Toxicity and accumulation of lead and cadmium in the land snail, Archachatina papyracea, in a tropical Alfisol from Southwestern Nigeria

Snails are an important link in the transfer of contaminants, especially metals in the food chain. Yet, few studies have examined the toxicity and accumulation of metals in snails in the tropics. This study assessed the toxicity and accumulation of two non-essential metals (cadmium and lead) to the tropical snail Archachatina papyracea. Specimens of the snail A. papyracea were exposed in a loamy soil collected from Ile-Ife, Nigeria and spiked with varying concentrations of Cd and Pb over 28 days. Survival and weight change of snails were monitored weekly, while tissue accumulation was assessed at the end of the 28-day period. Survival was a more sensitive endpoint than the weight change of snails. The Cd median lethal concentration (LC50) value was 93 ± 4.4 mg/kg, while the median effect concentration (EC50) for snail weight change was 131 ± 41 mg/kg. For Pb, LC50 value was 1121 ± 457 mg/kg, while the EC50 value for weight change was higher at 4541 ± 1180 mg/kg. Therefore, Cd was a factor of about 10 to 30 × more toxic than Pb, consistent with findings on the relative toxicity of Cd and Pb to other soil organisms, including earthworms, springtails, and mites. Although not included initially as an endpoint, egg production in the snails decreased with increasing Cd and Pb concentrations in the substrate. Metal analysis of the foot and visceral mass of surviving snails showed progressive accumulation of Cd and Pb as concentration increased, showing the tendency to use body residue of A. papyracea as an indicator of metal pollution. It further suggests the role of this snail species in above-ground metal transfer in the food chain and highlights the potential danger for human consumption.


