OCHRONA ŚRODOWISKA I ZASOBÓW NATURALNYCH VOL. 24, NO 2(56): 45–49 ENVIRONMENTAL PROTECTION AND NATURAL RESOURCES 2013 DOI 10.2478/oszn-2013-0015

Danuta Kowalczyk-Pecka*, Katarzyna Czepiel-Mil* The effect of accumulation of metals on selected physiological biomarkers in (Cochlodina) laminata (:) inhabiting urban biocenoses Wpływ akumulacji metali na wybrane biomarkery fizjologiczne Cochlodina (Cochlodina) laminata (Pulmonata:Clausiliidae) zasiedlających urbicenozy

* Dr Danuta Kowalczyk-Pecka, dr Katarzyna Czepiel-Mil, Departament of Zoology, Ecology and Wildlife Management, University of Life Science in Lublin, Akademicka 13St, 20-950 Lublin, phone: 81 4456962, e-mail: [email protected]

Keywords: Clausiliidae, Cochlodina, bioindicators, heavy metals, Enterobacteriaceae Słowa kluczowe: Clausiliidae, Cochlodina, biowskaźniki, metale ciężkie, Enterobacteriaceae

Abstract Streszczenie Using the small-sized snail species Cochlodina (Cochlodina) Wykorzystując powszechnie występujące w całej Europie, nie- laminata, commonly found throughout Europe, an analysis was wielkie rozmiarowo ślimaki Cochlodina (Cochlodina) laminata made of the accumulation of metals in the soft tissues and shells przeprowadzono analizę akumulacji metali w tkankach mięk- of these molluscs. Three locations were compared, all near cem- kich oraz w muszlach mięczaków. Porównano trzy lokalizacje eteries in Lublin, which were characterised by varying degrees of wyznaczone w okolicach lubelskich nekropolii i różniących się human impact. It was determined that this representative of the stopniem antropopresji. Zaliczono przedstawiciela Clausiliidae family Clausiliidae can be included in the set of Pulmonata taxa do użytecznego dla biomonitoringu panelu (zestawu) taksonów useful for biomonitoring, alongside Arianta, Cepaea, and Frutici- Pulmonata obok Arianta, Cepaea, Fruticicola. Stwierdzono cola. The soft tissues were found to have macro-concentration makrokoncentracyjne właściwości tkanek miękkich ślimaków properties for Zn, Cu, and Cd in relation to the values determined dla Zn, Cu oraz Cd w stosunku do wartości oznaczonych w gle- in the soil, while micro-concentration was observed in the case of bie. Mikrokoncentracja natomiast została oznaczona w przypad- Fe, Pb, and Cr, both in the soft tissues and in the shells. Pheno- ku Fe, Pb i Cr zarówno w tkankach miękkich, jak i w muszlach. typic differences were found in the Enterobacteriaceae bacteria Wykazano różnice fenotypowe bakterii Enterobacteriaceae associated with the digestive tract of the snails. związanych z układem pokarmowym badanych ślimaków.

© IOŚ-PIB

1. INTRODUCTION

Biomarkers of exposure to pollutants and its physiological con- may cite the labile physiology of such small in natural sequences in invertebrates have been the subject of research conditions. Nevertheless, analyses of selected factors indicat- for many years [Nahmani et al. 2006]. Bioindicator organisms ing morphological, biochemical, cytological, and histochemical include molluscs inhabiting both aquatic environments [Downs changes taking place in the bodies of snails living in their natural et al. 2001; Liang et al. 2004; Giarratano et al. 2010] and land habitats contribute vital information about the synergy between biotopes [Swaileh, Ezzughayyar 2001; Scheifler et al. 2002; Rad- these phenomena and external factors. Their comprehensive in- wan et al. 2010]. DNA strand breaks, adducts, and the presence terpretation may at times pose difficulties. of antioxidant enzymes, metallothionein, and heat shock proteins The aim of the study was to determine the metal accumulation have been proposed as molecular biomarkers of contact with a capacity of the small snails Cochlodina (Cochlodina) laminata, of pollutant in molluscs. Cytological biomarkers include damage the clade , which are rarely used in research on to reproductive cells, and physiological markers include heart the state of the environment (apart from faunistic research). The rate or the rate at which toxic substances are eliminated from bioaccumulation factor of the metals in the soft tissues and shells the organism [Nicholson, Lam 2005]. The usefulness of snails of the snails in relation to the content of these metals in the humus and slugs in environmental biomonitoring is still under discussion. layer of the soil they inhabit was used to analyse the three habitats Many analyses of the effects of harmful substances, e.g. heavy investigated with regard to the degree of availability and sorption metals [Yasoshima et al. 2001], pesticides, molluscicides, anti- of the metals. To search for parameters characterising this inver- bacterial agents [Coogan and La Point 2008], and other environ- tebrate population, and at the same time indicating the varying mental pollutants are conducted exclusively in vitro, on animals degrees of human impact in their habitats, Enterobacteriaceae mi- that were born in laboratory conditions, in order to ensure equiva- croflora associated with the digestive tract of the snails was anal- lent conditions for feeding and development. Arguments ques- ysed, and the susceptibility of the isolates to six chemotherapeutic tioning the usefulness of gastropods in ecotoxicological studies agents was compared.

