Camp. Biochem. Physiol.
Vol. IOOC,No. l/2, pp.89-93,
0306-4492/91 $3.00+ 0.00 0 1991PergamonPressplc
1991
Printedin Great Britain
THE EFFECTS OF ORGANIC AND INORGANIC POLLUTANTS ON INTRACELLULAR PHOSPHATE COMPOUNDS IN BLUE MUSSELS (MYTKUS EDULIS) TOREAUNAAS,SIGRUNEINARSON,*TIMOTHYE. SouTHoNt and KARL ERIK ZACHARIASSEN* The Research Foundation at the College of Arts and Science, Department of Ecotoxicology, The University of Trondheim, N-7055 Dragvoll, Norway (Received 1 October 1990) Abstract-1. The effects of sublethal concentrations of organic and inorganic pollutants on intracellular energy-rich phosphates in blue mussels, Mytilus edulis,were investigated by in viuo )‘P-NMR. 2. Formaldehyde (30 and 10mg/l), phenol, pyridine, mercury and cadmium gave marked reductions in phosphoarginine and, in some cases, the ATP amounts. The reduction in high-energy phosphate was accompanied by an increase in inorganic phosphate in all groups. 3. A “phosphorus index”, the product of the ratios between phosphoarginine and inorganic phosphate, and ATP and inorganic phosphate, is suggested, which might serve as an early warning (“alarm”) parameter in environmental monitoring. 4. Diversity in the responses to different pollutants make phosphorus compounds in M. edulisalso an interesting element in a finger print parameter system designed to distinguish between pollutants in the marine environment.
expressed as 2.19 mmol/kg wet weight (Wijsman, 1976). Addink and Veenhof (1975) confirmed these results and showed total animal values of 2.2 mmol ATP/kg wet weight in M. edulis. Enzymatic methods for the determination of highenergy phosphate compounds in animal tissues have been widely used. However, unless certain methodological precautions are taken, tissue excision and extraction might induce a breakdown of high-energy phosphate compounds (Van den Thillart et al., 1990). “P-NMR spectroscopy, as a technique for studying energy metabolism, is non-destructive to the organism, and the spectra of phosphorus compounds can be repeatedly obtained from intact tissues of live animals. Indeed, the effects of osmotic and hypoxic stress have already been studied in a marine mollusc (Hufiotis) by “P-NMR (Higashi et al., 1989). Exact quantitation of metabolites by NMR requires rather careful experimental procedures, but the relative contributions of phosphoarginine, ATP, inorganic phosphate and phosphodiesters to the total phosphorus signal can be easily determined (Gadian et al., 1979; Van den Thillart et al., 1990). In this investigation, the effects of exposure to sublethal concentrations of different organic and inorganic pollutants on phosphorus compounds of Myths edulis are described. The levels of phosphoarginine, ATP and inorganic phosphate were measured in vivo using 31P-NMR spectroscopy.
INTRODUCTION Toxic effects of environmental pollutants are due to disturbances of normal physiological functions of the affected organisms. Physiological parameters may be separated (classified) into two different categories, the regulated and the regulatory parameters (Zachariassen et al., 1991). Regulatory parameters, such as metabolic rate and energy turnover, vary according to the requirements of the organism in order to maintain regulated physiological parameters at a constant level. Thus, these parameters may display wide variations during normal cell functioning. Toxic effects of environmental pollutants on physiological parameters will probably be seen on regulatory parameters before they are manifest in effects on the regulated parameters. Environmental pollutants might affect regulatory physiological parameters in different ways. The energy turnover might increase or decrease as other physiological processes are influenced by the pollutant. Changes in the energy turnover and mitochondrial ATP production may thus be detected as changes in the amount of highenergy phosphate compounds. Phosphoarginine is believed to be the major phosphagen in the tissues of blue mussels, Myths edulis, and other marine invertebrates (Beis and Newsholme, 1975). Ansell (1974) showed that the ATP content of various species of bivalves is closely correlated with tissue dry weight, being 0.56mg ATP/lOOmg dry tissue for Myths edulis. Assuming that the dry weight is 20% of the wet weight, this amount can be
MATERIALSAND
METHODS
Blue mussels, Mytifusedulis,were collected from the tidal zone in Trondheimstjorden, Norway. The mussels were transported in thermo-containers to the laboratory and acclimated in 34.5% running seawater at 10°C for 4-6 days prior to the experiments. The mussels were not fed during the acclimation and experimental period.
