Biomonitoring of polycyclic aromatic hydrocarbons (PAH) and heavy metals Ni, V in sediments and anodonts of the Anzali Lagoon and applicability of netural red retention assay (NRR) as biomarkers of these pollutants

Biochemical ecotoxicology and biomarkers using are a new sciences that are used for biomonitoring in aquatic environment. Biomonitoring plays a vital role in strategies to identify, assess, and control contaminants. On the other hands in recent year's attention to polycyclic Aromatic Hydrocarbons (PAHs) and heavy metals increased in aquatic environments because of their carcinogenic and mutagenic properties combined with their nearly ubiquitous distribution in depositional environments by oil pollution or industrial waste waters. The present research aimed to assess PAHs and Ni, V levels in surface sediments and bivalves (Anodonta cygnea)and the effects of PAHs and heavy metals (Ni,V) on the hemocyte of the Anodonta cygnea were investigated in 2 stations (Mahrozeh, Selke in Anzali Lagoon, North of Iran). Samples were collected during at 2 different periods of the year, Dry and rain seasons, (June & September) and to confirm our first observations, Cage station is added. The bivalves hemocytes were monitored for membrane injury by NRR methods (neutral red retention assay). Heavy metal (Ni, V) concentrations were determined by Atomic Absorption in Anodonta cygnea and the sediments in Anzali Lagoon. The vanadium concentration in bivalves and sediments was ND(not detect )-0.4231 μg/g and 1.4381-306.9603 μg/g dry weight respectively. Nickel concentration in bivalves and sediments was 0.0231-1.3351, 0.4024-19.3561 μg/g dry weight respectively. PAHs concentrations were determined by GC-Mass in Anodonta cygnea and the sediments. Average concentration of PAHs is 115-373.788 ng/g dry weight in bivalves and average concentration of PAHs is 34.85-1339.839 ng/g dry weight in sediments. Bioaccumulation sediments factor(BASF) is high about PAHs (>1) and BASF is low for Ni, V (<1) . Internal Damage mechanisms of bivalves hemocytes (cell mortality, dye leakage, decreased membrane stability, are observed (Lowe Methods). Statistical analysis was used to explore the relationship between altered cellular and above contaminants. There are power and negative correlations between PAHs and NRR method for hemocytes in Anodonta cygnea (P<0.0005), but good correlation is not observed between Ni, V and NRR method for hemocytes in every time. This research indicates that the NRR assay is a useful screening technique able to discriminate polluted sites and at first we announce that Anodonta cygnea hemocytes are efficient biomarker for PAHs pollutants in fresh water.

The lysosome and lysozyme response in Chinese shrimp Fenneropenaeus chinensis to Vibrio anguillarum and laminarin stimulation

The effect of laminarin and Vibrio anguillarum on neutral red retention (NRR) time of lysosome and lysozyme activity in blood cells was investigated in Chinese shrimp, Fenneropenaeus chinensis. In addition the variation, of total haemocyte counts (THC) and differential haemocyte counts (DHC) were dtermined simultaneously The. results showed that the lysosome membrane stability was significantly elongated with the longest NRR time for 180 min at 3 h after lamianrin injection, (p<0.05). The lysozyme activity of haemocytes showed a moderate increase simultaneously. THC also increased and with a highest percentage of semi-granular cell counts at 3 h after laminarin injection. However, the NRR time sharply decreased with the shortest NRR time for 13 min at 1.5 h after V. anguillarum injection. Compared to the control group, the lysozyme activity obviously increased after injection, which was demonstrated at 1.5 h though the THC decreased. The percentage of hyaline cells increased obviously after V. anguillarum injection (p<0.05). The results suggested that the shrimp immune status could be evaluated by using lysosome membrane stability and lysozyme activity. (C) 2008 Elsevier B.V. All rights reserved.

