Methods to Identify Soil Microbial Bioindicators of Sustainable Management of Bioenergy Crops.

Here we describe a suite of methods to identify potential taxonomic and functional soil microbial indicators of soil quality and plant health in biofuel crops in various areas and land types. This approach draws on tools to assess microbial diversity, greenhouse gas fluxes, and soil physicochemical properties in bioenergy cropping systems. Integrative statistical models are then used to identify potential microbial indicators for sustainable management of bioenergy crops.

The explosive trinitrotoluene (TNT) induces gene expression of carbonyl reductase in the blue mussel (Mytilus spp.): a new promising biomarker for sea dumped war relicts?

Millions of tons of all kind of munitions, including mines, bombs and torpedoes have been dumped after World War II in the marine environment and do now pose a new threat to the seas worldwide. Beside the acute risk of unwanted detonation, there is a chronic risk of contamination, because the metal vessels corrode and the toxic and carcinogenic explosives (trinitrotoluene (TNT) and metabolites) leak into the environment. While the mechanism of toxicity and carcinogenicity of TNT and its derivatives occurs through its capability of inducing oxidative stress in the target biota, we had the idea if TNT can induce the gene expression of carbonyl reductase in blue mussels. Carbonyl reductases are members of the short-chain dehydrogenase/reductase (SDR) superfamily. They metabolize xenobiotics bearing carbonyl functions, but also endogenous signal molecules such as steroid hormones, prostaglandins, biogenic amines, as well as sugar and lipid peroxidation derived reactive carbonyls, the latter providing a defence mechanism against oxidative stress and reactive oxygen species (ROS). Here, we identified and cloned the gene coding for carbonyl reductase from the blue mussel Mytilus spp. by a bioinformatics approach. In both laboratory and field studies, we could show that TNT induces a strong and concentration-dependent induction of gene expression of carbonyl reductase in the blue mussel. Carbonyl reductase may thus serve as a biomarker for TNT exposure on a molecular level which is useful to detect TNT contaminations in the environment and to perform a risk assessment both for the ecosphere and the human seafood consumer.

Serum biomarkers from cell-based assays for AhRL and MIS strongly predicted the future development of diabetes in a large community-based prospective study in Korea.

Exposure to environment-polluting chemicals (EPC) is associated with the development of diabetes. Many EPCs exert toxic effects via aryl hydrocarbon receptor (AhR) and/or mitochondrial inhibition. Here we investigated if the levels of human exposure to a mixture of EPC and/or mitochondrial inhibitors could predict the development of diabetes in a prospective study, the Korean Genome and Epidemiological Study (KoGES). We analysed AhR ligands (AhRL) and mitochondria-inhibiting substances (MIS) in serum samples (n = 1,537), collected during the 2008 Ansung KoGES survey with a 4-year-follow-up. Serum AhRL, determined by the AhR-dependent luciferase reporter assay, represents the contamination level of AhR ligand mixture in serum. Serum levels of MIS, analysed indirectly by MIS-ATP or MIS-ROS, are the serum MIS-induced mitochondria inhibiting effects on ATP content or reactive oxygen species (ROS) production in the cultured cells. Among 919 normal subjects at baseline, 7.1% developed impaired glucose tolerance (IGT) and 1.6% diabetes after 4 years. At the baseline, diabetic and IGT sera displayed higher AhRL and MIS than normal sera, which correlated with indices of insulin resistance. When the subjects were classified according to ROC cut-off values, fully adjusted relative risks of diabetes development within 4 years were 7.60 (95% CI, 4.23-13.64), 4.27 (95% CI, 2.38-7.64), and 21.11 (95% CI, 8.46-52.67) for AhRL ≥ 2.70 pM, MIS-ATP ≤ 88.1%, and both, respectively. Gender analysis revealed that male subjects with AhRL ≥ 2.70 pM or MIS-ATP ≤ 88.1% showed higher risk than female subjects. High serum levels of AhRL and/or MIS strongly predict the future development of diabetes, suggesting that the accumulation of AhR ligands and/or mitochondrial inhibitors in body may play an important role in the pathogenesis of diabetes.

Urinary MicroRNAs in Environmental Health: Biomarkers of Emergent Kidney Injury and Disease.

