The contribution of the mud shrimp Callianassa subterranea (Decapoda: Thallassinidea) to sediment metabolism during oxygen deficiency in southern North Sea sediments

193

Journal of Sea Research 36 (3/4): 193-202 (1996)

THE CONTRIBUTION OF THE MUD SHRIMP CALLIANASSA SUBTERRANEA (DECAPODA: THALLASSINIDEA) TO SEDIMENT METABOLISM DURING OXYGEN DEFICIENCY IN SOUTHERN NORTH SEA SEDIMENTS MARTIN POWILLEIT 1 and GERHARD GRAF 2 l lnstitut for Ostseeforschung (lOW), SeestraBe 15, D-18119 Warnem#nde, Germany 2GEOMAR Forschungszentrum, WischhofstraBe 1-3, D-24148 Kiel, Germany

ABSTRACT Long-term experiments with single isolated specimens of C. subterranea (Montagu) in closed systems under anoxic conditions were conducted to measure CO 2 release and heat production as parameters for anaerobic metabolism. For small C. subterranea (48.3 - 97.6 mg dw) the rate of CO 2 release was estimated to be 1.69 to 3,63 p.mol CO2"g dw'l.h "1 and for large specimens (330.9 - 543.0 mg dw) 0.28 to 1.52 ~mol CO2"g dw'l.h "1 during the incubation period of 5 days (6°0). The rate of C O 2 release increased by a factor of up to 2 during the first three days and increased more rapidly later in this incubation period. Direct calorimetry revealed a decrease in the rate of heat production from 0.39 to 0.25 J.g dw'l.h "1 (Le. 64% of initial value) during long-term anoxia (110 h). Compared to normoxic values recalculated from literature data our results indicate a decrease in the heat production rate to 33% under prolonged anoxia. For a natural Callianassa population on station 'Schlicksandgrund' in the German Bight the rate of CO 2 release was calculated to be 8.0 ~mol.m'2.h "1 under anoxic conditions. T-.CO2 pore-water profiles on station 'Schlicksandgrund' were used to calculate a CO 2 flux from the sediment to the near-bottom water of 110 i~mol CO2"m'2,h "1 under normoxic and 338 pmol CO2'm'2.h "1 under hypoxic conditions. The anaerobic metabolic activity of the Callianassa population could account for about 7.3 (oxic conditions) to 2.4% (hypoxic conditions) of the above CO 2 fluxes.

Keywords: C. subterranea, Thallassinidea, sediment metabolism, anoxia, heat production, North Sea

1. INTRODUCTION The mud shrimp Callianassa subterranea (Montagu) inhabits a deep burrow system down to at least 40 cm sediment depth in southern North Sea sediments with densities between 21 and 78 ind-m -2 (Witbaard & Duineveld, 1989; KLinitzer et aL, 1992; Rowden & Jones, 1994; Forster & Graf, 1995). Due to its extreme excavation activity, which is also known for other thallassinidean shrimps (e.g. Dworschak, 1983), C. subterranea is responsible for a sediment turnover of 11 kg.m'2.y "1 (cf. Rowden & Jones, 1993). Recently the irrigation of C. subterranea was investigated by Forster & Graf (1995) in order to estimate its impact on oxygen flux into North Sea sediments. The dimensions of burrow system walls were calculated to create a secondary surface of 1.6 m2 per m2 sediment surface. With rhythmic strikes of its pleopods C. subterranea performs an 'intermittent pump-

ing', which can be divided into a highly regular irrigation (2.6 min breathing current and 40-min pauses) and a more intense irregular irrigation (less frequent). A time budget shows that only in the short periods of breathing currents and in periods of irregular irrigation (18% of total time) can oxygen be measured inside the burrows although present in the overlying water (using oxygen microsensors). At least 50% of the wall in C. subterranea burrows stays permanently anoxic, and it was concluded that the mud shrimp must be well adapted to such conditions (Forster & Graf, 1995). In its natural habitat, even if the near-bottom water is oxygenated, most of the time C. subterranea lives in hypoxic or even anoxic conditions interrupted by short oxic periods. We suggest that during extreme oxygen depletion (up to several weeks), as reported from southern North Sea sediments especially in the 1980s (e.g. Von Westernhagen et aL, 1986; Hickel et

194

M. POWILLEIT & G. GRAF

al., 1989; Duineveld et aL, 1991) such hypoxic conditions in the near-bottom water may even cause total anoxia in the burrow system for extended periods (days to weeks). Information on the ability of C. subterranea to survive prolonged anoxia in situ and on its metabolic activity under such conditions is lacking. Witbaard & Duineveld (1989) measured the oxygen consumption (respiration) of C. subterranea in a flow-through system under normoxic conditions and compared these rates to in situ oxygen measurements of the sediment surface in central North Sea sediments. They found that C. subterranea was responsible for 17% of the whole sediment oxygen demand (SOD), indicating that it is very important in the benthic community under normoxic conditions. CO 2 release and heat production have been measured together with quantitative determinations of anaerobic metabolites to quantify anaerobic metabolic activity of marine invertebrates. It was found that depending on the metabolic regulation capacity of an organism and on the biochemical pathway involved, the amount of metabolic end products and the rate of heat production differ (e.g. Zebe, 1982; Hammen, 1983; Gnaiger, 1983; Pamatmat, 1983; Sch6ttler et al., 1984; P6rtner et aL, 1984; Famme & Knudsen, 1983; Widdows, 1987; Oeschger, 1990; Hardewig et al., 1991; Oeschger et al., 1992; Fritsche & Von Oertzen, 1995). Carbon dioxide can also be used in calculations of the carbon flux in the sediment (e.g. Hargrave & Phillips, 1981; Andersen et aL, 1986; McNicol et aL, 1988; Kristensen et aL, 1991,1992). For a comparison of the metabolic activity of C. subterranea during anoxia with the total sediment metabolism, CO 2 pore-water profiles from a silty sediment in the southern North Sea were used to calculate CO 2 fluxes from the sediment to the near-bottom water under different oxygen regimes. These fluxes were compared to the rate of CO 2 release during anoxic incubation from the Callianassa population to determine the share of this dominant species in its natural habitat.

