The effect of hypoxia and anoxia on osmotic and ionic regulation in the brackish water isopod Saduria entomon (Linnaeus) from the Gulf of Gdansk (Southern Baltic).

ABSTRACT The experimental material for the investigations was collected from the deep zones of the Gulf of Gdansk. The experiments were performed at salinities of 3.0 PSU (96.87 mmol/kg), 7.3 PSU (235.73 mmol/kg), 15 practical salinity unit (PSU) (484.39 mmol/kg) and 25 PSU (807.32 mmol/kg) in conditions of hypoxia (saturation = 15% [O.sub.2]), anoxia (saturation <1% [O.sub.2]) and control conditions in aerated water (saturation = 100% [O.sub.2]), at a constant temperature of 10[degrees]C. Hypoxia (saturation = 15% [O.sub.2]) had no statistically significant effect (P > 0.05) on the osmoregulation of S. entomon in comparison with the control samples. Anoxia (saturation <1% [O.sub.2]) decreased the osmotic concentration of hemolymph in S.entomon. After 6 h only at salinity 3 PSU there was a statistically significant difference between the anoxic and control samples (P < 0.05). After 96 h there was a statistically significant tendency (P < 0.05) for the osmotic concentrations to decrease in anoxic conditions at all the salinities tested except 25 PSU. A tendency was observed for the [Na.sup.+] concentrations in the hemolymph to decrease in anoxic conditions, which was statistically significant at salinity 7.3 PSU (P < 0.05) after 96 h but not statistically significant at the other salinities tested. The results show a low concentration of [K.sup.+] in the hemolymph of S. entomon and slight fluctuations of [K.sup.+] concentrations, but at environmental salinities the [K.sup.+] concentration decreases under anoxic conditions but not to a statistically significant extent (P > 0.05).

KEY WORDS: anoxia, hypoxia, [K.sup.+] and [Na.sup.+] regulation, osmoregulation, S. entomon, Baltic isopod 

INTRODUCTION

The Gulf of Gdansk is one of the most eutrophicated areas of the Baltic Sea because of large content of organic matter. During long-term stagnation anoxia conditions appear at the bottom. Generally, oxygen conditions depend on season and water mass movement (Lysiak-Pastuszak 1995). Oxygen depletion occurs in the autumn-winter season, and in spring after strong inflow of oxygen-rich water from the North Sea, oxygen conditions under halocline become better (Cyberska et al. 1990).

Despite considerable human pressure and the gradual spreading of the zones of hypoxia and anoxia at the bottom of the Gulf of Gdansk (Southern Baltic), there do exist species capable of survival in difficult environmental conditions. One such species is Saduria entomon--the Baltic isopod, an important component of the Gulf of Gdansk zoobenthos (Janas et al. 2004).

The species shows very high tolerances to changes in environmental factors such as salinity and oxygen tension. S. entomon lives in the deeper waters in the Baltic, normally buried in the sediment (Croghan & Lockwood 1968).

Saduria entomon usually inhabits sandy-muddy bottoms, but can also survive on other substrates, as long as individual animals can burrow into them (Haahtela 1990). Tolerant to a broad range of salinity (0-40 PSU) (Bobowicz 1968), it is present in Baltic waters of salinities from 1-20 PSU (Haahtela 1990). S. entomon is also very tolerant of hypoxia and even of anoxia (Hagerman & Szaniawska 1988, 1990, 1991, 1992). Compared with other crustaceans, it can withstand anaerobic conditions (Normant et al. 1998, Normant & Szaniawska 2000), the L[T.sub.50] in anoxic conditions being 11 days (Kristoffersson & Pirkko-Leena Kuosa 1990).

S. entomon occurs at the depth from 22 to about 80-90 m in the Gulf of Gdansk. At the depth from 70-75 m to about 80-90 m oxygen amount decreases to 1 cm 3 [O.sub.2]x[dm.sup.-3]. On the bottom below 80 m depth, oxygen amount is lower than 1 [cm.sup.3] [O.sub.2]x[dm.sup.-3] (Witek 1995).

