E L S E V I E R Aquatic Botany 49 (1994) 183-192

Relationship between heavy metal accumulation and toxicity in Spirodela polyrhiza (L.) Schleid. and

Azolla pinnata R. Br.

J.P. Gaur ~'*, N. Noraho b, Y.S. C h a u h a n c

aCentre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221 005, India bBotany Department, Science College, Kohima 797 002, India

CBotany Department, North-Eastern Hill University, Shillong 793 014, India

Accepted 20 June 1994

Abstract

The accumulation ofCd, Cr, Co, Cu, Ni, Pb and Zn by Spirodelapolyrhiza (L.) Schleid. and Azolla pinnata R. Br. was directly related to the concentration of metals in the medium during a 4 day exposure period. The hierarchy of metal accumulation was N i > Z n > C o > C u > C d > P b > C r in S. polyrhiza and N i > Z n > C o = C d > C u > P b > C r in A. pinnata. All metals inhibited relative growth rates of test plants in a concentration- dependent manner. Depending on metal concentration in the medium, the hierarchy of metal toxicity was C d > C u = N i > C o > C r > Z n > P b for S. polyrhiza and Cd > Cr > Co > Cu > Ni > Pb > Zn for A. pinnata. The present work showed that the sulphur affinity hypothesis cannot be applied to explain variabilities in toxicities of test metals. When toxicity was evaluated in terms of metal concentration in plants the hierarchy of metal toxicity was C r > C d > C u > C o > N i > P b > Z n for S. polyrhiza and Cr > Cd > Cu > Co > Pb > Zn > Ni for A. pinnata.

1. Introduction

Increasing contamination of the aquatic environment by heavy metals poses a serious threat to biota (FSrstner and Wittmann, 1979). A great deal of informa- tion has accumulated on metal toxicity and accumulation in algae (De Filippis and Pallaghy, 1994). Notwithstanding, aquatic plants have been little explored in this regard despite the fact that they contribute significantly to nutrient and

* Corresponding author.

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184 J.P. Gaur et al. / Aquatic Botany 49 (1994) 183-192

energy dynamics of ponds and lakes. The available reports on macrophytes gen- erally describe either metal toxicity (Hutchinson and Czyrska, 1975; Seto ct al., 1979: Chigbo et al., 1982; Wang 1986, 1987; Porter and Francko, 1991 ) or ac- cumulation (Nakada et al., 1979: Nor and Chcng, 1986; Jain ct al., 1990: Benda and Kouba, 199 I; Kwan and Smith, 1991 ). Some workers have generated both metal accumulation and toxicity data in aquatic plants (Polar and Kiiciikcezzar, 1986; Charpenticr et al., 1987; Sela et al., 1989: Huebert and Shay, 1992). These latter investigations have tremendous value, as interpretation of toxicity data re- quires simultaneous metal accumulation data also (Davies, 1983 ). Few studies have compared the behaviour of several metals, and therefore it is difficult to draw conclusions about the relative toxicities of metals towards macrophytcs.

Species of Spirodela and AzoIIa are widely distributed and fast-growing aquatic vascular plants. It may be possible to use these plants in metal toxicity bioassays, or for removal of metals from wastewaters (Wang, 1986; Sela et al., 1989). This paper describes effects of seven heavy metals (Cd, Co, Cr, Cu, Ni, Pb and Zn) on Spirodela polyrhiza (L.) Schleid. and Azolla pinnata R. Br. The culture me- dium and test conditions were kept constant for various metal treatments so as to obtain comparable information. The effects of heavy metals on test plants were assessed by determining the relative growth rate (RGR). The efficiencies of test plants in accumulating test metals at their various external concentrations were also investigated.

2. Materials and methods

The test plants were collected from a waterlogged paddy field at Jakhama near Kohima (25.40°N, 94.08°E; 1460 m above mean sea-level). This area receives total annual rainfall of about 2000 mm and has a characteristic ferrugineous red soil. The test plants were cultivated in the laboratory using algal assay procedure (AAP) medium as recommended by Wang (1986). The medium was prepared using double-distilled water. The pH of the culture medium was 7.0. The stock cultures were grown at 25 _+ 1 °C under a 14 h light (photosynthetically active radiation (PAR) 45 gmol m -2 s - l ) and 10 h dark cycles. All experiments were conducted during 1992.

