Background, aim and scopeRia de Aveiro (Portugal) is a shallow coastal lagoon of high economic and ecological importance. Hardly any data on its chemical pollution by polar organic pollutants are available in literature. This study focused on the presence and sources of a series of phenolic endocrine-disrupting compounds (EDCs) in this area, including parabens, alkylphenolic compounds and bisphenol-A (BPA). A number of possible sources of pollution are present in the area, including the large harbours present in the lagoon, the city of Aveiro and the rivers discharging into the area. A recently constructed submarine wastewater outfall, located a few kilometres from the lagoon inlet has also been suggested as a possible source of pollution to Ria de Aveiro in several publications. The aim of the current field study was to investigate the occurrence and main sources of phenolic endocrine disruptors in Ria de Aveiro.Materials and methodsAn extensive sampling campaign was performed, with surface water and wastewater grab samples taken at over 50 locations, in duplicate on different days. Samples were treated using solid phase extraction and analysed by liquid chromatography tandem mass spectrometry.Results and discussionConcentrations in lagoon water were generally low: not exceeding 20 ng/L for most analytes. Levels in river water exceeded those in the lagoon by a factor 3 to 500 (o-phenylphenol (PhP) and nonylphenoxy ethoxy acetic acids (A9PEC), respectively), with concentrations up to 700 ng/L for BPA and 7,300 ng/L for A9PEC. Samples from the harbours showed EDC levels similar to those in the rest of the lagoon, but in the city of Aveiro, elevated concentrations were observed for alkylphenol ethoxylates (A9PEO), A9PEC, PhP and BPA. Wastewater effluents showed low levels for parabens, whilst alkylphenolic compounds reached several micrograms per litre. The effluents are discharged into the ocean via a submarine outfall, but as marine water near the outfall showed slightly elevated concentrations only for A9PEO, it does not seem to be a significant source of these EDCs for the area.ConclusionsAll the studied phenolic EDCs were detected in the study area, with high levels found in some of the rivers discharging into the lagoon, and generally low concentrations in the lagoon itself. The main sources for all investigated EDCs were the rivers Caster and Antuã which discharge into the lagoon. The city of Aveiro was identified as a secondary source. As the tidal water exchange volume is much larger than the freshwater input from the rivers, concentrations of phenolic EDCs remained low in the lagoon.Electronic supplementary materialThe online version of this article (doi:10.1007/s11356-009-0275-5) contains supplementary material, which is available to authorized users.

Samples of Nassarius reticulatus (L.) were obtained from May to July 2000 on the W Portuguese coast. N. reticulatus imposex (superimposition of male characters onto female prosobranchs) increased with the proximity of harbours. The percentage of imposex-affected females (%I) varied between 0 and 100%. The penis length index (PLI) and the relative penis length index (RPLI) were between 0 and 10.7 mm and 0.0 and 92.1%, respectively. The vas deferens sequence index (VDSI) ranged from 0 to 5. The average oviduct stage (AOS) varied between 0.0 and 1.3. Sterile females (22 specimens) were found at 8 of the 40 sampling stations with frequencies between 5 and 50% of the total females sampled at each site. Sterility occurred only in females highly affected with imposex. Tributyltin (TBT) and triphenyltin (TPT) female body burden varied from < 20 to 1368 and from <10 to 256 ng Sn g -1 dry weight (dry wt), respectively. The TPT residue was, on average, 18% of the TBT residue, and there was a highly significant correlation between TBT and lnTPT (r = 0.88, p < 0.001). Dibutyltin (DBT) and monobutyltin (MBT) female body burden varied from < 34 to 721 and from < 24 to 703 ng Sn g -1 dry wt, respectively. TBT represented the major fraction (34 to 60%) of the total butyltins, followed by DBT (24 to 40%) and MBT (10 to 31%). Highly significant correlations (p < 0.001) were established between the lnTBT female residue and the indices RPLI (r = 0.88), VDSI (5) (r = 0.81) and AOS (r = 0.72), as well as between TBT and percentage female sterility (%STER) (r = 0.61).KEY WORDS: Nassarius reticulatus · Imposex · Portugal · Tributyltin · Triphenyltin · SterilityResale or republication not permitted without written consent of the publisher

