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.     

Review: advances in electrochemical genosensors-based methods for monitoring blooms of toxic algae

Harmful algal blooms (HABs), which have expanded worldwide in their occurrence and frequency, are a serious menace to aquatic ecosystems and humans. The development of rapid, accurate and cost-effective detection systems for toxic algal monitoring in aquatic environments is urgently required. Although many efforts have been devoted to develop reliable tools to monitor the entire spectrum of existing toxic algae, a portable semi-automated system that enables HAB monitoring at a low cost is still not available for general purchase. This work reviews the challenges and opportunities in translating the remarkable progress of electrochemical genosensors-based methods towards practical in situ HAB monitoring applications. It is specifically focused on reviewing the optimised methods for a detection system based on a sandwich hybridisation assay (SHA) performed over transducer platforms of different materials, geometries and dimensions and presenting the diverse advantages and disadvantages among them. Probe design and specificity and optimisation of the genosensor in terms of hybridisation conditions and electrochemical signal are discussed as well as their long-term stability and storage and semi-automation attempts. With continuous innovation and attention to key challenges, we expect semi-automatic devices containing DNA-based electrochemical biosensors to have an important impact upon monitoring of serious HAB events.     

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.     

Toxicity of fluoranthene and its biodegradation metabolites to aquatic organisms

The toxicity of nine stable products of the biodegradation of fluoranthene with the pure bacterial strain Pasteurella sp. IFA was studied. For their quantification, an improved analytical procedure with two-step liquid-liquid extraction, derivatisation and gas chromatographic-mass spectrometric detection was used. Growth inhibition and immobility tests for fluoranthene and its metabolites were carried out using algae (Scenedesmus subspicatus), bacteria (Pseudomonas putida) and crustaceans (Daphnia magna and Thamnocephalus platyurus). Tests using the alga S. subspicatus revealed that with the exception of 9-hydroxyfluorene, which was only four times less toxic than fluoranthene, all the other metabolites were 37 to approximately 3000 times less toxic than the parent material. P. putida cells were resistant to fluoranthene and its primary metabolites, but were inhibited by low molecular weight intermediates, especially benzoic acid. Fluoranthene was not toxic to T. Platyurus, but was toxic to D. magna. Its primary metabolites (including 9-fluorenone and 9-hydroxyfluorene) were toxic to D. magna, and a low molecular weight metabolite (2-carboxybenzaldehyde) was highly toxic to T. platyurus.     

Snapping turtles - a biological screen for PCB's

Snapping turtles are capable of storing extremely high concentration of organochlorine compounds in their fat without any apparent detrimental effect. This tolerance, to high bioconcentration, permits a wide gradation between the extremes in pollution levels and facilitates the detection of extremely toxic substances present in trace amounts. Consequently snapping turtles provide an excellent biological screen for these compounds.     

Strengths and weaknesses of microarray approaches to detect Pseudo-nitzschia species in the field

The planktonic diatom genus Pseudo-nitzschia contains several genetically closely related species. Some of these can produce domoic acid, a potent neurotoxin. Thus, monitoring programs are needed to screen for the presence of these toxic species. Unfortunately, many are impossible to distinguish using light microscopy. Therefore, we assessed the applicability of microarray technology for detection of toxic and non-toxic Pseudo-nitzschia species in the Gulf of Naples (Mediterranean Sea). Here, 11 species have been detected, of which at least 5 are potentially toxic. A total of 49 genus- and species-specific DNA probes were designed in silico against the nuclear LSU and SSU rRNA of 19 species, and spotted on the microarray. The microarray was tested against total RNA of monoclonal cultures of eight species. Only three of the probes designed to be species-specific were indeed so within the limits of our experimental design. To assess the effectiveness of the microarray in detecting Pseudo-nitzschia species in environmental samples, we hybridized total RNA extracted from 11 seasonal plankton samples against microarray slides and compared the observed pattern with plankton counts in light microscopy and with expected hybridization patterns obtained with monoclonal cultures of the observed species. Presence of species in field samples generally resulted in signal patterns on the microarray as observed with RNA extracted from cultures of these species, but many a-specific signals appeared as well. Possible reasons for the numerous cross reactions are discussed. Calibration curves for Pseudo-nitzschia multistriata showed linear relationship between signal strength and cell number.     