Analytical-grade salts were used for accumulation and toxicity studies in the following forms: Cd as cadmium acetate; Co as cobalt (II) chloride; Cu as cop- per(II) chloride; Cr as chromium trioxide; Ni as nickel(II) chloride; Pb as lead (II) nitrate; Zn as zinc sulphate. For every experiment freshly prepared salt solutions were used, and each metal toxicity assay was carried out separately. As recommended (Wang, 1990), assays were carried out in Teflon beakers because glass beakers have a tendency to adsorb metals. Various concentrations of metals were added to medium in separate beakers. Test metals were used at the following concentrations; 0.75, 1.5, 7.5, 15.0 and 75.0 ]zM Zn; 0.24, 0.44, 2.3, 4.6, 24.0, 48.0 and 96.0 gM Pb; 0.85, 1.7, 8.5, 17.0 and 85.0 gM Ni; 0.75, 1.5, 7.5, 15.0 and 80.0 ]zM Cu; 0.95, 1.9, 9.5, 19.0, 95.0 and 192.0 #M Cr; 0.85, 1.7, 8.5, 17.0 and

J.P. Gaur et al. /Aquatic Botany 49 (1994) 183-192 185

85.0 pM Co; 0.45, 0.9, 4.5, 9.0 and 45.0/LM Cd. Precipitation of metals did not occur in the culture medium. Five replicates were considered for all concentra- tions. Each beaker (capacity 100 ml) contained 50 ml of medium supplemented with test metal. Ten healthy fronds of Spirodela or Azolla were weighed in a Sar- torius electronic balance, then transferred to beakers containing various concen- trations of test metal. All beakers were kept at 25 _+ 1 °C for 4 days, and were exposed to a 14 h light (PAR 45/maol m -2 s -~ ) and 10 h dark period. After a 4 day exposure, the test plants were harvested, kept on blotting paper for a few seconds and weighed. Growth was measured as the increase in fresh weight. The RGR was calculated by the equation (Greger ct al., 1991 )

R G R = (lnw2 - lnw= ) / t

where w~ and WE respectively represent the fresh weights at the beginning and end of the time interval t.

To compare the toxicity of test metals on RGR of test plants, the results were transformed to obtain ECso, i.e. the concentration of metals giving 50% effect or

0.3 ~ c C d

0.1

0.3 . . . . . . _ ~ _ . . . . . . -~c Co

0.1 i '~c I

0.3 1 Cr

0.1

T 0.3 "~ Oc ,Y Cu

I I I

0.3 Ni

0.1 I I I

0.3 . . . . . . C

I I

0.3 I- - - - ~c 0.1 ~ Zn

0 A i i i , =1 I 0.1 1.0 10 100

Meta l in medium ( pM)

Fig. 1. Relat ive ~ o w t h rate ( R G R day -z ) o f S. po[yrhiza in presence of various concentrations of metals in the medium. Broken lines show control. Da ta arc mcazts o f five replicates; vertical bars show SD.

186 J.P. Gaur et al. / Aquatic Botany 49 (1994) 183-192

0.3 ~ < . -~ c Co

0.1 I I

Cr 0.3

C u ~ _ _ j

0.1 1.0 10 100 Meto[ in medium (pM)

0.1

0.3

0 1 ¢'w

-~ c

I c

Fig. 2. RGR ofA. pinnata in presence of various concentrations of metals in the medium. Broken lines show control. Data are means of five replicates; vertical bars show SD.

inhibition. Linear regression analysis of RGR of test plants and the concentration of metal in medium or in plant was carded out. ECso was extrapolated by fitting a line to the dose (concentration in medium or plant) response (RGR) relation- ship by least-squares using log~o of the concentration as the independent variable (Vocke et al., 1980).

Relative efficiencies of S. polyrhiza and A. pinnata in accumulating test metals were also determined after a 4 day exposure. The test plants were treated with different concentrations of metals in a manner similar to that of toxicity bioas- says. Five replicates were considered for all treatments. The test plants were har- vested after a 4 day exposure to metals, and dried at 60°C in Teflon beakers until constant weights were obtained. The dried plant materials were subsequently di- gested following the method of Bates et al. ( 1982 ). Digestion was done with con- centrated nitric acid (BDH Aristar, Poole, UK), hydrogen peroxide (30% v/v) and double-distilled water in 1:1:3 ratio ( v / v / v ) over a hot plate until a clear

J.P. Gaur et al. /Aquatic Botany 49 (1994) 183-192 187

Table 1 ECso of various test metals for the relative growth rate ofS. polyrhiza and A. pinnata; ECso data are based on either metal content of medium or plant

ECr, o (/zM metal in medium) ECso (/zM metal g- i dry weight of plants)

S. polyrhiza A. pinnata S. polyrhiza A. pinnata

Cd 0.8+0.01 0.4+0.1 6.9+0.2 4.5+0.2 Co 2.3+0.1 4.1 +0.1 10.0+0.2 11.9+0.5 Cr 7.1 +0.1 25.0+0.1 3.0+0.1 3.2+0.2 Cu 1.8+0.1 4.2__.0.1 7.9+0.4 10.1 +0.3 Ni 1.8+0.1 5.1 +0.2 22.0_+ 1.5 32.6+ 1.2 Pb 18.0_+0.1 5.3+0.1 32.5+0.5 12.4+0.8 Zn 14.3+0.1 14.5+0.1 54.0-+ 1.5 16.7+ !.5

i Mean (n=5) +SD.

