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Effect of Ammonia on Survival and Adenylate Energy Charge in the ShrimpPalaemonetes varians
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY ARTICLE NO.
34, 103–108 (1996)
0050
Effect of Ammonia on Survival and Adenylate Energy Charge in the Shrimp Palaemonetes varians D. MARAZZA,1 PH. BORNENS,
AND
Y. LE GAL
Laboratoire de Biologie Marine, Colle`ge de France BP 225, 29182 Concarneau, France Received January 20, 1995
Shrimps Palaemonetes varians were submitted to lethal and sublethal concentrations of ammonia in order to measure lethality, ATP and adenylate nucleotide levels, and adenylate energy charge (AEC) index. LC50 was of about 3 mg/liter of ammonia. Adenylate measurements were performed over a period of 14 days and for two different concentrations. A population submitted to 0.5 mg/ liter of ammonia exhibited high survival and a marked consumption of ATP, whereas high mortality and disordered ATP metabolism are the characteristics of the population submitted to 3 mg/ liter of ammonia. An homeostatic model was applied in order to explain the significance of the AEC signal. Here, AEC is proposed as a measure of the limits of active response of an organism to environmental stress. q 1996 Academic Press, Inc.
INTRODUCTION
Various approaches and procedures have been proposed for environmental hazard assessment (Livingstone, 1982; Gray et al., 1991). Within this framework, the various techniques of biological monitoring proposed so far are based on different biological principles but have also different meanings. Mixed function oxydases enzymes (Stegeman et al., 1990), metallothioneins (Viarengo et al., 1988), mainly account, in a first approach, for the detoxification processes in response to the presence of environmental contaminants. Biochemical methods based on both systems are able to detect zones contaminated by hydrocarbons or by metals with a precision similar to or higher than that of chemical methods. However, it is not clear if the signals generated in this way are in reality informative about the health state and survival capacity of living organisms. Another aspect of these indexes is that they are reflecting only the presence of molecules or substances of a particular type: PAH, metals, etc. In these conditions, methods such as scope for growth (Bayne et al., 1988) appear very interesting as they have a global character and respond not only to specific contaminants but to a large series of natural or man1 Present address: Fondazione Flaminia, Piazza Kennedy 12, 48100 Ravenna, Italy.
induced stressing agents generally present in the environment at low concentrations. Such total or global indexes do have their natural place in any analytical approach of longterm effects of low level contaminants present in a marine environment (Howells et al., 1990). Indexes dealing with the evaluation of the general condition of organisms and applicable for laboratory tests as well as directly in situ have been proposed. Adenylate energy charge (AEC) is one of them. AEC is the ratio of the concentrations in ATP, ADP, and AMP and varies theoretically from 0 to 1. The value of AEC reflects the energy balance at a given time for an organism or a part of it (Atkinson and Walton, 1967). Whatever the biological system or the organism is, AEC reaches maximal values of 0.8–0.9. Outside stressing conditions, alterations of the environment (i.e., variations of temperature or salinity), presence of toxic substances, and pathological situations are followed by more or less pronounced and more or less permanent drops in the values of AEC. Due to more loose regulations and particularly to the low efficiency of the enzyme AMP deaminase, invertebrates are able to survive to lower energy charge (0.3– 0.4) than vertebrates (0.5–0.6) (Raffin et al., 1994). Ivanovici (1980) proposed the use of AEC as an index of environmental stress. AEC measurements have been performed either on organisms submitted to experimental contaminations in the laboratory or directly sampled in the natural environment (Din and Brooks, 1986; Sylvestre and Le Gal, 1987; Savary et al., 1989; Picado and Le Gal, 1990). Generally these measurements lead to the conclusion that AEC used either in the laboratory or in situ constitutes a sensitive mean of appreciating sublethal effects undergone by organisms living in polluted areas. However, a strict correlation between AEC index variations and survival is not always observed, and AEC cannot thus be considered as a direct health condition marker, although reflecting a response to environmental stress. Among the environmental stressors found in natural waters ammonia is present in both un-ionized (NH3) and ionized (NH4/) forms. The toxicity of ammonia to aquatic organisms is attributed to the un-ionized form (Thurston et al., 1981). The aqueous ammonia equilibrium is strongly
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dependent on pH and temperature and, to a lesser extent, upon salinity. The toxicity of ammonia to crustaceans have been studied by several authors (Chen and Lin, 1991; Chinn and Chen, 1987; Wajsbrot et al., 1990). Wajsbrot recommends a minimum of 96 hr bioassay on shrimp because of the heterogeneity of the molt stages in a shrimp population. In this paper, a study of the effect of sublethal concentrations of ammonia on AEC in the shrimp Palaemonetes varians is reported and, from the results, a reevaluation of the significance of AEC variations in relation with environmental stress is proposed. MATERIALS AND METHODS
The test organism is a euryhaline shrimp Palaemonetes varians purchased from TYMER aquaculture farm (44800 Le Croisic, France). Prior to any experimentation with ammonia the animals were acclimatized for 10 days to laboratory conditions. Two types of experimental devices were used. The first one is made from 12-liter tanks in which animals (20 individuals per tank) were exposed to various concentrations of ammonia. Water was changed every 24 hr. Following dissolution of NH4Cl, pH values varied form 8.16 (control) to 7.96 (3 g/liter of dissolved NH4Cl. The second experimental device comprised three isolated tanks containing 15 liters of sea water renewed daily. The concentrated solutions were renewed each day. Temperature was close to ambiant sea water (15–107C). Lighting followed the natural rhythm for the season (L: 16; D: 8). In all cases, shrimps were fed every 2 days with artificial pellets. Saturating oxygen and salinity (31‰) did not present significant variations during the experiments. Intoxication was obtained by adding NH4Cl in a concentration range comprised between 0.05 and 5 mg/liter. The corresponding final values of dissolved NH3 were calculated by using the equations of Bower and Bidwell (1978) and controlled by direct assay according to Harwood and Ku¨hn (1970). This method was adapted in order to chelate the excess Mg 2/ and Ca2/ ions present in sea water. Evaluation of mortality was performed by counting survivors every 24 hr. LC50 and LC99 were estimated according to Kooijman (1981). Adenylate nucleotides (ATP, ADP, and AMP) were measured by HPLC according to Leray (1979). Living shrimps were rapidly sampled and immediately immersed in liquid nitrogen before extraction in perchloric acid and treated according to Sylvestre (1988). Protein concentrations were estimated according to Lowry et al. (1951). RESULTS
Lethality was estimated over 96 hr in the shrimp P. varians for various concentrations of ammonia. The results pre-
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FIG. 1. Survival (% of control after 96 hr of experiment) of P. varians as a function of ammonia concentration.
