Blood and Urinary Metal Levels among Exclusive Marijuana Users in NHANES (2005–2018)

Background: Marijuana is the third most used drug in the world. Objectives: Because the cannabis plant is a known scavenger of metals, we hypothesized that individuals who use marijuana will have higher metal biomarker levels compared with those who do not use. Methods: We combined data from the National Health and Nutrition Examination Survey (2005–2018) for n=7,254 participants, classified by use: non-marijuana/non-tobacco, exclusive marijuana, exclusive tobacco, and dual marijuana and tobacco use. Five metals were measured in blood and 16 in urine using inductively coupled plasma mass spectrometry; urinary metals were adjusted for urinary creatinine. Results: Participants reporting exclusive marijuana use compared with non-marijuana/non-tobacco use had statistically significantly higher mean cadmium levels in blood [1.22μg/L (95% CI: 1.11, 1.34); p<0.001] and urine [1.18μg/g (95% CI: 1.0, 1.31); p=0.004] and statistically significantly higher mean lead levels in blood [1.27μg/dL (95% CI: 1.07, 1.50); p=0.006] and urine [1.21μg/g (95% CI: −0.006, 1.50); p=0.058]. Discussion: Our results suggest marijuana is a source of cadmium and lead exposure. Research regarding cannabis use and cannabis contaminants, particularly metals, should be conducted to address public health concerns related to the growing number of cannabis users. https://doi.org/10.1289/EHP12074


Introduction
Marijuana is the third most commonly used drug in the world behind tobacco and alcohol. 1 As of 2022, 21 states and Washington, DC, covering >50% of the U.S. population, have legalized recreational use of marijuana, and medical marijuana is legal in 38 states and Washington, DC. 2 However, because marijuana is still illegal at the federal level, regulation of contaminants in all cannabis-containing products remains piecemeal and there has been no guidance from federal regulatory agencies such as the U.S. Food and Drug Administration or the U.S. Environmental Protection Agency. 3etal and metalloid (henceforth collectively referred to as metal) contamination of marijuana products occurs during growth, production, and consumption, posing potential harmful effects to end users. 3The cannabis plant, from which marijuana is derived, is a known hyperaccumulator of metals present in water, soil, fertilizers, and pesticides. 4Unfiltered marijuana smoke contains high concentrations of metals 5 and vape delivery devices have shown metal leaching in cannabis aerosols. 6Although 28 states regulate inorganic arsenic (As), cadmium (Cd), lead (Pb), and total mercury (Hg) concentrations in marijuana products, regulation limits vary by metal and by state. 7Limited data exists for exposure to other metals, such as chromium (Cr), cobalt (Co), and nickel (Ni), that may come from relevant consumption practices. 8At the limits regulated by the states of California 9 and Colorado 10 (0:2 lg=g As, 0:2 lg=g Cd, 0:5 lg=g Pb, and 0:1 lg=g total Hg in inhalable marijuana products), consumers may be exposed to metal levels that have been shown to be associated with cardiopulmonary diseases, [11][12][13][14][15][16] neurodevelopmental effects, and cancer. 17,18ecause marijuana is relatively unregulated in an industry experiencing exponential growth, there is a need to understand contaminant exposures, including metals, associated with marijuana use.As of 2019, 48:2 million people, or 18% of Americans, report using marijuana at least once within the last year. 19espite the robust literature on metal biomarker levels among cigarette smokers [20][21][22] and growing evidence of metal contents in marijuana products, 7,[23][24][25] few studies have reported biomarker metal levels among marijuana users. 26In 2020, Ngueta et al.  reported an association between lifetime use of marijuana with urinary and blood Cd levels in the National Health and Nutrition Examination Survey (NHANES) 2009-2016.Other metals beyond Cd and the contribution of recent marijuana use have not been evaluated in NHANES or other populations.We hypothesized that individuals reporting current marijuana use (either exclusive marijuana or dual tobacco and marijuana use) would have higher levels of biospecimen metals than non-marijuana, non-tobacco users, reflecting internal dose.This secondary data analysis of all measured metals in NHANES, a representative sample of the U.S. population, compared participants' metal levels (5 metals in blood and 16 in urine) by categories of marijuana and tobacco use.Among exclusive marijuana users, we compared metal levels by days since last use.

Study Population
Led by the National Center for Health Statistics (NCHS) at the Centers for Disease Control and Prevention (CDC), NHANES is a biannual program of studies designed to assess the health and nutritional status of adults and children in the United States.NHANES is designed as a multiyear, stratified, clustered fourstage sample of noninstitutionalized civilians with fixed samplesize targets for sampling domains defined by age, sex, race and ethnicity, and socioeconomic status, with data released in 2-y cycles.Participants gave informed consent of the survey process and their rights as a participant, and the survey was approved by the NCHS Review Board. 27Questionnaires were administered inhome followed by standardized health examinations in specially equipped mobile examination centers.Publicly available, deidentified, and detailed health data sets are available on the NHANES website (https://wwwn.cdc.gov/nchs/nhanes/).We acquired NHANES data from seven 2-y cycles (2005-2018) to create a larger and more geographically diverse sample, including all cycles with available data on blood and urinary metals and detailed drug use.

