Characteristic Distribution of Ciguatoxins in the Edible Parts of a Grouper,Variola louti

Ciguatera fish poisoning (CFP) is one of the most frequently encountered seafood poisoning syndromes; it is caused by the consumption of marine finfish contaminated with ciguatoxins (CTXs). The majority of CFP cases result from eating fish flesh, but a traditional belief exists among people that the head and viscera are more toxic and should be avoided. Unlike the viscera, scientific data to support the legendary high toxicity of the head is scarce. We prepared tissue samples from the fillet, head, and eyes taken from five yellow-edged lyretail (Variola louti) individuals sourced from Okinawa, Japan, and analyzed the CTXs by LC-MS/MS. Three CTXs, namely, CTX1B, 52-epi-54-deoxyCTX1B, and 54-deoxyCTX1B, were confirmed in similar proportions. The toxins were distributed nearly evenly in the flesh, prepared separately from the fillet and head. Within the same individual specimen, the flesh in the fillet and the flesh from the head, tested separately, had the same level and composition of toxins. We, therefore, conclude that flesh samples for LC-MS/MS analysis can be taken from any part of the body. However, the tissue surrounding the eyeball displayed CTX levels two to four times higher than those of the flesh. The present study is the first to provide scientific data demonstrating the high toxicity of the eyes.     

Multiple ciguatoxins in the flesh of fish.

Most cases of ciguatera (fish poisoning) result from consumption of the flesh of fishes contaminated with ciguatoxin(s); however, the relatively low toxicity of ciguateric fish flesh has hindered attempts to identify these ciguatoxin(s). Utilising high performance liquid chromatography, mass spectroscopy and mouse bioassay signs we have determined that ciguatoxin-1 (MH+ m/z = 1112), ciguatoxin-2 and ciguatoxin-3 are the major ciguatoxins present in the flesh of ciguateric fish. Ciguatoxin-1, -2 and -3 were present in yields of 0.19, 0.09 and 0.02 microgram/kg flesh, respectively, in Scomberomorus commersoni; 0.08, 0.09 and 0.07 microgram/kg flesh, respectively, in Plectropomus spp. and; 0.67, 0.61 and 0.06 microgram/kg flesh, respectively, in Pomadasys maculatus. Two minor toxins, which may be further oxidised analogues of ciguatoxin-1 and ciguatoxin-2, were also identified. The presence of multiple ciguatoxins in fish flesh has important consequences for the detection of ciguateric fish and may be a contributing factor to the observed variability in the symptoms of ciguatera.     

First Report of Ciguatoxins in Two Starfish Species: Ophidiaster ophidianus and Marthasterias glacialis

Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk.     

A rapid, simplified enzyme immunoassay stick test for the detection of ciguatoxin and related polyethers from fish tissues

Y. Hokama A rapid, simplified enzyme immunoassay stick test for the detection of ciguatoxin and related polyethers from fish tissues. Toxicon23, 939–946. 1985. — A simplified and rapid enzyme immunoassay stick test is presented in this study. The salient feature of this test is the use of a coating (Liquid Paper) on the stick to adsorb the lipid ciguatoxin and its related polyether toxins onto the stick. This rapid solid phase stick test has been able to differentiate clinically implicated fishes (14 samples) from non-toxic (60 samples) with P < 0.005. Comparison of the slick test with the enzyme immunoassay procedure with the positive fish samples demonstrated a good correlation. All samples were positive by both procedures. Further comparison of the stick test, enzyme immunoassay and the mouse bioassay with unknown fish samples showed good relationships between the three procedures. Examination of 71 Seriola dumerili, a fish generally implicated in ciguatera poisoning, with the stick test procedure showed that 89% of the fishes were non-toxic and thus consumed without any incidence of ciguatera poisoning. Analysis of graded concentrations of ciguatoxin, in ng/ml of methanol, showed a typical immunological precipitation pattern. The stick test as conceived is simple and rapid, while retaining its sensitivity and specificity to detect ng levels of ciguatoxin and its related polyether toxins. It is suggested that the stick test will be valuable in the screening of ciguatoxin and related polyether toxins in contaminated fish tissues.     

