A Primer for EPA Employees:
Presenting Scientific Evidence
James A. Rogers
Office of General Counsel
Washington. D. C.
September, 1974
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Preface
This primer is the result of a dialogue between the Assistant
Administrator for Research and Development and the Assistant
Administrator for Enforcement and General Counsel requesting closer
interaction between the research and legal elements of EPA. The primer
was developed to supplement a seminar program held at the various
National Environmental Research Centers during the fall of 1974.
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TABLE OF CONTENTS
Page
1. Tut roduction 1
2. Types of Proceedings
a. Trials in Court 1
b. Administrative Trial-Type Hearings 2
c. Administrative Legislative Hearings 4
d. Presenting Direct Evidence 4
3. Discovery 5
4. Procedures of Laboratory Research and Field Investigations
Which Are Subject to Attack
a. Chain of Custody 8
b. Laboratory Research Techniques 10
5. What to Expect in Cross-Examination 13
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1. Introduction
This document is not intended to be used as a legal reference. The
purpose of this primer is to give practical guidance to scientists
as to what to expect when they become involved in some form of litigation
in which they are asked to present the results of their research or investi-
gation. The discussion is directed primarily at water pollution control
because most of the adversary type proceedings in which a scientist may be
called upon to testify will be related to water pollution and because most of
the practical lessons have been in this field. To avoid making this pre-
sentation unduly long, many generalizations have been made and fine points
of evidentiary rules, for example, have been ignored. The intent is to point
out in a general way what one will be asked by the government attorney and
on cross-examination so that laboratory or field investigation procedures
may be tailored to avoid the tragedy of having valuable scientific work
rendered less useful for failure to follow a protocol. The specific preparation
of a particular witness for a particular hearing, of course, necessarily must
take place with the government trial counsel in the time immediately before
he is to testify and is shaped largely by the substance of his testimony.
I have placed a great deal of reliance on actual examples of testimony,
both good and bad, in attempting to make points. The names of witnesses
have been deleted; none of them is an EPA employee.
2. Types of Proceedings
a. Trials in Court
The traditional way in which environmental issues are litigated is
in a courtroom, either federal or state. There have been hundreds of cases
in which the state or federal government brought actions against a polluter
either for violation of specific statutory or regulatory requirements or
for violation of some public nuisance concept. The Reserve Mining case is
the supreme example of this: the federal government based its claim for
relief on the pre-1972 Federal Water Pollution Control Act and the water
quality standards promulgated thereunder; the plaintiff states sued largely
on the bftslfl of public nuisances ("unreasonable interference with the public's
right, to use and enjoy tho onvtronmont").
Thero will be fewer court cases, involving water pollution at least,
in which expert witnesses will be called upon to testify. Or at least the
witnesses will be called upon to present less sophisticated proof than before.
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This is because the country is gradually moving to the National Permit
Discharge Elimination System (NPDES)lV under which most contested
facts will be resolved in hearings before the Agency instead of in trial
before a judge. Thus, whether the waste from a particular discharge will
interfere with oyster reproduction, and therefore what maximum effluent
discharge restrictions should be contained in the permit, is an issue which
will be addressed in hearings before the Agency's Administrative Law
Judges. If a discharger is violating its permit, the Justice Department --
or the State Attorney General if the NPDES program is being administered
by the State -- will bring an action. Here the issue will be simply whether
the effluent levels have exceeded the permit terms; it will be much like a
license violation case: the factual issue will be whether the permit was
violated -- basically a monitoring chore -- not whether deleterious effects
occur by discharging at that level. (This change in the burden of proof
was one of the major reasons for amending the Act to employ the permit
system).
Of course, even with the NPDES program, there will be court actions
and the basic rules of evidence for presentation of expert testimony will
come into play. These rules will be examined below in the section dealing
with adjudicatory administrative hearings.
b. Administrative Trial-Type Hearings
Increasingly EPA is holding administrative trial-type hearings.
Mention has already been made of the NPDES procedures. There also will
be a great need for expert testimony in FWPCA section 316(a) hearings in
which power companies will attempt to demonstrate that the "effluent
limitation proposed for the control of the thermal component of any dis-
charge from such source will require effluent limitations more stringent
than necessary to assure the protection and propagation of a balanced,
indigenous population of shellfish, fish, and wildlife in and on the body of
water into which the discharge is to be made...." The first of these
hearings will begin this fall.
The third section of the recently enacted water pollution legislation,
which has already generated substantial litigation, is section 307(a).
establishing effluent standards for toxic water pollutants. 2/ This section
In unusual In that Congress has called for a legislative rule-making hearing
to take on many of the trappings of a trial. Most importantly, the pro-
cedures for section 307(a) hearings call for cross-examination of witnesses.
The category of hearing which probably has thus far generated the
greatest workload for EPA scientists is the pesticide cancellation hearings.
These have been held for DDT and are being conducted for Aldrin/Dieldrin
I/The basic statutory framework is set out in sec. 402 of the Federal Water
Pollution Control Act, as amended.
2 /The first list of toxic water pollutants consisted of Aldrin/Dieldrin, DDT,
Benzidine, Cadmium, Mercury, Cyanide, PCB's, Endrin and Toxaphene.
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and Mirex under the Federal Insecticide Fungicide and Rodenticide Act
(FIFRA). Traditionally these battles last for months and are fought by
the manufacturer and EPA, although the parties also include environmental
groups, users, and smaller companies who package various formulations
using their own labels.
The rules for presenting the expert testimony in trials and adjudicatory
type administrative proceedings really differ little. In each situation the
scientist is asked to testify as to his knowledge on technical questions rele-
vant to the issues being tried. It may be helpful to remember that conclusions
and opinions generally are not permissible forms of testimony and that an
exception to this rule is made for expert testimony under the theory that lay-
men would be unable to draw conclusions in difficult technical areas with-
out the assistance of experts. But is only when the expert testifying is
truly expert in the field, is drawing upon his expertise in making a con-
clusion, and laymen (judge or jury), given the same facts, could not render
a conclusion, that his opinion testimony is permitted.
Except on rare occasions the expert will not be asked to render an
opinion on the ultimate question; for example, he will not be allowed to give
his opinion that the permit for a power plant discharge should call for a
mixing zone of 1500 feet. If he is a biologist he will, however, be allowed to
say what the effect of use of a 1500' mixing zone on the zooplankton would be.
The expert witness in his proper role is merely providing a part of the
technical base upon which decisions are made. For him to render a judg-
ment on questions in which other disciplines come into play is to enter
fields in which he is not expert and in which he cannot render assistance
to the trier of fact.
In the pesticide cancellation hearings, the Agency Administrative Law
Judges have been allowing scientists to state their views as to whether a
product should be banned, the ultimate question to be decided, but have
also said that they will not give this testimony great weight. In a court
trial, such testimony would not be allowed at all.
Perhaps the major difference between expert testimony in the court
trial and in an adjudicatory administrative proceeding is the extent to
which hearsay is allowed. Hearsay evidence is
... testimony in court, or written evidence
of n. Htntomont made out of court, th«
statement being olio red as an asHortiori
to show the truth of matters asserted therein,
and thus resting for its value on the credi-
bility of the out-of-court asserter. 3/
3/McCormick on Evidence, 2nd Ed., 1972, p. 584.
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It is important to remember that the hearsay rule applies to both oral and
written statements by an out-of-court party. In a traditional suit, then,
a witness testifying on the proper analytical methods for detecting
methoxychlor, for example, could not refer to a paper by another scientist
confirming the appropriateness of his methods if the purpose is to suggest
that the substance of that paper is true. Nor could a witness testify that
his results were confirmed by Dr. Jones, with whom he talked last week.
In administrative proceedings the hearsay rule is relaxed substantially.
