The toxic mechanism of silver nanoparticles (AgNPs) is still debating, partially because of the common co-occurrence and the lack of methods for separation of AgNPs and Ag(+) in biological matrices. For the first time, Triton-X 114-based cloud point extraction (CPE) was proposed to separate AgNPs and Ag(+) in the cell lysates of exposed HepG2 cells. Cell lysates were subjected to CPE after adding Na2S2O3, which facilitated the transfer of AgNPs into the nether Triton X-114-rich phase by salt effect and the preserve of Ag(+) in the upper aqueous phase through the formation of hydrophilic complex. Then the AgNP and Ag(+) contents in the exposed cells were determined by ICP-MS after microwave digestion of the two phases, respectively. Under the optimized conditions, over 67% of AgNPs in cell lysates were extracted into the Triton X-114-rich phase while 94% of Ag(+) remained in the aqueous phase, and the limits of detection for AgNPs and Ag(+) were 2.94 μg/L and 2.40 μg/L, respectively. This developed analytical method was applied to quantify the uptake of AgNPs to the HepG2 cells. After exposure to 10 mg/L AgNPs for 24 h, about 67.8 ng Ag were assimilated per 10(4) cells, in which about 10.3% silver existed as Ag(+). Compared to the pristine AgNPs (with 5.2% Ag(+)) for exposure, the higher ratio of Ag(+) to AgNPs in the exposed cells (10.3% Ag(+)) suggests the transformation of AgNPs into Ag(+) in the cells and/or the higher uptake rate of Ag(+) than that of AgNPs. Given that the toxicity of Ag(+) is much higher than that of AgNPs, the substantial content of Ag(+) in the exposed cells suggests that the contribution of Ag(+) should be taken into account in evaluating the toxicity of AgNPs to organisms, and previous results obtained by regarding the total Ag content in organisms as AgNPs should be reconsidered.

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Triton X-114 based cloud point extraction has been demonstrated to be an advantageous approach for the recovery of nanosized copper oxide (NCO) from water. The removal of NCO was influenced by the concentrations of TX-114 and salt, incubation temperature and time, as well as solution pH. With the addition of 0.3% (w/v) Triton X-114, over 88% of the spiked NCO was removed from wastewater after incubation at 35°C for 2 h and centrifugation, whereas over 85% of NCO was recovered after incubation at 28°C for 20 h by gravity phase separation, which is economical and energy-saving. This study suggests that the cloud point extraction technique has great potential in removal of nanomaterials from wastewater. . With the development of nanotechnology and the wide application of nanomaterials, engineered nanomaterials are also inevitably released into the aqueous environment. Recently the safety of engineered nanomaterials is increasingly concerned, as studies suggested that the toxicity of nanomaterials is not only from their own intrinsic toxicity, but also from their effects on the generation, transport and exposure of toxic substances [4,5].Therefore, we have to address the problem of removal of nanowaste with potential toxicity. A few procedures have been proposed for the recovery of nanomaterials from aqueous phase. C 60 fullerene was extracted by liquid-liquid extraction with toluene or by solid-phase extraction with octadecylsilyl [6,7]. Ag nanoparticles were recovered with anion exchange resin beads [8], whereas Au nanoparticles [9], CdTe quantum dots [10], and nanosized copper [1] were extracted with ionic liquids separately. These procedures were conducted on the basis of various mechanisms, thus a general, effective, economic, and mature method that is applicable to various nanomaterials is urgently needed. Recently, our group has discovered that Triton X-114 based cloud point extraction (CPE) provides a general, simple, and cost-effective route for reversible concentration/separation and dispersion of various nanomaterials in aqueous phase [11].The objective of this study is to evaluate the applicability of CPE for the recovery of nanomaterials in wastewater by using nanosized copper oxide (NCO) as a model. NCO is reported to widely exist in semiconductor industrial waste water with a considerable concentration [1].

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