Regional Studies in Marine Science 64 (2023) 103036

Contents lists available at ScienceDirect

Regional Studies in Marine Science

journal homepage: www.elsevier.com/locate/rsma

Toxicity assessment of Tetrodotoxin of commercially exploited

smooth blassop Lagocephalus inermis in south-eastern Arabian Sea
∗
Purbali Saha a,b , Sujitha Thomas a , , Murali Badanthadka c , Krupesha Sharma d ,
Michelle Mathias e
a
ICAR-Central Marine Fisheries Research Institute, Mangalore Regional Centre, Mangalore, Karnataka, 575 001, India
b
Department of Biosciences, Mangalore University, Mangalagangothri, Karnataka, 574 199, India
c
Nitte (Deemed to be University), NGSM Institute of Pharmaceutical Sciences (NGSMIPS), Department of Nitte University Centre for Animal Research
and Experimentation (NUCARE), Paneer Campus, Deralakatte, Mangalore 575 018, India
d
ICAR- Central Marine Fisheries Research Institute, Kochi, Kerala, 682 018, India
e
Department of Pathology, K.S. Hegde Medical Academy, Derelakatte, Mangalore, Karnataka, 575 018, India

article info a b s t r a c t

Article history: The toxicity assessment of Lagocephalus inermis, commonly known as smooth blassop, which is
Received 8 February 2023 commercially exploited in the south-eastern Arabian Sea, was conducted in this study, marking
Received in revised form 18 April 2023 the first of its kind for the region. The experiment involved testing control and standard samples
Accepted 25 May 2023
during pre-monsoon and post-monsoon seasons. Gross anatomical features were observed, followed
Available online 10 June 2023
by histopathological examination of hearts, livers, and kidneys of deceased mice to determine toxicity.
Keywords: High-performance liquid chromatography (HPLC) was used to analyze standard tetrodotoxin (TTX)
Indian coast concentrations, which showed characteristic peaks at retention times of 4.83 min (mins), 4.792
Mouse Bioassay min, 4.748 min, and 4.729 min for concentrations of 0.1 mg/ml, 0.08 mg/ml, 0.06 mg/ml, and 0.04
Neurotoxin mg/ml, respectively. However, the test samples consisting of liver, ovary, and muscle extracts did
Puffer fish not exhibit any characteristic peak indicating the presence of TTX in both seasons. Furthermore,
TTX bioassays conducted on the test samples did not show any toxicity symptoms, while standard test
mice exhibited characteristic symptoms of TTX poisoning. Histopathological examinations revealed
congestion, necrosis, degeneration, and hemorrhages in the liver, heart, and kidney tissues of the
standard sample, but no changes were observed in the test sample. Based on the findings, it was
concluded that Lagocephalus inermis, as exploited along the Karnataka coast, is not toxic at present.
However, considering that the bacteria responsible for the species’ toxicity may not be sufficiently
present to cause toxicity at the current time, it is imperative for Indian food sanitation authorities to
continuously monitor and assess the toxicity of pufferfish consumed in terms of food safety.
© 2023 Elsevier B.V. All rights reserved.

1. Introduction intoxication for a long time, and to date, there is no known

Tetrodotoxin (TTX) is an extremely potent marine toxin that
can be lethal to humans, occasionally resulting in poisoning and
even fatalities (Chew et al., 1983; Homaira et al., 2010). TTX poi-
soning most commonly occurs due to ingestion of toxic pufferfish,
although TTX has been found in various other marine organisms
as well. TTX has been detected in more than 20 species of puffer-
fish (Noguchi et al., 2006), and it is both water-soluble and heat
stable, meaning that cooking does not eliminate its toxicity; in
fact, it can even increase the toxic effect (Saoudi et al., 2007). The
toxin was initially discovered in 1909 in the ovaries of globefish
(Suehiro, 1994), but pufferfish have been known to cause human
∗ Corresponding author.
E-mail address: sujitha.thomas@icar.gov.in (S. Thomas).
antidote for TTX poisoning (Noguchi and Ebesu, 2001).
In the past, Tetrodotoxin (TTX) was primarily considered a
problem in Asia, particularly in Japan (Ansdell, 2019). However,
this belief has changed as TTX intoxication has been reported
in areas that were previously thought to be safe from TTX poi-
soning. The spread of TTX has been attributed to the global rise
in seawater temperatures (Danovaro et al., 2009). In pufferfish,
TTX is produced by bacterial species belonging to genera such
as Serratia, Aeromonas, Actinomycetes, Alteromonas, Pseudomonas,
and Vibrio, which are typically associated with pufferfish (Yan
et al., 2005). TTX is commonly accumulated in the ovaries, liver,
intestine, and skin of pufferfish, and it does not affect the appear-
ance or taste of the fish. Moreover, TTX is highly heat stable and
cannot be destroyed by smoking, cooking, freezing, or canning
(Saoudi et al., 2007).

