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In aquatic toxicity testing of engineered nanoparticles (ENPs) the process of agglomeration is very important as it may alter bioavailability and toxicity. In the present study, we aimed to identify test conditions that are favorable for maintaining stable ENP suspensions. We evaluated the influence of key environmental parameters: pH (2-12) and ionic strength using M7, Soft EPA (S EPA) medium, and Very Soft EPA (VS EPA) medium; and observed the influence of these parameters on zeta potential, zeta average, and acute immobilization of Daphnia magna for three different ENPs. Despite being sterically stabilized, test suspensions of silver (Ag) ENPs formed large agglomerates in both VS EPA and M7 media; and toxicity was found to be higher in VS EPA medium due to increased dissolution. Low-agglomerate suspensions for zinc oxide (ZnO) could be obtained at pH 7 in VS EPA medium, but the increase in dissolution caused higher toxicity than in M7 medium. Titanium dioxide (TiO2) ENPs had a point of zero charge in the range of pH 7-8. At pH 7 in VS EPA, agglomerates with smaller hydrodynamic diameters (~200nm) were present compared to the high ionic strength M7 medium where hydrodynamic diameters reached micrometer range. The stable suspensions of TiO2 ENPs caused immobilization of D. magna, 48-h EC50 value of 13.7mgL(-1) (95% CI, 2.4mg-79.1mgL(-1)); whereas no toxicity was seen in the unstable, highly agglomerated M7 medium suspensions, 48-h EC50 >100mgL(-1). The current study provides a preliminary approach for methodology in testing and assessing stability and toxicity of ENPs in aquatic toxicity tests of regulatory relevance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2014 SETAC.)

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 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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The ecotoxicity of three different sizes of titanium dioxide (TiO(2)) particles (primary particles sizes: 10, 30, and 300nm) to the freshwater green alga Pseudokirchneriella subcapitata was investigated in this study. Algal growth inhibition was found for all three particle types, but the physiological mode of action is not yet clear. It was possible to establish a concentration/dose-response relationship for the three particle sizes. Reproducibility, however, was affected by concentration-dependent aggregation of the nanoparticles, subsequent sedimentation, and possible attachment to vessel surfaces. It is also believed that heteroaggregation, driven by algal exopolymeric exudates, is occurring and could influence the concentration-response relationship. The ecotoxicity of cadmium to algae was investigated both in the presence and absence of 2mg/L TiO(2). The presence of TiO(2) in algal tests reduced the observed toxicity due to decreased bioavailability of cadmium resulting from sorption/complexation of Cd(2+) ions to the TiO(2) surface. However, for the 30nm TiO(2) nanoparticles, the observed growth inhibition was greater than what could be explained by the concentration of dissolved Cd(II) species, indicating a possible carrier effect, or combined toxic effect of TiO(2) nanoparticles and cadmium. These results emphasize the importance of systematic studies of nanoecotoxicological effects of different sizes of nanoparticles and underline the fact that, in addition to particle toxicity, potential interactions with existing environmental contaminants are also of crucial importance in assessing the potential environmental risks of nanoparticles.