Assessment of interactive acoustic deterrent devices set on trammel nets to reduce dolphin-fishery interactions in the Northern Tyrrhenian Sea.

Dolphin-fishery interaction is a worldwide issue affecting dolphins through bycatch and fishers through catch or gear damages. Concerning the Mediterranean Sea, problematic interactions mainly occur between common bottlenose dolphin and small-scale fisheries. Acoustic Deterrent Devices such as pingers, are one of the most widespread measures used in attempts to face this issue. Therefore, the efficiency of interactive pingers (DiD01) in protecting the trammel nets from dolphin interactions was assessed in the Northern Tyrrhenian Sea. From March to October 2021, a total of 139 fishing trials using nets with pingers (TEST) and without pingers (CTRL), respectively n = 97 and n = 42, were carried out. Non-parametric statistic of the Catch per Unit Effort, comparing control and test nets, was not significantly different (p > 0.05) using catches weights (CPUEW) while it was significant (p < 0.01) considering the number of individuals (CPUEN). Moreover, richness and relative abundance of species resulted statistically higher in test nets (p < 0.05). This finding suggests that the absence of dolphin in the neighbourhood of fishing areas thanks to the use of pingers increases the diversity of target species. Catch damages caused by dolphins were statistically higher in nets without pinger than in nets with pinger (p < 0.05). No dolphin bycatch was recorded during fishing operations.

Tree-rings analysis to reconstruct atmospheric mercury contamination at a historical mining site.

Mercury (Hg) is a global environmental concern due to its toxicity (especially high in methylated form) and the long-range distribution of its gaseous elemental form (GEM). Hg-contaminated areas, such as abandoned mining sites, pose intrinsic difficulties for their management and heavy monitoring costs. In these environments, plant-based solutions may play a key role in the ecosystem quality assessment and support remediation strategies, combining reliability and cost-effectiveness. In this study, we adopted a biomonitoring approach by using tree rings of four different species collected in the proximity of the mining-metallurgical area of Abbadia San Salvatore, central Italy, a major former Hg mining district whose reclamation is currently in progress. Our dendrochemical analysis was aimed at identifying the historical changes of local atmospheric Hg contamination and at singling out, for the first time in the study area, other potentially toxic elements (PTEs) associated with the past mining activity. Collected cores dated back to early as 1940 and provided the temporal patterns of atmospheric Hg emission vs the produced liquid quantities, so reconstructing the historical impact of the mining site on nearby terrestrial ecosystems and resident human population. Current GEM contamination was found about twenty times lower than that of the fully operational mine periods. From a first survey on other PTEs, thallium (Tl) and lead (Pb) appeared to be potentially associated with the mining activity, thus suggesting new working assumptions for further dendrochemical analyses and for the inclusion of Pb in human biomonitoring surveys of the Mt. Amiata area, actually not present in the control list. The results prompt a more thorough assessment by tracking for a longer time span a critical site that is an ideal open-field lab to study the ecophysiology of different tree species in relation to environmental behavior of PTEs for better-assessing wildlife and human exposures.

Air quality in post-mining towns: tracking potentially toxic elements using tree leaves.

In this study, leaves of the evergreen holm oak Quercus ilex were used to assess airborne contamination of potentially toxic elements (PTEs) at five towns located on the slopes of the Mt. Amiata (central Italy), an area with a long history of mining and, more recently, an important district for the industrial exploitation of geothermal energy. PTE composition and covariance of washed and unwashed Q. ilex leaves of three different ages (6, 12 and 24 month-old) were used to identify atmospheric inputs of PTEs at residential areas, evaluate long-term adsorption and retention of PTEs by the leaves, thus providing an indication of potential human exposure. Moreover, the determination of foliar concentrations of major elements (C, N, S and P) allowed an assessment of the nutritional status of the investigated urban tree stands which excluded the existence of stress condition caused by air pollution or other disturbances. Results indicated that overall Pb, Cu, and Cd concentration were low in the investigated urban sites, if compared with similar studies conducted in larger Italian cities, denoting a low contribution of vehicular traffic to the atmospheric pathway. The five urban settlements were characterized by a specific profile of elements (Al, Ba, Hg and Sb) enriched in unwashed leaves, resulting from the distinct geochemical characteristics of the area and from diffuse (i.e., urban activity) and point sources of PTEs emission (i.e., brownfields, geothermal power plants). The latter sources primarily govern the distribution of Hg, whose contamination was found to be very localized close to a major abandoned mining area. Our data provided quantitative evidence of the spectrum of PTEs potentially impacting resident population and may prove useful in support of follow-up instrumental monitoring campaigns of air quality, as well as for human health and ecological risk assessments.

