Occurrence of endocrine disruptor compounds in the estuary of the Iberian Douro River and nearby Porto Coast (NW Portugal)

Previous studies in the Douro River estuary, based on occasional sampling, showed the presence of several estrogenic disrupting chemicals (EDCs). In sequence, we hypothesized that such type of pollution was more likely an enduring issue than an occasional phenomenon, and that it may even affect recreational beaches in each side of the estuarine mouth. Thus to conclude about the continuous influx of EDCs, water samples were taken twice a day, once per a week, from March to May of 2009, at four sites within the estuary and at two sites in the coastline. After solid-phase extraction, the extracts were prepared for GC-MS analysis of 11 reference EDCs. These embraced natural and pharmaceutical estrogens (17β-estradiol, estrone and 17α-ethynylestradiol) and xenoestrogenic industrial pollutants (4-octylphenol, 4-nonylphenol, and their mono and diethoxylates and bisphenol A). Data showed the ubiquitous presence of potentially hazardous amounts of natural estrogens (particularly of estradiol, ca 5.5?ng?L^?1) and persistent organic pollutants such as nonylphenol mono (up to 550?ng?L^?1) and diethoxylate (up to 2000?ng?L^?1). It was concluded that the targeted area is continuously polluted by the assayed EDCs, and as a consequence, conditions exist for endocrine disturbance in the biota by chronic exposure to EDCs.     

Quantification of 17 endocrine disruptor compounds and their spatial and seasonal distribution in the Iberian Ave River and its coastline

The Ave River flows through the most urbanized and industrialized Porto district areas. Despite ovotestis was recently reported in male fish from the estuary, no data exist on the water levels of natural and pharmaceutical estrogens (17β-estradiol, estrone, and 17α-ethynylestradiol), xenoestrogenic industrial pollutants (4-octylphenol, 4-nonylphenol and their mono and diethoxylates, and bisphenol A), phytoestrogens (formononetin, biochanin A, daidzein, and genistein), and sitosterol. Absence of analyses applies to the river, estuary, and nearby coastline. Those compounds mimic the action of endogenous estradiol, being well-recognized endocrine disrupters (EDCs). To conclude about suspected influxes of estrogenic EDCs into the river and coastline, water samples were taken at eight sites every two months, during one year (2010), at low tide. Data showed ubiquitous presence of potentially hazardous amounts of estrogens (particularly ethynylestradiol, up to 10 ng L^?1), nonylphenol (up to 250 ng L^?1), and sitosterol (up to 6 μg L^?1), which helps explaining the ovotestis emergence in local fish. Also, because physicochemical parameters used in water quality evaluation, such as pH, dissolved oxygen, nitrates, and nitrites levels, were within legal limits – suggesting good quality – our study supports that assessment of human and environmental risks by targeting surface waters requires integrating EDCs monitoring in routine analyses.     

Genetic differentiation in three Sepia species (Mollusca: Cephalopoda) from Galician waters (north-west Iberian Peninsula)

The systematics of the genus Sepia is not yet clear. Morphological evidence has led to S. officinalis Linnaeus, 1758 being considered as belonging to the subgenus Sepia sensu stricto, and S. orbignyana Férussac, 1826 and S. elegans Blainville, 1827 as belonging to the subgenus Rhombosepion. Samples of 30 individuals of S. officinalis and S. orbignyana from both sides of an oceanographic boundary off the north-west Iberian Peninsula, and a sample of S. elegans from the northern side, were collected in 1993–1994. Allozyme electrophoresis for 32 presumptive loci revealed low levels of genetic variability for the three Sepia species (mean expected heterozygosity estimates were <0.052). No significant differences in allozyme frequencies were detected among populations of either S. officinalis or S. orbignyana. The genetic identities (I) of S. officinalis and S. orbignyana (I=0.12) and of S. elegans (I=0.13) were significantly different from that of S. orbignyana and S. elegans (I=0.49). The former are typical of values for confamilial genera, and a new generic status is proposed for the latter two species, which become Rhombosepion orbignyana (Férussac, 1826) and R. elegans (Blainville, 1827).     