Oxygen concentrations in the near-bottom water in the investigation period were always higher than 87% saturation (Forster, pers. comm.). Sediment cores for CO 2 pore-water measurements and specimens of C. subterranea were sampled using a box corer (0.25 m2). Sediment subsamples taken to a maximum depth of about 25 cm using Plexiglas cores with an inner diameter of 20 cm (314 cm 2) were transported to the laboratory at in situ temperature (6-16 °C). The overlying water was continuously aerated. Individuals of C. subterranea were either sieved from the sediment or collected by hand from box core sediments (Powilleit, 1991). 2.2. LABORATORY TREATMENT OF THE SAMPLES tn the laboratory cores were sectioned into 1-cm slices and the water content and the ZCO 2 concentration of centrifuged, particle-free pore water immediately analysed. Water content of the sediment and dry weight of C. subterranea were determined after 24 h heating at 60°C, as were organic content and ash-free dry weight after 24 h at 500°C. 2.3. DETERMINATION OF T.CO2 Determination of ZCO 2 was conducted using a non-dispersive infrared analyser (Beckman Ind. Model 880), and using a set-up described by Altenbach (1987) for measurements of carbon content in foraminifera. A closed, circulated airstream was continuously pumped through the IR-analyser. Prior to each measurement, all carbon dioxide and all water inside the system were removed by the absorbents ascarite and Mg(CLO4) 2. After injection of the sample (1 cm 3) and subsequent addition of 30% phosphoric acid, the released CO 2 was measured during a period of 5 to 8 min in which the carbon dioxide was evenly mixed inside the circulating gas. The CO 2 concentration measured was integrated over a maximum period of 3 min to obtain stable values. For further details see Forster et al. (1995).

2. MATERIAL AND METHODS 2.4. INCUBATION EXPERIMENTS 2.1. SAMPLING LOCATION Sediments and individuals of Callianassa subterranea were sampled four times between May 1989 and June 1990 at a silty-sand station 'Schlicksandgrund' about 10 nautical miles southwest of the island of Helgoland in the German Bight (southern North Sea, 54°01'N, 07°49'E). The benthic community was described as a Nucula nitidosa community (Salzwedel et al., 1985). Grain-size analysis yielded a silt-clay content (< 63 #m) of 21.8% and an organic matter content in the upper 20 cm of about 1.5% of sediment dry weight (Teucher, 1991). The sediment porosity in the upper 18 cm varied between 0.62 and 0.44 and the profile showed no pronounced gradient.

The course of CO 2 release was measured during 5-day incubation experiments in closed systems (cylindrical glass bottles) with different-sized C. subterranea (small individuals 73.0+_20.2 mg dw in -13-cm 3 bottles and large individuals 468.1+_94.7 mg dw in -60-cm 3 bottles) in June 1990 (in situ temperature of 10°C). After an adaptation period of several days the incubations were run in the dark at a temperature of 6 C+1 C. Prior to the incubation the oxygen of the incubation water was lowered to values _<15% oxygen saturation by N 2 purging. After a few hours there was no oxygen left inside the incubation bottles due to respiration by C. subterranea. One individual of C. subterranea was confined in each chamber, and

CALLIANASSA AND SEDIMENT METABOLISM

195

TABLE 1 Total amount of T-CO2 in incubation bottles with Callianassa subterranea and maximum bacterial impact ( #mol %CO2). incubation time

day 1 day 2 day 3 day 4 day 5

small individuals total T,CO2 max. microbial portion Ca-corrected

(uncorr.)

2.39 5.27 10.34 18.05 26.89

(2.39) (6.35) (15.53) (21.55) (33.40)

0.34 (14.2%) 0.61 (11.6%) 0.73 (7.1%) 1.18 (6.5%) 1.68 (6.2%)

daily subsamples of 1 cm 3 incubation water were taken by a gastight syringe to determine CO 2 concentrations. Additionally, at the beginning and at the end of each experiment 2 cm 3 were taken for determination of Ca concentrations (n = 5). In another parallel chamber the bacterial cells were counted in 1-cm 3 subsamples on each sampling date (without replicates). One blank chamber without animal was run as a control. The volumes of extracted subsamples were compensated by 1 cm 3 of deoxygenated seawater with known CO 2 concentration to continue anoxic conditions. This procedure was performed very quickly to avoid oxygen penetration into the incubation bottles. Moreover the surface area of the incubation chamber was very small so that diffusion of 0 2 into the chamber could be neglected. After incubation all large individuals were alive as could be seen by body movements. Only half of the small individuals showed reactions following mechanical stimulation after day 4 (only these data were used), the other half died between day 4 and 5.

large individuals total ~.C02 max. microbial portion Ca-corrected (uncorr.)

2.02 2.25 15.73 35.05 79.00

(2.02) (2.25) (21.49) (43.69) (93.38)

1.55 (76,7%) 2.80 (>100%) 3.27 (20.8%) 5.39 (15.4%) 7.66 (9.7%)

the 002 values were consequently lowered ('Ca-correction', Table 1). 2.6. BACTERIAL CELL NUMBERS To determine the impact of microbial heterotrophic activity on the amount of CO 2 in the incubation bottles we counted bacterial cell numbers at each sampling date. This was performed using the epifluorescent microscopy technique described by Meyer-Reil (1978). The preserved samples were sonicated to avoid aggregation of cells, stained with acridine orange, and filtrated with 0.2-p.m nucleopore filters before counting on a Zeiss Axiovert 35 (490 nm). From bacterial cell numbers the bacterial biomass was estimated assuming an idealized bacterium size (1 p.m length, 0.5 p.m width) and a conversion factor for volume to biomass of 1.06.10 -13 gC.#m -3 (Meyer-Reil, 1983). 2.7. HEAT PRODUCTION MEASUREMENTS

