Fishing to Design Inherently Safer Nanoparticles - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 278

between control and nanoparticle-exposed groups with a 0.10 significance level.
Control and nanoparticle-exposed groups are then statistically compared using one-
way analysis of variance (ANOVA) (SigmaStat, SPSS Inc., Chicago, IL). An
alternative statistical test, Kruskal-Wallis analysis of variance on ranks, is used
when several different groups are analyzed that have received different treatments or
if the raw data violate normality or equal variance assumptions. In addition, the lethal
concentration causing 50% embryonic mortality (LC 50 ) is calculated using probit and
sigmoidal regression analyses. The lowest concentration to elicit significant effects
(LOAEL) and the concentration at which no adverse effects are observed (NOAEL)
are determined using one-way ANOVA (SPSS Inc., Chicago, IL).
20.3.3 Uptake Determination
All embryos exposed to nanoparticles undergo uptake analysis whether or not the
nanoparticles induce significant effects (morphological or behavioral). After
uptake efficiency is determined, it can be correlated with the observed effects to
determine how dose metric relates to observed responses. These nanoparticles
continue onto tier 2. To achieve this, the amount of nanoparticles in individual
whole animals must be determined. In many cases, nanoparticles consist of metal
cores. In these situations, two types of quantification methods can be employed:
instrumental neutron activation analysis (INAA) or inductively coupled plasma
optical emission (ICP-OES)/mass spectrometry (ICP-MS). Both analytical
instruments are able to detect any element on the periodic table at trace levels.
Regardless of the instrumentation, three biological replicates are used, but for
either of the ICP instruments, additional three technical replicates are required due
to instrument variabilities.
The same volume and concentration required to produce significant abnormal
effects via waterborne exposure is used to determine actual uptake concentration via
INAA. Each instrument has its advantages and disadvantages. INAA is able to detect
the quantity of nanoparticles in individual embryonic zebrafish. Although this method
is accurate, technically easy to perform, and does not require pooling of animals, it is
also time consuming, expensive, and requires a radiation source, which is not
common at academic or commercial settings. In contrast, ICP-OES and ICP-MS
may not be as sensitive for certain metals and require embryo pooling in order to get a
significant signal (varies depending on the core element and noise to background
ratio). ICP-MS is three orders of magnitudemore sensitive than ICP-OES. However, it
is labor intensive and requires a reference standard for the nanoparticle core. In
addition, the matrix of choice for ICP instruments is HNO 3 to minimize background
signal interference. If ICP instruments are utilized, the analytical method must be
validated by completing a spike and recover assay (i.e., use unexposed embryos and
“spike” with the standards to ensure that the results can be replicated). In either INAA
or any ICP method, the remaining challenge is determining if the quantified
nanoparticles are integrated within the zebrafish or if it they are simply tightly
associated with the outside of the animal.