Assessing the Effects of Pesticides on the Soil Microbial Community: Advances, Standardization of Methods and the Need for a New Regulatory Framework

Upon their application pesticides end up in soil where they interact with the soil microbial community. Considering the pivotal role of soil microorganisms in ecosystem homeostasis and the growing evidence about their potential toxicity response to pesticide exposure, there is an urgent need to revisit the relevant regulatory framework. This is necessary in light of the enormous methodological and standardization advances in soil microbial ecology in the last 20 years and the outdated assessment scheme currently in place. In this chapter we highlight the key elements of a new risk assessment scheme including (a) the definition of microbial indicator groups like ammonia-oxidizing microorganisms and arbuscular mycorrhizal fungi (b) the parallel determination of the level and the duration of the exposure including transformation products (c) the need for implementation in environmental risk analysis of advanced and standardized tools. Based on all these a new tiered-risk assessment scheme is proposed. Emerging issues in soil microbial ecotoxicology are discussed including (a) the assessment of pesticide soil microbial toxicity at ecosystem level and (b) the assessment of the soil microbial toxicity of biopesticides, pesticide mixtures and pesticide transformation products on soil microorganisms. We conclude by highlighting emerging scientific questions that are expected to puzzle the soil microbial ecotoxicologists working with pesticides in the next decade. © 2021, The Author(s), under exclusive license to Springer Nature Switzerland AG

Pesticide soil microbial toxicity: Setting the scene for a new pesticide risk assessment for soil microorganisms (IUPAC Technical Report)

Pesticides constitute an integral part of modern agriculture. However, there are still concerns about their effects on non-target organisms. To address this the European Commission has imposed a stringent regulatory scheme for new pesticide compounds. Assessment of the aquatic toxicity of pesticides is based on a range of advanced tests. This does not apply to terrestrial ecosystems, where the toxicity of pesticides on soil microorganisms, is based on an outdated and crude test (N mineralization). This regulatory gap is reinforced by the recent methodological and standardization advances in soil microbial ecology. The inclusion of such standardized tools in a revised risk assessment scheme will enable the accurate estimation of the toxicity of pesticides on soil microorganisms and on associated ecosystem services. In this review we (i) summarize recent work in the assessment of the soil microbial toxicity of pesticides and point to ammonia-oxidizing microorganisms (AOM) and arbuscular mycorrhizal fungi (AMF) as most relevant bioindicator groups (ii) identify limitations in the experimental approaches used and propose mitigation solutions, (iii) identify scientific gaps and (iv) propose a new risk assessment procedure to assess the effects of pesticides on soil microorganisms © 2022 IUPAC & De Gruyter. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. For more information, please visit: http://creativecommons.org/licenses/by-nc-nd/4.0/ 2022

Towards a better pesticide policy for the European Union

This opinion article aims to foster the debate about pesticide legislation in the European Union (EU). Numerous formerly authorized and widely used pesticides are now banned in the EU because unexpected and unacceptable risks emerged after their initial introduction to the market. Throughout this time lapse, environmental quality and human health have been threatened by the use of these compounds. These hazards could have been prevented by a more responsive pesticide regulatory framework. This article provides detailed insights into the pros and cons of pesticides, and points out weaknesses of the current pesticide environmental risk assessment procedures. Possibilities for improving the robustness and reliability of the pesticide regulatory framework are discussed. © 2016 Elsevier B.V

Genome-Based Metabolic Reconstruction Unravels the Key Role of B12 in Methionine Auxotrophy of an Ortho-Phenylphenol-Degrading Sphingomonas haloaromaticamans

Auxotrophy to amino acids and vitamins is a common feature in the bacterial world shaping microbial communities through cross-feeding relations. The amino acid auxotrophy of pollutant-degrading bacteria could hamper their bioremediation potential, however, the underlying mechanisms of auxotrophy remain unexplored. We employed genome sequence-based metabolic reconstruction to identify potential mechanisms driving the amino acid auxotrophy of a Sphingomonas haloaromaticamans strain degrading the fungicide ortho-phenylphenol (OPP) and provided further verification for the identified mechanisms via in vitro bacterial assays. The analysis identified potential gaps in the biosynthesis of isoleucine, phenylalanine and tyrosine, while methionine biosynthesis was potentially effective, relying though in the presence of B12. Supplementation of the bacterium with the four amino acids in all possible combinations rescued its degrading capacity only with methionine. Genome sequence-based metabolic reconstruction and analysis suggested that the bacterium was incapable of de novo biosynthesis of B12 (missing genes for the construction of the corrin ring) but carried a complete salvage pathway for corrinoids uptake from the environment, transmembrane transportation and biosynthesis of B12. In line with this the bacterium maintained its degrading capacity and growth when supplied with environmentally relevant B12 concentrations (i.e., 0.1 ng ml–1). Using genome-based metabolic reconstruction and in vitro testing we unraveled the mechanism driving the auxotrophy of a pesticide-degrading S. haloaromaticamans. Further studies will investigate the corrinoids preferences of S. haloaromaticamans for optimum growth and OPP degradation. © Copyright © 2020 Perruchon, Vasileiadis, Papadopoulou and Karpouzas

The response of soil and phyllosphere microbial communities to repeated application of the fungicide iprodione: Accelerated biodegradation or toxicity?

