Biomonitoring of Bromine-Containing Trihalomethanes in the Pregnant Women: Urinary Levels and Association with Lifestyle Factors


 Trihalomethanes containing bromine (Br-THMs) are compounds formed from the reaction of organic and inorganic substances in drinking water with chlorine as a common disinfectant. Epidemiological evidence indicated various outcomes as a result of long-term exposure to these compounds. This study aims to investigate the concentration of urinary Br-THMs in a sample of third-trimester pregnant women and to determine its correlation with lifestyle factors. This cross-sectional study was conducted among 118 pregnant women in Isfahan, Iran. The third-trimester urine Br-THMs including dibromochloromethane (DBCM), bromodichloromethane (BDCM), and broomcorn (TBM) were measured using solid-phase micro-extraction gas chromatography. The distribution of urinary Br-THMs concentration was positively skewed then these variables were subjected to logarithmic transformation. Spearman ranks correlation coefficient, and two independent samples t-test were used to assess the association of urinary Br-THMs concentration with its demographic and lifestyle determinants.The findings showed that the mean (SD) concentrations of DBCM, BDCM, TBM and brominatede THMs were 14.76 (27.17), 10.25 (11.37), 4.96 (3.21) and 30.17 (40.80) ng/L, respectively. In addition, significant inverse correlation was found between the use of water storage tanks and Br-THMs (DBCM, BDCM, TBM and brominatede THMs, P-value = 0.005, 0.019, >0.001 and 0.007 < 0.05, respectively).In this study, among the quantitative and qualitative variables, the use of storage tanks had an inverse correlation with Br-THMs. In addition, the status of urinary concentration of Br-THMs in pregnant women was less than in the other countries in the same area. Improving water quality and using modern methods to eliminate disinfection by-products as well as improving lifestyle patterns are efficient methods to reduce exposure to these contaminants.


Introduction
Public health has been enhanced by disinfecting drinking water to prevent waterborne infections. Followed by disinfection by-products (DBPs) are created from the natural reaction of chlorine as the most popular disinfectant of drinking water with organic and inorganic substances in water. So that chlorination processes have produced more than 700 types of DBPs up to now (Deng et al., 2019).
Water ingestion or other activities such as swimming, showering, and bathing are the routes of exposure to THMs, through ingestion and non-ingestion, respectively (Andra et al., 2015). Also, adult women can be exposed to noningestion THMs by cleaning the house, such as washing dishes, cleaning surfaces, and toilets (Andra et al., 2015). Around 60 days accumulation of 100 mg/kg/day TCM, TBM, and BDCM and 400 mg/kg/day for TTHMs in mature rats caused neurotoxicity (Chen et al., 2019). Moreover, reproductive disorders in rodents have also been found based on exposure to high levels of DBPs according to toxicological evidence. For example, respiratory and oral exposure to THMs has resulted in low birth weight infants in rodents (Deng et al., 2019). Also, the carcinogenic and mutagenic effects of Br-THMs have been reported in rodents (Stayner et al., 2014).
Followed by cancer and reproductive implications in humans, including length of pregnancy, sperm quality, menstrual cycle, and pregnancy outcomes such as fetal growth, fetal loss, preterm delivery, and congenital anomaly, on epidemiological evidence, are the consequences of long-term exposure to THMs (Villanueva et al., 2011). How to use water in bathing, showering, and swimming, also water consumption pattern displays the degree of external exposure to THMs through tap water. Internal exposure to THMs can be assessed by measuring blood and urine biomarkers (Charisiadis et al., 2014). Different routes of exposure to THMs biomarkers are evaluated by measuring it in the urine and using lifestyle questionnaires. The remaining THMs in the kidneys and greater than in the liver are metabolized through the interaction of cytochrome (CY) P450 and the enzymatic system in two phases. The production of reactive oxygen types in the liver and kidneys by CYP2E1, followed by combination with glutathione in phase II, determines the Therefore, according to the available evidence based on the presence of these contaminants in the drinking water distribution system, this study aimed to investigate the concentration of urinary Br-THMs among third-trimester pregnant women in Isfahan as a sensitive and vulnerable group and their correlation with lifestyles such as how to consume drinking water and washing and bathing activities.

