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The influence of bulk and trace metals on the circadian rhythm of heart rates in freshwater crayfish, Astacus astacus
Pergamon
0025-326X(95)00029-1
Marine Pollution Bulletitt, Vol. 31, Nos I 3, pp. 87 92, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved (}025 326X/95 $9.50+(I.00
The Influence of Bulk and Trace Metals on the Circadian Rhythm of Heart Rates in Freshwater Crayfish, Astacus astacus B. STYRISHAVE*:~, A. D. RASMUSSEN* and M. H. D E P L E D G E t
*Ecotoxicology Group, Institute of Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark t Marine Biology & Ecotoxicology Research Group, Department of Biological Sciences, University of Plymouth, Drake Circus, PL 4 8AA, UK ~:Author to w h o m c o r r e s p o n d e n c e
should be addressed.
The freshwater crayfish A s t a c u s astacus is a nocturnal species known to express circadian rhythmicity in heart rate. With the aid of a Computer Aided Physiological MONitoring (CAPMON) system, the influence of potential hazards, such as trace metals, on the expression of circadian rhythmicity in heart rate was investigated. Effects of variations in salinity on circadian rhythmicity in heart rate were also investigated, since heart rates are known to be affected by such changes in some estuarine crustaceans. The influence of Hg 2+, Cu 2+ and NaCl on light driven (12:12 h, light:dark regime) circadian rhythmicity in heart rate was examined. Exposure to 0.1 mg Hg !-1 invariably increased heart rate by increasing heart rate during day time. This eventually resulted first in loss of rhythmicity and ultimately in death. The response to 8.0 mg Cu !-I seemed more complex with great inter-individual variation, involving increases as well as decreases in both day and night time heart rates. An increase in salinity from 0.09 mM NaC! to 24.0 mM NaCl seemed to decrease the expression of circadian rhythmicity in heart rate, primarily because heart rates remained low during the night. From the present experiments it appears that the presence of trace and bulk metals in the surrounding media influences the expression of circadian rhythmicity of heart rates in the crayfish A.
metal concentrations and salinity, on circadian rhythm expression. A study on the influence of mercury (Depledge, 1984) and one study on the influence of copper (Aagaard, 1991) on tidal rhythmicity of heart rate in the shore crab, Carcinus maenas, have been performed. In both cases, rhythms were shown to be perturbed. No such experiments have apparently been conducted for any freshwater species. In the case of salinity, Taylor (1977) found that for C. maenas, decreasing salinity increased heart rate in experiments performed during the day time, but there have been no similar investigations examining the influence of changes in salinity on the expression of circadian rhythmicity in heart rate. In some freshwater habitats close to mining activities salinity may be raised above normal freshwater levels and elevated trace metal concentrations may also be present. At present, very few studies have been conducted to examine the effects of such anthropogenic effects on freshwater fauna. In this study, the influences of Hg, Cu and salinity changes on the expression of circadian rhythmicity in heart rate of the freshwater crayfish, Astacus astacus, have been investigated.
astacus.
Crayfish were collected from a crayfish farm on the island of Funen, Denmark, and transported to Odense University. They were subsequently maintained in a constant temperature room (15°C) in a 12:12 h light:dark regime. The animals were fed fish pellets and carrots ad libitum. Six to ten days prior to experimentation, the animals were moved to individual 10 1 plastic containers with mixed freshwater (ASTM, 1980), and holders for infrared sensors were attached. Two to four days prior to experimentation, the sensors were placed in the holders (Depledge & Andersen, 1990). Circadian rhythmicity in heart rates were monitored by the use of a Computer Aided Physiological MONitoring (CAPMON) system (Depledge &
It is well established that freshwater crayfish express circadian rhythmicity in locomotor activity, respiration and heart rate (Fingerman & Lago, 1957; Page & Latimer, 1975; Pollard & Larimer, 1977) in association with general nocturnal behaviour. Expression of circadian rhythms is an integral part of behavioural adaptation to cyclical changes in the environment (Cukerzis, 1988). Few studies, however, have been concerned with the influence of environmental stress factors, such as trace
M a t e r i a l s and M e t h o d s
87
Marine Pollution Bulletin
