Does fish reproduction and metabolic activity influence metal levels in fish intestinal parasites, acanthocephalans, during fish spawning and post-spawning period?

Application of fish intestinal parasites, acanthocephalans, as bioindicators in metal exposure assessment usually involves estimation of their metal levels and bioconcentration factors. Metal levels in parasite final host, fishes, are influenced by fish physiology but there is no data for acanthocephalan metal levels. Gastrointestinal Zn, Fe, Mn, Cd, Ag levels in European chub (Squalius cephalus L.) from the Sava River were significantly higher during chub spawning (April/May) compared to the post-spawning period (September). In acanthocephalans (Pomphorhynchus laevis and Acanthocephalus anguillae) significantly higher metal levels during chub spawning were observed only for Zn in P. laevis. Bioconcentration factors were twice as high for Fe, Mn, Ag, Pb in the post-spawning period, probably as a consequence of lower gastrointestinal metal levels in fish rather than metal exposure. Therefore, bioconcentration factors should be interpreted with caution, due to their possible variability in relation to fish physiology. In addition, gastrointestinal Cu, Cd and Pb levels were lower in infected than uninfected chub, indicating that metal variability in fishes might be affected by the presence of acanthocephalans.

Distribution of Co, Cu, Fe, Mn, Se, Zn, and Cd among cytosolic proteins of different molecular masses in gills of European chub (Squalius cephalus L.).

The distribution of essential elements Co, Cu, Fe, Mn, Se, and Zn, and nonessential element Cd among cytosolic proteins of different molecular masses in the gills of European chub (Squalius cephalus) sampled in the moderately contaminated Sutla River in September of 2009, was studied after the protein separation by size exclusion high-performance liquid chromatography (SEC-HPLC), and the metal determination in the obtained fractions by high-resolution inductively coupled plasma mass spectrometry (HR ICP-MS). The aims of the study were to characterize the distribution profiles of metals within different protein categories in gills in the conditions of low metal exposure in the river water, and to compare them with the previously published hepatic profiles. The distribution profiles of analyzed metals were mainly characterized with several peaks. However, some observations could be emphasized: both Cu and Cd were eluted near metallothionein elution time; elution time of one of Co peaks could be associated with Co-containing compound cobalamin; increasing cytosolic Fe concentrations resulted in possible Fe binding to storage protein ferritin; both Mn and Zn had poorly resolved peaks covering wide ranges of molecular masses and indicating their binding to various proteins; both Zn and Se increased in protein fractions of molecular masses <5 kDa following their concentration increase in the gill cytosol; expected clear metallothionein peak was not observed for Zn. Comparison of gill profiles with previously published hepatic profiles revealed similar and in case of some elements (e.g., Co, Fe, Mn, and Se) almost identical distributions in both organs regarding elution times. On the contrary, heights of obtained peaks were different, indicating possible metal binding to the same proteins in the gills and liver, but in different proportions. The results obtained in this study can be used as a basis for comparison in monitoring studies, for identification of changes that would occur after exposure of chub to increased metal concentrations.

Relevance of biotic parameters in the assessment of the spatial distribution of gastrointestinal metal and protein levels during spawning period of European chub (Squalius cephalus L.).

The present field study, conducted during the spawning period (April/May) of European chub (Squalius cephalus L.) from the Sava River in Croatia, indicates that seasonal changes of fish physiological state might cause variability in gastrointestinal metal (Cd, Cu, Fe, Mn and Zn), total cytosolic protein and metallothionein (MT) levels. During the period of fish spawning and increased metabolic activity, a significant relationship with chub hepatosomatic index was evident for Fe and Mn in gastrointestinal tissue (r = 0.35 and 0.26, respectively) and in cytosolic fraction (r = 0.32 and 0.41, respectively) and for Zn and Fe in the gut content (r = 0.36 and 0.31, respectively). Total cytosolic protein and MT concentrations followed the same spatial distribution as Fe and Mn in all gastrointestinal fractions and as Zn in the sub-cellular fractions, with higher levels at upstream locations. Due to the role of essential metals in metabolic processes and gonad development, increased feeding and spawning activity in April/May resulted in higher gastrointestinal essential metal (Fe, Mn and Zn) and MT concentrations, which probably follow an increase in Zn concentrations, known as the primary MT inducer. Therefore, biotic factors should be considered as important confounding factors in metal exposure assessment, while their influence on gastrointestinal metal and protein levels should be interpreted depending on the season studied.

