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ReviewCite this article: Leduc AOHC, Munday PL,

Brown GE, Ferrari MCO. 2013 Effects of acidi-

fication on olfactory-mediated behaviour in

freshwater and marine ecosystems: a synthesis.

Phil Trans R Soc B 368: 20120447.

http://dx.doi.org/10.1098/rstb.2012.0447

One contribution of 10 to a Theme Issue

‘Ocean acidification and climate change:

advances in ecology and evolution’.

Subject Areas:behaviour, ecology, environmental science,

physiology

Keywords:climate change, ocean acidification, acid rain,

fish behaviour, olfaction, chemoreception

Author for correspondence:Antoine O. H. C. Leduc

e-mail: mirabiles@hotmail.com

& 2013 The Author(s) Published by the Royal Society. All rights reserved.

Effects of acidification on olfactory-mediated behaviour in freshwater andmarine ecosystems: a synthesis

Antoine O. H. C. Leduc1, Philip L. Munday2, Grant E. Brown3

and Maud C. O. Ferrari4

1Instistuto de Biologia, Universidade Federal da Bahia, Ondina, Salvador, Bahia, Brazil2ARC Centre of Excellence for Coral Reef Studies, School of Marine and Tropical Biology, James Cook University,Townsville, Queensland, Australia3Department of Biology, Concordia University, 7141 Sherbrooke St. West, Montreal, Quebec, Canada H4B 1R64Department of Biomedical Sciences, WCVM, University of Saskatchewan, Saskatoon, Saskatchewan,Canada S7N 5B4

For many aquatic organisms, olfactory-mediated behaviour is essential to

the maintenance of numerous fitness-enhancing activities, including fora-

ging, reproduction and predator avoidance. Studies in both freshwater and

marine ecosystems have demonstrated significant impacts of anthropogenic

acidification on olfactory abilities of fish and macroinvertebrates, leading to

impaired behavioural responses, with potentially far-reaching consequences

to population dynamics and community structure. Whereas the ecological

impacts of impaired olfactory-mediated behaviour may be similar between

freshwater and marine ecosystems, the underlying mechanisms are quite

distinct. In acidified freshwater, molecular change to chemical cues along

with reduced olfaction sensitivity appear to be the primary causes of olfac-

tory-mediated behavioural impairment. By contrast, experiments simulating

future ocean acidification suggest that interference of high CO2 with brain

neurotransmitter function is the primary cause for olfactory-mediated be-

havioural impairment in fish. Different physico-chemical characteristics

between marine and freshwater systems are probably responsible for these

distinct mechanisms of impairment, which, under globally rising CO2

levels, may lead to strikingly different consequences to olfaction. While fluc-

tuations in pH may occur in both freshwater and marine ecosystems, marine

habitat will remain alkaline despite future ocean acidification caused by

globally rising CO2 levels. In this synthesis, we argue that ecosystem-specific

mechanisms affecting olfaction need to be considered for effective manage-

ment and conservation practices.

1. IntroductionAnthropogenic acidification of lakes, rivers and the oceans can have dramatic and

far-reaching impacts on the structure and function of aquatic ecosystems [1–5].

Historically, the problem of acidification has mostly been associated with fresh-

water ecosystems affected by acid rain precipitation [6–8]. Decades of this

phenomenon have reduced the acid neutralizing capacity in many impacted

areas, leading to persisting vulnerability of freshwater ecosystems to future

changes of water pH [9,10]. However, attention has shifted towards the threat

of acidification in marine ecosystems, as increased CO2 uptake from the atmos-

phere is causing ocean pH to decline with a concomitant increase in dissolved

CO2 [5,11]. Although the mechanism of anthropogenic acidification differs

between freshwater and marine ecosystems (box 1), the underlying cause is the

same—the continued burning of fossil fuels (figure 1). Furthermore, the chemical

changes associated with acidification can lead to fundamental changes to bio-

logical and ecological processes in both freshwater and marine ecosystems,

including impacts on individual performance (e.g. reproductive output or

predator avoidance), species interactions and community structure [7,11,19].

Box 1. The acidifying processes.

Acidification of rivers and lakes occurs chiefly as a result of acidified rain or snow depositions. The process that results in the

formation of acid depositions begins with emissions into the atmosphere of sulfur dioxides and nitrogen oxides. These gases

are typically released following fossil fuel combustion and combine with water vapour to form sulfuric and nitric acids

(figure 1). When such precipitations fall, they have become highly acidic, typically with pH values of 5.6 or lower. In the Amer-

ican Northeast and Eastern Europe, decades of acid precipitations have depleted the soil buffering capacities such that any

further acidic depositions may have rapid and long-lasting effects on ambient water pH [12]. While in these regions emissions

of sulfur dioxide have generally decreased when compared with previous decades [13], globally they remain at relatively con-

stant levels (e.g. approx. 65 kton in 2000) from the increasing sulfur dioxide emitting countries [14,15]. As such, the problem of

acid rain is likely to expand to areas previously unaffected by this phenomenon.

Ocean acidification occurs through the uptake of additional CO2 from the atmosphere. Over 2 trillion tons of additional

CO2 (550 Gt carbon) has been released into the atmosphere by fossil fuel burning, cement production and land use changes

since the industrial revolution [16–18]. The partial pressure of CO2 at the surface ocean is proportional to that in the atmos-

phere, consequently more CO2 is taken up by the ocean as atmospheric CO2 levels rise. Additional CO2 reacts with water to

form carbonic acid (H2CO3), which quickly disassociates to form Hþ ions and bicarbonate (HCO32) ions. Excess Hþ ions

bond with CO322 ions producing more HCO3

2. Adding more CO2 to sea water thus increases aqueous CO2, bicarbonate

and hydrogen ions, the latter having the observed effect of lowering sea water pH, and decreases the abundance of carbonate

ions (figure 1).

