Toxicity of High Salinity Tannery Wastewater and Effects on Constructed Wetland Plants

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Toxicity of High Salinity TanneryWastewater and Effects on ConstructedWetland PlantsCristina S. C. Calheiros a , Gabriela Silva a , Paula V. B. Quitério a ,Luís F. C. Crispim b , Hans Brix c , Sandra C. Moura a & Paula M. L.Castro aa CBQF/Escola Superior de Biotecnologia, Universidade CatólicaPortuguesa, Rua Dr. António Bernardino de Almeida, Porto, Portugalb Centro Tecnológico das Industrias do Couro, São Pedro, Alcanena,Portugalc Department of Biological Sciences, Aarhus University, Ole WormsAllé, Aarhus C., Denmark

Available online: 15 Nov 2011

To cite this article: Cristina S. C. Calheiros, Gabriela Silva, Paula V. B. Quitério, Luís F. C. Crispim,Hans Brix, Sandra C. Moura & Paula M. L. Castro (2012): Toxicity of High Salinity Tannery Wastewaterand Effects on Constructed Wetland Plants, International Journal of Phytoremediation, 14:7, 669-680

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International Journal of Phytoremediation, 14:669–680, 2012Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2011.619233

TOXICITY OF HIGH SALINITY TANNERY WASTEWATERAND EFFECTS ON CONSTRUCTED WETLAND PLANTS

Cristina S. C. Calheiros,1 Gabriela Silva,1 Paula V. B. Quiterio,1

Luıs F. C. Crispim,2 Hans Brix,3 Sandra C. Moura,1

and Paula M. L. Castro1

1CBQF/Escola Superior de Biotecnologia, Universidade Catolica Portuguesa,Rua Dr. Antonio Bernardino de Almeida, Porto, Portugal2Centro Tecnologico das Industrias do Couro, Sao Pedro, Alcanena, Portugal3Department of Biological Sciences, Aarhus University, Ole Worms Alle,Aarhus C., Denmark

The toxicity of high salinity tannery wastewater produced after an activated sludge secondarytreatment on the germination and seedling growth of Trifolium pratense, a species usedas indicator in toxicity tests, was evaluated. Growth was inhibited by wastewater concentra-tions >25% and undiluted effluent caused a complete germination inhibition. Constructedwetlands (CWs) with Arundo donax or Sarcocornia fruticosa were envisaged to furtherpolish this wastewater. Selection of plant species to use in CWs for industrial wastewatertreatment is an important issue, since for a successful establishment they have to tolerate theoften harsh wastewater composition. For that, the effects of this wastewater on the growthof Arundo and Sarcocornia were assessed in pot assays. Plants were subject to differentwastewater contents (0/50/100%), and both were resilient to the imposed conditions. Arundohad higher growth rates and biomass than Sarcocornia and may therefore be the preferredspecies for use in CWs treating tannery wastewater. CWs planted with the above mentionedplants significantly decreased the toxicity of the wastewater, as effluent from the CWs outletstimulated the growth of Trifolium at concentrations <50%, and seed germination andgrowth even occurred in undiluted effluent.

KEY WORDS: Arundo donax, industrial wastewater, leather industry, Sarcocornia fruticosa,toxicity test with plants, Trifolium pratense

INTRODUCTION

Tannery wastewater has a complex composition and is potentially pollution intensive.The effluents from tannery industries may contain toxic, persistent or otherwise harmfulsubstances (EC 2003). High chemical oxygen demand (COD), organic nitrogen, chromium,NH4

+ and sulphide loads are typical of tannery effluents (INETI 2000). Also, certain streamsfrom the production process are hypersaline constituting a factor of environmental concernas well as jeopardizing the biological wastewater treatment (Lefebvre and Moletta 2006).

Address correspondence to Paula M. L. Castro, Escola Superior de Biotecnologia, Universidade CatolicaPortuguesa, Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto, Portugal. E-mail: [email protected]

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Constructed wetlands (CWs) are biological wastewater treatment systems that integrateseveral components, including plants and microorganisms, as major players (Stottmeisteret al. 2003; Vymazal et al. 2006). The type of plants and substrate used in a CW mayinfluence the microorganisms present (Calheiros et al. 2009b, 2009c), but the compositionof the wastewater may influence both plants and microorganisms (Klomjek and Nitisoravut2005). Plant selection is a crucial factor in CWs design for achieving sustainable andeffective systems, since different plant species respond differently to the same wastewater(Brix 1997; Klomjek and Nitisoravut 2005).