Andersen, 1990). With this system, individual heart rates can be monitored continuously in four (or a multiple of four) individuals simultaneously. The data are continuously logged onto a personal computer as beats per minute. In experiments with Hg and Cu, heart rates were monitored 3 days before the addition of metals. Control experiments were carried out with four individuals, recording heart rates continuously for 14 days in the conditions described earner but without exposure to metals or salinity changes. Other groups of four individuals were exposed individually to either 0.1 mg Hg 1-1 as HgCI2, or 8.0 mg Cu 1-t as CuCI2, for 19 days or until death ocurred. Another four individuals were exposed to increasing salinities: 0.09, 0.72, 6.0, and 24.0 mM NaC1 (i.e. maximum 1.40 g 1-1 NaC1) for a total of 24 days, i.e. 6 days at each salinity. The salinity was increased by addition of NaC1 to a reservoir chamber from which the experimental water was recycled. Care was taken not to disturb the test animals when metals and NaCI were added. All animals were starved during the experiments. The method of calculating d-values described by Atkinson & Parsons (1969) was used to analyse for progressive changes in circadian rhythmicity in metalexposed animals. The d-value describes the difference between average night heart rate and the average day heart rate corrected for standard deviations. One dvalue was calculated for each individual each day, before and during metal exposure. This method allowed determination of changes in circadian heart rate rhythms continuously over time for each individual. A d-value of 0 indicates no difference between night and day heart rates. Values above 1.96 indicate that heart rates at night were significantly higher (5% confidence level) than heart rates during the day, whereas d-values below -1.96 indicate significantly higher day time heart rates. The data obtained in the salinity experiments were analysed for changes in d-values and in the periodicity of the circadian heart rate rhythm using periodogram analysis, as described by Enright (1965), Vandenbussche (1969), and Williams & Naylor (1978). The periodogram is a further development of the 'BoysBallot' form-estimate technique, which plots the standard deviation (S) for each form-estimate for the same time series against increasing length of period. The value of the periodicity in the original data is indicated by the highest standard deviation (S) for the form-estimates. Both methods were used to analyse heart rate data for control experiments.
than heart rates during day time (d-values > 0) with a periodicity of 24 h. When exposed to 0.1 mg Hg 1-1, the general response appeared to be an increase in day time heart rates to values characteristic of night time. This was reflected in loss of rhythmicity and eventually death. Results for four individuals are shown in Fig. 3. The calculation of
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Fig. I Average heart rates of four individuals of the crayfish, A. astacus, recorded during 14 days with no food available. Black boxes indicate dark periods.
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Crayfish maintained in a 12:12 h light:dark regime for 14 days typically expressed circadian rhythmicity with elevated heart rates during the night compared to the day (Fig. 1). Periodogram and d-value analysis from this experiment are shown in Fig. 2. The methods clearly show heart rates during the night to be higher 88
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d-values shown in Fig. 4 illustrates loss of circadian rhythmicity in heart rate, as d-values decrease prior to death to levels close to or even below 0 after addition of mercury. The response in heart rate following exposure to 8.0 mg Cu 1-1 was more complex with great inter-individual variation, resulting in both increased day time heart rates and decreased night time heart rates. Rhythmicity was sometimes maintained until death occurred (Fig. 5). This is further illustrated in Fig. 6, where d-values after copper exposure are concentrated around 0 and for
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some individuals found to be above 1.96 prior to death, indicating that heart rate at night was significantly higher than heart rate during the day. One individual survived 19 days of exposure to 8.0 mg Cu 1-1 after which the experiment was curtailed. In Fig. 6 it can be seen that this individual had a significant circadian rhythmicity in heart rate at the end of the experiment. Average heart rates of four individuals during increased salinity exposure (from 0.09 to 24.0 mM) are illustrated in Fig. 7. This increase in salinity was associated with a fall in night time heart rates and thereby a decrease in the expression of rhythmicity in heart rate. A slight reduction in day time heart rates was also observed. The periodograms in Fig. 8 show that the 24 h periodicity gradually disappeared with increasing salinity, the 24 h rhythm only being significant at 0.09 mM NaCI. Furthermore, although decreases in d-values were observed during the 24 days of increasing salinities, these decreases were not significant.
Changes in d-values over time for the four individuals shown in Fig. 3. Vertical line indicates addition of 0.1 mg H g 1-1. Each symbol represents a different individual, Dotted lines indicate 95% confidence limits.
