EPA -660/3-74-003
May 1974
Ecological Research Series
Biologically Allowable
Thermal Pollution Limits
Office of Research and Development
U.S. Environmental Protection Agency
Washington. O.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1, Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formation,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
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EPA-660/3-74-003
May 1974
BIOLOGICALLY ALLOWABLE THERMAL POLLUTION LIMITS
PART I
By
W. Drost-Hansen
PART II
By
Dr. Anitra Thorhaug
Project 18050 DET
Program Element 1BA022
Project Officer
Dr. C. S. Hegre
"National Marine Water Quality Laboratory
South Ferry Road
Narragansett, Rhode Island 02882
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20102 - Price $1.20
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EPA Review Notice
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommen-
dation for use.
ii
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ABSTRACT
Part I
Literature and theoretical studies nave demonstrated the likely
existence of critical thermal transition regions for biological acti-
vity. Highly non-linear thermal effects, observed in many biological
systems, appear to be manifestations of higher-order phase transitions.
The origin of these transitions appears to be the vicinal water of the
cellular systems. As these thermal effects are manifestations of in-
trinsic structural changes in vicinal water, the effects are likely
invariants in terms of time and space. Thus, the corresponding
critical temperature regions may represent absolute, upper permissible
thermal pollution limits.
Part I I
Laboratory experiments, using some 18,000 individuals, have given
the most accurate account of thermal tolerances for marine estuarine
organisms to date. The organisms examined included the most important
macro-algae and larval stages of important food-chain organisms. The
expected Gaussian or skewed-Gaussian curve for lethal thermal limits
did not materialize. Instead an abrupt death point occurred often
within an interval of I°C and in many cases within 0.5°C resembling
a step function.
One of the most important conclusions from this data is that the
temperature tolerances obtained in the laboratory conformed closely
to those observed in the field. Thus, the field data (Perkison, in
iii
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preparation, Bader, et a_L, 1970) could be interpreted with more
validity as to the effect of temperature versus other environmental
factors. It should be emphasized that the laboratory upper thermal
limits of the algae were borne out in distribution in the field in
each case. The upper limits found in the laboratory for Halimeda,
PeniciIlus, and VaIonia were found to be the thermal limits in the
field. When sustained temperatures above those found as laboratory
survival limits were encountered at Turkey Point, these plants dis-
appeared. In addition, detailed laboratory observations on the
morphology of thermally stressed and thermally killed plants aided
field observations of "effected" areas.
The upper temperature limit for many of the plants examined as
well as the sensitive stage of the pink shrimp, crab megalops and
several carideans was 31 to 33°C. As previously stated, this was
corroborated in field investigation where the mean annual temperature
exceeded these limits near the mouth of the effluent canal at Turkey
Point. These critical temperatures are within I to 3°C of mean mid-
summer temperatures encountered under natural conditions. This
substantiates the hypothesis that tropical marine organisms live
closer to their upper lethal limit than do either temperate or
>
Arctic species.
This report (Parts I and II) are submitted in fulfillment of
Project Number 18085 DET contract under sponsorship
of the Water Quality Office, Environmental Protection Agency. Part I
*
of this work was partially sponsored by the U. S. Atomic Energy
Commission.
iv
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CONTENTS
PART I
Section
I
II
III
IV
V
VI
VII
VIII
PART II
Section
I
II
III
IV
V
VI
VII
Conclusions
Recommendations
Introduction
Background
Literature and Theoretical Studies
Water At Biological Interfaces
Discussion
Acknowledgements
Conclusions
Recommendations
Introduction
Methods
Results
Acknowledgements
Publications
Page
I - 1
1-2
1-3
1-6
1-7
1-8
1-32
1-36
II
II
II
II
II
II
II
1
3
4
6
12
38
39
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SECTION I
CONCLUSIONS
A. Abrupt anomalous and classically unpredicted changes occur
In the properties of cellular systems at a number of discrete tem-
perature ranges, including the ranges between 14 to 16 and between
29 to 32 C. A vast amount of data presented in the literature as
well as some experimental results obtained and reported separately
by Dr. Anitra Thorhaug, suggest to the present author that for many
(but not all) marine organisms - both plant and animal - upper
thermal limits frequently appear to be centered around 3I°C (within
+0.2°).
B. The degree of abruptness of thermal changes are too pro-
nounced to be accounted for in terms of classical ce'' physiology
by means of such mechanisms as protein denaturation, enzyme kinetics,
etc. Instead, it is proposed that the effects are due to cooperative
changes in the structure of cellular, vicinal water.
C. For some organisms, particularly fishes, prior acclimatization
may result in a change of the response tothermal stresses. However,
while such parameters as rate of growth or metabolism may show
optimum activity at somewhat different temperatures, depending
on acclimatization temperature, only relatively small changes are
usually observed in (long term) upper thermal limits, even after
extensive acclimatization.
I - 1
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SECTION (I
RECOMMENDATIONS
Continued research is recommended to delineate in some detail
the molecular basis for the effects of temperature on living or-
ganisms. From the review of the literature and the theoretical
studies reported here - together with the results from the experi-
mental studies and field work by the co-Principal Investigator,
reported elsewhere - it is suggested that until more information
can be gathered to fully describe the ecological effects of in-
creased temperatures, fixed maximum thermal pollution limits be
•specified through legislation. Specifically, it is proposed that
3I-32°C be considered an absolute upper maximum limit for lakes,
rivers, estuaries, and bay areas with low circulation. In view
of the near total lack of information regarding long-term genetic
effects on marine organisms of exposure to semi-lethal temperatures,
studies of these problems are strongly recommended in order to
evaluate the ultimate effects of thermal pollution. Mere measure-
ments of immediate, thermal death points and the effects of
these on local ecology are important, but the long-term effects
(over decades or even a century) may prove far more important
than additional immediate field-type studies of thermal pollution.
I - 2
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SECTION I I I
INTRODUCTI ON
The purpose of the present study was to attempt to delineate
biologically allowable thermal pollution limits. The project in-
cluded a laboratory study of temperature effects on certain or-
ganisms: this facet will be reported by Dr. Thorhaug.
Before proceeding, it is of interest to point out that the
annual, world-wide growth rate of energy production is continually
increasing. The (world) average rate of increase is presently
approximately 6% per year, but in certain countries (for instance,
Italy and Japan), the annual growth rate is now above 10* per
year. The impact of this energy input is perhaps most dramatically
illustrated by the prediction that in the not-too-distant future,
areas as large as 1,000 to 10,000 square kilometers (metropolitan
areas) may experience a man-made energy input of the same order
of magnitude as that due to the natural solar radiation influx.
It is little wonder, then, that man's waste heat production gives
cause for grave concern regarding the fate of our environment.
Effective means must be sought by which the localized, high inten-
sity energy production may be dissipated over sufficiently large
areas (or rather volumes) so as to minimize local heating effects -
as such local heating nearly always gives rise to detrimental effects
on the biosphere (and possibly some parallel detrimental effects on
the physical and chemical nature of the environment).
In a modern, coal-powered electric generating plant, each
kilowatt hour produced (3,400 B.T.U.) requires that about 6,000 BTUs
be dissipated by the cooling water of the heat exchangers. In other
I - 3
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words, essentially only one unit of energy is used beneficially
for each two units wasted. Actually, the ratio is even more
dismal than this in the sense that in a normal incandescent bulb, the
efficiency is very low; thus most of the energy converted is dissi-
pated as thermal energy rather than in the form of energy in the
desired frequency range (useful for practical illumination). Even
more distressing is the fact that nuclear power plants are less
efficient, with a waste of about 10,000 BTU per kilowatt hour
generated, and this problem is further amplified by the normally
much larger sizes of the nuclear, electric power plants.
In the present study the author has attempted to analyze the
effects of temperature on biological systems over the broadest
possible conceptual ranges in order to (A) seek empirical rules -
as general as possible - to describe effects of temperature on the
behavior and functioning of living systems. (B) To attempt to
explain the effects of temperature on biological systems in terms
of molecular processes, and thus achieve some fundamental under-
standing (as opposed to the currently practiced pragmatic but not
well-founded operational approaches). (C) Finally, it was hoped
that it would become possible to delineate "safe ranges" for per-
missible environmental temperature increases, and to determine if
"critical temperatures" for lethal or highly detrimental effects
could be established.
At the beginning of this study, it was already fairly apparent
that temperature exerts a unique influence on cellular systems via
structural effects on the water of the biological systems. Specifi-
cially, it was known (although not generally recognized) that much
of the water of cellular systems is capable of undergoing discrete
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structural changes over narrow temperature intervals, and that
these structural changes influence a number of processes on
the molecular level, occurring in all types of cellular systems.
The effects of temperature on biological systems - via the
structural changes in the "vicinal water" of cellular systems - is
only a specific example of structural changes in water near inter-
faces. Thus, many systems have served eminently weli to delineate
possible biological effects, while consisting primarily of reprodu-
cible, well-defined, physico-chemical materials.
Experimental evidence for the unique and sharply delineated
influence of temperature on cellular systems has bean discussed
in a number of publications, particularly by the present author.
Reference is made to these studies in the References and in the
List of Patents and Publications.
To supplement the theoretical and conceptual studies outlined
above, a laboratory study was carried out by Dr. Anitra Thorhaug,
the co-Principal Investigator of this study. The results obtained
from Dr. Thorhaug's work are presented independently in the report
to EPA.
I - 5
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SECTION IV
BACKGROUND
Experimental
A number of years ago, a practical device was developed for
the study of temperature effects on biological systems. A poly-
thermostat (sometimes referred to as a temperature gradient block)
was constructed by Drost-Hansen and Oppenheimer (I960). This de-
vice has proven highly useful and for some time a number of models
of polythermostats have been operated by the Principal Investigator
and his co-workers. Some of these instruments were used for the
study reported in a separate report by Dr. Anitra Thorhaug. In
passing, it is noted th-~-t a device of this type is manufactured
commercially in this country by Lab-Line, Inc., of Chicago, and a
somewhat larger, improved version is now available from Toyo Kagaku
Sangyo Co., Ltd., Tokyo, JAPAN, through Scientific Industries,
Minneola, New York. The rapid increase in the interest of deter-
mining very specific effects of temperature on biological systems
becomes apparent when it is realized that in a period of less than
18 months, no less than 70 such polythermostats were sold in Japan
alone.
I - 6
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SECTION V
LITERATURE AND THEORETICAL STUDIES
During this study, it became obvious that much information
is available in the literature, regarding effects of temperature
on biological systems, which has gone unnoticed and indeed un-
exploited in terms of the objectives of the present study (namely
attempting to delineate specific thermal pollution limits). The
results of.these studies, together with some earlier considerations
have been published in part, in an extensive article by the Principal
Investigator. The article deals, in general terms, with the struc-
ture and properties of water at biological interfaces, while one
Section is addressed directly to the problem of thermal pollution.
In the present context, we quote only some selected topics (as-
pects of metabolism and growth, germination, genetics and evolution,
hyperthermia, cell adhesion, thermal hysteresis effects, and thermal
pollution) from this paper.
I - 7
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SECTION VI
WATER AT BIOLOGICAL INTERFACES.
From "Chemistry" of the Cell Interface" Part B, edited by
H.D. Brown; Academic Press, N.T. 1971, with; publishers permission.
B. METABOLISM AND GROWTH
1. Distribution of Optimum and Lethal Temperatures
Metabolism and growth are complex physiological processes. Com-
plexity is taken here to mean the simultaneous and consecutive involve-
ment of a large array of individual chemical processes (reactions in the
classic sense, diffusion, active transport, etc.). In 1956 the present author
suggested that for such complex systems, temperature optima and minima
might be predicted from a simple consideration of the structural changes
in water.* One may assume that many reactions which might potentially
be rate determining in a complex biological system may undergo notable
changes at the temperatures of thermal anomalies. Based on. this as-
sumption, it was proposed that during evolution, biological systems have
tended to avoid the temperature regions near the sudden changes in the
(vicinal) water structure and, hence (at least in the case of the mammals),
have optimized body temperature as far away as possible from a lower and
a higher thermal anomaly (at 30° and 45°C, respectively). .Were the
thermal anomalies to occur exactly at 30° and.45°C, the body temperature
would then be expected to fall near 37.5°C. Figure 21 shows a histogram
of frequency of occurrence of body temperatures for approximately 160
mammals. It is seen that this distribution does, indeed, center very closely
around 38°C with a remarkably narrow distribution. (Note 37°C equals
98.6°F.) A small number of exceptions are indicated by the cross-hatched
area under the curve, around 27° to 34°C. This group includes the ant-
eater, the sloth, the echidna, the armadillo, and a few other species such
as the duckbill platypus. It seems legitimate in a first approximation to
neglect these exceptions because of the somewhat unusual nature of the
animals compared to most other mammals.
The distribution of l>ody temperatures of birds appears to be centered
*At that time it was felt that evidence was available foe the existence of thermal
anomalies in all properties of water and aqueous systems. As pointed out elsewhere
in this chapter (Section IH.C.l.d), it now appears that the anomalies are most pro-
nounced and possibly occur only in vicinal water rather than in bulk water.
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VI. STRUCTURE AND PROPERTIES OF WATER AT BIOLOGICAL INTERFACES 1 ] <)
around 41.5°C. It was proposed by Drost-Hansen (1965a) that the dis-
placement (by 3° or 4°) toward higher temperatures might be a con-
cession to the highest possible rate of energy production required for flight
(approximate Arrhenius type of activation). It is interesting to note that
both for all mammals and all birds studied, 45°C (±1°) appears to be an
absolute, upper thermal limit (lethal temperature). It is also interesting to
note that those birds that do not fly, such as the ostrich, the kiwi, and the
penguin, appear to have normal body temperatures around 38° to 39°C.
This would tend to substantiate the proposed explanation for the higher
body temperatures of birds. It is also well known that 30°C is a tempera-
60
50
40
120,
10-
Mommols
30
35 40
Tertp. (°C)
Flo. 21. Distribution of body temperatures of mammal (frequency distribution).
(Drost-Hansen, 1965ft, with permission from the New York Academy of Sciences.)
ture of considerable physiological importance in all mammals and birds.
This will be further discussed in the section on hypothermia.
By analogy with the reasoning presented above, it has been suggested
that an optimum might exist somewhere near the middle of the tempera-
tures between 45° and 60°C. Indeed, a majority of thermophilic bacteria
and thermophilic fungi-are known to possess optima around 53° to 55°C.
It is also well known that pasteurization temperatures usually tend to be
60° to 62°C; this suggests that the pasteurization temperature is a direct
manifestation of the structural changes in the vicinal water near this
temperature.
Finally, by the same type of argument it is proposed that optimum
activity may be encountered for a group of organisms (plants and ani-
mals) between 15° and 30°C. A large number of different types of
I - 9
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120 W. DROST-HANSEN
organisms appear to have optimum activity between 22° and 268C, includ-
ing many insects (though not all), many fishes, and soil bacteria. Thirty
degrees Centigrade is known to be an important temperature physiologi-
cally for both fishes and insects. For specific examples, see Drost-Hansen
(1965a).
Allowable ranges of temperatures for poikiiotherms are often 15° to
16°C—truly a vanishingly small range out of the total span of tempera-
tures in the universe. As will be discussed in .another section, thermal
adaptation may occur, but more frequently than not the adaptation is
merely a slight change in temperature of, say, optimum activity (for in-
stance, for growth or reproduction) rather than a notable change in the
low-temperature tolerance limit or the upper lethal temperature. How-
ever, for unicellular organisms, a special form of adaptation may take
place, namely, through the development of multiple optima for growth.
