Ecological changes as a plausible explanation for differences in uptake of contaminants between European perch and eelpout in a coastal area of the Baltic Sea.

Unexpected increasing trends in the concentration of contaminants in European perch (Perca fluviatilis) and in activity of ethoxyresorufin-O-deethylase (EROD) in European perch and eelpout (Zoarces viviparus) have been observed at a Swedish coastal reference site. This study uses data from different sources to investigate plausible explanations. The results showed that a change in diet and an improved overall condition coincide with an increase in mercury in European perch. Furthermore, an increase in several organic contaminants in European perch coincided with the introduction of an invasive deep-burrowing polychaete, which likely contributed to the release of contaminants through bioturbation. The increase in EROD-activity in both species seems to be related to contaminants that reach the fish through the water rather than the diet. The results show that for contaminants that are taken up via the diet, trends in contamination can be opposite for different species of fish in the same area.

A metabolic simulator for unmanned testing of breathing apparatuses in hyperbaric conditions.

BACKGROUND: A major part of testing of rebreather apparatuses for underwater diving focuses on the oxygen dosage system. METHODS: A metabolic simulator for testing breathing apparatuses was built and evaluated. Oxygen consumption was achieved through catalytic combustion of propene. With an admixture of carbon dioxide in the propene fuel, the system allowed the respiratory exchange ratio to be set freely within human variability and also made it possible to increase test pressures above the condensation pressure of propene. The system was tested by breathing ambient air in a pressure chamber with oxygen uptake (Vo2) ranging from 1-4 L · min(-1), tidal volume (VT) from 1-3 L, breathing frequency (f) of 20 and 25 breaths/min, and chamber pressures from 100 to 670 kPa. RESULTS: The measured end-tidal oxygen concentration (Fo2) was compared to calculated end-tidal Fo2. The largest average difference in end-tidal Fo2during atmospheric pressure conditions was 0.63%-points with a 0.28%-point average difference during the whole test. During hyperbaric conditions with pressures ranging from 100 to 670 kPa, the largest average difference in Fo2was 1.68%-points seen during compression from 100 kPa to 400 kPa and the average difference in Fo2during the whole test was 0.29%-points. CONCLUSION: In combination with a breathing simulator simulating tidal breathing, the system can be used for dynamic continuous testing of breathing equipment with changes in VT, f, Vo2, and pressure.

Environmental and health hazard ranking and assessment of plastic polymers based on chemical composition.

Plastics constitute a large material group with a global annual production that has doubled in 15 years (245 million tonnes in 2008). Plastics are present everywhere in society and the environment, especially the marine environment, where large amounts of plastic waste accumulate. The knowledge of human and environmental hazards and risks from chemicals associated with the diversity of plastic products is very limited. Most chemicals used for producing plastic polymers are derived from non-renewable crude oil, and several are hazardous. These may be released during the production, use and disposal of the plastic product. In this study the environmental and health hazards of chemicals used in 55 thermoplastic and thermosetting polymers were identified and compiled. A hazard ranking model was developed for the hazard classes and categories in the EU classification and labelling (CLP) regulation which is based on the UN Globally Harmonized System. The polymers were ranked based on monomer hazard classifications, and initial assessments were made. The polymers that ranked as most hazardous are made of monomers classified as mutagenic and/or carcinogenic (category 1A or 1B). These belong to the polymer families of polyurethanes, polyacrylonitriles, polyvinyl chloride, epoxy resins, and styrenic copolymers. All have a large global annual production (1-37 million tonnes). A considerable number of polymers (31 out of 55) are made of monomers that belong to the two worst of the ranking model's five hazard levels, i.e. levels IV-V. The polymers that are made of level IV monomers and have a large global annual production (1-5 million tonnes) are phenol formaldehyde resins, unsaturated polyesters, polycarbonate, polymethyl methacrylate, and urea-formaldehyde resins. This study has identified hazardous substances used in polymer production for which the risks should be evaluated for decisions on the need for risk reduction measures, substitution, or even phase out.

Biomarker analyses in caged and wild fish suggest exposure to pollutants in an urban area with a landfill.

