… on the chlorophyll concentration of i typha latifolia i plants, growing in a substrate...

The effect of heavy metals accumulation on the chlorophyllconcentration of Typha latifolia plants, growing in a substrate

containing sewage sludge compost and watered withmetaliferus water

Thrassyvoulos Manios a,*, Edward I. Stentiford b, Paul A. Millner c

a Department of Agricultural Technology, Technological Education Institute of Crete, Heraklion, 71110 Crete, Greeceb School of Civil Engineering, Leeds University, Leeds LS2 9JT, UK

c School of Biochemistry and Molecular Biology, Leeds University, Leeds LS2 9JT, UK

Received 20 April 2002; received in revised form 20 September 2002; accepted 15 December 2002

Abstract

Typha latifolia plants, commonly known as cattails, were grown in a mixture of sewage sludge compost, commercial

compost and perlite. Four groups (A, B, C and D) were irrigated (once every 2 weeks) with a solution containing

different concentrations of Cd, Cu, Ni, Pb and Zn, where in the fifth (group M) tap water was used. At the end of the 10

weeks experimental period the mean concentration of Ni, Cu and Zn in the roots and leaves of the plants in the four

groups was significantly larger to that of the plants of group M. A linear regression test satisfactorily correlated the

metals concentrations in the irrigation solutions with the metals concentration in the leaves and roots of groups A, B, C

and D. The concentration of total chlorophyll, chlorophyll a (chla) and chlorophyll b (chlb) in the leaves of the

developing plants was also monitored in 2 weeks intervals. Groups A, B, C and M presented an increasing

concentration of total chlorophyll, with time. In group D (stronger solution), the mean total chlorophyll concentration

was reduced from 1080.69 mg/g fresh weight (f.w.) in the 8th week to 715.14 mg/g f.w., in the 10th week, a probableevidence of inhibition. When statistically tested, it was suggested that there was no significant difference between the

mean chlorophyll values of the groups in each set of samples, concluding that no significant toxic action was imposed in

the plants by the metals. However, when similar statistical analysis was implemented in the ratios of chla and chlb, there

was significant reduction of the ratios in groups D plants, suggesting some increase in chlorophyll hydrolysis due to the

metals accumulation (toxic effect) in comparison with the other groups.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Typha latifolia ; Heavy metals; Sewage sludge compost; Wastewater; Constructed wetlands; Chlorophyll

* Corresponding author. Tel.: /30-810-379-400; fax: /30-810-318-204.E-mail address: [email protected] (T. Manios).

Ecological Engineering 20 (2003) 65/74

www.elsevier.com/locate/ecoleng

0925-8574/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0925-8574(03)00004-1

1. Introduction

Constructed reed beds or wetlands are claimed

to be low cost, low technology systems able to

treat a variety of wastewaters. In Europe such

systems have been successfully used for treating

domestic sewage for small communities (less than

2000 people equivalent; Green and Upton, 1994;

WRc and Severn Trent Water Plc, 1996; Obarska-Pempokowiak and Klimkowska, 1999). Some-

times such systems are used for the removal of

heavy metals from highways runoff (Mungur et

al., 1995) and acid mine drainage (Mitchel and

Karathanasis, 1995). Metals are removed from

wastewater by plant uptake, chemical precipitation

and ion exchange and adsorption to settled clay

and inorganic compounds (Gearheart, 1992; Mar-tin and Johnson, 1995; Obarska-Pempokowiak

and Klimkowska, 1999). It is likely that the

potential capacity of reed beds to remove metals

by plant uptake and harvesting will be small and

that the ultimate removal of metals from wetland

systems is probably most effectively achieved by

chemical precipitation (Gearheart, 1992; Mungur

et al., 1995; Mitchel and Karathanasis, 1995;Martin and Johnson, 1995; Obarska-Pempoko-

wiak and Klimkowska, 1999).