Mitchell and Houlahan (1946) reported distinct binodal distributions for
growth of a mutant of Neitrospora crassa. Somewhat similar results were
subsequently obtained by Oppenheimer and Drost-Hansen (1960) study-
ing a sulfate-rcducing bacterium. Later experiments (Schmidt and Drost-
Hansen, 1961) tended to suggest similar behavior for Escherichia coli.
More recently, Davey and Miller (1964) have also obtained very distinct
multiple optima for growth of a number of microorganisms. Four different
types of bacteria were used to cover the temperature range from 5° to
70°C; in all four cases, multiple growth optima were obtained. Oppenhei-
mer and Drost-Hansen (1960) suggested that such organisms might be
able to grow optimally in two different temperature intervals by utilizing
different metabolic pathways. Some preliminary evidence was obtained
for this proposition through a study of changes of pH of the medium on
which E. coli was grown and from a qualitative study of the pigmentation
in Serratia marcescens (Schmidt and Drost-Hansen, 1961).
2. Examples of Thermal Anomalies in Growth Processes
An interesting example of unusual temperature effects is described for
the rate of myeelial growth of a fungus (Waitea circinata), studied by
Agnihotri and Vaartaja (19691. These • authors found that the myeelial
growth of the fungus was strongly temperature dependent and, more-
over, that exudate from pines (Piling ce»ibro!rles) further stimulated this
myeelial growth. For both (without exudate as well as in the stimulated
mode of growth), myeelial growth showed indication? of an inflection
point near 15°C and a relatively notable drop in rate- of growth above
30°C. The same effect—a maximum in radial growth of the mycelium—
was obtained in the presence cf various initrient* such as aspa'rtic acid,
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VI. STRUCTURE AND PROPERTIES OF WATER AT BIOLOGICAL INTERFACES 121
malonic acid, glutamic acid, and arabinose, with notable drops in growth
above 30° C.
Recently, Walker (1969) has studied the effects of temperature in 1°
increments on the behavior of maize seedlings. The temperature range
covered was from 12° to 36°C, and several anomalies were observed. Thus,
significant irregularities in the concentration of many of the nutrients in
the shoots of the plants occurred near 15°C. Minor but persistent anom-
alies were also noted in total leaf length of the seedlings, and irregulari-
ties occurred in growth rate at 29° and 30°C. The author was not con-
vinced that all of the anomalies observed were real or whether some were
caused by experimental artifacts. However, an inspection of the data of,
for instance, dry weight of 23-day-old maize seedlings strongly suggests
an anomaly near 29° to 30°C. Walker correlates his observations with the
similar anomalous results by Davey and Miller (1964) at 15°C for the
uptake of potassium by wheat. Walker, although aware of the claim for
the existence of thermal anomalies in (vicinal) water, did not make any
conclusive association between the water structure changes and the ob-
served anomalies.
A good example of the abrupt change in growth at 30°C is shown in the
data by Buetow (1962), referred to by Farrcll and Rose (1967, p. 162, Fig.
BI. These results clearly show the dramatic change in the specific growth
rate of Euglena gracilis: a sharp maximum occurs in the vicinity of 28°
to 30°C.
Attention is called here to the monograph by Andrewartha and Birch
(1954) (in particular Part III, Chapter 6). A large number of examples are
discussed which clearly show that critical temperatures for many orga-
nisms frequently coincide with the thermal anomalies stressed in the
present chapter. It is impossible to go over all the examples discussed by
Andrewartha and Birch, but in particular the logistics curves are im-
portant (discussed in the section on "Weather: Temperature," Chapter 6,
pp. 129-205). In this connection, the frequency with which excellent agree-
ment can be achieved between some empirical or semitheoretical logistics
curves over the interval from about 15° to 30°C is noteworthy, as is the
frequent failure below 15_°C and almost invariable failure above 30°C.*
Levinson and Hyatt (1970) have studied the effects of temperature on
the activation, germination, and outgrowth of spores of Bacillus megate-
rium. The study is particularly interesting as it was designed specifically
to determine if there was evidence of thermal anomalies in these stages of
bacterial spore growth. Measurements were made at closely spaced tem-
peratures. The authors concluded that they "found no evidence of thermal
* It. is unfortunate that many authors have apparently chosen to study various
organisms "exactly" between 15° and 30°C.
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122 W. DROST-HAXSEN
discontinuities, or 'kinks' in these biological processes, hut we felt never-
theless that our data on the response of spores to small temperature in-
crements had sufficient intrinsic value to warrant publication." The nega-
tive conclusions drawn by Levinson and Hyatt is quite astounding in view
of the data reported. An inspection of their illustrations might equally
well have suggested that anomalies do occur. Thus, Fig. 4 in the paper by
Levinson and Hyatt suggests a distinct change jn slope near 16* to 18°C
for the germination temperature with a relatively' abrupt peak or change
in slope near 28° to 32°C. The authors felt these changes were not sig-
nificant but offer little additional information to substantiate this con-
clusion. The authors further note "there was some suggestion of a sharp
increase in germinability after heating at 56°C. However, as seen in the
semi-log plot (Figure 1) activation appeared to be an exponential func-
tion of activation temperatures from 52 to 60°C." The authors do not
point out, however, that above this temperature, the change in optical
density is practically constant over the range from approximately 62° to
78°C; again revealing a rather notable change in the vicinity of 60° to
62°C. The point intended here is not that the study by Levinson and
Hyatt provides strong evidence for the existence of thermal anomalies in
biological systems. Instead, it is merely emphasized that the findings by
these authors are not inconsistent with the notion of the occurrence of
thermal anomalies and that no other current theory for germination and
growth of spores is likely to predict the shape of the observed curves.
It is interesting that Levinson and Hyatt (1970) quote Thorley and
Wolf (1961) as having observed three temperature optima (near 3°, 25°,
and 41°C) for the germination of Bacillus cereus strain T. spores.
Levinson and Hyatt go on to "explain" that the multiple optima were at-
tributed by O'Connor and Halvorson (1961) to the use of a suboptimal
concentration of L-alanine. In other words, in the presence of sufficient
L-alanine there is no evidence of thermal anomalies in the response of the
spores to temperatures of germination. It appears that Levinson and
Hyatt have, indeed, missed the point: as stressed by Oppenheimer and
Drost-Hanscn (1960) and by Schmidt and Drost-Hansen (1961), it is on
minimal substrates that the multiple optima are to be expected. The fact
that the anomalies can be "swamped" by excess nutrient supply does not
explain away the nature of the growth on minimal media. In the latter
cases, limitations are imposed upon the organisms with respect to the
available metabolic pathways and the choice is limited, therefore, with the
result that the metabolites and/or appropriate enzymes are only those that
are most compatible with the structure of the vicinal water in the respec-
tive temperature ranges.
Observations of interesting anomalies around 15° (to 20°C) have been
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VI. STRUCTURE AXD PROPERTIES OF WATER AT BIOLOGICAL, INTERFACES
reported by Nishiyama. Because many of the papers by Nishiyama and co-
workers as well as other Japanese authors are not available in English
translation, we mention a number of these studies in some detail, based on
a recent personal communication to the present author from Nishiyama.
Nishiyama has been concerned with the effects of relatively low tem-
peratures on a number of plant phenomena (Nishiyama, 1969, 19701. In
the most recent article, "What is Between lo° and 20°C?" Xishiyama sug-
gests that general physiological (and pathological) changes occur in the
temperature range between 15° and 20"C. An inspection of the illustrations
in this article suggests to the present author that the rate of these changes
is frequently the greatest around 15° to 17°C. Nishiyama (1969) spe-
cifically proposes that the changes may be due to ;'the phase transition
point of water /crystal) in protoplasm." Further. Nishiyama (1970) has
suggested ''various crops are injured by low temperatures — below 15 to
20°C. One such example is a sterile type injury in rice plants (Figure 1-A
in our report, Nishiyama, 1969)." Although Nishiyama clearly recognizes
the importance of the role of water and the possibility that it may undergo
some type of phase transition, he also draws attention to the fact that "it
is to be noticed that the critical temperature varies with varieties and
conditions of cultivation. We must consider the participation of proto-
plasmic substances, such as proteins and lipids. other than the water it-
self." Nishiyama goes on to mention cold injury to plants discussed by
other Japanese researchers. Thus, injury to soybeans and red beans, and
a type of delayed injury in rice plants, is observed for low temperatures,
that is, temperatures below 15° (to 20°C).
Nishiyama has added several other examples in' support of thermal
anomalies in plant physiology and pathology. Thus, he states:
Dr. Yamashita et al. claim that there is a changeover temperature for day
length requirement [in "Control of Plant Flowering'' (Y. Goto, ed.), pp. 54-57.
Yokendo, Tokyo, 1968 (in Japanese with an English summary)]. The tempera-
ture was estimated about 175°C in several plant specie;!. Various fruits and
vegetables after harvest are susceptible to cooling below and about 15"C. These
include banana.* [T. Murata. Plant Phj/xiol. 22, 401 (I960)], oranges and lemons
[I. L. Eaks. Plnnl Phyxiol. 35, 632 (I960)], applet [A. C. Hulme el al.. J. Sd.
Food Agr. 15, 303 (1964)]. cucumbers [I. I.. Eaks and L. L. Morris, Plant
Physiol. 31, SOS (1956)]. cucumbers and pimentos [L. L. Morris and Platenius,
Proc. Amer. Soc. Hort. Sci. 36, 609 (193S)] and sweet potatoes [T. Mina-
mikawa et al.. Plant Physnol.2, 301 (1961)].
3. Thermal Classifications of Microorganisms
The traditional classification of bacteria into cryophiles, mesophiles,
and thcrrnophik-s may possibly be seen as a tendency for these groups of
organisms to exhibit maximum activity (usually optimal growth) bc-
I - 13
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124 W. DROST-HAXSEX
tween various consecutive thermal anomalies in the vicinal water. It
should be mentioned in this connection that one of the difficulties in
making a clear-cut distinction results from the fact that multiple tempera-
ture optima are often encountered. Thus, growth curves over an extended
temperature interval may show merely a broad and. at times, rather
flat peak around 30°C! The studios by Schmidt and Drost-Hansen (1961)
have suggested that this may result from considerable overlap of two
growth peaks (each with optima near 23° to 25° and 37° to 39°C, re-
spectively). Experimentally, we have noted that growth on "minimal
media'' tends to separate the overlapping peaks. Likewise, distinctly
binodal growth curves are sometimes seen in very old cultures—long after
the cessation of the logarithmic growth phase.
4. Thermal Conduction in Biological Systems
In connection with the problem of metabolic processes, the question
arises as to how the cell dissipates the lieat produced in'the cellular proc-
esses. Naturally, the component of the cell water which is more or less
bulk-like will have limited thermal conductivity (but rather high heat
capacity). However, once a steady state has been reached, the heat evolved
must be dissipated to maintain isothermal conditions in the homeothermic
organisms and in the poikilotherm organisms in "equilibrium" with the
surroundings. In this connection, recall that the heat conductivity of ice
is almost an order of magnitude greater than the heat conductivity of bulk
water. It seems eminently reasonable to suggest that the ordered water
of the cell interface facilitates the conduction of heat from the interior of
the cell to the surroundings. Heat conductivity studies of water between
closely spaced mica plates have been carried out in Russia by Metsik and
Aidanova (1966; also see Derjaguin, 1965)..These studies demonstrated
notably enhanced heat conductivity of vicinal water—as much as an in-
crease by 50 or more (for thicknesses less than 0.1 /») (see, also,"Section
VI.F.2 on hyperthcnnia).
It is interesting to speculate that shivering may reduce the amount of
ordered, structured water, somewhat similar to the breakdown, on agita-
tion, of "sot gels." In this fashion, the heat conductivity of the cellular
water might be reduced and thus minimize heat flow to the environment
upon cold exposure.
5. Xotes
As suggested by Oppenheimer and Drost-Hansen (I960),'temperature
adaptation may. indeed, take place. Thus, some bacteria have definite bi-
nodal distributions of growth rates as a function of temperature, with a
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VI. STRUCTURE AND PROPERTIES OF WATER AT BIOLOGICAL INTERFACES' 125
minimum near 30'C. As mentioned, similar results were obtained on a
mutant of Xeurospora crassa. The tentative proposal, by Oppenheimer
and the present author, is that in different temperature intervals those dif-
ferent metabolic pathways are chosen which are best "suited"' for the
organism at that temperature interval. Adaptation then may, in part, be
the proper choice of the substrate on which the organism is grown. Char-
acteristically, for bacterial studies, one gets the qualitative feeling that
thermal anomalies are enhanced when the organisms are maintained on a
"minimal medium." This would imply that the organism does not have the
normal "availability" of metabolic possibilities. It is also possible that
genuine adaptation—resulting from modification of, say, the controlling
protein structure—may be achieved through adaptation to a different
water structure in a different temperature interval. However, this is not a
likely possibility and is certainly not easily achieved. Hence, as will be
discussed in the section on paleozoogeography. it is undoubtedly correct
to say that the thermal anomalies at 15° and 30° (and perhaps 45°C) have,
in the past, imposed a significant "barrier'' leading to geographical zona-
tion of multicellular organisms, dependent on the local average lor maxi-
mum) temperature. Thus, in a sense, throughout evolution the water
structure change*; have imposed inviolable, "invariant" constraints. Stehli,
among others, has invoked this possibility in connection with paleozoo-
geographic studies (see Section VI,D,3).
As discussed in the present section, it appears that such phenomena as
body temperature of mammals, optimal temperatures for many organisms,
as well as maximum and lethal temperatures are determined by structural
changes in water (Drost-Hansen, 1956). Later (Drost-Hansen, 1965a), it
was more specifically noted that the interaction between the vicinal water
structure and the nature and conformation of the underlying substrate is
the result of mutual interactions:
T-lie cooperative action between many water molecules in the water clusters
of the solvent water may well be expected to influence drastically the rather
large amount of water associated with the proteins or membrane material. In
other words, the structural transitions in water may exert a direct and profound
influence on the immediate environment of the mat-romoiecules of the biologic
systems; the effects of the transitions are not merely "solvent effects'" mani-
fested by minute changes in the solvent viscosity, dielectric properties or
activity 1
C. GERMINATION
A vast amount of literature exists on the subject of germination (and
vernalization). It is interesting that these studies have often considered
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126 W. DHOST-HANSEN
the effects of temperature in some detail. However, as in a number of other
fields in biology, such as thermal adaptation, a vast number of complica-
tions occur due to other concomitant changes, such as changes in relative
humidity (water activity), light, and pressure. Hence, with the exception
of a relatively small number of studies, it is difficult or impossible to make
significant systematic comparisons between the structure (and thermal
anomalies) of vicinal water and the specific effects on the processes of
germination and %-ernalization. Obviously, the study of the influence of
water structure on these processes is further complicated by the fact that
frequently the systems have not been studied as a function of temperature
at closely spaced intervals, and the systems are, in addition, sensitive to
various electrolytes and nonelectrolytes1, which undoubtedly exert specific
influences through direct chemical interaction, for instance, with singular
functional groups in some controlling enzyme or at some membrane site.
A few examples of abrupt changes in germination rate? with tempera-
ture were discussed by Langridge and McWilliam in Rose's monograph
(1967, p. 244). The authors state ".. .the optimum temperatures for the
germination of most seeds fall between"15° and 30°G, although higher
optima (35° to 40°C) have been reported in tropical species, such as
Paspalum and Saccharuw." Also (Rose, 1967, p. 26], "...similarly, it
has been shown that potato tubers immediately after harvest are able to
sprout only within a narrow temperature range above *30°, which pre-
sumably protects them from premature sprouting in the autumn."
