question on which countries are doing things, I think that we
should probably look even closer to home. There are significant
differences between the regions and the states. Our region
appears a little bit behind several of the other regions. And
certainly, some states have been more able to use this in their
regulatory process. So you will see variations in biomonitoring
program applications over the next year or two. This is just a
matter of getting a program going with a lot of people involved.
Question: Are you looking at static testing as adequate?
Answer:
(Garde-
bring)
It's not the total answer, but we're certainly contemplating us-
ing it. I think we'll hear more about that as the day wears on.
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STATIC TEST USING FISH
Ronald Preston
U.S. EPA, Region III
Wheeling, West Virginia
My topic this morning is on static bioassay using fish, and I know my
attempts that follow in describing static bioassays will be unfamiliar
to some of you and will be very old hat to others. To those to whom it is
going to be a little bit new, all I can say is, well, welcome to the mystical
world of bioassay. I say that with some facetiousness because we frequently
run into the response that it is a very mysterious type of test, and some-
times we, as biologists, have some difficulty in accepting that response. I
think that if you can accept a test standard which is accepted by many people
and is a very, useful tool, then we don't have any trouble accepting the
static bioassay test.
The static test is a nondynamic test consisting simply of taking a
solution of a mixture, placing test organisms in it, and leaving it for a
specified period of time to observe the response of the test organisms.
Normally the mixture is not added to or taken away from during the test, and
once the test is set up it is left alone for the test period. There is
another type of static test that is called a static "renewal" test wherein,
on a specified periodic basis, a solution of the test concentration is added
to or exchanged with the original solution. There are several advantages and
disadvantages to the static test. The advantages meet many of the criteria
that we were into this morning. That is, it's simple, it's cheap, and
althpugh it requires trained technicians, it doesn't necessarily require the
highly trained PhD biologist. The disadvantages, of course, are that in the
static test, the toxicity that might be present may change during the test
period due to either degradation or volatility of the substance, absorption
of the toxicant, uptake of the toxicant by the organisms, or several other
factors. The flow-through test that you will hear about later takes away
those disadvantages. However, the basic experimental design and basic
requirements are well known for static tests, and I will refer you to many
excellent references. They are all very similar and describe the tests in
detail. These are: Standard Methods, ASTM, and the EPA documents on efflu-
ent toxicity testing.ORSANCO has a bioassay procedure and, of course, there
are many state agency documents describing static testing.
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When we are considering the performance of a static bioassay perhaps
it's worthwhile to understand what our objective is in looking at effluents.
It is simply this: The basic purpose of the effort of biomonitoring at the
point of discharge is to determine if acute toxicity to aquatic organisms
exists as a property of that discharge and if it does, then there may be a
potential impact in the receiving water. The next step would be to try to
evaluate that potential impact. In attempting to determine this objective
(which might be termed as a screening test) the static test serves well in
measuring this unknown property of effluent testing.
I think the best way I might be able to describe the static test, before
I refer to some of the requirements that are in all the standard references,
is to discuss some of the types of questions that we have received during the
performance of our program in Region III. We have been going on-site to
industrial and municipal treatment facilities and running some flow-through
and static tests. I think it might be good to repeat some of the questions
that we frequently run into from the industrial personnel. Once we get by
the question: Why are you there? (and I always refer to the statement that I
just made earlier, i.e., we are simply trying to determine the acute toxicity
of that discharge), the next question that we ordinarily get is: "What is
the test organism that you're using in the static bioassay and why is it the
fathead minnow? Why don't you use some indigenous form from the receiving
screen that's out there from our discharge? Why don't you use carp or blue
gill, or anything else? Basically we have a real problem with trying to use
wild stock as the test organism. You don't know anything about the quality
of the group of fish that you might collect, and you have a very difficult
time getting a constant size and age. You don't know the history of the
organism, or what it has been exposed to, and you do not have control over
the disease problems that might be inherent with the strain. There is a
whole host of variabilities that may play a role in evaluating the results of
the test, in addition to the difficulties of capturing the critters in
sufficient numbers to begin with. Therefore it's much better for the test
organism to be a fish that can be cultured, can be obtained commercially, and
that can be maintained in healthy stocks. We are trying to reduce all the
known variables, so the only variable that we would be measuring is the
quality of the effluent. By maintaining the tight control over the test
organism that is used, you have eliminated another area that could be dis-
tractive to the results of your test. As to why the fathead minnow, it can
be said that it is native to the midwest and eastern North America, and it is
the white rat, so to speak, of the aquatic testing world. There are a lot of
data available for the toxic response of this organism. It can be cultured
in the lab, and we have been able to use it successfully. There are other
organisms that probably are just as sufficient. Other fish such as blue
gill and channel catfish all meet the same kind of requirements, and in some
situations where there is a cold water environment, the trout meet these
criteria, also.
The next question that we run into frequently is: "We are going to use
the fathead minnow; where do we get the fathead minnow?" In our region, we
are attempting to develop a list of commercial sources for fathead minnow,
and, upon request, we would supply that list to anyone who is attempting to
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set up a bioassay program. We can't guarantee the quality of the fishes that
you want from these sources. We have, in our program, attempted to make use
of these commercial sources as well as other sources that we have available
to us, i.e., federal and state hatcheries. We have had some problems in
utilizing the commercial sources, and we have had some excellent results,
too. The point is: The fathead minnow is available, and the person or
industry who receives the fathead minnows must take extra precautions to be
sure that the quality of the fishes is well maintained. Another question
that we frequently get is: How are you sure they are fatheads? This brings
a chuckle from us too, because we didn't really expect that question. Just
recently we had a couple of industries bring that one up to us. My only
response at this time is: If you feel there may he some question about the
credibility of the source, that is, the species that you are getting from a
commercial source, I would simply recommend some local taxonomist to look at
a sample of the fish, make identifications, and provide the industry with a
statement of certification that these are indeed fathead minnows. As this
bioassay monitoring program goes down the road, regulatory agencies will have
the opportunity to visit industry or consulting labs that are performing
toxicity tests, and we will have the chance to make identifications ourselves.
Another question that comes up .- maybe this one comes first - is: How
much is this going to cost? You've heard some comments this morning about
some cost figures, and the guides that we have received from various contacts
range somewhere between $300 and $500 for a static test. And that means the
sample shipped to a central laboratory where the static bioassay is performed.
I would like to move on to some of the basic requirements in perform-
ing a static bioassay. What we are trying to emphasize, and no doubt in some
cases it will be overemphasized, is the quality control necessary to have a
good bioassay program. The reason that we need to emphasize this, I think,
is to reduce attempts by either a regulatory agency or a discharger who is
having bioassay performed to discredit the quality of data that is produced.
We are concerned with several areas of controls in the test. All of these
are described in the references that I mentioned earlier, and I simply want
to review them in this discussion today. We start off with facilities and
equipment, and one of the primary things that is recommended, of course, is
to have an adequate temperature control so you are reducing that variable
which can influence the results. You should have a well-ventilated facility.
It should be shielded from outside disturbances. In that regard, so you know
what I'm talking about here, it has been suggested in certain tests that
disturbances, such as laboratory people walking by highly visible test
containers, noise from machinery and other apparatus, and so forth, can
affect fish behavior and the test results. I think this is probably over-
emphasized, but we should try to control that condition. There may be
very specific additional facilities required for maintaining a stock of fish
for a long period of time. For instance, you may need to have apparatus for
continuous flow to your stock of fish in order to keep them healthy. You may
need a UV sterilizer for disease control or recirculatory system for treating
the water with charcoal filters. The materials that you use for water
transport, the construction material of the containers that you hold the fish
in during either the test or during holding times should be relatively inert.
-22-
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It has been recommended that 316 stainless steel, glass, teflon, or a similar
type of substance would minimize leaching, dissolution, or adsorption.
Another frequent item that's discussed is the size of the containers. This
is dependent upon the size of the test organisms and the loading specifica-
tions, which are adequately described in the references. We do not want to
have undue consumption of oxygen in the test container; therefore you need a
certain volume of the test solution depending upon the size of the organism.
Another area that's important to consider and one that needs to have
particular attention paid to it, if you're running frequent and intermittent
tests, is the cleaning procedure that must be performed between the static
tests that are run. The cleaning procedure, as simple as it may sound, needs
to be specified. Again, we're trying to reduce all of these variables down
to where the only variable is the quality of the effluent. The cleaning
procedure recommended is a simple washing with a detergent, rinse, use of a
solvent to remove any organics that might remain from the previous test,
dilute acid wash to remove reside metals from the previous test, and then
adequate rinsing.
When conducting the static test, normally more than one dilution or
concentration is set up, and then the question arises: Where do you get
your dilution water? Dilution water is acceptable if the healthy test
organisms survive in it throughout an acclimation period, that is, a period
of acclimatizing your fish to the dilution water prior to test, and if they
survive in that dilution water during the toxicity test without showing
signs of stress such as discoloration or unusual behavior. For effluent
toxicity testing, the dilution water should be a representative sample of the
receiving water and should be obtained from a point as close as possible to
hut upstream of and outside of the zone influenced by the effluent. It may
be practical to transport batches of water in tanks to the testing site if
the dilution water immediately upstream of your discharges is not acceptable.
Pretreatment of the dilution water should be limited to a basic
filtration through a nylon sieve having 2 mm or larger holes to remove
debris or to break up large floating or suspended solids. The water should
be obtained from the receiving water as close as possible to the time
the test starts. It should not be obtained more than 96 hours prior to
testing. If unacceptable, or if acceptable dilution water cannot be obtained
from the receiving water, some other uncontaminated, well-aerated surface or
ground water commercially available can be used. The water should have a
total hardness, total alkalinity, and specific conductance within 25 percent,
and pH within 0.2 units of the receiving water at the time of testing. If
a substitute dilution water cannot be obtained, reconstituted water may be
prepared and used for the dilutant.
Many of the items that I have mentioned about the loading of test
organisms for example, are described in the reference books on static bio-
assay. I refer you again to Standard Methods and the EPA manual. The
loading and static tests and test chambers should not exceed 0.8 g/1 at
temperatures of 20°C or less and not exceed 0.4 g/1 at temperatures above
20°C.
-23-
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There's always a question about dissolved oxygen. If you must aerate
to maintain a minimum level, and this does happen occasionally, the test
solution should not be permitted to fall below 40 percent saturation for warm
water species, and 60 pecent saturation for cold water species. If you do
aerate, then the exact methodology that you use should be detailed in a
report. Of course, organisms are not to be fed during the tests and there
should be some accompanying chemical and physical data of the solutions that
go with your report.
In summary, items that should go in your report describing the static
bioassay include the following:
• The name of the test method
• Investigator and laboratory and the date the test was conducted
• A detailed description of the effluent including its source,
date, and time of collection, composition, physical and chemical
properties, and variability
t The source of the dilution water, the date and time of its
collection, its chemical characteristics and description of
pretreatment
• Detailed information about the test organisms, including scien-
tific name, length and weight, age, lifestage, source, history,
observed diseases, treatments, and acclimation procedure used
t Detailed description of the test procedure, test chambers,
including the depth and volume of the solution, the day the test
was begun, the number of organisms per treatment, and the loading
• The definition of the adverse effect that you measured for the
test - was the data for immobility or whatever?
• A summary of general observations of other effects
• The number and percentage of organisms in each test chamber
including the control chambers that died or showed the effect
used to measure the toxicity
• How the LC50 was calculated should be described, and the EPA
may want the recommended procedure described in detail and
examples given
• Along with this should be any other relevant information that
applies to the static bioassay.
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STATIC TEST USING ALGAE
William Miller
U. S. EPA, Environmental Research Laboratory
Corvallis, Oregon
I am going to present a static test procedure that has been developed
by the Corvallis Environmental Research Laboratory. This test procedure is
an ecological test procedure that can be used to define a toxic response to
various compounds whether they be a single metal, a complex waste, or any
combination of interacting pollutants that enter the aquatic environment. I
would like to describe the development of this test from the basic foundation
to the complex application that we use today.
The test procedure was initiated in 1969 by a joint government indus-
try task force on eutrophication. The test was developed primarily as a
tool to define the interaction of nutrients in an aquatic environment and to
find out what effect might be obtained if you could regulate the nutrients
entering an ecosystem.
The key requirements that we adopted when we were developing the algal
assay test were:
• It should be designed so that technician-level personnel can
operate it
t The equipment and instrumentation should be modest and readily
obtainable. This is one area in which we may have failed (since
an electronic particle counter is necessary to conduct the test
accurately within an efficient time frame)
t It should be standardized to give reproducible results, and the
geographic location should not affect test results
t All results should be applied with judgement to real-world condi-
tions.
The test organisms that we chose for our algal assay test was Selenas-
trum capricornutum. It is a green unicellular alga that is easily cultured
-25-
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in the laboratory and is amenable to electronic particle counting. The
electronic particle counter was incorporated to measure cell density as
well as mean cell volume changes within the cells during the growth period.
The combination of the cell density measurements and the mean cell volume
changes can then be used in a calibration curve corresponding to dry weight,
which is the final product used in the test evaluation.
Two basic publications, developed in 1971, which form the foundation
of current assay research resulted from our initial efforts: an inter-
laboratory precision test of the provisional algal assay procedure, and a
standardized algal assay static test. (This static test has been further
refined (Miller, Greene, and Shirogama, 1973) and is included in Standard
Methods (1975) and is also in the ballot process with ASTM.)
One of the next things that we accomplished was to define the effects
of nutrients in 49 different lakes throughout the United States. Four basic
productivity groups were identified: low, moderate, moderately high, and
high productivity. We were able to classify the productivity potential of
the 49 lake samples sent to us by limnologists throughout the United States
through the use of a laboratory bioassay. In 23 of these lakes where we had
a wide background of limnological data, the algal assay test was able to
predict the productivity perfectly (Miller, Maloney, and Greene, 1974).
No single chemical test or biological measurement can be used to
define all the interactions regulating biological productivity in natural
waters. The relationship between algal assay bottle tests and measurements
of indigenous phytoplankton in Long Lake, Washington, has been reported
(Greene _et al., 1976). A high correlation (r = 0.95) between both mm^
indigenous p~h~ytoplankton/l and mg chlorophyll a/m3 (r = 0.93) and mg dry
weight S_^ capricornutum/1 was achieved when consideration was given to
whether the reservoir was stratified or homothermal.
The ratio of TSIN to Ortho-P content in test waters can be used as a
"guide" to nutrient limitations in natural waters. Waters containing N:P
ratios greater than 11:1 may be considered phosphorus limited. Those con-
taining N:P ratios less than 11:1 can be considered nitrogen limited for
algal growth. Confirmation of a nitrogen or phosphorus limitation prediction
is obtained by analysis of the assay response to singular and combined
nutrient (N,P) and/or chelator additions.
In most cases, the trophic status of lakes and impoundments is based
on their bioavaiTable nitrogen and phosphorus content. Those waters contain-
ing greater than 0.015 mg bioavailable P/l and 0.165 mg bioavailable N/l are
generally eutrophic.
It is important to note that, in the presence of adequate nitrogen
with phosphorus concentrations from 6 to 1,860 mg/1, 1 mg/1 of available
phosphorus will produce 0.43 mg dry weight of our test organism. Also,
1 mg/1 of available nitrogen (expressed as the summation of nitrite, nitrate,
and ammonia-N) supports 0.038 mg dry weight of our test organism.
-26-
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Some of our research indicated that the presence of toxicants pre-
vented certain waters from attaing the predicted yield or nutrient limitation
status.
Miller, Greene, and Shiroyama (l-976b), have reported that the inhibi-
tion of specific heavy metals upon the growth of _S_._ capricornutum may be
linear (0 to 100 percent) with the increase in zinc content of test waters,
but nonlinear for the increase in copper and cadmium content beyond 20 and
40 percent, respectively. These growth responses have established the
sensitivity of S_._ capricornutum to the bioreactive state of these heavy
metals. The greater than 95 percent Ij4 algistatic (inhibitory) response
of the test alga in these test waters is similar to that of sensitive indi-
genous species to accidental or recent discharges of heavy metals (an algi-
cidal response is verified when a subculture from an algistatic test water
fails to grow in assay medium). However, this inhibited response does not
necessarily reflect the growth potential of indigenous algae which have
evolved from long-term chronic exposure to heavy metals.
The study of heavy metal interaction in natural waters is complicated
by uncertainty of the form, concentration, and biological reactive state of
the metal. Thus, with few exceptions, the chemically analyzed heavy metal
content of a test water may not reflect the resultant biological interactions
and productivity in natural waters. The growth response of S. capricornutum
to conditions of heavy metal stress in natural waters is in essence a "biolo-
gical response model" of complex physical and chemical interactions. The
resultant biological response (maximum standing crop) is an integration of
the combined effects of solubility, ionic strength, metal concentration, and
contact time which regulate toxicity of the heavy metal to the test organism.
