Tissue-Level Biomarkers in Sentinel Slugs as Cost-Effective Toolsto Assess Metal Pollution in Soils

I. Marigomez,1 M. Kortabitarte,1 G. B. J. Dussart2

1 Biologia Zelularra Atala, Zoologia eta Animali Zelulen Dinamika Saila, Zientzi Fakultatea, Euskal Herriko Unibertsitatea, 644 PK E-48080 Bilbo,Basque Country2 Ecology Research Group, Canterbury Christ Church College, Canterbury, Kent, CT1 1QU England

Received: 2 December 1996/Accepted: 29 September 1997

Abstract. In previous laboratory experiments, slugs wereshown to be sensitive to metal pollution. Therefore, they mightbe invaluable instruments for biological assessment of soilpollution. The present investigation was carried out to validateprevious laboratory results in a field study. Slugs were collectedfrom an abandoned copper mine (Parys mountain top, PMT),from a site 7 km away from the mine (Parys mountain bottom,PMB), and from a clean site (Snowdonia Cwm Idwal, SCI) inWales in early July 1994. Whole soft body and digestive glandCd, Cu, and Zn concentrations were measured by means ofatomic absorption spectrophotometry (AAS). The digestivegland was the main tissue for metal accumulation, withsignificant differences in tissue metal levels between samplesfrom different sites. PMB presented the highest Cd and Znlevels and the highest Cu levels were found at PMT. In addition,metals were demonstratedin situby autometallography as blacksilver deposits (BSD) on histological sections of digestive glandtissue. The extent of BSD within lysosomes of digestive cellswas closely related to metal levels determined by AAS.Histochemistry revealed that Ca metabolism and structural andreserve connective tissues might be altered in slugs living inmetal-polluted soils. Finally, tissue-level biomarkers of biologi-cal effect [mean epithelial thickness (MET), mean diverticularradius (MDR), mean luminal radius (MLR), MET/MDR andMLR/MET] were quantified by image analysis of digestivegland histological sections stained with hematoxylin-eosin.MET and MDR values of slugs collected from SCI were high,while slugs from PMB presented low MLR/MET associatedwith environmental stress induced by metal exposure. Weconclude that exposure and effect biomarkers recorded insentinel slugs could be sensitive, quick, and cheap indices ofmetal pollution in soils. A Slug Watch monitoring programcould be developed similar to the Mussel Watch program,which is currently applied to assess environmental quality incoastal and estuarine areas.

Marigomezet al.(1996) suggested that slugs can be used in soilquality assessment in the way that other mollusks are used inmonitoring programs (i.e., Mussel Watch) to assess waterquality (Bayne 1989). Thus, slugs (Popham and D’Auria 1980)or equivalent terrestrial gastropods (Coughtrey and Martin1977; Russellet al.1981; Berger and Dallinger 1993) would besentinels (Slug Watch) where biomarkers of exposure topollutants and biological effect recorded at various levels ofbiological organization, including the cell and tissue levels,would indicate soil quality.

The digestive gland is a major site for metal accumulation interrestrial slugs and snails as demonstrated by means ofanalytical chemistry (Ireland 1984, 1994; Beeby and Richmond1987; Berger and Dallinger 1993). The accumulation of metalsin particular cellular compartments within the digestive glandand its association with metallothionein synthesis have alsobeen investigated (Schoettli and Seiler 1977; Ireland 1981,1984; Marigomezet al.1986a; Recioet al.1988a; Janssen andDallinger 1991). Some accurate techniques, such as analyticalchemistry of subcellular compartments and histochemistry ontissue sections, are currently available to assess environmentallevels of metals. Concretely, histochemistry is simpler, quicker,and cheaper than chemical analysis and might offer an alterna-tive for routine screening of metal accumulated in tissues ofsentinel slugs.

The lysosomes of the digestive cells are the first accumula-tion site for various metals in slugs (Marigo´mezet al. 1986a,1996; Recioet al. 1988a) and were proposed as a targetcompartment to detect the presence of abnormal levels ofmetals in the environment (Marigo´mez et al. 1996). On theother hand, exposure to extremely high levels of metals mayresult in an enhancement of excretory activity of digestive cellsfrom which protein-bound metals (Janssen and Dallinger 1991;Dallinger 1993, 1995) are released within lysosomes or residualbodies by processes of apocrine secretion and excess metals areeliminated via feces (Marigo´mezet al. 1986a, 1990a; Recioetal. 1988a; Ireland and Marigo´mez 1992). Such extreme expo-sure to metals would lead to significant changes in thehistological organization of the digestive gland, which affectsits accumulative capacity (Marigo´mezet al. 1990b, 1996). Inaddition, metals can also be localized in the lipofuscinegranules of the excretory cells (Marigo´mezet al. 1986a, 1996;Correspondence to:I. Marigomez

Arch. Environ. Contam. Toxicol. 34, 167–176 (1998) A R C H I V E S O F

EnvironmentalContaminationa n d Toxicologyr 1998 Springer-Verlag New York Inc.

