Zaham B.

Zaragoza December 9, 2017

Dev Bio Lab (MTh 3:00- 6:00)

Artemia salina bioassay

INTRODUCTION

Biological assays are methods for the estimation of nature, constitution, or potency of
a material by means of the reaction that follows its application to living matter(Panuganti,
2015). Moreover, biological assays or biological standardizations or simply bioassays are
methods used for estimation of the potency of substances by observing their pharmacological
effects on living animals (in vivo) or isolated tissues (in vitro) and comparing the effect of these
substances of unknown potency to the effect of a standard. Furthermore, it is a type of
scientific experiment typically conducted to measure the effects of a substance on a living
organism and is essential in the development of new drugs and in monitoring environmental
pollutants. It is based upon the use of biological responses as detection system for biologically
active substances (Panuganti, 2015). An example of a general bioassay tool is the tiny
crustacean brine shrimp, which is a rapid, inexpensive, in-house bioassay for screening and
fractionation monitoring various substances.
Artemia salina, commonly known as the brine shrimp, is a small crustacean, which has
been the subject of many studies. The brine shrimp lethality assay is considered one of the
most useful tools for the preliminary assessment of general toxicity (Ferraz Filha et al., 2012).
Any chemical that can be dissolved or dispersed into water can be studied in this bioassay:
plant extracts or other natural products, real or simulated hazardous waste, metal ions,
agricultural chemicals, food additives, home cleaning products, or pharmaceuticals
(Lieberman, 1999). The eggs of brine shrimps are readily available at low cost in pet shops as
a food for tropical fish, and they remain viable for years in the dry state (Meyer et al., 1982).

OBJECTIVES
The objective of this study is to compare previous studies in which they used Artemia
salina to assess general toxicity of various substances.
RESULTS AND DISCUSSIONS
Effects of titanium dioxide particle to Artemia salina (Ates et al., 2013)

Fig. 1. Phase contrast microscopy images of the TiO2 NPs inside Artemia nauplii and adults. The
images were taken 24 h after the start of exposure. The guts are completely empty in controls.
Aggregates of TiO2 are visible as a dark line inside the guts of the treatments.

Three different test concentrations (10, 50 and 100 mg/L) of TiO2 NPs were
administered to Artemia cultures. A control group was also setup without the test compound.
Studies were carried out in triplicate measurements in conical plastic containers (1-L and 2-L
inner volume). Exposures were conducted in 500 mL and 1500 mL seawater for Artemia
nauplii and adults, respectively.
Accumulation of TiO2 NPs on each group was observed under a phase contrast
microscope equipped with a digital camera. Compared with the controls, the guts of exposed
Artemia were filled particles (Fig. 1). The ingested TiO2 particles appeared as a long strip of
particles suggesting that even larger aggregates of TiO2 formed inside the guts. No significant
TiO2 was detected in the controls. Total TiO2 content increased with increasing concentration
and exposure time indicating a dose and time dependent accumulation that exhibited a plateau
beyond 24 h in suspensions of higher concentration (> 50 and 100 mg/L TiO2). This also lead
to significant mortality rate of the Artemia only after 96 hours of exposure of TiO2. This maybe
caused by lack of food intake due to accumulation of TiO2 NPs in the gut.
 Effects of -Fe2O3 nanoparticles to Artemia salina ( Wang et al., 2017)

Fig. 6. Ingestion of -Fe2O3-NPs (red arrows) by A. salina larvae. (A) The gut is empty in the control.
(B) A. salina larvae start to ingest -Fe2O3-NPs. (C) -Fe2O3-NPs is visible as a dark line inside the
gut of treatment. (DF) TEM characterization of intracorporal localization of -Fe2O3-NPs in A. salina
larvae. Scale bars in AC are of 300 m.

The toxicity test was performed by adding Artemia larvae to each well of 24-well plates
that contained 1 mL of -Fe2O3- NPs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L)
or supernatants. After exposure for 24 h, 20 surviving larvae were randomly selected from
each treatment for morphological and behavioral analysis.
The swimming speed of instar I, II and III larvae showed concentrationdependent
decreases following exposure to-Fe2O3-NPs suspensions, and was significantly decreased
in 100, 200, 400 and 600 mg/L. Interactions of NPs with A. salina also can be internal, such
as NPs intake. A. salina is non-selective filter feeder, and can ingest particles smaller than 50
m. In the study, uptake of -Fe2O3-NPs was checked under a microscope, and
representative images are shown in Fig. 2. The gut for the control was empty (Fig. 2.A), and
larvae started to ingest -Fe2O3-NPs (Fig. 2.B) following exposure to the NPs. Gradually, the
gut was almost entirely filled with -Fe2O3-NPs, verified by a dark line inside the gut (Fig. 2.C)
of the larvae. TEM was used to check the distribution of NPs in A. salina - Fe2O3-NPs were
visible within the nephridial duct (nd; Fig. 2.D), primary body cavity (pbc; Fig. 2.E) and intestine
(in; Fig. 2.F). causes significant changes in hatchability, mortality, and ethological,
morphological and biochemical parameters but only lasted for 24 hours. These toxic effects
were mediated by oxidative stress.
 Effects of oxidized multi-walled carbon nanotubes to A. salina (Zhu et al., 2017)

