Laia Quirós

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.

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.

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.

Blood biomarkers and contaminant levels in feathers and eggs to assess environmental hazards in heron nestlings from impacted sites in Ebro basin (NE Spain).

Blood biomarkers and levels of major pollutants in eggs and feathers were used to determine pollution effects in nestlings of the Purple Heron Ardea purpurea and the Little Egret Egretta garzetta, sampled on three Ebro River (NE Spain) areas: a reference site, a site affected by the effluents of a chlor-alkali industry and the river Delta. The two impacted heron populations showed mutually different pollutant and response patterns, suggesting different sources of contamination. In the population nesting near the chlor-alkali plant, elevated levels of hexachlorobenzene (HCB) and polychlorobiphenyls (PCBs) in eggs, and mercury in feathers in A. purpurea chicks were related with reduced blood antioxidant defenses and increased levels of micronuclei. In Ebro Delta, high levels of plasmatic lactate dehydrogenase in A. purpurea chicks and high frequency of micronuclei in blood of both species were tentatively associated with intensive agricultural activities taking place in the area. These results provide the first evidence of a biological response in heron chicks to the release of pollutants at a chlor-alkali plant.

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.

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.

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.

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.

Biological Effects of Chemical Pollution in Feral Fish and Shellfish Populations from Ebro River: From Molecular to Individual Level Responses

A multilevel approach, from whole animal to molecular level, was applied to the study of the biological impact of chemical pollution in fish and shellfish populations from the rivers Vero, Cinca, and from the Flix reservoir in the Ebro River. The analysis provided a general picture of the health status of the rivers and quantified the physiological effects of different pollutants originating in existing chemical plants discharging in the area. The data show that fish acclimated to very high concentrations of some toxicants, like mercury, whereas organochlorinated compounds (OCs) and poly bromo diphenyl ethers apparently induce permanent negative effects, including oxidative stress, poor condition and fertility, DNA damage, and liver and kidney histological anomalies. Toxic determinants appeared different for vertebrates and invertebrates and suggest that a key difference between both animal groups may be the presence of activable aryl hydrocarbon receptor (AhR), which only occurs in deuterostomata (Chordates, Echynoderma and alikes). The adverse biological effects were recorded up to 30–35 km downstream the different sources, and their distribution differed for OCs and for Hg. Intensive local agricultural practices, rather than pollution from the Ebro’s chemical plants, seem to account for adverse biological effects observed in the Ebro Delta.