Ciguatoxin-like toxicity distribution in flesh of amberjack (Seriola spp.) and dusky grouper (Epinephelus marginatus).

Ciguatoxins (CTXs) are marine neurotoxins that cause ciguatera poisoning (CP), mainly through the consumption of fish. The distribution of CTXs in fish is known to be unequal. Studies have shown that viscera accumulate more toxins than muscle, but little has been conducted on toxicity distribution in the flesh, which is the main edible part of fish, and the caudal muscle is also most commonly targeted for the monitoring of CTXs in the Canary Islands. At present, whether this sample is representative of the toxicity of an individual is undisclosed. This study aims to assess the distribution of CTXs in fish, considering different muscle samples, the liver, and gonads. To this end, tissues from four amberjacks (Seriola spp.) and four dusky groupers (Epinephelus marginatus), over 16.5 kg and captured in the Canary Islands, were analyzed by neuroblastoma-2a cell-based assay. Flesh samples were collected from the extraocular region (EM), head (HM), and different areas from the fillet (A-D). In the amberjack, the EM was the most toxic muscle (1.510 CTX1B Eq·g-1), followed by far for the caudal section of the fillet (D) (0.906 CTX1B Eq·g-1). In the dusky grouper flesh samples, D and EM showed the highest toxicity (0.279 and 0.273 CTX1B Eq·g-1). In both species, HM was one of the least toxic samples (0.421 and 0.166 CTX1B Eq·g-1). The liver stood out for its high CTX concentration (3.643 and 2.718 CTX1B Eq·g-1), as were the gonads (1.620 and 0.992 CTX1B Eq·g-1). According to these results, the caudal muscle next to the tail is a reliable part for use in determining the toxicity of fish flesh to guarantee its safe consumption. Additionally, the analysis of the liver and gonads could provide further information on doubtful specimens, and be used for CTX monitoring in areas with an unknown prevalence of ciguatera.

Metabolic Hepatic Disorders Caused by Ciguatoxins in Goldfish (Carassius auratus).

Ciguatera poisoning (CP) is a foodborne disease known for centuries; however, little research has been conducted on the effects of ciguatoxins (CTXs) on fish metabolism. The main objective of this study was to assess different hepatic compounds observed in goldfish (Carassius auratus) fed C-CTX1 using nuclear magnetic resonance (NMR)-based metabolomics. Thirteen goldfish were treated with C-CTX1-enriched flesh and sampled on days 1, 8, 15, 29, 36, and 43. On day 43, two individuals, referred to as 'Detox', were isolated until days 102 and 121 to evaluate the possible recovery after returning to a commercial feed. At each sampling, hepatic tissue was weighed to calculate the hepatosomatic index (HSI) and analyzed for the metabolomics study; animals fed toxic flesh showed a higher HSI, even greater in the 'Detox' individuals. Furthermore, altered concentrations of alanine, lactate, taurine, glucose, and glycogen were observed in animals with the toxic diet. These disturbances could be related to an increase in ammonium ion (NH4+) production. An increase in ammonia (NH3) concentration in water was observed in the aquarium where the fish ingested toxic meat compared to the non-toxic aquarium. All these changes may be rationalized by the relationship between CTXs and the glucose-alanine cycle.

Kinetics of the invasion of a non-phagocytic fish cell line, RTG-2 by Yersinia ruckeri serotype O1 biotype 1.

