Ecotoxicity of HfO2 and SiO2 Nanoparticles on Bacteria (anaerobic methane Archaea); Yeast (Candida albicans) and Biodegradability Tests

Nanoparticles (NPs) are wide class of materials that include particutae substance, which have one dimension less than 100 nm at least [Khan et al., 2019]. Different sizes, different structures, one-element or multi-element structure can be formed in different shapes and formats, or desired NPs have wide potential: in the short term in the textile, cosmetics and dye in the long-term medications are used in drug delivery systems to send the requested body [1-12]. NMOs often have special properties, which are more likely to induce hazardous effects compared to conventional materials (Wang, 2018). Also NMOs are widely used in the treatment of industrial wastewater [2,3]. This widespread production and use of nanoparticles in nature means intense accumulation. NMOs because of can easily be synthesized chemically and can easily be modifi ed consumer products ; industrial products , machinery industry , military applications, in wastewater treatment and medicine widely used [4-19]. In particular, the development of wastewater treatment technology that uses NP is seen as an alternative solution to the growing worldwide water pollution problems. Examples of this work in the treatment of heavy metals ; nano zinc oxide were used for the removal of copper from industrial waste water and the maximum adsorbition capacity obtained for nano-ZnO are 226 mg/g [5-21]. Among the inorganic oxide NPs, silica (SiO2), is among the most commonly utilized NMOs, and this oxide included in the Organization for Economic Cooperation and Development’s (OECD) priority list of NMOs requiring urgent testing for human health and environmental safety [6]. Hafnium oxide (HfO2) is a suitable replacement for silicon oxide. HfO2 has a dielectric constant of about 14, compared to silicon oxide with a dielectric constant of 3.9 [722]. Studies about the environmental toxicity of these NMOs is very limited. The most commonly used Nano-ZnO d creates high toxicity on bacteria (EC50 value for E.coli : 0,048 mg/l). Zinc ions (Zn+2) connect to bacterial cells and reported that damage Abstract

Citation: Sponza  to physiological function of the defeated cell to osmotic shock [8].
In this study the effects of increasing nano-SiO 2 and nano-HfO 2 concentrations (from 1 mg/l to 100 mg/l) were studied on two trophic levels (bacteria, yeast)and some toxicity analyses were performed to detect the EC 50 values (nanoparticle concentration inhibiting 50 % of the organisms).
Furthermore, their biodegradability tests were determined in an aquatic environment during 28 days based on the soluble COD concentrations.

Properties and preparation of NMOs
The environmental toxicity of nanoparticles was studied using three different NMOs. These nanoparticles are nano-SiO 2 (Sigma-Aldrich, 637238, Lot:MKBL8542V) and nano-HfO 2 (Sigma-Aldrich, 202118, Lot:MKBH3310V). The ranges of studied nanoparticles concentrations were determined by considering the acute toxicity test in the recent literature.

Toxicity tests
Yeast acute toxicity tests: Candida albicans from yeast was studied to study the toxic effect of nano-HfO2 and nano-SiO2.
Reference culture used this toxicity test that provided from Public Health Agency of Turkey. Lyophilized cultures were incubated in SD broth (sabouraud dextrose broth) during 4 hour at 30 ºC for both organisms, then they were transferred by pour plate technique to petri plates containing PDA (potato dextrose agar) and they were incubated during 72 hours at 30 ºC. When the organisms are in log phase (3,6×10 -5 cfu/ml) for C. albicans, the acute toxicity of NMOs were performed. NMOs stock solutions (100 mg/L) were sonicated during 60 min. From the stock NMO solutions serial dilutions were performed. 5 ml of the different NMOs dilutions and 1 ml of Candida albicans culture staying in log phase (5×10 -5 cfu/ml) for Candida albicans were placed on the steril tubes and they were incubated during 24h and 48h at 30ºC temperature. The numbers of yeast were enumerated and compared with control groups containing no NMOs. From the inhibiton percentages of the organisms, the values affecting the 50 % of the C.albicans was accepted as EC 50 values.
Anaerobic toxicity test ATA: Anaerobic toxicity assays (ATA) were performed at 35ºC and volume of 150 ml amber bottle reactors [9]. Anaerobic sludge used for this test was

