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A Review of Environmental Occurrence, Fate, Exposure, and Toxicity of Benzothiazoles Chunyang Liao, Un-Jung Kim, and Kurunthachalam Kannan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05493 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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Environmental Science & Technology

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A Review of Environmental Occurrence, Fate, Exposure, and Toxicity of

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Benzothiazoles

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Chunyang Liao 1, Un-Jung Kim 2, and Kurunthachalam Kannan 2,*

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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.

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State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for

Wadsworth Center, New York State Department of Health, and Department of Environmental

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Health Sciences, School of Public Health, State University of New York at Albany, Empire State

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Plaza, P.O. Box 509, Albany, New York 12201-0509, United States.

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*Corresponding author: K. Kannan

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Wadsworth Center

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Empire State Plaza, P.O. Box 509

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Albany, NY 12201-0509

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Tel: 1-518-474-0015

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Fax: 1-518-473-2895

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E-mail: [email protected]

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TOC ART

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Abstract: Benzothiazole and its derivatives (BTs) are high production volume chemicals that

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have been used for several decades in a large number of industrial and consumer products,

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including vulcanization accelerators, corrosion inhibitors, fungicides, herbicides, algicides, and

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ultraviolet (UV) light stabilizers. Several benzothiazole derivatives are used commercially, and

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widespread use of these chemicals has led to ubiquitous occurrence in diverse environmental

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compartments. BTs have been reported to be dermal sensitizers, respiratory tract irritants,

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endocrine disruptors, carcinogens, and genotoxicants. This article reviews occurrence and fate of

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a select group of BTs in the environment, as well as human exposure and toxicity. BTs have

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frequently been found in various environmental matrices at concentrations ranging from sub-

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ng/L (surface water) to several tens of µg/g (indoor dust). The use of BTs in a number of

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consumer products, especially in rubber products, has resulted in widespread human exposure.

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BTs undergo chemical, biological and photo-degradation in the environment, creating several

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transformation products. Of these, 2-thiocyanomethylthio-benzothiazole (2-SCNMeS-BTH) has

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been shown to be the most toxic. Epidemiological studies have shown excess risks of cancers,

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including bladder cancer, lung cancer and leukemia, among rubber factory workers, particularly

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those exposed to 2-mercapto-benzothiazole (2-SH-BTH). Human exposure to BTs continues to

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be a concern.

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Key words: Benzothiazoles; Occurrence; Degradation; Human exposure; Toxicity; Ecotoxicity

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1. Introduction

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Benzothiazoles (BTs) are a group of heterocyclic aromatic compounds with 1,3-thiazole

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ring fused to a benzene ring. The most commonly studied BTs include benzothiazole (BTH), 2-

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hydroxy-benzothiazole

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benzothiazole

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benzothiazole (2-SH-BTH), 2-thiocyanomethylthio-benzothiazole (2-SCNMeS-BTH), and 2-

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benzothiazole-sulfonic acid (2-SO3H-BTH) (1-3). As high production volume chemicals, BTH

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derivatives have been used in a variety of industrial and consumer products, including

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vulcanization accelerators in rubber production, fungicides in leather and paper production,

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corrosion inhibitors in antifreeze formulations, herbicides, algicides, and ultraviolet (UV) light

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stabilizers in textiles and plastics (4-10). Benzothiazole derivatives exhibit different kinds of

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biological activities, and in some cases, they have been employed as precursors in the production

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of pharmaceuticals for antitubercular, antimalarial, anticonvulsant, analgesic, anti-inflammatory,

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antidiabetic, and antitumor activities (11,12). Natural sources of BTs have been documented, and

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BTH is a constituent of tea leaves and tobacco smoke (13-15).