The response of the standard laboratory algal test organism to the
addition of Na2 EDTA, alone and in combination with nitrogen and phosphorus,
to heavy metal-laden test waters, has been shown to correlate (r = 0.82) with
indigenous phytoplankton standing crop (Greene et _§!_., 1978). The indigenous
phytoplankton growth in these waters can be attributed to: 1) adaptation to
their environment; 2) natural decomposition, and/or complexing of the heavy
metals by both organic and inorganic ligands; and 3) the presence of adequate
nutrients.
A case in point is the Spokane River. The Spokane River received
clarified wastewater from the Spokane Treatment Plant which was scheduled to
be upgraded to secondary treatment. The City of Spokane needed to determine
whether or not they should go to phosphorus removal as a treatment step and
what affect this nutrient reduction might have upon downstream productivity,
even though the Spokane River is also subject to upstream heavy metal inflow
from a smelter near Wallace, Idaho.
We started by defining the response of our laboratory test system to
specific heavy metals within the Spokane river system. We found that regard-
less of the algal species' interaction within a natural system, our labora-
tory test could define the growth response if the system was nutrient-regu-
lated. However, in the Spokane River, the indigenous organisms had 83 years
-27-
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to develop a tolerance to the heavy metals. .Therefore, the regulation that
was imposed upon the City of Spokane to actually go to phosphorus (nutrient)
removal was indeed justified since the heavy metals in this particular system
were not solely responsible for the deterioration of the environment. In
other systems we may find that the heavy metals are responsible and there-
fore we would have to attack the heavy metals entering the system.
How can the algal assay bottle test be used to assess the impact
of a complex waste? Since the components responsible for regulating the
biological productivity in natural waters are not necessarily identified
through chemical analysis, an alternate method is needed. The algal assay
bottle test can be used to indicate the presence of bioreactive components or
growth regulators within the waste. These components include (but are not
limited to) nitrogen, phosphorus, trace elements, heavy metals, herbicides,
and pesticides. The significance of the algal test is that both growth
stimulation and growth inhibition can be defined.
We evaluated 23 textile waste samples representing eight manufacturing
processes by seven assay techniques to define their toxic properties. The
bioassessment organisms included freshwater and marine algae, Crustacea,
fish, and mammals. A comparison of the sensitivity of these bioassays (Table
1) showed that the algal assay bottle test, using ^. capricornutum, was one
of the most sensitive tests used in the textile waste survey. This test not
only identified toxic wastes, but also those that were stimulatory (Shiroyama,
jt_ jil_., in preparation).
It is important to consider the following factors when designing an
assay protocol to evaluate the environmental impact of complex wastes:
§ The method of entry into the receiving water (i.e., direct
discharge after primary, secondary, or advanced waste treatment;
percolation through soils; etc.)
• The anticipated final concentration of the complex waste within
the receiving water.
t The degree to which the test waters are representative of those
receiving the candidate complex wastes.
In conclusion, the algal assay bottle test is a viable tool for the
study of nutrient limitation and heavy metal toxicity. It also shows great
potential for the evaluation of.complex wastes. The validation of the test
to define its sensitivity to broad classes of industrial wastes is of prime
importance. The biggest stumbling block in this validation is the evaluation
of the toxic and/or stimulatory effects of organic compounds. The reasons
for this are: 1) the safety factor in handling the compounds; 2) the vola-
tility and insoluble nature of these compounds; 3) the lack of knowledge
about the mode of interactions causing the toxicity; and, 4) the expense of
chemical identification of both the parent compound and its degradation
products within the test system.
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American Public Health Association. 1975. Standard Methods for the Exami-
nation of Hater and Waste Water. 14th Edition, p. 744-756.
Greene, J. C., R. A. Soltero, W. E. Miller, A. F. Gasperino, and T. Shiroyama
1976. The relationship of laboratory algal assays to measurements of
indigenous phytoplankton in Long Lake, Washington. In: Biostimulation
and Nutrient Assessment, E. J. Middlebrooks, D. H. Falkenborg, and T. E.
Maloney, eds.Ann Arbor Science, Ann Arbor, Michigan, pp. 93-126.
, W. E. Miller, T. Shiroyama, R. A. Soltero, and K. Putnam. 1978.
Use of laboratory cultures of Selenastrum, Anabaena and the indigenous
isolate Sphaerocystis to predict effects of nutrient and zinc interactions
upon phytoplankton growth in Long Lake, Washington. In: International
Symposium on Experimental Use of Algal Cultures in Limnology - Publication
of the Proceedings! Societas International is Limnologiae (S.I.L.) (Tn
press).
Miller, W. E., T. E. Maloney, and J. C. Greene. 1974. Algal productivity
in 49 lake waters as determined by algal assays. Water Research. 8:667-
679.
, J. C. Greene, and T. Shiroyama. 1976a. Application of algal assays
to define the effects of wastewate effluents upon algal growth in multiple
use river systems. In: Biostimulation and Nutrient Assessment, E. J.
Middlebrooks, D. H. Falkenborg, and T. E. Maloney, eds. Ann Arbor Science,
Ann Arbor, Michigan, p. 77-92.
, J. C. Greene, and T. Shiroyama. 1976b. Use of algal assays to
define trace-element limitation and heavy metal toxicity. In: Pro-
ceedings of the Symposium on Terrestrial and Aquatic Ecological Studies
of the Northwest. EWSC Press, Cheney, Washington, p. 317-325.
, J. C. Greene, and T. Shiroyama. 1978. The Selenastrum capricornutum
printz algal assay bottle test: experimental design, application and data
interpretation protocol. EPA-600/9-78-018. U.S. EPA, Corvallis, Oregon.
126 p.
Shiroyama T., E. A. Merwin, J.C. Greene, W. E. Miller, A. A. Leischman, and
H. A. Long. 1978. The comparative results of the AAP:BT to other
bioassay procedures in the determination of stimulatory/inhibitory
effects of textile wastewater effluents, (in preparation).
U.S. Environmental Protection Agency. 1969. Provisional assay procedure.
National Eutrophication Research Program, U.S. EPA, Corvallis, Oregon,
62^ p.
U.S. Environmental Protection Agency. 1971. Algal assay procedure: bottle
test. National Eutrophication Research Program, U.S. EPA, Corvallis,
Oregon. 82 p.
Weiss, C. M., and R. W. Helms. 1971. Inter-laboratory precision test.
National Eutrophication Research Program. U.S. EPA, Corvallis, Oregon
70 p.
-29-
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Table 1
COMPARATIVE RIOTEST RESPONSES FOR TEXTILE EFFLUENTS*t
Freshwater Ecology Series
Textile
Plant
A
B
C
D
E
F
G
H
J
c^ K
o L
' M
N
pa
R
S
T
U
V
M
X
Y
Z
Fathead
Mi nnow
(96-hr LC5(
% Secondary
Effluent
19.0
MATb
46.5
NAT
NAT
NAT
64.7
c
NAT
NAT
23.5
NAT
48.8
NAT
16.5
NAT
46.5
NAT
36.0
55.2
NAT
NAT
NAT
Daphnia Selenastrum
)) (48-hr EC50) (14-day EC50)
/ % Secondary % Secondary
Effluent Effluent
9.0
NAT
41.0
NAT
7.8
81.7
62.4
40% dead at
100% concen.
NAT
NAT
28.0
60.0
100% dead at
all dilutions
NAT
8.0
NSAd
NAT
12.1
9.4
6.3
NAT
NAT
42.6
11.3
____
< 2.0
___ _
7.8
12.0
< 2.0
__ _ _
8.8
__ —
1.0
._ — —
__ _..
15.5
Recommended
Interpretation
Selenastrum
20% Secondary
Effluent
%Il4 %Si4
53
—
—
__
956
—
—
92
-_
__
81
—
956
__
95
—
__
—
95
_«
— —
84
83
187
100
598
390
76
57
149
38
. _ _ _
382
1911
377
232
163
261
Marine Ecology Series
Sheepshead
Mi nnow
(96-hr LC50)
% Secondary
Effluent
62.0
NAT
69.5
f
NAT
NAT
NAT
f
f
NAT
NAT
f
47.5
f
f
NAT
68.0
NAT
f
37.5
NAT
f
f
Grass
Shrimp Algae
(96-hr LC50) (96-hr EC50)
% Secondary % Secondary
Effluent Effluent
21.2
NAT
12.8
f
NAT
NAT
NAT
f
f
NAT
NAT
f
26.3
f
f
NAT
34.5
NAT
f
19.6
NAT
f
f
f
g
90
f
10 to 50
85
59
f
f
77
1.7
f
2.3
9.0
f
g
70
g
94
50
g
f
f
a Sample inadvertently collected prior to settling pond. b No acute toxicity. c Diseased batch of
fish nullified this analysis. d No statistical analysis because heavy solids concentration obscured the
analysis; the sample did not appear to be acutely toxic. e 95% growth inhibition in 2% solution of secondary
effluent. f Analysis not performed on this sample. 9 Growth inhibition < 50% in 100% solution of
secondary effluent. * No chemical mutagen was detected by 10 microbial strains. t No rat mortality
after 14 days due to maximum dosage of 10~5 rn^/kg body weight (LD50). However, six samples (B, C, F, L, N, and S)
showed potential body weight effects, and sample P resulted in eye irritation.
-------
FLOW-THROUGH TEST USING FISH
William Peltier
U.S. FPA, Environmental Research Laboratory
Athens, Georgia
I looked at the program today and I noticed I was listed as being
located in Duluth, Minnesota, and I know that there are a lot of industrial
representatives in attendance today that have plants in Region IV and wish I
were in Duluth, Minnesota. This is because Region IV does have an aggressive
program, as some of you know, and, hopefully, today I can pass on some of
Region IV's experiences regarding the flow-through testing utilizing fish as
the test organism.
There are several types of flow-throughs; the first is a continuous
flow-through system, and that's exactly what it means. There is a contin-
uous flow of the pollutant mixed with a continuous flow of dilution water
that, after mixing together, goes to an aquarium containing fish. This
procedure can be accomplished in several ways. One is through the use of
metering pumps, where prescribed volumes of a pollutant and dilution water
can be delivered to a mixing chamber. Another approach is the use of a
constant water level head box of the type that many of you have seen in some
of the research laboratories or in some of the state mobile laboratories.
This second approach uses capillary tubes of different inside diameters
inserted in head boxes containing the pollutant and dilution water which
allows for a continuous regulated flow into a mixing chamber. In both
examples, following the mixing, the solution flows into the replicate test
tanks containing fish. These are two examples of a continuous flow-through
system.
The second type of flow-through system is the intermittent flow-
through system which is widely used and is the type used in Region IV. The
intermittent flow is patterned after the famous Mount and Brungs (1967)
proportional diluter system upon which we all have tried to improve over the
past years. It's like trying to build a better mouse trap. For instance,
Region IV has designed and built a total solenoid system which is described
and pictured in the EPA 1978 publication Methods for Measuring the Acute
Toxicity of Effluents to Fish and Invertebrates. This system will be shown
during theslide presentation andits operation explained to those people
that aren't familiar with this diluter system. In this system, flows to the
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proportional diluter can be adjusted to allow for the filling of the separate
dilution water and effluent chambers, and then when they are completely
filled, a liquid level switch triggers the emptying cycle. All chambers
empty into mixing chambers and from there flow into the aquariums containing
the test organisms. There is a delay in the flow of solution going to the
aquariums during the filling period. This delay varies anywhere from 2
minutes to 15 minutes and is usually dictated by the desired turnover time in
the aquariums.
The flow-through system has long been used in the fish toxicology
field. Applications of the system have been in the area of fish, inverte-
brate, algal, and bacterial testing. At the present time in Region IV,
flow-through tests are being used to determine the acute toxicity of efflu-
ents on fish species - acute meaning a short-term exposure period of usually
96 hours or less in duration. Flow-through tests also can be used for a
longer duration or chronic testing. Chronic testing may include exposure to
a pollutant for a portion of the fishes' life or may include the entire life
cycle. A life cycle test usually starts with embryos and continues through
life stages such as larval, juvenile, adult, and second generation embryo
larval stages. Of course this approach is time-consuming and very expensive.
In the past, most of the flow-through testing has been conducted using a
single chemical as the pollutant and determining its affect on fish. This
type of work has been conducted at universities, industries and federal
facilities. Flow-through testing has been used to establish safe limits or
no effects limits for chemicals such as pesticides, toxaphene, and endrin or
the heavy metals, zinc, copper, and mercury.
The use of the flow-through system can be taken a step further and
used in multiple chemical testing. For instance, with a slight modification
of the diluter system, it has been used to determine the interaction of
toxaphene when mixed with malathion. The main thrust in Region IV's toxicity
testing program is to determine the toxicity of complex effluents by utiliz-
ing the flow-through system. The system is very useful when one is dealing
with a "witch's brew" of compounds that are being discharged into the receiv-
ing water. The system is very useful in detecting wide variations in efflu-
ent concentrations during testing. In Region IV, effluents have been tested
where in excess of 60 organic compounds were identified in a single effluent;
however, they were not limited in the NPDES permit. At times, fish lethality
has occurred well into the test and has been attributed to a slug release of
effluent. Fortunately, because a flow-through system was being utilized,
these slug releases were detected by the fish dying.
The flow-through system can.also be used to determine the bioaccumula-
tion of individual compounds or compounds in effluents by fish when they are
exposed over a certain period of time to the pollutant. Fish tissues can
then be analyzed for residue accumulation. The test can be taken a step
further by sacrificing a portion of the fish for chemical analysis and then
flushing the aquariums containing the remaining fish with uncontaminated
water for a period of time. Following a period of exposure to the uncon-
taminated water, the remaining fish are sacrificed and the tissue analyzed
for the depuration of the pollutant. Also, the system can be utilized by
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industries as an on-line monitor to allow the permittee to continually
evaluate the effluent for changes which could adversely affect fish in the
aquarium and permit the industry to headoff potential fish kills in the
receiving water.
Additional uses can be made with slight modifications to the system to
study the avoidance of fish to various concentrations of pollutants, the fate
of pollutants such as degradation products, or decay rates. As you can see,
the flow-through system offers a wide application of uses in the area of
aquatic toxicology.
The following are advantages in using flow-through systems. First, in
addition to the acute test results, the duration of the exposure period can
be lengthened to derive chronic or sublethal results. Second, the problem
with maintaining a proper DO level can be reduced considerably in a flow-
through system. This problem is not completely eliminated because some
effluents have a very high biological oxygen demand or chemical oxygen
demand, and because of this, it has been necessary to aerate the solutions in
the aquariums in order to maintain suitable oxygen levels. However, for the
most part, in flow-through systems, the oxygen level is easily maintained
without the aid of aeration. As a result, larger fish can be tested or
increased numbers of fish can be tested for each concentration. Third, the
metabolic waste products from fish, which deplete the DO, are flushed from
the aquariums and do not cause the problems experienced in static tests.
Fourth, it is easier to achieve a steady-state chemical concentration in
a flow-through system when testing single compounds or mixtures of the
compounds. Fifth, in Region IV, approximately 50 to 75 percent of the
effluents were variable in their concentrations. As a result, the flow-
through system was useful in detecting slug discharges. It's the slug
discharges that usually cause the fish kills in our receiving waters.
Additional advantages are better control of the concentrations of
the volatile pollutants and better precision.
The following are some of the disadvantages. The first would be that
this sytem requires a higher degree of technical expertise to conduct the
test. Second, a greater degree of equipment sophistication is required as
will be seen from the slide presentation. Third, it requires copious
quantities of dilution water. Fourth, at times it can require much more
space. Finally, the most important disadvantage is cost. After polling 187
consulting firms located in Region IV during August 1979, it was established
that 27 of them had the capability for conducting toxicological studies.
Some firms provided information on the cost of various tests. Therefore,
some cost estimates are available at this time. An acute flow-through test
conducted at the consultant's laboratory on a single compound or an effluent
ranges between $700 and $1,000. The lowest estimate was $400 with the
highest at $1,500. For conducting acute flow-through tests in the field on
an industrial effluent, the consultant's cost ranged from $5,000 to $7,000
for an 8-10 day study. The lowest estimate was $3,000 and the highest,
$15,000.
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At this time, I would like to show a few slides. The first slide
(Figure 1) shows one of Region TV's mobile toxicity trailers set up at an
industrial site. In the trailer there are two solenoid flow-through systems.
During a test, a portion of the effluent is continually pumped from the
discharge ditch into the trailer. Dilution water is collected daily from the
receiving water upstream from the discharge outfall. The diluter water is
stored in a tank and then pumped into the trailer.
The second slide (Figure 2) shows the proportional diluter solenoid
system. The dilution water fills upper chambers; when the last chamber
fills, a liquid level switch triggers an electrical mechanism which opens the
solenoids, allowing the dilution water to flow into mixing chambers. Simi-
larly, the effluent goes into the small chambers located directly beneath
the dilution water chambers. The effluent in the chambers also empties into
the mixing chambers at the same time the dilution water is emptying into
them.
The third slide (Figure 3) shows what I would call a poor man's
diluter system. It is where a minimum of three solenoids are utilized and
the operation is based on a vacuum siphon system. The siphon system operates
very similarly to the total solenoid system; however, it is much less expen-
sive than the total solenoid system.
