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International Journal of
Phytoremediation
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Toxicity of High Salinity Tannery


Wastewater and Effects on Constructed
Wetland Plants
a

Cristina S. C. Calheiros , Gabriela Silva , Paula V. B. Quitrio ,


b

Lus F. C. Crispim , Hans Brix , Sandra C. Moura & Paula M. L.


Castro

CBQF/Escola Superior de Biotecnologia, Universidade Catlica


Portuguesa, Rua Dr. Antnio Bernardino de Almeida, Porto, Portugal
b

Centro Tecnolgico das Industrias do Couro, So Pedro, Alcanena,


Portugal
c

Department of Biological Sciences, Aarhus University, Ole Worms


All, Aarhus C., Denmark
Accepted author version posted online: 15 Nov 2011. Version of
record first published: 23 Apr 2012

To cite this article: Cristina S. C. Calheiros, Gabriela Silva, Paula V. B. Quitrio, Lus F. C. Crispim,
Hans Brix, Sandra C. Moura & Paula M. L. Castro (2012): Toxicity of High Salinity Tannery Wastewater
and Effects on Constructed Wetland Plants, International Journal of Phytoremediation, 14:7, 669-680
To link to this article: http://dx.doi.org/10.1080/15226514.2011.619233

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International Journal of Phytoremediation, 14:669680, 2012


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ISSN: 1522-6514 print / 1549-7879 online
DOI: 10.1080/15226514.2011.619233

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TOXICITY OF HIGH SALINITY TANNERY WASTEWATER


AND EFFECTS ON CONSTRUCTED WETLAND PLANTS
Cristina S. C. Calheiros,1 Gabriela Silva,1 Paula V. B. Quiterio,1
Lus F. C. Crispim,2 Hans Brix,3 Sandra C. Moura,1
and Paula M. L. Castro1
1

CBQF/Escola Superior de Biotecnologia, Universidade Catolica Portuguesa,


Rua Dr. Antonio Bernardino de Almeida, Porto, Portugal
2
Centro Tecnologico das Industrias do Couro, Sao Pedro, Alcanena, Portugal
3
Department of Biological Sciences, Aarhus University, Ole Worms Alle,
Aarhus C., Denmark
The toxicity of high salinity tannery wastewater produced after an activated sludge secondary
treatment on the germination and seedling growth of Trifolium pratense, a species used
as indicator in toxicity tests, was evaluated. Growth was inhibited by wastewater concentrations >25% and undiluted effluent caused a complete germination inhibition. Constructed
wetlands (CWs) with Arundo donax or Sarcocornia fruticosa were envisaged to further
polish this wastewater. Selection of plant species to use in CWs for industrial wastewater
treatment is an important issue, since for a successful establishment they have to tolerate the
often harsh wastewater composition. For that, the effects of this wastewater on the growth
of Arundo and Sarcocornia were assessed in pot assays. Plants were subject to different
wastewater contents (0/50/100%), and both were resilient to the imposed conditions. Arundo
had higher growth rates and biomass than Sarcocornia and may therefore be the preferred
species for use in CWs treating tannery wastewater. CWs planted with the above mentioned
plants significantly decreased the toxicity of the wastewater, as effluent from the CWs outlet
stimulated the growth of Trifolium at concentrations <50%, and seed germination and
growth 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 harmful
substances (EC 2003). High chemical oxygen demand (COD), organic nitrogen, chromium,
NH4 + and sulphide loads are typical of tannery effluents (INETI 2000). Also, certain streams
from the production process are hypersaline constituting a factor of environmental concern
as well as jeopardizing the biological wastewater treatment (Lefebvre and Moletta 2006).

