Determination of the targeted carbendazim metabolites in zebrafish water tank by liquid chromatography coupled to high-resolution mass spectrometry

ABSTRACT Carbendazim, a common pesticide, has shown signs of causing cancer, infertility and toxicity to organisms. Due to its intense use, this fungicide has become a persistent compound in the environment, raising the importance of better understanding its behaviour, metabolic pathways and effects on organisms. Zebrafish is considered an excellent animal model, being able to rapidly absorb compounds in water, mimicking what occurs in the aquatic environment. Therefore, the aim of this work was to evaluate carbendazim metabolites in zebrafish water tank using liquid chromatography coupled to a high-resolution mass spectrometer (LC-HRMS) to highlight analytical targets in order to monitor carbendazim exposure in aquatic environments. For this purpose, treatment, negative and stability groups were defined. In addition, water samples were collected from the tanks and analyses were carried out by LC-HRMS. The results allowed the putative annotation of 7 target metabolites. This study applied the analysis of zebrafish water tank for evaluation of target metabolites of carbendazim as a promising approach, since it is a much cleaner matrix than the usual biological matrices. These metabolites can ensure detectability and further strengthen confidence in monitoring carbendazim exposure in aquatic environments. To the best of our knowledge, there is no one study that has evaluated carbendazim metabolites produced by zebrafish, neither in the animal’s body nor in the water tank. This is the first report on the generation of carbendazim metabolites by zebrafish.


Introduction
Carbendazim is a broad-spectrum fungicide which is widely used in agriculture, painting, textile industry, leather and paper manufacturing in several countries [1].Because of its widespread use, carbendazim has been detected in water samples from watercourses, mainly in Brazil [2,3] and China [4,5].
Several studies have indicated that carbendazim exposure may contribute to tumour promotion, endocrine disruptions, and adverse effects on reproductive organs [ 6-11 ].Due to its high toxicity, carbendazim has been banned in Australia, the USA and several European Union (EU) countries [ 12-14 ], whereas it is still allowed in Brazil [15].The EU water quality guidelines allow carbendazim and other pesticides at a maximum concentration of 0.1 μg/L [16], while the Brazilian drinking water legislation permits a maximum concentration of 120 µg/L for carbendazim [15].
Most pesticides, as is the case with carbendazim, are toxic not only to target species, but also to a range of non-target species, such as fish.Since fish are exposed to xenobiotics via gills, skin and through their diet, numerous ecotoxicology studies involving fish have been conducted [17,18].Some of these studies indicate that carbendazim can induce hepatic dysregulation, changes in lipid metabolism and microbiota dysbiosis in adult zebrafish further to inducing oxidative stress, endocrine dysregulation and oxidative stress in the larval stage of zebrafish in addition to other effects [17,18].Furthermore, understanding how xenobiotics are metabolised by fish is important for monitoring exposure in the aquatic environment and for mitigating the effects of exposure [19].
Zebrafish (Danio rerio) is a classical and ideal model organism for toxicology studies and the adult zebrafish possess numerous advantages for studying xenobiotic metabolism [19].The ability of zebrafish to absorb agents administered in the water provides exposure of zebrafish to xenobiotics, thus mimicking what occurs in the aquatic environment, as well as combining the high-throughput of in vitro systems with the complexity of whole-organism studies [19,20].
Currently, there have been many studies using zebrafish as a model for the assessment of metabolites produced from environmental contaminants, such as pesticides [ 21-23 ]  illicit drugs [24], and pharmaceuticals [25].All of these studies have focused on the evaluation of metabolites in the zebrafish body.However, it is worth noting that the evaluation of metabolites in the zebrafish water tank has been shown to be a successful and advantageous approach for several xenobiotics [ 26-28 ].
Therefore, the aim of this work was to evaluate carbendazim metabolites in the zebrafish water tank using liquid chromatography coupled to a high-resolution mass spectrometer (LC-HRMS) to highlight analytical targets in order to monitor carbendazim exposure in aquatic environments.To the best of our knowledge, there is no one study that has evaluated carbendazim metabolites produced by zebrafish, neither in the animal's body nor in the water tank.This is the first report on the generation of carbendazim metabolites by zebrafish.