sented in Fig. 1 indicate a lethal concentration (LC50) for this period of about 3 mg/liter of ammonia (NH3). Adenylates were measured on survivor animals submitted respectively to 0, 0.5, and 3 mg/liter of ammonia for a period of 2 weeks (Table 1). At the end of this particular experiment a concentration of 3 mg/liter of ammonia resulted in 75% survival. The control (0 mg/liter of ammonia) exhibits the highest values for the total adenylate as well as for adenylate energy charge, which remains stable during the experimentation (Fig. 2a). In animals submitted to a concentration of 3 mg/liter of ammonia, lethality at the end of the experiments is in an acute range (25%). ATP is first recorded at a low level, but after 48 hr reaches the same values as that of the control. The same observation is made for total adenylates. The resulting AEC is comprised between 0.4 and 0.55 during the first 4 days and reaches 0.65 on the 14th day. In contrast, animals submitted to 0.5 mg/liter of ammonia (Fig. 2c) demonstrated no lethality after 2 weeks although adenylates and especially ATP stay at a very low level during the period of experimentation. AEC has a high initial value (first 48 hr) but drops sharply later. It is interesting to note that standard deviations of AEC in this assay are very much lower as in control and in experiments with 3 mg/liter of ammonia (Fig. 3). DISCUSSION
Shrimps Palaemonetes varians were intoxicated with different concentrations of ammonia. Levels of ammonia correspond to a range of high values when compared to that usually encountered in the sea but can be occasionally found in some polluted estuaries. When submitted to different stress conditions two series of animals have produced different biochemical signals. Controls exhibit high ATP, total adenylate, and AEC values. AEC values
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TABLE 1 Adenylate Nucleotides and Adenylate Energy Charge of P. varians Submitted to Ammonia for a Total Period of 14 Days Time of experiment (hrs) AEC
6 96 336
0.67 { 0.2 0.74 { 0.27 0.63 { 0.29
0.60 { 0.19 0.38 { 0.12 0.36 { 0.15
0.45 { 0.24 0.37 { 0.21 0.66 { 0.19
Total adenylates
6 96 336
423 { 53 330 { 62 620 { 84
51 { 11 168 { 15 224 { 25
91 { 15 385 { 41 416 { 66
Survival after 336 hr (%)
96%
100%
75%
Note. Each value is the average between four individuals for samples at 6 and 96 hr, six for samples at 336. ATP and total adenylates are in ng for mg of protein. CEA Å (ATP / 12 ADP)/(ATP / ADP / AMP).
can be compared to that (0.68) given by Sylvestre and Le Gal (1987) for Crangon crangon but are lower than that of 0.79 given by Giesy et al. (1981) in similar experiments. However, these values appear very dispersed, reflecting an heterogenous response of the population to an experimental stress (Picado and Le Gal, 1990). In contrast, animals submitted to 0.5 mg/liter of ammonia present an homogeneous response. The level of adenylates is very low level, although individuals are characterized by a high capacity of survival. Animals submitted to 3 mg/ liter of ammonia are characterized by a high mortality and an important variability of the values of biochemical parameters, at least during the first 4 – 5 days. These results have to be compared with those obtained from AEC measurements in organisms submitted to stressing agents of various natures and those provided by literature. A first group of experiments deals with the responses of organisms submitted to modifications of natural environmental parameters such as temperature, salinity, oxygen availability, or food supply. It is important to underline that in most of the cases, imposed modifications fall within the natural range of environmental parameters, so perturbations are easily sustained by organisms: mortality remains at a very low level and the control of stressing conditions can run over a long period of time. Biochemical responses are very coherent with the health state of organisms: to a perturbation always corresponds a signal of energy consumption (low level of adenylates, low AEC) and when returning to normal conditions, signals comparable to that of control were obtained (Ivanovici, 1980; Ellington, 1981; Rainer et al., 1979; Vetter and Hodson, 1982; Barthel, 1984; Sylvestre, 1988; Whitmore and Kindle, 1986). This represented an encouraging basis for a diagnostical AEC use. In a second series of experiments, animals were submitted to stress of anthropogenic origin and mainly to toxic substances. Here, experiments were running over short periods and were characterized by the use of lethal concentrations
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generating complex and contradictory responses. In some instances, a low AEC reflected an alteration of the vital functions and a direct correlation between mortality and AEC is observed (Sylvestre and Le Gal, 1987; Picado and Le Gal, 1990). Skjoldal and Bakke (1978), Bakke and Skjoldal (1979), Wijman (1976), Zaroogian and Johnson (1989), however, did not find any obvious correlation between the variation of AEC and the impact of mortality. Thus, the signal AEC does not reflect the amplitude of the perturbation to which the organism is submitted and similar levels are found for high or low concentrations of pollutants (Skjodal and Bakke, 1978; Bakke and Skjodal, 1979; Wijman, 1976; Schoffler, 1979; Giesy et al., 1981; Verschraegen et al., 1985). As a whole, these contradictory results lead, during the last decade, to abandon the use of AEC as an indice of the physiological state of an organism. However, these data can be further analyzed, considering two main types of situations. (i) If organisms are submitted to nonlethal stressing agents one can observe a lowering of the levels of AEC, ATP, and total adenylate nucleotides (type I signal). (ii) When a phenomenon of lethality appears, responses of individuals are complex and very differentiated (type II signal). This is true at the level of populations or at the level of species. The ecologically most sensitive species produce type II responses. The experiments of Buu and Le Gal (1989) studying the effect of a large range of concentrations of cadmium on cockles lead to the conclusion that type I signals are possible only for long period sustainable concentrations. Following this interpretation in the case of the present experiments, the population submitted to 0.5 mg/liter of ammonia exhibit a type I signal (no lethality, general and permanent lowering of all parameters). This concentration is a long period sustainable one. On the contrary, a population submitted to 3 mg/liter of ammonia exhibit very variable
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FIG. 2. Evolution of the concentration of (a) total adenylates, (b) ATP, and (c) adenylate energy charges of P. varians vs time for two different concentrations of ammonia. % —, baseline; h —, control; s —, 0.5 mg NH3/liter; L —, 3 mg NH3/liter.
