Distribution of essential and toxic metals in the terrestrial gastropod Arion ater

DISTRIBUTION OF ESSENTIAL A N D TOXIC METALS IN THE TERRESTRIAL GASTROPOD A R I O N A TER

M. P. IRELAND

Department of Zoology, University College oj Wales, Aberystwyth, Dyjed, Wales, Great Britain

ABSTRACT

A study was made of the metal concentrations in the slug Arion ater at two sites. In whole animals, all metals analysed, except magnesium, were higher in concentration at the Cwmystwyth site where high amounts o] lead, zinc and manganese but low concentrations of calcium and phosphate were Jound in vegetation. Individual tissue analyses showed high concentrations of magnesium, zinc, cadmium and phosphate in digestive gland and high concentrations oJ calcium in digestive gland and mantle. Lead and copper dM not concentrate to any extent in a specific tissue. Manganese was located mainly in mantle, skin and foot. Mucus secreted Jrom Cwmystwyth slugs contained high amounts of zinc and manganese, and granules isolated jrom the digestive gland, more zinc. Both extracts contained less calcium and magnesium than extracts from campus slugs. The results are discussed in relation to metal concentrations in vegetation and soil together with excretion and uptake mechanisms.

INTRODUCTION

Divalent metallic cations such as calcium and magnesium, as well as phosphate, are tound to be concentrated in the digestive gland of terrestrial gastropods in the lorm ot" intracellular granules (Wagge, 1951; Fretter, 1952; Abolin~-Krogis, 1961; Burton, 1972). Potentially toxic metals such as lead, copper and cadmium are also located in this organ (Meincke & Schaller, 1974; Coughtrey & Martin, 1976a) and, in the case of zinc, associated with the granules (Schoettli & Seiler, 1970). Concentrations of toxic and essential metals in the digestive gland and whole body of terrestrial gastropods are higher when the animals are in contact with elevated metal concentrations in the vegetation or external medium (Dexheimer, 1953; DeJorge & Haeser, 1968; Meincke & Schaller, 1974; Martin & Coughtrey, 1975, 1976; Coughtrey & Martin, 1976a, 1977). 271 Environ. Pollut. 0013-9327/79/0020-0271/$02"25 ~) Applied Science Publishers Ltd, England, 1979 Printed in Great Britain

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The purpose of the present work was to study the distribution of metals in the slug Arion ater collected from a relatively unpolluted site and from an area known to

contain high concentrations of lead, zinc and manganese (Ireland, 1979).

MATERIALS AND METHODS

According to Roach (197 !) the taxonomic status of the large British Arionidae is very confusing because of the great variability in various morphological features. He considered that the sub-species Arion ater ater and Arion ater ruJus (Quick, 1960) should be referred to as Arion ater. Further support tor the presence of a single species comes from the work of Evans (1976), who showed that electrophoretically the tissues of A. ater and A. ruJus are very similar. In the present study, the large Arionidae are referred to as A. ater. A rion ater and fresh vegetation, consisting mainly of grasses, were collected at two sites; one was situated near the disused Cwmystwyth lead and zinc mine in the Ystwyth Valley and was taken as the site containing high concentrations of lead, zinc and manganese. The second site was the University campus which was considered as relatively unpolluted with heavy metals. Slugs were collected on the same day at both sites lor comparative metal analysis. Animals used tor whole body analysis and mucus and digestive gland granule extraction were collected in July. Material used lor individual tissue analysis and metal content of vegetation was collected in August. Slugs were placed in large glass vessels at a relative humidity of 100~0 and a temperature of 16-18 °C. They were deprived of tood tor ten days to expel any material from the gut. Individual tissues were removed from slugs after anaesthesia in 15 ~o ethanol and rinsed in distilled water. Mantle consisted of the small area of skin above the anterior part of the body and the underlying pulpy calcareous secretion. The skin sample was made up of the rest of the ventral surface and the lateral surfaces of the body wall. Mucus was extracted from slugs by scraping the loot with a nickel spatula. The exudate was collected in pre-weighed flasks. Digestive gland granules were isolated by decapitating slugs and removing the gland. Alter separation of the gonad, the digestive gland was homogenised in 5 ml 10 3o Ficoll and centrifuged at 250 g tbr 15 min. The final pellet was washed three times in distilled water. Microscopical examination of the pellet revealed little cell debris. Pellets isolated from Cwmystwyth slugs were black and those from campus slugs, white. All samples were dried at 105 °C lor 48 h and extracted with concentrated nitric acid. Magnesium, manganese, zinc, copper, lead, cadmium and calcium were determined by atomic absorption analysis. No interference from calcium during lead determinations (Coughtrey & Martin, 1976b) or by sodium during cadmium determinations (Lener & Bibr, 1971) could be found using standards that covered

