Characterizing the antibiotic resistance genes in a river catchment: Influence of anthropogenic activities

Previous studies on environmental antibiotics resistance genes(ARGs) have focused on the pollution sources such as wastewater treatment plants, aquaculture and livestock farms,etc. Few of them had addressed this issue in a regional scale such as river catchment. Hence,the occurrence and abundances of 23 ARGs were investigated in surface water samples collected from 38 sites which located from the river source to estuary of the Beijiang River.Among them, 11 ARGs were frequently detected in this region and 5 ARGs(sul I, sul II, tet B,tet C, and tet W) were selected for their distribution pattern analysis. The abundances of the selected ARGs were higher in the upstream(8.70 × 10~6 copies/ng DNA) and downstream areas(3.17 × 10~6 copies/ng DNA) than those in the midstream areas(1.23 × 10~6 copies/ng DNA), which was positively correlated to the population density and number of pollution sources. Pollution sources of ARGs along the Beijiang River not only had a great impact on the abundances and diversity, but also on the distribution of specific ARGs in the water samples. Both sul I and sul II were likely originated from aquaculture farms and animal farms,tet W gene was possibly associated with the mining/metal melting industry and the electric waste disposal and tet C gene was commonly found in the area with multiple pollution sources.However, the abundance of tet B was not particularly related to anthropogenic impacts. These findings highlight the influence of pollution sources and density of population on the distribution and dissemination of ARGs at a regional scale.     

Biological methane production coupled with sulfur oxidation in a microbial electrosynthesis system without organic substrates

Methane is produced in a microbial electrosynthesis system (MES) without organic substrates. However, a relatively high applied voltage is required for the bioelectrical reactions. In this study, we demonstrated that electrotrophic methane production at the biocathode was achieved even at a very low voltage of 0.1 V in an MES, in which abiotic HS⁻ oxidized to SO₄²⁻ at the anodic carbon-cloth surface coated with platinum powder. In addition, microbial community analysis revealed the most probable pathway for methane production from electrons. First, electrotrophic H₂ was produced by syntrophic bacteria, such as Syntrophorhabdus, Syntrophobacter, Syntrophus, Leptolinea, and Aminicenantales, with the direct acceptance of electrons at the biocathode. Subsequently, most of the produced H₂ was converted to acetate by homoacetogens, such as Clostridium and Spirochaeta 2. In conclusion, the majority of the methane was indirectly produced by a large population of acetoclastic methanogens, namely Methanosaeta, via acetate. Further, hydrogenotrophic methanogens, including Methanobacterium and Methanolinea, produced methane via H₂.     

Strong ozone production at a rural site in theNorth China Plain: Mixed effects of urban plumesand biogenic emissions

Regional ozone (O₃) pollution has drawn increasing attention in China over the recent decade, but the contributions from urban pollution and biogenic emissions have not been clearly elucidated. To better understand the formation of the regional O₃ problem in the North China Plain (NCP), intensive field measurements of O₃ and related parameters were conducted at a rural site downwind of Ji'nan, the capital city of Shandong province, in the summer of 2013. Markedly severe O₃ pollution was recorded, with the O₃ mixing ratios exceeding the Chinese national ambient air quality standard on 28?days (a frequency of 78%) and with a maximum hourly value of 198 ppbv. Extensive regional transport of well-processed urban plumes to the site was identified. An observation-constrained chemical box model was deployed to evaluate in situ photochemical O₃ production on two episodes. The results show that the in situ formation accounted for approximately 46% of the observed O₃ accumulation, while the remainder (~ 54%) was contributed by regional transport of the O₃-laden urban plumes. The in situ ozone production was in a mixed controlled regime that reducing either NOx or VOCs would lead to a reduction of ozone formation. Biogenic VOCs played an important role in the local ozone formation. This study demonstrates the significant mixed effects of both anthropogenic pollution from urban zones and biogenic emission in rural areas on the regional O₃ pollution in the NCP region, and may have general applicability in facilitating the understanding of the formation of secondary pollution over China.     

