Butterflies as bioindicators of metal contamination.

Anthropogenic trace metal contamination has significantly increased and has caused many hazardous consequences for the ecosystems and human health. The Terni basin valley (Central Italy) shows a heavy load of pollutants from industrial activities, while the characteristic orography structure of the valley favours air stagnation, thus limiting air pollution dispersal. The present study conducted in 2014 aimed to determine the concentration of ten metals in five species of butterflies at nine sites in the Terni valley along a 21-km-long transect, including both relatively pristine and industrial areas. At sites where soil contamination was high for a given metal, such as for chromium as in the case of site 4 (the closest to the steel plant) and for lead as in the case of site 2 (contaminated by a firing range), higher levels of contamination were observed in the tissues of butterflies. We found a correlation between soil contamination and the concentration of Cr, Al and Sr in the tissues of some species of butterflies. The sensitivity to contamination differed among the five species; in particular, Coenonympha pamphilus was generally the species that revealed the highest concentrations of all the ten trace metals at the sites closer to the industrial area. It is known that C. pamphilus is a sedentary species and that its host plants are the Poaceae, capable of accumulating high quantities of metals in their rhizosphere region, thus providing the link with soil contamination. Therefore, monitoring the metal concentration levels in butterflies might be a good indicator and a control tool of environmental quality, specifically in areas affected by high anthropogenic pollution loads linked to a specific source.

Sex and seasonal differences in metal accumulation of selected tissues in red swamp crayfish from Lake Trasimeno (Umbria, Italy).

Concentrations of essential metals (Cu, Co, Cr, Fe, Ni, Mn, and Zn) and non-essential and toxic metals (Ag, Cd, Hg, and Pb) were quantified in hepatopancreas and abdominal muscle for 73 specimens (37 males and 36 females) of Procambarus clarkii from Lake Trasimeno (Italy) in a year's campaign (July 2018 to June 2019). Elemental concentrations determined in crayfish differed in tissues, seasons, and gender. Both sexes showed lower concentrations in abdominal muscle than in hepatopancreas. Essential metals accumulated in higher concentrations in both tissues than non-essential elements, while associations between essential metals were stronger, especially for females; however, a strong correlation was also observed between Cu and Ag in both sexes. No significant differences in concentrations were recorded between sexes for all seasons; contrarily, significant differences among the total seasonal concentrations (males plus females) were observed only for essential metals and for Ag. Seasonal Toxic Contamination Index (TCI) for Cr, Ni, Cu, Zn, Cd, Pb, and Hg were calculated to assess the toxicity level of metals in the sediments. TCI higher than 1 was detected for Ni only in summer, confirming the close connection between the fluctuating hydrological levels of the shallow Lake Trasimeno, strongly dependent on precipitation, and the metal contamination levels of sediments.

Applying Artificial Neural Networks to Oxidative Stress Biomarkers in Forager Honey Bees (Apis mellifera) for Ecological Assessment.

Insect pollinators provide an important ecosystem service that supports global biodiversity and environmental health. The study investigates the effects of the environmental matrix on six oxidative stress biomarkers in the honey bee Apis mellifera. Thirty-five apiaries located in urban, forested, and agricultural areas in Central Italy were sampled during the summer season. Enzyme activities in forager bees were analyzed using an artificial neural network, allowing the identification and representation of the apiary patterns in a Self-Organizing Map. The SOM nodes were correlated with the environmental parameters and tissue levels of eight heavy metals. The results indicated that the apiaries were not clustered according to their spatial distribution. Superoxide dismutase expressed a positive correlation with Cr and Mn concentrations; catalase with Zn, Mn, Fe, and daily maximum air temperature; glutathione S-transferase with Cr, Fe, and daily maximal air temperature; and glutathione reductase showed a negative correlation to Ni and Fe exposure. This study highlights the importance of exploring how environmental stressors affect these insects and the role of oxidative stress biomarkers. Artificial neural networks proved to be a powerful approach to untangle the complex relationships between the environment and oxidative stress biomarkers in honey bees. The application of SOM modeling offers a valuable means of assessing the potential effects of environmental pressures on honey bee populations.