Address correspondence to Paula M. L. Castro, Escola Superior de Biotecnologia, Universidade Catolica


Portuguesa, Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto, Portugal. E-mail: plcastro@esb.ucp.pt
669

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670

C. S. C. CALHEIROS ET AL.

Constructed wetlands (CWs) are biological wastewater treatment systems that integrate
several components, including plants and microorganisms, as major players (Stottmeister
et al. 2003; Vymazal et al. 2006). The type of plants and substrate used in a CW may
influence the microorganisms present (Calheiros et al. 2009b, 2009c), but the composition
of the wastewater may influence both plants and microorganisms (Klomjek and Nitisoravut
2005). Plant selection is a crucial factor in CWs design for achieving sustainable and
effective 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 relative
toxicity of an effluent or a specific substance (APHA 1998). Only a limited number of
substances can be analyzed, identified and quantified through chemical analysis of wastewater (OSPAR 2005), whereas a toxicity assessment constitutes an integrated measure of the
wastewater hazardousness. The whole effluent assessment (OSPAR 2005) is valuable when
applied to complex wastewaters since a wide variety of known and unknown substances
are tested as a whole, providing further insight in the understanding of the environmental
effects of their release or supporting the feasibility of a particular wastewater to be treated
by biological means. The plant toxicity test has been designed to assess the effects of
water contaminants on germination and seedling growth of emergent plants (APHA 1998).
Selected species for this purpose include Trifolium pratense (OECD 2006). This test has
been 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 plants
when facing complex wastewaters, such as high salinity tannery wastewater, is an important issue that quests for more investment; the present study was engaged to deepen this
knowledge. The aims of this study were, (1) to determine the toxicity of high salinity tannery wastewater collected from an activated sludge wastewater treatment plant
through the germination and seedling growth of a standard test species T. pratense, (2) to
assess the effect of this wastewater on the propagation and growth of two wetland plant
species used in CWs systems, Arundo donax and Sarcocornia fruticosa, and 3) to assess the
toxicity of the effluent from CWs systems polishing secondarily treated high salinity tannery
wastewater.

MATERIALS AND METHODS


Toxicity Tests with Trifolium pratense
The toxicity of the tannery wastewater was tested using the Trifolium pratense (red
clover) seed germination and seedling growth tests according to Standard Methods for
the Examination of Water and Wastewater (APHA 1998) and the Organization for Economic Co-operation and Development (OECD 2006). This toxicity test entails exposing
seeds of the plant to a control treatment and treatments with increasing concentrations of
the wastewater. All tests were carried out during 20 day incubation periods in a growth
chamber with a 16/8 h light/dark photoperiod, a light intensity of 450 mol m2 s1
photosynthetically active radiation, and a temperature of 2618 C. Different wastewater
concentrations were tested (10%, 25%, 50%, 75%, and 100%). The setup was conducted
as previously described by Calheiros et al. (2008). Briefly, for each trial, fifteen seeds of

