Authors: Mukhtar M, Herrel N, Amerasinghe FP, Ensink J, van der Hoek W, Konradsen F.

Journal: Southeast Asian J Trop Med Public Health. 2003 Mar;34(1):72-80.

International Water Management Institute, Lahore, Pakistan. m.mukhtar@cgiar.org

Abstract

Mosquito breeding within the wastewater irrigation system around the town of Haroonabad in the southern Punjab, Pakistan, was studied from July to September 2000 as part of a wider study of the costs and benefits of wastewater use in agriculture. The objective of this study was to assess the vector-borne human disease risks associated with mosquito species utilizing wastewater for breeding. Mosquito larvae were collected on a fortnightly basis from components of the wastewater disposal system and irrigated sites. In total, 133 samples were collected, about equally divided between agricultural sites and the wastewater disposal system. Overall, 17.3% of the samples were positive for Anopheles, 12.0% for Culex and 15.0% for Aedes. Four anopheline species, viz, Anopheles stephensi (84.3% of total anophelines), An. subpictus (11.8%), An. culicifacies (2.0%) and An. pulcherrimus (0.2%) were present, as were two species of Culex, viz, Cx. quinquefasciatus (66.5% of culicines) and Cx. tritaeniorhynchus (20.1%). Aedes were not identified to species level. The occurrence of different species was linked to particular habitats and habitat characteristics such as physical water condition, chemical water quality and the presence of fauna and flora. Anophelines and Aedes mosquitos were mainly collected during the month of July, while Culex were collected in September. The prevalence of established vectors of human diseases such as An. stephensi (malaria), Cx. tritaeniorhynchus (West Nile fever, Japanese encephalitis) and Cx. quinquefasciatus (Bancroftian filariasis, West Nile fever) in the wastewater system indicated that such habitats could contribute to vector-borne disease risks for human communities that are dependent upon wastewater use for their livelihoods. Wastewater disposal and irrigation systems provide a perennial source of water for vector mosquitos in semi-arid countries like Pakistan. Vector mosquitos exploit these sites if alternative breeding sites with better biological, physical, and chemical conditions are not abundant.

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INTRODUCTION

The use of urban wastewater in agricul-ture has become a widespread practice (WHO,1989). Wastewater provides a continuous sup-ply of water with high nutrient content. Thus,it serves as a reliable source of water andfertilizer and is one way to meet the growingdemand for food under conditions of increas-ing water scarcity. In developing countries,including the breadbaskets of China and India,approximately 80% of urban wastewater isused for irrigation (Cooper, 1991). Unfortu-nately, much of this is untreated or inadequatelytreated. Urban wastewater consists of sewageand industrial wastes that pose biological andchemical health risks for the irrigators andcommunities in prolonged contact with theuntreated wastewater, and for consumers ofcrops irrigated with wastewater.Pakistan has a semi-arid to arid climateand is facing severe water-shortage in the contextof a rapidly expanding human population. Thisincreases the scope for wastewater irrigation,which is already practised in almost all cities.Treatment systems have been constructed in some cities of Pakistan, but due to financialconstraints most of them are non-functional(Aftab, 1999).

Recently, periurban waste water irrigation around the city of Haroonabad in South Punjab, Pakistan, was studied with theobjective to describe the advantages and dis-advantages of waste water use in order to cometo a comprehensive cost-benefit analysis. Thework reported herein was a part of that project,and pertains to the potential of wastewater use systems to produce disease-transmitting mos-quitos. Negative health impacts could threatenthe sustainability of urban and periurban agriculture, but have not been adequately described. To the author’s knowledge, there havebeen no previous published studies on mos-quitos breeding in wastewater-irrigated sites in Pakistan.

