Unlocking secrets of microbial ecotoxicology: recent achievements and future challenges

Abstract Environmental pollution is one of the main challenges faced by humanity. By their ubiquity and vast range of metabolic capabilities, microorganisms are affected by pollution with consequences on their host organisms and on the functioning of their environment. They also play key roles in the fate of pollutants through the degradation, transformation, and transfer of organic or inorganic compounds. Thus, they are crucial for the development of nature-based solutions to reduce pollution and of bio-based solutions for environmental risk assessment of chemicals. At the intersection between microbial ecology, toxicology, and biogeochemistry, microbial ecotoxicology is a fast-expanding research area aiming to decipher the interactions between pollutants and microorganisms. This perspective paper gives an overview of the main research challenges identified by the Ecotoxicomic network within the emerging One Health framework and in the light of ongoing interest in biological approaches to environmental remediation and of the current state of the art in microbial ecology. We highlight prevailing knowledge gaps and pitfalls in exploring complex interactions among microorganisms and their environment in the context of chemical pollution and pinpoint areas of research where future efforts are needed.


Introduction
Ecotoxicology is defined as the "study of the toxic effects of chemical and physical agents on all living or ganisms, especiall y on populations and communities within defined ecosystems; it includes transfer pathways of these agents and their interactions with the envir onment", wher eas ecology is defined as the "br anc h of biology that studies the interactions between living organisms and all factors (including other organisms) in their envir onment.Suc h interactions encompass environmental factors that determine the distributions of living organisms" (Nordberg et al. 2009 ).In the Anthropocene , en vironmental pollution is omnipresent alongside other en vironmental factors .In order to understand the impacts of chemical pollution and their consequences on the interactions between organisms and their environment, ecotoxicology relies on existing ecological theories whereas in ecology, pollution is only one factor amongst many others.In this sense, ecotoxicology rather than ecology is r ele v ant for envir onmental r egulatory issues and for environmental risk assessment (ERA).In microbial ecology, this has led to the emergence of a fast-expanding researc h ar ea, micr obial ecotoxicology, at the intersection between microbial ecology , toxicology , and biogeochemistry that aims to decipher the interactions between pollutants and microorganisms at different organizational scales (Ghiglione et al. 2014(Ghiglione et al. , 2016 ) ). Interdisciplinarity is, thus both a k e y feature and a r equir ement in microbial ecotoxicology studies and for applications of ne wl y generated knowledge for toxicity assessment and environmental remediation.In this context, microbial ecotoxicology builds on a paradox in several ways (Fig. 1 ).First, it strives to yield insights about pollutant-driven impacts on ecosystem functioning at the global scale based on micrometer-scale processes.Second, in order to do so, it str ongl y r elies on existing knowledge and detailed analysis of individual model micr oor ganisms to c har acterize the response of complex microbial communities .Moreo ver, it is de v eloping an incr easing inter est in testing and a ppl ying concepts de v eloped for classical macr oecology, e.g.functional tr aits, toler ance, r esistance, functional r edundancy, r esilience, and connectivity (Cébron et al. 2021, Romillac and Santorufo 2021, Mony et al. 2022 ) through investigations to comprehend higher levels of organization at the community and ecosystem scale (Loreau et al. 2001 ).A better understanding of the complex interactions between micr obial comm unities and pollutants is essential for toxicity assessment and the implementation of sustainable bioremediation systems , i.e .the use of nature-based solutions to eliminate pollution.Of course, se v er al scientific c hallenges ar e still being activ el y tac kled to enable a wider use of micr oor ganisms in these fields (Peixoto et al. 2022 ).This also includes the de v elopment and application of new technologies and methods in microbial ecology to isolate and functionall y c har acterize a larger diversity of micr oor ganisms fr om envir onmental samples (Dur an et al. 2022 ).
To meet these ambitious expectations, microbial ecotoxicology will benefit from the EcotoxicoMic network ( https://ecotoxicomic. org/) born in France as a national network in 2013, which has now r eac hed an international dimension (Pesce et al. 2020a, Gallois et al. 2022 ).This perspective paper aims to present the main challenges and r esearc h opportunities identified for microbial ecotoxicology in light of the current state of the art.We focused on organic and inorganic chemical pollutants , lea ving aside topics associated with pathogens , antibiotics , and micr obiall y pr oduced toxins that w ould w arrant a specific discussion.The first two sections present the heart of microbial ecotoxicology and consider the impacts of pollutants on microbial biodiversity and functions and then the role of micr oor ganisms in pollutant tr ansformation, biodegr adation, and tr ansfer.The third section addresses the major challenge of linking the impact of pollution on micr oor ganisms with the functioning of hosts and ecosystems and possible consequences at a global scale.Finally, the fourth section provides an ov ervie w of curr ent a pplications of micr obial ecotoxicology for pr actical envir onmental assessment and bior emediation and associated challenges.Methods and technologies applied or considered in the field of microbial ecotoxicology today are discussed throughout the paper.

Impacts of pollutants on microbial biodiversity and functions
Micr oor ganisms ar e essential players of natur al ecosystems that cope with chemical and other environmental disturbances through the functions they perform (Delgado-Baquerizo et al. 2016, 2020, Cr avo-Laur eau et al. 2017, Borc hert et al. 2021 ).The r esponse of micr obial comm unities to disturbances is intrinsically linked to their diversity (Allison andMartiny 2008 , Tardy et al. 2014 ) (Fig. 2 ).For instance, more diverse communities provide greater functional redundancy (Birrer et al. 2017 ), thereby helping to maintain crucial functions e v en if the composition of the micr obial comm unity is alter ed (Her old et al. 2020, Walker et al. 2022 ).A major challenge faced by microbial ecotoxicologists and ecologists in polluted and pristine environments is linking taxonomic diversity to functionality.Although there is growing evidence that a loss in micr obial div ersity will ine vitabl y lead to a loss of multifunctionality (Delgado-Baquerizo et al. 2016, 2020, No y er et al. 2020, 2023 ), m uc h work is needed to better understand the consequences for ecosystem services on a global scale .T hus , considering the response of microbial communities at different levels of taxonomic diversity and functional redundancy has strong potential to help us better understand the effects of chemical disturbances in the environment, as discussed in the following.Fortunately, molecular tools and approaches to do this in more detail are now increasingly available and are continuously being de v eloped.

