Bioavailability and toxicity of cd to microorganisms and their activities in soil

khả năng dễ tiêu và độc tính của Cd lên vi sinh vật và hoạt động của chúng trong môi trường đất

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Advances in Environmental Research xx (2002) xxx–xxx

1093-0191/03/$ - see front matter 䊚 2002 Elsevier Science Ltd All rights reserved.

doi:10.1016/S1093-0191(02)00135-1

Bioavailability and toxicity of cadmium to microorganisms and

their activities in soil: a review

K Vig, M Megharaj* , N Sethunathan, R Naidu,1 1

CSIRO Land and Water, PMB 2, Glen Osmond, Adelaide, SA 5064, Australia

Received 28 January 2002; received in revised form 1 November 2002; accepted 13 November 2002

Abstract

Significant quantities of cadmium (Cd) have been added to the soils globally due to various anthropogenic

activities, raisingconcerns for environmental health Microorganisms play a unique role in the soil ecosystem, because

of their contributions to soil fertility Contrastingtrends, reported on the toxic effects of heavy metals includingCd

on soil microorganisms and their activities, are attributable to short-term studies often limited to a single soil type and conducted under controlled laboratory conditions There is a paucity of reliable field data on Cd alone, since most field studies on Cd-microorganism interactions in soils are based on sewage sludge containing multimetals and organic substances No single parameter can be used to generalize Cd toxicity and different parameters can provide contrastingresults A battery of relevant tests, rather than just one single assay, involvingimportant microbial activities should therefore be included in ecotoxicity studies The bioavailability of Cd and associated toxicity to soil biota vary with time, soil type, speciation, ageing, Cd-source, organisms and the environmental factors The available fraction or soil solution Cd, and not the total concentration of Cd, seems to correlate well with the toxicity parameters

䊚 2002 Elsevier Science Ltd All rights reserved

Keywords: Cadmium; Soil; Microorganisms; Bioavailability

1 Introduction

Cadmium(Cd), a potentially toxic heavy metal with

no known biological function, occurs widely in nature

in small amounts, with an average content of 0.2

mgkgy 1in the geosphere(Lindsay, 1979) The

concen-tration of Cd in rocks range from 0.1 to 11 mg kgy 1

Naturally occurringCd ores usually occur in association

with Zn ore sphalerite and are recovered as byproducts

of Zn mining Anthropogenic activities such as industrial

waste disposals, fertiliser application and sewage sludge

disposals on land have also led to accumulation of Cd

*Correspondingauthor Tel.: 8-8302-5044; fax:

q61-8-8302-3057.

E-mail address: megharaj.mallavarapu@unisa.edu.au

(M Megharaj).

Present address: Centre for Environmental Assessment and

1

Remediation, University of South Australia, Mawson Lakes,

SA 5095, Australia.

in soil and its leachingunder certain soil and environ-mental conditions(Alloway, 1990; Naidu et al., 1997),

with eventual increase in its concentration in food crops The FAOyWHO recommended maximum tolerable

intake of Cd is 70 mgdayy 1 With the estimated half-life of Cd in soil varyingbetween 15 and 1100 years

(Kabata Pendias and Pendias, 1992), its accumulation

in the environment and its entry into the food chain are

of great concern

Soil solution Cd is considered as the bioavailable form from the standpoint of its ecotoxicity The concen-tration of Cd in soil solution varies significantly with soil properties and nature of management practices imposed on the system by farmers and other land users

In soils with no anthropogenic inputs of Cd, soil solution concentrations could range from 0.3 to 22.5 mg ly 1

(Helmke, 1999), depending on the geological source of

the soil In agricultural soils, Cd concentrations rarely exceed 10 mgly 1in the soil solutions(Kookana et al.,

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2 K Vig et al / Advances in Environmental Research xx (2002) xxx–xxx

1999) The concentration of total dissolved Cd increases

with ionic strength and varies with the concentration of

dissolved organic carbon (Fotovat and Naidu, 1998)

More recent speciation studies by Krishnamurti and

Naidu (2000) demonstrated that over 60% of soil

solution Cd may be present in association with dissolved

organic matter

Cadmium, in association with chlorides, hydroxyl,

sulfhydryls and thiol groups, forms soluble complexes

and these complexes largely govern the biological

activ-ity of Cd

Given that soil microbiological activity has a great

potential as an early and sensitive indicator of stress in

soil(Brookes, 1995), our aim here is to (i) compile the

global information on Cd toxicity to soil microbes and

their activities; (ii) compare its toxicity between field

and laboratory studies; (iii) determine the relationship

between Cd bioavailability and toxicity; and(iv)

deter-mine the factors that control Cd bioavailability and

toxicity to soil microorganisms

2 Soil microorganisms and their significance

Soil serves as a habitat for diverse groups of

micro-organisms, comprised of algae, bacteria, fungi and other

organisms The major groups and their predominant

functions related to soil health and fertility are

highlight-ed below

2.1 Algae and cyanobacteria

Algae are the primary producers and form the base

of the food chain Algae account for 4–27% of the total

microbial biomass in the soil (McCann and Cullimore,

1979) and are involved in maintainingsoil fertility and

oxygen production (Bold and Wynne, 1978)

