Freshwater Ulva (Chlorophyta) as a bioaccumulator of selected heavy metals (Cd, Ni and Pb) and alkaline earth metals (Ca and Mg)

We analyzed the ability of freshwater taxa of the genus Ulva (Ulvaceae, Chlorophyta) to serve as bioindicators of metal in lakes and rivers. Changes in heavy metal (Ni, Cd and Pb) and alkaline earth metal (Ca and Mg) concentrations in freshwater Ulva thalli were investigated during the period from June to August 2010. The study was conducted in two ecosystems in Western Poland, the Malta lake (10 sites) and the Nielba river (six sites). Three components were collected for each sample, including water, sediment and Ulva thalli. The average concentrations of metals in the water sample and in the macroalgae decreased in the following order: Ca > Mg > Ni > Pb > Cd. The sediment revealed a slightly altered order: Ca > Mg > Pb > Ni > Cd. Ca and Mg were found at the highest concentrations in thalli due to the presence of carbonate on its surface. Among the examined heavy metals in thalli, Ni was in the highest concentration, and Cd found in the lowest concentration. There were statistically significant correlations between the levels of metals in macroalgae, water and sediment. Freshwater populations of Ulva exhibited a greater efficiency to bioaccumulate nickel as compared to species derived from marine ecosystems.     

Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils

Five bioassays (inhibition of lettuce germination and growth, earthworm mortality, inhibition of springtail population growth, avoidance by springtails) were compared, using four coke factory soils contaminated by PAHs and trace elements, before and after biotreatment. For each bioassay, several endpoints were combined in an ‘ecoscore’, a measure of test sensitivity. Ecoscores pooled over all tested bioassays revealed that most organisms were highly sensitive to the concentration of 3-ring PAHs. When four soils were combined, behavioural tests using the springtail Folsomia candida showed higher ecoscores, i.e. they were most sensitive to soil contamination. However, despite overall higher sensitivity of behavioural tests, which could be used for cheap and rapid assessment of soil toxicity, especially at low levels of contamination, some test endpoints were more sensitive than others, and this may differ from a soil to another, pointing to the need for a battery of bioassays when more itemized results are expected.     

Histopathological changes in the earthworm Eisenia andrei associated with the exposure to metals and radionuclides

Earthworms were exposed for 56 d to a contaminated soil, from an abandoned uranium mine, and to the natural reference soil LUFA 2.2. Histological changes in earthworm’s body wall (epidermis, circular and longitudinal muscles) and gastrointestinal tract (chloragogenous tissue and intestinal epithelium) were assessed, after 0, 14 and 56 d of exposure. Results have shown alterations in all the studied tissues after 14 d of exposure (except for the intestinal epithelium), yet more severe effects were registered after 56 d of exposure. Herein we report histopathological alterations as a good biomarker for the evaluation of soil quality. We also demonstrate that morphological changes in the body wall and gastrointestinal tract, are important endpoints that could be added to earthworm’s standardized tests, for the evaluation of soil toxicity, as part of the risk assessment of contaminated areas.     

Microcosm investigations of stormwater pond sediment toxicity to embryonic and larval amphibians: variation in sensitivity among species

Stormwater ponds have become common features of modern development and often represent significant amounts of open space in urbanized areas. Although stormwater ponds may provide habitat for wildlife, factors responsible for producing variation in wildlife use of ponds have received limited attention. To investigate the role of variation in species tolerances of pollutants in structuring pond-breeding amphibian assemblages, we exposed species tolerant (Bufo americanus) and not tolerant (Rana sylvatica) of urbanization to pond sediments in laboratory microcosms. Pond microcosms had elevated sediment metal levels and chloride water concentrations. Among R. sylvatica embryos, exposure to pond sediments resulted in 100% mortality. In contrast, B. americanus embryos and larvae experienced only sublethal effects (i.e., reduced size at metamorphosis) due to pond sediment exposure. Our results suggest variation in pollutant tolerance among early developmental stages of amphibians may act in concert with terrestrial habitat availability to structure amphibian assemblages associated with stormwater ponds.     

Naturally-assisted metal phytoextraction by Brassica carinata: Role ofroot exudates

Due to relatively high chelant dosages and potential environmental risks it is necessary to explore different approaches in the remediation of metal-contaminated soils. The present study focussed on the removal of metals (As, Cd, Cu, Pb and Zn) from a multiple metal-contaminated soil by growing Brassica carinata plants in succession to spontaneous metallicolous populations of Pinus pinaster, Plantago lanceolata and Silene paradoxa. The results showed that the growth of the metallicolous populations increased the extractable metal levels in the soil, which resulted in a higher accumulation of metals in the above-ground parts of B. carinata. Root exudates of the three metallicolous species were analysed to elucidate their possible role in the enhanced metal availability. The presence of metals stimulated the exudation of organic and phenolic acids as well as flavonoids. It was suggested that root exudates played an important role in solubilising metals in soil and in favouring their uptake by roots.     