Under that perspective an effort was made to

improve the substrates performance by using

materials containing large amounts of organic

mater as suggested by a number of researchers

(Gearheart, 1992; Mungur et al., 1995; Mitchel

and Karathanasis, 1995). Sewage sludge compostwas used in experiments conducted in the UK

using Typha latifolia plants and artificial waste-

water (Manios, 2000). The choice of sewage sludge

compost as part of the substrate was based in its

large composition of organic matter and the need

for investigating alternative applications for sew-

age sludge, under the large constrains imposed in

the agricultural use (EU Directive 86/278/EEC).The results indicated that the use of such material

could improve substantially the ability of a sub-

strate to retain heavy metals from wastewater

(Manios, 2000).

However, the use of sewage sludge compost with

large heavy metals concentration could result in an

increase in the metals accumulation in the plants

roots and leaves. Such a phenomenon may have aneffect in the plants development and health. Heavy

metals accumulation in the tissue of different

plants resulted in a decrease of the biomass and

the chlorophyll concentration in the leaves/stems

(Burzynski and Buczek, 1989; Ouzounidou et al.,

1992; Sharma and Gaur, 1995; Abdel-Basset et al.,

1995).

The main aim of this research was to study theeffect of heavy metals accumulation in the devel-

opment of T. latifolia plants growing in sewage

sludge compost containing substrate and watered

with artificial wastewater, by monitoring the

concentration of chlorophyll in their leaves during

a short period experiment. This would allow to

make a primary assumption of the combined effect

in the plants growth and health of the heavymetals existing in both the compost and the

wastewater.

2. Materials and methods

The sewage sludge compost was produced by

Thames Water Plc using a windrow system with

sewage sludge and straw on a 1:1 basis by volume

(v/v). The chemical characteristics of the sewage

sludge and the produced compost are shown inTable 1. The final material used in the pots was a

mixture of this compost with commercial peat

Table 1

Typical characteristics of the sewage sludge and the produced

compost (supplied by Thames Water Plc)

Parameters Sewage sludge Compost

Dry matter (% ww) 25.2 31

Volatile solids (% dw) 66.4 65

pH 6.7 7.9

Total-P (% dw) 2.3 2.6

Cu (mg/kg dw) 599 525

Zn (mg/kg dw) 728 825

Ni (mg/kg dw) 99 68

Cd (mg/kg dw) 1.2 1.5

Pb (mg/kg dw) 191 189

Cr (mg/kg dw) 134 118

Hg (mg/kg dw) 2.5 2.6

As (mg/kg dw) 2.5 1.9

Se (mg/kg dw) 2.0 1.9

T. Manios et al. / Ecological Engineering 20 (2003) 65/7466

based compost 25% v/v and perlite 25% v/v. The

use of peat and perlite was considered as necessary

in order to avoid any phytotoxic phenomena from

the compost. It is well established that mixtures

containing more than 50% v/v mature compost of

any origin can produce some kind of phytotoxicity

(Manios et al., 1989). The pH of the mixture was

7.1 and the concentrations of Cd, Cu, Ni, Pb and

Zn were 5.79/1.1, 5679/34.71, 479/6.82, 1309/12and 7459/21.50 mg/kg dw, respectively, (9/stan-dard deviation from analysing three samples).

A large number of small and healthy T. latifolia

plants (originally gathered from a local lagoon)

were left in the substrate for 6 weeks to adjust to

their new environment. At the end of that period

30 of them were selected for the experiment. The

plants and pots were separated into five groups

with six replicas (pots) in each. The selection was

based in the height and the number of leaves of

each plant in order to achieve a uniformity among

groups. Groups A, B, C and D were the groups,

which would be watered with the heavy metals

solution, and Group M which would be used as

blank and watered with tap water.

Table 2 shows the concentrations of metals used

for the different groups, ordinary tap water was

used to make up the solutions. These concentra-

tions are multiples of those found in domestic

wastewater and considerable higher than those

found in other types of wastewater, as for example,

acid mine drainage (Mitchel and Karathanasis,

1995). Such high concentrations would be suffi-

cient to create a substantial effect in the plants

development in the considerable short experimen-

tal period (Cheng et al., 2002).

