Morphology and Ontogeny of Statoliths in the Grooved Carpet Shell, Ruditapes decussatus.

Statoliths are nonskeletal calcified structures included in most invertebrates' gravireceptors. They have been identified and characterized in several gastropod and cephalopod molluscs and have proved to be very useful for age estimation, growth studies, and connectivity analysis, among other applications. Beyond the scarce available records on their occurrence in Class Bivalvia, statoliths are yet to be documented in the grooved carpet shell, Ruditapes decussatus, a species of high ecological and commercial value. An easy method for the extraction and processing of R. decussatus statoliths is described herein. The statolith growth was followed from the initial shell length (SL) of 2.5-3.5 mm (seed commercial size T1.5) for a period of 6 months in a nursery facility located in the Ria de Aveiro (an estuarine system in NW Portugal). The relationship between statolith diameter (StD) and SL follows the function StD=14.305 SL0.254 (N=173; r=0.855, p<0.001). All statoliths observed showed similar morphostructure and general chemistry: hard, translucent spheres of crystalline calcium oxalate (whewellite), with a central nucleus delimited by a growth check of 6.7±1.0 µm in diameter, possibly as a result of growth arrest during metamorphosis, a metamorphic ring, as described for their gastropod counterparts. Subsequent studies should validate this and will involve a search for the occurrence of additional checks that may potentially be present in older specimens and if they are, would open a new range of most promising applications for bivalve statoliths.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Crystallinity and microchemistry of Nassarius reticulatus (Caenogastropoda) statoliths: towards their structure stability and homogeneity.

Gastropod statoliths are spherical biocarbonates formed during their lifespan. The stability and homogeneity of these structures' mineral matrix was characterised along their radiuses, using Nassarius reticulatus as a model. Generally, they were proved to be bimineralic. Two of the three CaCO3 crystalline polymorphs occurring in biocarbonates - aragonite and calcite - coexist along statolith radiuses, aragonite being unequivocally the most abundant phase. The presence of a diffuse organic matrix was also perceived by the detection of a weak Raman band between 2800 and 3000 cm(-1) consistently observed along radiuses. Beyond the apparent stability and homogeneity, different crystalline orientations were disclosed by Raman spectroscopy. A change in the intensity pattern of the features related to the lattice and bending modes of aragonite between different radiuses give new insights for a possible spherulitic-like growth of these structures. As expected from the relative homogeneity of both mineral and organic signals, there was no pattern on the distribution of Ca, O, Na and S along radiuses. However, a higher concentration of Sr occurs in growth rings (known as winter tags), corroborating the already described negative correlation between the concentration of this element in statoliths and temperature. Despite the apparent stability and homogeneity of the matrix during its lifespan, the periodic distribution of Sr potentially influences a dissimilar incorporation of trace elements in increments and growth rings. Since gastropod statolith elemental fingerprinting was recently suggested as a new tool to monitor marine environmental changes, the pressing need for further studies on the incorporation of traces in these structures is highlighted.

Acute toxicity of tralopyril, capsaicin and triphenylborane pyridine to marine invertebrates.

A need for environmentally acceptable alternative antifouling (AF) biocides has arisen through restrictions in the use of many common biocides in the European Union through the Biocidal Product Regulation (Regulation EU No. 528/2012). Three such alternatives are triphenylborane pyridine (TPBP), tralopyril and capsaicin. This study aims at extending the available information on the toxicity of these three emerging AF biocides to key marine invertebrates. Here we investigate the toxicity of tralopyril and capsaicin to the early life stages of the mussel Mytilus galloprovincialis and the sea urchin Paracentrotus lividus and also of tralopyril, capsaicin and TPBP to the early life stages of the copepod Tisbe battagliai. The EC50 that causes abnormal development of mussel's D-veliger larvae and impairs the growth of sea urchin pluteus larvae are respectively 3.1 and 3.0 µg/L for tralopyril and 3,868 and 5,248 µg/L for capsaicin. Regarding the copepod T. battagliai, the LC50 was 0.9 µg/L for tralopyril, 1,252 µg/L for capsaicin and 14 µg/L for TPBP. The results obtained for the three substances are compared to a reference AF biocide, tributyltin (TBT), and their ecological risk evaluated. These compounds pose a lower environmental risk than TBT but still, our results suggest that tralopyril and TPBP may represent a considerable threat to the ecosystems.