Minerali-zation of algae releases nutrients which are utilized by

other organisms in the soil for their growth and

devel-opment Cyanobacteria are more widespread than other

free-livingmicroorganisms (Burns and Hardy, 1975)

and contribute to the nitrogen economy of agricultural

soils, because of their ability to fix atmospheric nitrogen

Since microalgae resemble higher plants in terms of

their intracellular organisation and cyanobacteria are

prokaryotes, bioassays with these groups can serve as

valuable indicators of toxic effects of pollutants to other

fundamentally similar cells(Megharaj et al., 2000a) In

spite of their importance, algae in the soil ecosystem

are the neglected group among soil microbiota

(Megh-araj et al., 2000a) However, the role of algae in

remediation of industrial wastewater and other

contam-inated resources through sorption and accumulation has

been gaining importance in recent years

2.2 Bacteria and other microbial populations in soil

Bacteria and fungi are probably the most widely studied groups of soil microorganisms In soil, fungi, although numerically much less abundant than bacteria, can account for twice the weight of bacteria and actinomycetes combined (Jenkinson and Ladd, 1981)

Soil fungi can occur free-living or in association with plant roots The best-known function of fungi is decom-position of complex compounds of plant and animal origins, such as cellulose, lignin, and chitin

3 Effect of Cd on microorganisms and their activities

Although toxic effects of heavy metals on soil micro-biota are well-recognized (Baath, 1989; Giller et al.,

1998), contrastingtrends have been reported in the

literature on the effect of Cd on soil microbiota These are largely because of the differences in soil types and source of metal contamination (e.g sewage sludge,

soluble metal salts) which would have profound effect

on Cd chemistry in soil and its impact on soil microbiota

Table 1 summarizes some information on the effects

of Cd on soil microorganisms In a recent study lasting over 180 days in our laboratory, we found that the concentration of soil solution Cd in freshly contaminated soil(3 mgCd kg soil) decreased exponentially withy 1

time to -0.0006 mg ly 1 within 50 days of ageing If the data, presented in Fig 1, reflect changes in Cd bioavailability under field conditions, then most of the short-term laboratory based studies have presumably overestimated the effects of Cd on soil microbiota when extrapolatingtheir findings to metal contaminated field soils Usually, any pollutant that permitted a full recov-ery of a microbial parameter within 30 days of exposure

is not considered a risk while any negative impact beyond 60 days is considered a significant risk(Domsch

et al., 1983)

3.1 Microbial population

Estimates of microbial population in soil by conven-tional plate count method have shown that bacteria are more sensitive to Cd than fungi Based on laboratory incubation experiments, the effect of Cd on the bacterial population depended on its concentration used and the soil type Even at environmentally unrealistic concentra-tions Cd was not toxic at all(1000 mgkg , Fritze ety 1

al., 2000) or toxic only for 4 weeks (5000 mgkg ,y 1

Kozdroj, 1995) to the population of heterotrophic

bac-teria in soils But in several other studies Cd was inhibitory to the bacterial population when spiked at much lower levels(Zibilske and Wagner, 1982; Dar and

Mishra, 1994; Dar, 1996) Fungal numbers increased

with increasing Cd concentration in both sewage

Effect of Cd on microbial populations and their activities (selected reports)

Soil type y treatment Cd (mgkg soil) y 1 % inhibition (y)y References

stimulation (q)

Field studies

Sandy loam soil (received sewage sludge

20 years ago ), pH 6.8, 1.9% C, 9% clay Cd 8.6, Ni 27, Cu 102, Zn 289 y , N-fix 25% Brookes et al (1986a)

Sandy loam soil (received sewage sludge

20 years ago ), pH 6.8, 1.9% C, 9% clay Cd 4.7, Ni 6.7, Cu 56.5, Zn 139.2 y , biomass 42–60% Brookes et al (1986b)

Clay-loam, pH 6.5, 26.3% clay Cd 60, Pb 1088, Zn 3049 y , cellulose decomp 7% Khan and Frankland (1984)

Oak forest near abandoned zinc smelter,

pH 5.0–6.2, 0.5–0.7% C Cd 26, Cu 15.0, Pb 21.6 and Zn 478

y , bacteria 86%, fungi 60%, actinomy-cetes 86%,nitrosomonas 94%, nitrobacter

40%, DHA 93%, urease 88% Pancholy et al (1975)

Lab amendments

pH 5.1–6.1, 1.5–2.9% OC, 10–21% clay CdCl 562 2 y , arylamidase 55–82% Acosta-Martinez and Tabatabai (2001)

pH 6.2–7.6, 2.7–5.3% OC, 26–34% clay 2810 y , arylsulfatase 23–55% AL-Khafaji and Tabatabai (1979)

pH 7.6, 3.2% OC, 30% clay 281 y , arylsulfatase 7%

Silt loam, pH 6.75, 1.8% C, 28% clay Cd (NO ) 10 3 2 y , denitr Bollagand Barabasz (1979)

Silt loam, (1.31% OC)q1% dry sludgeq

Sandy loam, pH 7.9, 0.47% OC; Loam,

pH 8.1, 1.6% OC; Clay-loam, pH 7.7,

y , CO 21–30%, biomass 17–25%, N- 2 min 42–53% Dar and Mishra (1994) Sandy loam, pH 7.9, 0.47% OC; Loam,

pH 8.1, 1.61% OC; Clay-loam, pH 7.7,

y , bacteria, biomass, DHA, alkaline phos-phatase, argammon Dar (1996) Sandy, pH 7.0, 1.6% OM, 2% clay CdCl 150 2 y , CO 9% 2 Doelman and Haanstra (1984)

