Marine Microbiota Responses to Shipping Scrubber Effluent Assessed at Community Structure and Function Endpoints.

The use of novel high-throughput sequencing (HTS) technologies to examine the responses of natural multidomain microbial communities to scrubber effluent discharges to the marine environment is still limited. Thus, we applied metabarcoding sequencing targeting the planktonic unicellular eukaryotic and prokaryotic fraction (phytoplankton, bacterioplankton, and protozooplankton) in mesocosm experiments with natural microbial communities from a polluted and an unpolluted site. Furthermore, metagenomic analysis revealed changes in the taxonomic and functional dominance of multidomain marine microbial communities after scrubber effluent additions. The results indicated a clear shift in the microbial communities after such additions, which favored bacterial taxa with known oil and polycyclic aromatic hydrocarbons (PAHs) biodegradation capacities. These bacteria exhibited high connectedness with planktonic unicellular eukaryotes employing variable trophic strategies, suggesting that environmentally relevant bacteria can influence eukaryotic community structure. Furthermore, Clusters of Orthologous Genes associated with pathways of PAHs and monocyclic hydrocarbon degradation increased in numbers at treatments with high scrubber effluent additions acutely. These genes are known to express enzymes acting at various substrates including PAHs. These indications, in combination with the abrupt decrease in the most abundant PAHs in the scrubber effluent below the limit of detection-much faster than their known half-lives-could point toward a bacterioplankton-initiated rapid ultimate biodegradation of the most abundant toxic contaminants of the scrubber effluent. The implementation of HTS could be a valuable tool to develop multilevel biodiversity indicators of the scrubber effluent impacts on the marine environment, which could lead to improved impact assessment. Environ Toxicol Chem 2024;43:1012-1029. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Rapid and sensitive method for the simultaneous determination of PAHs and alkyl-PAHs in scrubber water using HS-SPME-GC-MS/MS.

Scrubber water, a waste stream generated by ships exhaust gas cleaning systems, may pose a threat when released into the marine environment due to potential contamination with polycyclic aromatic hydrocarbons (PAHs) and their alkyl derivatives (alkyl-PAHs). This study aims to develop a reliable analytical procedure combining headspace solid-phase microextraction (HS-SPME) with gas chromatography coupled to triple quadrupole tandem mass spectrometry (GC-MS/MS) to simultaneously separate and determine target compounds in aqueous samples. Method validation demonstrated good linearity up to 200 ng L-1 (r2> 0.996) and low limits of detection (0.33 to 1.67 ng L-1, except for naphthalene at 3.3 ng L-1). The method shows good precision (RSD<20%) and satisfactory analytical recoveries. The methodology was successfully applied to scrubber water samples collected from a container ship and the results highlight the prevalence of naphthalene, phenanthrene, and their alkyl derivatives.•Rapid and reproducible HS-SPME-GC-MS/MS method for the analysis of PAHs and alkyl-PAHs in scrubber water.•The capacity of SPME to analyze both filtered and unfiltered samples was assessed, showing that the more hydrophobic PAHs may be lost during filtration.

Impacts of exhaust gas cleaning systems (EGCS) discharge waters on planktonic biological indicators.

Exhaust Gas Cleaning Systems (EGCS), operating in open-loop mode, continuously release acidic effluents (scrubber waters) to marine waters. Furthermore, scrubber waters contain high concentrations of metals, polycyclic aromatic hydrocarbons (PAHs), and alkylated PAHs, potentially affecting the plankton in the receiving waters. Toxicity tests evidenced significant impairments in planktonic indicators after acute, early-life stage, and long-term exposures to scrubber water produced by a vessel operating with high sulphur fuel. Acute effects on bacterial bioluminescence (Aliivibrio fischeri), algal growth (Phaeodactylum tricornutum, Dunaliella tertiolecta), and copepod survival (Acartia tonsa) were evident at 10 % and 20 % scrubber water, while larval development in mussels (Mytilus galloprovincialis) showed a 50 % reduction at ∼5 % scrubber water. Conversely, larval development and reproductive success of A. tonsa were severely affected at scrubber water concentrations ≤1.1 %, indicating the risk of severe impacts on copepod populations which in turn may result in impairment of the whole food web.

Graphene oxide addition to anaerobic digestion of waste activated sludge: Impact on methane production and removal of emerging contaminants.

