**4. Challenges of using NPs**

There are several problems related in in-situ transport processes of nano-particles which depict the reactivity loss of zero valent ion-particles with time [64, 65]. Apart from this, several researchers have found that the in-situ application of nanoparticles leads to clustering effect enhanced the deposition of nano-particles which blocks the pore of soil due to which difficulty faced to reach the targeted contaminated areas [66, 67]. Due to clogging effect of soil, filtration process becomes major issue in use of nZVI remediation. Besides this, NPs are denser than water molecules which lead to settle down of NPs in fluid medium, causing clogging effect [68].

Many studies have shown the effectiveness of nZVI application, but its effects to local microbes are still in early stages [69–72]. Lonf term effects and ethical issues related to use of NPs are hindrance to environmental agencies to implement NPs on

field scale. Hence, application of NPs primarily depends on regulatory affairs and policies of each country, that's why use of NPs is limited to European countries and nZVI extensively used in US [73–75].

There is a great concern related to use of NPs due to their negative effect on microbes. Number of studies has been done to address the toxicity of NPs with microbes. However, these studies have been performed on controlled laboratory conditions [76–81]. However, contrary results have also been reported with some studies showing inhibitory effects on microbes where as some of the studies have showing stimulatory effects of NPs [82].

Thus, till date the existing results are controversial and lacks proper studies on field level as these studies are based on type of NPs and tested microbes, pollutants and also, several studies have used specific culture media in controlled lab environment [83].
