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.

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.

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.

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.

Evaluating the Type II error rate in a sediment toxicity classification using the Reference Condition Approach.

Sediments from 71 river sites in Northern Spain were tested using the oligochaete Tubifex tubifex (Annelida, Clitellata) chronic bioassay. 47 sediments were identified as reference primarily from macroinvertebrate community characteristics. The data for the toxicological endpoints were examined using non-metric MDS. Probability ellipses were constructed around the reference sites in multidimensional space to establish a classification for assessing test-sediments into one of three categories (Non Toxic, Potentially Toxic, and Toxic). The construction of such probability ellipses sets the Type I error rate. However, we also wished to include in the decision process for identifying pass-fail boundaries the degree of disturbance required to be detected, and the likelihood of being wrong in detecting that disturbance (i.e. the Type II error). Setting the ellipse size to use based on Type I error does not include any consideration of the probability of Type II error. To do this, the toxicological response observed in the reference sediments was manipulated by simulating different degrees of disturbance (simpacted sediments), and measuring the Type II error rate for each set of the simpacted sediments. From this procedure, the frequency at each probability ellipse of identifying impairment using sediments with known level of disturbance is quantified. Thirteen levels of disturbance and seven probability ellipses were tested. Based on the results the decision boundary for Non Toxic and Potentially Toxic was set at the 80% probability ellipse, and the boundary for Potentially Toxic and Toxic at the 95% probability ellipse. Using this approach, 9 test sediments were classified as Toxic, 2 as Potentially Toxic, and 13 as Non Toxic.

Monitoring the sensitivity of the oligochaete Tubifex tubifex in laboratory cultures using three toxicants.

Acute toxicity tests were conducted to assess the sensitivity of laboratory cultured Tubifex tubifex (Annelida, Clitellata), as an intralaboratory quality assurance requirement for the test organisms used in sediment chronic bioassays. Worms were exposed to cadmium, copper, and chromium in water-only 96-h tests, in single metal exposure, over a three-year period. The lethal concentration for the x% of the exposed population (LC(x)), the No-Observable-Effect Concentration (NOEC) and the Lowest-Observable-Effect Concentration (LOEC) were estimated. Based on LC(50) values, the rank of toxicity was Cu(+2)>Cd(+2)>Cr(+6). Sensitivity remained relatively constant during the research period and was comparable with the sensitivity of the same culture stock examined more than 10 years ago. This observed consistency in sensitivity indicates that comparisons between different sediment chronic bioassays conducted during the study period, using T. tubifex worms from the same culture stock, was appropriate. It is necessary to control both worm biomass and age for the acute test in order to standardize the test-organism conditions in intralaboratory quality assurance programs.

Ecotoxicological assessment of effluents in the Basque country (Northern Spain) by acute and chronic toxicity tests using Daphnia magna straus.

Acute pass/fail, multi-concentration tests, and 3-brood chronic toxicity tests with Daphnia magna Straus (Cladocera, Crustacea) were used to characterise industrial and municipal effluents from various sources. The effluents that "passed" the pass/fail tests had 48-h EC50 values >100% and reproduction No Observed Effect Concentration (NOECs) > or =100%, except for one effluent that had a reproduction NOEC of 31.6%. The acute multi-concentration toxicity tests allowed a rapid classification of effluents from Very Toxic (48-h EC50 < 25%), to Non-Toxic (48-h EC50 >100%). The acute-to-chronic ratio (ACR: 48-h EC50 divided by the NOEC for reproduction) in the studied effluents ranged from 5 to about 100. From these results, we propose a step-wise protocol for assessing effluent toxicity. First, effluent is evaluated by means of simple and rapid pass/fail acute toxicity tests, to discriminate Non-Toxic from potentially Toxic effluents, thus facilitating the establishment of priority actions. Second, 48-h ECx is estimated to classify effluents on a toxicity scale from Non-Toxic to Very Toxic. Third, chronic multi-concentration tests are used to calculate reproduction NOECs. These parameters combined with data on effluent chemical composition, chemical and hydrological characteristics of receiving waters, and biological quality criteria can be jointly used for more rational regulatory practices and risk assessment of effluents.