AEC and ATP signals, classifiable in type II. Nevertheless, these results do not explain the correlation of type I and type II signals with the physiological state of organisms. The model of Stebbing (1981) which proposes an homeostatical-energetical approach can be used to take into account a generalized or nonspecific response such as the AEC. In this model, a toxicant induces a response in energy metabo-
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lism only under some conditions. The toxicant is perceived and counteracted by the control systems which, in each organism, render possible homeostasis: osmoregulatory systems, enzyme activities, detoxification, or sequestration processes, etc. Energetical response also is mediated by this system. First of all, the response of the control system depends on tested concentration: three levels of responses are
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EFFECT OF AMMONIA ON P. varians
FIG. 3. Homeostatical, physiological, and energetical response vs increasing concentration of a toxicant for an individual or for a species. For concentrations below C1 , the mortality is zero, for concentrations above C1 , mortality is increasing in proportion. (A) Counter response in three steps: normality, countereaction, and inhibition (redrawn from Stebbing, 1981). (B) Controlled state of physiological processes (redrawn from Stebbing, 1981). (C) AEC values for control, subletal, and letal concentrations; points represent different experiments. l, Picado (1990) on Cardium paucicostatum at different concentrations; n, Sylvestre (1988) on Mytilus edulis at different concentrations; X, Barthel (1984) on three different species for the same concentration but different mortalities; s, Sylvestre (1988) on two different species for the same concentration but different mortalities; m, our experiments.
predicted for a concentration spectra that ranges from 0 to lethal concentrations (Fig. 3). N (normality). At the lowest concentrations, the control systems do not perceive any change and no reaction occurs. Energetical metabolism and physiological processes are normal. C (counteraction). If the concentration is higher than a defined threshold, C0 , and less than a second threshold, C1 , the control system arises to maintain the preferred N level. Maintenance is costly in terms of energy, but this condition is sustainable for a long period of time. I (inhibition). The capacity to maintain a preferred physiological state is limited: up to threshold C1 the concentration of the toxicant can act as an inhibitor in many physiological systems, probably in the control system too; so nothing can be said about energy consumption. A state of bad health takes place in individuals which are correlated with a phenomenon of lethality at the level of the population. Energy is consumed to counteract the effects of the toxicant and this controlled process responds to the need of the principle of homeostasis. Nevertheless, the operational field of homeostatical control is limited to suboptimal concentrations. One can observe an intense and persistent consumption of energy only if the toxic concentration is in the range C0 – C1 ; out of this range there is no more control on toxic effects and energy is not spent with proficiency. If countereaction is marked by an intense consumption of adenylates then the type I and II signals have a physiological
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meaning that apply to Stebbing’s model. In the population submitted to 0.5 mg/liter of ammonia, type I signal is the consequence of an homeostatical effort; in the population submitted to 3 mg/liter of ammonia, type II signal reveals that there is no control on toxic effects and in fact mortality is relevant. Stebbing (1981) points out that the degree of control achieved is also dependent on how sensitive the control system is to small changes in whatever it regulates. This means a different response of each species for each kind of stressor. Effectively an interspecific AEC test demonstrates that the more resistant the species, the greater the sustainable range of concentration (CO-C1) where AEC lows is (Barthel, 1984). Sylvestre (1988) measure an AEC of 0.49 (mortality 0%) in Mytilus edulis (euroxic) and an AEC of 0.81 (mortality 40%) in Spisula ovalis (stenoxic) after a 24-hr exposure to air. Other points of agreement could be found when comparing theoretical reactions as predicted by Stebbing and measured AEC in function of time exposure, class age, and history of previous exposure. But further experiments have to be performed for this goal. In this context, the physiological meaning of AEC has to then be revised. (i) Appearance of a signal (drop in AEC) corresponds to the response of healthy organisms; perturbation is sustainable for a long period and there is no lethality. (ii) A scattered and chaotic signal is the indice that the capacity of response of individuals has been overstepped. There is then appearance of a phenomenon of lethality. CONCLUSION
AEC is not a signal of discomfort but an index of reaction or of counteraction to adverse situations. Appearance of the reaction signal is only possible within a given interval of intensity of the stressor. The study of this signal can thus indicate the limits of active response of an organism (counteractive capacity). REFERENCES Atkinson, D. E., and Walton, G. M. (1967). Adenosine triphosphate conservation in metabolic regulation. J. Biol. Chem. 242, 3239–3241. Bakke, T., and Skjoldal, H. R. (1979). Effects of toluene on survival, respiration and adenylate system of a marine isopode. Mar. Pollut. Bull. 10, 111–115. Barthel, D. (1984). Adenylate energy charge in three marine bivalve species during anoxia. Ophelia 23, 155–164. Bayne, B. L., Clarke, K. L., and Gray, J. S. (1988). Biological effect of pollutants: Results of a practical workshop. Mar. Ecol. Progr. Ser. 46, 1–278. Bower, C. E., and Bidwell, J. P. (1978). Ionization of ammonia in sea water. Effects of temperature, pH and salinity. J. Fish Res. Board Can. 35, 1012–1016.