Toxicity tests are useful for a variety of purposes, such as determining the relativetoxicity of an effluent or a specific substance (APHA 1998). Only a limited number ofsubstances can be analyzed, identified and quantified through chemical analysis of wastew-ater (OSPAR 2005), whereas a toxicity assessment constitutes an integrated measure of thewastewater hazardousness. The whole effluent assessment (OSPAR 2005) is valuable whenapplied to complex wastewaters since a wide variety of known and unknown substancesare tested as a whole, providing further insight in the understanding of the environmentaleffects of their release or supporting the feasibility of a particular wastewater to be treatedby biological means. The plant toxicity test has been designed to assess the effects ofwater contaminants on germination and seedling growth of emergent plants (APHA 1998).Selected species for this purpose include Trifolium pratense (OECD 2006). This test hasbeen used for the evaluation of wastewater toxicity in the tannery (Karunyal et al. 1994;Calheiros et al. 2008), textile (Rosa et al. 1999) and organic chemicals (Wang et al. 2001)sectors.

The lack of detailed research and information concerning the tolerance of plantswhen facing complex wastewaters, such as high salinity tannery wastewater, is an impor-tant issue that quests for more investment; the present study was engaged to deepen thisknowledge. The aims of this study were, (1) to determine the toxicity of high salin-ity tannery wastewater collected from an activated sludge wastewater treatment plantthrough the germination and seedling growth of a standard test species T. pratense, (2) toassess the effect of this wastewater on the propagation and growth of two wetland plantspecies used in CWs systems, Arundo donax and Sarcocornia fruticosa, and 3) to assess thetoxicity of the effluent from CWs systems polishing secondarily treated high salinity tannerywastewater.

MATERIALS AND METHODS

Toxicity Tests with Trifolium pratense

The toxicity of the tannery wastewater was tested using the Trifolium pratense (redclover) seed germination and seedling growth tests according to Standard Methods forthe Examination of Water and Wastewater (APHA 1998) and the Organization for Eco-nomic Co-operation and Development (OECD 2006). This toxicity test entails exposingseeds of the plant to a control treatment and treatments with increasing concentrations ofthe wastewater. All tests were carried out during 20 day incubation periods in a growthchamber with a 16/8 h light/dark photoperiod, a light intensity of 450 µmol m−2 s−1

photosynthetically active radiation, and a temperature of 26–18◦C. Different wastewaterconcentrations were tested (10%, 25%, 50%, 75%, and 100%). The setup was conductedas previously described by Calheiros et al. (2008). Briefly, for each trial, fifteen seeds of

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T. pratense (acquired from a local specialized shop) were placed in Petri dishes (85 mmdiameter, 15 mm height) filled with sand with a particle size ranging from 0.5 to 1.0 mm(AGS 0.5–1.0, from Areipor-Areias Portuguesas, Lda, Portugal). Thirty ml of the testedwastewater solutions were added to each of four replicate Petri dishes. Deionized waterwith an electrical conductivity <0.1 µS cm−1 was used as a control in all the experimentsand as dilution water for preparing the different concentrations of the wastewater tested.A seed was considered to be germinated when the length of the radicle was >5 mm. Seedgermination, root elongation, shoot length and biomass of the plants after drying at 70◦Cfor 48 h were recorded (Wallinga et al. 1989).

Tests were carried out with the effluent from a conventional secondary tannerywastewater treatment system (experiment I, sample A) and effluents from two one-yearold CWs systems established for further polishing of the wastewater (experiment II). Thetwo 48 m2 CWs were established as subsurface flow CWs and planted with Arundo donaxor Sarcocornia fruticosa in a 0.35 m deep substratum comprised of equal parts of expandedclay and washed sand. Wastewater from the inlet of CWs (sample B) and wastewater fromthe outlet of the A. donax planted CW (sample CWA) and the outlet of the S. fruticosaplanted CW (sample CWS) were used in the experiment.

