Effect of cadmium-bearing sewage sludge on crop plants and microorganisms in two different soils

Agriculture, Ecosystems and Environment, 20 ( 1988 ) 181-194

181

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Effect of Cadmium-bearing S e w a g e Sludge on Crop Plants and Microorganisms in Two Different Soils S. COPPOLA 1, S. DUMONTET 1., M. PONTONIO 1, G. BASILE 2 and P. MARINO 3

IIstituto di Microbiologia agraria, Universit~ di Napoli, 80055 Portici (Italy) 2Istituto di Chimica agraria, Universit~ di Napoli, 80055 Portici (Italy) 3Istituto Internazionale di Genetica e Biofisica Consiglio Nazionale delle Ricerche, 80100 Napoli (Italy) (Accepted for publication 14 January 1988)

ABSTRACT Coppola, S., Dumontet, S., Pontonio, M., Basile, G. and Marino, P., 1988. Effect of cadmiumbearing sewage sludge on crop plants and microorganisms in two different soils. Agr&. Ecosystems Environ., 20: 181-194. The metal-bonding capacities of two regional soils of southern Italy were evaluated. A volcanic soil and a sample of"terra rossa" were treated with sewage sludge spiked with CdS04 to obtain 0, 2, 4, 8 and 16 ppm of total cadmium in the soil. Cadmium uptake and cadmium effects on drymatter production of rye grass, spinach, dwarf bean and radish grown in the two soils and the effects of cadmium on soil microflora were investigated. Plant and microbial responses were considered in relation to three different analytical methods for evaluating the biologically-effective concentration of cadmium in soil.

INTRODUCTION

It is recognized that the heavy metals in sewage sludge are the biggest problem connected with the land disposal of this waste material. Mn, Zn, Cu and Co are essential elements, both to animals and plants; their application to agricultural land and introduction into the food chain could therefore be useful. However, frequent and repeated sludge applications may increase the concentration in the soil to toxic levels (Andersson, 1977a). On the other hand Hg, Cd, Cu, Ni and Pb are phytotoxic and their entrance into the food chain at elevated levels may be extremely harmful (Ryan et al., 1980; Murphy and Stuart, 1982; Nriagu, 1984). Moreover the simultaneous occurrence of more than one heavy metal in sludge at excessive levels can produce synergetic toxicity in soil (Smilde, 1982 ). *Present address: Istituto di Chimica Agraria, Universit~ della Basilicata, 85100 Potenza, Italy.

0167-8809/88/$03.50

© 1988 Elsevier Science Publishers B.V.

182

Biological availability of heavy metals is not directly correlated to their total concentration in the sludge bearing them or in the receiving soil (Andersson, 1977b). At the sludge pH value, metals are largely in an insoluble form. In the soil, metal mobility depends widely on chemical and physical characteristics of the soil-sludge system (Dhaese and Cottenie, 1979) as well as on soil microbial activity (Saxena and Howard, 1977; Kurek et al., 1982). Cation-exchange capacity, pH, organic matter, clay and moisture content affect heavymetal solubility and mobility in the soil (Haghiri, 1974; Levi-Minzi et al., 1976; Andersson, 1977b). pH regulates interactions between soil constituents and heavy metal; therefore heavy-metal availability could be controlled by preventing acidity increases (Dhaese and Cottenie, 1979). Cadmium is among the most dangerous metals in soil pollution because of its high toxicity, even at very low concentrations, and its high relative mobility (Andersson, 1976). Phosphorus fertilizers can also increase Cd availability levels in soil. They are generally Cd-contaminated, and moreover can free the metal by exchanging other ions with the soil cation exchange complex (Williams and Davis, 1973; Andersson, 1976). Soil microflora is another important factor able to affect heavy-metal mobility in the soil. Microorganisms are heavy-metal sensitive (Duxbury, 1981; Duxbury and Bicknell, 1983 ) and they can, in turn, affect both concentration and toxicity of metals. They are responsible for reduction of organo-metallic complexes, for precipitation after reaction with H2S produced by their metabolism, for chelation, absorption and transformation into innocuous forms (Saxena and Howard, 1977 ). Absorption by bacterial cells may play an important role in metal immobilization in soil (Kurek et al., 1982). The remaining available fraction of heavy metal can also exert adverse influences on soil microflora and inhibit many microbial activities indispensable to soil fertility. Nevertheless, the determination of such a fraction represents a very complex analytical problem, as interactions between soil components and heavy metals vary widely with soil type. Several a.nalytical procedures have been suggested, but correlation between their performance and biological response is inadequately investigated, with reference to the influence of soil type. Considering that, from a practical point of view, metal availability may be more interesting than total concentration in a soil, each method should be evaluated in many different soil types. Following a preliminary report (Coppola, 1983), this paper deals with the effects on plants and microorganisms of cadmium, added with sewage sludge, to two regional Italian soils. The biological results are considered by comparing the extraction efficiency of three different procedures.

