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Histopathological changes in snail, Pomacea canaliculata, exposed to sub-lethal copper sulfate concentrations
Ecotoxicology and Environmental Safety 122 (2015) 290–295
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Histopathological changes in snail, Pomacea canaliculata, exposed to sub-lethal copper sulfate concentrations Vipawee Dummee a,b, Phanwimol Tanhan c,n, Maleeya Kruatrachue a, Praneet Damrongphol a, Prayad Pokethitiyook a a
Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Interdisciplinary Graduate School of Earth System and Andaman Natural Disaster Management, Prince of Songkla University, Phuket Campus, Kathu, Phuket 83120, Thailand c Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, 50 Ngamwongwan Road, Chatujak, Bangkok 10900, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 February 2015 Received in revised form 11 August 2015 Accepted 11 August 2015 Available online 20 September 2015
The acute toxicity test of Cu including range-finding and definitive test, was performed on golden apple snails, Pomacea canaliculata. The median lethal concentrations (LC50) of Cu at exposure times of 24, 48, 72 and 96 h were 330, 223, 177 and 146 mg/L, respectively. P. canaliculata were exposed to Cu at 146 mg/L for 96 h to study bioaccumulation and histopathological alterations in various organs. Snails accumulated elevated levels of Cu in gill, and lesser amounts in the digestive tract, muscle, and digestive gland. Histopathological investigation revealed several alterations in the epithelia of gill, digestive tract (esophagus, intestine, rectum), and digestive gland. The most striking changes were observed in the epithelium of the gill in which there was loss of cilia, an increase in number of mucus cells, and degeneration of columnar cells. Similar changes occurred in digestive tract epithelium. The digestive gland showed moderate alterations, vacuolization and degeneration of cells and an increase in the number of basophilic cells. We concluded that, P. canaliculata has a great potential as a bioindicator for Cu, and a biomarker for monitoring Cu contamination in aquatic environment. & 2015 Elsevier Inc. All rights reserved.
Keywords: Histopathological changes Copper Sub-lethal condition LC50 Bioindicator Biomarker
1. Introduction Heavy metals encompass a wide range of substances that are non-degradable, highly persistent in the environment and which may be incorporated into the tissues of living organisms (Ortíz et al., 1999). Among heavy metals, Cu plays various physiological roles for a number of key metabolic enzymes in plants and animals (Watanabe et al., 1997). In gastropod mollusks, it is involved in hemocyanin metabolism (Gullick et al., 1981; White and Rainbow, 1985). However, increased concentration of Cu in organisms is potentially toxic since it interferes with numerous physiological processes. The major lethal effects of Cu in gastropod mollusks are disruption of the transporting surface epithelium and osmoregulation, eventually causing water accumulation in mollusk tissues (Cheng, 1979). High levels of Cu (4 200 mg/kg) were recently reported in the sediments of tributaries feeding into Beung Boraphet, a large freshwater lake in Nakhon Sawan province, Thailand (Dummee et al., 2012). These most likely resulted from the excessive use of n
Corresponding author. E-mail address: [email protected] (P. Tanhan).
http://dx.doi.org/10.1016/j.ecoenv.2015.08.010 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Cu-based fungicides, molluscicides, and pesticides in the rice paddies surrounding the tributaries (Dummee et al., 2012). Golden apple snails, Pomacea canaliculata, which were abundant in the area, contained high levels of Cu (81.8–127.8 mg/kg) in their visceral mass and foot. Several tools or biomarkers may be used to manage heavy metal contamination, such as toxicity assessment, histopathological evaluation, and biochemical assessment. Toxicity testing is an essential tool for assessing the effect and fate of toxicants in aquatic ecosystem, and for identifying suitable organisms as bioindicators (Shuhaimi-Othman et al., 2012). In addition, acute toxicity tests can help in the detection, evaluation, and abatement of pollution by providing reliable estimates of safe concentrations of toxicants (Ahsanullah et al., 1981). Histopathological alterations reveal effects of pollution on the tissues of exposed organisms, and provide early indications of toxicity (Sawasdee et al., 2011). Mollusks have long been regarded as promising bioindicator and biomonitoring subjects because they are abundant, highly tolerant to many pollutants, and exhibit high accumulation of heavy metals (Lau et al., 1998; Shuhaimi-Othman et al., 2012). Several studies reported on the acute and chronic toxicity of heavy metals, and attendant histopathological alterations in gastropod mollusks, such as Filopaludina martensi martensi (Jantataeme et al.,
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1996), Babylonia areolata (Supanopas et al., 2005; Tanhan et al., 2005), Melanoides tuberculata (Shuhaimi-Othman et al., 2012), Pomacea paludosa (T. Hoang et al., 2008; T.C. Hoang et al., 2008), and P. canaliculata (Dummee et al., 2012; Kruatrachue et al., 2011). In mollusks, the digestive gland is thought to play the most important role in metabolism of endogenous and xenobiotic compounds and is likely important in the storage and regulation of metals in terrestrial and aquatic gastropods. The introduced golden apple snail (P. canaliculata) is an agricultural pest in Asia and is now the most widespread snail species in Thailand where it is ubiquitous in aquatic habitats. They feed not only on rice but also on taro and lotus plants, causing much loss to the farmers. The present work aimed to assess the potential of P. canaliculata as a bioindicator and biomonitor of Cu in the aquatic environment. We investigated the acute toxicity in P. canaliculata exposed to Cu and evaluated Cu accumulation and associated histopathological manifestations in the soft tissues including the digestive organs.
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them in water (Otitoloju et al., 2009). 2.4. Accumulation study P. canaliculata were exposed to the median lethal concentration (LC50) for 96 h. Ten snails were placed in each of three replicates including controls. Two replicates were used to obtain the Cu accumulation analysis and the third was used for the histopathological study. After 96 h, the soft tissues were separated from shells, snail organs (gill, digestive tract and digestive gland) and head– foot (muscle) were isolated. The tissue samples were oven dried at 80 °C, and 0.5 g of each dried samples was digested with 67% conc. HNO3 (67%) according to APHA et al. (2011). The Cu concentrations were then determined using a flame atomic absorption spectrophotometer (FAAS; SpectrAA-55B). The Cu concentrations in each digested organ were determined by comparing its absorbance with known Cu standard solutions. The method detection limit was calculated by a standard procedure which is based on the analysis of ten samples of the matrix with the analyte. The lowest detection limit for Cu was 0.5 mg/g dw.
