The effects of H+ upon the gills of freshwater fish

D. G. MCDONALD
Department of Biology, McMaster University, Hamilton, Ont., Canada L8S 4 K l
Received October 7, 1982
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MCDONALD,
D. G. 1983. The effects of H + upon the gills of freshwater fish. Can. J . Zool. 61: 691-703.
The structure and function of the gills of freshwater fish are briefly summarized and the responses to low pH are reviewed, with
particular reference to the salmonid fishes. Major influences are seen upon ion and acid-base regulatory mechanisms at the gills
and upon mucous secretion and gill structure. Ionic imbalances which can be responsible for death at low pH are caused by
disturbances to both the active transport and the diffusional losses of Na+ and C1-. The disturbances, while not identical for the
two ions, are similar to the extent that the undirectional fluxes of both are dependent upon the severity and duration of the acid
exposure and upon the external calcium concentration. Calcium also has an important influence on the net flux of H+ across the
gills. A model is proposed for the interaction of c a 2 + and H+ on gill function. The physiological importance of mucous secretion
at low pH is discussed and the issue of whether acid-intolerant species can be successfully adapted for life at low pH is examined.

MCDONALD,
D. G. 1983. The effects of H + upon the gills of freshwater fish. Can. J. Zool. 61: 691-703.
La structure et le fonctionnement des branchies de poissons d'eau douce sont rCsumCs sommairement et leurs rCactions a un pH
faible ont fait I'objet d'une rCvision, en particulier chez les salmonidCs. Le pH faible semble affecter en particulier les
For personal use only.

mCcanismes de contrdle des ions et de 1'Cquilibre acide-base au niveau des branchies, de m2me que la sCcrCtion de mucus et la
structure des branchies. Le dCsCquilibre ionique qui peut entrainer la mort a un pH faible est causi par des traumatismes dans le
transport actif et par les pertes par diffusion des ions Na+ et C1-. Les traumatismes, bien que non identiques pour les deux ions,
sont semblables en ce que les mouvements non dirigks des deux ions dependent de la gravitC et de la durCe de I'exposition a I'acide
et de la concentration en calcium du milieu. Le calcium a Cgalement un effet important sur la circulation nette de H+ dans les
branchies. Un modele est proposk ici pour reprksenter I'interaction de c a 2 + et de H + sur les branchies. L'importance
physiologique de la sCcrition de mucus 21un pH faible de m2me que la possibilitk d'adapter des especes intolirantes 21I'acide 21un
mode de vie en pH faible font I'objet d'une discussion.
[Traduit par le journal]

Introduction
Acid rain and snowfall, primarily a consequence of
sulphur dioxide emissions from industrial sources, is
now prevalent in many parts of the world. The impact
upon the environment has been greatest in Scandinavia,
in the northeastern United States, and in eastern Canada,
areas where poorly buffered and, therefore, highly
sensitive watersheds are common. Fish dwelling in
these waters have, for several years, been experiencing
progressively increasing levels of acidity and as a result,
populations have decreased or are seriously threatened
in thousands of lakes (Schofield 1976; Muniz and
Leivestad 1980; Harvey et al. 1981). This serious
environmental problem has prompted a considerable
amount of research on the ecological and physiological
consequences of low pH to fish, research which has now
been quite thoroughly summarized in several reviews
(Schofield 1976; Leivestad et al. 1976; Harvey 1979;
Fromm 1980; Haines 1981; Harvey et al. 1981; Wood
and McDonald 1982). In light of the broad scope of
these earlier reviews, this one will focus upon the
responses of the gills of freshwater fish to low pH. The
point of view adopted here is that the gills are the
primary target of elevated hydrogen ion activity in the
environment. Thus, many of the effects of low pH upon
freshwater fish can be more fully understood by
appreciating the nature of the interference of H+ with
normal gill functions.
In order to review this subject, it is useful to start with
a brief summary of the structure and function of the gills.
Figure 1 has been prepared for this purpose and is a
distillation of information from several sources. For
more detailed information, the reader should consult
such reviews as Motais and Garcia-Romeu 1972, Maetz
1974, Maetz et al. 1976, Potts 1977, Kirschner 1979,
Evans 1980, and Rankin and Davenport 1981.
The gills of freshwater fish serve a variety of
functions. First and foremost, they are organs for the
exchange of respiratory gases, a function which
necessitates a large and permeable surface area. As a
consequence, the gills are also the main site for the
diffusional loss of ions (normally about 10% or less
occurs in the urine; McDonald and Wood 1981). The
principal components of these losses are sodium and
 CAN. J. ZOOL. VOL. 61, 1983

GlLL LAMELLAE
GILL ARCH GILL FILAMENTS 6
Water Flow
Pavement Cell

Afferent
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\\
, y( (kv-~fferent
M \ X Filament
Filament
Cartilage

BASO-LATERAL
C MEMBRANE
BASO-LATERAL APICAL
INNER OUTER APICAL
MEMBRANE MEMBRANE
PILLAR EPITHELIAL EPITHELIAL MEMBRANE
CELL LAYER
For personal use only.