Recioet al. 1988a) and rarely have been observed in calciumcells (Schoettli and Seiler 1977; Marigo´mezet al.1986a). Thus,the cell type composition of the digestive gland epithelium mayalso affect the accumulating capacity of the digestive gland(Marigomez et al. 1996) and therefore affect the relationshipbetween environmental levels of metals and those recorded insentinel slugs. There is some evidence of the changes in thedistribution and relative occurrence of cell types in response topollutants (Loweet al. 1981; Cajaravilleet al. 1990a, 1990b;Marigomez et al. 1990b, 1996). Moreover, the cell typecomposition of the mollusk digestive gland may change due tostressing factors other than pollution (Marigo´mezet al.1993).

In this context, the examination of changes in the histologicalorganization of the digestive gland tissue is useful to determinethe accumulating ability of a sentinel slug, and even to interpretcorrectly the data obtained by means of analytical chemistry.Moreover, the same samples might be sufficient to diagnosewhether the exposure to pollutants provokes a stress response,with further consequences likely for other levels of biologicalorganization (Marigo´mezet al.1990b, 1996). The thickening ofthe blood vessel walls and basal lamina underlining thedigestive tubules, the depletion of glycogen reserves and thealteration of the calcium metabolism have been described in avariety of gastropod mollusks on exposure to pollutants (Recioet al. 1988b; Ireland and Marigo´mez 1992; Cajaravilleet al.1990b; Marigo´mez et al. 1990b, 1993, 1996; Beeby 1993).These effects may be easily detected through routine qualitativeobservations at the light microscope. Nevertheless, if quantita-tive data are preferred, histological techniques may also providesimple, cheap, and quick estimators of the biological effect ofthe exposure to metallic pollutants, as explained below.

A series of works has demonstrated that the digestive glandtubules of mollusks are reduced in thickness in response to avariety of environmental stressors (Loweet al. 1981; Trippetal. 1984; Marigomezet al.1986b, 1993; Minniti 1987; Recioetal. 1988b; Vegaet al.1989; Cajaravilleet al.1992; Ireland andMarigomez 1992). Thus, the measure of such reduction hasbeen proposed as a biomarker of the biological effect ofsublethal exposure to pollutants. Most of these works have beenbased on marine mollusks, with the exceptions of Ireland andMarigomez (1992), Marigo´mezet al. (1986b, 1996), and Recioet al.(1988b), who recorded significant changes in the quantita-tive structure of digestive tubules of terrestrial gastropods onexposure to dietary metals. The mean epithelial thickness(MET) is reduced in mollusks exposed to sublethal concentra-tions of metals (Marigo´mez et al. 1986b, 1996; Vegaet al.1989; Ireland and Marigo´mez 1992) and other sources ofenvironmental stress (Loweet al.1981; Cajaravilleet al.1992),thus MET could be used as a tissue-level biomarker ofenvironmental stress. In addition, Vegaet al. (1989) proposedthat MET should be used simultaneously with other measuresof tubule morphology. Thus, the values of the mean diverticularradius (MDR) and the mean luminal radius (MLR), as well asthe ratios MLR/MET and MDR/MET, are routinely calculatedas measures of digestive tubule morphology. When changes inMET are related to either diverticular (MDR) or luminal (MLR)sizes, the attained MLR/MET and MET/MDR ratios exhibit ahigher sensitivity than MET alone (Vegaet al. 1989; Irelandand Marigomez 1992; Cajaravilleet al.1992; Marigomezet al.1992, 1993, 1996).

Summarizing, the digestive gland is the major site for metalaccumulation in slugs and shows histochemical and histologicalchanges that may be used as biomarkers of the biological effectof exposure to metals. In previous laboratory experiments, slugswere demonstrated to be useful sentinels for metal pollutionwhen the digestive gland was used as the target tissue forchemical, histological, and histochemical analyses. The presentinvestigation was carried out to validate in the field the resultspreviously achieved under controlled laboratory conditions.

Materials and Methods

Sample Collection and Processing

Up to 20 male slugs,Arion ater,were collected at once from each ofthree different localities in North Wales (Figure 1) in early July 1994:(a) a site close to the old copper mines (now inactive) in the top ofParys mountain (PMT); (b) a site in the bottom of Parys mountain(PMB), 7 km away from the mines, which receives drainage watersfrom the mining area; and (c) a nonpolluted site in a Natural Park(Snowdonia Cwm Idwal) far from the mining area (SCI). Slugs weretaken to the laboratory and maintained unfed for 48 h to homogenizetheir physiological condition and remove gut contents before chemicalanalyses.

