Ecotoxicity in the Reconquista River, Province of Buenos Aires, Argentina:A Preliminary StudyJorge Herkovits, Cristina Silvia Perez-Coil, and Francisco Damian HerkovitsPrograma de Seguridad Quimica, Instituto de Ciencias Ambientales y Salud, Fundaci6n PROSAMA, Buenos Aires, Argentina

The Reconquista River in Argentina i considered a cri al' river basin due to environ-mental degradtioWith itvlly of L547 u. them ateinre tan3 mioninh.0 tants

and 12,000industries. Using early-life-stage~~~~~~~~~~~. ests wit .f m..ru.. emryos.(thmost e of the nat )w dermined th water qualit at si sapling sons

river, theoxicitywas er than the alloable level ofwole dstnal effluent toxicity rcm-miended U.S. EPA. In a tiuaystrem, rry MoMa, te waer wa aout) times more

...P~~~~~~~~~~~~~~~~~..+t. ~~~~~~~~~~~~~. .. .. ..A

s wre°ff adip a of e heswobevd u m points ot tha ampibia eal-ftg txicity tsscould be appropriate

for 'gwa*sr~~~~~EcnAniaes and wae0ult~w* tesnilfrd timpr ,oreto

o'.nvironmental services, monitoring. and restoration purposes. IC-ry rw~ds;amphibia.., au. ic

w~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~o ifiE

toxicity1~ ;rgentina, B# wearswrs, early-life stage toxicity test~ rivst', taterqlty Eva ron

HakPerse- -0s8-g (1996)..

Most developing countries face the enor-mous challenge of improving living stan-dards against a backdrop of serious environ-mental and health problems. For instance,because streams and rivers have been consid-ered a convenient means of deaning and car-rying wastes away from the discharge points,most rivers in urban areas are highly loadedwith urban and industrial wastes (1,2).According to the Pan American HealthOrganization, less than 10% of the munici-palities in Latin America treat sewage ade-quately before emptying it into naturalwatercourses, and wastewater treatmentplants and sewers for industrial effluents areoften not working or nonexistent (3). Inaddition, pollution prevention, recycling,and chemical safety programs are scarce ornonexistent. As a result of these practices,South America pollutes nearly 11 times morefreshwater on a per capita basis than Europe(3). The untreated wastewater dumped intothe rivers in many cases becomes part of thewater sources downstream.

The Reconquista River Valley is locatedin the northern area of metropolitanBuenos Aires. It extends 1.547 km2 and hasmore than 3 million inhabitants. There arealso about 12,000 industries located in thearea, and therefore the major cause of pollu-tion is the high load of urban and industrialwastes poured into the river. The river usedto be dark-colored and bad smelling alongmost of its extension; it is well known thatmacroorganisms are alive only upstream. Itsflow varies between 69,000 and 1,700,000m3/day in cases of heavy rain (4-6).

Traditionally, the quality of liquideffluent discharges entering head watersand the river water itself has been con-trolled by comparing physical and chemicalparameters with reference parameters andpriority pollutant lists. But such an analyti-cal approach was recognized as unable toprotect the aquatic environment againsthazardous discharges (2). Regulatoryauthorities worldwide are showing increas-ing interest in using laboratory toxicity teststo assess the quality of river waters. Forinstance, the Organization for EconomicCooperation and Development recom-mended more than 10 years ago the adop-tion of toxicity testing as one factor in mak-ing decisions about water regulations (7,8).

Amphibians are useful indicators offreshwater contamination (9-11). Embryosof Bufo arenarum, a South American toad,due to their high sensitivity to environ-mental pollutants (12-15), could be veryuseful for ecotoxicological studies and forassessing the water quality needs of widlife(16). On the other hand, it has been estab-lished that there is a good predictive corre-lation between embryo-larval tests for mea-suring in-stream toxicity and estimatingchronic effects on aquatic biota (17).These studies indicate a good predictivecorrelation between embryo-larval survivaland independent ecological parameters,especially species richness (r = 0.96) anddiversity (r= 0.93).

