Luigi Viganò

First observation of intersex cyprinids in the Po River (Italy)

Barbel (Barbus plebejus, Cyprinidae) were captured in the Po River, upstream and downstream from the confluence of the Lambro River, a polluted tributary of the major Italian watercourse. The gonads of the two groups of barbel have been histologically examined, and only the downstream specimens showed histo-morphological alterations that can be related to the Lambro tributary as a source to the main river of endocrine disrupting chemicals, possibly with estrogenic effects. In fact, 50% of the barbel captured (8 of 16 fish) in the downstream reach showed intersex gonads.

Decabromodiphenyl ether (BDE-209) enters the food web of the River Po and is metabolically debrominated in resident cyprinid fishes.

Decabromodiphenyl ether (BDE-209), the primary constituent of a widely used flame retardant formulation, is present at relatively high levels in sediments and macroinvertebrates of the River Po. Since it was demonstrated that BDE-209 can be biotransformed to smaller and more toxic polybrominated dipheyl ethers (PBDEs), the main objective of this study was to assess whether the large quantities of BDE-209 present in the River Po are bioavailable to the higher levels of the food web and are biotransformed in feral fishes. To this aim, 23 cyprinids, mainly common carp, were analysed for the hepatic contents of PBDEs. Contrary to sediments and invertebrates of the same area, no fish sample contained detectable levels of BDE-209. All fishes contained typical PBDE representatives, e.g. BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154, but more importantly they contained three congeners, i.e. BDE-179, BDE-188 and BDE-202, which are not present in any technical formulations and are known products of BDE-209 debromination in fish. The age of carps had no effects on the bioaccumulation of PBDEs. Conversely, the contents of PCBs, which also were determined in the same fish samples, showed a positive correlation with age. Both groups of chemicals displayed a tendency to a higher contamination in male fish. This study shows that BDE-209 enters the food web of the River Po contributing to the load of lower brominated PBDEs and thus to the load of chemical stressors threatening the aquatic life of the major Italian watercourse.

Tracing endocrine disrupting chemicals in a coastal lagoon (Sacca di Goro, Italy): sediment contamination and bioaccumulation in Manila clams.

The Water Framework Directive, recently amended with new priority substances (2013/39/EU), is meant to regulate the health status of European aquatic environments, including transitional waters. Despite the ecological and economic importance of transitional water bodies and, in particular, of coastal lagoons, a relevant example of this type of environments, little is known about their contamination by priority substances, particularly by endocrine disrupting chemicals (EDCs). In this study, a wide array of priority substances, all with recognised disrupting properties, was investigated in the Sacca di Goro Lagoon (Adriatic Sea, Italy), which receives freshwater from the Po River after draining the most urbanised and industrialised Italian regions. Flame retardants, alkylphenols, bisphenol A, natural and synthetic steroids, personal care products and legacy pollutants were investigated both in sediments and in the clam Ruditapes philippinarum collected from three sites in the lagoon. Sediments showed that most of the chemicals analysed could reach the lagoon ecosystem but their concentrations were below existing quality guidelines. Clams essentially reflected this condition although some concern was raised by polybrominated diphenyl ethers (PBDEs): the limit for the sum of six congeners set for biota in the European Directive (2013/39/EU) to protect human health was exceeded 4-5 times. No significant biota-sediment accumulation factors (BSAFs) were calculated. Nonylphenol, tonalide, PBDE, polychlorinated biphenyls and bisphenol A were the most abundant chemicals in clam tissues.

Evaluation of microsomal and cytosolic biomarkers in a seven-day larval trout sediment toxicity test

Rainbow trout (Oncorhynchus mykiss) sac fry (larvae) were exposed to River Po sediments for 7 days. The sediments were collected in the River Po at two sites located upstream and downstream of the confluence of a polluted tributary, the River Lambro. An additional sediment treatment was also tested, in which trout larvae were kept from direct contact with the downstream sediment by interposing a Teflon net. Benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD), aminopyrine-N-demethylase (APDM) and UDP glucuronyl transferase (UDPGT) activities were found to be significantly induced in whole-body microsomal preparations of sac fry exposed to the downstream sediment. No significant modification was evident in any of the tested cytosolic biomarkers, i.e. glutathione reductase (GR) and glutathione peroxidase (GPx), glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD) and the content of nonprotein thiols (SH). With the exception of a slight induction of AHH enzyme activity, no difference could be found between fry exposed to control sediment and those screened from the downstream sediment, suggesting that direct contact with sediment was the major route of exposure to contaminants. This study demonstrates that several enzyme activities, which are known to occur in juvenile and adult rainbow trout, are also detectable at the sac-fry (larval) stage, and some of these activities can be induced by a short-term exposure to a contaminated sediment.

