Accumulation of aluminium by the freshwater crustacean Asellus aquaticus in neutral water

Environmental Pollution 106 (1999) 257±263

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Accumulation of aluminium by the freshwater crustacean Asellus aquaticus in neutral water R. Elangovan a, S. Ballance a, K.N. White a, C.R. McCrohan a,*, J.J. Powell b a

School of Biological Sciences, 1.124 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK b The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK Received 21 December 1998; accepted 4 May 1999

Abstract This study examined the accumulation of aluminium (Al), mostly as the insoluble (Al(OH)3) species, by the freshwater crustacean Asellus aquaticus at neutral pH. Animals were exposed to a range of Al concentrations (5±356 mg lÿ1) in three experiments. The ®rst two were of 30 and 50 days duration, respectively, followed by transfer of the A. aquaticus to water containing no Al for 20 days. The third used live and dead animals in order to investigate the contribution made by surface adsorption of Al to the total accumulated. Signi®cant accumulation of Al in the whole tissues occurred by day 10 in all animals in the 30- and 50- day experiment. Peak concentrations of Al were measured in animals between days 10 and 20 with high concentration factors ranging from 1.4104 to 5.5103. By day 30, accumulated Al had fallen but was still signi®cantly greater than the control in the 50- day exposure experiment. This 30- day increase followed by decreased accumulation of Al was repeated over the remaining exposure period (i.e. 30±50 days) although rates of uptake and loss and peak tissue levels of Al were higher. Proportionality between environmental (water) and tissue concentrations of Al occurred at day 20 but not at day 45. Signi®cantly more Al was accumulated by dead animals than live animals at all Al exposure concentrations. These results suggest that Al is available to the crustacean at neutral pH and that the cuticle may provide an important site of uptake. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Aluminium; Asellus aquaticus; Neutral pH; Bioaccumulation; Crustacean

1. Introduction Aluminium (Al) is mostly insoluble at neutral pH with typical dissolved concentrations seldom exceeding 50 mg lÿ1 (Baird, 1995; Driscoll and Postek, 1996). However, a recent study has shown that a number of large rivers in the UK contain levels of Al, mostly colloidal and particulate, up to 450 mg lÿ1 at circumneutral pH (7±8) (Dixon and Gardner, 1998). Although the entry of Al into the food chain at neutral pH is thought to be limited owing to its insolubility (Driscoll and Schecher, 1989), invertebrates (Asellus aquaticus, Erpobdella octoculata) taken from circumneutral freshwaters (River Irwell, Salford, UK) contain Al at similar concentrations to other trace metals, such as Cu and Zn (ca. 150 mg gÿ1; S. Ballance, unpublished results). Furthermore, in a laboratory * Corresponding author. Tel.: +44-161-275-5375; fax: +44-161275-5363. E-mail address: [email protected] (C.R. McCrohan)

study, we have shown that the pond snail, Lymnaea stagnalis, accumulates Al signi®cantly, when exposed to concentrations similar to those reported by Dixon and Gardener (1998) (Elangovan et al., 1997). It was suggested that insoluble Al is accumulated via the gut due to the ingestion of water during feeding. A subsequent study suggested that mucus secreted by L. stagnalis to facilitate locomotion binds large amounts of Al (Jugdaohsingh et al., 1998). As the snail ingests the mucus during grazing, this is likely to be an additional source of accumulated Al. In many species, gills are thought to be a further important route of uptake of Al (Pynnonen, 1990) but, as a pulmonate, L. stagnalis possesses lungs. These observations suggest that availability of Al at neutral pH to aquatic invertebrates may be in¯uenced markedly by feeding habits, mucus secretion and respiration. The isopod crustacean, Asellus aquaticus is a benthicdwelling detritivore which feeds on particulate organic matter (Williams, 1962). A. aquaticus breathes via gills located at the base of the thoracic appendages (Vandel,

