Journal of Thermal Biology 31
i
ler
, M
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Abstract
two different experimental methods. In the ??static meth-
od??, the temperature that causes 50% mortality is
determined from a plot of percent mortality at static
ture is increased or decreased gradually until a critical
levels of thermal tolerance is the rate at which temperature
is changed in the experiments. Rapid rates can produce
long lag times between environmental and body tempera-
ARTICLE IN PRESSture, which can overestimate thermal tolerance but may
also lead to variations in thermal tolerance in relation to
body size (Becker and Genoway, 1979; Lutterschmidt and
0306-4565/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jtherbio.2006.01.005

Corresponding author. Fax: +649 422 6113.
E-mail address: moracamilo@hotmail.com (C. Mora).The experimental determination of the thermal tolerance
of species is a common practice in the ?eld of thermal
biology. This is motivated not only by our interest in
understanding species? thermal physiology but also by our
current need to assess the biological effects of different
thermal phenomena such as climate change, El Nin?o, La
Nin?a, overwinters, etc. (Glynn and D?Croz, 1990; Bennett
et al., 1997; Addo et al., 2000; Beitinger et al., 2000; Mora
and Ospina, 2001, 2002; Kimball et al., 2004; Kimura,
2004). Thermal tolerance is commonly quanti?ed through
point is reached (e.g. equilibrium lost, death). In the
dynamic method the critical thermal maximum (CTMax)
or minimum is quanti?ed as the mean temperature at
which individual ?sh reach such critical points. The
dynamic method has been more broadly used because it
is easier to use, requires fewer animals, provide quick data
(Lutterschmidt and Hutchison, 1997) and its results are
more comparable to natural conditions (Bennett and Judd,
1992; see also Mora and Ospina, 2001).
One problem of using the dynamic method to determineor death. Here we carried out an experiment on a blenny species (Acantemblemaria hancocki; Pisces: Chaenopsidae) and reviewed the
literature to evaluate the extent to which variations in the rate at which temperature is increased in experimental trials affects thermal
tolerance of ?shes. For the blenny species, we found that thermal tolerance decreases signi?cantly from an intermediate heating rate of

1 1C/h towards quicker and slower heating rates. In the literature we found very few comparisons of thermal tolerance among heating
rates (i.e. eight ?sh species) and although such comparisons were done over narrow ranges of heating rates, overall they appear to follow
the pattern described for the blenny species. We discuss a variety of factors including variations in the levels of acclimation, energy use
and body quality among heating rates as the causes for this pattern. However, available data are still limited and further research will be
necessary to determine the generality and causes of the pattern we found here. Nevertheless, our results indicate the need for caution in
the extrapolation of thermal tolerance data when assessing the tolerance of organisms to environmental phenomena that vary in their
rates of warming.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Fishes; Thermal tolerance; Critical thermal maximum; Heating rates; Global warming; Dynamic method
1. Introduction temperatures whereas in the ??dynamic method??, tempera-Heat tolerance is commonly determined by exposing organisms to increasing temperatures until they show symptoms of thermal stressEffect of the rate of temperature
on the heat to
Camilo Moraa,

aLeigh Marine Laboratory, University of Auck
bSeccion de Biolog??a Marina, Universid
Received 24 August 2005(2006) 337?341
ncrease of the dynamic method
ance of ?shes
aria F. Mayab
, 349 Goat Island Road, Leigh, New Zealand
del Valle, A.A. 25360, Cali, Colombia
cepted 17 January 2006
www.elsevier.com/locate/jtherbio
Abstracts Database contained 197 citations referring to
ARTICLE IN PRESS
Th??critical thermal maximum??; of these only ?ve speci?cally
referred to ??heating and/or warming rates??. Out of these
?ve one did not provide any comparison of thermal
tolerances among heating rates (Gomez, 1990) and another
was based on the rate of temperature penetration in the
body of turtles (Webb and Witten, 1973). Our precarious
understanding about the effect of the heating rate on
thermal tolerance is particularly critical given the broad
range of heating rates used so far. Lutterschmidt and
Hutchison (1997) reviewing 604 papers on critical thermal
maximum reported that among studies heating rates have
varied between 10 1C/min to 1 1C per 3.5 days. Beitinger
et al. (2000), reviewing 80 publications on thermal
tolerance of North American ?shes reported heating rates
between 0.027 and 60 1C/h. Even recommendations for the
use of standard rates have been variable (e.g. 0.3 1C/min:
Becker and Genoway, 1979; 1 1C/min: Lutterschmidt and
Hutchison, 1997). Here we use different heating rates to
quantify the critical thermal maximum of a reef ?sh species
and compiled data from previous studies to search for
general patterns on the effect of heating rates on the
thermal tolerance of ?sh.