https://doi.org/10.1016/j.rsma.2023.103036
2352-4855/© 2023 Elsevier B.V. All rights reserved.
P. Saha, S. Thomas, M. Badanthadka et al.
Studies on the mechanism of TTX toxicity have revealed that
it binds to the sodium channels of muscles and nerves, resulting
in the inhibition of sodium ions through the channels, effectively
immobilizing the tissues (Denac et al., 2000). The effects of the
toxin on humans depend on the dosage. Early symptoms of TTX
poisoning include a tingling sensation of the lips and tongue,
headache, and vomiting, which can progress to numbness in the
muscles and loss of control and coordination. In severe cases,
victims may die of heart or respiratory failure (How et al., 2003).
Pufferfish poisoning has been predominantly reported in Japan,
where they are considered a highly attractive and favored deli-
cacy. Between 1886 and 1963, there were 6386 reported cases of
TTX poisoning in Japan, resulting in approximately 59% mortality
(Ansdell, 2019). However, increased awareness has led to the im-
plementation of various measures to monitor and minimize the
risks associated with pufferfish consumption (Kaku and Meier,
1995). Specialized training and licensing for chefs in handling
pufferfish have resulted in a decrease in poisoning cases and
deaths in the period of 1983–1992 (11%) compared to 1967–76
(Kaku and Meier, 1995).
In contrast, there have been no documented cases of TTX poi-
soning from the Indian subcontinent, which could be attributed
to traditional knowledge that discourages the consumption of
pufferfish due to the toxin. Researchers have primarily studied
TTX from the Indian subcontinent using methods such as Mouse
Bioassay (MBA) and analytical instruments such as HPLC, GC-MS,
TLC, and LC-MS. Pufferfish species studied from Indian waters
for the presence of TTX include Arothron hispidus, A. stellatus,
Chelonodon patoca, Lagocephalus inermis, L. lunaris, and Takifugu
oblongus (Tamele et al., 2019). Among these, the ovary of C. patoca
was found to be highly toxic (183 MU/g) (Ghosh et al., 2004),
followed by the ovary of T. oblongus (136 MU/g) (Indumathi and
Khora, 2017a), and the gonads of A. hispidus (132 MU/g) (Bra-
gadeeswaran et al., 2010). The liver and ovary of L. inermis were
found to be minimally toxic, with concentrations of 5.5 MU/g and
9.64 MU/g, respectively (Ghosh et al., 2004). However, there is a
lack of studies on TTX from L. inermis or any other species of
pufferfish from the west coast of India. Therefore, this study is
the first of its kind from the west coast of India, aiming to fill
the knowledge gap regarding the toxicity of pufferfish harvested
from the south-eastern Arabian Sea.
L. inermis has emerged as a valuable fishery resource and
is commercially exploited along the Karnataka coast, supply-
ing neighboring states. Therefore, understanding the presence
or absence of TTX and its concentration in L. inermis and other
pufferfish species becomes crucial for planning effective monitor-
ing strategies to ensure sustainable exploitation and formulating
policies for safe consumption of pufferfish.
Significance
Lagocephalus inermis, a fish species belonging to the Tetraodon-
tidae family, is well-known for its tetrodotoxin poisoning. As
commercial exploitation of L. inermis is limited to the Karnataka
coast in India, and is consumed, it is crucial to assess the species’
toxicity for safety purposes and ensure that the product is safe for
consumption. This study is the first of its kind conducted along
the west coast of India, where commercial exploitation of L. iner-
mis takes place. The findings of this study will provide valuable
information on the current toxicity levels of the species, and the
need for continuous monitoring and assessment of toxicity for
food safety reasons.
2. Materials and methods
2.1. Sample collection
Lagocephalus inermis samples were collected from the pre-
identified trawlers operating along the south-eastern Arabian Sea
2
Regional Studies in Marine Science 64 (2023) 103036
extending from Calicut in Kerala to Thane in Maharashtra with
the GPS coordinates 11.969◦ N 71.794◦ E to 18.821◦ N 74.932◦ E.
The samples were brought to the laboratory in the chilled con-
dition in the icebox. The samples collected were in the size range
of 20 cm to 30 cm in length because the significant catch in this
region is in this size group. After reaching the laboratory, each
specimen was cleaned thoroughly before dissection, cutting them
open using a sharp scissor and then the viscera were dissected
out. Samples of liver, muscle, and ovary were individually iso-
lated and stored for subsequent analysis. These specific organs
were chosen for the experiment due to the fact that muscle is
consumed by the public along this coast, and in marine puffer-
fish, the liver tissue is toxic throughout the year, except during
the spawning season when the ovaries are comparatively more
toxic (Bane et al., 2014). The specimens were then preserved
at −20 ◦ C until further investigation. Samples were collected
and preserved separately during both pre-monsoon and post-
monsoon periods in order to assess potential seasonal variations
in toxin concentration.
2.2. Extraction of TTX
To extract tetrodotoxin (TTX), samples of liver, muscle, and
ovary from L. inermis were collected (Indumathi and Khora, 2017b).
Approximately 1 g of tissue was taken from each sample, and up
