Fate of pharmaceutical residue in two Romanian rivers receiving treated water: Occurrence, distribution and risk assessment.

This study presents the first set of data on the removal of proton pump inhibitors (PPIs) and histamine H2 receptor antagonists (HRAs) and their transformation products in two Romanian wastewater treatment plants (WWTPs), as well as the impact of these organic pollutants on freshwater receiving effluents. The research investigated eight target pharmaceuticals and three metabolites using a newly developed and validated Liquid Chromatography - Mass Spectrometry (LC-MS/MS) method. The combined determination had a range of quantification limits varying from 0.13 ng/L to 0.18 ng/L for surface water and from 0.28 ng/L to 0.43 ng/L for wastewater. All analytes except cimetidine and 5-hydroxy-omeprazole were identified in water samples. The study found similar overall removal efficiencies for both WWTPs (43.2 % for Galati and 51.7 % for Ramnicu-Valcea). The research also showed that ranitidine and omeprazole could pose a low to high ecological risk to aquatic organisms. The findings suggest that the treatment stages used in the two Romanian WWTPs are insufficient to remove the target analytes completely, leading to environmental risks associated with the occurrence of pharmaceutical compounds in effluents and freshwater.

Occurrence and distribution of azole antifungal agents in eight urban Romanian waste water treatment plants.

Azole compounds are utilized to combat fungal infections in plants to protect them and also used for treating mycosis in humans. The LC-MS/MS method is a technique that combines liquid chromatography with tandem mass spectrometry for analysis of twelve azole compounds from wastewater (influent, effluent) and sewage sludge. The compounds were isolated from waste water using automatic extraction in the solid phase. Sludge samples were dried by lyophilization, after which they were subjected to ultrasound extraction with methanol. The quantification limits ranged from 0.3 ng/L (clotrimazole-CLO and prochloraz-PRO) to 1.5 ng/L (tetraconazole-TEB and penconazole-PEN), for wastewater samples and for sewage sludge, the LOQs ranged from 0.1 ng/g to 0.6 ng/g. High concentrations of climbazole-CLI (207-391 ng/L), tebuconazole (92-424 ng/L), and clotrimazole (6.9-93-ng/L) were observed in influent samples of the 8 urban wastewater treatment plants, followed by fluconazole (49.3-76.8 ng/L), and prochloraz (7.3-72 ng/L). The ∑Azoles had a maximum of 676 ng/L in the Galati effluent, followed by the Bucharest station 357 ng/L, and 345 ng/L in the Braila effluent. The highest value of the daily mass loading (input) level was observed for climbazole, 265 mg/day/1000 in Iasi station, followed by tebuconazole, 238 mg/day/1000 people in the Bucharest station, and 203 mg/day/1000 people for climbazole in the Targoviste station. The daily mass emission presented values between 0.7 and 247 mg/day/1000 people. The highest emissions were observed for climbazole, 247 mg/day/1000 people in Braila station; 174 mg/day/1000 people in the Iasi station and 129 mg/day/1000 people in the Bucharest station. The concentrations of climbazole detected in the effluent can present a high risk for the plants Lemna minor and Navicula pelliculosa. Clotrimazole may present a high risk to the plant Desmodesmus subspicatus and to the invertebrate Daphnia magna. PRO may present high risk to the invertebrate Mysidopsis Bahia.

Electrochemical System for Field Control of Hg2+ Concentration in Wastewater Samples.

The paper presents the validation of an electrochemical procedure for on-site Hg2+ ions determination in wastewater samples using a modified carbon screen-printed electrode (SPE) with a complexing polymeric film based on poly(2,2'-(ethane-1,2-diylbis((2-(azulen-2-ylamino)-2-oxoethyl)azanediyl))diacetic acid) (polyL). Using metal ions accumulation in an open circuit followed by anodic stripping voltammetry, the SPE-polyL electrode presents a linear range in the range of 20 µg/L to 150 µg/L, with a limit of detection (LOD) = 6 µg/L, limit of quantification (LOQ) = 20 µg/L, and an average measurement uncertainty of 26% of mercury ions. The results obtained in situ and in the laboratory using the SPE-polyL modified electrode were compared with those obtained by the atomic absorption spectrometry coupled with the cold vapor generation standardized method, with the average values indicating excellent recovery yields.

Bioavailability, Accumulation and Distribution of Toxic Metals (As, Cd, Ni and Pb) and Their Impact on Sinapis alba Plant Nutrient Metabolism.

