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Selecting appropriate ways of bringing engineered nanoparticles (ENP) into aqueous dispersion is a main obstacle for testing, and thus for understanding and evaluating, their potential adverse effects to the environment and human health. Using different methods to prepare (stock) dispersions of the same ENP may be a source of variation in the toxicity measured. Harmonization and standardization of dispersion methods applied in mammalian and ecotoxicity testing are needed to ensure a comparable data quality and to minimize test artifacts produced by modifications of ENP during the dispersion preparation process. Such harmonization and standardization will also enhance comparability among tests, labs, and studies on different types of ENP. The scope of this review was to critically discuss the essential parameters in dispersion protocols for ENP. The parameters are identified from individual scientific studies and from consensus reached in larger scale research projects and international organizations. A step-wise approach is proposed to develop tailored dispersion protocols for ecotoxicological and mammalian toxicological testing of ENP. The recommendations of this analysis may serve as a guide to researchers, companies, and regulators when selecting, developing, and evaluating the appropriateness of dispersion methods applied in mammalian and ecotoxicity testing. However, additional experimentation is needed to further document the protocol parameters and investigate to what extent different stock dispersion methods affect ecotoxicological and mammalian toxicological responses of ENP.

Effects and behaviour of nanomaterials in the environment depends on the materials' specific physical and chemical properties and for certain nanomaterials (e.g., Ag, ZnO and CuO) aqueous solubility is of outmost importance. The solubility of metals salts is normally described as a maximum dissolved concentration or by the solubility constant (Ksp). For nanomaterials it is essential to also assess solubility kinetics as nanomaterials will often not dissolve instantaneously upon contact with artificial aqueous media or natural waters. Dissolution kinetics will thereby influence their short and long-term environmental fate as well as laboratory test results. This highlights the need to evaluate and improve the reliability of methods applied to assess the solubility kinetics of nanomaterials. Based on existing OECD guidelines and guidance documents on aqueous dissolution of metals and metal compounds, the aim of this project was to conduct systematic experiments contributing to a better understanding of how nanomaterials solubility can be tested and evaluated under the auspice of the OECD framework. The focus was to evaluate the performance of three different techniques used to separate the dissolved and non-dissolved fraction of nanomaterials, namely: dialysis membranes (MWCO 8-10 and 50 kD), Diffusive Gradients in Thin films (DGT) and ultracentrifugation. The release of Cu ions from CuO nanoparticles (primary nominal size < 50nm) was used as a case study. Micron sized CuO was used as a "non-nano reference" and CuSO4 and CuCl2 were used as ionic Cu references. In a comparative study of the three methods we found a good reproducibility between replicates and a good recovery (88-99% of nominal concentrations). However, we observed a large difference in results between the three applied methods. Preliminary results showed that by using the DGT method the measured dissolved fraction was 5-7 times higher compared to using ultracentrifugation and dialysis membranes. Possible explanations include metal-specific interactions with the dialysis membranes as well as the DGT unit influencing the dissolution equilibrium by acting as a ‘sink’ for Cu ions. These are issues that require further investigations. Based on these first results we would recommend ultracentrifugation as the most suitable method as it also offers the advantage of being ‘material-independent’ when correctly adjusted for particle sizes and density.

Hydrophobic organic pollutants in urban wet weather discharges can accumulate in the sediments of receiving waters and may have adverse effects on the ecological system, especially on benthic organisms. Here, a novel method is developed for evaluating the bioavailability of such hydrophobic organic pollutants by considering the digestive guts in deposit-feeding polychaetes. We compared the amount of polycyclic aromatic hydrocarbons (PAHs) extracted by an organic solvent and by sodium dodecyl sulfate (SDS) solution (as a hypothetical digestive gut fluid of polychaetes) and interpreted the ratio of the two values as bioavailability. The sediment extracts were applied to bacterial acute toxicity tests and algal growth inhibition tests. Sediment samples were collected from an urban stream system receiving wet weather discharges. The bioavailability of the total amount of 12 PAHs in the sediments was in the range 14–38% based on the results from the GC/MS determination of the two different extracts. Lower molecular PAHs showed higher bioavailability compared to the higher molecular ones. The sediment extracts were shown to be toxic towards both algae and bacteria. The SDS extracts showed similar or higher toxicity in the two biotests compared to the organic solvent extracts in spite of their lower PAHs content.

Concentrations and isotopic compositions ( 13 C/ 12 C) of aromatic hydrocarbons were determined in eight samples obtained from the strongly anoxic part of the leachate plume downgradient from the Vejen Landfill (Denmark), where methanogenic, sulfate-reducing and iron-reducing conditions were observed. Despite the heterogeneous distribution of the compounds in the plume, the isotope fractionation proved that ethylbenzene and m / p -xylene were subject to significant biodegradation within the strongly anoxic plume. The isotope fractionation factors (αC) for the degradation of the m / p -xylene (1.0015) and ethylbenzene (1.0021) obtained from the field observations were similar to factors previously determined for the anaerobic degradation of toluene and o -xylene in laboratory experiments, and suggest that in situ biodegradation is one major process controlling the fate of these contaminants in this aquifer. The isotope fractionation determined for 1,2,4-trimethylbenzene and 2-ethyltoluene suggested in situ biodegradation; however, the isotopic composition did not correlate well with the respective concentration as expressed by the Rayleigh equation. Some other compounds (1,2,3-trimethylbenzene, o -xylene, naphthalene and fenchone) did not show significant enrichments in δ 13 C values along the flow path. The compound concentrations were too low for accurate isotope analyses of benzene, toluene, 1- and 2-methylnaphthalene, while interferences in the chromatography made it impossible to evaluate the isotopic composition for 4-ethyltoluene, 1,3,5-trimethylbenzene and camphor. In addition to demonstrating the potential of assessing isotopic fractionation as a means for documenting the in situ biodegradation of complex mixtures of aromatic hydrocarbons in leachate plumes, this study also illustrates the difficulties for data interpretation in complex plumes and high analytical uncertainties for isotope analysis of organic compounds in low concentration ranges.