P. A. Thompson (1969. 1970a,b) has studied the germination of seeds in
considerable detail by a variety of techniques. He has made use of a
thermogradient bar—a polythermostat somewhat similar to the one used
in the studies by Oppenheimer and Drost-Hansen (19GOi. In some cases,
the results are in excellent qualitative agreement with similar results ob-
tained for the rate of growth of a number of bacteria showing abrupt
changes near 15° and 30CC, for instance, for Silene tartarica and Silene
coeli-rosea. In other cases, vastly different temperature responses were
obtained in the sense that critical temperatures occurred, for instance, near
25° or 36°C depending on the geographical origin i and. hence, climate) of
the plants studied. There is little doubt that the study by Thompson will
prove a most important-contribution. He observed that a temperature dif-
ference as small as 0.5° may play a discriminating role in the germination
of seeds. Thompson also introduced alternating temperatures in order to
study an environment more nearly identical to that encountered in na-
ture. Furthermore, multiple growth optima were also encountered on oc-
casion. Thus, for Fragaria vesca Linn and, particularly, Ajuga reptans
Linn, multiple growth optima were observed with minima near 30°C. At
this point, attention is called in particular to Fig. 22 showing the pcrcent-
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VI. STRUCTURE AND PROPERTIES OF WATER AT BIOLOGICAL INTERFACES 127
age of germination curves as a function of temperature of two different
species of Fragaria vcsca Linn. The germination curves as a function of
temperature clearly exhibit binodal character with relative minima be-
tween 2G° and 29°C. This behavior strongly resembles the multiple tem-
perature optima obtained for the growth of a number of bacteria studied
by Drost-Hanscn and co-workers (Uppenheimer ami Drost-Hanscn, 1960;
Schmidt and Drost-Hansen, 19GU.
10 15 20 25 30 35 40
Temp (°C) olonq gradient bar
FIG. 22. Percent germination for two different species of Fragaria vesca Linn.
(P. A. Thompson. 1970b. with permission.)
Finally, attention is called to some studies of the effects of gibbcrellins
on the germination of some seeds. Thompson il9G9i concludes
There would appear to be no requirement for a close relationship between re-
sponding species irr taxonomic terms, nor is it ea.-y to find any similarities from
one to another, suggesting a common bond in terms of the conditions required
for germination in normal circumstances. Gibberellins will substitute for light
in dark-grown seed, for chilling treatments, and for fluctuating temperatures:
they will procure germination at temperatures normally too high and also
temperatures too low; and they will replace complex conditions for germination
such as the com^in^'ion of leaching :'.nd chilling required by the seed of A/ecf1-
nopsi cnnibricn. and the combination of light and fluctuating temperatures
rec|iiirod by l,>iri~>j>i>ne".<.
The fact that the gibbcrellins may act in .-uch a variety of ways and
mimic such vastly different functions suggest.- to the present author that
their effect is not based on a specific chemical reaction, such as interaction
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128 W. DROST-HANSEN
with one particular functional group in a controlling enzyme or substrate.
It is suggested, therefore, as an alternative hypothesis, that the effect is
due to some general influence and this most likely is through the action of
the gibberellins on the structure of the water vicinal to the site of control
of dormancy in the seed.
1. Vernalization
Vernalization is the induction of seeds to germinate after (often pro-
longed I exposure to low temperature. The subject is obviously of enormous
practical importance. The need for prior cooling, before germination can
take place, is the principal means whereby freshly discharged seeds from
a plant are prevented from germinating upon release in the autumn which
would expose the young plant to the cold of winter.
It is proposed here that vernalization is the relatively slow restructuring
(and probably the increased ordering) of water adjacent to some critical
component in the seed, probably a membrane or a protein. Only after the
vicinal water structure has changed to conform to the lower-temperature
range is the seed latently capable of germinating. Recall in this connection
that, whereas the substrate undoubtedly influences the nature of the
vicinal water, the converse must also hold true, namely, that the structure
of the vicinal water must influence the nature and conformation of the
underlying macromolccular substrate. That the process is .slow is perhaps
related to the thermal memory effect discussed in Section VI,J, probably
reflecting the difficulty in inducing order by merely removing available
thermal energy (thermal energy [kT] tends to disorder structures and,
conversely, a lowering in temperature increases the ordering).
Recently, Levitt (1969) has sun-eyed the growth and survival of
plants at extreme temperatures and presented a unified concept. Levitt
proposes that temperature exerts a controlling role in the response of
plants through the state of denaturation of the proteins. Specifically, he
proposes the simple scheme shown below for interrelations between the
native (N) and the denatured (D) forms of many enzymes:
above 40°C
N , D
below 40°C
and
below 10°C
N » D
above 10°C
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154 V. DROST-HAXSEN
H. HYPERTHERMIA
1. Upper Lethal Temperatures
As discussed in Sections YI,B,1 and 2, rather abrupt, critical upper
thermal limits are frequently encountered for various organisms. A direct
application of this notion is discussed, for example, in the section on
thermal pollution (Section VI,K). For mammals and birds, 44°-46°C
appears to be an absolute, upper thermal limit for survival. It should be
noted that Belding (1967) has suggested that a body temperature of 45°C
may be sustained by birds over relatively long periods of time without
injury, whereas a rise to about 47°C is lethal. In humans, body tempera-
tures above 433C (for instance, due to infectious diseases) is considered
highly dangerous and the prognosis is generally poor. However, when the
elevated body temperature is produced by external means, such as
diathermy or hot baths, the probability of survival is significantly in-
creased. This will be elaborated upon in the next section.
A rather detailed discussion of temperature effects on poikilotherms was
presented by Fry (.1967). The reader is referred to the article by Fry
for many interesting details, especially regarding definitions and methods
of determining thermal limits. Measurements are made by maintaining
organisms for various lengths of times at constant temperatures and
determining the time of death due to exposure to a particular tempera-
ture. Alternatively, the temperature may be recorded at which the animal
dies (or suffers loss of motor ability) in an environment of constantly
increasing temperature. The rate of temperature change now plays a
crucial (and ill-defined) role. Each approach has its advantages and dis-
advantages. The problem is not amenable to a description in terms of
physicochemical parameters because of its nonreversible, nonsteady state
aspects.
In connection with structural changes at 45°C a? the causative factor
in death, it can be questioned if evidence exists for the operation of only
one causative mechanism, or, if more than one direct cause is involved,
what are the different possibilities. Specifically, What is the mechanism
of thermal injury? Is it possible to determine the site or sites where water
structure change* may be the direct cause of death of the organism? Un-
fortunately, there appears to be very little information available on which
to base any judgment in this matter. Fry reports a few sets of data which
tend to suggest the occurrence of two different, distinct mechanisms of
death. Among possible mechanisms are failure of osmoregulation, in-
creased production of lactic acid (an unlikely effect), or asphyxia and/or
damage to the central nervous system. Fry mentions also that much of the
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VI. STBUCTURE AND PROPERTIES OF WATER AT BIOLOGICAL INTERFACES 155
work by investigators such as Precht, Prosser, and Ushakov tends to
suggest that the animal dies from the cessation of some regulatory
activity, rather than from "collapse of its cells."
2. Hyperthermia Therapy
Recently, von Ardenhe and co-workers (1965, 1966a,b) and Kirsch and
Schmidt (1966) have actively pursued the treatment of cancer by hyper-
thermia. Initially, the treatment consisted merely in heating the patient,
while more recently a "multi-step therapy" is employed, combining ex-
treme hyperthermia (heating of the patient's body to near 43° to 44°C)
with the prior, simultaneous, or subsequent administration of certain
drugs (pharmacons). With the development of more effective pharmacons,
the need for extreme hyperthermia has been reduced somewhat and
present therapy uses temperatures as low as 42° C.
The treatment of cancer, both experimentally and clinically," by high
temperature is by no means new. Cavaliere and co-workers (1967) have
discussed the heat sensitivity of cancer cells and reported both some
biochemical and some clinical studies. These authors mention that as early
as 1866, Busch described the complete disappearance of a histologically
proven sarcoma after the patient suffered two attacks of erysipelas (an
acute infection of the skin by a Group A hemolytic streptococci, char-
acterized by sharply delineated, red, swollen local areas with general fever
and malaise; temperatures as high as 42°C are often observed). The article
by Cavaliere and co-workers should be consulted for a number of
examples and a rather careful review of previous studies. It is interesting
and, in fact, impressive, that the previous studies as well as the work of
Cavaliere and co-workers and von Ardenne and co-workers clearly
demonstrate the increased heat sensitivity of malignant cells to tempera-
tures ranging from 43" to 44°C, Although Cavaliere and co-workers were
relatively successful in the clinical treatment of 22 cases of cancer of
the limbs, they present their findings with considerable reservation—
because, among other reasons, of the attendant clinical risk in any high-
temperature treatment as well as other complications.
No molecular mechanism has yet been postulated for the role that
temperature plays in that part.'of the original therapy which relied
primarily on the effects of temperature alone. It was suggested (Drost-
Hansen, 1966) that the effectiveness of the increased body temperature
depend? primarily on the structural transition near 45°C in water
associated with the cells (rather than, for instance, merely increasing the
activity of any administered pharmaeon). Burk and Woods (1967),
Olmstead (1966), and Szent-Gyorgyi (1965) have pointed out that cancer
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156 W. DROST-HAXSEN
cells generally have a much higher water content than normal cells. As
an example, ordinary liver cells possess about 67% water compared to
Rous sarcomas of chicken containing 939o water; in fact, there appears to
be a good correlation between malignancy and the water content of cancer
cells. It is now suggested that the molecular mechanism underlying the
effectiveness of the hyperthermia therapy may, in fact, be due to one or a
combination of several processes. The first of these possibilities involves
the greater ratio of bulk-like water to structured-water in the cancer cells
as compared to normal cells. Since vicinal water (and solutions) appear
to be stabilized near an interface (where it undergoes a transition at or
near 44° to 46°C), the cancer cells may be more susceptible to tempera-
tures in this vicinity than normal cells, merely because of the larger
amount of bulk-like water.* If the pharmacons administered tend to ac-
cumulate in the malignant cells, the effectiveness of these pharmacons
may be due to their ability to alter the water structure in such a fashion
that a lower transition temperature is obtained. Of course, the pharmacons
(such as alky latin g compounds) administered at the time of the treat-
ment will exert a direct influence on the biochemical processes involved in
the metabolism of the cancer cells. It is of interest to note that Burk and
Woods have shown that at 43°C and above, 9-a-fluoroprcdnisolone ac-
celerates loss of the Pasteur effect and metabolic death of the cells. In
addition, the Pasteur effect in cells of mouse melanoma S91 remains essen-
tially constant (for 1 to 2 hours) at any given temperature below 40°C.
However, above 43°C the aerobic acid production (glucolysis) increased
markedly more than the anerobic glucolysis. [See in this connection the
study by Haskins (1965) who investigated the effects of sterols on the
temperature tolerance in a fungus of the genus Pythium. ]
An alternative hypothesis is based on the assertion that cancer cells
probably present a far more disordered interface to the cell fluid than do
normal cells: since cancer cells generally are much less differentiated than
normal cell?, they do not present the intracellular water with the same
degree of stabilization near the interface that is provided by normal cells
(Szent-Gyorgyi, 1965). This effect would be particularly important near
the point where an abrupt transition in the water structure occurs.
Both of the mechanisms suggested above may play a role simultane-
ously. In general, the effect of elevated temperatures is to produce greater
instability in the water structures associated with the cells, and this effect
may be further enhanced near the critical transition point due to the
pharmacons administered at the time of treatment. The structural changes
in water are suggested here as an important factor iii the molecular process
* (See, however, discussion of the paradox of relatively invariant thermal transition
temperatures, Section III,C,1,2.)
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VI. STRUCTURE AND PROPERTIES OK WATER AT BIOLOGICAL INTERFACES 157
underlying the phenomenon of enhanced lethal effects of high body tem-
peratures, but this aspect is obviously only one factor in an exceedingly
complex molecular system and many other factors may play equally im-
portant roles,
In connection with hypcrthermia treatment, it is of interest to note that
sonic of the phcnylcncdianrines have been alleged to have a synergistic
effect in the hypcrthermia treatment of cancer. Apparently so does dopa.
The phenylencdiaiuines have the interesting property that their solubili-
ties increase extremely rapidly over very narrow temperature ranges.
Thus, conceivably, these compounds are highly "sensitive" to the structural
details of the aqueous environment. This would again suggest that a more
detailed understanding of the,effccts of various solutes on the structure of
water and, particularly, the structure of vicinal water may aid in predict-
ing the types of phannacons which may be most useful in the treatment
of cancer by multistep hypcrthermia therapy.
It is interesting to speculate on a rather simple mechanism for the in-
creased heat sensitivity of malignant cells over normal cells in terms of
intracejlular heat conductivity ('in the vicinity of 44° to 45°C). If it is
assumed that in hypcrthermia treatment, malignant cells and normal cells
are heated to the same temperature and if it is assumed that the meta-
bolic rates in these cells are roughly equal (but obviously not identical in
the two types of cells), it is seen that because of the greater amount of
ordinary water in the malignant cells, the local, internal temperature
and likely the "internal structural temperature" 'of the malignant cells
may be notably higher than that of the normal cell. Recall that the
malignant cell is generally characterized .by a considerable increase in the
amount of total water and this water is undoubtedly less structured
(more bulklike) than the water in normal cells. If, indeed, the ordered
water possesses higher thermal conductivities as suggested by Metsik and
Aidanova (1966), the normal cell will then be able to dissipate (by ther-
mal conduction) the energy produced, faster than the malignant cell.
Thus, the malignant cell will be subject internally to a somewhat higher,
local temperature. It may be by this mechanism that the heat sensitivity
of the malignant cells is enhanced.
Finally,' assuming again that the rate of energy production is approxi-
mately equal in normal and malignant cells, assume also the heat con-
ductivities may differ by a factor of 70 between ordered water (in normal
cells) and bulk water (in malignant cells), based on Metsik and Aida-
nova's data, rather large differences in internal temperatures may then be
expected for the two types of cells. Spanner (1954) has considered the
relation between the "heat of transfer" and the equivalent "osmotic" pres-
sure of the cells. The present author does not necessarily accept the de-
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.158 W. DROST-HANSEN
velopincnt on which Spanner's estimates arc made; however, if Spanner's
calculations are correct to an order of magnitude, it is interesting that a
temperature difference of 1°C produces an osmotic (thennomolecular pres-
sure effect) pressure difference of about 130 atm, where AP/AT ?a -132
atm/°C (note the minus sign!). Tims, a difference in temperature as slight
as 0.01 °C may then be expected to cause changes in the rate of water
permeation (driven by hydrostatic forces) equivalent to a pressure well
over 1 atm. Considering that the differences in "heat conductivity may be
as large as one and even two orders of magnitude, temperature differen-
tials of the order of 0.01 °C are not at all unlikely within any given system
of the cells presumed to be in isothermal (and isoosmolal) equilibrium.
In connection with the role of water in malignancy, attention is called
to an article by Apffel and Peters (1969). These authors discussed the
role of hydration of various macromolecules, in particular the glyco-
proteins, first noting that the glycoproteins may have distinctly varying
capacities to bind water. The degree of hydration appears to depend di-
rectly on the specific nature of the monosaccharides in the saccharides of
the glycoproteins and of the polysaccharides. They discuss the.general
phenomenon of tissue hydration in malignancy, noting the increase in
amounts of water, for instance in liver, during carcinogenesis and tumor
growth. The authors also consider, qualitatively, the conformational
changes that may result from differences in hydration, leading to signifi-
cant differences of interaction at the surface of a cell or a macromolecule.
Thus, "Hydration shells are proper to microorganisms and cells coated
with sialoglycoproteins, and to a much lesser degree, to solvatcd or dis-
persed macromolecules of that nature. Adsorption of sialoglycoproteins to
the surface of cells produces a unique situation where all the oligosac-
charide chains protrude into the medium in a single, committed direction.
