Toxics, Vol. 10, Pages 695: Sensitivity of Hydra vulgaris to Nanosilver for Environmental Applications

3.2. EcotoxicityFor the description of morphological alteration and regeneration capacity, we used the evaluation proposed by Wilby [29], by assigning a score from 1 to 10 based on the observed morphology of the specimens. For the morphological assay, the evaluation was performed every 24 h up to 96 h of exposure, while for the regeneration assay, it was performed after 96 h and after 7 days of exposure.The exposure of H. vulgaris to AgNPcitLcys up to 1000 µg/L showed minimal consequences for both morphology and regeneration, with the exception of a slight contraction of column and tentacles at 100 and 1000 µg/L (Figure 1, upper row; Figure 2A). In contrast, a dose-dependent reduction in morphological parameters was observed upon AgNO3 exposure, with organisms reaching the tulip shape at the highest concentration tested (100 µg/L) (Figure 1, lower row; Figure 2B). The higher toxicity of AgNO3 compared to AgNPs agrees with previous observations, as the ionic form is more available for biological uptake in short-term exposure, while the effects of AgNP depend on particle dissolution and uptake and usually need longer exposure times to be observed. Compared to available studies on AgNPs ecotoxicity on Hydra (Kang and Park, 2021; Auclair and Gagné, 2022), AgNPcitLcys exhibited lower toxicity, causing only mild morphological alterations (100–1000 µg/L) and no mortality. In the regeneration assay, both AgNPcitLcys- and AgNO3-exposed organisms showed a high regeneration rate, reaching a score of 8 or more after 7 days of exposure, even at the highest concentration tested (Figure 2C,D).The low acute ecotoxicity of AgNPcitLcys was confirmed for the freshwater cnidarian H. vulgaris, as also previously observed for freshwater and marine water microalgae and microcrustaceans [9]. The low release of Ag ions in Hydra medium, as shown by ICP-MS data, surely played a significant role in the observed reduced ecotoxicity, also based on the morphological effects observed upon exposure to AgNO3, confirming the sensitivity of Hydra to Ag. However, Kang and Park [30] measured high levels of Ag inside Hydra specimens exposed to sulfide-coated AgNPs (Ag2S-NPs), even if the nominal dissolution of AgNPs in the medium was as low as non-detectable (LOD not reported). This was associated with toxicity, both at the morphological and regeneration levels (with EC50 of 0.65 and 0.09 mg/L, respectively). The authors hypothesized the ingestion of Ag2S-NPs followed by dissolution, triggered by the acidic environment of the gastrovascular cavity [30].Previous findings showed effects upon AgNPcitLcys exposure despite the low dissolution levels [9]. This led to hypothesizing the occurrence of a nano-size-related toxic effect for AgNPcitLcys, especially when considering freshwater species. This hypothesis was supported by the low aggregation of AgNPcitLcys in freshwater media, which could have eased intracellular uptake and consequent dissolution and toxicity. In the current work, as shown by DLS data, AgNPcitLcys do undergo a moderate aggregation when dispersed in Hydra medium (HDd of 676 ± 10 nm), hence making an intracellular uptake less likely to happen. This could explain the almost total absence of toxicity observed for H. vulgaris compared to previously tested freshwater species.Although Ag levels were not measured in the whole body of Hydra and the possibility of ingestion or intracellular uptake cannot be ruled out, the low ecotoxicity seems to confirm the protective role of the double coating of citrate and L-cysteine in preventing Ag ion release and thus ecotoxicity [8,9].Among regenerated Hydra from AgNPcitLcys exposure, some appeared to be bending their tentacles towards the mouth, as shown in Figure 3A, a behavior usually observed during feeding. As a consequence of this observation and since no major disruption occurred for regenerated Hydra, a feeding assay was performed in order to check whether the regeneration in a polluted environment could have impaired tentacle functionality. The evaluation of the predation ability (Figure 4) showed a reduction in the number of eaten larvae only for H. vulgaris exposed to AgNO3 at the highest concentration (100 µg/L). Meanwhile, the number of larvae found dead and considered to be the result of a non-successful predatory attempt was higher than in the control group, in all exposed specimens, even if not statistically significant. The exposure of Hydra to both heavy metals and NPs was shown to alter the structure of battery cell complexes (BCCs) [31,32,33] and epithelio-muscular cells that house the cnidocytes and associated neurons in the tentacles, which are responsible for its predating behavior. The impairment of BCCs can affect the feeding behavior of H. vulgaris, which normally consists of the following steps: catching and killing of the prey by cnidocytes-armed tentacles, tentacle bending towards the mouth, mouth opening, and ingestion of the prey [34]. The falling off of caught preys could be caused by a reduced entangling ability of the tentacles following an alteration of BCCS. For AgNPcitLcys exposure, this was particularly emphasized at the lowest tested concentration (10 µg/L) (Figure 4A). Similarly, the freshwater microcrustacean Ceriodaphnia dubia was shown to suffer more from exposure to a lower concentration of AgNPcitLcys (1 µg/L) compared to an intermediate one (10 µg/L) [9]. AgNPs at lower concentrations (µg/L range) are subjected to reduced aggregation but usually undergo enhanced dissolution compared to higher AgNP concentrations (mg/L–g/L range). Moreover, lower aggregation corresponds to an increased number of AgNPs in their nano-size range, potentially able to cross cell membranes and exert a nano-related toxicity, as previously hypothesized for AgNPcitLcys [9]. This interplay of factors is reflected in toxicity results, with a non-linear trend of responses, and could be responsible for the occurrence of stronger toxic effects at lower concentrations.Furthermore, recent findings [25,26,35,36] have demonstrated that peptides extracted from Hydra basal disks, which are involved in the organism’s defense against bacterial attacks, show a natural ability to counteract AgNP toxicity towards different organisms and cells. Further investigations are thus encouraged to better understand the mechanisms behind such an ability for future biotechnological applications.

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