Influence of toxic metals on activity of acid and alkaline phosphatase enzymes in metal-contaminated landfill soils.
Abstract: A study was conducted to determine the effect of toxic metals on soil acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) enzyme activities in landfill soils. The enzyme activities were consistently higher in the landfill soils than in an uncontaminated alluvial soil. The landfill soils contained higher concentrations of metals (iron, manganese, cadmium, lead, zinc, copper) than did the alluvial soil. Enzyme activities were negatively correlated with the metals, with inhibition increasing with the bioavailability of the metals. It is suggested that the metals affected enzyme activities by behaving synergistically or additively with each other. Although the landfill soils had higher enzyme activities than the alluvial soil due to higher organic matter concentrations, the ratios of enzyme activity/organic carbon indicated that inhibition of enzyme synthesis and stability had occurred due to metal stress.

Additional keywords: phosphatase activity, metal, landfill soil.
Subject: Soils (Research)
Fills (Earthwork) (Research)
Authors: Roy, S.
Bhattacharyya, P.
Ghosh, A.K.
Pub Date: 05/01/2004
Publication: Name: Australian Journal of Soil Research Publisher: CSIRO Publishing Audience: Academic Format: Magazine/Journal Subject: Agricultural industry; Earth sciences Copyright: COPYRIGHT 2004 CSIRO Publishing ISSN: 0004-9573
Issue: Date: May, 2004 Source Volume: 42 Source Issue: 3
Topic: Event Code: 310 Science & research
Accession Number: 117988828
Full Text: Introduction

The preservation of soil quality is essential for sustainable agriculture. Organic matter is a vital attribute of soil quality. The scarcity of traditional manure and abundance of organic wastes, such as sewage sludge and municipal solid-waste compost, have led to the recycling of these organic wastes for use in crop production. According to Stratton et al. (1995), these wastes contain metals, which have the potential for contaminating the food chain. Several metals also have a detrimental effect on soil microbiological and biochemical parameters (Giller et al. 1998). The need for field studies lies in the fact that laboratory experiments, where usually large doses of metals are added only once, are very different from the field situation. In the field, pollution usually occurs for many years, with the soil microorganisms being challenged with a mixture of metals. Metals have very long half-lives, and once they enter the soil, they persist over a long period (McGrath 1987). Fresh additions of metals in the laboratory, therefore, do not model their long-term effects on soil microbial biomass and activity. The assay of soil enzymes can be a sensitive indicator for estimating the relative pollution effects of metals and other industrial pollutants in soil. The strong inhibition of a variety of enzyme activities in metal-polluted soils has been reported (Tyler 1974; Juma and Tabatabai 1977). In recent years, soil biochemical parameters have been seen to be early and sensitive indicators of soil stress and can be used to predict long-term trends in soil quality (Saviozzi et al. 2002).

Phosphorus availability may be the most limiting factor for plant growth. Much of the plant's demand for phosphorus is met by cycling of phosphorus in organic matter. Since plants can utilise only inorganic phosphorus (Stevenson 1986), organic phosphorus compounds, which mostly originate from plant roots, fungi, and soil microorganisms, must first be hydrolysed by phosphatase enzymes to the inorganic form (Dick et al. 1983). According to Nannipieri et al. (1990), phosphatase measured in soils reflects the activity of enzymes bound to soil colloids and humic substances, free phosphatases in the soil solution, and phosphatases associated with living and dead plant or microbial cells. Phosphatase activities can be a good indicator of the organic-phosphorus mineralisation potential and biological activity of soils (Dick et al. 1983).

