Interpretation of toxicity test results may be hampered when doubt exists about the actual exposure concentration. Processes that are responsible for differences between the nominal and the actual concentration in aqueous test systems may include sorption, precipitation, volatilization, chemical and biological degradation, and uptake into biological or test tissue. In this study, the use of a poly(dimethylsiloxane) (PDMS) film containing the test compound is introduced as a versatile technique for partition controlled delivery of hydrophobic compounds to aqueous toxicity tests. Two methods developed produced preloaded films, having toxicant added to the PDMS prepolymer solution before film deposition and curing, and postloaded films, which are created by the addition of toxicant in a solvent to an already-polymerized PDMS film. Preloaded films were generally more easily prepared, may better accommodate larger molecules, and have a higher capacity than postloaded films. Postloaded films provided film-solution partition coefficients with higher precision and allowed for the use of films from stock and thus for a more portable technique. Chemical analysis showed that equilibrium between films and the aqueous solution was established within 7-10 min and was maintained for a suite of aromatic compounds (log Kow ranging from 2.8 to 6.1). The reliability of the film technique was demonstrated by application to the Microtox bacterial toxicity tests of solutions of polycyclic aromatic hydrocarbons (PAHs).

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at non-cryogenic temperatures and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events – pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-type and mutant organisms and under stress conditions.

Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.