Impact of genetic diversity and inbreeding on the life-history of Chironomus midges over consecutive generations

We report results of a multigenerational experiment with Chironomus riparius. Two strains with a high and a low level of genetic variability were exposed to a low, environmentally relevant TBT concentration of 80 μg Sn kg⁻¹ sediment dw nominally (time weighted mean, based on measured concentrations: 4.5 μg Sn kg⁻¹ sediment dw), and various life history traits as well as genetic diversity were monitored for eleven consecutive generations. While TBT effects are hardly visible in the outbred and genetically diverse strain, the inbred and genetically impoverished strain shows a clearly reduced population growth rate compared to the control. Moreover, the impoverished strain shows an increase in fitness over time. Analyses of variation at five microsatellite loci revealed that the level of genetic variation is strongly reduced in the inbred compared to the outbred strain. Moreover, genetic diversity increases over time in the inbred strain. This finding explains the observed increase in fitness in both inbred lineages (control and TBT exposed). The results document that inbreeding and the level of genetic diversity might be of crucial importance in populations under pollution stress. Furthermore, ecotoxicological bioassays have to consider genetic diversity if results between laboratories should be comparable. Our data provides evidence that genetic diversity strongly contributes to the survival of a population exposed to chemical pollution.     

Variation in sensitivity to cadmium among genetically characterized laboratory strains of the midge Chironomus riparius

Estimating the extent of intraspecific variation in sensitivity to contaminant exposure is important in order to explain variation in the outcome of toxicity tests and to predict the effects of chemical stress on natural populations of plants and animals. However, only few studies provide evident data concerning intraspecific variation in life-history traits caused by a differential response to chemical stress. In this study, we compared the life-history response of six laboratory strains of the midge Chironomus riparius to cadmium exposure in a full life-cycle assay. In addition, the level of genetic variation in all strains was measured at five variable microsatellite loci. Several significant differences in life-history traits among the strains were observed in controls and cadmium treatments. The extent of variation among strains was largest at moderate cadmium concentration (0.42 mg Cd/kg dw). At increased Cd concentrations all strains showed similar levels of high mortality and reduced reproductive success. All strains exhibited considerable levels of genetic impoverishment compared to field populations. Strains with low genetic variation showed reduced fitness in the controls and were more susceptible to Cd exposure. For instance, no reproductive success in the lowest Cd treatment was observed for the strain with the lowest level of genetic diversity. In contrast, this Cd concentration had no negative effects on life-history traits of more variable strains. These results confirm recent findings, that inbreeding and reduced genetic variation influence the reaction of populations towards environmental stress. In addition, we show that the level of genetic variation and inbreeding directly influences the outcome of toxicity tests and contributes to the lack of reproducibility of test results among laboratories.     

The effects of dietary nickel on the detoxification enzymes, innate immunity and resistance to the fungus Beauveria bassiana in the larvae of the greater wax moth Galleria mellonella

In this study, we tested the effects of dietary nickel on the activity of glutathione S-transferase (GST), esterases, phenoloxidase, and encapsulation in the haemolymph of larvae of the greater wax moth Galleria mellonella. We also explored the effects of dietary nickel on larval resistance to infection by the fungus Beauveria bassiana. Larvae fed a low dose of nickel (10 μg g⁻¹) had significantly higher GST, phenoloxidase activity and encapsulation responses than controls fed on a nickel-free diet. We also found that larvae fed a sublethal dose of nickel (50 μg g⁻¹) had increased GST, esterase activity and encapsulation rates but decreased phenoloxidase activity. Although, a sublethal dose of dietary nickel enhanced innate immunity, we found that this reduced resistance against the real pathogen. Our results suggest that enhanced immunity and detoxification enzyme activity of insects may not be beneficial to resistance to fungal infection. It appears that there is a trade off between different resistance mechanisms in insects under different metal treatments.     

Multi-generation studies with Chironomus riparius--effects of low tributyltin concentrations on life history parameters and genetic diversity

While toxicological data are available for numerous chemicals from standard tests, little is known on effects of pollutants over several generations or regarding chronic effects of chemicals on genetic diversity. Within the experiments, effects of the model pollutant tributyltin (TBT) were investigated over eleven generations at a sublethal TBT concentration of 4.46 μg as Sn kg⁻¹ sediment dw on life-cycle parameters and genetic variability of Chironomus riparius. Moreover, the adaptation potential towards TBT was determined. This experimental design enables the identification of TBT effects on life-cycle parameters and the determination of a potential extinction risk caused by chronic exposure. Furthermore, effects on the genetic structure can be determined, which are not predictable based solely on knowledge of the toxic mode of action of the chemical.

