Plastic in the Environment: A Modern Type of Abiotic Stress for Plant Physiology.

In recent years, plastic pollution has become a growing environmental concern: more than 350 million tons of plastic material are produced annually. Although many efforts have been made to recycle waste, a significant proportion of these plastics contaminate and accumulate in the environment. A central point in plastic pollution is demonstrated by the evidence that plastic objects gradually and continuously split up into smaller pieces, thus producing subtle and invisible pollution caused by microplastics (MP) and nanoplastics (NP). The small dimensions of these particles allow for the diffusion of these contaminants in farmlands, forest, freshwater, and oceans worldwide, posing serious menaces to human, animal, and plant health. The uptake of MPs and NPs into plant cells seriously affects plant growth, development, and photosynthesis, finally limiting crop yields and endangering natural environmental biodiversity. Furthermore, nano- and microplastics-once adsorbed by plants-can easily enter the food chain, being highly toxic to animals and humans. This review addresses the impacts of MP and NP particles on plants in the terrestrial environment. In particular, we provide an overview here of the detrimental effects of photosynthetic injuries, oxidative stress, ROS production, and protein damage triggered by MN and NP in higher plants and, more specifically, in crops. The possible damage at the physiological and environmental levels is discussed.

Metal Release from Microplastics to Soil: Effects on Soil Enzymatic Activities and Spinach Production.

Microplastics (MPs) represent emergent pollutants in terrestrial ecosystems. Microplastics can cause the release of metal and damage to crop quality. The present research aimed to evaluate the effects of Mater-bi (Bio-MPs) and polyethylene (PE-MPs) MPs at different concentrations on soil properties and on the growth of Spinacia oleracea L. Plants were grown in 30 pots filled with soil mixed with 0.5, 1 and 2% d.w. of Bio-MPs and PE-MPs and in 5 pots filled only with soil, considered as controls (K). At the end of the vegetative cycle, the spinach plants were evaluated for the epigeal (EPI) and hypogeal (HYPO) biomasses and the ratio of HYPO/EPI was calculated. In the soil, the total and the available fractions of Cr, Cu, Ni and Pb and the hydrolase (HA), ß-glucosidase (ß-glu), dehydrogenase (DHA) and urease (U) activities were evaluated. The results revealed that the addition of Bio-MPs increased soil total Cr, Cu and Pb and available Cu concentrations, and the addition of PE-MPs increased Pb availability. In soil contaminated by both Bio-MPs and PE-MPs, HA and ß-glu activities were stimulated, whereas DHA activity was reduced. The HYPO and HYPO/EPI biomasses were reduced only in soils contaminated by the 2% Bio-MPs.

Photosynthetic, Molecular and Ultrastructural Characterization of Toxic Effects of Zinc in Caulerpa racemosa Indicate Promising Bioremediation Potentiality.

Caulerpaceae are unconventional green algae composed of multinucleated, single siphonous cells. The species of Caulerpa are acquiring major scientific interest for both their invasion in the Mediterranean ecological niche and for the production of valuable natural metabolites. Furthermore, the abilities of Caulerpa spp. in the biorecovery of polluted waters were recently investigated. Among heavy metal contaminants in marine systems, zinc (Zn) is considered a critical pollutant, progressively accumulating from plastic leachates. In this study, the responses of Caulerpa racemosa to different levels (5-10 mg L-1) of Zn were studied for 14 days under laboratory-controlled conditions. Effects of Zn were monitored by measuring the growth rate, photosynthetic efficiency and gene expression. Moreover, the ability of Caulerpa to remove Zn from seawater was monitored. Zn induced detrimental effects by decreasing the relative growth rate (RGR) and maximal PSII photochemical efficiency (Fv/Fm). Moreover, C. racemosa, grown in contaminated seawater, reduced the levels of Zn to a final concentration of 1.026 and 1.932 mg L-1 after 14 days, thus demonstrating efficient uptake. Therefore, our results characterized the effects of zinc on C. racemosa and the possible role of this alga as being effective in the bioremediation of marine seawater.

Does the Element Availability Change in Soils Exposed to Bioplastics and Plastics for Six Months?

