Detection of Azoxystrobin Fungicide and Metabolite Azoxystrobin-Acid in Pregnant Women and Children, Estimation of Daily Intake, and Evaluation of Placental and Lactational Transfer in Mice.

BACKGROUND: Azoxystrobin (AZ) is a broad-spectrum strobilurin fungicide that is used in agriculture and was recently added to mold- and mildew-resistant wallboards. AZ was found to have toxic effects in animals at embryonic stages and was listed as a frontline target for biomonitoring in children.
OBJECTIVES: This study investigated exposure to AZ in pregnant women and young children, whether AZ could be transferred from an exposed mother to offspring, and whether AZ or one of its primary metabolites, AZ-acid, was neurotoxic in vitro.
METHODS: We quantified AZ-acid, a sensitive indicator of AZ exposure, in urine samples collected from 8 pregnant women (12 urine samples) and 67 children (40-84 months old; 96 urine samples) with high-resolution mass spectrometry. Gestational and lactational transfer was assessed in C57Bl/6 mice. Neurotoxicity of AZ and AZ-acid was investigated in vitro with mouse cortical neuron cultures.
RESULTS: AZ-acid was present above the limit of quantification (0.01 ng/mL) in 100% of the urine samples from pregnant women and in 70% of the urine samples from children, with median concentration of 0.10 and 0.07 ng/mL, and maximal concentration of 2.70 and 6.32 ng/mL, respectively. Studies in mice revealed that AZ transferred from the mother to offspring during gestation by crossing the placenta and entered the developing brain. AZ was also transferred to offspring via lactation. High levels of cytotoxicity were observed in embryonic mouse cortical neurons at concentrations that modeled environmentally relevant exposures. DISCUSSION: Our study suggested that pregnant women and children were exposed to AZ, and at least 10% of the children (2 out of 20 that were evaluated at two ages) showed evidence of chronic exposure. Future studies are warranted to evaluate whether chronic AZ exposure affects human health and development. https://doi.org/10.1289/EHP9808.

Introduction

Azoxystrobin (AZ) is a broad-spectrum fungicide that is commonly applied to cereals, turfgrasses, grapevines, potatoes, fruits, nuts, and vegetable crops (Bartlett et al. 2002). More recently, AZ was added to some brands of mold- and mildew-resistant wallboards, to aid in the prevention of fungal growth (Cooper et al. 2020). Use of AZ is increasing, with an annual use estimated to be 1,000 tons in 2017 in the United States (U.S. Geological Survey 2017). Likely because of its widespread use, AZ was measured in crops and vegetables in Europe and fruits in Turkey (EFSA 2014; Golge and Kabak 2018), and from and environmental samples including surface water (Battaglin et al. 2011), groundwater (Reilly et al. 2012), and indoor dust (Cooper et al. 2020; Shin et al. 2020) in the United States.

An increasing number of experimental studies found that AZ has the potential to cause developmental toxicity and neurotoxicity. In cortical neuron cultures prepared from embryonic mice, AZ () induced reactive oxygen species (ROS) and caused transcriptional changes associated with autism, brain aging, and neurodegeneration (Pearson et al. 2016; Simon et al. 2019). In zebrafish, AZ altered apoptosis-related gene expression in larvae and caused oxidative stress in larvae and in adults (Cao et al. 2018). Following parental AZ exposure in zebrafish, a significantly higher incidence of mortality and malformations was observed in F1 offspring (Cao et al. 2019). These studies collectively suggest that AZ is toxic at embryonic stages.

AZ was identified as a major frontline target chemical for biomonitoring in the United States (Pellizzari et al. 2019). However, there is limited information as to whether humans, and particularly young children and pregnant mothers, are exposed to AZ. It is unclear whether AZ can be transferred from the mother to embryos in utero. Here, we measured the concentration of a biomarker of AZ exposure (AZ-acid) in the urine of pregnant women and in a separate cohort of children ranging from 40 to 84 months of age. The maternal transfer of AZ to embryos and weaning-age mice was also investigated.

Materials and Methods

Chemicals and Reagents

The chemicals and reagents used in the study are listed in the Table S1.

In Vitro S9 Incubation and Analysis

The in vitro S9 incubation was performed as described previously (Ashrap et al. 2017). In brief, human S9 () was incubated with AZ () to identify potential biotransformation products. Incubation of heat-deactivated S9 (pretreated at for 10 min) with AZ () was used as negative control. The reactions were performed in phosphate buffer (pH 7.4) containing EDTA, dithiothreitol, 20% glycerol, and NADPH regenerating system in amber glass vials ( per group). After incubation at for 1 h, the reaction was then quenched by adding ice-cold acetone.

The mixtures were extracted by adding ethyl acetate (EA), vortexed for 15 min and then sonicated for another 15 min. After repeating these steps twice, the organic layers were combined and evaporated to dryness, then redissolved in 20% acetonitrile (ACN) in water for analysis. Untargeted screening for AZ biotransformation products was performed by NanoLC-MS/MS analysis on an UltiMate 3000 RSLC Nano system coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer, using an EASY-Spray ion source for nano-electrospray (Thermo Fisher Scientific). Extractions were loaded into a C18 trapping column ( particle, i.d.) for 3 min, the flow rate was set to be , and 0.1% formic acid (FA) in double-distilled water () was used as a loading solvent. The trapped analytes were separated by an analytical column ( particle, i.d.) after 3 min trapping time. Liquid chromatography (LC) separation was performed at a flow rate of , and the gradient setting was listed in Table S2. The solvents used were solvent A (0.1% FA in ) and solvent B (0.1% FA in ACN). The parameter for full scan mass spectrometry (MS)/data-dependent (dd) MS2 mode is listed in Table S3.

