Genotoxic effects of old landfill leachate on HepG2 cells after nitration/ultrafiltration/reverse osmosis membrane treatment process.

Toxicity assessment of nitration/ultrafiltration/reverse osmosis (nitration/UF/RO) project, which has recently been widely used as an efficient process with applications in practical leachate treatment, was very limited. In the present study, DNA damage of leachates was investigated before and after the nitration/UF/RO process by a battery of assays with human hepatoma cells. Methyletrazolium assay showed a high cytotoxicity of 97.1% after being exposed to the highest concentration of raw leachate for 24 h, and a cytotoxicity of 26% in effluent at a concentration of 30% (v/v). Both comet assay (24 h) and γH2AX flow cytometer assay (3 h) showed increased levels of DNA damage in cells exposed to raw leachate and after nitration/UF-treated leachate followed by a significant increase of 7-ethoxyresorufin-O-deethylase activity. However, the effluent after nitration/UF/RO treatment showed no significant difference compared to negative control for γH2AX flow cytometer assay but slight DNA damage at concentrations of 20% and 30% (v/v) as well as increase of 7-ethoxyresorufin-O-deethylase. Analysis showed that nitration/UF/RO process exhibited high removal of physicochemical indexes and significant reduction of toxic and genotoxic effects of leachate, but still demands an improvement to reduce all possible negative risks to the environment and humans. Copyright © 2017 John Wiley & Sons, Ltd.

Exploration of the antioxidant system and photosynthetic system of a marine algicidal Bacillus and its effect on four harmful algal bloom species.

A novel marine bacterium, strain B1, initially showed 96.4% algicidal activity against Phaeocystis globosa. Under this situation, 3 other harmful algal species (Skeletonema costatum, Heterosigma akashiwo, and Prorocentrum donghaiense) were chosen to study the algicidal effects of strain B1, and the algicidal activities were 91.4%, 90.7%, and 90.6%, respectively. To explore the algicidal mechanism of strain B1 on these 4 harmful algal species, the characteristics of the antioxidant system and photosynthetic system were studied. Sensitivity to strain B1 supernatant, enzyme activity, and gene expression varied with algal species, while the algicidal patterns were similar. Strain B1 supernatant increased malondialdehyde contents; decreased chlorophyll a contents; changed total antioxidant and superoxide dismutase activity; and restrained psbA, psbD, and rbcL genes expression, which eventually resulted in the algal cells death. The algicidal procedure was observed using field emission scanning electron microscopy, which indicated that algal cells were lysed and cellular substances were released. These findings suggested that the antioxidant and photosynthetic system of these 4 algal species was destroyed under strain B1 supernatant stress. This is the first report to explore and compare the mechanism of a marine Bacillus against harmful algal bloom species of covered 4 phyla.

Spatial-temporal distribution of phthalate esters from riverine outlets of Pearl River Delta in China.

The aquatic environments of the Pearl River Delta (PRD) in China have been contaminated by various industrial chemicals from local industries. In this study, the spatial-temporal distribution of six priority phthalate esters (PAEs) in surface water and sediments from the PRD was investigated. The PAEs were detected with total concentrations (Σ6PAEs) ranging from 0.35 to 20.70 µg L⁻¹ in surface water and dry weight ranging from 0.88 to 5.69 µg g⁻¹ in sediments. The Σ6PAEs concentrations in surface water were higher in the wet season than those in the dry season, while the opposite pattern was observed in sediments. Di(2-ethylhexyl) phthalate (DEHP) was the most abundant congener, which was higher than those reported in the literature. Risk quotients for relevant aquatic organisms were obtained and showed that most of these PAEs, in particular, butyl benzyl phthalate, DEHP and di-n-octyl phthalate, have significant potential health and ecological risks for the aquatic environment studied.

Spatial and Seasonal Variations of Total Petroleum Hydrocarbon in Surface Water and Sediment in Pearl River Delta.