2. Materials and methods 2.5. Bioaccumulation efficiency 2.1. Chemical The stock of Cu solution (1000 μg/L) was prepared by dissolving Cu sulfate (hydrated form; CuSO4 5H2O) in deionized water according to APHA et al. (2011). Concentrations were expressed as micrograms of Cu ions (Cu2 þ ) per liter (μg/L) of solution. The stock solution was left for 1 day to equilibrate. Nominal Cu concentrations (0, 100, 150, 200, 250, 400, and 800 μg/L) were then prepared from the stock solution diluted to 1 L with deionized water in preacid washed aquarium.
Bioaccumulation is the process in which a chemical substance is absorbed in an organism by all routes of exposure including dietary and ambient environment sources (Arnot and Gobas, 2006). The bioaccumulation efficiency of Cu in P. canaliculata was evaluated using two parameters. First, the bioaccumulation capacity (BAC) was the ratio of the concentration of the metal in snail organs after 96 h of exposure to the level detected in the same organs of control snails (Otitoloju et al., 2009). Second, the bioconcentration factor (BCF) was the ratio of the metal concentration in snails to the concentration in water (T. Hoang et al., 2008).
2.2. Experimental animal 2.6. Histopathological study P. canaliculata eggs were collected from unpolluted natural ponds in Kanchanaburi province, Thailand. Eggs were hatched in the laboratory and juvenile snails were reared in the aerated aquaria with dechlorinated water at 25–27 °C and daily fed with lettuce until they were 2 months old. The uniform snails (shell length 2–3 cm) were used for acute toxicity tests and histopathological study. 2.3. Acute toxicity study The static (non-renewal, without food) technique was used for the acute toxicity test. The toxicity range-finding test consisted of a down-scale (100, 10, 1 and 0.10 μg/L of Cu) abbreviated static acute test in which groups of organisms were exposed to several widely spaced sample dilutions in a logarithmic series, and a control, for a period of 96 h. The definitive test was performed to follow-up the range-finding test. During the acclimatization (24 h) snails were fasted to prevent the binding of Cu to the accumulated feces (Ng et al., 2011). No mortality of P. canaliculata was detected during this period. One liter of each test solution (and controls) was added to 4 L glass aquaria at the nominal concentrations previously described. Three replicates for each concentration, including controls, were performed. Ten snails were placed in each aquarium and were not fed during the period of the bioassay. The dead and living snails were counted daily throughout the 96-h period. Dead snails were removed immediately to avoid the bacterial infection of other living snails. The percentage survival and mortality were calculated for each metal concentration. Snails were taken to be dead if there was no movement when the foot region was prodded with the metal needle or if there was no activity after 5 min of placing
After 96 h of exposure, the gill, digestive tract and digestive gland of P. canaliculata were dissected and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 12 h. Organs were washed with 0.1 M sodium cacodylate buffer three times before they were post-fixed in 2% osmium tetroxide (OsO4) for overnight. Samples were washed in 0.1 M sodium cacodylate buffer, dehydrated with the graded series of acetone, and embedded in aradite resin (Thophon et al., 2003). The semi-thin sections (1 μm) were prepared on an ultramicrotome, mounted on glass slides, stained with 1% toluidine blue in 1% borax, and examined under a light microscope (Olympus CH40). 2.7. Statistical analysis The dose–response data was analyzed by Probit analysis based on a computer program (SPSS version 15.0). The indices of toxicity measurement derived from this analysis were lethal concentrations (LC) at 25%, 50%, 75% and 99%. Analysis of variance (ANOVA) was carried out to compare the Cu accumulation in several treatments and organs mean at Po 0.05 level of significance (Thophon et al., 2003).
3. Results 3.1. Toxicity study The percentage mortality of P. canaliculata generally increased with increasing Cu concentration and exposure time (Table 1). A Cu concentration higher than 0.40 μg/L caused very high mortality
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Table 1 Percentage mortality of P. canaliculata exposed to various Cu concentrations. Cu concentration (μg/L)
0 100 150 200 250 400 800
Exposure time (h) 0 24
48
72
96
0 0 0 0 0 0 0
0 0 13.3 7 6.67 66.77 17.6 40.0 723.1 93.3 76.67 100
0 0 33.3 7 6.67 80.0 711.6 73.3 7 6.67 100 100
0 6.677 6.67 66.7 713.3 86.7 7 13.3 86.7 7 6.67 100 100
0 0 0 26.7 7 6.67 20.0 7 11.6 60.0 7 11.6 100
Table 2 Lethal concentration values and 95% confidence limits of Cu at different exposure times. Exposure time (h)
0 24 48 72 96
Lethal concentration (μg/L) LC25 (95% C.L.)
LC50 (95% C.L.) LC75 (95% C.L.)
LC99 (95% C.L.)
0 245 (200–288) 169 (58–225) 143 (113–163) 101 (0–156)
0 330 (281–410) 223 (149–381) 177 (154–201) 146 (0–241)
0 914 (642– 1932) 576 (345–26,481) 372 (296–620) 522 (284–a)
0 443 (366–626) 294 (221– 1129) 220 (195–266) 211 (115–1503,906)
C.L.–Confidence Limit. a
Concentration greater than 1024 mg/L.
(4 80%; Table 1) with sublethal effects occurring at lesser concentrations (100–250 μg/L). LC values showed a gradual decrease with an increase in exposure time (Table 2). The LC50 values of Cu were 330, 223, 177, and 146 mg/L for 24 h, 48 h, 72 h, and 96 h, respectively. 3.2. CU accumulation Copper accumulated significantly in the gill and digestive tract of P. canaliculata exposed to acute concentration (146 mg/L) for 96 h compared with control snails (Table 3). The highest Cu concentration was found in the gill (71.1 mg/g) with lesser concentrations in the digestive tract (47.1 mg/g) 4 muscle (38.6 mg/ g)4 digestive gland (30.7 mg/g). The BCF values also showed similar trend to those of Cu accumulation, i.e., gill (50.8) 4digestive tract (33.7) 4muscle (27.6) 4digestive gland (22.0) (Table 3). Snails accumulated Cu ions at levels about 1–2 times higher than those detected within organs of control snails (Table 3). 3.3. Histopathological study 3.3.1. Digestive gland The digestive gland or hepatopancreas of P. canaliculata was composed of numerous tubules, which consisted of digestive cells and scarce basophilic secretory cells (Fig. 1A). Hemolymph spaces Table 3 Cu accumulation in P. canaliculata. Cu concentration (mg/g)
Control Treated BAC BCF
Muscle
Gill
Digestive tract
Digestive gland
19.8 7 1.75 38.6 72.93 1.95 27.6
31.9 7 4.12 71.1 74.28 2.23 50.8
33.87 2.65 47.17 4.03 1.39 33.7
30.0 7 5.76 30.7 7 1.97 1.02 22.0
Data are means 7 S.E. Significant differences (Po 0.05) among concentrations were indicated by different letters. BCF: Bioconcentration factor; BAC: Bioaccumulation capacity.