The pollution of rivers in developingcountries is usually not evaluated from anecotoxicological point of view. The main

purpose of this study was to generate, bymeans of ecotoxicological data on theReconquista River basin, quantitative infor-mation on the water quality that could beused by the authorities as well as the generalpublic. The results of water quality assess-ment at six sampling stations were com-pared to the criteria maximum concentra-tion (CMC) for whole effluent toxicity rec-ommended by the U.S. EPA (17). Based onsusceptibility studies to toxicants in theReconquista River, performed in our labo-ratory with three native species [a fish(Cnesterodon decemmaculatus), a shrimp,collected at Rl, our reference upstreamsampling station, and toad embryos (Bufoarenarum)], this early-life toxicity test wasthe most sensitive and appropriate for eval-uating water quality at all sampling stations.Here we focus on the results obtained withBufo arenarum embryos. In addition, anoverview of the presence of macroorganisms(alive or dead) at the sampling stations isreported.

MethodsWe selected six sampling stations (Fig. 1):reference point upstream close to DiqueRuggero (RI), a reference point down-stream within 100 m of the discharge of theReconquista River into the Lujan River(R5), three sampling stations between RIand R5 (R2, R3, and R4), and one sam-pling station on a stream, Arroyo Moron(AM), about 300 m from its discharge intothe Reconquista River. All sampling sta-tions were located far away from industrialor municipal discharge points. Surfacewater was collected at each site near theshore in June 1994. We used a manualpump to collect 20 1 of water at a depth of0.5 m, transported the water to the labora-tory, and refrigerated it at 40C for process-ing. At each sampling station, the presence

Address correspondence to J. Herkovits, Instituto deCiencias Ambientales y Salud, Nicasio Orofio 710,1405 Buenos Aires, Argentina.The standardization of the toxicity test with Bufoarenarum embryos was performed with a grant fromthe Pan American Health Organization. This studyreceived support from the National Council ofScience and Technology (CONICET). Weacknowledge Marcelo Merino and Miguel AngelGomez Peral for their help during the sampling. Wethank Daniela Fuentes for secretarial assistance.Received 4 August 1995; accepted 20 November1995.

Volume 104, Number2, February 1996 * Environmental Health Perspectives186

Articles - Ecotoxicitv in Reconquista River, Argentina

Figure 1. Geographic localization of the Reconquista River basin showing the position of the sampling sta-tions (Rl, R2, AM, R3, R4, and R5).

of macroorganisms was recorded by captur-ing them with a net (diameter of the hole:0.2 mm) and by direct observation.

Ovulation of Bufo arenarum femaleswas induced by injecting a suspension of ahomologous hypophysis. The oocytes werefertilized in vitro with a sperm suspensionin 10% Holtfreter's solution (HS).Embryos obtained from three couples ofparents were maintained in HS at 20 ±2°C. Experiments were performed withembryos at the following developmentalstages: stage 13 (gastrulae), stage 18 (neu-romuscular activity), and stage 25 (com-plete operculum) (18). Starting at thesestages, we conducted 7-day embryo-larvalsurvival and growth renewal toxicity testusing duplicate batches of 10 embryosplaced in 10-cm glass petri dishes contain-ing 40 ml of water from each sampling sta-tion. In the case of embryos from the stage13 and 18, in addition to survival, werecorded malformations and anomaliesobserved using a binocular estereoscopicmicroscope. To obtain additional informa-tion on the water quality in samples withless toxic effects, the toxicity test performedwith embryos at stage 25 was extended upto 14 days. For each sample, eight dilutions

ranging between 80% and 30% were pre-pared using HS, and toxicity tests withembryos were conducted with these con-centrations simultaneously with the undi-luted samples and with control embryosmaintained in HS solution. For dilutions,we used HS, rather than water from thereference sampling point, in accordancewith the procedures of FETAX (19), thebest-known standard method for develop-mental toxicity tests with amphibians.

Conductivity was measured with aLuftman C400 conductivity meter, andpH was measured with a Luftman P300pH meter. These measurements providedinformation on the water quality neededfor the control solution in the toxicity test.Intralaboratory quality control methods forthe toxicity test based on reference toxi-cants were performed with cadmium, andthe minimum criteria of test acceptabilityincluding the prercent coefficient of varia-tion was conducted.