N a + d
ATP
7- K+ / Na+-TNa
BASAL
LAMINA

FIG. 1. Structure and function of the freshwater fish gill. (A) Horizontal section through the head showing gill arches with
filaments. Note direction of water flow. (B) Section of gill filament showing individual secondary lamellae. Note patterns of
blood and water flow and different cell types. The most common epithelial cells are the pavement or "respiratory" cells which
cover most of the suface of the lamellae and the filaments. The "chloride" cells, which are so called because of their staining
properties and their role in salt secretion in seawater (Keys and Willmer 1932), make up only about 7% of the gill surface area in
freshwater (Potts 1977). These large cells are confined primarily to the filament epithelium (Olson and Fromm 1973) where they
are deep set and largely overlain by pavement cells. Their function in freshwater is uncertain but excretion of organic molecules
(Masoni and Payan 1974) and active transport of calcium (Payan et al. 1981) have been suggested. Earlier reports of an important
role in NaCl absorption (e.g., Bierther 1970) have been disputed by Girard and Payan (1980). The third type are the mucous cells
which are abundant on the leading edge of the filaments (Laurent and Dune1 1980) a position enabling the dispersal of their
secretions by the branchial water flow. (C) Cross section through the lamellar epithelium showing cell layers. In the outer layer
the cells are firmly bound together by apical tight junctions and by desmosomes (Morgan and Tovell 1973; Sardet et al. 1979).
The connection with the inner layer is more tenuous and the result is that the basolateral border of the outer layer is often in direct
contact with extracellular fluid (Laurent and Dune1 1980). The apical surface of the outer layer is projected into many folds or
microvilli (Morgan and Tovell 1973; Olson and Fromm 1973). These microvilli, together with more prominent surface ridges on
the filament epithelium (Hossler et al. 1979; see Fig. 1B), likely serve to anchor a protective mucous layer to the outer gill surface
(Hughes 1980). (D) Model of ion diffusion and transport in the freshwater fish gill. In freshwater the lamellar layer of pavement
cells is likely responsible for most of the ~ a and
+ C1- active absorption from the medium (Girard and Payan 1980; Haswell et al.
1980). This transport is thought to operate largely by independent, electroneutral Na+/H+, NH4+ and Cl-/HC03-, OH-
exchanges localized on the apical membrane (Kirschner 1979). Ion-specific ATPases, responsible for transport, are membrane
bound to the basolateral membrane in the case of Na+ /K+ ATPase (Payan 1978) and possibly to the apical membrane in the case
of Cl-/HC03- ATPase (Kerstetter and Kirschner 1974). Ion diffusion, under normal circumstances is thought (Sardet et al. 1979)
to occur largely via a transcellular route (---), but paracellular movements (---) may also be important (Steen and
Stray-Pedersen 1975).
 chloride (Fig. 1D) , reflecting their predominance in
extracellular fluids, but lesser amounts of potassium,
calcium, and bicarbonate are lost as well. These ion
losses are compensated by active transport mechanisms
which are primarily localized in the gill epithelium
(Maetz 1974; Dacke 1979). At the gill, sodium uptake is
thought to be independent of chloride uptake (Maetz et
a1. 1976; Kirschener 1979; Evans 1980), with the uptake
of both occurring as a result of electroneutral exchanges;
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Na' /H' (or NH4') and Cl-/HC03- (or OH-) (Fig. 1D) .
Less is known of potassium and calcium uptake, but the
gill chloride cell (Fig. 1B) is thought to play an
important role in the latter (Payan et al. 1981).
Bicarbonate is a special case because of its importance to
C02 excretion and acid-base balance. Its uptake at the
gills is equivalent to the excretion of H' and both are
controlled by the branchial mechanism of acid-base
regulation. This mechanism is thought to operate by the
manipulation of the relative intensities of Na' and C1-
uptake, increased Na' uptake correcting an acidosis and
increased C1- uptake, an alkalosis (Cameron 1978;
Heisler 1982). In this, the gill assumes much of the role
that is the sole responsibility of the kidneys of terrestrial
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vertebrates. Finally, the gills are the principal site of
nitrogenous waste excretion. Over 90% of the ammonia
produced in the body is excreted by the gills (Maetz
1973; McDonald and Wood 1981) employing a
combination of ionic and nonionic diffusion (NH4' and
NH3) and exchange against Na' (Kormanik and
Cameron 1981). Thus, when one considers the
functional complexity of the fish gill together with its
structural delicacy and intimacy with the external
environment (Fig. I), it is easy to imagine that the
primary impact of H' will be upon the gills and that a
variety of disturbances will be produced.
On the basis of published work it is now possible to
propose a fairly detailed model for the responses of the
gill to elevated H' ,but the present information has some
limitations which should be considered. First, the full
range of acid pH levels that a fish species is likely to
encounter has not been thoroughly studied. Although
investigations to date have used pH levels ranging from
2.8 to 6.5 and have analyzed disturbances to various
aspects of gas exchange, acid-base, and ion regulation
(see reviews by Fromm 1980; Wood and McDonald
1982), much of the studies have concentrated on the
acutely to chronically toxic pH range of 3.5 to 4.5. In
particular, much less work has been done at the
presumed threshold for pH effects upon the gills, pH
levels in the range of 5.0 to 6.5. Secondly, in many
studies the concentrations of ions other than H' have not
been controlled or have not been specified, nor has
attention been paid to the prior acclimation history of the
fish. This is particularly important in the case of external
calcium, as discussed below. Thirdly, there are now
well-established interspecific differences in tolerance to
acid stress which tends to complicate comparisons
among species. Even within the salmonids, upon which
much of the work has been done, there appear to be
major differences. The brown trout, Salmo trutta, and
the rainbow trout, Salmo gairdneri, are apparently the
most sensitive to acid stress (Overrein et al. 1980)
whereas the brook trout, Salvelinus fontinalis, is
generally regarded as the least sensitive (Dunson and
Martin 1973; Daye and Garside 1975). Even greater acid
tolerances are seen among such teleosts as the white
sucker, Catostomus commersoni (Harvey 1979), the
perch, Perca sp. (Harvey 1979, Muniz and Leivestad
1980), the roach Rutilus rutilus (EIFAC (European Inland
Fisheries Advisory Commission) 1969), and the
characinid fishes of the Amazon Basin (Dunson et al.
1977). All of these fish species, as a rule, can survive at
pH levels lethal to most salmonids.
Despite the limitations of the present data, however,
seven major effects of pH upon the gills can be identified
(ranked in approximate order of their occurrence with
declining pH): inhibition of Na' and C1- uptake
mechanisms, increased ion permeability and diffusional
ion efflux, increased hydrogen ion permeation of the
gills, enhanced mucus production and release, mucus
coagulation and precipitation, inhibition of gas transfer
across the gills, and damage to and separation of gill
epithelial layers. This list has been compiled largely on
