The effect of environmental ammonia concentrations on the ion-exchange of shore crabs, Carcinus maenas (L.)

Camp. Eiochem. Physiol. Vol. 97C, No. 1, pp. 87-91, 1990 Printed in Great Britain

0306-4492/9033.00+ 0.00 0 1990Pergamon Press plc

THE EFFECT OF ENVIRONMENTAL AMMONIA CONCENTRATIONS ON THE ION-EXCHANGE OF SHORE CRABS, CARCINUS MAENAS (L.) D. H. SPAARGAREN Netherlands Institute for Sea Research, PO Box 59, 1790 AB Den Burg, Texel, The Netherlands [Telephone: 02220-19541; FAX (31) 02220-196741 (Received 30 March 1990) Abstract-l. Ion exchange characteristics (ion-permeability, net, passive and active ion fluxes) of adult shore crabs were studied in relation to experimentally-increased external ammonia concentrations. 2. Moderately-elevated ammonia concentrations (up to about 1 mmol/l NH:) induce an increase in the ion permeability and the salt fluxes across the body wall of Carcinw maenas. At still higher NH: concentrations, ion-permeability and ion fluxes are reduced again. 3. Active salt influx generally follows the same pattern as observed for whole animal permeability, probably because both parameters are strongly related to gill perfusion and gill ventilation. 4. Prolonged exposure to elevated ammonia concentrations in the environment is unfavourable, not because of the NH: toxicity, but because of the higher energy requirements associated with the higher salt fluxes.

INTRODUCIION

the alkaline ions Nat and K+ are in the formation of urine replaced by NH:, which prevents their loss. In aquatic animals a similar process could be shown to take place in a marine prawn, Penaeus japonicus, when water turnover is increased during exposure to hypotonic salinities (Spaargaren et al., 1982). The ammonia tolerance of most animal species is rather low. In invertebrates internal concentrations higher than 10 mmol/l can rarely be survived; vertebrates are generally even more sensitive (Campbell, 1973). The toxicity is related to its role in the stabilization of the pH of the body fluids but also to the interference with active Na+, Cl- and HCO; transport (e.g. Gupta et nl., 1977). In view of the toxicity of ammonia, elevated concentrations in the environment may be expected to be harmful. In the tissues ammonia is produced in the catabolism of proteins (amino acids). It is generally assumed that, in aquatic animals, ammonia is released to the environment at the same rate at which it is produced. This release can be a passive process as NH: in natural waters tends to be very low, related to the fact that NH: in an aerobic environment can easily be converted to oxidated nitrogen compounds (NO;, NO;). Improved stabilization of internal NH: concentrations can be obtained by the presence of mechanisms for active transport. Active transport of NH: has indeed been found in annelids and molluscs (Magnum et al., 1978) and a crustacean species (Spaargaren et al., 1982). This paper presents data on the permeability and the salt fluxes across the body wall (mainly through the gills) of shore crabs, Carcinus maenas, considering the question in what way the processes for ionic regulation are affected by increased NH; concentrations in the environment.

Shore crabs, Curcinus maenus (L.), are common inhabitants of the shallow waters alongside the Atlantic coasts. In estuarine areas, the animals are abundant in the waters, at low tide, left behind on tidal flats. Occasionally, the oxygen content of this environment may, especially during the night (in the absence of photosynthesis), fall to low values, due to the oxygen consumption of living organisms and of dead, decaying material. Concurrent with the fall in the oxygen content, the concentration of metabolic end-products, e.g. ammonia, total carbon dioxide content (TCOr = gaseous COZ, carbonic acid, bicarbonate and carbonate), will increase. The same may happen, to an even higher degree, in closed aquaculture systems containing high animal densities. The drop in the environmental oxygen concentration can readily be survived by this species (e.g. Herreid, 1980; Hill et al., 1989). Even in the complete absence of oxygen C. maenas can stay alive for periods of about two days. A previous study (Spaargaren, 1990a) showed that a rise in TCO, is also harmless as long as external TCO, concentrations remain lower than those in the blood, which, in natural circumstances, usually is the case. Elevated CO2 concentrations will, however, cause stress and can only be survived at the expense of metabolic energy. No information is available about the way in which the animals can withstand an increase in external ammonia concentrations. Ammonia is both a very useful and a very toxic substance. In the body fluids it plays a role in the stabilization of the pH, essential in governing the reaction rates of metabolic processes. In terrestrial animals it also plays a part in maintaining the alkali reserve: after the intake of a large quantity of water, 87

