Ivan C. Burkow

Patterns of growth and lipid deposition in cod (Gadus morhua L.) fed natural prey and fish-based feeds

Abstract Groups of cod were fed two natural prey types, prawn and herring, and two herring-based wet diets, one with low and the other with high energy density. Cod fed upon whole herring and enriched-herring paste showed the highest growth rates but tended to store increasing amounts of lipid in the liver. Cod fed upon the leanest diet, prawn, showed, however, better efficiencies of energy and nutrient conversion. The effect of decreasing food particle size, by mincing the herring, was to slightly impair growth and to substantially lower the efficiency of food conversion. The fatty acid compositions of the body lipids tended to reflect dietary input. The major lipid class in the muscle was the phospholipid whereas the main lipid storage depot consisted of triacylglycerols in the liver. There were some indications that either 20:5 n −3 was chain elongated to 22:6 n −3, or was selective utilized. Dietary 22:1 appeared to have been used as an energy source by the cod, a predator that naturally feeds on prey such as herring which are rich in these fatty acids.

Congener specific PCB and polychlorinated camphene (toxaphene) levels in Svalbard ringed seals (Phoca hispida) in relation to sex, age, condition and cytochrome P450 enzyme activity

Congener specific PCB and toxaphene (polychlorinated camphene, PCC) analyses were performed in seal blubber, collected in Svalbard, Norway. The concentration, body burden and metabolic index (PCB congener concentration in seal relative to their prey) were calculated. Multiple regression analyses were carried out to evaluate the influence of age, sex, blubber (as a percentage of total body weight) and cytochrome P450 activities on PCB and PCC levels. Levels of total PCBs found were five times higher than in ringed seals from the Canadian Arctic, corresponding with the relatively high contaminant levels in the European Arctic. The dominant PCB congeners (> 70% of the total PCBs measured) were 153, 138, 99, 180 and 101. The observed PCB and PCC accumulation patterns were very similar to patterns in seals from other studies, suggesting a large resemblance in contaminant metabolism. A decrease in the relative abundance of the lower chlorinated PCBs, was associated with higher concentrations of PCB 153. Since there was no indication for selective PCB excretion by lactating females, this suggests metabolism of these PCBs in ringed seals due to xenobiotic metabolising enzymes. The metabolic index confirmed the model of persistency of the different PCBs except for congener 128 and 138. These congeners, considered persistent in seals, could to some extent be metabolised in ringed seals. However, co-elution of PCB 138 with PCB 163 and of PCB 128 with TOX 50 possibly has resulted in an underestimation of the metabolic index for these congeners. Multiple regression analyses revealed a significant positive effect of age and a negative effect of the blubber content on the PCB concentrations. Since large fluctuations of body lipids occur between seasons in pinnipeds, PCB measurements should account for the total blubber content to avoid biased results. PCBs with vicinal H-atoms in the o, m or the m, p positions showed in addition a relation with cytochrome P450 enzyme activities. Surprisingly, no effect of sex on the PCB concentrations was observed, probably because female ringed seals, unlike other pinnipeds, continue feeding during lactation. This results in only small amounts of lipid and lipid-associated contaminants being mobilised from the blubber. Consequently, contaminant excretion with the milk will be low. Toxaphene concentrations found were low compared to levels found in the Canadian Arctic. Two congeners, TOX 26 and TOX 50 were predominant (15 and 18%, respectively of total toxaphene). There was no effect of sex, age, total blubber, or cytochrome P450 activities on the toxaphene levels. There was also no correlation between toxaphene and PCB levels, which may indicate differences in exposure and metabolism between these contaminants. Toxaphenes did not bioaccumulate to any substantial extent in ringed seals.

Gas chromatographic determination of synthetic musk compounds in Norwegian air samples

A new method is described for the simultaneous analysis of nitro and polycyclic musks in air samples. Additional considerations are given to the aspects of sample preparation and blank problems. For the first time concentration levels of synthetic musks in selected Norwegian air samples were determined. In a preliminary investigation using ambient air samples prepared for the determination of persistent organic pollutants, musk xylene concentrations were between 8 and 19 pg/m3. A new sample preparation for the simultaneous analysis of polycyclic and nitro musks is presented. Both GC-electron impact (EI) MS and GC-negative ion chemical ionisation (NICI) MS were compared as detection methods. GC–EI-MS is to be preferred for the determination of synthetic musks since GC–NICI-MS is not sensitive enough for the determination for polycyclic musks. Blank problems occurred which have to be carefully considered during the whole work-up procedure. One indoor air sample was taken in the laboratory where the clean-up of the samples took place to evaluate laboratory air contamination. A concentration for 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclo[g]-2-benzopyrane of 2470 pg/m3 was found using gas chromatographic separation coupled with mass spectrometric detection in the electron impact mode (GC–low-resolution EI-MS). A set of four ambient air samples taken nearby the institute facilities was analysed for both nitro and polycyclic musks. For polycyclic musks, concentrations up to 130 pg/m3 (GC–EI-MS) were determined and for musk xylene a concentration maximum in ambient air samples 54 pg/m3 (low resolution EI-MS) was found. Critical considerations about laboratory contamination, clean-up and analysis of ambient air samples are given.