Yersiniosis, caused by the fish pathogen Yersinia ruckeri, is a serious bacterial septicaemia affecting mainly salmonids worldwide. The acute infection may result in high mortality without apparent external disease signs, while the chronic one causes moderate to considerable mortality. Survivors of yersiniosis outbreaks become carriers. Y. ruckeri is able to adhere to, and to invade, phagocytic and non-phagocytic fish cells by using unknown molecular mechanisms. The aim of this study was to describe the kinetics of cell invasion by Y. ruckeri serotype O1 biotype 1 in a fish cell line (RTG-2) originating from rainbow trout gonads. The efficiency of invasion by Y. ruckeri was found to be temperature dependent, having a maximum at 20 °C. The bacterium was able to survive up to 96 h postinfection. The incubation of the cells at 4 °C and the pre-incubation of the bacteria with sugars or heat-inactivated antiserum significantly decreased the efficiency of invasion or even completely prevented the invasion of RTG-2 cells. These findings indicate that Y. ruckeri is capable of adhering to, entering and surviving within non-phagocytic cells, and that the intracellular environment may constitute a suitable niche for this pathogen that can favour the spread of infection and/or the maintenance of a carrier state of fish.

Global PBDE contamination in cetaceans. A critical review.

This review summarizes the most relevant information on PBDEs' occurrence and their impacts in cetaceans at global scale, with special attention on the species with the highest reported levels and therefore the most potentially impacted by the current and continuous release of these substances. This review also emphasizes the anthropogenic and environmental factors that could increase concentrations and associated risks for these species in the next future. High PBDE concentrations above the toxicity threshold and stationary trends have been related to continuous import of PBDE-containing products in cetaceans of Brazil and Australia, where PBDEs have never been produced. Non-decreasing levels documented in cetaceans from the Northwest Pacific Ocean might be linked to the increased e-waste import and ongoing production and use of deca-BDE that is still allowed in China. Moreover, high levels of PBDEs in some endangered species such as beluga whales (Delphinapterus leucas) in St. Lawrence Estuary and Southern Resident killer whales (Orcinus Orca) are influenced by the discharge of contaminated waters deriving from wastewater treatment plants. Climate change related processes such as enhanced long-range transport, re-emissions from secondary sources and shifts in migration habits could lead to greater exposure and accumulation of PBDEs in cetaceans, above all in those species living in the Arctic. In addition, increased rainfall could carry greater amount of contaminants to the marine environment, thereby, enhancing the exposure and accumulation especially for coastal species. Synergic effects of all these factors and ongoing emissions of PBDEs, expected to continue at least until 2050, could increase the degree of exposure and menace for cetacean populations. In this regard, it is necessary to improve current regulations on PBDEs and broader the knowledge about their toxicological effects, in order to assess health risks and support regulatory protection for cetacean species.

Ciguatoxin Detection in Flesh and Liver of Relevant Fish Species from the Canary Islands.

The Canary Islands are a ciguatoxin (CTX) hotspot with an established official monitoring for the detection of CTX in fish flesh from the authorised points of first sale. Fish caught by recreational fishermen are not officially tested and the consumption of toxic viscera or flesh could lead to ciguatera poisoning (CP). The objectives of this study were to determine the presence of CTX-like toxicity in relevant species from this archipelago, compare CTX levels in liver and flesh and examine possible factors involved in their toxicity. Sixty amberjack (Seriola spp.), 27 dusky grouper (Epinephelus marginatus), 11 black moray eels (Muraena helena) and 11 common two-banded seabream (Diplodus vulgaris) were analysed by cell-based assay (CBA) and Caribbean ciguatoxin-1 (C-CTX1) was detected by liquid chromatography mass spectrometry (LC-MS/MS) in all these species. Most of the liver displayed higher CTX levels than flesh and even individuals without detectable CTX in flesh exhibited hepatic toxicity. Black moray eels stand out for the large difference between CTX concentration in both tissues. None of the specimens with non-toxic liver showed toxicity in flesh. This is the first evidence of the presence of C-CTX1 in the common two-banded seabream and the first report of toxicity comparison between liver and muscle from relevant fish species captured in the Canary Islands.

Screening of New Potential Probiotics Strains against Photobacterium damselae Subsp. piscicida for Marine Aquaculture.

