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Radiocesium accumulation in the anuran frog, Rana tagoi tagoi, in forest ecosystems after the Fukushima Nuclear Power Plant accident

Amphibians are key components in forest food webs. When examining radioactive contamination in anurans, it is important to understand how radiocesium transfer occurs from lower to higher trophic levels in forest ecosystems. We investigated the
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  Radiocesium accumulation in the anuran frog,  Rana tagoi tagoi , inforest ecosystems after the Fukushima Nuclear Power Plant accident Teruhiko Takahara  a ,  * , Satoru Endo  b , Momo Takada  a , Yurika Oba  a , Wim Ikbal Nursal  a ,Takeshi Igawa  c , Hideyuki Doi  d , Toshihiro Yamada  a , Toshinori Okuda  a a Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8521, Japan b Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8527, Japan c Graduate School of International Development and Cooperation, Hiroshima University, 1-5-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8529, Japan d Institute for Sustainable Sciences and Development, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan a r t i c l e i n f o  Article history: Received 5 November 2014Received in revised form23 January 2015Accepted 24 January 2015Available online Keywords: ContaminationFNPPForest  󿬂 oorRadioactivityTago's brown frog a b s t r a c t Amphibians are key components in forest food webs. When examining radioactive contamination inanurans, it is important to understand how radiocesium transfer occurs from lower to higher trophiclevels in forest ecosystems. We investigated the activity concentration of radiocesium ( 134 Cs and  137 Cs) inTago's brown frog ( Rana tagoi tagoi ) captured on the forest  󿬂 oor approximately 2.5 years after theFukushima Nuclear Power Plant (FNPP) accident. We collected66  R. tagoi tagoi  at different distances fromthe FNPP. Radiocesium accumulation showed positive correlations with the air radiation dose rate andlitter contamination but not with distance from the FNPP. Whole-body radioactivity showed no corre-lation with body mass or length. Our results suggest that differences in the available food items result inlarge variability in individual contamination. Contamination level monitoring in terrestrial and aquaticamphibian is necessary for clarifying the processes and mechanisms of radiocesium transfer throughforest food webs. ©  2015 Elsevier Ltd. All rights reserved. 1. Introduction The March 2011 nuclear reactor accident at the FukushimaNuclear Power Plant (FNPP) in Japan resulted in the emission of largequantitiesofradioactivecontaminationintotheenvironment.Deciduous broad-leaved and evergreen coniferous forests occupymore than 70% of the land area of Fukushima Prefecture, andcontamination of the forest  󿬂 oor occurred as a result of the fallout(Fukushima Prefecture, 2012).Recent research indicated that radioactive fallout could havedifferenteffects on theforestdependingon distancefromthe FNPPand time after the FNPP accident (e.g., Hasegawa et al., 2013;Hayama et al., 2013). Ayabe et al. (2014) reported that radio- cesium concentrations in a forest  󿬂 oor-inhabiting web spider( Nephila clavata ) increased according to radiation dose rate, whichdepended on distance from the FNPP at 1.5 years after the FNPPaccident. Previous studies of aquatic habitats have mostly surveyedfreshwater 󿬁 shspecies(e.g.,Iguchietal.,2013;FukushimaandArai,2014; Yamamoto et al., 2014), although several other taxa havebeen examined (aquatic insects, Yoshimura and Akama, 2014;anuran tadpoles, Sakai et al., 2014). To clarify the overall  󿬂 ow anddistribution of radiocesium in a forest ecosystem, animals thatconnect the terrestrial and aquatic food webs must be carefullymonitored (Murakami et al., 2014).Most amphibians have complex life cycles, in which differentdevelopmental stages occupy different