685

Th e morphological eff ects of heavy metal stress on the nodulation ability of Rhizobium spp. and growth of white clover (Trifolium repens L.) were studied in the laboratory under controlled conditions. Fourteen topsoils were collected from an area with elevated metal concentrations (Cd, Zn, and Pb). White clover was cultivated using a specialized “rhizotron” method to observe the development of root and nodule characteristics. Results show eff ects of increasing heavy metal concentrations on nodulation development, especially the nodulation index (i.e., the number of nodules per gram of the total fresh biomass). A signifi cant decrease in nodulation index was observed at about 2.64 mg Cd kg–1, 300 mg Zn kg–1, and 130 mg Pb kg–1 in these soils. Th e sensitivity of the nodulation index in relation to other morphological characteristics is discussed further. It is proposed that the nodulation index of white clover is a suitable bioindicator of increased heavy metal concentrations in soil.

White Clover Nodulation Index in Heavy Metal Contaminated Soils–

A Potential Bioindicator

Nicolas Manier* and Annabelle Deram Université Droit et Santé de Lille

Kris Broos Flemish Institute for Technological Research

Franck-Olivier Denayer and Chantal Van Haluwyn Université Droit et Santé de Lille

Plants acquire N by assimilation of NO3

– and NH4+ from

the soil solution, or from atmospheric N2 fi xation through

a symbiotic association with N2–fi xing bacteria. Th is symbiotic

process of N2 fi xation is typical for leguminous plants that are

able to take up signifi cant amounts of N by forming nodules

on their roots in symbiosis with a particular bacteria species

called Rhizobium. Th ere are numerous factors that can aff ect this

relationship (De Ming and Martin, 1986; McGrath et al., 1988;

Forde and Lorenzo, 2001; Peralta-Videa et al., 2001; Wolf and

Rohrs, 2001; Tsvetkova and Georgiev, 2003; Broos et al., 2005a)

often resulting in stresses on the nodulation process, the N2–

fi xing ability, or an alteration in the structure of the root nodules.

Soil inorganic N content, acidic pH, and elevated heavy metal

concentrations in soil seem to be the most important factors that

can infl uence legume nodulation (Ye et al., 2001).

Th e toxicity of heavy metals on the nodulation process of legu-

minous plants has previously been examined in laboratory studies

(Obbard and Jones, 1993; Obbard et al., 1993). In general, increas-

ing concentrations of heavy metals in soil tend to reduce the nodu-

lation of leguminous plants (Van-Rossum et al., 1994; Ibekwe et

al., 1995; Zornoza et al., 2002). For example, Chen et al. (2003)

showed that nodulation of the roots of soybean (Glycine max L.)

was greatly inhibited by the addition of Cd, especially on the ad-

dition of 10 and 20 mg Cd kg–1 dry soil. Th e highest level of Cd

addition (20 mg Cd kg–1 dry soil) almost completely inhibited the

root nodulation, particularly at the seeding stage of the soybean. A

similar study showed that a relatively low number of nodules per

root of white lupin (Lupinus albus L.) was obtained with increasing

concentrations of Zn applied to the soil (Pastor et al., 2003). In a

more recent study, increasing levels of Cu2+ in solution have been

found to reduce root hair formation in cowpea (Vigna unguiculata

L.). Furthermore, the root hairs formed were also short, stubby, and

not curled (Kopittke et al., 2007). Th e authors concluded that nod-

ulation was more sensitive to increasing Cu2+ concentrations than

plant growth due to the reduction in potential infection sites.

Abbreviations: AFNOR, French Association for Normalization; CEC, cation exchange

capacity; ICP AES, Inductively Coupled Plasma–Atom Emission Spectrometry; ICP

MS, Inductively Coupled Plasma– Mass Spectrometry; PAR, photosynthetically active

radiation; RI, robustness index.

N. Manier, A. Deram, and F.-O. Denayer, Institut Lillois d’Ingénierie de la Santé (ILIS),

and C. Van Haluwyn, Département de Botanique, Université Droit et Santé de Lille, EA

2690, 42, rue Ambroise Paré, 59120 Loos, France; K. Broos, VITO, Flemish Institute for

Technological Research, Boeretang 200, B-2400 Mol, Belgium.

Copyright © 2009 by the American Society of Agronomy, Crop Science

Society of America, and Soil Science Society of America. All rights

reserved. No part of this periodical may be reproduced or transmitted

in any form or by any means, electronic or mechanical, including pho-

tocopying, recording, or any information storage and retrieval system,

without permission in writing from the publisher.