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the range o f s a m p l e s analysed. S a m p l e s used to e s t i m a t e calcium c o n t a i n e d 0.65 '~0 l a n t h a n u m c h l o r i d e to suppress iron a n d p h o s p h a t e interference. P h o s p h a t e was e s t i m a t e d using the m e t h o d described by R o c k s t e i n & H e r r o n (1951). D a t a were a n a l y s e d using a o n e - w a y analysis o f variance a n d the difference between m e a n s c o m p a r e d using D u n c a n ' s multiple range test. The 5')~, level o f significance was used in all tests.

RESULTS

WhoA, animals and regetation A n a l y s i s of A rion ater at the C w m y s t w y t h site showed that whole a n i m a l s had a greater c o n c e n t r a t i o n o f all metals investigated except m a g n e s i u m c o m p a r e d with the c a m p u s site slugs ( T a b l e 1). Metal analysis o f the vegetation at b o t h sites (Table 2) i n d i c a t e d that at C w m y s t w y t h there were higher a m o u n t s o f m a n g a n e s e , zinc a n d lead but lower c o n c e n t r a t i o n s o f calcium and p h o s p h a t e . TABLE 1 MEANS AND STANDARD ERRORS (SIX REPLICATES) OF METAL CONCENTRATIONS ( m g / g DRy WEIGHT) IN

Arion ater

Calcium Magnesium Phosphate Manganese Zinc Copper Lead Cadmium

Campus

Cwmystwyth

10.56 + 0.67 4-33 + 0.26 6.31 + 0.48 0.43 + 0-09 0.28 +_0.02 0-05 + 0.004 0.005 + 0.0009 0.002 + 0.0002

15.17 + 0.56 4.44 + 0.18 13-49 ___0.23 7.48 + 0-48 1.23 + 0.06 0.09 + 0-01 0.094 + 0.023 0.020 + 0.004

TABLE 2 MEANS AND STANDARD ERRORS (SIX REPLICATES) Ol~ METAL CONCENTRATIONS ( m g / g DRY WEIGHT) IN VEGETATION

Calcium Phosphate Manganese Zinc Copper Lead Cadmium

Campus

Cwmystwyth

4.70 + 0-17 2.44 + 0.07 0-152 _ 0.003 0.042 + 0.0007 0.009 + 0.0002 0.018 + 0.0005 0.0005 ___0.00001

0.944 +__0.014 0.465 + 0.025 0.207 + 0.003 0-060 + 0.0004 0.007 + 0.0003 0.342 + 0.0090 0.0004 + 0.00005

Individual tissue analysis T h e results are s u m m a r i s e d in T a b l e 3. Tissue metal analysis o f A. ater at b o t h sites showed t h a t the highest c o n c e n t r a t i o n s o f m a g n e s i u m , p h o s p h a t e , c a d m i u m

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and zinc were d e p o s i t e d in the digestive gland. The highest significant c o n c e n t r a t i o n o f m a n g a n e s e w a s l o c a t e d in the mantle, skin a n d foot. Lead, on the o t h e r h a n d , was not c o n c e n t r a t e d in a n y o f the tissues examined. A l t h o u g h n o t represented, the same conclusion can be d r a w n from d a t a if the metal c o n t e n t o f individual tissues is calculated. TABLE 3 MEANSANDSTANDARDERRORS(SIXREPLICATES)OFMETALCONCENTRATIONS(mg/g DRYWEIGHT)IN TISSUES OE ,4 rion ater Foot

Calcium Magnesium Phosphate Manganese Zinc Copper Lead Cadmium Calcium Magnesium Phosphate Manganese Zinc Copper Lead Cadmium

