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Effects of sublethal concentrations of copper and freshwater on behaviour in an estuarine gastropod Polinices sordidus
Volume IS/Number 3/March
1987
tlarme Pollution HuUetin, Vol. 18, No. 3, pp. 127-131. 1987.
0025-326X/87 S3.00+O.O0 © 1987 Pergamon Journals Ltd.
Printed in Great Britain.
Effects of Sublethal Concentrations of Copper and Freshwater on Behaviour in an Estuarine Gastropod Po linices sordidus J. M. HUGHES*, H. F. CHAPMAN* and R. L. KITCHING t *School of Australian Environmental Studiea~ Griffith University, Nathan, Queensland 4111, Australia *Department of Ecosystem Management, University of New England, Armidale, New South Wales 23M, Australia
This study investigated the effects of contamination by copper and freshwater on the behaviour of the estuarine gastropod Polinices sordidus. The burying response was inhibited by both pollutants. In the case of copper, it was suggested that this response after 30 min could reflect toxicity after 96 h, i.e. the 96 h LCs0 and the 30 min EDs0 were very similar. P. sordidus responded to freshwater at concentrations well below the 96 h LCs0 , suggesting that this species can detect sublethal concentrations of freshwater. Thus, for salinity, the 30 min EDs0 is not a reliable indicator of the 96 h LCs0. A linear decline in crawling activity was found in the presence of increasing concentrations of both pollutants. These results for both aspects of behaviour are discussed in the light of results reported earlier for a marine species Polinices incei.
Recently, the importance of the effects of sublethal levels of pollutants on behaviour has been recognized by a number of authors (Brown, 1976, 1982; Maclnnes & Thurlberg, 1973; Phelps et al., 1983). As Eagle (1981) points out, although a pollutant may not cause the death of an individual, it may cause serious impairment to behavioural responses. Some of these that have been investigated are burying behaviour in molluscs (Chapman et al., 1985; Mohlenberg & Kiorboe, 1983; Hargrave & Newcombe, 1973; Nagarajah et aL, 1985) and polychaetes (Mohlenberg & Kiorboe, 1983), crawling behaviour in gastropods (Nagarajah et al., 1985), general locomotor activity in fiddler crabs (Fingerman et al., 1979) and swimming speed in barnacle nauplii (Lang et al., 1980). In other papers we have reported the effects of copper and freshwater on the burying (Chapman et al., 1985) and crawling (Kitching et al., in review) behaviour of the intertidal marine gastropod Polinices incei
Philippi. For this species we found a close relationship between the 96 h LCs0 (mortality) and the 30 rain EDs0 for burying behaviour. We suggested that such a relationship, if it held for other species, would provide a quick indicator of toxicity effects. Further, for crawling, we reported that the length of track, left in the sediment by a standard number of snails after a given time, decreased with increasing concentrations of both copper and freshwater (Kitching et aL, in review). Polinices sordidus Swainson is a closely related snail which is found in more estuarine conditions than P. incei. It inhabits the sandflats and mangrove areas of Moreton Bay, south-east Queensland, where it can be collected in large numbers. Like P. incei, it is a predator and forages just below the sediment surface, leaving easily recognizable tracks. In this study we report effects of copper and freshwater on P. sordidus. Our aims were: (1) to determine whether the 30 min EDs0 for burying behaviour corresponded to the 96 h LC50 as in P. incei; (2) to determine whether the rate of crawling, as measured by total track length made by a set number of snails, was reduced by copper and freshwater contamination. As P. sordidus occurs naturally in more estuarine conditions than P incei, we expected this species to exhibit a greater tolerance to freshwater. Copper tolerance was expected to be similar between the two species. M a t e r i a l s and M e t h o d s
Snails were collected from Amity Point, North Stradbroke Is!and, south-east Queensland. After collection, they were held in seawater aquaria in the laboratory for 12-48 h before being used for experiments. They were not fed before or during experiments. Sand and seawater were collected from the same site as the snails. Glassware used in the experiments was washed thoroughly in detergent, rinsed with deionized water, 127
Marinc Pollution Bulletin
and further rinsed with dilute HC1 to remove any traces of copper. Aeration was maintained throughout the experiments, which were carried out at 18-200C.
Mortality Bioassays 96 h LCsos were determined for both copper and freshwater. These experiments were carried out using 18 c m × 3 0 cm glass aquaria containing 2 cm of sand and 5 1. of water. In each treatment, 20 snails were observed and compared with controls maintained in contaminant-free seawater. The condition of all snails was checked daily and dead animals were removed. Two criteria were used to determine 'death'. If the foot was extended, the animal was recorded as 'dead' if it did not respond to the touch of forceps. If the animal had retracted into the shell, it was recorded as 'dead' if it did not resist being pulled from the shell. Copper solutions used for all experiments were based on a stock preparation of 100 ppm of reagent grade copper sulphate (CuSO4.5H2O) in distilled water. Aliquots of this stock solution were used to obtain the test concentrations. These were: 0.33, 0.66, 1.00, 1.33, 1.66, and 2.00 gg g-¿ copper. All concentrations referred to in this paper are those at the start of the experiment and were determined using atomic weights. All concentrations were below 3.00 ppm, at which a precipitate of cupric hydroxide forms in seawater (Phelps etal., 1983). The range of salinities was obtained by diluting seawater with deionized water. Salinities used were: 5, 10, 15, 20, 25, and 32%o (100% seawater).