2.5. DETERMINATION OF CALCIUM Determination of Ca 2÷ concentrations was conducted according to a complexiometric titration method with EGTA (Tsunogai et aL, 1968) modified after Gra6hoff (1976). The relative precision was 0.35% (i.e. SD) of the measured values (n = 10). Anaerobic metabolites modify pH and may cause CaCO 3 dissolution. To correct the rates of CO 2 release for CaCO 3 in the incubation bottles, we measured initial and final concentrations of Ca 2+ in the incubation water. According to the reaction of calcium and bicarbonate to calcium carbonate (Le. equimolar ratio) the variations in Ca concentrations were multiplied by a factor of 1.0978 to alllow for associated CO 2 variations. During the experiments pH values of incubation water shifted from 8.2 to 6.8. Values of CO 2 release for these inorganic reactions were corrected by recalculating rates for the last three (large individuals) and four days (small individuals) of the incubation period. The measured increase of Ca concentrations in the incubation water indicates dissolution of CaCO 3 and

Measurements of the heat production of C. subterranea were conducted using a 10-fold multicalorimeter described by Graf et aL (1988). The volume of each chamber was 25 cm 3. Prior to experiments, specimens of C. subterranea (425+141 mg dw, n=3) were rinsed with particle-free seawater. One individual was placed in each chamber filled with deoxygenated, particle-free seawater. The experiments ran for 110 h mainly under anoxic conditions (about 15% oxygen saturation at the beginning of incubation). The incubation temperature was 7 C. Due to other measurements in the calorimeter and a long temperature equilibrium time when changing temperature in the large water reservoir of the calorimeter, there was a 1°C deviation in incubation temperature between different experiments. This was thought to have negligible effects on metabolic rate measurements in comparisons of results. The equilibration time of the calorimeter (of maximally 6 to 7 h after insertion of the sample) was no restriction in this kind of long-term experiment. After incubation all specimens showed reactions if they were mechanically stimulated.

196

M. POWILLEIT & G. GRAF

2.8. CALCULATION OF IN SITU CO 2 FLUXES CO 2 concentrations in centrifuged pore-water samples (1 cm 3) of sediment cores from station 'Schlicksandgrund' were determined immediately after sectioning of the core in the laboratory. Additionally the CO 2 concentration in the overlying water (about 0.5 cm above sediment surface) of each core was measured. Diffusive fluxes from the sediment into the near-bottom water were calculated according to Fick's First Law using the gradient close to the sediment water interface. The temperature-corrected diffusion coefficient, D O, was obtained by using the Stoke-Einstein relation for HCO~- in seawater (9°C/14°C, D O = 7.7/9.0.10 -6 cm2.sq; Li & Gregory, 1974). HCO 3- was considered to be the predominant diffusing species. The sediment-diffusion coefficient D s was calculated according to the following equation for sandy sediments (Berner, 1980; Dicke, 1986): D, = O2*Do = q)*D o

(eq. 1)

with 82 (tortuosity) substituted by porosity, ¢, calculated from water-content measurements to be 0.6. The CO 2 fluxes, J, were calculated according to: (eq. 2)

.I = --K BI 0 int*qb*D ~*~

where ~ is the porosity, D s is the sediment-diffusion coefficient of HCO3-, dC/dz the concentration gradient over the depth z, and corrected to an increased diffusive flux due to bioturbation in oxic and hypoxic situations in the near-bottom water taking a minimum

KBIO int. value of 1.2 at the sediment/water interface (derived from tracer experiments; see Forster et al., 1995). 3. RESULTS Single specimens of Callianassa subterranea were able to survive up to five days under anoxic conditions at 6 C at least in the two size classes 48.3 to 97.6 mg dw and 330.9 to 543.0 mg dw. The amount of CO 2 in the incubation water during five days of anoxic incubation of C. subterranea, corrected for inorganic CaCO 3 reactions, increased in both size classes, viz. from 2.39 to 26.89 ~mol CO 2 (means, n = 6, small individuals) and from 2.02 to 79.0 #mol CO 2 (means, n = 4, large individuals, Fig. 1). CO 2 concentrations in incubation bottles with small individuals increased by each sampling day, whereas in incubation bottles with large individuals a pronounced increase of CO 2 concentrations first occurred on day 3. Fig. 2 shows increased bacterial cell numbers in the incubation water during 5-day anoxic incubation from 18.105 cells.cm -3 on day 1 to 89.105 cells.cm -3 on day 5 (n = 1). Derived from these bacterial cell counts the potential heterotrophic CO 2 release during the other incubations may be estimated by assuming a very low net growth efficiency (P/C-ratio of 10%) for the heterotrophs. That means the bacteria convert only 10% of their food to biomass and 90% would be respired and released as CO 2 (this assumption includes the well-known net growth efficiency for aerobic conditions of about 30% and an additional loss in efficiency during anoxia). Even under this conserva-

~,,E 100

4

0

100 , CO 2

80

-concentration //~

4

~~,o,._6080 .1¢

4

3

_.._.I 1 _!

o ~ 60 C~ #-1 "6

E

t

40

®

4O

20

E ~ e.-

20

~.

~

J"

i 0

t 1

I 2

q 1 4 __1 1I

--4 --I

small speclrnens

I 3

incubationtime (days)

i 4

~ 5

0

I

1 2 incubation

3 time

4

5

(days)

Fig. 1. Absolute ~ncreases of .~CO2 in incubation bottles (with volumes of 13 and 60 cm3) during a 5-day anoxic incubation of small specimens (48 - 98 mg dw, mean values ± SD, n = 6) and of large specimens of Callianassa subterranea (331 - Fig. 2. Bacterial cell numbers ~n incubation water during a 543 mg dw, mean values + SD, n = 4). 5-day anoxic incubation of Callianassa subterranea (n = 1).

