Antimutagenic effect of neem leaves extract in freshwater fish, Channa punctatus evaluated by cytogenetic tests

Neem (Azadirachta indica), an indigenous plant commonly grown in India and its sub-continent is a multipurpose plant well known for its insecticidal and biomedical properties, however, its antimutagenic effects in vertebrate organisms are lacking. The present work is therefore, focused on possible antimutagenic potential of ethanolic extract of neem leaves evaluated on the clastogenicity induced by Pentachlorophenol (PCP) and 2, 4-dichlorophenoxyacetic acid (2,4-D) in freshwater fish, Channa punctatus used as a vertebrate model, by cytogenetic endpoints: chromosome aberration (CA) and micronucleus (MN) test. In the first set of experiment, fish were exposed by medium treatment to a single treatment of each chemical (PCP, 0.6 ppm; 2,4-D, 75 ppm; neem extract, 3 ppm) along with the controls. The chromosome preparations were made after processing kidney cells and micronucleus slides were prepared from peripheral blood at multiple duration (48, 72 and 96 h). PCP and 2,4-D when used alone, induced significant CA and MN in a time dependent manner. Neem extract did not show genotoxic potential in both assays. The maximum frequency of CA were recorded as 18.58% and 15.17%, while frequency of MN reached to 8.08% and 4.62% by PCP and 2,4-D respectively, after 96 h exposure. In the second set of experiment, three concentrations of neem extract (1, 2 and 3 ppm) were run simultaneously with the same concentration of PCP (0.6 ppm) and 2,4-D (75 ppm) for antimutagenicity estimates. In mixed treatment, neem extract significantly reduced the frequency of CA and MN. The reduction in the frequency of CA ranged from 40-75% and 45.4-83.3% and similar values for MN were 40.2-75.3% and 44.1-65.8% for PCP and 2,4-D respectively. Although the reductions were significant but not dependent on concentration and time intervals employed. Results suggested that under present experimental conditions, neem extract exhibit strong antimutagenic activity in this fish model, which could further contribute to study its benefit in humans.     

Response of antioxidant enzymes in freshwater fish populations (Leuciscus alburnoides complex) to inorganic pollutants exposure.

Evidence is accumulating indicating the importance of antioxidant enzyme activity measurements in eco-toxicological studies, as they may constitute markers for exposure to a large variety of pollutants. Variation of antioxidant enzymes, superoxide dismutase (SOD) and glutathione S-transferases (GST) and the effect of heavy metals and selenium exposure on these enzymes were investigated in the livers of Iberian endemic minnows (Leuciscus alburnoides complex) captured in a copper (Cu) mining area. Higher hepatic levels of copper and selenium were always observed in fish captured at the polluted site relative to the reference area population, reflecting the environmental monitoring results. A seasonal fluctuation in zinc and selenium levels for both populations was also observed which could be related to gonad maturation. The activity of SOD did not show significant regional alterations, but a seasonal variation occurred presumably associated with the Leuciscus life cycle. The GST activity was higher in the fish population from the polluted area (except in spring) and GST variability was associated with selenium and copper levels when both regions were compared. The increased GST activity was probably a metabolic adaptation to the continuous exposure to higher levels of those elements.     

Response of antioxidant enzymes in freshwater fish populations (Leuciscus alburnoides complex) to inorganic pollutants exposure

Evidence is accumulating indicating the importance of antioxidant enzyme activity measurements in eco-toxicological studies, as they may constitute markers for exposure to a large variety of pollutants. Variation of antioxidant enzymes, superoxide dismutase (SOD) and glutathione S-transferases (GST) and the effect of heavy metals and selenium exposure on these enzymes were investigated in the livers of Iberian endemic minnows (Leuciscus alburnoides complex) captured in a copper (Cu) mining area. Higher hepatic levels of copper and selenium were always observed in fish captured at the polluted site relative to the reference area population, reflecting the environmental monitoring results. A seasonal fluctuation in zinc and selenium levels for both populations was also observed which could be related to gonad maturation. The activity of SOD did not show significant regional alterations, but a seasonal variation occurred presumably associated with the Leuciscus life cycle. The GST activity was higher in the fish population from the polluted area (except in spring) and GST variability was associated with selenium and copper levels when both regions were compared. The increased GST activity was probably a metabolic adaptation to the continuous exposure to higher levels of those elements.     