the basis of observations on salmonid fishes, and it
should be kept in mind that it may apply only to this
group and to other, similarly intolerant, species.
However, as discussed in the last section of this review,
it does appear that H' may have similar types of effects
upon resistant species; the primary difference being
simply that a more severe exposure is required to
produce the effects.
Ionoregulatory disturbances at low pH
In this section I have included the ionoregulatory
responses of amphibian skin to low pH as these
responses have been more thoroughly examined,
particularly in vitro. Furthermore, much of the findings
should be relevant to fish since there are strong functional
similarities between the two transport epithelia (Motais
and Garcia-Romeu 1972; Kirschner 1979). From
several studies on amphibian skin (reviewed in Motais
and Garcia-Romeu 1972) it is evident that acidification
of the external medium causes an inhibition of the active
transport of ions across the epithelium. Furthermore,
sodium u take (J,F+) is apparently more readily
inhibited tffan chlonde uptake ( ~ 6 - both
) in vivo and in
vitro. At a pH of about 4.0, J?:+ inhibition is usually
about 50% complete (Garcia-Romeu et al. 1969;
Mandel 1978) while J-: is either unaffected (in vivo,
Garcia-Romeu et al. 1969) or is mildly stimulated (in
 694 CAN. I . ZOOL. VOL. 61, 1983
vitro, Ussing 1949). It is also evident that diffusional ion
losses increase at low pH but, as a rule, large increases
are not seen until pH levels are reached that are lower
than those inhibiting influx (Schoffeniels 1956;
Gonzalez et al. 1976). In toto, these observations can be
interpreted as evidence that the active transport
mechanisms for Na' and C1- are independent of one
another and also that ionic efflux at low pH occurs by
pathway(s) through the epithelium that are separate from
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the inward transport pathways. Further, the inhibition of
J?' and not of J;' can be explained by supposing that
fixed charges play an important role in the initial entry
step of both ions. Hydrogen ion titration of negative
charges in Na' -specific channels would act to restrain
the access of Na' to its transport mechanism but would
have little or no effect on neutral or positively charged
C1- channels (Motais and Garcia-Romeu 1972).
In the fish gill, the inhibitory effect of H+ on Na'
influx was first shown by Packer and Dunson (1970) and
has now been well established. Inhibition begins at pH
levels as high as pH 6.0 (Maetz 1973; McWilliams and
Potts 1978) and is typically nearly complete, in
acid-intolerant species, upon acute exposure to pH 4.0
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(Packer and Dunson 1970; McWilliams and Potts 1978;
McWilliams 1980a; McDonald et al. 1983). Effects on
Jgl- have been less thoroughly examined. However,
J:; was unaffected by pH 6.0 in goldfish, Carassius
auratus (Maetz 1973). Also, McWilliams (1980b),
quoting unpublished observations, indicated that a
reduction of pH from 7.0 to 4.0 actually reduced C1- loss
across the gills of brown trout by a factor of 2.5. The
latter is contrary to observations of marked plasma C1-
losses at pH 4.0 in a number of studies (Leivestad and
Muniz 1976; McDonald et al. 1980; Ultsch et al. 1981;
Booth et al. 1982) and a report of ~7;- inhibition at this
pH in rainbow trout, Salmo gairdneri (McDonald et al.
1983). Nevertheless, the initial inhibition of ~7;- in the
trout was less than that of JF' (McDonald et al. 1983)
which supports the view that the responses of the frog
skin and the fish gill to low pH are at least qualitatively
similar. The major diiferences are of a quantitative
nature; the ionic fluxes through frog skin appear to be
less sensitive to acid exposure than those of the fish gill
at least as far as salmonid fishes are concerned.
Whether ion uptake remains inhibited with continued
acid exposure has recently been explored in the fish gill.
The available evidence indicates that some recovery is
possible but the degree of recovery is dependent upon
pH, time, and upon the external calcium concentration.
McWilliams (l980a, 1980b) showed that J?:' in brown
trout recovered completely after 10 days at pH 6.0, but
recovery was only about 20% at pH 4.6 and virtually nil
at pH 4.0 by this time. McWilliam's experiments at pH
4.6 and 4.0 were conducted in very soft water (Ca2+ =
0.03 mequiv./L). Similarly, when rainbow trout were
maintained in soft water (ca2+ = 0.06 mequiv./L) !a
pH 4.0, there was very little recovery of J:
(McDonald et al. 1983). At higher c a 2 + levels (up to
5.7 mequiv. /L) recovery of J?:' was significantly
greater although it was not complete. [ca2+]and pH had
similar, although generally smaller, effects upon ~7;-
(McDonald et al. 1983).
With regard to the influence of H+ on the passive
efflux of ions (JZ' and ~ 2 : ~the~ )conclusion drawn from
frog skin studies is that low pH modifies the epithelium
from being characteristically "tight" to being "leaky." In
"tight" epithelia most of the Na' and C1- diffusive efflux
is thought to occur by a transcellular route, i.e. through
both the basolateral and apical membranes of the
epithelial cells (see Figs. 1C and 1D) while in "leaky" or
highly permeable epithelia such as gall bladder and
proximal kidney tubule, most of the efflux (>90%) is
actually paracellular, i.e. through the tight junctions
between epithelial cells (Erlij and Martinez-Palomo
1978). Gonzales et al. (1976), by following movements
of normally impermeant nonelectrolytes such as sucrose
and mannitol across the frog skin in vitro, concluded
that low pH (pH G3 .O) opened the paracellular pathway
to diffusion. Since tight-junction permeability is
restricted, at least in part, by calcium bound to fixed
negative charges in the intercellular cement (Oschman
1978), Gonzalez et al. proposed that Ca2+was liberated
from the epithelium by low pH titration, thus opening
the junctions to ionic efilux. In support of this
hypothesis they noted the appearance of Ca2+ in the
external medium upon acidification.
In the fish gill, the site and mode of action of H+ on
ion permeability is not known but large increases in
ionic efflux are typical of salmonids acutely exposed to
low pH (Fig. 2). This tends to argue for a paracellular
locus for the effects of H+ because the alternative, a
large increase in transcellular permeability, would
probably require modification to both the apical and the
basolateral membranes (Fig. 1C) .
Current studies indicate that low pH has similar,
though not identical, effects upon Na' and C1- efflux.
The threshold for stimulation of Na' efflux ( ~ 2 ' )in
salmonids is between pH 5.0 and 6.0 and the effect is
progressive with pH reduction (McWilliams and Potts
1978; McWilliams 1980b; McWilliams 1 9 8 2 ~ )At . pH
4.0, for example, J$ :' can be up to 10-fold higher than
normal (Packer and Dunson 1970). With influx virtually
completely inhibited at this pH the large JZ' resulted in
a net loss of body Na' of about 10%/h. A further
reduction to pH 3.0 increased the Na' loss to about
50%/h and very rapidly resulted in the death of the fish
(Packer and Dunson 1972).
The effect of low pH on JZ- has not been examined
as extensively. However, in rainbow trout, the acute
increases in JZ; upon exposure to pH 4.0 were similar
 to or greater than J~N.",' (McDonald et al. 1983). External
calcium, as discussed below, had a pronounced
influence on the degree to which ~ 2 ,increased
- with low
Jin
C
-
pH. 0
Y