Inhibition was determined as the effective concentration causing 50% inhibition(EC50) of seed germination and growth inhibition. For calculation of EC50 and 95% Con-fidence Intervals (CI) a statistical program using weighted nonlinear regression was used.The program assumes a logarithmic normal distribution of data, and for calculation ofconfidence limits it uses inverse estimation taking into account the covariance within thecontrol response (Christensen et al. 2009).

Toxicity Tests with A. donax and S. fruticosa

In order to consider the plants A. donax and S. fruticosa to be used in CWs cells, theirresilience was assessed through a toxicity test using the effluent from the tannery wastewatertreatment system. For that a pot trial with eighteen 20-L pots was setup. Half of the potswere planted with two similar-sized juvenile plants of A. donax and the other half withtwo juvenile plants of S. fruticosa. The pots were established with equal parts of expandedclay (Filtralite R© NR 3-8) and washed sand as substrate and were watered to the level justbelow the surface of the substrate. The solutions tested in triplicate were: T0- tap water(as a control), T1-50% tannery wastewater, and T2- 100% tannery wastewater. Wastewatersamples collected for feeding the pots were subject to physico-chemical analysis every2nd or 3rd week. Fresh solutions were prepared and added every week over a 9-monthperiod to maintain the same liquid level in the pots. Dilutions of the wastewater were madewith tap water. The pots were set up outdoors under a roof to protect the plants from highinsulation and precipitation. The air temperature varied between 12◦C and 34◦C during theexperiment period.

The performance of the plants were monitored regularly throughout the study. Plantswere visually inspected for toxicity signs such as chlorosis, necrosis, and malformed plants.The number of plants in each pot and the shoot height (measured from the substrate surfaceto the apex of the tallest shoot) were registered every second or third week. At the end ofthe trial, six circular discs with 10.5 mm diameter were cut from mature leaves of plants ineach pot and extracted in N,N′-dimethylformamide for chlorophyll analysis according toWellburn (1994). Plants were then harvested, rinsed in tap water and fractionated into roots,

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stems and leaves for dry biomass determination (70◦C for 48 h in an oven) and analysisof tissue concentrations of phosphorus (P) and nitrogen (N). Concentrations of N and P inleaves, stems and roots were analyzed by colorimetry (Helios Gamma, Unicam, Cambridge,UK) following the procedure of Wallinga et al. (1989). The growth inhibition (biomassbased) was calculated as the percent reduction in biomass in the treatments compared tothe control.

Wastewater Physico-Chemical Analysis

Grab samples of the wastewater used in the toxicity tests were analysed for thefollowing parameters using Standard Methods (APHA 1998): COD, biochemical oxygendemand (BOD5), total suspended solids (TSS), total dissolved solids (TDS), ammonium(NH4

+) and chloride (Cl−). pH, conductivity and temperature were registered on-site witha WTW handheld multi-parameter instrument 340i.

Statistics

Statistical analysis was performed using the software SPSS (SPSS Inc., Chicago, IL,USA; Version 12.0). The data concerning seed germination, seedling growth, chlorophyllcontent and P and N content were analyzed through one-way or two-way analysis ofvariance (ANOVA), according to the adequacy.

To detect the statistical significance of differences (p < 0.05) between means ofobservation, the Duncan test was performed. When applicable, values were presented asthe mean ± standard error.

RESULTS AND DISCUSSION

Wastewater Characteristic

The effluent used in this study originated from an activated sludge wastewater treat-ment plant treating tannery wastewater. The effluent from the activated sludge system wasfurther polished in two series of CWs cells before discharge. The characteristics of theeffluents from the activated sludge system as well as from CWs systems used in the toxicitytrials are shown in Table 1. The wastewater coming from the secondary treatment (samplesA, B, and C) is highly saline as shown by the high electric conductivity levels (EC 13.3–19.3mS cm−1) and high contents of dissolved solids (TDS 7794–12298 mg L−1). Besides that,the COD was often >150 mg L−1 which is not in compliance with the Portuguese legis-lation (Decreto-Lei n◦ 236/98 – 1 August). However, after the effluent had passed throughthe CWs series COD was reduced to levels below the discharge standard.