*Department of Zoology, The University of Trondheim, N-7055 Dragvoll, Norway. tSINTEF/UNIMED, The MR-Center, N-7034 Trondheim, Norway. 89
TORE AUNAAS et al.
90
After the acclimation period the mussels, ranging from 1.4 to 2.6 g tissue wet weight, were exposed for 96 hr to pure seawater (control) and sublethal concentrations of phenol (100 mg/l), formaldehyde (30, 10 and 1 mg/l), benzene (100 mg/l), pyridin (100 mg/l), mercury (0.52 mg/l), cadmium (1.84 mg/l) and zinc (2.40 mg/l). Heavy metals were added as dichloride salts to the seawater and the concentrations given refer to the metals in solution. The exposure of 5-8 individuals took place in aquaria at 10°C. The experiment also involved a flow exposure to benzene (100 mg/l) for 96 hr at 10°C. High sublethal exposure concentrations of the chemicals were established on the basis of 96-hr mortality experiments, carried out prior to the sublethal experiments. LC~~values of 96-hr exposures were approximately 450 mg/l phenol, 75 mg/l formaldehyde, 350 mg/l benzene, 450 mg/l pyridin, 1.5 mg/l mercury, 10 mg/l cadmium and 10 mg/l zinc. To keep the concentrations of the chemicals stable throughout the static exposures, the exposure media were exchanged once a day with newly-made solutions. Exposure solutions of pure seawater, and seawater with the different chemicals, were used as exposure media in the NMR
probe. NMR experiments were carried out on intact animals immersed in exposure solutions in a tube inside a 25mm NMR probe. The solutions were aerated from the top of the tube, and the temperature in the probe was controlled at 10°C during the experiments. The animals were kept in the tube for 2-2.5 hr. At the termination of the experiment, the mussels were removed from the tube and the tissue wet weights of total animals (without shells) were determined. “P-NMR spectra were obtained at 81 MHz on a Bruker MSL 200 spectrometer operated in the pulsed Fourier A
4
Fig. 1. In uivo “P-NMR spectra of M. edulis exposed for 96 hr at 10°C to (A) seawater (34.5%) and to (B) seawater (34.5%) with 0.52mg/l mercury. Peaks were identified by their chemical shifts in relation to standard spectra. The labelled resonances are those of (1) phosphomonoesters; (2) inorganic phosphate; (3) phosphodiesters; (4) phosphoarginine; (5) y-phosphate of ATP, (6) cc-phosphate of ATP and phosphate moiety of NAD; (7) /?-phosphate of ATP.
transform mode. For quantitation of the phosphorus compounds, fully relaxed spectra were accumulated with a recycle time of 20 set, a sweep width of 10,000 Hz and with a collection of 180 data acquisitions. Radiofrequency pulses of 90” (pulse width 301(sec) were employed in all experiments. Peak heights, corrected for the tissue wet weights, were used for the relative quantitation of the phosphorus compounds The P-ATP peak was used for quantitation of the amount of ATP. The metabolite levels were expressed as a percentage of the total phosphorus signal (phosphomonoesters + inorganic phosphate + phosphodiesters + phosphoarginine + ATP) as determined from the 3’P-NMR in viva spectra. Due to rather small sample sizes in the experimental groups, the underlying population from which the group of data are drawn, are assumed to be normally distributed. Student’s t-test was employed to test the significance of the data.
RESULTS In vivo “P-NMR spectra of Mytilus edulis exposed to 34.5o/ooseawater (control) and mercury
(0.52 mg/l) are presented in Fig. 1. The spectra show well-resolved resonances of phosphomonoesters, inorganic phosphate, phosphodiesters, phosphoarginine and the y-, GI-and b-phosphate atoms of ATP. In control mussels, the phosphoarginine peak height is typically 4-5 times greater than individual peaks from the other phosphorus compounds. In mussels exposed to mercury, the peak representing the inorganic phosphate is increased and dominates the spectrum. The height of the peak representing the phosphoarginine is markedly reduced by about the same magnitude as the increase in the peak height of the inorganic phosphate. Figure 2 shows the amount (%) of the total phosphorus compounds; of phosphoarginine, inorganic phosphate and ATP in M. edulis exposed to sublethal concentrations of different environmental pollutants. The values are corrected for the flesh wet weight of the mussels. Fully relaxed spectra from control mussels indicate that phosphoarginine normally makes up about 50% of the total NMR visible phosphorus pool in the mussels, while inorganic phosphate and ATP amount to 12 and 10% of the phosphorus pool, respectively. Significantly reduced amounts of phosphoarginine were registered in mussels exposed to formaldehyde (30 mg/l, P < 0.001 and 10 mg/l, P < O.OOl), phenol (lOOmg/l, P < O.OOl), pyridin (100 mg/l, P < O.OOl), mercury (0.52 mg/l, P < 0.001) and cadmium (1.84 mg/l, P < 0.001). Reduced amounts of phosphoarginine were, in all groups, followed by an increase in inorganic phosphate. Significantly reduced amounts of ATP (P < 0.001 and P < 0.01) were registered in groups of mussels exposed to 30 and 10 mg/l formaldehyde, phenol, cadmium and mercury (0.52mg/l). Although a marked (50%) reduction in the amount of phosphoarginine was registered in mussels exposed to pyridin, no significant reduction in the amount of ATP was registered in these animals. In contrast, cadmium, which reduced the phosphoarginine amounts by about 20%, caused a marked reduction (30%) in the amount of ATP.
