2542 Environmental Toxicology and Chemistry, Vol. 19, No. 10, pp. 2542–2550, 2000  2000 SETAC Printed in the USA 0730-7268/00 $9.00 + .00 INFLUENCE OF ZWITTERIONIC pH BUFFERS ON THE BIOAVAILABILITY AND TOXICITY OF COPPER TO THE ALGA AMPHIDINIUM CARTERAE M. TERESA S.D. VASCONCELOS,*² C RISTINA M.R. ALMEIDA,² O LGA M. LAGE,‡ and FILIPE SANSONETTY§ ²LAQUIPAI, Departamento de Quı ´mica da Faculdade de Cie ˆncias da Universidade do Porto, Rua do Campo Alegre 687, P4169-007 Porto, Portugal ‡Departamento de Bota ˆnica da Faculdade de Cie ˆncias da Universidade do Porto e IBMC, Rua do Campo Alegre 823, 4150 Porto, Portugal §Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Roberto Frias, 4200 Porto, Portugal ( Received 10 August 1999; Accepted 18 February 2000) Abstract—The culture and testing of algae frequently requires stabilization of pH levels to maintain the availability of trace metals. In this work we evaluated the influence of 25  10 -3 M N-[2-hydroxyethyl]piperazine-N'-[2-hydroxypropanesulfonic acid] (HEPPSO) and piperazine-N,N'-bis[2-hydroxypropanesulfonic acid] (POPSO) pH buffers, at pH 8.0, on the biological response of Amphidinium carterae Hulburt. Growth rate, copper uptake, mean cell size, mean cell complexity, and fluorescein labeling of algae cultures were studied in enriched solutions of artificial seawater (ESAWs) containing a range of total copper(II) concentrations between 23  10 -9 M (level before copper addition) and 15  10 -6 M. The ESAW (without a pH buffer, initial pH = 8.0) was used as the control medium. Initial free copper (Cu free ) was between 53  10 -15 and 72  10 -10 M, which represents ranges of concentrations typical of unimpacted and polluted surface seawater. These ranges were achieved using ehtylenediaminetetraacetic acid (EDTA) alone and in combination with the two buffers. Biological data endpoints were interpreted based on the cellular copper (Cu cellular , in fmol/cell) and on the estimated initial inorganic copper (Cu inorg = Cu free plus that bound with inorganic ligands) in the medium. At innocuous Cu inorg , up to 10  10 -9 M (Cu free  4  10 -11 M), the Cu cellular was independent of the presence of the buffer and very little dependent of Cu inorg . For Cu inorg up to 0.40  10 -9 M, the Cu cellular was approximately constant (0.1–0.2 fmol/ cell) and when Cu inorg ranged between 1.1  10 -9 M and 10  10 -9 M the Cu cellular was three times higher but also constant. The POPSO enhanced copper uptake and toxicity. The HEPPSO reduced uptake and toxicity of copper because of its enhanced binding capacity compared to POPSO. Both HEPPSO and POPSO altered copper effects on mean cell size, mean cell complexity and fluorescein labeling of A. carterae, but the inherent mechanisms differed between the two buffers. This present work demonstrated that the chemical binding affinity of HEPPSO and POPSO differed and that it resulted in altered uptake and effects of copper on algae. These observations have important implications in culture and testing of algae that require metals as micronutrients yet are sensitive to metals at concentrations that exceed basic metabolic needs. Keywords—Amphidinium carterae Copper speciation N-substituted aminosulfonic acids Copper bioavailability INTRODUCTION Cultures of marine microalgae in unbuffered synthetic me- dia may undergo pH changes during population growth due in part to the removal of dissolved CO 2 during photosynthesis and to nutrient absorption (and consequent ionic exchange) by the cells. Because of the high complexity of the chemical and biological systems, pH variations can strongly affect the results of studies of metal toxicity [1]. The control of pH in cultures of microorganisms can be achieved by bubbling CO 2 in the medium. However, this meth- od is not successful in some cases [2]. Classic buffers suitable for pH values close to 7 to 8 (e.g., phosphate; tris-(hydroxy- methyl)aminomethane [Tris]; glycylglycine [3]; tricine; bicine and glycinamide) have limited application because they can be toxic themselves or because they complex trace metals essential for growth [4,5]. A different class of buffers (N- substituted aminosulfonic acids) were introduced and studied by Good and collaborators since 1966 [4–6] and these buffers increasingly are being used in numerous biological and chem- ical applications [3,7–9]. These zwitterionic (i.e., ampholytic species or ions that have both a positive and a negative charge on the same species) buffers have been considered more suit- * To whom correspondence may be addressed (mtvascon@fc.up.pt). able than the classical buffers, because they are relatively free of undesirable effects [6]. Nevertheless, Ferguson et al. [6] stressed that it is quite impossible to find a buffer that does not have physiologic or other effects unrelated to pH stabili- zation. Therefore, the selection of an appropriate buffer should always be dependent on extensive testing [5] to make sure that the buffer meets the needs of the investigation. The objective of the present work was to investigate the influence of two zwitterionic buffers, N-[2-hydroxy- ethyl]piperazine-N'-[2-hydroxypropanesulfonic acid] (HEPPSO; pK a = 7.9 at 20 °C [6]) and piperazine-N,N'-bis[2-hydroxy- propanesulfonic acid] (POPSO; pK a = 7.85 at 20°C [6]) on the bioavailability and toxicity of copper to cultures of Am- phidinium carterae Hulburt. The study evaluated the biolog- ical behavior of A. carterae cultures in an enriched solution of artificial seawater (ESAW) [10], buffered (pH fixed at 8.0) with 25  10 -3 M HEPPSO or POPSO and, for comparison purposes, without a pH buffer (initial pH = 8.0, final pH = 7.8). The initial total copper concentrations (Cu total ) in the me- dia were varied (range 23  10 -9 to 15  10 -6 M) at levels higher than those typical of ambient marine conditions in open ocean (range 5  10 -10 to 5  10 -9 M [11]). However, because of the presence of strong copper-binding ligands in the media, free copper concentrations were much lower (between 53  10 -15 and 72  10 -10 M) and included the range considered