The major potential environmental impacts related to landfill leachate are pollution of groundwater and surface waters. Landfill leachate contains pollutants that can be categorized into four groups (dissolved organic matter, inorganic macrocomponents, heavy metals, and xenobiotic organic compounds). Existing data show high leachate concentrations of all components in the early acid phase due to strong decomposition and leaching. In the long methanogenic phase a more stable leachate, with lower concentrations and a low BOD/COD-ratio, is observed. Generally, very low concentrations of heavy metals are observed. In contrast, the concentration of ammonia does not decrease, and often constitutes a major long-term pollutant in leachate. A broad range of xenobiotic organic compounds is observed in landfill leachate. The long-term behavior of landfills with respect to changes in oxidation-reduction status is discussed based on theory and model simulations. It seems that the somewhere postulated enhanced release of accumulated heavy metals would not take place within the time frames of thousands of years. This is supported by a few laboratory investigations. The existing data and model evaluations indicate that the xenobiotic organic compounds in most cases do not constitute a major long-term problem. This may suggest that ammonia will be of most concern in the long run.

The present study investigated changes in suspension stability and ecotoxicity of engineered nanoparticles (ENPs) by addition of Suwannee River natural organic matter and aging of stock and test suspensions prior to testing. Acute toxicity tests of silver (Ag), zinc oxide (ZnO), and titanium dioxide (TiO2 ) ENPs with Daphnia magna were carried out following Organisation for Economic Co-operation and Development test guidelines. Daphnia magna was found to be very sensitive to Ag ENPs (48-h 50% effective concentration 33 μg L(-1) ), and aging of the test suspensions in M7 medium (up to 48 h) did not decrease toxicity significantly. Conversely, the presence of Suwannee River natural organic matter (NOM; 20 mg L(-1) ) completely alleviated Ag ENP toxicity in all testing scenarios and did not aid in stabilizing suspensions. In contrast, addition of Suwannee River NOM stabilized ZnO ENP suspensions and did not decrease toxicity. Aging for 48 h generated monotonous concentration-response curves in the presence and absence of Suwannee River NOM. At concentrations up to 100 mg L(-1) TiO2 ENPs did not cause immobilization of D. magna under any of the tested conditions. Presence of Suwannee River NOM caused agglomeration in stock suspensions. The authors' results suggest that aging and presence of Suwannee River NOM are important parameters in standard toxicity testing of ENPs, which in some cases may aid in gaining better control over the exposure conditions but in other cases might contribute to agglomeration or elimination of ENP toxicity. Therefore, modifications to the current guidelines for testing ENPs should be evaluated on a case-by-case basis. Environ Toxicol Chem 2015;34:497-506. © 2014 SETAC.

Despite substantial information on the acute toxicity of silver nanoparticles (AgNP) to aquatic organisms, little is known about their potential chronic effects and the applicability of current test guidelines for testing nanomaterials. The purpose of this study was to study the influence of food availability on toxicity. This was done through a series of Daphnia magna 21-day reproduction tests (OECD 211) using 30 nm citric acid stabilized AgNP aimed at studying the influence of food abundance on the reproductive toxicity of AgNP in D. magna. The experiments were carried out as static renewal tests with exposure concentrations from 10 to 50 mu g Ag/L, and test animals were fed green algae Pseudokirchneriella subcapitata in low and high food treatments. The endpoints recorded were survival, growth of parent animals and number of live neonates produced. Detrimental effects of AgNP on survival, growth and reproduction were observed in concentrations higher than 10 mu g Ag/L, whereas the animals exposed to 10 mu g Ag/L had larger body length and produced more offspring than controls at both food treatments. High food treatment resulted in higher animal survival, growth and reproduction compared to result found for low food treatment. (C) 2015 Elsevier B.V. All rights reserved.

This study presents a series of short-term studies (total duration 48 h) of uptake and depuration of engineered nanoparticles (ENP) in neonate Daphnia magna. Gold nanoparticles (Au NP) were used to study the influence of size, stabilizing agent and feeding on uptake and depuration kinetics and animal body burdens. 10 and 30 nm Au NP with different stabilizing agents [citrate (CIT) and mercaptoundecanoic acid (MUDA)] were tested in concentrations around 0.5 mg Au/L. Fast initial uptake was observed for all studied Au NP, with CIT stabilized Au NP showing similar rates independent of size and MUDA showing increased uptake for the smaller Au NP (MUDA 10 nm > CIT 10 nm, 30 nm > MUDA 30 nm). However, upon transfer to clean media no clear trend on depuration rates was found in terms of stabilizing agent or size. Independent of stabilizing agent, 10 nm Au NP resulted in higher residual whole-animal body burdens after 24 h depuration than 30 nm Au NP with residual body burdens about one order of magnitude higher of animals exposed to 10 nm Au NP. The presence of food (P. subcapitata) did not significantly affect the body burden after 24 h of exposure, but depuration was increased. While food addition is not necessary to ensure D. magna survival in the presented short-term test design, the influence of food on uptake and depuration kinetics is essential to consider in long term studies of ENP where food addition is necessary. This study demonstrates the feasibility of a short-term test design to assess the uptake and depuration of ENP in D. magna. The findings underlines that the assumptions behind the traditional way of quantifying bioconcentration are not fulfilled when ENPs are studied. Electronic supplementary material The online version of this article (doi:10.1007/s10646-014-1259-x) contains supplementary material, which is available to authorized users.

The aquatic ecotoxicity testing of nanoparticles is complicated by unstable exposure conditions resulting from various transformation processes of nanoparticles in aqueous suspensions. In this study, we investigated the influence of exposure timing on the algal test response to silver nanoparticles (AgNPs), by reducing the incubation time and by aging the AgNPs in algal medium prior to testing. The freshwater green algae Pseudokirchneriella subcapitata were exposed to AgNO3, NM-300 K (a representative AgNP) and citrate stabilized AgNPs from two different manufacturers (AgNP1 and AgNP2) in a standard algal growth inhibition test (ISO 8692:2004) for 48 h and a short-term (2 h) 14C-assimilation test. For AgNO3, similar responses were obtained in the two tests, whereas freshly prepared suspensions of citrate stabilized AgNPs were less toxic in the 2-h tests compared to the 48-h tests. The 2-h test was found applicable for dissolved silver, but yielded non-monotonous concentration–response relationships and poor reproducibility for freshly prepared AgNP suspensions. However, when aging AgNPs in algal medium 24 h prior to testing, clear concentration–response patterns emerged and reproducibility increased. Prolonged aging to 48 h increased toxicity in the 2-h tests whereas aging beyond 48 h reduced toxicity. Our results demonstrate that the outcome of algal toxicity testing of AgNPs is highly influenced not only by the test duration, but also by the time passed from the moment AgNPs are added to the test medium. This time-dependency should be considered when nanomaterial dispersion protocols for ecotoxicity testing are developed.