(Tri)butyltin biotic degradation rates and pathways in different compartments of a freshwater model ecosystem

Experiments were conducted in controlled temperate freshwater ecosystems (microcosms) to determine the persistence and biogeochemical dynamic of tributyltin (TBT) and its degradation products. TBT and its derivatives were monitored simultaneously for 23 days (552 h) in sediment-water systems, with or without macroorganisms (macrophytes: Elodea canadensis and gastropods: Lymnaea stagnalis). Biphasic TBT removal from the water column was significantly enhanced by the presence of biota. The persistence of TBT in biota was assessed by a kinetic approach of the different bioaccumulation pathways and associated metabolisms adopted by the snails and the macrophytes in response to the TBT contamination. Furthermore, sediment acted for the final sink for butyltins in both types of microcosms, with more than 70% of TBT and its metabolites recovered in this compartment after two weeks of exposure. Degradation pathways in sediments of both biotic and abiotic microcosms appeared to represent a key process in TBT cycle and were characterized by half-lives in the range of one month. Specific transformation and transfer pathways of TBT as reactional mechanisms are discussed and modelled assessing in detail the role of each compartment with regards to the fate of TBT in the model aquatic ecosystems.     

Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil

Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats.     

Monitoring of polychlorinated biphenyls (PCBs) and hexachlorocyclohexanes (HCH) in freshwater using the aquatic moss Cinclidotus danubicus

Concentrations of PCBs and α-, β, and γ-HCH have been measured in the aquatic moss Cinclidotus danubicus to examine its potential use an indicator of chlorinated organic pollutants in freshwaters. Samples of Cinclidotus were collected from an uncontaminated stretch of the Saône R. (France) and transplanted in the Durance R., which receives effluents of an insecticide factory. Mosses were sampled 13, 24 and 51 days after the transplant and analysed by capillary column gas chromatography. Concentrations in Cinclidotus transplanted 4 km downstream from the factory reached 0.30 μg g^?1 for PCBs, 2.37 μg g^?1 for α-HCH, 1.29 μg g^?1 for β-HCH and 0.50 μg g^?1 for γ-HCH; mean accumulation factors were 616 for α-HCH, 493 for β-HCH, 294 for γ-HCH and 4867 for PCBs. The preponderence of α- and β-HCH over the other pollutants investigated was also observed in water samples, which shows that aquatic mosses can be used as indicators of chlorinated organic pollutants in freshwaters.     

Biomonitoring of the genotoxic potential (micronucleus assay) and detoxifying activity (EROD induction) in the River Dadou (France), using the amphibian Xenopus laevis

Within the framework of a general survey of the water quality of the river Dadou (Tarn, France), different physico-chemical parameters were measured and an inventory of the fish population was made along the water course, around the Rassisse dam. With the aim of monitoring the potential genotoxic effects and the detoxifying activities induced in organisms exposed to the river water, two in vivo bioassays were performed in laboratory experiments, using larvae of the amphibian Xenopus laevis. The first was the micronucleus test, using red blood cells, and the second the assay of ethoxyresorufin-O-deethylase (EROD) induction in the liver of exposed animals. Eight water samples were taken from the river and at outlet points from the two major industrial activities of the studied section of the water course: a spar-fluor mine and a water treatment plant. Genotoxic impact and EROD induction were measured in the larvae. The effluent of the filter-washing process from the water treatment plant was found to be particularly genotoxic, even after dilution in pure reconstituted water, but no particular genotoxicity was found, either in Dadou river water, or in the effluents from the mine. On the other hand, most of the water samples tested produced a clear induction of EROD activity compared to the level of enzymatic activity found in the liver of larvae reared in the river water sampled upstream of the industrial activities. These results were interpreted taking into account (i) the high concentrations of pollutants (fluorine and manganese) measured in the river water, (ii) the very low population levels inventoried in the downstream section of the river and (iii) the possible interactions between the substances present in the river water, particularly the classical EROD inducers PAHs and PCBs.     