(2-OH-BTH),

(2-Me-BTH),

2-amino-benzothiazole

2-methylthio-benzothiazole

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(2-NH2-BTH),

(2-Me-S-BTH),

2-methyl2-mercapto-

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The extensive application of BTs in industrial and consumer products has led to

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widespread contamination in the environment. BTs are reported to occur in wastewater and other

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receiving water bodies, due to incomplete removal of these compounds in wastewater treatment

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plants (WWTPs) (5,7,16-18). The presence of BTH and its derivative 2-(4-morpholinyl)-BTH

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was reported in estuarine sediment and street runoff, and these chemicals were used as “tracers”

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of urban runoff (associated with rubber tire) (New-REF-159). The occurrence of BTH in sludge

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with a mean concentration of 50.2 µg/g, dry weight (dw) has been documented (19). BTH and

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three of its derivatives (2-OH-BTH, 2-NH2-BTH, and 2-Me-S-BTH) were also found in indoor

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air collected from various microenvironments, including offices, homes, laboratories,

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automobiles, garages, barbershops, and public places (20). Five BTH derivatives (BTH, 2-OH-

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BTH, 2-Me-S-BTH, 2-NH2-BTH and 2-SCNMeS-BTH) were detected in indoor dust samples

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from USA, China, Japan, and Korea (21). In short, due to their ubiquitous occurrence, human

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exposure to BTs is inevitable.

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In 1985, researchers observed the occurrence of BTH in human atherosclerotic aortas at a

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mean concentration of 10 ng/g (22). Since then, interest in the analysis of BTs in human

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specimens has increased, and recent studies have reported the presence of BTs in human urine

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samples from several countries (23). Furthermore, bioaccumulation of four BTs (BTH, 2-OH-

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BTH, 2-NH2-BTH, and 2-Me-S-BTH) in human adipose tissue has also been reported (24).

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Several studies have documented toxicities of BTs, which have been linked to mutagenicity in

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microorganisms (25) and carcinogenicity in humans (26,27). BTs have also been shown to be

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dermal sensitizers and respiratory tract irritants (28,29), and in vitro and in vivo assays have

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demonstrated endocrine disruption by BTs (30). The genotoxicity and cytotoxicity of nine BTs

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were determined by the SOS/umu test and high-content in vitro micronucleus test (31). Studies

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have also shown high acute toxicity of 2-SCNMeS-BTH and its transformation products in

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aquatic organisms, including algae, fish, and daphnia (32-34).

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BTs are contaminants of emerging concern. Recent studies reported the occurrence of

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BTs in artificial turf and health issues from chemicals present in artificial turf (New-REF-

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157,158). In this paper, we review the available literature on the occurrence, fate, and

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transformation of BTH and its major derivatives in the environment. Furthermore, studies

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reporting human exposure and the ecotoxicological effects of these toxicants are summarized.

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Finally, the knowledge gap and recommendations for the future research are described.

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2. Characteristics of benzothiazole and its major derivatives

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2.1. Physicochemical properties

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Benzothiazole is composed of a 5-membered 1,3-thiazole ring fused to a phenyl ring and

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its major derivatives are 2-substituted compounds. The chemical structures, select

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physicochemical properties, CAS number and abbreviations of BTH derivatives described in this

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paper are listed in Table 1. Log Kow values of BTs considered in this review have been predicted

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to range from -0.99 (2-SO3H-BTH) to 3.22 (2-Me-S-BTH), and their vapor pressures range from

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5.85 × 10-9 (2-SO3H-BTH) to 5.14 × 10-2 (2-Me-BTH) mm Hg (at 25 °C). BTs are thought to be

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slowly biodegradable and possess low volatility from water surfaces, with the exception of 2-

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SO3H-BTH, which has high water solubility (up to 2.55 × 105 mg/L at 25 °C). The reported half-

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lives of BTH, 2-OH-BTH, and 2,4-morpholino BTH in water were 15 days, 235 years, and

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>1000 years, respectively, at 25̊ C (4). The occurrence of BTs in dust, soil, sludge and sediment

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has been well documented (21,35-37). The bioconcentration factors (BCF) of BTs have been

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predicted to range from 3.16 (2-SO3H-BTH) to 69.3 (2-SCNMeS-BTH).

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2.2. Production and use

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Molecules encompassing a BTH moiety possess diverse biological properties (38), and

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the heterocyclic structure of BTH provides multiple functionality and opportunities for

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derivatization, making it a good starting material for other industrial chemicals.

