2.1 Industrial dyeing processes in the included factory

The textile dyeing processes in the selected factory are done in three steps, as observed during the walk-through survey. Each step is done in a special working section, but all are in the same open-air working place. The three steps are processing (preparation: of the fabric and select the appropriate dyes for each fabric type), dyeing (soaking the prepared fabrics in the dyes), and printing (finishing step, in which undesirable impurities are removed from the materials and print the required shapes).

2.2 Study design and sample size

A cross-sectional study was conducted between March 2022 to February 2023. All the workers in the three textile dyeing sections (n=147) were included in the study (Exposed group), and 130 unexposed workers were included. The unexposed workers were chosen from the workers in a wastewater treatment plant not occupationally exposed to dyes during their working day, The two groups of the exposed and unexposed workers were matched in their age, socioeconomic levels, and educational levels, and both worked in open-air working areas. After obtaining written consent from the included individuals, all were subjected to the questionnaire, clinical examination, and blood sampling.

2.2.1 The inclusion criteria

- Exposed workers occupationally exposed to textile dyeing processes for more than 5 years, and their ages between 25 and 50 years.

- Unexposed subjects were not occupationally exposed to chemicals used in textile dyeing processes and matched in age range and socioeconomic status of the exposed workers.

- The exposed and unexposed workers must be working during the day shift, and they must not take any vitamins in the last 6 months, especially vitamin D.

2.2.2 Exclusion criteria2.3 Environmental assessments

Environmental monitoring was done in an Egyptian textile factory. Polycyclic aromatic hydrocarbons (PAHs) in their particulates and vapor forms and volatile organic compounds (VOCs) samples were collected bi-weekly from three areas (printing area, processing area, and dyeing area). From the printing area, 30 samples from five sites, including the printing room, drawing and design office, printing machine site, roasting site, and customer reception desk. From the processing area, 30 samples from six sites, including ram-1, ram-2, and ram-3,4 (at the front and the end of the hall). Furthermore, from the dyeing area, 28 samples from 4 sites, including laundry 1 at the front of the hall (closed machine), laundry 1 at the end of the hall (open machine), laundry 2 at the front of the hall (open machine), and laundry 2 at the end of the hall (closed machine).

2.3.1 Polycyclic aromatic hydrocarbons (PAHs)

Polycyclic aromatic hydrocarbons (PAHs) in the textile factory were collected on Whatman grade GF/A glass microfiber filters [10,11,12,13], using the low volume sampler technique (The ARA N-FRM Sampler and BOECO Vacuum Pump R-300) with an average flow rate of 10–12 l/min. Firstly, the filters were impregnated in acetone for 24 h to remove all organic contamination, and then heated at 400 °C for four hours. The processed filters were saved in desiccators till used for sampling. After collecting suspended particulate matter (SPM) samples, sample filters were covered with aluminum foil and stored for 24 h in desiccators in darkness. SPM filters were weighed till obtained constant weight to estimate SPM concentration. SPM samples were used for PAHs analysis; each filter was transferred to a glass flask. Ultrasonic bath and DCM/n-hexane (10 ml of 1:1, v:v) were used for extraction of PAHs, three times and for 10 min at room temperature. A column filled with 10 gm silica gel (70–230 mesh, ASTM purchased from Merck) and 2.0 gm anhydrous sodium sulfate was used for extraction cleanup. A rotary evaporator was used to concentrate the obtained extraction. The gas chromatography technique (limit of detection ranged from 0.50 to 3.91 ng/m3) was used to identify and evaluate the levels of the 16 EPA PAHs, which were expressed in ng/m3. Standard solution mixture of 16 PAH compounds (Supelco, Inc., Bellefonte, PA, 2000 μg/ml for each PAH) [14].

2.3.2 Classification of PAHs according to molecular weight

In this study, the method developed by Patel et al. [15] was used for the classification of PAHs as low molecular weight (LMW) or 2 rings, which were more water-soluble, moderate molecular weight (MMW) or 3 and 4 rings, which were moderate water-soluble, and high molecular weight (HMW) or 5 and 6 rings, which were less water-soluble.