The potential uptake and trophic transfer of nanoparticles (NP) is not well understood so far and for ZnO NP the data presented in peer-reviewed literature is limited. In this paper the influence of surface functionalization on the uptake and depuration behavior of ZnO NP, ZnO-OH NP and ZnO-octyl NP in D. magna was studied. Bulk ZnO particles (≤5 μm) and ZnCl2 were used as references for uptake of particles and dissolved species of Zn, respectively. Furthermore, the trophic transfer of ZnO NP and ZnO-octyl NP from daphnids (Daphnia magna) to zebra fish (Danio rerio) was studied. For ZnO NP and ZnO-octyl NP fast uptakes in D. magna were observed, whereas no measurable uptake took place for ZnO-OH NP. Lower body burden of ZnCl2 was found compared to both ZnO NP and ZnO-octyl. Contrary, the body burden for bulk ZnO was higher than that of ZnO NP but lower than ZnO-octyl. The higher body burdens found for functionalized ZnO-octyl NP than for non-functionalized ZnO NP showed that that the functionalization of the NP has a high influence on the uptake and depuration behavior. Though no mortality was observed, the resulting body burdens were 9.6 times (ZnO NP) and 47 times (ZnO-octyl NP) higher than toxic levels reported for zinc in D. magna. Consequently, the zinc recovered in the animals was not solely due to soluble zinc, but agglomerates/aggregates of ZnO NP or ZnO-octyl NP contributed to the body burdens. The trophic transfer study showed uptake of both ZnO NP and ZnO-octyl NP reaching more than tenfold higher levels than those obtained through aqueous exposure in other studies. This study contributes to expand the available data on uptake behavior of differently functionalized ZnO NP in D. magna and the potential trophic transfer from zooplankton to fish.

The literature has been critically reviewed in order to assess the attenuation processes governing contaminants in leachate affected aquifers. Attenuation here refers to dilution, sorption, ion exchange, precipitation, redox reactions and degradation processes. With respect to contaminants, focus is on dissolved organic matter, xenobiotic organic compounds, inorganic macrocomponents as anions and cations, and heavy metals. Laboratory as well as field investigations are included. This review is an up-date of an earlier comprehensive review. The review shows that most leachate contamination plumes are relatively narrow and do not in terms of width exceed the width of the landfill. The concept of redox zones being present in the plume has been confirmed by the reported composition of the leachate contaminated groundwater at several landfills and constitutes an important framework for understanding the behavior of the contaminants in the plume as the leachate migrates away from the landfill. Diverse microbial communities have been identified in leachate plumes and are believed to be responsible for the redox processes. Dissolved organic C in the leachate, although it appears to be only slowly degradable when the volatile organic acids are gone, apparently acts as substrate for the microbial redox processes. Several xenobiotic organic compounds have been found to be degradable in leachate contaminated groundwater, but degradation rates under anaerobic redox conditions have only been determined in a few cases. Apparently, observations in actual plumes indicate more extensive degradation than has been documented in the laboratory. The behavior of cations in leachate plumes is strongly influenced by exchange with the sediment, although the sediment often is very coarse and sandy. Ammonium seems to be subject to anaerobic oxidation, but the mechanisms are not yet understood. Heavy metals do not seem to constitute a significant pollution problem at landfills, partly because the heavy metal concentrations in the leachate often are low, and partly because of strong attenuation by sorption and precipitation. Although complexation of heavy metals with dissolved organic matter is significant, the heavy metals are in most cases still strongly attenuated in leachate-polluted aquifers. The information available on attenuation processes has increased dramatically during the last 15 a, but the number of well-documented full scale leachate plumes are still few and primarily from sandy aquifers. Thus, the diversity of attenuation processes in leachate plumes is probably not yet fully understood. Apparently, the attenuation processes in leachate plumes may for many contaminants provide significant natural remediation, limiting the effects of the leachate on the groundwater to an area usually not exceeding 1000 m from the landfill.

The literature on nano(eco)toxicology is growing rapidly and has become increasingly difficult to interpret. We have developed a systematic tool called NanoRiskCat that can support companies and regulators in their first-tier assessment and communication on what they know about the hazard and exposure potential of consumer products containing engineered nanomaterials. The final outcome of NanoRiskCat is communicated in the form of a short-title describing the intended use and five colored dots. The first three dots refer to the qualitative exposure potential for professional end-users, consumers and the environment, whereas the last two refers to the hazard potential for humans and the environment. Each dot can be assigned one of four different colors, i.e. red, yellow, green, and gray indicating high, medium, low, and unknown, respectively. In this paper, we first introduce the criteria used to evaluate the exposure potential and the human and environmental hazards of specific uses of the nanoproduct. We then apply NanoRiskCat to eight different nanoproducts. The human and environmental exposure potential was found to be high (i.e., red) for many of the products due to direct application on skin and subsequent environmental release. In the NanoRiskCat evaluation, many of the nanomaterials achieve a red human and environmental hazard profile as there is compelling in vivo evidence to associate them with irreversible effects, e.g., carcinogenicity, respiratory, and cardiovascular effects, etc., in laboratory animals. A significant strength of NanoRiskCat is that it can be used even in cases where lack of data is prominent.

The aim of the present study was to describe the occurrence and distribution of toxicity related to organic chemical contaminants in the leachate plume downgradient of the Grindsted Landfill (Denmark). A total of 27 groundwater samples were preconcentrated by solid-phase extraction (SPE) using XAD-2 as the resin material. This treatment effectively eliminated sample matrix toxicity caused by inorganic salts and natural organic compounds and produced an aqueous concentrate of the nonvolatile chemical contaminants. The SPE extracts were tested in a battery of standardized short-term aquatic toxicity tests with luminescent bacteria (Vibrio fischeri), algae (Selenastrum capricornutum), and crustaceans (Daphnia magna). Additional genotoxicity tests were made using the umuC test (Salmonella typhimurium). Biotests with algae and luminescent bacteria were the most sensitive tests. On the basis of results with these two bioassays, it was concluded that SPE extracts of groundwater collected close to the landfill we...

