An 112-Days Experiment on Dietary Cadmium Retention in Hepatopancreas in Adult Cantareus aspersus Snails

GEORGE A. DRAGHICI1, CRISTINA A. DEHELEAN1, IULIA PINZARU1*, DESPINA M. BORDEAN2, GEORGETA POP2, DRAGOS V. NICA1 1Victor Babes University of Medicine and Pharmacy, Faculty of Pharmacy, 2 Eftimie Murgu Sq., 300041, Timisoara, Romania 2Banat University of Agricultural Sciences and Veterinary Medicine King Mihai I of Romania from Timisoara, Faculty of Food Processing Technology, 119 Calea Aradului, 300645, Timisoara, Romania


Cadmium (Cd) contamination in terrestrial environments is a major global issue; it increases year after
year with a massive impact on everyday life ranging from ecological sustainability and food security to human health. This trace metal (TM) occurs naturally in the Earth' crust with an abundance of 0.1 -0.5 mg/kg dry weight (mg/kg d. wt) [1].
Cadmium is a very hazardous pollutant due to several key reasons, primarily the long biological half-life (10-30 years), high accumulation potential along the terrestrial food webs and high toxicity at levels one-tenth that of lead (Pb), mercury (Hg) or aluminum (Al) [2,3].
There is limited knowledge about the kinetics of cadmium retention in gastropod models exposed for longterm (> 3 months) to elevated levels of cadmium [4]. The hepatopancreas, which is the molluskan homologous organ for both the mammalian liver and pancreas, represents the primary endpoint of Cd retention. In this context, the present experiment aimed to determine the level of dietary Cd which led to an increase in Cd retention in the snail hepatopancreas. Was used a continuous 112day exposure, multiple dose study design using newlymatured Cantareus aspersus snails as invertebrate model. Hepatopancreas cadmium level was assessed using Flame Atomic Absorption Spectrometry.
After being oven dried at 105°C, for 24 h, the hepatopancreas samples (between 1.2-1.5 g / each *email: iuliapinzaru@umft.ro sample) were weighed (to the nearest 0.01 mg) with an analytical balance (TP-214, Denver Instrument GmbH). Following their calcination in a muffle furnace (Nabertherm B150, Lilienthal) at 550°C, for 6 h, the ash obtained was dissolved with nitric acid using the wet digestion approach -as previously described for standard processing of animal samples [5].
Finally, the filtrates were brought to 25 mL using 10 mL 0.5N HNO 3 and cadmium concentrations were determined using an atomic absorption spectrometer (Varian AA240FS) fitted with a Cd-specific hollow-cathode lamp as the source of radiation. The results were expressed as mg/kg d.wt.

Statistical method
Differences in hepatopancreas cadmium among were analyzed using a Kruskal Wallis test, with posthoc Dunn's tests being applied in case of significant differences.

Results and discussions
Land snails (Pulmonata) have several ecological and biological attributes rendering them suitable study system for Cd hazard. These are: (i) ecologically associated with humans (i.e., synanthropic species), and also the most species-rich group of terrestrial mollusks; (ii) concentrate higher amounts of Cd than most terrestrial higher bilaterians (including commonly used toxicological models such as rats, mice, earthworms, or arthropods); (iii) sensitive to low level Cd exposure of contamination [6][7][8].
Cantareus aspersus (syn. Helix aspersa) was used as study system in this present work because it is the most often used terrestrial gastropod in environmental toxicology; it has a well-known biology [9] and is easily reared under laboratory conditions [10].
Commonly used animal models in toxicological studies (e.g. rats, mice, fish) have separate sexes, both of which have to be tested for providing relevant results on cadmium retention dynamics. Land snails, by contrast, are hermaphrodites; therefore, their use is a more costeffective alternative to vertebrate study systems in ecotoxicological studies on cadmium [11].
The median concentrations of Cd measured in the hepatopancreas were: (1) (fig. 1). The measured values in controls were within the same range with those assessed in one of our previous experiments [1], as well as with those determined in specimens sampled from unpolluted areas [15,16]. The maximum Cd levels observed here are also of interest from an ecotoxicological point of view since they are among the highest values ever reported in C. aspersus. Therefore, these values may be used as a benchmark for future studies investigating Cd retention in this gastropod species. Were identified significant differences in hepatopancreas Cd levels (Kruskal-Wallis, p< 0.001).
The measured values increased significantly compared to controls starting from the third Cd treatment (i.e. 0.2Cd treatment) onward, as shown by post hoc analyses using Dunn tests ( fig. 1). In a previous study, were investigated the retention of Cd in adult C. aspersus snails at similar exposure doses, but for shorter durations, namely 14, 28 and 56 days. Interestingly, the dose from which significant increases in hepatopancreas Cd were detected was similar [1].These results suggest the potential existence of threshold level below which these land snails are able to maintain relatively stable cadmium concentrations in the hepatopancreas, which is up to 5 mg/kg d. wt.
Starting from the third dose onward, the test snails concentrated Cd not only in a dose dependent manner, but also at levels far above the measured values in the food. This renders the hepatopancreas of mature snails, C. aspersus, as excellent endpoints for assessing Cd toxicity over a broad range of concentrations. Serving as a barometer for dietary Cd exposure ¯ the main path of Cd uptake not only in terrestrial gastropods [6], the hepatopancreas of this species of land snails can hence be reliably used for both active and pasive biomonitoring of environmental cadmium pollution.

Conclusions
The results of the present study support the use of hepatopancreas of mature snails, C. aspersus, as endpoints for assessing Cd accumulation and toxicity over a broad range of concentrations. Therefore, this gastropod species can be reliably used for both active and passive biomonitoring of environmental cadmium pollution.