Exclusion Criteria
Of the 70,190 NHANES participants from the combined 2005-2018 cycles, 10,921 participants had metals measured in blood and urine and available drug use questionnaire data.Individuals ≥18 years of age were included in our analysis.We excluded 2,928 individuals who were missing marijuana use, 337 missing serum cotinine, 335 missing urine As levels, 54 missing body mass index, 9 missing blood metals, and 4 missing urinary creatinine, leaving a total of 7,254 participants (Figure S1).

Blood Metals
As previously described, 28 whole blood specimens were collected at mobile examination centers, frozen at −20 C, and shipped to the CDC's Division of Laboratory Sciences, National Center for Environmental Health (NCEH) for analysis.Prior to analysis, whole blood samples were diluted: 1 part sample +1 part deionized water +48 parts diluent to solubilize blood components and aid aerosol generation for analysis.All metals were measured in whole blood using inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS).Cd, Pb, and total Hg were measured in whole blood for all included NHANES cycles (2005-2017).Manganese (Mn) and selenium (Se) were measured in whole blood starting in 2011 and every cycle thereafter (Table S1).Total Hg was measured only in women of childbearing age and 1-to −5-y-old children.There were no changes in equipment, laboratory methods, or laboratory site in any cycle.Lower limits of detection declined in 2013 but remained steady thereafter (Table S2).Values below the limit of detection (LOD) were imputed, with the lower level of detection (LLOD) divided by the square root of 2. The percentage of participants with metal levels below the LOD are reported in Table S2, and they range between 0% for Pb, Mn, and Se and 12% for Cd and Hg.All blood metals were reported as levels in micrograms of metal per liter of blood, or per deciliter of blood for Pb.
High-performance liquid chromatography (HPLC) coupled to ICP-DRC-MS was used to detect the As species arsenobetaine (Ab), and dimethylarsinic acid (DMA).All As species were measured at each of the NHANES cycles included (Table S2).Urine samples were diluted 1:9 with 2% (vol/vol) double-distilled nitric acid containing gallium or tellurium for internal standardization.Values below the LOD were imputed, with the LLOD divided by the square root of 2. Total As and DMA species were recalibrated to remove the contribution of Ab, an indicator of seafood consumption. 31We used the recalibrated tAs and DMA as measures of inorganic As and internal dose.All urinary metals and metal species were corrected for urine dilution using individual urinary creatinine and reported as levels in micrograms of metal per gram of creatinine.

Marijuana and Tobacco Use Categorization
We used four NHANES variables to define exclusive marijuana and tobacco use: a) current cigarette smoking, b) serum cotinine levels, c) self-reported ever marijuana use, and d) recent marijuana use.Exclusive tobacco use was defined by individuals who either answered "yes" to "Do you now smoke cigarettes?" (SMQ040) or whose serum cotinine levels were >10 ng=mL (LBXCOT). 32on-tobacco use was defined by individuals who either answered "no" to now smoking cigarettes or whose serum cotinine levels were ≤10 ng=mL.Reclassification of self-reported smoking status by serum cotinine levels increased the number of smokers from 1,745 to 2,207 (Table S3).Any individuals with missing selfreported smoking status or serum cotinine were removed from analysis.Serum cotinine was measured by an isotope-dilution HPLC/atmospheric pressure chemical ionization tandem MS (ID HPLC-APCI MS/MS) method, as previously described. 33xclusive marijuana use was defined by individuals who had answered "yes" to both "Ever used marijuana or hashish?" (DUQ200) and had used marijuana within the last 30 d, as derived from the variables "Last time used marijuana" (DUQ220Q) and the unit of time at which the individual last used marijuana in days, months, weeks, or years (DUQ220U).Non-marijuana use was defined by individuals who either answered "no" to ever using marijuana or hashish or had not used marijuana in the past 30 d.We categorized individuals into four types of use: a) non-marijuana/nontobacco use (never user of marijuana or former user who had not used marijuana in >30 d and no tobacco use or serum cotinine ≤10 ng=mL), b) exclusive marijuana use (current marijuana use who had used within the last 30 d and self-reported not currently smoking cigarettes or serum cotinine levels of ≤10 ng=mL), c) exclusive tobacco use (either self-reported current cigarette smoking or serum cotinine level >10 ng=mL who had not used marijuana within the last 30 d), or d) dual use (self-reported current marijuana use who had used within the last 30 d and either self-reported current cigarette smoking or had serum cotinine levels of >10 ng=mL).Hereafter, we refer to these categories as non-marijuana/nontobacco use, exclusive marijuana use, exclusive tobacco use, or dual use (Table S4).
For our analysis on time since last use among exclusive marijuana users, we restricted analyses to exclusive marijuana use, and categorized recent marijuana use into four groups: individuals who had never used marijuana or had not used marijuana in over a year (reference group), and individuals who had exclusively used marijuana within the last 7, 8-30, or 31-365 d (Table S4).information.Race and ethnicity was classified as non-Hispanic White, non-Hispanic Black, Mexican American, Other Hispanic, and Other races (including multiple races), based on self-identified race and ethnicity as originally designated by NHANES."Other races" included all other races, including non-Hispanic Asian individuals and those reporting multiple races.In the NHANES cycles 2011-2014, non-Hispanic Asian individuals were oversampled using a new race and ethnicity variable (RIDRETH3).Because this variable was included only during half of the study period, we used the original race and ethnicity classification (RIDRETH1).Coexposure to other metal exposures (e.g., diet) may differ by race and ethnicity and was therefore used for adjustment in our models. 34We reclassified education as less than a high school education, a high school education or General Education Development (GED) equivalent, or more than a high school education.Household income categories were categorized as $0-$24,999, $25,000-$54,999, $55,000-$74,999, and ≥$75,000.Body mass index (BMI; in kilograms per meter cubed) was measured at the mobile examination center.Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), 35,36 which estimates GFR for the specified categories of race, sex, and serum creatinine in micromoles per liter.eGFR is a measure of glomerular function.GFR can influence metal excretion in urine and was therefore used for adjustment in our models.

