Chronic tribasic copper chloride exposure induces rat liver damage by disrupting the mitophagy and apoptosis pathways.

Despite the fact that copper (Cu) is a vital micronutrient to maintain body function, high doses of Cu through environmental exposure damage various organs, especially the liver, which is the main metabolic organ. To investigate the influence of long-term Cu-induced toxicity on mitophagy and apoptosis in rat liver, 96 seven-month-old male Sprague-Dawley rats were fed TBCC for 24 weeks. The results revealed that exposure to high Cu concentrations could promote oxidative stress liver injury by increasing the hepatic function index (ALT, AST and ALP) and MDA content, while reducing the activity of antioxidant enzymes (T-SOD, GSH-Px and CAT) related to oxidative stress. Consistent with histopathological observations, proper dietary Cu (15-60 mg/kg) could improve antioxidant stress levels and induce a dose-dependent increase in the mRNA expression of mitophagy-related genes, whereas a high Cu concentration (120 mg/kg) could cause severe liver impairment and ultrastructural changes and a reduction in mitophagosomes, accompanied by downregulation of Atg5, Beclin1, Pink1, Parkin, NIX, P62 and LC3B. The expression of apoptosis-related genes (Bax, Bax/Bcl-2, Caspase3, Cytc and p53) and proteins (Caspase3 and p53) was upregulated with the addition of dietary Cu. The results demonstrated that an appropriate dose of TBCC could improve liver function by promoting mitophagy and Cu enzymes that play antioxidative roles, while the accumulation of excess Cu could induce liver lesions by enhancing apoptosis and inhibiting mitophagy pathways.

Disposable luciferase-based microfluidic chip for rapid assay of water pollution.

In the present study, we demonstrate the use of a disposable luciferase-based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3-dihydroxybenzene and 1,4-benzoquinone for the proposed microfluidic biosensor measured 3 µM, 15 mM, and 2 µM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers.

Effect of different glycation agents on Cu(II) binding to human serum albumin, studied by liquid chromatography, nitrogen microwave-plasma atomic-emission spectrometry, inductively-coupled-plasma mass spectrometry, and high-resolution molecular-mass spectrometry.

The ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)-DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)-DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation.

Effects of two copper compounds on Microcystis aeruginosa cell density, membrane integrity, and microcystin release.

Microcystin release following Microcystis aeruginosa cell lysis after copper-based algaecide treatment is often cited as a concern leading to restricted use of algaecide in restoration of natural water resources. To examine this concern, bench-scale experiments were conducted to study responses of M. aeruginosa to 8-day copper exposures as copper sulfate and copper-ethanolamine (Cu-EA). M. aeruginosa UTEX 2385 was cultured in BG11 medium to cell density of 10(6)cells/mL with total and extracellular microcystin of 93 and 53µg/L, respectively. Exposures of copper concentration ranged from 40 to 1000µgCu/L. Cell membrane integrity was indicated by erythrosine B. In the end of experiment, total microcystin and cell density in untreated control (313µg/L and 10(7)cells/mL) was 3.3 and 10 times greater than pretreatment value, respectively. Minimum amount of copper required to reduce M. aeruginosa population within 8 days was 160µgCu/L as copper sulfate and 80µgCu/L as Cu-EA, where total and extracellular microcystin concentrations (47 and 44µg/L for copper sulfate; 56 and 44µg/L for Cu-EA) were degraded with degradation rate coefficient 0.1 day(-1) and were less than pretreatment values. Given a copper concentration at 80µgCu/L as Cu-EA, M. aeruginosa cells were intact and less microcystin were released compared to treatments at 160-1000µgCu/L, where lysed cells and relatively greater microcystin release were observed. Based on the laboratory results, a minimum amount of copper required for reducing M. aeruginosa population could decrease total microcystin concentration and not compromise cells and minimize microcystin release.

Sources and levels of copper affect liver copper profile, intestinal morphology and cecal microbiota population of broiler chickens fed wheat-soybean meal diets.