Fig. 3. Uptake and excretion of O-MWCNTs/FITC-MWCNTs by A. salina. (A) Gut of newly hatched larva
in the control is hazy due to the digestive tract is not fully formed. (BeD) After exposure, O-MWCNTs
are gradually accumulated in the guts. (E) The accumulated O-MWCNTs are excreted by A. salina
following transfer to FNSW. (F) OMWCNTs are not completely excreted at 72 h (exposure for 24 h in
FNSW). (G) A. salina in the control show faint self-fluorescent in the head. (HeJ) After exposure to
FITCMWCNTs, fluorescence intensity increases markedly over time until 48 h. After transfer to FNSW,
the accumulated FITC-MWCNTs are excreted by A. salina (K), and a small quantity of FITC-MWCNTs
is residual in A. salina (L). Scale bars: 300 mm.

The toxicity test was performed by adding Artemia larvae to each well of 24-well plates
that contained 1 mL of O-MWCNTs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) or
supernatants. After exposure for 24 h, 20 surviving larvae were randomly selected from each
treatment for morphological and behavioral analysis.
The uptake of MWCNTs by A. salina was checked, and representative images are shown
in Fig. 3. Gut of newly hatched larva in the control (Fig. 3.A) was hazy due to the digestive
tract is not fully formed. After exposure for 12 h, O-MWCNTs gradually accumulated in the gut
(Fig. 3.B). The guts were almost entirely filled with O-MWCNTs at 24 (Fig. 3.C) and 48 h (Fig.
3.D), manifested by a dark line inside the guts. After transferred in FNSW, the accumulated
O-MWCNTs were excreted by A. salina (Fig. 3.E). However, the accumulated O-MWCNTs
were not completely excreted at 72 h (exposure for 24 h in FNSW; Fig. 3.F). For exposure to
FITC-MWCNTs, A. salina in the control showed faint self-fluorescent in the head (Fig. 3.G).
After exposure, fluorescence intensity increased markedly over time until 48 h (Fig. 3.HeJ).
Interestingly, as shown in Fig. 3.H, the gut was half-baked. That was because that the mouth
and anus of larvae are not yet completely opened, and the digestive tract is not fully formed
at the early stage of instar I. After transferred in FNSW, similar excretion (Fig. 3.K) and residual
(Fig. 3.L) were observed as OMWCNTs. The results indicated that MWCNTs can move in and
out of A. salina in accordance to the external concentration. Besides, the accumulated O-
MWCNTs were not completely excreted at 72 h most likely due to the formation of larger
aggregates inside the guts (Ates et al., 2013). The study also revealed the mortality rates of
the larvae which were 47.3%, 74.4% and 69.9% for instar I, II and III following exposure to
600 mg/L, indicating that O-MWCNTs possess a lower toxicity file.

CONCLUSION
The results of the studies showed similar trends such as direct relationship between
the mortality rate and the toxin concentration. However, their mortality rate differs. TiO2 NPs
were nontoxic to Artemia since the extended exposure to 96 h was the only time that mortality
was induced. The death maybe due to the lack of food intake due to accumulation of TiO2
NPs in the gut. -Fe2O3-NPs and O-MWCNT both have short-term effects on A. salina. -
Fe2O3-NPs only lasted for 24h while O-MWCNT lasted for 48h which make both of them safe
for commercial purposes.

LITERATURE CITED
Ferraz Filha, Z.S., Lombardi, J.A., Guzzo, L.S., Sade-Guimares, D.A., 2012. Brine shrimp
(Artemia salina Leach) bioassay of extracts from Lychnophoriopsis candelabrum and
different Lychnophora species. Rev. Bras. Plantas Med. 14, 358361.
https://doi.org/10.1590/S1516-05722012000200016
Lieberman, M., 1999. A Brine Shrimp Bioassay for Measuring Toxicity and Remediation of
Chemicals. https://doi.org/10.1021/ed076p1689
Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D., L. Nicols and
McLaughlin, J., 1982. Brine Shrimp: A Convenient General Bioassay for Active Plant
Constituents. https://doi.org/10.1055/s-2007-971236
Panuganti, S.J., 2015. Principles Involved in Bioassay by different Methods: A Mini-Review.
Res. Rev. J. Biol. 3, 21.