Because of the close association thus brought about, there is a tendency
toward gelification. The resulting semi-rigid shell of hydration water is
tantamount to a volume forbidden to many solutes, depending on their
size, charge and shape." Finally, Apffel and Peters call attention to the
note by Good (1967) who also stressed the importance of hydration phe-
nomena superimposed on charge-charge interactions between cells or be-
tween cells and macromolccular solutes. It is particularly interesting, as
noted by Apffel and Peters (1969) that Good suggests that "at interfaces,
as between cells and medium, the hydration of charged ions is more
stable because there is less thermal gyration and less mechanical dis-
turbance- [see Good, 1967]." It should be observed in this context, however,
that the type of hydration discussed by Apffcl and Peters (and by Good,
1967) is primarily the very direct (and energetically strong) interactions
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VI. STRUCTURE AXD PROPERTIES OF WATER AT BIOLOGICAL INTERFACES 159
between ionic sites and the water molecules (or strong dipole-dipole
interactions). In this chapter, stress is placed instead on the (very likely I
much weaker, but possibly far more extensive hydration phenomena in-
volving energetically only slightly different states of water. However,
the basic idea regarding the stabilization of water structure near an inter-
face suggested by Good (and quoted by Apffel and Peters) is the same as
the one advocated in this chapter, namely, the reduced "thermal gyration
and less mechanical disturbance'' which results from the "momentum
sink effect." Xo doubt, the study of the hydration of cell surfaces and
macromolecular solutes will prove an important requirement for further
advance in a detailed molecular understanding of the role of serum pro-
teins in cancer.
I. CELL ADHESION
Adhesion in general and cell adhesion in particular are extremely com-
plex phenomena. Attention is called here only to the types of interactions
that do not depend on attractive forces deriving from functional groups of
the membrane materials. Pethica (1961) has reviewed the type of forces
which may exist between cell surfaces; these forces include attractive as
well as repulsive forces. In addition to such forces as chemical bonds be-
tween the opposing surfaces, ion pairing, image forces, and van der Waals
forces, Pethica mentioned a "hindrance" to attraction due to steric barriers
such as "inert capsules and solvated layers." Pethica points out that the
latter do not actually represent a force "except that the entropy effect due
to the mutual disordering of adsorbed layers, as the surface is ap-
proached, might be regarded as a force. The effect of adsorbed inert layers
may more usually be to increase in range between otherwise active groups,
and to attenuate the attractions between the surfaces to the point where
reversible collisions can take place."
As discussed in Section III,CM.a, Peschel and Adlfinger (1967) have
determined the disjoining pressure between surfaces (specifically, quartz
surfaces), and the major feature of their results may probably be gen-
eralized to cell surfaces, at least to the extent that anomalous temperature
dependencies may be expected in any quantitative data on cell adhesion.
Pethica has considered the role of image forces in the attraction between
cells in solution. For this purpo.-e, the cells were modeled as two thick
planes of material with much lower dielectric constants. Using the ex-
pression (applicable to a structureless dielectric material of dielectric
constant t\. IVlhiru calculate the osmotic pres.-ure (*•/) from the free
energy (A(/> i'xprr«-ioii:
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160 W. DROST-HANSEN
A
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VI. STRUCTURE AND PROPERTIES OF WATER AT BIOLOGICAL INTERFACES 161
of adsorption of platelets onto a standard glass surface). For both proc-
esses, Abdullah suggests that "some platelet aggregating substances act by
increasing ice-likeness (ordered structure) of water around platelets7' and
that the active, initial process is followed by a "chain reaction" which re-
sults in the accretion of many iayers of platelets onto the first-formed
layer. Abdullah measured the effects of various nonelectrolytes on platelet
suspensions, following the degree of aggregation optically. Very interesting
results were obtained with a number of normal aliphatic alcohols (pen-
tanol, hexanol, octanol, and decanol), two tetraalkylammonium salts, and
argon and xenon (in oxygen-rich mixtures). From the data obtained,
Abdullah suggested th:
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162 \T. DROST-HAXSEX
naroic parameters to be measured. Yet, in kinetic studies, memory effects,
or at least time-dependent behavior, is noted in some instances. Character-
istically, practically all liquids, water in particular, may be significantly
supercooled, whereas the ice lattice (or crystalline hydrates) apparently
never superheats. Clay suspensions, once agitated, may "reset" at rest or
at low shearing rates, whereas an initial disturbance can lead to the imme-
diate disruption of the prevalent structure (which is causing the gel
rigidity).
Recently, the present author and his co-workers have had ample op-
portunity to note strong time-dependent variations. Among these have
been memory effects (or at least, time-dependent effects) in the properties
of membranes. Thus, thermal anomalies have, from time to time, been ob-
served in membrane properties when studied during slow heating—either
through discrete increments of temperature or using continuously variable
temperatures. Likewise, Kerr (1970), working in the author's laboratory,
has observed thermal anomalies in the viscous damping of a water-filled
vibrating quartz capillary. The anomalies are particularly pronounced
during heating. The anomalies are sometimes completely absent (or nota-
bly displaced) upon repeating the measurements with decreasing tempera-
tures. The most significant contribution, however, to the study of the
possible existence of thermal hysteresis has come from the work by Bach
(1971). Bach observed a memory effect in the structural properties of
water deposited on a silver (or silver oxide) surface, using a differential
thermal analyzer (DTA), and on glass, using a vapor phase osmometer
in a differential manner.
It is not difficult to propose an explanation for time-dependent effects.
As temperature is increased and thermal energy thus enhanced, any or-
dered matrix or array is readily disturbed into a more disordered state
(i.e., a state of higher entropy). However, upon cooling, removal of a "like
amount" of thermal energy does not readily cause a reordering of the sys-
tem into the original order of the crystalline lattice. It is obviously far
easier to disrupt and disorder a lattice than to perform the converse: to
induce a specific order by merely lowering the available thermal energy
fluctuation. Characteristically, thermal hysteresis phenomena have been
observed especially with aqueous systems near interfaces ("although super-
cooling does illustrate a bulk phenomenon of similar type). Near 15° and
30°C. for instance, water in biological systems (or at or near almost any
aqueous interface) will undergo a phase transition, as discussed in previous
sections. However, since as increase in temperature (for instance, from
28° to 33°C) will have resulted in a disruption of a structured matrix, it is
possible, and sometimes likely, that :\ similar decrease in temperature may
not reversibly lead to the ''same7' change in biological functions—at least.
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VI. STRUCTURE AND PROPERTIES OF WATER AT BIOLOGICAL INTERFACES 163
not the same change at the same rate of change. Thus, thermal hysteresis
must be expected in living systems also.
In summary, near a biological interface, water structures are stabilized
which differ from the bulk structures. Disruption of these structures by
increasing the temperature is readily achieved. However, structures that
are stable at low temperature may not readily be reformed upon lowering
the temperature; thus, the possibility exists for a significant lack of "sym-
metry" in the behavior of biological systems under temperature cycling.
The attention of the experimental biologist to this possibility may likely
prove rewarding. It is also of interest to note that the clathrate formed by
hydroquinone and argon is "stable" (or, rather, may be kept in a bottle
nearly indefinitely) once formed, although at room temperature its (equi-
librium) vapor pressure is several atmospheres. The reason for this (meta)
stability is the high energy of activation required to break a number of
H-bonds in this hydroquinone lattice in order to release the trapped argon
(see van der Waals and Platteeuw, 1959).
It is interesting that in many studies, particularly on biological system,
the experimentally observed errors often tend to be larger in the vicinity
of the temperatures of the thermal anomalies. Based on the studies by
Bach (1969), Kerr (1970), and Thorhaug (1971) (all formerly working
in the author's laboratory), it is suggested that this may be related to
thermal hysteresis effects. Bach, in particular, has proposed that whether
or not the system has been cooled or heated immediately prior to an ex-
periment may influence the physical states attained. Thus, the possibility
exists that a particular experiment near the temperature of one of the
thermal anomalies may find the system in question in one of two states,
corresponding, respectively, to either the structure which is stable above
the transition temperature or the structure stable in the lower temperature
range. Since these states will have different properties (for instance, reac-
tion rates), it is not to be wondered at that the scatter in these cases occa-
sionally are larger than toward the middle of each temperature interval.
K. THERMAL POLLUTION
The general question of thermal pollution is obviously not immediately
related to the specific discussion of the structural and functional role of
water near the cell surface (and in biological systems in general). Further-
more, the question of thermal pollution is an exceedingly complicated one,
but not merely in the ordinary sense of complications as they are en-
countered in the study of any biological phenomenon, say, metabolism. In
the case of thermal pollution, factors enter which are extraneous to a con-
X - 28
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164 JT. DHOST-HAXSEX
ccptually homogeneous approach to the problem. Thus, as an example, the
effects of elevated temperatures will influence the entire life cycle of any
of the multitude of organisms making up the ecological network and in-
clude as well additional "external" factor:; such as environmental tempera-
ture fluctuations (frequency and amplitude of variations) and attendant
changes due to additional stresses such as salinity fluctuations, chemical
pollutants, and politicians. However, one specific aspect of thermal effects
on the stnicture and properties of water near interfaces may play a
singularly important role in determining the overall response of the entire
ecosystem. What is implied here obviously is the abrupt and likely rela-
tively invariant constraints imposed on any biological system due to the
sudden changes in vicinal water structure at the temperatures of the
thermal anomalies; hence, from this point (and this point only) is dis-
cussed the more obvious aspects of the possible existence of guidelines
for allowable thermal pollution limits.
Fundamental to the problem of delineating permissible temperature in-
tervals for biological organisms—a problem of crucial significance in any
thermal pollution study—is the simple statement (Drost-Hansen, 1965a)
that "If we are correct in assessing the importance of the structural
changes in water for the behavior of biologic systems, it may be possible
to delineate ranges of environmental temperatures that are conducive to
life." It is, indeed, this idea which was elaborated upon subsequently in
the paper by the present author (Drost-Hansen. 1969c) on thermal pollu-
tion limits.
In connection with the effects of temperature on biological systems in
nature as distinct from laboratory studies, we must take into account the
effects of varying temperature. This undoubtedly plays a crucial role, as
is already known from both marine biological studies as well as physio-
logical studies, for instance, on land plants. The overall dynamics are
further complicated by variations in light intensity, availability of in-
organic nutrients, etc. The question here is whether or not it is more appro-
priate to be concerned with the extremes of temperature rather than the
average temperatures. Certainly, a ''steady'1 temperature of 28°C could
conceivably be compatible with growth and reproduction of an organism.
but even relatively short-time excursions of ±4° from this average (to
temperatures between 24° and 32°C) might lead to catastrophic results in
the narrow temperature range from, say, 30° to 32°C.
For marine fishes, the probably critical nature of temperatures around
30°C was carefully reviewed by DeSylva (1969).
Elsewhere the present author (Drost-Hanson, 1969e) has emphasized
that in thermal pollution studies it is necessary to be concerned with the
effects of temperature on each of the different stages of life development.
I - 29
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VI. STRUCTURE AND PROPERTIES OF WATER AT BIOLOGICAL INTERFACES 165
A simple example of the different requirements for optimal development is
illustrated in the study by Calabrcse (1909). In this study the effects of
the salinity and temperature on tome marine bivalves were studied in co.n-
sidcrable detail. Specifically, Calnhresc studied the effects of temperature
and salinity on the development of embryos and larvae of Mulinia lateralis
over wide ranges of the two variables. The percent of embryos developing
normally shows a notable peak as a function of salinity at approximately
25 ppt. This sensitivity to electrolytes is paralleled with a notable maxi-
mum in the number of embryos which developed normally as a function
of temperature. The survival of larvae and the number of eggs developing
normally both showed maxima in the range between 15° and 30°C with
precipitous decreases in normal development above 30°C. It is interesting
also that the percent survival of the larvae as a function of the combined
effects of salinity and temperature shows a rather wide range of survival:
between temperatures of 7.5° and 27.5°C, and for salinities ranging-as high
as 35 ppt and as low as (10 to) 15 ppt. However, the percent increase in
the mean length of these larvae as a function of the change in the same
parameters showed a notably more restricted domain of optimum develop-
ment, namely, between temperatures of 7.5° and 22.5°C and salinities be-
tween 20 and 35 ppt.
The abrupt changes in biological functioning, which have been men-
tioned in this chapter, near the temperatures of the thermal anomalies
likely play a crucial role in thermal pollution. The literature provides a
vast number of such examples of abrupt changes near 15° and 30°C. See,
for instance, the cases discussed in the paper on thermal pollution by the
present author (Drost-Hansen, 1969el. Figure 30 shows the percentage of
normal development, compared to the development of major anomalies, in
the frog (Rana cyanophlyctis). It is seen that, above approximately 33°
and below 15°C,.nonc of the progeny develops normally, whereas 100%
normal development occurs between approximately 21° and 31 °C. Un-
doubtedly this is not a unique example. Compare, for instance, the dis-
cussion in Section VI.D.2 on rates of mutation (chromosome aberrations).
Furthermore, it should be stressed again that the effects of tempera-
ture in an ecological system must be fully concerned with the effects of
temperature on all stagcs'oT the life cycle, ranging from the egg and sperm
stages through the development of mature individuals (Drost-Hansen,
1969c): "obviously, even if only one. life stage is sensitive to the tempera-
ture changes around the thermal anomalies, the ecological significance
may be great."
In summary, then, it is suggested that significant and, in fact,'possibly
disastrous results may occur to the ecology of a particular area should
the temperature for any length of time exceed the temperature of one of the
I - 30
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166
W. DROST-HANSEN
thermal anomalies (the anomaly which occurs above the range of optimum
temperature for the majority of organisms in that locale). This suggests
that it may be possible, on the basis of purely physicochemical observa-
tions, to propose rather clearly delineated limits for thermal pollution.
This is particularly tine in cases such as in subtropical and tropical cli-
mates where the temperature may already be close to 30°C.
100
90-
80-
E 70-
I 60-
| 30-
z 20-
10-
Progeny of
• Normal T * Normal /
oNormol t *Risfon i
8 8
8 8
Major
defects
Fie. 30. Development of major anomalies in the frog (Ratio cyanophlyctis) as a
function of temperature. (Data by DasGupta and Grewal, 1968.)
I - 31
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SECTION VI I
DISCUSSION
A number of points are discussed in this section, representing
some important conclusions which can be drawn from all the material
presented in this report regarding the possibility of defining
specific upper thermal pollution limits.
Perhaps, above all, it must be stressed that the temperature
effects on biological systems are highly non-linear. This is most
notably reflected in the degree of abruptness observed in the
thermal death ranges of organisms. The main point to note is that
gradual thermal death of a population of organisms does not occur
when the organisms are exposed to varying temperatures. In other
words, the lethal limits do not extend over ranges of, say, 5 to 10 ,
as might have been expected were upper thermal limits best described
by, for instance, a Gaussian distribution curve. CMore sophisticated
"bell-shaped" curves are required and such have been employed in the
past by the Principal Investigator, particularly in connection with
organisms for which multiple growth ranges are observed!]. Thermal
death appears not to be merely a manifestation of some protein de-
naturation process; instead, the degree of abruptness observed sug-
gests the operation of a higher-order phase transition of some ex-
tensively structured elements. The vicinal water of cells is
eminently we I I cast for this role. For many organisms, particularly
algae, but others as well (crab megalopa and certain carideans), the
data by Dr. A. Thorhaug appear to prove this point particularly well.
