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Anomalous tolerance to low pH in the estuarine killifish Fundulus heteroclitus
Conrp E~oclwm Ph.wo/
Vol
94C. No
0306-4492/89$3 00 + 0.00 ,(_‘1989Pergamon Press plc
I. pp 169-172, 1989
PrInted ,n Great Bntam
ANOMALOUS TOLERANCE TO LOW pH IN THE ESTUARINE KILLIFISH FUNDULUS HETEROCLITUS RICHARD J. GONZALEZ, CHARLES H. MASON and WILLIAM A. DUNSON Department of Biology, The Pennsylvama State University, 208 Mueller Laboratory. University PA 16802, U.S.A.
Telephone:
Park,
814-865-2461
(Receiz:ed 30 January 1989) Abstract-l. The euryhaline fish Fundulus heteroclitus has an incipient lethal pH between 3.75 and 4.0 in fresh water. 2. Fish exposed to pH 3.5 m sea water or fresh water died in about 3 hr. and had greatly elevated or depressed body sodium concentrations, respectively. The direction and degree of change m body sodium level depended on the sochum dlffuslon gradient between the environment and the fish. This is the first time that the death of fish m sea water at low pH has been shown to be associated with hypernatremla. 3. Yet. sodmm fluxes during the first hour of exposure to pH 3 5 in water of 3.5 or 35 ppt salinity were not different from controls, and body and plasma sodium concentration did not change during 2 hr exposure to pH 3.5. This Initial Insensitivity of gdl so&urn regulation to blockage by low pH is quite different from the response of previously studied freshwater fish 4. The degree of acid tolerance dlsplayed by F. heterochtus is surprising considering its estuarine habits. This paradoxical tolerance appears to be a secondary consequence of its ability to adjust sodium balance m relation to rapid changes in salinity
INTRODUCTION
(loss of equilibrium which precedes death) m acidified water of high and low salinities to determine if H+ affects membrane permeability similarly in reverse diffusion gradients. In freshwater fish, gill permeability increases greatly almost immediately upon exposure to low pH (Gonzalez and Dunson, 1987, 1989a,b). Changes in gill permeability in sea water during acid exposure have not been measured previously. In addition. we measured unidirectional sodium fluxes during acute acid exposure as a direct indication of the changes in gill permeability that occur during exposure to low pH. Finally, we followed changes in body and plasma sodium levels during short term acid exposure in sea water as an indication of the time sequence of permeability changes.
Studies of the osmoregulatory mechanisms of the euryhaline killifish (Fundulus heteroclitus) indicate that it possesses remarkably flexible branchial responses to salinity changes. It is able to survive direct transfer from sea water to fresh water and maintain a plasma sodium concentration of about 150 mM (Potts and Evans, 1967; Evans, 1980). It appeared to us that the osmoregulatory adaptations of F. heteroclitus that are necessary for survival in some estuaries (subject to rapid changes in salinity) might fortuitously confer resistance to low pH. Of course estuarine habitats rarely. if ever, experience low environmental pHs of natural origin. Euryhaline fish transferred from sea water to fresh water face the same rapid net diffusive loss of sodium as a fresh water fish does when exposed to low pH. The first objective of this study was to assess the degree of acid tolerance of F. heteroclitus by measuring cumulative mortality during prolonged exposure to various pHs in fresh water. This indicated that the incipient lethal pH for this species was lower than that of many freshwater fish which have been examined. Our second objective was to assess for the first time the mechanism of toxicity of acidity in sea water to reveal the source of this unusual and unexpected level of tolerance to low pH. The primary toxic action of H+ in fresh water is disruption of sodium balance which results from increases in sodium permeability of the gills. the main organ of sodium regulation (McDonald, 1983a). We took advantage of the fact that F. heterochtus is euryhaline to expose them to low pH in reverse diffusion gradients (in 35 ppt and 3.5 ppt) in order to closely examine characteristics of membrane permeability during acid exposure. We measured body sodium concentration at rollover
MATERIAL AND METHODS
E.rperimental animals F. heferoclitus were collected
from tidal creeks on Chincoteague Island, Virginia, and were held there in local sea water (salinity of 28-30 ppt) or m artlficlal sea water made from Instant Ocean brand sea salts (35 ppt) in Pennsylvania. Fish were fed frozen brine shrimp and/or Tetramm once daily until 24 hr before experimentation; they were not fed during testing.
