The Fate of α-Pinene in Sediments of a WetlandPolluted by Bleached Pulp Mill Effluent: Is It a New Clueon the “Carlos Anwandter” Nature Sanctuary Wetland Case,Valdivia, South of Chile?

Hernán Palma-Fleming & Magay Foitzick &

Ximena Palma-Larrea & Eduardo Quiroz-Reyes

Received: 26 October 2012 /Accepted: 22 August 2013 /Published online: 11 September 2013# Springer Science+Business Media Dordrecht 2013

Abstract The volatile monoterpene α-pinene has beenmeasured in sediments of a selected area at “CarlosAnwandter” Nature Sanctuary, a Ramsar protected wet-land located at the northwest of Valdivia City, south-central Chile. The ecosystem was seriously damaged byan uncontrolled liquid emission of a pulp mill (CELCO-Arauco) located about 15 km upstream of Rio Crucesduring 2004. Exploratory data analysis was applied toanalytical data collected from sediment samples, havingfound α-pinene as a reiterative chemical at relativelyhigh concentrations in some specific areas of the wet-land. The decrease of the total concentration ofα-pinenein the area under study is coincident with a point con-tamination that occurred during 2004 (12,240 ng g−1)showing a decay in 2005 (7,890 ng g−1) and middle of2006 (4,060 ng g−1). The following years, last 2006,2008, and 2009, show a relatively constant concentra-tion with a clear tendency toward baseline levels(2,460–2,640 ng g−1). Since the decrease of concentra-tions of α-pinene in sediments in the period 2004–2009shows an opposite trend as compared to the surface areaincrease of pine and eucalyptus plantation in Region de

Los Rios, and not having found potential sources of α-pinene by anthropogenic activities other than the pulpmill in the area under study, it may be finally concludedthat this compound did not enter the bodies of waterfrom a gradual and natural process; instead there is abase to sustain anthropogenic input. α-Pinene in sedi-ments may be a plausible chemical tracer capable ofdetecting pollution events over time and its impacts inaquatic ecosystems as well as changes in aquatic eco-systems produced by improperly treated pulp mill liquidemissions that use pine and eucalyptus species.

Keywords α-Pinene .Wetlands . Nature sanctuary .

Pulp mill emissions . Sediment contamination

1 Introduction

The toxic effect of bleached pulp mills on the sur-rounding environment is well documented in the liter-ature (McMaster et al. 2003; Environment 2003). Kraftpulp bleaching activities have produced important dis-turbances in aquatic and terrestrial life, where the mainenvironmental problems are emissions into bodies ofwater and the atmosphere, waste management, andenergy consumption (Strömvall and Petersson 1993;Juuti et al. 1990; McDonald et al. 2002; Hynynen et al.2004). Effluents carry out a number of varied naturaland unnatural chemicals like resin acids, phytosterols,fatty acids, terpenes, organochlorines, and a number of

Water Air Soil Pollut (2013) 224:1729DOI 10.1007/s11270-013-1729-4

H. Palma-Fleming (*) :M. Foitzick :X. Palma-Larrea :E. Quiroz-ReyesInstituto de Ciencias Químicas,Universidad Austral de Chile,Campus Isla Teja, Valdivia, Chilee-mail: hernanpalma@uach.cl

E. Quiroz-Reyese-mail: equiroz@uach.cl

uncharacterized compounds that come from raw mate-rials or additives used in the production process, gen-erating associated chronic and acute toxic effects onthe aquatic biota (Munkittrick et al. 1992; Sibley et al.1997, 1998; Lacorte et al. 2003; Hewitt et al. 2003;Oakes et al. 2003; Hynynen et al. 2004; Orrego et al.2009; López et al. 2011). The chemical composition ofliquid residues depends on the type of wood employed,the pulp processing, and cellulose bleaching (Lacorteet al. 2003; Kukkola et al. 2011). Turpentine oil is atoxic by-product of the processing of cellulose pulp,and its greater weight of toxicity comes from α-pinene,which constitutes about 60 % of the composition of theoil (Mercier et al. 2009). It has been reported that α-pinene and β-pinene found in pulp wastewater fromkraft pulp paper industries are toxic to brine shrimp(Artemia salina) (Tambosi et al. 2006). In addition,monoterpenes are potent germination inhibitors andsuppress early root growth (Zunino and Zygadlo2004; Nishida et al. 2005; Singh et al. 2006). Amongmonoterpenes, α-pinene inhibits growth and inducesoxidative stress in roots (Abrahim et al. 2000; Singhet al. 2006), and disrupts energy metabolism by actingas an uncoupler of oxidative phosphorylation andinhibiting the electron transport chain (Abrahim et al.2000, 2003).