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TOXICITY OF HIGH SALINITY TANNERY WASTEWATER

671

T. pratense (acquired from a local specialized shop) were placed in Petri dishes (85 mm
diameter, 15 mm height) filled with sand with a particle size ranging from 0.5 to 1.0 mm
(AGS 0.51.0, from Areipor-Areias Portuguesas, Lda, Portugal). Thirty ml of the tested
wastewater solutions were added to each of four replicate Petri dishes. Deionized water
with an electrical conductivity <0.1 S cm1 was used as a control in all the experiments
and 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. Seed
germination, root elongation, shoot length and biomass of the plants after drying at 70 C
for 48 h were recorded (Wallinga et al. 1989).
Tests were carried out with the effluent from a conventional secondary tannery
wastewater treatment system (experiment I, sample A) and effluents from two one-year
old CWs systems established for further polishing of the wastewater (experiment II). The
two 48 m2 CWs were established as subsurface flow CWs and planted with Arundo donax
or Sarcocornia fruticosa in a 0.35 m deep substratum comprised of equal parts of expanded
clay and washed sand. Wastewater from the inlet of CWs (sample B) and wastewater from
the outlet of the A. donax planted CW (sample CWA) and the outlet of the S. fruticosa
planted 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% Confidence Intervals (CI) a statistical program using weighted nonlinear regression was used.
The program assumes a logarithmic normal distribution of data, and for calculation of
confidence limits it uses inverse estimation taking into account the covariance within the
control 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, their
resilience was assessed through a toxicity test using the effluent from the tannery wastewater
treatment system. For that a pot trial with eighteen 20-L pots was setup. Half of the pots
were planted with two similar-sized juvenile plants of A. donax and the other half with
two juvenile plants of S. fruticosa. The pots were established with equal parts of expanded
R
NR 3-8) and washed sand as substrate and were watered to the level just
clay (Filtralite
below 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. Wastewater
samples collected for feeding the pots were subject to physico-chemical analysis every
2nd or 3rd week. Fresh solutions were prepared and added every week over a 9-month
period to maintain the same liquid level in the pots. Dilutions of the wastewater were made
with tap water. The pots were set up outdoors under a roof to protect the plants from high
insulation and precipitation. The air temperature varied between 12 C and 34 C during the
experiment period.
The performance of the plants were monitored regularly throughout the study. Plants
were 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 surface
to the apex of the tallest shoot) were registered every second or third week. At the end of
the trial, six circular discs with 10.5 mm diameter were cut from mature leaves of plants in
each pot and extracted in N,N -dimethylformamide for chlorophyll analysis according to
Wellburn (1994). Plants were then harvested, rinsed in tap water and fractionated into roots,

672

C. S. C. CALHEIROS ET AL.

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

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Wastewater Physico-Chemical Analysis


Grab samples of the wastewater used in the toxicity tests were analysed for the
following parameters using Standard Methods (APHA 1998): COD, biochemical oxygen
demand (BOD5 ), total suspended solids (TSS), total dissolved solids (TDS), ammonium
(NH4 +) and chloride (Cl). pH, conductivity and temperature were registered on-site with
a 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, chlorophyll
content and P and N content were analyzed through one-way or two-way analysis of
variance (ANOVA), according to the adequacy.
To detect the statistical significance of differences (p < 0.05) between means of
observation, the Duncan test was performed. When applicable, values were presented as
the mean standard error.
RESULTS AND DISCUSSION
Wastewater Characteristic
The effluent used in this study originated from an activated sludge wastewater treatment plant treating tannery wastewater. The effluent from the activated sludge system was
further polished in two series of CWs cells before discharge. The characteristics of the
effluents from the activated sludge system as well as from CWs systems used in the toxicity
trials are shown in Table 1. The wastewater coming from the secondary treatment (samples
A, B, and C) is highly saline as shown by the high electric conductivity levels (EC 13.319.3
mS cm1) and high contents of dissolved solids (TDS 779412298 mg L1). Besides that,
the COD was often >150 mg L1 which is not in compliance with the Portuguese legislation (Decreto-Lei n 236/98 1 August). However, after the effluent had passed through
the 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 conventional activated sludge wastewater treatment system in order to assess its feasibility
and adequacy for further polishing in a plant-based CW treatment system. At a wastewater
concentration of 50% the germination rate of the T. pratense was significantly reduced, and
at 100% no germination occurred (Table 2). Other authors dealing with tannery wastewater
have reported complete germination inhibition for concentrations above 50% (Karunyal

TOXICITY OF HIGH SALINITY TANNERY WASTEWATER

673

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

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Parameters
pH
Conductivity (mS cm1)
TDS (mg L1)
COD (mg L1)
BOD5 (mg L1)
TSS (mg L1))
NH4 + (mg L1)
Cl (mg L1)

Experiment II

Experiment I
Aa

Ba

CWAa

CWSa

Pot trial
Ca (SE)