Sandy peat, pH 4.4, 12.8% OM 400 y , CO 10% 2

Sandy, pH 7.0, 1.6% OM CdCl 150 2 y , urease 10% 6 weeks Doelman and Haanstra (1986)

Sandy peat, pH 4.4, 12.8% OM 1980 y , urease 10% 6 weeks

40 y , urease 10% 1.5 years Calcareous soil pH 7.4, 5.4% OC, 4.3%

Ca equiv, 23.5% clay; CdCl 10 2

q , nitrate production 15–16% in both the

Non-calcareous soil pH 7.6, 2.6% OC,

1.2% Ca equiv, 18.6% clay 100 and 500 y , nitrate production 13–37%

Silty, pH 5.6, 2.6% OC, 28% clay 562 y , amidase 6% Frankenberger and Tabatabai, (1981)

Loamy, pH 7.0, 3.2% OC, 30% clay

Podzolized sandy soil with 3 cm thick

humus, pH 3.8, 50.3% C CdCl 200–4000 2 y , CO 15–58% 2 Fritze et al (1995)

Forest humus, pH 3.95, 52% C CdO or CdCl 2 y , CO 16–24% 2 Fritze et al (2000)

1000qPumice NE, biomass Sandy loam soil, pH 5.4, 1.65% OC, 16%

Sandy loam soil, pH 5.4, 1.4% OC, 16%

Agricultural sandy loam, pH 7.8, 4% OM CdSO 1800 y , biomass 78%, DNA 36% Griffiths et al (1997)

Table 1(Continued)

Soil type y treatment Cd (mgkg soil) y 1 % inhibition (y)y References

stimulation (q) Sandy, pH 7.0, 1.6% OM, 2% clay; CdCl 55 2 q , glutamic acid decomp Haanstra and Doelman (1984)

Silty loam, pH 7.7, 2.4% OM, 19% clay; 55 q , glutamic acid decomp

Clay, pH 7.5, 3.2% OM, 60% clay; 55 NE, glutamic acid decomp

Sandy peat, pH 4.4, 12.8% OM, 5% clay 1000 NE, glutamic acid decomp

Clayey loam, pH 8.52, 0.87% C CdSO 2248 4 q , N-min 28% Hassen et al (1998)

5 different soils: River sand, Gley, Gray

lowland, Andosol and Humic Andosol

5 different soils—River sand, Gley, Gray

lowland, Andosol and Humic Andosol

y , CO , N-min, bacteria, actinomycetes, 2 proteinase Hattori (1989)

q , fungal, b-glucosidase Gley soil, pH 5.8, 96.4% sand, 1.3% silt,

2.2% clayq1% glucose ycellulose CdCl 11.24–1124 2 y , CO 2 Hattori (1991)

q , fungi, ATP

NE, bacteria Gley soil, pH 5.8, 96.4% sand, 1.3% silt,

2.2% clayq2% sewage sludge; CdCl 1124 2 y , bacteria, Gley Hattori (1992)

y , CO both the soils 2

q , bacteria, Andosol Andosol, pH 6.4, 36.3% sand, 36.1% silt,

27.6% clayq2% sewage sludge q , fungus both the soils

Brown earth-loamy sand, pH 4.6, 12.3%

clay; CdCl 10–100 2 y , cellulose decomp 13.5–35% Khan and Frankland (1984)

Brown earth-loamy sand, pH 5.4, 10.3%

Sandy loam from long-term liming

experi-ment, pH 4.5–7.0 CdSO 3.1–4.3 4 NE, biomass Knight et al (1997)

Red soil, pH 4.51, 1.74% OM, 1.08%

Sandy loam, pH 6.5, 12.6% OC, 8% clay,

12% silt, 80% sand CdCl 5000 2

y , heterotrophic bacteria initially fol-lowed by similar levels after 4 weeks Kozdroj (1995)

q , argammon Forest soil, pH 4.8, 2.3% OC, 87% sand,

8% silt, 5% clay CdSO 500 4 y , CO , DHA, ATP, acid phosphatase 2 Landi et al (2000)

NE, biomass

Forest litter from forest floor, pH 3.4–4.3 CdCl 250 2 y , CO 14% 2 Laskowski et al (1994)

Cd yCa or CdyMgor CdyK

Cdqcitrate 562 y , N-fix 19%

Agricultural, pH 5.8–7.8, 2.6–5.5% C,

23–34% clay CdSO 562 4 y , nitr 67–94% Liangand Tabatabai (1978)

McKenney and Vriesacker (1985)

Table 1(Continued)

Soil type y treatment Cd (mgkg soil) y 1 % inhibition (y)y References

stimulation (q)

Montepaldi soil, pH 8.1, 66% sand, 21%

silt, 13% clay, 1.7% TOC; CdSO 3–4000 4 y , ATP, DHA, urease Moreno et al (2001)

Castelporziano soil, pH 4.8, 87.5% sand,

8% silt, 4.6% clay, 2.3% TOC Montepaldi )Castelporziano

Forest litter MOR—brown podzolized, pH

Forest litter MULL—rendzina pH 3.8–4.5,

Phaeosem, pH 6.9, 2.2% OM; Cd (CH COO) 1.7–228.8 3 2 y , CO 2 Reber (1989)

