Toxicogenomics of the Freshwater Oligochaete, Tubifex tubifex (Annelida, Clitellata), in Acute Water-Only Exposure to Arsenic.

A toxicogenomic approach was used for toxicity evaluation of arsenic in the aquatic environment, and differential gene expression was investigated from 24 h and 96 h water-only acute toxicity tests with the aquatic oligochaete, Tubifex tubifex (Annelida, Clitellata). Several toxicological endpoints (survival and autotomy) of the oligochaete and tissue residues were measured, and dose-response modelling of gene expression data was studied. A reference transcriptome of the aquatic oligochaete, T. tubifex, was reconstructed for the first time, and genes related to cell stress response (Hsc70, Hsp10, Hsp60, and Hsp83), energy metabolism (COX1), oxidative stress (Cat, GSR, and MnSOD), and the genes involved in the homeostasis of organisms (CaM, RpS13, and UBE2) were identified and characterised. The potential use of the genes identified for risk assessment in freshwater ecosystems as early biomarkers of arsenic toxicity is discussed.

Developing As and Cu Tissue Residue Thresholds to Attain the Good Ecological Status of Rivers in Mining Areas.

The study was performed on residue-effects datasets from polluted and unpolluted sites in the Nalón River basin (northern Spain). The effects were measured in terms of alteration of field macroinvertebrate communities, and measured as ecological status scores, and number of families and abundance of Ephemeroptera, Plecoptera and Trichoptera (EPT). Non-linear regression models of the field-measured tissue residues in 10 taxa related to the ecological status of the macroinvertebrate communities were used to derive effective tissue residues (ERs). These were estimated for the good/moderate boundary defined by the ecological quality ratio (EQRs) score and for the 50% reduction of EQR and EPT metrics. As, Cu, Hg and Se ERs were calculated for several macroinvertebrate taxa with different feeding styles. The ER dataset allowed us to estimate As and Cu hazardous concentrations (HC), using species sensitivity distribution models, and were interpreted as community thresholds. Further studies for Hg and Se are needed to complete the database required for HC estimation. The reliability and differences of the several thresholds were tested in a risk assessment using a tissue-residue approach (TRA) conducted with field organisms from Cauxa Creek, a tributary from the same basin exposed to high levels of metals in the sediments due to gold mining activities. This risk assessment identified that As and Cu tissue residues satisfactorily explained the reduction in the ecological status of the macroinvertebrate assemblages. Our results indicate that TRA can help in setting future environmental quality standards for the protection of aquatic biota.

Bioaccumulation and chronic toxicity of arsenic and zinc in the aquatic oligochaetes Branchiura sowerbyi and Tubifex tubifex (Annelida, Clitellata).

Oligochaetes feed on bulk sediment and penetrate the sediment through the construction of burrows, making them especially vulnerable to sediment metal contamination. However, the few oligochaete species that have been tested to date are almost exclusively temperate test species. Although the warmwater adapted species Branchiura sowerbyi has been indicated as a promising candidate for tropical sediment toxicity testing, few (especially chronic) studies have been conducted so far to confirm this. Therefore, the aim of the present study was to evaluate the bioaccumulation and chronic 28d lethal and sublethal toxicity of arsenic (As) and zinc (Zn) to both the warmwater-adapted B. sowerbyi and the coldwater-adapted oligochaete Tubifex tubifex for comparison. Arsenic was more toxic to both oligochaete species than Zn. Inter- and intra-species variability in toxicity values of the two test species and other benthic invertebrates was within an order of magnitude. However, B. sowerbyi was the most sensitive species to As even for sediment concentration (EC50: 36.6 ± 2.1 µg/g and 147.1 ± 21.7 µg/g, for B. sowerbyi and T. tubifex, respectively) and for tissue concentration (ER50: 9.2 ± 0.9 µg/g and 887.0 ± 35.0 µg/g, for B. sowerbyi and T. tubifex, respectively). Finally, the Tissue Residue-effects Approach (TRA) using Effective Tissue Residues appears to be a promising way forward in advancing in this since it considers internal body concentrations.

Proposal of integrative scores and biomonitor selection for metal bioaccumulation risk assessment in mine-impacted rivers.