PVC pots were used with an average diameter of

200 mm and a height of 200 mm, with a usable

volume of 5.0 l. The trays were large enough to

retain any drainage water enabling it to be

reabsorbed by the soil in the pot. Each pot of

each group was given 1 l of the groups solution

every 2 weeks. The solution was added slowly to

the surface of the soil taking care not to spill any

on the leaves or outside the pot. In total five

waterings (week 0, 2, 4, 6 and 8) with the heavy

metal solutions took place over a period of 10

weeks and six sets of leaves tissue samples

(sampling in week 0, 2, 4, 6, 8 and 10) for each

group were gathered. Each set was combined by a

sample from each plant of each group (total of 30

samples per set). The samples were collected from

leaves of similar age and development, about 1/2cm2 and wrapped into marked aluminium foil.

They were then flash frozen in liquid nitrogen and

either stored at /20 8C, or analysed directly.Total chlorophyll concentration is a unifying

parameter for indicating the effect of specific

interventions. However, it is important to record

changes in the two components of chlorophyll,

chlorophyll a (chla) and chlorophyll b (chlb) and

especially their ratio. This is due to the fact that

heavy metals could affect each component at a

different level creating changes in some part of

plants physiology and not in others. Concentra-

tions of chla and chlb and total chlorophyll (total

Table 2

Concentration of heavy metals in each experimental groups solution and the solutions pH

Cd (mg/l)

[Cd(NO3)24H2O]

Cu (mg/l)

[Cu(NO3)25H2O]

Ni (mg/l)

[Ni(NO3)26H2O]

Pb (mg/l)

[Pb(NO3)2]

Zn (mg/l)

[Zn(NO3)26H2O]

pH

Group

M

/ / / / / 7.53

Group

A

0.5 10 5 5 10 6.55

Group

B

1 20 10 10 20 6.32

Group

C

2 40 20 20 40 6.30

Group

D

4 80 40 40 80 6.15

T. Manios et al. / Ecological Engineering 20 (2003) 65/74 67

chl/chla/chlb), were calculated using the meth-odology developed by Arnon (1949).

At the end of the tenth week the plants were

carefully uprooted, washed thoroughly with water

and soap and rinsed twice with distilled water in

order to remove any soil particles from the

substrate. Such particle could effect considerable

the measured metals concentration, due to the

presence of sewage sludge in the substrate. Afterwashing, the roots and leaves were separated and

put in weighed paper bags and dried (80 8C for 72h). One gram of the leaves tissue (six replicas per

group), were put in special digestion tubes (Buchi

430 Digestor). Concentrated (97%) nitric acid was

put in the tubes and the tubes were retained for 24

h at room temperature. After this initial digestion

the samples were digested for 4 h at a range oftemperatures. For the first hour the temperature

was 100 8C, for the second hour 150 8C andfinally 200 8C for 2 h (Sposito et al., 1983). Theremaining liquid, which most times was about one

quarter of the original acid dilution, was filtered

using Whatman GF/C paper filters. De-ionised

water was added until the new solution reached the

volume of the acid originally used (25 ml). Thesamples were then analysed using an atomic

absorption spectrophotometer (A.A.S., Spectra

AA-10).

In order to evaluate statistically any significant

differences among mean values, a single factor

ANOVA test was used. In all tests the significance

level at which we evaluated critical values differ-

ences was 5%. Linear regression was used forevaluating the effect of metals concentration in the

watering solutions in the mean metals concentra-

tion in the plants biomass.

3. Results and discussion

As the experiment progressed, the amount of

metals present in each pot increased in all groups,with the exemption of the blank, for which

remained the same and equal to the original

amount of metals existing in the substrate. Re-

spectively, and according to Tables 3 and 4, the

mean concentration of Cu, Ni and Zn in the roots

and leaves of the plants, at the end of the

experiment, was larger in groups A, B, C and D

compared with group M. This difference was

significant (5% level) according to a single factor

ANOVA test, in both roots and leaves/stems of the

plants, among all five groups. Since the use of

different solutions for the irrigation of the plants

was the only notable difference among the groups,

is safe to suggest that the differentiation in the

concentration of metals in the plants biomass

should be correlated with the watering pattern.