183 MATERIALS AND METHODS

Soils The characteristics of the two soils assayed are listed in Table I. The first is a fine-textured volcanic soil, developed on the yellow tuff of Posillipo (Naples), with a high percentage of vitreous material and very low clay content. It is the typical soil of the N - N W area of Naples (Campi Flegrei) and of the Islands of Ischia and Procida. Similar soil can also be found in some volcanic zones of Lazio. The second soil is a typical "terra rossa" from Castellana (Province of Bari), rich in iron oxides, with a high percentage of non-expansible kaolinite-type clay. "Terra rossa" can be found in Italy in large areas of Puglie, Lazio, Venezia-Giulia, Toscana, Liguria, Sardegua and in some areas of Southern France, Spain, Greece, Yugoslavia, Algeria and Israel. Before the trials, pH (KC1), C.E.C., clay and organic matter (dichromate method), soil texture and initial heavy-metal contents of the soils were determined. Routine analytical methods were used. Metals were assayed by atomicabsorption spectrophotometry (Perkin Elmer) after nitric-perchloric digestion.

Sludge The sludge came from the urban waste-water treatment plant of Torte del Greco, Villa Inglese (25 000 inhabitants), near Naples, and it had the composition reported in Table II.

Experimental design Cd was added to sludge as CdSO4 to obtain mixtures in the soil of each pot, with 100 g of dry matter of sludge and 0, 2, 4, 8 and 16 p p m of Cd per kg of airTABLE I Physical and chemical characteristics of the soils used in the experiments

pH Clay (%) Silt (%) Humus (%) CEC (meq× 100 g) Cadmium (ppm) Zinc (ppm) Copper (ppm) Manganese (ppm) Iron (ppm)

Volcanic

Terra rossa

6.4 7.70 16.80 1.20 9.72 0.15 107 12 518 18 600

6.6 69.30 20.30 1.00 28.88 0.15 134 11 959 34 100

184 TABLE II Chemical composition of the sewage sludge used in the experiment Percentage Ash Dry matter Nitrogen Potassium Phosphorus

36.43 22.00 6.41 1.07 1.95

ppm Cadmium Nickel Chromium Copper Lead Zinc

< <

0.1 0.1 77 512 581 2931

dried soil. The spiked sludge was mixed with the soil of each pot 2 weeks after the CdS04 treatment to establish equilibrium between sludge and Cd. Eight kg of soil was placed in normal plastic pots. The soils were fertilized with 100 mg of NH4NO3, 100 mg of Ca(H2PO4)2, 180 mg of KC1 and 50 mg of MgSO4 per kg of air-dried soil. Test plants were sown 2 weeks after the sludge addition to soil. Trials were carried out with the accumulator plant Lolium perenne L. cv. '524' and a sequence of sensitive plants: (1) Spinacia olaracea L. cv. 'Rijk Zwaan Dynamd'; (2) Phaseolus vulgaris L. cv. 'Cascade'; (3) Raphanus sativus L. cv. 'French Breakfast'. Three pots per experimental condition were sown with rye grass seeds (50 g m -2). Three pots per experimental condition were firstly sown with spinach (500 g m-2). When the longest leaf had reached 15-20 cm in length, crops were harvested and the same pots were sown with Phaseolus vulgaris (120 seeds m-2). Dwarf beans were grown until economic maturity, when the pods were harvested. The pots were therefore sown with radish (500 g of seeds m -2) and storage roots harvested when the largest was about 3 g. For the rye grass, 4 successive cuts were taken when the longest leaf blades were 20-25 cm in length. Grass was cut 2 cm above the soil surface. After the first two cuttings 50 mg of N per kg of soil was added, and for every further cutting 50 mg N, 25 mg P2Os and 50 mg K20. The dry-matter production a n d Cd uptake were calculated on cumulated data from the 4 cuts. The yield of dried (85°C) leaves per pot and the mean yield (fresh weight) per plant of pods and storage roots for bean and radish, respectively, were determined. Only the storage roots of the radish were washed free of soil with water before analysis. Cadmium content of plant material has been determined after nitric-perchloric digestion.

