Analysis of micronucleated erythrocytes in heron nestlings from reference and impacted sites in the Ebro basin (N.E. Spain).

The frequency of micronuclei (MN) in peripheral erythrocytes was tested for 59 heron nestlings (Ardea purpurea, Egretta garzetta and Bubulcus ibis) sampled at two areas (polluted and reference) on the River Ebro (NE Spain) and at its Delta during Spring 2006. Flow-cytometry analysis revealed higher (three- to six-fold) MN counts in samples from the most polluted site relative to samples from the reference area. Samples from the Delta showed intermediate values. Age, morphometric parameters (weight, tarsus size and bill-head length) and maturation status showed no significant differences among the different populations for each species; nor were they correlated with MN levels. The data suggest that elevated levels of MN in chicks in impacted areas reflected the chemical pollution of their nesting sites. The use of nestlings for this assay appears to be a convenient, non-destructive method to assess the impact of pollution in natural bird populations.

Distribution and biological impact of dioxin-like compounds in risk zones along the Ebro River basin (Spain).

The aim of this investigation was to evaluate the environmental impact associated to PCDDs/Fs and dioxin-like PCBs in the Ebro River basin. Sediments and fish from several species were sampled at three sites with different historical pollution records, including the Barbastro area with different industrial activities, and the Flix and Monzón sites, associated to heavy organochlorine compound pollution. Seventeen toxic PCDDs/Fs and 12 dioxin-like PCBs were analyzed by GC-MS. The results obtained indicated significant accumulation of dioxin-like PCBs, but not PCDDs/Fs, in sediments and fish at the Flix site compared to the other sites. Concomitantly, cytochrome p450 1A (CYP1A) expression, a known indicator for pollution by dioxins and dioxin-like PCBs, was significantly elevated in barbel (Barbus graellsii) from the Flix site, compared to the population from the Barbastro site. CYP1A expression correlated with the concentration of dioxin-like PCBs in the fish fat, whereas no significant correlation was found with PCDDs/Fs concentrations. Our data suggest a significant biological impact at the Flix site, closely related to the presence of dioxin-like PCBs, whereas the PCDDs/Fs contribution to this impact appears to be non-significant, at least in the studied sites.

Effects on growth and biochemical responses in juvenile gilthead seabream 'Sparus aurata' after long-term dietary exposure to low levels of dioxins.

The farmed fish gilthead seabream (Sparus aurata) were fed with a dry feed spiked with a low level (23 ng WHO-TEQ/kg of feed) polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) mixture in order to assess bioaccumulation of these contaminants in the muscle and liver tissues after long-term exposure (approximately 390 days). Furthermore, effects on fish growth, feeding and on the response of some biochemical markers (induction of the CYP1A dependent EROD activity, the conjugating enzyme GST, the antioxidant enzymes CAT, t-GPX and DTD, lipid peroxidation and the AhR gene expression) were also evaluated. After feeding with the spiked dry feed for 3 months the PCDD/F concentrations in the exposed fish were 5.50 pg WHO-TEQ/g fresh weight (f.w.) in flesh and 8.45 pg WHO-TEQ/g f.w in liver tissue, which are approximately 24-fold and 14-fold higher than background levels, respectively. However, a progressive increase in PCDD/F levels was not found during the rest of the exposure period. Differences in fish growth were not observed between dioxin-exposed and non-exposed animals and, in addition, no mortalities were recorded attributable to the dioxin intake. Significant increases in the EROD activity, as well as in AhR gene expression were observed in liver after approximately 300 days of exposure. However, no effect on the antioxidant enzymes CAT and t-GPX was found.

Environmental monitoring by gene expression biomarkers in Barbus graellsii: laboratory and field studies.

Barbel (Barbus graellsii) is a freshwater fish used as a sentinel species in environmental monitoring programmes and ecotoxicological studies in northern Spain, particularly in the Ebro River basin, the largest freshwater resource in Spain. We developed specific primers for the quantification of CYP1A and metallothionein (MT)-1 and -2 gene expression by QRT-PCR in barbel in order to assess their suitability in biological effect monitoring of dioxin-like compounds and metals. All three genes responded as expected in laboratory tests, using model inducers. Hepatic CYP1A mRNA levels showed a twofold induction in fish injected intraperitoneally with beta-naphthoflavone, whereas MT-1 and MT-2 gene expression was strongly induced by cadmium (15- and 13-fold, respectively) and mercury (five- and eightfold). Barbel populations from different sites on the Ebro River basin showed a good correlation between the historical records of organochlorine pollution, CYP1A expression levels and EROD activity. Nevertheless, although metallothionein protein levels in the liver of wild fish correlated with hepatic levels of mercury, MT-1 and MT-2 gene expression did not correlate with the mercury content or with the concentration of metals in sediments from the sites inhabited by the fish. These results demonstrate the utility of barbel CYP1A-mRNA expression, but not that of MT-1 or MT-2, as a biomarker in field studies. The tools and protocols developed here are likely to apply to other species of the genus Barbus, with some 700 species distributed throughout most of the Old World.

