Snails are widely used as environmental monitors for pesticides, PAHs, PCBs, PBDEs, OCPs, glyphosate, glufosinate and heavy metals. Various snail species have been studied, such as Indothais gradata (Proum et al., 2016), Papillifera papillaris (Emilia et al., 2016), Helix aspersa (Abdel-Halim et al., 2013 ; Viard et al., 2004), Pomacea canaliculata (Dummee et al., 2012 ; Ramli et al., 2019 ; Wu et al., 2019), Achatina fulica (Cho et al., 2019) and Cantareus apertus (Mleiki et al., 2016). Among these species, Helix aspera was used to investigate herbicides (glyphosate and glufosinate) and fungicides (cymoxanil, folpet, tebuconazole and pyraclostrobin) contamination using fluorescence detection HPLC and GC-MS respectively (Druart et al.
, 2011) while analysis of polycyclic aromatic compounds was done using liquid chromatography coupled to a ?uorimetric detector (Sverdrup et al., 2006).
In addition, it has been confirmed that the apple snail Pomacea canaliculate is identified as bioindicators for many environmental pollutants, indicating both the level and the profile of pollutants, as well as for persistent organic pollutants (POPs) (Fu et al., 2011; Harmon and Wiley, 2010) as for metals and organometallic compounds (Campoy-Diaz et al.
, 2018; Cueto et al., 2013; Giraud-Billoud et al., 2018). Their ecology and biology can be classified as having most of the essential characteristics of an ideal bioindicator, such as bioaccumulation potential, ease of collection, limited range of movement, short life span and wide distribution (Tanabe and Subramanian, 2006). Apple snails occupy a significant trophic position and are widely dispersed in bounty in abundance in local ecosystems, and they are a popular source of food for various animals like fish, birds andhumans. Apple snails are easy to collect compared to common animals used as bioindicators, such as fish or birds, and their range of movement is comparatively narrow over their lifetime.
Apple snail’s lifespan is usually in the range of 1-4 years, and the size and whorls of the snail could easily identify age. Therefore, it is beneficial to use apple snails to evaluate the pollution of the environment.
It is also noted that LC was the main analytical process used to analyze POPs in combination with a fluorometric detector to analyze polycyclic aromatic hydrocarbons (Beach et al., 2009; Sverdrup et al., 2006) and to mass spectrometry to analyze polychlorinated biphenyls (Storelli et al., 2014). However, the techniques of GC-MS could be used. Analysis of polychlorinated biphenyls and fungicides (cymoxanil, folpet, tebuconazole and pyraclostrobin) using gas chromatographs coupled with mass spectrometer techniques was presented by Fu et al. (2011) and Druart et al. (2011) respectively (Druart et al., 2011; Fu et al., 2011).
On the other side, the atomic absorption spectrophotometer, inductively coupled plasma-mass spectrometry and inductively coupled plasma-emission spectrophotometry are the most widely used methods for the determination of heavy metals in environmental samples (Sastre et al., 2002). In addition, the process of microwave-assisted acid digestion has also been widely reported. It is a closed system providing high temperature and pressure in closed vessels, depending on various parameters such as the digestion temperature, time, and the chemical reactive used. The nature of the matrix to be studied determines the choice of acids or the combination of acids used as reagents. (Durand et al., 2016).
3. Presence and detection of pollutants in snails
3.1. Snail pollution by metallic trace elements (MTE)
Metal trace elements (MTEs) are part of trace elements family and constitute only 0.6% of the total elements. Their denomination is due to their low concentration, which usually does not exceed 1000 mg.kg-1 naturally in soils (Cr?mazy et al., 2019; Emilia et al., 2016). Among these MTEs are cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), zinc (Zn) classified in the metal class and arsenic (As) and antimony (Sb) in the metalloid group. Due to their harmful effects on the environment and their classification as carcinogenic or dangerous to human health, their presence in the environment and soil is a significant source of concern. However, three metals (cadmium (Cd), lead (Pb) and mercury (Hg)) have been identified as priority dangerous substances in Decision 2455/2001/EC of the European Council. (INERIS, 2006; Cheng and Yap, 2015; Emilia et al., 2016).
