Biotransformation Studies Research Articles

Biotransforming the "Forever Chemicals": Trends and Insights from Microbiological Studies on PFAS.

Per- and polyfluoroalkyl substances (PFAS) are recalcitrant contaminants of emerging concern. Research efforts have been dedicated to PFAS microbial biotransformation in the hopes of developing treatment technologies using microorganisms as catalysts. Here, we performed a meta-analysis by extracting and standardizing quantitative data from 97 microbial PFAS biotransformation studies and comparing outcomes via statistical tests. This meta-analysis indicated that the likelihood of PFAS biotransformation was higher under aerobic conditions, in experiments with defined or axenic cultures, when high concentrations of PFAS were used, and when PFAS contained fewer fluorine atoms in the molecule. This meta-analysis also documented that PFAS biotransformation depends on chain length, chain branching geometries, and headgroup chemistry. We found that the literature is scarce or lacking in (i) anaerobic PFAS biotransformation experiments with well-defined electron acceptors, electron donors, carbon sources, and oxidation-reduction potentials, (ii) analyses of PFAS biotransformation products, and (iii) analyses to identify microorganisms and enzymes responsible for PFAS biotransformation. To date, most biotransformation research emphasis has been on 8:2 fluorotelomer alcohol (8:2 FTOH), 6:2 fluorotelomer alcohol (6:2 FTOH), perfluorooctanesulfonic acid (PFOS), and perfluorooctanoic acid (PFOA). A wide array of PFAS remains to be tested for their potential to biotransform.

Isolation, identification and mutants generation of isolate 04 for its improved bioconversion of ferulic acid to vanillin

Vanillin is widely used as a food flavouring, scent ingredient in cosmetics and perfumes, as an intermediate in agrochemicals & medicines and also as a nutraceutical. Serial dilution of sediment sample yielded 32 strains, among which three isolates A2, A7, and 04 reported here were characterized at molecular level by 16S rDNA sequencing. They were identified and similar to Pseudomonas aeruginosa (94.97%), Acinetobacter baumannii (99.93%) and Lysinibacillus macroides (98.67%) respectively based on its 16S rDNA sequences. Among these three strains, isolate 04 was with high ferulic acid (FA) to vanillin bioconversion activity. Vanillin concentration was determined by the Thiobarbituric acid (TBA) method and analysed by UV-spectrophotometry, which showed 11.63 ± 0.05 mg/ml vanillin after optimization studies. HPLC with UV detector was also used for the characterization of the vanillin formed, which showed peak at 8.793 rt. To improve FA to vanillin bioconversion, physical mutagenesis was performed on isolate 04, which generated three mutants (1, 2, and 3). These three mutants were subject to FA to vanillin biotransformation study. TBA method and UV spectrophotometric analysis showed a higher conversion by mutant 2 than other two mutants, 1 and 3. Mutant 2 formed almost two-fold vanillin i.e. 19.78 ± 0.05 mg/ml when compared to its wild strain, isolate 04. Hence, this mutant may be further subjected to optimization studies and exploited for industrial biotransformation process.

An engineered dual-functional L-DOPA decarboxylase enables a minimized hydroxytyrosol cascade

An engineered dual-functional L-DOPA decarboxylase enables a minimized hydroxytyrosol cascade

Turkish Astragalus Species: Botanical Aspects, Secondary Metabolites, and Biotransformation.

