Maite Martinez-Madrid

Selective feeding by the aquatic oligochaete Tubifex tubifex (Tubificidae, Clitellata)

The particle size distribution of faecal pellets produced by the tubificid worm Tubifex tubifex in laboratory culture, was measured with a Coulter® Multisizer. The faecal material from worms cultured in a range of sediments was composed of particles with a mean diameter of less than 63 μm, and only a few isolated larger particles were found by microscopic analysis. This suggests that this species actively selects the silt-clay fraction, avoiding larger sand particles. A more detailed analysis of faeces revealed that about 75%, by volume, was composed of particles with a mean diameter < 25μm, and the mode was < 10μm. T. tubifex fed selectively on the organic rich particles of the sediment, and this feeding was independent of particle size. Measurement of the organic content of faeces (measured as % loss on ignition) showed that they had a consistently higher organic content than the sediment, considered as whole sediments or the <63 μm sieved fraction. On the basis of these results, we hypothesise that this species exhibits two levels of selectivity in its feeding behaviour. Thus selection is primarily based on particle size, avoiding the ingestion of sand particles and also, on the preferential selection of particles associated with organic material, within the fine (silt-clay) fraction of the sediment.

Sediment Toxicity Bioassays for Assessment of Contaminated Sites in the Nervion River (Northern Spain). 2. Tubifex tubifex Reproduction Sediment Bioassay

Toxicity assessment based on sediment chronic bioassays with the aquatic worm Tubifex tubifex was performed at ten contaminated sites in the industrial area of Bilbao (Northern Spain). One control and three reference sites were also included. Tubifex bioassay measures both survival and reproduction impairment. These endpoints have been contrasted and discussed in relation to somatic growth and both individual and total biomass of cocoons. Survival was only affected at one site which was heavily contaminated by organic compounds, mainly PAHs. A group of four severely ecotoxic sediments was characterised by a drastic reduction in number and size of cocoons, and adult somatic growth. In other group of sediments, some significant increases were found for these variables. It is suggested that these increases represent an effect of hormesis. An index of reproductive effort was used to integrate the relationship between somatic growth and reproduction. Values of reproductive effort at the test sediments were lower than those at the control sediment, suggesting a conservative strategy of oligochaete worms which consisted in an investment into somatic line (growth) at the expenses of offspring. Rates of food consumption which were estimated from egestion rates, were low at the contaminated sites. This fact could be related to the low production levels found at these sites and may reflect avoidance feeding behaviour of the oligochaete worms within the sediments. At some reference sites, high production could have resulted from high nutritional quality of sediments, or to an hormetic effect due to low concentration of some chemicals. Results are discussed in relation to toxicity data from sediment three-brood bioassay with Daphnia magna Straus performed separately on the same sediments.

Sediment Toxicity Bioassays for Assessment of Contaminated Sites in the Nervion River (Northern Spain). 1. Three-Brood Sediment Chronic Bioassay of Daphnia Magna Straus

The ecotoxicity of sediments from ten contaminated sites of the Nervion river catchment were assessed with the three-brood chronic sediment bioassay of Daphnia magna. Three additional locations were used as reference sites and one site as control for every bioassay. Survival, number and biomass of neonates and final adult biomass were measured as chronic ecotoxicity endpoints. High mortality of daphnids was observed at one site where sediment was heavily contaminated by metals. At the remaining sites only sublethal reproductive responses were observed, including inhibition of maturity and hence of reproduction, brood delay, reduction in number of neonates and lower adult final biomass. Hormetic responses in adult final biomass and number of neonates per brood were observed where toxicant concentrations were low or were not bioavailable. Female fecundity increased with size in the controls but the reproductive effort is lower in daphnids exposed to contaminated sediments. This was interpreted as an auto-conservative strategy of the species.

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.

Toxicity of Santander Bay sediments to the euryhaline freshwater oligochaete Limnodrilus hoffmeisteri