Non-biological inhibition-based sensing (NIBS) demonstrated for the detection of toxic arsenic compounds

This work demonstrates the success of a recently developed technique in chemical amplification, non-biological inhibition-based sensing (NIBS), for the detection of toxic arsenic compounds. Screening for toxic arsenic compounds is especially important due to their prevalence in wastewater and water sources. The detection method presented in this work amplifies the chemical response of toxic arsenic compounds by developing a sensor chemistry where the analyte inhibits, rather than enhances, the rate of a catalytic reaction. This technique mimics the work done with enzyme inhibition; however, using non-biological molecules allows for selective detection without the shelf-life issue associated with biological molecules. Using NIBS we find that we can enhance the sensitivity of the system by two orders of magnitude with no apparent loss in selectivity. This work demonstrates the versatility of NIBS, showing that the technique can be of general use for the detection of toxic compounds.     

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.     

Comprehensive two-dimensional gas chromatography time of flight mass spectrometry (GC×GC-TOFMS) for environmental forensic investigations in developing countries

The disposal and dumping of toxic waste is a matter of growing concern in developing countries, including South Africa. Frequently these countries do not possess access to gas chromatography-high resolution mass spectrometry (GC-HRMS) for the determination of persistent organic pollutants (POPs). This publication describes an alternative approach to the investigation of toxic waste using comprehensive gas chromatography coupled to time of flight mass spectrometry (GC × GC-TOFMS). The technology permits both comprehensive screening of toxic samples for numerous classes of organic pollutants and also quantitative analysis for the individual compounds. This paper describes the use of this technique by analysing samples obtained from a hazardous waste treatment facility in South Africa. After sampling and extraction the samples were analysed for polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and four dioxin-like non-ortho substituted polychlorinated biphenyls (PCBs). The quantitative values, as well as detection limits, obtained using the GC × GC-TOFMS methodology compares well with those obtained using GC-HRMS; the accepted benchmark technology for this analysis. Although GC × GC-TOFMS is not a target compound analytical technique (as is GC-HRMS), it is possible to obtain information on numerous other classes of organic pollutants present in the samples in one analytical run. This is not possible with GC-HRMS. Several different column combinations have been investigated for handling very complex waste samples and suggestions are presented for the most suitable combination.     

Regional background monitoring of PBT compounds. The comparison of the results from measurements and modelling

A comparison of the modelling results of persistent, bioaccumulative and toxic (PBT) chemicals is presented with measurements. Contribution will present mean annual concentrations calculated and observed at EMEP stations and their ratios. The comparison of the calculated results with older results indicates that the model modification improved the agreement with measurement data. PBT compounds in ambient air are monitored in the area of Kosetice observatory (professional observatory of the Czech Hydrometeorological Institute located in south Bohemia). Calculated and measured mean annual concentrations of PBTs in precipitation, soil, vegetation and their ratios are presented. It should be mentioned that the number of measurements in such compartments as seawater, soil and vegetation is insufficient for model verification at present. The agreement between results from MSC-East models and results from long-term regional air background monitoring in Central Europe is good.     

Optical remote measurement of toxic gases.

Enactment of the Clean Air Act Amendments (CAAA) of 1990 has resulted in increased ambient air monitoring needs for industry, some of which may be met efficiently using open-path optical remote sensing techniques. These techniques include Fourier transform spectroscopy, differential optical absorption spectroscopy, laser long-path absorption, differential absorption lidar, and gas cell correlation spectroscopy. With this regulatory impetus, it is an opportune time to consider applying these technologies to the remote and/or path-averaged measurement and monitoring of toxic gases covered by the CAAA. This article reviews the optical remote sensing technology and literature for that application.     

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.     

