The quantitative real-time PCR applications in the monitoring of marine harmful algal bloom (HAB) species

In the last decade, various molecular methods (e.g., fluorescent hybridization assay, sandwich hybridization assay, automatized biosensor detection, real-time PCR assay) have been developed and implemented for accurate and specific identification and estimation of marine toxic microalgal species. This review focuses on the recent quantitative real-time PCR (qrt-PCR) technology developed for the control and monitoring of the most important taxonomic phytoplankton groups producing biotoxins with relevant negative impact on human health, the marine environment, and related economic activities. The high specificity and sensitivity of the qrt-PCR methods determined by the adequate choice of the genomic target gene, nucleic acid purification protocol, quantification through the standard curve, and type of chemical detection method make them highly efficient and therefore applicable to harmful algal bloom phenomena. Recent development of qrt-PCR-based assays using the target gene of toxins, such as saxitoxin compounds, has allowed more precise quantification of toxigenic species (i.e., Alexandrium catenella) abundance. These studies focus only on toxin-producing species in the marine environment. Therefore, qrt-PCR technology seems to offer the advantages of understanding the ecology of harmful algal bloom species and facilitating the management of their outbreaks.     

An evaluation of the applicability of microarrays for monitoring toxic algae in Irish coastal waters

The applicability of microarrays to monitor harmful algae across a broad range of ecological niches and toxic species responsible for harmful algal events has been one of the key tasks in the EU Seventh Framework Programme (FP7)-funded Microarrays for the Detection of Toxic Algae project. The technique has a strong potential for improving speed and accuracy of the identification of harmful algae and their toxins to assist monitoring programmes. Water samples were collected from a number of coastal sites around Ireland, including several that are used in the Irish National Phytoplankton and Biotoxin Monitoring Programme. Ribosomal RNA was extracted from filtered field samples, labelled with a fluorescent dye, and hybridised to probes spotted in a microarray format on a glass slide. The fluorescent signal intensity of the hybridisation to >120 probes on the chip was analysed and compared with actual field counts. There was a general agreement between cell counts and microarray signal. Results are presented for field samples taken from a range of stations along the Irish coastline known for harmful algal events during the first field trial (July 2009–April 2010).     

Sensitive time-resolved fluoroimmunoassay for quantitative determination of clothianidin in agricultural samples

Europium (Eu³⁺)-labeled antibody was used as a fluorescent label to develop a highly sensitive time-resolved fluoroimmunoassay (TRFIA) for determination of clothianidin residues in agricultural samples. Toward this goal, the Eu³⁺-labeled polyclonal antibody and goat anti-rabbit antibody were prepared for developing and evaluating direct competitive TRFIA (dc-TRFIA) and indirect competitive TRFIA (ic-TRFIA). Under optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (LOD, IC₁₀) of clothianidin were 9.20 and 0.0909 μg/L for the dc-TRFIA and 2.07 and 0.0220 μg/L for the ic-TRFIA, respectively. The ic-TRFIA has no obvious cross-reactivity with the analogues of clothianidin except for dinotefuran. The average recoveries of clothianidin from spiked water, soil, cabbage, and rice samples were estimated to range from 74.1 to 115.9 %, with relative standard deviations of 3.3 to 11.7 %. The results of TRFIA for the blind samples were largely consistent with gas chromatography (R ²?=?0.9902). The optimized ic-TRFIA might become a sensitive and satisfactory analytical method for the quantitative monitoring of clothianidin residues in agricultural samples.     

Introduction to project MIDTAL: its methods and samples from Arcachon Bay, France

Microalgae worldwide regularly cause harmful effects, considered from the human perspective, in that they cause health problems and economic damage to fisheries and tourism. Cyanobacteria cause similar problems in freshwaters. These episodes encompass a broad range of phenomena collectively referred to as “harmful algal blooms” (HABs). For adequate management of these phenomena, monitoring of microalgae is required. However, effective monitoring is time-consuming because cell morphology as determined by light microscopy may be insufficient to give definitive species and toxin attribution. In the European Union FP7 project MIDTAL (Microarrays for the Detection of Toxic Algae), we achieved rapid species identification using rRNA genes as the target. These regions can be targeted for probe design to recognise species or even strains. We also included antibody reactions to specific toxins produced by these microalgae because, even when cell numbers are low, toxins can be present and can accumulate in the shellfish. Microarrays are the state-of-the-art technology in molecular biology for the processing of bulk samples for detection of target RNA/DNA sequences. After 36 months, we have completed RNA-cell number–signal intensity calibration curves for 18 HAB species and the analysis of monthly field samples from five locations from year 1. Results from one location, Arcachon Bay (France), are reported here and compared favourably with cell counts in most cases. In general, the microarray was more sensitive than the cell counts, and this is likely a reflection in the difference in water volume analysed with the volume filtered for the microarray an order of magnitude greater.     