From these experiments it is evident that crayfish express light associated circadian rhythmicity in their heart rate (Figs 1 and 2). This rhythmicity persists for at least 14 days in the laboratory and starvation caused no apparent changes in this pattern. Unpublished data (Styrishave) show that A. astacus can maintain a high degree of circadian rhythmicity in heart rate (d-values > 1.96) for at least 21 days with no food available. Consequently, this suggests that the effects on heart rate 89
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similar to those of night heart rates. Consequently, rhythmicity was lost, resulting in d-values approaching 0 (Fig. 4). This type of response has not been observed previously in freshwater species, but for C. maenas it has been found that exposure to 0.05 mg Hg 1-~ resulted in loss of endogenous tidal rhythmicity in heart rate (Depledge, 1984). The rhythm in C. maenas was lost in a similar manner by a general increase in heart rate, primarily at low tide. It is evident from Fig. 5 that the response to 8.0 mg Cu 1-1 was less consistent than was the case with mercury, resulting in four different types of responses. These involve changes in both night and day heart rates. In contrast to the case of mercury exposure, circadian rhythmicity in heart rate is not necessarily lost prior to death when the crayfish are exposed to copper. Exposing C. maenas to 1.0 mg Cu 1-I, Aagaard (1991) found a consistent increase in heart rates and a loss of tidal rhythm. This is. similar to the loss of circadian rhythm observed for mercury in the present study, but is in contrast to the response observed for copper in A. astacus. It is possible that this more variable response to copper reflects the fact that copper is an essential metal, unlike mercury (Depledge & Rainbow, 1990). The physiological processes involved in uptake, handling and excretion of copper may therefore be more complex. When exposed to 0.09 mM NaC1, the crayfish expressed circadian rhythmicity in heart rate. To some extent (but not significantly), this was also the case for animals exposed to 0.72 mM NaCI. When the salinity was
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Fig. 8 Periodogram analysis of the average heart rates of the four individuals shown in Fig. 7. A: 0.09 mM NaCl; B: 0.72 mM NaCl; C: 6.0 mM NaCl; D: 24.0 mM NaCl. Symbols as in Fig. 2. 91
Marine Pollution Bulletin
raised to 6 or 24 mM NaCI, well above the salinities normally encountered by crayfish, circadian rhythmicity decreased as a result of a general lowering of night heart rates (Figs 7 and 8). In the study by Taylor (1977), C. rnaenas was shown to increase its day heart rates with decreasing salinity (from 34 to 12%o). Even though the present experiment was conducted with increasing salinity, the data presented here are generally in agreement with experiments involving C. maenas. It must be mentioned, however, that the main changes in heart rate in the present study were associated with changes in heart rate at night, whereas Taylor (1977) only measured day rates. A decrease in heart rate at night during exposure to increasing salinity has also been observed in C. maenas using the CAPMON system (Rasmussen, unpublished data). The expression of nocturnal behaviour (and circadian rhythmicity in related parameters such as heart rate, oxygen consumption, etc.) is considered to be of high adaptive value to crayfish in natural habitats, for example by minimizing predation from animals which hunt by sight (Cukerzis, 1988). High trace metal concentrations and salinity changes seem to have the potential to influence the expression of circadian rhythmicity in heart rate and probably nocturnal behaviour. This again may influence the Darwinian fitness of crayfish in habitats exposed to such external perturbations, for example in coal mining areas or in other heavily industrialized areas where the anthropogenic output of trace metals, as well as NaC1, may be high. We are grateful to Jorn F. Andersen and Rorkjaer Krebsebrug for supply of crayfish, and to Alf Aagaard for assistance on periodogram analysis. Aagaard, A. (1991). Undersogelse af Adfserdsmaessige og Fysiologiske Rytmer hos Carcinus maenas Eksponeret til Kobber ved brug af Computerteknik. Master Thesis. Institute of Biology, Odense University, Denmark.