Astragalus is a widespread genus comprising approximately 3500 species, both annual and perennial, found across Asia, Europe, Africa, and the Americas. In Turkey, it is represented by 63 sections and 485 taxa with a high endemism ratio (51%). In traditional medicine, the roots of various Astragalus species represent very old and well-known drugs used for antiperspirant, diuretic, and tonic purposes, as well as for the treatment of nephritis, diabetes, leukemia, and uterine cancer. The genus Astragalus is the richest source of cycloartane-type compounds, which display a diverse range of bioactivities, such as wound healing, immunomodulatory, antitumor, hepatoprotective, antimutagenic, antiviral, and antiprotozoal activities. Moreover, cycloastragenol, the main sapogenol of many cycloartane-type glycosides found in the Astragalus genus, has gained attention as a potent telomerase activator over the past decade. The preparation of cycloastragenol derivatives could be significant in the near future due to their unique bioactivity. This review covers the botanical aspects of Astragalus L., as well as the phytochemical and biological activity studies conducted on Turkish Astragalus species, with a special focus on cycloartenols. It contains 36 articles reporting the phytochemistry of 29 Astragalus species and 111 new compounds, including 104 triterpene saponins. In addition to the phytochemical studies, this review summarizes the biotransformation studies on Astragalus cycloartanes via endophytic fungi isolated from the tissues of Astragalus species.

A Brief Review of Radiolabelling Nucleic Acid-Based Molecules for Tracking and Monitoring.

The rise of nucleic acid-based therapeutics continues apace. At the same time, the need for radiolabelled oligonucleotides for determination of spatial distribution is increasing. Complex molecular structures with mostly multiple charges and low solubility in organic solvents increase the challenge of integrating radionuclides. In preclinical research, it is important to understand the fate of new drug candidates in biodistribution studies, target binding or biotransformation studies. Depending on a specific question, the selection of a respective radiolabelling strategy is crucial. Radiometals for molecular imaging with positron emission tomography or single-photon computed tomography generally require an attached chelating agent for stable complexation of the metal with the oligonucleotide, whereas labelling using carbon-11/-14 or tritium allows incorporation of the radioisotope into the native structure without altering it. Moreover, the suitability of direct radiolabelling of the oligonucleotide of interest or indirect radiolabelling, for example, by a two-step pretargeting approach, for the study design requires consideration. This review focuses on the challenges of radiolabelling nucleic acid-based molecules with beta-plus, gamma and beta-minus emitters and their use for tracking and monitoring.

Structure-Metabolism Relationships of Benzimidazole Derivatives with anti-Trypanosoma cruzi Activity for Chagas Disease.

This study introduces further insights from the hit-to-lead optimization process involving a series of benzimidazole derivatives acting as inhibitors of the cruzain enzyme, which targets Trypanosoma cruzi, the causative parasite of Chagas disease. Here, we present the design, synthesis and biological evaluation of 30 new compounds as a third generation of benzimidazole analogues with trypanocidal activity, aiming to enhance our understanding of their pharmacokinetic profiles and establish a structure-metabolism relationships within the series. The design of these new analogues was guided by the analysis of previous pharmacokinetic results, considering identified metabolic sites and biotransformation studies. This optimization resulted in the discovery of two compounds (42 e and 49 b) exhibiting enhanced metabolic stability, anti-Trypanosoma cruzi activity compared to benznidazole (the reference drug for Chagas disease), as well as being non-cruzain inhibitors, and demonstrating a satisfactory in vitro pharmacokinetic profile. These findings unveil a new subclass of aminobenzimidazole and rigid compounds, which offer potential for further exploration in the quest for discovering novel classes of antichagasic compounds.

Elucidating the potential role of microorganisms in postmortem biotransformation: a comparison of clonazolam and its metabolite in postmortem and DUID cases.