A field study in the Pearl River Delta of China was conducted in order to describe to the spatial and seasonal variation of occurrence and concentrations of total petroleum hydrocarbon (TPH) in surface water and sediments. Petroleum hydrocarbons and isoprenoid alkanes were quantified by UV spectroscopy and gas chromatography with a mass selective detector. The concentrations of TPH ranged from 4.3 to 68.7 µg L(-1) in surface water, and from 66.6 to 1445 µg g(-1) in surface sediments. The ratios of pristine to phytane suggested that the main sources of TPH in the sediment were petroleum importation. The highest concentrations of TPH were present in the spring season. When compared with results from previous studies, it can be concluded that the Pearl River Delta was moderately polluted by TPH. No statistically significant correlations were observed between the concentrations of TPH in surface water and sediments.

Algicidal metabolites produced by Bacillus sp. strain B1 against Phaeocystis globosa.

The bloom of Phaeocystis globosa has broken out frequently in the coastal areas of China in recent years, which has led to substantial economic losses. This study shows that Bacillus sp. strain B1, which was previously identified by our group, is effective in regulating P. globosa by excreting active metabolites. Heat stability, pH stability and molecular weight range of the algicidal compounds from strain B1 were measured and the results demonstrated that the algicidal activities of these compounds were not affected by pH or temperature variation. The algicidal compounds extracted with methanol were isolated and purified by ODS-A column chromatography and HPLC. The algicidal compounds corresponding to peaks 2-5 eluted from HPLC were further analysed by quadrupole time-of-flight mass spectrometry (Q-TOF-MS). PeakView™ Software determined the compounds corresponding to peaks 2-5 to be L-histidine, o-tyrosine, N-acetylhistamine and urocanic acid on the basis of the accurate mass information, the isotopic pattern and MS-MS spectra. Furthermore, these compounds were also able to eliminate Skeletonema costatum, Prorocentrum donghaiense and Heterosigma akashiwo. This is the first report of bacteria-derived algicidal compounds being identified only by Q-TOF-MS and PeakView™ Software, and these compounds may be used as the constituents of algicides in the future.

Spatial-temporal distribution and potential ecological risk assessment of nonylphenol and octylphenol in riverine outlets of Pearl River Delta, China.

The aquatic environments of the Pearl River Delta in Southern China are subjected to contamination with various industrial chemicals from local industries. In this paper, the occurrence, seasonal variation and spatial distribution of alkylphenol octylphenol (OP) and nonylphenol (NP) in river surface water and sediments in the runoff outlets of the Pearl River Delta were investigated. NP and OP were detected in all water and sediment samples and their mean concentrations in surface water during the dry season ranged from 810 to 3366 ng/L and 85.5 to 581 ng/L, respectively, and those in sediments ranged from 14.2 to 95.2 ng/g dw and 0.4 to 3.0 ng/g dw, respectively. In surface water, much higher concentrations were detected in the dry season than those in the wet season. In sediments, the concentrations in the dry season were also mostly higher. High concentrations of NP and OP were found in Humen outlet, likely due to high levels of domestic and industrial wastewater discharges. An ecological risk assessment with the use of hazard quotient (HQ) was also carried out and the HQ values ranged from 3.6×10(-5) to 35 and 64% of samples gave a HQ>1, indicating that the current levels of NP and OP pose a significant risk to the relevant aquatic organisms in the region.

The effects of extracellular algicidal compounds of Bacillus sp. B1 on Heterosigma akashiwo: a metabolomics approach.

Heterosigma akashiwo (H. akashiwo), a harmful algal species, has been a global environmental problem. Extracellular algicidal compounds (EACs) extracted from Bacillus sp. B1 exhibited algicidal effects against H. akashiwo. However, little is known about the algicidal mechanism and metabolic process. In this study, metabolomics and physiological analyses were combined to investigate the cellular responses of H. akashiwo when treated with EACs. The results indicated that EACs at 10% (vEACs/vsample) showed more than 90% inhibition of H. akashiwo. EAC treatment resulted in excessive reactive oxygen species (ROS) production in algal cells, causing stress responses such as inhibition of photosynthetic pigment synthesis, reduction of sugar synthesis, imbalance of osmotic pressure in the cell membrane, disruption of cell size and morphology, and eventual cell death. The results reveal the underlying mechanism of the algicidal process and provide new insights into algae-bacteria interactions and the application of metabolomics to algal research.