45 Danuta Kowalczyk-Pecka, Katarzyna Czepiel-Mil

2. MATERIAL AND METHODS the zones of inhibition were measured and the results interpreted according to the recommendations of the producer of the disks. The snails were collected by hand in July 2012 over a period of one week. Study sites characterised by varying degrees of human 3. RESULTS AND DISCUSSION impact were chosen near three of the largest cemeteries in Lub- lin. These were the Roman Catholic Cemetery located on Droga Cochlodina (Cochlodina) laminata [Montagu 1803], the plaited Męczenników Majdanka (51 ° 22’43” N; 22 ° 61’04”E) – designated door snail, occurs in nearly all of Europe [Uit de Weerd, Gitten- as L I, the Roman Catholic Cemetery on ul. Lipowa (51°24’36”N; berger 2013]. In Poland it is one of the most common door snails 22 ° 55’30”E), which is the oldest and best-known cemetery com- and can be found all over the country [Wąsowski, Penkowski plex in eastern Poland – L II, and the Roman Catholic Cemetery 2003]. It is a forest species, mainly inhabiting deciduous – ide- on ul. Unicka (51 ° 15’42”N; 22 ° 34’44”E) – L III. The locations were ally beech – forests, less often mixed forests, and sporadically ranked according to increasing levels of heavy metal contamination. coniferous forests. It can also be found in old, neglected munici- From each habitat, 20 Cochlodina (Cochlodina) laminata individu- pal parks and in cemeteries. It lives in litter, under tree bark, and als were selected, identified according to a key by Wiktor [2004], among stones. It is often seen after rain, gliding along tree trunks, with similar shell size (average height 17 mm) and body weight rocks, and gravestones. Despite its widespread occurrence, it is (average 0.063 g). Due to their small size, the snail bodies were rarely the subject of research on the spread of environmental pol- separated into soft tissues – mainly reproductive organs, the hepa- lutants. Door snails are typically thermophilic snails. Unlike many topancreas, and foot tissues – and shells. other domestic gastropod families, they do not leave their winter Samples of the snail parts and of soil collected from the humus shelters until May, and return to them as early as the beginning of layer (20 samples from each location) were subjected to the proce- September [Wiktor 2004]. Their relatively short period of activity in dures and analysis for metal content described in a previous paper the open and their long period of feeding in litter may allow door [Kowalczyk-Pecka 2009]. The snails burrow in the soil and feed on snails to be included among concentrators of pollutants, including plant detritus they find there, and also take in soil mineral particles metals taken in together with food or though direct contact of the together with pollutants. Metal contents were analysed by atomic foot with the soil. absorption spectrometry at the Central Analytical Laboratory of the Comparison of the bioaccumulation factor at the sites investigated University of Life Sciences in Lublin. The results were converted compared to the content of the metals in the soil showed high vari- to μg metal per g DW of the sample. The bioaccumulation factor of ation in BAF values depending on the type of metal, the habitat, the metals in the bodies of the snails was determined in relation to and the part of the snail (Tables 1–3). their content in soil. The statistical analysis took into account the The bioaccumulation factor calculated in most of the samples total content of the metals in all of the soft tissues. in relation to the quantity of the metals in the soil was far lower Snail faeces were prepared for microbiological testing in sterile than the BAF calculated previously by other authors for metals conditions. Intestinal contents were transferred to LB enrichment in snail tissues in relation to their accumulation in plant leaves broth (Difco) and then to SS and MacConkey media. Following in- from the habitats studied [Scheifer et al. 2002]. This indicates cubation on the selective media, bacterial colonies were randomly that the transfer of metals to snails from soil is slower or simply selected for further identification and analysis. lower than transfer from plants. Particularly high values indicat- The entire collection of isolated strains was analysed biochemi- ing macro-concentration were noted for zinc and copper in the cally and identified using commercial API 20E kits (bioMérieux). soft tissues, and somewhat lower values for cadmium in all habi- Susceptibility to chemotherapeutic agents was determined by the tats. The BAF for iron, chromium, and lead in the soft tissues disk-diffusion method according to Bauer et al. (1966), on Mueller- in relation to their content in the soil samples indicate micro- Hinton medium (Difco), using bioMérieux disks (30 strains of each concentration [Dallinger et al. 2001]. In the case of the shells, on location). The following chemotherapeutic agents were used to the other hand, the only macro-concentration observed was in determine the drug susceptibility of the isolates: the β-lactam anti- the case of copper at location I. It is easier to obtain individual biotic ampicillin (AM at a concentration of 10 μg/ml), the aminogly- internal organs e.g. the digestive tract, the hepatopancreas, re- coside streptomycin (S 10 IU), tetracycline (TE 30 IU), nitrofuran- productive organs, and others, from gastropod taxa of greater toin (FM 300 IU), chloramphenicol (C 30 μg/ml), and trimethoprim/ size, with larger internal organs. When the amount of metal ac- sulfamethoxazole (SXT 1.25 + 23.75 μg/ml). Following incubation, cumulated in the hepatopancreas and the amount in the other