An improved procedure for quantification of results from the umuC tests for genotoxicity is presented. The calculation method better separates toxic growth inhibition (cytotoxicity) from genotoxic effects than currently used methods and therefore, greatly extends the applicability of genotoxicity tests on environmental samples. The basic principle is to normalize the genotoxic response compensating for both decreasing biomass and growth rate reduction that results from cytotoxicity. The improved method and the currently used method was compared for umuC tests on the pure compounds: methylmethanesulfonate (MMS), N-methyl-N'-nitro-N-nitroguanidine (MNNG), sodium azide (NaN3), and 4-nitroquinoline-1-oxide (4-NQO). For compounds with no or low cytotoxicity, the two calculation methods gave practically identical results, while for highly cytotoxic compounds, the traditional method overestimated genotoxicity. umuC tests were also carried out on leachate polluted groundwater sampled downgradient of a landfill (Grindsted, Denmark). All polluted samples showed high cytotoxicity concomitant with high genotoxicity when the results were quantified in the traditional way. The new method showed that these results were in fact false positive, as the apparent genotoxicity was a result of cytotoxicity. Based on the mathematical analysis leading to the improved procedure for correction for cytotoxicity, it is suggested to alter the present test design of the umuC test in order to obtain well-defined exposure concentrations as well as mathematical consistency in the quantification of genotoxicity.

Nanosilver: Safety, health and environmental effects and role in antimicrobial resistance Philippe Hartemann, Peter Hoet, Ana Proykova, Teresa Fernandes, Anders Baun, Wim De Jong, Juliane Filser, Arne Hensten, Carsten Kneuer, Jean-Yves Maillard, Hannu Norppa, Martin Scheringer, Susan Wijnhoven European Commission, Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) Corresponding author :. Proykova, A. (SANTE-C2-SCIENTIFICCOMMITTEES@ec.europa.eu)

The aerobic aquatic biodegradability of pentachlorophenol (PCP) was studied in standard Organization for Economic Cooperation and Development/International Organization for Standardization shake flask screening tests for ready biodegradability and in more environmentally realistic surface water die-away tests with low chemical concentrations. Screening tests were conducted with either activated sludge or supernatant from settled activated sludge. Concentrations of activated sludge were 30 and 0.3 mg suspended solids per liter. Sludges were either freshly collected or initially preexposed to PCP. With supernatant, only low inoculum levels were used (0.5 ml/L). Some surface water tests were carried out with sediment amendment. Added chemical concentrations ranged from 1 to 74,000 μg/L. At nontoxic concentrations, PCP was rapidly biodegradable after an adaptation period of about 10 to 20 d. At concentrations below about 10 μg/L, degradation rates decreased, and the shape of the degradation curves indicated a shift from growth-linked degradation to non-growth-linked degradation. Addition of sediment to surface water reduced the acclimation periods but did not influence the subsequent degradation rate. Preexposure of activated sludge to PCP drastically eliminated the acclimation period and increased the tolerance of the sludge to PCP toxicity. With PCP used as a model compound, the results show that a battery of simple shake flask tests is suitable for provision of a detailed characterization of the biodegradability chemical compounds.

Information related to the potential environmental exposure of engineered nanomaterials (ENMs) in the solid waste management phase is extremely scarce. In this paper, we define nanowaste as separately collected or collectable waste materials which are or contain ENMs, and we present a five-step framework for the systematic assessment of ENM exposure during nanowaste management. The framework includes deriving EOL nanoproducts and evaluating the physicochemical properties of the nanostructure, matrix properties and nanowaste treatment processes as well as transformation processes and environment releases, eventually leading to a final assessment of potential ENM exposure. The proposed framework was applied to three selected nanoproducts: nanosilver polyester textile, nanoTiO2 sunscreen lotion and carbon nanotube tennis racquets. We found that the potential global environmental exposure of ENMs associated with these three products was an estimated 0.5–143 Mg/year, which can also be characterised qualitatively as medium, medium, low, respectively. Specific challenges remain and should be subject to further research: (1) analytical techniques for the characterisation of nanowaste and its transformation during waste treatment processes, (2) mechanisms for the release of ENMs, (3) the quantification of nanowaste amounts at the regional scale, (4) a definition of acceptable limit values for exposure to ENMs from nanowaste and (5) the reporting of nanowaste generation data. Electronic supplementary material The online version of this article (doi:10.1007/s11051-014-2394-2) contains supplementary material, which is available to authorized users.

During the last couple of years the use of nanoparticles (NP) has dramatically increased. Zinc oxide nanoparticles (ZnO NP) have a wide range of applications e.g. in personal care products, paints and semi conductors. However, only a limited number of studies have so far investigated the ecotoxicity of ZnO NP and to our knowledge the bioaccumulation behavior in regards to difference in functionalization of ZnO NP has not been studied previously. In this study, experiments with trophic transfer using crustaceans (Daphnia magna) as food source for zebrafish (Danio rerio) was carried out to test if changes in functionalization of ZnO NP would affect the bioaccumulation behavior compared to ZnO NP. D. magna was exposed to pristine and functionalized ZnO NP in concentrations considered non-lethal in a 6341 Daphnia immobilization test. Bioconcentration studies with D. magna included a 24h uptake and 24h depuration phase and it was found that steady state with regards to body burden was reached after 24h uptake. The trophic transfer studies were carried out as 14 days of uptake feeding with pre-exposed D. magna and 7 days of depuration feeding with non-exposed D.magna. For the trophic transfer studies,5 days old D. magna were exposed to 1 mg/L ZnO NP (ZnO NP and ZnO-C8H17 NP) for 24h before feeding to D. rerio. This was done at a daily rate corresponding to 8% wet weight of the D. rerio. Food (D. magna) not eaten after 2 hours was removed and the resulting exposure loading was corrected for in the data treatment. The tested ZnO NP was of same primary size (35 nm) but with different functionalizations (ZnO and ZnO-C8H17). Characterization included ICP-MS, DLS, BET and TEM. Results show a fast uptake of ZnO NP in D. rerio reaching steady state after 5 days of exposure yielding a total body burden of 887±184 mg Zn/kg dry weight. A fast depuration (k2=-0.13 d-1) was observed reaching steady state after 3 days of depuration. The calculated BioMagnificationFactor (BMF) was calculated to 0.15 and a biological half-life time of 5.3 days. In contrast, ZnO-C8H17 showed linear uptake in D.rerio during the 14 days of uptake thus not reaching steady state. A total body burden of 2169±414 mg Zn/kg dry weight was observed past 14 days of uptake. However, the depuration rate was faster (k2=-0.32 d-1) compared to ZnO NP. The calculated BMF was 0.42 and a biological half-life time of 2.2 days. The studies demonstrate the feasibility of conducting bioconcentration and trophic transfer studies with NP and the results indicate that functionalizing of NP may affect the uptake and depuration of NP in aquatic organisms.