Composition of bacterial and archaeal communities in freshwater sediments with different contamination levels (Lake Geneva, Switzerland)

The aim of this study was to compare the composition of bacterial and archaeal communities in contaminated sediments (Vidy Bay) with uncontaminated sediments (Ouchy area) of Lake Geneva using 16S rRNA clone libraries. Sediments of both sites were analysed for physicochemical characteristics including porewater composition, organic carbon, and heavy metals. Results show high concentrations of contaminants in sediments from Vidy. Particularly, high contents of fresh organic matter and nutrients led to intense mineralisation, which was dominated by sulphate-reduction and methanogenesis. The bacterial diversity in Vidy sediments was significantly different from the communities in the uncontaminated sediments. Phylogenetic analysis revealed a large proportion of Betaproteobacteria clones in Vidy sediments related to Dechloromonas sp., a group of dechlorinating and contaminant degrading bacteria. Deltaproteobacteria, including clones related to sulphate-reducing bacteria and Fe(III)-reducing bacteria (Geobacter sp.) were also more abundant in the contaminated sediments. The archaeal communities consisted essentially of methanogenic Euryarchaeota, mainly found in the contaminated sediments rich in organic matter. Multiple factor analysis revealed that the microbial community composition and the environmental variables were correlated at the two sites, which suggests that in addition to environmental parameters, pollution may be one of the factors affecting microbial community structure.     

Mechanisms of bioaccumulation of a herbicide (atrazine, s-triazine) in a freshwater mollusc (Ancylus fluviatilis müll.) and a fish (Coregonus fera jurine)

The herbicide atrazine was used to study mechanisms of bioaccumulation in a freshwater mollusc (Ancylus fluviatilis) and a fish (Coregonus fera). Measurements were performed with non-labelled and [¹⁴C]ring-labelled atrazine as well as tritiated water. The kinetics of accumulation indicate that atrazine is accumulated via gills, and that other organs are contaminated via blood.The exchange of atrazine was investigated at the equilibrium state of accumulation. The total residue in organisms is subject to an exchange process; the rates of atrazine accumulation and atrazine exchange are similar. Investigations of the water balance revealed that water exchange in organisms is the main mechanism of atrazine accumulation and exchange.     

红鳍东方鲀抗苗勒氏管激素(AMH)基因在不同发育时期的组织表达

为研究抗苗勒氏管激素(AMH)基因在红鳍东方鲀Takifugu rubripes各组织和不同发育时期的表达情况以及在性别分化和性腺发育等过程中的作用,利用荧光实时定量PCR(qRT-PCR)技术对红鳍东方鲀成鱼的肾脏、脑、心脏、脾脏、肝脏、精巢和卵巢等组织进行了amh基因的表达分析以及不同发育时期(23、30、40、60、80日龄及2龄和3龄鱼)红鳍东方鲀个体水平上amh基因的表达分析。结果表明:红鳍东方鲀3龄成鱼脾脏、肾脏和肝脏中的amh基因表达量极显著高于其他组织(P<0.01),在精巢和心脏中的表达量极显著高于卵巢和脑(P<0.01),其中脾脏中表达量最高,脑中表达量最低;红鳍东方鲀23日龄、30日龄、2龄和3龄鱼中,雄鱼amh基因的表达量极显著高于同期雌鱼(P<0.01),而60日龄和80日龄时雌鱼表达量显著高于雄鱼(P<0.05),40日龄时雌、雄鱼的表达量无显著性差异(P>0.05),3龄时雌、雄个体性腺中amh基因的表达量均显著高于2龄鱼(P<0.05);雄性幼鱼amh基因的表达量从23~30日龄呈增长趋势,且在30日龄时达到最高,随后逐渐降低,而雌鱼整体amh基因的表达量也呈现先升高后降低的趋势,80日龄时雌鱼amh基因表达量最高。研究表明,amh基因在红鳍东方鲀的性腺发育和性别分化过程中发挥着一定的作用。     

氟苯尼考对三疣梭子蟹不同组织中ABC转运蛋白基因(ABCG)表达量的影响

为研究氟苯尼考(FLR)对三疣梭子蟹Portunus trituberculatus不同组织中ABC转运蛋白基因(ABCG)表达量的影响,采用RACE法进行了三疣梭子蟹ABCG基因克隆并对其生物学进行了分析,采用荧光定量PCR法进行了不同浓度(20、40、80 mg/kg)氟苯尼考对三疣梭子蟹肝胰腺、鳃和肌肉中ABCG相对表达量的影响研究。结果表明:三疣梭子蟹ABCG基因全长c DNA序列为2473 bp,共编码578个氨基酸;三疣梭子蟹ABCG不含信号肽,具ABC转运家族蛋白的典型结构域,包括1个高度保守的核苷酸结构域(NBD)和1个疏水性跨膜区(TMD),为半转运子;同源性及系统进化分析表明,三疣梭子蟹ABCG与中国对虾、凡纳滨对虾的ABCG聚为一支,具有较高的亲缘关系;不同浓度的氟苯尼考对三疣梭子蟹肝胰腺、鳃、肌肉中ABCG表达均具有诱导作用,且呈时间剂量效应,由此推测,三疣梭子蟹ABCG蛋白参与氟苯尼考的代谢转运过程。本研究结果可为揭示甲壳动物ABCG转运蛋白对抗生素的转运机制提供参考依据。     