Data sets were deconvoluted and aligned using Progenesis QI (Waters) as described previously with little modification (Hu et al. 2019). We set the initial and final retention times to be 5 and 35 min, respectively. Principal component analysis (PCA) based on the aligned peak list using soft independent modeling by class analogy () (version 12.0; Umetrics) was applied to differentiate the chemicals between the inactive (control, S9-) and active S9 samples. Peaks with a fold change of () in the AZ-treated group in comparison with the control group were selected. We generated a database of potential biotransformation products of AZ based on the structure of the parent chemical; molecular weights (MWs) were predicted with ChemDraw (PerkinElmer) (Figure S1). For the peaks with MW that matched with chemicals in our database, the peaks were then analyzed by a targeted parallel reaction monitoring mode, and the samples were analyzed with the same instrument conditions as described above. The standards of AZ-acid and R402173 were purchased to compare their mass spectra with unknowns for structure confirmation.

Animal Experiments

Animal protocols in this study were approved by the Institutional Animal Care and Use Committee of The University of North Carolina at Chapel Hill (UNC Chapel Hill). C57Bl/6 mice (Jackson Labs) were maintained on a light:dark cycle (12 h:12 h) and given food and water ad libitum. Matings were set up with one male and two females per breeding cage. Mice used in the experiments were randomly assigned to control (mice treated with corn oil) and exposure groups (mice treated with AZ dissolved in corn oil). Corn oil was used as the solvent because we previously found that pyraclostrobin, a related strobilurin fungicide (a group of natural products and their synthetic analogs), was soluble in corn oil but was insoluble in aqueous solutions (Tuttle et al. 2019). The dosing method was oral gavage, which mimics the potential main exposure pathways of AZ dietary ingestion and indoor dust inhalation, most of which is ultimately ingested in nasal secretions (Wilson et al. 2013).

Animal experiment 1: Nine-wk-old male ( per group) and female ( per group) mice were treated with corn oil or AZ (0.0002, 0.02, and ) by oral gavage. The lowest dose was based on data indicating that 0–6-month-old children () ingest an estimated of dust per day (Phillips et al. 2015; ExpoFIRST Scenarios Tool: https://www.epa.gov/expobox) and in house dust. This AZ dust concentration was based on the highest measured value in dust samples collected from North Carolina homes in 2014–2016 (Cooper et al. 2020), giving an estimated exposure of . Mouse urine was collected before and 1, 3, and 24 h after oral gavage. Mice were handled by grasping from behind and positioning over foil to collect urine, which was then transferred to vials. Urine samples were stored at until analysis.

Animal experiment 2: Female mice (9–12 wk old) were bred overnight, and the copulatory plug-positive females were identified as embryonic (E) 0.5. Pregnant dams were single housed and treated with corn oil () or AZ at a concentration of () from E0.5 to E14.5 once daily by oral gavage. On E14.5, dams were humanely euthanized by swift decapitation, and then the placenta and cortex from the dams and embryos were collected.

Animal experiment 3: Dams (9–12 wk-old) were treated with corn oil () or AZ (, ) from postnatal day (PND) 1 to PND 12 once daily by oral gavage. On PND12, dams and pups were sacrificed by decapitation, then cerebral cortices were collected for further analysis. There were 6–8 embryos/pups per dam. The cerebral cortices and placenta from each embryo/pup were collected for AZ/AZ-acid analyses. All the samples collected above were analyzed separately.

Human Urine Collection

Urine samples were derived from two different study populations. Prenatal specimens were collected from a pilot and feasibility study conducted at UNC Chapel Hill over a 7-month period. To be eligible to participate, pregnancies could not be the result of in vitro fertilization, and no first degree relative could have autism, intellectual disability, schizophrenia, or bipolar disorder. Women’s urine samples were first collected between 8 and 15 wk gestation at enrollment; then repeated samples were collected at 18–24 wk or . Following informed consent, prenatal spot urine samples were collected during routine prenatal care appointments from eight women (IRB 18-0889) receiving routine prenatal care at the UNC Medical Center. For four participants, samples were subsequently collected at a later time in pregnancy during their routine prenatal care appointments (Table S4 and S5). Spot urines were collected in a sterile polypropylene collection cup and kept at until processing at the UNC Biospecimen Processing Facility. Aliquots were frozen at . In general, sample processing occurred within 2 h of collection (average 81 min); however, one sample experienced a delay in processing (2 d) and was frozen prior to processing, due to an unanticipated closure of the processing facility. The characteristics of the population are listed in Table S4. Maternal urine samples were collected between April 2019 and October 2019. Samples from children were collected during an ongoing longitudinal study of brain development (IRB 16-1943) (Howell et al. 2019). Eligibility criteria included gestational age between 37 and 42 completed weeks gestation, birthweight appropriate for gestational age, and absence of major pregnancy and delivery complications, and no first-degree relative could have autism, intellectual disability, schizophrenia, or bipolar disorder. Children were identified through existing birth record and registries maintained at UNC Chapel Hill, as well as from local community day care centers and the postpartum floor of UNC Chapel Hill. Following informed consent, spot urine samples were collected from 67 children (40–84 months old) during follow-up appointments. For 20 participants, additional samples were collected 9–12 months after the first collection (Tables S4 and S6). A sterile toilet hat was placed into the toilet to collect urine, which was then poured into sterile polypropylene collection cups. Urine was kept at until processing, and then aliquoted and stored at . Average time between collection and processing was 18 h. These studies were approved by the UNC institutional review board (18-0889; 16-1943). Child urine samples were collected between July 2017 and February 2020.