Sandy hortisol, pH 7.0, 2.6% OM; Acidic

cambisol, pH 5.6, 1.6% OM

Agricultural soil, 1.3% OC Cd (NO ) 150 3 2 y , DHA 48% Rogers and Li (1985)

Fir needle litter, 78% OM CdCl 1000 2

y , CO 24%, cellulase 29%, amylase 2

NE, invertase, xylanase, b-glucosidase, polyphenoloxidase

pH (CaCl ) 4.4–6.6, 0.9–2.8% C, 1–17% 2

clay; CdCl 200 2 y , potential nit rate 50–80% Smolders et al (2001)

pH 4.6–7.0, 1.99–5.32% C, 24–36% clay CdSO 2810 4 y , pyrophosphatase 19–50% Stott et al (1985)

Surface soils, pH 5.1–7.8, 2.6–5.5% OC,

Agricultural, pH 5.1 29 y , CO 36% 2 Walter and Stadelman (1979)

Sandy luvisol, pH (CaCl ) 6.0, 0.9% OC, 2

7% clay CdCl 2 (single or successive dose) 100 y , CO , DHA 2 Wilke (1991)

Silt loam (Alfisol), pH 6.0, 2.1% OM CdCl 2 (spiked in sewage sludge) 0.1 y , ATP 50% Zibilske and Wagner (1982)

1.0 altered fungal community

NE, no effect; OC, organic carbon; OM, organic matter; N-fix, nitrogen fixation; arg ammon, arginine ammonification; nit, nitrification; decomp, decomposition; DHA, dehydrogenase.

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Fig 1 Effect of ageing on soil solution Cd in a Xeralf from

South Australia (Naidu, unpublished).

amended (Hattori, 1989) and unamended (Hattori,

1991) soils, spiked with Cd (0–1124 mgkg ) It wasy 1

the amount of water-soluble Cd, and not the total Cd

applied that determined its impact on the

microorgan-isms(Hattori, 1992)

Most of the published work on metal toxicity to algae

is based on studies conducted with aquatic algae, while

information on terrestrial algae is limited

Cyanobacter-ial colonization and autotrophic nitrogen fixation have

been shown to decrease in a metal contaminated soil

(EDTA extractable soil metal concentrations of Cd, 3;

Ni, 2.5; Cu, 20; Zn, 50 mgkgy 1) even 20 years after

the last metal inputs through sewage sludge were made

(Brookes et al., 1986a) But, the effect of Cd, applied

alone, on soil algal populations is little understood

3.2 Microbial biomass

Microbial biomass serves as a pool of nutrients and

is a sensitive indicator of microbial changes in soil

Generally, microbial biomass in soils, measured by the

fumigation extraction method, is not adversely affected

by Cd even at abnormally high concentrations of 500–

1000 mgCd kgy 1(Fritze et al., 2000; Landi et al.,

2000) But, Cd, applied at 500 mgkg , albeit innocu-y 1

ous to microbial biomass as estimated by the fumigation

extraction method, effected a decrease in ATP content

(Landi et al., 2000) Evidently, microbial biomass may

not always be a reliable indicator of metal stress

(Megharaj et al., 2000b) A distinct decrease in the

ATP content was caused by Cd (Moreno et al., 2001)

whereas Hattori (1991) reported an increase in ATP

content followingincreased Cd addition to soil The

reason for such contrastingeffects is not clear

The metabolic quotient (qCO ), the ratio between2

respiration and the microbial biomass, has also been

used as an ecophysiological indicator of heavy metal

stress in soil (Anderson and Domsch, 1990; Brookes,

1995) qCO decreased in the heavily Cd contaminated2

soil over that in control duringthe first 2-d incubation

(Landi et al., 2000) Reports on qCO values in metal2

contaminated soils have been contradictory, with higher values in contaminated soils than that in uncontaminated soils in some reports (Chander and Brookes, 1991;

Moreno et al., 1999) and vice versa in other reports (Baath et al., 1991; Insam et al., 1996; Landi et al.,

2000) It is appropriate to use qCO as an indicator for2

assessingmetal effects in not very dissimilar soils

(Insam et al., 1996)

In a recent study(Renella et al., 2002), Cd, applied

singly and in combination, at 12 mg kgy 1 (4 times the

EU limit), was the least toxic amongthe three metals

Cu, Cd and Zn But, the concentrations of metals used

in this study were different (Cd, 12 mgkgy 1 soil; Cu,

140 mgkg ; Zn, 300 mgkgy 1 y 1 soil), makingthe

comparison of the acute effects of these metals in this short-incubation study difficult

3.3 Microbial community structure

Gross measurements of microbial diversity have been used to assess environmental stress (Atlas, 1984), but

such studies are hampered by problems of sampling, extraction and culturingleadingto bias towards certain groups within microbial communities Pollution may lead to a decrease in microbial diversity due to the extinction of species which lack sufficient tolerance to the stress imposed, and enhanced population of other species which thrive under stress(Atlas, 1984)