Zebrafish maintenance and exposure to carbendazim
This study was approved by the Ethics Committee on the Use of Animals of the Federal University of Ouro Preto (Protocol number 7061120319).Adult zebrafish (Danio rerio) (approximately 2 cm) were acquired from a local provider in Muriaé located in Minas Gerais state.The fish were equally distributed in male and female.They were maintained in 3.5 L aerated water tanks at 26 ± 2°C and under a 14 h light/10 h dark cycle.The fish were fed with Basic® ration (Alcon, Camboriú, Brazil) once a day, except in the day before euthanasia when they were kept fasting for 24 h.Before experimentation, fish were acclimated to water tanks for 14 days.Once a week, the pH and the concentration of ammonia, nitrite, and dissolved O 2 were evaluated using colorimetric tests (Alcon, Camboriú, Brazil).Three treatment tanks (AT) with 20 fish each were treated with carbendazim at 120 µg/L, and the stability control (AS) without fish but with carbendazim at 120 µg/L was used as a control for carbendazim stability.The dosage of 120 µg/L was adopted because it is the maximum value allowed in water according to Brazilian legislation [15].
A single tank with 20 fish and DMSO 0.006% was used as a negative control (AN).For biotransformation evaluation, two samples containing 10 mL of each tank were collected at 0 h, 1 h, 3 h, 5 h, 8 h, 10 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h.

Water sample preparation
The water sample preparation was adapted from the method developed by Sardela [29].Both samples from each collect point were prepared as follows.First, a 10 uL aliquot of each sample was transferred to a vial and the internal standard was added to achieve a concentration of 100 µg/L.Then, 80 µL of β-Glucuronidase from E. coli was added, and all samples were incubated in a 50°C water bath for 1 hour aiming at cleavage of glucuronide conjugates.After enzymatic hydrolysis, the samples were loaded onto SPE cartridges.Previously, the cartridges were inserted into a vacuum manifold and conditioned with methanol (2 mL) followed by water (2 mL).Thereafter, the analytes were eluted with 2 mL methanol: formic acid (95:5 v/v) and 1 mL of methanol: ammonium hydroxide (95:5 v/v).
The eluates were dried under nitrogen flow and reconstituted in 60 µL of water: methanol (7:3 v/v) with 0.1% formic acid.Finally, this reconstituted extract was added to the 10 µL aliquot and analysed by LC-HRMS.

Instrumental analysis
The water samples were analysed by liquid chromatography (LC) coupled to a highresolution mass spectrometry (HRMS).The LC system consisted of a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany) and the HRMS was a Q-Exactive™ Orbitrap (Thermo Fisher Scientific, Bremen, Germany).The LC separation was performed using a reversed phase column (Syncronis-Thermo C18 50 mm × 2.1 mm × 1.7 μm) maintained at 40°C.Mobile phase A (water, 0.1% formic acid and 5 mM ammonium formate) and mobile phase B (methanol with 0.1% formic acid) were employed in the following gradient of mobile phase B: 0 min, 5%; 0.2 min, 5%; 0.5 min, 10%; 1 min, 25%; 7 min, 90%; 11 min, 100%; 13 min, 100%; 13.1 min, 5%; 15 min, 5%.The injection volume was 5 μL and the flow rate was 300 μl/min.The mass spectrometer (HRMS) operated in positive mode ionisation using electrospray ionisation source (ESI).Full-MS and Full-MS/data dependent acquisition (DDA)-MS2 scan modes analyses were performed at a resolution of 70,000 (arbitrary unit) and a mass range of 100 to 1000 m/z.The acquisitions were adjusted to the following parameters: spray voltage was set to 2.90 kV, capillary temperature was 380°C and the applied S-lens radio frequency (RF) level was 80 (arbitrary unit).Nitrogen sheath and auxiliary gas flow rates were set to 60 and 20 (arbitrary unit), respectively.The automatic gain control was set to 1e6 (arbitrary unit), maximum injection time was set to 100 ms; isolation window of 2.0 m/z; loop count of 10 and top N was set to 10.The instrument was calibrated in positive mode using the calibration solutions provided by the manufacturer (Thermo Fisher Scientific, Bremen, Germany) to ensure mass accuracies below 6 ppm.