Aquatic toxicology of engineered nanoparticles is challenged by methodological difficulties stemming partly from highly dynamic and poorly understood behavior of nanoparticles in biological test systems. In this paper scientific and technical challenges of testing not readily soluble nanoparticles in standardised algal growth inhibition tests are highlighted with specific focus on biomass quantification methods. This is illustrated through tests with TiO2 and Au nanoparticles, for which cell-nanoparticle interactions and behavior was studied during incubation. Au NP coating layers changed over time and TiO2 nanoparticle aggregation/agglomeration increased as a function of concentration. Three biomass surrogate measuring techniques were evaluated (coulter counting, cell counting in haemocytometer, and fluorescence of pigment extracts) and out of these the fluorometric methods was found to be most suitable. Background correction was identified as a key issue for biomass quantification, complicated by algae-particle interactions and nanoparticle transformation. Optimisation of the method is needed to reduce further particle interference on measurements.

The number of available studies on the acute effects of silver nanoparticles (AgNP) on aquatic organisms has increased dramatically in recent years, but there is still very limited information available on chronic effects. In this study, a series of Daphnia magna 21-days reproduction test (OECD 211) were performed using 30 nm citric acid stabilized AgNP. The aim of the study was, besides providing data for the chronic toxicity of AgNP, to study the influence of the food availability on the reproductive toxicity of AgNP in Daphnia magna. The exposure concentrations applied ranged from 2 to 50 μgAg/L, and the test animals were fed with green algae Pseudokirchneriella subcapitata in amounts of approximately 0.12 mgC/Daphnia/day for standard treatment or 0.36 mgC/Daphnia/day for high food availability treatment. The parameters of interest were survival, growth rate of mother animals, days to first live offspring, and number of neonates produced. The data obtained from the chronic tests are intended for modeling using the Dynamic Energy Budget (DEB) theory, which will hopefully provide information on growth and reproduction strategy of the test animals. The concentrations of silver in the test medium over time were measured by ICP-OES and the size of the particles in the medium was measured with DLS.With standard food treatment no significant effects were observed for concentrations lower than 10 μgAg/L. The animals exposed to 10 μgAg/L were slightly bigger and produced more offspring in comparison to controls, whereas concentrations above 10 μgAg/L resulted in inhibition of growth and reproduction as well as an increased mortality.The addition of higher amounts of food showed a beneficial effect on animal survival, growth and reproduction. Similar as in normal food availability treatment, animals exposed to 10 μgAg/L showed enhanced reproduction compared to controls, however the toxic effects on growth and reproduction appeared in concentrations above 20 μgAg/L.A reference test was performed with silver nitrate in concentrations ranging from 0.25 to 1.5 μgAg/L. Mortality was increasing in concentrations above 0.25 μgAg/L, but other than that no significant effects were observed of any of the tested exposure concentrations compared to the control.

In recent years, the ecotoxicity of silver nanoparticles (AgNPs) has been studied intensively due to their high toxicity and extensive use in consumer products. However, the field of aquatic nanotoxicology is generally challenged by poor reproducibility, lack of dose-response relationships, and difficulties in controlling and/or describing the characteristics of the tested NPs. These issues may be related to the widespread approach of using freshly prepared stock solutions for ecotoxicity testing, as the introduction of NPs into aqueous media initiates time-dependent processes that possibly interfere with the toxicity testing, e.g. dissolution, speciation, aggregation, sedimentation and interactions with media components. The aim of this study was to investigate whether suspension of AgNPs in test media 24h prior to algal toxicity testing (a pre-suspension step) affects the toxicity and the reproducibility of the test. Ultimately, the aim is better control of the AgNPs in the algal test system and improved prerequisites for describing their toxicity to alga. The underlying hypothesis is that a large part in the variability of AgNPs toxicity to algae can be explained by the kinetics of dissolution and speciation of Ag ions in the test media. To reduce the amount of time in which changes to NPs may occur during testing, the exposure period was minimized. A recently proposed short-term (2h) algal test was applied, using 14C incorporation during photosynthesis as toxic endpoint [1]. For citrate coated spherical AgNPs with a nominal size of 30 nm, the resulting dose-response relationships from tests without (A) and with (B) the pre-suspension step are illustrated in figure 1. Without the pre-suspension step, poorly reproducible results were obtained and it was not possible to produce comparable EC50 values from the three test runs. Introduction of the presuspension step resulted in a higher degree of reproducibility and in more comparable EC50 values, indicating better control of the processes affecting AgNPs during the 2h testing. Moreover, the algal toxicity of AgNPs increased when extending the pre-suspension step period from 24 to 48h, suggesting that ionic dissolution of AgNPs into the media takes part in AgNP toxicity. Our results stress the importance of dealing with the time-dependent processes that NPs undergo in aquatic media when investigating their toxicity.

The use of engineered nanomaterials (ENM) is increasing. Especially, zinc oxide nanoparticles (ZnO NP) are now widely used in a range of consumer goods e.g. car tires, sunscreens and catalysts for various processes. The increasing use will inevitably result in ENM being released to the environment. One release pathway could be to soils through e.g. biosolids. While much attention has been on aquatic exposure less is known about the behavior of ENM in soils. Especially, the adsorption and desorption properties of ENM onto different soil types are lacking. In this study, the OECD guideline 106 for adsorption and desorption was used to evaluate the behavior of ZnO NP in two different standardized soils. The standardized soils tested were LUFA 2.2 and 2.3. The applied concentration was 3.35 mg Zn/L and samples were taken at 2, 4, 6 and 24 hours. The ZnO NP had a primary particle size of 35 nm and was characterized by ICP-MS, DLS, BET and TEM. During the test a very rapid adsorption of the ZnO NP was observed on both soils reaching equilibrium within the first two hours of the test. The equilibrium concentration was found to be 2.02±0.2 mg Zn/L and 0.29±0.10 mg Zn/L for LUFA 2.2 and LUFA 2.3 respectively. The adsorption capacity was found to be 389.5 L/kg and 177.97 L/kg for LUFA 2.2 and LUFA 2.3 respectively. The adsorbed ZnO NP desorped rapidly within the first 6 hours of exposure to clean media. At 24 hours the desorbed fraction could not be determined statistically different from 6 hours. Consequently, it was proposed that the remaining Zn was bound as inner-sphere complexes. The linearized Freundlich adsorption isotherm was fitted to the data and it was observed that the adsorbed amount of Zn increased with increasing concentrations. For the desorption isotherm it was found that the rate of desorption increased more than the adsorption with increasing concentrations.