CALLIANASSA AND SEDIMENT METABOLISM tive assumption the possible bacterial impact accounted for maximum values of 20.8% of the measured CO 2 in our experiments with small specimens and at least for the last three incubation days in experiments with large specimens. For the first two incubation days with large specimens the bacterial impact could theoretically account for a significant part of the CO 2 measured (Table 1). The rates of CO 2 release during five days of anoxic incubation of C. subterranea showed an increase in both size classes, viz. from 1.69 to 3.63 p.mol CO2"g dw'l.h "1 (means, n = 6, small individuals) and from 0.28 to 1.52 p.mol CO2"g dw-l.h -1 (means, n = 4, large individuals) between initial and final values (Fig. 3). Adapting these values to single individuals of C. subterranea, rates of 0.24+0.05 for small specimens and of 0.68+0.15 p.mol'ind-l.h -1 (means + SD) for large specimens were calculated. On the first two days all specimens showed a slight decrease of CO 2 release per unit of body weight. During this initial period there has to be a change in the metabolic pathway from aerobic to anaerobic metabolism of C. subterranea which obviously does not result in an enhanced rate of CO 2 release. Rates on day 3 increased up to a factor of 2 compared with initial values. These rates were used to estimate the in situ impact of the mud shrimps on CO2 fluxes. Rates later in the incubation period increased more rapidly and exceeded rates of aerobic CO 2 release recalculated from literature data (Fig. 3). Visual observations during the incubation and calorimetric measurements (indirectly) indeed 5,0

I

0 0 2 -release

l

4,0 3,0 O F,1 2,0

E ..i 1,0 0,0

I

2

I

3 4 incubation time (days)

I

5

Fig. 3. Rates of T_.CO2 release (in p,mol per g dw per h) during a 5-day anoxic incubation of small specimens (48 - 98 mg dw, means _+SD, n = 4) and large specimens (331 - 543 mg dw, means + SD, n = 6) of Callianassa subterranea. Arrows indicate rates of aerobic T_.CO2 release for two mean size classes of the mud shrimp (49 and 285 mg afdw) as recalculated from oxygen consumption rates measured during short-term incubations in a flow-through system (Witbaard & Duineveld, 1989) assuming a RQ of 1.0.

197

showed physical activity of the incubated specimens: C. subterranea periodically started to strike with its pleopods, probably to bring some oxygen into the simulated burrow lumen represented by the incubation chamber during experimentation. The recorded calorimetric values of the metabolic heat production by large C. subterranea (425 +141 mg dw) during anoxic incubations are calculated means on a daily basis. Some irregular bursts of heat production were mainly detectable during the first 24 hours and may indicate mechanical activity of the shrimps perhaps due to irrigation activity. Furthermore thermograms showed only low reduction of the metabolic rate during extended periods of time. Initial values during the first 24 hours, possibly including some oxygen metabolism and a change in the metabolic pathway towards anaerobic reactions, were 0.39+0.03 J.g dw -1.h-1. Values of later anoxic incubation periods (after 72 hours) were 0.25+0.12 J.g dw-l.h -1 (means _+ SD, n = 3). This reduction of the metabolic rate (64% of the initial value) was not significant (t-test). CO2-measurements in pore-water samples of station 'Schlicksandgrund' in different seasons of the investigation period down to 18 cm sediment depth showed only minor seasonal variations. A typical pore-water profile (oxic conditions in the near-bottom water) from May 1989 is shown in Fig. 4 with CO 2 concentrations between 2.6 and 3.8 mmol.dm -~. A pronounced gradient with a difference of 0.55 mmol.dm -3 could only be found between the near-bottom water (0.5 cm above sediment surface) and the first sediment horizon (0-1 cm). The calculated CO2 flux, characterizing oxic conditions in the near-bottom water, according to the profile shown in Fig. 4 and equation 2, was 110 #mol.m-2-h -1 (9°C). Due to seasonal variations in these concentration gradients (0.67 + 0.10 mmol.dm -3, mean + SD, Powilleit, 1991), the corresponding temperature-corrected fluxes ranged between 110 and 172 p.mol.m-2.h-1. Under hypoxic conditions, simulated in the laboratory, these CO 2 fluxes increased up to 338 #mol.m'2.h -1 (cf. Forster et aL, 1995) as indicated by the steeper pore-water gradient, amounting to a difference of 1.45 mmol.dm 3 in the upper 1 cm of the sediment (Fig. 4). 4. DISCUSSION 4.1. FACTORS INFLUENCING EXPERIMENTAL DATA As shown in our incubation experiments there are some factors which may contribute to the CO 2 release other than the biological activity of Callianassa subterranea: it seems reasonable that the main source of errors are inorganic reactions of the calcium carbonate system in the incubation water as a result of pH shifts (resulting in apparently higher rates of CO 2 release). Measurements of calcium ion concentrations could be used for corrections because

198

M. POWILLEIT & G. GRAF

CO2 (mmol*dm -3) o.o

2.0

4.0

80

6.0 I

o.o

I

lO.0 I

2.0 E

4.0

r-

6.0

oxic

hypoxic

8.o E

E ej

10.o 12.o 14.0 16.o 18.o

Fig. 4. CO 2 pore-water profiles from a silty-sand station in the southern North Sea ('Schlicksandgrund') at 35 m water depth. The oxic profile representsa typical in situ situation in periods of oxygen saturation in the near-bottom water. The hypoxic profile is derived from an artifically induced oxygen depletion in the laboratory and representsperiods of oxygen deficiencies which periodically occur in southern North Sea sediments (cf. Forster et al., 1995). The concentration gradients between the bottom water and the upper cm of sediment were used to calculate CO2 fluxes. most of the carbonates in seawater exist as calcium carbonate and only minor parts as magnesium or sodium carbonates (GraBhoff, 1976). If the increase in calcium concentration during experimentation is a result of such inorganic reactions this will lead to an overestimation of the CO 2 pool (up to 1/5 of the measured CO2 concentration changes, Table 1). In any kind of respiration experiment with macrofauna the role of bacteria has to be taken into account. In our experiments the unknown efficiency of the nucleopore filters could be a source of error and the heterotrophic bacteria directly attached to the surface of C. subterranea could be a source of CO 2. Only during the initial phase, and especially in experiments with large specimens, could this bacterial impact theoretically be a considerable part of the measured CO 2 changes. But after three days even the maximum estimation of the heterotrophic impact does not change our results substantially (Table 1). Nevertheless there are some uncertainties in the assumption of a net growth efficiency of 10% (viz. unknown efficiency in fermentation processes). Groenendaal (1980) and Von Oertzen (1984) pointed out some critical factors in determining survival times and respiration rates under anoxia using closed systems, such as: elevated metabolic rates at the beginning of the experiment due to manipulation

