The effect of heterogeneous copper micro-supplementation on fatty acid profiles in the tissues of snails Helix pomatia (Gastropoda Pulmonata)

Ecological Indicators 76 (2017) 335–343

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The effect of heterogeneous copper micro-supplementation on fatty acid profiles in the tissues of snails Helix pomatia (Gastropoda Pulmonata) Danuta Kowalczyk-Pecka a,∗ , Edyta Kowalczuk-Vasilev b , Stanisław Pecka b a

Department of Zoology, Animal Ecology and Wildlife Management, Poland Institute of Animal Nutrition and Bromatology, Faculty of Biology, Animal Sciences and Bioeconomy, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland b

a r t i c l e

i n f o

Article history: Received 18 July 2016 Received in revised form 24 January 2017 Accepted 26 January 2017 Available online 7 February 2017 Keywords: Copper Chelates Fatty acid Snail Biomarker

a b s t r a c t We analyzed the interesting phenomenon of the increase in the content of polyunsaturated fatty acids (PUFAs) in the foot tissues and hepatopancreas of Helix pomatia L. snails resulting from administration of five microdoses of copper (0.0001, 0.00025, 0.0005, 0.00075, or 0.001 mg/ml) in three forms: pure salt solution, EDTA, or lysine chelates. The snails received the copper solution per os in the dose of 10 ␮l twice a week. The experiment lasted 12 weeks. The content of PUFAs increased as the supplementation with copper increased up to 0.001 mg/ml. This trend was observed in both analyzed tissue types − foot and hepatopancreas. The greatest accumulation of copper was observed in the groups receiving the lysine chelate. Copper added to the diet of the snails significantly increased the individual (C18:2 n-6, C18:3 n-3, C20:2, C20:4 n-6, C20:5 n-3, C22:4 n-6, and C22:5 n-3) and total share of PUFAs. In all treatments, very strong (r ∼ 1) negative correlations (p ≤ 0.01) were found between Cu contained in the diet and the individual saturated FA or their sums in the tissues. The results proved that determination of FA profile in snails can be used in ecotoxicological research as a reliable test of the effect of trace doses of stressors. The results analyzed indicate that the micro addition of copper induced an evidently positive stimulation of metabolic transformations of fatty acids in the snails. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Due to their common occurrence in the natural environment and their susceptibility to accumulate contaminants, gastropods play an important role in biomonitoring and ecotoxicology (Beeby and Richmond, 2011; Côte et al., 2015; Das and Khangarot, 2011; Dummee et al., 2015; Notten et al., 2006, 2008). These organisms are good research material in both natural and laboratory conditions (Bourioug et al., 2015; Kowalczyk-Pecka et al., 2015; Yasoshima et al., 2001). Previous laboratory studies on mollusks as bioindicator organisms were carried out with the use of high doses of pollutants, e.g. metals, and focused on observation of pronounced responses to stress (Cortet et al., 1999; Manzl et al., 2004). The kinetics of accumulation of metals in the bodies of mollusks and

∗ Corresponding author at: Danuta Kowalczyk-Pecka. Department of Zoology, Animal Ecology and Wildlife Management, Faculty of Biology, Animal Sciences and Bioeconomy, University of Life Sciences in Lublin, Akademicka 13, Lublin, 20-950, Poland. E-mail address: [email protected] (D. Kowalczyk-Pecka). http://dx.doi.org/10.1016/j.ecolind.2017.01.031 1470-160X/© 2017 Elsevier Ltd. All rights reserved.

detoxification continue to be a subject of discussion and research (Berandah et al., 2010; Coeurdassier et al., 2010; Menta and Parisi, 2001; Radwan et al., 2010a; Ramskov et al., 2015). Metals are accumulated actively or passively by organisms and may be stored in the body or excreted from the each organism has a specific strategy of protection from harmful effects of metals (Ardestani et al., 2014). Gastropods accumulate substantial quantities of pollutants in their bodies, often without lethal consequences (Holan et al., 2017; Itziou and Dimitriadis, 2012; Jordaens et al., 2006a,b). The mechanism of changes induced by environmental stressors is mainly based on stimulation of oxidative reactions (Chandran et al., 2005), which in turn entail diverse negative physiological effects, including lipid peroxidation manifested by saturation of fatty acids (El-Shenawy et al., 2012). Copper may have an inhibitory effect on the growth of fungi, bacteria, and some animal life forms, particularly those that inhabit ecosystems, causing mortality in many land and aquatic invertebrates. Copper belongs to the category of borderline metals and is highly toxic to invertebrates, particularly. Due to their natural occurrence, copper compounds are used in organic breeding

