CHEMICAL ENGINEERING TRANSACTIONS
VOL. 51, 2016
A publication of
The Italian Association
of Chemical Engineering
Online at www.aidic.it/cet
Guest Editors: Tichun Wang, Hongyang Zhang, Lei Tian
Copyright © 2016, AIDIC Servizi S.r.l.,
ISBN 978-88-95608-43-3; ISSN 2283-9216
The Susceptibility of Soil Microbial Metabolism and
Diazotroph Functional Groups to Silica Nanoparticles
Hankui Chaia, Jun Yao*,a,Mijia Zhua, Brunello Ceccantib
a School of Civil & Environmental Engineering, National International Cooperation Base on Environment and Energy,
University of Science and Technology Beijing, 100083 Beijing, PR China
b Institute of Ecosystem Studies (ISE), Italian National Research Council (CNR), Pisa, Italy
yaojun@ustb.edu.cn
The agro-ecological effect of silica nanoparticles on soil metabolism has been investigated by
microcalorimetry with specific enzymatic tests (urease, catalase and fluorescein diacetate hydrolase, FDA)
and diazothroph account. Three SNP doses (10, 25 and 50 mg kg-1) corresponding to each dimension (9, 12
and 40 nm) were used. The thermodynamic parameters obtained from the power-time curves showed an
increase, with some exception, of total heat output, microbial growth rate constant (k) and heat output peak
(Pmax) by increasing SNP dose, indicating a general stimulation of microbial populations. Urease and FDA
activity, except catalase, showed similar positive size-dose response thus supporting microcalorimetric
analysis. However, no direct effects on diazotrophs were found. The results confirmed the capability of SNP to
maintain or enhance nitrogenous nutrient availability and to promote microbial deoxygenation of the soil
microenvironments. SNP did not depress total microbial biomass (no changes in FDA) or impair plant nutrition
and the diazotroph taxa.
1. Introduction
Engineered nanoparticles (NP) are now becoming a significant fraction of the material in global economy.
Their application in the fields of material sciences, industrial engineering and medical care has increased in
last few years owing to their special and novel physical and chemical properties (Tso et al., 2010, Keller et al.,
2013; Wehling et al., 2013). The reduction of particle size to nanoscale dimension not only provides benefits to
diverse medical and technological areas but also poses potential risks for human health and ecosystem safety
once dispersed into the environment (Dominique et al., 2013, Arne et al., 2013). Now NP pollution is an
increasing environmental problem and worldwide concern (Yotova et al., 2015; Feng et al., 2015; Wu et al.,
2014; Li and Zhang, 2014). Silicon is the second most abundant element on the earth’s crust, but silica
nanoparticles (SNP) may be not beneficial for many organisms as their bulk (Schaller et al., 2013). Previous
research on phytotoxicity of SNP showed that they were non-toxic (Lee et al., 2009). By contrast, serious
damage was detected in pulmonary and angiocarpy of adult mice after inhalation of SNP (Chen et al., 2008).
Disperse SNP caused cell membrane damage of Saccharomyces cerevisiae (García et al., 2011). SNP
showed size-dependent toxicity for microorganisms which increased by decreasing particle sizes (Wehling et
al., 2013). SNP were also found to exhibit size-dependent toxicity toward the alga (Chlorella kessleri,
Pseudokirchneriella subcapitata) (Fujiwara et al., 2008, Van et al., 2011).
The abuse of NP will cause accumulation over time in the form of aggregates and colloids, producing an
unpredictable anthropogenic waste in the agroecosystem (Thul et al., 2013). Emissions of NP into soils
represent up to about a quarter of the material flows, mostly from disposal of biosolids to land, thus
highlighting the importance of understanding the impact of NP on agriculture soils (Keller et al., 2013).