Effects of pollutants on “F in M. edulis
91
60 60 40 30 20 10 0 60
50
40
30
20 10
0
t6 * 8
,,IT 14
4
C
T _
Fig. 2. Changes in levels of (A) phosphoarginine, (B) inorganic phosphate and (C) ATP in edulis exposed to different environmental pollutants for 96 hr at 10°C. The metabolite levels were obtained by measuring peak heights from the spectra, and are expressed as a percentage of the total phosphorus signal (phosphomon~sters f inorganic phosphate + phosphodiesters + phosphoar~nine + ATP) hr 3’P-NMR in U&O spectra. Values are presented as mean + SD. The number of individuals in each group is given above the bars. *P < 0.01 and **P < 0.001 with respect to the control. M.
Exposure to benzene (100 mg/l), both static and in a flow-system, did not seem to affect the amounts of individua1 phosphorus compounds in the mussels.
CBP1lmc,1-~--0
This also seemed to be the situation for 96hr exposure to zinc (2.4 mgjl) and to the low concentration (1 mg/l) of formaldehyde tested.
TOREAUNM et al.
92
Fig. 3. Changes in the “phosphorus index”, expressed as the product of the ratios between phosphoarginine and inorganic phosphate, and ATP and inorganic phosphate, following 96-hr exposure to sublethal concentrations of different environmental pollutants at 10°C. Values are presented as mean f SD. The number of individuals in each group is given above the bars, lP < 0.01 and **P< 0.001 with respect to the control.
DISCUSSION
Seawater containing formaldehyde (30 and 10 mg/l), phenol (100 mg/l), pyridin (100 mg/l), mercury (OS2mg/l) and cadmium (1.84mg/l) is demonstrated to strongly affect phosphorus compounds in Myths edulis. The effects on the phosphorus compounds in mussels exposed to these chemicals were correlated to observed clinical effects,
such as reduced byssal thread formation (formaldehyde, phenol, heavy metals), shedding of byssal threads (phenol, pyridin) and, in sequences of the exposure time, abnormal shell closure (formaldehyde, pyridin, heavy metals) or shell opening (phenol). Exposure to benzene (100 mg/l), formaldehyde (1 mg/l) and zinc (2.4 mg/l) does not seem to affect the phosphorus compounds in the mussels to any degree. Alterations in behaviour and appearance of mussels exposed to this concentration of formaldehyde and to zinc were not observed. This indicates that the concentrations used were non-toxic to the mussels at acute exposure. This assumption is supported by the fact that no effects were found on oxygen consumption rates, transmembrane distribution of inorganic ions or concentrations of individual free amino acids in these mussels (Olsen et al., 1990; Zachariassen et al., 1990). The sublethal exposure concentration used for benzene (100 mg/l) is not far from the 96-hr ~q,, value (approx. 350 mg/l). Thus, the non-response of phosphate compounds in mussels exposed to benzene indicates that benzene affects other parameters than those directly concerned with the energy metabolism. Phosphoarginine seems to be the phosphate compound in M. edulis which is the most sensitive to environmental pollutants. A majority of the pollutants, which gave mortality at concentrations close to the sublethal exposures, also gave a reduction of the
phosphoarginine amounts after the sublethal exposures. The capacity to display effects of sublethal concentrations during short-time exposure makes phosphoarginine a candidate for being an “alarm parameter” in environmental monitoring (Zachariassen et al., 1991). In the majority of the affected groups, severe reductions in the amount of phosphoarginine were followed by a reduction in the amount of ATP. Thus, the phosphoarginine probably functions as an energy reservoir to maintain the amount of ATP during a high-energy turnover and/or when the ATP production is restricted. In this context phosphoarginine seems to act as a regulatory parameter whose purpose is to keep ATP amounts unaffected by the pollutants. The use of changes observed in the phosphorus compounds in mussels exposed to pollutants as an “alarm” parameter might be a powerful tool in environmental monitoring. The “energy charge” has been used as an index of the energy status of the organism