Risk management strategy to increase the safety of workers in the nanomaterials industry

Journal: Journal of Hazardous Materials

Risk Management Strategy to Increase the Safety of Workers in the Nanomaterials Industry

Min-Pei Ling

, Wei-Chao Lin

, Chia-Chyuan Liu

, Yi-Shiao Huang

, Miao-Ju Chueh

, Tung- Sheng Shih

a

Department of Health Risk Management, China Medical University, Taichung 40402, Taiwan, ROC.

b

Department of Cosmetic Science, Chia Nan University of Pharmacy and Science, Tainan 71710, Taiwan, ROC.

c

Industrial Safety and Health Association of Taiwan, Taipei 11670, Taiwan, ROC.

d

Institute of Occupational Safety and Health, Council of Labor Affairs, Taipei 22143, Taiwan, ROC.

*Address correspondence to Min-Pei Ling, Department of Health Risk Management, China

Medical University, Taichung, Taiwan 40402, tel: +886-4-2205-3366; ROC; fax: 886-4-

22070429; lingmp@mail.cmu.edu.tw.

ABSTRACT

In recent years, many engineered nanomaterials (NMs) have been produced, but increasing research has revealed that these may have toxicities far greater than conventional materials and cause significant adverse health effects. At present, there is insufficient data to determine the permissible concentrations of NMs in the workplace. There is also a lack of toxicity data and environmental monitoring results relating to complete health risk assessment. In view of this, we believe that workers in the NMs industry should be provided with simple and practical risk management strategy to ensure occupational health and safety. In this study, we developed a risk management strategy based on the precautionary risk management (PRM).

The risk of the engineered NMs manufacturing plants can be divided into three levels based on aspect identification, solubility tests, dermal absorption, and cytotoxic analyses. The risk management strategies include aspects relating to technology control, engineering control, personal protective equipment, and monitoring of the working environment for each level.

Here we report the first case in which a simple and practical risk management strategy applying in specific engineered NMs manufacturing plants. We are confident that our risk management strategy can be effectively reduced workers risks for engineered NM industries.

Keywords: Nanomaterials; Risk management; Solubility; Dermal absorption; Cytotoxic

1. Introduction

Nanotechnology has become increasingly important in recent scientific and technological developments and has been considered as the new industrial revolution of the 21

century [1].

Much research has been conducted on different types of engineered nanomaterials (NMs) that

possess special physical and chemical properties distinct from regular materials. This research

has mostly focused on identifying and characterizing both the effects and properties of such

NMs. American National Standards Institute [1] defined engineered NMs as a type of particle with at least one dimension smaller than 100 nm, which includes engineered NMs, biological NMs, and ambient ultrafine particles. Engineered NMs are now being manufactured and used in many products and are particles engineered by humans with specific physicochemical compositions and structures on the nanoscale to exploit properties and functions associated with its dimensions. There has been an abundance of research investigation on different aspects of the properties and functions of engineered NMs, which has given rise to a better understanding of their potential applications and continued extensive development.

However, recent studies show that engineered NMs may be hazardous to human health, and some have suggested that toxicity associated with nanoparticles are possibly greater than that of microparticles and particles of larger scales [24]. The greater toxicity of NMs may perhaps be explained by the small size effect, surface and interface effect, quantum size effect, and macroscopic quantum tunnel effect. It is possible that change in engineered NM properties application might lead to changes in potential toxicity, but these relationships have not been well investigated.

The exposure of the human to engineered NMs is thought to cause the transference of

engineered NM into body via the skin, respiratory tract, or gastrointestinal tract, but the effect

of engineered NM uptake and translocation is not completely understood [5]. Recently, a

number of reports on NM toxicity have mentioned the importance of managing the risks

associated with occupational exposure to engineered NMs, since workers in these industries

are especially susceptible to high dose exposure [2,3,6,7]. The toxic effects of engineered

NMs come from their unique physicochemical characteristics, including the size, shape,

surface area, surface chemistry, reactivity, and solubility of these materials. Many of the

toxicological studies on occupational exposure to engineered NMs have provided information

on aerosol-related pulmonary diseases, cell inflammation, cytokine production, and oxidative

stress in humans [812].