to 10 g was taken for TTX extraction from each organ. Tissues
were homogenized using a tissue homogenizer with 50 ml of
0.1% acetic acid in distilled water. The homogenized samples were
then boiled at 50 ◦ C in a hot water bath for 10 min. After cooling,
the homogenates were centrifuged at 3000 rpm for 10 min. The
supernatant was collected and stored, and the procedure was
repeated three times. The collected supernatants were combined,
filtered using a Whatman glass microfiber filter, and stored at
−20 ◦ C (Khora, 1991).
2.3. High-Performance Liquid Chromatography (HPLC) specifications
The samples were tested for the presence of TTX using HPLC
(Shimadzu SPD-M20A), (Indumathi and Khora, 2017b) with the
oven temperature maintained at 30 ◦ C. Shim-pack solar 5 µm
C18 column with 4.6 mm × 250 mm dimension was used, and
0.04% phosphoric acid was used as the mobile phase with a 0.5
ml/min flow rate. Photodiode Array Detector (PDA) was used to
prominence the test samples with the wavelength set at 195 nm.
The standard TTX (1 mg) was dissolved in 10 ml of distilled water
to prepare the standard stock solution (0.1 mg/ml), which was
then diluted further into 0.08 mg/ml, 0.06 mg/ml, and 0.04 mg/ml
for obtaining the standard graph (Qun et al., 2005).
2.4. Experimental animals for mouse bioassay
To support and further confirm the result obtained from HPLC,
mouse bioassay was done. This study was conducted at NGSM
Institute of Pharmaceutical Sciences (NGSMIPS), Department of
NITTE University Centre for Animal Research and Experimenta-
tion (NUCARE). Swiss albino mice ranging between 18–22 g were
used for the study. The experiment was approved by Institutional
Animal Ethics Committee with the protocol no: NGSMIPS/IAEC/
MARCH-2019/130. All experiments were carried out following
CPCSEA (Committee for the Purpose of Control and Supervision
of Experiment on Animals) guidelines.
P. Saha, S. Thomas, M. Badanthadka et al.
Fig. 1. Euthanization and dissection of the experimental mice for the collection of organs (brain, liver, heart and kidney) to perform the histopathology.
2.5. Experimental set up for mouse bioassay
The experimental mice were divided into five groups con-
sisting of three mice in each group. They were acclimatized in
the cages for a few days before the experiment started. The first
group was the control, the second received the standard toxin,
the third received the liver extracts, the fourth group received the
ovary extracts, and the fifth received the muscle extracts. All the
samples were injected intraperitoneally into the mice. The dose-
volume was calculated according to the weight of the respective
mice with the maximum permissible limit of 10 ml/kg as per
the guideline given by CPCSEA Standard Operating Procedures for
Institutional Animal Ethics Committee (IAEC). The control mice
received 0.1% acetic acid as a vehicle. After the injection, animals
were observed for toxic symptoms and mortality for a maximum
of 6 h before euthanasia (Yu, 2003; Indumathi and Khora, 2017b)
2.6. Histopathological examination
The brain, heart, liver, and kidney of all the mice after their
death/euthanization were dissected out to ascertain the his-
tological changes (Fig. 1). Tissues were fixed in 10% neutral
buffered formalin for 24 h and then processed further using
routine methods. Following dehydration, clearing and paraffin
infiltration, paraffin-embedded tissues were cut at 5 µm thick-
ness using a semi-automatic rotary microtome (Slaoui and Fiette,
2011). Sections were stained with Hematoxylin and Eosin (H&E).
Stained sections were observed under the light microscope, and
photographs were taken (Lefkowitch, 1987; Indumathi and Khora,
2017b).
3
Regional Studies in Marine Science 64 (2023) 103036
3. Results
3.1. High performance liquid chromatography
The standard TTX showed characteristic peaks for the four
concentrations with the retention times being 4.83 minutes
(min), 4.792 mins, 4.748 mins and 4.729 mins for 0.1 mg/ml, 0.08
mg/ml, 0.06 mg/ml and 0.04 mg/ml respectively (Fig. 2). Inter-
estingly, the test samples consisting of liver, ovary and muscle
extracts did not show any characteristic peak for the presence
of TTX. All the peaks in the test samples of both post-monsoon
(Fig. 3) and pre-monsoon season (Fig. 4) started after a retention
time of 5 mins, indicating the absence of TTX in the samples
during the study period. To confirm the findings of the HPLC,
mouse bioassay (MBA) was done using the same test samples.
3.2. Mouse bioassay
The first group was the control and, as expected, did not show
any adverse symptoms, and microscopic examination of the cells
in various organs also showed no changes from normal cells.
The second group received the standard toxin and showed symp-
toms of toxicity before death. The mice showed hyperactivity by
running inside the cage for the initial few seconds, followed by
shivering, paralysis, and paleness of the eyes before suffocating to
death. The time of death was noted for each mouse. They were
subjected to different concentrations of standard TTX. The mice
injected with 0.1 mg/ml, 0.05 mg/ml and 0.025 mg/ml concentra-
tion of TTX died after 1.2 seconds (s), 30 s and 62 s respectively.
The mice injected with the test samples (TTX extracted from liver,
ovary, and muscles of L. inermis) did not show any symptoms of
P. Saha, S. Thomas, M. Badanthadka et al. Regional Studies in Marine Science 64 (2023) 103036