In a first set of 10 slugs, the digestive gland was excised and fixed informalin, paraffin-embedded, and sectioned at 8 µm for furtherhistological observations and histochemical analyses. A second set of10 slugs was processed for chemical analysis of metals. The digestivegland was excised and both digestive gland and remainder tissues wereprocessed separately. After dissection, soft tissues were rinsed indistilled water, then dried at 120°C for 48 h until constant weight wasreached (Ireland 1981). Dry weights were recorded as digestive glanddry weight (mg) and remainder tissue dry weight (mg). Total dryweight (mg) was calculated as the sum of both partial dry weights.Finally, a digestive gland index (DG index) was calculated as the ratiobetween both partial dry weights.

Chemical Analysis

Dried tissues were digested in concentrated nitric acid, diluted with 0.1M nitric acid, and analyzed by atomic absorption spectrophotometry(Perkin Elmer 2280 spectrophotometer) with simultaneous backgroundcorrection and a sensitivity of 0.3 mg/L. Merck standard solutions werediluted in 0.1 M nitric acid for calibration (Ireland 1981). Three metalswere analyzed: cadmium (Cd), copper (Cu), and zinc (Zn).

Histochemical Analysis

Autometallography:Aset of paraffin sections was stained by autometal-lography, a procedure to demonstrate metals in tissue sections as blacksilver deposits (BSD), which is based on the principles of photography(Danscher 1984). Paraffin sections were dewaxed in xylene, hydratedin ethanol-water mixtures, and left in an oven at 40°C until completelydried. Then, tissue sections were covered with a photographic emulsion(Ilford Nuclear Emulsion L4) under safety light conditions. Afterdrying under completely dark conditions (over 30 min) sections wererinsed in developer bath for 15 min, rinsed in stop bath for 1 min, andfinally rinsed in a fixative bath for 10 min. Sections were mounted inKaiser’s gelatin and sealed with nails’ protector (Soto 1995).

Conventional Metal Histochemistry:Conventional metal histochemis-try was also applied on paraffin sections. The rubeanic acid method was

168 I. Marigomezet al.

applied for the histochemical localization of Cu (Marigo´mez et al.1986a). Copper appears as dark green deposits. The dithizone methodwas applied for the histochemical localization of Zn (Recioet al.1988a). Zinc appears as red purple or light red. The cobalt nitratemethod of Stoeltzner was applied for the histochemical localization ofCa (Marigomez et al. 1986a). Calcium phosphates, carbonates, andoxalates are stained as deep brown to black deposits. Alizarin red wasalso used to localize Ca (Marigo´mezet al.1986a). Calcium phosphatesare stained reddish orange. Control sets were performed for all thehistochemical reactions.

Carbohydrate Histochemistry:Alcian blue (pH 2.5) was used todemonstrate acid mucopolysaccharides on histological sections. Best’scarmine staining was applied to visualize glycogen as pink deposits(Ireland and Marigo´mez 1992).

Quantitative Histological Analyses

A set of paraffin sections was stained with hematoxylin-eosin. Draw-ings of digestive tubule sections were made at a final3650 magnifica-tion with the aid of a drawing tube attachment onto a Nikon ‘‘Optiphot’’microscope. According to previous statistical analyses, five tubulesections in five histological sections, with at least 50 µm distancebetween each other, were drawn and measured by the planimetricprocedure called geometrical transformation after introduction oftubule section profiles by a scanner (Scanjet IIP, Hewlett Packard)attached to a 486 PC computer (Eduskan-93 Software Package)(Marigomezet al.1996). Five parameters were calculated (Vegaet al.1989): MET, mean epithelial thickness; MDR, mean diverticularradius; MLR, mean luminal radius; MLR/MET and MET/MDR.

Data Analysis

The significance of the differences between groups in the biometriccharacteristics, metal concentration, and planimetric parameters wasanalyzed by means of one-way analysis of variance (ANOVA) and

subsequent Duncan’s test to compare pairs of means (SPSS/PC1statistical package; SPSS Inc., Microsoft Co.).

Results

The digestive gland of slugs always exhibited much highermetal concentrations (µg metal/g dry flesh wt) than the remain-der tissue (Figure 2, Table 1). Significant differences werefound between sites. Parys mountain bottom, which receivesdrainage waters from the Cu mining area, presented the highestCd and Zn concentrations in the digestive gland. Copperconcentration, however, was higher in Parys mountain top (theCu mining area) than in the other two sampling sites (Figure 2).

