Renato Bacchetta

DDT contamination in Lake Maggiore (N. Italy) and effects on zebra mussel spawning.

Zebra mussel (Dreissena polymorpha) is commonly used as contaminant bioindicator in Europe and North America. We used the zebra mussel to follow DDT pollution trends from 1996 to 1997 in Pallanza bay, Lake Maggiore, near the inlet of the River Toce, after a DDT-manufacturing plant discharging residues into a tributary of the River Toce had closed down. DDT contamination fell off sharply outside the bay, but tissue concentrations of the parent compound and residues remained high in molluscs sampled within the bay a year later. Molluscs collected in Pallanza bay in June 1997 released gametes earlier than those sampled at a nearby reference station. Histological studies showed that a significant percentage of these specimens showed marked oocyte degeneration suggesting that DDTs have endocrine-disrupting effects in this species.

Nano-sized CuO, TiO2 and ZnO affect Xenopus laevis development

Abstract The teratogenic potential of commercially available copper oxide (CuO), titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) was evaluated using the standardized FETAX test. After characterization of NP suspensions by TEM, DLS and AAS, histopathological screening and advanced confocal and energy-filtered electron microscopy techniques were used to characterize the induced lesions and to track NPs in tissues. Except for nCuO, which was found to be weakly embryolethal only at the highest concentration tested, the NPs did not cause mortality at concentrations up to 500 mg/L. However, they induced significant malformation rates, and the gut was observed to be the main target organ. CuO NPs exhibited the highest teratogenic potential, although no specific terata were observed. ZnO NPs caused the most severe lesions to the intestinal barrier, allowing NPs to reach the underlying tissues. TiO2 NPs showed mild embryotoxicity, and it is possible that this substance could be associated with hidden biological effects. Ions from dissolved nCuO contributed greatly to the observed embryotoxic effects, but those from nZnO did not, suggesting that their mechanisms of action may be different.

Axial-skeletal defects caused by Carbaryl in Xenopus laevis embryos.

Embryotoxic effects of Carbaryl (CB), a widely used carbamate insecticide, was evaluated by modified Frog Embryo Teratogenesis Assay-Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. X. laevis embryos were exposed to 1, 2, 4, 8, 16 and 24 mg/L CB from stage 8 to stage 47. From an estimated LC50 of 20.28 mg/L and TC50 of 8.43 mg/L a TI of 2.41 was derived, indicating that CB is to be considered teratogenic for X. laevis embryos. The most characteristic terata, classified as abnormal tail flexure, involved a significant percentage of larvae from 1 mg/L CB onward, reaching 100% at 24 mg/L CB. Histopathological screening revealed tail musculature and notochord as the main targets for CB. Skeletal muscle lesions consisted of myotomes reduced in size, showing myocytes with disorganized contractile systems and irregular myosepta, coupled with disarranged myocyte apexes. Notochords from CB exposed larvae appeared wavy or bent, with irregular connective sheaths and histologically characterized by protrusions of fibrous matrix and inclusions of ectopic cell masses. This axial-skeletal damage was hypothesized to be related both to the inhibition of acetylcholinesterase, with consequent muscular tetanic spasms, and to disorders in the organization of the connective tissue matrix surrounding the notochord.

DDT polluted meltwater affects reproduction in the mussel Dreissena polymorpha

The zebra mussel Dreissena polymorpha was used to follow the recently reported DDT pollution of Lake Iseo (N. Italy). Histopathological analyses were performed on mussels sampled from March 2005 to April 2006, when high DDT levels were found, and results were compared to those from mussels sampled in 2001/2002, before the pollution event. During the 2005/2006 reproductive season, the first male gamete release happened one month later than the onset of spawning in females who showed a high number of specimens with degenerating oocytes, despite a regular pattern of gametogenesis. These results indicated a disrupting action of DDT on the mechanisms involved in sperm release, and a disturbance in the gametogenic phases of the ovary. Pathological pictures in the digestive gland of many mussels from both 2001/2002 and 2005/2006 have also been observed, but DDT pollution is unable to explain the presence of pathological fields in mussels during 2001/2002, for which a previously reported contamination seems to be the main cause. A possible role of DDT in skewing the sex ratio towards a predominance of females was also discussed, considering the high number of females sampled in 2005/2006.

The agrochemical fungicide triadimefon induces abnormalities in Xenopus laevis embryos.

The dysmorphogenic activity of the triazole fungicide triadimefon (FON) has been evaluated using Xenopus laevis development. Embryos, exposed to 500 μM FON during early neurulation phases (stages 13-17, Nieuwkoop and Faber), were allowed to develop until stage 47. Larvae revealed serious craniofacial defects, bent forebrain, and abnormal hindbrain segmentation. CRABP and CYP26 (markers related to retinoic acid homeostasis) gene and protein expression and protein distribution have been evaluated at stage 17 and at the end of the branchial arch morphogenesis (stage 27) by real-time PCR, western blot and whole-mount immunostaining. A significant increase of CYP26 transcript has been observed at both embryonic stages. A co-localization of the two markers has been observed at the cephalic region. Embryos exposed to FON showed abnormal distribution of positive tissues. Due to the strict similarity of these results with those previously described in rodents, a FON-related alteration of mechanism conserved during vertebrate evolution is suggested.