Therefore, more data are required about the behavior of NP in soil and their interactions with microorganisms
especially under intensive cultivation. Heat transfer in conjunction with other specific bio-tests has been
successfully applied to assess the impacts of environmental hazardous pollutants on soil microbial metabolism
(Mahmoudi and Mejri, 2015; Hongyang,2015; Biserni and Garai, 2016). In microcalorimetry, the heat evolution
is positively correlated to the amount of glucose degradation, microbial biomass and activity in glucose-
DOI: 10.3303/CET1651014
Please cite this article as: Chai H.K., Yao J., Zhu M.J., Ceccanti B., 2016, The susceptibility of soil microbial metabolism and diazotroph
functional groups to silica nanoparticles, Chemical Engineering Transactions, 51, 79-84 DOI:10.3303/CET1651014
79
addition calorimetric assays (Kimura and Takahashi, 1985). In addition, developing new approaches to assess
soil enzyme functional diversity is necessary for our comprehending of the relations between resource
availability, microbial community structure and function, and ecosystem processes (Caldwell, 2005). As such,
this study focuses on the effects of various sizes of SNP on microbial metabolism in agricultural soil. The
thermogenic metabolic energy flux, enzymatic activities (urease, catalase, fluorescein diacetate hydrolase)
and soil diazotroph number were systematically monitored as they are markers fertility and health.
2. Materials and methods
2.1 Soil analysis
The experimental loam soil was collected on the top (2–5 cm) of an arable field planted with winter wheat from
Hebei Province, China. The soil was thoroughly sieved < 2mm, air dried and kept at 4 ºC in refrigerator before
using. The properties of soil is pH7.2, sand/silt/clay 13.3/70.8/15.9, CEC (cmol kg-1) 7.6, total N (µg g-1) 0.8,
total organic C (mg g-1)15.4, available P (µg g-1) 13.2, available K (µg g-1) 96.7, field capacity (v/v) 0.32.
2.2 Preparation of soil with nanoparticle
Three commercial SNP amorphous powders at ca. 9, 12, 40 nm with > 99.9% purity were obtained from
Boyugaoke Inc. (Beijing, China). The surface area were 300 ± 30 m2 g-1 (9nm), 200 ± 20 m2 g-1 (12 nm) and
110 ± 25 m2 g-1 (40nm), respectively. The size of SNP were determined by scanning electron microscopy and
the surface area of SNP was examined using the BET method.
SNP were suspended in distilled water and sonicated to achieve a homogeneous mixture before adding to the
soil samples in a test-microcosm. Each microcosm consisted of 50 g of soil in 200 mL sterile plastic bottles
and NP mixtures were added to achieve a final concentration of 10, 25, 50 mg kg-1, respectively. Microcosm
without SNP was used as control. Three triplicates of each microcosm were incubated at 25 ºC for one month
and soil moisture was adjusted at 25% of water holding capacity by adding sterile deionized water.
2.3 Microcalorimetric analysis
An isothermal TAM III multi-channel microcalorimeter (TA instruments, New Castle, DE, USA) was used to
record the metabolic thermogenetic flux curves. Each 4.0 ml stainless steel ampoule was loaded with 1 g soil
containing SNP. Then 200 µL nutrient solution containing 5.0 mg glucose and 5.0 mg ammonium sulphate
was added to the soil. The calorimetric parameters such as total heat output (Qtotal), microbial growth constant
(k), peak time and the height of peak was calculated from the power-time curves. Qtotal was computed by
integrating the power-time curves. A classical equation is used to fit the exponential growth phase, lnPt =lnP0 +
kt, where t is the time, Pt is the output of power at time t, and the P0 is the power at the initial stage of the
exponential growth (Guo et al., 2012).
2.4 Measurement of soil enzyme activity and diazotroph counting
The activity of urease was measured using colorimetric determination of ammonium released from urea
hydrolysis (Zhuang et al., 2011, Guo et al., 2012). Catalase activity was determined by back-titrating residual
H2O2 with 0.1 mol L-1 KMnO4 solution (Du et al., 2011). The FDA hydrolytic activity was carried out at 490 nm
as absorption of the hydrolysis product fluorescein (Schnürer and Rosswall, 1982). Soil samples (5 g) were
placed in Erlenmeyer flasks containing sterile water for shaking (180 rpm, 30 min) and followed by continuous
dilutions for plate counting. Viable counts of soil diazotroph were performed on Ashby Mannitol Phosphate
Agar incubated 28 ºC for 7 days. All bioassays conducted with materials were in accordance with national and
institutional guidelines for the protection of human subjects and animal welfare.