Effect of acanthocephalan infection on metal, total protein and metallothionein concentrations in European chub from a Sava River section with low metal contamination.

In the present study, the importance of considering fish intestinal parasites i.e. the acanthocephalans in metal exposure assessment was estimated under low metal contamination conditions. Two acanthocephalan species, Pomphorhynchus laevis and Acanthocephalus anguillae were examined in 59 specimens of European chub (Squalius cephalus L.) sampled at 5 locations along the Sava River, Croatia. Concentrations of essential (Cu, Mn) and non-essential (Ag, Cd, Pb) metals were higher in intestinal parasites than chub gastrointestinal tissue, but levels of essential metals Fe and Zn were comparable or lower in parasites, respectively. The highest accumulation in both acanthocephalan species was found for non-essential metals and followed the order: Ag>Pb>Cd. Higher infection intensity with P. laevis allowed us to present their spatial metal distribution and evaluate the influence of P. laevis on metal levels and sub-cellular biological responses (total protein and metallothionein levels) in the host infected with P. laevis. Even in the river section with low metal contamination, parasitism affected metal levels, resulting in lower Cu, Cd and Pb concentrations in chub infected with P. laevis than in uninfected chub. Although total protein and metallothionein levels remained constant in infected and uninfected chub, acanthocephalans should be considered a potential confounding factor in metal exposure assessments. Moreover, P. laevis-chub system can be suggested as an appropriate tool in biomonitoring, since in both species increased Cu and Cd concentrations towards the downstream locations were found. Higher Cu and Cd levels in P. laevis suggest acanthocephalans to be sensitive bioindicators if low metal levels have to be detected.

Assessment of general condition of fish inhabiting a moderately contaminated aquatic environment.

The assessment of general condition of fish in the moderately contaminated aquatic environment was performed on the European chub (Squalius cephalus) caught in September 2009 in the Sutla River in Croatia. Although increases of the contaminants in this river (trace and macro elements, bacteria), as well as physico-chemical changes (decreased oxygen saturation, increased conductivity), were still within the environmentally acceptable limits, their concurrent presence in the river water possibly could have induced stress in aquatic organisms. Several biometric parameters, metallothionein (MT), and total cytosolic protein concentrations in chub liver and gills were determined as indicators of chub condition. Microbiological and parasitological analyses were performed with the aim to evaluate chub predisposition for bacterial bioconcentration and parasitic infections. At upstream river sections with decreased oxygen saturation (~50%), decreased Fulton condition indices were observed (FCI: 0.94 g cm(-3)), whereas gonadosomatic (GSI: 2.4%), hepatosomatic (HSI: 1.31%), and gill indices (1.3%) were increased compared to oxygen rich downstream river sections (dissolved oxygen ~90%; FCI: 1.02 g cm(-3); GSI: 0.6%; HIS: ~1.08%; gill index: 1.0%). Slight increase of MT concentrations in both organs at upstream (gills: 1.67 mg g(-1); liver: 1.63 mg g(-1)) compared to downstream sites (gills: 1.56 mg g(-1); liver: 1.23 mg g(-1)), could not be explained by induction caused by increased metal levels in the river water, but presumably by physiological changes caused by general stress due to low oxygen saturation. In addition, at the sampling site characterized by inorganic and fecal contamination, increased incidence of bacterial bioconcentration in internal organs (liver, spleen, kidney) was observed, as well as decrease of intestinal parasitic infections, which is a common finding for metal-contaminated waters. Based on our results, it could be concluded that even moderate contamination of river water by multiple contaminants could result in unfavourable living conditions and cause detectable stress for aquatic organisms.

Distribution of selected essential (Co, Cu, Fe, Mn, Mo, Se, and Zn) and nonessential (Cd, Pb) trace elements among protein fractions from hepatic cytosol of European chub (Squalius cephalus L.).