At the EPA laboratory in Athens, Region IV is evaluating the Reckman
Microtox, which some of you may have read or heard about. I want to make one
thing perfectly clear, and that is Region IV is evaluating the instrument for
Beckman - we are not endorsing or selling the instrument. For the past 18
months, we have been testing complex effluents to determine the 24-hour LCBOs
(lethal concentration which is lethal to 50 percent of the test organisms)
for fish and daphnids and comparing the results with a 5-minute EC50s (effec-
tive concentration which causes an effect on 50 percent of the test organisms)
obtained from the Microtox unit. The principle of the test is based on
the use of freeze-dried, saltwater, luminescent bacteria which are recon-
stituted in a buffer solution. An aliquot of cells are then placed in a
cuvette which in turn is placed into a chamber in order to measure the light
output from the bacteria. Then the effluent is added and the light output
is measured again for either a decrease, increase, or no change in the light
output. When testing various effluent dilutions, an EC50 can be calculated.
The EC50 in this case would be the concentration that would reduce the light
by 50 percent. I would just like to briefly say that, from our observations
and the limited data, the unit has some advantages and some disadvantages.
The following are the advantages of the Microtox. Its evaluations are
very rapid; it takes about an hour to test a control and five effluent
concentrations. While the results from the static fish and Daphnia test are
not available for 24 hours, the data from the Microtox unit is available in
one hour. When using a standard toxicant, the results from the unit are
fairly accurate and provide good reproducibility. The unit is very compact
and fits nicely on a standard laboratory bench. A direct digital display
readout is standard, or a recorder may be attached for continuous recording.
-34-
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CO
en
Figure 1. Mobile toxicity trailer set up at an industrial site (Region IV).
-------
Figure 2. Proportional diluter solenoid system.
-36-
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Figure 3. Solenoid, vacuum siphon diluter system,
-37-
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The following are disadvantages of the Microtox. First, the highest
concentration that can be tested at the present time is 50 percent. The
reason for this limitation is the test requires a 0.5 ml addition of bacteria
cells to 0.5 ml of test solution which results in the 50 percent test solu-
tion. Therefore, the highest concentration that can be tested is a 50
percent effluent concentration. Second, the color of effluents poses a
problem due to certain colors masking the luminescent output from the bac-
teria. Therefore, a reduction in light may be due to either the pollutant's
effect on the bacteria or due to the color absorbing the light. Third, some
tests have been providing false negatives. False negatives are defined as
where lethality has occurred in the tests with fish and/or invertebrates, but
the Microtox test results have indicated no significant change or an increase
in the luminescence from the bacteria. Therefore, if you are using the unit
as the sole tool in determining an effect, you're going to be misled in some
cases. So far, between 10 and 20 percent of the effluents tested have
resulted in false negatives. Fourth, fresh water effluents require that
sodium chloride be added to bring the salinity up to 20 parts per thousand
prior to testing. The reason for this is the bacteria used in the test is a
saltwater species. Therefore, there is some question from the regulatory
aspect on the validity of the results obtained from testing an effluent
sample to which some compound has been added that may change the effluent
chemical characteristics. Representatives from Beckman Instruments are fully
aware of all the disadvantages and are working to resolve the problems. They
have indicated recently that they may have a breakthrough in the color
problem, and may also have a technique for increasing the effluent concen-
tration tested. In Region IV, the Microtox unit will certainly not replace
fish toxicity tests in the regulatory requirements; however, once the prob-
lems are resolved, it may be used as an additional tool in the aquatic
toxicologist's tool box for screening effluents for toxicity. Also, keep in
mind that the findings from the evaluators testing pure compounds may be
entirely different than what Region IV has found with the complex effluents.
Also, there is one last topic I would like to cover briefly which
involves an instrument which was developed and is being used in Europe to
monitor toxic effluents. Region IV^ is presently evaluating this instru-
ment which is called TOXIGUARD and is manufactured by EUR-Control. The
company has an office in Decatur, Georgia. The Europeans are using it as an
on-line monitor of raw waste before to the wastewater goes into a treatment
system. The instrument utilizes bacteria growing on multiple plates with a
small continuous flow of wastewater passing through an aeration chamber and
then through a chamber containing the bacteria growing on the multiple
plates. If the wastewater is not toxic and the bacteria are not stressed,
then normal biological activity occurs and the DO in the wastewater is
removed. As the wastewater exits the chamber, it passes across a DO probe
which monitors the DO level. If there is zero DO, then the bacteria are not
stressed, and it is assumed that the wastewater is not toxic. However, in
the event the DO begins to increase and it approaches a pre-determined
milligrams per liter, an alarm bell goes off, and the operator can divert the
of November 1979, Region IV is no longer evaluating the TOXIGUARD.
-38-
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wastewater to a back-up lagoon so it won't kill the bacteria in the treatment
system. We're starting to evaluate the instrument using complex effluents
and comparing the results with the results obtained from acute toxicity tests
with fish.
LITERATURE CITED
Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish
toxicological studies. Water Res. 1:21-29.
U.S. Environmental Protection Agency. 1975. Methods for measuring the acute
toxicity of effluents to aquatic organisms. Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio.
The author is presently an aquatic biologist with EPA Region IV and is respon-
sible for the aquatic toxicology program. His office is located in Athens,
Georgia 30605, and he can be contacted at the following phone numbers:
FTS-250-2294 or Comm. 404/546-2294.
-39-
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BIOCONCENTRATION TESTS FOR EFFLUENTS
Gilman D. Veith
U.S. EPA Environmental Research Laboratory
Duluth, Minnesota
One of the objectives of biomonitoring programs for complex effluents
is to select the most cost-effective set of tests which provide reasonable
assurance that potential hazards of a discharge have been evaluated. Stan-
dards for BOD have been in use for a long time, yet it is obvious that many
highly hazardous chemicals could be discharged even though the BOD standards
are met.
Although recent studies indicate that most effluents receiving proper
secondary treatment show little acute toxicity to- aquatic organisms, acute
tests are the best experience tests to conduct on an effluent after BOD has
been removed. The next level of concern is the chronic toxicity of the waste
stream which could be determined from the more costly embryo-larval test.
Finally, even if testing showed no hazard from the above tests, chemicals
such as PCB, hexachlorobenzene, etc. could be discharged at quantities great
enough to cause hazardous residues in fish in the receiving waters. The
reason is that these chemicals are extremely bioaccumulative and will be
found as concentrated residues even when the concentration in the water is
below a toxic level. Consequently, there is a need for a test for bioaccumu-
lable chemicals and a need for a strategy for using the test in an appro-
priate manner.
First of all, let me just define some terms. The bioconcentration
process is the accumulation of chemicals in the body of an animal over and
above the ambient concentration. In the case of fish, it's simply the
amount in the fish tissue compared to the water environment or the food.
There's been an argument in the literature whether fish bioaccumulate more
through the gills by respiratory uptake and partitioning into the fish, or
through the food chain. The answer is that both mechanisms are important,
and the question of how important depends on the environment in a very
predictable way. The bioaccumulation process is the sum of bioconcentration
and biomagnification; biomagnification is that which is accumulated from
ingesting the chemical, and bioconcentration is that which fish can accumu-
late through the gills when placed in water with that chemical dissolved in
it.
-40-
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The bioconcentration test is conducted by placing approximately 35
fish such as the fathead minnow in a large aquarium. The chemical or effluent
is continuously added, and composite samples of five fish are removed after 2,
4, 8, 16, and 32 days for analysis. It is generally observed that the chemi-
cal is accumulated in a manner simulated by a first-order process which gives
a rapid initial uptake followed by a slower rate and finally a relatively
constant residue concentration (Figure 1). This point is called the steady-
state concentration for the exposure and the bioconcentration factor is
calculated by dividing the steady-state residue by the water concentration.
Reasearch has shown that the ratio between the fish residue and the
water concentration of a chemical, i.e., the bioconcentration factor, is
constant over a wide range of water concentrations. This means that the
steady-state residue in fish can be calculated from the water concentration
if the latter is known. Also, the hioconcentration factor for chemicals
varies from less than one to greater than 100,000. Chemicals which have
small bioconcentration factors generally reach steady-state in fish within
the first few days of exposure whereas those with high hioconcentration
factors may take several weeks or may never reach a true steady-state con-
dition. Figure 1 shows the uptake of hexachlorobenzene in various age groups
of fathead minnows. Although it appears that a steady-state is reached, a
closer examination shows that the residue continually increases over the
115-day exposure.
The research we have completed at the Environmental Research Laboratory
- Duluth suggests that a 30-day exposure is the longest period of exposure
needed to assess the bioaccumulation potential even though the actual accumu-
lation in the environment may be slightly higher for the higher bioaccumulable
chemicals. Moreover, the test can be readily adapted for effluent tests
using either live cages or a single aquarium receiving the effluent. We have
conducted bioconcentration tests concurrently with toxicity tests by analyzing
surviving fish from toxicity tests for residues. While the kinds of experi-
ments give a rapid indication of the presence of highly bioaccumulative
chemicals, they may not give the same bioconcentration factors obtained from
the longer bioconcentration test.
Because bioconcentration tests on complex effluents require a quali-
tative and quantitative chemical analyses of residues in the exposed fish,
the cost of the tests in a monitoring program is still rather substantial.
Consequently, we have developed a screening test which is based on the use of
bioconcentration factors but reduces the overall costs. We tested chemicals
which were representative of a wide range of chemicals and found that the
bioconcentration factor could be related to the n-octanol/water partition
coefficient as shown in Figure 2. The octanol/water partition coefficient
is a measure of the fat solubility of chemicals and is generally inversely
related to water solubility. Chemicals such as detergent builders, solvents,
phenolics, etc. have a high water solubility and a corresponding low biocon-
centration factor. Chemicals such as the chlorinated pesticides, PCB's, and
brominated flame retardants have a very low water solubility and a corres-
ponding high bioconcentration factor.
-41-
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I
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• Newly hatched fry
o 30-day fry
A 90-day juvenile
• Adults
20 30 40 50 60 70 80
Exposure Time (days)
90
100 110 I2O
Figure 1. Accumulation of hexachlorobenzene by fathead minnows of four age groups.
-------
CO
"8 5
O>
-8
CM
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U_
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GO
0
0
Log BCF=0.85 Log P-0.70
R =0.947
N=59
• Fathead minnow
D Rainbow trout
A Bluegill
234567
Log P (n-octanol/water)
8
Figure 2. Relationship between the bioconcentration factor of 54 organic chemicals
in fish to the log P of the chemical.
-------
This correlation is useful because it will permit estimates of biocon-
centration factors for chemicals without testing. Only if those chemicals
which have a large partition coefficient are being used in a plant would a
detailed bioconcentration test be justified to determine if such chemicals
are leaking out of the plant. Since the log P can be estimated from struc-
ture, the overall estimate of bioaccumulation potential can be achieved at
the screening level at low cost. We have found that chemicals with partition
coefficients less than 1000 are unimportant from a bioaccumulation viewpoint,
and bioconcentration tests should be limited to those chemicals most likely
to be bioaccumulative. Since the majority of industrial chemicals belong to
this group, these screening relationships may substantially reduce the cost
of testing.
If the effluent or wastewater is so complex that it is impossible to
list the possible chemicals, it is still possible to screen the effluent for
bioaccumulative chemicals to determine if the bioconcentration test is
justified. In the EPA report EPA-600/3-78-049 (available from NTIS), we
showed that the partition coefficient of chemicals can be determined from the
retention time of the chemical on a reverse-phase liquid chromatography
column. Consequently, the retention time of the "peaks" on this column can
be used to estimate log P and therefore the bioconcentration factor of
chemicals, even though the identity of the "peaks" is not known. Screening
for chemicals of high bioaccumulation potential is translated to screening
for chemicals which have a long retention time on the reverse-phase column.
Since water is used as a solvent, effluent samples can be injected directly
onto the column or a precolumn and effluent screened within twenty minutes.
If "peaks", or chemicals, are detected which have a long retention time, it
is highly probable they will have a large log P and a large bioconcentration
factor. If none are detected, it is unlikely a bioconcentration test will
provide useful information concerning the hazards of the effluent.
DISCUSSION
Statement
by Glen
Pratt, EPA
Chicago
Question:
Let me just clarify one question that was asked earlier today on
natural toxicants in an industry's intake water. If you have a
material in your intake water and if you do not add any contami-
you are not responsible for it in
is that if you take water from one
put it into another waterway, you
a lower quality than the receiving
nants to it in your process,
your effluent. The exception
waterway or from a well and
would be liable if it were of
waterway.
In the last session a question was asked regarding what other
jurisdictions were doing with respect to biomonitoring. I might
point out that in Canada there is a requirement in the regula-
tions to have industry do bioassay tests, basically 6-hour 1X50
flow-through tests. Also, to carry on with a question that was
raised this morning which addressed the increase in the amount of
biomonitoring that's going to be requested by the U.S. from the
-44-
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Answer:
Question:
Answer:
Question:
Answer:
state agency, I've got a problem with lab availability now, and
it may be a problem in the future. Also what kind of quality
control problems will there be as a result of the increased
workload?
Are you asking will laboratories be available?
The problem is that now in Illinois I don't know of more than two
or three sources that are available commercially to do biomoni-
toring. For example, if 30 industries have biomonitoring require-
ments within their permit, how are they going to get the moni-
toring accomplished? We will get to the point where labs start
to spring up, and then what kind of quality assurance are we
going to get from these labs?
I don't have too much concern with the first problem of lab
availability so long as the regulatory agencies don't impose
unreasonable deadlines in doing this. I think you'll be sur-
prised to find how many labs really are now able to do these
tests, and I can assure you that, if the need is there, those who
are willing to do them for a fee will also appear. This is
state-of-the-art technology rather than research tools at this
stage. As far as quality control is concerned, there are a
number of round-robin tests being performed right now to measure
the precision of these various tests. There are activities by
the Office of Toxic Substances and probably state agencies, too,
to develop good laboratory practice manuals and offer laboratory
certification. So I think there's a lot of activity in the area
of quality control.
We're looking
what criteria
for any given
at a lot of bioassay tests today,
is used in determining which test
waste stream?
and I'm wondering
might be suitable
It's my view that this should be based on an industry-by-industry,
pi ant-by-plant basis. What we're trying to do today is to give
you the broad range of tests that are available. In our work
and the state's work with industries, we need to look back at
the specific processes in the plant. This is the one reason
why we believe it's tied with process evaluation. Are you
looking at a type of compound in the plant that you think would
tend to be a long-term bioaccumulative material?
Are you looking at a process in a plant that produces only an
intermediate compound which is consumed within the plant? When
Don Mount says you can't generalize, we totally agree, and this
is why we're not having a broad spectrum requirement that every
industry must do test A, B, or 2 from Column A and one from
saying that it should be a pi ant-by-
are in an industry that doesn't have
Column B. Instead, we're
plant evaluation. If you
biologists on its staff, you might want to get together with
-45-
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either the state agency or a biological consulting firm to see
which particular test would be suitable for your given facility.
Answer #2: In the way of clarification, in case it slipped by some of you,
the bioconcentration tests that Gil described is directed at a
phenomenon or an effect that we're concerned about at levels
below those concentrations causing toxicity. So if you have a
direct toxicity problem, you may not be concerned with the
bioaccumulation test at this point. I think it is clear from
everyone I've talked to that it is not prudent to require all of
these tests by any means, but rather to select those that are
most appropriate to the problem that you have.
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SEDIMENT BIOASSAY
Max Anderson
Central Regional Laboratory
U.S. FPA, Region V
Chicago, Illinois
Bayliss (Rock) Prater
Aqua Tech Environmental Consultants, Inc.
P.O. Box 76
Mel more, Ohio 44845
Max Anderson
The need for a sediment bioassay in Region V became quite apparent in
1976 and 1977 when we encountered an increase in dredging activities in the
harbors and streams around the Great Lakes. At that time, Rock Prater was
working for the Central Regional Laboratory on a special assignment. Through
a combined effort, we adapted an apparatus that was originally described by
Cal Fremling of Wannona State College and have been using that as our sedi-
ment bioassay chamber. The chamber is depicted in Figure 1.
The chamber is constructed entirely of glass and is a closed cycle
system. An aerator forces air and water to circulate throughout the system.
The sediment is deposited in the upper part along with the organisms that are
being tested. The small cup on the top left side is used for the Daphnia
bioassay. We place other organisms within the chamber itself. If we're
using fish as our test organisms, they are placed in one of the larger glass
jars. The cost of this particular apparatus runs between $30 and $50, so
it's not very expensive and it works quite well.
We have adapted and enlarged this apparatus at the Central Regional
Lab for use in long-term biomagnification work that is just getting started.
Since we're only using fish in this particular study we find that we can
maintain them for a longer period in the larger unit. The principle is
exactly the same as the smaller unit but it is constructed of a 10 gallon
aquarium on the top and two 5-gallon aquariums underneath (Figure 2).
In Figure 3 we have two tandem units together. Figure 4 shows a bank of
units in our environmental chamber. We estimate that the cost for setting up
-47-
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*****
Figure 1. Schematic diagram of a closed-cycle sediment
bioassay chamber.