Snails are well known for the bioaccumulation of these pollutants (C?urdassier et al., 2002; Viard et al., 2004). In fact, it has been observed that about 68% of the cadmium (Cd), 90% of the copper (Cu), 43% of the lead (Pb), and 60% of the zinc (Zn) ingested were accumulated in Helix aspera snails ‘ soft tissue, shells, and faeces that were analyzed by flame atomic absorption spectrometry after mineralization and extraction using concentrated HNO3. Snails seem to be more important pathways for transport along the food chains of Cu and Cd than Zn and Pb and are not able of depositing large quantities of metals in their shells. Resistance to the appearance of effects associated with the accumulation of metals by snails may cause their predators to be contaminated (Laskowski and Hopkin, 1996). However, the snail has many predators, including vertebrates, such as birds, small mammals, reptiles and invertebrates, such as carabids (Liew and Schilthuizen, 2014). Staikou and Lazaridou emphasize the role of snails in the transfer of material and energy from producers to higher trophic levels (Staikou and Lazaridou, 2013), suggesting their potential involvement in the transfer of metallic pollutants along trophic chains (Dar et al., 2019; Hispard et al., 2008). Human consumption of the Helix aspersa snail is also small (15 tons of canned food in France in 2008) relative to the Bourgogne Helix pomatia snail (876 tons of canned food in France in 2008) (Druart et al., 2011).
In addition, metals in wet and dry atmospheric deposition are absorbed by mosses through passive cation exchange processes and airborne particle detection (Bargagli, 2016), while metal absorption can occur through various pathways in snails, such as algae ingestion, lichens, mosses, herbs, soil particles, inhalation and contact with wall surfaces. Metabolism and detoxification processes of cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), zinc (Zn) and mercury (Hg) are conducted in the digestive gland, and their bioaccumulation is mainly due to the compartmentalization in intracellular granules or vesicles in the digestive gland or other organs such as the foot or the albumen gland (Boshoff et al., 2013; Regoli et al., 2006). Soft snail tissues purged of their intestine content have low metal concentrations and their chemical composition may provide a reliable image of environmental metal pollution. For example, lead (Pb) concentrations in Italian urban environments can be very high in vehicle-suspended street dust, and this metal could have a relatively small impact on urban food chains, particularly in black crusts formed on monuments (Barca et al., 2014).
Specially cadmium (Cd), lead (Pb), zinc (Zn) under alkaline soils increases the adsorption and precipitation of trace metals. A higher percentage of metals will be present in their ionic state as pH decreases. In order to successfully bind to exchange sites, such metal ions should interact with additional cations such as hydrogen (H+), calcium (Ca2 +), aluminum (Al3+), iron (Fe2+) and magnesium (Mg2+) (Bakircioglu et al., 2011). Metals may therefore become more bioaccessible for plant uptake at lower pH levels. (Bakircioglu et al., 2011). Pauget et al. demonstrated a significant correlation between physicochemical soil properties (pH and cation exchange capacity) and metal accumulation by Cantareus aspersus (Pauget et al., 2012). Nonetheless, based on the MLR, it was not possible to conclude with confidence that soil metal and physicochemical properties contributed to the accumulation of metal in the digestive gland, although the physicochemical characteristics of the soil may have indirectly affected the accumulation of metals in snails (Cepaea nemoralis) and determined the accumulation of metals in plants (Urtica dioica) as well as snails by their digestive exposure (C?urdassier et al., 2002; Gimbert et al., 2006). Boshoff et al. (Boshoff et al., 2013), Notten et al. (Notten et al., 2006) and Boshoff et al. (Boshoff et al., 2015) reported different levels of cadmium (Cd) such as 33.93-148.40 mg kg-1, 94mg kg-1 and 60-150 mg kg-1 respectively in several studies on differential organ metal levels in Cepaea nemoralis. The absorption of cadmium (Cd) as a non-essential element is not regulated at a specific level or less efficiently regulated than essential elements (Tchounwou et al., 2012). Higher concentrations were observed in the helix aspersa snail fed on a rich diet of cadmium (Cd) (Scheifler et al., 2002) by binding to metallothionein-like proteins present in the digestive gland that accumulate metal at higher levels without severe effects. (Gimbert et al., 2006; Manzl et al., 2004; Nica et al., 2013). For snail physiological functioning, copper (Cu) and zinc (Zn) are required and their absorption is controlled until a threshold level is exceeded(Nica et al., 2012). Snails specifically require high amounts of copper as an element of hemocyanin that is transformed and assimilated after accumulation (Manzl et al., 2004). Snails can maintain zinc in tissues for essential functions at low levels and can affect feeding and development at high concentrations(Swaileh and Ezzughayyar, 2001).