The aim of this work was to investigate the effect of hypoxia on osmoregulation and of anoxia on ionic and osmotic regulation in S. entomon at different salinities: earlier work examined only the effect of anoxia on ionoregulation in S. entomon (Hagerman & Szaniawska 1991) and only at one environmental salinity, namely, 7 PSU, The present study focuses on the ionic concentrations of [Na.sup.+] and [K.sup.+] in the hemolymph of S. entomon at salinities of 3.0 PSU (96.87 mmol/ kg), 7.3 PSU (235.73 mmol/kg), 15.0 PSU (484.39 mmol/kg), and 25.0 PSU (807.32 mmol/kg), and also on the effect of anoxia on osmoregulation in this species.

MATERIALS AND METHODS

Isopods S. entomon, were collected from the Gulf of Gdansk (Fig. 1) and transferred to the laboratory, where they were acclimatized to the ambient conditions for one week to allow the metabolic processes to stabilize (Einarson 1993, Funge-Smith et al. 1995). Fed with dry Daphnia sp., the animals were kept in containers with sandy sediment and aerated water at the environmental salinity (7.3 PSU). These in turn were placed in thermostatted aquaria at a constant temperature of 10[degrees]C.

[FIGURE 1 OMITTED]

Following acclimatization, the S. entomon specimens were moved directly to hypoxic (saturation = 15% [O.sub.2]) and anoxic (<1% oxygen saturation) waters of various salinities: 3.0 PSU (96.87 mmol/kg), 7.3 PSU (235.73 mmol/kg), 15 PSU (484.39 mmol/kg) and 25 PSU (807.32 mmol/kg). Hypoxic (15% oxygen saturation) and anoxic (<1% oxygen saturation) conditions were produced by purging the water with gaseous nitrogen piped into hermetically sealed containers, like in investigations performed under anaerobic conditions (Gamble 1971, Hagerman & Uglow 1982, Hagerman & Szaniawska 1988; 1991, Scholz & Zerbst-Boroffka 1998). Oxygen levels were monitored with a Microprocessor Oximeter OXI 96, the salinity with an LF 196 conductometer. The various salinities were prepared by diluting Atlantic water or Baltic water from the Gulf of Gdansk with distilled water. Control experiments in aerated water were conducted simultaneously. Hemolymph was sampled from five specimens for each experimental salinity/ oxygen combination; individual animals were used once only. Prior to the determination of the osmotic concentrations of hemolymph, animals were placed abdominally on blotting paper and gently dried, after which hemolymph was extracted from the heart with a syringe (Percy 1985). The hemolymph was then transferred to:

(a) glass capillary tubes (the hemolymph was situated between layers of liquid paraffin wax) for measuring osmotic concentrations; these were determined according to Ramsay's method (1949), appropriately modified according to the melting point of hemolymph (Dobrzycka & Szaniawska 1995);

(b) 1.5 mL Eppendorf test tubes for determining [Na.sup.+] and [K.sup.+] concentrations. For this purpose, the hemolymph was diluted 4 x with deionizer water, after which the levels of these ions were determined in an AVL 982-S Electrolyte Analyzer using ion-selective electrodes (Tamura et al. 1983, Moody et al. 1989, Spichiger-Keller 1998).

The hemolymph samples were collected in the refrigerator before measuring the osmotic concentrations and in the freezer before measuring the ionic values.

The nonparametric Kolmogorov-Smirnov test (P < 0.05) was used to determine the level of significance of the differences between the stressed and control samples. The Kolmogorov-Smirnov test is the best for comparing the very similar samples from stressed and control conditions with known size of samples (5).

RESULTS

The effect of hypoxia (15% oxygen saturation, T = 10[degrees]C, S = 3.0 PSU (96.87 mmol/kg), 7.3 PSU (235.73 mmol/kg), 15 PSU (484.39 mmol/kg), and 25 PSU (807.32 mmol/kg)).

Behavior

In the first minutes of the experiment some of the S. entomon specimens emerged from the sediment and remained on the surface. There was a distinct raising of the telsons in conjunction with intensive ventilatory movements. Later the animals became quite inactive, as if "weightless." They seemed to be suspended in the water. After 30 min a few animals began to burrow into the sediment, whereas others remained on its surface.