92
Aldrich, J. C. (1975). Individual variability in oxygen consumption rates of fed and starved Cancer pagurus and Maia squinado. Comp. Biochem. Physiol. 51A, 175-183. Ansell, D. A. (1973). Changes in oxygen consumption, heart rate and ventilation accompanying starvation in the decapod crustacean Cancer pagurus. Neth. J. Sea Res. 6,455-475. ASTM Method (American Society for Testing and Materials) (1980). Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians. In Annual Book of ASTM Standards, Vol. 11.4, pp. 285-309. Philadelphia, USA. Alkinson, R. J. A. & Parsons, A. J. (1969). Seasonal patterns of migration and locomotor rhythmicity in populations of Carcinus. 7th European Symposium on Marine Biology. Neth. J. Sea Res. 7, 81-93. Cukerzis, J. M. (1988). Astacus astacus in Europe. In Freshwater Crayfish: Biology, Management and Exploitation (D. M. Holdich & R. S. Lowery, eds), pp. 309-340. Cambridge University Press, UK. Depledge, M. H. (1984). Disruption of endogenous rhythms in Carcinus maenas (L.) following exposure to mercury pollution. Comp. Biochem. Physiol. 78A, 375-379. Depledge, M. H. & Andersen, B. B. (1990). A computer-aided physiological monitoring system for continuous, long term recording of cardiac activity in selected invertebrates. Comp. Biochem. Physiol. 96A, 473-477. Depledge, M. H. & Rainbow, E S. (1990), Models of regulation and accumulation of trace metals in marine invertebrates. Comp. Biochem. Physiol. 97C, 1-7. Entight, J. T. (1965). The search for rhythmicity in biological timeseries. J. Theor. Biol. 8,426-468. Fingerman, M. & Lago, D. A. (1957). Endogenous twenty-four hour rhythm of locomotor activity and oxygen consumption in the freshwater crayfish Orconectes clypeatus. American Midland Naturalist 58, 383-393. Page, T. L. & Latimer, J. L. (1975). Neural control of circadian rhythmicity in the crayfish. I. The locomotor activity rhythm. J. Comp. Physiol. 97, 59-80. Pollard, T. G. & Latimer, J. L. (1977). Circadian rhythmicity of heart rate in the crayfish Procambarus clarkii. Comp. Biochem. Physiol. 57A, 221-226. Taylor, A. C. (1977). The respiratory responses of Carcinus maenas (L.) to changes in environmental salinity. J. Exp. Mar. Biol. Ecol. 29, 197-210. Vandenbussche, E. (1969). The detection of periodicities in time series. I. Calculation of a periodogram and of a correlogram. Psychol. Belg. IX-I, 59-77. Williams, J. A. & Naylor, E. (1978). A procedure for the assessment of significance of rhythmicity in time-series data. Int. J. Chronobiol. 5, 435-444.
0025-326X(95)00029-1
Marine Pollution Bulletitt, Vol. 31, Nos I 3, pp. 87 92, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved (}025 326X/95 $9.50+(I.00
The Influence of Bulk and Trace Metals on the Circadian Rhythm of Heart Rates in Freshwater Crayfish, Astacus astacus B. STYRISHAVE*:~, A. D. RASMUSSEN* and M. H. D E P L E D G E t
*Ecotoxicology Group, Institute of Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark t Marine Biology & Ecotoxicology Research Group, Department of Biological Sciences, University of Plymouth, Drake Circus, PL 4 8AA, UK ~:Author to w h o m c o r r e s p o n d e n c e
should be addressed.
The freshwater crayfish A s t a c u s astacus is a nocturnal species known to express circadian rhythmicity in heart rate. With the aid of a Computer Aided Physiological MONitoring (CAPMON) system, the influence of potential hazards, such as trace metals, on the expression of circadian rhythmicity in heart rate was investigated. Effects of variations in salinity on circadian rhythmicity in heart rate were also investigated, since heart rates are known to be affected by such changes in some estuarine crustaceans. The influence of Hg 2+, Cu 2+ and NaCl on light driven (12:12 h, light:dark regime) circadian rhythmicity in heart rate was examined. Exposure to 0.1 mg Hg !-1 invariably increased heart rate by increasing heart rate during day time. This eventually resulted first in loss of rhythmicity and ultimately in death. The response to 8.0 mg Cu !-I seemed more complex with great inter-individual variation, involving increases as well as decreases in both day and night time heart rates. An increase in salinity from 0.09 mM NaC! to 24.0 mM NaCl seemed to decrease the expression of circadian rhythmicity in heart rate, primarily because heart rates remained low during the night. From the present experiments it appears that the presence of trace and bulk metals in the surrounding media influences the expression of circadian rhythmicity of heart rates in the crayfish A.
