Association between Glyphosate Exposure and Erythrograms in a Representative Sample of US Adults: NHANES 2013-2014

doi:10.21203/rs.3.rs-2746470/v1

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Association between Glyphosate Exposure and Erythrograms in a Representative Sample of US Adults: NHANES 2013-2014

https://doi.org/10.21203/rs.3.rs-2746470/v1

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published 20 Jul, 2023

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Glyphosate, being the most commonly utilized herbicide globally, and a growing body of experimental research has linked its exposure to red blood cell impairment. However, the potential toxicity of glyphosate exposure on erythrocytes in the general population remains poorly understood. Therefore, we analyzed data from the 2013–2014 National Health and Nutrition Examination Survey (NHANES) of 1466 adults (≥ 18 years) to explore the potential relationship between glyphosate exposure and erythrocyte profiles. Our results indicated a significant negative association between urinary glyphosate levels and hemoglobin (Hb) and hematocrit (Hct) in multiple regression analysis, with ß coefficients of -0.157 (S.E. = 0.055, P = 0.012) and − 0.431 (S.E. = 0.195, P = 0.043), respectively. Additionally, the odds ratio showed a significant increase in individuals with anemia with a one-unit increase in ln-glyphosate levels (odds ratio = 1.523 (95% CI = 1.301–1.783), P < 0.001 in the final model). The negative correlation between glyphosate and Hb was more pronounced in subjects aged over 60 years, non-Hispanic white ethnicity, lower income, and those with a body mass index (BMI) < 25 and ≥ 30. In conclusion, our results offer initial evidence of a plausible link between glyphosate exposure and anemia among the adult populace in the United States. However, further research is necessary to comprehend the underlying mechanisms and clinical implications of this link.

Anemia

Erythrograms

Glyphosate

Glyphosate-based herbicides (GBH)

Hemoglobin

National Health and Nutrition Examination Survey (NHANES)

Glyphosate functions as a competitive inhibitor for the 5-enolpyruvylshikimate-3-phosphate synthase, which is crucial for the biosynthesis of aromatic amino acids in plants and microorganisms (Peillex &Pelletier 2020). Since 1974, glyphosate has been utilized as the primary active component in herbicides. Glyphosate based herbicides (GBH) are a mixture of glyphosate and various adjuvants, which are mainly surfactants that aid in the herbicide's penetration into plants and increase its effectiveness (Williams et al. 2000). Due to their high efficacy in managing weed growth, Glyphosate and GBH have become the most widely used herbicides across the globe (Benbrook 2016). Humans can potentially be exposed to Glyphosate and GBH through multiple pathways, such as dermal contact, inhalation, and oral ingestion (Ospina et al. 2022). The elimination half-life of glyphosate in humans typically falls within the range of 5.5 to 10 hours. Notably, the primary routes of excretion are fecal excretion and urinary excretion, accounting for 90% and 6% of elimination, respectively, and occurring within a 48-hour timeframe (Zoller et al. 2020). The available evidence suggests that there has been a notable increase in both the frequency and level of glyphosate exposure in the United States over time. (Mills et al. 2017).

Glyphosate has historically been considered safe as it targets an enzyme that is not present in animals. Nonetheless, in recent years, mounting apprehension has emerged regarding the potential adverse health impacts of glyphosate and its derivatives. Numerous studies have connected glyphosate exposure to a variety of health hazards, including cancer, congenital disabilities, and reproductive issues (de Araujo et al. 2016). These discoveries prompted the International Agency for Research on Cancer to classify glyphosate as a likely human carcinogen in 2015 (Davoren &Schiestl 2018). Glyphosate and its formulations have been found to have varying cellular effects depending on the cell type and tested concentrations (Peillex &Pelletier 2020). Studies have shown glyphosate and GBH have exhibited dose-dependent cytotoxicity and genotoxicity at environmentally-relevant concentrations in human cell culture (Vanlaeys et al. 2018). Several experimental investigations have explored the potential toxic effects of glyphosate and GBH on erythrocytes and hematological parameters. In vitro studies indicate that these compounds may cause damage to erythrocytes, inducing oxidative stress, hemolysis, and hemoglobin oxidation (Kwiatkowska et al. 2014, Pieniążek et al. 2004, Rodrigues et al. 2011). Exposure to glyphosate and GBH can lead to decreased red blood cells (RBC) counts, changes in parameters of erythrograms, increased inflammation, and toxic to bone marrow in animal models (Bojarski et al. 2022, Mzengereza et al. 2020, Prasad et al. 2009, Rodrigues et al. 2011)