0.50, 0.75, and 1.0 ppm. Cu ++ and these were prepared in the same way as those for the mortality bioassays. The salinities used were: 5, 10, 15, 20, 25, and 32%0 S. For both copper and salinity, two tanks were set up at each concentration, i.e. 10 for copper and 12 for salinity. Five snails were placed on the sediment surface in each tank. In order to assess the amount of crawling in each tank, the sand surface was photographed at approximately 12 h intervals on each of 4 consecutive days. After each photograph had been taken, the sand was smoothed over by hand. The use of a photographic stand ensured that photographs were taken from the same position above each tank. The total length of tracks was determined from the photographs. These represent the total length of track recorded over the 4 days after collection. The length of track was measured using an opisometer and corrected to actual length by allowing for the scale reduction of the photographs. For both copper and salinity, regression analysis was used to assess the relationship between pollutant concentration and the total length of track.
Results
Mortality Bioassays The LCs0 for copper was 0.77+0.35 gg g-I and that for freshwater was 8.0+1.8%o S. Dosage response curves are shown in Figs l(c) and 2(c).
Assays of Burying Behaviour Assays of Burying Behaviour A similar experimental set-up was used to examine the effects of copper and freshwater on burying behaviour. In each test, 20 snails were placed on the sediment surface. After 30 min, the number of snails buried was recorded. Those not buried were usually retracted into their shells. They were recorded as showing the 'stress response'. Animals were left in the containers for 24 h, after which they were recovered from the sediment and again placed on the sediment surface. Again, the number buried after 30 rain was recorded. This data was used to calculate the 24 h ED50 (which was the concentration at which 50% of individuals would be showing the stress response). This was compared with the 30 rain EDs0 to identify any evidence of acclimation to the pollutants. We used the same concentrations of copper and freshwater as in the mortality experiments. Both mortality and burying behaviour bioassays were analysed using probits (Finney, 1952). We calculated the 96 h LCso and EDs0s for 30 min and 24 h, as well as 95% confidence limits.
Effects of CrawlingBehaviour 30 cm × 60 cm glass aquaria were used for crawling experiments. Before use, they were cleaned with EXTRAN300 and rinsed with dilute HCI. 2 cm of clean sand from the collection site was placed in each aquarium before it was filled with 25 1. of clean seawater. The copper concentrations used were: 0.0, 0.25, 128
The burying response of P. sordidus was inhibited significantly by copper. There was a significant difference in proportion of animals buried between the control and 0.33 gg g-i of copper, with higher levels of significance at higher copper concentrations (Table 1, Fig. 1). There was little difference (p > 0.05) between the 96 h LCs~, the 30 min EDs0 and the 24 h EDs0, all ranging between 0.7 and 1.1 gg g-~ Cu (Table 2). There was, however, a tendency not to show the stress response until a slightly higher concentration than that which would eventually kill them. This effect was more obvious initially. After 24 h, the EDs0 was almost identical to the 96 h LCso. Note here that in P. incei the reverse tendency was observed. If anything, they showed a stress response (after 30 rain) at concentrations below that which would eventually kill them. As the percentage of freshwater increased, the burying response of P. sordidus was inhibited (Fig. 2). After 30 rain, some animals exhibited the stress response at salinities as high as 25%o and the number showing stress increased with decreasing salinity (Table 1). After 24 h, a very different result was observed. Stressed animals were only recorded at salinities below 15%o and most were below 10%o (Table 1). This result is shown more clearly when EDsos are compared. The EDs~~after 30 rain was significantly higher than after 24 h (p < 0.05), indicating that many fewer animals were showing the stress response in the second test. The other important result is that, contrary to the result for copper, the stress response was observed at freshwater
Volume IS/Number 3, March 1987 (a)
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Salinity (%oS)
Fig. 1 Response of P. sordidusto copper contamination (a) Percentage of animals stressed after 30 min (b) Percentage of animals stressed after 24 h (c) Percentage mortality after 96 h
Fig. 2 Response of P. sordidus to freshwater contamination (a) Percentage of animals stressed after 30 min (b) Percentage of animals stressed after 24 h (c) Percentage mortality after 96 h
TABLE 1 Results of tests of proportions (Freund, 1979) comparing proportion of animals responding in contaminated tanks with proportion responding in control tanks 30 min ED~II Proportion responding
24 h EDs,
Result of comparison with control (Z value)
Proportion responding
96 h LCs,
Result of comparison with control