In recent years the effects of silver nanoparticles(AgNP) has become one of the most studied areas of in nanoecotoxicology. Several studies have concluded that the toxicity of AgNP to freshwater organisms may be explained by the release of ionic silver. In this study, a series of experiments with the freshwater green algae Pseudokirschneriella subcapitata were carried out to falsify the hypothesis: “The toxicity of silver nanoparticles towards algae is solely caused by the monovalent silver ion”. These experiments were based on PHREEQC modeling of silver ion behavior (added as AgNO3) in 72h OECD algal growth inhibition tests and a newly developed 4-h photosynthesis inhibition algal test. The tested AgNPs were of the same primary size (30 nm) but with different capping agents (citrate, mercaptoundecanoic acid, …MARINA). Characterization included ICP-OES, DLS, NTA, and TEM before and after incubation. Results show that the toxicity of AgNO3 and citrate-stabilized AgNP decreased significantly when the pH was lowered from 8.2 to 7.0. This is in contrast to PHREEQC predictions, but in agreement with the biotic ligand theory. The poster will focus a trends in changes in toxicity of AgNP as a result modifications of test conditions (pH, addition of mono-valent ions and chelator) benchmarked against changes predicted and observed for AgNO3.

Based on previously published hydroponic plant, planktonic bacterial, and soil microbial community research, manufactured nanomaterial (MNM) environmental buildup could profoundly alter soil-based food crop quality and yield. However, thus far, no single study has at once examined the full implications, as no studies have involved growing plants to full maturity in MNM-contaminated field soil. We have done so for soybean, a major global commodity crop, using farm soil amended with two high-production metal oxide MNMs (nano-CeO(2) and -ZnO). The results provide a clear, but unfortunate, view of what could arise over the long term: (i) for nano-ZnO, component metal was taken up and distributed throughout edible plant tissues; (ii) for nano-CeO(2), plant growth and yield diminished, but also (iii) nitrogen fixation--a major ecosystem service of leguminous crops--was shut down at high nano-CeO(2) concentration. Juxtaposed against widespread land application of wastewater treatment biosolids to food crops, these findings forewarn of agriculturally associated human and environmental risks from the accelerating use of MNMs.

The European Commission should be regulating nanosilver, not asking for yet another report on its impact on health and the environment.

After learning about the potential risks associated with various specific nanomaterials, concerns have been raised about adequacy of existing regulation in Europe and what should be done to address any potential regulatory gaps related to nanomaterials. Understanding the limitations of the current regulation in regard to nanomaterials is a starting point in a democratic and transparent process towards adapting existing laws and facilitating an informed discussion about which kind of regulatory options best address the identified limitations. In the following we will introduce key pieces of European legislation affecting nanomaterials, analyze their limitations, and provide a number of recommendations on how these can be overcome. We find that, although nanomaterials are in principle covered by the scope of many of the existing legislative frameworks, it is often unclear, if current regulations are actually applicable when it comes to specific nanomaterials and their diverse applications. Main limitations seem to be: that requirements to do safety evaluations are triggered by production volumes by tonnage not tailored to the nanoscale, the profound lack of (eco)toxicological data, and that thresholds values and occupational exposure limits cannot be established with existing methodologies.

There is an urgent need for simple and yet robust scientific methods to evaluate the potential of occupational exposure related to the production and application of nanomaterials. A number of alternatives to traditional exposure assessment have recently been explored and proposed for nanomaterials. Examples of these include the "Control Banding Nanotool" developed to assess and control the risks of nanomaterials, the more holistic "Swiss precautionary matrix", and the first order quantitative risk assessment tool, NanoSafer. Here we review these and other tools and we discuss various elements of the tools (input data requirements, exposure evaluation and handling to reduce exposure) as well as specific pros and cons. Most of the tools provide a transparent and comprehensible approach to assess occupational exposure, but the majority of them are based on purely qualitative considerations about occupational settings. A few methods include specific advice on risk management going well beyond what is normally considered in traditional exposure assessment. A disadvantage in most of the existing concepts is that their data requirements are fairly high. In some cases the technical and scientific procedures to determine them is inconclusive or non-existing. Some of the concepts are furthermore based on purely theoretical considerations and too time-consuming to apply in reality. We provide a set of recommendations for what regulators and risk assessors need to consider before selecting and applying one or the other tool in a given situation and call for further application and development of these tools in the support regulatory decision-making. The aim should be to develop a tiered approach with a purely qualitative, a semi-quantitative, and purely quantitative tool, respectively which can be employed depending on available data and user background. See figure 1 for an illustration of how this could be envisioned in regard to the on-going development of the Danish NanoSafer tool.

While it is generally agreed that successful strategies to address the health and environmental impacts of engineered nanomaterials (NM) should consider the well-established frameworks for conducting life cycle assessment (LCA) and risk assessment (RA), scientific research and specific guidance on how to practically apply these methods are still very much under development. This paper evaluates how research efforts have applied LCA and RA together for NM, particularly reflecting on previous experiences with applying these methods to chemicals. Through a literature review and a separate analysis of research focused on applying LCA and RA together for NM, it appears that current research efforts have taken into account some key “lessons learned” from previous experience with chemicals while many key challenges remain for practically applying these methods to NM. We identified two main approaches for using these methods together for NM: “LC-based RA” (traditional RA applied in a life cycle perspective) and “RA-complemented LCA” (conventional LCA supplemented by RA in specific life cycle steps). Hence, the latter is the only identified approach which genuinely combines LC- and RA-based methods for NM-risk research efforts to date as the former is rather a continuation of normal RA according to standard assessment procedures (e.g. REACH). Both these approaches along with recommendations for using LCA and RA together for NM are similar to those made previously for chemicals, and thus, there does not appear to be much progress made specific for NM. We have identified one issue in particular that may be specific for NM when applying LCA and RA at this time: the need to establish proper dose metrics within both methods., JRC.I.4-Nanobiosciences