The effect of graphene oxide on the anaerobic digestion of waste activated sludge was investigated at two graphene oxide concentrations (0.025 and 0.075 g graphene oxide per g volatile solids) using biochemical methane potential tests. The occurrence of 36 pharmaceuticals was monitored in the solid and liquid phases before and after the anaerobic treatment. The addition of graphene oxide improved the removal of most pharmaceuticals detected, even those that are considered persistent to biological degradation, such as azithromycin, carbamazepine, and diclofenac. No significant differences were observed in the final specific methane production without graphene oxide and with the lowest graphene oxide concentration, yet the highest graphene oxide concentration partially inhibited methane production. The relative abundance of antibiotic resistance genes was not affected by the graphene oxide addition. Finally, significant changes in the microbial community including bacteria and archaea were detected with graphene oxide addition.

Antibiotics, antibiotic resistance and associated risk in natural springs from an agroecosystem environment.

This study investigates the occurrence, transport, and risks associated to antibiotic residues, antibiotic resistance genes (ARGs) and antibiotic resistant Escherichia coli (AR-E. coli) in eleven natural springs in an agroecosystem environment with intense livestock production, where groundwater nitrate concentration usually sets above 50 mg L-1. Out of 23 multiple-class antibiotics monitored, tetracycline and sulfonamide residues were the most ubiquitous, and they were detected at concentrations ranging from ng L-1 to µg L-1. Five ARGs were monitored, conferring resistance to the antibiotic classes of major use in livestock production. Thus, genes conferring resistance to sulfonamides (sul1 and sul2) and tetracyclines (tetW) as well as a gene proxy for anthropogenic pollution (intI1) were present in most springs. sul1 was the most abundant, with absolute concentrations ranging from 4 × 102 to 5.6 × 106 gene copies L-1 water. AR-E. coli showing resistance to sulfonamides and tetracyclines was also detected, with a prevalence up to approximately 40 % in some sites but with poor correlations with the concentration of antibiotic residues and ARGs. The occurrence of antibiotics, ARGs and AR-E. coli was characterized by large seasonal variations which were mostly associated to both hydrological factors and reactive transport processes. Finally, a risk assessment approach pointed out towards low risk for both the groundwater environment and human health, when spring water is used for direct human consumption, associated with the occurrence of antibiotics, ARGs and AR-E. coli. However, long-term effects cannot be neglected, and proper actions must be taken to preserve groundwater quality.

Occurrence of veterinary drugs and resistance genes during anaerobic digestion of poultry and cattle manures.

In the present paper, the mesophilic (35 °C) and thermophilic (55 °C) biomethanization of poultry and cattle manures were investigated using biochemical methane potential (BMP) tests. Specific methane production (SMP), 24 pharmaceutical compounds (PhACs), and five antibiotic resistance genes (ARGs) (blaKPC, ermB, qnrS, sul1 and tetW) together with the microbial community were analyzed. Mesophilic BMP tests resulted in the highest SMP when poultry manure was used (285.5 mL CH4/g VSS with poultry vs 239.6 mL CH4/g VSS with cattle manure) while thermophilic temperatures led to the highest SMP with cattle manure (231.2 mL CH4/g VSS with poultry vs 238.0 mL CH4/g VSS with cattle manure). Higher removals of veterinary pharmaceuticals were detected at 55 °C with both manures indicating that thermophilic digestion is better suited for the removal of these compounds. Tylosin, tilmicosin, chlortetracycline, and sulfamethoxazole presented removals higher than 50%, being the first two completely removed under mesophilic and thermophilic conditions. When comparing the relative abundance of ARGs at the end of each treatment, the most significant removal was found for qnrS which was not detected after the anaerobic treatment. The remaining ARGs did not suffer significant changes. Finally, microbial composition analysis showed that temperature affected the final microbial population more than the microorganisms present in the substrate or inoculum.

Groundwater antibiotic pollution and its relationship with dissolved organic matter: Identification and environmental implications.

The occurrence of veterinary antibiotics and hydro-chemical parameters in eleven natural springs in a livestock production area is evaluated, jointly with the characterization of their DOM fingerprint by Orbitrap HRMS. Tetracycline and sulfonamide antibiotics were ubiquitous in all sites, and they were detected at low ng L-1 concentrations, except for doxycycline, that was present at µg L-1 in one location. DOM analysis revealed that most molecular formulas were CHO compounds (49 %-68 %), with a remarkable percentage containing nitrogen and sulphur (16 %-23 % and 11 %-24 %, respectively). Major DOM components were phenolic and highly unsaturated compounds (~90 %), typical for soil-derived organic matter, while approximately 11 % were unsaturated aliphatic, suggesting that springs may be susceptible to anthropogenic contamination sources. Comparing the DOM fingerprint among sites, the spring showing the most different profile was the one with surface water interaction and characterized by having lower CHO and higher CHOS formulas and aliphatic compounds. Correlations between antibiotics and DOM showed that tetracyclines positively correlate with unsaturated oxygen-rich substances, while sulfonamides relate with aliphatic and unsaturated oxygen-poor compounds. This indicates that the fate of different antibiotics will be controlled by the type of DOM present in groundwater.