of the animal, animal size in relation to the volume of the test chamber, toxicity and accumulation of metabolic end products, and the addition of buffers and/or antibiotics. Compared to the typical in situ situation, some stress by the side effects mentioned above might possibly have reduced the ability of C. subterranea to decrease its metabolic activity during anoxia. To avoid an overestimation of the CO 2 release of the mud shrimp and its consequent impact on CO 2 fluxes (biodegradation of organic carbon), which probably became more pronounced later in the incubation period (day 4 and 5), only the CO 2 release data obtained on day 3 were used in further calculations. 4.2. ANAEROBIC METABOLISM AND BEHAVIOURAL STRATEGY IN CALLIANASSA SUBTERRANEA In contrast to most other taxa, crustacean species that are able to tolerate hypoxia and anoxia are very actively regulating organisms and do not show any pronounced decreases in metabolic activity under hypoxia and anoxia of up to 48 h (Pamatmat, 1978; Pritchard & Eddy, 1979; Zebe, 1982; G&de, 1983). On day 4 of the incubation period the smaller specimens of C. subterranea were probably near their anaerobic capacity and were highly stressed. Half of the original test individuals died between days 4 and 5. The in situ depth distribution of the mud shrimp reflected the fact that smaller individuals always inhabited the more oxygenated upper sediment horizons (up to 10 cm sediment depth), whereas larger specimens lived in burrows much deeper in the sediment (e.g. Forster & Graf, 1995). The size dependence of the metabolic rate (Le. the different tolerance to anoxia), which was also found for carbon dioxide in this study, could be one possible explanation for this in situ depth distribution pattern As known for other crustaceans, the main anaerobic pathway during initial hypoxic and anoxic incubation periods is the glycolytic way, which results in the formation of L-lactate as the main end product (De Zwaan & Putzer, 1985; Taylor & Spicer, 1987). Theoretically there should be no direct liberation of CO 2 in this reaction (De Zwaan, 1977; Wieser, 1986), which is called 'functional anaerobiosis' by Zebe et al. (1980). This pathway may cause the small decrease in CO 2 release observed in the first two days of the present experiments. For the related thallassinidean species Upogebia pugettensis and Callianassa californiensis there is some information on their anaerobic metabolism in the form of quantitative determinations of anaerobic end products (Thompson & Pritchard, 1969; Pritchard & Eddy, 1979; Zebe, 1982). These two species survive three to five days at complete anoxia at 10°C. In C. californiensis the main substrate in anaerobic metabolism after 12 and 24 h was glycogen and the corresponding main end product L-lactate. Small

CALLIANASSA AND SEDIMENT METABOLISM quantities of aspartate are also utilized; minor end products are alanine and succinate (Zebe, 1982). In long-term experiments with another crustacean species, the isopod Saduria (Mesideothea) entomon (L.), which inhabits hypoxic soft bottoms in the Baltic Sea, Hagerman & Szaniawska (1990) found alanine to be the main metabolic end product. Rates of oxygen consumption for C. subterranea from Witbaard & Duineveld (1989) allow us to calculate (using a conversion factor of 0.47 J.p.mol oxygen consumed) an equivalent heat- production rate due to aerobic carbohydrate oxidation of 0.75 J-g dw -l.h -1. Using direct calorimetry, rates in the initial hypoxic phase of the present study were 52% of this calculated value for normoxic conditions. Literature data on another crustacean species, the blue crab Callinectes sapidus, which is known to have a low tolerance to anoxia and little metabolic regulation capacity, demonstrated that its heat production rates (QH) were half the fully aerobic rates at 22.4% oxygen saturation, and these rates are depressed in seawater at 25% air saturation to 32% of their metabolic rate at normoxia. There was no anaerobic shutdown of heat production, and QH during anoxia was 7 to 25% of aerobic rates (Hammen, 1983; Stickle et aL, 1989). The low reduction of QH during prolonged anoxia to 64% of hypoxic or 33% of normoxic values indicates that C. subterranea has a high rate of heat production even under relatively long-term anoxia compared to other crustaceans (Carcinus maenas <20% during one day of anoxia), and other marine invertebrates such as polychaetes (20%), some bivalves (2%) or a priapulid species (<1%) (Widdows, 1987; Oeschger, 1990; Hill et aL, 1991; Oeschger et aL, 1992; Fritsche & Von Oertzen, 1995). Despite the availability of oxygen throughout the investigation period in the near-bottom water (oxic conditions) of the natural habitat studied, C. subterranea usually has to deal with hypoxic or anoxic conditions inside the burrow lumen. The results presented here show a considerable amount of CO 2 release and heat production during long-term anoxia in C. subterranea compared to normoxic rates (viz. 33% of aerobic heat production and 41 to 60% of carbon dioxide release recalculated from oxygen consumption rates measured during short-term incubations by Witbaard & Duineveld, 1989, Fig. 3). One possible explanation could be the anaerobic breakdown of the amino acid aspartate to the end product alanine, in which CO 2 is liberated (De Zwaan, 1983). This pathway could be classified as part of the 'environmental anaerobiosis' according to Zebe et aL (1980), which seems to be a very successful strategy to overcome long-term anaerobiosis (e.g. Pamatmat, t980; Gnaiger, 1983; Oeschger, 1990; Oeschger et aL, 1992). We suggest that C. subterranea first deals with anoxia by attempting to increase irrigation in their burrows, as already described in related species by e.g. Paterson & Thorne (1993) (which probably leads to lactate forma-

