Artistic forms and complexity.

We discuss the inter-relationship between various concepts of complexity by introducing a complexity 'triangle' featuring objective complexity, subjective complexity and social complexity. Their connections are explored using visual and musical compositions of art. As examples, we quantify the complexity embedded within the paintings of the Jackson Pollock and the musical works of Johann Sebastian Bach. We discuss the challenges inherent in comparisons of the spatial patterns created by Pollock and the sonic patterns created by Bach, including the differing roles that time plays in these investigations. Our results draw attention to some common intriguing characteristics suggesting 'universality' and conjecturing that the fractal nature of art might have an intrinsic value of more general significance.

Bioaccumulation of persistent organic pollutants in female common dolphins (Delphinus delphis) and harbour porpoises (Phocoena phocoena) from western European seas: geographical trends, causal factors and effects on reproduction and mortality.

Concentrations of polychlorinated biphenyls (PCBs) in blubber of female common dolphins and harbour porpoises from the Atlantic coast of Europe were frequently above the threshold at which effects on reproduction could be expected, in 40% and 47% of cases respectively. This rose to 74% for porpoises from the southern North Sea. PCB concentrations were also high in southern North Sea fish. The average pregnancy rate recorded in porpoises (42%) in the study area was lower than in the western Atlantic but that in common dolphins (25%) was similar to that of the western Atlantic population. Porpoises that died from disease or parasitic infection had higher concentrations of persistent organic pollutants (POPs) than animals dying from other causes. Few of the common dolphins sampled had died from disease or parasitic infection. POP profiles in common dolphin blubber were related to individual feeding history while those in porpoises were more strongly related to condition.

Biological and ecological factors related to trace element levels in harbour porpoises (Phocoena phocoena) from European waters.

Selected trace elements (Cd, Cu, Hg, Se, Zn) were measured in the kidneys and the liver of 104 harbour porpoises (Phocoena phocoena) stranded along the coasts of France, Galicia (Spain), Ireland, Scotland (UK), and the Netherlands. Generally, relatively low concentrations of toxic elements were encountered in the tissues of European porpoises, except for two individuals, which displayed high hepatic Hg concentrations. Also, elevated Cd levels obtained in Scottish porpoises could be related to their feeding preferences and this result suggests an increase of the proportion of cephalopods in their diet with latitude. Moreover, significant geographical differences were seen in hepatic Zn concentrations; the elevated Zn concentrations displayed by porpoises from the Netherlands may relate their poor health status. Variation in metal concentrations within porpoises from the North Sea is likely to reflect a long-term segregation between animals from northern (Scotland) and southern areas (the Netherlands), making trace elements powerful ecological tracers.

Organotin contamination in the Atlantic Ocean off the Iberian Peninsula in relation to shipping.

Imposex in female snails, a bioindicator of TBT contamination, and the presence of organotins in snails' tissue and sediments were studied at nine sites off the western Iberian Peninsula. The study was part of a European project (acronym HIC-TBT) co-financed by the EU-LIFE programme, intending to investigate and communicate the impact of organotins from ships in marine ecosystems. Snails and sediments were sampled during two cruises in May/June 1999 and in January 2000 in areas of high, intermediate and low-shipping density. Imposex was found in female snails from several sampling sites, some of which had an imposex incidence of 100%. Differences in sensitivity were found between species; hence comparison of imposex levels between locations where different species were collected was not straightforward. Total organotin concentrations in sediments (sum of butyl and phenyltin compounds) ranged from 21 to 185 ng Sn g(-1) with higher values for most sites sampled in the vicinity of shipping lanes. Organotin concentration in snails' tissue ranged from <5 to 196 ng Sn g(-1), which are similar to those found in snails from other offshore areas contaminated by TBT. Overall, these results give further support to the recent ban on the use of organotin based antifouling paints to all ship size.

Treatment of ballast water; how to test a system with a modular concept?