MATERIALS AND METHODS
Study area
The study was carried out in an agricultural area irrigated with untreated urban waste-water around the city of Haroonabad in the Bahawalnagar district of Punjab Province,Pakistan. The city (population 63,000) is situ-ated at the edge of the Cholistan Desert(72.08оE, 29.9оN) at an altitude of 90 m abovemean sea level. The mean annual maximum and minimum temperatures of the area are46ą2šC and 9ą2šC respectively and the aver-age annual precipitation is 160 mm. The watertable ranged from 1.5-2.5 m below the soilsurface, but ground water was not used forirrigation due to its high salinity. The total area receiving wastewater was 127 ha. The maincrops were vegetables, such as round gourds,cauliflower, brinjal (aubergine) and tomatoes.Other field crops, such as cotton, wheat, sug-arcane and maize were also grown.Wastewater systemThe wastewater disposal system inHaroonabad consisted of a primary disposalsite northeast of the city where wastewater waspumped to fields through field watercourses.A secondary pumping well was located at theend of one field watercourse, 1.2 km from themain pumping site. Here, a mobile pump wasinstalled to shift water to other field water-courses, which were slightly elevated. An olddisposal system that was constructed in 1960but that had been non-functional since 1979,was located in the same area (Fig 1). Thecomponents of this system included grid chambers, sieve chamber and collecting chambers.In the original system, waste water fromHaroonabad City was channeled to the grid chamber to remove large solid wastes, then tothe sieve chamber to remove finer wastes, andfinally to the collecting chambers from which water was pumped onto fields for irrigation.Even at present, sometimes wastewater ispumped into the collecting chambers, particu-larly in the rainy season when there is excesswater standing on fields and when there is alsoa large flow of wastewater from the city. Thus the water in the collecting chambers consistedof a mixture of ground water, rainwater andwastewater. The grid chamber, too, regularly received wastewater by surface run-off fromsurrounding fields, but this water was not usedfor agriculture because of its high salinity.Larval collectionsMosquito larvae were collected on afortnightly basis from July to September 2000,between 8.00 hr and midday, from 10 selected sites within the study area. This period coin-cided with the monsoon season, which isgenerally the peak breeding time for mosqui-tos. For larval collection, all water bodies weredivided into two main categories, ie, (A)agricultural sites, comprising of field watercourses, irrigated fields, pumping wells anddrainage ponds; and (B) the old and non functional wastewater disposal system, includingthe grid chamber, sieve chamber and collectingchambers. Sometimes, water was not present in the field watercourse or the irrigated fieldsduring the harvesting period, in August. Thedrainage pond was sampled only once becauseit was dry during most of the study period.The components of the old disposal systemwere sampled regularly. The surface of eachsampled site was estimated in m2. The samples were collected by using a standard 350 mlaluminum dipper and dipping at the rate of sixdips per m2 of surface area for smaller habitats.For larger ones, a “sample” representing 30dips was taken within a 5m2 area (ie equivalentto 6 dips per m2). For sites with a surface areabetween 5-10 m2, one sample was collected,while for 11-20 m2, two samples were taken,and so on (Herrel et al, 2001). The collectedlarvae were preserved in vials containing 70%isopropyl for later identification. The 3rd and4th instar larvae were identified using the keysof Amerasinghe et al (2001), Glick (1992),Harbach (1988), Rao (1984) and Reuben et al(1994) for the identification of Anopheles andCulex immature stages. Representative sampleswere reared in the laboratory to the adult stagefor the confirmation of species. Other mos-quito groups were only identified up to thegeneric level. Early instars and damaged larvaewere counted but not identified.Habitat characteristics, such as vegetationand fauna, were noted on each sampling oc-casion. Fauna were sub-divided into predatorsand non-predators, with the former categorycomprising fish, water bugs (Diplonychus sp,Hemiptera: Notonectidae), water beetles andwater beetle larvae (Dytiscus sp, Coleoptera:Dytiscidae), damselfly larvae (Agrion sp,Odonata: Agrionidae), dragonfly larvae (Pantalasp, Odonata: Libellulidae), water scorpions(Nepa sp, Hemiptera: Nepidae), water boatmen(Corixa sp, Hemiptera: Corixidae) and back-swimmers (Notonecta sp, Hemiptera: Notonec-tidae). The group of non-predators includedChirinomid larvae, mayfly larvae, water fleas,snails and worms. The substratum was classi-fied as either soil or cement. Water was re-corded as flowing or standing, and its physicalcondition assessed by eye as colored/foul-smell-ing and colorless/foul-smelling. Light condi-tions at sampling sites were recorded as ex-posed, partially shaded or shaded. The chemicalwater quality parameters measured were dis-solved oxygen (DO, in mg/l, Hach DO 175Meter), electro-conductivity (EC, in mS/cm, ;;; yyy ;;;;;; QQQQQQ ;;;; yyyy DPMWWCPWRAWastewater irrigated area.CCSCGCPSPakistan sindhBlochistanPunjabNWFP NHaroonabad Fig 1–CC = Collecting chamber; SC = Seiver chamber; GC = Grid chamber; PW = Pumping well; DP = Drainagepond; PS = Pumping station; MWWC = Main wastewater channels; RA = Residential area.