In the proceedings held to date before EPA administrative law judges,
hearsay expert testimony has been allowed if there is a "nexus between
the witness's expertise and the subject of the paper -- authored by
another --to which he wishes to refer. The witness in the hearing room
must, however, be prepared to stand some cross-examination on the
document. Thus, if he cannot say whether the analytical methods used by
the other investigator were acceptable, he may not be allowed to use the
paper.
c. Administrative Legislative Hearing
Quick mention is made of those administrative proceedings in which
"generic" rules are being considered. This may be in an EPA rule-making
hearing or in federal or state legislative proceedings in which proposed
statutes are being debated. There is usually only informational questioning
not in an adversary setting. Often scientists appear in panels and most
of the time the bulk of the testimony has been prepared in advance.
d. Presenting Direct Evidence
The direct testimony in a court trial is usually given orally, often
with reference to a written report and always with access to written factual
data upon which the expert is relying in rendering his conclusions. And
almost always the direct testimony relates to the effects of the discharges
from a specific plant or outfall.
In administrative proceedings quite often now the direct testimony is
in written narrative form and only the cross-examination is done orally.
Thnro urn many ndvunLaftOH to this: the witness and his lawyer can be
sure that the important points are covered, and difficult concepts can bo
presented with more precision than is usually possible in oral testimony.
The opposition is usually given a week or two to study the document
before the witness appears. This allows them to narrow the areas of
cross-examination and to prepare for the often intricate questioning of
the scientific data. It allows the cross-examiner to have his own expert
go over the material with a fine-toothed comb. The end result is a
more organized hearing. It also allows the hearing to go forward without
the necessity of elaborate "discovery, " since the tender of written direct
testimony well in advance of the hearing serves the basic purposes of
pre-trial discovery: avoidance of surprise. <
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Unfortunately, one of the by-products of the use of written direct
testimony which is entered into the record without reading, is a feeling
by some witnesses that their testimony did not hold up well. This is
because the experienced cross-examining attorney chooses to question
the \\ itness only on points on which he thinks the witness is not capable
of {jiving firm, well documented answers. Thus witnesses have gone
nn entire day without being asked to discuss their basic research.
Nexertheless, that research will be used if it is adequately presented
in the written testimony.
More than any other problem encountered by lil'A trial lawyers is
Uie natural resistance on the part of scientists to write complete narratives
rather than short precis of their work. There may bo an assumption that
whatever the rules at the hearing they will get to elaborate orally on the
presentation. Tn several instances the opposition attorneys have not
eross-examined at. all because otherwise dangerous witnesses did not
present a statement worthy of the underlying research or investigations.
The rule to remember in writing direct testimony is to put on paper
everything you want to say. It is far easier for your lawyer to cut you
hack if you said too much than it is for him to interpolate in a difficult
scientific area. An example of a good written presentation is attached as
Appendix A. Appendix 13 is an example of a statement that, while short,
attempts to say too much (see the last paragraph). Note in the well written
statement that reference is made to Appendix A, B, C, etc. Documentary
evidence relevant to the witness's presentation is usually physically
at (.ached to the written testimony and referred to in the body of the
presentation. Sometimes these attachments are referred to as exhibits
rather than appendices.
It has often been said that the direct testimony of an expert witness
consists of four parts: (a) his qualifications (by education and/or
experience) as an expert, (b) the material from which he fashions his
opinion, (c) the process or reasoning by which he gets from the material
at hand to his conclusion or opinion, and (d) the conclusion or opinion itself.
Usually there is little dispute over an expert's personal background and that
information comes in without question. In many cases the presentation of
raw data itself or with a clear statistical explanation is enough for one to
draw a conclusion, and logical step-by-step delineation of how the experi-
ment was conducted or how the field samples were nnaly/cd is vital to show-
case this data. Italher than belabor points (b)-(d) in abstract terms, actual
examples will be presented in the later discussion of cross-examination to
show what should and should not be done.
:*. Discovery
Discovery is a general term used to describe the process by which one
side in litigation finds out what the factual basis for the other side's case
is. In federal or state court actions there are several procedures by which
this can be accomplished. The most frequently used procedure is the taking
of oral depositions. Under this procedure, the potential witness is placed
under oath before a court reporter and asked a wide range of questions
designed to prepare the opposing lawyer for his testimony at the trial.
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The deposition is also an opportunity for the opposition to ask about reports,
memos, maps, lab books, pictures, etc., which the "deponent" knows of
or may have in his possession and which he does not intend to use in the
trial, i. e., material which the other side may wish to use. By use of a
subpoena duces tecum (very roughly "you are ordered to appear and bring
all the following documents with you") the opposing party can force an EPA
scientist to collect all material arguably applicable to the issue. The lawyer
may precede the "noticing" of a deposition by filing a motion to inspect all
the documents related to the question. This helps prepare him to take the
deposition.
Another less often used procedure is the use of written questions served
upon the opposition and to be answered under oath (interrogatories). Some-
times this is used to initiate discovery by asking "who are the scientists
who have any knowledge on this subject" or "where are your freshwater
laboratories located,' or "who have you consulted in bringing this lawsuit"9
Scientists are virtually united in their horror of the all powerful dis-
covery procedures, drafted and enacted by lawyers, which can force them
to pholoduplicate massive amounts of material. Some lawyers have argued
that, unlike conspiring executives in an antitrust case, scientists should not'
be put through the ordeal of having filing cabinets raided. Actually, the
Federal Rules of Civil Procedure, applicable in federal courts but adopted
intact by most states, provide for restricted discovery of an expert's data.
Rule 26(b)(l) states the basic rule:
Parties may obtain discovery regarding
any matter, not privileged, which is
relevant to the subject matter involved
in the pending action, whether it relates
to the claim or defense of the party seeking
discovery or to the claim or defense of any
other party, including the existence, des-
cription, nature, custody, condition and
location of any books, documents, or other
tangible things and the identity and location
of persons having knowledge of any dis-
coverable matter. It is not ground Tor
<>l)jccl,ion thiil I he Information Hoiitflil. will
be inadmissible al. the trial IF I ho Infor-
mation sought appears reasonably calculated
to lead to the discovery of admissible evidence.
l''rom this base, the Rules in section 26(b)(4) set forth an exception for
experts:
Discovery of facts known and opinions held
by experts, otherwise discoverable under
the provisions of subdivision (b)(l) of
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tliis rule and acquired or developed in an-
ticipation oi' litigation or i'or trial, may
be obtained only as follows:
(A)(i) A party may through interroga-
tories require any other party to identify
each person whom the other party expects
to call as an expert witness at trial, to
stale the subject matter on which the ex-
pert is expected to testify, and to state
Hie substance of the facts and opinions to
which the expert is expected to testify
and a summary of the grounds for each
opinion, (ii) Upon motion, the court may
order L'urther discovery by other means,
subject lo such restrictions as to scope
and such provisions, pursuant to sub-
division (b)(4)(C) of this rule, concerning
fees and expenses as the court may deem
appropriate. " (Emphasis supplied )
Wh.il the Kules giveth (in the form of protection to scientists) the trial
judges usually taketh away, in response to motions to have full discovery
of expert witnesses and documents. The theory advanced by most of these
judges is that in large complex cases, in order not to unduly drag out the
trial, it is essential to have the parties do the exploratory questioning
prior to trial. It must be remembered that most civil litigation in the
United States is between two private parties and may involve one or two
experts at the most; the EPA scientist is likely to appear, if at all, in
a major suit or hearing in which the government and a large business
are the parties and in which a dozen or more experts will testify. In
such situations it is unlikely that discovery will be restricted.