Development of sound criteria for metal and metalloid bioaccumulation risk assessment in river basins affected by mining activities is a necessary tool to protect the aquatic communities. The aim of this study is to propose integrative scores for tissue residues that are suitable for surveillance programs and readily interpreted in terms of risk assessment in mining impacted rivers. Tissue residues of 7 trace metals and 2 metalloids were measured in ten macroinvertebrate taxa from the Nalón River basin (Spain), affected by Hg, Cu and Au mining activities. Compared with reference sites, biomonitor taxa from Hg and Au mining districts showed the highest bioaccumulation. However, low or non-significant bioaccumulation was found in sites influenced by historical Cu mining. Multivariate analyses (ANOSIM) performed on individual taxa revealed significant differences in tissue residues between sites classified according to their ecological status. The bioaccumulation risk assessment was based on the average ratio of the actual metal tissue residues in each macroinvertebrate taxon to the corresponding Ecological Threshold tissue concentration (Tissue residue Ratio to Threshold, TRT). The suitability of the biomonitors was evaluated using linear regression models fitted to the relationships between TRT scores and site sediment pollution or ecological status scores. Biomonitor selection also considered differences in invertebrate functional traits, which can influence metal and metalloid bioavailability. Site bioaccumulation risk was assessed on an Integrated Tissue concentration score (INTISS), calculated over a selection of the most relevant chemicals (As, Cu and Hg) and 3 biomonitor taxa (Baetidae, Hydropsychidae, Microdrile oligochaetes) comprising a set of feeding styles. Based on INTISS, it was possible to predict community alteration scores, using linear regression models. A comparison of site bioaccumulation and ecological status assessments based on the departure from reference conditions showed that operational monitoring programs in basins impaired by mining can be optimized by combining both approaches.

Changes in invertebrate community composition allow for consistent interpretation of biodiversity loss in ecological status assessment.

Biological communities change in response to human alteration. The response of individual taxa and the community can be used to establish preventive criteria to halt further biodiversity deterioration. Here we explore how consistent are the boundaries between Good and Moderate ecological status derived from classification systems used in North-NW Spain: NORThern Spain Indicators system (NORTI), River type specific multimetric (METI) and Iberian Bio-monitoring Working Party (IBMWP), by using common interpretation of normative definitions of Water Framework Directive. We applied the three classifications to a monitoring dataset of Nalón River basin, comprising samples from different stream types and reference conditions. We applied Threshold Indicator Taxa ANalysis to the invertebrate community along the most relevant environmental pressures and biological impairment gradients represented by the Ecological Quality Ratio (EQR) scores of the classification systems. Only NORTI provided a true community ecological threshold and the change point (cp) 95% quantile (Q95%) range of 0.760 was assumed to be the boundary from Good to Moderate (G/M) status, used to standardize the number of taxa loss in all systems. Since the average number of taxa at reference sites was 34, the estimated loss of sensitive taxa was up to 97.1% in IBMWP, 73.5% in METI and 52.9% in NORTI when passing from Good to Moderate status, revealing very permissive boundaries. The loss of common sensitive taxa in NORTI at Q95% was used as G/M threshold and applied to the other classifications, resulting all in a common biodiversity loss of 21% of sensitive taxa richness at values of NORTI-EQR = 0.760, METI-EQR = 0.818 and IBMWP-EQR = 0.753. Results indicate that significant community changes along pressure gradients allow for establishing quantitative criteria consistent with normative definitions. This understanding derived from Directive monitoring programs can assess the risk that invertebrate communities face in terms of species loss derived from anthropogenic pressures.

Exposure to heavy metal-contaminated sediments disrupts gene expression, lipid profile, and life history traits in the midge Chironomus riparius.