7.46
14.84
7794
103
27
42
1.5
3100

7.85
17.14
11293
200
42
120
2.5
5900

7.86
16.22
10085
72
14
47
1.1
5375

7.62
16.17
10030
71
12
40
1.0
5325

7.85 0.06
16.97 0.35
11009 224
209 14
41 3
77 8
5.0 1.3
5650 302

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

et al. 1994; Calheiros et al. 2008). The shoot length was also significantly reduced at
wastewater concentrations of 50% and higher, but the root length was significantly reduced
already at 25% wastewater. The estimated effective concentrations resulting in 50% inhibition (EC50 ) were higher for seed germination (59%) than for shoot and root length (47
and 44%, respectively). This shows that growth parameters were more sensitive to high
salinity tannery wastewater than seed germination. The high level of toxicity associated
with the wastewater might be related to the high salinity of the wastewater, but other compounds discharged from the tannery process probably also have toxic effects. The tannery
industrys productive cycle has an assertive influence on the variability of wastewater composition. Lower EC50 values of 6% have been reported for effluents coming directly from
the 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 after
exposure to wastewater originating from the outlet of a tannery wastewater treatment plant, and calculated
effective concentrations giving 50% inhibition (EC50 )
Wastewater
concentration
0%
10%
25%
50%
75%
100%
EC50 (%)
(95% CI)

Germination
(%)

Shoot length
(mm)

Root length
(mm)

Biomass
(g)

90 2cd
93 3cd
80 2c
63 6b
20 4a
0
59
(5663)

43 1b
44 1b
42 3b
16 2a
13 2a
0
47
(4055)

34 3c
35 1c
27 1b
14 1a
10 2a
0
44
(3950)

0.396
0.439
0.262
0.247
0.117
0

Note: Means of four observations ( 1 standard error) followed by the same letters within each column are not
significantly different according to Duncans multiple range test at the level of p < 0.05. The data were analyzed
without including the concentration 100%.
CI: 95% confidence intervals.
No EC50 data for biomass as the nonlinear regression model could not fit the data.

674

C. S. C. CALHEIROS ET AL.

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

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Toxicity Assessment in A. donax and S. fruticosa


It is important to assess the feasibility of plant species to be used in CWs for treating
saline wastewater, since the treatment may be compromised if the plant does not establish. Klomjek and Nitisoravut (2005) have tested eight emergent plant species (Typha
angustifolia, Cyperus corymbosus, Brachiaria mutica, Digitaria bicornis, Vetiveria zizaniodes, Spartina patens, Leptochloa fusca, and Echinodorus cordifolius) in CWs systems
for 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 (1416 mS
cm1) 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. fruticosa
exposed 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 4
times compared to the initial number of shoots; 15 in the control, 19 at 50%, and 21 at
100% wastewater. The number of S. fruticosa shoots doubled during the trial; 12 at the
control, 13 at 50%, and 12 at 100% wastewater. The plant height was in general highest
for plants grown in 50% wastewater, followed by plants in 100% wastewater and then the
control. Particularly by the end of the trial the differences between the control and plants
exposed to the wastewater were very obvious. A. donax reached heights up to 1 m and
S. fruticosa heights of 0.3 m. The difference between the species is due to their intrinsic
characteristics (Bell 1997). The concentrations of pigments in the leaves were also affected
by the treatments. Chlorophyll a and b content in A. donax leaves and S. fruticosa plants
is shown in Table 3. A. donax had significant higher concentrations in leaves of plants
grown in 100% wastewater, followed by 50% and by the control. This may be caused by
the fact that the chlorophyll concentrations are expressed on a fresh weight basis, as a
common response of plants exposed to salinity stress is to reduce the water content of the
tissue. 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 of
Sarcocornia fruticosa after 9 months growth in different concentrations of tannery wastewater
Chlorophyll (mg g1 fresh weight)
Plant species
Sarcocornia fruticosa

