Bentos, CA

Gómez, Sergio E.

Villar, Carlos Alberto

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Bull. Environ. Contam. Toxicol. (2000) 65:465-469 1 Environmental© 2000 Springer-Verlag New York Inc. ContaminationDOI: 10.1007/s001280000147 and ToxicologyLethal Concentration of Cu in the Neotropical FishCnesterodon decemmaculatus (Pisces, Cyprinodontiformes)C. A. Villar,' S. E. Gomez,' ' 2 C. A. Bentos2' Limnology Institute "Dr. R. A. Ringuelet" (UNLP-CONICET), C. C. 712 (1900),La Plata, Argentina2 Argentine Museum of Natural Sciences "Bernardino Rivadavia," Av. Angel Gallardo470 (1405), Buenos Aires, ArgentinaReceived: 22 October 1999/Accepted: 19 June 2000Copper is a classic limiting factor of fishes, as it is both essential and toxic. Insmall amounts the element is a vital micronutrient needed for haemoglobinsynthesis and a major component of cytochrome oxidase. Indeed, most fishes willselectively accumulate copper from surrounding water independent of actualconcentrations. As concentrations exceed metabolic requirements, however,copper becomes injurious to fishes, and ultimately reaches lethal levels ifconcentrations continue to increase.Most research on fish toxicity was carried out on the holartic fauna, while metaltoxicity on neotropical fish remains mainly unreported. Cnesterodondecemmaculatus, a widely distributed endemic small fish of neotropical America,which fulfils requested conditions for toxicity tests, has been used in water qualityassays (De la Torre et al. 1997; Garcia et al. 1998), in Zn toxicity tests (Gomez etal. 1998), and to evaluate its resistance to changes in water pH (Gomez andToresani 1998). The present contribution assesses Cu lethal concentration (LC50)on C. decemmaculatus by means of static acute bioassays in natural water.Obtained results will be used to stablish sublethal concentrations for chronicbioassays.MATERIALS AND METHODSAcute static bioassays were performed following Ward and Parrish (1982) andSprague (1990). Lethal tests were conducted using 150 adults of C.decemmaculatus (not sexed) captured in a small lake without any point sourcecontamination. Fishes were acclimated in the laboratory with test water (24-26°C,pH of 7.2-8.0) for four weeks prior to experimental use. Composition of the waterused in the bioassays (Table 1) resembles mean water characteristics of naturalenvironments populated by C. decemmaculatus.During acclimation animals were fed with a daily ration of ca. 3% body weight ofcommercial fish food Tetra Werke®. The fish were split up into seven groups.The effect of CuSO45H2O on fish was tested in 2 L Pyrex glass chambers atcontrolled temperature, with natural light and artificial aeration. The test wasCorrespondence to: C. A. Villar465Table 1. Physical and chemical composition of the test water.Cond pH DOC Ca + Mg2 Na+ K+ Cl" HCO3 SO4µS cm' mg L - 'Test water 312 7.7 1.3 21.0 3.7 29.5 3.9 25.0 59.5 48.6performed with one control chamber and 7 different Cu doses: 600, 300, 100, 50,30, 5 and 1 µg L- ' nominal concentrations. Each chamber contained a group of 10specimens. Bioassays with 50 and 30 µg Cu L"' were tested in 20 L chamberscontaining 60 and 40 specimens, respectively, as they were part of a chronic Cuexposure experience. Cu concentration at each chamber was attained by pipettingfrom a stock solution of 1 g Cu L" I . Total Cu concentrations were measured at thebegining and at the end of the bioassays. Final Cu concentrations were roughly5.5-8.9 % lower. Average total Cu concentrations were 469, 251, 110, 49, 30, 3and <2 jig L"', respectively. Statistical analysis was performed on the averagemeasured concentrations. The number of fish alive was registered twice a dayduring 96 hr. All the fishes were measured and weighed at the end of thebioassays. Average fish load at each chamber was 0.48 g L"'. During theexperiment fish were not fed.The median lethal concentration (LC50) at 24, 48, 72 and 96 hr were