Proportion responding
Result of comparison with control
COPPER CONCENTRATION (ggg ') o.00
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SAL[N1TY (%,,) 32 25 20 15 10 5 0
1.01
* indicates proportion significantly different from control (p < 0.05) t indicates proportion significantly different from control (p < 0.01 ) 129
Marine Pollution Bulletin
concentrations significantly lower than those that would eventually cause mortality. This result was much more marked in P. sordidus than had been recorded earlier for P. incei, where the 30 min EDs0 and the 96 h LCs0 were not significantly different (Chapman et al., 1985). Effects on Crawling Figure 3 shows the results from the crawling experiments. For both copper and freshwater, there was a significant reduction in crawling with increasing concentrations of pollutant. The regression equations were: Copper: y = - - 1 8 6 3 x + 2328, r = 0.841, p < 0.01 Salinity: y = 120x+ 388.8, r = 0.945, p < 0.01 Discussion
P. sordidus burying behaviour was affected by both copper and freshwater. One important difference between P. sordidus and P. incei was the behaviour of the 'stressed' or unburied individuals. P. sordidus individuals retracted into their shells with their opercula tightly shut, thus removing themselves from their external environment, or at least postponing the need to adjust to the new conditions. In P. incei, stressed individuals often remained with their feet fully extended, apparently unable to move across the sediment surface or to bury (Chapman et al., 1985). Obviously, such behaviour will only increase the speed with which they are affected by any pollutants in their environment. Because of this behavioural difference between the two species, one might expect that P. sordidus would take longer to die than P. incei in polluted conditions. This did not appear to be the case as, after 96 h, roughly the same number of the two species had died in both copper and freshwater experiments. Responses to freshwater were interesting in that the actual LCs0, although slightly below that recorded earlier for P. incei (Chapman et aL, 1985), was not significantly different from that of its fully marine relative. The nature of the burying response was quite different though. At first, individuals of P. sordidus retracted into their shells and did not bury at around 20%o, a salinity much higher than that which causes mortality after 96 h. This may be an adaptive response to periodic changes in salinity which occur in the
natural environment, especially daily salinity changes associated with tides. Possibly, retraction into the shell allows the animal to adjust more slowly to changing conditions (Barnes, 1974). Because P. sordidus regularly has to deal with high levels of freshwater in its natural environment, it may be more sensitive to salinity changes. This would mean that it would respond earlier than P. incei, which rarely has to face salinity levels below 30%0. As the 24 h EDs0 shows, there is evidence that the animals adjust over time and, in the median salinities, many do not show the stress response after 24 h. Unfortunately, no data on acclimation in P. incei was available, but as there was little difference between the 96 h LCs0 and the 30 min EDs0, any significant acclimation in that species seems unlikely. The response of P. sordidus to copper was very similar to that reported earlier for P. incei, with no significant difference between the 30 min EDs0 and the 96 h LCs0 (Chapman et al., 1985). Also, there was little evidence of acclimation after 24 h. Other studies (e.g. Mohlenberg & Kiorboe, 1983) have examined burying behaviour and have demonstrated that it is affected by the presence of pollutants. However, LC~0 values have not been presented, so it is not possible to determine whether alterations in burying behaviour occur at concentrations similar to the LCs0s in species other than Polinices. Our results suggest that, in Polinices, the burying response after 30 min is a reliable indicator of the 96 h LCs0 for pollutants which the species does not encounter in the natural environment. However, when the species has become adapted to a particular pollutant, such as freshwater in the case of P. sordidus, behavioural changes may occur much earlier. Obviously, these suggestions need to be investigated in other species and for a wide range of pollutants. As found previously for P. incei (Kitching et aL, in review), both freshwater and copper had significant effects on the crawling behaviour of /3. sordidus. The reduction in total track length represents a combination of two variables: the number of animals crawling and their rate of movement. These reductions in movement were recorded at pollutant concentrations considerably lower than the 96 h LCs0s. Thus, they represent sublethal effects on the behaviour of these animals, which (b)
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Fig. 3 Effect of (a) freshwater and (b) copper contamination on the crawling behaviour of P. sordidus, y-axis represents the total length of track made by five snails over 96 h.
