Abstract
The analysis of (bio)available copper in complex environmental settings, including biological test media, is a challenging task. In this study, we demonstrated the potential of a recombinant Pseudomonas fluorescens-based biosensor for bioavailability analysis of CuSO4 and CuO nanoparticles (nano-CuO) in seventeen different ecotoxicological and microbiologial test media. In parallel, free Cu in these test media was analysed using Cu-ion selective electrode (Cu-ISE). In the case of CuSO4, both free and bioavailable Cu decreased greatly with increasing concentration of organics and phosphates in the tested media. A good correlation between free and bioavailable Cu was observed (r = 0.854, p < 0.01) indicating that the free Cu content in biological test media may be a reasonably good predictor for the toxicity of CuSO4. As a proof, it was demonstrated that when eleven EC50 values for CuSO4 from different organisms in different test media were normalized for the free Cu in these media, the difference in these EC50 values was decreased from 4 to 1.8 orders of magnitude. Thus, toxicity of CuSO4 to these organisms was attributed to the properties of the test media rather than to inherent differences in sensitivity between the test organisms. Differently from CuSO4, the amount of free and bioavailable Cu in nano-CuO spiked media was not significantly correlated with the concentration of organics in the test media. Thus, the speciation of nano-CuO in toxicological test systems was not only determined by the complexation of Cu ions but also by differential dissolution of nano-CuO in different test conditions leading to a new speciation equilibrium. In addition, a substantial fraction of nano-CuO that was not detectable by Cu-ISE (i.e., not present as free Cu-ions) was bioavailable to Cu-biosensor bacteria. Thus, in environmental hazard analysis of (nano) particulate materials, biosensor analysis may be more informative than other analytical techniques. Our results demonstrate that bacterial Cu-biosensors either in combination with other analytical/speciation techniques or on their own, may serve as a rapid (eco)toxicological screening method.
Highlights
Copper is a microelement necessary for various vital functions, but at the same time one of the most toxic heavy metals for aquatic organisms, acting adversely already at sub-ppm concentrations
The media selection included various standard ecotoxicological test media: 2% NaCl, algal medium, two artificial freshwaters
Common media used for cultivation of microorganisms were included: LB medium, malt extract (ME) and YPD (Yeast Peptone Dextrose medium) both used for the cultivation of yeasts (Table 1)
Summary
Copper is a microelement necessary for various vital functions, but at the same time one of the most toxic heavy metals for aquatic organisms (e.g., crustaceans, algae, bacteria [1]), acting adversely already at sub-ppm concentrations. There has been a considerable effort in developing simple bioassays that may be used for direct assessment of heavy metal bioavailability An example of such bioassays are microbial biosensors [13,14], which allow specific detection of bioavailable metals through a highly sensitive biorecognition process followed by induction of a measurable signal, e.g., bioluminescence [15]. We performed a comprehensive comparison between free and bioavailable copper (applied as CuSO4 and nano-CuO) determined using a Cu ion-selective electrode (Cu-ISE) and a. The results for Cu speciation in the selected test media were used to calculate the amount of free ions at reported experimentally determined toxicity values (E(I)C50) for different aquatic test organisms
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