2. Materials and methods
The blenny ?sh Acantemblemaria hancocki Myers and
Reid, 1936 (Pisces, Chaenopsidae) was the focal species of
this paper. This is a common species of rocky shores in the
Eastern Paci?c where it inhabits mainly dead barnacles.
Fish of similar size (average total length ? 3.1 cm,
SD ? 0.3) were collected with hand nets and clove oil in
Tabogilla Island (Panama) and then transported within
20min to the laboratory of the Smithsonian Tropical
Research Institute in Naos Panama. All ?sh collections
were done during one day and the experiment started the
day after. Collected ?sh were randomly placed into 20 l
aquaria (?ve ?sh per aquarium) (water temperature in all
aquaria was the same as in the ?eld: 24 1C). A total of 21Hutchison, 1997; but see Ospina and Mora, 2004). In
contrast, slower heating rates allow ?sh to acclimatize to
new temperatures, which could increase thermal tolerance
(Cocking, 1959; Beitinger et al., 2000). However, if heating
rates are too slow they could reduce tolerance as
temperature could have more time to exert its lethal effects
(Cocking, 1959; Beitinger et al., 2000). Finally, in experi-
mental animals, stress hormones are known to increase
with time of captivity (Pankhurst and Sharples, 1992); if
stress affects thermal tolerance, then one would expect
thermal tolerance to reduce at slower heating rates due to
the increased captivity time at such heating rates.
Despite the potential incidence of the chosen heating rate
on the levels of thermal tolerance this has been poorly
studied. As an example, until 2004 the Cambridge Scienti?c
C. Mora, M.F. Maya / Journal of338aquaria were deployed for ?ve heating rates (60 1C/h, 1 1C/h,
1 1C/12 h, 1 1C/24 h, 1 1C/48 h) and two sets of controlswith ambient temperature. We used three replicate aquaria
for each of the treatments and controls.
We estimate any effect of captivity time (and/or its
associated stress) by determining the thermal tolerance of
?sh with a single heating rate of 1 1C/h at different times
during the duration of this experiment (days 1, 8 and 25).
The data of thermal tolerance with a heating rate of 1 1C/h
obtained the day after ?sh collections was used for the
comparison among heating rates but also for this analysis
on the effect of captivity time (i.e. day 1 of captivity).
Thermal tolerance for the two other captivity times was
determined using the two sets of control aquaria 8 and 25
days after the start of the experiment (day 25 was the last
day of the experiment). Thermal tolerance was quanti?ed
as the mean temperature at which individual ?sh show
erratic swimming. This critical point is widely used as it
represents conditions that will ultimately lead to death
(Beitinger et al., 2000).
Fish were fed once per day with frozen artemia (food
ration of 0.1ml of artemia per ?sh). Filtered seawater was
supplied constantly to ensure 
100% water interchange
per day. Aeration was also constant to keep high oxygen
concentrations and a continuous mixing of water. All sides
of the aquaria, but the frontal side, were covered with
Styrofoam to avoid heat lost and changes of temperature in
the aquaria through their interface with the surrounding
medium. Each aquarium was equipped with an electronic
control of temperature (Autonics TZN4S), a sensor of
temperature (Autoniz PT100) and a 300W heater.
With this setup water temperature was controlled with
0.1 1C accuracy. These electronic controls were also
designed to control water temperature inertia, which
can expose ?sh to undesired thermal effects (here inertia
refers to increases in water temperature beyond a desire
point when temperature is gradually increased and
not to the rate of temperature penetration in the body of
?shes). To do so, the electronic controls switched on
and off the heaters before reaching the desire temperature
so that temperature never exceeded the desire limit. Note
that broader inertias are often present at quicker heating
rates as temperature cannot be easily stopped at the desire
point when it is being quickly increased. The dif?culty of
keeping constant heating rates throughout an experiment
has been an important critique to the dynamic method of
quantifying thermal tolerance of ?shes (Cocking, 1959)
and it is a key assumption in the interpretation of its
results (Becker and Genoway, 1979; Lutterschmidt and
Hutchison, 1997).