185

Microbiological investigations The ability of control and Cd-treated soils to mineralize organic C was evaluated by measuring the C02 release during the incubation of each soil sample, amended with 5% of wheat straw as C source, 0.005% of urea N and 0.005% of NO3-N. C02-free air was passed through the incubation vessel, and the effluent gas was bubbled through 0.5 N NaOH to trap the C02 produced by the samples. The rates of the process were determined by precipitating the carbonate with 15% BaC12 and titrating the excess base with HC1, in, the presence of thymolphthalein as an indicator. Nitrifications was measured after 15 mg of NH4-N was added per 100 g of control and treated soil, then proceeding to regular analysis of NH4-N (Nessler reagent), NO2-N (Griess reagent) and NO3-N (AgS04 Phenoldisulphonic-acid method, according to Stewart et al., 1975). Nitrogenase activity of soil samples was assayed through the technique of reduction of acetylene to ethylene. Soil samples were amended with 1% of mannitol, transferred in serum bottles, flushed several times with N2 then filled with 0.7 atm of He, 0.2 atm of 02 and 0.1 atm of C2H2 and incubated for 48 h. Measurements were made by gas-chromatographic determination (Fractovap Carlo Erba equipped with Porapak R column), and computed according to Hardy and Holsten (1977). All the above-mentioned microbial activities were studied by incubation of samples at room temperature. Moreover, water content of soil was carefully brought to 50% of the saturation value. Isolation of Azotobacter strains from soil was performed on plates of Beijerinck agar (K2HPO4, 0.2 g; CaC03, 5 g; agar-agar, 18 g; mannitol, 20 g; deionized water, 1000 ml). The ability of the isolated strains to exhibit nitrogenase activity in the presence of 0-1.6 ppm of Cd (supplied as CdSO4) has been assayed in a medium with the following composition (g l-1): mannitol, 10; K2HPO4, 0.64; KH2P04, 0.16; NaC1, 0.2; MgS04, 0.2; CaC12, 0.1; FeSO4, 0.0025; H3B03, 0.0029; COS04, 0.0025; CuSO4, 0.0001; MnC12, 0.00009; N~2Mo04, 0.0025; ZnS04, 0.0021. The most probable number (MPN) of ammonifiers, ammonia-oxidizers, nitrite-oxidizers, aerobic and anaerobic free-living nitrogen fixers was determined according to Pochon and Tardieux (1962), but the medium for ammonifiers was supplied with Casaminoacids (Difco Lab, U.S.A.) and the positivity of nitrification was tested by Merckoquant strips (10020, E. Merck, Darmstadt). Extraction of ATP from soil was carried out according to Eiland (1979). Measurements of ATP content were performed with ATP Bioluminescence HS kit (Biochemia, Mannheim) and liquid spectrophotometer (Tri-Carb 2001, Packard).

