Ricki R. Rosenfeldt

Biological surface coating and molting inhibition as mechanisms of TiO2 nanoparticle toxicity in Daphnia magna.

The production and use of nanoparticles (NP) has steadily increased within the last decade; however, knowledge about risks of NP to human health and ecosystems is still scarce. Common knowledge concerning NP effects on freshwater organisms is largely limited to standard short-term (≤48 h) toxicity tests, which lack both NP fate characterization and an understanding of the mechanisms underlying toxicity. Employing slightly longer exposure times (72 to 96 h), we found that suspensions of nanosized (∼100 nm initial mean diameter) titanium dioxide (nTiO2) led to toxicity in Daphnia magna at nominal concentrations of 3.8 (72-h EC50) and 0.73 mg/L (96-h EC50). However, nTiO2 disappeared quickly from the ISO-medium water phase, resulting in toxicity levels as low as 0.24 mg/L (96-h EC50) based on measured concentrations. Moreover, we showed that nTiO2 (∼100 nm) is significantly more toxic than non-nanosized TiO2 (∼200 nm) prepared from the same stock suspension. Most importantly, we hypothesized a mechanistic chain of events for nTiO2 toxicity in D. magna that involves the coating of the organism surface with nTiO2 combined with a molting disruption. Neonate D. magna (≤6 h) exposed to 2 mg/L nTiO2 exhibited a “biological surface coating” that disappeared within 36 h, during which the first molting was successfully managed by 100% of the exposed organisms. Continued exposure up to 96 h led to a renewed formation of the surface coating and significantly reduced the molting rate to 10%, resulting in 90% mortality. Because coating of aquatic organisms by manmade NP might be ubiquitous in nature, this form of physical NP toxicity might result in widespread negative impacts on environmental health.

Heavy Metal Uptake and Toxicity in the Presence of Titanium Dioxide Nanoparticles: A Factorial Approach Using Daphnia magna.

Unintentionally released titanium dioxide nanoparticles (nTiO2) may co-occur in aquatic environments together with other stressors, such as, metal ions. The effects of P25-nTiO2 on the toxicity and uptake of the elements silver (Ag), arsenic (As) and copper (Cu) were assessed by applying a factorial test design. The test design consisted of two developmental stages of Daphnia magna, two levels of nTiO2 (0 versus 2 mg/L) as well as seven nominal test concentrations of the respective element. The presence of nTiO2 increased Ag toxicity for juveniles as indicated by a 40% lower 72-h EC50, while the toxicities of As and Cu were reduced by up to 80%. This reduction was even more pronounced for Cu in the presence of dissolved organic carbon (i.e., seaweed extract) and nTiO2. This outcome coincides with the body burden of the elements, which was elevated 2-fold for Ag and decreased 14-fold for Cu in the presence of nTiO2. Although the underlying mechanisms could not be uncovered, the data suggest that the carrier function of nTiO2 plays a central role. However, to understand the processes and mechanisms occurring in the field due to the presence of nTiO2 further systematic investigations considering environmental variables and nanoparticle characteristics are required.

Nanoparticle toxicity in Daphnia magna reproduction studies: The importance of test design

The increasing use of titanium dioxide nanoparticles (nTiO(2)) inevitably results in their release into the environment, raising concerns about potential adverse effects in wildlife. By following standard test protocols, several studies investigated the ecotoxicity of nTiO(2) among others to Daphnia magna. These studies indicated a large variability - several orders of magnitude - in the response variables. However, other factors, like nanoparticle characteristics and test design, potentially triggering these differences, were largely ignored. Therefore, the present study assessed the chronic ecotoxicity of two nTiO(2) products with varying crystalline structure (A-100; P25) to D. magna. A semi-static and a flow-through exposure scenario were compared, ensuring that both contained environmentally relevant concentrations of dissolved organic carbon. Utilizing the semi-static test design, a concentration as low as 0.06 mg/L A-100 (∼330 nm) significantly reduced the reproduction of daphnia indicating environmental risk. In contrast, no implication in the number of released offspring was observed during the flow-through experiment with A-100 (∼140 nm). Likewise, P25 (∼130 nm) did not adversely affect reproduction irrespective of the test design utilized. Given the present studys results, the particle size, the product composition, i.e. the crystalline structure, and the accumulation of nTiO(2) at the bottom of the test vessel - the latter is relevant for a semi-static test design - may be suggested as factors potentially triggering differences in nTiO(2) toxicity to D. magna. Hence, these factors should be considered to improve environmental risk assessment of nanoparticles.