Evidence for the Range Expansion of Ciguatera in French Polynesia: A Revisit of the 2009 Mass-Poisoning Outbreak in Rapa Island (Australes Archipelago)

Ciguatera poisoning (CP) results from the consumption of seafood contaminated with ciguatoxins (CTXs). This disease is highly prevalent in French Polynesia with several well-identified hotspots. Rapa Island, the southernmost inhabited island in the country, was reportedly free of CP until 2007. This study describes the integrated approach used to investigate the etiology of a fatal mass-poisoning outbreak that occurred in Rapa in 2009. Symptoms reported in patients were evocative of ciguatera. Several Gambierdiscus field samples collected from benthic assemblages tested positive by the receptor binding assay (RBA). Additionally, the toxicity screening of ≈250 fish by RBA indicated ≈78% of fish could contain CTXs. The presence of CTXs in fish was confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential link between climate change and this range expansion of ciguatera to a subtropical locale of French Polynesia was also examined based on the analysis of temperature time-series data. Results are indicative of a global warming trend in Rapa area. A five-fold reduction in incidence rates was observed between 2009 and 2012, which was due in part to self-regulating behavior among individuals (avoidance of particular fish species and areas). Such observations underscore the prominent role played by community outreach in ciguatera risk management.     

Analysis of Acanthurus triostegus for marine toxins by the stick enzyme immunoassay and mouse bioassay.

The herbivorous convict tang or surgeon fish, Acanthurus triostegus, collected at different locations in the Hawaiian islands, was analyzed for toxicity using a mouse bioassay and a stick enzyme immunoassay (S-EIA) developed to detect ciguatoxin and closely related polyethers. Results of the S-EIA test indicated that about 94% of the samples gave negative readings, while 6% of the fish were positive and thus considered toxic. Fish samples from each location were pooled and the flesh and viscera were successively extracted with hexane, methanol, and water and tested in a mouse bioassay. About one-third of the methanol soluble fractions killed mice within 20 min, 70% killed within 4 hr, and 85% killed within 48 hr. About 40% of the hexane-soluble fractions killed mice within 24 hr and 55% killed within 48 hr. The water-soluble extracts of the flesh and viscera of fish taken from three locations showed relatively high toxicity. Common symptoms for all fractions included convulsions and jumping (especially just prior to death), respiratory distress, hind leg to complete paralysis, loss of body tone, and tremors. Analysis of the data suggests that in the flesh and viscera of A. triostegus there are at least three different nervous system-type toxins, most of which did not appear to react to the S-EIA test.     

Ciguatera Poisoning: Clinical and Immunological Aspects

Abstract

Ciguatera poisoning refers to a type of fish intoxication following consumption of fishes contaminated with ciguatoxin (CTX). The latter toxin originates in a dinoflagellate designated Gambierdiscus toxicus, passes through herbivorous and then to carnivorous fishes along the food chain, ultimately to man. Ciguatera poisoning is unlike tetrodotoxin or scombroid fish poisonings. The Eormer is due to a toxin isolated From puffer fish and the latter to histamine-like degradation products due to bacterial spoilage of fish fissues. Ten minutes Lo 24 hr after consumption of a CTX contaminated fish, clinical symptoms involving the gastroinlestinal, neurological and cardiovascular systems are manifested. The neurological symptoms may persist for munths in some individuals. Ciguatera poisoning is rarely fatal (less than 0.5%). Therapy at best is symptomatic with no specific drugs. Detection in fisti tissues depends on the mouse test Eollowing extraction of the toxin in organic solvents and more recently an immunological approach using specific anti-CTX monoclonal antibody. The stick test is rapid, simple, and specific and requires nu laboratory equipment. It is hoped to adapt the stick test for wide scale use in screening fishes for markets in the endemic areas of the tropical regions.     

