See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/263210855 Effect of mixtures of metals on the spotted Babylon snail (Babylonia areolata) under different temperature conditions Article in Toxicological and Environmental Chemistry · September 2013 DOI: 10.1080/02772248.2014.881077 CITATIONS READS 36 authors, including: Taufik Hayimad Universiti Malaysia Terengganu PUBLICATIONS 9 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: ovarian maturation of mud spiny lobster View project All content following this page was uploaded by Taufik Hayimad on 29 March 2017 The user has requested enhancement of the downloaded file This article was downloaded by: [Vikrant Vedamanikam] On: 04 February 2014, At: 17:38 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Toxicological & Environmental Chemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gtec20 Effect of mixtures of metals on the spotted Babylon snail (Babylonia areolata) under different temperature conditions a V.J Vedamanikam & T Hayimad a a Institute of Oceanography, University Malaysia Terengganu, Kuala Terengganu, Malaysia Accepted author version posted online: 08 Jan 2014.Published online: 31 Jan 2014 To cite this article: V.J Vedamanikam & T Hayimad , Toxicological & Environmental Chemistry (2014): Effect of mixtures of metals on the spotted Babylon snail (Babylonia areolata) under different temperature conditions, Toxicological & Environmental Chemistry, DOI: 10.1080/02772248.2014.881077 To link to this article: http://dx.doi.org/10.1080/02772248.2014.881077 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the 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is expressly forbidden Terms & Downloaded by [Vikrant Vedamanikam] at 17:38 04 February 2014 Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions Toxicological & Environmental Chemistry, 2014 http://dx.doi.org/10.1080/02772248.2014.881077 Effect of mixtures of metals on the spotted Babylon snail (Babylonia areolata) under different temperature conditions V.J Vedamanikam and T Hayimad* Institute of Oceanography, University Malaysia Terengganu, Kuala Terengganu, Malaysia Downloaded by [Vikrant Vedamanikam] at 17:38 04 February 2014 (Received 19 November 2013; accepted 26 December 2013) A study was conducted on the Babylon snail (Babylonia areolata) to examine the effects of copper (Cu), cadmium (Cd), nickel (Ni), and zinc (Zn) on different life stages of this gastropod Metal toxicity significantly varied according to the life stage of the snail The different LC50 values obtained were 0.51, 5.49, 0.31, and 0.2 ppm for Cu, Zn, Cd, and Ni for the larval stage and 4.98, 15.19, 0.91, and 1.21 ppm at the juvenile stage and 8.54, 17.52, 1.14, and 1.44 ppm in the adult stage Studies were also conducted on the effects of dual metal concentrations and experiments were repeated with temperature as a variable Results demonstrated that metal toxicity values were altered depending on the metals involved in the combination as well as temperature under which the experiment was conducted Keywords: Babylonia areolata; toxicity; heavy metals; dual metal combinations; temperature variation Introduction In the natural environment organisms are subjected to a variety of pollutants sometimes singularly and most to mixtures Toxicity testing of heavy metals both acute and chronic is well established Various marine organisms such as fish, bivalve, shrimp, and gastropod were examined and monitored to determine effects of the heavy metal contamination on these organisms (Chan, 1995; Hashmi, Mustafa, and Tariq 2002; Neuberger-Cywiak, Achituv, and Garcia 2003; Gammon, Turner, and Brown 2009) These studies were carried out predominantly with a single metal in each experiment In reality, organisms in nature undergo exposure to the effect of the metal mixtures which cannot be predicted from the effects of individual toxicants (Utgikar et al 2004) The toxic effects of metal mixtures were studied in various organisms and different combination of metals showed different adverse effects The mixture of cobalt–cadmium, cadmium–zinc, cadmium–lead and copper–lead displayed antagonistic effects, while cobalt–copper and zinc–lead showed synergistic effects in a bactericidal study (Fulladosa, Murat, and Villaescusa 2005) Data indicated that cadmium (Cd) became