Despite the concern about anthropogenic heavy metal accumulation, there remain few multi-level ecotoxicological studies to evaluate their effects in fluvial ecosystems. The toxicity of field-collected sediments exhibiting a gradient of heavy metal contamination (Cd, Pb, and Zn) was assessed in Chironomus riparius. For this purpose, larvae were exposed throughout their entire life cycle to these sediments, and toxic effects were measured at different levels of biological organization, from the molecular (lipidomic analysis and transcriptional profile) to the whole organism response (respiration rate, shape markers, and emergence rate). Alterations in the activity of relevant genes, as well as an increase of storage lipids and decrease in membrane fluidity, were detected in larvae exposed to the most contaminated sediments. Moreover, reduced larval and adult mass, decrease of larval respiration rate, and delayed emergence were observed, along with increased mentum and mandible size in larvae and decreased wing loading in adults. This study points out the deleterious effects of heavy metal exposure at various levels of biological organization and provides some clues regarding the mode of toxic action. This integrative approach provides new insights into the multi-level effects on aquatic insects exposed to heavy metal mixtures in field sediments, providing useful tools for ecological risk assessment in freshwater ecosystems.

Derivation of sediment Hg quality standards based on ecological assessment in river basins.

The Environmental Quality Standards (EQS) directive was an important improvement of long-term water quality monitoring at the European level, leading to the use of sediments and biota as relevant matrices for assessing priority substances under the European Water Framework directive. Currently, commonly accepted sediment EQS for Hg are missing in Europe. In this study we present a new, tiered approach to deriving sediment quality standards for Hg: the derivation of Predicted No-Effect Concentration (PNEC) from data in the literature, followed by adjusting values at regional scale, using ecological field data (macroinvertebrate community assessment) and field sediment ecotoxicity bioassays. The limited set of effect data available for Hg spiked-sediment ecotoxicity tests has resulted in unreliable PNEC values for sediment quality assessment. Field reference sites (n = 40) where the macroinvertebrate community status was assessed as High or Good were used to define the ecological background and threshold levels in sediments in northern Spain. Sediment QS developed in other areas were not suitable for specific basins in our study area, since they were within the range of our Hg background levels. Temporary sediment Quality Standards (QS) for Hg were developed for the Nalón River basin (where several mining districts occur), using field effect-based approaches such as sediment ecotoxicity data from Tubifex tubifex chronic bioassays and ecological assessment of macroinvertebrate communities. A proposal for Hg quality assessment in freshwater sediments of northern Spain is made based on ecologically relevant QS values, providing benchmark values for No-Effect and Effect Hg sediment concentrations.

Seed Carotenoid and Tocochromanol Composition of Wild Fabaceae Species Is Shaped by Phylogeny and Ecological Factors.

Carotenoids distribution and function in seeds have been very scarcely studied, notwithstanding their pivotal roles in plants that include photosynthesis and phytohormone synthesis, pigmentation, membrane stabilization and antioxidant activity. Their relationship with tocochromanols, whose critical role in maintaining seed viability has already been evidenced, and with chlorophylls, whose retention in mature seed is thought to have negative effects on storability, remain also unexplored. Here, we aimed at elucidating seed carotenoids relationship with tocochromanols and chlorophylls with regard to phylogenetic and ecological traits and at understanding their changes during germination. The composition and distribution of carotenoids were investigated in seeds of a wide range of wild species across the Fabaceae (the second-most economically important family after the Poaceae). Photosynthetic pigments and tocochromanols were analyzed by HPLC in mature dry seeds of 50 species representative of 5 subfamilies within the Fabaceae (including taxa that represent all continents, biomes and life forms within the family) and at key timepoints during seedling establishment in three species representative of distinct clades. Total-carotenoids content positively correlated with tocopherols in the basal subfamilies Detarioideae, Cercidoideae, and Dialioideae, and with chlorophylls in the Papilionoideae. Papilionoideae lacked tocotrienols and had the highest total-carotenoids, chlorophyll and γ-tocopherol contents. Interestingly, lutein epoxide was present in 72% of the species including several herbs from different subfamilies. Overall, species original from temperate biomes presented higher carotenoids and lower tocochromanols levels than those from tropical biomes. Also shrub species showed higher carotenoids content than herbs and trees. During germination, total content of photosynthetic pigments increased in parallel to changes in relative abundance of carotenoids: zeaxanthin and anteraxanthin decreased and ß-carotene augmented. Notably, the highest contents of nutritionally valuable carotenoids were found in Papilionoideae subfamily to which all pulses of socio-economic importance belong. The major differences in carotenoids and tocochromanols composition across the Fabaceae are apparently related to phylogeny in conjunction with ecological traits such as biome and growth form.