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Buu, B., and Le Gal, Y. (1989). Re´ponse de la coque Cardium edule a` l’intoxication par le cadmium. Oceanis 15, 591–597. Chen, J. C., and Lin, C. Y. (1991). Lethal doses of ammonia on Penaeus chinensis larvae. Bull. Inst. Zool. Academia Sinica 30(4), 289–297. Chinn, T. S., and Chen, J. C. (1987). Acute toxicity of ammonia to larva of the tiger prawn Penaeus monodon. Aquaculture 96, 247–253. Din, Z. B., and Brooks, J. M. (1986). Use of adenylate energy charge as a physiological indicator in toxicity experiments. Bull. Environ. Contam. Toxicol. 36, 1–8. Ellington, W. R. (1981). Effect of anoxia on the adenylates and energy charge in the sea anemone Bunodosoma cavernata. Physiol. Zool. 54, 415–422. Giesy, J. P., Denzers, R., Duke, C. S., and Dickson, G. W. (1981). Phosphoadenylate concentrations and energy charge into freshwater crustaceans: Responses to physical and chemical stressors. Verh. Intern. Limnol. 21, 205–220. Gray, J. S., Calamari, D., Duce, R., Portman, J. E., Wells, P. G., and Windom, H. L. (1991). Scientifically based strategies for marine environmental protection and management. Mar. Pollut. Bull. 22, 432–440. Harwood, J. E., and Ku¨hn, A. L. (1970). A colorimetric method for ammonia in natural waters. In Water Research, pp. 805–811. Pergamon Press, NY. Howells, G., Calamari, D., Gray, J., and Wells, P. G. (1990). An analytical approach to assessment of long term effects of low levels of contaminants in the marine environment. Mar. Pollut. Bull. 21, 371–375. Ivanovici, A. M. (1980). Adenylate energy charge: An evaluation of applicability to assessment of pollution effects and directions for future research. Rapp. P.V. Reun. Com. Int. Explor. Mer. 179, 23–28. Kooijman, S. A. L. M. (1981). Parametric analysis of mortality rates in bioassays. Water Res. 15, 107–119. Leray, C. (1979). Patterns of purine nucleotides in fish erythrocytes. Comp. Biochem. Physiol. 64B, 77–82. Livingstone, D. R. (1982). General biochemical indexes of sublethal stress. Mar. Pollut. Bull. 13, 261–263. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Picado, A. M., and Le Gal, Y. (1990). Assessment of industrial sewage impacts by adenylate energy charge measurements in the bivalve Cerastoderma edule. Ecotox. Environ. Safety 19, 1–7. Raffin, J. P., Thebault, M. T., Picado, A. M., Mendonc¸a, E., and Le Gal, Y. (1994). Modelization of coordinated changes of the adenylate energy charge and the ATP/ADP ratio: Use in ecophysiological studies. In preparation. Rainer, S. F., Ivanovici, A. M., and Wadley, V. A. (1979). Effects of reduced salinity on adenylate energy charge in three estuarine molluscs. Mar. Biol. 54, 91–99.
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Savary, A., Sylvestre, C., Sheader, M., Le Gal, Y., and Lockwood, A. P. M. (1989). Stress studies on the commun cockle (Cerastoderma edule) in Southampton water. Topics Mar. Biol. 53, 89–95. Schoffler, V. (1979). On the anaerobic metabolism of three species of Nereis (Annelida). Mar. Ecol. Prog. Ser. 1, 249–254. Skjoldal, H. R., and Bakke, T. (1978). Relationship between ATP and energy charge during lethal metabolic stress of the marine isopode Cirolana borealis. J. Biol. Chem. 253, 3355–3356. Stebbing, A. R. D. (1981). Are bioassays any use? Mar. Pollut. Bull. 19, 542–545. Stegeman, J. J., Renton, K. W., Woodin, B. R., Zhang, Y. S., and Addison, R. F. (1990). Experimental and environmental induction of cytochrome P450 E. in fish from Bermuda waters. J. Exp. Mar. Biol. Ecol. 138, 49– 68. Sylvestre, C. (1988). La charge e´nerge´tique adenylique: Possibilite´s d’utilisation dans la gestion de l’environnement marin. Thesis University Western Brittany, Brest, 125 pp. Sylvestre, C., and Le Gal, Y. (1987). In situ measurements of adenylate energy charge and assessment of pollution. Mar. Pollut. Bull. 18, 36– 39. Thurston, R. V., Russo, R. J., and Vinogradov, G. A. (1981). Ammonia toxicity to fish. Effect of the pH on the toxicity of the un-ionized ammonia species. Environ. Sci. Technol. 15, 837–840. Verschraegen, K., Herman, P. M. J., Van Gaunsbeke, D., and Braekman, A. (1985). Measurement of the adenylate energy charge in Nereis diversicolor and Nephtys sp.: Evaluation of the usefullness of AEC in pollution monitoring. Mar. Biol. 86, 233–240. Vetter, R. D., and Hodson, R. E. (1982). Use of adenylate concentration and adenylate energy charge as indicators of hypoxic stress in estuarine fishes. Can. J. Fish Aquat. Sci. 39, 535–541. Viarengo, A., Mancinelli, G., Martino, G., Pertica, G., and Mazzucotelli, A. (1988). Integrated cellular stress indices in trace of pollutants, metal contamination: Critical evaluation in a field study (B. L. Bayne, K. D. Clarke, and J. S. Gray, Eds.), Mar. Ecol. Prog. Ser. 46, 65–70. Wajsbrot, N., Gasith, A., Krom, M. D., and Somocha, T. M. (1990). Effect of dissolved and the molt stage on the acute toxicity of ammonia to juvenile green tiger prawn Penaeus semisulcatus. Environ. Toxicol. Chem. 9, 497–504. Whitmore et Kindle (1986). Biochemical indicators of thermal stress in Tilapia aurea. J. Fish. Biol. 29, 243–255. Wijman, T. C. M. (1976). Adenosine phosphates and energy charge in different tissues of M. edulis under aerobic and anaerobic conditions. J. Comp. Physiol. 107, 129–140. Zaroogian, G. E., and Johnson, M. (1989). Application of adenylate energy charge and adenine nucleotide measurements as indicators of stress in Nephtys incisa treated with dredged material. Bull. Environ. Contam. Toxicol. 43, 261–270.