Upper temperature tolerance data among heating rates
and captivity times in A. hancocki were presented
graphically and compared via ANOVA. Finally, we
searched the literature for studies that have speci?cally
analyzed variations in thermal tolerance among heating
rates. Our search was based on the indexing databases of
Biological Sciences and Biology Digest from Cambridge
ermal Biology 31 (2006) 337?341Scienti?c Abstracts and from references cited in the papers
found.
3. Results
None of the ?sh held in captivity in control aquaria died
or showed symptoms of stress (e.g. unusual behaviors or
changes in normal coloration) during the experiment. The
effect of captivity stress on ?sh tolerance was opposite to
expected (i.e. tolerance decreasing over time) as thermal
tolerance actually increased with increasing captivity time.
There were signi?cant differences in thermal tolerance
among different lengths of captivity (F ? 4:3, po0:01).
Fishes held in captivity for 25 days showed higher thermal
tolerances than ?shes held in captivity for 1 or 8 days
(Tukey test, po0:01) (Fig. 1). However, all the variation in
thermal tolerance among captivity lengths was less than
1 1C (Fig. 1).
A. hancocki showed the maximum tolerance at a heating
rate of 1 1C/h (i.e. CTMax ? 36.6 1C, 70.15 SD) and
ARTICLE IN PRESS
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Fig. 2. Relationship between heating rates and thermal tolerance to high
temperatures in different ?sh species. Data is shown as means. Numbers
between parentheses indicate the temperature of acclimation in the
different studies. In plot A, data among aquaria were identical; therefore
statistics were calculated combining the data of the 15 ?sh used for each
heating rate; in this plot vertical bars represent standard deviations of that
data. (a) Acantemblemaria hancocki (This study), (b) Salmo salar (Elliot
and Elliot, 1995), (c) Salmo trutta (Elliot and Elliot, 1995), (d)
Oncorhynchus kisutch (Becker and Genoway, 1979), (e) Lepomis macro-
C. Mora, M.F. Maya / Journal of Thermal Biology 31 (2006) 337?341 339decreased from this point towards slower and faster
heating rates [at 60 1C/h CTMax ? 35.7 (70.6 SD) and
at 0.02 1C/h CTMax ? 34.3 (70.27 SD)] (Fig. 2(A)).
Because the data on thermal tolerance showed no
homogeneity of variances (Levine?s test F ? 27:9,
po0:0001; Cochran 0.67, po0:0001) (Fig. 2(A)) we also
analyzed variations in thermal tolerance among heating
rates with the use of a non-parametric analysis of variance
(i.e. Kruskal-Wallis ANOVA). With the use of both
parametric and non-parametric tests, thermal tolerance
was signi?cantly different among heating rates (F ? 123,
po0:0001; H ? 75, po0:0001): all heating rates but the
two slowest ones were signi?cantly different from each
other at po0:001 with the use of a Tukey test with both
parametric and ranked data.
Comparisons in thermal tolerance among heating rates
were found for eight ?sh species (Fig. 2). None of these
studies determined thermal tolerance over a range of
heating rates as broad as the one analyzed in this study.
The most recent study on the effect of heating rates, Elliot
36.2
36.5
36.8
37.1
1050 15 20 25 30
Days of captivity
Cr
iti
ca
l t
he
rm
a
l m
ax
im
u
m
 (?
C)
Fig. 1. Effect of captivity time on the thermal tolerance of the blenny ?sh
Acantemblemaria hancocki. Thermal tolerance was determined at different
times during the duration of the experiment with a unique heating rate of
1 1C/h. Thermal tolerance is expressed as the mean temperature at which
?sh showed erratic swimming (7SD). For each captivity time, there were
three replicate aquariums with ?ve ?sh each. Data among aquaria were
identical; therefore statistics were calculated combining the data of the 15
?sh used for each captivity time.
chirus (Cox, 1974; Becker and Genoway, 1979), (f) Rutilus rutilus (Cocking
1959) and (g) Other species.