Are any materials privileged and not subject to disclosure? Increas-
ingly the answer is, virtually nothing. Memos between researchers in a
laboratory, draft reports, memos of telephone calls, and letters have all
been held to be discoverable. Only a very limited category of documents
which fell! in the category of attorney "work product" are privileged. What
I'sills under this heading cannot he .staled with precision bill, they arc
e:.:-ent i:il l\ llio.se an.'il vses of the l;iw mid/or fi,icln produced by Ihe attorney
i>r Ml lii.-: ill reclinn in prcp:ir:il Ion specifically I'or Ihlt-i I it ig'allon. Thin
author has seen very lew works of :i scientist which have fallen within
this category. That your work is probably susceptible lo discovery should
not be a deterrent to candor: no one can be faulled for slating what he
knows. Hut it should be a deterrent for hastily formed opinions or personal
continents which may be misunderstood if taken out of context.
No rigid distinction can be drawn in the above; discussion between
trials in courts and administrative proceedings because increasingly
lawyers have used the freedom of Information Act (I''OTA) lo obtain
those documents discoverable under court rules. The RPA regulations
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on the FOIA reveal how this can be done. Once having complied with the
mechanical requirements of 40 CFR Part 2, such as making a request
in writing at the right office, a party is entitled to review and copy all
materials except those
1. -- specifically exempted from disclosure by [some
other] statute.
'2. -- trade secrets and commercial or financial in-
formation obtained from a person and privileged
or confidential.
3. -- interagency or intra-agency memorandums or letters
which would not be available by law to a party other
than an agency in litigation with the agency.
4. -- geological and geophysical information and data,
including maps, concerning wells.
Emphasis has been added to category 3 to show why lawyers can argue
thai llic broad rules of the Federal Rules of Civil Procedure should be
employed in FOTA interpretations. The complete list of exemptions are
in 40 CTR §2. 105(a). If there is doubt whether all or part of the requested
material should be disclosed, the procedures set forth in 40 CFR §§2. 104 -
2. 107 should be followed.
-L Procedures of Laboratory Research and Kield Investigations Which
Arc Subject to Attack
a. Chain of Custody
The scientist or technician who fills water bottles in a stream just
,bolo\\ a potential deteiulanl's outfall must take precautions to insure that
at trial the sample bottle lie refers to can be shown to correspond to a
sample taken at a certain time and a certain place. The often elaborately
stated rules of chain of custody arc nothing more than a means of guaran-
teeing the integrity of the identification of field samples. IVlcCormick's
Handbook of the Law of Evidence states simply that the expert witness must
be ahle to trace the chain of custody "with sufficient completeness to render
it improbable that the original item lias either been exrhanged with another
or been contaminated or tampered with. " This requirement must be
met before the evidence eon be received at all; it does not simply affect
the \\eig~ht to be given to the evidence.
The Legal Support Division of the Office of Enforcement and General
Counsel in May of l!)7'J prepared a rather detailed jjfuldc for scientists
gathering field samples. What follows is largely taken from that primer.
Stream anil effluent samples should be obtained by using standard field
sampling techniques. The chain of custody record tag should be attached
to I he sample container at the lime the sample is collected and should
ronl;\in (lie following information: s;uiiple number, dale and time taken,
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source of sample (include type of sample and name of firm), the pre-
servative and analyses required, name of person taking sample and
witnesses. An actual tag is shown in Appendix C, and a sample trans-
mittal sheet is reproduced in Appendix D. The pre-filled side of the
card should be signed, timed and dated by the person sampling. The
sample container should then be scaled with a pre-printcd, gummed
seal containing the Agency's designation, date and sampler's signature.
The seal should cover the string or wire tie of the chain of custody tag
so that the tag cannot be removed and the container cannot be opened
without breaking the seal. The tags and seals must be filled out legibly
in ballpoint (waterproof ink).
Blank samples should be collected in containers with and without
preservatives so that laboratory analyses can be performed to show that
there was no container contamination. A bound field notebook, or log,
should be used to record field measurements and other pertinent information
necessary to refresh the sampler's memory in the event he later becomes a
witness in an enforcement proceeding. A separate set of field notebooks
should be maintained for each survey and stored in a safe place where they
can be protected and accounted for at all times. A standard format should
be established to minimize field entries and should include the date, time,
survey, type of samples taken, volume of each sample, type of analysis,
sample numbers, preservatives, sample location, field measurements such
as temperature, conductivity, DO, pH, and any other pertinent information
or observations. The entries should then be signed by the field sampler.
The responsibility for preparing and retaining field notebooks during and
after the survey should be assigned to a survey coordinator, or his designated
representative.
The field sampler is responsible for the care and custody of the samples
collected until properly dispatched to the receiving laboratory or turned over
to an assigned custodian. He must assure that each container is in his
physical possession or in his view at all times, or stored in a locked place
where no one can tamper with it.
Color slides or photographs are sometimes taken of the outfall sample
location and any visible water pollution in the vicinity. Written docu-
mentation on the back of the photo should include the signature of the
photographer, lime. dale, and site location. Photographs of this nature,
which may be lined us evidence, should be handled according to the established
chain of cnslody procedures.
When transfer-ring the possession of samples, the transferee must sign
and record the date and time on the chain of custody record tag. Custody
transfers, if made to a sample custodian in the field, should be recorded
for each individual sample. Every person who takes custody must fill in a
standardi/.ed "Receipt of Sample" form (see Appendix C). To prevent undue
proliferation of custody cards, the number of custodians in the chain of
possession should be as few as possible.
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Mailed packages should be registered with return receipt requested. If
packages are sent by common carrier, a Government Bill of Lading should be
obtained. Receipts from post offices, and bills of lading or other common
carrier receipts should be sent to and retained by the laboratory custodians
as part of the permanent chain of custody documentation.
The laboratory should designate an employee or employees as a sample
custodian. In addition, the laboratory should designate a clean, dry,
isolated room that can be securely locked from the outside as a "sample
storage security area. " The sample custodian must maintain a permanent
log book in which he records, for each sample, the person delivering the
sample, the person receiving the sample, date and time received, source
of sample, sample number, how transmitted to lab, and a number assigned
to each sample by the laboratory. A standardized format should be established
for log book entries.
Samples should be handled by the minimum possible number of persons.
Distribution of samples to laboratory personnel who are to perform analyses
should be made only by the custodian. The custodian should enter into the
log the laboratory sample number, time and data, and the signature of the
person to whom the samples were given.
Laboratory personnel are responsible for the care and custody of the
sample once it is handed over to them and should be prepared to testify that
the sample was in their possession and view or securely locked up at all
times from the moment it was received from the custodian until the tests
were run. Once the sample testing is completed, the unused portion of
the sample, together with all identifying tags and seals, should be returned
to the custodian who will make appropriate entries in his log. The returned
tagged sample should be retained in the sample room until it is required
for trial. Strip charts and other testing documentation also should be turned
over to the custodian.
b. Laboratory Research Techniques
Volumes have been written on proper laboratory techniques, so
there will be no attempt, here to indicate in even a general way what pro-
rrilmvs should be lollovvril In examining 11 particular substance. The
purpose of IhlH MIT! Ion i;i more lo cnmhaMl'/.r Ihe role proper (or arguably
improper) .sampling l.crhnU|uc plays In a case. II' a lawyer dolci'mlnos
that an expert witness con do harm lo his client's rase, and that the
subs tan ec of what the witness has to say is probably correct, or at least
difficult to attack, then he will attempt to cast doubt upon the analytical
methods employed by that scientist. It is imperative that accepted
laboratory techniques be followed to the letter and that if the methods
are not presented in depth in the research paper itself, at least detailed
records arc kept so that questions going to those methods can be answered.
The increasing number of environmental disputes has generated a lawyer-
specialist who (a) knows where to find consultants and (b) knows how to
use their expertise., in ways which can seriously disci-edit researchers
who are not carciul.
10
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Improper cleanup before use of gas chromatography, failure to run
blanks or controls, failure to measure other possible stresses on the
organisms beside the test toxicant, have either totally impeached or
seriously questioned scientific work.
The statistical significance of test results is often taken for granted,
yot. several witnesses who have appeared in recent EPA hearings have had
their published work seriously questioned by skillful use of desk calculators
and accepted statistical analyses. Normit, probit and "t" tests are now
common terms in lengthy proceedings.