Sample Preparation and Analysis

Urine samples ( for human urine and for mouse urine) were spiked with d4-AZ (). Samples were adjusted to pH 6.5 with sodium acetate buffer (, pH 5.0) and then treated with ( in sodium acetate buffer). Samples were incubated at for 2 h and diluted with sodium acetate buffer. The ISOLUTE C18 solid phase extraction (SPE) cartridge was prewashed with 5% in methanol (MeOH) and sodium acetate buffer. After urine samples were loaded, the column was washed with 5% MeOH in sodium acetate buffer and then eluted by adding 5% in MeOH. The extracts were collected and evaporated to dryness and then dissolved in 20% ACN in water ( for human urine, for mouse urine). The specific gravity (SG) of human urine was measured using a Reichert TS Meter Model TS400 Refractometer (Reichert Technologies). To account for urinary dilution, we corrected for SG using the following formula: , where is the SG-corrected AZ/AZ-acid concentration; and is the observed urinary AZ/AZ-acid concentration.

Mouse brain (about ) and placenta (about ) samples were freeze dried and ground to a fine powder with mortar and pestle. Afterward, d4-AZ () was spiked in, and the samples were incubated at room temperature for 0.5 h. Next, ACN was added to each sample, and the samples were sonicated for 5 min, vortexed for 15 min, and centrifuged at 3,500 rpm for 5 min. The extracts were then transferred to a new glass vial. After repeating this step twice, the organic layers were combined and stored at . After overnight incubation, the extracts were centrifuged at 12,000 rpm for 10 min at . Supernatants were transferred to new vials and evaporated to dryness and dissolved in 5% MeOH in sodium acetate buffer. The extraction was loaded onto the ISOLUTE C18 SPE cartridge, and the procedures for washing and eluting the SPE cartridge were the same as described above for urine samples.

Analysis of AZ and AZ-acid was performed using a Vanquish UHPLC system coupled with Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer, and a Heated Electrospray Ionization probe was used as an ionization source (Thermo Fisher Scientific). AZ and AZ-acid were detected by parallel reaction monitoring for the following targeted precursors: AZ m/z 404.12, AZ-acid m/z 390.11, and d4-AZ m/z 408.15. LC Separation was achieved by using an ACQUITY UPLC HSS T3 column ( particle, i.d.). A solvent system consisting of 0.1% formic acid in (solvent A) and 0.1% formic acid in ACN (solvent B) was used for LC separation at a flow rate of . The gradient setting was following: 30% B for 2 min, from 30% to 95% B in 5 min, held at 95% B for 2 min, 95% to 30% B in 0.5 min, and held at 30% B for 2.5 min before next injection. The parallel reaction monitoring MS2 scan resolution was set at 35,000 with automatic gain control target of and maximum fill time of 200 ms. The isolation window of precursor ions was set to 2.0 m/z and normalized collision energy of 18 for fragmentation. The HESI ion source condition was used the default settings for the flow rate at .

When analyzing the urine, brain and placenta samples, matrix spiked recoveries were estimated by spiking in the d4-AZ and AZ-acid to the analytical procedure at the concentrations of 0.05, 0.25, and . The recoveries and matrix effects for the urine, brain, and placenta samples are listed in Table S7. The recoveries were procedural blank subtracted. In urine samples, recoveries for AZ and AZ-acid ranged from 90% to 103% and 96% to 111%, respectively. In brain samples, recoveries for AZ and AZ-acid ranged from 87% to 96%, and 90% to 97%, respectively. In placenta samples, recoveries for AZ and AZ-acid ranged from 83% to 91%, and 87% to 91%, respectively. Procedural blank samples were prepared and analyzed. For the urine samples, the procedural blank samples were prepared in water. For the brain and placenta samples, procedural blank samples were prepared in ACN. All the extraction steps described for the urine samples, brain samples, and placenta samples were followed as described above. AZ was detected in the procedural blank samples ( for each matrix) with the average of ; AZ-acid was not detected from these procedural blank samples. The concentrations of all the samples were blank corrected using the average level measured in the procedural blanks. Calibration curves of AZ and AZ-acid were calculated with a concentration series of 0.01, 0.05, 0.1, 0.5, 1, 5, and , and the calibration curves had an of . Limits of quantification (LOQs) for AZ and AZ-acid were 0.003 and , respectively.