Population genetic structure of the prosobranch Nassarius reticulatus (L.) in a ria seascape (NW Iberian Peninsula) as revealed by RAPD analysis

Randomly amplified polymorphic DNA (RAPD) banding patterns were compared between samples of the netted dogwhelk Nassarius reticulatus from 11 locations along the NW Iberian Peninsula coast. To detect if rias (estuaries formed by drowned river valleys) might promote genetic differentiation, five sampling sites were located within a ria (ria of Muros) and the remaining six were scattered along open-coast areas at increasing distances from the ria mouth. Population differentiation statistics (Φ-values) were estimated using a hierarchical analysis of molecular variance (AMOVA) with samples sorted into two groups: open-coast and ria populations. Despite a high potential to disperse, AMOVA demonstrated a modest, statistically significant genetic heterogeneity among N. reticulatus populations. Most of the genetic structure resided in differences among open-coast populations; ria populations were genetically homogeneous. No obvious geographical pattern was detected for the pairwise genetic distances (non-metric multidimensional scaling; UPGMA tree; Mantel test). Unlike previous studies with other species at a variety of estuarine systems other than rias, there was no evidence that the ria of Muros may enhance the genetic divergence of N. reticulatus populations. This discrepancy is discussed in relation to the biological features of the species (high dispersal potential and a preference for mid-low estuarine habitat) and the strong hydrographic connectivity between ria and neighbouring off-shore waters.     

Scales of variation in otolith elemental chemistry of juvenile staghorn sculpin (<Emphasis Type="Italic">Leptocottus armatus</Emphasis>) in three Pacific Northwest estuaries

Although distinct otolith elemental signatures are often observed in fish collected from different estuaries, significant differences are also observed among sites within estuaries. Variation at these smaller spatial scales is not well quantified and has the potential to lead to inappropriate interpretations of otolith elemental data. To quantify variation at multiple scales, the otolith elemental composition (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, and Pb:Ca) of juvenile staghorn sculpin (Leptocottus armatus Girard, 1854) collected from five sites within three estuaries, the Columbia River (two sites) and Coos Bay (one site), Oregon, and Humboldt Bay, California (two sites), was examined. Using laser ablation-inductively coupled plasma mass spectrometry, each otolith was sampled at three zones: (1) within the primordium, which represents the egg and early larval periods; (2) at the outer edge, which represents the juvenile period just prior to collection; and (3) midway between the primordial and edge samples, which represents the late larval and early juvenile period. There were significant differences in otolith metal-to-calcium ratios at all scales examined. Using multi-element otolith signatures, fish were classified to estuary and site within estuary with relatively high levels of accuracy (av = 70–90%). The largest differences in metal-to-calcium ratios were observed between sites within estuaries (<5 km apart) and the smallest differences were observed among otolith zones. Variation in otolith chemistry may be used to provide information on probable habitat use by estuarine fish but studies must be carefully designed. Electronic supplementary material  Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Elemental composition of otolith growth marks in three geographically separated populations of European hake (Merluccius merluccius)

The composition of the opaque and translucent bands in the otoliths of European hake (Merluccius merluccius) from three different populations (Celtic Sea, Galicia shelf and Cretan Sea) was analysed with wavelength dispersive spectrometry (WDS) and differences in Ca, Sr and Na were compared. The translucent bands (annual marks and checks altogether) were significantly richer in Sr and Ca and significantly poorer in Na than opaque bands. Results support the idea of a more compact arrangement of crystals in translucent bands compared to opaque. Analyses did not help to discriminate between putative annual marks and checks. Clear differences in the range of Sr/Ca variation across the otolith as well as in the composition of the otolith core were observed among the three populations. As a result it was possible to discriminate the natal origin of each fish using the composition of the otolith core in Ca, Sr and Na. Nevertheless, the overall pattern across the otolith was similar among the three populations suggesting the existence of ontogenetic trends. Variation of Na/Ca ratios was largely uninformative. Based on these results, a conceptual model for hake is proposed which integrates the different sources of otolith Sr/Ca variation: ambient Sr/Ca, endogenous cues (ontogeny) and exogenous cues (environment).     

