387 Environmental Toxicology and Chemistry, Vol. 27, No. 2, pp. 387–396, 2008  2008 SETAC Printed in the USA 0730-7268/08 $12.00 + .00 EFFECT OF PROLONGED EXPOSITION TO PULP MILL EFFLUENTS ON THE INVASIVE AQUATIC PLANT EGERIA DENSA AND OTHER PRIMARY PRODUCERS: A MESOCOSM APPROACH ALVARO T. PALMA,*† MARCELO G. SILVA,† CARLOS A. MUN ˜ OZ,† CAROLINA CARTES,‡ and FABIA ´ N M. JAKSIC† †Pontiﬁcia Universidad Cato ´lica de Chile and Center for Advanced Studies in Ecology and Biodiversity (CASEB), Alameda 340 Casilla 114-D, Santiago, Chile ‡DSS Ambiente, Ernesto Pinto Lagarrigue 2C, San Pedro de la Paz, Chile ( Received 9 March 2007; Accepted 2 August 2007) Abstract—The recent disappearance of the aquatic plant Egeria densa, a Brazilian native invasive species, from a wetland in southern Chile prompted several efforts to unveil the origin of this phenomenon. Because these changes occurred by the time a newly built pulp mill started its operations in the area, a reasonable doubt for a cause–effect relationship is plausible. We implemented a mesocosm approach to directly evaluate the effect of treated pulp mill efﬂuents (PMEs) on several growth-related parameters of E. densa as well as other primary producers. We hypothesize that efﬂuent, at a dilution similar to that detected in the zone of the wetland where the negative environmental impacts were evident, has a signiﬁcant negative effect on this aquatic plant as well as on other primary producers inside a mesocosm system. After a prolonged (months) exposure to both 0% PME with pure river water and a 4 to 5% (v/v) dilution of treated PME, no effect on E. densa was measured. Furthermore, plants exposed to efﬂuent exhibited a signiﬁcantly greater general growth rate. Coincidently, chlorophyll a concentration in the water column and periphyton biomass also changed over time, but without any pattern attributable to the efﬂuent. Values of the autotrophic index obtained from the periphyton growth pattern did not suggest enrichment of the system with organic matter. Our results only refer to the direct effect of mill efﬂuents on several biotic responses, but they represent an important advance toward generation of the scientiﬁc knowledge necessary to understand how the ecosystem functions while receiving this and other unquantiﬁed sources of water. Keywords—Mesocosm Primary producers Egeria densa Pulp mill INTRODUCTION The increasing need to better understand the ecological effect of human activity on ecosystems has prompted the use of speciﬁcally designed approaches, particularly in aquatic en- vironments, and this has led to the design and implementation of enclosed experimental ecosystems (i.e., mesocosms) as re- search tools [1,2]. Mesocosms have become widely used and accepted tools in experimental ecology, because they provide a means of conducting ecosystem-level experiments under rep- licated, controlled, and repeatable conditions while maintain- ing some environmental realism [3]. Among the human-related agents that affect aquatic ecosystems, both freshwater and ma- rine, those associated with the paper and pulp mill industry are of growing importance. During the past few decades, this activity has experienced a signiﬁcant boost worldwide, and it is expected to increase by 77% from 1995 to 2020 [4]. During the past two decades, signiﬁcant efforts have been imple- mented to reduce the impact of this activity on the receiving environment [5], and advances have been made in assessing the effect of pulp mill efﬂuents (PMEs) on aquatic systems [6]. It remains important, however, to determine unequivocally the direct effect of this industrial activity on local receiving ecosystems. Although difﬁcult, particularly because receiving aquatic systems often are exposed to multiple stressors [7], the ability to understand the relative effect of individual factors represents a step toward improving our ability to make valid, ecologically based predictions and, thus, contribute with reg- * To whom correspondence may be addressed (apalma@bio.puc.cl). Published on the Web 10/02/2007. ulatory guidelines [8,9]. Artiﬁcial stream technologies (here- after referred to as mesocosms) have been used successfully to achieve those goals in river systems [10–12]. The relatively large size and ﬂow-through characteristics of this experimental approach allows the control of exposure conditions while maintaining environmental realism [6]. Continuous ﬂow is considered to be more appropriate than static experiments with no exchange of test water, because in nature, organisms are continuously exposed to renewed solutions. Hence, the use of mesocosms allows the isolation of factors that are hypothesized to have negative effects on identiﬁed ecosystem end points. In 2004 and 2005, important ecosystem-level changes were detected in one of Chile’s most important wetland systems: The Carlos Anwandter Nature Sanctuary in the southern prov- ince of Valdivia. During this period, the sanctuary suffered a process of rapid environmental degradation, including the sud- den, massive die-off and migration of a colony of black-necked swans (Cygnus melancoryphus). This and other bird species in the wetlands rely, in large part, on the aquatic plant Egeria densa, a species originally from Brazil that serves as a major food source. This plant species also experienced a severe die- off, a factor that has been considered to be the main cause for the disappearance of birds in this area. These negative impacts occurred by the time the newly installed pulp mill owned by Celulosa Arauco y Constitucio ´n S.A. (hereafter referred to as CELCO) started its operations some 32 km upstream from the sanctuary. Although directly linking this industrial activity with the above-mentioned ecosystem changes is tempting, our aim in the present study was to understand the speciﬁc effect of the PMEs on E. densa as well as on other primary producers