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For many aquatic organisms, one of the main conse-

quences of non-lethal acidification of aquatic ecosystems

may be the disruption of olfaction and chemosensory abilities

[20,21]. The detection of chemical cues and signals by olfac-

tory and gustatory receptors plays a central role in the

decision-making process [22], especially in structurally com-

plex or turbid environments or under low light conditions

[23–25]. For instance, homing [26,27], microhabitat choice

[28,29], mate selection [30,31], kin recognition [32], food

location [33], dominance interactions [34] and predator avoid-

ance [35,36] are all mediated by the detection of chemical

cues, at least to some extent. Any interference with the func-

tioning of normal chemosensory responses could reduce the

ability to perceive basic environmental stimuli or public

information that is critical for long-term survival. Conse-

quently, impaired olfactory-mediated responses caused by

acidification in freshwater and marine ecosystems may have

significant impacts on the ecology and life histories of

many aquatic organisms. Experiments with freshwater fish

have demonstrated a range of behavioural and ecological

changes associated with the impairment of olfaction and

chemical cues detection under weakly acidified conditions

(approx. pH 6.0) [20,37,38]. Recently, a very similar range of

behavioural changes has been observed in experiments

with marine fish exposed to predicted future changes in

ocean pH due to rising atmospheric CO2 [39,40]. However,

despite the remarkable similarity in many of the behavioural

responses, the underlying mechanisms appear to be entirely

different [37,41]. In this review, we compare and contrast the

effects of acidification on olfactory-mediated behaviour in

freshwater and marine organisms and present evidence for

significant dissimilarities between the underlying mechanisms

of action. Numerous studies demonstrating these effects

have used fish as a model organism, whereas studies using

invertebrates are by comparison much rarer. Reflecting this

asymmetry, we mostly used fish examples to explore this ques-

tion and included invertebrates examples when available. We

then consider how projected increases in atmospheric CO2

may affect marine and freshwater organisms. Identifying the

consequences of acidification on olfactory-mediated behav-

iour, along with an understanding of the mechanisms at play

become critical for applying effective management and

conservation practices in freshwater and marine ecosystems.

2. Ecological effects of acidification on olfaction(a) Freshwater ecosystemsExperiments in freshwater have demonstrated that the ability

of fishes to detect and respond to chemical cues of injured

conspecifics (termed ‘alarm cues’ [24,25]) may be disrupted

under weakly acidified conditions. Alarm cues are particu-

larly useful for studying chemosensory abilities in fishes as

they trigger innate, readily measurable overt responses

[24,42]. Exposure to alarm cues can elicit adaptive predator

avoidance behaviour, such as reduced activity levels and

increased hiding or tighter group cohesion [43]. These behav-

ioural responses to alarm cues have been observed in a range

of freshwater fishes, including fathead minnows (Pimephalespromelas), finescale dace (Phoxinus neogaeus), pumpkinseed

(Lepomis gibbosus) and rainbow trout (Oncorhynchus mykiss)

[20,37,44]. However, predator avoidance behaviour was

diminished or absent when these alarm cues were presented

in experimental treatments acidified using a minute amount

of sulfuric acid (often a major contributor to freshwater acid-

ification) [20,38,45]. Furthermore, behavioural responses to

basic chemical stimuli from food (amino acids) were signifi-

cantly reduced under a similar range of pHs (e.g. 5.0–6.0)

in fathead minnows and Atlantic salmon (Salmo salar)

[38,46]. Under field conditions, experiments revealed a simi-

lar absence of response to chemosensory cues in brook

trout (Salvelinus fontinalis) and Atlantic salmon exposed to

conspecific alarm cues in acidic streams (pH range; 5.8–

6.3), whereas avoidance responses were retained in neutral

pH streams (pH range; 6.8–7.4) [20,45,47,48]. Likewise, episo-

dic acidifying events may cause short-term loss of

chemosensory abilities in freshwater fish. Field tests demon-

strated that following acid rain precipitations, the response of

Atlantic salmon to alarm cues did not differ from their

response to a control stimulus of stream water (at approx. pH

6.2) [45]. Following subsequent flushing of the acid pulses,

the response of the salmon returned to pre-acidification levels

SO2+H2O

SO2

CO2

ocean

NOxwet dry

acid depositions

lake

H2SO4

NOx+H2O HNO3

CO2+H2O H2CO3 HCO3–+H+

HCO3–H++CO3

2–

Figure 1. Anthropogenic sources of marine (left) and freshwater ecosystem acidification (right). Ocean acidification occurs through the uptake of additional CO2 from theatmosphere, the CO2 converting into carbonic acid as it reacts with seawater. In freshwater systems, acidifying depositions occur following the release of nitrogen oxides(NOx) and sulfur dioxide (SO2) into the atmosphere, mainly from the combustion of fossil fuels, which then may fall in dry or wet form. (Online version in colour.)

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(approx. pH 6.9) and they exhibited strong predator avoidance

behaviour in the presence of the alarm cues.

Together, these experiments and observations demonstrate

that even relatively small changes in pH (approx. 0.5 unit)

can dramatically affect both the feeding and antipredator

responses of freshwater fishes.

Such demonstrated loss of adaptive alarm behaviour

under weakly acidic conditions is probably due to an

inability to detect and thus respond to alarm cues. Indeed,

following the pairing of aversive alarm cues stimulus with

a novel odour, wild Atlantic salmon tested in neutral streams

subsequently responded to the novel odour as a dangerous

stimulus [49]. However, following the same procedure, simi-

lar learned responses did not occur for salmon conditioned

and tested in acidic streams, suggesting a pH-mediated

inability to perceive the information of risk that is normally

associated with the alarm cues [50]. Under acidified con-

ditions, the lack of antipredator response towards the alarm

cues probably prevents the learned association of risk with

the novel predator odour, resulting in failed learning. Thus,

the retention of and the ability to generalize acquired preda-

tor recognition may be significantly impaired under weakly

acidic conditions [51,52].

Failure to appropriately respond to chemical cues could

significantly increase mortality rates of prey and potentially

alter population dynamics. The ecological consequence of

pH-mediated chemosensory impairment to chemical alarm

cues was demonstrated in staged encounters between rainbow

trout prey and a predator, the largemouth bass (Micropterussalmoides). Bass captured trout faster and in greater proportion

when trout alarm cues introduced in test tanks were acidified

(pH 6.0) compared with treatments were alarm cues were

introduced using neutral pH water [45]. Thus, prey ‘naivety’

arising from acid-mediated chemosensory impairment led to

greater predation cost. In addition to alarm behaviour, weak

acidification may affect other behavioural activities in fresh-

water fishes. Suppression of spawning [53], nest digging [54]

and migration behaviour [55] were demonstrated in land-

locked sockeye salmon (Oncorhynchus nerka) exposed to

weak levels of acidification (i.e. pH 6.1–6.3). Thus, non-lethal

anthropogenic acidification may affect a wide range of behav-

iour in freshwater fishes, with significant effects on their life

histories and role in the ecosystem. Although providing

important insights to the potential ecological consequences of

acidity-mediated maladaptive behaviour, the studies men-

tioned above were conducted under controlled laboratory

conditions. Future studies would further this knowledge,

while providing ecologically relevant data, by conducting

investigations under field conditions.