Results and DiscussionThe biological integrity of the ReconquistaRiver was evaluated by the presence ofmacroorganisms (alive or dead) at each sam-pling station. At the upstream reference

sample station (RI), a large number ofshrimp were collected, while at R2, manydead fish, an average of 10 within 10 m ofthe shore, were counted. No living macroor-ganisms were observed from this samplingstation downstream. These observationsindicate the high degradation of the waterquality in this ecosystem. There have beensome previous chemical, physical, and bio-logical integrity studies on the ReconquistaRiver which also indicated the poor waterquality of this river. For instance, the river issubstantially anaerobic, with a mean of bio-logical oxygen demand of approximately 60mg/I, varying between 30 mg/l and 100mg/l (5). In addition, some data on ammo-nia, hardness, pH, cations, and pollutantslike heavy metals and pesticides (5,6,20-22),as well as a partial inventory of the biota, areavailable (23).

Although most EPA chronic toxicitytests have been shortened to 7 days byfocusing on the most sensitive life-cyclestages (17), because our study is focused onsurface water toxicity and based on ourprevious experience (24), we extended theobservations the stage 25 embryos up to 14days. Due to the very poor water quality inthe Reconquista River, the 7-day toxicitytest was sufficient for most of the purposesof this study. In Figure 2, the profile ofexposure-toxicity of the water from the sixsampling stations is shown. The concentra-tion-exposure-response results obtained forAM, which had the worst water quality ofthe sampling stations, is shown in Figure 3.These results illustrate the high sensitivityof the amphibian embryo to the toxicity ofthe river water. It is noteworthy that in aprevious study conducted in our laboratoryinvolving the early-life stage Bufoarenarum, we found that, in the case of cer-tain leachates, up to 0.1% of a dilution canbe detected (24).

Because toxicity involves an inverse rela-tionship to effective concentration (EC; thelower the EC the higher the toxicity), it ishelpful to translate concentration-based tox-icity measurements into toxic units. Thenumber of toxic units was defined as 100divided by the EC measured: in the case ofacute toxicity (TUa) = 100/LC50. for chron-ic toxicity (TUe) = 100/no-observed effectlevel (17). For whole (industrial) effluent,EPA recommended a maximal value of 0.3TUa and 1.0 TUc for the most sensitive ofat least three test species in both cases.These values were used as references for allthe sampling stations studied. The toxicityof the water from the Reconquista Riverbasin on Bufo arenarum embryos (stage 25),expressed as TUa and TUc, is presented inFigures 4 and 5. These data demonstratethat the water sampled was about 10 times

Environmental Health Perspectives * Volume 104, Number 2, February 1996 187

more toxic than the maximal value allowedby the EPA for industrial effluents (17).

90 ........The toxicity of water from the different80 | lligllgilgsamplingstations was compared to the "tox-

icity" of that from a reference sampling sta-70 || | gag w mxg g tion, RI, using the short-term chronic toxi-

60 ~~~~~~~~~~~~~~~~~~~~~~citytest (7 days) and was equal to the values6 RI reported in Figure 5. When the exposure50 AM ' ~~ period is extended, the sensitivity of the test

so40Ji 0 |84 increases (Fig. 6), but in any case it is note-40 R5 ntrol 1X_Xg g_gg Xworthy that the profile obtained in extended

30 l o _E [ 1 [ l _tests is similar to that obtained based on theEPA criteria for whole industrial effluents.

20 Although the use of earlier develop-

10 m.ental stages (stages 13 and 18), provides,0afm I so1 WIN_{ -meaningful additional information on the0 teratological potential of the water contam-0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ination and on water quality, the results do

2. Survival of Bufo arenarum embryos maintained in water samples from the Reconquista River not reflect a significantly higher susceptibil-ity than the mortality exerted on Bufo are-narum embryos at stage 25. The evaluationof these earlier stages is by far more detailedand includes a precise record of malforma-

100 tions and other developmental anomalies.90 , The main malformations found were axial

i i M% incurvation, retarded growth, reduced80 - 3 40% body size, microcephaly, and fin anomalies.