The use of mosquitoes (Aedes aegypti) to detect ciguatoxin in surgeon fishes (Ctenochaetus striatus)

A new animal assay to detect ciguatoxin in fishes was developed. Mosquitoes were subjected to intrathoracic injection of serial dilutions of ciguatoxin crude extracts. Toxicity of the fishes was expressed as the mosquito LD50 (g of flesh per mosquito). A significant correlation between the mosquito bioassay and the mouse bioassay performed on the same extracts was shown (r = 0.72, n = 80). We were able to detect 96% of the toxic fishes, while 91% of the fishes non-toxic in mice were also non-toxic in mosquitoes.     

Ciguatera fish poisoning on the West Africa Coast: An emerging risk in the Canary Islands (Spain)

Ciguatera fish poisoning (CFP) is endemic in certain tropical and subtropical regions of the world. CFP had not been described on the West Africa Coast until a 2004 outbreak in the Canary Islands. In 2008–2009, two additional outbreaks of ciguatera occurred. Individuals afflicted had consumed lesser amberjack (Seriola rivoliana) captured from nearby waters. Caribbean ciguatoxin-1 (C-CTX-1) was confirmed in fish samples by LC-MS/MS. Ciguatoxic fish in this region may pose a new health risk for the seafood consumer.     

Analysis of Acanthurus triostegus for marine toxins by the stick enzyme immunoassay and mouse bioassay

The herbivorous convict tang or surgeon fish, Acanthurus triostegus, collected at different locations in the Hawaiian islands, was analyzed for toxicity using a mouse bioassay and a stick enzyme immunoassay (S-EIA) developed to detect ciguatoxin and closely related polyethers. Results of the S-EIA test indicated that about 94% of the samples gave negative readings, while 6% of the fish were positive and thus considered toxic. Fish samples from each location were pooled and the flesh and viscera were successively extracted with hexane, methanol, and water and tested in a mouse bioassay. About one-third of the methanol soluble fractions killed mice within 20 min, 70% killed within 4 hr, and 85% killed within 48 hr. About 40% of the hexane-soluble fractions killed mice within 24 hr and 55% killed within 48 hr. The water-soluble extracts of the flesh and viscera of fish taken from three locations showed relatively high toxicity. Common symptoms for all fractions included convulsions and jumping (especially just prior to death), respiratory distress, hind leg to complete paralysis, loss of body tone, and tremors. Analysis of the data suggests that in the flesh and viscera of A. triostegus there are at least three different nervous system-type toxins, most of which did not appear to react to the S-EIA test.     

Extraction and LC-MS/MS Analysis of Ciguatoxins: A Semi-Targeted Approach Designed for Fish of Unknown Origin

Ciguatoxins (CTXs) are polyether marine biotoxins that can cause ciguatera poisoning (CP) after the consumption of fish or invertebrates containing sub ppb levels; concentrations that present a challenge for current extraction and analysis methods. Here, a newly developed and (partly) validated single-day extraction protocol is presented. First, the fish sample is broken-down by enzymatic digestion, followed by extraction and extract clean-up by defatting and two solid-phase extractions. Final extracts were investigated using two different CTX-analysis methods; an in vitro cytotoxicity assay (N2a-assay) and by LC-MS/MS. Validation was performed for both fillet and freeze-dried samples of snapper, parrotfish, and grouper spiked with CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, and CTX3C. Based on recovery rates (35–88%) and matrix effects (66–116%) determined by LC-MS/MS, the enzyme protocol is applicable to various matrices. The protocol was applied to naturally contaminated fish tissue (Lutjanus bohar) obtained during a CP incident in Germany. Several potential CTX congeners were identified by a two-tier LC-MS/MS approach (screening of sodium adducts, high-resolution or low-resolution confirmation via ammonium adducts). Inclusion of >30 known CTX congeners into the LC-MS/MS methods and single-day sample preparation make the method suitable for analysis of ciguatera suspect samples at sub ppb levels also with undisclosed CTX profiles.     