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1943; Babula, 1979). In common with other crustacea, A. aquaticus does not secrete mucus to assist in locomotion, although small amounts may be produced by the gills. This organism, therefore, provides an opportunity to investigate further the role of mucus in accumulation of Al. The accumulation of Al in A. aquaticus at neutral pH was examined and compared with our previous study of L. stagnalis, using identical experimental conditions and exposure concentrations. The aim was to compare accumulation and loss pro®les of Al in the two organisms and relate these to di€erences in feeding behaviour and physiology. A further possibility was that Al is adsorbed onto the exoskeleton in arthropods, thus contributing to the total amount associated with the animal. Therefore, an experiment was carried out in which dead animals were exposed to Al and accumulation compared to that of live animals. 2. Materials and methods Adult (4±6 mm) A. aquaticus were obtained from either a eutrophic lake in north Cheshire, UK (Rostherne Mere; National Grid Reference SK 936 987) or the River Irwell at Salford, UK (National Grid Reference SD 823 993). The animals were maintained in highdensity plastic aquaria containing either 10 (Experiments 1 and 2) or 3 L (Experiment 3) of water of known composition (`standard snail water', SSW; Thomas et al., 1975). The acclimation time (15 days), light/dark regime (14/10 h) and temperature (10 C) were identical to our previous studies of L. stagnalis (Elangovan et al., 1997). A. aquaticus were fed ad libitum ash leaves, aged for 15 days in SSW to allow colonisation of the microbial community essential to provide the correct balance of nutrients (Graca et al., 1993). For Experiments 1 and 2, A. aquaticus were exposed to 0 (control), 100, 300, 400 and 500 mglÿ1 added Al, prepared from Al(NO3)3.9H20 in acidi®ed SSW (pH <3), for 30 days, and 0, 100, 300 and 500 mg lÿ1 for 50 days, respectively. The tanks were aerated to maintain the particulate Al in suspension and the SSW, Al and ash leaves renewed every 2 days. The pH was measured daily and maintained at 7.0‹0.5 by the addition of 1 M HNO3 or NaOH. Animals were sampled at 10-day intervals during the 30-day exposure experiment, but this was decreased to 5 days in the subsequent 50-day experiment to allow for a greater resolution of the changes in Al accumulation observed in the 30-day study. After either 30 or 50 days, the remaining A. aquaticus were transferred to uncontaminated SSW for a further 20 days and sampled, as before, at either 10 or 5 days. Five samples, each containing 15 animals, were removed at each time interval. The SSW and ash leaves were analysed at 2-day intervals throughout the 50- and

70-day period. Ash leaves were rinsed with double-distilled water prior to analysis. For Experiment 3, tanks contained either 30 live or 30 dead A. aquaticus exposed to either 0 (control), 100 or 500 mg lÿ1 added Al for 48 h. Animals were killed by prior exposure to liquid nitrogen. These six sets of conditions were replicated in three tanks each. Animals were sampled in groups of eight (i.e three groups per tank) at 48 h. They were not fed during the experiment. A. aquaticus that had been fed during Experiments 1 and 2 were allowed to defecate overnight in aerated SSW and then rinsed brie¯y with 0.1 M HNO3 to remove adsorbed Al. The Al content of these animals was measured by graphite furnace atomic absorption spectroscopy as described by Elangovan et al. (1997). Animals from Experiment 3 were rinsed in double-distilled water and digested in 3 ml 30% `Aristar' (BDH Ltd, Cambridge, UK) H2O2 and 3 ml `SpA' (Romil Ltd, Poole, UK) concentrated HNO3. Diluted digests were analysed by inductively coupled plasma optical emission spectroscopy (ICPOES). Whole organisms only were analysed as it is not possible to remove individual organs without including nearby tissues that are likely to contain di€erent amounts of Al. Any mortalities were noted during the course of the experiment. 2.1. Statistical analysis For the ®rst two experiments, two-way factorial analysis of variance (ANOVA) was employed to compare the e€ects of time of exposure and exposure concentrations, and their interaction, on the amount of Al in the body. Regression analysis was used to examine the relationship (proportionality) between environmental and tissue concentrations of Al. In Experiment 3, the amount of Al associated with the tissues in relation to environmental exposure and between living and dead A. aquaticus was compared using the Mann±Whitney U-test. A signi®cance level of at least p<0.05 was used in all analyses. Concentration factors of accumulated Al are expressed as a ratio of the mean concentration in the tissue (mg gÿ1 dry wt) to the mean concentration in the water (mg mlÿ1). 3. Results 3.1. Experiments 1 and 2 3.1.1. Al in SSW and food An average of 8, 46, 44 and 45% of the Al in the SSW was lost from the water column 2 days after the addition of 100, 300, 400 and 500 mg lÿ1 to the 48 mg lÿ1 present as a contaminant in the SSW. The average amount of Al, taken as the mean of the initial and ®nal

