The Toxicological Analysis and Toxicological Risk Assessment of Chosen Elemental Impurities (Ag, Au, Co, Cr, Cs, Li, Mo, Se, and Sr) in Green Tea (Camellia sinensis (L.)) Infusions

Our study’s objective is to evaluate the potential health effects of elemental impurities (Ag, Au, Co, Cr, Cs, Li, Mo, Se, and Sr) found in green tea infusions (Camellia sinensis (L.)). The ICP-MS-based methodology was employed for elemental analysis and a detailed health risk evaluation based on weekly consumption (μg/L of infusion/week). The provisional tolerable weekly intake (PTWI), established by the Joint FAO Expert Committee on infusion/week/month based on existing literature data, was then compared to the subjects with data from the available literature. The exposure of the study items to Co ranged from 0.07904 to 0.85421 μg/day. On the contrary, the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines state that the established permitted daily exposure PDE (oral exposure) for Co is 50 μg/day. The PDE of lithium is around 560 μg/day, and according to our study, the estimated daily exposure of the evaluated products to Li fell between 0.0185 and 0.7170 μg/day. Our research also revealed modest concentrations of Cs (0.399–2.654 μg/L), Mo (0.0113–0.107 μg/L), and Sr (9.848–22.331 μg/L) in infusions. The recognized PDE for molybdenum is approximately 3400 μg/day. Only two samples contained silver, and when considering daily consumption, the predicted daily exposure to Ag is between 4.4994 and 0.03122 μg/day. The amounts of all evaluated elements in a daily dose of green tea infusions should not harm the consumer’s health. Further considerations should take aspects, such as constant change and environmental pollution, into account.


Introduction
People have been using plant extracts for health purposes for centuries, especially in the form of tea, due to their easy and accessible administration and the possibility of applying them in various situations [1]. These characteristics make tea the second most consumed beverage globally, after water [2]. Green tea was the first to be discovered. It contains large amounts of polyphenols and caffeine (2-5%, depending on the age of the leaf), as well as minerals and trace amounts of vitamins, amino acids, and carbohydrates [3]. Because green tea is not fermented, it contains much more natural substances in its fresh leaves and shows fewer vitamin losses than other types of tea [4]. Green tea has healthpromoting effects due to its high content of polyphenols in fresh leaves, especially flavanols, which account for 30% of the dry weight [5]. Furthermore, green tea exceeds the catechin content of all known dietary sources, such as apples, red grapes or chocolate [6]. The both the inhalation and oral pathways. Trivalent chromium is a vital mineral for human nutrition [29]. If a significant amount of chromium(III) appears in living cells, there may be a potential threat of genotoxicity. However, regular metabolism and cell function prevent it. Persistent exposure to trivalent chromium leads to weight loss, anaemia, liver failure, and renal failure [30]. On the other hand, caesium naturally occurs primarily in the form of 133 Cs isotopes. Caesium is an exciting, but undervalued element, with 11 other major radioactive isotopes that can harm humans [31]. Caesium side symptoms include cardiac arrhythmias, hypokalaemia, fainting, convulsions, and cardiac arrest. There is no complete understanding of caesium functions [32]. Lithium is another engaging element that is used as a therapeutic agent for humans. An example is lithium salts, used to treat mania, recurrent unipolar depression, and affective problems in people with bipolar disorder [33]. An excess of lithium increases the chances of hypothyroidism and polyuria, a condition in which the body excretes excessive amounts of urine, decreased weight gain, and hyperparathyroidism [34]. In the case of toxicity associated with molybdenum consumption, there is little difference between animals treated with toxic concentrations and those that are molybdenum deficient. Exceeding the range triggers respiratory symptoms and increases neutrophils and lymphocytes [35]. Therefore, it is crucial to precisely control molybdenum content in the diet [36]. Selenium is a crucial trace element for the existence of many species and ensures the proper functioning of enzyme systems. Its most important function is to create a powerful antioxidant, an enzyme called glutathione peroxidase, which protects red blood cells and cell membranes from the damaging effects of free radicals [37]. Continuous overexposure to selenium can lead to chronic intoxication called selenosis, and is characterised primarily by epidermal and neurological effects that involve unpredictable gait and paralysis [25]. Since strontium is treated by the body similarly to calcium, it can be incorporated into the structure of the bone. Most strontium taken up is quickly excreted, and 20-30% is retained in the skeletal system [38]. Due to easy absorption and permanent incorporation into the body, radioactive isotopes from waste products of nuclear technologies are particularly dangerous, mainly 90 Sr, which persists in a contaminated environment for a long time due to its half-life of nearly 29 years [39]. These isotopes can be inhaled with dust, although they enter the body mainly through food. It may increase the risk of bone cancer and leukaemia [40]. Our article aimed to identify and evaluate the toxicological risks to humans of novel and traditional elemental impurities (Ag, Au, Co, Cr, Cs, Li, Mo, Se, and Sr) in green tea infusions (n = 12) accessible in the Polish market. The first step in our studies was to determine the investigated EI and present the raw results obtained as an EI profile. The weekly consumption of green tea was then estimated (µg/L of infusion/week), and the weekly intake was assumed to be approximately 6 L of tea per week according to [41]. The next step was to estimate the weekly consumption per body weight (µg/L of infusion/week/bw). Based on the weekly intake of weekly green tea per person (~70 kg bw) compared to the provisional tolerable weekly intake (PTWI), established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Furthermore, an individual toxicological risk assessment was performed for elements that do not include PTWI values.
Green tea is among the most consumed single-ingredient teas worldwide [25]. Despite the widely studied beneficial effects of green tea, some contaminants in tea leaves may lead to human health risks when drinking tea. In the context of exposure to green tea, this problem seems very basic, but is extremely important due to the lack of a comprehensive human toxicological risk assessment (TRA) within scientific works of NEIs and traditional EIs in green tea. Only one article on this topic in the scientific literature refers to infusions of green tea [42]. Therefore, we decided to estimate the exposure to NEIs and traditional EIs listed in the final tea infusions, and assess the safety of drinking green tea and the associated health risks. For this purpose, complete and well-designed TRA of the listed elements in green tea infusions have been developed.