NMR spectral assignments of two sterols from a soft coral Sinularia brassica.

Two new steroids, named 4α,22-dimethyl-Cholest-22-en-3ß-ol (1) and 4α-methyl-Cholest-7,22-dien-3ß-ol (2), along with two known steroids, were isolated from the soft coral Sinularia brassica. The structures of the new compounds were determined on the basis of extensive spectroscopic data (MS, (1)H and (13)C NMR, (1)H-(1)H COSY, (13)C-(1)H COSY, HMBC and NOESY) analysis.

Effects of two algicidal substances, ortho-tyrosine and urocanic acid, on the growth and physiology of Heterosoigma akashiwo.

Heterosigma akashiwo is a commonly found harmful microalgae, however, there are only few studies on its control using algicidal components particularly those identified from algicidal bacteria. In our previous study, ortho-tyrosine and urocanic acid identified from Bacillus sp. B1 showed a significantly high algicidal effect on H. akashiwo. The growth inhibition rates of H. akashiwo after 96 h of treatment with 300 µg/mL o-tyrosine and 500 µg/mL urocanic acid were 91.06% and 88.07%, respectively. Through non-destructive testing by Pulse Amplitude Modulation fluorometry and flow cytometer, the effects of o-tyrosine and urocanic acid on H. akashiwo PS II and physiological parameters (cell volume, mitochondrial membrane potential, and membrane permeability) were estimated. This study shows that o-tyrosine affected the photosynthesis system of H. akashiwo, decreased the mitochondrial membrane potential, and increased the membrane permeability of the algal cells. Treatment with urocanic acid decreased the mitochondrial membrane potential, resulting in the inhibition of algal cell growth and reproduction, but had little effect on membrane permeability and photosynthetic system. Our results may imply that when uridine degrades, surviving H. akashiwo cells may be reactivated. Therefore, o-tyrosine and urocanic acid have the potential to become new biological algicides, which can effectively control the growth of H. akashiwo.

Effects of combined toxicity of methamphetamine and ketamine on apoptosis, oxidative stress and genotoxicity in HepG2 cells.

Methamphetamine (MA) and ketamine (KET) are widely abused drugs individually. Previous surveys have revealed that the combined consumption of MA and KET were prevalent in illicit drugs abusers. However, few studies on the toxic effects induced by the combination of MA and KET have been reported. In this study, combined treatments were carried out using 3 × 3 full factorial design to determine the combined effects of MA and KET on apoptosis, oxidative stress and genotoxicity in HepG2 cells. Higher apoptosis and oxidative damage were observed in the MA treatments groups. Compared with control groups, the maximum apoptotic rate and level of malondialdehyde were ∼7.7 fold and ∼5.5 fold respectively. The mechanism that excessive oxidative stress resulted in cell apoptosis and DNA damage was inferred. For the joint effects, synergistic or additive interactions were found at different biological endpoints for various combinations, likely due to the mechanism in which MA promotes the metabolism of KET, which together provokes even greater oxidative stress. In conclusion, synergistic or additive interactions between MA and KET enhance cytotoxicity, oxidative damage and genotoxicity in HepG2 cells more than either of the drugs alone, which implies higher risk for abusers when exposed to the polydrug situation.

Combined algicidal effect of urocanic acid, N-acetylhistamine and l-histidine to harmful alga Phaeocystis globosa.