Published in J. Environ. Qual. 38:685–692 (2009).

doi:10.2134/jeq2008.0013

Received 10 Jan 2008.

*Corresponding author (nmanier@gmail.com).

© ASA, CSSA, SSSA

677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS: HEAVY METALS IN THE ENVIRONMENT

686 Journal of Environmental Quality • Volume 38 • March–April 2009

Due to the sensitivity of the nodulation process toward in-

creasing heavy metal concentrations, it may be possible to use

the nodulation as a bioindicator for soil quality. Several studies

concerning legume nodulation process and heavy metals based

on a fi eld-experiment (nonhydroponic culture system) have

been published (McGrath et al., 1988; Ibekwe et al., 1995;

Chaudri et al., 1993, 2008). Nevertheless they mainly deal

with the symbiotic Rhizobia population in soil. Studies con-

cerning the morphology of root nodules and heavy metals are

still few and generally based on hydroponic culture systems,

inoculated with a specifi c symbiotic bacteria and literature is

generally related to monocontamination with Cd or Zn (Zor-

noza et al., 2002; Chen et al., 2003; Pastor et al., 2003).

Th erefore, we decided to study the development and the mor-

phological variations in nodulation signs of white clover when

grown in soils contaminated with a mixture of heavy metals.

Consequently, the aims of the study are: (i) to assess the

ability of white clover to grow in anthropogenic soils contami-

nated with heavy metals and (ii) to observe the variability of

nodulation characteristics in contaminated soils through a di-

rect approach, without inoculation (i.e., using the indigenous

population of rhizobia present in each soil). In conclusion we

discuss the sensitivity and the robustness of the nodulation

index as an ecotoxicological endpoint, to use this index as a

bioindicator of heavy metal contaminated soils.

Materials and Methods

Site Selection and Soil Sampling ProceduresTh e study was performed in northern France and the study

site was selected because of high soil concentrations of heavy

metals (Cd, Zn, and Pb) owing to the proximity of a smelter.

Within a radius of 4 to 5 km around this smelter, the metal

concentrations in the topsoil reached up to 350 mg Cd kg–1

dry soil, 40,000 mg Zn kg–1 dry soil and 8000 mg Pb kg–1 dry

soil (Deram et al., 2006). Th e Cd concentrations in most of

the soils exceeded the regional background, which is approxi-

mately 0.40 mg Cd kg–1 soil (Sterckeman et al., 2007). Four-

teen sampling sites were selected taking into account the heavy

metal concentrations in the soil, the distance from the smelter,

the prevailing winds and the site characteristics (Fig. 1) to cre-

ate a soil dataset with a heavy metal gradient. At each sampling

site, one bulk soil sample (20 by 20 cm) was collected to a

depth of 15 cm. Fourteen soil samples were divided into four

diff erent groups (Gp1, Gp2, Gp3, Gp4) based on heavy metal

concentrations. Gp1 includes the uncontaminated soils with

metal concentrations <1 mg Cd kg–1 dry soil, 200 mg Zn kg–1

dry soil and 100 mg Pb kg–1 dry soil; Gp2 the slightly contami-

nated soils, with metal values ranging from 1 to 5 mg Cd kg–1

dry soil, 200 to 400 mg Zn kg–1 dry soil and 100 to 300 mg

Pb kg–1 dry soil; Gp3 the relatively heavily contaminated soils,

with metal values ranging from 5 to 10 mg Cd kg–1 dry soil,

400 to 800 mg Zn kg–1 dry soil and 300 to 600 mg Pb kg–1 dry

soil; and Gp4 is composed of the most heavily contaminated

soils, with metals concentrations more than 10 mg Cd kg–1 dry

soil, 1000 mg Zn kg–1 dry soil, and 800 mg Pb kg–1 dry soil.

Soil AnalysesTh e certifi ed Laboratory of Soil Analysis of the French Nation-

al Institute for Agricultural Research (Arras, France) determined

the physical and chemical characteristics of soil samples. All the

procedures listed below were in full compliance with the Norme

Française (NF) and NF ISO standard procedures published by the

French Association for Normalization (AFNOR, 1994).