Hazelnut and Walnut Nutshell Features as Emerging Added-Value Byproducts of the Nut Industry: A Review.

The hard-shelled seed industry plays an important role in the global agricultural economy. In fact, only considering hazelnut and walnut, the global nut supply is over 5.6 tons. As a result considerable amounts are produced year by year, burnt or discarded as waste, bypassing a potential source of valuable compounds or features. This review deals with the recent scientific literature on their chemical composition as well as functional applications as an approach to sustain the utilization of the main byproduct derived from industry. Indeed, nutshells have received great interest due to their lignin, antioxidant, physical and mechanical features. It was found that these properties vary among cultivars and localities of plantation, influencing physical and structural features. The inconsistencies regarding the above-mentioned properties of nutshells lead to exploring the status of hazelnut and walnut shell applications in sustainable bio-economy chains. In fact, in terms of potential applications, the state of the art links their use to the construction industry and the manufacture of materials, such as resin or plastic composites, particleboards or construction panels, or vital infrastructure and as a filler in cement pavements. However, their current use continues bypassing their great antioxidant potential and their interesting chemical and mechanical features.

Bio-based resources: systemic & circular solutions for (agro)environmental services.

The global promotion of decarbonisation through the circular solutions and (re)use of bio-based resources (BBR), i.e. waste streams, notably from the agricultural, forest and municipal sectors has steadily increased in recent decades. Among the transformative solutions offered by BBR, biosolids (BS), biochars (BC), and bioashes (BA) specifically attract scientific attention due to their highly complex organo-mineral matrices, which present significant potential for recovery in the agro-/forest-ecosystems. These materials enhance various soil (i) chemical (pH, macro/micro nutrient concentrations, organic matter content), (ii) physical (porosity, water-air relations, compaction) or (iii) microbial (diversity, activity) properties. Furthermore, some of transformed BBR contribute to a multitude of environmental services such as the remediation of contaminated sites and wastewater treatment, employing cost-effective and eco-friendly approaches that align with circular economy/waste management principles, ultimately contributing to climate change mitigation. However, several challenges impede the widespread utilization/transformation of BBR, including technological limitations in processing and application, concerns about contamination (e.g., PAHs, PCBs, micro/nano plastics present in BS), toxicity issues (e.g., heavy metals in BA or nanoparticles in BC), and regulatory constraints (e.g., non-uniform regulations governing the reuse of BA and BS). Addressing these challenges demands an interdisciplinary and intersectoral approach to fully unlock the potential of BBR in sustainable decarbonisation efforts.

Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus.

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.

Antioxidants in Shell and Nut Yield Components after Ca, Mg and K Preharvest Spraying on Hazelnut Plantations in Southern Chile.

To determine the effects of pre-harvest calcium (Ca), magnesium (Mg) and potassium (K) spraying on the antioxidant activity and capacity of hazelnut (Corylus avellana L.) shells, as an approach to sustain the utilization of the main residue derived from this industry, four commercial hazelnut (Tonda di Giffoni) orchards located in Southern Chile (Cunco, Gorbea, Perquenco and Radal), during the 2018/19 season were sprayed three times with five combinations of Ca (300 and 600 mg L-1), Mg (300 and 600 mg L-1) and K (300 and 600 mg L-1). Yield components were determined in harvested whole nuts, whereas Ca, Mg and K concentrations, as well as total phenolic compounds, free radical scavenging antioxidant activity, and oxygen radical absorbance capacity, were determined in shells. All spray treatments with both Ca, Mg and K combinations showed a significant interaction between locality and treatment (L × T) on increased stabilized nut yield (kg ha-1) in comparison with the control treatments, whereas nut quality was differentially affected by treatment and orchard locality, thus confirming a significant L × T relationship regarding nut length and kernel yield. However, locality showed a major effect on kernels and shells. A significant relationship was determined between locality and the Ca, Mg and K spraying (L × T) concerning antioxidant compounds such as phenolics, whose amounts exceeded those of the control treatments by three times. Antioxidant capacity and activity in shells showed a significant L × T relationship, and nutshells collected from Cunco showed remarkably (3-4 times) higher levels of these factors than the other evaluated localities. Interactions between spray treatment and orchard location were responsible for the different values obtained in the experiments, confirming the influence of the environment on the efficacy of the treatments. Finally, these shells are worth further study as an interesting residue of the hazelnut industry due to their nutritional and antioxidant properties.