Engineered NMs that have special properties and diverse applications might lead to different levels of occupational health problems. Since this is fairly recent technology, the lack of good epidemiological and toxicological data on engineered NMs leads to difficulty in determining permissible exposure limits for risk assessment of different occupational environments. So far, there has not been enough information on experimental data can be applied into traditional health risk assessment models (e.g. hazard identification, exposure analysis, effect analysis, and risk characterization) for evaluating engineered NM associated health risks. Therefore, our current knowledge on the health risk of engineered NMs is incomplete [3,13,14]. For the above-mentioned reasons, we cannot apply traditional health risk assessment models to specific engineered NMs at present. However, it is important to provide an easy and practical method to manage occupational exposure to those who are in directly contact with engineered NMs.

As suggested by Erdely et al. [15], Kandliker et al. [16], Bartis and Landress [17],

Maynard and Kuempel [18], Oberdörster et al. [19], determining the toxicity of engineered

NMs can be done by selected toxicity screening processes on the basis of number

concentration, size distribution, shape, surface area, surface chemistry, surface charge,

composition, crystal structure, solubility, porosity, and agglomeration state. Luther [20] also

proposed the concept of a preliminary scheme for risk management to describe the level of

health risk a specific type of engineered NMs might cause. This involves considering

production volume, aerosol release conditions, solubility, shape, size distribution,

toxicological screening, and ecotoxicological screening of engineered NMs in manufacturing

plants. Similarly, Bartis and Landree [17] emphasized that despite the large number of

methods for testing and evaluation of engineered NM toxicity currently being developed,

many studies have generally agreed that the overall goal should be to roughly predict and

classify the potential toxicity of engineered NMs based on their material properties.

Occupational exposure limits have to take into account technology controls, engineering controls, the use of personal protective equipment, and monitoring of the working environment to ensure safe levels of occupational exposure are maintained. In recent years, more and more literatures also suggest the alternative risk management approaches [21,22]

and implementable classification schemes [2326] were discussed for reduce the risk of human NM exposures. However, these studies did not discuss how to apply the approaches and classification schemes in a specific case study.

The purpose of this study was: (i) to propose a precautionary risk management (PRM) method for classifying engineered NMs with diverse properties into three different levels by analysis of engineered NM concentration, aspect, size distribution, solubility, dermal absorption, and toxicity; (ii) to apply the above-mentioned classification system to several engineered NMs manufacturing plants; (iii) to devise risk management strategies for occupational safety and health protection guidelines based on the PRM classification results.

2. Materials and methods

2.1. Precautionary risk management

Traditional risk assessment in general comprises several components that include hazard identification, exposure analysis, effect analysis, and risk characterization. The risk assessment framework is a complex process that involves the integration of information across a range of domains such as engineered NMs-related characteristics, product usage and disposal, exposure routes, environmental monitoring, occupational monitoring, transport, toxic effect, susceptibility extrapolation models, and threshold value calculation [14,16].

Hazard identification depends on the diverse characteristics of engineered NM chemical

composition, particle size, structure, properties, and coatings. Exposure analysis includes

engineered NM product usage and routes of entry, while effect analysis includes the uptake, distribution, metabolism, and excretion of engineered NMs in humans. Risk characterization of engineered NMs is associated with the likelihood of effects, the nature of effects, and the effectiveness of controls [27]. However, there are insufficient studies describing effective and practical methods for determining exposure, establishing toxicity, or evaluating the risks of engineered NMs at present.

Given the large number of different engineered NMs currently being developed, this study proposed to classify the potential toxicity of engineered NMs based on their material properties [18]. The precautionary scheme applies where scientific evidence is insufficient, inconclusive, or uncertain for potentially harmful health. Luther [20] proposed the concept of a preliminary scheme for risk management, which can be used to determine risk and risk rankings for various engineered NMs. Risk factors that can give an initial estimation of potential risk for engineered NMs are production volume, aerosol release condition, solubility, shape, size distribution, and toxicological screening of engineered NMs. These risk factors were also proposed by Brouwer [2], Bergamaschi [3], Nel et al. [4], Schulte et al. [6], Schulte and Salamanca-Buentello [7], Erdely et al. [15], Kandlikar [16], Bartis and Landree [17], Maynard and Kuempel [18], and Oberdörster et al. [19] study.