Clonazolam is a designer triazolobenzodiazepine first synthesized in 1971 and is primarily used for its anxiolytic and sedative effects. It became a drug of misuse in 2012 and is known for its high potency and long duration of effect. Previous studies of nitrobenzodiazepines, such as nitrazepam, clonazepam, and flunitrazepam, as well as their metabolites, have demonstrated that bacterial species native to the gastrointestinal tract and active during postmortem (PM) decomposition are capable of affecting positivity and compound-to-metabolite ratios. Further studies have not been performed with clonazolam; however, it possesses the nitro functional group necessary for this biotransformation. To understand whether clonazolam may be similarly affected, PM cases (n = 288) and driving under the influence of drugs (DUID, n = 54) cases, positive for 8-aminoclonazolam reported by NMS Laboratories from 2020 to 2023, were selected for inclusion in this study. Concentrations of clonazolam and 8-aminoclonazolam were evaluated, and concurrent identification of parent drugs and their metabolites occurred less frequently in PM cases (n = 1, 0.30% of cases) than in DUID cases (n = 21, 38% of cases). The clonazolam concentration in one PM case was 13 ng/mL. In DUID cases, the median clonazolam concentration was 4.0 ng/mL and ranged from 2.0 to 10 ng/mL. 8-Aminoclonazolam had median concentrations of 13 and 19 ng/mL, with ranges 2.0-580 and 2.8-59 ng/mL for PM and DUID cases, respectively. Due to the ever-changing landscape of the designer benzodiazepine market, in vitro studies of PM microbial biotransformation of clonazolam are unavailable. The data reported herein provide valuable information in the absence of such studies and represent an alternative method of investigating this phenomenon as a potential cause of parent nitrobenzodiazepine to metabolite conversion.

Biotransformation of rifampicin by Aspergillus niger and antimicrobial activity of proposed metabolites.

Drug biotransformation studies emerges as an alternative to pharmacological investigations of metabolites, development of new drug candidates with reduced investment and most efficient production. The present study aims to evaluate the capacity of biotransformation of rifampicin by the filamentous fungus Aspergillus niger ATCC 9029. After incubation for 312 h, the drug was metabolized to two molecules: an isomer (m/z 455) and the rifampicin quinone (m/z 821). The monitoring of metabolite formation was performed by high-performance liquid chromatography, followed by their identification through ultra-high-performance liquid chromatography coupled to tandem mass spectrometer. In vitro antimicrobial activity of the proposed metabolites was evaluated against Staphylococus aureus microorganism, resulting in the loss of inhibitory activity when compared with the standards, with minimum inhibitory concentration of 7.5μg/ml. The significant biotransformation power of the ATCC 9029 strain of A. niger was confirmed in this study, making this strain a candidate for pilot studies in fermentation tanks for the enzymatic metabolization of the antimicrobial rifampicin. The unprecedented result allows us to conclude that the prospect of new biotransforming strains in species of anemophilic fungi is a promising choice.

Miniaturized Extraction Techniques in Drug Biotransformation Studies by using Endophytic Fungi Cunninghamella elegans

In addition to predicting biotransformation in humans, drug biotransformation studies are important because they can generate active metabolites or new intermediates with possible use by the pharmaceutical industry. Endophytic fungi of the genus Cunninghamella can metabolize many drugs in a similar way to humans. The analysis of these metabolites requires prior treatment of the samples in order to obtain compatibility with the detection system and the separation technique. This work aimed to study the biotransformation of the drugs duloxetine (DLX), citalopram (CIT) and amlodipine (ANL) by endophytic fungi Cunninghamella elegans ATCC 9245, the development of micro extraction methods in the context of green chemistry, and the validation of the analytical methods for drugs and their respective metabolites. Bioanalytical method by HPLC was successfully developed and validated for both drugs (DLX, CIT and ANL). The metabolites of DLX and CIT obtained by biotransformation studies were not detected in the conditions of this study, but it was possible to visualize one additional peak in the chromatogram analysis of ANL indicate that this drug has been metabolized by Cunninghamella elegans. Furthermore, it has been developed two liquid-liquid micro extraction methods achieving success by removing drugs in both techniques, with recovery greater than 90%. The use of Quality by Design (QbD) through the experimental design provided the best conditions for carrying out biotransformation and micro extraction studies of drugs from the fungal matrix.