199

tion), and secondly, if anoxia continues, the mud shrimp could use the 'environmental anaerobiosis' pathway via aspartate-alanine while slightly reducing the metabolic rate, or even maintaining aerobic metabolic rates. Verification of the suggestion about the anaerobic pathway involved in C. subterranea awaits future determination of anaerobic metabolites. 4.3. IMPACT ON IN SITUCO 2 FLUXES The calculation of diffusive carbon fluxes from 002 gradients between sediment pore water and bottom water is a frequently used method (e.g. Balzer, 1989; McNichol et aL, 1988; Andersen et al., 1986; Forster et aL, 1995). Our CO 2 fluxes were lower than fluxes measured by other authors in nearshore sediments with similar sediment types but other macrofauna composition (e.g. Andersen et al., 1986). Direct measurements of CO 2 fluxes derived from incubation experiments with similar sediments of the Kattegat and Belt Sea region also yielded higher rates of more than 500 p.mol CO2.m'2.h "1, which was thought to be a result of advective transport processes by macrofauna via irrigation and burrowing, and a stimulation of decay in sediment organic matter (e.g. Andersen & Kristensen, 1992). Taking into account the irrigation activity of C. subterranea at our study site, which definitely has an advective component in addition to diffusive processes, the in situ CO 2 flux into the near-bottom water might be higher even though we enhanced the calculated flux by using a KBIO int. of 1.2 at the sediment/water interface (see Forster et aL, 1995). Subsequently this would to some extent decrease the impact of the mud shrimp on fluxes. Unfortunately we did not find any published data on CO 2 fluxes at our study site to compare with. The porosity in the sediment also significantly affects these fluxes. Because of uncertainties in porosity determinations in the upper centimetre of the sediment (the porosity is far higher near the sediment-water interface, up to 0.9), positive deviations in the calculated fluxes in the range of 50% are possible (Forster, 1991). This would also decrease our estimates of the impact of C. subterranea on carbon fluxes. Seasonal temperature differences of 10 °C did not affect the measured concentration profiles significantly. In January 1990 (6°C) fluxes were calculated to be 141 # m ol CO2"m"2.h- 1 and in August 1989 (16 °C) 172. Theoretically metabolism of organisms should be approximately double between 6 and 16°C (according to Q+0) and more rapid mineralization processes in the sediment should increase concentration gradients. We suggest a simultaneous increase in the macrobenthic bioturbative activity resulting in enhanced advective transport processes from the sediment into the near-bottom water which may cover the temperature effect.

200

M. POWILLEIT & G. GRAF

TABLE 2 Impact of Callianassa subterranea on CO 2 fluxes on station 'Schlicksandgrund' (southern North Sea). 1) conditions in the near-bottom water layer, 2) calculated from CO2 release on day 3 of the anoxic incubation period and extrapolated to natural densities of 42 ind.m-2 . CO2 flux ( pmol.m-2.h- 1) oxic1) 110

rate of CO2 release of Callianassa_population2) ( ~tmol.m-2.h - 11 hypoxic 1) 338

However, taking a study site's natural mud shrimp density to be 42 ind.m -2 (21 ind.m -2 for each size class is in the range of densities given in the literature) and data of the CO 2 release under anoxic conditions (day 3 of the incubation period, see Fig. 3) for both size classes of the shrimp, we calculate a rate of 8.0 pmol CO2.m-2.h -1 (i.e. 3.2 _+2.0 for small and 4.8 _+2.4 for large specimens) for the Callianassa population in southern North Sea sediments (Table 2). Applying these results to the calculated total CO 2 fluxes in these sediments, we calculate a share of 7.3 and 2.4% of the oxic and hypoxic fluxes, respectively. If we extrapolate our results to the Oyster Ground region in the central North Sea assuming a mean density of C. subterranea of 60 ind.m -2 (30 ind.m -2 for each of our size classes) and a flux of 225 #mol CO2.m-2.h -1 equal to a mean spring 0 2 flux measured by De Wilde et aL (1984), which would represent mainly aerobic mineralization processes, the Callianassa population would account for 5.1% of this flux. A comparison of these results with the total sediment metabolism under oxic conditions, where the Callianassa population accounted for about 17% of the SOD at the Oyster Ground (De Wilde et al., 1984; Witbaard & Duineveld, 1989), shows that the high anaerobic capacity of C. subterranea could theoretically make up a considerable part (about 1/3) of this value under oxic and hypoxic in situ conditions in the near-bottom water and anoxic conditions in the sediment. Acknowtedgements.--This work was funded by a research graduate grant of the Christian-Albrechts-Universitb.t, Kiel (state of Schleswig-Holstein, Germany) and was part of the Ph.D. thesis by M. Powilleit at the Institut for Meereskunde, Kiel. Our work was conducted within a national research project funded by the German Federal Ministry of Research and Technology. Thanks are due to D. Schiedek, R. Schneider, and three unknown referees for very helpful comments on earlier versions of the manuscript. We acknowledge the help of S. Forster, S. JAhmlich, J. Kitlar, W. Queisser, M. Teucher, and W. Ziebis during sampling. This is publication no. 234 of the Baltic Sea Research Institute. 5. REFERENCES AItenbach, A.V.,1987. The measurements of organic carbon in foraminifera.--J. Foram. Res. 17: 106-109. Anderson, L.G., P.O.J. Hall, A. Iverfeldt, M.M. Rutgers Van der Loeff, B. Sundby & S.F.G. Westerlund, 1986. Benthic respiration measured by total carbonate produc-