Soil enzymes can consist of intracellular and extracellular components. Extracellular enzymes like acid and alkaline phosphatase can be stabilised in a 3-dimensional network of organo-mineral complexes and maintain their activities (Burns 1982). The activities of these extracellular enzymes are, however, affected by soil constituents (Gianfreda and Bollag 1994), pH, and trace metals (Eivazi and Tabatabai 1990), because these factors modify the conformation of the enzymes (Geiger et al. 1998). Furthermore, Eivazi and Tabatabai (1990) assumed that metal ions might inhibit enzyme reactions: (i) by complexing with the substrate, (ii) by combining with the protein-active group of the enzymes, or (iii) by reacting with the enzyme-substrate complex. Therefore, it is imperative to consider soil properties along with the metals in order to examine the influence of the metals on soil acid and alkaline phosphatase activities.

The disposal of huge amounts of solid wastes, both domestic and industrial, in any metropolitan city is a global problem. In Kolkata, about 3500 tonnes of solid wastes are generated daily and dumped in the low-lying areas situated at the eastern outskirt of the city, locally known as dhapa. Cultivation of vegetables with municipal solid waste and sewage water, either full or in part, at dhapa has continued for about a century. Olaniya et al. (1995) reported that the soils of the dhapa landfill were more heavily contaminated with metals than normal arable land and are subjected to severe anthropogenic stress and are potentially hazardous in nature.

A short-term study with municipal solid-waste compost revealed that there was no detrimental influence on soil microbial and biochemical parameters (Bhattacharyya et al. 2001), but studies on long-term effects are scarce. In the absence of any data on phosphatase activity in landfill soils, and dhapa soil in particular, the objective of this work was to ascertain whether metals in the landfill soil are a threat to the soil environment for sustainable agriculture. Both acid and alkaline phosphatase activities were determined and comparisons made with activities in uncontaminated arable land. The importance of studying these activities lies in their vital role in the phosphorus cycle and their sensitivity towards heavy metals (Juma and Tabatabai 1978).

Materials and methods

Soils were collected during the early summer season from a normal alluvial soil tract (S1) covered with grassland and 5 different zones (S2, S3, S4, S5, S6) of dhapa landfill area that were originally on the same alluvial soil type. A description of the landfill and agricultural land is presented in Table 1. The sampling locations were about 700 m apart. Five surface soil samples (0-15 cm) from each zone (total 30 samples) were collected with a spade and brought to the laboratory in sealed polythene bags and passed through a 2-mm sieve. The sampling was designed to be representative of the different sites.

Physico-chemical analyses were carried out with air-dried soil samples. The pH was determined in a 1:2.5 soil:water suspension, whereas sand, silt, and clay percentages were measured by the International Pipette Method (Piper 1966). Organic carbon (C) was determined as in Nelson and Sommers (1982) and total nitrogen according to the method of Sankaram (1966). Available phosphorus (Olsen P) was estimated according to Black (1965). Total, DTPA-extractable, and water-soluble metals were determined by the methods of Page et al. (1982), Lindsay and Norvell (1978), and Ma and Uren (1998), respectively. Acid and alkaline phosphatase activities of the soils were estimated by the methods of Tabatabai and Bremner (1969) with field-moist samples. The data given in the table were mean of triplicate laboratory analyses.

Statistical analyses were performed with the help of the IRRISTAT statistical package (version 3/93) developed by IRRI, Philippines.

Results and discussion

Physico-chemical analysis

The landfill soils contained a much higher percentage of sand (44-69%), but a lesser percentage of silt and clay, than the alluvial soil; soil texture varied from sandy clay loam to clay (Table 2). All soil samples were slightly alkaline. Organic C varied significantly among the soils and was 4-7 times higher in the landfill soils than in the alluvial soil. The difference in organic C among the landfill soils seemed to be related to their history of land filling with the organic-rich solid waste. The landfill soils also contained significantly higher amounts of total N and available phosphorus than the alluvial soil. The landfill soils had higher CEC than alluvial soil. The status of metals in the soils is given in Table 3. The landfill soils had significantly higher concentrations of total, DTPA-extractable and water-soluble iron, manganese, cadmium, lead, zinc, and copper than the alluvial soil. There were also significant variations in the metal concentrations among the landfill soils.