Slugs were larger in the control site (Snowdonia Cwm Idwal)than in the metal-polluted sites. Moreover, slugs from thecopper mining area exhibited the lowest whole body anddigestive gland dry weights (Figure 3a, Table 1). The differ-ences in dry weight might be expected to affect the values ofmetal concentration in flesh, but interestingly, the DG indexwas lower in Parys mountain bottom than in either of the othertwo sampling points, which did not differ between each other(Figure 3b).

Autometallography showed significant differences in theextent of BSD within lysosomes of digestive cells as well asalong the basal membrane of the digestive tubules (Figure4a–f). The digestive cells of slugs from the control site werealmost devoid of BSD while the lipofuscins within vacuoles ofexcretory cells and some few residual bodies of digestive cellsexhibited moderately conspicuous BSD (Figure 4a, b). Al-though the connective tissue layer around the digestive tubuleswas not significantly stained (Figure 4b), BSD were localized inthe connective tissue layer around the blood vessel (Figure 4a).A lowering in the BSD extent around blood vessels and anincrease in the BSD extent in the basal lamina of the digestivetubules were observed in slugs from Parys mountain bottom

Fig. 1. Location of the three sampling areas in North Wales (Great Britain)

169Tissue-Level Biomarkers in Sentinel Slugs

compared with those from the control site (Figure 4c, d). Inaddition, more conspicuous BSD appeared in digestive celllysosomes and residual bodies while lipofuscins in excretorycells exhibited scarce BSD (Figure 4d). Finally, a very strongreaction was recorded in the digestive gland tissue of slugs fromthe Cu mining area while blood vessels appeared almost devoidof BSD (Figure 4e). Digestive cell lysosomes and residualbodies showed a very strong autometallographic reaction(Figure 4e–f). Lipofuscins in the vacuoles of excretory cellsusually lacked BSD (Figure 4e–f). Calcium cells exhibitedeither a strong presence of BSD or no BSD (Figure 4f). In fact,calcium cells only bore BSD in slugs from the Cu mining area(Figure 4f), seldom in those from Parys mountain bottom(Figure 4d) and never in those from the control site (Figure 4b).Finally, abundant BSD were found in vesicles occupying thelumen of the excretory duct and stomach (Figure 4f). Thesevesicles resulted from processes of merocrine and apocrineextrusion from digestive and excretory cells (Figure 4f).

Histochemistry revealed the presence of more Ca in Leydigcells of slugs from the two metal-polluted sites than in thosefrom the control site (Figure 5) but, conversely, Ca was moreabundant in calcium cells of slugs collected from the controlsite than in those collected from the metal-polluted sites (Figure5). In slugs from the control site, Leydig cells did not showsignificant positive reaction with Stoeltzner’s or Alizarin redtechniques (Figure 5a, c). Calcium cells, however, exhibitedmoderate reaction products after staining with either cobaltnitrate (Figure 5a) or Alizarin red (Figure 5c). Occasionally,some weak positive reaction was found in digestive celllysosomes and excretory cell lipofuscins after staining withcobalt nitrate (Figure 5a). A different picture was found in slugscollected from the Cu mining area (Figure 5b, d). These slugsexhibited calcium cells devoid of histochemically demonstrableCa and Leydig cells with a strong positive reaction product afterboth Stoeltzner’s and alizarin red staining (Figure 5b, d). An

Fig. 2. Concentrations of metals in the tissues of slugs collected fromParys Mountain Bottom (PMB), Parys Mountain Top (PMT), andSnowdonia Cwm Idwal (SCI). Asterisks indicate that the correspond-ing mean value is significantly dissimilar to the other two (p, 0.05)according to Duncan’s test. Labels between brackets indicate the groupwith dissimilar mean value in those instances where only partialdissimilarities are significant. Truncated mean values are given on thetop of each bar to assist comparisons

Table 1. Summary of the one-way ANOVAs carried out to assess thevariability between sampling sites (Parys Mountain Top, Cu mine;Parys Mountain Bottom, vicinity receiving drainage waters; andSnowdonia Cwm Idwal, control site) for variables recorded in maleslugs,Arion ater. d.f. (between)5 2; d.f. (residual). 15

Variable P (F ratio)

Whole body Cu conc 0.002Whole body Zn conc 0.006Whole body Cd conc 0.002Digestive gland Cu conc , 0.001Digestive gland Zn conc 0.011Digestive gland Cd conc , 0.001Remainder tissue Cu conc 0.032Remainder tissue Zn conc , 0.001Remainder tissue Cd conc 0.060(NS)Whole body dry wt 0.040Digestive gland dry wt 0.024Digestive gland index 0.325(NS)Mean epithelial thickness , 0.001Mean luminal radius 0.237(NS)Mean diverticular radius 0.001

d.f., degrees of freedom(NS), not significant variability between sites

Fig. 3. Whole body and digestive gland dry weights (a) and DigestiveGland Index (b) of slugs collected from PMB, PMT, and SCI. Asterisksindicate that the corresponding mean value is significantly dissimilar tothe other two (p, 0.05) according to Duncan’s test. Labels betweenbrackets indicate the group with dissimilar mean value in thoseinstances where only partial dissimilarities are significant. Truncatedmean values are given on the top of each bar to assist comparisons

170 I. Marigomezet al.

intermediate situation was found in slugs collected from Parysmountain bottom.