The importance of tackling taxonomic diversity in microbial ecotoxicology
For both pristine and polluted en vironments , whether terrestrial, aquatic, or aerial, taxonomic alpha and beta div ersities hav e been extensiv el y studied in bacterial communities.Other types of micr oor ganisms suc h as micr oeukaryotes (with the exception of diatoms in aquatic systems), archaea, viruses, and fungi remain less inv estigated, particularl y in lotic and aerial ecosystems.Simultaneously studying alpha and beta diversities of se v er al domains of life through metabarcoding can help to better understand how comm unities r espond to disturbances (Delgado-Baquerizo et al. 2016, 2020, No y er et al. 2020, 2023 ).For example, recent work on micr obial comm unities of fr eshwater sediments experimentall y exposed to copper has shown concomitant effects of the metal on the structure of bacterial communities (A-RISA method) and their functional potential.Ho w e v er, r esponses wer e v ariable ov er time during this c hr onic 21-day exposur e: continuous effects during the experiment on some catabolic activities ( β-glucosidase and phosphatase activities), resilience of other activities (denitrification and phosphatase activity), or time-lagged (respiration), while the bacterial structure remained impacted throughout the experiment.These results show the need for further study of these ecotoxicological processes on the diversity/function nexus, in particular the temporal dynamics of ecotoxicological effects (Mahamoud et al. 2018 ).
Environmental DN A (eDN A) metabar coding enables to e v aluate taxonomic diversity of bacteria or fungi, but rarely considers the whole micr obial comm unity.Ho w e v er, eDN A metabar coding a ppr oac hes ar e r a pidl y e volving, making it possible, e.g. to e v aluate the diversity of microeukaryotes such as in marine environments impacted by offshore gas platforms (Cordier et al. 2019 ).Methodologically, nucleic acid (NA)-based approaches are the most widely used methods for high-throughput characterization of microbial communities at the taxonomic level (Fig. 3 ).Targeting DNA (who is present and potentially doing what) versus RNA (who is active now) will estimate different fractions of the community in a given environment and provide complementary information (Argudo et al. 2020 ).Many sets of 'universal' primer pairs targeting several domains have been designed to sequence amplicons and analyse microbial diversity.Ho w ever, they are often biased against less dominant groups (Francioli et al. 2021, Tahon et al. 2021 ).T hus , car efull y c hosen domain-specific primers (e.g.Tahon et al. 2021, Tapolczai et al. 2021 ) remain the best available choice to provide detailed cov er a ge of the taxonomic diversity of a domain of interest.
Identifying the roles and importance of e v ery type of microorganism in any given polluted environment and under any physicochemical condition is very challenging.T hus , microbial diversity Figur e 1. T he multiple scales of microbial ecotoxicology, at molecular , cellular , community (including interactions), and ecosystem levels.in polluted environments is usually compared with that of reference pristine environments or along a pollutant gradient, or by monitoring changes in microbial composition before and after chemical disturbance .T his a ppr oac h has been a pplied in numerous studies in order to gain insights on the toxicity of pollutants and the r esistance, r esilience, toler ance, and ada ptation of micr obial communities to pollutants (Fig. 2 ; Morin et al. 2009, Lemmel et al. 2019a, No y er et al. 2020, 2023 ).In so doing, certain taxa have been identified whose presence or absence in a polluted environment, or whose sensitivity to chemical exposure, sho w ed potential as indicator species for use in ERA (No y er et al. 2020, 2023, Lemmel et al. 2021, Bourhane et al. 2022, Veloso et al. 2023 ).This is further discussed in the dedicated section.
Ne v ertheless, comparisons of microbial diversity between different samples have several limitations.Some are technical and intrinsic to the applied methods (see Fig. 3 ) while others include the difficulty of securing r efer ence pristine samples .T his can be overcome using long-term observatories for envir onmental r esearc h.As an example, the SOERE PRO (Système d'Observation et d'expérimentation sur le long terme pour la Rec herc he en Envir onnement) dedicated to the study of organic residues in agriculture soils provides experimental devices to obtain long-term monitoring of the impact of pollutants associated with organic amendments compared with unamended sites.Another example of long-term microbial observatories is the International Long Term Ecological Resear ch Netw ork (IL-Figur e 2. P ossible outcomes in microbial community responses to pollutant disturbance with respect to a function of interest.(A) Model community initially composed of seven equally abundant taxa, some of which are capable of performing the function of inter est (gr een star).This function may be dir ectl y associated with pollutant transformation, with another specific function (e.g.nitrogen fixation or nitrification), or with a widely distributed function (e.g.oxygen r espir ation).(B) Selected r esponse pr ofiles of micr obial comm unities to disturbance (lightning) in terms of the function of interest (solid green line, left y -axis) and of taxonomic diversity (dashed black line, right y -axis).(C) Examples of microbial communities compatible with the differ ent r esponse pr ofiles sho wn in (B) follo wing pollution disturbance of the model community sho wn in (A).Appar ent functional r esistance to chemical pollution may involve loss of taxonomic diversity or functional redundancy, which will be detrimental for ecosystem functioning in the long-term.Functional resilience , i.e .the reco very of a particular micr obiall y determined function following pollution disturbance , ma y featur e c hanges in the taxonomic profile of the microbial community acting on the pollutant and gain of the functional ability to transform or degrade the pollutant by a resistant taxon through horizontal gene transfer, as shown.Of course, other comm unity r esponses ar e also possible, suc h as gain of a degradation function upon pollution disturbance without a ppar ent c hange of taxonomic div ersity.TER) that counts 28 sites.In the same way, microbial ecotoxicology could benefit from longitudinal r efer ence databases (Martínez Arbas et al. 2021 ; https:// www6.inrae.fr/valor-pro _ eng/ French-Observatory-on-Organic-Residues/Objectives ).Ho w ever, discriminating against potential effects of other environmental parameters (as potential confounding factors) such as pH, temper atur e, or moisture in accounting for the observed changes in microbial communities exposed to pollutants remains a challenge.