Akin to fish olfaction abilities, experimental evidence

demonstrated that freshwater invertebrates may, likewise,

be affected by acidification. In crayfishes, Orconectes virilisand Procambarus acutus, weakly acidified conditions (pH

5.8) had no effect on feeding behaviour to an amino acids

mixture but more severe acidification levels (pH 4.5 and

3.5) led to significant reductions of this response [56]. Inter-

estingly, in this experiment the amount of food consumed

was not affected by acidification, suggesting that compared

with fishes, these crustaceans may have higher tolerance

towards acidification. Similar olfaction impairment was

also demonstrated in the crayfish Cambarus baroni, which

showed reduced ability to detect a food source at pH 4.5 [57].

(b) Marine ecosystemsAtmospheric CO2 has increased from approximately 280 ppm

at the start of the industrial revolution to over 390 ppm today,

and there has been a corresponding 0.1 pH unit decline in

ocean pH [16]. Depending on emission scenarios, CO2 levels

in the atmosphere could exceed 900 ppm by the end of the cen-

tury [58]. This would cause the pH of the ocean to drop another

0.3–0.4 units. Under such scenarios of future oceanic acidifica-

tion, the ability of larval orange clownfish (Amphiprion percula)

to discriminate between olfactory cues involved in the selection

of suitable settlement sites was tested [21]. Larval clownfish

reared in control seawater (today’s pH of 8.15) had the

ability to discriminate between chemical cues of suitable

versus unsuitable settlement sites and between kin and non-

kin. However, when larvae were reared from hatching under

conditions simulating near-future CO2-induced ocean acidifi-

cation (pH 7.8 and 1050 ppm CO2), this discriminatory

ability was lost and larvae became attracted to olfactory stimuli

that they normally avoided. Similarly, five-lined cardinalfish

(Cheilodipterus quinquelineatus) exposed to future conditions

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of CO2 demonstrated significantly reduced abilities to discrimi-

nate between odours of their home site versus a foreign site

[59]. In another study, aquarium-reared larval clownfish

under acidified conditions became attracted to the odour of pre-

dators and were unable to distinguish between the chemical

cues of predators and non-predators [60]. Further experiments

demonstrated that clownfish and wild-caught damselfish

(Pomacentrus wardi) reared under high CO2 treatments (700

and 850 ppm CO2) for only 2–4 days also displayed impaired

olfactory-mediated behaviour, whereby CO2-treated fish

initially avoided predator odour, as did control fish, but sub-

sequently became attracted to this odour [61].

Similar to the loss of alarm response in freshwater fishes,

laboratory tests revealed that juvenile damselfish exposed to

CO2-acidified conditions for 4 days exhibited a decreased

response to conspecific chemical alarm cues [62]. The response

of predators to prey alarm cues was also affected, with a

common predator of juvenile damselfishes, the brown dotty-

back (Pseudochromis fuscus), spending approximately 20% less

time in a water stream containing prey odour following

exposure to near-future CO2-acidification conditions [62,63].

Reduced attraction to prey odour by the predator did not, how-

ever, offset an increased risk of predation in prey fish exposed

to elevated CO2. Mortality rates of small damselfishes were

almost twice that of controls when a high CO2-exposed dotty-

back interacted with high CO2-exposed damselfish in a

mesocosm experiment [62].

Field tests conducted in natural habitat revealed signifi-

cant ecological outcomes of behavioural changes caused by

predicted ocean acidification. Juvenile damselfish exposed

to CO2-acidified conditions and then placed on patches of

coral habitats were more active and ventured further from

shelter than juveniles exposed to current-day CO2 levels

[61,62]. This riskier behaviour was associated with increased

mortality from predation when compared with control fish

[61,62]. Reduced olfaction abilities and other cognitive

impairment that occurs at elevated CO2 levels [41,64,65]

also appear to affect navigation and homing of reef fish

under natural conditions. For instance, cardinalfish uses

olfactory cues and visual landmarks to aid in navigation to

daytime resting sites after nocturnal foraging, but cardinal-

fish reared under elevated CO2 treatments displayed a

22–31% reduction in homing success compared with control

fish when released at 200 m from home sites [66]. Despite

showing home-site fidelity, high CO2-treated fish were

more active and ventured further from the reef compared

with control fish [66], which would be likely to increase their

risk of predation. Given that the persistence of most coastal

species depends on the ability of larvae to find suitable settle-

ment sites and avoid predators at the end of their pelagic phase,

the effects of ocean acidification on habitat selection and pred-

ator avoidance behaviour may have significant consequences

for the replenishment of fish populations. Furthermore, vari-

ation in sensitivity to acidification among prey species [67]

and shifts in prey preference by predators [62] could lead to

far-reaching effects on reef fish community structure.

In addition to predator–prey interactions and homing

behaviour, changes in seawater pH may also have conse-

quences on mate choice and reproductive behaviour. For

instance, females may choose between males using major

histocompatibility complex (MHC) alleles, which may be

perceivable through scent [68,69]. In a laboratory choice exper-

iment, gravid female stickleback (Gasterosteus aculeatus) were

more attracted to male olfactory cues when pH was raised

from 8.0 to 9.5 using NaOH [70]. In pipefish (Syngnathustyphle), a similar pH increase through Na2CO3 addition

reduced the number of pregnant males [71]. Reducing seawater

pH to 7.5 with CO2 had no effect on mating propensity in the

pipefish [71], however the duration of exposure to acidified

conditions (less than 5 h total) was probably insufficient for

any behavioural effects of elevated CO2 to be induced [61].

While these examples demonstrated effects of seawater alkalini-

zation through eutrophication, rather than its acidification by

rising CO2 levels, they underscore that changes in seawater

pH may alter important processes in sexual selection. At pre-

sent, a gap in knowledge exists limiting our ability to predict

if near-future ocean acidification scenarios will have similar

effects on mate choice.