habitats (usually aquaticand terrestrial) and consume different food sources. Amphibiansareamajorfaunalconstituentofforestecosystems,wheretheyactas both higher consumers and as prey (e.g., for snakes and  󿬁 sh)(Whiles et al., 2006). Adult amphibians have thin, permeable skin,whichcan makethemsensitivetoradiation exposureinterrestrialhabitats. Amphibian larvae feed mainly on algae and detritus;larval stages serve as important indicators of bioaccumulation of trace elements and transport of contaminants (e.g., heavy metals)in ecosystems (Unrine et al., 2007). Therefore, examination of radiocesium accumulation in amphibians can facilitate under-standing of the circulation of this contaminant in forest ecosys-tems, including terrestrial and aquatic environments (Stark et al., *  Corresponding author. E-mail address: (T. Takahara). Contents lists available at ScienceDirect Environmental Pollution journal homepage: ©  2015 Elsevier Ltd. All rights reserved. Environmental Pollution 199 (2015) 89 e 94  2004). To date, few studies have investigated radiocesium accu-mulation in amphibians after the FNPP accident (but see Sakaiet al., 2014).To clarify the relationship between radiocesium concentrationin amphibians and conditions in the surrounding forest ecosystem2.5 years after the FNPP accident, we investigated net quantities of  134 Cs and  137 Cs accumulated inTago's brown frogs,  Rana tagoi tagoi (Amphibia: Ranidae) at different sampling sites in Fukushima. R. tagoi tagoi  is endemic to Japan and is widely distributed onHonshu, Shikoku, and Kyushu islands. This species inhabitsmountainous regions and feed on crickets, spiders, and land snails(GorisandMaeda,2004).Because  R. tagoi tagoi  alsoinhabitsforestsinFukushima,itisasuitableindicatorspeciesforevaluatinguptakeand transfer of radiocesium.To understand radioactive accumulation processes in frogsapproximately 2.5 years after the FNPP accident, we tested thefollowing hypotheses: (1) radiocesium accumulation in frogs in-creases according to distance from the FNPP; (2) radiocesiumaccumulation varies according to air radiation dose rates and littercontamination at frog sampling sites; and (3) whole-body radio-cesium in frogs is positively correlated with body size.We  󿬁 rst examined whether the concentration of   134 Cs/ 137 Cs inindividual frogs was correlated with air radiation dose rate andlitter contamination at each sampling site, and with distance fromtheFNPP.Next, weevaluatedtherelationshipbetweenwhole-bodyradiocesium and body size (mass and length) in frogs. Finally, wediscuss the potential role of frogs in circulating radiocesium be-tween terrestrial and aquatic habitats in forest ecosystems. 2. Methods  2.1. Field survey Our sampling sites were distributed in Minami-Soma City(22.6 e 25.1 km from the FNPP), Iitate Village (37.8 e 39.7 km), andSoma City (42.5 e 45.3 km) in Fukushima Prefecture (Fig. 1). Wecollected 66  R. tagoi tagoi  (juveniles or adults) by hand or with ahand-net from July 31 to August 6, 2013. Frogs were placed indi-viduallyintoplasticbags(UnipakMark-D)andimmediatelytreatedby low-temperature anesthesia on ice in a cooling box, and werepreserved at  <  15   C.During the  󿬁 eld survey, we attached a personal dosimeter(PM1610, Polimaster Ltd., Belarus) 1 m above the ground andrecorded air radiation dose rates of gamma radiation ( m Sv $ h  1 ) ateach sampling site, consistent with other studies (e.g., Hasegawaet al., 2013; Ayabe et al., 2014; Yoshimura and Akama, 2014).These dose rates were presented as average values of measure-ments made at 3 e 5 locations in which frogs were caught in eachsampling site. Litter and soils (0 e 5 cm depth) had a strong positivecorrelation, and radiocesium concentrations in litter samples weremuch higher than those of soil samples (Takada et al., in prepara-tion). Thus, only litter samples were collected at locations wherefrogs were caught. The average values