Investigation of mixing ratio and restraint condition effects on alkali–silica gel pressure in mortar by using a uniaxial alkali–silica gel pressure measuring device (ASGPM-D)

A pressure of alkali silica gel which is the product of alkali–silica reaction causes pattern crack development and ultimately failure in cement based materials. This paper examines the measurement of alkali–silica gel pressure in cylindrical mortar specimen by a new test device (alkali–silica gel pressure measuring device, ASGPM-D) and investigates also the effects of the test medium on test results. Specimens of various sizes and geometries were tested to develop standard specimen size for ASGPM test method. The most suitable specimen with diameter of 35 mm and 250 mm in length was selected by optimizing the experimental results. Furthermore, the highest alkali–silica pressure value was recorded on a specimen which has 0.45 w/c and 74–150 μm grain size in an 80 °C 1 N NaOH solution. Also, swelling pressures of four reactive aggregates (opal, chert, basalt and chalcedony) were determined under two distinct restraint stages (1.5 kN and 3 kN).     

Study on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chloro-phenoxyacetic sodium (MCPA sodium) in natural agriculture-soils of Fuzhou, China using capillary electrophoresis

A new method of analyzing trace 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2-methy-4-chloro-lphenoxyacetic sodium (MCPA sodium) in soils by capillary electrophoresis (CE) has been developed in this study. The optimum analytical conditions including chemical component and concentration of buffer solution, pH, separation voltage and sample injection time were studied in detail. Under the optimum conditions, 2,4-D and MCPA sodium in soils can be speedy separated and determined within 20 min with detection limits of 0.15 µg/g (2,4-D) and 0.25 µg/g (MCPA sodium) , a RSD (n = 6) < 5% and a recovery > 89%.With the help of analytical method developed in this study, the degradations of 2,4-D and MCPA sodium in natural agriculture-soils of Fuzhou were studied. The experimental results indicated that the degradations of 2,4-D and MCPA sodium follow first-order kinetics with degradation constants of 0.1509 day^-1 (2,4-D) and 0.2722 day^-1 (MCPA sodium) respectively. The degradation half-life were calculated to be 4.6 days (2,4-D) and 2.6 days (MCPA sodium) at 27 °C, implied that 2,4-D and MCPA sodium can be speedy degraded in natural agriculture-soils of Fuzhou, China.     

Zero Nuisance Piggeries: Long-term performance of MBR (membrane bioreactor) for dilute swine wastewater treatment using submerged membrane bioreactor in semi-industrial scale

Effective aerobic/anoxic treatment of piggery manure wastewater was achieved in a real farm scale using a small piggery (72 pigs) with reuse of the treated water. The experimental procedure was followed for 9 months. Fresh manure (FM) is formed by daily flush on piggeries and biologically treated after centrifuge pre-treatment. For upgrade liquid/solid separation and pathogen retention in biological treatment, a membrane system was used with the aim of effluent reuse in flush. Despite an evolution of FM through time, centrifuge pre-treatment and bioreactor performances stayed at high level. An elimination of 86% of the suspended solids occurred through pre-treatment, and nitrogen and COD biological degradation remains at 90% all time long. Moreover, interestingly about half of the soluble part of phosphorus (20% of the global phosphorus content) was biologically removed via the recirculation between the anoxic and the aerobic tank which acted as an intermittent aerobic/anoxic sequence. A part of COD was proved not biodegradable and was accumulated via the reuse of the treated water for flushing purpose. This accumulation justifies washing of the biomass between two runs in purpose to enhance the treated water quality and also to meet the membrane tolerance. The membrane was proved reliable as far as the maintenance procedure was respected. Maintenance cleaning had to be operated as soon as the TransMembrane Pressure (TMP) achieved 50 mbar and curative washing was necessary if the TMP increased over 90 mbar or between 2 runs. The temperature was proved to influence both the bioactivity and the membrane fouling kinetic. Finally, it was demonstrated that the process was sustainable for long-term management of swine wastewater at semi-industrial scale.     