Cumularitre mortahty m water oflowpH Fish were exposed to fresh water (Chincoteague tap water: 2 04mM Na. 0.53 mM K. 0.45 mM Ca. 0.33 mM Mg) of various pHs (3.0. 3.25, 3.5. 3.75, 4.0) andmomtored until death. The fish were allowed to acclimate to fresh water for 3 days prior to testmg. At the start of the exposure period, fish were transferred to 20 I aquaria (12 per aquarium, 24 at pH 4.0) containing the acidified me&a. pHs were monitored with a Beckman model 3500 pH meter, and adjusted with H,SO, as needed. Fish were removed when opercular movements had ceased and they no longer 169
RICHARD J. GONZALEZ et al.
170 responded to proddmg. after 158 hr.
The experiment
was terminated
EfleciJ of low pH on body and plasma sodium concentration Body sodium concentration was measured in F. heteroc.hfu.cexposed to water of 3.5, 12.5 and 35 ppt salinity at pH 3.5 The lowest and highest salinities were approximately one-third and 3 ttmes the sodium concentration of fish plasma. respecttvely. A salimty of 12.5 ppt which contained sodmm roughly isosmottc wtth plasma provtded a test of the effects of low pH on the fish without a sodium diffusion gradient. Immediately before the exposure period began, 15 fish were removed from the holding tank containing sea water and rinsed in tap water for 1min to remove excess salts that may have adhered to the skin. These control fish were wetghed. dried for 24 hr at IOO’C. weighed agam, and drssolved in concentrated HNO,. The resulting solution was diluted and analyzed for sodium with a Perkin-Elmer model 3280 Atomic Absorptton Spectrophotometer. Forty-five fish were then randomly divided among three 201 aquaria contammg the test media and pH was monitored regularly; It was adjusted as needed with dilute H,SO,. The fish were removed at rollover (loss of equthbrmm), weighed and analyzed for sodium as before. Rollover ts the point precedmg death when fish can no longer maintain equilibrium and cannot recover If transferred to a neutral pH medium (Beamtsh. 1972) Time to rollover 1s highly correlated wrth time to death (Swarts rr al.. 1978); removal of fish at this point was a precaution against changes in body sodium concentration which can occur tmmedrately after death (Dunson PI u/. 1977) Body and plasma sodium concentrations of F. heteroclilus were also measured after 0.5, 1 and 2 hr exposure to sea water at pH 3.5 prior to rollover. Fifteen fish placed in a 20 1 aquarium containing sea water were allowed to remain undisturbed overnight, The following morning, 3 fish were removed from the tank as controls, decapitated, and a blood sample was collected from the dorsal blood vessel m a heparmtzed captllary tube. The blood sample was centrrfuged and the plasma was collected and analyzed for sodium. The fish were weighed and analyzed for sodium. The pH of the tank was then lowered to 3.5, 4 fish were removed at each of the prescribed times, and they were analyzed for body and plasma sodium concentration.
Sodium influx (J,,) and efflux (J,,,,) were measured m sea water at pH 7.15 (control) and upon exposure to pH 3.5. To measure J,, , IO fish were placed m mdivtdual 250 ml baths (5 control. 5 low pH) containing sea water with 15 kBq ‘JNaCl/ml. A 5 ml sample was removed from each bath just prior to the introduction of the fish and again after 1 hr. Bath samples were analyzed for gamma radiation with a NaI crystal connected to a Canberra Series 30 multi-channel analyzer, and for soldium with the atomic absorption spectrophotometer. The fish were removed from the baths after the second sample was taken and analyzed for sodium. J,, was calculated from the disappearance of “Na from the bath using a two-compartment equation (Stokes and Dunson. 1982). To measure J,,,, 10 fish were placed in a 11 loading bath containing sea water with about 50 kBq “NaCliml for 1 hr. They were rinsed in unlabeled sea water for 1 min and placed in individual 250 ml baths (5 control, 5 test). A 5 ml sample was removed after 1 hr and assayed for gamma radiation and sodium concentration. The fish were removed, rinsed, placed in a test tube, and assayed for gamma radiation for 5 min. They were then weighed and analyzed for sodium. J,,, was calculated from the disappearance of Z4Na from the fish using the two-compartment equatton
4.0 #a-e 1
10 Log
100
time
(hj
Fig. 1. Cumulative mortality of F. heteroclitus exposed to various pHs (as labeled) in fresh water. All fish were acclimated to fresh water for 3 days before exposure. The survivors were removed after 158 hr. n = 12 for each pH except 4.0 where it was 24. Wet mass = 3.1 f. 1.1 g to 5.5 * 2.9 g. Statistical analysis All results are reported as means k SE. Significant dtfferences between means were determined by r-test or analysis of variance and multiple comparisons (Bonferroni’s method) with an overall significance level of P < 0.05
RESULTS Cumulatit~e mortality
in fresh
water of low pH
All F. heteroclitus exposed to pH 3.15 died in less then 40 hr (Fig. I), but those pH 4.0 experienced 8% mortality over appears that the incipient lethal pH (50% for this species lies between pH 3.75 and
and below exposed to 158 hr. It mortality) 4.0.