Plastic sheets are widely used in farming soil to improve the productivity of cultures. Due to their absorption capacity, plastic sheets can alter element and metal content in soils, and in turn affect soil properties. The use of biodegradable films is an attractive eco-sustainable alternative approach to overcome the environmental pollution problems due to the use of plastic films but their impacts on soil are scarcely studied. The aim of the research was to evaluate the impact of conventional plastic and bioplastic sheets on total and available concentrations of elements (Al, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, and Zn) in soils. The research was performed in mesocosm trials, filled with soil covered by conventional plastic and bioplastic sheets. After six months of exposure, soils were characterized for pH, water content, concentrations of organic and total carbon and total nitrogen, and total and available Al, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, and Zn element concentrations. The results highlighted that soils covered by bioplastic sheets showed higher total and available concentrations of elements and higher contamination factors, suggesting that bioplastic sheets represented a source of metals or a less-effective sink to these background metals in soils, compared to conventional plastic ones.

Impact of prescribed burning, mowing and abandonment on a Mediterranean grassland: A 5-year multi-kingdom comparison.

Mediterranean grasslands are semi-natural, fire-prone, species-rich ecosystems that have been maintained for centuries through a combination of fire, grazing, and mowing. Over the past half century, however, grasslands have faced numerous threats, including the abandonment of traditional agro-pastoral practices. Our hypothesis was that mowing and prescribed burning are management practices potentially effective in counteracting the reduction of plant diversity triggered by land abandonment. However, the long-term effects of such management practices on plant communities and soil microbiota in Mediterranean grassland remain poorly studied. Here, we conducted a 5-year field experiment comparing prescribed fire, vegetation mowing, and abandonment in a fire-prone Mediterranean grassland in southern Italy in order to evaluate the capability of such management strategies to counteract the detrimental impacts of land abandonment on plant diversity and the associated increase of wildfire. We combined vegetation analysis and soil chemical characterization and several microbiota analyses, including microbial biomass and respiration, arthropod community, and high-throughput sequencing of bacterial and eukaryotic rRNA gene markers. Burning and mowing significantly increased plant species richness and diversity compared to abandonment plots, reducing the abundance of perennial tall grasses in favour of short-lived species. Standing litter followed the same trend, being 3.8-fold greater and largely composed of grass remains in the abandoned compared to burnt and mowed plots. In the soil, prescribed burning caused significant increase in pH, a reduction in organic carbon, total N, and cation exchange capacity. Diversity and taxonomic composition of bacterial and fungal microbiota was affected by burning and mowing treatments. Abandonment caused shifts of microbiota towards a fungal-dominated system, composed of late successional fungi of the Basidiomycota. Fast-growing and putative fungal pathogens were more abundant under burnt and mowed treatments. Soil arthropods were influenced by vegetation and microbiota changes, being strongly reduced in mowed plots. Our study demonstrated that grassland abandonment promotes the spread of tall grasses, reducing plant diversity and increasing the risk of wildfire, while prescribed burning and mowing are effective in counteracting such negative effects.

Soil Biological Responses under Different Vegetation Types in Mediterranean Area.

The knowledge of the effects of fire on soil properties is of particular concern in Mediterranean areas, where the effects of vegetation type are still scarce also. This research aimed: to assess the properties of burnt soils under different vegetation types; to highlight the soil abiotic properties driving the soil microbial biomass and activity under each vegetation type; to compare the biological response in unburnt and burnt soils under the same vegetation type, and between unburnt and burnt soils under different vegetation types. The soils were collected at a Mediterranean area where a large wildfire caused a 50% loss of the previous vegetation types (holm oak: HO, pine: P, black locust: BL, and herbs: H), and were characterized by abiotic (pH, water, and organic matter contents; N concentrations; and C/N ratios) and biotic (microbial and fungal biomasses, microbial respiration, soil metabolic quotient, and hydrolase and dehydrogenase activities) properties. The biological response was evaluated by the Integrative Biological Responses (IBR) index. Before the fire, organic matter and N contents were significantly higher in P than H soils. After the fire, significant increases of pH, organic matter, C/N ratio, microbial biomass and respiration, and hydrolase and dehydrogenase activities were observed in all the soils, especially under HO. In conclusion, the post-fire soil conditions were less favorable for microorganisms, as the IBR index decreased when compared to the pre-fire conditions.

Impact of Anthropic Activities on Soil Quality under Different Land Uses.