In situ monitoring of urban air in Southern Italy with the Tradescantia micronucleus bioassay and semipermeable membrane devices (SPMDs)

This study was designed to assess toxic and genotoxic compounds in the urban air of Caserta, South Italy using cuttings from the plant Tradescantia #4430. In situ monitoring of gaseous pollutans was made at 17 sampling points in two seasons of the year. Genotoxicity was evaluated by recording the micronuclei in meiotic pollen mother cells (Trad-MCN assay). In addition, the passive sampler semipermeable membrane devices (SPMDs) were deployed at the sampling points with a significant increase in micronuclei frequency. SPMDs concentrated priority organic pollutants were identified by high performance liquid chromatography and gas chromatography, while toxicity and mutagenesis were assessed on the bacterium Vibrio fischeri using the Microtox and Mutatox systems respectively. Significant toxic and mutagenic effects were observed at different points on the town grid and SPMDs effectively concentrated trace contaminants. The relationship between what was present in the air sampled by SPMDs and the micronuclei frequency was also explored.     

Development of a comprehensive analytical method for the determination of chlorinated paraffins in spruce needles applied in passive air sampling

Conifer needles are used for the monitoring of atmospheric persistent organic pollutants. The objective of the present study was to develop a method for the detection of airborne chlorinated paraffins (CPs) using spruce needles as a passive sampler. The method is based on liquid extraction of the cuticular wax layer followed by chromatographic fractionation and detection of CPs using two different GCMS techniques. Total CP concentrations (sum of short (SCCP), medium (MCCP) and long chain CPs (LCCP)) were determined by EI-MS/MS. SCCP and MCCP levels as well as congener group patterns (n-alkane chain length, chlorine content) could be evaluated using ECNI-LRMS. For the first time, data on environmental airborne CPs on spruce needles taken within the Monitoring Network in the Alpine Region for Persistent and other Organic Pollutants (MONARPOP) are presented providing evidence that spruce needles are a suitable passive sampling system for the monitoring of atmospheric CPs.     

Protocol for early detection and evaluation of inhibitory wastewater using combined aerobic respirometric and anaerobic batch techniques.

Faced with the task of treating significant volumes of complex industrial wastewaters, the biological components of municipal wastewater treatment plants are operating under the risk of toxic or inhibitory contaminants from the industrial effluents that may be detrimental to their operation. This might lead to undesirable effluent toxicity and/or result in permit violations. Therefore, there is a need for upset early warning systems that can protect full-scale plants from toxic or inhibitory constituents in the incoming wastewaters. This study focused on the development of a protocol for rapid detection of potentially toxic inhibitory or toxic wastewaters using combined aerobic respirometric and anaerobic batch techniques. Aerobic respirometers equipped with automated data acquisition systems were used as potential early warning devices. The inhibition effect on carbon and nitrogen oxidation was assessed. The degree of inhibition was evaluated as the concentration causing 50% reduction in microbial activity, which was estimated by an inhibition model. Anaerobic toxicity assays were also conducted to evaluate the inhibitory effects of the toxic compounds to anaerobic inocula obtained from a master culture reactor fed with ethanol. The developed protocol for early detection of toxicity was validated using wastewater samples from a biotechnology industry and a food processing industry, and pure chemicals such as furfural and phenol. Varying degrees of sensitivity were observed in the study when different groups of microorganisms, wastewater samples, and chemicals were tested. The comparison of aerobic and anaerobic inhibition suggested the importance of using both aerobic and anaerobic cultures to maximize the necessary sensitivity of the protocol.     

Organic tracers identification as a convenient strategy in industrial landfills monitoring

Today, a significant environmental problem is due to the presence of industrial landfills, some of them old, which can release toxic compounds into the environment (Chemosphere 42 (2001) 415; Chemosphere 31 (1995) 3455). The identification of directly linked to the leachate tracers of contamination in the wells around the landfills, as early markers of a potential risk for the environment and/or man, is a good strategy for an early evaluation of contamination. Our protocol developed for wells water characterization is based on solid phase extraction combined with GC-MS and HPLC-MS/MS.In this way a list of organic contaminants of a specific industrial landfill is obtained, giving a solid basis also for the risk assessment. Here we present some specific examples and the use of these pollutants for environmental monitoring.     