I000

o Cd A Co

- - • C r

~: O Cu 100 • Ni

.E 10

.= U x

1 I I I 0.1 1 10 1 0 0

Met,,! in medium ( p M )

Fig. 3. Metal concentration in S. polyrhiza as a function of metal level in the medium. Data are means of five replicates.

solution o f abou t 0.5 ml was left. This solution was m a d e to 5 ml by the addi t ion o f 2% ( v / v ) ni tr ic acid, and t ransfer red to a plastic tube. The digested samples were analysed for meta l content with a Pe rk in -E lmer (Norwalk, CT, USA) a tomic absorp t ion spec t ropho tomete r (Model 2380) .

3. Results

Morphologica l s y m p t o m s o f metal toxici ty were evident in test plants. High levels o f meta ls generally caused yellowing and disintegrat ion o f fronds. Only Cd

188 J.P. Gaur et al. /Aquatic Botany 49 (1994) 183-192

~100 =L

.il 10 .£

0

0 Cd & Co • Cr O Cu • Ni

• Pb • Zn

1 I I 0.1 1 10 100

Metal in med ium ( p M )

Fig. 4. Metal concentration in A. pinnata as a function of metal concentration in the medium. Data are means of five replicates.

(9.0/zM) and Cu (7.5 gM ) caused root shedding in S. polyrhiza and A. pinnata. Heavy metals lowered the RGR of test plants in a concentration-dependent man- ner (see Fig. 1 for S. polyrhiza and Fig. 2 for A. pinnata). Cd caused most pro- nounced inhibition of RGR even at its lowest tested concentration (0.45 #M). Other metals, however, elicited toxic effects at much higher concentrations. Pb and Zn were least inhibitory to test organisms. The data on RGR of test plants were utilized to calculate ECso of various test metals. ECso values for the RGR of S. polyrhiza and A. pinnata are presented in Table 1. RGR was found to be neg- atively correlated with metal concentration in the medium. A. pinnata showed greater sensitivity to the metals in comparison with S. polyrhiza. The order of toxicity of metals towards RGR was Cd > Cu = Ni > Co > Cr > Zn > Pb for S. po- lyrhiza and Cd > Cr > Co > Cu > Ni > Pb > Zn for A. pinnata.

The ability ofS. polyrhiza and A. pinnata to accumulate Cd, Co, Cr, Cu, Ni, Pb and Zn was tested at different external concentrations of these metals after a 4 day exposure period. Metal accumulation data for S. polyrhiza and A. pinnata are presented in Figs. 3 and 4, respectively. The accumulation of heavy metals by test plants was dependent on metal concentration in the medium. Both test plants accumulated Ni maximally, and Cr was accumulated the least. A highly signifi- cant positive correlation was observed between metal concentration in plant and metal concentration in the medium for S. polyrhiza and A. pinnata (data not shown). The calculation of concentration of metal in plants causing 50% inhibi- tion of RGR (ECso) showed the following hierarchies of toxicities: C r > C d > C u > C o > N i > P b > Z n in S. polyrhiza and C r > C d > C u > C o > Pb > Zn > Ni in A. pinnata.

J.P. Gaur et al. / Aquatic Botany 49 (1994) 183-192 189

4. Discussion

All test metals (Cd, Co, Cr, Cu, Ni, Pb and Zn) were found to reduce the growth of S. polyrhiza and A. pinnata. Thus, present findings agree with previous work on metal toxicity to species of Lemna (Hutchinson and Czyrska, 1975: Nasu and Kugimoto, 1981; Jain et al., 1990: Huebert and Shay, 1991, 1992) and to other aquatic plants (Hutchinson and Czyrska, 1975; Sela et al., 1989). Charpentier ct al. (1987) observed stimulation of growth of Spirodela polyrhiza at low concen- trations (0.02-0. I mg I -~ ) of Cd; however, this phenomenon was not observed in the present study despite the fact that the same species was employed.