70 0~~~~~~~~~~~~s 60% .These are unespecific malformations pro--- AiM 70% [ duced by different xenobiotics (25-27).A* Control At the screening level, hazard assess-

ment of environmental toxicants is often500 based on a few laboratory tests in single

40 ~~~~~~~~~~~~~~~~~~species.Safety factors are applied toaccount for the many uncertainties inher-

30 ent in extrapolating from one species to

20~~~~~~~~~~~~~~~~~another, from acute to chronic exposures,and from single species to ecosystems

10 __ (7,8,28). Our results seem to confirm that_ within single-species protocols, amphibian

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 embryos could be among the most sensitivetO environmental pollutants (13,14,29).

D 3. Concentration-response relationships obtained with Bufo arenarum embryos in the Moron Athough the shrimp we collected inR1n sample. Atog th .hipw olce nR

were rather sensitive to the degradation ofthe water quality in Reconquista River

1.6 j Ntc ..... .iTA (data not shown), this species seems to be_<'_ .* . .,,adapted to the high salinity at the reference

14 7 W~~~~~ ... ....~~~i sampling station (RI) where it was collect-.. .. ....... ... .R2 .-..2is

C.a

le0

C)Sa=

MRV Rl R2 AM R3 R4 RS

Sampling station

reference value Figure 5. Ecotoxicity of Reconquista River basinexpressed in chronic toxicity units. MRV, EPA

Figure 4. Ecotoxicity of Reconquista River basin maximal reference value; AM, Arroyo Mor6nexpressed in acute toxic units. stream.

R1 R2 AM R3 R4 R5

Sampling station

Figure 6. Ecotoxicity of Reconquista River basinexpressed in reference chronic toxicity units. AM,Arroyo Mor6n stream.

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Articles - Herkovits et al.

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Articles * Ecotoxicity in Reconquista River, Argentina

ed and therefore does not survive in lowosmolarity conditions found in the lowerpart of the river (the mixing zone with thewater of Lujan River). It is also noteworthythat the test performed with Cnesterodondecemmaculatus only detected toxicity with-in 7 days for water obtained from AM, thesampling station with highest pollution.The difference in susceptibility betweenfish and amphibian embryos was morethan one order of magnitude. These resultsare in agreement with the high susceptibili-ty of amphibians to xenobiotics(13,14,29,30) support increasing concernsabout the decline of amphibian popula-tions (9,10,31), which seems to be relatedto the water quality needs of wildlife partic-ularly neglected for these species (16).

The primary objective of this study wasto evaluate the possibility of determiningpollution by using a relatively simple bioas-say procedure performed with native speciesand to identify the most appropriate test forwater quality-based toxicants evaluation(control) for the Reconquista River ecosys-tem. By focusing on the most sensitive life-cycle stages, the duration of EPA chronictoxicity tests for water quality-based toxi-cants control has been shortened to 7 days(17). Our study points out that although a7-day toxicity test with Bufo arenarumembryos seems to be appropriate for watertoxicity screening purposes, at least for thehigh pollution found at most of the samplingstations, it is worthwhile to extend the test to14 days of exposure to detect toxicity levelsthat could be significant for wildlife. In thisrespect, ecotoxicological studies should becustomized and interpreted carefully to pro-vide meaningful information (32).

River systems play key roles in ecologi-cal interactions, and in the regulation andmaintenance of biodiversity in the land-scape (33). These interactions include theatmosphere and groundwater. Humansocieties rely on functions of ecosystemsthat are essential to the quality of humanlife, including provision of water and food,the decomposition of sewage, and thereplenishment of breatheable air (34).Evaluations of the environmental impact ofhuman activities as well as strategies forriver conservation should be considered amain priority for the environmental andhuman health. The idea that rivers are aconvenient means of cleaning and carryingpollution away from the discharge pointshould be urgently and properly reconsid-ered worldwide in order to protect waterquality, ecosystems, and human health.Our results support the use of toxicity testswith amphibian embryos, as they couldaccurately indicate water quality forwildlife protection purposes.

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