Frank Seitz

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.

Titanium dioxide nanoparticles detoxify pirimicarb under UV irradiation at ambient intensities.

Titanium dioxide nanoparticles (nTiO₂) form reactive oxygen species (ROS) under irradiation by ultraviolet light (UV). This known photocatalytic activity may finally affect the presence and toxicity of organic environmental chemicals, which have not yet been studied at ambient UV intensity. The authors used a three-factorial design to evaluate the interaction of the carbamate insecticide pirimicarb (initial nominal concentration, 20 µg/L), ambient UV irradiation (40 W/m² for 15 min), and nTiO₂(~100 nm; 2.0 mg/L). Pirimicarb, pirimicarb × UV, and pirimicarb × nTiO₂ treatments revealed a median immobilization of Daphnia magna after 72 h ranging between 70 and 80%. This effect seemed to be caused by the initial nominal pirimicarb concentration. However, UV irradiation before an exposure of daphnids in the presence of 2.0 mg nTiO₂/L reduced pirimicarb concentrations to values below the limit of quantification, likely because of the formation of ROS. This reduction was associated with an almost complete removal of toxicity for D. magna. Furthermore, during a second experiment, 0.2 mg nTiO₂/L in combination with 15 min UV irradiation reduced pirimicarb concentrations by approximately 30%. These results indicate a detoxification and therefore remediation potential of the combined application of nTiO₂ and UV irradiation at ambient levels. This potential has not been documented to date in surface waters, where nTiO₂ concentrations in the low to medium µg/L range may occur.

Ecotoxicological evaluation of three tertiary wastewater treatment techniques via meta-analysis and feeding bioassays using Gammarus fossarum.

Advanced treatment techniques, like ozone, activated carbon and TiO(2) in combination with UV, are proposed to improve removal efficiency of micropollutants during wastewater treatment. In a meta-analysis of peer-reviewed literature, we found significantly reduced overall ecotoxicity of municipal wastewaters treated with either ozone (n=667) or activated carbon (=113), while TiO(2) and UV was not yet assessed. As comparative investigations regarding the detoxification potential of these advanced treatment techniques in municipal wastewater are scarce, we assessed them in four separate Gammarus-feeding trials with 20 replicates per treatment. These bioassays indicate that ozone concentrations of approximately 0.8mg ozone/mg DOC may produce toxic transformation products. However, referred effects are removed if higher ozone concentrations are used (1.3mg ozone/mg DOC). Moreover, the application of 1g TiO(2)/l and ambient UV consistently reduced ecotoxicity. Although activated carbon may remove besides micropollutants also nutrients, which seemed to mask its detoxification potential, this treatment technique reduced the ecotoxicity of the wastewater following its amendment with nutrients. Hence, all three advanced treatment techniques are suitable to reduce the ecotoxicity of municipal wastewater mediated by micropollutants and may hence help to meet the requirements of the European Water Framework Directive.

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.