Efficient Bio-Oxidation of Cellobiose with Engineered Gluconobacter oxydans to Provide Highly Concentrated Cellobionic Acid

Cellobionic acid (CBA) can be obtained through the oxidation of cellobiose, the monomer of cellulose. CBA serves as a plant-based alternative to its stereoisomer lactobionic acid, which is used in the pharmaceutical, cosmetic, and food industries. Gluconobacter oxydans is a well-established whole-cell biocatalyst with membrane-bound dehydrogenases (mDH) for regio-specific oxidations. As G. oxydans wildtype cells show low cellobiose oxidation activities, the glucose mDH from Pseudomonas taetrolens was overexpressed in G. oxydans BP9, a multi mDH deletion strain. Whole-cell biotransformation studies were performed with resting cells of the engineered G. oxydans in stirred tank bioreactors. Initial biomass specific cellobionate formation rates increased with increasing cellobiose concentrations up to 190 g L−1, and were constant until the solubility limit. The maximal volumetric CBA formation rates and the oxygen uptake rates increased linearly with the concentration of engineered G. oxydans. This enables the estimation of the maximum biocatalyst concentration limited by the maximum oxygen transfer rate of any bioreactor. Thus, 5.2 g L−1 G. oxydans was sufficient to produce 502 g L−1 CBA with >99% yield in a simple aerobic batch process. The highly concentrated CBA will reduce downstream processing costs considerably after cell separation.

Identification and synthesis of metabolites of the new 4.5-dihydroisoxazol-5-carboxamide derivate

Introduction: 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4-methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2-oxazole-5-carboxamide is new antirheumatic drug. It is necessary to identify and synthesize the biotransformation products for its complete pharmacokinetic study. Materials and Methods: A biotransformation study was carried out by intraperitoneal administration of the drug to Wistar rats and Soviet Chinchilla breed rabbits. Animal blood sampling was performed before the injection and 0.5 h, 1 h, 2 h, 4 h, 24 h after the injection of the investigated compound. The samples were immediately centrifuged for plasma separation. Urine was simultaneously collected from rats before the administration and at intervals of 0-2 h, 2-4 h, 4-6 h, 6-24 h after administration, faeces – before administration and at intervals of 0-12 h and 12-24 h after administration. The samples were analyzed by HPLC-MS/MS after immediate preparation by adding acetonitrile. Results and Discussion: 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-4,5-dihydro-1,2-oxazole-5-carboxylic acid and 4-methoxy-3-(trifluoromethyl)aniline – hydrolysis products of the active substance were found during the analysis of plasma, urine and fecal samples. The 4,5-dihydro-1,2-oxazole-5-carboxylic acid derivative has been synthesized. The second metabolite is a raw material for production of active pharmaceutical substance. During comparative tests, no significant difference between the retention times, ratio areas of chromatographic peaks at the main MRM-transitions and mass spectra of these metabolites on chromatograms of standard and animal samples was found, which indicates the correct identification of biotransformation products. Conclusion: The studied drug undergoes biotransformation by hydrolysis to form two main metabolites: 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-4,5-dihydro-1,2-oxazole-5-carboxylic acid and 4-methoxy-3-(trifluoromethyl)aniline. The structure of the metabolites was confirmed by comparison with the synthesized standard samples using HPLC-MS/MS.

Comparative study of trastuzumab modification analysis using mono/multi-epitope affinity technology with LC-QTOF-MS

Comparative study of trastuzumab modification analysis using mono/multi-epitope affinity technology with LC-QTOF-MS

Antimicrobial Properties of Flavonoid Derivatives with Bromine, Chlorine, and Nitro Group Obtained by Chemical Synthesis and Biotransformation Studies.