Soils collected were dried at temperature below 40°C and

physically characterized for their particle size distribution (clay,

fi ne and coarse silt, fi ne and coarse sand). Th e particle size deter-

mination was performed according to the NF X 31–107 standard,

after destruction of organic matter by hydrogen peroxide and the

separation of the diff erent classes of particles using sedimentation

(particles <50 μm) and sieving (particles >50 μm). Soil pH was de-

termined in deionized water with a soil-to-water ratio of 1:2 (NF

ISO 10390). Organic matter was determined by dry combustion

or sulfo-chromic acid oxidation (when CaCO3 > 50 g kg–1) (NF

ISO 10694) and total N concentrations were measured accord-

ing to the NF ISO 13878 standard, which consists of burning

samples at 1000°C in presence of O2 and subsequently measuring

reduced N amounts by gas chromatography with a catharometer

detector. Th e cation exchange capacity (CEC) was determined af-

ter percolation of 1 mol L–1 ammonium acetate solution at pH 7

(NF X 31–130). Magnesium and potassium were extracted in a

Fig. 1. Map showing the sampling location. A rectangle with slashes shows the location of Metaleurop factory, fi lled circles shows the location of soil samples, and circle with x shows the location of spoil heap.

Manier et al.: White Clover Nodulation Index in Contaminated Soils 687

1 mol L–1 ammonium acetate solution according to NF X31–108.

Magnesium concentrations were measured using a fl ame atomic

absorption spectrophotometer and K concentrations using a fl ame

emission absorption spectrophotometer. Ammonium and nitrate

were determined after extraction with a 0.5 mol L–1 KCl solution,

by spectrometry (continuous fl ow methods) at 660 nm for am-

monium and 540 nm for nitrate.

Total Cd, Zn, and Pb concentrations in soils were measured

after microwave digestion. Th us, 300 mg of 315 μm-sieved soils

were digested in a mixture of HF/HClO4 (2:4, v/v). After fi ltra-

tion (0.45 μm), digestion products were adjusted to a volume of

25 mL with ultra-pure water. Total metal concentrations were

measured using inductively coupled plasma– mass spectrometry

(ICP–MS) for Pb and Cd and inductively coupled plasma–atom

emission spectrometry (ICP–AES) for Zn (NF EN 11885). In

addition, the CaCl2 (0.01 mmol L–1) extractable fractions of Cd,

Pb, and Zn were determined using ICP–MS.

Plant Material and Culture ProtocolWhite clover seeds used were from the same stock, homo-

geneous in size and stored in a dark cold chamber. Seeds were

soaked in distilled water for 6 h at room temperature. Th e seeds

were then maintained for 2 d at 21°C ± 2°C on fi lter paper

regularly moistened with distilled water.

Culture System: RhizotronA specialized plant growth container was constructed (Fig. 2).

A corrugated polyvinyl propionate (PVP) plate (50 by 50 cm)

was soldered with a Plexiglass plate of the same size. Th is system

is called “rhizotron” and consists of seven translucent columns

(50 by 3 by 2.5 cm). Th e base of each column is obstructed

with carded cotton. Collected soil samples were sieved (2.5 mm)

fi eld moist. Subsamples of sieved soils were added in three suc-

cessive columns and considered as three replicates. Two random

soil samples were added in each rhizotron. Seven rhizotrons were

used for this experiment. Th is system, fi rst used in our laboratory,

allows the plant to develop and permits the direct observation of

root elongation and nodulation development for several months.

No inoculation with the bacteria Rhizobium leguminosarum bio-

var trifolii was implemented.

Experiment DesignTh e experiment consisted of 14 diff erent treatments (i.e., 14

soils) each three with replicates (42 columns in total). Pregermi-

nated seeds with 0.5-cm primary roots were placed in the top

centimeter of each soil column of the rhizotron (three seeds per

column, 126 seeds in total). Th e rhizotrons were then placed

in a controlled environmental growth chamber with light-dark

cycles of 16/8 h day/night at 21 ± 2°C. White light was pro-

vided by Sylvania cool-white 20 W F20T12/CW tubes, with

photosynthetically active radiation (PAR) of 1000 mol m–2 s–1

(maximum) during the day. Rhizotrons were randomized using

a random number table every second day within the growth

cabinet. All plants were watered with 15 mL of tap water ev-

ery second day. No nutrient applications were made during the

growth of the cultures. Plants were harvested after 11 wk of

growth. Roots were washed free from soil with deionized water

for several minutes. Both aerial parts and roots were blotted dry

on fi lter paper. Total and shoot fresh biomass were weighed and

root fresh biomass was then calculated. Subsequently, the fi nal

Fig. 2. Sketch of the rhizotron culture system.