The algicidal compounds produced by Bacillus sp. strain B1 against Phaeocystis globosa, one of the main red-tide algae, were isolated and identified in a previous study as urocanic acid (uro), l-histidine (his) and N-acetylhistamine (ace). The 96 h median effective concentration EC50 values indicated the algicidal effect order of uro (8 µg mL-1) > ace (16 µg mL-1) > his (23 µg mL-1). The interaction between uro and ace had a synergistic effect on Phaeocystis globosa, accelerated the increase in its intracellular reactive oxygen species (ROS) levels, and further decreased the activities of antioxidases after 96 h, causing destruction of cell membrane integrity and nuclear structure. However, the other two binary mixtures uro + his and ace + his were both antagonistic to Phaeocystis globosa. The increase in the level of ROS indicated that the algal cells suffered from oxidative damage. The surplus ROS induced the increase in malondialdehyde (MDA) content and activities of antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), all of which reached maxima after 72 h treatment. Transmission electron microscopy (TEM) analysis revealed that these nitrogen-containing compounds caused destruction of cell membrane integrity, chloroplasts and nuclear structure. The present study will provide useful information for the combined effect of algicidal compounds on the harmful alga Phaeocystis globosa. This is the first report to explore single and combined algicidal effects of three nitrogen-containing compounds against the harmful alga Phaeocystis globosa.

Adverse effect of nano-TiO2 on the marine macroalgae Gracilaria lemaneiformis (Gracilariales, Rhodophyta): growth and antioxidant activity.

Macroalgae, the major contributor of primary productivity in coastal seas, contribute to the material cycle and energy flow in marine ecosystems. The purpose of this work was to evaluate the toxic effect of nano-TiO2 on the growth and antioxidant activity of Gracilaria lemaneiformis. An obvious inhibition of growth was observed in this study. The algae exposed to nano-TiO2 showed a negative growth rate at 20 mg L-1 and 40 mg L-1 during the 15 days exposure. The concentration of soluble protein increased slightly during the first 3 days of exposure, but it gradually diminished thereafter due to the high concentrations of nano-TiO2 and to prolonged exposure. Nano-TiO2 caused oxidative damage in G. lemaneiformis; superoxide anions accumulated, and nitrate reductase activity decreased linearly with the increase in nano-TiO2. Furthermore, extracts of G. lemaneiformis can scavenge DPPH· and hydroxyl radicals for their antioxidant capacity. However, the capacity to scavenge DPPH· and hydroxyl radicals in vitro decreased slightly with the increase in nano-TiO2. The results from this work imply that macroalgae can be an effective biomarker of nano-TiO2 contamination and can be useful indicators to evaluate the oxidative damage of increasing pollutants in marine ecosystems.

Toxic effect of nonylphenol on the marine macroalgae Gracilaria lemaneiformis (Gracilariales, Rhodophyta): antioxidant system and antitumor activity.

The objective of the present work was to evaluate the toxic effect of nonylphenol (NP) on the antioxidant response and antitumor activity of Gracilaria lemaneiformis. An obvious oxidative damage was observed in this study. The thallus exposed to NP showed 1.2-2.0-fold increase in lipid peroxide and displayed a maximum level of 16.58 µmol g-1 Fw on 0.6 mg L-1 for 15-day exposure. The activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) enhanced significantly by 1.1-3.2-fold and subsequently diminished at the high concentrations and prolonged exposure. The results of DNA damage in comet assay also supported that NP was obviously toxic on G. lemaneiformis with increasing the percentage of tail DNA in a dose-dependent manner. Furthermore, the ethanol extract of G. lemaneiformis (EEGL) did exhibit antitumor potential against HepG-2 cells. While decreased in cell inhibition, ROS generation, apoptosis, and caspase-3 in HepG-2 cells treated with the EEGL were observed when G. lemaneiformis was exposed to NP for 15 days, and which were related to exposure concentration of NP. These suggested that NP has strongly toxic effect on the antitumor activity of G. lemaneiformis. The results revealed in this study imply that macroalgae can be useful biomarkers to evaluate marine pollutions.

Effects of individual and combined toxicity of bisphenol A, dibutyl phthalate and cadmium on oxidative stress and genotoxicity in HepG 2 cells.

