Capillary electrophoresis finger print technique (CE-SSCP): an alternative tool for the monitoring activities of HAB species in Baja California Sur Costal

In Mexican waters, there is no a formal and well-established monitoring program of harmful algal blooms (HAB) events. Until now, most of the work has been focused on the characterization of organisms present in certain communities. Therefore, the development of new techniques for the rapid detection of HAB species is necessary. Capillary electrophoresis finger print technique (CE-SSCP) is a fingerprinting technique based on the identification of different conformers dependent of its base composition. This technique, coupled with capillary electrophoresis, has been used to compare and identify different conformers. The aim of this study was to determine if CE-SSCP analysis of ribosomal RNA (rRNA) gene fragments could be used for a rapid identification of toxic and harmful HAB species to improve monitoring activities along the coasts of Baja California Sur, Mexico.Three different highly variable regions of the 18S and 28S rRNA genes were chosen and their suitability for the discrimination of different dinoflagellate species was assessed by CE-SSCP.The CE-SSCP results obtained for the LSU D7 fragment has demonstrated that this technique with this gene region could be useful for the identification of the ten dinoflagellates species of different genera.We have shown that this method can be used to discriminate species and the next step will be to apply it to natural samples to achieve our goal of molecular monitoring for toxic algae in Mexican waters. This strategy will offer an option to improve an early warning system of HAB events for coastal BCS, allowing the possible implementation of mitigation strategies. A monitoring program of HAB species using molecular methods will permit the analysis of several samples in a short period of time, without the pressure of counting with a taxonomic expert in phytoplankton taxonomy.     

Shifts and stasis in marine HAB monitoring in New Zealand

This review article outlines harmful algal bloom (HAB) monitoring practices in New Zealand and highlights the shift from light microscope (LM)-based identification and quantification of the early 1990s to the use of molecular tools to support the HAB monitoring programmes two decades later. Published research and available client information from the monitoring programmes have been reviewed; HAB events and programme changes are highlighted. The current HAB monitoring practices allow for rapid determination of potential biotoxin issues for the shellfish industry and of potential ichthyotoxic events for finfish farmers. The use of molecular tools, including quantitative PCR, has improved risk assessments for those HAB species that are difficult to differentiate to species level using LM. This has enabled rapid feedback to aquaculture managers during HAB events. Tests for biotoxins in flesh remain the regulatory tools for commercially harvested shellfish, but this is supported by the weekly phytoplankton monitoring data. Recreational (non-commercial) shellfish harvesting and commercial finfish aquaculture rely solely on phytoplankton monitoring to assess the biotoxin risk. HAB monitoring in New Zealand continues to maintain internationally recognised standards, and the government-funded research programmes feed the latest knowledge and technical methods into the programmes. The early dependence on light microscopy continues but is now supported by molecular tools, with a view to employing multi-species detection systems in the future. The traditional mouse bioassay test has been fully replaced by chemical tests.     

Seasonal and annual dynamics of harmful algae and algal toxins revealed through weekly monitoring at two coastal ocean sites off southern California, USA

Reports of toxic harmful algal blooms (HABs) attributed to the diatom Pseudo-nitzschia spp. have been increasing in California during the last several decades. Whether this increase can be attributed to enhanced awareness and monitoring or to a dramatic upswing in the development of HAB events remains unresolved. Given these uncertainties, the ability to accurately and rapidly identify an emerging HAB event is of high importance. Monitoring of HAB species and other pertinent chemical/physical parameters at two piers in southern California, Newport and Redondo Beach, was used to investigate the development of a site-specific bloom definition for identifying emerging domoic acid (DA) events. Emphasis was given to abundances of the Pseudo-nitzschia seriata size category of Pseudo-nitzschia due to the prevalence of this size class in the region. P. seriata bloom thresholds were established for each location based on deviations from their respective long-term mean abundances, allowing the identification of major and minor blooms. Sixty-five percent of blooms identified at Newport Beach coincided with measurable DA concentrations, while 36 % of blooms at Redondo Beach coincided with measurable DA. Bloom definitions allowed for increased specificity in multiple regression analysis of environmental forcing factors significant to the presence of DA and P. seriata. The strongest relationship identified was between P. seriata abundances 2 weeks following upwelling events at Newport Beach.     