Statistical Analysis
To evaluate the characteristics of the study population, we compared participant sociodemographic characteristics, self-reported marijuana and tobacco use, and biomarkers of marijuana and tobacco use across categories of marijuana use using analyses of variance, t-tests, and v 2 tests.To estimate the arithmetic mean difference of biomarker metal concentrations by marijuana use, we built generalized linear models with the gamma distribution and log link function for right-skewed metal concentrations.Groups based on recent marijuana and tobacco use as described above were modeled as categorical independent variables, and blood and urine metal levels were modeled as continuous dependent variables.8][39] Adjustments included age, sex, race and ethnicity, education, eGFR, and NHANES cycle year.We assessed effect modification of the association between exclusive marijuana and or tobacco use with urinary blood and urine metal levels by subgroups of age, sex, and race and ethnicity.

Sensitivity Analyses
We conducted several sensitivity analyses.Owing to the limitations of the drug use questionnaire, among participants reporting exclusive marijuana use, we evaluated frequency of marijuana use as a sensitivity analysis using DUQ219, "When you did use marijuana, how many pipes or joints did you smoke per day?," and DUQ230, "In the last 30 days, how many days did you use marijuana?"We conducted a sensitivity analysis without adjustment for race and ethnicity in our models.Urinary creatinine levels reflect urine dilution and can vary by age, sex, and other characteristics. 40Although the influence of kidney function on urinary creatinine levels is generally small and urinary creatinine is commonly used to correct urine albumin, a marker of kidney damage for urine dilution, we also conducted a sensitivity analysis without adjustment for urinary creatinine.We removed eGFR from our models to assess any difference in estimates, particularly for blood metals.We further adjusted our models for serum cotinine to determine whether tobacco use, use of an unaccounted-for nicotine product, or secondhand tobacco exposure may be the source of metal exposures.In addition, we assessed the effect of former cigarette smoking on urinary Cd levels by analyzing ever and never cigarette smokers separately where ever cigarette smokers were those who reported smoking at least 100 cigarettes in their lifetime and never cigarette smokers were those who did not (SMQ020).Finally, we conducted sensitivity analyses, further adjusting for seafood consumption using dietary recall or by further adjusting for urinary Ab levels to account for any metal exposure from seafood in the diet.
Combined NHANES survey cycles and weights produced estimates representative of the U.S. civilian noninstitutionalized population at the midpoint of the combined survey period.We constructed new sample weights for the combined cycles (7 cycles, 2005-2018) by multiplying 2-y subsample A weights for environmental chemicals (WTSA2YR) by 1/7 (for 7 NHANES cycles) as described. 41,42Model estimates are population-weighted arithmetic mean differences with 95% confidence intervals (CIs) compared with the reference group (non-marijuana/non-tobacco use).All central tendency estimates and proportions were population weighted.Data analysis was performed in R (version 3.1.3;R Development Core Team) using the nhanesA, 43 tidyverse, 44 and survey packages 45 to account for the complex survey design and sampling weights.

