253 Environmental Toxicology and Chemistry, Vol. 24, No. 2, pp. 253–260, 2005  2005 SETAC Printed in the USA 0730-7268/05 $12.00 + .00 Environmental Chemistry TOXICITY ASSESSMENT OF MONO-SUBSTITUTED BENZENES AND PHENOLS USING A PSEUDOMONAS INITIAL OXYGEN UPTAKE ASSAY DED-SHIH HUANG,* THOU-JEN WHANG,FEI-CHEN CHENG,YA-PING WU,YI-TING WANG,WEN-I LUO, and YANE-SHIH WANG Department of Chemistry, National Cheng Kung University, Tainan, Taiwan 701 ( Received 26 April 2004; Accepted 3 June 2004) Abstract—A methodology is presented for assessing the toxicity of chemical substances through their inhibitory action toward the Pseudomonas initial oxygen uptake (PIOU) rate. The current studies reveal that the PIOU assay is rapid, cost-efficient, and easy to perform. The oxygen uptake rate was found to be associated with a putative benzoate transporter and highly dependent on benzoate concentration. The putative benzoate transporter has been shown to follow Michaelis–Menten kinetics. Most phenols were found to be noncompetitive inhibitors of the benzoate transporter. The inhibition constant (K i ) of these noncompetitive inhibitors can be related to the concentration causing 50% oxygen uptake inhibition in Pseudomonas putida. Modeling these data by using the response–surface approach leads to the development of a quantitative structure–activity relationship (QSAR) for the toxicity of phenols ((1/K i ) =-0.435 (0.038) lowest-unoccupied-molecular orbital + 0.517 (0.027)log K OW - 2.340 (0.068), n = 49, r 2 = 0.930, s = 0.107, = 0.926, F = 303.1). A comparison of QSAR models derived from the K i data of the PIOU method 2 r adj and the toxicity data of 40-h Tetrahymena pyrifomis growth inhibition assay (Tetratox) indicated that there was a high correlation between the two approaches (r 2 = 0.925). Keywords—Quantitative structure–activity relationship Toxicity Benzoate transport Pseudomonas putida INTRODUCTION Among the principal natural pollutant transformation pro- cesses, microbial degradation may be the only approach that can completely remove chemical pollutants from subsurface waters and soils. Benzoate and its derivatives are the major key metabolites of biodegradation of alkyl benzenes via either aerobic [1,2] or anaerobic [3] pathways. On the other hand, members of the genus Pseudomonas are perhaps the best- characterized degraders of aromatic compounds [4,5]. It might be possible that any chemical that inhibits the benzoate deg- radation process of Pseudomonas would adversely impact en- vironments where exposure to such compounds occurs. Thus, screening of these chemicals by using cost-efficient, sensitive, and rapid tests to assess their potential hazards to soil or aquatic life might be important to society. Toward this end, this work was conducted to evaluate the inhibitory effect of toxicants on the initial oxygen uptake kinetics of a culture of Pseudo- monas putida mt-2 (American Type Culture Collection [ATCC] 23973, Rockville, MD, USA). Pseudomonas putida mt-2 has been identified as ‘‘Pseu- domonas arvilla (ATCC 33015)’’ [6], which contains both metapyrocatechase and pyrocatechase activities [7]. Strain mt- 2 was discovered to harbor TOL plasmid pWW0, which en- codes broad substrate-specific enzymes that covert the initial growth substrate to either benzoate or a substituted benzoate [2,8]. The pWW0 plasmid also encodes toluate 1,2-dioxygen- ase [9] and cis-diol dehydrogenase, which transforms meta- and para-substituted benzoates to the corresponding cis-dihy- droxyhexadienes and catechols. In addition, the plasmid-en- coding catechol catabolism enzymes catalyze a meta-ring cleavage of catechol and subsequent reactions, leading to the * To whom correspondence may be addressed (dedshih@mail.ncku.edu.tw). formation of tricarboxylic acid cycle intermediates (the meta pathway). Strain KT2440 (derivative of strain mt-2), which lacks the TOL plasmid, also converts benzoate to catechol by using chromosomally encoded enzymes [10,11]. Catechol is then cleaved by an intradiol ring-cleavage (ortho cleavage) diox- ygenase, and catabolism proceeds via the -ketoadipate path- way [12]. Thus, benzoate degradation in P. putida mt-2 can proceed via the plasmid-encoded meta-cleavage pathway or the chromosomally encoded ortho-cleavage pathway. Benzo- ate catabolism is regulated by cross talk between two different regulators. One is the plasmid-encoded XylS regulator [13,14], whereas the other is the chromosome-encoded BenR [11,15]. Although the conversion of benzoate to tricarboxylic acid cycle intermediates has been studied intensively, very little is known about the mechanism of benzoate transport. Aromatic acids can diffuse across biological membranes, making transport theoretically unnecessary. However, a number of studies have inferred the existence of specific transport systems for aromatic acids and related compounds [10,15–19]. The benK and benF genes almost certainly encode a benzoate permease and porin, respectively [11], but the mechanism of benzoate transport has not been investigated. Nevertheless, benK from Acinetobacter sp. strain ADP1, which encodes a benzoate transporter, has been characterized [20]. We had reported previously that the relative toxicity of phenol and monochlorophenols to Pseudomonas can be ana- lyzed by oxygen uptake rates and can be evaluated from the relative 1/K i values, where K i is the inhibition constant [21,22]. Similarly, in this work, the Michaelis–Menten kinetic model has been used to analyze the initial rate of oxygen uptake by P. putida in the presence of benzoate and its inhibitor. The kinetics of inhibition served to distinguish between competi- tive and noncompetitive inhibition. With competitive inhibi-