Evaluation of toxicity and lethal concentration (LC50) of silver and selenium nanoparticle in different life stages of the fish Tenualosa ilish (Hamilton 1822)
Mahnaz Sadat Sadeghi1* and Sadegh Peery2
1Department of Marine Biology, Islamic Azad University, Iran
2Department of Marine Biology, Khoramshahr University, Iran
Submission: February 14, 2018; Published: June 14, 2018
*Correspondence author: Mahnaz Sadat Sadeghi, Department of Marine Biology, Marine Science and Technology Faculty, Tehran North Branch, Tehran, Iran.
How to cite this article: Mahnaz S S, Sadegh P. Evaluation of toxicity and lethal concentration (LC50) of silver and selenium nanoparticle in different life stages of the fish Tenualosa ilish (Hamilton 1822). Oceanogr Fish Open Access J. 2018; 7(5): 555722. DOI:10.19080/OFOAJ.2018.07.555722
The anadromous Tenualosa ilish is the most economically important fish in the world and consequently in Iran. The national fish of Iranian contributes about 12% of the total fish production and about 1% of GDP. About 4,500,000 people are directly involved with the catching for livelihood; around four to five million people are indirectly involved with the trade. The present study aimed to estimate 96hLC50s of silver and selenium on LC50 of nanoparticle of silver and selenium in different life stages of the fish T. ilish in the environment experimental. The LC50 for silver in four stages of fish T. ilish were larvae (0.23ppm), fry (0.45ppm), juvenile (0.90ppm) and fingerling (1.45ppm), respectively. There was significance difference between LC50 levels in four stages of fish species (P<0.05>) and the LC50 for selenium in four stages of fish T. ilish were larvae (0.89ppm), fry (0.95ppm), juvenile (1.12ppm) and fingerling (1.65ppm), respectively. There was significance difference between LC50 levels in four stages of fish species (P<0.05>). Variability in acute toxicity even in a single species and single toxicant depending on the size, age and condition of the test species along with experimental factors. Finally, in order of the toxicity of heavy metal in different stage of fish were larvae > fry > juvenile > fingerling, respectively. In conclusion, the toxicity tended to elevate with decreasing fish size.
Keywords: Nanotechnology; Heavy metal; Nanoparticle; LC50sub>; Tenualosa ilish
The aquatic environment makes up the major part of our environment and resources. Therefore, its safety is directly related to human health. The excessive contamination of aquatic ecosystems has evoked major environmental and health concerns worldwide because the aquatic environment is the ultimate recipient of pollutants produced by natural and anthropogenic sources .
Heavy metals are defined as metallic elements that have a relatively high density compared to water. With the assumption that heaviness and toxicity are inter-related, heavy metals also include metalloids, such as silver and selenium that are able to induce toxicity at low level of exposure . In recent years, there has been an increasing ecological and global public health concern associated with environmental contamination by these metals. Also, human exposure has risen dramatically as a result of an exponential increase of their use in several industrial, agricultural, domestic and technological applications. Reported
sources of heavy metals in the environment include geogenic, industrial, agricultural, pharmaceutical, domestic effluents, and atmospheric sources. Environmental pollution is very prominent in point source areas such as mining, foundries and smelters, and other metal-based industrial operations [1,3].
Heavy metals become toxic when they are not metabolized by the body and accumulate in the soft tissues . Heavy metals may enter the human body via food, water, air, or absorption through the skin in agriculture, manufacturing, pharmaceutical, industrial, or residential settings. Industrial exposure is common in adults. Ingestion is the most common route in children . Children may develop toxic levels from normal hand-to-mouth activity (ie, coming in contact with contaminated soil or eating objects that are not food such as dirt or paint chips). Less common routes of exposure include a radiological procedure, inappropriate dosing or monitoring during intravenous (parenteral) nutrition, a broken thermometer, or a suicide or homicide attempt .
