Physiological parameters in ecotoxicology

0306-4492/91 $3.00 + 0.00 0 I99 1 Pergamon Press plc

Camp. Biochem. Physiol.Vol. IOOC, No. l/Z, pp. 77-79, 1991

Printed in Great Britain

PHYSIOLOGICAL

PARAMETERS

IN ECOTOXICOLOGY

K. E. ZACHARIASSEN,* T. AUNAAS,~J.

F. BBRSETH,~S. EINARSON,* T. NORDTIJG,~A. OLsENt and G. SKJERvBt *Department of Zoology, The University of Trondheim, 7055 Dragvoll, Norway; and TDepartment of Ecotoxicology, The Research Foundation at the College of Arts and Science, The University of Trondheim, 7055 Dragvoll, Norway (Received 1 October

1990)

Abstract-l. Regulated physiological parameters are normally maintained at a constant level by regulatory mechanisms. Acute toxic effects develop whenever a pollutant causes a regulated parameter to be displaced beyond tolerated limits, and thus, regulated parameters may be convenient toxicity parameters. The present study indicates that ApNa+across the adductor muscle membrane of Mytilus edulis is a regulated parameter, and that injuries develop whenever this parameter drops below -7000 J/mole. 2. Regulatory physiological parameters may display quick and substantial changes when regulatory mechanisms are activated to counteract variations in the regulated parameters. Thus, regulatory parameters may be used as sensitive alarm parameters in environmental monitoring. The present results indicate that the phosphate index [(ATP x P-arginine)/(P,)2], metabolic rate and strombine may be used as alarm parameters. 3. The combined response of all parameters may provide a pollutant-specific fingerprint in environmental monitoring.

The responsiveness of regulatory parameters implies that they may have a great potential as “alarm parameters” for early warning purposes in environmental monitoring. The ideal “alarm parameter” responds quickly to the lowest possible concentrations of the broadest possible array of environmental pollutants. The combined response of several parameters may even be specific to the various pollutants, and thus provide an “effect fingerprint”. The purpose of this study has been to search for physiological parameters to use in toxicity tests and environmental monitoring of marine environments. The study was focused on the electrochemical potential difference of sodium (ApNa+), across the cell membrane of the posterior adductor muscle of blue mussels (Mytilus edulis). ApNa+ is high due to the action of the membrane bound sodium-potassium pump, which requires a substantial fraction (70%) of the total cellular ATP production even in ectotherms (Florey, 1966).

INTRODUCTION Toxic effects of pollutants are due to a disturbance of the normal physiological functions of the affected organisms. Thus, physiological studies are required to understand why toxic pollutants are toxic. Studies on physiological parameters may also be useful in environmental monitoring, particularly to detect the presence of a pollutant before manifest ecological effects develop. When physiological parameters are used for this purpose, it is important to distinguish between regulated and regulatory parameters. Regulated parameters are usually important for a wide range of physiological processes, and organisms seek to maintain such parameters at constant levels. Wellknown examples of regulated parameters are the deep-body temperature of homeotherms and the plasma sodium concentration of vertebrates. Injury and death following exposure to environmental pollutants are likely to be due to a displacement of one or more regulated parameters beyond the tolerated range. However, before regulated physiological parameters are altered by exposure to a toxic agent, regulatory parameters display marked changes to maintain the regulated parameters at the optimal levels. Subsequently, regulatory parameters may vary considerably, and will also be more sensitive to pollutants than the regulated parameters. Examples of regulatory parameters are the metabolic rate of endotherms, which may increase substantially to maintain a constant deep-body temperature during thermal stress, and the rate of sodium excretion from vertebrates, which may vary to maintain the plasma sodium concentration constant. Also levels of hormones and metabolites may conveniently be defined as regulatory parameters.

MATERIALSAND METHODS Blue musssels were obtained from a commercial farm in the Trondheimsfjord area in mid-Norway. Toxicity tests with formalin were run for up to 240 hr and standard exposures to the pollutants were made for 96 hr at 6°C in 35%0seawater. Initial series of lethality tests were run to establish concentrations of exposure chemicals causing a high, but sublethal stress. High sublethal concentrations were then used to study the effects of the various pollutants on the physiological parameters in the standard exposures. Solute concentrations were measured in hemolymph and muscle tissue, inorganic ions by flame photometry and potentiometric chloridometry, and free amino acids on an HPLC instrument as described by Aunaas et al. (1989). 77

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Fig. 1. Transmembrane electrochemical potential difference of sodium (x(-l)) in the posterior adductor muscle of Mytiius edulis exposed to 60ppm formalin for different periods of time, and after 96 hr recovery in full seawater. Dead animals are indicated by +. &,a+ was calculated from the transmembrane concentration quotient of sodium and the membrane potential, estimated as described by Bsrseth et al. (1990). Metabolic rate was measured by an oxygen electrode in a flow-through system, whereas cellular phosphates were measured by P-NMR. RESULTS