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mollusks and crops (Capinera and Dickens, 2016). Copper restricts the migration of snails by acting as an irritant. Elevated copper concentrations have been demonstrated to cause dysfunction of biochemical and physiological mechanisms in invertebrates (Atli and Grosell, 2016; Kulac et al., 2013). Thus, Cu is used as an agent limiting food intake in mollusks, inhibiting growth, and having molluscicidal effects. The major lethal effects of Cu in snails include disruption of the transporting surface epithelium and osmoregulation and a possible increase in water accumulation in tissues (Hoang et al., 2008). On the other hand, copper is essential for snail physiological functioning. Snails need elevated amounts of Cu as a constituent of hemocyanin which is transformed after accumulation (Boshoff et al., 2015). The search of biomarkers of chemical agents in biomarker organisms is still a current issue. It aims to simplify the methods and reduce the cost of analysis while maintaining their accuracy, reliability, and repeatability. Biomarkers can be any changes, including structural or functional changes, in induced biochemical cellular processes that can be measured or visualized in another way. Fatty acids have not been considered as biomarkers of chemical stress so far. The hypothesis of the research assumed that subthreshold doses of copper as a xenobiotic substance affect the fatty acid profile in snails, therefore, determination of their fatty acid profile could be used in routine ecotoxicological tests as a useful biomarker of snail exposure to even slight xenobiotic pollution. For this aim, the experiment was designed to assess the effect of five microdoses of copper administered in three forms: inorganic (pure salt solution) and organic (EDTA and lysine chelates) as a dietary supplement for Helix pomatia L. on chosen biomarker fatty acids determined in the foot tissues and hepatopancreas of the snails.

2. Material and methods 2.1. Experiment design The Helix pomatia L. snails used in the study were obtained from the naturalized laboratory population maintained at the Department of Zoology, Animal Ecology and Wildlife Management of the University of Life Sciences in Lublin on a diet prepared according to Ligaszewski et al. (2008) with slight modification; i.e. without soybean oil, which could affect the natural profile of fatty acids (FA). Adult snails with a well-defined lip on the shell, weighing 23.0 ± 1.0 g and having shells 40.0 ± 1.0 mm in diameter, were sampled from the laboratory population. After they had been thoroughly rinsed with water, the snails were placed individually in perforated plastic (PE-HD) containers (15 × 15 × 5 cm) and then transferred to a phytotron chamber (BIOGENET; 20 ◦ C, RH = 90%, photoperiod 18 h L/6 h D) for further experiments. The experiment involved 170 snails divided into 17 groups, 10 snails in each. To determine the possible impact of the phytotron chamber environment on the FA profile of the snails, an initial control group (Initial) was also included in the experiment. The snails from the initial group were sampled from the laboratory population and left without food for 48 h to clear the digestive tract, and then frozen at −70 ◦ C for further biochemical analysis. The snails from the remaining 16 groups were kept in the BIOGENET for 12 weeks. The animals were fed twice a week with a medium prepared according to Laskowski and Hopkin (1996) with slight modification (without any fungicide, which is a xenobiotic and might affect the FA profile). The medium contained 1 g of agar (Difco), 3 g of dried and powdered carrot root (Daucus carota L.), 0.5 g of milk powder (SM Siedlce) + 0.5 g wheat bran, and 0.01 g CaCO3 (BDH Ltd, UK); it was suspended in doubledistilled water to obtain 100 ml of medium. A volume of 15 ml of

the medium was poured into Petri dishes and left to solidify before individual feeding. This volume of the medium was estimated to be consumed completely and was therefore optimal. The laboratory control group of snails (Control) received the medium twice a week, with the addition of 10 ␮l of pure redistilled water (per os). The other 15 experimental groups of snails were exposed to copper administered each time the medium was given to the snails. The snails received per os 10 ␮l of copper solution per individual snail (with a pipette). Copper was introduced in 3 forms: a solution containing Cu2+ ions (CuSO4 , POCH S.A.), a solution containing a copper EDTA chelate (POCH S.A.), and a solution containing a copper lysine chelate (POCH S.A.). All of the solutions were prepared at concentrations of 0.0001, 0.00025, 0.0005, 0.00075, or 0.001 mg of Cu/ml redistilled H2 O (groups designated as 0.1, 0.25, 0.5, 0.75 and 1.0, depending on the group). In this way, 15 experimental groups were established: snails receiving the solutions of copper sulfate (groups designated as Cu 0.1, Cu 0.25, Cu 0.5, Cu 0.75, and Cu 1.0, respectively), snails receiving the solutions of copper EDTA chelate (groups designated as Cu + EDTA 0.1, Cu + EDTA 0.25, Cu + EDTA 0.5, Cu + EDTA 0.75, and Cu + EDTA 1.0, respectively), and snails receiving the solutions of copper lysine chelate (groups designated as Cu + Lys 0.1, Cu + Lys 0.25, Cu + Lys 0.5, Cu + Lys 0.75, and Cu + Lys 1.0, respectively). After 12 weeks, the snails from all groups were left without food for 48 h to clear the digestive tract and then frozen at −70 ◦ C for further biochemical analysis. 2.2. Copper analysis in the tissues Tissue samples from the hepatopancreas and foot of the snails were dried for 18 h at 80 ◦ C until a constant dry weight was obtained. Each sample was weighed to the nearest 0.0001 mg and then 200 mg was placed in 5 ml of 50% HNO3 (according to Beeby and Richmond, 2002) and heated in a heating block at 80 ◦ C for 2 h and then at 210 ◦ C for 2.5 h to mineralize the sample. After cooling, the suspension was filtered (Whatman 541 filter paper) and distilled, deionized water was added to attain a volume of 25 ml. Deionized water prepared in a deionizer Direct Q5 was used throughout the experiment. The same procedure was carried out for the medium supplemented with agar that was given to the snails. The copper content was analyzed by atomic absorption spectrometry at the Central Laboratory of Agroecology of the University of Life Sciences in Lublin which is accredited by the Polish Centre for Accreditation (no. AB 1375). This analytical facility and their quality control system are certified under PN-EN ISO/IEC 17025:2005. The content of Cu was determined using the FAAS flame atomic absorption spectroscopy (FAAS) with a SOLAAR 939/959 spectrophotometer (Unicam, Cambridge, UK) according to Polish Norm PN-EN ISO 6869:2002. Cu was detected at ␭ = 324.8 nm, with 4 mA and 0.5 nm spectral bandpass (LOD 10 ppm). Detection limit was 0.01 ␮g g−1 . The accepted recoveries for spiked samples ranged from 90.0% to 110.5%. The standard solutions of the metal were provided by Merck (Darmstadt, Germany) and their working standards were used. The reference material NIST SRM 2976 (freeze-dried mussel tissue) was analyzed in order to evaluate the quality of the analytical procedures. Blank samples, containing only nitric acid, were used to check a possible contamination as well. To compare the depositions of metals, the results were converted to ␮g per g of dry weight of tissue. 2.3. Obtaining fatty acid methyl esters After initial preparation and lyophilization of the analyzed material, lipids were extracted from the medium, as well as from the hepatopancreas and foot of each snail with a Soxhlet extractor (VELP SCIENTIFICA ser 148 Solvent Exractor). 50 mg of lipids were collected to obtain fatty acids. Fatty acid esters were obtained