Spatial and temporal variability of phytoplankton biomass,primary production and community structure in the Pontevedra Ria (NW Iberian Peninsula): oceanographic periods and possible response to environmental changes

The spatial distribution of phytoplankton assemblages, chlorophyll, primary production and physical and chemical parameters were studied in the Pontevedra Ria in Galicia (NW Iberian Peninsula) from October 1997 to October 1998. In addition to the usual oceanographic periods described for the Galician Coast, two other periods were observed: a Prebloom or winter bloom, occurring during calm, sunny days in winter and a Continental period, related to the allochthonous intrusion of low salinity water from the Miño River in late spring. The phytoplankton biomass and production in both periods reached values of up to 145 mgChl-a m^?2 and 3.6 gC m^?2 day^?1, respectively, which were similar to those found in summer upwelling blooms. Throughout the year, the phytoplankton biomass and primary production gradients along the ria’s axis were highly dependent on the balance between upwelling and runoff. When the latter prevailed, increased values were measured toward the inner ria, while the opposite pattern was observed during summer upwelling blooms. According to projections derived from climate models and the analysis of wind patterns, temperature and precipitation trends in the area, a drop in the productivity of the ria would be expected as a result of reduced upwelling intensity in summer and decreasing rainfall in spring. In any case the estuarine part of the ria would be the most seriously affected.     

Testing the Stochastic Dynamic Methodology (StDM) as a management tool in a shallow temperate estuary of south Europe (Mondego, Portugal)

A long-term monitoring program has been carried out since the early 1990s in the Mondego estuary, on Portugal's west coast, which is presently under heavy human pressure. In this shallow warm-temperate estuary, a significant macroalgal proliferation has been observed, which is a clear sign of nutrient enrichment. As a result of competition with algae, the extension of the seagrass meadows (mainly Zostera noltii) has been reduced. The present paper examined the applicability of a holistic Stochastic Dynamic Methodology (StDM) in predicting the tendencies of trophic key-components (macrophytes, macroalgae, benthic macroinvertebrate and wading birds) as a response to the changes in estuarine environmental conditions. The StDM is a sequential modelling process developed in order to predict the ecological status of changed ecosystems, from which management strategies can be designed. The data used in the dynamic model construction included true gradients of environmental changes and was sampled from January 1993 to September 1995 and from December 1998 to December 2005. The dynamic model developed was preceded by a conventional multivariate statistical procedure performed to discriminate the significant relationships between the selected ecological components. The model validation was based on independent data collected from January 1996 to January 1997 and from February 1999 to April 2000 for all the state variables considered. Overall, the simulation results are encouraging since they seem to demonstrate the StDM reliability in capturing the trophic dynamics of the studied estuary, by predicting the behavioural pattern for the most part of the components selected, with a focus on the Zostera noltii meadows recovery after the implementation of important management measures.     

Exposure of ground water to alachlor,atrazine and metolachlor in maize areas of ribatejo and oeste (Portugal)

In irrigated maize areas of an important Portuguese agricultural area, Ribatejo and Oeste Region, alachlor, atrazine and metolachlor were detected in ground water.

During the study performed from 1996 to 1998 atrazine was the herbicide that showed the highest frequency of detection. In the 177 ground water samples collected 62% were contaminated with atrazine, 30% with alachlor and 12% with metolachlor. All these herbicides were detected both in ground water for human consumption and for irrigation, in some cases above 0.1 ug/L. The maximum levels quantified were 13μg/L for alachlor, 30μg/L for atrazine and 56 μg/L for metolachlor.

Seasonal variation of residues in ground water it is also presented through several examples of studies performed during the period 1991–1999.     

