## 4 Discussion  
### Cu Is the Most Toxic Metal  
The results showed that the mortality of the snails increased as they were exposed to increasing concentrations of metals or for longer durations. Cu was the most toxic metal. The snails were most susceptible to Cu with the lowest LC\({}_{50}\) values compared to other metals. Comparing the LC\({}_{50}\) values of Cd and Cu for _P. insularum_ with those of other snail species (>10 species), including mussels, clams, sea anemones, cockles and shrimp, revealed that _P. insularum_ was more sensitive to Cu than Cd, Ni, Pb and Zn. This is well indicated in the different species of bivalves and gastropods from the literature (Tables 2-6). Even though juvenile snails were more sensitive to the five metals, Cu was shown to be the most toxic metal for both juvenile and adult snails, when compared to Ni, Pb, Cd and Zn. This is consistently correlative with research on the toxicity of heavy metals to freshwater organisms. For instance, the rank order of toxicity of some heavy metals to _Daphnia magma_ was Cu > Zn > Cd > Pb > Ni (48 h) [35]; for rainbow trout (_Salmo gairdneri_) it was Cu > Zn > Cd > Pb > Ni (96 h) [35]; for amphibian tad-poles (_Bufo melanostictus_) it was Cu > Cd > Zn > Ni (96 h) [36]; and for _Lymnaea luteola_ it was Cu > Cd > Ni > Zn (72 h) [28]. The findings of the present study showed that the LC\({}_{50}\) values in the five metals significantly decreased (\(p<0.05\)) from 48-h to 72-h periods in both juvenile and adult snails. This indicated that the longer period of toxicity testing resulted in the snails being more sensitive to the five metal toxicities. [PERSON] et al. [37] reported that the LC\({}_{50}\) values of Cu in _Gammasulex_ decreased from 0.047 to 0.037 after 48-h and 96-h periods, respectively. Similarly, they also found that the LC\({}_{50}\) values of Cu in _Chironomus triparius_ decreased from 1.20 to 0.70 after 48- and 96-h periods, respectively. Using _P. canaliculata_ as the test organism, [PERSON] et al. [38] demonstrated that the LC\({}_{50}\) values of Cu exposure periods of 4, 48, 72 and 96 h were 0.330, 0.223, 0.177 and 0.146 mg/L, respectively. This indicates a decreasing order of LC\({}_{50}\) values with increasing Cu exposure period. All of these data demonstrated that the longer the duration of exposure, the more sensitive the invertebrates to pollutants. [PERSON] et al. [39] showed that the 96-h LC\({}_{50}\) value of Cu in _Lymnaea stagnalis_ was 31 g/L, indicating a moderate acute sensitivity to Cu. However, the projected EC\({}_{20}\) value (the median effective concentration of a substance to 20% of test organisms) for Cu after a 30-day chronic exposure of juvenile _L. stagnalis_ to Cu was 1.8 mg/L, making it the most sensitive organism to Cu investigated to date. In a different experiment with adult freshwater snails, _Melanoides tuberculata_, [PERSON] et al. [9] observed an increase in the median lethal times (LT\({}_{50}\)) and concentrations (LC\({}_{50}\)) of eight metals after four days of laboratory exposure. Cu was the most hazardous metal to _M. tuberculosis_, followed by Cd, Zn, Pb, Ni, Fe, Mn and Al. Several investigations suggested that _Pomacea_ snails were efficient bioindicators for Cu and Cd. _Pomacea canaliculata_ has the capacity to acquire Cu from a variety of metals (20, 30, 45, 67.5 and 101.3 mg/L), but demonstrated behavioral control at Cu concentrations of 67.5 and 101.3 mg/L, as determined by [PERSON] and [PERSON] [40]. This provided evidence for using the golden apple snail (whole tissue analysis) as a sublethal Cu biomonitor (0-45 mg/L). Additionally, [PERSON] et al. [41] reported acute toxicity of Cu and Cd on the hepatopancreas cells of _Helix pomatia_ (toxicity of Cu \(>\) Cd). [PERSON] and [PERSON] [42] showed that CuCO\({}_{3}\) was toxic to apple snails (_Pomacea puludosa_) due to the fact that Cu concentrations were higher in living snails than in dead snails. Their results indicated that apple snails could excrete deposited Cu [38].