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 fluorescence (LIF) spectroscopy, fluorescence imaging and a radiotracer method. The vibronic band intensities of the LIF spectra indicated that the microenvironment of pyrene in the nematodes was similar to a low-polarity solvent, and thus provided direct evidence that pyrene was accumulated in lipid-rich areas inside the nematodes. The concentration of pyrene in the nematodes was estimated from the monomer/excimer fluorescence intensity ratio. Results from this method were in fair agreement with results using C-14 labeled pyrene for measuring pyrene bioconcentration. Preliminary results indicated that LIF measurements of pyrene may be possible even in single nematodes. Fluorescence microscopic observations revealed that pyrene was not adsorbed on the outside of the organisms, but was strongly concentrated in restricted areas inside the worms. In the second part of the study, the effects of six different humic substances (HS) on the bioconcentration of pyrene were investigated and sorption coefficients (K-DOC) calculated from reductions in bioconcentration (K-DOC(biol)) were compared with sorption coefficients measured with a fluorescence quenching technique (K-DOC(flu)). The results of these two different experimental methods agreed well (with K-DOC(BIOL) being slightly lower than K-DOC(flu)), indicating that the fraction of pyrene that was determined as freely dissolved by the fluorescence quenching method was comparable to the bioavailable fraction.