Fig. 2. Graph showing the HPLC results of standard Tetrodotoxin with standard peaks and retention time in different concentrations: (1) 0.1 mg/ml; (2) 0.08 mg/ml:
(3) 0.06 mg/ml; and (4) 0.04 mg/ml.

Fig. 3. HPLC results of the test samples showing no peak at the retention time of the standard tetrodotoxin during the pre-monsoon season: (1) Sample from liver
of L. inermis; (2) Sample from ovary of L. inermis and; (3) Sample from muscle of L. inermis.

4
P. Saha, S. Thomas, M. Badanthadka et al. Regional Studies in Marine Science 64 (2023) 103036

Fig. 4. HPLC results of the test samples showing no peak at the retention time of the standard tetrodotoxin during the post-monsoon season: (1) Sample from liver
of L. inermis; (2) Sample from ovary of L. inermis and; (3) Sample from muscle of L. inermis.

Table 1 vacuolation and necrosis of myocytes were observed in the heart.

Lethality data of standard TTX and organ extracts. Extensive congestion, haemorrhage, tubular dilation, and necrosis
Sl. No. Test drug No of animals Mortality were observed in the kidney. Acute congestion, focal necrotic
1 0.1% acetic acid (Control) 3 0/3 hepatocytes, hyperplasia of the bile ducts, increased activity of
2 Standard TTX 3 3/3
kupffer cells, and accumulation of inflammatory cells around the
3 Liver extracts 3 0/3
4 Ovary extracts 3 0/3 central vein were observed in the liver (Fig. 6).
5 Muscle extracts 3 0/3
3.3.3. Effect of the test extracts
There were no changes in the brain tissues except for mild
congestion. The muscle tissues of the heart appeared normal.
toxicity like that of the symptoms shown by the mice injected
Mild congestion and tubular dilation could be seen in the kidney.
with standard TTX. Since organ extract was challenged, animals
Mild congestion, inflammation, vacuolation, haemorrhage and
did not show any mortality, confirming the result obtained from
increased activity of Kupferr cells were observed in the liver of
HPLC. As there were no symptoms of toxicity noted in the mice,
mice injected with liver, muscle and ovary extracts (Figs. 7–9).
after 6 h of observation, they were euthanized to collect organs
(Table 1).
4. Discussion
3.3. Histopathological examination
The consumption of pufferfishes can pose a severe threat of
A detailed account of the histopathological examination on food poisoning to coastal nations. Based on a literature review,
the brain, kidney, heart, and liver of each mouse injected with this study hypothesized that L. inermis is toxic, and the main
control, standard, liver, muscle, and ovary extracts are given objective was to determine the seasonal variations in its con-
below: centration. A study conducted in Japan found that the liver of
L. inermis was highly toxic, with a concentration of 1230 MU/g,
3.3.1. Effect of the control while the skin and muscle had minimal toxicity, with concentra-
The typical appearance of the granular and molecular layer tions below 5 MU/g (Nagashima et al., 2012). In India, a study on
with the normal distribution of the cortical neuron could be seen the seasonal toxicity profiles of L. lunaris, Chelonodonpatoca, Tak-
in the brain. In the heart, normal cardiac muscle fibers could ifugu oblongus, and L. inermis was conducted on the east coast, and
be seen. Similarly, normal liver parenchyma with hepatocytes it was found that L. inermis was the least toxic species compared
arranged radially around the central vein and normal kidney to other species in the same region (Ghosh et al., 2004). In the
tubules and glomeruli could be seen (Fig. 5). above study, the liver samples of L. inermis during pre-monsoon
had a toxin level of 4.6 ± 1.2 MU/g, which increased to 4.9 ± 0.8
3.3.2. Effect of the standard tetrodotoxin MU/g during post-monsoon. A similar trend was observed in
Mice injected with standard TTX showed congestion, necrosis, the ovary samples (Ghosh et al., 2004). Pufferfish toxicity levels
reactive gliosis, and granuloma in the brain tissues. Extensive are classified as strongly toxic (≥1000 MU/g tissue), moderately
congestion and degeneration of the cardiac muscle fibers with toxic (100–999 MU/g tissue), weakly toxic (10–99 MU/g tissue),
5
P. Saha, S. Thomas, M. Badanthadka et al. Regional Studies in Marine Science 64 (2023) 103036

Fig. 5. Images of histopathological slides showing the normal arrangement of cells in Control mice group- (1) Brain (2) Heart (3) Kidney (4) Liver.