Hach EC 20 Meter) and pH (Hanna Instru-ments). In-situ measurements were not possiblein the case of collecting chambers and the sievechamber, and readings were made from a freshlycollected water sample in the dipper itself.All water bodies in the study area wereclassified into the following categories:(1) Field watercourse: earthen watercoursethat delivers wastewater to fields. Flowing water.This category also includes pools in the wa-tercourse.(2) Irrigated field: field that has beendeliberately inundated with wastewater for cul-tivation purposes. Includes field pools createdafter a wastewater irrigation turn. Standingwater.(3) Drainage pond: pond to which excesswastewater is diverted and which also receivesordinary irrigation canal water. Surface area:> 10,000 m2. Water stands for a relatively longtime and is relatively clean. Standing water.(4) Grid chamber: deep, non-functionaldisposal structure where wastewater and ground-water accumulate. Covered by thick emergentvegetation. Surface area: 5 m2. Standing water.(5) Seiver chamber: deep and rectangularconcrete basin with grid, part of the non-func-tional disposal works. Surface area: 2 m2.Standing water, with heavy algal cover.(6) Collecting chambers: two round con-crete basins with perpendicular walls, parts ofthe non-functional disposal works. Mixture ofground, rain and wastewater. Surface area: 50m2. Standing water, with heavy algal cover andfloating debris.(7) Pumping well: brick structure and plas-tered walls where water is collected to bepumped to another watercourse. Thick grassesand floating debris. Surface area: 1.5 m2.Standing water.RESULTSOut of 133 collected samples, 53.4% (n= 77) were from agricultural sites and 46.6%(n = 56) were from components of the waste-water disposal system. Most of the sampleswere taken from irrigated fields and collectingchambers, ie, 39.1% (n = 52) and 31.6% (n= 42) respectively, while 9.8% (n = 13) ofsamples came from field watercourses. Thissampling effort represents the availability ofwater at different sites. Overall, 17.3% (n =23) of samples were positive for Anopheles,12.0% (n = 16) for Culex and 15.0% (n = 20)for Aedes mosquitos. Details of species col-lected in different habitats are provided inTable 1. Of the 1,338 anopheline larvae ex-amined, 92.0% (n = 1,222) were found in thecollecting chambers, followed by the pumpingwell, from where 6.1% (n = 81) were col-lected. Anopheles stephensi was the dominantspecies, comprising 84.3% (n = 1,120) of thetotal anophelines; An. subpictus comprised11.8% (n = 156), An. culicifacies 2.0% (n =27), and An. pulcherrimus 0.2% (n = 2). Themain breeding sites for all Anopheles specieswere the collecting chambers, from where 96.9%of An. stephensi, 69.2% of An. subpictus, and94.1 % of An. culicifacies were collected. Thepumping well-produced 1.6% of An. stephensiand 25.6% of An. subpictus. Culex larvaeoccurred predominantly in the pumping wells(96.8%, n = 2,857) and at low levels in thecollecting chambers (2.0%, n = 59) and gridchambers (1.1%, n = 32). Culex quinquefasciatuswas the dominant species, comprising 66.5%(n = 1,964) of the total Culex larvae collected.The other identified species present was Cx.tritaeniorhynchus, which comprised 20.1% (n= 594). In the case of Aedes, 79.2% (n = 909)of a total of 1,148 immatures was collectedfrom the grid chamber, 13.2% (n = 152) fromthe pumping well and 7.1% from the collectingchambers (Table 1).The present study was of short duration,only three months (July-September 2000), butdifferent mosquito groups showed high popu-lation densities during different months. Ofthe total 1,338 anophelines, 88.0% (1,178) werecollected during the month of July. Most ofthe Aedes (65.6%, n = 764) and Culex (75.4%,n = 2,227) were collected during the monthsof July and September, respectively. Similarly,
