Plastic debris in lakes and reservoirs.

Plastic debris is thought to be widespread in freshwater ecosystems globally1. However, a lack of comprehensive and comparable data makes rigorous assessment of its distribution challenging2,3. Here we present a standardized cross-national survey that assesses the abundance and type of plastic debris (>250 µm) in freshwater ecosystems. We sample surface waters of 38 lakes and reservoirs, distributed across gradients of geographical position and limnological attributes, with the aim to identify factors associated with an increased observation of plastics. We find plastic debris in all studied lakes and reservoirs, suggesting that these ecosystems play a key role in the plastic-pollution cycle. Our results indicate that two types of lakes are particularly vulnerable to plastic contamination: lakes and reservoirs in densely populated and urbanized areas and large lakes and reservoirs with elevated deposition areas, long water-retention times and high levels of anthropogenic influence. Plastic concentrations vary widely among lakes; in the most polluted, concentrations reach or even exceed those reported in the subtropical oceanic gyres, marine areas collecting large amounts of debris4. Our findings highlight the importance of including lakes and reservoirs when addressing plastic pollution, in the context of pollution management and for the continued provision of lake ecosystem services.

Pb(ii) binding to the brain specific mammalian metallothionein isoform MT3 and its isolated αMT3 and ßMT3 domains.

The toxicity of lead, one of the most ubiquitous toxic metals, is well known. Some of its pathological effects are related to its preference for the sulfhydryl groups of proteins. Metallothioneins (MT) are a particular family of metalloproteins characterized by their high Cys content that, among other functions, are linked to the detoxification of heavy metals. In mammals, 4 MT isoforms have been found. The MT3 isoform, also called "neuronal growth inhibitory factor", is mainly synthesized in the brain and contains several structural differences that may contribute to important functional differences between it and other MT isoforms. The abilities of recombinant MT3 and its individual αMT3 and ßMT3 fragments to bind Pb(ii) have been investigated here, under different pH conditions, by means of spectroscopy, mass spectrometry and isothermal titration calorimetry. The results obtained show that the binding of Pb(ii) to the intact MT3 protein is relatively unaffected by pH, while the individual domains interact with Pb(ii) in a pH-sensitive manner. The mass spectrometry data reveal the evolution with time of the initially formed Pb-MT complexes. In the case of the full length protein, Pb(ii) remains bound for a long period of time. With the isolated fragments, the lead is eventually released. The Pb-species formed depend on the amount of Pb(ii) present in solution. The thermodynamic data recorded, as measured by ITC, for the replacement of Zn(ii) by Pb(ii) in reactions with Zn-MT3, Zn-αMT3 and Zn-ßMT3 are all similar, and in all cases, the displacement of Zn(ii) by Pb(ii) is thermodynamically favorable. Zn-Replete and Pb-replete MT3 have distinctive circular dichroism spectra, suggestive of structural differences with different metallation status.

Effects of short-term exposure to the DDT metabolite p,p'-DDE on bone tissue in male common frog (Rana temporaria).

Experimental studies as well as studies in free-ranging animals have shown that endocrine-disrupting chemicals (EDCs) impair bone tissue composition and strength. The aim of the present study was to expand our studies on bone tissue in a new group of animals by investigating whether bone tissue in frogs is an additional potential target of EDCs. Adult male European common frogs (Rana temporaria) were divided into 5 groups (n = 20) and injected (sc, single injection) with p,p'-DDE, a total dose of 0.01, 0.1, 1, or 10 mg of p,p'-DDE/kg body weight, respectively. A control group was treated with the vehicle (corn oil). Two weeks after injection the frogs were euthanized and samples taken. The diaphysis of the excised left femur was scanned using peripheral quantitative computed tomography (pQCT) and cortical variables, such as cortical bone mineral density (BMD), cortical cross-sectional area (CSA), and periosteal circumference, were determined. In addition, biomechanical three-point bending of the bones was conducted, with the load being applied to the same point as where the pQCT measurement was performed. The results from the pQCT measurements show that bone tissue in male frogs exposed to p,p'-DDE is negatively affected. A significant decrease in cortical BMD at the diaphysis was observed in frogs exposed to 1 mg p,p'-DDE. However, the biomechanical testing of the bones showed no significant differences between exposed and control group. Although this is the only study performed to date examining the possible relationships between EDCs and negative effects on frog bones, it supports both previous experimental findings in rodents and findings in free-ranging animals.

Effects of p,p'-DDE on retinoid homeostasis and sex hormones of adult male European common frogs (Rana temporaria).

Reports of declining numbers of species and individuals of amphibians in most parts of the world have caused great concern. Several causative factors have been linked to this amphibian decline, and increased environmental pollution related to pesticide use seems to be one important factor. Persistent organic pollutants may act as endocrine disrupters, and thereby exert adverse effects on development (metamorphosis, growth and sexual differentiation) in amphibians. During periodic events, such as spring snowmelt, amphibians may be exposed to acute high levels of pesticides. 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene (p,p'-DDE) is a pesticide that is still found in wetlands and soils. In order to study the effects of p,p'-DDE on susceptible amphibian endocrine systems, adult male European common frogs (Rana temporaria) were exposed to different doses of p,p'-DDE (0, 0.01, 0.1, 1 and 10 mg p,p'-DDE/kg body mass) for 14 d and sex hormone levels (testosterone and 17beta-estradiol) in plasma and retinoid concentrations (retinol and retinyl palmitate) in liver were determined. The results showed a significant variation in the liver retinol concentration at increasing doses of p,p'-DDE, suggesting that p,p'-DDE may interfere in the hepatic metabolism of retinol in adult frogs. Lack of effects on sex hormones may indicate that after arousal from hibernation males are relatively resistant to adverse reproductive effects of p,p'-DDE.