Fig. 6. Images of histopathological slides showing the affected cells of the organs from the mice injected with standard TTX
(1) Brain—Severe tissue destruction (thick arrow) in the brain accompanied by vacuolation (thin arrow) and haemorrhage (arrowhead)
(2) Heart—Extensive haemorrhage (thick arrow), blood congestion (thin arrow) and vacuolation (arrowhead) in the myocardium of the heart
(3) Kidney—Section of the kidney showing severe congestion (thick arrow) and haemorrhage (star)
(4) Liver—Section of the liver showing focal necrosis (thick arrow) and nuclear pyknosis (thin arrow).

and non-toxic (<10 MU/g tissue) from a food safety perspective
(Nagashima et al., 2012).
Although there have been reports of mild toxicity in L. inermis
along the east coast (Ghosh et al., 2004), the findings of the
present study, which utilized HPLC and mouse bioassay, revealed
6
no detectable levels of TTX in all sampled organs during both
pre-monsoon and post-monsoon seasons. Test samples injected
into mice did not show any symptoms of toxicity. These results
are consistent with a previous study by Ghosh et al. (2004) that
found TTX levels in L. inermis to be near the threshold of non-
P. Saha, S. Thomas, M. Badanthadka et al. Regional Studies in Marine Science 64 (2023) 103036

Fig. 7. Images of histopathological slides showing the cells of the organs from the mice injected with test sample extracted from Liver L. inermis
(1)- Brain—Normal appearance of the brain cells
(2) Heart—Normal appearance of the myocytes
(3)- Kidney—Section showing vacuolation of the kidney cells
(4)- Liver—Mild inflammation of the liver cells could be seen.

Fig. 8. Images of histopathological slides showing the cells of the organs from the mice injected with test sample extracted from ovary L. inermis.
(1) Brain—Normal appearance of the brain cells
(2) Heart—Normal appearance of the heart muscle fibers
(3) Kidney—Mild vacuolation and congestion could be seen
(4) Liver—Mild congestion and inflammation of the cells could be seen.