Fig. 1. Digestive gland of P. canaliculata: (A) digestive gland epithelium of control snail showing numerous columnar digestive cells (DC) and a few pyramidal basophilic cells (BC) surrounding the lumen (L). Numerous digestive vacuoles (V) are present in the digestive cell. Notice dark granules (dg) with well-defined membrane. (B) and (C) Digestive gland of treated snail (B) narrow lumen with secretion and vacuolization of digestive cells (DC). (C) Loss of membrane around dark granule (dg) and increase in size of basophilic cell (BC) were observed.
were present among the tubules. Digestive cells were characterized by the presence of numerous vacuoles and oval dark granules of variable size and electron density. Basophilic cells contained homogeneously electron-dense secretory vesicles of varying size with a few dark granules. In treated snails, secretions of the digestive gland tubule in the lumen increased concurrently (Fig. 1B)
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mucus vacuoles and a basal nucleus (Fig. 3A and C). The histopathological alterations in Cu-exposed P. canaliculata, were similar in all three organs. The most striking change was the increase in number of mucus cells (Fig. 3B and D) while loss of cilia was observed in some surface areas of the epithelium. Dilation of the nucleus with the loss of heterochromatin was observed in the epithelial cells of esophagus (Fig. 3B).
4. Discussion 4.1. Cu toxicity and accumulation
Fig. 2. Gill of P. canaliculata: (A) gill filament of control snail showing columnar cells (CC) and a few mucous cells (MC). Notice numerous hemocytes (Hc) in hemolymph space. (B) Gill filament of treated snail showing increase in number of mucus cell (MC) and degeneration of columnar cell (CC).
with the increase in size of basophilic cells (Fig. 1C). The digestive cells underwent vacuolization and the dark granules lost their well-defined membranes (Fig. 1B). In addition, narrowing of the tubular lumen was also observed (Fig. 1B). 3.3.2. Gill The gill of P. canaliculata was composed of numerous gill filaments with long cilia. The gill epithelium contained ciliated-columnar cells, each with a dense oval nucleus and some mucussecreting goblet cells (Fig. 2A). Numerous hemocytes were found in the hemolymph space (Fig. 2A). Histopathological alterations observed in P. canaliculata were an increase in number of mucussecreting goblet cells and the degeneration of columnar cells (Fig. 2B). 3.3.3. Digestive tract The digestive tract of P. canaliculata consisted of the esophagus, intestine, and rectum. The esophagus was lined by pseudostratified columnar epithelium (Fig. 3A) while that of intestine and rectum was of the simple columnar type (Fig. 3C). The columnar epithelium consisted of two cell types: columnar cells and mucussecreting goblet cells. The columnar cells contained light acidophilic granules and an ellipsoidal nucleus rich in heterochromatin (Fig. 3A). The mucus cells were characterized by the presence of
The lethal concentration (LC) values of P. canaliculata exposed to Cu showed a gradual decrease with the increase in exposure time. The 96-h LC50 of Cu was 146 mg/L, similar to the 96-h LC50 of Cu in the Florida apple snail, P. paludosa (140 mg/L) (Rogevich et al., 2008) and in M. tuberculata, another freshwater prosobranch (140 mg/L) (Shuhaimi-Othman et al., 2012). However, P. canaliculata showed less sensitivity to Cu compared to pulmonate gastropods. LC50 values for Lymnaea stagnalis and Biomphalaria glabrata were 24.90 and 40 mg/L, respectively (Bellavere and Gorbi, 1981; Ng et al., 2011). Prosobranch snails have a tightly sealed operculum that allows them to withstand desiccation and apparently also increases their tolerance to metals (Mitchell et al., 2007). In addition, differences in sensitivity to Cu may be due to differences in Cu metabolism in snails such as the excretion or storage of Cu (Farid, 2005). It is also difficult to compare the toxicity values across studies because of differences in the characteristics of the tested waters (especially water hardness, pH, and temperature), species, ages, and sizes of snails tested (Shuhaimi-Othman et al., 2012). Most Cu in P. canaliculata accumulated in the viscera or digestive tract and digestive gland ( 80%) and foot ( 20%). Previous studies also reported that up to 60–80% of Cu was found in the viscera and 20–40% in the foot of snails such as Florida apple snail P. paludosa (T.C. Hoang et al., 2008), pond snail L. stagnalis (Desouky, 2006) and garden snail Helix aspersa (Laskowski and Hopkin, 1996). In addition, T. Hoang et al. (2008) found that the percentage of Cu accumulation in visceral organs of P. paludosa was greater than that in the foot. Among the visceral organs, stomach or digestive glands accumulated the highest concentration of Cu and the levels were lesser in kidney and liver, respectively (Pyatt et al., 2003). Several mechanisms may account for the high Cu concentration found in the soft tissues of snails. Cu can form complexes with carbonate content in the snail and enter the snail as Cu carbonate (Hoang and Rand, 2009). Carbonate is required for shell development while Cu is accumulated in the soft 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) (Phillips and Rainbow, 1989; Simkiss, 1981). The high Cu burden found in the soft tissues of P. canaliculata implies the likelihood of Cu transfer through the food chain. Gastropods and bivalve mollusks have long been known to accumulate exceedingly high levels of heavy metals. Our data clearly demonstrated that P. canaliculata accumulated and concentrated the highest Cu concentration in the gill (71.09 mg/g), followed by digestive tract (47.12 mg/g), foot muscle (38.63 mg/g) and digestive gland (30.74 mg/g). The gill was suggested to have a central role in Cu uptake in mussels Mytilus edulis (Al-Subiai et al., 2011), in another bivalve, Meretrix casta (Nambisan et al., 1977), and in the gastropod Busycon canaliculatum (Betzer and Pilson, 1974). From the previous studies, gill and digestive gland were reported to be the important target organs for metal accumulation (AbdAllah and Moustafa, 2002; Supanopas et al., 2005; Tanhan
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Fig. 3. Digestive tract of P. canaliculata: (A) and (C) ciliated epithelium of control snail showing columnar cell (CC) and mucus cell (MC) in esophagus (A) and intestine (C). (B) and (D) Changes in the digestive tract epithelium include increase in number of mucus cell (MC), dilation of nucleus (N) with the loss of heterochromatin in the columnar cell (CC) of esophagus. Ag, acidophilic granule; Ci, cilia.