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).     

Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment

Pesticides are released intentionally into the environment and, through various processes, contaminate the environment. Three of the main classes of pesticides that pose a serious problem are organochlorines, organophosphates and carbamates. While pesticides are associated with many health effects, there is a lack of monitoring data on these contaminants. Traditional chromatographic methods are effective for the analysis of pesticides in the environment, but have limitations and prevent adequate monitoring. Enzymatic methods have been promoted for many years as an alternative method of detection of these pesticides. The main enzymes that have been utilised in this regard have been acetylcholinesterase, butyrylcholinesterase, alkaline phosphatase, organophosphorus hydrolase and tyrosinase. The enzymatic methods are based on the activation or inhibition of the enzyme by a pesticide which is proportional to the concentration of the pesticide. Research on enzymatic methods of detection, as well as some of the problems and challenges associated with these methods, is extensively discussed in this review. These methods can serve as a tool for screening large samples which can be followed up with the more traditional chromatographic methods of analysis.     

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.     

Note: steps taken to optimise probe specificity and signal intensity prior to field validation of the MIDTAL (Microarray for the Detection of Toxic Algae)

A microarray for the detection of toxic algal species was developed in the European Union 7th Framework project MIDTAL. We initially tested all available fluorescence in situ hybridisation probes for toxic algae, which are normally designed to a length of 18 nt, and found that in most cases the signal was rather weak or all probes designed from the second half of the molecule were inaccessible in a microarray format because of secondary structure of the ribosomal RNA molecule We modified the length of the probes, the fragmentation of the rRNA, the stringency of the washing buffers and the length of the spacer molecules linking the probes to the glass surface of the microarray. Because of the secondary structure of the rRNA molecule, regions of the molecule can be difficult to access by the probes. Each of these modifications has improved probe accessibility and probe specificity to reduce false positives.     

Water quality guidelines for chemicals: learning lessons to deliver meaningful environmental metrics

Many jurisdictions around the globe have well-developed regulatory frameworks for the derivation and implementation of water quality guidelines (WQGs) or their equivalent (e.g. environmental quality standards, criteria, objectives or limits). However, a great many more still do not have such frameworks and are looking to introduce practical methods to manage chemical exposures in aquatic ecosystems. There is a potential opportunity for learning and sharing of data and information between experts from different jurisdictions in order to deliver efficient and effective methods to manage potential aquatic risks, including the considerable reduction in the need for aquatic toxicity testing and the rapid identification of common challenges. This paper reports the outputs of an international workshop with representatives from 14 countries held in Hong Kong in December 2011. The aim of the workshop and this paper was to identify ‘good practice’ in the development of WQGs to deliver to a range of environmental management goals. However, it is important to broaden this consideration to cover often overlooked facets of implementable WQGs, such as demonstrable field validation (i.e. does the WQG protect what it is supposed to?), fit for purpose of monitoring frameworks (often an on-going cost) and finally how are these monitoring data used to support management decisions in a manner that is transparent and understandable to stakeholders. It is clear that regulators and the regulated community have numerous pressures and constraints on their resources. Therefore, the final section of this paper addresses potential areas of collaboration and harmonisation. Such approaches could deliver a consistent foundation from which to assess potential chemical aquatic risks, including, for example, the adoption of bioavailability-based approaches for metals, whilst reducing administrative and technical burdens in jurisdictions.     

Evaluation of the MIDTAL microarray chip for monitoring toxic microalgae in the Orkney Islands, U.K.

