Springer-Verlag 1997
Marine Biology (1997) 127: 411– 418
A. S. C. Schmitt · R. F. Uglow
Effects of ambient ammonia levels on blood ammonia, ammonia excretion and heart and scaphognathite rates of Nephrops norvegicus
Received: 20 May 1996 / Accepted: 1 July 1996
Abstract During commercial handling of Nephrops norvegicus (L.) there are a number of situations when the prawns may be exposed to very high ambient ammonia levels. These experiments evaluated the effects of increased levels of ambient total ammonia (TA = NH3 + NH4+) on blood ammonia, ammonia efflux rates and on the cardio-ventilatory performance of N. norvegicus. When prawns were taken from <1 to 2000 lmol TA l)1 medium, blood TA concentrations increased rapidly for the first 2 h but tended to drop thereafter. Original blood TA levels were restored 6 h after the prawns were transferred back from seawater containing 2000 to <1 lmol TA l)1. Sudden exposure to 500, 1000, 2000 or 4000 lmol TA l)1 medium induced blood TA concentrations to increase respectively to 50, 30, 33 and 36% of external concentrations (normally, internal TA values are much higher than external levels). Immediately after transfer back to seawater with low ammonia concentration ( <1 lmol TA l)1), excretion rates were higher than those of control prawns, and the absolute amounts of TA excreted were considerably higher than those calculated to have accumulated in the haemolymph. Heart rate (HR) and scaphognathite rate (SR) were not altered when prawns were subjected to sudden alterations in ambient ammonia ( <1 to 2000 to <1 lmol TA l)1). When water ammonia concentrations were altered more gradually, both rates increased, but only at 4000 lmol TA l)1. These results show that N. norvegicus is able to remove ammonia from the haemolymph and/ or transform ammonia into some other substance when subjected to increased levels of ambient ammonia. Pos-
Communicated by L. Hagerman, Helsingør A.S.C. Schmitt1 · R.F. Uglow Department of Applied Biology, The University of Hull, Kingston upon Hull, HU6 7RX, UK Present address: (&) Departmento de Cieˆncias Fisiolo´gicas, Universidade do Rio Grande, CP 474 Rio Grande RS, 96201-900 Brazil
1
sible mechanisms involved (e.g. active transport across the gills; storage in some other tissue; glutamate synthesis) are discussed.
Introduction Ammoniotelism is a characteristic of aquatic crustaceans. Ammonia is normally formed during various catabolic reactions and is easily excreted across the gills, by diffusional movement and/or ionic exchange mechanisms, without any further processing (Kleiner 1981; Kormanik and Cameron 1981; Evans and Cameron 1986; Regnault 1987). Amounts and rates of ammonia production and excretion may be influenced by nutritional status, moult stage, activity levels, salinity and temperature (Needham 1957; Regnault 1987; Hunter and Uglow 1993a), and the comprehension of such factors is important in predicting and controlling ambient ammonia levels in aquaculture. Ambient ammonia levels in the marine and freshwater environments are usually very low, and the ammonia excreted by the animals under such circumstances rarely poses a problem for them. The same may not be true when the animals are held in artificial environments, such as aquaculture or the vivier tanks used during live marketing and transport, where ammonia levels may occasionally rise and compromise the quality of the livestock. According to Campbell (1991) most of the toxic effects of ammonia are related to pH changes that affect enzyme activity, and such effects are known to cause increased mortality and lowered growth rates in several species of crustaceans (Wickins 1976; Armstrong et al. 1978; Provenzano 1983; Chen et al. 1990; Chen and Lin 1991, 1992; Lin et al. 1993). Toxicity may be affected by environmental and physiological factors. Chen and Lin (1991) found that median lethal concentration (LC50) values for ammonia in Penaeus penicillatus were ca. 80% lower at salinity levels of 25 psu than at 34 psu. According to Wajsbrot et al. (1990), P. semisulcatus is twice as sensitive to ammonia when exposed to 27% saturation
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of dissolved oxygen than to 96% saturation, and mortality rates were influenced by moult stage. An understanding of the physiological mechanisms used to cope with progressive or sudden increases in water and/or blood ammonia levels is also very important, but, to date, these aspects have not been thoroughly investigated (see Regnault 1992, 1994). Nephrops norvegicus is fished extensively in northern European waters, and part of the catch is transported and marketed alive. During such situations, the prawns may be held in poorly designed vivier tank systems in which water quality control is inefficient or absent and ammonia may accumulate. Freshly caught N. norvegicus may also be put into systems which already contain high levels of ammonia, although the effects of such conditions are unknown. The prawns may be held in these systems for periods of time varying from a few hours to several days, as the dealers may have to wait until a sufficient number of prawns is available to be transported. According to R.F. Uglow (personal observation), dissolved ammonia levels in such situations may reach 2000 lmol TA l)1; values ranging from 800 to 4300 lmol TA l)1 were observed by Schmitt (1995) in tanks used to hold live Homarus gammarus and Cancer pagurus. Unfortunately, such data are very scarce, as there is a general fear amongst the dealers that valuable information may be passed to potential competitors. The present experiments were designed to evaluate some of the responses of N. norvegicus to increased concentrations of ammonia in the holding water.
