Biomonitorization of concentrations of 28 elements in serum and urine among workers exposed to indium compounds

Nan Liu; Yi Guan; Bin Li; Sanqiao Yao

doi:10.1371/journal.pone.0246943

. 2021 Feb 22;16(2):e0246943. doi: 10.1371/journal.pone.0246943

Biomonitorization of concentrations of 28 elements in serum and urine among workers exposed to indium compounds

Nan Liu ¹, Yi Guan ¹, Bin Li ², Sanqiao Yao ^1,^3,^*

Editor: Yi Hu⁴

PMCID: PMC7899351 PMID: 33617552

Abstract

Many studies have documented the abnormal concentrations of metals/metalloids in serum or urine of occupational workers, but no works systematically analysed the concentrations of elements in serum or urine of indium-exposed workers. This study was aimed to assess 28 elements in serum and urine from 57 individuals with occupational exposure to indium and its compounds. Control subjects were 63 workers without metal exposure. We collected information on occupation and lifestyle habits by questionnaire. Biological samples were collected to quantify elements by inductive coupled plasma-mass spectrometer. Air in the breathing zones was drawn at flow rates of 1.5–3 L/min for a sampling period of 6 to 8 h, using a Model BFC-35 pump. The average ambient indium level was 0.078 mg/m³. Serum/urine Indium levels were significantly higher in indium-exposed workers than in controls (P < 0.01). Moreover, serum/urine indium concentrations in the group with 6–14 years and ≥15 years of employment were significantly higher than those with ≤5 employment years(P < 0.05). Ten of the other 27 elements/metals measured were higher in serum/urine in indium-exposed workers compared to the controls (aluminum, beryllium, cadmium, cesium, chromium, lithium, manganese, magnesium, molybdenum and vanadium). Zinc levels in serum/urine were significantly decreased in the indium-exposed workers. Additionally, other elements/metals were higher in one specimen (serum or urine) but lower in the other (Selenium was lower in serum but higher in urine in the indium-exposed workers compared with the controls; likewise Thallium and Rubidium were higher in serum but lower in urine). Linear regression analyses, revealed significant correlations between serum and urine for indium, aluminum, arsenic, barium, cadmium, cesium, cobalt, selenium, silver, and zinc (P < 0.05). These data suggest that occupational exposure to indium and its compounds may disturb the homeostasis of trace elements in systemic circulation, indium concentrations in serum or urine appear reflective of workers’ exposure to ambient indium and their years of working, respectively. The serum/urine levels of essential metals are modified by exposure to indium in occupationally exposed workers. Further studies including larger sample size and more kinds of biological sample are needed to validate our findings.

Introduction

Indium is relatively rare element found in ores of zinc, copper, and tin and has been used in flat-panel displays, optoelectronic devices, and photovoltaic cells for decades. Occupational exposure to indium and its compounds can result in potentially fatal indium lung disease including pulmonary alveolar proteinosis that may progress to fibrosis [1–7]. In the process of indium smelting, there are occupational hazards such as dust, lead, arsenic, cadmium, indium, zinc, hydrogen arsenide, various acids and bases, noise, high temperature. Workers are likely to be exposed to various metals/metalloids and elements simultaneously in their working environment. Lead, cadmium, and arsenic were reported to be the main hazardous metals/metalloids. They cause serious damage to many target organs in human bodies through the mechanism of inflammation, production of oxidative stress, and interference with essential elements. The interactions among these toxic metals/metalloids are extremely complex and some effects which have not been observed in single constituent exposure may occur. On the other hand, copper, and zinc are essential trace elements with regulatory, immunologic, and antioxidant functions, but when they are beyond or below a certain serum concentration, they may cause a threat to human health. Under the exposure to multiple metals/metalloids which may compete with or regulate the function of the essential elements in human bodies, they may cause additive, synergistic, or antagonistic effects, or even new effects may occur [8]. To date, no studies on indium occupational workers concerned trace elements. Indium pulmonary toxicity may be associated with its interaction with other essential trace elements. Although there are some reports on levels of indium in blood or urine during production process, the question as to how low-level, long-term exposure to indium and its compounds may affect blood or urine levels of trace metals is unanswered [9]. Therefore, it is important to know and measure trace elements status in indium-exposed workers because the alterations in the content may play an important role in the pathogenesis of pulmonary alveolar proteinosis and related metabolic risk factors.

This study will analyze 28 elements in two different human biological materials (serum/urine) by inductively coupled plasma-mass spectrometer (ICP-MS), and assess the classical fluids (serum and urine) for biomonitoring the internal dose of individuals occupationally exposed to indium and its compounds. Because the indium-exposed workers were typically population occupationally exposed to indium compounds and toxic metals, they are particularly suitable for biological monitoring programs to measure trends of occupational exposure with early biological effects and to explore their dose-response relationships. In particular, a risk assessment methods should be also adopted, given that this population may present an increased health risk to pulmonary alveolar proteinosis disease. Hence this study provides values that may be useful for comparisons in future studies, or in addressing occupational health challenges associated with indium and its compounds exposure. The purpose of this study was to investigate whether or not chronically occupational exposure to indium and its compounds altered the homeostasis of essential trace elements in biological fluids.

Subjects and methods

Study population

Subjects

This was an exploratory study that used data obtained during a health check by a cross-sectional study between February 21 and March 15, 2015. The subjects were workers in indium ingot production plant from Guangxi, China who were mainly exposed to indium metal with a potentially high level, but also to In₂SO₄, In₂O₃, and to a lesser extent, indium chloride [InCl₃] with a potentially high level of exposure. During operation, the indium-exposed workers wore dust-free clothes, while maintenance workers wore full face filter cotton, goggles and protective gloves. The control subjects in this study were a random sample of all candidates derived from another nearby factory, who were office workers and had no history of occupational exposure to indium and other metals. The selected control subjects were matched to the age, gender, average employment history, smoking status and drinking habits with indium-exposed workers.

The selection criteria of the study population for this study were:(1) no history of occupational exposure to other metals; (2) no supplement of trace elements; (3) complete information of outcome measured value (both in serum and urine); and (4) excluding outliers of each measured value. In this study, new occurrence of hypomagnesemia was defined as outliers, and thus observation in worker who already had outliers was excluded. Finally, the data analysis included a total of 120 subjects, which comprised of 57 indium-exposed workers and 63 controls.

Patient and public involvement

Characteristics of the sampling method used to collect the biological samples selected for this study have been described previously [10]. An interview with a questionnaire was completed by trained interviewers to obtain detailed information on personal information, occupational history, job description, lifestyle information (smoking, alcohol consumption, dietary habits), and personal medical history. Written consent was obtained from the subjects who participated in this study and they were also informed of their right to withdraw anytime. The proposal was previously approved by the Ethics Review Committee of the North China University of Science and Technology (Approval No.15080). All study participants in both indium occupational workers and control groups at the time of interview had no reported exposure to other toxic substances, radiation therapy, autoimmune disease, or substance abuse (such as antibiotics).

Potential confounders and sources of bias

As potential confounding factors, data on age, gender, smoking status, and drinking habits were obtained from the interview with a questionnaire. We collected data of indium-handling workers who wore protective equipment, but controls have no data for protective equipment. In addition, the missing and incorrect values (i.e., new occurrence of hypomagnesemia, among others) were reconfirmed with indium ingot production plant and another nearby factory, and tried to exclude selection bias.

Sample preparation and analysis

Workplace air concentrations of indium dust

Based on the indium production process, four different locations in the workplace were identified as the monitor sites. Air samplers were placed in each workplace. The workplace air was drawn at a flow rate of 1.5-3L/min for a sampling period of 6 h or 8 h, except during lunchtime, using a Model BFC-35 pump equipped with a mixed cellulose ester membrane filter (a diameter of 37 mm, a cut size of 0.8 μm). The samples were collected for two more times at each monitoring site. Workers who wore the personal sampling devices were asked to record the contents of their jobs during the work periods.

A glass fiber filter on which indium-containing particulate matter had been collected was placed into a flask containing 20 ml of mixed acid (nitric acid: ultrapure water = 1:1) and subjected to ultrasonic application for 60 min, and digested on a hot plate at 120°C for 60 min. The digested sample was diluted to 10ml with ultrapure water and then subjected to analysis. All analyses were performed in a blinded manner, and the results were confirmed by replication on the following day.

A standard solution of indium for inductively coupled plasma mass spectrometer (ICP-MS) (Agilent 7500a, Agilent Technology, USA) was used for preparation of a calibration curve for the quantitation. The quantitation limit of the airborne indium concentration was estimated to be 0.045 μg In/m³. This estimate is ~15% of the acceptable indium exposure limit of 0.0003 mg/m³.

Sample preparation and elements analysis

Sample collection and processing were carried out in local clinics. Five milliliters of venous blood was collected in a pro-coagulation tube, let stand at room temperature for 15 min, and centrifuged at 12,000 rpm for 10 min to separate serum, and immediately transferred to 2 mL frozen pipe. A spot 10-mL urine sample collected from each subject at the end of a work shift have been described previously [10]. All samples were stored at -80°C until analysis. All test tubes used in the study were free of metal contamination.

