Effect of pH, EDTA, and Anions on Heavy Metal Toxicity Toward a Bioluminescent Cyanobacterial Bioreporter Ismael Rodea-Palomares Æ Coral Gonza ´lez-Garcı ´a Æ Francisco Legane ´s Æ Francisca Ferna ´ndez-Pin ˜as Received: 20 November 2008 / Accepted: 22 December 2008 / Published online: 24 January 2009 Ó Springer Science+Business Media, LLC 2009 Abstract The bioavailability and therefore toxicity of a metal depends on the chemical species present in a par- ticular environment. We evaluated the effect of a series of factors that could potentially modify metal speciation on the toxicity of Hg, Cu, Zn, and Cd toward a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 with cloned lux operon of luminescent terrestrial bacterium Photorhabdus luminescens. The strain, denoted as Anabaena CPB4337, showed a high constitutive lumi- nescence with no need to add exogenous aldehyde. The tested factors were pH, EDTA (as organic ligand), and anions PO 4 3– , CO 3 2– , and Cl – . Chemical modeling and correlation analyses were used to predict metal speciation and link it with toxicity. In general, metal toxicity signif- icantly correlated to the predicted metal free-ion concentration, although Zn–EDTA complexes and certain Hg chloro-complexes could also exhibit some toxicity to cyanobacteria. An interesting feature of metal toxicity to strain Anabaena CPB4337 was that low amounts of PO 4 3– and CO 3 2– increased metal toxicity; this effect could not be related to significant changes in metal speciation and could be attributed to a modulating effect of these anions on metal/uptake toxicity. The combination of toxicity studies that take into account a range of factors that might mod- ulate metal toxicity with chemical modeling to predict changes in metal speciation might be useful for interpreting complex toxicity data. Finally, this cyanobacterial biore- porter, due to its ecological relevance as a primary producer, could be used as a tool for toxicity assessment in freshwater environments. Available freshwater resources are polluted by industrial effluents, domestic and commercial sewage, as well as mine drainage, agricultural runoff and litter. Among water pollutants, heavy metals are priority toxicants that pose potential risks to human health and the environment. The evaluation of heavy metal contamination traditionally relies on highly sensitive and specific physical and chem- ical techniques such as atomic absorption spectroscopy or mass spectrometry; however, such methods are not able to distinguish between available (potentially hazardous to biological systems) and nonavailable (potentially nonhaz- ardous) fractions of metals that exist in the environment in inert or complexed forms. Toxicity bioassays and biosen- sors might complement physical and chemical methods by detecting the toxicity related with bioavailable metals in environmental samples, effectively integrating the com- plexity of environmental factors (pH, redox potential, exchangeable cations, biological activity, etc.) that con- tribute to bioavailability (Ko ¨hler et al. 2000). Bioavailability is strongly affected by the speciation of a metal in a particular environment. Water chemistry parameters such as alkalinity, pH, salinity, hardness, phosphates, or ionic strength (Cook et al. 2000; Ho et al. I. Rodea-Palomares and C. Gonza ´lez-Garcı ´a contributed equally to this work. I. Rodea-Palomares  C. Gonza ´lez-Garcı ´a  F. Legane ´s  F. Ferna ´ndez-Pin ˜as (&) Departamento de Biologı ´a, Facultad de Ciencias, Universidad Auto ´noma de Madrid, Cantoblanco, 28049 Madrid, Spain e-mail: francisca.pina@uam.es Present Address: C. Gonza ´lez-Garcı ´a Instituto de Salud Carlos III, Centro Nacional de Microbiologı ´a, Regulacio ´n Ge ´nica, Crta de Majadahonda-Pozuelo Km 2, 28220 Madrid, Spain 123 Arch Environ Contam Toxicol (2009) 57:477–487 DOI 10.1007/s00244-008-9280-9