Investigation of Some Freshwater Snails as Bioindicator for Heavy Metals

Investigation of Some Freshwater Snails as Bioindicator for Heavy Metals

The term “heavy metals” was in use as far back as 1817, when Gmelin divided elements into non-metals, light metals and heavy metals (Habashi, 2009). Over the past two decades, the term “heavy metals” has been used increasingly in various publications and in legislation related to chemical hazards and the safe use of chemicals (Duffus, 2001). Criteria adopted to define heavy metals have included density, atomic weight, atomic number, or periodic table position (Duffus, 2002).

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Density criteria range from 3.5 g/cm3 to above 7 g/cm3. Atomic weight definitions start at greater than 22.98 to greater than 40 (Hawkes, 1997). Atomic numbers of heavy metals are given as greater than 20, sometimes this is capped at 92. Hawkes suggested referring to heavy metals as all the metals in Groups 3 to 16 that are in periods 4 and greater (Hawkes, 1997). In other words, the term “heavy metals” has been used inconsistently and has reflected inconsistency in the scientific literature. This practice has led to general confusion regarding the significance and generally accepted definition of the term and it has become imperative to review the usage that has developed for the term, paying particular attention to its relationship to fundamental chemistry (Duffus, 2001).

The term “Heavy metals” is often used as a group name for metals and semimetals (metalloids) that have been associated with contamination and potential toxicity or ecotoxicity (Duffus, 2001). Heavy metals are among the major concerns in waste water treatment since human (anthropogenic) activities carried out in a quest to uplift man’s living condition directly or indirectly produce chemical pollutants which severely affect the condition of aquatic ecosystems worldwide and are often derived from heavy metal operations such as electroplating, battery factories and mining operations (Salchi et al., 2008).

Pollutants such as heavy metals can be bioconcentrated by aquatic biota such as macrophytes, macro invertebrates and fish (USEPA, 1991). Bioconcentration measurements refer to the monitoring of uptake and retention of pollutants like heavy metals in the organs or tissues of organisms. Bioconcentration takes place if the rate of uptake of pollutants by organisms exceeds the rate of elimination or excretion (Spacie and Hamelink, 1985). Plants are exposed to heavy metals through the uptake of water; animals eat these plants; ingestion of plant- and animal-based foods is the largest sources of heavy metals in humans (Radojevic and Bashkin, 1999). Absorption through skin contact, for example from contact with soil, is another potential source of heavy metal contamination (Qu et al., 2014). Commonly encountered heavy metals are chromium, cobalt, nickel, lead, copper, zinc, silver, selenium, arsenic, mercury, thallium, etc (Bánfalvi, 2011).

A freshwater snail is one kind of freshwater mollusc, the other kind being freshwater clams and mussels, i.e. freshwater bivalves. Specifically a freshwater snail is a gastropod that lives in a watery non-marine (freshwater) habitat (Strong et al., 2008). According to present classification efforts, there are about 4,000 species of freshwater gastropods and at least 33–38 independent lineages of gastropods have successfully colonized freshwater environment (Colgan et al., 2011)

A bioindicator is an organism whose presence or absence in an environment indicates conditions or the presence of a contaminating substance or as a plant or an animal which reveals the presence of a substance in its vicinity by showing some typical symptoms which can be distinguished from the effects of other natural or anthropogenic stresses (Mhatre, 1991). To reveal the presence of pollutants and to measure their toxic effect, biological indicators can be used which are suitable for prediction of the expectable toxic influence of known or unknown substances.

Freshwater molluscs are among the most thoroughly investigated bioindicator organisms due to their special benefits, one of which is that they accumulate metals in their bodies (Lopez-Artiguez et al., 1989). Heavy metals such as arsenic (As), mercury (Hg), cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), iron (Fe), manganese (Mn), zinc (Zn) etc. do not degrade in general; as they are hard to metabolize (process and eliminate), (Pezzarossa et al., 2011) therefore, they accumulate in organisms and throughout the trophic chain. Accumulation in living organisms leads to concentrations several orders of magnitude higher than those of the surrounding water (Casas et al., 2008).


To investigate freshwater snails sold in Abakaliki for the presence of heavy metals

Specific Objectives
To indicate and analyse the tissues of freshwater snails for bioaccumulation of heavy metals.
To investigate freshwater habitats in Abakaliki and test for the presence of heavy metals.
To confirm that commonly available species of freshwater snails in these investigated freshwater habitats accumulate heavy metals and therefore are bioindicators of heavy metals.
To sensitize and enlighten the ignorant populace on the adverse effect of frequent snail consumption.
To discourage the drinking of water from these freshwater habitats as they pose a very great health risk to their consumers.
To possibly bring to the barest minimum their fixation in the aquatic ecosystems by discouraging the channeling of wastes from industries producing heavy metals into aquatic ecosystems.