Anthropization often leads to land use transformation, causing deep changes to soil properties and its quality. Land use change could be an environmental and socioeconomic problem, as it impacts soil quality and ecosystem services. There is an urgent need to understand the pressures affecting soil quality. The aim of the work is to quantify the impact of different land uses on soil abiotic and biotic properties and on its quality. To achieve the aims, soils from different land uses (forest, urban and agricultural) were collected in the surroundings of Naples and analyzed for pH, water content, contents of C and N, C/N ratio and total and available concentrations of Cu, Ni and Pb, microbial and fungal biomasses, basal respiration and metabolic quotient. Then, a soil quality index (SQI) was calculated for each land use. The results showed that soil abiotic and biotic properties of the agricultural sites differed from those of forest and urban sites. At agricultural sites, microbial abundances decreased due to low amount of C and N and to high amount of Cu and Pb. This caused low use efficiency of energetic substrates and a reduced soil quality of agricultural sites as compared to forest and urban sites.

Combined Effects of Wildfire and Vegetation Cover Type on Volcanic Soil (Functions and Properties) in A Mediterranean Region: Comparison of Two Soil Quality Indices.

Mediterranean regions are the most impacted by fire in Europe. The effects of fire on soil greatly vary according to several factors such as vegetation cover type, but they are scarcely studied. Therefore, this research aimed at evaluating the combined impacts of fire and vegetation on single soil characteristics and on the overall soil quality and functionality through two soil quality indices, simple additive (SQI) and a weighted function (SQIFUNCT). In order to reach the aims, burnt and unburnt soils were collected under different vegetation cover types (herbs and shrubs, black locust, pine and holm oak) within the Vesuvius National Park. The soils were analyzed for the main abiotic (water and organic matter content, total C, N, Ca, K, Cu and Pb concentrations, C/N ratio) and biotic (microbial and fungal biomasses, basal respiration, ß-glucosidase activity) characteristics. On the basis of the investigated soil characteristics, several soil functions (water retention, nutrient supply, contamination content, microorganism habitat and activities), and the soil quality indices were calculated. The results showed that the impact of fire on soil quality and functionality was mediated by the vegetation cover type. In fact, fire occurrence led to a decrease in water and C/N ratio under herbs, a decrease in C concentration under holm oak and a decrease in Cu and Pb concentrations under pine. Although the soil characteristics showed significant changes according to vegetation cover types and fire occurrence, both the additive and weighted function soil quality indices did not significantly vary according to both fire occurrence and the vegetation cover type. Among the different vegetation cover types, pine was the most impacted one.

Do Wildfires Cause Changes in Soil Quality in the Short Term?

Wildfires have high frequency and intensity in the Mediterranean ecosystems that deeply modify the soil abiotic (i.e., pH, contents of water, organic matter and elements) and biotic properties (i.e., biomass and activity). In 2017, an intense wildfire occurred inside the Vesuvius National Park (Southern Italy), destroying approximately 50% of the existing plant cover. So, the research aimed to evaluate the fire effects on soil quality through single soil abiotic and biotic indicators and through an integrated index (SQI). To achieve the aim, soil samples were collected inside the Vesuvius National Park at 12 sampling field points before fire (BF) and after fire (AF). The findings highlighted that in AF soil, the contents of water and total carbon, element availability, respiration and the dehydrogenase activity were lower than in BF soil; in contrast, pH and hydrolase activity were significantly higher in AF soil. The microbial biomass and activity were affected by Al, Cr and Cu availability in both BF and AF soils. Despite the variations in each investigated soil abiotic and biotic property that occurred in AF soil, the overall soil quality did not significantly differ as compared to that calculated for the BF soil. The findings provide a contribution to the baseline definition of the properties and quality of burnt soil and highlight the short-term effects of fire on volcanic soil in the Mediterranean area.

The fate of cigarette butts in different environments: Decay rate, chemical changes and ecotoxicity revealed by a 5-years decomposition experiment.

Cigarette butts (CBs) are the most common litter item on Earth but no long-term studies evaluate their fate and ecological effects. Here, the role of nitrogen (N) availability and microbiome composition on CBs decomposition were investigated by a 5-years experiment carried out without soil, in park grassland and sand dune. During decomposition, CBs chemical changes was assessed by both 13C CPMAS NMR and LC-MS, physical structure by scanning electron microscope and ecotoxicity by Aliivibrio fischeri and Raphidocelis subcapitata. Microbiota was investigated by high-throughput sequencing of bacterial and eukaryotic rRNA gene markers. CBs followed a three-step decomposition process: at the early stage (∼30 days) CBs lost ∼15.2% of their mass. During the subsequent two years CBs decomposed very slowly, taking thereafter different trajectories depending on N availability and microbiome composition. Without soil CBs showed minor chemical and morphological changes. Over grassland soil a consistent N transfer occurs that, after de-acetylation, promote CBs transformation into an amorphous material rich in aliphatic compounds. In sand dune we found a rich fungal microbiota able to decompose CBs, even before the occurrence of de-acetylation. CBs ecotoxicity was highest immediately after smoking. However, for R. subcapitata toxicity remained high after two and five years of decomposition.