Introduction
Metal pollution has become a great source of concern, especially through contamination of soil and groundwater via point and non-point sources (Edwards, 2002). Metals can persist in the soil for a long period of time, hence creating long-term hazards. Environmental contamination with metals results in accumulation and subsequent toxicity to plants (Zayed et al. 1998;Gimmler et al. 2002), microbes, and invertebrates (Otitoloju et al. 2009;Abdel Gawad 2018a). In metal-contaminated industrial soils, reports of loss of invertebrate species diversity are commonly reported (Spurgeon et al. 1994;Jones 1991). In earthworms, heavy metals can cause spermatozoa damage thereby reducing the earthworm sperm count (Cikutovic et al. 1993;Reinecke and Reinecke 1997), which negatively affects reproduction (Spurgeon et al. 1994). Abnormal environmental metal concentrations have been reported to adversely affect the feeding, growth, reproduction, and general physiology of molluscs (de Calventi 1965;Calabresse et al. 1977).
One way by which organisms interact with metals in the environment is through bioaccumulation. Metals are nondegradable and tend to bioaccumulate in soil biota. For example, Owojori and Siciliano (2012) reported a substantial accumulation of cadmium and lead in soil mite, Oppia nitens. In two genera of earthworms, Amynthas and Metaphire exposed to a metal-contaminated site, cadmium's bioaccumulation factor was reported to be more than ten (Wang et al. 2018). In a study where snails, Helix aspersa, were Communicated by Bruno Nunes. exposed to a metal-polluted site and compared to a non-polluted site, a substantial accumulation of copper, zinc, lead, and cadmium was reported (Gomot de Vaufleury and Pihan 2000). Similar results were reported for the aquatic snails Theodoxus niloticus which significantly accumulated lead, zinc, and iron and for Corbicula fluminalis which accumulated cadmium from a metal contaminated water compared to control (Abdel Gawad, 2006Gawad, , 2018b. The bioaccumulation potential of soil biota, demonstrates that bioaccumulation is a major process through which increased levels of metals are transferred across food chains, creating ecological and public health problems. Therefore, it is important to always determine the capacity for bioaccumulation and the toxic effect of heavy metals on organisms, especially edible ones like snails, to assess potential risks to human health. Moreover, it is important to note that molluscs can accumulate higher metal ion concentrations than other invertebrate groups (Beaby and Eaves 1983;Fishar and Ali, 2005;Phuong, 2014).
In a study of invertebrates in the vicinity of the Avonmouth smelting works (UK), the snail, Helix aspersa, was not found in regions of extremely high metal pollution that are close to the factory (Jones 1991). One way to explain this phenomenon was that there was direct poisoning of snails by metals ingested with food. However, in laboratory experiments, where snails and slugs were exposed to very high heavy metal concentrations through food, they have proved to be tolerant (Marigomez et al. 1986). The authors did not find any effect of Zn or Cu in diets on the mortality of the slug Arionater at concentrations as high as 1000 mg/kg. Similarly, Russell et al. (1981) found no mortality in Helix aspersa that were fed on a diet containing up to 1000 mg Cd/kg food. This high tolerance in the molluscs is possibly due to the efficient binding of metal ions by metallothioneins (Taylor et al., 1988) and deposition in insoluble intracellular granules (Howard et al. 1981). For example, Beeby and Richmond (1989) suggested that the snail shell could act as a temporary "sink" for Pb. Terrestrial molluscs may also possess other physiological mechanisms to regulate metal assimilation from food sources, e.g., by storing in hepatopancreas and elimination through feces (Berger and Dallinger 1989).
Most of these studies were conducted with temperate land snail species, and very few studies have been conducted on the toxicity of metals to tropical land snail species. Notably, among the snail species studied in the tropics are the giant land snail, Archachatina marginata. Their metal bioaccumulation tendencies in the field have been widely reported (Wegwu and Wigwe 2006;Ebenso and Ologhobo 2008). Some studies have also reported metal toxicity to A. marginata (Otitoloju et al. 2009). Ugokwe et al. (2020) also studied the effect of contamination on another tropical land snail species, Limicolaria aurora, where it reported induction of oxidative stress enzymes when exposed to waste leachates containing metals compared to the non-exposed snails. Archachatina papyracea is another known giant land snail species, but metal toxicity to this snail and metal accumulation are largely unknown. In Nigeria and many other countries, Pb and Cd are introduced into the environment through paint flakes, burning of fossil fuels which contain these metals as anti-knocking additives, and through metal mining activities (Yakeem and Onifade, 2012;Adeyi and Babalola 2017;Yahaya et al. 2021). Therefore, metal contamination of the environment is a plausible occurrence through these activities, especially in sites close to these industrial activities. Snails purchased in the local market and eaten in these regions are often gathered from the wild and farmlands (Babatunde et al. 2019); however, studies have shown that snails gathered from farmlands close to industrial activities have a high accumulation of metals (Onuoha et al. 2016). Therefore, it is important to assess the effect of some metals on the toxicity and accumulation of local snail species in the area.
The land snail Archachatina papyracea is often found in garden soils throughout Nigeria. Therefore, a study assessing the effects of Pb and Cd in the local population of snails may be critical to assessing the environmental and human impact of the metal contamination in an area. For this study, we assessed the accumulation of Pb and Cd, and the toxicity of these metals based on survival and weight change to the tropical snail species, A. papyracera. The choice of heavy metals, Pb and Cd, used for this study was because Pb and Cd, though non-essential metals, are often found in high concentrations in abandoned mining and smelting sites and most industrial activities such as in the auto-mechanic industry (Dudka et al. 1995;Nica et al., 2015). For example, Abidemi, 2011) reported excess of 2400 mg/kg of Pb in some soils close to auto-mechanic industry in Nigeria. Moreover, compared with other metals, the toxicity of Cd and Pb can be very high (Owojori and Siciliano 2012;Zhiyou et al., 2016;Mahmutovic et al. 2018).

