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

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

Physico-chemical determinants of the toxicity of silica nanoparticles

Nanomaterials are already impacting on virtually all sectors of industrial and domestic products (cosmetics, clothing, personal care, sporting goods, sunscreens, filtration, electronics and computers, food and beverage), and their production and marketing will increase in the coming years.Silica nanoparticles (SiO2-NPs) are produced on an industrial scale and are an addition to a growing number of commercial products. SiO2-NPs also have great potential for a variety of diagnostic and therapeutic applications in medicine. Contrary to the well-studied crystalline micron-sized silica, relatively little information exists on the toxicity of its amorphous and nano-size forms. Because nanoparticles possess novel properties, kinetics and unusual bioactivity, their potential biological effects may differ greatly from those of micron-size bulk materials. The objectives of this project is to determine: (1) which physico-chemical characteristics of nanoparticles direct the toxicity;(2) which mechanisms are involved in the toxicity of silica nanoparticles.The strategy of the presented doctoral project was to conduct toxicity studies with a very carefully characterized model material silicon dioxide (silica) nanoparticles (SiO2-NPs). The general (cyto)toxicity of these particles was assessed and two specific mechanisms were Investigated; oxidative stress and endothelial dysfunction.The effect that monodisperse amorphous spherical silica particles of different sizes have on the viability of endothelial cells (EA.hy926 cell line) was investigated. The results indicate that exposure to silica nanoparticles causes cytotoxic damage (lactate dehydrogenase (LDH) release) and a decrease in cell survival (tetrazolium reduction, MTT assay) in the EA.hy926 cell line in a dose- and size-related manner. Concentrations leading to a 50% reduction in cell viability (TC50) for the ranged from 33 µg/cm² for the smallest particles (14 nm diameter) to 1087 µg/cm² for 335 nm diameter particles. The smaller particles also appear to affect the exposed cells faster, by necrosis, compared to the bigger particles. We showed that the surface area of monodisperse amorphous silica nanoparticles is an important parameter in determining the toxicity.A similar study has been undertaken using nanosized zeolite particles (crystalline silica particles). The synthesis of nanozeolites Y and A resulted in particle sizes of 25-100 nm and an adequate colloidal stability for in vitro cytotoxicity experiments. The cytotoxic response of macrophages, epithelial and endothelial cells to these nanocrystals was also assessed by the MTT assay and the LDH leakage assay. After 24h of exposure, no significant cytotoxic activity was detected for zeolite doses up to 500 μg/ml.Addition of fetal calf serum to the cell culture medium during exposure did not significantly change this low response. The nanozeolites are of low toxicity compared with monodisperse amorphous silica nanoparticles of similar size (60 nm). These results may contribute to the application of nanozeolites for purposes such as medical imaging, sensing materials, low-k films and molecular separation processes.The association of oxidative stress with SiO2-NPs induced cytotoxicity in human endothelial cells was studied with pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and iron-doped nanosilica (19 nm; SFe). We found significant modifications in GSSG/total glutathione ratio and MDA and HAE concentrations only in cells treated with SFe nanoparticles. Significantly higher HO-1 mRNA expression was found in endothelial cells after 6h treatment with S16 or SFe nanoparticles; no such up-regulation was seen with S60 particles. Our study demonstrates that cytotoxicity occurs in endothelial cells exposed to pure SiO2-NPs in the absence of oxidative stress.There is evidence suggesting that nanoparticles can induce endothelial dysfunction, which may lead to vascular diseases. The present study was undertaken to examine the effect of amorphous (monodisperse) SiO2-NPs of different sizes (28, 59 and 174 nm) on endothelial (EA.hy 926 cell line or primary hPAEC)cell function, in the absence or presence of a previously established in vitro human airway model consisting of triple cell co-cultures. At non-toxic concentrations, a significant increase (up to 2-fold) in U937 cell adhesion was observed. Exposure to all three SiO2-NPs induced expression of ICAM-1 but no VCAM-1 in the EA.hy 926 and ICAM-1 and VCAM-1 in hPAEC cultures. Experiments performed with fluorescently labeled amorphous monodisperse SiO2-NPs (24 nm) showed the uptake of the nanoparticles into the cytoplasm of both EC tested. We concluded that exposure of human endothelial cells to amorphous silica nanoparticles enhances adhesive properties of the studied cells.TABLE OF CONTENT List of abbreviations General introduction and objectives 1 Chapter 1: The nanosilica hazard: another variable entity 31 Chapter 2: Size-Dependent Cytotoxicity of Monodisperse Silica Nanoparticles in Human Endothelial Cells 65 Chapter 3: Investigation of the Cytotoxicity of Nanozeolites A and Y 83 Chapter 4: Oxidative stress is not associated with the cytotoxicity of pure amorphous silica nanoparticles 113 Chapter 5: Adhesive properties of human endothelial cells exposed to amorphous silica nanoparticles 141 General discussion, conclusions and future perspectives 159 Summary 187 Samenvatting 191 Short curriculum vitae and list of publications 195nrpages: 200status: publishe