Mortality

No mortality of S. entomon individuals was recorded during the experiments.

Osmotic Concentrations

The levels of osmotic concentrations after an experimental period of 120 h hypoxia (Fig. 2).

Control conditions versus hypoxic conditions (100% oxygen saturation, T = 10[degrees]C, S = 3.0 PSU [96,87 mmol/kg], 7.3 PSU [235,73 mmol/kg], 15 PSU [484,39 mmol/kg] and 25 PSU [807,32 mmol/kg]).

Behavior

The animals remained buried in the sandy sediment; only the tips of the telsons protruded above the surface.

Mortality

No mortality of S. entomon was recorded during any of the control experiments.

Osmotic Concentrations

Osmotic concentrations after 120 h are presented in Figure 2. The differences between the osmotic concentrations under hypoxic (15% oxygen saturation) and control conditions after both 6 h and 120 h were statistically insignificant (P > 0.05).

[FIGURE 2 OMITTED]

In these investigations S. entomon exhibited a hyperosmotic body fluid level at lower salinities and a slightly hypoosmotic level at higher salinities.

The effect of anoxia (<1% oxygen saturation, T = 10[degrees]C, S = 3.0 PSU [96.87 mmol/kg], 7.3 PSU [235.73 mmol/kg], 15 PSU [484.39 mmol/kg] and 25 PSU [807.32 mmol/kg]).

Behavior

Initially, some animals emerged onto the surface of the sediment, and ventilatory movements intensified. Later, they began to respond to the anaerobic conditions by becoming immobile, remaining suspended in the water as if in a state of weightlessness. Afterwards, some S. entomon started to burrow back into the sediment, leaving just their telsons exposed. The animals remaining on the surface of the sediment became completely immobile.

Mortality

There was no mortality after 6 and 120 h of the experiments.

Osmotic Concentrations

After an experimental period of 6 h, the lack of oxygen did not substantially affect osmoregulation, but it did tend to lower the osmotic concentration in the stressed, anaerobic environment as compared with the controlled, aerated conditions. After 6 h only at salinity 3 PSU there was a statistically significant difference between the anoxic and control conditions (P < 0.05).

After 96 h of experiments, there was distinct tendency for the osmotic concentrations of hemolymph to decrease under anoxic conditions. The differences between osmotic concentrations under anoxic and controlled conditions were statistically significant (P < 0.05) at all the salinities except 25 PSU (Fig. 3).

[FIGURE 3 OMITTED]

[Na.sup.+] Concentrations

After 6 h of experiments [Na.sup.+] concentrations in hemolymph did not differ substantially between the various levels of oxygen. After 96 h the [Na.sup.+] concentrations in S. entomon hemolymph under anaerobic conditions were lower than in the control samples (Fig. 4).

[FIGURE 4 OMITTED]

[K.sup.+] Concentrations

A relatively low concentration of [K.sup.+] was determined in the hemolymph of S. entomon. The results show small fluctuations of the [K.sup.+] level (Fig. 5).

[FIGURE 5 OMITTED]

Generally, [K.sup.+] concentrations of hemolymph of S. entomon after 6 h under anoxic conditions were lower than under control conditions.

After 96 h, the [K.sup.+] level under anoxic conditions tended to fall in comparison with the control.

Control conditions versus anoxic conditions (100% oxygen saturation, T = 10[degrees]C, S = 3.0 PSU [96.87 mmol/kg], 7.3 PSU [235.73 mmol/kg], 15 PSU [484.39 mmol/kg], and 25 PSU [807.32 mmol/kg]).

Behavior

The animals remained buried in the sediment, with only the tips of their telsons protruding above the surface of the sediments.

Mortality

No mortality was recorded at any of the tested salinities under the control conditions.

Osmotic Concentrations

Osmotic concentrations of hemolymph after 96 h are presented in Figure 3. In these experiments S. entomon exhibited hyperregulation at the lower salinities and hyporegulation at the higher ones.