Pesticides interact with microorganisms in various ways with the outcome being negative or positive for the soil microbiota. Pesticides' effects on soil microorganisms have been studied extensively in soil but not in other pesticides-exposed microbial habitats like the phyllosphere. We tested the hypothesis that soil and phyllosphere support distinct microbial communities, but exhibit a similar response (accelerated biodegradation or toxicity) to repeated exposure to the fungicide iprodione. Pepper plants received four repeated foliage or soil applications of iprodione, which accelerated its degradation in soil (DT50-1st = 1.23 and DT50-4th = 0.48 days) and on plant leaves (DT50-1st > 365 and DT50-4th = 5.95 days). The composition of the epiphytic and soil bacterial and fungal communities, determined by amplicon sequencing, was significantly altered by iprodione. The archaeal epiphytic and soil communities responded differently; the former showed no response to iprodione. Three iprodione-degrading Paenarthrobacter strains were isolated from soil and phyllosphere. They hydrolyzed iprodione to 3,5-dichloraniline via the formation of 3,5-dichlorophenyl-carboxiamide and 3,5-dichlorophenylurea-acetate, a pathway shared by other soil-derived arthrobacters implying a phylogenetic specialization in iprodione biotransformation. Our results suggest that iprodione-repeated application could affect soil and epiphytic microbial communities with implications for the homeostasis of the plant-soil system and agricultural production. © 2020 FEMS 2020

Expanding the use of biobeds: Degradation and adsorption of pesticides contained in effluents from seed-coating, bulb disinfestation and fruit-packaging activities

Agro-food industries that use pesticides constitute significant point sources for the contamination of natural water resources. Despite that, little is known about the treatment of their pesticide-contaminated effluents. Biobeds could be a possible solution for the depuration of these effluents. In this context, we explored the degradation and adsorption of pesticides used in seed-coating (carboxin (CBX), metalaxyl-M (MET-M), fluxapyroxad (FLX), fludioxonil (FLD)), bulb-dipping (chlorothalonil (CHT), thiabendazole (TBZ), FLD) and fruit-packaging activities (FLD) in a biomixture, used as biobed packing material, and in soil. The degradation of pesticides was tested individually and in mixtures relevant to their industrial use, while FLD was also tested at different concentrations (10, 20, and 150 mg kg−1) representing its use in the different industries. CBX, FLD, and CHT, when applied individually, and all other pesticides when applied in mixtures, degraded more rapidly in biomixture than in soil. In most cases pesticides application in mixtures retarded their degradation. This was more pronounced in soil than in biomixture, especially for MET-M and FLD. CHT had the most prominent inhibitory effect on the degradation of TBZ and FLD. FLD degradation showed a dose-dependent pattern (DT50 42.4 days at 10 mg kg−1 and 107.6 days at 150 mg kg−1). All pesticides showed higher adsorption affinity in the biomixture (Kf = 3.23–123.3 g mL−1) compared to soil (Kf = 1.15–31.2 g mL−1). We provide initial evidence for the potential of the tested biomixture to remove pesticides contained in effluents produced by different agro-industrial activities. Tests in full-scale biobeds packed with this biomixture will unravel their full depuration potential for the treatment of these agro-industrial effluents. © 2019 Elsevier Lt

Isolation and characterization of soil bacteria able to rapidly degrade the organophosphorus nematicide fosthiazate

Foshtiazate is an organophosphorus nematicide commonly used in protected crops and potato plantations. It is toxic to mammals, birds and honeybees, it is persistent in certain soils and can be transported to water resources. Recent studies by our group demonstrated, for the first time, the development of enhanced biodegradation of fosthiazate in agricultural soils. However, the micro-organisms driving this process are still unknown. We aimed to isolate soil bacteria responsible for the enhanced biodegradation of fosthiazate and assess their degradation potential against high concentrations of the nematicide. Enrichment cultures led to the isolation of two bacterial cultures actively degrading fosthiazate. Denaturating Gradient Gel Electrophoresis analysis revealed that they were composed of a single phylotype, identified via 16S rRNA cloning and phylogenetic analysis as Variovorax boronicumulans. This strain showed high degradation potential against fosthiazate. It degraded up to 100 mg l−1 in liquid cultures (DT50 = 11·2 days), whereas its degrading capacity was reduced at higher concentration levels (500 mg l−1, DT50 = 20 days). This is the first report for the isolation of a fosthiazate-degrading bacterium, which showed high potential for use in future biodepuration and bioremediation applications. Significance and Impact of the Study: This study reported for the first time the isolation and molecular identification of bacteria able to rapidly degrade the organophosphorus nematicide fosthiazate; one of the few synthetic nematicides still available on the global market. Further tests demonstrated the high capacity of the isolated strain to degrade high concentrations of fosthiazate suggesting its high potential for future bioremediation applications in contaminated environmental sites, considering high acute toxicity and high persistence and mobility of fosthiazate in acidic and low in organic matter content soils. © 2018 The Society for Applied Microbiolog