Estimation of AZ Daily Intake

To estimate the daily intake of AZ from AZ-acid excretion, the following formula, described in (Hoffman et al. 2017), was used: Urine output was not evaluated in our study. We used published estimates for children of this age, which is for children (Valentin 2002). The normal urine output for an adult is ; thus, we used for the pregnant women (Valentin 2002). The urine excretion fraction of AZ converted to AZ-acid has not been evaluated in humans; therefore, we used urine excretion of AZ that was obtained from a rat study (WHO/FAO 2011). In this study, rats were treated with C14-pyrimidinyl-labeled AZ, and the excreted radioactivity in urine was 10.2% for male and 17.9% for female (WHO/FAO 2011). We likewise found that female mice excreted more AZ metabolite than males excreted (see below).

Cortical Neuron Culture and Live/Dead Assay

Primary mouse cortical neuron cultures were prepared as described previously from E14.5 C57Bl/6 mice (Huang et al. 2012). In brief, cerebral cortices were digested with papain () and DNase I () at 37°C for 30 min; then 5% FBS in neurobasal medium was added to deactivate the papain. After washing with Hank’s balanced salt solution, the isolated cells were added to seeding medium (Neurobasal medium, B27, Antibiotic-Antimycotic and Glutamax). The live/dead cells assay was performed as described previously with some modifications (Pearson et al. 2016). Briefly, cells were seeded at a density of cells per well in poly D-lysine–coated 96-well plates. On day in vitro (DIV) 7, half of the cell culture medium was replaced with AZ/AZ-acid at concentration in feeding medium (Neurobasal medium, B27, 5-fluoro-2-deoxyuridine, and Glutamax), to achieve the final concentration of 1, 10, 100, 1,000, . On DIV 14, half of the media was removed and replaced with feeding medium containing SYTOX Green (). After incubation at for 0.5 h, cells were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde in 0.1 M PBS for 10 min. Cells were imaged using a high-content microscope (IN Cell Analyzer; GE Healthcare). Four images were scanned per well, and ImageJ was used to quantify the number of live and dead cells.

Immunofluorescence Staining

To analyze the number of neurons after AZ and AZ-acid treatment, cells were cultured and treated as described above, and on DIV 14, cells were fixed and then incubated with blocking buffer (PBS with 2% normal donkey serum and 0.5% Triton X-100) at room temperature for 30 min, followed by staining with neuronal nuclear antigen (NeuN; 1:1,000; ABN78, EMD Millipore) for 2 h. Cells were rinsed with PBS before applying AlexaFluor-conjugated donkey secondary antibody (1:1,000; A31573, Invitrogen) and DAPI (1:4,000). After a 1 h incubation with the secondary antibody, cells were washed three times with PBS and then observed by high-content microscopy as described above.

Statistical Analysis

All results are presented as . For mouse and neuron culture experiments, analysis of variance followed by post hoc Dunnett’s test was performed to determine the statistical significance of the results. The animal data were analyzed by using the litter as the statistical unit to minimize potential confounds associated with the litter effect. All offspring were used for each litter and analyzed by applying the nonlinear mixed-effects model approach (Jiménez and Zylka 2021). For studies with human urine samples, the differences between groups were compared using the Mann-Whitney test. Results with were considered to be statistically significant. Values below LOQ were replaced with a value of LOQ/2 for statistical analyses (Antweiler and Taylor, 2008).

Results

Identification of AZ Metabolites

AZ is rapidly metabolized with a half-life of 14–21 h in rats (EFSA 2010). For chemicals that are rapidly metabolized, exposure can be ascertained by detecting metabolites of the parent chemical. To identify metabolites of AZ, we incubated human liver S9 fractions with AZ, and as a control heat-denatured S9 fractions with AZ, and then we analyzed these samples using nontargeted MS. The spectral data of all samples were analyzed by PCA and plotted (Figure S2). There was a distinct separation between the inactive (control, S9-) and active S9 samples, suggesting that AZ was biotransformed by the active S9 fraction. To narrow the list of candidate metabolites, we focused on 113 chemical peaks that were 5-fold or more abundant in the active S9 vs. inactive S9 samples (supplemental Excel file). We compared the m/z of these 113 chemicals to a database of potential AZ biotransformation products that we generated (Figure S1). The MW of two peaks (m/z 390.10794, RT 12.98; m/z 361.1158, RT 10.26) matched chemicals 7, 8, and 9 in the database (chemical 7 and 8 have the same MW). Two of these chemicals (7 and 9) were commercially available. After comparing the RT and tandem mass spectrometry of these two standards with the unknowns, only one peak (m/z 390.10794, RT 12.98) matched well with standard chemical R234886, also known as AZ-acid. These human liver S9 experiments indicated that AZ-acid was a metabolite of AZ (Figure 1 and Figure S3).

Figure 1.

Chromatogram (A) and mass spectra (B) of azoxystrobin biotransformation product azoxystrobin-acid.

Detection of AZ and AZ-Acid in Mice

To evaluate whether AZ and/or AZ-acid could be used to quantify AZ exposure in vivo, we orally gavaged (p.o.) groups of male and female mice with corn oil or with AZ at three doses (0.0002, 0.02, , in corn oil). We found that AZ and AZ-acid were undetectable in the vehicle-only group and in all exposure groups at the 0 h (preexposure) time point (Figure 2A). In contrast, the urine concentrations of AZ and AZ-acid in treated mice varied in a dose- and time-dependent manner (Figure 2A,B; Tables S8 and S9). Of note, AZ-acid was detected in mouse urine samples at much higher concentrations than the parent chemical (Figure 2B relative to Figure 2A), suggesting that AZ is rapidly biotransformed in mice and that AZ-acid could serve as a sensitive readout of AZ exposure.