Bisphenol A, dibutyl phthalate and cadmium can be found in environment simultaneously. Several studies suggested that they had genotoxic effect. In this study, mono-exposure and co-exposure treatments, designed by 3 × 3 full factorial, were established to determine the individual toxicity and binary mixtures' combined effects on the oxidative stress and genotoxicity in HepG 2 cells. The highest oxidative damage was observed in the Cd treatments groups. Compared with control groups, the maximum level of reactive oxygen species and malondialdehyde were ∼1.4 fold and ∼2.22 fold respectively. And a minimum level of superoxide dismutase activity was found with the decrease of 43%. The mechanism that excessive oxidative stress led to the DNA damage was inferred. However, cells treated with BPA showed the worst DNA damage rather than Cd, which may because Cd mainly damages DNA repairing mechanism. For the joint effect, different interactions can be found in different biological endpoints for different combinations since different mechanisms have been clarified in mixture toxicity studies. It is sure that the co-exposure groups enhanced cytotoxicity, oxidative stress and genotoxicity compared to the mono-exposures. Synergistic and additive interactions were considered, which means greater threat to organisms when exposed to multiple estrogenic endocrine disruptors.

Estrogenicity assessment of membrane concentrates from landfill leachate treated by the UV-Fenton process using a human breast carcinoma cell line.

Membrane concentrates (MCs) are generated when membranes are used to concentrate landfill leachate. It contains high concentrations of inorganic and organic environmental pollutants, which are highly toxic and carcinogenic. In this paper, the proliferation effect (PE) from MC before and after treatment with the UV-Fenton process was assessed using the human breast carcinoma cell line MCF-7. The highest value of 116% was found at 5% (v/v) concentration after a 10 min reaction. Phthalic acid esters (PAEs) play an important role in the MC estrogenicity. Estrogen simulation solutions (ESS) of PAEs were prepared to simulate the changes in estrogenic active substances during the UV-Fenton process. The ESS degradation conformed to the first-order kinetics model. The estrogenicity decreased after an initial increase until it acted in a non-estrogenic manner. Convincingly, the intermediates were determined by GC/MS, and the estrogenicity was assessed during the degradation process. The estrogenicity was highly related to the generation of intermediates and the PAE concentration. The results provide guidance for UV-Fenton application in MC estrogenicity reduction.

Phthalate esters in water and surface sediments of the Pearl River Estuary: distribution, ecological, and human health risks.

The Pearl River Estuary (PRE) is vulnerable due to the increasingly serious environmental pollution, such as phthalate esters (PAEs) contaminants, from the Pearl River Delta (PRD). The concentrations of six US Environmental Protection Agency (USEPA) priority PAEs in water and surface sediments collected from the PRD's six main estuaries in spring, summer, and winter 2013 were measured by GC-MS. Total PAEs (∑6PAEs) concentrations were from 0.5 to 28.1 µg/L and from 0.88 to 13.6 µg/g (dry weight (DW)) in water and surface sediments, respectively. The highest concentration was detected in summer. Higher concentrations of PAEs were found in Yamen (YM) and Humen (HM) areas than the other areas. Bis(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP) were the dominant PAEs in the investigated areas, contributing between 61 and 95 % of the PAEs in water and from 85 to 98 % in surface sediments. Based on risk quotients (RQs), DEHP posed greater ecological risks to the studied aquatic environments than other measured compounds. Little human health risk from the target PAEs was identified.

Photosynthetic inhibition and oxidative stress to the toxic Phaeocystis globosa caused by a diketopiperazine isolated from products of algicidal bacterium metabolism.