Furthermore, snails living in areas highly contaminated with metals also exhibit morphological and physiological changes due to higher energy costs associated with detoxification and excretion processes (Radwan et al., 2010; Regoli et al., 2006). As the strength of the shell increases slightly, there were no significant morphological changes in the strength and thickness of the shell that could be related to the interaction between site and time even when exposed to contaminated soil. Similarly there are no detected effects of metal toxicity such as cadmium (Cd) and lead (Pb) on snail shell size or weight (Jordaens et al., 2006; Mourier et al., 2011). Although adult snails have a fully developed shell, variation in wild population shell morphology can occur (Gomot de Vaufleury and Pihan, 2000).
Snails have mechanisms for metabolizing, exporting storing and excreting metals, and these mechanisms are activated through a process called acclimatization, such as transfer to new environment, physiological changes resulting from experimental stressors or response to stress due to new restricted microcosm conditions (Bighiu et al., 2017). Accumulated metals will reach a stable state after a specified exposure period and they will not be able to interfere with biochemical reactions (Rainbow, 2007; Tchounwou et al., 2012).
Several studies found that metal exposure (arsenic, lead, and cadmium) caused more severe effects at both relatively high and low dose levels, so these effects are influenced by dosage, exposure duration and genetic factors. kidney failure was caused by human exposure to a mixture of metals, such as cadmium and inorganic arsenic relative with the exposure to each of these elements alone (Nordberg et al., 2005; Wang and Fowler, 2008). Digestion with HNO3 and H2O2 mixture (Abdel Gawad, 2018; Dummee et al., 2012; Emilia et al., 2016; Rybak et al., 2012; Santos et al., 2009) followed by the inductively coupled plasma mass spectrometer (ICP-MS) (Cui et al., 2012; Mleiki et al., 2016; Rybak et al., 2012; Santos et al., 2009; Yin et al., 2014) was the main multi-residue extraction and analytical procedure of heavy metals from the different snail species used.
3.2. Snail pollution by organic pollutants
The high use of pesticides as a result of the increased plant protection practices and global human population growth has contributed in pesticide contamination of snails. They are rarely used individually in agriculture and are used in combination at specific times during crop production. (Ngowi et al., 2007). As a consequence of usage and application, the presence of pesticides overlaps in space and time. (Smiley et al., 2014). Snails are widely used in ecotoxicological studies to investigate pesticide exposure effects that have been observed in response to both individual and combined exposure to pesticides (Hock and Poulin, 2012; Mora et al., 2011). The effects of pesticides and their accumulation via different routes on snails that affecting their response, are influenced by several factors such as, the chemical mode of action (Staley et al., 2015), the sensitivity of species to pesticide (Lushchak et al., 2018) and their exposure period(Damalas and Eleftherohorinos, 2011). Exposure can cause direct effects on all levels of biological organization, while the mode of action of toxicant mainly determines which group of organisms is affected. Several characteristic of snails such as, fertility (Coutellec et al., 2008), survival (Qiu et al., 2011), and movement (Perez et al., 2009) can be quantified to estimate the fitness of snails that decreases in response to environmentally related stress (Coutellec et al., 2008), including physicochemical parameters (Elias and Bernot, 2017) and pesticides contamination (Coutellec and Lagadic, 2006). In addition, the effects of pesticides on snails can either directly affect their egestion and movement or indirectly affect food webs and prey-predator interactions. Lower egestion rates for snails when exposed to pesticides can reduce the availability of nitrogen and carbon, thus affecting nutrient fluxes and algal biomass. (Fink and Elert, 2006). In addition to the mode of action of pesticide, it is important to consider the sensitivity of organisms to pesticides coupled with different periods of exposure in order to understand the potential adverse ecological effects of co-occurring pesticides on ecosystems. The ecology and biology of snails reflect most of the essential characteristics of an ideal bioindicator, i.e. bioaccumulation capacity, wide distribution and ease of sampling (Hall et al., 2009), and these advantages make the snail field an effective bioindicator for pollutants and other organic chemicals such as Pomacea canaliculata (Fu et al., 2011; Koch et al., 2013; Mart?n et al., 2019).
Snails used as environmental biomonitors to manage the air quality were evaluated and were proved to be efficient, accessible, inexpensive, and non-toxic. Due to the wide distribution in the environment, the ability to accumulate several types of organic pollutants in the body without being harmful and facility in adaptation and manipulation in the laboratory, as well as its sensitivity to genotoxicity assays, snails could be the best choice for biomonitoring pollutants monitoring such as pesticides, PAHs, PCBs and heavy metals. The use of snails seems particularly in heavy-metal monitoring as these latest species are in contact especially with soils highly contaminated by these pollutants and will reach a stable state after a determined period of exposure and they will not be able to interfere with biochemical reactions.
For all these reasons, the use of snails as matrix is highly recommended in order to monitor a wide class of environmental pollutants that might be present in the environment.