There are two aspects of the effect of pH on the ionic C

'r
permeability of fish gills which are noteworthy since
they provide some insights into the mechanism of action -3
.>
of H+. Firstly, the effect of H+ appears to be quite u
a
=a

similar to that exhibited by two polyvalent cations, CI

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lanthanum ( ~ a " , on goldfish; Eddy and Bath 1979) and Jout

poly-L-lysine (PLL; on rainbow trout; Greenwald and
Kirschner 1976). These substances, at concentrations of
2.0 mmol/L and 0.001 mmol/L, respectively, cause 5-
to 10-fold increases in both JZ' and JZ-.The effect of
h3+ is particularly interesting because ~ a is ~thought
+ to
specifically bind to and precipitate glycoproteins in the
intercellular cement (Oschman 1978) and in this respect
may be operating as a c a 2 + antagonist. These findings J in
reinforce the view that H+ increases the permeability of
the gills by displacing c a 2 + from the paracellular diffu-
sion pathway.
The second feature which H+ , ~ a ~and + PLL
, share is Jout
that the increase in ion pemeability which they produce
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declines rapidly with time (Greenwald and Kirschner

1976; Eddy and Bath 1979; McDonald et al. 1983). For
~ a " and PLL, normal permeability was restored by
about 4 h while at pH 4.0 (or 0.1 mmol/LH+), ion
efflux fell from 6 x to 2 x normal by 4 h and was normal
within 40 h (McDonald et al. 1983; see Fig. 2). The
mechanism by which the increase in permeability is
rapidly reversed with the continued presence of the
stressor is unknown. It is unlikely to be due to any
significant change in the ion diffusion gradient as plasma
ion levels decline too slowly (Eddy and Bath 1979;
McDonald et al. 1980). It could have a hormonal basis;
there is now evidence that secretion of the ionoregula-
tory hormones, cortisol (Aschom 1979) and prolactin
(Notter et al. 1976), is stimulated by acid exposure but
again, hormonal effects usually require a minimum of
several hours before they are expressed. Another
possibility may be a restiction of perfusion of lamellar
surfaces thereby reducing ion losses (i.e., a change in
functional surface area rather than a true change in
permeability). 'In this regard, Jackson and Fromm
(1980) report an immediate increase in vasomotor tone
in the isolated trout gill upon acid exposure (pH 3.5).
Although the significance of this observation is
uncertain it does suggest that gill tissue is capable of
rapid adjustments. Finally, the epithelial cells them-
selves may respond directly to restrict the ion loss. Fibrillar
elements in the desmosomes, the organelles which seal
epithelial cells together (Fig. 1C) are contractile and
thus may be able to reduce the spacing between cells and
thereby restrict the paracellular diffusion channels
(Geise 1979).
FIG.2. A possible model for the pH and time dependence of
unidirectional ion fluxes (Jin and J,,,) across the gills of
freshwater fish. Shaded area indicates net. ion flux. Note the
rapid recovery of J,,, and the slower and only partial recovery
of Jin.Values extrapolated from measurements on Na+ fluxes
in rainbow trout in high Ca2+ water (5.7 mequiv. /L) at pH 4 . 0
and 7 . 0 (McDonald et al. 1983). A plot for C1- fluxes would
show a similar pattern except for smaller disturbances in hi h
k+
c a 2 + and larger disturbances in low ~ a ? . Also, in low Ca
there would be less recovery in both J:" and J ~ A - . At +40 h,
no values are shown below pH 4 . 0 because the rainbow trout
would not have survived to this point. See text for further
details.
Whatever the mechanisms for the reversal of the
permeability increase, the fact that it occurs indicates
that the rate of diffusional efflux of ions across the gills
will be strongly time dependent, as well as being pH
dependent. Similar conditions also apply to the
inhibition of influx, as previously discussed. The
complex nature of these effects is suggested by Fig. 2,
which is a model based on Na+ movements in the
rainbow trout (McDonald et al. 1983). Since the
inhibition of J$'- and the recovery of J,S:- are not
identical to that of Na+ (McDonald et al. 1983), the
 696 CAN. J. ZOOL. VOL. 61, 1983
complete picture for ion fluxes at the gill will be more
complex than indicated in Fig. 2. Furthermore, an even
greater level of complexity is added when the effects of
external calcium are considered. Not only does calcium
reduction increase the net losses of ions across the gills
at low pH (McDonald 1983) but it affects Na+ and C1-
movements in qualitatively different ways as discussed
below.
The influence of external calcium
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In an ecological context, water-borne calcium is
important to fish populations since waters low in
calcium and other dissolved minerals are generally low
in productivity (Willoughby 1976) and by virtue of their
low buffering capacity (i.e., low [CaC03]) are
susceptible to acidification. Calcium is also of
considerable physiological importance. It has well-
known effects on the stability and permeability of
biological membranes (Schoeffeniels 1967) and when
added to water will improve the survival of marine fish
in freshwater (Carrier and Evans 1976) and that of
freshwater fish in acidified water (Leivestad et al. 1980;
McDonald et al. 1980; Brown 1981; McDonald 1983).
For personal use only.
In fact, in a survey of 700 acid lakes (pH 4.5 and 5.5) in
southern Norway, Wright and Snekvik (1978)
concluded that calcium was at least as critical to fishery
status as was pH.
To understand the ameliorative effects of calcium at
low pH it is of value to first examine its normal
interactions with gill function. In neutral waters calcium
probably exerts most of its effect as a result of being
bound to the gills. While it is likely that the binding
activity for calcium varies with species it is generally
true that continued washing of animals in deionized
water (Eddy 1975) or the use of chelating agents
(Cuthbert and Maetz 1972) is required before the effects
of calcium removal from the medium are expressed.
Furthermore, calcium is probably bound to at least two
separate sites involved in ion regulation: to the tight
junctions, as previously discussed in reference to frog