Evaluation of tourism impact on soil metal accumulation through single and integrated indices.

Recently, surface soils of remote or protected areas, that receive pollutants from the surroundings or in situ activities through dry and wet deposition, exceed the baseline content of heavy metals. In the last decades, the use of single and integrated indices is a powerful tool to process, analyze and convey information about metal accumulation degree for decision makers to better manage protected areas. Single indices provide information about only one metal, whereas the integrated ones give a holistic evaluation. The aim of the research was to assess the temporal trends of three single (Contamination Factor, Enrichment Factor and Geo-accumulation Index) and two integrated (Pollution Load Index, Nemerow index) indices in order to evaluate if the touristic impact caused soil metal (Cr, Cu, Ni and Pb) accumulation. In autumn 2016 and in spring 2016 and 2017, the surface soils (0-10 cm) were collected at eight sites inside the Vesuvius National Park (Southern Italy), characterized by intense tourism from spring to autumn. The metal concentrations were measured in the soils and used to calculate the indices. In addition, the Ecological Risk Factor was calculated and the phytotoxicological assays were performed. The findings showed that the surface soils of the Vesuvius National Park were polluted by Cr, Cu, Ni and Pb, according to both single and integrated indices. The touristic impact would seem to be the main cause of soil metal accumulation, as the highest values of the calculated indices were detected for samples collected at the end of the touristic season and the lowest at the beginning of the touristic season. Anyway, Cu and Pb would seem also to derive by ex situ anthropogenic sources, whereas Cr and Ni also by natural sources, such as spontaneous fires and substrate weathering. Finally, the soils showed phytotoxic effects and low ecological risks.

Quercus ilex L. leaves as filters of air Cd, Cr, Cu, Ni and Pb.

The study of particulate matter pollution in urban areas is of great concern due its adverse effects on human health. Roadside vegetation, intercepting and filtering it, contributes to improve air quality. The aim of the research was to investigate the capability of Q. ilex leaves, already known good biomonitors of air quality, in filtering air metals. Besides, the main derivation (i.e. air or root uptake) of the investigated metals in leaf tissue was evaluated. The concentrations of Cd, Cr, Cu, Ni and Pb were measured in three groups of Q. ilex leaves (unwashed, water washed and chloroform washed). Besides, several leaf traits (i.e. length, width, petiole length, leaf area, leaf mass area and thickness) were evaluated. The findings highlighted that Cd, Cr, Cu, Ni and Pb in Q. ilex leaves exceeded the chemical fingerprint. In particular, Cd and Pb appeared the main contaminants of the investigated area as also after water washing of the leaves, their concentrations exceeded the chemical fingerprint. Ni, Cr and Pb appeared to be accumulated on leaf deposit; whereas, Cd appeared mainly adsorbed to leaf cuticle. Higher leaf width, lower leaf area and shorter petiole favoured leaf metal accumulation. Root uptake and translocation to leaves cannot be excluded for Cr and Ni; whereas, leaf Cu content would seem to depend on both leaf deposit and soil content. In conclusion, Q. ilex can be useful in filtering air metal pollution, especially for Ni, Cd, Cr and Pb, and improving air quality.

Overall plant responses to Cd and Pb metal stress in maize: Growth pattern, ultrastructure, and photosynthetic activity.

This study provides a full description of the responses of the crop energy plant Zea mays to stress induced by Cd and Pb, in view of a possible extensive use in phytoattenuation of metal-polluted soils. In this perspective, (i) the uptake capability in root and shoot, (ii) the changes in growth pattern and cytological traits, and (iii) the photosynthetic efficiency based on photochemistry and the level of key proteins were investigated in hydroponic cultures. Both metals were uptaken by maize, with a translocation factor higher for Cd than Pb, but only Cd-treated plants showed a reduced growth compared to control (i.e., a lower leaf number and a reduced plant height), with a biomass loss up to 40%, at the highest concentration of metal (10-3 M). The observation of cytological traits highlighted ultrastructural damages in the chloroplasts of Cd-treated plants. A decline of Rubisco and D1 was observed in plants under Cd stress, while a relevant increase of the same proteins was found in Pb-treated plants, along with an increase of chlorophyll content. Fluorescent emission measurements indicated that both metals induced an increase of NPQ, but only Cd at the highest concentration determined a significant decline of Fv/Fm. These results indicate a different response of Z. mays to individual metals, with Pb triggering a compensative response and Cd inducing severe morpho-physiological alterations at all investigated levels. Therefore, Z. mays could be successfully exploited in phytoattenuation of Pb-polluted soil, but only at very low concentrations of Cd to avoid severe plant damages and biomass loss.