A variety of methods were successfully applied to examine the efficacy of a modular ballast water system according to the standards as adopted by the International Maritime Organization. The ballast water treatment system had a capacity of 530 m3 h(-1) consisted of a pump system, a hydrocyclone, a 50 microm mesh-size self-cleaning filter and an installation for the addition of a chemical disinfectant (PERACLEAN Ocean). The land-based testing facility used natural sea water of high turbidity during the spring phytoplankton bloom. The mesozooplankton fraction was inspected with a standard binocular. Larger zooplankton were effectively removed with the filter; the smaller sized fraction containing larvae and nauplia were killed after chemical treatment. The phytoplankton component was monitored using flow cytometry. The huge colonies of the phytoplankton Phaeocystis globosa were disrupted in the hydrocyclone liberating the colony cells which passed as single cells through the filter. These cells remained viable but were finally killed in the secondary (chemical) step. Bacteria also passed all mechanical treatment steps unharmed but were killed in the final step. Viability tests with SYTOX Green, which were specifically designed for phytoplankton, showed that mechanical treatment did not affect the percentage of viable cells a short-term, but after several hours the viable cell counts dropped down to 70%. Phytoplankton cells recovered within a single day and formed a new dense bloom rapidly. The bacteriostatic component of the chemical disinfectant (H2O2) remained present for several days preventing regrowth of bacteria for up to 15 days after addition. In conclusion, the IMO standards were met using the modular ballast water treatment unit and the applied instruments and assays were effective and rapid tools to qualify and quantify the organisms present as well as their viability.

Location and characterization of the suramin binding sites of human serum albumin.

The objective of the present study was to investigate the location of the high-affinity suramin binding sites on the human serum albumin molecule. For this purpose, circular dichroism and equilibrium dialysis experiments were performed on the interaction between suramin and a large peptic and a large tryptic fragment of albumin, the former comprising domains one and two of the albumin structure and the latter domains two and three. The equilibrium dialysis experiments revealed that albumin and the fragments have a comparable total affinity for suramin. Furthermore, all three proteins display a similar pH dependence of the unbound fraction of suramin. The circular dichroism experiments revealed that only the suramin-albumin and the suramin-peptic fragment complexes can undergo the pH dependent neutral-to-base or N-B conformational change, whereas the suramin-tryptic fragment complex lacks this ability. It is likely that the main parts of the high-affinity binding sites for suramin are located in domain two of the albumin molecule. The nature of these binding sites is discussed. The deprotonation of histidine and other positively charged residues taking part in salt bridges between suramin and albumin is, in all probability, the main cause of the decrease in affinity of suramin for albumin as the pH is raised from 6 to 9.

A model for the bioaccumulation of chlorobiphenyl congeners in marine mammals.

The behaviour of chlorobiphenyls in marine mammals is best described by a pharmacokinetic model where the blood acts as the central transport compartment between the external environment and a number of peripheral organs, each maintaining a dynamic balance with the concentrations in the blood. Thus, blood samples can be a useful tool in monitoring programmes of chlorobiphenyl concentrations. Differences in the chlorobiphenyl patterns between seals and fish could be explained by the structure-biotransformation relationship developed in an experimental study. A harbour porpoise (Phocoena) seemed also able to metabolize chlorobiphenyl congeners with vicinal hydrogen atoms in the meta and para positions and two ortho-Cl atoms. Because the ratios between persistent and metabolizable congeners differed between specimens, it was not possible to derive 'dioxin type' toxic equivalents from concentrations of congeners occurring at much higher concentrations by calculation of their ratios.

Polybrominated biphenyl and diphenylether flame retardants: analysis, toxicity, and environmental occurrence.

Data on two classes of brominated polyaromatic flame retardants are reviewed with emphasis on analytical aspects, occurrence, fate, and toxicity in the environment. Concentrations of brominated fire retardants are quantified as equivalents of commercial mixtures. Because different congeners behave differently in the environment and show large differences in toxicity, future studies would benefit from the availability of analytical standards of individual congeners. The main environmental properties and mechanisms of toxicity of the PBBs and PBDEs are similar to those of the structurally related PCBs and dibenzodioxins. Although the present concentrations of brominated fire retardants do not yet appear to represent a major environmental risk in marine food chains, their replacement by environmentally less harmful alternatives is recommended.

Immunochemical and catalytic characterization of hepatic microsomal cytochrome P450 in the sperm whale (Physeter macrocephalus).