Effects of chemical disturbances on microbial functions-the need for a wider assessment
Dir ectl y measuring k e y ecosystem functions , when possible , is another way to assess the toxic effects of chemical pollutants on living micr oor ganisms or comm unities (e.g.see r e vie w in Morin and Artigas 2023 for aquatic microbial communities).This can be ac hie v ed by dir ectl y monitoring pr ocesses in situ suc h as organic matter degradation by using litter bags (Lecerf et al. 2021 ) or microbial activities (respiration, enzymatic tests, photosynthesis , flux measurements , and so on) at the field scale (Bungau et al. 2021 ).Microbial respiration and denitrification have been widely used to account for chemical disturbances (Wakelin et al. 2013, Bérard et al. 2016, Lyautey et al. 2021 ) as well as other widespread enzyme activities such as urease , β-glucosidase , leucine aminopeptidase , acid phosphatase , and fluorescein diacetate hydr ol ysis activities (Fei et al. 2020, Lyautey et al. 2021, Li et al. 2022 ).The expression of activities fr om micr obial functional guilds such as nitrifiers, which are less diverse functional groups, pr ovides other highl y r ele v ant indicators to account for disturbances since their lo w er functional redundancy can lead to more deleterious consequences on ecosystem functioning (see dedicated section) (Simonin et al. 2016, Lu et al. 2022 ).
NA-based a ppr oac hes also yield r ele v ant data on micr obial ecosystem functions and link the presence and e v en the expr ession of genes associated with chemical toxicity or pollutant degradation or tr ansformation.Ne v ertheless, the same limitations as those mentioned in the pr e vious section for taxonomy-associated genes a ppl y.Mor eov er, in gene-specific PCR-based studies, readouts will be limited to genes with pr ov en functional associations and related sequences that are amplified with the chosen PCR primers (Simonin et al. 2016 ).More generally, a significant knowledge gap remains regarding the impact of pollutants on microbial functions.Mor e tr aditional molecular methods suc h as quantitative PCR (qPCR) and microarrays are also widely used to search for specific w ell-kno wn functions suc h as hydr ocarbon degr adation (Yergeau et al. 2009 ).These approaches have also evolved with methods such as digital PCR, which was found to be a ppr opriate to detect and quantify sequences of genes coding for the resistance to pollutants in biofilms (Kimbell et al. 2021 ).For a large proportion of the studies reported so far, investigated functions are dir ectl y linked to the pollutant of inter est, suc h as genes involved in their degradation or transformation and corresponding metabolic pathwa ys , especially if they are distributed across a wide range of microbial taxa.In such cases, the linkage between pollutants and functions and the associated taxa that accomplish them r eadil y leads to the definition of new eukaryotic and/or prokaryotic indicators among enriched taxa in polluted en vironments .Ho w ever, assessing the impact of new or emerging pollutants for which micr obial r esponses ar e not y et w ell-investigated or understood is challenging.Yet we now have molecular tools that may help to decipher new metabolic pathways (see next section).Emphasis should now also extend to taxa or micr obial gr oups poorl y inv estigated or ne wl y discov er ed that ar e involv ed in k e y ecosystem functions but not necessarily directly involved in the dissipation of chemical pollutants such as comammox and anammox bacteria.These bacteria are involved in a single-step production of nitr ate fr om ammonium, and in the pr oduction of nitr ogen gas fr om ammonium and nitrite (or nitr ate), r espectiv el y (Li et al. 2021 , Madeira andde Araújo 2021 ).
Fortunatel y, the incr easing use of meta-omics a ppr oac hes has begun to overcome the focus on well-c har acterized genes and pathwa ys .Indeed, meta-omics make it possible to in vestigate , based on a unique shotgun sequencing experiment, the dynamics of all genes present or expressed in an environmental sample without a priori on the genes involv ed.Shotgun meta genomic sequencing (MGS) provides comprehensive information on the DNA present in a given sample, but requires extensive bioinformatic posteriori data analysis and is more expensive than previously mentioned methods (Ranjan et al. 2016, Douglas 2021 ).Advances in metagenome-assembled genomes (MAGs) technology, such as long-read sequencing and single-cell metagenomics, can improve the quality of MGS data.These technical advancements may lead to the discovery of specific genes responding to the presence of c hemicals (Ac hermann et al. 2020 ) that once c har acterized functionally and tested through environmental ecotoxicology studies could become k e y bioindicators for microbial ecoto xicology.Nevertheless, methodology is not the only scientific bolt for studying diversity and function but also the way we connect it or not among life domains, which is very tricky, and the usual lack of quantitative estimation when using barcoding methods (relative and not absolute abundances).
Mor e fundamentall y, the lac k of dir ect corr espondence between taxonomic identity and a given function of interest limits the use of taxonomy-based investigations to c har acterize the micr obial r esponse to c hemical pollution in micr obial ecotoxicology.Mor eov er, w ork with DN A itself does not allow to gain insights into the physiological and metabolic state of micr oor ganisms , while mRNA reco very remains challenging for some environmental samples and may limit the e v aluation of in situ expr ession of micr obial functions .Along the same lines , de v elopment of microbial metabolomics and increased knowledge of k e y metabolic pathways altered by responding to chemical pollution and of specific pollutant transformation pathways will help define new additional potential readouts for microbial ecotoxicology (Muller et al. 2018, Muller 2019 ) (see section entitled "Microbial roles in pollutant fate and tr ansfer").Gr aduall y, this ar ea will also benefit from new and ongoing advances in emerging experimental a ppr oac hes (Malla et al. 2018 ) to help in environmental assessment and in the de v elopment of ne w r emediation str ategies (see the dedicated section).
In par allel, se v er al r ecent initiativ es aiming at de v eloping an ecology-inspired conceptual framework to define microbial functional tr aits hav e emer ged (Westoby et al. 2021 ).They are also intended to be used in the c har acterization of ecosystem functioning under different environmental conditions (Virta and Teittinen 2022 ), with applications for the toxicological assessment of chemical pollutants (Martini et al. 2021 ).Several easy-to-use tools or databases providing potential functional information such as PICRUSt2 (Douglas et al. 2020), Tax4Fun (Asshauer et al. 2015 ), bactoTr aits (Cébr on et al. 2021 for bacteria), FUNGuild (Nguyen et al. 2016 ), and FungalTraits (Põlme et al. 2020 for fungi) have been reported for this purpose (see Fig. 3 ).They can help assign functions or traits based on taxonomic identities and help identify bioindicators for ERA.Ho w e v er, the infer ence of functionality fr om taxonomic div ersity r emains c hallenging, particularl y concerning pollutant degradation.Indeed, it is common that within the same bacterial species, some strains can degrade or transform and others not.Mor eov er, the lac k of functionall y c har acterized r efer ence micr oor ganisms of known genomic sequences, as well as the large proportion of genes with unknown functions in sequence databases, still limits the application of such tools for robust prediction of ecosystem functioning from taxonomic diversity.Although the number of available traits is still limited, it will be pr ogr essiv el y enric hed with ongoing pr ogr ess in this ar ea, in particular for the large number of taxa for which a corresponding set of traits usable for environmental assessment is still lacking.Some biases also persist due to the fact that these tools are generall y mor e extensiv el y de v eloped for bacteria than other micr oorganisms such as fungi or algae (Berg et al. 2020, Douglas 2021 ).The use of micr oarr a ys (e .g. Geochips; He et al. 2010 ) allowing to target thousands of functional genes could help in identifying which functions are impacted by pollutants (He et al. 2012 ).Recently, a ne w gener al and mor e po w erful gener ation of bioc hip has been introduced in order to link microbial genes/populations to ecosystem functions (Shi et al. 2019 ).

Applying fundamental concepts in ecology to micr obial ecoto xicology-potential benefits
Excitingl y, the emer ging r ene wed emphasis on anal ysis of ecosystem functioning fuelled by functional genomic a ppr oac hes now enables us to a ppl y classical fundamental questions and concepts of macroecology to the microbial compartment (Muller 2019 ).This seems of particular r ele v ance for micr obial ecotoxicology.Indeed, k e y issues in the assessment of toler ance, r esistance, or ada ptation of ecosystems to chemical stress, and of their resilience, can now be addressed for the microbial compartment as well.For the c har acterization of ecosystem functions, it is now possible to inv estigate the r ele v ance not onl y of the pr esence or absence of specific genes or taxa but also of the co-occurrence or e v en of the interactions of specific sets of genes and/or taxa and their dynamics upon exposure to chemicals.While this research area is still in its infancy, se v er al important studies hav e r ecentl y been r eported, and display the great potential of the corresponding findings as bioindication tools for microbial ecotoxicology.Emerging initiatives using ecology-inspired approaches (Virta et al 2020 a) and omics (for r e vie w see Sene vir atne et al. 2020 ), suc h as the combination of meta genomics, metatr anscriptomics, meta pr oteomics, and metabolomics , pro vide new insights into the functional networks that arise , e .g. following pollution or during biodegradation of pollutants (Muller et al. 2018 ).For instance, Herold et al., ( 2020 ) demonstr ated thr ough a m ulti-omics a ppr oac h that r esistance and r esilience pr operties of waste water tr eatment plant comm unities to a disturbance depended on phenotypic plasticity and niche complementarity.
On the other hand, because not e v ery ecosystem function is fav oured b y higher comm unity div ersity, the combination of complementary experimental a ppr oac hes, including omics (see Fig. 3 ), together with a ppr opriate statistical or mac hine learning methods may allow accurate assessment of changes in alpha diversity along with the underlying stochastic-deterministic assembl y pr ocesses.Recent adv ances in mac hine deep learning a ppr oac hes (e.g. using r andom for est or deep convolutional neural networks) can help to elucidate relationships between the composition of the microbiome and its functions or to monitor changes in the composition of the microbiome in response to environmental stresses (Hernández Medina et al. 2022 ).Deep learning can also be applied for image analyses to study the morphometry of microbial taxa and is being de v eloped for diatoms , algae , fungi, and bacteria (Kloster et al. 2020, Picek et al. 2022, Xu et al. 2022, Venkataramanan et al. 2023 ).These tools are complementary to molecular biology a ppr oac hes and morphology-based taxonomy in microbial ecotoxicology studies, due to their potential to characterize the effects and fate of pollutants at the ecosystem scale and the taxonomic , beha viour al, and mor phometric r esponses of micr obial comm unities (in particular protists and microalgae) to pollutants.Also noteworthy is the recent development of image analysis and spectral imaging tools (associated even more recently with deep learning) to study changes in ecosystems (e.g.r emote sensing a pplied to aquatic envir onmental monitoring; Li et al. 2020, Sagan et al. 2020 ).Finally, the development of mechanistic computational models to analyse the dynamics of complex micr obial inter actions at differ ent le v els is also pr omising (Henry et al. 2016 , Niarakis andHelikar 2021 ).
In this way, both taxonomic and functional knowledge of micr obial comm unities in an envir onment of inter est may inform on the nature and extent of toxic effects of pollutants, and on the capacity of the ecosystem to functionally recover from pollutant exposure (Fig. 2 ).Such a combination of experimental and adv anced anal ytical methods was found essential to understand the impact of pollutant disturbance on complex microbial communities in activated sludge bioreactors (Santillan et al. 2019 ) and on bacterial diversity along a river-to-estuary gradient (Meziti et al. 2016 ).Notabl y, pollutants may adv ersel y affect micr obial functional and phylogenetic diversity through cascading effects on bioc hemical pr ocesses (Meena et al. 2020 ).While likel y v ery significant, the effects of multiple ecological interactions and associated unsuspected links between diversity and function are still r ar el y described.
The use of structural equation modelling (SEM; Xiao et al. 2021 ), machine-learning algorithms and metabolic models, such as Flux-Based Analysis (FBA; Cuevas et al. 2016 ), may provide a cum ulativ e understanding of the direct effects of pollutants on micr obial div ersity and functions, as well as of the cascading effects between inter acting or ganisms in an holobiont or in an ecosystem.These a ppr oac hes may be useful to test and e v aluate m ultiv ariate causal r elationships (Fan et al. 2016 ), as shown by Simonin et al. ( 2016 ).SEM has also already been combined with machinelearning algorithms (particularly random forests) to provide insight into the direct and indirect effects of different stressors and diversity on the ecosystem multifunctionality (Delgado-Baquerizo et al. 2016, 2020 ).