Results
The characteristics of NHANES participants by marijuana and tobacco use categories are shown in Table 1 (characteristics by recent use among exclusive marijuana users are shown in Table S5).In comparison with non-marijuana/non-tobacco use, participants reporting exclusive marijuana use were on average younger, more likely non-Hispanic White males, and had lower BMI.Exclusive marijuana use was associated with reporting more than a high school education and a higher income, and 40% had reported formerly smoking cigarettes in their lifetime.Of individuals who used neither marijuana nor tobacco currently, 47% had used marijuana in their lifetime.In unadjusted analysis, blood and urinary metals were lower, except for Cd and Hg in blood, and Sr and Tl in urine, in individuals who reported exclusive marijuana use compared with non-marijuana/non-tobacco use (Table 2; Table S6).
In fully adjusted analyses, we found that blood Cd and Pb levels were higher in participants reporting exclusive marijuana use, exclusive tobacco use, and dual use as compared with nonmarijuana/non-tobacco use (Figure 1; Table S7).We found 1:22 lg=L (95% CI: 1.11, 1.34; p < 0:001) higher blood Cd levels and 1:27 lg=dL (95% CI: 1.07, 1.50; p = 0:006) higher blood Pb levels in participants reporting exclusive marijuana use compared with non-marijuana/non-tobacco use when adjusting for age, sex, race and ethnicity, education, and NHANES cycle year.These results were confirmed in urine where exclusive marijuana use was associated with 1:18 lg=g (95% CI: 1.06, 1.31; p = 0:004) higher urinary Cd levels and 1:21 lg=g (95% CI: 0.99, 1.50; p = 0:06) higher urinary Pb levels compared with non-marijuana/non-tobacco use (Figure 2; Table S8).Exclusive marijuana use was associated with 1:34 lg=L (95% CI: 1.03, 1.73; p = 0:03) higher total blood Hg level.We found that exclusive tobacco use was associated with higher blood levels of Cd and Pb; higher urinary levels of Sb, Ba, Cd, Pb, and U; and lower urinary levels of Mo compared with nonmarijuana/non-tobacco use.Dual tobacco and marijuana use was also associated with higher blood levels of Cd and Pb and higher urinary levels of Cd, Pb, and U compared with non-marijuana/nontobacco use.
To assess metal levels by recent marijuana use, we restricted the sample to only those individuals who had used marijuana Environmental Health Perspectives 087019-3 131(8) August 2023 within the last year (n = 569) and non-marijuana/non-tobacco users (n = 4,455), excluding exclusive tobacco and dual users (see Tables S5 and S6 for descriptive statistics).In fully adjusted analyses, individuals who had used marijuana within the last 7 d had higher Cd and Pb levels in both blood and urine compared with those who did not use marijuana or had not used marijuana in more than a year.We found 1:23 lg=L (95% CI: 1.12, 1.35; p < 0:001) higher Cd and 1:39 lg=dL (95% CI: 1.11, 1.75; p = 0:005) higher Pb concentrations in blood among those who had used in the last week as compared with those who do not use marijuana or had not used marijuana within the last year (Figure 3; Table S9).These results were confirmed in urine with 1:20 lg=g (95% CI: 1.03, 1.39; p = 0:02) higher Cd and a 1:31 lg=g (95% CI: 1.01, 1.70; p = 0:045) higher Pb levels compared with non-marijuana use (Figure 4; Table S10).As time since last use increased, mean Cd and Pb levels in blood and urine were lower.Marijuana use within the last 7 d was associated with 1:40 lg=L (95% CI: 1.06, 1.85; p = 0:02) higher total blood Hg level.
In sensitivity analyses, we found that associations for Cd and Pb levels in both urine and blood were higher among individuals >30 years of age (Figures S2 and S3).Exclusive marijuana users who were females had higher blood and urinary Cd levels than males.However, males who used tobacco and reported dual use had higher levels of blood and urinary Cd.Associations for blood and urinary Cd levels were stronger among non-Hispanic White individuals than other race and ethnicity groups (Figures S4 and  S5).In addition, we evaluated the frequency of marijuana use among exclusive marijuana users, modeling the number of joints or pipes smoked per day.Consistent with our primary analysis on time since last use among exclusive marijuana users, we found similar patterns for the number of joints or pipes smoked per day.Those who reported using more than three to five joints or pipes per day had had higher levels of blood Cd and those reporting six or more joints or pipes per day had higher levels of blood and urinary Pb (Figures S6 and S7).In addition, we evaluated models without adjustment for race and ethnicity and found little difference in the results (Figures S8-S11).In sensitivity analyses, we used urine metals not accounting for urine dilution using urinary creatinine, and urine metals adjusted for urine creatinine in the model (instead of dividing) and found little to no change in the results (Figures S12 and S13).Similarly, we removed the adjustment for eGFR and found little difference in the results (Figures S14-S17).We added cotinine to our models to assess any potential confounding of tobacco and secondhand smoke exposure.The associations were attenuated only in those who smoked and those reporting dual use, although these results remained significant, with no changes for recent exclusive marijuana use (Figures S18-S21).To further assess potential confounding by former cigarette use, we stratified by self-reported ever/never cigarette use and found that effect estimates among never cigarette smokers were attenuated for urinary metals; however, results remained consistent for blood metals (Figures S22 and S23).Finally, adjustment for potential metal exposures from seafood consumption showed an attenuation for the association between recent exclusive marijuana use and Hg levels (Figures S24-S27).