The “Carlos Anwandter” Nature Sanctuary of theCruces River of Valdivia, a protected wetland under theRamsar Convention, was remarkably impacted by anunknown contamination during the period March–April 2004 (Fig. 1). The environmental disaster pro-voked massive emigration and death of the black-necked swans (Cygnus melancoryphus) and abruptdisappearance of their main food “luchecillo” (Egeriadensa). The Final Report of the investigationconducted by the Universidad Austral de Chile con-cluded that the main cause of the ecological disasterwas the kraft pulp mill industry “Celulosa Arauco yConstitución” (CELCO-Arauco), located at San Josede la Mariquina, about 15 km to the northeast of thesanctuary (Fig. 1) (CONAMA-UACH 2005; DiMarzio and McInnes 2005; World Wide Fund forNature WWF 2005). Nevertheless, there remain doubtsin relation to the factors that caused E. densa to disap-pear. It has been claimed by Ramirez et al. (2006) thathigh UV-B radiation would be the cause of E. densadecay. However, high level of iron was claimed in theUniversidad Austral Final Report to be the main causeof the ecological disaster. In addition, sulfate at high

concentration was reported by Mulsow and Grandjean(2006) as the main cause of E. densa disappearance. Inthe last case, the proposal is controversial since bothexperimental design and theoretical aspects related tochemical equilibrium have been proposed to be erro-neous by Harding et al. (2007) and Soto-Gamboa et al.(2007).

α-Pinene was detected at significant levels in sedi-ments through exploratory data analysis (EDA)(Morrison 2000; Johnson 2002) from data collectedby the Universidad Austral of Chile investigations onthe Carlos Anwandter Wetland several months after theecosystem was degraded. In this EDA, α-pinene wasindicated as a potential chemical tracer of intentional orunintentional environmental contamination by the pulppaper industry CELCO-Arauco that was using pineand eucalyptus species. α-Pinene concentrations weremeasured in sediments in specific areas of the impactedwetland in the period 2005–2006 and 2008–2009, in-cluding relevant results of α-pinene concentrations insediments collected during 2004 in some stations a fewmonths after the emigration and death of the black-necked swans.

The aim of this study was to determine the temporaland spatial variations of α-pinene concentrations insediments from selected area of the Carlos AnwandterNature Sanctuary of Valdivia, south-central Chile, in theperiod 2004–2009 and to discuss its possible origineither natural or anthropogenic that may have occurredby liquid or air emissions into the wetland, as well as itspossible use as a chemical tracer of contamination by thecellulose pulp processing industry.

2 Materials and Methods

2.1 Study Area

The Carlos Anwandter Nature Sanctuary is a naturalwetland bathed by freshwaters of the Cruces River,Nanihue, Cudico, Pichoy, Cayumapu, and San Ramon.The site covers approximately 4,900 ha and is locatedimmediately to the northwest of the city of Valdivia,south-central Chile (Región de Los Ríos) (39°41′S,73°11′W) (Fig. 1a). It is listed under the RamsarDirectory of Wetlands of International Importance since1981 to protect species likeGallinula melanops (taguita),Fulica armillata (Vieillot) (tagua), Coscoroba coscoroba(Coscoroba swan), Plegadis chihi (white-faced ibis),

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Ardea cocoi (white-necked heron), Lutra provocax(otter), and C. melancoryphus (black-necked swan),among many others. The study area covers samplingstations located at Cruces River and two of the main

tributaries, Nanihue and Cudico, as well as some otherspecific sites located in the wetland and the connectiveestuarine system (Calle-Calle and Valdivia Rivers, andthe Corral Bay) (Fig. 1b).