In this study, we reconstructed and developed a precautionary risk management (PRM)

to investigate the exposure and toxicities associated with different engineered NM types in

workers adopted from Luther [20] in Fig. 1. We used the concept of classification which

involved three different tiers based on the PRM and further divided into liquid, colloid, and

powder NMs for different toxicological screening test. The different exposure routes

(inhalation and dermal exposure) of entry into human during production, processing, and

handing were also considered in this study. This included routes of exposure such as by

inhalation or dermal exposure (Tier 1), aspect identification (to distinguish between fibers and

particles) (Tier 2), and toxicological screening for dermal absorption, solubility, and cytotoxicity (Tier 3).

2.2. NM exposure routes for workers (Tier 1)

The earliest and most extensive exposure to engineered NM is likely to occur in engineered NM manufacturing plants. Recently, reports have emphasized the importance of managing occupational engineered NMs exposure as workers generating engineered NMs are at especially high risk given their exposure to particularly high doses [2,3,17,2830]. While inhalation, ingestion, and skin penetration are the potential exposure routes for engineered NMs, occupational engineered NM inhalation has perhaps received the most attention [31].

Oberdöster et al. [32] pointed out that healthy skin is sufficient to stop the absorption of NMs through the dermis; but when the skin is damaged, NMs may then be capable of crossing through the epidermis into the dermis and entering the circulation. Tinkle et al. [33] similarly proved that NMs did not penetrate the epidermis of flat and undamaged skin. However flexing of normal skin, such as bending at the wrist, did allow engineered NM penetration into the dermis.

Recent literature has demonstrated that engineered NMs need to have fulfilled certain

criteria before being able to cause adverse health effects, although findings have been

controversial in this regard [28,34,35]. Some studies have shown that the inhalation of

airborne engineered NMs is dependent on the amount of engineered NMs released into the air

during production, changes in its physicochemical properties while in the air, and the

likelihood of the engineered NMs being inhaled [36,37]. Evaluation of the risk factors for

exposure requires measurement of the aerosol properties of engineered NM size, aspect, and

surface area [27]. Moreover, skin can be exposed to engineered NMs through non-intentional

dermal contact with anthropomorphic substances generated during engineered NM

manufacturing. Working with engineered NMs in colloid or liquid media without adequate barrier protection (e.g. gloves) increases the probability of skin exposure [30]. In this study, the first tier in the classification was to determine the significance of inhalation or dermal exposure routes during engineered NM production, processing, and handling based on P (Fig.

1). Particle size distributions were measured with an Engine Exhaust Particle Sizer (EEPS- 3090, TSI Inc.) with five minutes of continuous sampling in each engineered NM manufacturing plant. This instrument was able to rapidly measure particle size distributions in the nanometer range.

2.3. Aspect identification for NMs (Tier 2)

This step involved laboratory analysis of NMs provided by each of the manufacturing plants. Although engineered NMs are characterized by physicochemical structures smaller than approximately 100 nm, exposure to particles composed of engineered NMs, such as aggregates of engineered NMs ranging from a few nanometers to several micrometers in diameter, can cause similar effects [27]. These nanostructured materials are potentially of concern if they deposit within the human body and have nanostructure-influenced toxicity (e.g. small diameters, high surface area, or disaggregation into smaller particles after deposition) [27,38]. Oberdörster et al. [19] described shape, size distribution, agglomeration state, and chemical composition of engineered NMs as key characteristics considered essential in their physicochemical characterization. These properties depend on the size, shape, and structure of engineered NMs at the nanoscale. In this study, the second tier involved distinguishing fibers and particles from engineered NMs based on PRM (Fig. 1).

Engineered NMs were morphologically identified using a field emission transmission electron

microscopy (FETEM; JEOL JEM-2100F), while engineered NM elemental composition was

determined by energy dispersive spectrometers (EDS) with copper grids.

2.4. Toxicological screening of NMs (Tier 3)

Safety evaluation of engineered NMs is an essential process. In this study, this mainly involved laboratory analysis on NM from the four manufacturing plants. Both in vitro and in vivo methods can be used for toxicity assessments of engineered NMs. Because in vivo experiments using animal models are expensive and slow, Luther [20] advocates the use of the low-cost, high-throughput in vitro assays, which do not have reduced efficiency or reliability compared to in vivo methods in the PRM. Luther [20] also indicated that the toxicological screening should be capable of studying the relationship between deposited particles and acute/chronic inflammation to determine which aspects of surface area (and other possible parameters) are best predictors of adverse health effects in the PRA. Ideally, the test method should be capable of evaluating the relationship between colloid, liquid, and powder engineered NM and dermal absorption, solubility, and cell viability to determine the known and unknown toxicities. In this study, this third tier involved screening for engineered NM toxicity following airborne or contact exposure to workers for particles greater than 5 m length or smaller than 100 nm diameter.