from 1 to 6 points at eachsampling site are presented (the number of sites varied accordingto available survey time).The permit to enter the surveyareawas obtained from the localgovernment (Iitate Village). Noti 󿬁 cation of the  󿬁 eld survey wasaccepted by the Iwaki District Forest Of  󿬁 ce, and the permits wereobtained from private landowners as needed for each study site. Inaddition, ethical practices were approved by the Animal ResearchCommittee of Hiroshima University. The study was performed ac-cording to the guidelines for experimental vertebrate animals(Hiroshima University, Law No.102,1 Mar. 2012).  2.2. Sample treatments of frogs and litter  Frogs were thawed at room temperature (for approximately20 min), and the surface skin of each frog was  󿬂 ushed fully withdistilled water and blotted with paper wipes to remove adheredsubstances (e.g., plant material or soil). Each frog was weighed tothe nearest 0.01 g using an electronic balance (CPA Analytical Bal-anceCPA224S,SartoriusAG,Germany),andsnout-to-ventlengthasbody length was measured to the nearest 0.01 mm using a digitalcaliper(CodeNo.500-151, Mitutoyo,Kawasaki,Japan).Toeliminategut contents that contained radiocesium, the internal organs (e.g.,stomachandintestines)wereremovedusingscissorsandtweezers.We also collected one toe from each frog for DNA analysis (toeswere stored at   30   C until analysis). After weighing again todetermine wet weight for radiocesium measurement, the speci-mens were dried at 60   C for  > 4 d. The dried specimens wereground using an agate mortar and pestle, and body dry weight wasmeasured.Eachpowderedsamplewaspreservedina10-mLplastic Fig. 1.  Map of the locations of sampling sites and the Fukushima Nuclear Power Plant (FNPP). Open circles indicate sites where frogs ( Rana tagoi tagoi ) were collected. T. Takahara et al. / Environmental Pollution 199 (2015) 89 e 94 90  tube prior to radiocesium measurements. Littersamples were ovendried at 70  C for 42 h. The samples werehomogenized and packedinto 100-mL polystyrene containers (U-8) prior to radiocesiummeasurements.  2.3. Identi  󿬁 cation of frog species Two congeneric species ( Rana japonica  and  Rana ornativentris )sometimes distribute sympatrically or parapatrically with  R. tagoitagoi  in Fukushima Prefecture. We con 󿬁 rmed correct identi 󿬁 cationof the samples using polymerase chain reaction e restriction frag-ment length polymorphism (PCR-RFLP) (Igawa et al., 2015). Thismethod utilizes species-speci 󿬁 c restriction enzyme digestion sitesin nucleotide fragments ampli 󿬁 ed by PCR. We identi 󿬁 ed a  Rana japonicus- speci 󿬁 c  Spe I restriction site and  R. ornativentris -speci 󿬁 cand  R. tagoi tagoi- speci 󿬁 c  Hph I restriction sites in the nucleotidesequence of the mitochondrial 16S rRNA gene by comparing ourdata with 16S rRNA data deposited in GenBank. In brief, weampli 󿬁 ed the partial region of 16S rRNA, including these species-speci 󿬁 c restriction sites, and digested the ampli 󿬁 ed fragments by Spe I and  Hph I separately. We then con 󿬁 rmed the  R. tagoi tagoi- speci 󿬁 c fragment pattern digested with  Hph I by electrophoresis onagarose gel. Total genomic DNA of each individual was extractedfrom clipped toes using a DNeasy Blood and Tissue Kit (Qiagen,Hilden, Germany) following the manufacturer's instructions. PCR ampli 󿬁 cation of the partial 16S rRNA gene was performed in a 20- m L volume containing 10- m L of EmeraldAmp MAX PCR Master Mix(TaKaRa), 1  m L of genomic DNA solution, and 2  m L of the 10  m Mprimer pair F51 (5 0 -CCCGCCTGTTTACCAAAAACAT-3 0 ) and R51 (5 0 -GGTCTGAACTCAGATCACGTA-3 0 ) (Sumida et al., 2002). Thermalcycling was performed under the following conditions: 95   C for3 min, followed by 35 cycles of 95   C for 30 s, 55   C for 30 s, and Fig. 2.  