Study population
This cross-sectional study was conducted to measure the concentration of urinary Br-THMs. The target group consisted of 118 pregnant women in the third trimester, who participated in the PERSIAN Birth Cohort and lived in different parts of Isfahan city, Iran (Ref: DOI: https://doi.org/10.1332/175795920X16062247639874).
The locations of living areas of participants are given in Fig. 1. The informed consent form was provided to participants regarding the type of study, purpose, and method of urine sampling. Also, all stages of this study were performed after the approval of the ethics committee of Isfahan University of Medical Sciences. Inclusion criteria in this study for participants include living in Isfahan for at least one year or more, the third trimester of pregnancy, and no history of chronic diseases. Subsequently, smoking and alcohol consumption were considered exclusion criteria.

Questionnaire
To determine the correlation between the concentration of urinary Br-THMs with lifestyle factors and demographic characteristics such as age, education, and BMI the prepared questionnaires in the birth cohort were used that its validity and reliability had been con rmed.
In addition, all the questions of the questionnaire were asked under face-to-face interviews by the trained questioner. Personal information of pregnant women including age and occupation and their physical characteristics such as height, the third-trimester weight of each month, and body mass index regularly during prenatal care according to appointments scheduled was requested by a trained health expert and was measured using calibrated instruments and recorded electronically. BMI is equivalent to weight divided by height squared. (Kang et al., 2016). Education level was also classi ed into 5 groups: primary school, junior school, high school, and academic. In addition, study participants were selected from different areas of Isfahan. Moreover, information on how to use drinking water daily or weekly or related activities were collected in the form of questions such as drinking water source, amount of water used per day, consumption of boiled water, use of refrigerator, frequency and duration of washing dishes, clothes and baths.

Urine Sampling and Analysis
In this study, morning spot urine samples of 118 pregnant women in the third trimester were randomly collected in the referral hospitals of Isfahan University of Medical Sciences, Isfahan, Iran. Urine sampling was performed by sterile polypropylene containers by the individual and nally, the samples were transferred to the laboratory of the Health school under cold conditions and kept at -20°C until use.

Extraction method
The solid-phase microextraction method was used to extract species from urine tissue. The used ber was PDMS-CAR with a thickness of 75 µm. 0.5 mL of the urine sample was poured into a 12 mL glass container with a septum lid. 1.2 g of sodium chloride with a magnet was added to the container and the lid was closed and equilibrated at room temperature at 1250 rpm. After 5 min of equilibrium time, the ber was exposed through the septum to the upper space of the sample and the extraction was performed for 15 min. Decoding was performed at 240°C for 45 sec inside a gas chromatographic injection chamber.

Chromatographic method and device conditions
Gas chromatography-mass spectrometer was used to isolate and measure the species. The gas chromatograph used was the FISONS-8000 (8060) and the mass spectrometer used was the Hewlett Packard-5972. Supelco column (SLB-5 MS) with a length of 30 m and a static phase thickness of 0.25 µm and a diameter of 0.25 mm was used for separation. Oven temperature programming: 45°C for 5 min -increase the temperature to 100 ° C with a slope of 20 °p er minute and stay at 100°C for 1 min. Limit of detection (LOD) for DCBM, CDBM, and TBM was calculated 5, 6, and 3 ng/L, respectively. The terms of the mass spectrometer can be seen in Table 1. . Quantitative continuous data were reported as mean (SD) or geometric mean and minimum and maximum. Qualitative variables were also reported as frequency and percentage. The normality of urinary Br-THMs concentration was evaluated using the Kolmogorov-Smirnov test and Q-Q plot. The distribution of urinary Br-THMs concentration was positively skewed. Then these variables were subjected to logarithmic transformation. Person or Spearman ranks correlation coe cients were used to assess the association of urinary Br-THMs concentration with demographic and lifestyle-related variables of study participants. Two independent samples t-test was used for comparing urinary Br-THMs concentration between categories of qualitative lifestyle-related variables.

Characteristics of the study population
The demographic and lifestyle characteristics of 118 pregnant women in the present study are shown in Table 2  Other information about washing dishes and clothes, as well as cleaning the house, including the number of times (per day or week) and duration (min), is given in Table 2. Table 3 shows

Urinary concentration of Br-THMs and related factors
No signi cant dose-response relationship was observed through correlation coe cient (r s ) between all of the variables (P-value > 0.05) in Table 4. The variables were age, education, weight, BMI, water, and tea consumption, and frequency and time of washing clothes, dishes, and bathing. The effect of the storage tank on the concentration of three contaminants has shown a signi cant relationship (Pvalue < 0.05). Other parameters in Table 5, such as boiling water, level of education, use of dishwasher, and gloves did not show a signi cant relationship with the concentration of all three contaminants (P-value > 0.05).