The modification of the metabolic behaviour of the whole microbial community should reflect a shift in its quantitative and qualitative composition Biologsoil community fingerprints, based on inoculation of Biolog plates with soil suspensions, have been used to deter-mine the changes in soil biochemical properties insti-gated by heavy metals (Knight et al., 1997; Fritze et

al., 2000) However, the reproducibility of the results

obtained is governed by the inoculum density and microscale heterogeneity, which may make Biolog assay unsuitable for comparingenvironmental samples from different origins Another approach to detect the changes

in the microbial community is to determine the phos-pholipid fatty acid(PLFA) composition in the soil This

method is based on the extraction and identification of signature lipid biomarkers from the cell membranes of microorganisms Its quantitative analysis can provide taxonomic information at the species and subspecies level and serves as a measure of the viable biomass Evidence from field studies suggests that under long-term metal stress there is a change in the genetic structure of the soil microbial community, without nec-essarily any increase in the metal tolerance (Giller et

al., 1998) Fritze et al (2000) reported an increase in

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K Vig et al / Advances in Environmental Research xx (2002) xxx–xxx

the actinomycete PLFA signatures and a decrease in the

fungal PLFA signatures in laboratory-incubated forest

soils amended with 400 and 1000 mgCd kgy 1 (as

CdCl or CdO2 ) Griffiths et al (1997) also reported a

shift in microbial communities in an agricultural sandy

loam soil, spiked with CdSO at 1800 mgkgy 1soil, by

4

%GqC profiles, DNA hybridization and PLFA patterns

Generally, actinomycetes are more resistant to Cd than

Gram negative and Gram positive bacteria in that order

(Doelman, 1986) There is a need for more research in

species diversity in Cd polluted soils

3.4 Microbial activities

Microbial activities may reflect the functions of total

(respiration) or specific (nitrification) groups of

micro-organisms in the soils(Domsch et al., 1983) The most

commonly studied microbial reactions impacted by

inor-ganic or orinor-ganic pollutants include: C-mineralization,

N-mineralization, CO production and enzyme activities.2

3.4.1 Mineralization of carbon

Mineralization of organic carbon to CO commonly2

known as ‘soil respiration’ is a good index of total

activity of microflora involved in organic matter

decom-position (Anderson, 1982) Therefore, soil respiration

has been the most studied parameter on the effects of

metals on microbial activities in soil (Baath, 1989)

Total soil respiration reflects the metabolic behaviour of

a range of microorganisms, which are not equally

sensitive to pollutants Tests based on this parameter are

therefore not always valid(Vallaeys et al., 1997)

Usually, there is no clear trend between metal

contam-ination and respiration in agricultural soils(Giller et al.,

1998), whereas negative effects have been reported in

forest soils (Baath, 1989) Heavy metals may reduce

the substrate availability for soil respiration by forming

complexes with the substrates or by killingthe

micro-organisms (Landi et al., 2000) Even lower levels (10

mgCd kgy 1 soil) of spiked Cd can decrease CO

2

evolution from an acid sandy loam soil (Gupta et al.,

1984) There are also reports (Niklinska et al., 1998;

Landi et al., 2000) of inhibition of carbon mineralization

by Cd but only at unrealistically high levels(400–500

mgCd kgy 1 soil) The impact of pollutants on soil

respiration differs with soil type The level of Cd

required to produce a 9% decrease in CO evolution2

was 150 mgkgy 1 in a sandy soil (pH 7.0, organic

matter 1.6%) and 400 mgkgy 1 in a sandy peat soil

(pH 4.4, organic matter 12.8%) (Doelman and Haanstra,

1984) The inhibitory effect of Cd on microbial

respi-ration in forest litter soil was noticed only when

amend-ed with nutrients such as Ca, Mgor K (Laskowski et

al., 1994), probably due to its increased solubility

(Schierl et al., 1986) In another study on the effects of

different forms of Cd on soil respiration, Fritze et al

(2000) observed a similar inhibitory effect on

respira-tion in forest humus soil spiked with 1000 mg

Cd kgas CdO or CdCl Evidently, in a sandy loamy 1

2

soil (planted to wheat) spiked with 100 mgCd kg ,y 1

Cd, added in insoluble forms as carbonate and oxide, was less toxic to cellulose decomposition than Cd added

as sulfate or chloride (Khan and Frankland, 1984)

Hattori (1989) reported a negative correlation between

the amount of C mineralized and the concentration of water-soluble Cd in Cd- and sludge-amended soils Thus, the toxicity of Cd varies with the soil type and the nature of Cd salt

3.4.2 Nitrogen mineralization

The impact of metals on nitrogen mineralization rates has been studied extensively Nitrification in the field appears to be a sensitive indicator of metal pollution

(Baath, 1989) In laboratory incubation studies, Cd was

generally inhibitory to nitrification in soils(Necker and

Kunze, 1986; Hattori, 1989) However, the inhibitory

effect varied with the soil and test conditions Liang and Tabatabai (1978) reported an inhibition of

nitrifi-cation at 562 mgCd kgy 1 in soils Likewise, nitrifica-tion, but not ammonificanitrifica-tion, was inhibited by 500 mg

Cd kgand abovey 1 (Bewley and Stotzky, 1983)

Cad-mium, applied at 50 mgkg , adversely affected nitro-y 1

gen mineralization to a greater extent in a sandy loam soil than that in a clay-loam soil (Dar and Mishra,

