Since reproductive parameters are common endpoints in pollution monitoring, the description of the spermatogenesis of Lipophrys pholis, a recently proposed sentinel species, is of critical importance. Mature males were used to determine the annual maturation cycle, using a stereological approach. The spermatogenic cycle was classified into three stages: early spermatogenesis (May), mid spermatogenesis (September), and spawning (November and January). The testes were asynchronously arranged with various cell types observable during all seasons, although in different proportions. Gonadosomatic index and spermatozoa proportions confirmed the established reproductive season. Differences between the sampled populations and those from septentrional locations are discussed, namely the absence of ''resting'' and ''spent'' periods.

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Information on the genital morphology of male and female Lipophrys pholis is provided, as well as for two other sympatric blenniid species, Coryphoblennius galerita and Lipophrys trigloides. The use of non-invasive sex determination procedures described may be extremely useful not only for ecological studies but also as a proxy for the detection of environmental exposure to endocrine disruptive chemicals, given that blennies have been proposed as potential sentinel species for chemical contamination.

The recent advances in molecular biology techniques have prompted the use of vitellogenin (VTG) gene expression as a sensitive and reliable indicator of estrogenic chemicals (EC) exposure. However, data on the dynamic response of the different VTGs genes upon EC exposure is still poorly understood, particularly in sentinel fish species used in field monitoring studies. Hence, the present study aimed at developing a sensitive real-time PCR assay for determining the response of VTG I and II in the recently proposed marine sentinel species Lipophrys pholis upon exposure to the model EC 17α-ethinylestradiol (EE2). The findings of the laboratory study indicate that L. pholis VTG I proved to be not only more inducible but also more sensitive to EE2 exposure than VTG II, for the same range of concentrations. In fact, VTG I gene induction was 475-fold higher than VTG II at 15 ng/L EE2, and 13-fold at 5 ng/L EE2. Overall, the findings of the present study indicate that in the field, expression of VTG I in L. pholis should be preferentially used in the screening of EC exposure because of its higher sensitivity. Furthermore, the present study favors L. pholis integration in monitoring programs associated with EC's pollution within the European water policy legislation.