Silver nanoparticles (AgNPs) are widely used worldwide as
spectrally selective coatings for solar energy absorption, chemical
catalysts and especially for antimicrobial sterilisation . After
their discharge, AgNPs will most likely enter the ecosystems and
may produce a physiological response in many animals, possibly
altering their fitness, and ultimately changing their densities or
community populations. Open access literature regarding the
toxicity of nanoparticles is still emerging, and gaps still exist in
our knowledge of this area. The mechanism of toxicity of silver
NPs in fish has not been determined. An enhanced toxicity of
nanoparticulate silver, compared to silver ions (Ag+), may result
from their shape and/or size, the release of silver ions (silver ions
are well known to be toxic to aquatic organisms) or a combination
of both [7-9].
Yeo and Kang examined the rate of photosynthesis in
zebrafish embryogenesis exposed to silver NPs or silver ions in
both the presence and the absence of cysteine (which binds free
silver ions) and showed that silver NPs were more toxic than
silver ions, based on the concentration of ions present, requiring
a higher concentration of cysteine to eliminate the toxicity.
Wu et al.  studied the effects of silver nanoparticles on the
development and histopathology of Japanese medaka (Oryzias
latipes) and after observing many changes, suggested further
research on toxicity of silver nanoparticle in fish species. Yeo &
Kang confirmed fatal effects of nanometer sized silver materials
on zebrafish embryogenesis. Also, Solatni et al.  reported
the toxic effect of nanosilver particles on hatchability of rainbow
trout (Oncorhynchus mykiss) eggs and the survival of the produced
Ishikawa et al.  studied the effects of selenium
nanoparticles on the histopathology of Goldfish (Carassius
auratus). They suggested further research on toxicity of selenium
nanoparticle in fish species. Karasu et al.  studied the effects
of selenium nanoparticles on the histopathology on zebrafish.
Also, Kamunde et al.  reported the toxic effect of nanoselenium
particles on hatchability of zebrafish.
The LD50 is the dose of a given chemical product (pesticide,…)
which causes the mortality of 50% of a group of test animals in a
specified period. It is commonly used in bioassays assessments
to measure the acute toxicity of a chemical active ingredient.
The lower the LD50 value the more toxic the chemical . These
calculations are based on two important assumptions. The first
assumption is that the exposure time associated with the specified
LC50 is sufficient to allow almost complete chemical equilibration
between the fish and the water. The second assumption is that
the specified LC50, the minimum LC50 that kills the fish during
the associated exposure interval . Fortunately, most reliable
LC50 satisfy these two assumptions . The 96h LC50 tests are
conducted to measure the susceptibility and survival potential of
organisms to particular toxic substances such as oils pollution.
Higher LC50 values are less toxic because greater concentrations
are required to produce 50% mortality in organisms .
Tenualosa ilisha is a species of fish in the herring family
(Clupeidae), and a popular food fish in South Asia. It is also the
national fish of Iran. The national fish of Iranian contributes
about 12% of the total fish production and about 1% of GDP .
About 4,500,000 people are directly involved with the catching
for livelihood; around four to five million people are indirectly
involved with the trade. The average annual landing of this species
in 2006 was 4989.83t and constituted about 15% of Khouzestan
Province total commercial fish landing. During 2008, 4645t of
T. ilisha was landed in the Khouzestan Province (Northwest of
Persian Gulf). The present study aims to investigate the LC50 of
selenium and silver in four life stages of, Tenualosa ilish. This fish
species was chosen for its economic important, easily transported
and maintained in the laboratory.
The fish used in the present experiments (Anadromous fish,
Tenualosa ilisha) were reared in the outdoor pond of NIOF fish
farm. Four Different life stages were picked after 5, 15, 30, 45
and 90 days (larvae, fry, juvenile and fingerlings, respectively.)
from the hatching. Fish stages and they were transported to the
laboratory in containers containing water from the same pond.
They have been acclimated for a day in a stock tank. Table 1 show
length and weight in different stage fish species T. ilish.