PURPOSE OF REVIEW: There is a critical need for sensitive biomarkers of renal disease and progression. Micro(mi)RNAs are attractive as next-generation biomarkers in kidney disease, particularly as urine miRNAs can inform kidney function and cellular integrity. This review summarizes recent epidemiologic and toxicologic advances using urinary miRNAs and exosomal miRNAs as novel biomarkers of chemical exposure and of kidney damage and disease. RECENT FINDINGS: Urine miRNA biomarkers offer improved stability over protein in stored samples, relative ease of collection and quantitation, and conserved sequence homology across species. Particularly in the case of emergent environmental health threats such as chronic kidney disease of unknown origin, urinary miRNAs hold promise as biomarkers of disease and/or exposure. We present evidence to address scientific knowledge gaps, comment on the relevance of urine-derived miRNAs in environmental health research, and discuss limitations and recommendations for future directions needed to advance miRNA biomarker strategies.

DNA Methylation-Based Biomarkers of Environmental Exposures for Human Population Studies.

PURPOSE OF REVIEW: This manuscript orients the reader to the underlying motivations of environmental biomarker development for human population studies and provides the foundation for applying these novel biomarkers in future research. In this review, we focus our attention on the DNA methylation-based biomarkers of (i) smoking, among adults and pregnant women, (ii) lifetime cannabis use, (iii) alcohol consumption, and (iv) cumulative exposure to lead. RECENT FINDINGS: Prior environmental exposures and lifestyle modulate DNA methylation levels. Exposure-related DNA methylation changes can either be persistent or reversible once the exposure is no longer present, and this combination of both persistent and reversible changes has essential value for biomarker development. Here, we present available biomarkers representing past and cumulative exposures using individual DNA methylation profiles. In the present work, we describe how the field of environmental epigenetics can leverage machine learning algorithms to develop exposure biomarkers and reduce problems of misreporting exposures or limited access technology. We emphasize the crucial role of the individual DNA methylation profiles in those predictions, providing a summary of each biomarker, and highlighting their advantages, and limitations. Future research can cautiously leverage these DNA methylation-based biomarkers to understand the onset and progression of diseases.

Structural and functional differentiation of bacterial communities in post-coal mining reclamation soils of South Africa: bioindicators of soil ecosystem restoration.

Soil microbial communities are suitable soil ecosystem health indicators due to their sensitivity to management practices and role in soil ecosystem processes. Presently, information on structural and functional differentiation of bacterial communities in post-coal mining reclamation soils of South Africa is sparse. Here, bacterial communities in three post-coal mining reclamation soils were investigated using community-level physiological profiling (CLPP), enzyme activities, and next-generation sequencing of 16S rRNA gene. Inferences were drawn in reference to adjacent unmined soils. CLPP-based species diversity and proportionality did not differ significantly (P > 0.05) whereas activities of ß-glucosidase, urease and phosphatases were significantly (P < 0.05) influenced by site and soil history (reclaimed vs unmined). Bacterial communities were influenced (PERMANOVA, P < 0.05) by soil history and site differences, with several phylotypes differentially abundant between soils. Contrastingly, predicted functional capabilities of bacterial communities were not different (PERMANOVA, P > 0.05), suggesting redundancy in bacterial community functions between reclamation and unmined soils. Silt content, bulk density, pH, electrical conductivity, Na and Ca significantly influenced soil bacterial communities. Overall, results indicate that bacterial community structure reflects underlying differences between soil ecosystems, and suggest the restoration of bacterial diversity and functions over chronological age in reclamation soils.

Assessing legacy and endocrine disrupting pollutants in Boston Harbor with transcriptomic biomarkers.

Within monitoring frameworks, biomarkers provide several benefits because they serve as intermediates between pollutant exposure and effects, and integrate the responses of contaminants that operate through the same mechanism of action. This study was designed to verify the use of transcriptomic biomarkers developed in our prior work (i.e., Coastal Biosensor of Endocrine Disruption; C-BED assay) on Mytilus edulis and identify additional biomarkers for legacy pollutants. M. edulis were collected from a reference site in Pemaquid, ME, USA and deployed by the Massachusetts Water Resources Authority (MWRA) at locations in and outside Boston Harbor, MA, USA: including (1) Boston Inner Harbor (IH), (2) the current outfall (OS), (3) 1 km away from the current outfall (LNB), and (4) Deer Island (DI), the site where untreated wastewater was formerly discharged into the bay. Differential gene expression was quantified with a high density microarray. Seven genes significantly correlated with whole tissue concentration of PAHs, and six genes significantly correlated with whole body concentrations of PCBs, two groups of legacy contaminants that were elevated at stations IH, OS, and DI. Enrichment analysis indicated that IH mussels had the highest induction of stress response genes, which correlated with the higher levels of contaminants measured at this site. Based on the C-BED assay gene analysis, stations IH and OS exhibited signs of endocrine disruption, which were further confirmed by incorporating the results for the C-BED assay within the Integrated Biomarker Response (IBR) approach. This study successfully demonstrated the potential use of transcriptomic biomarkers within a monitoring program to identify the presence and organismal responses to endocrine disrupting and legacy contaminant classes.