Samples
From June 2022 to September 2022, green tea samples (n = 12) were purchased from stores in Rzeszów, Kraków, Toruń, Gdańsk, and Poznań in Poland. The samples analysed came in various forms, such as raw materials (in the form of leaves or needles) and tea bag containers (20-25 pieces per box; 1.4-2.0 g of raw materials). Samples were coded in a random order (GT1, GT2, etc.). Table 1 shows the characteristics of the examined green tea samples. To minimise potential impurities (elemental impurities require a specific condition in the laboratory) from other sources, all sampling procedures were performed at the analytical and clinical purity in the Bioelement Laboratory of the Collegium Medicum of the Jagiellonian University in Kraków. Furthermore, plastic equipment was applied to avoid impurities during the study. Additionally, laboratory glass equipment (volumetric flasks) was kept overnight in a 10% solution of nitric acid (HNO 3 ), rinsed with distilled water, and dried in the air before analysis. Additional processing (e.g., homogenisation and digestion) was not required, because all samples were liquid samples (tea infusion). Consequently, in situ analysis was applied at the measurement stage.

Chemicals
In this research, nine elements (Ag, Au, Co, Cr, Cs, Li, Mo, Se, and Sr) were analysed, and two multi-element stock solutions (CHECL 01.13632.0100 and Merck 01.10580.0100) containing Ag (10.

Instrumentation and Determination of Elements
The determination of elements (Ag, Au, Co, Cr, Cs, Li, Mo, Se, and Sr) was performed using an ICP-MS method, that uses argon gas (plasma) to convert the sample into the ionization state for elements that are then separated, measured and investigated using a mass spectrometer. In our study, we applied an Elan DRC-e spectrometer (PerkinElmer, Waltham, MA, USA) [43]. We used simultaneous multi-element detection mode. The plasma excitation power was 1150 W; the plasma gas, carrier gas, and composition gas flow rates were 15.0 L/min, 1.1 L/min, and 1.0 L/min, respectively. All experimental conditions are summarised in Table 2. All details regarding analytical calibration and quality control are described in Supplementary Materials S1 (SM S1). The green tea infusion process was carried out according to the information in Table 1 (infusion process, raw materials, and infusion time). First, an appropriate amount of green tea was poured into a beaker, and then ultrapure, demineralized, boiling water was poured over it (according to the manufacturer's recommendations). The mixture was then covered for 3 to 8 min (according to the tea manufacturer's recommendation), to ensure sufficient immersion [44]. After the injection, the solution was decanted and chilled to room temperature until determination.