Discussion
In this nationally representative study of NHANES participants, we found higher levels of Cd and Pb in blood and urine among participants reporting exclusive marijuana use when compared with participants who neither used marijuana nor tobacco.Cd and Pb levels were also higher in exclusive marijuana users who reported using marijuana within the last 7 d, with metal levels being lower with increased time since last use.Cd biomarker levels were higher in exclusive tobacco users compared with exclusive marijuana users, either because of differences in frequency of use or differences in Cd levels in the tobacco and cannabis plants themselves.However, blood and urinary Pb levels among those who exclusively used marijuana and those who exclusively used tobacco were similar.Dual marijuana and tobacco users also had higher levels of Cd and Pb compared with participants who used neither marijuana nor tobacco.Taken together, these observations suggest that marijuana use is an important and underrecognized source of Cd and Pb exposure independent of tobacco use and that chronic marijuana use may contribute to adverse health effects associated with chronic, low-level metal exposure.
One other study assessed blood and urinary Cd levels in individuals who use marijuana in the 2009-2016 NHANES cycles.Ngueta et al. found that marijuana users had higher Cd levels, and that Cd levels were positively associated with increased frequency and duration of marijuana use. 26Although the work of Ngueta et al. focused on the relationship between lifetime marijuana use and Cd biomarkers, our study was further able to identify differences in metal levels, including but not limited to Cd, with increasing time since last use among individuals who had recently used marijuana.Furthermore, our analysis more stringently defined exclusive marijuana use by removing underlying metal exposures from tobacco use and assessing multiple metals in addition to Cd.Because tobacco is the predominant source of Cd exposure among cigarette smokers, 11 we also evaluated differences in Cd levels stratified by former smoking status, a more robust indicator of past tobacco use than serum cotinine levels.We found that the relationship between exclusive marijuana use and blood Cd levels were the same regardless of former smoking behavior.However, for urinary Cd, the association between exclusive marijuana users and Cd was consistent across smoking status, but it was stronger for former smokers.7][48] As in our former smoking behavior analysis, urinary Cd was higher among individuals >30 years of age,  e Total Arsenic and DMA were recalibrated for arsenobetaine to remove exposure from seafood consumption.
f Cadmium, manganese, total mercury, lead, and selenium were measured in whole blood (lg=L).
g Lead was measured in whole blood and is reported as lg=dL.
Environmental Health Perspectives 087019-5 131(8) August 2023 whereas blood Cd was not different between age groups, likely because individuals who used marijuana were younger and thus had a lower body burden of Cd.Females who used marijuana had higher urinary Cd levels compared with males, which has been previously reported and may be explained by differences in divalent metal transporter (DMT1) expression. 49We did not find clear differences in urinary metal levels by race and ethnicity, with the exception that participants in the "Other races" group had lower levels of metals than those who did not use marijuana.However, associations between marijuana use and blood Cd levels were somewhat stronger among non-Hispanic White participants.
Although urinary Pb levels are reported to be poorly correlated with low levels of environmental exposures, 50 we found that blood and urinary Pb levels were both consistently higher among individuals who exclusively used marijuana compared with nonusers.Pb is a heavy metal known to be detrimental to human health, particularly associated with adverse neurodevelopmental effects in children and cardiovascular disease in adults 17 ; health organizations acknowledge that there is no safe level of Pb exposure. 51Pb is persistent in the environment, 52 and poses potential risks from exposure.Our results indicate that marijuana use was one of these unregulated sources of exposure.Contrary to our study, a recent laboratory-based study of aerosols found no Pb in marijuana smoke. 5However, the authors reported that the marijuana used in their study was grown in a controlled and contaminant-regulated environment.Thus, our results suggest that not all marijuana is grown in this way and may be contaminated with metals such as Pb.Lower quality fertilizers and irrigation water or soils contaminated with Pb may also contribute to higher Pb levels in the cannabis plant.Finally, a recent study found higher concentrations of Pb in vape aerosols from tobacco/nicotine e-cigarettes, indicating that vaporization may cause metal leaching from vape devices (Ni, Cr, Pb, and zinc), supporting that method of marijuana use may be relevant to our findings. 8However, method of use was not collected in NHANES, and information concerning metals in cannabis vape aerosols is limited.S8 for all model estimates.The reference group of non-marijuana/non-tobacco use included individuals who had not used marijuana within the last 30 d and individuals who self-reported they did not currently smoke cigarettes or those with serum cotinine levels of ≤10 ng=mL (n = 4,666).Exclusive marijuana use included those who had used marijuana within the last 30 d and had serum cotinine levels of <10 ng=mL (n = 358).Exclusive tobacco use included those who either self-reported currently smoking cigarettes or had a serum cotinine level of >10 ng=mL and had not used marijuana within the last 30 d (n = 1,511).Dual users included both individuals who had used marijuana within the last 30 d and either currently smoked cigarettes or had serum cotinine levels of >10 ng=mL (n = 719).Models were adjusted for age, sex, race, education, eGFR, and NHANES cycle year.tAs and DMA were recalibrated for arsenobetaine to remove exposure from seafood consumption.Note: Ba, barium; Cd, cadmium; CI, confidence interval; Co, cobalt; Cs, cesium; DMA, dimethylarsinic acid; eGFR, estimated glomerular filtration rate; Mn, manganese; Mo, molybdenum; NHANES, National Health and Nutrition Examination Survey; Pb, lead; Sb, antimony; Sn, tin; Sr, strontium; tAs, total arsenic; Tl, thallium; U, uranium; W, tungsten.
