A long-term case of residual pollution is studied after 20 years since the largest mining accident in Spain (the Aznalcóllar spill) happened. This pollution is manifested through a surface zoning consisting of bare soils (B0), sparsely vegetated soils (B1), and densely vegetated and recovered soils (B2). A biopiles treatment with a mixture of contaminated soils (B0 and B1) with recovered soils (B2) at 50% (w/w), and vermicompost addition (50 tons ha-1) was evaluated. To assess the effectiveness of treatments, total, water-soluble, and bioavailable fractions of the most polluting elements in the zone (Cu, Zn, As, Pb, Cd, and Sb) was analyzed. To evaluate the potential risk of contamination for the ecosystem, a bioassay with earthworm Eisenia andrei was carried out. Twenty years after the accident, there are still soils where total As and Pb exceed the regulatory levels and water-soluble Zn and As exceed the toxicity guidelines. According to toxicity bioassay, weight variation and juvenile production of earthworms showed an improvement after biopiles treatment, with values that trend to be similar to those of recovered soils. The only bioaccumulated element in earthworms was Cd (BAF>1), both in polluted as in treated soils, which indicates the possible existence of exclusion mechanisms of the other pollutants by earthworms. The comparison between biopiles and polluted soils showed no significant differences for the bioaccumulation factor of trace elements, with the exception of Zn and Cu, which slightly increased after treatment. According to our results, biopiles treatment combined with vermicompost addition is a good technique for the recovery of residual contaminated areas, by the improvement of soil properties and the reduction of the potential toxicity; anyway, monitoring of soils and organisms is needed to prevent the increase of bioavailability of some potentially pollutant elements over time.

In the present study, we evaluate our previously developed non-destructive soil algal toxicity method using species from a different class of algae; Class Trebouxiophyceae (Chlorella vulgaris and Chlorella sorokiniana), and directly measure the photosynthetic activity of these species adsorbed onto the soil as a new toxicity endpoint. This study shows that non-destructive soil algal toxicity method is applicable to non-specific test species, including those of Class Trebouxiophyceae as well as Class Chlorophyceae (Chlorococcum infusionum and Chlamydomonas reinhardtii). Furthermore, by performing photosynthesis image analysis, we verify that it is possible to measure the photosynthetic activity of soil algae Chlorella vulgaris adsorbed onto soils without the need to extract algal cells from the soil. We propose that the non-destructive soil algal toxicity method represents a novel technique for 1) evaluating pollutants in soil using non-specific algae and 2) conveniently and rapidly assessing the photosynthetic activity of soil algae Chlorella vulgaris adsorbed onto soil as a new toxicity endpoint.

The bioremediation of historic industrial contaminated sites is a complex process. Co-contamination, often with lead which was commonly added to gasoline until 16 years ago is one of the biggest challenges affecting the clean-up of these sites. In this study, the effect of heavy metals, as co-contaminant, together with total petroleum hydrocarbons (TPH) is reported, in terms of remaining soil toxicity and the structure of the microbial communities. Contaminated soil samples from a relatively hot and dry climate in Western Australia were collected (n = 27). Analysis of soils showed the presence of both contaminants, TPHs and heavy metals. The Microtox test confirmed that their co-presence elevated the remaining ecotoxicity. Toxicity was correlated with the presence of lead, zinc and TPH (0.893, 0.599 and 0.488), respectively, assessed using Pearson Correlation coefficient factor. Next Generation Sequencing of soil bacterial 16S rRNA, revealed a lack of dominate genera; however, despite the variation in soil type, a few genera including Azospirillum spp. and Conexibacter were present in most soil samples (85% and 82% of all soils, respectively). Likewise, many genera of hydrocarbon-degrading bacteria were identified in all soil samples. Streptomyces spp. was presented in 93% of the samples with abundance between 7% and 40%. In contrast, Acinetobacter spp. was found in only one sample but was a dominant member of (45%) of the microbial community. In addition, some bacterial genera were correlated to the presence of the heavy metals, such as Geodermatophilus spp., Rhodovibrio spp. and Rubrobacter spp. which were correlated with copper, lead and zinc, respectively. This study concludes that TPH and heavy metal co-contamination significantly elevated the associated toxicity. This is an important consideration when carrying out risk assessment associated with natural attenuation. This study also improves knowledge about the dynamics of microbial communities in mixed contamination scenarios.

Methylparaben, which is known to be an endocrine-disrupting chemical, is added to various personal care products, including cosmetics, and is also used as a food preservative and in pharmaceuticals. However, information on the toxicity of methylparaben in soil ecosystems is limited. Furthermore, unlike other substances such as metals and pesticides, there is no regulation of levels or safe concentrations of methylparaben in soil ecosystems. Therefore, the aims of this study were to evaluate the toxicity of methylparaben on soil species and to derive hazardous concentration (HC) values with respect soil ecosystem protection. We conducted acute bioassays on eight species within six taxonomic groups and chronic bioassays on five species within four taxonomic groups. On the basis of the results obtained, we derived an acute HC₅ value of 44 mg/kg soil and a corresponding chronic value of 27 mg/kg soil for methylparaben using species sensitivity distribution methodology following Australian and New Zealand guidelines. Given that there has been no proposed standard value for methylparaben in soil in any country, it was not possible to compare the HC values calculated in this study with regulation standard levels. Nevertheless, to our knowledge, this study is first to assess the toxicity of methylparaben against soil-inhabiting species and to estimate acute and chronic HCs for soil fauna and flora. The results of this study will provide valuable fundamental data for the establishment of acceptable levels of methylparaben in soil.