Study of the Mandibular Bone Microstructure and Blood Minerals Bioavailability in Rainbow Trout (Oncorhynchus mykiss, Walbaum 1792) from Freshwater.

Farmed salmonids show alterations in bone structure that result in skeletal deformities during formation, repair, and regeneration processes, with loss of mineralization at the level of the axial skeleton, mainly the head and spine, affecting their quality of life and even causing death. Despite improving factors, such as farming conditions, diets, and genetics, bone alterations appear more frequently in farmed fish than in wild fish. Thus, we used SEM-EDX, and TGA-DSC to study bone mineralization in farmed and wild rainbow trouts. As expected, we found significant differences in the nutritional parameters of farmed and wild fish (p < 0.05). Microstructural analyses indicated that farmed fish have a more robust mineral structure (p < 0.05), confirming the differences in mineralization and microstructure between both groups. However, the mechanisms regulating absorption and distribution in the organism and their effect on bone mineralization remain to be known. In our study, the combined use of techniques such as SEM-EDX and TGA-DSC allows a clearer assessment and detailed characterization beneficial to understanding the relationship between diet control and bone microstructure.

L-NAME and L-arginine differentially ameliorate cigarette smoke-induced emphysema in mice.

Nitric oxide (NO) represents one of the most important intra- and extracellular mediators and takes part in both biologic and pathologic processes. This study aimed to verify the treatment with an NO inhibitor and an NO substrate in pulmonary emphysema induced by cigarette smoke (CS) in a murine model. We compared N-acetylcysteine (NAC), a precursor of glutathione, to G-nitro-L-arginine-methyl ester or L-NAME (LN), which is an NO inhibitor, and to l-arginine (LA), which is a substrate for NO formation. Mice were divided into several groups: control, CS, CS + LN, CS + LA, and CS + NAC. Control and CS groups were treated daily with a vehicle, while CS + LN, CS + LA, and CS + NAC groups were treated daily with LN (60 mg/kg), LA (120 mg/kg) and NAC (200 mg/kg), respectively. The bronchoalveolar lavage was analyzed and the lungs were removed for histological and biochemical analysis. CS increases neutrophil number. Neutrophil number was lowest in CS + LN, followed by CS + LA. The lungs of CS + LN, CS + LA and CS + NAC mice were protected compared to the lungs of CS mice, but not equal to the quality of lungs in control mice. The CS group also exhibited increased oxidative stress, which was also present in the CS + LN group and to a lesser extent in the CS + LA group. Tissue inhibitor of metalloproteinase 1 and 2 increased in the CS + LN group and to a lesser extent in the CS + LA group relative to the control group. These results suggest that LN and LA treatment protected the mouse lung from CS. However, NAC treatment was more than LN and LA. We suggest that the protection conferred by LN treatment requires a balance between proteases and antiproteases, and that protection conferred by LA treatment involves the balance between oxidants and antioxidants.

An innovative statistical approach for analysing non-continuous variables in environmental monitoring: assessing temporal trends of TBT pollution.

The current work presents an innovative statistical approach to model ordinal variables in environmental monitoring studies. An ordinal variable has values that can only be compared as "less", "equal" or "greater" and it is not possible to have information about the size of the difference between two particular values. The example of ordinal variable under this study is the vas deferens sequence (VDS) used in imposex (superimposition of male sexual characters onto prosobranch females) field assessment programmes for monitoring tributyltin (TBT) pollution. The statistical methodology presented here is the ordered logit regression model. It assumes that the VDS is an ordinal variable whose values match up a process of imposex development that can be considered continuous in both biological and statistical senses and can be described by a latent non-observable continuous variable. This model was applied to the case study of Nucella lapillus imposex monitoring surveys conducted in the Portuguese coast between 2003 and 2008 to evaluate the temporal evolution of TBT pollution in this country. In order to produce more reliable conclusions, the proposed model includes covariates that may influence the imposex response besides TBT (e.g. the shell size). The model also provides an analysis of the environmental risk associated to TBT pollution by estimating the probability of the occurrence of females with VDS ≥ 2 in each year, according to OSPAR criteria. We consider that the proposed application of this statistical methodology has a great potential in environmental monitoring whenever there is the need to model variables that can only be assessed through an ordinal scale of values.