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34, 103–108 (1996)
0050
Effect of Ammonia on Survival and Adenylate Energy Charge in the Shrimp Palaemonetes varians D. MARAZZA,1 PH. BORNENS,
AND
Y. LE GAL
Laboratoire de Biologie Marine, Colle`ge de France BP 225, 29182 Concarneau, France Received January 20, 1995
Shrimps Palaemonetes varians were submitted to lethal and sublethal concentrations of ammonia in order to measure lethality, ATP and adenylate nucleotide levels, and adenylate energy charge (AEC) index. LC50 was of about 3 mg/liter of ammonia. Adenylate measurements were performed over a period of 14 days and for two different concentrations. A population submitted to 0.5 mg/ liter of ammonia exhibited high survival and a marked consumption of ATP, whereas high mortality and disordered ATP metabolism are the characteristics of the population submitted to 3 mg/ liter of ammonia. An homeostatic model was applied in order to explain the significance of the AEC signal. Here, AEC is proposed as a measure of the limits of active response of an organism to environmental stress. q 1996 Academic Press, Inc.
INTRODUCTION
Various approaches and procedures have been proposed for environmental hazard assessment (Livingstone, 1982; Gray et al., 1991). Within this framework, the various techniques of biological monitoring proposed so far are based on different biological principles but have also different meanings. Mixed function oxydases enzymes (Stegeman et al., 1990), metallothioneins (Viarengo et al., 1988), mainly account, in a first approach, for the detoxification processes in response to the presence of environmental contaminants. Biochemical methods based on both systems are able to detect zones contaminated by hydrocarbons or by metals with a precision similar to or higher than that of chemical methods. However, it is not clear if the signals generated in this way are in reality informative about the health state and survival capacity of living organisms. Another aspect of these indexes is that they are reflecting only the presence of molecules or substances of a particular type: PAH, metals, etc. In these conditions, methods such as scope for growth (Bayne et al., 1988) appear very interesting as they have a global character and respond not only to specific contaminants but to a large series of natural or man1 Present address: Fondazione Flaminia, Piazza Kennedy 12, 48100 Ravenna, Italy.
induced stressing agents generally present in the environment at low concentrations. Such total or global indexes do have their natural place in any analytical approach of longterm effects of low level contaminants present in a marine environment (Howells et al., 1990). Indexes dealing with the evaluation of the general condition of organisms and applicable for laboratory tests as well as directly in situ have been proposed. Adenylate energy charge (AEC) is one of them. AEC is the ratio of the concentrations in ATP, ADP, and AMP and varies theoretically from 0 to 1. The value of AEC reflects the energy balance at a given time for an organism or a part of it (Atkinson and Walton, 1967). Whatever the biological system or the organism is, AEC reaches maximal values of 0.8–0.9. Outside stressing conditions, alterations of the environment (i.e., variations of temperature or salinity), presence of toxic substances, and pathological situations are followed by more or less pronounced and more or less permanent drops in the values of AEC. Due to more loose regulations and particularly to the low efficiency of the enzyme AMP deaminase, invertebrates are able to survive to lower energy charge (0.3– 0.4) than vertebrates (0.5–0.6) (Raffin et al., 1994). Ivanovici (1980) proposed the use of AEC as an index of environmental stress. AEC measurements have been performed either on organisms submitted to experimental contaminations in the laboratory or directly sampled in the natural environment (Din and Brooks, 1986; Sylvestre and Le Gal, 1987; Savary et al., 1989; Picado and Le Gal, 1990). Generally these measurements lead to the conclusion that AEC used either in the laboratory or in situ constitutes a sensitive mean of appreciating sublethal effects undergone by organisms living in polluted areas. However, a strict correlation between AEC index variations and survival is not always observed, and AEC cannot thus be considered as a direct health condition marker, although reflecting a response to environmental stress. Among the environmental stressors found in natural waters ammonia is present in both un-ionized (NH3) and ionized (NH4/) forms. The toxicity of ammonia to aquatic organisms is attributed to the un-ionized form (Thurston et al., 1981). The aqueous ammonia equilibrium is strongly
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0147-6513/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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dependent on pH and temperature and, to a lesser extent, upon salinity. The toxicity of ammonia to crustaceans have been studied by several authors (Chen and Lin, 1991; Chinn and Chen, 1987; Wajsbrot et al., 1990). Wajsbrot recommends a minimum of 96 hr bioassay on shrimp because of the heterogeneity of the molt stages in a shrimp population. In this paper, a study of the effect of sublethal concentrations of ammonia on AEC in the shrimp Palaemonetes varians is reported and, from the results, a reevaluation of the significance of AEC variations in relation with environmental stress is proposed. MATERIALS AND METHODS
The test organism is a euryhaline shrimp Palaemonetes varians purchased from TYMER aquaculture farm (44800 Le Croisic, France). Prior to any experimentation with ammonia the animals were acclimatized for 10 days to laboratory conditions. Two types of experimental devices were used. The first one is made from 12-liter tanks in which animals (20 individuals per tank) were exposed to various concentrations of ammonia. Water was changed every 24 hr. Following dissolution of NH4Cl, pH values varied form 8.16 (control) to 7.96 (3 g/liter of dissolved NH4Cl. The second experimental device comprised three isolated tanks containing 15 liters of sea water renewed daily. The concentrated solutions were renewed each day. Temperature was close to ambiant sea water (15–107C). Lighting followed the natural rhythm for the season (L: 16; D: 8). In all cases, shrimps were fed every 2 days with artificial pellets. Saturating oxygen and salinity (31‰) did not present significant variations during the experiments. Intoxication was obtained by adding NH4Cl in a concentration range comprised between 0.05 and 5 mg/liter. The corresponding final values of dissolved NH3 were calculated by using the equations of Bower and Bidwell (1978) and controlled by direct assay according to Harwood and Ku¨hn (1970). This method was adapted in order to chelate the excess Mg 2/ and Ca2/ ions present in sea water. Evaluation of mortality was performed by counting survivors every 24 hr. LC50 and LC99 were estimated according to Kooijman (1981). Adenylate nucleotides (ATP, ADP, and AMP) were measured by HPLC according to Leray (1979). Living shrimps were rapidly sampled and immediately immersed in liquid nitrogen before extraction in perchloric acid and treated according to Sylvestre (1988). Protein concentrations were estimated according to Lowry et al. (1951). RESULTS
Lethality was estimated over 96 hr in the shrimp P. varians for various concentrations of ammonia. The results pre-
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FIG. 1. Survival (% of control after 96 hr of experiment) of P. varians as a function of ammonia concentration.