Table 1. Comparison of amounts of accumulated metals in the soil and in the soft tissues and shells of Cochlodina (Cochlodina) laminata (μg • g-1 DW) and the bioaccumulation factor (BAF) of the metals in relation to the soil – location I Location I Metal Soil 0–10 cm Soft tissues BAF Shell BAF Zn 62.55 ± 3.82 573.28 ± 10.21 9.16 101.98 ± 3.99 1.63 Cu 7.18 ± 0.53 75.67 ± 5.02 10.53 17.76 ± 0.98 2.47 Fe 3358.04 ± 62.92 227.33 ± 6.98 0.067 233.83 ± 5.21 0.069 Cr 4.95 ± 0.42 0.46 ± 0.03 0.092 0.67 ± 0.03 0.135 Pb 0.72 ± 0.03 0.025 ± 0.002 0.034 0.078 ± 0.003 0.108 Cd 0.115 ± 0.009 0.43 ± 0.0.21 3.74 0.16 ± 0.011 1.39

pH • H2O of soil 7.3 ± 0.03 Organic matter (%) 19.7 ± 0.7 Designations for locations are given in “Material and methods” DW – dry weight, values are means ± S.D., n = 20

46 THE EFFECT OF ACCUMULATION OF METALS ON SELECTED PHYSIOLOGICAL BIOMARKERS IN COCHLODINA (COCHLODINA) LAMINATA (PULMONATA:CLAUSILIIDAE) INHABITING URBAN BIOCENOSES

Table 2. Comparison of amounts of accumulated metals in the soil and in the soft tissues and shells of Cochlodina (Cochlodina) laminata (μg • g-1 DW) and the bioaccumulation factor (BAF) of the metals in relation to the soil – location II Location II Metal Soil 0–10 cm Soft tissues BAF Shell BAF Zn 157.91 ± 8.22 864.21 ± 15.77 5.47 118.45 ± 6.26 0.751 Cu 11.04 ± 0.76 86.33 ± 6.56 7.81 22.01 ± 1.97 1.99 Fe 4878.11 ± 75.09 259.54 ± 13.44 0.053 564.44 ± 13.45 0.115 Cr 6.43 ± 0.51 0.71 ± 0.04 0.110 1.02 ± 0.13 0.158 Pb 0.86 ± 0.04 0.130 ± 0.011 0.151 0.105 ± 0.015 0.122 Cd 0.137 ± 0.010 0.502 ± 0.038 3.66 0.088 ± 0.004 0.642

pH∙H2O of soil 6.8 ± 0.03 Organic matter (%) 18.3 ± 0.10 * Designations for locations are given in “Material and methods” DW – dry weight, values are means ± S.D., n = 20

Table 3. Comparison of amounts of accumulated metals in the soil and in the soft tissues and shells of Cochlodina (Cochlodina) laminata (μg • g-1 DW) and the bioaccumulation factor (BAF) of the metals in relation to the soil – location III Location III Metal Soil 0–10 cm Soft tissues BAF Shell BAF Zn 271.82 ± 8.54 695.28 ± 26.65 2.56 131.65 ± 5.22 0.484 Cu 15.33 ± 1.02 101.52 ± 8.97 6.62 24.81 ± 1.12 1.62 Fe 6273.06 ± 62.11 291.88 ± 13.01 0.046 816.34 ± 14.99 0.130 Cr 7.09 ± 0.51 1.32 ± 0.08 0.186 1.99 ± 0.21 0.281 Pb 1.19 ± 0.09 0.298 ± 0.017 0.250 0.19 ± 0.014 0.159 Cd 0.172 ± 0.011 0.596 ± 0.029 3.47 0.045 ± 0.002 0.262

pH∙H2O of soil 6.5 ± 0.04 Organic matter (%) 15.5 ± 0.6 Designations for locations are given in “Material and methods” DW – dry weight, values are means ± S.D., n = 20