Table 1Numbers of mosquitos collected in different components of the wastewater system. Agricultural sitesWastewater disposal systemTotalFWCIFDPPWCCSCGCNo. of samples1352664277133Percentage9.839.14.54.531.65.35.3100.0An. stephensi200181,0861311,120An. subpictus0004010844156An. culicifacies00001611027An. pulcherrimus00002002Unidentified00023100033Anopheles sub total200811,2222851,338Cx. tritaeniorhynchus01050855129594Cx. quinquefasciatus0201,9554031,964Unidentified000394000394Culex sub total0302,857591322,952Aedes spp4201528109091,148Grand total6503,3232,665299465,438CC = Collecting chamber; DP = Drainage pond; FWC = Field watercourse; GC = Grid chamber; IF = Irrigatedfield; PW = Pumping well; SC = Seiver chamber. different species showed high population den-sities in different months: for instance, 92.3%(n = 1,078) of An. stephensi and 96.6% (n =1,898) of Cx. quinquefasciatus, were collectedduring the months of July and September,respectively (Table 2).Biophysical conditions in breeding habi-tats are summarized in Table 3. Vegetation waspresent in almost all habitats, but the presenceof predators and non-predators fluctuated widely.Water conditions, too, varied widely betweenhabitats, as did dissolved oxygen (DO) andelectrical conductivity (EC). However, pH wasmore uniform, fluctuating within the alkalinerange. Most anophelines occurred in collectingchambers, which were characterized by stand-ing water with high proportions of predators,non-predators, colorless/foul-smelling water, andrelatively high levels of DO and EC. In con-trast, most of the Culex spp were collectedTable 2Abundance of wastewater-breeding mosquito larvae by month. MonthsSpeciesJulyAugustSeptemberAn. stephensi0.74ą2.21 (n=1,078)0.06ą0.23 (n=23)0.03ą0.09 (n=19)An. subpictus0.05ą0.07 (n=71)0.00ą0.00 (n=0)0.13ą0.62 (n=80)An. culicifacies0.02ą0.15 (n=26)0.01ą0.06 (n=01)0.00ą0.00 (n=00)Cx. tritaeniorhynchus0.87ą4.34 (n=449)0.31ą1.42 (n=111)0.09ą0.55 (n=34)Cx. quinquefasciatus0.01ą0.05 (n=10)0.16ą0.69 (n=56)4.79ą31.76 (n=1,898)Aedes spp0.82ą2.54 (n=764)0.29ą1.12 (n=259)0.34ą1.54 (n=125)Note: The values are mean per dip ą SD, with the number of identified immatures in parentheses.Unidentified first and second instar larvae and damaged immatures were omitted from computation.
from the pumping well. The key characteristicsof this habitat were standing water with amainly colored and foul-smelling condition,lower proportions of predators, and low DOand EC. Most of the Aedes larvae were col-lected from the grid chamber. This standingwater habitat was characterized by the pres-ence of vegetation, predators and non-preda-tors in all samples, a roughly even split be-tween colored and colorless foul water, and thelowest DO and highest EC values recorded inthe study (Table 3).DISCUSSIONThe study showed that Anopheles, Culexand Aedes mosquitos bred in various compo-nents of the wastewater irrigation system atHaroonabad. Species within each of thesethree mosquito genera were found to be as-sociated with specific breeding habitat loca-tions and environmental characteristics.Anophelines were attracted to the collectingchambers, whereas Culex and Aedes wereattracted to the pumping well and grid cham-ber, respectively. The collecting chambers andgrid chamber were part of a non-functional oldwastewater disposal system, while the pump-ing well was a functional part of the waste-water irrigation system. Irrigated fields werealways mosquito-negative. This was probablydue to the percolation of water (within a day)through the porous soil, which resulted in therapid elimination of potential mosquito breed-ing sites. Field watercourses were also nega-tive, probably due to the continuous flow ofwater that would wash away eggs and imma-ture stages.The results of this study provided someinteresting contrasts with previous findings,indicating that An. culicifacies, An. stephensi,An. subpictus and An. pulcherrimus, collec-tively, preferred clear or turbid non-foul breed-ing water (Herrel et al, 2001; Reisen et al,1981; Talibi and Qureshi, 1956; Ansari andNasir 1955; Ansari and Shah, 1950). Under thearid conditions of the study area, it was clearthat foul water habitats were exploited by thesespecies. However, only one species, An.stephensi, appeared to breed prolifically in thesefoul water habitats, with An. subpictus a dis-tant second. The other two species occurredin trivial numbers and the wastewater systemwas apparently a relatively minor componentof their overall