Soil element fractions affect phytotoxicity, microbial biomass and activity in volcanic areas.

Soil quality is strongly affected by microbial biomass that is involved in organic matter mineralization and the supply of nutrients to plants. The effects of trace elements on soil microbial biomass and activity are still controversial, and the contents of the elements in different forms, more than the total amounts, may affect soil microbial community. Volcanic soils are peculiar environments because of their chemical characteristics. Therefore, the aims of this research were to evaluate in volcanic soils: i) the elemental composition; ii) the elemental availability; and iii) the effects of elemental fractions on soil microbial biomass and activity. In order to reach the aims, the BCR sequential extraction method was applied in order to separate 22 elements in different soil fractions: acid soluble, reducible, oxidisable and residual. The studied biological parameters were: microbial and fungal biomasses, soil respiration, metabolic quotient, coefficient of endogenous mineralization, dehydrogenase and hydrolase activities, and phytotoxicity. Among the investigated elements, Al, B, Ba, Ca, Cr, Fe, K, Mg, Mn, Na, Ni, Ti, V and Zn were scarcely available; Cd appeared to be the most ready available element; Zn was mainly present in the acid soluble and in the residual fractions. Microbial biomass and activity appeared to be mainly affected by the reducible and oxidasable fractions of the investigated elements more than the acid soluble or residual ones. With the exception of La and V, the elemental content in the various fractions would seem to stimulate the microbial biomass and activity. Finally, the investigated volcanic soils showed phytotoxic properties.

Total and fraction content of elements in volcanic soil: Natural or anthropogenic derivation.

Soil element composition derives from parent material disaggregation during pedogenesis and weathering processes but also by anthropogenic inputs. Elements are present in soils in different chemical forms that affect their availability and mobility. The aim of the study was to evaluate the main derivation, natural or anthropogenic, of elements in the soils of the Vesuvius National Park (a natural environment strongly affected by human impacts). Besides, the effects of age of the lava from which soils derive, different vegetation covers, traffic fluxes along the two roads connecting the Vesuvius crater and altitudes of the sites on the pseudo-total element concentrations and on their contents in different fraction of soil were investigated. To reach the aims, BCR (Bureau Commun de Référence) sequential extraction was performed in order to determine the distribution of elements into: acid-soluble, reducible, oxidizable and residual fractions. The relationship between the main environmental media and distribution of elements was discussed using non-metric multidimensional scaling (NMDS). The findings showed that, with the exception of Cd, Cu, Pb and Zn that would seem to derive also from human activities, the other investigated elements (Al, As, B, Ba, Ca, Cd, Cr, Cu, Fe, K, La, Mg, Mn, Na, Ni, P, Pb, Si, Ti, V, W and Zn) mainly had a natural derivation. Among the investigated elements, only Cd could represent a potential high risk for the studied andosols. The highest element accumulations in the soils at low altitude could be attributable to an integrated effect of plant cover, vicinity of downtowns and traffic flux. The acid-soluble fraction of elements appeared more linked to lava age; the reducible and oxidizable ones to plant cover; the residual one to the chemical composition of the parent material that gave origin to the soils.

Heavy metal and polycyclic aromatic hydrocarbon concentrations in Quercus ilex L. leaves fit an a priori subdivision in site typologies based on human management.

Concentrations of four heavy metals (HMs) (Cd, Cr, Fe, Pb) and four polycyclic aromatic hydrocarbons (PAHs) (fluoranthene, phenanthrene, chrysene, benzo[a]pyrene) in Quercus ilex L. leaves collected at the Campania Region (Southern Italy) in previous air biomonitoring studies were employed to (1) test the correspondence with an a priori site subdivision (remote, periurban, and urban) and (2) evaluate long temporal trends of HM (approximately 20 years) and PAH (approximately 10 years) air contaminations. Overall, Q. ilex leaf HM and PAH concentrations resulted along the gradient: remote < periurban < urban sites, reflecting the a priori subdivision based on human management. Over a long time, although a clear decrease of leaf Pb, chrysene, fluoranthene, and phenanthrene concentrations occurred at the urban sites, a high contamination level persists.