Association of selected essential (Co, Cu, Fe, Mn, Mo, Se, and Zn) and nonessential (Cd, Pb) trace elements with cytosolic proteins of different molecular masses was described for the liver of European chub (Squalius cephalus) from weakly contaminated Sutla River in Croatia. The principal aim was to establish basic trace element distributions among protein fractions characteristic for the fish living in the conditions of low metal exposure in the water. The fractionation of chub hepatic cytosols was carried out by size exclusion high performance liquid chromatography (SE-HPLC; Superdex™ 200 10/300 GL column), and measurements were performed by high resolution inductively coupled plasma mass spectrometry (HR ICP-MS). Elution profiles of essential elements were mostly characterized by broad peaks covering wide range of molecular masses, as a sign of incorporation of essential elements in various proteins within hepatic cytosol. Exceptions were Cu and Fe, with elution profiles characterized by sharp, narrow peaks indicating their probable association with specific proteins, metallothionein (MT), and ferritin, respectively. The main feature of the elution profile of nonessential metal Cd was also single sharp, narrow peak, coinciding with MT elution time, and indicating almost complete Cd detoxification by MT under the conditions of weak metal exposure in the water (dissolved Cd concentration ≤0.3 µg L(-1)). Contrary, nonessential metal Pb was observed to bind to wide spectrum of proteins, mostly of medium molecular masses (30-100 kDa), after exposure to dissolved Pb concentration of ~1 µg L(-1). The obtained information within this study presents the starting point for identification and characterization of specific metal/metalloid-binding proteins in chub hepatic cytosol, which could be further used as markers of metal/metalloid exposure or effect on fish.

Multielement analysis in the fish hepatic cytosol as a screening tool in the monitoring of natural waters.

The possibility of direct measurement of trace elements in hepatic cytosol of European chub (Squalius cephalus) by high-resolution inductively coupled plasma mass spectrometry (HR ICP-MS) after cytosol dilution with Milli-Q water and subsequent acidification was investigated. Due to low detection limits of this procedure, determination of 13 elements (As, Cd, Co, Cu, Fe, Mn, Mo, Pb, Sb, Sn, Sr, V and Zn) was possible in the chub hepatic cytosol, exhibiting excellent measurement repeatability in duplicates. Some of these elements were also measured by HR ICP-MS in acid digested cytosols (Cd, Co, Cu, Fe, Mn, Mo, Sr, V and Zn). Good agreement of the results obtained after sample dilution and sample digestion indicated that complex organic matrix of hepatic cytosol did not affect measurement reliability. Cytosolic concentrations of 13 trace elements in the chub liver were quantified in the following order: Fe, Zn>Cu>Mn>Mo>Sr, V, Cd>Co>As, Pb>Sn>Sb. Unlike Cd, Cu, Fe, Mn and Zn for which the cytosolic concentrations were previously reported after measurement by AAS, cytosolic concentrations of eight additional trace elements characteristic for the liver of chubs inhabiting the low contaminated river water were reported here for the first time (in nanogrammes per gramme)-Mo, 136.8-183.6; Sr, 32.7-63.0; V, 17.5-69.0; Co, 24.3-30.7; As, 9.9-29.5; Pb, 5.8-35.6; Sn, 5.5-12.4; and Sb, 0.9-2.6. The simultaneous measurement of large number of trace elements in the cytosolic fractions of fish tissues, which comprise potentially metal-sensitive sub-cellular pools, could be beneficial as a screening tool in the monitoring of natural waters, because it would enable timely recognition of increased fish exposure to metals.

Lead concentration increase in the hepatic and gill soluble fractions of European chub (Squalius cephalus)-an indicator of increased Pb exposure from the river water.

PURPOSE: To examine if chronic exposure of feral fish to elevated Pb concentrations in the river water (up to 1 µg L(-1)), which are still lower than European recommendations for dissolved Pb in surface waters (7.2 µg L(-1); EPCEU (Official J L 348:84, 2008)), would result in Pb accumulation in selected fish tissues. METHODS: Lead concentrations were determined by use of HR ICP-MS in the gill and hepatic soluble fractions of European chub (Squalius cephalus) caught in the Sutla River (Croatia-Slovenia). RESULTS: At the site with increased dissolved Pb in the river water, soluble gill Pb levels (17.3 µg L(-1)) were approximately 20 times higher compared to uncontaminated sites (0.85 µg L(-1)), whereas the ratio between contaminated (18.1 µg L(-1)) and uncontaminated sites (1.17 µg L(-1)) was lower for liver (15.5). Physiological variability of basal Pb concentrations in soluble gill and hepatic fractions associated to fish size, condition, sex, or age was not observed, excluding the possibility that Pb increase in chub tissues at contaminated site could be the consequence of studied biotic parameters. However, in both tissues of Pb-exposed specimens, females accumulated somewhat more Pb than males, making female chubs potentially more susceptible to possible toxic effects. CONCLUSIONS: The fact that Pb increase in gill and hepatic soluble fractions of the European chub was not caused by biotic factors and was spatially restricted to one site with increased dissolved Pb concentration in the river water points to the applicability of this parameter as early indicator of Pb exposure in monitoring of natural waters.