Materials and methods Nephrops norvegicus (L.) were creel-caught off the west Scottish coast (57° 38′ N; 5° 51′ W) at depths of 150 to 250 m. Prawns weighing 20.4 to 36.2 g (mean ± SE: 28.9 ± 0.4 g) were wrapped with seawater-soaked tissue paper and transported to Hull University inside polystyrene boxes supplied with ice packs. They were kept in running seawater (12 °C, 34 psu) supplied with aeration and biological filters and under low intensity light. Small lengths of plastic tubing (60 to 70 mm diam) and fine sand were placed in the stock aquaria, providing substitute burrows and substratum for the prawns. The prawns remained in this system for at least 60 d prior to experiments and they were fed twice-weekly ad libitum with mussel flesh until 48 h before the experiments. All experiments were performed inside temperature-controlled rooms at 12 °C and 75% relative humidity. Total ammonia (TA) concentrations in the stock tanks were <1 lmol l)1, and increased TA concentrations were obtained by adding NH4Cl (Anal-R grade) to seawater. Water samples were taken from all the solutions used, and their TA contents did not differ by more than 1% of calculated values. The effects of ambient ammonia on blood ammonia concentrations of Nephrops norvegicus were studied in two separate experiments: Experiment 1. 120 prawns from the stock tanks were transferred (20 individuals each) to 6 plastic containers (35 × 45 cm, base) with 30 litre of ammonia-spiked seawater (2000 lmol l)1). Blood samples were collected from individuals from the stock tank and from 60 individuals (10 each time) at 10, 30, 60, 120, 240 and 360 min following transfer to the plastic containers. Immediately after that, the remaining 60 prawns in the plastic containers were returned to the stock tank and the sampling procedure repeated.
Experiment 2. 20 prawns were placed in each of 5 plastic containers (35 × 45 cm, base) each provided with 30 litre of a different concentration of ammonia-spiked seawater (0, 500, 1000, 2000 or 4000 lmol l)1). Initial blood samples were taken from 10 prawns at each concentration after 9 h exposure. The remaining 10 individuals at each concentration were placed individually in rectangular plastic containers (12 × 22 cm, base) each supplied with 1.5 litre of seawater from the stock tank, and their TA efflux rates were then monitored. Water samples (1.5 ml each) were collected from each of the individual experimental containers after 0, 15, 30, 60, 120, 310 and 400 min. Final haemolymph samples were then collected from these individuals. Calculated TA efflux rates were based on the difference between the concentrations obtained for two consecutive (timed) samples. Heart rate (HR) and scaphognathite rate (SR) were measured in a flow-through system consisting of a header tank, a sump, a water pump and 10 small (4 litre each) containers each supplied with a lid in which inlet and outlet valves were set. Seawater was passed through the small containers and driven into the sump, where aeration was provided. The water was then pumped into the header tank and back to the small containers. The total volume of the system was 122 litre in the first experiment and 130 litre in the second experiment, and the flow rate through each individual container was 1.9 litre min)1. Prawns were individually placed in the small containers, and the electrode wires passed through a Suba-seal placed on the lid. Water TA concentrations were altered without handling or interfering with the prawns at any time. The effects of ambient ammonia on Nephrops norvegicus HR and SR were analysed in two different situations: Experiment 3. Nephrops norvegicus were placed in the flow-through system, and ambient TA concentration was progressively increased over a period of 6 h. Such increases were performed by adding preweighed amounts of NH4Cl to the sump every 20 min. HR and SR were registered every hour. After 6 h, the TA level was lowered to 70 lmol l)1, and HR and SR were measured 1 h later. The water TA level was lowered by replacing the water in the sump and header tank with clean seawater whilst the containers were closed. Then the outlets of the small containers were disconnected from the system and flushed with clean seawater from the header tank for ca. 10 min. Experiment 4. Water TA concentrations were increased from control conditions to 2000 lmol l)1 by adding NH4Cl to the sump and header tank and allowing the system to recirculate. After 24 h, the water TA concentration was lowered to 15 lmol l)1 using the same procedure described for the previous experiment. HR and SR were monitored during 9 h following each alteration of water TA levels (increase and decrease). Haemolymph samples (200 ll) were collected via the