Environmental Engineering Reference
In-Depth Information
More recently, efforts have been focused on the development and validation of new alternative test systems (sensitive,
specific, rapid) for toxicological research that will reduce, replace, or refine animal use. Model systems under development
include nonmammalian species, transgenic species, genetically engineered
in vitro
cell systems, microchip array technology,
and computer-based predictive toxicology models [70]. Fish and amphibian embryo models are gaining increasing popularity
in the area of toxicology, both in research and potential regulatory application. The fish and amphibian embryo models provide
an ethically acceptable small-scale analysis system with the complexity of a complete organism.
The OECd is an intergovernmental organization in which representatives from 34 industrialized countries of North and
South America, Europe, Asia, and the Pacific region, as well as the European Commission, meet to coordinate and harmonize
policies, discuss issues of mutual concern, and work together to respond to international problems such as NM ecotoxicity. The
OECd's WPMN was established in 2006 to promote international cooperation in human health and environmental safety
aspects of manufactured NMs. The OECd program has focused on generating appropriate methods and strategies to ensure
potential safety issues through the following approaches:
• Establishing an OECd database on manufactured NMs to inform and analyze research activities and strategies on environ-
mental, human health, and safety issues
• Testing specific NMs for human health and safety evaluation, while ensuring appropriate testing methods (
in vivo
and
in vitro
).
Some standards of OECd ecotoxicity
in vivo
tests include water flea acute (
Daphnia magna
), where the dosing method is
natural water by 48 h and the test end point is the half maximal effective concentration (EC
50
) [75]
. D. magna
is an organism
widely used as an indicator in aquatic environmental risk assessment because
Daphnia
filter large volumes of water and water-
suspended particles. It also plays an important role in freshwater food chains [76, 77]. These features make
D. magna
a partic-
ularly useful test animal for assessing the accumulation of NMs, because their uptake in this organism could result in transfer
throughout the food chain.
D. magna
may be grown in artificial freshwater (Ca + Mg hardness 2.5 mM, pH 6.5-7.1) with a
photoperiod of 16:8 light:dark at 20 ± 2°C. The population is fed three times a week with a green algae culture of
Scenedusmus
sp.
(dominant species),
Monoraphidium contortum
, and
Selenastrum capricornutum
. Organisms used in tests must be 5-7 days
old at the beginning of the experiments.
For fish acute (
Zebrafish
), the test medium is natural water by 96 h and the test end point is 50% of the maximum lethal
concentration (LC
50
). In the case of prolonged fish toxicity (
Zebrafish
), the study is monitored for 14 days following EC/LC
50
until the test end point. There are
in vivo
assays using birds, such as bird dietary toxicity, where the doses are applied in the basal
diet for 8 days and the test end point is LC
50
. Other
in vivo
assays include other fish species, honey bee, earthworms, and plants.
These are some examples of
in vivo
assays approved by the OECd [68].
Aditionally, studies to evaluate how NMs may affect the different development stages of plants are also an easy alternative
to assess their potential environmental effects. For example, the germination and growing of seeds of
Lactuca sativa
were tested
by yang and Watts [78]
. L. sativa
is one species that is used and recommended by the Environmental Protection Agency (EPA)
regularly for measuring pesticide and toxic substances in the environment. The germination average rate is usually 85%, and
the seeds have to be stored in dry and dark places at room temperature. Initially, the seeds are wetted in a bleach solution (10%
from commercial product) for 10 min to eliminate biological contaminants. The seeds are then rinsed three times and set up for
germination immediately. In a plastic tray (transparent), squares of 2.25 cm
2
are drawn to accommodate the seeds in each inter-
section. The number of seeds will depend on how many substances should be tested and must be by triplicate. The system will
include the NM to be tested in solution at different concentrations (i.e., 0.1, 0.5, 0.75 mg/ml) in sterile deionized water. All
positive and negative controls must be considered.
The seeds are incubated by 168 h using a photoperiod of 12 h (light and dark) and temperature at 25°C ± 0.5°C. Observation
and counting registration of germination is recorded each 24 h. The root size (mm) is also registered using a vernier scale and
the exposed seeds are compared with the nonexposed ones. The elongation root during the exposure is calculated using the
formula
ER L
=
−
L
treated
non-treated
RRG ER
=
−
ER
sample
control
where L
treated
and L
non-treated
are the length of roots with or without treatment, respectively. The relative root growth (RRg) is
calculated according to Schildknecht and de Campos-vidal (2002, cited in Ref. [70]).