88

D. H. SPAARGAREN MATERULS

AND METHODS

the straight line which can be obtained by autocorrelation of subsequent measurements according to a method described in detail elsewhere (Spaargaren, 1990b). From the permeability, determining the passive flux (J,), and the net flux (J,), the active flux can be derived as the difference between J, ‘and J,. Permeability and flux data were determined in relation to

Shore crabs, Carcinus maenas (L.), were collected in the Western part of the Dutch Wadden Sea, near the island of Texel. In the laboratory, the animals, males as well as non-ovigerous females, with weights varying between 15 and 60 g, were placed in natural seawater (salinity about 28%). For the measurements, individual specimens were taken from their storage container, wiped dry and weighed (to the nearest mg) and transferred to a measuring chamber containing a known volume (200 ml) of seawater with a salinity equal to that to which the animals were adapted. After a few minutes of acclimatization, the salinity in the measuring chamber was abruptly decreased by replacing a certain part of the medium volume with a mixture of demineralized water and 0.5 mol/l NH,Cl solution. The volumes of demineralized water and NH,Cl solution, replacing a certain medium volume, were calculated by solving two simultaneous equations. One of them described a selected chloride quantity in the medium as being the sum of the chloride quantity already present in the remaining medium and that present in the NH,Cl solution added. A second equation described in similar way a selected NH: concentration as the sum of the quantity present in the remaining medium and that present in the added NH,Cl solution. The two equations can then be solved for the unknown volumes composing the added mixture. After replacing a certain medium volume with the diluted NH,Cl solution, and a short incubation time (about 2 min), needed for complete mixing, the medium salinity was recorded automatically during 90 set periods with a sampling frequency of 1 measurement per second. Medium salinity was measured by means of a flow-through conductivity electrode (Philips, type PW 9513) connected to a Wayne Kerr (type B642) conductivity bridge, reaching a sensitivitv of four to five digits. The conductivity cell was continuously flushed with the medium using a centrifugal water pump (Eheim) at a rate of 4 l/min. The time course of the change in medium conductivity yields information on the passive salt permeability (P,) of the body wall and the net salt flux (J,) at the start of the measuring period. These two quantities follow from the slope and the Y-intercept of Whole

animal

pwmeabilitv

(ml/s)

Active

salt

external NH: concentration, medium salinity and time after the change of the medium composition. RESULTS

Time dependence of the exchange characteristics

When, in the presence of external NH: concentrations between 0.2 and 2.0 mmol/l, the exchange characteristics are determined repetitively (each 2 min after a 4% decrease in medium salinity) then it appears that shortly after a salinity decrease each quantity shows drastic changes. Permeability (Fig. la), after remaining constant during the first few minutes, rises rapidly to about twice its original value. This is rather unexpected as, at the hyper-ionic NH: concentrations, concurrent with the increase in permeability the passive influx of NH: will be facilitated. In the absence of elevated NH: concentrations, permeability does not increase after a decrease in external salinity, but, on the contrary, always shows (e.g. Spaargaren, 1990) a (temporary) reduction. This reduction seems more functional as it decreases the salt loss at the lowered salinity. A possible explanation for the increase in permeability, after a salinity reduction and in the presence of elevated NH2 concentrations, will be given below. The active salt influx (Fig. 1b), shortly after changing the medium composition, appears to be very low, even slightly negative (which indicates an active salt loss). In the course of time, the active influx (very similar to permeability) rises. With the rise in active influx, the net salt efflux (Fig. lc), which is, of course, Influx

Net

(umol/s)

salt

efflux

(umol/s)

, 6-

4-

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a

0.00’ 0

I

2

4

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,

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(mid

Fig. 1. Whole animal permeability (a), active ion influx (b) and net salt efflux of Carcinus maenas exposed to medium NH: concentrations between 0.2 and 2.0mmol/l in relation to time after a 4%0 decrease in external salinity. In this, and all subsequent figures, the numbers in parenthesis next to the symbols refer to the number of observations. The vertical lines through the symbols represent the standard errors of the means. Temperature 20°C.