2.3.3 Estimation of VOCs in the workplaces

The NIOSH standard method number 2549 was used for sampling and analysis of VOCs from the work environment. In this method, activated charcoal tube type ORBOTM-32 activated coconut charcoal (20/40) was used for VOCs air sampling according to NIOSH, 1996 [16]. Atmospheric air was drawn through tubes, by using a calibrated vacuum pump with a flow rate of 0.2 l/min (low volume sampler method: The ARA N-FRM Sampler and BOECO Vacuum Pump R-300). Sample tubes were stored in special plastic bags and kept in a freezer till processed not more than 15 days. Carbon disulfide (CS2) was used for VOCs extraction. 2.0 ml of CS2 was added to the loaded charcoal, and then a mechanical shaker was used for shaking. It stood for at least one hour to obtain the final solution. A Gas chromatography technique (limit of detection ranged from 0.001 to 0.02 mg/m3) was used for identifying and evaluating the concentrations of the individual VOCs [16].

2.4 Tools2.4.1 Questionnaire

Personal interviews were conducted with all the included subjects to fulfill personal, history, family history of cancers, and medical questionnaire, including bladder cancer symptoms (in the form of unpainful hematuria occurring suddenly, problems emptying the bladder, false sensation of a full bladder, burning feeling when passing urine, pain while urinating, lower abdominal or back pain), [17]. The questionnaire also thoroughly asked about exposure to environmental and occupational pollutants: hours of working per day, use of personal protective equipment (PPE), hours of exposure to sunlight, any drug taken, or vitamins, especially vitamin D.

2.4.2 Clinical examination

An occupational medicine specialist conducted abdominal-pelvic medical examinations for all the included persons to detect if there was any swelling, masses, or stiffness [17, 18], but per rectum examination was not done for the workers, as all of the included workers refused that.

2.4.3 Blood sampling and analysis

A 5.0-ml venous blood was obtained from each participant. A portion of the blood was placed in a clean tube and centrifuged to separate the serum. The tumor biomarkers bladder tumor antigen (BTA), nuclear matrix protein 22 (NMP-22), and the serum 25-hydroxy vitamin D were measured by quantitative sandwich enzyme immunoassay technique (ELISA) using ELISA kit from SinoGeneClon Biotech Co., Ltd. The specimens required for this kit are serum, plasma, and other biological fluids (SinoGeneClon Biotech www.sinogeneclon.com). In the present work, the serum of the included subjects was used.

The concentration of BTA in the samples was determined by comparing the O.D. of the samples to the standard curve (https://www.sinogeneclon.com/plus/view.php?aid=690).

The concentration of NMP-22 in the samples was determined by comparing the O.D. of the samples to the standard curve (https://www.sinogeneclon.com/plus/view.php?aid=4447).

Serum 25-hydroxy vitamin D (25-OH vitamin D) was measured by ELISA kit from EDI Epitope Diagnostics, Inc. The kit is designed, developed, and produced for the quantitative measurement of total 25-OH vitamin D in serum utilizing the competitive immunoassay technique. This assay utilizes a monoclonal antibody that binds to both 25-OH vitamin D2 and 25-OH vitamin D3 equally.

The concentration of a total 25-OH vitamin D in samples was determined directly from this calibration curve (https://www.sinogeneclon.com/plus/view.php?aid=2414).

2.5 Statistical analysis

The collected data were statistically analyzed using the SPSS package for Windows version 23. The quantitative data was presented as mean ± standard deviation (SD).

The limit of detection (LOD) was calculated based on the standard deviation (σ) of PAHs concentrations and the slope of the calibration curve (s) at levels approximating the LOD according to the formula [19]:

$$\text= 3.3(\upsigma /\text),$$

The smoking index (SI) was calculated for each smoker in both exposed and unexposed groups according to the number of cigarettes smoked per day (CPD) and number of years of tobacco use through the following formula [20]:

$$\text=\text\times \text$$

The comparisons were done first between the exposed and the unexposed workers using the independent t-test, and then, between the exposed workers according to their different tasks using Analysis of variance (ANOVA) and least significant differences (LSD) as a post-hoc test. Pearson’s correlation coefficient was used to test the relationships of the results. The level of significance was at P value < 0.05.