Epidemiology and Clinical Features of Ciguatera Fish Poisoning in Hong Kong

In the present review, the main objective was to describe the epidemiology and clinical features of ciguatera fish poisoning in Hong Kong. From 1989 to 2008, the annual incidence of ciguatera varied between 3.3 and 64.9 (median 10.2) per million people. The groupers have replaced the snappers as the most important cause of ciguatera. Pacific-ciguatoxins (CTX) are most commonly present in reef fish samples implicated in ciguatera outbreaks. In affected subjects, the gastrointestinal symptoms often subside within days, whereas the neurological symptoms can persist for weeks or even months. Bradycardia and hypotension, which can be life-threatening, are common. Treatment of ciguatera is primarily supportive and symptomatic. Intravenous mannitol (1 g/kg) has also been suggested. To prevent ciguatera outbreaks, the public should be educated to avoid eating large coral reef fishes, especially the CTX-rich parts. A Code of Practice on Import and Sale of Live Marine Fish for Human Consumption for Prevention and Control of Ciguatera Fish Poisoning was introduced from 2004 to 2013. The Food Safety Ordinance with a tracing mechanism came into full effect in February 2012. The Government would be able to trace the sources of the fishes more effectively and take prompt action when dealing with ciguatera incidents.     

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding.

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for > 90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and < 900 Da were suspected to contribute to the overall toxicity.     

Ciguatoxin-Producing Dinoflagellate Gambierdiscus in the Beibu Gulf: First Report of Toxic Gambierdiscus in Chinese Waters

Ciguatera poisoning is mainly caused by the consumption of reef fish that have accumulated ciguatoxins (CTXs) produced by the benthic dinoflagellates Gambierdiscus and Fukuyoa. China has a long history of problems with ciguatera, but research on ciguatera causative organisms is very limited, especially in the Beibu Gulf, where coral reefs have been degraded significantly and CTXs in reef fish have exceeded food safety guidelines. Here, five strains of Gambierdiscus spp. were collected from Weizhou Island, a ciguatera hotspot in the Beibu Gulf, and identified by light and scanning electron microscopy and phylogenetic analyses based on large and small subunit rDNA sequences. Strains showed typical morphological characteristics of Gambierdiscus caribaeus, exhibiting a smooth thecal surface, rectangular-shaped 2′, almost symmetric 4″, and a large and broad posterior intercalary plate. They clustered in the phylogenetic tree with G. caribaeus from other locations. Therefore, these five strains belonged to G. caribaeus, a globally distributed Gambierdiscus species. Toxicity was determined through the mouse neuroblastoma assay and ranged from 0 to 5.40 fg CTX3C eq cell⁻¹. The low level of toxicity of G. caribaeus in Weizhou Island, with CTX-contaminated fish above the regulatory level in the previous study, suggests that the long-term presence of low toxicity G. caribaeus might lead to the bioaccumulation of CTXs in fish, which can reach dangerous CTX levels. Alternatively, other highly-toxic, non-sampled strains could be present in these waters. This is the first report on toxic Gambierdiscus from the Beibu Gulf and Chinese waters and will provide a basis for further research determining effective strategies for ciguatera management in the area.     

Isolation of Prorocentrum lima (Syn. Exuviaella lima) and diarrhetic shellfish poisoning (DSP) risk assessment in the Gulf of California, Mexico.

A benthic toxic dinoflagellate identified as Prorocentrum lima (Syn. Exuviaella lima), and designated as strain PRL-1, was isolated from the coast of El Pardito (Coyote) Island in Baja California Sur, Mexico, after a fisherman poisoning incident involving consumption of liver from Lutjanus colorado, and Mycteroperca prionura fish. Purification and culturing was done in ES-Si medium, under 12:12 light/dark cycle (4 x 20 W cool-white fluorescent lamps), at 22 degrees C and constant stirring during 28 days. Whole cells were toxic to Artemia franciscana and its methanolic extract to mouse and to the marine yeast Debaryomyces hansenii. Chromatographic analysis (TLC and HPLC-MS) of such extract indicated an unusual proportion (1:2) okadaic acid (OA) and dinophysistoxin-1 (DTX-1). Estimated total toxin content by mouse bioassay (based on OA toxicity) was 19 pg/cell, a value significantly higher than that found by HPLC-MS (about 5.2 pg/cell, taking into account OA and DTX-1 only), suggesting that additional toxic components of unidentified nature are detected with the bioassay. This is the first report of a successful isolation and culturing of a toxic dinoflagellate from the Gulf of California, Mexico.     

