Acute Toxicity of Metals: Cu, Pb, Cd, Hg and Zn on Marine Molluscs, Cerithedia cingulata G., and Modiolus philippinarum H.

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1 Indian Journal of Geo-Marine Sciences Vol. 41(2), April 2012, pp Acute Toxicity of Metals: Cu, Pb, Cd, Hg and Zn on Marine Molluscs, Cerithedia cingulata G., and Modiolus philippinarum H. Ramakritinan C M*, Rathishri Chandurvelan & A K Kumaraguru Department of Marine and Coastal Studies, School of Energy, Environment and Natural Resources, Madurai Kamaraj University, Madurai , Tamilnadu, India. *[ ramakritinan@gmail.com; ramakritinan@yahoo.com] Received 03 March 2011; revised 24 June 2011 Present study was intended to compare the acute static renewal toxicity tests on marine snail, Cerithidae cingulata and bivalve, Modiolus philippinarum that were performed with Cu, Cd, Pb, Zn and Hg. The 96 h LC 50 values for C.cingulata were 0.521, 9.193, , and mg.l -1 and the 96 h LC 50 values for M.philippinarum were 0.023, 0.221, 2.876, and mg.l -1 in respective metals. This study indicated that mercury was highly toxic to both C.cingulata and M.philippinarum. While lead was resistant metal to both marine molluscs tested. Order of increasing toxicity of metals to C.cingulata was Hg > Cu > Zn> Cd > Pd and to M.philippinarum was Hg > Cu > Cd > Zn > Pb. Safe level concentrations for all 5 metals were also calculated. The LC 50 values of the present study may be useful in deriving marine water quality standards in the coastal waters of Gulf of Mannar. [Keywords: Acute toxicity, snail, bivalve, heavy metals, LC 50 ] Introduction Pollution in the aquatic environment by heavy metals has received considerable attention 1,2 Owing to their toxicity at very low levels, persistence in the environment and their ability to accumulate in animal tissues. It is well known that metal toxicants seldom occur individually in the aquatic environment. Marine invertebrates especially marine molluscs such as bivalves and snails are used as test species for conducting toxicity experiments since these are mostly in benthic habitat and filter feeders and some are found in the rocky shores 3. Despite the considerable amount of information available on the effects of heavy metals on marine snails and bivalves, reports on acute toxic effects of heavy metals to Cerithidea cingulata and Modiolus philippinarum are quite limited 4-7. Present study was investigated the acute toxic effect of marine molluscs, Cerithidea cingulata (snail) and Modiolus philippinarum (bivalve) to heavy metals under static renewal bioassay tests. Materials and Methods: Two species of marine molluscs such as Cerithedia cingulata (Snail) and Modiolus philippinarum (Bivalve) were selected to find out the heavy metal levels of selected metals. Adult C. cingulata of uniform size (23 ± 0.5 mm Length) were collected by hand from the intertidal region of the muddy shores of Mandapam coast of the Palk Bay (N & E ). They were transported to the laboratory immediately in plastic buckets with natural seawater and mud and acclimated to room temperature in circular aquaria ( cm) containing filtered seawater. Adult M. philippinarum (10 ± 2 mm Length) were collected from the intertidal rocky shore of Pudumadam coast of the Gulf of Mannar (N & E ) and transported to the laboratory immediately and acclimated to room temperature in circular aquaria (60 30) containing filtered seawater. The collected animals were acclimated for a period of 7 days in an ambient room condition. During the acclimation period, mixed culture of phytoplankton was given as food and water was aerated gently. Physico-chemical characterization of the filtered seawater was estimated using standard protocol 8 and the essential parameters such as temperature, ph, salinity, dissolved oxygen, BOD, COD and Total Organic Carbon were 29.2 C, 8, 33, 5.5mg.l -1, mg.l -1, 3.45 mg.l -1 and 0.25% respectively. Nutrient such as nitrite, nitrate, phosphate and silicates were mg.l -1,