In marine invertebrates, acidification has been shown to

detrimentally affect behavioural choices at very low pH. In

hermit crabs (Pagurus bernhardus), finding and selecting the

gastropod shells they inhabit is conducted via tactile, visual

and chemical assessment. Choosing an optimal shell is vital

as it provides protection against environmental extremes

and predators. Under highly acidified conditions (pH 6.8 and

12 000 ppm CO2), crabs were less likely to change from a sub-

optimal to an optimal shell than those in untreated seawater,

while the time required to change shells significantly increased

[72]. Additionally, at this reduced pH level, hermit crabs were

less successful in locating an odour source and had lower

locomotory activity compared with hermit craps in untreated

seawater [73]. Thus, reductions in seawater pH may have disrup-

tive consequence on resource assessment and gathering along

with the decision-making processes of hermit crabs. Whether

these impaired response patterns occur at more representative

future levels of ocean acidification (CO2 levels , 1000 ppm)

and are generalizable in marine invertebrates is unknown.

3. Mechanisms of behavioural impairmentAlthough olfaction impairment in freshwater and marine

organisms exposed to acidified conditions may have very

similar consequences on behaviour (table 1), translating into

equivalently significant ecological costs (e.g. increased pre-

dation, reduced recruitment), given the different modes of

acidification between freshwater and marine habitat, the mech-

anisms affecting olfactory/chemosensory response appear to

be entirely distinct.

(a) Freshwater ecosystemsIn freshwater, acidification may affect fish olfaction by two

non-mutually exclusive mechanisms. First, reduced chemo-

sensory abilities observed in acidified conditions can stem

from molecular change to chemical cues, essentially render-

ing them as ‘non-functional’ [37]. For example, the alarm

cues of Ostariophysan fishes probably consist of a suite of

active compounds such as purine-N-oxides [30,68] and gly-

cosaminoglycan chondroitins [79]. Analytical chemical work

has suggested that under acidic conditions, one of these

active components, hypoxanthine-3-N-oxide is converted to

6,8-dioxypurine with a loss of the 3-N-oxide functional

group [80,81] (figure 2). This suggests that acute exposures

to acidic conditions might result in the non-reversible loss of

the N–O functional group, reducing the effectiveness of the

alarm cue to elicit an adaptive alarm response [82]. It is

Table 1. Effects of acidification/alkalinization on olfactory/chemosensory abilities and its consequences in fish and invertebrate species. PC, post-conditioning;RTE, reciprocal transfer experiment.

species type of cuebehaviouralresponse

treatment/environmentalchange and range consequence references

freshwater

Pimephales promelas food stimulus food searching acidification (H2SO4) [38]

pH 6.5 neutral

pH 6.0 negative

Poecilia sphenops food stimulus food searching acidification (H2SO4) [74]

pH 6.0 neutral

pH 5.0 negative

Salmo salar L-serine odour

discrimination

acidification (HNO3 and

H2SO4)

[46]

pH 5.1 negative

alanine — pH 5.1 positive

Onchorynchus mykiss L-serine electro-

physiological

response

acidification (H2SO4)

metal dilution (Al2)

[75]

pH 4.7 negative

pH 4.7,

20 mmol l21 Al2negative

Orconectes virilisa amino acids

mixture

food searching acidification (H2SO4) [56]

Procambarus acutusa pH 6.8 neutral

pH 5.8 neutral

pH 4.5 negative

pH 3.5 negative

Cambarus bartonia food stimulus food searching acidification (H2SO4)b [57]

pH 7.5 neutral

pH 4.5 negative

pH 7.5 intermediate

S. salar testosterone,

ovulated female

urine

electro-

physiological

response

acidification (H2SO4) [76]

pH 6.5 – 3.5 increasingly

negative

alkalinization (NaOH)

pH 8.5 – 9.5 increasingly

negative

Pimephales promelas,

Phoxinus neogaeus

conspecific skin

extract,

alarm response acidification (H2SO4)

pH 6.0

negative [37]

hypoxanthine-3-N-

oxide

— — negative

Lepomis gibbosus conspecific skin

extract,

alarm response acidification (H2SO4)

pH 6.0

negative [44]

congener skin

extract,

— — negative

hypoxanthine-3-N-

oxide

— — negative

(Continued.)

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Table 1. (Continued.)

species type of cuebehaviouralresponse

treatment/environmentalchange and range consequence references

O. mykiss conspecific skin

extract

alarm response acidification (H2SO4)

pH 6.0

negative [20]

Salvelinus fontinalis conspecific skin

extract

— stream acidificationc

pH � 6.1

negative

S. salar conspecific skin

extract

alarm response acidic rainc

pH � 6.2

negative [45]

O. mykiss — survival to

predator

acidification (H2SO4)

pH 6.0

negative

S. salar conspecific skin

extract

alarm response stream acidificationc

pH � 5.8 – 6.1

negative [47]

S. salar conspecific skin

extract, lemon

odour

acquired predator

recognition

stream acidificationc [50]

conditioning pH � 6.1 negative

1 day PC — negative

O. mykiss conspecific skin

extract, predator

odour

acquired predator

recognition

acidification (H2SO4) [51]

conditioning pH 6.0 negative

2 days PC — negative

7 days PC — negative

S. salar conspecific skin

extract

alarm response stream acidificationc [77]

RTE pH � 6.1 negative

— pH � 7.3 neutral

marine

Gasterosteus aculeatus male odour mate choice alkalinization (NaOH) [70]

pH 9.5 positive

Pomacentrus moluccensis,

P. amboinensis,

P. chrysurus

habitat odour habitat choice elevated carbon dioxide

700, 850 ppm CO2

negative [59]

Cheilodipterus

quinquelineatus

habitat odour habitat choice elevated carbon dioxide

550 – 950 ppm CO2

negative [66]

— habitat odour homing response elevated carbon dioxidec

550 – 950 ppm CO2

negative

Amphiprion percula habitat odour,

conspecific

odour

habitat choice/

homing

acidification, elevated

carbon dioxide

[21]

pH 7.8, 1050 ppm CO2 negative

Pagurus bernhardusa gastropod shell

cues

resources

assessment

acidification, elevated

carbon dioxide

[72]

pH 6.8, 12 000 ppm CO2 negative

Pagurus bernhardusb food odour food searching acidification, elevated

carbon dioxide

[73]

pH 6.8, 12 000 matmCO2 negative

A. percula predator odour avoidance acidification, elevated

carbon dioxide

[60]

pH 7.8, 1000 ppm CO2 negative

(Continued.)