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according to PN-EN ISO 12966-1:2015-01 and PN-EN ISO 129662:2011 standards and AOAC Official Method 969.33 (1969). The ester samples were analyzed using a Varian 3800 gas chromatograph with a FID detector and a CP-Wax 52CBWCOT Fused Silica capillary column (length: 60 m, inner diameter: 0.25 mm). The initial temperature for the analysis was 120 ◦ C for 5 min and the final temperature was 210 ◦ C. The injector temperature was 260 ◦ C and the detector temperature was 260 ◦ C. The hydrogen flow rate was 30 ml/min, air flow − 300 ml/min, and helium flow − 1.4 ml/min. The volume of the injected sample was 1 ␮l. The results for the percentage content of fatty acids in the sample were obtained using Star GC Workstation Version 6.30. In addition to the analysis of tissue fatty acids and as a control of the dietary input of FA, the medium FA profile was analyzed as well (Table S1). From a pool of 56 fatty acids analyzed in the tissues of the foot and hepatopancreas of the snails, the FAs proposed as biomarkers for further studies were selected in two steps. Initially, 21 FAs that were mostly affected by the copper treatment were chosen from the pool of 56 FAs (Table S2–S3). Next, to simplify the methodology, 12 FAs were selected. The selection criteria included the greatest percentage among the 21 FAs chosen in the first step and their significant role in physiological processes. The final set of the biomarker FAs comprised C16:0; C18:0; C23:0; C18:1 n-9; C20:1 n-9; C18:2 n-6; C18:3 n-3; C20:2; C20:4 n-6; C20:5 n-3; C22:4 n-6; and C22:5 n-3. The sums of saturated fatty acids (SFAs), iso-SFAs, anteiso-SFAs, monounsaturated (MUFAs), polyunsaturated (PUFAs), n-6, and n-3 fatty acids and their ratios were taken into consideration as well. 2.4. Statistical analysis All the data were analyzed with Statistica software ver. 10 (StatSoft, 2011, License No. AXAP307F818708FA-K). Normality was assessed using the Kolmogorov-Smirnov test, and Levene’s homogeneity of variance test was applied to examine the equality of variances. Two-way ANOVA was used to assess the effect of the dose (0.0001, 0.00025, 0.0005, 0.00075, or 0.001 mg/ml) and the source of the microelement (sulfate, EDTA or lysine) and their interaction on the content of each FA. One-way ANOVA and Tukey’s multiple range tests were performed to compare all the experimental groups and the control one. Pearson correlation coefficients between the experimental factors and the FA content were also calculated at a significance level of p ≤ 0.01. 3. Results The comparison of copper content in the tissues of the both control groups of animals (Initial) and (Control) showed only slight differences in the copper content in the tissues and the fatty acid profile of the foot and hepatopancreas. The supplementation of the snails’ diets with copper significantly increased the copper concentration in both analyzed tissues, the foot and hepatopancreas, compared to the control, and it increased linearly with the dose of the additive (p ≤ 0.01) (Fig. 1A–B). However, the bioavailability of copper depended of its source. Compared to the other two sources of copper, the highest accumulation of this microelement was noted in the groups receiving the lysine chelate (Cu + Lys), which was particularly evident in the hepatopancreas (Fig. 1A–B). Statistical analysis confirmed a significant (p ≤ 0.01) impact of the dose (groups 0.1, 0.25, 0.5, 0.75, or 1.0) and the source of the microelement (sulfate, EDTA, or lysine) on the Cu concentration. The strong relationship between the dose of copper in the diet and its concentration in the tissues was confirmed by Pearson’s correlation analysis. The results confirm a very strong statistically significant (p ≤ 0.01) correlation between the copper