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concentration (Elangovan et al., 1997), was 44, 81, 196, 247 and 314 mg lÿ1 for control, 100, 300, 400 and 500 mg lÿ1 added Al, respectively, in the 30-day experiment. A similar rate of loss of Al resulted in an average of 47, 88, 186 and 356 mg lÿ1 for control, 100, 300 and 500 mg lÿ1 added Al, respectively, in the 50-day experiment. The concentration of Al associated with the ash leaves was low in all treatments. The amount of Al increased with exposure concentration (Table 1) but proportionality (i.e. a direct correlation between concentration in the leaves and in the water) was not observed. 3.1.2. Al in A. aquaticus during 30 days exposure to added metal (Experiment 1) Signi®cantly more Al was associated with A. aquaticus exposed to all concentrations of added Al compared to control by day 10 (Fig. 1). There were highly signi®cant di€erences between exposure times and treatments (p<0.001). Interaction e€ects (timetreatment) were also highly signi®cant (p<0.001) (Table 2). The concentration of Al in all animals fell by between 93 and 61% from the maximum during the ®nal 10 days of exposure although all remained signi®cantly above the control (Fig. 1). Regression analysis revealed no proportionality between body and environmental concentrations at day 10 but proportionality was observed at day 20 (Fig. 2). In line with this observation, inspection of the individual ®rst-order interactions (not shown) failed to show any consistent pattern. Maximum concentration factors compared to SSW of between 7.4103 and 1.3104 were observed in A. aquaticus after 20 days exposure to 81, 247 and 314 mg Al lÿ1 and 9.7103 after 10 days exposure to 196 mg Al lÿ1, falling to 4.9102 ± 5.1103 at the end of the 30 days. 3.1.3. Aluminium in A. aquaticus during 50 days exposure to added metal (Experiment 2) Signi®cant amounts of Al were found in A. aquaticus in all treatments by day 5 (Fig. 3). Again, highly signi®cant main and interaction e€ects (p<000.1) were found (Table 2). Concentrations in animals exposed to the lower amounts of added Al rose initially at a much Table 1 Concentration of Al in ash leaves (mg gÿ1 dry wt) from the experimental containers after 2 days exposurea Mean concentration of Al (mg lÿ1) in the water

Mean concentration in ash leaves (mg gÿ1) (‹ SE, n=5)

44 (control) 81 196 247 314

68‹2.1 88‹4 77‹5.7 90‹2.7 102‹6.5

a

Data from Experiment 1.

Fig. 1. Al concentration in Asellus aquaticus (mean‹SE, n=5) exposed to an average of: ^, 44 (control); &, 81; ~, 196; , 247; *, 314 mg Al lÿ1 for 30 days and uncontaminated standard snail water (SSW) for a further 20 days.

faster rate (151 and 209 mg dayÿ1 in 88 and 186 mg Al lÿ1, respectively) than those exposed to the highest amount (93 mg dayÿ1 in 356 mg lÿ1). In common with the 30-day experiment, Al in A. aquaticus exposed to the two lower exposure concentrations peaked at 10 days while those subject to the highest amount peaked at day 20 (animals were not exposed to the intermediate concentration of 400 mg lÿ1 added Al in the 50-day experiment). The amount of Al in animals from all treatments fell by between 60 and 47% from the maximum by day 30, reaching levels close to, but still signi®cantly above, the control (Fig. 3). Following day 30 in all treatments, the concentration of Al increased signi®cantly again; levels continued to rise until day 45 (Fig. 3). The rate of rise increased progressively with exposure concentration, and was faster (99±212 mg dayÿ1) than that between day 0 and day 20 (87±104 mg dayÿ1). In all cases, much more Al was associated with animals by day 45 than days 10±15. Regression analysis revealed proportionality between body and environmental concentrations at day 20