pereiopod sinus using disposable syringes and needles. Measurements of blood pH were made immediately after collection using a JP PHM2 pHmeter and a Whatman protein-resistant microelectrode. Haemolymph samples (50 ll) for glucose analysis were mixed with 6% ice-cold perchloric acid (1:1). Such samples were centrifuged for 5 min at 10 000 rpm (7200 ×g), and their glucose contents were enzymatically determined using glucose-oxidase kits from Sigma Chemicals (Cat. No. 510). The colour produced was read at 540 nm using a LKB-Biochrom (Ultrospec 4050) spectrophotometer. Total dissolved ammonia (NH4+ + NH3) concentrations in the water and haemolymph were measured with a flow injection/gas diffusion technique adapted from Clinch et al. (1988) and Hunter and Uglow (1993b). This technique consists of a carrier stream of NaOH (0.01 M) separated from an indicator solution (Bromothymol Blue, 0.5 g l)1) by a gas permeable membrane (PTFE). All ammonia in the samples is converted to gaseous NH3 which diffuses across the membrane and reacts with the indicator to produce a pH-dependent colour change that is detected by a photometer. A calibration curve is made using different concentrations
413 of (NH4)2SO4. Haemolymph samples were diluted (1:19) with a saline solution (9 g l)1 NaCl) before analysis. HR and SR were measured according to a modified impedance technique (Dyer and Uglow 1977). Electrodes attached to the prawns were made using silver-plated solid copper wires with KYNAR insulation (0.25 mm diam). Electrodes were affixed with cyanoacrilate glue on both sides and on the anterior part of the cephalothorax for SR and HR measurements, respectively. Heart electrodes were introduced into the body through a small hole (0.25 mm diam) drilled above the heart. Each scaphognathite electrode was hooked over the margin of the cephalothorax so that the cut cross-sectional area of the electrode was positioned near the scaphognathite. Prawns were left to settle in the flow-through system for 48 h before experiments. The homogeneity of variances of the groups was tested using the Levene test. All variables were analysed with one-way ANOVA. When significant differences were detected, the Tukey multiple range test was applied to identify which groups differed. All statistical analyses were performed at the 0.05 level of significance.
Results All prawns used in these experiments survived exposure to increased ambient ammonia concentrations. Figure 1 shows the results obtained for blood ammonia concentrations and blood pH following transfer of Nephrops norvegicus from normal seawater ( < 1 lmol TA l)1) to enriched seawater (2000 lmol TA l)1) (Experiment 1). Blood TA levels increased significantly (P < 0.05) from 111.43 ± 5.54 to 806.94 ± 65.77 lmol TA l)1 within 10 min of transfer, and maximum values (1180 ± 64.14 lmol TA l)1) were measured at 2 h following transfer. Blood TA levels then decreased significantly (P < 0.05) over the following 4 h but were still significantly higher (P < 0.05) than the original values. Blood pH dropped significantly (P < 0.05) 0.15 pH units to a minimum value measured after the first hour following transfer but progressively regained original pH values during the remaining 5 h of exposure (Fig. 1). After 6 h of exposure to enriched medium, the N. norvegicus were transferred back to clean seawater which, after 10 min, resulted in a significant drop (P < 0.05) in mean blood TA level from 807.31 ± 34.55 to 392.18 ± 27.70 lmol TA l)1. Blood TA values then reFig. 1 Nephrops norvegicus. Haemolymph total ammonia (TA) concentrations (triangles) and pH (circles) following exposure to ammonia-enriched (2000 lmol TA l)1) seawater (closed symbols) and following return to clean ( < 1 lmol TA l)1) seawater (open symbols). Values are given as means ± SE for n = 9 or 10 in each case
mained relatively stable but dropped significantly (P < 0.05) once again after 4 h (Fig. 1). The blood pH remained unchanged during the first 10 min in clean seawater but then rapidly dropped (P < 0.05) over the following 20 min and reached a minimum value 2 h following transfer to clean seawater. For the remainder of the measuring period, the blood pH rose steadily and, after 6 h, the value was not significantly different (P > 0.05) from the original value measured. Table 1 summarises the data obtained on blood TA and glucose levels and pH values when groups of prawns were transferred from clean seawater to seawater enriched with various levels of ammonia (Experiment 2). Blood TA levels increased significantly (P < 0.05) in all groups after 9 h exposure. When returned to clean seawater, mean blood ammonia levels dropped and although original values were not regained in the 5 h recovery period, the values attained