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substituted BTH derivatives are synthesized for industrial and consumer applications (See the

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Supporting Information for details; 39-41). The estimated annual production of BTH derivatives

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used in the production of rubber in Western Europe in the 1980s was approximately 38,000 tons

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(5), while the production of BTH in the United States in 1993 was estimated to be 4.5-450 tons

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(2). Considering the broad spectrum of biological activities, a wide variety of BTH derivatives

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are produced for use as biocides and corrosion inhibitors. However, their main use is in

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vulcanization accelerators in rubber manufacture, and 2-mercaptobenzothiazole (2-SH-BTH) is

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the most widely used compound for this purpose. The annual production of 2-SH-BTH in the

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United States in 2012 was between 227 and 454 tons (42), while estimates of annual 2-SH-BTH

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production in Western Europe in the early 2000s were in excess of 40,000 tons (43). Exact

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production information for other BTH derivatives is not available, but since BTH can be

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derivatized to yield a wide range of biologically active compounds, it is expected that these

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chemicals are very high production volume chemicals. Although production rates for each of the

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BTH derivatives are likely to differ, based on existing figures, it seems reasonable to assume that

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hundreds of thousands of tons are produced annually worldwide.

The 2-

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In rubber vulcanization, 2-morpholinothiobenzothiazole (a BTH derivative widely used

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in vulcanization) is added in the amounts of over 1% by weight of rubber (5,6). The most

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important products of the rubber industry are automobile tires, which account for 2/3 of the total

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rubber production, while the remaining rubber is used in “rubber goods”. BTs are also used as

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corrosion inhibitors in antifreezes and cooling liquids (5,6). 2-substituted BTs are used as

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herbicides, algicides, slimicides in paper and pulp industry, and fungicides in lumber and leather

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industry (7,13,44-46). BTs are also utilized as photosensitizers and constituents of azo dyes, and

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as UV light stabilizers in textiles and plastics (8-10,18).

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Some BTH derivatives are also used in medicinal chemistry due to their broad-spectrum

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biological activities (11,12) and the efficacy of BTH derivatives against cancer has been

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documented (See the Supporting Information for details; 47-54). Although a BTH moiety is

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embedded in several therapeutic drugs, the actual production volume of BTH for medicinal use

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is not known. It is also interesting to note that each of the derivatives can have different

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biological activities; while some are toxic (as pesticides), others are therapeutic (as anticancer

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drugs).

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3. Occurrence, fate and human exposure

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3.1. Water

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In the 1980s, the U.S. EPA estimated that over 500 tons of 2-SH-BTH was being released

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into the environment through direct and indirect discharges and breakdown of accelerators in

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discarded rubber products (55). A survey of 1300 organic chemicals in 20 surface water samples

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collected from Tianjin, an industrial city in northern China, showed high concentrations of BTH

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(mean: 1.88 µg/L; detection frequency: 20%), 2-Me-BTH (0.534 µg/L, 85%), and 2-Me-S-BTH

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(2.47 µg/L, 85%) (Table 2) (56). Similarly, high concentrations of BTH (range: 0.06-2500 µg/L;

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detection frequency: 100%), 2-Me-BTH (nd-28.0 µg/L, 75%), and 2-Me-S-BTH (0.01-650 µg/L,

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100%) were also found in surface waters from an estuarine system in Kerala, India. The

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occurrence of BTH, 2-Me-BTH and 2-Me-S-BTH in waters was an indicator for sources 7

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originating from tire and rubber products (57). BTH was reported to occur in river water samples

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(100%) from the Schwarzbach watershed in Germany at concentrations of 0.058 to 0.856 µg/L

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(58). BTH, 2-OH-BTH, and 2-NH2-BTH were also detected in river water samples collected

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from Catalonia, Spain, at concentrations on the order of a few to several tens of ng/L (59) (Table

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2). Widespread occurrence of BTs was reported in Pearl River Delta, China, with concentrations

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of BTH and 2-Me-S-BTH in the range of 0.00048-3.19 µg/L (detection frequency: 100%) and

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0.00135-0.413 µg/L (100%), respectively (7). Flux of BTs to rivers from urban runoff was

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identified, and elevated levels of BTH (range: 0.378-1.21 µg/L; detection frequency: 100%) and

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2-OH-BTH (0.721-6.91 µg/L, 100%) were detected in urban runoff (Table 2) (4).