688 Journal of Environmental Quality • Volume 38 • March–April 2009

root elongation (length of taproot after uprooting the plants)

and the fi nal shoot elongation (length of peduncle) were mea-

sured. Nodulation of white clover was estimated according to

diff erent characteristics described in literature (Ibekwe et al.,

1995; Smith, 1997; Rebah et al., 2002; Pastor et al., 2003).

Th erefore, nodule number, size, and pigmentation (pink or

white) were recorded on primary and secondary roots of each

plant. Th e number of nodules was then converted into the

“nodulation index” according to the weight of plant, for ex-

ample, the nodulation index was calculated as the number of

nodules per gram of total fresh biomass.

Data AnalysesData were subjected to one-way ANOVA followed by a

post hoc multiple comparison of means using the LSD test

(signifi cance level P < 0.05). Additional step by step multiple

regressions (forward) were used to seek relationships between

the nodulation index and one or more soil variables. All the

statistical tests were performed using the software program

STATISTICA (version 5.1.97). Th e robustness index (RI) is

defi ned as the reciprocal of the variation coeffi cient of the end-

point (mean/standard deviation) among all uncontaminated

soils (Broos et al., 2005b).

Results

Physicochemical and Agronomical Characteristics of SoilsParticle size distribution and physicochemical characteris-

tics of the soils are summarized in Table 1. Th e soils of Gp2,

Gp3, and Gp4 were dominated by silt and clay (41% of clay

and fi ne silt and 32% of coarse silt). Texture analysis showed a

signifi cantly higher sand percentage (P < 0.01) in soils of Gp1

(up to 51% of fi ne and coarse sand). Soil pH values ranged

from 6.9 to 8.5. Generally soils were slightly alkaline. Average

pH values observed in the study groups were not statistically

diff erent (P > 0.05). Total N concentrations ranged from 0.7

to 4.2 g kg–1, concentrations of each study group were not sig-

nifi cantly diff erent (P > 0.05), with an average concentration

of 2.4 ± 1.0 g kg–1. Th e NH4+ levels varied within each group,

especially in Gp1 which varied from 2.65 to 9.19 mg N kg–1.

Nevertheless, statistical analyses did not reveal a diff erence be-

tween the groups considered (P > 0.05) with the average NH4

+

concentration being 5.6 ± 2.1 mg N kg–1. In contrast, NO3

–

concentrations were heterogeneous in the diff erent groups and

were signifi cantly smaller in soils from Gp1 compared to soils

from Gp3 (P = 0.024) and Gp4 (P = 0.004). Th e soil NO3

– lev-

el from Gp1 was 0.7 ± 0.1 mg N kg–1 vs.17.8 ± 13.7 mg N kg–1

for Gp3 and 22.2 ± 19.2 mg N kg–1 for Gp4.

Heavy Metal ConcentrationsTotal and CaCl

2 extractable concentrations of Cd, Pb, and

Zn are summarized in Table 2. Th e soils showed a large gradi-

ent of total metal concentrations with Cd ranging from 0.5

to 93 mg kg–1, Pb from 46 to 3780 mg kg–1, and Zn from

112 to 4840 mg kg–1. A large gradient of extractable metal

was also observed in our study with Cd ranging from 0.004 to

2.15 mg kg–1, Pb from 0.003 to 0.426 mg kg–1, and Zn from

0.02 to 12.4 mg kg–1.

Plant Growth and Nodulation DevelopmentTh e parameters of the development of white clover are sum-

marized in the Table 3. Using the rhizotron system, we were

able to observe the development of roots and nodules over

the course of the experiment. On average, shoot elongation

was signifi cantly higher in Gp2 (9.3 ± 3.6 cm) compared to

Gp1 (5.6 ± 1.1 cm) and Gp4 (6.02 ± 3.49 cm). On the other

hand, there were no signifi cant diff erences in root elongation

between the diff erent groups (P > 0.05) after 11 wk of culture

in experimental conditions and an average root length of 7.82

± 4.51 cm was measured.

Th e fi rst nodules appeared during the sixth week of growth;

however this timescale was not appropriate to observe diff er-

ences between the plants of each treatment. All individuals (ex-

cept individuals from soil 10 and 7) developed at least one or

two nodules during the seventh and eighth week and most of

the nodules observed were developing during the 9th and 10th

week. Diff erences in nodule numbers only seemed to appear at

the 10th or 11th week of growth. Th erefore, we suggest that

the observation must be done after 11 wk of growth. Th ere

were, however, no diff erences in the pigmentation and nodu-

lation size in all other experimental conditions. Th e majority

of samples showed small white nodules with the exception of

soil 17 (in Gp2) where some pink nodules could be observed.