Soil collection and preparation for experiment
The soil sample used was a loamy soil collected from Ede Road, in Ile-Ife, Nigeria. They were collected by scraping the first 5-cm topsoil from the site. The soil was oven-dried for 7 days to defaunate it, and the physicochemical properties of the soils such as soil texture, water holding capacity (WHC), pH, and organic matter (OM) were subsequently determined using methods described by Jaabiri Kamoun et al. (2018). The soil was re-moistened to 50% of its maximum water holding capacity (WHC) and used for the acclimatization of the snails to laboratory conditions. During acclimatization, the snails were fed with fresh vegetables collected from a site where no pesticides were used at the Obafemi Awolowo University Ile Ife, Nigeria. The physicochemical properties are summarized (Table 1).

Metal background levels in the soil.
The metal background levels of the soil were determined using methods described by Maboeta et al. (2003). One gram of soil was at 106 °C for 48 h after which the soil was sieved to 500 µm. The sieved soils were then digested with 10 mL of 55% HNO 3 and left for 24 h. The samples were then heated for 2 h at 60 °C and 1 h at 130 °C. The samples were then allowed to cool before adding 5 mL of HClO (perchloric acid) and heated for 1 h at 130 °C. The samples were allowed to cool again, and water was added before heating again at 130 °C until white fumes were observed. The samples were then allowed to cool and were filtered with 0.45µm syringe filters before analyzing with Varian AA-1275 flame atomic absorption spectrophotometer (AAS) at the Central Science Laboratory, Obafemi Awolowo University, Ile-Ife.

Test animals and acclimatization
The test animals used in the laboratory experiments were specimens of the indigenous land snail, Archachatina papyracea. The snails were purchased from the Kings market, Ile-Ife, Nigeria. The snails within weight ranges of 8.5-10.5 g were selected for all the bioassays. The live snails were taken to the laboratory to acclimatize to laboratory conditions (28 ± 2 °C; 80-95 ± 2% relative humidity) in a plastic bucket terrarium (200 cm × 80 cm × 30 cm) containing a layer of moist humus soil as the substrate for at least 7 days before commencement of bioassays. The bucket terrarium was covered with a lid to prevent the snails from crawling out. The lids were perforated for proper ventilation. The snails were fed ad libitum on fresh pawpaw leaves (Carica papaya). Unconsumed food and fecal material were removed daily to prevent contamination. The snails used throughout the experiment were obtained from the same source to reduce the variability of the biotype.

Test procedures.
The metals used for this experiment are lead added as lead sulphate (PbSO 4 .5H 2 O; purity, 98%) and cadmium added as cadmium chloride (CdCl 2 .2½H 2 O). Pre-determined amounts of the metals were weighed using an analytical balance and made up to a fixed volume by adding an appropriate volume of distilled water as diluents to achieve a stock solution of known strength. The resultant stock was then serially diluted to obtain solutions of the required concentration. The concentrations of the Pb and Cd used were as follows: (1500, 3000, 6000, 12,000, mg/kg Pb) and (15, 30, 60, and 120 mg/kg Cd). The concentrations used were chosen after a range-finding test. Each treatment soil (2400 g) was split into 3 equal portions and was put into different containers. Each of the samples containing 800 g dry weight soil was placed in a properly labeled cylindrical plastic vessel of 4L and allowed to equilibrate for 7 days before snails were introduced. Ten snails per container were used in each exposure regime and were introduced into the relevant test soil by placing them on the soil surface. The test containers were covered with a perforated lid to limit water loss due to evaporation and kept in 16 h light and 8 h dark at 28 ± 2 °C for 28 days. To maintain the snails during the exposure period in the test medium, the snails in each container were fed weekly with 100 g of Carica papaya (pawpaw) leaves. Uneaten food materials were removed weekly before adding new food items. Sampling was done weekly for weight change and survival. On the last day of the experiment, surviving snails were de-shelled and used for the metal analysis.