186

Determination of the available cadmium in the soil Each soil sample was extracted by the three methods shown below. (1) Determination of mobile and mobilizable Cd concentrations by means of progressive acidification with HNO3. A soil sample of 20 g was suspended in about 80 ml of distilled water and automatically titrated with 0.1 N HNO3. Titrations were carried out at pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0. The suspensions were brought up to 100 ml in volume and filtered after 1 h of equilibration at each pH. The filtrates were acidified with HNO3 to a final concentration of about 2%, and then absorption-atomic-spectrophotometer assayed for the Cd content. (2) Determination of exchangeable Cd through 0.1 M NaNO3 solution. Solution of NaNO3 was utilized in three different ratios of soil: 1:2.5, 1:5 and 1:10. A sample of 20 g of air-dried soil was weighed in a 250-ml polyethylene bottle and shaken for 2 h with the 0.1 M NaNO~ solution. Filtrates were collected into other polyethylene bottles. (3) Extraction with 0.5 M NH4-acetate+0.02 M EDTA. A sample of 20 g of air-dried soil in a 250-ml polyethylene bottle was added to 100 ml of a solution containing 38.5 g of NH4-acetate, 25 ml of glacial acetic acid and 5.845 g of EDTA in 1 1 at pH 4.65. After shaking for 30 min, suspensions were filtered and collected into a polyethylene bottle. RESULTS

Table III shows the results for the yields of rye grass, spinach, dwarf beans and radish, grown in volcanic soil and in "terra rossa". The effect of Cd is proportional to the amount of metal added and is always statistically significant, with the exception of the radish in volcanic soil. Rye grass, which is Cd-resistant and accumulating, has the highest yield in volcanic soil, where only the 0 ppm treatment is different from the others. No depressive effects on yield are noticeable in "terra rossa" up to 4 ppm of Cd added. Spinach, which is Cd-sensitive and accumulating, shows fast yield decreases in both soils, but faster in the volcanic one. In this soil, the depressive effect seems to be present at a 4 ppm dose and at 2 ppm in "terra rossa". Dwarf bean has a similar dry-matter reduction in both soils, but negative Cd influence starts at 4 ppm in volcanic soil and at 8 ppm in "terra rossa". Radish cultivated in volcanic soil seems unaffected by Cd at all doses and in "terra rossa" a depressive effect on yield is shown at 16 ppm only. Table IV contains Cd-uptake values for rye grass and spinach. The edible parts of dwarf bean and radish show a meaaurable uptake only at 8 and 16 ppm of Cd added in volcanic soil. In "terra rossa" there is no detectable uptake for the dwarf bean at all doses of Cd, while the radish accumulates a detectable amount of metal at 8 and 16 ppm.

187 TABLE III Dry-matter production (g dry matter per pot) Crops

Cd added (ppm) 0

2

4

8

16.0±1.7 1.5±0.8 50.2±9.9 8.2±3.1

15.3±1.5 0.9±1.7 34.0±9.8 7.8±0.8

15.0±2.0 13.5±2.7 0.3±0.0 0.1±0.1 32.6±14.1 17.0±3.7 6.8±2.0 6.2±1.2

16.6±1.9 3.6±0.5 27.3±1.5 10.1±1.0

12.1±3.2 2.7±0.3 25.7±2.6 9.8±0.2

12.0±2.3 2.2±0.7 11.4±3.1 9.8±1.1

11.2±1.5 1.2±0.2 10.0±2.5 6.7±0.6

16.0 1.5

15.3 0.9

15.0 0.3

13.5 0.1

62.4

50.2

34.0

32.6

17.0

17.4 3.7 27.8 10.2

16.6 3.6 27.3 10.1

12.1 2.7 25.7 9.8

12.0 2.2 11.4 9.8

11.2 1.2 10.0 6.7

Volcanic soil Rye grass (a)' 20.1 ± 1.72 Spinach (a) 1.8 ± 1.0 Dwarf bean (b) 62.4±14.3 Radish (c) 8.3±0.7 Terra rossa Rye grass (a) 17.4 ± 2.2 Spinach (b) 3.7 ± 0.5 Dwarf bean (b) 27.8±1.7 Radish (b) 10.2 ± 0.5 Mean comparison (ls.d., P = 5% ) Volcanic soil Rye grass 20.1 Spinach 1.8 Dwarf bean Terra rossa Rye grass Spinach Dwarf bean Radish

16

~(a), P = 5 % ; (b) P = 1%; (c) P - - n o t significant. 2Mean of three replicates _ standard deviation.