Environmental Health Perspectives 087019-6 131(8) August 2023 Total blood Hg levels were higher among participants reporting exclusive marijuana use and were lower with increasing time since last use.Total blood Hg levels were measured only in women of childbearing age (16-49 years of age) and 1-to 5-yold children in NHANES; however, individuals <18 years of age were excluded from our analysis.Total blood Hg is a robust measure of short-term total Hg exposure, including inorganic and organic forms. 53Diet, and in particular, fish consumption, is the primary source of organic Hg exposure in the United States. 54ur study indicates marijuana may be a source of Hg exposure.However, recent fish consumption may be confounding these results.In our sensitivity analyses, we found that adjusting for seafood consumption did attenuate the estimates for total blood Hg in exclusive marijuana users.Although these estimates were no longer significant, the estimates were still positive.
Tobacco is a documented source of metal exposure. 55In our study, we found that exclusive tobacco use was associated with higher levels of Sb, Ba, Cd, Pb, W, and U.As in the cannabis plant, Cd and Pb hyperaccumulate in tobacco plants. 56,57In addition, Cd and Pb are reported to have a high transfer rate from tobacco plant to cigarette smoke (Cd: 81%-90%; Pb 46%-60%) and are found at higher levels in the lung tissue of individuals who smoke cigarettes. 58Tobacco smoke is the main source of Cd exposure followed by consumption of food for the nonsmoking general population. 59Tobacco smoking is estimated to increase overall Cd exposure by 15%-30%, although there are discrepancies in reported percentage differences. 60In our study, we found that exclusive tobacco users had urinary Cd levels (0:75 lg=g) three times higher than those of exclusive marijuana users (0:18 lg=g).Dual users had similarly higher levels of urinary Cd compared with exclusive tobacco users (0:64 lg=g and 0:75 lg=g, respectively).The general population is exposed to Pb from drinking water, food, air and indoor dust. 61Unlike Cd, tobacco smoke is not the primary source of Pb.However, we found that exclusive tobacco users had 26% higher blood Pb levels than exclusive marijuana users.Dual users, however, had much higher blood Pb levels of 0:64 lg=dL, indicating that cumulative exposures may increase blood Pb levels, as previously reported with Pb from diet. 62,63Ba has been measured in tobacco plants at high levels (123:0 lg=g), but was reported to have a lower rate of transfer to cigarette smoke. 58,64Similar to our results, Badea et al. recently found higher levels of Sb and Sr  measured in blood serum of participants recruited from Romania who smoke cigarettes. 65W and U have been reported as harmful constituents of cigarette smoke, 66 but in an earlier study of NHANES (1999-2004), the authors did not find a significant difference in urinary W levels in participants who did and did not smoke. 67Thus, our study of NHANES 2005-2018 provides updated evidence of metal exposures from cigarette smoking.
To the best of our knowledge, this is the largest known study on biomarkers of metal exposure in participants who exclusively use marijuana in a representative population of U.S. adults.We combined seven NHANES cycles to include 7,254 participants and evaluated 17 metal biomarkers measured in blood and urine in groups of marijuana use.Previous studies have measured metals in marijuana plants, products, or marijuana smoke; here, we used robust biomarkers of metal exposure and internal dose to explicitly quantify blood and urine metal levels among real-world marijuana users.Our study provides rationale to design cohort studies to investigate metal exposures and their health effects among marijuana users.Finally, we used a stringent definition of marijuana use to include current marijuana use and exclude any current cigarette smokers by self-report and serum cotinine to remove potential confounding by tobacco smoke exposure.Serum cotinine is a shortterm marker of tobacco exposure and cannot account for long-term exposure.However, sensitivity analyses adjusted for cotinine and subgroup analysis by former smoking were robust when accounting for potential exposure misclassification from long-term tobacco use.
Limitations of the study include the cross-sectional design, small sample of exclusive marijuana users, recall bias, social desirability bias, and potential for exposure misclassification.Because of the cross-sectional design, we can only estimate associations, not causation, between metal biomarker levels and marijuana use, and thus further work is required to corroborate the findings of our study.Participants in states where marijuana use is legal may be more likely to report marijuana use more accurately than those where marijuana use is still criminalized.Similarly, participants, particularly Black and Hispanic populations, historically targeted for illicit drug use may be disinclined to report marijuana use despite decriminalization, which may explain our small sample of individuals who exclusively used marijuana.Although the landscape of marijuana use is changing rapidly, the NHANES drug use questionnaire did not include method of use, such as vape, combustibles, and edibles, and thus we were unable to determine the difference in metal concentrations by method of use.Data from cohorts such as the Population Assessment of Tobacco and Health 68 or other more contemporary cohorts designed to study cannabis use may help elucidate differences in contaminants by type of marijuana and its origin and methods of use, particularly products growing in popularity, such as cannabis vape, consumables, and use of cannabidiol (CBD) oil.The combination of analytical chemistry analysis, in vitro and in vivo experiments, and epidemiological research will ultimately be needed to adequately answer how the use of marijuana products contributes to metal exposure and the resulting health effects in the U.S. population.Our results highlight the need to design contemporary studies to examine real-world marijuana use and related exposures in the rapidly growing marijuana industry.
We found overall associations between internal metal levels and exclusive marijuana use, highlighting the relevance of marijuana for metal exposure and the importance of follow-up studies to identify the long-term implications of these exposures.Future investigations of cannabis contaminants must assess other contaminants of concern and potential health effects to inform regulatory, industry and other key stakeholders, to safeguard public health and address safety concerns related to the growing use of cannabis in the United States.