et al., 2005). The present study showed that the gill accumulated more Cu than the digestive gland after 96 h of exposure to LC50 concentration of Cu. This was probably due to the short time of exposure (4 days) to metal in this study. Gills are frequent targets of environmental pollutants because they are the main interface between the organism and its environment (Rajalakshmi and Mohandas, 2005). Metals enter organisms via passive diffusion at the gill membranes along a concentration gradient (Grosell et al., 2002). 4.2. Histopathological study The accumulation of Cu in P. canaliculata organs was reflected in easily discernible histological parameters. All organs (gill, digestive tract, digestive gland) showed histological alterations but they were most severe in the gill and digestive tract. More severe alterations, such as the degeneration of columnar cells, creating large gaps, were observed in the epithelium of the gill and intestine. These alterations were also reported in P. canaliculata exposed to contaminated sediments (Cr, Fe, Cu, Zn, Mn) from Mae Klong tributaries (Kruatrachue et al., 2011) and from Beung Boraphet tributaries (Dummee et al., 2012), B. areolata exposed to Cd and Pb (Supanopas et al., 2005; Tanhan et al., 2005) and Marisa cornuarietis exposed to Cu and Li (Sawasdee et al., 2011). The increase in number of mucus cells and mucus production could be explained in terms of a defense and detoxification strategy (Triebskorn, 1989). Production of mucus may arrest metals which are then excreted to the environment (Sze and Lee, 1995), or may dilute the concentration of the toxicant (Triebskorn, 1989). Since the gill is a vital organ in oxygen uptake, enhanced production of
mucus might be a first reaction to mechanically protect the epithelia. However, the production of mucus may be a trade-off against oxygen transport capacity and therefore may decrease the vitality of the snail (Osterauer et al., 2010). In mollusks, the digestive gland plays a major role in contaminant uptake and accumulation, intracellular food digestion, and metabolism of inorganic and organic chemicals (Marigómez et al., 2002; Usheva et al., 2006). Histopathological changes in the epithelium of digestive gland of P. canaliculata after Cu exposure (an increase in size of basophilic cells, tubular secretion, vacuolization of digestive cells, and narrowing of the tubular lumen) were similar to findings reported in other species of gastropods exposed to toxicants, e.g., M. cornuarietis exposed to PtCl2, Cu and Li (Osterauer et al., 2010; Sawasdee et al., 2011). The vacuolization of digestive cells is commonly observed as a cellular response of aquatic invertebrates affected by metal toxicity (AbdAllah and Moustafa, 2002; Najle et al., 2000) and believed to be related to cellular detoxification (Remedios Rubio et al., 1993). In addition, damage to the digestive gland may lower the ability of snails to digest food (Osterauer et al., 2010).
5. Conclusion The present study demonstrated the ability of the golden apple snails, P. canaliculata to serve as a bioindicator for Cu, and a biomarker for monitoring Cu contamination in aquatic environment. The snails can serve as a bioindicator due to the following reasons: (1) they are ubiquitous in aquatic habitats as an important component of freshwater food web, (2) they are resistant to various
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heavy metals, (3) they can accumulate many heavy metals such as Cu, Cr, Fe, Mn and Zn (Dummee et al., 2012; Kruatrachue et al., 2011; Sawasdee et al., 2011), (4) they are easy to harvest, (5) they showed a high correlated (R2 40.900) between metal concentrations in soil and their tissue (Dummee et al., 2012). In addition, we demonstrated that an LC50 value of Cu (146 mg/L) caused histopathological alterations in the gill, digestive tract and digestive gland of the snail which were positively correlated with Cu exposure, enabling P. canaliculata to serve as the biomarker for monitoring Cu contamination in aquatic environment.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments This study was funded by the Center for Environmental Health, Toxicology and Management of Chemicals under Science and Technology Postgraduate Education and Research Office (PERDO) of the Ministry of Education, Thailand; Faculty of Science, Mahidol University. We also thank Associate Professor Philip D. Round for editing the manuscript.
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toxicity to the Florida apple snail (Pomacea paludosa): implications in Everglades restoration. Environ. Pollut. 154, 338–347. Jantataeme, S., et al., 1996. Acute toxicity and bioaccumulation of lead in the snail, Filopaludina (Siamopaludina) martensi martensi (Frauenfeldt). J. Sci. Soc. Thail. 22, 237–247. Kruatrachue, M., et al., 2011. Histopathological effects of contaminated sediments on golden apple snail (Pomacea canaliculata, Lamarck 1822). Bull. Environ. Contam. Toxicol. 86, 610–614. Laskowski, R., Hopkin, S.P., 1996. Accumulation of Zn, Cu, Pb and Cd in the garden snail (Helix aspersa): implications for predators. Environ. Pollut. 91, 289–297. Lau, S., et al., 1998. Accumulation of heavy metals in freshwater molluscs. Sci. Total Environ. 214, 113–121. Marigómez, I., et al., 2002. Cellular and subcellular distribution of metals in molluscs. Microsc. Res. Tech. 56, 358–392. Mitchell, A.J., et al., 2007. The effect of chemical treatments on red-rim melania Melanoides tuberculata, an exotic aquatic snail that serves as a vector of trematodes to fish and other species in the USA. North Am. J. Fish. Manage. 27, 1287–1293. Najle, R., et al., 2000. Histopathology of the digestive gland of an Antarctic limpet exposed to cadmium. Sci. Total Environ. 247, 263–268. Nambisan, P.N.K., et al., 1977. On the uptake of copper (II) by Meretrix casta (Cheminitz), an indicator species of metal pollution. Curr. Sci. 46, 437–440. Ng, T.Y.T., et al., 2011. Mechanisms of waterborne Cu toxicity to the pond snail Lymnaea stagnalis: physiology and Cu bioavailability. Ecotoxicol. Environ. Saf. 74, 1471–1479. Ortíz, J.B., et al., 1999. Quantification and histopathological alterations produced by sublethal concentrations of copper in Fundulus heteroclitus. Cienc. Mar. 25, 119–143. Osterauer, R., et al., 2010. Histopathological alterations and induction of hsp70 in ramshorn snail (Marisa cornuarietis) and zebrafish (Danio rerio) embryos after exposure to PtCl2. Aquat. Toxicol. 