skin and to the Na+-transport channels in the apical
membrane. The evidence for these loci is that calcium
removal from the external medium provokes 5- to
10-fold increases in J%+ (increased junctional per;
meability?) and somewhat smaller increases in 4:"
(Cuthbert and Maetz 1972; Eddy 1975). The latter
presumably occurs because the access of Na+ to its
transport channel is increased. This is supported by
observations on the action of amiloride; a highly specific
sodium channel blocker in a wide variety of epithelia
(Benos 1982). Amiloride inhibition will not occur
without the presence of external ca2+ (Cuthbert and
Wong 1972) which suggests that calcium is required for
amiloride binding and thus is normally present in the
Na+ channel.
Calcium appears to have a somewhat smaller
influence on C1- movements. The effect of c a 2 + upon
J$'- has not been studied but ~$1,- is not affected as
much by Ca2+ removal as is JZ' (Eddy 1975) nor is it
reduced to the same extent by c a 2 +addition to deionized
water (Maetz and Bornancin 1975). Nevertheless, in
goldfish kept in deionized water, plasma chloride losses
did occur, although they were not as large as the sodium
losses (de Renzis and Maetz 1973).
Although the removal of external calcium causes
ionic disturbances, fish eventually reach a new
equilibrium at lower plasma ion levels (de Renzis and
Maetz 1973; Olivereau et al. 1980) and some species
such as the eel Anguilla anguilla and the goldfish are
apparently capable of indefinite survival in this
environment (Olivereau et al. 1980). Furthermore, if
fish are maintained in calcium-depleted but otherwise
normal freshwater they are usually able to fully recover
their original ion balance (McDonald et al. 1980;
McDonald 1983). This recovery process (which
requires at least a week and may require much longer
depending on species and on water ion levels;
McDonald et al. 1980; Olivereau et al. 1980) appears,
from recent studies on the rainbow trout (McDonald et
al. 1983), to involve primarily a gradual reduction in ion
permeability rather than a stimulation of ion uptake.
This conclusion is based on the observation that following
1-2 weeks of acclimation to low ca2+ (0.06
mequiv. /L) the gill turnover rates for Na+ and C1-,
measured at a constant Na Cl,,, of 0.3 mmol/L, were
similar to or actually lower than those measured in
animals in high c a 2 + (5.7 mequiv. /L). The mecha-
nism(~)responsible for this branchial adjustment to low
ca2+ are not fully known, but an enhanced secretion of
the pituitary hormone prolactin may play an important
role (Wendelaar Bonga 1978; Olivereau et al. 1980).
Suggestions as to the precise role of prolactin in this
instance have included a direct effect upon diffusion
channel permeability (Bern 1975) and an increase in the
calcium binding capacity of the gills (Wendelaar Bonga
1978). The latter implies that prolactin, in essence, may
be creating with low Ca2+ acclimation, a high calcium
microenvironment at the surface of the gills.
In view of the above, it is not surprising that external
calcium will modulate the effects of hydrogen ions on
ionoregulating mechanisms and that the effect will
depend upon ca2+ acclimation history. Acute reduction
of both Ca2+ and pH in brown trout increased JZ'
substantially more than that caused by pH reduction
alone (McWilliams 1 9 8 2 ~ )When,
. however, fish had
been first acclimated to low calcium and then acid
exposed the effects of external ca2+ were much less
pronounced. J,;' considerably elevated in rainbow
trout upon initial exposure to pH 4.0, was virtually
unaffected by water-borne c a 2 + over a nearly 100-fold
 range in concentration (McDonald et al. 1983).
Nevertheless, effects of Ca2+were still evident; animals
acclimated to low Ca2+ showed initially a substantially
greater ;:J than animals acclimated to high c a 2 + and,
although;:J and ::J: eventually declined to normal
levels, irrespective of [Ca2+], there was a persistent
inhibition of J?' and JE1-that was more pronounced at
low ca2+. These two effects, the initially higher J&lt-
and the greater inhibition of active ion uptake, are thus
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together responsible for the elevated plasma ion losses
which occur in acidified soft water compared with hard
water (McDonald et al. 1980; McDonald 1983;
Leivestad et al. 1980) and also likely account for the
increased mortality in this environment (McDonald
1983).
The question thus arises as to the precise nature of the
interaction of Ca2+and H+ upon NaCl fluxes across the
gills. While it is likely that this interaction is complex
and not constant with time, it is possible to advance
some plausible explanations for its various components.
A probable explanation for the initially large passive
ionic effluxes at low pH is that H+ displaces c a 2 + from
the gill paracellular diffusional channels, as previously
For personal use only.
discussed. Indeed, exposing fish to deionized water
produces plasma ionic and acid-base disturbances (cf.
de Renzis and Maetz 1973) very similar in nature to
those produced by low pH exposure, particularly when
the latter is conducted in hard water (McDonald et al.
1980). Under the circumstance where both external
[ca2+] and pH are low, then the displacement of
membrane-bound Ca2+and, therefore, ion losses would
be increased. While prior acclimation to low c a 2 + will
reduce this effect, it cannot abolish it entirely because
ca2+ is likely to retain an important control over
membrane permeability. The observation that low
ca2+-acclimated animals initially lose Cl- more rapidly
at low pH than high ca2+-acclimated animals can also
be explained by supposing that diffusion channels are
physically restructured in the process of low Ca2+
acclimation. For example, these channels may be
altered from being cation selective (possessed of a high
density of negatively charged residues) to being neutral
or anion selective. This change, which would be distinct
from any increase in gill Ca2+-binding activity that
might be occurring with low Ca2+ acclimation, would
specifically favor C1- diffusion when the channels were
opened by low pH exposure.
Finally, the observation that low :a2+ prevents or
limits the recovery of ion uptake (JEa and JE-) during
low pH exposure is more difficult to explain. The effect
