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Glyphosate (Gly) is a broad-spectrum herbicide widely used thanks to its high efficiency and low toxicity. However, evidence exists of its toxic effects on non-target organisms. Among these, the animals inhabiting agricultural fields are particularly threatened. Recent studies demonstrated that exposure to Gly markedly affected the morphophysiology of the liver and testis of the Italian field lizard Podarcis siculus. The present study aimed to investigate the effects of the herbicide on the female reproductive system of this lizard in order to have a full picture of Gly-induced reproductive impairment. The animals were exposed to 0.05 and 0.5 μg/kg of pure Gly by gavage for 3 weeks. The results demonstrated that Gly, at both doses tested, profoundly interfered with ovarian function. It induced germ cells’ recruitment and altered follicular anatomy by anticipating apoptotic regression of the pyriform cells. It also induced thecal fibrosis and affected oocyte cytoplasm and zona pellucida organizations. At the functional levels, Gly stimulated the synthesis of estrogen receptors, suggesting a serious endocrine-disrupting effect. Overall, the follicular alterations, combined with those found at the level of the seminiferous tubules in males, suggest serious damage to the reproductive fitness of these non-target organisms, which over time could lead to a decline in survival.

The growing environmental spreading of food synthetic dyes and bio-colors have the potential for altering organisms’ redox states. Here, three model species for aquatic pollution trials, Cucumis sativus seeds, Artemia salina cysts, and Danio rerio embryos, were short-term exposed to a fixed concentration of the artificial red E124, and two red bio-colors, cochineal E120, and vegan red (VEGR). In the animal models, we evaluated the total reactive oxygen species (ROS) and the susceptibility to in vitro oxidative stress, and in C. sativus, H2O2 production and antioxidant capacity. We also measured organismal performance indices (routine oxygen consumption in the animal models, dark oxygen consumption, and photosynthetic efficiency in C. sativus). In C. sativus, only E124 increased ROS and affected dark oxygen consumption and photosynthetic efficiency, while all dyes enhanced the antioxidant defenses. In the A. salina nauplii, all dyes increased ROS, while E120 and E124 reduced the susceptibility to oxidative stress. In D. rerio, treatments did not affect ROS content, and reduced oxidative stress susceptibility. Our data show that red food dyes affect the redox state of the developing organisms, in which ROS plays a significant role. We suggest a potentially toxic role for red food dyes with environmentally relevant consequences.

Benzodiazepines, psychotropic drugs used for the treatment of insomnia and anxiety, are worldwide one of the most prescribed treatments [1]. The massive use results in the release of their active principles and metabolites in the wastewater, where they persist since not eliminated by sewage treatments [2]. Consequently, they accumulate in effluent waters [3] reaching concentrations ranging from µg/L to ng/L. For these reasons, BZPs are being considered emerging contaminants [4] and represent, even at low concentrations, a potential environmental hazard, especially for aquatic species [5]. Bioaccumulation is already reported in marine invertebrates and vertebrates as significant effects on behavior, gene expression and enzymes activity [6,7]. The fate of BZDs in the aquatic environment is still not fully clear and so are the effects on non-target species which may come accidentally into contact with these drugs. In this study, we investigated the influence of the benzodiazepine delorazepam on Xenopus laevis embryogenesis. Embryos were exposed to an environmental concentration (1 μg/L)[8] and, to mimic the simultaneous exposure to multiple BZDs occurring in nature, to 5 and 10 times higher (5 and 10 μg/L) concentrations. Results demonstrated that delorazepam reduces vitality, with decreased heart rate and motility, induces marked cephalic and abdominal edema, and causes alterations in the gut. At the molecular level, the increase of ROS production is observed together with an altered expression of developmental genes and the production of pro-inflammatory cytokines. A significant increase in ATP-binding cassette activity is also observed, as an attempt to improve drug clearance. The resulting stressful condition significantly impairs embryos development and threatens their survival. Similar effects should be expected also in embryos belonging to other aquatic species that have not been yet considered targets for benzodiazepines.

