Environmental Engineering Reference
In-Depth Information
health risks. One problem with
in vitro
assays is that the results that are obtained in this study cannot guarantee biocompatibility
in vivo
, and therefore data from
in vitro
studies may be misleading and will require verification through animal evaluations [30].
In order to understand the impact of NMs on the environment and living systems, several specific methods have been devel-
oped. They can be grouped into four categories: (1) chemical and physical characterization; (2) microbiological assays;
(3)
in vitro
assays; and (4)
in vivo
assays.
30.2.1
chemical and Physical characterization
very sophisticated and specialized analytical instrumentation has been developed to obtain some fundamental physical
information about NMs that we desire to study and is already available in major facilities around the globe [31]. Several well-
established techniques are also available such as scanning or transmission electron microscopy (SEM, TEM) to obtain precise
information about the size, morphology, and chemical composition (when energy-dispersive x-ray (Edx) detectors are avail-
able) of NMs. With dynamic light scattering (dLS) instrumentation it is possible to determine the hydrodynamic radii of the
nanoparticles when dispersed in a liquid, and it is possible to study the influence of pH on the surficial charge (Zeta potential),
the nature of the solvent, its temperature, the effect of capping agents and detergents in the stabilization of nanoparticles, the
stability against time, pH, and temperature, and to understand the kinetics of aggregation in a solution. The specific area of
powdered materials can be obtained by using Brunauer-Emmett-Teller (BET) analyzers; thermal stability and transformation
may be determined by thermal gravimetric analyzers (TgAn); and chemical composition and presence of contaminants can be
determined by atomic absorption spectrophotometry (AASp) or inductively coupled plasma mass spectrometry (ICP-MS).
Other spectroscopies such as visible-ultraviolet, infrared, or Raman may also be useful in defining the existence of organic or
inorganic coatings, chemical modifications in the surface, and chemical identity, among other characteristics.
Of course, once the chemical and physical analyses show some relevant data,
in vitro
and
in vivo
studies may give us complemen-
tary information. Biotests using bacterial, cells or tissue cultures, animal models (mice, rats, rabbits, dogs, fishes, etc.), and ecotoxicity
models (
Micropterus salmoides
,
Caenorhabditis elegans
,
Daphnia magna
,
Ceriodaphnia dubia
, common fruit fly,
Drosophila mela-
nogaster
, and some invertebrates and small vertebrates) are currently among the most used in research laboratories around the world.
30.2.2
microbiological assays
different approaches can be used to assess bacterial toxicity using well-characterized materials and standard bacterial assay
systems. It is possible to examine the effects of nanoparticle concentration, particle size, exposure time, growth medium, and
pH on the growth and viability of bacterial cells like
Escherichia coli
,
Bacillus subtilis
, and
Shewanella oneidensis
. Among
other methods to assess NM bacterial toxicity, we can mention the following:
Disk diffusion tests.
Bacterial sensitivity to different-sized NMs is tested by disk diffusion test as described by Ruparelia
[32]. Small filter paper disks of uniform size (i.e., 6 mm diameter) are placed separately in each of the different nanopar-
ticle suspensions for 5 min; then the disks are carefully removed using sterile forceps. After the bacterial suspension
(100 µl of 10
4
-10
5
CFU ml
−1
) is uniformly plated on Luria-Bertani (LB) agar plates or other rich media, a disk containing
nanoparticles is placed at the center of each plate and the plate is incubated at 37°C for 18 h. The average diameter of the
inhibition zone (dIZ) surrounding the disks is measured to determine inhibition. This simple method gives us an idea as
to whether some NMs have any activity; however, sometimes one might need to use minimum media in place of rich
media (i.e., M9 media) to see if any effect is present.
Determination of minimum inhibitory concentrations (MIC).
The MIC is defined as the lowest concentration of a compound
that inhibits the growth of an organism [33]. The MIC test can be determined for
E. coli
in LB medium at pH 7.2 and/or
in M9 minimal medium at pH 6.4 [34], 7.2, and 7.8. For
B. subtilis
, MIC can be tested in LB and minimal media at pH
7.2 [35], and for
S. oneidensis
in LB and horse blood agar (HBA) minimal media at pH 7.2 [36]. Reactions are carried
out in test tubes containing 5 ml of the logarithmic-phase (Od
600
~0.098) bacterial cultures and different-sized nanopar-
ticles at various concentrations (i.e., 50, 100, and 150 mg/l). Tubes with sterile media containing no nanoparticles and
nanoparticles only served as controls. Samples are incubated on a shaker (200 rpm) at 37°C (
E. coli
and
B. subtilis
) or
30°C (
S. oneidensis
), with growth monitored by obtaining measurements of the optical density at 600 nm (Od
600
) every
30 min for 8 h. In the end, the last tube with no growth corresponds to the MIC of that compound.
Colony-forming unit (CFU) measurements.
Studies of
E. coli
and
B. subtilis
viability are performed in liquid cultures at a
nanoparticle concentration of 100 mg/l (or the proper concentration according to the NM). Aliquots are taken at 0, 1, 5,
and 24 h and serially diluted in the appropriate minimal medium, and the dilutions are seeded on LB agar plates. After
overnight incubation at 37°C, the numbers of CFU are counted manually.
