Use of endoparasites, Ligula intestinalis plerercoid in ...

association in monitoring increasing heavy metalpollution in lacusterine environment. This hostparasiteassemblage was analyzed at different sites toidentify the variable metal exposure in the coastalzone of Lake Victoria.2. Materials and methods2.1 Study area and sampling sitesLake Victoria, the second largest freshwater bodyin the world (area 68,800 km 2 ), is generally shallow(mean depth 40 m) and lies in a catchment of about184,000 km 2 , shared by three riparian states (Kenya,Tanzania and Uganda). Site selection in Kenya wasbased on the anthropogenic activity profiles along thecoastal zones (Fig. 1). Site 1 (Kisumu City) has apopulation of about 1.1 Million and is a centre ofurban development with various industries anddrainage of intense agriculture. Site 2 (Kendu-Bay) isa rural agricultural area without fertilizer inputs. Site 3(Karungu) receives drainage from small gold mines.Site 4 (Port Victoria) is a rural area, receiving waterfrom the River Nzoia, containing effluents from twosugar factories and a paper mill factory situated about100-150 km upstream from the Lake.L. intestinalis involves three hosts, a crustacean(copepod), a fish (e.g. R. argentea) and a bird (e.g.pied Kingfisher or Cormorant). Copepods carryinfective stages of the parasite, which are transferredto the fish R. argentea and subsequently to the fishconsumers in the coastal zone of the Lake Victoria.Accumulated metals are thus transferred via the samefood chain.Fish samples were obtained from local fishermenon two sampling occasions in June 2006 from the sitesS1, S2, S3 and S4 using a beam trawl with 5 mmstretched mesh; fish was attracted in the night bylamps. The catch data are presented in Table 1. Tominimize contamination, all materials used in theexperiment were previously washed in ultra purewater. Fish were weighed (to the nearest 0.1 g),measured (folk length in mm) and dissected.Dissection of the fish was done on the shore usingstainless steel instrument pre-cleaned in doubledistilled water. In parasitized fish, parasites wereremoved using stainless steal instruments, counted,weighed and stored in glass vials and later transferredto Teflon vials and freeze dried, awaiting furtheranalysis. Both parasitized and unparasitized fish werebagged and frozen until processed in the laboratoryusing metal-free techniques in the Netherlands.2.3 Metal enrichmentsWater from Lake Victoria was enriched using Pb,Cd, Cr and Cu. The concentration of the specific metalapplied were 10 times the initial concentration of theLake water in site 1. The parasitized and nonparasitized fish were then transferred to the water andleft in the solution for 96 h.Fig. 1. Map of Lake Victoria basin (Kenya) showing thesampling sites2.2 Fish and endoparasite collectionThe fish host chosen in this study was a cyprinid, R.argentea (Pellegrin, 1904). This fish occurs in a highabundance in Lake Victoria, more than any otherspecies. During the past eight years it has composedbetween 37 – 45% of the commercial fish catch(Wanink, 1999). Due to its relatively low price, itdominates as a source of protein among the poor lakeshore communities, mainly because other larger fishspecies are preferred for trade, including export for thecountry’s foreign exchange earning. The life cycle of2.4 Metal analysisThe frozen samples were thawed, crushed andhomogenized using a Fritsch, Pulverisette 5, planetarymill (Fritsch GmbH Laborgerate, Idar-Oberstein,Germany) for 5 minutes at 400 rpm. About 0.5 g ofsample were accurately weighed in Telflon (© polytetra-fluor-etheen(PTFE), DuPont) high pressurevessels. Then 4.0 ml concentrated nitric acid (65%),1.0 ml concentrated hydrochloric acid (37%) and 1.0ml ultra pure water was added to the samples. Sixsamples were placed in the carousel of a PaarMicrowave oven (Anton Paar GmbH – Graz –Austria).Digestion took place at 200°C and 70 bar pressureduring 15 minutes. After cooling the obtained clearsolutions were quantitatively poured in 50mlvolumetric flasks and diluted to the mark with ultrapure demineralized water. Finally the diluted solutionswere transferred into acid cleaned polyethylene bottles.All elements were determined by means of inductivelycoupled plasma-optical emission spectrometry (PerkinElmer Optima 3000 XL, ICP-OES) using the PEcalibration standards. The concentrations werecalculated as mg kg -1 dry weight. The quality of theanalytical process was controlled by the analysis ofIAEA MA-A-3/TM certified standard referencematerial of shrimp. Measured values deviated lessthan 10% from the certified values.2