1994) Interestingly, there was a significant decrease (14%) in nitrification in a soil (pH 6.6) spiked with 2

mgCd kgy 1 (Smolders et al., 2001) Also, there was

evidence that Cd promoted nitrification at 10 mgkgy 1

and inhibited it at 100 mgkgy 1in both calcareous and non-calcareous soils(Dusek, 1995) Nitrogen

minerali-zation was stimulated under field conditions by Cd as opposed to inhibition under the laboratory incubation

(Necker and Kunze, 1986), however, the concentration

used in field was less than that in the laboratory The stimulation under long-term impact may be due to the slow development of an adapted microflora tolerant to Cd

Cadmium has also been shown to be inhibitory to denitrification in a silt loam soil at 10 mgkgy 1(Bollag

and Barabasz, 1979), and in clay and sandy loam soils,

but only at )50 mgkgy 1 soil(McKenney and

Vrie-sacker, 1985), probably due to its decreased

bioavailability

3.4.3 Enzymes

Soil enzyme activity is used as a sensitive indicator

of the effect of pollutants, includingmetals in soils

(Giller et al., 1998) In some of the soil enzyme assays,

abiotic activity, however, originates from dead cells, which cannot readily be distinguished from the enzy-matic activity of the livingcells(Burns, 1982)

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Heavy metals can reduce enzyme activity by

inter-actingwith the enzyme–substrate complex, denaturing

the enzyme protein, interactingwith its active sites

(Nannipieri, 1994) or by affectingthe synthesis of the

enzymes within the microbial cells Metal-induced

changes in the community structure can also modify the

enzyme activity(Nannipieri, 1994) When Cd binds to

active sites, enzymes such as alkaline phosphatase are

inactivated and metabolism is disrupted (McGrath,

1999) Cadmium, besides beingan enzyme inhibitor,

can have deleterious effects on membrane structure and

function by bindingto the ligands such as phosphate

and the cysteinyl and histidyl groups of proteins(Collins

and Stotzky, 1989) The effect of Cd on the soil enzymes

varies with the enzyme studied and the soil type as

shown in Table 1

Dar (1996) reported a decrease in dehydrogenase

activity(DHA) and alkaline phosphatase activity at 50

mgCd kgy 1 in a laboratory study with different soil

types Landi et al.(2000) found a negative effect of Cd

on DHA, but at a much higher concentration of 500 mg

Cd kgwhereas acid phosphatase activity decreased aty 1

50 mgCd kg The concentration of Cdy 1 (mgkg )y 1

required to cause a 10% decrease in urease activity after

6 week incubation amounted to 150 in sandy loam soil,

360 in silt loam, 950 in clay soil and 1980 in sandy

peat soils (Doelman and Haanstra, 1986) The Cd

concentration required to cause a similar inhibitory

effect on phosphatase activity, was 10 times greater in

clay soil than in sandy soil (Doelman and Haanstra,

1989) Rogers and Li (1985) found that Cd was 4 times

more toxic to DHA in unamended soil than in soil

amended with 1% alfalfa Alfalfa amendment apparently

increased the microbial population resistant to Cd or

decreased its availability The toxicity of Cd to urease

and dehydrogenase activities decreased in soil with low

pH (4.8) and high organic carbon (2.3%) (Moreno et

al., 2001)

3.4.4 Biodegradation of organic contaminants

Bioaugmentation with focus on using metal-resistant

microorganisms for promoting the degradation of

organ-ic contaminants by morgan-icroorganisms is an emerging area

of research in the remediation of cocontaminated sites

Toxicity of metals such as Cd to soil microorganisms

adversely affected the biodegradation of organic

con-taminants in cocontaminated soil (Said and Lewis,

1991) But, in a dual bioaugmentation strategy by

coinoculation, four Cd-resistant bacterial isolates were

used to reduce the soluble concentrations of Cd and

then allow the degradation of 2,4-dichlorophenoxyacetic

acid (2,4-D) by a Cd-sensitive 2,4-D-degrading

bacte-rium, Ralstonia eutropha JMP134 in a cocontaminated

soil (Roane et al., 2001) None of the four

metal-resistant bacteria could degrade 2,4-D, but each of these

isolates supported the degradation of 500-mg2,4-D

mly 1by R eutropha JMP134 Cadmium detoxification

by the metal-resistant bacteria involved a plasmid-independent intracellular mechanism

4 Field studies

As mentioned earlier, data related to field studies with Cd alone are negligible Field studies with Cd come mainly from industrially contaminated sites or from agricultural fields amended with sludge containing multimetals or exposed to extensive fertilizer applica-tions Baath (1989) and Giller et al (1998) have

extensively reviewed these aspects of metal toxicity in forest and agricultural soils As in laboratory amend-ments, toxicity observed in the field also varies with the soil type as shown in Table 1

5 Possible reasons for variability in Cd toxicity data

Reports on the ecotoxicity of Cd, like other metals,

on biodiversity and microbial activities have seldom been consistent for many reasons Some of the discrep-ancies in the literature on ecotoxicity of Cd in soils may be related to bioavailability, sensitivity of micro-organisms, and the methodologies used

5.1 Bioavailability of Cd

A major factor governing the toxicity of a metal in soil is its bioavailability Bioavailability is considered as the fraction of the total contaminant in the interstitial water and soil particles that is available to the receptor organism This suggests that there is a continuum between 0 bioavailability and 100% bioavailability and within this spectrum the pool of contaminant available

to receptor organisms may vary depending on the nature

of organisms and the perturbations imposed by the environment (Naidu et al., 2000) There has been

limited work on bioavailability of Cd and its relationship

to ecotoxicity in soils spiked with Cd Bioavailability

of Cd is largely governed by (i) soil type, (ii) Cd

speciation, (iii) ageing, (iv) nature of Cd applied and (v) nature of microorganisms