-48-
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Figure 3. TWO tandem closed-cycle bioassay units (Central Regional Laboratory,
Region V).
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one of these unit would be about $75 to $100, which is relatively inexpensive
if you're interested in a long-term biomagnification study.
I would like now to turn the time over to Rock Prater who is going to
discuss the application of this sediment bioassay apparatus based upon some
extensive work that he has done.
-52-
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Rock Prater
It never fails to amaze me that, after 21 years of teaching, I still
get nervous standing in front of a group. Not to use this platform as a
business opportunity, but as of last Friday, I am no longer with Heidelberg
College; I'm with a consulting firm that's doing primarily sediment bioassay
work.
As Donald Mount said in his introduction this morning, there are two
accepted approaches to determining the condition of an aquatic ecosystem -
animal counts in standing crop and identification of chemical parameters.
Although the ecosystem might contain an acceptable set of chemical para-
meters, there are situations in which no animals are present. Unfortunately,
the impact of the sediments in such a situation is usually overlooked.
This particular apparatus that we are currently using for sediment
bioassay is quite basic; however, I think that the data that we have accumu-
lated after investigating harbors on all the major lakes, with the exception
of Lake Ontario, shows that it seems to be working quite well. The apparatus
still needs refinement; we are soliciting a grant to continue development.
Mr. Robert Hoke, who is also with Aqua Tech, will be a valuable asset since
he has just finished three years of graduate research in this area.
We have used this apparatus in both riverine systems and lakes.
Figure 1 shows a small creek which receives effluents from four major
industries. One of these, the "oil refinery", was being taken to task by
EPA for thermal pollution of this stream. We took samples above and below
the major outfalls to determine what the bottom type was like.
We used three organisms in this bioassay - Hexagenia limbata, Asellus
communis and Daphnia. Hexagenia limbata is a burrowing mayfly which is
especially usefulff you're going to look at the effects of sediment.
Asellus communis is an isopod that lives at the interface, and Daphnia is a
water flea which lives within the water column. In other studies we usually
use fish.
Figure 2 indicates the toxicity to the three major species that we
used. Proceeding downstream from the control station we get 100 percent
mortality during the 96 hours, a small amount of recovery, and then an in-
crease. Put what does this mean in relationship to the refinery? Should
they be forced to eliminate the thermal discharge when indeed the sediment
types that currently exist (and we did profiles on these) might prevent the
existence of a healthy ecosystem? In addition, the seiche effect on this
system would prevent any scouring so that the return of a healthy ecosystem
may take a number of years or in fact may never occur. This is a typical
example of the need for a sediment bioassay. If we simply look at the water
column, or at the standing crop of organisms, and ignore the sediment type
we may be amiss.
-53-
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Maumee Bay
Oil Refinery (0.8 km)
Station 0.4 km-
Sediment 5
Station 1.3 km-
Sediment 4
Glass Mfg. (10.9 km)
Water Treatment Plant (4.9km)
Station 6.8 km-
Sediment 3
Oil Refinery (8.7 km)
'Station 9.3 km-
Sediment 2
Control Station 13.0 km-
Sediment 1
Sediment Station
and Number
Figure 1. Sediment stations and industry locations
on Otter Creek, Ohio (not to scale).
-54-
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-55-
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This technique can also be used in making management decisions. We
are currently under contract with the Corps and private industry to study a
number of harbors. The situation that exists in Indiana Harbor is a good
example since it is generally recognized that Indiana Harbor is a toxic
environment.
If the harbor is dredged, how do we dispose of the dredged material?
Should we dump it into the open lake, or should we take it upland? The
sediment bioassay can be used to help answer this question.
In Figures 3 and 4, it is evident that the toxicity of the sediments
decreases as we go from the harbor to the open lake. Since the toxicity of
the dredged material varies between locations, the disposal technique should
vary accordingly. If the alternative is to dispose of the toxic material
upland, it still must be capped. The most reasonable approach, then, would
then be to use the less toxic material as the cap, since it would have to be
dredged anyway.
Another example is Ashtabula Harbor. We did a study of the upper
layers only. Using this sediment bioassay apparatus, we determined that
these upper layers were only moderately polluted or nonpolluted. The results
led me to conclude that the dredged material should be allowed to be disposed
of in the open lake, which is what I would normally recommend for moderately
polluted or nonpolluted sediments. However, EPA did not agree.. They wanted
to know if the toxicity changed with different depths of the sludge.
So, in the next series of samples we looked at the toxicity at differ-
ent levels. According to the results presented in Figure 5, the top layer
of site 6 (Ash-6T) was more toxic than the bottom layer (Ash-6B). However,
at site 2, the top layer was less toxic than the bottom layer. If you must
decide how deep to dredge and where to put the dredged material, this type of
information is a necessity. If the top 6 feet are not very toxic but the
underlying sediments are, it makes sense to dredge to the six foot level,
dispose of the nontoxic material in the open lake and do something else
with the underlying material.
Unfortunately, Figure 5 indicates that the replicability is not
as good as we would like. But anyone who has done chemical analysis on
sediments will admit that replicates can vary considerably.
Basically, that is the sediment bioassay program that we are using to
determine what we are going to do with dredged spoils from harbors and ship
slips in the Great Lakes. It is my opinion that there will be more sediment
bioassay work down the road. Although it needs refining, it is a very
substantial technique.
-56-
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100 -
Pimephales promelas
_ Hexagenia limbata
= Daphnia magna
Sediment IH-1
Sediment IH-2
Sediment IH-3
Sediment IH-4
B
Sediment IH-5
A I B
Sediment IH-6
Sediment Numbers (Sites)
Figure 3. Percent mortality of Hexagenia limbata, Daphnia magna, and
Pimephales promelas during a 96-hour sediment bioassay of
Indiana Harbor, Indiana, 1978.
-57-
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«••
"5
100-1
90-
80-
70-
60-
50-
40 H
30-
20-
10-
H Pimephales promelas
_ Hexagenia limbata
= Daphnia magna
00
0 0 S 000
Sediment IH-7 Sediment IH-8 Sediment IH-9 Sediment IH-11
A I B
Sediment IH-12
A | B
Sediment IH-13
Sediment Numbers (Sites)
Figure 4. Percent mortality of Hexagenia limbata, Daphnia magna, and
Pimephales promelas during a 96-hour sediment bioassay of
Indiana Harbor, Indiana, 1978.
-58-
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Sediment
Sites
Ash-4B
Ash-5
Ash-6T
Ash-6B
Ash Beach
A
0
I I I 1 I I I I L 1
B (Replicate)
0
P. promelas
H. limbata
D. magna
i i I i i i i I I I
100 90 80 70 60 50 40 30 20
10 0 10 20 30 40 50 60 70 80 90 100
Mortality (%)
Figure 5. Percent mortality of Pimephales promelas, Hexagenia limbata, and Daphnia magna
from a 96-hour sediment bioassay of Ashtabula Harbor illustrating different
toxicity for surface samples (T) and bottom sediments (B).
-------
DISCUSSION
Question:
Answer:
Question:
Answer:
Question:
Answer:
What is the time of the exposure?
96 hours, basically. What we tried to do was develop a procedure
that you can do in a short period of time because there are about
126 harbors in Region V that are proposed to be dredged. It's
not practical to run six to eight week tests on 100 or more
harbors. So what we need is some sort of test that's a red flag.
I'm not proposing that this be used in lieu of bulk
chemical analysis and bioassay of the elutriate. I
should be used in conjunction with them. We've just
elutriate chemistry and bioassay argument to rest. I
has been working on a lengthy statistical analysis using a large
data base to see when elutriate chemistry and bulk chemistry is
analysis or
m saying it
put the old
believe Bob
important and how they relate
hours? That just seems to be
speaking, if you've got very toxic sediment, you'd get the
same results in 24.
to percent mortality. Why 96
accepted exposure. Generally
If you're looking
apparatus?
at sediment, why do you have the flow-through
You get a high BOD with sediments. If dissolved oxygen were to
drop down to 1 part per million, let's say, and the critters die,
you can't say it's because of the sediment toxicity. So the
recycling is important in maintaining dissolved oxygen within a
reasonable level. Very seldom does it drop down below 5 parts
per million. Plus, if you're in a riverine system, we can almost
duplicate the flow because you can change the calibration on the
apparatus and actually have a flow-through system that might
approach the CFS's; or you can change the flow across the pattern
in the sediment. Primarily, though, we use continuous flow to
maintain dissolved oxygen.
Are your critters
yourself?
available from stock or do you cultivate them
from the Toledo
are quite readily
The last study we did, we got the Daphnia
Zoo. Daphnia magna we had Dr. Kreiger do; they
available. Pimephales, the cosmopolitan that was talked about
this morning, is readily available here. As for the quality, we
get ours in Newtown, as long as we're doing governmental work.
The Hexagenia are called wigglers, and we get them from a
bait shop at $7 per thousand. The proprietor thinks I'm the
greatest fisherman in the world, I guess. Asellus communis -
that's another story. We maintain a culture unit and keep these.
The biggest hassle is in the winter; you've got to be prepared
for the winter.
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Question: About the relationship betv/een toxicity with Daphm'a and toxicity
with your other test animals - it appears as if the Daphnia was
more sensitive most of the time. Is there any advantage to using
an additional three species or should you just go with that?
Answer: Section 404 says that Thou Shalt use three. So you must use mul-
tiple species, number one. And I think that we got the spectrum.
Daphnia is very sensitive, and Pimephales I think you'd have to
hit with a sledge hammer. I think the other two species fall
somewhere in between. Daphm'a is very erratic; it is very tough.
Sometimes we use clean sand controls and get a 30% kill with
Daphnia. Maybe they're eating the sand grains, I don't know.
Question: Does your sedimentation take into consideration acclimation?
Answer: Yes it does.
Question: How long do you acclimate?
Answer: Generally, 24 hours. We've found that we get a recolonization in
the sediment very similar to the recolonization that existed in
the sediment when we took it. In other words, we've looked at
the different horizons after we put it in and let it settle out.
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TENTATIVE GUIDELINES FOR
FLOW-THROUGH EARLY LIFE STAGE TOXICITY TESTS WITH FATHEAD MINNOWS
FOR USE IN THE U.S. EPA, OTS-ORD ROUND ROBIN TEST
Donald I. Mount
U.S. EPA Environmental Research Laboratory
Duluth, Minnesota
The following draft protocol for an embryo-larval test is being
evaluated for adoption. Users should bear in mind that changes will be
made before finalization.
Tests as described in this method estimate chronic toxicities as
measured in life cycle tests in a high percentage of the comparisons that
currently can be made with the existing data base.
1. In an Early Life Stage Toxicity Test with fathead minnows,
organisms are exposed to toxicant during part of the embryonic stage,
all of the larval stage, and part of the juvenile stage. The orga-
nisms are examined for statistically significant reductions in percent
hatch, percent survival, and weight in order to determine upper and
lower chronic values.
A lower chronic value is the highest tested concentration (a) in
an acceptable chronic test, (b) which did not cause the occurrence
(which was statistically significantly different from the control at
the 95 percent level) of any specified adverse effect, and (c) below
which no tested concentration caused such an occurrence.
An upper chronic value is the lowest tested concentration (a) in
an acceptable chronic test, (b) which caused the occurrence (which was
statistically significantly different from the control at the 95
percent level) of any specified adverse effect and (c) above which all
tested concentrations caused such an occurrence.
2. Not enough information is currently available concerning early
life stage tests with fathead minnows to allow precise specification
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of details for most aspects of the test. Enough such tests have been
conducted and enough aspects have been studied, however, to indicate
that these guidelines are appropriate. A prudent course of action for
anyone planning to conduct such tests would be to initially conduct a
test with no toxicant to gain experience and to determine if the
requirements of items 10, 11, 19, 20, 25 and 26 are met using the
planned water, food, procedures, etc. General information on such
things as apparatus, dilution water, toxicant, randomization of test
chambers and organisms, and methods for chemical analyses, can be
found in Draft #10 of the proposed ASTM Standard Practice for Conduc-
ting Acute Toxicity Tests with Fishes. Macroinvertebrates. and Amphib-
ians.
3. Tests should be conducted with at least five toxicant concentra-
tions in a geometric series and at least one control treatment. The
concentration of toxicant in each treatment, except for highest
concentration and the control treatment, should usually be 50 percent
of that in the next higher one.
4. If a solvent other than water is used to prepare test solutions,
a solvent control (at the highest solvent concentration present in any
other treatment) is required in addition to the regular control,
unless such a control has already been tested in the same water with
the same species of fish, food, and test procedure and the water
quality has not changed significantly. A concentration of solvent is
acceptable only if it is (or has been) shown that that concentration
or a higher one does not cause a difference (increase or decrease in
any of the kinds of data specified in item 27) from control organisms
that is significant at the 95 percent level using a two-tailed t-test.
5. For each treatment (toxicant concentration and control) there
must be at least two replicate test chambers each containing one or
more embryo cups with at least 50 embryos divided equally between the
embryo cups at the beginning of the test.
6. Two test chambers have been used routinely:
a. Twenty fish have been tested in a chamber which is 16 cm x 44 cm
x 18 cm high with a 16 cm x 18 cm, 40 mesh stainless steel screen
6 cm from one end, with a water depth of 12.8 cm and with a flow
rate of 190 ml/min.
b. Fifteen fish have been tested in a chamber which is 6.5 cm x 17.5
cm x 9.5 cm high with a 6.5 cm x 9.5 cm, 40 mesh stainless steel
screen 2 cm from one end, with a water depth of 4.4 cm and with a
flow rate of 15 ml/min.
All of the above are inside dimensions. In both test chambers the
water depth is controlled by a standpipe located in the smaller
screened compartment with the test solution entering at the other
end of the test chamber.
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7. Embryo cups should be glass cylinders about 4.5 cm inside dia-
meter and about 7 cm high with 40 mesh nylon or stainless steel screen
glued to the bottom. The embryo cups must be suspended in the test
chamber is such a way as to ensure that the organisms are always
submerged and that test solution regularly flows into and out of the
cup without agitating the organisms too vigorously. A rocker arm
apparatus driven by a 2 rpm motor and having a vertical travel dis-
tance of 2.5 to 4.0 cm has been successfully used, as have self-
starting siphons that cause the level of solution in the test chamber
to rise and fall.
8. An acceptable dilution water for early life stage toxicity tests
with fathead minnows is one in which the species will survive, grow,
and reproduce satisfactorily.
9. A 16-hour light and 8-hour dark photoperiod should be provided.
A 15- to 30-minute transition period at "lights on" and "lights off"
may be desirable. Light intensities from 10 to 100 lumens at the
water surface have been used successfully, but the intensity should be
about the same for all test chambers. Light should be provided by
wide-spectrum (color rendering index > 90) fluorescent lamps.
10. Tests should be conducted at 25°C. The temperature in each
test chamber should be between 24 and 26°C at all times and must
be between 20 and 28°C at all times. If the water is heated, pre-
cautions should be taken to assure the supersaturation of dissolved
gases is avoided and total dissolved gases should he measured at least
once during the test in the water entering the control treatment.
11. The dissolved oxygen concentration should be between 75 and
100 percent saturation at all times in all test chambers. At no
time during the test should one test chamber have a dissolved oxygen
concentration that is more than 1.1 times the dissolved oxygen concen-
tration occurring in another tank at the same time.
12. The flow rate of test solution through the test chambers must be
great enough to maintain the dissolved oxygen concentration (see items
11 and 22) and to ensure that the toxicant concentrations are not
decreased significantly due to uptake by test organisms and material
on the sides and bottoms of the chambers.
13. A test begins when embryos in embryo cups are placed in test
solution and ends 32 days later.
14. Embryos and fish should not be treated to cure or prevent disease
or fungus before or during a test.
15. Embryos should be obtained from a fathead minnow stock culture
maintained at 25°C and a dissolved oxygen concentration between 75
and 100 percent saturation with a 16-hour light and 8-hour dark
photoperiod. Frozen adult brine shrimp have been successfully used as
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a food for adult fathead minnows. The most eggs have been obtained in
a 30 cm x 60 cm x 30 cm deep chamber with a water depth of 15 cm when
15 cm x 30 cm quadrants are formed with stainless steel screen and one
male, one female and one or two substrates are placed in each quadrant.
Half-round spawning substrates with an inside diameter of 7.5 cm and a
length of 7.5 cm have been used successfully.
16. The afternoon before a test is to begin, all of the substrates
should be removed from an appropriate number of tanks in the stock
culture unit and should be replaced about the time the lights are
turned on the next morning. Enough (at least three) substrates with
embryos on them should be removed six hours later and soaked in
dilution water for two hours. For each individual substrate the
embryos should be gently separated and removed and visually examined
using a dissecting scope or a magnifying viewer. Empty shells and
undeveloped and opaque embryos should be discarded. If less than 50
percent of the embryos from a substrate appear to be healthy and
fertile, all the embryos from that substrate should be discarded.