Altered hepatic retinol and CYP26 levels in adult European common frogs (Rana temporaria) exposed to p,p'-DDE.

The effects of environmental contamination on amphibians are of particular concern because there are reports of declining numbers of species and individuals in most parts of the world during the last 50 years. During the last decade there has been increased focus on the role of persistent organic pollutants as retinoid (vitamin A) disrupters, and their effects on development, growth and sexual differentiation. To study the effects of p,p'-DDE, one of the most persistent metabolites of the pesticide DDT, on retinol homeostasis, we subcutaneously exposed adult male European common frogs (Rana temporaria) to different doses of p,p'-DDE (0, 0.01, 0.1, 1 and 10 mg/kg body mass) and studied the effect of a short term exposure (14 days) on hepatic retinoid levels and CYP26 gene and protein expression. Hepatic retinol concentrations, CYP26 gene and protein levels were analysed using HPLC, quantitative RT-PCR and indirect ELISA, respectively. Our results showed a significant p,p'-DDE dose-specific increase in the hepatic retinol concentration. CYP26 gene and protein expression were reduced in an apparent p,p'-DDE dose-specific manner. The results suggest that p,p'-DDE may interfere with the hepatic metabolism of retinol in adult frogs by decreasing CYP26 expression patterns.

A study of the Pb(II) binding to recombinant mouse Zn(7)-metallothionein 1 and its domains by ESI TOF MS.

This work reports, for the first time, a complete analysis of the binding of Pb(II) ions to mammalian Zn-metallothionein (MT) and its individual domains (alpha and beta) by means of ESI TOF MS. The use of highly pure recombinant Zn-MT and high resolution MS allowed the identification of the different heteronuclear Pb,Zn-MT and homonuclear Pb-MT complexes formed by Zn/Pb replacement in the initial Zn-MT complexes at pH 7. The results obtained showed, both for the whole Zn(7)-MT and for its individual Zn(4)-alpha and Zn(3)-beta fragments, a partial substitution of the Zn initially bound to give rise to the formation of both Pb,Zn-MT and Pb-MT species. In the case of the whole Zn(7)-MT, the addition of seven equivalents of Pb(II) give rise to the formation of several Pb(x)Zn(y)-MT species (x+y=7 or 8) as well as Pb(8)-MT and Pb(9)-MT coexisting with the initial Zn(7)-MT species. The addition of a five-fold excess of Pb(II) showed the formation of species containing a higher amount of lead(II), Pb(x)Zn(y)-MT species (x=8-12, y=0-1). In both cases, the evolution with time showed the disappearance of the species containing lead and concomitantly only detection of the initial Zn-MT clusters within 48h. This behaviour was also observed for the Zn(4)-alpha and Zn(3)-beta aggregates, confirming the reversibility of the Pb(2+) displacement of Zn(2+) ions in the three forms of MT. A similar result was obtained at acidic pH, when the apo-form was the major species in solution, this therefore confirming the low affinity of MT for Pb(II), even in the absence of zinc. The results obtained were consistent with the increase of MT synthesis in response to Pb(2+) administered to rats reported in the literature, since the initial release of Zn(II) would promote the biosynthesis of MT, in agreement with the function of this ion as primary activator of the MTF-1 transcription factor; and could explain the difficulty of recovering homonuclear Pb-MT species from lead-treated organisms.

Mercury(II) binding to metallothioneins. Variables governing the formation and structural features of the mammalian Hg-MT species.

With the aim of extending our knowledge on the reaction pathways of Zn-metallothionein (MT) and apo-MT species in the presence of Hg(II), we monitored the titration of Zn7-MT, Zn4-alphaMT and Zn3-betaMT proteins, at pH 7 and 3, with either HgCl2 or Hg(ClO4)2 by CD and UV-vis spectroscopy. Detailed analysis of the optical data revealed that standard variables, such as the pH of the solution, the binding ability of the counter-ion (chloride or perchlorate), and the time elapsed between subsequent additions of Hg(II) to the protein, play a determinant role in the stoichiometry, stereochemistry and degree of folding of the Hg-MT species. Despite the fact that the effect of these variables is unquestionable, it is difficult to generalize. Overall, it can be concluded that the reaction conditions [pH, time elapsed between subsequent additions of Hg(II) to the protein] affect the structural properties more substantially than the stoichiometry of the Hg-MT species, and that the role of the counter-ion becomes particularly apparent on the structure of overloaded Hg-MT.

Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins.

To elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by electronic absorption and CD spectroscopies the stepwise addition of MeHgCl stock solution to mammalian Zn(7)-MT1 and the isolated Zn(4)-alphaMT1 and Zn(3)-betaMT1 fragments. The incorporation of MeHg(+) into Zn(7)-MT and Zn(3)-betaMT entails total displacement of Zn(II) and unfolding of the protein. However, both features are only partial for Zn(4)-alphaMT. The different behavior observed for this fragment, whether isolated or constituting one of the two domains of Zn(7)-MT, indicates interdomain interactions in the whole protein. Overall, the binding properties of Zn(7)-MT, Zn(4)-alphaMT and Zn(3)-betaMT toward MeHg(+) are unprecedented. In addition, the sequestration of MeHg(+) by Zn(7)-MT and the concomitant release of Zn(II) are probably two of the main contributions in the detoxifying role of mammalian MT.