On intensive fish farms, 10% of the population dies exclusively from pathogens, and Photobacterium damselae subsp. Piscicida (Ph. damselae subsp. Piscicida), the bacteria causing pasteurellosis in marine aquaculture, is one of the major pathogens involved. The objective of this study was to obtain new probiotic strains against pasteurellosis in order to limit the use of chemotherapy, avoiding the environmental repercussions generated by the abusive use of these products. In this study, 122 strains were isolated from the gills and intestines of different marine fish species and were later evaluated in vitro to demonstrate the production of antagonistic effects, the production of antibacterial substances, adhesion and growth to mucus, resistance to bile and resistance to pH gradients, as well as its harmlessness and the dynamic of expression of immune-related genes by real-time PCR after administration of the potential probiotic in the fish diet. Only 1/122 strains showed excellent results to be considered as a potential probiotic strain and continue its characterization against Ph. damselae subsp. piscicida to determine its protective effect and elucidating in future studies its use as a possible probiotic strain for marine aquaculture.

A Study on the Pathological Effects of Trypanorhyncha Cestodes in Dusky Groupers Epinephelus marginatus from the Canary Islands.

Trypanorhyncha are cestodes commonly infecting marine fish. Numerous studies have detailed the biology of Trypanorhyncha species, but information on the pathological changes produced by these parasites is limited. Dusky groupers are keystone species necessary for the preservation of several marine ecosystems. Considering their vulnerable state of conservation and the efforts being made to culture them, identification of the effects caused by Trypanorhyncha is vital. Here, we aimed to determine the prevalence and pathological changes produced by Trypanorhyncha in dusky groupers from the Canary Islands. The prevalence of trypanorhynch plerocerci was 96%. Grossly, in the abdominal cavity, there were numerous larvae-filled cysts and nodules. These were embedded in abundant fibrosis, producing visceral adhesions. Histologically, affecting the peritoneum, stomach, and intestine there were numerous degenerated encysted plerocerci and extensive deposition of mature connective tissue. These findings indicate that Trypanorhyncha is highly prevalent in adult dusky groupers from the Canary Islands, producing a progressive and chronic response. Furthermore, fish immune system appears to attempt to eliminate the parasites through fibrous encapsulation. Nonetheless, extensive fibrosis may have a detrimental impact on fish health when adjacent cells or tissues are compressed and their functions impaired.

Accumulation of C-CTX1 in Muscle Tissue of Goldfish (Carassius auratus) by Dietary Experience.

Ciguatoxins (CTXs) are produced by dinoflagellates usually present in tropical and subtropical waters. These toxins are bioaccumulated and transformed in fish causing ciguatera fish poisoning (CFP) in humans. Few trials have been performed to understand how CTXs are incorporated into fish. This study developed an experimental model of goldfish (Carassius auratus) fed flesh contaminated with Caribbean ciguatoxin (C-CTX1). Fourteen goldfish were fed 0.014 ng CTX1B (Eq. g-1 of body weight) daily, and control goldfish received non-toxic flesh. CTX presence was determined by a cell-based assay on days 1, 8, 15, 29, 36, 43, and 84. Toxicity was detected in muscle from the second sampling and then seemed to stabilize at ~0.03 ng CTX1B Eq. g-1. After two weeks, all experimental goldfish developed lethargy and loss of brightness, but only two of them displayed erratic swimming and jerking movements near the sixth sampling. One of these fish had its toxic diet replaced by commercial food for 60 more days; the fish showed recovery signs within the first weeks and no CTX activity was detected. These results indicate that C-CTX1 could accumulate in goldfish muscle tissue and produce toxic symptoms, but also remarked on the detoxification and recovery capacity of this species.

Retrospective Study of Fishery Interactions in Stranded Cetaceans, Canary Islands.

