Table 2.

Summary of studies conducted on the effect of nZVI on microbial communities.

Community studied	nZVI characteristics	nZVI concentration	Experimental conditions	Measured biological parameters	Methods	Effect	Reference
Agricultural land	nZVI (NANOFER 25S*)	34 g Fe0/kg	72 h-exposure, shaking, aerobic.	Community composition	FISH	Significant shifts in community composition.	Fajardo et al., 2012
	< 50 nm			Gene quantification and expression level (nirS, narG, gyrA)	RT-qPCR	No effect on denitrifying populations and gene expression level.
Brownfield soil	nZVI (NANOFER 25S*)	100 g/kg	13 d-exposure.	Community composition	FISH	Increase in β- and decrease in ε-proteobacteria.	Fajardo et al., 2015
				Gene expression level (nirS, narG, KatB, pykA)	RT-qPCR	Overexpression of katB, and underexpression of pykA. No change in narG and nir S expression.
Arable land sandy, loam, and clay soil	Home-made CMC-nZVI	0.27 g Fe0/kg	4 month-exposure, 15°C, dark.	Community composition	PLFA profiles	Community composition shifts higher in sandy soil. Shift in community functionality only in clay soil.	Pawlett et al., 2013
	10 ± 5.2 nm			Community functional profile	MSIR	Reduction of Gram – bacteria and AMF.
				Microbial biomass	Fumigation-extraction
Arable land soil	PAA-nZVI (Golder Associates Inc., USA)	10 g/kg	28 d-exposure, 25°C, dark	Community composition	DGGE	Shifts in Community composition.	Tilston et al., 2013
	12.5 ±0.3 nm			Chloroaromatic mineralizing microbial population activity	14C-based MPN technique ([14C]-UL-2,4-D substrate)	Chloroaromatic biodegradation activity reduced.
Loamy-sand, and loam soil	nZVI (NANOFER 25S*)	17 g/kg	7 d-exposure, 21 ±0.5°C, dark.	Community composition	FISH	No major change in community composition.	Sacca et al., 2014b
	<50 nm			Gene expression level (katB, and pykA)	RT-qPCR	Overexpression of katB and pykA.
Aquifer sediment	PAA-nZVI, bare-nZVI (Toda Kogyo Corp., Japan)	1,500 mg/l	250 d-exposure, shaking, 23 ±2°C, dark.	Community composition	DGGE	Significant shift in microbial community composition.	Kirschling et al., 2010
	27.5 nm			Microbial population dynamics (bacterial and archaeal 16S rDNA, dsrA gene)	qPCR	Increase of SRB and methanogens.
Aquifer sediment	nZVI (Toda Kogyo Corp., Japan)	500-3,000 mg/l	130 d-exposure, 16±2°C, dark, anaerobic	Community composition	16S environmental cloning-sequencing	Shifts in microbial community.	Kumar et al., 2014
	70-100 nm
Activated sludge	Home-made bare-nZVI	20-200 mg/l	9 h-exposure, SBR, anaerobic/aerobic	Community composition	Pyrosequencing	No drastic changes in community composition.	Wu et al., 2013
	SSA: 20.71 m2.g-1			nZVI cytotoxicity	Intracellular	ROS increased with increasing nZVI doses.
				Microbial activity	ROS and LDH	No effect of nZVI on LDH release.
				Sludge surface integrity	ATP cellular content	ATP content decreased with increasing nZVI doses.
					SEM/EDS	Slight damage of sludge surface at 200 mg/l.
Activated sludge	nZVI (NANOFER 25S*)	0.1 20 mg/l	56 d-exposure, SBR, 20°C	Community composition	Pyrosequencing	No significant shifts in community composition.	Ma et al., 2015
	46 ± 10 nm

Abbreviations: AMF, arbuscular mycorrhizal Fungi; CMC, carboxyl methyl cellulose; DGGE, denaturing gradient gel electrophoresis; EDS, energy dispersive X-ray spectroscopy; FISH, fluorescent in situ hybridization; LDH, lactate dehydrogenase; MSIR, multiple substrate-induced respiration; PAA, polyacrylic acid; PLFA, phospholipid fatty acid; ROS, reactive oxygen species ; RT-qPCR, reverse transcription-quantitative PCR; SBR, sequencing batch reactor; SEM, scanning electron microscopy; SRB, sulfate-reducing bacteria. GirA, katB, narG, nirS, and pykA genes encode for gyrase (DNA replication enzyme), catalase (cellular detoxification enzyme), nitrate reductase, nitrite reductase, and pyruvate kinase (glycolysis enzyme), respectively.

^*NANOFER 25S: nZVI coated with polyethylene glycol sorbitant monostearate with a percentage Fe⁰ of 14-18% (NANOIRON s.r.o. , Czech Republic).