Spinach shows a Cd concentration statistically different for every dose in volcanic soil, while in "terra rossa" the value obtained at 4 ppm is not different from that at 8 ppm. The amount of Cd in the spinach leaf at 8 ppm in the volcanic soil is slightly, but statistically different from the value obtained at 16 ppm. In "terra rossa", on the contrary, concentrations at 4 and 8 ppm are about half the value of that at 16 ppm. The two soils also show different patterns for the dwarf bean Cd concentration. In "terra rossa" there is no significant uptake but in volcanic soil there is 0.5/~g of Cd per g of dry matter at 8 ppm and 1.8 #g at 16 ppm. Radish has 6.3/~g of metal per g of dry matter at 8 ppm and 6.9/lg at 16 ppm in volcanic soil. In "terra rossa" Cd has concentration of 4/lg g-1 only at 16 ppm. The results confirm the already known behaviour of the test-plants utilized in these experiments, as regards the effects of heavy metals. At the same time, differences between the two soils have been pointed out. In fact, the higher clay

188

TABLE IV Cadmium concentration in plant tissues (/lg Cd mg-1 dry matter) Crops

Cd added (ppm) 0

Volcanicsoil Ryegrass (a) 1 >0.1 Spinach (a) 1.7±1.02 Dwarfbean >0.1 Radish >0.1 Te~arossa Ryegrass (a) >0.1 Spinach (a) 0.4±0.2 Dwarfbean >0.1 Radish >0.1 Mean comparison (ls.d., P = 5 % ) Volcanic soil Rye grass > 0.1 Spinach 1.7 Terra rossa Rye grass > 0.1 Spinach 0.4

2

4

8

16

13.4±0.5 12.6±0.7 >0.1 >0.1

25.4±2.8 25.3±0.1 >0.1 >0.1

38.6±4.0 53.7±0.7 0.5±0,0 6.3±0.6

17.4±3.0 15.8±3.2 >0.1 >0.1

28.2±2.9 29.5±2.0 >0.1 >0.1

31.5±9.3 27.2±2.0 >0.1 1.6±0.1

58.3±4.8 59.8±4.2 >0.1 4.0±0.2

13.4 12.6

25.4 25.3

38.6 53.7

68.2 55.3

17.4 15.8

28.2 29.5

31.5 27.2

58.3 59.8

68.2±13.4 55.3±0.6 1.8±0.1 6.9±0.3

1 (a) P = 1%.

2Mean of three replicates, ± standard deviation.

content of "terra rossa" seems to reduce Cd availability and metal influence on plant growth. Soil microbial activities were evaluated at the end of the rye-grass crop test to get more complete information about the biological effect of Cd in the two soils in order to achieve a better comparison with analytical detection of bioavailable metal. The quantification of soil biomass by ATP measurement has shown very little difference among the treatments. ATP concentrations detected in the various samples are reported in Table V. An adverse influence caused by 16 ppm of Cd only occurs in the volcanic soil. But such a negative effect has not been confirmed through the evaluation of soil respiration. In both soils, mineralization of organic C ran at a similar rate, unaffected by the metal concentration. The cumulative release of CO2 reached 5000 mg per kg of soil after 20 days of incubation, about 10 000 after 35 days, from all the samples, in the experimental conditions indicated. Results from the analyses for functional groups of soil microorganisms are reported in Table VI. Some influences of Cd on the microbial populations taken into account, are more evident in the volcanic soil than in "terra rossa". Am-

189 TABLE V Average amount of ATP in samples of soils treated with increasing quantities of cadmium Cd added (ppm) to softs

ng A T P g- dry soil

Volcanic 0 2 4 8 16

32 19 33 44 6

Terra rossa 0 2 4 8 16

21 11 23 38 27

TABLE VI Microbial counts of two Italian soils treated with different amounts of cadmium. (Log. of the most probable number of viable cells g-1 dry soil) Cd added (ppm)

Volcanic soil Ammonifiers Ammonium-oxidizers Nitrite-oxidizers Free-living N2 fixers Aerobic Anaerobic Terra rossa Ammonifiers Ammonium-oxidizers Nitrite-oxidizers Free-living N2 fixers Aerobic Anaerobic

