Cyanotoxins: methods and approaches for their analysis and detection

Cyanotoxins are secondary metabolites produced by cyanobacteria, a group of photosynthetic prokaryota especially found in freshwater. In favourable conditions (i.e. high nutrient levels, light intensity, water temperature), cyanobacteria can form blooms, a natural phenomenon characterised by an algal biomass accumulation and the possible release of cyanotoxins in water ecosystems. Toxins represent an emerging threats for the aquatic organisms which can bioaccumulate these compounds and transfer them throughout the food chain to wildlife and humans. Other ways of exposure for humans include the oral, dermal and inhalation route. The consumption of contaminated drinking water, skin contact and swallowing water during recreational activities are among the most frequently reasons for human poisonings caused by cyanotoxins. The associated symptoms usually range from severe headache to fever, respiratory paralysis and in rare case, death. The World Health Organization (WHO) has issued a provisional guideline value of 1 µg/L in drinking water for Microcystin-LR (MC-LR), the most toxic, widespread and common toxin in water supplies. Due to the lack of complete toxicological data for a range of cyanotoxins, their concentration in drinking water is not yet well regulated even in countries belonging to the European Union (EU). In this report, attention is focused on the methodologies commonly used to detect cyanotoxins in water environments. These applications can be grouped in: I) microscopy analysis II) physicochemical methods III) molecular-based methods IV) biochemical-based methods V) chemical methods. Each technique shows specific limitations in terms of sensitivity, reliability and limit of detection. The choice of the best one to use is determined in accordance with the information they provide, the availability of facilities and the technical expertise of the operators. Most of the research about cyanotoxins has been mainly focused on microcystins (MCs). The other cyanotoxins have been much less investigated and more tools need to be developed to overcome this problem. Notwithstanding there is no a single analytical application able to detect all cyanotoxin variants in an environmental sample. Some current methods described in this report show great promise in terms of being simple, cost-effective, specific and sensitive for the analysis of a defined toxin.JRC.D.2-Water and Marine Resource

Review of the 1st Watch List under the Water Framework Directive and recommendations for the 2nd Watch List

The surface water Watch List (WL) under the Water Framework Directive (WFD) is a mechanism for obtaining high-quality Union-wide monitoring data on potential water pollutants for the purpose of determining the risk they pose and thus whether Environmental Quality Standards (EQS) should be set for them at EU level. According to the EQS Directive (article 8b) , this list should be updated every 2 years. The main objectives of this report are: • To present an overview of the data gathered during the 1st year of monitoring of the 1st WL (also called WL dataset in this report), • To assess whether this WL dataset is sufficient to determine the risk posed by the WL substances, and consequently to determine whether any of these substances can be taken out of the WL, • To propose new substance(s) to be included in the second WL, using the information and results from the latest review of the list of priority substances, as well as any other relevant information available at the time of this report. The executive summary first explains the context for the assessment. Then, mirroring the report itself, it presents an overview of the WL dataset for the different WL substances, it specifies the criteria for taking substances out of the WL and the substances proposed on the basis of these criteria, and finally it presents the criteria for including new substances in the WL and the new proposed WL candidates.JRC.D.2-Water and Marine Resource

Microbiological parameters under the Drinking Water Directive.

In November 1998, the European Council adopted a directive, the Drinking Water Directive (DWD), concerning the quality of water intended for human consumption. It includes a certain number of microbiological, chemical or physical criteria or parameters to monitor, to ensure that i) it is “clean”, ii) the distribution network is safe and iii) to react promptly in case of contamination (Directive 98/83/EC)1. The Directive has been implemented by Member States, but its approach to monitoring quality at the point of consumption uses parameters determined over 20 years ago. After the submission of the European’s citizens’ initiative “Right2Water” to the Commission in December 2013, the Commission invited Member States to improve the access to a minimum water supply and the management of water in a sustainable manner. In 2017, following the WHO recommendations2, the Drinking Water Directive (DWD) was revised either for the microbiological or chemical parameters (RECAST DWD). Among the first ones, somatic coliphage (virus infecting Escherichia coli) has been proposed as new parameter, while bacterium Clostridium perfringens (C. perfringens) and its spores are already included in the Directive. The present report provides an overview on the current knowledge of these two microbiological parameters, their biological characterisations, relevance and suitability as indicators for human faecal contamination in the drinking water treatment. Finally, the report illustrates the available and standardised methods for their detection in water, listing as well the new and most promising ones with advantages/disadvantages and costs. Furthermore, the report provides a list of recommendations in order to elucidate the role of the two microbiological parameters for drinking water quality management.JRC.D.2-Water and Marine Resource