Bioassay
On each sampling occasion, weight change and mortality were assessed, while Pb and Cd accumulation were only assessed at day 28. The snails were rinsed in deionized water to remove soils or dirt from their shells and visible skin prior to weighing. Weight change was determined by collectively weighing all snails in each container (i.e., per replicate) and comparing the mean weight with initial values. Mortality was assessed by stimulating the snail with a blunt probe, and the snail was judged dead if no response could be observed. To determine the amount of metal accumulated, one snail per container was selected and was placed on moist filter paper in petri dishes for 24 h at 20 °C to allow depuration of their gut contents. Afterwards, they were de-shelled and frozen individually for metal analysis. The procedure for the metal extraction and analysis has been described by Maboeta et al. (2003). This was done by acid digestion with HNO 3 and spectrophotometric analysis.

Lead and cadmium accumulation analyses
The acid digested snail samples and three blanks were analyzed for Pb and Cd by a Varian AA-1275 flame atomic absorption spectrophotometer (AAS) at the Central Science Laboratory, Obafemi Awolowo University, Ile-Ife. To determine the Pb and Cd concentrations in the acid digested snail samples, the absorbances of Pb and Cd standards (Perkin Elmer Pure) were compared to the concentrations in the digested samples. For quality control, blanks (HNO 3 acid with no digested snail sample), duplicates, and calibration standards were run for every five samples.

Statistics
The mortality and weight change data were presented as mean ± standard deviation (SD). The survival was assessed as lethal concentration. The lethal concentrations at which 50% mortality of the snail population was observed (LC50) for each metal was calculated using the Trimmed Spearman Karber (TSK) method (USEPA 1993). The effective concentrations of the metals causing 50% or 25% reduction in snail weight (EC50) were estimated with non-linear regression models (3-parameter logistic models) in R (Ritz 2016).

Survival of the snails after exposure to Pb and Cd
The mortality of the snails increased with increasing concentration of Pb (Fig. 1). The numbers of snail in the control experiment remained relatively constant (≥ 90% survival) throughout the experiment. At day 7, no noticeable difference in survival was found for all Pb concentrations. However, by day 14 through day 28, a progressive effect of Pb on the survival of the snails was obvious. This progressive effect of Pb on the snails was evident in the LC50 values recorded after day 14 (2786 ± 852 mg/kg), which was higher than at day 28 (1121 ± 457 mg/kg) ( Table 2). For Cd, at day 7, the dose-dependent effect of Cd was observed earlier than for Pb, as Cd had more than 20% effect on mortality (LC25 = 84 ± 8.50 mg/kg) at the highest exposure concentration. As the duration of exposure increased from 7 to 14 days, the toxicity of Cd to the snails increased (Fig. 2). The toxic response of the snails to Cd was not different from day 14 to day 28, and the LC50 values were also similar at day 14 and day 28 (86 ± 3.5 and 93 ± 4.4 mg/kg, respectively) ( Table 2).

Weight change of snails exposed to Pb and Cd
Similar to the effect on survival at day 7, there was no substantial effect on weight change at day 7, as the concentration of Pb increased (Fig. 3). As from day 14, dosedependent effect of Pb was seen on the weight change of the snails with the highest effect found at day 28. There is a drastic change in weight of the animals from day 7 to 28 ( Fig. 3). There was no effect of Cd on the weight of the snails up until day 7 at all the concentrations tested (Fig. 4). However, about 25% effect on snail weight started showing from day 14 but only at the highest concentration of Cd. Toxicity of Cd on snail weight did not change as the duration of exposure increased from 14 to 28 days (EC25 ~ 120 mg/kg) (Table 3).

Other observations of the snails indicative of toxicity
From day 7, the snails were frequently seen clinging to the upper part of the container devoid of soil as a form of avoidance. At the end of the experiment, the number and size of eggs produced by snails in the contaminated soils were fewer and smaller than those in the control soils. Furthermore, when the snails were deshelled in readiness for accumulation assay, the hemolymph of the snails in the contaminated soils was thick and dark in color compared to light-colored hemolymph of snails in the control soils.