Liver samples from three live-stranded adult male sperm whales, that could be sampled and frozen in liquid nitrogen within 18 h post mortem, provided an opportunity to learn more about the basic properties of their cytochrome P450 (CYP) system. All samples were catalytically active and showed sharp bands of the different CYP enzymes after Western blotting, indicating that degradation of the proteins was negligible. All three sperm whales showed a similar immunochemical CYP pattern: bands of CYP1A1/2, CYP3A and CYP4A were present, but CYP2B1/2 was not detected. Significant biotransformation of the polychlorinated aromatic hydrocarbons 4, 4'-dichlorobiphenyl (CB-15), 2,7-dichlorodibenzodioxin and 1,2,3,4,8-pentadibenzofuran was measured in an in vitro biotransformation assay. In contrast, 3,3',4,4'-tetrachlorobiphenyl (CB-77) and two chlorobornanes (CHB-32 and CHB-62) occurring in the insectide toxaphene(R), were not metabolised.

Indications for the involvement of a CYP3A-like iso-enzyme in the metabolism of chlorobornane (Toxaphene) congeners in seals from inhibition studies with liver microsomes.

The different isoforms of the cytochrome P450 (CYP) system can metabolise a suite of classes of lipophilic, anthropogenic compounds. The bioaccumulative potential as well as the toxicity of xenobiotics may be significantly altered in the process. To compare the metabolic ability of different wildlife species, it is important to identify the different iso-enzymes of CYP, which are responsible for the metabolism of different classes of compounds. This can be achieved with in vitro incubation assays. In the present study, preparations of hepatic microsomes of a harbour seal (Phoca vitulina) and a grey seal (Halichoerus grypus) demonstrated that the chlorobornane (CHB) congeners CHB-32 and -62 were metabolised enzymatically to their hydroxylated derivatives. These derivatives were partially characterised by their NCI mass-spectra. Inhibition studies were carried out to identify the specific CYP isoform(s) responsible for the metabolism of CHB-32 and -62. Ketoconazole has been shown to inhibit CYP3A enzymes in human and rat studies. In this study, ketoconazole caused concentration-dependent inhibition of metabolism of CHB-32 and -62, reaching 80% at the 1.0 microM treatment level. Ellipticine (1.0 microM), which has been shown to inhibit CYP1A1/2, also inhibited CHB-32 and -62 metabolism in the microsomes of grey seal, but to a much lower degree of less than 10 and 24%, respectively. In the same experiment the metabolism of 4,4'-dichlorobiphenyl was already inhibited 70% by ellipticine treatment at the same concentration. This non-ortho substituted PCB congener can easily attain a planar molecular configuration, and therefore served as a model CYP1A substrate. Inhibition of chlorobornane metabolism was not observed after the addition of goat anti-rat CYP2B antibodies or Aldrin, which is a model CYP2B substrate in rat. Cautious interpretation is advised for results obtained with so-called selective competitive inhibitors. Regardless, these studies indicated for the first time the possible involvement a CYP3A isoform in the mediation of chlorobornane metabolism in seals. The immunochemical cross-reactivity of mouse, rabbit or sheep anti-rat antibodies in the hepatic microsomes of harbour seal confirmed the presence of CYP1A1/2, CYP1A1, CYP2B1/2, CYP3A and CYP4A isoenzymes. Enantioselective metabolism by the microsomes of harbour seal was observed for both CHB-32 and -62. Stereochemical preferences of biotransformation enzymes can have an influence on the environmental distribution of both enantiomers of optically active compounds.

Residues of chlorinated biphenyl congeners and pesticides in brain and liver of the oystercatcher (Haematopus ostralegis) in relation to age, sex and biotransformation capacity.

Polychlorinated biphenyl concentrations in brain and liver oystercatchers, Haematopus ostralegis, increased similarly in males and females until sexual maturation. The concentrations were higher in adult males than in subadults, but for females the concentrations were lower in adults than in subadults. Absolute PCB, dieldrin and DDE concentrations were higher in liver than in brain. Compared with these compounds, alpha-HCH was relatively enriched in brain. No significant relation between pesticide concentrations and age or sex could be established. Despite differences in absolute PCB concentrations, the PCB patterns were very similar between all categories of birds and between brain and liver of the same animals. Only in the brain of juvenile males were some congeners present in relatively higher concentrations than in the brain of subadults and adults. Oystercatchers are able to metabolize CB congeners with vicinal H-atoms in the meta and para positions irrespective of the number of ortho-chlorines present. However, their ability to metabolize congeners with vicinal H-atoms in the ortho and meta positions is limited.

Changes in levels of hepatic biotransformation enzymes and haemoglobin levels in female plaice (Pleuronectes platessa) after oral administration of a technical polychlorinated biphenyl mixture (Clophen A40).