Extended suspect screening to identify contaminants of emerging concern in riverine and coastal ecosystems and assessment of environmental risks.

A suspect screening methodology was developed for the fast and reliable identification of 360 contaminants of emerging concern (CECs) of anthropogenic origin in the vulnerable area of the Ebro Delta (Catalonia, Spain) and to track for potential contamination sources. The suspect screening methodology was combined with a risk assessment approach to prioritize the most ecologically relevant CECs. Out of the 360 suspects, 37 compounds were tentatively identified, 22 of which were fully confirmed using isotopically labelled standards. The detected suspect compounds included pesticides, pharmaceuticals, personal care products, stimulants and their metabolites. Pesticides were more ubiquitous in irrigation and drainage channels, while pharmaceuticals, stimulants, and personal care products were the most common in effluent wastewaters, in the receiving freshwater systems as well as in the marine environment. Ten compounds were found to be of high ecological concern, including the pharmaceuticals telmisartan, venlafaxine, and carbamazepine, the herbicides terbuthylazine, desethylterbuthylazine, and terbutryn, the fungicides azoxystrobin, tebuconazole and prochloraz and the insecticide tebufenozide. These compounds could be used as markers of anthropogenic contamination in riverine and coastal ecosystems.

Identification of organic contaminants in vinasse and in soil and groundwater from fertigated sugarcane crop areas using target and suspect screening strategies.

This work evaluated for the first time the sustainability of vinasse reuse as a fertilizer in sugarcane crops by assessing the occurrence of organic contaminants and their potential for dissemination to soils and groundwater in fertigated areas. A comprehensive screening of organic contaminants was performed in vinasse, soil and groundwater using target analysis, to investigate the occurrence of multiple-class antibiotics, in combination with suspect screening using NORMAN Digital Sample Freezing Platform. Even though antibiotics are used in the ethanol production process and were expected to be ubiquitous contaminants, they were not detected in any of the samples. Nevertheless, the HRMS-based wide-scope suspect screening (including >7800 substances such as pharmaceuticals, agrochemicals, preservatives and industrial chemicals) allowed the tentative identification of 56 compounds, mostly pesticides, food additives, industrial and naturally occurring substances. Results showed no overlap between the compounds detected in vinasse and environmental samples, suggesting that the pollutants found in soil and groundwater might come from alternative sources other than vinasse reuse.

Pressurized Liquid Extraction (PLE) and QuEChERS evaluation for the analysis of antibiotics in agricultural soils.

Vinasse, a liquid waste which originates from the production of ethanol fuel from sugarcane, has been widely used as soil amendment in Brazil. An important concern that arises from vinasse reuse is the dissemination of antibiotics to the environment through crop soils. This work evaluated the performance of Pressurized Liquid Extraction (PLE) and QuEChERS (quick, easy, cheap, effective, rugged and safe) to extract several multiple-class antibiotics, such as cephalosporins, fluoroquinolones, ionophores, lincosamides, macrolides, quinolones, streptogramin, sulfonamides, tetracyclines and others, from agricultural soils. The performance of several parameters was evaluated for both PLE and QuEChERS, such as the extraction temperature (for PLE), solvents composition, pH and the addition of EDTA. Both methods were able to extract most target antibiotics. However, QuEChERS showed higher recoveries for macrolides and nitroimidazoles, while PLE was more suitable for fluoroquinolones and ionophores (i.e. monensin). The use of citrate-phosphate buffer at pH 7.0, in combination with methanol for PLE and with acetonitrile for QuEChERS, provided the highest antibiotic recoveries for both methods. The use of EDTA did not increase antibiotic recovery rates for QuEChERS, while the temperature had almost no influence on the extraction efficiency in PLE.•Citrate-phosphate buffer at pH 7.0 provided higher antibiotic recoveries for QuEChERS and PLE.•The combination buffer-methanol provided higher recoveries for PLE.•QuEChERS and PLE methods were able to extract most of the target antibiotics.