less toxic when combined with other metals, and lead (Pb) seemed to be less toxic in the presence of Cd Otituluju (2002) reported that zinc (Zn) reduced the adverse effects of Cd and copper (Cu) in a study conducted on the periwinkle, gastropod (Tympanotonus fuscatus) Synergistic effects of Zn with other metals were reported in biting midge (Chironomus plumosus) tested with different metal mixtures (Vedamanikam and Shazilli 2009) It was found that the toxicity of Zn was increased when combined with chromium (Cr), nickel (Ni), mercury (Hg), and Cd (Vedamanikam and Shazilli 2009) This study reported further that *Corresponding author Email: thedoctoroftime@gmail.com Ó 2014 Taylor & Francis V.J Vedamanikam and T Hayimad Downloaded by [Vikrant Vedamanikam] at 17:38 04 February 2014 silver, Pb, Cu, and manganese reduced the toxicity of Zn (antagonistic effects) Thus, the effects of metal mixtures need to be studied to better understand how an organism responds to a variety of metals Methodology To study the effects of different metals on Babylonia areolata, two definitive bioassays were conducted The first bioassay was a 96-hr median lethal concentration test to determine the 96-hr LC50 values for the different metals selected The second bioassay again consisted of a 96-hr LC50, but in this case, temperature was a variable and tests were conducted at different fixed temperature values Four metal salts were selected for this investigation – zinc chloride, nickel (II) chloride, copper (II) chloride, and cadmium (II) chloride All metal salts were of analytical grade supplied by MERCK, Germany Stock solutions of 1000 mg/L were prepared for each metal A range-finding experiment was conducted to determine the appropriate concentrations of the four different metals for further toxicity studies on adult B areolata All tests were conducted with three replicates and controls Test containers with volumes of 600 ml were selected for execution of different bioassays Each chamber was filled with 400 ml filtered sea water To each test container, a single adult was added, until each chamber had 10 adults, each adult randomly selected to prevent any bias For each metal, six test containers were allocated of which five were for metal concentrations and one a control; each test had three replicates Water quality parameters (temperature, salinity, pH, and dissolved oxygen) were monitored throughout the 96-hr period At termination of each test, mortality data were compiled and 96-hr LC50 values calculated using the trimmed Spearman–Karber toxicity program (Hamilton, Russo, and Thurston 1977) Animals were considered dead when all movement ceased and organisms exhibited nil response to gentle stimulation This was carried out by gently touching the organism with a glass rod and observing the effect of stimulation on the organism The concentrations tested in for adult B areolata for different metals are presented in Table Concentrations listed are the nominal values and measured values The concentrations were measured with a SpectrAA 220 Fast Sequential Atomic Absorption Spectrophotometer (AAS) (manufactured by Varian - South Queensferry, Edinburgh, Scotland, UK) Prior to running the sample water, a calibration curve was obtained by running different concentrations of the various analytical standards (seven concentrations and a blank) through the AAS Further quality assurance was obtained with the use of the standard reference materials Cd, Cu, Ni, and Zn Standards for AAS (Fluka product codes 51994, 38996, 42242, and 18827, respectively) The metal recovery percentage from the standard was 99.87% for Cd, 98.81% for Cu, 99.24% for Ni, and 99.85% for Zn The effect of temperature on the 96-hr LC50 of the four different metals on B areolata was explored The median lethal toxicity experiments were again conducted under conditions of fixed temperature The temperatures selected were 10, 15, 20, 23, 25, 28, 30, 35, and 40 C The heavy metal concentration range for each metal tested remained the same for the different temperatures The concentrations used in each median lethal toxicity test are provided in Table Temperatures of 10, 15, and 20 C were maintained with the help of a temperature control cabinet, while the higher temperatures were maintained with the help of heating elements (Askol-IP68) Temperature fluctuations were observed to be Ỉ0.5 C For each temperature, a median lethal toxicity test was conducted for each metal The main part of this investigation was carried out in two phases Phase was to obtain baseline data on effects of metal mixtures at a constant temperature of 25 C Toxicological & Environmental Chemistry Table Concentrations of heavy metals used in the 96-hr LC50 experiment Concentrations utilized in the bioassay (mg/L) (measured concentrations are in parenthesis) Metal Downloaded by [Vikrant Vedamanikam] at 17:38 04 February 2014 Zinc Nickel Copper Cadmium 0 0 10.0 (9.0Ỉ 0.5) 15.0 (14.9Ỉ0.1) 25.0 (23.9Ỉ0.5) 5.0 (4.9Ỉ0.1) 15.0 (14.8Ỉ0.1) 20.0 (19.5Ỉ0.3) 30.0 (28.9Ỉ0.5) 10.0 (9.0Ỉ0.2) 20.0 (18.9Ỉ0.3) 25.0 (24.1Ỉ0.5) 35.0 (34.9Ỉ0.02) 15.0 (14.9Ỉ0.1) 25.0 (26.1Ỉ0.5) 30.0 (29.8Ỉ0.1) 40.0 (40.1Ỉ0.02) 20.0 (21.1Ỉ0.4) 30.0 (29.8Ỉ0.1) 35.0 (34.9Ỉ0.02) 45.0 (44.9Ỉ 0.1) 25.0 (24.1Ỉ0.5) Copper was used as the main metal and paired with Cd, Ni, and Zn Variations in LC50 value would show if Cu was becoming more toxic or less toxic with the addition of the second metal The mixtures were tested against three life stages of the spotted Babylon snail, the larva, juvenile, and adult, the concentrations of heavy metals used for each life stage are found in Tables and Methodology used in the combination of metal followed Vedamanikam and Shazilli (2009) method PPMỵPPM In this method, actual test concentrations are used in the mixtures rather than percentage of LC50 values This allows direct comparisons to be made as to whether a combination is more toxic than the individual metal The basic set-up of the experiment is the same as the standard LC50 test with the exception that a second metal is then added The 96-hr LC50 values were calculated based on individual metal concentrations and variation between the new value and the original LC50values, i.e the LC50 values obtained in the aquatic toxicity test versus the LC50 values obtained from the mixture All the tests were monitored at test initiation for water quality – temperature, salinity, dissolved oxygen, and pH (Table 4) Results and discussion The toxicity tests of metal mixtures showed 96-hr LC50 values of each metal in every stage of B areolata life cycle varied with the individual heavy metal With Cu case, 96hr LC50 value for the individual metal in larva, juvenile, and adult stage were 0.51, 4.98, and 8.54 ppm, respectively However, when paired with either Cd, Ni, or Zn, the LC50 Table Nominal concentrations of metal used in the metal mixtures experiment for different life stages of B areolata Treatment Life stage Larval stage Juvenile and adult stage Copper ỵ cadmium 0.10 0.50 1.00 5.00 10.00 15.00 1.00 5.00 10.00 15.00 20.00 25.00 0.01 0.05 0.10 0.50 1.00 5.00 0.10 0.50 1.00 1.50 2.00 2.50 Copper ỵ nickel 0.10 0.50 1.00 5.00 10.00 15.00 1.00 5.00 10.00 15.00 20.00 25.00 0.01 0.05 0.10 0.50 1.00 5.00 0.50 1.00 1.50 2.00 2.50 3.00 Copper ỵ zinc 0.10 0.50 1.00 5.00 10.00 15.00 1.00 5.00 10.00 15.00 20.00 25.00 0.50 1.00 5.00 10.00 15.00 20.00 5.00 10.00 15.00 20.00 25.00 30.00 V.J Vedamanikam and T Hayimad Table 96-hr LC50 values of B areolata in all stages exposed to individual and mixtures of heavy metals LC50 values relative to single metal (ppm) Type of metals Downloaded by [Vikrant Vedamanikam] at 17:38 04 February 2014 Stage Cd Cu Ni Zn 95% lower 95% upper confidence limit confidence limit Larva Single Cu Zn Cd Ni Mixtures Cuỵ Cd Cuỵ Ni Cuỵ Zn Juvenile Single Cu Zn Cd Ni Mixtures Cuỵ Cd Cuỵ Ni Cuỵ Zn 0.51 0.31 0.05 0.50 – 1.01 – 0.70 4.98 – 15.19 – 0.91 – 1.21 – 0.41 4.11 – 6.46 – 7.13 – – – 0.20 – 0.10 – – – – – – 1.22 – – 5.49 – – – – 2.12 – – – – – – 12.84 0.36 4.03 0.19 0.11 0.03/0.35 0.53/0.05 0.43/1.34 3.34 13.44 0.71 1.07 0.23/2.30 4.16/1.08 5.29/11.06 0.71 7.50 0.53 0.34 0.07/0.71 1.95/0.20 1.12/3.36 7.42 17.16 1.17 1.37 0.73/7.33 10.05/1.21 9.60/14.91 Adult Single 8.54 17.52 1.14 1.44 6.75 – – – – – – – 1.60 19.45 – – – – – – – 6.27 15.67 0.81 1.26 0.44/4.39 7.63/1.39 9.78/15.47 11.65 19.58 1.60 1.64 1.04/10.36 12.67/1.84 19.35/24.45 Cu Zn Cd Ni Mixtures Cuỵ Cd Cuỵ Ni Cuỵ Zn 0.67 9.83 13.75 values were 0.5, 1.01, and 0.7 ppm in larva; 4.11, 6.46, and 7.13 ppm in juvenile; and 6.75, 9.83, and 13.75 ppm in adult stage A similar trend was seen for other dual