were not significantly different (P > 0.05) from that of the control group which was subjected to similar handling procedures but held in low ammonia seawater all the time. Blood pH increased significantly (P < 0.05) in the groups transferred to 500, 1000 and 2000 lmol TA l)1 medium but, after return to clean seawater, only the blood pH of the group treated with 2000 lmol TA l)1 dropped significantly (P < 0.05) (Table 1). When exposed to 4000 lmol TA l)1 seawater, the blood pH dropped to a value significantly lower than the control group and increased significantly when returned to clean seawater (P < 0.05 in each case). Hyperglycaemia occurred in all groups when first removed from the stock tank and placed in the experimental containers under ammonia-enriched conditions (Table 1). When transferred to the smaller containers containing clean seawater, the blood glucose levels dropped to values not significantly different from original values (P < 0.05 in all cases). Weight-specific TA efflux rates immediately after transfer from 500, 1000, 2000 and 4000 lmol TA l)1 to < 1 lmol TA l)1 medium were respectively 1.65 ± 0.11, 1.98 ± 0.13, 3.14 ± 0.38 and 6.46 ± 0.51 lmol g)1 h)1, and all these values were significantly higher (P < 0.05)
414 Table 1 Nephrops norvegicus. Glucose, pH and total ammonia (TA) concentrations of the blood of prawns exposed for 9 h to various concentrations of ammonia and 5 h after subsequent transfer to low ammonia ( < 1 lmol TA l)1) seawater. Control groups were kept in low ammonia seawater at all times. The absolute blood TA levels refer to the accumulated values above blood TA of control prawns. Values are means ± SE of n = 9 or 10 in each case (TAin/TAout relation between haemolymph and external TA concentrations) Experimental conditions
Glucose (mg 100 ml)1)
pH
TA (lmol l)1)
Prawns from the stock tank Control prawns for ammonia exposure Control prawns for recovery 500 lmol l)1
2.23 ± 0.44 7.39 ± 1.59 3.28 ± 0.74 6.45 ± 1.51 4.74 ± 1.41 9.64 ± 1.57 3.52 ± 0.77 6.03 ± 1.88 2.31 ± 0.44 12.76 ± 3.15 4.78 ± 1.26
7.69 ± 0.01 7.82 ± 0.03 7.84 ± 0.03 7.86 ± 0.02 7.83 ± 0.02 7.89 ± 0.01 7.82 ± 0.02 7.81 ± 0.01 7.72 ± 0.02 7.63 ± 0.03 7.77 ± 0.03
91.26 ± 5.63 104.92 ± 13.01 174.59 ± 16.95 284.70 ± 28.66 203.11 ± 16.06 336.81 ± 12.93 253.04 ± 27.10 606.38 ± 33.04 164.87 ± 12.62 1456.18 ± 57.55 255.41 ± 33.09
500 to < 1 lmol l
)1
1000 lmol l)1 1000 to < 1 lmol l)1 2000 lmol l)1 2000 to < 1 lmol l)1 4000 lmol l)1 4000 to < 1 lmol l)1
than that of the control group (0.53 ± 0.14 lmol g)1 h)1) (Fig. 2). TA efflux rates of all experimental groups subsequently decreased and, apart from the group treated with 4000 lmol TA l)1, the values of TA excreted between 0.5 and 1 h after transfer were not significantly different (P > 0.05) from that of the control group. TA excreted by prawns transferred from 4000 lmol TA l)1
TAin/TAout (%)
TA (lmol prawn)1)
56.94
1.31 ± 0.21
33.68
1.69 ± 0.09
30.00
3.66 ± 0.24
36.40
9.86 ± 0.42
medium reached control values between 1 and 2 h after transfer (P > 0.05). The absolute values of TA excreted by each individual, in excess of that excreted by the control group, are shown in Table 2. The HR and SR of prawns experiencing progressive increases in water TA concentrations (Experiment 3) are shown in Fig. 3. Initial HR and SR were respectively 43.40 ± 7.02 and 88.17 ± 8.92 beats min)1 and they increased significantly (P < 0.05) to 83.27 ± 5.84 and 171.99 ± 23.90 beats min)1 when water TA reached 4000 lmol TA l)1. Following the water TA concentra-
Table 2 Nephrops norvegicus. Absolute values of excreted ammonia (lmol prawn)1) in excess of that excreted by the control group following 9 h exposure to various ammonia concentrations and subsequent transfer to clean seawater ( < 1 lmol l)1). Values are given as means ± SE of n = 9 or 10 Time after transfer (h)
Exposure concentration(lmol l)1): 500
1000
2000
4000
0.25
7.89 ± 0.86 11.79 ± 1.23 14.43 ± 1.81 19.70 ± 3.18 27.32 ± 4.72 30.91 ± 5.61
9.53 ± 1.04 14.43 ± 1.75 17.27 ± 2.35 21.48 ± 4.37 29.99 ± 5.63 34.56 ± 23.29
15.43 ± 2.18 21.31 ± 2.36 27.79 ± 3.55 34.26 ± 5.69 36.14 ± 8.34 32.41 ± 10.06
38.29 ± 3.27 51.86 ± 4.06 62.53 ± 5.47 73.44 ± 6.50 87.38 ± 9.70 86.38 ± 10.17
0.5 1 Fig. 2 Nephrops norvegicus. Weight-specific total ammonia (TA) efflux rates following 9 h exposure to seawater containing 500 (j), 1000 (m), 2000 (s) and 4000 (n) lmol TA l)1 and following return to clean ( < 1 lmol TA l)1) seawater. Control prawns (d) were kept in low TA seawater at all times. Values are given as means ± SE for n = 9 or 10 in each case
2 3.5 5
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altered (P > 0.05) throughout the experimental time, despite an apparent trend for rates to increase initially.