淡水水体麻痹性贝毒污染的现状

麻痹性贝毒是一类水溶性的神经毒素,造成神经细胞电压敏感性钠离子通道(VSSC)高亲和力障碍,与细胞膜离子结合后引起细胞膜内外正常离子的流动失衡,造成膜电位反常,使人瘫痪.麻痹性贝毒的污染及其产毒藻已成为一个受人关注的公共健康问题.综述麻痹性贝毒的理化性质和毒性特点、淡水水体麻痹性贝毒污染及其5种淡水产毒蓝藻,并对淡水中PSP防治工作提出建议.     

Effects of zeolites on cultures of marine micro-algae

GOAL, SCOPE AND BACKGROUND: The cation-exchange capacity of zeolites is well known and has been increasingly explored in different fields with both economic and environmental successes. In aquatic medium with low salinity, zeolites have found multiple applications. However, a review of the literature on the applications of zeolites in salt waters found relatively few articles, including some recently published papers. The purpose of this review is to present the state-of-the-art on applications of using zeolites for amending the trace elemental contents of salt water as well as the implications of this property for promoting marine micro-algal growth. MAIN FEATURES: This paper deals with the following features: Sorption capacity of zeolites including 1. application of zeolites in saltwater, 2. the role of silicon and zeolites on cultures of micro-algae, and 3. the role of organically chelated trace metals. RESULTS: The following competing factors have been identified as effects of zeolites on algal growth in salt water: (i) ammonia decrease: growth inhibition reduced; (ii) macro-nutrients increase, mainly silicon: stimulation of silicon-dependent algae; (iii) trace metals increase (desorption from zeolites) or decrease (adsorption): inhibition or stimulation, depending on the nature of the element and its concentration; and, (iv) changes in the chelating organics exudation: inhibition or stimulation of growth, depending on the (a) nature of the complexed element; (b) bioavailability of the complex; and (c) concentration of the elements simultaneously present in inorganic forms. DISCUSSION: Zeolites have been capable of stimulating the growth of the silicon-demanding marine micro-algae, like diatoms, mainly because they can act as a silicon buffer in seawater. Zeolites can also influence the yield of non-silicon-demanding algae, because the changes they can cause (liberation and adsorption of trace elements) in the composition of the medium. CONCLUSIONS: Zeolites have been capable of stimulating the growth of the marine micro-algae. However, the extent of ion exchange between zeolite and seawater, which conditions the effects, will depend on several factors: (1) initial metal concentration in seawater; (2) levels of trace metals in the zeolites (contaminants); (3) characteristics of the zeolites in terms of both ion-exchange capacity and specific affinities for the different cations; (4) quantity of zeolite per litre of solution; (5) pH and (6) response of the organism in terms of liberation of organic ligands. RECOMMENDATIONS AND PERSPECTIVES: RECOMMENDATIONS: Therefore, a previous investigation in each particular case is recommended, in order to select the zeolitic characteristics and concentrations that will maximize the algal yield. PERSPECTIVES: Stimulation of phytoplankton growth can be economically relevant since phytoplankton constitutes the basis of the marine food webs and is required in fish farming nurseries in the marine aquaculture industry. Zeolites are cheap, only small amounts (few milligrams per liter of culture) are required and the addition of some micro-nutrients may be omitted. Therefore, the inclusion of zeolites in algal cultures in aquaculture may have economic advantages.     

Use of Seawater in Flue Gas Desulfurization

For seasite power plants using seawater for condenser cooling, Bechtel developed a new process to remove better than 90 percent of flue gas SO₂ using seawater and lime. Tests demonstrate that marine life is not affected by the effluent from this unique scrubbing process; therefore, the aqueous effluent containing reacted products (gypsum) in solution at low concentration is suitable for discharge to the sea.     