Effects of silver nanoparticle properties, media pH and dissolved organic matter on toxicity to Daphnia magna

Studies assessing the acute and chronic toxicity of silver nanoparticle (nAg) materials rarely consider potential implications of environmental variables. In order to increase our understanding in this respect, we investigated the acute and chronic effects of various nAg materials on Daphnia magna. Thereby, different nanoparticle size classes with a citrate coating (20-, ~30-, 60- as well as 100-nm nAg) and one size class without any coating (140 nm) were tested, considering at the same time two pH levels (6.5 and 8.0) as well as the absence or presence of dissolved organic matter (DOM; <0.1 or 8.0 mg total organic carbon/L). Results display a reduced toxicity of nAg in media with higher pH and the presence of DOM as well as increasing initial particle size, if similarly coated. This suggests that the associated fraction of Ag species <2 nm (including Ag(+)) is driving the nAg toxicity. This hypothesis is supported by normalizing the 48-h EC50-values to Ag species <2 nm, which displays comparable toxicity estimates for the majority of the nAg materials assessed. It may therefore be concluded that a combination of both the particle characteristics, i.e. its initial size and surface coating, and environmental factors trigger the toxicity of ion-releasing nanoparticles.

Effects of nano-TiO2 in combination with ambient UV-irradiation on a leaf shredding amphipod

Production and use of engineered nanoparticles, such as titanium dioxide nanoparticles (nTiO(2)), is increasing worldwide, enhancing their probability to enter aquatic environments. However, direct effects of nTiO(2) as well as ecotoxicological consequences due to the interactions of nTiO(2) with environmental factors like ultraviolet (UV) irradiation on representatives of detrital food webs have not been assessed so far. Hence, the present study displayed for the first time adverse sublethal effects of nTiO(2) at concentrations as low as 0.2 mg L(-1) on the leaf shredding amphipod Gammarus fossarum both in presence and absence of ambient UV-irradiation following a 7-d exposure. In absence of UV-irradiation, however, the effects seemed to be driven by accumulation of nTiO(2) at the bottom of the test vessels to which the gammarids were potentially exposed. The adverse sublethal and lethal effects on gammarids caused by the combined application of nTiO(2) and ambient UV-irradiation are suggested to be driven by the formation of reactive oxygen species. In conclusion, both the accumulation of nTiO(2) at the bottom of the test vessel and the UV induced formation of reactive oxygen species clearly affected its ecotoxicity, which is recommended for consideration in the environmental risk assessment of nanoparticles.

Titanium Dioxide Nanoparticles Increase Sensitivity in the Next Generation of the Water Flea Daphnia magna

The nanoparticle industry is expected to become a trillion dollar business in the near future. Therefore, the unintentional introduction of nanoparticles into the environment is increasingly likely. However, currently applied risk-assessment practices require further adaptation to accommodate the intrinsic nature of engineered nanoparticles. Combining a chronic flow-through exposure system with subsequent acute toxicity tests for the standard test organism Daphnia magna, we found that juvenile offspring of adults that were previously exposed to titanium dioxide nanoparticles exhibit a significantly increased sensitivity to titanium dioxide nanoparticles compared with the offspring of unexposed adults, as displayed by lower 96 h-EC50 values. This observation is particularly remarkable because adults exhibited no differences among treatments in terms of typically assessed endpoints, such as sensitivity, number of offspring, or energy reserves. Hence, the present study suggests that ecotoxicological research requires further development to include the assessment of the environmental risks of nanoparticles for the next and hence not directly exposed generation, which is currently not included in standard test protocols.

Size-, surface- and crystalline structure composition-related effects of titanium dioxide nanoparticles during their aquatic life cycle.

Nanoparticle toxicity depends amongst others on particle characteristics and nanoparticle behavior during their aquatic life cycle. Aquatic organisms may be exposed to nanoparticle agglomerates of varying size, while lager agglomerates after settling rather affect benthic organisms. In this context, the present study systematically examined the role of particle characteristics, i.e. crystalline structure composition (anatase as well as mixture of anatase-rutile), initial particle size (55-, 100-, and 140-nm) and surface area, in the toxicity of titanium dioxide nanoparticles (nTiO2) to the pelagic filter feeder Daphnia magna (n = 4) and the benthic amphipod Gammarus fossarum (n = 30). Smaller initial particle sizes (i.e. 55-nm) and anatase based particles showed an approximately 90% lower Daphnia EC50-value compared to its respective counterpart. Most importantly, particle surface normalized EC50-values significantly differed for nanoparticles equal to or below 100 nm in size from 140-nm sized particles. Hence, these data suggest that the reactive initial surface area may explain the ecotoxicological potential of different particle size classes only if their size is smaller or around 100 nm. In contrast to Daphnia, Gammarus was not affected by nTiO2 concentrations of up to 5.00 mg/L, irrespective of their characteristics. This indicates fundamental differences in the toxicity of nTiO2 during its aquatic life cycle mediated by alterations in their characteristics over time.