Figure 2.

Concentration of (A) azoxystrobin and (B) azoxystrobin-acid in mouse urine after oral administration of azoxystrobin at the indicated concentrations ( per group, with 6 male and 6 female). Concentration of (C) azoxystrobin and (D) azoxystrobin-acid after treating urine samples with ( per group, with 6 male and 6 female). Values were shown in Table S8 and S9. Results are expressed as . *(), **() indicate values significantly different from vehicle-treated controls. Analysis of variance followed by post hoc Dunnett’s test was performed to determine the statistical significance of the results. Note: SEM, standard error of the mean.

Metabolites are often excreted as conjugate forms in urine, and these conjugates cannot readily be detected due to their size and chemical characteristics. Thus, to further improve detection sensitivity, we treated mouse urine samples with to hydrolyze potential glucuronide biotransformation products of AZ and AZ-acid. Urine concentrations of AZ were similar in untreated (Figure 2A) and enzyme–treated samples (Figure 2C). In contrast, the concentrations of AZ-acid were higher in enzyme-treated urine samples (compare Figure 2D with Figure 2B). These data suggested that some AZ-acid exists as a glucuronide conjugate in mouse urine. We treated all subsequent urine samples with to maximize AZ-acid detection sensitivity.

Drug and chemical metabolism and toxicokinetics are known to differ between males and females (Gochfeld 2017), so we next evaluated AZ and AZ-acid concentration in male and female urine samples separately from the exposure group (Figure 3; all samples were ). There were no significant differences in AZ concentration between males and females, except for the 3-h time point (Figure 3A). The concentration of AZ in female urine samples was , which was significantly higher than that in the male urine samples (). In contrast, the AZ-acid concentration was significantly higher in female urine samples at the 1 h ( vs. ) and 3 h ( vs. ) time points and significantly lower at the 24 h time point ( vs. ).

Figure 3.

Urine (A) azoxystrobin and (B) azoxystrobin-acid concentration separated by sex. Male () and female () mice were orally gavaged with azoxystrobin and urine samples were treated with prior to analysis. Results are expressed as . Data can be found in the “Results” section. * (), ** () indicate values significantly different from vehicle-treated controls. Analysis of variance followed by post hoc Dunnett’s test was performed to determine the statistical significance of the results. Note: SEM, standard error of the mean.

AZ Exposure Measurements in Pregnant Women and Young Children

To determine if humans are exposed to AZ, urine concentrations of AZ and AZ-acid from 40–84-month-old children (96 samples from 67 participants) and pregnant women (12 samples from 8 participants) were measured. AZ was not detected in urine samples from pregnant women, but it was detected in 10.4% (10 out of 96) of the urine samples from children. The SG-adjusted geometric mean concentration of AZ was , with a range from (Table S10). AZ-acid was detected in all of the urine samples from the pregnant women (Table S5) and 70% of the urine samples from the children (67 out of 96) (Table S6). In pregnant women, the SG-adjusted geometric mean concentration of AZ-acid was , with a range from (Table 1; Table S5). In children, the SG-adjusted geometric mean concentration of AZ-acid was , with a range from . Based on the urine concentration of AZ-acid from the individuals in our study population, the average daily intake level of AZ was estimated to be for children and for pregnant women (Table 1). The maximum estimated daily intake of AZ was .

Table 1

Urinary concentration of azoxystrobin-acid (ng/mL, specific gravity corrected) and estimated daily intake of azoxystrobin by pregnant women and children (ng/kg/d).

Variable	n (M/F)	Detection (%)	GM±GSD	Range	25th	50th	75th	95th	Average estimated daily intake	Maximum estimated daily intake
Samples from pregnant women	12a (0/12)	100 (12/12)	0.09±2.13	0.03–2.70	0.04	0.07	0.14	1.33	75.6	669.4
Samples from children	96b (50/43c)	70.0 (67/96)	0.09±2.53	<LOQd-6.32	<LOQ	0.10	0.24	1.21	112.6	2,305.0
Children
40–50 months	36 (16/18)	69.4 (25/36)	0.09±3.22	<LOQ−6.32	<LOQ	0.15	0.23	2.11	132.9	2,305.0
51–60 months	29 (19/10)	72.4 (21/29)	0.09±1.64	<LOQ−2.39	<LOQ	0.10	0.29	1.04	97.3	871.7
61–70 months	16 (8/8)	68.8 (11/16)	0.08±2.85	<LOQ−5.38	<LOQ	0.11	0.27	1.73	159.4	1,961.1
71–84 months	15 (7/7)	64.7 (10/15)	0.05±1.28	<LOQ−0.82	<LOQ	0.06	0.12	0.71	43.4	243.9
Males	50	66.0 (33/50)	0.08±2.25	<LOQ−5.38	<LOQ	0.09	0.26	1.04	117.8	1,961.1
Females	43	73.9 (32/43)	0.09±2.90	<LOQ−3.19	<LOQ	0.12	0.22	1.62	58.6	663.5

Note: F, female; LOQ, limit of quantification; M, male.

12 urine samples from 8 pregnant women, for some participants samples were collected at different gestation weeks (Table S5).

A total of 96 urine samples from 67 children for some participants samples were collected 9–12 months after the first collection (Table S6).