Toxicological Risk Assessment
For a complex toxicological risk assessment, an appropriate strategy, consisting of three crucial steps, was applied. Table 3 shows the characteristics of the applied toxicological risk assessment. This strategy was based on our previously published article on mint tea [45]. Table 3. Description of the applied toxicological risk assessment.
Step Description Depending on weekly tea consumption per person compared to PTWI, weekly intake was evaluated according to body weight (µg/L of infusion/week/bw), using the equation: EWIBW = EWI/BW, where EWI is the estimated weekly intake (µg/L of infusion/week), and BW is the average body weight (approximately 70 kg bw) (kg).

Statistical Analysis
Data were analysed, and graphs were generated using the OriginLab 2010 statistical software. Data processing and basic descriptive calculations, compilation, and storage of the collected data at the laboratory stage were done using Excel 2010 (Microsoft Office), licensed by Rzeszów University. The results of five independent replications are expressed as relative standard errors (RSD, %).

The NEI and Traditional EI Profiles of All Investigated Green Tea Samples
The concentration (µg/L) of all investigated elemental impurities (Ag, Au, Co, Cr, Cs, Li, Mo, Se, and Sr) in all samples (n = 12; GT1-GT12) are shown in Figures 1-7 as NEI and traditional EI profiles by plots, as box diagrams. All the green tea samples analysed generally contained investigated elements at different concentrations. The descriptive statistics (minimum, maximum, mean, RSD) are shown in Table 4.
the collected data at the laboratory stage were done using Excel 2010 (Microsoft Offic licensed by Rzeszów University. The results of five independent replications are e pressed as relative standard errors (RSD, %).

The NEI and Traditional EI Profiles of all Investigated Green Tea Samples
The concentration (μg/L) of all investigated elemental impurities (Ag, Au, Co, Cr, C Li, Mo, Se, and Sr) in all samples (n = 12; GT1-GT12) are shown in Figures 1-7 as NEI an traditional EI profiles by plots, as box diagrams. All the green tea samples analysed ge erally contained investigated elements at different concentrations. The descriptive stat tics (minimum, maximum, mean, RSD) are shown in Table 4.                   All analysed green tea infusions contained all elemental impurities except silver. This element was present only in two samples; that is, GT1: 0.364 ± 0.05 µg/L and GT3: 15.748 ± 0.09 µg/L. Surprisingly, the Ag concentration in the GT3 sample was enormously high (15.748 ± 0.09 µg/L). In seven samples (GT1, GT2, GT3, GT4, GT5, GT9, and GT12), Au was present at relatively low concentrations (0.105-0.0830 µg/L). Figure 1 shows that Sr

The Toxicological Risk Assessment
As described in Table 3, the second step of our toxicological risk assessment was the weekly EI assessment (based on consumption scenarios). Because there are many consumption scenarios, this was difficult. The worst scenario (WC) is usually applied to toxicological risk assessments, i.e., the highest possible frequency of weekly tea consumption. Assuming that the average consumer drinks 3-10 cups of green tea daily, the weekly intake of the elements investigated from the infusion of green tea is estimated in Table 5. The last step was estimating the weekly intake based on the weight and weekly consumption of green tea. The weekly intake of each element in the investigated samples was calculated by dividing it by 70 kg (average adult weight recommended by EFSA [50]). The results obtained are shown in Table 5. Table 5. The estimation of the weekly intake (µg/week) in the range of 600-2000 mL, and weekly intake per body weight estimation of the examined elements based on the consumption of green tea.

