Improving Plant-Based Genotoxicity Bioassay for Trace Metal-Contaminated Water: Insights from Myriophyllum Aquaticum (Vell.) Verdc. and Cd.

In this work, we evaluated whether the species Myriophyllum aquaticum (Vell.) Verdc. can be a promising material for devising reliable ecotoxicological tests for Cd contaminated waters. Plants of M. aquaticum were exposed to Cd, using different concentrations and exposure times, in order to address as many possible effects as possible of its presence. Plant growth and Cd accumulation were monitored along the treatment period and Cd genotoxicity was assessed by analyzing Cd-induced changes in the AFLP ngerprinting proles. Root and shoot growth was reduced already at the lowest Cd concentration used (1 mg L -1 ). Shoots showed a higher Cd sensitivity and a lower accumulation, thus being chosen as the more suitable organ for the genotoxic analysis. DNA variation was observed starting from 2.5 mg L -1 , indicating that the metal-induced depression of plant growth at the lower concentration did not necessarily imply a genotoxic effect. Similar results were obtained in the time-dependent experiment, since Cd effect on DNA ngerprinting prole was observed after three days of exposure and without a signicant growth decrease growth. Therefore, our results showed that M. aquaticum proved to be a suitable model system for the investigation of Cd genotoxicity through AFLP ngerprinting prole, whereas the more classic eco-toxicological tests based only on biometric parameters could underestimate the risk associated to undetected Cd genotoxicity.


Introduction
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 scienti cally-sound and cost-e cient 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 ll 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, shes, microorganisms, plants and algae, even though some of them can be di cult 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 rst 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 rst 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 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, classi ed 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 scienti c 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 ngerprinting pro les, 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.  The pots were maintained in the growth chamber for all the duration of the treatment, removing plants from the test solution after 3, 7, 14 and 21 days of exposure.

Growth measurement and determination of Cd concentration
Root and shoot length and fresh weight of each plant were measured prior and after Cd treatment to evaluate plant response to Cd concentration and time exposure in terms of increment in plant growth. Subsequently, each sample was incubated in ice-cold (4°C) Pb(NO 3

DNA isolation and AFLP protocol
A portion of shoot material (apex) was sampled from each plant, after growth measurements, from both experiment 1 and 2. 30 mg of each dried plant portion were ground in a mortar with sterile sand. The DNA was extracted by using the 2xCTAB protocol (Doyle and Doyle 1990). The quality and quantity of the extracted DNA was checked by a spectrometric survey that used a Bio-Photometer (Eppendorf). The AFLP analysis followed standard procedure with minor modi cations (Coppi et al. 2018 and references therein). In order to screen the primers combination that produces the most informative, readable, and repeatable pro les, three primer pair combinations (Table 1) were tested on at least of three individuals from each sampling group. Table 1 List of primer tested for the AFLP analysis. Fluorescent labelling and sequence with selective extension (in brackets) were also added.

Plant growth and Cd accumulation
Data on plant growth and Cd accumulation in roots and shoots of M. aquaticum after Cd exposure are reported in Fig. 1A, B. The presence of the metal induced a signi cant reduction in both root and shoot length increment starting from the lowest concentration used. A signi cant tting of root and shoot growth data to a logistic dose-response equation was obtained (P < 0.001, R = 0.96 for roots, P < 0.001, R = 0.91 for shoots). For a quantitative estimation of Cd effect on plant growth, the parameter EC 50ext was calculated on external Cd concentration. The obtained values were 2.5 ± 0.5 mg L − 1 for roots and 2.0 ± 0.5 mg L − 1 for shoots, and were not signi cantly different between them.
Cadmium concentrations in both roots and shoots of M. aquaticum increased with increasing metal exposure (Fig. 1B), reaching values of about 1600 and 1200 µg g − 1 d.w., respectively, at the highest level of exposure used. Cadmium accumulation in the roots was always signi cantly higher than in the shoots (at least p < 0.05).
The parameter EC 50 was calculated also on the basis of internal Cd concentration (EC 50int ), using root and shoot accumulation data. As for external Cd concentration, the data tting gave signi cant results for a logistic dose-response relationship (P < 0.001, R = 0.97 for roots, P < 0.0001, R = 0.91 for shoots) and the value of EC 50int was signi cantly higher for roots (974 ± 34 µg g − 1 d.w.) than for shoots (785 ± 71 µg g − 1 d.w.).
In the experiment 2, plant growth and Cd accumulation at a xed Cd concentration (2.5 mg L − 1 ) was monitored over different exposure times, until 21 days ( Fig. 2A, B). The presence of Cd in the solution started to produce a signi cant reduction in root and shoot growth after seven days of exposure ( Fig. 2A).
Regarding Cd accumulation, both roots and shoots showed an increase in metal concentration with time, following a saturating trend. Starting from seven days of exposure, Cd concentrations were always signi cantly higher in roots than in shoots (at least p < 0.01), reaching values about 2.5-fold higher after 14 and 21 days.
3.2Analysis of AFLP pro les AFLP analysis was successfully performed on a total of 124 samples (52 from the experiment 1 and 72 from the experiment 2), all of which produced a peculiar ngerprint pro le. As for the experiment 1, the primer combinations produced a total of 154 polymorphic fragments, 84 from the famEcoRI (TAC) /MseI (ATG) and 70 from the hexEcoRI (ACG) /MseI (ATG) . A total of 191 fragments were obtained for the experiment 2, 88 from the famEcoRI (TAC) /MseI (ATG) and 103 from the hexEcoRI (ACG) /MseI (ATG) . The overall level of genetic diversity within sampling groups was higher for the controls than the treatments (0.257 and 0.190 respectively). Concerning the experiment 1, the levels of genetic diversity within each sampling group varied from 0.256 to 0.161, for TREAT1 and TREAT10 respectively ( Table 2). The genomic template stability analysis showed a consistent reduction of the percentage of polymorphic loci for the group of treated samples excluding TREAT1 (Fig. 3A, p < 0.001). The number of SpL were higher for the group formed by controls and TREAT1 compared to the TREAT2.5, TREAT5 and TREAT10 (24 and 8 respectively). The genetic diversity within each sampling group varied from 0.326 for CONT21G and 0.153 for T03G in the experiment 2 ( Table 2). The analysis of genomic template stability con rmed the results of experiment 1, showing a reduction of the percentage of polymorphic loci for the treated group (Fig. 3B, p < 0.001). The number of SpL were 17 for the control group, whereas no speci c loci were showed for the treated ones.
As for the AMOVA, the greatest percentage of the total genetic variation was due to among sampling group differences (52.38%, p < 0.001). Among all hypothetical cluster of sampling groups, the one formed by control and the treated with 1 mg L − 1 of Cd (CONT-TREAT1), and the group of plant exposed to 2.5, 5 and 10 mg L − 1 (TREAT2.5-TREAT5-TREAT10), accounted for the highest percentage of among-groups variation 63.38 %; P < 0.0001; Table 3). The higher level of total genetic variation in the experiment 2 was for within sampling group (74.8%, p < 0.022). Among the hypothetical grouping of sampling groups, control plants vs treated plants accounted for the high percentage of variation among groups (see Table 3).

