The experimental set-up.

<p>The flow-through testing apparatus, showing four experimental units (volume, 500 mL) with five <i>D. magna</i> each. Approximately 40 mL of the test medium was introduced every other hour slightly below the surface of the water. The old test medium was passively discharged using hydrostatic pressure at the bottom of each vessel. A fine mesh screen (0.1 mm) prevented the loss of recently hatched juvenile daphnids.</p

Titanium Dioxide Nanoparticles Increase Sensitivity in the Next Generation of the Water Flea <em>Daphnia magna</em>

<div><p>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 <em>Daphnia magna</em>, 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.</p> </div

Experimental design.

<p>(<i>A</i>) Schematic diagram illustrating the experimental procedure of each of the five sets of experiments conducted. (<i>B</i>) Visualised experimental procedure for the assessment of the “early exposure” hypothesis (second set of experiments).</p

The sensitivity of juveniles released by nTiO₂-exposed adults.

<p>The cumulative mean (±95% CIs) difference in sensitivity – in terms of 96 h-EC50 values – of the offspring (fifth brood) released by adults exposed to 0.02 or 2.00 mg nTiO2/L and offspring released by control (uncontaminated) daphnids is displayed using the standardised effect size Cohen’s <i>d</i>. (<i>A</i>) The cumulative effect sizes for all bioassays conducted (n = 7) with the fifth brood during the first and second set of experiments. (<i>B</i>) The cumulative effect sizes for acute toxicity experiments conducted with offspring released in the control medium by adults previously exposed to the above-mentioned nTiO2 concentrations during the second set of experiments (n = 3). The statistical significance of a cumulative effect is highlighted by an asterisk (*). Negative effect sizes indicate increased toxicity.</p

The sensitivity of juveniles released during the third set of experiments.

<p>96 h- EC₅₀ values with respective 95% CIs of the fifth brood released by adults exposed to 0.00 and 2.00 mg/L P25-nTiO₂ during the third set of experiments, which considered exclusively potential implication of nTiO₂ exposure within the brood pouch. No statistically significant difference among treatments was observed.</p

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

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 <i>Daphnia magna</i>, 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

The sensitivity of juveniles released by adults exposed to different nTiO₂ products.

<p>Sensitivity, displayed as percent relative to the 96 h-EC₅₀ of the respective control, of the fifth brood released by adult <i>D. magna</i> exposed to different nTiO₂ treatments using the products P25 or A-100. The data displayed for P25 represent the weighted mean values of the seven experiments (first and second set of experiments), each with four replicates per treatment, whereas the 96 h-EC₅₀ for the offspring released from the control parents was 3.13 mg/L nTiO₂. For the product A-100, the results of one experiment with four replicates of pre-treatment are displayed (fourth set of experiments). In this situation, the 96 h-EC₅₀ for the offspring released from the control parents was 1.98 mg/L nTiO₂. The error bars and dashed lines indicate the standard error. The dashed lines are related to the control. Asterisks (*) denote significant differences between a treatment and the respective control.</p

Scanning electron microscope image.

<p>An image of the size-homogenised, stable nTiO₂ suspension of the product P25 taken by an scanning electron microscope using 100,000-fold magnification (Hitachi SU8030).</p

Acute toxicity of aged nTiO2.

<p>(A) 96-h EC50 values (half maximal effective concentration; ± 95% CI) of nTiO2 aged for 0, 1, 3 or 6 d in Milli-Q with (■) or without (□) NOM. (B) 96-h EC50 values (± 95% CI) of nTiO2 previously aged for 0, 1, 3 or 6 d in ASTM with (●) and without (○) NOM. 96-h EC50 values followed by different lower case letters are significantly different.</p

Particle uptake of <i>Daphnia</i>.

<p>Schematic draft of the preferably ingested particle size range of <i>Daphnia magna</i>.</p