Ecotoxicological Analyses of Springs of a Brazilian Northeast Conservation Unit.

Ecotoxicological assessment is an useful tool to evaluate toxicity of pollutants on aquatic organisms. In the present study, we evaluated the lethal effect, micronucleus frequency and histological changes in gills of fish species Danio rerio exposed to water from two sources of a Conservation Unit. We performed acute tests, Micronucleus Test in blood cells and histological analysis of gills. As results, the springs had no lethal toxic effects on the species. Nuclear alterations were present in erythrocytes in organisms exposed to water from the two sampled sites. As well, we found moderate to severe gill abnormalities generating the impairment of respiratory vital functions of these organisms. This study indicates that the water of the springs does not present lethal toxic effect but damage in gills for species from short exposure time.

Root microbiomes as indicators of seagrass health.

The development of early warning indicators that identify ecosystem stress is a priority for improving ecosystem management. As microbial communities respond rapidly to environmental disturbance, monitoring their composition could prove one such early indicator of environmental stress. We combined 16S rRNA gene sequencing of the seagrass root microbiome of Halophila ovalis with seagrass health metrics (biomass, productivity and Fsulphide) to develop microbial indicators for seagrass condition across the Swan-Canning Estuary and the Leschenault Estuary (south-west Western Australia); the former had experienced an unseasonal rainfall event leading to declines in seagrass health. Microbial indicators detected sites of potential stress that other seagrass health metrics failed to detect. Genera that were more abundant in 'healthy' seagrasses included putative methylotrophic bacteria (e.g. Methylotenera and Methylophaga), iron cycling bacteria (e.g. Deferrisoma and Geothermobacter) and N2 fixing bacteria (e.g. Rhizobium). Conversely, genera that were more abundant in 'stressed' seagrasses were dominated by putative sulphur-cycling bacteria, both sulphide-oxidising (e.g. Candidatus Thiodiazotropha and Candidatus Electrothrix) and sulphate-reducing (e.g. SEEP-SRB1, Desulfomonile and Desulfonema). The sensitivity of the microbial indicators developed here highlights their potential to be further developed for use in adaptive seagrass management, and emphasises their capacity to be effective early warning indicators of stress.

MiR-122-5p and miR-326-3p: Potential novel biomarkers for early detection of cadmium exposure.

Cadmium is a common environmental and occupational pollutant and can produce toxic effects in a range of organs, especially in kidneys, after long-term exposure. MicroRNAs are ideal candidate biomarkers for various types of disorders, including renal diseases. In this study, we profiled the global miRNA expressions in rat kidneys using miRNA microarrays and found a collection of differentially expressed miRNAs induced by cadmium exposure. Among all of the candidate miRNAs, we identified miR-122-5p and miR-326-3p as early biomarkers for cadmium-induced nephrotoxicity. The two-miRNA signature was validated by quantitative real-time PCR in HK-2 and NRK-52E cells, rat kidney, serum and urine samples, and serum of an occupational population. Our results indicate that miR-122-5p and miR-326-3p may be potential biomarkers for cadmium exposure.

czcD gene from Bacillus megaterium and Microbacterium liquefaciens as a potential nickel-vanadium soil pollution biomarker.

Metals are among the most prevalent pollutants released into the environment. For these reasons, the use of biomarkers for environmental monitoring of individuals and populations exposed to metal pollution has gained considerable attention, offering fast and sensitive detection of chemical stress in organisms. There are different metal resistance genes in bacteria that can be used as biomarkers, including cation diffusion facilitators carrying metal ions; the prototype is the cobalt-zinc-cadmium transporter (czcD). The present study reports the expression changes in the czcD gene in Bacillus megaterium and Microbacterium liquefaciens under nickel and vanadium exposure by real-time polymerase chain reaction. The nickel-vanadium-resistant strains of B. megaterium and M. liquefaciens used in this study were isolated from mine tailings in Guanajuato, Mexico. The czcD gene showed high expression under exposure to 200 ppm of Ni and 200 ppm of V during the logarithmic growth phase of M. liquefaciens in PHGII liquid media. In contrast, no changes were observed in B. megaterium during logarithmic and stationary growth, perhaps due to the gene having differential expression during the growth phases. The expression profiles obtained for czcD show the possibility of using this gene from M. liquefaciens as a biomarker of nickel and vanadium pollution in microorganisms.