Toxicity of Effluent from the Activated Sludge System

In experiment 1 the wastewater toxicity was evaluated after passing through the con-ventional activated sludge wastewater treatment system in order to assess its feasibilityand adequacy for further polishing in a plant-based CW treatment system. At a wastewaterconcentration of 50% the germination rate of the T. pratense was significantly reduced, andat 100% no germination occurred (Table 2). Other authors dealing with tannery wastewaterhave reported complete germination inhibition for concentrations above 50% (Karunyal

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Table 1 Characteristics of the wastewater used in the three toxicity trials

Experiment IIExperiment I Pot trial

Parameters Aa Ba CWAa CWSa Ca (±SE)

pH 7.46 7.85 7.86 7.62 7.85 ± 0.06Conductivity (mS cm−1) 14.84 17.14 16.22 16.17 16.97 ± 0.35TDS (mg L−1) 7794 11293 10085 10030 11009 ± 224COD (mg L−1) 103 200 72 71 209 ± 14BOD5 (mg L−1) 27 42 14 12 41 ± 3TSS (mg L−1)) 42 120 47 40 77 ± 8NH4

+ (mg L−1) 1.5 2.5 1.1 1.0 5.0 ± 1.3Cl− (mg L−1) 3100 5900 5375 5325 5650 ± 302

aWastewater samples: A- from wastewater treatment plant outlet. B- from wastewater treatment plant outlet.CWA—from constructed wetlands planted with Arundo donax outlet. CWS- from constructed wetlands plantedwith Sarcocornia fruticosa outlet. C- average value (n = 16 ± 1 standard error) from wastewater treatment plantoutlet

et al. 1994; Calheiros et al. 2008). The shoot length was also significantly reduced atwastewater concentrations of 50% and higher, but the root length was significantly reducedalready at 25% wastewater. The estimated effective concentrations resulting in 50% inhi-bition (EC50) were higher for seed germination (59%) than for shoot and root length (47and 44%, respectively). This shows that growth parameters were more sensitive to highsalinity tannery wastewater than seed germination. The high level of toxicity associatedwith the wastewater might be related to the high salinity of the wastewater, but other com-pounds discharged from the tannery process probably also have toxic effects. The tanneryindustry’s productive cycle has an assertive influence on the variability of wastewater com-position. Lower EC50 values of 6% have been reported for effluents coming directly fromthe production process (Calheiros et al. 2008). The inhibition of germination and growth

Table 2 Experiment I: Seed germination, shoot length, root length and biomass of Trifolium pratense afterexposure to wastewater originating from the outlet of a tannery wastewater treatment plant, and calculatedeffective concentrations giving 50% inhibition (EC50)

Wastewater Germination Shoot length Root length Biomassconcentration (%) (mm) (mm) (g)

0% 90 ± 2cd 43 ± 1b 34 ± 3c 0.39610% 93 ± 3cd 44 ± 1b 35 ± 1c 0.43925% 80 ± 2c 42 ± 3b 27 ± 1b 0.26250% 63 ± 6b 16 ± 2a 14 ± 1a 0.24775% 20 ± 4a 13 ± 2a 10 ± 2a 0.117100% 0 0 0 0EC50 (%) 59 47 44(95% CI)∗ (56–63) (40–55) (39–50)

Note: Means of four observations (± 1 standard error) followed by the same letters within each column are notsignificantly different according to Duncan’s multiple range test at the level of p < 0.05. The data were analyzedwithout including the concentration 100%.

∗CI: 95% confidence intervals.No EC50 data for biomass as the nonlinear regression model could not fit the data.

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of the indicator species T. pratense indicate that the effluent would also potentially affectthe plants in CWs systems established to further treat the effluent.

Toxicity Assessment in A. donax and S. fruticosa

It is important to assess the feasibility of plant species to be used in CWs for treatingsaline wastewater, since the treatment may be compromised if the plant does not estab-lish. Klomjek and Nitisoravut (2005) have tested eight emergent plant species (Typhaangustifolia, Cyperus corymbosus, Brachiaria mutica, Digitaria bicornis, Vetiveria zizan-iodes, Spartina patens, Leptochloa fusca, and Echinodorus cordifolius) in CWs systemsfor salinity tolerance by spiking municipal wastewater with NaCl. They have found that E.cordifolius and V. zizaniodes are not tolerant to the saline conditions imposed (14–16 mScm−1) and B. mutica dies after the completion of the experiment.