What follows is an excerpt from part of the Aldrin/Dieldrin proceeding.
It is not one of the several examples in which the witness was totally trapped
by improper methods; it is a more typical case in which a "question" is
raised in the mind of the trier of fact:
Q. First of all, I would like to discuss the
methodology that you employed in this partic-
ular experiment. In particular, I would like
to discuss the reliability and the weight to
which you give to the levels of dieldrin and
aldrin that you found... I would like to focus
on the methodology.
In particular I want to ask you, Dr.
whether in the techniques that you employed
for analyzing the presence of aldrin and/or
dieldrin, whether you used any separation
techniques, or so-called clean-up techniques,
in order to eliminate the presence of DDE, or
PCB, or any other artifacts which could have
caused interference on the GLC columns, and,
therefore, exaggerated or made too large the
results which you found for aldrin and/or
dieldrin ?
A. Really there are two components to the
qup.stion. One is the sampling and one is the
in-house aiinlyRifl of the sample.
You arc asking once the sample Is in-house,
and in a correctly identified manner, how it
is analyzed?
Q. That is correct.
A. In this particular investigation, some of
the peculiarities of saltwater chemistry said
it really wasn't that necessary to go through
elaborate separation schemes with the type of
gas chromatography, the type of detector that
11
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was employed. We did use different columns so
we wouldn't catch any of these places where
one type of compound overlaps another, or one
reacts in a column and produce a spurious peak
of one sort or another.
Tn other types of work, sometimes medium clean-
up, extensive cleanup, might be needed, but not
in this case.
Q. Are you saying that because the samples
were taken from saltwater, in this case it
was actual seawater, wasn't it --
A. That is correct.
Q. -- that there were no artifacts that
could have been present in the seawater?
A. Oh, there may have been many arti-
facts. But using the particular column,
the inlet design, the type of detector,
the sensitivity settings, the thermal
settings, flow rates, all of those para-
meters, there was no interference at this
point. There were lots of other items
that could be seen on some of the chroma-
tograms, but they weren't of interest for
this particular paper.
Q. Did you separate the PCB's from your
sample?
A. I really don't recall in this partic-
ular case.
Q. You don't recall whether you used
separation techniques'
A. No. I .H. mr rrstuto that.. I do not
rrrull whcMirr Ilic roiidlLloiiH won- Htlrh
thai I'C'll'n wuiv occurring wil.li tlio detec-
tor sensitivities, et cetera. Tn other
words, tho conditions under which the
instrument was set up. There was no
specific procedures, again going through
column separations and things of this
sort, to try to separate out different
groups of compounds in this case.
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A. No. Let me restate that. I do not
recall whether the conditions were such
that PCB's were occurring with the detec-
tor sensitivities, et cetera. In other
words, the conditions under which the
instrument was set up. There was no
specific procedures, again going through
column separations and things of this
sort, to try to separate out different
groups of compounds in this case.
Q. You said earlier, I believe, that
there was no specific separation of PCB's9
A. In this case.
Q. In this case. Can you state --
A. In other words, we were not looking for
PCB's.
Q. I understand that.
Now can you state that it was your belief
that there were no PCB's in the samples
that you took ?
A. No, I have no real feeling one way or
the other as to what might have been.
II must be emphasized that a judge cannot easily determine what is
"harmless analytical error"; as a lawyer in a strange field, he must rely
upon certain procedures which others in the field have called the standard
methods for analysis. If the witness cannot tick off the requisite pro-
cedures he should be prepared to explain why he used a different method,
and preferably bo able to point to some published work which sanctions the
method he used. Than* is an aura of "peer acceptability" that surrounds
published work which iloun not attach to unpublished research. If at all
poMrtlblo. Urn oxl.ru I lino und olTnrl should he much; to publish your work,
preferably not Just In an LIP A circular. Although prohuhly unjustlt'lod,
the greater weight given by lawyers and judges to glossy papered finished
reports will no doubt continue.
5. What to Expect in Cross-Examination
When scientists think of trials or administrative proceedings in which
they are to appear they may not think of the purpose of the hearing, or even
the purpose of his testimony. They may not think of the novel scientific
legal issues involved. Often, their main concern is how bad cross-examina-
will be. To some scientists cross-examination is a forceful wrenching from
the world of the reasonable and polite to the world in which word games pre-
vail over accepted fact. In some trials, unfortunately, this has been true.
13
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but a witness can control the cross-examination to a remarkable extent by
being adequately prepared. Most oi% this preparation should be directed by
his lawyer, but there are some general points which apply to most situations.
The following guidelines have been used in preparing witnesses for the
headquarters hearings on pesticides and section 307(a) of the FWPCA:
1. You have no obligation to answer a question which you
do not feel qualified to answer. You are not a defendant
in a criminal trial required to answer. An "I am not
qualified to answer that" or "I do not have enough facts
to answer that" is perfectly acceptable.
2. Do not be lured into areas beyond your field.
3. Ask for clarification of a question if you have any
doubt what is being asked.
4. When a hypothetical question is posed, make sure all
elements of the hypothetical needed for you to be able
to answer are present.
5. Take your time in responding to questions.
6. Do not elaborate beyond what is necessary to give a
complete answer --on the other hand, do not allow
yourself to fall into trap of giving an "out of context"
answer --an answer which, in and of itself, is true but
which has a misleading implication if further comment is
not given. If you cannot answer with a "yes" or "no",
make it plain you need to qualify your answer.
7. You may be asked to comment on works of other
scientists you do not know or have not read recently --
e. g. , "I show you this list of pesticide residue figures
from Iowa -- aren't they awfully low?" You probably
need to know how the research was conducted, the
details of the methods, before you can comment accurately.
U. Don't respond to a challenge by boasting.
i). Don't try to rondur mivjor Horiolul dodHloiiM ("nil
pesticides art; bad or corporations mislead Uu> public").
10. You may be confronted with statements made by you at
an earlier date which arc too broad. If those statements
were your personal opinions and not your professional
scientific opinion you should say so. Scientists are
allowed personal opinions but are allowed to testify
in court in opinion and conclusion form only as to
matters within their scientific realm, upon which a lay-
man would be unqualified.
14
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11. Don't get angry at the interrogator if he becomes
arrogant or insulting. This invariably is because
he dosen't have any way to crack your testimony
scientifically and is trying to rattle you. Allow
your lawyer to attempt to put him in his place.
12. The good lawyer will not ask a question in an
opposing party's witness' strongest ground. Do not
feel upset if you are not challenged on work you
want to discuss.
13. Don't be drawn into an argument with opposing
counsel. He isn't being called to testify.
14. If you feel discomfort, ask the judge for a recess.
Don't use this an an excuse -- your counsel will ask
for a recess if he sees you need a chance to collect
your thoughts. Only for necessity will the court
interrupt a cross-examination.
15. Most importantly, remember you know more about
what you are talking about than anyone else in the court-
room. Your "home ground" is your data --do not
stray too far from it.
There have been notable examples in each major administrative hearing
held by EPA or court trial in which EPA was a party, of witnesses who have
fallen into one or more of the traps mentioned above.
The ideal expert witness has facetiously been characterized by some as
a white haired gentleman with a pipe and elbow patched tweed sport coat who
understates most answers he gives and never changes his mood of academic
detachment. This picture is not altogether misleading, for the best expert
witnesses seem to be those who are never caught exaggerating, never lower
themselves to the rancor of the hearing room, and never deviate from their
area of expertise. Judge E. Barrett Prettyman gives this advice to experts:
Don't argue. Don't fence. Don't guess.
Don't make wisecracks. Don't, lake sides.