Impact of pyrethroids and organochlorine pesticides residue on IGF-1 and CYP1A genes expression and muscle protein patterns of cultured Mugil capito.

This study aimed to assess the levels of pyrethroids and organochlorine residues in the tissues of cultured Mugil capito and in water samples obtained from three different sites (Al-Hamol, Al-Riad and Sidi Salem; referred to as Area 1, Area 2, and Area 3, respectively) in the Delta region, Egypt. The study also assessed the biochemical markers in exposed mullet and evaluated the impact of these residues on the expression of insulin-like growth factor 1 (IGF-1) in muscle and cytochrome P4501A (CYP1A) in liver tissues using qRT-PCR and SDS-PAGE methods. The results revealed that pesticide residue levels in the water were variable, but were lower than detected levels in fish. Significant (P < 0.05) differences were found across the three study areas in terms of serum ALT, but the serum AST level was not significantly (P > 0.05) elevated in all study regions. Serum creatinine and urea levels were significantly (P < 0.05) elevated in area 3. Furthermore, glutathione and malondialdehyde concentrations significantly increased (P < 0.05) in liver tissues in area 3. Using the qRT-PCR technique, the results revealed that the expression level of IGF-1 was most significant in area 3, while the expression level of CYP1A was most significant in area 1. The protein profile showed some differences in band numbers and molecular weights of protein bands across different regions. Overall, the alteration in biochemical parameters revealed pesticide interference with the metabolic processes of fish. Furthermore, the pesticide pollution had an effect on the expression of IGF-1 and CYP1A genes and led to changes in the protein profile. Therefore, these markers can be used to monitor fish distress following exposure to the pollutant.

MicroRNAs as Potential Biomarkers of Insecticide Exposure: A Review.

Insecticides are key weapons for the control of pests. Large scale use of insecticides is harmful to the ecosystem, which is made up of a wide range of species and environments. MicroRNAs (miRNAs) are a class of endogenous single-stranded noncoding small RNAs in length of 20-24 nucleotides (nt), which extensively regulate expression of genes at transcriptional and post-transcriptional levels. The current research on miRNA-induced insecticide resistance reveals that dysregulated miRNAs cause significant changes in detoxification genes, particularly cytochrome P450s. Meanwhile, insecticide-induced changes in miRNAs are related to the decline of honeybees and threatened the development of zebrafish and other animals. Additionally, miRNAs are involved in insecticide-induced cytotoxicity, and dysregulated miRNAs are associated with human occupational and environmental exposure to insecticides. Therefore, miRNAs are valuable novel biomarkers of insecticide exposure, and they are potential factors to explain the toxicological effects of insecticides.

Population imaging of neural activity in awake behaving mice.

A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here we describe a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and is compatible with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable several-fold over previously published fully genetically encoded reagents1-8. Under conventional one-photon microscopy, SomArchon enables the routine population analysis of around 13 neurons at once, in multiple brain regions (cortex, hippocampus, and striatum) of head-fixed, awake, behaving mice. Using SomArchon, we detected both positive and negative responses of striatal neurons during movement, as previously reported by electrophysiology but not easily detected using modern calcium imaging techniques9-11, highlighting the power of voltage imaging to reveal bidirectional modulation. We also examined how spikes relate to the subthreshold theta oscillations of individual hippocampal neurons, with SomArchon showing that the spikes of individual neurons are more phase-locked to their own subthreshold theta oscillations than to local field potential theta oscillations. Thus, SomArchon reports both spikes and subthreshold voltage dynamics in awake, behaving mice.

Potential bacterial bioindicators of urban pollution in mangroves.