The 28 elements analyzed in this work were aluminum (Al), arsenic (As), barium (Ba), beryllium (Be), bismuth (Bi), cadmium (Cd), calcium (Ca), cesium (Cs), chromium (Cr), cobalt (Co), copper (Cu), indium (In), iron (Fe), lead (Pb), lithium (Li), manganese (Mn), magnesium (Mg), molybdenum (Mo), nickel (Ni), potassium (K), rubidium (Rb), selenium (Se), silver (Ag), sodium (Na), strontium (Sr), thallium (Tl), vanadium (V), and zinc (Zn).

At the time of sample analysis, serum and urine samples were brought to room temperature. An aliquot of 500 μL serum and urine samples was diluted with a solution containing 0.1% (V/V) Triton-X-100 (Sigma, USA) and 1% ultrapure concentrated nitric acid (Sigma, USA) to a 5 mL total volume. Prior dilution of each sample was critical in order to obtain the best results. The samples were then quantified by ICP-MS using freshly made multi-element stock solution on the day of analysis. The instrument parameters are as followed: nebulizer carrier gas flow, 1.10 L/min; sample depth, 4.8 mm; RF power, 1450 W; sampler/skimmer, nickel; CeO⁺/Ce⁺, <0.5%; ¹⁴⁰Ce¹⁶O/¹⁴⁰Ce, <2%. Calibration was performed using a certified reference standard (Agilent, USA). Validity of the calibration curve was evaluated by analyzing the standards from the same source after every two hours injection. The calibration curve was considered valid if the observed concentration of the independent standard was within 10% of the expected concentration. We also measured the recovery of each sample at random. The internal standard and the limits of detection (LOD) for these 28 elements were shown in Table 1.

Table 1. The limits of detection for these 28 elements.

Element	Internal standard	Limits of detection (LOD)	Element	Internal standard	Limits of detection (LOD)
²⁷Al	⁴⁵ Sc	0.108 μg/L	⁷Li	⁴⁵ Sc	0.025 μg/L
⁸⁵As	⁷² Ge	0.032 μg/L	⁵⁵Mn	⁴⁵ Sc	0.014 μg/L
¹³⁷Ba	¹⁰³ Rh	0.041 μg/L	²⁴Mg	⁴⁵ Sc	0.795 μg/L
⁹Be	⁴⁵ Sc	0.007 μg/L	⁹⁵Mo	⁸⁹ Y	0.057 μg/L
²⁰⁹Bi	¹⁰³ Rh	0.014 μg/L	⁶⁰Ni	⁷² Ge	0.042 μg/L
¹¹⁴Cd	¹⁰³ Rh	0.075 μg/L	³⁹K	⁴⁵ Sc	4.220 μg/L
⁴⁰Ca	⁴⁵ Sc	1.202 μg/L	⁸⁹Rb	⁸⁹ Y	0.053 μg/L
¹³³Cs	¹⁰³ Rh	0.061 μg/L	⁸²Se	⁸⁹ Y	0.142 μg/L
⁵²Cr	⁴⁵ Sc	0.039 μg/L	¹⁰⁸Ag	¹⁰³ Rh	0.036 μg/L
⁵⁹Co	⁴⁵ Sc	0.015 μg/L	²³Na	⁴⁵ Sc	2.560 μg/L
⁶³Cu	⁷² Ge	2.526 μg/L	⁸⁸Sr	⁸⁹ Y	0.009 μg/L
¹¹⁵In	¹⁰³ Rh	0.053 μg/L	²⁰⁵Tl	¹⁰³ Rh	0.037 μg/L
⁵⁶Fe	⁴⁵ Sc	4.068 μg/L	⁵¹V	⁴⁵ Sc	0.079 μg/L
²⁰⁸Pb	¹⁰³ Rh	0.019 μg/L	⁶⁶Zn	⁷² Ge	2.431 μg/L

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Statistical analyses

All statistical analyses were conducted with SPSS 17.0 software. All data are presented as the mean ± SD unless otherwise stated. Differences between two groups were evaluated for significance using Welch’s t test for datas. The differences between two means were analyzed by independent samples T test. Associations between serum and urine concentrations of elements were analyzed by a linear regression. The standardized regression coefficient was used to compare the effect of different independent variables (serum and urine for indium, aluminum, arsenic, barium, cadmium, cesium, cobalt, selenium, silver, and zinc) on dependent variables. To relate indium concentration with trace element concentrations variables, we used multiple linear regression and included age, length of service, gender, smoking and alcohol consumption as co-variates. Age and length of service were adjusted as continuous random variables, while gender, smoking and alcohol consumption were categorical variables. The results which includes all covariates with p-values less than 0.05 are reported. All statistical tests are two-sided with a significance level of 0.05.

Results

Demographics of the study population

The data of 138 subjects (included 69 indium-exposed workers and 69 control subjects) were analyzed. The subjects selection flowchart are summarized in Fig 1. Initially, 8 indium-exposed workers and 5 controls were excluded owing to oral calcium tablets and oral iron supplement; 4 workers with missing biological samples were then excluded. One observation had outliers outcome were excluded. Finally, data of elemental analysis from 120 workers of serum and urine were analyzed.

The demographic characteristics are summarized in Table 2. The distribution of smoking status was significantly different between the 57 indium-exposed workers and 63 unexposed controls. The exposed workers included less smokers than the controls. There were no significant differences in age, sex distribution, average employment history and drinking habits between the exposed workers and controls.

Table 2. Demographics of the study population.

		Controls (n = 63)	Workers (n = 57)
Age (years)	Mean ± SD	39.32 ± 11.37	37.82 ± 9.34
Sex	Male	46 (73.0)	41 (71.9)
	Female	17 (27.0)	16 (28.1)
Smoking	Non-smoker	32 (50.8)	36 (63.2)
	Smoker	31 (49.2)	21 (36.8)
Drinking	Non-drinker	43 (68.3)	39 (68.4)
	Drinker	20 (31.7)	18 (31.6)
Employment period (years)	Mean ± SD	8.97 ± 8.39	8.31 ± 7.54

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Ambient indium concentrations

The airborne indium levels in the indium ingot production plant varied from 0.001 to 1.12 mg/m³ (median indium level, 0.008 mg/m³). The average ambient air level of indium was 0.078 ± 0.065 mg/m³. In the control group, however, none of the airborne samples reached a quantifiable level of indium.

Determination of 28 elements in serum and urine

The distribution of indium and other 27 elements levels in serum is shown in Table 3. Serum concentrations of the 28 elements varied from 26.38 ng/L (Be) to 346.72 mg/L (Na). Eleven of the 28 (39%) elements showed approximately equal concentrations in serum of the workers and controls. These included As, Ba, Bi, Ca, Co, Fe, Ni, K, Ag, Na and Sr. The serum concentrations of indium in workers and controls were (39.26±25.57) and (4.93±2.76) μg/L (P <0.01), respectively. Analyses revealed that there were 28% increase in serum concentration of Al, 31% increase in Be, 27% increase in Cd, 24% increase in Cs, 55% increase in Cr, 19% increase in Cu, 50% increase in Pb, 16% increase in Li, 35% increase in Mn, 7% increase in Mg, 15% increase in Mo, 23% increase in Rb, 36% increase in Tl, 27% increase in V, and 29% decrease in Zn, 19% decrease in Se among indium-exposed workers in comparison to those in controls.

Table 3. Concentrations of 28 elements in serum and urine of workers and control subjects.