[Na.sup.+] Concentrations

After 6 h the differences between samples from the anoxic and control conditions were statistically insignificant (P > 0.05). After 96 h the only statistically significant difference between the anoxic and control conditions was for 7.3 PSU (P < 0.05) (Fig. 4).

[K.sup.+] Concentrations

After 6 h and after 96 h differences between [K.sup.+] concentrations under anoxia and under control conditions were not statistically significant (P > 0.05). The effect of anoxia (<1% oxygen saturation) on [K.sup.+] concentration in the hemolymph of S. entomon after 96 h of the experiment is visible (Fig. 5).

DISCUSSION

Osmotic concentration in S. entomon has been investigated by Bogucki (1932), Bobowicz (1968, 1970), Percy (1985) and Dobrzycka & Szaniawska (1995). Many papers have also focused on the ionic composition of S. entomon, for example, Lockwood & Croghan (1957), Croghan & Lockwood (1968), Lockwood et al. (1976). Even so, some effects of factors disturbing ionic and osmotic regulation in this species are less well known. These problems have been studied only by Hagerman & Szaniawska (1991), who examined the ionic regulation of this species under anoxic conditions.

The fact that hypoxia and anoxia occur in the Gulf of Gdafisk, where this species is common, seemed sufficient justification for investigating the effect of hypoxia and anoxia on osmoregulation. Additionally, the fact that hypoxia and anoxia at the bottom has strong impact on the structure of biocenosis case that this subject is very important to investigate.

In low oxygen conditions of the environment only a few groups of organisms are able to survive. The largest groups are bivalves (Theede et al. 1969) and species such as the priapulid Halicryptus spinulosus (Oeschger et al. 1992). Crustaceans are generally not very tolerant of hypoxia and anoxia, but S. entomon is the species of very high resistance to such factors in the environment (Hagerman & Szaniawska 1990, Vismann 1991). This species is able to reduce its metabolic level in anoxia down to very low levels, in this way economizing energy expenditure. During 40 h of anoxia S. entomon can gradually decreased its heat production to 5% to 16% of aerobic level, demonstrating the high adaptation of this species to changeable oxygen conditions in the Baltic Sea (Normant et al. 1998). In comparison: Halicryptus spinulosus decreases its total metabolism after 48 h of anoxia to 26% and after 2 wk to 2% of the aerobic level (Oeschger et al. 1992). The values obtained for S. entomon are similar to those found in bivalve species, such as Mytilus edulis and Modiolus demissus, which are able to decrease their metabolic rate to <5% of the normoxic value (Pamatmat 1979, Hammen 1983). The major substrate for anaerobic metabolism of this species is glycogen. Glycogen content in S. entomon is the highest during winter months. In this season glycogen equals about 34% of the total dry mass of carbohydrates (Normant & Szaniawska 1996). This is much more than in other crustaceans from the Gulf of Gdansk.

The osmotic concentrations in S. entomon specimens under conditions of hypoxia fluctuated little in comparison with the control specimens, which were kept in well-aerated conditions.

Effects of anoxia was statistically significant after 6 h of experiment only at the lowest salinity (3 PSU). After 96 h there was a statistically significant tendency (P < 0.05) for the osmotic concentrations to decrease in anoxic conditions at all the salinities tested, except 25 PSU.

Osmoregulation in the light of these investigations seems to be a stable metabolic process. Crustaceans, as long as it is possible, maintain a stable level of hemolymph concentration.

The effect of hypoxia and anoxia on osmotic concentrations of another crustacean from the Gulf of Gdansk--Corophium volutator explained the same pathway: very stable level of osmotic concentration, but mentioned species is less tolerant to long-time anoxia--it was not possible to determine long-term effect of anoxia because of high mortality of the animals (Dobrzycka-Krahel & Szaniawska 2005).

Analyses of the [Na.sup.+] and [K.sup.+] concentrations in the hemolymph of S. entomon indicate that the [Na.sup.+] concentrations of the former are considerable this ion appears to play a crucial role in ionic regulation. In this study, the concentration of [Na.sup.+] (in environmental salinity = 7.3 PSU) = 172.67 mmol/L: was found to be relatively similar in comparison the [Na.sup.+] concentration in Baltic S. entomon as determined by Croghan & Lockwood (1968) = 200 mmol/L, and in the investigations made by Hagerman & Szaniawska (1991) = 300 mmol/L.