In the last few decades, increasing attention has been paid to the potential ecological effects of heavy metals concentrations in the environment and has become of great concern due to their highly bioaccumulative nature, persistent behavior and higher toxicity (Gupta and Singh, 2011). These metals biomagnify in the food chain and impose various toxic effects on both aquatic organisms and their consumers since the absorption of high concentrations of these metals by commercial species might prove harmful to man and the productivity of freshwater organisms may be affected. Hence toxicity occurs along the food chain when the contaminated species or substance is consumed or eaten by an organism on the higher trophic level (Heng et al, 2004). Freshwater snails/molluscs reflect the higher degree of environmental contamination by heavy metals and are the most useful bioindicator tools due to their seeming reflection of environmental heavy metal contamination (Gundacker, 1999, Pihan et al., 2000).
Several studies and research work have been cited to establish and evaluate this relationship between the conformation of heavy metals and their impact on aquatic organisms.

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Gupta and Singh, (2011) showed that anthropogenic activity makes a significant contribution to the total aquatic burden of toxic metals by both point and non-point source contamination usually arising from agricultural, industrial and urban effluents that reach the coast by way of waterways, surface runoff and precipitation. Lopez-Artiguez et al., (1989) studied some bivalves and showed that analysed water and investigated molluscs’ soft parts and shells indicated the accumulation of heavy metals in high concentrations in their bodies, and so proved they could be useful as bioindicators for heavy metals. Adeyeye, (1996) in his study of the composition of three different snails in Nigeria asserted that Snails play an important role in nature with diverse functions and may act as sensitive indicator of environmental change. Regolli et al., (2005) in their study about Prooxidant effects of extremely low frequency electromagnetic fields (ELF-EM) in the land snail Helix aspersa observed that a snail’s intoxication is a result of feeding with contaminated plants in polluted regions.

Accumulated pollutants could be transported through different routes by hemolymph and blood cells in the organs e.g. hepatopancreas, which is a gland that support the food digestion and is the main organ for detoxification and metabolism. Tarun Balain et al., (2011) in their study on hematological and biochemical changes found out that high dose of toxicants resulted in hypoglycemia to snails. In fact the authors concluded that the effects of toxicants in glicemy depend on the dose, so e.g. high doses of agrochemicals reduce blood glucose concentration, whereas low doses result in hyperglycemia. Related to cholesterol concentration in hemolymph, Nedjoud Grara, (2012) found a significant reduction of lipids in the snails, treated with heavy metals, and this proportion depended on the concentrations of heavy metals. On the other hand Cheng Wan Hee, (2008) by analyzing the heavy metal concentration in snail (Nerita lineate) soft tissues, shell and operculum from a region contaminated with heavy metals, found high level of Cd, Cu, Fe, Ni, Pb and Zn concentration, derived from different paths. Casas et al., (2008) in a study to show the relationship between metal and concentration in water and metal content of molluscs proved that heavy metals such as arsenic (As), mercury (Hg), cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), iron (Fe), manganese (Mn), zinc (Zn) do not degrade in general therefore accumulate throughout the trophic chain in several orders of magnitude higher than those of the surrounding water.

They further proved that the accumulation depended on factors some of which had environmental origin such as temperature, pH, salinity, etc whereas others were related to biological factors like age, sex, sexual maturity stage, etc. Gryseels, (1989) in studying the relevance of Schistosomiasis for public health showed that most intermediate hosts for human Schistosoma parasite belonged to three genera; Biomphalaria, Oncomelania and Bulinus. The study also proved that Biomphalaria and Bulinus were aquatic snails that live under water and cannot usually survive elsewhere. A report of World Health Organization (WHO) study group, (1995) showed that eggs of the Schistosoma parasite were present in freshwaters where snails inhabit and penetrate the soft body of a suitable snail where recurring asexual reproduction takes place until thousands of cercaria breakout of the snail into the water.