Elements	Biological sample	Controls (n = 63)				Workers (n = 57)
Elements	Biological sample	Mean± SD	Range	Median	5-95th percentiles	Mean± SD	Range	Median	5-95th percentiles
Al (μg/L)	Serum	7.82±2.51	3.10–13.96	7.59	4.42–12.94	10.01±3.35^**	5.88–21.39	9.04	6.24–18.41
	Urine	17.04±5.03	1.75–22.56	18.77	5.10–21.75	19.92±6.53^**	4.81–40.16	17.91	13.19–35.27
As (μg/L)	Serum	4.73±3.46	0.31–14.98	4.31	0.52–11.12	5.40±3.70	0.43–13.06	5.53	0.89–12.42
	Urine	3.62±3.21	0.18–17.86	2.85	0.34 = 9.86	8.33±4.51^**	0.52–19.06	8.03	1.38–15.90
Ba (μg/L)	Serum	7.99±6.57	2.20–36.59	6.12	2.59–25.21	9.98±5.89	3.67–42.22	8.97	4.27–20.34
	Urine	3.01±0.87	2.10–4.79	2.69	2.11–4.40	3.00±0.83	2.14–4.92	2.60	2.17–4.65
Be (ng/L)	Serum	26.38±11.84	3.80–58.40	26.25	8.24–48.58	34.62±21.59^*	7.10–147.5	31.23	11.38–69.58
	Urine	17.75±3.46	13.30–29.90	17.10	13.44–24.54	19.80±3.74^**	14.20–29.20	19.10	14.38–26.50
Bi (μg/L)	Serum	15.61±13.56	1.23–76.93	12.71	3.12–47.21	30.00±26.11	3.39–100.43	20.78	5.03–94.58
	Urine	130.05±33.51	76.80–256.50	123.30	85.68–198.24	150.01±53.77^*	67.50–281.70	141.00	81.39–249.28
Cd (μg/L)	Serum	0.77±0.35	0.33–1.99	0.71	0.35–1.46	0.98±0.37^**	0.42–1.92	0.91	0.43–1.65
	Urine	0.28±0.21	0.07–1.13	0.20	0.08–0.77	0.38±0.30^*	0.12–1.67	0.25	0.14–1.15
Ca (mg/L)	Serum	104.33±36.23	62.15–222.05	88.80	64.59–206.13	94.98±36.85	46.05–220.55	86.23	47.64–187.53
	Urine	80.62±21.84	52.19–141.12	74.96	52.89–124.20	94.76±31.62^**	51.34–152.15	82.73	53.09–150.28
Cs (μg/L)	Serum	4.33±1.12	2.56–8.55	4.12	2.86–6.61	5.35±2.61^**	2.25–16.05	4.70	2.71–10.78
	Urine	3.23±1.03	2.10–7.24	3.23	2.14–4.86	4.01±1.73^**	2.01–8.84	3.50	2.09–7.43
Cr (μg/L)	Serum	0.86±0.31	0.43–1.72	0.75	0.50–1.53	1.33±0.53^**	0.43–2.77	1.42	0.49–2.48
	Urine	1.13±0.87	0.20–4.37	1.02	1.24–3.66	2.58±1.38^**	0.31–6.47	2.36	0.60–5.49
Co (μg/L)	Serum	1.54±0.62	0.75–3.57	1.39	0.84–2.48	1.75±0.56	0.92–3.36	1.80	0.92–2.63
	Urine	0.29±0.18	0.10–1.05	0.26	0.10–0.84	1.22±0.54^**	0.33–2.60	1.19	0.35–2.29
Cu (μg/L)	Serum	0.83±0.14	0.65–1.46	0.81	0.67–1.10	0.99±0.32^**	0.68–2.13	0.86	0.72–1.97
	Urine	31.92±12.39	10.71–60.26	25.87	15.13–54.11	52.28±25.19^**	10.12–105.70	51.13	12.43–104.13
In (ng/L)	Serum	4.93±2.76	0.93–13.64	4.36	0.98–10.38	39.26±25.57^**	11.86–137.63	32.93	14.78–97.99
	Urine	2.34±1.77	0.01–8.61	1.81	0.11–5.02	9.24±10.31^**	0.07–54.93	7.24	0.29–31.90
Fe (mg/L)	Serum	4.34±0.56	2.74–5.88	4.37	3.11–5.11	5.99±1.71	3.00–9.68	6.05	3.57–9.58
	Urine	1.54±0.55	1.03–3.11	1.19	1.10–2.38	2.70±1.88^**	1.11–8.98	2.09	1.13–8.19
Pb (μg/L)	Serum	6.86±3.58	2.36–21.92	6.13	2.97–15.43	10.29±6.23^**	3.32–38.55	8.54	3.96–25.12
	Urine	0.97±0.44	0.09–2.37	1.14	0.21–1.37	1.07±0.99	0.06–7.19	1.03	0.13–2.33
Li (μg/L)	Serum	38.27±14.76	29.93–130.00	35.10	31.07–47.66	44.45±7.26^**	34.58–69.80	42.30	35.39–57.02
	Urine	37.04±13.72	17.05–79.60	36.10	18.35–68.80	49.84±18.96^**	22.35–120.25	43.85	28.96–85.64
Mn (μg/L)	Serum	7.41±2.74	3.41–15.92	7.22	3.67–13.76	9.97±3.79^**	4.47–24.05	8.77	5.79–17.13
	Urine	2.04±1.30	1.01–5.68	1.22	1.04–4.87	4.54±2.04^**	1.06–9.11	4.42	1.52–8.49
Mg (mg/L)	Serum	18.74±2.47	14.42–26.26	18.85	14.85–23.54	19.99±3.45^*	14.41–28.35	19.22	15.02–26.05
	Urine	21.01±3.57	12.16–28.35	22.12	14.52–25.38	22.83±4.93^*	10.26–38.30	22.26	14.61–31.85
Mo (μg/L)	Serum	5.44±1.03	3.24–7.99	5.36	4.03–7.55	6.28±1.40^**	4.16–8.89	6.09	4.27–8.81
	Urine	18.51±4.00	10.24–26.79	18.12	11.31–25.10	44.01±27.37^**	10.01–97.53	37.80	10.49–97.22
Ni (μg/L)	Serum	8.51±3.45	3.03–19.57	8.01	3.52–14.99	9.84±4.50	3.82–23.62	8.45	4.69–20.35
	Urine	8.14±4.16	2.08–17.56	7.21	2.60–16.62	9.04±4.91	2.05–21.96	8.28	2.90–21.50
K (mg/L)	Serum	145.38±28.88	90.18–189.33	148.10	99.07–184.82	144.23±27.66	93.55–183.43	156.70	96.76–177.02
	Urine	212.87±46.47	140.90–315.10	204.83	145.94–306.40	220.06±41.58	144.50–323.00	218.25	155.55–304.30
Rb (mg/L)	Serum	2.73±0.46	1.93–3.90	2.60	2.14–3.63	3.35±1.09^**	1.92–6.89	3.09	2.06–6.25
	Urine	3.39±0.70	1.72–5.77	3.32	2.22–4.62	2.73±0.49^**	1.56–3.99	2.80	1.59–3.60
Se (μg/L)	Serum	84.67±17.52	42.90–144.13	82.93	55.55–115.42	68.16±12.16^**	39.00–99.70	69.98	48.83–90.07
	Urine	10.08±3.31	3.61–19.97	11.11	4.46–14.17	21.65±10.26^**	4.10–44.23	21.21	5.51–42.05
Ag (μg/L)	Serum	0.05±0.02	0.002–0.13	0.05	0.01–0.09	0.06±0.03	0.003–0.17	0.05	0.01–0.14
	Urine	0.10±0.03	0.04–0.20	0.11	0.04–0.14	0.14±0.05^**	0.04–0.25	0.14	0.06–0.23
Na (mg/L)	Serum	339.19±26.38	291.01–394.97	338.02	293.98–382.21	346.72±34.46	289.57–398.97	351.77	291.02–392.62
	Urine	924.07±185.46	609.10–1840.00	904.25	713.44–1163.80	901.07±130.01	717.56–1294.00	913.85	722.90–1107.83
Sr (μg/L)	Serum	30.06±19.99	8.59–92.20	26.68	9.70–85.01	30.72±19.19	8.05–85.98	25.4	10.54–74.59
	Urine	12.64±2.58	8.05–18.63	12.56	8.38–17.27	13.52±2.71	8.03–20.36	13.67	9.23–17.96
Tl (μg/L)	Serum	0.11±0.07	0.01–0.33	0.11	0.02–0.23	0.15±0.10^*	0.02–0.43	0.13	0.04–0.40
	Urine	0.13±0.02	0.09–0.20	0.12	0.09–0.17	0.09±0.02^^	0.05–0.16	0.09	0.06–0.14
V (μg/L)	Serum	31.56±4.05	19.5–42.73	31.65	24.54–38.65	40.10±8.07^**	26.90–60.83	39.23	28.03–59.26
	Urine	21.00±2.68	15.54–25.90	21.54	16.04–24.65	23.77±3.81^**	16.70–32.81	23.89	17.63–31.50
Zn (mg/L)	Serum	1.27±0.26	0.85–2.04	1.24	0.89–1.79	0.90±0.22^**	0.46–1.59	882.73	0.61–1.37
	Urine	982.47±54.77	904.66–1136.14	966.10	910.49–1108.40	687.62±156.77^**	442.20–999.76	689.80	465.56–983.30

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Note

*p<0.05

** p<0.01 compared with controls.

Urine concentrations of 28 elements in workers and controls were summarized in Table 3. Twenty-two of the 28 (79%) elements showed significantly different concentrations in the urine of workers and controls. In comparison to control subjects, the data showed statistically significant increases in urine concentrations of In (295%), Al (17%), As (130%), Be (12%), Bi (15%), Cd (36%), Ca (18%), Cs (24%), Cr (128%), Co (321%), Cu (64%), Fe (75%), Li (35%), Mn (123%), Mg (9%), Mo (138%), Se (115%), Ag (40%), V (17%), and significant decreases in Rb (19%), Tl (31%), Zn (30%).

Effect of employment years and concentrations of indium in serum and urine

A linear regression analysis was used to explore whether concentrations of indium in serum or urine changed as the function of worker’s duration of exposure. The employment years of indium-exposed worker were further divided into ≤5 year, between 6 years and 14 years, and ≥15 years. Among the indium-exposed workers, significant increases in serum and urine indium concentrations were observed when the years of employment exceeded 6 years (P < 0.05) (Table 4). There was no statistically significant difference between the groups of 6–14 years of employment and ≥15 years of employment.

Table 4. Serum indium and urine indium concentrations among workers with different length of service.

Group	n	Serum indium (μg/L)	Urine indium (ng/L)
Group	n	Mean± SD (Range)	Mean± SD (Range)
≤ 5 years	30	30.61±12.31 (11.86–58.68)	5.90±4.38 (0.07–13.90)
6–14 years	15	46.00±32.88 (17.60–137.63)^*	11.57±14.28 (0.72–54.93)^*
≥ 15 years	12	52.45±33.14 (27.03–137.25)^**	14.67±12.71 (4.33–46.89)^**

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Note

*p<0.05

** p<0.01 compared with workers of ≤5 years.

Correlation analysis of elements in serum or urine

When serum concentrations of In, Al, As, Ba, Cd, Cs, Co, Se, Ag, and Zn were performed correlation analysis with urine concentrations of the same element, significant correlations were observed for In (r = 0.935, P < 0.01), Al (r = 0.602, P < 0.01), As (r = 0.499, P < 0.01), Ba (r = 0.699, P < 0.01), Cd (r = 0.476, P < 0.01), Cs (r = 0.869, P < 0.01), Co (r = 0.325, P < 0.05), Se (r = 0.441, P < 0.01), Ag (r = 0.317, P < 0.05), and Zn (r = 0.441, P < 0.05), but not for Be, Bi, Ca, Cr, Cu, Fe, Pb, Li, Mn, Mg, Mo, Ni, K, Rb, Na, Sr, Tl, V (Fig 2).