In order to support this theory a linear regres-

sion test was used to correlate the concentration of

each metal in the irrigation solutions with the

relevant concentration in the roots and leaves/

stems, through the groups. The results indicated

that there was a strong linear relationship among

these variables, with the minimum r2 value higher

than 0.7 (Table 5). Based in these two statistical

Table 3

Mean metals concentration (mg/kg d.w.) in the roots of the

plants of each group at the end of the experiment

Group Cu Ni Zn

M 40.009/14.14a 30.009/8.16a 293.339/28.09a

A 46.679/12.47b 38.339/12.13ab 300.009/20.00a

B 45.009/9.58b 45.009/15.00b 330.009/21.60b

C 60.009/10.00c 51.679/10.67b 361.679/36.25bc

D 93.339/12.47d 55.009/9.57b 391.679/19.51c

Six replicate per group, (9/) S.D. The mean values followedby different superscripts within each column indicate that they

were significantly different at a probability level of 0.05

according to ANOVA test.

Table 4

Mean Cu, Ni and Zn concentration (mg/kg d.w.) in the leaves

of the plants of each group at the end of the experiment

Group Cu Ni Zn

M 9.179/3.44a 17.509/6.92a 34.189/15.38a

A 10.839/4.48ab 21.679/8.98ab 48.339/12.14ab

B 10.839/5.34ab 25.009/9.58ab 58.339/10.68b

C 14.179/4.48b 27.679/4.53b 55.839/8.38b

D 15.009/7.64b 27.509/3.82b 60.839/13.04b

Six replicate per group, (9/) S.D. The mean values followedby different superscripts within each column indicate that they

were significantly different at a probability level of 0.05

according to ANOVA test.


tests is safe to suggest that the metals accumulated

in the roots-leaves origin mostly from the artificial

wastewater for three reasons: (a) the metals in the

wastewater were in the easily absorbable by the

plants form of diluted inorganic salts, (b) the

majority of the metals in the compost were

retained by the colloids delaying their release inthe water solution, and (c) the short duration of

the experiment did not allow the plants adequate

time in order to absorb metals from the substrate.

The concentrations of metals in the roots and

leaves, as presented in Tables 3 and 4, are some of

the larger recorded in literature for cattail plants

(Mungur et al., 1995; Ye et al., 1997a).

According to Burzynski and Buczek (1989),Sharma and Gaur (1995), Salt et al. (1995),

Abdel-Basset et al. (1995), Rai et al. (1995) and

Ewais (1997), there is a threshold of tolerance in

each plant to the heavy metals accumulation. For

a number of environmentally, physiologically and

genetically determined reasons this threshold is

different among plants species. When this limit is

crossed then the toxic effect of the metals in theplants, takes its toll.

If the amount of metals accumulated in the

tissue of the leaves/stems had crossed the tolerance

threshold of the T. latifolia plants in groups A, B,

C and D, then there should have been some

decrease of the total chlorophyll concentration

(Gadallah, 1994; Sharma and Gaur, 1995). Figs.

1/3 suggest that in groups A, B, C and M, thechlorophyll concentration increases with time. In

Fig. 2 and for group D the mean chlorophyll

concentration from 1080.69 mg/g fresh weight(f.w.) in the eighth week, dropped in 715.14 mg/gf.w. 2 weeks and a watering later. This could be an

indication of some inhibition of growth. However,

when a single factor ANOVA test (5% significance

level) was used, suggested that there was no

significant difference among the mean values of

the five groups, in all six set of samples. This can

be interpreted as a failure of the accumulated

metals to effect the chlorophyll concentration, in

the leaves.

According to Gadallah (1994), Drazkiewicz(1994), Abdel-Basset et al. (1995), Sharma and

Gaur (1995) and Ewais (1997), changes in the

concentration of chl a and b and particularly

changes in their ratio are an equal important

parameter, which should always been taken under

consideration when estimating the effect of an

environmental parameter (as irrigation with meta-

liferous wastewater) in plants. Ewais (1997) usedCyperus difformis L., Chenopodium ambrosiodes L.

and Digitaria sanguinolis L., Sharma and Gaur

(1995) used Lemna polyrrhiza (duckweed) and

Abdel-Basset et al. (1995) used two algae species

(Chlorella fusca and Kirchneriella lunaris ) to

evaluate the effect of heavy metals in total

chlorophyll concentration. All three agreed that

heavy metals accumulation, responsible for thereduction of total chlorophyll concentration, had a

similar negative effect in the ratio of chla to chlb.