3. Results and discussions
3.1 Dose-size effects of silica nanoparticles
Figure 1 depicts the thermal output curves of soil samples containing various concentrations and sizes of
SNP. Two major peaks (Pmax1 at the time Pt1 and Pmax2 at the time Pt2) were observed in the thermograms.
These peaks showed a tendency to appear at shorter times with the increase in SNP concentration and even
shorter than the untreated controls. However, no significant differences of Pt were observed by changing size
of SNP. In general, Pmax1 is smaller than Pmax2. Both Pmax1 and Pmax2 increases with the concentration of SNP.
SNP is likely to express indirect positive effects on microbial metabolism, acting as a biochemical concentrator
of substrates at the surface (Dinesh et al., 2012), thus, allowing dormant populations to benefit from the
enriched surrounding habitat to grow faster than the control. Qtotal increases with the increase in the
concentration of SNP whereas k does not change much with the increase in dose and size of SNP. The
average relative values of Pmax1 showed a regular increase with increasing SNP size; Pmax2, even showing
values higher than Pmax1, also showed a flattening at 9-12 nm and a dropping in the 40 nm test. The percent
increase of Pmax1 and Pmax2 had the same order of magnitude at 9-12 nm, while at 40 nm size showed an
80
opposite trend, i.e., maximum stimulation at short time Pt1 (8-10 h) and lower metabolic rates at longer Pt2 (30
h) time, comparatively to the percent increase control.
0 10 20 30 40 50 60 70
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 50 60 70
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 50 60 70
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 50 60 70
0
50
100
150
200
250
300
350
400
450
P
o
w
e
r(
W
)
Time (h)
control
(A)
P
o
w
e
r(
W
)
Time (h)
9 nm (10 mg kg
1
)
9 nm (25 mg kg
1
)
9 nm (50 mg kg
1
)
(B)
P
o
w
e
r(
W
)
Time (h)
12 nm (10 mg kg
1
)
12 nm (25 mg kg
1
)
12 nm (50 mg kg
1
)
(C)
P
o
w
e
r(
W
)
Time (h)
40 nm (10 mg kg
-1
)
40 nm (25 mg kg
-1
)
40 nm (50 mg kg
-1
)
(D)
Figure 1: Thermogenesis metabolism curves of soil microorganism spiked with various sized of silica
nanoparticles: (A) control, (B) 9, (C) 12, and (D) 40 nm
Kinetics of glucose degradation is proportional to kinetics of microbial growth (Castello et al., 2014). For this
reason, the microbial growth rate constant k is considered as the apparent degradation rate of the substrate
(Barja and Núñez, 1999). Therefore, k was regarded as a sensitive parameter expressing minor changes in
the mean life adaptability of the many microbial populations and in their response to the stressing effects
caused (Koga et al., 2003). It has been found that generally NP with different sizes caused different
environmental effects at equivalent concentrations (Gao et al., 2008). This variability in microbial activities of
the soil might have been caused by superoxide and other reactive oxygen species (Yang et al., 2010). SNP
were found to exhibit size-dependent toxicity toward the alga (Fujiwara et al., 2008). It also has been
demonstrated that the cytotoxicity of SNP with the same morphology was strongly related to the particle size
(Napierska et al., 2009). Most of studies have proved that metal oxide NP display effect of size-dependent
effectiveness and efficiency on bacteria (Ma et al., 2013).