Hemelraad et al., (1986, 1987 and 1988) published a series of papers concerned with the effect of cadmium on freshwater clams. Hemelraad et al., (1990) studied the effects of cadmium on freshwater clams Anodonta cygnea and the interaction of cadmium with the essential elements sodium, potassium, calcium, mercury, iron, zinc. Abdel and Farag, (1991) studied the rate of bioaccumulation in Edku lake and El-Fayomy (1994) found that the marine clam Cardium edula in Lake Manzala accumulated more heavy metals than the examined fishes. Zhou, (2008) reported that the snails accumulate metals to high concentrations than any other group of invertebrate and demonstrated the ability as potential bioindicator. Bioaccumulation of metals in snail species has been reported by several investigators for the past two decades. Reed-Judkins et al., (1998) studied bioaccumulation of heavy metals in Lymnaea stagnalis. Snyman et al. (2000) studied bioaccumulation of metals in snail Helix aspersa. Enzemonye et al., (2006) studied bioaccumulation of heavy metals (Cu, Zn, Fe) in freshwater snail (Pliaovota; Oliver 1804) from Ikpoba River of south Nigeria. Kim and Kim (2007) studied heavy metals accumulation in Oxyloma hirasei from the Upo wetland. Jan et al., (2011) studied metal accumulation in pond snail Lymnaea stagnalis in freshwater. Astani et al., (2012) studied bioaccumulation of heavy metals in soft body tissues of gastropods Thais mutabilis and sediments from intertidal zone of Bandar Abbas. Bryan et al., (1980) proved that although at suitable concentrations, some heavy metals such as copper and zinc were important for life; there also formed an important group of enzyme inhibitors when natural levels were exceeded. Svobodova et al., (2002) in his study proved that generally, sediments can accumulate large amount of heavy metals and become their main reservoir in the wetlands. Kim and Kim, (2006) showed that when heavy metals have accumulated in sediments, it can affect concentrations of heavy metals in the organisms that dwell in those sediments.

Howard and Nickless, (1977) demonstrated that bivalves accumulate more cadmium than crustaceans and much more than fish. Moreover, bivalves and other molluscs were found to be critical groups in the biological transport, Engel and Fowler, (1979); Frazier, (1979). Bivalve molluscs were also reported by Zadory, (1984); Hemelraad et al., (1986 and 1987) to withstand remarkably high metal concentrations in their environment. Cadmium, Zinc, Nickel, Copper, Lead and Mercury are accumulated to high tissue levels in both freshwater and marine mussels. Yager and Harry, (1963) in studying the uptake of radioactive heavy metals like zinc, cadmium and copper asserted that heavy metal deteriorates cell dynamics and damages cell membranes and tissues. Waykar and Petare, (2013) during their recent study, observed that the magnitude of heavy metal accumulation in snails tissues depend upon type of heavy metal and the species of the snail.

Valavanidis and Vlachogianni, (2010) studied and observed that the differences in tissues metal concentrations between snail species might be due to variation in reproductive condition, genotype of the animal, difference in metabolic rate, body weight, trophic position, presence or absence of enzyme system that can degrade the pollutants. Herg et al., (2001) in their study observed higher levels of trace metals in the viscera of the freshwater snail as an indication that ingestion of both sediment and algae might be the primary source of uptake. Kraak et al., (1993) also asserted that the regulatory capacity of the snail is accountable to high concentration of all the trace metals in the viscera and shell that is organ specific. The shell of the freshwater snail Melanoides tuberculata contained lower tissue concentration of the trace metals in accordance with the observation of Laskowski and Hopkin, (1996) and Heng, (2004). Campbell and Evans, (1987) demonstrated that Pb is strongly bound to humic acids and therefore less bio-available to the unionid Elliptio complanata.

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Timmer-mans et al., (1989) observed also very low Pb contents in the invertebrates at the Maarsseveen Lake system in the Netherlands. They concluded that Pb had probably limited availability in the study area. Kraak et al., (1993) made evident that the Cu regulation capacity of U. pictorum was organ- specific: gill, digestive gland and mantle showed higher accumulation capacity for Cu than gonads and kidney. Salanki and Van-Balogh, (1989) revealed that the gill of Anodonta cygnea showed the longest half-depuration time for Cu and Pb, and were therefore recommended as the best biomonitoring organs.

Properties Required Of an Indicator Organism for Heavy Metals
It is important that indicator organisms should not only be good accumulators of metals and of reasonable size (for easy analysis) but should reflect the changing availability of metals in some phase of the environment in this case, the sediments. The ability of some organisms to maintain relatively constant metal concentrations in their tissues clearly makes them unsuitable as indicators. Ideal indicator organisms should in addition be;
Present wherever and whenever heavy metals are present
Relatively stationary
Long lived
Available at all times of the year.
General Science of Snails

Phylum: Mollusca;

Class: Gastropoda

Phylum Mollusca is one kind of an invertebrate (i.e. animals that lack a back bone) (Campbell & Reece, 2005). The Mollusca consists of snails and slugs, oysters and clams, octopuses, and squids. Most of the molluscs are living in marine habitats; however, some of them inhabit fresh land and waters. They have a soft body and most of them are protected by a hard shell. Some of them, though, have lost their shells through years of evolution (Campbell & Reece, 2005). The mollusca body has three main parts; a muscular foot for moving around, a visceral mass that contains most of their organs, and they also have a mantle. Molluscs in general have separate sexes, but snails, however, are hermaphroditic (Campbell & Reece, 2005). One of the eight classes of the phylum Mollusca is Gastropoda which contains snails and slugs. Gastropoda are marine, freshwater or terrestrial living organisms.