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concentration in the diet and in both tissues (foot and hepatopancreas). Similarly, the introduction of copper to the snails’ diets strongly affected the fat composition of the foot and hepatopancreas. Among the analyzed fatty acids, the greatest volatility and sensitivity to the changes in the diet were shown by palmitic (C16:0), stearic (C18:0), tricosylic (C23:0), oleic (C18:1 n-9), eicosanoic (C20:1 n9), linolenic (C18:2 n-6), ␣-linolenic (C18:3 n-3), eicosadienoic (C20:2), eicosatetraenoic (C20:4 n-6), eicosapentaenoic (C20:5 n3), adrenic (C22:4 n-6), and docosapentaenoic (C22:5 n-3) fatty acid, which were proposed as biomarkers. Irrespective of the source (inorganic − sulfate or organic − EDTA and lysine chelates), copper added to the diet caused significant changes in the fatty acid profile in the snails (Fig. 2A–D; 3A–C; 4A–H). The share of saturated FAs (SFAs) in the Cu supplemented snails decreased sharply, especially in the sulfate and lysine Cu treatments. In all treatments, very strong (r ∼ 1) negative correlations (p ≤ 0.01) were found between Cu contained in the diet and saturated FAs in the foot and the hepatopancreas (Fig. 5A–B; Table S4–S6). Similarly, the content of copper accumulated in both tissues was negatively correlated with the proportion of saturated fatty acids, with the strongest correlation coefficient noted in the lysine groups. All correlations were statistically significant (p ≤ 0.01). Substantial changes were also observed in the total share of monounsaturated fatty acids (MUFAs) and in the proportions of oleic (C18:1 n-9) and eicosanoic (C20:1 n-9) in the snail tissues. In general, the addition of copper at a dose greater than 0.00025 mg/ml diminished their total content in hepatopancreas by nearly 50%. Similar tendencies were observed in the foot, although the changes were not as evident as in the HP (Fig. 3A–C; Table S2–S3). As in the case of SFAs, the MUFA content was also negatively correlated with the Cu content (Fig. 5A–B). As a result of changes in the proportions of SFA and MUFA, a significant increase (p ≤ 0.01) in the share of polyunsaturated fatty acids (PUFAs) was noted in the snails from the experimental groups, in comparison with the control ones. Statistical analysis confirmed the significant (p ≤ 0.01) impact of the dose (groups 0.1, 0.25, 0.5, 0.75, or 1.0) and the source of the microelement (sulfate, EDTA, or lysine) on the FA profile in the snail tissues. The share of most of the detected PUFA increased along with the increasing Cu dose, which was confirmed by Pearson’s correlation analysis (Fig. 5A–B; Table S4–S6). All of the PUFAs selected as biomarkers were very strongly positively correlated with the dietary copper level (p ≤ 0.01) as well as the level of copper accumulated in the tissues. The strongest, nearly linear increase (r ∼ 1) was observed in the C18:3 FA in all experimental groups in both the foot and the hepatopancreas (Fig. 4B). Generally, the lowest share of the C18:2 n-6 and C18:3 n-3 FAs was noted in the EDTA chelate groups, compared to the other Cu sources. In turn, substantial variation was noted in the content of long-chain polyunsaturated fatty acids (over 20-carbon FAs) depending on the dose or tissue. A spectacular increase was observed in the share of eicosadienoic (C20:2) and eicosatetraenoic (C20:4 n-6) FAs in all experimental groups, especially in the fat of the hepatopancreas. A positive effect was also observed in the case of eicosapentaenoic (C20:5 n-3, EPA) and docosapentaenoic (C22:5 n-3, DPA) acids (Fig. 4E and G; Table S2S3). Generally, a pronounced influence on the share of EPA and DPA was noted in the groups receiving the higher amounts of copper, irrespective of its source.

4. Discussion Both the exposure time and the dose of xenobiotics affect its accumulation and induce physiological changes. Apparently, even brief exposure to low doses of xenobiotics may cause an explicit

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Fig. 1. The figures (A–B) present the relation between Cu dietary level and its bioaccumulation in foot tissue (A) and in hepatopancreas tissue (B) of Helix pomatia snails [␮g g−1 of DM of tissue].

Fig. 2. The figures (A–D) presents the content of saturated fatty acids in foot (solid line) and hepatopancreas (dotted line) of snails exposed to different forms and doses of copper (the transversal line show mean, and whiskers show standard deviation).