99, 100–107. Otitoloju, A.A., et al., 2009. Histopathology and bioaccumulation of heavy metals (Cu & Pb) in the giant land snail, Archachatina marginata (Swainson). Open Environ. Pollut. Toxicol. J. 1, 79–88. Phillips, D.J.H., Rainbow, P.S., 1989. Strategies of trace metal sequestration in aquatic organisms. Mar. Environ. Res. 28, 207–210. Pyatt, F.B., et al., 2003. Copper bioaccumulation by the freshwater snail Lymnaea peregra: a toxicological marker of environmental and human health? Environ. Toxicol. Chem. 22, 561–564. Rajalakshmi, S., Mohandas, A., 2005. Copper-induced changes in tissue enzyme activity in a freshwater mussel. Ecotoxicol. Environ. Saf. 62, 140–143. Remedios Rubio, M., et al., 1993. Effects of cadmium exposure on the ultrastructure of hepatopancreatic cells of Thais haemastoma (Gastropoda, Prosobranchia). Mar. Environ. Res. 35, 47–51. Rogevich, E.C., et al., 2008. The effects of water quality and age on the acute toxicity of copper to the Florida apple snail, Pomacea paludosa. Arch. Environ. Contam. Toxicol. 54, 690–696. Sawasdee, B., et al., 2011. Histopathological effects of copper and lithium in the ramshorn snail, Marisa cornuarietis (Gastropoda, Prosobranchia). Chemosphere 85, 1033–1039. Shuhaimi-Othman, M., et al., 2012. Toxicity of metals to a freshwater snail, Melanoides tuberculata. Sci. World J. 2012, 10. Simkiss, K., 1981. Cellular discrimination processes in metal accumulating cells. J. Exp. Biol. 94, 317–327. Supanopas, P., et al., 2005. Acute and subchronic toxicity of lead to the spotted babylon, Babylonia areolata (Neogastropoda, Buccinidae). J. Shellfish Res. 24, 91–98. Sze, P.W.C., Lee, S.Y., 1995. The potential role of mucus in the depuration of copper from the mussels Perna viridis (L.) and Septifer virgatus (Wiegmann). Mar. Pollut. Bull. 31, 390–393. Tanhan, P., et al., 2005. Histopathological alterations in the edible snail, Babylonia areolata (spotted babylon), in acute and subchronic cadmium poisoning. Environ. Toxicol. 20, 142–149. Thophon, S., et al., 2003. Histopathological alterations of white seabass, Lates calcarifer, in acute and subchronic cadmium exposure. Environ. Pollut. 121, 307–320. Triebskorn, R., 1989. Ultrastructural changes in the digestive system of Deroceras reticulatum (Mollusca: Gastropoda) induced by lethal and sublethal concentrations of the carbamate molluscicide cloethocarb. Malacologia 32, 89–106. Usheva, L.N., et al., 2006. Histopathology of the digestive gland of the bivalve mollusk Crenomytilus grayanus (Dunker, 1853) from southwestern Peter the Great Bay, Sea of Japan. Russ. J. Mar. Biol. 32, 166–172. Watanabe, T., et al., 1997. Trace minerals in fish nutrition. Aquaculture 151, 185–207. White, S.L., Rainbow, P.S., 1985. On the metabolic requirements for copper and zinc in molluscs and crustaceans. Mar. Environ. Res. 16, 215–229.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Histopathological changes in snail, Pomacea canaliculata, exposed to sub-lethal copper sulfate concentrations Vipawee Dummee a,b, Phanwimol Tanhan c,n, Maleeya Kruatrachue a, Praneet Damrongphol a, Prayad Pokethitiyook a a
Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Interdisciplinary Graduate School of Earth System and Andaman Natural Disaster Management, Prince of Songkla University, Phuket Campus, Kathu, Phuket 83120, Thailand c Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, 50 Ngamwongwan Road, Chatujak, Bangkok 10900, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 February 2015 Received in revised form 11 August 2015 Accepted 11 August 2015 Available online 20 September 2015
The acute toxicity test of Cu including range-finding and definitive test, was performed on golden apple snails, Pomacea canaliculata. The median lethal concentrations (LC50) of Cu at exposure times of 24, 48, 72 and 96 h were 330, 223, 177 and 146 mg/L, respectively. P. canaliculata were exposed to Cu at 146 mg/L for 96 h to study bioaccumulation and histopathological alterations in various organs. Snails accumulated elevated levels of Cu in gill, and lesser amounts in the digestive tract, muscle, and digestive gland. Histopathological investigation revealed several alterations in the epithelia of gill, digestive tract (esophagus, intestine, rectum), and digestive gland. The most striking changes were observed in the epithelium of the gill in which there was loss of cilia, an increase in number of mucus cells, and degeneration of columnar cells. Similar changes occurred in digestive tract epithelium. The digestive gland showed moderate alterations, vacuolization and degeneration of cells and an increase in the number of basophilic cells. We concluded that, P. canaliculata has a great potential as a bioindicator for Cu, and a biomarker for monitoring Cu contamination in aquatic environment. & 2015 Elsevier Inc. All rights reserved.
Keywords: Histopathological changes Copper Sub-lethal condition LC50 Bioindicator Biomarker
1. Introduction Heavy metals encompass a wide range of substances that are non-degradable, highly persistent in the environment and which may be incorporated into the tissues of living organisms (Ortíz et al., 1999). Among heavy metals, Cu plays various physiological roles for a number of key metabolic enzymes in plants and animals (Watanabe et al., 1997). In gastropod mollusks, it is involved in hemocyanin metabolism (Gullick et al., 1981; White and Rainbow, 1985). However, increased concentration of Cu in organisms is potentially toxic since it interferes with numerous physiological processes. The major lethal effects of Cu in gastropod mollusks are disruption of the transporting surface epithelium and osmoregulation, eventually causing water accumulation in mollusk tissues (Cheng, 1979). High levels of Cu (4 200 mg/kg) were recently reported in the sediments of tributaries feeding into Beung Boraphet, a large freshwater lake in Nakhon Sawan province, Thailand (Dummee et al., 2012). These most likely resulted from the excessive use of n
Corresponding author. E-mail address: [email protected] (P. Tanhan).