by several authors. This index involves the molar fractions of ATP, ADP and AMP. An alternative index of the energy status is the phosphorylation potential, ([ATP])/([ADP] x [pi]), which is directly related to the free energy available from ATP. Both these indices involve the components ADP and AMP, which cannot be quantified by the NMR method. Thus, unless additional analyses on tissue extracts of the phosphorus compounds are performed, neither of these indices could possibly be used to apply the data from the NMR studies. However, the main changes in the NMR visible phosphorus compounds (reduced amounts of phosphoarginine and ATP and increased amounts of inorganic phosphorus) might provide information about the energy status of the organism. This may be expressed in a special “phosphorus index”, defined as the product of the ratios between the amounts of phosphoarginine and inorganic
Effects of pollutants on “P in M. edulis phosphate, and ATP and inorganic phosphate, (P,,,/P,) x (ATP/P,). Presented in terms of this index (Fig. 3), the results show significantly reduced values for those mussels exposed to pollutants which display a clinically correlated toxic effect and no changes in the value for pollutants which display no toxic effects. The index should, however, be thoroughly examined before being broadly used in environmental monitoring, especially in the light of the non-response on exposure to benzene. Mussels exposed to high concentrations of formaldehyde and mercury display marked effects on energy-requiring physiological processes, which were observed as a reduced ApNa+ in the posterior adductor muscle (Einarson et al., 1990; Zachariassen et al., 1991). The reduced levels of high-energy phosphorus compounds are likely to be the reason for the reduced adductor muscle of the &.,a+ in the posterior mussels. studies were financed by Fina Exploration Norway u.a.s. through the BECTOS Project (Biological Effects of Chemical Treatment of Oilspills at Sea) and by the Norwegian Research Council for Science and Technology (NTNF). Acknowledgement-These
REFERENCES
Addink A. D. F. and Veenhof P. R. (1975) Regulation of mitochondrial matrix enzymes in Mytitus edulis L. Proceedings of the 9th European Marine Biology Symposium (Edited by Barnes H.), pp. 109-119. University
Press, Aberdeen. Ansell A. N. (1974) In Annual Report of the Scottish Marine Biological Association. p. 29.
93
Beis I. and Newsholme E. A. (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Bi0chem.J. 152, 23-32. Einarson S.. Aunaas T.. Berseth J. F.. Nordtuz T.. Olsen A.. Skjsrvla ‘G. and Zachariassen K: E. (1990) Effects of organic and inorganic pollutants on electrochemical potential difference of sodium in blue mussels (Mytifus edulis). Abstr. 12th ESCPB Conference, Utrecht, The Netherlands, 1990. Gadian D. G., Radda G. K., Richards R. E. and Seeley P. J. (1979) 31P-NMR in living tissues: the road from a promising to an important tool in biology. In Biological Applications of Nuclear Magnetic Resonance (Edited by Shulman R. G.), pp. 463-535. Academic Press, New York. Higashi R. M., Fan T. W. and MacDonald J. M. (1989) Monitoring of metabolic responses of intact Haliotis (Abalones) under salinity stress by “P surface-probe localized NMR. J. exp. Zool. 249, 35&356. Olsen A., Aunaas T., Borseth J. R., Einarson S., Nordtug T., Skjierve G. and Zachariassen K. E. (1990) Chemically induced alterations of the free amino acid pool in the posterior adductor muscle of MytiZus edulis. Abstr. 12th ESCPB Conference, Utrecht, The Netherlands, 1990. Thillart G. van den, Waarde A. van, Muller H. J., Erkelens C. and Lugtenburg J. (1990) Determination of highenergy phosphate compounds in fish muscle: “P-NMR spectroscopy and enzymatic methods. Comp. Biochem. Physiol. MB, 789-795. Wijsman T. C. M. (1976) Adenosine phosphates and energy charge in different tissues of Mytilus edults L. under aerobic and anaerobic conditions. J. camp. Physiol. 107, 129-140.
Zachariassen K. E., Aunaas T., BeTseth J. F., Einarson S., Nordtug T., Olsen A. and Skjervo G. (1991) Physiological parameters in ecotoxicology. Camp. Biochem. Physiol. IOOC, 77.-79.