Acid mucopolysaccharides (MPS) were, according to thealcian blue stain, more abundant in both the basal lamina of

digestive tubules and the connective tissue layer surroundingthe Leydig cells of slugs from the Cu mining area than in thosefrom the control site (Figure 6a–c). Calcium cells and Leydigcells were found to be more positive after alcian blue staining in

Fig. 4. Autometallographical demonstration of metals (as black silver deposits, BSD) on paraffin sections of the digestive gland of slugs. (a, c, e)General views of tissue sections of slugs collected from SCI (a), PMB (c), and PMT (e). Scale bars: 80 µm. (b, d, f ) Details of digestive tubule sec-tions of slugs from SCI (b), PMB (d), and PMT (f). Scale bars (b–f): 30 µm. Scale bar (d): 50 µm. T, digestive tubule; V, blood vessel; D, digestivecell; C, calcium cell; E, excretory cell; L, Leydig cell; B, basal lamina (histological sense); arrowheads, BSD

Fig. 5. Calcium histochemistry on paraffin sections of the digestive gland of sentinel slugs. (a–b) Detail of tissue sections stained with the Stoelt-zner’s technique in slugs from SCI (a), and PMT (b). (c–d) Detail of tissue sections stained with alizarin red in slugs from SCI (c) and PMT (d).Scale bars: 40 µm. T, digestive tubule; V, blood vessel; D, digestive cell; C, calcium cell; E, excretory cell; L, Leydig cell; arrowheads, Ca

171Tissue-Level Biomarkers in Sentinel Slugs

the Cu mining area than in Parys mountain bottom; the weakestreaction products were found in these cell types of slugs fromthe control site (Figure 6a–c). Conversely, the levels of acidMPS in digestive cell lysosomes where higher in slugs from thecontrol site than in those from the metal-polluted sites (Figure6a–c). Similarly, the lowest glycogen levels in Leydig cellswere found in slugs from the metal-polluted sites, according tothe Best’s carmine stain (Figure 6d, e). Concomitant to the lossof histochemically demonstrable glycogen in Leydig cells, aslight increase in glycogen levels was envisaged in calciumspherules of calcium cells (Figure 6e).

Finally, the tissue-level biomarkers of biological effect, MET,and MDR of slugs collected from the control site weresignificantly higher than those of slugs collected from themetal-polluted sites (Figure 7a, Table 1). Accordingly, slugsfrom the control site presented significantly lower MLR/METvalues than those from the metal-polluted sites (Figure 7b).Moreover, no significant difference in the quantitative structureof the digestive tubules was found between slugs from the Cumining area and Parys mountain bottom (Figure 7). However,some qualitative differences in the histological organization ofthe digestive gland were observed between slugs from the threelocalities (Figure 8). The digestive tubules of slugs from thecontrol site contained abundant digestive cells, scarce torelatively conspicuous excretory cells (varying between individu-als), and scarce calcium cells (Figure 8a, b). In slugs from Parysmountain bottom, excretory and calcium cells increased inrelative numbers in comparison with the control site and, in

addition, digestive cells were active in excretion of cytoplasmicgranules (Figure 8c, d). This change is much more marked inslugs from the Cu mining area, where digestive cells wereactively excretory and exhibited a conspicuous degree ofvacuolization and where calcium cells comprised a large extentof the digestive tubule epithelium (Figure 8e, f).

Discussion

Ancient mines provide useful field laboratories for investigat-ing the capacity of soil organisms as bioindicators or sentinelsof metal pollution. Dallingeret al. (1989) found well-definedgradients of Cd and Zn pollution around an old lead mining areawhich were significantly related to the metal burdens ofbioindicator slugs,Arion lusitanicus.In the present work, theabandoned Cu mines in Parys mountain provided wild slugswith Cu, Cd, and Zn burdens different from those found in anear control site (Snowdonia Cwm Idwal). The Cu mining areaat Parys mountain top is highly polluted with Cu while amoderate to high Cd and Zn pollution is present at Parysmountain bottom as a result of drainage water from the Cumine. Previous laboratory experiments demonstrated that boththe metal body burdens and the degree of biological effectincrease on exposure to metals (Beeby and Richmond 1987;Berger and Dallinger 1993; Marigo´mez et al. 1986a, 1986b,1993; Recioet al. 1988a, 1988b; Ireland 1994). Our resultsconfirm the existence of a significant association between metal