8.0

impact of C. subt. on CO2 fluxes (%) oxic 1) 7.3

hypoxic1) 2.4

tion.--Limnot. Oceanogr. 31:319-329. Andersen, F.(3. & E. Kristensen, 1992. The importance of benthic macrofauna in decomposition of microalgae in a coastal sediment.--Limnol. Oceanogr. 37: 1392-1403. Balzer, W., 1989. Chemische Reaktionen und Transportprozesse in oberfl~ichennahen Sedimenten borealer und polarer Meeresgebiete. Habil. Schrift, Univ. Kiel: 1-312. Berner, R.A., 1980. Early diagenesis, a theoretical approach. Princeton Univ. Press: 1-241. De Wilde, P.A.W.J., E.M. Berghuis & A. Kok, 1984. Structure and energy demand of the benthic community of the Oyster Ground, central North Sea.--Neth. J. Sea Res. 18: 143-159. De Zwaan, A. 1977. Anaerobic energy metabolism m bivalve molluscs.--Oceanogr, mar. Biol. ann. Rev. 15: 103-187. - - , 1983. Carbohydrate catabolism in bivalves. In: K.M. Wilbur. The Mollusca. Volume 1: Metabolic biochemistry and molecular biomechanics. Academic Press, New York: 137-175. De Zwaan, A. & V. Putzer, 1985. Metabolic adaptations of intertidal invertebrates to environmental hypoxia (a comparison of environmental anoxia to exercise anoxia).--Symp. Soc exp. Biol. 39- 33-62. Dicke, M., 1986. Vertikale Austauschkoeffizienten und PorenwasserfluB an der Sediment/Wasser-Grenzflb.che. Ph.D. thesis, Univ. Kiel: 1-164. Duineveld, G.C.A., A. KiJnitzer, U. Niermann, P.A.W.J. De Wilde & J. Gray, 1991. The macrobenthos of the North Sea.--Neth. J. Sea Res. 28: 53-65. Dworschak, P.C., 1983. The biology of Upogebia pusitla (Petagna) (Decapoda, Thallassinidea) 1. The burrows.--P.S.Z.N.I. Mar. Ecol. 4: 19-43. Famine, P. & J. Knudsen, 1983. Transitory activities of metabolism, carbon dioxide production and the release of dissolved organic carbon by the mussel Mytilus edulis (L.), following periods of self-induced anaerobiosis.--Mar. Biol. Letters 4:183-192. Forster, S. & G. Graf, 1995. Impact of irrigation on oxygen flux into the sediment: intermittent pumping by Callianassa subterranea and 'piston pumping' by Lanice con. chilega.--Mar. Biol. 123: 335-346. Forster, S., G. Graf, J. Kitlar & M. Powilleit, 1995. Effects of bioturbation in oxic and hypoxic conditions: a microcosm experiment with a North Sea sediment community.--Mar. Ecol. Prog. Ser. 116: 153-161. Fritzsche, D. & J.-A. Von Oertzen, 1995. Metabolic responses to changing environmental conditions in the brackish water polychaetes Marenzelleria viridis and Hediste diversicolor.--Mar. Biol. 121 : 693-699. Ga.de, G., 1983. Energy metabolism of arthropods and molluscs during environmental and functional anaerobiosis.--J, exp. Zool. 228: 415-429. Gnaiger, E., 1983. Heat dissipation and energetic efficiency

CALLIANASSA AND SEDIMENT METABOLISM

in animal anoxibiosis: Economy contra power.--J, exp. Zool. 228' 471-490. Graf, G., V. Martens, W. Queisser, P. Weinholz & A. Altenbach, 1988. A multicalorimeter for the study of biological activity in marine sediments.--Mar. Ecol. Prog. Ser. 46" 201-204. GraBhoff, K., 1976. Methods of sea water analysis. Verlag Chemie, Weinheim: 1-317. Groenendaal, M., 1980. Tolerance of the lugworm (Arenicola marina) to sulphide.--Neth. J. Sea Res. 14: 200-207. Hagermann, L. & A. Szaniawska, 1990. Anaerobic metabolic strategy of the glacial relict isopod Saduria (Mesidotea) entomon.--Mar. Ecol. Prog. Ser. 59" 91-96. Hammen, C.S., 1983. Direct calorimetry of marine invertebrates entering anoxic stages.--J, exp. Zool. 228: 397-403. Hardewig, I., M. Addink, M.K. Grieshaber, H.O. PSrtner & G. Van den Thillart, 1991. Metabolic rates at different oxygen levels determined by direct and indirect calorimetry in the oxyconformer Sipunculus nudus.-~J, exp. Biol. 157: 143-160. Hargrave, B.T. & G.A. Phillips, 1981. Annual in situ carbon dioxide and oxygen flux across a subtidal marine sediment.--Estuar, coast. Shelf Sci. 12" 725-737. HickeI,W., E. Bauernfeind, U. Niermann & H. Von Westernhagen, 1989. Oxygen deficiency in the south-eastern North Sea: sources and biological effects.--Ber. Biol. Anst. Helgoland 4; 1-148. Hill, A.D., A.C. Taylor & R.H.C. Strang, 1991. Physiological and metabolic response of the shore crab Carcinus maenas (L.) during environmental anoxia and subsequent recovery.~, exp. mar. Biol. Ecol. 150" 31-50. Kristensen, E., M. Holmer & N. Bussarawit, 1991. Benthic metabolism and sulfate reduction in a southeast Asian mangrove swamp.--Mar. Ecol. Prog. Ser. 73:93-103. Kristensen, E., F. Qstergaard Andersen & H. Blackburn, 1992. Effects of benthic macrofauna and temperature on degradation of macroalgal detritus: the fate of organic carbon.--Limnol. Oceanogr. 37:1404-1419. K~nitzer, A., D. Basford, J.A. Craeymeersch, J.M. Dewarumez, J. DSrjes, G.C.A. Duineveld, A. Eleftheriou, C. Help, P. Herman, P. Kingston, U. Niermann, E. Rachor, H. Rumohr & P.A.W.J. De Wilde, 1992. The benthic infauna of the North Sea: species distribution and assemblages.--ICES J. mar. Sci. 49: 127-143. Li, J.-H. & S. Gregory, 1974. Diffusion of ions in sea water and in deep-sea sediments.~eochim. Cosmochim. Acta 38" 703-714. McNichol, A.P., C. Lee & E.R.M. Druffel, 1988. Carbon cycling in coastal sediments: 1. A quantitative estimate of the remineralization of organic carbon in the sediments of Buzzards Bay, MA.--Geochim. Cosmochim. Acta 52" 1531-1543. Meyer-Reil, L.-A. 1978. Autoradiography and epifuorescence microscopy combined for the determination of number and spektrum of actively metabolizing bacteria in natural waters.--Appl, environm. Microbiol. 36: 506-512. ,1983. Benthic response to sedimentation events during autumn to spring at a shallow water station in the Western Kiel Bight. I1. Analysis of benthic bacterial populations.-- Mar. Biol. 77: 247-256. Oeschger, R., 1990. Long-term anaerobiosis in sublittoral marine invertebrates from the Western Baltic Sea:

