High Lipid Content of Prey Fish and n-3 PUFA Peroxidation Impair the Thiamine Status of Feeding-Migrating Atlantic Salmon (Salmo salar) and Is Reflected in Hepatic Biochemical Indices.

Signs of impaired thiamine (vitamin B1) status in feeding-migrating Atlantic salmon (Salmo salar) were studied in three Baltic Sea areas, which differ in the proportion and nutritional composition of prey fish sprat (Sprattus sprattus) and herring (Clupea harengus). The concentration of n-3 polyunsaturated fatty acids (n-3 PUFAs) increased in salmon with dietary lipids and n-3 PUFAs, and the hepatic peroxidation product malondialdehyde (MDA) concentration increased exponentially with increasing n-3 PUFA and docosahexaenoic acid (DHA, 22:6n-3) concentration, whereas hepatic total thiamine concentration, a sensitive indicator of thiamine status, decreased with the increase in both body lipid and n-3 PUFA or DHA concentration. The hepatic glucose 6-phosphate dehydrogenase activity was suppressed by high dietary lipids. In salmon muscle and in prey fish, the proportion of thiamine pyrophosphate increased, and that of free thiamine decreased, with increasing body lipid content or PUFAs, or merely DHA. The thiamine status of salmon was impaired mainly due to the peroxidation of n-3 PUFAs, whereas lipids as a source of metabolic energy had less effect. Organochlorines or general oxidative stress did not affect the thiamine status. The amount of lipids, and, specifically, their long-chain n-3 PUFAs, are thus responsible for generating thiamine deficiency, and not a prey fish species per se.

Distribution of perfluoroalkyl acids in fish species from the Baltic Sea and freshwaters in Finland.

Occurrence and distribution of perfluoroalkyl acids (PFAAs), a sub-category of per- and polyfluoroalkyl substances (PFASs), is widespread in the environment. Food, especially fish meat, is a major pathway via which humans are exposed to PFAAs. As fish is an integral part of Nordic diet, therefore, in this study, several fish species, caught in selected Baltic Sea basins and freshwater bodies of Finland, were analysed for PFAAs. Perfluorooctane sulfonate (PFOS) was detected in all Baltic Sea fish samples and in >80% fish samples from freshwaters. PFOS contributed between 46 and 100% to the total PFAA concentration in Baltic Sea fish samples and between 19 and 28% in fish samples from freshwaters. Geographically, concentration ratios of PFOS to other PFAAs differed between fish from the Baltic Sea and Finnish lakes suggesting that distribution of PFAAs differ in these environments. Results were compared with current safety thresholds - environmental quality standard for biota (EQSbiota) set by the European Commission and a group tolerable weekly intake (TWI) for the sum of four PFASs (∑PFAS-4) i.e. perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexane sulfonate (PFHxS) and PFOS, recommended by the European Food Authority (EFSA). EQSbiota compliance was observed for PFOS in all species except smelt caught in the Baltic Sea and also in the River Aurajoki, where smelt had migrated from the Baltic Sea for spawning. Moderate consumption of most Baltic fishes (200 g week-1) results in an exceedance of the new TWI (4.4 ng kg-1 body weight week-1) for ∑PFAS-4.

How to preserve and handle fish liver samples to conserve RNA integrity.

As transcriptomic studies are becoming more and more common, it is important to ensure that the RNA used in the analyses is of good quality. The RNA integrity may be compromised by storage temperature or freeze-thaw cycles, but these have not been well studied in poikilothermic fishes. This work studied the effects of tissue storage time and temperature, and freeze-thaw cycles of tissue and extracted RNA on RNA integrity in brown trout (Salmo trutta L.) liver. The storage time and temperature had an effect on RNA integrity, but RNA suitable for quantitative reverse transcription PCR (RT-qPCR) (RIN > 7) was still obtained from samples preserved at - 20 °C for 6 months. Freeze-thaw cycles of tissue or RNA did not compromise the integrity of RNA. RNA degradation had an effect on RT-qPCR results, and the effect depended on gene. The RT-qPCR analysis of historical samples from a bleached kraft pulp mill effluent exposure in 1984 revealed no significant cyp1a induction. Recommendations are given for the preservation and handling procedures of samples designated for transcriptomic analyses.

Fatty acid signatures connect thiamine deficiency with the diet of the Atlantic salmon (Salmo salar) feeding in the Baltic Sea.