metal combinations Statistical analysis of data showed a significant difference in the LC50 values (f ¼ 0.002) The larval stage was also observed to be the most sensitive life stage of the Babylon snail especially when exposed to dual combinations of Ni ỵ Cu (LC50 Table Water quality parameters tested at 20 and 32 C for copper toxicity tests conducted on B areolata Condition Temperature ( C) Max Min Median Standard deviation 20.05 20.00 20.025 0.035355 Max Min Median Standard deviation 32.00 31.05 31.525 0.67175 Salinity (ppt) Dissolved oxygen (ppm) pH 20 C 36.61 32.50 34.74 1.34 8.23 5.35 6.23 0.88 8.25 7.88 8.05 0.10 32 C 36.66 32.93 34.91 1.19 9.20 3.98 5.53 2.01 7.87 6.66 7.51 0.36 Downloaded by [Vikrant Vedamanikam] at 17:38 04 February 2014 Toxicological & Environmental Chemistry Figure 96-hr LC50 values trend of metal mixture compared with normal temperature in larva (A), juvenile (B), and adult (C) stages 0.1 ppm) and Cd ỵ Cu (LC50 0.05 ppm) Adults were observed to be the least sensitive (Table 3) When temperature was used as a variable it was observed that the 96-hr LC50 values varied, either increasing or decreasing depending upon the case (Figure 1) The results showed that temperature played a role in either raising or lowering the toxicity of the metal Downloaded by [Vikrant Vedamanikam] at 17:38 04 February 2014 V.J Vedamanikam and T Hayimad mixtures At the highest temperature tested (32 C), 96-hr LC50 values could not be calculated for several combinations as thermal toxicity came into effect Dual metal toxicity was been studied by different investigators Otitoluju (2002) examined the effects of Zn and Cu in the periwinkle juvenile, Tympanotonus fuscatus, observing antagonistic effects of Cu in combination, a similar reaction as seen in this experiment Similar results were observed by others (Fulladosa, Murat, and Villaescusa 2005; Utgikar et al 2004) The synergistic and antagonistic effects noted in this study may be due to Cu enhancing the absorption of other metals or in the case of Zn decreasing absorption of other metals as reported by Xu et al (2011) The present study demonstrated that non-essential heavy metals are more toxic to the snail than essential metals in all experiments within this study Nott and Langston (1993) suggested that marine gastropod (Littoriana littorea) might detoxify essential metal such as Zn via feces Copper plays an important role in many enzyme systems of gastropods, especially for hemocyanin loading While Cd, which is the most toxic metal in the present study, was found to disrupt respiration, feeding, and activities level of these organisms Cadmium is also known to induce a shift from aerobic to anaerobic metabolic pathway (Moolman, Van Vuren, and Wepener, 2007) Data thus indicate that metal mixtures exert adverse effects on the snail but due to the variability in responsiveness it is not possible to utilize this model as a biomonitor for effects in other organisms Acknowledgments The authors would like to acknowledge the Fundamental Research Grants System (FRGS 59180) for funding this project References Chan, K.M 1995 “Metallothionein: Potential Biomarker for Monitoring Heavy Metal Pollution in Fish Around Hong Kong.” Marine Pollution Bulletin 31: 411–415 Fulladosa, E., J.C Murat, and I Villaescusa 2005 “Study on the Toxicity of Binary Equitoxic Mixture of Metals Using the Luminescent Bacteria, Vibrio fischeri as a Biological Target.” Chemosphere 58: 551–557 Gammon, M., A Turner, and M Brown 2009 “Accumulation of Cu and Zn in Discarded Antifouling Paint Particles by the Marine Gastropod, Litterina Littorea.” Estuarine, 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stats ... http://dx.doi.org/10.1080/02772248.2014.881077 Effect of mixtures of metals on the spotted Babylon snail (Babylonia areolata) under different temperature conditions V.J Vedamanikam and T Hayimad* Institute of Oceanography, University... toxicity of Zn (antagonistic effects) Thus, the effects of metal mixtures need to be studied to better understand how an organism responds to a variety of metals Methodology To study the effects of. .. for Zn The effect of temperature on the 96-hr LC50 of the four different metals on B areolata was explored The median lethal toxicity experiments were again conducted under conditions of fixed