Water quality of medium size watercourse under baseflow conditions: the case study of river Sutla in Croatia.

The study on medium size river Sutla in Croatia indicated considerable water contamination at specific sites during the baseflow period, probably associated to low flow-rate (0.73-68.8 m3 s(-1)), and consequently low dilution capacity of this river. Various aspects of contamination were observed: increased conductivity to 1,000 microS cm(-1), decreased dissolved oxygen level to 50%, 4-5 degrees C increased water temperature, increased concentrations of several dissolved trace elements (e.g., maximal values of Li: 45.4 microg l(-1); Rb: 10.4 microg l(-1); Mo: 20.1 microg l(-1); Cd: 0.31 microg l(-1); Sn: 30.2 microg l(-1); Sb: 11.8 microg l(-1); Pb: 1.18 microg l(-1); Ti: 1.03 microg l(-1); Mn: 261.1 microg l(-1); and Fe: 80.5 microg l(-1)) and macro elements (e.g., maximal values of Na: 107.5 mg l(-1); and K: 17.3 mg l(-1)), as well as moderate or even critical fecal (E. coli: 4,888 MPN/100 ml; total coliforms: 45,307 MPN/100 ml; enterococci: 1,303 MPN/100 ml) and organic pollution (heterotrophic bacteria: 94,000 cfu/ml). Although metal concentrations still have not exceeded the limits considered as hazardous for aquatic life or eventually for human health, the observed prominent increases of both metal concentrations and bacterial counts in the river water should be considered as a warning and incentive to protect the small and medium size rivers from the future deterioration, as recommended by EU Water Framework Directive.

The impact of fish spawning on metal and protein levels in gastrointestinal cytosol of indigenous European chub.

The study presented here reports for the first time cytosolic metal and protein levels in the gastrointestinal tissue of field-collected European chub (Squalius cephalus), living in low metal-contaminated river section. In two sub-cellular fractions (untreated cytosol and heat-treated cytosol), isolated from the whole gastrointestinal tract of indigenous chubs collected during spawning (April/May 2006) and post-spawning period (September 2006), seasonal or gender related differences of trace metals and proteins were determined. In both fractions, cytosol and heat-treated cytosol, metal levels decrease as follows: Zn>Fe>Cu>Mn>Cd and are significantly higher during the spawning period. Besides that, the level of heat-sensitive proteins (metalloenzymes) in cytosolic fractions is also significantly higher during the spawning period in both male and female specimens. Higher condition indices of chubs in the spawning period imply higher feeding activity, energy reserves and recent growth of indigenous chubs. Metallothionein levels, determined in the heat-treated cytosol, are comparable in gastrointestinal tract of feral chubs collected in both seasons and confine the background metallothionein levels, on average 3mgg(-)(1) wet tissue. Chub spawning has been identified as a confounding factor, having an impact on cytosolic metal and protein levels, presumably due to enhanced food supply (higher condition indices) and fish spawning (higher gonadosomatic indices in some male specimens).

Assessment of low-level metal contamination using the Mediterranean mussel gills as the indicator tissue.

PURPOSE: The aim of this study was to compare the level of metal contamination in two bays in the middle part of the Eastern Adriatic coastal zone in Croatia using the gills of mussels Mytilus galloprovincialis as indicator tissue. Despite the existing sources of contamination, previous studies with caged mussels only indicated moderate metal contamination of the Kastela Bay, contrary to the Trogir Bay in which marina and shipyard present a probable source of Cu- and Zn-contamination. METHODS: The measurements of metallothioneins (MTs) and metals that induce MT synthesis (Cu, Zn, and Cd) were performed in the heat-treated gill cytosol and total proteins (TPs) in the untreated gill cytosol. MTs were determined by differential pulse voltammetry, Cu and Zn by flame atomic absorption spectrometry (AAS), Cd by graphite furnace AAS, and TPs by Bradford spectrophotometric procedure. RESULTS: The results collected in four sampling campaigns (autumn periods from 2001 to 2004) indicated that MT levels in mussel gills (expressed on dry mass basis 2.3+/-0.3 mg g(-1)) were comparable with basal levels reported in the literature (2.5+/-0.8 mg g(-1)). Observed interindividual, temporal, and spatial MT variability could be associated with different confounding factors, such as the time of sampling, total protein concentration, and mussel size rather than cytosolic levels of Cu and Zn. Metal levels, expressed on wet mass basis, in the heat-treated gill cytosol ranged from 1.33 to 11.31 microg g(-1) for Zn, from 0.72 to 2.96 microg g(-1) for Cu, and from 0.036 to 0.100 microg g(-1) for Cd. The highest Zn level was measured at Vranjic (Kastela Bay)-the site influenced by untreated domestic wastewater, while somewhat increased Zn and the highest Cu levels were found at marina and shipyard locations (Trogir Bay). The highest Cd level was measured at Inavinil (Kastela Bay). CONCLUSIONS: The observed association of gill MT levels with several biotic and abiotic factors limits its use as the biomarker of low-level metal exposure. Therefore, the use of the metal concentrations in the heat-treated gill cytosol of Mediterranean mussels should be considered for the assessment of the low-level metal contamination of coastal marine areas.