Specificity of LSU rRNA-targeted oligonucleotide probes for Pseudo-nitzschia species tested through dot-blot hybridisation

In the scope of the development of a microarray PhyloChip for the detection of toxic phytoplankton species, we designed a large series of probes specific against targets in the nuclear large subunit (LSU) rRNA of a range of Pseudo-nitzschia species and spotted these onto the microarray. Hybridisation with rRNA extracted from monoclonal cultures and from plankton samples revealed many cross-reactions. In the present work, we tested the functionality and specificity of 23 of these probes designed against ten of the species, using a dot-blot procedure. In this case, probe specificity is tested against the target region in PCR products of the LSU rRNA gene marker region blotted on nitrocellulose filters. Each filter was incubated with a species-specific oligoprobe. Eleven of the tested probes showed specific responses, identifying seven Pseudo-nitzschia species. The other probes showed non-specific responses or did not respond at all. Results of dot-blot hybridisations are more specific than those obtained with the microarray approach and the possible reasons for this are discussed.     

Concentration of Organochlorine Pollutants in Surface Waters of the Central European Biosphere Reserve Krivoklatsko (8 pp)

Background, Aim and Scope The article is focused on dioxin, furan, PCB and organochlorine pesticide monitoring in the surface waters of the Central European, protected natural reserve Krivoklatsko, under the UNESCO programme Man and Biosphere. Persistent compounds are presently transported via different means throughout the entire world. This contamination varies significantly between sites. This raises the question of what constitutes the naturally occurring background levels of POPs in natural, unpolluted areas, but which are close to industrialised regions. Information of real background POP contamination can be of high value for risk assessment management of those sites evidently polluted and for the defining of de-contamination limits. Preserved areas should not be seen as isolated regions in which the impacts of human activities and natural factors are either unexpected or overlooked. Every ambient region, even those protected by a law or other means, are still closely connected to neighbouring human developed and impacted areas, and are therefore subject to this anthropogenic contamination. These areas adjacent to natural reserves are sources of diverse substances, via entry of air, water, soil and/or biota. After an extended period of industrial activities, organochlorine pollutants, even those emitted in trace concentrations have reached detectable levels. For future research and for the assessment of environmental changes, present levels of contamination would be of high importance. This work publishes data of the contamination with organochlorine pollutants of this natural region, where biodiversity and ecological functions are of the highest order. Materials and Methods: Semipermeable membrane devices (SPMDs) were utilised as the sampling system. SPMDs were deployed in two small creeks and one water reservoir selected in the central part of the Krivoklatsko Natural Reserve, where it could be expected that any possible contamination by POPs would be lowest. The exposed SPMDs were analysed both for chemical contents of POPs and for toxicity properties. The chemical analyses of dibenzo-dioxins, dibenzo-furans, PCBs and OCPs were analysed by GC/MS/MS on GCQ or PolarisQ (Thermoquest). Toxicity bioassays were performed on the alga Desmodesmus subspicatus, bacteria Vibrio fischeri and crustacean Daphnia magna. All toxicity data were expressed as the effective volume Vtox. Vtox is a toxicity parameter, the determination of which is independent of SPMD deployment time and pre-treatment dilution (unlike, for example, the EC50 of the SPMD extract). Results: The following chemical parameters were monitored: 1) tetra, penta, hexa and hepta dibenzo-p-dioxins and furans; 2) all those detectable from tri- through deca-polychloriated biphenyls (PCBs) and 3) a group of organochlorine pesticides: hexachlorobenzene and isomers of hexachlorocyclohexane, DDE, DDD and DDT. The concentrations of dioxins and furans on the assessed sites varied from under detection levels up to 7 pg.l-1; PCBs were detected in a sum concentration up to 2.8 ng.l-1; and organochlorine pesticides up to 346 pg.l-1. The responses of bioassays used were very low, with the values obtained for Vtox being under 0.03 l/d. Discussion: Toxicity testing showed no toxicity responses, demonstrating that the system used is in coherence with the ecological status of the assessed sites. Values of Vtox were under the critical value – showing no toxicity. The PCA of chemical analysis data and toxicity responses resulted in no correlations between these two groups of parameters. This demonstrated that the present level of contamination has had no direct adverse effects on the biota. Conclusions: The concentration values of six EPA-listed, toxic dioxins and sums of tetra-hepta dioxins; nine EPA toxic dibenzofurans and the sums of tetra-hepta bibenzofurans are presented together with all tri-deka PCBs and organochlorine pesticides (alfa-, beta-, gama-, delta-HCH, HCB, opDDE, ppDDE, opDDD, ppDDD, opDDT, ppDDT). These values represent possible current regional natural background values of these substances monitored within the Central European region, with no recorded adverse effects on the freshwater ecosystem (up until the present time). Recommendations and Perspectives: Assessment of dioxins, furans and other organochlorine compounds within natural reserves can be important for the monitoring of human-induced impacts on preserved areas. No systematic monitoring of these substances in areas not directly affected by industry has generally been realised. There is a paucity of data of the presence of any of these substances within natural regions. Further monitoring of contamination of both soil and biota by dioxins and furans in preserve regions is needed and can be used for future monitoring of man-made activities and/or accidents. Semipermeable membrane devices proved to be a very good sampling system for the monitoring of trace concentrations of ambient organochlorine compounds. Toxicity evaluation using the Vtox concept demonstrated that those localities assessed expressed no toxicity.     