They have an asymmetric body usually with a coiled shell, if a shell is present, and a foot for locomotion and a radula (Campbell & Reece, 2005). Several of the different Gastropods have a single, spiral shell which often is conical but sometimes even flat. They have a rippling motion of their foot to by means of cilia. Most of the gastropods graze on algae or plants with their radula, and most of marine living gastropods have gills (Campbell & Reece, 2005), except the Pulmonata, who has developed a pallial lung and can breathe air (Madsen, 1985a). The class Gastropoda comprises about three quarters of all Molluscs, and includes several subclasses including Pulmonata (Madsen, 1985b). All of the investigated snails are Pulmonates, which is a genus of Gastropoda.

Biomonitoring of Aquatic Environment
Among aquatic organisms suitable for biological monitoring, molluscs occupy a prominent place and they are often used both for passive and active biomonitoring and in hazard and risk assessment (Salanki, 1986). In recent years, researchers have focused their attention on the identification of other possible bioindicators for trace metal pollution, such as the gastropod molluscs. Several biomonitoring studies for the heavy metals pollution in aquatic ecosystem, have been carried out in past two decades using different mollusc species like, gastropod mollusc Bembicium nanum (Gay and Maher, 2003) Donax trunculus and Chamelea gallina (Usero et al., 2003), bivalve: Pyganodon grandis (Bonneris et al., 2005), Crassostrea angulata, Scrobicularia plana, Palameon longirostris, Uca tangeri, Melicertus kerathurus (Blasco et al., 1999), Crassostrea virginica (Apeti et al., 2005), Radix ovata and Viviparus spp. (Gundacker, 2000), Rapana venosa and Neverita didyma (Lee et al., 2006). This is because it is necessary to identify a wider range of bioindicators and thus expand current understanding of different bioaccumulation strategies for trace metals. Both essential and non-essential trace elements are known to be highly accumulated by invertebrates, in particular by a variety of molluscs’ species. Aquatic molluscs seem to reflect ambient metal contamination and are therefore widely used as bioindicator organisms. Undoubtedly, aquatic molluscs are amongst the most thoroughly investigated bioindicator organisms.

The translocation of sentinel species, mainly mussels from a reference site to the study areas has been demonstrated as a useful strategy for the assessment of water quality in coastal and estuarine environments, either through bioaccumulation or biomarkers analysis (Fowler et al., 2004). Body size, condition index and tidal height also affects the concentrations of As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn in Mytilus edulis and findings suggested that the body weight was inversely related to metal concentrations and for Cd, Mn, Pb and Zn the regression was affected by tidal height. Except for As, Fe and Mn metal concentrations were inversely related to physiological status though no differences between essential and non-essential metals were recorded. Thus it is recommended that stringent measures during sampling for biomonitoring or metal concentrations at each location must be normalized to a common body size, condition index and tidal height (Mubiana et al., 2006). As even closely related species may exhibit different accumulation strategies for trace elements, there is a need to identify widespread cosmopolitan biomonitors to allow intra-specific comparison of accumulated metals concentrations over large geographical areas. Considering that bioaccumulation of heavy metals is highly site dependent, it was of general interest to test the suitability of molluscs as metal bioaccumulators in the moderately polluted waters. Different feeding habits in mollusc may influence metal bioaccumulation for example the prosobranch grazer Viviparus species is both a deposit-feeder and facultative suspension-feeder. In this specie, the bypobranchial gland beneath the gill coats the filtered particles with mucus; food particles are then transported towards the mouth and ingested (Gundacker, 2000).

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Various metal accumulating bivalve and gastropod species show a high presence and abundance in marine and freshwater riverine ecosystems therefore they are suitable for different monitoring projects. Mussels can accumulate and integrate concentrations of several metals in seawater for relatively long intervals. They also assimilate trace metals from their food and from the ingestion of inorganic particulate material (Philips, 1977). The digestive gland of bivalves is a target organ for the accumulation of metals, furthermore, the lysosomes of the digestive cells are generally considered as target organelles while the gills have also been shown to accumulate various heavy metals either in the field or in the laboratory.