Fig. 6. Carbohydrate histochemistry on paraffin sections of the digestive gland of sentinel slugs. (a–d) Tissue sections stained with alcian blue foracid MPS in slugs from SCI (a, b), PMB (c), and PMT (d). Scale bars: 30 µm. (e–f) Tissue sections stained with Best’s carmine for glycogen inslugs from SCI (e), and PMB (f). Scale bars: 30 µm. CH, excretory channel; T, digestive tubule; V, blood vessel; D, digestive cell; C, calcium cell;E, excretory cell; L, Leydig cell; arrowheads, carbohydrate

172 I. Marigomezet al.

load and biological characteristics of slugs even in the complex-ity of the field. Thus, it seems that the use of slugs in monitoringprograms based on the biomarker approach is a valuablestrategy for the assessment of metal pollution in soils. However,a feasible ‘‘Slug Watch’’ would still require a huge researcheffort before it could be routinely applied in the way MusselWatch monitoring programs (Bayne 1989) are currently beingcarried out in water quality assessment. Nevertheless, the dataavailable at present suggest that a close parallel exists betweenthe sentinel abilities of slugs in soil and mussels in water, aswell as between the methodological approaches to be applied inboth soils and water. In addition, the general biology of slugs iswell known (South 1992).

The digestive gland of bivalve mollusks is known to be atarget organ in environmental pollution studies. Previous labo-ratory results also confirm the ability of the digestive gland ofslugs to accumulate environmental toxicants and respond tochanges in the degree of environmental quality (Ireland 1981,1984; Marigomez et al. 1986b, 1996; Recioet al. 1988b;Triebskorn et al. 1989). It is not surprising then that thedigestive gland tissue exhibited a higher metal concentrationthan the remainder tissues in all the localities studied herein.Moreover, the metal content of the digestive gland tissue inslugs is significantly related to the environmental levels ofmetals as previously reported in the laboratory (Ireland 1981;Russellet al.1981). In the case of the marine environment, suchrelationship has been also demonstrated in field studies (Regoliand Orlando 1994; Sotoet al.1995).

Accordingly, the metal concentration in the digestive glandof bioindicator slugs might be used as an index of theenvironmental levels of bioavailable metals. However, weshould be aware that these concentrations may change season-ally (Ireland 1984), with age and size (Williamson 1980;Ireland 1994) or in response to environmental stressors (Irelandand Marigomez 1992), due to changes in either mass or cellularcomposition of the digestive gland (as reported in slugs underlaboratory exposure conditions [Marigo´mezet al.1996] and inmussels in field studies [Sotoet al.1995]). Slugs were larger atthe reference site than at the metal-polluted sites. Whole bodyand digestive gland dry weights were severely reduced in slugsfrom the Cu mining area compared with the other two samplingsites. The DG index was lower in Parys mountain bottom thanin the Cu mine and the reference site. Due to the effect of suchvariation on metal concentration values irrespective of changesin metal bioavailability, some alternative techniques such asx-ray microprobe analysis (Hopkinet al.1989) and histochem-istry (Marigomezet al. 1986a, 1996; Recioet al. 1988b) havebeen proposed to assess the level of exposure to metallicpollutants in terrestrial environments.

Particularly, autometallography is a histochemical techniqueable to detect metal traces (Danscher 1984), which provides avaluable screening method to detect exposure to metal in slugs(Marigomezet al.1996). It has been suggested that unnecessarychemical analysis can be avoided in water quality assessmentwhenever BSD are not significantly conspicuous in targetcompartments (i.e. digestive lysosomes) of the digestive glandof sentinel mussels (Soto 1995). The visualization of metals asBSD in sentinel mussel tissues is cost-effective because it isextremely cheap and not time-consuming. The present observa-tions suggest a similar use of autometallography for thescreening of metal pollution in soils by examining the digestivegland of sentinel slugs by light microscope.

In slugs from the reference site, the lipofuscins of excretorycells and some few residual bodies of digestive cells exhibitedmoderate to low levels of autometallographically demonstrablemetals. In addition, conspicuous BSD were localized in theconnective tissue layer around the blood vessel. This picturewould correspond to the reference background level and clearlydiffers from those observed in slugs from metal-polluted sites.Low BSD extent around blood vessels and within lipofuscins ofexcretory cells and moderate to high BSD extent in thelysosomes and basal lamina of the digestive cells were observedin slugs from Parys mountain bottom, the site moderatelypolluted with Cd and Zn. In slugs from the Cu mining area,which exhibited the highest levels of Cu in their tissues, bloodvessels and excretory cell lipofuscins appeared almost devoidof BSD, whereas digestive cell lysosomes, residual bodies, andnumerous calcium cells showed a very intense autometallo-graphic reaction product.