Super dosing copper (Cu) has long been used as an alternative to antibiotic growth-promoters in broiler chickens' diet to improve gut health. This study was designed to compare nutritional and growth-promoting levels of Cu hydroxychloride (CH) with CuSO4 on gut health bio-markers and liver mineral profile of broiler chickens. Ross 308 chicks (n = 864) were randomly assigned to eight treatments, as basal diet containing no supplemental Cu; the basal diet with 15 or 200 mg/kg Cu as CuSO4; or 15, 50, 100, 150 or 200 mg/kg Cu from CH. The highest liver Cu content was observed in birds fed the diets with 200 mg/kg CuSO4 (P < 0.01). Serum FITC-d concentration as the leaky gut marker, and liver malondialdehyde concentration were not affected. Copper level or source had no effect on cecal short chain fatty acid and the mRNA expression of five jejunal genes involved in gut integrity. Negative linear responses of Cu were observed on Lactobacillus (P = 0.032), Bacteroides (P = 0.033), and Enterobacteriaceae (P = 0.028) counts. The jejunal villus height increased in birds fed CH at 200 and 100 mg/kg (P < 0.05). Increasing Cu levels, linearly and quadratically (P < 0.001), increased Cu excretion.

Effects of dietary copper supplementation on production performance and plasma biochemical parameters in broiler chickens.

A study was conducted to estimate the effect of copper (Cu) supplementation on growth performance and biochemical profiles of blood and meat in broiler chickens. A total of 240 d-old broiler chicks (Vencobb-100) were randomly divided into 12 groups, each of 20 chicks (4 treatments x 3 replicates). The basal diet (T1) contained 215 g kg⁻¹ crude protein (CP), 12·76 MJ kg⁻¹ ME, 32 g kg⁻¹ total calcium and 5 g kg⁻¹ total phosphorus. T2, T3 and T4 were formulated to contain an additional 75, 150 and 250 mg Cu kg⁻¹ diet, respectively. Copper sulphate pentahydrate (CuSO4, 5H2O) was used as the source of Cu. Significant reductions in plasma total cholesterol and triglyceride, and an elevated concentration of HDL-cholesterol, were observed in the chickens fed with 250 mg Cu kg⁻¹ (T4) of feed at the 3rd and 6th week of the experiment. Total cholesterol in meat decreased significantly in the birds fed with dietary Cu at 250 mg kg⁻¹ (T4) of feed. Growth performance was measured in terms of live weight gain, cumulative feed intake and feed conversion ratio at the end of d 21 and d 42 of the experiment, and the result was found to be commercially beneficial for the chickens receiving 150 mg Cu kg⁻¹ (T3) of diet. The concentration of Cu in breast muscle and liver increased significantly at the end of experiment. From this study it can be concluded that supplementation with dietary Cu may be beneficial for production performance and plasma biochemical characteristics of broiler chickens.

Chronic Copper Exposure Induces Hypospermatogenesis in Mice by Increasing Apoptosis Without Affecting Testosterone Secretion.

Chronic copper exposure impaired spermatogenesis in adult male mice. The aim of this study was to determine whether chronic copper exposure can induce apoptosis of testicular cell and hypospermatogenesis via disturbing testosterone synthesis in adult male mice. In the present study, sixty CD-1 male mice were randomly divided into four groups, and were continuously administered for 8 weeks by oral gavage with copper sulfate at a dose of 0, 25, 100, and 150 mg/kg/day, respectively. We determined the content of serum and testicular copper, testicular coefficient, testicular histopathology, sperm count and motility, the mRNA and protein levels of Caspase-3, Bax, and Bcl-2, Leydig cell count, testosterone content, testosterone synthetase, and testosterone synthesis-related genes. The results showed that the copper levels in serum increased in a dose-dependent manner, and the copper levels in testes were significantly related to serum copper levels. Male mice given copper sulfate 100 and 150 dosage groups showed significant decreased in sperm motility and sperm number as well as increased in testes damage, and there was no significant change in testicular coefficient in the four groups. The mRNA levels of Bcl-2 decreased and Caspase-3 increased in 150 dosage group, and Bax increased in two higher dosage groups. Meanwhile, Caspase-3 and Bax proteins increased in 150 dosage group, and Bcl-2 protein decreased in three copper treatment groups. Nevertheless, there were no differences on the levels of testosterone content and testosterone synthetase of 3ß-HSD, 17ß-HSD, 17α-Hyd, and 20α-Hyd, mRNA levels of Cyp11a1, Cyp17a1, and Star, and quantity of Leydig cells in four groups. Overall, these data showed that chronic copper exposure led to copper residues in the testes, and the doses of 100 and 150 mg/kg/day copper sulfate may induce hypospermatogenesis by increasing apoptosis without affecting testosterone secretion.

