Maria Faye Stephanie J. Cantago Zoology 131.

1 A
BS Biology 4 December 7, 2017

Effects of different pollutants to Artemia sp.

INTRODUCTION

Nanoscience is a novel science that is being developed to probe and manipulate matter
on the scale of single atoms and molecules (Rajabi et al., 2015). Nanotechnology is the use
of nanoscience to design NMs (nanomaterials) and NPs (nanoparticles), with structural
components between 1 and 100 nanometers; it is thought to be one of the key technologies
of the 21st century (Lloyd et al., 2005; Yadac et al., 2011). The physicochemical characteristics
of NPs in biological systems can vary. Some potential hazards have been identified in the life
cycle of NMs and NPs (Drobne, 2007; Ikramullah et al., 2013). Growing research and
development in nanotechnology have resulted in the identification of many unique properties
of nanomaterials such as enhanced magnetic, catalytic, optical, electrical, and mechanical
properties when compared to conventional formulations of the same materials (Arora et al.,
2012). While nanoparticles have a wide variety of functions, there has been increasing issues
and debate amongst the regulatory and scientific community regarding the fate of
nanoparticles in biological systems and associated side effects these agents might have on
living organisms (Sharma et al., 2012; Hsieh et al., 2012).

According to Rajabi and colleagues (2015), nanotoxicology research is applied to

various fields including biology and pathology, but typically to pharmacology and to the use of
NMs and nanodevices for diagnostic and therapeutic purposes. They concluded that the key
goal for toxicologists is to identify in vitro and in vivo assays accurately reflecting the ability of
NPs to induce toxic effects in the humans and in the environment.

Marine ecosystems are extremely important, as these ultimately receive the run-off and
wastewaters from domestic and industrial sources (Baun et al. 2008). Artemia (brine shrimp)
is zooplankton that is used to feed larval fishes (Sorgeloos, 1980). Artemia present one
common characteristic, that is, their strong adaptability to hypersaline environments, such as
permanent salt lakes, coastal lagoons, and man-made salt pans. They play an important role
in the energy flow of the food chain in marine environment (Vanhaeke and Persoone, 1981;
Sanchez-Fortun et al., 1997). Artemia sp. is used in toxicology poses a reasonable number of
answerable questions, namely, practical considerations of laboratory culture and attainment
of cyst, ecological relevance, systematic use, and practical conditions of maintenance and
sustainability of laboratory conditions of animal model (Blaste, 1998).
OBJECTIVE

The goal of this study is to compare the different morphological effects of exposure to
high CO2 leading to seawater acidification, silver nanoparticles and other metal oxide
nanoparticles to Artemia sp. presented in previous studies.

RESULTS AND DISCUSSION

Nano-sized polystyrene effects on Artemia franciscana larvae by Bergami, et al.,

2015

Fig. 1. Effects of 40 nm PS-COOH and 50 nm PS-NH2 on brine shrimp A. franciscana nauplii at 48h:
(a) control in NSW; (b, c) accumulation of green fluorescent PS-COOH (25 mg/ml) and (e, f) blue
fluorescent PS-NH2 (25 mg/ml) inside the digestive trait; (d) detail of fecal pellet containing PS-COOH
aggregates. Images are representative of three independent experiments. Scale bar: 100 m.

In order to assess acute toxicity of anionic and cationic PSNPs (40 nmPS COOH and
50 nmPS-NH2), brine shrimp larvae were exposed to NPs suspension in NSW for 48 hand
observed formortality and NPs accumulation according to the standard APAT IRSA CNR 8060
method (CNR, 2003) (Fig. 1a andb).

Light microscopy of larvae at 12 hours of exposure clearly showed uptake of PS-COOH

aggregates at all tested concentration (5- 100um/ml) which are absent in the control. A
massive sequestration inside the gut lumen was evident for both PSNPs (5100 mg/ml) at48h
(nauplius stage), as shown in Fig. 1(a and b). A further confirmation of the nature of aggregates
was given by fluorescent microscopy which revealed green fluorescence in the gut of PS-
COOH exposed larvae as well as blue fluorescence of PS-NH2 NPs exposed ones (Fig. 1c
and d). By continuous recording after 48 h of exposure, some of them were lately excreted as
fecal pellets but not a full release of aggregates present in the gut lumen was observed (Fig.
S2).

Fig. 2. Effects of 50 nm PS-NH2 on brine shrimp Artemia franciscana nauplii at 48h. Upper images
showing control in NSW: detail of clear (a)sensorial antennules, (b) antennae and (c) abdomen. Lower
images showing PS-NH2 (50 g/ml) exposed nauplii (48h): detail of nanoplastics attached to
(d)sensorial antennules, (e) sensory hairs of the antennae and (f) abdomen region. Aggregates of PS-
NH2 are indicated by black arrows. Images are representative of three independent experiments.
Scalebar: 100 m.