Fig. 1 a Sampling sites in the Carlos Anwandter NatureSanctuary Wetland located at Valdivia, XIV Region,south-central Chile. b Sampling sites used as reference for

baseline concentrations of α-pinene in sediments of theValdivia Estuarine System during 2004

Water Air Soil Pollut (2013) 224:1729 Page 3 of 11, 1729

2.2 Sampling

Sediment samples were taken in November 2005, June2006, November 2006, December 2008, andSeptember 2009 at the Carlos Anwandter NatureSanctuary in Valdivia, XIV Region. Fifteen samplingstations were selected at Fuerte San Luis (S1) (39°36′18″S, 73°08′55″W), Entrada Canal (S2) (39°37′38″S,73°10′27″W), Santa María (S3) (39°39′59″S, 73°10′44″W), Nanihue 1 (S4) (39°37′14″S, 73°08′13″W),Nanihue 2 (S5) (39°37′29″S, 73°08′38″W), Nanihue3 (S6) (39°38′09″S, 73°08′58″W), Nanihue 4 (S7)(39°38′51″S, 73°09′24″W), Nanihue 5 (S8) (39°39′14″S, 73°09′50″W), Nanihue 6 (S9) (39°39′41″S,73°09′41″W), Confluencia (S10) (39°39′52″S, 73°09′52″W), Cudico 1 (S11) (39°38′41″S, 73°07′38″W),Cudico 2 (S12) (39°39′06″S, 73°08′09″W), Cudico 3(S13) (39°39′08″S, 73°08′35″W), Cudico 4 (S14)(39°39′37″S, 73°09′29″W), and Bajo Rialejo (S15)(39°43′18″S, 73°13′18″W) (Fig. 1a). Triplicate surfacesediment samples were collected at each samplingstation using an Eckman grab, stored at low tempera-ture in a cooler, and then transported to the laboratoryand immediately frozen until analysis (U.S. EPA2001). Sampling sites were selected according to theinformation obtained from the EDA applied to thesamples collected during 2004 and 2005. The highestconcentrations of α-pinene in sediments were ob-served in the Nanihue tributary.

2.3 Analytical Procedure

All solvents used for extraction and cleanup proce-dures were of nanograde quality (Merck). Glass distil-lation was used when solvent quality did not meet therequirement of purity specified by standard operationprocedures. Certified standards of cyclohexanone(Merck), menthol (Aldrich Chemistry), α-pinene, andβ-pinene were used for qualitative and quantitativepurposes (Fluka Analytical).

An HP 6890 PLUS gas chromatograph coupled toan HP 5973 mass selective detector (Hewlett-Packard,Palo Alto, CA, USA) and a Focus gas chromatographcoupled to a DSQ II mass spectrometer (ThermoScientific) were used for α-pinene analysis. The injec-tor temperature was maintained at 280 °C in a pulsed,splitless mode. A GC temperature program ramp from70 °C for 4 min and then at a rate of 10 °C/min up to300 °C was used to afford the best separation of α-

pinene using a capillary HP-5 MS column (Hewlett-Packard, Palo Alto, CA, USA) and a capillary Rtx-5MS (Restek, PA, CA, USA) column, both30 m×0.25 mm ID×0.25 μm film thickness of 5 %diphenyl/95 % dimethyl polysiloxane.

Sediment samples (7–10 g) were previously driedwith anhydrous sodium sulfate and then extracted withhexane as described by the method MSE-3570 (U.S.EPA 2002). The hexane extracts were rota-evaporatedto reduce the volume to approximately 4 mL, and thentiny copper foils were added to eliminate sulfur inter-ference overnight. The hexane extracts were then con-centrated to 0.5–1.0-mL volume under nitrogen flux,and menthol or cyclohexanone was added as internalstandards for quantitative analysis and recovery calcu-lations. Calculated limit of detection was 20 ng g−1.

Results were validated by applying the internal stan-dard addition method in real samples and analyticalblanks. Analytical recoveries were between 75 and 110%.