Genetic variety was determined via microsatellite analysis, measuring individual length differences of highly variable satellite DNA fragments. For the identification of changes in tolerances towards the stressor, acute and chronic toxicity experiments were conducted.

During the multi-generation study, significant effects on development and reproduction were determined. For some generations, the emergence was significantly (p < 0.05) delayed under TBT exposure. Reproduction seems to be a sensitive parameter as well, whereby females laid significantly larger egg masses (p < 0.05) in the latter generations. TBT did not affect the population growth rate nor the genetic variability, while clear deviations from the Hardy–Weinberg equilibrium appeared. The study also provides strong evidence for the acquirement of a higher tolerance towards the stressor in the TBT-exposed group.     

Highly charged swelling mica reduces Cu bioavailability in Cu-contaminated soils

This is the first test of a highly charged swelling mica's (Na-2-mica) ability to reduce the plant-absorbed Cu in Cu-contaminated soils from Chile. Perennial ryegrass (Lolium perenne L.) was grown in two acid soils (Sector 2: pH 4.2, total Cu = 172 mg Cu kg⁻¹ and Sector 3: pH 4.2, total Cu = 112 mg Cu kg⁻¹) amended with 0.5% and 1% (w/w) mica, and 1% (w/w) montmorillonite. At 10 weeks of growth, both mica treatments decreased the shoot Cu of ryegrass grown in Sector 2 producing shoot Cu concentrations above 21-22 mg Cu kg⁻¹ (the phytotoxicity threshold for that species), yet the mica treatments did not reduce shoot Cu concentrations when grown in Sector 3, which were at a typical level. The mica treatments improved shoot growth in Sector 3 by reducing free and extractable Cu to low enough levels where other nutrients could compete for plant absorption and translocation. In addition, the mica treatments improved root growth in both soils, and the 1% mica treatment reduced root Cu in both soils. This swelling mica warrants further testing of its ability to assist re-vegetation and reduce Cu bioavailability in Cu-contaminated surface soils.     

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.     

Food utilization and growth of cutworm Spodoptera litura Fabricius larvae exposed to nickel,and its effect on reproductive potential

Food utilization and growth of the 5th and 6th instar Spodoptera litura Fabricius larvae, and its effect on reproduction potential was evaluated by feeding larvae diets with different doses of Ni for 3 generations. Dose-dependent relationships between Ni levels and food consumption and growth were variable with different larval developmental period and Ni exposure duration. RCR, AD and RGR of the 6th instar larvae were much more affected by Ni exposure than those of 5th instar larvae, and the effects were strongest in the 3rd generation. It was found that RCR was significantly stimulated after 1 and 20 mg kg⁻¹ Ni exposure, while AD was significantly inhibited after 1, 5, 10 and 40 mg kg⁻¹ Ni exposure. However, lower levels of Ni (?5 mg kg⁻¹) significantly increased and higher levels of Ni (?10 mg kg⁻¹) significantly decreased RGR. In 3 successive generations, 10 mg kg⁻¹ Ni significantly increased the ECI and ECD of the 5th instar larvae, and 5 mg kg⁻¹ Ni significantly increased the ECD of the 6th instar larvae. However, ECD were all significantly inhibited with 20 mg kg⁻¹ Ni exposure. Results also revealed that durations of larvae were shortened at low levels of Ni, but extended at high levels of Ni. Fecundity was inhibited by the highest Ni doses in each generation, while improved by low Ni doses in the 3rd generation. Hatching rates in all treatments were significantly decreased in a Ni dose-dependent manner. Study indicated that effects of Ni on these parameters were predominant with the increasing Ni exposure period.     

Effects of experimental nitrogen additions on plant diversity in tropical forests of contrasting disturbance regimes in southern China

Responses of understory plant diversity to nitrogen (N) additions were investigated in reforested forests of contrasting disturbance regimes in southern China from 2003 to 2008: disturbed forest (with harvesting of understory vegetation and litter) and rehabilitated forest (without harvesting). Experimental additions of N were administered as the following treatments: Control, 50 kg N ha⁻¹ yr⁻¹, and 100 kg N ha⁻¹ yr⁻¹. Nitrogen additions did not significantly affect understory plant richness, density, and cover in the disturbed forest. Similarly, no significant response was found for canopy closure in this forest. In the rehabilitated forest, species richness and density showed no significant response to N additions; however, understory cover decreased significantly in the N-treated plots, largely a function of a significant increase in canopy closure. Our results suggest that responses of plant diversity to N deposition may vary with different land-use history, and rehabilitated forests may be more sensitive to N deposition.     