130
Volume 18/Number 3/March 1987 TABLE 2
96 h LC~.. 30 rain EDs. and 24 h EDs. for P sordidus exposed to different concentrations of copper and freshwater. Results for P. incei arc included for comparison and are taken from Chapman et al., 1985. o 3 0 m i n E D s , + 9 5 % 2 4 h E D 5,+95Yo o 9 6 h L C5c}+95Yo confidence limits confidence limits confidence limits COPPER
(~gg-')
P. sordidus P. incei
0.77 + 0.35 1.17 5:0.49
1.09 + 0.24 0.95 + 0.22
0.87:t:0.38
8.05:1.8 10.75:2.1
19.2 + 4.2 13.2+2.2
12.5+0.01
FRESHWATER
(%0) P. sordidus P, incei
may reduce their prey-searching efficiency in the field and result in serious long-term effects which would not be predicted merely from LCs0 information. By using the regression lines to calculate the pollutant concentrations at which track length would be reduced to zero (i.e. where there would be no animals crawling), it is possible to compare the responses of the two species. As expected, crawling in P. sordidus was less affected by freshwater than it was in P. incei. For P. sordidus, the x-intercept was 3.2%o, and for P. incei it was 5.7%o (Kitching et al., in review). There was also a difference between the two species for copper, with an x-intercept of 2.11 p.g g-i in P. incei (Kitching et al., in review) and only 1.25 ~tg g-~ for P. sordidus, implying that copper has a greater effect on crawling rate in P. sordidus than it does in P. incei. There could be a number of factors involved here. For example, Kitching et al., (1986) report a significant batch effect, i.e. differences in crawling rate between batches of P. incei collected at different times. Given these problems, we considered it was not meaningful to attempt to compare responses between the two species any further. Instead, we are following up the suggestions arising from these experiments by examining the crawling behaviour of individual snails in arenas. We are testing for differences in various components of movement in different pollutant concentrations.
This work was carried out under Marine Science and Technologies Grant No. 83/1 343 I. We are most grateful to this body for assistance.
Barnes, R. S. K. (1974). Estuarine Biology. Arnold, London Brown, A. C. (1976). Toxicity studies on marine animals. South African Journal of Science 72, 197-199. Brown, A. C. (1982). Pollution and the sandy-beach whelk Bullia. Trans. Roy. S. Afr. 44,555-562. Chapman, H. E, Hughes, J. M. & Kitching, R. L. (1985). Burying response of an intertidal gastropod to freshwater and copper contamination. Mar. Polha. Bull. 16,442-445. Chapman. P. M. & Lang, E. R. (1983). The use of bioassays as part of a comprehensive approach to marine pollution assessment. Mar. Pollut. BulL 14, 81-84. Eagle, G. A. (1981). Study of sublethal effects of trace metals on marine organisms--the need for some standardization. Mar. Env. Res. 5, 181-194. Fingermaa, S. W., van Meter, C. & Fingerman, M. (1979). Increased spontaneous locomotor activity in the fiddler crab Uca P,tcilator after exposure to sublethal concentration of DDT. Bull. Environ. Contain. Toxicol. 21, 11-16. Finney, D. J. (1952). Probit Analysis: A Statistical Treatment of the Sigmold Response Curve. Cambridge University Press, London. Freund, J. E. (1979). Modern Elementa~ Statistics. Prentice Hall, London. Hargrave, B. T. & Newcombe, C. P. (1973). Crawling and respiration as indices of sublethal effects of oil and a dispersant on an intertidal snail Littorina littorea. J. Fish. Res. Board Canada 30, 1789-1792. Kitching, R. L., Hughes, J. M. & Chapman, H. F. (0000). Tidal rhythms in activity in the intertidal gastropod Polinices incei Philippi. J. Ethol. (in review). Kitching, R. L., Chapman, H. F. & Hughes, J. M. (0000). Levels of activity as indiators of sublethal impacts of copper and freshwater contamination in the gastropod Polinices incei Philippi. Mar. Env. Res. (in review). Lang, W. H., Forward, R. B., Miller, D. C. & Marcy, M. (1980). Acute toxicity and sublethal behavioural effects of copper on barnacle nauplii Balanus improvistas'. Mar. Biol. 58, 139-145. Maclnnes, J. R. & Thurlberg, F. P. (1973). Effects of metals on the behaviour and oxygen consumption of the mudsnail. Mar. Pollut. Bull. 4, 85-186. Mohlenberg, F. &. Kiorboe, T. (1983). Burrowing and avoidance behaviour in marine organisms exposed to pesticide contaminated sediment. Mar. Pollut. Bull. 14, 57-60. Nagarajah, N., Antonette Sophia, A. J. & Balasubramanian, T. (1985). Behaviour of some intertidal molluscs exposed to water soluble fractions of diesel. Mar. Pollut. Bull. 16,267-271. Phelps, H. L., Handy, J. T., Pearson, W. H. & Apts, C. W. (1983). Clam burrowing behaviour: inhibition by copper-enriched sediment. Mar. Pollut. Bull. 14,452-455.
131
1987
tlarme Pollution HuUetin, Vol. 18, No. 3, pp. 127-131. 1987.
0025-326X/87 S3.00+O.O0 © 1987 Pergamon Journals Ltd.
Printed in Great Britain.
Effects of Sublethal Concentrations of Copper and Freshwater on Behaviour in an Estuarine Gastropod Po linices sordidus J. M. HUGHES*, H. F. CHAPMAN* and R. L. KITCHING t *School of Australian Environmental Studiea~ Griffith University, Nathan, Queensland 4111, Australia *Department of Ecosystem Management, University of New England, Armidale, New South Wales 23M, Australia
This study investigated the effects of contamination by copper and freshwater on the behaviour of the estuarine gastropod Polinices sordidus. The burying response was inhibited by both pollutants. In the case of copper, it was suggested that this response after 30 min could reflect toxicity after 96 h, i.e. the 96 h LCs0 and the 30 min EDs0 were very similar. P. sordidus responded to freshwater at concentrations well below the 96 h LCs0 , suggesting that this species can detect sublethal concentrations of freshwater. Thus, for salinity, the 30 min EDs0 is not a reliable indicator of the 96 h LCs0. A linear decline in crawling activity was found in the presence of increasing concentrations of both pollutants. These results for both aspects of behaviour are discussed in the light of results reported earlier for a marine species Polinices incei.