Micr obial r oles in pollutant fa te and tr ansfer
The c hr onic or r epeated exposur e of micr oor ganisms to c hemical pollutants can lead them to de v elop dir ect and indir ect metabolic or detoxification pathways to degr ade, tr ansform, or accum ulate them.Degr adation or tr ansformation of pollutants can be considered as an ecological function beneficial for the environment contributing to reduce their persistence and consequentl y, exposur e and toxicity to w ar ds living or ganisms.Micr obial activities can also affect pollution fate by releasing toxic elements from their carrier phases e.g. release of arsenic or mercury through redox reactions and mineral solubilization (Hellal et al. 2015, Héry et al. 2015 ).Unfortunatel y, some micr obial activities can lead to the formation of compounds with greater toxicity [e.g.perchloroethene (PCE) to 1,2-dic hlor oethene (DCE), and vin yl c hloride (VC); Adrian and Löffler 2016 ].With the continual emergence of new synthetic chemicals and the remaining knowledge gaps about historical ones, many questions remain on the roles of microorganisms in pollutant fate and transfer.In particular, there is a need to identify the major microbial actors involved in the degradation and trans-formation of synthetic chemicals in situ .Innovative approaches based on holistic, m ultidisciplinary, and integr ating ne w tec hnologies (Fig. 3 ) are also needed to cope with the complexity of interactions between microbes, and between microbes and their environment (biotic and abiotic factors) and pollutants.

Pollutants as a selecti v e force
The range of currently known chemical pollutants, whether natural (metals and metalloids, hydrocarbons) or anthropogenic (pesticides , plastics , pharmaceuticals , etc) is extr emel y v ast and in continuous expansion due to the constant release of new molecules and the production of a myriad of intermediate degradation metabolites.Although there are many studies on their tr ansformation or degr adation (P ar ales and Haddoc k 2004 , Gadd 2010 , Dur an and Cr avo-Laur eau 2016 , Hidalgo et al. 2020 ) ther e ar e still man y knowledge ga ps to be filled on the micr obial mec hanisms involved in these reactions.
Metallic compounds can accumulate in the environment due to human activities (e.g.Cu and Zn in a gricultur e, Hg and As in mining, and so on).They can be biotransformed by enzymatic r eactions (oxidation, r eduction, methylation) (Fig. 4 ).These reactions can be a defensive mechanism (e.g.Hg reduction encoded by the mer operon; Barkay et al. 2003 ) or a consequence of metabolic activity (e.g.As(V) or Fe(III) reduction).Apart from these wellknown examples, microbial interactions with metals or metalloids of emerging concern remain poorly documented.For example, knowledge on the uptake, efflux, and redox transformation pathways of antimony is limited (Deng et al. 2021 ).Recentl y, ne w pr actices hav e led to an incr ease in the use of r ar e earth elements and metal nanoparticles in different sectors (e.g. a gricultur e, r emediation, cosmetics, batteries, and so on).Ho w e v er, the r ole of micr oor ganisms on the fate of these elements in the environment r emains poorl y understood (Xie et al. 2017, Cr ampon et al. 2018, Eymard-Vernain et al. 2018 ).
For many of the ever-expanding range of organic pollutants, there is no data on their biodegradability or transformation potential.Man y or ganic pollutants can be dir ectl y tr ansformed by micr oor ganisms and ar e used as a carbon source (e.g.pol ycyclic ar omatic hydr ocarbons (PAH) degr adation) and/or as electr on donors or acceptors (e.g.organohalid respiration of chloroethenes).They can also be tr ansformed indir ectl y thr ough cometabolic r eactions (e.g.c hlor oethenes; Dolinová et al. 2016, Zhang et al. 2019 ) (Fig. 4 ).Man y degr adation pathways and the corr esponding genes ar e well-c har acterized, yet micr obes degr ading (ne w) emer ging organic compounds need to be identified and their metabolic pathways further studied in order to de v elop ada pted r emediation strategies .T he use of next-generation physiology approaches that are independent from a priori knowledge of genomic information could allow to focus on cellular functions (Hatzenpichler et al. 2020 ).These a ppr oac hes combine micr obial phenotype pr obing, high throughput cell sorting, and downstream techniques such as single-cell sequencing, targeted cultivation (e.g.culturomics; (Martiny 2019, Almeida et al. 2022 ), or complementary microscopy or ima ging anal yses (Fig. 3 ).A c hange of perspectiv e is also r equired to favour understanding of in situ situations rather than of pur e str ains exposed to one compound (or family of compounds).Indeed, degradation can involve microbes interacting in a consortium (e .g. syntrophy; T homas et al. 2019 ) and many other biotic and abiotic factors can sim ultaneousl y influence degr adation (Chishti et al. 2021, Yuan et al. 2021 ).Understanding in situ reactions will contribute to identifying ERA indicators and adapting bioremediation to the environmental context.An emerging topic related to microorganism-pollutant interactions is the occurrence of increasingly complex associations of different compounds and their consequences for microbial communities and their transformation potential.Phenotypic probing appr oac hes suc h as SIP (stable isotope pr obing), will allow the identification of micr oor ganisms activ el y involv ed in specific metabolic pr ocesses suc h as the degr adation of or ganic pollutants (Lemmel et al. 2019b ).A gener al effort has also been made in r ecent years to impr ov e isolation of micr obial str ains fr om understudied taxa (Chaudhary et al. 2019 ), (since this remains the approach of choice to study their metabolism and particularly their role in the transformation of pollutants) and potentially use them for bioremediation purposes (Fig. 3 ).Ho w ever, it is beginning to be possible to predict the modes of action, effects, behaviour , and transformation of pollutants in silico (Han et al. 2019, Singh et al. 2021 ).This type of a ppr oac h would make it possible to assess ecotoxicity and fate of new molecules more quickly and comprehensively, e v en if the prediction of effects of pollutant mixtur es r emains a challenge.