Cadmium Bioaccumulation in Aquatic Oligochaetes Using a Biodynamic Model: A Review of Values of Physiological Parameters and Model Validation Using Laboratory and Field Bioaccumulation Data.

This study reviews certain physiological digestive parameters in the literature that could be used to predict tissue residues in aquatic oligochaetes using the biodynamic model. Predictions were evaluated with independently measured Cd bioaccumulation data in sediment bioassays and field oligochaetes. The parameter review focused on three species commonly used in ecotoxicity testing and bioaccumulation studies: Tubifex tubifex (Tt), Limnodrilus hoffmeisteri (Lh) and Lumbriculus variegatus (Lv). Median Ingestion rates (g g-1 d-1, dw) at unpolluted conditions were 7.8 (Tt), 24.5 (Lh) and 11.5 (Lv), while results were lower (1.7-2.4) at polluted conditions. Assimilation efficiencies ranged from 3.4-19.6% (Tt), 2.7-16.1% (Lh), and 10.9-25.6% (Lv). The biodynamic model accurately predicted Cd tissue concentration in T. tubifex exposed to spiked sediments in laboratory bioassays. Comparisons of predicted vs. measured Cd tissue concentration in bioassays or field aquatic oligochaetes suggest that the biodynamic model can predict Cd tissue concentration within a factor of five in 81.3% of cases, across a range of measured tissue concentrations from 0.1 to 100 µg Cd g-1 dw. Predictions can be refined by using physiological parameter values that have been measured under varying environmental conditions (e.g. temperature, dissolved oxygen). The model can underestimate tissue concentration by up to one order of magnitude when worms are exposed to highly contaminated sediments. Contrarily, predictions overestimate tissue concentration by up to two orders of magnitude when the measured Cd < 0.1 µg g-1 dw, although in most cases these predictions do not fail bioaccumulation-based risk assessments, using a tissue threshold value of 1.5 µg Cd g-1 dw.

Baseline tissue concentrations of metal in aquatic oligochaetes: Field and laboratory approaches.

Metal tissue residue evaluation in benthic macroinvertebrates is an important component of an integrated approach to ecological risk assessment of metals and metalloids in the Nalón River basin (North Spain), where historic mining activities took place. The purpose of this study was to know the baseline tissue concentration of 7 metals (Cd, Cu, Cr, Hg, Ni, Pb, and Zn) and one metalloid (As) in aquatic oligochaetes, sediment burrower organisms, representative of the collector-gatherer functional feeding group in the macroinvertebrate community. Metal concentration was measured in sediment and field aquatic oligochaetes at several reference (minimally disturbed) sites of the Nalón River basin, selected following Water Framework Directive criteria. Metal tissue residues were measured separately in field microdriles and lumbricids and compared with tissue concentrations measured in the aquatic oligochaete Tubifex tubifex exposed to reference sediments from the Nalón and other Cantabrian River basins in 28-d chronic laboratory bioassays. Metal tissue residues in bioassay organisms attained usually higher levels than in field worms, in special for As, Cu, Hg and Zn, although metal levels were within the same order of magnitude. The baseline values for metals were calculated from 90th percentile (P90) values in field aquatic oligochaetes (microdriles and lumbricids). The P90 for Hg, As and Zn could efficiently discriminate Toxic and Non-Toxic sites, while baseline values calculated for the other metals deserve further research due either to the low range of values found in the present study, or to the regulation of the metal body concentration, as in the case of Cu.

Heavy metal concentration in feathers of Little Egret (Egretta garzetta) nestlings in three coastal breeding colonies in Spain.