Despite PS-NH2 (5100 mg/ml) also accumulated alike in brine shrimp swimming
larvae at 48h, many specimen showed empty digestive traits (Fig. 1d and e). Clearly, these
cationic nanoplastics were stuck to the external surface of the swimming larvae (Fig. 2c) and
in particular to the sensorial appendages, as shown in Fig. 2 d- f. The presence of PS-NH2 on
the appendages was found to noticeably hampering brine shrimp larvae natation at 48 h and
thus probably limiting their ability of feeding. Brine shrimp A.franciscana creates feeding
currents while swimming in order to ingest waterborne particles (Ruppert etal.,2004).
Therefore, longer exposure scenarios will aim to evaluate the outcome of these sublethal
observed effects and predict possible consequences at organismal and population level by
disrupting behavior, feeding and growth.
 Effects of Silver Nanoparticles in Brine Shrimp Artemia sp. by Arulvasa et al.,
2013

Fig. 3. Morphological variations of Artemia nauplii treated with silver nanoparticle observed using
inverted phase contrast microscope (20x magnification): (a) control, (b) 2 nM concentration of AgNPs,
(c) LC50 concentration (10 nM) of AgNPs, and (d) 12 nM concentration of AgNPs.

In this study, the Artemia nauplii were treated with various nanomolar concentrations
of AgNPs such as 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, and 12nMand after 48 hours of treatment
they were observed under the phase contrast microscope and results were photographed. The
results showed that in control the animal did not show any trace of aggregation; the mouth
parts and the gut region appeared clear (Figure 3.a). In minimum concentration (2 nM) the
aggregation of silver nanoparticles was found around the mouth parts and some regions of the
gut (Figure 3.b), whereas in higher concentration (10 nM), the entire gut region was
accumulated with silver nanoparticles (Figure 3.c). And in maximum concentration (12 nM) the
gut was completely filled with silver nanoparticle; due to the effect of high toxicity of silver
nanoparticles the tissue of the animal started to degrade (Figure 3.d).

The observations were in accordance with in vitro toxicity study on silver nanoparticle,
where higher concentrations had more apoptotic cells and necrotic cells than lower
concentrations of silver nanoparticle. In order to study the occurrence of apoptosis, Artemia
nauplii treated with various concentrations of silver nanoparticles were stained using acridine
orange and observed under the fluorescence microscope. At higher concentration such as 12
nM, Artemia nauplii exhibited bright green spots all over the body (Figure 4.d). In lower
concentrations, only few green spots were visible in the animal body (Figures 4.b and 4.c).
 Figure 4. Acridine orange stained Artemia nauplii treated with silver nanoparticles observed using
fluorescence microscope (20x magnification): (a) control, (b) 2 nM concentration of AgNPs, (c) LC50
concentration (10 nM) of AgNPs, and (d) 12 nM concentration of AgNPs.

In the control there was no appearance of green spots in animal body, as there were no
apoptotic cells damaged due to silver nanoparticle (Figure 4.a). This is due to the condition
that as the concentration increases the amount of cell damage increases and the emission of
fluorescence by acridine orange stain is proportional to the amount of necrotic cells due to the
effect of toxicity of silver nanoparticles. Hence, in the animal body the region that emits
fluorescence reveals the condition of apoptosis due to toxicity of silver nanoparticles.

Selected metal oxide nanoparticles effects on Artemia salina larvae by

Gambardella et al., 2013

Fig. 5. Representative microscopy images of the A. salina larvae revealing the MO-NPs inside the gut
after 48 h of the control and with exposure to 0.01 mg/mL MO-NPs (as indicated). Note that the gut is
empty in the control group (0) compared to the treated samples. Bar, 400 m.
 The toxicity test was performed by adding A. salina larvae to each well of a 24-well
polystyrene plate that contained 1ml of filtered natural seawater with suspensions of different
concentrations of the MO-NPs (0.01, 0.1, 1.o mg/ml).
The microscopy observations showed that aggregation of the MO-NPs occurred after
48 h of exposure in all of the suspensions in the filtered natural seawater. In addition, larvae
accumulated the aggregates of MO-NPs and the ingested MO-NPs appeared to be
compressed inside the larval gut (Fig. 5). Ce and Fe levels accumulated within 48 h in larvae
exposed to CeO2 and Fe3O4 NPs were statistically different from the controls. It is also good
to note that during the study, many larvae died in the SnO2 and CeO2 but not in Fe3O4 although
they are not statistically significant.