ARTICLE IN PRESS
Thand Elliot (1995) using two Salmo species, determined
thermal tolerance using slow heating rates and showed
trends remarkably similar to ours with those rates (Figs.
2(B) and (C)). Unfortunately, the trend observed with
Salmo species did not provide information on the side of
quicker heating rates (Figs. 2(B) and (C)). Another recent
study, Chung (1997) using Cyprinodon dearborni, analyzed
quick but not slow heating rates and showed a declining
trend in thermal tolerance similar to ours on that spectrum
of the heating rates (Fig. 2(G)). Older studies commonly
used quicker heating rates to determine thermal tolerance
and all showed patterns of variation opposite to ours. That
is, thermal tolerance increasing with quicker heating rates
(Cox, 1974; Becker and Genoway, 1979) (Figs. 2(D)?(F)).
An interesting aspect of the comparison between older and
more recent studies is that among recent studies tempera-
ture of acclimation yielded only minimal variations in
thermal tolerance at similar heating rates (Figs. 2(B) and
(C)) while such differences were more pronounced among
old studies (Figs. 2(D)?(G)).
4. Discussion
Determining the levels of tolerance to temperature of
marine organisms is critical to assess the extent to which
climate change and other thermal phenomena will affect
mortality rates and the abundance and distribution of
species. In this regard, out of the available approaches, the
dynamic method of determining thermal tolerance in
aquatic organisms seems to provide the most reliable data
for comparative purposes with environmental conditions
(see Bennett and Judd, 1992; Mora and Ospina, 2001). Yet
its use will require proper understanding of the factors
likely to affect thermal tolerance in experimental trials. In
this study, we have shown that the speed of the heating rate
is a key factor in?uencing levels of thermal tolerance in
?shes.
The common pattern among recent studies is for thermal
tolerance to decrease from an intermediate heating rate (i.e.

1 1C/h) towards faster and slower rates. Cocking (1959)
suggested that heating rates affect not only the time a ?sh
have for acclimation but also the time they are exposed to
lethal temperatures. Maximum tolerance gain due to
acclimation is then likely to occur at the heating rate at
which ?sh achieve full acclimation (Cocking, 1959). In our
results such a rate appears to be 1 1C/h given that at this
rate several of the analyzed species showed their maximum
tolerance. For ectotherms, full acclimation to high
temperatures can be achieved within only few hours
(Hutchison, 1976; Chung, 1985, 1995, 2001 Segnini et al.,
1993; Lutterschmidt and Hutchison, 1997) suggesting that
the increased tolerance at a heating rate of 1 1C/h could
indeed obey to the fact that at this rate ?sh achieve full
acclimation to the change in temperature they were
exposed to. Reduced levels of tolerance toward faster
C. Mora, M.F. Maya / Journal of340heating rates could be similarly explained by ??inappropri-
ate?? acclimation to the quick change in temperature.Barker et al. (1981) and Hutchison and Murphy (1985) also
suggest that rapid changes in temperature can induce heat-
shocks effects leading to underestimation of upper thermal
limits (Lutterschmidt and Hutchison, 1997).
Cocking?s (1959) argued that heating rates slower than
the rate for complete acclimation could have harmful
effects on ?sh as they will have longer exposure to the
lethal effects of any given temperature. This in turn could
explain the reduced thermal tolerance observed in ?sh
exposed to heating rates slower than 1 1C/h. An additional
and/or complementary explanation for the reduced toler-
ance at slow heating rates is the reduction of energetic
reserves and/or body deterioration at high temperatures
even if temperatures are non-lethal. Elevated temperatures
are known to increase metabolism and energetic demands
(Hochachka and Somero, 1973). If food supply is kept
constant (as in our case) body condition is likely then to
deteriorate over time. In fact, we noted that after few days
of exposure to slow heating rates ?sh were more voracious
and bonier than ?sh in control aquaria highlighting their
increased food demands and diminished body condition.