Arundo donax

Tannery wastewater (%)

a/b-ratio

0%
50%
100%
0%
50%
100%

0.21 0.01 NS
0.21 0.02 NS
0.20 0.01 NS
0.78 0.03 a
1.07 0.04 b
1.40 0.07 c

0.06 0.002 NS
0.10 0.01 NS
0.09 0.01 NS
0.27 0.01 a
0.63 0.04 b
0.88 0.05 c

3.43 0.15
2.09 0.06
2.24 0.07
2.86 0.04
0.80 0.10
1.78 0.19

Note: Means of 18 observations ( 1 standard error) followed by the same letter within each column and
species are not significantly different according to Duncans multiple range test at the level of p < 0.05. NS = not
significant

247

0
282

205

171

150

130

10

86

72

15

65

57

20

53

10

44

25

37

12

31

30

23

14

S. fruticosa height (cm)

(a) 35

Sampling day (d)

(b) 35

25

30 0%

50%

25 Shoots in 0% pots

Shoots in 50% pots

100%

20

Number of shoots

A. donax height (cm)

Shoots in 100% pots

20

15

15

10

10

5
282

247

205

171

130

150

86

65

72

57

44

53

37

31

0
1

23

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675

Number of shoots

TOXICITY OF HIGH SALINITY TANNERY WASTEWATER

Sampling day (d)


0%

50%

100%

Shoots in 0% pots

Shoots in 50% pots

Shoots in 100% pots

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

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


content.
In Table 4 the average P and N concentrations in the plant tissues are shown. There
was no significant difference in the concentrations of N and P in S. fruticosa exposed to
different wastewater concentrations, but there were significant differences for A. donax in
control pot and exposed to 50 and 100% wastewater, as plants watered with wastewater
always had significant higher tissue concentrations of N and P. Also, growth stimulation
of both plants when subject to 50100% wastewater occurred (increases of 3040% for
Sarcocornia and 70138% for Arundo).

676

C. S. C. CALHEIROS ET AL.

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

Plant species
Sarcocornia fruticosa

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Arundo donax

Tannery
wastewater (%)

Tissue-N (mg N g1
dry weight)

Tissue-P (mg P g1
dry weight)

0%
50%
100%
0%
50%
100%

5.17 0.48 NS
6.33 1.58 NS
5.80 0.97 NS
2.89 0.31 a
4.67 0.60 b
5.00 0.55 b

1.43 0.14 NS
1.35 0.28 NS
0.96 0.33 NS
0.23 0.03 a
0.49 0.03 b
0.58 0.05 b

Note: Means of plant material comprised in three pots ( 1 standard error) followed by the same letter within
each column and plant species are not significantly different according to Duncans multiple range test at the level
of 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 saline
aquaculture effluent (Brown et al. 1999). On the other hand, A. donax recently has been
reported 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 been
used in CW for sewage post-treatment (El Hamouri et al. 2007). The tested emergent plants
turned out to be quite resilient and appropriate for the purpose of polishing high salinity
wastewater 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 CWs
systems (CWA and CWS) was better than at the inlet as there was a reduction in several
parameters: 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 significant
influence on seed germination, shoot and root development of T. pratense (Table 5). As the
concentration of wastewater increased, inhibition of germination and growth also increased.
No germination occurred in wastewater from the CWs inlet (100%), while in effluents from
the CWs outlets (100%) some germination always occurred. Wastewater from the CW
outlets only inhibited seed germination significantly at the highest concentration tested (75%
and 100%). The shoot and root growth were actually promoted at wastewater concentrations
of 10% to 50% as compared to the control.
As in Experiment 1, the growth inhibition of the wastewater coming from the tannery
wastewater treatment plant and entering the CWs, was seen at lower concentrations (25 to
100%). The germination and development of the T. pratense that were exposed to lower
levels of the wastewater were in general better than in the controls. This stimulatory effect
of low wastewater concentrations might be due to nutrients present in the tannery effluent as
has also previously been reported (Calheiros et al. 2008). Stimulatory effects with greater
biomass of plants exposed to raw textile effluent than in controls has previously been
reported by Rosa et al. (1999), who have attributed the stimulatory effects to the nutrients
present in that effluent.