An unexpectedly high frequency of skeletal deformations in brown trout has previously been observed in the brook Vallkärrabäcken in southern Sweden. Environmental pollutants from storm water and leachate from an old landfill have been suggested as responsible for the observed deformations. Biomarkers in farmed rainbow trout, placed in tanks with water supplied from the brook, were used to investigate if exposure to pollutants may induce toxic responses in fish. Furthermore, biomarkers were also measured in wild brown trout that were caught in the brook. The most important finding was that the hepatic ethoxyresorufin-O-deethylase (EROD) activity was five to seven times higher for rainbow trout and brown trout in exposed areas compared to reference sites (P<0.001). Analyses of bile in rainbow trout showed that the concentration of PAH-metabolites was two to three times higher (P<0.001) in the exposed areas. However, due to their smaller size and the feeding status, only insufficient amounts of bile could be retrieved from the wild brown trout. The study provides evidence for pollution in parts of Vallkärrabäcken. It is therefore possible that the previously observed high frequency of skeletal damage have been caused by pollutants. The methodology with farmed rainbow trout in flow through tanks worked well and provided more information about the occurrence of pollutants in Vallkärrabäcken than the data from brown trout. The main reasons for this were that the size and the feeding status of the fish could be controlled. This allowed a total of 21 biomarkers to be analyzed in farmed rainbow trout compared to only five in wild brown trout. Furthermore, the use of farmed fish eliminates the risk of migration, which may otherwise bias the data when wild fish are used.

Spatial and annual variation to define the normal range of biological endpoints: an example with biomarkers in perch.

A signal from a biological endpoint can be considered evidence of environmental impact only if it is known that similar signals are unlikely to occur naturally. Using multiple reference sites, the normal range can be estimated. This can be defined as the span that includes 95% of the reference sites. If an investigated site is outside this range, it is interpreted as evidence of impact. The choice of reference sites is thus important for the outcome of the study. Here, biomarker levels in perch (Perca fluviatilis) at a potentially impacted site were compared to different types of reference data. The potentially impacted site was located close to a densely populated area. Four reference sites were located in relatively undisturbed areas on the Swedish Baltic coast. Furthermore, historical data from one of the reference sites were included. The present study showed that multiple reference sites are needed to avoid the risk of interpreting natural variation as impact. The number of reference sites needed depends on the desired level of statistical power. An alternative to multiple reference sites may be to use historical data to estimate the natural variation. However, historical data can include temporal variation due to factors that may not be relevant for the hypothesis that is tested, e.g., climatic variation or changed background levels of pollution.

Experiences from a biomarker study on farmed rainbow trout (Oncorhynchus mykiss) used for environmental monitoring in a Swedish river.

Biomarkers in fish may serve as a useful tool for evaluating the pollution load in the environment and for early warning signals about new environmental threats. By employing a strategy with fish that are caged or reared in tanks, problems linked to migration and feeding status can be reduced or eliminated. Such a strategy, however, also may introduce other confounding factors linked to, for example, hierarchical behavior or disease outbreaks. In the present study, rainbow trout (Oncorhynchus mykiss) were reared in plastic tanks at four sites in the Göta älv river system (plus one external reference) in western Sweden during 2006 and 2007. Because of low population density and high water turnover in the area, pollution levels are expected to be low. Therefore, this should be a good test for the sensitivity of the methodology. Several significant differences were found between sites as well as between years, such as ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbon (PAH) metabolites in bile, and concentration of inorganic ions in blood plasma, but it also was seen that factors other than pollution could be contributing to these differences. The condition factor (CF) varied between sites, possibly because of differences in feeding resulting from variations in water turbidity. Furthermore, even the small differences in CF that were found within sites correlated significantly to several of the biomarkers. It was shown that PAHs likely are the most important EROD inducers in Göta älv and that variation over time is greater than variation between sites. Because CF differed between sites despite a standardized feeding ration, starving of the fish during the exposure period should be considered for future studies.

Analyses of perch (Perca fluviatilis) bile suggest increasing exposure to PAHs and other pollutants in a reference area on the Swedish Baltic coast.