This occurs due to a faster hydrolysis ratio of chla

compared with chl b when plants are under stress

(Schoch and Brown, 1987; Drazkiewicz, 1994;

Abdel-Basset et al., 1995).

Table 6 presents the mean ratios of chla to chlb

in all five groups for all six set of samples. A singlefactor ANOVA test was used in order to evaluate

the significance (5% level) of the different between

the ratios. For the first five sets of samples

(columns-week 0/week 8) in Table 6, there wasno significance difference indicating no signifi-

cance effect of the metals in the plant. For the

sixth set (week 10) there was a significant differ-

Table 5

The r2 values of the linear regression correlation between the concentration of Cu, Ni and Zn in the irrigation solutions and their mean

concentration in the leaves and roots of groups A/D

Concentration of metals in the solutions

Cu Ni Zn

Mean concentration of metals in the roots 0.813 0.979 0.998

Mean concentration of metals in the leaves 0.869 0.860 0.700


ence, which should be considered as an evidence of

some effect of continues waterings in the plants.

However, this can not indicate which solution (A/D) or water (M) was responsible.

As presented in Table 6, there is no significance

difference in the ratio for groups A/C and M,between the consecutive waterings (rows-week 0/week 10) suggesting no effect of the irrigation

solution and water, their chlorophyll production

and health. On the contrary in group D, there is a

significant difference (ANOVA) in the ratios of

chla and chlb, among the six sets of samples,

suggesting a significant effect of the five waterings

with the stronger solution (Table 2) in the plants,

health and development.These results are in agreement with relevant

research by Burzynski and Buczek (1989), Sharma

and Gaur (1995), Abdel-Basset et al. (1995), Salt et

al. (1995), Rai et al. (1995), Ewais (1997) and

Manios et al., (2002), suggesting that for the plants

used in this experiment (T. latifolia ) and under the

conditions of growth (sewage sludge in the sub-

strate, metals solution for irrigation) the metals

accumulation became toxic only in group D and

only after the last watering. In all other groups

plants did not present any toxic symptom which

Fig. 1. Changes in the concentration of total chlorophyll (j), chla ( ) and chlb (I) in the plants of groups A and B.


could be correlated with the metals accumulation

in the plants biomass. On the contrary the

chlorophyll concentration was increasing with

time phenomenon, which can be explained

through the following assumptions:The tolerance threshold of the T. latifolia plants

was not reached in those groups, during the

experimental period. Mitchel and Karathanasis

(1995) and Ye et al. (1997a) in relevant experi-

ments with T. latifolia plants indicated the in-

creased tolerance of the plant in both the presence

and the accumulation of heavy metals. None of the

authors determined a specific threshold since that

would be affected by the age and development

stage of the plant, the concentration of metals in

substrate and solution, the different metals in the

solution and their independent concentration, the

pH of the solution and the environmental condi-

tions. It is safe, though, to suggest, based on

literature and the above presented results, that for

the plants in Groups A, B, C and M, this threshold

was not reached.

The concentration of metals in the solutions was

not very large, providing the plants with an

amount of metals which would accelerate their

growth instead of inhibiting it. Most of the metals

used in the solution are necessary, in some small

amounts, for plants development (Streit and

Fig. 2. Changes in the concentration of total chlorophyll (j), chla ( ) and chlb (I) in the plants of groups C and D.


Stumm, 1993; Salt et al., 1995). The concentration

of the metals in Groups A, B and especially C

could not be considered as small. It could be

suggested that in the beginning of the experiment

the metals offered in the plants did help them to

develop better by covering their needs in easily

absorbed micronutrients. As time progressed, the

amount of metals added in each pot exceeded by

much the necessary amount of metals for the

development of the plants. As so, this parameter

should not be regarded as substantial.