7
P. Saha, S. Thomas, M. Badanthadka et al.
Fig. 9. Images of histopathological slides showing the cells of the organs from the mice injected with test sample extracted from muscle L. inermis.
(1) Brain—Normal appearance of the brain cells
(2) Heart—Normal appearance of the myocytes
(3) Kidney—Mild vacuolation and congestion could be seen in the kidney
(4) Liver—The liver cells appeared normal.
toxicity (Nagashima et al., 2012). Furthermore, HPLC analysis
in the present study also did not detect the presence of TTX.
Therefore, it can be inferred that L. inermis from the west coast of
India can be considered non-toxic, as no detectable levels of TTX
were observed in the study.
The liver and kidneys are vital organs for xenobiotic excretion,
detoxification, storage, and metabolism, and they are susceptible
to damages (Omar, 2013). In the present study, necrosis, conges-
tion, and inflammation of the hepatic cells were also observed
when injected with standard TTX, while it was normal in con-
trol. Mild changes observed in the test samples could be due to
any organic compounds present in the extract. The relevance of
the liver as a marker for pathological alterations has long been
recognized, and liver damage is the most commonly reported
histological reaction to organic substances (Metcalfe, 1998). The
studies showed that in the kidney’s treated with standard TTX
showed different signs of histopathological deformations; how-
ever, such deformations were absent in the test samples. The
absence of apparent histopathological deformations in the organs
treated with the extracts also supports results of HPLC for the L.
inermis not being toxic along the coast during the present study.
When it comes to food safety, it is imperative to thoroughly
evaluate the toxicity of pufferfish before considering them safe for
consumption. Incidents of poisoning resulting from the consump-
tion of pufferfish have been widely reported worldwide, although
information from India is limited or unavailable. This may be due
to inadequate documentation or the fact that pufferfish has not
traditionally been consumed in India. However, in recent times,
pufferfish has been increasingly caught as by-catch in trawlers
across the country and has emerged as a commercially exploited
resource, particularly along the west coast of India, as reported
by Thomas et al. (2009) and Saha et al. (2019). Therefore, this
study was conducted to gain insights into the toxicity profile of
pufferfish, considering its growing commercial importance along
the west coast of India
8
Regional Studies in Marine Science 64 (2023) 103036
Research on the origin of Tetrodotoxin (TTX) has demon-
strated that it accumulates in the bodies of organisms through
the food chain, originating from TTX-producing bacteria, rather
than being produced by the TTX-bearing organisms themselves
(Arakawa et al., 2010). However, the specific mechanism of trans-
fer, accumulation, and elimination of TTX in pufferfishes remains
unclear. Recent studies have shown that toxic pufferfishes can
naturally lose their toxicity when kept in an environment devoid
of TTX-producing bacteria (Arakawa et al., 2010). In Japan, due
to comprehensive investigations into the toxicity and biology
of puffers, as well as increased attention to pufferfish selection
and preparation, there has been a decline in fatal cases of TTX
intoxication (Hashimoto, 1979).
Based on interviews and interactions with fishers along the
Karnataka coast, it is seen that there have been no recorded
fatalities associated with the consumption of Lagocephalus iner-
mis, commonly known as the smooth pufferfish. Fishers at the
Mangalore fishing harbour hold the belief that L. inermis is non-
toxic, and our study results have confirmed this. Extracts from
tissues of L. inermis collected from the Karnataka coast showed no
toxicity in mice and were also analyzed using high-performance
liquid chromatography (HPLC), which further supported our find-
ings. During the study period from 2016 to 2019, no detectable
levels of Tetrodotoxin (TTX) were found in L. inermis speci-
mens, possibly due to the absence or low levels of TTX-producing
bacteria such as Serratia, Aeromonas, Actinomycetes, Alteromonas,
Pseudomonas, and Vibrio spp. in the areas where this species is
found along the Karnataka coast.
Continuous monitoring of L. inermis for toxicity is essential due
to the potential for future toxicity when a sufficient quantity of
TTX-producing bacteria is available in the ecosystem. Therefore,
frequent monitoring studies are necessary in this area and other
coastal states in India to ensure that the flesh of L. inermis can
be consumed without posing any risk to humans. It is important
to note that pufferfish toxicity is influenced by various factors
P. Saha, S. Thomas, M. Badanthadka et al.
such as feeding behavior, reproductive stages, and geographic
distribution, as reported by Matsui et al. (1982), Sabrah et al.
(2006), and Katikou et al. (2009). In fact, this species has been
found to be toxic in other parts of the world, as reported by
Nagashima et al. (2012). Thus, the consumption of L. inermis can
be considered safe as long as the bacteria responsible for TTX
production are absent or not present in sufficient numbers to
induce toxicity.
Furthermore, it is worth mentioning that TTX exists in various
analogues, and many scientists have attempted to study these
analogues. However, due to limited availability of standards, their
studies are constrained. Although some researchers have artifi-
cially synthesized standards, their availability is also limited, as