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Table 2 Analysis of variance (ANOVA) of the interaction between treatments and with exposure timea Source of variation

SS

df

Experiment 1 Treatment Time Treatmenttime Within

6,369,563 30,510,473 17,874,250 230,286.5

4 4 16 100

Total

54,984,573

124

Experiment 2 Treatment Time Treatmenttime Within

44,017,249 82,600,353 43,049,742 4,033,402

3 13 39 224

173,700,746

279

Total a

MS

F

p-value

F crit

1,592,391 7,627,618 1,117,141 2302.9

691.5 3312.2 485.1

< 0.001 < 0.001 < 0.001

2.5 2.5 1.75

1,467,2416 6,353,873 1,103,839 180,006.3

814.9 352.9 61.3

< 0.001 < 0.001 < 0.001

2.6 1.8 1.5

Data from Experiments 1 and 2.

Signi®cant loss of Al occurred between days 45 and 50 at all exposure concentrations although body concentrations remained well above the control. Maximum concentration factors compared to SSW of 1.4104, 8.9103 and 5.5103 were recorded in A. aquaticus between 10 and 20 days exposure to, respectively, 88, 186 and 356 mg Al lÿ1, falling to 1.2103 to 5.9103 by 30 days. Signi®cantly higher (p<0.05) concentration factors were observed at day 45, ranging from 2.3104 in animals exposed to 88 mg Al lÿ1 to 1.0104 in 356 mg Al lÿ1. Loss of Al resulted in lower concentration factors by day 50 (1.9104, 7.2103 and 5.4103 in 88, 186 and 356 mg Al lÿ1 respectively).

Fig. 2. Relationship between the concentration of Al in Asellus aquaticus and in water, after 20 days exposure to an average of: ^, 44 (control); &, 81; ~, 196; , 247; *, 314 mg Al lÿ1.

(Fig. 4(a)). At day 45 no proportionality was observed at Al concentrations greater than 200 mg lÿ1 which means that either the Al is less bioavailable or the animal is showing regulation (Fig. 4(b)). Body concentrations were not proportional (p>0.05) to environmental levels at day 30 when the smallest amounts of Al were present.

3.1.4. Al in A. aquaticus following transfer to SSW with no added metal (Experiments 1 and 2) Signi®cant loss of Al occurred from A. aquaticus previously exposed to added Al for both 30 days (Fig. 1) and 50 days (Fig. 3). However, the amount of Al in animals remained signi®cantly above control in all cases, both after 30 and 50 days. A. aquaticus exposed to Al for 50 days contained more metal, as a proportion of the control, after 20 days in SSW with no added metal than those exposed for 30 days. A larger proportion of the Al was released by A. aquaticus exposed to Al for 50 days (cf. Figs. 1 and 3). However, body concentrations were higher in the 50-day experiment due to the Al peak occurring 5 days before transfer to SSW with no added Al, compared to 10 days in the 30-day study. 3.2. Experiment 3 3.2.1. Al in SSW An average of 50, 47 and 78% of the Al in the SSW was lost from the water column over 48 h following the addition of 0 (control), 100 and 500 mg Al lÿ1 to that present as a contaminant in the SSW. This corresponded

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maximum mortality of 27% was observed in animals exposed to added Al. Ten per cent of the live animals exposed to both 88 and 289 mg Al lÿ1 in Experiment 3 died by 48 h. 4. Discussion

Fig. 3. Al concentration in Asellus aquaticus (mean‹SE, n=5) exposed to an average of: ^, 47 (control); &, 88; ~, 186; *, 356 mg Al lÿ1 for 50 days and uncontaminated standard snail water (SSW) for a further 20 days.