Although experimental studies suggest that glyphosate and GBH may have harmful effects on erythrocytes, there is limited understanding of their association with erythrocyte parameters in humans. In an occupational study, a significant difference in RBC count was observed among glyphosate-exposed workers compared to non-exposed staff when glyphosate concentration in the air was below 9.40 mg/m³ (Zhang et al. 2019). However, there is no research on the relationship between glyphosate and erythrocyte profiles in the general population. Erythrograms are standard clinical tools utilized to assess the general health of RBC and identify a spectrum of erythrocyte diseases. To augment our comprehension of the correlation between glyphosate levels and erythrograms, we recruited participants from the 2013–2014 National Health and Nutrition Examination Survey (NHANES), which includes extensive data on urinary glyphosate levels and erythrograms.

Study population

The NHANES is a biennial national survey that selects a representative sample of US citizens. The survey methodology and consent forms can be accessed on the NHANES website (CDC 2016a). For our study, we selected individuals aged 18 years and above who had measurements of glyphosate exposure and erythrograms, along with necessary demographic data from NHANES 2013–2014. We included a total of 1466 participants aged between 18–80 years in our analysis.

Measurement of urinary glyphosate levels:

In the NHANES 2013–2014 survey, urinary glyphosate concentrations were quantified in a representative sample of one-third of participants aged 6 years and older using a highly sensitive and specific two-dimensional on-line ion chromatography coupled with tandem mass spectrometry method with isotope dilution for quantification (Schütze et al. 2021). Quality control and quality assurance measures were strictly adhered to during the analytical measurements. For concentrations below the limits of detection (LOD), a value was imputed using the square root of 2 times the LOD value, as per NHANES protocol. The detailed analytical method is available on the official NHANES website (CDC 2022).

Erythrograms

RBC counts indicate the quantity of RBC in a given volume of blood, while hemoglobin (Hb) levels measure the weight of Hb in a corresponding volume of blood. Hematocrit (Hct), expressed as a percentage, reflects the volume of RBC in the blood. Mean corpuscular volume (MCV) is used to determine the average size of RBC by dividing HCT by RBC counts. Mean corpuscular hemoglobin (MCH) reflects the average weight of Hb in each RBC and is calculated by dividing Hb by the number of RBC in a given volume of blood. Mean corpuscular hemoglobin concentration (MCHC) is used to determine the concentration of Hb in each RBC by dividing Hb by HCT. Red cell distribution width (RDW) shows the deviation in the volume of RBC and is calculated by dividing the standard deviation of MCV by MCV and multiplying by 100, expressed as a percentage. The detailed analytical method is available on the official NHANES website (CDC 2016b).

Covariates

The NHANES website indicates that skilled personnel utilizing standardized procedures conducted data collection at all study sites. Sociodemographic information, such as age, sex, and race/ethnicity, was gathered during the household interview. To determine body mass index (BMI), weight in kilograms was divided by height in meters squared. Participants were categorized as active smokers, environmentally tobacco smoke (ETS) exposed, or non-smokers based on the results of a smoking questionnaire (CDC 2016c). An alcohol consumption questionnaire was utilized, and participants were asked whether they had consumed at least 12 alcoholic beverages in the preceding year, and their responses were dichotomized. During NHANES 2013–2014, individuals who were at least 1 year old were questioned whether they had received treatment for anemia in the previous 3 months. Chronic kidney disease is defined as an estimated glomerular filtration rate of less than 60 ml/min per 1.73 square meters (Yan et al. 2021). The criteria used to define anemia are Hb concentrations of less than 12 gm/dL for females and less than 13 gm/dL for males in whole blood.