Thiamine (vitamin B1) deficiency in salmonids related to a lipid-rich fish diet causes offspring mortality in the yolk-sac fry phase. A low free thiamine (THIAM) concentration in eggs is an indication of this syndrome. Thiamine deficiency of salmon (Salmo salar) feeding in the Baltic Sea, called M74, was connected to the principal prey fish and feeding area using fatty acid (FA) signature analysis. The FAs of feeding salmon from two areas of the Baltic Sea, the Baltic Proper (57°10' 19°30') and the Bothnian Sea (61°30' 20°00') in 2004, reflected the principal prey species in these areas, sprat (Sprattus sprattus) and herring (Clupea harengus), respectively. Arachidonic acid (ARA, 20:4n-6) and 18:1n-7 indicated dietary herring, 18:1n-9 dietary sprat and 14:0 feeding in the Baltic Proper. The muscle FA profile of non-M74 female spawners of the River Simojoki in a year (1998) with a moderate M74 incidence and salmon of a non-M74 year (2004) reflected herring FAs, whereas the FAs in the M74 year and specifically in M74 females displayed characteristics of sprat. In the M74 year, the THIAM concentration had the strongest positive correlation with the proportion of muscle ARA, and the strongest negative correlations with 14:0 and the ratios 18:1n-9/ARA and 14:0/ARA. Thus, ARA along with 14:0 and these ratios were the most sensitive FA indicators of the dietary species and origin of the M74 syndrome. Despite the pre-spawning fasting, tissue FA signatures were consequently able to connect dietary sprat in the Baltic Proper with thiamine deficiency in Baltic salmon.

Oxygen and carbon isoscapes for the Baltic Sea: Testing their applicability in fish migration studies.

Conventional tags applied to individuals have been used to investigate animal movement, but these methods require tagged individuals be recaptured. Maps of regional isotopic variability known as "isoscapes" offer potential for various applications in migration research without tagging wherein isotope values of tissues are compared to environmental isotope values. In this study, we present the spatial variability in oxygen (δ18OH2O) and dissolved inorganic carbon (δ13 CDIC) isotope values of Baltic Sea water. We also provide an example of how these isoscapes can reveal locations of individual animal via spatial probability surface maps, using the high-resolution salmon otolith isotope data from salmon during their sea-feeding phase in the Baltic Sea. A clear latitudinal and vertical gradient was found for both δ18OH2O and δ13 CDIC values. The difference between summer and winter in the Baltic Sea δ18OH2O values was only slight, whereas δ13 CDIC values exhibited substantial seasonal variability related to algal productivity. Salmon otolith δ18Ooto and δ13Coto values showed clear differences between feeding areas and seasons. Our example demonstrates that dual isotope approach offers great potential for estimating probable fish habitats once issues in model parameterization have been resolved.

Organohalogen concentrations and feeding status in Atlantic salmon (Salmo salar L.) of the Baltic Sea during the spawning run.

Changes in the concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in Baltic salmon muscle were studied during the spawning migration from the southern Baltic Sea to rivers flowing into the northern Gulf of Bothnia and during the spawning period. The aim was to obtain information to facilitate the arrangement of salmon fisheries such that the human dioxin intake is taken into account. The EC maximum allowable total toxic equivalent concentration (WHO-TEQPCDD/F+PCB) was exceeded in the muscle of the majority of the migrating salmon, except in the Baltic Proper. The fresh-weight-based concentrations of all toxicant groups in salmon tended to be the lowest in the Baltic Proper and the Northern Quark, and all toxicant concentrations, except PCDDs and PCDFs, were significantly higher in the spawning salmon than in the salmon caught during the spawning run. The fat content of the salmon muscle decreased by 60% during the spawning run, and the lipid-based total toxicant concentrations were consequently 4.2-6.2 times higher during the spawning period than during the spawning migration. However, the toxicants were concentrated just before spawning, and thus there is no essential difference related to whether the salmon are caught in the sea or the recreational river fishery.

Biomagnification of organohalogens in Atlantic salmon (Salmo salar) from its main prey species in three areas of the Baltic Sea.

Factors affecting the biomagnification of organohalogens in Baltic salmon from sprat, herring and three-spined stickleback were assessed in three feeding areas. Second sea-year salmon contained (in fresh weight of whole fish) 79-250ngg(-1) polychlorinated biphenyls (ΣPCB), 0.9-2.7pgg(-1) dibenzo-p-dioxins (ΣPCDD), 8-19pgg(-1) dibenzofurans (ΣPCDF), 96-246pgg(-1) coplanar PCBs, 2.4-3.6ngg(-1) polybrominated diphenylethers (ΣPBDE), and 39-136ngg(-1) Σ(indicator) PCB6. The EU limits for WHO toxic equivalent concentrations in fish feed were already exceeded in one-year-old sprat and herring and were exceeded many-fold in older age groups. The differences in the biomagnification rates of organohalogens in salmon appeared to be related to the feeding area, principal prey species, and the fat content and growth rate of the prey species.