The results of the uptake studies performed while varying the initial concentra- tion of heavy metals showed that S. polyrhiza and A. pinnata have tremendous ability to remove metal ions from solutions over a wide range of concentrations. The positive relationship between the degree of metal accumulation by test plants and external metal concentration was statistically significant in all the treat- ments. Thus, present findings are in agreement with previous reports on metal accumulation (Landolt, 1986; Hagcmcyer and Waisel, 1989: Kwan and Smith, 1991 ). None the less, Charpentier et al. (1987) could not find a significant rela- tionship between Cd concentration in A. polyrhiza and Cd concentration of the external environments. It has been often suggested that bioaccumulation of a par- ticular metal is a function of its ionic level, and is not linearly related to total concentrations in waters rich in natural metal chelators (De Filippis and Pal- laghy, 1994). The present work, however, shows that the accumulation ofCd, Cr, Co, Cu, Ni, Pb and Zn in test plants was directly related to total metal concentra- tion in the culture medium. This relationship was probably obtained because a chelator-free inorganic medium was used for cultivating S. polyrhiza and A. pin- nata. It should, howcvcr, be kept in mind that similar experiments with natural waters must define the ionic concentrations of heavy metals. S. polyrhiza and A. pinnata accumulated Ni maximally and Cr minimally. No effort was made to differentiate metal accumulation in different plant parts, although Charpenticr ct al. (1987) and Scla ctal. (1989) observed greater accumulation of metals in roots in comparison with other parts of floating macrophytcs. This is perhaps the reason why root senescence and shedding occurred in S. polyrhiza and A. pinnata after exposure to Cd and Cu, which were perhaps accumulated maximally in thc roots of test plants.

Depending on the metal concentration in the medium, thc order of metal tox- icity to S. polyrhiza was Cd > Cu-- Ni > Co > Cr > Zn > Pb and the order of metal accumulation was Ni > Zn > Co > Cu > Cd > Pb > Cr. In A. pinnata, however, the orders were Cd > Cr > Co > Cu > Ni > Pb > Zn for metal toxicity to RGR and Ni> Zn > Co = Cd > Cu > Pb > Cr for metal accumulation. Although not accu- mulated maximally in both test plants, Cd exerted most toxic effects at high ex- ternal concentrations. Pb was not accumulated much by the plants and was found to be the least toxic. Zn was not very toxic to test plants despite the fact that test plants could accumulate high concentrations of this metal. The hierarchy of metal toxicity, based on metal accumulation by test plants, showed an interesting trend.

190 J.P. Gaur et aL /Aquatic Botany 49 (1994) 183-192

Despite accumulation of very low levels of Cr in test plants, this metal proved to be the most toxic. A. pinnata showed Cr toxicity at a far lower external Cr con- centration than S. polyrhiza.

The primary toxicity mechanisms of different metals may be as different as their chemical properties, especially valence, ion radius and capacity to form or- ganic complexes (Barcelo and Poschenrieder, 1990). Fisher and Jones (1981 ) found a highly significant correlation of metal toxicities with ( 1 ) insolubility of corresponding metal sulphides, and (2) stability constants for metal complexes formed with sulphur-containing amino acids. According to these workers, there could be a common mechanism of metal action in all living organisms, and the differences in metal toxicities may be due to variable affinities of metal ions for sulphur complexation. If this hypothesis is valid, metals having a greater affinity for sulphur (e.g. Cu, Pb) should be more toxic than those having lesser affinity for sulphur (e.g. Cd). This hypothesis is not acceptable, as Cd was found to be more toxic to test plants than Cu or Pb.

It has been often observed that toxicity of a metal depends on the concentra- tion of its ionic species (De Filippis and Pallaghy, 1994 ). Natural waters contain several complexing agents (such as humic acid and fulvic acid), which can che- late metals and thereby reduce the concentration of free metal ions. Thus, mea- surement of total amount of metals in a water may not provide useful informa- tion. The problem can be resolved either by measuring concentration of ionic species of metals or by measuring metal concentration in plants (Davies, 1983 ). The former option may not be rewarding, as evidence for uptake of metal chelates does exist (Taylor and Foy, 1985). The present work shows that the measure- ment of metal content of plants may allow the assessment of toxic effects likely to be caused by metals. This approach should prove useful in metal toxicity bioas- says of natural waters as well.

Acknowledgements

Professor D.T. Khathing allowed the use of atomic absorption spectrophoto- meter at RSIC, North-Eastern Hill University, Shillong. One of the authors (N.N.) thanks the Principal, Science College, Kohima, for help in granting him a study leave.

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