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Table 1. (Continued.)

species type of cuebehaviouralresponse

treatment/environmentalchange and range consequence references

Plectropomus leopardus predator odour antipredator elevated carbon dioxide

700 – 850 matm CO2

negative [78]

A. percula predator odour predator

discrimination

elevated carbon dioxide

900 matm CO2

negative [41]

900 matm CO2, GABA-A

receptor antagonist

neutral

A. percula predator odour antipredator elevated carbon dioxide [61]

550 ppm CO2 neutral

700, 850 ppm CO2 negative

Pomacentrus wardi live predators antipredator elevated carbon dioxidec

700 ppm CO2 neutral

850 ppm CO2 negative

P. moluccensis,

P. amboinensis,

P. nagasakiensis,

P. chrysurus

live predator predator

avoidance

elevated carbon dioxide

700 matm CO2

negative [62]

Pseudochromis fuscus live prey prey selection elevated carbon dioxide

700 matm CO2 reversed

preferred

size

selection

P. moluccensis,

P. chrysurus,

P. amboinensis

P. nagasakiensis

conspecific skin

extract

antipredator elevated carbon dioxide

700 – 850 ppm CO2

negative [67]

P. chrysurus live predators survival elevated carbon dioxidec

700 – 850 ppm CO2

negative

Pseudochromis fuscus prey skin extract prey odour

discrimination

acidification, elevated

carbon dioxide

[63]

pH 8.0, 630 matm CO2 negative

pH 7.8, 950 matm CO2 negative

Pomacentrus amboinensis conspecific skin

extract, predator

odour

acquired predator

recognition

acidification, elevated carbon

dioxide

[65]

pH 8.0, 700 ppm CO2 negative

pH 7.9, 850 ppm CO2 negativeaInvertebrate.bConducted sequentially.cExperiments conducted under field conditions.

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unknown, but likely that changes in pH also affect chondroi-

tin functionality as well. In keeping with the hypothesis of

an irreversible molecular change to the chemical cue, short-

term (48 h) changes in water pH, from neutral to weakly

acidic and back to neutral, were well matched with the ability

of fish to detect and respond to the alarm cues [37]. This exper-

imental design suggested that no permanent olfactory damage

or significant stress could account for the loss of response

observed at the acidity level tested (pH 6.0). Similarly, a

reverse transplant experiment, in which Atlantic salmon

from either neutral or acidic streams were alternatively tested

in their natal stream or in a stream of a different pH, demon-

strated that the rearing pH was not directly affecting salmon

chemosensory detection abilities. After a 24 h period of acclim-

ation in stream enclosures, the response to chemical alarm cues

was detectable in all salmon (reared in neutral or acidic

streams) when tested under neutral but not under acidic

streams [77]. Thus, under acidic condition, the alarm cues

regulatorychanges in fish:elevated HCO3

–

reduced Cl–

maintained H+

hyperpolarizing currentwhen binding of GABA to theGABA-A receptor leads to inflow of Cl– and/or HCO3

–

into neurons

depolarizing current when binding of GABA to theGABA-A receptor leads to outflow of Cl– and/or HCO3

–

from neurons

alteredgradients of Cl–

and/or HCO3–

over neuronalmembranes

O(a)

(b)

O

O

H

H

(i) (ii)

NH

H

OH

N

+ +

–

Cl–

HCO3–

Cl–

HCO3–

GABA binding can beantagonized experimentallywith gabazine

GABA

future seawater:elevated pCO2

elevated H+

N

N

N

NNN

Figure 2. Potential mechanisms leading to olfactory-mediated behavioural impairment in freshwater (a) and marine fishes (b). (a) In the Ostariophysi alarm cuemolecule (hypoxanthine-3-N-oxide), at neutral pH the acid – base equilibrium heavily favours the deprotonated (N – O2) form (i). However, as the pH decreases, theacid – base equilibrium shifts in favour of the hydroxy form (ii), decreasing the relative concentration of the deprotonated active form of the chemical signal.Modified from Brown et al. [61]. (b) In marine fish, physiological interferences arising from the hyperpolarization of the neurotransmitter membrane of theGABA-A receptor occur when exposed to elevated CO2-acidified conditions. Adapted from Nilsson et al. [39]. (Online version in colour.)

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appear to undergo molecular changes and become either com-

pletely degraded (i.e. non-functional) and unrecognizable, or

the concentration of its active component is reduced below a

threshold necessary to trigger an adaptive alarm response [44].

A second mechanism by which olfaction and chemosen-

sory abilities may be affected stems from physiological

disruptions of the fishes’ olfactory organs, whereby reduced

sensitivity or affinity to fish olfactory receptors may occur

[74,76,83]. In rainbow trout, fathead minnow and Atlantic

salmon, reduced abilities to respond to amino acids or ovulated

female urine were demonstrated at approx. pH 6.0 [38,46,76].

Similar to the pH-dependent response observed for chemical

alarm cues, the feeding response to amino acids was well

matched with short-term changes in water pH. Indeed, Atlan-

tic salmon were attracted to glycine under neutral pH (7.6) but

became indifferent under lower tank pH (5.1), while following

a return to neutrality, salmon olfactory attraction towards

glycine was re-activated [46]. Importantly, these short-term

effects may be magnified in heavily acidified conditions (i.e.

pH , 6.0) under which the release of monomeric ions (Al2,

Cuþ) may further interfere with olfactory epithelia, leading to

lasting chemosensory impairment [75,76].

Intriguingly, in freshwater systems, the ability of fishes to

detect chemical cues may not be steadily affected by acidifica-

tion. For instance, the time required of gold mollies (Poeciliasphenops) to locate food sources was not significantly different

between pH 6.0 and pH 7.9 [74]. Likewise, the pH of a novel

odour was not a critical factor for its subsequent recognition

as a danger stimulus by juvenile rainbow trout, as long as it

was paired with chemical alarm cues that were left unacidified

[84]. Thus, in freshwater, acidification does not appear to sys-

tematically affect fish chemosensory abilities and olfactory

receptor sensitivity. However, as described earlier, the chemical

nature of the alarm cues appears to be modified, thereby redu-

cing their effectiveness to trigger a response [37]. For this later

mechanism of impairment, a threshold for chemosensory detec-

tion has been shown to occur at a pH of approximately 6.3,

whereby rainbow trout failed to respond to chemical alarm

cues [85]. Likewise, electro-physiological recordings of Atlantic

salmon olfactory epithelia were significantly reduced at a pH of

5.5 [76]. Several variables could affect such a potential threshold

of chemosensory impairment, including the species under

investigation along with the chemical compounds tested. How-

ever, experimental results performed close to these thresholds

indicate that chemosensory impairment may be readily

reversed following a return of ambient water to neutral pH

[37,46,76,85], thereby suggesting that weak acidification does

not have lasting physiological consequences to fish olfaction.