0

2

4

8

16

8.4 4.3 4.4

8.4 3.9 3.9

7.6 3.5 2.4

5.6 3.4 1.6

4.9 3.6 1.4

1.4 3.9

1.4 3.6

1.4 3.4

1.4 2.9

1.4 2.4

6.0 3.6 3.4

5.6 3.6 3.4

4.9 3.4 3.4

4.8 2.4 3.4

4.2 2.4 3.4

2.2 2.6

2.0 2.5

2.0 2.2

1.9 2.0

1.6 2.0

monifiers seem to be the most sensitive to the action of the metal, as their counts dramatically decrease with increase in the Cd added, especially in the volcanic soil. These conclusions arewidely confirmed by the time-courses of ammonification previously reported (Coppola, 1983). Among nitrifiers, only nitrite-oxidizers are depressed by Cd-ions in volcanic soil. However, nitrifica-

190

tion was uninhibited in all samples during incubation and 97-99% of soil inorganic nitrogen was detected as NO3-N after 30 days. Neither aerobic nor einaerobic free-living N2-fixing microorganisms were particularly numerous in the two soils, consistent with the low humus content. Microbial counts for these groups were not significantly affected by Cd. On the other hand, soil dinitrogen fixation, gas-chromatographically measured by C2H2-C2H4 assay, was considerably inhibited by increasing Cd-dose. In Table VII results are reported as grams of N2 fixed per hectare and per day per kg of dry soil. The metal influence appears particularly dramatic in "terra rossa", where N2-fixat±on is reduced by 2 p p m of Cd, to 60% of the control sample. Since all the other biological responses reported in this study have shown higher biologically-effective concentrations of Cd in the volcanic soil than in "terra rossa", the adverse influence exerted by Cd upon N2 fixers in "terra rossa" can only be explained by attributing a particular Cd-sensitivity to the microbial strains in this type of soil. Fifty strains of Azotobacter chroococcum, the most representative free-living N2 fixer in our soil, were isolated. Half of these strains came from the volcanic soil and half from "terra rossa". They were assayed for nitrogenase activities affected by Cd in the medium. The results, reported in Fig. 1, establish that the strains from the volcanic soil exhibit a greater Cd-resistance than the strains from "terra rossa": about 50% of the former are indeed able to fix N2 in the presence of 1.6 ppm of Cd in the medium, while only 30% of strains isolated from "terra rossa" tolerate 0.8 p p m of metal. A previous experiment, carried out in the same conditions but without mannitol in the medium, showed no C2H2 reduction at any Cd dose. The analytical TABLE VII Cadmium effect on non-symbiotic N2 fixation Cd added (ppm) to soils Volc~ic 0 2 4 8 16 Terra rossa 0 2 4 8 16

mg N2 day-1 kg= 1 dry soil

16.9±1.01 16.1±3.0 12.9±0.7 3.4±2.2 3.1±0.8 33.3±4.0 17.8±0.9 13.9±3.7 4.6±0.2 0.7±0.1

1Mean of three replicates _ standard deviation.

191

z~ x

o

100

_m n 1

m "iC

75

~

so

1 II

•

25

1 0

0,1

0,2

0,4

C d 2+ o o n c e n t r a l : i o n s

0.8

1.6

(pprn)

Fig. 1. Percentage of isolated Azotobacter strains able to fix nitrogen at different cadmium concentrations in the medium. [-1= Volcanic soil; [~ = Terra rossa.

evaluation through different methods of biologically-available Cd concentration in the two soils, was made at the end of rye-grass crop test. The comparison between the Cd added and the Cd detected by the various methods can always be described by linear equations. The differences among the 3 methods, concern the quantities of metal extracted. The ammonium acetate + EDTA method extracts more Cd from both soils, while sodium nitrate solutions never extract an amount of Cd higher than 0.1 pg g- 1 soil from "terra rossa" and only very low quantities at 4, 8 and 16 ppm of added Cd in volcanic soil (Fig. 2). By the progressive acidification method {Fig. 3), the amounts of Cd extracted decrease as pH increases. Levels of metal extracted are always higher in the volcanic soil than in "terra rossa". Also at pH 6, detectable amounts of Cd are extracted from the volcanic soil amended with 4, 8 and 16 ppm. On the contrary, detectable amounts of metal are extractable at pH 4.5 from "terra rossa" treated with 8 and 16 ppm of Cd only. These results are reported as Cd-ions extracted/Cd-ions added in Fig. 3. The ammonium acetate + EDTA method allows the extraction of the highest quantities of Cd, but it does not seem to reflect the different metal-bonding capacities of the two soils, and the differences between the Cd extracted from the two soils at the various total concentrations are inconsistent with soil behaviour. On the other hand, NaNO3 solutions are often unable to extract Cd, even when the metal effects on plant growth and quality can be evaluated. For the two soils studied, the progressive-acidification method seems to be able to indicate the diversity of the two soils showing "terra rossa" bonds higher quantities of metal than the volcanic soil.