Algal bloom and its economic impact

Harmful algal blooms (HABs) represent a natural phenomena caused by a mass proliferation of phytoplankton (cyanobacteria, diatoms, dinoflagellates) in waterbodies. Blooms can be harmful for the environment, human health and aquatic life due to the production of nocive toxins and the consequences of accumulated biomass (oxygen depletion). These blooms are occurring with increased regularity in marine and freshwater ecosystems and the reasons for their substantial intensification can be associated with a set of physical, chemical and biological factors including climate changes and anthropogenic impacts. Many bloom episodes have significant impacts on socio-economic systems. Fish mortality, illnesses caused by the consumption of contaminated seafood and the reluctance of consumers to purchase fish during HABs episodes represent only some of the economic impacts of HABs. The aim of this report is to evaluate the economic losses caused by HABs in different sectors. This was achieved by collecting data that exist in the technical literature and group them into four categories: (1) human health impacts; (2) fishery impacts; (3) tourism and recreation impacts; (4) monitoring and management costs. The data analysed refer to both marine and freshwater HABs. Among the sectors examined in this study, human health impacts appear less investigated than the other three categories. This is probably caused by the difficulty to assess the direct effects of toxins on human health because of the wide range of symptoms they can induce. Looking at the data, the interest in mitigating the economic losses associated with blooms is particularly demonstrated by studies aimed to develop monitoring and management strategies to reduce HABs episodes. Indeed, the water monitoring, when accompanied by appropriate management actions, can assure the mitigation of ongoing HABs and the reduction of negative impacts. During data collection, it has been more difficult to find economic data about blooms in Europe than in United States of America (USA). A reason may be the lack of European reports or publically available data about HABs and their socio-economic impacts. Much studies still have to be performed in this field, but the reported increase in HABs frequency will surely increase not only scientific analysis about HABs but also economic studies to report whether safeguards taken have succeeded in mitigating the economic impact associated with blooms.JRC.H.1-Water Resource

Increased circulating levels of vitamin D binding protein in MS patients

Vitamin D (vitD) low status is currently considered a main environmental factor in multiple sclerosis (MS) etiology and pathogenesis. VitD and its metabolites are highly hydrophobic and circulate mostly bound to the vitamin D binding protein (DBP) and with lower affinity to albumin, while less than 1\% are in a free form. The aim of this study was to investigate whether the circulating levels of either of the two vitD plasma carriers and/or their relationship are altered in MS. We measured DBP and albumin plasma levels in 28 MS patients and 24 healthy controls. MS patients were found to have higher DBP levels than healthy subjects. Concomitant interferon beta therapy did not influence DBP concentration, and the difference with the control group was significant in both females and males. No significant correlation between DBP and albumin levels was observed either in healthy controls or in patients. These observations suggest the involvement of DBP in the patho-physiology of MS

Modes of action of the current Priority Substances list under the Water Framework Directive and other substances of interest