In the present study, plant growth and number of shoots of A. donax and S. fruticosaexposed to the saline tannery wastewater effluent were assessed during a nine month period(Figure 1). In general, plants developed and proliferated without showing signs of toxicity.By the end of the pot experiment the number of shoots of A. donax had increased 3 to 4times compared to the initial number of shoots; 15 in the control, 19 at 50%, and 21 at100% wastewater. The number of S. fruticosa shoots doubled during the trial; 12 at thecontrol, 13 at 50%, and 12 at 100% wastewater. The plant height was in general highestfor plants grown in 50% wastewater, followed by plants in 100% wastewater and then thecontrol. Particularly by the end of the trial the differences between the control and plantsexposed to the wastewater were very obvious. A. donax reached heights up to 1 m andS. fruticosa heights of 0.3 m. The difference between the species is due to their intrinsiccharacteristics (Bell 1997). The concentrations of pigments in the leaves were also affectedby the treatments. Chlorophyll a and b content in A. donax leaves and S. fruticosa plantsis shown in Table 3. A. donax had significant higher concentrations in leaves of plantsgrown in 100% wastewater, followed by 50% and by the control. This may be caused bythe fact that the chlorophyll concentrations are expressed on a fresh weight basis, as acommon response of plants exposed to salinity stress is to reduce the water content of thetissue. A low water content will result in relatively high concentrations expressed on a fresh

Table 3 Average concentrations of chlorophyll a and b and their ratio in leaves of Arundo donax and shoots ofSarcocornia fruticosa after 9 months growth in different concentrations of tannery wastewater

Chlorophyll (mg g−1 fresh weight)

Plant species Tannery wastewater (%) a b a/b-ratio

Sarcocornia fruticosa 0% 0.21 ± 0.01 NS 0.06 ± 0.002 NS 3.43 ± 0.1550% 0.21 ± 0.02 NS 0.10 ± 0.01 NS 2.09 ± 0.06

100% 0.20 ± 0.01 NS 0.09 ± 0.01 NS 2.24 ± 0.07Arundo donax 0% 0.78 ± 0.03 a 0.27 ± 0.01 a 2.86 ± 0.04

50% 1.07 ± 0.04 b 0.63 ± 0.04 b 0.80 ± 0.10100% 1.40 ± 0.07 c 0.88 ± 0.05 c 1.78 ± 0.19

Note: Means of 18 observations (± 1 standard error) followed by the same letter within each column andspecies are not significantly different according to Duncan’s multiple range test at the level of p < 0.05. NS = notsignificant

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Figure 1 Average plant height (n = 12) and number of shoots of a) Arundo donax and b) Sarcocornia fruticosaduring the 9 month trial period. Plants were grown in triplicate pots and watered with different concentrationsof tannery wastewater. Number of shoots in: 0% wastewater ( ), 50% wastewater ( ) and 100%wastewater ( ). Plants height in: 0% wastewater ( ), 50% wastewater ( ) and 100% wastew-ater ( ).

weight basis. Concerning S. fruticosa no significant differences were seen in chlorophyllcontent.

In Table 4 the average P and N concentrations in the plant tissues are shown. Therewas no significant difference in the concentrations of N and P in S. fruticosa exposed todifferent wastewater concentrations, but there were significant differences for A. donax incontrol pot and exposed to 50 and 100% wastewater, as plants watered with wastewateralways had significant higher tissue concentrations of N and P. Also, growth stimulationof both plants when subject to 50–100% wastewater occurred (increases of 30–40% forSarcocornia and 70–138% for Arundo).

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Table 4 Average phosphorus (P) and nitrogen (N) concentrations in the plant tissues collected from the potsexperiment with plants grown at different concentrations of tannery wastewater

Tannery Tissue-N (mg N g−1 Tissue-P (mg P g−1

Plant species wastewater (%) dry weight) dry weight)

Sarcocornia fruticosa 0% 5.17 ± 0.48 NS 1.43 ± 0.14 NS50% 6.33 ± 1.58 NS 1.35 ± 0.28 NS

100% 5.80 ± 0.97 NS 0.96 ± 0.33 NSArundo donax 0% 2.89 ± 0.31 a 0.23 ± 0.03 a

50% 4.67 ± 0.60 b 0.49 ± 0.03 b100% 5.00 ± 0.55 b 0.58 ± 0.05 b

Note: Means of plant material comprised in three pots (± 1 standard error) followed by the same letter withineach column and plant species are not significantly different according to Duncan’s multiple range test at the levelof p < 0.05. NS = not significant.