Relationship between radiocesium concentration in  R. tagoi tagoi  and distance from the Fukushima Nuclear Power Plant (FNPP) (a), air radiation dose rate (b), and radi-ocesium concentration ( 134 þ 137 Cs) of litter (c) at each sampling site. Regression lines in (b) and (c) showed signi 󿬁 cant trends by GLM ( P   <  0.05). In (c), data for four frogs wereeliminated from the analysis because litter could not be collected at the sampling site. T. Takahara et al. / Environmental Pollution 199 (2015) 89 e 94  91  72   C for 30 s. PCR products were digested with  Spe I and  Hph Iseparately and visualized by electrophoresis on 2.0% agarose gel.  2.4. Radiocesium measurements Radiocesium concentrations (Bq $ g  1 ) in the powdered frogsamples were individually measured using a gamma spectrometerwith a low-background well-type germanium (Ge) detector (SeikoEG & G, GWL 120230-S, Japan) (Shizuma et al., 1992) and correctedto a wet-weight basis. The uncertainty of the radiocesium mea-surements was approximately 5% (sample height within tube: > 0.5 cm) or 10 e 15% ( < 0.5 cm). Radiocesium concentrations indried litter samples (Bq $ g  1 ) were measured using a GEM serieshigh-purity (HP) Ge coaxial detector system (ADCAM-100; EC  & GORTEC Company, USA). The uncertainty of the radiocesiummeasurements was approximately 10%. The radiocesium concen-trationofallsampleswascorrectedforradioactivedecaytothe 󿬁 rstsampling day of this survey.  2.5. Statistical analysis We used a general linear model (GLM) (Venables and Ripley,2002) with Gaussian error distribution to estimate the relation-ship between  134 Cs or  137 Cs concentrations in  R. tagoi tagoi  and  󿬁 vefactors:distancefromtheFNPP,airradiationdoserate,radiocesiumcontamination level of litter, frog wet weight, and body length. The 134 Cs or  137 Cs concentrations in frogs and litter were log 10 -trans-formed prior to GLM to normalize the values based on the Sha-piro e Wilk normality test ( a  ¼  0.05). Statistical signi 󿬁 cance wasde 󿬁 ned as  P  < 0.05. All statistical analyses were performed using R version 3.1.0 (R Core Team, 2014). 3. Results Therelationshipsbetween 134 Csand 137 Csconcentrationinfrogsand distance from the FNPP were not signi 󿬁 cant (GLM,  134 Cs:  Table 1 Resultsofgenerallinearmodelswithbinomialerrordistribution(SE,standarderror)forconcentration of   134 Cs (a) and  137 Cs (b) in  R. tagoi tagoi . FNPP, FukushimaNuclearPower Plant. Bold font indicates signi 󿬁 cant  t  -values ( P   <  0.05).Coef  󿬁 cient SE  t  -value(a)(Intercept) 1.37 7.46  10  1 1.829Distance from FNPP 2.29  10  2 1.34  10  2 1.707Air radiation dose rate 1.94  10  1 9.27  10  2 2.088 Log 10  (litter contamination) 2.02  10  6 8.13  10  7 2.478 Frog wet weight 2.01  10  2 6.33  10  2 0.317Body length 1.29  10  2 1.27  10  2 1.018(b)(Intercept) 9.91  10  1 7.39  10  1 1.342Distance from FNPP 2.35  10  2 1.33  10  2 1.772Air radiation dose rate 1.95  10  1 9.18  10  2 2.119 Log 10  (litter contamination) 2.04  10  6 8.05  10  7 2.537 Frog wet weight 1.36  10  2 6.27  10  2 0.216Body length 1.38  10  2 1.26  10  2 1.100 Fig. 3.  Relationship between radiocesium concentration and wet weight (a), or body length (b) of   R. tagoi tagoi  specimens at each sampling site. Closed circles ( n  ¼  9) representfrogs collected from sampling sites with higher air radiation dose rates (2.46 e 5.27  m Sv $ h  1 ); open circles ( n ¼ 57) represent frogs collected at lower sites (0.52 e 1.37  m Sv $ h  1 ). Therelationships were not signi 󿬁 cant (GLM,  P  > 0.05). Arrows represent frogs captured at highly contaminated site but having low rates of radiocesium accumulation, or frogs capturedat low-contamination sites but having high radiocesium levels. T. Takahara et al. / Environmental Pollution 199 (2015) 89 e 94 92  P   ¼  0.082,  137 Cs:  P   ¼  0.093; Fig. 2a) (Table 1). On the other hand, radiocesium concentrations were positively correlated with airradiation dose rate ( 134 Cs:  P  ¼ 0.039, 137 Cs:  P  ¼ 0.041; Fig. 2b), andlittercontaminationlevel( 134 Cs: P  ¼ 0.014, 137 Cs: P  ¼ 0.002;Fig.2c)(Table 