Estimating cetacean interactions with fishery activities is challenging. Bycatch and chronic entanglements are responsible for thousands of cetacean deaths per year globally. This study represents the first systematic approach to the postmortem investigation of fishery interactions in stranded cetaceans in the Canary Islands. We retrospectively studied 586 cases necropsied between January 2000 and December 2018. Of the cases with a known cause of death, 7.4% (32/453) were due to fishery interactions, and the Atlantic spotted dolphin (Stenella frontalis) was the most affected species [46.9% (15/32)]. Three types of fishery interactions were recognized by gross findings: bycatch [65.6% (21/32)], chronic entanglements [18.8% (6/32)], and fishermen aggression [15.6% (5/32)]. Among the bycaught cases, we differentiated the dolphins that died because of ingestion of longline hooks [23.8% (5/21)] from those that died because of fishing net entrapments [76.2% (16/21)], including dolphins that presumably died at depth due to peracute underwater entrapment (PUE) [37.5% (6/16)], dolphins that were hauled out alive and suffered additional trauma during handling [43.8% (7/16)], and those that were released alive but became stranded and died because of fishery interactions [18.7% (3/16)]. Gross and histologic findings of animals in each group were presented and compared. The histological approach confirmed gross lesions and excluded other possible causes of death. Cetaceans in good-fair body condition and shallow diving species were significantly more affected by fishery interactions, in agreement with the literature. Low rates of fishery interactions have been described, compared with other regions. However, within the last few years, sightings of entangled live whales, especially the minke whale (Balaenoptera acutorostrata) and Bryde's whale (B. edeni), have increased. This study contributes to further improvement of the evaluation of different types of fishery interactions and may facilitate the enforcement of future conservation policies to preserve cetacean populations in the Canary Islands.

Presence of CTXs in moray eels and dusky groupers in the marine environment of the Canary Islands.

Local population frequently consumes moray eels and dusky groupers from the Canary Islands. These species are top predators and the interactions between them include predation but also, in some cases, collaborative hunting. These fish are well known to cause ciguatera (CFP) outbreaks in several marine areas such as Japan, Hawaii, French Polynesia and Caribe. Groupers have been involved in CFP events in the Canary Islands, however, moray eels have not yet been well studied in this regard. The present research seeks to describe the finding of a black moray in the stomach of a positive dusky grouper during its necropsy, and to clarify the implication of groupers and moray eels in the food webs, accumulating CTXs in the Canarian environment. The study also updates statistics on the presence of toxic groupers in this archipelago. For these purposes, 248 grouper samples from the CFP official control in the Canary Islands (2018-2019) were analysed and 36 moray eels (5 species) were collected under the EuroCigua project and one was obtained during a dusky grouper necropsy. All samples were analysed with the Neuro-2a cell-based assay (CBA) to evidence CTX-like toxicity. Regarding the necropsied grouper and the moray eel found in its stomach content, the LCMS/MS method allowed the identification and quantification of CCTX1 in both fish at similar levels while none of the P-CTXs for which standards were available were detected. Among groupers, 25.4 % displayed CTX-like toxicity with differences between islands. For moray eels 38.9 % showed toxicity, involving 4 species. Black moray exhibited a high proportion of positives (9/12) and a positive correlation was found between CTX-like toxicity quantification and the black moray weight. Regarding the grouper, and the moray eel found in its stomach, the LCMS/MS method allowed the identification and quantification of C-CTX1 in both fish at similar levels. This found suggests a trophic interaction between these species and their role in maintaining CTXs in the Canary waters where local population commonly demand those species for consumption. The island of El Hierro stands out above all the other Canary Islands with the concerning percentage of positive grouper samples and the high CTX toxicity levels obtained in moray eel specimens analysed in this marine area. This is the first report of CTX-like toxicity in flesh of moray eels fished in the Canary archipelago and the confirmation of the presence of C-CTX1 by LCMS/MS in a black moray from this marine area.

Predictive score and probability of CTX-like toxicity in fish samples from the official control of ciguatera in the Canary Islands.

