Regulatory trends of organophosphate and pyrethroid pesticides in cannabis and applications of the Comparative Toxicogenomics Database and Caenorhabditis elegans

Abstract

Organophosphate and pyrethroid pesticides are common contaminants in cannabis. Due to the status of cannabis as an illicit Schedule I substance at the federal level, there are no unified national guidelines in the United States to mitigate the health risk of pesticide exposure in cannabis. Here, we examined the change in the state-level regulations of organophosphate and pyrethroid pesticides in cannabis. The medians of pyrethroid and organophosphate pesticides specified by each state-level jurisdiction increased from zero pesticide in 2019 to 4.5 pyrethroid and 7 organophosphate pesticides in 2023, respectively. Next, we evaluated the potential connections between pyrethroids, organophosphates, cannabinoids, and Parkinson's disease using the Comparative Toxicogenomics Database (CTD). Eleven pyrethroids, 30 organophosphates, and 14 cannabinoids were associated with 95 genes to form 3,237 inferred and curated Chemical-Gene-Phenotype-Disease tetramers. Using a behavioral repulsion assay with the whole organism model Caenorhabditis elegans, we examined the effect of cannabinoids and insecticides on depleting dopamine synthesis. Exposure to chlorpyrifos and permethrin, but not Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), results in dose-dependent effects on 1-nonanol repulsive behaviors in C. elegans, indicating dopaminergic neurotoxicity (P < 0.01). Dose-dependent effects of chlorpyrifos are different in the presence of Δ9-THC and CBD (P < 0.001). As a proof of concept, this study demonstrated how to use new approach methodologies such as C. elegans and the CTD to inform further testing and pesticide regulations in cannabis by chemical class.

Keywords: Caenorhabditis elegans; cannabis; new approach methodology; pesticide; regulatory policy; systems biology.

Fig. 1.

Histograms of the number of organophosphate (left) and pyrethroid pesticides (right) listed by each jurisdiction in the regulatory documents for legalized medical or recreational cannabis in 2019 and 2023. The dashed lines indicate the median number of pesticides regulated in each class for each year.

Fig. 2.

Box plot of the regulatory action levels of organophosphate and pyrethroid pesticides ranked by the number of mentions in state-level regulatory documents for flower/inhalable products in 2019 and 2023. The top 11 pesticides’ action levels for 2019 and 2023 are plotted in a strip plot on a log 10 scale in ppm with a boxplot background to show the distribution of regulatory action levels for each pesticide. Each chemical name is colored based on the chemical class (orange for pyrethroids, purple for organophosphates) with the number of action levels for each year listed to the right. The upper boxplots for each pesticide show the action levels in 2019, whereas the lower plots for each pesticide show the action levels in 2023.

Fig. 3.

(A, B) Quantitative bipartite network showing the chemical–gene connections between (A) cannabinoids and organophosphate pesticides and (B) cannabinoids and pyrethroid pesticides in the CTD. The chemical–gene connections were quantified by the number of curated and inferred tetramers connecting organophosphate pesticides, pyrethroid pesticides, and cannabinoids to Parkinson’s disease through genes and phenotypes in the CTD. The connections for each pesticide class were passed into Gephi—a network analysis and visualization application—to create a bimodal network color-coded by the gene’s biological function according to KEGG pathways and the Reactome Knowledgebase.

Fig. 4.

One-way ANOVAs showing exposure to chlorpyrifos and permethrin (A and B), but not THC and CBD (C and D), results in dose-dependent effects on 1-nonanol repulsive behaviors of Caenorhabditis elegans (n = 36 to 103). Each dose is compared with control using Dunnett’s post hoc test.

Fig. 5.

Two-way ANOVAs showing different dose-dependent effects of chlorpyrifos on 1-nonanol repulsive behaviors of Caenorhabditis elegans with THC, CBD, and no cannabinoid (n = 93 to 103). The first factor of the 2-factor ANOVA consisted of 4 doses with 0, 7.5, 15, and 50 µM for chlorpyrifos. The second factor consisted of 3 groups—100 µM Δ9-THC, 100 µM CBD, and ethanol (i.e. no cannabinoid) as control. Chlorpyrifos doses and cannabinoid groups showed a statistically significant interaction (P < 0.001). Each chlorpyrifos dose was compared with control in each cannabinoid group using pairwise comparison with Bonferroni’s multiple testing correction. The asterisks (***) denote P < 0.001.