b
Exclusive marijuana use includes those who had used marijuana within the last 30 d and had serum cotinine levels of <10 ng=mL (n = 358).c Exclusive tobacco use includes those who either self-reported currently smoking cigarettes or had a serum cotinine level of >10 ng=mL and had not used marijuana within the last 30 d (n = 1,511).d Dual use includes both individuals who had used marijuana within the last 30 d and either currently smoked cigarettes or had serum cotinine levels of >10 ng=mL (n = 719).

Figure 1 .
Figure 1.Arithmetic mean differences and 95% CIs in blood metal concentrations (lg=L or lg=dL for Pb) across categories of exclusive marijuana use (circle), exclusive tobacco use (square), and dual use (triangle), as compared with non-marijuana/non-tobacco use (reference), among 7,254 NHANES participants (2005-2018).See TableS7for all model estimates.The reference group of non-marijuana/non-tobacco use included individuals who had not used marijuana within the last 30 d and individuals who self-reported they did not currently smoke cigarettes or those with serum cotinine levels of ≤10 ng=mL (n = 4,666).Exclusive marijuana use included those who have used marijuana within the last 30 d and had serum cotinine levels of <10 ng=mL (n = 358).Exclusive tobacco use included those who either selfreported currently smoking cigarettes or had a serum cotinine level of >10 ng=mL and had not used marijuana within the last 30 d (n = 1,511).Dual users included both individuals who had used marijuana within the last 30 d and either currently smoked cigarettes or had serum cotinine levels of >10 ng=mL (n = 719).Models were adjusted for age, sex, race, education, eGFR, and NHANES cycle year.Note: Cd, cadmium; CI, confidence interval; eGFR, estimated glomerular filtration rate; Hg, mercury; Mn, manganese; NHANES, National Health and Nutrition Examination Survey; Pb, lead; Se, selenium.

Figure 2 .
Figure 2. Arithmetic mean differences and 95% CIs in urinary metal levels (lg=g creatinine) across categories of exclusive marijuana use (circle), exclusive tobacco use (square), and dual use (triangle), as compared with non-marijuana/non-tobacco use (reference), among 7,254 NHANES participants (2005-2018).See TableS8for all model estimates.The reference group of non-marijuana/non-tobacco use included individuals who had not used marijuana within the last 30 d and individuals who self-reported they did not currently smoke cigarettes or those with serum cotinine levels of ≤10 ng=mL (n = 4,666).Exclusive marijuana use included those who had used marijuana within the last 30 d and had serum cotinine levels of <10 ng=mL (n = 358).Exclusive tobacco use included those who either self-reported currently smoking cigarettes or had a serum cotinine level of >10 ng=mL and had not used marijuana within the last 30 d (n = 1,511).Dual users included both individuals who had used marijuana within the last 30 d and either currently smoked cigarettes or had serum cotinine levels of >10 ng=mL (n = 719).Models were adjusted for age, sex, race, education, eGFR, and NHANES cycle year.tAs and DMA were recalibrated for arsenobetaine to remove exposure from seafood consumption.Note: Ba, barium; Cd, cadmium; CI, confidence interval; Co, cobalt; Cs, cesium; DMA, dimethylarsinic acid; eGFR, estimated glomerular filtration rate; Mn, manganese; Mo, molybdenum; NHANES, National Health and Nutrition Examination Survey; Pb, lead; Sb, antimony; Sn, tin; Sr, strontium; tAs, total arsenic; Tl, thallium; U, uranium; W, tungsten.

