Effects of elevated temperature on osmoregulation and stress responses in Atlantic salmon Salmo salar smolts in fresh water and seawater.

Smolting in Atlantic salmon Salmo salar is a critical life-history stage that is preparatory for downstream migration and entry to seawater that is regulated by abiotic variables including photoperiod and temperature. The present study was undertaken to determine the interaction of temperature and salinity on salinity tolerance, gill osmoregulatory proteins and cellular and endocrine stress in S. salar smolts. Fish were exposed to rapid changes in temperature (from 14 to 17, 20 and 24°C) in fresh water (FW) and seawater (SW), with and without prior acclimation and sampled after 2 and 8 days. Fish exposed simultaneously to SW and 24°C experienced 100% mortality, whereas no mortality occurred in any of the other groups. The highest temperature also resulted in poor ion regulation in SW with or without prior SW acclimation, whereas no substantial effect was observed in FW. Gill Na+ -K+ -ATPase (NKA) activity increased in SW fish compared to FW fish and decreased with high temperature in both FW and SW. Gill Nkaα1a abundance was high in FW and Nkaα1b and Na+ -K+ -2Cl- cotransporter high in SW, but all three were lower at the highest temperature. Gill Hsp70 levels were elevated in FW and SW at the highest temperature and increased with increasing temperature 2 days following direct transfer to SW. Plasma cortisol levels were elevated in SW at the highest temperature. Our results indicate that there is an important interaction of salinity and elevated temperature on osmoregulatory performance and the cellular stress response in S. salar, with an apparent threshold for osmoregulatory failure in SW above 20°C.

Evaluation of the microscopic distribution of florfenicol in feed pellets for salmon by Fourier Transform infrared imaging and multivariate analysis.

Fourier Transform infrared imaging and multivariate analysis were used to identify, at the microscopic level, the presence of florfenicol (FF), a heavily-used antibiotic in the salmon industry, supplied to fishes in feed pellets for the treatment of salmonid rickettsial septicemia (SRS). The FF distribution was evaluated using Principal Component Analysis (PCA) and Augmented Multivariate Curve Resolution with Alternating Least Squares (augmented MCR-ALS) on the spectra obtained from images with pixel sizes of 6.25 µm × 6.25 µm and 1.56 µm × 1.56 µm, in different zones of feed pellets. Since the concentration of the drug was 3.44 mg FF/g pellet, this is the first report showing the powerful ability of the used of spectroscopic techniques and multivariate analysis, especially the augmented MCR-ALS, to describe the FF distribution in both the surface and inner parts of feed pellets at low concentration, in a complex matrix and at the microscopic level. The results allow monitoring the incorporation of the drug into the feed pellets.

Prevalence, Variability and Bioconcentration of Saxitoxin-Group in Different Marine Species Present in the Food Chain.

The saxitoxin-group (STX-group) corresponds to toxic metabolites produced by cyanobacteria and dinoflagellates of the genera Alexandrium, Gymnodinium, and Pyrodinium. Over the last decade, it has been possible to extrapolate the areas contaminated with the STX-group worldwide, including Chile, a phenomenon that has affected ≈35% of the Southern Pacific coast territory, generating a high economic impact. The objective of this research was to study the toxicity of the STX-group in all aquatic organisms (bivalves, algae, echinoderms, crustaceans, tunicates, cephalopods, gastropods, and fish) present in areas with a variable presence of harmful algal blooms (HABs). Then, the toxic profiles of each species and dose of STX equivalents ingested by a 60 kg person from 400 g of shellfish were determined to establish the health risk assessment. The toxins with the highest prevalence detected were gonyautoxin-4/1 (GTX4/GTX1), gonyautoxin-3/2 (GTX3/GTX2), neosaxitoxin (neoSTX), decarbamoylsaxitoxin (dcSTX), and saxitoxin (STX), with average concentrations of 400, 2800, 280, 200, and 2000 µg kg-1 respectively, a species-specific variability, dependent on the evaluated tissue, which demonstrates the biotransformation of the analogues in the trophic transfer with a predominance of α-epimers in all toxic profiles. The identification in multiple vectors, as well as in unregulated species, suggests that a risk assessment and risk management update are required; also, chemical and specific analyses for the detection of all analogues associated with the STX-group need to be established.