TOXICITY OF HIGH SALINITY TANNERY WASTEWATER

677

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

Sample origin

Wastewater
concentration

Germination
(%)

Shoot
length (mm)

Root
length (mm)

Biomass
(g)

0%
10%
25%
50%
75%
100%
10%
25%
50%
75%
100%
10%
25%
50%
75%
100%

85 2 ef
86 3 ef
81 4 de
73 1 d
31 4 b
0
89 2 ef
83 3 ef
80 2 de
54 2 c
14 2 a
91 2 f
85 2 ef
81 4 de
61 5 c
20 2 a

54 0.4 f
58 1 g
49 1 e
43 1 d
38 0.4 c
0
67 2 h
64 2 h
53 1 f
40 1 cd
17 0.4 a
73 1 i
67 1 h
60 2 g
48 1 e
21 1 b

34 3 c
40 2 d
35 3 cd
30 0.4 bc
26 2 b
0
65 4 fg
60 1 f
48 0.3 e
34 1 c
15 1 a
69 1 g
65 2 fg
50 1 e
31 1 bc
19 1 a

0.447 0.038
0.514 0.022
0.337 0.018
0.295 0.013
0.103 0.013
0
0.739 0.066
0.692 0.045
0.633 0.049
0.294 0.023
0.050 0.007
0.783 0.009
0.715 0.025
0.678 0.009
0.320 0.016
0.074 0.003

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Control
CWs inlet

Sarcocornia CW outlet

Arundo CW outlet

Sample 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 not
significantly different according to Duncans 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 was
found 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 for
the 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 been
treated 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 be
higher than for instance for a CW system that is used for secondary treatment. Calheiros
et al. (2008) have reported that the EC50 values of wastewater from a secondary treatment
are 28% to 41%. It is however difficult to compare results from the literature with those
obtained in the present study as different test species and different incubation conditions
and times may have been used (Rosa et al. 1999).
The application of CWs systems for the treatment of tannery wastewater has been
addressed previously and in general CWs systems have proved to be effective (Calheiros
et al. 2008, 2009a) although the salinity issue has not been assessed before. The toxicity
studies reported here show that the water quality was significantly improved by CWs
systems, 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 well
when fed with the secondary treated tannery wastewater, and showed resilience to the harsh
conditions.

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C. S. C. CALHEIROS ET AL.

(A) Germination
CW inlet
Sarco outlet
Arundo outlet
(B) Shoot length
CW inlet

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Sarco outlet
Arundo outlet
(C) Root length
CW inlet
Sarco outlet
Arundo outlet
(D) Biomass
CW inlet
Sarco outlet
Arundo outlet

20

40

60
80
EC50 (%)

100

120

140

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

CONCLUSIONS
Effluent from a conventional activated sludge system treating high salinity tannery
wastewater 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 CWs
systems planted with A. donax or S. fruticosa. Effluent from the CWs systems actually
stimulated growth of T. pratense at concentrations below 50%, and seed germination and
growth even occurred in undiluted effluent.
EC50 , the effective concentration causing 50% inhibition of T. pratense seed germination
and biomass production, was significantly lower for the CWs inlet wastewater (68% and
50%, respectively) than for the CWs effluent (83% and 75%, respectively).

TOXICITY OF HIGH SALINITY TANNERY WASTEWATER

679

Both A. donax and S. fruticosa were resilient to the high salinity conditions imposed
by 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 treating
tannery wastewater.

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ACKNOWLEDGMENTS
This work was supported by the AdI, PRIME - IDEIA Programme, through the Project
n. 70/00324 - Planticurt, including grants to Gabriela Silva and Paula V.B. Quiterio.
Cristina S.C. Calheiros wishes to thank a research grant from Fundaca o para a Ciencia
e Tecnologia (FCT), Portugal (SFRH/BPD/63204/2009).
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