On the other hand, the nodulation index decreased with in-

creasing concentrations of heavy metals in soil (Fig. 3). Th e

quantity of nodules per gram of total fresh biomass recorded

here within each contaminated group (Gp2–Gp4) signifi cantly

decreased (P < 0.01) compared to the uncontaminated group

(120 ± 66 nodules per gram of total fresh biomass). Whereas

there is no signifi cant diff erence between the nodulation index

in Gp2 and Gp3 (P > 0.05), the nodulation index in Gp4 (13

± 30 nodules per gram of total fresh biomass) is signifi cantly

lower than all other groups (P < 0.01) and even 8.5-fold lower

compared to the nodulation index in Gp1. Th e high variability

in the nodulation index from Gp1 can be explained by one

replicate of soil 23 which developed much more nodules (242)

than the other replicates (45–133).

DiscussionFirst of all, it should be acknowledged that our approach has

certain limitations that could be addressed in future work of

this type: in particular, it is possible that the use of the Rhizo-

tron system may have infl uenced our results by aff ecting plant

growth and/or health (note the low biomasses observed for all

plants at the end of the experiment), despite previous work

in this laboratory indicating no such problems (Langeureau-

Leman, 1999). In addition, the watering protocole we adopted

should perhaps be revised to better respond to the needs of

the plants as they grow, and also to permit this type of study

among diff erent soil types. Nevertheless, the use of the Rhizo-

tron system facilitated the measurement of parameters such as

Manier et al.: White Clover Nodulation Index in Contaminated Soils 689

root growth and nodule development in a nondestructive man-

ner on plants as they grew, permitting the accurate observa-

tion of nodule appearance and development over time, and has

great potential for future studies of this type. In addition, the

normalizing of conditions under which plants were grown is an

important factor in experiments of this type so that even if the

conditions were not entirely “optimal,” each exposure group

was nonetheless subject to those same conditions.

Table 1. Particle size distribution and selected soil physicochemical characteristics of all 14 soils. Soil samples are divided into four groups (Gp1–Gp4) with increasing metal concentrations.

Soil group

Soil sample

Clay, <2 μm

Fine silt, 2–20 μm

Large silt, 20–50 μm

Fine sand, 50–200 μm

Large sand, 200–2000 μm Total N NO

3−N NH

4+N pH, water CEC† MgO K

2O C/N

––––––––––––––––––––––––%–––––––––––––––––––––––– g kg–1 –––mg kg–1––– cmol kg–1 ––g kg–1––Gp1 23 21.0 19.0 19.6 13.9 26.5 2.13 0.69 2.65 8.53 9.81 0.60 0.21 31.60

24 13.3 14.5 11.2 11.8 49.2 4.15 0.74 9.19 7.26 10.00 0.47 0.29 39.10

Gp2 4 19.4 22.4 41.2 8.3 8.7 3.21 21.60 5.55 7.18 16.10 0.19 0.24 19.30

8 20.3 20.8 38.4 15.1 5.4 1.20 4.84 3.49 8.18 12.10 0.17 0.19 15.50

12 18.4 20.9 41.5 14.5 4.7 1.63 8.35 3.89 6.86 13.10 0.21 0.37 16.00

17 30.0 22.2 24.1 9.8 13.9 1.96 3.46 6.40 6.85 17.60 0.52 0.21 13.20

21 22.6 22.5 40.5 8.8 5.6 2.30 30.90 5.60 7.83 15.80 0.36 1.54 12.50

Gp3 5 21.4 21.5 38.0 11.0 8.1 3.34 32.30 9.46 7.80 14.60 0.22 0.80 16.50

15 15.7 17.6 29.6 11.2 25.9 1.63 20.30 3.84 8.09 8.88 0.11 0.20 13.50

16 20.2 12.6 22.8 35.6 8.8 2.08 0.86 5.72 7.17 14.30 0.21 0.48 17.20

Gp4 1 24.0 23.3 29.5 14.5 8.7 1.37 6.60 2.89 8.22 10.50 0.19 0.21 22.00

2 24.0 20.6 37.3 12.6 5.5 3.29 44.70 7.49 7.43 19.00 0.32 0.79 14.40

7 31.0 27.6 27.3 10.2 3.9 0.74 0.65 3.16 8.25 13.40 0.27 0.25 19.70

10 15.6 17.2 27.3 11.0 28.9 4.02 31.80 5.44 7.96 17.50 0.28 0.25 21.20

† CEC = cation exchange capacity.