Discussion
For complex toxicological risk assessment, we have implemented appropriate strategies composed of three key steps ( Table 3). The first step was the analysis of the preliminary results of the determination of the elemental impurities (EIs) of the green tea infusion (GT1-GT12) investigated as NEI, based on the ICP-MS method, with the EI profile and descriptive statistics (minimum, maximum, average). In this phase, we demonstrated that all green tea infusions (impurities profile: Figure 1, and normal distribution curve boxes: Figures 2-7 . Surprisingly, Ag concentration in the GT3 sample was enormously high (15.748 ± 0.09 µg/L). Furthermore, seven samples (GT1, GT2, GT3, GT4, GT5, GT9, and GT12) contained Au, but were relatively low (0.105-0.0830 µg/L). This article is the first in the scientific literature to determine the selected elements of the green tea infusion (raw results). We tried to compare our results (µg/L) with data from the literature on selected elements. Only one article [51] described the determination of the same elements, but only four of the elements (Co = 1.715 µg/L, Cs = 1.515 µg/L, Mo = 0.043 µg/L, Sr = 7.25 µg/L) coincided. The results are very comparable for the first three elements, and the only significant difference is in the case of strontium (in our case, Sr = 17.763 µg/L). The difference may result from comparing the average values based on the product ranges of the variables and the products analysed (n = 12). There are several articles in the scientific literature in which the elements were also determined. However, compared to our article, dry matter and green tea leaves were used as samples for analysis, and the data were presented in µg/g, µg/kg and mg/kg [52][53][54][55][56][57]. In the second and third steps of toxicological risk assessment, the weekly intake (µg/week) in the 600-2000 ml range, and the weekly intake per body weight depending on green tea consumption, were examined (Table 5). Since most of the EIs in the investigation (Ag, Au, Co, Cr, Cs, Li, Mo, and Sr) did not have an established value of PTWI, individual health risk assessments were evaluated. The results indicate that daily EI concentrations should not represent a health risk to consumers after consuming green tea infusions from the products available on the Polish market. The possible assessment of health risks did not show any health hazards to consumers for weekly exposure only for Se.

Silver
Silver is a naturally occurring element. Silver can exist in various forms, such as soluble silver compounds, insoluble compounds, or in the form of metallic silver. Soluble silver compounds can potentially cause adverse effects on the human body, because they are more easily absorbed than metallic or insoluble silver. In the example of overexposure to silver nitrate, symptoms include diarrhoea, stomach irritation, breathing problems, or a drop in blood pressure. After prolonged inhalation or ingestion of soluble silver compounds or colloidal silver, the most familiar characteristic, irreversible discolouration of the skin (argyria) and/or eyes (argyrosis), may appear [58]. With reference to the daily consumption (about 250 ml of green tea infusion daily), the estimated daily exposure to Ag was approximately 0.03122-4.4994 µg/day. According to the ICH guideline Q3D (R1) on elemental contaminants, the established permitted daily exposure (PDE and oral exposure) for Ag is 167 µg/day [59]. This means that drinking green tea is safe. Silver was present only in two samples (GT1: 0.364 ± 0.05 µg/L and GT3: 15.748 ± 0.09, µg/L).

Gold
Gold was present in all samples (0.105-0.0830 µg/L), but in seven at relatively low concentrations. Toxicity studies have shown that Au tends to exhibit relatively little, if any, toxicity, since many cytotoxicity studies show that gold is non-toxic [60]. The gastrointestinal tract partially absorbs elemental gold or released ions. Organs such as the liver, heart, kidneys, and lungs receive gold. Gold is primarily expelled in the urine after ingestion. There are only a few studies on the oral toxicity of elemental gold. According to Hadrup et al., rats in their investigation were unaffected by a single dose of 2000 mg of nanoparticles/kg body weight, suggesting that elemental gold has low acute toxicity [61]. According to the ICH Q3D guideline (R1), the PDE for Au of 134 µg/day, Au impurities are not hazardous to human health and are not pollutants with a crucial concentration in the environment. The study showed that you could take 0.00049-0.02074 µg/day of Au when drinking green tea (250 mL of green tea infusion per day), which is safe for health.

Cobalt
Cobalt has magnetic properties and promotes oxidation and reduction reactions. Exposure to cobalt and its compounds causes adverse health effects, such as carcinogenicity in humans [62]. Cobalt impurities were observed in all samples (0.580-2.989 µg/L). There was no Co assessment in the Joint FAO/WHO Expert Committee on Food Additives (JECFA) database. The established PDE (oral exposure) for Co by the ICH Q3D guideline (R1) is 50 µg/day [59]. Compared to the findings of this investigation, the Co impurities in green tea are low. Providing the body with such an amount of cobalt, by consuming the green tea available in Poland, does not threaten human health. The estimated daily exposure of the body to this element is between 0.07904 and 0.85421 µg/day (approximately 250 mL of green tea infusion per day). Co impurities are negligible compared to the 50 g/day (oral exposure) Co established PDE by the ICH Q3D (R1) guideline [59], and do not pose a risk to people.