Despite their ecological and socioeconomic importance, mangroves are among the most threatened tropical environments in the world. In the past two decades, the world's mangrove degradation and loss were estimated to lie between an 35% and >80%. However, appropriate bioindicators for assessing the impact of external factors, and for differentiating polluted from unpolluted areas are still scarce. Here, we determine the physicochemical profiles of the soils of two mangroves, one exposed to and one not exposed to anthropogenic factors. By metagenomic analysis based on 16S rRNA, we generated the bacterial diversity profiles of the soils and estimated their functional profiles. Our results showed that the two examined mangrove forests differed significantly in the physicochemical properties of the soils, especially regarding organic carbon, phosphorus and metal content, as well as in their microbial communities, which was likely caused by anthropogenic pollution. The physicochemical differences between the soils explained 76% of the differential bacterial composition, and 64% depended solely on gradients of phosphorus, metal ions and potassium. We found two genera JL-ETNP-Z39 and TA06 exclusively in polluted and non-polluted mangroves, respectively. Additionally, the polluted mangrove was enriched in Gemmatimonadetes, Cyanobacteria, Chloroflexi, Firmicutes, Acidobacteria, and Nitrospirae. A total of 77 genera were affected by anthropic contamination, of which we propose 33 as bioindicators; 26 enriched, and 7 depleted upon pollution.

Evaluation of gene expression of different molecular biomarkers of stress response as an effect of copper exposure on the earthworm EIsenia Andrei.

The paper reports the results of a laboratory test on the bioaccumulation and toxicological effects of sub-lethal soil concentration of copper, a widely used fungicide in organic farming, on DNA damage, a critical marker increasingly used in ecotoxicology in the earthworm Eisenia andrei. In the same experimental setting we evaluated gene expression of classical biomarker of stress induced by xenobiotic. [Heat Shock Protein 70 (HSP70) and Metallothionein (MET)], as well as genes coding for enzymes involved in detoxification of reactive oxygen species [Superoxide dismutase (SOD) and catalase (CAT)]. Additionally, expression of genes involved in the immune response were investigated: a Toll-like receptor (TLR), a receptor with cytolytic activity named Cytolytic Factor (CCF) and two antimicrobial peptides, fetidin (FET) and lysenin (LYS). Results showed significant time-dependent bioaccumulation of Cu and DNA damage at concentrations remarkably lower than those found in most agricultural soils worldwide. MET was increased as was FET and TLR. The present work gives new insights into the mechanisms of sub-lethal toxicity of copper as an environmental pollutant and in the identification of novel sub-lethal biomarkers of cellular response to the stressor such as immune response genes.

Evaluation of cytotoxic potential of chlorpyrifos using Lens culinaris Med as efficient bioindicator.

The aim of this study was to evaluate the cytotoxic effect of different concentrations of chlorpyrifos (CPF), using L. culinaris apical cells as a biological indicator. L. culinaris seeds were exposed to different concentrations of chlorpyrifos (0, 1, 3, 5, 7, 8, 10 and 15 mg L-1) and a control solution based on distilled water. Subsequently, root growth was measured during 24, 48 and 72 h. Therefore, the mitotic index (MI) and the number of cellular abnormalities were determined at 72 h. According to the obtained results, a decrease in root size was observed in the concentrations of T5 (8 mg L-1) and T6 (10 mg L-1). On the other hand, it was evidenced that, through all the evaluated concentrations, the inhibition of mitosis in the concentrations of T5 (8 mg L-1), T6 (10 mg L-1) and T7 (15 mg L-1) was greater than 50%. Additionally, a variety of chromosomal abnormalities were reported, such as Micronuclei, sticky chromosomes in anaphase, chromosome disruption, irregular anaphase, nucleus absence, nuclear lesions, chromosomes grouped in metaphase, anaphase bridges, metaphase sticky chromosomes, present in all concentrations evaluated. Consequently, the presence of micronuclei in the concentrations of 8 mg L-1, 10 mg L-1 and 15 mg L-1 indicates that the CPF is a highly cytotoxic substance to L. culinaris. Therefore, L. culinaris is a plant species that offers a feasible experimental model to be implemented in laboratory studies with the purpose to evaluate the cytotoxic effect of pesticides.

Absence of mutations in the human interferon alpha-2b gene in workers chronically exposed to ionising radiation.

Individuals chronically exposed to low-level ionising radiation (IR) run the risk of harmful and long-term adverse health effects, including gene mutations and cancer development. The search for reliable biomarkers of IR exposure in human population is still of great interest, as they may have a great implementation potential for the surveillance of occupationally exposed individuals. In this context, and considering previous literature, this study aimed to identify mutations in the human interferon alpha-2b (hIFNα-2b) as a potential biomarker of occupational chronic low-dose IR exposure linking low-IR exposure to the effects on haematopoiesis and reduced immunity. The analysis was performed in the genomic DNA of 51 uranium miners and 38 controls from Kazakhstan, and in 21 medical radiology workers and 21 controls from Italy. hIFNα-2b gene mutations were analysed with the real-time polymerase chain reaction (PCR) or Sanger sequencing. However, none of the investigated workers had the hIFNα-2b mutation. This finding highlights the need for further research to identify biomarkers for early detection of health effects associated with chronic low-dose IR exposure.