2 142 INDIAN J. MAR. SCI., VOL. 41, NO. 2, APRIL mg.l -1, mg.l -1 and mg.l -1 and the levels of heavy metals such as copper, cadmium, lead, zinc and arsenic were 0.25 µg.l -1, 3.1 µg.l -1, µg.l -1, µg.l -1 and µg.l -1 and the mercury was below detection limit. Copper sulphate [CuSO 2 5H 2 O], lead nitrate [Pb(NO 2 ) 2 ], cadmium chloride [CdCl 2 2.5H 2 O], zinc chloride [ZnCl 2 ] and mercury chloride [HgCl 2 ] were used for conducting the heavy metal toxicity studies. Stock solutions of heavy metals were prepared by dissolving the appropriate quantity of metal salts in Milli-Q-Water in 1000 ml glass volumetric flasks. These treated as Main Stock Solutions (MSS). They were stored in a refrigerator until use. From the MSS, Sub-Stock Solution (SSS) of each metal was prepared daily and used for preparation of required concentrations of toxicant. Glassware used for storage stock of solutions had previously been washed using 10% nitric acid and well rinsed with distilled water. Acute static renewal toxicity tests were conducted the method described by Sprague 9 and USEPA 10. Preliminary toxicity tests for each metal were conducted on C. cingulata and M. philippinarum and the animals were starved one day prior to the bioassay tests. A group of 10 well acclimated C. cingulata and M. philippinarum (uniform size) were separately introduced in circular glass aquaria (60 30 cm) containing 5 litres of test medium. For preliminary range finding tests, the test concentrations of Cu, Cd, Pb, Zn and Hg were made in a logarithmic scale i.e., 1000, 100, 10, 1, 0.1, 0.01 and mg.l -1 using previously filtered seawater. Replicates were maintained in each test concentration and also in seawater control. Range finding tests were conducted for a total period of 48 hours and the mortality was observed. During observation, dead animals were removed immediately from the test chambers. Tests were conducted in an ambient room temperature. All experiments were carried out at 28±2 C, salinity 30 (ppt) under a 12:12 light: dark regime without aeration. During the test, the essential water quality parameters such as temperature, salinity, dissolved oxygen and ph of control as well as test media were estimated at beginning i.e., 0 h and at the end of a day i.e., 24 to ensure the acceptability of the tests. Median lethal concentration (LC 50 ) values of C.cingulata and M.philippinarum exposed to heavy metals were calculated by Probit Analysis 11 using computer programme 12. After range finding tests, the definitive toxicity tests were conducted using narrow range of concentrations (geometrical scale) in order to find out the LC 50 values. Selected concentrations for C.cingulata were 0.12, 0.24, 0.48, 0.96, 1.92 mgcu.l -1, 3.4, 6.8, 13.7, 27.4, 54.8 mgcd.l -1, 5.45, 10.9, 21.8, 43.7, 87.4 mgpb.l -1, 3.0, 6.0, 12.0, 24.0, 48.0 mgzn.l -1 and 0.012, 0.025, 0.050, 0.100, mghg.l -1. For M.phillipinarum, the concentrations were 0.01, 0.02, 0.04, 0.08, 0.16mgCu.l -1, 0.929, 1.858, 3.716, 7.432, mgcd.l -1, 0.06, 0.12, 0.24, 0.48, 0.96 mgpb.l -1, 1.015, 2.030, 4.061, 8.122, mgzn.l -1 and 0.002, 0.004, 0.008, 0.017, mghg.l -1. Further the test protocols as described in the range finding tests were followed for the respective animals and toxicant. All test media were renewed daily with freshly prepared ones in order to provide a constant effect of the toxicant on test animals. During the renewal, at least 80% of the test medium was renewed and freshly prepared toxicant was added. During the period of bioassay tests, the organisms were starved and the medium was not aerated. Mortality was observed at 24, 48, 72 and 96 hours of exposure and during observation, dead ones were removed immediately from the test chamber. Animals were considered