Kvädöfjärden on the Swedish Baltic coast has been used as a reference station in the Swedish National Marine Monitoring Programme since 1988. During this time an increasing trend in ethoxyresorufin-O-deetylase (EROD) activity and a decreasing trend in gonad size for female perch have been observed. It is known that EROD activity respond to exposure to certain groups of pollutants, including polycyclic aromatic hydrocarbons (PAHs). In the present study, perch bile from two years with high (2006) and low (2003) EROD levels, respectively, were analyzed. The concentration of PAH metabolites in bile was semi-quantified using fixed wavelength fluorescence. Furthermore, toxicity of the bile was tested on Daphnia magna. The total concentration of PAH metabolites and toxicity to D. magna was higher in 2006 than in 2003. Furthermore, there was a higher proportion of combustion-type PAHs in bile from 2006. The concentration of PAH metabolites in bile correlated to the EROD activity of the fish from which the bile was taken. There was no correlation between the concentrations of PAH metabolites and bile toxicity. Therefore, other pollutants than PAHs may also have been elevated in 2006. The results suggest that increasing exposure to PAHs can be causing the time trend in EROD activity. It is known that PAHs can have a negative effect on gonad development. Therefore, the decreasing trend in gonad size may also be caused by increasing exposure to PAHs.

Evaluation of long-term biomarker data from perch (Perca fluviatilis) in the Baltic Sea suggests increasing exposure to environmental pollutants.

Since 1988, biomarkers in female perch (Perca fluviatilis) have been analyzed at a reference site on the Swedish Baltic coast. Strong time trends toward increasing hepatic ethoxyresorufin-O-deethylase (EROD) activity and reduced gonadosomatic index (GSI) have been observed. This could be caused by pollutants as well as other factors, such as increasing water temperature or reduced mean age of sampled fish. Correlation analyses were used to find the most probable explanation for the time trends. The time trends were still significant for EROD (p < 0.001) and GSI (p < 0.001) when the correlations were controlled for age. Furthermore, increasing water temperature could not explain the time trends. Exposure to pollutants through runoff from land was found to be probable, because mean flow rate in a nearby river during the last 20 d before sampling correlated to EROD activity (p < 0.01). In addition, the sum of EROD activities during the life time of the perch (ERODlife) correlated significantly with GSI (p < 0.001). This suggests that perch exposed to more EROD-inducing chemicals during their lifetime have reduced or delayed gonad development. The time trend in GSI and the correlation between ERODlife and GSI were supported by data from a site in the Bothnian Bay (northern Baltic Sea; p < 0.05). The results indicate that increased rain fall (climate change) can affect the distribution and bioavailability of chemicals in coastal areas. The link between EROD and gonad size supports the common assumption that biochemical biomarkers can act as early warning signals for effects on higher levels, which commonly is difficult to show. The significant results can probably be attributed to the unique 20-year data set.

Calretinin expression in the differential diagnosis of human ameloblastoma and keratocystic odontogenic tumor.

Ameloblastoma is a benign, locally aggressive epithelial odontogenic tumor that has the potential to become malignant and produce metastasis to distant sites such as lungs and kidneys. The histologic presentation can be, in some instances, mistaken for keratocystic odontogenic tumor (KCOT) (formerly known as odontogenic keratocyst). The expression of calretinin [calbindin2 (CALB2)] was investigated on both ameloblastoma and KCOT. Nineteen cases of ameloblastoma and 17 cases of KCOT were stained with calretinin antiserum 18-0211 (Zymed, San Francisco, CA). All cases (100%) of ameloblastoma showed positive calretinin staining, restricted to the neoplastic epithelial component and none (0%) of the 17 KCOTs showed positive calretinin staining. Gene expression profiling of ameloblastomas showed CALB2 expressed in the basal cell layer of columnar cells resembling preameloblasts, in all 5 of the ameloblastomas evaluated. Taken together, the results of this study strongly support calretinin as a useful immunohistochemical marker for ameloblastoma and malignant ameloblastoma and it can also be used in the differential diagnosis of KCOT.