A complex network of microbial interactions and collabor a tions
Although many studies have been carried out on single microorganisms exposed to a particular pollutant, holistic a ppr oac hes ar e now needed to better understand the different levels and means of biotic inter actions.Indeed, biotic inter actions can occur within a domain or between domains and also include trophic interactions or host-microbiome interactions (Adamovsky et al. 2018 ) (see dedicated section).Unr av elling these complex interactions in situ is a major challenge for microbial ecotoxicology.For example, positiv e inter actions between fungi and bacteria have recently been demonstrated (Álvarez-Barragán et al. 2022 ) where bacteria can be dispersed in PAH-polluted environments via fungal hyphae , allowing to o vercome barriers and promote accessibility to PAHs.Other examples are the cascades of redox conditions that lead to the dehalogenation of PCE to ethylene by promoting optimal conditions for halorespiring bacteria such as Dehalococcoides sp.(Hellal et al. 2021 ), or the total degradation of PAH by a microbial consortium in successive degradation steps (Thomas et al. 2019 ) (Fig. 4 ).
Biodegr adation and tr ansformation r eactions ar e also tightl y controlled by environmental factors.A better understanding of how envir onmental, physico-c hemical, and oper ational (in a bior emediation context) par ameters driv e micr obial div ersity and activity is r equir ed to de v elop effectiv e and robust bioremediation str ategies (Lar oc he et al. 2018 ), as w ell as ho w it impacts pollutant bioavailability and speciation (Barral-Fraga et al. 2020 ).In a context of global c hange, this r einforces the importance of combining laboratory and in situ approaches for more realistic conditions and ecological r ele v ance, and of de v eloping models for biogeoc hemical pr ocesses allowing to disentangle between correlation and causality.

Consequences on pollutant behaviour and transfer
Microbial activity can impact the mobility of metals and metalloids through the dissolution or the precipitation of metalbearing minerals (Dong et al. 2022 ).Dissolution of metal-bearing minerals will contribute to impacting previously pristine environments .Con versely, the immobilization of toxic elements by pr ecipitation or adsor ption r esults in natur al attenuation of the pollution (Egal et al. 2010 ).This is of particular importance in continuums (soil/coastal marine environments) or at the interface between different conditions (o xic/ano xic) or compartments (water/sediments) (Héry et al. 2014, Hellal et al. 2015, Zhang et al. 2020 ).Pollutants as well as micr oor ganisms can also be tr ansferr ed fr om one envir onment to another (Châtillon et al. 2023 ).For example, adsorption of metals to the surface of microplastics (Liu et al. 2021 ) can impact their fate since microplastics act as vectors of metallic pollutants and attached micr oor ganisms to w ar ds aquatic envir onments or or ganisms (Wang et al. 2021 ).Recentl y, it has been suggested that suc h complex interactions may also promote the transport and diffusion of antibiotic-resistance genes in the aquatic environment (Marathe and Bank 2022 ).

Specificities of experimental microbial ecotoxicology in deciphering the role of microorganisms in pollutant biotr ansforma tion and transfer
Curr ent knowledge ga ps on biotr ansformation pr ocesses under contr olled labor atory conditions or under envir onmentall y r ele v ant conditions lead microbial ecotoxicologists to innovate at the experimental le v el.Simplified micr obial experimental systems have been particularly useful to address ecological questions allowing for experimental controls (see reviews by Jessup et al. 2004Jessup et al. , 2005 , Cr avo-Laur eau and Dur an 2014 ).Futur e r esearc h should now r eac h beyond these r elativ el y simple models and attempt to address the complexity of the r eal world.This issue of upscaling is a major challenge (Bonnineau et al. 2021, Guasch et al. 2022 ).Transdisciplinarity has always been central in microbial ecotoxicology and is now also taking on board new technologies in chemistry and biology, in particular for investigations at different scales, dynamics and levels of complexity, in order to impr ov e our understanding of the fate and transfer of pollutants in the environment (Fig. 3 ).In the future, identifying and referencing the degradation or biotransformation pathways of pollutants and products/metabolites will be essential to better understand all the chemical entities (exposome) presented to microbial communities in a situation of interest.An ideal microbial ecotoxicology database should be compr ehensiv e, interdisciplinary, and multiscale, and include data on microbial diversity and functions , metabolites , metabolic pathwa ys , physico-chemical conditions , chemicals , and pollutants .Ho w ever, much w ork remains to be done to de v elop these databases and make them usable.
Being able to estimate the contribution of micr obial comm unities to the transformation and fate of toxic compounds will allow a better estimation of the persistence of pollutants (half-life, dT50) in natural environments (see dedicated section).The identification of families of compounds and chemical structures that are mor e easil y degr aded by micr obial comm unities or less likely to be bioaccumulated (and thus transferred through the trophic chain) will help provide guidelines for the design of new green chemicals that should have a reduced impact on ecosystems.A better understanding of the mechanisms involved in the interactions between micr oor ganisms and pollutants is thus a pr er equisite for the development of effective and sustainable bioremediation strategies in the future (see dedicated section).

Linking impacts on microbial communities to impacts and risks for ecosystem and host functioning
As illustrated in the first section, important conceptual and methodological adv ances hav e been ac hie v ed in the last decades to assess the effects of pollutants on micr obial div ersity and functions in polluted ecosystems (Pesce et al. 2020b , Morin andArtigas 2023 ).These advances have also made it possible to study pollutant effects on the interactions occurring between various animal or plant organisms and symbiotic micr oor ganisms, in-cluding e.g.microbiomes (Duperron et al. 2020 ) and rhizosphere micr obial comm unities (Barr a Car acciolo and Ter enzi 2021 ).Yet some authors recognize the importance of microbial ecotoxicology r esearc h in other fields such as animal conservation biology (Tr e v elline et al. 2019 ) or human and animal health (Adamovsky et al. 2018, Greenspan et al. 2022 ) underlining the importance of studying the links between microbial communities and their hosts (Fig. 5 ).

A lack of knowledge due to the difficulty of assessing complex interactions
Knowledge of the consequences of ecotoxicological effects on micr obial comm unities at the scale of ecosystems or symbiotic partners is still scarce.As an exception, many studies have dealt with the effects of pollutants on the interactions between microorganisms and plants.Ho w e v er, these studies mainly aimed to improve a gr onomic pr actices (e.g.selection of plant gr owth-pr omoting rhizobacteria strains resistant to pesticides, for inoculation in conv entional a gricultur e and compensation of the inhibition of natural symbioses; Ahemad and Khan 2010 ), or phytoremediation (e.g.use of micr oor ganisms to impr ov e the uptake capacity of plants for metals; Yang et al. 2022 ).Yet, there is still a lack of studies assessing this kind of interaction in an ecotoxicological fr ame work.
This limited knowledge is primarily explained by the fact that concepts and methods in microbial ecology for linking microbial communities to ecosystem functions are still in their infancy (Orland et al. 2019, Morris et al. 2020, Codello et al. 2023 ).Despite this, it is now well-recognized that microbiomes are affected by the same threats as their hosts, with environmental pollution among the most important (Tr e v elline et al. 2019 ).First, pollution alters the composition of envir onmental micr obial comm unities (see pr e vious sections) from which the host can build up its microbiome.Second, pollutant toxicity can also directly alter the host-associated microbiomes by incr easing r esistance (Lapanje et al. 2010 ) or tolerance (Costa et al. 2016 ) to pollutants, or by decreasing microbial diversity and, consequently, causing the loss of functions (Kakumanu et al. 2016 ) potentially important to the host (Fig. 2 ).Third, host microbiomes can respond to pollution by transforming pollutants into more toxic metabolites affecting the host (Piny ay ev et al. 2011, Claus et al. 2016 ).Mor eov er, se v er al studies suggest that a loss of microbial diversity (Tardy et al. 2014, Delgado-Baquerizo et al. 2016, 2020, Lafor est-La pointe et al. 2017 ) or micr obial inter actions (Wa gg et al. 2019 ) can impair ecosystem multifunctionality.Ho w ever, examining the relationships between micr obial comm unity structur e and ecosystem (Gr aham et al. 2016 ) or host (Adamovsky et al. 2018, Duperron et al. 2020 ) functioning remains challenging.This is first due to the existence of high functional redundancy within micr obial comm unities including gut microbiota (Mo y a and Ferrer 2016 ).Of particular interest in this regard is Allison and Martiny's ( 2008 ) conceptual appr oac h to how disturbances may or may not alter ecosystem processes through microbial functions .T heir model is based on levels and patterns of functional redundancy and made clear the lack of data on the links between microbial ph ylogeny, ph ysiological tr aits, and r esponses to disturbance.Second, it is extr emel y difficult if not impossible to have a reference point of the pristine environment or holobiont, with which to perform comparisons in an ecoto xicological context.When stud ying host-shelter ed comm unities, the lack of pristine habitat can translate into lack of knowledge about what a eubiotic (versus dysbiotic) host-associated microbiome is.