calculatedusing the LC50 Calculation Program (Harrass 1986). This program calculates anLC50 estimate with confidence limits based on data from toxicity tests using threemethods: Probit analysis, Moving Average, and trimmed Spearman-Karber. LC50concentrations obtained by Probit analysis were related with time by means of anasymptotic function to obtain an estimate of LC50 at infinite time. The bioassaywith Cu concentration below 2 µg L"' was asigned a value of 1 µg L"' for thestatistical analysis.Water pH (Orion 250 pH meter), conductivity (Luftman conductance meter), andHCO3" ((iran titration), Na+ and K+ (flame photometry), Cl" (silver nitratetitration), SO42" (turbidimetry), Cat and Mgt (flame atomic absorption)concentrations were determined at the beginning of the test (APHA 1998).Dissolved organic carbon (DOC) was determined after GOLTERMAN et al. (1978).Cu concentrations were assessed by flame atomic absorption (Buck 200A)following Bettinelli et al. (1989). Samples with Cu concentrations lower than 40µg L"' were concentrated in a cation exchange resin (Amberlite IRC50), and lattereluted back with 2M11NO3. The limit of detection for Cu was 2 µg L"'.RESULTS AND DISCUSSIONTable 2 shows the measured Cu concentrations in water and the % mortality atdifferent times. No mortality was observed at the control, 3 and <2 µg Cu L"'doses. Table 3 shows the LC50 values as a function of exposure time.The LC50 decreased exponentially with time (p<0.05), towards an asymptotic466value attained at about 96 hr, as is normally observed (Ward and Parrish 1982;Sprague 1990). Mean LC50 (96hr) obtained by the three methods was 155 .tg CuL" 1 . Figures obtained by Probit analysis were related with time (t in hr) leading tothe following equation:LC50 =101.6 + 5309 . f 1 R2 =0.97resulting in an estimated LC50(.) of 102 µg L -1 (Fig. 1) .Table 2. Average Cu concentrations (Cu in µg L-'), water temperature (T in °C),number (N), average standard length (STL in mm) and average weight (Win g,range between brackets) of fish and percent mortality (M in %) at different times(hr) in each Cu dose assayed.Cu T N STL W M(24hr) M(48hr) M(72hr) M(96hr)469 24.2-27.0 10 20.6 0.4 (0.36-0.42) 80 80 100 100251 24.0-26.3 10 18.8 0.1 (0.06-0.14) 20 50 50 50110 24.2-27.0 10 19.2 0.1 (0.06-0.12) 20 30 50 5049 24.5-26.0 60 16.3 0.07 (0.05-0.13) 1.7 1.7 5 6.730 24.3-26.9 40 15.0 0.05 (0.04-0.07) 2.5 2.5 2.5 2.53 24.5-25.5 10 20.2 0.13 (0.10-0.15) 0 0 0 0<2 24.5-25.5 10 19.8 0.12 (0.09-0.14) 0 0 0 0During the first hours of exposition above 100 µg Cu L' 1 , fish increased the use ofaquatic surface respiration, showing evidence of the afection of the respiratorysystem. High doses of Cu cause death by the combination of hypoxemia due toimpaired oxygen uptake by the gills, and dysfunctions in ionorregulation (Heath1995). After this initial phase, fish activity returned to normality.Table 3. Median lethal concentration (LC50, µg L'') at different exposure time,calculated by three different methods. In each case lower and upper confidencelimits are indicated (LL and UL).Time (hr) 24 48 72 96ProbitLC50 318 231 160 157LL 209 165 121 118UL 659 390 242 238Moving averageLC50 342 219 154 151LL 214 155 122 119UL 1296 365 214 210Spearman-KarberLC50 139 96 157 156LL 98 62 103 102UL 198 148 241 2404674000C)J 200I0 24 48 72 96Time (hr)Figure 1. LC50 values calculated by Probit analysis (dots) and confidence limits(vertical bars) plotted as a function of exposure time (hr). Continuous linerepresent the fitted asymptotic function. Dotted line represent asymptotic value ofLC50(oc).Because Cu toxicity in water is influenced by water hardness, alkalinity, pI, anddissolved organic matter, and experiments on fish toxicity are carried out at awide range of different experimental conditions, caution should be taken whencomparing LC50 data from the literature. LC50s of C. decemmaculatus resemblerather low when compared, at similar experimental