1).The relationship between whole-body radioactivity and bodysizeisshowninFig.3.Thewholebodyradioactivityof  134 Csor 137 Csof frogs did not signi 󿬁 cantly increase according to the degrees of thefrogwetweight(GLM, 134 Cs: P  ¼ 0.829, 137 Cs: P  ¼ 0.752;Fig.3a)(Table 1). The relationships between whole body radioactivity of  134 Cs or  137 Cs in frogs and body length were also not signi 󿬁 cant( 134 Cs: P  ¼ 0.276, 137 Cs: P  ¼ 0.313;Fig.3b)(Table1).Thewholebody radioactivity( 134 þ 137 Cs)ofthefrogcollectedatthesiteswithhigherair radiation dose rates was about 10 times as high as that of thelower sites [higher sites: 27.7  ±  6.8 (n  ¼  9); lower sites: 2.6  ±  0.4(n ¼ 57); means  ±  SE (Bq $ g  1 )]. 4. Discussion In this paper, we showed that the frog  R. tagoi tagoi , which in-habits forest ecosystems, accumulated high concentrations of radiocesium. Amphibians are key components of food webs inforest ecosystems because of their dual roles as higher consumersand as prey for top predators (Regester et al., 2006). Thus, the highcontamination levels in  R. tagoi tagoi  suggest that radiocesiumreleased from the FNPP accident is transferred through the foodweb and reaches higher trophic levels in forest ecosystems.The frogs accumulated more radiocesium at sampling sites withhigher air radiation dose rates and litter contamination. In thisstudy, however, the litter samples were only collected once at twoof the sampling areas due to the limitation of survey times. Furtherstudy is needed to increase the numberof sampling points in orderto improve accuracy in determination of litter contaminationwithin the survey areas. In addition, we did not detect a positiverelationship between distance from the FNPP and accumulatedradiocesium, although the relationship showed a positive trend.This result suggests that radioactive contamination is not alwayshomogeneously distributed with distance from the FNPP.While Ayabe et al. (2014) reported a signi 󿬁 cant relationshipbetween bodysize and contamination level in the spider  N. clavata, we observed a positive, but not signi 󿬁 cant, relationship betweenaccumulated radiocesium and body size in  R. tagoi tagoi . Thisprobably was related to the fact that contamination levels in frogscollected at sites with higher air radiation dose rates wereapproximately10-fold higher than those at the lower sites. Thus, inthe future,it is necessary tocollect more individuals per group(i.e.,with high and low air radiation dose rates), and to evaluate therelationship between accumulated radiocesium and body size of frogs in each group.Consideration of the multiple connections (e.g., terrestrial vs.aquatic) among food webs is important for clarifying the radio-cesium cycle in forest ecosystems (Murakami et al., 2014). Somefrogs collectedin this studywere females that contained eggs (datanot shown). Thus, frogs with higher radiocesium accumulation arepredictedtostronglyin 󿬂 uencecontaminant 󿬂 owfromterrestrialtoaquatic systems through oviposition and maternal transfer of radioactive cesium to eggs, as observed for other contaminants(Metts et al., 2013). On the other hand, some tadpoles feed onsediment detritus, which is likely to be highly polluted (Fukushimaand Arai, 2014; Yoshimura and Akama, 2014). In fact, Sakai et al.(2014) reported that local differences in radiocesium concentra-tions in rice paddy sediments could in 󿬂 uence the accumulation of radiocesium in  Pelophylax porosa porosa  tadpoles. Therefore, uponmetamorphosis, larvae of amphibians (i.e., frogs and salamanders)might transfer radiocesium from aquatic to terrestrial ecosystems.In the future, it is necessary to clarify contamination levels in am-phibians in both terrestrial and aquatic life stages.However, radiocesium accumulated in frogs was not alwaysre 󿬂 ected in site contamination levels (see arrows in Fig. 3). Forexample, local hot (and low) spots can occur within samplingsites. Ayabe