Occurrence and assessment of environmental risks of endocrine disrupting compounds in drinking, surface and wastewaters in Serbia.

The present study is the first comprehensive monitoring of 13 selected endocrine disrupting compounds (EDCs) in untreated urban and industrial wastewater in Serbia to assess their impact on the Danube River basin and associated freshwaters used as sources for drinking water production in the area. Results showed that natural and synthetic estrogens were present in surface and wastewater at concentrations ranging from 0.1 to 64.8 ng L-1. Nevertheless, they were not detected in drinking water. For alkylphenols concentrations ranged from 1.1 to 78.3 ng L-1 in wastewater and from 0.1 to 37.2 ng L-1 in surface water, while in drinking water concentrations varied from 0.4 to 7.9 ng L-1. Bisphenol A (BPA) was the most abundant compound in all water types, with frequencies of detection ranging from 57% in drinking water, to 70% in surface and 84% in wastewater. Potential environmental risks were characterized by calculating the risk quotients (RQs) and the estrogenic activity of EDCs in waste, surface and drinking water samples, as an indicator of their potential detrimental effects. RQ values of estrone (E1) and estradiol (E2) were the highest, exceeding the threshold value of 1 in 60% of wastewater samples, while in surface water E1 displayed potential risks in only two samples. Total estrogenic activity (EEQt) surpassed the threshold of 1 ng E2 L-1 in about 67% of wastewater samples, and in 3 surface water samples. In drinking water, EEQt was below 1 ng L-1 in all samples.

Pharmaceuticals in source separated sanitation systems: Fecal sludge and blackwater treatment.

This study investigated, for the first time, the occurrence and fate of 29 multiple-class pharmaceuticals (PhACs) in two source separated sanitation systems based on: (i) batch experiments for the anaerobic digestion (AD) of fecal sludge under mesophilic (37 °C) and thermophilic (52 °C) conditions, and (ii) a full-scale blackwater treatment plant using wet composting and sanitation with urea addition. Results revealed high concentrations of PhACs in raw fecal sludge and blackwater samples, with concentrations up to hundreds of µg L-1 and µg kg-1 dry weight (dw) in liquid and solid fractions, respectively. For mesophilic and thermophilic treatments in the batch experiments, average PhACs removal rates of 31% and 45%, respectively, were observed. The average removal efficiency was slightly better for the full-scale blackwater treatment, with 49% average removal, and few compounds, such as atenolol, valsartan and hydrochlorothiazide, showed almost complete degradation. In the AD treatments, no significant differences were observed between mesophilic and thermophilic conditions. For the full-scale blackwater treatment, the aerobic wet composting step proved to be the most efficient in PhACs reduction, while urea addition had an almost negligible effect for most PhACs, except for citalopram, venlafaxine, oxazepam, valsartan and atorvastatin, for which minor reductions (on average 25%) were observed. Even though both treatment systems reduced initial PhACs loads considerably, significant PhAC concentrations remained in the treated effluents, indicating that fecal sludge and blackwater fertilizations could be a relevant vector for dissemination of PhACs into agricultural fields and thus the environment.

Pharmaceuticals removal in an on-farm pig slurry treatment plant based on solid-liquid separation and nitrification-denitrification systems.

The fate and degradation of 28 multiple-class veterinary pharmaceuticals in an on-farm pig slurry treatment plant based on solid-liquid separation and a nitrification-denitrification (NDN) sequence batch reactor (SBR) were evaluated for the first time. The pharmaceuticals detected at the highest concentrations in raw pig slurries belonged to the group of tetracycline antibiotics. Fluoroquinolone, lincosamide and pleuromutilin antibiotics and other drugs such as flubendazole and flunixin were also frequently detected. After solid-liquid separation, target compounds were distributed in an average of 64% onto the liquid fraction. Pharmaceuticals distributed in this fraction were removed in an average of almost 50% after being treated in NDN-SBR. Lincomycin was the compound with the highest removal percentage, reaching 100% reduction, while tetracyclines and fluoroquinolones showed moderate removal percentages (50 and 40%, respectively). Regarding nitrogen removal, NDN-SBR reduced a 77% of the content of this nutrient in the liquid slurry fraction.

Fate of pharmaceuticals and antibiotic resistance genes in a full-scale on-farm livestock waste treatment plant.