Table 2. Total and extractable metal concentrations for all 14 soil samples. Soil samples are divided into four groups (Gp1–Gp4) with increasing metal concentrations.

Soil group Soil sample Total Zn Total Pb Total Cd Extractable Zn Extractable Pb Extractable Cd

–––––––––––––––––––––––––––––––––––––mg kg–1–––––––––––––––––––––––––––––––––––––Gp1 23 112.0 46.0 0.47 0.02 0.01 0.01

24 180.0 66.6 0.79 0.13 0.01 0.02

Gp2 4 258.0 144.0 2.45 0.50 0.01 0.04

8 270.0 123.0 2.68 0.08 0.01 0.03

12 383.0 119.0 2.76 3.74 0.01 0.12

17 266.0 137.0 3.35 1.91 0.01 0.17

21 326.0 129.0 1.94 1.21 0.01 0.03

Gp3 5 586.0 293.0 4.94 0.35 0.02 0.04

15 322.0 324.0 5.14 0.17 0.03 0.06

16 451.0 302.0 5.17 1.69 0.03 0.15

Gp4 1 2380.0 2480.0 19.40 1.81 0.40 0.03

2 910.0 707.0 13.20 1.36 0.07 0.15

7 2730.0 2180.0 92.60 1.42 0.43 2.15

10 4840.0 3780.0 16.40 1.50 0.24 0.09

Table 3. Development of white clover in all soils. Both shoot and root development and root nodulation parameters are presented. Results are presented into four groups (Gp1–Gp4) in accordance to the increasing metal concentrations of the soils.

Soil group Soil sample Shoot development Root development Total fresh biomass

Root nodulation

Nodulation index Pigmentation Size

cm, mean ± SD cm, mean ± SD g, mean ± SD mean ± SD mm

Gp1 23 6.2 ± 0.7 8.7 ± 1.1 0.08 ± 0.03 129.2 ± 101.6 White 1

24 4. 9 ± 1.0 7.2 ± 1.6 0.04 ± 0.03 111.3 ± 21.4 White 1

Gp2 4 6.8 ± 1.6 6.2 ± 2.9 0.03 ± 0.02 11.4 ± 19.8 White <1

8 10. 7 ± 1.5 7.1 ± 1.6 0.23 ± 0.06 73.7 ± 54.4 White 1

12 4.5 ± 1.4 4.6 ± 1.5 0.05 ± 0.01 96.9 ± 11.9 White 1

17 13.5 ± 3.6 16.1 ± 10.8 0.35 ± 0.11 40.0 ± 12.6 White and pink 1

21 9.4 ± 1.2 8.1 ± 2.2 0.22 ± 0.04 30.5 ± 7.1 White 1

Gp3 5 7.4 ± 1.4 8.6 ± 10.0 0.11 ± 0.02 63.4 ± 29.7 Pink 1

15 4.7 ± 0.3 4.2 ± 0.8 0.05 ± 0.01 75.4 ± 27.2 White 1

16 8.6 ± 1.8 9.6 ± 4.4 0.22 ± 0.18 24.4 ± 10.8 White 1

Gp4 1 4.9 ± 0.1 4.7 ± 1.4 0.10 ± 0.04 10.8 ± 4.0 White 1

2 10 ± 4.9 9.1 ± 3.4 0.22 ± 0.18 42.9 ± 51.4 White 1

7 5.5 ± 0.9 9.8 ± 4.6 0.06 ± 0.02 0 0 – –

10 3.3 ± 0.8 3.5 ± 0.9 0.01 ± 0.01 0 0 – –

690 Journal of Environmental Quality • Volume 38 • March–April 2009

From our data, it is clear that plant growth (especially root

growth) was less sensitive to the heavy metal contamination

compared to the nodulation process in our experiment. Brad-

shaw (1952) observed that heavy metals have a direct and last-

ing eff ect limiting the root development of nontolerant plants.

In our experiment, root development was not signifi cantly af-

fected by soil contaminated with heavy metals, although we

observed shorter roots in some individuals exposed to the most

contaminated soils (Gp4), and the comments above concern-

ing the possible limitations of the Rhizotron should also be

taken into account when interpreting these data.