Production of monoclonal antibody and application in indirect competitive ELISA for detecting okadaic acid and dinophytoxin-1 in seafood

Background, aim, and scope

Okadaic acid (OA) and analogues of dinophysistoxin (DTX) are key diarrheic shellfish poisoning (DSP) toxins, which possibly arouse DSP symptoms by consuming the contaminated shellfish. Because of the stable toxicity in high temperature and the long-term carcinogenicity, the outbreaks of DSP related to consumption of bivalve mollusks contaminated by DSP toxins pose a hazard to public health. Therefore, it is worth developing a fast and reliable analytical method for the detection of OA and analogues in shellfish. In this paper, an indirect competitive enzyme-linked immunosorbent assay (ELISA) (icELISA) for detecting OA and DTX-1 in seafood was developed based on monoclonal antibody (McAb).

Methods

The OA was conjugated to human immunoglobulin G (IgG) and bovine serum albumin (BSA) by the active ester method as the immune antigen and the detective antigen. The spleen cells from BALB/c mice immunized with OA-IgG were fused with SP2/0 myeloma cells. A hybridoma cell line, which secreted McAb against OA, was selected by ??limiting dilution?? cloning. An icELISA was developed based on immobilized conjugate (OA-BSA) competing the McAb with the free OA in seafood sample.

Results

A hybridoma cell line, which secreted IgG1 subclass monoclonal antibody (McAb) against OA, was selected. The IC₅₀ of the McAb for OA and dinophytoxin-1 (DTX-1) were 4.40 and 3.89 ng/mL, respectively. Based on the McAb, an indirect competitive ELISA for detection of OA and DTX-1 in seafood was developed. The regression equation was y?=?54.713x???25.879 with a coefficient correlation of R ²?=?0.9729. The linear range and the limit of detection were 0.4?C12.5 and 0.45 ng/mL, respectively. The average recovery of OA and DTX-1 spiked shellfish was 82.29% with the coefficient of variation of 7.67%.

Conclusion

The developed icELISA is a fast, sensitive, and convenient assay for detecting of total amount of OA and DTX-1 in seafood.     

Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris

Adverse effects of manufactured nickel oxide nanoparticles on the microalgae Chlorellavulgaris were determined by algal growth-inhibition test and morphological observation via transmission electron microscopy (TEM). Results showed that the NiO nanoparticles had severe impacts on the algae, with 72 h EC(50) values of 32.28 mg NiOL(-1). Under the stress of NiO nanoparticles, C. vulgaris cells showed plasmolysis, cytomembrane breakage and thylakoids disorder. NiO nanoparticles aggregated and deposited in algal culture media. The presence of algal cells accelerated aggregation of nanoparticles. Moreover, about 0.14% ionic Ni was released when NiO NPs were added into seawater. The attachment of aggregates to algal cell surface and the presence of released ionic Ni were likely responsible for the toxic effects. Interestingly, some NiO nanoparticles were reduced to zero valence nickel as determined by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis. The maximum ratios of nickel reduction was achieved at 72 h of exposure, in accordance with the time-course of changes in soluble protein content of treated C. vulgaris, implying that some proteins of algae are involved in the process. Our results indicate that the toxicity and bioavailability of NiO nanoparticles to marine algae are reduced by aggregation and reduction of NiO. Thus, marine algae have the potential for usage in nano-pollution bio-remediation in aquatic system.     

In situ measurement of isoprene in the marine air and surface seawater from the western North Pacific

Isoprene (2-methyl-1,3-butadiene) was measured on board of R/V Mirai for eight air samples and 14 seawater samples collected in the western North Pacific during ACE-Asia campaign (from 18 to 26 May 2001). The measurements were conducted in situ using a cryo-focus/gas chromatography/mass spectrometry (Cryo/GC/MS). Concentrations of isoprene ranged from 7.2 to 110 parts-per-trillion (pptv) in the marine air, and ranged from below 12 to 94 pmol l⁻¹ in the seawater. Based on these results, sea-to-air fluxes of isoprene were calculated to be 184 and 300 nmol m⁻² day⁻¹ for two samples, and the upper limits of the fluxes were also calculated to be from 32 to 300 nmol m⁻² day⁻¹. Atmospheric isoprene concentrations cannot be explained only by the flux from the seawater. Thus, the concentrations of isoprene in the marine air in western North Pacific should be significantly affected by terrestrial vegetational emission and subsequent long-range atmospheric transport of isoprene.     