It is of interest, in passing, to observe that such an effect WE?
essentially predicted as long ago as I960 by Drost-Hansen and Oppen-
I - 32
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heimer, and amplified upon by the Principal Investigator (I965a):
"the cooperative action between many water molecules in
the water clusters of the solvent water may we I I be ex-
pected to influence drastically the rather large amount
of water associated with the proteins of membrane ma-
terial. In other words, the structural transitions in
water may exert a direct and profound influence on the
immediate environment of the macromolecules of the bio-
logical systems; the effect of the transitions are not
merely 'solvent effects', manifested by minute changes
in the solvent viscosity, dielectric properties, or
act i v i ty!'
Some forms of organisms in various stages may possess distinctly
different, upper thermal death limits. !n a later study, the Prin-
ciple Investigator (1969) specifically noted that it is also "impor-
tant to recognize that the study of the effects of temperature in
pollution should be concerned with the effects of temperature on
a I I stages of the life cycles, ranging from egg and sperm stages
through the developed, mature individuals. Obviously, even if only
one Iife stage is sensitive to the temperature changes around the
thermal anomalies, the ecological significance may be great".
For an interesting analysis of thermal death mechanism, see the
discussion of enthalpy-entropy compensation phenomena in the arti-
cles by Lumry and Rajender (1970), Drost-Hansen (1971), and most
recently, Drost-Hansen (1972). A quantitative understanding of
thermal death is beginning to emerge from such studies, particularly
related to the entropy-enthalpy compensation phenomenon for large
macromolecules in aqueous systems. Note also in the laboratory
study reported by Dr. A. Thorhaug that the upper thermal limits
for species of Valonia appear relatively independent of the sali-
nity (whthin "normal ranges"): nearly identical maximum thermal
death points (at 3I.5°C) are observed for 32 0/00 and 25 0/00
I - 33
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salinity. This is important for the following reason: the thermal
anomalies responsible for the abrupt changes in cellular function-
ing depend on intrinsic aspects of water structure, and the present
author has, on several occasions, demonstrated these to be relatively
independent of the concentration and nature of solutes present.
Thus, the temperatures of the thermal anomalies appear to be indepen-
dent of the concentration of electrolytes over ranges from zero to
near I molar, and for non-electrolytes, independent of the concen-
tration up to even higher concentrations. However, note that dis-
tinctly toxic compounds exert a non-linear effect when coupled with
thermal stresses.
In passing, attention is called once more to the highly non-
linear effects occurring at the critical temperature ranges where
the structure of vicinal water undergoes changes. As these effects
are clearly manifested in the functioning of cellular systems, it is
proposed that the concept of "degree-days", often used in ecological
studies, be either abandoned or at least restricted carefully to
areas of study where it is certain that none of the limits for
thermal anomalies have been transgressed. The reason is obviously
that outside these thermal limits, irreversible effects are en-
countered. Thus, in the language of physics, the processes are not
conservative, and a summation of product of temperature and time
becomes meaningless. Note that especially for terrestrial organisms,
ambient temperatures may not necessarily be a sufficient guideline to
whether or not the "degree-days" concept is applicable. Thus, in
dark-materials (for instance, dark shells of insects or strongly
absorbing leaves) local temperatures may well exceed ambient tem-
peratures significantly.
I -34
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Thermal death points remain somewhat ill-defined, because
of the non-linear aspects discussed above. However, very short
term exposure to temperatures which would ordinarily be lethal
may be tolerated by some organisms. However, It appears reason-
able to suggest that such short term exposure to high temperature
may present hazards which are not immediately observable. Thus,
semi-lethal temperatures on mutation rates, recombination and
replication of bacterial viruses (in a cell containing a provirus)
are among the topics which deserve considerable further study.
I - 35
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SECTION VIII
ACKNOWLEDGEMENTS
The Principal Investigator gratefully acknowledges the support
of this Project by the Water Quality Office, Environmental Protection
Agency, specifically the help provided by Dr. J. D. Allen, and C. S.
Hegre, Grant Project Officer.
I - 36
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SECTION I
CONCLUSIONS
1. Certain of the macro-algae in the tropical marine estuary Biscayne
Bay appear to have their upper temperature limits between 31 and 33°C.
The intertidal Acetabularia had an upper limit between 38 to 39°C.
2. Instead of the expected Gaussian or skewed-Gaussian curve for lethal
thermal limits, abrupt death points occurred within 1 to 2°C.
3. Some of the sensitive larval forms of important commercial inverte-
brates (nauplius stage of the pink shrimp and megalops stage of the stone
crab) have upper lethal limits near 31°C, while the limits are between
34 and 37°C for other larval stages of these same organisms. The post-
larvae appear to have higher temperature limits. '
4. Depending on the species, caridean shrimp, important intermediate
food chain organisms, have upper temperature limits from 31 to 37°C.
5. Temperature limits were time dependent; however, an "equilibrium"
temperature was found beyond which prolonged exposure no longer caused
death.
6. The laboratory data for macro-algae was in complete agreement with
field data collected under a separate research program.* In both studies,
Penicillus capitatus was more temperature sensitive than Halimeda incrassata
and both were far more sensitive to temperature than Acetabularia crenulata.
In the laboratory, Laurencia poiteii demonstrated pronounced death at 32°C
and all experimental plants perished at 34°C. In the field no Laurencia
existed where water temperature of 34°C persisted, yet it was common in
areas where the temperature remained below 32°C as represented by Roesslert
* Research supported by the Atomic Energy Commission, the Florida Power &
Light Company and the National Oceanographic & Atmospheric Administration (Sea Grant)
+ Research report to FWQA (EPA) DI FWPCA-18050 DFS.
II-l
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7. In extensive studies conducted on five species of Valonia from parts
of the tropics where mean annual temperature varied from 22.5 to 28.5°C,
no shift in temperature tolerance was apparent. This observation is quite
different from some of the fishes and indicates that long-term acclimation
is not possible in this relatively primative marine algae.
8. In the tropical and sub-tropical bays of Southeast Florida where the
energy system is benthic rather than planktonic and nektonic it appears
that most organisms including the primary producers and important members
of the food chain are living precariously close to their upper thermal
limits. Thus man's activities in the utilization of the Bay and coastal
area must be under constant surveillance and thermal modifications must be
kept to a minimum. Because of the tight thermal constrictions other adda-
tives to the marine environment must also be taken under thorough study
and quite probably sharply limited.
II-2
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SECTION II
RECOMMENDATIONS
This program was a careful laboratory study of thermal limits of impor-
tant tropical marine estuarine organisms. In general, it was seen that the
first trophic level organisms, the macro-algae had upper temperature limits
quite close to the normal summer temperatures. These limits were abrupt
with organisms living within 1°C of a lethal temperature. The several in-
vertebrates examined appeared to have somewhat higher temperature limits,
although sensitive stages in the pink shrimp and stone crab had limits near
31°C. It is recommended that temperature limits of other important food
chain organisms of which time did not permit adequate studies, be observed.
These include the Florida rock lobster, blue crab and important fishes in
both adult and larval forms. In the preceeding studies, highly interesting
behavioral patterns appeared in the near lethal temperature ranges of the
stone crab and pink shrimp. Aggressive behavioral repetoires replaced the
normal patterns and the larvae began attacking their cospecifics. Obviously,
such a phenomena could have serious consequences to an ecosystem. Further
investigation of these behavioral aspects appears highly desirable at this
time.
In summary, from our present evidence of detailed laboratory studies
of important tropical marine plants and animals, we would recommend that
water exceeding 2.0*C of summer (June through September) bay temperatures
would be detrimental to the macro-algae and some of the sensitive larval
stages. Further research on temperature limits of other invertebrates and
fishes is recommended.
II-3
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SECTION III
INTRODUCTION
To begin with it must be said that the temperature limits of tropical
estuarine organisms have had scant attention in the past. Thus, in order to
predict the effects of elevated temperatures on these organisms, either by
natural means or by man-made heated effluents, basic information is essential,
In field conditions many factors such as salinity, organic composition,
alkalinity, reduction potential and metalic concentration may be changing
simultaneously, making it difficult at least or impossible in the extreme
to separate with confidence the sole effect of elevated temperature. The
problem that has plagued the analysist has been the lack of information on
the precise point at which heat death occurs. It is not yet perfectly clear
whether the lethal temperature observed in the field is the mean temperature
over a given period of time or the highest temperature encountered and the
period of exposure to this temperature. However, for practical purposes
thermal limits can be defined.
In an effort to help overcome these problems, laboratory investigations
were conducted to examine the effect of temperature alone on the viability
of selected tropical and subtropical organisms. The experiments concerned
only those organisms at significant trophic levels, thus allowing a close
integration with readily observable field information. Investigators in
the experimental program were also involved in field studies, thus, field
data was directly related to laboratory observations and laboratory experi-
mental designs were constantly improved and directed toward important field
observations. In addition, results from the benthic trawling work aided the
laboratory studies and came to very similar conclusions. (EPA report by
Roessler WP-01351-01-A1 and DI FWPCA-18050 DFU),
II-4
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The organisms investigated were selected from the many found living in
Biscayne Bay and Card Sound. Over 18,000 individual organisms were examined
CD
in order to determine the temperature effect on their viability. Lethal
temperature limits were determined for 27 different species and life stages.
Nine species of plants, all important to the benthic community, were studied.
Also, the early life stages of two commercially important species were observed
under identical laboratory conditions, namely: the stone crab (Menippe mercen-
aria) and the pink shrimp (Penaeus duorarum). Five species of caridean shrimp,
important members of the food chain residing at an intermediate trophic level,
were also studied; they are: Tozeuma carolinense. Periclemenes americanus.
Falaemonetes intermedius. Leander tenuicornig^ and Hippolyte sp.
It is essential that the test organism to be used in the controlled
laboratory experiments be in optimum condition in order to assure that the
data is related to the effect of temperature on "healthy" organisms. Thus,
great precautions were taken to ensure that only specimens in apparently per-
fect health were used. For the algae, a preliminary laboratory study of
growth and health was conducted. The fact that growth and reproductive rates
did not differ significantly in the field and in the laboratory encourages
the belief that normal cells were being used. The crabs and pink shrimp were
obtained from the University of Miami Sea Grant mariculture facility with the
cooperation of Drs. Yang, Idyll, and Tabb.
II-5
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SECTION IV
METHODS
The Polythermostat
The basic instrument used in the controlled temperature experiments was
an aluminum bar bored to fit glass tubes, heated at one end and cooled at the
other to provide the desired temperature. Selected organisms were placed in
each tube and held at the observed temperature for the desired time and kept
under near constant surveillance. Improvements over short or long cycles,
providing aeration for adequate oxygen for animal experiments and a constant
accurate temperature readout for each tube.
Specifically, the polythermostat is a block of aluminum (6* x 3" x 9")
precision bored to fit 24 sets of 19 x 150 mm glass cuvettes. The holes
were spaced every 2.5 cm starting 24 cm from both ends of the bar. Twenty-
four 3/32 inch thermocouple fittings were also bored in the block 0.5 cm
from each tube (permitting temperature monitoring on each set of tubes);
ten holes were bored for thermometers. One end of the block was heated with
two strip heaters (750 and 400 watts) and the other end cooled by pumping a
50:50 mixture of ethylene glycol and water at -10°C through cooling fins cut
into the aluminum bar. A 55-gallon drum containing the glycol-I^O mixture
was cooled by a constant flow portable cooling unit. The mixture was pumped
through 1/4 inch copper refrigeration tubing to the cold end of the tempera-
ture gradient bar. Both ends of the bar were temperature regulated to +0.15°C
by two electric mercury thermoregulators inserted directly into the bar, one
at each end. These were, in turn, controlled by a special relay variable
transformer circuit. Recording accuracy was better than +0.05°C.
Insulation was found to be critical for maintaining the desired tempera-
ture gradient in the laboratory. Three inches of styrofoam sheeting was placed
on the bottom and sides of the bar and the entire assembly mounted in a wooden
II-6
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casing. Strips of 1/4 inch styrofoam were fitted on the top of the bar.
Laboratory airconditioning was kept at 22°C for best results.
For fluctuating temperature experiments, a tripper switch was hooked
into the circuit with the polythermostat. The switch could turn the machine
on for a given length of time and then turn off, and the chart recorder would
give the thermal history of each set of tubes during the specific time period.
Also, the amount of heat produced could be\aried and thus, the extremes of
the fluctuating temperature regime by resetting the mercury thermoregulators.
Fast and slow cycling can thus be accomplished.
Bubbling was supplied with an aquarium pump, with the air passing through
as an interconnected system of aquarium gang valves connected by plastic
tubing to disposable Pasteur pipettes. The pipettes were inserted through
corks and into the cuvettes containing the experimental organisms; penetra-
tion into the seawater was controlled. Under rates of bubbling ample to main-
tain the organisms, using this system, no temperature error or variability
was observed.
Using two or three polythermostats at the same time permitted the fine
discrimination over a large temperature range, for example one polythermo-
stat could be set from 10 - 40°C; the other from 25 - 35°C also, one broad
temperature range and one narrow finely divided one could be observed. In
short, many combinations of temperature ranges from 0 - 100°C could be selected;
therefore, the system provided a way to set up finely divided and accurate
temperature gradients for the purpose of examining the effects of both fluctu-
ating and constant temperatures on living processes.
Plant and Animal Culture
The experiments during the past year were designed to hold the organisms
at optimum conditions prior to the experiments and during the experiments at
essentially the same conditions while varying the temperature. This required
II-7
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knowledge of the culture methods and physiology of each organisms used. For
this reason, organisms on which work had already been accomplished were chosen.
Since pink shrimp and stone crabs, both important commercial species, have
been reared in the University of Miami Sea Grant facilities (Tabb and Yang,
personal communication), their tolerances were studied. The caridean shrimp
Tozeuma has been kept in culture by Ewald (1965 and 1969) and is an extremely
hardy organism. All collected caridean shrimp were handled with great care
and those demonstrating any ill effects of captivity or showing damage were
discarded.
Single cell green marine alga, Valonia. has been grown for 70 years and
its culture conditions are well known. The laboratory growth of the other
green and red species was a continuation of earlier work; the methodological
details are given in Thorhaug (1965).
Pink Shrimp (Penaeus duorarum) Culture: The basic culture methods used
in this investigation were those of Dobkin (1961) as refined by Idyll, et al
(1967). Prior to each experiment, seawater (31°/oo) was filtered through a
Whatman #50 Millipore filter and 17 ml was placed in each of 60 test tubes;
these were then put in the polythermostat to attain temperature equilibrium.
During this period air bubbling was initiated and all necessary adjustments
made to assure proper aeration. The temperature intervals chosen for the
experiments were approximately 1°C. The range for the nauplii was 10.0 to
38.0°C; for the protozoea 25.0 to 43.0°C; for the mysis 10.0 to 41.0°C; and
for the post-larvae 10.0 to 41.0°C. Replicate tests were run for each stage.
The nauplii were obtained from spawning females collected in the field.
The identification of stages of nauplii and protozoea development were taken
from Dobkin (1961). The eggs were allowed to hatch under optimum conditions
and the most active selected. The more developed stages were obtained from
specimens raised at the University of Miami Sea Grant shrimp mariculture
II-8
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facility. They were transported directly to the laboratory in oxygenated water.
Stone Crab (Menippe mercenaria) Culture; Ovigerous females of the species
M. mercenaria were kept in 10-gallon glass tanks with flowing seawater at a
salinity of 30.0°/oo and a temperature of 27°C. When the larvae were released
by the female they were collected and transferred to 4-gallon glass tanks
equipped with air stones. The water was changed daily and the salinity 30.0°/oo
and temperature (24.0°C) recorded. The larvae were fed daily with freshly
hatched Artemia from San Francisco. Following this phase of their growth which
was under the supervision of Dr. Yang of the School of Marine and Atmospheric
Science, they wer.e. transported to the experimental laboratory in 5-gallon
plastic jugs :and maintained in 15-gallon all glass aquaria. Heaters kept the
temperature at 24°C +1.0°C. The water was changed daily; the salinity during
the experimental period averaged 33.9°/oo +_ 0.20°/oo. After one day in aquaria,
ten zoea were removed with a pipette and immediately placed in a test tube
containing 17.0 ml of freshly filtered seawater with salinity of 33.9/oo.