Efects of low pH on body and plasma sodium concen tration
Body sodium concentration of fish at rollover in low pH was related to the sodium concentration of the external medium. Fish exposed to sea water at pH 3.5 rolled over in about 3 hr and their body sodium concentrations were about 2.4 times greater than control levels (Table 1). Those exposed to water of 3.5 ppt salinity at pH 3.5 also rolled over in about 3 hr, but in contrast they experienced a 54% decline in body sodium concentration. There was no change in the body sodium concentration of fish kept in 12.5 ppt salinity at pH 3.5, and none of the fish had lost equilibrium when they were removed after 29 hr. Measurement of body and plasma sodium concentration prior to rollover after 0.5, 1 and 2 hr exposure to pH 3.45 sea water revealed no significant differences from those at 0 hr (Table 2). Table I. The effect of low pH at drtTerent salinrtres on body sodrum concentratron of F. heteroclirus PH 7.15 35 3.5 3.5
Sahmty (PPt) 35 0 35 0 12 5 3.5
Body [Na] (pmol/g wet mass) 667kl2 158.229 I 60.8 f 1.9* 30.7 f I 0
Values are means f-SE (n = 15 Per treatment). Fish were removed at rollover or after 29 hr (*). Wet mass = 1.5* 0. I g
Tolerance to low pH Table 2. Body and plasma so&urn concentratmns of F heteroclirus exposed to sea water (35 ppt) at pH 3 5 over a 2 hr period and removed prior to rollover Time Chr)
Plasma (Na] fmM)
0.0 0.5 1.0 20
I SO.8i 2.4
Body pal ( p mol/g wet mass)
65.4 61.6 66.1 74.8
1699i1.7 188.8 II3 7 1780+ 108
* 5.1 & 5.0 ir I.6 + 2. I
Values are means i SE (n = 4 per sample) Wet mass=28iO5g
Sodium fluxes J,, and JO,, of F. heteroclitus in sea water at pH 3.45 were not different from controls at pH 7.15 (Table 3). J,, and Jout of fish at low and high pH were not statistically
different
from each other indicating
the fish were in or near equilibrium exposure period.
during
that
the
DISCUSSION F. heteroc~jtus demonstrates a surprising degree of acid tolerance despite the low probabiIity that it ever experiences acidic conditions in estuaries. With an incipient lethal pH between 3.75 and 4.0, it appears that F. heteroclitus is much more tolerant of low pH than many freshwater species in similarly hard water. For example, the incipient lethal pH for common guppies (Poeciiia reticulata) was about pW 4.75 (Dunson et al., 1977). In soft water, the common shiner (Notropis cornutus) and rainbow trout (Saimo gairdneri) survived only 4.6 and 6.5 hr, respectively, at pH 4.0 (Freda and McDonald, 1988). This unexpected level of acid tolerance in F. heteroclitus may be a consequence of its osmoregulatory adaptations for estuarine life. Estuaries vary greatly in salinity, both temporally and spatially, and F. heteroclitus are able to move freely without regard to salinity as a result of their ability to regulate sodium. Gills of F. heteroclitus in sea water have an exceptionally high permeability to sodium and turn over sodium at least 100 times faster than a freshwater fish does (Potts and Evans, 1967). Within 15 min of exposure to fresh water, sodium fluxes of F. ~teteroel~t~ decline to about 5% of their rate in sea water, a striking change in permeability. Such a tight control of sodium permeability of the gills may play a key role in the resistance of the sodium fluxes to low pH. JI, in sea water was unaltered during the first hour of exposure to pH 3.5 (Table 3), and body and plasma sodium concentration of fish exposed to sea water of low pH were not changed during the first 2 hr of exposure. Numerous studies have shown that sodium permeability of the gills of freshwater fish increase Table 3 The effect of low pH on sodium influx (J,.) and effiux (J,,,) of F. hefe~o~lir~~ placed in sea water (35 ppt) for 1 hi pm&g P’-’ 7 I5
35
wet mass hr
J,, 30.0rt 4.1 28.8f 12.9
JOYI 41.3 * 5.3 58 4 f 23.6
Values are means I SE fn = 5 for each treatment) wet mass=21 *02g
171
