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Biological responses and contaminant levels in biological tissues were investigated in fish specimens of grass goby (Zosterisessor ophiocephalus) collected in spring 2000 in a moderately polluted brackish ecosystem, the Orbetello lagoon (southern coast of Tuscany, Italy). The complexity of harmful effects that contaminants may cause in fish over time was assessed. General condition indices (somatic liver index, and gonadal somatic index), cytochrome P450 enzymes, acetylcholinesterase activity and ovarian morphology were integrated with the content of organochlorines (OCs-hexachlorobenzene, DDTs and polychlorinated biphenyls) in liver and ovaries and p-nonylphenol (NP) and lower ethoxylate (NPE 1-2) content in muscle and ovaries as well. Samples collected from the western basin of the lagoon near the Orbetello sewage treatment plant had somatic liver index values and two P450 activities significantly higher than those collected from the same basin at the Albegna river delta and in the Ansedonia canal in the eastern basin (P < 0.05). On the other hand, while muscle content of p-NP and NPE 1-2 was significantly higher in samples from the Orbetello and Albegna river sites than in samples from the Ansedonia canal site, OC levels in liver (DDTs > PCBs > HCB) were particularly lower in samples from STP compared to levels detected in sites AC and AR. Light microscopy of ovaries revealed that although the gonads were sexually mature, the diameter of the oocytes and eggs was smaller than that commonly reported. High levels of OCs and NPs were, in fact, observed in grass goby ovaries, and the differences observed between acetylcholinesterase activities among investigated sites suggest exposure to insecticides as well. The integration of biological responses and contaminant tissue content reveals that certain areas of the lagoon are selectively impacted by various human activities and helps in distinguishing relationships between biological responses and contaminant load in the lagoon environment.