is probably not related to the displacement of c a 2 +from
Na+ transport channels as this displacement increases
JP' at neutral pH (Eddy 1975) and indeed, does so
under mildly acid conditions in soft water (pH 5.5, Ca2+
S 0.5 mequiv./L; McWilliams 1 9 8 2 ~ ) Since . both
JP' and J$'- are inhibited at low pH and remain so at low
ca2+ and low pH, it is more likely that external c a 2 +
exerts a general effect upon membrane stability and
upon the structure of proteins in the ion transport
channels. However, ,the nature of this effect remains to
be elucidated.
Acid-base disturbances at low pH
Low pH environments are likely to interfere with
acid-base regulation by the gills in two important ways.
Firstly, the inhibition of active ion transport will disrupt
Na+/H+ and Clh/HC03- exchanges and thus disable
rapid responses by fish to blood acid-base disturbances
(cf. Heisler 1982). Although the kidney remains quite
competent in acid-base regulation at low pH
(McDonald and Wood 1981) it cannot fully replace the
function of the gills as it responds very slowly to acid
challenge (Wood and Caldwell 1978). Secondly and
more importantly, the gills can act as a major route for
H+ ion permeation of the body fluids when external [H+]
is elevated. McWilliams and Potts (1978) recorded
marked changes in transepithelial potential in brown
trout when pH was reduced from pH 7.0 to 3.5 and
concluded that, in fact, the gills were about 1000-2000
times more permeable to H+ than Na'. This high
permeability has been confirmed with recent measure-
ments of a substantial net influx of H+ (J:~:) across the
gills at pH 4.0 (Ultsch et al. 1981;McDonald and Wood
1981; McDonald et al. 1983) and of the development of
a blood acidosis (Neville 1979; Packer 1979; McDonald
etal. 1980;Ultsch etal. 1981). AtpH 3.5 the J,Hd inthe
carp, Cyprinus carpio, was even higher than at pH 4.0
and the resulting acidosis more severe (Ultsch et al.
1981). In this instance the acidosis was likely of
sufficient magnitude to have been the direct cause of
death.
The net influx of H+ at low pH is similar to Jd;',- and
';5
: in that it declines with continued low pH exposure
(Ultsch et al. 1981; McDonald et al. 1983). This
suggests that there is a time-dependent reduction in
epithelial permeability to H'. This interpretation,
however, is complicated by the fact that branchial
ammonia excretion also increases substantially during
acid exposure (McDonald 1983; McDonald et al. 1983).
Since a variable proportion of this excretion can occur as
NH4+ as opposed to NH3, and since NH4+ and H+
excretion are equivalent, then NH4+ excretion could
have accounted for the decline in.::J This question
cannot be further resolved at the present time because it
is not possible to separately measure NH3 and N H ~ +
excretion nor, in fact, to measure the undirectional
fluxes of H+. It should be noted, however, that since
JP' remained low in the acid-exposed trout, particularly
in soft water (McDonald et al. 1983), that the
 698 CAN. J . ZOOL. VOL. 61, 1983
contribution of Nat/NH4+ exchange (Fig. 1D) to Ht
excretion would be small.
External calcium, in addition to its effects upon ~ a '
and Cl- regulation, also has an effect upon J,$at low
pH. Over relati~elylong-term exposures (4-5 days at
pH 4.0-4.5) ,J: was lower in rainbow trout acclimated
to soft water than in animals acclimated to hard water
(McDonald 1983). In the former, J:;declined to zero in
1-2 days and there was virtually no detectable blood
Can. J. Zool. Downloaded from www.nrcresearchpress.com by RICE UNIVERSITY on 08/08/17
acid-base disturbance, while in the latter a significant
acidosis gradually developed that was similar in propor-
tion to that caused by severe exercise (Heisler 1982).
These results suggest that the gills of fish in low pH soft
water are, in fact, less permeable to H+ than those of fish
in low pH hard water, particularly since there is no evi-
dence for increased branchial or renal H+ excretion in the
former (McDonald 1983; McDonald et al. 1983). The
basis for this phenomenon is uncertain but it may be related
to the higher chloride permeability in soft water. If, in-
deed, the diffusion channels in the low ca2+-acclimated
animal are no longer cation selective then these channels
would favor equimolar ~ a and + C1- efflux and the
inward diffusion of H+ would be less favored.
For personal use only.
Certainly, there is a good correlation between the net
movements of Na+ and C1- and the net movements of
H+. In soft water acclimated and acid-exposed trout the
low ,J: was correlated with nearly equimolar net losses
of Na+ and Cl-, while in hard water the high J z w a s
correlated with a large':J: and a small ~2:- (McDonald
1983). These are not surprising findings in themselves
because charge balance across the gills must be
maintained. However, they do serve to emphasize the
important conclusion that ion and H+ movements are
interrelated at the gills and, also, that hydrogen ions
need not permeate to the body fluids in order to have
toxic effects upon fish.
Mucus and low pH
One of the first and now extensively documented
responses of fish to acid exposure is the increased release
of mucus from cells in the skin and gill surfaces. The
threshold for this response appears to be below pH 5.6
(Daye and Garside 1976) while at pH levels of less than
4.0 mucus typically appears rapidly as a visible film on
the body surface (Westfall 1945; Beamish 1972; Ultsch
and Gros 1979) and also accumulates to a considerable
extent on the gills, particularly in the interlamellar
spaces (Plonka and Neff 1969; Daye and Garside 1976;
Muniz and Leivestad 1980). The rapid appearance of
mucus at low pH is probably due to both enhanced
release and to the precipitation of the protein
components in the secretion (Westfall 1945). A number
of authors (see Fromm 1980 for review) have suggested
that a thick mucous layer upon the gills would impede
oxygen transfer and thus would contribute to a severe
tissue hypoxia. This conclusion has been supported by
measurements of much reduced oxygen consumption at
low pH (Packer and Dunson 1972; Ultsch et al. 1980)
and by evidence for restriction of oxygen diffusion
by mucus (Ultsch and Gros 1979). While it seems
likely that gas transport failure is an important