The assessment of natural causes of metallothionein variability in the gills of European chub (Squaliuscephalus L.).

The possible causes of the variability of gill metallothionein (MT) levels were studied on 182 specimens of 2- and 3-year old European chub (Squaliuscephalus L.) from the Sava River in Croatia. The most pronounced differences in MT levels were obtained between three sampling campaigns, and especially between periods with presumably different metabolic activities (April/May 2006 vs. September 2005 and 2006). Next to the probable influence of metabolic activity on MT level, the correlation analysis indicated a significant association between MTs and the fish size. Differences between males and females, as well as between mature and non-mature fish, were not observed in the young group of studied chub even in the spring reproductive season. Based on the analysis of the site-specific MT variability, it could be concluded that, under the conditions of low dissolved metal concentrations in the river water (as reported for the Sava River), MTs seem to be more affected by different biotic factors than by metal exposure. Therefore, MTs measured in this study were considered as the constitutive gill MT levels of young European chub. The constitutive MT ranges were defined separately for the season of lower metabolic rate (autumn: 1.31-2.00 mg g(-1)) and the season of higher metabolic activity (spring: 2.15-2.95 mg g(-1)).

Protein and metal concentrations in two fractions of hepatic cytosol of the European chub (Squalius cephalus L.).

Indigenous specimens of European chub were caught at five sites in Sava River during two successive non-reproductive periods and used as bioindicator organisms for the assessment of metal accumulation. Liver was selected as a target organ and the metal concentrations (Zn, Cu, Fe, Mn, and Cd) were determined in two fractions of hepatic cytosol, namely in untreated (S50) and heat-treated (HT S50) fraction. In addition, the concentrations of total cytosolic proteins were measured in untreated (S50) fractions, whereas the concentrations of specific metal-binding proteins, i.e. metallothioneins, were determined in the heat-treated fractions. Concentrations of Zn and Fe were significantly higher in the untreated compared to heat-treated cytosol, because these metals are associated to the heat-sensitive metalloenzyme and high molecular mass proteins. On the contrary, Cu, Mn and Cd were predominantly present in the heat-treated cytosol, which contains metallothioneins. In both fractions of hepatic cytosol, nonessential metal Cd showed statistically significant increase from the upstream sites (median 5 ng mL(-1)) toward the downstream site Jasenovac (median 11 ng mL(-1)). The metallothionein concentrations, on the contrary, did not differ between sites, probably due to low dissolved and labile concentrations of metals known as metallothionein inducers (Zn, Cu, and especially Cd) in river water. Therefore, Cd concentrations in hepatic cytosol of European chub can be recommended as an early-warning marker of fish chronic exposure to Cd from combined sources, water and ingested food.

Seasonal variation of the metal (Zn, Fe, Mn) and metallothionein concentrations in the liver cytosol of the European chub (Squalius cephalus L.).

Seasonal variations of biometric parameters as well as metal and protein concentrations were analyzed in the liver cytosol of indigenous European chub (Squalius cephalus L.). Fish sampling was carried out in the reproductive (spring) and non-reproductive period (autumn) in order to define parameters which are seasonally dependent. The specimens caught in the spring period had higher biometric (liver mass, condition and hepatosomatic indices) and some biochemical parameters (metallothionein (MT) and Mn) that are characteristic for the reproductive period. Contrary, Fe hepatic cytosolic concentrations were significantly lower in specimens from spring than in autumn period. Total cytosolic proteins, Cu and Cd concentrations were not seasonally dependent. Range of constitutive levels was determined for total cytosolic proteins (17.7 to 24.7 mg ml(-1)) from the whole set of data. Constitutive range of seasonally dependent parameters MT (207 to 337 microg ml(-1)) and Mn (0.11 to 0.19 microg ml(-1)) is based on the data from non-reproductive period. In order to avoid the interference of reproductive cycle on the level of selected biochemical parameters, it is recommended to perform chub sampling during non-reproductive period.