Stock solutions of the heavy metals selenium and silver were
prepared by using pure grad of CuSO4.5H2O and HgCl2. During the
experiments, test solutions of selenium and silver were freshly
prepared from the stock solutions. Test solutions of Se ranged
from 0.5 to 5ppm, while Hg varied from 0.05 to 2ppm for all 4 life
All samples were acclimated for 1 week in 15 aerated fiberglass
tanks at 25 °C under a constant 12:12h light:dark photoperiod.
Acclimatized fish were fed daily with a formulated feed. Dead fish
were immediately removed with special plastic forceps to avoid
possible deterioration of the water quality.
Silver tested concentrations were 0, 0.1, 0.5, 1.00 and 1.5ppm
of silver, and selenium tested concentrations were 0, 0.1, 0.2, 0.3
and 0.5ppm of selenium. Groups of 21 fish were exposed for 96h
in fiberglass tank. Test medium was not renewed during the assay
and no food was provided to the animals. Values of mortalities
were measured at time 0, 24, 48, 72 and 96h.
Acute toxicity tests were carried out in order to calculate
the 96h LC50 for metals, based on the study conducted by Hotos and Vlahos. Mortality was recorded after 24, 48, 72 and 96h and
LC50 values and its confidence limits (95% CLs) were calculated.
Percentages of fish mortality were calculated for each metal
concentration at 24, 48, 72 and 96h of exposure.
Also, LC50 values were calculated from the data obtained
in acute toxicity bioassays, using Finney’s method of ‘probit
analysis’ and with SPSS computer statistical software. In Finney’s
method, the LC50 value is derived by fitting a regression equation
arithmetically and also by graphical interpolation by taking
logarithms of the test chemical concentration on the x-axis and
the probit value of percentage mortality on the y-axis.
The 95% CLs of the LC50 values obtained by Finney’s method
was calculated with the formula of Mohapatra and Rengarajan.
Probit transformation adjusts mortality data to an assumed
normal population distribution that results in a straight line.
Probit transformation is derived from the normal equivalent
deviate approach developed by Tort who proposed measuring the
probability of responses (i.e. proportion dying) on a transformed
scale based in terms of percentage of population or the SDs from
the mean of the normal curve.
All controls resulted in low mortalities, fewer than 5%, which
indicated the acceptability of the experiments. The mortality of
roach for silver and selenium was examined during the exposure
times at 24, 48, 72 and 96h in Tables 2 & 3, respectively. With
increasing concentration, fish exposed during the period of 24–
96h had significantly increased number of dead individual. There
were significant differences in number of dead fish between the
duration of 24 and 96h in each exposure.
The LC50 for silver in four stages of fish T. ilish were larvae
(0.23), fry (0.45), juvenile (0.90) and fingerling (1.45), respectively.
There was significance difference between LC50 levels in four
stages of fish species (P<0.05>). The highest of Toxicity of silver
were detected fingerling stage of fish species. Considering the
silver bioassay, the lowest concentration causing 100% of fish
mortality was 1.00mg/l at 96h, while the highest concentration
causing no fish mortality was 1.5mg/l at 96h.
The LC50 for selenium in four stages of fish T. ilish were
larvae (0.89), fry (0.95), juvenile (1.12) and fingerling (1.65),
respectively. There was significance difference between LC50 levels
in four stages of fish species (P<0.05>). The highest of Toxicity of
silver were detected fingerling stage of fish species. Considering
the selenium bioassay, the lowest concentration causing 100% of
fish mortality was 0.3mg/l at 96h, while the highest concentration
causing no fish mortality was 0.5mg/l at 96h.
Variability in acute toxicity even in a single species and single
toxicant depending on the size, age and condition of the test
species along with experimental factors. The differences in acute
toxicity may be due to changes in water quality and test species. It
is evident from the results that the heavy metal concentration has
a direct effect on the LC50 values of the respective fish. LC50 values
indicated that silver is more toxic than others. LC50s obtained in
the present study were compared with corresponding values that
have been published in the literature for other species of fish.