Algicidal bacteria have been turned out to be available for inhibiting Phaeocystis globosa which frequently caused harmful algal blooms and threatened to economic development and ecological balance. A marine bacterium Bacillus sp. Ts-12 exhibited significant algicidal activity against P. globosa by indirect attack. In present study, an algicidal compound was isolated by silica gel column, Sephadex G-15 column and HPLC, further identified as hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, cyclo-(Pro-Gly), by GC-MS and (1)H-NMR. Cyclo-(Pro-Gly) significantly increased the level of reactive oxygen species (ROS) within P. globosa cells, further activating the enzymatic and non-enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and ascorbic acid (AsA). The increase in methane dicarboxylic aldehyde (MDA) content showed that the surplus ROS induced lipid peroxidation on membrane system. Transmission electron microscope (TEM) and flow cytometry (FCM) analysis revealed that cyclo-(Pro-Gly) caused reduction of Chl-a content, destruction of cell membrane integrity, chloroplasts and nuclear structure. Real-time PCR assay showed that the transcriptions of photosynthesis related genes (psbA, psbD, rbcL) were significantly inhibited. This study indicated that cyclo-(Pro-Gly) from marine Bacillus sp. Ts-12 exerted photosynthetic inhibition and oxidative stress to P. globosa and eventually led to the algal cells lysis. This algicidal compound might be potential bio-agent for controlling P. globosa red tide.

Evaluation of toxicity and estrogenicity of the landfill-concentrated leachate during advanced oxidation treatment: chemical analyses and bioanalytical tools.

Landfill-concentrated leachate from membrane separation processes is a potential pollution source for the surroundings. In this study, the toxicity and estrogenicity potentials of concentrated leachate prior to and during UV-Fenton and Fenton treatments were assessed by a combination of chemical (di (2-ethylhexyl) phthalate and dibutyl phthalate were chosen as targets) and biological (Daphnia magna, Chlorella vulgaris, and E-screen assay) analyses. Removal efficiencies of measured di (2-ethylhexyl) phthalate and dibutyl phthalate were more than 97 % after treatment with the two methods. Biological tests showed acute toxicity effects on D. magna tests in untreated concentrated leachate samples, whereas acute toxicity on C. vulgaris tests was not observed. Both treatment methods were found to be efficient in reducing acute toxicity effects on D. magna tests. The E-screen test showed concentrated leachate had significant estrogenicity, UV-Fenton and Fenton treatment, especially the former, were effective methods for reducing estrogenicity of concentrated leachate. The EEQchem (estradiol equivalent concentration) of all samples could only explain 0.218-5.31 % range of the EEQbio. These results showed that UV-Fenton reagent could be considered as a suitable method for treatment of concentrated leachate, and the importance of the application of an integrated (biological + chemical) analytical approach for a comprehensive evaluation of treatment suitability.

Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line.

Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates.

[Effects of Algicidal Substance on Phaeocystis globosa and Its Fatty Acids by the Simulation Experiment].

In order to evaluate the effects of algicidal substance on Phaeocystis globosa (P. globosa) and its algal toxin-fatty acids, the changes of chlorophyll-a, pH, dissolved oxygen, permanganate index and N, P concentration were evaluated by the simulation experiment. Fatty acids composition in P. globosa was detected by GC-MS. After adding algicidal substance in simulative water with the volume ratio 1: 100, the levels of chlorophyll-a, pH and permanganate index were reduced, while the concentrations of dissolved oxygen and N, P were increased significantly within 14 days. Comparing with control group after 14 days, pH was reduced to 7. 51 from 8. 50, chlorophyll-a and permanganate index were reduced by 82. 3% (P < 0. 05) and 55. 2% (P < 0. 01), respectively. Dissolved oxygen was significantly increased by 29. 5% (P < 0. 05). The concentrations of NH4+ -N, NO2- -N, NO3- -N and PO(4)3- -P were respectively 0. 46, 1. 50 , 6. 24 and 1. 30 times higher than that in control group. 14 days after the addition of algicidal substance, the total fatty acids of P. globosa were reduced by 83. 4%. The major fatty acids C18:2, C16:0, and C18:1, were reduced by 100%, 97. 7% and 85. 4% (P <0. 01), respectively. Our results indicated that algicidal substance from Bacillus sp. BI can effectively inhibit the growth of P. globosa and reduce the concentration of algal toxin-fatty acid in the simulation experiment. This study provides a theoretical basis for ecological safety of algicidal substance form Bacillus sp. strain Bl.