Nucella lapillus L. imposex levels after legislation prohibiting TBT antifoulants: temporal trends from 2003 to 2008 along the Portuguese coast.

Nucella lapillus (dog whelk) imposex levels were assessed along the mainland Portuguese coast in 2006 and 2008 and were compared with available data from 2003 for the same area. Given that specimen size has been described as a factor inducing variation in some of the imposex assessment indices, and thus resulting in less reliable results, new monitoring and data analysis procedures are described and applied to study change of imposex levels from 2003 to 2008. A significant decline in imposex intensity was observed in the study area during the study period, and the Portuguese coast ecological status (under the terms defined by the OSPAR Commission) notably improved after 2003, confirming the effectiveness of the Regulation (EC) No. 782/2003 in reducing TBT pollution. Nevertheless, N. lapillus populations are still extensively affected by imposex, and fresh TBT inputs were detected in seawater throughout the coast in 2006. These recent inputs are attributed to vessels still carrying TBT antifoulants applied before 2003, as their presence in vessels was only forbidden in 2008. Considering that Regulation (EC) No. 782/2003 is an anticipation of the IMO global ban entered into force in September 2008, a worldwide-scale decrease in TBT pollution can be expected in the near future.

Combined use of Nassarius reticulatus imposex and statolith age determination for tracking temporal evolution of TBT pollution in the NW Portuguese continental shelf.

Spatial and temporal trends of tributyltin (TBT) pollution on the northern continental shelf of Portugal (5-34 m depth) were assessed using Nassarius reticulatus as a bioindicator. Imposex levels were determined in 2006 and 2010, and integrated with age readings from statoliths for a better chronological analysis of the data. Females affected with imposex were detected in 84% of the sites surveyed in 2006, with levels higher than the Ecological Quality Objective (EcoQO) set by the OSPAR Commission for N. reticulatus (VDSI < 0.3) in approximately half of those sites. In 2010, imposex was only detected in 20% of the surveyed sites and, when detected, the VDSI were lower than the EcoQO, except for one location. The comparison between the current results and the levels previously reported for 2004 and 2005 in the same offshore area evidences a clear decline of imposex in 2010 that confirms the efficacy of Regulation (EC) No. 782/2003 and the IMO AFS-Convention (entered into force in 2003 and 2008, respectively). Moreover, the age of the whelks used in the monitoring is estimated in 3-5 years, indicating that actual TBT water contamination in 2010 may in fact be lower than that extrapolated from the imposex levels. We conclude that the legislation was indeed very effective in reducing TBT pollution in the NW Portuguese continental shelf, not only in terms of the magnitude but also in terms of fast field repercussion.

Long-term monitoring of Nucella lapillus imposex in Ria de Aveiro (Portugal): When will a full recovery happen?

Nucella lapillus imposex has been monitored in Ria de Aveiro (Portugal) in order to understand the evolution of organotin (OT) pollution after the EU ban on OT-based antifouling systems (OT-AFS). A rapid decrease of imposex occurred, but a countercurrent tendency upsurged recently (2018-2019) with a slight increase of imposex at some sites, instead of a continuous decline towards a complete recovery. In 2018 the vas deferens sequence index ranged between 0.6 and 1.0 across stations and the percentage of females with imposex varied from 63 to 100%. In 2019 these parameters varied between 0.4 and 1.0 and 37-100%, while the concentration of butyltins (TBT, DBT, MBT) in the dogwhelk tissues were < 1 ng Sn g-1 dw. Although N. lapillus imposex levels are low they are indicative of OT pollution. The full recovery of N. lapillus imposex in the future is uncertain and a cause for concern.