The degree of studied fish to lower concentrations of
selenium may be attributed to the altered physiological response
of every species to the specific metal and the level of solubility
of metals. The fish exposed to selenium can compensate for the
pollutant. If it cannot successfully compensate for contaminant
effects, an altered physiological stage may be reached in which
the fish species continues to function and, in extreme cases, the
acclimation response may be exhausted with a subsequent effect
The susceptibility of fish species to a particular heavy metal
is a very important factor for LC50 levels. The fish that is highly
susceptible to the toxicity of one metal may be less or even
nonsusceptible to the toxicity of another metal at the same level
of that metal in the ecosystem. Also, the metal which is highly toxic
to a fish species at low concentration may be less or even nontoxic
to other species at the same or even higher concentration.
Because of the lack of available data on the effects of selenium on
the respective LC50 values of all studied species, the results of the
present study have not been compared with those of other studies
and discussed accordingly. However, some justifications have
been provided following various studies.
Silver is highly toxic to aquatic organisms and interacts with
numerous inorganic and organic compounds which affect its
bioavailability and toxicity to aquatic biota. Its toxicity depends
on environmental factors that change through time and space (e.g.
temperature and water quality) and on the affected organism’s
species, age, size, and reproductive condition . Figure 1 shows
the toxicity patterns of silver to fish, T. ilish, at different life stages
(larva, fry, juvenile and fingerling). Estimation of the median
lethal concentrations of silver showed that the 96h LC50s were 0.1,
0.5, 1.00 and 1.5ppm, respectively. It can be noticed that the LC50s
in the 1st 3 length categories were similar and the 4th category was
the highest one which means that the tolerance of fish increases at
the 4th length category than the others, also indicated that the 1st
stages were more venerable than the older stages.
Few studies examined the relation between fish size and the
silver toxicity for finfish. Howarth and Sprague reported that
silver toxicity to rainbow trout, Oncorhynchus mykiss, decreased
with fish growth, and the toxicity for 0.71g fish was three times
higher than that for 10g in freshwater. Hedayati et al. reported
that silver toxicity to Rutilus rutilus decreased with fish growth,
and the toxicity for 2gr fish was four times higher than that for
10g in freshwater. They reported that LC50 for silver in Rutilus rutilus was 0.36ppm of silver. Also, Hedayati et al. reported that
the toxicity for 3gr fish was three times higher than that for 9g
in fish Acanthopagrus latus. They reported that LC50 for silver in
Rutilus rutilus was 0.45ppm of silver.
There were numerous studies examining the silver toxicity
to fresh and marine fish, 96h LC50 reported a range between 2 to
5ppm and 0.1 to 15ppm, respectively . Straus  recorded
a range of (0.23 to 28.39ppm) within a total alkalinity of 11-
215mg/l CaCO3, and stated that the Se sulfate can be extremely
toxic to fish in water of low alkalinity. In the same manner the
mean total alkalinity in the present study condition was 218mg/l
CaCO3. As well as, more studies recorded that Cu is less toxic in
hard water than in soft water [16,18]. For fresh water fish, Oliva et
al.  reported 0.35ppm silver as 96h LC50 for juvenile singales,
0.25 for chequered rainbow, 0.14 for black striped rainbow
and 0.021ppm for fly specked hardhead. This indicates that the
present studied species was more tolerance to silver toxicity than
other species studied.
Silver is classified as one of the most toxic metals, which are
introduced into the natural environment by human interferences. Inorganic silver is the most common form of the metal released in
the environment by industries, presenting a stronger acute effect
on fish tissues than that of its organic form . Figure 2 shows
the toxicity patterns of selenium to the different studied stages
of the fish T. ilish. 96h Hg LC50 were 0.1, 0.2, 0.3 and 0.5ppm for
larva, fry, juvenile and fingerling stages, respectively. It was found
that the toxicity of selenium was significantly decreased (r=0.98,
p≤0.05) with increasing the fish life stage (Figure 3). Unlike the
present study, some studies on other aquatic fish species indicated
that selenium toxicity did not change significantly with varying
Hedayati et al. reported that selenium toxicity to Rutilus rutilus
decreased with fish growth, and the toxicity for 2gr fish was three
times higher than that for 10g in freshwater. They reported that
LC50 for selenium in Rutilus rutilus was 0.87ppm. Also, Hedayati et
al. reported that the toxicity for 5gr fish was five times higher than
that for 20g in fish Acanthopagrus latus. They reported that LC50
for silver in Rutilus rutilus was 1.37ppm.
