Sample Pretreatment Method for Determination of Indicator Polychlorinated Biphenyls in Seafood using Ultrasonic Extraction Followed by Dispersive Solid-Phase Extraction and Gas Chromatography - Electron Capture Detection.

A simple, rapid and novel method has been developed and validated for the determination of seven indicator polychlorinated biphenyls (PCBs) in seafood samples by gas chromatography coupled to electron capture detector. Freeze-dried samples were done first ultrasonic extraction by n-hexane:methylene chloride:acetone (3:1:1, v/v), and then one-step clean-up (dispersive solid-phase extraction clean-up) or two-step clean-up (concentrated sulfuric acid purification and dispersive solid-phase extraction clean-up) was selected according to the lipid contents of the samples, if the lipid content was no more than 1%, one-step clean-up was used, otherwise, two-step clean-up was chose. The linearity of this method ranged from 1.25 to 100 µg/L, with regression coefficients ranging between 0.9991 and 0.9998. The limits of detection were in low ng/g level, ranging between 0.005 and 0.0076 ng/g (wet weight). The recoveries of spiked seven PCBs with external calibration method at different concentration levels in Pseudosciaena polyactis, Penaeus vannamei and Sinonovacula constricta were in the range of 78-105%, 73-110% and 75-107%, respectively, and with relative standard deviations of 3.3-5.1%, 3.5-6.3% and 3.4-5.1% (n = 5), respectively. The performance of the proposed method was also compared with traditional soxhlet extraction and column chromatography clean-up on the same real seafood samples and comparable efficiencies were obtained. It is concluded that this method can be successfully applied for the determination of PCBs in different seafood samples.

Primary secondary amine as a sorbent material in dispersive solid-phase extraction clean-up for the determination of indicator polychlorinated biphenyls in environmental water samples by gas chromatography with electron capture detection.

A simple, rapid, and novel method has been developed and validated for determination of seven indicator polychlorinated biphenyls in water samples by gas chromatography with electron capture detection. 1 L of water samples containing 30 g of anhydrous sodium sulfate was first liquid-liquid extracted with an automated Jipad-6XB vertical oscillator using n-hexane/dichloromethane (1:1, v/v). The concentrated extract was cleaned up by dispersive solid-phase extraction with 100 mg of primary secondary amine as sorbent material. The linearity of this method ranged from 1.25 to 100 µg/L, with regression coefficients ranging between 0.9994 and 0.9999. The limits of detection were in the ng/L level, ranging between 0.2 and 0.3 ng/L. The recoveries of seven spiked polychlorinated biphenyls with external calibration method at different concentration levels in tap water, lake water, and sea water were in the ranges of 85-112, 76-116, and 72-108%, respectively, and with relative standard deviations of 3.3-4.5, 3.4-5.6, and 3.1-4.8% (n = 5), respectively. The performance of the proposed method was compared with traditional liquid-liquid extraction and solid-phase extraction clean-up methods, and comparable efficiencies were obtained. It is concluded that this method can be successfully applied for the determination of polychlorinated biphenyls in different water samples.

Microcystin-LR Induced Apoptosis in Rat Sertoli Cells via the Mitochondrial Caspase-Dependent Pathway: Role of Reactive Oxygen Species.

Microcystins (MCs), the secondary metabolites of blue-green algae, are ubiquitous and major cyanotoxin contaminants. Besides the hepatopancreas/liver, the reproductive system is regarded as the most important target organ for MCs. Although reactive oxygen species (ROS) have been implicated in MCs-induced reproductive toxicity, the role of MCs in this pathway remains unclear. In the present study, Sertoli cells were employed to investigate apoptotic death involved in male reproductive toxicity of microcystin-LR (MC-LR). After exposure to various concentrations of MC-LR for 24 h, the growth of Sertoli cells was concentration-dependently decreased with an IC50 of ~32 µg/mL. Mitochondria-mediated apoptotic changes were observed in Sertoli cells exposed to 8, 16, and 32 µg/mL MC-LR including the increased expression of caspase pathway proteins, collapse of mitochondrial membrane potential (MMP), and generation of ROS. Pretreatment with a global caspase inhibitor was found to depress the activation of caspases, and eventually increased the survival rate of Sertoli cells, implying that the mitochondrial caspases pathway is involved in MC-LR-induced apoptosis. Furthermore, N-acetyl-l-cysteine attenuated the MC-LR-induced intracellular ROS generation, MMP collapse and cytochrome c release, resulting in the inhibition of apoptosis. Taken together, the observed results suggested that MC-LR induced apoptotic death of Sertoli cells by the activation of mitochondrial caspases cascade, while its effects on the ROS-mediated signaling pathway may contribute toward the initiation of mitochondrial dysfunction.

N-acetylcysteine protects Chinese Hamster ovary cells from oxidative injury and apoptosis induced by microcystin-LR.

This study aimed to investigate the MC-LR induced oxidative injury and apoptosis in Chinese hamster ovary (CHO) cells, and the protective effects of N-acetylcysteine (NAC) on these cells. Cell viability was determined by MTT assay after exposure to NAC at various concentrations (0, 1, 5, 10, 20, 30, 40, 50, 60 and 80 mmol/L) alone, or NAC (0, 1 and 5 mmol/L) plus MC-LR (0, 2.5, 5 and 10 µg/ml) for 24 h. The reactive oxygen species (ROS) in CHO cells were measured by DCFH-DA, mitochondrial membrane potential (MMP) by fluorescence probe JC-1 staining, and apoptosis index determined by Annexin V-PI staining. Results showed, following exposure to NAC alone for 24 h, cell viability remains higher than 80% at 1 and 5 mmol/L. After exposure to NAC at different concentrations plus MC-LR, cell viability increased, ROS decreased, MMP elevated, and apoptosis index reduced to a certain extent. In conclusion, MC-LR may induce the apoptosis of CHO cells by inducing ROS production which is protected by NAC.

[Feeding habits of Trachurus japonicus in the East China Sea].

A total of 453 Trachurus japonicus specimens with a fork length (FL) of 46-250 mm were sampled in the seasonal light seine net surveys in the East China Sea in May, August, and November, 2008 and in February, 2009. The stomach contents of the specimens were analyzed, and the seasonal and ontogenetic variations in the feeding habits of the T. japonicus were examined by the Kruskal-Wallis test, chi-squared test, and cluster analysis. There were 124 prey species (including not identified) ingested by the T. japonicus, among which, planktonic crustaceans and small-scale marine fish made up the dominant prey groups. According to the percentage index of relative importance (IRI%), Bregmaceros macclellandi was the most dominant prey, accounting for 39.2%, followed by Macrura mysis larva (18.4%), brachyura zoea larva (7.6%), and Euphausia pacifica (6.6%). The feeding intensity of the T. japonicus varied significantly with its FL and season, being the highest for the T. japonicus with a FL of 140-159 mm, higher for the T. japonicus juveniles with a FL of 45-99 mm, while lower for the T. japonicus of other size classes, and the highest in spring and the lowest in winter. Cluster analysis revealed there was an abrupt change in the diet composition for the T. japonicus with a FL of about 100 mm FL. The average trophic level of the T. japonicus in four seasons was 3.51, indicating that the T. japonicus in the East China Sea was of low-level carnivores feeding on plankton and nekton.