Fixed wavelength fluorescence to detect PAH metabolites in fish bile: increased statistical power with an alternative dilution method.

Polyaromatic hydrocarbons (PAHs) are a group of environmental toxicants mainly emitted from diffuse sources. Fixed wavelength fluorescence of fish bile is a simple screening method that allows environmental monitoring of PAH exposure to fish. One drawback with this method is that the results can be biased by the presence of a so-called inner filter effect. This effect can be reduced by dilution of the bile samples. However, bile samples differ in density and extensive dilution may cause increased measurement error. An alternative method is to adjust the dilution rate to the density of each bile sample. Here, both methods have been applied to bile samples collected from caged rainbow trout. The bile density was estimated using biliverdin. The results show that the variance within experimental groups is reduced when the dilution rate is adjusted to the bile density, resulting in increased statistical power to detect differences when applied for environmental monitoring.

Influence of feeding procedure on biomarkers in caged rainbow trout (Oncorhynchus mykiss) used in environmental monitoring.

Biomarkers in feral fish can be a useful tool for environmental monitoring of aquatic ecosystems. Drawbacks, however, are that suitable fish species are not always available and that natural variations can bias the results. An alternative strategy is to use farmed fish placed in cages. There is, however, still a risk that factors other than pollution level could have an impact on the biomarkers and the observed responses in the fish. The present study evaluates the effects of feeding procedure on biomarkers in caged fish. Two feeding rations (2% and 8% weekly feeding) have been examined for a large number of biomarkers in caged rainbow trout (Oncorhynchus mykiss). Significant effects of feeding rations were found on hepatic ethoxyresorufin-O-deethylase (EROD) and catalase activity, PAH metabolites in bile, plasma ion concentrations and the presence of immature red blood cells. The influence on EROD activity and PAH metabolites seems to be caused by elevated uptake of pollutants when feeding ratios are higher. The effects on other biochemical and physiological variables are more likely caused by stress due to insufficient feeding. According to these results, valid comparison of fish groups in environmental monitoring requires standardized feeding levels.

Long-term monitoring of the health status of female perch (Perca fluviatilis) in the Baltic Sea shows decreased gonad weight and increased hepatic EROD activity.

Annual investigations of the health status of female perch (Perca fluviatilis) were performed during 8-13 years in order to detect long-term, large-scale changes in the Baltic Sea. These background data are also meant to be used as baselines in recipient investigations at various point sources in the Baltic Sea. Fish were sampled at three coastal sites in Sweden: two in the Baltic Proper (since 1988) and one in the Bothnian Bay (since 1993). In all, 19 biochemical, physiological and histopathological variables were measured. By reflecting central functions of life, such as red blood cell functions, immune defence, liver function, biotransformation processes, intermediary metabolism, ion balance, and reproduction, the selected variables give a good picture of the health status of the fish. The most important observations were strong trends towards decreased gonadosomatic index (relative gonad size) and increased hepatic EROD activity in female perch in the Baltic Proper. Most of the other variables varied little between the years. The reduced gonad size and the EROD induction indicate that factors in the environment, e.g. the occurrence of pollutants, affect important physiological functions, such as reproduction and biotransformation processes, in fish even in a reference area in the Baltic Proper. It is urgent to make further studies in order to identify which pollutant(s) or other possible cause(s) are responsible for the observed biological effects.

Expression of plasminogen activator inhibitor-1, urokinase receptor and laminin gamma-2 chain is an early coordinated event in incipient oral squamous cell carcinoma.