http://dx.doi.org/10.1016/j.ecoenv.2015.08.010 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Cu-based fungicides, molluscicides, and pesticides in the rice paddies surrounding the tributaries (Dummee et al., 2012). Golden apple snails, Pomacea canaliculata, which were abundant in the area, contained high levels of Cu (81.8–127.8 mg/kg) in their visceral mass and foot. Several tools or biomarkers may be used to manage heavy metal contamination, such as toxicity assessment, histopathological evaluation, and biochemical assessment. Toxicity testing is an essential tool for assessing the effect and fate of toxicants in aquatic ecosystem, and for identifying suitable organisms as bioindicators (Shuhaimi-Othman et al., 2012). In addition, acute toxicity tests can help in the detection, evaluation, and abatement of pollution by providing reliable estimates of safe concentrations of toxicants (Ahsanullah et al., 1981). Histopathological alterations reveal effects of pollution on the tissues of exposed organisms, and provide early indications of toxicity (Sawasdee et al., 2011). Mollusks have long been regarded as promising bioindicator and biomonitoring subjects because they are abundant, highly tolerant to many pollutants, and exhibit high accumulation of heavy metals (Lau et al., 1998; Shuhaimi-Othman et al., 2012). Several studies reported on the acute and chronic toxicity of heavy metals, and attendant histopathological alterations in gastropod mollusks, such as Filopaludina martensi martensi (Jantataeme et al.,
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1996), Babylonia areolata (Supanopas et al., 2005; Tanhan et al., 2005), Melanoides tuberculata (Shuhaimi-Othman et al., 2012), Pomacea paludosa (T. Hoang et al., 2008; T.C. Hoang et al., 2008), and P. canaliculata (Dummee et al., 2012; Kruatrachue et al., 2011). In mollusks, the digestive gland is thought to play the most important role in metabolism of endogenous and xenobiotic compounds and is likely important in the storage and regulation of metals in terrestrial and aquatic gastropods. The introduced golden apple snail (P. canaliculata) is an agricultural pest in Asia and is now the most widespread snail species in Thailand where it is ubiquitous in aquatic habitats. They feed not only on rice but also on taro and lotus plants, causing much loss to the farmers. The present work aimed to assess the potential of P. canaliculata as a bioindicator and biomonitor of Cu in the aquatic environment. We investigated the acute toxicity in P. canaliculata exposed to Cu and evaluated Cu accumulation and associated histopathological manifestations in the soft tissues including the digestive organs.
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them in water (Otitoloju et al., 2009). 2.4. Accumulation study P. canaliculata were exposed to the median lethal concentration (LC50) for 96 h. Ten snails were placed in each of three replicates including controls. Two replicates were used to obtain the Cu accumulation analysis and the third was used for the histopathological study. After 96 h, the soft tissues were separated from shells, snail organs (gill, digestive tract and digestive gland) and head– foot (muscle) were isolated. The tissue samples were oven dried at 80 °C, and 0.5 g of each dried samples was digested with 67% conc. HNO3 (67%) according to APHA et al. (2011). The Cu concentrations were then determined using a flame atomic absorption spectrophotometer (FAAS; SpectrAA-55B). The Cu concentrations in each digested organ were determined by comparing its absorbance with known Cu standard solutions. The method detection limit was calculated by a standard procedure which is based on the analysis of ten samples of the matrix with the analyte. The lowest detection limit for Cu was 0.5 mg/g dw.
2. Materials and methods 2.5. Bioaccumulation efficiency 2.1. Chemical The stock of Cu solution (1000 μg/L) was prepared by dissolving Cu sulfate (hydrated form; CuSO4 5H2O) in deionized water according to APHA et al. (2011). Concentrations were expressed as micrograms of Cu ions (Cu2 þ ) per liter (μg/L) of solution. The stock solution was left for 1 day to equilibrate. Nominal Cu concentrations (0, 100, 150, 200, 250, 400, and 800 μg/L) were then prepared from the stock solution diluted to 1 L with deionized water in preacid washed aquarium.
Bioaccumulation is the process in which a chemical substance is absorbed in an organism by all routes of exposure including dietary and ambient environment sources (Arnot and Gobas, 2006). The bioaccumulation efficiency of Cu in P. canaliculata was evaluated using two parameters. First, the bioaccumulation capacity (BAC) was the ratio of the concentration of the metal in snail organs after 96 h of exposure to the level detected in the same organs of control snails (Otitoloju et al., 2009). Second, the bioconcentration factor (BCF) was the ratio of the metal concentration in snails to the concentration in water (T. Hoang et al., 2008).
2.2. Experimental animal 2.6. Histopathological study P. canaliculata eggs were collected from unpolluted natural ponds in Kanchanaburi province, Thailand. Eggs were hatched in the laboratory and juvenile snails were reared in the aerated aquaria with dechlorinated water at 25–27 °C and daily fed with lettuce until they were 2 months old. The uniform snails (shell length 2–3 cm) were used for acute toxicity tests and histopathological study. 2.3. Acute toxicity study The static (non-renewal, without food) technique was used for the acute toxicity test. The toxicity range-finding test consisted of a down-scale (100, 10, 1 and 0.10 μg/L of Cu) abbreviated static acute test in which groups of organisms were exposed to several widely spaced sample dilutions in a logarithmic series, and a control, for a period of 96 h. The definitive test was performed to follow-up the range-finding test. During the acclimatization (24 h) snails were fasted to prevent the binding of Cu to the accumulated feces (Ng et al., 2011). No mortality of P. canaliculata was detected during this period. One liter of each test solution (and controls) was added to 4 L glass aquaria at the nominal concentrations previously described. Three replicates for each concentration, including controls, were performed. Ten snails were placed in each aquarium and were not fed during the period of the bioassay. The dead and living snails were counted daily throughout the 96-h period. Dead snails were removed immediately to avoid the bacterial infection of other living snails. The percentage survival and mortality were calculated for each metal concentration. Snails were taken to be dead if there was no movement when the foot region was prodded with the metal needle or if there was no activity after 5 min of placing
After 96 h of exposure, the gill, digestive tract and digestive gland of P. canaliculata were dissected and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 12 h. Organs were washed with 0.1 M sodium cacodylate buffer three times before they were post-fixed in 2% osmium tetroxide (OsO4) for overnight. Samples were washed in 0.1 M sodium cacodylate buffer, dehydrated with the graded series of acetone, and embedded in aradite resin (Thophon et al., 2003). The semi-thin sections (1 μm) were prepared on an ultramicrotome, mounted on glass slides, stained with 1% toluidine blue in 1% borax, and examined under a light microscope (Olympus CH40). 2.7. Statistical analysis The dose–response data was analyzed by Probit analysis based on a computer program (SPSS version 15.0). The indices of toxicity measurement derived from this analysis were lethal concentrations (LC) at 25%, 50%, 75% and 99%. Analysis of variance (ANOVA) was carried out to compare the Cu accumulation in several treatments and organs mean at Po 0.05 level of significance (Thophon et al., 2003).