Sulfate reduction behavior in pressure-bearing leachate saturated zone

Attention should be paid to the sulfate reduction behavior in a pressure-bearing leachate saturated zone. In this study, within the relative pressure range of 0–0.6 MPa, the ambient temperature with the highest sulfate reduction rate of 50°C was selected to explore the difference in sulfate reduction behavior in a pressure-bearing leachate saturated zone. The results showed that the sulfate reduction rate might further increase with an increase in pressure; however, owing to the effect of pressure increase, the generated hydrogen sulfide (H₂S) could not be released on time, thereby decreasing its highest concentration by approximately 85%, and the duration extended to about two times that of the atmospheric pressure. Microbial community structure and functional gene abundance analyses showed that the community distribution of sulfate-reducing bacteria was significantly affected by pressure conditions, and there was a negative correlation between disulfide reductase B (dsrB) gene abundance and H₂S release rate. Other sulfate reduction processes that do not require disulfide reductase A (dsrA) and dsrB genes may be the key pathways affecting the sulfate reduction rate in the pressure-bearing leachate saturated zone. This study improves the understanding of sulfate reduction in landfills as well as provides a theoretical basis for the operation and management of landfills.     

Copper stimulates the incidence of antibiotic resistance, metal resistance and potential pathogens in the gut of black soldier fly larvae

The black soldier fly larvae (BSFL) have been successfully applied to treat various organic wastes. However, the impacts of heavy metals on antibiotic resistance in the BSFL guts are poorly understood. Here, we investigated the effect of copper (exposure concentrations of 0, 100 and 800 mg/kg) on the antibiotic and metal resistance profiles in BSFL guts. A total of 83 antibiotic resistance genes (ARGs), 18 mobile genetic elements (MGEs) and 6 metal resistance genes (MRGs) were observed in larval gut samples. Exposure to Cu remarkably reduced the diversity of ARGs and MGEs, but significantly enhanced the abundances of gut-associated ARGs and MRGs. The levels of MRGs copA, czcA and pbrT were dramatically strengthened after Cu exposure as compared with CK (increased by 2.8–13.5 times). Genera Enterococcus acted as the most predominant potential host of multiple ARG, MGE and MRG subtypes. Meanwhile, high exposure to Cu aggravated the enrichment of potential pathogens in BSFL guts, especially for Escherichia, Enterococcus and Salmonella species. The mantel test and procrustes analysis revealed that the gut microbial communities could be a key determinant for antibiotic and metal resistance. However, no significant positive links were observed between MGEs and ARGs or MRGs, possibly suggesting that MGEs did not play a crucial role in shaping the ARGs or MRGs in BSFL guts under the stress of Cu. These findings extend our understanding on the impact of heavy metals on the gut-associated antibiotic and metal resistome of BSFL.     

Stable-isotope probing of bacterial community for dissolved inorganic carbon utilization in Microcystis aeruginosa-dominated eutrophic water

Dissolved inorganic carbon (DIC) is an important source of carbon in aquatic ecosystems, especially under conditions of increased frequency of cyanobacterial bloom. However, the importance of bacteria in direct or indirect utilization of DIC has been widely overlooked in eutrophic freshwater. To identify the functional bacteria that can actively utilize DIC in eutrophic freshwater during cyanobacterial bloom, stable-isotope probing (SIP) experiments were conducted on eutrophic river water with or without inoculation with cyanobacteria (Microcystis aeruginosa). Our 16S rRNA sequencing results revealed the significance of Betaproteobacteria, with similar relative abundance as Alphaproteobacteria, in the active assimilation of H¹³CO₃^? into their DNA directly or indirectly, which include autotrophic genera Betaproteobacterial ammonia-oxidizing bacteria. Other bacterial groups containing autotrophic members, e.g. Planctomycetes and Nitrospira, also presented higher abundance among free-living bacteria in water without cyanobacteria. Microcystis aggregates showed a preference for some specific bacterial members that may utilize H¹³CO₃^? metabolized by Microcystis as organic matter, e.g. Bacteroidetes (Cytophagales, Sphingobacteriales), and microcystin-degrading bacteria Betaproteobacteria (Paucibacter/Burkholderiaceae). This study provides some valuable information regarding the functional bacteria that can actively utilize DIC in eutrophic freshwater.     