almost immediately upon exposure to low pH (Gonzalez and Dunson, 1987, 1989a,b; McDonald, 1983b). Despite the initial resistance of sodium fluxes to the toxicity of low pH, fish removed at rollover (after about 3 hr) experienced significant changes in body sodium concentration directly related to the diffusion gradient (Table 1). This suggests that the initial resistance of sodium fiuxes to low pH eventually fails and death results. That disruption of sodium regulation is the major cause of death in fish exposed to low pH in high or low salinity is indicated by the fact that the group exposed to low pH in 12.5 ppt salinity did not experience changes in body sodium concentration (Table l), and did not die within a 29 hr test period. It has been proposed that in waters of low pH that were high in calcium death resulted from acid/base disturbances, not disruption of sodium regulation (McDonald et al., 1980). This does not appear to be the case for F. heteroclitus at low pH m sea water (10 mM Ca, 52mM Mg). Body sodium concentrations of F. heteroc~itus exposed to sea water of low pH were slgni~cantly elevated, while those placed in water of 3.5 ppt salimty were depressed greatly. Although both groups died in about the same amount of time, those in sea water gained 2.5 times more sodium than those in the dilute water lost. The difference can be explained by differences in the diffusion gradients. The difference in sodium concentration between 35 ppt sea water and plasma is approximately 2.7 times greater than the difference between the plasma and water of 3.5 ppt salinity. It appears from these data that H+ causes a symmetrical mcrease in permeability of the gills to sodium, and sodium moved in or out depending on the existing gradient. In summary, F. heterocirtus was unexpectedly more tolerant to low pH than many freshwater species. We suggest that this tolerance results from evolutionary adaptations for osmoregulation in estuaries. These fish maintain tight control of the sodium permeability of their gills allowing them to regulate sodium m the face of rapid salinity change. Depending on the pH, this tight control may help delay or even prevent increases in gill permeability that otherwise would result in detrimental changes in body sodium concentration and eventual death. REFERENCES Beamish R. J. (1972) Lethal pH for the white sucker Castasromus commersoni (Lacepede). Trans. Am. Ash. sot. 101, 355-358. Dunson W. A., Swarts F. and Silvestri M. (1977) Exceptional tolerance to low pH of some tropical blackwater fish. J. Exp. Zool. 201, IS?-I62. Evans D. H. (1980) Kinetic studies of ion transport by fish gill epitheiium. Am. J. Physiai. 238, RX%-Ri30. _ Freda J. and McDonald D. G. (1988) Phvsiolonical correlates of interspecdic variations in a&d iolerance in fish. J. Exp. Biol. 136, 243-258. Gonzalez R. J. and Dunson W. A. (1987) Adaptations of sodium balance to low pH in a sunfish (Enneacanthus obesus) from naturally acichc waters. J. Comp. Physiol. 157B, 555-566.
172
RICHARD J. GONZALEZ et al.
Gonzalez R. J. and Dunson W. A. (1989a) Acclimatron of sodtum regulatton to low pH and the role of calcium m the actd tolerant sunfish Enneacanrhus obesus. Physiol. Zool. 62, 977-992 Gonzalez R. J. and Dunson W A. (1989b) Differences in low pH tolerance among closely related sunfish of the genus Enneacanthus. Enr;. Biol. Fish. In press. McDonald D. G (1983a) The effects of H+ upon the grlls of freshwater fish Can. J. Zoo/. 61, 691-703. McDonald D. G. (1983b) The mteractron of pH and calcium on the phystology of the rambow trout, Salmo gawdnerz. I, Branchial and renal net ion and Hi fluxes. J E.xp. Biol 102, 123m 140. McDonald D. G . Hobe H and Wood C. M. (1980) The
influence of calcium on the phystological responses of the rainbow trout, Sulmo gairdneri, to low envtronmental pH J. Exp. Biol. 88, 1099131. Potts W. T. W. and Evans D. H. (1967) Sodium and chlortde balance in the killifish, Fund&s heteroclirus. Biol. Bull 133, 411425. Stokes G. D. and Dunson W. A. (1982) Permeability and channel characteristrcs of reptilian skin. Am J. Phwiol. 242, F681LF689. Swarts F. A.. Dunson W. A. and Wrtght J. E. (1977) Genettc and environmental factors involved in increased reststance of brook trout to sulphuric acid solutions and mine acid polluted waters. Trans. Am Fish Sot 107, 651457.