3. Results 3.1. Toxicity study The percentage mortality of P. canaliculata generally increased with increasing Cu concentration and exposure time (Table 1). A Cu concentration higher than 0.40 μg/L caused very high mortality
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Table 1 Percentage mortality of P. canaliculata exposed to various Cu concentrations. Cu concentration (μg/L)
0 100 150 200 250 400 800
Exposure time (h) 0 24
48
72
96
0 0 0 0 0 0 0
0 0 13.3 7 6.67 66.77 17.6 40.0 723.1 93.3 76.67 100
0 0 33.3 7 6.67 80.0 711.6 73.3 7 6.67 100 100
0 6.677 6.67 66.7 713.3 86.7 7 13.3 86.7 7 6.67 100 100
0 0 0 26.7 7 6.67 20.0 7 11.6 60.0 7 11.6 100
Table 2 Lethal concentration values and 95% confidence limits of Cu at different exposure times. Exposure time (h)
0 24 48 72 96
Lethal concentration (μg/L) LC25 (95% C.L.)
LC50 (95% C.L.) LC75 (95% C.L.)
LC99 (95% C.L.)
0 245 (200–288) 169 (58–225) 143 (113–163) 101 (0–156)
0 330 (281–410) 223 (149–381) 177 (154–201) 146 (0–241)
0 914 (642– 1932) 576 (345–26,481) 372 (296–620) 522 (284–a)
0 443 (366–626) 294 (221– 1129) 220 (195–266) 211 (115–1503,906)
C.L.–Confidence Limit. a
Concentration greater than 1024 mg/L.
(4 80%; Table 1) with sublethal effects occurring at lesser concentrations (100–250 μg/L). LC values showed a gradual decrease with an increase in exposure time (Table 2). The LC50 values of Cu were 330, 223, 177, and 146 mg/L for 24 h, 48 h, 72 h, and 96 h, respectively. 3.2. CU accumulation Copper accumulated significantly in the gill and digestive tract of P. canaliculata exposed to acute concentration (146 mg/L) for 96 h compared with control snails (Table 3). The highest Cu concentration was found in the gill (71.1 mg/g) with lesser concentrations in the digestive tract (47.1 mg/g) 4 muscle (38.6 mg/ g)4 digestive gland (30.7 mg/g). The BCF values also showed similar trend to those of Cu accumulation, i.e., gill (50.8) 4digestive tract (33.7) 4muscle (27.6) 4digestive gland (22.0) (Table 3). Snails accumulated Cu ions at levels about 1–2 times higher than those detected within organs of control snails (Table 3). 3.3. Histopathological study 3.3.1. Digestive gland The digestive gland or hepatopancreas of P. canaliculata was composed of numerous tubules, which consisted of digestive cells and scarce basophilic secretory cells (Fig. 1A). Hemolymph spaces Table 3 Cu accumulation in P. canaliculata. Cu concentration (mg/g)
Control Treated BAC BCF
Muscle
Gill
Digestive tract
Digestive gland
19.8 7 1.75 38.6 72.93 1.95 27.6
31.9 7 4.12 71.1 74.28 2.23 50.8
33.87 2.65 47.17 4.03 1.39 33.7
30.0 7 5.76 30.7 7 1.97 1.02 22.0
Data are means 7 S.E. Significant differences (Po 0.05) among concentrations were indicated by different letters. BCF: Bioconcentration factor; BAC: Bioaccumulation capacity.
Fig. 1. Digestive gland of P. canaliculata: (A) digestive gland epithelium of control snail showing numerous columnar digestive cells (DC) and a few pyramidal basophilic cells (BC) surrounding the lumen (L). Numerous digestive vacuoles (V) are present in the digestive cell. Notice dark granules (dg) with well-defined membrane. (B) and (C) Digestive gland of treated snail (B) narrow lumen with secretion and vacuolization of digestive cells (DC). (C) Loss of membrane around dark granule (dg) and increase in size of basophilic cell (BC) were observed.
were present among the tubules. Digestive cells were characterized by the presence of numerous vacuoles and oval dark granules of variable size and electron density. Basophilic cells contained homogeneously electron-dense secretory vesicles of varying size with a few dark granules. In treated snails, secretions of the digestive gland tubule in the lumen increased concurrently (Fig. 1B)
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mucus vacuoles and a basal nucleus (Fig. 3A and C). The histopathological alterations in Cu-exposed P. canaliculata, were similar in all three organs. The most striking change was the increase in number of mucus cells (Fig. 3B and D) while loss of cilia was observed in some surface areas of the epithelium. Dilation of the nucleus with the loss of heterochromatin was observed in the epithelial cells of esophagus (Fig. 3B).
4. Discussion 4.1. Cu toxicity and accumulation
Fig. 2. Gill of P. canaliculata: (A) gill filament of control snail showing columnar cells (CC) and a few mucous cells (MC). Notice numerous hemocytes (Hc) in hemolymph space. (B) Gill filament of treated snail showing increase in number of mucus cell (MC) and degeneration of columnar cell (CC).
with the increase in size of basophilic cells (Fig. 1C). The digestive cells underwent vacuolization and the dark granules lost their well-defined membranes (Fig. 1B). In addition, narrowing of the tubular lumen was also observed (Fig. 1B). 3.3.2. Gill The gill of P. canaliculata was composed of numerous gill filaments with long cilia. The gill epithelium contained ciliated-columnar cells, each with a dense oval nucleus and some mucussecreting goblet cells (Fig. 2A). Numerous hemocytes were found in the hemolymph space (Fig. 2A). Histopathological alterations observed in P. canaliculata were an increase in number of mucussecreting goblet cells and the degeneration of columnar cells (Fig. 2B). 3.3.3. Digestive tract The digestive tract of P. canaliculata consisted of the esophagus, intestine, and rectum. The esophagus was lined by pseudostratified columnar epithelium (Fig. 3A) while that of intestine and rectum was of the simple columnar type (Fig. 3C). The columnar epithelium consisted of two cell types: columnar cells and mucussecreting goblet cells. The columnar cells contained light acidophilic granules and an ellipsoidal nucleus rich in heterochromatin (Fig. 3A). The mucus cells were characterized by the presence of
The lethal concentration (LC) values of P. canaliculata exposed to Cu showed a gradual decrease with the increase in exposure time. The 96-h LC50 of Cu was 146 mg/L, similar to the 96-h LC50 of Cu in the Florida apple snail, P. paludosa (140 mg/L) (Rogevich et al., 2008) and in M. tuberculata, another freshwater prosobranch (140 mg/L) (Shuhaimi-Othman et al., 2012). However, P. canaliculata showed less sensitivity to Cu compared to pulmonate gastropods. LC50 values for Lymnaea stagnalis and Biomphalaria glabrata were 24.90 and 40 mg/L, respectively (Bellavere and Gorbi, 1981; Ng et al., 2011). Prosobranch snails have a tightly sealed operculum that allows them to withstand desiccation and apparently also increases their tolerance to metals (Mitchell et al., 2007). In addition, differences in sensitivity to Cu may be due to differences in Cu metabolism in snails such as the excretion or storage of Cu (Farid, 2005). It is also difficult to compare the toxicity values across studies because of differences in the characteristics of the tested waters (especially water hardness, pH, and temperature), species, ages, and sizes of snails tested (Shuhaimi-Othman et al., 2012). Most Cu in