Female plaice (Pleuronectes platessa) were orally dosed with a gelatin capsule containing a solution of the technical PCB mixture Clophen A40 in sunflower oil. They were compared to plaice injected with a gelatin capsule containing only the sunflower oil at 10 and 16 days after injection. Even at 16 days after injection, the increase in concentrations of individual CB congeners in muscle was proportional to their contribution in Clophen A40. Biochemical effects are related to increases in concentrations of well-separable CB congeners in muscle, which increased by factors between 1.6 and 64 compared to the reference group of fish. Of both sampling points, total cytochrome P-450 levels were higher than the control groups, but surprisingly ethoxyresorufin-O-deethylase (EROD) activities did not differ between the groups. Enzyme-linked immunosorbent assay (ELISA) showed increased concentrations of the inducible cytochrome P-450IA1 in PCB-treated fish. The apparent lack of EROD induction may be due to competitive substrate inhibition by certain CB congeners present in the sample. The activity of glutathione-S-transferase (GST) (with CDNB as model substrate) was significantly elevated by PCB-treatment at day 16, but not at day 10. A longer time interval between injection with PCBs and induction of GST compared to P-450 monooxygenase activities has been reported earlier and may indicate that in fish both groups of enzymes are regulated individually and not as an [Arylhydrocarbon] gene battery as appears to be the case in mammals. Haemoglobin concentrations and MCHC were decreased in fish treated with Clophen A40. Haematocrit values did not differ between groups of fish.

Toxic and biochemical effects of 3,3',4,4'-tetrachlorobiphenyl (CB-77) and clophen A50 on eider duckling (Somateria mollissima) in a semi-field experiment.

In this study the possible toxic and biochemical effects of one intraperitoneal dose of 5 or 50 mg kg(-1) of 3,3',4,4'-tetrachlorobiphenyl (CB-77) or 50 or 200 mg kg(-1) of Clophen A50 (CloA50) on 28-day-old eider ducklings (Somateria mollissima) were investigated. After ten days, no significant differences could be observed in any of the toxic and biochemical parameters studied, apart from ethoxyresorufin (EROD) activity, when comparing group average values of the dosed and control animals. However, significant correlations were observed at day 10 after exposure between the individual internal PCB concentration and body weight gain and beak length growth (negative correlations in the CloA50 groups); relative liver weight and cytochrome P4501A activity (positive correlations in CB-77 and CloA50 groups); plasma thyroid-hormone and hepatic retinoid levels (negative correlations in CB-77 groups); and plasma retinol levels and the ratio plasma retin/hepatic retinyl palmitate (positive correlations in CB-77 groups only). Animal activity was significantly reduced in the group that received 50 mg CB-77 kg(-1). These observations indicate that eider ducks are a sensitive species to PCB toxicity and may be at risk for development of adverse health effects in relatively highly contaminated areas such as the Waddenzee.

The use of microsomal in vitro assay to study phase I biotransformation of chlorobornanes (Toxaphene) in marine mammals and birds. Possible consequences of biotransformation for bioaccumulation and genotoxicity.

The factors determining the bioaccumulation of lipophilic compounds in wildlife are often poorly understood, partly because it is difficult to do in vivo experiments with animals such as marine mammals and birds. To evaluate the role of phase I biotransformation in the bioaccumulation process of chlorobornanes (toxaphene), this was studied in in vitro assays with hepatic microsomes of animals that could be sampled shortly after death. The capacity of microsomes to metabolise a technical toxaphene mixture decreased in the order Phoca vitulina (harbour seal) >> Lagenorhynchus albirostris (whitebeaked dolphin) approximately equal to Diomedea immutabilis (Laysan albatross) > Physeter macrocephalus (sperm whale). Harbour seal microsomes metabolised the chlorobornane (CHB) congeners CHB-32 and CHB-62; whitebeaked dolphin and Laysan albatross microsomes only metabolised CHB-32. Metabolism of CHB-26 and CHB-50 was never observed. The negative chemical ionisation (NCI-) mass spectra of some of the hydroxylated metabolites were obtained. The number of peaks in the toxaphene residues of wildlife extracts decreased in the order of increasing in-vitro biotransformation capacity. Thus, the results of the in vitro assays and residue analysis were in accordance, although assays with microsomes of more individuals of the same species are required for a more general conclusion at the species level. Finally, the effect of in vitro biotransformation was evaluated in terms of the genotoxic potential using the Mutatox assay. Only technical toxaphene and CHB-32 were genotoxic in the direct assay, whereas the addition of rat S9 fraction or microsomes of harbour seal and albatross decreased the genotoxic response. Thus, organisms with a low ability to metabolise chlorobornanes, such as whales, may be most affected by the carcinogenic properties of toxaphene. A hypothetical reaction which fits the experimental results is discussed. Based on these results it is concluded that in vitro assays with microsomes of wildlife animals which died a natural cause can act as a valuable tool to assess the occurrence and effects of phase I metabolism. Some precautions are discussed, that should be taken to reduce the chance of false negative results.