Method validation
The method was validated according to US EPA [30] and INMETRO [31] guidelines.Specificity, linearity, quantitation limit, repeatability, reproducibility, robustness, carryover and recovery were the parameters evaluated.
Specificity was assessed by analysing five blank samples, while ensuring the absence of any interfering signal at the same retention time as the analyte.The linearity was evaluated by the coefficient of determination (R 2 ) from the linear regression and by the Grubbs test.For this, six concentration levels (5; 50; 150; 300; 500 and 800 µg/L) of carbendazim solution were injected in triplicates for constructing the analytical curve.Subsequently, the limit of quantitation (LOQ) was established as the lowest concentration at which carbendazim could be detected with a signal-to-noise ratio (S/N) of peak areas greater than 10.Repeatability was evaluated using seven blank samples spiked with carbendazim at 150 µg/L.The repeatability evaluation was based on the relative standard deviation (RSD) of peak areas.Likewise, the reproducibility assessment was performed by analysing these seven blank samples spiked with carbendazim at 150 µg/L on the day following the repeatability analyses.
The robustness was evaluated by reconstitution of three dried extracts on the day after SPE procedure, followed by RSD calculation of peak areas.Then, the evaluation was carried out by calculating the relative error between RSD for repeatability and RSD for robustness.Carryover could be assessed by analysing a sample spiked with 300 µg/L carbendazim between two blank samples.Recovery was evaluated by adding carbendazim at 150 µg/L in seven blank samples before extraction and in seven blank samples after extraction.The recovery (%) was calculated according to equation (1) and RSD of peak areas among the seven replicates according to Sardela [29].

Detection of metabolites for monitoring carbendazim exposure
First, the exact masses of those metabolites already described in the literature or target metabolites [ 32-37 ] were calculated using Thermo Xcalibur software (version 3.0.63;Thermo Fisher Scientific, Bremen, Germany).For metabolite assignments, the molecular formulas were calculated using Thermo Xcalibur software (version 3.0.63;Thermo Fisher Scientific, Bremen, Germany) through an elemental composition calculator.These molecular formulas have been proposed according to the agreement of theoretical isotopic pattern and lower mass error.
The corresponding exact masses of the precursor ions were evaluated by searching for the in full-MS scan mode.Subsequently, the MS/MS spectra generated by full-MS/DDA-MS2 were also evaluated to verify the product ions.

Analytical method validation
The analytical method was validated to ensure reliable evaluation of carbendazim biotransformation allowing its detection and quantification in water samples.The method showed enough specificity, since no interfering signal was detected at the same retention time as carbendazim.Regarding linearity, the analytical curve yielded R2 of 0.9905 and no outliers were detected.Thereafter, the limit of quantitation (LOQ) was defined as the lower level of the analytical curve (5 µg/L) which exhibited a S/N ratio greater than 10.
Repeatability and reproducibility were both suitable, resulting in an RSD of 4.4 and 4.8%, respectively.The method proved to be robust, as the reconstituted samples the day after SPE procedure yielded an RSD of 4.98%.
This analytical method also showed enough recovery (108.6%) and no carryover was detected indicating that there was no risk of sample contamination by a previously analysed sample.