Single embryos with no fungus or partial shells attached are prefer-
able, although embryos with some fungus or partial shells attached and
clumps of two or three embryos (with or without separation) have been
used successfully. An approximately equal number of acceptable
embryos from one substrate should be impartially distributed to each
embryo cup and the process repeated for at least two more substrates
until the proper number of embryos have been placed in each cup to
give at least 50 embryos per treatment. The embryo cups should be
standing in dilution water when the embryos are being distributed and
then the cups should be randomly placed in the test chambers.
17. Twenty-four hours after they are placed in the embryo cups, the
embryos should be visually examined under a dissecting scope or magni-
fying viewer and all dead embryos discarded. Embryos that are alive
but heavily fungused should also be discarded. Forty-eight hours
after the start of the exposure, all dead and heavily fungused embryos
should be removed and the remaining healthy, fertile embryos randomly
reduced to the required number (at least 30 per treatment). If more
than about 25 percent of embryos in the control treatment are dis-
carded within the first 48 hours of the test because they are dead or
heavily fungused, the test should probably be restarted. Each day
thereafter dead embryos should be discarded.
18. In each treatment, when hatching is about 90 percent completed or
48 hours after first hatch in that treatment, the live young fish
should be counted and released into the test chambers. Unhatched
embryos should be left in the cups and released into the test chamber
when they hatch. The range of time to hatch in each cup should be
recorded.
19. A test should be terminated if the average percent hatch in any
control treatment is less than 50 percent or if the percent hatch in
any control embryo cup is more than 1.6 times that in another control
embryo cup.
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20. The flow rate, size of the test chamber and the amount of food
added should be such that the average weight of the controls at the
end of the test would not be significantly greater if only half as
many fish were tested.
21. Each test chamber containing live fish over 2 days old must be
fed live, newly-hatched brine shrimp at least twice a day at least
6 hours apart (or three times a day about 4 hours apart) on days
2 to 5 after hatch and at least 5 days a week thereafter. They must
be fed at least once a day on all other days. Other food may also be
provided in addition to the above. The amount of food provided to
each chamber may be proportional to the number and size of fish in the
chamber, but each chamber must be treated in a comparable manner.
Quantifying the amount of live, newly-hatched brine shrimp to be fed
is difficult, but the fish should not be overfed or underfed too
much. A large buildup of food on the bottom of the chamber is a sign
of overfeeding. A sign of not feeding enough of the right kind of
food is that in a sideview the abdomen does not protrude.
22. Test chambers should be cleaned often enough to maintain the
dissolved oxygen concentration (see items 11 and 12) and to ensure
that the toxicant concentrations are not decreased significantly due
to sorption by matter on the bottom and sides. In most tests if the
organisms are not overfed too much and the flow rate is not too low,
removing debris from the bottom once or twice a week should be ade-
quate. With some toxicants that promote growth of bacteria, the sides
and bottoms should be cleaned more often. Debris can be removed with
a large pipette and rubber bulb or by siphoning into a white bucket.
The pipette or bucket should be examined to ensure that no live fish
is discarded.
23. Temperatures should be recorded in all test chambers once at the
beginning of the test and once near the middle of the test. In
addition, temperatures should be recorded at least hourly in one test
chamber throughout the test.
The dissolved oxygen concentration should be measured in each
treatment near the 1st, 21st and 28th days of the test.
Hardness, pH, alkalinity, and acidity should be measured once a
week in the control treatment and once in the highest toxicant concen-
tration.
The concentration of toxicant should be measured at least twice a
week in each treatment.
24. Dead fish should be removed and recorded when observed. At a
minimum, live fish should be counted 11, 18, 25 and 32 days after the
beginning of the test. The fish should not be fed for the last 24
hours prior to termination. At termination the number of fish that
are visibly (without the use of dissecting scope or magnifying viewer)
-66-
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grossly abnormal in either swimming behavior or physical appearance
should be determined. Also at termination the weight (wet, blotted
dry) of each fish that was alive at the end of the test should be
determined. 'If the fish exposed to toxicant appear to be edematous
compared to control fish, determination of dry rather thin wet
weight is desirable.
25. A test is not acceptable if the average survival, of the controls
at the end of the test is less than 80 percent or if survival in any
control chamber is less than 70 percent.
26. A test is not acceptable if the relative standard deviation (RSD
= 100 times the standard deviation divided by the mean) of the weights
of the fish that were alive at the end of the test in any control test
chamber is greater than 40 percent.
27. Data to be statistically analyzed are:
• Percent normal hatch
• Percent survival at end of test (based on fry, not embryos)
• Percent normal at end of test (based on fry, not embryos)
• Weights of individual fish that were alive at end of test.
28. Dichotomous data (live-dead, normal-abnormal) should be analyzed
using contingency tables or log linear techniques. For weight data,
the individual fish are used as the replicates unless a two-tailed
F-test indicates that differences between replicate test chambers are
not negligible. Weight data may be analyzed using Bartletts' test and
one-way analysis of variance, but to obtain information concerning the
upper and lower chronic values, Dunnett's procedure (Steel and Torrie,
Principles and Procedures of Statistics. 1960, p. Ill) should be used
to identify treatment means that are statistically significantly
different from the controls at the 95 percent level.
References
Benoit, D. A. and R. W. Carlson. 1977. Spawning success of fathead minnows on
seclected artificial substrates. Prog. Fish-Cult. 39:67-69.
Flickinger, S. A. 1969. Determination of sexes in the fathead minnow.
Trans. Amer. Fish. Soc. 198:526-527.
Gast, M. H. and W. A. Brungs. 1973. A procedure for separating eggs of the
fathead minnow. Prog. Fish-Cult. 35:54.
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May, R. C. 1970. Feeding larval marine fishes in the laboratory: a review.
Calif. Mar. Res. Comm., California Cooperative Oceanic Fisheries
Investigations Report 14:76-83.
This tentative procedure was written by Charles Stephan with the help of many
members of the staff of the Environmental Research Laboratory in Duluth,
Minnesota.
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EFFLUENT GUIDELINES LIMITATIONS AND LETHAL UNITS
Kenneth J. Macek
EG & G, Bionomics, Inc.
Wareham, Massachusetts
I've been asked to talk a little bit about the toxic unit concept. It
seems to me, from some of the questions that have been asked, that there
still seems to be a certain amount of confusion in the minds of some people
here about what exactly this is all about and why you should be concerned
about it. Therefore, I'll take just a minute to review a few concepts that
led me to go through some of the thought processes which I did in preparing
this paper.
Obviously, the requirement for measuring toxicity is here to stay
whether people in the agency who have to deal with the problem or people in
the industry who have to live with the problem like it or not. Another
thing that's just as clear is that the reliance on analytical chemistry
measurements in the permit program has not adequately addressed the question
of toxicity, primarily because constitutents not routinely monitored can, and
do, contribute to toxicity. However, I think it's also obvious that increas-
ing the analytical burden of the permittee is not really the answer, despite
the fact that you might feel you'd rather make a few more measurements than
deal with this nebulous area called "biomonitoring". I think in the long
run, as Dr. Mount suggested this morning, we'll find that the cost and the
effort related to doing it biologically and toxicologically are probably
going to be less than continuing to increase the already complex and signi-
ficant analytical costs.
But more important are the toxicological reasons why analytical mea-
surements aren't the answer. Even if we did measure everything that was
in the waste, we'd have to know the toxicity to the aquatic organisms of
each of those constituents. If any of you have looked at the proposed water
quality criteria documents being published under the consent decree, you'll
find that even for some very, very common compounds like benzene and chloro-
form, we don't have toxicity data for aquatic organisms. As the chemicals in
your waste become more and more unique or more and more characteristic of a
particular industry, the less and less likely it is that such data exists.
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Furthermore, even if we had such data, we would then need data on all
of the combinations of constituents that could be in the wastewater and the
interactions toxicologically that could occur there. So it's clear that
measuring all of these constituents is not the answer - we can't do it, we
don't have half of the toxicity data required, and even if we did, we would
still have a long way to go. So the simplest approach is to measure the
toxicity of the whole waste, and that is what I think this meeting is all
about.
I'll spend a little bit of time talking about toxic units although
I'm not sure in my own mind where we can go with it at this point. Back in
1971, a group working in the San Francisco Bay area led by Esvelt and cowork-
ers at the University of California completed a comprehensive study of the
toxicity of municipal wastes to aquatic organisms. As a result of these
efforts they proposed measuring, monitoring, and regulating toxicity as we
have historically regulated conventional pollutants such as solids, BOD and
COD. In its most simplistic form, they proposed to measure toxicity, estab-
lish a discharge limitation in the form of a mass emission rate, and apply
treatment technology designed to "remove toxicity" in order to meet the
reasonably established limitation.
I guess I better go into a little detail here because this toxic unit
concept can be confusing if you try to make it anything more than it is.
All it represents is a simple alternative way of expressing an LC50.
Basically what they proposed was that LC50 values, expressed normally
as mass/volume or volume/volume concentrations, be translated into what
would represent a relative toxicity "concentration" which could be expressed
as toxic units. The concept of toxic units is well established in the
literature at this time. In its simplest form, if you have a constituent
like copper whose LC50 to some organism is 2 yg/1, and you have 10 yg/1 of
copper in your waste, the copper is said to contribute 5 toxic units to the
toxicity of the waste. When you have an effluent, for which the effective
concentration of the "constituent" is 100 percent, the number of toxic units
in the effluent is simply the LC50 for that effluent to some organism divided
by 100. The quotient thus represents a unitless measure of toxicity arbi-
trarily called the toxicity "concentration" and expressed as toxic units.
Unfortunately, the use of the term concentration can be misleading as the
calculation yields toxic units, a measure independent of any specific volume
- it's 2 per liter, 2 per gallon, 2 per million gallons, whatever. They
further propose, with respect to the mass emission rate, that the product of
the toxicity concentration (in toxic units per some unit volume) in the
wastewater times the flow rate of the wastewater in the same unit volume per
unit time would yield a mass emission rate in toxic units per unit time.
The authors recognized that, if toxicity was to be considered in water
quality management in a manner similar to conventional pollutants, they had
to evaluate the effectiveness of conventional and advanced treatment technolo-
gies to reduce toxicity. They also recognized that, since a toxic unit was a
test-specific parameter, the tests had to be fairly standardized and repro-
ducible. This latter point is particularly important if, in fact, the
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concept of toxic units ever gets into the NPDES program. I'll elaborate on
that in a few minutes.
I'd like to give you just a brief outline of the results of Esvelt's
program. They worked primarily with five municipal waste effluents, and the
mean LC50 value of all samples tested ranged from 40 to 60 percent effluent.
These LC50 values translate to 1.5 to 2.5 toxic units. Furthermore, they
found that the mean relative standard deviation derived from repeating tests
24 times on the same source of effluent was about +20 percent. Neither of
the above findings should be particularly startling, as relatively low
toxicity and relatively little variability in the toxicity of municipal waste
would not be unexpected.
They went on to point out that when they evaluated the mass emission
rate for a single effluent tested daily for eight consecutive days, they
found that, although the absolute LC50 value and the absolute flow rates could
vary by as much as 100 percent, the mean mass emission rate for toxicity had
a relative standard deviation of only 15 percent.
Unfortunately they didn't go on to say whether this was coincidence or
whether they had some fundamental basis for assuming there is a real relation-
ship here. I certainly don't know what the relationship would be, and I sus-
pect it's more coincidence.
They went on to look at the reproducibility of these toxicity tests in
their program, and they found that the replication of results was excellent
with a relative standard deviation of only 5 percent. Now that's a lot
better than I've ever done, and I suspect it's a lot better than most other
people in the room have done. Again, I don't know what the reasons for those
numbers are, but in my opinion I believe a more representative number of
reproducibility would be something on the order of 15 to 20 percent.
They also went on to look at whether one could treat toxicity to
reduce it. They looked at biological treatment as you would in a municipal
waste system, and they found that even for those wastes that were relatively
non-toxic to begin with (1 to 2 toxic units), biological treatment reduced
what little toxicity was there by more than 75 percent.
Now that's all I'm going to say about the toxic unit concept. It's
just another way of expressing the toxicity which one measures by a bioassay.
I'm a little concerned about using it in a mass emission sense because,
even in this limited program, they showed that the same waste could vary
by jf 20 percent. If you believe that the reproducibility of the bioassay is
+_ 20 percent, then you've got a propagation of errors there that makes it
very hard to live with a single mass emission rate number.
Now I'd like to tell you about some studies that we conducted for
EPA to further evaluate this toxic unit concept and how it might apply to
industrial waste treatment facilities and NPDES programs.
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Based on the results of the study by Esvelt and his coworkers, and
motivated by a desire to control toxicity of wastewaters discharged into
surface waters, the EPA was concerned with answering several questions about
this potential approach. The questions to be answered in our program were
concerned with (1) the efficacy of treatment systems for removing toxicity in
industrial wastes; (2) the variability of the toxicity of both the influent
to and the effluent from industrial waste treatment systems; (3) the required
frequency of sampling for enforcement and compliance monitoring; and (4) the
effect of species selection for monitoring. EG&G, Bionomics, using mobile
laboratory facilities, visited seven industrial sites (comprised of one
organic chemical facility, five pesticide facilities - selected because of
their anticipated relatively high toxicity, and one pulp and paper facility).
The sites were also selected because they represented various types of
treatment technologies essentially equivalent to BPT, and in some cases
technologies more advanced than BPT. In order to investigate some of those
areas of interest to the agency, we conducted 24-hour static toxicity tests
with fathead minnows using batch wastewater samples collected twice a day for
10 consecutive days. Also, in order to investigate the effect of species
selection, we concurrently tested Daphnia magna on alternate days. And I'd
like now to show you some of that data.
The first data set is the results, expressed in toxic units, of tests
with fathead minnows at a pulp mill of International Paper Company in George-
town, South Carolina (Table 1). There are several things to note about these
data. First is the relatively low toxicity of the influent to the treatment
system. Second, and perhaps more important, is the relatively low varia-
bility in the toxicity of the influent to the treatment system. The third is
the complete removal of toxicity by the treatment system. Lastly, we find
that the comparability of the results from the two species tested seems
reasonable. Now this is obviously a pretty clean plant.
The next data set is from an organic chemicals plant, Union Carbide's
plant in Charleston, West Virginia (Table 2). Significant aspects of these
data are the slightly higher toxicity of the influent; the occasional order
of magnitude excursions in toxicity of the influent; the complete removal
of toxicity as a result of treatment even in the face of these relatively
large excursions; and again the comparability of the daphnid data to the
fathead minnow and the ability of either species to detect the rather large
excursions in the toxicity in the influent. In this case the treatment
system did not entirely remove the toxicity, although I must caution you that
measurements of toxic units less than unity are tentative to say the least.
You have to extrapolate when you don't have a complete 100 percent mortality
or mortality above 50 percent to try to get at these numbers, and I wouldn't
put a lot stock in toxic units less than unity.
The last set of data is from a pesticide manufacturing plant operated
by Diamond Shamrock in Green Bayou, Texas (Table 3). This plant is charac-
terized by a relatively high variability in the toxicity of the influent to
the treatment system, with excursions exceeding two orders of magnitude; on
the other hand you get absolutely zero variability in the effluent from the
treatment system. So there is a rather remarkably consistent degree of
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Table 1
TOXIC UNIT CONCENTRATION IN THE EFFLUENT TO. AND EFFLUENT FROM
THE INTERNATIONAL PAPER COMPANY INDUSTRIAL WASTE TREATMENT PLANT
Sample
AM-1
PM-1
AM-2
PM-2
AM-3
PM-3
AM-4
PM-4
AM-5
PM-5
AM-6
PM-6
AM-7
PM-7
AM-8
PM-8
AM-9
PM-9
AM-10
PM-10
Mean
(S.D.)
GEORGETOWN
Minnow
, SOUTH CAROLINA
Influent Effluent
1.9
2.9
2.4
2.9
2.9
2.4
2.4
1.9
1.9
2.4
2.4
4.3
2.4
2.4
4.3
4.8
2.4
2.9
3.7
>8.3
2.8
0.8
0
0
0
0
0
0
0
-
0
0
0
0
0
0
0
0
0
0
0
0
Daphnid
Influent Effluent
<1.5 0
2.0 0
-
-
1.8 0
1.2 0
-
-
1.3 0
1.4 0
-
-
1.3 0
<1.5 0
-
-
2.4 0
2.4 0
-
-
1.7
0.5
-73-
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Table 2
TOXIC UNIT CONCENTRATION IN THE EFFLUENT TO, AND EFFLUENT FROM
THE UNION CARBIDE
INDUSTRIAL
CHARLESTON, WEST
Minnow
Sample
AM-1
PM-1
AM-2
PM-2
AM-3
PM-3
AM-4
PM-4
AM-5
PM-5
AM-6
PM-6
AM-7
PM-7
AM-8
PM-8
AM-9
PM-9
AM-10
PM-10
Mean
(S.D.)