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3.2. Wastewater

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Municipal and industrial wastewater discharge is a significant source of BTs released into

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the environment. Significant concentrations of BTs in municipal WWTPs and household

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wastewater have been reported in Germany (5). BTH, 2-OH-BTH, 2-SH-BTH, 2-Me-S-BTH,

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and 2-SO3H-BTH were found in wastewater, and the dominant and frequently detected

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analogues were 2-SO3H-BTH, BTH and 2-Me-S-BTH (Table 2). The mean concentrations of 2-

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SO3H-BTH were in the range of 0.84 (household wastewater) to 2.25 µg/L (WWTP effluent),

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which were one to two orders of magnitude higher than those found for 2-SH-BTH and 2-Me-S-

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BTH (Table 2). Other researchers (61) reported the occurrence of BTH, 2-OH-BTH, 2-NH2-

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BTH, 2-SH-BTH, 2-Me-S-BTH, and 2-SO3H-BTH in influent and effluent from a German

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tannery wastewater treatment system. The mean concentrations ranged from below the detection

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limit (2-OH-BTH) to 747 µg/L (2-SH-BTH) in influent and from 0.39 (2-NH2-BTH) to 76.8

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µg/L (2-SO3H-BTH) in effluent (Table 2). Use of 2-SCNMeS-BTH as a fungicide in the tanning

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process was found to be the source for BTs in tannery wastewater. The authors estimated that 90-

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95% of BTs were removed in the biological wastewater treatment (61), but a similar study of

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tannery wastewater found increased concentrations of 2-OH-BTH and 2-SO3H-BTH after

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treatment, indicating these compounds were formed during treatment (62). Elevated levels of

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BTs were also found in domestic wastewater samples from WWTPs in India. The mean

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concentrations of BTH detected in influent and effluent (49.3 and 19.5 µg/L) were 4-10 times

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higher than those found for 2-OH-BTH (0.423 and 0.145 µg/L) and 2-Me-S-BTH (0.999 and

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0.513 µg/L) (Table 2). The removal rates of BTs in these WWTPs were in the range of 40-52%,

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which were lower than the values reported for the tannery wastewater treatment system in

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Germany (90-95% for BTs, including BTH, 2-OH-BTH, 2-NH2-BTH, 2-SH-BTH, 2-Me-S-BTH,

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and 2-SO3H-BTH) (19,61). Asimakopoulos et al. (18) investigated the occurrence and removal

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efficiencies of BTs in a WWTP in Athens, Greece. BTH, 2-OH-BTH, 2-NH2-BTH, and 2-Me-S-

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BTH were detected in dissolved and particulate phases of both influent and effluent. The

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concentrations of BTs in the dissolved phase were in the range of nd-30.2 µg/L, which were one

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to two orders of magnitude higher than concentrations in the particulate phase (nd-0.21 µg/L)

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(Table 2). It was estimated in that study that >64% of BTs were removed in the activated sludge

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treatment process.

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3.3. Sediment and sludge

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Relatively few studies are available on the occurrence of BTs in sediment. 2-OH-BTH

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was detected in surface sediment samples collected from Rhode Island, USA, at concentrations

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ranging from below the detection limit to 31.0 µg/kg dw (4). The Swedish Environmental

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Protection Agency conducted a nationwide survey on the concentrations of select biocides,

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including 2-SH-BTH, in environmental matrices in Sweden. 2-SH-BTH was found in lake

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sediment from Stockholm at concentrations of up to 70 µg/kg dw (63). Other studies have

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reported the occurrence of BTs in WWTP sludge in several countries (18, 19, 36, 64-68). 2-OH-

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BTH and 2-Me-S-BTH were found in sludge from WWTPs in Catalonia, Spain, at

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concentrations ranging from nd-0.181 µg/kg dw and nd-0.040 µg/kg dw, respectively (Table 2)

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(36). BTH, 2-OH-BTH, 2-Me-S-BTH and 2-SO3H-BTH were found in sludge from a WWTP in

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Koblenz, Germany, at concentrations ranging from 0.157 to 0.326 µg/kg dw with 2-SO3H-BTH

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and 2-OH-BTH as the major derivatives (67). BTH, 2-OH-BTH, and 2-Me-S-BTH were detected

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frequently (>93%) at mean concentrations of 0.086, 0.189, and 0.052 µg/kg dw, respectively, in

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dewatered sewage sludge collected from a WWTP in Athens, Greece (68) (Table 2).