Don't got. IrrUntod. Think 1'ii'Hl.. then
speak. IT you do know tht» miHwrr to
a question, say so. Tf you do not know
the answer but have an opinion or belief
on the subject based on information, say
exactly that and let the hearing officer
decide whether you shall or shall not give
such information as you have. Tf a 'yes
or no' answer to a question is demanded but
you think that a qualification should be
made to any such answer, give the "yes or
no" and at once request permission to ex-
plain your answer. Don't worry about the
15
-------
effect an answer may have. Don't worry
about being bulldozed or embarrassed;
counsel will protect you. If you know
the answer to a question, state it as pre-
cisely and succinctly as you can. The
best protection against extensive cross-
examination is to be brief, absolutely
accurate, and entirely calm.
The skillful witness also knows when to concede a point, even if it
reflects poorly on his work. To struggle with a lawyer on a line of
questioning, only to agree with him later, highlights the concession and
places the other answers of the witness in an unfavorable light. What
follows is the aftermath of a cross-examination on a point on which a
witness refused to yield until the last possible moment. The expert
then became argumentative and refused to answer questions clearly
within his area of expertise. The questions deal with possible sources
dieldrin found along the Atlantic coast:
Q. Looking at Table 5, I notice that New York
is the most frequent reporter of residues of
dieldrin in mollusks. Are you able to account
for that1'
A. No; that is an interesting observation,
but I am not able to account for it.
Q. Why is it interesting?
A. It just interests me as a person.
. Q. What does it suggest to you?
A. I have no further comment.
Q. Refer to the New York section of the
paper. This begins at page 303.
A. YOH.
Q. You will notice llio HitOH of the moni-
toring stations are fringed arounH the
island of Long Island, not notorious as
one of the world's great feed corn granaries.
Does that suggest anything to you '•»
A. I am not in position to comment on that.
Q. You are not even in position to comment
on whether or not these sites are adjacent
to urban areas ?
16
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A. No comment.
Q. No comment'
A. No.
Q. Are you able to comment, for example,
with respect to page 304 and let's say, for
example, the Mamaroneck data which shows
residues, if you allow a subjective judgment,
for example, in 1967, a fairly constant rate
throughout the year and tell us whether or
not that indicates to you that these are
agricultural or nonagricultural sources ?
A. No, I have no basis for comment.
Q. Let's go back to page 243 and notice in
the next column of Table 5 that Georgia is the
state reflecting the maximum value in ppb.
Are you able to comment about that?
A. No, I am not.
Q. If you will turn to the Georgia section and
particularly the Lazareth Creek data, Station
Number 1, for example; are you able to advise
us as to the existence of one or more wool treat-
ment plants on this creek?
A. No, I am not.
To some people, giving testimony as an expert witness is a challenging
experience which starts the adrenalin pumping and prompts an attempt to
answer all questions which are posed. A good lawyer will endeavor to draw
an export away from his area of expertise to a topic on which the witness
knowH enough to want to answer the questions but not enough to avoid being
Inippiul. The wltnoHH also ran ho led Into this unfortunate situation by a
Hlenl. Mini lawyer who wlnh l.o prove n point by forcing the witness to
"cxpmul ;i Htlio upon this expertise. " The example which follows IH ol' a
witness who rose to bait offered by the interrogator. The witness, who
was a rhemisl, had just presented data on the runoff of pesticides from a
cornfield during a heavy rain.
Q. Over the course of five years. Doctor,
how many days would you expect that kind
of rainfall to occur of that intensity? Did
you have any way of making an estimate ?
Iowa weather?
(
A. Yes, I could make an estimate.
17
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Q. Out of five years, what would your
estimate be?
A. Well, I won't be numerical.
Q. Well, could you try -- how many days?
A. With considerable frequency. It is not
uncommon. Several times a year, at the
appropriate seasons; sometimes a couple
of times a week it's happened.
*******************
Q. Would you identify that for the record
and tell me what you see, whether you
recognize that9
(indicating.)
A. Yes, I recognize it. It is a publication,
1969, by the Iowa Academy of Sciences,
entitled, "Water Resources of Iowa. "
Q. Now I direct your attention to figure 8,
done in exactly the same method.
A I understand this figure, Doctor, and I
ask you to correct me if I am incorrect,
we can expect a four-inch rainfall in a
24-hour period once in five years; is that
correct9
A. Yes, sir.
Q. Thank you.
Tli<> socond example of a witness leaving his area of knowledge was
probohly tilt* fault of MH IftwyorH. who assisted in t.lu> drafting of an ovorly-
broud written statement. The wllncsH was uttamplliiK l.o rebut an WPA
position in the Aldrin/Dieldrin hearings that much if not most of the
residues of these pesticides come from agricultural runoff rather than
point sources. Shell Chemical Company was attempting to show that sloppy
handling by formulation and fertilizer blenders was the cause of the pollu-
tion. (If this were so, the argument goes, EPA could reduce pollution
measurably by enforcement actions against certain plants and would not
need to ban the pesticide. Another more immediate purpose was to throw
doubt upon the EPA studies showing high residues in those agricultural
areas in which Aldrin is used.) A company chemist was put in the
uncomfortable position of carrying this torch:
18
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Q. Are any of your publications related
to the material you talk about in your
statement9
A. No.
Q. So to shorten this up you have never
published in the fields of -- stop me if
you have, I am just going to read a list,
aquatic toxicology, kinetics of aldrin-
dieldrin degradation, the adsorption of
aldrin-dieldrin to soil particles, erosion
problems, the fate and effect of aldrin-
dieldrin in fresh water moving stream
environment, or the relationship between
turbidity and aldrin-dieldrin concentrations
in a moving fresh water stream.
Have you ever published in those areas?
A. No.
Q. Do you know how many tons of soil leave
an average American corn field according to
the U.S. Department of Agriculture?
A. No.
Q. Don't you think that would be a good
figure to have in mind when you are
talking about the relative pollution of Iowa
corn streams 9
*******************
A. I don't see the need to know that figure.
*******************
Q. Did yon huv
-------
Q. You have no data on how far it would
travel in a highly turbid drainage ditch
or turbid Iowa stream of 500 cfs, do you?
A. No.
Q. Doctor, do you have any example of a
number in parts per million or pounds per
day for any formulating plant in the Midwest
at any time of the year 9
A. No.
Q. Do you have any number for the pounds
per day or parts per million from any municipal
outfall in the Midwest?
A. No.
Q. Dr. _ , have you been in any of the
eight major Shell formulating plants in the
United States ?
A. No.
##**#*#***#***#*#**
Q. Let me run to Figure C, the map of
fertilizer blenders, and so on. I take it
you have no knowledge of whether the
formulators on that map ever discharged
a drop of dieldrin to the water, is that
correct?
Q. T mean In normal operations.
A. I liavo no (MTHorml knnwlcclf/r.
Q. And you have no knowledge of any type
of numbers in parts per millions or pounds
per day from any of these plants?
A. No.
Q. So, you do not know if they arc polluting
the water in Iowa or not, basically, do you?
They could be all closed systems for all you
know, right?
20
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A. Right.
There are, unfortunately, many examples of expert witnesses who have
violated one or more of the fundamental rules for presenting evidence. The
chances of doing so, however, are far less if the potential witness has viewed
at least a day or more of the proceedings prior to giving testimony. This
accomplishes several things: it gives the "tone" of the hearing, it usually
indicates what general type of questions to expect, and most of all, it
reassures the witness. If you are called upon to testify you should make every
effort to arrive enough before your appearance to view the proceedings.
Unfortunately, simple fatigue can undo the best of research. Experts
have likened giving testimony before good lawyers to a lengthy oral dis-
sertation defense without the usual opportunity to give complete answers.
By the end of a day of hard questioning, the witness1 concentration and the
precision of the answers fall off markedly. Good lawyers may save the most
aggressive and most important questioning for after the midafternoon break.
It is also at this time that the skillfully phrased leading question has its
greatest effect. Lawyers are not allowed to "lead" their own witnesses, but
may phrase long rhetorical questions when facing witnesses for the opposition.