Discussion
Our results provided useful and novel information for the direct quanti cation of the genotoxic effect of Cd-polluted waters by using AFLP ngerprinting on M. aquaticum. accumulation, thus suggesting different sensibility to the internal metal concentrations. Since Cd accumulation was higher in roots than in shoots, this latter organ proved to be more susceptible to Cd toxicity (as quanti ed by its signi cantly lower EC 50int in respect to the one of the roots). This outcome cannot be ignored in optimizing the tests, therefore shoots were chosen for the genotoxic analysis, to get more reliable results on the effect of Cd. For the devising of eco-toxicological tests, previous screenings need to be undertaken to choose the most suitable organ. Generally, roots of land plants are used for practical reasons (Aksoy, 2017), thus raising doubts not only about their validity as the most sensitive part but also on the reliability of the exposure methods. Obviously, plants exposed to Cd-contaminated waters are mostly the macrophytes, with the whole body in contact with the pollutant. This feature cannot be neglected and makes the effects of the metal on such organisms hardly comparable with those on land plants.
Comparing the biometrical data to the molecular information, the rst treatment concentration at which both the effect on growth and on DNA variation were contemporarily signi cant was as high as 2. Given the above-mentioned double result of the concentration 2.5, the effect of this same Cd amount on growth, accumulation and AFLP pro les was evaluated in a time-dependent experiment.
Concerning plant elongation and metal concentration in roots and shoots, experiment 2 con rmed the same results observed in the concentration-dependant one. The two organs showed a similar Cd-induced decrease in growth (around 70% reduction of length increment at the end of the experiment for both the organs), nonetheless shoots displayed a lower metal accumulation than roots and, accordingly, a higher sensitivity to the presence of the metal inside its tissues. Therefore, shoots con rmed to represent a better candidate than roots for eco-toxicological tests. In both organs, a signi cant Cd-induced decrease in growth was present after seven days. However, plant Cd accumulation occurred already after three days, but apparently at harmless concentration for growth. Comparing to the molecular data, again an out-ofsync scenario appeared, this time with an unexpected vice-versa. Actually, Cd treatment was able to induce variation in the DNA ngerprinting pro les, evidencing a genotoxic effect, already after three days and without a signi cant growth depression. Therefore, for shorter times and higher doses in respect to the critical threshold of experiment 1, the harmful effect of the metal on the plant genome was already present, thus corroborating the unreliability of eco-toxicological tests based only on plant growth already supposed by the results of experiment 1. Particularly, the AMOVA analysis showed that the large portion of the molecular variance is signi cantly explained by keeping the control samples separated from the treated ones. Also, the identi cation of 17 loci speci c for the control plants con rmed that the genotoxic effect of 2.5 mg L − 1 Cd treatment may become evident from the rst days of the experiment. As in previous studies, DNA-ngerprinting pro les generated from plants exposed to ascending concentrations of phytotoxic inorganic substances revealed appearing and disappearing bands in comparison to control samples (Liu et al. 2009). Moreover, in our experiment, AFLP pro les variation induced by time-dependent Cd exposure was re ected by changes in DNA pro les and by reduction of the genome template stability after only three days, indicating a high-sensitivity of the molecular approach in the identi cation of genotoxic effects of medium-low concentration of Cd in water. Our results corroborate other earlier studies (Labra et al. 2003) reporting the high sensitivity of molecular approach than classic genotoxic trials, since they are capable of detecting DNA changes that may not manifest themselves as biometrical mutations.

Conclusions
Myriophyllum aquaticum revealed to be a suitable model system for the investigation of Cd genotoxicity through AFLP ngerprinting pro le. Our joint experiments in concentration and in time allowed to identify two of the combinations of dose (2.5 mg L − 1 ) and exposure (3 days) for Cd-polluted waters beyond which concern for environmental genotoxic danger could arise. Moreover, our results showed that a metal-induced reduction of plant growth does not necessarily imply a DNA damage and, more worryingly, vice versa. Consequently, there is the urgent need to implement those more classic eco-toxicological tests based only on plant growth. Such tests, despite being easy to use and cost-effective, can underestimate the risk associated to not detected Cd-induced mutations in the genome of living organisms, that can have concerning implications in the long term.

Declarations
Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.  Values are mean ± standard error. The signi cant effect of Cd treatment in respect to the respective control is indicated by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001) Figure 3