(b) Marine ecosystemsIn contrast to the findings in freshwater fishes, CO2-induced

acidification affects the behavioural response of marine fish

to chemical cues, but does not appear to affect the cue

itself. Orange clownfish larvae reared in control conditions

(pH 8.15) but immediately tested in acidified conditions

(pH 7.6) exhibited the same odour choices as larvae reared

and immediately tested in control water. Larvae reared at

pH 7.6 but tested in control water did not respond to any

olfactory stimuli, just as larvae reared and tested at pH 7.6

water did not respond to odours [21]. This demonstrates

that rearing the fish in acidified conditions causes the behav-

ioural changes, and that the pH of seawater does not seem to

affect the cue, at least for a change in pH of 0.5 unit. Likewise,

using a classical conditioning design, pre-settlement damsel-

fish (Pomacentrus amboinensis) reared in control water were

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able to associate a novel predator odour as a stimulus of

danger when the novel odour was paired with innately aver-

sive alarm cues. However, this ability was compromised in

damselfish larvae reared in acidified water for 4 days, despite

holding the recognition phases of the novel predator odour in

control water [86]. Overall, these results show that exposures

to CO2-acidified water affect the olfactory system and that

the observed changes are not caused by an immediate inter-

ference to the fishes’ olfactory capabilities or chemical

modification of the odour cues.

The mechanism accounting for a loss of olfaction and

chemosensory abilities in marine fishes exposed to acidified

conditions appears to stem from a disruption of the normal

Cl– and/or HCO3– gradients over neuronal membranes,

causing some GABA-A receptors to become excitatory (depolar-

izing) rather than inhibitory (hyperpolarizing) [41]. The

GABA-A receptor, which is the major inhibitory neurotrans-

mitter receptor in vertebrate brains, normally hyperpolarizes

neuronal membranes by opening a channel that leads to

an influx of Cl– and HCO3–. When exposed to high CO2,

marine fishes regulate their acid–base balance by accumulating

HCO3–, with compensatory reductions in Cl– [87]. In the brain,

this may lead to disturbances of the normal Cl2 and/or HCO3–

gradients in some neurons, resulting in gradients that favour

influx rather than out flux of these anions. Changes in HCO3–

and Cl– levels during high CO2 exposures thereby affect

behaviour and cause dramatic shifts in sensory choices [41].

Consequently, it is not the change in seawater pH that impairs

olfaction and chemosensory abilities in marine fishes but

rather the long-term exposure to elevated levels of environ-

mental CO2. This is a critical distinction between the effects of

acidification in freshwater systems that occurs as a result of min-

eral acids, and acidification in marine systems, which is

occurring owing to increased uptake of atmospheric CO2.

Aside from olfaction, neurotransmitter disruption caused

by elevated CO2 levels has far-ranging effects on fishes’ be-

haviour. For instance, elevated CO2 levels led to increased

activity levels in fish [61,78] and interfered with auditory

preferences, whereby no avoidance behaviour occurred

during playbacks of predator-rich daytime reef recordings

[88]. Furthermore, elevated CO2 repressed the degree of be-

havioural lateralization, with individual propensity to turn

left or right becoming random under elevated CO2 [64].

Interestingly, treatments using a specific GABA-A receptor

antagonist (gabazine) administered 30 min before behaviou-

ral assays restored behavioural responses in clownfish,

(A. percula) that had been reared for 11 days in acidified con-

ditions. When tested prior to administration of the receptor

antagonist, the fish exhibited abnormal responses to chemical

cues for a predatory fish. By contrast, they displayed similar

olfaction abilities as fish reared in control conditions follow-

ing the administration of the receptor antagonist, indicating

that altered GABA-A receptor activity mediates the effects

of high CO2 on neural function in marine fishes, including

reversal of olfactory responses [41]. Although neurophysiologi-

cal stress triggered by high CO2 may lead to olfactory-mediated

maladaptive behaviour, it is unknown to what extent the

concomitant changes in pH predicted under future ocean acid-

ification scenarios may directly affect the chemosensory cues.

Behavioural tests conducted under a range of pH while keeping

CO2 constant would resolve this question.

As it pertains to organisms living in brackish conditions

(e.g. estuarine ecosystems), little is known about the potential

effects of acidification on olfactory-mediated behaviour. Euro-

pean eels (Anguilla Anguilla) demonstrated a surprisingly

high level of physiological tolerance towards hypercapnia

[89,90]. Although these experiments were conducted in fresh-

water, such tolerance to hypercapnia is greater than what

can be expected from most teleost fish. Studies conducted on

Atlantic salmon demonstrated that osmotic stress might be

amplified when exposed to acidification [91–93]. As such, it

is not well known how the chemosensory ability of organisms

found in brackish waters will be affected by acidification.

Likewise, for aquatic invertebrates little is known regarding

the mechanisms affecting chemosensorial abilities under

freshwater or marine acidification.

4. Why the difference in response mechanisms?A critical question arising from the research conducted to date

in freshwater and marine ecosystems is why different mechan-

isms should be responsible for otherwise similar effects of

acidification on olfactory responses in these two ecosystems.

In what way are freshwater and marine ecosystems fundamen-

tally different in their respective acidification processes that

affect olfactory functions in aquatic organisms? Answering

these questions is important for predicting the effects of differ-

ent forms of anthropogenic acidification in aquatic systems.