Figure 1 shows that increasing exposure time to 60 ppm formalin leads to an increasing depression of Api++. During 96 hr of recovery in pollutant-free seawater, ApLNa+ may return to normal. When Ap(,,+ dropped below -7000 J/mole, the ability to reverse

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Fig. 2. Semi-quantitative presentation of effects on physiological parameters (metabolic rate = poz, phosphate index [ATP x P-arginine/(P,)*] = PI, transmembrane electrochemical potential difference of sodium = ApNar, strombine = Str, and free amino acids) of Mytilus edulis exposed to various environmental pollutants. Increased and reduced parameter values compared to control animals are indicated by bars pointing respectively up and down from the horizontal base-line. Length of bars indicates magnitude of response. Short bar: change by factor ~2. Medium sized bar: change by a factor between 2 and 5. Long bar: change by a factor ~5. x : substances causing lethality.

the reduction during recovery seemed to be lost. Under these conditions ApNa+continued to drop, and the animals died or were moribund. Figure 2 is a summary of the effects of sublethal exposures to a number of organic (30 ppm formalin, 300ppm phenol, 100ppm benzene) and inorganic (2 ppm of ZnCl,, CdCl, and HgCl,) substances. The results reveal that the levels of energy-rich phosphates were markedly depressed following exposure to formalin, phenol, cadmium and mercury, whereas Apr.,*+was depressed only after exposure to formalin, benzene and mercury. The latter were also the chemicals that lead to clinical injury and death. Intracellular levels of free amino acids were reduced in those mussels which displayed a reduced value of &a+. Cellular strombine levels were markedly increased in mussels exposed to formalin, benzene and mercury. The results in Fig. 2 also show that the combined response of the parameters was specific to the respective pollutants. DISCUSSION

The transmembrane electrochemical potential difference of sodium (ApCNa+)in the adductor muscle of Mytilus edulis displays only very moderate variations during the year, and when the animals are exposed to different temperatures and medium salinities (Aunaas, unpublished results). The stability of ApNa+ indicates that it is a regulated parameter. The results (Fig. 1) reveal a clear correlation between the effects on ApLNa+ and the clinical effects of formalin, in that injuries develop whenever ApNa+ drops below a certain minimum value. Thus, Ap,,, may be used as a toxicity parameter for formalin. The results of the standard tests (Fig. 2) reveal that &,,+ is depressed by a number of organic and inorganic chemicals. These chemicals are also the ones causing acute lethality, indicating that a depression of ApNa+ may be the cause of the acute toxicity of a wide variety of chemicals. The maintenance of A,r+,%+ depends on a number of cellular processes, such as an adequate supply of oxygen, an intact metabolic system for production of ATP, an intact NA+/K+-ATPase, and a low membrane permeability to sodium. Chemical agents which interact at any. of these points disturb the mechanisms that mamtam ApNa+. Whenever the compensatory capacity of these mechanisms is surpassed, Apu,,+ will start to drop, ultimately leading to death of the organism, possibly as a consequence of a collapse in sodium-dependent cellular calcium extrusion (Ganong, 1987). Thus, ApNa+may be used as a clinically relevant toxicity parameter for a broad array of environmental pollutants. However, changes in regulatory parameters such as metabolic rate and energy-rich phosphates are seen before ApNa+is affected. The high sensitivity of these parameters to potentially toxic chemicals implies that these regulatory parameters may have an interesting potential as “alarm parameters” in environmental monitoring. Also the amino acid-related compound, strombine, that is accumulated to high levels following exposure to a variety of toxic chemicals (Fig. 2), may be used.

Parameters in toxicology

The combined response of the parameters presented in Fig. 2 varies from one pollutant to another. The combined effects are likely to reflect the specific mechanisms of action of the various toxic chemicals, and may thus be used to obtain information on these mechanisms. They may also be considered as physiological “effect fingerprints”, characteristic of the respective toxic agents, By including a sufficient number of parameters, a high specificity may be obtained. It is possible that the “effect fingerprints” in subIethally affected organisms may provide information for qualitative identification of poliutants in situations where their identity is not known. Acknowledgements-The present study is part of the BECTOS Research Program which is financed by FINA Exploration Norway u.a.s., and of the project “Physiologica Parameters in Marine Environmental Monitoring”, which is financed by the Norwegian Research

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REFERENCES

Aunaas T., Borseth J. F., Denstad J.-P., Ekker M., Jenssen B. M., Jorgensen L., Olsen A., Schmid R. and Zachariassen K. E. (Editor) (1989) Biological Effects of Chemical Treatment of Oilspills at Sea. Report from the BECTOSprogram 19851989. The University of Trondheim, Norway. Borseth J. F., Aunaas T., Nordtug T., Olsen A., Skjervnr G. and Zachariassen K. E. (1990) Method to determine intracellular ionic concentration and membrane potential of animal tissues. (In preparation.) Florey E. (1966) An Introduction to General and Comparative Animal Physiology. 713 pp. W. B. Saunders, Philadelphia, PA. Ganong W. F. (1987) Review of Medical Physiology. 13th Edn. Appleton & Lange, Prenti~-Hall Intemationa~, Englewood Cliffs, NJ.