Skin

Mantle

Digestive gland

Gonad

Intestine

Campus 26.39+3.29 27.48+1.60 59.51+7.29 58.13+6.66 23.25+4.08 29.88+1.99 4.60+0.48 4.35+0.25 5-20+0.12 14.04+0.84 2.45+0.22 2.11+0.14 4-05+0.23 3.34+0-08 2.88+0.07 46.98+5.63 5-72+0.18 3.46+0.19 0.31 +0.04 0.46+0.05 0.56+0.06 0.08+0.01 0.08+0.01 0.I9+0.01 0.099+0.007 0-179+0.024 0.190+0.015 3.20+0.19 0.162+0.015 0.115+0.01 0.049+0.006 0.041 +0.005 0-026+0.002 0.044+0.005 0.018+0.003 0.013+0.001 ND ND ND 0.011 +0.001 0.012+0.001 0.017+0.005 ND ND ND 0.010+0.025 ND ND Cwmystwyth 18-89+ 1-34 21.14+ 1.92 25.57+2.48 32-94+5.66 5.45+0.46 20.19+2.87 4.46+0.23 4.85+0.24 6.98+0.14 12-20+1.74 1.45+0.09 1.93+0.11 6.23+0.08 4.84+0.15 3.56+0-21 27.22+5.11 6.24+0.25 3.58+0.39 26.08+2.81 23-07_+3.06 30.11 _+4.54 0-21-t-0.02 0.79_+0,07 0.44_+0.07 0.273_+0.017 0.338_+0.036 0.497_+0.034 7.13+0.80 0.096_+0.007 0.196_+0.018 0.028 _+0-003 0.031 _+0.001 0.030 _+0.002 0.072 _+0-018 0.076 _+0.008 0.026 _+0.002 0.129+0.012 0.106_+0.023 ND 0.129_+0.061 0.041 _+0.013 0.176_+0.024 ND ND ND 0.067+0.025 ND ND

ND = none detected. At the site where there were significantly more metals except magnesium in whole animals (Cwmystwyth), calcium c o n c e n t r a t i o n was highest in the digestive gland and mantle, but m a n t l e metal c o n c e n t r a t i o n was not significantly different from skin, foot or intestine. The highest c o n c e n t r a t i o n o f c o p p e r was located in the digestive gland and g o n a d . C a l c i u m c o n c e n t r a t i o n in slugs collected at the c a m p u s site was highest in the digestive gland a n d mantle with a general d i s t r i b u t i o n in the other tissues. F o o t , digestive gland and skin c o n t a i n e d the highest c o n c e n t r a t i o n o f copper. M u c u s and granules

There was significantly m o r e calcium, m a g n e s i u m a n d c o p p e r in m u c u s extracted from c a m p u s slugs a n d m o r e zinc and m a n g a n e s e in C w m y s t w y t h A . ater. G r a n u l e s isolated from the digestive g l a n d o f C w m y s t w y t h slugs c o n t a i n e d higher c o n c e n t r a t i o n s o f zinc while isolated granules from c a m p u s slugs c o n t a i n e d significantly m o r e calcium, m a g n e s i u m and p h o s p h a t e (Table 4).

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ESSENTIAL AND TOXIC METALS IN Arion ater

TABLE 4 MEANSAND STANDARDERRORS(FIVE REPLICATES)OF METALCONCENTRATIONS(mg/g DRY WEIGHT)IN MUCUS AND GRANULES ISOLATED FROM THE DIGESTIVE GLAND OF hrion ater

Campus

Cwmystwyth Mucus

Calcium Magnesium Phosphate Manganese Zinc Copper Lead Cadmium

24.71 + 1.66 12.02 _ 0.77 1.63 0 . 1 4 0.053 + 0.005 0.234+0.023 0.406 + 0.031 ND ND

Calcium Magnesium Phosphate Manganese Zinc Copper Lead Cadmiu m

269-59+ 12.86 25.78 ___1.69 99.34 + 10-22 0.238 + 0.021 6.81 + 1-07 0.038 _ 0.005 ND ND

18.43+0.65 7.68 _ 0.47 1.91 +0.07 0-566 + 0.059 0.381 +0.016 0.148 + 0-019 ND ND Granules 45.34+ 12.71 10.10 + 2.45 42.79 + 10.25 0.318 + 0.052 16.32+3.34 ND ND ND