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3.4. Food and wine

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BTs are used in biocides, food flavorings, and cork stoppers (69-72). BTH is used as a

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flavoring substance in foods at levels up to 0.5 µg/g in non-alcoholic and alcoholic beverages,

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soft and hard candy, baked goods, meat products, gravies, soups, milk products, and cheese.

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Food products containing BTH include papaya (0.004 µg/g), cooked asparagus (0.02 µg/g), malt

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whiskey (0.006 µg/g), and cocoa (trace) (73), and it also naturally occurs in tea leaves and

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tobacco smoke (13-15). BTH was found in wine samples (100%) from a study conducted in Italy

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at concentrations ranging from 0.24 to 1.09 µg/L (69). Prat et al. (70) reported the occurrence of

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BTH in cork stoppers used for sparkling wine. The concentrations of BTH in still wines ranged

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from 1.0 to 14.1 µg/L (mean: 5.2 µg/L), which were higher than those found in Italian wines in

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another study (range: 0.24-1.09 µg/L) (69). The concentrations of BTH in sparkling wines

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ranged from 3.20 to 5.58 µg/L (Table 2). BTH levels in wine were not dependent on grape

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variety, aging or storage (71,72). To investigate the migration of BTH and 2-SH-BTH from

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rubber utensils used in contact with food, Barnes et al. (74) analyzed 236 food samples that were

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in contact with rubber and found that the concentrations of both chemicals in food samples were

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below the detection limits of 5-43 ng/g.

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3.5. Crumb rubber and artificial turf

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Recycled rubber tires used in playgrounds and commercial pavers in Spain were found to

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contain BTs (77). BTH was found in all recycled tire samples at concentrations ranging from

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0.47 to 39.9 µg/g (mean: 9.60 µg/g), which were lower than those of 2-Me-S-BTH (range: 72-

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398, mean: 195 µg/g). BTH was also found in all commercial pavers purchased from local stores,

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at concentrations of 19.6 to 158 µg/g (mean: 95.6 µg/g) (Table 2). Simcox et al. (82) measured

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concentrations of semi-volatile and volatile organic compounds including BTH in indoor and

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outdoor air of synthetic turf crumb rubber fields in Connecticut, USA. BTH concentrations in

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outdoor air samples were in the range of 98% with Rhodococcus opacus

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and Rhodococcus pyridinovorans (66,98,104). The fastest degradation of BTH was observed

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with Rhodococcus erythropolis, which almost completely degraded 3 mM of BTH in 1.5 hr (97),

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while Rhodococcus opacus and Rhodococcus pyridinovorans required more than 2 weeks to

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degrade 98% of 10 mg/L of BTH (66). The average biodegradation rates (k) of 2-OH-BTH in

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continuous flow moving bed biofilm reactor and activated sludge were 1.78-4.74 d-1 and 2.41-

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3.36 d-1, respectively (105). 2-SH-BTH and 2-Me-S-BTH were originally thought to be

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recalcitrant (43,101), but Pseudomonas sp. was able to transform 2-SH-BTH and 2-Me-S-BTH

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through oxygenase, hydroxylase, methyltransferase activities (96,104).