The Water Framework Directive 2000/60/EC (WFD) has established a strategy for water protection that includes specific measures for pollution control to achieve good chemical and ecological status at European level. There is a need to review the approach to the current listing of priority substances (PS) under the WFD and to the current assessment of the chemical status, and consider eventually a wider range of chemical substances that could be covered in future monitoring programmes. Overall, the aim is to assess the water status more holistically and understand which the real effects are caused by the sum of the chemical substances present in the aquatic environment (including emerging pollutants /other substances of interest, metabolites and transformation products). The assessment of chemical status should be improved and linked with ecological status where relevant. Hundreds of different substances may co-occur, and even if most are present at very small concentrations they could exert a toxic effect on aquatic organisms (Carvalho et al. 2014) exposed for their entire life cycle and indirectly on human health (via food and drinking water consumption). Some of the substances in the current list of Priority Substances and in the first Watch List are considered in groups (e.g. brominated diphenylethers, neonicotinoid insecticides), but the overall approach to chemical pollution is otherwise based on the regulation of single substances. It has become increasingly clear that the risks from the vast number of chemical substances present in the environment cannot be adequately controlled on this basis. The Commission acknowledges the need to consider the potential toxic effects of mixtures of chemicals (EC COM(2012)252, 7th EAP). The challenge is to find a way of capturing a true picture of the chemical status of water bodies based on standards and methods that assess the presence of an adequate range of representative chemical effect types or modes of action (MoA), for example. The knowledge on the MoA is an important driver for linking exposure to chemicals to their effects in the aquatic environment, and therefore for development and application of the scientific methodologies for the assessment of combined effects of chemicals - the effect-based methods (EBM). The EBM, including biomarkers and bioassays, can target different levels of biological organisation in the aquatic environment, such as individual and/or sub-organism, community, and population levels (Carvalho et al. 2014, Ann-Sofie Wernersson et al. 2014). It is however much less clear how these EBM can be used to capture (predictively) the indirect effects that might occur in humans following long-term chronic exposure to pollutants via the aquatic environment. The use of effect-based monitoring approaches, complementary to chemical analysis, could allow assessing chemical status more holistically (rather than with a limited but ever-growing list of individual substances). The use of the EBM offers also the advantage of overcoming analytical difficulties (Kunz et al. 2015) and reducing monitoring costs by screening. To become a credible complement to chemical monitoring information, however, a better understanding of the capabilities and gaps of available EBM is needed. This report, based on a comprehensive literature study, reviews the current PS list and other substances of interest, considering their MoA(s). The review of data from the open sources clearly identified few groups of toxicological endpoints, with the majority driven by non-specific mechanisms (e.g. oxidative stress, activation of metabolizing / detoxifying pathways, histopathology, and others), and few groups with more specific biochemical / physiological pathways (photosynthesis inhibition, acetylcholinesterase inhibition, presence of PAHs metabolites, expression of metallothioneins). The majority of current PS and other substances of interest can be grouped, based on few common toxicological endpoints, and biomarkers are available for determining the concentrations and/or effects of some groups of substances. The identified biomarkers of effect seem to be however in general not very specific. There is clearly no “one size fits all” bioassay / EBM that could provide the toxicological potency of every PS and other substances of interest and their mixture toward all aquatic organisms in all water bodies, but rather a battery of bioassays that should be selected as “fit for purpose”. In addition, the present report allowed identification of uncertainty and inconsistency in observations, and thus identified areas where future investigations can be best directed. The present knowledge about MoA(s) remains limited, especially for the emerging substances of concern, such as pyrethroids and neonicotinoides.JRC.D.2-Water and Marine Resource

Analytical methods for substances in the Watch List under the Water Framework Directive

The JRC organised in March 2018 a technical workshop on the analysis of the existing and the three new watch list (WL) substances of the Water Framework Directive (WFD) in order to share experience and knowledge on analytical methods and to identify the obstacles to reaching the environmental quality standards (EQS) or predicted no effect concentrations (PNEC). The results from the first year of monitoring the substances of the 1st WL showed that some countries have found it very difficult to reach a satisfactory analytical limit of quantification (LOQ) for five out of the 17 substances (17-alpha-ethinylestradiol (EE2), 17-beta-estradiol (E2), azithromycin, imidacloprid, and methiocarb). The most difficult WL substance to analyse is EE2 with its very low EQS value of 0.035 ng/L. Five countries have reported for EE2 LOQs ≤ EQS, and other four countries a close LOQ (0.05 - 0.1 ng/L). However, the workshop participants (experts from Member States and Island, Switzerland and Turkey) stressed that the analysis of EE2 at ultra trace levels is complicated and work intensive because a good clean-up procedure and a very clean and sensitive LC-MS/MS instrument are necessary. The analytical methods for the three new WL substances amoxicillin, ciprofloxacin, and metaflumizone were discussed at the workshop and optimized methods for the water sample preservation and analysis for them, based on solid-phase extraction (SPE) followed by liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS) analysis, are presented in this report. The workshop showed that many Member States (MS) are using today direct LC-MS/MS injection techniques. Direct LC-MS/MS injection should however only be performed for water soluble substances with low KOW and Kd(sed) values for which partitioning to suspended particulate matter (SPM) can be neglected. A literature study revealed that EE2 and E2 are strongly adsorbed to particulate matter and therefore have to be analysed in whole water samples. Moreover, around 20 % of ciprofloxacin is present in the particulate water phase. Therefore, analysis of the dissolved water phase by direct LC-MS/MS injection could deliver for this substance acceptable results similar to whole water analysis. However, an experimental analysis should be performed to confirm the comparability. Finally, some MS asked to specify in detail the different methods for the determination or calculation of the Limit of Quantification (LOQ), because the available general guidelines are not sufficient for a uniform approach. It remained unclear whether the LOQs of the presented methods are derived from appropriate real surface water samples with or without standard addition, or from calibration curves. For a better harmonisation of the methods, a consensus on the determination of the LOQ is necessary, which needs a thorough discussion on the minimum requirements for sample preparation and analysis, i.e. representative surface water matrix, standard addition, recovery, reproducibility, and multiplication factor.JRC.D.2-Water and Marine Resource