This study presents the behavior of white mustard seedlings Sinapis alba grown for three months in laboratory polluted soil containing As, Cd, Ni and Pb. Four different experiments were performed in which As was combined with the other three toxic metals in different combinations (As, AsCd, AsCdNi, AsCdNiPb), keeping the same concentrations of As and Cd in all tests and following the national soil quality regulations. The effects of these metals were monitored by the analytical control of metal concentrations in soil and plants, bioavailability tests of mobile metal fractions using three different extracting solutions (DTPA + TEA + CaCl2-DTPA, DTPA + CaCl2-CAT, and CH3COONH4 + EDTA-EDTA) and calculation of bioaccumulation and translocation factors. Additionally, micro, and macro-nutrients both in soil and plant (root, stem, leaves, flowers and seeds) were analyzed in order to evaluate the impact of toxic metals on plant nutrient metabolism. Metals were significantly and differently accumulated in the plant tissues, especially under AsCdNi and AsCdNiPb treatments. Significant differences (p < 0.05) in the concentration of both As and Cd were highlighted. Translocation could be influenced by the presence of other toxic metals, such as Cd, but also of essential metals, through the competition and antagonism processes existing in plant tissues. Significantly, more Cd and Ni levels were detected in leaves and flowers. Cd was also detected in seeds above the WHO limit, but the results are not statistically significant (p > 0.05). The extraction of metallic nutrients (Zn, Cu, Mn, Ni, Mg, K, Fe, Ca, Cr) in the plant was not influenced by the presence of toxic metal combinations, on the contrary, their translocation was more efficient in the aerial parts of the plants. No phytotoxic effects were recorded during the exposure period. The most efficient methods of metal extraction from soil were for As-CAT; Cd-all methods; Pb and Ni-DTPA. The Pearson correlations (r) between applied extraction methods and metal detection in plants showed positive correlations for all toxic metals as follows: As-CAT > DTPA > EDTA, Cd-DTPA > CAT > EDTA, Ni-EDTA = DTPA > CAT, Pb-EDTA = DTPA = CAT). The results revealed that Sinapis alba has a good ability to accumulate the most bioavailable metals Cd and Ni, to stabilize As at the root level and to block Pb in soil.

Toxic Metals (As, Cd, Ni, Pb) Impact in the Most Common Medicinal Plant (Mentha piperita).

This study aimed to evaluate the behavior of Mentha piperita under Cd, Pb, Ni, and As soil contamination and their transfer from soil in plants as well as translocation in the roots/stems/leaves system compared with a control without metal addition. The mint seedlings were exposed for a three-month period using two metal mixtures in the same concentrations such as AsCd and AsCdNiPb (23.7 mg/kg As, 5 mg/kg Cd, 136 mg/kg Ni, and 95 mg/kg Pb). The results of metal concentration in plants showed that Cd, Ni, and Pb were accumulated in different parts of the plant, except for As. In plants organs, the order of metal accumulation was roots > stems > leaves. No significant impact on the growth, development, and chlorophyll content compared to the control was observed in the first month of exposure. After three months of exposure, phytotoxic effects occurred. Generally, the transfer coefficients and translocation factors values were less than 1, indicating that Mentha piperita immobilized the metals in root. The laboratory experiments highlighted that for a short period of time, Mentha piperita has the capacity to stabilize the metals at the root level and was a metal-tolerant plant when using a garden rich-substrate.

Surgical Treatment of an Aneurysmal Bone Cyst with Avascular Bone Graft.

Background: Aneurysmal bone cyst is a solitary bone tumor, expansile and lytic most often seen in the second decade of life, more frequently in men than in women (2: 1). They can occur in any bone, most common in the metaphysis of the long bones of the lower limbs. Although it is a benign tumor formation, aneurysmal cysts may have an aggressive local evolution and can cause a significant decrease in bone strength. The pacient may present local pain, the appearance of local deformation due to a tumor mass or occurrence of pathological fractures. Traditionally these lesions were treated surgically (curettage or resection and bone grafting) with a relapse rate of about 20%. Because bone resection may lead to bone defects, deformations or damage in the affected limb's function, lately the preferred treatement percutaneous sclerotherapy using fibrosing alcoholic agents. CASE REPORT: We present the case of a 14 year old pacient submitted for pain and deformity at the distal third of the right forearm with insidious onset and exacerbated lately. Following clinical investigations, laboratory and histopathology he was diagnosed with aneurysmal bone cyst of the right ulna. Since sclerotherapy is not available in our clinic, we initially performed an excisional biopsy with curettage of the lesion. Because the tumor still had an aggressive postoperative evolution, we decided for a bone resection and reconstruction using an avascular peroneal graft. Postoperative, the patient presents a favorable short and medium term evolution, the disappearance of pain and resumed function of the affected segment. Radiologically bone graft integration can be observed, with no evidence of local recurrence. CONCLUSION: Although modern tehniques for treating anurysmal bone cyst include either injecting fibrosing alcoholic agents or resection and grafting using vascular bone graft, the traditional tehnique described by Merle d'Aubigne which implies the usage of avascular bone graft is still heplful, leading to succesful results especially in the upper limbs.

