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The aim of the study was to develop fast and accurate method for assessment of intracellular level of cadmium (Cd) and thallium (Tl), and to establish accumulation of the metals in the cells. HepG2 cells were treated with Cd or Tl (1.0 or 10.0 mg/L; 24 h) and level of Cd or Tl was assessed. ICP-MS was applied and the method was optimized and validated. Correlation coefficient (R2) for Cd was 0.9999 with intercept 0.0732 while for Tl was 1.00009 with intercept −0.1497, and limit of detection (LOD) for Cd was 0.020 μg/L and for Tl 0.097 μg/L. Both metals, Cd and Tl, accumulate in the cells in concentration-dependent manner. However, higher uptake of Cd in comparison to Tl was observed. Cells treated with the same concentration of the metal (1.0 mg/L) accumulated 10.0% of Cd and 1.0% of Tl. Higher uptake of Cd than Tl can explain higher toxicity of Cd toward HepG2 cells. Obtained results imply to the importance of monitoring the level of metals in the cells in order to connect changes at the molecular level with exposure to specific metal.

Acrylamide is a monomer widely used as an intermediate in the production of organic chemicals, e.g. polyacrylamides (PAMs). Since PAMs are low cost chemicals with applications in various industries and waste- and drinking water treatment, a certain amount of non-polymerised acrylamide is expected to end up in waterways. PAMs are non-toxic but acrylamide induces neurotoxic effects in humans and genotoxic, reproductive, and carcinogenic effects in laboratory animals. In order to evaluate the effect of acrylamide on freshwater organisms, bioassays were conducted on four species: algae Desmodesmus subspicatus and Pseudokirchneriella subcapitata, duckweed Lemna minor and water flea Daphnia magna according to ISO (International Organization for Standardisation) standardised methods. This approach ensures the evaluation of acrylamide toxicity on organisms with different levels of organisation and the comparability of results, and it examines the value of using a battery of low-cost standardised bioassays in the monitoring of pollution and contamination of aquatic ecosystems. These results showed that EC50 values were lower for Desmodesmus subspicatus and Pseudokirchneriella subcapitata than for Daphnia magna and Lemna minor, which suggests an increased sensitivity of algae to acrylamide. According to the toxic unit approach, the values estimated by the Lemna minor and Daphnia magna bioassays, classify acrylamide as slightly toxic (TU=0-1; Class 1). The results obtained from algal bioassays (Desmodesmus subspicatus and Pseudokirchneriella subcapitata) revealed the toxic effect of acrylamide (TU=1-10; Class 2) on these organisms.

The proliferation of silver nanoparticle (AgNP) production and use owing to their antimicrobial properties justifies the need to examine the resulting environmental impacts. The discharge of biocidal nanoparticles to water bodies may pose a threat to aquatic species. This study evaluated the effects of citrate-coated AgNPs on the standardized test organism Daphnia magna Straus clone MBP996 by means of biochemical biomarker response. AgNP toxicity was compared against the toxic effect of Ag(+). The toxicity endpoints were calculated based upon measured Ag concentrations in exposure media. For AgNPs, the NOAEC and LOAEC values at 48 h were 5 and 7 μg Ag/L, respectively, while these values were 0.5 and 1 μg Ag/L, respectively, for Ag(+). The EC50 at 48 h was computed to be 12.4 ± 0.6 and 2.6 ± 0.1 μg Ag/L for AgNPs and Ag(+), respectively, with 95 % confidence intervals of 12.1-12.8 and 2.3-2.8 μg Ag/L, respectively. These results indicate significant less toxicity of AgNP compared to free Ag(+) ions. Five biomarkers were evaluated in Daphnia magna neonates after acute exposure to Ag(+) or AgNPs, including glutathione (GSH) level, reactive oxygen species (ROS) content, and catalase (CAT), acetylcholinesterase (AChE), and superoxide dismutase (SOD) activity. AgNPs induced toxicity and oxidative stress responses in D. magna neonates at tenfold higher concentrations than Ag. Biochemical methods revealed a clear increase in AChE activity, decreased ROS level, increased GSH level and CAT activity, but no significant changes in SOD activity. As Ag(+) may dissolve from AgNPs, these two types of Ag could act synergistically and produce a greater toxic response. The observed remarkably high toxicity of AgNPs (in the parts-per-billion range) to crustaceans indicates that these organisms are a vulnerable link in the aquatic food chain with regard to contamination by nanosilver. Graphical Abstract ᅟ.

Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 μmol L-1), ZnCl2 (25 or 50 μmol L-1), and CuCl2 (2.5 or 5 μmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F’m) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 μmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 μmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms.