Statistics

In this study, measurements of glyphosate levels were obtained in units of µg/L or µg/g creatinine. The natural logarithm of glyphosate was used to determine the exponential mean and standard deviation for various subgroups. Statistical analyses were conducted using a two-tailed Student's t-test and one-way analysis of variance. To evaluate the relationship between the erythrograms, which were used as the dependent variable, and ln-glyphosate, the independent variable, a complex sample of the general linear model was utilized. Two models were utilized for covariate adjustment. Model 1 adjusted for various demographic factors such as age, sex, ethnicity, BMI, smoking, alcohol consumption, household income, and urinary creatinine. Model 2 included additional adjustments for chronic renal failure and treatment for anemia in the previous three months. Urinary creatinine was treated as a separate independent variable based on prior research, instead of being adjusted for hydration (Barr et al. 2005, O'Brien et al. 2016). To minimize the potential for model-dependent associations, a result was deemed statistically significant only if it was significant in both Models 1 and 2 (Lang et al. 2008, Lin et al. 2009). For logistic regression analyses examining the potential relationship between anemia and glyphosate, model 2 was used for covariate adjustment in the complex sample. Sampling weights were applied based on recommended protocols outlined on the NHANES website (CDC 2005). The natural logarithm of glyphosate and urinary creatinine were utilized in the analysis due to their non-normal distributions. All statistical analyses were conducted using SPSS version 20 (SPSS Inc., Chicago, Illinois, USA). The significance level was set at P < 0.05.

The study population had an average age (SD) of 48.16 (18.31) years, with 80.2% of individuals exhibiting detectable levels of glyphosate. The mean levels (SD) of glyphosate were found to be 0.55 (0.54) µg/L. As presented in Table 1, the geometric means of glyphosate across different subgroups indicate that urinary glyphosate levels were higher in men, older individuals, non-Hispanic Black, and those with higher BMI. Moreover, glyphosate levels (creatinine-corrected) were higher in women, older individuals, non-Hispanic white, and non-smokers.

Table 1

The geometric means (S.E.) of urinary glyphosate levels in different demographic subgroups
	No.	Glyphosate (µg/L)	P value	Glyphosate (µg/g creatinine)	P value
Total	1466	0.41 (1.02)		0.43 (1.02)
Sex			0.003		< 0.001
Men	710	0.43 (1.03)		0.37 (1.03)
Women	756	0.38 (1.03)		0.49 (1.03)
Age (years)			< 0.001		< 0.001
18–40	521	0.38 (1.03)		0.34 (1.03)
40–59	486	0.37 (1.03)		0.43 (1.03)
≥ 60	459	0.47 (1.04)		0.55 (1.04)
Ethnicity			0.001		< 0.001
Mexican-American	196	0.38 (1.05)		0.39 (1.06)
Other Hispanic	127	0.39 (1.07)		0.42 (1.07)
Non-Hispanic white	692	0.41 (1.03)		0.47 (1.03)
Non-Hispanic black	257	0.47 (1.05)		0.37 (1.05)
Non-Hispanic Asian	148	0.34 (1.07)		0.44 (1.07)
Other ethnicity	46	0.36 (1.10)		0.34 (1.12)
Household income (USD)			0.342		0.556
< 4500	679	0.41 (1.03)		0.43 (1.03)
≥ 4500	787	0.40 (1.03)		0.42 (1.03)
Body mass index (kg/m²)			0.008		0.440
< 25	448	0.37 (1.03)		0.44 (1.04)
25–30	465	0.41 (1.04)		0.43 (1.04)
≥ 30	553	0.43 (1.03)		0.42 (1.03)
Smoking status			0.083		0.002
Non-smoker	895	0.41 (1.03)		0.45 (1.02)
ETS	216	0.36 (1.05)		0.39 (1.05)
Current smoker	355	0.41 (1.04)		0.40 (1.04)
Alcohol consumption (drink/year)			0.917		0.230
< 12	438	0.40 (1.04)		0.44 (1.04)
≥ 12	1028	0.40 (1.02)		0.42 (1.02)
Tested by Student’s 2-tailed t-test or by one-way analysis of variance
Abbreviations. ETS, environmental tobacco smoke