Temporal evolution of imposex and butyltin contamination in Gemophos viverratus from São Vicente (Cabo Verde) - a countercurrent trend on the world scenario.

Organotin (OT) based Antifouling Systems (AFS) were globally banned by the AFS Convention since 2008, but the Republic of Cabo Verde did not ratify this Convention, nor did it develop a national legislation to control OT-AFS. Gemophos viverratus imposex and butyltin tissue contamination were assessed around the São Vicente Island (Cabo Verde) in 2019 and compared with the data available from 2012. The vas deferens sequence index (VDSI), the relative penis length (RPL), the percentage of females with imposex (%I) and the percentage of sterilized females (%S) in 2019 ranged from 0 to 4.0, 0-84.4%, 0-100% and 0-5.1%, respectively, whilst TBT reached maximum values of ≈30 ng TBT-Sn g-1 dw in the whelk tissues. These values are very similar to those registered in 2012, which indicates that TBT pollution has not decreased over the years, in contrast to the declining trend observed worldwide.

Organ-related cigarette smoke-induced oxidative stress is strain-dependent.

BACKGROUND: Cigarette smoke (CS) is associated with oxidative stress in several organs because it contains high concentrations of free radicals and reactive oxygen species. Experimental models, using different strains, provide important insights into the genetic basis of diseases. This study sought to identify, in different mouse strains, the organ that is most-susceptible to CS-induced oxidative stress to obtain an optimized experimental animal model of oxidative injury induced by CS. MATERIAL/METHODS: Male Swiss, DBA/2, C3H, BALB/c, and C57BL/6 mice were exposed to CS 3 times a day (4 cigarettes per session) for 60 consecutive days. Control groups from the same strains were sham-treated. Protein content, malondialdehyde level, myeloperoxidase activity, and nitrite level were assayed in lung, liver, kidney, and brain from all strains. Catalase and glutathione peroxidase activities were measured. Analyses of data were done by using a 1-way ANOVA with Bonferroni's post-test (P<.05). RESULTS: Cigarette smoke exposure resulted in distinct, organ-specific responses among strains. The survival rate of DBA/2 mice was lowest. BALB/c and C57BL/6 strains were more-susceptible to oxidative damage in the lung and liver. C3H and C57BL/6 mice were more-susceptible to oxidative damage in the brain. No renal oxidative damage was seen. CONCLUSIONS: Mouse strains and individual organs display a range of susceptibilities to CS-induced oxidative stress. BALB/c and C57BL/6 strains appear to be the best choices as experimental models for studying CS effects on liver and lung, and C3H and C57BL/6 strains for CS-effects on the brain.

Imposex in Bolinus brandaris from the Ria formosa lagoon (southern Portugal): usefulness of "single-site baselines" for environmental monitoring.

The present study aimed at creating a so called "single-site baseline" (SSB) of imposex in the purple dye murex (Bolinus brandaris) from the Ria Formosa lagoon (Algarve coast, southern Portugal), at the moment that the IMO Antifouling System Convention (banning globally the use of organotin compounds in antifouling paints on ships' hulls) entered into force (September 2008). In the context of monitoring long-term temporal trends of pollution levels, SSBs are proposed whenever it is difficult to obtain adequate numbers of bioindicator specimens in most of the study area or if significant short-term variability in imposex levels is likely to occur. In order to deal with these constraints, this study created a SSB by sampling on a monthly-basis (from October 2008 to September 2009) in a single site considered to be representative of the Ria Formosa as a whole (vicinities of the Culatra Island) and where B. brandaris is abundant all year round. The SSB provided a good characterisation of the monthly variation in the imposex indices, which ranged throughout the year between 72.7 < I% < 100; 0.08 < FPLI < 1.73; 1.20 < RPLI < 23.27; 1.29 < VDSI < 3.54. Only a SSB could have detected this large variability, which otherwise might have been unnoticed and would have produced biased temporal and spatial comparisons, leading to erroneous interpretations of environmental monitoring data. Therefore, this SSB of imposex in B. brandaris constitutes valuable reference data to assess the future trend of organotin pollution in the Ria Formosa, after the benchmark date of the complete eradication of organotin from antifouling agents was implemented worldwide.