When referring to size and pigmentation of nodules, Chen

et al. (2003) observed that soybean [Glycine max (L.) Merr.]

developed large and pink root nodules when growing on un-

contaminated control soils, but developed small and white

nodules when growing on Cd-contaminated soils. Th is is a

nodulation pattern typically found when a legume is presented

with an ineff ective Rhizobium strain. Perhaps unsurprisingly,

our observations were inconsistent with the results obtained by

Chen et al. (2003) in soybean, but most of the observed nod-

ules were morphologically similar to those described in white

clover by McGrath et al. (1988) growing in both contaminated

and uncontaminated soils, that is, small (1 mm) and white.

Regarding the number of nodules observed, our results were

in agreement with the results from Chen et al. (2003) with

a decrease in the number of root nodules being concomitant

with an increase in heavy metal soil concentrations. Chen et al.

(2003) observed a total absence of soybean root nodules for 20

mg Cd kg–1 soil; in our experiment an absence of nodulation

was recorded in the two most heavily contaminated soils (soils 7

and 10). In these soils, Cd concentrations (respectively 19 and

93 mg Cd kg–1 dry soil) exceeded by factors of 41 (soil 10) and

230 (soil 7) the regional background (mean of 0.40 mg Cd kg–1

dry soil; Sterckeman et al., 2007). Zinc and Pb concentrations

in these soils were also in excess of the regional background

concentrations by factors of 59 and 32, respectively. Chaudri

et al. (1993) and Broos et al. (2005a) showed that Rhizobium leguminosarum bv. trifolii is sensitive to elevated Zn concentra-

tions in soils. For example, Broos et al. (2005a) showed that

the number of rhizobia in soil declined by 50% compared with

their uncontaminated control soil at 233 mg Zn kg–1 dry soil

(pH 5.6) and at 876 mg Zn kg–1 dry soil (pH of 6.3; pH mea-

sured in 0.01 CaCl2). Given that the pH of all our soils is much

closer to the latter, it should not come as a surprise that we

fi nd only negative eff ects on the nodulation index in the most

contaminated soils of Gp4 (ranging from 910–4840 mg Zn

kg–1 soil). It was also demonstrated by Broos et al. (2005a) that

at these elevated concentrations of Zn the number of rhizobia

in soils was greatly reduced, which will clearly have an eff ect on

the nodulation index. Th erefore, the decreases in nodulation

index in soils along the gradient from Gp2, Gp3, and Gp4,

and the complete absence of nodules in certain soils from Gp4,

is most likely due to a strongly decreased population of rhizo-

bia (<103 cells g–1 soil).

It is known that nodulation of leguminous species could be

infl uenced by increased levels of inorganic N; excessive nitrate

Fig. 3. (a, b, c). Nodulation index vs. total metal concentrations in soils (a: Cd; b: Zn; c: Pb). Each experiment point represents the mean of results obtained from nine diff erent plants. The solid line represents the log regression.

Manier et al.: White Clover Nodulation Index in Contaminated Soils 691

is reported to block or delay the nodulation process at a num-

ber of diff erent stages, including both rhizobial infection (by

inhibiting root hair curling and infection thread formation)

and nodule development (Forde and Lorenzo, 2001; Vassileva

et al., 1997). A strongly suppressive eff ect of combined N (es-

pecially NO3

–) on nodulation is observed, because legumes will

use this combined N as a N source in preference to forming the

N2–fi xing symbiosis. When focusing on the nodulation index

obtained in soils of Gp2, we observed that those individuals

with fewer nodules came from the soils with the most elevated

NO3

–concentrations (soils 4 and 21). However, when looking

at the complete dataset, the relationship between nodule devel-

opment and NO3– was not apparent, since concentrations of

NO3

– in soils of Gp2, Gp3, and Gp4 were higher than the con-

centrations in soils from Gp1. Hence, although the combined

N in our soils may have an infl uence on the nodulation index,

we cannot conclusively show this within our dataset due to an

overlaying eff ect of increased toxicity of the heavy metals.