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Several studies (43,99,100,104,106) have shown that microbial degradation of BTH

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yields 2-OH-BTH, which is further hydroxylated to 2,6-dihydroxy-BTH. The formation of 2-

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OH-BTH from either 2-SO3H-BTH or BTH has also been observed. In addition, 2,6-dihydroxy-

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BTH is catalyzed by monooxygenase into catechol (dihydroxy benzene) and dicarboxylic acid by

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catechol 1,2-dioxygenase (100). In general, BTH, 2-SO3H-BTH and 2-OH-BTH are

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biodegradable through enzymatic transformation of microorganisms. 2-NH2-BTH, 2-SH-BTH

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and 2-Me-S-BTH appear less amenable for biodegradation, but can be partially biodegradable

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either through one of the enzymatic pathways (e.g., hydroxylation or methylation) or non-

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enzymatic processes in activated sludge (107). 2-SH-BTH has been suggested as an inhibitor of

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biodegradation of BTH, 2-OH-BTH and 2-SO3H-BTH by Rhodococcus erythropolis (106) in

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sludge collected from rubber industry WWTP (108,109). 2-SH-BTH has been observed to

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undergo biomethylation, leading to the formation of 2-Me-S-BTH (110, 111). The

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biodegradation pathways of BTs are shown in Fig. 1-B.

383 384

4.3. Photodegradation

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BTs have been shown to undergo photolysis under sunlight (110,112), with more than

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50% of initial amount of BTs photo-degraded after 30 days of sunlight exposure (112). Since

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both BTH and 2-OH-BTH absorb sunlight very weakly at wavelengths >290 nm, direct

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photolysis of these compounds is expected to be slow (4). Brownlee et al. reported that 2-SH-

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BTH photodegrades to produce 2-OH-BTH and BTH (110), and 2-(4-morpholinyl)-BTH is

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similarly expected to degrade into BTH (113,114). Studies investigating the degradation of 2-

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NH2-BTH by combined (photo)chemical degradation and biodegradation showed the formation

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of new BTH metabolites which were not observed from a single degradation mechanism

393

(45,102,103). The degradation pathway of 2-NH2-BTH is shown in Fig 2.

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A limited number of studies have reported on the removal of BTs in WWTPs. Matamoros

395

et al. reported removal efficiencies of BTH, 2-OH-BTH and 2-Me-S-BTH from 0 to 80% in

396

conventional activated sludge system, and from 83 to 90% in constructed wetlands (17). Another

397

study reported much lower removal efficiencies of BTH in conventional activated sludge

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treatment (5 to 28%) (5). Moving bed biofilm reactor (MBBR) displayed greater removal

399

efficiency for 2-OH-BTH than activated sludge, which was probably related to higher residence

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time of attached biomass for biodegradation of 2-OH-BTH (105). Significant removal of 2-

401

SO3H-BTH was reported (65%) in membrane bioreactor (MBR) (115). In activated sludge

402

treatment (5,6), 2-SO3H-BTH and 2-Me-S-BTH levels increased by 20% and 160%,

403

respectively, derived from biomethylation of 2-SH-BTH into 2-Me-S-BTH.

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5. Toxicity

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5.1. In vitro studies

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Studies have documented a variety of toxic in vitro effects of BTs in animals and humans.

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Researchers reported modulation of thyroid hormone activity by 2-SH-BTH, 5-chloro-2-

409

mercapto-BTH, 2-NH2-BTH, 2-OH-BTH, and 2-Me-S-BTH (30). Hornung and colleagues

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examined inhibition of thyroid peroxidase in vitro with porcine thyroid glands, and observed 18

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inhibitory potency occurring in this order: 2-SH-BTH = 5-chloro-2-mercapto-BTH > 2-NH2-

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BTH > BTH, with IC50 values of 12, 13, 1200, and 10000 µM, respectively. However, no

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inhibitory activity was found for 2-OH-BTH and 2-Me-S-BTH (Table 5) (30). Genotoxicity and

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cytotoxicity of BTH, 2-chloro-BTH, 2-bromo-BTH, 2-fluoro-BTH, 2-Me-BTH, 2-SH-BTH, 2-

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NH2-BTH, 2-OH-BTH and 2-Me-S-BTH have also been studied (31). One comprehensive study

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observed dose-dependent cytotoxicity of BTs on Salmonella typhimurium TA1535/pSK1002 and

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on two human gastric and lung carcinoma epithelial cells (MGC-803 and A549), with median

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lethal concentrations (LC50) in the range of 19 mg/L (2-SH-BTH in Salmonella) to 270 mg/L (2-

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chloro-BTH in A549). BTH, 2-OH-BTH and 2-NH2-BTH were cytotoxic to S. typhimurium

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TA1535/pSK1002, and all the tested BTs, with the exception of BTH and 2-fluoro-BTH, were

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more cytotoxic to bacteria than to human carcinoma cells. The genotoxicity of nine BTs was

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examined by SOS/umu test using S. typhimurium TA1535/pSK1002. Except for BTH, 2-fluoro-

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BTH and 2-SH-BTH, all other tested BTs, at concentrations less than the LC50 values, induced

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DNA- and/or chromosomal damage (Table 5) (31).

