792 Environmental Toxicology and Chemistry, Vol. 17, No. 5, pp. 792–797, 1998  1998 SETAC Printed in the USA 0730-7268/98 $6.00 + .00 EFFECT OF pH ON THE TOXICITY AND BIODEGRADATION OF PENTACHLOROPHENOL BY SPHINGOMONAS SP. STRAIN P5 IN NUTRISTAT CULTURE MICHIEL RUTGERS,*² S ANDER VAN BOMMEL,² A NTON M. BREURE,² J OHAN G. VAN ANDEL,² and WOUTER A. DUETZ‡ ²Laboratory for Ecotoxicology, National Institute of Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, P.O. Box 1, 3720 BA Bilthoven, The Netherlands ‡Institute of Biotechnology, E.T.H. Honggerberg, HTP, CH-8093 Zu ¨rich, Switzerland (Received 11 April 1997; Accepted 21 August 1997) Abstract—A polychlorinated-phenol degrading bacterium, Sphingomonas sp. strain P5, was grown in nutristat culture (i.e., a continuous culture at a controlled substrate concentration) with pentachlorophenol (PCP) as the sole carbon and energy source. During steady state conditions, the effect of the medium pH on the growth of strain P5 on PCP was established. At lower pH values PCP exhibited a stronger toxicity than at higher pH values. Inhibition of the growth of strain P5 by PCP was correlated to the concentration of the undissociated phenol in the system, rather than to the dissociated or total PCP concentration. The results indicate that acidification of natural environments may enhance the toxicity of chlorophenols and suggest that treatments to increase environmental pH may reduce risk of chlorophenol toxicity at acidified sites. Keywords—Polychlorophenol Continuous culture Steady state Toxicity pH INTRODUCTION The release of biocides such as chlorinated phenols by hu- man activities can be a threat to public health and the envi- ronment. Pentachlorophenol (PCP) has been widely used as a wood preservative and is often found at wood-treatment fa- cilities [1]. Chlorinated phenols, including PCP, are susceptible to microbial attack and can be used as carbon and energy source [2,3]. Pentachlorophenol is a weak acid (pK a is 4.7) and exists in two chemical forms in aqueous solution: a relatively apolar and slightly soluble phenol (PCP 0 ) and the dissociated form, a highly soluble phenolate ion (PCP - ) 0 - + 3 PCP + HO 5 PCP + HO (1) (aq) 2 (aq) 3 (aq) In an aqueous system the equilibrium can be calculated from the pK a of PCP (4.7) - 0 pH = p K + log([PCP ]/[PCP ]) (2) a or rearranged 0 pH-pK -1 a [PCP ] = [PCP ]  (1 + 10 ) (3) tot where PCP tot is total PCP. At pH 7 the equilibrium shifts to the right, that is 99.4% of the total amount of PCP is in the dissociated form (PCP - ). The PCP - can form weak bonds with metal ions, such as K + , Na + , Ca 2+ , and Mg 2+ , depending on the ionic strength of the solution, but not depending seriously on the pH [4]. These complexes are not considered in this study because the con- centration of NaPCP (the most abundant species) does not vary * To whom correspondence may be addressed (michiel.rutgers@rivm.nl). with pH and is not higher than about 2% of the total amount of PCP in mineral-salts medium [5]. Generally, the phenolic structure (PCP 0 ) is thought to be more toxic than the charged analogue (PCP - ) due its tendency to sorb to biological membranes, the principle target of toxic action [6–11]. Accordingly, it might be expected that the tox- icity of PCP is enhanced when the pH is lowered. Acidification of the environment is currently a topic of interest and therefore knowledge of pH-dependent toxicity of PCP and other weak acid toxicants should be valuable for risk assessment proce- dures. In this study, we examined the effect of pH on the bio- degradation rate and the toxicity of PCP using a PCP-degrad- ing microbial population. For this we used a continuous culture with control of the substrate (toxicant) concentration, a so- called nutristat, to establish chronic exposure of the microbes to PCP [12]. Chronic exposure is achieved during highly re- producible steady state conditions and resembles natural con- ditions better than acute exposure experiments. Our emphasis was to test the hypothesis that PCP 0 , rather than the charged PCP - , was the toxicant. MATERIALS AND METHODS Chemicals The chemicals were purchased from the following sup- pliers: pentachlorophenol (PCP) from Fluka (Buchs, Switzer- land), 2,4,6-trichlorophenol (2,4,6-TCP) from Sigma (Zwijnd- recht, The Netherlands), and 2,4-dichlorophenol (2,4-DCP) and 2,6-dichlorophenol (2,6-DCP) from Merck (Darmstadt, Germany). The chemical formulations contained more than 98% of the requested compound (pure analytical quality).