Using biobeds for the treatment of fungicide-contaminated effluents from various agro-food processing industries: Microbiome responses and mobile genetic element dynamics

Agro-food processing industries generate large amounts of pesticide-contaminated effluents that pose a significant environmental threat if managed improperly. Biopurification systems like biobeds could be utilized for the depuration of these effluents although direct evidence for their efficiency are still lacking. We employed a column leaching experiment with pilot biobeds to (i) assess the depuration potential of biobeds against fungicide-contaminated effluents from seed-producing (carboxin, metalaxyl-M, fluxapyroxad), bulb-handling (thiabendazole, fludioxonil and chlorothalonil) and fruit-packaging (fludioxonil, imazalil) industries, (ii) to monitor microbial succession via amplicon sequencing and (iii) to determine the presence and dynamics of mobile genetic elements like intl1, IS1071, IncP-1 and IncP-1ε often associated with the transposition of pesticide-degrading genes. Biobeds could effectively retain (adsorbed but extractable with organic solvents) and dissipate (degraded and/or not extractable with organic solvents) the fungicides that were contained in the agro-industrial effluents with 93.1–99.98% removal efficiency in all cases. Lipophilic substances like fluxapyroxad were mostly retained in the biobed while more polar substances like metalaxyl-M and carboxin were mostly dissipated or showed higher leaching potential like metalaxyl-M. Biobeds supported a bacterial and fungal community that was not affected by fungicide application but showed clear temporal patterns in the different biobed horizons. This was most probably driven by the establishment of microaerophilic conditions upon water saturation of biobeds, as supported by the significant increase in the abundance of facultative or strict anaerobes like Chloroflexi/Anaerolinae, Acidibacter and Myxococcota. Wastewater application did not affect the dynamics of mobile genetic elements in biobeds whose abundance (intl1, IS1071, IncP-1ε) showed significant increases with time. Our findings suggest that biobeds could effectively decontaminate fungicide-contaminated effluents produced by agro-food industries and support a rather resilient microbial community. © 2022 Elsevier B.V

Isolation and characterisation of a Sphingomonas strain able to degrade the fungicide ortho-phenylphenol

BACKGROUND: Ortho-phenylphenol (OPP) is a fungicide used in fruit packaging plants for the control of fungal infestations during storage. Its application leads to the production of large wastewater volumes which according to the European legislation should be treated on site. In spite of this, no efficient treatment systems are currently available, and the development of biological systems based on tailored-made pesticide-degrading inocula for the treatment of these wastewaters is an appealing solution. RESULTS: Enrichment cultures from a soil collected from a wastewater disposal site resulted in the isolation of a pure Sphingomonas haloaromaticamans strain P3 able to degrade rapidly OPP and use it as an energy source. Its degrading capacity was dependent on the external supply of amino acids or on the presence of other bacteria that did not contribute to fungicide degradation. The isolated S. haloaromaticamans strain was able to metabolise up to 150mgL-1 of OPP within 7 days, in a wide range of pH (4.5-9) and temperatures (4-37°C), and in the presence of other pesticides (thiabendazole and diphenylamine) co-used in the fruit packaging industry. CONCLUSION: Overall, the OPP-degrading bacterium isolated showed high potential for use in future biodepuration treatment systems and bioremediation strategies. © 2015 Society of Chemical Industry

Effects of organic loading rate and hydraulic retention time on bioaugmentation performance to tackle ammonia inhibition in anaerobic digestion

Three continuously stirred-tank reactors fed with manure operating under high ammonia levels (5.0 g NH3-N L−1) and with increased organic loading rate (OLR), (2.09 R1, 3.02 R2 and 4.0 R3 g VS L−1 d−1), achieved through glucose amendment in R2 and R3, were inoculated with an ammonia-acclimatized microbial culture. Successful bioaugmentation was endured only in R2 and R3, both reactors characterized by high OLR, resulting in 19.6 and 24.5% increase in methane production, respectively. The high OLRs in these reactors favored the co-occurrence of the hydrogenotrophic (Methanobacteriaceae), methylotrophic (Methanomethylophilaceae) and aceticlastic methanogenic pathways. The latter was supported by the successful establishment of ammonium-tolerant Methanosarcina, prevailing in the inoculum. Oppositely in R1, the low OLR prevented the establishment of Methanosarcina, leading to an exclusive hydrogenotrophic methanogenesis and reduced methane production. HRT shortening resulted in limited effect on biomethane performance, indicating a well establishment of the introduced bioaugmentation culture in the reactors. © 2021 Elsevier Lt