2.4 Statistical Analysis

Data analysis was performed for temporal and spatialcomparisons. Tukey’s test for multiple comparisons aswell the Kruskal–Wallis test for a one-tailed ANOVAon ranges was applied.

3 Results

Data collected from sediment sample analysis duringthe period 2005–2009 and results of some relevantsampling stations during 2004 are also included inTable 1. The total concentration of α-pinene, as indi-cator of net mass per year, has been assumed as thesummation of the concentration at each sampling sitein the area under study. When considering the sevensampling sites for which data were collected during2004, the total concentration of α-pinene correspondedto a concentration of 12,240 ng g−1; however, a de-crease to 7,890 ng g−1 was observed during 2005 (butincludes new sampling stations). The following years,2006, 2008, and 2009 for which more sampling siteswere added, a relatively constant concentration andeven lower concentration than that in 2005 can beobserved, showing a variation between 2,460 and2,640 ng g−1.

Temporal variations of concentration in samplingstations that comprise a similar and remarkable trend

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of α-pinene fate are shown in Fig. 2. Campaigns in 2004and 2005 are also included for some stations, and sincethe greatest concentrations were registered in the Nanihuetributary during these 2 years, more attention has beenput on this area to assess possible origins of α-pinene.Unfortunately, most of the samples collected during 2007were lost by a fire incident in December 2007 thatresulted in a complete destruction of the Faculty ofScience, Universidad Austral de Chile at Valdivia.

The spatial variability of α-pinene concentrationsfrom 2004 to 2009 is shown in Fig. 3. ANOVA wasapplied to compare the spatial and temporal variabilitybetween groups of different sample sizes. Data analysisfor sampling points in the geographical area outside thewetland, except Cabo Blanco, and used as reference forbaseline concentrations of α-pinene in sediments of theValdivia Estuarine System in 2004 were as follows:Punta Castillito (20 ng g−1), Carboneros (60 ng g−1),Corral Bay (150 ng g−1), and Cabo Blanco (<20 ng g−1)(Fig. 1b). Nonhomogeneous space distribution of α-pinene can be observed from years 2004 to June 2006.During 2004, the distribution shows two critical pointsof high concentration, S4 (5,580 ng g−1) and S9(4,560 ng g−1). However, during 2005, these samplingpoints show an abrupt decrease roundly by a factor of 10in Nanihue 1 and a factor of 2 in Nanihue 6. Otherstations like Fuerte San Luis and Santa Maria showsimilar behavior. The following campaign, June 2006,shows a similar tendency to dissipation ofα-pinene witha marked uneven spatial distribution; during November2006, however, the spatial distribution becomes morehomogeneous, maintaining this tendency until the 2009campaign. Nevertheless, there were significant differ-ences in concentration (p<0.05) among sites for allcampaigns. The distribution of α-pinene in stations S4

and S9 is of particular interest since the former acts as a“containing wall” of water flux carrying particulatematter; in other words, this particular point is the pointwhere the tiny and weakwater flow coming from the topof the Nanihue tributary is slowed down by the wetland.S9, which is located at the mouth of the tributary,receives particulate matter coming from the bottom ofthe wetland, i.e., from the junction of the Cruces andValdivia Rivers mainly by rising tides.

The granular material of sediments from the wetlandis a relatively evenly distributed type of material be-tween silt and very fine sand (sediment analysis carriedout by the Institute of Environmental and EvolutionSciences-Faculty of Science-Universidad Austral deChile). Most of the samples collected from the areaunder study in the period 2004–2009 were the type ofsediments with grain size distribution roundly 60–70 % fine sand. The Carlos Anwandter wetland ischaracterized as a microtidal system with estimatedrecovery times of approximately 100–200 years whereparticle size distribution (PSD) shows no significantdifferences among surface and deep sediments (30–40-cm deep), indicating no significant temporal variation.It is also characterized by a high organic matter content(60–80 %) and PSD between 4 and 5 phi (very finesand) (Reinhardt et al. 2010). According to this, PSDshould not be correlated to the temporal neither to thespatial variation ofα-pinene content, at least in the areaof this particular study. The temporal distribution of α-pinene from 2004 to 2009 shows a notorious decreaseof concentrations, particularly at stations S4 (5,580(2004) to 200 ng g−1 (2009)) and S9 (4,560 (2004) to120 ng g−1 (2009)). However, there are several otherstations that show similar trends like Nanihue 1 and 6whose tendency lines are shown in Fig. 2. Even though