Determination of Oxytetracycline from Salmon Muscle and Skin by Derivative Spectrophotometry.

A method was developed for the identification and quantification of oxytetracycline residues present in salmon muscle and skin using UV-Vis derivative spectrophotometry. With this method, it was possible to reduce the number of steps in the procedure typically required for instrumental analysis of a sample. The spectral variables, order of the derivative, scale factor, smoothing factor, and analytical wavelength were optimized using standard solutions of oxytetracycline dissolved in 900 mg/L oxalic acid in methanol. The matrix effect was significant; therefore, quantification for oxytetracycline residues was carried out using drug-free salmon muscle and skin samples fortified with oxytetracycline. The LOD and LOQ were found to be 271 and 903 µg/kg, respectively. The precision and accuracy of the method were validated using drug-free salmon muscle and skin tissues fortified at three different concentrations (8, 16, and 32 mg/kg) on 3 different days. The recoveries at all fortified concentrations were between 90 and 105%, and RSDs in all cases were less than 6.5%. This method can be used to screen out compliant samples and thereby reduce the number of suspect positive samples that will require further confirmatory analysis.

Phosphorus and osmium as elemental tags for the determination of global DNA methylation--a novel application of high performance liquid chromatography inductively coupled plasma mass spectrometry in epigenetic studies.

The hyphenation of high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) is proposed in this work as a novel approach for the evaluation of DNA methylation, defined as the ratio between methylated cytosine and total cytosine bases in DNA. In the first part, reversed phase separation of 5-methyl-2'-deoxycytidine monophosphate (5mdCMP) and four deoxynucleotides with specific ICP-MS detection on (31)P had been explored. In further development, selective labeling of 5-methylcytosine in ssDNA was carried out using potassium osmate (K(2)OsO(4)) in the presence of strong oxidant (K(3)Fe(CN)(6)) and N,N,N',N'-tetramethylethylenediamine (TEMED). The sample was then cleaned up and introduced to size exclusion chromatography-ICP-MS for specific detection at (31)P and (189)Os and for evaluation of the molar ratio between Os and P eluted in DNA molecular mass fraction. The quantification of the two elemental tags was achieved by external calibration with phosphoric acid and Os(VI)-TEMED, respectively. The amount of Os in DNA fraction corresponded to methylated cytosines, while P signal was directly proportional to the total amount of DNA and could be recalculated to the amount of cytosine bases. The two procedures were tested by analyzing salmon testes DNA and a commercial oligonucleotide of known composition. For comparative purposes, these same samples were digested to deoxynucleosides and analyzed by reversed phase HPLC with spectrophotometric detection (DAD) at 280 nm. The results obtained using two procedures based on ICP-MS detection were in good agreement and also in agreement with the results obtained by HPLC-DAD procedure. In conclusion, ICP-MS specific detection at internal or external element tags seems to be an interesting alternative for the evaluation of global DNA in epigenetic studies.

PCBs and PBDEs in wild Chinook salmon (Oncorhynchus tshawytscha) in the Northern Patagonia, Chile.

This paper documents the accumulation and emerging levels of Persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) found in the tissues of the migrating salmon species Oncorhynchus tshawytscha (Chinook salmon) in the Chilean Patagonia area. Even though salmon are not native to the southern hemisphere, reports indicate that Chinook salmon in the last few years have performed natural free-living style cycling, returning to rivers in the southern Patagonia area. Our study seeks to determine the presence and levels of PCB's and PBDE's in wild Chinook salmon in the northern part of the Chilean Patagonia, analyzing their relation with physiological parameters. Fish were sampled at the end of their entire life cycle when they returned to two principal rivers in the Aysen region in southern Chile. A number of fish (12) were sacrificed in situ and muscle samples were taken for PCBs measurements (sum 44 congeners) by gas chromatography electron capture detector (GC-ECD) and a number of PBDEs congeners, by gas chromatography mass spectrometry with negative chemical ionization detection (GC-MS NCI). Observed levels and patterns were characterized by concentrations of these POPs for PCBs ranging between 78 and 25.5 ng g(-1) wet weight and for PBDEs ranging between 272 and 1046 pg g(-1) wet weight, respectively. These ranges are among same levels reported in this same species in the northern hemisphere.