Renata Behra

Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi

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.

Bioavailability of silver nanoparticles and ions: from a chemical and biochemical perspective

Owing to their antimicrobial properties, silver nanoparticles (NPs) are the most commonly used engineered nanomaterial for use in a wide array of consumer and medical applications. Many discussions are currently ongoing as to whether or not exposure of silver NPs to the ecosystem (i.e. plants and animals) may be conceived as harmful or not. Metallic silver, if released into the environment, can undergo chemical and biochemical conversion which strongly influence its availability towards any biological system. During this process, in the presence of moisture, silver can be oxidized resulting in the release of silver ions. To date, it is still debatable as to whether any biological impact of nanosized silver is relative to either its size, or to its ionic constitution. The aim of this review therefore is to provide a comprehensive, interdisciplinary overview—for biologists, chemists, toxicologists as well as physicists—regarding the production of silver NPs, its (as well as in their ionic form) chemical and biochemical behaviours towards/within a multitude of relative and realistic biological environments and also how such interactions may be correlated across a plethora of different biological organisms.

Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench.

Long-term effects of copper on the structure of freshwater periphyton communities and their tolerance to copper, zinc, nickel and silver

Abstract A community adapted to elevated ambient levels of a particular pollutant is expected, compared to a non-exposed community, to display an increased tolerance to that pollutant. The potential of tolerance measurements as a method to detect metal-induced structural impacts at the community level is poorly known. Particularly, the determination of increased tolerance to various metals may confound conclusions related to the causes of the impact. In this study the effects of long-term copper exposure on the community structure of freshwater periphyton, and the short-term community tolerance of photosynthesis to copper, zinc, nickel and silver were determined. Using an outdoor flow-through aquaria system, we carried out long-term exposure of freshwater periphyton communities to copper (0, 0.05, 0.1, 0.5, 1 and 5 μM copper). After 12 weeks we examined how the copper exposure affected the taxonomic composition, photosynthesis rate and tolerance thereof to copper, zinc, nickel and silver. Effects included changes in the distribution of algal classes from a community dominated by Cyanophyceae to one dominated by Chlorophyta. The relative abundance of Oocystis nephrocytioides increased from less than 1% in the control aquaria to 56% in the 5 μM copper treatments. Except at the highest copper exposure, communities did not significantly differ in their photosynthesis rate, although the short-term tolerance of photosynthesis to metals was affected by the copper treatments. Significant increases in tolerance to copper were found in communities previously exposed to ≥0.1 μM copper concentrations. Communities exposed to copper also displayed an increased co-tolerance to zinc, nickel and silver. These observations suggest that copper-induced structural impacts on periphyton communities can be evidenced as an increased tolerance to copper. However, because of the occurrence of co-tolerance, the identification of the metals that have induced the structural and tolerance changes may require metal determinations in organisms.

METAL-INDUCED REACTIVE OXYGEN SPECIES PRODUCTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation‐sensitive reporter dye, 5‐(and‐6)‐carboxy‐2′,7′‐dihydrodifluorofluorescein diacetate (H2DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10−6 to 10−9 M, except in the case of Fe(III), which was effective at 10−18 M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations.

Phytochelatin induction, cadmium accumulation, and algal sensitivity to free cadmium ion in Scenedesmus vacuolatus

Phytochelatins are small, intracellular, metal-binding polypeptides produced by algae on exposure to increased metal concentration in their environment. The aim of the present study was to examine the relationship between phytochelatin concentration, bioaccumulated metal, and sensitivity of algal growth on cadmium exposure. For that purpose, intracellular cadmium concentration and thiol (glutathione, gamma-glutamylcysteine [gammaGluCys], and phytochelatins [PCn]) content were determined in the freshwater green alga Scenedesmus vacuolatus exposed to growth-inhibitory and noninhibitory concentrations of free Cd2+ in the range from 10(-14) to 10(-7) M. The algal growth rate was optimal up to a free Cd2+ concentration of 10(-9) M and then decreased by 40% at higher concentrations. The intracellular cadmium content increased sharply from 0.22 to 746 amol/cell over this free Cd2+ range. At the lowest Cd2+ concentration (control), glutathione was the only detectable thiol (127 amol/cell). With increasing Cd2+, formation of gammaGluCys and phytochelatins from PC2 to PC6 were observed. The predominant oligomer was PC3, with 42 amol/cell at the highest Cd2+ concentration (10(-7) M). The ratio of the concentration of total thiol groups to intracellular cadmium was important for maintaining optimal growth. In contrast, thiol groups from phytochelatins were never measured in excess to intracellular cadmium content.