Statistical analysis
The acute toxicity of NMOs to organisms with increasing doses has been studied by the statistical analysis of inhibiton of organims whether it is time-dependent or dose-dependent.
The relationships between the variables of time and inhibition percentages were investigated with multiple regression analysis using the ANOVA program (JMP 10). r 2 and p (<0.05) parameters were used to describe the statistical signifi cance between dependent and independent variables.

Eff ect of NMOs to Yeast -Candida albicans
During 24 h and 48 h incubation period C.albicans colonies were exposed to increasing NMOs concentrations (1 mg/L; 10 mg/L; 50 mg/L and 100 mg/L). C.albicans colonies were enumerated and were calculated as percent inhibition compared to the control groups.  Almost complete killing of 99.5 % of C.albicans was observed [17]. Kasemets and coworkers (2011) found that the EC 50 value of yeast -Saccharomyces cerevisiae was 131 mg/l for nano-ZnO after 24 hours [18].

Results of Anaerobic Toxicity Assay (ATA)
Anaerobic toxicity assay was performed on methane production during 24 h and 48 h incubation periods at different NMOs concentrations. In this test methane gas production of each assay bottle were measured and inhibition percentages were calculated with the control groups no containing NMOs. The ATA test results indicated that increasing of NMOs doses caused adverse effect to methane productions from the anaerobic Archaea (Table 3). After 24 and 48 h incubation period, there is low toxicity to anaerobic methane Archaea for 1 mg/L nano-HfO 2 (I=0.35 %; I=1.79 %, respectively). As the nano-HfO 2 doses increased, the toxicity were increased 0.35 % to 14.23 % for 24 h and 1.79 % to 13.97 % for 48h (Table  3). Otherwise nano-SiO2 is very toxic to anaerobic methane Archaea as seen in Table 3. After 48 h exposed to nano-SiO2, bacteria were inhibited 100 % at 100 mg/L nano-SiO2 dose ( Table 3). ANOVA tests statistics for anaerobic toxicity assay (ATA) tests revealed that there is a linear relationship between NMOs concentrations and incubation period (for nano-HfO 2 , Rsquare=0.095; for nano-SiO 2 , Rsquare=0,92). It was found that regression analysis between time (for nano-HfO 2 , p=0,0119<0,05; for nano-SiO 2 , p=0,0143< 0,05) and doses (for nano-HfO 2 , p=0,0003< 0,05 ; for nano-SiO 2 , p=0,0101< 0,05) was signifi cant as a results of ANOVA tests (=0,05).   Table 3: Methane production and inhibition percentages of anaerobic Archaea after exposed to NMOs during 24h and 48h.

Concentrations
(mg/L)  Nano-ZnO is more toxic for all bacteria and fungus species as compare to other NMOs (nano-SiO2, nano-TiO2) but the building materials biodegradated with NMOs [10].

Biodegradability of NMOs
of Natural and Applied Sciences. Also, the author acknowledged The Scientifi c and Technological Research council of Turkey (TUBITAK) for fi nancial support (2210-C).

Conclusıons
In conclusion, among two nano-metal oxides (nano-SiO 2 , nano-HfO 2 ) investigated in this study it was found that the less toxic NMO is nano-HfO 2 to yeast -Candida albicans because of the highest EC 50 value (250.16 mg/L) after 48 hours exposure time. While the more toxic NMO is nano-SiO2 because of 100 % inhibition rate after 48 h at 100 mg/L doses, the less toxic NMO is nano-HfO2 (I=13,97 %, after 48 h). The easiest biodegradable nanoparticles is nano-HfO 2 due to the highest removal effi ciencies (77.1 %, at 10 mg/L concentration).