Vol
94C. No
0306-4492/89$3 00 + 0.00 ,(_‘1989Pergamon Press plc
I. pp 169-172, 1989
PrInted ,n Great Bntam
ANOMALOUS TOLERANCE TO LOW pH IN THE ESTUARINE KILLIFISH FUNDULUS HETEROCLITUS RICHARD J. GONZALEZ, CHARLES H. MASON and WILLIAM A. DUNSON Department of Biology, The Pennsylvama State University, 208 Mueller Laboratory. University PA 16802, U.S.A.
Telephone:
Park,
814-865-2461
(Receiz:ed 30 January 1989) Abstract-l. The euryhaline fish Fundulus heteroclitus has an incipient lethal pH between 3.75 and 4.0 in fresh water. 2. Fish exposed to pH 3.5 m sea water or fresh water died in about 3 hr. and had greatly elevated or depressed body sodium concentrations, respectively. The direction and degree of change m body sodium level depended on the sochum dlffuslon gradient between the environment and the fish. This is the first time that the death of fish m sea water at low pH has been shown to be associated with hypernatremla. 3. Yet. sodmm fluxes during the first hour of exposure to pH 3 5 in water of 3.5 or 35 ppt salinity were not different from controls, and body and plasma sodium concentration did not change during 2 hr exposure to pH 3.5. This Initial Insensitivity of gdl so&urn regulation to blockage by low pH is quite different from the response of previously studied freshwater fish 4. The degree of acid tolerance dlsplayed by F. heterochtus is surprising considering its estuarine habits. This paradoxical tolerance appears to be a secondary consequence of its ability to adjust sodium balance m relation to rapid changes in salinity
INTRODUCTION
(loss of equilibrium which precedes death) m acidified water of high and low salinities to determine if H+ affects membrane permeability similarly in reverse diffusion gradients. In freshwater fish, gill permeability increases greatly almost immediately upon exposure to low pH (Gonzalez and Dunson, 1987, 1989a,b). Changes in gill permeability in sea water during acid exposure have not been measured previously. In addition. we measured unidirectional sodium fluxes during acute acid exposure as a direct indication of the changes in gill permeability that occur during exposure to low pH. Finally, we followed changes in body and plasma sodium levels during short term acid exposure in sea water as an indication of the time sequence of permeability changes.
Studies of the osmoregulatory mechanisms of the euryhaline killifish (Fundulus heteroclitus) indicate that it possesses remarkably flexible branchial responses to salinity changes. It is able to survive direct transfer from sea water to fresh water and maintain a plasma sodium concentration of about 150 mM (Potts and Evans, 1967; Evans, 1980). It appeared to us that the osmoregulatory adaptations of F. heteroclitus that are necessary for survival in some estuaries (subject to rapid changes in salinity) might fortuitously confer resistance to low pH. Of course estuarine habitats rarely. if ever, experience low environmental pHs of natural origin. Euryhaline fish transferred from sea water to fresh water face the same rapid net diffusive loss of sodium as a fresh water fish does when exposed to low pH. The first objective of this study was to assess the degree of acid tolerance of F. heteroclitus by measuring cumulative mortality during prolonged exposure to various pHs in fresh water. This indicated that the incipient lethal pH for this species was lower than that of many freshwater fish which have been examined. Our second objective was to assess for the first time the mechanism of toxicity of acidity in sea water to reveal the source of this unusual and unexpected level of tolerance to low pH. The primary toxic action of H+ in fresh water is disruption of sodium balance which results from increases in sodium permeability of the gills. the main organ of sodium regulation (McDonald, 1983a). We took advantage of the fact that F. heterochtus is euryhaline to expose them to low pH in reverse diffusion gradients (in 35 ppt and 3.5 ppt) in order to closely examine characteristics of membrane permeability during acid exposure. We measured body sodium concentration at rollover
MATERIAL AND METHODS
E.rperimental animals F. heferoclitus were collected
from tidal creeks on Chincoteague Island, Virginia, and were held there in local sea water (salinity of 28-30 ppt) or m artlficlal sea water made from Instant Ocean brand sea salts (35 ppt) in Pennsylvania. Fish were fed frozen brine shrimp and/or Tetramm once daily until 24 hr before experimentation; they were not fed during testing.