Female resistance and duration of mate-guarding in three aquatic peracarids (Crustacea)

Both theoretical and empirical studies have treated mate-guarding in aquatic Crustacea purely as a male decision problem. However, male and female interests are rarely identical, as implied by observations of female resistance against guarding attempts. We tested experimentally the occurrence of sexual conflict over guarding duration in three crustacean species: Idotea baltica, Asellus aquaticus (Isopoda), and Gammarus zaddachi (Amphipoda). Specifically, we manipulated, by osmotic stress or a neuromuscular blocking agent, the female's ability to resist guarding attempts. Female manipulation, by both methods, roughly doubled precopula duration in I. baltica (Figs. 1 and 2) showing that female resistance effectively diminishes guarding duration. However, in A. aquaticus and G. zaddachi female manipulation had no effect on guarding duration, which also was longer than in I. baltica (Fig. 2). This implies either that male and female interests are equal or that the conflict is resolved according to the male interest in these species. The lack of female resistance in such species allows long precopulatory guarding. In I. baltica we also manipulated, by osmotic stress and by clipping nails, male ability to hold the female. These treatments had no effect on guarding duration (Figs. 1 and 2). Male size tended to correlate positively with guarding duration in control groups, but not in female manipulation groups (Fig. 3). Thus, conflict is mainly resolved according to the female interest in I. baltica. Results in this species also suggest that female resistance selects for large male size. Consequently, mechanisms of sexual selection may differ considerably between species with otherwise comparable mating patterns.     

Nestmate recognition in three species of stenogastrine wasps (Hymenoptera,Vespidae)

The capacity to recognise a conspecific intruder was investigated in Parischnogaster jacobsoni, Liostenogaster flavolineata and L. vechti, three species of primitively social wasps of the subfamily Stenogastrinae. Results of behavioural experiments carried out in the field showed that females of all three species react pacifically if presented with female nestmates, but aggressively reject an intruder from a conspecific colony. As L. flavolineata and L. vechti both build large clusters of nests, often very close to each other, the recognition capacity among females from different nests, but in the same conspecific cluster, was also investigated. Females of both species were more aggressive towards females from a different colony in the same cluster than towards their female nestmates. Additional experiments on L. flavolineata showed that there was no difference in reaction towards females from colonies nearer or further from the tested colony but within the same cluster, nor towards females from a different cluster. The capacity to recognise an alien conspecific nest containing immature brood was investigated in P. jacobsoni. Adult females of this species, invited to land on an alien nest which had experimentally been exchanged for their own, accepted the new nest and partially destroyed the immature brood. The behaviour of the females when they land on an alien nest, however, suggests that they do recognise the nest as foreign. Acceptance of foreign nests coupled with low immature brood destruction is probably due to the high energetic costs of egg-deposition and larval rearing in stenogastrine wasps. These results suggest that nestmate recognition in these wasps is very efficient, even though they belong to the most primitive subfamily of social wasps. Received: 16 April 1996/Accepted after revision: 9 August 1996     

Toxicity of the venom in three neotropical Crematogaster ants (Formicidae: Myrmicinae)

Bioassays were performed to investigate and compare the toxic properties of the contact venoms of three Neotropical Crematogaster species (C. sp. prox. abstinens, C. distans and C. brevispinosa rochai), whose main venom compounds are chemically different (long chain derivatives linked to an aldehyde or a primary acetate, and furanocembrenoid diterpenes, respectively). Different quantities of venom were topically applied on the bodies of three target insect species (workers of the termite Nasutitermes ephratae, workers of the ant Solenopsis sp., “media” and “major” workers of the ant Cephalotes pusillus). The toxicity of the venom greatly varied according to the Crematogaster species, the venom of C. b. rochai showing the highest toxic activity towards all target species. The sensitivity of the target species also greatly varied, the workers of N. ephratae showing a great sensitivity to the venoms, while the heavily armoured workers of the ant C. pusillus, particularly “major” ones, were resistant. Cuticle thickness was found to be a key factor in the resistance to the toxic effects of Crematogaster venom. Sensitivity of the target workers was also directly related to the amount of venom applied to their cuticle. Workers of C. distans were resistant to topical application of their own venom, while C. sp. prox. abstinens and C. b. rochai were not. In all cases, the venoms were slow acting, as several hours to one or even several days were needed to obtain a significant mortality. The main biological activities of the Crematogaster venom could be related to more immediate repellent properties, as recently shown with the European species C. scutellaris.     

