Humic acids are large, complex, organic molecules which are ubiquitous components of aquatic environments as products of degradation of plant material. In aqueous solution they form microvesicles. As many teleost pheromones are steroidal in nature, we hypothesised that they would preferentially dissolve in the organic, hydrophobic core of these vesicles instead of in water and therefore be unavailable for detection. This would have obvious and profound effects on many aspects of fish biology. To test this hypothesis we recorded electro-olfactogram (EOG) response of the goldfish (Carassius auratus) olfactory epithelium to the pheromones 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (1720 beta-P), its sulphated conjugate (1720 beta-P-SO(4)) and prostaglandin F(2alpha) (PGF(2alpha)), all at 10(-11) to 10(-8) M, in the absence and presence of humic acids (1-1000 m x gl(-1)). At nearly all concentrations of humic acid tested, there was a significant attenuation of the amplitude of the initial (phasic) response to 1720 beta-P compared to 1720 beta-P alone. At higher concentrations of humic acid, the EOG response to 1720 beta-P was often completely obliterated, suggesting that the concentration of the pheromone available to the olfactory epithelium was below the threshold of detection. Exposure of the olfactory epithelium to humic acid did not cause any short-term loss of sensitivity to 1720 beta-P per se. Furthermore, simultaneous recording of electro-encephalograms from the olfactory bulb demonstrated that the nervous activity evoked by the same concentration of 1720 beta-P was less intense in the presence of humic acid than its absence. PGF(2alpha) is non-steroidal and much more soluble in water. In contrast to 1720 beta-P, only the higher concentrations of humic acid (100 and 1000 mg x l(-1)) significantly diminished the EOG amplitude. 1720 beta-P-SO(4) is detected via a distinct olfactory mechanism to the free form. Given that the sulphate group increases the water solubility, we predicted that the effect of humic acid would be reduced. However, the effect of humic acid on EOG amplitude in response to 1720 beta-P-SO(4) was similar to that of the free form. We suggest that the steroid portion of the molecule adsorbs onto the surface of the humic acid microvesicles and is still effectively unavailable for olfactory detection. In conclusion, humic acid may significantly reduce the concentration of 1720 beta-P and 1720 beta-P-SO(4) available for detection by Carassius auratus in natural environments. Furthermore, as many teleost pheromones are steroid derivatives, this phenomenon may be applicable to chemical communication systems in teleosts in general.

http://www.ncbi.nlm.nih.gov/pubmed/12200084?dopt=Abstract