Role of soluble zinc in ZnO nanoparticle cytotoxicity in Daphnia magna: A morphological approach

The role of soluble zinc has been determined in Daphnia magna by a morphological approach, integrating a previous paper in which the ultrastructural damages to gut epithelial cells have been studied after ZnO nanoparticles exposure. In the present paper, the toxicity and morphological effects of soluble zinc from ZnSO4 have been determined in a 48-h acute exposure test. Daphnids have been exposed to six nominal zinc concentrations (0.075, 0.15, 0.3, 0.6, 1.2, and 2.4mg Zn/L) and then fixed for microscopic analyses. Data from the acute toxicity tests gave an EC50 value of 0.99mg/L and showed that no immobilization appeared up to 0.3mg Zn/L. Ultrastructural analyses of samples from the two highest concentrations showed large vacuolar structures, swelling of mitochondria, multilamellar bodies, and a great number of autophagy vacuoles. These findings have been compared to those from our previous study, and similarities and/or differences discussed. Based on the overall results it can be concluded that dissolved zinc ions played a key role in ZnO nanoparticle toxicity and that the morphological approach is an extremely useful tool for comparing toxicological effects as well. A possible common toxic mechanism of soluble zinc and ZnO nanoparticles was also proposed.

Is the amphibian X. laevis WEC a good alternative method to rodent WEC teratogenicity assay? The example of the three triazole derivative fungicides Triadimefon, Tebuconazole, Cyproconazole

The aim of the present work is the assessment of teratogenic effects of three triazole-derived fungicides (Triadimefon, FON, Tebuconazole, TEBU, Cyproconazole, CYPRO) on rat and Xenopus laevis embryos cultured in vitro. Rat embryos, exposed to FON 31.25-250μM, CYPRO 31.25-62.5μM and to TEBU 62.5-250μM, showed specific malformations (fusions) at the level of the first and second branchial arches, with a concentration-dependent increase of severity of malformative pictures. After immunostaining, the ectomesenchyme has been identified as the target tissue. X. laevis larvae showed, at the same concentrations, specific malformations at the level of cartilaginous element derived from the first and second branchial arch ectomesenchyme. This work indicates the three tested triazoles as teratogenic both in rodents and in amphibian, inducing ectomesenchymal abnormalities, and suggests, at least for this class of molecules, the X. laevis method as adequate alternative model for teratogenic screening.

Histological studies on the zebra mussel Dreissena polymorpha reproduction from a DDT contaminated area in Lake Maggiore (N. Italy)

Histological techniques were used to monitor Dreissena polymorpha reproductive biology during 2001 in Lake Maggiore (N. Italy). Considering the DDT pollution which occurred during the mid 1990s in this lake and the high contamination level registered in 2001, we focused on the course of spawning events in zebra mussels and possible modified reproductive behavior induced by this pollution. Specimens were collected from the lake at Baveno as the polluted station, and at Villa Taranto as the control site. At Villa Taranto, morphological analyses of gonadal changes showed a gamete developing and maturing period until April, spawning phases from May to August, the restart of gametogenesis in females and an inactive period in males, immediately after the end of spawnings. This pattern was perturbed at Baveno where the mussels showed a delay in oocyte maturation with a significant percentage of specimens showing oocyte degeneration. Moreover, the onset of gamete release seemed to shift from May to July in the males at Baveno. Our findings agreed with the well recognized endocrine disrupting properties of DDT, even though the physiological mechanisms involved must be studied further.

Do Nanoparticle Physico-Chemical Properties and Developmental Exposure Window Influence Nano ZnO Embryotoxicity in Xenopus laevis?

The growing global production of zinc oxide nanoparticles (ZnONPs) suggests a realistic increase in the environmental exposure to such a nanomaterial, making the knowledge of its biological reactivity and its safe-by-design synthesis mandatory. In this study, the embryotoxicity of ZnONPs (1–100 mg/L) specifically synthesized for industrial purposes with different sizes, shapes (round, rod) and surface coatings (PEG, PVP) was tested using the frog embryo teratogenesis assay-Xenopus (FETAX) to identify potential target tissues and the most sensitive developmental stages. The ZnONPs did not cause embryolethality, but induced a high incidence of malformations, in particular misfolded gut and abdominal edema. Smaller, round NPs were more effective than the bigger, rod ones, and PEGylation determined a reduction in embryotoxicity. Ingestion appeared to be the most relevant exposure route. Only the embryos exposed from the stomodeum opening showed anatomical and histological lesions to the intestine, mainly referable to a swelling of paracellular spaces among enterocytes. In conclusion, ZnONPs differing in shape and surface coating displayed similar toxicity in X. laevis embryos and shared the same target organ. Nevertheless, we cannot exclude that the physico-chemical characteristics may influence the severity of such effects. Further research efforts are mandatory to ensure the synthesis of safer nano-ZnO-containing products.

Sexual maturity in young Dreissena polymorpha from Lake Como (N. Italy)

Summary The reproductive biology of the zebra mussel Dreissena polymorpha was studied during 1999 in a natural population living in Lake Como (N. Italy), with particular reference to the differences in behavior between young and adult mussels. Histological analysis of gonads was used to study the gametogenic cycles and to evaluate when the young reached sexual maturity. Signs of active gametogenesis were detected in ≥4 mm shell length mussels. Mature gametes and the first spawning events were observed in ≥5 mm males and in ≥6 mm females. The gonad feature of ≥7 mm mussels was similar to that of the adults. Size frequency distributions revealed that the new generation (0+) first spawned in the year following birth, but only after winter. The length of larval time seemed to vary greatly depending on the spawning period, in addition to the environmental conditions. The first settled mussels ≥500 μm in shell length were observed 3–4 months after the first depositions. Water temperature seemed to be the most important factor which regulated sexual phenomena of both young and adult mussels. Its role in the onset of the spawning events was confirmed, but it is important to point out that temperature is not enough to trigger reproduction since gamete release is impossible without sufficient energy, especially for young mussels.