Toward climate change impact: Vectors carrying viral infections

Global warming has allowed mosquitoes, ticks and other disease-bearing insects to proliferate, adapt to different temperatures, migrate to places that have become warmer and adapt to new niches and new areas (e.g. urban area). More humans are therefore exposed to viral infections such as Dengue fever, Zika, Chikungunya, West Nile fever, Yellow fever and Tick-borne encephalitis. All these diseases are transmitted by mosquitoes with the exception of Tick-borne encephalitis that is caused by tick-borne encephalitis virus transmitted to humans predominantly by tick bites. For many of these diseases, there are as yet no specific vaccines or antiviral agents. In urban areas, due to the temperature changes (i.e. warm temperature in winter time) and the egg resistance, they are active in all seasons of the year. The reduction of potential breeding sites (e.g fountains, ponds, water-filled containers) can decrease mosquito abundance, however an early warning system would mitigate the risk. Insecticides have been used widely in control programs against mosquito populations, but negative effects on human and environmental health due to their toxicity have been well documented. Environmentally-sustainable measures are required to control mosquitoes without relying on widespread pesticide applications but rather based on surveillance data. While a better control of mosquito population is advocated, it’s also important to warn that it would be unwise to remove mosquitoes completely from the ecosystem. They are part of the food chain for some species, and pollinate many plants. Wiping them out completely could have negative effects on nature, and consequently on humans.JRC.D.2-Water and Marine Resource

State of the art on the contribution of water to antimicrobial resistance

The antimicrobial resistance (AMR) is defined as the ability of microorganisms to withstand the effects of antibiotics and is considered a universal threat to humans, animals and the environment. The resistance mechanisms developed by bacteria originate from either the overuse of antibiotics in medical care and animal farming or from the spread of resistant genes among microorganisms. Worldwide, 700.000 people die annually from resistant infections and this means that if no action is taken, the estimated annual deaths attributable to AMR will be 10 million by 2050. The aim of this report is to discuss the mechanisms of antibiotic action and antibiotic resistance focusing on potential effects in water. Waterbodies have been indeed recognised as a significant reservoir of antibiotics and antibiotic resistance genes (ARG). They contribute to the interchange of resistance genes between pathogenic and non-pathogenic bacteria and they may favour the maintenance of the resistance in the environment. In this report, a review of the global scientific literature was conducted to show the levels of antibiotics in waste water treatment plants (WWTP), surface waters, agricultural runoff and drinking waters The most frequently monitored antibiotics in WWTP were sulfamethoxazole, ciprofloxacin and trimethoprim, while the most important substances in surface waters were erythromycin, sulfamethoxazole, trimethoprim and sulfamethazine.In parallel, a European database has been consulted to identify the antibiotics monitored in inland surface waters and the co-occurrence of heavy metals and antibiotic resistance in bacteria is discussed. It is indeed known that the chemical environmental pollution caused by heavy metals like silver (Ag), copper (Cu) or zinc (Zn) can co-select for antibiotic resistance. Antibiotics have been frequently detected in different aquatic environments within urban water cycles (waste, surface and drinking water) and, even if the levels are low (in the range of ng/L to µg/L), they could promote the acquisition of resistance by gene transfer between bacteria. The spread of AMR may be however constrained if general safety measures are taken to improve the effectiveness of wastewater treatment processes and to control the use of antibiotics in animal husbandry and in human medical practices. Additionally, new research must be conducted to understand the relationship between antibiotics’ concentration and the selection of resistance determinants in order to experimentally define the minimal concentration of antibiotics (as single and mixture) that induces resistance in bacteria. This factor should be then also considered in the evaluation of the risk assessment of antibiotics in water in order to define their environmental impactJRC.D.2-Water and Marine Resource

Algal blooms and their socio-economic impact

Algal blooms are a natural phenomena caused by a rapid growth of aquatic algae in waterbodies. Bloom formation is influenced by light intensity, water temperature, pH, climate change, water flow, water column stability and anthropogenic modifications of aquatic environment including nutrient over-enrichment (eutrophication). Indeed, in the last years, an increasing trend of blooms caused by cyanobacteria (photosynthetic bacteria) have been reported in freshwater due to the warmer temperatures. During an algal bloom, the water colour could vary from green to brown, red or yellow depending on the organism causing the bloom and the concentration of the organism. Not all algal blooms are dense enough to induce water discoloration and blooms of algae that discolour the water have traditionally been called “red tides”, whether toxic or not. The occurrence of blooms in aquatic environment is considered an environmental problem of serious concern due to direct and indirect negative impacts on different sectors such as fishery, tourism and public health.JRC.D.2-Water and Marine Resource