Time course of xenobiotic biotransformation enzyme activities of rainbow trout caged in the River Po

Juvenile rainbow trout were exposed to River Po water in cages suspended upstream and downstream from the entry of the River Lambro, a minor and heavily polluted tributary. A third group of trout was held at the laboratory and served as a control. Fish were sampled after 7, 15 and 30 days of exposure. Benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD), aminopyrine-N-demethylase (APDM), UDP glucuronyl transferase (UDPGT), glutathione transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) activities were measured along with the contents of protein and non-protein thiols (SHP and SHNP) in trout liver. With the exception of GST activity and SHP content which remained unchanged in both the exposure groups, all the biomarkers showed significantly altered values. Fish exposed at the upstream site showed minor modifications and with the apparent contribution of a doubled flow of the River Po. On the contrary, the downstream-stretch of water induced earlier and higher responses of the biomarkers, confirming the higher degree of contamination in this area, where polycyclic aromatic hydrocarbons (PAH), or related compounds were suspected to act as inducers. Also for this group of organisms, an apparent relationship was found between the temporarily doubled river flow and the level of those biomarkers which can react more rapidly.

Exposing native cyprinid (Barbus plebejus) juveniles to river sediments leads to gonadal alterations, genotoxic effects and thyroid disruption

Juveniles (50 days post hatch) of a native cyprinid fish (Barbus plebejus) were exposed for 7 months to sediments from the River Lambro, a polluted tributary impairing the quality of the River Po for tens of kilometers from their confluence. Sediments were collected upstream of the city of Milan and downstream at the closure of the drainage basin of the River Lambro. Chemical analyses revealed the presence of a complex mixture of bioavailable endocrine-active chemicals, with higher exposure levels in the downstream section of the tributary. Mainly characterized by brominated flame retardants, alkylphenols, polychlorinated biphenyls, and minor co-occurring personal care products and natural hormones, the sediment contamination induced reproductive disorders, as well as other forms of endocrine disruption and toxicity. In particular, exposed male barbel exhibited higher biliary PAH-like metabolites, overexpression of the cyp1a gene, vitellogenin production in all specimens, the presence of oocytes (up to 22% intersex), degenerative alterations in their testis, liver fat vacuolization, a marked depression of total thyroxine (T4) and triiodothyronine (T3) plasma levels, and genotoxic damages determined as hepatic DNA adducts. These results clearly demonstrate that Lambro sediments alone are responsible for recognizable changes in the structure and function of the reproductive and, in general, the endocrine system of a native fish species. In the real environment, exposure to waterborne and food-web sources of chemicals are responsible for additional toxic loads, and the present findings thus provide evidence for a causal role of this tributary in the severe decline observed in barbel in recent decades and raise concern that the fish community of the River Po is exposed to endocrine-mediated health effects along tens of kilometres of its course.

Updating and validation of a daphnia toxicity model for benzene derivatives

Abstract A QSAR model for predicting the acute toxicity to Daphnia of mono-substituted benzenes (MBs) has been updated and validated experimentally. The model had been previously constructed by using a training set of eight MBs, which were selected by a statistical design out of a series of 100 and subsequently tested on their toxicity. For updating the model, a recently developed data analytic method based on PLS (Partial Least Squares) has been applied. For its validation, a total number of thirteen MBs have been tested under the same conditions as used for the training set. The selection of the validation compounds was carried out with a view to identifying potential structural and mechanistic outliers. The study allowed us to clarify the range of the model and to assess the accuracy of the predictions for non-tested MBs. The agreement between actual and predicted toxicities was good for the validation compounds ( Q 2 = 0.89); it was also acceptable for other MBs whose toxicities were reported in the literature.

Hepatic gene expression profiles of a non-model cyprinid (Barbus plebejus) chronically exposed to river sediments