Liver histopathology of the sharptooth catfish Clarias gariepinus as a biomarker of aquatic pollution

This paper reports on a comparative perspective of liver histopathological data of the sharptooth catfish Clarias gariepinus. The data was collected from a spectrum of relatively un-impacted and isolated, to polluted, eutrophic freshwater ecosystems. Results were compared between regional areas, by combining data from freshwater systems which has a similar pollution status and/or is located within the same geographical region. Measurements included necropsy observations, semi-quantitative liver histopathology (Liver Index), and selected biometrical indices. The aim was to establish whether the results of these measurements would differ between, and/or reflect the pollution status of, the different freshwater aquatic ecosystems. The histopathological analysis showed a higher prevalence of toxicopathic non-neoplastic, and pre-neoplastic alterations in C. gariepinus from the polluted sites. We also found a significant difference between the Liver Index, hepatosomatic index, and condition factor values of fish inhabiting impoundments known to be polluted, compared to the same species from the selected reference sites. Fish from polluted sites also had more macroscopic liver abnormalities. The results suggest that the liver histopathology of this bio-indicator fish species could be a useful biomarker of freshwater aquatic pollution.     

Environmental conditions enhance toxicant effects in larvae of the ground beetle Pterostichus oblongopunctatus (Coleoptera: Carabidae)

The wide geographical distribution of ground beetles Pterostichus oblongopunctatus makes them very likely to be exposed to several environmental stressors at the same time. These could include both climatic stress and exposure to chemicals. Our previous studies demonstrated that the combined effect of nickel (Ni) and chlorpyrifos (CHP) was temperature (T)-dependent in adult P. oblongopunctatus. Frequently the different developmental stages of an organism are differently sensitive to single stressors, and for a number of reasons, such as differences in exposure routes, their interactions may also take different forms. Because of this, we studied the effects of the same factors on the beetle larvae. The results showed that all factors, as well as their interactions, influenced larvae survival. The synergistic effect of Ni and CPF was temperature-dependent and the effect of Ni × T interaction on the proportion of emerged imagines indicated stronger toxicity of Ni at 25 °C than at 10 °C.     

The use of oxygen consumption and ammonium excretion to evaluate the toxicity of cadmium on Farfantepenaeus paulensis with respect to salinity

The main purpose of the present study was to detect the acute toxicity of cadmium (Cd) in F. paulensis and to investigate its effect on oxygen consumption and ammonium excretion different salinities. First, we examined the acute toxicity of Cd in F. paulensis at 24, 48, 72, and 96-h lethal concentration (LC50). Cd was significantly more toxic at 5 salinity than at 20 and 36. The oxygen consumption and ammonium excretion were estimated through experiments performed on each of the twelve possible combinations of three salinities (36, 20 and 5), at temperature 20 °C. Cd showed a reduction in oxygen consumption at 5 salinity, the results show that the oxygen consumption decreases with respect to the Cd concentration. At the highest Cd concentration employed (2 mg L⁻¹), the salinity 5 and the temperature at 20 °C, oxygen consumption decreases 53.7% in relation to the control. In addition, after separate exposure to Cd, elevation in ammonium excretion was obtained, wish were 72%, 65% and 95% higher than the control, respectively. The results show that Cd is more toxic to F. paulensis at lower salinities.     

Eisenia fetida avoidance behavior as a tool for assessing the efficiency of remediation of Pb, Zn and Cd polluted soil

Remediation by means of soil leaching with ethylenediaminetetraacetic acid (EDTA) is capable of extracting the most labile soil fractions, leaving the residual metals in biologically non-available forms. We evaluated the feasibility of the standardized earthworm (Eisenia fetida) avoidance test for assessing the efficiency of soil remediation of Pb, Zn and Cd polluted soil. Chemical extraction tests (six-step sequential extraction, toxicity characteristic leaching procedure, physiologically based extraction test, diethylenediaminepentaacetic acid extraction) indicated that the mobility, oral bioaccessibility and phytoavailability of Pb, Zn and Cd were consistently reduced. However, the avoidance test showed no significant avoidance of polluted soil in favor of that which had been remediated. Pb, Zn and Cd accumulation in E. fetida mirrored the decreasing pattern of metal potential bioavailability gained by leaching the soil with increasing EDTA concentrations. The calculated bioaccumulation factors indicated the possibility of underestimating the metal bioavailability in soil using chemical extraction tests.     