The role of hydrostatic pressure on developmental stages of Pomatoceros lamarcki (Polychaeta: Serpulidae) exposed to water accommodated fractions of crude oil and positive genotoxins at simulated depths of 1000-3000 m

The effect of high hydrostatic pressures on the ecotoxicological profile of pollutants is an unexplored research area. Using Pomatoceros lamarcki as a surrogate organism for this eco-barotoxicological study, it was found that in a 48 h larval bioassay with water accommodated fractions (WAF) of crude oil of up to 15.1 mg L⁻¹ (total hydrocarbon content) and hydrostatic pressures up to 300 bar (3000 m), an additive response was found (p < 0.001) rather than any synergism (p = 0.881). Comprehensive cytogenetic analysis of 6-h (15 °C) embryos exposed to WAF (0.19 mg L⁻¹) at 100 bar showed no effects on mitotic fidelity or cell division rate over the 1 bar treatment. However, embryo's treated with the clastogen mitomycin-c at 100 bar exhibited a significant increase in mitotic aberrations over 1 bar treated as was the case with hypo/hypersaline treatments (p < 0.05). Conversely, an increase in hydrostatic pressure actually reduced the effects of spindle inhibition by the aneugen colchicine (p < 0.05).     

Does nitrogen deposition increase forest production? The role of phosphorus

Effects of elevated N deposition on forest aboveground biomass were evaluated using long-term data from N addition experiments and from forest observation plots in Switzerland. N addition experiments with saplings were established both on calcareous and on acidic soils, in 3 plots with Fagus sylvatica and in 4 plots with Picea abies. The treatments were conducted during 15 years and consisted of additions of dry NH₄NO₃ at rates of 0, 10, 20, 40, 80, and 160 kg N ha⁻¹ yr⁻¹. The same tree species were observed in permanent forest observation plots covering the time span between 1984 and 2007, at modeled N deposition rates of 12-46 kg N ha⁻¹ yr⁻¹. Experimental N addition resulted in either no change or in a decreased shoot growth and in a reduced phosphorus concentration in the foliage in all experimental plots. In the forest, a decrease of foliar P concentration was observed between 1984 and 2007, resulting in insufficient concentrations in 71% and 67% of the Fagus and Picea plots, respectively, and in an increasing N:P ratio in Fagus. Stem increment decreased during the observation period even if corrected for age. Forest observations suggest an increasing P limitation in Swiss forests especially in Fagus which is accompanied by a growth decrease whereas the N addition experiments support the hypothesis that elevated N deposition is an important cause for this development.     

Microtubule integrity and cell viability under metal (Cu,Ni and Cr) stress in the seagrass Cymodocea nodosa

The effects of increasing Cu, Ni and Cr concentrations (0.5, 5, 10, 20 and 40 mg L⁻¹) on microtubule organization and the viability of leaf cells of the seagrass Cymodocea nodosa for 13 consecutive days were investigated under laboratory conditions. Increased oblique microtubule orientation, microtubule depolymerization at the 5–40 mg L⁻¹ Ni treatments after 3 d of exposure, and a complete microtubule depolymerization at all Ni treatments after 5 d were observed. Cu depolymerised microtubules after three to 7 d of exposure, while Cr caused an extensive microtubule bundling after 9 or 11 d of exposure, depending on metal dosage. Fluorescence intensity measurements further consolidated the above phenomena. Cell death, occurring at later time than microtubule disturbance, was also observed at all Cu and Ni treatments and at the 10–40 mg L⁻¹ Cr treatments and adding to the above quantification of the number of dead cells clearly showed that only a portion of the cell population studied died. The data presented, being the first assessment of microtubule disturbance in seagrasses, indicate that microtubules in seagrass leaf cells could be used as a valuable and early marker of metal-induced stress in biomonitoring programmes.     

Strong links between metal contamination, habitat modification and estuarine larval fish distributions

Changes to larval fish assemblages may have far reaching ecological impacts. Correlations between habitat modification, contamination and marine larval fish communities have rarely been assessed in situ. We investigated links between the large-scale distribution of stressors and larval fish assemblages in estuarine environments. Larval fish communities were sampled using a benthic sled within the inner and outer zones of three heavily modified and three relatively unmodified estuaries. Larval abundances were significantly greater in modified estuaries, and there were trends towards greater diversity in these systems. Differences in larval community composition were strongly related to sediment metal levels and reduced seagrass cover. The differences observed were driven by two abundant species, Paedogobius kimurai and Ambassis jacksoniensis, which occurred in large numbers almost exclusively in highly contaminated and pristine locations respectively. These findings suggest that contamination and habitat alteration manifest in substantial differences in the composition of estuarine larval fish assemblages.     