breeding habitat palette. Pre-viously, Krishnan (1961) found An. stephensiassociated with polluted water habitats in thewinter season, when there was low rainfall andless irrigation practices in the Punjab. In contrast,our study was carried out during the hot summermonths. It seems that the species can exploitfoul water habitats under both environmentalTable 3Selected physical, chemical and biological characteristics of habitats sampled. HabitatVegetation PredatorsNon-Colorless Coloredpredators & foul & foulDOECpHIrrigated field10048315850.95.07.8Field water course1008698920.94.27.8Drainage pond1000001001.212.38.2Pumping well1005010017830.72.17.3Collecting chamber100987986145.814.38.7Siever chamber8610010014864.98.98.1Grid chamber10010010043570.221.38.9Values for vegetation, fauna and water condition are percentages of occurrence; DO = Dissolved oxygen(mg/l); EC = Electrical conductivity (mS/cm); values for DO, EC, and pH are means.FaunaWater conditionWater quality

extremes. Anopheles stephensi has been con-sidered a malaria vector in parts of Asia,including the Indo-Pakistan subcontinent andthe Persian Gulf (Rao, 1984; Manouchehri etal, 1976). An. stephensi was naturally infestedwith plasmodia in urban Karachi (Rehman andMuttlib, 1967) and also in rural Punjab (Pervezand Shah, 1989). Recent evidence fromSheikhupura, in northern Punjab, suggests thatAn. stephensi may be a more important vectorthan previously believed. It was five timesmore prevalent than An. culicifacies, and P.falciparum malaria cases peaked after An.culicifacies had disappeared, but when An.stephensi was present (Rowland et al, 2000).Three morphological variants of An. stephensiare presently recognized (Sweet and Rao, 1937;Subbarao et al, 1987), and it is still not clearwhich variant(s) are good and poor vectors. Itwould be interesting to investigate whetherbreeding in foul and non-foul water is a variant-dependent behavioral trait.The next most prevalent anopheline, An.subpictus, was mainly recorded from foul waterin collecting chambers and also from thepumping well. This is consistent with previousresearch in the Punjab that showed it to breedin foul and polluted water in septic tanks,street pools and street drains in a rural irrigatedvillage ecosystem (Herrel et al, 2001). Thespecies also has been recorded from extremelysaline, highly silted habitats (Ansari and Nasir,1955), and is generally considered to be ableto tolerate a wide range of physio-chemicalconditions, including a high organic mattercontent (Herrel et al, 2001; Reisen et al, 1981).Anopheles subpictus exists as a complex offour sibling species whose breeding ecologiesare largely unknown, apart from the fact thatsibling B generally breeds in saline water(Suguna et al, 1994). As in the case of An.stephensi, it is not known whether foul waterbreeding in An. subpictus is related to siblingspecies preferences.Anopheles culicifacies occurred infre-quently in the wastewater habitats. Only 27 immatures were collected, and these, too, onlyfrom the collecting chamber and seiver cham-ber. Interestingly, these two habitats had the highest DO values among all the sampledhabitats. Previous research in South Asia(Amerasinghe et al, 1995; Reisen et al, 1981;Russell and Rao, 1942) has shown the breed-ing of this species to be closely linked to waterwith a high DO content. The present resultsconfirm this association, even in breeding waterthat is physically foul-smelling and has gen-erally lower DO levels than unpolluted water.Culex quinque fasciatus and Cx. tritaenio-rhynchus occurred primarily in the pumpingwell, characterized by colored foul water witha low DO content. Both species are pollution-tolerant, and their existence in such habitatsis well documented in the literature. For in-stance, Carlson and Knight (1987) recordedextremely high Cx. quinquefasciatus popula-tions in wastewater treatment ponds in Florida.The WHO (1995) reported that stagnant pol-luted water bodies are also a favored breedinghabitat of Cx. pipiens fatigans and Cx.tritaeniorhynchus. In Pakistan, Reisen et al(1981) and Aslamkhan and Salman (1969)reported that Cx. tritaeniorhynchus was