3.2 Enzymatic activities
Soil enzyme activity is considered as direct indicators of the soil community to metabolic demand and
available nutrients. It has been argued that maintaining critical functions may ultimately be more important
than maintaining taxonomic diversity in soil microbial communities (Caldwell, 2005). Urease activity in soil
originated from soil microbes containing urease and approximately 17-77 % soil bacteria and 78-98 % soil
fungi have capacity to hydrolyze urea into ammonia. Table 1 displays the effect of SNP on the soil urease
activity. It is found that the soil urease activity significantly (p < 0.05) increases with increase concentration of
SNP but to a lesser extent with the increase in particle size. The highest urease activity was found in
coincidence of the highest SNP dose and size, contrarily to the trend observed for the thermodynamic
parameters Pmax2. It seems that the soil catalase activity does not affect too much by the dose of SNP (p >
0.05). There is also not much difference in the soil catalase activity for difference sizes of SNP. Since catalase
is not affected by the size and dose of SNP, it could be a good candidate for assessing oxidation–reduction
potential of stressed soils. By the contrast, FDA is significantly stimulated by SNP concentration (p < 0.01) at
all sizes. FDA shows higher activity with the increase in the concentration of SNP. The trends are similar for
each type of SNP. At 10 mg kg-1 SNP, the effect is very small and is not significantly different (p > 0.05) from
that of the control. Our results are different to that of ZnO NP which can inhibit soil protease, catalase,
dehydrogenase, phosphatase and peroxidase (Du et al., 2011, Kim et al., 2011).
3.3 Viable counting of soil diazotroph
Soil diazotroph plays a critical role in the nitrogen enrichment for plant growth as promoters of nitrogen fixation
and stimulators of the nitrogen cycle in soils, which is also a good criterion for assessing soil ecosystem
quality and plant health. These bacteria can contribute to 10-50 % of the total N requirement of wheat
81
(Kennedy and Islam, 2001). Figure 3 depicts the effect of SNP on soil diazoroph. The colony forming units of
soil diazotroph are not affected by the SNP. There is no sign of inhibition or stimulation effect of SNP in terms
of size and concentration on the number of diazotroph bacteria. However, other soils contaminated with ZnO
and TiO2 NP resulted in the reduction of N-fixation capacity of soils N-fixing microorganisms (Priester et al.,
2012). Several studies reported other NP show inhibition effect on the N-cycling bacteria and N-fixation rate of
aquatic organism (Yang et al., 2013). It is obvious that our SNP do not exhibit inhibition effect on the soil
microbes which are different from other types of NP.
Table 1: Effect of silica nanoparticles on the soil enzyme activities (A) urease, (B) catalase and (C) FDA
hydrolysis (Urease, mg NH4+-N g-1soil 24 h-1; Catalase, mL KMnO4 g-1 soil 0.5 h-1; FDA Hydrolysis, mg
Fluorescein g
-1
soil 0.5 h
-1
)
Size control 10 mg kg-1 25 mg kg-1 50 mg kg-1
9 nm
0.98
0.97 1.03 1.18
Urease 12 nm 1.03 1.08 1.17
40 nm 1.18 1.20 1.25
9 nm
1.14
1.14 1.17 1.23
Catalase 12 nm 1.13 1.16 1.19
40 nm 1.13 1.18 1.22
9 nm
13.01
13.74 16.25 19.65
FDA
Hydrolysis 12 nm 13.44 19.27 20.58
40 nm 14.28 18.48 19.86
9nm 12nm 40nm
0
5
10
15
20
25
control 10 mg kg
1
25 mg kg
1
50 mg kg
1
N
u
m
b
e
r
o
f
th
e
s
o
il
d
ia
z
o
tr
o
p
h
(
1
0
5
C
F
U
g
1
s
o
il
)
Figure 3: Effect of silica nanoparticles on the soil diazotroph
4. Conclusion
Microcalorimetry and biomarkers of metabolic processes related to soil fertility, i.e., urease, catalase, FDA,
and diazotroph are used to estabilsh an efficient and fast screening methodology for NP. The reason for
strengthening microcalorimetry with additional biomarkers, arises from the general knowledge and the wide
scientific consensus that the total thermal effect observed under microcalorimetric tests is the result of the
catabolic degradation of a substrate with little anabolic reactions contribute to the final state of equilibrium. The
combination of easily executable, efficient and reproducible analytical tests such as the enzymatic assays and
microcalorimetric analysis that gives a continuous and longer monitoring of the metabolic processes, are very
promising for revealing many and still unknown reasons of microbial biomass activity and structure. Such
methodology, before being systematically applied in many agro-ecosystems, must be affined and validated on
a wide range of cultivated and uncultivated soils. It has been demonstrated that the effect of SNP is strongly
related to the dose and particle size but smaller SNP only showed minor effects.