The toxicity and toxic potential of fly ash from municipal incinerators assessed by means of a fish early life stage test

The toxicity and toxic potential of fly ash were assessed, using rainbow trout yolk sac fry. In contrast to fly ash itself, extracts of fly ash were extremely toxic, producing typical toxicopathological features of TCDD-intoxication. By comparison with earlier experiments using pure 2,3,7,8-TCDD, the toxic potential of fly ash was roughly estimated to be 75–125 ng.g^?1 toxic aequivalents TCDD. Obviously, this toxic potential is attributable for a minor part to 2,3,7,8-TCDD and for the greater part to the other chlorinated dioxin congeners and the dibenzofurans, present in fly ash.     

Importance of heterocylic aromatic compounds in monitored natural attenuation for coal tar contaminated aquifers: A review

NSO heterocycles (HET) are typical constituents of coal tars. However, HET are not yet routinely monitored, although HET are relatively toxic coal tar constituents. The main objectives of the study is therefore to review previous studies and to analyse HET at coal tar polluted sites in order to assess the relevance of HET as part of monitored natural attenuation (MNA) or any other long-term monitoring programme. Hence, natural attenuation of typical HET (indole, quinoline, carbazole, acridine, methylquinolines, thiophene, benzothiophene, dibenzothiophene, benzofuran, dibenzofuran, methylbenzofurans, dimethylbenzofurans and xanthene) were studied at three different field sites in Germany. Compound-specific plume lengths were determined for all main contaminant groups (BTEX, PAH and HET). The results show that the observed plume lengths are site-specific and are above 250m, but less than 1000m. The latter, i.e. the upper limit, however mainly depends on the level of investigation, the considered compound, the lowest measured concentration and/or the achieved compound-specific detection limit and therefore cannot be unequivocally defined. All downstream contaminant plumes exhibited HET concentrations above typical PAH concentrations indicating that some HET are generally persistent towards biodegradation compared to other coal tar constituents, which results in comparatively increased field-derived half-lives of HET. Additionally, this study provides a review on physicochemical and toxicological parameters of HET. For three well investigated sites in Germany, the biodegradation of HET is quantified using the centre line method (CLM) for the evaluation of bulk attenuation rate constants. The results of the present and previous studies suggest that implementation of a comprehensive monitoring programme for heterocyclic aromatic compounds is relevant at sites, if MNA is considered in risk assessment and for remediation.