to a respective mean Al concentration in the water column of 5, 88 and 289 mg lÿ1. 3.2.2. Al in live and dead A. aquaticus following 48 h exposure to added metal After 48 h, both live and dead A. aquaticus contained signi®cantly higher concentrations of Al than controls at both exposure concentrations (p<0.004) (Fig. 5). Signi®cantly more Al was associated with animals exposed to 289 mg Al lÿ1 than those exposed to 88 mg Al lÿ1 (p<0.006). Dead animals, however, had a signi®cantly higher body concentration of Al than live animals at all exposure concentrations including controls (p<0.002). 3.3. Mortality Mortality during the course of the experiments was low in all treatments. Between 10 and 14% of control A. aquaticus died by the end of Experiments 1 and 2. A

Although Al is generally insoluble in the pH range 6.0±8.0, the metal is accumulated signi®cantly by a number of molluscs (Pynnonen, 1990; Elangovan et al., 1997) in near-neutral freshwater. Concentration factors are similar to those measured for trace metals such as Cu and Zn (reviewed by Phillips and Rainbow, 1993). Asellus is known to accumulate trace metals such as Pb and Cu from both food and water (e.g. Brown, 1977; Lewis and McIntosh, 1986; Van Hattum et al., 1993). However, the present study suggests that Al associated with A. aquaticus following exposure may be largely accounted for by passive adsorption onto the cuticle at least over the ®rst 48 h; the amount of Al measured in dead animals was signi®cantly greater than that in live animals. This indicates that in this species the cuticle may provide a barrier to Al uptake into soft tissues. An unexpected ®nding was that the rate of accumulation of Al following initial exposure was higher in animals exposed to the lower concentrations of Al in the water. This may be due to the characteristic aqueous chemistry of Al polymerisation at neutral pH. The rate of precipitation of Al is slower at lower added concentrations (100 mg lÿ1) compared to higher concentrations (Ballance et al., 1999), allowing higher levels of Al to remain in the water column than might be predicted thermodynamically. Such kinetic e€ects are important in the environment where ¯uctuating conditions mean that true equilibria are unlikely to be attained. The di€erent feeding habits of L. stagnalis and A. aquaticus may give some clue as to the di€erences in their apparent accumulation of Al into soft tissues. L. stagnalis is a surface grazer, taking in large amounts of mucus, presumably with much associated Al. A. aquaticus feeds on decaying leaf material; in our experiments, little Al was found in the ash leaf food source. However, other explanations may also apply. For example, di€erences in acidity in the gut and in the quantity and composition of mucus secreted by the gut could a€ect uptake of Al from food and from any water directly ingested. Exposure of A. aquaticus to Al over 50 days revealed two peaks of accumulated Al at about 15 and 45 days, with concentrations falling almost to control levels at day 30. The mechanism underlying this is unknown. One possible explanation is synchronised moulting in the population, as observed in marine crustacea (Williams, 1979; Iribarne et al., 1995), leading to loss of Al associated with the cuticle.

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Fig. 4. (a) Relationship between the concentration of Al in Asellus aquaticus and in water, after 20 days exposure to an average of: ^, 47 (control); &, 88; ~, 186; *, 356 mg Al lÿ1. (b) Relationship between the concentration of Al in Asellus aquaticus and in water, after 45 days exposure to an average of: ^, 47 (control); &, 88; ~, 186; *, 356 mg Al lÿ1.

Fig. 5. Al concentration in dead (&) and live (&) Asellus aquaticus (mean‹SE, n=9) after 48 h exposure to an average of 5, 88 and 289 mg Al lÿ1.