Hutchison (1976) argued that body condition and energetic
reserves are likely to affect thermal tolerance in ?shes. This
in turn could have reduced tolerance at slow heating rates
and/or contributed to the effects of slow heating rates
argued by Cocking (1959) and pointed above. Note that
stress due to captivity time during long experiments is
unlikely to explain the reduced thermal tolerance with slow
heating rates since we found that captivity time did not
diminish thermal tolerance (Fig. 1).
However, the pattern of decreasing thermal tolerance
towards slow and quick heating rates was not without
exceptions. Speci?cally, we found a set of papers reporting
increasing thermal tolerance among quick heating rates
(Figs. 2(E)?(F)). Interestingly, however, was the fact that
all these cases occurred among old studies. Although the
mentioned differences may relate to differences in the
species used, it may also conform to technical advances in
the control of water temperature (e.g. control of water
temperature inertia, which is stronger at quicker heating
rates, which were commonly used among old studies).
Dif?culties in maintaining constant heating rates have been
previously mentioned as a limitation of old studies
(Cocking, 1959). Compared to more recent papers, old
studies also showed stronger effects of acclimation on
thermal tolerance with the use of similar heating rates. This
suggests that methodological differences between these
groups of studies may have indeed played an important
role in explaining the opposite results shown by old studies
about the effects of heating rates on thermal tolerance.
Previous studies have argued the need for standardiza-
tion of fast heating rates for determining heat tolerance in
?shes (e.g. 0.3 1C/min: Becker and Genoway, 1979; 1 1C/
min: Lutterschmidt and Hutchison, 1997). Such rates have
been argued to be rapid enough to avoid the effects
ermal Biology 31 (2006) 337?341acclimation and slow enough to prevent lags between water
and ?sh body temperature during a trial. However, the
speeds of acclimation are known to vary among species
(Chung, 1985, 1995, 2001; Segnini et al., 1993; Luttersch-
midt and Hutchison, 1997) and so are the rates of
temperature penetration in the body of ?shes given
variations in the ratio area/volume among species and in
the actual body size of species. This suggests that for
Chung, K.S., 1995. Thermal acclimation rate of the tropical long-
whiskered cat?sh Pimelodella chagresi to high temperature. Caribbean
J. Sci. 31, 154?156.
Chung, K.S., 1997. Tolerancia te?rmica del pez tropical Cyprinodon
dearborni (Atheriniformes, Cyprinodontidae) a diversas tasas de
calentamiento y salinidades. Rev. Biol. Trop. 45, 1541?1545.
Cocking, A.W., 1959. The effect of high temperatures on roach (Rutilus
ARTICLE IN PRESS
C. Mora, M.F. Maya / Journal of Thermal Biology 31 (2006) 337?341 341standard heating rates could have been premature. If the
pattern we describe here holds for other species, for
comparative purposes it would be more recommendable
to use heating rates at which species show similar responses
(e.g. the in?ection point at which tolerance declines
towards fast and slow heating rates). This will require
studies to start reporting thermal tolerance over broad
heating rates. Such data would allow more useful extra-
polations of thermal tolerance data to thermal phenomena
given the possibility of accounting for variations in the
rates of warming of different environmental phenomena.
Acknowledgments
C.M. was supported by a STRI Predoctoral Fellowship,
and by OGS and NSERC through a grant awarded to P.F.
Sale. Thanks to D.R. Robertson for advice and to E. Pen?a,
A. Domingo and the staff at the Smithsonian Tropical
Research Institute in Panama for collaboration in ?eld
collections and laboratory work.
References
Addo, A., Chown, S.L., Gaston, K.J., 2000. Thermal tolerance, climatic
variability and latitude. Proc. Roy. Soc. Lond. B 267, 739?745.
Barker, S.C., Townsend, D.W., Hacunda, J.S., 1981. Mortalities of
Atlantic hearing, Clupea h. harengus, smooth ?ounder, Liopsetta
putnami, and rainbow smelt, Osmerus mordax, larvae expose to acute
thermal shock. Fish. Bull. 79, 198?200.
Becker, C.D., Genoway, R.G., 1979. Evaluation of the critical thermal
maximum for determining thermal tolerance of freshwater ?sh. Env.
Biol. Fish. 4, 245?256.
Beitinger, T.L., Bennett, W.A., McCauley, R.W., 2000. Temperature
tolerance of North American freshwater ?shes exposed to dynamic
changes in temperature. Environ. Biol. Fish. 58, 237?275.