Sources and transport of persistent pollutants to the Arctic.

During the last two decades the importance of long-range transport as a contaminant source for the pristine Arctic regions has been proven. Models are developed in order to predict spatial, seasonal and structural dependent patterns in the distribution of antropogenic pollutants. The most accepted theory today describes the transport of semi-volatile persistent organic pollutants as a temperature and weather dependent repeated deposition and remobilization process between the atmosphere and the ocean and land surface, with a final deposition in the cold northern region. Results also indicate possible new local sources of pesticides in the European Arctic in addition to the long-range transport from more temperate areas.

Phase I and phase II enzyme activities in Ringed seals (Phoca hispida): characterization of hepatic cytochrome P450 by activity patterns, inhibition studies, mRNA analyses, and western blotting

Abstract Hepatic phase I and phase II enzymes play an important role in contaminant metabolism in mammals. Knowledge of these enzymes is essential since their presence and activity determines the potential biological effects of contaminant exposure. In this study activities of hepatic phase I enzymes (cytochrome P450 (CYP)) and phase II enzymes (UDP glucuronosyl transferase (UDPGT) and glutathione S -transferase (GST)) in Ringed seals ( Phoca hispida ) were assessed. In addition, CYP enzymes were characterized using catalytic activities, selective inhibitors, mRNA analyses, as well as Western blotting. Both UDPGT and GST activities were present, indicating that these seals may form the reactive methylsulfonated PCB metabolites. The results from the CYP characterization showed ethoxyresorufin- O -deethylation (EROD) and caffeine demethylation activity, while the pentoxyresorufin- O -depenthylation activity was low. The activity towards testosterone resulted in several hydroxy-metabolites. Based on these activity studies the presence of CYP1A, CYP3A, but not CYP2B was insinuated. The inhibition of EROD and caffeine demethylation by α -naphthoflavone but not by furafylline suggested that in this seal species only one CYP1A enzyme was present. This was supported by the results from the mRNA measurements and Western blots. Only one mRNA band cross-hybridized with human CYP1A cDNA probes at the rat CYP1A1 position, while also one protein band, cross reacting with anti-rat CYP1A, was detected. The selective inhibition of the formation of the testosterone 2 β - and 6 β -hydroxy metabolites by ketoconazole supported the suggestion that the formation of these metabolites was mediated by CYP3A. The mRNA measurements and the results from the Western blots confirmed these results. The Northern blots showed cross hybridization with human CYP3A cDNA, while in the Western blots one protein band cross-reacting with anti-rat CYP3A was detected. No cross hybridization with rat CYP2B1/2 cDNA was observed. However, the Western blots showed a band cross-reacting with anti-rat CYP2B, suggesting the presence of a CYP2B-like protein. In conclusion, this study has shown that Ringed seal liver contains multiple forms of CYP as well as phase II enzymes, showing different catalytic activities, i.e. EROD, caffeine- N -demethylation, and testosterone hydroxylation at different positions. Only one CYP1A isoform seemed to be present as well as a CYP3A-like isoform. Although the catalytic activities and mRNA analyses did not indicate the presence of a CYP2B-like protein in Ringed seals, the Western blots suggested the presence of a CYP2B-like enzyme. However, its functional significance remains unclear.

Analysis of HO-PCBs and PCP in blood plasma from individuals with high PCB exposure living on the Chukotka Peninsula in the Russian Arctic

A trace analytical method is presented for the analysis of hydroxylated polychlorinated biphenyl metabolites (HO-PCBs) and pentachlorophenol (PCP) in human plasma. The described methodology is a modification of a previously validated method used for PCB and organochlorine pesticide analysis. The modified method enables the combined analysis of phenolic and neutral halogenated compounds. A tandem Florisil column is used for separating the HO-PCBs and PCP from the neutral fraction, instead of the more common chemical partitioning. In the same step the neutral fraction is purified for GC analysis. The extraction of the HO-PCBs and PCP was found to be highly dependent on sufficient acidification of the sample and the polarity of the extracting solvent. Analysis of plasma samples gave recovery rates for (13)C(6)-PCP and (13)C(12)-4-HO-CB 187 of 64 and 72%, respectively. The limit of detection ranged between 2-20 pg g(-1) plasma for the HO-PCBs and 5 pg g(-1) plasma for PCP. No matrix interferences were observed in the chromatograms. In plasma samples (n = 15) from the native Chukchi people in Uelen (Russian Arctic), a population with high PCB exposure, the median ratio of sum HO-PCBs to sum PCBs was as high as 0.4 and the sum HO-PCBs and PCBs were significantly correlated (r(2) > 0.7, p < 0.01). The median sum HO-PCBs (10 congeners) was 5920 pg g(-1) plasma with 4-HO-CB 107 as the dominating congener (median: 1670 pg g(-1) plasma). The median PCP level was measured at 642 pg g(-1) plasma.