In this study, we characterized the gene expression responses of the Padanian barbel (Barbus plebejus), a native benthivorous cyprinid with a very compromised presence within the fish community of the River Po. Barbel juveniles were exposed in the laboratory to two river sediments reflecting an upstream/downstream gradient of increasing contamination and collected from one of the most anthropized tributaries of the River Po. After 7months of exposure, hepatic transcriptional changes that were diagnostic of sediment exposure were assessed. We investigated a set of 24 genes involved in xenobiotic biotransformation (cyp1a, gstα, ugt), antioxidant defense (gpx, sod, cat, hsp70), trace metal exposure (mt-I, mt-II), DNA repair (xpa, xpc), apoptosis (bax, casp3), growth (igf2), and steroid (erα, erβ1, erβ2, ar, vtg) and thyroid (dio1, dio2, trα, trβ, nis) hormone signaling pathways. In a consistent overall picture, the results showed that long-term sediment exposure mainly increased the levels of mRNAs encoding proteins involved in xenobiotic metabolism, oxidative stress defense, repair of DNA damage and activation of the apoptotic process. Transcript up-regulation of three receptor genes (erβ2, ar, trβ), likely representing compensatory responses to antagonistic/toxic effects, was also observed, confirming the exposure to disruptors of the reproductive and thyroidal axes. In contrast to expectations, a few genes showed no response (e.g., casp3) or even downregulation (vtg), further suggesting that the timing of exposure/assessment, potential compensatory effects or post-transcriptional modifications interact to modify the gene expression profiles, particularly during exposure to mixtures of contaminants.

Phosphorus content in a deep river sediment core as a tracer of long-term (1962–2011) anthropogenic impacts: A lesson from the Milan metropolitan area

Reconstructions of past fluvial contamination through the analysis of deep sediment cores are rarely reported in literature. We examined the phosphorus fractions in a deep (2.6 m) sediment core of the Lambro River downstream of the highly anthropized Milan metropolitan area and upstream of the Po river the main Italian watercourse. The core covered the period 1962-2011. Total phosphorus concentrations resulted typical of a strongly impacted environment (4788 mg P kg DW-1 on average) with the highest concentrations related to the 1960s (7639 mg P kg DW-1) reflecting the period of maximum demographic growth. Afterwards, phosphorus concentrations decreased thanks to the infrastructural and legislative initiatives carried out in the 1980s and the 1990s to reduce the impact of urban point sources. Subsequently, total phosphorus concentrations stabilized on values around 3000 mg P kg DW-1 and did not diminish further, even after the second phase of infrastructural interventions carried out in the second half of the 2000s. This was related to the increasing relative impact of the combined sewer overflows in the sewage system and to the strong phosphorus enrichment of the basin. Most of the phosphorus was in inorganic forms (86% of the total) that have been identified as the final target of the domestic effluent inputs. The contribution of organic phosphorus was lower but constant over the period 1962-2011. It likely originated from the agricultural areas located south of the city of Milan. In conclusion, this study underlines how past interventions have been effective in reducing urban point sources but it also highlights the current difficulties related to the growing importance of other sources influenced by the surface runoff (i.e., combined sewer overflows and agriculture). The study also emphasizes a general phosphorus enrichment of the Lambro River basin and its impact on the Po River and the Adriatic Sea.

Gene expression and genotoxicity in Manila clam (Ruditapes philippinarum) modulated by sediment contamination and lagoon dynamics in the Po river delta

The lagoons of the Po River delta are potentially exposed to complex mixtures of contaminants, nevertheless, there is a substantial lack of information about the biological effects of these contaminants in the Po delta lagoons. These environments are highly dynamic and the interactions between chemical and environmental stressors could prevent the proper identification of biological effects and their causes. In this study, we aimed to disentangle such interactions focusing on Manila clams, previously exposed to six lagoons of the Po delta, adopting three complementary tools: a) the detailed description via modelling techniques of lagoon dynamics for salinity and water temperature; b) the response sensitivity of a number of target genes (ahr, cyp4, ρ-gst, σ-gst, hsp22, hsp70, hsp90, ikb, dbh, ach, cat, Mn-sod, Cu/Zn-sod, cyp-a, flp, grx, TrxP) investigated in clam digestive glands by Real Time PCR; and c) the relevance of DNA adducts determined in clams as markers of exposure to genotoxic chemicals. The lagoons showed specific dynamics, and two of them (Marinetta and Canarin) could induce osmotic stress. A group of genes (ahr, cyp4, Mn-sod, σ-gst, hsp-22, cyp-a, TrxP) seemed to be associated with overall lagoon characteristics as may be described by salinity and its variations. Lagoon modelling and a second group of genes (hsp70, hsp90, cat, ikb, ach, grx, Cu/Zn-sod) also suggested that moderate increases of river discharge may imply worse exposure conditions. Oxidative stress seemed to be associated with such events but it was slightly evident also under normal exposure conditions. DNA adduct formation was mainly associated with overwhelmed antioxidant defences (e.g. low Cu/Zn-sod) or seemingly with their lack of response in due time. In Po delta lagoons, Manila clam can be affected by chemical and environmental factors which can contribute to induce oxidative stress, DNA adduct formation and, ultimately, to affect clam condition and health.