Anaerobic oxidation of methane coupled to sulfate reduction: Consortium characteristics and application in co-removal of H2S and methane

Anaerobic sludge from a sewage treatment plant was used to acclimatize microbial colonies capable of anaerobic oxidation of methane (AOM) coupled to sulfate reduction. Clone libraries and fluorescence in situ hybridization were used to investigate the microbial population. Sulfate-reducing bacteria (SRB) (e.g., Desulfotomaculum arcticum and Desulfobulbus propionicus) and anaerobic methanotrophic archaea (ANME) (e.g., Methanosaeta sp. and Methanolinea sp.) coexisted in the enrichment. The archaeal and bacterial cells were randomly or evenly distributed throughout the consortia. Accompanied by sulfate reduction, methane was oxidized anaerobically by the consortia of methane-oxidizing archaea and SRB. Moreover, CH₄ and SO₄^2 ? were consumed by methanotrophs and sulfate reducers with CO₂ and H₂S as products. The H₃CSH produced by methanotrophy was an intermediate product during the process. The methanotrophic enrichment was inoculated in a down-flow biofilter for the treatment of methane and H₂S from a landfill site. On average, 93.33% of H₂S and 10.71% of methane was successfully reduced in the biofilter. This study tries to provide effective method for the synergistic treatment of waste gas containing sulfur compounds and CH₄.     

Atmospheric Particle Hygroscopicity and the Influence by Oxidation State of Organic Aerosols in Urban Beijing

A hygroscopic tandem differential mobility analyser (H-TDMA) was used to observe the size-resolved hygroscopic characteristics of submicron particles in January and April 2018 in urban Beijing. The probability distribution of the hygroscopic growth factor (HGF-PDF) in winter and spring usually showed a bimodal pattern, with more hygroscopic mode (MH) being more dominant. The seasonal variation in particle hygroscopicity was related to the origin of air mass, which received polluted southerly air masses in spring and clean northwesterly air masses in winter. Particles showed stronger hygroscopic behaviour during heavy pollution episodes (HPEs) with elevated concentrations of secondary aerosols, especially higher mass fraction of nitrate, which were indicated using the PM_2.5 (particulate matter with diameter below 2.5 µm) mass concentration normalised by CO mass concentration. The hygroscopic parameter (κ) values were calculated using H-TDMA (κ_htdma) and chemical composition (κ_chem). The closure study showed that κ_chem was overestimated in winter afternoon when compared with κ_htdma, because the organic particle hygroscopic parameter (κ_org) was overestimated in the calculations. It was influenced by the presence of a high concentration of hydrocarbon-like organic aerosol (HOA) with a weak water uptake ability. A positive relationship was observed between κ_org and the ratio of oxygenated organic aerosol (OOA) and HOA, thereby indicating that the strong oxidation state enhanced the hygroscopicity of the particles. This study revealed the effect of local emission sources and secondary aerosol formation processes on particle hygroscopicity, which is of great significance for understanding the pollution formation mechanism in the North China Plain.     

Importance of nutrient availability for soluble microbial products formation during a famine period of activated sludge: Evidence from multiple analyses

Much remains unknown about compositional variations in soluble microbial products (SMP) with the shift of the substrate condition from a feast to a famine phase in biological treatment systems. This study demonstrated that the formation of SMP could be suppressed by up to 75% during the famine phase with the addition of essential nutrients. In contrast, presence of electron acceptor did not play any significant role during the stress condition, showing the similar amounts of SMP (r?=?0.98, p?<?0.05) formation between the bioreactors supplied with air and N₂. The SMP formed in the famine phase was more bio-refractory in the famine versus the feast phase with a linear correlation shown between the production and their aromatic structures in the composition (R²?>?0.95). The fluorescence excitation–emission matrix coupled with parallel factor analysis (EEM-PARAFAC) revealed the presence of four different fluorescent components, including two protein-like (C1 and C4), fulvic-like (C2), and humic-like (C3) components, in the SMP and bEPS formed at different conditions. Both C1 and C4 showed increasing trends (R²?>?0.95) with the length of starvation in the bioreactors without essential nutrients. Nutrient availability was found to be a key factor to quench the production of large-sized biopolymers. This study provides a wealth of information on operation conditions of activated sludge treatment systems to minimize large sized SMP molecules (particularly proteins), which typically exert many environmental concerns to effluent organic matter quality.     