The freshwater euryhaline oligochaete Limnodrilus hoffmeisteri was selected for use in bioassays with polluted sediment from Santander Bay. It is easy to culture; is tolerant of low to moderate, up to 15‰, salinity; and is common in oligohaline conditions in European and North American estuaries. Worms were collected from an estuarine population and kept in unpolluted sediment for between 2 and 4 weeks, under laboratory conditions, at 7–8.5‰ salinity and 22.5 °C. Sediment from different sites in Santander Bay were sieved through 250 μm mesh and adjusted to a salinity of approx. 7‰ prior to the bioassays, either by adding sea water or dechlorinated tap water to the overlying water. High levels of ammonia in some sediment, which may confound results in ecotoxicity bioassays, were reduced by oxidation of the sediments in shallow trays. Sediment bioassays were performed with sexually mature Limnodrilus hoffmeisteri worms in 250-ml beakers, with a 1:3 ratio of sediment:water and 4 worms per baker. Endpoints in the 14-day bioassay were % mortality, adult final biomass, % adults that have shown resorption of the clitellum, number of cocoons, and burrowing behaviour. It was possible to rank the sites according to their toxicity using both mortality rates and sublethal effects. The control site had the following values for the endpoints: 5% mortality, ( \(\overline{x}\pm {\rm sd}\)) 2.40 ± 1.52 cocoons per beaker and 1.271 ± 0.470 mg dw adult final biomass. The most toxic sediment resulted in 65% mortality, resorption of the clitellum in 67% of the adults, no production of cocoons and a low final biomass (\(\overline{x}\pm {\rm sd}\)=0.681 ± 0.489 mg dw per adult). A second site had high mortality (60%) and no reproduction, although resorption of the clitellum did not occur in surviving animals. The remaining sites showed similar mortality (35–42%), and at only one of them was low reproduction observed (0.8 ± 0.447 cocoons per beaker). Behavioural effects, measured as length of galleries in a fixed area of the test-vessel at the end of the bioassay, were significant compared with control at only one site. Multivariate analysis showed the mortality gradient to be the strongest, with a second unassociated gradient representing clitellum resorption. The mortality gradient was associated with Cu and Zn concentration, and PAH and Pb possibly with resorption.

Sediment Toxicity and Bioaccumulation Assessment in Abandoned Copper and Mercury Mining Areas of the Nalón River Basin (Spain)

Sediment toxicity and metal bioaccumulation were assessed at sites affected by historical copper (Cu) and mercury (Hg) mining activities in the Nalón River basin, Asturias, Spain. Toxicity assessment of stream sediments was based on a 28-day oligochaete Tubifex tubifex sediment bioassay, which allowed the classification of sites into three levels of toxicity: 11 sites were classified as nontoxic (including Cu mine sites), three sites as potentially toxic, and seven sites as toxic (all located in Hg mine districts). The greatest levels of arsenic (As), chromium, Hg, lead (Pb), and zinc (Zn) in T. tubifex were measured at sites affected by Hg mining and the highest Cu levels in tissues at Cu mining sites. Chronic toxicity responses were best explained by As and Hg sediment concentrations and by As, Pb, and Zn tissue residues. Residue levels of As, Hg, Zn, and Pb were successfully used to predict sediment chronic toxicity and estimate effective tissue residues.

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.

Effects of three chemicals on the survival and reproduction of the oligochaete worm Enchytraeus coronatus in chronic toxicity tests

Summary Using Enchytraeus coronatus as a test organism, we have measured the effects of Derosal (carbendazim as active ingredient), 4-nitrophenol and potassium dichromate on survival (Lethal Concentration: LC 50 ) and on several sublethal variables (Effective Concentrations: EC 10 , EC 50 and the No Observed Effective Concentration: NOEC), in chronic agar tests. Worms were also exposed to carbendazim and potassium dichromate in a soil toxicity test (Enchytraeid Reproduction Test, ERT). Sublethal variables measured in agar tests (number of juveniles and cocoons per adult, number of eggs per cocoon, percent of hatching and adult biomass) were used to facilitate interpretation of the causes of reproductive inhibition of the number of juveniles in the ERT soil toxicity test. Effective concentrations (EC) were estimated using Probit and logistic models. The later provided a better fit for the response of the worms exposed to carbendazim in agar test, in which an increase of the number of juveniles with respect to control was found at low doses. The number of juveniles per adult was the most sensitive variable measured for the three substances in the agar toxicity tests. The reduction in the number of juveniles could be explained mainly by a negative effect on cocoon hatching due to carbendazim (4-week NOEC juveniles/adult = 0.32 mg l —1 , 4-week EC 10 Reproductive inhibition (RI) = 0.37mg l —1 ), a result probably related to failure of embryonic development. The effect of 4-nitrophenol on the number of juveniles (4-week NOEC = 0.32mg l —1 , 4-week EC 10 (RI) = 0.25 mg l —1 ) was interpreted as a consequence of a reduction in the number of eggs per cocoon, associated with a reduction in parental biomass. Reproductive inhibition (RI) in worms exposed to potassium dichromate (4-week NOEC juveniles/adult = 0.32mg l —1 , 4-week EC 10 (RI) = 0.40 mg l —1 ) was explained by a reduction in cocoon production, which apparently was not related to the loss of parental biomass but rather to a failure of worm reproductive physiology. The results of this study provide useful information regarding the effects of carbendazim, 4-nitrophenol and potassium dichromate on worm reproduction biology and contribute to our understanding of the effects of these substances measured in ERT soil toxicity tests.

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.

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.