Carbendazim biotransformation
The putative carbendazim metabolites already described in the literature were investigated by searching for the corresponding exact masses in full-MS scan mode.Once detected the exact mass of the precursor ion, the MS/MS spectrum in full-MS/DDA-MS 2 was evaluated aiming to verify the product ions.Hence, the annotation of 7 metabolites could be suggested (Figure S1).The retention times, molecular formulas and experimental accurate masses of precursor and product ions, as well as error values in parts per million (ppm) are shown in Table 1.
In the LC-HRMS method, the retention time for carbendazim was 4.67 min, providing an excellent peak resolution in a satisfactory time (Figure S2A).From the full-MS scan mode, it was possible to detect the exact mass of m/z 192.07703 [M + H] + , corresponding to protonated carbendazim.Evaluating the MS/MS spectrum generated by full-MS/DDA-MS 2 , it was observed that the product ions were detected at m/z 160.05069 and at m/z 132.05597, representing the loss of methoxyl (-HOCH 3 ) and carbonyl (-CO) groups, respectively (Figure S2B).The MS/MS spectrum (Figure 1(a)) of 5-hydroxycarbendazim (5-HBC) shows a precursor ion at m/z 208.07183, and product ions at m/z 176.04568 and m/z 149.05951.The product ion at m/z 176.04568 could be observed as base peak and represents the loss of methoxyl group (-HOCH 3 ), as well as the product ion at m/z 149.05951 represents the carbonyl loss.Both product ions at m/z 176.04568 and 149.05951 correspond to the ions at m/z 160.05069 and 132.05597, observed on the MS/MS spectrum of carbendazim, added with a hydroxyl group.Hence, this indicates that hydroxylation has occurred on the benzimidazole.
Regarding the MS/MS spectrum of methyl 5-sulfatecarbendazim (5-HBC-S) (Figure 1(b)), a precursor ion at m/z 288.02859 and product ions at m/z 208.07191 and 176.04576 could be observed.The product ion at m/z 208.07191 represents a mass loss of 80 with respect to the precursor ion, corresponding to sulphate (-SO 3 ) loss.Thus, it was evident that a sulphation reaction occurred at the hydroxyl group of the 5-HBC metabolite.Subsequently, there was a loss of the methoxyl group (-HOCH 3 ) resulting in the product ion at m/z 176.04576, which confirmed the sulphation reaction in the 5-HBC metabolite.
Another carbendazim metabolite detected in this study was 2-aminobenzimidazole (2-AB).The MS/MS spectrum (Figure 1(c)) shows a precursor ion at m/z 134.07126 corresponding to the metabolite 2-AB, which has been generated from carbendazim biotransformation resulting in the elimination of methoxycarbonyl group.Owing to the high chemical stability of the 2-AB molecule, the collision energy applied was not sufficient to cause its fragmentation, so any product ion could be detected in the MS/ MS spectrum.However, it is possible to suggest the annotation of 2-AB since the experimental mass error was very low (−0.01 ppm).
Furthermore, the metabolites aminohydroxybenzimidazole and 2-hydroxybenzimidazole (2-HB) were detected, resulting from N-oxidation reaction and oxidative deamination, respectively.Their full-MS spectra showed a precursor ion at m/z 150.06638 for aminohydroxybenzimidazole (Figure S3A) and a precursor ion at m/z 135.05547 for 2-HB (Figure S3B).Nevertheless, it was not possible to evaluate their product ions spectra as the signal intensities of both metabolites were too low to trigger data-dependent acquisition of the product ion spectrum.It is worth noting that to our knowledge, this is the first work suggesting the generation of the metabolite aminohydroxybenzimidazole and the undetermined hydroxy position is indicated by tilde.
Ultimately, evaluating the MS/MS spectra of 1,2-diaminobenzene (Figure S3C), it is possible to detect a precursor ion at m/z 109.07643generated from cleavage of the benzimidazole ring.However, due to chemical stability, the collision energy has not been enough to result in fragmentation, making it impossible to detect product ions in the MS/ MS spectrum.Similarly, MS/MS spectrum of 1,2-dihydroxybenzene (Figure S3D) shows a precursor ion at m/z 111.04436 and no product ion could be detected either.

Carbendazim metabolic profile
The metabolic profile of carbendazim was evaluated by determining the generated metabolites followed by estimating this generation through the ratio between the metabolite chromatographic peak area and the internal standard chromatographic peak area over the collection time (0-168 h) (Figure 2).In addition, the production of these metabolites in treatment tanks (AT), stability control (AS) and negative control (AN) was also considered.
Regarding the samples from AT, it was evident that carbendazim concentration was decreasing over time, mainly after 8 h exposure (Figure 2).Similarly, the metabolites 5-HBC and 2-AB were generated in the first hours, but showed a significant increase from 8 h onwards, particularly after 96 h.The metabolite 5-HBC started to decrease after 120 h, just when the 5-HBC-S began to be generated, thus confirming that 5-HBC-S resulted from the sulphation reaction on the hydroxyl group of 5-HBC.
The metabolites aminohydroxybenzimidazole, 2-HB, 1,2-diaminobenzene and 1,2-hydroxybenzene were found to be generated in the first few hours and in much lower concentrations than the others.The aminohydroxybenzimidazole and 2-HB metabolites exhibited an increased concentration over time, reaching a maximum at 144 h with a subsequent decrease.On the other hand, 1,2-diaminobenzene showed the highest generation at 8 h followed by a decrease, whereas 1,2-dihydroxybenzene showed a maximum at 168 h.