Vol. IOOC,No. l/2, pp.89-93,
0306-4492/91 $3.00+ 0.00 0 1991PergamonPressplc
1991
Printedin Great Britain
THE EFFECTS OF ORGANIC AND INORGANIC POLLUTANTS ON INTRACELLULAR PHOSPHATE COMPOUNDS IN BLUE MUSSELS (MYTKUS EDULIS) TOREAUNAAS,SIGRUNEINARSON,*TIMOTHYE. SouTHoNt and KARL ERIK ZACHARIASSEN* The Research Foundation at the College of Arts and Science, Department of Ecotoxicology, The University of Trondheim, N-7055 Dragvoll, Norway (Received 1 October 1990) Abstract-1. The effects of sublethal concentrations of organic and inorganic pollutants on intracellular energy-rich phosphates in blue mussels, Mytilus edulis,were investigated by in viuo )‘P-NMR. 2. Formaldehyde (30 and 10mg/l), phenol, pyridine, mercury and cadmium gave marked reductions in phosphoarginine and, in some cases, the ATP amounts. The reduction in high-energy phosphate was accompanied by an increase in inorganic phosphate in all groups. 3. A “phosphorus index”, the product of the ratios between phosphoarginine and inorganic phosphate, and ATP and inorganic phosphate, is suggested, which might serve as an early warning (“alarm”) parameter in environmental monitoring. 4. Diversity in the responses to different pollutants make phosphorus compounds in M. edulisalso an interesting element in a finger print parameter system designed to distinguish between pollutants in the marine environment.
expressed as 2.19 mmol/kg wet weight (Wijsman, 1976). Addink and Veenhof (1975) confirmed these results and showed total animal values of 2.2 mmol ATP/kg wet weight in M. edulis. Enzymatic methods for the determination of highenergy phosphate compounds in animal tissues have been widely used. However, unless certain methodological precautions are taken, tissue excision and extraction might induce a breakdown of high-energy phosphate compounds (Van den Thillart et al., 1990). “P-NMR spectroscopy, as a technique for studying energy metabolism, is non-destructive to the organism, and the spectra of phosphorus compounds can be repeatedly obtained from intact tissues of live animals. Indeed, the effects of osmotic and hypoxic stress have already been studied in a marine mollusc (Hufiotis) by “P-NMR (Higashi et al., 1989). Exact quantitation of metabolites by NMR requires rather careful experimental procedures, but the relative contributions of phosphoarginine, ATP, inorganic phosphate and phosphodiesters to the total phosphorus signal can be easily determined (Gadian et al., 1979; Van den Thillart et al., 1990). In this investigation, the effects of exposure to sublethal concentrations of different organic and inorganic pollutants on phosphorus compounds of Myths edulis are described. The levels of phosphoarginine, ATP and inorganic phosphate were measured in vivo using 31P-NMR spectroscopy.
INTRODUCTION Toxic effects of environmental pollutants are due to disturbances of normal physiological functions of the affected organisms. Physiological parameters may be separated (classified) into two different categories, the regulated and the regulatory parameters (Zachariassen et al., 1991). Regulatory parameters, such as metabolic rate and energy turnover, vary according to the requirements of the organism in order to maintain regulated physiological parameters at a constant level. Thus, these parameters may display wide variations during normal cell functioning. Toxic effects of environmental pollutants on physiological parameters will probably be seen on regulatory parameters before they are manifest in effects on the regulated parameters. Environmental pollutants might affect regulatory physiological parameters in different ways. The energy turnover might increase or decrease as other physiological processes are influenced by the pollutant. Changes in the energy turnover and mitochondrial ATP production may thus be detected as changes in the amount of highenergy phosphate compounds. Phosphoarginine is believed to be the major phosphagen in the tissues of blue mussels, Myths edulis, and other marine invertebrates (Beis and Newsholme, 1975). Ansell (1974) showed that the ATP content of various species of bivalves is closely correlated with tissue dry weight, being 0.56mg ATP/lOOmg dry tissue for Myths edulis. Assuming that the dry weight is 20% of the wet weight, this amount can be
MATERIALSAND
METHODS
Blue mussels, Mytifusedulis,were collected from the tidal zone in Trondheimstjorden, Norway. The mussels were transported in thermo-containers to the laboratory and acclimated in 34.5% running seawater at 10°C for 4-6 days prior to the experiments. The mussels were not fed during the acclimation and experimental period.