The results of the present work show that the [Na.sup.+] concentrations tend to decrease in anoxic conditions. The fact that a lack of oxygen decreases the level of [Na.sup.+] in anoxia, was first observed by Hagerman & Szaniawska (1991), because earlier investigations were concerned with the effect of hypoxic conditions on ionic regulation in organisms.

The [Na.sup.+] concentration in the hemolymph is linked with the [H.sup.+] and N[H.sub.4.sup.+] balance (Mantel & Farmer 1983). Under long-time anoxic conditions, the ammonium concentration in hemolymph increases, because S. entomon produces alanine as an anaerobic end product (Hagerman & Szaniawska 1990), which probably brings about changes in the hemolymph buffer system of S. entomon.

Glycogen utilization (as mentioned earlier) is reflected in increased blood glucose levels during anoxia. Contrary to most crustaceans, alanine was an important anaerobic end product in inactive, long-term anoxia exposed S. entomon. Hemolymph alanine increased steadily with time and was, quantitatively, the most important end product after 50 h of anoxia. The reduction of glycolytic flux after 120 h anoxia (a reduction of anaerobic metabolic rate) might be affected by the inhibitory effect of alanine on the enzyme pyruvate kinase. This would enable Saduria to extend the tolerance period of anoxia because the energy resources would last longer (Hagerman & Szaniawska 1992).

Even very small increases in [p.sub.w][0.sub.2] from anoxia to just 4% to 5% saturation caused re-establishment of the high [Na.sup.+] concentration found in normoxia (Hagerman & Szaniawska 1991).

The low [K.sup.+] level indicates that this ion plays little part in this process. The [K.sup.+] concentration (in environmental salinity = 7.3 PSU) in this study was 6.93 mmol/L, whereas [K.sup.+] in the hemolymph of S. entomon as determined by Croghan & Lockwood (1968) was about twice as high as that determined by Hagerman & Szaniawska (1991) = 2.3 mmol/L.

Ionic regulation in hypoxic/anoxic conditions has been investigated many times in hyper/hyporegulators, for example in Crangon crangon (Hagerman & Uglow 1982), Palaemon adspersus (Hagerman & Ugtow 1981), or S. entomon (Hagerman & Szaniawska 1991). The results of these studies show that these animals attempt to maintain an aerobic metabolism and maintain a normal ionic balance for as long as they can. S. entomon maintains a stable respiratory rate up to 10 Torr, but below this value, the increasing accumulation of anaerobic products (Hagerman & Szaniawska 1990) causes the ionic regulation to be disturbed. This may indicate that aerobic metabolism of S. entomon is lower than in other crustaceans (Hagerman & Szaniawska 1988); this is confirmed by the adaptational abilities of this species.

Thus, these studies showed that the isopod S. entomon is very well adapted to survive long-time anoxia.

CONCLUSION

Osmoregulation of S. entomon specimens under hypoxic and anoxic conditions demonstrates the high resistance of the species to oxygen depletion. Anoxia is a factor whose effect intensifies with time. Whereas the [Na.sup.+] level in the hemolymph in S. entomon did generally tend to fall in conditions of anoxia, the differences between the control and anoxic samples were statistically not significant P > 0.05 (except for salinity 7.3 PSU after 96 h of experiments). [K.sup.+] levels did not fluctuate under anoxic conditions; [K.sup.+] concentrations were relatively low.

ACKNOWLEDGMENT

This research was supported by grant BALTDER SPB 127/ E-335/SPB/5PR UE/DZ 78/2003-2005.

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ALDONA DOBRZYCKA-KRAHEL * AND ANNA SZANIAWSKA

Institute of Oceanography, University of Gdansk, Al. Marszalka J. Pilsudskiego 46, 81-378 Gdynia, Poland

* Corresponding author. E-mail: [e-mail]