Discussion
Fig. 3 Nephrops norvegicus. Heart and scaphognathite rates (means ± SE for n = 8 in each case) during progressive increase of ambient ammonia levels and after return to low ammonia concentration seawater. Values indicated by an asterisk are significantly higher than time zero (P < 0.05)
tion reduction to 70 lmol TA l)1, HR and SR still remained significantly higher (P < 0.05) than initial values. The results of Experiment 4, when the prawns were transferred from < 1 to 2000 lmol TA l)1 medium and back to < 1 lmol TA l)1 medium are shown in Fig. 4. Initial HR and SR rates were respectively 44.67 ± 8.73 and 96.15 ± 18.49 beats min)1 and were not significantly
Fig. 4 Nephrops norvegicus. Mean heart rate (d) and scaphognathite rate (s) following exposure to (a) ammonia-enriched (2000 lmol TA l)1) seawater and (b) following return to low ammonia seawater. Values are given as means ± SE for n = 8 in each case
By virtue of its small molecular size and high solubility, ammonia can diffuse rapidly across biological membranes (Schmidt-Nielsen 1983), and when crustaceans are exposed to media with dissolved ammonia levels greater than those occurring in the blood, an influx-induced elevation of blood ammonia levels may occur. Chen and Kou (1993) measured ammonia influx in Penaeus monodon exposed to various ambient ammonia levels and showed that influx rates were directly related to water pH. In our studies, a 9 h exposure period to various ammonia-enriched media induced elevated blood TA levels but, in all cases, blood TA levels failed to equilibrate with ambient levels. In all the media with TA concentrations higher than 500 lmol TA l)1, the prawns were able to maintain internal TA values at ca. 30% of the ambient levels (Table 1). This suggests strongly that regulatory mechanisms were operating to remove and/or transform the accumulating blood ammonia. Further evidence of a regulatory system is provided by the data summarised in Fig. 1, which show that, on exposure to ammonia-enriched medium (2000 lmol TA l)1), the blood ammonia rose from 5.6 to 59% of the ambient levels after 2 h but dropped to 40% ambient TA levels after 6 h. When prawns were returned to clean seawater after exposure to TA-enriched media, their recovery was rapid but not immediate, with TA efflux rates reaching those of control prawns within 1 h (Fig. 2). Most of the ammonia accumulated in the blood had apparently disappeared within 10 min of the recovery period, and blood TA levels then remained constant until decreasing further after 4 h. This period of stable, supranormal blood TA levels in normal medium may reflect a period when accumulated ammonia was being removed from other tissues into the haemolymph or when other forms of nitrogen were being reconverted to ammonia. Ammonia may be removed from the haemolymph by excretion or by storage in some other tissue. Excretion of ammonia against a concentration gradient has been shown to occur and is clear evidence of the presence of ionic exchange systems involving NH4+ (Kormanik and Cameron 1981). Active transport of ammonia across the gills of water-breathing animals may take place via basolateral or apical Na+/NH4+ exchange (Evans and Cameron 1986). If such mechanisms were responsible for maintaining low internal TA levels, then an increased energy supply may be needed to cope with the requirements of intense ionic exchange. In the present studies, however, blood glucose levels do not appear affected by exposure to ammonia-enriched media. The hyperglycaemic response found occurred also in the control prawns subjected to similar handling procedures, and it was most probably caused by the stress due to mutual
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interaction in the containers. After transference to the individual containers the blood glucose levels returned to normal. Furthermore, Chen et al. (1993) found that ammonia effluxes were less than influxes when Penaeus chinensis were exposed to ca. 700 and 1400 lmol TA l)1 seawater, demonstrating that active transport mechanisms could not match the inward diffusion from the external environment at such concentrations. The TA content of the blood of each prawn exposed to an enriched medium was calculated on the basis of blood volume = 30% fresh body weight (Table 1) and the amount of TA excreted by each individual following return to clean seawater was also calculated (Table 2). These data reveal that the amount excreted during recovery exceeded by far that accumulated in the blood during exposure. The values shown in Table 1 refer only to TA accumulated in the haemolymph but, even if TA accumulated equally among all tissues, internal TA accumulated would still be lower than that excreted during recovery. This is an indication that ammonia was either stored elsewhere or, more likely, was transformed to some other nitrogenous compound during exposure. Regnault (1994) raised the possibility that emersed crabs may store ammonia in some fluid compartment or in the acidic gut fluids. A further possibility could be the amination of ammonia as a detoxification process. Ammonia may be used to produce glutamate (Claybrook 1983) and the reversible reaction (ammonia formation and glutamate synthesis) is controlled by the activity of the enzyme glutamate dehydrogenase (GDH), (Batrel and Regnault 1985; King et al. 1985; Regnault 1987; Regnault and Batrel 1987). An increased GDH activity towards glutamate synthesis was found in emersed Cancer pagurus which, presumably, were unable to excrete ammonia in the absence of gill chamber ventilation (Regnault 1992). Some or all of these mechanisms may have occurred in the present experiments as a means of reducing the effects of ammonia influx, but further research on GDH activity in Nephrops norvegicus is necessary to elucidate this aspect. If no regulatory mechanism existed and internal and external ammonia levels were in equilibrium, the theoretical values of the excess blood ammonia content of prawns exposed to the media mentioned above, over that of the control group would be 7.68, 16.74, 40.65 and 90.28 lmol prawn)1 l)1. These theoretical values for the 500 and 1000 lmol TA l)1 groups are still less than the quantities of ammonia excreted during recovery. It would appear that, at these exposure levels, ammonia was removed from the haemolymph creating a gradient by which more ammonia diffused in. At the higher concentrations it may be that the stored or transformed ammonia, accumulated under exposure, was less rapidly reconverted to ammonia during recovery. Blood pH may be affected by a variety of environmental and physiological factors, and branchial excretion of acid-base equivalents is one of the mechanisms used to regulate pH (Truchot 1983; Wheatly and Henry 1992; for review see also Truchot 1994). Sudden influxes
of ammonia may affect this balance as NH3 entering the body may cause alkalosis – capturing H+ to form NH4+. This did not happen in the present experiments, and the small transient decreases in blood pH shown in Fig. 1 may have been the result of metabolic and/or respiratory acidosis at such times. Cardiac and ventilatory beat activity changes have been measured in association with physiological responses of crustaceans to altered environmental conditions in a number of studies. Temperature has been shown to have a direct effect on such rates, and the temperature coefficient (Q10) values obtained generally approach those which are considered to typify physiological processes (2 to 3) (deFur and Mangum 1979; Spaargaren and Achituv 1977). Perfect acclimation to a changed temperature may also occur, as shown in Calinectes sapidus (Burton et al. 1980). Salinity-induced rate changes have also been found, but such responses appear to be variable (deFur and Mangum 1979) and may be influenced by the rate at which the alteration is affected (Dyer and Uglow 1980). Ambient PwO2 (water oxygen tension) may affect PaO2 and PvO2 (post branchial and pre branchial blood oxygen tensions, respectively), and compensatory adjustments of the circulatory and ventilatory systems have been described (Coyer 1977; Taylor et al. 1973; Uglow 1973; Wilkens et al. 1984; Hagerman and Uglow 1985). The majority of species studied have shown a bradycardia or unchanged heart rates accompanied by increased scaphognathite rates until a critical PwO2 is achieved, and increased hypoxia beyond these levels results in pronounced beat retardations. Such adjustments are, presumably, related to the oxygen-carrying performances of the blood and, ultimately, to the oxygen consumption of the animals. As shown by Angersbach and Decker (1978) on Astacus leptodactylus, an increase in scaphognathite beat rate can increase PaO2 . Ambient ammonia concentrations may also be expected to have an impact on cardioventilatory