*Department of Zoology, The University of Trondheim, N-7055 Dragvoll, Norway. tSINTEF/UNIMED, The MR-Center, N-7034 Trondheim, Norway. 89
TORE AUNAAS et al.
90
After the acclimation period the mussels, ranging from 1.4 to 2.6 g tissue wet weight, were exposed for 96 hr to pure seawater (control) and sublethal concentrations of phenol (100 mg/l), formaldehyde (30, 10 and 1 mg/l), benzene (100 mg/l), pyridin (100 mg/l), mercury (0.52 mg/l), cadmium (1.84 mg/l) and zinc (2.40 mg/l). Heavy metals were added as dichloride salts to the seawater and the concentrations given refer to the metals in solution. The exposure of 5-8 individuals took place in aquaria at 10°C. The experiment also involved a flow exposure to benzene (100 mg/l) for 96 hr at 10°C. High sublethal exposure concentrations of the chemicals were established on the basis of 96-hr mortality experiments, carried out prior to the sublethal experiments. LC~~values of 96-hr exposures were approximately 450 mg/l phenol, 75 mg/l formaldehyde, 350 mg/l benzene, 450 mg/l pyridin, 1.5 mg/l mercury, 10 mg/l cadmium and 10 mg/l zinc. To keep the concentrations of the chemicals stable throughout the static exposures, the exposure media were exchanged once a day with newly-made solutions. Exposure solutions of pure seawater, and seawater with the different chemicals, were used as exposure media in the NMR
probe. NMR experiments were carried out on intact animals immersed in exposure solutions in a tube inside a 25mm NMR probe. The solutions were aerated from the top of the tube, and the temperature in the probe was controlled at 10°C during the experiments. The animals were kept in the tube for 2-2.5 hr. At the termination of the experiment, the mussels were removed from the tube and the tissue wet weights of total animals (without shells) were determined. “P-NMR spectra were obtained at 81 MHz on a Bruker MSL 200 spectrometer operated in the pulsed Fourier A
4
Fig. 1. In uivo “P-NMR spectra of M. edulis exposed for 96 hr at 10°C to (A) seawater (34.5%) and to (B) seawater (34.5%) with 0.52mg/l mercury. Peaks were identified by their chemical shifts in relation to standard spectra. The labelled resonances are those of (1) phosphomonoesters; (2) inorganic phosphate; (3) phosphodiesters; (4) phosphoarginine; (5) y-phosphate of ATP, (6) cc-phosphate of ATP and phosphate moiety of NAD; (7) /?-phosphate of ATP.
transform mode. For quantitation of the phosphorus compounds, fully relaxed spectra were accumulated with a recycle time of 20 set, a sweep width of 10,000 Hz and with a collection of 180 data acquisitions. Radiofrequency pulses of 90” (pulse width 301(sec) were employed in all experiments. Peak heights, corrected for the tissue wet weights, were used for the relative quantitation of the phosphorus compounds The P-ATP peak was used for quantitation of the amount of ATP. The metabolite levels were expressed as a percentage of the total phosphorus signal (phosphomonoesters + inorganic phosphate + phosphodiesters + phosphoarginine + ATP) as determined from the 3’P-NMR in viva spectra. Due to rather small sample sizes in the experimental groups, the underlying population from which the group of data are drawn, are assumed to be normally distributed. Student’s t-test was employed to test the significance of the data.
RESULTS In vivo “P-NMR spectra of Mytilus edulis exposed to 34.5o/ooseawater (control) and mercury
(0.52 mg/l) are presented in Fig. 1. The spectra show well-resolved resonances of phosphomonoesters, inorganic phosphate, phosphodiesters, phosphoarginine and the y-, GI-and b-phosphate atoms of ATP. In control mussels, the phosphoarginine peak height is typically 4-5 times greater than individual peaks from the other phosphorus compounds. In mussels exposed to mercury, the peak representing the inorganic phosphate is increased and dominates the spectrum. The height of the peak representing the phosphoarginine is markedly reduced by about the same magnitude as the increase in the peak height of the inorganic phosphate. Figure 2 shows the amount (%) of the total phosphorus compounds; of phosphoarginine, inorganic phosphate and ATP in M. edulis exposed to sublethal concentrations of different environmental pollutants. The values are corrected for the flesh wet weight of the mussels. Fully relaxed spectra from control mussels indicate that phosphoarginine normally makes up about 50% of the total NMR visible phosphorus pool in the mussels, while inorganic phosphate and ATP amount to 12 and 10% of the phosphorus pool, respectively. Significantly reduced amounts of phosphoarginine were registered in mussels exposed to formaldehyde (30 mg/l, P < 0.001 and 10 mg/l, P < O.OOl), phenol (lOOmg/l, P < O.OOl), pyridin (100 mg/l, P < O.OOl), mercury (0.52 mg/l, P < 0.001) and cadmium (1.84 mg/l, P < 0.001). Reduced amounts of phosphoarginine were, in all groups, followed by an increase in inorganic phosphate. Significantly reduced amounts of ATP (P < 0.001 and P < 0.01) were registered in groups of mussels exposed to 30 and 10 mg/l formaldehyde, phenol, cadmium and mercury (0.52mg/l). Although a marked (50%) reduction in the amount of phosphoarginine was registered in mussels exposed to pyridin, no significant reduction in the amount of ATP was registered in these animals. In contrast, cadmium, which reduced the phosphoarginine amounts by about 20%, caused a marked reduction (30%) in the amount of ATP.