Acknowledgments
Financial supports from the International Joint Key Project of the Chinese Ministry of Science and Technology
82
(2010DFB23160), International Joint Key Project of the National Natural Science Foundation of China
(40920134003), National Natural Science Foundation of China (41273092), Overseas, Hong Kong and Macau
Young Scholars Collaborative Research Fund (41328005), and National Outstanding Youth Research
Foundation of China (40925010) are gratefully acknowledged.
Reference
Arne, K., Tim, R., Marc, S. & Ulrich, K. 2013. Modified setup of 20-L-sphere for the determination of safety
characteristics of nano powders. Chemical Engineering Transactions, 31, 805-810. DOI:
10.3303/CET1331135.
Barja, I. & Núñez, L. 1999. Microcalorimetric measurements of the influence of glucose concentration on
microbial activity in soils. Soil Biology and Biochemistry, 31, 441-447. DOI: 10.1016/S0038-
0717(98)00149-7.
Biserni, C. & Garai, M. 2016. ENERGY BALANCE AND SECOND LAW ANALYSIS APPLIED TO
BUILDINGS: AN OPPORTUNITY FOR BEJAN’S THEORY. International Journal of Heat and Technology,
34, S185-S187. DOI: 10.18280/ijht.34S125.
Caldwell, B. A. 2005. Enzyme activities as a component of soil biodiversity: a review. Pedobiologia, 49, 637-
644. DOI: 10.1016/j.pedobi.2005.06.003.
Castello, D., Kruse, A. & Fiori, L. 2014. Supercritical water gasification of glucose/phenol mixtures as model
compounds for ligno-cellulosic biomass. Chemical Engineering Transactions, 37, 193-198.
DOI:10.3303/CET1437029.
Chen, Z., Meng, H., Xing, G., Yuan, H., Zhao, F., Liu, R., Chang, X., Gao, X., Wang, T. & Jia, G. 2008. Age-
related differences in pulmonary and cardiovascular responses to SiO2 nanoparticle inhalation:
nanotoxicity has susceptible population. Environmental Science & Technology, 42, 8985-8992. DOI:
10.1021/es800975u.
Dinesh, R., Anandaraj, M., Srinivasan, V. & Hamza, S. 2012. Engineered nanoparticles in the soil and their
potential implications to microbial activity. Geoderma, 173–174, 19-27. DOI:
10.1016/j.geoderma.2011.12.018.
Dominique, F., Guillaume, F., Alexis, V., François, H. & Emeric, F. 2013. Nanomaterials risk assessment in
the process industries: evaluation and application of current control banding methods. Chemical
Engineering Transactions, 31, 949-954. DOI: 10.3303/CET1331159.
Du, W., Sun, Y., Ji, R., Zhu, J., Wub, J. & Guo, H. 2011. TiO2 and ZnO nanoparticles negatively affect wheat
growth and soil enzyme activities in agricultural soil. J. Environ. Monit, 13, 822-828. DOI:
10.1039/C0EM00611D.
Feng, G., Du, J., Zhu, W. & Qiu, Z. 2015. Study on constitutive model for root system of korshinsk peashrub in
axial tension. International Journal Bioautomation, 19, 563-574.