reported by Kishi et al. (1972), Nishikawa et al. (2002a,b, 2003,
2004)), and Ohyabu et al. (2003). The minor changes observed
in the organ tissues of mice injected with test samples, as com-
pared to the control in the present study, could potentially be
attributed to the presence of analogues of TTX. However, the
detailed investigation of this aspect was beyond the scope of this
study.
Top of Form
Furthermore, it is crucial to note that climate change has
emerged as one of the foremost driving factors behind marine
processes and ecosystem changes. Studies have reported signif-
icant alterations in marine species’ phenology, abundance, and
distribution, as highlighted by Alabia et al. (2015) and Poloczan-
ska et al. (2016). Consequently, there is a possibility of an in-
crease in TTX-producing bacteria in the ecosystem, which may
potentially induce toxicity in L. inermis in the future. This study
underscores the need for more comprehensive investigation of
regional variations in muscle toxicity of edible pufferfish species.
Therefore, continuous monitoring and assessment of pufferfish
toxicity by Indian food sanitation authorities from a food safety
perspective is imperative.
CRediT authorship contribution statement
Purbali Saha: Concept, Design, Literature search, Experi-
mental studies, Data analysis, Manuscript preparation, Critically
revised the manuscript for intellectual content. Sujitha
Thomas: Concept, Design, Literature search, Experimental
studies, Data analysis, Manuscript preparation, Critically revised
the manuscript for intellectual content. Murali Badanthadka:
Design, Experimental studies, Manuscript editing, Critically
revised the manuscript for intellectual content. Krupesha
Sharma: Interpretation of histological slides, Manuscript
editing, Critically revised the manuscript for intellectual
content. Michelle Mathias: Interpretation of histological slides,
Manuscript editing, Critically revised the manuscript for
intellectual content.
Declaration of competing interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Data availability
Data will be made available on request.
9
Regional Studies in Marine Science 64 (2023) 103036
Acknowledgments
Authors would like to thank the Director, ICAR-CMFRI and
Head, ICAR-CMFRI, Mangalore Regional Centre for their contin-
uous support and guidance. The authors would also like to thank
NICRA-ICAR and Principal Investigator of NICRA Project. The first
author would like to express her gratitude to Department of
Biosciences, Mangalore University, Mangaluru. We would also
like to thank Mr. G.D. Nataraja for his help during sampling. The
help rendered by Madhura, Meghashree, Varsha and Vinitha for
smooth running of the experiment is duly acknowledged.
All authors approved the version of the manuscript to be
published
Funding
This research was supported by National Innovations on Cli-
mate Resilient Agriculture (NICRA) project funded by Indian
Council of Agricultural Research, Ministry of Agriculture and
Farmers Welfare (Project code- 11-4/2017).
Disclaimer
The results presented in this study are solely the research find-
ings of the authors who have worked on the pufferfish resource
commercially exploited along Karnataka, India coast.
References
Alabia, I.D., Saitoh, S., Mugo, R., Igarashi, H., Ishikawa, Y., Usui, N., Kamachi, M.,
Awaji, T., Seito, M., 2015. Seasonal potential fishing ground prediction of
neon flying squid (Ommastrephesbartramii) in the western and central North
Pacific. Fish. Oceanogr. 24, 190–203.
Ansdell, V., 2019. Seafood poisoning. In: Keystone, J.S., Kozarsky, P.E., Con-
nor, B.A., Nothdurft, H.D., Mendelson, M., Leder, K. (Eds.), Travel Medicine,
fourth ed. Elsevier, London, pp. 449–456.
Arakawa, O., Hwang, D.-F., Taniyama, S., Takatani, T., 2010. Toxins of pufferfish
that cause human intoxications. In: Ishimatsu, A., Lie, H.-J. (Eds.), Coastal
Environmental and Ecosystem Issues of the East China Sea. Terrapub and
Nagasaki University, Tokyo, Japan, pp. 227–244.
Bane, V., Lehane, M., Dikshit, M., O’Riordan, A., Furey, A., 2014. Tetrodotoxin:
chemistry, toxicity, source, distribution and detection. Toxins 6, 693–755.
Bragadeeswaran, S., Therasa, D., Prabhu, K., Kathiresan, K., 2010. Biomedical and
pharmacological potential of tetrodotoxin-producing bacteria isolated from
marine pufferfishArothronhispidus (Muller, 1841). J. Venom. Anim. Toxins
Incl. Trop. Dis. 16, 421–431.
Chew, S.K., Goh, C.H., Wang, K.W., Mah, P.K., Tan, B.Y., 1983. Puffer fish
(Tetrodotoxin) poisoning: clinical report and role of anti-cholinesterase drugs
in therapy. Singapore Med. J. 24, 168–171.
Danovaro, R., Fonda, U.S., Pusceddu, A., 2009. Climate change and the potential
spreading of marine mucilage and microbial pathogens in the Mediterranean
Sea. PLoS One 4, 1–8.
Denac, H., Mevissen, M., Scholtysik, G., 2000. Structure, function and phar-
macology of voltage-gated sodium channels. Naunyn-Schmiedeberg’s Arch.
Pharmacol. 362, 453–479.
Ghosh, S., Hazra, A.K., Banerjee, S., Mukherjee, B., 2004. The seasonal toxico-
logical profile of four puffer fish species collected along Bengal coast, India.
Indian J. Mar. Sci. 33 (3), 276–280.
Hashimoto, Y., 1979. Marine Toxins and Other Bioactive Marine Metabolites.
Japanese Scientific Society Press, Tokyo, Japan.
Homaira, N., Rahman, M., Luby, S., Rahman, M., Haider, M.S., Faruque, L.I.,
Khan, D., Parveen, S., Gurley, E., 2010. Multiple outbreaks of puffer fish
intoxication in Bangladesh, 2008. Am. J. Trop. Med. Hyg. 83 (2), 440–444.
How, C.-K., Chern, C.-H., Huang, Y.-C., Wang, L.-M., Lee, C.-H., 2003. Tetrodotoxin