Preliminary assessment of total dissolved trace metal concentrations in Sava River water.

This study provides the preliminary data set for total dissolved trace metal concentrations in the surface water of the Sava River in Croatia and the assessment of Sava River water quality status. The highest levels of total dissolved metals were observed for Fe, Mn, and Zn (12.6 +/- 7.8 mirog L(-1), 3.44 +/- 3.95 mirog L(-1), and 2.27 +/- 1.53 mirog L(-1), respectively), the intermediate concentrations for Ni, Cu, and Cr (0.59 +/- 0.14 mirog L(-1), 0.54 +/- 0.14 mirog L(-1), and 0.32 +/- 0.06 mirog L(-1), respectively), and the lowest levels for Co, Pb, and Cd (0.064 +/- 0.022 mirog L(-1), 0.055 +/- 0.051 mirog L(-1), and 0.011 +/- 0.004 mirog L(-1), respectively). The results refer to the grab water samples taken at five sites in the period from March to June, 2006. For four trace metals (Mn, Pb, Zn, and Fe), the high temporal variability within one season was observed. It can present a problem for reliable evaluation of total dissolved concentrations of these metals in the river water, if the assessment is based on the occasional grab water sampling. The comparison of results obtained in this study with previously reported data for several unpolluted rivers indicated that Sava River water reflects a certain anthropogenic impact. However, according to the levels proposed by European regulations, it still can be classified as water containing total dissolved trace metals in concentrations not significantly above the natural level.

Combined use of bioindicators and passive samplers for the assessment of river water contamination with metals.

In autumn 2005, the site-specific variability of cytosolic metal and protein concentrations in gills of European chub from the Sava River could be mostly associated with gill mass variability. In spring 2006, the correlations of metals and proteins with gill mass were mainly nonsignificant, and their site-specific variability could be presumably associated with river water pollution. The spring cytosolic concentrations of Zn, Cu, and Mn did not differ significantly between the sites (medians: 8.37-11.34 microg ml(-1), 68.2-86.2 ng ml(-1), and 55.9-68.6 ng ml(-1), respectively). Increased cytosolic Cd concentrations were obtained at Oborovo and Lukavec Posavski (median: 4.01 ng ml(-1))-the sites influenced by pollution sources from two major urban areas-compared with the remaining sampling sites (median: 1.93 ng ml(-1)). Cytosolic Fe concentrations were almost two times higher at Jasenovac (median: 11.98 microg ml(-1)) compared with the concentrations at Sava-Zagreb (median: 6.72 microg ml(-1)). Labile Fe concentrations measured in river water with passive samplers indicated that cytosolic Fe concentrations in the spring possibly reflected water-borne Fe uptake. The spring cytosolic protein concentrations decreased from upstream (Otok Samoborski: 27.2 +/- 5.6 mg ml(-1)) toward the downstream sites (Lukavec Posavski: 14.8 +/- 2.8 mg ml(-1)), possibly because of the influence of organic pollution and water toxicity. The spring period seems to be a more appropriate season for the assessment of the river water pollution if chub gills are used as the target organ.

The influence of the season and the biotic factors on the cytosolic metal concentrations in the gills of the European chub (Leuciscus cephalus L.).

The influence of the season and the biotic factors (age and gill mass) on metal and protein levels in the gill cytosol of the young chubs (2 and 3 years old) was studied in Sava River in autumn 2005 and spring 2006. The obtained results are the first reported cytosolic concentrations of five metals and proteins for the chub gills. The average levels in autumn and spring, respectively, for total cytosolic proteins were 11.2 and 19.9 mg ml(-1), for Zn 6.3 and 10.3 microg ml(-1), for Fe 3.9 and 9.6 microg ml(-1), for Cu 68.4 and 79.0 ng ml(-1), for Mn 55.0 and 63.5 ng ml(-1), and for Cd 2.9 and 3.6 ng ml(-1). The influence of the gill mass on both the protein and the metal levels was observed, but it was seasonally dependent. In autumn, positive correlations were obtained between the gill mass and four parameters (total proteins, Mn, Zn and Fe), and negative with Cu. Contrary, in spring, even negative correlations of total proteins and some metals with the gill mass were observed. The proposed explanation for the different dependence of metal levels on the gill mass in autumn and spring was the seasonal difference in feeding intensity and metabolic rate, with presumably faster metabolism and water filtration through gills in spring. This hypothesis was further supported by the statistically significantly higher concentrations of the total proteins, Zn and Fe, as well as the Fulton condition indices in the spring period.

