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

Abstract

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 ($0.01\text{ng}/\mathrm{mL}$) 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 $0.07\text{ng}/\mathrm{mL}$, and maximal concentration of 2.70 and $6.32\text{ng}/\mathrm{mL}$, 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.

Figure 1.

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

Figure 2.

Concentration of (A) azoxystrobin and (B) azoxystrobin-acid in mouse urine after oral administration of azoxystrobin at the indicated concentrations ($n=12$ per group, with 6 male and 6 female). Concentration of (C) azoxystrobin and (D) azoxystrobin-acid after treating urine samples with $\mathrm{\beta }\text{-glucuronidase}$ ($n=12$ per group, with 6 male and 6 female). Values were shown in Table S8 and S9. Results are expressed as $\text{means}\pm \text{SEM}$. *($p<0.05$), **($p<0.01$) 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.

Figure 3.

Urine (A) azoxystrobin and (B) azoxystrobin-acid concentration separated by sex. Male ($n=6$) and female ($n=6$) mice were orally gavaged with $2\mathrm{mg}/\mathrm{kg}$ azoxystrobin and urine samples were treated with $\mathrm{\beta }\text{-glucuronidase}$ prior to analysis. Results are expressed as $\text{means}\pm \text{SEM}$. Data can be found in the “Results” section. * ($p<0.05$), ** ($p<0.01$) 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.

Figure 4.

Urine azoxystrobin-acid concentration in children sampled twice ($n=20$). The second urine samples were collected 9–12 months after the first samples. Data can be found in Table S6.

Figure 5.

Concentration of azoxystrobin and azoxystrobin-acid in (A) dam ($n=3/\text{group}$) and (B) embryo’s brain ($n=21-23/\text{group}$), and (C) placenta ($n=21-23/\text{group}$) after dams were treated with $2\mathrm{mg}/\mathrm{kg}$ azoxystrobin from E0.5-E14.5 relative to vehicle-treated control group. (D) Concentration of azoxystrobin in pup’s brain via lactational exposure ($n=18-19/\text{group}$) compared with the concentration of azoxystrobin in dam’s brain ($n=4/\text{group}$); dotted line represents the level of azoxystrobin in embryo’s brain shown in (B). Results are expressed as $\text{means}\pm \text{SEM}$. *($p<0.05$), **($p<0.01$) 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.

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 $\mathrm{10,000}\text{\hspace{0.17em}nM}$. (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 $\mathrm{1,000}\text{\hspace{0.17em}nM}$ azoxystrobin. Values are $\text{means}\pm \text{SEM}$ ($n=4$). Data can be found in “Results” section. *($p<0.05$), **($p<0.01$) 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.