A previous study suggested that adsorption of some metals, including Al, onto the exoskeleton of larval chironomids makes a contribution to total body burden at near-neutral pH (Krantzburg and Stokes, 1988). The cuticle is likely to be colonised by capsular polysaccharide-producing bacteria that could enhance the adsorption of Al onto the surface of A. aquaticus. An increase in the growth of such bacteria following death may explain why signi®cantly more Al was associated with dead than live animals exposed to the same concentration of metal. Experiment 3 was conducted over a short time scale (48 h) during which live animals were not fed. During the period immediately following moulting, it is likely that some Al may be accumulated by the soft tissues. Colloidal Al associated with the food and water is also likely to be ingested during feeding as is the case for Pb associated with particulates (Van Huttem et al., 1993). In addition, many crustaceans consume their shed cuticle (Dall and Moriarty, 1983) and consequently any bound metal. Brown (1977) found that Pb is present partly in the hepatopancreas and partly in the cuticle and underlying tissues of Asellus meridianus. The equivalent organ to the hepatopancreas in molluscs is the digestive gland which is an important `sink' for Al in the land snail Helix pomatia (Brooks and White, 1995) and the freshwater snail L. stagnalis (Elangovan et al., 1997). Moulting may result not only in the loss of any Al associated with the cuticle but also from the underlying soft tissues.

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In the freshwater cray®sh, Procambarus clarkii, it is known that the stimulatory moult hormone 20-hydroxyecdysone results in the loss of Cu and Zn from the hepatopancreas (Torreblanca et al., 1996). Hormonal changes associated with this organ may facilitate the loss of stored Al in A. aquaticus. Accumulated Al is a potential in¯uence on invertebrate populations in natural neutral aquatic systems, for example through e€ects on behaviour (Truscott et al., 1995). A further possibility is that aquatic macroinvertebrates may provide a route of entry of Al into the wider food chain as they are an important source of food to many ®sh and some birds such as the dipper Cinclus sp.. However, mucus is likely to comprise a major barrier in the gastrointestinal uptake of insoluble Al in higher animals (vertebrates; Whitehead et al., 1996). Indeed, birds fed a diet containing elevated levels of Al (1500 mg gÿ1) accumulated only trace levels of the metal (Scheuhammer, 1996). Furthermore, a reduction in mass and thickness of egg shells from birds exposed to acidic waters was not due to accumulation of Al from the invertebrate food web (Ormerod et al., 1998). Acknowledgements R. Elangovan was supported by the Indian Government. S. Ballance is supported by the BBSRC, UK. We thank Dr. R.S. Callow for statistical advice. References Babula, A., 1979. Structure of the respiratory organs of the freshwater isopod Asellus aquaticus (L., Crustacea). Bulletin. Societe des Amis des Sciences et des Lettres de Poznan 19, 75±82. Baird, C., 1995. Environmental Chemistry, W.H. Freeman, New York. Ballance, S., White, K.N., McCrohan, C.R., Powell, J.J., 1999. In¯uence of extracellular polysaccharides on the behaviour and bioavailability of aluminium at neutral freshwaters. In: Lovejoy, D.A. (Ed.), Heavy Metals in the Environment: An Integrated Approach. Proceedings of the 1st International Conference on Metals in the Environment, Vilnius, October 1997. Institute of Geology, Vilnius, pp. 261±265. Brooks, A.W., White, K.N., 1995. The localisation of aluminium in the digestive gland of the terrestrial snail Helix aspersa. Tissue & Cell 27, 61±72. Brown, B.E., 1977. Uptake of copper and lead by a metal tolerant isopod Asellus meridianus (Rac.). Freshwater Biology 7, 235±244. Dall, W., Moriarty, D.J.W., 1983. Functional aspects of nutrition and digestion. In: Bliss, D.E., Mantel, L.H. (Eds.), The Biology of Crustacea. Vol. 5. Internal Anatomy and Physiological Regulation. Academic Press, London, pp. 215±261. Dixon, E., Gardner, M., 1998. Reactive aluminium in UK surface waters. Chemical Speciation and Bioavailability 10, 11±17. Driscoll, C.T., Postek, M., 1996. The chemistry of aluminium in surface waters. In: Sposito, G. (Ed.), The Environmental Chemistry of Aluminium, 2nd ed. Lewis, London, pp. 363±418.

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