These often begin with "I take it we can agree that... " or "I assume you
are aware that... " or some form of a lead-in which calls for a yes or no
answer to an often lengthy proposition. The prepared cross-examiner will
know where he wants to go, and roughly how many leading or hypothetical
questions it will take to get there. In most cases the final answer will not
be the conclusion the witness anticipated when he conducted his research, i. e.,
it may be a consistent extrapolation from his original work. Or it may be
a conclusion not truly in line with the data, but the inevitable result of
the skillful questioning.
The latter result, most frustrating to good scientists, can happen when
the leading or hypothetical questions are 95% accurate and the respondent
is either too tired or too timid to demand the correction of the 5%. As
any scientist knows, a 5% error compounded several times leads to sub-
stantial deviation: this simply is what happens when a witness is not
careful with leading questions. He should demand that all elements of a
hypothetical question he needs to reply are indeed included in the question
or that all elements of M (fading question do indeed reflect tho state of
facts. This training host romon from urinal fxprrlonrr, but Intensive;
mock cross-examination by his own lawyer can give a fair idea of what
to expect.
It is often a good idea at the end of a day of hearing or trial for
attorney and witness to review the past testimony in addition to preparing
for likely cross-examination to come the next day. Witnesses and their
lawyers often disagree as to what was said, or how it was interpreted, or
whether that was really what the witness wanted to say. If there has been
testimony that could be misinterpreted or was simply mis-spoken, the
government attorney should try to correct the misimpression by well
phrased "re-direct question. These are traditionally questions which
21
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deal with issues raised in the cross-examination, not with "new matters".
It is helpful to trial counsel if the witness keeps a mental note of areas
of cross-examination in which he feels he needs to say more, and if the
witness can suggest appropriate questions to his lawyer.
In some of the bigger trials and trial-type administrative hearings
in which EPA has been a party, a daily transcript is made and is usually
available to the parties four or five hours after the close of the day's
hearings. Reference to the actual recorded answers, of course, greatly
facilitate the correction of misimpressions and the protection of a precise
record.
22
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Appendix A
STATEMENT OF
My name is I am a physiologist at the Fish-
Pesticide Research Laboratory of the Bureau of Sport Fisheries and
Wildlife, U. S. Department of the Interior at Columbia, Missouri. My
duties there involve supervisory physiological, histopathological, and
nutritional research dealing with the chronic effects of environmental
pollutants on the physiology and biochemistry of fishes. I have been
employed by the Fish-Pesticide Research Laboratory since 1969.
My educational background is as follows: I received my B. A. in
Biology from Southwestern at Memphis, Memphis, Tennessee, in 1967,
my M. A. in Zoology in 1969 and my Ph. D. in Agricultural Chemistry
in 1971 both from the University of Missouri at Columbia.
My testimony will be taken from three papers which I coauthored.
These are:
1. "Serum Amino Acids in Rainbow Trout (Salmo gairdneri)
as Affected by DDT and Dieldrin" which appeared in Comparative Big-
chemistry and Physiology in 1971, Volume 38B, pages 373 to 377. This
is attached as Appendix A.
2. "Ammonia Detoxifying Mechanisms of Rainbow Trout Altered
by Dietary Dieldrin", a manuscript accepted for publication in Toxicology
and Applied Pharmacology. This is attached as Appendix B.
3. "Phenylalanine Metabolism Altered by Dietary Dieldrin",
appeared in Nature, Vol. 238, pages 462 to 463 in 1972. This is attached
as Appendix TT.
My papers iloal witli the effoct of dicldrin on amino arid metabolism,
ammonia detoxifying merlmninmH. und phonylalanino and phenylketo arid
motahollsrn of rainbow trout.
D1ELDIUN AND AMINO ACID MET A HOLISM
Amino acids are organic molecules which are utilized for energy as
well as for molecular components of proteins. Proteins are exceedingly
important to all living organisms; they serve both structural and functional
capacities in living organisms. The twenty naturally occurring amino
acids normally found in organisms arc considered important building blocks
for life processes. Thus recognizing the importance of amino acids in bio-
logical systems, we initiated studies to elucidate the effect of dieldrin,
a common environmental contaminant, on amino acid metablism of rainbow
trout.
-------
Each of three groups of rainbow trout was fed a diet containing 7.1 ug
DDT/gm food, 7.1 ug dieldrin/gm food, or a diet containing neither DDT or
dieldrin (control group). The dieldrin dosage was 143 ug/kg body weight
per day. The fish were fed in three separate 570 liter fiberglass tanks
for 140 days. After 140 days, whole body residue analyses were performed
on 4 fish from each group, serum amino acids were analyzed from 6 fish
in each group, and 12 trout from each group were subjected to forced
swimming to determine the effects of DDT and dieldrin on serum amino
acids after the trout were exercised. Six fish from each group were
analyzed after 6 hours and 6 fish after 24 hours of forced swimming. The
fish were exercised by placing them in a stamina tunnel which forces the
trout to swim against a current of water. The velocity of water was 2
ft/sec, which is similar to velocities encountered by trout in many natural
streams. Appendix A contains the references for the methods used.
Table 1, Appendix A, presents the results of this study.
The concentration of each amino acid in the control group, except
alanine, decreased after the fish were exercised. It is assumed that this
is a result of the fish's energy needs, i. e., amino acids were being
utilized in response to forced swimming.
Dieldrin altered the concentrations of 11 amino acids. The level
of seven amino acids and the total amino acids concentration were ele-
vated, while the concentration of four other amino acids were decreased
by dieldrin. The effect of dieldrin could have been on the amino acid in
question or perhaps on a metabolic pathway which affects a particular amino
acid via a "feedback" mechanism.
There was a significant interaction between dieldrin treatment and
forced swimming. The concentration of fewer amino acids were significantly
lower after exercise in the dieldrin group than in the control group. Leucine
and serine did not decrease in either of the exercised dieldrin groups, whereas
in the control exercised group both amino acids significantly decreased. The
concentration of methionine, hydroxyproline and aspartate increased in the
dieldrin exercised group, but decreased in the control exercised group.
The accumulation of these five amino acids in the serum suggests that the
utilization was inhibited by dieldrin. The mechanism that caused these
dieUlrln-Induced chn.ntfpfl is unknown, but our results can be considered
Indicative of the Hubl.li1, biochemical oH'oets of dioldrin that may alter
rainbow trout in our aquatic environment.
DDT also had a significant effect on amino acid metabolism, but these
results will not be discussed in this testimony.
DTELDR1N AND AMMONIA DETOXIFICATION
The second study involved the relationship between dieldrin and ammonia
metabolism in fish. Ammonia is a natural end-product of protein and amino
acid metabolism, and it manifests a strong cytotoxicity in living cells. The
following biosynthetic reactions are responsible for detoxifying and maintaining
low, non-toxic levels of ammonia in fish:
-------
Reaction 1
cx-oxorlutarate + NADH + H+ + NHj^ ^ > Glutamate + NAJ) + H^O
Glutamate
Hehydrogen as e
(P.DH)
Reaction 2
Glutamnto + ATP + NI!,'*' ^ ^ Glutamine + ADP + Orthophosphate
Glutamine
Synthetase
(GS)
Reaction 1 occurs in both the liver and brain of fish, whereas the second
occurs only in the brain. Thus, the brain has both biosynthetic reactions,
and the liver has only one. Regulation of these two reactions is extremely
important in maintaining low, non-toxic, ammonia levels in fish. This
study was initiated to elucidate the effects of dieldrin on the ammonia
detoxifying mechanisms and brain amino acid metabolism of rainbow trout.