These observations agree with previous findings. Accordingto histochemical and autometallographical data, Hg (Marigo´-mezet al. 1996), Zn (Recioet al. 1988a) and Pb (unpublisheddata) become primarily accumulated in the digestive celllysosomes of slugs. Digestive cell lysosomes are target compart-ments involved in metal handling in mollusks (Dallinger 1993;Marigomez et al. 1995). Digestive cells of slugs, like otherterrestrial and aquatic mollusks, excrete metal-rich lysosomesby processes of merocrine, apocrine, or holocrine extrusion,depending on the level of metal exposure (Marigo´mez et al.

Fig. 7. Tissue-level biomarkers of environmental stress based on thequantitative structure of the digestive tubules of slugs collected fromPMB, PMT and SCI. (a) Mean epithelial thickness (MET), meanluminal radius (MLR), and mean diverticular radius (MDR) as absoluteparameters in µm. (b) MLR/MET and MET/MDR are relative param-eters relating absolute parameters to each other. Asterisks indicate thatthe corresponding mean value is significantly dissimilar to the othertwo (p , 0.05) according to Duncan’s test. Labels between bracketsindicate the group with dissimilar mean value in those instances whereonly partial dissimilarities are significant. Truncated mean values aregiven on the top of each bar to assist comparisons

173Tissue-Level Biomarkers in Sentinel Slugs

1990a, 1996; Dallinger 1993, 1995; Soto 1995). By contrast, Cuis mainly accumulated in calcium cells, as evidenced by theabundant BSD found in slugs living in the Cu mining area. Thisobservation is controversial because Cu is known to accumulatewithin digestive lysosomes of most mollusks (Harrison andBerger 1982; Soto 1995). However, by using conventional Cuhistochemistry with rubeanic acid, Marigo´mez et al. (1986a)found that Cu accumulates in the calcium cells ofA. ater.Therefore it seems that calcium cells are target accumulatingsites for Cu in this particular species. Whether this phenomenonis general for terrestrial gastropods or not is a question thatdeserves further research. It has also been demonstrated that Cuaccumulates in digestive cell lysosomes in marine gastropods(Soto 1995), therefore, its accumulation in calcium cells may beassociated with life in soil rather than with phylogeneticrestrictions. Nevertheless, Cu can also be excreted from thedigestive epithelium by releasing calcium cells or calcium cellportions from the epithelium, and thus Cu exposure may exertan antagonistic effect on Ca metabolism, not observed onexposure to any other metal (Marigo´mezet al.1986a).

Thus, Ca metabolism may be altered in slugs living in Cupolluted soils, as previously reported in laboratory experiments(Marigomez et al. 1986b). More Ca occurred in Leydig cellsand less in calcium cells in slugs collected from the Cu miningarea than in those from the reference site, suggesting that Ca ismobilized when Cu is accumulated in calcium cells. Interest-ingly, excessive dietary Ca provokes Cu mobilization from thedigestive gland to the foot in terrestrial gastropods (Ireland and

Marigomez 1992), the competition between Ca and Cu in thedigestive gland of gastropods being a well-documented phenom-enon (Mason and Simkiss 1983). It seems that the reservoir forCu in terrestrial gastropods is the foot tissue (Berger andDallinger 1989) and thus it is likely that calcium cells partici-pate in the physiological mobilization of Cu between organs.Some authors have proposed a close collaboration betweenthese cells and connective tissue cells (i.e.Leydig cells).

Even if the metals are accumulated in different sites, a uniquepattern of change takes place in the digestive gland epithelium(Cajaravilleet al. 1990a). Basal membranes are thickened inresponse to metal pollution and mucopolysaccharides areredistributed in different cell compartments in stressed terres-trial gastropods (Recioet al. 1988b; Ireland and Marigo´mez1992; Marigomezet al. 1996). Digestive cells are first vacuol-ized and then reduced in numbers, calcium cells becomehypertrophied, excretory cells gain relative abundance, epithe-lium thickness decreases, and blood cells and migrating connec-tive tissue cells accumulate around digestive tubules. Thealterations in digestive cells might indicate a pathological,degenerative process associated with changes in the structure ofthe well-developed lysosomal vacuolar system as interpreted inthe early 1980s (Loweet al. 1981; Tripp et al. 1984). Bycontrast, some evidence suggests that a role replacementoccurs: digestive cells would be almost depleted while calciumcells would become more active in enzyme secretion forextracellular digestion and blood cells and connective tissuecells would contribute to the handling of Ca, Cu, and the toxic