contributor to death at acutely toxic pH levels (pH S3.5)
it should be pointed out that extensive damage to the gill
lamellae, with separation of the outer epithelial layer,
also occurs (Daye and Garside 1976). The latter would
more profoundly interfere with gas exchange because
the diffusion distance, water to blood, would be greatly
increased (Hughes 1980). Thus, it still remains to be
established whether mucous accumulation alone has any
harmful effects upon gill function.
At less toxic pH levels (pH 24.0) the enhanccd
release of mucus is continued by the proliferation of
mucous cells in the skin (Zuchelkowski et al. 1981) and
the gills (Daye and Garside 1976). Since mucous
secretion is a common response not only to H+ but also
to a number of other toxicants such as trace metals
(Lewis and Lewis 1971; Varanasi and Markey 1978;
Muniz and Leivestad 1980; Lock and Van Overbeeke
1981) and organics (Christie and Battle 1963) it is
worthwhile examining what the physiological benefits
of this secretion to gill function, if any, might be.
Under normal circumstances the gills of freshwater
fish are covered with a thin layer of mucus (see Fig. 1C).
This layer consists of a water-soluble complex of
glycoproteins (Harris and Hunt 1973) which, because of
the presence of sialic acid residues, will have a net
negative charge (i.e., be polyanionic) at neutral pH
(Kirschner 1978). Suggestions as to the function of
mucus have included a defensive role, both against
abrasion damage and against bacterial pathogens (by
virtue of the presence of immunoglobins and
antibody-like activity; Harris and Hunt 1973), and a role
in osmoregulation. Three separate osmoregulatory
functions for the mucous layer have been proposed: that
it is a diffusion barrier to ion and water movement but
not, perhaps, to respiratory gases (Hughes 1980); that by
virtue of its polyanionic nature it will tend to concentrate
cations and thus aid their active inward transport
(Kirschner 1978), and that it has the property of being
able to specifically bind calcium (Wendelaar Bonga
1978). Marsha11 (1978) demonstrated in vitro that the
ability of mucus to impede ion diffusion was not
impressive; mucus had permeation characteristics
similar to that of water. Nevertheless, Shephard (1982),
using cation-selective microelectrodes, showed that the
mucous layer overlying fish esophageal epithelium was
an unstirred layer supporting a stable portion of the
transepithelial ionic gradient. Also Bierther (1970)
demonstrated, by histochemical methods, the accumula-
tion of both sodium and chloride in this layer on the gills.
 The implication of these findings is that the ionic
activities "seen" by the apical transport pumps are likely
to be higher than those of the external medium and this
would clearly assist ion uptake. How much higher is
difficult to estimate and, as Shephard (1982) points out,
will depend upon the thickness of the layer, the
distribution and the dissociation of the charged groups,
and the electrochemical equilibria established within the
layer.
Can. J. Zool. Downloaded from www.nrcresearchpress.com by RICE UNIVERSITY on 08/08/17
These conditions will, of course, also influence the
concentration of calcium at the gill surface but, in view
of the importance of calcium to permeability control of
the gills (see above) special provisions for its binding
might be expected. Marshall (1978) has shown that the
exchange capacity of mucus is, indeed, higher for ca2'
than for Na' and Chartier (1973) was able to isolate a
protein fraction in branchial mucus that had specific
calcium binding properties. In later studies, Chartier et
al. (1977) demonstrated that the calcium-binding
activity of the external surface could be significantly
increased by the removal of the Stannius corpuscles.
This observation raises the possibility that ionic
disturbances can be corrected by a mechanism involving
For personal use only.
an increased production and secretion of mucus,
particularly if the result is an increased calcium
concentration at the gill surface.
It then follows .that mucous secretion at low pH may
well be a highly appropriate physiological response.
However, it shouid be remembered that much of the
function of mucus may depend upon its polyanionic
nature. If this is the case, then acidification of the
medium will titrate the charged residues and will
profoundly interfere with their ion binding properties.
This aspect of the effect of pH has not been examined in
any detail but recently Miller and MacKay (1982)
showed that fish mucous solutions, which bind copper at
neutral pH, lose that binding capacity entirely at pH 3.5.
Thus the question of the physiological benefit of mucous
secretion at low pH is still not resolved.
Epithelial damage at low pH
Daye and Garside (1976) have provided the only
detailed study of the effects of pH on gill morphology.
They showed, in brook trout exposed to various pH
levels for 7 days (or until death), that the threshold for
gill injury was between pH 5.6 and 5.2 and that, not
surprisingly, the gill was the most sensitive of the
exposed surfaces. At pH 5.2 the principal effect was a
separation of the epithelial layers of the gill lamellae
which became progressively greater with increased acid
stress. Since brook trout are capable of prolonged
survival at pH levels below the threshold for injury,
Daye and Garside (1976) suggested that an equilibrium
was established between cell destruction and replace-
ment. While there is no direct evidence for this, there is
some support for the view that short-term sublethal acid
exposure has relatively little permanent effect. Lloyd
and Jordan (1964) found that rainbow trout, which had
lost equilibrium after 24 h at pH 3.8, recovered
completely when transferred to water of normal pH.
Similarly, McDonald er al. (1983) have shown that J:"'
in this species returned immediately to normal after 2
days at pH 4.0. Since Na' uptake is possibly the most
sensitive indicator of acid stress, this finding suggests
that the gill damage was of a relatively minor and
reversible nature. Nevertheless, at more acutely toxic
pH levels, irreversible gill damage probably does occur.
Beamish (1972) exposed white suckers to pH levels of
3.0 to 3.8 and found that once a visible surface film of