Fig 2 — Data were analyzed by linear correlation with 95% confidence interval, with a correlation coefficient (r) = 0.935 and P < 0.01.

With regard to the degree to which element concentrations changed in serum and urine between indium-exposed workers and controls, the percentage of changes in serum In (696% increase vs. controls), Al (28%), Be (31%), V (27%), were larger than those in urine In (295%), Al (17%), Be (12%), V (17%); the changes of serum Cd (27%), Cr (55%), Cu (19%), Li (16%), Mn (35%), Mg (7%), Mo (15%) and Zn (29%), however, were smaller than those in urine Cd (36%), Cr (128%), Cu (64%), Li (35%), Mn (123%), Mg (9%), Mo (138%) and Zn (30%); the changes of Cs content in serum and urine were the same (24%); Se declined in serum(19%) but rose in urine(115%) in the indium-exposed workers compared with the controls; likewise Tl and Rb rose in serum(36%, 23%) but declined in urine(31%, 19%). Therefore, we further analyzed the correlation between changes of 15 elements (Al, Be, Cd, Cs, Cr, Cu, Li, Mn, Mg, Mo, V, Zn, Se, Tl and Rb) concentrations and indium in the same biological fluid (serum and urine) (Table 5). There were significant positive correlations between In and Be, Cd, Cr, Zn in serum as well as in urine. There was significant positive association between In and Se in urine, however, In concentrations were inversely associated with those of Se in serum. There were no significant correlations between In and Cs, Cu, Li, Mn, Mg, Mo, Tl, Rb in either serum or urine.

Table 5. Correlation coefficients between indium and other elements in serum and urine of indium-exposed workers.

	Serum	Urine
Al—In	r = 0.398 (P > 0.05)	r = 0.436 (P < 0.01)
Be—In	r = 0.406 (P < 0.01)	r = 0.425 (P < 0.01)
Cd—In	r = 0.473 (P < 0.01)	r = 0.626 (P < 0.01)
Cr—In	r = 0.582 (P < 0.01)	r = 0.456 (P < 0.01)
Se—In	r = -0.593 (P < 0.01)	r = 0.326 (P < 0.05)
V—In	r = 0.331 (P < 0.05)	r = 0.032 (P > 0.05)
Zn—In	r = 0.735 (P < 0.01)	r = 0.547 (P < 0.01)

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Note: Data from In-exposed workers were combined and analyzed by linear regression.

Table 6 shows multiple linear regression analyses for indium concentration (log transformed) in serum and urine adjusted for age, length of service, gender, smoking and alcohol consumption, all elements concentration in serum and urine.

Table 6. Forward stepwise multiple linear regression analysis of elements concentration (logtransformed) in serum and urine studied.

	Biological sample	Adj R²	Predictor	B	P	Partial correlation
Indium	serum	0.532	As	0.296	0.005	0.369
			Be	0.225	0.017	0.319
			Cd	0.245	0.011	0.338
			Cr	0.277	0.008	0.350
			Zn	0.087	0.000	0.735
	urine	0.381	age	0.250	0.018	0.323
			As	0.242	0.031	0.289
			Cd	21.870	0.000	0.626
			Cs	-1.844	0.002	-0.409
			Mg	-0.402	0.040	-0.283
			Zn	0.355	0.001	0.418

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Note: The following variables entered into the models: age; length of service; gender (0: male; 1: female); smoking (1: yes; 0: no); alcohol consumption (0: no; 1; yes); only indium and elements values with p-values that are significant are presented; all elements concentration in serum and urine.

Discussion

The main source of indium in the working environment is the manufacturing process of melting raw materials which releases indium to the atmosphere around the furnace as industrial dust [11, 12]. The data presented in this report clearly show that indium was emitted in the fume produced during the production process. The highest concentration of airborne indium surrounding the workers’ breathing zones inside the workshop exceeded the permissible concentration-time weighted average (PC-TWA, 0.1 mg/m³) by more than eleven times. Cynthia’s group in USA reported a similar finding [13]. By using an air sampler with a 37-mm, 0.8-μm MCE filters, they found that the airborne indium level was about 0.072 mg/m³ during cleaning an indium-tin oxide (ITO) evaporation chamber. Kristin et al. have also reported that air indium concentrations varied from 0.0004 to 0.108 mg/m³ during the production process [14]. The values in our study appeared to be higher than that detected by Kristin’s team. The mean value of airborne indium in our study was 0.078 mg/m³, and ranged from 0.001 to 1.12 mg/m³. The results are consistent with Ichiro’s observations (AM indium concentration was 0.095 mg indium/m³ as total dust, and ranged from 0.00040 to 1.27 mg In/m³) and support the occupational risk of workers exposed to airborne indium [15]. Owing to respiratory tract is the main way of workers exposed to indium and its compounds, it is necessary to adopt perfect ventilation and dust removal facilities in the workplace. At the same time, it is a duty to improve the awareness of occupational health protection of enterprise manager and workers, and scientifically to prevent the occupational hazards of indium and its compounds.

At present, some researchers believe that the concentration of indium in serum or urine can be used as a biological indicator or biomarker of indium exposure [16–19]. The indium-exposed workers did show a significantly higher indium level in serum or urine as compared to control subjects [20]. Makiko has summarized that heavy indium exposure is a risk factor for emphysema, which can lead to a severity level, such as emphysematous changes have been highlighted as a long-term adverse effect on lungs in indium-exposed individuals with serum indium 20 μg/L in their 5-year follow-up study as well as a separate 8-year follow-up study, even after adjusting for age, duration since initial indium exposure, and smoking history [21]. A recent epidemiological study including 87 cases of ITO workers further suggests that plasma indium concentration reflected cumulative respirable indium exposure, which was associated with clinical, functional, and serum biomarkers of lung disease [14]. Serum or urine indium levels may reasonably indicate the status of systemic indium following recent exposure among the active indium-exposed workers. Therefore, this study used serum and urine samples as a vivo biomonitoring of indium and its compounds. The results from this study showed that long-term, chronic exposure to indium and its compounds had a significantly impact on level of serum and urine indium. Serum and urine concentrations of indium among workers were about 8-fold and 4-fold greater than those detected in controls. The increases in levels of serum and urine indium appeared to be related exposure time (Table 3). When the employment years of indium-exposed worker were stratified into three groups, compared with the group of ≤5 employment years, significant increases in serum indium and urine indium concentrations were observed in the group with 6–14 years and ≥15 years of employment. It is suggested that the increase of work-length could cause the increase levels of indium in serum and urine.

Another finding is the fact that exposure to indium and its compounds also had effects on serum concentration of As, Ba, Be, Cd, Cr, Se, V, and Zn, i.e., an increase in serum As, Ba, Be, Cd, Cr, V, and a decrease in serum Se, Zn. The same is true for urine concentrations of Al, As, Be, Cd, Cr, Se, and Zn, i.e., an increase in urine Al, As, Be, Cd, Cr, Se, and a decrease in urine Zn. Exposure to indium compounds resulted in altered levels of In, Al, Be, Cd, Cs, Cr, Li, Mn, Mg, Mo, V, Se and Zn in serum and urine of indium exposed workers. A comprehensive analysis of the changes in the concentration of these elements in serum and urine, it appeared that element concentrations, particularly Be, Cd, Cr, Se, and Zn, are significantly association with the serum indium and urine indium. This association could partly be attributed to the possible action of indium on trace elements metabolism. However, the fact that the indium production process contains more metals including lead, arsenic, cadmium, zinc suggests that the elevated serum and urine concentrations of these elements may be a direct result of the overexposure to these metals in the fume. It is also possible that differences in toxicokinetics among different years of employment groups may contribute to higher serum levels of these metals. Perhaps the higher exposure in the indium exposed workers will really make this group of workers more vulnerable to metal toxicities, after all, our previous animal experiments have found that the trace elements imbalance in rats exposed to ITO exposure rats [22].

Our results demonstrate that measurement of elements concentrations in both serum and paired urine rather than only in serum or urine are important to get insights into the roles of these elements in disease. The results showed that serum indium concentrations were significantly correlated with serum Be, Cd, Cr, Se, Zn, respectively; the same was true for urine indium and urine Be, Cd, Cr, Se, Zn. Among metals of health concern, Se and Zn are essential trace elements and involved in numerous biochemical processes that support life, but yet their deficiency or overload is detrimental to human health [23]. Results of the present study indicate that workers chronically exposed to indium may decrease serum Se and Zn level compared to the control group. Since there is no homeostatic control of indium in the human body, it is toxic trace element. We speculate that when high amounts of indium are introduced into the body, it may affect the normal metabolism of Zn or Se in human body, and then may replace Zn or Se at the key enzyme sites causing metabolic disorders, and eventually lead to a significant decrease in serum Zn or Se levels in workers chronically exposed to indium. The previous research of our group shows that following chronic exposure of ITO to rats, the Zn content decreases in lung and the Cu content increases in lung. Since both Cu and Zn are actively participating in cellular redox reactions, changes in the homeostasis of these two metals in serum and urine of indium-exposed workers may aggravate the damage of reactive oxygen species to cells, and ultimately contribute to the pulmonary toxicity caused by indium and its compounds [24, 25]. The causal correlation between the element (Se and Zn, for example) content and disease is complicated, and so far we did not know why concentrations of Se and Zn were perturbed in indium-exposed workers. But the low levels of Se and Zn in the indium occupational workers are alarming. Trace metals such as As, Be, Cd and Cr, on the other hand, are toxic metals with no beneficial health effects and have been recognized as human or animal carcinogens by International Agency for Research on Cancer [26–28]. The effect of occupational exposure to indium on the metabolism of toxic metals appears to be poorly understood. However, results of the present study indicate that exposure to indium may influence serum and urine levels of As, Be, Cd and Cr. Therefore, their interactions with indium may affect various fundamental biological processes, including intra- and intercellular signaling, apoptosis, and ionic transportation. Besides, we don’t known whether toxic metals could influence indium’s absorption, distribution, deposition, and excretion processes.