The enrichment of n−3 polyunsaturated fatty acids using aminopropyl solid phase extraction columns

A rapid, simple and reliable method is described for the preparation of concentrates of methyl or ethyl esters of n−3 polyunsaturated fatty acids by solid phase extraction using aminopropyl bonded silica columns. After applying mixtures of fatty acid esters in hexane, saturated and monounsaturated fatty acid esters are preferentially eluted with hexane whereas polyunsaturated fatty acids (PUFA) can subsequently be eluted with dichloromethane. Concentrates containing 80–90% n−3 PUFA can thus be obtained using fish oil fatty acids esters as a starting material.

Cytochrome P450-mediated enzyme activities and polychlorinated biphenyl accumulation in harp seal (Phoca groenlandica)

The presence and activities of hepatic cytochrome P450 (CYP) isoforms in Barents Sea harp seals (n=13) were studied using catalytic activities, selective inhibitors, and western blots. In addition the polychlorinated biphenyl (PCB) burden was measured in both the blubber and the food of the seals. The CYP activities and CYP isoforms present were related to the blubber PCB load and the relative presence of each PCB congener in seals relative to their food (metabolic index). The CYP activities measured were 2‐7 times higher than in ringed and harp seals from other studies, suggesting induction due to contaminant exposure. However, PCB burdens were surprisingly low and were not related to the CYP activities. Probably these high activities were due to exposure to other contaminants or due to the diet consisting mainly of crustaceans, which are rich in carotenes. Carotenes have been shown to be potent CYP inducers in several laboratory animals. The CYP studies indicated the presence of CYP1A, -2B and -3A isoforms. A decrease in the relative presence of a number of PCB congeners in seal blubber relative to their food, supposed to be due to CYP-mediated metabolism, corresponded with the metabolic capabilities of the CYP isoforms found. # 1999 Elsevier Science Ltd. All rights reserved.

Influence of Lipid Status on Tissue Distribution of the Persistent Organic Pollutant Octachlorostyrene in Arctic Charr (Salvelinus alpinus)

Abstract The tissue distribution of the lipophilic persistent pollutant octachlorostyrene (OCS) in relation to the amount, composition and localization of body lipids was investigated in two groups of Arctic charr, which before OCS administration had been held on a restricted feed ration (lean charr) or fed to satiation (fat charr). Lean charr were only a little over a third of the size of the fat charr, with most tissues and organs having a substantially lower concentration of triacylglycerols but only a slightly lower concentration of phospholipids and cholesterol than those of fat charr. No differences were found between the two groups, regarding the size or lipid content and composition of the brain. The tissue concentration of OCS was positively correlated with the concentration of triacylglycerols and negatively correlated with phospholipids and cholesterol. The proportion of the total body burden of OCS deposited in extra-adipose tissues was higher in the lean charr (28%) than in fat charr (13%). The mean OCS concentration in the brain of the lean charr was almost four times higher than in fat charr. The results indicate an increased risk of xenobiotic-associated stress when storage lipids are depleted, for example, during the period in which anadromous salmonid fish undergo the morphological, physiological and behavioral pre-adaptations necessary for a successful seaward migration.

Human plasma levels of POPs, and diet among native people from Uelen, Chukotka

Some of the people living in the Chukotka Peninsula of Russia depend heavily on marine mammals, but little is known of the exact dietary patterns and plasma levels of POPs among these populations. In this study, POPs levels in plasma from 50 participants from the isolated community of Uelen (Bering Strait) were determined and related to dietary information obtained through a food frequency questionnaire. The intake of marine mammals was high and the combined intake of blubber from walrus, seal and whale was a significant predictor (p < 0.01) of plasma concentrations of sum PCBs and borderline for sum CDs (p = 0.02) and sum DDTs (p = 0.04). There was a significant gender difference in the levels of POPs, and among women there was a significant increase with age. Extensive breastfeeding and lower blubber intake among women could be possible explanations for this gender difference. Despite the high intake of blubber the plasma levels of PCBs and DDTs were lower than some of those reported for the East Coast of Greenland. The geometric mean values for sum PCBs (17 congeners) and sum DDTs were 1316 ng g(-1) lipids and 563 ng g(-1) lipids, respectively. PCB 163, which partly co-eluted with PCB 138, was found in high concentrations (40% of PCB 138). This raises questions regarding the validity of using PCB 138 and PCB 153 to calculate the level of Arochlor 1260. The geometric mean of sum CDs was 518 ng g(-1) lipids. Concentrations of beta-HCH (geometric mean; 410 ng g(-1) lipids) were higher than observed for other native populations depending on marine mammals. Transportation of beta-HCH by ocean currents through the Bering Strait into the Arctic Ocean or regional point sources might explain these elevated levels.