A noninvasive test of exposition to toxicants: quantitative analysis of cytochrome P4501A expression in fish scales.

Elevated expression of cytochrome P4501A (CYP1A) is an established biomarker for exposition to a wide range of toxicants, particularly for dioxin and structurally similar compounds. Expression of CYP1A usually is analyzed in internal organs, which involves dissection of the specimen. To avoid unnecessary animal killing, we present here an alternative method based on the monitoring of CYP1A expression in fish scales. Using beta-naphthoflavone (BNF; 50 mg/kg body wt, intraperitoneal injection) as inducer in goldfish (Carassius auratus), we monitored levels of CYP1A mRNA both in scales and liver of treated and control specimens. Treatment with BNF resulted in a similar induction of CYP1A gene in both tissues, although scales responded faster (at 8 h after treatment) than liver (between 24 and 48 h). The scale-based test has the unique advantage of allowing sequential testing in the same specimen, which facilitates analysis of the time course of CYP1A induction and allows the study of individual variability. The method implies minimal suffering of the animals, because it only requires removal of a moderate (n = 1-3) number of scales for each time point. This nondestructive, fast, and relatively inexpensive test for toxic exposure therefore is suitable for environmental monitoring and food safety control programs in which specimen preservation is required.

Detection and evaluation of endocrine-disruption activity in water samples from Portuguese rivers.

Water samples (n = 183) from Portuguese rivers were tested for the presence of endocrine disruptors using the recombinant yeast assay (RYA) combined with chemical identification of compounds having endocrine-disruption properties by liquid chromatography coupled to mass spectrometry. Ten selected locations were sampled monthly for a period of 20 months, from April 2001 to December 2002. More than 90% of samples showed either no detectable or low levels of estrogenicity (<0.1 ng/L of estradiol equivalents). The remaining samples (17 in total, 9.3%) showed estrogenicity values ranging from 0.1 to 1.7 ng/L of estradiol equivalents; only two samples showed values greater than 1 ng/L of estradiol equivalents. Most highly estrogenic samples (13 of 17 samples) originated in five sampling sites clustered in two zones near Porto and Lisbon. Chemical analysis detected alkylphenolic compounds (octyl- and nonylphenol plus nonylphenol ethoxylates) in all samples, albeit at concentrations less than 1 microg/L for each compound in 80% of samples. Total analyte concentration exceeded 10 microg/L in only 10 samples, with all but one of those originating from only two sampling sites. In these two locations, a good correlation was observed between the concentrations of octylphenol, nonylphenol, and to a lesser extent, bisphenol A in the samples and their estrogenicity values as calculated by RYA. We conclude that estrogenic activity can be explained by alkylphenol contamination in only these sites; for the remainder, we propose that pesticides and urban waste may be the main factors responsible for estrogenic contamination.

Background fish feminization effects in European remote sites.

Human activity has spread trace amounts of chemically stable endocrine-disrupting pollutants throughout the biosphere. These compounds have generated a background level of estrogenic activity that needs to be assessed. Fish are adequate sentinels for feminization effects as male specimens are more sensitive than humans to exogenous estrogenic compounds. High mountain lakes, the most distant environments of continental areas, only receive semi-volatile compounds from atmospheric deposition. We analyzed the expression levels of estrogen-regulated genes in male fish from these mountain lakes in Europe. Incipient feminization involving expression of estrogen receptor and zona radiata genes revealed a widespread diffuse estrogenic impact. This effect was correlated with the concentrations of some organochlorine compounds in fish and was consistent with the persistent occurrence of these tropospheric pollutants in the most remote planet regions. These results should be of general concern given the increasing endocrine disruption effects in human populations.

Integrated biological and chemical analysis of organochlorine compound pollution and of its biological effects in a riverine system downstream the discharge point.