performance, as ammonia too may enter or exit the blood via the branchial tissue. In these studies, both the HR and SR were ultimately altered by water TA concentrations but only at concentrations of 2400 lmol TA l)1 and above (Fig. 3). Even when organ rates were monitored immediately after an increase in ambient TA to 2000 lmol TA l)1, changes in HR and SR were not apparent (Fig. 4). Nephrops norvegicus is a burrowing species found principally associated with fine deposits, which indicate a general lack of water currents; such species may be expected to be tolerant of low PwO2 and high TA concentrations in their burrows. This may be why only very high TA concentrations affected the HR and SR; such responses have little or no ecological relevance as these extreme TA levels are unlikely to occur naturally. However, N. norvegicus is a species of considerable commercial importance and is frequently marketed alive. Very high TA levels may be found if the prawns are held in poorly designed systems or at high densities. The elevated HR and SR levels measured at high TA levels possibly reflect an altered metabolism
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caused by the toxic effects of ammonia. The maintenance of elevated organ beat rates 1 h after ambient TA was brought back to normal levels reinforces this view, as excess blood ammonia is comparatively rapidly flushed from the blood. Besides, elevated organ rates are just as likely to enhance influxes in high ambient TA media as effluxes in low ambient TA media. These data on cardioventilatory responses to very high levels of dissolved ammonia are preliminary, and further work on ammonia fluxes in association with the activity of such organs is necessary to evaluate their physiological relevance. Nephrops norvegicus showed great ability to adapt and compensate for high levels of ambient ammonia through mechanisms to remove and/or store ammonia as a different compound. It must be noted, however, that the prawns may be exposed to other stress factors (e.g. emersion and hypoxia) during live marketing, and the physiological effects of such factors in combination with exposure to high levels of ammonia on N. norvegicus are presently unknown. Hence, the use of systems that minimise the accumulation of ammonia in the holding water should be strongly recommended. Acknowledgements We wish to thank Mr M. McDonald, Mr S. McDonald, from the ‘‘Sealgair Mara’’ fishing vessel, and Mr D. McRae for kindly supplying the prawns used in these experiments. Mr A.S.C. Schmitt is a fellow of CAPES – Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nivel Superior – Brazil.
References Angersbach D, Decker H (1978) Oxygen transport in crayfish blood: effect of thermal acclimation, and short-term fluctuations related to ventilation and cardiac performance. J comp Physiol 123: 105–112 Armstrong DA, Chippendale D, Knight AW, Colt JE (1978) Interaction of ionised and un-ionised ammonia on short term survival and growth of prawn larvae, Macrobrachium rosenbergii. Biol Bull mar biol Lab, Woods Hole 154: 15–31 Batrel Y, Regnault M (1985) Metabolic pathways of ammoniogenesis in the shrimp Crangon crangon L.: possible role of glutamate dehydrogenase. Comp Biochem Physiol 82B(2): 217– 222 Burton DT, Richardson LB, Moore CJ (1980) Cardiac frequency compensation responses of adult blue crabs (Calinectes sapidus Rathbun) exposed to moderate temperature increases. Comp Biochem Physiol 65A: 259–263 Campbell JW (1991) Excretory nitrogen metabolism. In: Prosser CL (ed) Comparative animal physiology. Wiley-Liss Inc., New York Chen JC, Kou YZ (1993) Accumulation of ammonia in the haemolymph of Penaeus monodon exposed to ambient ammonia. Aquaculture, Amsterdam 109: 177–185 Chen JC, Lin CY (1991) Lethal effects of ammonia and nitrite on Penaeus penicillatus juveniles at two salinity levels. Comp Biochem Physiol 100C(3): 477–482 Chen JC, Lin CY (1992) Effects of ammonia on growth and moulting of Penaeus monodon juveniles. Comp Biochem Physiol 101C(3): 449–452 Chen JC, Nan FH, Cheng SY, Sheen SS (1993) Effects of ambient ammonia on ammonia-N and protein concentrations in hemolymph and ammonia-N excretion of Penaeus chinensis. Mar Ecol Prog Ser 98: 203–208