adominant species in pools and ponds as theygrew older and more polluted in the summer;a similar phenomenon occurred with Cx. pipiensfatigans in the winter. Culex tritaeniorhynchusis a vector of Japanese encephalitis in Southand Southeast Asia and Japan, and also a vectorof West Nile virus in Pakistan and India (Peirisand Amerasinghe, 1994). Culex quinquefasciatusis well known to be a major vector of lym-phatic filariasis throughout the tropics, andalso has been incriminated in the transmission of West Nile virus in Pakistan and India (Peirisand Amerasinghe, 1994). Historically, cases ofhuman filariasis have been reported in Paki-stan among repatriated refugees from Bangla-desh, although transmission to indigenousPunjabis remains to be documented (Aslamkhanand Pervez, 1981). West Nile fever is generallya mild febrile infection, but recent outbreaksof a more severe form of West Nile virus, thathave resulted in significant human case fatali-ties in Europe, Pakistan, the Middle East andthe United States (Petersen and Roehrig, 2001),are of concern. Among major zoonotic hostsof the virus are migratory birds that could potentially carry virulent strains into new areassuch as Pakistan. Wastewater systems thatgenerate potential vectors, such as Cx.quinquefasciatus, and Cx. tritaeniorhynchus,and also attract aggregations of birds under thearid climatic conditions of the region, couldincrease the risks of transmission to humans.In our study, Aedes mosquitos were mainlyrecorded from the grid chamber and from thepumping well to some extent. Since speciesidentifications were not done, we cannot com-ment on their potential disease implications.However, there is little doubt that they wouldconstitute a nuisance hazard to humans andlivestock around the wastewater system.One of the limitations of this preliminarystudy was its short duration, which precludedthe recording of seasonal trends in breeding.However we did observe that within the 3-month span of the study, An. stephensi and An.culicifacies were collected mainly during July,An. subpictus in July and September, Cx.tritaeniorhynchus in July-August, and Cx.quinquefasciatus in September. The Aedes sppdecreased from July to September. Previousresearch in Pakistan and the neighboring In-dian Punjab show Cx. tritaeniorhynchus andCx. quinquefasciatus peak around these sametimes, whereas for An. culicifacies, the peakabundance was during September-December,and for An. stephensi no clear seasonal trendwas observed (Ansari and Nasir, 1955;Aslamkhan and Salman, 1969; Reisen, 1978;Reisen and Milby, 1986; Reisen et al, 1976;1982). The extent to which wastewater habitatsare seasonally utilized by mosquitos is an aspectthat needs further investigation. Overall, how-ever, this short study highlighted the fact thatthe wastewater system in Pakistan generatedsubstantial populations of mosquitos, such asAn. stephensi (malaria), Cx. tritaeniorhynchus(West Nile fever, Japanese encephalitis) andCx. quinquefasciatus (Bancroftian filariasis, WestNile fever), thereby contributing to vector-borne disease risks to human communitiesdependent upon wastewater use for a liveli-hood. Amelioration of this mosquito problemcould be achieved by covering the smallernon-functional components of the wastewatersystem so that mosquitos do not have directaccess to the water within, flushing the com-ponents of the functional system frequently towash away larvae, and regular removal offloating debris and marginal and emergentvegetation from the open components of thesystem.ACKNOWLEDGEMENTSThe authors thank Tariq Nazir, AsimMunawar and Tipu Naveed for their dedicatedfield assistance; Najaf Ali Khan for data entryand management; and Dr M Aslamkhan, forproviding literature and useful discussions. Helpfrom Dr M Yousuf, Head of the Departmentof Agricultural Entomology, University ofAgriculture, Faisalabad, Pakistan, for the iden-tification of non-dipterous insect fauna ofwastewater also is highly appreciated. The IWMIstudies on irrigation and health in the southernPunjab are supported by the Canadian Inter-national Development Agency (CIDA).

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