CONCLUSION

The studies suggest that PSNPs might pose a risk to marine zooplankton as a result
of exposure to nanoplastics at the concentrations tested here. Nano-sized PS might be able
to impair food uptake (feeding), behavior (motility) and physiology (multiple molting) of brine
shrimp larvae A. franciscana with consequences not only at organism and population level but
on the overall ecosystem based on the keyrole of zooplankton on marine food webs. The
toxicity study of silver nanoparticles in Zebra fish revealed that the accumulation of silver in
the body of the specimen degrades its tissues. Lastly, the exposure of A. salina larvae to the
selected MO-NPs did not induce significant mortality, although the NPs accumulated in the
gut. However, behavioral and biochemical changes occurred after the exposure.

There are many pollutants that we dont know about that play an important role to the
life of the marine organisms. These may cause subtle changes to them or even death that is
why we must use the technology we have now to understand and lessen this problem.

LITERATURE CITED

Arora S, Rajwade JM, Paknikar KM. (2012). Nanotoxicology and in vitro studies: the
need of the hour. Toxicol Appl Pharmacol. 258:15165.
Arulvasu, C., Jennifer, S., Prabhu, D. and Chandhirasekar, D. (2014). Toxicity Effect
of Silver Nanoparticles in Brine ShrimpArtemia.
Baun, A., Hartmann, N. B., Grieger, K., & Kusk, K. O. (2008). Ecotoxicity of
engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for
future toxicity testing. Ecotoxicology, 17, 387395.
C. Blaise (1998). Microbiotesting: an expanding field in aquatic toxicology,
Ecotoxicology and Environmental Safety, vol. 40, no. 1-2, pp. 115119
CNR, A.I., (2003). Metodo 8060 di valutazione della tossicit acuta con Artemia sp.,
Manuali e Linee Guida-Metodi analitici per le acque, Vol. 3. APAT IRSA CNR, pp. 10431049.
 Drobne D. Nanotoxicology for safe and sustainable nanotechnology. Arh Hig Rada
Toksikol. 2007;58:4718.
Gambardella, C., Mesari, T., Milivojevi, T., Sepi, K., Gallus, L., Carbone, S.,
Ferrando, S. and Faimali, M. (2014). Effects of selected metal oxide nanoparticles on Artemia
salina larvae: evaluation of mortality and behavioural and biochemical
responses. Environmental Monitoring and Assessment, 186(7), pp.4249-4259.
Hsieh SF, Bello D, Schmidt DF, Pal AK, Rogers EJ. (2012). Biological oxidative
damage by carbon nanotubes: fingerprint or footprint? Nanotoxicology. 6:6176.
Ikramullah A, Salve D, Pai G, Rathore M, Joshi D. (2013). In vitro cytotoxicity testing
of silver nano-particals in lymphocyte and sperm cells. Ind J Fund Appl Life Sci. 3:447.
Lloyd SM, Lave LB, Matthews HS. (2005). Life cycle benefits of using nanotechnology
to stabilize platinum-group metal particles in automotive catalysts. Environ Sci Technol.
39:138492.
Sorgeloos P.,(1980) Availability of reference Artemia cysts, Marine Ecology
Progress Series, vol. 3, pp. 363364.
Vanhaecke P. and Persoone G.,(1981) Report on an intercalibration exercise on a
short-term standard toxicity test with Artemia nauplii (ARC-test), INSERM 106: 359-376.
Rajabi, S., Ramazani, A., Hamidi, M. and Naji, T. (2015). Artemia salina as a model
organism in toxicity assessment of nanoparticles. DARU Journal of Pharmaceutical Sciences,
23(1).
Ruppert, E.E., Fox, R.S., Barnes, R.D. (2004). Invertebrate Zoology: A Functional
Evolutionary Approach. Thomson-Brooks/Cole, Australia.
Sanchez-Fortun S., Sanz F., Santa-Mara A. et al., (1997) Acute sensitivity of three
age classes of Artemia salina larvae to seven chlorinated solvents, Bulletin of Environmental
Contamination and Toxicology, vol. 59, no. 3, pp. 445451.
Sharma A, Madhunapantula SV, Robertson GP. (2012). Toxicological considerations
when creating nanoparticle-based drugs and drug delivery systems. Expert Opin Drug Metab
Toxicol. 8:4769.
Yadav A, Ghune M, Jain DK. (2011). Nano-medicine based drug delivery system. J
Advanced Pharm Educ Res. 1:20113.