Each of five groups of rainbow trout was fed a diet containing 0, 0. 36
1.08, 3.6 or 10.8 Hg dieldrin/gm of food. This equals to 0, 14, 43, 143 or
430 Ug dieldrin/kg body weight of fish per day. The trout were fed daily
rations equivalent to 4% of their body weight per day for 240 days, after
which liver, blood and brain were taken for biochemical analyses. Each
group of fish was weighed monthly and feeding rates were adjusted accordingly
during the exposure.
The activity of the enzymes glutamate-oxaloacetate transaminase (GOT),
glutamate-pyruvate transaminase (GPT), and glutamate dehydrogenase (GDH)
were measured in both the liver and brain of fish from each group after 240
days. Also, glutamine synthetase activity (GS) (measured as glutamine
transferable) was measured in the brain of fish from each group. The
methodology, results, statistics, and discussion of data are presented in
Appniullx M. A nummary of tho data ftloiiR with implications will be presented
in this testimony.
The whole-body dieldrin residues after 240 days of exposure were 0. 39,
0.62, 2.0, and 5.86 yg/gm (ppm) in the 14, 43, 143, and 430yg/kg dosage
groups, respectively. The dieldrin residues resulting from the three lowest
dosages were in the range of those reported in fish from the National
Pesticide Monitoring Program. We therefore suggest that results from this
study are indicative of biochemical effects of dieldrin that could be occurring
in fish in our aquatic environment.
-------
Liver GOT and GPT activities were not significantly altered by dioldrin
except for GOT in the highest dosage group. These two enzymes are indicative
of the amount of ammonia being transaminated by the liver. These results
suggest that dieldrin did not alter the transamination of amino nitrogen in
the liver. However, brain GOT and GPT activities were significantly decreased
by dieldrin except for GOT activity in the lowest dosage group. Decreased
activity of these enzymes could cause a concomitant increase in concentra-
tions of the amino acids aspartate and alanine. Analyses of the concentration
of free amino acids in the brain confirmed that both aspartate and alanine
concentrations were significantly increased in fish from the two highest
dosage groups, but not in those fed less than 143yg/kg/day (3. 6 yg/gm food).
Nine of the 16 amino acids measured in the brain were altered in the 143
log/gm group, whereas 12 of the 16 amino acids were altered in the 430p g/kg
group. The significance of these individual changes in amino acid concentra-
tions is not completely understood, but we can conclude that brain amino
acid metabolism was significantly altered by chronic, dietary dieldrin
exposure.
The enzymes concerned directly with ammonia detoxification, GDH and
GS, were significantly altered by dieldrin. Liver GDH was significantly
stimulated by all dieldrin dosages, which suggests that the ammonia detox-
ifying capabilities of the liver were increased. Because GDH is located
exclusively in the mitochondrial matrix, these data indicate that mito-
chondrial metabolism was stimulated by dieldrin.
Electron microscopic analyses of liver cells showing changes in mito-
chondrial morphology give further evidence that dieldrin has an effect at
this site. An electron photomicrograph representative of the control group
(0 dosage) is shown in Figure 1, Appendix B. In comparison, the lowest
dieldrin dosage caused swelling of mitochondria with no apparent disruption
of membranes (Figure 2, Appendix B). However, the highest dieldrin treat-
ment caused more pronounced effects on the mitochondrial (Figure 3, Appendix
B). The mitochondria were swollen, mitochondrial membranes were dis-
rupted, and the matrices of several mitochondrial appear severely damaged.
The electron microscopic examinations were correlated with the altered GDH
activity, and our interpretation offers an explanation as to why the lowest
dieldrin-treated group had slightly greater GDH activity than the highest
treated group. The mitochondria of the low dosage group were swollen,
which suggests stimulated mitochondrial metabolism, whereas those of the
high dosage group were not only swollen, but the outer membranes were
disrupted whic.h results In the mitochondria being iimr.tlvu.
Hrain GI31I activity was decreased significantly h.y all doses of dicldrin.
Tin; Inhibition of brain GDI! suggests decreased ammonia detoxification, which
could have increased ammonia concentrations in the brain. However, dicldrin
had no effect on brain ammonia concentrations. In contrast, serum ammonia
increased significantly at the two highest doses of dicldrin. Because brain
ammonia is metabolized sequentially by glutamatc dehydrogenase and glutaminc
synthctase, we feel that the inhibition of GDH activity placed a greater load
of ammonia on the glutamine synthetase system. In all groups exposed to
dicldrin, glutamine synthetase activity (as assayed by glutamine transferase)
was stimulated, and compensated for the loss in ammonia detoxifying function
-------
performed by GDH. Thus,- glutamine synthetase in the brain of dieldrin-
dosed fish in this study is likely responsible for maintaining brain levels
of ammonia within physiological limits. When this compensating mechanism
is exhausted or exceeded, then the toxic effects of ammonia are perhaps
manifested.
The dieldrin-induced change in brain GDH activity of rainbow trout may
account for the stimulation of brain 'glutamine synthetase activity, increase
in serum ammonia, and the increase in liver GDH activity. The ammonia
liberated from the brain in the low dosage group (14pg/day) did not result in
a detectable increase in the concentrations of either brain or serum ammonia.
This suggested that the ammonia liberated due to decreased brain GDH activity
was either bound by brain glutamine synthetase or transported from the brain
to blood, and then either excreted or detoxified by the liver. The ammonia
liberated from the brain probably accounted for the stimulated liver GDH
activity. In the second lowest dosage group (43/day), this same trend was
apparent. The two highest doses of dieldrin (143 and 430 yg/day) caused an
increase in serum ammonia, which suggests that the excretion and liver
detoxification capabilities were being exceeded. Although the excess ammonia
load was not toxic to the trout, it caused the ammonia detoxifying mechanisms
to be continually taxed.
The implication that brain ammonia detoxifying mechanisms of fish play
such an important role in maintaining ammonia levels within physiological
limits demonstrates the impact that dieldrin could have on fish and their
ability to adapt to their environment. However, the most serious implica-
tions on fish involve the interaction of dieldrin and other environmental
chemicals that may also alter ammonia metabolism, as well as the inter-
action of dieldrin and elevated ammonia concentrations in water. Ammonia
is one of the most common poisons discharged into the aquatic environment
and has been a subject of much concern in fish toxicology. Results from
our study suggest that fish carrying body burdens of dieldrin would be less
tolerant to increased levels of ammonia in water.
DIELDRIN AND PHENYLALANINE METABOLISM
Kurlhor research was done with rainbow trout which involved determining
tluj I'l'I'ortH of dietary illoldrtn on the metabolism of one particular ami no acid,
plioii.vliUuiilnp. The ronrnntrutlon ol' phunylalnninn WUH altered by diolclrin in
our I'lrnt study. Clifintfos lit the muluhnlIt-tin of pIxMiylrtlunliift havo boon rclatod
to altered activity of brain enzymes and mental deficiency in mammals.
Although little is known about the situation in fish, we evaluated the effects
of dietary dieldrin on metabolic pathways of phenylalanine in rainbow trout
(Salmo gairdneri).
In this experiment, we fed each of 5 groups of rainbow trout diets con-
taining dieldrin at the following concentration: 0, 0.36, 1.08, 3.6 or 10.8
ug dieldrin/gm food. This corresponded to 0, 14, 43, 143, or 430yg dieldrin/
kg body weight/day, respectively. The fish were fed daily rations equivalent
to 4% of their body weight for 300 days. The trout were weighed each month,
and we adjusted the feeding rates accordingly.
-------
At the end of the experiment liver phenylalanine hydroxylase, serum
phenylalanine and urinary phenylpyruvic acid were measured. The techniques
used in this study are noted in Appendix C.
Growth rates were not affected during the 300 day exposure period.
Whole body dieldrin residues after 300 days were 0.41, 0.79, 2.10 and
6. 23yg/gm (ppm) in the 14, 43, 143, and 430 yg/kg groups, respectively.
It is important to note that residues in fish at the three lowest exposure
levels were in the range reported for fish in the aquatic environment by
the National Pesticide Monitoring Program.