Sex information was missing for 2 participants in 40–50-month-old group, and 1 participant in 71–84-month-old group.

The LOQ is for azoxystrobin-acid.

We next separated the urine samples from children into four groups: 40–50, 51–60, 61–70, and 71–84 months old (Table 1). Sex-related differences in urinary concentrations of AZ-acid were also examined for each age group. In general, no significant sex-related or age-related differences in urinary AZ-acid concentrations were observed for any age group. Additional urine samples were collected from a subset of the participants (10 male and 10 female) at a later 9–12-month follow-up visit. This dual collection permitted comparison of urinary AZ-acid concentrations in the same individuals at two different times. For most participants, the level of urinary AZ-acid was lower (Figure 4; Table S6), suggesting that AZ exposure decreased between the first and second urine collection. AZ-acid levels were relatively constant and above the mean in two participants, suggestive of chronic exposure.

Figure 4.

Urine azoxystrobin-acid concentration in children sampled twice (). The second urine samples were collected 9–12 months after the first samples. Data can be found in Table S6.

Evaluate Maternal Transfer of AZ to Offspring

Lipophilic chemicals partition into the placenta and milk, raising the possibility that AZ might transfer from exposed females to offspring during gestation and lactation. To evaluate gestational transfer, we treated pregnant mice with corn oil or AZ (p.o., daily) from E0.5 to 14.5. The urine concentration of AZ-acid in mice exposed acutely at was similar to that in human urine [ at 24 h time point in mice (Table S9); maximal in humans (Table 1)]. Thus, we used the exposure for subsequent mouse experiments to maintain AZ levels within environmentally relevant exposure levels. We then collected placenta and cerebral cortices from the dams and embryos at E14.5 and quantified AZ and AZ-acid levels. No AZ or AZ-acid was detected in the control groups (Figure 5A–C). In contrast, AZ and AZ-acid were detected in the cerebral cortices of the dams and embryos in the exposure groups (Figure 5A,B; tissues collected 1 h post dosing). AZ levels were similar in the cerebral cortices of dams and embryos ( and , respectively), and there were no significant differences in AZ levels in the cortex when embryos were analyzed separately by sex at this time point, (male: vs. female ; Figure S4A). AZ-acid levels in the cerebral cortex were also similar between dams and embryos ( and , respectively). AZ levels were significantly (3.6-fold) higher than AZ-acid in cortices dissected from embryos. AZ and AZ-acid were also detected in the placenta, although the ratio of AZ to AZ-acid differed relative to the brain. In the placenta, the concentration of AZ-acid was 7.1-fold higher than AZ ( vs. , respectively) (Figure 5C). AZ exposure did not alter the body weight (BW) of the dams (control: ; AZ exposure: ), the number of embryos (control: ; AZ exposure: ) or weight of the embryos (control: ; AZ exposure: ) when compared with the control groups (Figure S4 B–D).

Figure 5.

Concentration of azoxystrobin and azoxystrobin-acid in (A) dam () and (B) embryo’s brain (), and (C) placenta () after dams were treated with azoxystrobin from E0.5-E14.5 relative to vehicle-treated control group. (D) Concentration of azoxystrobin in pup’s brain via lactational exposure () compared with the concentration of azoxystrobin in dam’s brain (); dotted line represents the level of azoxystrobin in embryo’s brain shown in (B). Results are expressed as . *(), **() indicate values significantly different from vehicle-treated controls. Analysis of variance followed by post hoc Dunnett’s test was performed to determine the statistical significance of the results. Note: SEM, standard error of the mean.

To evaluate lactational transfer, dams were treated with corn oil or AZ () from PND 1 to PND 12. Cerebral cortices from the dams and pups were then collected on PND 12. No AZ or AZ-acid was detected in the corn oil control group (Figure 5D). In the treated group, the level of AZ-acid was in dams’ brains, and the level of AZ-acid in pups’ brains was below LOQ (Figure S5). AZ was detected in the dams’ brains and pups’ brains ( vs. , respectively). Lactational AZ exposure did not alter the BW (control: ; AZ exposure: ) or cortex weight of the pups (control: ; AZ exposure: ) (Figure S6).

Measurement of AZ-Induced Embryonic Cortical Neuron Death in Culture

AZ was detectable in the cerebral cortex of dams, embryos, and pups (average concentration in dam and embryo was , equal to AZ), so we next sought to determine whether AZ or AZ-acid had adverse effects on cultured cortical neurons at various concentrations. Using SYTOX green live/dead staining, we found that cells exposed to AZ at concentrations of 10, 100, 1,000, and exhibited significantly more cell death after 7 d exposure in comparison with control, and the percent of dead cells was , , , and , respectively (Figure 6A). We also exposed cortical neuron cultures to AZ-acid at multiple concentrations. We found that AZ-acid-treated cells died at 1,000 and , and the percent of dead cells was , and , respectively.

Figure 6.

Cell death of cultured mouse cortical neurons following treatment with azoxystrobin and azoxystrobin-acid at the concentration of 1, 10, 100, 1,000, and . (A) Percentage of dead cells based on SYTOX green labeling and (B) NeuN positive cells after treatment (for 7 d) with azoxystrobin or azoxystrobin-acid (vehicle-subtracted; the percentage of cell death in vehicle control group was 7.3%). (C, D) NeuN staining of mouse cortical neuron cultures treated with DMSO or azoxystrobin. Values are (). Data can be found in “Results” section. *(), **() indicate values significantly different from vehicle-treated controls. Analysis of variance followed by post hoc Dunnett’s test was performed to determine the statistical significance of the results. Note: SEM, standard error of the mean.

