## 2 Materials and Methods  
### Sample Preparations and Laboratory Experiments  
The snails, _P. insularum_, were collected from Universiti Putra Malaysia's Lake (N 02\({}^{\circ}\)59\({}^{\prime}\)58.84\({}^{\prime\prime}\), E 101\({}^{\circ}\)42\({}^{\prime}\)39.42\({}^{\prime\prime}\)), on the 11 March 2007. The lake is considered unpolluted with low Cu concentration in the surface sediments (27.7 mg/kg dry weight) and with good surface water quality parameters; temperature (32.6 \({}^{\circ}\)C), conductivity (77.9 \(\upmu\)S/cm), total dissolved solids (0.05 mg/L), dissolved oxygen (7.56 mg/L), pH (6.61) and turbidity (0.00 NTU) (unpublished data). For acclimation, a maximum of 100 juveniles and 100 adult snails were kept per aquarium (30 cm length \(\times\) 19 cm with \(\times\) 18 cm height containing 6000 mL). During the acclimation period, the surface water quality parameters of the dechlorinated tap water in the plastic aquarium tanks ranged from 28.0 to 31.0 \({}^{\circ}\)C for temperature, 1.00-5.00 \(\upmu\)S/cm for conductivity, 0.01-0.03 mg/L for total dissolved solids, 6.50-7.50 mg/L for dissolved oxygen, 6.70-7.10 for pH and 0.00 NTU for turbidity (unpublished data). Each aquarium was also regularly aerated to give a moderate airflow, in which the snails were acclimatized to laboratory settings for three days. To eliminate any potential bias in the experiments, the snails were divided into two groups, namely juvenile (shell lengths: 0.50 to 0.70 cm) and adult (shell lengths: 1.50 to 2.20 cm) snails for the experimental toxicity study. Prior to static toxicity testing, range-finding-tests following the standard methods [27] were conducted to determine the critical range, which is defined as the interval between the lowest concentration of metals that kills very few or none of the experimental snails and the highest concentration that kills the majority or all of the experimental snails during the experimental period. The purpose of these tests was to determine the median lethal doses (LC\({}_{50}\)) of each metal in the snails exposed for 24, 48, 72 and 96 h (h). Following the range-finding-tests, five nominal concentrations of Cu, Cd, Zn, Pb and Ni were chosen (Table S1). Metal solutions were prepared by diluting the metal stock solutions with dechlorinated tap water to respective nominal concentrations of the five metals. Dechlorinated tap water was used as the negative control. The control samples comprised snails which had not been exposed to any metal solutions and were treated with the identical experimental conditions as the experimental samples. The tests were carried out under static conditions, without renewal of the solution until the end of the experiment. The control and metal-treated groups each consisted of two replicates of 10 healthy snails in a plastic aquarium tank of 21 cm length \(\times\) 13 cm with \(\times\) 11.5 cm height, containing 1000 mL of the respective nominal concentrations of the five metals. During the experimental exposure period, the surface water quality parameters in the plastic aquarium tanks were 28.0-30.2 \({}^{\circ}\)C for temperature, 1.00-3.00 \(\upmu\)S/cm for conductivity, 0.01-0.05 mg/L for total dissolved solids, 6.50-7.50 mg/L for dissolved oxygen, 6.50-7.05 for pH and 0.00 NTU for turbidity (unpublished data). The standard stock solutions (100 mg/L) of Cd, Cu, Ni, Pb and Zn, were prepared from analytical grade metallic salts of CdCl\({}_{2}\).25H\({}_{2}\)O, CuSO\({}_{4}\).5H\({}_{2}\)O, NiSO\({}_{4}\).6H\({}_{2}\)O, Pb(NO\({}_{3}\))\({}_{2}\) and ZnSO\({}_{4}\).7H\({}_{2}\)O, respectively (Merck, Darmstadt, Germany), by diluting with deionized water in 1 L volumetric flasks. Acute Cu, Cd, Zn, Pb and Ni toxicity experiments were performed for a four-day (96-h) period using juvenile and adult snails, obtained from stocking tanks. The snail death rates were determined at 24-, 48-, 72- and 96-h periods. The deceased snails were removed from the experimental tanks at each period.