Silver nanoparticles (AgNPs) are the dominating nanomaterial in various consumer products. To enhance nanoparticle useful properties, different surface coatings are used, which affect physico-chemical characteristics of AgNPs. As plants can have a significant role in accumulation and biodistribution of many environmentally released substances, they are also very likely to be influenced by AgNPs, serving as a potential pathway for transport and bioaccumulation of AgNPs into food chains. In this study we investigated the phytotoxicity of AgNO3 and three types of laboratory-synthesized AgNPs with different surface coatings [citrate, polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB)] on tobacco (Nicotiana tabacum L.), agriculturally interesting and frequently used model plant. In vitro grown 3-months old plants were treated with 25, 50, 75, 100 and 150 microM of AgNO3, AgNPs-citrate, AgNPs-PVP and AgNPs-CTAB. After seven days chlorophyll fluorescence parameters, concentration of photosynthetic pigments and Ag uptake were determined. Treatments with AgNO3 and all types of AgNPs resulted with the increased Ag uptake in the leaves. Positively charged AgNPs-CTAB lowered most parameters of chlorophyll fluorescence, while at higher concentrations decreased the concentration of photosynthetic pigments, including chlorophylls, thus indicating strong negative influence on photosynthetic apparatus. Treatments with AgNO3 and AgNPs-PVP, NPs with the low negative charge, decreased the non-photochemical quenching and negatively influenced majority of photosynthetic pigments, but only at higher concentrations. Negatively charged AgNPs-citrate exhibited the weakest impact by only increasing the non-photochemical quenching and concentration of lutein. We can conclude that the phytotoxicity of AgNPs is correlated with their surface coating and overall surface charge.

Discharge of silver nanoparticles (AgNP) from textiles and cosmetics, todays major application areas for metallic AgNP, into wastewater is inevitable. Transformation in wastewater treatment plants (WWTP) will determine the impact of AgNP on aquatic and terrestrial environments. This study aimed to determine the behavior of AgNP in the sludge at the WWTP by adressing the stability of citrate- coated AgNPs and their interactions with laundry-relevant surfactants (sodium dodecyl sulphate (SDS) and dodecylammonium chloride (DDACl)). The stability of the AgNPs was shown to depend on the charge and concentration of the adsorbed surfactants. The presence of DDACl prevented the formation of AgNP agglomerates even at higher ionic strength. In order to determine the effect of AgNP on biodegradation of laundry surfactants, we employed standardized method for water quality and HRN- EN-ISO-10707:2000 (Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds). Additionally, we evaluated the inhibitory effect of AgNPs on the growth of activated sludge microorganisms (ASMs)). Results showed that AgNPs were toxic to ASMs at very high concentration (>50 mg/L). However, biodegradability of SDS, DDACl and commercial laundry detergent were inhibited by more than 5% and 20% in the presence of 0.1 and 1 mg/L AgNP, respectively. It can be concluded that AgNP discharged to the wastewater stream will influenced overall biodegradability of organic matter at various degrees in the sewer system. Such knowledge is important as it may influence the subsequent transport of AgNPs through different chemical transients and thus their potential bioavailability and toxicity.

The objective of the present study was to investigate the effects of cadmium-zinc (Cd-Zn) interactions on their uptake, oxidative damage of cell macromolecules (lipids, proteins, DNA) and activities of antioxidative enzymes in tobacco seedlings as well as roots and leaves of adult plants. Seedlings and plants were exposed to Cd (10 µM and 15 µM) and Zn (25 µM and 50 µM) as well as their combinations (10 µM or 15 µM Cd with either 25 µM or 50 µM Zn). Measurement of metal accumulation exhibited that Zn had mostly positive effect on Cd uptake in roots and seedlings, while Cd had antagonistic effect on Zn uptake in leaves and roots. According to examined oxidative stress parameters, in seedlings and roots individual Cd treatments induced oxidative damage, which was less prominent in combined treatments, indicating that the presence of Zn alleviates oxidative stress. However, DNA damage found in seedlings, and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity recorded in both seedlings and roots, after individual Zn treatments, indicate that Zn accumulation could impose toxic effects. In leaves, oxidative stress was found after exposure to Cd either alone or in combination with Zn, thus implying that in this tissue Zn did not have alleviating effects. In conclusion, results obtained in different tobacco tissues suggest tissue-dependent Cd-Zn interactions, which resulted in activation of different mechanisms involved in the protection against metal stress.