To specifically evaluate neurotoxicity, we quantified the number of neurons in vehicle and AZ/AZ-acid treated cultures. We detected significantly less cells in cultures treated with 10, 100, 1,000, and AZ, and the percent of cells was , , , and , respectively. Cells exposed to AZ-acid at concentrations of 1,000 and also had lower percentages of cells to and in comparison with control.

Discussion

Pesticide metabolites, when detected in urine, provide strong evidence of exposure to the parent pesticide(s). In our present study, we identified AZ-acid as a key metabolite of AZ in human S9 liver fractions. We also developed a sensitive mass spectrometry assay to detect AZ-acid in urine and other tissues. We found that AZ-acid was commonly detected in urine samples from 67 children between the ages of 40 and 84 months and 8 pregnant adults, suggesting exposure to AZ is widespread in the sampled population. We also investigated the toxicokinetic properties of AZ in mice. We found that AZ could be transferred from exposed mothers to their offspring prenatally and postnatally, and that AZ led to cell death of neurons in culture at relatively low concentrations. Such information is needed to inform future toxicological research and will facilitate risk management of this strobilurin fungicide.

In mice, the urinary concentration of AZ-acid was higher than that of AZ following oral AZ dosing. The higher concentration of AZ-acid relative to AZ suggested that AZ-acid could serve as a more sensitive readout of exposure relative to the parent pesticide. We cannot exclude the possibility that humans are also exposed to AZ-acid, although it is unclear how such exposure might occur. AZ-acid is not used as a pesticide (it was at least less toxic than AZ, based on our cell viability results as shown in Figure 6), and humans are unlikely to consume water directly from natural aquatic environments, where AZ-acid was detected as a microbial biotransformation product (Singh et al. 2010). In mice, AZ levels were largely similar between the sexes over time (Figure 3A), our data suggested that AZ was metabolized at similar rates in males and females, but the excretion rate of AZ-acid was relatively high in female mice, which was consistent with a previous report using rats and radiolabeled AZ (WHO/FAO 2008).

In our study, urinary AZ-acid was detected in 70% (67 out of 96) of children and 100% (12 out of 12) of all pregnant women sampled. These results suggested that AZ is biotransformed to AZ-acid in humans and that AZ-acid could be used to evaluate AZ exposure in the general population. These AZ-acid detection frequencies were much higher than those found in prior studies that measured the parent compound (AZ) in human blood and urine samples. Specifically, Chang et al. detected AZ in blood from 3% (6/200) of the participants (adult population in China) with a concentration that ranged from to (Chang et al. 2017). Gallo et al. detected AZ in urine from 10% of the participants (1 out of 10; healthy adults) (Gallo et al. 2021), which is similar to our findings (Table S10). The lower detection rate of AZ relative to AZ-acid in urine could be due to the rapid metabolism of AZ to AZ-acid in rats and mice (EFSA 2010). As noted in the “Introduction” section, AZ usage has been increasing year over year (U.S. Geological Survey 2017), and AZ was added to PURPLE® branded mold- and mildew-resistant wallboard beginning around 2004 (U.S. EPA 2004). The AZ-containing wallboard is increasingly being used in new construction and renovations in the United States (Cooper et al. 2020). Our study participants were sampled in the United States and samples were collected more recently than in the Chang et al. study (Chang et al. 2017), which could contribute to higher detection frequency and concentration.

We collected repeated urine samples from a subset of the children, allowing us to evaluate whether AZ exposure varied in specific individuals over time. Most of the urine samples collected in a 9–12 month follow-up had lower AZ-acid levels. However, AZ-acid levels were relatively constant and above the mean in two participants. Further studies will be needed to evaluate temporal and spatial variability over time. When compared with other commonly detected pesticides in children 6–11 y of age (Oulhote and Bouchard 2013), the level of AZ-acid was similar in magnitude to metabolites of pyrethroid pesticides but lower than nonspecific dialkyl phosphate metabolites of organophosphorous pesticides (Table S11). It should be noted that these metabolites represent several different parent chemicals of pyrethroids and organophosphates. In contrast, AZ-acid is specifically indicative of AZ-exposure and not broadly indicative of strobilurin fungicide exposure. It is unlikely that eating conventionally grown crops results in high chronic levels of exposure, because only trace amounts of AZ were typically detected on nonorganic crops in the United States, such as cilantro, spinach, kale, and sweet potato (USDA 2020). However, AZ-containing wallboards appear to release AZ into indoor dust samples with the concentration of to 10,590 ng/g, based on the highest measured values from dust samples (Cooper et al. 2020). These dust samples were collected from North Carolina homes in 2014–2016 and hence may underestimate the present-day exposures from new construction, which increasingly incorporates AZ-containing wallboard (Cooper et al. 2020). Overall, our data suggested that AZ is an emerging environmental exposure in humans, and future biomonitoring of AZ and AZ-acid is warranted.