The search for new substances of natural origin, such as flavonoids, is necessary in the fight against the growing number of diseases and bacterial resistance to antibiotics. In our research, we wanted to check the influence of flavonoids with chlorine or bromine atoms and a nitro group on pathogenic and probiotic bacteria. We synthesized flavonoids using Claisen-Schmidt condensation and its modifications, and through biotransformation via entomopathogenic filamentous fungi, we obtained their glycoside derivatives. Biotransformation yielded two new flavonoid glycosides: 8-amino-6-chloroflavone 4'-O-β-D-(4″-O-methyl)-glucopyranoside and 6-bromo-8-nitroflavone 4'-O-β-D-(4″-O-methyl)-glucopyranoside. Subsequently, we checked the antimicrobial properties of the aforementioned aglycon flavonoid compounds against pathogenic and probiotic bacteria and yeast. Our studies revealed that flavones have superior inhibitory effects compared to chalcones and flavanones. Notably, 6-chloro-8-nitroflavone showed potent inhibitory activity against pathogenic bacteria. Conversely, flavanones 6-chloro-8-nitroflavanone and 6-bromo-8-nitroflavanone stimulated the growth of probiotic bacteria (Lactobacillus acidophilus and Pediococcus pentosaceus). Our research has shown that the presence of chlorine, bromine, and nitro groups has a significant effect on their antimicrobial properties.

Using Network Analysis and Predictive Functional Analysis to Explore the Fluorotelomer Biotransformation Potential of Soil Microbial Communities.

Microbial transformation of per- and polyfluoroalkyl substances (PFAS), including fluorotelomer-derived PFAS, by native microbial communities in the environment has been widely documented. However, few studies have identified the key microorganisms and their roles during the PFAS biotransformation processes. This study was undertaken to gain more insight into the structure and function of soil microbial communities that are relevant to PFAS biotransformation. We collected 16S rRNA gene sequencing data from 8:2 fluorotelomer alcohol and 6:2 fluorotelomer sulfonate biotransformation studies conducted in soil microcosms under various redox conditions. Through co-occurrence network analysis, several genera, including Variovorax, Rhodococcus, and Cupriavidus, were found to likely play important roles in the biotransformation of fluorotelomers. Additionally, a metagenomic prediction approach (PICRUSt2) identified functional genes, including 6-oxocyclohex-1-ene-carbonyl-CoA hydrolase, cyclohexa-1,5-dienecarbonyl-CoA hydratase, and a fluoride-proton antiporter gene, that may be involved in defluorination. This study pioneers the application of these bioinformatics tools in the analysis of PFAS biotransformation-related sequencing data. Our findings serve as a foundational reference for investigating enzymatic mechanisms of microbial defluorination that may facilitate the development of efficient microbial consortia and/or pure microbial strains for PFAS biotransformation.

Production of geraniol and geranic acid: High value compounds of citral biosynthetic pathway from lemongrass oil using Aspergillus niger AUD-C2 as catalyst

Microbial biotransformation of lemongrass oil to geraniol and geranic acid, high value compounds of citral biosynthetic pathway has been reported. Biotransformation studies were carried out using a fungus Aspergillus niger strain AUD-C2 isolated from the rhizospheric soil of lemongrass, as catalyst. Three different methodologies using culture broth, spore suspension and cell-free extract, have been utilized for biotransformation and successfully demonstrated at litre scale in bioreactors. Overall 55% geraniol and 75% geranic acid were obtained by biotransformation of lemongrass oil in 48 h using spore suspension and cell free extract, respectively.

Biotransformation of 6:2 fluorotelomer sulfonate and microbial community dynamics in water-saturated one-dimensional flow-through columns

Biotransformation of 6:2 fluorotelomer sulfonate and microbial community dynamics in water-saturated one-dimensional flow-through columns

Potential of measured relative shifts in collision cross section values for biotransformation studies.