This research identifies factors associated with the contamination by ciguatoxins (CTXs) in a population of fish and proposes a predictive score of the presence of CTX-like toxicity in amberjack samples from the official control program of ciguatera in the Canary Islands of the Directorate-General (DG) Fisheries (Canary Government). Out of the 970 samples of fish studied, 177 (18.2%) samples showed CTX-like toxicity. The fish were classified according to the species, amberjack (Seriola dumerili and S. rivoliana) (n = 793), dusky grouper (Epinephelus marginatus) (n = 145) and wahoo (Acanthocybium solandri) (n = 32). The data were separated by species category and statistically examined, resulting in 137 (17.3%) amberjack and 39 (26.9%) grouper samples showing CTX-like toxicity; regarding wahoo species, only 1 toxic sample (3.1%) was found. According to fishing location the contamination rates suggested grouping the islands in four clusters; namely: {El Hierro: HI; La Gomera: LG; La Palma: LP}, {Gran Canaria: GC; Tenerife: TF}, {Fuerteventura: FU} and {Lanzarote: LZ}. For the amberjack species, the multivariate logistic regression showed the factors that maintained independent association with the outcome, which were the warm season (OR = 3.617; 95% CI = 1.249-10.474), the weight (per kg, 1.102; 95% CI = 1.069-1.136) and the island of fish catching. A prediction score was obtained for the probability of contamination by CTX in amberjack fish samples. The area under de curve (AUC) obtained using the validation data was 0.747 (95% CI = 0.662-0.833). Regarding grouper species, the island of fishing was the only factor that showed significant differences associated with the presence of CTX-like toxicity. We provide herein data for a better management and prediction of ciguatera in the Canary Islands, suggesting a review of the minimum limits of fish weight established by the Canary Government for the control program.

Mercury and selenium status of bottlenose dolphins (Tursiops truncatus): A study in stranded animals on the Canary Islands.

The mercury (Hg) level in the marine environment has tripled in recent decades, becoming a great concern because of its high toxic potential. This study reports Hg and selenium (Se) status, and the first Se/Hg molar ratio assessment in bottlenose dolphins (Tursiops truncatus) inhabiting the waters of the Canary Islands. Total Hg and Se concentrations were determined in the blubber and liver collected from 30 specimens stranded along the coasts of the archipelago from 1997 to 2013. The median values for total Hg in the blubber and liver were 80.83 and 223.77 µg g(-1) dry weight (dw), and the median levels for Se in both tissues were 7.29 and 68.63 µg g(-1) dw, respectively. Hg concentrations in the liver were lower than 100 µg g(-1) wet weight (ww), comparable to those obtained in bottlenose dolphins from the North Sea, the Western Atlantic Ocean and several locations in the Pacific Ocean. The Mediterranean Sea and South of Australia are the most contaminated areas for both elements in this cetacean species. In addition, it must be stressed that the levels of Hg and Se in the liver showed an increasing trend with the age of the animals. As expected, a strong positive correlation between Hg and Se was observed (rs=0.960). Surprisingly, both younger and older specimens had a Se/Hg molar ratio different from 1, suggesting that these individuals may be at greater toxicological risk for high concentrations of both elements or a deficiency of Se without a protective action against Hg toxicity.

Plasma levels of pollutants are much higher in loggerhead turtle populations from the Adriatic Sea than in those from open waters (Eastern Atlantic Ocean).

In this paper we determined the levels of 63 environmental contaminants, including organic (PCBs, organochlorine pesticides, and PAHs) and inorganic (As, Cd, Cu, Pb, Hg and Zn) compounds in the blood of loggerhead turtles (Caretta caretta) from two comparable populations that inhabit distinct geographic areas: the Adriatic Sea (Mediterranean basin) and the Canary Islands (Eastern Atlantic Ocean). All animals were sampled at the end of a period of rehabilitation in centers of wildlife recovery, before being released back into the wild, so they can be considered to be in good health condition. The dual purpose of this paper is to provide reliable data on the current levels of contamination of this species in these geographic areas, and secondly to compare the results of both populations, as it has been reported that marine biota inhabiting the Mediterranean basin is exposed to much higher pollution levels than that which inhabit in other areas of the planet. According to our results it is found that current levels of contamination by organic compounds are considerably higher in Adriatic turtles than in the Atlantic ones (∑PCBs, 28.45 vs. 1.12ng/ml; ∑OCPs, 1.63 vs. 0.19ng/ml; ∑PAHs, 13.39 vs. 4.91ng/ml; p<0.001 in all cases). This is the first time that levels of PAHs are reported in the Adriatic loggerheads. With respect to inorganic contaminants, although the differences were not as great, the Adriatic turtles appear to have higher levels of some of the most toxic elements such as mercury (5.74 vs. 7.59µg/ml, p<0.01). The results of this study confirm that the concentrations are larger in turtles from the Mediterranean, probably related to the high degree of anthropogenic pressure in this basin, and thus they are more likely to suffer adverse effects related to contaminants.