A useful way to contextualize the cause of an apparently

similar impairment to chemosensory functions between

freshwater and marine ecosystems involves comparing their

inherent characteristics of acidification. Freshwater ecosys-

tems often have a pH that is near neutrality but can also be

acidic or alkaline. By contrast, seawater always remains alka-

line. Thus, it might not be the change in pH level but rather

the absolute pH value that determines the non-reversible

changes in chemical alarm cues and impairs olfactory neur-

ons in freshwater fishes, but not in marine fishes. In

freshwater systems, behavioural impairment has been

shown to start occurring in mildly acidic conditions (6.3–

6.5), becoming very evident as pH decreases [38,76,87]. By

contrast, predicted ocean acidification will bring seawater

pH to a more acidic value, but it will still remain alkaline

(i.e. above pH 7.0). In fact, ocean acidification experiments

never get close to the pH levels under which olfaction is

impaired in freshwater. Thus, compared with marine fishes,

freshwater fishes may tend to live in more acidic conditions

and therefore closer to the threshold under which the struc-

ture of the chemical alarm cues is changed [37,85] or the

sensitivity of olfactory neurons is affected [38,46,76]. The

chemical bonds of alarm cue molecules, and probably other

odour molecules, are probably sensitive to the absolute pH

they experience, not just a change in pH per se [51,52]. There-

fore, the reason why studies on acidification in freshwater

have found that changes to chemical stimuli are responsible

for altered behaviour, whereas this does not appear to be

the case in marine studies, is probably that they are working

in different parts of the pH scale (i.e. weakly acidic freshwater

versus weakly alkaline ocean water).

In addition to absolute pH levels, the absolute pCO2 level

experienced is also critical for understanding the potential

causal mechanism of olfactory-mediated maladaptive behav-

iour detected in fish. In ocean acidification studies, both the

pH and pCO2 are manipulated, whereas in freshwater acidi-

fication studies only pH is manipulated. This reflects the

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different mechanism of acidification in these two ecosystems,

and there are important considerations that emerge from

these differences in experimental approach. First, although

the pH of seawater is usually much higher than that of fresh-

water when both are in equilibrium with the atmosphere

(and thus have approximately the same pCO2), freshwater

studies have not manipulated pCO2 levels. Consequently, it

is impossible to conclude that freshwater fishes are more tol-

erant to acidification than marine fishes simply because their

behaviour is not affected at the pH levels at which marine

fishes are highly affected. Secondly, if pCO2 remains rela-

tively unchanged in freshwater that is acidified by acids,

because any CO2 generated is rapidly equilibrated with the

atmosphere, then a similar mechanism of behavioural

change cannot be expected to occur in freshwater fishes com-

pared with marine fishes that are exposed to increased pCO2.

At identical pH levels, there is evidence that CO2 has greater

negative impacts on aquatic organisms when compared with

mineral acids [94,95]. However, further research is needed to

carefully elucidate the various effects of pH and pCO2 in

ocean acidification research. Considering freshwater fishes,

it is unknown whether their sensitivity to increasing pCO2

is the same as in marine fishes. It is possible that freshwater

fishes might be less sensitive to future changes in pCO2

than marine fishes, but to our knowledge this hypothesis

has not been tested.

5. Environmental variability and adaptationsIn order to understand the potential consequences of anthro-

pogenic acidification on olfactory functions of freshwater and

marine organisms, it is necessary to consider the spatial and

temporal variation of pH and pCO2 in their native habitats.

Species living in environments that naturally experience sig-

nificant variation in pH and/or pCO2 may be adapted to

this variation, and thus may be more tolerant of anthropo-

genic acidification compared with species living in more

stable pH and pCO2 environments [96,97]. In freshwater eco-

systems, the magnitude of pH changes may be substantial.

For example, short-term rainfall events (i.e. approx. 30 mm)

may be sufficient to reduce ambient acidity by 0.2–0.6 pH

units [45,98] whereas major rain precipitation events may

have more drastic effects on ambient acidity levels [99].

Additionally, in fresh and brackish water with poor buffering

capacity, daily fluctuations of nearly 1 pH unit may occur as a

function of changing dissolved CO2 concentrations driven by

algal metabolism and growth [100,101]. Likewise, in some

marine habitats, pH gradients can be quite steep. For example,

pH typically fluctuates on a diel cycle in shallow coral reef

habitats, changing by more than 0.5 units in some locations

[102,103]. The cause of this variation in pH is a change in the

balance between the respiratory CO2 production and photo-

synthetic CO2 consumption of reef organisms, including

corals and their symbiotic algae. On shallow reef flats that

have restricted water exchange with the open ocean, pCO2

may vary by over 1000 matm over 24 h, driving the variation

in pH seen in these habitats [104,105]. Variations in pH and

pCO2 are generally much less in reef habitats with greater

water exchange, but may still shift by more than 0.2 units

within a few hours [106], whereas areas of upwelling and

coastal habitats can see sharp pH declines that last for days

to weeks (approx. 0.4 pH units) [102,107]. Thus, significant

shifts in pH may occur in many freshwater and some marine

habitats, and this could influence the tolerance to acidification

of species living within these particular habitats.

There is evidence for adaptation to acidified habitats in

freshwater ecosystems. Natural factors (e.g. rain or snowmelt,

high decomposition rates, sulfur oxides/sulfuric acids

released from volcanic activity and forest fires) may create

extended or even permanent acidification of many freshwater

habitats [3,12,108,109]. Associated with these conditions,

several freshwater fish species have evolved physiological

tolerance towards extreme acidification levels [108,110].

Whether fish in these habitats also experience impaired

olfactory-mediated behavioural responses or have evolved

tolerance is yet to be tested. Interestingly, a shift in sensitivity

of other sensory modalities was shown to occur in juvenile

Atlantic salmon inhabiting weakly acidic streams, displaying

greater reliance on vision to mediate local predation risks

[48]. This suggests that differences in antipredator responses

may occur and be linked to the perceived level of information

available from different sensory modalities (e.g. from losing

chemical information under acidic conditions). However, it

might not simply be the range over which pH varies that is

important for tolerance to acidification, but also the duration

of pH and pCO2 shifts might be critically important. For

example, considerable fluctuations in pH and pCO2 may

take place in shallow coral reef habitat, but they occur on a

regular diel cycle. Low pH and high pCO2 occur at night

for a few hours, and the opposite takes place during the

day [103–105]. These short-term environmental fluctuations

appear to be of insufficient duration to have led to the evol-

ution of tolerance to low pH or high CO2 for extended

periods (days) [41,111]. Indeed, maladaptive olfactory-

mediated behaviour only starts to occur after at least 24 h

exposure to high CO2 [61] in marine fishes. Consequently, a

few hours of high CO2 exposure overnight [104,112] is

probably of insufficient duration to lead to any discernable

effects. A 2–4 day exposure to CO2 conditions similar to

that briefly experienced overnight is enough to cause

impaired behavioural responses in fish [61], indicating that

these short-term exposures to elevated CO2 have not pro-

duced adaptations that overcome longer term exposure to

elevated pCO2.