UVR sensitivity of Chaoborus larvae

In small temperate lakes, predation by fish generally regulates the species structure and abundance of larval Chaoborus. Yet, Chaoborus abundance may also vary appreciably among lakes with no fish. Many fishless lakes in Sudbury, Ontario, have transparent waters. This raises the possibility that low abundance of Chaoborus in such lakes may be attributable to UVR-induced mortality. To determine whether UVR affects Chaoborus survival, we performed 6 in situ experiments over 2 to 4 day periods at 4 depths in Ruth-Roy Lake (a clear fishless lake with few Chaoborus). Third and fourth instar C. punctipennis were randomly allocated to 3 treatments: quartz (UVR+ PAR), OP3 acrylite (PAR only) and dark controls. Survival under UVR+PAR was significantly reduced in comparison with the other treatments. Survival under PAR only was high, and did not differ from the dark controls. Time to death increased with incubation depth and larval stage. These results suggest that the small Chaoborus population in Ruth-Roy Lake, and perhaps in other fishless, clear lakes may be attributed to UVR-induced mortality.     

Phenogenetic response of silver birch populations and half-sib families to elevated ozone and ultraviolet-B radiation at juvenile age

Phenogenetic response of silver birch populations and half-sib families to separate and combined elevated ozone (O₃) concentrations and ultraviolet-B (UV-B) radiation dozes was studied at juvenile age in the climatic chambers. Significant population and family effects were found for seedling height, lamina width, and leaf damage. The exposure to UV-B radiation decreased genetic variation at the stage of seed germination. Complex exposure to UV-B and O₃ caused an increase of genetic variation at the stage of intensive seedling growth: seedling height genetic variation in separate treatments increased from 23.7–38.6 to 33.7–65.7%, the increase for lamina width was from 10.2–13.9 to 13.6–31.8%. Different populations and families demonstrated differing response to elevated complex UV-B and O₃ exposure. Changes of genetic intra-population variation were population-specific. Such changes in genetic variation under the impact of stressors can alter adaptation, stability, and competitive ability of regenerating populations in a hardly predictive way.     

Polyaniline nanofibers: acute toxicity and teratogenic effect on Rhinella arenarum embryos

The fate and effect of nanomaterials in the environment is of paramount importance towards the technological application of the materials. This work shows the ecotoxicological potential of polyaniline (PANI) nanofibers in the larvae Rhinella arenarum by means of AMPHITOX test. Acute toxicity of PANI nanofibers towards embryos of the common South American toad R. arenarum (Anura: bufonidae) was evaluated in the premetamorphosis (stage 25) larvae. The exposure of R. arenarum larvae to at dose of 150, 250 and 400 mg L⁻¹ resulted in 100% viability within 96 h exposure. The embryos at 2-4 blastomers stage (early life stage teratogenic test) revealed that embryos were not killed and no teratogenic effects were observed when embryos were incubated with PANI nanofibers (150 and 250 mg L⁻¹), while only a growth retardation of embryos was induced at levels of 250 mg PANI nanofibers L⁻¹. On the other hand, at 400 mg L⁻¹ concentration, a reduction in the body length of larvae and tail malformation was observed. This results suggest that a concentration-dependent toxicity is operative, typified by phenotypes that had abnormal body axes. The presence of PANI nanofibers in gut contents and its excretion by larval stages of R. arenarum was confirmed by UV-visible spectroscopy.     

Triclosan affects the microbial community in simulated sewage-drain-field soil and slows down xenobiotic degradation

Effects of the common antibacterial agent triclosan on microbial communities and degradation of domestic xenobiotics were studied in simulated sewage-drain-field soil. Cultivable microbial populations decreased 22-fold in the presence of 4 mg kg⁻¹ of triclosan, and triclosan-resistant Pseudomonas strains were strongly enriched. Exposure to triclosan also changed the general metabolic profile (Ecoplate substrate profiling) and the general profile (T-RFLP) of the microbial community. Triclosan degradation was slow at all concentrations tested (0.33-81 mg kg⁻¹) during 50-days of incubation. Mineralization experiments (¹⁴C-tracers) and chemical analyses (LC-MS/MS) showed that the persistence of a linear alkylbenzene sulfonate (LAS) and a common analgesic (ibuprofen) increased with increasing triclosan concentrations (0.16-100 mg kg⁻¹). The largest effect was seen for LAS mineralization which was severely reduced by 0.16 mg kg⁻¹ of triclosan. Our findings indicate that environmentally realistic concentrations of triclosan may affect the efficiency of biodegradation in percolation systems.     