poisoning. Am. J. Emerg. Med. 21, 51–54.
Indumathi, S.M., Khora, S.S., 2017a. Seasonal toxicity assessment of Oblong
Blowfish (Takifuguoblongus) from Tamil Nadu coast, India. J. Appl. Pharm.
Sci. 7 (3), 188–193.
Indumathi, S.M., Khora, S.S., 2017b. Toxicity assessment and screening of
tetrodotoxin in the oblong blowfish (Takifuguoblongus) from the Tamil Nadu
Coast of Bay of Bengal, India. Asian Pac. J. Trop. Med. 10 (3), 278–284.
Kaku, N., Meier, J., 1995. Clinical toxicology of fugu poisoning. In: Meier, J.,
White, J. (Eds.), Handbook of Clinical Toxicology of Animal Venoms and
Poisons. CRC Press, Boca Raton, FL, pp. 75–83.
P. Saha, S. Thomas, M. Badanthadka et al.
Katikou, P., Georgantelis, D., Sinouris, N., Ptesi, A., Fotaras, T., 2009. First report
on toxicity assessment of the Lessepsian migrant puffer fish Lagocephaluss-
celeratus (Gmelin, 1789) from European waters (Agean Sea, Greece). Toxicon
54, 50–55.
Khora, S.S., 1991. Toxicity Studies on Puffer Fish from Tropical Waters (D. Ag.
Thesis). Tohuku University, Sendai, Japan.
Kishi, Y., Aratani, M., Fukuyama, T., Nakatsubo, F., Goto, T., Inoue, S., Tanino, H.,
Sugiura, S., Kakoi, H., 1972. Synthetic studies on tetrodotoxin and related
compounds. III. Stereospecific synthesis of an equivalent of acetylated
tetrodamine. J. Am. Chem. Soc. 94, 9217–9219.
Lefkowitch, H.J., 1987. Histopathology of Disease. Churchill Living Stone
Publisher, New York.
Matsui, T., Sato, H., Hamada, S., Shimuzu, C., 1982. Local variation of toxicity of
the puffer fish Fuguniphobles. Bull. Jpn. Soc. Sci. Fish. 48, 1179.
Metcalfe, C.D., 1998. Toxicopathic responses to organic compounds. In: Leather-
land, J.F., Woo, P.T.K. (Eds.), Fish Diseases and Disorders: Non- Infectious
Disorders, second ed. CABI publishing, UK and USA, pp. 133–162.
Nagashima, Y., Matsumoto, T., Kadoyama, K., Ishizaki, S., Taniyama, S.,
Takatani, T., Arakawa, O., Terayama, M., 2012. Tetrodotoxin poisoning due to
Smooth-backed Blowfish Lagocephalusinermis and toxicity of L. inermis caught
off the Kyushu Coast, Japan. J. Food Hyg. Soc. Jpn. 53 (2), 85–90.
Nishikawa, T., Asai, M., Isobe, M., 2002a. Asymmetric total synthesis of 11-
deoxytetrodotoxin, a naturally occurring congener. J. Am. Chem. Soc. 124,
7847–7852.
Nishikawa, T., Urabe, D., Yoshida, K., Iwabuchi, T., Asai, M., Isobe, M., 2002b.
Stereocontrolled synthesis of 8, 11-dideoxytetrodotoxin, unnatural analogue
of puffer fish toxin. Org. Lett. 4, 2679–2682.
Nishikawa, T., Urabe, D., Yoshida, K., Iwabuchi, T., Asai, M., Isobe, M., 2003. Total
syntheses of 11-deoxytetrodotoxin and 8, 11-dideoxytetrodotoxin. Pure Appl.
Chem. 75, 251–257.
Nishikawa, T., Urabe, D., Yoshida, K., Iwabuchi, T., Asai, M., Isobe, M., 2004. Stere-
ocontrolled synthesis of 8, 11-dideoxytetrodotoxin, an unnatural analogue of
puffer fish toxin. Chem. Eur. J. 10, 452–462.
Noguchi, T., Arakawa, O., Takatani, T., 2006. TTX accumulation in pufferfish—
Review. Comp. Biochem. Physiol. D 1, 145–152.
Noguchi, T., Ebesu, J.S.M., 2001. Puffer poisoning: Epidemiology and treatment.
J. Toxicol. 20, 1–10.
Ohyabu, N., Nishikawa, T., Isobe, M., 2003. First asymmetric total synthesis of
tetrodotoxin. J. Am. Chem. Soc. 125, 8798–8805.
10
Regional Studies in Marine Science 64 (2023) 103036
Omar, A.M.S., 2013. Histopathological and physiological effects of liver and
kidney in rats exposed to cadmium and ethanol. Glob. Adv. Res. J. Environ.
Sci. Toxicol. 2 (3), 93–106.
Poloczanska, E.S., Burrows, M.T., Brown, C.J., Molinos, J.G., Halpern, B.S., Hoegh-
Guldberg, O., Kappel, C.V., Moore, P.J., Richardson, A.J., Schoeman, D.S.,
Sydeman, W.J., 2016. Responses of marine organisms to climate change
across oceans. Front. Mar. Sci. 3 (62), http://dx.doi.org/10.3389/fmars.2016.
00062.
Qun, Y., Yu, P.H.F., Zhong, L.H., 2005. Detection of tetrodotoxin and bacte-
rial production by Serratiamarcescens. World J. Microbiol. Biotechnol. 21,
1255–1258.
Sabrah, M.M., El-Ganainy, A.A., Zaky, M.A., 2006. Biology and toxicity of the
puffer fish Lagocephalussceleratus (Gmelin, 1789) from the Gulf of Suez.
Egypt. J. Aquat. Res. 32 (1), 283–297.
Saha, P., Thomas, S., Salian, T.S., Dineshbabu, A.P., Rohit, P., Nataraja, G.D.,
2019. Fishery and GIS based spatio-temporal distribution analysis of smooth
blaasopLagocephalusinermis, in south-eastern Arabian Sea. Turk. J. Fish. Aquat.
Sci. 20 (4), 267–278.
Saoudi, M., Rabeh, F.B., Jammoussi, K., Abdelmouleh, A., Belbahri, L., Feki, A.E.,
2007. Biochemical and physiological responses in Wistar rat after adminis-
tration of puffer fish (Lagocephaluslagocephalus) flesh. J. Food Agric. Environ.
5, 107–111.
Slaoui, M., Fiette, L., 2011. Histopathological procedures: from tissue sampling
to histopathological evaluation. Methods Mol. Biol. 691, 69–82. http://dx.doi.
org/10.1007/978-1-60761-849-2_4.
Suehiro, M., 1994. Historical review on chemical and medical studies of globefish
toxin before World War II. Jpn. Soc. Hist. Pharm. 29, 428–434.
Tamele, I.J., Silva, M., Vasconcelos, V., 2019. The incidence of tetrodotoxin and
its analogs in the Indian Ocean and the Red Sea. Mar. Drugs 17 (28),
http://dx.doi.org/10.3390/md17010028.
Thomas, S., Kemparaju, S., Sampath Kumar, G., 2009. Puffer fish Lagocephalusin-
ermis, an emerging fishery along Mangalore coast of Karnataka. Mar. Fish.
Infor. Serv. T & E Ser. 200, 23–24.
Yan, Q., Yu, P.H.-F., Li, H.-Z., 2005. Detection of tetrodotoxin and bacte-
rial production by Serratiamarcescens. World J. Microbiol. Biotechnol. 21,
1255–1258.
Yu, C.F., 2003. A Comprehensive Study of the Hong Kong Puffer Fishes and their
Toxins (Ph.D. Dissertation). Hong Kong Polytechnic University, Hong Kong.