www.nature.com/scientificreports OPEN Efects of brewing conditions on infusible fuoride levels in tea and herbal products and probabilistic health risk assessment Nattha Pattaravisitsate1, Athit Phetrak2*, Thammanitchpol Denpetkul2, Suthirat Kittipongvises3 & Keisuke Kuroda4 Excessive ingestion of fuorides might adversely afect the health of humans. Hence, this study aimed to investigate the concentrations of infusible fuoride in fve diferent types of tea and herbal products; additionally, the probabilistic health risks associated with the ingestion of fuoride in drinking tea and herbal products were estimated. The highest and lowest concentrations of infusible fuoride were detected in black and white tea, respectively. On average, the highest amount of infusible fuoride was extracted following a short brewing time of 5 min in the case of black tea (2.54 mg/L), herbal tea (0.40 mg/L), and white tea (0.21 mg/L). The level of infusible fuoride during brewing was inversely associated with the leaf size of the tea and herbal products. Furthermore, the type of water used infuenced the release of infusible fuoride; purifed water yielded lower amounts of infused fuoride. The fndings of the probabilistic health risk assessment indicated that the consumption of black tea can increase the fuoride intake leading to chronic exposure. Thus, the health risk posed by fuoride intake from drinking tea needs to be evaluated in more details in the future. Appropriate measures for health risk mitigation need to be implemented to minimize the total body burden of fuorides in humans. Fluorine is the 13th most abundant element in the Earth’s crust (0.054% by weight) and is mostly present in the form of fuoride in various minerals. Fluoride is an essential micronutrient, and an appropriate amount of intake of this mineral, daily, can prevent the development of dental caries. However, numerous studies have indicated that excessive fuoride ingestion over a long period can lead to potentially severe dental and skeleton fuorosis, hypertension, damage to the neurological system, and a lower intelligence quotient 1–5. Furthermore, it might have an adverse impact on the male reproductive system6. Drinking water is the main source of fuoride intake in humans7. Approximately 90% of fuoride in drink- ing water can be absorbed in the human digestive system 7. In Tailand, the guideline value of fuoride in drinking water is regulated at 0.7 mg/L, which is lower than that reported by the World Health Organization (WHO; < 1.5 mg/L)8. Tis was done to prevent the development of fuorosis in the country9. Nonetheless, the number of cases of dental fuorosis among children in Bangkok has been consistently increasing over the past two decades (18.4% as of 2014), possibly because of the intake of fuoride supplements 10, thus indicating that the potential risk of excess fuoride consumption in Tailand might be a serious public health concern. Tea (Camellia sinensis) is one of the most popular nonalcoholic beverages extensively consumed by health- conscious people in many societies11,12. Te numerous types of teas available in the market could be broadly categorized into four types depending on the number of oxidation steps required during processing: black tea (fully oxidized), green tea (nonoxidized), oolong tea (semioxidized), and white tea (lightly oxidized)13–15. Herbal tea products, in which some contain the leaves of Camellia sinensis11, are also widely popular among consumers in Tailand and across the world16. Te consumption of tea daily is increasing, and large amounts are consumed by 1Research Ofce, Faculty of Dentistry, Mahidol University, Bangkok, Thailand. 2Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 3Environmental Research Institute, Chulalongkorn University, Bangkok, Thailand. 4Department of Environmental and Civil Engineering, Toyama Prefectural University, Imizu, Japan. *email: [email protected] Scientifc Reports | (2021) 11:14115 | https://doi.org/10.1038/s41598-021-93548-3 1 Vol.:(0123456789) www.nature.com/scientificreports/ Equations Description · · · = C×DI×EF×ED Chronic Daily Intake (CDI) BW×AT C is the fuoride concentration in tea infusion (mg/L) DI is the average daily intake rate of tea (L/day) EF is the exposure frequency (day(s)/year) · · = CDI ED is the exposure duration (year) Hazard Quotient (HQ) RfD An HQ of < 1 indicates an insignifcant risk level, whereas an HQ of > 1 implies a BW is body weight (kg) potential noncancer-causing health impact AT is the averaging time (days) CDI is the chronic daily intake (mg/kg/day) RfD is the reference dose (mg/kg/day) Table 1. Equations used to estimate the Human Exposure and Health Risk28. adults (nearly 1 L per person per day on average) in oriental countries11. Te positive health benefts of tea include the presence of nutrients and minerals, antioxidant activity, favorable taste, aroma, and anticancer properties17–19. Tea contains a greater amount of fuoride than other