Metal background level of soil and accumulation of Pb and Cd in the snails
The background level of Pb was 0.369 ± 0.004 mg/kg of soil and Cd was 0.473 ± 0.015. In terms of metal accumulation, no dose-response effect was seen in the accumulation of Pb in the snails (Fig. 5), but exposed snails had higher concentrations of Pb than unexposed snails. The Pb content of snails in any of the exposed soil was at least 35 times higher than those in the unexposed soil (Fig. 5a).
Conversely, in the case of Cd exposure, the accumulation followed a dose-response pattern except at the highest concentration (120 mg/kg) of exposure (Fig. 5b).

Discussion
This study demonstrated that Pb and Cd, two contrasting metals, are toxic to the tropical snail A. papyracea exposed to concentrations likely to be found in the environment. Although some studies have reported the toxicity of these two metals to other species of snails, the toxicity they reported was lower than in our study, suggesting A. papyracea may be more sensitive than these species. Sahraoui et al., 2021) reported 20% H. aspersa mortality at 3300 mg of Pb/kg and 40% mortality at 2000 mg of Cd/kg of soil.
In the same study, EC50 on growth ranged from 2397 to 4804 mg of Pb/kg and 327 to 591 mg of Cd/kg of soil. Coeurdassier et al., 2000) reported EC50 of about 1000 mg of Cd/kg of soil. However, studies on snails exposed to contaminated food reported higher values-EC50 > 13,900 mg of Pb/kg in food (Laskowski and Hopkin 1996a;Gomot-De Vaufleury 2000), suggesting that exposure via soil may be a more important pathway for metal toxicity to snails.
Cadmium (Cd) was more toxic than Pb in this study. In many ecotoxicological studies, Cd is usually the most toxic metal to soil organisms (Abdel Gawad 2006;Zhiyou et al. 2016;Owojori and Siciliano 2015). The comparatively lower Pb toxicity may be due to its low ability to cross membranes of exposed organisms. Studies have also shown that animals  can accumulate Pb without any adverse effect (Otitoloju et al. 2009). It appears strange that the EC50 for weight change is higher than the LC50 for survival, but this could have been caused by the fact that only surviving snails were used to estimate weight change, and therefore, the surviving snails could have been those avoiding the Pb in the substrate. Avoidance mechanisms, where soil organisms move away from contaminated food or soil, are well pronounced (Owojori et al. 2009). Also, Laskowski and Hopkin (1996a, b) reported that the intrinsic growth rate of H. aspersa populations was influenced more by delayed sexual maturity than by fecundity inhibition. The EC20 fecundity of Pb on H. aspersa was 6140 ppm and for Cd was 120 ppm (Laskowski and Hopkin, 1996b), values close to what we found in this study for effect on growth at that effect level. Apart from data on the general trend in survival and weight change of snails after exposure to Pb and Cd, other significant changes in the behavior or physiology of the exposed snails were observed. These behavioral and physiological observations of the snails were not particularly measured since it was beyond the scope of the experiment; we reported the observations, nevertheless. For example, the animals tended to move away from the contaminated soils to the upper part of the container. The number of eggs produced was fewer and smaller when compared to those in the control experiment. Carbone and Faggio (2019) reported that H. aspersa did not transfer metals to their eggs, but the eggs were smaller in size (Beeby and Richmond 2001;Carbone and Faggio 2019). Although we did not assess metal deposition in the eggs, the eggs produced in contaminated soils were smaller than eggs from the control soil. It is possible that the snails fed poorly, resulting in smaller egg production. For example, snails, Alinda biplicata, exposed to high Cd concentration of about 2692 µg/g started feeding poorly at the onset of the study to avoid metal uptake, and they stopped consuming food by the end of the study (Pedrini-Martha et al. 2020). Many snails exposed to deleterious metal levels have been reported to avoid metal uptake by reducing food consumption (Laskowski and Hopkin, 1996a;Notten et al. 2006;El-Gendy et al. 2011). Another observation of the snails in our study was that the hemolymph of the snails in the control soils were light, with slight change in color than the ones in the exposed soils, which were thick with darkened coloration. Otitoloju et al. (2009) reported the thickening and inflammation of hepatic tubules of the hepatocytes of A. marginata exposed to copper and lead.