It has been widely recognized that pesticides represent a potential threat in aquatic ecosystems. However, the knowledge on the genotoxicity of pesticides to fish is still limited. Moreover, genotoxic studies have been almost exclusively focused on the active ingredients, whereas the effect of adjuvants is frequently ignored. Hence, the present study addressed the herbicide Roundup®, evaluating the relative contribution of the active ingredient (glyphosate) and the surfactant (polyethoxylated amine; POEA) to the genotoxicity of the commercial formulation on Anguilla anguilla. Fish were exposed to equivalent concentrations of Roundup® (58, 116 μg L⁻¹), glyphosate (17.9, 35.7 μg L⁻¹) and POEA (9.3, 18.6 μg L⁻¹), during 1 and 3 days. The comet assay was applied to blood cells, either as the standard procedure, or with an extra step involving DNA lesion-specific repair enzymes in an attempt to clarify DNA damaging mechanisms. The results confirmed the genotoxicity of Roundup®, also demonstrating the genotoxic potential of glyphosate and POEA individually. Though both components contributed to the overall genotoxicity of the pesticide formulation, the sum of their individual effects was never observed, pointing out an antagonistic interaction. Although POEA is far from being considered biologically inert, it did not increase the risk associated to glyphosate when the two were combined. The analysis of oxidatively induced breaks suggested that oxidation of DNA bases was not a dominant mechanism of damage. The present findings highlighted the risk posed to fish populations by the assessed chemicals, jointly or individually, emphasizing the need to define regulatory thresholds for all the formulation components and recommending, in particular, the revision of the hazard classification of POEA.

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Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this 'nonmodel' species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator-activated receptor (PPAR) pathways. Application of rosiglitazone, a well-known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT-induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR-responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption.

The prosobranchs Nucella lapillus, Nassarius reticulatus, Hydrobia ulvae and Littorina littorea were sampled from May to July 1998 at 45 stations in the Ria de Aveiro (NW Portugal) and the adjacent coastal zone. The distribution patterns of these populations were related to the salinity gradients. The tributyltin (TBT) concentration in the water was determined for 17 stations spread over the study area during a neap tide in July 1998 and varied from 9 to 42 ng Sn l

The reproductive cycle of Nassarius (Hinia) reticulatus was investigated in the Ria de Aveiro on the north-west Portuguese coast during 1992 and 1993. A clear seasonal pattern was observed with the beginning of gametogenesis in August followed by ripening of the gonads in January and February and spawning from February to July. The increase of water temperature above 11°C in February 1993 was associated presumably with the release of gametes. Recruitment inside the Ria of juveniles with 6·5 mm mean shell height was detected in February 1993 and these individuals were spawned in the previous year. Males of N. reticulatus exhibited a considerable variation in length of the penis throughout the reproductive cycle. The accuracy of the use of relative penis length (RPL) index for intersite comparisons of imposex is discussed and the magnitude of the error that might be involved when comparisons are made using populations in different phases of the reproductive cycle is evaluated.

The present study aimed at analysing the monthly variation in penis length (PL) during the reproductive cycle of the purple dye murex (Bolinus brandaris). Two hypotheses were tested: PL variation reflects male maturation and reproductive activity; PL variation bias the calculation of imposex indices based on penis measurement (RPLI and RPSI). Sampling was performed during 1 year in a population from Ria Formosa lagoon (Algarve coast, southern Portugal) with high incidence of imposex. Penis-bearing individuals were measured for shell length (SL) and PL, and two bio-physiological indices were calculated for both sexes, the general condition index (K) and the gonadosomatic index (GSI). PL presented monthly variation in both sexes, but while female PL showed small and random oscillation, male PL exhibited significant variation throughout the reproductive cycle (as evidenced by the similar and synchronous trends between male PL and GSI). These findings have implications both for reproduction studies and imposex monitoring: calculation of standardised PL (PL/SL) allows for gender identification without killing the organism and constitutes a useful penial index for assessing male maturation and reproductive activity; RPLI and RPSI should be applied cautiously in spatial and temporal comparisons of imposex severity in B. brandaris.