Levels and profiles of POPs (organochlorine pesticides, PCBs, and PAHs) in free-ranging common bottlenose dolphins of the Canary Islands, Spain.

The effect of anthropogenic pollution in marine mammals worldwide has become an important issue due to the high concentrations found in many areas. The present study represents the first report of pollutants in free-ranging cetaceans from the Canary Islands, where there are 12 marine Special Areas of Conservation (SACs), because of the presence of bottlenose dolphins (Tursiops truncatus). We selected this resident population of dolphins as a bioindicator to gain knowledge concerning the toxicological status of the cetaceans of this protected area. In 64 biopsy samples of live free-ranging animals sampled from 2003 to 2011, we determined the concentrations of 18 polychlorinated biphenyls (PCBs), 23 organochlorine pesticides (OCPs) and 16 polycyclic aromatic hydrocarbons (PAHs). We found high levels of many of these pollutants, and some of them were detectable in 100% of the samples. The median value for ∑OCPs was 57,104 ng g(-1) lipid weight (lw), and the dichlorodiphenyldichloroethylene (p,p'-DDE) accounted for 70% of this amount. Among PCBs, congeners 180, 153 and 138 were predominant (82% of ∑PCBs; median = 30,783 ng g(-1) lw). Concerning the analyzed PAHs, the total median burden was 13,598 ng g(-1) lw, and phenanthrene was the compound measured at the highest concentration followed by pyrene and by naphthalene. Surprisingly, we have found that organohalogen pollutants exhibit an upward trend in recent years of sampling. Thus, according to the guidelines outlined in the EU's Marine Strategy Framework Directive, further monitoring studies in Canary Islands are required to contribute to the conservation of the resident populations of marine mammals in this region.

Assessment of the levels of polycyclic aromatic hydrocarbons and organochlorine contaminants in bottlenose dolphins (Tursiops truncatus) from the Eastern Atlantic Ocean.

The concentrations of 18 polychlorinated biphenyls (PCBs), 23 organochlorine pesticides (OCPs), and 16 polycyclic aromatic hydrocarbons (PAHs) were determined in the blubber and liver of 27 bottlenose dolphins (Tursiops truncatus) stranded along the Canary Islands coasts from 1997 to 2011. DDTs (mean of 60,960 and 445 ng/g lw., respectively) and PCBs (mean of 47,168 and 628 ng/g lw., respectively) were the predominant compounds in both tissues. Among PCBs the highly chlorinated PCB 180, 153 and 138 were the predominant congeners. We found a p,p'-DDE/∑DDTs ratio of 0.87 in blubber and 0.88 in liver, which is indicative of DDT ageing. All the samples showed detectable values of any of the 16 PAH studied. Phenanthrene was the most frequently detected and at the highest concentration. According to our results, concentrations of OCPs, and especially PCBs, are still at toxicologically relevant levels in blubber of bottlenose dolphins of this geographical area.

Fatal systemic morbillivirus infection in bottlenose dolphin, canary islands, Spain.

A systemic morbillivirus infection was diagnosed postmortem in a juvenile bottlenose dolphin stranded in the eastern North Atlantic Ocean in 2005. Sequence analysis of a conserved fragment of the morbillivirus phosphoprotein gene indicated that the virus is closely related to dolphin morbillivirus recently reported in striped dolphins in the Mediterranean Sea.