Nineteen such tubes were than placed in the polythermostat and aerated. No
change in salinity was observed in any of the test tubes in the polythermo-
stat during a 24 hour period of monitoring. The temperature of each tube in
the polythermostat was constant to +0.10°C. The number of zoea alive in each
tube was recorded at four hour intervals. Larval stages were determined
according to Porter (1960).
Morphological Criteria of Death
Despite common notions, it is often not too easy to determine when an
organism is dead or dying; definitions are vague or non-existent. At times,
the transition from living to the dead is almost imperceptible, in other in-
stances it proceeds slowly but with noticeable clarity and in some cases, as
with the sporulation of Valonia. it is shockingly sudden. All species used
in this investigation were observed over extended periods under a variety of
H-9
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conditions and the following morphological criteria have been developed.
Halimeda; (1) A color change from deep green to pastel green to pale
yellow-green to white. All these may exist in small sections of a completely
healthy specimen but when terminal segments are dramatically lighter than
proximal ones death is indicated. (2) Individual segments crack easily.
(3) Separation of segments on slight touching or shaking of tube. (4) Loss
of turgor with a rubbery flexibility to branches, basal stalks,.and the en-
tire plant. (5) Care must be taken to note original condition of healthy
plants which may be quite pale, with individual dead or damaged terminal
segments, broken branches, etc., but with full turgor, and to individually
observe changes from this point on.
Penicillus; (1) Color change from a healthy dark green to pale green
to yellow green and then white, especially the filaments. (2) Loss of turgor
of filaments. (3) Stalk becomes rubbery and then brittle. (4) Actual decay
of plant with filaments decaying first, then interior of stipe.
Acetabularia; (1) Loss of color, change from green to white. (2) Spor-
ulation and spores released from cap. (3) Breaking away of cap from stipe
and decay of stipe.
Valonia; (1) Outright plasmolysis which is not reversible. (2) The
formation of aplanospores. (3) Separation of plasma membrane from outer
cellulose membrane forming a gap especially in medium and small cells.
(4) The development of patchy grid-like reticulations on the cell wall.
(5) Change from a dark green homogenous opaqueness or translucence to a
spotty or complete transparency. (6) Loss of positive turgor; concavity
may be introduced on the cell surface by slight pressure. The cells may
not have plasmolyzed. (7) A loss of sheen to the cell wall.
Laurencia; (1) The foremost criteria is the condition of the vege-
tative buds on the tips, including color, shape, and degree of translucency
when viewed under low power of a dissecting scope. Death caused the buds to
II-10
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become opaque and lighter in color, and to swell. (2) Secondary indications
are color changes on the stem and slightly bloated -appearance. When 50% of
the buds were dead the entire tip was called dead. Biebl (1962a) used
staining techniques and noted that the cells swelled upon thermal death.
This is interesting because Valonia cells among others shrink upon thermal
death.
Crustaceans; (1) The cessation of swimming or other characteristic
motion of a given stage (e.g. "whirring"of zoeae) after a two-minute obser-
vation. (2) Lack of movement of antennae, antennules and limbs. Occasional
twitches must be looked for after the two-minute observation. (3) Lack of
movement of appendages associated with respiration and feeding (i.e. mouth
parts). (4) No telson flexing; often the last movement of a dying specimen
are twitches of the telson. (5) Cessation of heartbeat; this readily observ-
able in nearly all larval stages and sufficiently transparent adult forms.
(6) A loss of body translucence; many larvae and some adults turn to opaque
white. (7) Gross color change; crustaceans often turn pink rapidly upon
thermal death due to carotenoid changes; this is often accompanied by an un-
natural curling of the abdomen. (8) The formation of a mucus-like shroud;
this is observed mostly in larval forms.
Molluscs; In the snail Nassarius vibex the appearance of death differs
for low and high temperatures. (1) When heated, the snails cease to cling
to the sides but instead lay in the bottom with their foot, antenna and
siphon even more extended than usual. (2) At high temperatures the foot
loses its gloss and becomes dull colored and limp and the animal doesn't
move when prodded. (3) At less extreme temperatures the animal lies on the
bottom extended from shell with''only siphon motion observable. (4) At low
temperatures death is evidenced by the animals not clinging to the walls of
the tubes and pulling back into their shells rather than extending. (5) The
operculum cover is closed and the siphon barely visible.
II-11
-------
SECTION V
RESULTS
Halimeda incrassata
An earlier investigation (Thorhaug, 1965) showed that Halimeda incrassata
could be successfully grown under laboratory conditions with rates of growth
close to those in the field. In view of this and the fact that this ubiquitous
algae is very abundant in Biscayne Bay and Card Sound it made an excellent
experimental plant. Specimens were obtained from the field and gently cleaned
to remove epiphytes and debris. The results'of three experiments indicated
that exposure of eight days at temperatures from 32.9 and 34.8°C caused death
(see Table 1). Field studies indicated that those stations at which the temp-
erature rose above average daily temperatures of 33°C or a measured mid-day
temperature of 32.6°C produced no young Halimeda and the general condition of
the algae began to deteriorate. Temperatures rose to this level in late May
and early June, 1971. When the temperature dropped below 30°C, Halimeda began
to recolonize. Earlier information obtained by Dr. Zieman was not available
at this time, but will be used in future comparisons.
Acclimation studies were attempted by holding Halimeda in a controlled
environment for two weeks. One group of plants held at 15°C had upper lethal
temperature limits between 33.2 and 34.7°C. A group held at 30°C had upper
limits between 32.6 to 34.2°C. Obviously, before valid statements on acclima-
tion can be made, one must investigate various acclimation periods ranging from
days to several generations. However, these preliminary results, coupled with
experimental data on Valonia macrophysa (to be presented later) suggest that
little if any acclimation occurs with some tropical algae; if anything those
plants held for extended periods close to their upper thermal limits have a
lower lethal limit than those held at lower temperatures. This is different
than what was seen in some fishes (Brett, 1956).
11-12
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Penicillus capitatus
The Thalassia community contained an abundance of Penicillus. Specimens
of P_. capitatus from the Florida Keys were used in five temperature tolerance
experiments that ranged from 3 to 12 days duration (see Figure 1 and Table 1).
As an additional control, Penicillus plants were held in the polythermostat
at 24°C for eight weeks; they continued to be in excellent health. Previous
laboratory studies demonstrated that specimens and their clones could be held
for a year or more (Thorhaug, 1965). The temperature tolerance experiments
showed that after eight days Penicillus kept at temperatures below 31.5°C
were all alive while those held above 34.7°C were dead. This compared well
with the field studies at Turkey Point where Penicillus was stressed or non-
healthy when the temperature in May and early June rose to 32°C. In June,
1970, 95% of the adults were dead at stations SE 1, 16, 24, 26 and 35. There
was growth renewal only after the temperatures fell below 31°C in the fall
of 1970; however, stations SE 1, 24 and 26 did not attain the previous
abundance. This observation was in agreement with laboratory experiments
which showed H. incrassata withstood temperatures slightly higher than did
Penicillus capitatus.
H-13
-------
100
75
50
0-
*
10 15 20 25 30 35 40
TEMPERATURE (°C )
FIGURE 1 Percent alive versus temperature after 8 to 10 days for Peniclllus
capitatus from the Florida Keys. Each spot represents 10 organisms.
-------
Acetabularia crenulata
Specimens were taken from the field attached to small rocks. The rhizoids
were carefully detached with needles and held for several days before use in
order to insure that the alga was not damaged in handling. When kept under care-
fully controlled conditions the plants reacted favorably to transplanting and
detaching. Thirty replicate tubes each containing five specimens of Acetabularia
crenulata were held at temperatures between 10 and 45°C. Between 38.1 and 39.1°C
the specimens were no longer able to survive. One might well expect the lethal
temperature of Acetabularia to be higher than that seen for any of the other
algae since it is an intertidal form. Such plants and animals are well known
to be very resistant to many physical stresses including temperature. These
data are summarized in Table 1.
Valonia
Since 1891 when Meyer performed the first physiological experiments with
Valonia. it has been used as an indicator of marine algal physiological proper-
ties by many investigators. This plant is a large singel-celled, tropical ben-
thic green algae found only in the marine environment and can attain a diameter
of more than 10 cm. Because of its large size, morphological observations which
indicate cell death are relatively easy. Valonia grows in Biscayne Bay, Card
Sound and in the waters of the Florida Keys as a part of the abundant green algal
community. In addition, Valonia is a member of the Order Siphonales which in-
cludes the important estuarine algal families of Caulerpaceae. Codiaceae and
Vaucheraceae. These families include most of the major macroalgae in Biscayne
Bay and Card Sound (Caulerpa, Avainvillea, Halimeda, Penicillus, Udotea, Rhipo-
cephalus, Chamaedris and Dictosphaera), hence Valonia may provide useful extra-
polation.
For these above reasons, it was decided to use Valonia as the tool to study
many of the details of thermal stress. It was most thoroughly investigated
II-15
-------
during this study and many of the findings are applicable to Penicillust Halimeda,
Acetabularia, Laurencia and even Thalassia. The understanding of the gradual pro-
cess of heat death by observing these giant cells was invaluable for comprehending
the events in this field.
One very important consideration in thermal stress studies is the ability of
and ease to which an organism can acclimate to changing conditions. In order to
investigate this, five species of Valonia from eight locations were used. The
organisms were: V. ;macrophysa1 V_. ventricosa. V_. utricularis, V_. ocellata and
V_. aegrophilia. The cells were collected from Biscayne Bay, the Florida Keys,
the Dry Tortugas in the moat at Fort Jefferson, Puerto Rico, (La Parreguera),
Jamaica, (Port Royal), Curacao, (Pescadera Baai), Bermuda, (St. Georges) and
Venezuela (Cumana) . They were flown directly to Miami and immediately used in »
the experiments. Other algae collected locally were maintained in the laboratory
under culture conditions resembling the natural habitat in an aquaria outside the
laboratory that had continuously running seawater percolating up through the sand
and rock on the bottom (Thorhaug, 1965) .
Valonia macrophysa: A number of experiments, including all the acclimation
studies, were conducted using this species. A summary of the results is given
in Figure 2 and in Table 1.
In one set of experiments different sized cells of each of three species of
Valonia (macrophysa, ventricosa and utricularis) were compared to test if there
were differences in temperature tolerances between different sizes (age) of cells
within a species. We concluded that temperature tolerance was not dependent on
cell size in any of the three species. Naturally, as in all these experiments,
encrusting growth was removed from the plants and only healthy cells were selected.
The cells in the polythermostat were observed at appropriate intervals, the light
was kept at less than one foot candle and the light-dark periods were 14 hours
and 10 hours, respectively*
11-16
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I
M
«J
100
80
60
40
20-
15
20 25 30 35 40
TEMPERATURE (°C )
45 50
FIGURE
Irreversible plasmolysis versus temperature after 3 days for
Valonla macrophysa cells from various parts of the Eastern American
tropics. Each point represents 25 to 100 cells.
-------
Two experiments were conducted using cells from Biscayne Bay and the Florida
Keys. The first consisted of two replicates of 19 sets of six cells held at tem-
peratures ranging from 7.0 to 36.6°C for a period of three days, at a salinity of
32°/oo. The cells maintained a healthy condition between 15 and 31.5°C. Irre-
versible plasmolysis occurred abruptly below 14°C and above 33.5°C. Death began
at 15°C and 31.5°C. A similar experiment conducted at a lowered salinity (25°/oo)
gave the same thermal tolerance limits.
Another experiment used 16 cells held at 30 different temperatures ranging
from 8.0 to 38.2°C for a period of five days. The temperature interval was 1.1°C,
a more closely-spaced interval than used in the previous experiment. During the
first 24 hours all cells remained healthy; after 48 hours of exposure above 31.5°C
they began to show distress. On the third day, complete irreversible plasmolysis
occurred below 15°C and above 33.5°C, partial mortality took place at 13.3 and
31.5°C. No change occurred over a two week period.
To determine if acclimation due to long-term growth at various temperature
regimes in the tropics would cause the thermal limits to change, experiments were
conducted utilizing V_. macrophysa from Puerto Rico where the mean water tempera-
ture was 28.5°C. Nineteen sets of 24 cells each were held at temperatures rang-
ing from 7.9 to 38.1°C for 72 hours. Almost all the cells had undergone irre-
versible plasmolysis at temperatures below 15.6C and above 29.7°C. Partial
mortality was observed between 14.6 and 15.6°C and between 29.7 and 30.7°C. All
cells appeared healthy at the intermediate temperatures.
Since acclimation at the warmer temperatures of Puerto Rico did not affect
the lethal limits of Valonia the effects of acclimation on cells living in a
cooler area were tested using Bermuda specimens where the mean annual temperature
was 22.6°C. In one polythermostat, sets of 16 cells each were held at tempera-
tures ranging from 8.0 to 38.0°C at intervals of 1.5°C. The results showed that
11-18
-------
below 13.9°C and above 33.6°C, all the cells died after three days. In the second
polythermostat 30 sets of 13 cells each were placed at 0.33°C intervals between
24 and 34°C. The results show that between 32.0 and 32.6°C more than 50% of the
cells died after three days.
Cells from the Dry Tortugas, located at the tip of the Florida Keys and close
to Yucutan, where the mean annual temperature is 27.0°C, were examined. In one
polythermostat 19 sets of 12 cells each were held between 9.8 and 36.8°C. After
five days death occurred in all cells held below 12.3°C and above 32.8°C. In a
second trial, four sets of 10 cells were tested in the range of 29.3 to 32.8°C.
Between 31.3 and 31.6°C more than 50% of the cells died after five days.
Two final acclimation experiments were run using specimens from Biscayne
Bay. Over 500 cells were held for 10 and 14 days at 30 and 15°C. Subsequently,
they were jlaced in a polythermostat and held for 160 hours. One cell of each
sample survived at 33.4°C; all cells died above this temperature. At lower
temperatures, 14% or less mortality occurred in the 30°C acclimated cells with
1% or less occurring in the cell acclimated at 15°C. The critical interval was
32.3 to 33.4°C for those acclimated at 30°C and 33.4 to 34.5°C for those at
15°C. It should be observed that those cells held for extended periods (acclim-
ated) at the higher temperature not only had a lower upper thermal limit but
also had a much higher mortality at "normal" or "optimal" temperatures. This
observation matched that found with the Valonia from Puerto Rico where the mean
annual temperature was 28.5°C, the highest for all specimens examined. These
results were remarkably close to those using "non-acclimated cells" and suggested
that the algal thermal limit was very closely confined with little possibility
for acclimation. This, of course, is in variance with what is known about
bacteria and fishes. In addition, it strongly indicated that although the thermal
limit appeared abruptly, the organisms were under severe thermal stress at
temperatures below the death point and that exposure to slightly higher temperatures
for short periods will prove fatal.
11-19
-------
Valonia utricularis; Comparative experiments were conducted utilizing
V,. utricularis specimens from two locations, Bermuda and the Florida Keys near
Miami. Two sets of 13 cells from Bermuda were held at 30 different temperatures
ranging from 8.6 to 37.1°C for five days. The results showed that those cells
exposed to temperatures below 13°C and above 31.0°C died within three days.
For the Florida Keys specimens, 30 sets of 10 cells each were held at tempera-
tures ranging from 26.6 to 32.7°C. Within the range of 31.0 to 31.4°C there
was over 50% mortality after five days (see Figure 3 and Table 1). The
similarity of temperature tolerance for cells from the two areas is obvious;
there is also good agreement with the thermal tolerance of V_. macrophysa.