Chen JC, Ting YY, Lin JN, Lin MN (1990) Lethal effects of ammonia and nitrite on Penaeus chinensis juveniles. Mar Biol 107: 427–431 Claybrook DL (1983) Nitrogen metabolism. In: Bliss DE (ed) Biology of Crustacea. Vol. 5. Academic Press, New York Clinch JR, Worsfold PJ, Sweeting FW (1988) An automated spectrophotometric field monitor for water quality parameters: determination of ammonia. Analytica chim Acta 214: 401–407 Coyer PE (1977) Responses of heart and scaphognathite rates in Cancer borealis and C. irroratus to hypoxia. Comp Biochem Physiol 56A: 165–167 deFur PL, Mangum CP (1979) The effects of environmental variables on the heart rates of invertebrates. Comp Biochem Physiol 62A: 283–294 Dyer MF, Uglow RF (1977) On a technique for monitoring heart and scaphognathite activities in Natantia. J exp mar Biol Ecol 27: 117–124 Dyer MF, Uglow RF (1980) Cardiac and ventilatory changes associated with altered salinity in Crangon crangon (L.). Ophelia 19(2): 117–125 Evans DH, Cameron JN (1986) Gill ammonia transport. J exp Zool 239: 17–23 Hagerman L, Uglow RF (1985) Effects of hypoxia on the respiratory and circulatory regulation of Nephrops norvegicus. Mar Biol 87: 273–278 Hunter DA, Uglow RF (1993a) Moult stage-dependant variability of haemolymph ammonia and total protein levels of Crangon crangon (L.) (Crustacea, Decapoda). Ophelia 37(1): 41–50 Hunter DA, Uglow RF (1993b) A technique for the measurement of total ammonia in small volumes of seawater and haemolymph. Ophelia 37(1): 31–40 King FD, Cucci TL, Bidigare RR (1985) A pathway of nitrogen metabolism in marine decapod crabs. Comp Biochem Physiol 80B(3): 401–403 Kleiner D (1981) The transport of NH3 and NH4+ across biological membranes. Biochim biophys Acta 639: 41–52 Kormanik GA, Cameron JN (1981) Ammonia excretion in animals that breath water: a review. Mar Biol Lett 2: 11–23 Lin HP, Thuet P, Trilles JP, Mounet-Guillaume R, Charmantier G (1993) Effects of ammonia on survival and osmoregulation of various development stages of the shrimp Penaeus japonicus. Mar Biol 117: 591–598 Needham AE (1957) Factors affecting nitrogen-excretion in Carcinides maenas (Pennant). Physiologia comp Oecol 4: 209– 239 Provenzano AJ Jr (1983) Culture of crustaceans: general principles. In: Bliss DE (ed) Biology of Crustacea. Vol. 10. Academic Press, New York Regnault M (1987) Nitrogen excretion in marine and fresh-water Crustacea. Biol Rev 62: 1–24 Regnault M (1992) Effect of air exposure on nitrogen metabolism in the crab Cancer pagurus. J exp Zool 264: 372–380 Regnault M (1994) Effect of air exposure on ammonia excretion and ammonia content of branchial water of the crab Cancer pagurus. J exp Zool 268: 208–217 Regnault M, Batrel Y (1987) Glutamate dehydrogenase of the shrimp Crangon crangon L. effects of shrimp weight and season upon its activity in the oxidative and reductive function. Comp Biochem Physiol 86B(3): 525–530 Schmidt-Nielsen K (1983) Animal physiology: adaptation and environment. Cambridge University Press, Cambridge Schmitt ASC (1995) Aspects of the physiology of some crustacean species with particular reference to their live marketing. PhD thesis, The University of Hull, Kingston upon Hull, UK Spaargaren DH, Achituv Y (1977) On the heart rate response to rapid temperature changes in various marine and brackish water crustaceans. Neth J Sea Res 11(1): 107–117 Taylor EW, Butler PJ, Sherlock PJ (1973) The respiratory and cardiovascular changes associated with the emersion response of Carcinus maenas (L.) during environmental hypoxia, at three different temperatures. J comp Physiol 86: 95–115
418 Truchot JP (1983) Regulation of acid-base balance In: Bliss DE (ed) Biology of Crustacea. Vol. 5. Academic Press, New York Truchot JP (1994) Acid-base homeostasis in aquatic animals exposed to natural and perturbed environments. Belg J Zool 124: 61–74 Uglow RF (1973) Some effects of acute oxygen changes on heart and scaphognathite activity in some portunid crabs. Neth J Sea Res 7: 447–454 Wajsbrot N, Gasith A, Krom MD, Samocha TM (1990) Effects of dissolved oxygen and the moult stage on the acute toxicity of
ammonia to juvenile green tiger prawn Penaeus semisulcatus. Envir Toxic Chem 9: 497–504 Wheatly MG, Henry RP (1992) Extracellular and intracellular acid-base regulation in crustaceans. J exp Zool 263: 127–142 Wickins JF (1976) Prawn biology and culture. Oceanogr mar Biol A Rev 14: 435–507 Wilkens JL, Wilkes PRH, Evans J (1984) Analysis of the scaphognathite ventilatory pump in the shore crab Carcinus maenas. II. Pumping efficiency and metabolic cost. J exp Biol 113: 69–81