Discussion
An area of present cross-sectional research about investigating the concentration of Br-THMs in urine as a biomarker of exposure to drinking water disinfection by-products, in pregnant women was done in Isfahan, Iran, as the rst study.
In the previous studies, the concentration of total trihalomethanes (TTHMs) has been identi ed in the water of Isfahan city (  In our study, according to statistical reports, the effective source of receiving Br-THMs in pregnant women in Isfahan was the use of a water storage tank (p < 0.05) (Tables 5). That showed a signi cant negative correlation with the concentration of urinary Br-THMs in the participants.
Using a tank is a common way for storage of water in the residential house. In this study, the use of water tanks, which are usually made of plastic, with a negative correlation, reduced the amount of DCBM, BDCM, and TBM.
Scienti c research by Cao et al. showed that the application of polyethylene pipes has been effective on water quality over time by sorption contaminants, especially THMs (Cao et al., 2020). Also, according to the study of Slavik et al., increasing the volume of the storage tank has reduced the amount of disinfection residual, thus affecting the formation of disinfection by-products (Slavik et al., 2020).
In Table 2, in addition to tap water, participants have used other sources, such as bottled water, for drinking; but most of them had a higher average consumption of tap water than other sources. According to the analysis of Charisiadis et al., the amount of THMs in water bottles against tap water was non-detectable (Charisiadis et al., 2013).
Although there was an excessive daily intake of drinking water with an average of 7.14 glasses per day, urinary levels of THMs were reduced because of dilution of the THMs mass (Charisiadis et al., 2013). As a result, according to Table 4, there is no signi cant relationship between the urinary concentration of these contaminants and water intake (p > 0.05).
Also, Table 4  In another evaluation, participants' tea consumption did not have a signi cant correlation with brominated THMs.
According to Fakour et al., the use of boiling water and preserving the water temperature at 100°C for a long time, as well as more use of teapot instead of the ask as tea containers, reduces chlorine by 5-19% via better output evaporated volatile compounds into the atmosphere (Fakour and Lo, 2019). In Iran, based on the culture, tea is consumed as a common beverage during the day and for a long time, and also using teapot is usual but our data in this study did not agree with the ndings of this researcher (25).
In Table 5, for other qualitative parameters, we did not observe a signi cant relationship between the concentration of Br-THMs and the use of household water puri ers, boiled water, dishwasher, gloves, and nally the type of detergent.
Carrasco-Turigas et al. stated that the use of household reverse osmosis lters affect the removal of THMs containing bromine as non-volatile compounds, moreover boiling through conventional heating such as electric kettles by creating big bubbles and equally energy had a signi cant in uence on the chloroform removal (Carrasco-Turigas et al., 2013).

Study strengths and limitations
The strengths of this study include the population-based design, high-quality urinary Br-THMs assays.
One of the strong limitations of this study was the small statistical population. According to previous studies, despite long-term sampling, increased the identi cation of THMs compounds, especially chloroform, which has high volatility (Gängler et al., 2017), but the agreement of the participants to attend in this study depended on the short sampling time. In addition, the majority of participants have not much contribution in household tasks due to being in the third trimester of pregnancy.
The spot urine sampling may not be the best biomarker of maternal Br-THMs exposure and does not control the effect of other pollution during pregnancy.

Conclusion
Among the participants of this study, the use of storage tanks inversely showed a signi cant correlation. There was no evidence of signi cance between other quantitative and qualitative parameters. Comparison of Br-THMs concentrations in global studies showed that the amount of Br-THMs in women's urine in this study was dramatically less than some Asian and Asian-European countries in urine samples and was more than some American and other Asian countries in blood samples. Lifestyle and the way of consuming water between different countries have created different external and internal exposures.
Our ndings show that information extracted from a combination of external and internal exposure can be effective in identifying and managing the health outcomes of environmental exposure. Furthermore, larger sample size is necessary to more suitably and signi cantly correlate the concentration of contaminants with lifestyle factors.

Declarations
Authors' contributions BD contributed in the concept, conducting the study, and drafting the manuscript.AF conducted the statistical analysis and revision of the manuscript. MA contributed in the concept, conducting the study, and revising the manuscript. NB contributed in conducting the study and revising the manuscript. ShD contributed in conducting the study and revising the manuscript. RK contributed in the concept, conducting the study, and revising the manuscript.

Figure 1
The locations of living areas of participants