Unlike most marine systems, fluctuations in pCO2 may

occur for extended periods (e.g. weeks to months) in some

freshwater systems. In large rivers and lakes, O2 saturation

may naturally range from anoxia to super-saturation as a

result of seasonal phenomena [113,114]. Environmental

hypercapnia is common in some freshwater systems, with

the most extreme examples being tropical aquatic environ-

ments [115]. Many freshwater fish have become tolerant to

these O2 fluctuations. In highly productive systems in

which CO2 and O2 are usually inversely related [116], it is

likely that they may also have become tolerant to high CO2.

Thus, if freshwater fish can tolerate periods of lower O2 ten-

sion than most marine fishes, then they may also have

become more tolerant to higher pCO2 tension [117]. Although

complete or significant degrees of physiological pH compen-

sation during hypercapnia have been demonstrated in both

freshwater and marine fish species [118–120], freshwater

fishes were shown to have adapted to the most extreme con-

ditions [117]. Therefore, compared with marine ecosystems,

the extended periods of pH and pCO2 shifts occurring in

freshwater may have set the stage for greater tolerance of

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freshwater fish towards acidification and reduced their

susceptibility to rising atmospheric CO2. Interestingly, signifi-

cant interspecific differences in the sensitivity of reef fishes

to CO2-induced acidification exists [67]. Thus, not all species

will be affected in the same way by acidification, probably

resulting in shifts in assemblage structure favouring more

tolerant species.

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6. Management implications and conclusionsConsidering that species and ecosystem-specific differences

exist in olfactory-mediated responses of fish to acidification,

understanding these critical dissimilarities becomes important

for dealing with the ecological consequences of near-future

acidification in freshwater and marine systems. Management

strategies in a high CO2 world are clearly dissimilar between

freshwater and marine ecosystems. In freshwater, short-term

(and local) mitigations of the effects of acidification may

include the liming of rivers or lakes. This technique by which

ambient acidified water pH may be partially neutralized has

been extensively and successfully used in Scandinavia [121].

Furthermore, emissions of sulfur dioxides and nitrogen

oxides, which drive freshwater acidification, can be curtailed

over relatively short timeframes (e.g. years to decades), as evi-

denced by the success in reducing these atmospheric pollutants

in Northern America and Europe [13]. Consequently, there are

local and regional management strategies that can be used to

help combat the impacts of freshwater acidification. Within

freshwater ecosystems, hatchery-reared fishes are commonly

stocked into natural waterways as part of population recovery

and maintenance programmes. Recent work suggests that the

survival of hatchery-reared fishes may be lower than that of

natural populations [122], partly owing to their inability to

respond to natural predators. This naivety to novel predators

could be compounded if hatchery-reared fishes are stocked

into weakly acidic conditions, under which the ability to rely

on innately aversive chemical alarm cues is reduced [24,42].

Hence, ambient pH or the timing of episodic acidification

events should be included as a variable in stocking efforts to

minimize such predator advantage [123].

By contrast, for marine ecosystems no local solution exists

to temper the effects of acidification on the behavioural

impairment observed in fishes. A strategy of globally limiting

atmospheric CO2 increase is at present the only solution to

ocean acidification, from which freshwater ecosystems

would also benefit following reductions in the severity of

acid rain [7]. A major concern is that atmospheric CO2

levels are set to exceed 500 ppm by 2050 and are on track

to reach at least 900 ppm by 2100 [17,58]. Consequently, in

the second half of this century, the surface ocean will experi-

ence CO2 levels that are known to significantly impair the

behaviour of some marine fishes, unless significant and sus-

tained reductions in global CO2 emission are achieved. Given

the significant interspecific differences in the sensitivity of

reef fishes to CO2-induced acidification [67], not all species

will be affected the same way by future ocean acidification,

probably resulting in shifts in community structure favouring

more tolerant species. Further tests on species-specific response

to acidification will be necessary for managers to predict

which species might be at greatest risk and concentrate conser-

vation efforts on these species. Limiting other stressors and

impacts, for instance fishing mortality, may be one way to

help maintain viable populations of sensitive species and to

reduce the impacts of acidification on assemblage structure

and ecosystem function.

A major unknown in predicting the future impacts of

acidification, in both freshwater and marine ecosystems, is

the capacity for adaptation to lower pH and/or higher

pCO2. The different mechanisms of impaired olfactory-

mediated behaviour in freshwater (e.g. degradation of che-

mosensory molecules, reduced olfactory/chemosensorial

sensitivity) and in marine (e.g. altered neurotransmitter func-

tion) fishes (and potentially in invertebrates) might lead to

different prospects for adaptation. Little is known regarding

the potential of freshwater fishes’ olfactory systems to adapt

to the loss of relevant chemical cues in low pH water. It is

possible that there could be selection favouring individuals

that respond most strongly to low levels of olfactory cues; how-

ever, other sensory modalities (i.e. vision) may also play a

heightened role if chemical cues become deactivated [38,48].

In marine habitats, adaptation through selection on individual

variation in CO2 sensitivity could occur. Variation in the level

of behavioural impairment exhibited by individual reef fishes

exposed to elevated pCO2 has been observed in some studies,

with some individuals apparently more resistant to the effects

of elevated CO2 than others [61]. Furthermore, natural selection

favouring CO2 tolerant individuals has recently been demon-

strated under natural conditions [111]. Consequently, there is

clearly the potential for natural variation in sensitivity among

individuals to lead to genetic adaptation in marine fishes.

Understanding the adaptive potential of marine fishes to elev-

ated CO2 and the speed at which adaptation might occur will

be critical in predicting the impacts of acidification on marine

ecosystems. Ultimately, an evolutionary perspective will be

required to assess the likely ecosystem effects of acidification

in both freshwater and marine ecosystems.

Acknowledgements. The authors wish to thank Howard Browman fororganizing ‘Workshop on acidification in aquatic environments:what can marine science learn from limnological studies of acidrain?’ from which this work stemmed. We also wish to thank thereviewers of this manuscript for providing constructive comments.

Funding statement. This work was supported by a grant from Coordena-cao de Aperfeicoamento de Pessoal de Nıvel Superior (CAPES) PVEto A.O.H.C.L. P.L.M. is supported by the Australian Research Coun-cil. M.C.O.F. is supported by the Natural Sciences and EngineeringResearch Council of Canada.

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