Cancer cell invasion is facilitated by extracellular matrix degrading proteases such as plasmin. We have studied the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) together with the gamma2-chain of laminin-5 (lam-gamma2) by immunohistochemistry in 20 cases with incipient oral squamous cell carcinoma (SCC). PAI-1-positive neoplastic cells located at the tip of the putative invasive front of grade 1 (incipient) carcinoma were seen in 16 of the 20 cases (75%), whereas adjacent normal and dysplastic epithelium was PAI-1-negative. Clusters of putative invasive neoplastic cells located in the lamina propria were PAI-1-positive in areas with grade 2 incipient carcinoma as were invasive cancer cells in areas of grade 3-4 invasive carcinoma. uPAR immunoreactivity was strongly expressed in numerous stromal cells in the carcinoma area in all 20 lesions, while a few uPAR-positive stromal cells were found in areas with normal and dysplastic epithelium. uPAR-positive neoplastic cell islands located at the front of the lesions were seen in 15 of the 20 cases. The expression pattern of lam-gamma2 was very similar to that of PAI-1; however, lam-gamma2-positive neoplastic cells were only detected in 11 of the 20 cases (55%) in areas of grade 1 incipient carcinoma. Direct comparison of the 3 components revealed colocalization in neoplastic cell islands in both incipient and invasive SCC. Our results suggest that PAI-1 is a novel potential marker of initial invasion in oral SCC, and that the coordinated expression of PAI-1 with uPAR and lam-gamma2 sustain the features of the early invasive cancer cells.

The effect of different holding conditions for environmental monitoring with caged rainbow trout (Oncorhynchus mykiss).

Biomarkers in fish can be a useful tool for environmental monitoring of aquatic ecosystems when diffuse pollution is becoming more important and new chemicals are being created continuously. There are, however, a number of drawbacks with this method. Because of environmental variability, health status of wild fish populations may differ between years, leading to unrepresentative results in long term studies. Furthermore, genetic or adaptive differences between populations complicate the interpretation of studies on different sites. The use of farmed fish, placed in cages, can reduce these problems. However, experimental conditions are likely to differ between sites. For practical reasons it may, e.g., be neccesary to use different types of caging. Here, the use of net cages and flow through tanks has been compared for a number of biomarkers. Rainbow trout (Oncorhynchus mykiss) were placed in net cages and flow through tanks in the river Göta Alv, in western Sweden, during three different periods in 2004 and 2005. No differences between types of caging were found for any biomarker. Therefore, the results suggest that net cages and flow through tanks can be used and compared in environmental monitoring using biomarkers in caged rainbow trout. However, efforts should be taken to reduce differences in experimental conditions, e.g., light intensity and feeding levels.

Bone graft and implants in a patient with systemic mastocytosis.

BACKGROUND: Systemic mastocytosis (mast-cell proliferation in various organs, including the skeleton) is a rare disease. Reports on mastocytosis that affects facial bones are few. The bone lesions may be osteolytic or sclerotic. PURPOSE: To describe (for the first time) bone grafting followed by dental implant treatment yielding a good result in a patient with systemic mastocytosis. MATERIALS AND METHODS: A bone graft was performed on a 60-year-old woman with systemic mastocytosis. Dental implant treatment was performed 13 weeks after sclerotic bone of the iliac crest was grafted to the maxillary sinus bilaterally. A microimplant was installed simultaneously with the dental implants and was removed 6 months later for histomorphometric evaluation. Bone biopsy specimens from the donor site of the sclerotic iliac crest and later from the remodeled maxillary bone graft were histologically analyzed. A clinical examination of the patient with regard to her mastocytosis was performed by a dermatologist. The patient was followed up after 3 years. RESULTS: Bone grafting and dental implant treatment were successful, and the patient's clinical and radiologic situation was stable after 3 years. Histologic examination of the bone grafted from the iliac crest showed sclerotic lesions in the bone and a dense infiltration of mast cells. The bone graft seemed to remodel initially in a normal way in the maxillary sinus. However, computed tomography 3 years later showed regions of sclerosis in the remodeled maxillary bone. These lesions now had a pattern similar to the adjacent facial bone. Both the microimplant and the dental implants integrated well. Bone-implant contact measured on the microimplants was 20% higher in this actual case, compared to that of patients previously treated and grafted with the same technique. CONCLUSIONS: There are many clinical implications to be considered when treating this group of patients. Bone grafting, remodeling of the bone, and dental implant installation were successful in this patient with systemic mastocytosis and signs of osteosclerosis. Installation of microimplants in patients with pathologic bone conditions may allow successful dental implant treatment.