Enzyme activities

Plant roots are major producers of acid phosphatase enzymes (Speir and Cowling 1991), but they do not produce alkaline phosphatase. Alkaline phosphatase enzymes originate from soil bacteria, fungi, and fauna (Tarafdar and Claassen 1988). Soil microbes can produce and release large amounts of extracellular phosphatase due to their large combined biomass, high metabolic activity, and short life cycles (Speir and Ross 1978).

In high-pH soils, alkaline phosphatase generally exceeds acid phosphatase activity (Eivazi and Tabatabai 1977). In our alkaline soils, alkaline phosphatase activity was 1.3-2.1 times higher than acid phosphatase activity. The higher enzyme activities recorded in the landfill soils (Table 4) than the alluvial soil could be attributed to their higher organic C concentrations (Table 2). The negative influence of metals on enzyme activities (Tyler 1981) was masked in the landfill soils because of their high organic matter content and, probably, high microbial biomass, and hence high enzyme activities. The variation in enzyme activities in the landfill soils is related to the interplay of physico-chemical properties as well as the bioavailability of the metals. Possibly, the metal form(s) in the landfill soils were not sufficiently bioavailable to adversely affect the microbial populations. Chander et al. (2001) emphasised that apart from metals, other factors such as soil organic matter also regulate the amount of microbial biomass, and hence enzyme activity, in soil.

Expressing enzyme activities on an organic C basis can give an improved understanding of metal stress (Tscherko and Kandeller 1997). Dick (1994) also had suggested relating soil enzyme activities to soil organic C. On this basis, activities were higher in the alluvial soil (S1) than in the landfill soils (Table 4). Our study therefore suggested that, although the landfill soils had higher enzyme activities than the alluvial soil due to their high organic matter concentrations, there was actually an inhibition of enzyme synthesis and stability due to metal stress.

Relationships between enzyme activities and other soil properties

Regression analysis of the mean values of data from each of the 5 landfill sites showed that the coefficient of determination ([R.sup.2]) of the relationships between acid phosphatase activity and organic C or pH, as separate variables, were 0.41 and 0.94, respectively. When activity values were regressed on both organic C and pH, the [R.sup.2] increased significantly to 0.98. The [R.sup.2] values for alkaline phosphatase activity with organic C, pH, and organic C combined with pH were 0.45, 0.90, and 0.98, respectively. Frankenberger and Dick (1983) and Gianfreda and Bollag (1994) also found the same relationships. These positive relationships indicate that the enzyme activities may be primarily associated with the soil organic fraction and stabilised as enzyme-organic matter complexes.

Both acid and alkaline phosphatase activities were positively correlated with Olsen-extractable P (r = 0.85, 0.95 respectively; P < 0.05). Cbunderova and Zubets (1969) (cited in Speir and Ross 1978) reported increased phosphatase activity with increasing P fertilization until the soluble P content of the soil reached 200 mg/kg soil. Phosphatase activity declined and disappeared completely at critical soluble P contents of 600-800 mg P/kg soil. In our study, Olsen-extractable P ranged from 18 to 85 mg/kg soil, which was low enough not to retard activity. The high correlation coefficients (r = 0.98; P < 0.05) between acid and alkaline phosphatase activities in our soils were probably due to the fact that both enzymes were correlated with organic C concentrations. This result was consistent with the findings of Frankenberger and Dick (1983).

Influence of metals on the enzyme activities

Metal ions are assumed to inactivate enzymes by reacting with sulfhydril groups, a reaction analogous to the formation of a metal sulfide. Sulfhydril groups in enzymes serve as integral parts of the catalytically active sites or as groups involved in maintaining the correct structural relationship of the enzyme protein (Juma and Tabatabai 1977). Metals can reduce enzyme activity by interacting with the enzyme substrate complex, by denaturing the enzyme protein, or interacting with the protein active groups (Nannipieri 1995). They can also affect the synthesis of the enzymes by the microbial cells. Metals in soils are present in various forms due to interactions with various soil components; therefore, the total concentrations in soils cannot provide a precise index for evaluating their influence on soil microorganisms and enzyme activities (Kunito et al. 2001). Water, and DTPA- and EDTA-type extractants are more popular reagents to extract bioavailable metals (Kabata-Pendias and Pendias 1992).