living if movement was exhibited after gentle stimulation. Tests were rejected when the control mortality exceeded 10% 10. The LC 50 values at each exposure period, 95% Confidence Interval values, Chi-Square values, slopes and intercepts were obtained using probit analysis 11 computer programme 12. Safe concentrations of the metals were determined by the methods of Kameswara-Rao 13 and Miller & Miller 14 following the application factor, 1/100 th of the 96 h LC 50 values. Results and Discussion: Mortality of test animals was positively correlated with increasing concentration of metals as well as increasing exposure period. As may be expected, the greater the metal concentration, the greater is the mortality. Similar reports on the mortality of marine molluscs such as bivalves, oysters, mussels, snails and clam were observed in relation to heavy metal toxicity 6, Lakshmanan and Nambisan 18 have reported that, the rapid uptake of Hg was higher when the green mussel, Perna viridis was exposed at higher concentration of Hg. It suggests that, the toxic effect was more at higher concentration and also increasing

3 RAMAKRITINAN et al.: ACUTE TOXICITY OF METALS 143 exposure time. It can be seen that lethal time decreased as concentration of metal increased. While M.philippinarum exposed to Cu at 48 and 72 h and Zn at 72 h; C.cingulata exposed to Cd at 48 and 96 h found partial mortalities i.e., the lower metal concentration found to have higher mortality. This may be due to individual sensitivity of animal and health status. Primary criterion of a toxicity test is the survival after exposure to contaminated and uncontaminated (Control) waters None of the control animals died, demonstrating that the holding facilities and handling techniques were acceptable for conducting such tests, as required in the standard EPA/COE protocol where mean survival should be 90% 19. In the present study, no mortality was noticed in animal exposed in seawater only medium. The calculated LC 50 values for marine molluscs, C. cingulata and M. philippinarum decreased with increasing exposure time (Table 1). The 96 h LC 50 values for C.cingulata exposed to Cu, Cd, Pb, Zn and Hg were 0.521, 9.193, , and 0.053mg.l -1 respectively and the 96 h LC 50 values for M.philippinarum exposed to Cu, Cd, Pb, Zn and Hg were 0.023, 0.221, 2.876, and 0.007mg.l -1 respectively. Present study indicated that mercury was highly toxic to C.cingulata and M.philippinarum, while lead was resistant metal to both marine molluscs tested. Based on the acute toxicity test carried out in the present study both C.cingulata and M.philippinarum was shown to be most sensitive to mercury and most resistant to lead. Eisler 22 has reported that the LC 50 values of adult soft shell clam Mya arenaria exposed to copper, cadmium, zinc, lead, manganese and nickel were 0.035, 0.150, 1.55, 8.80, and >50.0 mg.l -1 respectively at 30 (ppt) (22 C) during 168 h under static bioassay tests. This concluded that except copper and lead, the LC 50 values of Cd and Zn found to be more in the present study when compared to the LC 50 values of Mya arenaria 22. Cheung et al. 23 reported that the 96-h LC 50 values for Cu, Cd and Zn for sandy shore scavenging gastropod Nassarius festivus were 0.36, 1.52 and 1.76 mg.l 1 respectively. The present study showed that mercury was highly toxic to both marine molluscs. On comparison, the Table 1 Parameters of acute lethal toxicity bioassays with selected metals for marine snail C.cingulata and bivalve M. philippinarum exposed under static bioassay tests. Metals used Exposure (h) LC 50 (mg.l -1 ) Snail C. cingulata 95% Confidence Interval Safe Conc. (µg.l -1 ) Chisquare calculated* LC 50 (mg.l -1 ) Bivalve M. philippinarum 95% Confidence Interval Conc. Safe (µg.l -1 ) Chi-square calculated* Copper Cadmium Lead Zinc Mercury *Chi-Square tabular value at 0.05 level = 7.815