The tolerance threshold was pushed upwards

due to the existence of a growth acceleration factor

in the solution. Wong et al. (1997) irrigated

Aegiceras corniculatum with wastewater of differ-

ent strength, containing heavy metals. The plants

watered with the lower concentration wastewater

produced higher yield than the blank and any

other group. The authors explain this positive

effect due to the nutrients existing in the waste-

water. The plants supported by the easily absorbed

micro and macronutrients were able to overcome

the toxic effect produced by the low heavy metals

concentration. However, when the amount of

metals became larger, then the yield was decreased

as the plant could not overcome the metals

toxicity. In this specific experiment the toxic

threshold of the heavy metals for Aegiceras

Fig. 3. Changes in the concentration of total chlorophyll (j), chla ( ) and chlb (I) in the plants of group M.

Table 6

The mean ratios of chla to chlb concentration in all five groups for all six sets of samples

Group

M A B C D

Week 0 2.8679/0.014a* 2.8659/0.029a* 2.8789/0.007a* 2.8679/0.040a* 2.8909/0.017a*

Week 2 2.8179/0.097a* 2.8689/0.014a* 2.8629/0.012a* 2.8559/0.082a* 2.8749/0.010a*

Week 4 2.8779/0.09a* 2.8889/0.014a* 2.8339/0.045a* 2.8709/0.008a* 2.8099/0.132ab*

Week 6 2.7369/0.232ab* 2.8459/0.014a* 2.8079/0.038a* 2.8239/0.024a* 2.8369/0.025a*

Week 8 2.6179/0.174b* 2.7909/0.009ab 2.7209/0.306a* 2.8329/0.079a 2.7809/0.062b

Week 10 2.7679/0.067ab* 2.7629/0.055b* 2.7299/0.116a* 2.8479/0.032a 2.5819/0.150c$

Six replicate per group, (9/) S.D. The mean values followed by different superscripts letters within each column indicate that theywere significantly different at a probability level of 0.05 according to ANOVA test. The mean values followed by different superscripts

symbols within each row indicate that they were significantly different at a probability level of 0.05 according to ANOVA test.


corniculatum, was pushed upwards by the exis-tence of the nutrients (growth factor). The same

effect has been recorded by Sharma and Gaur

(1995) when working with Lemma polyrrhiza

(duckweed) plants and Gadallah (1994) when

working with Phaseolous vulgaris (bush bean).

In this experiment the growth factor offered to

the plants was the NO3 diluted in the artificial

wastewater through the metals containing saltsused for the solutions production. In Groups A/C the toxicity of the metals absorbed by the plants

was overruled by the accelerated growth of the

plants due to the nitrogen availability. In Group D

the concentration of the metals became so large,

that the increased presence of nitrogen could not

any more balance the toxicity, and chlorophyll

production after the fifth consecutive watering waseffected. Similar results were presented by Ouzou-

nidou et al. (1992), Ye et al. (1997a,b), Ewais

(1997) and Wong et al. (1997).

4. Conclusions

The sewage sludge compost used as a substrate

component in this experiment was highly contami-nated with heavy metals which however did not

impose any significant effect in the development

and growth of the cattails (T. latifolia ) plants.

There was a significant increase in the metals

concentration in the plants tissue (roots and

leaves), which was sufficiently correlated with the

metals in the watering solutions and not with the

metals in the substrate. There was some inhibitionin the plants growth (noticed through a reduction

in chla to chlb ratio) recorded in the plants

irrigated with the stronger of the watering solu-

tions. In the same plants the larger heavy metals

concentrations were also recorded. This toxicity

was significantly correlated with the watering

pattern and not the substrate. In conclusion it is

safe to suggest that there were no evidence ofincompatibility in the use of sewage sludge and T.

latifolia plants in wetlands treating metaliferus

wastewater. Additionally monitoring total chl

concentration and chla to chlb ratio can be used

as an early warning systems for the toxic effect of

metals accumulation in plants.

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The effect of heavy metals accumulation on the chlorophyll concentration of Typha latifolia plants, growing in a substrate contIntroductionMaterials and methodsResults and discussionConclusionsReferences

… on the chlorophyll concentration of i typha latifolia i plants, growing in a substrate...

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