Regression analyses reported in Table 2 suggest that a one-unit increase in ln-glyphosate levels was negatively correlated with both Hb and Hct, with ß coefficients of -0.157 (S.E. = 0.055, P = 0.012) and − 0.431 (S.E. = 0.195, P = 0.043), respectively. An overview of the Hb and Hct across quartiles of urinary glyphosate in multiple linear regression models is presented in Fig. 1. The findings of the study reveal that in model 2, both Hb and Hct exhibit a statistically significant decrease with increasing glyphosate quartiles, with P values of 0.028 and 0.036, respectively.

Table 2

Linear regression coefficients (standard error) of erythrograms with one unit increase in ln- glyphosate (µg/L) in multiple linear regression models, with results weighted for sampling strategy.
	ln- Glyphosate (µg/L)
	Model 1		Model 2
	ß coeff (S.E.)	P value	ß coeff (S.E.)	P value
RBC (million/uL)	-0.033 (0.022)	0.151	-0.032 (0.020)	0.140
Hb (gm/dL)	-0.173 (0.058)	0.009	-0.157 (0.055)	0.012
Hct (%)	-0.463 (0.200)	0.035	-0.431 (0.195)	0.043
MCV (fL)	-0.370 (0.195)	0.077	-0.339 (0.219)	0.143
MCH (pg/cell)	-0.165 (0.078)	0.051	-0.144 (0.078)	0.084
MCHC (gm/dL)	-0.046 (0.073)	0.537	-0.034 (0.068)	0.629
RDW (%)	-0.085 (0.062)	0.196	0.054 (0.052)	0.317
Model 1 adjusted for age, gender, ethnicity, smoking status, drinking status, household income, BMI, and urinary creatinine
Model 2 adjusted for model 1 plus chronic kidney disease, and treatment for anemia in three months,
Unweighted no: 1465; Population size: 209123002

Logistic regression analyses reported in Tables 3 and 4 assess the association between glyphosate and anemia. The odds ratios (ORs) of anemia were significantly lower with a one-unit increase in ln-transformed glyphosate in both models (OR = 1.523, 95% CI = 1.301–1.783, P = < 0.001 in model 2). Table 4 further illustrates the negative correlation between glyphosate and Hb in different subgroups of study subjects. This correlation was found to be more pronounced in subjects aged over 60 years, those of non-Hispanic white ethnicity, those with lower income, and those with a BMI < 25 and ≥ 30.

Table 3

Odds ratios (95% confidence interval [C.I.]) of anemia with one unit increase in ln-glyphosate (µg/L) in logistic regression models, with results weighted for sampling strategy. (n = 1454)
	Odds ratio	95% C.I.		P value
		Lower	Upper
Model 1	1.526	1.294	1.800	< 0.001
Model 2	1.523	1.301	1.783	< 0.001
Model 1 adjusted for age, gender, ethnicity, smoking status, drinking status, household income, BMI, and urinary creatinine
Model 2 adjusted for model 1 plus chronic kidney disease, and treatment for anemia in three months,
Abbreviations:
Definition of anemia: females with whole blood hemoglobin concentrations < 12 gm/dL and males with whole blood hemoglobin concentrations < 13 gm/dL.