As Smith (1997) has noted, it is necessary to use a statistical

modeling process to separate the eff ects of metals on the nodu-

lation index from other edaphic factors. Consequently, as a fi rst

step, a single linear regression was performed to show the infl u-

ence relatively, of edaphic variables on nodulation index. Th e

clay content, CEC, total Zn, total Pb, total Cd, extractable Pb,

and extractable Cd all signifi cantly decreased the nodulation

index, whereas coarse sand content increased with increasing

nodulation index: nevertheless, R2 values were typically very

low (ranging from 0.10–0.23). Th ese analyses underlined the

infl uence of particle size distribution of the soils on the nodu-

lation index, especially the clay content and the percentage of

coarse sand in the soil, and highlighted the relative infl uence of

heavy metals on the nodulation index. Th is simple regression

analysis was followed by a step by step (forward) linear regres-

sion analysis to estimate the individual eff ects of the variables

on the nodulation index, as well as their simultaneous eff ect,

to estimate which variable has the greatest explanatory power

on the observed nodulation index. Th e most signifi cant model

described the nodulation index for N predictors (with N rang-

ing from 1–5): the best model (cross R2 = 0.51; P < 0.001)

was obtained with fi ve variables including the summed total

metal concentration (total Zn, total Pb, total Cd), coarse sand

percentage, CEC, MgO content, and the percentage of clay in

the soil. It is important to underline that even if we take into

consideration all fi ve parameters used by the model, only 50%

of the variation in the nodulation index was explained.

Finally, the sensitivity and the robustness of the nodulation

index as an ecotoxicological endpoint was tested to investigate

the possible application of this index as a bioindicator for heavy

metal contaminated soils. Sensitivity and robustness to the pol-

lutant are often used as important selection criteria for bioas-

says in ecotoxicological studies (Giller et al., 1998; Broos et

al., 2005b) and the ecotoxicological endpoints used need to be

both reproducible and consistent. Th is implies that the bioin-

dicator should be sensitive to the pollutant yet robust toward

changes in other soil characteristics. Concerning soils contami-

nated with heavy metals, robustness of the response observed

is key factor because physicochemical characteristics can dif-

fer greatly among samples and this may have an infl uence on

the responses observed. We investigated the sensitivity and the

robustness of shoot development, root elongation and nodula-

tion index; that is, biological parameters potentially infl uenced

by heavy metal toxicity.

As demonstrated above, shoot and root development of

white clover do not seem to be sensitive parameters toward

increasing metal concentrations in soils, however the robust-

ness index (RI) for shoot development and root elongation

(RI = 9.1 and 8.2, respectively) are high, showing that these

endpoints are very constant among uncontaminated soils. Th e

nodulation index appeared to be a more sensitive measure of

soil contamination by heavy metals and we observed a signifi -

cant decrease in the nodulation index at approximately 300 mg

Zn kg–1, 130 mg Pb kg–1, and 2.64 mg Cd kg–1 soil. On the

other hand, the nodulation index appears to be less robust

compared to the other parameters measured here (RI = 2.3).

Although our RI value was determined based on only two un-

contaminated soils, which is not a large replication, this RI for

the nodulation index is very similar to that found by Broos et

al. (2005b) for the N2–fi xation bioassay in white clover (RI cal-

culated with 14 diff erent uncontaminated soils). Nevertheless,

further work is necessary, using a larger group of uncontami-

nated soils with a large range of physicochemical characteris-

tics, to confi rm this RI and to conclude if the nodulation index

can be considered as suffi ciently robust a bioindicator of metal

contamination in soils.

ConclusionsTh e sensitivity of the nodulation process to soil contamina-

tion by heavy metals was examined using a specialized Rhizo-

tron method. Despite the potential limitations of this method,

it was possible to observe the development of roots and legume

nodules growing in diff erent soils with diff erent concentrations

of heavy metals. Data obtained show that the nodulation in-

dex of Trifolium repens L. was sensitive toward increasing heavy

metal concentrations in soils with a mixture of heavy metals

(signifi cant decrease in nodulation index at approximately

300 mg Zn kg–1, 130 mg Pb kg–1, and 2.64 mg Cd kg–1 dry

soil). Subject to confi rmation of the robustness of this bioin-

dicator, its higher sensitivity compared with the classical shoot

or root elongation indicators make the nodulation index a po-

tentially useful bioindicator for the assessment of the quality of

heavy metal contaminated soils.

AcknowledgmentsWe gratefully thank the French government for their

fi nancial support for this research program (ANR funds), which

was also subsidized by the European Regional Development

funds, to Mike Howsam for help with the English and to the

two anonymous reviewers whose comments at the reviewing

stage of this manuscript greatly enhanced its clarity.

692 Journal of Environmental Quality • Volume 38 • March–April 2009

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