Chironomid mouthpart deformities as indicator of environmental quality: a case study in Lake Trasimeno (Italy).

The aim of the present study was to monitor mouthpart deformities of Chironomus plumosus (Insecta-Diptera) in Lake Trasimeno (2000-2010) in relation with sediment contamination by heavy metals, which is one of the main causes of deformity induction. In Lake Trasimeno, concentrations of heavy metals in the investigated littoral zone were low, in comparison with those of the central area. By contrast, the incidence of deformities was much higher in the littoral station (2006-2009) than in the central area. In the littoral zone, the deformities progressively decreased and by 2010 reached values similar to those observed in the central area (2000-2002, 2010). This decrease was mainly due to the reduction of severe deformities, thus indicating an environmental quality recovery of this littoral sector. Chemical analysis revealed that the higher incidence of deformities in the littoral zone was not caused by heavy metal contamination, unless they may have contributed to a joint action with other toxicants. The present study on Lake Trasimeno provided additional evidence that chironomid mouthpart deformities can be used in biomonitoring programs to evaluate sediment contamination by toxicants. Since mouthpart deformities may also be induced by compounds not included in routine chemical analyses, they may better reflect sediment quality than chemical analysis alone.

Sources and trends of trace elements and polycyclic aromatic hydrocarbons in a shallow lake in the Mediterranean area from sediment archives of the Anthropocene.

In this study, the anthropogenic contamination in Trasimeno lake (Central Italy) was investigated using three sediment cores spanning over the last 150 years (Anthropocene) to identify the primary sources of pollution and quantify the level of contaminant enrichment in the basin. First, based on the relative cumulative frequency and linear regression methods, we obtained a geochemical baseline for the lake using the deeper parts of the sediment cores. The geochemical baseline allowed us to determine the values of trace elements enrichment factors. On this knowledge, as a second result, we were able to reconstruct the natural sources and the anthropogenic impact on the lake with a biennial resolution. This goal has been obtained by combining different inorganic and organic chemical proxies such as trace elements, polycyclic aromatic hydrocarbons, and lead isotope ratios and exploiting both principal component and factor analysis to associate chemical proxies to human-driven contamination processes. Five different groups of elements have been identified, one of which is of natural origin and four of anthropogenic origin. In particular, it was possible to identify the times and impacts of the industrial activities during the Second World War, which dispersed heavy metals in sediments. Moreover, we found evidence of the recent human activities that have characterized the surroundings of the basin, such as Pb inputs related to the use of gasoline and the enrichment of certain elements generally used in agricultural activities (such as P, Cu, and Mn) due to the development of this sector in the last 40 years.

Population Ecology and Genetic Diversity of the Invasive Alien Species Procambarus clarkii in Lake Trasimeno (Italy).

The deliberate or accidental introduction of invasive alien species (IAS) causes negative ecological and economic impacts altering ecosystem processes, imperiling native species and causing damage to human endeavors. A monthly monitoring program was performed in Lake Trasimeno (Central Italy) from July 2018 to July 2019 in order to provide an upgrade of the population ecology of Procambarus clarkii and to assess the genetic diversity by analyzing the relationships among mitochondrial DNA diversity. Our results confirmed that P. clarkii is well acclimatized in the lake, revealing a stable population structure favored by the resources and conditions typical of this ecosystem, which seem to be optimal for the maintenance of the species. Four distinct mitochondrial haplotypes were detected, but one of them was clearly overrepresented (76%), suggesting that a single predominant introduction event may have occurred in this area, likely followed by secondary events. The identification of the typical genetic variants provides a better understanding of the evolutionary scenarios of P. clarkii in this biotope and it can be helpful in management plans concerning the expanding populations of this invasive alien species.

Evaluation of geochemical baselines and metal enrichment factor values through high ecological quality reference points: a novel methodological approach.