Physiological and cellular responses in two populations of the mussel Perna perna collected at different sites from the Coast of São Paulo, Brazil

The physiological conditions of mussels from Ubatuba and Santos and also of organisms transplanted from Ubatuba to Santos were studied by using different techniques. Assays for lysosomal stability were conducted on the haemolymph. Heart rate activity was monitored for 6h. The embryonic development of larvae obtained from the collected mussels was analysed. For all the compared groups of mussels, no significant differences were observed for the cardiac activity monitoring and the embryonic bioassays. The mean Neutral Red (NR) retention time was similar for the animals from Santos and Ubatuba, whereas the organisms transplanted to Santos showed a reduction in the retention time of the dye, indicating damage in the lysosomal membranes. These differences were possibly due to environmental factors, but further investigations are required to confirm this hypothesis.

Pharmaceutical residues in aquatic systems: mode of action and effects on mussel physiology

Pharmaceutical residues contaminate aquatic ecosystems as a result of their widespread human and veterinary usage. Since continuously released and not efficiently removed, certain pharmaceuticals exhibit pseudo-persistence thus generating concerns for the health of aquatic wildlife. This work aimed at assessing on mussels Mytilus galloprovincialis, under laboratory conditions, the effects of three pharmaceuticals, carbamazepine (antiepileptic), propranolol (β-blocker) and oxytetracycline (antibiotic), to evaluate if the human-based mode of action of these molecules is conserved in invertebrates. Furthermore, in the framework of the European MEECE Programme, mussels were exposed to oxytetracycline and copper at increasing temperatures, simulating variations due to climate changes. The effects of these compounds were assessed evaluating a battery of biomarkers, the expression of HSP70 proteins and changes in cAMP-related parameters. A decrease in lysosomal membrane stability, induction of oxidative stress, alterations of cAMP-dependent pathway and the induction of defense mechanisms were observed indicating the development of a stress syndrome, and a worsening in mussels health status. Data obtained in MEECE Programme confirmed that the toxicity of substances can be enhanced following changes in temperature. The alterations observed were obtained after exposure to pharmaceuticals at concentrations sometimes lower than those detected in the aquatic environment. Hence, further research is advisable regarding subtle effects of pharmaceuticals on non-target organisms. Furthermore, results obtained during a research stay in the laboratories of Cádiz University (Spain) are presented. The project aimed at measuring possible effects of polluted sediments in Algeciras Bay (Spain) and in Cádiz Bay, by assessing different physiological parameters in caged crabs Carcinus maenas and clams Ruditapes decussatus exposed in situ for 28 days. The neutral red retention assay was adapted to these species and proved to be a sensitive screening tool for the assessment of sediment quality.

Valutazione del rischio ecologico dei residui di farmaci nel suolo: analisi sperimentale della tossicità della carbamazepina sul lombrico Eisenia andrei

