Sub-cellular biomarkers provide a sensitive tool in environmental monitoring as they are able to detect environmental perturbations at an early stage before a whole animal, physiological or community response becomes manifested.A sub-cellular biomarker for the detection of estuarine sediment contamination by a in vitro staining method, employing the weak base, neutral red, was developed for use with the euryhaline endobenthic polychaete Nereis diversicolor, O.F. Müller.Coelomocytes extracted from the coelomic cavity of rag worms from different sites into a physiological saline were allowed to adhere to a microscope slide before administration of neutral red dye. As a result of its weak alkaline and cationic character the dye was readily taken up and retained by lysosomes. Observation of dye leakage from lysosomes to the cytosol has permitted the quantification of the degree of lysosomal membrane pathology caused by increased sequestration of pollutants or natural induced stress.The assay enabled the detection of site specific differences: Rag worms from Aveton Gifford had the lowest neutral red retention times as a result of low salinity at that site. Animals from the moderately contaminated site St. Johns Lake and the grossly heavy metal polluted site, Restronguet Creek had similar NRR times. Nereis from the secluded Erme estuary were in the healthiest state.In vitro exposures of coelomocytes to different copper concentrations did not reveal a dose response relationship, possibly because the concentrations used lay below a threshold level and because the inter-animal variability was very high.