Detection of paralytic shellfish poisoning (PSP) toxins in shellfish tissue using MIST Alert, a new rapid test, in parallel with the regulatory AOAC mouse bioassay.

In parallel trials with the mouse bioassay, MIST Alert for Paralytic Shellfish Poisoning (PSP), a rapid diagnostic test for PSP, detected 100% of the toxic extracts in over 2100 regulatory samples. Toxic extracts contained at least 80 microg saxitoxin equivalents (STX equiv.) in 100 g of shellfish tissue, or more, as measured by the regulatory AOAC mouse bioassay. Only one potentially toxic sample, which contained 78 and 86 microg STX equiv./100 g shellfish tissue in two different mouse bioassays, was recorded as negative in one replicate of MIST Alert. All other toxic extracts among more than 2100 regulatory shellfish tissue samples were detected by MIST Alert for PSP. The MIST Alert for PSP also detected the majority of extracts containing PSP toxin greater than 32 microg STX equiv./100 g, which is the mouse bioassay detection limit. The MIST Alert for PSP gave a false positive result compared to the mouse bioassay at an average rate of about 14% over all sites, although some differences were seen between sites. Further analysis by high performance liquid chromatography (HPLC) of the (false positive) extracts showed that many contained PSP toxicity in the range of 20-40 microg STX equiv./100 g, below the level detectable by the mouse bioassay. The MIST Alert for PSP gave false positive results from extracts containing less than 20 microg STX equiv./100 g shellfish tissue only about 6% of the time. The PSP family of toxin analogues can occur in any combination in naturally contaminated shellfish tissue and the antibody mixture in the MIST Alert tests detect each of the different PSP toxin analogues with different efficacy. It is therefore impossible to provide an exact detection limit for the MIST Alert that would be applicable for all possible toxin profiles. Through the experience of comparison testing with the regulatory mouse bioassay in many parts of the world, with over 2100 different samples, the MIST Alert for PSP has proven its ability to detect all types of profiles of the PSP toxin analogues. The detection limit for MIST Alert for PSP was about 40 microg STX equiv./100 g for the 'average' profile of PSP toxin analogues. Since the detection limit depends on the toxin profile in the individual extract, it will also vary depending on the profile of analogues most commonly found at each geographic location. This was observed in our study. Over all sites in the trials, approximately 5% of samples below 40 microg STX equiv./100 g were positive, and 5% of samples between 40-80 microg STX equiv./100 g were negative. This is a reflection of the different analogue profiles found in naturally contaminated extracts, even after acid hydrolysis using the AOAC extraction method.     

Human fatality associated with Pacific ciguatoxin contaminated fish

Ciguatera is a food poisoning identified as the principal risk factor in the consumption of tropical fish in Oceania. The syndrome, which follows ingestion of ciguatoxin-contaminated ciguateric fishes, is characterised by an array of gastrointestinal and neurological features. In this report we examine forensic samples associated with a human fatality using a ³H-brevetoxin binding assay and reversed-phase HPLC/MS and HPLC/MS/MS. Three Pacific ciguatoxins (P-CTX) were detected in the implicated fish flesh sample by LC-MS/MS, implicating multiple P-CTXs in the fatal case. Additionally, ciguatoxin was identified in a liver sample obtained at post-mortem. The level of ciguatoxin detected (0.14 ppb P-CTX-1 equivalents by binding assay) indicated that at least 10% of the ingested P-CTX-1 remained in the human liver 6 days after the toxic fish was consumed. This study confirms the potential of tropical reef fish to accumulate sufficient P-CTX to be lethal to humans, especially if the liver and viscera are consumed as part of the meal.     

