Neutral red is a weakly cationic dye that is soluble in water, has a low toxicity for almost all classes of organisms, and has been used as a histological and vital stain since the early twentieth century. Estimating the volume of water cleared of indicator material by suspension feeding bivalves (clearance or filtration rate) was one of the earliest applications of neutral red; however, less than thirty studies have applied this methodology since it was first described in 1954. The feeding/clearance rate is used as a sensitive indicator of physiological stress and is therefore an early-warning tool that is suitable for monitoring the ecological status of water bodies. The aim of our study was to optimise a clearance index based on neutral red solution by addressing the effect of i) the acidifying pH; ii) the holding temperature before spectrophotometric reading; and the time iii) before and iv) after the acidification of solutions of neutral red used to carry out clearance assays. Furthermore, as a case study we fine-tuned the clearance assay for the edible estuarine bivalve, Cerastoderma edule.


The results showed that there were no statistical differences as regards the absorbance of neutral red solutions holding at 4 or 20 ºC or a solution acidified between the ranges of pH 4-5. However, the absorbance significantly decreased as the pH increased to pH 6. The time before acidification had no significant effect on absorbance. Once the neutral red solution is acidified, the absorbance decreases over time, signifying that the absorbance should be read in the first 24 h.


The concentration of neutral red used in the experiences should be sufficient to allow final concentrations of over 0.5 mg/L after the clearance period, since we observed that the sensitivity of this protocol decreased at low concentrations.


In the case of C. edule, the optimum clearance conditions per individual were found to be 100 ml of 4 mg/L of neutral red dye during a 30 min period in dark conditions. A bioassay using a clearance index of C. edule based on this simple colorimetric technique would appear to be a potential tool for implementation in environmental monitoring programmes for water quality assessment in accordance with European directives. We trust that the new harmonised protocol will become a widely used and cost-effective means to monitor the clearance index as an indicator of physiological stress for bivalves.


 This study was the first step in developing an in situ bioassay with this species.


This study is published in:

Martinez-Haro M, Pais-Costa AJ, Verdelhos T, Marques JC, Acevedo P. 2016. Optimising a clearance index based on neutral red as an indicator of physiological stress for bivalves. Ecological Indicators 71:514-521 (link)



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