Valonia yentricosa; Specimens of this third species from the Florida Keys,
Curacao and Jamaica were examined; the results are shown in Figure 4 and
Table 1. For the Florida Keys specimens, 19 sets of six cells each were
held at temperature intervals between 7.7 and 38.9°C. After three days of expos-
ure, over 50% of the cells underwent irreversible plasmolysis below 14.3°C and
above 33.0°C. Cells from Curacao, where the mean annual temperature is 24.5°C,
were held between 9.7 and 36.9°C in 19 groups of six each. The cells were unable
to survive a six day exposure below 12.1°C and above 31.5°C. Three additional
trials using Curacao cells showed that this species had a lower tolerance limit
of 14.5°C and an upper limit of 33.0°C with death beginning at 31.5°C. Cells
from Cumana, Venezuela had a 100% mortality below 15.5°C after five days. The
upper critical limit was between 29.1 and 31.9°C.
Nineteen sets of 17 cells each collected in Jamaican water, where the mean
annual temperature is 27.4°C, were held between 9.7 and 37.0°C. The results
showed that below 12.2°C and above 31.5°C more than 50£ of the cells were unable
to survive after five days. Irreversible plasmolysis began to take place at
13.8 and 29.9 °C; cells held between 23 and 26°C for a period of three weeks
11-20
-------
V
N3
CO
O
<0
#
Uj
•5
^
KJ
ki
^
0
25
50
75
100
4
_ «e • • •• •
— . A m
• • *
o •
•
^•^•w
M^lV
IM^*
g ^5
te
FIGURE
30 32
TEMPERATURE (°C )
Irreversible plasmolysis versus temperature for VaIonia
after 5 days exposure to the given temperature. Each
25 cells.
-------
H
N>
NJ
100
80
60
40
20-
0
15
20 25 30 35 40
TEMPERATURE (°C )
45 50
FIGURE
Percentage survival after 3 days for Valonla ventricosa cells from
various parts of the Eastern American tropics. Each point represents
25 to 100 cells.
-------
remained healthy. These limits are very similar to those found for V_. ventricosa
for Curacao and only tenths of a degree from the Florida Keys specimens. The
striking similarity of the upper death limits of the V_. macrophysa. V_. ventricosa
and V_. utricularis is also obvious.
Valonia ocellata; Cells from the Florida Keys were tested over the tempera-
ture range of 8.1 to 40°C. After a three day exposure to temperature below 14.7
or above 34.0°C all cells died; those from Curacao had a very similar limit of
34.6°C. Temperature intolerance began at 32.8°C (see Table 1).
Valonia aegrophilia; This is a very small, relatively rare species collected
from the Dry Tortugas. Nineteen sets of 32 cells each were held between 9.5 and
37.0°C. After three days cells survived ceased below 10.5 and above 33.0°C. The
cells began to die at 12.0 and 31.4°C (see Table 1).
Laurencia
The red algae, Laurencia poitei. is found in many tropical and subtropical
waters and is a dominant species in Biscayne Bay and Card Sound. It exists in
non-attached clumps of single strands and masses which move freely with the tide
and current except when caught on projections of the bottom. It is not known
whether herbivores use it directly as food but it does form a significant portion
of the biomass and thus is a major contributor to the bottom detritus. In addi-
tion, it provides a substrate for many algae and sessile animals as well as
shelter for small fish, polychaetes, molluscs and crustaceans. The color of the
plant ranges from a light yellow to a dark purple-red; in summer it tends towards
lighter colors.
The algae were collected by hand from the Card Sound and brought back in
large plastic containers equipped with aeration systems. Debris, animals, and
other foreign matter were gently removed by mechanical cleaning in running sea-
water. The plants were held in five gallon glass tanks, the water was changed
every two days and the salinity, pH, and temperature recorded.
II-23
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A plant tip (6 to 10 cm) was placed in each of 48 cuvettes containing 20 ml
of filtered seawater; the salinity, pH, and appearance of the tip were noted.
The temperature gradient used was from 6 to 45°C for a period of 10 days. The
tips were wxamined daily and the water replaced with water of the same temperature.
Three trials showed that at the end of 10 days more than 80% of the cells held
below 30.1°C were healthy; even those held at 6.3°C were alive. At temperatures
from 31.7 to 33.3°C less than 40% of the tips were living and above 34.9°C all
were dead. Due to the difficulty in establishing indications of the morphological
death point, the upper tolerance can only be expressed as a range of 31.7 to
34.9°C. This information is presented in Figure 5 and Table 1 and agrees
with field data which indicates that no healthy Laurencia occurred above a temp-
erature averaging 33°C for 10 or more days. The benthic biology studies show
that the animal populations closely associated with Laurencia became less abun-
dant after sustained periods with average daily temperatures in excess of 33°C.
In addition, these values agree with Biebl (1962) for Laurencia poitei held at
32 to 35°C for 12 hours.
Pink Shrimp^
The pink shrimp, Penaeus duQjrarum, is a major member of the animal community
of Biscayne Bay and forms one of the most important commercial fisheries in
Florida. Juvenile shrimp leave the area and migrate to the spawning grounds,
the offspring return as four spine larvae, settle to the bottom and grow to
juveniles. Fresh larvae from three different spawning periods were used in
these experiments. Nine individuals were placed in each tube for the nauplii
experiment, 10 for the protozoea, six for the mysis and 25 to 40 for the post-
larval experiments. The data is summarized in Table 1.
11-24
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V
NJ
100
80
60
40-
20-
0-
15
20
25 30
35
40
45 50
TEMPERATURE (°C )
FIGURE
Percentage survival for Laurencia poitei after 10 days exposure to
the given temperature. Each point represents 7 plants.
-------
Nauplii: Over 500 individuals were used in this experiment and exposed to
a temperature range of 10 to 38.3°C; the nauplii held below 15°C and above 37°C
for 12 hours were unable to survive. Abnormally vigorous swimming activity was
observed at 35°C; this may be a response to stress condition. After 18 hours
the temperature tolerance was lower, with one exception 50% of the nauplii or
less survived at 33.0°C and under 15°C. Between 15 and 23°C many individuals
rested on the bottom of the tubes and required prodding to elicit movement.
Between 24 and 30°C the organisms swam actively and remained in a healthy
condition.
The nauplii metamorphosed into protozoea only between 25.0 and 31.5°C. In
the temperature range of 23.0 to 32.6°C the shrimp attained the fifth nauplii
stage, above and below these temperatures development was negligible; the ability
to develop decreased more rapidly at the hot temperature. This has been dis-
cussed in detail by Thorhaug (1970).
First Protozoea: Nineteen tubes, each containing 10 individuals, were
held in a temperature gradient of 28 to 42°C for 18 hours. All specimens held
above 37.6°C,died; death became evident at 37.0°C.
Third Protozoea: Nineteen groups of organisms were subjected to a temper-
ature range of 12 to 43°C. After 17 hours all specimens kept above 37.7°C were
dead. Exposure for 22 hours above 36.7°C caused total mortality; death to
individuals began at 35.7°C.
Third Mysis: Nineteen sets of third mysis stage were held in a temperature
range of 12.6 to 42.0°C. After 20 hours all specimens held above 36.9°C were
dead, while all those below 35.9°C were alive. All individuals remained in the
third mysis stage. After 34 hours 100% mortality occurred above 36.9°C, thus
preventing development to the first post-larval stage. Death was first observed
at 34.9°C.
11-26
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First Posit-Larvae; Nineteen groups of first post-larvae were held between
temperatures of 12.6 to 47.0°C for 25 hours. All specimens kept above 37.8°C
were dead; all those retained below 33.5°C survived. Survival of 60-70% was
attained at 34.9 to 36.3°C.
Second Post-Larvae; Two experiments were conducted; in the first, seven
tubes each containing 22 second pest-larvae were subjected to a temperature
range of 36.7 to 41.0°C. After 45 minutes all specimens kept above and at
39.1°C died while the shrimp at 38.5eC and below had 100% survival. An exposure
of 128 minutes proved fatal to all individuals maintained at and above 38.5°C,
while those at and below 37.9°C remained alive. In the second experiment, 30
post-larvae were placed in seven tubes ranging from 36.7 to 41.0°C. Temperatures
lethal to all individuals were 38.5 and 33.0°C, for exposures of 30 minutes and
2.5 hours respectively. All post-larvae survived 37.2°C.
Juvenile; Young shrimp were aquired from an autumn brood hatched at the
University of Miami Sea Grant mariculture facility at Turkey Point. Because of
the large size of the shrimp, only one individual was put in each cuvette. The
critical temperature for a two hour exposure was 37.9 to 39.6*?C; decreasing to a
range of 36.0 to 37.9°C after 16.5 to 24 hours. All specimens kept above 40.7°C
died within five minutes and the lower temperature limit has been set tentatively
at 12.8°C but this requires further investigation.
Stone Crab
s
The stone crab, Menippe mercenaria. a shallow water burrowing organism, is
a significant contributor to the commercial fisheries of Biscayne Bay. The female
carries a spongy mass of eggs which, upon hatching, become planktonic. Larvae
settle to the bottom in the megalops stage and thence grow to adults. In addi-
tion 1-9 comprising a fishery in themselves, they are part of the food chain for
many fish. A series of experiments using the polythermostat were conducted using
11-27
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eggs; first, second and third zoea; megalops and juvenile stages. The results
are tabulated in Table 1.
Eggs: In the first of a series of experiments 19 tubes containing six
eggs each were held at temperatures ranging from 10 to 50°C. After 40 hours of
exposure at 29.1 to 36.3°C hatching into the first zoeal stage was observed.
No hatching occurred below 29.1°C and the eggs failed to survive above 36.3°C.
In the temperature range of 23.4 to 3j6.3°C normal development of the eggs to
the first zoea was observed after 70 to 90 hours exposure. The eggs appeared
to be tolerant of cold as they remained alive after 280 hours at 12.6°C.
First Zoea; Over 315 individuals, were examined over a temperature range
of 29.0 to 50.0°C for 36 hours (See Figure 6). After 20 minutes all individuals
were dead above 42.8°C; those kept below 40.3°C remained alive. A two hour
exposure lowered the upper lethal limit; all zoea above 40.3°C died; 39.1°C
proved compatible. The downward trend continued as expected after a 3.5 hour
period; no zoea were alive above 38.0°C while those kept below 34.5°C were actively
swimming. After 36 hours the lethal temperature decreased to 36.7°C; 100% re-
mained alive under 34.5°C as was the case after only 3.5 hours. A second experi-
ment of nine hours duration showed that temperatures above 37.8°C were fatal
while 33S0°C was tolerated for the same time period; the 50% survival point was
*'•*•••".
34.8°C. A compilation of all data indicates that the upper lethal temperature
for Menipj)^ first zoea ranged from 36.1 to 37.4°C.
Second Zoea; Four replicates of 19 tubes with 10 individuals per tube held
for 13 hours over a range of 25 to 46°C demonstrated the same general trend as
shown for the first zoea. After four minutes all individuals died above 43.8°C;
below 42.8°C life was sustained. Twenty-six minutes later the lethal temperature
was lowered by 1°C. Temperatures over 38.5 and 37.5°C were fatal to all indivi-
duals after 3.25 hours and 13 hours respectively; life was sustained at a temp-
erature of 1°C lower. A later 22 hour experiment showed that the second zoea
11-28
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M
CO
VO
100
80
60
40
20-
0-
FIGURE
15
20 25 30 35 40 45 50
TEMPERATURE (°C )
Percentage survival of Menippe mercenarla first zoea after twenty-
four hour survival versus temperature. Each point represents 20
crabs.
-------
could not tolerate temperatures above 37.3°C for such an extended period; while
about 90% could take less than 36.1°C. The 50% survival point was between 36.1
and 37.3°C, almost identical with the first zoea experiment.
Third Zoea; Nineteen tubes each containing 10 third zoea were subjected to
temperatures ranging from 28.0 to 46.0°C for 23 hours. After five hours no zoea
were alive above 37.6°C; survival was 80% at 36.9°C and 100% at 32.9°C. After
23 hours no third zoea remained alive above 36.7°C. During these experiments
an increase in activity apparently related to increased temperatures was noted.
Temperatures slightly below the lethal level appeared to increase cannibalistic
activity but further behavioral observations are required to define the signifi-
cance of this phenomenon.
Megalops; This stage is by far the most difficult one to raise and to
handle experimentally. Although few specimens were observed, the data is quite"
impressive (see Figure 7). The first experiment lasted for 24 hours; fifth
zoea were placed in the polythermostat tubes just prior to metamorphosis into
megalops. Those individuals maintained in the temperature range of 16.7 to 30.5°C
achieved the megalops characteristics. An additional experiment showed that 23
hours above 30.5°C was lethal. It is not known whether death was due directly
to temperature or indirectly because the elevated temperature prevented meta-
morphis.
Juvenile: One experiment was performed using a brood of ft'lly metamorphosed
juvenile M. mercenaria. placed singly in cuvettes to avoid damage from hostile
behavior. One-hundred percent survival was maintained between 12.6 and 37.0°C
over a period of 42 hours. Death occurred at 42.7°C within 15 minutes, 41.3°C
within 29 minutes, 40.3°C within 44 minutes and at 38.0°C after four hours of
exposure.
H-30
-------
too
80
60
3 40
20
0
FIGURE
5 10 15 20 25 30 35 40 45 50
TEMPERATURE °C
7 Percent alive versus temperature after 24 hours for Menippe mercenaria
megalopa stage. Each dot represents 2 specimens.
-------
Caridean Shrimp
As adults these shrimps comprise part of the benthos of Biscayne Bay and as
larvae they are important members of the planktonic community. They are reported
to live in the Thalassia community which includes macroalgae such as Halimeda,
Penicillus and Laurencia. These small shrimp are important members of the food
chain, they are eaten by stone crabs and young fish, especially the sciaenids
such as sea trout, red drum and silver perch. The results of these experiments
are summarized in Table 1.
Tozeuma carolenenses: The taxonomy, distribution and ecology of this shrimp
treated by Ewald (1969). In an experiment with mostly gravid specimens, those
kept at 39.5°C were killed after 20 minutes; after four hours 100% mortality was
observed at 33.9°C and above. After 48 hours the critical temperature decreased
to 32.8°C. In an additional test, all specimens kept at and above 38.7°C died
in less than 18 minutes, while after 24.5 hours all shrimp held at 34.3°C were
dead. After 128 hours exposure the critical temperature was 32.8°C. Another set
of experiments with a 72 hour duration showed that after 47 hours all specimens
held at temperatures above 33.9°C were dead and only 14% survived between 30.6
and 32.3°C. After 72 hours the critical temperature was the same but stress
was evident at lower temperatures. Less than 50% survival was recorded between
29.0 and 32.3°C while below 29.0°C at least 80% of the shrimp survived.
Palaemonetes intermedius; Mature females were obtained from Matheson Hammock
Beach during April, May, June and July. They were placed carefully in a 30 gallon
plastic container supplied with air from a portable air pump and transported to
the laboratory. Before being used as test specimens, they were maintained in a
15 gallon all glass aquaria for 24 hours at 25CC. Control organisms lived for
more than two weeks at 25°C. Five experiments were performed, each utilized 19
sets of tubes containing two individuals held at temperatures varying from 10.0
to 45.0°C. All animals kept below 36.2°C survived; those above 37.8°C died. No
difference in temperature tolerance was observed between the April and July
TT_oo
specimens. •"
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Periclimenes species: These shrimp were obtained in Thalassia beds at
Bear Cut; the collecting and holding procedures were similar to those used for
P_. intermedius. The shrimp were conditioned at a salinity of 36.2°/oo over a
temperature range of 24.0 to 26.0°C and were fed one Oppenheimer pellet per
shrimp per day. Under these conditions the control animals lived more than two
weeks. R>r the experiment one shrimp was placed in each of the 38 polythermo-
stat tubes; the temperature range was 1.0 to 45.0°C at 2°C intervals. This
experiment was replicated 15 times with freshly obtained specimens in order to
obtain statistical significance. The results showed that the animals lived
adequately between 14.0 and 35.0°C, but 100% mortality occurred below 14.0°C
and above 37.6°C. No difference was observed in the thermal tolerances be-
tween individuals collected in April and those in July (see Figure 8).