Table 4

Linear regression coefficients (standard error) of Hb (gm/dL) per unit increase in ln glyphosate (g/L) in subpopulation, with results weighted for sampling strategy
	Unweighted no./ Population size	Adjusted β (SE)	P value
Gender
Men	710/102184440	-0.159 (0.095)	0.115
Women	755/106938562	-0.133 (0.078)	0.108
Age, years
20–40	521/78758631	-0.113 (0.070)	0.126
40–59	485/73819844	-0.066 (0.098)	0.511
≥60	155/21075447	-0.338 (0.063)	< 0.001
Ethnicity
Non-Hispanic white	692/142417028	-0.149 (0.082)	0.088
Other	773/66705973	-0.188 (0.066)	0.012
Annual household income
≤ $4500	687/78547726	-0.298 (0.049)	< 0.001
> $4500	787/130575276	-0.057 (0.098)	0.568
Body mass index (kg/m²)
<25	447/62574646	-0.187 (0.087)	0.048
25–30	465/65357735	-0.021 (0.106)	0.849
≥30	553/81190621	-0.285 (0.050)	< 0.001
Adjusted for model 2

Our study employed a representative cohort of the general U.S. adult population and has revealed a significant negative correlation between urinary glyphosate levels and both Hb and Hct. Furthermore, our findings suggest a notable increase in the odds of anemia in individuals with higher glyphosate levels. The negative correlation between glyphosate and Hb was particularly pronounced in subgroups of subjects aged over 60 years, of ethnicities other than non-Hispanic white, with lower income, and those with BMI < 25 and ≥ 30. This study presents initial evidence of a potential association between glyphosate exposure and anemia in the general adult population. The possible establishment of a causal relationship is of great concern, as it highlights the potential adverse impact of glyphosate exposure on erythrocytes in American adults. The significance of this research is attributed to its use of dependable and extensive data from the NHANES database and the incorporation of a representative sample of American adults aged 18 years and above.

In this study of 1466 individuals aged 18–80 years, detectable levels of glyphosate were found in 80.2% of participants. Similarly, another NHANES survey of individuals aged six years and above found glyphosate residues in 81.2% of participants (Ospina et al. 2022). These high detection rates suggest that exposure to glyphosate is inevitable during routine daily activities. One study indicated that glyphosate concentrations vary based on fasting status, while another found a positive correlation between consumption of whole-grain bread and increased urinary glyphosate levels (Ashley-Martin et al. 2023, Ospina et al. 2022). These findings suggest that individuals with elevated glyphosate levels may have been exposed to a diet that frequently undergoes herbicide application. The present study also found that urinary creatinine corrected glyphosate levels are significantly higher in women, advanced ages, non-Hispanic white, and non-smokers. It is well documented that demographic characteristics can influence an individual's exposure to environmental contaminants, including glyphosate (Shea 2003). In a study using data from NHANES 2013–2016, the amount of fruit and vegetable intake was higher in women, those with advanced ages, and non-smokers, which are commonly exposed to glyphosate as a herbicide (Kolakowski et al. 2020, Martin et al. 2019). Further study is warranted to determine the specific reasons for differences in glyphosate exposure among subpopulations.

There have been numerous experimental studies investigating the effects of glyphosate and GBH on erythrocytes. For instance, an in vitro study discovered that exposure to glyphosate and GBHs at concentrations of 0.25 mmol led to the production of reactive oxygen species in human erythrocytes, without altering their size or shape (Kwiatkowska et al. 2014). Another study found that GBH induced an increase in methemoglobin levels, lipid peroxidation products, and hemolysis at concentrations of 500 ppm and 1500 ppm, while glyphosate caused an increase in the levels of methemoglobin and lipid peroxidation only at a higher dose of 1000 ppm, and hemolysis was observed at 1500 ppm. GBH were more likely to affect erythrocyte function than glyphosate, possibly due to the properties of additives (Pieniążek et al. 2004). Moreover, one study demonstrated that GBH caused 100% hemolysis within the concentration limit recommended for agricultural purposes (approximately 200 ppm of Roundup) (Rodrigues et al. 2011).

