Long-Term Exposure of the Red Cherry Shrimp Neocaridina davidi to Diclofenac: Impact on Survival, Growth, and Reproductive Potential.

The current study was aimed at studying the long-term effects of diclofenac on the freshwater shrimp Neocaridina davidi, concerning survival, somatic growth, and reproduction. In this study, both ovigerous females and males of this species were exposed for 63 d to 0 (control), 0.1, or 1 mg/L of diclofenac. At the highest concentration, significant mortality was detected, and the somatic growth of females was significantly decreased. The percentage of females with a second spawn, observable from day 45, significantly increased at 1 mg/L, while the time between spawns was significantly reduced at both concentrations assayed. However, the gonadal analysis made at the end of the assay in the surviving females showed a significantly lower proportion of advanced oocytes in females exposed to 1 mg/L, as compared to control. Concerning hatching, the percentage of ovigerous females that could have successful hatching was reduced at 1 mg/L of diclofenac, especially for the first spawn. For the second spawn, the low number of juveniles hatched from females exposed to 1 mg/L also showed a significantly higher incidence of morphological abnormalities, such as hydropsy and underdeveloped appendages. Taken together, these results showed that even when diclofenac was able to produce earlier spawns, the reproductive output of each spawn was reduced.

Imbalances in the male reproductive function of the estuarine crab Neohelice granulata, caused by glyphosate.

The effect of glyphosate, both pure and formulated (Roundup Ultramax®), was evaluated on males of the estuarine crab Neohelice granulata, by means of both in vivo and in vitro assays. The in vivo assays comprised the exposure for 30 d to 1 mg/L of the herbicide, until finally assessing weight gain, levels of energy reserves, sperm number per spermatophore, proportion of abnormal spermatophores, and sperm viability. At the end of this assay, significant (p < 0.05) decrease in weight gain and muscle protein levels was detected by effect of both pure and formulated glyphosate. In spermatophores from the vas deferens, a significant (p < 0.05) decrease of the sperm count was observed by effect of Roundup, while a significant incidence (p < 0.05) of abnormal spermatophores was observed either with glyphosate or with Roundup treatment. No changes were seen in the spermatophore area or in vas deferens secretions. Since no sperm mortality was induced by the formulated herbicide, we propose a probable inhibiting effect on spermatogenesis might explain the observed sperm count decrease. In this sense, an in vitro assay was designed by incubating testes and vasa deferentia with Roundup, in order to corroborate the possible interference of glyphosate with the secretion of the androgenic gland hormone that controls the spermatogenesis, in the presence or absence of the androgenic gland. Although the herbicide per se was able to reduce the sperm count to some extent, the increase in the number of spermatozoa/spermatophore produced by the co-incubation with the androgenic gland was completely reverted by the addition of Roundup (1 mg/L of glyphosate a.e.), suggesting that an inhibition on the secretion and/or transduction of the androgenic gland hormone could be taking place.

Effects of glyphosate on sperm quality of the estuarine crab Neohelice granulata, under the "One Health" perspective.

This study was aimed at evaluating the effects of the commercial formulation Roundup Ultramax® on sperm mortality and viability, in terms of the capacity of spermatozoids (spz) to trigger the acrosome reaction (AR), using the estuarine crab Neohelice granulata as a model. To this, an in vivo assay comprising 100 days duration was carried out, on a control group and two groups exposed to the formulation (0.01 mg/L and 0.2 mg/L of glyphosate) under controlled conditions of photoperiod, feeding, and temperature. At the end of the assay, the right vas deferens (VD, proximal and middle portion) was dissected, and after homogenizing it in calcium-free saline solution, the acrosome reaction was induced in the phase containing the spz. In each treatment, the percentage of spz with total and partial AR was calculated, as well as that of dead spz. Compared to the control, crabs exposed to the herbicide showed a significant decrease in spz with full AR, together with an increase in the percentage of spz with partial AR. Furthermore, spz mortality was significantly higher in both glyphosate concentrations compared to the control, in a concentration-dependent manner. On the other hand, abnormal spermatophores, showing expanded walls and coalescence, were observed in a significant percentage in the left VD of the groups treated with the herbicide. The results obtained are compared with those from other studies on several invertebrate and vertebrate species that found inhibition of the AR and abnormal sperm, together with inhibition of spermatogenesis, endocrine disruption, and reduced sperm motility by effect of pure glyphosate and/or different glyphosate formulations. In summary, the available evidence highlights the possible impact of glyphosate on sperm quality, in a wide variety of species.

Interference of an atrazine commercial formulation with the endocrine control of ovarian growth exerted by the eyestalks.

Atrazine is currently one of the most used herbicides worldwide. We tested the possible effect of the widely used herbicide atrazine on the endocrine control of ovarian growth exerted by the neurohormones secreted at the eyestalk of the estuarine crab Neohelice granulata. For this, both in vivo and in vitro assays were carried out. The in vivo assay comprised the exposure for 1 month to 3 mg/L of a commercial formulation containing 90% of atrazine as active ingredient (Gesaprim 90 WDG®, Syngenta) on three categories of females: intact, ablated of one eyestalk, and ablated of both eyestalks. At the end of the assay, only the intact females showed a significant (p < 0.05) decrease in both content of vitellogenic ovarian proteins and proportion of vitellogenic oocytes, compared to a concurrent control. The results of the in vitro incubation of ovarian pieces with the eventual addition to the incubation medium of eyestalk tissue and/or atrazine at 3 mg/L showed a significant (p < 0.05) decrease in the proportion of vitellogenic oocytes only when atrazine and eyestalk tissue were added. Taken together, these results strongly suggest that the assayed atrazine formulation may act as an endocrine disruptor at the eyestalk level, by altering the normal secretion of some eyestalk hormone, therefore inhibiting ovarian growth.