The probabilistic ecological risk assessment using terrestrial toxicity data has been mainly based on microfauna or mesofauna. Soil algae, which are food source for microfauna and mesofauna, may be alternatively used for assessing soil toxicity. However, there are no internationally recommended guidelines for soil algal bioassays, and the collection of algae from the test soils has some limitations. In this study, we suggested the paper-disc method as an easy-to-use alternative. This method has been widely used for testing the antibacterial toxicity of various chemicals in agar media by measuring the diameter of the inhibition zone around the disc. We adapted the paper-disc method for screening the toxicity of copper (Cu) and nickel (Ni) to the soil alga Chlorococcum infusionum using various evaluation endpoints, such as growth zone, chlorophyll fluorescence, and photosynthetic activity. Chlorophyll fluorescence and photosynthetic activity decreased with the increasing concentrations of Cu+2 or Ni+2 contaminated soils. Algal growth zone was analyzed visually and showed similar results to those of chlorophyll fluorescence. The direct ethanol extraction method and indirect culture medium extraction method were similarly effective; however, the former was easier to perform, while the latter might facilitate the analysis of additional endpoints in future studies. Overall, the results suggested that the paper-disc method was not only a user-friendly assay for screening soil toxicity, but also effective due to its association with indirect soil quality indicators.

Diclofenac is widely used as nonsteroidal anti-inflammatory drug leaving residues in the environment. To investigate effects on terrestrial ecosystems, we measured dissipation rate in soil and investigated ecotoxicological and transcriptome-wide responses in Folsomia candida. Exposure for 4 weeks to diclofenac reduced both survival and reproduction of F. candida in a dose-dependent manner. At concentrations ≥200 mg/kg soil diclofenac remained stable in the soil during a 21-day incubation period. Microarrays examined transcriptional changes at low and high diclofenac exposure concentrations. The results indicated that development and growth were severely hampered and immunity-related genes, mainly directed against bacteria and fungi, were significantly up-regulated. Furthermore, neural metabolic processes were significantly affected only at the high concentration. We conclude that diclofenac is toxic to non-target soil invertebrates, although its mode of action is different from the mammalian toxicity. The genetic markers proposed in this study may be promising early markers for diclofenac ecotoxicity.

A phenanthrene-degrading bacterium, Sphingomonas paucimobilis EPA505 was used to construct two fluorescence-based reporter strains. Strain D harboring gfp gene was constructed to generate green fluorescence when the strain started to biodegrade phenanthrene. Strain S possessing gef gene was designed to die once phenanthrene biodegradation was initiated and thus to lose green fluorescence when visualized by a live/dead cell staining. Confocal laser scanning microscopic observation followed by image analysis demonstrates that the fluorescence intensity generated by strain D increased and the intensity by strain S decreased linearly at the phenanthrene concentration of up to 200 mg/L. Such quantitative increase and decrease of fluorescence intensity in strain D (i.e., from 1 to 11.90 ± 0.72) and strain S (from 1 to 0.40 ± 0.07) were also evident in the presence of Ottawa sand spiked with the phenanthrene up to 1000 mg/kg. The potential use of the reporter strains in quantitatively determining biodegradable or toxic phenanthrene was discussed.

Numerous reports have indicated that hydrophobic organic compound bioaccessibility in sediment and soil can be determined by extraction using aqueous hydroxypropyl-β-cyclodextrin (HPCD) solutions. This study establishes the compatibility of HPCD with Selenastrum capricornutum and assesses whether its presence influences the toxicity of reference toxicants. Algal growth inhibition (72 h) showed no significant (P > 0.05) difference at HPCD concentrations up to and including 20 mM. HPCD presence did not influence the toxicity of the inorganic reference toxicant (ZnSO4), with IC50 values of 0.82 μM and 0.85 μM, in the presence and absence of HPCD (20 mM), respectively. However, HPCD presence (20 mM) reduced the toxicity of 2,4-dichlorophenol and the herbicides diuron and isoproturon. These reductions were attributed to inclusion complex formation between the toxicants and the HPCD cavity. Liberation of complexed toxicants, by sample manipulation prior to toxicity assessment, is proposed to provide a sensitive, high throughput, bioassay that reflects compound bioaccessibility. Compatibility of the biomimetic HPCD extraction method with algal cell growth inhibition bioassays to assess toxicity of reference toxicants and environmental relevant herbicides.

Propylparaben is widely used as a preservative in pharmaceuticals and personal care products and is ultimately excreted by the human body. Thus, propylparaben reaches sewage and enters the soil environment by sludge fertilization and wastewater irrigation. However, there are few existing studies on the toxicity and risks of such chemicals in terrestrial environments. In this study, a multispecies bioassay for propylparaben was performed and protective concentrations (PCs) were derived based on toxicity values by probabilistic ecological risk assessment. Acute and chronic bioassays were conducted on 11 species in eight taxonomic groups (Magnoliopsida, Liliopsida, Clitellata, Entognatha, Entomobryomorpha, Chromadorea, Chlorophyceae, Trebouxiophyceae). Based on the toxicity values calculated, the PC₉₅ values for acute and chronic SSDs were 13 and 6 mg/kg dry soil, respectively. Toxicity varied among taxa, with soil algae emerging as the most sensitive to propylparaben. This may be attributable to differences in exposure pathways among species. The exposure pathway of propylparaben can be altered by adsorption to soil particles. As parabens are presently under-regulated globally in terms of their environmental effects, our findings can serve as the basis to propose standard values for environmental protection.