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

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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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ACS Paragon Plus Environment


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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.

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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

388

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

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(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

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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

400

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.

404 405

5. Toxicity

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5.1. In vitro studies

407

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

408

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

413

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

418

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

422

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

423

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

424

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

425

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

426

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

427

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

428

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

429

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

430

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

431

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

432

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

433

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

19



434

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)

435

(118).

436

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

437

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

438

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

439

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

440

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

441

extracts, potentiated the GABA responses significantly.

442 443

5.2. In vivo studies

444

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

445

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

446

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

447

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

448

100-7500 mg/kg (Table 6).

449

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

450

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

451

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

452

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

453

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

454

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

455

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

456

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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458

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

459

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

460

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

461

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

462

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

463

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

464

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

465

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

466

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

467

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

468

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

469

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

470

vivo with X. laevis tadpoles (Table 6).

471

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

472

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

473

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

474

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

475

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

476

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

477

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

478

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

479

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

480

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

21



481

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

482

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

484

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

485

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

486

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

487

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

488

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

489

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

490

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

491

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

492

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

493

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

495

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

496

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

497

(130,131).

498 499

5.3. Ecotoxicity in aquatic organisms

500

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

501

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

502

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

503

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

504

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


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505

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

506

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

507

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).

509

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

510

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

511

0.15 mg/L for duckweed (Lemna gibba) to

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

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