Abundance of antibiotic resistance genes and their association with bacterial communities in activated sludge of wastewater treatment plants: Geographical distribution and network analysis

Wastewater treatment plants(WWTPs) are deemed reservoirs of antibiotic resistance genes(ARGs). Bacterial phylogeny can shape the resistome in activated sludge. However, the co-occurrence and interaction of ARGs abundance and bacterial communities in different WWTPs located at continental scales are still not comprehensively understood. Here, we applied quantitative PCR and Miseq sequence approaches to unveil the changing profiles of ARGs(sul1, sul2, tet W, tet Q, tet X), int I1 gene, and bacterial communities in 18 geographically distributed WWTPs. The results showed that the average relative abundance of sul1 and sul2 genes were 2.08 × 10~(-1) and 1.32 × 10~(-1) copies/16 S rRNA copies, respectively. The abundance of tet W gene was positively correlated with the Shannon diversity index(H′), while both studied sul genes had significant positive relationship with the int I1 gene. The highest average relative abundances of sul1, sul2, tet X, and int I1 genes were found in south region and oxidation ditch system. Network analysis found that 16 bacterial genera co-occurred with tet W gene. Co-occurrence patterns were revealed distinct community interactions between aerobic/anoxic/aerobic and oxidation ditch systems. The redundancy analysis model plot of the bacterial community composition clearly demonstrated that the sludge samples were significant differences among those from the different geographical areas,and the shifts in bacterial community composition were correlated with ARGs. Together,these findings from the present study will highlight the potential risks of ARGs and bacterial populations carrying these ARGs, and enable the development of suitable technique to control the dissemination of ARGs from WWTPs into aquatic environments.     

Toxic effects of acetochlor, methamidophos and their combination on nifH gene in soil

Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis （DGGE） and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity （both in gene richness and diversity index values） and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patterns showed that nifH gene composition which had been affected by low concentration of acetochlor （50 mg/kg） recovered firstly. Methamidophos reduced nifH gene richness that except at 4 weeks. The medium concentration of methamidophos （150 mg/kg） caused the most apparent changes in nifH gene diversity at the first week while the high concentration of methamidophos （250 mg/kg） produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes （bands） responded differently to the impact of agrochemicals： four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha- and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.     

Arsenic uptake by arbuscular mycorrhizal maize （Zea mays L.） grown in an arsenic-contaminated soil with added phosphorus

The effects of arbuscular mycorrhizal (AM) fungus (Glomus mosseae) and phosphorus (P) addition (100 mg/kg soil) on arsenic (As) uptake by maize plants (Zea mays L.) from an As-contaminated soil were examined in a glasshouse experiment.Non-mycorrhizal and zero-P addition controls were included.Plant biomass and concentrations and uptake of As,P,and other nutrients,AM colonization,root lengths,and hyphal length densities were determined.The results indicated that addition of P significantly inhibited root colonization and development of extraradical mycelium.Root length and dry weight both increased markedly with mycorrhizal colonization under the zero-P treatments,but shoot and root biomass of AM plants was depressed by P application.AM fungal inoculation decreased shoot As concentrations when no P was added,and shoot and root As concentrations of AM plants increased 2.6 and 1.4 times with P addition,respectively.Shoot and root uptake of P,Mn,Cu,and Zn increased,but shoot Fe uptake decreased by 44.6%,with inoculation, when P was added.P addition reduced shoot P,Fe,Mn,Cu,and Zn uptake of AM plants,but increased root Fe and Mn uptake of the nonmycorrhizal ones.AM colonization therefore appeared to enhance plant tolerance to As in low P soil,and have some potential for the phytostabilization of As-contaminated soil,however,P application may introduce additional environmental risk by increasing soil As mobility.     