Hydrobia ulvae imposex levels at Ria de Aveiro (NW Portugal) between 1998 and 2007: a counter-current bioindicator?

Imposex expression in prosobranch gastropods has been widely used as a biomarker of tributyltin (TBT) pollution. Estuaries have been described as the most affected areas by this problem since they usually enclose the main TBT sources--ports, dockyards and marinas--resulting from the compound's application as a biocide in antifouling paints on ships. Using Hydrobia ulvae as a bioindicator, the current work addresses the most reliable methods to reduce the influence of critical variables, such as the animals' size, on imposex levels assessment for TBT pollution monitoring and presents its temporal trends from 1998 to 2007 in Ria de Aveiro (NW Portugal) to evaluate the effectiveness of recent legislation applied to reduce TBT environmental levels. H. ulvae imposex levels did not decrease in this estuarine system during the last decade despite the implementation of the EU Regulation No. 782/2003. Instead, there was a global significant increase in the percentage of females affected by imposex and a slight increase of the vas deferens sequence index (VDSI), contrasting with what has been described for other bioindicators in the same study area. These results show that different biology/ecology traits determine distinct routes of TBT uptake and/or bioaccumulation, pointing the importance of choosing the bioindicator depending on the compartment that is being monitored (sediment vs. water). Sediment ingestion as feeding habit is discussed and pointed as a reason to choose H. ulvae as a bioindicator of TBT pollution persistence in sediment. It is therefore predicted that the response of different prosobranch species around the world may diverge according to the compartment that is being monitored and that female masculinisation may not be completely eradicated in the near future due to TBT persistence in sediments.

Factors affecting RPSI in imposex monitoring studies using Nucella lapillus (L.) as bioindicator.

Nucella lapillus (L.) is a marine gastropod mollusc widely used as a bioindicator of TBT pollution in the North Atlantic coastlines. The species reproductive cycle and the male penis length seasonal/spatial variation were studied at a single site at Aveiro seashore (NW Portugal) between December 2005 and June 2007. The main objective of this work is to assess if the "Relative Penis Size Index" (RPSI)--an important imposex assessment index--varies seasonally and spatially in the same sampling site and how this can affect results obtained in imposex monitoring programmes. Animals able to reproduce were found every month but a seasonal pattern in N. lapillus reproductive cycle was evident. Female gametogenic maturation varied seasonally and a decrease in capsule gland volume and condition factor occurred in late summer/early autumn. The gametogenesis in males did not show a significant seasonal variation as in females but the condition factor, penis length, amount of sperm and prostate volume also diminished in late summer/early autumn. On the other hand, males that were close to egg capsules clusters had larger penises than those far away from clusters. The temporal and spatial male penis length variation introduces a bias on imposex assessment results when using RPSI and the magnitude of the error involved is evaluated for different TBT pollution levels scenarios. We consider that RPSI provides interesting and complementary information that should not be excluded from monitoring programmes, but temporal or spatial comparisons of imposex should be based on other more reliable imposex indices like the VDSI.

Chemical and biological characterization of estrogenicity in effluents from WWTPs in Ria de Aveiro (NW Portugal).

Effluents from wastewater treatment plants (WWTPs) are responsible for the input of estrogenic contaminants into aquatic ecosystems, leading to widespread effects in wildlife. In the present work, levels of estrone (E1), 17alpha- and 17beta-estradiol (E2), 17alpha-ethinylestradiol (EE2), bisphenol A (BPA), and nonylphenol (NP) were quantified in effluents from WWTPs located in Ria de Aveiro (NW Portugal), as well as in the final effluent discharged into the Atlantic Ocean through the S. Jacinto submarine outfall. Reference sites, located at the entrance of the estuarine system and at the seaside, were also included. Samples were collected under summer (June 2005) and winter (February 2006) conditions. For the summer survey samples, estrogenicity and androgenicity were evaluated using the yeast estrogen screen (YES) and the yeast androgen screen (YAS) assay. Estrone levels varied from 0.5 to 85 ng/L in the summer survey and between