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Fig. 3. The figures (A–C) presents the content of monounsaturated fatty acids in foot (solid line) and hepatopancreas (dotted line) of snails exposed to different forms and doses of copper (the transversal line show mean, and whiskers show standard deviation).

physiological effect. In turn, long-term exposure to even higher doses may stimulate the body’s tolerance (Bjerregaard et al., 2015). Tolerance to contaminants can differentiate even closely related taxa, most likely due to individual responses at the cellular or molecular level (Pirro de and Marshall, 2005). The high tolerance of land gastropods to metals in a polluted environment may be related to their high capacity of accumulation but also isolation of metals in their tissues in the form of silicon capsules. Similarly, detoxifying calcium granules of basophilic cells in snail tissues are one of the intracellular ways of metal binding. Copper can form complexes with carbonate in the snail body. Carbonate is required for shell development whereas copper is accumulated in the tissues. Some snail species can store excess Cu by sequestering the metals in forms that are either metabolically available (as metallothionein) or unavailable (as phosphate granules) (Dummee et al., 2015). The produced specific metallothionein isoforms strongly bind metals. The function of copper-specific metallothionein is associated with the role of copper as a component of hemocyanin (Gomot de Vaufleury and Pihan, 2000). It appears that the different organs (foot and hepatopancreas) respond to contact with xenobiotics at different times and to a varying extent depending on e.g. the rate of metabolism. The unique properties of the mollusk hepatopancreas result from its function and capability of synthesis of enzymes as well as detoxification and storage of xenobiotics. It is known to be one of the important sites of multiple oxidative reactions and contains high basal levels of antioxidant enzymes. The different reaction of the foot tissue, observed also in our study, may be due to its basal levels of metabolism parameters that were sufficient to tolerate Cu (Atli and Grosell, 2016). Gomot and Pihan (1997) found that differences in accumulation of metals in field conditions are associated with different

food preferences of individual snail species and interspecific differences in metabolism. Analyses conducted as part of a microcosm field study are being increasingly published (Boshoff et al., 2015). Results obtained in breeding in laboratory conditions allow tracking the dynamics of accumulation of microdoses of metals, while eliminating the variability of environmental conditions. The ultimate level of metal accumulation is influenced by the dose, form of administration, analyzed tissue, length of the administration period, availability, animal age, breeding conditions, and feed components. Only the first three parameters are the variables in our study. The main hypothesis of the present study was to check the influence of copper microdoses on the fatty acid profiles in the tissues of land snails. Based on the literature and our own experience, we assumed that copper acts as a xenobiotic. The results of the experiment revealed that minimal doses of copper did not inflict serious physiological or even lethal damage in snails, but mobilized beneficial metabolic transformations in the organism instead. The microdoses of copper used in the study were too low to have a detrimental effect on the physiological functions. However, they were sufficient to cause a change in the profile of fatty acids. Copper in snails undergoes a specific form of accumulation and elimination. Among four metals analyzed by Beeby and Richmond (2002), copper taken in with food was not stored for a long period and was removed most quickly from snail tissues. There was no reflection of long-term exposure to copper in the foot tissues or hepatopancreas, which is not the main reservoir of Cu in Gastropoda, in contrast to Zn, Cd, and Pb. However, the results of the present study indicate the bioconcentration of copper microdoses in the tissues, confirmed by the Pearson’s correlation analysis which showed almost linear relationship (r ∼ 0.98). Thus, the dynamic of the concentration and distribution of copper did not

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Fig. 4. The figures (A–H) presents the content of polyunsaturated fatty acids in foot (solid line) and hepatopancreas (dotted line) of snails exposed to different forms and doses of copper (the transversal line show mean, and whiskers show standard deviation).

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Fig. 5. The average values of Pearson correlation coefficient (r) between the content of copper (Cu) supplemented in the diet (irrespective of the source) and the Cu concentration in the foot tissues and the share of fatty acids (FA) in foot of snails (A) and the Cu concentration in the hepatopancreas (HP) and the share of fatty acids (FA) in HP of snails (B); all correlation coefficients are statistically significant at the level p ≤ 0.01.

affect the doses and animals tested. The differences in copper accumulation may result from small differences between the analyzed tissues types of snails and their abilities in the excretion or storage of copper. Cu, like Fe, acts as a catalyst for the Fenton or Haber-Weiss reactions, facilitating the conversion of superoxide anion and hydrogen peroxide to the hydroxyl radical. Oxidative stress may lead to DNA damage, lipid peroxidation, and protein modification (Atli and Grosell, 2016; Radwan et al., 2010a,b). The differences observed in the level of fatty acid saturation between the tissues foot and hepatopancreas may be due in part to the different content of polyunsaturated fatty acids in these organs and the different levels of cellular respiration (Correia et al., 2003; Misra et al., 2002). The analysis of fatty acid profile of snails as physiological parameters was neglected for many years. However, the fatty acid profile can be very precise and sensitive biomarker induced by xenobiotics. In this study we considered the fatty acid profile as a biomarker of exposure to copper. We were expecting an increased saturation of fatty acids as a peroxidative reaction. It turned out that copper microsupplementation at the dosages 0.0001, 0.00025, 0.0005, 0.00075, 0.001 mg/ml administered 2 times per week in a volume of 10 ␮l for 12 weeks, has increased the amount of PUFAs. Perhaps the observed response was due to the fact that xenobiotic − copper was applied not once, but periodically, over a relatively long period of time and at minimal doses (0.01–0.001 ␮g). Nevertheless, taking into account the protecting role of PUFAs in cell membrane integrity (Misra et al., 2002; Vijayavel et al., 2007), and the fact that some of the PUFAs exhibit antibacterial activity (Kowalczyk-Pecka and Puchalski, 2008) these findings might be considered as beneficial. However, further studies are needed to explain those mechanisms in details. Mollusks as a group have a unique composition of sterols and fatty acids (Özogul et al., 2005). Among polyunsaturated fatty acids