metal concentrations and salinity, on circadian rhythm expression. A study on the influence of mercury (Depledge, 1984) and one study on the influence of copper (Aagaard, 1991) on tidal rhythmicity of heart rate in the shore crab, Carcinus maenas, have been performed. In both cases, rhythms were shown to be perturbed. No such experiments have apparently been conducted for any freshwater species. In the case of salinity, Taylor (1977) found that for C. maenas, decreasing salinity increased heart rate in experiments performed during the day time, but there have been no similar investigations examining the influence of changes in salinity on the expression of circadian rhythmicity in heart rate. In some freshwater habitats close to mining activities salinity may be raised above normal freshwater levels and elevated trace metal concentrations may also be present. At present, very few studies have been conducted to examine the effects of such anthropogenic effects on freshwater fauna. In this study, the influences of Hg, Cu and salinity changes on the expression of circadian rhythmicity in heart rate of the freshwater crayfish, Astacus astacus, have been investigated.
astacus.
Crayfish were collected from a crayfish farm on the island of Funen, Denmark, and transported to Odense University. They were subsequently maintained in a constant temperature room (15°C) in a 12:12 h light:dark regime. The animals were fed fish pellets and carrots ad libitum. Six to ten days prior to experimentation, the animals were moved to individual 10 1 plastic containers with mixed freshwater (ASTM, 1980), and holders for infrared sensors were attached. Two to four days prior to experimentation, the sensors were placed in the holders (Depledge & Andersen, 1990). Circadian rhythmicity in heart rates were monitored by the use of a Computer Aided Physiological MONitoring (CAPMON) system (Depledge &
It is well established that freshwater crayfish express circadian rhythmicity in locomotor activity, respiration and heart rate (Fingerman & Lago, 1957; Page & Latimer, 1975; Pollard & Larimer, 1977) in association with general nocturnal behaviour. Expression of circadian rhythms is an integral part of behavioural adaptation to cyclical changes in the environment (Cukerzis, 1988). Few studies, however, have been concerned with the influence of environmental stress factors, such as trace
M a t e r i a l s and M e t h o d s
87
Marine Pollution Bulletin
Andersen, 1990). With this system, individual heart rates can be monitored continuously in four (or a multiple of four) individuals simultaneously. The data are continuously logged onto a personal computer as beats per minute. In experiments with Hg and Cu, heart rates were monitored 3 days before the addition of metals. Control experiments were carried out with four individuals, recording heart rates continuously for 14 days in the conditions described earner but without exposure to metals or salinity changes. Other groups of four individuals were exposed individually to either 0.1 mg Hg 1-1 as HgCI2, or 8.0 mg Cu 1-t as CuCI2, for 19 days or until death ocurred. Another four individuals were exposed to increasing salinities: 0.09, 0.72, 6.0, and 24.0 mM NaC1 (i.e. maximum 1.40 g 1-1 NaC1) for a total of 24 days, i.e. 6 days at each salinity. The salinity was increased by addition of NaC1 to a reservoir chamber from which the experimental water was recycled. Care was taken not to disturb the test animals when metals and NaCI were added. All animals were starved during the experiments. The method of calculating d-values described by Atkinson & Parsons (1969) was used to analyse for progressive changes in circadian rhythmicity in metalexposed animals. The d-value describes the difference between average night heart rate and the average day heart rate corrected for standard deviations. One dvalue was calculated for each individual each day, before and during metal exposure. This method allowed determination of changes in circadian heart rate rhythms continuously over time for each individual. A d-value of 0 indicates no difference between night and day heart rates. Values above 1.96 indicate that heart rates at night were significantly higher (5% confidence level) than heart rates during the day, whereas d-values below -1.96 indicate significantly higher day time heart rates. The data obtained in the salinity experiments were analysed for changes in d-values and in the periodicity of the circadian heart rate rhythm using periodogram analysis, as described by Enright (1965), Vandenbussche (1969), and Williams & Naylor (1978). The periodogram is a further development of the 'BoysBallot' form-estimate technique, which plots the standard deviation (S) for each form-estimate for the same time series against increasing length of period. The value of the periodicity in the original data is indicated by the highest standard deviation (S) for the form-estimates. Both methods were used to analyse heart rate data for control experiments.