The snails accumulated Pb and Cd to varying degrees where Pb was constant at all exposed concentrations and Cd accumulated in a dose-response pattern except for 120 mg/ kg. At 120 mg/kg, Cd accumulation declined possibly due to increased excretion of metals or increased avoidance of the highly contaminated soil. We did not investigate further on the reason which was beyond the scope of the study. However, in general the snails substantially accumulated Pb and Cd, which was consistent with many studies that reported significant metal accumulation by snails (Nica et al. 2012;Pedrini-Martha et al. 2020). Compared to A. marginata, our study showed that A. papyracea accumulated more Pb (38 µg/g) and Cd (8 µg/g) than observed in field studies with A. marginata. For example, Pb in the range of 0.77 mg/kg to 7.51 µg/g were reported in snails (A. marginata) sampled from nine sites in southern Nigeria (Chukwujindu et al. 2008). Ebenso and Ologhobo (2008) reported Pb accumulation ranging from 0.14 to 8.42 µg/g in A. marginata exposed to lead vehicular fumes. A. marginata collected from the field in six Nigerian states accumulated averages of 0.8 µg/g of Pb and 0.6 µg/g of Cd per snail (Wegwu and Wigwe 2006). However, Otitoloju et al. (2009) in a laboratory study reported a higher accumulation of Pb (67 µg/g) in A. marginata than A. papyracea (38 µg/g) in our study. The high accumulation observed by Otitoloju et al. (2009) was probably because it is a laboratory study as the present study, and the method of application showed that both food and soil could have been dosed. However, in our study, only soil was dosed. Moreover, the increased accumulation of Pb or Cd in the laboratory studies showed that laboratory studies might over-exaggerate the accumulation of metals by snails in the wild, perhaps due to bioavailability differences or increased propensity of snails in the wild to avoid contamination owing to a larger landscape. Land snails belonging to the family Achatinidae are native to Africa (Awodiran et al. 2013). Many snail genera such as Archachatina belonging to this family are often consumed as food in Nigeria. Their meat is proteinous with low cholesterol and contains vital minerals making them nutritious (Awodiran et al. 2013;Onuoha et al. 2016). Therefore, snails can also be a source of human health concern in Nigeria if contaminated snails are consumed (Onuoha et al. 2016). This study has revealed that the non-essential metals, Pb and Cd, are harmful to the snail Archachatina papyracea and that this snail species could accumulate both metals in their tissues. Since the snails accumulated Pb and Cd in this laboratory experiment, it is an indication that snails in the wild could also accumulate Pb or Cd if their environment is contaminated with these metals. Therefore, snails picked in these areas and sold in the market might pose a severe health risk to humans. For example, the metal concentrations in the soil exceeded the limit of 1.5 mg/kg for Pb and 3 mg/kg for Cd (WHO 1990), and the metal concentrations in the snails exceeded the daily ingestion reference dose of 0.0035 mg/ kg per day for Pb and 0.001 mg/kg for Cd (Joseph et al., 2021). Due to a possible health impact from consuming contaminated snails, great care should be taken when such snails are bought in the market. For example, snails should be purged by not feeding for about 5 days before consuming the snails to detoxify pathogens and chemicals like metals (Antwi 2009). Apart from the human health risk associated with consuming contaminated snails, the ecological status and preserving their biodiversity are also vital. We hereby advocate that more metal accumulation and toxicity studies on snails need to be conducted for tropical snail species.

Conclusion
In this study, A papyracea (based on survival and growth) is more sensitive to Pb and Cd compared to most studies on other snail species. A papyracea also accumulates Pb and Cd to a higher degree than most snail species exposed to similar concentrations reported in literature. Physiological effect such as darkened hemolymph and sublethal manifestations such as decreased egg size and production were observed in the exposed snails. Moreover, because of the propensity of A. papyracea to accumulate substantial concentration of metals from the environment, purging of the snails is advised prior to human consumption. The study showed that A. papyracea is suitable for use as a soil biological indicator of toxic metals in the tropics, such as in Nigeria.