Chromium
Chromium (Cr) is a "hazy" chemical element regarding human health. Although it is a crucial micronutrient, it is also linked to several diseases and toxic effects, including carcinogenicity. We still do not fully understand how Cr and its components work in humans. The chromium content in tested teas varied between 7.121 and 10.993 µg/L. Chromium PDE is described in the ICH guideline Q3D (R1), which is 10700 µg/day [59]. In the tested green teas, the Cr concentration was within the range of 7.12-10.99 µg/L. According to our estimates, drinking green tea does not pose a risk of chromium toxicity.

Caesium
Caesium was present in each tested sample, in concentrations between 0.399 and 2.654 µg/L. The knowledge of the metabolism and toxicity of caesium is limited [63]. Oral consumption of caesium chloride has been widely promoted based on a hypothesis called "high-pH cancer treatment" [64]. This element is excreted by the kidneys in humans. The biokinetic model gives the following percentages: urine at 85%, faeces at 13%, and sweat at 2% [65]. Taking into account the content of caesium in green tea infusions tested in Poland, its consumption appears safe for the human body. The ICH Q3D (R1) guideline and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) database lack Cs papers. As a result, it is impossible to compare the data obtained, although the low concentration of this element suggests no significant health problems for humans.

Lithium
A popular and successful treatment for mood disorders is lithium. Although there has been concern about its safety, there is insufficient evidence for side effects. The lithium content in the tested samples varied between 0.205 and 2.667 µg/L. Lithium can be harmful if used in excess or in situations with a risk of fluid or sodium deficit. When drinking green tea, the risk of lithium toxicity is not significant [34]. Consuming green tea infusions does not pose a risk associated with the toxic effects of lithium. The established PDE (oral exposure) for Li by the ICH Q3D guideline (R1) is based on human experience with this element, and is approximately 560 µg/day. Since the expected daily exposure to Li in the items under investigation (approximately 250 mL of green tea infusion per day) ranges from 0.0185 to 0.7170 µg/day, there is no potential issue with Li exposure after green tea consumption.

Molybdenum
Very little is known about the effects of Mo on human health. This trace element is essential for both animals and plants. In mammals, molybdenum is present as a component of some metalloflavoproteins. Molybdenum is present in drinking water in the range of 0.11-6.2 or 0-20 µg L −1 [66]. Our studies found low molybdenum concentrations in green tea infusions (0.0113-0.107 µg/L). From a regulatory point of view, this element only has a PDE (oral) value of 3400 g/day [59]. No risk is associated with green tea infusions, because the Mo concentration range in the infusions tested was between 0.002177 and 0.02488 µg/day.

Strontium
The estimated exposure to strontium in the green tea infusions investigated available in Poland was in the range of 9.848-22.331 µg/L. Strontium had the highest concentrations across all samples. However, our results compared to the value of PDE (oral) for this element, described in the ICH Q3D (R1) guideline, which is 120 µg/day, are relatively low; therefore, strontium concentrations do not pose a health risk, including drinking green tea infusions available in Poland.

Selenium
Selenium is poisonous at high concentrations, and several examples of apparent toxicity and fatalities after acute poisoning have been documented [67]. The trace element selenium (Se) is essential for synthesising amino acids, such as selenocysteine and selenomethionine. We discuss the acute hazardous effects, including exposure and concentrations in the blood and urine, linked to mortality [67]. The selenium content in the tested green tea samples was in the range of 0.067-0.308 µg/L. With the regular consumption of green tea, selenium contamination does not threaten the human body health. The PTWI for selenium is 66 µg/kg bw/week, meaning drinking 600-2000 mL of green tea covered by the study is safe for the human body [68].

Conclusions
Our developed EI research health risk assessment strategy provides pioneering data (Ag, Au, Co, Cr, Cs, Li, Mo, Se, and Sr) and can be helpful for additional research and manufacturers. Furthermore, well-designed health risk assessment methods will be valuable and essential for public health and environmental studies. As these environmental