2.4.1. Dermal absorption test

Skin is a vital protective organ for the body and is made up of three layers: the

epidermis, the dermis, and subcutaneous tissues. The outermost layer of the epidermis is the

stratum corneum, consisting of formed by keratinocytes. This layer plays the most important

role in the absorption or penetration of most chemicals. In this study, we adopted two skin

exposure techniques based on the NIOSH standard, the transdermal franz diffusion cell drive

console and tape stripping. For the former technique, excised porcine skin was used as a

model for human skin, a widespread practice which has been supported by numerous studies

[39,40] confirming the similarities in histology, morphology, and permeability with human skin. The nano-Ag colloid and nano-Ag liquid samples flowed with fixed rates through tubes through the detection slot to a fraction collector. Percutaneous absorption tests were run for 18 hours. After this, the porcine skin and remaining fluids were carefully collected and analyzed using flame atomic absorption spectrometry (FAAS) to determine the amounts of Ag. Ag concentrations of the remaining fluids and skins were then added and compared with the original amounts.

For the tape stripping technique, nano-Ag liquid and nano-Ag colloid fluid at concentrations of 20 μg/mL and 300 μg/mL respectively were applied evenly on the human skins for 2 hours. After this, tapes with areas of 5 cm

(2.5 cm × 2.0 cm) were patched on the human skins and subjected to pressure by applying a rolling pin over it for 15 times. This was followed by immediate tape stripping. Stripping was repeated 5 times, with each taking up 6-8 layers of the stratum corneum, thereby yielding about 30-40 cell layers of the stratum corneum. The stripping tapes were subjected to digestion and analyzed via inductively coupled plasma mass spectrometry (ICP/MS) to quantify Ag contents.

2.4.2. Solubility test

In 2002, Kreyling et al. [41] pointed out that insoluble NMs not absorbed by the intestines accumulate and remain in the liver and spleen, potentially contributing to cardiovascular or lung disease. In 2004, Borm et al. [42] performed animal experiments showing that insoluble NMs induced the formation of fibrosis, neoplastic lesions, and lung tumors. Tomellini and de Villepin [43] also stated that from a toxicology perspective, insoluble NMs exert toxic effects on organisms due to their unique characteristics.

As part of our investigation, we sought to determine the solubility of various engineered

NMs in blood and facilitate appropriate risk reduction strategies. Engineered NMs were first

dissolved in saline (0.9%) as an initial measurement of their solubility in blood. In this study, 2.5 mg engineered NM samples were dissolved in 5 mL of normal saline (pH 7.4) inside test tubes according to Itoh et al. [44] study. Tubes were placed on magnetic stirrers to maintain the temperature and provide mixing for 30 minutes. Subsequently, solutions were observed after 10, 30, and 60 minutes. If a NM did not dissolve after 60 minutes, it was assumed that that particular NM could not be dissolved rapidly in this study. For saturated NM solutions, solutions were subjected to another 10 minutes of stirring using a magnetic stirrer, before being strained through a 0.2 µm filter and nitrification. The saturation of the solution was measured with graphite furnace atomic absorption spectrometry (GFAAS) to provide a preliminary assessment of solubility.

2.4.3. Cell viability assay

Human skin fibroblasts (Hs68 cells) and murine hepatic cells (BNL CL.2 cells) used in this study were obtained from American-Type Culture Collection (ATCC, Rockville, MD).

The cells were grown in DMEM containing 10% (v/v) FBS, 0.12% NaHCO

, penicillin (100 U/mL), streptomycin (100 g/mL) and 5% CO

in an incubator at 37

C. The cells had been passaged for 20 times (p20) before purchasing, and were only used up to 40 passages to avoid a phenotypic drift.

Before use, Hs68 cells and BNL CL.2 cells were digested by 0.25% trypsin, and cell

numbers counted, before being diluted into cell suspensions at a density of 5 × 10

/mL in

complete medium. They were then seeded into 24-well plates at 1 mL/well. After being

cultured for 24 hours, cells were immediately treated with various doses of ionic liquids for

another 24, 48, and 72 hours. The effect of different treatments on cell viability was assessed

by the modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)

colorimetric assay [45]. The MTT assay assesses the ability of viable cells to reduce MTT

from a yellow water-soluble dye to a dark blue insoluble formazan product. MTT was dissolved in phosphate-buffered saline (PBS) at a concentration of 5 mg/mL and added to the cell culture to a final concentration of 100 L/mL. After 1 hour, the medium was removed and the remaining MTT crystals were dissolved in 1 mL DMSO. Quantitative colorimetric assay at 492 nm was performed using a spectrophotometer.