Food supplements adulterated with analogues pose a pharmacological and toxicological health risk. The scale at which erectile dysfunction (ED) medicines are obtained outside of the official health system rivals and possibly exceeds legitimate sales. The legitimate health systems were dominated by the EMA and FDA approved ED medicines ; Viagra® (sildenafil), Cialis® (tadalafil) and Stendra® (avanafil). According to literature a high-risk segment of this market is occupied by adulterated food supplements. We studied food supplements (capsules, pastes and drinks) purchased from Croatian market or as a row material on sildenafil, tadalafil and avanafil content. Substances were determined by high-pressure liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS-MS). For each substance two transitions was monitored, using protonated molecules as precursor ions and sildenafil D8 as internal standard. The drugs were isolated from food remedies using simple methanol extraction. The target drug was separated using reversed-phase liquid chromatography on chromatographic column Zorbax SB C18 (150 mm × 2.1 mm, 3.5 μm) with an isocratic elution using acetonitrile – 0.1% formic acid mobile phase at a flow rate of 0.2 mL/min. 59 samples were analysed in period from 2012 till 2014. 46% of samples were collected by sanitary inspection and others by private companies. The study showed that sildenafil and tadalafil were detected in 12 samples in range (

The effects of 10 μmol L-1 and 15 μmol L-1 cadmium (Cd), a nonessential toxic element and 25 μmol L-1 and 50 μmol L-1 zinc (Zn), an essential micronutrient, on proteins and glycoproteins of Nicotiana tabacum L. seedlings and plants were investigated after exposure to each metal alone or to their combinations. Changes in only few polypeptides related to heavy metal treatments were observed in tobacco seedlings and leaves of adult plants, while the greatest change in total soluble protein pattern was observed in plant roots. Differences between control and treated tobacco tissues were more pronounced in the glycoprotein pattern, which was analysed by application of different lectins. The majority of the detected glycoproteins in leaves and roots of adult plants can be considered as a result of enhanced glycosylation due to heavy metal stress. The difference in glycoproteins between Cd and Zn application on tobacco seedlings and adult plants could not be determined since enhanced glycosylation was noticed after treatment with either metal alone or in combination. Therefore, it can be concluded that both metals induced N- and Oglycosylation as a result of changed environmental conditions.

The effects of 5 μM cadmium (Cd), a non-essential toxic element and 25 and 50 μM zinc (Zn), an essential micronutrient, were investigated in aquatic plant Lemna minor L. after 4 and 7 days of exposure to each metal alone or to their combinations. Both metals showed tendency to accumulate with time, but when present in combination, they reduced uptake of each other. Cd treatment increased the lipid peroxidation and protein oxidation indicating appearance of oxidative stress. However, Zn supplementation in either concentration reduced values of both parameters, while exposure to Zn alone resulted in elevated level of lipid peroxidation and protein oxidation but only on the 7th day. Enhanced DNA damage, which was found on the 4th day in plants treated with Cd alone or in combination with Zn, was reduced on the 7th day in combined treatments. Higher catalase activity obtained in all treated plants on the 4th day of experiment was reduced in Zn-treated plants, but remained high in plants exposed to Cd alone or in combination with Zn after 7 days. Cd exposure resulted in higher peroxidase activity, while Zn addition prominently reduced peroxidase activity in the plants subjected to Cd stress. In conclusion, Cd induced more pronounced oxidative stress and DNA damage than Zn in applied concentrations. Combined treatments showed lower values of oxidative stress parameters— lipid peroxidation, protein oxidation and peroxidase activity as well as lower DNA damage, which indicates alleviating effect of Zn on oxidative stress in Cd-treated plants.

Effect of Copper on the Toxicity and Genotoxicity of Cadmium in Duckweed (Lemna Minor L.)We investigated interactions between copper (in the concentrations of 2.5 μmol L-1 and 5 μmol L-1) and cadmium (5 μmol L-1) in common duckweed (Lemna minor L.) by exposing it to either metal or to their combinations for four or seven days. Their uptake increased with time, but it was lower in plants treated with combinations of metals than in plants treated with either metal given alone. In separate treatments, either metal increased malondialdehyde (MDA) level and catalase and peroxidase activity. Both induced DNA damage, but copper did it only after 7 days of treatment. On day 4, the combination of cadmium and 5 μmol L-1 copper additionally increased MDA as well as catalase and peroxidase activity. In contrast, on day 7, MDA dropped in plants treated with combinations of metals, and especially with 2.5 μmol L-1 copper plus cadmium. In these plants, catalase activity was higher than in copper treated plants. Peroxidase activity increased after treatment with cadmium and 2.5 μmol L-1 copper but decreased in plants treated with cadmium and 5 μmol L-1 copper. Compared to copper alone, combinations of metals enhanced DNA damage after 4 days of treatment but it dropped on day 7. In conclusion, either metal given alone was toxic/genotoxic and caused oxidative stress. On day 4 of combined treatment, the higher copper concentration was more toxic than either metal alone. In contrast, on day 7 of combined treatment, the lower copper concentration showed lower oxidative and DNA damage. These complex interactions can not be explained by simple antagonism and/or synergism. Further studies should go in that direction.