High-field 1H T(1) and T(2) NMR relaxation time measurements of H2O in homeopathic preparations of quartz, sulfur, and copper sulfate.

Quantitative meta-analyses of randomized clinical trials investigating the specific therapeutic efficacy of homeopathic remedies yielded statistically significant differences compared to placebo. Since the remedies used contained mostly only very low concentrations of pharmacologically active compounds, these effects cannot be accounted for within the framework of current pharmacology. Theories to explain clinical effects of homeopathic remedies are partially based upon changes in diluent structure. To investigate the latter, we measured for the first time high-field (600/500 MHz) 1HT(1) and T(2) nuclear magnetic resonance relaxation times of H2O in homeopathic preparations with concurrent contamination control by inductively coupled plasma mass spectrometry (ICP-MS). Homeopathic preparations of quartz (10c-30c, n = 21, corresponding to iterative dilutions of 100(-10)-100(-30)), sulfur (13x-30x, n = 18, 10(-13)-10(-30)), and copper sulfate (11c-30c, n = 20, 100(-11)-100(-30)) were compared to n = 10 independent controls each (analogously agitated dilution medium) in randomized and blinded experiments. In none of the samples, the concentration of any element analyzed by ICP-MS exceeded 10 ppb. In the first measurement series (600 MHz), there was a significant increase in T(1) for all samples as a function of time, and there were no significant differences between homeopathic potencies and controls. In the second measurement series (500 MHz) 1 year after preparation, we observed statistically significant increased T(1) relaxation times for homeopathic sulfur preparations compared to controls. Fifteen out of 18 correlations between sample triplicates were higher for controls than for homeopathic preparations. No conclusive explanation for these phenomena can be given at present. Possible hypotheses involve differential leaching from the measurement vessel walls or a change in water molecule dynamics, i.e., in rotational correlation time and/or diffusion. Homeopathic preparations thus may exhibit specific physicochemical properties that need to be determined in detail in future investigations.

Differential toxicity to Cd, Pb, and Cu in dragonfly larvae (Insecta: Odonata).

Odonate larvae are important organisms in aquatic ecosystems but have been rarely studied in laboratory toxicity tests. Only a few previous studies have been conducted on odonates and their responses to heavy metals. We exposed two species of libellulid larvae (Anisoptera: Libellulidae) to equimolar concentrations of cadmium, lead, or copper in 7-day survival tests. Larvae were tolerant of high concentrations of cadmium and lead, as no significant decrease in survival was observed at exposures as high as 0.893 and 2.232 mM, respectively. In contrast, larvae were more sensitive to copper exposure, demonstrating significantly decreased survival to exposures as low as 2.360 microM. In whole animal samples, larvae accumulated very high concentrations (>1000 microg/g dry weight) of all three metals in an exposure-related manner. Much of this accumulation could probably be attributed to adsorption or accumulation of metal within the exoskeleton, because odonate larvae are known to sequester metals into this material. Our results were generally consistent with previous observations indicating that odonates are tolerant to metal exposures, even in comparison with other aquatic invertebrates. However, there are few studies that have used odonates in toxicity tests and compared these organisms to other aquatic life. Based on their abundance and their simple requirements in the laboratory, we believe that odonate larvae can be useful toxicological model organisms.

A single chamber packed bed microbial fuel cell biosensor for measuring organic content of wastewater.

This study reports an investigation of the effect of the anode surface area on the performance of a single chamber microbial fuel cell (SCMFC) based biosensor for measuring the organic content of wastewater. A packed bed of graphite granules was used as the anode. The surface area of the anode was changed by altering the granule bed thickness (0.3 cm and 1 cm). The anode surface area was found to play a role in the dynamic response of the system. For a granule bed thickness of 1 cm and with an external resistance of 500 Omega, the response time (defined as the time required to achieve 95% of the steady-state current) was reduced by approximately 65% in comparison to a SCMFC biosensor with a carbon cloth anode.