Cumularitre mortahty m water oflowpH Fish were exposed to fresh water (Chincoteague tap water: 2 04mM Na. 0.53 mM K. 0.45 mM Ca. 0.33 mM Mg) of various pHs (3.0. 3.25, 3.5. 3.75, 4.0) andmomtored until death. The fish were allowed to acclimate to fresh water for 3 days prior to testmg. At the start of the exposure period, fish were transferred to 20 I aquaria (12 per aquarium, 24 at pH 4.0) containing the acidified me&a. pHs were monitored with a Beckman model 3500 pH meter, and adjusted with H,SO, as needed. Fish were removed when opercular movements had ceased and they no longer 169
RICHARD J. GONZALEZ et al.
170 responded to proddmg. after 158 hr.
The experiment
was terminated
EfleciJ of low pH on body and plasma sodium concentration Body sodium concentration was measured in F. heteroc.hfu.cexposed to water of 3.5, 12.5 and 35 ppt salinity at pH 3.5 The lowest and highest salinities were approximately one-third and 3 ttmes the sodium concentration of fish plasma. respecttvely. A salimty of 12.5 ppt which contained sodmm roughly isosmottc wtth plasma provtded a test of the effects of low pH on the fish without a sodium diffusion gradient. Immediately before the exposure period began, 15 fish were removed from the holding tank containing sea water and rinsed in tap water for 1min to remove excess salts that may have adhered to the skin. These control fish were wetghed. dried for 24 hr at IOO’C. weighed agam, and drssolved in concentrated HNO,. The resulting solution was diluted and analyzed for sodium with a Perkin-Elmer model 3280 Atomic Absorptton Spectrophotometer. Forty-five fish were then randomly divided among three 201 aquaria contammg the test media and pH was monitored regularly; It was adjusted as needed with dilute H,SO,. The fish were removed at rollover (loss of equthbrmm), weighed and analyzed for sodium as before. Rollover ts the point precedmg death when fish can no longer maintain equilibrium and cannot recover If transferred to a neutral pH medium (Beamtsh. 1972) Time to rollover 1s highly correlated wrth time to death (Swarts rr al.. 1978); removal of fish at this point was a precaution against changes in body sodium concentration which can occur tmmedrately after death (Dunson PI u/. 1977) Body and plasma sodium concentrations of F. heteroclilus were also measured after 0.5, 1 and 2 hr exposure to sea water at pH 3.5 prior to rollover. Fifteen fish placed in a 20 1 aquarium containing sea water were allowed to remain undisturbed overnight, The following morning, 3 fish were removed from the tank as controls, decapitated, and a blood sample was collected from the dorsal blood vessel m a heparmtzed captllary tube. The blood sample was centrrfuged and the plasma was collected and analyzed for sodium. The fish were weighed and analyzed for sodium. The pH of the tank was then lowered to 3.5, 4 fish were removed at each of the prescribed times, and they were analyzed for body and plasma sodium concentration.
Sodium influx (J,,) and efflux (J,,,,) were measured m sea water at pH 7.15 (control) and upon exposure to pH 3.5. To measure J,, , IO fish were placed m mdivtdual 250 ml baths (5 control. 5 low pH) containing sea water with 15 kBq ‘JNaCl/ml. A 5 ml sample was removed from each bath just prior to the introduction of the fish and again after 1 hr. Bath samples were analyzed for gamma radiation with a NaI crystal connected to a Canberra Series 30 multi-channel analyzer, and for soldium with the atomic absorption spectrophotometer. The fish were removed from the baths after the second sample was taken and analyzed for sodium. J,, was calculated from the disappearance of “Na from the bath using a two-compartment equation (Stokes and Dunson. 1982). To measure J,,,, 10 fish were placed in a 11 loading bath containing sea water with about 50 kBq “NaCliml for 1 hr. They were rinsed in unlabeled sea water for 1 min and placed in individual 250 ml baths (5 control, 5 test). A 5 ml sample was removed after 1 hr and assayed for gamma radiation and sodium concentration. The fish were removed, rinsed, placed in a test tube, and assayed for gamma radiation for 5 min. They were then weighed and analyzed for sodium. J,,, was calculated from the disappearance of Z4Na from the fish using the two-compartment equatton
4.0 #a-e 1
10 Log
100
time
(hj
Fig. 1. Cumulative mortality of F. heteroclitus exposed to various pHs (as labeled) in fresh water. All fish were acclimated to fresh water for 3 days before exposure. The survivors were removed after 158 hr. n = 12 for each pH except 4.0 where it was 24. Wet mass = 3.1 f. 1.1 g to 5.5 * 2.9 g. Statistical analysis All results are reported as means k SE. Significant dtfferences between means were determined by r-test or analysis of variance and multiple comparisons (Bonferroni’s method) with an overall significance level of P < 0.05
RESULTS Cumulatit~e mortality
in fresh
water of low pH
All F. heteroclitus exposed to pH 3.15 died in less then 40 hr (Fig. I), but those pH 4.0 experienced 8% mortality over appears that the incipient lethal pH (50% for this species lies between pH 3.75 and
and below exposed to 158 hr. It mortality) 4.0.