Accumulation and effects of nodularin from a single and repeated oral doses of cyanobacterium Nodularia spumigena on flounder (Platichthys flesus L.).

Nodularin (NODLN) is a cyclic pentapeptide hepatotoxin produced by the cyanobacterium Nodularia spumigena, which occurs regularly in the Baltic Sea during the summer season. In this study flounder (Platichthys flesus L.) was orally exposed to NODLN either as a single dose or as three repeated doses 3 days apart. Liver and bile samples of the fish were taken 4 days after the last dose. Liver glutathione-S-transferase (GST) activity was also measured and the histopathology of the liver was investigated. The liver of the exposed fish was analyzed by liquid chromatography-mass spectrometry for NODLN concentration. The content of NODLN-like compounds in the bile was analyzed by enzyme-linked immunosorbent assay. NODLN exposure caused slightly incoherent liver architecture and degenerative cell changes in both groups. The mean liver GST activity was significantly higher in the repeatedly dosed flounders than in the singly dosed flounders or in the control. In conclusion, the significantly lower NODLN concentration and the increased GST activity in the liver of the repeatedly dosed flounders compared to the singly dosed flounders suggest that NODLN is rapidly detoxificated. The absence of NODLN glutathione conjugates and the low concentrations of NODLN-like compounds in the bile indicate that detoxification products disintegrate or they are rapidly excreted.

Use of biliary PAH metabolites as a biomarker of pollution in fish from the Baltic Sea.

During field campaigns of the BEEP project (Biological Effects of Environmental Pollution in Marine Coastal Ecosystems) in 2001-2002, metabolites of polycyclic aromatic hydrocarbons (PAHs) were determined in bile samples from three fish species, flounder (Platichthys flesus), perch (Perca fluviatilis) and eelpout (Zoarces viviparus), from four separate areas in the Baltic Sea. Two determination methods were applied: fixed wavelength fluorescence (FF) for pyrene-type metabolites and high-performance liquid chromatography with fluorescence detection (HPLC). There was a good correlation between the FF method and 1-OH pyrene determined by HPLC. Normalisation of the FF data for absorbance at 380 nm or bile protein concentrations greatly increased variance in one third and decreased it in two thirds of the cases and resulted in a loss of significant differences (protein normalisation) between the sampling stations, but normalisation of the HPLC data had little effect on the results. The biliary PAH metabolite content was usually higher in males than in females. In perch and eelpout the biliary PAH contents were at similar levels, whereas in flounder the levels were lower. The sampling areas arranged in decreasing order of biliary PAH contents were: Wismar Bay > Gulf of Gdansk > Lithuanian coast > Kvadofjärden (reference area). It is concluded that FF with un-normalised data is a reliable and simple method for monitoring purposes and only one sex of a selected species should be used.

The susceptibility of early developmental phases of an acid-tolerant and acid-sensitive fish species to acidity and aluminum.

The mortality of roach (Rutilus rutilus) embryos increased during the early embryonic phase following exposure to Al and acidity from pH 6.7 to 5.0. No roach hatched at pH<5.00 or at pH 5.00 in the presence of Al. By contrast, increased embryonic mortality in pike (Esox lucius) occurred mainly during and after the normal hatching period due to the acidity-dependent prolongation and inhibition of hatching at pH 4.00. Newly hatched fry of both species were more sensitive to Al than the embryos. A reduction in the development of yolk-sac fry occurred in pike at pH 4.00 and in roach at pH 5.00, and in the presence of Al at pH< or =5.00 and pH< or =5.75, respectively. Thus, the most susceptible early developmental phase to acidity differed between the acid-sensitive roach and the acid-tolerant pike, although the newly hatched fry was the developmental phase most sensitive to Al for both species.

Effects of temperature on the recovery of juvenile grayling (Thymallus thymallus) from exposure to Al+Fe.

One-summer-old graylings (Thymallus thymallus) were exposed for 6 days to water containing a mixture of 1 mg l(-1) Fe and 100 microg l(-1) Al at pH 5.5, with or without 15 mg l(-1) humic acid. A pH of 6.9 was used as a control. The experiment was performed at two test temperatures, 13 and 3 degrees C. After 1 week of exposure, half of the fish in the tank were taken for oxygen measurement and tissue sampling. The remaining half were maintained in metal-free water for a further week to assess their recovery at the two test temperatures. Fifty percent of the fish died under metal exposure at 13 degrees C, but none at 3 degrees C. Despite of the lack of mortalities at the lower temperature, surviving fish at both test temperatures suffered similar gill damage (adherence of lamellae), disturbances in ion regulation, increases in haematocrit value (Hct) and haemoglobin (Hb) concentration and reductions in oxygen consumption. The addition of humic acid prevented these changes. The reduced plasma chloride concentration, increased blood Hct value and Hb concentration recovered completely at 13 degrees C, whereas the lowered oxygen consumption recovered less completely, which may be attributed to the remaining minor gill damage (lamellar hypertrophy). At the lower temperature, the adherence of lamellae persisted after the recovery period, and oxygen consumption, therefore, remained at a very low level. The decreased plasma chloride concentration was also unable to recover. The accumulation of Al inside the gill tissue was greater at low temperature. We conclude that the acute toxicity of a mixture of Fe and Al to grayling and their subsequent ability to recovery is dependent on the exposure temperature.