2.5. Risk management strategies in NMs industry

At the same time that nanotechnology evolves, it is important to consolidate and establish effective occupational safety and risk management strategies based on PRMs to minimize risk to manufacturing workers who come into direct contact with engineered NMs.

In this study, occupational safety and risk management strategies were constructed according to the different levels of the PRM. These strategies include technology control, engineering control, personal protective equipment, and working environment monitoring. Technology control refers the arrangement of engineering and technology to remove potential hazards from mechanical equipment, manufacturing processes, raw materials and factory facilities and other operating environments. Engineering control refers to the adoption of additional protective methods such as through engineering or technical means to prevent and limit sources of risk when such hazards cannot be removed. Personal protective equipment may take the form of breathing apparatuses, gloves or protective clothing worn by workers [46].

Monitoring of the working environment is also important and refers to both exposure

monitoring and special health examinations. These ensure the safety of the operating

environment and periodic special health examinations allow for the early detection and

prevention of disease in workers. The four management strategies proposed above were made

based on the PRM of Fig. 1. Strategies are classified according to the characteristics of the

NMs produced by the various manufacturing plants and are summarized in Table II.

3. Results and discussion

3.1. Application of the risk management strategy for NMs industry

This study selected four manufacturing plants that produced nano-sized zinc oxide (nano-ZnO) powder, multi-walled carbon nanotube (MWCNT) powder, nano-sized silver (nano-Ag) colloid, and nano-Ag liquid as examples, and according to the PRM framework (Fig. 1) to describe workplace risk associated with NMs in these four plants. Based on these assessments, we then devised different risk management strategies for workers in these workplaces according to risk levels. These selected engineered NMs manufacturing plants were certified having the capability to manufacture NM products in Taiwan.

According to the PRM in Fig. 1, exposure routes measurement of tier 1, EEPS-3090 was used to measure powdered NMs, produced in the manufacturing plant to quantify particle size and number concentration of dispersed NMs in the atmosphere. In this study, we used to measure the nano-ZnO powder and MWCNT powder in the air under three conditions:

background (no operation), starting operations, and after one hour of continuous operation of the nano-ZnO powder and MWCNT powder manufacturing plants, respectively. Aspect identification of tier 2 follows the assessment of exposure routes in tier 1. This study adopted FETEM-EDS to measure the shape, size distribution (Fig. 3), and composition (Fig. 4) of NMs from the nano-ZnO powder, nano-Ag colloid, and nano-Ag liquid manufacturing plants.

The size distribution was best fitted to the measured data, and the selected lognormal (LN)

distribution had the chi-square (

) and Kolmogorov-Smirnov (K-S) goodness-of-fit. For

toxicological screening of tier 3, the dermal absorption analysis can only be performed for

liquid or colloid NMs. As such, only nano-Ag colloid and nano-Ag liquid underwent this

analysis. The solubility analysis should be undertaken for nanopowders such as nano-ZnO

powder and MWCNT powder in this study. If the NMs are absorbed through the skin or

dissolved easily, we need to proceed with cell viability assay follows the analysis of solubility and dermal absorption. The designated risk management strategies (Table I) can be determined according to different site measurement and laboratory results based on the PRM.

3.2. MWCNT powder manufacturing plant

3.2.1. Classification criteria-based PRM of MWCNT powder: first-level

Fig. 2A shows the results of the MWCNT powder manufacturing plant. When the plant was not in operation, almost no NM was found in the indoor environment of the plant. After commencement of operations, NMs 1-5 nm in size was measurable at approximately 550 particles/cm

. This was increased to 900 particles/cm

with particle diameter of about 1-3 nm after 1 hour of operation. Although this concentration increased over time, it was relatively low compared to the environment, such as at heavy traffic sites. Because of low concentrations during production, as shown in Fig. 2A, workers at the MWCNT powder manufacturing plant were unlikely to inhale MWCNT powder. Besides, according to our observations in the field, the workers cannot direct skin contact with MWCNT powder during handling due to they wear protective gloves in a confined production system. In other words, workers in the MWCNT powder manufacturing plant did not have a high level of exposure to MWCNT powder during production, processing, and handling. Therefore, MWCNT powder was classified as requiring only a first-level risk management strategy to ensure occupational health and safety (Fig. 6A).