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Aryl hydrocarbon receptor (AhR)-mediated activity of leachates from rubber tire was

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examined by He et al. in recombinant mouse hepatoma (Hepa1c1c7) cell-based CALUX

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(H1L1.1c2 and H1L6.1c2) and CAFLUX (H1G1.1c3) assays (117). The tire extracts activated

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both AhR binding and AhR-dependent gene expression, and several chemicals contained in the

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extract, including 2-Me-S-BTH and 2-SH-BTH, were identified as potent AhR agonists. Further

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studies with a structurally diverse BTs demonstrated that many of these chemicals activated AhR

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signaling pathways, identifying BTs as a new class of AhR agonists. In another study,

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researchers evaluated the ability of BTH, 2-Me-BTH, and 2-OH-BTH to bind and activate

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human estrogen receptor (ER) and AhR in yeast recombinant cell assays. The EC50 values were

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in the range of 5.5 to >10 mg/L for ER and 6.9 to 11.0 mg/L for AhR, respectively (Table 5)

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(118).

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Hossain and colleagues studied BTH’s role in the neurotoxicity of coffee extracts by

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altering γ-aminobutyric acid type A (GABAA) receptors expressed in Xenopus oocytes (119).

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The aqueous extract of coffee inhibited the GABA responses in a dose-dependent manner, while

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the lipophilic extract of coffee showed slight potentiation of the responses at low doses (0.1-0.4

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µL/mL) and inhibition at high doses (0.5-0.8 µL/mL). BTH, one of the components in coffee

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extracts, potentiated the GABA responses significantly.

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5.2. In vivo studies

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Ginsberg et al. reported acute toxicity of BTs that varied from animal to animal; the

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median lethal dose (LD50) in rat of BTH was reported at 380-900 mg/kg for oral exposures and

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95-200 mg/kg for intravenous, intraperitoneal, or dermal administration (120). The acute toxicity

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of 2-SH-BTH was somewhat lower than that of BTH, with the oral LD50 in rats in the range of

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100-7500 mg/kg (Table 6).

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In an evaluation of food additive toxicity performed by the World Health Organization,

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rats that were orally exposed to a single dose of BTH (5.1 mg/kg/d) for 90 days exhibited no

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alterations in blood chemical parameters, organ weights and histopathology. Thus, the oral dose

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of 5.1 mg/kg/d was considered to be at the “no observed adverse effect level” (NOAEL) (121).

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However, a 20-month dietary study in mice exposed to 2-SH-BTH showed renal morphological

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alterations at doses ≥58 mg/kg/d; the NOAEL value was reported at 14 mg/kg/d (Table 6) (122).

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Dermatitis and irritation (allergic) reactions to BTs in skin and sensory organs have also

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been documented. Allergic and dermatitis reactions in humans exposed topically to BTH were

457

reported as early as 1931 (123). Similarly, contact dermatitis and skin sensitization induced by 20

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2-SH-BTH in humans and rodents have been demonstrated (28,124). Irritation of nose and throat

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has been reported in asphalt-rubber workers involved in laying pavements, which was thought to

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be due to the presence of BTH in asphalt-rubber (125). Sensory and pulmonary irritation as well

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as allergic reactions of BTs were illustrated in mice and ex vivo lymph node cell models,

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respectively (Table 6) (See the Supporting Information for details; 29,126).