et al. (2014) also reported that radiocesium concen-trations in the web spider varied widely among individuals. Infact, in our preliminary survey, some points of soil/littercontamination with high concentrations of radioactivity (i.e., hotspots) occurred even within local survey plots (Takada et al., inpreparation). Prey animals such as earthworms can feed onhighly contaminated litter on the forest  󿬂 oor (Hasegawa et al.,2013), and frogs often consume such prey. Determination of thecontamination levels of potential  R. tagoi tagoi  food sources insampling sites is needed.In conclusion, we studied the relationships between radio-cesium contamination of   R. tagoi tagoi , air radiation dose rates, andlittercontaminationinforest 󿬂 oorhabitatsapproximately2.5yearsafter the FNPP accident. We suggest that these frogs play animportant role in circulation of radiocesium between terrestrialandaquatic habitats inthese ecosystems. It is necessarytoevaluate 󿬂 uctuations in radiocesium accumulation in organisms, includingamphibian species, by long-term monitoring.  Acknowledgments WethanktheIwakiDistrictForestOf  󿬁 ceforsupportingour 󿬁 eldsurvey. We thank Drs. T. Gomi and M. Sakai at Tokyo University of Agriculture and Technology for their useful comments for sampletreatments. We are also grateful to Drs. H. Deguchi, E. Oguri, M.Ishitani, and H. Hanada at Hiroshima University for their kindadvice regarding the study topic. This study was supported byPhoenix Leader Education Program (Hiroshima Initiative) for Re-naissance from Radiation Disaster, Organization of the LeadingGraduate Education Program. References Ayabe, Y., Kanasashi, T., Hijii, N., Takenaka, C., 2014. Radiocesium contamination of the web spider  Nephila clavata  (Nephilidae: Arachnida) 1.5 years after theFukushima Dai-ichi Nuclear Power Plant accident. J. Environ. Radioact. 127,105 e 110.Fukushima Prefecture, 2012. Fukushima Prefecture Radioactivity MeasurementMap. Fukushima, T., Arai, H., 2014. Radiocesium contamination of lake sediments and  󿬁 shfollowing the Fukushima nuclear accident and their partition coef  󿬁 cient. InlandWaters 4, 204 e 214.Goris, R.C., Maeda, N., 2004. Guide to the Amphibians and Reptiles of Japan. Krieger Publishing Company, Florida.Hasegawa, M., Ito, M.T., Kaneko, S., Kiyono, Y., Ikeda, S., Makino, S., 2013. Radio-cesium concentrations in epigeic earthworms at various distances from theFukushima Nuclear Power Plant 6 months after the 2011 accident. J. Environ.Radioact. 126, 8 e 13.Hayama, S., Nakiri, S., Nakanishi, S., Ishii, N., Uno, T., Kato, T., Konno, F.,Kawamoto, Y., Tsuchida, S., Ochiai, K., Omi, T., 2013. Concentration of radio- cesium in the wild Japanese monkey ( Macaca fuscata ) over the  󿬁 rst 15 monthsafter the Fukushima Daiichi nuclear disaster. PLoS One 8, e68530.Igawa, T., Komaki, S., Takahara, T., Sumida, M., 2015. Development and validation of PCR-RFLP assay to identify three brown frogs of the true frog genus Rana. Curr. Herpetol. 34, 1 e 6.Iguchi, K., Fujimoto, K., Kaeriyama, H., Tomiya, A., Enomoto, M., Abe, S., Ishida, T.,2013. Cesium-137 discharge into the freshwater  󿬁 shery ground of grazing  󿬁 sh,ayu  Plecoglossus altivelis  after the March 2011 Fukushima nuclear accident. Fish. Sci. 79, 983 e 988.Metts, B.S., Buhlmann, K.A., Tuberville, T.D., Scott, D.E., Hopkins, W.A., 2013.Maternal transfer of contaminants and reduced reproductive success of southern toads ( Bufo  [  Anaxyrus ]  terrestris ) exposed to coal combustion waste.Environ. Sci. Technol. 47, 2846 e 2853.Murakami, M., Ohte, N., Suzuki, T., Ishii, N., Igarashi, Y., Tanoi, K., 2014. Biologicalproliferation of cesium-137 through the detrital food chain in a forestecosystem in Japan. Sci. Rep. 4, 3599.R Core Team, 2014. R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. T. Takahara et al. / Environmental Pollution 199 (2015) 89 e 94  93
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