Accumulation and resistance to copper of two biotypes of Cynodon dactylon.

The effects of copper accumulation and resistance in two biotypes of Cynodon dactylon were studied. Results showed that at a low concentration of copper (<100 mg/kg), the growth of Cynodon dactylon was generally unaffected. As copper concentration increased, negative effects on the growth of Cynodon dactylon became apparent. The critical concentration at which the plant exhibited poisoning symptoms was different for the two biotypes of Cynodon dactylon. At 500 mg/kg copper concentration in soil, the biotype from the polluted area showed significantly higher tolerance of copper than the biotype from the unpolluted area.

Chemical residuals in the environment and on chicken carcasses associated with scalding chickens in an acidic, copper sulfate-based commercial sanitizer during poultry processing.

A study was conducted to determine if an acidic, copper sulfate-based commercial sanitizer evolves into surrounding air when introduced in a commercial poultry scalder. Two identical slaughter lines in a large poultry processing plant were used. One scalder was dosed with the sanitizer, and the other one was not. Four air samples were collected per day over the treated and control lines by an impingement method for 4 days for each of the two lines. Results showed that dosing the scalder with an acidic, copper sulfate-based commercial sanitizer resulted in no significant increase (P < or = 0.05) in the levels of the chemical components (sulfuric acid, ammonium sulfate, and copper sulfate) in the air above the scalder. Another study was conducted to evaluate the effect of the addition of this sanitizer during scalding on chemical residuals on chicken skin, meat, and fat. Five chickens were tagged and scalded in commercial scald water (136 degrees F [57.8 degrees C]), and five chickens were tagged and scalded in scald water (136 degrees F) treated with an acidic, copper sulfate-based commercial sanitizer as above. The chickens for both the control and treated lines were allowed to continue down the processing line and be exposed to the various washes (inside and outside bird washer and on-line reprocessing system) and the chiller. Skin, breast meat, and fat were removed from each carcass and analyzed. Three repetitions were conducted. Results demonstrated that there was no significant difference (P < or = 0.05) in the amount of sulfuric acid, ammonium sulfate, or copper sulfate recovered from these carcass parts.

Impact of copper sulfate application at an urban Brazilian reservoir: A geostatistical and ecotoxicological approach.

A landscape ecotoxicology approach was used to assess the spatial distribution of copper in the recent bottom sediment (surficial sediment) of a Brazilian subtropical reservoir (the Guarapiranga reservoir) and its potential ecotoxicological impacts on the reservoir ecosystem and the local society. We discuss the policies and procedures that have been employed for the management of this reservoir over the past four decades. Spatial heterogeneity in the reservoir was evaluated by means of sampling design and statistical analysis based on kriging spatial interpolation. The sediment copper concentrations have been converted into qualitative categories in order to interpret the reservoir quality and the impacts of management policies. This conversion followed the Canadian Water Framework Directive (WFD) ecotoxicological concentration levels approach, employing sediment quality guidelines (SQGs). The SQG values were applied as the copper concentration thresholds for quantitative-qualitative conversion of data for the surficial sediment of the Guarapiranga. The SQGs used were as follows: a) interim sediment quality guideline (ISQG), b) probable effect level (PEL), and c) regional reference value (RRV). The quantitative results showed that the spatial distribution of copper in the recent bottom sediment reflected the reservoir's management policy and the copper application protocol, and that the copper concentrations varied considerably, ranging from virtually-zero to in excess of 3gcopper/kgds. The qualitative results demonstrated that the recent bottom sediment was predominantly in a bad or very bad condition, and could therefore have impacts on the local society and the ecosystem. It could be concluded that the management policy for this reservoir was mainly determined by the desire to minimize short-term costs, disregarding long-term socioeconomic and environmental consequences.

Modification of chitosan/calcium alginate/Fe3O4 hydrogel microsphere for enhancement of Cu(II) adsorption.