Harmful or nuisance algal blooms can cause economic damage to fisheries and tourism. Additionally, toxins produced by harmful algae and ingested via contaminated shellfish can be potentially fatal to humans. The seas around the Orkney Islands, UK currently hold a number of toxic algal species which cause shellfishery closures in most years. Extensive and costly monitoring programs are carried out to detect harmful microalgae before they reach action levels. However, the ability to distinguish between toxic and non-toxic strains of some algae is not possible using these methods. The microarrays for the detection of toxic algae (MIDTAL) microarray contains rRNA probes for toxic algal species/strains which have been adapted and optimized for microarray use. In order to investigate the use of the chip for monitoring in the Orkney Islands, samples were collected between 2009 and 2011 from Brings Deep, Scapa Flow, Orkney Islands, UK; RNA was extracted and hybridized with generation 2 and 3.1 of the chip. The data were then compared to cell counts performed under light microscopy and in the case of Alexandrium tamarense to qPCR data targeting the saxitoxin gene and the LSU-rRNA gene. A good agreement between cell numbers and microarray signal was found for A. tamarense, Pseudo-nitzschia sp., Dinophysis sp. (r?<?0.5, for all) in addition to this there the chip successfully detected a large bloom of Karenia mikimotoi (r?<?0.70) in August and September 2011. Overall, there was good improvement in probe signal between generation 2 and generation 3.1 of the chip with much less variability and more consistent results and better correlation between the probes. The chip performed well for A. tamarense group I signal to cell numbers in calibrations (r?>?0.9). However, in field samples, this correlation was slightly lower suggesting interactions between all species in the sample may affect signal. Overall, the chip showed it could identify the presence of target species in field samples although some work is needed to improve the quantitative nature of the chip before it would be suitable for monitoring in the Orkney Islands.     

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.     

The potential environmental risks of pharmaceuticals in Vietnamese aquatic systems: case study of antibiotics and synthetic hormones

Presently, many pharmaceuticals are listed as emerging contaminants since they are considered to be great potential threats to environmental ecosystems. These contaminants, thus, present significant research interest due to their extensive use and their physicochemical and toxicological properties. This review discusses a whole range of findings that address various aspects of the usage, occurrence, and potentially environmental risks of pharmaceuticals released from various anthropogenic sources, with emphasis on the aquatic systems in Vietnam. The published information and collected data on the usage and occurrence of antibiotics and synthetic hormone in effluents and aquatic systems of Vietnam is reported. This is followed by a potential ecological risk assessment of these pollutants. The extensive use of antibiotics and synthetic hormones in Vietnam could cause the discharge and accumulation of these contaminants in the aquatic systems and potentially poses serious risks for ecosystems. Vietnam is known to have extensively used antibiotics and synthetic hormones, so these contaminants are inevitably detected in aquatic systems. Thus, an appropriate monitoring program of these contaminants is urgently needed in order to mitigate their negative effects and protect the ecosystems.     

Determination of brevetoxin in recent marine sediments

Harmful algal blooms (HAB) of Karenia brevis (K. brevis) produce a suite of lipid soluble polyether brevetoxins, known to cause environmental, health and economic ill effects. There is evidence that K. brevis has increased in abundance over the past 50 years, but the dataset is incomplete. The objective of this paper was to analyze sediment from an area where K. brevis blooms have occurred and investigate if these compounds are incorporated into the underlying sediment, thus potentially allowing the use of brevetoxins as an indicator of past K. Brevis blooms. The results from LC-ESI-MS-MS analyses of brevetoxin analogs detected in surficial sediments from three sites (Fort Meyers Beach [FMB], Big Hickory Pass [BHP] and Big Carlos Pass [BCP]) along the Southwest Florida coastline with prior HAB history are promising. The analogs detected from BHP sediments were PbTx-2 and PbTx-3 with values of 0.81 and 3.1 ng g(-1) dry sediment, respectively. The detected PbTx-2 from BCP was 3.6 ng g(-1) dry sediment, while the detected PbTx-3 from BCP was 9.7 ng g(-1) dry sediment. PbTx-3 was only detected at the FMB site (2.7 ng g(-1) dry sediment). The detection of brevetoxins in recent sediments where K. brevis have occurred indicates brevetoxin incorporation into marine sediments.     

