## 4 Discussion
In our study, the DO concentration was kept under control (>7 mg/L and 100% oxygen saturation), and the observed effects cannot be attributed to significant changes in this variable (Table 1). The neutral red retention assay has been and is widely used to determine lysosomal membrane stability in invertebrate hemocytes, with special reference to bivalves [60; 61]. Threshold values used as assessment criteria for lysosomal membrane stability using the NRRA have been determined from _Mytilus_ sp. on the bases of numerous studies performed around the UK and other European waters, including Mediterranean waters [39; 62]. There is a limited number of studies investigating LMS in _C. gallina_ hemocytes [52; 54; 63]. To our knowledge, there are no LMS data in the literature obtained by using the NRRA in the hemocytes of _D. trunculus_. In the absence of specific evaluation criteria for these species, absolute values for LMS measurements in _Mytilus_ sp. could be assumed to be comparable. In this sense, NRRT shorter than the EAC level suggested the organism were severely stressed and probably exhibiting pathology. An NRRT shorter than the BAC level but longer than the EAC level was considered to represent stressed but compensating organism. Concerning _C. gallina_, neutral red retention times within the lysosomal compartment of the small granulocytes were in general longer (mean time higher than 50 min for all treatments) than the retention times reported on an earlier field study performed in Adriatic waters with the same species (NRRT \(\leq\) 30 min in spring (20 \({}^{\circ}\)C salinity of 35) and autumn (16 \({}^{\circ}\)C salinity of 36)) [52]. According to the assessment criteria established for this biomarker, our results point out that the treatments performed with _C. gallina_ caused less cellular stress compared to _D. trunculus_, since the organisms showed retention times below the environmental assessment criteria established for this biomarker (EAC \(<\) 50 min) in none of the treatments [62]. In general, for both species, the environmental conditions that led to greater LMS were those at a medium temperature (20 \({}^{\circ}\)C) at the three studied salinity ranges. In other bivalves, temperatures close to 20 \({}^{\circ}\)C have been found to be optimal for maintaining hemocyte lysosomal stability, with membrane stability being decreased both when increasing and decreasing the temperature [61; 64]. In addition, in the case of _C. gallina_, organisms exposed to the lowest temperature (12 \({}^{\circ}\)C) and salinity 32-33 also presented high stability. For _D. trunculus_ at 20 \({}^{\circ}\)C and salinity 32-33 and 37-38, the NRRTs were higher than the background assessment criteria value, while at the temperature 27.5 \({}^{\circ}\)C, at these same salinities, the NRRTs obtained were lower than the EAC value. [PERSON] et al. [65] observed that mussels exposed to aquatic hyperthermia conditions (28 \({}^{\circ}\)C) for 48 h were able to recover LMS values. In our current investigation, prolonged exposure to elevated temperatures (27.5 \({}^{\circ}\)C) combined with a salinity of 27-28 resulted in 100% mortality in _D. trunculus_ after 14 days. These results suggest that 27.5 \({}^{\circ}\)C could be the threshold temperature level for this species at low salinities. Moreover, in the surviving treatments at that temperature (salinity 32-33 and 37-38), the lowest LMS and the lowest CI values of all treatments were observed. The cellular responses analyzed revealed extremely severe stress in the organisms of those treatments 27.5 \({}^{\circ}\)C/32-33 and 27.5 \({}^{\circ}\)C/37-38 (N NRT of 28.1 \(\pm\) 12.7 min and 42.2 \(\pm\) 9.9 min, respectively) and the lowest mean %LMS values (20.8 \(\pm\) 7.0 and 36.9 \(\pm\) 5.4, respectively). Overall, the results of this study indicated that LMS responses were more affected by temperature than salinity, as NRRTs greater than 50 min at 27.5 \({}^{\circ}\)C were never detected, regardless of salinity. In the case of _C.