The concentration of phenylalanine in blood was increased by all
dosages of dieldrin. The effects of dieldrin on serum phenylalanine are
given in Figure 2, Appendix C.
Liver phenylalanine hydroxylase is an enzyme which converts phenylalanine
to tyrosine. This enzyme is responsible for maintaining the normal concen-
tration of blood phenylalanine. Our study showed that all doses of dieldrin
decreased the activity of this enzyme (Figure 1, Appendix C). These results
are consistent with our finding of increased serum phenylalanine, i. e., the
enzyme was less active and phenylalanine was not being converted to tyrosine.
Dieldrin1 s effect on phenylalanine hydroxylase activity appeared to be
persistent, for the liver enzyme activities in the group given the largest
and smallest doses of dieldrin were still significantly (P<0.05) lower than
the control group after the trout were fed control diets (0 dosage) for 4
months after the initial 300-day exposure. The half-life of dieldrin in
trout has been reported to be 44 days. Thus, after approximately three
half-lives, the enzyme activity was still decreased.
Urinary phenylpyruvic acid, a phenylketo acid metabolite of phenylalanine,
increased in concentration in the groups receiving the three highest dosages
of dieldrin. There was a significant correlation between decreased phenylalanine
hydroxylase activity and increased urine phenylpyruvic acid concentration among
the various dosage groups (r=0. 860, P<0.01). Thus, as dieldrin caused a
decrease in hydroxylase activity, there was a concomitant increase in the
urinary phenylketo acid metabolite.
IMirnylkwtourta Is an Inherited dot'oct in phen.ylalanine metabolism of
nu\mm:ilH Hmrar.toH/.rd by an Inhibition of phonylalanlnc hydroxylasc, Increased
blood phcnylalunine, Increased urinary phenylketo adds, and mental deficiency.
Our study indicated that dieldrin has a marked effect on phenylalanine metabolism
and can induce the biochemical manifestations of phenylketouria; however, the
effects of dieldrin on learning ability in fish remain to be tested.
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Appendix B
My name is _^___^ _ and I am currently assistant professor of
environmental studies at ~ _ My formal educa-
tion includes BSc and MA degrees from the University of Missouri and a
PhD degree from Montana State University. I have had experience as a
research assistant at the University of Missouri and as a pollution biologist
with the Tennessee Game and Fish Commission. A list of my research
contributions and a curriculum vitae are attached for your review. The
information I will present below stems from a research project conducted
at the University of Missouri during the late 1960's under the supervision
of . This work dealt with the effects of selected
pesticides (including dieldrin) on planktonic algae.
As you know, all life on this planet depends upon the energy of the
sun which is "fixed" or converted into a useable form via the activity of
green plants. Our goal in this research project was to determine if
certain chemicals, which were widely used at the time of the project had
any effect upon the ability of green plants to perform their important
role of energy fixation. We choose a green algae (Scenedesmus quadricauda)
as the test organism. Our experimental design consisted of exposing
laboratory cultures of this plankton organism to various concentrations
of specific pesticides and over a period of approximately 10 days determining
what influence, if any, that chemical had on the ability of the test organism
to grow (i. e. , increase in numbers within the cultures) and to fix energy.
The methods used during this study were such that sublethal effects of any
of the tested compounds could be determined. In other words, the test
organism did not have to turn brown and die in order to determine whether
or not the compound under investigation had an adverse effect.
Growth was measured simply by counting the number of plankton algae
cells in a 1 ml aliquot of the control and pesticide treated cultures at two
day intervals. The rate of energy fixation (photosynthesis) was determined
with the aid of radioactive carbon-14 (C1 **) in the form of sodium carbonate.
Aliquots of each culture were withdrawn at two day intervals and incubated
with C1 ''for four hours . The cells were filtered, washed, dried and the
amount of radlourtlvo carbon which Imd been "fixod" was determined by
liquid scintillation.
A summary of the results from the dieldrin part of the study are
presented in Table T.
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Table I. Percentage differences cell number, carbon assimilation and
biomass between dieldrin treated cultures and controls at
0, 2, 4, 6, 8, and 10 days. Carbon assimilation expressed
as unit volume = cpm C1 per 50 ml of culture. * = significant
difference (p = 0.05, N=4).
Time in Days
0.1 mg/1 o 2 i» 6 8 10
Cell number -32» -23* -Ztf* -17* -0
Unit volume +S + l -20* -l»2» -25 +35
Biomass -22
1.0 mg/1
Cell number -37* -2Q* -3R* -23* -11
Unit volume +3 + 1 -33* -51* -32* +33
Biomass -3P
These data show the percentage change of treated cultures from controls
as to the number of cells, the amount of carbon-14 taken up and the biomass.
Thus, by the second day of the experiment, the number of cells in the dieldrin
treated cultures (0.1 mg/1) was 32% less than the control cultures while the
amount of radioactive carbon being assimilated was about the same (1% greater
than controls). At a concentration of 1.0 mg/1 on this same day, the number
of cells was 37% less than the control cultures and again carbon uptake was
about the same (1% greater than controls). By the sixth day of the experi-
ment cell number at the lower concentration (0. 1 mg/1) was 26% below the
controls and the rate of carbon fixation was 42% less than the control cul-
tures. At the higher concentration of 1.0 mg/1 the number of cells in the
I routed cultures wore 3H% lower than In the control cultures and the rate of
carbon uptake !>!% below the control rule. Ry the ond of llie experiment.
total biomass was 22% lower than control cultures in the 0. I mg/1 treatment
group and 32% lower in the 1.0 mg/1 group.
My conclusion from this information is that the compound dieldrin has
an adverse effect on the green plankton algae S. quadricauda. There was a
significant decrease in the growth and energy fixing abilities of the test
organism. Total biomass of the treated cultures was lower than controls at
the end of the test period.
c
T t'col that dieldrin has fairly widespread effects. As other witnesses
have testified, this compound affects many diverse types of organisms. As
I have found, members of the plant kingdom are adversely affected. The
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ramification of this is that dieldrin may have a very subtle yet widespread
influence on an ecosystem. I therefore would support a complete ban on
the pesticide dieldrin while encouraging an increased research effort into
more species-specific pest control methods.
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Appendix C
)
CHAIN OF CUSTODY RECORD
ENVIRONMENTAL PROTECTION AGENCY
SAMPLE NO. TIME TAKEN DATE TAKEN
SOURCE OF SAMPLE PRESERVATIVE
NAME OF PERSON TAKING SAMPLE
WITNESS (ES) TO TAKING SAMPLE
ANALYSIS REQUIRED:
Front
ll
°w
*(J
JW
a
RECEIPT OF
SAMPLE
1 DISPATCH OF
SAMPLE
I hereby certify that: I received this sample and
HispnsoH nf -If as nnt-pd hplow.
RECEIVED FROM
DISPOSITION OF SAMPLE
DATE REC'D TIME REC'D
SIGNATURE
I hereby certify that I received this sample and
disposed of It as notud below.
RKCK1VK1) FROM
DISPOSITION OF SAMPLE
1 hereby certify that I o
dispatched it as shown be
DATE OBTAIND TIME OBTAIND
)ATE REC'U TIME REC'I)
SIGNATURE
btained this sample and
low.
SOURCE
DATE DISPATED TIME UISPATED METHOD OF SHIPMENT
SENT TO
SIGNATURE
Back
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Appendix D
SAMPLE TRANSMITTAL SHEET
TO: (Laboratory Name & Address)
FROM: (Field custodian or Field Sampler)
Sample No. Lab Number Preservative Analysis Required
To be completed in field:
Prepared by: Date:
Signature
Field Notebook No. Time:
To be completed by Laboratory:
Received by: Date:
Time:
Distribution: Orig. & copy - Accompany shipment
1 copy - mail directly to Laboratory
1 copy - mail to Data Management
1 copy - Survey Coordinator Field Files
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