Efects of low pH on body and plasma sodium concen tration
Body sodium concentration of fish at rollover in low pH was related to the sodium concentration of the external medium. Fish exposed to sea water at pH 3.5 rolled over in about 3 hr and their body sodium concentrations were about 2.4 times greater than control levels (Table 1). Those exposed to water of 3.5 ppt salinity at pH 3.5 also rolled over in about 3 hr, but in contrast they experienced a 54% decline in body sodium concentration. There was no change in the body sodium concentration of fish kept in 12.5 ppt salinity at pH 3.5, and none of the fish had lost equilibrium when they were removed after 29 hr. Measurement of body and plasma sodium concentration prior to rollover after 0.5, 1 and 2 hr exposure to pH 3.45 sea water revealed no significant differences from those at 0 hr (Table 2). Table I. The effect of low pH at drtTerent salinrtres on body sodrum concentratron of F. heteroclirus PH 7.15 35 3.5 3.5
Sahmty (PPt) 35 0 35 0 12 5 3.5
Body [Na] (pmol/g wet mass) 667kl2 158.229 I 60.8 f 1.9* 30.7 f I 0
Values are means f-SE (n = 15 Per treatment). Fish were removed at rollover or after 29 hr (*). Wet mass = 1.5* 0. I g
Tolerance to low pH Table 2. Body and plasma so&urn concentratmns of F heteroclirus exposed to sea water (35 ppt) at pH 3 5 over a 2 hr period and removed prior to rollover Time Chr)
Plasma (Na] fmM)
0.0 0.5 1.0 20
I SO.8i 2.4
Body pal ( p mol/g wet mass)
65.4 61.6 66.1 74.8
1699i1.7 188.8 II3 7 1780+ 108
* 5.1 & 5.0 ir I.6 + 2. I
Values are means i SE (n = 4 per sample) Wet mass=28iO5g
Sodium fluxes J,, and JO,, of F. heteroclitus in sea water at pH 3.45 were not different from controls at pH 7.15 (Table 3). J,, and Jout of fish at low and high pH were not statistically
different
from each other indicating
the fish were in or near equilibrium exposure period.
during
that
the
DISCUSSION F. heteroc~jtus demonstrates a surprising degree of acid tolerance despite the low probabiIity that it ever experiences acidic conditions in estuaries. With an incipient lethal pH between 3.75 and 4.0, it appears that F. heteroclitus is much more tolerant of low pH than many freshwater species in similarly hard water. For example, the incipient lethal pH for common guppies (Poeciiia reticulata) was about pW 4.75 (Dunson et al., 1977). In soft water, the common shiner (Notropis cornutus) and rainbow trout (Saimo gairdneri) survived only 4.6 and 6.5 hr, respectively, at pH 4.0 (Freda and McDonald, 1988). This unexpected level of acid tolerance in F. heteroclitus may be a consequence of its osmoregulatory adaptations for estuarine life. Estuaries vary greatly in salinity, both temporally and spatially, and F. heteroclitus are able to move freely without regard to salinity as a result of their ability to regulate sodium. Gills of F. heteroclitus in sea water have an exceptionally high permeability to sodium and turn over sodium at least 100 times faster than a freshwater fish does (Potts and Evans, 1967). Within 15 min of exposure to fresh water, sodium fluxes of F. ~teteroel~t~ decline to about 5% of their rate in sea water, a striking change in permeability. Such a tight control of sodium permeability of the gills may play a key role in the resistance of the sodium fluxes to low pH. JI, in sea water was unaltered during the first hour of exposure to pH 3.5 (Table 3), and body and plasma sodium concentration of fish exposed to sea water of low pH were not changed during the first 2 hr of exposure. Numerous studies have shown that sodium permeability of the gills of freshwater fish increase Table 3 The effect of low pH on sodium influx (J,.) and effiux (J,,,) of F. hefe~o~lir~~ placed in sea water (35 ppt) for 1 hi pm&g P’-’ 7 I5
35
wet mass hr
J,, 30.0rt 4.1 28.8f 12.9
JOYI 41.3 * 5.3 58 4 f 23.6
Values are means I SE fn = 5 for each treatment) wet mass=21 *02g
171