in snails, linoleic (C18:2 n-6) and arachidonic (C20:4 n-6) acids have been found to be the most frequent n-6 acids, while linolenic (C18:3 n-3) and eicosapentaenoic (C20:5 n-3) were dominant among n-3 acids (Zhu et al., 1994). In the present study, a significant amount of C22:5 n-3, beside the above-mentioned acids, was noted as well. Interestingly, in our studies (Kowalczyk-Pecka et al., 2016, 2017), in contrast to previous reports, the examined snails demonstrated a high content of non-methylene interrupted dienoic fatty acids (NMDI) such as C20:2. Highly unsaturated fatty acids such as 20and 22-carbon FA of the n-3 family are present in small quantities (Allen et al., 2001; Freites et al., 2002; Pirini et al., 2007). The analysis of the results obtained in this study yielded a set of 12 biomarker acids undergoing significant changes in contact with five microdoses of copper. The selection criteria included the greatest percentage among the FA profile and their significant role in physiological processes. The quantitative changes noted in the fatty acid profiles are a reflection of changes in the cellular metabolism initiated by an external factor, i.e. the microdoses of copper. The changes involved the content of marker fatty acids, with a tendency towards an increase in polyunsaturated acids, and the decrease in the n-6 to n-3 ratio with the increasing microdoses of copper. 5. Conclusion The supplementation of copper in the inorganic (sulfate) or the organic forms (EDTA and lysine chelates) to the snails’ diet had a significant effect on the fatty acid profile in both the foot and hepatopancreas tissues. The results indicate that the microaddition of copper induced an evidently positive stimulation of metabolic transformations of fatty acids in the snails Helix pomatia. It may be concluded that the doses of 0.0001–0.001 mg Cu/ml did not induce saturation of the fatty acids. Furthermore, an increase in PUFAs was observed in the snail tissues. The significant changes in the

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snail fatty acid profiles in response to the microdoses of copper, manifested by FA fluctuations, proved that the selected fatty acids (C16:0; C18:0; C23:0; C18:1 n-9; C20:1 n-9; C18:2 n-6; C18:3 n3; C20:2; C20:4 n-6; C20:5 n-3; C22:4 n-6; and C22:5 n-3) might be considered as physiological biomarkers. This may be the result of highly diverse regulatory processes at many levels of organization, beginning with regulation of induction of gene expression. This provides an incentive to conduct further detailed research on the relationships determining lipid metabolism in land mollusks. Future research on the relationships between microdoses of metals and saturation of fatty acids in mollusks should attempt to find a direct relationship between the final physiological effect and the level of the metal in the tissues. In order to use the proposed supplementation in practice, the dosages should be optimized according to the species specificity and biological rhythm of snails as well as factors that could cause significant changes in the antioxidant system. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ecolind.2017. 01.031. References Allen, C.E., Tyler, P.A., van Dover, C.L., 2001. Lipid composition of hydrothermal vent clam Calyptogena pacifica (Mollusca: Bivalvia) as trophic indicator. J. Mar. Biol. Ass. UK 81, 817–821. Ardestani, M.M., van Straalen, N.M., van Gestel, C.A.M., 2014. Uptake and elimination kinetics of metals in soil invertebrates: a review. Environ. Poll. 193, 277–295. Atli, G., Grosell, M., 2016. Characterization and response of antioxidant systems in the tissues of the freshwater pond snail (Lymnaea stagnalis) during acute copper exposure. Aquat. Toxicol. 176, 38–44. Beeby, A., Richmond, L., 2002. Evaluating Helix aspersa as a sentinel for mapping metal pollution. Ecol. Indic. 1, 261–270. Beeby, A., Richmond, L., 2011. Sources of variation in the assimilation of lead by a common gastropod sentinel Cantareus aspersus. Sci. Total Environ. 409, 5499–5504. Berandah, F.E., Kong, Y.C., Ismail, A., 2010. Bioaccumulation and distribution of heavy metals (Cd, Cu, Fe, Ni, Pb and Zn) in the different tissues of Chicoreus capucinus Lamarck (Mollusca: Muricidae) collected from Sungai Janggut Kuala Langat, Malaysia. Environ. Asia 3 (1), 65–71. Bjerregaard, P., Andersen, C.B.I., Andersen, O., 2015. Ecotoxicology of Metals—Sources, Transport, and Effects on the Ecosystem. In: Nordberg, G.F., Fowler, B.A., Nordberg, M. (Eds.), Handbook on the Toxicology of Metals. , 4th edition. Elsevier, Academic Press, pp. 425–459. Boshoff, M., Jordaens, K., Baguet, S., Bervoets, L., 2015. Trace metal transfer in a soil–plant–snail microcosm field experiment and biomarker responses in snailsl. Ecol. Indic. 48, 636–648. Bourioug, M., Gimbert, F., Alaoui-Sehmer, L., Benbrahim, M., Aleya, L., Alaoui-Sossé, B., 2015. Sewage sludge application in a plantation: effects on trace metal transfer in soil–plant–snail continuum. Sci. Total Environ. 502, 309–314. Capinera, J.L., Dickens, K., 2016. Some effect of copper-based fungicides on plant-feeding terrestrial molluscs: a role for repelents in mollusc management. Crop Prot. 83, 76–82. Chandran, R., Sivakumar, A.A., Mohandass, S., Aruchami, M., 2005. Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod, Achatina fulica. Comp. Biochem. Phys. C 140, 422–426. Coeurdassier, M., Scheifler, R., Mench, M., Crini, N., Vangronsveld, J., de Vaufleury, A., 2010. Arsenic transfer and impacts on snails exposed to stabilized and untreated As-contaminated soils. Environ. Poll. 158, 2078–2083. Cortet, J., Gomot-De Vauflery, A., Poinsot-Balaguer, N., Gomot, L., Texier, C., Cluzeau, D., 1999. The use of invertebrate soil fauna in monitoring pollutant effects. Eur. J. Soil Biol. 35 (3), 115–134. Correia, A.D., Costa, M.H., Luis, O.J., Livingstone, D.R., 2003. Age-related changes in antioxidant enzyme activities, fatty acid composition and lipid peroxidation in whole body Gammarus locusta (Crustacea: Amphipoda). J. Exp. Mar. Biol. Ecol. 289, 83–101.