3.2.2. First-level risk management strategy

According to the results in Fig. 6A, the MWCNT powder manufacturing plant needs to

implement the first-level risk management strategy to protect workers. First-level risk

management strategies involve the basic protective measures in the technology control,

engineering control, personal protective equipment, and monitoring of the working environment to protect workers. We suggested that the MWCNT powder manufacturing plant can adopt wet operations to replace dry operations for technology control. Wet operations reduce the inhalation exposure risk to NMs. Because the basic process improvements and ventilation system are sufficient, the engineering control aspects do not require extra equipment for improvement. The use of N95 filtering respirators, latex gloves, and overall clothing are examples of personal protective equipment. Monitoring of the working environment can be divided into exposure monitoring and special health examinations. The former is general monitoring and the latter would involve a general occupational health evaluation.

3.3. Nano-Ag colloid and Nano-Ag liquid manufacturing plants

3.3.1. Classification criteria-based PRM of Nano-Ag colloid and Nano-Ag liquid: second- level

Although it was unlikely that nano-Ag colloid and nano-Ag liquid could enter the body

via inhalation, workers at the respective manufacturing plants who do not wear protective

gloves are likely to have skin contact with these both materials according to our observations

in the field. Therefore, both these plants were required to proceed to the next step of aspect

identification. Results in Figs. 3A and 3B show nano-Ag colloid to have a particle size

distribution of LN(9.87 nm, 1.92), while nano-Ag liquid had a particle size distribution of

LN(13.04 nm, 1.57). The composition analysis of the two engineered NMs is summarized in

Figs. 4A and 4B. Among these results, the silver (Ag) contents were almost 100% and around

87.25% in nano-Ag liquid and nano-Ag colloid, respectively. Composition analysis results

showed that the NMs did not contain other components to influence the toxicological

screening of tier 3.

According to the PRM in Fig. 1, dermal absorption analysis can only be performed for liquid or colloid NMs. As such, only nano-Ag colloid and nano-Ag liquid underwent this analysis. After 18 hours of diffusion in the percutaneous absorption analysis, the Ag content in the collected fluid was very low, indicating that nano-Ag did not penetrate the stratum corneum. Following this, the skin and remaining samples were carefully removed and analyzed via FAAS to determine Ag contents. The amount of Ag content in the remaining fluids and skin were then compared with the original solution. The recovery rate for the Ag content was noted to be above 90%. Results showed that when the particle size of nano-Ag in the solution was between 3 nm to 40 nm, the rate of percutaneous absorption was very slow.

This also confirmed that the penetration phenomenon was not significant.

Results of the tape stripping analysis showed that the distribution of nano-Ag colloid and nano-Ag liquid was predominantly on the second and first strippings of the stratum corneum, respectively. This phenomenon occurred for both low concentration 20 μg/mL and high concentration 300 μg/mL, despite the differences in their concentration. The maximum number of stratum corneum layers reached for both nano-Ag liquid and nano-Ag colloid was about 20. These results implied that the possibility of nano-Ag entering the body is low during a short period of exposure. In addition, the experiments also showed that organic modifiers affected the infiltration of nano-Ag. When a less polar solvent such as isopropyl alcohol was used, the penetration rate was higher than for other polar solvents. Therefore, Nano-Ag colloid and Nano-Ag liquid were classified as requiring the second-level risk management strategy to ensure occupational health and safety (Figs. 6B and 6C).

Because nano-Ag colloid is in gel form, exposure via inhalation is unlikely during the

production process. However, workers who failed to wear protective gloves were more likely

to have skin contact with nano-Ag colloid. Aspect identification of tier 2 was therefore

required. These results showed that the aspect ratio and diameter of nano-Ag colloid was less

than 100:1 and less than 100 nm respectively (Fig. 3A). The composition analysis revealed that nano-Ag colloid contained about 87.25% Ag. After tier 2 assessment, nano-Ag colloid progressed to toxicological screening in tier 3. Dermal absorption analysis confirmed that Nano-Ag colloid was not easily absorbed into the body through skin. As a result, the manufacturing plant only needs to implement a second-level risk management strategy (Fig.