Upscaling in microbial ecotoxicology: limits, pitfalls, and possible solutions
Besides the difficulties in extr a polating micr obial ecotoxicological responses to the ecosystem scale, it is important to emphasize that ecosystem processes and functions are not only driven by micr oor ganisms but also by abiotic factors and/or by biological processes carried out by macroorganisms (van der Plas 2019 ).Indeed, a potential limitation of up-scaling from microbial ecotoxicology is that it may be difficult to accur atel y pr edict the effects of toxic chemicals on ecosystems or holobionts (Duperron et al. 2020 ) based solely on their effects on micr oor ganisms.Different classifications of ecosystem functions are available in the liter atur e (e.g.Pettor elli et al. 2018, Garland et al. 2021, Pesce et al. 2023 ) and most of them involve microorganisms which are sometimes the major contributors.One of the best examples is the pr ominent r ole of micr oor ganisms in nutrient cycling (Garland et al. 2021 ).The effects of chemical pollutants on the capabilities of micr oor ganisms to contribute to nutrient cycling ar e widel y studied in soil and aquatic environments using a combination of various approaches (from molecular to potential or effective activity measurements; see Fig. 3 ).Ho w ever, these assessments ar e gener all y carried out in small-scale laboratory studies or at the scale of microhabitats .T his severely limits the possibility of upscaling to the ecosystem le v el, especiall y if complex interactions with k e y envir onmental and/or nonmicr obial biological fac-tors are not taken into consideration (van der Plas 2019 ), and lead to unpredictable cascading effects.Besides the issue of spatial heter ogeneity, tempor ality also needs to be taken into consideration depending on the capacity (or not) of microbial communities to cope with chemical and other environmental stresses (e.g. according to their ada ptation, r esistance, and r esilience ca pacities; Allison and Martiny 2008 ; Fig. 2 ).Moreover, it is important to note that some categories of functions are not or only minimall y consider ed in micr obial ecotoxicology e v en though they str ongl y involv e micr oor ganisms (e.g.soil/sediment formation or erosion; based on the classification proposed by Pettorelli et al. 2018 ).T hus , assessing the impact of pollutants on the functioning of ecosystems through the prism of the response of microbial communities to these chemicals probably requires a paradigm shift.Indeed, r ather than addr essing this issue primaril y fr om a micr obial perspectiv e, it would be r ele v ant to a ppr oac h it fr om the perspective of ecosystem function by determining the most r ele v ant study scale .T hus , taking the abo ve example of nutrient cycling, it seems necessary to de v elop a ppr oac hes that combine measurements at the scale of micr obial comm unities (exoenzyme production, catabolic activities, and so on) with others carried out at the scale of ecosystems such as measurements of nutrient flo ws.F rom this point of view, the ecosystem services approach of Hayes et al., ( 2018 ) based on logic chains (linking direct ecotoxic impacts, via secondary interactions, to impacts on ecosystem pr ocesses/pr operties) seems pr omising for tar geting the ar eas of r esearc h to be de v eloped in order to better quantify the effects of pollutants on ecosystem services .Hence , in their case study (i.e.responses of ecosystems to soil copper pollution), the authors emphasized the need to focus on se v er al micr obial activities (i.e.organic matter decomposition and nutrient cycling) to take into account the role of microbial communities in ecosystem functions (Ha yes et al. 2018 ).T hese shifts in scale in relation to an ecosystem function perspective suggest that it is necessary to manage these microbial ecotoxicology issues through a strong interdisciplinary a ppr oac h.For example , the abo ve-mentioned ecosystem function of erosion limitation, which is likely to be impacted by pollutants, r equir es the combination of microbial ecotoxicology measurements with physical measurements (Gerbersdorf et al. 2005, Crouzet et al. 2019 ).
As mentioned abo ve , microbial communities , being an integral part of the ecosystem, are subject to other factors than chemical pollutants.We ther efor e highlight the importance of taking m ultistress into account in microbial ecotoxicology studies, reflecting not only the reality of pollutant mixtures but also the reality of climate change (Zandalinas et al. 2021 ).This latter high-stakes subject is beginning to be considered by the scientific community (Luo et al. 2021, Courcoul et al. 2022 ) and recent work is also attempting to address this issue by integrating microorganisms into different ecosystem le v els (O'Brien et al. 2022, Vijayaraj et al. 2022 ).

Microorganisms as a tool for environmental risk assessment and bioremediation
This section aims to illustrate the range of existing applications of micr obial ecotoxicology wher e micr oor ganisms ar e used as tools for ERA (limiting the scope to EU for standardized and normalized tests) and bioremediation, what potentially limits their application and where future developments and opportunities lie (Fig. 6 ).

Microorganisms for ERA
Despite the recognized importance of microbial communities in numerous ecological functions supporting ecosystem services and a large number of r eported micr obial-based methods (Bouchez et al. 2016 ), only a few tools based on microorganisms have been standardized and used for ERA (Table 1 ).Some of them, such as the single-species tests Ames, Microtox, or the microalgal test ar e mainl y used for a priori ERA, to predict hazards and assess risks before a new active compound is brought onto the market.Others such as the Biological Diatom Index are applied for a posteriori ERA to assess the ecotoxicological impacts of chemical residues in the environment.Complementary approaches have to be applied to integrate microorganisms from aquatic and soil ecosystems into ERA (Escher et al. 2023 ).