Ion exchange in Carcinus tnaenas highest immediately after the decrease in external salinity, starts to decrease. Hence, the increase in passive salt loss, evoked by the rise in permeability, is strongly counteracted by the simultaneous increase in active influx. During the first period there still remains a net salt loss, but this net efflux decreases continuously, eventually to reach zero (when the animals are adapted to the lowered salinity). This is the same pattern as observed in the absence of elevated NH: concentrations. When the animals are adapting to a decrease in salinity, the net salt efflux decreases in the course of time. Control animals show a decrease in net efflux from about 3 pmol/sec to about 1.8 pmol/sec during the first 8 min after a 4% change in salinity. In NH:-enriched media, the net efflux falls from about 6 pmol/sec to about 4 pmol/sec (Fig. lc). At elevated external ammonia concentrations the net efflux values remain higher. The differences (of about a factor 2) must be due to the higher passive salt effluxes associated with higher permeabilities. The higher salt fluxes indicate that elevated NH: concentrations will be unfavourable to the animals, because the higher active salt fluxes will require more metabolic energy. Salinity dependence of permeability and fluxes

Within the test range of salinities (15-25’%), in which the total ion concentration of the extracellular body fluids is strongly stabilized, permeabilities show a tendency to rise with increasing salinities, but, considering the high variability, the effect of external salinity on ion permeability is not very clear (Fig. 2a). The permeabilities of animals exposed to elevated NH: concentrations remain high compared to those observed in control animals. Active salt influx (Fig. 2b) tends to rise at decreasing salinities, to reach a maximum at about 16%. The

Whole

animal

permeability

(ml/s)

4;

:tivo

salt

89

relationship between active influx and salinity therefore reflects the concentration gradient across the body wall at the various salinities. The net salt efflux (Fig. 2c) is found to be positively, almost linearly, related to external salinity. This is the normal response, also observed in the absence of elevated ammonia concentrations. The net flux values, however, are again about twice as high as in control animals. In the salinity range tested (12-24%) Carcinus maenas shows a fairly strong hypertonic regulation of the extracellular body fluids (e.g. Zanders, 1980). This implies that at decreasing salinities the concentration gradient across the body wall increases. The passive efflux, being the product of the concentration gradient and the permeability, will therefore also increase towards lower salinities. Although the animals loose more salts by passive diffusion, the active influx is increased to such an extent that the net salt effluxes become reduced at lower salinities. The effects of elevated medium NH:

concentrations

Figure 3 summarizes the effects of elevated NH: concentrations on the ion exchange characteristics of C. maenas. Up to external NH: concentrations of about 0.7 mol/l, permeabilities remain more or less constant. At higher external NH: concentrations, permeability strongly increases to reach a maximum at [NH:], concentrations of about 1 mmol/l. At still higher external NH: concentrations permeability decreases again: at a medium ammonium concentration of about 2 mmol/l, permeability is again as low as in control conditions. Active influx (Fig. 3b), closely related to permeability, shows a similar pattern. The net fluxes (Fig. 3c) show a slight, but, considering the high variability, insignificant increase at increasing medium ammonium concentrations.

influx

NIrst salt

(umol/s)

efflux

8-

krmol/s)

?-

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6-

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a

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1

I

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1

12

14

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16

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1

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-2 -

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12

Medium

salinity

I

I

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,

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(ppt)

Fig. 2. Whole animal permeability (a), active ion influx (b) and net salt efflux of Carcinus maenasexposed to medium NH: concentrations between 0.2 and 2.0 mmol/l after a 4% decrease in external salinity as a function of the final medium salinity.

D. H.

90 Whole animal

.2sr

permeability

(ml/s)

15

Active

SPAARGAREN

salt

influx

NCIt

(umol/s)

salt

6.S-

efflux

(umol/s)

o-

5-

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am

riu

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: ‘lom

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0.0

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.5

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2.0

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.5

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I

1.0

1.5

1

2.0

2.5

(mmol/l)

Fig. 3. Whole animal permeability (a), active ion influx (b) and net salt efflux of Carcinus maenas after a 4%0 decrease in external salinity as a function of external NH: concentrations.