sented in Fig. 1 indicate a lethal concentration (LC50) for this period of about 3 mg/liter of ammonia (NH3). Adenylates were measured on survivor animals submitted respectively to 0, 0.5, and 3 mg/liter of ammonia for a period of 2 weeks (Table 1). At the end of this particular experiment a concentration of 3 mg/liter of ammonia resulted in 75% survival. The control (0 mg/liter of ammonia) exhibits the highest values for the total adenylate as well as for adenylate energy charge, which remains stable during the experimentation (Fig. 2a). In animals submitted to a concentration of 3 mg/liter of ammonia, lethality at the end of the experiments is in an acute range (25%). ATP is first recorded at a low level, but after 48 hr reaches the same values as that of the control. The same observation is made for total adenylates. The resulting AEC is comprised between 0.4 and 0.55 during the first 4 days and reaches 0.65 on the 14th day. In contrast, animals submitted to 0.5 mg/liter of ammonia (Fig. 2c) demonstrated no lethality after 2 weeks although adenylates and especially ATP stay at a very low level during the period of experimentation. AEC has a high initial value (first 48 hr) but drops sharply later. It is interesting to note that standard deviations of AEC in this assay are very much lower as in control and in experiments with 3 mg/liter of ammonia (Fig. 3). DISCUSSION
Shrimps Palaemonetes varians were intoxicated with different concentrations of ammonia. Levels of ammonia correspond to a range of high values when compared to that usually encountered in the sea but can be occasionally found in some polluted estuaries. When submitted to different stress conditions two series of animals have produced different biochemical signals. Controls exhibit high ATP, total adenylate, and AEC values. AEC values
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TABLE 1 Adenylate Nucleotides and Adenylate Energy Charge of P. varians Submitted to Ammonia for a Total Period of 14 Days Time of experiment (hrs) AEC
6 96 336
0.67 { 0.2 0.74 { 0.27 0.63 { 0.29
0.60 { 0.19 0.38 { 0.12 0.36 { 0.15
0.45 { 0.24 0.37 { 0.21 0.66 { 0.19
Total adenylates
6 96 336
423 { 53 330 { 62 620 { 84
51 { 11 168 { 15 224 { 25
91 { 15 385 { 41 416 { 66
Survival after 336 hr (%)
96%
100%
75%
Note. Each value is the average between four individuals for samples at 6 and 96 hr, six for samples at 336. ATP and total adenylates are in ng for mg of protein. CEA Å (ATP / 12 ADP)/(ATP / ADP / AMP).
can be compared to that (0.68) given by Sylvestre and Le Gal (1987) for Crangon crangon but are lower than that of 0.79 given by Giesy et al. (1981) in similar experiments. However, these values appear very dispersed, reflecting an heterogenous response of the population to an experimental stress (Picado and Le Gal, 1990). In contrast, animals submitted to 0.5 mg/liter of ammonia present an homogeneous response. The level of adenylates is very low level, although individuals are characterized by a high capacity of survival. Animals submitted to 3 mg/ liter of ammonia are characterized by a high mortality and an important variability of the values of biochemical parameters, at least during the first 4 – 5 days. These results have to be compared with those obtained from AEC measurements in organisms submitted to stressing agents of various natures and those provided by literature. A first group of experiments deals with the responses of organisms submitted to modifications of natural environmental parameters such as temperature, salinity, oxygen availability, or food supply. It is important to underline that in most of the cases, imposed modifications fall within the natural range of environmental parameters, so perturbations are easily sustained by organisms: mortality remains at a very low level and the control of stressing conditions can run over a long period of time. Biochemical responses are very coherent with the health state of organisms: to a perturbation always corresponds a signal of energy consumption (low level of adenylates, low AEC) and when returning to normal conditions, signals comparable to that of control were obtained (Ivanovici, 1980; Ellington, 1981; Rainer et al., 1979; Vetter and Hodson, 1982; Barthel, 1984; Sylvestre, 1988; Whitmore and Kindle, 1986). This represented an encouraging basis for a diagnostical AEC use. In a second series of experiments, animals were submitted to stress of anthropogenic origin and mainly to toxic substances. Here, experiments were running over short periods and were characterized by the use of lethal concentrations
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generating complex and contradictory responses. In some instances, a low AEC reflected an alteration of the vital functions and a direct correlation between mortality and AEC is observed (Sylvestre and Le Gal, 1987; Picado and Le Gal, 1990). Skjoldal and Bakke (1978), Bakke and Skjoldal (1979), Wijman (1976), Zaroogian and Johnson (1989), however, did not find any obvious correlation between the variation of AEC and the impact of mortality. Thus, the signal AEC does not reflect the amplitude of the perturbation to which the organism is submitted and similar levels are found for high or low concentrations of pollutants (Skjodal and Bakke, 1978; Bakke and Skjodal, 1979; Wijman, 1976; Schoffler, 1979; Giesy et al., 1981; Verschraegen et al., 1985). As a whole, these contradictory results lead, during the last decade, to abandon the use of AEC as an indice of the physiological state of an organism. However, these data can be further analyzed, considering two main types of situations. (i) If organisms are submitted to nonlethal stressing agents one can observe a lowering of the levels of AEC, ATP, and total adenylate nucleotides (type I signal). (ii) When a phenomenon of lethality appears, responses of individuals are complex and very differentiated (type II signal). This is true at the level of populations or at the level of species. The ecologically most sensitive species produce type II responses. The experiments of Buu and Le Gal (1989) studying the effect of a large range of concentrations of cadmium on cockles lead to the conclusion that type I signals are possible only for long period sustainable concentrations. Following this interpretation in the case of the present experiments, the population submitted to 0.5 mg/liter of ammonia exhibit a type I signal (no lethality, general and permanent lowering of all parameters). This concentration is a long period sustainable one. On the contrary, a population submitted to 3 mg/liter of ammonia exhibit very variable
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FIG. 2. Evolution of the concentration of (a) total adenylates, (b) ATP, and (c) adenylate energy charges of P. varians vs time for two different concentrations of ammonia. % —, baseline; h —, control; s —, 0.5 mg NH3/liter; L —, 3 mg NH3/liter.