than heart rates during day time (d-values > 0) with a periodicity of 24 h. When exposed to 0.1 mg Hg 1-1, the general response appeared to be an increase in day time heart rates to values characteristic of night time. This was reflected in loss of rhythmicity and eventually death. Results for four individuals are shown in Fig. 3. The calculation of
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Crayfish maintained in a 12:12 h light:dark regime for 14 days typically expressed circadian rhythmicity with elevated heart rates during the night compared to the day (Fig. 1). Periodogram and d-value analysis from this experiment are shown in Fig. 2. The methods clearly show heart rates during the night to be higher 88
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d-values shown in Fig. 4 illustrates loss of circadian rhythmicity in heart rate, as d-values decrease prior to death to levels close to or even below 0 after addition of mercury. The response in heart rate following exposure to 8.0 mg Cu 1-1 was more complex with great inter-individual variation, resulting in both increased day time heart rates and decreased night time heart rates. Rhythmicity was sometimes maintained until death occurred (Fig. 5). This is further illustrated in Fig. 6, where d-values after copper exposure are concentrated around 0 and for
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some individuals found to be above 1.96 prior to death, indicating that heart rate at night was significantly higher than heart rate during the day. One individual survived 19 days of exposure to 8.0 mg Cu 1-1 after which the experiment was curtailed. In Fig. 6 it can be seen that this individual had a significant circadian rhythmicity in heart rate at the end of the experiment. Average heart rates of four individuals during increased salinity exposure (from 0.09 to 24.0 mM) are illustrated in Fig. 7. This increase in salinity was associated with a fall in night time heart rates and thereby a decrease in the expression of rhythmicity in heart rate. A slight reduction in day time heart rates was also observed. The periodograms in Fig. 8 show that the 24 h periodicity gradually disappeared with increasing salinity, the 24 h rhythm only being significant at 0.09 mM NaCI. Furthermore, although decreases in d-values were observed during the 24 days of increasing salinities, these decreases were not significant.
Changes in d-values over time for the four individuals shown in Fig. 3. Vertical line indicates addition of 0.1 mg H g 1-1. Each symbol represents a different individual, Dotted lines indicate 95% confidence limits.
From these experiments it is evident that crayfish express light associated circadian rhythmicity in their heart rate (Figs 1 and 2). This rhythmicity persists for at least 14 days in the laboratory and starvation caused no apparent changes in this pattern. Unpublished data (Styrishave) show that A. astacus can maintain a high degree of circadian rhythmicity in heart rate (d-values > 1.96) for at least 21 days with no food available. Consequently, this suggests that the effects on heart rate 89
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similar to those of night heart rates. Consequently, rhythmicity was lost, resulting in d-values approaching 0 (Fig. 4). This type of response has not been observed previously in freshwater species, but for C. maenas it has been found that exposure to 0.05 mg Hg 1-~ resulted in loss of endogenous tidal rhythmicity in heart rate (Depledge, 1984). The rhythm in C. maenas was lost in a similar manner by a general increase in heart rate, primarily at low tide. It is evident from Fig. 5 that the response to 8.0 mg Cu 1-1 was less consistent than was the case with mercury, resulting in four different types of responses. These involve changes in both night and day heart rates. In contrast to the case of mercury exposure, circadian rhythmicity in heart rate is not necessarily lost prior to death when the crayfish are exposed to copper. Exposing C. maenas to 1.0 mg Cu 1-I, Aagaard (1991) found a consistent increase in heart rates and a loss of tidal rhythm. This is. similar to the loss of circadian rhythm observed for mercury in the present study, but is in contrast to the response observed for copper in A. astacus. It is possible that this more variable response to copper reflects the fact that copper is an essential metal, unlike mercury (Depledge & Rainbow, 1990). The physiological processes involved in uptake, handling and excretion of copper may therefore be more complex. When exposed to 0.09 mM NaC1, the crayfish expressed circadian rhythmicity in heart rate. To some extent (but not significantly), this was also the case for animals exposed to 0.72 mM NaCI. When the salinity was