Ecotoxicological assessment of metal-polluted urban soils using bioassays with three soil invertebrates.

This study aimed at assessing the quality of urban soils by integrating chemical and ecotoxicological approaches. Soils from five sites in downtown Naples, Italy, were sampled and characterized for physical-chemical properties and total and water-extractable metal concentrations. Bioassays with Eisenia andrei, Enchytraeus crypticus and Folsomia candida were performed to assess toxicity of the soils, using survival, reproduction and growth as the endpoints. Metal bioaccumulation in the animals was also measured. The properties and metal concentrations of the soils strongly differed. Metal bioaccumulation was related with total metal concentrations in soil and was highest in E. crypticus, which was more sensitive than E. andrei and F. candida. Responses of the three species to the investigated soils seemed due to both metal contamination and soil properties.

Soil invertebrates as bioindicators of urban soil quality.

This study aimed at relating the abundance and diversity of invertebrate communities of urban soils to chemical and physical soil characteristics and to identify the taxa most sensitive or tolerant to soil stressors. The invertebrate community of five urban soils in Naples, Italy, was sampled. To assess soil quality invertebrate community indices (Shannon, Simpson, Menhinick and Pielou indices), Acarina/Collembola ratios, and the soil biological quality index (QBS) were calculated. The chemical and physical characteristics of the soils strongly differed. Abundance rather than taxa richness of invertebrates were more affected by soil characteristics. The community was more abundant and diverse in the soils with high organic matter and water content and low metal (Cu, Pb, Zn) concentrations. The taxa more resistant to the urban environment included Acarina, Enchytraeids, Collembola and Nematoda. Collembolans appeared particularly sensitive to changing soil properties. Among the investigated indices, QBS seems most appropriate for soil quality assessment.

Assessment of the effects of Cr, Cu, Ni and Pb soil contamination by ecotoxicological tests.

This study aimed to assess soil quality by chemical and ecotoxicological investigations and to check the correspondence between soil metal concentrations and ecotoxicity. For these purposes, surface soils collected at four adjacent roadside urban parks and at a former industrial area were characterized for C/N, organic matter content, texture, and pH. Cr, Cu, Ni and Pb, chosen among the most representative soil metal contaminants, were measured as total content and as available and water soluble fractions. In addition, the total concentrations of the investigated metals were used to calculate two chemical indices: the contamination and the potential ecological risk factors. The toxicity of the investigated soils was evaluated by an ecotoxicity test battery carried out on both soil samples (Vibrio fischeri, Heterocypris incongruens and Sinapis alba) and elutriates (Vibrio fischeri, Daphnia magna and Selenastrum capricornutum). The findings, both by the chemical and ecotoxicological approaches, would suggest that the soils with high metal contamination pose ecological risks. On the other hand, moderately metal contaminated soils did not exclude soil ecotoxicity. In fact, toxic effects were also highlighted in soils with low metal content, toxicity being affected by metal availability and soil characteristics. Moreover, the results suggest the importance of using a battery of tests to assess soil ecotoxicity.

Suitability of lysosomal membrane stability in Eisenia fetida as biomarker of soil copper contamination.

Accumulated metals in soils negatively affect dwelling organisms. Earthworms, which are widespread and perform various essential functions, are able to accumulate metals that can damage the coelomic cells. The aim of this work was to evaluate the effect on Eisenia fetida lysosomal membrane stability both during and after copper exposure, and finally to link this to internal concentrations. E. fetida specimens were exposed to a reference soil and two Cu-spiked soils (35 and 350 mg kg(-1) d.w.) for 14 days (uptake period) and then transferred into the reference soil for other 18 days (elimination period). After 3 days of uptake, internal Cu concentrations increased and were higher in the specimens exposed to soils spiked with 350 mg Cu kg(-1) d.w. After 2 days of elimination, a strong decrease of internal Cu concentrations was always observed. The lysosomal membrane stability, measured as neutral red retention-times, was approximately 50 min for the earthworms exposed to the reference soils, whereas it decreased, at the end of the uptake period, to 21 and 13 min, respectively, for the organisms exposed to soils spiked with 35 and 350 mg Cu kg(-1) d.w. A full recovery of the lysosomal membrane stability was reached after 14 and 18 days of the elimination period, respectively, for the organisms exposed to soils spiked with 35 and 350 mg Cu kg(-1) d.w. The neutral-red assay would seem a good biomarker since the lysosomal membrane stability of E. fetida appeared to respond rapidly and strongly to soil copper contamination.