Effect of metal accumulation on metallothionein level and condition of the periwinkle Littorina littorea along the Scheldt estuary (the Netherlands)

Metal (i.e. Ag, As, Ca, Cd, Co, Cu, Mn, Pb and Zn) and metallothionein (MT) concentrations in the soft tissue of Littorina littorea were measured along the heavily polluted Western Scheldt (WS) and relatively clean Eastern Scheldt (ES) estuary. Along the WS metal and MT levels in periwinkles reflected the known downstream decreasing pollution gradient. Surprisingly in ES animals As, Mn and Zn concentrations decreased from east to west reflecting past pollution. Compared to the WS metal concentrations of ES periwinkles were significantly lower and both estuaries were maximally discriminated from each other based on their Cd soft tissue concentration using a canonical discriminant analysis. Furthermore, no overall difference was found in MT levels among animals from both estuaries. Using previously obtained condition data (i.e. dry/wet weight ratio and lipid content) the relation between soft tissue metal concentration (i.e. Cd, Cu and Zn) and fitness indicators (i.e. MT and condition data) was examined using a canonical correlation analysis. Periwinkles with a high metal load (i.e. Cd and Zn) also had high MT levels but were in a relatively poor condition.     

Arsenic accumulation by the aquatic fern Azolla: comparison of arsenate uptake, speciation and efflux by A. caroliniana and A. filiculoides

This study investigates As accumulation and tolerance of the aquatic fern Azolla. Fifty strains of Azolla showed a large variation in As accumulation. The highest- and lowest-accumulating ferns among the 50 strains were chosen for further investigations. Azolla caroliniana accumulated two times more As than Azolla filiculoides owing to a higher influx velocity for arsenate. A. filiculoides was more resistant to external arsenate due to a lower uptake. Both strains showed a similar degree of tolerance to internal As. Arsenate and arsenite were the dominant As species in both Azolla strains, with methlyated As species accounting for <5% of the total As. A. filiculoides had a higher proportion of arsenite than A. caroliniana. Both strains effluxed more arsenate than arsenite, and the amount of As efflux was proportional to the amount of As accumulation. The potential of growing Azolla in paddy fields to reduce As transfer from soil and water to rice should be further evaluated.     

The exposition of a calcareous Mediterranean soil to toxic concentrations of Cr, Cd and Pb produces changes in the microbiota mainly related to differential metal bioavailability

The involvement of the bacterial community of an agricultural Mediterranean calcareous soil in relation to several heavy metals has been studied in microcosms under controlled laboratory conditions. Soil samples were artificially polluted with Cr(VI), Cd(II) and Pb(II) at concentrations ranging from 0.1 to 5000 mg kg⁻¹ and incubated along 28 d. The lowest concentrations with significant effects in soil respirometry were 10 mg kg⁻¹ Cr and 1000 mg kg⁻¹ Cd and Pb. However, only treatments showing more than 40% inhibition of respirometric activity led to significant changes in bacterial composition, as indicated by PCR-DGGE analyses. Presumable Cr- and Cd-resistant bacteria were detected in polluted microcosms, but development of the microbiota was severely impaired at the highest amendments of both metals. Results also showed that bioavailability is an important factor determining the impact of the heavy metals assayed, and even an inverted potential toxicity ranking could be achieved if their soluble fraction is considered instead of the total concentration. Moreover, multiresistant bacteria were isolated from Cr-polluted soil microcosms, some of them showing the capacity to reduce Cr(VI) concentrations between 26% and 84% of the initial value. Potentially useful strains for bioremediation were related to Arthrobacter crystallopoietes, Stenotrophomonas maltophilia and several species of Bacillus.     

Integration of biological responses from a suite of bioassays for the Venice Lagoon (Italy) through sediment toxicity index - Part A: Development and comparison of two methodological approaches

Marine and coastal quality assessment, based on test batteries involving a wide array of endpoints, organisms and test matrices, needs for setting up toxicity indices that integrate multiple toxicological measures for decision-making processes and that classify the continuous toxicity response into discrete categories according to the European Water Framework Directive.Two toxicity indices were developed for the lagoon environment such as the Venice Lagoon. Stepwise procedure included: the construction of a database that identified test-matrix pairs (indicators); the selection of a minimum number of ecotoxicological indicators, called toxicological core metrics (CMs-tox) on the basis of specific criteria; the development of toxicity scores for each CM-tox; the integration of the CMs-tox into two indices, the Toxicity Effect Index (TEI), based on the transformation of Toxic Unit (TU) data that were integrated as logarithmic sum, and the Weighted Average Toxicity Index (WATI), starting from toxicity classes integrated as weighted mean. Results from the indices are compared; advantages and drawbacks of both approaches are discussed.     