Discussion
The evaluation of carbendazim biotransformation by zebrafish was performed by analysing aliquot samples collected from treatment (AT), stability (AS) and negative (AN) tanks in full-MS and full-MS/data-dependent acquisition (DDA)-MS 2 scan modes, both in positive ionisation mode.It is noteworthy that in DDA mode, the precursor ions detected with accurate mass are directed to the quadrupole and fragmented in the higher-energy C-trap dissociation cell (HCD).The resulting product ions are then sent to the Orbitrap where their masses are accurately measured [38].Therefore, this acquisition mode enables obtaining data able to increase the reliability of metabolites annotation, especially for novel ones.
Zebrafish have the ability to perform phase I metabolic reactions, such as N-oxidation and hydroxylation, as well as phase II reactions, such as sulphation, in order to conjugate metabolites resulting from phase I with hydrophilic compounds [20,39].The metabolic enzymes involved in these processes are highly conserved in zebrafish compared to mammals [39].Phase I reactions are mostly executed by cytochrome P450 (CYP450) enzymes, which occur in humans and have orthologs in zebrafish, such as the CYP1A and CYP3C1-4 enzymes that play a remarkable role in oxidative metabolism [40] and might be responsible for generation of the metabolites 5-HBC and aminohydroxybenzimidazole. Likewise, the sulphation (phase II reaction) performed by sulfotransferases enzymes, also identified in zebrafish [20], might be responsible for the generation of the metabolite 5-HBC-S.
The possibility these metabolites have been generated by bacteria was rejected or ignored, since the ammonia and nitrite levels were maintained below of the recommended values.One of the ways of removing ammonia excreted by fish is by autotrophic bacterial conversion of ammonia in nitrate through nitrification.Nitrification occurs in two steps: oxidation of ammonia to nitrite by ammonia-oxidising bacteria (AOB) and oxidation of nitrite into nitrate by nitrite-oxidising bacteria (NOB).AOB and NOB collectively termed as nitrifying bacteria are found in soil, freshwater and marine environments [42].Therefore, low levels of ammonia and nitrite means that there was not significative bacterial activity in the tanks.

Conclusions
Carbendazim is ubiquitous in the environment and chronic exposure even at low dose over lifetime is a reality.This study applied the analysis of zebrafish water tank for evaluation of target metabolites of carbendazim as a promising approach, since it is a much cleaner matrix than the usual biological matrices.Thus, this approach can be potentially applied to the evaluation of other xenobiotics, especially those environmentally relevant that might be present in aquatic matrices affecting non-target species, such as fish.
The putatively assigned metabolites, mainly 5-HBC and 5-HBC-S, could be used as potential analytical targets and could enable the monitoring of carbendazim exposure.It became evident that the metabolite generation by zebrafish starts after 8 h and the greatest amount of the produced metabolites occurs after 120 h, with a maximum generation at 168 h after the exposure.Therefore, these metabolites can ensure detectability and further strengthen the confidence in monitoring carbendazim exposure in a short period after exposure or even after a longer period, up to one week.

Figure 1 .
Figure 1.Mass Spectrum in full-scan and full-scan DDA-MS 2 modes for the 5-HBC (a), 5-HBC-S (b) and 2-AB (c) metabolites identified in the water tank.

Figure 2 .
Figure 2. Carbendazim biotransformation profile (a).Magnification of the metabolites in Fig.A to view details (b).

Figure 3 .
Figure 3. Production of metabolites during the first 168 hours of exposure based on data from the treatment and stability groups.