Taken together, we believe that element analysis from serum and urine provide an additional means for assessment of indium concentrations in indium occupational workers. Trace metal elements play an important role in biological processes by coordinating enzymatic reactions or by affecting the permeability of cell membranes, among others. Since exposure to indium and its compounds apparently alters the homeostasis of these trace elements, future studies should investigate the importance of these trace metals in indium-associated pulmonary toxicity.

Limitations of the study results

This study has limitations without doubt. First, a larger sample size and more kinds of biological sample are desirable for a more accurate estimation, the impact of random errors might be large owing to the small sample size. Second, false positive rate may have resulted owing to the multiple statistical testing problem. Adjustment of p-values (and confidence intervals) is usually unrealistic when large numbers of statistical tests are performed in an exploratory analysis. In such situations, it is difficult to accurately quantify the total number of tests performed and their interconnectedness, and the adjusted threshold for statistical significance is very small (and the false-negative rate is very high) because of the large number of tests performed. Therefore, it is important to interpret results cautiously rather than trying to accurately determine the true significance level of the p-value. Third, the blood indium level of local residents was unknown. In this study, the control subjects would have measurable indium in serum and urine. The population in our research is in Guangxi, which is the largest indium production base in China, and there are many local production and processing enterprises. Although there was no occupational indium exposure in the control subjects, the indium pollution in river or soil was not ruled out. Meanwhile, because the background of indium exposure was similar for indium-exposed workers and controls, it was likely to have no or minimal impact on the analysis. Our future studies will be directed to overcome these limitations.

Generalization of the study results

Because only factories and workers from one region of China were included in this study, the scope of the results may be limited by the geographical characteristics. Furthermore, because data were collected cross-sectionally, the change of trace elements in urine can only reflect the recent exposure, we can’t infer the influence of diet and other changes on the elements in urine. Therefore, these results should be generalized only after careful consideration.

Conclusions

To the best of our knowledge, the current research represents the first scientific contribution reporting levels of 28 elements in two biological samples across indium-exposed population. The results of this study provide the information regarding serum and urine concentrations of elements in workers of indium ingot production in China. The serum and urine levels of some elements are modified by exposure to indium in occupationally exposed workers. We showed that the concentrations of 13 elements were increased or decreased in serum and urine compared to control groups, suggesting that the abnormal expression of these elements may have a potential effect on the lung toxicity induced by indium and its compounds. These findings shall be useful for future research to monitoring occupational and environmental exposure and to compare the indium exposure levels within China and around the world.

Supporting information

S1 Data

(SAV)

Click here for additional data file.^{(66.8KB, sav)}

S1 Questionnaire

(DOC)

Click here for additional data file.^{(60.5KB, doc)}

Acknowledgments

The authors gratefully acknowledge the technical assistance of Cheng Juan who is in Institute for Occupational Health and Poison Control under the China Center for Disease Control and Prevention (CDC).

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by National Public Welfare Health Industry Scientific Research (No. 201402021) and Graduate Innovation Project (No. 2019B17).

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PLoS One. doi: 10.1371/journal.pone.0246943.r001

Decision Letter 0

Yi Hu

4 Aug 2020

PONE-D-20-20542

Biomonitorization of Concentrations of 28 Elements in Serum and Urine among Workers Exposed to Indium Compounds

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript measures indium and 27 other elements/metals via mass spec. in workers at an indium ingot production facility and includes a comparison/control group of workers at a nearby facility in China (industry for comparison group not identified – but considered unexposed to indium). Area and personal air sampling were also performed at the indium ingot production facility. For the indium-exposed group, serum and urine indium was stratified by tenure. All elements/metals were compared in the serum and urine of indium-exposed workers and the comparison group. Correlations between serum and urine levels were measured for all elements/metals. The four components of the study are outlined below.

• Mean indium measured from air sampling was 78 μg/L.

• Indium in serum and urine increased dramatically by tenure.

• Some of the 27 elements/metals measured were higher in serum and urine in the indium-exposed workers compared to the control group (Aluminum, Beryllium, Cadmium, Chromium, Copper, Lithium, Magnesium, and Molybdenum), Zinc was lower in serum and urine in the indium-exposed workers than the control, and other elements/metals were higher in one specimen (serum or urine) but lower in the other in the indium-exposed workers (serum Selenium lower in indium-exposed workers, urine Selenium higher in indium-exposed workers; serum Thallium and Rubidium higher in indium-exposed workers than control group, urine Thallium and Rubidium lower in indium-exposed workers than control group).

• A number of elements had positive correlations with serum and urine levels, selenium had a negative correlation, and other elements’ serum and urine levels did not correlate.

The manuscript is well written and warrants publication. Consider a follow up health study in the future if that is a possibility. A few additional considerations below:

Abstract:

When listing elements in Results, be sure to specify serum, urine, or both.

Methods:

Demographics are more appropriate in the Results section

‘Were non-alcohol drinkers’ > ‘did not drink alcohol’

Discussion:

Kristin et al. > Cummings et al.

Still should address how controls could have that level of serum indium? Environmental contamination from proximity to indium facility?

Can you include any thoughts on why Thallium and Rubidium would be higher in blood but lower in urine in the indium-exposed workers compared with the controls? Likewise Selenium lower in blood but higher in urine?

‘At present, it is uncertain whether the concentration of indium in serum or urine can be used as a biological indicator or biomarkers of indium exposure’ I don’t think this is true, I would say they can be used as biological indicators of exposure?

Reviewer #2: [Overall Comment]

Thank you for the opportunity to review the article "Biomonitorization of Concentrations of 28 Elements in Serum and Urine among Workers Exposed to Indium Compounes (PONE-D-20-20542)". Since indium-exposed worker has become important health issue, an effective prevention is an urgent matter. Exposure will need to be prevented before health problems appear. It would also be helpful to know the biokinetics of metals. This study will provide important insights into the prevention of these metal-related health problems. However, there are a few concerns with this paper. I hope the authors will consider the matters I have listed.

1. Some paragraph in the Introduction section and the Discussion section is not paragraph writing. The authors should try to write one topic per one paragraph.

2. There seems to be a paucity of cited references. Please do a more careful review of previous studies.

3. A large number of statistical tests have been performed. Therefore, it is necessary to control for the family-wise error rate. There are 120 subjects in the analysis and 133 p-values in the tables and charts. This do clearly cause errors.

4. The Discussion section is very long and redundant. It needs to be more concise.

5. Please check the STROBE statement. Then, please check your manuscript using the STROBE checklist.

6. Please check your English grammar.

[Comment]

I show the page and line number of the Word file (manuscript.docx), "quote from the text", and my comments.

7. Page 1, Line 16, "27 elements": I think "27" is wrong. Is it "28 elements" ?

8. Page 1, Line 23, "Ambient indium levels ... 78.41 μg/m3.": The Abstract-Method section don't contains the method that the ambient indium level measure. Please add the method.

9. Page 1, Lines 25-26, "the variation in ... among study populations.": there are no results of analysis to support the association between serum/urine indium level and airborne indium level. Please provide the results of the analysis that support this.

10. Page 2, Line 33, "Linear regression analysis": Is the "r" you are presenting here a standardized regression coefficient ? If so, please describe that.

11. Page 3, Lines 77-79, "we have validated ... (ICP-MS)." : Please cite the authors' validated results, if they report them.

12. Page 3, Lines 81-84, "To the best ... indium-exposed population.": I believe this should be included in the Discussion section.

13. Page 3, Line 85, "toxic metals": The indium-exposed worker be exposed to any other toxic metal, too? If so, what other metals besides indium will the indium-exposed worker be exposed to? Please describe that.

14. Page 4, Lines 90-92, "Hence this study ... its compounds exposure.": I believe this should be included in the Discussion section.

15. Page 4, Lines 100-115: I believe some of this part should be included in the Result section.

16. Page 4, Line 100: Did the exposed workers wear personal protective equipment? Please describe.

17. Page 4, Lines 107-110, "The control subjects ... and other metals.": Please describe more details on how the control subjects were recruited. Are they volunteers? Or were they selected at random? Or were they selected by the researcher to be similar to the exposed workers?

18. Page 4, Lines 113-114, "The two groups ... no significace differences": No statistical test at baseline is required. Please check the STROBE statement.

19. Page 5, Line 118 & Line 123-124: Similar overlap. Please merge them together.

20. Page 5, Line 124-125: Please include approval number.

21. Page 6, Line 148, "~10%": This is 15%, isn't it? (0.045 μg/0.0003 mg = 0.000045 mg/0.0003 mg = 0.15). If so, please replace "~10%" with "15%".