Influent
6.7
10.5
4.3
5.6
6.7
8.3
4.0
6.7
3.6
3.6
4.5
7.7
50.0
6.3
13.3
8.3
4.5
38.5
3.6
5.6
10.2
11.7
Effluent
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
WASTE TREATMENT PLANT
VIRGINIA
Daphnid
Influent Effluent
4.8 0
5.9 0
-
-
3.8 0
4.8 0
-
-
5.3 0
7.1 0.6
-
-
55.6 0
7.7 0.6
-
-
2.0 0.6
21.3 0.8
-
-
11.8
15.4
-74-
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Table 3
TOXIC UNIT CONCENTRATION IN THE EFFLUENT TO. AND EFFLUENT FROM
THE DIAMOND SHAMROCK
INDUSTRIAL WASTE
TREATMENT PLANT
GREEN BAYOU, TEXAS
Minnow
Sample
AM-1
PM-1
AM-2
PM-2
AM-3
PM-3
AM-4
PM-4
AM- 5
PM-5
AM-6
PM-6
AM-7
PM-7
AM-8
PM-8
AM-9
PM-9
AM-10
PM-10
Mean
(S.D.)
Influent
>400
794
37
952
19
20
47
>2000
12
19
400
24
30
24
1785
1785
19
24
>2000
588
373
645
Effluent
1.2
1.2
1.2
1.2
1.1
1.1
1.2
1.1
1.2
1.1
1.1
1.1
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
0.05
Daphnid
Influent Effluent
535 3.1
>1700 2.5
-
-
-
-
-
-
26 2.5
42 2.5
-
-
96 2.5
71 3.1
-
-
24 2.5
28 2.5
-
-
133 2.6
199 0.25
-75-
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removal of toxicity even in the face of unusually high excursions. Also,
these data again suggest that if you had to decide whether to use one species
or the other, I don't really think it makes much difference. Certainly from
an economy of scale, the Daphnia would seem to be a much more attractive
organism.
Now very quickly some other data sets were very similar. For example,
the treatment system at a pesticide plant operated by Monsanto in Muscatine,
Iowa removed an average of 96 to 98 percent of the toxicity of the waste
going into the system. And the mean toxic unit concentration of the effluent
for the treatment system for fatheads was 1.5 +_ 0.3, and for daphids it was
1.3 +_ 0.2 - essentially the same numbers.
The treatment system at Monsanto's pesticide plant in Luling, LA
removed 92 percent of the toxicity, leaving an average of about 6.0 toxic
units.
Now, what can we do with all this information? I'd like to take just
a few minutes to give you my thoughts. Let me preface these comments by
emphasizing the fact that the following thoughts are my own and should not be
construed as representing in anyway the conclusions or philosophy of the
agency, nor any of the companies participating in the project.
It seems to me that data on the toxicity and variability of influent
to a treatment system should be considered by the industry in the same light
as any engineering data for the design and operation of a treatment system.
Similarly, such data should be of interest to the agency only as it relates
to an assessment of the technological capability to remove toxicity - that
is, is it feasible, is it practical, is it economical, and so forth.
It further seems to me that, from the standpoint of regulating
toxicity, it is the data regarding the toxicity of the effluent from the
treatment system which should be of prime consideration. With that in mind
I'd like to take a look at a summary of these data (Table 4). These are the
mean number of toxic units in the plant effluents which we evaluated to both
fathead minnow and daphnid. The important thing to note here is the rela-
tive standard deviation of the mean. You can see it runs as high as 26%
in one case and as low as 4 percent in another, but that's fairly uncharac-
teristic. A value between 10 and 20 percent is a good number. These data
suggest that the toxicity of discharges from treatment systems does not vary
dramatically when the system is up and functioning and that approximately 95
percent of the time a bioassay should produce results within 25 to 50 percent
of a mean value, assuming a level of reproducibility in bioassays of 20
percent. This observation relates to the toxic unit limitation. It appears
that any toxicity limitation, or "lethality baseline", included in a permit
would of necessity have to be incorporated in the form of a "range" of
acceptable values. I don't see how you could pick a single number and say
"thou shalt not ever violate it". The data further suggests that perhaps one
could expect slightly less variability using daphnids, although I'm not sure
those differences are significant. I personally believe that the utility of
toxicity tests lies in providing data for establishing the permit conditions
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Table 4
MEAN (RELATIVE STANDARD DEVIATION. %) TOXIC UNIT CONCENTRATION
IN THE EFFLUENT FROM INDUSTRIAL WASTE TREATMENT SYSTEMS
PLANT
Georgetown, SC
Charlestown, WV
Muscatine, IA
Green Bayou, TX
Kansas City, MO
Luling, LA
Laporie, TX
TYPE
Paper
Organ ic s
Pesticide
Pesticide
Pesticide
Pesticide
Pesticide
MINNOW
0
0
1.5 (20%)
1.2 (4%)
3.8 (26%)
6.0 (23%)
2.4 (12%)
DAPHNID
0
0
1.3 (15%)
2.6 (10%)
4.4 (9%)
3.2 (22%)
-
-77-
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relative to toxicity. However, I have serious questions about the utility of
such tests for monitoring compliance with those permit conditions. Now let
me go on to explain this last statement.
I think that bioassays could provide significant information on the
efficacy of treatment systems for removing toxicity. Clearly they can provide
information on what I call the "residual acute toxicity" of the wastewater
and on normal variability in toxicity of a typical discharge remaining after
treatment, thus providing relevant information regarding dilution required in
a stream to meet water quality standards. I think a program similar to the
ones we've described here would provide data on the small and random fluctua-
tions in effluent quality associated with the regular operation of an indus-
trial plant and its effluent control equipment, thus defining a range of LC50
values which might be considered "normal". I think if you couple this
approach with the embryo-larval test that Dr. Mount described earlier, one
could even use bioassays to evaluate the potential hazard associated with the
long term chronic effects of "residual toxicity" of a typical discharge
after treatment on receiving water organisms.
I strongly urge both the agency and industry to utilize reliable
toxicity testing with aquatic organisms, where appropriate for toxicolo-
gically characterizing a waste stream for the purpose of establishing permit
conditions with regard to toxicity, and the required degree of control of
toxicity.
However, I do have some problem with the concept of requiring industry
to use bioassays as a monitoring tool. An effective monitoring tool should
be one which has the capacity to detect most or all excessive pollutant dis-
charges to the environment. Now clearly bioassays have the capacity to do
this as demonstrated by some of the data we've just looked at. However,
the monitoring system should produce information on three critical variables:
the frequency, intensity, and duration of significantly elevated pollutant
discharges. Now although bioassays have the ability to detect the intensity,
they don't tell us much about the frequency or the duration. I believe that
bioassays could provide such information if utilized frequently enough.
However, the costs of using bioassays with sufficient frequency (real-time,
hourly, or daily) would clearly be prohibitive, and I think such monitoring
could be more effectively done using a surrogate physical or chemical para-
meter for which changes can be correlated with, or indicative of, changes in
toxicity.
For example, there is some data that suggests that with certain wastes
there is a straight correlation between the pH of the waste as it comes out
of the treatment system and toxicity. There may be a future in using some-
thing like a Microtox system which can be correlated to toxicity as a routine
monitoring tool. Now the reason I say this is because of the frequency with
which I think we'd have to do bioassays to effectively monitor effluent
toxicity. I base this observation on a recently published study on a paper
mill in British Columbia by Nemetz and Drechsler. This represents the most
complete and comprehensive analysis of sampling design in effluent monitoring
which I've ever seen. In it they concluded that "composite sampling even at
frequent intervals...performs the ironic function of apparently measuring
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elevated pollutant releases without detecting them." As a result, they
conclude that composite sampling fails to provide either of the essential
functions of an effective monitoring system: (1) a high representational
accuracy of the effluent profile; or (2) the detection of most or all pollu-
tant releases with potential serious environmental consequences. They
recommend that various considerations be given to the cessation of composite
sampling in effluent monitoring programs and their replacement by frequent
grab sampling and sequential analysis. How frequent is the .question. They
found that with a paper mill where they were measuring a parameter having a
mean of X, with an N of 120,000, and a relative standard deviation of 50
percent (similar to bioassay data) that the duration of 95 percent of the
"spills" was less than 4 hours. As I interpret their data, they further
estimated that if one took instantaneous grab samples every day, one would
still not detect 99 out of every 100 "spills". Clearly then, monitoring
using bioassays is not a feasible method of "monitoring" toxicity. I'm not
suggesting that bioassays have no place during the life of a permit. I'm
clearly suggesting that their place is in the establishment of the permit
conditions. But in addition, if one has evidence to suggest that, or expect
that, the quality of the discharge may be significantly different from the
typical discharge, either due to changes in the inputs to the system or in
the operation of the system, it might be prudent to monitor the toxicity of
the discharges using bioassays at these times. However, I see little utility
in arbitrarily predetermined, periodic (e.g., quarterly or annual) toxicity
testing except to reassure everyone that the system is working as it should
on the day and at the time the sample was taken.
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APPLICABILITY OF THE AMES TEST IN BIOMONITORING
Larry Claxton
U.S. EPA, Environmental Toxicology Division
Research Triangle Park, North Carolina
Most of the things that you've heard so far deal with assays that
detect mortality, and/or morbidity in different test organisms. This assay
that we'll be describing detects changes that occur in hereditary material.
In other words, the question that we'll be asking is: Do the chemicals in
effluents that are tested alone, when reaching hereditary material, cause
changes in the hereditary material that can be inherited? Do chemical
effluents have the capability of causing hereditary diseases or if inter-
actions occur in what are called somatic cells of the body, cells other than
the gonadal tissue cells, could this interaction possibly cause cancer?
The test that we'll be describing today is commonly called, and most
people know of it as, the Ames test. It's used for a variety of reasons.
There are several reasons why people might like to use it. It is rapid and
inexpensive. It is a first level indication of genotoxicity rather than
mortality or morbidity, and we can use it to set priorities in further geno-
toxicity testing. Something that's very important to many of you is that we
can use this test coupled with chemistry, technology, and a variety of other
things in order to direct the development of technology and to help those who
are in technology development to know what types of active compounds are
coming out of the system.
There are two assumptions I think that you should be aware of that
this test is based upon. First, we are assuming, and this is generally
accepted in the scientific world today, that the structure of DNA, not
chromosomes, but DNA structure, is the same for all organisms, except
for some viruses. Second, gene mutations that occur at the DNA level
will occur generally by the same molecular mechanisms and therefore be
inherited in the same way. There are some debatable issues within that.
The Ames test is a test run with a bacterium called Salmonella typhi-
murium. Figure 1 is an outline of how the test is run. It's a technically
simple test to perform; however, it gets more complicated when you get into
the laboratory. Essentially what we do is we have a soft agar overlay that
is melted. Into this overlay we add the bacteria indicator organism, the
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chemical or substance or effluent that is to be tested, and we, on occasions,
will also add in a mammalian activation system. We refer to this activation
system as 59 because it is a 9,OOOG supernatant microsome-containing fraction.
This is used to activate promutagens and procarcinogens to an active form.
After these are combined together into the agar overlay and overlaid onto the
plate, they are incubated for 48 hours. These are derived bacterial strains
with a mutation within the histidine locus. They mutate "in reverse" to the
wild type, which means that they can grow in the selective media and when that
reversion occurs we get a response. We plate approximately 10^ bacteria
per plate; therefore, on a control plate we'll find a few spontaneous mutant
colonies that will grow. If our substance is mutagenic and has no killing,
we'll see increasing numbers of colonies with increasing dosage. So there-
fore, we tested a variety of doses; we usually do this in our laboratory in
triplicate at each dose. We run the proper positive and negative controls
and usually, if we have enough material, replicate the experiment. So we get
a fairly large amount of data coming out of this.
Bruce Ames in developing this system recognized, as most geneticists
do today, that there is a variety of gene mutations that can occur; there-
fore, he developed five different tester strains of bacteria. Each tester
strain has a specific type of mutation incorporated and other characteristics
that are important to the people conducting the test. But generally, for a
complete screening, at least these five strains and sometimes other strains,
if you're in a research mode, could be used.
There are some major variables that can occur within a system. We are
maintaining a biological organism over time, frozen, and yet even with that
there are some natural variations within subcultures. This makes the use of
positive and negative controls, the checks that Ames describes for his
strains, mandatory for essentially every test. There are also areas of
variation within the mammalian activation system, but the greatest variation
that's brought into the test is usually due to the physical and chemical
characteristics of the substance that's to be tested.
One of the topics I was asked to present is the equipment that's
generally needed. Table 1 is a list of this equipment. A laminar flow
biological safety hood is needed because we do use known carcinogens and
mutagens which are hazardous to humans and the safety factors within the
laboratory are very stringent. Much of the remaining list also involves
safety. We allow no mouth pipetting, the area has to be properly exhausted,
and so forth. This can be quite expensive. These are optional types of
equipment that you can use which would increase the use of your manpower,
and there is other common biological equipment that might be needed.
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S-9 MICROSOMES
00
ro
i
BACTERIA
CHEMICAL
SOFT AGAR OVERLAY
CONTROL
PLATE WITH
OVERLAY
RESULTS:
INCREASING DOSAGE
(MUTAGENIC WITH NO KILLING)
INCUBATE
48 HOURS, 37°C
Figure 1. Schematic diagram of the plate incorporation (Ames) test.
-------
Table 1
EQUIPMENT
• Laminar Flow Biological Safety Hood
t Automatic Pi pets
• Properly Exhausted Incubators and Work Areas
• (Automatic Colony Counter)
• (Automatic Plate Pourer)
• Other Equipment Common to Microbiological Laboratory
Two things that I forgot to add onto this list are very important.
Most people prepare their own mammalian microsomal activation, and so
if you do that, you'll have to plan also on having a rodent colony and
biochemical laboratory backup to check those preparations.
When it gets to you, you're probably not interested as much in the
first part as in how we interpret the data that's coming out of the Ames
test. I'd like to run very quickly through some aspects that are generally
accepted with the Ames test. A positive response (Table 2) is usually easy
to ascertain because positive responses are usually dramatic in the vast
majority of cases that we've dealt with. A positive dose is defined in the
following manner: There's at least a 2.5 fold increase in revertant colony
counts over the spontaneous or control level. When you plot the dose re-
sponse from the zero dose up to your highest dose, there's usually a very
regular type of dose response curve. As I mentioned, we always run specific
controls for these particular strains. We expect the proper response in each
of those controls. Another thing that we can do in the system is that after
we find some mutants, in order to make sure that we're not getting a false
positive, we can check these mutant colonies to make sure that they truly are
mutants. So, this is what we would define as a definite positive response.
Table 2
DATA INTERPRETATION
Positive Response
t At Least 2.5 x Increase
• "Regular" Dose Response Curve
t Expected Response for all Controls
t Check for True Reversion
__
-------
One of the somewhat more difficult jobs, however, is assigning a
negative response. Table 3 gives the criteria that we use in our laboratory
for assigning a negative response. This may vary a little bit according to
whom you talk to, but this is pretty acceptable now to most people. We
expect, within our laboratory, at least five doses at 1/2-log intervals. So
we're exploring quite a dose range when we do the test. The top dose we
expect to be either toxic or greater than or equal to 5 mg per plate. This
amount is chosen because it's physically about the largest amount that you
could normally put into the test. We also expect the proper responses from
all controls, of course. We expect the doses to be replicated within the
experiment or we expect replicate experiments - either would be acceptable.
And we expect the major tester strains to be used and the proper steril-
ity controls to be completed. If you find that all of these components are
completed and you have no dose response -- there's no activity other than
spontaneous — you can he confident in saying that the Ames test indication
is negative.
Table 3
DATA INTERPRETATION
Assigning A Negative Response:
• At Least 5 Doses At Half-Log Invervals
t Top Dose is Toxic or Fquals or Greater than 5 mg/Plate
t Fxpected Responses from all the Proper Controls
• At Least Replicate Dose Responses and/or Experiments
• Tested in 5 Major Tester Strains
t Proper Sterility Controls
That leaves a little bit of data that's left with a questionable re-
sponse (Table 4). Sometimes you'll see an increased response with dose but
it doesn't ever reach the 2.5 fold level. Sometimes the response will
approach that point. Because of the toxicity that tends to mask the response
after that point, you can't force the response to go past a 2.5 fold in-
crease. So this leaves you with a questionable result. This uncertainty can
usually be removed by replicate experiments. Sometimes you will see at
certain doses that, indeed, you have a count that's greater than a 2.5 fold
increase, but the data is very irregular. We would generally call this a
negative, qualify, and explain it.
-84-
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Table 4
DATA INTERPRETATION
Questionable Responses:
1. Increase Response With Dose, But Never Exceeds
Spontaneous Level by 2.5 Fold
2. Irregular Response Curve With One or More Responses
Greater Than 2.5 x Spontaneous
There are many different ways, and Table 5 lists some of the ways,
that you may see the data summarized. When you get out the data from one
experiment in the Ames test, you're looking at many strains of bacteria done
in triplicate at five doses or more each. Then with a whole series of
controls, you're looking at over 200 pieces of data in one test; and we can
run three to four tests a week in one laboratory with just two technicians.
So there's a vast amount of data, and people, including us, tend to summarize
data in a variety of ways. So there are a variety of ways to summarize data;
however, we tend to be very careful in comparing data by using only specific
activity or a statistical modeling curve.