This study investigated, for the first time, the distribution and fate of 28 multiple-class veterinary pharmaceuticals and antibiotics (PhACs), and their corresponding antibiotic resistance genes (ARGs), in a full-scale on-farm livestock waste treatment plant. The plant relies on several technologies, including: anaerobic digestion (AD), solid-liquid separation, and two stages reverse osmosis (RO) of the liquid digestate. Tetracycline, fluoroquinolone, lincosamide and pleuromutilin antibiotics, together with anti-helmintic (flubendazole) and anti-inflammatory (flunixin) drugs were the most frequently detected compounds in livestock waste and in slaughterhouse sludge. This last fraction is used as co-substrate in the AD process and showed to be an important input source of PhACs and ARGs. In terms of treatment performance, AD exhibited moderate to low PhACs and ARGs reduction, while a large fraction (<50%) of the PhACs present in the digestate were distributed onto the solid fraction, after solid-liquid separation. Both solid and liquid digestates had relatively high copy numbers of ARGs. Finally, RO showed high rejection percentages for all PhACs (<90%), with concentrations in the low ng L-1 range in permeates, for most target PhACs. Nevertheless, moderate copy numbers of ARGs were detected in permeates.

Veterinary pharmaceuticals and antibiotics in manure and slurry and their fate in amended agricultural soils: Findings from an experimental field site (Baix Empordà, NE Catalonia).

The fate and transport of 34 veterinary pharmaceuticals (PhACs) is investigated in swine slurry and dairy cattle manure-amended agricultural soils, from an experimental field site, by using both analytical and modelled data. Potential differences on PhACs fate, attributed to the application of distinct swine slurry fractions (total, solid, and liquid), are herein assessed for the first time. Surface and deep soil layers, up to a depth of 120 cm, were analyzed at different periods after an annual fertilization event. Using input data representing typical agricultural soil conditions and the PhACs concentration measured in organic fertilizers the transport of these pollutants was modelled for a period of 10 years, including the monitored annual fertilization event. Fluoroquinolone, tetracycline and pleuromutilin antibiotics, together with anti-helmintics and analgesic and anti-inflammatories, were detected in manure-amended soils, at average concentrations ranging from 0.078 to 150 µg/kg dw in surface layers, with the highest levels found in the fields fertilized with the swine slurry solid fraction. Even though severe disagreements were observed between experimental and simulated PhACs concentrations along the soil column, both approaches pointed out that target compounds strongly adsorb onto surface layers, showing limited mobility along the soil profile. Thus, repeated manure and slurry fertilizations will contribute in building up persistent PhACs residues in the uppermost layers of the soil, while leaching will be a minor process governing their fate towards the subsurface. The ecotoxicological risks posed by the occurrence of PhACs in soils were estimated to be low for terrestrial organisms. Nevertheless the antibiotic enrofloxacin showed some potential to induce negative effects to crops.

Critical review: Grand challenges in assessing the adverse effects of contaminants of emerging concern on aquatic food webs.

Much progress has been made in the past few decades in understanding the sources, transport, fate, and biological effects of contaminants of emerging concern (CECs) in aquatic ecosystems. Despite these advancements, significant obstacles still prevent comprehensive assessments of the environmental risks associated with the presence of CECs. Many of these obstacles center around the extrapolation of effects of single chemicals observed in the laboratory or effects found in individual organisms or species in the field to impacts of multiple stressors on aquatic food webs. In the present review, we identify 5 challenges that must be addressed to promote studies of CECs from singular exposure events to multispecies aquatic food web interactions. There needs to be: 1) more detailed information on the complexity of mixtures of CECs in the aquatic environment, 2) a greater understanding of the sublethal effects of CECs on a wide range of aquatic organisms, 3) an ascertaining of the biological consequences of variable duration CEC exposures within and across generations in aquatic species, 4) a linkage of multiple stressors with CEC exposure in aquatic systems, and 5) a documenting of the trophic consequences of CEC exposure across aquatic food webs. We examine the current literature to show how these challenges can be addressed to fill knowledge gaps. Environ Toxicol Chem 2019;38:46-60. © 2018 SETAC.

Pharmaceuticals as chemical markers of wastewater contamination in the vulnerable area of the Ebro Delta (Spain).