赤子爱胜蚓(Eisenia foetida)对三种土壤Zn、Pb有效态含量的影响

以褐土(Cinnamonsoil)、潮棕壤(Aquicbrownsoil)和酸性棕壤(Acidbrownsoil)为供试土壤,通过培养实验,研究了土壤中重金属Zn、Pb对赤子爱胜蚓(Eiseniafoetida)生长率的影响,赤子爱胜蚓(Eiseniafoetida)对不同土壤中有效态Zn、Pb含量的影响,并与野生环境中生长的灰暗异唇蚓(Allolobaphoracaliginosa)进行比较。结果表明:重金属的污染明显抑制了赤子爱胜蚓的生长;赤子爱胜蚓可以显著提高潮棕壤中DTPA-Pb的含量,对DTPA-Zn的含量影响不大;加赤子爱胜蚓,褐土中DTPA-Zn和Pb的含量显著高于加灰暗异唇蚓,潮棕壤中DTPA-Zn的含量和酸性棕壤中大DTPA-Pb的含量则相反。     

土壤中三种盐对油菜富集镉的影响

为了利用被镉污染的盐土,通过实验分析镉污染盐土中三种盐对油菜（Brassica napus）富集镉影响的差异性,探明不同类型镉污染盐土上种植油菜的植物修复效果。以镉超累积植物油菜为研究植物,通过温室盆栽土培试验,将油菜在含有不同质量分数盐（wS：0 g·kg^-1,2 g·kg^-1,4 g·kg^-1,6 g·kg^-1）的含镉（wCd：10 mg·kg^-1）土壤中培养60天,研究油菜对镉的生物富集因子、植株内地上部分和根部镉的质量分数变化。选择土壤中三种主要盐的类型,即氯化钠、硫酸钠和碳酸钠作为分析和研究对象。结果表明,含碳酸钠的土壤对油菜吸收镉有抑制作用,含硫酸钠的土壤对油菜吸收镉也有抑制作用,但效果没有碳酸钠的土壤大,含氯化钠的土壤对油菜吸收镉的影响不显著,只是在高质量分数时对油菜吸收镉有一些促进作用。在含不同类型盐的土壤中,不同土壤盐对油菜富集镉的能力也有显著的差异,土壤中氯化钠对油菜根部富集镉的能力没有显著影响,而对油菜地上部分富集镉的能力有一定的促进作用;土壤中的碳酸钠对油菜地上和地下部分富集镉的能力都有显著的抑制作用,不利于油菜对镉的富集。     

Determination and occurrence of endocrine disrupting compounds,pharmaceuticals and personal care products in fish (Morone saxatilis)

Endocrine disrupting compounds (EDCs), pharmaceuticals and personal care products (PPCPs) have attracted much attention due to widespread contamination in aquatic environment. In this study, we determined 13 EDCs and PPCPs in fish blood, bile and muscle by using gas chromatography-mass spectrometry (GC-MS). The limits of quantitation (LOQ) were in the ranges of 0.23–2.54, 0.22–2.36 ng·mL⁻¹, and 0.24–2.57 ng·g⁻¹ dry weight (dw) for fish blood, bile and muscle, respectively. Recoveries of target compounds spiked into sample matrices and passed through the entire analytical procedure ranged from 65% to 95%, from 60% to 92% and from 62% to 91% for blood, bile and muscle, respectively. The methods were applied to the analysis of fish from a lake in California. Target compounds were relatively low in bile, and only bisphenol A (BPA) and diclofenac were measurable near the LOQ. Seven of 13 compounds were detected in blood, with total concentrations up to 39 ng·mL⁻¹. Only BPA was frequently found in muscle, with mean concentration of 7.26 ng·g⁻¹ dw. The estimated daily intake of BPA through fish consumption for U.S. resident was significantly lower than the tolerable daily intake recommended by the European Food Safety Authority. This study showed that the exposure to the bisphenol A from fish diet is unlikely to pose a health risk.     