Surface and ground waters characterization in Tuscany (Italy) by using algal bioassay and pesticide determinations: comparative evaluation of the results and hazard assessment of the pesticides impact on primary productivity

Algal bioassays and pesticide analysis were joined in Tuscany regional monitoring of surface and ground waters. Results were compared. Waters affected algal growth in any of the following ways: stimulation, inhibition, stimulation at low concentrations and inhibition at higher concentrations. Most of ground waters inhibited algal growth whereas only a little percentage were contaminated with pesticide. Most of surface waters stimulated algal growth and a higher percentage were contaminated with pesticides. In waters with inhibitory effect toxicological parameters (EC50, EC25, EC10 and NOEC) were measured and compared; EC25 values were very close to NOEC values. Hazard to aquatic primary productivity for each pesticide found during chemical monitoring was evaluated; for this aim the margin of safety (MOS) was calculated by using algal toxicity and the pesticide concentration measured.     

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.     

Seasonal variation of sediment toxicity in the Rivers Dommel and Elbe

Contaminated sediment in the river basin has become a source of pollution with increasing importance to the aquatic ecosystem downstream. To monitor the temporal changes of the sediment bound contaminants in the River Elbe and the River Dommel monthly toxicity tests were applied to layered sediment and river water samples over the course of 10 months. There is an indication that contaminated sediments upstream adversely affected sediments downstream, but this process did not cause a continuous increase of sediment toxicity. A clear decrease of toxic effects in water and upper layer sediment was observed at the River Elbe station in spring related to high water discharge and algal blooms. The less obvious variation of sediment toxicity in the River Dommel could be explained by stable hydrological conditions. Future monitoring programmes should promote a more frequent and intensive sampling regime during these particular events for ecotoxicological evaluation.     

Environmental toxicity monitoring using electrochemical biosensing systems

Environmental monitoring faces the challenge of measuring an increasing number of analytes at ever decreasing concentrations. Since not all species of a given analyte have the same detrimental impact on the environment, new analytical devices and techniques are required to distinguish between the different species of a pollutant or different groups of pollutants. This paper describes analytical techniques based on biomaterials that are toxically sensitive to pollutants. This approach permits the biomonitoring of certain compounds by looking at their toxic properties. Although these techniques are based on a sound analytical strategy, their applications are limited because most of the interactions between the biological material and the analyte are irreversible. Additionally, the immobilised biological material has a limited stability. Several biomonitoring strategies based on electrochemical biosensing are discussed here and how to recover the bioactivity of biosensing system, both in discrete and automated procedures, is also reviewed.     

Microarray testing for the presence of toxic algae monitoring programme in Galicia (NW Spain)

Rapid and reliable detection of harmful algae in coastal areas and shellfish farms is an important requirement of monitoring programmes. Monitoring of toxic algae by means of traditional methods, i.e., light microscopy, can be time consuming when many samples have to be routinely analysed. Reliable species identification requires expensive equipment and trained personnel to carry out the analyses. However, all techniques for the monitoring of harmful algae usually require transportation of samples to specialised laboratories. In many monitoring laboratories, results are usually obtained within five working days after receiving the sample and therefore preventative measures are not always possible. Molecular technologies are rapidly improving the detection of phytoplankton and their toxins and the speed at which the results can be obtained. Assays are based on the discrimination of the genetic differences of the different species and species-specific probes can be designed. Such probes have been adapted to a microarray or phylochip format and assessed in several EU monitoring sites. Microarray results are presented for 1 year of field samples validated with cell counts from concentrated samples taken during toxic events from the weekly sampling of the Galician Monitoring Programme done by INTECMAR. The Galician monitoring laboratory does their own counting and their results are posted on their web site within 24 h. There was good correlation between cells present and microarray signals. In the few cases of false negatives, these can be attributed to poor RNA extraction of the target species, viz. Prorocentrum or Dinophysis. Where potential false positives were encountered, the smaller volume taken for cell counts as compared to the upto 300 times more volume taken for RNA extraction for the microarray is likely the cause for these differences, making the microarray more sensitive. The microarray was able to provide better species resolution in Alexandrium and Pseudo-nitzschia. In all cases, the toxins recovered by the toxin array were matched by target species in the array or in the cell counts.     