Effects of pollutants on “F in M. edulis
91
60 60 40 30 20 10 0 60
50
40
30
20 10
0
t6 * 8
,,IT 14
4
C
T _
Fig. 2. Changes in levels of (A) phosphoarginine, (B) inorganic phosphate and (C) ATP in edulis exposed to different environmental pollutants for 96 hr at 10°C. The metabolite levels were obtained by measuring peak heights from the spectra, and are expressed as a percentage of the total phosphorus signal (phosphomon~sters f inorganic phosphate + phosphodiesters + phosphoar~nine + ATP) hr 3’P-NMR in U&O spectra. Values are presented as mean + SD. The number of individuals in each group is given above the bars. *P < 0.01 and **P < 0.001 with respect to the control. M.
Exposure to benzene (100 mg/l), both static and in a flow-system, did not seem to affect the amounts of individua1 phosphorus compounds in the mussels.
CBP1lmc,1-~--0
This also seemed to be the situation for 96hr exposure to zinc (2.4 mgjl) and to the low concentration (1 mg/l) of formaldehyde tested.
TOREAUNM et al.
92
Fig. 3. Changes in the “phosphorus index”, expressed as the product of the ratios between phosphoarginine and inorganic phosphate, and ATP and inorganic phosphate, following 96-hr exposure to sublethal concentrations of different environmental pollutants at 10°C. Values are presented as mean f SD. The number of individuals in each group is given above the bars, lP < 0.01 and **P< 0.001 with respect to the control.
DISCUSSION
Seawater containing formaldehyde (30 and 10 mg/l), phenol (100 mg/l), pyridin (100 mg/l), mercury (OS2mg/l) and cadmium (1.84mg/l) is demonstrated to strongly affect phosphorus compounds in Myths edulis. The effects on the phosphorus compounds in mussels exposed to these chemicals were correlated to observed clinical effects,
such as reduced byssal thread formation (formaldehyde, phenol, heavy metals), shedding of byssal threads (phenol, pyridin) and, in sequences of the exposure time, abnormal shell closure (formaldehyde, pyridin, heavy metals) or shell opening (phenol). Exposure to benzene (100 mg/l), formaldehyde (1 mg/l) and zinc (2.4 mg/l) does not seem to affect the phosphorus compounds in the mussels to any degree. Alterations in behaviour and appearance of mussels exposed to this concentration of formaldehyde and to zinc were not observed. This indicates that the concentrations used were non-toxic to the mussels at acute exposure. This assumption is supported by the fact that no effects were found on oxygen consumption rates, transmembrane distribution of inorganic ions or concentrations of individual free amino acids in these mussels (Olsen et al., 1990; Zachariassen et al., 1990). The sublethal exposure concentration used for benzene (100 mg/l) is not far from the 96-hr ~q,, value (approx. 350 mg/l). Thus, the non-response of phosphate compounds in mussels exposed to benzene indicates that benzene affects other parameters than those directly concerned with the energy metabolism. Phosphoarginine seems to be the phosphate compound in M. edulis which is the most sensitive to environmental pollutants. A majority of the pollutants, which gave mortality at concentrations close to the sublethal exposures, also gave a reduction of the
phosphoarginine amounts after the sublethal exposures. The capacity to display effects of sublethal concentrations during short-time exposure makes phosphoarginine a candidate for being an “alarm parameter” in environmental monitoring (Zachariassen et al., 1991). In the majority of the affected groups, severe reductions in the amount of phosphoarginine were followed by a reduction in the amount of ATP. Thus, the phosphoarginine probably functions as an energy reservoir to maintain the amount of ATP during a high-energy turnover and/or when the ATP production is restricted. In this context phosphoarginine seems to act as a regulatory parameter whose purpose is to keep ATP amounts unaffected by the pollutants. The use of changes observed in the phosphorus compounds in mussels exposed to pollutants as an “alarm” parameter might be a powerful tool in environmental monitoring. The “energy charge” has been used as an index of the energy status of the organism