Fig. 8. Paraffin sections of the digestive gland of sentinel slugs collected from SCI (a, b), PMB (c, d), and PMT (e, f), stained with hematoxylin-eosin to illustrate the morphology of the digestive tubules as well as the cell type composition of the digestive epithelium. Scale bar (a, c, e): 100µm. Scale bar (b, d, f ): 30 µm. Note the increase in the relative abundance of calcium and excretory cells from SCI to PMT, as well as the concomi-tant reduction of both diverticular size and epithelial thickness. In order to illustrate the different distribution of basic cell types in the digestivegland epithelium, tissue areas with dominant excretory, and/or calcium cells are shown within circles. D, digestive cell; C, calcium cell; E, excre-tory cell

174 I. Marigomezet al.

metals (Marigo´mezet al. 1986a, 1986b, 1990b, 1996; Irelandand Marigomez 1992). Thus, it appears that a different steadystate is reached in the digestive gland tissue of mollusks afterexperimental exposure to sublethal levels of metals. Thequestion then would be whether this so-called steady state,reached under laboratory conditions at relatively short exposureperiods, also occurs in wild populations chronically exposed tosublethal levels of toxic metals.

As a matter of fact, differences in the histological organiza-tion of the digestive gland were observed between slugs fromthe three localities. The digestive tubules of slugs from thecontrol site at Snowdonia contained abundant digestive cells,relatively less abundant excretory cells, and scarce calciumcells. In slugs from Parys mountain bottom, and more markedlyin those from the Cu mining area at Parys mountain top,excretory and calcium cells increased in relative numbers incomparison with Snowdonia slugs. In addition, digestive cellswere active in excretion of cytoplasmic granules and exhibiteda certain vacuolization. Acid MPS levels in various basallaminae of the digestive gland tissue exhibited a decreasinggradient from the Cu mining area to the reference site togetherwith an increasing gradient in digestive cell lysosomes. Bycontrast, glycogen levels were lower within Leydig cells andhigher in calcium cells of slugs from the reference site than inthose from the metal-polluted sites.

In addition, the tissue-level biomarkers of biological effect,MET and MDR values, of slugs collected from SnowdoniaCwm Idwal were significantly higher than those collected fromthe metal-polluted sites. Moreover, slugs from the reference sitepresented significantly lower MLR/MET values than thosefrom Parys mountain; no significant difference in the quantita-tive structure of the digestive tubules was found between slugsfrom the Cu mining area (high Cu pollution) and from the areareceiving drainage waters from the Cu mine (moderate Cd andZn pollution). These results are in agreement with previouslaboratory investigations that related low MET and highMLR/MET values with stress induced by metal exposure interrestrial gastropods (Marigo´mezet al.1986a,b, 1996; Recioetal. 1988a,b; Ireland and Marigo´mez 1992). Such changes seemto be due to a complex response that includes at least (1)alterations in the functioning of the digestive epithelium, whichsecretes metals instead of absorbing food; and (2) replacementof cell types (Marigo´mezet al.1986b, 1996; Recioet al.1988b;Ireland and Marigo´mez 1992), as discussed above.

Unfortunately, we cannot say at present what is normal andwhat is not regarding the values of MET and related parameters.An extensive data base is needed. By now, only punctual datafrom laboratory studies are available. In addition, most avail-able data are not comparable in absolute terms because theywere obtained by different analytical procedures (Marigo´mezetal. 1986b, 1996; Recioet al.1988b). Exceptionally, the presentresults can be compared with those obtained by Marigo´mezetal. (1996), where MET values in control slugs range between31.5 µm at the start of the experiment (somehow stressedthrough experimental handling) and 28.5 µm at the end, whilethe treatment with 1,000 µg Hg/g food produced a reductionbeyond 26 µm (, 82%). In the present study, similar valueshave been recorded. MET ranged between 42 µm at the controlsite and 29 µm (69%) at the Cu-polluted site. This comparison isof limited value, but these values might constitute the firstconsistent data of the above mentioned data base.

Conclusion

It seems that exposure to metals does not provoke a degenera-tive pathological change but an adaptive process that enablesslugs to inhabit chronically polluted sites by dramaticallymodifying the cell and tissue organization of their digestivegland. Thus, histochemical and histological observations andtissue-level biomarkers recorded in this organ might provide asensitive, quick, and cheap indication of the degree of metalpollution in soils in combination with other measures. Finally,we want to emphasize the value of developing a Slug Watchmonitoring program, based on the biomarker approach, aidedwith the experience of the Mussel Watch program currentlyapplied to assess environmental quality in coastal and estuarineareas.

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