mucus became evident the animals could not be revived.
Furthermore, the fact that gill injury has apparently such
a low threshold suggests that a gradual deterioration of
the epithelium is likely to occur with long-term sublethal
acid exposure. This may be one of the major reasons
why attempts to acclimate fish to low pH have so far
proven largely unsuccessful (see below).
Adaptation to low pH
Many species of fish are adapted for life in acid
waters. The Amazon basin, for example, supports an
abundant aquatic fauna despite the fact that the waters
are ion poor and often acidic (pH 4.0 to 5.0; Heisler
1980). Furthermore, many species of fish from these
waters have an exceptional tolerance of acid levels
which would be acutely lethal to most temperate-zone
teleosts . The cardinal tetra, Cheirodon axelrodi, is, for
example, able to survive immediate transfer to pH 3.5
for an indefinite period (Dunson et al. 1977). The
physiological basis of this acid tolerance requires more
thorough investigation, but an important component is
likely to be the acid resistance of gill mechanisms of ion
regulation. Dunson et al. (1977) showed while that the
net salt losses in cardinal tetras, which were probably
responsible for their death, were comparable to those in
a less acid-tolerant species, the guppy (Poecilia
reticulata), a much lower pH (3.0 vs. 3.75) was required
to produce such losses. Furthermore, McWilliams
(1982b) has shown that Na' transport and diffusional
loss in the acid-tolerant perch, Perca jluviatilis, were
completely insensitive to pH reduction down to a level
of pH 4.0. Exposing the less tolerant salmonids to this
pH, as discussed, will produce virtually comppte
inhibition of JP' and several fold increases in J?tt .
This acid tolerance has raised the possibility that less
tolerant animals can be gradually acclimated to low pH.
Several studies have now addressed this question with
variable, but largely negative, results. In the rainbow
trout, for example, some adaptive changes did occur
during pH 4.0 exposure: the passive efflux of ions
returned to normal levels by 40 h and there was a partial
 700
recovery of ion uptake in hard water by this time
(McDonald et al. 1983). However, plasma ion levels
did not begin to return toward normal levels even after 5
days' exposure to pH 4.0-4.5 (McDonald et al. 1980)
and, thus, body ion balance was not being restored.
McWilliams (1980b), on the other hand, did show
evidence of fairly complete recovery of ion levels in
brown trout after 3-6 weeks at pH 6.0. Furthermore,
when these animals were subsequently exposed to pH
Can. J. Zool. Downloaded from www.nrcresearchpress.com by RICE UNIVERSITY on 08/08/17
4.0, Na' influx and efflux were less affected than in
animals previously maintained at pH 7.0. However, an
important criterion for successful low pH acclimation is
increased resistance to lethal pH and it can be argued that
the brown trout would not have passed this test. To date,
only one study, by Trojnar (1977) on eggs and alevins,
has shown improved lethal resistance following low pH
exposure. The remaining studies, using sublethal
acclimation times ranging from 2 h to 12 months have
shown either no effect upon resistance time (Lloyd and
Jordan 1964; Mount 1973; Falk and Dunson 1977; Daye
1980; Gerking and Lee 1982) or deleterious effects
(Swarts et al. 1978). Furthermore, the recent study of
Gerking and Lee (1982) showed that the desert pupfish,
For personal use only.
Cyprinodon nevadensis, maintained for 14 weeks at pH
6.3, failed to recover its reproductive capacity upon
return to neutral waters, thus indicating that some
permanent damage had occurred.
The conclusion that one can draw is that while sensitive
species such as trout can probably survive relatively
indefinitely under mild acid stress (i.e., pH 2 5 . 9 , they
probably do so at added cost. The greater proportion of
this cost, under routine circumstances, can be expected
to arise from the maintenance of gill function, this being
the primary target of environmental H'. Above-average
energy expenditures may be required for such activities
as active ion transport (e .g . , continued H' competition
with ~ a at+ the apical membrane may necessitate the
synthesis of additional transport ATPases, in order to
maintain ~ a balance),
+ . for the maintenance of a thicker
mucous coat upon the gills, and for an increased turnover
of gill epithelial cells to replace damaged tissues.
there may be disturbances
occurring as a of minor gill which,
though relatively minor in nature and difficult to detect,
may, nevertheless, impair the animal's ability to
perform costly activities (e.g ., exercise, reproduction)
and defend against added stress (e.g., episodic low pH
excursions at time of snowmelt). Two examples which
come to mind are subtle cardiovascular disturbances
related to net ion loss (cf. Milligan and Wood 1982) and
gradual skeletal demineralization and reproductive
failures associated with impaired calcium homeostasis
(Beamish et al. 1975).
Thus it remains an open question as to whether
attempts to acclimate animals to live at low pH will ever
have much success. Certainly, the Amazonian fishes are
CAN. J. ZOOL. VOL. 61, 1983
clear evidence that such adaptation is possible but it is
uncertain whether low pH tolerance would, in fact, arise
from generations of exposure to chronically reduced pH.
A study by Robinson et al. ( 1976) on brook trout strains
has shown that, indeed, inheritable differences in low pH
tolerance do exist, but points out that this tolerance arose
not from selection for low pH resistance but rather from
selection for other desirable traits. In any case, studies
by both Robinson et al. (1976) and Swarts et al. (1978)
indicate that such selection is likely to require many
generations and is, therefore, unlikely to occur as
rapidly as the rate at which sensitive freshwater
environments are becoming critically acidic in the wild.
Acknowledgements
The author's research quoted here has been supported
by grants from the Natural Sciences and Engineering
Research Council of Canada and from Fisheries and
Oceans, Canada. Thanks are extended to the coauthors
of this work: Dr. Chris Wood, Dr. Dick Walker, Peter
Wilkes, and Helve Hobe for many useful discussions.
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