AEC and ATP signals, classifiable in type II. Nevertheless, these results do not explain the correlation of type I and type II signals with the physiological state of organisms. The model of Stebbing (1981) which proposes an homeostatical-energetical approach can be used to take into account a generalized or nonspecific response such as the AEC. In this model, a toxicant induces a response in energy metabo-
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lism only under some conditions. The toxicant is perceived and counteracted by the control systems which, in each organism, render possible homeostasis: osmoregulatory systems, enzyme activities, detoxification, or sequestration processes, etc. Energetical response also is mediated by this system. First of all, the response of the control system depends on tested concentration: three levels of responses are
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FIG. 3. Homeostatical, physiological, and energetical response vs increasing concentration of a toxicant for an individual or for a species. For concentrations below C1 , the mortality is zero, for concentrations above C1 , mortality is increasing in proportion. (A) Counter response in three steps: normality, countereaction, and inhibition (redrawn from Stebbing, 1981). (B) Controlled state of physiological processes (redrawn from Stebbing, 1981). (C) AEC values for control, subletal, and letal concentrations; points represent different experiments. l, Picado (1990) on Cardium paucicostatum at different concentrations; n, Sylvestre (1988) on Mytilus edulis at different concentrations; X, Barthel (1984) on three different species for the same concentration but different mortalities; s, Sylvestre (1988) on two different species for the same concentration but different mortalities; m, our experiments.
predicted for a concentration spectra that ranges from 0 to lethal concentrations (Fig. 3). N (normality). At the lowest concentrations, the control systems do not perceive any change and no reaction occurs. Energetical metabolism and physiological processes are normal. C (counteraction). If the concentration is higher than a defined threshold, C0 , and less than a second threshold, C1 , the control system arises to maintain the preferred N level. Maintenance is costly in terms of energy, but this condition is sustainable for a long period of time. I (inhibition). The capacity to maintain a preferred physiological state is limited: up to threshold C1 the concentration of the toxicant can act as an inhibitor in many physiological systems, probably in the control system too; so nothing can be said about energy consumption. A state of bad health takes place in individuals which are correlated with a phenomenon of lethality at the level of the population. Energy is consumed to counteract the effects of the toxicant and this controlled process responds to the need of the principle of homeostasis. Nevertheless, the operational field of homeostatical control is limited to suboptimal concentrations. One can observe an intense and persistent consumption of energy only if the toxic concentration is in the range C0 – C1 ; out of this range there is no more control on toxic effects and energy is not spent with proficiency. If countereaction is marked by an intense consumption of adenylates then the type I and II signals have a physiological
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meaning that apply to Stebbing’s model. In the population submitted to 0.5 mg/liter of ammonia, type I signal is the consequence of an homeostatical effort; in the population submitted to 3 mg/liter of ammonia, type II signal reveals that there is no control on toxic effects and in fact mortality is relevant. Stebbing (1981) points out that the degree of control achieved is also dependent on how sensitive the control system is to small changes in whatever it regulates. This means a different response of each species for each kind of stressor. Effectively an interspecific AEC test demonstrates that the more resistant the species, the greater the sustainable range of concentration (CO-C1) where AEC lows is (Barthel, 1984). Sylvestre (1988) measure an AEC of 0.49 (mortality 0%) in Mytilus edulis (euroxic) and an AEC of 0.81 (mortality 40%) in Spisula ovalis (stenoxic) after a 24-hr exposure to air. Other points of agreement could be found when comparing theoretical reactions as predicted by Stebbing and measured AEC in function of time exposure, class age, and history of previous exposure. But further experiments have to be performed for this goal. In this context, the physiological meaning of AEC has to then be revised. (i) Appearance of a signal (drop in AEC) corresponds to the response of healthy organisms; perturbation is sustainable for a long period and there is no lethality. (ii) A scattered and chaotic signal is the indice that the capacity of response of individuals has been overstepped. There is then appearance of a phenomenon of lethality. CONCLUSION
AEC is not a signal of discomfort but an index of reaction or of counteraction to adverse situations. Appearance of the reaction signal is only possible within a given interval of intensity of the stressor. The study of this signal can thus indicate the limits of active response of an organism (counteractive capacity). REFERENCES Atkinson, D. E., and Walton, G. M. (1967). Adenosine triphosphate conservation in metabolic regulation. J. Biol. Chem. 242, 3239–3241. Bakke, T., and Skjoldal, H. R. (1979). Effects of toluene on survival, respiration and adenylate system of a marine isopode. Mar. Pollut. Bull. 10, 111–115. Barthel, D. (1984). Adenylate energy charge in three marine bivalve species during anoxia. Ophelia 23, 155–164. Bayne, B. L., Clarke, K. L., and Gray, J. S. (1988). Biological effect of pollutants: Results of a practical workshop. Mar. Ecol. Progr. Ser. 46, 1–278. Bower, C. E., and Bidwell, J. P. (1978). Ionization of ammonia in sea water. Effects of temperature, pH and salinity. J. Fish Res. Board Can. 35, 1012–1016.