ADSORPTION AND UPTAKE OF COPPER BY THE GREEN ALGA SCENEDESMUS SUBSPICATUS (CHLOROPHYTA)1

Copper (II) accumulation has been investigated in the green alga Scenedesmus subspicatus G. Brinkmann considering both adsorption and uptake kinetics. Experiments were conducted in a Cu‐ and PH‐buffered medium at different free Cu2+ concentrations that were neither growth limiting nor toxic. We distinguished between adsorption on the cell surface and intracellular uptake by extracting copper from the cells with EDTA. Data from short‐term experiments were compared with data obtained from experiments under steady state conditions. The accumulation of Cu can be described by two processes, an initial fast adsorption occurring within a minute followed by a slower intracellular uptake. Metal uptake followed Michaelis‐Menten kinetics and is mediated by two systems, one with low and the other with high affinity. The maximum uptake rates (1.30 × 10−‐10 mol·[g dry wt algae]−1· min−1, 3.67 × 10−‐9 mol·[g dry wt algae]−1·min−1), and the half‐saturation constants (6.84 × 10−‐14 M, 2.82 × 10−‐12 M) for the two uptake systems were determined using the Lineweaver‐Burk plot. The calculated maximum concentration of binding sites on the surface of the algae is initially higher (9.0 × 10−‐6 mol Cu.[g dry wt algae]−1) than under steady state conditions (2.9 × 10−‐6 mol Cu·[g dry wt algae]−1). This suggests that the initial binding to the algal surface comprises the binding to specific transport ligands as well as to inert adsorption sites. The conditional stability constant of the Cu binding to surface ligands was calculated as log KCu= 11.0 at pH 7.9. This freshwater alga has a high ability to accumulate Cu, reflecting its adaptation to the bioavailable concentration of copper.

Colloidal stability of carbonate-coated silver nanoparticles in synthetic and natural freshwater.

To gain important information on fate, mobility, and bioavailability of silver nanoparticles (AgNP) in aquatic systems, the influence of pH, ionic strength, and humic substances on the stability of carbonate-coated AgNP (average diameter 29 nm) was systematically investigated in 10 mM carbonate and 10 mM MOPS buffer, and in filtered natural freshwater. Changes in the physicochemical properties of AgNP were measured using nanoparticle tracking analysis, dynamic light scattering, and ultraviolet-visible spectroscopy. According to the pH-dependent carbonate speciation, below pH 4 the negatively charged surface of AgNP became positive and increased agglomeration was observed. Electrolyte concentrations above 2 mM Ca(2+) and 100 mM Na(+) enhanced AgNP agglomeration in the synthetic media. In the considered concentration range of humic substances, no relevant changes in the AgNP agglomeration state were measured. Agglomeration of AgNP exposed in filtered natural freshwater was observed to be primarily controlled by the electrolyte type and concentration. Moreover, agglomerated AgNP were still detected after 7 days of exposure. Consequently, slow sedimentation and high mobility of agglomerated AgNP could be expected under the considered natural conditions. A critical evaluation of the different methods used is presented as well.

Toxicity of inorganic and methylated arsenic to algal communities from lakes along an arsenic contamination gradient

Abstract The toxicity of arsenate (As(V)), arsenite (As(III)), monomethylarsonic (MMAA) and dimethylarsinic acid (DMAA) to natural algal assemblages from lakes within the Aberjona watershed having different arsenic concentrations was determined by a short-term photosynthesis assay. Total arsenic concentrations in the studied lakes ranged from 1.5×10 −8 to 1.9×10 −7 M. The toxicity of the arsenic species generally decreased in the order of As(V)>As(III)>DMAA for all lakes. Toxicity of As(V) to phytoplankton collected from relatively unpolluted Horn Pond was highest (EC 50 =3×10 −7 M), whereas algae from the polluted Upper Mystic Lake were more tolerant (EC 50 =6×10 −6 M) and those from Halls Brook Storage Area, a highly contaminated lake, were tolerant to As(V) up to 10 −3 M. The sensitivities of the different algal communities to As(III) were similar (EC 50 value=5×10 −5 M). MMAA was as toxic as As(V) in the unpolluted system (Horn Pond). However, photosynthesis of lakes from contaminated lakes was slightly enhanced by MMAA. DMAA concentration in the range of 10 −5 M was strongly enhancing to short-term CO 2 fixation rates of all phytoplankton assemblages; activity increased up to 600% compared to control values. Algae from contaminated sites appear to have adapted to higher As(V) and MMAA concentrations, whereas algae from the unpolluted lake remained sensitive. As(III) represented no chemical stress to the algal assemblages as measured by the assays. The role of DMAA is unclear. Considering the concentrations and the toxicity of As(V) and MMAA in unpolluted systems, one must conclude that As(V) is the major arsenical environmental hazard, however, MMAA might also pose a potential risk in unpolluted systems.

Intracellular Silver Accumulation in Chlamydomonas reinhardtii upon Exposure to Carbonate Coated Silver Nanoparticles and Silver Nitrate

The intracellular silver accumulation ({Ag}(in)) upon exposure to carbonate coated silver nanoparticles (AgNP, 0.5-10 μM, average diameter 29 nm) and silver nitrate (20-500 nM) was examined in the wild type and in the cell wall free mutant of the green alga Chlamydomonas reinhardtii at pH 7.5. The {Ag}(in) was measured over time up to 1 h after a wash procedure to remove silver ions (Ag(+)) and AgNP from the algal cell surface. The {Ag}(in) increased with increasing exposure time and with increasing AgNP and AgNO(3) concentrations in the exposure media, reaching steady-state concentrations between 10(-5) and 10(-3) mol L(cell)(-1). According to estimated kinetic parameters, high Ag(+) bioconcentration factors were calculated (>10(3) L L(cell)(-1)). Higher accumulation rate constants were assessed in the cell wall free mutant, indicating a protective role of the cell wall in limiting Ag(+) uptake. The bioavailability of AgNP was calculated to be low in both strains relative to Ag(+), suggesting that AgNP internalization across the cell membrane was limited.