The use of engineered nanoparticles (e.g. in industrial applications and consumer products) is increasing. Consequently, these particles will be released into the aquatic environment. Through aggregation/agglomeration and sedimentation, sediments are expected ultimately to be sinks for nanoparticles. Both in the water phase and in the sediments engineered nanoparticles will mix and interact with other environmental pollutants, including metals. In this study the toxicity of cadmium to two freshwater organisms, water column crustacean Daphnia magna and sediment oligochaete Lumbriculus variegatus , was investigated both in the absence and presence of titanium dioxide (TiO 2 ) nanoparticles (P25 Evonic Degussa, d : 30 nm). The uptake of cadmium in sub-lethal concentrations was also studied in the absence and presence of 2 mg/L TiO 2 nanoparticles. Formation of larger nanoparticles aggregates/agglomerates was observed and sizes varied depending on media composition (358 ± 13 nm in US EPA moderately hard synthetic freshwater and 1218 ± 7 nm in Elendt M7). TiO 2 nanoparticles are potential carriers for cadmium and it was found that 25% and 6% of the total cadmium mass in the test system for L. variegatus and D. magna tests were associated to suspended TiO 2 particles, respectively. μXRF (micro X-ray fluorescence) analysis confirmed the uptake of TiO 2 in the gut of D. magna . For L. variegatus μXRF analysis indicated attachment of TiO 2 nanoparticles to the organism surface as well as a discrete distribution within the organisms. Though exact localisation in this organism was more difficult to assess, the uptake seems to be within the coelomic cavity. Results show that the overall body burden and toxicity of cadmium to L. variegatus was unchanged by addition of TiO 2 nanoparticles, showing that cadmium adsorption to TiO 2 nanoparticles did not affect overall bioavailability. Despite facilitated uptake of cadmium by TiO 2 nanoparticles in D. magna , resulting in increased total cadmium body burden, no change in toxicity was observed.

In this study, aged aqueous suspensions of C 60 (nC 60 ) were investigated in the respirometric OECD test for ready biodegradability. Two suspensions of nC 60 were prepared by stirring and aged under indirect exposure to sunlight for 36 months. ATR-FTIR analyses confirmed the presence of C 60 -structures in the suspensions. Samples of the nC 60 suspensions (20 mg/l) were inoculated with activated sludge (30 mgTSS/L) and incubated in a mineral medium under aerobic conditions. Since no mineralisation of nC 60 was observed after 28 days of incubation, 5 mg/l sodium acetate was added to the media. After additional 20 days, no mineralisation of nC 60 was observed. However, within a few days sodium acetate was completely mineralised, showing that the biomass was not inhibited by the presence of nC 60 . Based on results from this simple approach, aged nC 60 can be classified as not ready biodegradable according to the standard OECD test procedure.

Stormwater in urban areas comprises of a substantial part of the urban water cycle, dominating the flow in many small urban streams, and the pollution levels are sizeable. No stormwater quality criteria were found here and no European or national emission limit values exist. Stormwater pollutants however are present in levels exceeding most of the regulated surface water quality criteria and environmental quality standards. Therefore catchment characterisation is needed to chose suitable treatment prior to discharge into receiving surface waters, as the mixing may be insufficient in small streams.

The use of nanoscaled zero-valent iron particles (nZVI) to remediate contaminated soil and groundwater has received increasing amounts of attention within the last decade, primarily due to its potential for broader application, higher reactivity, and cost-effectiveness compared to conventional zero-valent iron applications and other in situ methods. However, the potential environmental risks of nZVI in in situ field scale applications are largely unknown at the present and traditional environmental risk assessment approaches are not yet able to be completed. Therefore, it may not yet be fully clear how to consider the environmental benefits and risks of nZVI for in situ applications. This analysis therefore addresses the challenges of comprehensively considering and weighing the expected environmental benefits and potential risks of this emerging environmentally-beneficial nanotechnology, particularly relevant for environmental engineers, scientists, and decision makers. We find that most of the benefits of using nZVI are based on near-term considerations, and large data gaps currently exist within almost all aspects of environmental exposure and effect assessments. We also find that while a wide range of decision support tools and frameworks alternative to risk assessment are currently available, a thorough evaluation of these should be undertaken in the near future to assess their full relevancy for nZVI at specific sites. Due to the absence of data in environmental risk evaluations, we apply a 'best' and 'worst' case scenario evaluation as a first step to qualitatively evaluate the current state-of-knowledge regarding the potential environmental risks of nZVI. The result of this preliminary qualitative evaluation indicates that at present, there are no significant grounds on which to form the basis that nZVI currently poses a significant, apparent risk to the environment, although the majority of the most serious criteria (i.e. potential for persistency, bioaccumulation, toxicity) are generally unknown. We recommend that in cases where nZVI may be chosen as the 'best' treatment option, short and long-term environmental monitoring is actively employed at these sites. We furthermore recommend the continued development of responsible nZVI innovation and better facilitated information exchange between nZVI developers, nano-risk researchers, remediation industry, and decision makers.

Lemna spp. have been shown to be up to 1000 times more sensitive to some sulfonylurea herbicides (SUs) than the green alga (e.g., P. subcapitata) which is commonly used as a representative organism for aquatic primary producers in environmental risk assessments. When the compounds are evaluated according to the EU Water Framework Directive, the resulting Water Quality Standards (WQSs) are below the analytical quantification limit, making it difficult to verify compliance with the limit values. However, several methodological concerns may be raised in relation to the very low effect concentrations reported for SUs in duckweed tests in the literature. Therefore, we decided to further investigate the effects of four SUs on Lemna gibba by studying how different test parameters affected the toxicity of the SUs. The SUs used were metsulfuron methyl, thifensulfuron methyl, flupyrsulfuron methyl and rimsulfuron. The following parameters were varied during testing: pH, exposure duration, temperature and light/dark cycle. Preliminary results show that a decrease in pH causes an increase in toxicity for all compounds. Exposure to a high concentration for 24 hours caused a reduction in growth rate, from which the plants recovered within 5 days. Difficulties in maintaining the concentration of the test substances were encountered due to a rapid hydrolysis of certain SUs in aqueous solution. These preliminary results raise the question whether the presently used standard Lemna tests are suitable for setting limit values for SUs or if more detailed information should be gained by taking methodological considerations into account.