赤泥复合剂絮凝处理太湖水华藻与海洋赤潮藻

研究了一种新型赤泥复合剂对太湖夏季水华原水、实验室培养海洋赤潮藻及模拟海水的絮凝除藻降浊性能。结果表明,赤泥复合剂具有良好的除藻降浊效果,对太湖水华藻去除率为97.3%,原水浊度去除率为95%;对海洋小球藻和三角褐指藻2种赤潮藻去除率分别高达99.1%和98.9%;对2种模拟海水的最高除浊率分别为96.3%和95.3%,其剩余浊度均低于4 NTU,从而为治理淡水水华与海洋赤潮提供一种新技术,同时为赤泥综合利用提供一种新途径。     

Monitoring of the booster biocide dichlofluanid in water and marine sediment of Greek marinas

Dichlofluanid (N-dichlorofluoromethylthio-N'-dimethyl-N-phenylsulphamide) is used as booster biocide in antifouling paints. The occurrence of dichlofluanid and its metabolite DMSA (N'-dimethyl-N-phenyl-sulphamide) was monitored in seawater and marine sediment from three Greek marinas. Seawater and sediment samples were collected at three representative positions and one suspected hotspot in each marina and shipped to the laboratory for chemical analysis. As part of the project, an analytical method had been developed and validated. Furthermore, some additional experiments were carried out to investigate the potential contribution of paint particle bound dichlofluanid on the total concentration in the sediment. As expected, given its known high hydrolytic degradation rate, no detectable concentrations of dichlofluanid were measured in any of the seawater samples. DMSA was detected in seawater samples at very low concentrations varying from <3 ng l(-1) (LOD) to 36 ng l(-1). During method validation, it had already been demonstrated that dichlofluanid is unstable in sediment and can therefore only be determined as its metabolite DMSA. In a separate experiment, in which marine sediment was spiked with artificial paint particles containing dichlofluanid and then analysed according to the validated method, it was demonstrated that if there is any dichlofluanid originating from paint particles, this would be determined as DMSA. No DMSA was detected in any of the sediment samples. It could therefore be concluded that there were no significant concentrations of dichlofluanid in the sediment samples.     

Phytoplankton dynamics with a special emphasis on harmful algal blooms in the Mar Piccolo of Taranto (Ionian Sea,Italy)

The response of phytoplankton assemblages to the closure of urban sewage outfalls (USOs) was examined for the Mar Piccolo of Taranto (Mediterranean Sea), a productive semi-enclosed coastal marine ecosystem devoted to shellfish farming. Phytoplankton dynamics were investigated in relation to environmental variables, with a particular emphasis on harmful algal blooms (HABs). Recent analyses evidenced a general reduction of the inorganic nutrient loads, except for nitrates and silicates. Also phytoplankton biomass (chlorophyll a) and abundances were characterized by a decrease of the values, except for the inner area of the basin (second inlet). The phytoplankton composition changed, with nano-sized species, indicators of oligotrophic conditions, becoming dominant over micro-sized species. If the closure of the USOs affected phytoplankton dynamics, however, it did not preserve the Mar Piccolo from HABs and anoxia crises. About 25 harmful species have been detected throughout the years, such as the potentially domoic acid producers Pseudo-nitzschia cf. galaxiae and P seudo-nitzschia cf. multistriata, identified for the first time in these waters. The presence of HABs represents a threat for human health and aquaculture. Urgent initiatives are needed to improve the communication with authorities responsible for environmental protection, economic development, and public health for a sustainable mussel culture in the Mar Piccolo.     

Semi-specific Microbacterium phyllosphaerae-based microbial sensor for biochemical oxygen demand measurements in dairy wastewater

Although the long incubation time of biochemical oxygen demand (BOD₇) measurements has been addressed by the use of microbial biosensors, the resulting sensor-BOD values gained from the measurements with specific industrial wastewaters still underestimates the BOD value of such samples. This research aims to provide fast and more accurate BOD measurements in the dairy wastewater samples. Unlike municipal wastewater, wastewater from the dairy industry contains many substrates that are not easily accessible to a majority of microorganisms. Therefore, a bacterial culture, Microbacterium phyllosphaerae, isolated from dairy wastewater was used to construct a semi-specific microbial biosensor. A universal microbial biosensor based on Pseudomonas fluorescens, which has a wide substrate spectrum but is nonspecific to dairy wastewater, was used as a comparison. BOD biosensors were calibrated with OECD synthetic wastewater, and experiments with different synthetic and actual wastewater samples were carried out. Results show that the semi-specific M. phyllosphaerae-based microbial biosensor is more sensitive towards wastewaters that contain milk derivates and butter whey than the P. fluorescens-based biosensor. Although the M. phyllosphaerae biosensor underestimates the BOD₇ value of actual dairy wastewaters by 25–32 %, this bacterial culture is more suitable for BOD monitoring in dairy wastewater than P. fluorescens, which underestimated the same samples by 46–61 %.     