From the previous studies, Ishikawa et al.  recorded
0.45ppm 96h LC50 for Oreochromis leucostictus fingerlings
which was compare to the present study (Table 4). In the
context, Ramamurthi et al. recorded Hg LC50 0.87ppm for Tilapia
mossambicus. As well as, many studies recorded a range of 0.045–
0.21ppm selenium LC50 for different fish species which was more
or less lower than the present study . It was indicated that the
present examined species was more tolerant than the previous
studied species. For both of the present studied metals (selenium
and silver), it can be noticed that the toxicity tended to elevate
with decreasing fish size, this mean that the earlier stages were
more sensitive than the older one. Grosell et al.  stated that
the size was an effective factor for acute toxicity in fresh water organisms. It has been mentioned that small fish or younger
organisms were more susceptible to metal poisoning than the
larger or more mature fish. Thongra-ar et al.  and Furuta et
al.  found that the seabass larvae were more sensitive to Hg
toxicity than the juvenile stages.
By comparing the toxicity of selenium and silver in the present
study (Figure 3), it can be stated that the silver is more toxic than
selenium during the 1st 2 life stages, where they recorded the
same toxicity during the last 2 life stages with slight increase of Se
toxicity at the last stage. Gooley and Shyong and Chen found that
selenium was more toxic than silver to Acrosscheilus paradoxus
and Zacco barbata, respectively. Hedayati et al. reported that silver
toxicity to Rutilus rutilus was more toxic than other metal and LC50
of silver was higher than selenium, cobalt and silver. Also, Verep et
al. and Vieira et al. reported that silver toxicity to pomatoschistus
microps and Rainbow trouts was higher than other heavy metal.
The results of toxicity test of silver and selenium in current
study were compared to those reported for fish in the Persian
Gulf and different regions of the world (Table 4). In general, the
LC50 of silver and selenium in our study were higher than those
in Oreochromis leucostictus by Ramamurthi et al. Tenualosa ilisha
by Verma et al. Oreochromis leucostictus by Chen and Tilapia
mossambicus in study of Grosell et al. . The LC50 of silver
and selenium in Tenualosa ilisha in this study was higher than all
studies that present in Table 4, expect in Euryglossa orientalis in
study by Hosseini et al.
However, in the current study, the LC50 values vary from each
species and the accumulation of heavy metals in the body of
fish depends upon several factors, it is evident from the present
study that concentrations of selenium and physiological response
affect the LC50 values of fish. It may be due to the increased
resistance of carp to the selenium through acclimatization.
During acclimatization, some proteins are released in the body
of fish and detoxify the metal ions. This may cause higher levels
of heavy metals being required to cause effects, resulting in
higher LC50 amounts. The selection of heavy metals may be an
important tool for the assessment of the effects of pollutants
in aquatic ecosystems; both metals used in our experiment
demonstrated their potential for use in bioassays. In conclusion,
comparing the sensitivity of these metals to common reference
toxicants, we suggest using Roach for toxicity determinations
as a suitable model of ecotoxicological studies. Clearly, there is
a need to conduct further studies with specific contaminants on
this species to assess its suitability for detecting toxicity, as well as
experiments involving a complex mixture of pollutants, in order to
determine aquatic ecosystems monitoring program.
Special thanks are due to Dr Shahram Valizadeh Moejezi for
help with statistical analysis and Moemen Ali and Malek Ali Peery
for field assistance. This work was funded by 2 Iranian National
Institute for Oceanography (INIO) and Environment Protection
Institute of Tehran, Iran.