In this study, we propose a new approach to estimate geochemical local baselines and enrichment factor values for metals in riverine sediments. The goal is to describe catchment areas characterized by intensive and spread anthropogenic activities, for which it is challenging to identify undisturbed sites to utilize as reference. The case study is the Nestore river basin (Central Italy). Our approach is based on the use of ecological quality as a criterion to select the reference points in the normalization processes of metal baselines. The rationale is to assume that the sediments with a better environmental quality are anthropogenically least impaired. On these grounds, we detected geochemical local baselines and enrichment factor values of various metals (Ca, Co, Cr, Cu, Mn, Ni, Pb, Se, Sr, and Zn). Also, this approach allowed highlighting a major level of pollution for the most downstream site of Nestore river and its left tributaries.

Cross-sectional study to identify risk factors associated with the occurrence of antimicrobial resistance genes in honey bees Apis mellifera) in Umbria, Central Italy.

The use antimicrobials for therapeutic and metaphylactic purpose in humans and agriculture exerts selective pressure on animal and environmental microbiota resulting in the survival and spread of antimicrobial resistance genes among bacteria and subsequent development of resistance in bacteria. Previous studies have shown that honey bees' microbiota (Apis mellifera) can accumulate antimicrobial resistance genes in their microbiome and act as collectors and disseminators of resistance genes. The aim of this study was to investigate to what extent honey bees act as reservoir of select antimicrobial resistance genes. This study was conducted on 35 groups of bees. Bees were collected from 35 sites in Umbria, Italy. PCR was used to screen pooled ground bees' specimens for genes that code for resistance against antimicrobials that are commonly used in humans and in veterinary medicine including aminoglycosides (aph), beta-lactams (blaZ), tetracycline (tetM) and sulphonamides (sul1 and sul2). Twenty-four samples out of 35 (68.57%) were positive for at least one antimicrobial resistance gene. Two samples were positive for the aph, 5.71%; eight for blaZ, 22.86%; three for tetM, 8.57%; ten for sul1, 28.57% and eighteen for sul2, 51.43%. Positivity to more than one antimicrobial resistance gene was observed in nine samples, 25.71%. The multivariate analysis identified "presence of farms nearby" as the factor most closely related to PCR positivity. Honey bees (Apis mellifera) from Umbria, Italy, carry antimicrobial resistance genes and can be used as indicators of the presence of resistance genes in the environment.

Occurrence of actinosporean stages (Myxozoa) in the Nera River system (Umbria, central Italy).

We investigate the oligochaetes populations in the Nera River systems (central Italy) in order to highlight the occurrence of actinosporean stages in this natural environment. The survey was carried out during 2 years of monitoring 24 stations upstream and downstream the 12 trout farm along the rivers. In four of the 24 sites, oligochaetes releasing Echinactinomyxon and Aurantiactinomyxon types spore were collected. The actinospores observed were described morphologically, by transmission electron microscopy and by molecular analysis. Histological observations of the infected oligochaetes were carried out. A single morphotype of the collective group Echinactinomyxon were detected showing the presence of branches at the distal end of the caudal processes. The morphological and morphometrical characters showed that the Echinactinomyxon type herein described is a novel phenotype. This data has to be confirmed by the molecular comparison with sequences not yet available in GenBank. The molecular analysis of the Aurantiactinomyxon type, showed high genetic similarity (99.8%) with the two alternate stages of Chloromyxum truttae even if our type showed some differences in morphology stressing the importance of combining the two methods to avoid misidentifications. This is the first survey carried out in natural environment in Italy. Further analyses on fish population are required in order to detect myxosporean stage in the vertebrate hosts and confirm the molecular data.

Analysis of the causes of the seizure and destruction of carcasses and organs in a slaughterhouse in central Italy in the 2010-2016 period.