Lichen transplants as indicators of gaseous elemental mercury concentrations.

Lichens play an important role in the biogeochemical cycling of mercury (Hg) and are commonly used as indicators of Hg enrichment in remote and anthropogenically impacted environments. To assess their capacity for Hg uptake and accumulation, we determined the concentration of gaseous elemental mercury (GEM) in air and the concentration of total Hg (THg) in transplanted thalli of two lichen species. Lichen transplants and passive air samplers (PASs) were concurrently deployed, side by side, at 10 sites within an abandoned mining area, characterized by large gradients in atmospheric Hg contamination. Highly variable time-weighted GEM concentrations determined by the PASs, ranging from 17 to 4,200 ng/m3, were mirrored by generally high Hg concentrations in transplanted thalli of both Xanthoria parietina (174-8,800 ng/g) and Evernia prunastri (143-5,500 ng/g). Hg concentrations in the two species co-varied linearly indicating about 60% greater Hg accumulation in X. parietina than in E. prunastri. Whereas Hg uptake in the fruticose E. prunastri increased linearly with GEM, a power law equation with a fractional exponent described the uptake in the foliose X. parietina. Extrapolating the relationships observed here to higher GEM levels yielded concentrations in lichen that agree very well with those measured in an earlier fumigation experiment performed under laboratory-controlled conditions. The uptake model of X. parietina was further verified by correctly estimating GEM concentrations from the THg measured in autochthonous thalli collected from the urban area adjacent to the mine site. Passive sampling can effectively provide time-weighted data of suitable spatial resolution to quantitatively describe GEM assimilation by lichens. Therefore, the combined use of passive sampling and lichen transplants can contribute to a more comprehensive understanding of the role of lichens, and potentially also of other cryptogams, in the deposition of atmospheric Hg to terrestrial ecosystems.

Assessment of the effectiveness of a novel BioFilm-Membrane BioReactor oil-polluted wastewater treatment technology by applying biomarkers in the mussel Mytilus galloprovincialis.

Petrochemical industries and oil refineries are sources of hazardous chemicals into the aquatic environments, and often a leading cause of reduced oxygen availability, thus resulting in adverse effects in biota. This study is an expansion of our previous work on the assessment of the BioFilm-Membrane Bioreactor (BF-MBR) to mitigate the impact of oil-polluted wastewater on marine environments. Specifically, this study evaluated the reduction of selected chemical constituents (hydrocarbons and trace metals) and toxicity related to hypoxia and DNA damage to mussels Mytilus galloprovincialis, before and after treatment of oil-polluted wastewater with the BF-MBR. The application of a multidisciplinary approach provided evidence of the efficiency of BF-MBR to significantly reducing the pollutants load from oily contaminated seawaters. As result, the health status of mussels was preserved by a hypoxic condition due to oily pollutants, as evidenced by the modulation in the gene expression of HIF-1α and PHD and changes in the level of hypotaurine and taurine. Moreover, ameliorative effects in the energy metabolism were also found in mussel gills showing increased levels of glycogen, glucose and ATP, as well as a mitigated genotoxicity was revealed by the Micronucleus and Comet assays. Overall, findings from this study support the use of the BF-MBR as a promising treatment biotechnology to avoid or limiting the compromise of marine environments from oil pollution.

Accumulation and Release of Mercury in the Lichen Evernia prunastri (L.) Ach.

This study investigated the dynamics of the accumulation and release of Hg2+ in lichens, using Evernia prunastri (L.) Ach. as a model species. Thalli were incubated with solutions containing 1, 10, and 100 µM Hg2+ and then exposed for 1, 2, 3, 6, 12, 18, and 24 months at the Botanical Garden of the University of Siena (a location free from local Hg sources). Lichen samples accumulated Hg proportionally to the exposure concentration, and after the exposure, reductions over time were evident, already starting from 1-2 months. After 24 months, samples released 72-74 (healthy thalli) to 94% (unhealthy thalli) of the accumulated Hg, but control values of untreated samples were never reached. Depending on the Hg content after the exposure, stable decreased concentrations were reached after 6-24 months. The results of this study highlight the ability of the lichen E. prunastri to reflect rapidly increasing environmental Hg concentrations, as well as to indicate an ameliorated situation (e.g., the closure of an Hg source). However, we have found evidence that an acute pollution episode can influence the content of Hg in lichens for several years.