201

Halicryptus spinulosus (Priapulida), Astarte borealis and Arctica islandica (Bivalvia).--Mar. Biol. Prog. Ser. 59" 133-143. Oeschger, R., H. Peper, G. Graf & H. Theede, 1992. Metabolic responses of Halicryptus spinulosus (Priapulida) to reduced oxygen levels and a n o x i a . ~ , exp. mar. Biol. Ecol. 162" 229-241. Pamatmat, M.M., 1978. Oxygen uptake and heat production in a metabolic conformer (Littorina irroata) and a metabolic regulator (Uca pugnax).--Mar. Biol. 48" 317-325. - - , 1980. Facultative anaerobiosis of benthos. In: K.R. Tenore & B.C. Coull. Marine benthic dynamics. Univ. South Carolina Press, Colombia: 69-91. - - , 1983. Measuring aerobic and anaerobic metabolism of benthic infauna under natural conditions.--J, exp. Zool. 228" 405-413. Paterson, B.D. & M.J. Thorne, 1993. The effect of oxygen tension on the swimmeret rate of Callianassa australiensis and C. arenosa (Crustacea, Decapoda, Thallassinidea).--Mar. Behav. Physiol. 24" 15-24. PSrtner, H.-O., U. Kreutzer, B. Siegmund, N. Heisler & M.K. Grieshaber, 1984. Metabolic adaptation of the intertidal worm Sipunculus nudus to functional and enviromental hypoxia. --Mar. Biol. 79" 237-247. Powilleit, M., 1991. CO2-Messungen zur Untersuchung des anaeroben Stoffwechsels benthischer Evertebraten und zur Aktivit&tsbestimmung der gesamten Sediment-Lebensgemeinschaft.--Ber. Inst. Meeresk., Kiel Univ. 220" 1-103. Pritchard, A.W. & S. Eddy, 1979. Lactate formation in Callianassa cafiforniensis and Upogebia pugettensis (Crustacea: Thallassinidae).--Mar. Biol. 63: 249-253. Rowden, A.A. & M.B. Jones, 1993. Critical evaluation of sediment turnover estimates for Callianassidae (Decapoda: Thallassinidae).--J. exp. mar. Biol. Ecol. 173: 265-272. --, 1994. A contribution to the biology of the burrowing mud shrimp, Callianassa subterranea (Decapoda: Thallassinidae).--J. mar. Biol. Ass. U.K. 74: 623-635. Salzwedel, H., E. Rachor & D. Gerdes, 1985. Benthic macrofauna communities in the German Bight.--VerSff. Inst. Meeresforsch. Bremerhaven 20" 199-267. SchSttler, U., B. Surholt & E. Zebe, 1984. Anaerobic metabolism in Arenicola marina and Nereis diversicolor during low tide.--Mar. Biol. 81" 69-73. Stickle, W.B., M.A. Kapper, L.-L. Liu, E. Gnaiger & S.Y. Wang, 1989. Metabolic adaptations of several species of crustaceans and molluscs to hypoxia: tolerance and microcalorimetric studies.--Biol. Bull. 177" 303-312. Taylor, A.C. & J.I. Spicer, 1987. Metabolic responses of the prawns Palaemon elegans and P. serratus (Crustacea: Decapoda) to acute hypoxia and anoxia.--Mar. Biol. 95" 521-530. Teucher, M., 1991. Luminophoren und ein neues Bildauswertungssystem zur Darstellung des bioturbaten Partikeltransportes in marinen Sedimenten.--Ber. Inst. Meeresk., Kiel Univ., 212: 1-69. Thompsen, R.K. & A.W. Pritchard, 1969. Respiratopy adaptations of two burrowing crustaceans, Callianassa carlforniensis and Upogebia pugettensis (Decapoda: Thalassinidae).--Biol. Bull. Mar. Biol. Lab., Woods Hole 136" 274-287. Tsunogai, S., M. Nishimura & S. Nakaya, 1968. Complexometric titration of calcium in the presence of larger

202

M. POWILLEIT & G. GRAF

amounts of magnesium.--Talanta 15" 385-390. Von Oertzen, A., 1984. Open and closed systems - their suitability for functional respiration measurements.--Limnologia 15: 541- 545. Von Westernhagen, H., W. Hickel, E. Bauernfeind, U. Niermann & I. Kr6ncke, 1986. Sources and effects of oxygen deficiencies in the south-eastern North Sea.--Ophelia 26: 457-473. Widdows, J., 1987. Applications of calorimetric methods in ecological studies. In: A.M. James. Thermal and energetic studies of cellular biological systems. Wright Publishers, Bristol: 182-215. Wieser, W., 1986. Bioenergetik: Energietransformationen bei Organismen. Thieme, Stuttgart, New York: 1-245. Witbaard, R. & G.C.A. Duineveld, 1989. Some aspects of the biology and ecology of the burrowing shrimp

Callianassa subterranea (Montagu) (Thalassinidea) from the southern North Sea.--Sarsia 74: 209-219. Zebe, E., 1982. Anaerobic metabolism in Upogebia pugettensis and Caflianassa californiensis (Crustacea, Thalassinidea).--Comp. Biochem. Physiol. 72 B: 613-617. Zebe, E., MK. Grieshaber & U. Schottler, 1980. Biotopbedingte und funktionsbedingte Anaerobiose. Der Energiestoffwechsel wirbelloser Tiere bei Sauerstoffmangel.--Biologie in unserer Zeit 10: 175-182 (accepted 4 June 1996)