Three-dimensional analysis of the swimming behavior of Daphnia magna exposed to nanosized titanium dioxide.

Due to their surface characteristics, nanosized titanium dioxide particles (nTiO2) tend to adhere to biological surfaces and we thus hypothesize that they may alter the swimming performance and behavior of motile aquatic organisms. However, no suitable approaches to address these impairments in swimming behavior as a result of nanoparticle exposure are available. Water fleas Daphnia magna exposed to 5 and 20 mg/L nTiO2 (61 nm; polydispersity index: 0.157 in 17.46 mg/L stock suspension) for 96 h showed a significantly (p<0.05) reduced growth rate compared to a 1-mg/L treatment and the control. Using three-dimensional video observations of swimming trajectories, we observed a treatment-dependent swarming of D. magna in the center of the test vessels during the initial phase of the exposure period. Ensemble mean swimming velocities increased with increasing body length of D. magna, but were significantly reduced in comparison to the control in all treatments after 96 h of exposure. Spectral analysis of swimming velocities revealed that high-frequency variance, which we consider as a measure of swimming activity, was significantly reduced in the 5- and 20-mg/L treatments. The results highlight the potential of detailed swimming analysis of D. magna for the evaluation of sub-lethal mechanical stress mechanisms resulting from biological surface coating and thus for evaluating the effects of nanoparticles in the aquatic environment.

Inorganic fungicides as routinely applied in organic and conventional agriculture can increase palatability but reduce microbial decomposition of leaf litter

Summary The application of fungicides is considered an indispensable measure to secure crop production. These substances, however, may unintentionally enter surface waters via run-off, potentially affecting the microbial community. To assess such risks adequately, authorities recently called for suitable test designs involving relevant aquatic micro-organisms. We assessed the structural and functional responses of leaf-associated microbial communities, which play a key role in the breakdown of allochthonous leaf material in streams, towards the inorganic fungicides copper (Cu) and elemental sulphur (S). These substances are of particular interest as they are authorized for both conventional and organic farming in many countries of the world. We used the food choice of the amphipod shredder Gammarus fossarum (indicative for micro-organism-mediated leaf palatability) as well as microbial leaf decomposition as functional endpoints. Moreover, the leaf-associated microbial communities were characterized by means of bacterial density, fungal biomass and community composition facilitating mechanistic understanding of the observed functional effects. While Gammarus preferred Cu-exposed leaves over unexposed ones, microbial leaf decomposition was reduced by both Cu and S (up to 30%). Furthermore, Cu exposure decreased bacterial densities (up to 60%), stimulated the growth of leaf-associated fungi (up to 100%) and altered fungal community composition, while S did not affect any of the assessed structural endpoints. Synthesis and applications. We observed both structural and functional changes in leaf-associated microbial communities at inorganic fungicide concentrations realistic for surface water bodies influenced by conventional and organic farming. Our data hence justify a careful re-evaluation of the environmental safety of the agricultural use of these compounds. Moreover, inclusion of an experimental design similar to the one used in this study in lower tier environmental risk assessments of antimicrobial compounds may aid to safeguard the integrity of aquatic microbial communities and the functions they provide.

Nanosized Titanium Dioxide Reduces Copper Toxicity—The Role of Organic Material and the Crystalline Phase

Titanium dioxide nanoparticles (nTiO2) are expected to interact with natural substances and other chemicals in the environment, however little is known about their combined effects. Therefore, this study assessed the toxicity of copper (Cu) in combination with varying crystalline phases (anatase, rutile, and the mixture) of nTiO2 and differing organic materials on Daphnia magna. The nanoparticles reduced the Cu-toxicity depending on the product (0.3- to 2-fold higher 48-h EC50). This decrease in toxicity coincided with a lowered Cu-concentration in the water column, which was driven by the adsorption of Cu to nTiO2-depending on available surface area and structure-and their subsequent sedimentation. In the presence of organic material and nTiO2, the Cu-toxicity was further reduced (up to 7-fold higher 48-h EC50). This observation can be explained by a reduced Cu-bioavailability as a result of complexation and adsorption by the organic material and nTiO2, respectively. Thus, the crystalline phase composition, which is determining the surface area and structure of nTiO2, seems to be of major importance for the toxicity reduction of heavy metals, while the influence of the organic materials was mainly driven by the quantity and quality of humic substances.