Modeling heavy metal release in the epiphytic lichen Evernia prunastri.

In this study, the release of Cu2+ and Zn2+ was investigated and modeled in the epiphytic lichen Evernia prunastri. Samples were incubated with solutions containing these metals at ecologically relevant concentrations (10 and 100 µM) and then transplanted to a remote area and retrieved after 1, 2, 3, 6, 12, and 18 months. The results showed that, after 12 months, all samples faced similar metal reductions of ca. 80-85%, but after this period, all the involved processes seem to be no longer capable of generating further reductions. These results suggest that the lichen E. prunastri can provide information about environmental improvements after exposure to high or very high pollution levels in a relatively short period of time.

Combined use of native and transplanted moss for post-mining characterization of metal(loid) river contamination.

Abandoned mine sites are a cause of great environmental concern, being potential sources of toxic elements for adjacent aquatic ecosystems with intrinsic difficulties for their management (i.e. episodic nature of pollution, technical difficulties and high costs of monitoring, remoteness). Aquatic macrophytes can find effective application in these situations, providing cost-effective data for instream water quality assessment. In this study, native and transplanted specimens of the aquatic moss Platyhypnidium riparioides were used to evaluate metal(loid) contamination in a river receiving multiple acidic and metalliferous drainages from sulphide mineralized areas and derelict mines. Analysis of native P. riparioides thalli was used to identify, in the upland course of the river, the pattern of contamination (As, Cd, Cu, Pb and Zn) which was related to the geo-environmental features of the watershed and the nearby historical mining areas. Attenuation of metal(loid) availability in the lowland river, apparently due to eco-hydrological and physic-chemical processes, was also highlighted by spatial trends of concentrations data of native and transplanted moss. The latter, deployed for 21 days at specific stretches of the river and in a tributary hydrologically connected with a dismissed mine, supported the identification of point sources (i.e. mine effluents, metallurgical waste piles amassed on the banks of the river) and the reckoning of their quantitative impact on different segments of the watercourse. By exploring multi-elemental and native-to-transplant relationships, differences in metal(loid) accumulative capacities were recognized between sampled thalli and exposed moss bags in relation to the severity of the contamination. The observed discrepancy in the accumulation of As, Fe, Ni and Pb in highly contaminated areas between native and transplanted moss of P. riparioides raises questions on the possible competing mechanisms of element uptake and retention. These findings prompt studies to discern possible limitations of the transplanting moss technique under extreme stream-quality conditions.

Assessing the Effect of Contaminated and Restored Marine Sediments in Different Experimental Mesocosms Using an Integrated Approach and Mytilus galloprovincialis as a Model.

The research presented here was conducted to ascertain the effectiveness of recovery technologies in remediating a compromised marine environment. The multidisciplinary approach aims to integrate traditional chemical-physical analysis and to assess the biological parameters of Mytilus galloprovincialis within different experimental mesocosms (W, G, and B). In particular, this system was designed to reproduce sediment resuspension in a marine environment, which is thought to be one cause of contaminant release. The study combined morphological and ultrastructural observations with DNA damage assessment and mRNA expression of those genes involved in cellular stress responses. The tissues of mussels maintained in the polluted mesocosm showed a higher accumulation of Pb and Hg than in those maintained in restored mesocosm. This observation correlates well with mRNA expression of MT10 and data on DNA damage. The outcome of the biological evaluation consolidates the chemical characterization and supports the concept that the remediation method should be evaluated at an early stage, both to analytically determine the reduction of toxic components and to assess its ultimate impact on the biological system.

Trace elements levels in muscle and liver of a rarely investigated large pelagic fish: The Mediterranean spearfish Tetrapturus belone (Rafinesque, 1810).