22. Page 6, Line 151, "local clinics": Please provide a little more detail about "local clinics".

23. Page 6, Line 155-156, "Nan et al, 2017": Incorrect citation style.

24. Page 6, Line 156 "℃until": Insert a space between "C" and "u", please.

25. Pages 6-7, Lines 172-184: Please put them in a table, they are difficult to read in the main text.

26. Page 7, Line 188, "Student's t test": Since the variance is unknown, it is Welch's t test is better than Student's t test.

27. Page 7, Line 188, "paired samples": In the present study, I believe the subjects were not paired. If indium-exposed workers and control workers were paired, please describe how did the authors make paired subjects.

28. Page 7, Lines 189-190, "ANOVA":In the Result section, I couldn't find the results of the ANOVA. Please describe more clearly the method and result of ANOVA.

29. Page 7, 191-192, "by a linear regression": A multiple regression analysis has also been done, but it is not listed in Method section. Please stated it. And, please describe an explanation that the standardized regression coefficients were calculated.

30. Page 7, Lines 196-197, "No biological sample ... level of indium.": The first sentence of paragraph is the sentence of the paragraph's topic. In this paragraph, the first sentence should be a brief description of the airborne indium level. Please rewrite the paragraph to make it more clear.

Reviewer #3: This study provides valuable data on indium concentrations from indium-exposed workers’ serum and urine. The authors also point out that indium affects different trace elements’ homeostasis in the systemic circulation. The paper is well organized and written. However, minor revision is needed, and the following concerns need to be addressed before the paper can be accepted for publication.

1. The author should elaborate on why serum and urine samples were selected for in vivo biomonitoring of indium and its compounds. What are the limitations of using only fluid samples as bio-monitor? Why not use blood or hair samples as a bio-monitor for those metal measurements?

2. Please include a paragraph about the quality assurance of your results.

3. p.16; It might be better to use the same units, such as µg/m3 (or mg/m3), that are consistent with your data and the critical literature concentrations.

4. “When high amounts of indium are introduced into the body, it maybe replace Zn or Se at the key enzyme sites causing metabolic disorders.” The author should provide more information for supporting this statement. Which physical or chemical-depended mechanisms might affect the decrease of serum Zn or Se under indium exposed?

5. “When indium-exposed worker’s years of employment were stratified into three employment groups, serum indium and urine indium concentrations in the group with 6-14 years and ≥15 years of employment were significantly higher than those with less than five employment years, suggesting a work-length related increase in serum and urine indium.” Could the authors conclude that indium’s residence time in vivo is quite long that cause indium more hazard to human health?

6. A fine-tuning in English is required.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: Yes: Toshiharu Mitsuhashi

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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PLoS One. 2021 Feb 22;16(2):e0246943. doi: 10.1371/journal.pone.0246943.r002

Author response to Decision Letter 0

13 Sep 2020

Reply to revision

Responses to Editor:

Thank you for the opportunity to revise my paper. An explanation of the additional requirements below:

1.I read the PLOS ONE style templates carefully, and ensure that my manuscript meets PLOS ONE's style requirements.

2.Thank you very much for pointing out some problems. In my revision, I cite all my sources (including my own works), and make certain appropriate readjustments to some texts. But the author still wants to explain the difference between our paper and the following previous publications.

Publication 1：Title: Alteration of saliva and serum concentrations of manganese, copper, zinc, cadmium and lead among career welders. The differences between our paper and the publication are research objects (indium-exposed workers are different from career welders), biological samples (serum and urine rather than saliva), and analysis elements (28 elements are far more than 5 elements).

Publication 2：Title: Occupational Exposure to Welding Fume among Welders: Alterations of Manganese, Iron, Zinc, Copper, and Lead in Body Fluids and the Oxidative Stress Status. The differences between our paper and the publication are research objects (indium-exposed workers are different from welders), the treatment method of biological samples (the digestion solution direct dilution method is simpler and more accurate than microwave digestion), and analysis elements (28 elements are far more than 5 elements).

Publication 3：Title: Trace elements and carcinogenicity: a subject in review. The differences between our paper and the publication are article type (research article is completely different from review article in essence), main idea of the article (one is to study the detection and analysis of 28 elements in indium-exposed workers, the other is to study the relationship between trace elements and carcinogenicity).

Publication 4：Title: Inhibition of the WNT/β-catenin pathway by fine particulate matter in haze: Roles of metals and polycyclic aromatic hydrocarbons. The author has never read this publication. Moreover, through the abstract of that article, I found that the research content of that article is metal and polycyclic aromatic hydrocarbons in haze. The detection objects of our paper are indium-exposed workers, which is essentially different from that article.

3.I have attached a questionnaire as part of this study. I upload this as a separate file labeled 'Questionnaire'.

4.The corresponding author has an ORCID iD and has completed the Authorization process and have been logged in to Editorial Manager.

Response to Reviewers:

Response Reviewer #1:

Abstract:

When listing the elements in the results, the author has carefully revised the specific samples of each element, such as serum, urine or both.

Methods:

Demographics have been changed to the Results section.

In our study, individuals who had consumed alcohol more than twice a week during the previous 6 months were defined as drinkers.

Discussion:

In order to control the workers' serum indium level, we should control the environmental contamination from indium facilities and do a good job of workers' personal protection. Therefore, the author address solutions at the end of the first paragraph of the discussion.

Our results show that, some elements/metals were higher in one specimen (serum or urine) but lower in the other in indium-exposed workers, this increase or decrease could partly be attributed to the possible action of indium on trace elements metabolism. Perhaps the higher exposure in the indium exposed workers will really make this group of workers more vulnerable to metal toxicities.

‘At present, it is uncertain whether the concentration of indium in serum or urine can be used as a biological indicator or biomarkers of indium exposure’: this statement is not correct, the author has corrected this sentence in the discussion section.

Response Reviewer #2:

1.The author has made some modifications to some paragraphs in the Introduction section and the Discussion section.

2.By carefully reviewing previous studies, the author increases the number of cited references.

3.I'm very sorry, I didn't find the error you said. Please point it out.

4.The author has made some concise modifications to the discussion part.

5.The author submitted a Research Checklist.

6.Thank you for the opportunity to check my English grammar.

7.This study was aimed to assess inddium and other 27 elements in serum and urine. The total is 28 elements.

8.The author has added the method of the ambient indium level measure in the Abstract-Method section.

9. "the variation in ... among study populations.": this conclusion is inaccurate and has been deleted.

10."Linear regression analysis": the "r" was a standardized regression coefficient. The standardized regression coefficient refers to the regression coefficient calculated after the data is standardized (minus the mean value and dividing the variance). It can eliminate the influence of dimension and order of magnitude after the standardized data, so that different variables are comparable. Therefore, the standardized regression coefficient can be used to compare the effect of different independent variables on dependent variables.

11."we have validated ... (ICP-MS)." : in this part of the content, the author's expression may be wrong, there is no “validated results” here. The author has made corrections in the paper.

12. "To the best ... indium-exposed population.": this part has been changed to the Discussion section.

13."toxic metals": the indium-exposed workers were exposed to other toxic metal, too. It can be seen from the first paragraph of the Introduction section, “In the process of indium smelting, there are occupational hazards such as dust, lead, arsenic, cadmium, indium, zinc, hydrogen arsenide, various acids and bases, noise, high temperature.”.

14."Hence this study ... its compounds exposure.": This sentence is repeated with the Discussion section and has been deleted.

15.Demography has been placed in the Results section.

16.During operation, the exposed workers wear dust-free clothes, while maintenance workers wear full face filter cotton, goggles and protective gloves.

17.The control subjects were office workers from another factory who were not exposed to occupational hazards. They were volunteers, and were matched with the exposed workers for age, sex, and smoking habits.

18.General characteristics of the 120 study populations in the control and indium exposure group are summarized in Table 1. The distribution of age and frequencies of sex, smoking and drinking were not significantly different among the two groups.

Table 1 General characteristics and lifestyle habits in study populations.

Variable Controls Wokers statistics P

Number 63 57

Age (year,±s) 39.32 ± 11.37 37.82 ± 9.34 0.77 0.44a

Sex (male/female,%) 46/17(73) 41/16(72) 0.02 0.89b

Smoking (yes/no, %) 31/32(49) 21/36(37) 1.86 0.17c

Drinking (yes/no, %) 20/43(32) 18/39(32) 0.00 0.98c

Years of exposure (year,±s) 8.97 ± 8.39 8.31 ± 7.54 0.45 0.65a

a Ln-transformed for statistical inference. b One-way ANOVA. c χ2 test.

19.The author has merged the overlap together.

20.The author has added the approval number.

21.Yes, it is 15%, the author has corrected the mistake.

22.The “local clinics” refers to the local occupational disease prevention and control hospital where the indium factory is located.

23.The author has corrected the incorrect citation style.

24.The author has inserted a space between "℃" and "u".

25.The author has put the information of 28 elements in Table 1.

26.The author has used Welch's t test to analyze the data.

27.The author has changed the description of the error.

28.The differences between two means were analyzed by independent samples T test.

29.A multiple regression analysis has listed in Method section,and stated it.

30.The author has rewrited the paragraph to brief description of the airborne indium level.