Bennett, W.A., Judd, F.W., 1992. Comparison of methods for determining
low temperatures tolerance, experimental with Pin?sh, Lagodon
rhomboides. Copeia 1992, 1059?1065.
Bennett, W., Currier, R.J., Beitinger, T.L., 1997. Cold tolerance and
potential overwinter of red-bellied piranha, Pygocentrus nattereri, in
the United States. Trans. Am. Fish. Soc. 126, 841?849.
Chung, K.S., 1985. Adaptabilidad de Oreochromis mossambicus (Peters)
1852 a los cambios de temperatura. Acta Cient???ca Venezolana 36,
180?190.rate. J. Exp. Biol. 36, 217?226.
Chung, K.S., 2001. Critical thermal maxima and acclimation rate of the
tropical guppy Poecilla reticulata. Hydrobiologia 462, 253?257.
Cox, D.K., 1974. Effects of three heating rates on the critical thermal
maximum of bluegill. In: Gibbons, J.W., Sharitz, R.R. (Eds.), Thermal
Ecology. US Atomic Energy Commission, Savannah, GA,
pp. 158?163.
Elliot, J.M., Elliot, J.A., 1995. The effect of the rate of temperature
increase on the critical thermal maximum for parr of Atlantic salmon
and Brown trout. J. Fish Biol. 47, 917?919.
Glynn, P.W., D?Croz, L.D., 1990. Experimental evidence for high
temperatures stress as the cause of El Nin?o-coincident coral mortality.
Coral Reefs 8, 181?191.
Gomez, S.E., 1990. Some thermal ecophysiological observations on the
cat?sh Hatcheria macraei (Girard, 1855) (Pisces, Trichomycteridae).
Biota Osorno 6, 85?89.
Hochachka, P.W., Somero, G.N., 1973. Strategies and biochemical
adaptation. Saunders Company Publishers, Philadelphia.
Hutchison, V., 1976. Factors in?uencing thermal tolerance of individual
organisms. In: Esch, G.W., McFarlane, R., (Eds.), Symposium Series
of the National Technical Information Service, Spring?led, VA,
pp. 10?26.
Hutchison, V., Murphy, K., 1985. Behavioral thermoregulation in the
salamander Necturus maculosus after heat shock. Comp. Biochem.
Physiol. A. 82, 391?394.
Kimball, M.E., Miller, J.M., Whit?eld, P.E., Hare, J.A., 2004. Thermal
tolerance and potential distribution of invasive lion?sh (Pteroios
volitans/miles complex) on the east coast of the United States. Mar.
Ecol. Prog. Ser. 283, 269?278.
Kimura, M.T., 2004. Cold and heat tolerance of drosophilid ?ies with
reference to their latitudinal distributions. Oecologia 140, 442?449.
Lutterschmidt, W.I., Hutchison, V.H., 1997. The critical thermal
maximum: history and critique. Can. J. Zool. 75, 1561?1574.
Mora, C., Ospina, F., 2001. Thermal tolerance and potential impact of sea
warming on reef ?shes from Gorgona Island (Eastern Paci?c ocean).
Mar. Biol. 139, 765?769.
Mora, C., Ospina, F., 2002. Experimental effects of La Nina cold
temperatures on the survival of reef ?shes from Gorgona Island
(Tropical Eastern Paci?c). Mar. Biol. 141, 789?793.
Ospina, F., Mora, C., 2004. Effect of body size on reef ?sh tolerance
to extreme low and high temperatures. Environ. Biol. Fish. 70,
339?343.
Pankhurst, N.W., Sharples, D.F., 1992. Effects of capture and con?ne-
ment on plasma cortisol levels in the snapper Pagrus auratus. Aust.
J. Freswater Res. 43, 334?346.
Segnini, B.M., Chung, K.S., Ciurcina, P., 1993. Tasa de aclimatacio?n de
temperatura de Mugil curema. Rev. Biol. Trop. 41, 59?62.
Webb, G.J., Witten, G.J., 1973. Critical thermal maxima of turtles,
validity of body temperature. Comp. Biochem. Physiol. 45, 829?832.comparative purposes the recommendation of using rutilus). II: The effect of temperature increasing at a known constant