The enzyme activities of the landfill soils were negatively correlated with the different fractions of all the metals, being lowest for total concentrations and highest for water-soluble concentrations (Table 5). Hattori (1992) also found that water-soluble forms of metals were more toxic than insoluble forms. Among the metals studied, DTPA-extractable copper and cadmium were most toxic, as reflected by their comparatively high negative correlation values with both enzyme activities. It has been generally recognised that copper and cadmium are more toxic than the other metals (Hiroki 1992). However, it is difficult to determine whether copper and cadmium strongly affected the soil enzyme activities individually, because there were highly positive correlations (P < 0.05) among all the metals studied (r values ranged from 0.88 to 0.99; mean 0.97) and the changes in the concentrations of the metals in the field were similar to each other (Table 3). It is thus suggested that the metals affected enzyme activities in combination with each other. Beckett and Davis (1978) previously found that when metals were present in a mixture they behaved synergistically or additively.

Conclusion

The landfill soils were heavily contaminated with metals. Although the landfill soils had higher enzyme activities than the alluvial soil because of their high organic matter content, inhibition of enzyme synthesis and stability due to metal stress is, nevertheless, indicated.

References

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S. Roy (A), P. Bhattacharyya (B,D), and A. K. Ghosh (C)

(A) CRIJAF, Nilganj, Barrackpur-743 101, West Bengal, India.

(B) NBSS & LUP (ICAR), Block-DK, Sector-II, Salt Lake City, Kolkata-700 091, West Bengal, India.

(C) NBSS & LUP (ICAR), Block-DK, Sector-II, Salt Lake City, Kolkata-700 091, West Bengal, India.

(D) Corresponding author; email: b_pradip@rediffmail.com

Manuscript received 27 March 2003, accepted 19 December 2003
Table 1. Description of the landfill and agricultural land

Sample no.   Description of sites of soil samples

S1           Arable grassland; not adjacent to the landfill site
S2           Sewage-water-fed brinjal-growing agricultural land
S3           Brinjal-growing agricultural land with annual garbage
             dumping
S4           Brinjal-growing agricultural land where garbage has not
             been dumped for the last 20 years
S5           Brinjal-growing agricultural land where garbage has not
             been dumped for the last 50 years
S6           Agricultural land where fresh garbage is supplied
             regularly; the land has not been cropped

Table 2. Physico-chemical parameters of the alluvial and landfill soils

Soil code           Sand   Silt   Clay                     pH (1:2.5
                     (%)    (%)    (%)   Texture             water)

S1                   38     22     40    Clay                 7.2
S2                   44     21     35    Clay loam            7.3
S3                   69      6     25    Sandy clay loam      7.5
S4                   62     13     25    Sandy clay loam      7.6
S5                   65      7     28    Sandy clay loam      7.7
S6                   68     12     20    Sandy clay loam      7.3
Mean                 58     14     29    --                   7.4
l.s.d. (P = 0.05)   0.3    0.3    0.3    --                   0.3

Soil code           Organic C   Total N    C/N    Available P
                     (g/kg)      (g/kg)   ratio     (mg/kg)

S1                     7.4        0.68    10.9         18
S2                    30.7        2.53    12.1         72
S3                    44.6        3.86    11.6         67
S4                    34.5        3.21    10.7         58
S5                    32.6        3.13    10.4         54
S6                    55.6        4.8     11.6         85
Mean                  34.2        3.0     11.2         59
l.s.d. (P = 0.05)      3.0        0.1     --          0.3

Table 3. Total, DTPA-soluble, and water-soluble metal concentrations
(mg/kg) of the alluvial and landfill soils

BDL, below detection limit

                            Iron                 Manganese
Soil
code                Total   DTPA   Water   Total   DTPA   Water