P. canaliculata accumulated in the viscera or digestive tract and digestive gland ( 80%) and foot ( 20%). Previous studies also reported that up to 60–80% of Cu was found in the viscera and 20–40% in the foot of snails such as Florida apple snail P. paludosa (T.C. Hoang et al., 2008), pond snail L. stagnalis (Desouky, 2006) and garden snail Helix aspersa (Laskowski and Hopkin, 1996). In addition, T. Hoang et al. (2008) found that the percentage of Cu accumulation in visceral organs of P. paludosa was greater than that in the foot. Among the visceral organs, stomach or digestive glands accumulated the highest concentration of Cu and the levels were lesser in kidney and liver, respectively (Pyatt et al., 2003). Several mechanisms may account for the high Cu concentration found in the soft tissues of snails. Cu can form complexes with carbonate content in the snail and enter the snail as Cu carbonate (Hoang and Rand, 2009). Carbonate is required for shell development while Cu is accumulated in the soft 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) (Phillips and Rainbow, 1989; Simkiss, 1981). The high Cu burden found in the soft tissues of P. canaliculata implies the likelihood of Cu transfer through the food chain. Gastropods and bivalve mollusks have long been known to accumulate exceedingly high levels of heavy metals. Our data clearly demonstrated that P. canaliculata accumulated and concentrated the highest Cu concentration in the gill (71.09 mg/g), followed by digestive tract (47.12 mg/g), foot muscle (38.63 mg/g) and digestive gland (30.74 mg/g). The gill was suggested to have a central role in Cu uptake in mussels Mytilus edulis (Al-Subiai et al., 2011), in another bivalve, Meretrix casta (Nambisan et al., 1977), and in the gastropod Busycon canaliculatum (Betzer and Pilson, 1974). From the previous studies, gill and digestive gland were reported to be the important target organs for metal accumulation (AbdAllah and Moustafa, 2002; Supanopas et al., 2005; Tanhan
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Fig. 3. Digestive tract of P. canaliculata: (A) and (C) ciliated epithelium of control snail showing columnar cell (CC) and mucus cell (MC) in esophagus (A) and intestine (C). (B) and (D) Changes in the digestive tract epithelium include increase in number of mucus cell (MC), dilation of nucleus (N) with the loss of heterochromatin in the columnar cell (CC) of esophagus. Ag, acidophilic granule; Ci, cilia.
et al., 2005). The present study showed that the gill accumulated more Cu than the digestive gland after 96 h of exposure to LC50 concentration of Cu. This was probably due to the short time of exposure (4 days) to metal in this study. Gills are frequent targets of environmental pollutants because they are the main interface between the organism and its environment (Rajalakshmi and Mohandas, 2005). Metals enter organisms via passive diffusion at the gill membranes along a concentration gradient (Grosell et al., 2002). 4.2. Histopathological study The accumulation of Cu in P. canaliculata organs was reflected in easily discernible histological parameters. All organs (gill, digestive tract, digestive gland) showed histological alterations but they were most severe in the gill and digestive tract. More severe alterations, such as the degeneration of columnar cells, creating large gaps, were observed in the epithelium of the gill and intestine. These alterations were also reported in P. canaliculata exposed to contaminated sediments (Cr, Fe, Cu, Zn, Mn) from Mae Klong tributaries (Kruatrachue et al., 2011) and from Beung Boraphet tributaries (Dummee et al., 2012), B. areolata exposed to Cd and Pb (Supanopas et al., 2005; Tanhan et al., 2005) and Marisa cornuarietis exposed to Cu and Li (Sawasdee et al., 2011). The increase in number of mucus cells and mucus production could be explained in terms of a defense and detoxification strategy (Triebskorn, 1989). Production of mucus may arrest metals which are then excreted to the environment (Sze and Lee, 1995), or may dilute the concentration of the toxicant (Triebskorn, 1989). Since the gill is a vital organ in oxygen uptake, enhanced production of
mucus might be a first reaction to mechanically protect the epithelia. However, the production of mucus may be a trade-off against oxygen transport capacity and therefore may decrease the vitality of the snail (Osterauer et al., 2010). In mollusks, the digestive gland plays a major role in contaminant uptake and accumulation, intracellular food digestion, and metabolism of inorganic and organic chemicals (Marigómez et al., 2002; Usheva et al., 2006). Histopathological changes in the epithelium of digestive gland of P. canaliculata after Cu exposure (an increase in size of basophilic cells, tubular secretion, vacuolization of digestive cells, and narrowing of the tubular lumen) were similar to findings reported in other species of gastropods exposed to toxicants, e.g., M. cornuarietis exposed to PtCl2, Cu and Li (Osterauer et al., 2010; Sawasdee et al., 2011). The vacuolization of digestive cells is commonly observed as a cellular response of aquatic invertebrates affected by metal toxicity (AbdAllah and Moustafa, 2002; Najle et al., 2000) and believed to be related to cellular detoxification (Remedios Rubio et al., 1993). In addition, damage to the digestive gland may lower the ability of snails to digest food (Osterauer et al., 2010).
5. Conclusion The present study demonstrated the ability of the golden apple snails, P. canaliculata to serve as a bioindicator for Cu, and a biomarker for monitoring Cu contamination in aquatic environment. The snails can serve as a bioindicator due to the following reasons: (1) they are ubiquitous in aquatic habitats as an important component of freshwater food web, (2) they are resistant to various
V. Dummee et al. / Ecotoxicology and Environmental Safety 122 (2015) 290–295
heavy metals, (3) they can accumulate many heavy metals such as Cu, Cr, Fe, Mn and Zn (Dummee et al., 2012; Kruatrachue et al., 2011; Sawasdee et al., 2011), (4) they are easy to harvest, (5) they showed a high correlated (R2 40.900) between metal concentrations in soil and their tissue (Dummee et al., 2012). In addition, we demonstrated that an LC50 value of Cu (146 mg/L) caused histopathological alterations in the gill, digestive tract and digestive gland of the snail which were positively correlated with Cu exposure, enabling P. canaliculata to serve as the biomarker for monitoring Cu contamination in aquatic environment.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments This study was funded by the Center for Environmental Health, Toxicology and Management of Chemicals under Science and Technology Postgraduate Education and Research Office (PERDO) of the Ministry of Education, Thailand; Faculty of Science, Mahidol University. We also thank Associate Professor Philip D. Round for editing the manuscript.
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