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Thyroid hormone-related effects of BTs have been demonstrated in vitro, ex vivo and in

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vivo studies by Hornung et al. (30). Thyroid glands, dissected from Xenopus laevis tadpoles,

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were cultured in 96-well plates and exposed ex vivo to graded concentrations (0.001-10000 µM)

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of several BTs. The researchers observed inhibition of thyroxine (T4) release, with inhibition

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potency in this order: 2-SH-BTH (median inhibitory concentration, IC50: 3 µM) > 5-chloro-2-

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mercapto-BTH (30 µM) > 2-OH-BTH (100 µM) > 2-NH2-BTH (100-300 µM) > 2-Me-S-BTH

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(300 µM) > BTH (457 µM). The inhibitory effect of BTs on T4 release was further verified in

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vivo with X. laevis tadpoles (Table 6).

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In another study, oral exposure to a single dose of BTH at 1 mM/kg/d in rats for 5 days

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increased the activities of phase I metabolic and conjugation enzymes (15). Oral exposure of

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mice to BTH (400 mg/kg/d) for 5 days followed by intraperitoneal injection with acetaminophen

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(400 mg/kg) increased serum alanine aminotransferase activities, which suggested BTH-

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mediated potentiation of the hepatotoxicity by acetaminophen. This was explained by potent

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induction of phases II metabolizing enzymes by BTH (15).

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The developmental and reproductive effects of BTs have also been examined in some

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studies. Dietary exposure to a single dose of 2-SH-BTH in rats reduced body weight, with the

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lowest observed adverse effect level (LOAEL) reported at 357 mg/kg/d. However, gavage

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exposure to 2-SH-BTH showed no effect on body weight, and a NOAEL value of 300 mg/kg/d

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was suggested (122). Another study using rats showed that a gavage dose as high as 2200

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mg/kg/d of 2-SH-BTH in dams did not induce congenital malformations during gestation (122)

483

(Table 6). When 2-SH-BTH was administrated to rats at 200 mg/kg/d, by daily intraperitoneal

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injection on days 1-15 of gestation, no marked maternal toxicity, fetal toxicity, or teratogenicity

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was found (127). However, another study found fetotoxic and teratogenic effects of 2-SH-BTH

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in chicken embryos that were injected with 2-SH-BTH at 0.1-2 µmol/egg into the air sac for 11

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days. Malformations including defects in eye, neck, and back, as well as open coelom, were

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observed in 20% of the chicken embryos (128).

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Carcinogenicity of 2-SH-BTH was examined by the National Toxicology Program of the

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U.S. (129). Following oral exposure of rats to a single dose of 2-SH-BTH (188 or 375 mg/kg/d

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for males and 375 or 750 mg/kg/d for females) for 2 years, a variety of gender-specific tumors

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were reported. These included adrenal gland and pituitary gland tumors for both genders, and

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pancreas, preputial gland, and leukemia tumors for males only. Gavage doses of 375 or 750

494

mg/kg/d in mice resulted in liver tumor in females only. However, the results of two studies on

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mutagenicity of BTs, as tested with the Ames Salmonella typhimurium and L5178Y mouse

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lymphoma cell mutation assays, suggested that neither BTH nor 2-Me-BTH was mutagenic

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(130,131).

498 499

5.3. Ecotoxicity in aquatic organisms

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A few studies have examined toxic effects of BTs in aquatic organisms. BTs occur in

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aquatic environments as the result of roadside runoff, wastewater and industrial discharges.

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Although sorption and bioaccumulation potentials of BTs are thought to be low due to their high

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water solubility, BTs are still expected to be present in water columns. Bioaccumulation and

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depuration of 16 organic compounds, including BTH and 2-Me-S-BTH, were compared in three 22

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species of leeches (Dina dubia, Erpobdella punctate, and Helobdella stagnalis). The

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bioconcentration factors (BCFs) of BTH and 2-Me-S-BTH found in this study were in the range

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of 100-400, and the half-lives of 2-Me-S-BTH and BTH in leeches were 1-2.5 and 7 days,

508

respectively (132).

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The U.S. EPA conducted an investigation on acute toxicities of 2-SCNMeS-BTH in fish,

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invertebrates, and aquatic plants. No observed effect concentrations (NOEC) were reported from

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0.15 mg/L for duckweed (Lemna gibba) to