To our knowledge S. fruticosa has not yet been tested for use in CWs systems,although other halophytes have been investigated for example for the treatment of salineaquaculture effluent (Brown et al. 1999). On the other hand, A. donax recently has beenreported to tolerate high concentrations of heavy metals (Cd and Ni) (Papazoglou 2007),has been used to treat water contaminated with arsenic (Mirza et al. 2010) and has beenused in CW for sewage post-treatment (El Hamouri et al. 2007). The tested emergent plantsturned out to be quite resilient and appropriate for the purpose of polishing high salinitywastewater in CWs.

Toxicity of Wastewater Before and After Treatment in Planted CWs

In experiment II the wastewater toxicity after passing through CWs planted with A.donax and S. fruticosa was evaluated. The wastewater quality at the outlet of the CWssystems (CWA and CWS) was better than at the inlet as there was a reduction in severalparameters: COD (65%), BOD5 (70%), TSS (66%), NH4

+ (60%), Cl− (10%), and TDS(11%) (Table 1). Salinity had, as expected, not decreased significantly.

The wastewater concentration and its origin (inlet and CWs outlets) had a significantinfluence on seed germination, shoot and root development of T. pratense (Table 5). As theconcentration of wastewater increased, inhibition of germination and growth also increased.No germination occurred in wastewater from the CWs inlet (100%), while in effluents fromthe CWs outlets (100%) some germination always occurred. Wastewater from the CWoutlets only inhibited seed germination significantly at the highest concentration tested (75%and 100%). The shoot and root growth were actually promoted at wastewater concentrationsof 10% to 50% as compared to the control.

As in Experiment 1, the growth inhibition of the wastewater coming from the tannerywastewater treatment plant and entering the CWs, was seen at lower concentrations (25 to100%). The germination and development of the T. pratense that were exposed to lowerlevels of the wastewater were in general better than in the controls. This stimulatory effectof low wastewater concentrations might be due to nutrients present in the tannery effluent ashas also previously been reported (Calheiros et al. 2008). Stimulatory effects with greaterbiomass of plants exposed to raw textile effluent than in controls has previously beenreported by Rosa et al. (1999), who have attributed the stimulatory effects to the nutrientspresent in that effluent.

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Table 5 Experiment II: Seed germination, shoot length, root length and biomass of Trifolium pratense afterexposure to wastewater originating from the inlet and the outlets of the two constructed wetlands planted withSarcocornia fruticosa and Arundo donax, respectively, and results of 2-way ANOVA

Wastewater Germination Shoot Root BiomassSample origin concentration (%) length (mm) length (mm) (g)

Control 0% 85 ± 2 ef 54 ± 0.4 f 34 ± 3 c 0.447 ± 0.038CWs inlet 10% 86 ± 3 ef 58 ± 1 g 40 ± 2 d 0.514 ± 0.022

25% 81 ± 4 de 49 ± 1 e 35 ± 3 cd 0.337 ± 0.01850% 73 ± 1 d 43 ± 1 d 30 ± 0.4 bc 0.295 ± 0.01375% 31 ± 4 b 38 ± 0.4 c 26 ± 2 b 0.103 ± 0.013

100% 0 0 0 0Sarcocornia CW outlet 10% 89 ± 2 ef 67 ± 2 h 65 ± 4 fg 0.739 ± 0.066

25% 83 ± 3 ef 64 ± 2 h 60 ± 1 f 0.692 ± 0.04550% 80 ± 2 de 53 ± 1 f 48 ± 0.3 e 0.633 ± 0.04975% 54 ± 2 c 40 ± 1 cd 34 ± 1 c 0.294 ± 0.023

100% 14 ± 2 a 17 ± 0.4 a 15 ± 1 a 0.050 ± 0.007Arundo CW outlet 10% 91 ± 2 f 73 ± 1 i 69 ± 1 g 0.783 ± 0.009

25% 85 ± 2 ef 67 ± 1 h 65 ± 2 fg 0.715 ± 0.02550% 81 ± 4 de 60 ± 2 g 50 ± 1 e 0.678 ± 0.00975% 61 ± 5 c 48 ± 1 e 31 ± 1 bc 0.320 ± 0.016