We determined levels of mercury, cadmium, lead and selenium in muscle and liver of 29 specimens of a large pelagic fish rarely investigated, the Mediterranean spearfish Tetrapturus belone (Raf., 1810). The following element concentration ranking (mean ± S.D.; espressed in mg/kg dry weight) was recorded in muscle: Hg (3.401 ± 1.908) > Se (1.727 ± 0.232) > Pb (0.532 ± 0.322) > Cd (0.019 ± 0.015), and Se (6.577 ± 1.789) > Cd (5.815 ± 3.038) > Hg (2.698 ± 2.214) > Pb (0.661 ± 1.334) in liver. Levels of Hg, Se and Cd were compared to those reported for other Istiophoridae from oceanic areas and for other large predators of Mediterranean Sea, like swordfish and tuna. Organotropism of trace elements and their relation to size was discussed. Ecophysiological considerations regarding the Se-Hg relationship as well as Se-Cd indicate a possible detoxification mechanism. The implications for human consumption are briefly discussed.

Drawing the baseline of trace element levels in the vulnerable Mediterranean osprey Pandion haliaetus: variations by breeding location, habitats, and egg components.

Due to its peculiarity to accumulate environmental contaminants, the osprey Pandion haliaetus is a sentinel species for the biomonitoring of contaminants in aquatic ecosystems. Despite this, no information on trace element concentration exists for the Mediterranean area, where relict and vulnerable osprey populations exist. We evaluated the geographical patterns of heavy metals and selenium in osprey eggs from three different populations of the Mediterranean basin (Balearic Islands, Corsica, and Tuscany), to identify any possible contaminant sources. Pattern of metal concentration followed the order: Fe > Zn > Cu > Se > Hg > Pb > Cd. Differences in contaminant concentrations between habitats and among egg components were found. Egg content and inner membrane showed higher mercury levels (1.06 ± 0.89 and 0.67 ± 0.62 mg/kg dw, respectively) than those recorded in the eggshell. Mercury concentration was ca. two times higher in marine than in wetland samples, and even higher (3.6 times) when referred to the eggshell. Cu, Fe, Zn, and Se had higher concentration in the inner membrane. We stress how the choice of the biological material can have significant implications for the correct evaluation of contamination. Our study represents a first regional scale survey for the vulnerable Mediterranean osprey populations and provides baseline data for their long-term biomonitoring.

Exposure to a nanosilver-enabled consumer product results in similar accumulation and toxicity of silver nanoparticles in the marine mussel Mytilus galloprovincialis.

The incorporation of silver nanoparticles (AgNPs) in commercial products is increasing rapidly. The consequent release of AgNPs into domestic and industrial wastewater raises environmental concerns due to their anti-microbial properties and toxicity to non-target aquatic organisms. The aim of the present study was to investigate the effects of nanArgen™ (Nanotek S.A.), a AgNP-enabled consumer product, in the marine bivalve Mytilus galloprovincialis. Two environmentally relevant concentrations of nanArgen™ (1 and 10 µg/L) were tested in vivo for 96 h, and Ag was quantified in mussel soft tissue and natural seawater (NSW). nanArgen™ suspensions were characterized via TEM, SEM, EDS, DLS, and UV-vis optical analysis. Several molecular and biochemical responses were investigated in exposed mussels: lysosomal membrane stability by Neutral Red Retention Time (NRRT) assay; micronucleus (MN) frequency in hemocytes; metallothionein (MT) protein content and gene expression (mt10 and mt20); catalase (CAT) and glutathione-S-transferase (GST) activities; malondialdehyde (MDA) accumulation in digestive glands; and efflux activity of ATP-binding cassette transport proteins (ABC) in gill biopsies. SEM, TEM and DLS analyses confirmed the presence of well-defined AgNPs in nanArgen™ which were roughly spherical with an average particle size of approx. 30 ± 10 nm. DLS analysis revealed the formation of AgNP aggregates in nanArgen™ suspension in NSW (Z-average of 547.80 ± 90.23 nm; PDI of 0.044). A significant concentration-dependent accumulation of Ag was found in mussels' whole soft tissue in agreement with a concentration-dependent decrease in NRRT and an increase of MN frequency in hemocytes and GST activities in digestive glands. A significant increase in MDA levels and MT via both molecular and biochemical tests, were also observed but only at the highest nanArgen™ concentration (10 µg/L). No changes were observed in CAT activities. ABC efflux activities in gill biopsies showed a significant decrease (p < 0.05) only at the lowest concentration (1 µg/L). On such basis, nanArgen™ is shown to be able to induce toxicity and Ag accumulation in marine mussels similarly to AgNPs and in short-term exposure conditions at environmentally relevant concentrations. AgNP-enabled products, instead of pristine AgNPs, should be the focus of future ecotoxicity studies in order to address any risks associated to their widespread use, disposal and uncontrolled release into the aquatic environment for non target species.