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples.

Microbial source tracking (MST) methods measure fecal contamination levels and identify possible sources using quantitative PCR (qPCR) that targets host-associated fecal microorganisms. To date, most established MST assays for human sources, especially bacterial markers, have shown some nonhuman host cross-reactions. Recently developed assays, such as the crAssphage CPQ_056, Lachnospiraceae Lachno3, and Bacteroides BacV6-21, have more limited information on host sensitivity and host specificity for human or sewage sources, particularly in countries other than the United States. In this study, we rigorously evaluated six sewage-associated MST assays (i.e., Bacteroides HF183, human adenovirus [HAdV], human polyomavirus [HPyV], crAssphage CPQ_056, Lachno3, and BacV6-21) to show advantages and disadvantages of their applications for MST. A total of 29 human and 3 sewage samples and 360 nonhuman fecal samples across 14 hosts collected from a subtropical region of Australia were tested for marker host specificity, host sensitivity, and concentrations. All sewage samples were positive for all six marker genes tested in this study. Bacterial markers were more prevalent than viral markers in human feces. Testing against animal hosts showed human feces (or sewage)-associated marker gene specificity was HAdV (1.00) > HPyV (0.99) > crAssphage CPQ_056 (0.98) > HF183 (0.96) > Lachno3 (0.95) > BacV6-21 (0.90), with marker concentrations in some animal fecal samples being 3 to 5 orders of magnitude lower than those in sewage. When considering host specificity, sensitivity, and concentrations in source samples, the HF183, Lachno3, and crAssphage CPQ_056 tests were the most suitable assays in this study for sewage contamination tracking in subtropical waters of Australia.IMPORTANCE Large financial investments are required to remediate fecal contamination sources in waterways, and accurate results from field studies are crucial to build confidence in MST approaches. Host specificity and sensitivity are two main performance characteristics for consideration when choosing MST assays. Ongoing efforts for marker assay validation will improve interpretation of results and could shed light on patterns of occurrence in nontarget hosts that might explain the underlying drivers of cross-reaction of certain markers. For field applications, caution should be taken to choose appropriate MST marker genes and assays based on available host specificity and sensitivity data and background knowledge of the contaminating sources in the study area. Since many waterborne pathogens are viruses, employing both viral and bacterial markers in investigations could provide insight into contamination dynamics and ecological behavior in the environment. Therefore, combined usage of marker assays is recommended for more accurate and informative sewage contamination detection and fecal source resolution.

Polyester-derived microfibre impacts on the soil-dwelling earthworm Lumbricus terrestris.

Microplastic (MP) pollution is everywhere. In terrestrial environments, microfibres (MFs) generated from textile laundering are believed to form a significant component of MPs entering soils, mainly through sewage sludge and compost applications. The aim of this study was to assess the effect of MFs on a keystone soil organism. We exposed the earthworm Lumbricus terrestris to soil with polyester MFs incorporated at rates of 0, 0.1 and 1.0 %w/w MF for a period of 35 days (in the dark at 15 °C; n = 4 for each treatment). Dried plant litter was applied at the soil surface as a food source for the earthworms. We assessed earthworm vitality through mortality, weight change, depurate production and MF avoidance testing. In addition, we measured stress biomarker responses via the expression of metallothionein-2 (mt-2), heat shock protein (hsp70) and superoxide dismutase (sod-1). Our results showed that exposure and ingestion of MFs (as evidenced by subsequent retrieval of MFs within earthworm depurates) were not lethal to earthworms, nor did earthworms actively avoid MFs. However, earthworms in the MF1.0% treatment showed a 1.5-fold lower cast production, a 24.3-fold increase in expression of mt-2 (p < 0.001) and a 9.9-fold decline in hsp70 expression (p < 0.001). Further analysis of soil and MF samples indicated that metal content was not a contributor to the biomarker results. Given that burrowing and feeding behaviour, as well as molecular genetic biomarkers, were modulated in earthworms exposed to MFs, our study highlights potential implications for soil ecosystem processes due to MF contamination.