192 16

w

•o U "0

8

g o

o ,4

---O

0

2

4

8 Tot;ol

16

C d 2+ i n s o i l

[ppm)

Fig. 2. Cadmium detected by different extractants, o = Volcanic soil, H N 0 3 p H 2.5; y = 0.03 + 0.71x, R2>0.99; . = T e r r a rossa, H N 0 3 p H 2.5, y=O.66+O.37x, R2=0.99; Z~ =Volcanic soil, Ammon i u m acetate + EDTA, y = 0.216 + 0.86x, R 2 = 0.97; • = Terra rossa, A m m o n i u m acetate + EDTA, y= 1.04+ 0.76x, R2=0.97; [] =Volcanic soil, NAN03.

0. 0. w "0

U '0 II "0 "0 <

E D.

•o

u

1.0

0.8

0.6

0.`4

"0 O tO

0.2

O

O

;5

.o

g%

;o

`45

5:0

pN

Fig. 3. Extraction of cadmium from two soils through the progressive-acidification (HNO3) method. o =Volcanic soil, y = 5 . 0 8 - 0 •78x , R2=0.94; • = T e r r a rossa, y = 3 . 1 9 -°'81x, R2=0.91.

193 CONCLUSIONS The influence of Cd added to soil with sewage sludge upon physiology and biomass of plants and microorganisms depends on the metal dose, as well as on the type of so~l, plant and microorganism. This study on two typical, neutral soils of southern Italy shows that metal uptake by plants is the most suitable phenomenon to describe the different behaviour of soil responsible for biologically-active Cd availability. Cd uptake was always lower in crops grown on "terra rossa" than on the volcanic soil. Only spinach, as a sensitive and accumulating plant, has exhibited comparable metal uptake in both the soils. The evaluation of soil microbial biomass through ATP has pointed out an adverse Cd influence at 16 p p m and in volcanic soil only. Among the various soil microbial groups, ammonifiers and their activity were particularly affected by Cd, showing progressive decrease as the metal concentration increased, mainly in the volcanic soil. A particularly depressive effect of Cd on free-living aerobic N2 fixers of "terra rossa" was evident. The metal sensitivity of Azotobacter strains isolated from this soil were greater in comparison with strains isolated from the volcanic soil, where lack of clay content probably induces a selection of more metal-resistant strains in natural conditions. Concerning the chemical assessment of biologically-available Cd in soil, different results were obtained through the three methods used. The extraction with 0.1 M NaNO3 from neutral soil, poor in organic matter, in agreement with the results of Sauerbeck and Stypereck (1985), gives low estimates of Cd. On the other.hand, the extraction with ammonium acetate -b EDTA probably produces overestimated values, since the amounts are near to the total metal content. In our experiments, metal bonding capacities in the volcanic soil and in "terra rossa" are well described by the progressive-acidification method, Nevertheless, according to the results presented by Sauerback and Stypereck (1985) and by-Haeni and Gupta (1984), it must be considered that, for the type of soils assayed in this study, better correlations between plant response and analytical evaluations could be achieved through extraction with 0.1 M CaC12 solutions. A direct biological verification of the toxicity levels of a metal, polluting a specific natural ecosystem, seems inevitable, however, as some microbiological soil properties can be adversely affected by heavy metal before the higher plants. ACKNOWLEDGEMENTS This work was supported by a grant of the Consiglio Nazionale delle Ricerche, Rome.

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