Expression of metal-responsive genes.

<p>Transcripts levels analysis by qPCR in liver (black bars) and gills (white bars). Transcript accumulation of (<b>A</b>) <i>Cytochrome P450 1A</i> (<i>CYP1A</i>), and (<b>B</b>) <i>Metallothionein</i> (<i>MT</i>). The genes were analyzed by qPCR in gills and liver, and normalized as function of the levels of endogenous control <i>β-Actin</i> gene. FW0, fresh water; FW0.5, FW plus 0.5 mg·L⁻¹ CuO; FW5, FW plus 5 mg·L⁻¹ CuO; SW0, increasing salinity; SW0.5, SW plus 0.5 mg·L⁻¹ CuO; SW5, SW plus 5 mg·L⁻¹ CuO. Results expressed as percentage of relative quantification. Significant different means by Anova on ranks analysis are shown with ‡ (<i>P</i> value), and significant differences between FW-SW at the same CuO NP concentration (Dunn's post test, <i>P</i><0.05) are shown with §. Asterisks represent significant difference compared to the respective FW0 control by Wilcoxon Signed Rank test (*, <i>P</i><0.05; ** <i>P</i><0.025). <i>n</i>×treatment = 5.</p

Cu-content of liver and gills after CuO NP exposure at different salinities.

<p>Values (ng of Cu per mg of dry weight) are depicted as mean ± SEM. FW0, fresh water; FW0.5, FW plus 0.5 mg·L⁻¹ CuO; FW5, FW plus 5 mg·L⁻¹ CuO; SW0, increasing salinity; SW0.5, SW plus 0.5 mg·L⁻¹ CuO; SW5, SW plus 5 mg·L⁻¹ CuO. Significant different means by Anova analysis are shown with ‡ (<i>P</i> value). Letters denote groups showing non significant differences by Tukey's post-test (<i>P</i><0.05). <i>n</i>×treatment = 5.</p

Characterization of CuO NP used in the present study.

<p>(A) Representative transmission electron microscopy (TEM) images of the CuO NP at two magnifications. The polydisperse size distribution of CuO NP (n = 245) determined from TEM images is shown in the insert. The average size of the NP is shown (mean ± <i>SD</i>). (B) X-Ray diffraction (XRD) pattern of the NP. The X, and Y axes of the XRD represents the angles (2θ) of incident X-ray beam and the corresponding diffraction peak intensity.</p

Sublethal Effects of CuO Nanoparticles on Mozambique Tilapia (<i>Oreochromis mossambicus</i>) Are Modulated by Environmental Salinity

<div><p>The increasing use of manufactured nanoparticles (NP) in different applications has triggered the need to understand their putative ecotoxicological effects in the environment. Copper oxide nanoparticles (CuO NP) are toxic, and induce oxidative stress and other pathophysiological conditions. The unique properties of NP can change depending on the characteristics of the media they are suspended in, altering the impact on their toxicity to aquatic organisms in different environments. Here, Mozambique tilapia (<i>O. mossambicus</i>) were exposed to flame synthesized CuO NP (0.5 and 5 mg·L⁻¹) in two environmental contexts: (a) constant freshwater (FW) and (b) stepwise increase in environmental salinity (SW). Sublethal effects of CuO NP were monitored and used to dermine exposure endpoints. Fish exposed to 5 mg·L⁻¹ CuO in SW showed an opercular ventilation rate increase, whereas fish exposed to 5 mg·L⁻¹ in FW showed a milder response. Different effects of CuO NP on antioxidant enzyme activities, accumulation of transcripts for metal-responsive genes, GSH∶GSSG ratio, and Cu content in fish gill and liver also demonstrate that additive osmotic stress modulates CuO NP toxicity. We conclude that the toxicity of CuO NP depends on the particular environmental context and that salinity is an important factor for modulating NP toxicity in fish.</p></div

Glutathione levels in livers and gills of CuO NP exposed fish in different salinities.

<p>Total glutathione (GSH+GSSG, top panel); Oxidized glutathione (GSSG) and reduced glutathione (GSH) levels (center panel). The ratio GSH/GSSH is shown in bottom panel. FW0, fresh water; FW0.5, FW plus 0.5 mg·L⁻¹ CuO; FW5, FW plus 5 mg·L⁻¹ CuO; SW0, increasing salinity; SW0.5, SW plus 0.5 mg·L⁻¹ CuO; SW5, SW plus 5 mg·L⁻¹ CuO. Expressed as mean nmol.mg tissue⁻¹ ± <i>SD</i>. Groups with significant different means by Anova analysis are shown with ‡ (<i>P</i> value). Letters denote groups showing non significant differences by Tukey's post-test (<i>P</i><0.05). <i>n</i>×treatment = 5.</p

Experimental conditions during CuO NP exposure.

<p>(<b>A</b>) Salinity changes regime for FW and SW tanks. The FW tanks were kept in constant freshwater (0.1 ppt, gray triangles), while in SW tanks, salinity was increased in a stepwise manner every day (black squares), by addition of increasing amounts of sea salt. Salinity increase steps were of 7 ppt/day, except for the first day when the increase was of 3 ppt. (<b>B</b>) Opercular ventilation rate (OVR) in experimental tanks, measured as opercular beats/minute and expressed as the daily mean per tank (n = 7). The dotted line represent the 25% increment selected as experimental end point. Asterisks denote significant differences relative to the FW0 per day (<i>P</i><0.05). Top: FW0, fresh water; FW0.5, FW plus 0.5 mg·L⁻¹ CuO; FW5, FW plus 5 mg·L⁻¹ CuO; Bottom: SW0, increasing salinity; SW0.5, SW plus 0.5 mg·L⁻¹ CuO; SW5, SW plus 5 mg·L⁻¹ CuO. <i>n</i>×tank = 7.</p

BET and Zeta Potential of CuO NP.

<p>(<b>A</b>) BET isotherm of the CuO NP (powder). (<b>B</b>) Zeta Potential (ZP) of the NP sample taken from FW5 tanks over the time. Day 0 represents the ZP of the NP right after mixing to the fresh water of the fish tank. The ZP of the NP remained fairly constant though out the experiments. The dotted line showed as a guide.</p