Response Reviewer #3:

1.The author has explained in the second paragraph of the Discussion section why serum and urine samples were selected for in vivo biomonitoring of indium and its compounds.

2.The author has attached a paragraph about the quality assurance of our results in the Method section.

3.The author has changed the unit of the airborne indium level into the same units, such as mg / m3.

4.As requested, the author has elaborated on this statement in the fifth paragraph of the Discussion section.

5.Through these research results, the author couldnot conclude that indium’s residence time in vivo is quite long that cause indium more hazard to human health. The author can only prove that the longer the working years of workers, the longer the exposure time of indium, resulting in the higher content of indium in serum or urine, which may cause more hazard to human health; however, this does not mean that the long accumulation time of indium in the body causes harm to human body.

6.I had readjusted my English grammar. If you feel there are any problems, please let me know as soon as possible, and I will try my best to adjust it.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(152.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0246943.r003

Decision Letter 1

Yi Hu

7 Oct 2020

PONE-D-20-20542R1

Biomonitorization of Concentrations of 28 Elements in Serum and Urine among Workers Exposed to Indium Compounds

PLOS ONE

Dear Dr. yao,

Thank you for submitting your manuscript to PLOS ONE. While most reviewers' comments have been addressed, I would ask the authors to address the issue about the measured indium levels in the control subjects - it suggests background indium exposure or previous occupational exposure, but indium is not a trace element and we don't expect dietary exposure, but at the same time, there have not been large studies to measure blood indium in unexposed populations. Still, it is worth a sentence or two in the discussion.

We invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 21 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Yi Hu

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: The manuscript is much improved and most reviewer comments were addressed. The lingering question I think should be addressed is why the control subjects would have measurable indium in blood?

Reviewer #2: [Overall comment]

Thank you for revising the manuscript. Revisions have been made in a number of places and I believe that the research paper becomes more qualified for the readers.

However, I still have concerns about some points I have ever pointed. There are also some new concerns that should be pointed out in the revised manuscript. Please review and revise the manuscript.

[Comment continued from last time; comment number x-r1]

*3-r1. multiplicity of statistical tests

I apologize for my earlier point, which was poorly worded. I will be more specific below.

In a statistical test, there is a 5% chance of p<0.05, even for comparisons that are not truly different (i.e., Type I error). So, for example, if there are 50 reported p-values, the probability that one or more of them will be p<0.05 by only chance is about 92%.

In this study, more than 100 p-values were calculated (for example, although not reported, I think we calculated p-values even for metals that are not marked with "*" or "**" in Table 2. ). With such a large number of calculated p-values, it is impossible to distinguish between cases where there is a truly significant difference and cases where the difference appears to be significant by type I errors.

In this study, both cases of p-values are considered to be mixed. In other words, some of the comparisons that resulted in p<0.05 in this study are just coincidental.

Please make the following two points on this issue

a) Please address it statistically.

b) Please consider this issue in the Discussion section.

Reference: the number of p-values

Table 2=56, Table 3=4, Table 4=54, Table 5=11, Figure 1=10. that is to say, there are at least 135 p-values in this manuscript.

*5-r1. Check List

Thank you for completing and submitting the Check List (SRQR guideline for Qualitative research). However, this study is not a qualitative research, so this checklist is not appropriate. Therefore, I believe that this checklist is inappropriate.

I think it would be better to use STROBE (https://www.strobe-statement.org/index.php?id=strobe-home).

*10-r1. standardized regression coefficient

Thank you for describing r. I've always known what standardized regression coefficient is. What I wanted to point out in comment #10 is that it was not stated in the Method section that "standardized regression coefficients are reported in linear regression analysis".

Please add and explain this description in the Method section.

*16-r1. personal protective equipment

Thank you for describing your personal protective equipment.

Please explain this in the Method section (Subject) as well as in the rebuttal letter.

*17-r1. Control subject

Thank you for explaining the controls (non-exposed group). Please explain this information in the body of the manuscript as well as in the rebuttal letter.

You mentioned that you matched workers at the indium plant (exposed group) and controls (non-exposed group) with age, gender and smoking history, but the number of person is 57:63. Why is this? Please add an explanation to the main text.

And, there is a difference in the percentage of smokers between the groups, even though they were matched. Please add an explanation for this in the main text.

*18-r1 Comparison between workers and controls

Table 1 in rebuttal letter is also required for the manuscript, so please add it.

However, I believe that statistical tests should be removed. If the controls is matched to the workers, there must be no significant difference in age, gender, and smoking habits, which are not required to be tested.

And, the significant test in baseline demographic data is not recommended in the STROBE statement (JP Vandenbroucke et al, 2007, https://pubmed.ncbi.nlm.nih.gov/18049195/). The following is a quote from JP Vandenbroucke 2007, p.822.

"Inferential measures such as standard errors and confidence intervals should not be used to describe the variability of characteristics, and significance tests should be avoided in descriptive tables. Also, P values are not an appropriate criterion for selecting which confounders to adjust for in analysis; even small differences in a confounder that has a strong effect on the outcome can be important."

[New comments]

31. Limitation

Please add the limitations of this study in the Discussion section.

32. Duplicate expressions.

The following are duplicates, please merge them.

Page 6, Lines 162-163: "P values less than 0.05 indicate statistical significance."

Page 6, Lines 169-170: "All statistical tests are two-sided with a significance level of 0.05."

33. emotional expression

It is best to avoid expressing emotions in academic papers.

Page 11, Line 292: "More surprising"

Page 12, Line 329: "Noticeably"

34. Description to be included in the Result section

Page 11, Line 312 - Page 12, Line 326: "It was also found that ... Se (115%) and Zn (30%). "

Much of this paragraph is the description of results and should not be in the Discussion section. Please minimize the description of results in the Discussion section and put the description of results in the Results section.

35. Conclusion section

Page 13, Lines 370-371: "broad indium-exposed population"

In this study, 57 subjects were included in the study, so I do not think it can be "broad". Please delete it or replace it with a different word.

36. Typo and unformal word

Page 6, Line 169: "p-val-ues": Is it typo of "p-values" ?

Page 13, Line 347: "correlationship": The word does not appear to be a formal expression (e.g., it is not listed in the Oxford dictionary). I recommend replacing it with "correlation".

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Toshiharu Mitsuhashi

PLoS One. 2021 Feb 22;16(2):e0246943. doi: 10.1371/journal.pone.0246943.r004

Author response to Decision Letter 1

19 Oct 2020

Responses to Academic Editor:

Thank you for the opportunity to revise my paper. I am honored to have the opportunity to talk about the indium levels measured by the subjects in the control group. Through consulting the previous literature, the author found that the geometric mean of indium in blood of general population was lower than the detection limit. Therefore, when the present results appeared, the author thought that there was something wrong with the detection. After careful analysis and verification, there is no problem with our detection method, and the selected control population has no previous occupational exposure. As a result, we analyzed that the possible reason was background indium exposure. The population in our research is in Liuzhou City, Guangxi Province, China, which is the richest and most abundant indium resources in China. They are mainly distributed along the river. Although there was no occupational indium exposure in the control population, the indium pollution in river or soil was not ruled out. If possible, we would like to study the serum or urine indium levels of local non occupational indium-exposed populations.

DOI：dx.doi.org/10.17504/protocols.io.bne2mbge

Response to Reviewers:

Response Reviewer #1:

why the control subjects would have measurable indium in blood?

Thank you for the opportunity to talk about the indium levels measured by the subjects in the control subjects. Indium is not a trace element and the author found that the geometric mean of indium in blood of general population was lower than the detection limit by consulting the literature. But at the same time, there have not been large studies to measure blood indium in unexposed populations. Therefore, up to now, there are very few reports on the level of indium in normal human serum or blood. First of all, the author can confirm that the detection method in this paper is appropriate and the selected control population has no previous occupational exposure. As a result, we analyzed that the possible reason was background indium exposure. The population in our research is in Liuzhou City, Guangxi Province, China, which is the richest and most abundant indium resources in China. Guangxi has become the largest indium production base in China due to its advantaged indium resources, and is known as "indium capital" in the world. Those indium bearing mine are mainly distributed along the river and there are many local production and processing enterprises. Although there was no occupational indium exposure in the control population, the indium pollution in river or soil was not ruled out. Of course, this is only our speculation. If possible, we would like to study the serum or urine indium levels of local non-occupational indium-exposed populations.

Response Reviewer #2:

Thank you for the opportunity to revise my paper. An explanation of the points is below:

1.*3-r1. multiplicity of statistical tests

Thank you very much for your detailed description of the statistical problems in our study. The inevitable limitation of our study is that the sample size is too small. In order to distinguish between cases where there is a truly significant difference and cases where the difference appears to be significant by type I errors, the author reduced the number of calculated p-values. Therefore, the author makes some modifications in the analysis of the Results section (Table 5).

2.*5-r1. Check List

Thank you for sending me a link to the STROBE checklist. The author has carefully read each checklist item of the STROBE checklist to use with this article.

3.*10-r1. standardized regression coefficient

The author has added and explained this description in the Method section.

4.*16-r1. personal protective equipment

The author has explained this in the Method section (Subject).

5.*17-r1. Control subject

Information on the control subjects has been supplemented in the body of the manuscript.

The author has added an explanation about the selection criteria of the study population and added an explanation for the difference in the percentage of smokers between the groups.

6.*18-r1 Comparison between workers and control

The author has added this Table for the manuscript.

[New comments]

7.Limitation

The author has added the limitations of this study in the Abstract and Discussion section.