N D = none detected. DISCUSSION

Because of the numerous parameters which may affect the metal content of molluscs such as size, age, sex, environmental metal levels and metal antagonism (SchultzBaldes, 1973; Martin & Flegal, 1975; Boyden, 1977; Coughtrey & Martin, 1977; Ireland, in press), it is sometimes difficult to compare the metal content of the same species at different sites. According to Williamson (in press), in his study of the snail Cepaea hortensis at one site, cadmium, zinc and lead decrease with increasing weight of the animals. Cadmium accumulation is dependent on age (Williamson, 1979). In the present study, the slugs taken at the campus site were heavier (campus, 1974 _+ 182 mg dry weight; Cwmystwyth, 355 + 30 mg dry weight) and the concentrations of cadmium, zinc and lead lowerl No comparison was made between metal concentrations and slug weight at one site. As far as possible, the ages of A. ater were comparable at both sites: slugs collected in the field at the same time of year are of the same age because the life span ot" A. ater is 11- 12 months (Lusis, 1961 ; Smith, 1966). The differences in metal content of the whole slugs at the two sites may be reflected by the concentrations of metals in the vegetation (Martin & Coughtrey, 1975). Despite the variations in the metal concentration of whole A. ater at the two sites, the distribution of metals within the various tissues was similar, irrespective of the magnitude of the metal concentration. With the exception of calcium and magnesium, which are always high in digestive gland (Burton, 1972), other divalent

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cations have been reported to be concentrated in this organ (DeJorge & Haeser, 1968; Schoettli & Seiler, 1970; Meincke & Schaller, 1974; Coughtrey & Martin, 1976a; Williamson, in press). In the present study, zinc and cadmium were deposited in relatively high concentrations in the digestive gland. The zinc in the digestive gland was associated with granules. A similar association of zinc with digestive gland granules has been reported in Arion rujus (Schoettli & Seiler, 1970). Cadmium was not associated with digestive gland granules in A. ater. Contrary to the findings in Helix spp. (Meincke & Schaller, 1974; Coughtrey & Martin, 1976a) and in C. hortensis (Williamson, in press), lead was not located in high concentrations in the digestive gland of A. ater. A comparative study of lead in the tissues of C. hortensis (Williamson, in press) and A. ater, when the amount of lead in the digestive gland was 120-129/~g/g dry weight, showed the concentration of lead in the gonad material to be similar in both species (41/~g/g dry weight). The concentration of metal in the foot and intestine was considerably higher in A. ater. It is difficult to speculate on the reasons for this difference in metal distribution in the two species since very little is known about the uptake, storage and metabolism of heavy metals in terrestrial gastropods. High concentrations of lead have been reported to occur in rat and earthworm intestines after treatment with the metal (Gruden & Stanti6, 1975; Ireland & Richards, 1977). It was concluded by Forester (1977) that the intestine of the snail Helix pomatia is principally involved in the uptake of water. Theretbre, if"divalent cations are not transported across the epithelium of the gut, they may concentrate in the intestinal wall. According to Simkiss & Wilbur (1977) cations may be taken up and excreted across the epidermis of the toot in snails. At Cwmystwyth, the concentration of lead in the soil (Ireland, 1975) and vegetation is high while the concentration of calcium is low. It has previously been reported that low concentrations of calcium in the soil at Cwmystwyth may enhance lead uptake in earthworms (Ireland, 1979). No data are available on the metal content of the soil from the habitat of C. hortensis but the concentration of lead in the living vegetation is less than one-sixth of that found in living vegetation from Cwmystwyth (Williamson, 1979). Manganese is found in high concentrations in the tissues producing mucus and at much higher amounts in Cwmystwyth slugs where the concentration of manganese is high in vegetation and soil (Ireland, 1979). It is tentatively suggested that mucus may be the site of excretion of manganese since high concentrations are found in mucus and it is not stored in association with digestive gland granules. Contamination of the mucus by urine (Simkiss & Wilbur, 1977) is unlikely since manganese is also located in mantle and skin where less urine contamination could occur.

Despite the high concentrations of calcium and phosphate in the whole body of slugs from Cwmystwyth, the amounts of calcium in mucus and of phosphate in digestive gland granules are low. This apparent anomaly could be the result of low metal values in the vegetation affecting the amount of metal stored within the

ESSENTIAL AND TOXIC METALS IN Arion a t e r

277

animal. DeJorge & Haeser (1968) have shown that the snail Strophocheilus oblongus musculus, when given a choice, will only take vegetation containing high concentrations of calcium, and high concentrations of calcium are secreted in the mucus.

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