Recently, the importance of the effects of sublethal levels of pollutants on behaviour has been recognized by a number of authors (Brown, 1976, 1982; Maclnnes & Thurlberg, 1973; Phelps et al., 1983). As Eagle (1981) points out, although a pollutant may not cause the death of an individual, it may cause serious impairment to behavioural responses. Some of these that have been investigated are burying behaviour in molluscs (Chapman et al., 1985; Mohlenberg & Kiorboe, 1983; Hargrave & Newcombe, 1973; Nagarajah et aL, 1985) and polychaetes (Mohlenberg & Kiorboe, 1983), crawling behaviour in gastropods (Nagarajah et al., 1985), general locomotor activity in fiddler crabs (Fingerman et al., 1979) and swimming speed in barnacle nauplii (Lang et al., 1980). In other papers we have reported the effects of copper and freshwater on the burying (Chapman et al., 1985) and crawling (Kitching et al., in review) behaviour of the intertidal marine gastropod Polinices incei
Philippi. For this species we found a close relationship between the 96 h LCs0 (mortality) and the 30 rain EDs0 for burying behaviour. We suggested that such a relationship, if it held for other species, would provide a quick indicator of toxicity effects. Further, for crawling, we reported that the length of track, left in the sediment by a standard number of snails after a given time, decreased with increasing concentrations of both copper and freshwater (Kitching et aL, in review). Polinices sordidus Swainson is a closely related snail which is found in more estuarine conditions than P. incei. It inhabits the sandflats and mangrove areas of Moreton Bay, south-east Queensland, where it can be collected in large numbers. Like P. incei, it is a predator and forages just below the sediment surface, leaving easily recognizable tracks. In this study we report effects of copper and freshwater on P. sordidus. Our aims were: (1) to determine whether the 30 min EDs0 for burying behaviour corresponded to the 96 h LC50 as in P. incei; (2) to determine whether the rate of crawling, as measured by total track length made by a set number of snails, was reduced by copper and freshwater contamination. As P. sordidus occurs naturally in more estuarine conditions than P incei, we expected this species to exhibit a greater tolerance to freshwater. Copper tolerance was expected to be similar between the two species. M a t e r i a l s and M e t h o d s
Snails were collected from Amity Point, North Stradbroke Is!and, south-east Queensland. After collection, they were held in seawater aquaria in the laboratory for 12-48 h before being used for experiments. They were not fed before or during experiments. Sand and seawater were collected from the same site as the snails. Glassware used in the experiments was washed thoroughly in detergent, rinsed with deionized water, 127
Marinc Pollution Bulletin
and further rinsed with dilute HC1 to remove any traces of copper. Aeration was maintained throughout the experiments, which were carried out at 18-200C.
Mortality Bioassays 96 h LCsos were determined for both copper and freshwater. These experiments were carried out using 18 c m × 3 0 cm glass aquaria containing 2 cm of sand and 5 1. of water. In each treatment, 20 snails were observed and compared with controls maintained in contaminant-free seawater. The condition of all snails was checked daily and dead animals were removed. Two criteria were used to determine 'death'. If the foot was extended, the animal was recorded as 'dead' if it did not respond to the touch of forceps. If the animal had retracted into the shell, it was recorded as 'dead' if it did not resist being pulled from the shell. Copper solutions used for all experiments were based on a stock preparation of 100 ppm of reagent grade copper sulphate (CuSO4.5H2O) in distilled water. Aliquots of this stock solution were used to obtain the test concentrations. These were: 0.33, 0.66, 1.00, 1.33, 1.66, and 2.00 gg g-¿ copper. All concentrations referred to in this paper are those at the start of the experiment and were determined using atomic weights. All concentrations were below 3.00 ppm, at which a precipitate of cupric hydroxide forms in seawater (Phelps etal., 1983). The range of salinities was obtained by diluting seawater with deionized water. Salinities used were: 5, 10, 15, 20, 25, and 32%o (100% seawater).
0.50, 0.75, and 1.0 ppm. Cu ++ and these were prepared in the same way as those for the mortality bioassays. The salinities used were: 5, 10, 15, 20, 25, and 32%0 S. For both copper and salinity, two tanks were set up at each concentration, i.e. 10 for copper and 12 for salinity. Five snails were placed on the sediment surface in each tank. In order to assess the amount of crawling in each tank, the sand surface was photographed at approximately 12 h intervals on each of 4 consecutive days. After each photograph had been taken, the sand was smoothed over by hand. The use of a photographic stand ensured that photographs were taken from the same position above each tank. The total length of tracks was determined from the photographs. These represent the total length of track recorded over the 4 days after collection. The length of track was measured using an opisometer and corrected to actual length by allowing for the scale reduction of the photographs. For both copper and salinity, regression analysis was used to assess the relationship between pollutant concentration and the total length of track.