Figure 3 .
Figure 3. Arithmetic mean differences and 95% CIs in blood metal concentrations (lg=L or lg=dL for Pb) across categories of time since last use among exclusive marijuana users who had used within the last 7 d (circle), the last 8-30 d (square), and the last 31-365 d (triangle), as compared with participants who used neither marijuana nor tobacco or who had not used marijuana in >1 y (reference), n = 5,024 participants NHANES (2005-2018).See Table S9 for all model estimates.The reference group of nonuse included individuals who used neither marijuana nor tobacco or who had not used marijuana in >1 y (n = 4,455).Groups of individuals who had recently used marijuana included those who had used within the last 7 d (n = 226), those who had used within the last 7-30 d (n = 132), or those who had used within the last 31-365 d (n = 211).Individuals who self-reported cigarette smoking or had serum cotinine levels of >10 ng=mL were excluded.Models were adjusted for age, sex, race, education, eGFR, and NHANES cycle year.Values of Hg include only women of childbearing age.Note: Cd, cadmium; CI, confidence interval; eGFR, estimated glomerular filtration rate; Hg, mercury; Mn, manganese; NHANES, National Health and Nutrition Examination Survey; Pb, lead; Se, selenium.

Figure 4 .
Figure 4. Arithmetic mean differences and 95% CIs in urinary metal levels (lg=g) across categories of time since last use among exclusive marijuana users who had used within the last 7 d (circle), the last 8-30 d (square), and the last 31-365 d (triangle), as compared with participants who used neither marijuana nor tobacco or who had not used marijuana in >1 y (reference), n = 5,024 participants NHANES (2005-2018).See TableS10for all model estimates.The reference group of nonuse included individuals who used neither marijuana nor tobacco or who had not used marijuana in >1 y (n = 4,455).Groups of individuals who had recently used marijuana included those who had used within the last 7 d (n = 226), those who had used within the last 8-30 d (n = 132), or those who had used within the last 31-365 d (n = 211).Individuals who self-reported cigarette smoking or had serum cotinine levels of >10 ng=mL were excluded.Models were adjusted for age, sex, race, education, eGFR, and NHANES cycle year.tAs and DMA were recalibrated for arsenobetaine to remove exposure from seafood consumption.Note: Ba, barium; Cd, cadmium; CI, confidence interval; Co, cobalt; Cs, cesium; DMA, dimethylarsinic acid; eGFR, estimated glomerular filtration rate; Mn, manganese; Mo, molybdenum; NHANES, National Health and Nutrition Examination Survey; Pb, lead; Sb, antimony; Sn, tin; Sr, strontium; tAs, total arsenic; Tl, thallium; U, uranium; W, tungsten.

Table 1 .
Participant characteristics across categories of non-marijuana/non-tobacco use (n = 4,666), exclusive marijuana use (n = 358), exclusive tobacco use (n = 1,511), and dual use (n = 719), among 7,254 NHANES participants (2005-2018).Non-marijuana/non-tobacco use a Exclusive marijuana use b Exclusive tobacco use c Dual use d Note: Analyses conducted using survey package to account for NHANES complex sampling design and weights; percentages are weighted and indicate population percentages.Continuous variables are reported as mean ± SD and categorical variables as number (population-weighted percentage).Note: BMI, body mass index; eGFR, estimated glomerular filtration rate; GED, General Education Development (U.S. high school diploma alternative); NHANES, National Health and Nutrition Examination Survey; SD, standard deviation.aThe reference group of non-marijuana/non-tobacco use includes individuals who had not used marijuana within the last 30 d and individuals who self-reported they did not currently smoke cigarettes or those with serum cotinine levels of ≤10 ng=mL (n = 4,666).bExclusive marijuana use includes those who had used marijuana within the last 30 d and had serum cotinine levels of <10 ng=mL (n = 358).cExclusive tobacco use includes those who either self-reported currently smoking cigarettes or had a serum cotinine level of >10 ng=mL and had not used marijuana within the last 30 d (n = 1,511).dDual use includes both individuals who had used marijuana within the last 30 d and either currently smoked cigarettes or had serum cotinine levels of >10 ng=mL (n = 719).eEver smoker is a participant who self-reported smoking at least 100 cigarettes in their lifetime.f Current smoker is a participant who self-reported currently smoking cigarettes.g Cotinine smoker is a participant with serum cotinine values of >10 lg=L.h Ever cannabis use is a participant who self-reported having ever used marijuana.
Note: DMA, dimethyl arsenic acid, NHANES, National Health and Nutrition Examination Survey.aThereference group of non-marijuana/non-tobacco use includes individuals who had not used marijuana within the last 30 d and individuals who self-reported they did not currently smoke cigarettes or those with serum cotinine levels of ≤10 ng=mL (n = 4,666).