6B).

As nano-Ag liquid is in liquid form, inhalation of nano-Ag into the body during the production process is unlikely to occur. However, workers who failed to wear protective gloves were again more likely to have skin contact with the nano-Ag liquid. Progression to tier 2 aspect identification showed an aspect ratio of less than 100:1 and diameter of less than 100 nm (Fig. 3B). Toxicological screening of tier 3 involved dermal absorption analysis, which proved it was difficult for nano-Ag liquid to be absorbed into the body through the skin. As a result, the plant needed to implement a second-level risk management strategy to ensure occupational health and safety of its workers (Fig. 6D). This result consistent with Nano-Ag colloid and in fact, both plants were recommended second-level risk management strategies.

3.3.2. Second-level risk management strategy

Figs. 6B and 6C indicate that the Nano-Ag colloid and Nano-Ag liquid plants were required to implement second-level risk management strategies to protect the workers following assessment of their NMs. It would be replace dry operation with wet operation or use the confined production process system with dry operations as part of technology control.

Engineering control measures might involve assessing suitable design parameters of

production equipment, while personal protective equipment could again include dust

protection masks, chemical protective gloves, and protective clothing. For the working

environment monitoring, reparable dust monitoring and health risk evaluation for dust operations should be conducted periodically.

Manufacturing plants with a second-level risk management strategy that produce nano- powder should consider a confined production process system. The ventilation system of these sites should be associated with a hood. One could consider the partial vacuum and evaluate the necessity of installing partial exhaust systems.

3.4. Nano-ZnO powder manufacturing plant

3.4.1. Classification criteria-based PRM of Nano-ZnO powder: third-level

Fig. 2B illustrates the probability density functions of nano-Zn concentration during background (no operation), starting operations, and after one hour of continuous operation in the nano-Zn powder manufacturing plant. When the nano-ZnO powder manufacturing plant was not operating, the concentration of NM in the air was very low at around 430 particles/cm

, with particle sizes range falling between 1-30 nm. After commencing operations of nano-ZnO powder production, the maximum concentration measured was 6,200 particles/cm

with a mean particle size of about 7 nm. This further increased after one hour of operation, with maximum concentrations of 13,000 particles/cm

and particle size of 5 nm being recorded. Because of the high concentration during production, as shown in Fig. 2B, workers in the nano-ZnO powder manufacturing plant were likely to inhale nano-ZnO powder.

Hence, according to the PRM in Fig. 1, the next step for this case should be aspect

identification. Fig. 3C shows that nano-ZnO powder particle size distribution was LN(25.06

nm, 1.47). The composition analysis of the Zn content was almost 100% in nano-ZnO powder

(Fig. 4C). Due to the diameter was less than 100 nm and the aspect ratios was less than 100:1,

toxicological screening was to be undertaken as part of the next step.

The nano-ZnO powder was analyzed in toxicological screening. Results showed that nano-ZnO powder had a solubility of 0.0066mg/L after 30 minutes of stirring and 60 minutes remaining stationary. This concentration of Zn was obtained by analyzing the saturated nano- ZnO powder in solution and appeared to be very low. From observation alone throughout the process, it was obvious that nano-ZnO powder did not dissolve quickly. The cell cytotoxicity assay was to be undertaken as part of the next step.

Fig. 5 describes the dose-responses relationship of viability in Hs68 cells and BNL CL.2 cells to the nano-ZnO powder. The result of cell cytotoxicity assay are expressed as IC50 values (g various nano-compounds per mL) for comparison. Incubation with nano-ZnO powder for 24 h led to marked increased of cell cytotoxicity in both Hs68 cells and BNL CL.2 cells (43  3 g/mL and 58  5 g/mL). The experimental results showed that the toxicity of nano-ZnO powder was higher in both Hs68 cells and BNL CL.2 cells. The IC50 of nano-ZnO powder was higher than the recommended IC50 of 100 g/mL in this study. This implied that nano-ZnO powder is toxic to organisms. By contrast, although nano-Ag colloid, nano-Ag liquid, and MWCNT powder did not require cell cytotoxicity assay, they were also subjected to analyze. These results showed that nano-Ag colloid and nano-Ag liquid had only weak effects on cell cytoxicity in both cells (IC50 = ~1000 g/mL). The toxicity of MWCNT powder was below detection limits.