This study evaluated the occurrence and distribution of 81 pharmaceutically active compounds (PhACs) in the vulnerable area of the Ebro Delta region (Catalonia, Spain), to assess the environmental impact of wastewater treatment plants discharge to coastal environments. The occurrence of PhACs was followed along the wastewater-recipient water-sediment chain until they reach estuaries and the Mediterranean Sea. Water and sediment samples were collected in an integrated way at different sampling points covering three different seasons in reaches of the Ebro River located upstream and downstream from wastewater treatment plants (WWTPs), surrounding channels, estuaries, and the associated receiving seawater. 28 out of the 57 compounds detected in effluent wastewater were positively identified in estuary and seawaters, revealing that WWTP discharges are an important source of contamination in coastal environments and that PhACs are suitable markers of urban contamination in these areas. The substances with the highest frequency of detection belonged to the groups of analgesics/anti-inflammatories (acetaminophen, salicylic acid), antihypertensives (valsartan), psychiatric drugs (carbamazepine), and antibiotics (clarithromycin, trimethoprim). In general, a decrease in concentration was observed from inland sampling points towards the Mediterranean Sea, resulting from a dilution in the recipient marine water bodies. A reduced number of PhACs, at concentrations ranging from 0.1 to 12.5 ng g-1 dry weight (d.w.) was detected in sediment samples, indicating that sorption is a minor natural attenuation pathway for these compounds. Finally, a prioritization strategy, based on the compounds concentration and frequency of detection in seawater, removal efficiency in WWTP, bioaccumulation potential, toxicity to marine organisms and persistency, was used to highlight the PhACs of major ecological concern and that could be used as relevant indicators of wastewater contamination in coastal environments.

Mass fluxes per capita of organic contaminants from on-site sewage treatment facilities.

This study is the first attempt to quantify environmental fluxes per capita of organic contaminants discharged from on-site sewage treatment facilities (OSSFs) in affected recipients. Five sites were monitored around the River Fyris in Sweden: three mainly affected by OSSFs and two mainly affected by municipal sewage treatment plants (STPs). Gas chromatography-mass spectrometry was used to determine environmental concentrations of 30 anthropogenic contaminants, including organophosphorus compounds, rubber and plastic additives, UV stabilizers, fragrances, surfactant ingredients and polycyclic aromatic hydrocarbons. Uni- and multivariate statistical analysis of the most frequently detected contaminants showed that median fluxes per capita of tris(1,3-dichloro-2-propyl) phosphate, tris(1-chloro-2-propyl) phosphate, tris(2-chloroethyl) phosphate, and n-butylbenzene sulfonamide were similar at OSSF and STP sites, but the mass fluxes per capita of tris-(2-butoxyethyl) phosphate, 2-(methylthio)benzothiazole, and galaxolide, were significantly lower (∼2-3-fold) at OSSF sites than at STP sites (Mann-Whitney, α = 0.05). Differences between these sites were larger in samples collected in summer and autumn than in samples collected in winter. Deviations likely originated from differences in fate processes and distances between source and sampling sites. Further studies are needed to characterize mass fluxes per capita of contaminants in waters that directly receive discharges from OSSFs.

Potential of biochar filters for onsite sewage treatment: Adsorption and biological degradation of pharmaceuticals in laboratory filters with active, inactive and no biofilm.

This study investigated the potential of biochar filters as a replacement or complement for sand filters for removal of pharmaceutically active compounds (PhACs) from wastewater in onsite sewage facilities (OSSF). Specifically, the study investigated the effects of biodegradation, adsorption and a combination of these processes on removal of four model PhACs from wastewater in biochar filters operated under hydraulic loading conditions mimicking those found in onsite infiltration beds. Concentrations and removal of the four PhACs (i.e. carbamazepine, metoprolol, ranitidine and caffeine) were investigated over 22weeks in four treatments: biochar (BC) with active or inactive biofilm (BC-active-biofilm, BC-inactive-biofilm), biochar without biofilm (BC-no-biofilm) and sand with active biofilm (Sand-active-biofilm). The adsorption of carbamazepine was high in BC-no-biofilm (99% removal after 22weeks), while biodegradation was very low in Sand-active-biofilm (7% removal after 22weeks). Removal of carbamazepine in BC-active-biofilm was high and stable over the 22weeks (>98%), showing a significant role of biofilm in filter biogeneration. However, carbamazepine removal declined over time in BC-inactive-biofilm, from 99% in week 13 to 73% in week 22. Metoprolol was poorly degraded in Sand-active-biofilm (37% after 22weeks), while adsorption seemed to be the major pathway for removal of metoprolol in biochar. Ranitidine and caffeine were efficiently removed by either adsorption (97% and 98%, respectively, after 22weeks) or biodegradation (99% and >99%, respectively, after 22weeks). In conclusion, biochar is a promising filter medium for OSSF, especially for persistent PhACs such as carbamazepine and metoprolol.