The colonial ardeid Little Egret (Egretta garzetta), which is is protected under the European Birds Directive (2009/147/EC), can be a reliable bioindicator of aquatic environmental pollution. Concentrations of the heavy metals Cd, Cr, Cu, Hg, Ni, Pb and Zn in nestling feathers were assessed for three different breeding colonies of Little Egret on the Spanish coast during 2013 (5 individuals in Urdaibai, 10 in Santoña and 26 in Odiel). There were no significant differences in mean tissue residues of Cd, Ni, Pb and Zn between the colonies; however, mean concentration of Hg in Odiel nestlings was approximately three times lower than that of the other colonies, while Cr and Cu were significantly higher. In general, Little Egret nestlings from the three study sites had low levels of most of the measured metals, and thus the breeding populations did not appear to be at risk from heavy metal pollution. Baseline metal concentration in feathers derived from this study and calculated as the 90th percentile values were: 0.02 µg Cd g(−1) dw, 0.42 µg Cr g(−1) dw, 1.63 µg Hg g(−1) dw, 0.40 µg Pb g(−1) dw and 122 µg Zn g(−1) dw. However, mean Cu residues attained relatively high levels (17.6­26.9 µg Cu g(−1) dw) compared with data reported elsewhere, which raises concern and indicates a need for further research.

Influences of sediment geochemistry on metal accumulation rates and toxicity in the aquatic oligochaete Tubifex tubifex.

Metal bioaccumulation and toxicity in the aquatic oligochaete Tubifex tubifex exposed to three metal-contaminated field-sediments was studied in order to assess whether sediment-geochemistry (AVS, TOC) plays a major role in influencing these parameters, and to assess if the biodynamic concept can be used to explain observed effects in T. tubifex tissue residues and/or toxicity. An active autotomy promotion was observed in three studied sediments at different time points and reproduction impairment could be inferred in T. tubifex exposed to two of the tested sites after 28 days. The present study showed that sediment metal concentration and tissue residues followed significant regression models for four essential metals (Cu, Co, Ni and Zn) and one non-essential metal (Pb). Organic content normalization for As also showed a significant relationship with As tissue residue. Porewater was also revealed to be an important source of metal uptake for essential metals (e.g. Cu, Ni and Zn) and for As, but AVS content was not relevant for metal uptake in T. tubifex in studied sediments. Under the biodynamic concept, it was shown that influx rate from food (IF, sediment ingestion) in T. tubifex, in a range of sediment geochemistry, was able to predict metal bioaccumulation, especially of the essential metals Cu, Ni and Zn, and for the non-essential metal Pb. Additionally, IF appeared to be a better predictor for metal bioaccumulation in T. tubifex compared to sediment geochemistry normalization.

Toxicity and critical body residues of Cd, Cu and Cr in the aquatic oligochaete Tubifex tubifex (Müller) based on lethal and sublethal effects.

The aim of the present study was to estimate critical body residues (CBRs) of three metals [cadmium (Cd), copper (Cu), chromium (Cr)] in the aquatic oligochaete Tubifex tubifex based on lethal (LBR) and sublethal effects (CBR), and to discuss the relevance of the exposure to sediment for deriving CBR. Toxicity parameters (LC50, EC50, LBR50 and CBR50) were estimated for each metal by means of data on survival and on several sublethal variables measured in short-term (4 days), water-only exposures and in long-term, chronic (14 and 28 days) exposures using metal-spiked sediment. Sublethal endpoints included autotomy in short-term exposure, as well as reproduction and growth in chronic bioassays. LBR50 and CBR50 were 3-6 times higher in sediment than in water-only exposure to Cd and about 2-11 times higher for Cu, depending on the measured endpoint; however, for Cr these parameters varied only by a factor of 1.2. Cu and Cr LBR50 and CBR50 values in 96 h water-only exposure were very similar (survival 2.39 µmol Cu g(-1) dw, 2.73 µmol Cr g(-1) dw; autotomy 0.53 µmol Cu g(-1) dw, 0.78 µmol Cr g(-1) dw). However, in metal-spiked sediments, 28 d CBR50 values for autotomy, reproduction and growth ranged 6.76-29.54 µmol g(-1) dw for Cd, 3.88-6.23 µmol g(-1) dw for Cu, 0.65 µmol g(-1) dw for Cr (calculated only on total number of young). Exposure conditions (time and presence/absence of sediment) seem to be influential in deriving metal CBR values of Cd and Cu, while appear to be irrelevant for Cr. Thus, CBR approach for metals is complex and tissue residue-toxicity relationship is not directly applicable so far.