A level change in mutagenicity of Japanese tap water over the past 12 yr

A relative comparison study of mutagenicity in Japanese tap water was conducted for 1993 and 2005 surveys. It intended to assess the effects of advanced water treatment installations to water works, improvement of raw water quality and improvement of residual HOCl concentration controlling. Sampling points (taps) were the same in both surveys. The results of 245 samples obtained by the Ames Salmonella mutagenicity test (Ames test) were analyzed. The Ames tests were conducted by using Salmonella typhimurium TA98 and TA100 strains with and without exogenous activation (S9). With the exception of TA100-S9, the other conditions needed no discussion as a factor in the mutagenicity level change. The average mutagenicity in 1993 and 2005 under the conditions of TA100-S9 were 2600 and 1100 net revertant L⁻¹, respectively. This indicated that the mutagenicity level of Japanese tap water decreased during the 12-yr period. Particularly a remarkable decrease in mutagenicity was observed in the water works where the advanced water treatments were installed during the 12-yr period. The advanced water treatments were effective in decreasing the mutagenicity of tap water. Mutagenicity also decreased in the water works with conventional water treatments; the improvement of residual HOCl concentration controlling was also considered to be effective in decreasing the mutagenicity of tap water.     

Assessment of bacterial communities and characterization of lead-resistant bacteria in the rhizosphere soils of metal-tolerant Chenopodium ambrosioides grown on lead-zinc mine tailings

Bacterial communities in the rhizosphere soils of metal tolerant and accumulating Chenopodium ambrosioides grown in highly and moderately lead-zinc mine tailings contaminated-soils as well as the adjacent soils with low metal contamination were characterized by using cultivation-independent and cultivation techniques. A total of 69, 73, and 83 bacterial operational taxonomic units (OTUs) having 84.8-100% similarity with the closest match in the database were detected among high, moderate, and low-contamination soil clone libraries, respectively. These OTUs had a Shannon diversity index value in the range of 4.06-4.30. There were 9, 10, and 14 bacterial genera specific to high, moderate, and low metal-contaminated soil clone libraries, respectively. Phylogenetic analysis showed that the Pb-resistant isolates belonged to 8 genera. Pseudomonas and Arthrobacter were predominant among the isolates. Most of the isolates (82-86%) produced indole acetic acid and siderophores. More strains from the highly metal-contaminated soil produced 1-aminocyclopropane-1-carboxylate deaminase than the strains from the moderately and lowly metal-contaminated soils. In experiments involving canola grown in quartz sand containing 200 mg kg⁻¹ of Pb, inoculation with the isolated Paenibacillusjamilae HTb8 and Pseudomonas sp. GTa5 was found to significantly increase the above-ground tissues dry weight (ranging from 19% to 36%) and Pb uptake (ranging from 30% to 40%) compared to the uninoculated control. These results show that C. ambrosioides harbor different metal-resistant bacterial communities in their rhizosphere soils and the isolates expressing plant growth promoting traits may be exploited for improving the phytoextraction efficiency of Pb-polluted environment.     

Reduction of selenite to red elemental selenium by moderately halotolerant Bacillus megaterium strains isolated from Bhitarkanika mangrove soil and characterization of reduced product

Two Gram (+) bacterial strains, BSB6 and BSB12, showing resistance and potential for Se(IV) reduction among 26 moderately halotolerant isolates from the Bhitarkanika mangrove soil were characterized by biochemical and 16S rDNA sequence analyses. Both of them were strictly aerobic and able to grow in a wide range of pH (4-11), temperature (4-40 °C) and salt concentration (4-12%) having an optimum growth at 37 °C, pH ∼7.5 and 7% salt (NaCl). The biochemical characteristics and 16S rDNA sequence analysis of BSB6 and BSB12 showed the closest phylogenetic similarity with the species Bacillus megaterium. Both the strains effectively reduced Se(IV) and complete reduction of selenite (up to 0.25 mM) was achieved within 40 h. SEM with energy dispersive X-ray and TEM analyses revealed the formation of nano size spherical selenium particles in and around the bacterial cells which were also supported by the confocal micrograph study. The UV-Vis diffuse reflectance spectra and XRD of selenium precipitates revealed that the selenium particles are in the nanometric range and crystalline in nature. These bacterial strains may be exploited further for bioremediation process of Se(IV) at relatively high salt concentrations and green synthesis of selenium nanoparticles.