Hippolyte: This is a hitherto undescribed species existing in Biscayne
Bay and adjacent waters. Gravid females were collected and handled as de-
scribed earlier. One experiment showed an upper tolerance limit of 35.5°C after
one hour and 32.1°C after 5.5 hours. In another run there was 100% mortality
in shrimp kept at temperatures above 34.7°C after 90 minutes. An upper critical
level was noted at 32.8°C after 48 hours and did not change for the remainder
of the five day experiment. A lower critical level of 10.0°C was observed but
only after five days exposure.
Leander tenuicornis; This is a robust, predatory caridean shrimp. Held
at temperatures above 38.7°C all individuals died within 15 minutes, those held
above 35.5°C were dead within 5.5 hours. Thus Leander may be more temperature
tolerant than Hippolyte sp.
Molluscs
The small snail, Nassarius vibex is an important part of the intertidal com-
munity. It is a saprotroph, feeds on dead animal tissue, and is equipped with a
chemosensory apparatus which enables the sensing of food at great distances. It
11-33
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i
OJ
700-
80
60
40
20
0
0 5 10 15 20 25 30 35 40 45
TEMPERATURE °C
FIGURE 8 Percentage survival versus temperature for Perlclimenes sp. after
168 hours exposure. Each point represents 6 organisms.
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exists between the high and low tide marks on mud or other suitably soft substrates
and spends its time with the shell just beneath the mud and the siphon projecting
upward into the water. This species would be expected to have a higher tempera-
ture tolerance than open water organisms due to its intertidal adaptation (Newell,
1970).
The snails were collected in mud flat tidal pools at low tide just to the
north of the Miami Seaquarium, placed in a mud bottom holding tank and observed
for several days. Specimens were then put in cuvettes with 22 mis of filtered
seawater (changed twice daily). After one hour in the polythermostat all
Nassarius held above 46.7°C were dead. All specimens held above 40.2°C for 24
hours and 37.5°C for 72 hours expired; the lower limit was 8.0°C. Some signs of
stress were observed above 32.2°C; however, upon reducing the temperature they
became vigorous. This information is similar to that found by Professor H. Moore.
11-35
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TABLE 1
UPPER TEMPERATURE LIMITS OF SELECTED TROPICAL
ESTUARINE ORGANISMS IN THE LABORATORY
Time of
Organism Exposure
I. Plants
1. Halimeda incrassata 8 days
acclimated 15°C 15 days
acclimated 30°C 15 days
2. Penicillus capitatus 8 days
3. Acetabularia crenulata 4 days
4. Valonia ventricosa 72 hrs.
5. V_. macrophysa 42 hrs.
acclimated 30°C 72 hrs.
acclimated 15°C 72 hrs.
6. _V. utricularis 120 hrs.
7. J£. aegrophilia 72 hrs.
8. V. ocellata 72 hrs.
9. Laurencia poitei 10 days
Upper Lethal
Limit in °C
32.9 -
34,
32,
31.5 -
38
30.0
32.0
32.6
33
31.0
31.4
32.8
31.7
1 -
2 -
34.8
36.6
34.6
34.
39.
31.
.7
.1
.5
- 33.6
34.2
34.7
31.4
33.0
34.0
34.9
No. of
Organisms
152
41
40
159
600
9725*
144
II. Invertebrate Larvae
Penaeus duorarum nauplii
P^. duorarum 1st protozoea
]P. duorarum 3rd protozoea
P_. duorarum 3rd mysis
jP. duoraruro 1st postlarvae
P_. duorarum late juvenile
Menippe mercenaria eggs
ti. aercenaria 1st zoea
M. mercenaria 2nd zoea
M. mercenaria 5th zoea
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
12.
£L fflercenaria megalopa
M. mercenaria zoea/mega.
M. aercenaria mega./juve.
22 hrs.
18 hrs.
17 hrs.
72 hrs.
1 hr.
40 hrs.
40 hrs.
24 hrs.
91 hrs.
44 hrs.
16 hrs.
24 hrs.
24 hrs.
30.5
36.0
36.8
36.8
37.9
36.3
36.3
34.4
33.1
34.7
36.0
30.5
28.9
- 31.5
- 37.6
- 37.8
- 37.8
- 40.7
- 38.5
- 38.5
- 36.0
- 34.2
- 35.5
- 37.0
- 31.4
- 30.5
2159
3886
III. Caridean Shrimp
1. Tozeuma carolenensis 72 hrs.
2. Palaemonetes intermedius 72 hrs.
3. Paraclimenes sp. 168 hrs.
4. Hippolyte sp. 48 hrs.
5. Leander tenuicornis 24 hrs.
32.3
36.2
36.1
31.0
34.4
33.9
37.8
37.6
32.8
35.5
768
570
190
66
24
IV. Mollusca (intertidal)
1. Nassarius vibex
72 hrs.
37.5 - 40.2
48
*Valonia of all species
TOTAL
18,572
11-36
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TEMPERATURE (°C)
28 29 30 31 32 33 34 35 36 37 38 39 40
Plants
Halimeda inerassata
acclimated 15°C
acclimated 30°C
Penicillus capitatus
Acetabularia crenulata
Valonia ventricosa
y_, macrophys a
acclimated 30°C
acclimated 15°C
V. utricularis
V_. aegrophilia
_V. ocellata
Laurencia poitei
Invertebrate Larvae
Penaeus duorarum nauplii
P_. duorarum 1st protozoea
P_. duorarum 3rd protozoea
P_. duorarum 3rd mysis
£. duorarum 1st postlarvae
P_. duorarum late juvenile
Henippe mercenaria eggs
M. mercenaria 1st zoea
|J. mercenaria 2nd zoea
M. mercenaria 5th zoea
M. mercenaria megalopa
M. mercenaria zoea/megalopa
M. mercenaria megalopa/juvenile
Carldean Shrimp
Tozeuma carolenensis
Palaemonetes^ interroedlus
Paraclimenes sp.
Hippolyte sp.
Leander tenuicornis
Mollusca (intertidal)
Nassarius vibex
FIGURE
28 29 30 31 32 33 34 35 36 37 38 39 40
Upper temperature limits of selected tropical estuarine organisms
in laboratory investigations. Black line indicates near 100%
survival, dotted line indicates interval to near complete mortality,
See Table XI-1 for details of time and numbers of test organisms.
11-37
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SECTION VI
ACKNOWLEDGMENTS
The preceeding laboratory results were accomplished with the help of
Dr. Thomas Devany, Dr. Marcela Fernandez, and Mr. Steven Bach. The larval
specimens obtained from Drs. Yang, Idyll and Tabb were invaluable in the
success of that aspect of the program. The benthic animal field work of
Dr. Martin Roessler was a constantly valuable influence for comparative
study. Many conferences with Dr. Roessler are gratefully acknowledged.
Likewise conferences on the benthic field program of Mr. Lee Purkinson
(FWQA-EPA) were deeply appreciated. Dr. Hilary Moore's laboratory studies
(FWQA [DIWP-01433]) on the thermal and salinity limits of selected Biscayne
Bay invertebrates were useful points of comparison.
The support of this project by the Water Quality Office,
Environmental Protection Agency and advice provided by Dr. C. Hegre, Grant
Project Officer, as well as Dr. J. Praeger, is acknowledged with sincere
thanks.
This work was partially supported by the U. S. Atomic Energy Commission.
11-38
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PUBLICATIONS AND PATENTS
1. Thorhaug, A., "Temperature effects on the membrane potential of Valonia."
Third International Congress of Biophysics. Boston (1969).
2. Thorhaug, A., "Temperature limits of five species of Valonia." Journal
of Phycology. 6, pp 27 (1970).
3. Thorhaug, A., "Temperature effects on Valonia bioelectric potential,"
Biochem. et Biophys. Acta.
4. Thorhaug, A., "A temperature controlled perfusion technique for a single
cell marine alga," Proc. First European Biophys. Congress (1971)
5. Thorhaug, A., T. Devany and B. Murphy, "Refining shrimp culture methods:
the effect of temperature." Proc. Gulf Carib. Fish. Instit., 23, pp 31-38.
6. Thorhaug, A. and A. Katchalsky, "The role of thermoosmosis on marine
macroalgae." Proc. VII International Seaweed Symposium (in press) (1972).
7. Thorhaug, A., T. Devany and S. Pepper, "The effect of temperature on
Penicillus capitatus survival in laboratory and field investigations."
J. Phycol.. 7, pp 5-6 (1971).
8. Thorhaug, A., K. F. Kellar and S. A. Bach, "Temperature tolerance of
several important marine crustaceans." Florida Academy of Science (1972).
Additional publications not sponsored by EPA, but adding to the biological
literature of thermal effects in Biscayne Bay - Card Sound, Florida.
9. Thorhaug, A., R. G. Bader and M. Roessler, "Thermal effects on a tropical
marine estuary." FAO Symposium on Marine Pollution. Rome. (1970).
10. Thorhaug, A., R. Stearns, "A field study of marine grasses in a tropical
marine estuary before and after heated effluents." Amer. Jr. Botany
65(5):412-413 (1971).
11. Thorhaug, A., R. D. Stearns, "A preliminary field and laboratory study of
physiological aspects of growth and reproduction of Thalassia testudinum."
Am. J. Bot. 59: 670 (1972).
12. Thorhaug, A., J. Garcia-Gomez, "Preliminary laboratory and field growth
studies of the Laurencia complex." J. Phycol. 8(S):10.
13. Thorhaug, A., K. F. Kellar, "Laboratory and field growth studies of four
green calcareous algae. I. Preliminary results." J. Phycol. 8(S):10.
14. Thorhaug, A., T. H. Thorhaug, "The diving botanist." Oceans 6(l):73-76.
(1972).
15; Thorhaug, A., Marcela Fernandez, "Bioelectric potential measurements of
living membranes as indicators of thermal pollution." Proc. J. Electro-
chem. Soc. (1972).
11-39
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16. 1972 (with D. W. Hood, E. Kelley, et al.) "Contamination and coastal
pollution." pp. 146-186. IN: The Water's Edge: Critical Problems of
the Coastal Zone. B. H. Ketchem (ed.) M.I.T. Press, Cambridge.
17. 1972 (with J. Armstrong, at al^.) "Coastal management and planning."
pp 246-304. IN: The Water's Edge: Critical Problems of the Coastal
Zone. B. H. Ketchem (ed.) M.I.T. Press, Cambridge.
IN PRESS;
18. (with R. D. Stearns and S. Pepper) "Effect of heat on Thalassia
testudinum in Biscayne Bay." Florida Academy of Science.
19. (with K. F. Kellar and S. A. Bach) "Temperature tolerance of several
important marine crustaceans." Florida Academy of Science.
20. (with R. Stearns) "Preliminary field observations on the sexual repro-
duction stages of Thalassia testudinum in south Biscayne Bay and Card
Sound, Florida."
21. "Stress responses in coastal marine organisms." IN; B. Ketchem (ed.)
Critical Problems of the Marine Coastal Zone. M.I.T. Press, Cambridge.
22. (with Robert D. Stearns) "An ecological study of Thalassia testudinum
in unstressed and thermally stressed estuaries." Ecology.
23. "Ecological investigations of the macroalgae in Biscayne Bay and Card
Sound, Florida." I. Preliminary investigation of the red algal complex.
J. Phycol.
24. "Ecological investigations of the macroalgae in Biscayne Bay and Card
Sound, Florida." II. Preliminary investigation of the green algae.
J. Phycol.
25. "The effect of temperature on the grasses and macroalgae." IN; An
Ecological Study of South Biscayne Bay and Card Sound, Florida. R. G.
Bader and M. A. Roessler (eds.) Univ. Press.
26. (with R. Stearns) "A comparison of Thalassia testudinum populations
in an estuary before and after the opening of a thermal effluent."
Am. J. Bot. 60.
27. (with S. D. Bach) "Production of important green and red benthic
macrophytes in an estuary before and after the opening of a thermal
effluent canal." J. Phycol. 9(5).
28. (with M. Fernandez) "The effect of various temperature gradients on
the flux of water through a Valonia membrane system. J. Phycol. 9(5).
29. (with R. G. Bader, M. A. Roessler, et_ al^) "The environmental impact
of a power plant on a subtropical bay." Trans. Am. Nuclear Soc.
30. (with G. Voss) "The effect of thermal effluents on the biology of
Biscayne Bay and Card Sound, Florida."
11-40 *US. GOVERNMENT PRINTING OFFICE: W4 S46-319/393 '1-3
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
$* Report It a.
W
4. Title
"BIOLOGICALLY ALLOWABLE THERMAL POLLUTION LIMITS1'
7. Author.(s)
W. Drost-Hansen and Anitra Thorhaug
$. Organization
University of Miami, Coral Gables, Fla.
5. Report Date
6.
8. Performing Qrg*nit»trQn
Report ft a.
lit. /!,)•'£ . ,".' J
EPA 18050 DET
n.
13.
EPA 18050 DET
i«*? THJP'T^V
Period Covered
32,
Or giuif ?
is. supplementary Notes Environmental Protection Agency report
Number EPA-660/3-74-003, May 1974
16. Abstract Literature -and theoretical studies have demonstrated the likely existence of
critical thermal transition regions for biological activity. Highly nonlinear thermal
effects, appear;to be manifestations of higher-order phase transitions most likely in the
vicinal water of the cellular systems.The effects are likelyinvariants in time and space
rhus, the corresponding critical temperature regions may represent absolute, upper per-
missible thermal pollution limits .Laboratory experiments, using some 18,000 individuals
have yielded the most accurate thermal tolerances to date for marine estuarine organisms
(Including macro-algae and larval stages of Important food-chain organisms). Gaussian (or
skewed-Gaussian) curves for lethal thermal limits;were not: observed. Instead an abrupt
death point occurred, often within an interval of 0.5 to 1°C. The temperature tolerances
obtained in the laboratory conformed closely to those observed in the field. Thus upper
limits found in the laboratory for Halimeda. Penicillus. and Valonia were found to be the
thermal limits in the field.At Turkey Point, these plants disappeared above the thermal
limits. The upper temperature limit for many of the plants examined, as well as the sensi-
tive stage of the pink shrimp, crab megalops (fedseveral carideans, was 31 to 33°C..Thls
critical temperature region is within 1 to 3° C of mean mid-summer temperatures sub-
stantiating the'hypothesis that tropical marine organisms live closer to their upper le-
thal limit than do either temperate or Arctic species. '
17a. Descriptors •
^Environmental Effects, *Molecular structure, *Physico-chemical properties, ^Temperature,
*Thermal properties, *Water, *Water structure, Ecology, Pood-chain organisms.
17b. Identifiers '
*Aqueous solutions, *Growth rate, *Heat resistance, interfaces. Colloids, Electrolytes,
Enthalpy, Entropy, Enzymes, Hydration, Hydrogen bonding, Lipids, Membranes, Metabolism,
Proteins, Halimeda, Penicillus. Valonia. pink Shrimp, crab megalobs.
17c. CO WRR Field^ Group 05C .
Id.
Send To:
A fiftractor \J. Droflt—Hannen
nztitutian
University of Miami, Coral Gables, Fla.
WRS'C" 'C>«! ;ftfv JIINF 1871)
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