Table 1 Concentration ofα-pinene (10−3 ng g−1 dw) in sediments collected from 15 sampling stations at the Carlos Anwandter Wetland,Valdivia, during the period 2004–2009

Campaigns Sampling stations Total

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15

Dec. 23, 2004 0.71 – 0.69 5.58 0.16 – – – 4.56 – 0.06 0.48 – – – 12.24

Nov. 28, 2005 0.09 0.27 0.23 0.68 0.21 0.10 1.02 0.26 2.72 1.43 0.16 0.31 – – 0.40 7.89

June 16, 2006 – – 0.38 0.06 0.20 0.92 0.38 1.24 0.11 0.45 0.20 0.04 0.112 – 4.06

Nov. 17, 2006 0.37 0.21 0.16 0.21 0.15 0.26 0.10 0.09 0.16 0.10 0.12 0.13 0.05 0.1 0.44 2.64

Dec. 10, 2008 0.03 0.1 0.09 0.21 0.17 0.09 0.47 0.53 0.31 0.07 0.19 0.07 0.03 0.07 0.12 2.54

Sept. 02, 2009 0.07 0.22 0.29 0.20 0.08 0.06 0.06 0.27 0.12 0.08 0.17 0.21 0.41 0.08 0.14 2.46

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the concentration of α-pinene was not measured for allstations during 2004, the total concentration was12,240 ng g−1, and a decrease of about 36 % wasobserved during 2005 (7,890 ng g−1). However, a de-crease of the concentration of about 70 % was observedfor samples measured during the middle of 2006 (June16th). The following campaigns (November 2006,

December 2008, and September 2009) show a constanttotal concentration of α-pinene that ranges from 2,460to 2,640 ng g−1, which represent roundly a 90 % de-crease with respect to the observed value during 2004.

Tukey’s test for multiple comparisons was applied tothe data, showing that the levels of concentrations of α-pinene corresponding to the years 2004 and 2005 are

Fig. 2 α-Pinene fate in sediments from the sampling stations that shows significant trends located at the Carlos Anwandter NatureSanctuary Wetland, Valdivia, south-central Chile during the period 2004–2009

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significantly different (p<0.05) within a 95 % confidenceinterval as compared to those measured during 2006,2008, and 2009. However, Kruskal–Wallis analysis fornonparametric data shows that there are no significantdifferences (p>0.05) among data of 2008 and 2009.

4 Discussion

Generally, data collected for each sampling show somespatial variations of α-pinene concentrations between

2005 and 2009, which may be probably due to smoothtidal oscillations of the estuarine system and that maybe responsible for redistribution of α-pinene in the areaunder study (Fig. 3). Attention has been paid to theabrupt decrease of α-pinene concentrations in sedi-ments from 2004 to the following years in theNanihue tributary, even though some other samplingsites located at the Cruces River exhibit the samepattern. The fate trend behavior of α-pinene is coinci-dent with the presence of the event that released a highdischarge of particulate material and chemicals to the

Fig. 3 Spatial distribution of α-pinene in sediments of the Nanihue, Cudico, and Cruces Rivers located at the Carlos Anwandter NatureSanctuary Wetland, Valdivia, south-central Chile, in the period 2004–2009

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Carlos Anwandter Nature Sanctuary by the pulpmill plant (CELCO-Arauco) between April andOctober 2004.