First ecotoxicological assessment of Caretta caretta (Linnaeus, 1758) in the Mediterranean Sea using an integrated nondestructive protocol.

The loggerhead sea turtle (Caretta caretta) is a long-lived carnivorous reptile included in the IUCN Red List. The IUCN assessment for this species underlines the lack of information regarding pollution and pathogens and indicates as a priority effort to investigate and reduce the impacts of these threats. In this contest, the aim of our study was to conduct the first ecotoxicological assessment of this species in the Mediterranean Sea using a nondestructive integrated methodology. We set up and applied a monitoring protocol which includes endpoints, such as CYP1A, LPO, ENA assay, B esterases, never investigated before in this species. Seventy-five loggerhead turtles were sampled in a nondestructive way in Italian Sea Turtle Rescue Centers or free-ranging along the Spanish coasts. Blood, skin and carapace samples were used to test biomarker responses (CYP1A, VTG, LPO, Comet and ENA assay, BChE, GGT) and contaminant (OCs, PAHs, Pb, Cd, Hg) levels. Elaboration of experimental results was carried out taking also into consideration different age classes of the specimens. Among the main results obtained we should underline the statistically significant correlation between carcinogenic PAHs in blood and DNA fragmentation as well as between Cd in carapace and GGT in plasma. We also evidenced as youngest animals showed significantly higher DNA fragmentations, BChE inhibition and increase of GGT. Older specimens showed the highest levels of erythrocyte nuclear abnormalities which may indicate a long term toxicological stress. This study contributed to expand the knowledge about the ecotoxicology of C. caretta in the Mediterranean. The nondestructive protocol could also be applied to other marine ecosystems and other sea turtle species.

Organisms in wall ecosystems as biomonitors of metal deposition and bioavailability in urban environments.

Vegetated walls are common structures in urban environments, and aiming to test the hypothesis that the biogenic crusts and plant and animal communities inhabiting these vertical surfaces can be more reliable indicators of atmospheric metal deposition than plants or animals inhabiting urban soils, we analyzed the chemical composition of the wall crusts, moss cushions (Tortula muralis) and the shells, soft tissues and feces of the stonework snail Papillifera papillaris collected in three small towns in Tuscany (Central Italy). Crusts and mosses from the same stones or bricks indicated that Cd, Pb, and Zn are the main pollutants released by vehicular traffic, while Hg and Cu probably originate from other sources. The soft tissues of P. papillaris (purged of the gut contents) showed as well higher Cd, Pb, and Zn and lower Hg concentrations at more traffic-affected sites, while data from shells and feces suggested that this species probably ingests large amounts of Al, Cr, Fe, Mn, and Pb, and avoids eating mosses. Most lithophilic elements and Pb are scarcely absorbed in the snail digestive tract and soft tissues mainly accumulate Cd and essential elements such as Cu and Mn. This study definitively confirms the extraordinary Mn bioaccumulation in P. papillaris soft tissues and reports extraordinary Mn levels also in the shell. The shells also contain unusually high Cu, Fe, and Zn concentrations and this bioaccumulation likely remains after death, potentially providing a historical record of the snail exposure to metals over lifetime.

Organotropism of persistent organic pollutants and heavy metals in the Greenland shark Somniosus microcephalus in NE Greenland.

The Greenland shark Somniosus microcephalus is an opportunistic feeder, a top predator, and a very long-lived species. The brain, liver, red and white muscle, gonads, fat, skin, pancreas, and spleen of Greenland sharks from NE Greenland fjords were analysed for PCBs, PCDDs/DFs, PBDEs; DDT isomers; HCH isomers; dieldrin; endrin; HCB; Cd, Hg, Pb, and Se. PCBs (2.01-103 ng/g wet wt) and PBDEs (7.9-3050 pg/g wet wt) were detected in most of the samples. PCDDs/DFs showed high values when detected. DDTs, HCB and HCHs were only detected in some tissues. The ΣTEQ was 5.76 pg/g in muscle. Cadmium mainly accumulated in the pancreas and liver (19.6 and 10.7 mg/kg dry wt, respectively); mercury in red muscle (4.10-6.91 mg/kg dry wt); selenium in the pancreas (3.57 mg/kg dry wt) and spleen (1.95 mg/kg dry wt); lead in the skin (0.358 mg/kgd ry wt). The selenium-mercury ratio in the liver was also evaluated.