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Marine Pollution Bulletin
raised to 6 or 24 mM NaCI, well above the salinities normally encountered by crayfish, circadian rhythmicity decreased as a result of a general lowering of night heart rates (Figs 7 and 8). In the study by Taylor (1977), C. rnaenas was shown to increase its day heart rates with decreasing salinity (from 34 to 12%o). Even though the present experiment was conducted with increasing salinity, the data presented here are generally in agreement with experiments involving C. maenas. It must be mentioned, however, that the main changes in heart rate in the present study were associated with changes in heart rate at night, whereas Taylor (1977) only measured day rates. A decrease in heart rate at night during exposure to increasing salinity has also been observed in C. maenas using the CAPMON system (Rasmussen, unpublished data). The expression of nocturnal behaviour (and circadian rhythmicity in related parameters such as heart rate, oxygen consumption, etc.) is considered to be of high adaptive value to crayfish in natural habitats, for example by minimizing predation from animals which hunt by sight (Cukerzis, 1988). High trace metal concentrations and salinity changes seem to have the potential to influence the expression of circadian rhythmicity in heart rate and probably nocturnal behaviour. This again may influence the Darwinian fitness of crayfish in habitats exposed to such external perturbations, for example in coal mining areas or in other heavily industrialized areas where the anthropogenic output of trace metals, as well as NaC1, may be high. We are grateful to Jorn F. Andersen and Rorkjaer Krebsebrug for supply of crayfish, and to Alf Aagaard for assistance on periodogram analysis. Aagaard, A. (1991). Undersogelse af Adfserdsmaessige og Fysiologiske Rytmer hos Carcinus maenas Eksponeret til Kobber ved brug af Computerteknik. Master Thesis. Institute of Biology, Odense University, Denmark.
92
Aldrich, J. C. (1975). Individual variability in oxygen consumption rates of fed and starved Cancer pagurus and Maia squinado. Comp. Biochem. Physiol. 51A, 175-183. Ansell, D. A. (1973). Changes in oxygen consumption, heart rate and ventilation accompanying starvation in the decapod crustacean Cancer pagurus. Neth. J. Sea Res. 6,455-475. ASTM Method (American Society for Testing and Materials) (1980). Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians. In Annual Book of ASTM Standards, Vol. 11.4, pp. 285-309. Philadelphia, USA. Alkinson, R. J. A. & Parsons, A. J. (1969). Seasonal patterns of migration and locomotor rhythmicity in populations of Carcinus. 7th European Symposium on Marine Biology. Neth. J. Sea Res. 7, 81-93. Cukerzis, J. M. (1988). Astacus astacus in Europe. In Freshwater Crayfish: Biology, Management and Exploitation (D. M. Holdich & R. S. Lowery, eds), pp. 309-340. Cambridge University Press, UK. Depledge, M. H. (1984). Disruption of endogenous rhythms in Carcinus maenas (L.) following exposure to mercury pollution. Comp. Biochem. Physiol. 78A, 375-379. Depledge, M. H. & Andersen, B. B. (1990). A computer-aided physiological monitoring system for continuous, long term recording of cardiac activity in selected invertebrates. Comp. Biochem. Physiol. 96A, 473-477. Depledge, M. H. & Rainbow, E S. (1990), Models of regulation and accumulation of trace metals in marine invertebrates. Comp. Biochem. Physiol. 97C, 1-7. Entight, J. T. (1965). The search for rhythmicity in biological timeseries. J. Theor. Biol. 8,426-468. Fingerman, M. & Lago, D. A. (1957). Endogenous twenty-four hour rhythm of locomotor activity and oxygen consumption in the freshwater crayfish Orconectes clypeatus. American Midland Naturalist 58, 383-393. Page, T. L. & Latimer, J. L. (1975). Neural control of circadian rhythmicity in the crayfish. I. The locomotor activity rhythm. J. Comp. Physiol. 97, 59-80. Pollard, T. G. & Latimer, J. L. (1977). Circadian rhythmicity of heart rate in the crayfish Procambarus clarkii. Comp. Biochem. Physiol. 57A, 221-226. Taylor, A. C. (1977). The respiratory responses of Carcinus maenas (L.) to changes in environmental salinity. J. Exp. Mar. Biol. Ecol. 29, 197-210. Vandenbussche, E. (1969). The detection of periodicities in time series. I. Calculation of a periodogram and of a correlogram. Psychol. Belg. IX-I, 59-77. Williams, J. A. & Naylor, E. (1978). A procedure for the assessment of significance of rhythmicity in time-series data. Int. J. Chronobiol. 5, 435-444.