As seen in Fig. 6D, it was shown during the tier 1 assessment that workers in the Nano- ZnO powder manufacturing plant were more susceptible to powder inhalation based on measurements using the EEPS-3090 (Fig. 2B). Aspect identification in tier 2 further discovered that the aspect ratio and particle size of nano-ZnO powder was less than 100:1 and 100 nm respectively (Fig. 3C). The purity of the Zn also approached 100% (Fig. 4C).

Toxicological screening as part of tier 3 assessment involved measuring solubility, which

showed that nano-Zn powder did not dissolve rapidly. This implied that the powder was likely

to be harmful inside the human body. A cytotoxic analysis was then performed which confirmed the toxicity of nano-Zn powder (Fig. 5). Given these results, the Nano-ZnO powder manufacturing plant would require the implementation of a third-level risk management strategy to ensure the occupational health and safety of its workers.

3.4.2. Third-level risk management strategy

As shown in Fig. 6D, the nano-ZnO powder manufacturing plant required a third-level risk management strategy (Table I) for the occupational health and safety of its workers. For technology control, it was suggested in this study that the plant adopt wet operations with a confined production process system and a view to evaluate suitable design parameters for production equipment simultaneously. The ventilation system should also be installed with a hood with consideration to a partial vacuum or the installation of local exhaust. Personal protective equipments should take the form of full protection masks, rubber or non-porous gloves, and high-level protective clothing. For measures relating to monitoring of the working environment, it was recommended that the site carry out particle size and concentration monitoring for specific high risk on site locations, and health risk evaluation for dust operations as well as screening with abdominal ultrasound examination.

4. Conclusions

The application of NMs has been widely increasing. However, related data for risk

assessment is extremely inadequate. Preventive measurements are required in nano-industries

to ensure safety to workers who come into direct contact with NMs. Additionally, there needs

to be a simple and practical management scheme for minimizing risk. In order to implement a

feasible health risk management scheme in nano-industries, this study integrated a relatively

simple risk management strategy (Fig. 1) by applying some simple parameters as a reference

for different levels of management, and establishing a preliminary classification in risk management standards based on the level of risk. This classification first distinguishes characteristics of the NMs and provides different control managements strategies according the level of risk. It focuses on the particular NM industry and provides a set of complete management strategies aimed at reducing workplace risk to operational personnel. The different level NMs according to their characteristics are summarized as follows.

The characteristics of first-level NMs are as follows: (1) NMs are not exposed to the body do not enter via inhalation or skin contact during processing and handling. A typical operation is wet and confined the production process that does not result in respiratory or skin exposure. Contact with NMs is extremely minimal. Workplaces producing this type of NM are classified as first-level. (2) The NM is exposed to the body directly or via respiratory or skin contact but the NM is not harmful to humans due to its size. The aspect ratio is less than 100:1 and its diameter is larger than 100 nm. Alternatively, the aspect ratio is greater than 100:1, but the length is less than 5 m. NMs that meet these criteria are listed as first-level.

The characteristics of second-level NMs are as follows: (1) Workers are directly exposed to such NMs. Its aspect ratio and particle size is less than 100:1 and smaller than 100 nm respectively. Alternatively, its aspect ratio may be larger than 100:1, but its length is larger than 5 μm. In addition, the NM is able to dissolve rapidly (thereby implying that NMs entering the body can dissolve quickly and be excreted from the body) or cannot be absorbed through the skin. These characteristics make up second-level NMs. (2) NMs that do not dissolve quickly but can be absorbed through the skin are classified as second-level if toxicological screening indicates that it has low toxicity.

The characteristics of third-level NMs are as follows: (1) This NM is in direct exposure

to workers. It does not dissolve quickly or is absorbed through the skin. Toxicity tests either

show that it is toxic or cannot show that its toxicity is low. Such NMs are managed via a

third-level approach.

On-site assessment of NMs using physiochemical and cytotoxic analysis can help identify potential risks associated with NM production for each manufacturing plant.

Subsequent and, appropriate safety management measurements can then be established for these risks. Such measures include technology control, engineering control, personal protective equipment, and monitoring of the working environment. This strategy is economical and provides efficient risk management of the workplace for various NM manufacturing plants. This also allows practical management measures to be implemented according to demands. This approach can provide a complete management strategy specialized to NM industry, and is able to minimize workplace risks to operational personnel.

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