Côte, J., Bouétard, A., Pronost, Y., Besnard, A.-L., Coke, M., Piquet, F., Caquet, T., Coutellec, M.-A., 2015. Genetic variation of Lymnaea stagnalis tolerance to copper: a test of selection hypotheses and its relevance for ecological risk assessment. Environ. Poll. 205, 209–217. Das, S., Khangarot, B.S., 2011. Bioaccumulation of copper and toxic effects on feeding, growth, fecundity and development of pond snail Lymnaea luteola L. J. Hazard. Mater. 185, 295–305. Dummee, V., Tanhan, P., Kruatrachue, M., Damrongphol, P., Pokethitiyook, P., 2015. Histopathological changes in snail, Pomacea canaliculata, exposed to sub-lethal copper sulfate concentrations. Ecotoxicol. Environ. Saf. 122, 290–295. El-Shenawy, N.S., Mohammadden, A., Al-Fahmie, Z.H., 2012. Using the enzymatic and non-enzymatic antioxidant defence system of the land snail Eobania vermiculata as biomarkers of terrestrial heavy metal pollution. Ecotoxicol. Environ. Saf. 84, 347–354. Freites, L., Fernandez-Reiriz, M.J., Labarta, U., 2002. Fatty acid profiles of Mytilus galloprovincialis (Lmk) mussel of subtidal and rocky shore origin. Comp. Biochem. Phys. B 132, 453–461. Gomot, A., Pihan, F., 1997. Comparison of the bioaccumulation capacities of copper and zinc in two snail subspecies (Helix). Ecotoxicol. Environ. Saf. 38, 85–94. Gomot de Vaufleury, A., Pihan, F., 2000. Growing snails used as sentinels to evaluate terrestrial environment contamination by trace elements. Chemosphere 40, 275–284. Hoang, T.C., Rogevich, E.C., Rand, G.M., Gardinali, P.R., Frakes, R.A., Bargar, T.A., 2008. Copper desorption in flooded agricultural soils and toxicity to the Florida apple snail (Pomacea paludosa): implications in Everglades restoration. Environ. Poll. 154, 338–347. Holan, J.R., King, C.K., Sfiligoj, B.J., Davis, A.R., 2017. Toxicity of copper to three common subantarctic marine gastropods. Ecotoxicol. Environ. Saf. 136, 70–77. Itziou, A., Dimitriadis, V.K., 2012. Multiple pollution biomarker application on tissues of Eobania vermiculata during two periods characterized by augment and reduced snail activity. Ecotoxicol. Environ. Saf. 86, 13–22. Jordaens, K., de Wolf, H., van Houtte, N., Vandecasteele, B., Backeljau, T., 2006a. Genetic variation in two land snails, Cepaea nemoralis and Succinea putris (Gastropoda, Pulmonata), from sites differing in heavy metal content. Genetica 128, 227–239. Jordaens, K., de Wolf, H., Vandecasteele, B., Blust, R., Backeljau, T., 2006b. Associations between shell strength, shell morphology and heavy metals in the land snail Cepaea nemoralis (Gastropoda: Helicidae). Sci. Total Environ. 363, 285–293. Kowalczyk-Pecka, D., Puchalski, A., 2008. Potential interaction between the Cepaea nemoralis wild snail and Citrobacter spp bacteria. Vet. Med. 64 (6), 786–790. Kowalczyk-Pecka, D., Stryjecki, R., Czepiel-Mil, K., 2015. Comparison of the bioaccumulation potential of selected metals in the tissues of three invertebrate taxa: Cepaea, Lumbricus and Geotrupes. Teka (Archives) Commis. Prot. Form. Nat. Environ.–OL PAN 12, 31–37. Kowalczyk-Pecka, D., Pecka, S., Kowalczuk-Vasilev, E., 2016. Selected fatty acids as biomarkers of exposure to microdoses of molluscicides in snails Helix pomatia (Gastropoda Pulmonata). Environ. Pollut., http://dx.doi.org/10.1016/j.envpol. 2016.12.068. Kowalczyk-Pecka, D., Pecka, S., Kowalczuk-Vasilev, E., 2017. Changes in fatty acid metabolism induced by varied micro-supplementation with zinc in snails Helix pomatia (Gastropoda Pulmonata). Ecotoxicol. Environ. Saf. 138, 223–230. Kulac, B., Atli, G., Canli, M., 2013. Response of ATPases in the osmoregulatory tissues of freshwater fish Oreochromis niloticus exposed to copper in increased salinity. Fish Physiol. Biochem. 39, 391–401. Laskowski, R., Hopkin, S.P., 1996. Effect of Zn, Cu, Pb and Cd on fitness In snails (Helix aspersa). Ecotoxicol. Environ. Saf. 34, 59–69. Ligaszewski, M. Łysak, A., Mach-Paluszkiewicz, Z., 2008. Principles of breeding and rearing Helix pomatia snail. Manual Implementation Ed. IZ-PIB, Cracow, i-12/08, pp. 1–14. Manzl, C., Krumschnabel, G., Schwarzbaum, P.J., Dallinger, R., 2004. Acute toxicity of cadmium and copper in hepatopancreas cells from the Roman snail (Helix pomatia). Comp. Biochem. Phys. C 138, 45–52. Menta, C., Parisi, V., 2001. Metal concentration in Helix pomatia, Helix aspersa, and Arion rufus: a comparative study. Environ. Poll. 115, 205–208. Misra, K.K., Shkrob, I., Rakshit, S., Dembitsky, V.M., 2002. Variability in fatty acids and fatty aldehydes in different organs of two prosobranch gastropod mollusks. Biochem. Syst. Ecol. 30, 749–761. Notten, M.J.M., Oosthoek, A.J.P., Rozema, J., Aerts, R., 2006. Heavy metal pollution affects consumption and reproduction of the landsnail Cepaea nemoralis fed on naturally polluted Urtica dioica leaves. Ecotoxicology 15, 295–304. Notten, M.J.M., Walraven, N., Beets, C.J., Vroon, P., Rozema, J., Aerts, R., 2008. Investigating the origin of Pb pollution in a terrestrial soil-plant-snail food chain by means of Pb isotope ratios. Appl. Geochem. 23, 1581–1593. Özogul, Y., Özogul, F., Olgunoglu, A.I., 2005. Fatty acid profile and mineral content of the wild snail (Helix pomatia) from the region of the south of the Turkey. Eur. Food Res. Technol. 221, 547–549. Pirini, M., Manuzzi, M.P., Pagliarani, A., Trombetti, F., Borgatti, A.R., Ventrella, V., 2007. Changes in fatty acid composition of Mytilus galloprovincialis (Lmk) fed on microalgal and wheat germ diets. Comp. Biochem. Phys. B 147, 616–626. Pirro de, M., Marshall, D.J., 2005. Phylogenetic differences in cardiac activity, metal accumulation and mortality of limpets exposed to copper: a prosobranch-pulmonate comparison. J. Exp. Mar. Biol. Ecol. 322, 29–37. Radwan, M.A., El-Gendy, S., Gad, A.F., 2010a. Biomarkers of oxidative stress in the land snail, Theba pisana for assessing ecotoxicological effects of urban metal pollution. Chemosphere 79, 40–46.

