In vivo laser-induced fluorescence detection of pyrene in nematodes and determination of pyrene binding constants for humic substances by fluorescence quenching and bioconcentration experiments

The bioconcentration of pyrene by bacterivorous thread worms (nematodes) of the species Caenorhabditis elegans was studied with laser-induced fluoresc ence (LIF) spectroscopy, fluorescence imaging and a radiotracer method. The vibronic band intensities of the LIF spectra indicated that the microenvir onment of pyrene in the nematodes was similar to a low-polarity solvent, an d thus provided direct evidence that pyrene was accumulated in lipid-rich a reas inside the nematodes. The concentration of pyrene in the nematodes was estimated from the monomer/excimer fluorescence intensity ratio. Results f rom this method were in fair agreement with results using C-14 labeled pyre ne for measuring pyrene bioconcentration. Preliminary results indicated tha t LIF measurements of pyrene may be possible even in single nematodes. Fluo rescence microscopic observations revealed that pyrene was not adsorbed on the outside of the organisms, but was strongly concentrated in restricted a reas inside the worms. In the second part of the study, the effects of six different humic substances (HS) on the bioconcentration of pyrene were inve stigated and sorption coefficients (K-DOC) calculated from reductions in bi oconcentration (K-DOC(biol)) were compared with sorption coefficients measu red with a fluorescence quenching technique (K-DOC(flu)). The results of th ese two different experimental methods agreed well (with K-DOC(BIOL) being slightly lower than K-DOC(flu)), indicating that the fraction of pyrene tha t was determined as freely dissolved by the fluorescence quenching method w as comparable to the bioavailable fraction.