La carbamazepina fu commercializzata a partire dagli anni Sessanta; è un analgesico anticonvulsivante e specifico per la nevralgia del trigemino ed è uno dei principali farmaci usati nel trattamento dell’epilessia. La sua azione più nota a livello del sistema nervoso è quella di rallentare il recupero dei canali al sodio, sebbene abbia anche effetti metabolici importanti interferendo con il ciclo degli inositoli e con la GSK-3 (glicogeno sintasi-chinasi 3). Tale sostanza è sotto la lente d’ingrandimento sia per le sue caratteristiche chimico-fisiche (vedi la sua alta persistenza in ambiente) sia per la sua alta tossicità per la salute umana. Le sue proprietà terapeutiche spesso sono accompagnate da effetti collaterali sia nei pazienti che assumono direttamente il medicinale, sia negli organismi non-bersaglio che vengono a contatto con i residui ed i metaboliti del farmaco in ambiente. Le principali fonti di contaminazione dell’ambiente sono rappresentate dagli scarichi domestici, urbani, ospedalieri ed industriali e dagli effluenti di impianti di depurazione. Inoltre, l’uso irriguo di acque contenenti residui del farmaco oppure fenomeni di esondazione di corpi idrici contaminati contribuiscono ampiamente alla distribuzione di questo composto nei suoli. La matrice suolo ha avuto relativamente poca attenzione per quanto riguarda gli effetti dell’inquinamento sugli organismi in generale, ed in particolare non vi sono studi sui farmaci. Il presupposto di questo studio dunque è stato quello di mettere a punto una metodologia volta a valutare gli effetti all’esposizione del farmaco carbamazepina su organismi bioindicatori, i lombrichi della specie Eisenia andrei. Il seguente progetto è durato da Maggio 2012 a Febbraio 2013, periodo in cui sono stati effettuati saggi sub cronici per valutare l’effetto di suoli sperimentalmente contaminati con il farmaco sui parametri del ciclo vitale del lombrico (accrescimento, mortalità e riproduzione) e su una serie di biomarker cellulari (neutral red retention assay, accumulo lisosomiale di lipofuscine, accumulo lisosomiale di lipidi neutri insaturi, attività dell’enzima acetilcolinsterasi, attività dell’enzima catalasi, attività dell’ enzima glutatione-S-transferasi e concentrazione di malondialdeide). I risultati ottenuti mostrano che la carbamazepina non ha effetti sui parametri del ciclo vitale. Per quanto riguarda i parametri fisiologici si notano tuttavia dei risultati diversi. L’accumulo lisosomiale di lipofuscine e lipidi neutri indica che il metabolismo dei vermi risulta in qualche modo alterato dall’esposizione alla carbamazepina alle concentrazioni saggiate. Queste alterazioni potrebbero essere spiegate da un effetto di tipo ossidante; infatti i due biomarker oltre a rappresentare un segnale di alterazione metabolica rappresentano anche un indicazione di perossidazione lipidica. Queste osservazioni meritano di essere approfondite studiando il bioaccumulo e la degradazione della carbamazepina nei suoli, che potrebbero essere alla base della diversità di risultati rispetto alla tossicità evidenziata negli organismi acquatici. A fronte della consapevolezza dei rischi potenziali dovuti alla presenza di farmaci nelle acque e nel suolo, molto resta da fare per ampliare le conoscenze su questa tipologia di contaminazione, in particolare nei campi del monitoraggio e del comportamento ambientale, degli studi ecotossicologici e delle procedure e tecnologie idonee a limitare la loro immissione nell’ambiente.

Neutral red uptake (NRU) entseguaren optimizazioa lur-zizareen sistema immuneko zelulen kultibo primarioetan

Azken urteotan eman diren kutsatzaile desberdinen gehiegizko isurpen edo erabilpena dela eta, lurzoruetan metal astunak metatu dira eta horrek , kate trofikoaren oinarrian dauden organismoen gainean k altea k sorrarazi d itu. Eisenia fetida zizarea espezie zent inela gisa erabili da lur entsegu ekotoxikologi koet an . Esperimentu honetan Eisenia fetida - ren jariakin zelomikoan dauden eta babes funtzioa duten zelomozitoak pH desberdinetara edo Kadmio bezalako metalera esposatzeak Neutral Red (NR) edo gorri neutro tindatzaile kationikoaren harreran eta fagoz itosi ahalmenean , eragina duen aztertu nahi izan da. Esperimentua in vitro egin zen 3R filosofia jarraituz. Alde batetik, pH desberdinen eragin ari dagokio nez gorri neutroaren harrera ( NRU ) entsegua n erantzun bimodal bat behatu zen eta beste alde bate tik, f agozitatzeko ahalmenari dagokiola zelomozitoak Eagle medioan kultibatzean ez zen aldaketa esangarria ikusi kadmioa ren, kontr olaren eta kontrol positi boaren artean. Bai ordea L - 1 5 medioarekin, non kontrol posit iboa k beste biak baino absorbantzia handiagoa erakutsi zuen. Emaitza hauek erakusten dute zelomozitoe n mintza gradualki kaltetzen doala pH neutrotik aldentzen doan heinean eta puntu batetik aurrera NRU emendatzen da zelomozito batzuen (amebozitoen) fagozitosia aktibatzen d el a ko .