A priori ERA: illustration with the case of pesticides
In Europe, the a priori ERA of active ingredients in pesticides is conducted in compliance with the 1107/2009/EC dir ectiv e, whic h authorizes market delivery.For soil microorganisms, ERA of activ e ingr edients solel y r elies on the assessment of their effects on nitrogen (OECD 216; OECD 2000a ) and carbon (OECD 217; OECD 2000b ) mineralization.In order to better protect soil ecosystem services, EFSA (European Food Safety Authority 2013 ) proposed to set up a series of specific protection goals including the protection of functional groups of microorganisms.Seven years later EFSA ( 2017 ) proposed a set of endpoints to be considered for the protection of in-soil living organisms, including nitrifiers, a micr obial guild involv ed in the N-cycle (Ockleford et al. 2017 ), and arbuscular mycorrhiza fungi (AMF), which form an obligate symbiosis with most higher plants.Ho w e v er, despite these two scientific opinions (EFSA 2013(EFSA , 2017 ) ), specific protection goals and criteria have not yet been implemented in the ERA of active ingredients.One reason for this is that the endpoints proposed to fulfill the specific protection goals of k e y soil ecological functions are criticized (Sweeney et al. 2022 ), with some authors pointing out the difficulties in concluding on the origin of observed effects (direct or indirect) on these endpoints after pesticide exposure (Karpouzas et al. 2014 ).
In addition, there is still a need for r esearc h to better define effect thresholds based on the acquisition of the normal operating range (NOR) of each microbial endpoint in order to consider their possible r ecov ery following the dissipation of the activ e ingr edients and of their degradation products (Brock et al. 2018 ).The inter actions that micr oor ganisms hav e among them and with their host were recently shown to have an impact on pollutants fate (cometabolism) and to xicity.In ad dition, it is now time to move from a priori assessment of the effect of a single pur e activ e ingredient on a single species or function to a posteriori assessment of complex environmental situations where complex mixtures of c hemical r esidues of differ ent origins ar e often found.To this end, microbial biosensors, defined as analytical devices combining living micr oor ganisms as a sensing element with a process of integration of the metabolic or physiological state through a transducer, could be suitable to tackle this challenge .T he toxicity of se v er al or ganic and inor ganic pollutants has been inv estigated using either microbial cell fuel biosensors, associated with micr obes fr om activ ated sludge, biofilm, or specific species (Dávila et al. 2011, Zhou et al. 2017, Uria et al. 2020 ), or reporter biosensors, using recombinant microbial strains (Jia et al. 2012, Durand et al. 2016 ).

A posteriori ERA
Integr ativ e methods ar e r equir ed to dia gnose in situ toxicity in order to impr ov e the a posteriori ERA of v arious pollutants (Table 1 ).The PICT a ppr oac h is r ecognized as a r ele v ant method to demonstr ate the dir ect causality between envir onmental pollutant pr essure and in situ response of microbial communities in both aquatic (Tlili et al. 2016 ) or soil environments (Campillo-Cora et al. 2021 ).Ho w e v er, the a pplication of PICT still faces se v er al c hallenges that deserve further investigation.First, establishing the NOR of tolerance of microbial communities is one main issue requiring large r efer ence datasets that remains poorly tackled (but see Blanck et al. 2003, Campillo-Cora et al. 2021 ).Second, there is a need to de v elop and validate via ring-testing standardized protocols based on published methods (from in situ sampling to modelling and inter pr etation of lab toxicity test r esults).Third, ne w methods ar e r equir ed to increase the diversity of considered pollutants and microbial functions used as endpoints in the toxicity tests to e v aluate toler ance le v els.Fourth, guidelines need to be elaborated to inter pr et comm unity toler ance with r efer ence to the toler ance baseline (Campillo-Cora et al. 2021 ).Finally, new knowledge should be acquired to understand the processes involved in PICT responses (influence of confounding factors, cotolerance processes, etc; Tlili et al. 2016 ).In addition, the costs of adaptation of microbial communities to pollutants (e.g.secondary effects on microbial diversity and ecological functions; Tlili et al. 2011, Bérard et al. 2016, Pesce et al. 2020b ) should be studied to give clues on how to transform PICT responses into an assessment of ecological risks and   ( 1998 ).
ecotoxicological effects at the microbial community and ecosystem le v els.The Triad a ppr oac h, whic h combines toxicity testing and chemical and ecological data of a site to determine the effect of pollution on the ecosystem, can also be viewed as a promising tool (Gutiérr ez et al. 2015, Klimk owicz-P awlas et al. 2019 ).Although this ISO standard lists se v er al standardized methods for measuring each of the identified risk types, the use of nonstandardized methods can be emplo y ed.Due to their ubiquity and different ecological roles in the environment, microbial communities could constitute good indicators to consider in this a ppr oac h.
Curr entl y, onl y micr oalgae ar e consider ed in the EU Water Fr ame work Dir ectiv e for the calculation of indices based on diatoms and phytoplankton.Although these indices have gained importance for the assessment of the ecological quality of aquatic ecosystems (Venkatac hala pathy and Karthik e y an 2015 , Lav oie et al. 2018 ), their output remains poorly informative with regards to the ecological effects of a large variety of pollutants.Such indices could be further de v eloped to take into account the impacts of pollutants.In combination with r ele v ant pollutant monitoring studies, the incr easing a pplication of diatom DN A metabar coding will help to monitor the effects of pollutants on diatoms (Tapolczai et al. 2019, Maitland et al. 2020 ).This is likely to be also applicable to other microbial groups and in different types of ecosystems and environmental compartments due to the concomitant and ongoing impr ov ement of sampling and analytical methods for c har acterizing envir onmental pollution by a lar ge v ariety of chemicals (Hollender et al. 2017 ) and of eDNA studies (Seymour 2019 ).A fe w years a go, Bouc hez et al. ( 2016) defined the le v el of operability of several molecular microbial indicators according to eac h envir onmental matrix (i.e .soil, sediments , water, atmosphere, and wastes) for envir onmental dia gnosis.For the ERA of herbicides in soil, some authors suggest searching for tolerant and/or sensitive populations of nontarget microorganisms that nonetheless carry the enzyme specifically targeted by the active ingredient (Petric et al. 2016, Thiour-Mauprivez et al. 2019 ).Monitoring the quality of aquatic environments could also be done by integrating bacteria naturally present in the aquatic compartment to propose a new generation of microbial biosensors (Zhou et al. 2017, Jiang et al. 2018, Fang et al. 2020 ).

Microorganisms as tools for assessing ecosystem functioning
Ensuring environmental and human health protection requires pr eserving or r estoring ecosystem functioning and their ca pabilities to provide services.As noted pr e viousl y, the ubiquity of micr oor ganisms and the numerous ecological functions they perform make them essential key drivers that must be protected in order to ensure the continued functioning of ecosystems and preserve the One Health concept.In 2023, an EU directive on soil protection is on the v er ge of being adopted by the European Commission 17 years after its first proposal.In the meantime, several EU countries are already conducting national soil surveys to monitor changes in abiotic and biotic parameters, including endpoints related to soil functions .Nonetheless , the implementation of microbial endpoints in national soil surve ys de pends on the availability of standardized methods.Ho w e v er, despite pr ogresses done (Thiele-Bruhn 2021 ) there is still a huge challenge to pr ovide ne w standardized a ppr oac hes, r efer ence bioindicators and guidelines related to soil microorganisms (Djemiel et al. 2022 ).This observation is striking with regards to the EU water framework dir ectiv e, the most significant European water legislation to date, whic h onl y considers diatom biodiv ersity as a micr obial endpoint to assess the biological quality of water bodies, and toxicity tests on microalgae to establish environmental quality standards to c hemicals, micr obial functions or other micr oor ganisms being totall y disr egar ded (P esce et al. 2020a ).