Relationship between biomarker responses and contaminant concentration in selected tissues of flounder (Platichthys flesus) from the Polish coastal area of the Baltic Sea

Previous studies in the Gulf of Gdańsk discussed the responses of selected enzymatic biomarkers to the contaminant gradient in fish and mussels. In the present study, flounder muscle and liver tissues were analyzed for polychlorinated biphenyls (PCB congeners: 28, 52, 101, 118, 138, 153 and 180), organochlorine pesticides (HCHs, HCB and DDTs), and trace metals (Pb, Cd, Zn, Cu, Hg, Cr). An attempt was made to identify the relationship between the measured enzymatic biomarker responses (cholinesterases, malic enzyme, isocitrate dehydrogenase, glutathione S-transferase) and contaminant concentrations in selected flounder tissues. The observed differences in enzymatic biomarker levels suggest that chronic exposure to low-concentration mixtures of contaminants may be occurring in the studied area. However, no conclusive evidence was found of a clear link between the biomarker responses and contaminant concentrations in flounder tissues

Intracellular oxidative stress caused by nanoparticle: what do we measure with dichlorofluorescein assay?

status: publishe

How physico-chemical characteristics of nanoparticles cause their toxicity: complex and unresolved interrelations

The increased use of and interest in nanoparticles (NPs) have resulted in an enormous amount of NPs with different compositions and physico-chemical properties. These unique properties not only determine their utility for (bio-medical) applications, but also their toxicity. Recently, "nano-researchers" became aware of the importance of determining the characteristics since they might be predictors of their toxicity. Currently, we face a large set of (non-coordinated) experiments with miscellaneous objectives resulting in a large quantity of available (and often incomplete) data, which hamper the unraveling of the complex interrelated NP characteristics with experimental results. Here, we try to link different critical physico-chemical characteristics separately with toxicity observed in both in vitro and in vivo models.status: publishe

The nanosilica hazard: another variable entity

Silica nanoparticles (SNPs) are produced on an industrial scale and are an addition to a growing number of commercial products. SNPs also have great potential for a variety of diagnostic and therapeutic applications in medicine. Contrary to the well-studied crystalline micron-sized silica, relatively little information exists on the toxicity of its amorphous and nano-size forms. Because nanoparticles possess novel properties, kinetics and unusual bioactivity, their potential biological effects may differ greatly from those of micron-size bulk materials. In this review, we summarize the physico-chemical properties of the different nano-sized silica materials that can affect their interaction with biological systems, with a specific emphasis on inhalation exposure. We discuss recent in vitro and in vivo investigations into the toxicity of nanosilica, both crystalline and amorphous. Most of the in vitro studies of SNPs report results of cellular uptake, size-and dose-dependent cytotoxicity, increased reactive oxygen species levels and pro-inflammatory stimulation. Evidence from a limited number of in vivo studies demonstrates largely reversible lung inflammation, granuloma formation and focal emphysema, with no progressive lung fibrosis. Clearly, more research with standardized materials is needed to enable comparison of experimental data for the different forms of nanosilicas and to establish which physico-chemical properties are responsible for the observed toxicity of SNPs.status: publishe

The nanosilica hazard : another variable entity.

Silica nanoparticles (SNPs) are produced on an industrial scale and are an addition to a growing number of commercial products. SNPs also have great potential for a variety of diagnostic and therapeutic applications in medicine. Contrary to the well-studied crystalline micron-sized silica, relatively little information exists on the toxicity of its amorphous and nano-size forms. Because nanoparticles possess novel properties, kinetics and unusual bioactivity, their potential biological effects may differ greatly from those of micron-size bulk materials. In this review, we summarize the physico-chemical properties of the different nano-sized silica materials that can affect their interaction with biological systems, with a specific emphasis on inhalation exposure. We discuss recent in vitro and in vivo investigations into the toxicity of nanosilica, both crystalline and amorphous. Most of the in vitro studies of SNPs report results of cellular uptake, size- and dose-dependent cytotoxicity, increased reactive oxygen species levels and pro-inflammatory stimulation. Evidence from a limited number of in vivo studies demonstrates largely reversible lung inflammation, granuloma formation and focal emphysema, with no progressive lung fibrosis. Clearly, more research with standardized materials is needed to enable comparison of experimental data for the different forms of nanosilicas and to establish which physico-chemical properties are responsible for the observed toxicity of SNPs