Investigations on the adsorption behavior of Neutral Red on mercaptoethane sulfonate protected gold nanoparticles

A detailed investigation on the adsorption behavior of Neutral Red (NR) molecules on mercaptoethane sulfonate-monolayer protected gold clusters (MES-MPCs) has been conducted by the spectroscopic method. It is found that cationic NR molecules are adsorbed on the negatively charged MPCs surfaces via electrostatic attractive forces. The absorption study shows that the optical properties of NR molecules are significantly influenced upon the adsorption. Based on the electrostatic adsorption nature and the excellent stability of MES-MPCs against the electrolytes, this association can be released by the addition of electrolyte salts, which can be monitored by both absorption and fluorescence spectroscopy. In addition, dication Ca2+ is found to be more effective in the release of NR than univalent Na+. Moreover, the MES-MPCs exert energy transfer quenching of NR fluorescence by both static and dynamic quenching. However, static quenching seems to be the dominating quenching mechanism. Furthermore, this energy transfer quenching exhibits strong dependence of Au core size, and 5.0 nm MPCs show stronger ability in quenching the NR fluorescence than that of 2.7 nm MPCs.

In vitro study on the binding of neutral red to bovine serum albumin by molecular spectroscopy

In this paper, the binding of neutral red (NR) to bovine serum albumin (BSA) under physiological conditions has been studied by spectroscopy method including fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The Stern-Volmer fluorescence quenching constant (K-SV), binding constant (K-b) and the number of binding sites (It) were measured by fluorescence quenching method. Fluorescence experiments were also performed at different ionic strengths. It was found K-SV was ionic strength dependent, which indicated the electrostatic interactions were part of the binding forces. The distance r between donor (BSA) and acceptor (NR) was obtained according to Foster's non-radiative energy transfer theory. CD spectroscopy and FT-IR spectroscopy were used to investigate the structural information of BSA molecules on the binding of NR, and the results showed no change of BSA conformation in our experimental conditions.

A temperature-dependent interaction of neutral red with calf thymus DNA

Neutral red (NR) is used as a probe to study the temperature and concentration dependent interaction of a cationic dye with nucleic acid. A temperature-dependent interaction of NR with calf thymus DNA (CT DNA) has been studied by differential pulse voltammetry (DPV), UV-Visible absorption, circular dichroism (CD) and fluorescence spectroscopy. The experimental results of increasing peak current, changes in the UV-Visible absorption and fluorescence spectra of NR and decreasing the induced circular dichroism (ICD) intensity show that (i) the binding mode of NR molecules is changed from intercalating into DNA base pairs to aggregating along the DNA double helix and (ii) the orientation of NR chromophore in DNA double helix is also changed with the temperature.

Lysossomal membrane stability of the brown mussel Perna perna (Linnaeus) (Mollusca, Bivalvia) exposed to the anionic surfactant Linear Alkylbenzene Sulphonate (LAS).

The effects of the Linear Alkylbenzene Sulphonate (LAS) were evaluated on the mussel Perna perna (Linnaeus, 1758), using a cellular level biomarker. The Neutral Red Retention Time (NRRT) assay was used to estimate effects at cellular levels. Significant effects were observed for the NRRT assay, even in low concentrations. The effects at cellular level were progressive, suggesting that the organisms are not capable to recover of such increasing effects. Additionally, the results show that the levels of LAS observed for Brazilian coastal waters may chronically affect the biota.