This paper helps bridge the gap between scientists and other stakeholders in the areas of human and environmental risk management of chemicals and engineered nanomaterials. This connection is needed due to the evolution of stakeholder awareness and scientific progress related to human and environmental health which involves complex methodological demands on risk management. At the same time, the available scientific knowledge is also becoming more scattered across multiple scientific disciplines. Hence, the understanding of potentially risky situations is increasingly multifaceted, which again challenges risk assessors in terms of giving the 'right' relative priority to the multitude of contributing risk factors. A critical issue is therefore to develop procedures that can identify and evaluate worst case risk conditions which may be input to risk level predictions. Therefore, this paper suggests a conceptual modelling procedure that is able to define appropriate worst case conditions in complex risk management. The result of the analysis is an assembly of system models, denoted the Worst Case Definition (WCD) model, to set up and evaluate the conditions of multi-dimensional risk identification and risk quantification. The model can help optimize risk assessment planning by initial screening level analyses and guiding quantitative assessment in relation to knowledge needs for better decision support concerning environmental and human health protection or risk reduction. The WCD model facilitates the evaluation of fundamental uncertainty using knowledge mapping principles and techniques in a way that can improve a complete uncertainty analysis. Ultimately, the WCD is applicable for describing risk contributing factors in relation to many different types of risk management problems since it transparently and effectively handles assumptions and definitions and allows the integration of different forms of knowledge, thereby supporting the inclusion of multifaceted risk components in cumulative risk management.

NanoImpactNet is a European Commission Framework Programme 7 (FP7) funded project that provides a forum for the discussion of current opinions on nanomaterials in relation to human and environmental issues. In September 2008, in Zurich, a NanoImpactNet environmental workshop focused on three key questions: 1. What properties should be characterised for nanomaterials used in environmental and ecotoxicology studies? 2. What reference materials should be developed for use in environmental and ecotoxicological studies? 3. Is it possible to group different nanomaterials into categories for consideration in environmental studies? Such questions have been, at least partially, addressed by other projects/workshops especially in relation to human health effects. Such projects provide a useful basis on which this workshop was based, but in this particular case these questions were reformulated in order to focus specifically on environmental studies. The workshop participants, through a series of discussion and reflection sessions, generated the conclusions listed below. The physicochemical characterisation information identified as important for environmental studies included measures of aggregation/agglomeration/dispersability, size, dissolution (solubility), surface area, surface charge, surface chemistry/composition, with the assumption that chemical composition would already be known. There is a need to have test materials for ecotoxicology, and several substances are potentially useful, including TiO(2) nanoparticles, polystyrene beads labelled with fluorescent dyes, and silver nanoparticles. Some of these test materials could then be developed into certified reference materials over time. No clear consensus was reached regarding the classification of nanomaterials into categories to aid environmental studies, except that a chemistry-based classification system was a reasonable starting point, with some modifications. It was suggested, that additional work may be required to derive criteria that can be used to generate such categories, that would also include aspects of the material structure and physical behaviour.

Exposure assessment is crucial for risk assessment for nanomaterials. We propose a framework to aid exposure assessment in consumer products. We determined the location of the nanomaterials and the chemical identify of the 580 products listed in the inventory maintained by the Woodrow Wilson International Center for Scholars. It was found that in 19% of the products the nanomaterial were nanoparticles bound to the surfaces. Nanoparticles suspended in liquids were used in 37% of the products, whereas 13% used nanoparticles suspended in solids. One percent were powders containing free potentially airborne nanoparticles. Based on the location of the nanostructure we were able to further group the products into categories of: (1) Expected to cause exposure; (2) May cause exposure; and (3) No expected exposure to the consumer. Most products fall into the category of expected exposure, but we were not able to complete the quantitative exposure assessment mainly due to the lack of information on the concentration of the nanomaterial in the products — a problem that regulators and industry will have to address if we are to have realistic exposure assessment in the future. To illustrate the workability of our procedure, we applied it to a product scenario — the application of sun lotion — using best estimates available and/or worst case assumptions.

Along with the possibilities of lighter and stronger materials, clean water and sustainable energy, the development of nanomedicine is one of the primary reasons why huge amounts of public funding are currently going into the development of nanotechnology worldwide. The environmental risks of conventional medical products did not receive much attention until the 1990s; however, the potential environmental side effects have come under increased scientific and regulatory scrutiny in the last decade. In this Editorial, we will take a closer look at scientific and regulatory issues related to the environmental risks of nanomedical products and we will discuss some of the challenges that need to be addressed to ensure that nanomedicines are not only safe for humans but also for the environment. Although the field of nanotechnology is very broad in scope, most attention with regard to human health and environmental risks has evolved around engineered nanoparticles (ENPs), such as C60, carbon nanotubes, metals, metaloxides and quantum dots [1]. At the same time, potential risks related to dendrimers and liposomes have only been raised to a lesser extent. The application of ENPs for medical use offers immense benefits within areas, such as diagnosis, targeted drug delivery and drug development [101]. However, the use of ENPs in nanomedicine has not been subject to much regulatory scrutiny because existing laws and regulatory instruments are believed to also cover medical products based on nanotechnology. The extensive testing requirements prior to marketing of medicine may also contribute to the notion that the potentially negative effects will be discovered prior to marketing, that patients are adequately informed about negative side effects and that the benefits outweigh the risks or the adverse effects, should such be found to occur.

A new technology will only be successful if those promoting it can show that it is safe, but history is littered with examples of promising technologies that never fulfilled their true potential and/or caused untold damage because early warnings about safety problems were ignored. The nanotechnology community stands to benefit by learning lessons from this history.

15 páginas, 4 figuras, 1 tabla.-- From the issue entitled "Special issue on Ecotoxicology, Chemistry and Risk Assessment of Nanoparticles. Guest Editors: Richard Handy and Richard Owen".-- et al., Developments in nanotechnology are leading to a rapid proliferation of new materials that are likely to become a source of engineered nanoparticles (ENPs) to the environment, where their possible ecotoxicological impacts remain unknown. The surface properties of ENPs are of essential importance for their aggregation behavior, and thus for their mobility in aquatic and terrestrial systems and for their interactions with algae, plants and, fungi. Interactions of ENPs with natural organic matter have to be considered as well, as those will alter the ENPs aggregation behavior in surface waters or in soils. Cells of plants, algae, and fungi possess cell walls that constitute a primary site for interaction and a barrier for the entrance of ENPs. Mechanisms allowing ENPs to pass through cell walls and membranes are as yet poorly understood. Inside cells, ENPs might directly provoke alterations of membranes and other cell structures and molecules, as well as protective mechanisms. Indirect effects of ENPs depend on their chemical and physical properties and may include physical restraints (clogging effects), solubilization of toxic ENP compounds, or production of reactive oxygen species. Many questions regarding the bioavailability of ENPs, their uptake by algae, plants, and fungi and the toxicity mechanisms remain to be elucidated.