Results
Mortality Bioassays The LCs0 for copper was 0.77+0.35 gg g-I and that for freshwater was 8.0+1.8%o S. Dosage response curves are shown in Figs l(c) and 2(c).
Assays of Burying Behaviour Assays of Burying Behaviour A similar experimental set-up was used to examine the effects of copper and freshwater on burying behaviour. In each test, 20 snails were placed on the sediment surface. After 30 min, the number of snails buried was recorded. Those not buried were usually retracted into their shells. They were recorded as showing the 'stress response'. Animals were left in the containers for 24 h, after which they were recovered from the sediment and again placed on the sediment surface. Again, the number buried after 30 rain was recorded. This data was used to calculate the 24 h ED50 (which was the concentration at which 50% of individuals would be showing the stress response). This was compared with the 30 rain EDs0 to identify any evidence of acclimation to the pollutants. We used the same concentrations of copper and freshwater as in the mortality experiments. Both mortality and burying behaviour bioassays were analysed using probits (Finney, 1952). We calculated the 96 h LCso and EDs0s for 30 min and 24 h, as well as 95% confidence limits.
Effects of CrawlingBehaviour 30 cm × 60 cm glass aquaria were used for crawling experiments. Before use, they were cleaned with EXTRAN300 and rinsed with dilute HCI. 2 cm of clean sand from the collection site was placed in each aquarium before it was filled with 25 1. of clean seawater. The copper concentrations used were: 0.0, 0.25, 128
The burying response of P. sordidus was inhibited significantly by copper. There was a significant difference in proportion of animals buried between the control and 0.33 gg g-i of copper, with higher levels of significance at higher copper concentrations (Table 1, Fig. 1). There was little difference (p > 0.05) between the 96 h LCs~, the 30 min EDs0 and the 24 h EDs0, all ranging between 0.7 and 1.1 gg g-~ Cu (Table 2). There was, however, a tendency not to show the stress response until a slightly higher concentration than that which would eventually kill them. This effect was more obvious initially. After 24 h, the EDs0 was almost identical to the 96 h LCso. Note here that in P. incei the reverse tendency was observed. If anything, they showed a stress response (after 30 rain) at concentrations below that which would eventually kill them. As the percentage of freshwater increased, the burying response of P. sordidus was inhibited (Fig. 2). After 30 rain, some animals exhibited the stress response at salinities as high as 25%o and the number showing stress increased with decreasing salinity (Table 1). After 24 h, a very different result was observed. Stressed animals were only recorded at salinities below 15%o and most were below 10%o (Table 1). This result is shown more clearly when EDsos are compared. The EDs~~after 30 rain was significantly higher than after 24 h (p < 0.05), indicating that many fewer animals were showing the stress response in the second test. The other important result is that, contrary to the result for copper, the stress response was observed at freshwater
Volume IS/Number 3, March 1987 (a)
(a)
100 u)
•o
" 60 o n
100" 80
6o
(n I~" 60.
~) 40
,~ 40.
-~ 20
20
--
0
¢,,
0 . 3 .6 1 1.33 1.66 2 Copper concentration (ug g-l)
30
35
25
20 1; 10 Salinity (%oS)
;
20 1~ ~b
;
o~
A
o~
(b)
(b) 100.
100 IP
80-
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c0
60
60-
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;
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Os, 35
28
Salinity (%os)
(c)
(e)
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80
8o
60
(~ 60
40
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o
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20 0
30
g-l)
0
.3~3 .66
0
"1 1.33 1 . 6 6 : 2
35
3o
25 20 1~ lb
Copper concentration (ug g-l)
Salinity (%oS)
Fig. 1 Response of P. sordidusto copper contamination (a) Percentage of animals stressed after 30 min (b) Percentage of animals stressed after 24 h (c) Percentage mortality after 96 h
Fig. 2 Response of P. sordidus to freshwater contamination (a) Percentage of animals stressed after 30 min (b) Percentage of animals stressed after 24 h (c) Percentage mortality after 96 h
TABLE 1 Results of tests of proportions (Freund, 1979) comparing proportion of animals responding in contaminated tanks with proportion responding in control tanks 30 min ED~II Proportion responding
24 h EDs,
Result of comparison with control (Z value)
Proportion responding
96 h LCs,
Result of comparison with control
Proportion responding
Result of comparison with control
COPPER CONCENTRATION (ggg ') o.00
(I.05
o.25
-
0.00
(/.33 0.66 1.0(1 1.33 1.66 2.00
0.35 0.35 0.6(/ 0.50 0.70 0.75
2.37* 2.37* 3.71 ? 3.197 4.24t 4.52?