Establishing a public health analytical service based on chemical methods for detecting and quantifying Pacific ciguatoxin in fish samples

A referee analysis method for the detection and quantification of Pacific ciguatoxins in fish flesh has recently been established by the public health analytical laboratory for the State of Queensland, Australia. Fifty-six fish samples were analysed, which included 10 fillets purchased as negative controls. P-CTX-1 was identified in 27 samples, and P-CTX-2 and P-CTX-3 were found in 26 of those samples. The range of P-CTX-1 concentrations was 0.04-11.4 μg/kg fish flesh; coefficient of variation from 90 replicate analyses was 7.4%. A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method utilising a rapid methanol extraction and clean-up is reliable and reproducible, with the detection limit at 0.03 μg/kg fish flesh. Some matrix effects are evident, with fish oil content a likely signal suppression factor. Species identification of samples by DNA sequence analysis revealed some evidence of fish substitution or inadvertent misidentification, which may have implications for the management and prevention of ciguatera poisoning. Blinded inspection of case notes from suspect ciguatera poisoning cases showed that reporting of ciguatera-related paraesthesias was highly predictable for the presence of ciguatoxins in analysed fish, with 13 of 14 expected cases having consumed fish that contained P-CTX-1 (p < 0.001, Fishers Exact Test).     

Paralytic shellfish poisoning toxin profile of mussels Perna perna from southern Atlantic coasts of Morocco

During the monitoring programme of harmful algal blooms established along the south Atlantic coast of Morocco, a bimonthly determination of harmful algae and phycotoxins analysis in Perna perna was carried out from May 2003 to December 2004. Results of mouse bioassay (in organs and whole flesh) showed a seasonal evolution of paralytic shellfish poisoning (PSP) toxin. The mussel's contamination was associated with the occurrence in water of Alexandrium minutum.The PSP toxin profile obtained with high-performance liquid chromatography (HPLC/FD) revealed the dominance of gonyautoxins GTX2 and GTX3 and a minority of GTX1, GTX4 and saxitoxin (STX). This profile explains that the toxicity was mainly associated with A. minutum.     

Neurotoxicity in rats induced by the poisonous dreamfish (Sarpa salpa)

Abstract

Context: Consumption of Sarpa salpa Linn. (Sparidae) in certain periods of the year is inadvisable because it can cause central nervous system disorders resulting in sea food poisoning.

Aims: The present study assesses the cytotoxic effects of compounds, not-yet identified, present in the organ extracts of S. salpa, collected in autumn, the period corresponding to the peak in human health problems.

Materials and methods: The toxicity was assessed by mouse bioassay of aqueous extract of the fish organs. Wistar rats received daily extracts of different organs of S. salpa by gastric gavage for 7?d (0.3?mL of extract/100?g body weight BW). The dose of tissue extracts of viscera, liver, brain, and flesh of S. salpa administered to rats was as follows: 172, 313, 2050, and 2660?mg/kg BW, respectively. No deaths occurred during the period of treatment.

Results: The lethal dose (LD₅₀) determined for the crude ciguatoxin (neurotoxins) extracts of viscera, liver, brain, and flesh of S. salpa was as follows: 1.2, 2.2, 14.4, and 18.6?g/kg mouse, respectively. Changes in locomotor activity during the first 2?h and failure in breathing and no evident signs of gastrointestinal problems were recorded. We observed (1) induction of oxidative stress, indicated by an increase in lipid peroxidation (TBARS) in groups that received extracts of liver (+425%) or viscera (+433%), and a significant decrease in antioxidant enzyme activities (SOD, CAT, and GPx) in cerebral cortex tissue by 13%, 25%, and 25% (LT: animals receiving liver extracts) and by 16%, 26%, and 27% (VT: animals receiving viscera extracts), respectively. In contrast, the administration of extracts of flesh and brain induced an increase in antioxidant enzyme activities (SOD, CAT, and GPx) in cerebral cortex tissue by 26%, 23%, and 44% (FT: flesh extract) and 28%, 24%, and 46% (BT: brain extract), respectively; (2) a significant decrease for acetylcholinesterase (AChE) activity in cerebral cortex was recorded in FT, BT, LT, and VT by 27, 34, 58, and 78%, respectively. Moreover, a significant decrease of AChE activity in plasma was recorded in FT, BT, LT, and VT by 16, 21, 38, and 48%, respectively; (3) the histological findings confirmed the biochemical results.

Conclusions: Liver and especially the visceral part of S. salpa presented toxicity, which clearly indicates the danger of using this fish as food.