Metallothionein in intestine of red mullet, Mullus barbatus as a biomarker of copper exposure in the coastal marine areas.

Dietary metal exposure in fish is studied in native red mullet, Mullus barbatus, involving metallothionein (MT) response in the intestine, a primary site for metal uptake. Intestinal MTs (microg mg(-1) proteins), total proteins (mgml(-1) S50), Cu, Zn, Fe, Mn and Cd (microg ml(-1) S50) are considered as indicators of metal exposure in specimens from near-shore and off-shore locations of the Eastern Adriatic Sea. Metallothionein and Cu are statistically significantly higher in 68 specimens from the Kastela Bay, a near-shore area (30.0+/-6.71microg MT mg(-1) proteins; 0.30+/-0.08microg Cu ml(-1) S50), in comparison to 51 specimens from off-shore area, Solta Island (25.9+/-4.21microg MT mg(-1) proteins; 0.26+/-0.09microg Cu ml(-1) S50). Cytosolic Cu, a MT inducer, is significantly correlated (p<0.05, r=0.44) with MT in specimens from the Kastela Bay, independent on fish biometry. Our study indicates that cytosolic intestinal Cu and MT represent a measurable biochemical response in red mullet specimens, dwelling above sediments contaminated with Cu.

Metal exposure assessment in native fish, Mullus barbatus L., from the Eastern Adriatic Sea.

Metallothionein (MT) and metal (Zn, Cu, Fe, Mn, Cd) association in the cytosol of liver, kidney and intestine was studied on native Mullus barbatus specimens. Investigated parameters of all specimens (1 to 8-year-old) were related to the fish length, which reflects fish growth and aging. Cytosolic metal concentrations in liver, kidney and intestine are tissue specific but in general decrease as follows: Fe approximately Zn>Cu>Mn>Cd. Metallothionein levels are also tissue specific with the highest level in intestine, followed by kidney and liver. Percentage partition of MT and Zn in liver, kidney and intestine cytosol of red mullet is comparable. Associations of MT and metals that induce MT synthesis exist in liver for MT/Zn (r=0.28) and MT/Cu (r=0.26) and in intestine for MT/Cu (r=0.38). Unlike essential metals, there is no significant correlation between MT and the toxic metal Cd, what is ascribed to its very low cytosolic concentration. The most pronounced size-related accumulation is found for the toxic metal Cd in liver cytosol (r=0.69), indicating chronic Cd accumulation. Negative correlation between metals (Cd, Zn, Fe, Mn) and the condition factor also indicates chronic metal effects and their metabolic impact.

Age- and tissue-dependent metallothionein and cytosolic metal distribution in a native Mediterranean fish, Mullus barbatus, from the Eastern Adriatic Sea.

The levels of metallothionein (MT), a biomarker of metal exposure, and of cytosolic metals (Zn, Cu, Cd), known as MT inducers, were investigated as variables of age (1 to 8 years) and tissue mass (liver, kidney, brain) of red mullet (Mullus barbatus). Within the age from 1 to 8 years the most significant increase is evident for cytosolic Cd in liver (43-fold) and in kidney (5-fold). MT and essential metals are constant with age or slightly increased. Over the growth period, statistically significant MT and metal increase is evident only between 1 and 6-8 years old specimens, while for Cd in liver and kidney cytosol significant increase already exists at 4 years old specimens. Metal distribution in all tissues follows the order: Zn>Cu>Cd, with even 500-800 times lower Cd levels than essential metal levels. Consequently, MTs follow the levels of essential metals, Zn and Cu, indicating MT involvement in homeostasis of essential metals. In contrast to kidney and brain, hepatic MT levels are not age-dependent. Inclusion of hepatic MT measurements and the associated cytosolic metals will be useful in the assessment of long-term metal effects in demersal fish M. barbatus.