0.10 0.65 0.75 0.95 0.90 (I.95
1.25 2.54* 3.16t 4.524.16t 4.52?
0.20 0.50 0.80 0.65 0.80 0.85
2.11" 3.65t 5.I6t 4.38? 5.16t 5.44t
0.00 0.35 0.50 0.45 0.85 1.00 1.00
2.91 t 3.65? 3.41f 5.44? 6.32t 6.32?
0.00 0.00 0.00 0.05 0.95 1.0(1 1.00
1.01 5.72? 6.32? 6.32?
0.00 0.00 0.00 0.05 0.70 I).85
2.11" 5.48?
SAL[N1TY (%,,) 32 25 20 15 10 5 0
1.01
* indicates proportion significantly different from control (p < 0.05) t indicates proportion significantly different from control (p < 0.01 ) 129
Marine Pollution Bulletin
concentrations significantly lower than those that would eventually cause mortality. This result was much more marked in P. sordidus than had been recorded earlier for P. incei, where the 30 min EDs0 and the 96 h LCs0 were not significantly different (Chapman et al., 1985). Effects on Crawling Figure 3 shows the results from the crawling experiments. For both copper and freshwater, there was a significant reduction in crawling with increasing concentrations of pollutant. The regression equations were: Copper: y = - - 1 8 6 3 x + 2328, r = 0.841, p < 0.01 Salinity: y = 120x+ 388.8, r = 0.945, p < 0.01 Discussion
P. sordidus burying behaviour was affected by both copper and freshwater. One important difference between P. sordidus and P. incei was the behaviour of the 'stressed' or unburied individuals. P. sordidus individuals retracted into their shells with their opercula tightly shut, thus removing themselves from their external environment, or at least postponing the need to adjust to the new conditions. In P. incei, stressed individuals often remained with their feet fully extended, apparently unable to move across the sediment surface or to bury (Chapman et al., 1985). Obviously, such behaviour will only increase the speed with which they are affected by any pollutants in their environment. Because of this behavioural difference between the two species, one might expect that P. sordidus would take longer to die than P. incei in polluted conditions. This did not appear to be the case as, after 96 h, roughly the same number of the two species had died in both copper and freshwater experiments. Responses to freshwater were interesting in that the actual LCs0, although slightly below that recorded earlier for P. incei (Chapman et aL, 1985), was not significantly different from that of its fully marine relative. The nature of the burying response was quite different though. At first, individuals of P. sordidus retracted into their shells and did not bury at around 20%o, a salinity much higher than that which causes mortality after 96 h. This may be an adaptive response to periodic changes in salinity which occur in the
natural environment, especially daily salinity changes associated with tides. Possibly, retraction into the shell allows the animal to adjust more slowly to changing conditions (Barnes, 1974). Because P. sordidus regularly has to deal with high levels of freshwater in its natural environment, it may be more sensitive to salinity changes. This would mean that it would respond earlier than P. incei, which rarely has to face salinity levels below 30%0. As the 24 h EDs0 shows, there is evidence that the animals adjust over time and, in the median salinities, many do not show the stress response after 24 h. Unfortunately, no data on acclimation in P. incei was available, but as there was little difference between the 96 h LCs0 and the 30 min EDs0, any significant acclimation in that species seems unlikely. The response of P. sordidus to copper was very similar to that reported earlier for P. incei, with no significant difference between the 30 min EDs0 and the 96 h LCs0 (Chapman et al., 1985). Also, there was little evidence of acclimation after 24 h. Other studies (e.g. Mohlenberg & Kiorboe, 1983) have examined burying behaviour and have demonstrated that it is affected by the presence of pollutants. However, LC~0 values have not been presented, so it is not possible to determine whether alterations in burying behaviour occur at concentrations similar to the LCs0s in species other than Polinices. Our results suggest that, in Polinices, the burying response after 30 min is a reliable indicator of the 96 h LCs0 for pollutants which the species does not encounter in the natural environment. However, when the species has become adapted to a particular pollutant, such as freshwater in the case of P. sordidus, behavioural changes may occur much earlier. Obviously, these suggestions need to be investigated in other species and for a wide range of pollutants. As found previously for P. incei (Kitching et aL, in review), both freshwater and copper had significant effects on the crawling behaviour of /3. sordidus. The reduction in total track length represents a combination of two variables: the number of animals crawling and their rate of movement. These reductions in movement were recorded at pollutant concentrations considerably lower than the 96 h LCs0s. Thus, they represent sublethal effects on the behaviour of these animals, which (b)
Ca)
~'3000
~" 3000"
u v
o
2500 2000
2500O
~ 2000'
t-
1500
"~ 1500"
= 1000
=~1000 "
t.-
•
•
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500
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o
•
o
0
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i
.25
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.75
110
Copper concentration (ug g-l)
Fig. 3 Effect of (a) freshwater and (b) copper contamination on the crawling behaviour of P. sordidus, y-axis represents the total length of track made by five snails over 96 h.