Temporal induction pattern of hepatic cytochrome P450 1A in thermally acclimated dab (Limanda limanda) treated with 3,3',4,4'-tetrachlorobiphenyl (CB77).

Mature male dab (Limanda limanda) acclimated at 10 degrees and 16 degrees C were orally administered a single dose of 0.5 mg/kg 3,3',4,4'-tetrachlorobiphenyl (CB77). At both temperatures, levels of cytochrome P450 1A (CYP1A) protein and 7-ethoxyresorufin O-deethylase (EROD) activity showed a two to six fold induction 40 days after CB77 treatment compared to control groups. Maximum responses of both EROD activity and CYP1A protein for the warm-acclimated fish were observed at 5 days after treatment. For the cold-acclimated fish a slow, progressive elevation for both EROD activity and CYP1A protein was observed and maximum responses were measured 40 days after treatment. Absolute EROD activity and CYP1A protein levels of fish from both temperatures were equally high at 40 days after treatment. Since in the control groups EROD activity and CYP1A protein levels were higher in the cold-acclimated fish, the magnitude of induction was higher in the warm acclimated ones. The highest concentrations of CB77 in muscle of fish from both temperatures were found at 5 and 10 days after treatment. The liver somatic index (LSI) showed 1.5 fold significantly higher values for the fish acclimated at 10 degrees C.

Dose- and time-dependent formation of biliary benzo[a]pyrene metabolites in the marine flatfish dab (Limanda limanda).

Polycyclic aromatic hydrocarbons (PAHs) are abundant pollutants, and many PAHs are carcinogenic, but only after metabolic activation. Benzo[a]pyrene (BaP) is among the most carcinogenic PAHs. The dose and time response of two enzymes involved in BaP metabolism and the amounts of BaP metabolites excreted into the bile were evaluated in an experiment with dab (Limanda limanda). Ninety dab were exposed orally to one of five doses of BaP (0, 0.08, 0.4, 2, or 10 mg/kg) and sampled at 3, 6, or 12 d after exposure. None of the doses studied caused significant induction of either microsomal ethoxyresorufin-O-deethylase (EROD). which reflects cytochrome P450 1A (CYP1A) activity, or cytosolic glutathione-S-transferase activity (GST). Concentrations of biliary BaP metabolites significantly increased with dose and significantly decreased with time after exposure. It is concluded that biliary BaP metabolites provide a much more sensitive method than EROD (CYP1A) or GST activity to monitor recent exposure to PAHs in dab.

Cytochrome P450 differences in normal and imposex-affected female whelk Buccinum undatum from the open North Sea.

Normal and imposex-affected female Buccinum undatum were sampled from the open North Sea at three locations, one with low, and two with high shipping densities. Cytochrome P450 components and P450 aromatase activity were determined in the microsomal fractions isolated from pooled digestive gland/gonads. Cytochrome P450 aromatase activity was significantly higher (P < 0.05) in normal females collected in the low shipping density area (1,325 +/- 295 fmol/h/mg protein) than levels from imposex animals from a high shipping density area (620 +/- 287 fmol/h/mg protein). A negative correlation was found between aromatase activity and organotin body burden (r = -0.99). Levels of CYP450, cytochrome b5 and NADPH cytochrome c reductase activity did not show differences among groups. This is the first field evidence of depressed aromatase activity in imposex affected females, although additional research under laboratory controlled conditions is required to fully understand the mechanisms underlying the development of imposex in this species.

The expression of CYP1A, vitellogenin and zona radiata proteins in Atlantic salmon (Salmo salar) after oral dosing with two commercial PBDE flame retardant mixtures: absence of short-term responses.

The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and cta-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days I and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n = 5 for each group) and 14 (n = 6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYPIA were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17beta for Vtg and Zrp expression, and beta-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.