8.Duplicate expressions.

The author has merged the duplicate expressions.

9. emotional expression

The author has changed the way of expression.

10.Description to be included in the Result section

The author has put the description of results in the Results section

11.Conclusion section

The author has deleted “broad”.

12.Typo and unformal word

I'm very sorry. This is the author's mistake. The author has corrected it.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(18.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0246943.r005

Decision Letter 2

Yi Hu

2 Nov 2020

PONE-D-20-20542R2

Biomonitorization of Concentrations of 28 Elements in Serum and Urine among Workers Exposed to Indium Compounds

PLOS ONE

Dear Dr. yao,

Thank you for submitting your manuscript to PLOS ONE. Please fully address the reviewer's concerns before submitting the revised manuscript for re-consideration.

Please submit your revised manuscript by Dec 17 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Yi Hu

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: Please check the attached PDF file in detail.

Thank you for the second revision of the manuscript. More revisions have been made in more places and we believe that the research will be more qualified to the reader.

However, we still have concerns about some of the comments. Also, there are some points that should be pointed out in the revised manuscript. We ask that you please review and revise the manuscript.

It would also help me to complete this review more quickly if the rebuttal letter could be more specific as to which parts (Pages and Lines) of the manuscript have been changed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Toshiharu Mitsuhashi

Attachment

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PLoS One. 2021 Feb 22;16(2):e0246943. doi: 10.1371/journal.pone.0246943.r006

Author response to Decision Letter 2

30 Dec 2020

Responses to Academic Editor:

Thank you for the opportunity to revise my paper.

Response to Reviewers:

Response Reviewer #2:

Thank you for the third opportunity to revise my paper. An explanation of the points is below:

[Ongoing concerns]

*3-r2. Multiple statistical testing Problem (False positive rate will be extremely high)

Thank you very much for your careful explanation and explanation of this problem, so that the author has the opportunity to learn a lot of analysis methods.

The author carefully interpreted the p value, added that “this is an exploratory study” in the Method section, added it as a Limitation in the Discussion section.

*13-r2. Are the study subjects exposed to any other toxic metals?

The selection criteria for the study population in this study were indeed no history of occupational exposure to other metals. The author’s response stated "the indium-exposed workers were exposed to other toxic metals, too." , which means that lead, arsenic, cadmium and other toxic metals exist in the process of indium smelting, workers may also be exposed to lead, arsenic, cadmium and other toxic metals when they are exposed to indium. However, this does not mean that the indium-exposed workers we chose are also occupationally exposed to other toxic metals.

*17-r2 How to select the study subjects

Please see the flow diagram.

*17-r2-(1). How to select non-exposed persons

I'm sorry, maybe there is something wrong with my language expression in my rebuttal letter (R1). The study subjects (= Indium-exposed workers + unexposed workers) are not volunteers. The Indium-exposed workers were selected from an indium ingot production plant (Guangxi, China) who were mainly exposed to indium metal. The unexposed workers (Control subjects) were a random sample of all volunteers derived from another nearby factory, who were office workers and had no history of occupational exposure to indium and other metals. The selected control subjects were matched to the age, sex distribution, average employment history, smoking status and drinking habits with indium-exposed workers.

Respond to the clarification noted in Rev-com(R1) comment No *17-r1: The study subjects were initially matched by inclusion criteria, the number of person is 69:69; but after a series of exclusion criteria, there were some differences between indium-exposed workers and control subjects (the number of person is 57:63).

*17-r2-(2). Number of excluded subjects.

The author described the number of subjects excluded because of criteria (3) and (4).

*17-r2-(3). Definition of Outlier

In our analysis of serum elements of all study subjects, we found that, the serum magnesium level of one control worker was abnormally low (3.8 mg/L). But the worker did not know about his problem. After our discovery, he went to the hospital for further examination and found that he had hypomagnesemia. Thus observations in this worker who had outliers outcome were excluded.

*17-r2-(4). Need for flow diagram

The author saw the explanation in STROBE 13(c) Consider the Use of a Flow Diagram; and supplemented a flow diagram.

Figure 1. Flow of Indium-exposed workers and unexposed workers in the study.

*17-r2-(5). Selection Bias and Generalizability

The author added “Potential confounders and sources of bias” in the Method section, added “Limitations and Generalization of the study results” in the Discussion section.

[Concerns of the newly additional parts]

37. about Table 2.

What the author wants to express is "employment period (years)". Changes have been made in Table 2.

38. about Table 5.

After checking and verifying Table 3, the author found some errors and corrected them in Table 5.

39. position of the Limitation

The author has listed the limitations in the Discussion section.

40. the Terms used in the selection criteria.

The author has changed the word "parameter" to "measured value".

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(79.3KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0246943.r007

Decision Letter 3

Yi Hu

18 Jan 2021

PONE-D-20-20542R3

Biomonitorization of Concentrations of 28 Elements in Serum and Urine among Workers Exposed to Indium Compounds

PLOS ONE

Dear Dr. yao,

Please submit your revised manuscript by Mar 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Yi Hu

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: Thank you for the third revision of the manuscript. More revisions have been made in more places and we believe that the research will be more qualified to the reader.

The major concerns have already been fixed, but I have only two comments on minor points.

[Minor concerns]

41. The English word "volunteer" should be avoided.

According to the Rebuttal letter, it states that the research subjects are not volunteers. However, in the text, there is the following statement.

(Page 4, Lines 10-11, Study population Section)

"The control subjects in this study were a random sample of all volunteers derived from another nearby factory ,"

The term "volunteers" is used here. If they are not volunteers, then another word should be used.

Self-selection bias may occur in studies using volunteers and should be discussed in the text. To make it clear that this study does not require that consideration, I would recommend the English word "volunteer" should be avoided.

For example, "candidates" would be one choice of word to replace it.

42. Elemental symbols or English words.

With the exception of indium, most elements are described in the main text using element symbols. For example, "Al" is used instead of "aluminum".

However, in the following sections, English words are used instead of element symbols.

(Page 15, Lines 11-13, Result section)

"Selenium declined in serum (19%) ... But declined in urine (31%, 19%)."

To avoid confusion for the reader, I would recommend using the elemental symbols instead of the English words as in the other sections.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Toshiharu Mitsuhashi

PLoS One. 2021 Feb 22;16(2):e0246943. doi: 10.1371/journal.pone.0246943.r008

Author response to Decision Letter 3

27 Jan 2021

Reply to revision

Responses to Academic Editor:

Thank you for the opportunity to revise my paper.

Response to Reviewers:

Response Reviewer #2:

Thank you very much for your patient guidance and careful revision of this paper, so that the author has the opportunity to learn a lot of writing and analysis methods. An explanation of the points is below:

[Minor concerns]

41. The English word "volunteer" should be avoided.

The author has changed the word "volunteer" to "candidates".

42. Elemental symbols or English words.

The author has used the elemental symbols instead of the English words.

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(31.5KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0246943.r009

Decision Letter 4

Yi Hu

29 Jan 2021

Biomonitorization of Concentrations of 28 Elements in Serum and Urine among Workers Exposed to Indium Compounds

PONE-D-20-20542R4

Dear Dr. yao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Yi Hu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0246943.r010

Acceptance letter

Yi Hu

11 Feb 2021

PONE-D-20-20542R4

Biomonitorization of Concentrations of 28 Elements in Serum and Urine among Workers Exposed to Indium Compounds

Dear Dr. Yao:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Yi Hu

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Data

(SAV)

Click here for additional data file.^{(66.8KB, sav)}

S1 Questionnaire

(DOC)

Click here for additional data file.^{(60.5KB, doc)}

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Submitted filename: Response to Reviewers.docx

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Submitted filename: Response to Reviewers.docx

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Attachment

Submitted filename: Reviewer_Comment.pdf

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Attachment

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Biomonitorization of concentrations of 28 elements in serum and urine among workers exposed to indium compounds

Nan Liu

Yi Guan

Bin Li

Sanqiao Yao

Roles

Abstract

Introduction

Subjects and methods

Study population

Subjects

Patient and public involvement

Potential confounders and sources of bias

Sample preparation and analysis

Workplace air concentrations of indium dust

Sample preparation and elements analysis

Table 1. The limits of detection for these 28 elements.

Statistical analyses

Results

Demographics of the study population

Fig 1. Flow of indium-exposed workers and unexposed workers in the study.

Table 2. Demographics of the study population.

Ambient indium concentrations

Determination of 28 elements in serum and urine

Table 3. Concentrations of 28 elements in serum and urine of workers and control subjects.

Effect of employment years and concentrations of indium in serum and urine

Table 4. Serum indium and urine indium concentrations among workers with different length of service.

Correlation analysis of elements in serum or urine

Fig 2. Correlation between serum indium and urine indium among 57 workers.

Table 5. Correlation coefficients between indium and other elements in serum and urine of indium-exposed workers.

Table 6. Forward stepwise multiple linear regression analysis of elements concentration (logtransformed) in serum and urine studied.

Discussion

Limitations of the study results

Generalization of the study results

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Yi Hu

Roles

Author response to Decision Letter 0

Decision Letter 1

Yi Hu

Roles

Author response to Decision Letter 1

Decision Letter 2

Yi Hu

Roles

Author response to Decision Letter 2

Decision Letter 3

Yi Hu

Roles

Author response to Decision Letter 3

Decision Letter 4

Yi Hu

Roles

Acceptance letter

Yi Hu

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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