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Buu, B., and Le Gal, Y. (1989). Re´ponse de la coque Cardium edule a` l’intoxication par le cadmium. Oceanis 15, 591–597. Chen, J. C., and Lin, C. Y. (1991). Lethal doses of ammonia on Penaeus chinensis larvae. Bull. Inst. Zool. Academia Sinica 30(4), 289–297. Chinn, T. S., and Chen, J. C. (1987). Acute toxicity of ammonia to larva of the tiger prawn Penaeus monodon. Aquaculture 96, 247–253. Din, Z. B., and Brooks, J. M. (1986). Use of adenylate energy charge as a physiological indicator in toxicity experiments. Bull. Environ. Contam. Toxicol. 36, 1–8. Ellington, W. R. (1981). Effect of anoxia on the adenylates and energy charge in the sea anemone Bunodosoma cavernata. Physiol. Zool. 54, 415–422. Giesy, J. P., Denzers, R., Duke, C. S., and Dickson, G. W. (1981). Phosphoadenylate concentrations and energy charge into freshwater crustaceans: Responses to physical and chemical stressors. Verh. Intern. Limnol. 21, 205–220. Gray, J. S., Calamari, D., Duce, R., Portman, J. E., Wells, P. G., and Windom, H. L. (1991). Scientifically based strategies for marine environmental protection and management. Mar. Pollut. Bull. 22, 432–440. Harwood, J. E., and Ku¨hn, A. L. (1970). A colorimetric method for ammonia in natural waters. In Water Research, pp. 805–811. Pergamon Press, NY. Howells, G., Calamari, D., Gray, J., and Wells, P. G. (1990). An analytical approach to assessment of long term effects of low levels of contaminants in the marine environment. Mar. Pollut. Bull. 21, 371–375. Ivanovici, A. M. (1980). Adenylate energy charge: An evaluation of applicability to assessment of pollution effects and directions for future research. Rapp. P.V. Reun. Com. Int. Explor. Mer. 179, 23–28. Kooijman, S. A. L. M. (1981). Parametric analysis of mortality rates in bioassays. Water Res. 15, 107–119. Leray, C. (1979). Patterns of purine nucleotides in fish erythrocytes. Comp. Biochem. Physiol. 64B, 77–82. Livingstone, D. R. (1982). General biochemical indexes of sublethal stress. Mar. Pollut. Bull. 13, 261–263. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Picado, A. M., and Le Gal, Y. (1990). Assessment of industrial sewage impacts by adenylate energy charge measurements in the bivalve Cerastoderma edule. Ecotox. Environ. Safety 19, 1–7. Raffin, J. P., Thebault, M. T., Picado, A. M., Mendonc¸a, E., and Le Gal, Y. (1994). Modelization of coordinated changes of the adenylate energy charge and the ATP/ADP ratio: Use in ecophysiological studies. In preparation. Rainer, S. F., Ivanovici, A. M., and Wadley, V. A. (1979). Effects of reduced salinity on adenylate energy charge in three estuarine molluscs. Mar. Biol. 54, 91–99.
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Savary, A., Sylvestre, C., Sheader, M., Le Gal, Y., and Lockwood, A. P. M. (1989). Stress studies on the commun cockle (Cerastoderma edule) in Southampton water. Topics Mar. Biol. 53, 89–95. Schoffler, V. (1979). On the anaerobic metabolism of three species of Nereis (Annelida). Mar. Ecol. Prog. Ser. 1, 249–254. Skjoldal, H. R., and Bakke, T. (1978). Relationship between ATP and energy charge during lethal metabolic stress of the marine isopode Cirolana borealis. J. Biol. Chem. 253, 3355–3356. Stebbing, A. R. D. (1981). Are bioassays any use? Mar. Pollut. Bull. 19, 542–545. Stegeman, J. J., Renton, K. W., Woodin, B. R., Zhang, Y. S., and Addison, R. F. (1990). Experimental and environmental induction of cytochrome P450 E. in fish from Bermuda waters. J. Exp. Mar. Biol. Ecol. 138, 49– 68. Sylvestre, C. (1988). La charge e´nerge´tique adenylique: Possibilite´s d’utilisation dans la gestion de l’environnement marin. Thesis University Western Brittany, Brest, 125 pp. Sylvestre, C., and Le Gal, Y. (1987). In situ measurements of adenylate energy charge and assessment of pollution. Mar. Pollut. Bull. 18, 36– 39. Thurston, R. V., Russo, R. J., and Vinogradov, G. A. (1981). Ammonia toxicity to fish. Effect of the pH on the toxicity of the un-ionized ammonia species. Environ. Sci. Technol. 15, 837–840. Verschraegen, K., Herman, P. M. J., Van Gaunsbeke, D., and Braekman, A. (1985). Measurement of the adenylate energy charge in Nereis diversicolor and Nephtys sp.: Evaluation of the usefullness of AEC in pollution monitoring. Mar. Biol. 86, 233–240. Vetter, R. D., and Hodson, R. E. (1982). Use of adenylate concentration and adenylate energy charge as indicators of hypoxic stress in estuarine fishes. Can. J. Fish Aquat. Sci. 39, 535–541. Viarengo, A., Mancinelli, G., Martino, G., Pertica, G., and Mazzucotelli, A. (1988). Integrated cellular stress indices in trace of pollutants, metal contamination: Critical evaluation in a field study (B. L. Bayne, K. D. Clarke, and J. S. Gray, Eds.), Mar. Ecol. Prog. Ser. 46, 65–70. Wajsbrot, N., Gasith, A., Krom, M. D., and Somocha, T. M. (1990). Effect of dissolved and the molt stage on the acute toxicity of ammonia to juvenile green tiger prawn Penaeus semisulcatus. Environ. Toxicol. Chem. 9, 497–504. Whitmore et Kindle (1986). Biochemical indicators of thermal stress in Tilapia aurea. J. Fish. Biol. 29, 243–255. Wijman, T. C. M. (1976). Adenosine phosphates and energy charge in different tissues of M. edulis under aerobic and anaerobic conditions. J. Comp. Physiol. 107, 129–140. Zaroogian, G. E., and Johnson, M. (1989). Application of adenylate energy charge and adenine nucleotide measurements as indicators of stress in Nephtys incisa treated with dredged material. Bull. Environ. Contam. Toxicol. 43, 261–270.
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