Further support for AZ as an emerging environmental exposure comes from our estimates of daily AZ intake, which ranged from for children and from for pregnant women. This upper estimate of daily intake was higher than our initial approximation (see “Methods” section), which was based on AZ levels in house dust collected in 2014–2016 (Cooper et al. 2020). The urine samples in our present study were collected more recently (see “Methods” section), suggesting a need to expand dust sampling to newer homes to better estimate present-day AZ exposure. Among these child participants, the average estimated daily intake was , which is lower than the acceptable daily intake of AZ () estimated in a previous report (WHO 2019) but 24- to 97-fold higher than intake reported in a previous study that estimated dietary daily AZ intake of for children between 2 and10 y old (Winter 2015). This previous study assumed that the primary source of exposure to AZ was through the diet. Our much higher exposure estimate, based on real-world sampling data in humans, reinforces the possibility that exposure pathways beyond food exist for AZ.

It should also be noted that the acceptable daily intake of AZ was based on toxicological studies, most of which used aqueous solvents that do not dissolve strobilurin fungicides (Tuttle et al. 2019). A case study used new approach methodology (NAM) data to conclude that AZ is not a neurotoxicant (Hougaard Bennekou et al. 2020). However, it may be necessary to revise the acceptable daily intake estimate of AZ and other strobilurin fungicides, after performing new developmental and adult neurotoxicological studies that, at a minimum, use fully solubilized fungicides.

Our data suggest that represents an environmentally relevant exposure dose range for chronic studies. This range is based on our finding that human urinary AZ-acid levels reached , which was similar to urinary AZ-acid levels in mice 1 h after oral dosing with AZ and was similar to urinary AZ-acid levels 24 h after oral dosing with AZ.

We found that AZ accumulated in the cerebral cortex of embryonic and weanling mice when the mother was exposed to AZ, demonstrating AZ could be transferred from exposed dams to offspring during the prenatal and postnatal periods via the placenta and breast milk, respectively. The ratio of AZ/AZ-acid in embryonic cortex was 8.44, and the ratio was 0.15 in placenta, suggesting a high placental transfer efficiency of AZ. The most common mechanism for xenobiotics to cross the placenta is passive diffusion. The MW of AZ and AZ-acid are 403.4 Da and 389.4 Da, respectively, suggesting that both chemicals could cross the placenta (Myllynen et al. 2007). We observed that the concentration of AZ in the cerebral cortex of embryonic mice was significantly higher than that of AZ-acid, suggesting a relatively high placental transfer efficiency of AZ, possibly reflective of the higher lipophilicity of AZ (Log ) relative to AZ-acid (Log ).

We also found evidence of lactational transfer of AZ into the brain of weanling pups, although the concentration of AZ following lactational transfer was lower than that of the placental transfer. This finding could reflect the low Log of AZ (Log ). Others previously found that environmental chemicals with relative high Log enter breast milk. Chemicals such as 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (Log ), 2,2′,4,4′,5,5′-hexachlorobiphenyl (Log ), p,p′-dichlorodiphenyldichloroethylene (Log ) were reported to be transferred to offspring almost exclusively through milk, rather than by placental transfer (Lyche et al. 2004; Phillips et al. 2016; You et al. 1999).

AZ is a highly effective fungicide (quinone outside inhibitor) that inhibits mitochondrial complex III and blocks electron transport (Bartlett et al. 2002). Mitochondrial complex III-deficient mice showed superoxide-dependent damage to cortical brain regions (Diaz et al. 2012; Esser et al. 2014). Previous studies found that AZ induced ROS production in embryonic cortical neuron cultures with an of , when treated acutely (Pearson et al. 2016). The intracellular accumulation of ROS can trigger the oxidation of proteins, lipids, and nucleic acids and ultimately causes cell death (Lewén et al. 2000). In the present study, we found that AZ accumulated in the cerebral cortex of embryonic and early postnatal mice at a concentration of following chronic exposure. AZ at a concentration of induced cell death in cultured cortical neurons. Although these cultures are primarily made up of cortical neurons (Pearson et al. 2016), the SYTOX Green live/dead stain does not discriminate between neurons and nonneuronal cells. We further used NeuN, a well-established neuronal marker (Wolf et al. 1996) to specifically evaluate neurotoxicity of AZ. AZ also decreased the number of cells at concentration of . Collectively, these data show that AZ, induced cell death in cultured cortical neurons in a concentration-dependent manner, consistent with prior studies (Pearson et al. 2016; Simon et al. 2019). Moreover, these data suggest that AZ had deleterious effects at concentrations that were reached in the embryonic and adult brain during chronic AZ exposure. These data reinforce the possibility that chronic exposure to AZ has the potential to negatively impact the brain.

Our results should be interpreted in the context of several limitations: Our estimated daily intake was based on the molar fraction of AZ to AZ metabolites obtained from an animal study; however, human excretion could vary considerably. The urine output estimate we used was based on prior research by other groups. Urine output may differ for pregnant women in different gestation weeks. We did not measure the urine output from the participants in our study. Finally, a relatively small number of participants were sampled in our study, and all resided within close proximity to a tertiary care medical center. Therefore, exposures may be different in geographically distinct areas. Studies of nationally representative populations are needed. Our study shows that AZ-acid could serve as a more sensitive readout of AZ exposure and could be incorporated into future epidemiological studies, to evaluate more rigorously the extent to which AZ exposure affects health outcomes in human populations.

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