Fertilization and embryonic development of whitefish (Coregonus lavaretus lavaretus) in acidic low-ionic-strength water with aluminum.

The fertilization rate and extent of the cortical reaction of anadromous whitefish decreased with increasing acidity (0.034 mmol Ca(2+)L(-1)); at pH 4.5 the fertilization rate was 46% of that at pH 6.5. Aluminum (250 microg L(-1)) decreased the percentage fertilization independently of pH and reduced the cortical reaction magnitude at all pH levels. In eggs at pH 5.0, especially at pH 4.5, and in solutions with Al, the growth of embryos was impaired by a perivitelline space that was too narrow. The proportion of embryos with a curved tail tip and those with blood circulation failure increased with decreasing pH. No fry hatched at pH 4.5 and those few that hatched at pH 5.0 did not survive. No fry survived Al-containing water at pH 5.5 and survival was decreased at pH 6.5 with Al. It is concluded that for successful recruitment of anadromous whitefish, pH should preferably remain >5.5 in dilute soft water when toxic concentrations of Al are present.

Reproduction, blood and plasma parameters and gill histology of vendace (Coregonus albula L.) in long-term exposure to acidity and aluminum.

Vendace were exposed to pHs 4.75 and 5.25 with or without added aluminum (200 microg=7.4 micromol AlL(-1)) from late endogenous vitellogenesis in July through the spawning period. At the normal time of spawning, when 48% of the control females had already released their eggs, 50% of females at pH 4.75+Al had completely unovulated oocytes. The final proportions of completely ovulated females were 14%, 36%, 25%, 61%, and 81% at pH 4.75+Al, pH 4.75, pH 5.25+Al, pH 5.25, and in the control group, respectively. Delayed testes regression was seen in males at pH 4.75+Al. A clear decrease in plasma Na(+) and Cl(-) and an increase in blood glucose concentration was detected only near spawning time, from October to November, coincident with Al accumulation inside the gill tissue. It is concluded that seasonal changes, probably related to reproductive physiology or to the decrease in water temperature, are associated with the increase in Al toxicity in vendace.

Acute effects and bioaccumulation of nodularin in sea trout (Salmo trutta m. trutta L.) exposed orally to Nodularia spumigena under laboratory conditions.

Nodularin (NODLN) is a cyclic pentapeptide hepatotoxin that is regularly produced in high amounts by the cyanobacterium Nodularia spumigena in the Baltic Sea, and can bioaccumulate in Baltic biota. Baltic sea trout (Salmo trutta m. trutta L.) were exposed orally to a single dose of food containing NODLN (125 mg/kg ww) from N. spumigena (strain AV1, from the Baltic Sea). The level of exposure was 210-620 (average 440) microg NODLN per kg bw. Based on an 8-day survey under laboratory conditions, NODLN-like compounds accumulated in trout liver, with increasing liver concentrations (from 19 microg/kg on day 1 up to 1200 microg/kg on day 8 as measured with the EnviroLogix ELISA kit) during the experiment. Thus, accumulation of NODLN-like compounds in liver increased from 0.05% of the total NODLN dose administered on day 1 to 0.53% on day 8. However, the ELISA test kit is also sensitive to metabolites of algal hepatotoxins. In the HPLC chromatograms, no NODLN peak was detected after 24 h that also suggested NODLN absorbed in trout was metabolized or bound rapidly. According to ELISA, NODLN-like compounds also accumulated in trout muscle in lower quantities (from 125 to 34 microg/kg dw). Histopathology revealed complete loss of liver architecture after 1-2 days of the single oral dose. From day 4 to 8, there was partial recovery of liver cells. NODLN did not affect thiamine levels or water content of trout liver. The results showed that NODLN rapidly induces severe but reversible liver damage. Apparently NODLN accumulated in trout liver from cyanobacteria in the intestine, but was detoxified rapidly. On the basis of discrepancies between the histopathology and ELISA, and on the other hand, between the HPLC and ELISA methods, analysis of NODLN and its metabolites in biological tissue needs to be improved.