Animal studies have also been conducted to investigate the effects of glyphosate and GBH on erythrocytes. One such study examined the sub-lethal toxicity of glyphosate herbicide on Koi carp fingerlings at water concentrations of 3.6 mg/L and 6.6 mg/L, and found that exposure to glyphosate resulted in a notable reduction in RBC counts, Hb, Hct, and MCHC, along with an elevation in MCV and MCH (Mzengereza et al. 2020). Moreover, a study investigating the impact of GBH on common carp by exposing them to water concentrations equivalent to 1 and 5 mg/L of glyphosate, found a decrease in RBC count and an increase in inflammation in the exposed carp. The study suggests that hematological parameters are more sensitive and reliable indicators of Roundup exposure in common carp than other tested parameters (Bojarski et al. 2022). Another study reported that oral administration of 375 mg/kg of GBH for 56 days induced severe anemia, altered platelets and white blood cells, and caused inflammation and oxidative stress in mice. However, supplementation with vitamin B12 attenuated these effects, suggesting a potential role of antioxidants in managing GBH-induced toxicity (Ngatuni et al. 2022). Additionally, a study demonstrated that intraperitoneal administration of glyphosate at levels of 25 and 50 mg/kg is clastogenic and cytotoxic to mouse bone marrow, potentially leading to decreased erythrocyte production in the mice (Prasad et al. 2009). These findings provide evidence that glyphosate and GBH can have harmful effects on erythrocytes, which may lead to anemia and inflammation in animals.

Most research investigating the impact of glyphosate exposure on humans has focused on the consequences of intentional or unintentional poisoning. One case report highlighted a woman who suffered from hypoxemia, hyperkalemia, hypotension, and hemoconcentration after accidentally consuming 100 mL of GBH (Ozaki et al. 2017). In 2015, the European Food Safety Authority conducted a review to evaluate the potential risks associated with glyphosate usage and established several toxicological endpoints based on laboratory research conducted on rabbits (Soares et al. 2021). Current epidemiological evidence suggests that the usage of glyphosate at recommended concentrations is improbable to result in detrimental health effects, including changes in hematological parameters (Agostini et al. 2020). While there are a limited number of epidemiological studies examining the potential negative impact of glyphosate/GBH exposure on red blood cells, one occupational study compared 178 workers exposed to glyphosate with 203 non-exposed administrative staff. The study concluded that there were no significant differences in the abnormal rate of blood routine between the two groups. However, when the time-weighted average concentration of glyphosate in the air was below 9.40 mg/m³, there was a notable difference in the platelet distribution width and RBC count results between the two groups (Zhang et al. 2019). Using data from NHANES, the present study discovered a negative correlation between glyphosate levels and Hb as well as Hct. Considering the widespread detection of urinary glyphosate and the routine application of herbicides on crops consumed by individuals (Ashley-Martin et al. 2023, Ospina et al. 2022), it is possible that the exposure dosage of glyphosate and GBH in U.S. adults surpasses the anticipated levels and may lead to detrimental effects on erythrocytes. Exposure to glyphosate has been linked to the development of anemia through potential mechanisms such as hemolysis and bone marrow suppression in animal studies (Kwiatkowska et al. 2014, Prasad et al. 2009). Moreover, glyphosate exposure may disrupt the gut microbiome, which has been linked to iron status in the gut (Hu et al. 2021, Paganini et al. 2016, Yilmaz &Li 2018). Our investigation provides preliminary evidence that there may be an association between exposure to glyphosate and erythrograms in adults.