4 144 INDIAN J. MAR. SCI., VOL. 41, NO. 2, APRIL 2012 Table 2 The LC 50 /EC 50 values of marine molluscs exposed to metals under acute bioassay tests. Species Life Stages Test Duration (h) Test System LC/EC 50 concentration (µg.l -1 ) References Copper Clam- Donax faba 10-15mm 96 S, U 930 Sommanee 6 Clam-Donax faba 17-20mm 96 S, M 120 Ong and Din 17 Snail-Cerithedia cingulata Adult 96 S-R, U 521 Present study Bivalve-Modiolus philippinarum Adult 96 S-R, U 23 Present study Cadmium Anadara granosa (Blood cockle) 9 ± 2mm 96 S, M 950 ± 440 Ong and Din 17 Clam-Donax faba 21 ± 1mm 96 S, M 1,100 ± 310 Ong and Din 17 Snail-Cerithedia cingulata Adult 96 S-R, U 9,193 Present study Bivalve-Modiolus philippinarum Adult 96 S-R, U 221 Present study Lead Pacific oyster-crassostrea gigas Embryos 48 S, U 758 Martin et al. 5 Quahog clam-mercenaria mercenaria Larva 96 S, U 780 Calabrese and Nelson 4 Soft shell clam-mya arenaria Adult 96 S, U 27,000 Eisler 22 Blue mussel-mytilus edulis Larva 96 S, U 476 Martin et al. 5 Snail-Cerithedia cingulata Adult 96 S-R, U 15,507 Present study Bivalve-Modiolus philippinarum Adult 96 S-R, U 2,876 Present study Zinc Mud snail-nassarius obsoletus Adult 96 S, U 50,000 Eisler and Hennekey 16 Soft-shell clam-mya arenaria Adult 96 S,U 7,700 Eisler and Hennekey 16 Soft-shell clam-mya arenaria Adult 96 S,U 5,200 Eisler 22 Snail-Cerithedia cingulata Adult 96 S-R, U 8,990 Present study Bivalve-Modiolus philippinarum Adult 96 S-R, U 2,337 Present study Mercury Clams-Donax faba 10-15mm 96 S 160 Sommanee 5 Hard clam-mercenaria mercenaria Embryo 48 S, U 4.8 Calabrese and Nelson 4 Softshell clam-mya arenaria Adult 96 S, U 400 Eisler and Hennekey 16 Mud snail-nassarius obsoletus Adult 96 S, U 32,000 Eisler and Hennekey 16 Snail-Cerithedia cingulata Adult 96 S-R, U 53 Present study Bivalve-Modiolus philippinarum Adult 96 S-R, U 7 Present study S = static; S-R = static-renewal; FT = flow-through; M = measured; U = unmeasured; NR = not reported. 96h LC 50 values of M.philippinarum exposed to all 5 tested metals were less than that of C.cingulata, it means that C.cingulata has more resistant. Based on the LC 50 values, C.cingulata was resistant to all five metals i.e., Cu, Cd, Pb, Zn and Hg than that of M.philippinarum. The order of increasing toxicity of metals to C.cingulata was Hg > Cu > Zn> Cd > Pd and to M.philippinarum was Hg > Cu > Cd > Zn > Pb. Whereas Martin et al. 5 found mercury and copper to be about equally toxic to M. edulis embryo and the rank of order of toxicity was Hg and Cu > Ag > Zn > Pb > Ni > Cd > Ar > Cr > Se. The LC 50 values for heavy metals for other marine molluscs are also given in Table 2. The LC 50 values of the present study may be useful in deriving marine water quality standards in the coastal waters of Gulf of Mannar. On comparison with the LC 50 values for copper, cadmium, lead, zinc and mercury recorded in other marine molluscs are not similar to those recorded in this study. These differences may be attributed to different collection sites and periods, different species, different experimental salinities, different size test chambers, different laboratory conditions and different geographical regions. Generally marine molluscs including bivalves and oysters have been used as indicator organisms of marine pollution. The development of corrective action plan is mainly dependent on the criterion or safe concentration of metals that is protective to snails, bivalves and other marine molluscs. The calculated safe concentrations for C. cingulata were 5.2, 91.9, 155.1, 89.9 and 0.5 µg.l -1 for Cu, Cd, Pb, Zn and Hg respectively and for M. philippinarum were 0.2, 2.2, 28.8, 23.4 and 0.07 µg.l -1 at respective metal. Concentration of metal above these levels can have adverse effects to the survival of C. cingulata and

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