Extracellular trypsin increases ASIC1a selectivity for monovalent versus divalent cations.

Sustained proton activation of native ASIC channels in primary sensory neurons or HEK293 cells leads to a reduction in the peak amplitude of transient inward currents and the progressive development of a persistent component, which hinders titration experiments in pharmacological studies. Here we report that extracellular trypsin applied for 5 min at 10-45 microg/ml and/or a short exposure to high Ca2+ (75 mM for less than 1 min) alleviate the persistent component, improving reproducibility of acid-elicited transients. Selectivity measurements performed in current clamp mode, in essentially bi-ionic conditions, prove that these two treatments decrease hASIC1a permeability for divalent but not for monovalent cations, producing a significant change in P(Na)/P(Ca) from 8.2+/-2.1 (mean+/-S.D.) to 26.0+/-7.8 (trypsin) or 24.5+/-11.1 (high Ca2+). The slope conductance of the unit inward Ca2+ transient was also lowered from 5.7 to 2.7 pS after trypsin.

Comparative study of different methods for detection of toxic and other enzymatic factors in Vibrio cholerae strains.

The purpose of this work was to characterize the toxin profile and the presence of other virulence factors involved in the pathogenesis and biology of 13 V. cholerae O1 (11 clinical cases and 2 waters) and 6 V. cholerae non O1 strains (4 clinical cases and 2 waters) using genetic (PCR), immunological (RPLA), biochemical (NAD degradation, haemolysis, Kanagawa phenomenon, caseinase, lecithinase, mucinase, amylase, esculine hydrolysis) and cell culture (Vero E6, HEp-2) assays. The results indicated a concordance between PCR-RPLA (84%), PCR-NAD (73%) and RPLA-NAD (84%) methods. The sensitivity of RPLA and NAD degradation methods were comparable to PCR in detecting CT in Vibrio cholerae O1 strains. Although NAD degradation method was not exclusively specific for the CT detection, it proved its usefulness in screening certain virulent, CT-negative clones of V. cholerae. The cytotoxic effect on Vero E6 cells, enzyme production (Kanagawa haemolysins, lecithinase, caseinase, esculine hydrolysis) as well as adherence ability on inert substrate proved to be much more constant in V. cholerae non O1 (CT- negative) than in V. cholerae O1 (CT-positive). All V. cholerae non O1 strains isolated in diarrheal cases were Kanagawa positive. This complex of virulence factors detected in V. cholerae non O1 strains could probably contribute during interepidemic periods to human-to-human transmission and to greater resistance as compared to O1 strains in the environment.

[Study of antibiotic influence on adherence capacity of gram positive and gram negative bacteria to the cellular substrate]. / Studiul influentei antibioticelor asupra capacitatii de aderenta a bacteriilor gram-pozitive si gram-negative la substratul celular.

Bacterial adherence to the cellular substrate (skin and mucosa) represents a precondition of infectious pathology. It was demonstrated that bacteria which adhere and form biofilms on catheters and other inert materials used in medicine are resistant to the therapeutic antibiotic concentrations being protected by the biofilm mathrix and generating severe and hard to treat infections. There are only few studies on the influence of antibiotics on the bacterial adhesins synthesis and bacterial adherence to the cellular substrate. The purpose of this study was to investigate the influence of subinhibitory concentrations of antibiotics on adherence capacity of Listeria monocytogenes, Vibrio cholerae and Aeromonas hydrophyla to the cellular substrate represented by HEp-2 cells. Suspensions (approximately 10(10) cells/ml) of bacterial cultures developed on solid media were incubated for 30 minutes in the presence of subinhibitory concentrations of penicillin, ampicillin, amoxicilin with clavulanic acid, ceftazidim, norfloxacin, kanamycin, chloramphenicole and vancomycin. Study of bacterial adherence to the cellular substrate was done by Cravioto's modified method. The quantitative evaluation of adherence/invasion capacity of bacterial suspensions pretreated with antibiotics was done by comparing the adherence/invasion index with controls without antibiotics. Penicillin, amoxicillin with clavulanic acid and vancomycin have significantly stimulated the adherence of Listeria monocytogenes strain and inhibited the adherence of Vibrio cholerae and Aeromonas hydrophyla strains. Ampicillin and chloramphenicole exhibited no significant effect on bacterial adherence capacity. The influence of kanamycin, ceftazidim and norfloxacin could not be interpreted due to the occurrence of a severe cytotoxic effect manifested by cell monolayer detaching, probably due to the action of antibiotic suspensions or to the increase of bacterial virulence under the selective pressure of the antibiotic.