Pollution in riverine systems, along with its biological effects, may propagate downstream even at considerable distances. We analyzed the organochlorine compound (OC) pollution in a section of the low Ebro River (Northeast Spain) downstream a long-operating chlor-alkali plant. Maximal levels of OCs and of their associated dioxin-like biological activity occurred in residue samples from the plant, and persisted in river sediments some 40km downstream (Xerta site). Biological analysis at multiple organization levels in local carp (Cyprinus carpio, EROD, Cyp1A mRNA expression in the liver, hepatosomatic index, condition factor, and micronuclei index in peripheral blood) showed a similar pattern, with a maximal impact in Ascó, few kilometers downstream the plant, and a clear reduction at Xerta. This combination of chemical, molecular, cellular and physiological data allowed the precise assessment of the negative impact of the chlor-alkali plant on the quality of river sediments and on fish, and suggests that sediments may be a reservoir for toxic substances even in dynamic environments like rivers.

Physiological responses to mercury in feral carp populations inhabiting the low Ebro River (NE Spain), a historically contaminated site.

The low Ebro River course (Northeast Spain) is historically affected by mercury pollution due to a chlor-alkali plant operating at the town of Flix for more than a century. River sediments analysed during the last 10 years showed high mercury levels in the river section starting just downstream the factory and spanning some 90km, down to the river delta. The possible environmental impact was studied by a combination of field and laboratory studies. Mercury concentrations in liver, kidney and muscle of feral carp (Cyprinus carpio) sampled downstream Flix were one to two orders of magnitude higher than those from carps sampled upstream Flix. Elevated levels of mercury in these samples associated with significant increases on the concentration of reduced glutathione (GSH) in liver and on mRNA expression of two metallothionein genes, MT1 and MT2, in kidney and, partially, in scales, but not in liver. Conversely, no biochemical evidence for oxidative stress or DNA damage was found in these tissues. Non-contaminated carps subjected to intraperitoneal mercury injection resulted in a 20-fold increase of MT1 and MT2 mRNA levels in carp kidney, with minimal changes in liver levels. Our data suggests the coordinate increase of metallothionein mRNA in kidney and of GSH in liver constitutes an excellent marker of exposure to sub-toxic mercury levels in carps. This study also demonstrates that apparently healthy fish populations may exceed the mercury contamination acceptable for human consumption.

Physiological response to persistent organic pollutants in fish from mountain lakes: analysis of CYP1A gene expression in natural populations of Salmo trutta.

Cytochrome p450 1A (CYP1A) gene expression in fish liver increases upon exposure to a variety of chemical compounds, including organochlorine compounds (OCs) and polycyclic aromatic hydrocarbons (PAHs). To use this physiological response as a marker of environmental impact, we developed and validated a set of primers to quantify CYP1A expression by qRT-PCR in the brown trout, Salmo trutta. These primers were used to explore the natural variability of CYP1A expression in 8 isolated populations (65 samples) from European remote lakes, in a geographical distribution encompassing the Tyrolean Alps, Pyrenees, Rila, Tatras, and Norwegian and Scottish mountains. CYP1A expression values varied more than 2 orders of magnitude among samples, with strong variations within each population. CYP1A expression values were significantly elevated in Tatras and Pyrenees fish populations, whereas the lowest median values were found in populations from the Tyrolean Alps and Rila. These values correlated with the content of different environmentally relevant pollutants in the sediments of the lakes harboring each fish population, particularly with HCB and 4,4'-DDE contents. To our knowledge, this works represents a first report of a physiological response linked to persistent organic pollutants in fish from mountain lakes.

Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish.

The presence of the female-specific yolk protein precursor vitellogenin in blood and liver from male fish is widely used as an indicator of endocrine disruption. We studied the induction of vitellogenin mRNA in liver from several species of fish, both maintained in fish tanks or captured in the wild. Our procedure requires minute amounts of liver samples (down to 50 mg), and can be applied to field samples if the appropriate RNA-stabilisation agent is used. We used reverse-transcriptase PCR and quantitative real-time PCR for detection and precise quantitation of vitellogenin mRNA levels. Male mummichog (Fundulus heteroclitus) exposed to 17 beta-estradiol contained levels of vitellogenin mRNA up to 30 times higher than in untreated females and treatment with nonylphenol resulted in a weak but consistent induction of this transcript. We also studied levels of vitellogenin mRNA in a population of common carp (Cyprinus carpio) from the Anoia river, a river known for its high levels of estrogenic alkylphenols. The results were consistent with recorded data for fish from this sampling site. Finally, we also detected vitellogenin mRNA in barbs (Barbus graellsi), a species for which no vitellogenin sequence was available. The use of mRNA quantitation techniques for analysis of feral and cultured fish of different species opens the possibility of more precise detection and further control of the noxious effects of contaminants on the local fauna exposed to them.