Table 4. Enterobacteriaceae taxa isolated from Cochlodina (Cochlodina) laminata from the three locations investigated Abbreviation Bacteria taxon LI LII LIII Ecl Enterobacter cloacae + + + Ei Enterobacter intermedium - + - Es Enterobacter sacazakii + + - Ha Hafnia alvei + + + Kp Klebsiella pneumoniae - - + Ck/a Citrobacter koseri/amalonaticus - + + Cb Citrobacter braakii + + + Cy Citrobacter youngae - + - Cf Citrobacter freundii + - + Ba Buttiauxella agrestis + + + Ev Escherichia vulneris + - - Ah Aerobacter hydrophila - + +

soft tissues are given separately, in the case of copper the values ors may potentially affect expression of various phenotypic traits in from the hepatopancreas are found to bring down the average. these bacteria [Kowalczyk-Pecka, Puchalski 2008]. Seven bacteria This is because snails eliminate copper from the digestive gland species were observed in snails from location I, 8 in location II, and very quickly, which is not true of the other tissues. Zinc, on the 9 in location III. The absence of Salmonella or Shigella bacteria in other hand, is accumulated to a significant extent both in the he- the snails from the habitats investigated does not indicate that the patopancreas and in the other soft tissues, so when the average snails are of no epidemiological importance, as bacteria taxa were is calculated, a high accumulation result is obtained. isolated whose pathogenic role has been increasingly emphasised, Research conducted thus far on Citrobacter isolated from faecal e.g. Hafnia alvei [Katzenellenbogen et al. 2001], Bautiauxella agres- samples of various land snail taxa indicate that environmental stress- tis [Fihman et al. 2002], and Citrobacter [Nawaz et al. 2008] (Table 4).

47 Danuta Kowalczyk-Pecka, Katarzyna Czepiel-Mil

Quantitative and qualitative changes in the micro-organisms isolated variation in susceptibility and resistance in the isolates from the three from the snails may also depend on oxidative mechanisms activated in locations was noted after the use of chloramphenicol. In the case each organism under the influence of negative environmental factors. of the other drugs, the appearance of many resistant strains was A phenotypic, and most likely genotypic, characterisation of the group noted. Similar observations have previously been made for Arianta comprising Enterobacteriaceae taxa isolated from snails from all three arbustorum snails [Kowalczyk-Pecka, unpublished data]. In the case habitats can provide information regarding physiological changes in of Cochlodina (Cochlodina) laminata, the increasing amount of ac- the bodies of snails induced by pollutants in their environment. cumulated metals in the snail tissues accompanying increasing con- Among the chemotherapeutic agents applied, the trimethoprim/sul- tamination of the habitats also leads to increased expression of traits phonamides combination proved to be the most effective. The least of resistance to chemotherapeutic agents (Table 5).

Table 5. Drug susceptibility (%) of Enterobacteriaceae strains isolated from the experimental groups of Cochlodina (Cochlodina) laminata CMT LI LII LIII S I R S I R S I R C 9.2 55.4 35.4 12.2 52.0 35.8 11.3 52.5 36.2 T 63.6 27.3 9.1 51.5 32.2 16.3 43 27.3 28.6 SXT 100 0 0 100 0 0 100 0 0 S 0 18.2 81.8 0 17.4 82.6 0 14.3 85.7 FM 60.6 36.4 4.0 42.8 57.2 0 14.3 85.7 0 AM 27.3 54.5 18.2 18.6 59.9 21.5 14.3 57.1 28.6 CMT – chemotherapeutic agent LI, LII, LIII – designations for locations are given in “Material and methods” S – sensitive, I – intermediately susceptible, R – resistant, C – Chloramphenicol (30), T – Tetracycline (30), SXT – Trimethopr.+Sulphmethox. (1.25+23.75) S – Streptomycin (10), FM – Nitrofurantoin (300), AM – Ampicillin (10) Figures in brackets denote amount of chemotherapeutic agent in disk

In terms of the epidemiology of Enterobacteriaceae infections, the metabolic conversion in the snails – or direct inductors at the mo- appearance of multiresistant strains, i.e. those which are resistant lecular level. When reading the value of the bioaccumulation factor to numerous antibiotics, is a cause for concern. of the metals in the bodies of snails from natural populations, we should not expect to find linear dependencies as contamination 4. SUMMARY AND CONCLUSIONS levels increase. An interdisciplinary search for dependencies be- tween coexisting environmental factors is necessary. By selecting partially isolated natural habitats of invertebrates, in- cluding snails, within cities or other areas with a substantial influx of ACKNOWLEDGEMENTS pollutants, it is possible to single out parameters indicating chang- es in the biology of bioindicator organisms. Knowing the mecha- We would like to express our heartfelt thanks to Stanisław nism of resistance to particular chemotherapeutic agents and the Pecka of the Department of Nutrition of the University location of the genes responsible for it, increasing amounts of met- of Life Sciences in Lublin for his invaluable assistance in pre- als in tissues may be presumed to function as indirect – following paring this publication.

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