Whole cell hybridisation for monitoring harmful marine microalgae

Fluorescence in situ hybridisation (FISH) is a powerful molecular biological tool to detect and enumerate harmful microorganism in the marine environment. Different FISH methods are available, and especially in combination with automated counting techniques, the potential for a routine monitoring of harmful marine microalgae is attainable. Various oligonucleotide probes are developed for detecting harmful microalgae. However, FISH-based methods are not yet regularly included in monitoring programmes tracking the presence of harmful marine microalgae. A limitation factor of the FISH technique is the currently available number of suited fluorochromes attached to the FISH probes to detect various harmful species in one environmental sample at a time. However, coupled automated techniques, like flow cytometry or solid-phase cytometry, can facilitate the analysis of numerous field samples and help to overcome this drawback. A great benefit of FISH in contrast to other molecular biological detection methods for harmful marine microalgae is the direct visualisation of the hybridised target cells, which are not permitted in cell free formats, like DNA depending analysis methods. Therefore, an additional validation of the FISH-generated results is simultaneously given.     

Oxidative stress and detoxification biomarker responses in aquatic freshwater vertebrates exposed to microcystins and cyanobacterial biomass

Cyanobacterial blooms represent a serious threat to the aquatic environment. Among other effects, biochemical markers have been studied in aquatic vertebrates after exposures to toxic cyanobacteria. Some parameters such as protein phosphatases may serve as selective markers of exposure to microcystins, but under natural conditions, fish are exposed to complex mixtures, which affect the overall biomarker response. This review aims to provide a critical summary of biomarker responses in aquatic vertebrates (mostly fish) to toxic cyanobacteria with a special focus on detoxification and oxidative stress. Detoxification biomarkers such as glutathione (GSH) and glutathione-S-transferase (GST) showed very high variability with poor general trends. Often, stimulations and/or inhibitions and/or no effects at GSH or GST have been reported, even within a single study, depending on many variables, including time, dose, tissue, species, etc. Most of the oxidative stress biomarkers (e.g., superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) provided more consistent responses, but only lipid peroxidation (LPO) seemed to fulfill the criteria needed for biomarkers, i.e., a sufficiently long half-life and systematic response. Indeed, reviewed papers demonstrated that toxic cyanobacteria systematically elevate levels of LPO, which indicates the important role of oxidative damage in cyanobacterial toxicity. In summary, the measurement of biochemical changes under laboratory conditions may provide information on the mode of toxic action. However, comparison of different studies is very difficult, and the practical use of detoxification or oxidative stress biomarkers as diagnostic tools or early warnings of cyanobacterial toxicity is questionable.     

垃圾焚烧飞灰有毒重金属固化稳定技术研究综述

采用焚烧法处理生活垃圾正呈快速增长的趋势,焚烧过程产生的飞灰中所含有的大量有毒重金属对自然环境和人类生存都造成极大影响。阐述了目前常用的几种固化稳定垃圾焚烧飞灰有毒重金属的方法,综合分析了其固化特点和优缺点,并初步探索了一种低能耗环境下实现高温熔融处置技术的有效途径,从而为垃圾焚烧飞灰高温固化稳定有毒重金属的实施提供一定的理论基础和技术支持。