Over the past decades, intensive industrialization and agriculture have increased considerably the release of various toxic compounds into air, soil and water, causing many environmental problems (Khan and Ghouri 2011). Among the most common contaminants, trace metals are of serious concern, since, unlike organic toxins, they are non-biodegradable and accumulate in the environmental matrixes (Ali et al. 2013). Water, the key vital resource for natural ecosystems and human life, is subjected to a continuous anthropogenic input of these elements, that can thus freely reach any kind of biota (Schwarzenbach et al. 2006). Therefore, the development of scientifically-sound and cost-efficient tools for the contaminant monitoring is a priority for the overall protection of all living organisms, along with the pressing necessity to restore the polluted aquatic ecosystems and to clean the contaminated wastewaters.
Among the monitoring tools, effect-based methods, such as bioassays and biomarkers, are widely employed because their unique ability to fill the gap between chemical pollution and ecological status, covering a broad range of exposure times and toxicity mechanisms in diverse biological systems (Brack et al. 2017). Test organisms include invertebrates, fishes, microorganisms, plants and algae, even though some of them can be difficult to handle and their use may be ethically objectionable. Other systems, such as mammalian cells, are expensive and results are not always consistent (Hassan et al. 2016). Nonetheless, the use of aquatic plants as biological models in eco-toxicological tests is limited if compared to animals, although contaminants mainly enter the ecosystem through such organisms, that are the first and obligate step of the trophic chains (Ceschin et al. 2020).
Over the last years, increasing attention has been paid to bioassays for the assessment of trace metal genotoxic effects, since the interaction with nucleic acids is considered one of the primary causes of the toxicity of such elements (Kleinjans and van Schooten 2002; Zhu and Costa 2020). Actually, only if the conduction of genotoxicity assays is added to the analysis of conventional water quality parameters, the presence of mutagens in water is considered to be reliably assessed (Ohe et al. 2004). As for the test organisms, plants are regarded as ideal assay systems for screening and monitoring mutagens in the environment, providing vital information from the viewpoint of preserving biodiversity and ecological resources (Panda and Panda 2002; Aksoy 2017). Plant organisms are affected by water pollution earlier than other organisms, since they are the first interface between abiotic and biotic constituents of an ecosystem and, therefore, considered as early warning systems, essential for intercepting contaminations in advance (Ceschin et al. 2020). The development of molecular biology has led to several PCR-based techniques used to evaluate DNA damage in toxicological studies with plants as model systems. Those techniques include analysis of microsatellite markers (Monteiro et al. 2009), random amplified polymorphic DNA assay (Liu et al. 2009; Surgun-Acar et al. 2018) and analysis of amplified fragment length polymorphism (AFLP). In plant research, AFLP is already a powerful tool with broad applications in population genetics, linkage mapping, phylogeny, and biogeography (Meudt and Clarke 2007). More recently, AFLP has been used to screen plant genomic DNA for evidence of mutational events induced by environmental contaminants (Labra et al. 2003; Xue-mei et al. 2006; Aina et al. 2007; Coppi et al. 2018). In addition, AFLP can offer the possibility of detecting large portions of the genome at the populational level at relatively low cost (Caballero et al. 2013).
Among the most dangerous mutagens, cadmium (Cd) is one of the trace elements arising more concern for the environment and human health (ATSDR 2005), thus needing to be extensively studied and monitored for its public health effects (ATSDR 2015; USEPA 2015). The genotoxicity of Cd, classified as human carcinogen (IARC 2016), is supposed to derive from its direct binding to DNA, possibly at adenine, guanine and thymine (Hossain and Hug 2002), or direct inhibition of DNA mismatch repair (Jin et al. 2003). The Cd genotoxic effect may also be indirect, through generation of reactive oxygen species, which may then damage nucleic acids (Apel and Hirt 2004; Valverde and Rojas 2001). Several studies have demonstrated Cd-induced micronuclei formation, chromosomal aberrations or DNA base damage (Beyersmann and Hartwig 2008). Therefore, the assessment of genotoxicity of metals like Cd is an important topic in environmental research, with the increasing need of devising ecotoxicological tests sensitive to both concentration and exposure time to capture and address any possible effect of its presence. To this aim, we performed a toxicity test to assess if the species Myriophyllum aquaticum (Vell.) Verdc. can be a promising model system to propose to the eco-toxicologist scientific community in the view of devising reliable bioassays for Cd genotoxicity in waters. Myriophyllum aquaticum is a macrophyte native from Tropical and Subtropical America. We chose this species because of its unique advantages of yearly availability, large occurrence, easy to handle and to grow without the need of sterile conditions or expensive materials. Furthermore, an aquatic species can represent per se a more suitable model system for the evaluation of the genotoxic potentiality of contaminated waters, whereas generally such kinds of bioassays are unconcernedly performed on land plants. In addition, this plant can be easily propagated in a vegetative way, thus giving a living material with low level of genetic differentiation, that should be more reliable in revealing variation in its genetic structure when exposed to Cd. Considering M. aquaticum as a promising model system, we assessed the changes caused by toxic Cd concentration on its AFLP fingerprinting profiles, at different doses and times of exposure. We moreover tested if one of the response traits of plants to the exposure of toxic concentration of Cd (e.g. plant growth), is time-coupled to evident genotoxic effect. Therefore, our results could provide fundamental information not only to devise more reliable eco-toxicological tests but also on the still poorly known Cd genotoxic effects on plants.