D. Kowalczyk-Pecka et al. / Ecological Indicators 76 (2017) 335–343 Radwan, M.A., El-Gendy, S., Gad, A.F., 2010b. Oxidative stress biomarkers in the digestive gland of Theba pisana exposed to heavy metals. Arch. Environ. Contam. Toxicol. 58, 828–835. Ramskov, T., Croteau, M.-N., Forbes, V.E., Selck, H., 2015. Bikinetics of different-shaped copper oxide nanoparticles in the freshwater gastropod, Potamopyrgus antipodarum. Aquat. Toxicol. 163, 71–80. Vijayavel, K., Gopalakrishnana, S., Balasubramanian, M.P., 2007. Sublethal effect of silver and chromium in the green mussel Perna viridis with reference to alteration in oxygen uptake, filtration rate and membrane bound ATPase system as biomarkers. Chemosphere 69, 979–986.

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Yasoshima, M., Matsuo, M., Kuno, A., Takano, B., 2001. Studies on intake of heavy metals by Bradybaena similaris, land snails, by XAFS measurement. J. Synchrotron. Rad. 8, 969–971. Zhu, N., Dai, X., Lin, D.S., Connor, W.E., 1994. The lipids of slugs and snail: evolution, diet and biosynthesis. Lipids 29 (12), 869–875.