Copper in drinking water causes a significant environmental problem. Composite material based on alginate hydrogel has been gaining attention in the field of Cu(II) adsorption. However, alginate-based hydrogel exhibits poor mechanical property and relative low adsorption capacity which limit their application. The present study is devoted to the modification of chitosan/calcium alginate/Fe3O4 (CAF) hydrogel microsphere by NaOH solution for enhancement of Cu(II) adsorption. Results reveal that modification of CAF via NaOH solution significantly improves the mechanical strength and Cu2+ adsorption capacity of pristine materials. FTIR and XRD analysis confirms that CAF and newly prepared materials (NACAF) are successfully prepared. SEM and EDX are employed to analyze the surface morphology and elemental composition, respectively, both before and after their loading with Cu2+. XPS study demonstrates adsorption mechanism is based on chelation and ion-exchange. Compressive stress-strain curves demonstrate NACAF has better mechanical performance than CAF. The adsorption kinetics of the two adsorbents follow a pseudo-second-order model. The equilibrium data were best described by Langmuir isotherm model, and the estimated maximum equilibrium sorption capacity, q m,is 261.31 mg/g for the NACAF, which is larger than that of CAF (145.39 mg/g). Hence, NACAF shows excellent mechanical strength and high sorption capacity for Cu2+. It has great potential for Cu(II) removal in aqueous solutions.

Noninvasive rapid bacteria-killing and acceleration of wound healing through photothermal/photodynamic/copper ion synergistic action of a hybrid hydrogel.

Bacterial infection often delays healing of wounded tissues and so it is essential to improve the antibacterial efficiency in situ. In this work, a hybrid hydrogel composed of 3-(trimethoxysilyl)propyl methacrylate (MPS, 97%) and mesoporous silica (mSiO2) modified CuS nanoparticles (NPs) is synthesized by radical polymerization. The materials possess excellent and controllable photothermal and photodynamic properties under 808 nm near-infrared (NIR) light irradiation as well as an antibacterial efficacy of 99.80% and 99.94% against Staphylococcus aureus and Escherichia coli within 10 min, respectively. The excellent performance stems from the combined effects of hyperthermia, radical oxygen species, and released copper ions produced during NIR irradiation of CuS NPs. Moreover, the released copper ions stimulate fibroblast proliferation and angiogenesis and the intrinsic volume transition of the hydrogel composed of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) controls the release rate of copper ions during NIR light irradiation leading to both antibacterial effects and skin tissue regeneration.

Effect of copper sulphate treatment on natural phytoplanktonic communities.

Copper sulphate treatment is widely used as a global and empirical method to remove or control phytoplankton blooms without precise description of the impact on phytoplanktonic populations. The effects of two copper sulphate treatments on natural phytoplanktonic communities sampled in the spring and summer seasons, were assessed by indoor mesocosm experiments. The initial copper-complexing capacity of each water sample was evaluated before each treatment. The copper concentrations applied were 80 microg l(-1) and 160 microg l(-1) of copper, below and above the water complexation capacity, respectively. The phytoplanktonic biomass recovered within a few days after treatment. The highest copper concentration, which generated a highly toxic environment, caused a global decrease in phytoplankton diversity, and led to the development and dominance of nanophytoplanktonic Chlorophyceae. In mesocosms treated with 80 microg l(-1) of copper, the effect on phytoplanktonic community size-class structure and composition was dependent on seasonal variation. This could be related to differences in community composition, and thus to species sensitivity to copper and to differences in copper bioavailability between spring and summer. Both treatments significantly affected cyanobacterial biomass and caused changes in the size-class structure and composition of phytoplanktonic communities which may imply modifications of the ecosystem structure and function.

Hepatocyte nuclear structure and subcellular distribution of copper in zebrafish Brachydanio rerio and roach Rutilus rutilus (Teleostei, Cyprinidae) exposed to copper sulphate.

Copper is a trace element essential to life, but also a heavy metal with toxic effect clearly demonstrated. Cu induced perturbations in fish liver are well documented but the variability of the reported results is large. In this study two cyprinids, zebrafish and roach, were exposed to copper. Reported histocytological changes are either adaptative or degenerative depending on fish species, concentration of metal, and duration of exposure. Hepatic subcellular distribution of copper was determined by X-ray microanalysis in control and Cu-exposed roach and zebrafish. Sublethal copper sulphate contamination induced the development of a particular nucleolar alteration forming a network or honeycomb like structure in liver. This perturbation is observable in almost all the hepatocytes of zebrafish and roach exposed to copper for a minimum of 4 days of exposure. It seemed to concern more precisely the pars fibrosa. X-ray microanalysis showed that the appearance of network nucleolus was in relation to a Cu accumulation. Cu deposit was well located in the network as pars granulosa and nucloplasm showed very lower metal concentrations. The origin and consequence of network structure in nucleolus was discussed.