almost immediately upon exposure to low pH (Gonzalez and Dunson, 1987, 1989a,b; McDonald, 1983b). Despite the initial resistance of sodium fluxes to the toxicity of low pH, fish removed at rollover (after about 3 hr) experienced significant changes in body sodium concentration directly related to the diffusion gradient (Table 1). This suggests that the initial resistance of sodium fiuxes to low pH eventually fails and death results. That disruption of sodium regulation is the major cause of death in fish exposed to low pH in high or low salinity is indicated by the fact that the group exposed to low pH in 12.5 ppt salinity did not experience changes in body sodium concentration (Table l), and did not die within a 29 hr test period. It has been proposed that in waters of low pH that were high in calcium death resulted from acid/base disturbances, not disruption of sodium regulation (McDonald et al., 1980). This does not appear to be the case for F. heteroclitus at low pH m sea water (10 mM Ca, 52mM Mg). Body sodium concentrations of F. heteroc~itus exposed to sea water of low pH were slgni~cantly elevated, while those placed in water of 3.5 ppt salimty were depressed greatly. Although both groups died in about the same amount of time, those in sea water gained 2.5 times more sodium than those in the dilute water lost. The difference can be explained by differences in the diffusion gradients. The difference in sodium concentration between 35 ppt sea water and plasma is approximately 2.7 times greater than the difference between the plasma and water of 3.5 ppt salinity. It appears from these data that H+ causes a symmetrical mcrease in permeability of the gills to sodium, and sodium moved in or out depending on the existing gradient. In summary, F. heterocirtus was unexpectedly more tolerant to low pH than many freshwater species. We suggest that this tolerance results from evolutionary adaptations for osmoregulation in estuaries. These fish maintain tight control of the sodium permeability of their gills allowing them to regulate sodium m the face of rapid salinity change. Depending on the pH, this tight control may help delay or even prevent increases in gill permeability that otherwise would result in detrimental changes in body sodium concentration and eventual death. REFERENCES Beamish R. J. (1972) Lethal pH for the white sucker Castasromus commersoni (Lacepede). Trans. Am. Ash. sot. 101, 355-358. Dunson W. A., Swarts F. and Silvestri M. (1977) Exceptional tolerance to low pH of some tropical blackwater fish. J. Exp. Zool. 201, IS?-I62. Evans D. H. (1980) Kinetic studies of ion transport by fish gill epitheiium. Am. J. Physiai. 238, RX%-Ri30. _ Freda J. and McDonald D. G. (1988) Phvsiolonical correlates of interspecdic variations in a&d iolerance in fish. J. Exp. Biol. 136, 243-258. Gonzalez R. J. and Dunson W. A. (1987) Adaptations of sodium balance to low pH in a sunfish (Enneacanthus obesus) from naturally acichc waters. J. Comp. Physiol. 157B, 555-566.
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Gonzalez R. J. and Dunson W. A. (1989a) Acclimatron of sodtum regulatton to low pH and the role of calcium m the actd tolerant sunfish Enneacanrhus obesus. Physiol. Zool. 62, 977-992 Gonzalez R. J. and Dunson W A. (1989b) Differences in low pH tolerance among closely related sunfish of the genus Enneacanthus. Enr;. Biol. Fish. In press. McDonald D. G (1983a) The effects of H+ upon the grlls of freshwater fish Can. J. Zoo/. 61, 691-703. McDonald D. G. (1983b) The mteractron of pH and calcium on the phystology of the rambow trout, Salmo gawdnerz. I, Branchial and renal net ion and Hi fluxes. J E.xp. Biol 102, 123m 140. McDonald D. G . Hobe H and Wood C. M. (1980) The
influence of calcium on the phystological responses of the rainbow trout, Sulmo gairdneri, to low envtronmental pH J. Exp. Biol. 88, 1099131. Potts W. T. W. and Evans D. H. (1967) Sodium and chlortde balance in the killifish, Fund&s heteroclirus. Biol. Bull 133, 411425. Stokes G. D. and Dunson W. A. (1982) Permeability and channel characteristrcs of reptilian skin. Am J. Phwiol. 242, F681LF689. Swarts F. A.. Dunson W. A. and Wrtght J. E. (1977) Genettc and environmental factors involved in increased reststance of brook trout to sulphuric acid solutions and mine acid polluted waters. Trans. Am Fish Sot 107, 651457.