plants, predominantly because it is an efcient fuoride accumulator20. Most of the fuoride in tea (nearly 98%) is accumulated in the leaves, particularly the mature leaves 21, and can be easily released during tea infusion22,23. Hence, the ingestion of fuoride from tea can lead to potential detrimental health efects. According to previous studies 13,22,24,25, diferent types of tea, the brew- ing times, the tea leaf size, and the type of water used for tea infusion could infuence the infusion of fuoride. However, the efects of these factors on releasable fuoride during infusion have not been evaluated in detail. Terefore, the present study aimed to investigate the efects of diferent conditions of tea infusion, such as the type of tea used, the brewing time, the leaf size, and the type of water used, on the concentration of releasable fuoride from the infusion. Additionally, the associated probabilistic health risk following exposure to fuoride in drinking was evaluated using the Monte Carlo simulation. Materials and methods Collection and preparation of the tea and herbal products. Sixteen brands (locally made and imported) of tea and herbal products (fve black tea, three green tea, three oolong tea, two white tea, and three herbal tea products) were purchased from a market in Bangkok, Tailand, in January 2019 (Table S1; as shown in supplementary information (SI)). Approximately 200 g of each tea and herbal product was collected and indi- vidually stored in airtight plastic bags (referred to as uncrushed tea samples). Approximately 100 g of each type of tea was crushed using an electrical grinder and individually packed into plastic bags. Te ground samples were passed through a sieve (mesh size, 20) and stored in cleaned containers (hereafer referred to as crushed tea). Te lengths of the tea leave in the uncrushed tea samples (n = 20) were carefully measured using a ruler. Dynamic light scattering was used to analyze the apparent hydrodynamic diameters of tiny tea particles ranging between 0.1 and 1000 µm in suspension (Partica mini LA-350, Horiba, Japan). Efect of tea infusion on fuoride release into the solution. Batch experiments of the infusion pro- cess were conducted using the one bottle point technique to investigate the levels of fuoride released from the tea and herbal products. Te efects of the types of tea, brewing times (5, 10, and 20 min), leaf size (uncrushed and crushed tea samples), and types of water (distilled, ultrapure, water treated by reverse osmosis membrane system [RO], tap, bottled, and bottled mineral) were evaluated using a method similar to that described by Mahvi et al. (2006)26. Briefy, the dried tea sample (1.00 ± 0.02 g) was placed in a cotton bag, which was then placed in a beaker (100 mL) containing 50 mL of boiling water (2% by w/v). Te prepared container was covered with a watch glass, and the tea was continuously brewed at ~ 100 °C for 5, 10, or 20 min. Subsequently, the tea bag was taken out, and the obtained solution was passed through a flter paper (pore size, 11 µm; Whatman No. 1, UK). A solution was prepared by adding water to a fnal volume of 50 mL, at room temperature. Te total infusible fuoride concentration in the tea solution was quantifed using a fuoride ion-selective electrode (VSTAR 40A, Orion, USA) as described previously by Kaophun et al. (2018)27. Each experiment was conducted in triplicate, and the average value ± the standard deviation (SD) was reported as the fnal result. Estimation of health risk. Health risk assessment is one of the methods used to indicate the possible harmful efects of environmental pollutant exposure on human health 28. Te equations used to evaluate the noncarcinogenic health risks of fuoride exposure from tea consumption in the present study are shown in Table 128 among individuals belonging to diferent age groups (children, 0–10 years; teenagers, 11–20 years; and adults, 21–70 years)22. Te probabilistic health risk assessment was conducted using Monte Carlo simulations with 10,000 iterations. Additionally, sensitivity analyses were conducted using the Oracle Crystal Ball sofware v.11.1.2.4.850 to determine the input parameters with the highest impact on the calculation of the probabilistic health risk assessment. Statistical analysis, quality control, and quality assurance. Te descriptive statistics were calcu- lated using Microsof Ofce Excel 2016. Te normality of the data sets was tested using the Shapiro–Wilk test. Due to non-normal distribution of the data sets, the nonparametric Mann–Whitney U test was used for pair- wise comparisons using the SPSS statistical package (version 18.0). Spearman’s rank correlation analysis was used to investigate the correlations between the parameters (quality of water) used for infusion and infusible fuoride concentrations. A p-value of < 0.05 was considered signifcant. Scientifc Reports | (2021) 11:14115 | https://doi.org/10.1038/s41598-021-93548-3 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 1.