Biological effects of palytoxin-like compounds from Ostreopsis cf. ovata: A multibiomarkers approach with mussels Mytilus galloprovincialis

Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans.The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis. Organisms were sampled during various phases of a O. cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na⁺/K⁺-ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage).Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O. cf. ovata. These organisms exhibited a marked inhibition of the Na⁺/K⁺-ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms.     

The algal toxicity of silver engineered nanoparticles and detoxification by exopolymeric substances

In this study, we report that silver ions (Ag⁺) from the oxidative dissolution of silver engineered nanoparticles (Ag-ENs) determined the EN toxicity to the marine diatom Thalassiosira weissflogii. Most of the Ag-ENs formed non-toxic aggregates (>0.22 μm) in seawater. When the free Ag⁺ concentration ([Ag⁺]_F) was greatly reduced by diafiltration or thiol complexation, no toxicity was observed, even though the Ag-ENs were better dispersed in the presence of thiols with up to 1.08 × 10⁻⁵ M Ag-ENs found in the <0.22 μm fraction, which are orders of magnitude higher than predicted for the natural aquatic environment. The secretion of polysaccharide-rich algal exopolymeric substances (EPS) significantly increased at increasing [Ag⁺]_F. Both dissolved and particulate polysaccharide concentrations were higher for nutrient-limited cells, coinciding with their higher Ag⁺ tolerance, suggesting that EPS may be involved in Ag⁺ detoxification.     

Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy

Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella — where two morphologically similar species impossible to separate by light microscopy were distinguished — in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring.     

水处理工艺去除藻毒素的效果探讨

探讨了饮用水处理的各工艺过程对藻毒素的去除作用,并根据前期已进行的实验对各工艺的优化组合进行了讨论,认为以气浮代替沉淀,并附之以粉末活性炭吸附预处理可有效地去除藻毒素;对没有条件改造沉淀池的水厂可暂时采用预氯化和粉末活性炭吸附组合的工艺也可达到较好的效果;对生物预处理工艺进行适当的强化,可达到对有机物、藻类、藻毒素等物质的有效去除.     

Lethal effects on different marine organisms, associated with sediment�Cseawater acidification deriving from CO2 leakage

CO(2) leakages during carbon capture and storage in sub-seabed geological structures could produce potential impacts on the marine environment. To study lethal effects on marine organisms attributable to CO(2) seawater acidification, a bubbling CO(2) system was designed enabling a battery of different tests to be conducted, under laboratory conditions, employing various pH treatments (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5). Assays were performed of three exposure routes (seawater, whole sediment, and sediment elutriate). Individuals of the clam (Ruditapes philippinarum) and early-life stages of the gilthead seabream, Sparus aurata, were exposed for 10?days and 72?h, respectively, to acidified clean seawater. S. aurata larvae were also exposed to acidified elutriate samples, and polychaete organisms of the specie Hediste diversicolor and clams R. philippinarum were also exposed for 10?days to estuarine whole sediment. In the fish larvae elutriate test, 100?% mortality was recorded at pH?6.0, after 48?h of exposure. Similar results were obtained in the clam sediment exposure test. In the other organisms, significant mortality (p?相似文献   

GPR-Analyzer: a simple tool for quantitative analysis of hierarchical multispecies microarrays

Monitoring of marine microalgae is important to predict and manage harmful algae blooms. It currently relies mainly on light-microscopic identification and enumeration of algal cells, yet several molecular tools are currently being developed to complement traditional methods. MIcroarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a hierarchical multispecies microarray as one of these tools. Prototype arrays are currently being tested with field samples, yet the analysis of the large quantities of data generated by these arrays presents a challenge as suitable analysis tools or protocols are scarce. This paper proposes a two-part protocol for the analysis of the MIDTAL and other hierarchical multispecies arrays: Signal-to-noise ratios can be used to determine the presence or absence of signals and to identify potential false-positives considering parallel and hierarchical probes. In addition, normalized total signal intensities are recommended for comparisons between microarrays and in order to relate signals for specific probes to cell concentrations using external calibration curves. Hybridization- and probe-specific detection limits can be calculated to help evaluate negative results. The suggested analyses were implemented in “GPR-Analyzer”, a platform-independent and graphical user interface-based application, enabling non-specialist users to quickly and quantitatively analyze hierarchical multispecies microarrays. It is available online at http://folk.uio.no/edvardse/gpranalyzer.