S1                  1320    34.8    1.4     292     20     1.8
S2                  1332    52.9    5.2     312     38     3.6
S3                  1344    78.9    6.1     378     55     4.9
S4                  1343    86.9    6.7     396     59     5.2
S5                  1347    87.6    6.7     380     62     5.3
S6                  1346    82.4    6.4     385     58     4.9
Mean                1339    70.6    5.4     357     49     4.3
l.s.d. (P = 0.05)      3     0.3    0.3       3      3     0.3

                          Cadmium                  Lead
Soil
code                Total   DTPA   Water   Total   DTPA   Water

S1                   1.1     0.2    BDL      94     17     0.4
S2                   5.2     1.2    BDL     192     29     1.1
S3                   6.6     1.6    BDL     368     38     1.4
S4                   6.9     2.1    BDL     398     40     1.7
S5                     7       2    BDL     379     42     1.9
S6                   6.8     1.7    BDL     372     40     1.6
Mean                 5.6     1.5    --      301     34     1.4
l.s.d. (P = 0.05)    0.3     0.3    --        3      3     0.3

                            Zinc                  Copper
Soil
code                Total   DTPA   Water   Total   DTPA   Water

S1                   165    13.9    0.5      52    13.8    0.5
S2                   194    31.8    1.8     168    31.9    1.2
S3                   248    41.9      3     208    39.2    1.7
S4                   257    47.8    3.7     222    42.8      2
S5                   261    48.9    3.8     228    41.9    2.3
S6                   254    45.8    3.4     224    40.1    1.8
Mean                 230    38.4    2.7     184    35.0    1.6
l.s.d. (P = 0.05)      3     0.3    0.2       2     0.3    0.2

Table 4. Acid and alkaline phosphatase activities and their ratios with
organic C in the alluvial and landfill soils

Soil                   Phosphatase activity       Acid phosphatase
code                ([micro]g p-nitrophenol/g     activity/alkaline
                        oven-dry soil.h at      phosphatase activity
                           37[degrees]C)

                         Acid   Alkaline

S1                        262      344                   1.3
S2                        396      618                   1.6
S3                        356      602                   1.7
S4                        290      592                   2.0
S5                        272      578                   2.1
S6                        435      638                   1.5
Mean                      335      562                   2
l.s.d. (P = 0.05)           3        3                   --

Soil                   [10.sup.-4] x Acid       [10.sup.-4] x Alkaline
code                  phosphatase activity/      phosphatase activity/
                       organic C ([micro]g        organic C ([micro]g
                    p-nitrophenol/h.[micro]g   p-nitrophenol/h.[micro]g
                           organic C)                 organic C)

S1                             354                        465
S2                             129                        201
S3                              80                        135
S4                              84                        172
S5                              83                        177
S6                              78                        115
Mean                           135                        211
l.s.d. (P = 0.05)               --                         --

Table 5. Correlation coefficients (r) between enzyme activities
and metals

DTPA and water represent metal-soluble in DTPA and water,
respectively

Metal              Acid         Alkaline
                phosphatase   phosphatase

Fe      Total     -0.34         -0.27
        DTPA      -0.52 **      -0.42 *
        Water     -0.57 **      -0.45 *
Mn      Total     -0.40 *       -0.29
        DTPA      -0.50 **      -0.41 *
        Water     -0.58 **      -0.49 *
Cd      Total     -0.47 *       -0.39 *
        DTPA      -0.72 **      -0.59 **
Pb      Total     -0.45 *       -0.35
        DTPA      -0.48 *       -0.39 *
        Water     -0.64 **      -0.53 **
Zn      Total     -0.46 *       -0.37
        DTPA      -0.54 **      -0.42 *
        Water     -0.56 **      -0.44 *
Cu      Total     -0.42 *       -0.31
        DTPA      -0.58 **      -0.46 *
        Water     -0.71 **      -0.62 **

* P <0.05; ** P <0.01.
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