Environmental pollutants such as metals pose serious risks to many aquatic organisms. Accordingly, a great deal of previous research that has characterized physiological mechanisms of toxicity in animals exposed to contaminants. Relationships between ambient geochemistry, watershed land-use and trace metal (Cu, Zn, and Pb) concentrations in three types of invertebrate aquatic molluscs, odonates, and composite were established (Reiner and Sparling, 2001).

Metallothioneins are cysteine rich, low molecular weight, heat stable proteins that bind to metals such as Cd, Cu and Zn. Metallothioneins in aquatic invertebrates play an important role in the homeostasis of essential metals like Cu and Zn and detoxification of excess amount of essential and non essential metals such as Cd, therefore extensively used as biomarkers (Rainbow et al., 2000). Moreover, MT levels are also known to be related to the fitness status and health of organisms. The literature on metallothioneins (MT) and metallothionein-like proteins (MTLP) in aquatic invertebrates is large and increasing. Metallothionein like proteins appear to play an important role in mediating metal uptake and hence accumulation, therefore metallothionein has been assayed in a range of aquatic animal tissues as an indicator of metal exposure. MTs can be induced by the essential metals Cu and Zn and the non-essential metals Cd, Ag and Hg in both vertebrates and invertebrates, but their induction is variable (Urena et al., 2010). Against this background of variability, MTs do appear to play roles both in the routine metabolic handling of essential Cu and Zn, but also in the detoxification of excess amounts intra-cellularly of these metals and of non-essential Cd, Ag and Hg. Different isoforms of MT play different physiological roles, and the dependence on MT in detoxification processes varies environmentally and between zoological groups.

Need for Biomonitoring of Aquatic Environment
Chemical analysis of the environment matrix such as water, sediment is the most direct approach to reveal the heavy metal pollution status in the environment, while it cannot afford the powerful evidence on the integrated influence and possible toxicity of such pollution on the organisms and ecosystem. Biomonitoring is a scientific technique for assessing environment including human exposures to natural and synthetic chemicals, based on sampling and analysis of an individual organism’s tissues and fluids. The results of these measurements provide information about the amounts of natural and manmade chemicals that have entered and remained in the organisms and the corresponding effects induced. Due to consistency between the selected organisms and the corresponding living space, biomonitoring can directly offer the data on the potential effects and actual integrated toxicities of pollutants, reflecting the corresponding deleterious degree in the environment (Zhoua et al., 2008).
An important approach to assessment of risk from environmental and occupational exposures is biomonitoring which provides an estimate of the total dose absorbed and gives indirect access to determination of target site concentrations.

To reveal the presence of pollutants and to measure their toxic effect biological indicators can be used. Active and passive monitoring are two general approaches to assess the pollutants and their toxic effects at different levels from species to community level of any ecosystem. In passive monitoring degradation of the ecosystem, elimination of sensitive species and reduction of biodiversity can be revealed as adverse consequences of pollution at the level of populations, while at the level of individual’s accumulation of toxic substances in specimen, in organs and tissues indicative of pollution in the environment can be traced. In active monitoring the response of artificial or modified populations, behavioral patterns of specimen, specific function of organs like movement, feeding, respiration, reproduction and the neural regulation, as well as cellular and subcellular events are studied under the effect of toxic substances.

In biomonitoring surveys, the toxic elements arsenic, cadmium, chromium, cobalt, lead and nickel etc are used as examples to illustrate the disturbing factors in the interpretation of biomonitoring results (Christensen, 1995). The accumulation of trace elements in aquatic consumers is of interest to environmental scientists concerned with the fate and effect of contaminants, as well as to ecologists interested in food web dynamics and trace metal biogeochemical cycles to assess the toxic impact or distribution of contaminants. It is necessary to understand how elements move through aquatic food webs. Understanding the means by which aquatic organisms accumulate trace metals from their environment is complicated by the existence of both soluble and dietary sources. For many aquatic invertebrates, trophic transfer accounts for a major portion of total trace element accumulation. In the field, the ecotoxicological approach is very difficult for evaluation of the impact of heavy metals in an aquatic environment, due to the complexity of interrelationships between organisms and the ecosystem.

However, field studies can enable assessment of the long-term effect on organisms of heavy metals. The underlying regulator of metal concentrations accumulated by animals in tissue is the balance between accumulation and elimination (both of which vary according to the organism’s accumulation strategy and diluting body growth) (Colac et al., 2006)

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