Ion mobility spectrometry-mass spectrometry (IMS-MS) separates gas phase ions due to differences in drift time from which reproducible and analyte-specific collision cross section (CCS) values can be derived. Internally conducted in vitro and in vivo metabolism (biotransformation) studies indicated repetitive shifts in measured CCS values (CCSmeas) between parent drugs and their metabolites. Hence, the purpose of the present article was (i)to investigate if such relative shifts in CCSmeas were biotransformation-specific and (ii) tohighlight their potential benefits for biotransformation studies. First, mean CCSmeas values of 165 compounds were determined (up to n = 3) using a travelling wave IMS-MS device with nitrogen as drift gas (TWCCSN2, meas). Further comparison with their predicted values (TWCCSN2, pred, Waters CCSonDemand) resulted in a mean absolute error of 5.1%. Second, a reduced data set (n = 139) was utilized to create compound pairs (n = 86) covering eight common types of phase I and II biotransformations. Constant, discriminative, and almost non-overlapping relative shifts in mean TWCCSN2,meas were obtained for demethylation (- 6.5 ± 2.1 Å2), oxygenation (hydroxylation + 3.8 ± 1.4 Å2, N-oxidation + 3.4 ± 3.3 Å2), acetylation (+ 13.5 ± 1.9 Å2), sulfation (+ 17.9 ± 4.4 Å2), glucuronidation (N-linked: + 41.7 ± 7.5 Å2, O-linked: + 38.1 ± 8.9 Å2), and glutathione conjugation (+ 49.2 ± 13.2 Å2). Consequently, we propose to consider such relative shifts in TWCCSN2, meas (rather than absolute values) as well for metabolite assignment/confirmation complementing the conventional approach to associate changes in mass-to-charge (m/z) values between a parent drug and its metabolite(s). Moreover, the comparison of relative shifts in TWCCSN2, meas significantly simplifies the mapping of metabolites into metabolic pathways as demonstrated.

Uptake and Biotransformation of the Tire Rubber-derived Contaminants 6-PPD and 6-PPD Quinone in the Zebrafish Embryo (Danio rerio).

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD) is a widely used antioxidant in tire rubber known to enter the aquatic environment via road runoff. The associated transformation product (TP) 6-PPD quinone (6-PPDQ) causes extreme acute toxicity in some fish species (e.g., coho salmon). To interpret the species-specific toxicity, information about biotransformation products of 6-PPDQ would be relevant. This study investigated toxicokinetics of 6-PPD and 6-PPDQ in the zebrafish embryo (ZFE) model. Over 96 h of exposure, 6-PPD and 6-PPDQ accumulated in the ZFE with concentration factors ranging from 140 to 2500 for 6-PPD and 70 to 220 for 6-PPDQ. A total of 22 TPs of 6-PPD and 12 TPs of 6-PPDQ were tentatively identified using liquid chromatography coupled to high-resolution mass spectrometry. After 96 h of exposure to 6-PPD, the TPs of 6-PPD comprised 47% of the total peak area (TPA), with 4-hydroxydiphenylamine being the most prominent in the ZFE. Upon 6-PPDQ exposure, >95% of 6-PPDQ taken up in the ZFE was biotransformed, with 6-PPDQ + O + glucuronide dominating (>80% of the TPA). Among other TPs of 6-PPD, a reactive N-phenyl-p-benzoquinone imine was found. The knowledge of TPs of 6-PPD and 6-PPDQ from this study may support biotransformation studies in other organisms.

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies.

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising "humanised" in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine.

Finding a way out? Comprehensive biotransformation study of novel fluorinated surfactants

In this study the environmental fate of two novel trifluoromethoxy-substituted surfactants with respectively an ether or thioether linkage were investigated, of which the design aimed for less persistency and complete mineralization. Long-term microbial transformation studies under aerobic conditions in activated sludge-wastewater medium were performed for 126 days. A semi-closed experimental system with a trapping sorbent was selected to avoid losses of possible volatile transformation products (TPs). The changes in the concentration of the surfactants and their expected TPs were monitored by target analysis using liquid chromatography–tandem mass spectrometry. Significant decrease in the concentration of the surfactants was observed over the incubation period. The main detected TPs were short-chained carboxylic acids (CAs), including a CA with two fluorinated carbon atoms representing the last product prior to mineralization. High stability of these CAs and lack in the formation of inorganic fluoride over the incubation time was however observed. Consequently, unequivocal final mineralization of the investigated surfactants could not be confirmed. Regarding the mass balance, the total amount of detected substances achieved only 30–37% of the expected concentration at the end of the incubation time. The reason of the incomplete mass balance should be further investigated.