Table 5
DATA INTERPRETATION
Methods of Data Summary:
1. Fold Increase or Mutagenic Index
2. Net Response (In Revertants Per Plate)
3. Specific Activity
4. Specific Activity Fold Increase
5. Specific Response Activity
6. Maximum Response
7. Statistical Modeling Curve
For those who are involved in water effluents, there are some associ-
ated techniques (Table 6) that turn out to be very important: concentration,
extraction, and fractionation. There are a variety of concentration proce-
dures, and most of you are probably quite familiar with these. And then there
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are extraction procedures. Within water samples from effluent sites you also
find particulates, and this is where extraction can become very important.
Particulate matter generally interferes with the test. Particles make plates
very hard to read, to count, and so forth. So generally what we do is
extract the organics off the particulate matter by different methods and then
place the extract into the test system. Fractionation can be important if
you're going into technology development and want to know what compounds or
what types of compounds are causing the activity.
Table 6
ASSOCIATED TECHNOLOGIES
1. Concentration
A. Adsorbants (XAD)
B. Lyophilization
C. Reverse Osmosis
D. Filtration (Particulate)
E. Extraction
2. Extraction
A. Solvent Systems
B. Sonication
C. Physiological
3. Fractionation
There is a variety of other tests that I'll just review very quickly
that are essentially modifications of the Ames test - the original Ames test.
One is a simple spot test (Figure 2) where the bacteria and activation system
are first put on a plate, the substance is spotted in the center, and you get
either a negative or a positive in which you had a zone of killing and a zone
of mutation and outside of that another zone of spontaneous mutation. For
many of the things that you'll be dealing with, this will not work too well.
Many of the organics that you test will not diffuse readily in agar, and so
the spot test has limited applicability when you concentrate the organics.
There's a liquid suspension test (Figure 3) which is important
because it not only gives you a revertant number, but it also gives you the
survival number. For a geneticist this can turn out to be very important
because he's looking at the number of hits on a particular gene in a parti-
cular organism. Very seldom, unless you are in a research mode, would you
want to use this test. There are a couple of occasions, however, if you're
working with some specific types of compounds such as nitrosamines, when you
may want to go to a test such as this.
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S-9 MICROSOMES
ft BACTERIA
PIPET WITH CHEMICAL
DISC
SOFT AGAR OVERLAY
PLATE WITH OVERLAY
00
-J
I
INCUBATE 48 HOURS, 37°C
RESULTS:
ZONE OF KILLING
ZONE OF MUTATION
ZONE OF SPONTANEOUS
MUTATION
CONTROL
NEGATIVE
POSITIVE
Figure 2. Schematic diagram of the spot test.
-------
00
CO
BACTERIA
SOLVENT
CONTROL
S-9
CONTROL
INCUBATE
I
MUTANT PLATES
CHEMICAL IN SOLVENT
TEST
1
SERIAL DILUTION
H SOFT AGAR H
I OVERLAY TO PLATE I
^3 i I
INCUBATE
CONTROL
SURVIVAL PLATES
Figure 3. Schematic diagram of the liquid suspension test.
-------
Something that's very similar but more rapid than the suspension test
is the preincubation test (Figure 4) which is just a shortened modification
of the suspension test. It is also good for testing compounds such as the
nitrosamines.
A modification that we've developed to the spot test is the well test
(Figure 5) in which we can test two chemicals - a solvent control and a posi-
tive control - with and without activation on one plate.
I've run through quite a bit very rapidly - it's really about a 2 hour
lecture what I've tried to do in about 20 minutes here. I brought along two
articles that you might be interested in. One of them describes the Ames
test in more detail - some of the pitfalls and some of the uses of the Ames
test. The other is a much more general and lay-like publication that des-
cribes short term tests for carcinogens, mutagens, and other genotoxic
agents.
DISCUSSION
Question: I am a plant engineer of a medium sized steel plant on the shore
of Lake Erie at Lorraine. I am impressed by the enthusiasm
and great mental capacity devoted to the goal here of preserving
the long term existence of aquatic organisms. From where I sit,
I am also interested in preserving the existence of an organism.
The organism I think of is my plant. It employs about 7,000 to
8,000 people; about 30 to 40 percent of the members of the
community depend on its employment. And gentlemen, that organism
finds itself in a heavily polluted environment. What this
pollution is and who the polluters are of this environment that
makes it so difficult for these organisms to survive, I leave to
everybody's imagination here. I can take some inflation, taxa-
tion, and here comes the 30-day regulation. I was one of the
unfortunate cosignatories of a $500,000 order a few months ago,
and that money is going to give us nothing but a book or maybe
two books that went to a consulting firm to tell us just what
this plant, this medium sized steel plant, would have to do to
comply with BAT. Now we've been through BPT, we've been through
NPDES, we've been through air problems, and now we are facing
BAT. I want to be sure that now that you've invited me to this
conference, when I get back I ge.t the right message to my plant
management and to the corporate staff that we work with, and it
poses a little question to me. In a few months we are going to
get out a few nice little books which will say this is what we'll
offer to the regulating agencies as our BAT approach. Now, my
question to you gentlemen is, in developing your emerging science
of the effects of toxicity on aquatic organisms, are you going to
give us your virginity - the industry your virginity - when RAT
time and negotiations and decisions come around to land on
something that meets the long term requirements economically. My
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BACTERIA
ID
O
S-9
i
CHEMICAL
^INCUBATE
INCUBATE
1
RESULTS:
f
SOFTAGAR
OVERLAY
PLATE WITH OVERLAY
CONTROL
INCREASING DOSAGE
Figure 4. Schematic diagram of the preincubation test.
-------
S-9 MICROSOMES
SOLVENT
CONTROL
CHEMICAL
A
>£>
POSITIVE
CONTROL
NEGATIVE
CONTROL
CHEMICAL A-
CHEMICAL POSITIVE
B WITH
ACTIVATION
PLATE WITH WELLS
J
BACTERIA IN
SOFT AGAR
PLATE WITH WELLS AND OVERLAY
RESULTS:
POSITIVE
CONTROL
CHEMICAL B:
POSITIVE
WITHOUT
ACTIVATION
t
Figure 5. Schematic diagram of the well test.
-------
second question is this: Water is an essential medium that
supports life. We started by chemical analysis, we've gone to
toxicity of aquatic organisms. Now there are organisms that live
in air. Next year are we going to be analyzing the effect of
toxicity of air on the variety of organisms such as butterflies,
mosquitos, sparrows, etc.? Maybe you gentlemen are missing a big
bet. I would appreciate a reply to both of these questions.
Answer: I'm not sure I picked up the question. I heard the comment.
Question: You know what we are talking about as far as BAT is concerned.
Are you going to spring an air problem on us just as you sprung
the water problem on us?
Answer: The main reason we had the session now and sponsored a session
about two years ago called "Industry Takes Initiative" was to
encourage industry to take a look at the processes and to avoid
as much as possible end of the line treatment by looking at
process modification and changing the way things are made.
You're saying you know what may come out in BAT or what may come
out of guidelines in Washington. But we're trying to say that,
although there may be some numbers that come out of the guide-
lines in Washington, the national numbers will not cover every-
thing. The specific problems at each facility must be considered
individually. For instance, at a steel plant, the general type
of toxicants is known - such as polynuclear hydrocarbons from a
coke operation. In fact, Congress and our agency are already
changing the requirements in many industries where they have, you
might say, backed off in the BAT area and have gone to Best
Conventional Treatment. And I agree with them because they are
eliminating the need to install expensive pollution control
equipment that really has little effect for the cost.
The purpose of this session is to get you, the industry, to
start considering the toxicity testing described here today. If
you look at processes in your own plant and current treatment
technology, then combine that with some toxicity testing, you can
determine and install the treatment technology that will elimi-
nate some as yet unregulated toxicants in addition to "standard"
toxicants and put yourself ahead of the game. But you must first
evaluate your individual process to predict future requirements.
So I think that's an answer to the first question as far as that
goes. And as far as the second question goes, I don't know about
measuring butterflies, but we are in fact significantly concerned
about air emissions to the extent that some get into the water.
don't think that there would be that much
for instance, municipal sludge treatment
beautiful example of delaying the intro-
Now in your facility I
of a problem, but say
and incineration is a
duction of a toxicant to the receiving waters. By incinerating a
toxicant removed at a treatment plant, you may just volatilize it
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so it can fall back into the water. It's estimated, I think,
that 80 to 90 percent of the PCBs going into Lake Frie and Lake
Michigan are from the air not from the water. You need to look
at what you're doing with your waste, and if you're incinerating
something, are you really destroying it or are you only volati-
lizing it? And so you may need to be concerned about air emis-
sions. And certainly the RCRA requirements I think fit very
strongly into this.
Question: Excuse me. I want to go back to that. He says that we should
look into it but back with Mr. Macek - he didn't say how fre-
quently we had to test. Like with your wastewater system, how
often do we have to run a bioassay? Daily, weekly, monthly,
bimonthly, are you going to set up some sort of system?
Answer: There have been several people here today who have said you
cannot set a time and say if you sample it every day or every
week or every month that you will thereby resolve your problem.
You're going to have to know, you're going to have to learn what
your system is and learn how frequently you have to test in order
to get the answer. In some systems it may turn out that there is
just no change over any reasonable amount of time, so you could
sample on a very infrequent basis. But there are other systems
that change on an hourly basis, so there is really no way you can
set up a sampling program whereby you sample infrequently and not
miss certain things that will happen. You would have to have
almost a continuous test in some cases or find some other way to
measure to tie it down.
Answer: Just to further clarify. It would be very easy to come out and
say that every industry will run a 24-hour test. Every industry
will run a 96-hour test. And, this regulatory-wise, would be
much easier. Maybe that's what you all wanted to hear us say
today. But we're trying to say, which is I think a little more
difficult for both of us, that we and the states want to work
with you and for you to go ahead on your own to look at your
specific facility. You may have only one product line, but we
have plants where there are several thousand products in one
plant. We would much rather work with you to try to gear the
requirements or gear the testing to your specific operation, or
have the states v/ork with you if you're going ahead on your own,
or have your consultant work with you. We're not trying to avoid
setting numbers; it's just that there's so much variability from
one type of industry to another that I think it would be unfair
to you for us to propose to the region an absolute set of guide-
lines. And so I hope that we're not sounding like we're putting
you off by failing to give you specific numbers without going to
a particular class.
Answer: What Ken's saying is we're taking the hard way. It is much more
difficult from our standpoint to work to try to sit down and
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figure out which tests should be run on what frequency, at what
facility and how to use the results than it is to say every
discharger in the Region shall have to run one 96-hour test or
one 24-hour test on 10 percent effluent or 100 percent effluent
and if he gets this score he is thereby failed or passed. That
is an easy way - an easy approach which does not yield an answer.
And we just don't need any more nonanswers.
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RAPID ASSESSMENT METHODS (FISH COUGH RESPONSE, FTC.)
Robert Drummond
U.S. EPA, Environmental Research Laboratory
Duluth, Minnesota
Rapid assessment methods are not a new thing as people have been
researching this area for several years. For example, there is an EPA
publication called Short-Term Tests for Health and Ecological Effects.
A number of tests are outlined in this publication such as fresh water
algal assay bottle tests; acute fish toxicity tests; subchronic embryo-
larval tests; chronic fish toxicity tests with flag fish; fish avoidance
tests; acute invertebrate toxicity tests; subchronic invertebrate tox-
icity tests; acute plant toxicity tests; and acute static and acute flow-
through tests with marine fish. Some of the endpoints of these tests,
their strengths, weaknesses, status of development, application, and some
idea of the cost of the test and people to contact are also discussed.
Other recent publications are concerned primarily with fish as a tool
for biomonitoring effluents. Some of these papers are: A Computerized
System for Monitoring Fish Activity (Virginia Polytechnic Institute, Vir-
ginia) ; A Continued Biological Monitoring System to Predict Chronic Effects
of Toxicants (Virginia Polytechnic Institute. Virginia); Fish Locomotor'
Behavior Patterns as a Monitoring Tool (Morgan, South Africa); Fish Activity
Monitors (Water Research Center. England); Ventilatory Patterns of Fish Using
a Microcomputer Monitoring System (U.S. Army MedicalCenter,Maryland); An
Automated Biological Monitoring Facility for Rapid Assessment of Industrial
Effluents(VirginiaPolytechnic Institute,Virginia);Bluegill Respiratory
Activity~and Prediction of Chronic Toxicity (Proctor and Gamble, Cincinnati,
Ohio); and Biomonitoring with Fish:An Aid to Industrial Effluent and
Surface Water Quality Control (Morgan, South Africa).Our particular labora-
tory(U.S.EPA,Environmental Research Laboratory-Duluth), has been involved
in studies that are summarized in publications titled Procedures for Measuring
Cough Rates of Fish and The Fish Cough Response: A Method for Evaluating the
Quality of Treated Complex Effluents.
In order for behavioral responses of fish to be useful for biomoni-
toring effluents the test procedure must be inexpensive, simple enough to
be carried out by non-technical personnel in a short period of time, and the
endpoint being measured must have some relevance to the animal's ability to
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live and function properly. Of the various behavioral endpoints being
considered, five appear to be closely allied to the animal's well-being and
meet these other criteria. They are: 1) reproductive habits, 2) feeding
patterns/habits, 3) fear responses, 4) the righting reflex, and 5) respira-
tion. In a further breakdown, respiratory tests, in my opinion, are the most
applicable and advantageous as an endpoint for monitoring effluents for a
variety of reasons.
The term respiration as used here is an overall term denoting gill
purge activity (cough rates), ventilation rate/amplitude, oxygen consumption,
or any combination of the above. Advantages of using fish respiration as an
endpoint become clear when one reviews the abundance of literature that has
been published showing that numerous toxicants/effluents affect fish respira-
tion rates. Fish act as an integrator of toxicity - synergistic or antago-
nistic effects of an effluent will be reflected in terms of changes in
respiration rates. These changes occur within minutes or hours at lethal
concentrations. At sublethal concentrations it may take 1 to 3 days for a
change to appear. Thus, the response appears quickly. Another advantage
is that the response is graded to the concentration. The intensity of the
response can be used as a predictor of adverse effects. Lastly, the test is
adaptable to both flow-through and recirculating bioassays. The recirculating
bioassay approach has a big advantage in that hundreds of effluent samples
(5 to 8 gallons) can be shipped to a central point for testing. The small
volume of effluent required and speed at which respiratory changes appear
make this an attractive approach for screening out those effluents that are
of concern and need further testing.
We are now at a point where it is no longer necessary to observe fish
directly to tabulate changes in respiratory rates. These data can be collec-
ted, tabulated, and analyzed automatically using computers or other automatic
data processing devices. The automated approach allows more data to be
collected and processed accurately at an affordable cost.
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TEST ORGANISM ACQUISITION AND CULTURING IN THE LAB
Charles Steiner
Central Regional Laboratory
U.S. EPA, Region V
Chicago, Illinois
Good afternoon. The purpose of this discussion is to cover some of
the major considerations for culturing or acquiring organisms. For this
discussion, I'll use the working definitions that culturing organisms is the
rearing of these test organisms for the entire 1ifecycle, that is, the
entire reproductive cycle within the lab, while acquiring organisms is
receiving them from an outside source and holding within the lab for a dura-
tion of time. The purpose of both techniques is to provide test organisms in
which as many variables as possible are controlled with the ideal situation
being that the only variable between the organisms is the test variable. The
achievable goal, on the other hand, is to provide test organisms which are
healthy, relatively free of pollutants, of known age, and which are physio-
logically representative of the species. Two of the general points to
consider in culturing and holding organisms are that the holding or rearing
facilities should be of a size which is adequate for the number and species
of organism being utilized and the water supply to the culture unit should be
of high quality and be delivered through inert piping. This is not to say
that one must have a pristine water source for a culture unit. There are
successful units now operating which use deep well water, river water, lake
water, and even chlorinated tap water.
Many factors produce stress in organisms in the lab. Stress-producing
factors should be avoided. The organisms should be shielded from any excess
noise. The loading capacity of the facility should not be exceeded, and
proper diet should be maintained at all times. Also, one must consider
that for some organisms special environmental requirements are necessary
such as flowing water or fluctuating water levels.
As was mentioned earlier this morning, the recommended construction
materials for toxicity testing are teflon, glass, and number 316 stainless
steel. Fortunately, holding facilities are not so limited in the choice of
materials. For many years, concrete raceways and troughs were used for
holding fish within the lab. These facilities have the disadvantage that
they are permanent and do not allow flexibility within the lab. Recently,
however, fiberglass tanks are more in line with what is needed because they
are movable, durable, and versatile. And additionally, these units can be
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rigged to be self-cleaning, which is a real plus for anyone that has to
work with the animals.
Table 1 is a list of the recommended test species and test tempera-
tures. It includes also the salt water organisms, which I am not addressing.
I would like to say that there are occasions when an organism would be used
for a test that is not included in this list. In the case of an effluent
toxicity test sometimes it may be desirable to use a sensitive species that
is indigenous to the receiving water and/or is commercially or recreationally
important to the receiving strain.