130
Volume 18/Number 3/March 1987 TABLE 2
96 h LC~.. 30 rain EDs. and 24 h EDs. for P sordidus exposed to different concentrations of copper and freshwater. Results for P. incei arc included for comparison and are taken from Chapman et al., 1985. o 3 0 m i n E D s , + 9 5 % 2 4 h E D 5,+95Yo o 9 6 h L C5c}+95Yo confidence limits confidence limits confidence limits COPPER
(~gg-')
P. sordidus P. incei
0.77 + 0.35 1.17 5:0.49
1.09 + 0.24 0.95 + 0.22
0.87:t:0.38
8.05:1.8 10.75:2.1
19.2 + 4.2 13.2+2.2
12.5+0.01
FRESHWATER
(%0) P. sordidus P, incei
may reduce their prey-searching efficiency in the field and result in serious long-term effects which would not be predicted merely from LCs0 information. By using the regression lines to calculate the pollutant concentrations at which track length would be reduced to zero (i.e. where there would be no animals crawling), it is possible to compare the responses of the two species. As expected, crawling in P. sordidus was less affected by freshwater than it was in P. incei. For P. sordidus, the x-intercept was 3.2%o, and for P. incei it was 5.7%o (Kitching et al., in review). There was also a difference between the two species for copper, with an x-intercept of 2.11 p.g g-i in P. incei (Kitching et al., in review) and only 1.25 ~tg g-~ for P. sordidus, implying that copper has a greater effect on crawling rate in P. sordidus than it does in P. incei. There could be a number of factors involved here. For example, Kitching et al., (1986) report a significant batch effect, i.e. differences in crawling rate between batches of P. incei collected at different times. Given these problems, we considered it was not meaningful to attempt to compare responses between the two species any further. Instead, we are following up the suggestions arising from these experiments by examining the crawling behaviour of individual snails in arenas. We are testing for differences in various components of movement in different pollutant concentrations.
This work was carried out under Marine Science and Technologies Grant No. 83/1 343 I. We are most grateful to this body for assistance.
Barnes, R. S. K. (1974). Estuarine Biology. Arnold, London Brown, A. C. (1976). Toxicity studies on marine animals. South African Journal of Science 72, 197-199. Brown, A. C. (1982). Pollution and the sandy-beach whelk Bullia. Trans. Roy. S. Afr. 44,555-562. Chapman, H. E, Hughes, J. M. & Kitching, R. L. (1985). Burying response of an intertidal gastropod to freshwater and copper contamination. Mar. Polha. Bull. 16,442-445. Chapman. P. M. & Lang, E. R. (1983). The use of bioassays as part of a comprehensive approach to marine pollution assessment. Mar. Pollut. BulL 14, 81-84. Eagle, G. A. (1981). Study of sublethal effects of trace metals on marine organisms--the need for some standardization. Mar. Env. Res. 5, 181-194. Fingermaa, S. W., van Meter, C. & Fingerman, M. (1979). Increased spontaneous locomotor activity in the fiddler crab Uca P,tcilator after exposure to sublethal concentration of DDT. Bull. Environ. Contain. Toxicol. 21, 11-16. Finney, D. J. (1952). Probit Analysis: A Statistical Treatment of the Sigmold Response Curve. Cambridge University Press, London. Freund, J. E. (1979). Modern Elementa~ Statistics. Prentice Hall, London. Hargrave, B. T. & Newcombe, C. P. (1973). Crawling and respiration as indices of sublethal effects of oil and a dispersant on an intertidal snail Littorina littorea. J. Fish. Res. Board Canada 30, 1789-1792. Kitching, R. L., Hughes, J. M. & Chapman, H. F. (0000). Tidal rhythms in activity in the intertidal gastropod Polinices incei Philippi. J. Ethol. (in review). Kitching, R. L., Chapman, H. F. & Hughes, J. M. (0000). Levels of activity as indiators of sublethal impacts of copper and freshwater contamination in the gastropod Polinices incei Philippi. Mar. Env. Res. (in review). Lang, W. H., Forward, R. B., Miller, D. C. & Marcy, M. (1980). Acute toxicity and sublethal behavioural effects of copper on barnacle nauplii Balanus improvistas'. Mar. Biol. 58, 139-145. Maclnnes, J. R. & Thurlberg, F. P. (1973). Effects of metals on the behaviour and oxygen consumption of the mudsnail. Mar. Pollut. Bull. 4, 85-186. Mohlenberg, F. &. Kiorboe, T. (1983). Burrowing and avoidance behaviour in marine organisms exposed to pesticide contaminated sediment. Mar. Pollut. Bull. 14, 57-60. Nagarajah, N., Antonette Sophia, A. J. & Balasubramanian, T. (1985). Behaviour of some intertidal molluscs exposed to water soluble fractions of diesel. Mar. Pollut. Bull. 16,267-271. Phelps, H. L., Handy, J. T., Pearson, W. H. & Apts, C. W. (1983). Clam burrowing behaviour: inhibition by copper-enriched sediment. Mar. Pollut. Bull. 14,452-455.
131