Benzodiazepines, psychotropics drugs used for treating sleep disorders, anxiety and epilepsy, represent a major class of emerging water pollutants. As occurs for other pharmaceutical residues, they are not efficiently degraded during sewage treatment and persist in effluent waters. Bioaccumulation is already reported in fish and small crustaceans, but the impact and consequences on other "non-target" aquatic species are still unclear and nowadays of great interest. In this study, we investigated the effects of a pharmaceutical preparation containing the benzodiazepine delorazepam on the embryogenesis of Xenopus laevis, amphibian model species, taxa at high risk of exposure to water contaminants. Environmental (1 μg/L) and two higher (5 and 10 μg/L) concentrations were tested on tadpoles up to stage 45/46. Results demonstrate that delorazepam interferes with embryo development and that the effects are prevalently dose-dependent. Delorazepam reduces vitality by decreasing heart rate and motility, induces marked cephalic and abdominal edema, as well as intestinal and retinal defects. At the molecular level, delorazepam increases ROS production, modifies the expression of some master developmental genes and pro-inflammatory cytokines. The resulting stress condition significantly affects embryos' development and threatens their survival. Similar effects should be expected as well in embryos belonging to other aquatic species that have not been yet considered targets for these pharmaceutical residues.

The abuse of artificial food dyes and the evidence that they harm human health recently prompted a significant effort to introduce vegan substitutes prepared from fruits and vegetables. Not much information, however, has been collected on their possible effects on aquatic and terrestrial ecosystems once released as waste in surface waters. For this purpose, we analyzed the effects of a vegan red (VEG) preparation (concentration 1.2 g/L) on three rapidly proliferating models for terrestrial and aquatic ecosystem contamination. In particular, in vitro cells cultures (exposure for 24 h), Artemia salina nauplii and Cucumis sativus seedlings (exposure 5 days). A comparison was made with the effects exerted by the two dyes that vegan red is intended to replace: an animal dye, cochineal E120 and an artificial dye E124. The analyses of conventional endpoints, indicative of cell proliferation, differentiation, and growth rate, demonstrate that the three dyes affect development and that the vegan substitute is as unsafe as the E124 and E120. Vegan red in fact impairs cell growth in in vitro cells, delays naupliar hatching and early growth in Artemia, and reduces shoot/root biomass in Cucumis. Marked hyperplasia and hypertrophy of mesophyll are also observed in Cucumis leaves. Substitution in food and beverages, therefore, should be carefully reconsidered to avoid unnecessary environmental contamination.

Spermatogenesis is a genetically driven differentiation process that occurs in the testis and leads to the formation of spermatozoa. This process is extensively studied in several experimental models, particularly in vertebrates that share the morphological structure and functionality of the mammalian testis. Although reptiles are not generally considered biological models, the lizard Podarcis siculus has represented a suitable organism for the study of spermatogenesis over the years. In this lizard, the process of spermatogenesis is regulated by the interaction between systemic factors such as gonadotropins and local factors, i.e., molecules produced by the somatic and germinal cells of the testis. Many exogenous substances are able to alter the production of these regulative factors, thus altering the course of spermatogenesis, and P. siculus has proven to be an excellent model for studying the effects of various endogenous or exogenous substances on mechanisms underlying spermatogenesis. This review summarizes the available data on the effects of different substances on the control of spermatogenesis, highlighting the induced morphological and molecular alterations. Overall, the data show that sex hormone levels as well as the final stages of spermatogenesis are most affected by an imbalance of endogenous compounds or contamination by environmental pollutants. This is helpful for the male individual, since the damage, not affecting the spermatogonial stem cells, can be considered transient and not irreversible.

In aquatic organisms, cadmium exposure occurs from ovum to death and the route of absorption is particularly wide, being represented by skin, gills and gastrointestinal tract, through which contaminated water and/or preys are ingested. It is known that cadmium interferes with the gut; however, less information is available on cadmium effects on an important component of the gut, namely goblet cells, specialized in mucus synthesis. In the present work, we studied the effects of two sublethal cadmium concentrations on the gut mucosa of Danio rerio. Particular attention was paid to changes in the distribution of glycan residues, and in metallothionein expression in intestinal cells. The results show that cadmium interferes with gut mucosa and goblet cells features. The effects are dose- and site-dependent, the anterior gut being more markedly affected than the midgut. Cadmium modifies the presence and/or distribution of glycans in the brush border and cytoplasm of enterocytes and in the goblet cells’ cytoplasm and alters the metallothionein expression and localization. The results suggest a significant interference of cadmium with mucosal efficiency, representing a health risk for the organism in direct contact with contamination and indirectly for the trophic chain.