Meat safety and hygiene are fundamental issues for producers and endusers. To achieve these goals, the inspection of carcasses and organs at slaughter is critical. The results of post-mortem inspection are the basis for planning preventive measures against consumer risks and for limiting economic losses. In this retrospective study, the causes of the seizure and destruction of carcasses and organs were analysed at a slaughterhouse in central Italy from 2010 to 2016. 436,646 slaughtered animals were taken into consideration, for a total of 61,799 seizures (73.29% pigs, 23.87% cattle, 2.77% sheep and 0.07% horses). The organs or groups of organs that most frequently showed lesions in pigs were liver (72.38%), heart (10.77%) and pluck (10.20%); in cattle, lungs (64.86%), liver (31.20%) and stomachs (11.63%); in sheep, liver (77.15%), pluck (18.70%) and lung (3.80%); in horses, liver (75.56%), kidney (68.89%) and lung (31.11%). Among the diagnoses, parasitic diseases of the liver (ascariasis and distomatosis) were especially frequent in all species, followed by pericarditis and polyserositis in pigs and diseases affecting the respiratory system in cattle. The data obtained show that postmortem inspection is of fundamental importance for limiting risks for the consumer and ensuring meat safety. It is also evident, even more than ten years after the coming into force of the so-called hygiene package regulations, that the slaughterhouse can still act as an epidemiological observatory to provide the data needed for the development of plans for the control and eradication of the most frequent diseases in the area.

Invertebrate diversity in relation to chemical pollution in an Umbrian stream system (Italy).

We used self-organizing maps (SOM, neural network) to bring out patterns of benthic macroinvertebrate diversity in relation to river pollution. Fourteen stations were sampled over various seasons in the Nestore drainage basin (Central Italy) and characterized for macroinvertebrate communities, nutrient and heavy metal concentrations. Physicochemical variables were introduced into a SOM previously trained with macroinvertebrate data. Patterns of communities matched spatial and seasonal changes in environmental conditions, including water chemistry related to economic activities in the catchment. Although our analyses did not allow us to establish the specific effect of any given environmental parameter upon macroinvertebrate community composition based on the field study, they enabled us to map the ecological health of river ecosystems in a readily interpretable manner.

Induction of mouthpart deformities in chironomid larvae exposed to contaminated sediments.

The aim of the present study was to improve the cause-effect relationship between toxicant exposure and chironomid mouthpart deformities, by linking induction of mouthpart deformities to contaminated field sediments, metal mixtures and a mutagenic polycyclic aromatic compound metabolite (acridone). Mouthpart deformities in Chironomus riparius larvae were induced by both the heavy metal mixture and by acridone. A clear correlation between metal concentrations in the sediment and deformities incidence was only observed when the contaminated field sediments were left out of the analysis, probably because these natural sediments contained other toxic compounds, which could be responsible for a higher incidence of deformities than predicted by the measured metal concentrations only. The present study clearly improved the cause-effect relationship between toxicant exposure and the induction of mouthpart deformities. It is concluded that the incidence of mouthpart deformities may better reflect the potential toxicity of contaminated sediments than chemical analysis.

Sediment toxicity and deformities of chironomid larvae in Lake Piediluco (Central Italy).

The chemical analysis of the bottom sediments of the Lake Piediluco (Central Italy) has been carried out in order to individuate the potential correlation between the sediment toxicity and the high incidence of mouthpart deformities in chironomid larvae (biological indicators) found in this lake. The environmental contamination has been analyzed by determining the concentrations of the main heavy metals (lead, copper, cadmium, chromium, zinc and nickel), and the concentrations of organic compounds of anthropic source: PAHs, NPPs and OCPs. Heavy metals concentrations have pointed out a non-elevated contamination grade for the Lake Piediluco. The highest level of metals has been detected in the western area that feels the effect of the continuous tributaries incoming load. Also, concerning PAHs, NPPs and OCPs the lake does not present high values of pollution. The highest concentrations of the organic toxicants has been observed in the eastern sector of the lake, which presents typical lentic characteristics. A clear relationship has not found between the toxic substances present in the lacustrine sediments and the deformities incidence for chironomid larvae, which represent an index of environmental alteration. Probably, the mouthpart deformities found in the chironomid larvae of Chironomus plumosus are affected by a synergic action due to the whole toxic mixture present in the sediments of the Lake Piediluco.