[Factors influencing the capacity of cellular substrate adherence of vibrio cholerae O1 and non O1 strains]. / Studiul factorilor care influenteaza capacitatea de aderenta la substratul celular a tulpinilor de Vibrio cholerae O1 si non O1.

Bacterial adherence to eukariotic cells represents an important step of tissue colonization and is mediated by specific molecules called adhesins. Bacterial adherence to cellular substrate is a very complex process consisting in specific interactions between the surface of host cell and bacterial cell surface respectively. Adherence to cellular substrate confers selective advantages to bacterial cells, as: rapid growth rate by shorter lag period and protection against antibodies and lysozime. Adherence and colonization of small bowel represent the early steps of cholera infection (1, 2). The purposes of this study were to characterize the adherence ability of 46 Vibrio cholerae O1 and non O1 strains with different sources of isolation (acute diarrhea, water sources) to HEp-2 cell; to determine the influence of different factors (culture media, bacterial culture growth phase, proteolytic enzymes, carbohydrates and polyvalent agglutinant anti V. cholerae O1 serum) on the bacterial adherence capacity. Adherence capacity was assayed using the qualitative Cravioto's method. The adherence ability was appreciated by semi quantitative ("+", "++" and "+++") and quantitative assays. The adherence pattern of the tested strains was predominantly a diffuse one. The agar medium proved to be the most appropriate for the early and maximal expression of adhesion molecules, by comparison with nutritive broth and alkaline peptone water. Manose in different concentrations (1% and 3%) inhibited the adherence ability, demonstrating the role of manose-sensitive haemagglutinating fimbriae (MSHA) in mediating the adherence of V. cholerae strains to cellular substrate. Trypsine has no notable effect on the adherence ability, suggesting that the major V. cholerae adhesion molecules are not essentially of protein nature, so that the afimbrial adhesins could also play an important role in bacterial adhesion to eukariotic cells. The agglutinant polyvalent anti-V. cholerae O1 serum had the most significant inhibitory effect on the adherence ability, which was completely abolished in the presence of sub-agglutinant dilutions of serum titer (1/60-1/120) and partially reduced at titers ranging from 1/240 to 1/920. This inhibitory effect could be explained by bacterial agglutination, but also by the specific blocking of some surface structure implicated in the adherence process (i.e. lipopolysaccharides, as demonstrated by the inhibitory effect of sub-agglutinant serum titers). The inhibitory effect of polyvalent anti-V. cholerae O1 serum was limited to O1, but was not evident for the non O1 serogroups, demonstrating that the serum antibodies are acting on serogroup specific antigenic fractions.

[ Study of exo-enzymatic profile and cytotoxic effect of bacterial culture supernatants in different growth phase]. / Studiul profilului exo-enzimatic si al citotoxicitatii supernatantelor de culturi bacteriene în diferite faze de crestere.

Bacterial quorum-sensing represents an ubiquitary regulating system in which the pheromones (small molecules with different chemical structures, i.e. homoserin-lactones, octapeptides, aminoacids) act as extracellular mediators of signaling and intercellular communication. This chemical system is implicated in the regulation of different physiological processes dependent on the cell density (i.e. biolumniscence, virulence factors expression, sporulation, conjugation, antibiotic secretion etc). It is also mentioned in the literature the implication of bacterial pheromones in the modulation of eukariotic cells division rate. The objectives of this study were: a) to determine the exo-enzymatic profile of bacterial cultures in different growth phase in order to establish potential relationships between the phenotypic expression of some virulence factors on one side and the growth phase and bacterial culture density, on the other side; b) to determine de cytotoxic effect and the influence of bacterial culture supernatants on the HEp-2 cell division rate. Supernatants of bacterial cultures in nutrient broth of 2, 5 and 24 hrs of Staphylococcus aureus and Proteus sp. were tested directly and also, after thermic inactivation (at 100 degrees C, for 5 minutes) for the presence of different enzymatic activities known as virulence factors (spot and Kanagawa haemolysins, CAMP-like factor, caseinase, amilase, lipase, lecithinase, mucinase, DNA-ase). The exo-enzymatic profile of bacterial cultures of 2 and 5 hrs proved to be similar, the tested supernatants exhibiting haemolytic activity, and for Staphylococcus aureus, amilase and caseinase activities. Supernatants of and 5 hrs bacterial cultures exhibited also cytotoxic effect on HEp-2 cells. Supernatants of bacterial cultures of 24 hrs exhibited neither enzymatic activities, nor cytotoxic effect on HEp-2 cells, probably due to the inhibition of phenotypic expression of enzymatic activities at high bacterial densities through the activation of the quorum-sensing system. Bacterial supernatants did not significantly influence the HEp-2 cells division rate.