Sex-specific energy requirements in nestlings of an extremely sexually size dimorphic bird, the European sparrowhawk (Accipiter nisus)

Allocation of parental investment is predicted to be equal at the population level between both sexes of offspring, and should lead to sex ratio biases in species that exhibit a sex-difference in parental care. Sex-differences in parental care are rarely quantified. We measured daily energy expenditure in free-living nestlings of the extremely sexually size dimorphic European sparrowhawk (Accipiter nisus), using the doubly labelled water method. These data were combined with measured growth characteristics to estimate daily and total metabolised energy intake of male and female young during the nestling stage. Females reached an asymptotic body mass 1.6 times higher than males. This resulted in a total metabolised energy an estimated 1.4 times higher for the nestling stage. Furthermore, we observed a decline in daily metabolised energy with an increase in brood size, which was significantly stronger for females than for males. These results are discussed in the context of Fishers equal allocation theory. Empirical evidence of a sex ratio bias at the end of parental care, with an overall excess of males, is lacking in this species. Consequently, our data do not support the idea of equal allocation between the sexes. The observed sex difference in daily metabolised energy in response to brood size may give scope for sex ratio bias at the level of the individual brood.     

Measurement of parental investment and sex allocation in the European beewolf Philanthus triangulum F. (Hymenoptera: Sphecidae)

Fisher’s 1930 theory of sex allocation predicts a population-wide 1:1 ratio of parental investment. We tested this prediction in the European beewolf, a sphecid wasp that hunts for honeybees as larval food. Because the method to quantify parental investment is of crucial importance, we compared the suitability of several different investment measures. Female/male cost ratios were determined from a sample and the total investment in sons and daughters was calculated. In addition, the actual number of prey items for sons and daughters was directly determined by excavating nests and counting the cuticle remains of the prey. Though mortality was high (70%), it had only a weak effect on the estimate of the investment ratio. Based on commonly used measures like fresh and dry weight of emerged adults, the investment ratio did not deviate from Fisher’s prediction of equal investment. However, progeny weight considerably underestimates investment in males and investment in large progeny. Measures that reflect the allocation of resources more directly (amount of provisions, brood cell volume) revealed a significant male bias and thus contradicted Fisher’s theory. Three kinds of explanation are discussed. First, non-adaptive explanations are unlikely. Second, from the spectrum of alternative adaptive theories, only models that assume a non-linear relationship between amount of investment and progeny fitness seem to be relevant for the study species. Third, though the number of prey in a brood cell seems to be a rather good measure of parental investment in European beewolves, some problems in measuring parental investment remain. These problems are of broad significance. Received: 17 June 1999 / Received in revised form: 6 July 1999 / Accepted: 11 July 1999     

Chemical determinants of resistance in winter-dormant seedlings of European white birch (Betula pendula) to browsing by the mountain hare

Summary A sample of one-year-old seedlings of European white birch (Betula pendula) was analyzed to determine the content of sugars, phenolics and terpenoid compounds. Two vertical segments of each seedling were analyzed separately. The number of resin droplets, which correlates strongly and negatively with feeding by the mountain hare, was also counted on the bark of experimental seedlings. The variation in the palatability of birch seedlings to mountain hare was determined primarily by the most abundant terpenoid, papyriferic acid. On the other hand, the resistance to hare feeding at the seedling bases, which are frequently attacked by voles, appeared to be dependent on other components, apparently phenolic substances. Sugars did not affect the resistance of the tested seedlings. The variation among experimental seedlings was much greater for secondary substances, especially terpenoid compounds, than for sugars. It is suggested that this high variation in protective compounds may be an adaptive trait selected for by the feeding of generalist herbivores.