by several authors. This index involves the molar fractions of ATP, ADP and AMP. An alternative index of the energy status is the phosphorylation potential, ([ATP])/([ADP] x [pi]), which is directly related to the free energy available from ATP. Both these indices involve the components ADP and AMP, which cannot be quantified by the NMR method. Thus, unless additional analyses on tissue extracts of the phosphorus compounds are performed, neither of these indices could possibly be used to apply the data from the NMR studies. However, the main changes in the NMR visible phosphorus compounds (reduced amounts of phosphoarginine and ATP and increased amounts of inorganic phosphorus) might provide information about the energy status of the organism. This may be expressed in a special “phosphorus index”, defined as the product of the ratios between the amounts of phosphoarginine and inorganic
Effects of pollutants on “P in M. edulis phosphate, and ATP and inorganic phosphate, (P,,,/P,) x (ATP/P,). Presented in terms of this index (Fig. 3), the results show significantly reduced values for those mussels exposed to pollutants which display a clinically correlated toxic effect and no changes in the value for pollutants which display no toxic effects. The index should, however, be thoroughly examined before being broadly used in environmental monitoring, especially in the light of the non-response on exposure to benzene. Mussels exposed to high concentrations of formaldehyde and mercury display marked effects on energy-requiring physiological processes, which were observed as a reduced ApNa+ in the posterior adductor muscle (Einarson et al., 1990; Zachariassen et al., 1991). The reduced levels of high-energy phosphorus compounds are likely to be the reason for the reduced adductor muscle of the &.,a+ in the posterior mussels. studies were financed by Fina Exploration Norway u.a.s. through the BECTOS Project (Biological Effects of Chemical Treatment of Oilspills at Sea) and by the Norwegian Research Council for Science and Technology (NTNF). Acknowledgement-These
REFERENCES
Addink A. D. F. and Veenhof P. R. (1975) Regulation of mitochondrial matrix enzymes in Mytitus edulis L. Proceedings of the 9th European Marine Biology Symposium (Edited by Barnes H.), pp. 109-119. University
Press, Aberdeen. Ansell A. N. (1974) In Annual Report of the Scottish Marine Biological Association. p. 29.
93
Beis I. and Newsholme E. A. (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Bi0chem.J. 152, 23-32. Einarson S.. Aunaas T.. Berseth J. F.. Nordtuz T.. Olsen A.. Skjsrvla ‘G. and Zachariassen K: E. (1990) Effects of organic and inorganic pollutants on electrochemical potential difference of sodium in blue mussels (Mytifus edulis). Abstr. 12th ESCPB Conference, Utrecht, The Netherlands, 1990. Gadian D. G., Radda G. K., Richards R. E. and Seeley P. J. (1979) 31P-NMR in living tissues: the road from a promising to an important tool in biology. In Biological Applications of Nuclear Magnetic Resonance (Edited by Shulman R. G.), pp. 463-535. Academic Press, New York. Higashi R. M., Fan T. W. and MacDonald J. M. (1989) Monitoring of metabolic responses of intact Haliotis (Abalones) under salinity stress by “P surface-probe localized NMR. J. exp. Zool. 249, 35&356. Olsen A., Aunaas T., Borseth J. R., Einarson S., Nordtug T., Skjierve G. and Zachariassen K. E. (1990) Chemically induced alterations of the free amino acid pool in the posterior adductor muscle of MytiZus edulis. Abstr. 12th ESCPB Conference, Utrecht, The Netherlands, 1990. Thillart G. van den, Waarde A. van, Muller H. J., Erkelens C. and Lugtenburg J. (1990) Determination of highenergy phosphate compounds in fish muscle: “P-NMR spectroscopy and enzymatic methods. Comp. Biochem. Physiol. MB, 789-795. Wijsman T. C. M. (1976) Adenosine phosphates and energy charge in different tissues of Mytilus edults L. under aerobic and anaerobic conditions. J. camp. Physiol. 107, 129-140.
Zachariassen K. E., Aunaas T., BeTseth J. F., Einarson S., Nordtug T., Olsen A. and Skjervo G. (1991) Physiological parameters in ecotoxicology. Camp. Biochem. Physiol. IOOC, 77.-79.