Fujiwara, K., Suematsu, H., Kiyomiya, E., Aoki, M., Sato, M. & Moritoki, N. 2008. Size-dependent toxicity of
silica nano-particles to Chlorella kessleri. Journal of Environmental Science and Health, Part A, 43, 1167-
1173. DOI: 10.1080/10934520802171675.
Gao, F., Liu, C., Qu, C., Zheng, L., Yang, F., Su, M. & Hong, F. 2008. Was improvement of spinach growth by
nano-TiO2 treatment related to the changes of Rubisco activase? Biometals, 21, 211-217. DOI:
10.1007/s10534-007-9110-y.
García, C., Field, A., Otero, L. & Sierra, R. 2011. Low toxicity of HfO2, SiO2, Al2O3 and CeO2 nanoparticles
to the yeast, Saccharomyces cerevisiae. Journal Of Hazardous Materials, 192, 1572-1579. DOI:
10.1016/j.jhazmat.2011.06.081.
Guo, H., Yao, J., Cai, M., Qian, Y., Guo, Y., Richnow, H. H., Blake, R. E., Doni, S. & Ceccanti, B. 2012.
Effects of petroleum contamination on soil microbial numbers, metabolic activity and urease activity.
Chemosphere, 87, 1273-1280. DOI: 10.1016/j.chemosphere.2012.01.034.
Hongyang, Z. 2015. Thermodynamic property of concrete and temperature field analysis of the base plate of
intake tower during construction period. International Journal of Heat and Technology, 33, 145-154. DOI:
10.18280/ijht.330120.
Keller, A., Mcferran, S., Lazareva, A. & Suh, S. 2013. Global life cycle releases of engineered nanomaterials.
Journal of Nanoparticle Research, 15, 1-17. DOI: 10.1007/s11051-013-1692-4.
Kennedy, I. & Islam, N. 2001. The current and potential contribution of asymbiotic nitrogen fixation to nitrogen
requirements on farms: a review. Animal Production Science, 41, 447-457. DOI: 10.1071/EA00081.
Kim, S., Kim, J. & Lee, I. 2011. Effects of Zn and ZnO nanoparticles and Zn2+ on soil enzyme activity and
bioaccumulation of Zn in Cucumis sativus. Chemistry and Ecology, 27, 49-55. DOI:
10.1080/02757540.2010.529074.
83
Kimura, T. & Takahashi, K. 1985. Calorimetric studies of soil microbes: quantitative relation between heat
evolution during microbial degradation of glucose and changes in microbial activity in soil. Journal of
general microbiology, 131, 3083-3089. DOI: 10.1099/00221287-131-11-3083.
Koga, K., Sueh1ro, Y., Matsuoka, S.-T. & Takahashi, K. 2003. Evaluation of growth activity of microbes in tea
field soil using microbial calorimetry. Journal Of Bioscience and Bioengineering, 95, 429-434. DOI:
10.1016/S1389-1723(03)80040-3.
Lee, C. W., Mahendra, S., Zodrow, K., Li, D., Tsai, Y. C., Braam, J. & Alvarez, P. J. J. 2009. Developmental
phytotoxicity of metal oxide nanoparticles to Arabidopsis thaliana. Environmental Toxicology and
Chemistry, 29, 669-675. DOI: 10.1002/etc.58.
Li, B. & Zhang, N. 2014. Stability analysis and controlling scheme optimization on roadway driven along goaf
of fully mechanized top coal caving. Environmental and Earth Sciences Research Journal, 1, 17-22. DOI:
10.18280/eesrj.010104.
Ma, H., Williams, P. L. & Diamond, S. A. 2013. Ecotoxicity of manufactured ZnO nanoparticles – A review.
Environmental Pollution, 172, 76-85. DOI: 10.1016/j.envpol.2012.08.011.
Mahmoudi, A. & Mejri, I. 2015. Analysis of conduction-radiation heat transfer with variable thermal conductivity
and variable refractive index: application of the lattice boltzmann method. International Journal of Heat and
Technology, 33, 1-8. DOI: 10.18280/ijht.330101.
Napierska, D., Thomassen, L. C. J., Rabolli, V., Lison, D., Gonzalez, L., Kirsch-Volders, M., Martens, J. A. &
Hoet, P. H. 2009. Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial
cells. Small, 5, 846-853. DOI: 10.1002/smll.200800461.