Our study found that the relationship between glyphosate exposure and Hb was more pronounced in individuals who were older, had ethnic backgrounds other than non-Hispanic whites, had lower incomes, and had a BMI < 25 and ≥ 30. To our knowledge, no studies have been conducted that investigate the potential differences in glyphosate's effects among various ethnic groups and socioeconomic statuses. Nonetheless, discrepancies in genetics, lifestyle factors such as diet, occupation, and environment could all play a role in the varying levels of glyphosate toxicity observed among individuals from different ethnic backgrounds and income levels. Prior epidemiological investigations have demonstrated that obesity may disturb iron homeostasis through chronic inflammation, leading to iron deficiency anemia (Alshwaiyat et al. 2021, Qin et al. 2013), while low body weight is also a risk factor for anemia (Sumarmi et al. 2016). Glyphosate has also been found to modify the gut microbiome, which can affect nutrient metabolism and energy expenditure (Aoun et al. 2020, Hu et al. 2021). It is plausible that there is a complex interplay and synergistic impact between glyphosate and BMI on anemia. Our research has identified demographic subgroups that may be vulnerable to the adverse effects of glyphosate exposure on erythrocytes. Further inquiry is required to comprehend the potential mechanisms behind such differences.

There are several limitations to consider when interpreting the findings of this study. Firstly, it is important to note that the observed negative association between glyphosate levels and Hb/Hct does not imply causation. Other variables, such as dietary factors, iron status, or additional environmental exposures, may also influence this correlation. Secondly, since this study used a cross-sectional design, it only provides a snapshot of glyphosate exposure and hemoglobin levels at a specific time and cannot establish a causal relationship. Therefore, further research, including longitudinal studies that track participants over time, is necessary to verify and expand our understanding of the potential relationship between glyphosate exposure and erythrocytes. Thirdly, the sample size was limited to NHANES 2013–2014 data, which may have restricted the scope of the analysis and could have affected the reliability of the findings. Lastly, the study only included adult participants from the United States, which may limit the generalizability of the results to other populations and regions.

After conducting our research on a representative cohort of adults in the United States, we found important evidence of a correlation between urinary glyphosate levels and erythrograms. Specifically, our study showed a significant negative association between glyphosate exposure and Hb and Hct levels. Furthermore, our results indicated that the likelihood of anemia was significantly higher in individuals with higher glyphosate levels. While further investigation is necessary to determine the causality and clinical significance of these findings, our study highlights the importance of continued research on the potential toxic effects of glyphosate on erythrocytes in adult populations. By informing public health policies and regulatory decisions related to glyphosate use, such research has the potential to contribute to the protection of human health.

ETS: Environmental tobacco smoke

GBH: Glyphosate based herbicides

Hb: Hemoglobin

HCT: Hematocrit

LOD: Limits of detection

MCH: Mean corpuscular hemoglobin

MCHC: Hemoglobin concentration

MCV: Mean corpuscular volume

NHANES: National Health and Nutrition Examination Survey

RBC: Red blood cells

RDW: Red blood cell distribution width

Ethical approval: This study was approved by the Ethics Committee of the En Chu Kong hospital (ECKH_W11203).

Consent to participate: Informed consent was obtained from all individual participants included in the study.

Consent to publish: All enrolled subjects signed informed consent regarding publishing their data.

Funding: This study was funded by grants from the Ministry of Science and Technology of Taiwan NSC 110-2314-B-385-001-MY3.

Competing interests: The authors declare that there are no competing financial interests.

Author’s contributions: Ming-Hao Chang and Pei-Lun Chu were both involved in the literature search and writing of the paper. Chi-Kang Wang contributed to the statistical analysis. Chien-Yu Lin played a pivotal role in the development of the hypothesis setting and provided final approval of the paper's revision

Availability of data and materials: The datasets analyzed during the current study are available at the NHANES website (https://www.cdc.gov/nchs/nhanes/index.htm (accessed on 27 March 2023)

Acknowledgments: We would like to thank everyone who has contributed to NHANES' progress. Our appreciation extends to the anonymous participants who have made this study possible.

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Association between Glyphosate Exposure and Erythrograms in a Representative Sample of US Adults: NHANES 2013-2014

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Measurement of urinary glyphosate levels:

Erythrograms

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