Let us now move to the test organisms themselves. The test organisms
can be broken into two groups: the fish and the invertebrates. Fish may be
obtained from three different sources. They may either be reared in the lab,
obtained from a hatchery, or they may be collected from the wild population.
Table 1
RECOMMENDED SPECIES AND TEST TEMPERATURES
Species Test Temperature (°C)a
Freshwater
Vertebrates
Coho salmon, Oncorhynchus kisutch 12
Rainbow trout, Sal mo gai?dneri 12
Brook trout, Salvelinus fontinalis 12
Goldfish, Carassius auratus 22
Fathead minnow, Pimephales promelas 22
Channel catfish, Ictalurus punctatus 22
Bluegill, Lepomis macrocHTrus 22
Invertebrates9
Daphnids, Daphm'a magna or D^_ pulex 17
Amphipods, Gammarus lacustris, G. fasciatus, 17
or G^ pseudolimnaeus 17
Crayfish, Orconectes sp., Cambarus sp., Procambarus 22
sp., or Pacifastacus leniusculus 22
Stoneflies, Pteronarcys sp. 12
Mayflies, Baetis sp. or Ephemerella sp. Hexagenia 17
limbata or H^ bilineata 22
Midges, Chironomus sp. 22
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Table 1
RECOMMENDED SPECIES AND TEST TEMPERATURES (Continued)^
Species Test Temperature (°C)a
Marine and Estuarine
Vertebrates
Sheepshead minnow, Cyprinodon variegatus 22
Mummichog, Fundulus heteroclitus 22
Longnose killifish, Fundulus sTmilis 22
Silverside, Menidia sp. 22
Threespine stickleback, Casterosteus aculeatus 22
Pinfish, Lagodon rhomboides 22
Spot, Leiostomus xanthurus 22
Shiner perch, Cymatogaster aggregata 12
Pacific staghorn sculpin. Leptocottus armatus 12
Sanddab, Citharichthys stigmaeus 12
Flounder. Paralichthys dentatus, P. lethostigma 22
English sole. Parophrys vetulus 12
Marine and estuarine
Invertebrates9
Shrimp, Penaeus setiferus, P. duorarum, or 22
P. aztecus
Grass shrimp, Palaemonetes sp. 22
Shrimp, Crangon sp. 22
Oceanic shrimp, Pandalus jordani 12
Blue crab, Callinectes sapidus 22
Dungeness crab, Cancer magister 12
Mysid shrimp, Mysidopsis sp., Neomysis sp. 22
Atlantic oyster, Crassostrea virginica 22
Pacific oyster, Crassostrea gigas 20
aFreshwater amphipods, daphnids, and midge larvae and shrimp should be
cultured and tested at the recommended test temperature. Other inverte-
brates should be held and tested within 5°C of the temperature of the water
from which they were obtained. If the recommended test temperature is not
within this range, they should be tested at the temperature from the series
7, 12, 17, 22, and 27°C that is closest to the recommended test temperature
and is within the allowed range.
Material obtained from: Methods for Measuring the Acute Toxicity of Efflu-
ents to Aquatic Organisms.U.S.EnvironmentalProtectionAgency,1978.
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I'd like to address the collection from the wild population first. As
was mentioned this morning, I would put collection from the wild population
as the last resort in the case of fish, for the reasons that the life history
is not known, the fish population may be diseased, the population may be
hybridized, or it may already be contaminated with pollutants. Fish reared
in the laboratory, on the other hand, present a real source and a continuous
source of fish for most laboratories. As I stated earlier, the water supply
should be of high quality, but is not limited to a source such as a deep
well. An example is our own laboratory; our only source of water is chlori-
nated tap water supply. What we did was take the hot and cold water taps,
put on a mixing valve to regulate the temperature and send the water through
an activated carbon filter. The carbon filter is capable of removing the
total residual chlorine down to less than five parts per billion and the unit
itself has a capacity of 5,000 gallons a day, which is a lot more than most
of you would ever anticipate using.
The advantages to maintaining a culture are many. As I mentioned
earlier, it provides a continuous supply of fish of all stages. This in-
cludes eggs and allows for testing of the more sensitive life stages, a topic
which was covered earlier. The life history of the fish from a culture unit
is documented, and the fish remain relatively stress-free. But I think the
most important aspect of having a rearing unit is that the fish are main-
tained in a disease-free state. As some of you well know, there is nothing
more frustrating than starting a bioassay and having your control fish start
dying off because of a disease problem. However, along with the advantages
of the culture unit, there are some disadvantages. I feel that these are
minor and probably are far outweighed by the advantages. The first disadvan-
tage is that there is a moderate initial cost for setting up a culture unit.
It would be hard for me to describe the entire unit we've got at the lab, but
before we moved to our new facility, we set up an entire culture unit in a
room for about $700. And the second disadvantage of the culture unit is that
there is a need for continuous manpower supply to maintain the organisms.
The second source of fish I mentioned was hatchery fish. I would
suggest that if you start looking for hatchery fish, one thing to consider is
that the hatchery selected should be one which promotes the inbreeding of the
parent stock to maintain the stock, rather than to replenish the parent stock
from an outside source. Therefore I believe a hatchery should be picked in
which the hatchery fish are several generations removed from the wild popula-
tion to assure a better supply of test organisms.
There are some advantages to receiving fish from a hatchery. One, a
lot of you may be limited in space, and a rearing unit does take up some
space. But by the same token, a holding unit will also take up some floor
space. Usually one can receive more species of fish from a hatchery than one
can rear in the lab. The second advantage over a culture unit is that there
is less time spent in maintaining the stock. You only need someone to work
on the fish when you have them in the lab. There are several disadvantages
to receiving hatchery fish. The desired size or age of fish that you may
want may not be available at the time you need to do your testing. Transport
from the hatchery to the lab causes stress on the organisms, and also there
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has to be some arrangement made for transport of the fish. Sometimes long-
term planning is required to allow the hatchery to know the species and
the number of fish that will be needed. But I think the most important point
here is that fish received from an outside source have the potential to be
diseased. Therefore, hatchery-reared fish must first be quarantined at least
10 days, preferably 21, before used as test organisms. Additionally, it may
be advisable to give the fish a prophylactic disease treatment upon arrival
in the lab. At this point I'd like to comment that on the disease treatment
in hatchery fish, rather the disease prevention of fish received from an
outside source. Table 2 includes the recommended prophylactic and thera-
peutic disease treatments, and Figure 1 is a copy of the procedure we use at
Central Regional Lab. And I'd like to emphasize that any fish that we
receive from an outside source are given a prophylactic disease treatment.
One thing I tried to do was find some sources of hatchery fish.
Commonly, we go to federal and state hatcheries. Unfortunately, these
facilities are not available to everyone in the audience. And so, in digging
around, I did find some information. One possible contact for getting test
fish would be the American Fish Farmer Federation. Their administrative
offices are in Lonok, Arkansas, and they can put you in touch with bait
dealers in your area. Another source may be to obtain a copy of the Annual
Buyer's Guide published by the magazine Commercial Fish Farmer where they
have listed all their members under the type of services provided.
The maintenance of any fish for testing should follow the strictest
quality control practices and a log should be kept on all the aspects of
rearing or maintaining these test organisms. I had thought about talking
about the actual maintenance practices, but I don't think that time would
allow me to cover all the points. I took Table 3 from a publication by the
Sport Fish and Wildlife Service that showed the organic contamination of
fish food. This is something that should be considered in your background
information.
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Table 2
RECOMMENDED PROPHYLACTIC AND THERAPEUTIC
TREATMENTS FOR FRESHWATER FISH*
Disease
Chemical
Concentration
(mg/1)
Duration ofc
Treatment
External
Bacteria
Oxytetracycline hydrochloride
(water soluble)
Procaine Penicillin G in
25& 30-60 min
(3ml /I 00 gal) 48-72 hrs
Dihydrostreptomycin sulfate
solution (Franklin Lab, Denver, CO)
Benzalkonium chloride
(HYAMINE 1622R)
l_2b
30-60 min
Monogenetic
trematodes
fungi, and
external
protozoa^
Parasitic
copepjods
Nitrofurazone (water mix)
Neomycin sulfate
Formalin plus zinc-free
malachite green oxalate
Formalin
Potassium permanganate
Sodium chloride
DEXONR (35% Active Ingredient)
Trichlorfon
(MASOTENR)
3-5b
25
25
0.1
150-250
2-6
15000-30000
2000-4000
20
0.25b
30-60 min
30-60 min
1-2 hrs
30-60 min
30-60 min
5-10 min dip
(e)
30-60 min
Continuous^
aThis table indicates the order of preference of treatments that have been
reported to be effective, but their efficacy against diseases and toxicity
to fish may be altered by temperature or water quality. Caution: test
treatments on small lots of fish before making large-scale applications.
Fish should not be treated the first day they are in the facility.
Material obtained from: Methods for Measuring the Acute Toxicity of Efflu-
ents to Aquatic Organisms.U.S.EnvironmentalProtectionAgency,1978.
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C. DISEASE PREVENTION TREATMENT.
All fish shall be given preventive treatment for disease or parasites
upon receipt from the hatchery. Treat the fish in the holding tank in
the following day-by-day sequence and do not stop aeration at any time.
Day 1: Two to three days before the fish arrive, fill the holding tank with
nonchlorinated water to the 5-gallon level and mark the level per-
manently with waterproof marker. Add 45 more gallons of nonchlori-
nated water and mark the 50-gallon level. Fill three storage con-
tainers with nonchlorinated water and bring to room temperature.
Aerate the water continuously. (Caution: Be sure to dechlorinate
with a carbon filter if water contains chlorine).
Day 3: Fish arrival. Without opening, float the plastic shipping bag (water
and fish) in the tank and acclimate to tank temperature for 1-2
hours before releasing the fish to tank water.
Day 4: (a) Add 5 grams ACS grade potassium permanganate (KMn04) to 450
milliliters distilled or deionized water. When dissolved dilute
up to 500 milliliters. This is a 1% solution of KMn04. This
solution is used on days 4 and 5, and any remaining solution is
then discarded.
(b) Add 190 mill il Hers of the KMn04 solution to the holding
tank containing 50 gallons of water. (Caution: Stir while
adding the MNn04 to achieve a uniform solution.) Hold the fish
in the KMn04 solution for 1/2 hour. Do not exceed the 1/2-hour
limit, but continue the treatment to that time even if the fish
begin to surface or otherwise seem stressed.
(c) After 25 minutes of treatment, begin to drain the holding
tank to the 5-gallon mark. At 30 minutes add water from the
storage container to bring the tank volume to 50 gallons.
(d) Add to the holding tank 1-2 milliliters of 0.1 N^ sodium
thiosulfate (HazS&z'SHz®) solution, or until the water turns
yellow-brown. (To prepare 0.1 N_ sodium thiosulfate solution
dissolve 25 grams sodium thiosulfate in volumetric flask and
dilute to 1 liter with distilled or deionized water.) Color
change is slow, so do not be concerned if the water does not
become yellow-brown immediately. Refill storage container with
non-chlorinated water. Be sure water is at room temperature
before it is used on day 5.
Figure 1. Disease-prevention procedure used at Central Regional
Laboratory, U.S. EPA Region V, Chicago.
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Day 5: Repeat steps (b), (c), and (d) from day 4. It is not necessary
to refill the storage containers on this day.
Day 6: (a) Add 4,750 milligrams of tetracycline (the material from nine-
teen 250-milligram capsules) to 500 milliliters of warm tap
water. Shake until dissolved. Pour this solution into the
holding tank and mix gently. Leave the fish in this solution.
Fish are fed for the first time on this day.
(b) Fill storage containers with effluent-receiving water for
acclimation (Section II, D).
Day 7: Feed fish and clean bottom of tank with siphon. Aerate water.
Material obtained from: ORSANCO 24-Hour Bioassay. Ohio River Water Sanita
tion Commission, 1974.
Figure 1. Disease-prevention procedure used at Central Regional
Laboratory, U.S. EPA Region V, Chicago (continued).
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Table 3
SELECTED CONTAMINANTS DETECTED IN DIETS ANALYZED INTERMITTENTLY
AT FPRL FROM AUGUST, 1972 - AUGUST, 1973
Residues (ug/g)
Diets
Oregon Moist
Glenco
Clark
Silver Cup
(2 samples)
EWOS
(3 samples)
h- »
S Colorado State Diets
1 (9 samples)
BSFW Hatchery Diets
(2 samples)
Purina Catfish Chow
DDTa PCBb
0.06 0.30
0.11 0.30
0.11 0.20
0.08- 0.20-
0.17 0.32
0.15- 0.20-
0.39 0.30
0.11- 0.10-
0.84 2.80
0.12- 0.20-
0.19 0.30
-
Hexchl oro-
benzene
(HCB)
_d
-
-
mm
0.06
0.008-
0.046
_
0.003
—
-
Total
Organo-
chlorine Phthalate0
Dieldrin Endrin
-
-
-
• *•
0.01
0.01-
0.02 0.01
0.01-
0.30 0.01
.m .
0.01
0.01
Content
0.36
0.41
0.31
0.37
0.555
0.361-
0.766
0.213-
3.953
0.32-
0.50
0.01
esters
-
-
-
_
-
•V
3.0
^
-
_
-
-
Reference Research Diet - <0.1 - - - <0.1
(4 samples)
Minimum Detection
Limits
a All DDT analogs.
b All polychlorinated
0 Di-n-butyl phthalate
d None detected.
Table Obtained From:
0.005 0.1
biphenyls, but usual
and di-2-ethylhexyl
0.0001
0.01 0.01
0.1-
0.5
ly AroclorR 1254 and 1260.
phthalate.
Acquisition and Culture of Research
Fish: Rainbow Trout,
Fathead Minnows,
Channel
Catfish and BluegiTTs.U.S. Bureau of Sport Fisheries and Wildlife, 1975.
-------
The last topic that I was going to talk on was invertebrates. And I
saved this for last because there's not much, information I can give you.
With the exception of Daphnia, invertebrates are difficult to maintain in the
laboratory for any extended length of time because they have very precise
environmental requirements. For the most part, individuals seeking these
invertebrates are required to develop their own sources - that means going
out and scouring the streams in your area. The reason for this is that, for
the most part, invertebrates do not ship well, and it's hard to maintain good
quality up until the test time. The exception in the invertebrate field are
the Daphnia. Daphnia can be obtained from many sources. If one is looking
to start his own Daphnia culture, small numbers of animals can be obtained
from several biological supply houses. In most cases it's best to let the
people know that you are looking for a particular species and what they are
to be used for so that you can guarantee proper identification. On the other
hand, if one is looking for large numbers of Daphnia, I know of at least one
supply facility in Minneapolis, Minnesota, Aquatic Life, that shipped units
of Daphnia between 12,000 and 15,000 in number for around $50, including
the air freight.
In summary, I would like to say that this has been a general discus-
sion on rearing and holding, and I would reemphasize that good quality
control practices are needed to maintain the test organisms. If this is
done, it makes the whole job of running bioassay tests a lot simpler.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
PPA
3. RECIPIENT'S ACCESSION NO.
TITLE AND SUBTITLE
Proceedings of the Seminar on Biological
Monitoring and its use in the NPDES Permit
Program
5. REPORT DATE
Mav 1983 ("Issuing Date!
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
Timothy O'Mara
Orville E. Macomber, Editors
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Center for Environmental Research Information
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
10. PROGRAM ELEMENT NO.
11.c6NtRACf,rGRANf NO.
Inhouse
2. SPONSORING AGENCY NAME AND ADDRESS
Center for Environmental Research Information
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
13. TYPE OF REPORT AND PERIOD COVERED
10/2/79
14. SPONSORING AGENCY CODE
EPA/600/00
5. SUPPLEMENTARY NOTES
seminar was conducted in cooperation with the Enforcement and
Surveillance $ Analysis Divisions US EPA Region V, Chicago, IL.
Contact: Orville Macomber f513l 684-7394 - _-_
6. A8STRAC
This seminar brought together representatives from industry, state and
local governments and biological monitoring consulting firms to present the
U.S. Environmental Protection Agency, Region V perspective on biomonitoring
requirements for the National Pollutant Discharge Elimination System (NPDES)
permit activities.
A limited number of industries have the potential for the discharge of
toxicants and they will be required to conduct special testing, monitoring
and evaluation of their effluents by utilizing biological testing techniques.
"This seminar was held to clarify the methods and uses of biomonitoring and
its application to setting limits in NPDES permits. Presentations were made
concerning biological monitoring phytoplanktons, zooplanktons, macroinver-
tebrates, fish, bacteria, etc. The range of tests include static and flow-
through bioassays, including tests for bioaccumulation, Ames tests for
mutagenicity, and some rapid assessment methods such as the fish cough
response test.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lOENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport/
Unclassified
21. NO. OF PAGES
111
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Perm 2220-1 (R«v. 4-77) PREVIOUS EDITION is OBSOLET
E107
US GOVERNMENT PRINTING OFFICE 1980-657-146/5673
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