5.1.1 Soil type

Physico-chemical and biological properties of the soil play major roles in the availability of the metal to the organisms Change in pH affects the sorption of Cd by soils and thereby its concentration in soil solution

(Naidu et al., 1997) Increases in soil pH decrease Cd

in solution or make it less available Organic matter and clay content of soil can also significantly influence the concentration of soil solution Cd The effect of soil contamination with Cd on microbial processes

(phos-phatase activity(Doelman and Haanstra, 1989)), urease

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K Vig et al / Advances in Environmental Research xx (2002) xxx–xxx

activity (Doelman and Haanstra, 1986), as described

earlier under Section 3.4.3) is generally not of great

magnitude especially in clay soil, probably due to its

low bioavailabilty (Khan et al., 1997) Application of

Cd (55–400 mgkg ) delayed the decomposition ofy 1

glutamic acid in both sandy and silty loam soils, but

not in clay and sandy peat soils(Haanstra and Doelman,

1984) The mineralization of carbon and nitrogen in

different soils were related to the amount of

water-soluble Cd in soil(Hattori, 1989, 1992) Evidently, it is

not the amount of metal spiked in soil that causes

toxicity, but the amount ‘available’ Complexation of

Cd with organics andyor the formation of insoluble

chelates was a possible reason for its low bioavailability

in clay-loam and loam soils(Lighthart et al., 1983; Dar,

1996) For the microorganisms, the free divalent ion,

considered as the toxic species of Cd, in soil solution

is the readily bioavailable fraction Clay is the dominant

abiotic component in soils that decreases the toxicity of

Cd, followed by organic matter content(Kuo and Baker,

1980; Doelman and Haanstra, 1984) Babich and

Stotz-ky (1977) also reported decreased toxicity of Cd to

several fungi in soil with an increasing content of

montmorillonite Presence of different mineral nutrients,

cations or anions in the soil can also alter the toxicity

of Cd by complexation, sorption or desorption processes

(Naidu et al., 1994, 1997; Bolan et al., 1999) Because

soil is such a complex system, it is difficult to make

broad generalizations on the effect of ligands in solution

on the sorption of Cd (Harter and Naidu, 1995) and

hence on the availability of Cd However, various

studies suggest that bioavailable fraction of Cd in soil

decreases with time and with increase in pH, clay and

organic matter contents in the soil

5.1.2 Speciation

The total metal content of a soil is distributed among

all the possible chemical forms(speciation) in the solid,

liquid or the biotic phases of the soil (Krishnamurti,

2000) Speciation of the metal ion in the soil solution

may play a significant role in its bioavailability

Only few studies have tried to identify the particular

species of the Cd, which may contribute to its

bioavail-ability The availability of particulate-bound Cd in a

soil–plant system decreased in the order: exchangeable

carbonates metal–organic complexes organics

-Fe and Mn oxides -mineral lattices (Krishnamurti et

al., 1995) Comparison of the toxicity of pore water in

two soils spiked with Cd(NO ) to a soil alga, Chloro-3 2

coccum sp clearly showed that organically complexed

Cd is bioavailable and contributed to the toxicity to the

alga(Krishnamurti, unpublished data) This contradicts

the long-held notion that Cd–organic complexes are not

bioavailable to soil biota There is a need for more

research on speciation in relation to toxicity to

micro-organisms and their activities

5.1.3 Ageing

The bioavailability of a heavy metal decreases with the duration of its contact with soil(Naidu et al., 2003)

due to its decreased desorbability over time, as

illustrat-ed with soil solution Cd in Fig 1 The concentration of soil solution Cd in a freshly contaminated soil decreased exponentially from 3 mgkgy 1 soil at the start to negligible levels within 50 days of ageing (Naidu,

unpublished data)

A decrease in solution Cd can lead to its decreased toxicity to microorganisms (Hattori, 1989, 1992; Dar

and Mishra, 1994; Dar, 1996) In a recent laboratory

incubation study(Vig, unpublished data), toxicity of Cd

to nitrate reductase decreased with time in a sandy loam soil at 100 mgCd kg In contrast, Doelman andy 1

Haanstra (1986) reported an increase in toxicity of Cd

to urease activity with time(1.5 years) in a laboratory

study with 5 soils spiked with 55–8000 mgCd kgy 1

soil, probably due to increased mobility of Cd by chelation(Doelman and Haanstra, 1984)

5.1.4 Nature of Cd applied

Usually, studies on Cd toxicity have been conducted usingCd salts such as CdCl ,2 CdSO4 and

Cd(CH COO) The nature of the anions can modify3 2

the toxicity of Cd, as, for instance, the nitrate seems to counteract the negative effect of Cd on soil respiration

(Saviozzi et al., 1997) Soil respiration was inhibited

by CdCl and CdSO , and not by Cd2 4 (NO ) (Rother et3 2

al., 1982) Acetates of Cd, Pb and Zn were less toxic

than the respective sulfates to ammonification and nitri-fication Cadmium acetate (14–91%) effected a more

pronounced decrease in microbial biomass carbon than CdCl (6–14%) when spiked at 0–100 mgCd kgy 1 2