Impact of atrazine and nitrogen fertilizers on the sorption of chlorotoluron in soil and model sorbents

Sorption of chlorotoluron in ammonium sulfate, urea and atrazine multi-solutes system was investigated by batch experiments. The results showed application of nitrogen fertilizers to the soil could affect the behavior of chlorotoluron. At the same concentration of N, sorption of chlorotoluron decreased as the concentration of atrazine increased on the day 0 and 6 in soil, respectively. The sorption of chlorotoluron increased from 0 to 6 d when soils were preincubated with deionized water, ammonium sulfate and urea solution for 6 d. That indicated incubation time was one of the most important factors for the sorption of chlorotoluron in nitrogen fertilizers treatments. The individual sorption isotherms of chlorotoluron in rubbery polymer and silica were strictly linear in single solute system, but there were competition sorption between pesticides or between pesticides and nitrogen fertilizers. That indicated the sorption taken place by concurrent solid-phase dissolution mechanism and sorption on the interface of water-organic matter or water-mineral matter.     

Degradation of metabolites accumulated of benzo[a]pyrene by coupling Penicillium Chrysogenum with KMnO4

Several main metabolites of benzo[a]pyrene （BaP） formed by Penicillium chrysogenum, Benzo[a]pyrene-1,6-quinone （BP 1,6- quinone）, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene （BP 7,8-diol）, 3-hydroxybenzo[a]pyrene （3-OHBP）, were identified by high-performance liquid chromatography （HPLC）. The three metabolites were liable to be accumulated and were hardly further metabolized because of their toxicity to microorganisms. However, their further degradation was essential for the complete degradation of BaP. To enhance their degradation, two methods, degradation by coupling Penicillium chrysogenum with KMnO4 and degradation only by Penicillium chrysogenum, were compared; Meanwhile, the parameters of degradation in the superior method were optimized. The results showed that （1） the method of coupling Penicillium chrysogenum with KMnO4 was better and was the first method to be used in the degradation of BaP and its metabolites; （2） the metabolite, BP 1,6-quinone was the most liable to be accumulated in pure cultures; （3） the effect of degradation was the best when the concentration of KMnO4 in the cultures was 0.01% （w/v）, concentration of the three compounds was 5 mg/L and pH was 6.2. Based on the experimental results, a novel concept with regard to the bioremediation of BaP-contaminated environment was discussed, considering the influence on environmental toxicity of the accumulated metabolites.     

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.     

Removal of heavy metals from sewage sludge by low costing chemical method and recycling in agriculture

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Isolation and preliminary characterization of a 3-chlorobenzoate degrading bacteria

A study was conducted to compare the diversity of 2-, 3-, and 4-chlorobenzoate degraders in two pristine soils and one contaminated sewage sludge. These samples contained strikingly different populations of mono-chlorobenzoate degraders. Although fewer cultures were isolated in the uncontaminated soils than contaminated one, the ability of microbial populations to mineralize chlorobenzoate was widespread. The 3- and 4-chlorobenzoate degraders were more diverse than the 2-chlorobenzoate degraders. One of the strains isolated from the sewage sludge was obtained. Based on its phenotype, chemotaxonomic properties and 16S rRNA gene, the organism S-7 was classified as Rhodococcus erythropolis. The strain can grow at temperature from 4 to 37℃. It can utilize several （halo）aromatic compounds. Moreover, strain S-7 can grow and use 3-chlorobenzoate as sole carbon source in a temperatures range of 10-30℃ with stoichiometric release of chloride ions. The psychrotolerant ability was significant for bioremediation in low temperature regions. Catechol and chlorocatechol 1,2-dioxygenase activities were present in cell free extracts of the strain, but no （chloro）catechol 2,3- dioxygenase activities was detected. Spectral conversion assays with extracts from R. erythropolis S-7 showed accumulation of a compound with a similar UV spectrum as chloro-cis,cis-muconate from 3-chlorobenzoate. On the basis of these results, we proposed that S-7 degraded 3-chlorobenzoate through the modified ortho-cleave pathway.     