Priester, J. H., Ge, Y., Mielke, R. E., Horst, A. M., Moritz, S. C., Espinosa, K., Gelb, J., Walker, S. L., Nisbet,
R. M. & An, Y.-J. 2012. Soybean susceptibility to manufactured nanomaterials with evidence for food
quality and soil fertility interruption. Proceedings of the National Academy of Sciences, 109, E2451-E2456.
DOI: 10.1073/pnas.1205431109.
Schaller, J., Brackhage, C., Paasch, S., Brunner, E., Bäucker, E. & Dudel, E. G. 2013. Silica uptake from
nanoparticles and silica condensation state in different tissues of Phragmites australis. Science Of The
Total Environment, 442, 6-9. DOI: 10.1016/j.scitotenv.2012.10.016.
Schnürer, J. & Rosswall, T. 1982. Fluorescein diacetate hydrolysis as a measure of total microbial activity in
soil and litter. Applied and Environmental Microbiology, 43, 1256-1261. DOI: 0099-2240/82/061256-
06$02.00/0.
Thul, S. T., Sarangi, B. K. & Pandey, R. A. 2013. Nanotechnology in agroecosystem: implications on plant
productivity and its soil environment. Expert Opin. Environ. Biol., 2, 1-7. DOI: 10.4172/2325-
9655.1000101.
Tso, C., Zhung, C., Shih, Y., Tseng, Y., Wu, S. & Doong, R. 2010. Stability of metal oxide nanoparticles in
aqueous solutions. Water Science and Technology, 61, 127-133. DOI: 10.2166/wst.2010.787.
Van, K., Karel, C., Ramirez, S., Van Der Meeren, P., Smagghe, G. & Janssen, C. R. 2011. Influence of
alumina coating on characteristics and effects of SiO2 nanoparticles in algal growth inhibition assays at
various pH and organic matter contents. Environment International, 37, 1118-1125. DOI:
10.1016/j.envint.2011.02.009.
Wehling, J., Volkmann, E., Grieb, T., Rosenauer, A., Maas, M., Treccani, L. & Rezwan, K. 2013. A critical
study: Assessment of the effect of silica particles from 15 to 500 nm on bacterial viability. Environmental
Pollution, 176, 292-299. DOI: 10.1016/j.envpol.2013.02.001.
Wu, F., Wang, C., Li, B., Li, Q., Li, B., Du, W., Wang, Y. & Chen, Y. 2014. Effect of eco-physiological factors
on the tobacco potassium content in huili county, sichuang province, china Environmental and Earth
Sciences Research Journal, 1, 1-6. DOI: 10.18280/eesrj.010101.
Yang, X., Liu, J., He, H., Zhou, L., Gong, C., Wang, X., Yang, L., Yuan, J., Huang, H. & He, L. 2010. SiO2
nanoparticles induce cytotoxicity and protein expression alteration in HaCaT cells. Part Fibre Toxicol, 7, 1-
12. DOI: 10.1186/1743-8977-7-1.
Yang, Y., Wang, J., Xiu, Z. & Alvarez, P. J. 2013. Impacts of silver nanoparticles on cellular and transcriptional
activity of nitrogen-cycling bacteria. Environmental Toxicology and Chemistry, 32, 1488-1494. DOI:
10.1002/etc.2230.
Yotova, L., Hassaan, A. & Yaneva, S. 2015. Covalent immobilization of peroxidase onto hybrid membranes for
the construction of optical biosensor. International Journal Bioautomation, 19, 563-574.
Zhuang, R., Chen, H., Yao, J., Li, Z., Burnet, J. E. & Choi, M. M. 2011. Impact of beta-cypermethrin on soil
microbial community associated with its bioavailability: a combined study by isothermal microcalorimetry
and enzyme assay techniques. Journal Of Hazardous Materials, 189, 323-328. DOI:
10.1016/j.jhazmat.2011.02.034.
84