(Khan et al., 1997) The low toxicity of CdCl was2

probably due to the fixation of the major part of the Cd

in the soil But, acetate in Cd(CH COO) acted as a3 2

metal-complexingligand, thereby reducingthe adsorp-tionyfixation of the applied Cd on the soil constituents (Elliott and Denneny, 1982) and increasingits

bioavail-ability and toxicity to biomass

5.1.5 Nature of microorganisms

The bioavailability of Cd, like that of other metals,

in soils is governed by the nature of microorganisms and microbially mediated processes Sorption of Cd to

a Gram positive soil bacterium, Rhodococcus erythro-polis A177 was caused mostly by its bindingto the cell

wall, with a minor part beingtaken up inside the cells

(Plette et al., 1999) Bindingof Cd to the cell walls

depended on its solution concentration, pH, calcium concentration and ionic strength Bacterial sorption of

Cd was more pronounced in sandy soil than in clay soil under similar conditions (total Cd, pH and calcium

concentration)

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10 K Vig et al / Advances in Environmental Research xx (2002) xxx–xxx

Fig 2 A generalized diagram showing heavy metal sensitivity to different components of soil biota.

Soil microorganisms can alter the metal

bioavailabil-ity by releasingspecific compounds that form complex

with Cd andyor by changing the soil pH Rayner and

Sadler (1989) demonstrated complexation of Cd to

polyphosphate granules in the cell membrane and

intra-cellular Cd bindingproteins in a tolerant strain of

Pseudomonas putida, grown in a defined medium

con-taining3 mM Cd2q Mechanisms of complexingCd

have evolved over time to allow the cell to survive high

intracellular Cd concentrations Extracellular

accumula-tion of metals on microbial cells generally involves

either adsorption of metals to the polysaccharide

coat-ing, or adsorption to binding sites such as carboxyl,

phosphate, sulfahydryl or hydroxyl groups on the cell

surface There is also considerable evidence of microbial

methylation of Cd, As, Se, Hgand Pb by bacteria of

common genera, such as Bacillus and Pseudomonas

(Hughes and Poole, 1989) However, as the studies

involvingmicrobial transformations have been

per-formed mainly in vitro with pure cultures, the effect of

these processes on metal bioavailability and dynamics

in soils is not clear Physiology of the microbes

includ-ingthe transport of metals across the membranes are

often not considered duringthe assessment of

contami-nant bioavailability The parameters that control the

membrane transport of contaminants are species-specific

and therefore, the same bioavailable pool of

contami-nants in soils may not necessarily result in the same

rates of impact on different microorganisms

Competitive interactions between livingcells and

ligands and the physiological status of the organism

should also be considered in the relationship between

toxicity and free ion metal concentration (Campbell et

al., 2000) Toxic responses of a genetically modified,

luminescent bacterium,Escherichia coli revealed a

pos-itive relationship between free metal ion activity and

toxicity for Cu, but not for Zn and Cd The stable

chloride complexes appeared to contribute to the toxicity

of Cd under the test conditions Nederlof and Van

Riemsdijk (1995) attributed the sorption of metal ions

by soil organisms to the competition for binding of that

metal ion by all reactive soil components(includingthe

organisms) In addition, the total amount of bioavailable

metal in soil is influenced by complexation with

dis-solved organic and inorganic ligands

The toxicity of Cd to the mineralization of 14 C–ace-tate to14C–CO byPseudomonas putida MT 2 increased

2

with increasingpH of soil pore water in model system

(Vanbeelen and Fleurenkemila, 1997) Plette et al (1996) reported a similar increase in the metal toxicity

with increasingpH to soil bacteria in a nutrient medium

A close relationship existed between the amount of metals that can be bound by the organism and the amount of metal that can potentially cause an effect Metal toxicity was closely related to the bindingof metal ions to membrane proteins, prior to membrane transport Evidence suggested that speciation and bioa-vailability of Cd govern the effects of this metal on the biota in the system

Microbial activity in the rhizosphere of plants is several orders of magnitude greater than that in the bulk soil Root exudates can lower the rhizosphere soil pH generally by one or two units over that in bulk soil Such changes in pH along with exudation of organic ligands from plant roots may impact metal bioavailabil-ity (Naidu et al., 2003) Since microorganisms are

known to mobilize Cd in soils (Chanmugathas and

Bollag, 1987) intense microbial interactions in the

rhizosphere may lead to increased availability of metals

5.2 Tolerance and adaptation of microorganisms

The effects of chemicals on microorganisms are difficult to evaluate due to the differential sensitivity of microorganisms (Fig 2), complexity of the population

dynamics, multitude of species, and conditions used for toxicity assays Microorganisms within species of the same genus or within strains of the same species can differ in their sensitivity to metals Giller et al.(1993)

demonstrated thatRhizobium meliloti was less sensitive,

in terms of growth, to Cd thanR leguminosarum and

R loti.

Tolerance mechanisms in microorganisms toward spe-cific metals often include the bindingof metals by cell wall or by proteins and extracellular polymers, forma-tion of insoluble metal sulfides, volatilizaforma-tion and enhanced export from cell (Hughes and Poole, 1989)

The microbes also influence the metal availability by changing the pH, metal valence, chelation and other mechanisms(Francis, 1990) Microbes can sorb metals