Relative bioavailability of copper in tribasic copper chloride to copper in copper sulfate for laying hens based on egg yolk and feather copper concentrations.

This experiment was conducted to determine the relative bioavailability (RBV) of Cu in tribasic copper chloride (TBCC) to Cu in copper sulfate (monohydrate form; CuSO4·H2O) for layer diets based on egg yolk and feather Cu concentrations. A total of 252, 72-wk-old Hy-Line Brown laying hens were allotted to 1 of 7 treatments with 6 replicates consisting of 6 hens per replicate in a completely randomized design. Hens were fed corn-soybean meal-based basal diets supplemented with 0 (basal), 100, 200, or 300 mg/kg Cu from CuSO4 or TBCC for 4 wk. Results indicated that egg production, egg weight, and egg mass were not affected by dietary treatments. However, increasing inclusion levels of Cu in diets from CuSO4 decreased (P < 0.05) feed conversion ratio (FCR), whereas increasing inclusion levels of Cu in diets from TBCC did not affect FCR, indicating significant interaction (P < 0.05). Increasing inclusion levels of Cu from TBCC or CuSO4 increased (P < 0.05) Cu concentrations of egg yolk and feathers. Feather Cu concentrations were greater (P < 0.01) for hens fed diets containing CuSO4 than for hens fed diets containing TBCC. The values for the RBV of Cu in TBCC to Cu in CuSO4 based on log10 transformed egg yolk and feather Cu concentrations were 107.4% and 69.5%, respectively. These values for the RBV of Cu in TBCC did not differ from Cu in CuSO4 (100%). The RBV measured in egg yolk did not differ from the RBV measured in feather. In conclusion, the RBV of Cu in TBCC to Cu in CuSO4 can be determined using Cu concentrations of egg yolk and feathers although the values depend largely on target tissues of laying hens. For a practical application, however, the RBV value of Cu in TBCC to Cu in CuSO4 could be 88.5% when the RBV values determined using egg yolk and feather Cu concentrations were averaged.

Terrestrial avoidance behaviour tests as screening tool to assess soil contamination.

To assess soil quality and risk assessment, bioassays can be useful tools to gauge the potential toxicity of contaminants focusing on their bioavailable fraction. A rapid and sublethal avoidance behaviour test was used as a screening tool with the earthworm Eisenia andrei and the isopod Porcellionides pruinosus, where organisms were exposed during 48 h to several chemicals (lindane, dimethoate and copper sulphate, for isopods and carbendazim, benomyl, dimethoate and copper sulphate for earthworms). Both species were also exposed to soils from an abandoned mine. For all bioassays a statistical approach was used to derive EC50 values. Isopods and earthworms were able to perceive the presence of toxic compounds and escaping from contaminated to clean soil. Furthermore the behaviour parameter was equally or more sensitive then other sublethal parameters (e.g. reproduction or growth), expressing the advantages of Avoidance Behaviour Tests as screening tools in ERA.

Exploiting external reflection FTIR spectroscopy for the in-situ identification of pigments and binders in illuminated manuscripts. Brochantite and posnjakite as a case study.

In the present work, the use of portable instrumentation allowing in-situ reflection FTIR analyses is exploited to identify the coloring matters of northern-Italian illuminations dating to the XVI century. In order to build a database of spectra, reference paint samples were prepared spreading the pigments on parchment with two different binders, i.e. gum arabic and egg white, used in antiquity. Pigments for the database were chosen considering their use in the Middle Ages and in the Renaissance and their response in the mid- and near-IR region. The reflection FTIR spectra obtained resulted to be dominated by the specular reflection component, allowing the use of the Kramers-Kronig transform to convert them to the more conventional absorbance FTIR spectra. Several pigments could thus be identified in ancient illuminations, even if some green details showed a spectral pattern different with respect to the most common commercial green pigments of the database. Therefore, in addition, basic copper sulfates brochantite and posnjakite were synthesized and characterized. In three green details, posnjakite was identified, both as a pure compound and together with malachite.