This is the first report on the presence of α-pinene insediments of fluvial and estuarine ecosystems thatcould be related to pulp mill black liquor discharges.Button and Jüttner (1989) reported the presence ofterpene compounds in marine waters of Alaska(13.6 μg L−1), where α- and β-pinene are the maincomponents of the mixture, and they concluded thatboth compounds were essentially of terrestrial origin.Many terpenes of marine algae have been detectedhowever volatiles like α-pinene and β-pinene havenever been reported in mixtures of terpenes of marineorigin. Since conifers contain high amounts of α- andβ-pinene, it appears that both volatile monoterpenes insediments of aquatic ecosystem would mainly have aterrestrial origin (Gershenzon and Dudareva 2007).

A significant increase of pine and eucalyptus plan-tations (roundly 60 %) has occurred in an area com-prising the Region de Los Ríos in the period 1998–2009 (INFOR 2010; CONAF 2008). These data wouldmean a significant environmental impact coming fromair emissions of the volatile monoterpenes α- and β-pinene. Rolon and Rocha (2011) have suggested thatthe presence of pines influence directly or indirectlythe aquatic macrophyte structure, producing a reduc-tion of its richness and changing the species composi-tion of natural ponds in the southeast of Brazil. Eventhough Los Rios Region is threatened by forestry plan-tations, no changes of land use in either plantations ofpine or eucalyptus in the area close to the Río CrucesWetland were detected that could generate suspicionsabout direct effects on E. densa. However, the increaseof pine and eucalyptus plantations in the outer area ofthe wetland opposes the tendency to the decrease of thecontent of α-pinene in sediments observed during theperiod 2004–2009. In other words, an increase and nota decrease of pinenes in sediments in the wetlandconcomitant with the increase of pine plantations andeucalyptus in the last decade should be noted.

The detection of α-pinene but not β-pinene in sedi-ments in this study may be explained by consideringtheir physicochemical properties; indeed, α-pinene ex-hibits a better potential of bioconcentration in lipophylicsubstrates (bioconcentration factor (BCF)=1,040; watersolubility at 25 °C, Cw=4.07 mg L−1; octanol–waterpartition coefficient, Kow=27,542; and a Henry’s Lawconstant, Hpc=13,590), as compared to β-pinene

(BCF=320, Cw=7.06 mg L−1, Kow=14,454, andHpc=6,826) (McDonald et al. 2002; Copolovici andNiinemets 2005; Taherpour et al. 2011). These differ-ences may explain not only the absence of β-pinene insediments over time but also the absence of some otheraccompanying volatile monoterpenes, which is the casewhen pollutants are introduced into aquatic ecosystemsby certain events. Since the speed of foliar emissions ofmonoterpene-generating plants is highly dependent onthe partition between air, water, and lipid phase, α-pinene has advantage over other volatile compoundsrelative to air emission volumes (Copolovici andNiinemets 2005, 2007); in this context, there is suffi-cient basis to consider that could display a greater ten-dency to accumulate in sediments over time, given thedynamic behavior of this chemical from the leaf oforigin until its final deposition in the sediments ofaquatic ecosystems, provided that its origin correspondsto natural runoff or air inputs and a significant increaseof pine and eucalyptus plantation should be occurring inthe area under study (Strömvall and Petersson 1993).

There are no reports in the literature about the levelsof concentration and toxicity of α-pinene in sedimentsover macrophyte plants in wetlands or natural ponds,but its synergistic action when present together withother pollutants is known (Mercier et al. 2009). Furtherresearch on α-pinene toxicity on E. densa and otheraquatic organisms should be necessary to assess itspotential damage on sensitive aquatic environmentalecosystems. α-Pinene concentration of 460 ng g−1 wasmeasured in a single and seriously damaged specimenof E. densa rescued in December 2005 from the im-pacted area. However, samples of the aquatic plantcollected from a non-impacted area in the Calle-CalleRiver (39°48′52.5″S, 73°13′51.2″W) outside theCarlos Anwandter Sanctuary in 2010 had a mean con-centration range of 50–70 ng g−1.

During 2007, a new case of a punctual contamina-tion of kraft pulp mill emissions occurred in Chile, butthis time by LICANTEL, a cellulose plant in theMataquito River, VII Region (Region del Maule). Inthis case, there was massive death of fish and manyother aquatic species. Liver and gill samples analyzedby our laboratory showed that the organisms weredamaged by a liquid emission into the river thatcontained high levels of α- and β-pinene among manyother chemicals. The industry admitted that it wasguilty: an accidental release of black liquor containingturpentine oil impacted the fragile ecosystem.