Long-term continuous monitoring of the dissolved CO2 performed by using a new device in groundwater of the Mt. Etna (southern Italy)

We present a new device for continuous monitoring of the concentration of CO₂ dissolved in water. The device consists of a tube made of a polymeric semi-permeable membrane connected to an infrared gas analyser (IRGA) and a pump. Several laboratory experiments were performed to set the best operating condition and test the accuracy of measurements. We used the device for performing 20 months of continuous monitoring of dissolved CO₂ concentration (DCC) in groundwater within a drainage gallery at Mt. Etna. The monitored groundwater intercepts the Pernicana Fault, along which degassing is observed related to volcano-tectonic activity. The acquired data were compared with continuous and discrete data obtained using existing methods. The measurements of DCC resulted in some period of the year well correlated with air temperature. We also found that long-term trends, as well as short-term variations, are probably linked to the dynamics of volcanic activity and/or perturbations in the local or regional stress fields.     

The effect of anoxia and anaerobia on ciliate community in biological nutrient removal systems using laboratory-scale sequencing batch reactors (SBRs)

Little is known about the effect of anaerobic and anoxic stages on the protozoan community in the activated sludge process and how this subsequently affects performance. Using a laboratory-scale BNR system the effect of different periods of anoxia on both the protozoan community and performance efficiency have been examined. Four SBRs were operated at two cycles per day using a range of combined anoxic/anaerobic periods (0, 60, 120 and 200 min). Effluent quality (TOC, BOD, TP, TN, NH₄-N, NO₃-N and NO₂-N), sludge settleability and ciliate community (species diversity and abundance) were analysed over a periods of up to 24 days of operation. The species richness and total abundance of ciliates were found to decrease with longer anoxic/anaerobic periods. Both, positive and negative significant correlations between the abundance of certain species and the period of anoxia was observed (e.g. Opercularia microdiscum, Epicarchesium granulatum), although other species (i.e. Acineria uncinata, Epistylis sp.) were unaffected by exposure to anoxia. In the laboratory-scale units, the 60 min anoxic/anaerobic period resulted in good process performance (TOC and BOD removal of 97-98% respectively), nitrification (80-90%), denitrification (52%) but poor levels of biological P-removal (12%); with the protozoan community moderately affected but still diverse with high abundances. Increasing the length of anoxia to up to 200 min did not enhance denitrification although P-removal rates increased to between 22 and 33%; however, ciliate species richness and total abundance both decreased and sludge settleability became poorer. The study shows that activated sludge ciliate protozoa display a range of tolerances to anoxia that result in altered ciliate communities depending on the length of combined anoxic/anaerobic periods within the treatment process. It is recommended that anoxic/anaerobic periods should be optimised to sustain the protozoan community while achieving maximum performance and nutrient removal.     

The acute effect of bunker C oil and an oil dispersant on: 1 Serum glucose, serum sodium and gill morphology in both freshwater and seawater acclimated rainbow trout (Salmo gairdneri)

Bunker C oil and an oil dispersant were tested for physiological stress on both freshwater and saltwater acclimated rainbow trout. Both compounds tended to reduce serum glucose levels with Bunker C causing the more significant decrease (P < 0.08), indicating a possible dysfunction of the kidney. The freshwater treatment group showed a significant decrease in sodium levels (P < 0.01) when treated with a dispersant, while under similar conditions, saltwater acclimated fish show a very marked increase in serum sodium concentrations (P < 0.025). Those fluctuations in sodium levels are resultant from direct interference with the energy activated sodium transport systems of the gills. Microphotographs of gill filaments and lamellae show severe damage caused by the dispersant and dispersant/oil mixture with less impairment resultant from Bunker C exposure.     

A study of external mass transfer effect on biodegradation of phenol using low‐density polyethylene immobilized Bacillus flexus GS1 IIT (BHU) in a packed bed bioreactor

The impact of external mass transport on the biodegradation rate of phenol in a packed bed bioreactor (PBBR) was studied. A potential bacterial species, Bacillus flexus GS1 IIT (BHU), was isolated from the petroleum‐contaminated soil. Low‐density polyethylene (LDPE) immobilized with the B. flexus GS1 IIT (BHU) was used as packing material in the PBBR. The PBBR was operated by varying the inlet feed flow rate from 4 to 10 mL/min, and the corresponding degradation rate coefficients were found to be in the range of 0.119–0.157 L/g h. In addition, the highest removal rate of phenol was obtained to be 1.305 mg/g h at an inlet feed rate of 10 mL/min. The external mass transfer was studied using the model . A new empirical correlation for the biodegradation of phenol in the PBBR was developed after the evaluation at various values of K and n.