conditions, to fishes oftenutilised in toxicity tests. In the upper range of the literature data, fingerlings ofIctalurus punctatus (catfish) showed a LC50 96 hr of 730 µg Cu L"' (Straus andTucker 1993), and adults of the same specie a LC50 48 hr of 28 mg Cu L -1(Stouthart et al. 1996), while juveniles of Tilapia nilotica showed LC50 72 hr of58.3 mg Cu L" 1 (Somsiri 1982). In the lower range of the literature data,Noemacheilus rupicola showed a LC50 96 hr of 230 µg Cu L"1 (Joshi andPrakash-Semwal 1990), and Acrossocheilus paradoxus a LC50 96hr of 26 µg CuL"', being the sensitivity of the last specie similar to that of Salmo sp. (Chen andYuan 1994).Gomez et al. (1998) observed that C. decemmaculatus showed a comparativelylarge resistance to zinc injury, being the LC50 96 hr of 87.8 mg Zn L 4 , andattributed it to the remarkable euritopic characteristic of this fish. Nevertheless,the apparent resistance to zinc injury is not shared with Cu toxicity. Present resultsshow that Cu is much more toxic to C. decemmaculatus than Zn, in coincidencewith previous works with other fish species (Chen and Yuan 1994).468Acknowledgements. This work was supported by grants from IFS, CONICET andPablo Cassari Foundation. Thanks to R Ferriz for technical assistance and usefulcomments, and to C Bonetto for the critical review of the manuscript.REFERENCESAPHA (1998) Standard Methods for the examination of water and wastewater.Amer Publ Health Assoc, WashingtonBettinelli M, Baroni U, Pastorelli N (1989) Determinazione diretta di elementi intraccia in matrici ambientali mediante imprego dell' ETA-AAS Zeeman emidificazione di matrice. In: Minoia C, Caroli S (ed) Applicazionidell'ETA-AAS Zeeman nel laboratorio chimico e tossicologico, vol. 1Eddizioni Libreria Cortina, Padova, p 49-81Chen H, YuanY (1994) Acute toxicity of copper, cadmium and zinc to freshwaterfish Acrossocheilus paradoxus. Acta Zool Taiwan 5:45-60De la Torre FR, Demichelis SO, Ferrari L, Salibian A (1997) Toxicity ofReconquista River water: Bioassays with juvenile Cnesterodondecemmaculatus. Bull Environ Contain Toxicol 58:558-565Garcia ME, Demichelis SO, de la Torre FR, Ferrari L (1998) Freshwater toxicityto Cnesterodon sp: bioassays with water from the Reconquista River. VerhIntemat Verein Limnol 26:1216-1218Golterman H, Clymo R, Ohnstad M (1978) Methods for the physical and chemicalexamination of freshwaters. Blackwell Sci Pub, OxfordG6mez SE, Toresani NI (1998) Nivel minimo letal de pH en Cnesterodondecemmaculatus (Jenyns, 1842), (Pisces, Atheriniformes). Rev Mus Arg CsNat `Bernardino Rivadavia", Hydrobiologia 7:63-67G6mez SE, Villar CA, Bonetto C (1998) Zinc toxicity in the fish Cnesterodondecemmaculatus in the Parana. River and Rio de la Plata Estuary. EnvirPollut 99:159-165Harrass M (1986) LC50 calculation program. version 2.0Heath AG (1995) Water pollution and fish physiology. Lewis Publishers, NewYorkJoshi SN, Prakash-Semwal V (1990) Toxicity of zinc sulphate and coppersulphate to a hill-stream cobitid fish Noemacheilus rupicola Indian J Ecol17:148-150Somsiri C (1982) Acute toxicity of mercury, copper and zinc to the Nile tilapia.Thai Fish Gaz 35:313-318Sprague JB (1990) Aquatic toxicology. In: Schreck CB, Moyle PB (ed) Methodsfor fish biology, vol 15. American Fisheries Society, Maryland p 491-528Stouthart XJ, Haans JLM, Lock RAC, Bonga SEW (1996) Effects of water pH oncopper toxicity to early life stages of the common carp (Cyprinus carpio).Environ Toxicol Chem 5:376-383Straus DL, Tucker, CS (1993) Acute toxicity of copper sulphate and chelatedcopper to channel catfish Ictalurus punctatus. J World Aquacult Soc24:390-395Ward GS, Parrish PR (1982) Manual de m6todos de investigaci6n del medioambiente acuitico. Parte 6. Ensayos de toxicidad. FAO, Doc T8c Pesca 185469

Lethal Concentration of Cu in the Neotropical Fish Cnesterodon Decemmaculatus (Pisces, Cyprinodontiformes)

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