Perfluorinated compounds in blood of Caretta caretta from the Mediterranean Sea.

Perfluorinated compounds (PFCs), widely used for their hydro-oil repellent properties, are almost non-degradable in the environment; there is scientific evidence that indicate bioaccumulation. They represent a threat to many organisms, because they are toxic and are endocrine disruptors. Scientific studies have demonstrated the presence of PFCs in blood and liver samples of fish, turtles, birds and mammals of marine ecosystems in different geographical areas. The aim of this study was to determine the distribution of PFOS and PFOA in blood samples of the marine turtle Caretta caretta, using a minimally invasive sampling procedure. 49 blood samples of marine turtle, taken from several Italian marine turtle rescue centers, were analyzed. While PFOA was never detected, measurable concentrations of PFOS were found in 15 blood samples; the values show a range from 1.14 ng/g to 28.51 ng/g (wet wt.). No differences between groups of samples taken from different areas were found.

Mercury, lead and copper in feathers and excreta of small passerine species in relation to foraging guilds and age of feathers.

In the present study excreta and feathers of five species of adult passerine birds from Montepulciano wetland (Siena, Italy) were assayed for trace elements between January and August 2006. Lead concentrations varied from 16.31 to 26.50 mg/kg and were found strictly related to the age of feathers. Copper levels were found to be high mainly in insectivorous birds (9.68 mg/kg) and were probably influenced by local use of copper-based agricultural fungicides. Mercury accumulation in feathers varied between species from 0.08 to 0.73 mg/kg. The role offeeding habits on trace elements levels in feathers and excreta is discussed.

Cadmium, lead, and mercury levels in feathers of small passerine birds: noninvasive sampling strategy.

Bird feathers have been widely used as a nondestructive biological material for monitoring heavy metals. Sources of metals taken up by feathers include diet (metals are incorporated during feather formation), preening, and direct contact with metals in water, air, dust, and plants. In the literature, data regarding the origin of trace elements in feathers are not univocal. Only in the vast literature concerning mercury (as methyl mercury) has endogenous origin been determined. In the present study, we investigate cadmium, lead, and mercury levels in feathers of prey of Falco eleonorae in relation to the ecological characteristics (molt, habitat, and contamination by soil) of the different species. Cluster analysis identified two main groups of species. Differences and correlations within and between groups identified by cluster analysis were then checked by nonparametric statistical analysis. The results showed that mercury levels had a pattern significantly different from those of cadmium and lead, which in turn showed a significant positive correlation, suggesting different origins. Nests of F. eleonorae proved to be a good source for feathers of small trans-Saharan passerines collected by a noninvasive method. They provided abundant feathers of the various species in a relatively small area--in this case, the falcon colony on the Isle of San Pietro, Sardinia, Italy.

Heavy metals in flamingos (Phoenicopterus ruber) from Italian wetlands: the problem of ingestion of lead shot.

Waterbirds are exposed to many contaminants, including lead from ingestion of shot and fishing sinkers. Lead poisoning had never been reported in flamingos wintering in Italian wetlands. Our investigation stems from a case of four flamingos found dead in Tuscany in 2002 with numerous lead shot in their gizzards. We therefore considered other specimens found dead in different Italian wetlands. Many lead shot found in gizzards and lead tissue concentrations confirmed the hypothesis of lead poisoning in two of the seven specimens analysed: concentrations in liver, kidney, and bone were 361.3, 265.09, and 43.31mug/g d.w., respectively. Lead organotropism was typical of acute poisoning. Cadmium and mercury were also determined, and found to be in line with what little data are available on this species in the literature. Although Italy recently endorsed the African-Eurasian Waterbird Agreement (AEWA) prohibiting use of lead shot for hunting in wetlands, our results reveal a first case of lead shot poisoning in flamingos wintering in Italian wetlands. This evidence sounds a further warning of the problem of spent lead shot in countries where hunting in wetlands is not strictly regulated.