Microorganisms as nature-based solutions for pollution treatment
The scientific community faces several challenges to enable more widespread use of microorganisms for bioremediation of contaminated en vironmental matrices .Based on our fundamental understanding of the biodegradation or biotransformation of pollutants (see dedicated section), many recent examples demonstrate the effectiveness of using microorganisms for the bioremediation of soils and waters polluted by hydrocarbons or organohalides (Mc-Carty et al. 2020 , Naeem andQazi 2020 ).Ho w e v er, ther e is still a long way to go to be able to propose bioremediation techniques for emerging or recalcitrant organic pollutants such as (micro-) plastics or PFAS, whose kinetics and degradation pathways are still understudied (Zhou et al. 2022 ).Although less commonly applied to metallic pollutants (particularly at large scale), bioremediation can effectiv el y r emov e metals or metalloids fr om mine waste , soils , or waters through immobilization or transformation processes (Rahman and Singh 2020, Jacob et al. 2022, Nivetha et al. 2023 ).For example, bacterially mediated treatment of arsenic-rich acid mine waters has r ecentl y been successfull y up-scaled fr om the lab to the field (Diaz-Vanegas et al. 2022 ).Mor eov er, phytoextraction of metals is more developed and can be efficiently impr ov ed thr ough the action of micr obes suc h as mycorrhizal fungi or plant gr owth pr omoting (PGP) bacteria that could enhance the speed and quantity of metal uptake by plants (Kazemalilou et al. 2020 ).
The different bioremediation approaches applied to soils or (gr ound)water ar e natur al attenuation, bioaugmentation, biostim ulation, and rhizostim ulation (Khan et al. 2004 ).Natural attenuation, whic h r equir es that pollutants ar e being immobilized or degraded by natural processes (biotic or abiotic) without any human intervention, can be slow compared to bioaugmentation or biostimulation, and require long-term monitoring (Khan et al. 2004 ).Bioaugmentation consists of growing selected micr oor ganisms with a known ability to degrade or transform a target pollutant.Although its effectiveness depends on the survival and development of inoculated strains, bioaugmentation can be effective, fast, and affordable as a 'green' clean-up option (Nw ankw egu et al. 2022 ).A pitfall for bioaugmentation is the unforeseen interactions on added degrading strains with autochthonous microbes (Yu et al. 2005 ).Biostimulation aims to overcome factors limiting the activity of autoc hthonous micr oor ganisms thr ough the supply of nutrients (nitrogen source, electron acceptors/donors, and so on), surfactants, and/or oxygen.This r equir es a good knowledge of the indigenous communities and their physiological and metabolic needs .T he optimal C/N/P ratio and bioa vailability of pollutant must be determined and adjusted, and the stimulation of other populations that can out-compete the target microorganisms is not excluded (Adams et al. 2015 ).
In addition to the fact that bioremediation methods may be slo w er than more conventional physico-chemical approaches, microbial activity is also under the complex and tight influence of man y envir onmental factors, and ther efor e, difficult to pr edict (Bala et al. 2022 ).To impr ov e the reliability and the sustainability of bioremediation performance in situ , it is now crucial to better understand the factors driving microbial activity (Lar oc he et al. 2018, Diaz-Vanegas et al. 2022 ).
Another use of micr oor ganisms that r epr esents an innov ativ e exploitation of microbe-metal interactions is the recovery of critical metals from secondary sources .T his results in environmental clean-up and contributes to recycling.As cost is often an important obstacle for r emediation, str ategies integr ating bior emediation and r ecov ery of pollutants of economic inter est suc h as metals hold gr eat pr omise for the environmental and economic sectors (Guezennec et al. 2015, Bryan et al. 2020, Hubau et al. 2020, Gavrilescu 2022 ).
These pr e vious examples highlight the persisting fundamental need to de v elop ne w isolation a ppr oac hes (bacterial tr a pping, ne w cultur e media, and high thr oughput cultur omics) in order to have a greater diversity of microbial strains degrading or transforming pollutants for bioaugmentation applications.One future challenge is to explore the higher potential of microbial consortia rather than individual strains and to be able to conserve these consortia and their properties on the long-term.In addition, impr ov ed methods ar e also needed to scr een, c har acterize, pr oduce, form ulate, test, and v alidate degr ading inoculants for cleaning up polluted soils (Duran et al. 2022 ).From a functional point of view, understanding the dynamics of microbial communities in these systems and how they can be stable and effective over time will be essential to engineer well-built and sustainable bioremediation systems .T his in turn will help with the demands of the ecological transition to address identified planetary boundaries (Persson et al. 2022, Arp et al. 2023 ).The application of integrativ e a ppr oac hes could be useful in this respect, as shown in a recent study by Hellal et al. ( 2021 ) on the monitoring of in situ natural attenuation of a multipolluted aquifer.Enrichment, isolation and pr eserv ation of efficient micr obial str ains ca pable of degr ading v arious or ganic pollutants is still necessary.The creation of an open repository of adequately characterized degrading strains could facilitate the choice of the most effective isolates depending on the pollutant to be biodegraded and the physico-chemical conditions of the environment to be r emediated.A fe w r ecent initiativ es ar e w orking to w ar ds this goal (e.g. the EU Horizon project MIBIREM), in particular on the pr eserv ation of microbial consortia whose pr eserv ation, stability, and maintenance of activity over time remains a challenge.

Concluding remarks
The anthropocene is characterized by global chemical pollution as underlined by the International Panel on Chemical Pollution (IPCP).Micr obial comm unities, thr ough their r esponses to exposure to pollutants and their biotransformation capabilities, repr esent sensitiv e bioindicators for r e v ealing the ecological quality of the environment and are promising actors for the remediation of polluted en vironments .Consequently, microbial ecotoxicology has become a k e y(stone) area for scientific research as it fills the knowledge gaps necessary to implement a strategy taking on board micr obial comm unities in order to monitor and implement the 'One Health' agenda.One of the challenges for microbial eco-toxicologists is now to embed their work on the fate and effects of pollutants in a perspective that simultaneously embraces ecosystem taxonomy and functions , ecosystem services , and naturebased solutions (Peixoto et al. 2022 , Lemke andDeSalle 2023 ), and whic h integr ates m ultistr ess situations r elated to global c hanges (Sabater et al. 2019 ).In addition to these new knowledge inputs and contributions, microbial ecotoxicologists will also assist in the definition of NOR and threshold values of acceptable effects of pollutants .T his knowledge can then be integrated and proposed to stakeholders for implementation in new regulations more protective of One Health.To ac hie v e this objectiv e, micr obial ecotoxicologists will r epr esent a driving force to propose innovative concepts, a ppr oac hes and (standardized) methods, open science data sets and scientific expertise that can be further mobilized by socio-economic partners.To increase their visibility and their impact, microbial ecotoxicologists will have a k e y role in further promoting and developing interdisciplinarity.Furthermore, they will have the responsibility of training a new generation of scientists aware of the importance of microbial communities within the 'One Health' fr ame work and ca pable of scientific mediation to w ar ds diverse players of the society, in particular stakeholders, politicians, and elected r epr esentativ es who hav e in their hands the po w er to implement ne w r egulations and impulse ne w dir ections in favour of a more sustainable world.
Taken together, the methodological challenges identified in order to adequately assess the biological effects of chemical pollution will r equir e mor e and impr ov ed integr ativ e studies .T hese will cover a larger diversity of microbial groups, more directly link the microbiome to its function, and combine novel and/or traditional methods with statistical and modelling a ppr oac hes.All these tools hold str ong pr omise for the field of microbial ecoto xicology, as the y will allow the c har acterization of the effects and fate of toxic chemicals at the ecosystem scale as well as the taxonomic, functional, and morphometric responses of microbial communities.In turn, this should also allow easier consideration of space and time in environmental studies in the futur e, thr ough long-term monitoring and original experimental designs considering the complexity of real-world en vironments .

Figure 3 .
Figure 3. Ov ervie w of the current gaps and limitations of the main methods for studying microorganisms (di versity, acti vities) at different levels of complexity and perspectives for data interpretation and diagnostic tool development.

Figur e 4 .
Figur e 4.An o v ervie w of micr obial tr ansformation and degr adation mec hanisms of metals and metalloids (left) and or ganic molecules (right).

Figure 5 .
Figure 5. Illustration of the current paradigm shift and how impacts of pollutants on microbial community diversity and functions can in turn affect host and ecosystem functioning.

Figure 6 .
Figure 6.Illustration of different applications of microorganisms as tools for ERA and bioremediation.

Table 1 .
Some existing tools based on microbial ecotoxicology concept for ERA, presented by context of application.