DISCUSSION Ammonium chloride solutions, used to increase the medium ammonia levels, are slightly acid. As the NH: concentrations used in the experiments remain low ( c 2.0 mmol/l), and the pH of seawater is buffered fairly well, the addition of ammonium chloride solutions to the media never measurably affected the pH of the media. The observed effects of external NH: on the ion exchange characteristics of Cur&us can therefore be ascribed merely to the effect increased NH: concentrations. With medium NH: concentrations between zero and 2.5 mmol/l C. maenas stabilizes the internal ammonia concentration between 0.25 and 0.55 mol/l (Spaargaren, 1982a). Internally-produced ammonia can be excreted passively up to external ammonia concentrations of about 0.28 mmol/l. At higher concentrations passive diffusional loss is not possible any longer. Ammonia is entering passively from the medium, but, internally it is detoxified to urea. The rapid increase in active influx, within the first 8 min after a medium change, demonstrates that the salt transport mechanism is not blocked by the presence of elevated ammonia concentrations. It also has a clear adaptive function in protecting the animal against rapid changes of the internal solute concentrations: despite the higher permeabilities, the net salt efflux (Fig. lc) decreases rapidly in the course of time. The net efflux, however, remains higher than in the absence of elevated NH,+ concentrations. Very likely, gill perfusion and gill ventilation are involved in the rapid increase of permeability and active influx after a medium change. Gill ventilation and perfusion can change almost instantaneously (e.g. Uglow, 1973; Cumberlidge and Uglow, 1977a,b) and can also strongly influence salt exchange (Spaargaren, 1981, 1982b). Any increase in permeability will result in a higher passive ion transport. This seems a disadvantage,

as for stabilizing the internal solute concentrations, a higher, energy consuming, active influx will also be required. At slightly elevated [NH:], levels, however, it may also help to remove excessive NH: from the body fluids. At higher external ammonia concentrations, exceeding those present internally, an increase in permeability and water turn-over will improve the passive loss of urea. The strong increase in permeability (Fig. 3a), found at [NH:], around 0.7 mmol/l might therefore be useful. The concurrent rise in active transport (Fig. 3b) effectively counteracts the increased passive salt loss. At very high external ammonia concentrations (> 2 mmol/l, which are not likely to occur in nature) the detoxication mechanism cannot handle the increased passive ammonia influx any longer and both permeability and active influx become reduced. Generally, active transport and permeability are found to be closely related to each other, whereas the net fluxes remain independent on permeabilities. The close connection between permeability and active transport (also reported earlier: Spaargaren, 1990b) may be explained from the fact that both quantities strongly depend on the ventilation and perfusion of the gills. In the gills, together with the antenna1 glands, most of the salt exchange between the animals and their environment takes place. Permeability, being defined as the passive salt flux per unit of concentration gradient across the body wall, not only depends on the structural properties of the body wall (e.g. pore size, pore density), but it is also strongly dependent on the perfusion of the gills by cardiac activicr as well as on ventilation of the gill chambers by scaphognathite movements. Active transport uses metabolic energy to move ions against an existing concentration gradient and is also dependent on the perfusion of the gills for the supply of energy-rich substrates and on gill ventilation for the supply of oxygen. An increase in external ammonia concentration could be expected to be unfavourable to the animals

Ion exchange in Carcinus maenas (by the inhibition of ammonia removal and by interference with the pH regulation of the body fluids). The results obtained confirm this expectation, but also clarify that the negative effects can be counteracted effectively. The net salt effluxes at elevated ammonia levels in the medium are about twice as high as compared to the values in normal seawater. Between 0.5 and 2.0 mmol/l, however, a further increase can be prevented (the increase in net e!lIux remains insignificant). The increase in active salt influx requires a higher energy consumption, but, as long as elevated ammonia concentrations in the medium remain temporary (as in nature they usually do) they can be survived. In closed aquaculture systems NH: concentrations of the water can rise very easily by the accumulation of excretion products. The results give reason to keep NH: concentrations in such systems as low as possible. REFERENCES

Campbell J. W. (1973) Nitrogen excretion. In Comparative Animal Physiology (Edited by Presser C. L.) pp. 279-316. Saunders, London. Cumberlidge N. and Uglow R. F. (1977a) Heart and scaphognatithe activity in the shore crab, Carcinus maenas (L.). J. exp. mar. Biol. Ecol. 28, 87-107. Cumberlidge N. and Uglow R. F. (1977b) Size, temperature and scaphognathite frequency dependent variations of ventilation volumes in Carcinus maenas (L.). J. exp. mar. Biol. Ecol. 30, 85-93.

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Hill A. D., Taylor A. C. and Strang R. H. (1989) Anaerobic metabolism in the shore crab, Carcinw maenas. SEB Edinburgh Meeting Abstracts p. 109. April, 1989. Magnum C. P., Dykens J. A., Henry R. P. and Polites G. (1978) The excretion of NHa+ and its ouabain sensitivity in aquatic annelids and mollusks. J. exp. Zool. 203,151-157.

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