Single and joint effects of pesticides and mercury on soil urease

The influence of two pesticides including chlorimuron-ethyl and furadan and mercury （Hg） on urease activity in 4 soils （meadow burozem and phaeozem） was investigated. The soils were exposed to various concentrations of the two pesticides and Hg individually and simultaneously. Results showed that there was a close relationship between urease activity and organic matter content in soil. Chlorimuron-ethyl and furadan could both activate urease in the 4 soils. The maximum increment of urease activity by chlorimuronethyl was up to 14%-18%. There was almost an equal increase （up to 13%-21%） in the urease activity by furadan. On the contrary, Hg markedly inhibited soil urease activity. A logarithmic equation was used to describe the relationship （P〈0.05） between the concentration of Hg and the activity of soil urease in the 4 tested soils. Semi-effect dose （ED50） values by the stress of Hg based on the inhibition of soil urease in the 4 soils were 88, 5.5, 24 and 20 mg/kg, respectively, according to the calculation of the corresponding equations. The interactive effect of chlorimuron-ethyl or furadan with metal Hg on soil urease was mainly synergic at the highest tested concentrations.     

Organochlorine pesticides in soils under different land usage in the Taihu Lake region, China

A field study was conducted in the Taihu Lake region, China in 2004 to reveal the organochlorine pesticide concentrations in soils after the ban of these substances in the year 1983. Thirteen organochlorine pesticides (OCPs) were analyzed in soils from paddy field, tree land and fallow land. Total organochlorine pesticide residues were higher in agricultural soils than in uncultivated fallow land soils. Among all the pesticides, ΣDDX (DDD, DDE and DDT) had the highest concentration for all the soil samples, ranging from 3.10 ng/g to 166.55 ng/g with a mean value of 57.04 ng/g and followed by ΣHCH, ranging from 0.73 ng/g to 60.97 ng/g with a mean value of 24.06 ng/g. Dieldrin, endrin, HCB and α-endosulfan were also found in soils with less than 15 ng/g. Ratios of p,p'-(DDD DDE)/DDT in soils under three land usages were: paddy field ＞ tree land ＞ fallow land, indicating that land usage inlfuenced the degradation of DDT in soils. Ratios of p,p'-(DDD DDE)/DDT ＞1, showing aged residues of DDTs in soils of the Taihu Lake region. The results were discussed with data from a former study that showed very low actual concentrations of HCH and DDT in soils in the Taihu Lake region, but according to the chemical half-lives and their concentrations in soils in 1980s, the concentration of DDT in soils seemed to be underestimated. In any case our data show that the ban on the use of HCH and DDT resulted in a tremendous reduction of these pesticide residues in soils, but there are still high amounts of pesticide residues in soils, which need more remediation processes.     

Alterations of organ histopathology and metallolhionein mRNA expression in silver barb, Puntius gonionotus during subchronic cadmium exposure

Common silver barb,Puntius gonionotus,exposed to the nominal concentration of 0.06 mg/L Cd for 60 d,were assessed for histopathological alterations(gills,liver and kidney),metal accumulation,and metallothionein(MT)mRNA expression.Fish exhibited pathological symptoms such as hypertrophy and hyperplasia of primary and secondary gill lamellae,vacuolization in hepatocytes,and prominent tubular and glomerular damage in the kidney.In addition,kidney accumulated the highest content of cadmium,more than gills and liver.Expression of MT mRNA was increased in both liver and kidney of treated fish.Hepatic MT levels remained high after fish were removed to Cd-free water.In contrast,MT expression in kidney was peaked after 28 d of treatment and drastically dropped when fish were removed to Cd-free water.The high concentrations of Cd in hepatic tissues indicated an accumulation site or permanent damage on this tissue.