100% 20 ± 2 a 21 ± 1 b 19 ± 1 a 0.074 ± 0.003Sample origin (S) ∗∗∗ ∗∗∗ ∗∗∗Wastewater concentration (C) ∗∗∗ ∗∗∗ ∗∗∗Interaction (S × C) ∗∗ ∗∗∗ ∗∗∗

Note: Means of four observations (± 1 standard error) followed by the same letters within each column are notsignificantly different according to Duncan’s multiple range test at the level of p < 0.05. For two-way ANOVA:∗∗p < 0.01; ∗∗∗p < 0.001. The data were analyzed without including the concentration 100% for CWs inlet.

The effective concentration causing 50% inhibition (EC50) of seed germination wasfound to be between 81% and 85%, values much higher than at the inlet (68%) (Figure 2).Also for biomass production, the EC50 was much lower for the CWs inlet (50%) than forthe CWs outlets (74 and 75%). On the contrary, there was no difference between EC50

for shoot and root length (Figure 2). In the present study the wastewater tested have beentreated in a conventional activated sludge system and thereafter polished in CWs systems.The toxicity of the wastewater, and hence the EC50 values, are therefore expected to behigher than for instance for a CW system that is used for secondary treatment. Calheiroset al. (2008) have reported that the EC50 values of wastewater from a secondary treatmentare 28% to 41%. It is however difficult to compare results from the literature with thoseobtained in the present study as different test species and different incubation conditionsand times may have been used (Rosa et al. 1999).

The application of CWs systems for the treatment of tannery wastewater has beenaddressed previously and in general CWs systems have proved to be effective (Calheiroset al. 2008, 2009a) although the salinity issue has not been assessed before. The toxicitystudies reported here show that the water quality was significantly improved by CWssystems, and the toxicity of the effluent decreased by passing through the CWs systems.The pot trials showed that both plant species tested, A. donax and S. fruticosa, grew wellwhen fed with the secondary treated tannery wastewater, and showed resilience to the harshconditions.

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EC50 (%)0 20 40 60 80 100 120 140

CW inlet

Sarco outlet

Arundo outlet

CW inlet

Sarco outlet

Arundo outlet

CW inlet

Sarco outlet

Arundo outlet

CW inlet

Sarco outlet

Arundo outlet

(A) Germination

(B) Shoot length

(C) Root length

(D) Biomass

Figure 2 Calculated effective wastewater concentrations (%) giving 50% inhibition (EC50 ± 95% confidenceintervals) of Trifolium pratense (a) seed germination, (b) shoot length, (c) root length, and (d) biomass productionduring 20-days toxicity tests. Wastewater tested are inlet to the constructed wetland systems (CW inlet), effluentfrom the Sarcocornia fruticosa planted constructed wetland (Sarco outlet) and effluent from the Arundo donaxplanted constructed wetland (Arundo outlet).

CONCLUSIONS

• Effluent from a conventional activated sludge system treating high salinity tannerywastewater caused growth inhibition of T. pratense at concentrations higher than 25%.Undiluted effluent caused a complete germination inhibition.

• The toxicity of the wastewater decreased significantly when it passed through CWssystems planted with A. donax or S. fruticosa. Effluent from the CWs systems actuallystimulated growth of T. pratense at concentrations below 50%, and seed germination andgrowth even occurred in undiluted effluent.

• EC50, the effective concentration causing 50% inhibition of T. pratense seed germinationand biomass production, was significantly lower for the CWs inlet wastewater (68% and50%, respectively) than for the CWs effluent (83% and 75%, respectively).

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TOXICITY OF HIGH SALINITY TANNERY WASTEWATER 679

• Both A. donax and S. fruticosa were resilient to the high salinity conditions imposedby the tannery wastewater. A. donax had much higher growth rates and biomass than S.fruticosa and may therefore be the preferred species for use in CWs systems treatingtannery wastewater.

ACKNOWLEDGMENTS

This work was supported by the AdI, PRIME - IDEIA Programme, through the Projectn. ◦ 70/00324 - Planticurt, including grants to Gabriela Silva and Paula V.B. Quiterio.

Cristina S.C. Calheiros wishes to thank a research grant from Fundacao para a Cienciae Tecnologia (FCT), Portugal (SFRH/BPD/63204/2009).

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