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The spatial–temporal distribution model is usuallyan important information that may be used to establishcause–effect relationships, especially when looking forthe origin of a given pollution point. The particulargeomorphologic characteristics along the Cruces Riverin the section between CELCO-Arauco and the wet-land (approximately 15-km long), where the sedimen-tary material found at the bottom of the river rangesfrom gravel to stones, make it impossible to model thespatial distribution. As a result, the temporal modelingin this particular case has an important weight factor asit has been discussed previously.

α-Pinene and β-pinene are the main constituents ofthe essential oils of Pinus radiata (22 % α-pinene and35 % β-pinene) and Eucalyptus globulus (20 % α-pinene and 0.5 % β-pinene) (Sacchetti et al. 2005).However, the composition of turpentine sulfate as asubproduct of the chemical pulping of pines containmainly 60–65 % α-pinene and 25–35 % β-pinene, butit can considerably change according to the speciesfrom which it is extracted and the extracting tempera-ture (Thiel and Hendricks 2004; Bero et al. 2011).Probably, α-pinene was introduced into the wetlandtogether with other contaminants in an uncontrolledliquid emission containing black liquor. CELCO plantuses approximately 2,240,000 m3 year−1 of P. radiataand 563,000 m3 year−1 of eucalyptus. The absence ofβ-pinene in sediments in the Rio Cruces wetland a yearafter the ecological disaster could also be explained ifthe liquid emission contained essential oils from E.globulus in addition to the difference with respect toα-pinene in the physicochemical properties and tur-pentine sulfate composition.

The event that occurred in the wetland needs essen-tially a different approach to establish a cause–effectrelationship if compared to the Mataquito River caseduring 2007, from a forensic point of view: biota andsediment samples were collected a long time after thewetland was environmentally impacted, while in thesecond case, samples were collected and analyzed im-mediately after the liquid emission provoked the disas-ter, and in this case, β-pinene could be certainlydetected.

5 Conclusions

α-Pinene was detected in sediments on Rio CrucesNature Sanctuary Wetland located at Valdivia, south-

central Chile in the period 2004–2009. Temporal andspatial distributions of α-pinene in a selected area thatencloses the Nanihue, Cudico, and Cruces Rivers at theupper site of the sanctuary were determined, showing adecline of total concentration levels from 2004(12,240 ng g−1) to 2005 (7,890 ng g−1) and middle of2006 (4,060 ng g−1). The following years, last 2006,2008, and 2009, show a relatively constant concentra-tion with a clear tendency toward baseline levels(2,460–2,640 ng g−1). The most affected area turnedout to be the Nanihue tributary, where the most abruptconcentration decline was observed. This observationis concomitant with the environmental disaster thatoccurred during 2004. Since the decrease of α-pineneconcentrations in sediments in the period 2004–2009shows an opposite trend as compared to the surfacearea increase of pine and eucalyptus plantation in LosRios Region, and not having found potential sources ofα-pinene by anthropogenic activities in the area understudy other than pulp mill activities, it may be finallyconcluded that this compound did not enter the bodiesof water from a gradual and natural process. On thecontrary, there are more bases to hold that there was ashock due to successive punctual releases of pulp milleffluent into the Carlos Anwandter Sanctuary that in-cluded black liquors generated by the CELCO-Araucoplant during 2004 located in San José de la Mariquina.α-Pinene in sediments may be a plausible chemicaltracer capable of detecting pollution events over timeand its impacts in aquatic ecosystems as well aschanges in aquatic ecosystems produced by improperlytreated pulp mill liquid emissions that use pine andeucalyptus.

Acknowledgments This work was supported by theUniversidad Austral de Chile, project DID-2008-07. The finan-cial support for this study is gratefully acknowledged. The au-thors of this study want to thank to Dr. Daisaku Ikeda, Presidentof SGI, for his valuable support on the protection of the envi-ronment and human life.

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