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: A case study

This report describes an investigation of genotoxic effects in medicinal leech (Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.     

Accumulation of trace elements in the peripheral and central parts of two species of epiphytic lichens transplanted to a polluted site in Portugal

This paper compares the dynamics, i.e. the rates of change in element concentrations of young and older lichen thallus parts, of one foliose and one fruticose lichen, during a transplant experiment to a polluted site. Both lichen parts respond to environmental changes. Here, differential accumulation suggests that differential constitution leads to differential uptake and release, and/or the overall behaviour is partly due to internal translocation and regulation mechanisms within the whole lichen. For thallus parts, internal translocation should be taken into account as one more factor affecting lichen “memory length”. Young parts of the thallus presented higher rates of change, but different lichen parts accumulate different elements to different extents. Therefore tissue selection in monitoring may depend on the element of interest, and cannot be made into a generalized approach in survey set-ups: the choice depends on the element.     

Trace element accumulation in woody plants of the Guadiamar Valley, SW Spain: a large-scale phytomanagement case study

Phytomanagement employs vegetation and soil amendments to reduce the environmental risk posed by contaminated sites. We investigated the distribution of trace elements in soils and woody plants from a large phytomanaged site, the Guadiamar Valley (SW Spain), 7 years after a mine spill, which contaminated the area in 1998. At spill-affected sites, topsoils (0-25 cm) had elevated concentrations of As (129 mg kg(-1)), Bi (1.64 mg kg(-1)), Cd (1.44 mg kg(-1)), Cu (115 mg kg(-1)), Pb (210 mg kg(-1)), Sb (13.8 mg kg(-1)), Tl (1.17 mg kg(-1)) and Zn (457 mg kg(-1)). Trace element concentrations in the studied species were, on average, within the normal ranges for higher plants. An exception was white poplar (Populus alba), which accumulated Cd and Zn in leaves up to 3 and 410 mg kg(-1) respectively. We discuss the results with regard to the phytomanagement of trace element contaminated sites.     

Chronic exposure to environmental stressors induces fluctuating asymmetry in shrews inhabiting protected Mediterranean sites

Many ecotoxicological studies have addressed the effects of contaminant exposure at various levels of biological organization. However, little information exists on the effects of toxicants on wildlife populations. Here we examined exposure of populations of the greater white-toothed shrew Crocidura russula (Soricomorpha, Soricidae) occupying two protected Mediterranean sites (a polluted area, the Ebro Delta, and a control site, Garraf Massif). Bioaccumulation of selected elements (Pb, Hg, Cd, Zn, Cu, Fe, Mn, Cr, Mo, Sr, Ba, and B), a body condition index (BCI) and fluctuating asymmetry (FA) were used to assess the chronic exposure to environmental pollution. BCI was correlated neither to metal concentrations nor to FA, suggesting that this fitness measure only reflects environmental disturbances at a local level. However, shrews from the polluted area showed higher concentrations of metals and metalloids (Pb, Hg, B, and Sr) and greater shape FA than specimens from the reference area. A correlation between FA was found for both first and second principal component vectors suggesting that developmental instability increases as a result of exposure to multiple pollutants. Our results corroborate the suitability of C. russula as a bioindicator of environmental quality and show that FA is an appropriate index to examine impact of developmental stressors in populations inhabiting disturbed areas.     

Flow cytometric measurement of the clearance rate in the blue mussel Mytilus edulis and the development of a new individual exposure system for aquatic immunotoxicological studies

Animals in poor health condition are not relevant biological models. The current study focused on the use of the clearance rate of Mytilus edulis to assess the gross physiological condition of individuals maintained in stressful experimental conditions. This approach was developed in a new, highly controlled experimental exposure device designed to investigate individual responses in aquatic ecotoxicological studies. Both clearance rate values and immune parameters analysis indicated that the health condition of mussels kept in 50ml tubes for 24h or 48h was not altered compared to controls, while most parameters were depressed after 72h. Moreover, this study confirms the relevance of flow cytometric for the measurement of clearance rate compared to techniques utilizing microscopy. Current results prompted us to perform further 24h chemical exposure using this "in tubo" device.     

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: Linking biomarkers to behaviour

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC₅₀s of copper and mercury (dissolved throughout metal concentrations) at 96 h were 568 μg L⁻¹ and 62 μg L⁻¹, respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 μg L⁻¹ for copper and 3 μg L⁻¹ for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 μg L⁻¹ for copper and from 3 to 25 μg L⁻¹ for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.