Integrated assessment of multilevel biomarker responses and chemical analysis in Mussels from Sao Sebastiao, São Paulo, Brazil

The aim of the present study was to investigate the presence of contaminants in the mussel Perna perna from Sao Sebastiao Channel, São Paulo, Brazil, and to evaluate the effects of these contaminants on these organisms at biochemical (catalase [CAT], glutathione-S-transferase [GST], and cholinesterase [ChE]), cellular (neutral red retention time [NRRT] assay), and physiological (cardiac monitoring) levels. Two sampling surveys were performed (winter of 2001 and summer of 2002) at six stations along the channel: Cigarras, station 1; late Clube de Ilhabela, station 2; Oil Terminal, station 3; Toque Toque, station 4; Ponta da Sela, station 5 (reference station); and Taubate, station 6. Differences in CAT activity were observed between mussels from stations 3 and 5 during the winter, but no differences were detected in the summer. No differences in GST activity were found among stations during the winter, although animals from station 3 showed higher activity during the summer. The ChE activity was significantly higher in the mussels from stations I and 2 during the winter and from stations I and 3 during the summer. Organisms from stations I through 4 showed statistically lower NRRT in both seasons. Similar heart rates were observed in the mussels from all stations. Hydrocarbons were detected in organisms from all the stations in both seasons. During the winter, higher polycyclic aromatic hydrocarbon (PAH) levels were observed in organisms from station 3, whereas during the summer, higher levels of metals were found in organisms from stations 1, 3, and 4. The multivariate analyses showed a strong influence of PAHs on the winter biological results, but metals showed higher influence on these responses in the summer, indicating multiple contaminant sources.

Avaliação citotóxica de Amoxilina e Clavulanato de Potássio em mexilhões Perna perna

Compostos farmacêuticos são identificados em matrizes ambientais em ordens de grandeza de ng.L-1 a μg.L-1. Dentre os fármacos, os antibióticos têm recebido atenção especial devido aos problemas que podem causar à biota aquática. O objetivo do presente estudo foi avaliar a citotoxicidade de Amoxicilina e Clavulanato de Potássio isolados e em associação para mexilhões Perna perna utilizando o ensaio do tempo de retenção do corante vermelho neutro, que avalia a estabilidade da membrana lisossômica de hemócitos dos organismos teste. A Amoxicilina causou citotoxicidade aos mexilhões nas concentrações de: CEO: 1 ng.L-1, CI25-24h: 0,44 ng.L-1, CI25-48h: 1,19 ng.L-1 e CI25-72h: 0,85 ng.L-1, o Clavulanato de Potássio foi citotóxico nas concentrações de: 10 ng.L-1 em 24h e 50 ng.L-1 e 100 ng.L-1 em 48h e 72h. Os valores de concentração inibitória foram de CI25-24h: 3,11 ng.L-1, CI25-48h: 3,45 ng.L-1 e CI25-72h: 3,43 ng.L-1. No ensaio realizado com a associação dos fármacos todas as concentrações foram citotóxicas aos mexilhões em 48h e em 72h apenas 40 ng.L-1 de Amoxicilina + 10 ng.L-1 de Clavulanato de Potássio e 200 ng.L-1 de Amoxicilina + 50 ng.L-1 de Clavulanato de Potássio. As concentrações inibitórias foram: CI25-48h: 1,67 ng.L-1 e CI25-72h: 1,36 ng.L-1 a partir dos dados de Amoxicilina e CI25-48h: 0,42 ng.L-1 e CI25-72h: 0,34 ng.L-1 a partir dos dados de Clavulanato de Potássio.

Lysossomal membrane stability of the brown mussel Perna perna (Linnaeus) (Mollusca, Bivalvia) exposed to the anionic surfactant Linear Alkylbenzene Sulphonate (LAS).

The effects of the Linear Alkylbenzene Sulphonate (LAS) were evaluated on the mussel Perna perna (Linnaeus, 1758), using a cellular level biomarker. The Neutral Red Retention Time (NRRT) assay was used to estimate effects at cellular levels. Significant effects were observed for the NRRT assay, even in low concentrations. The effects at cellular level were progressive, suggesting that the organisms are not capable to recover of such increasing effects. Additionally, the results show that the levels of LAS observed for Brazilian coastal waters may chronically affect the biota.