Limitations of soil enzymes as indicators of soil pollution
C. Trasar-Cepeda
a,*, M.C. Leiro´s
b, S. Seoane
b, F. Gil-Sotres
ba
Departamento de Bioquı´mica del Suelo, Instituto de Investigaciones Agrobiolo´gicas de Galicia, Consejo Superior de Investigaciones Cientı´ficas, Apartado 122, E-15080 Santiago de Compostela, Spain
b
Departamento de Edafologı´a y Quı´mica Agrı´cola, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain Accepted 3 May 2000
Abstract
Soil enzyme activities are considered to be sensitive to pollution and have been proposed as indicators for measuring the degree of soil degradation. In this work we found that in three galician soils exposed to various degrees of pollution by tanning effluent, hydrocarbons or landfill effluent, the changes in the activities of individual enzyme did not allow precise quantification of soil degradation. Thus, the enzymatic activities in polluted soils with respect to that in control soils was between 37 and 260% for phosphomonoesterase, between
16 and 250% forb-glucosidase, between 28 and 194% for urease and between 24 and 251% for dehydrogenase. The degree of degradation
was, however, clearly shown in all cases by the ratio Nc/Nk, where Nk is Kjeldahl nitrogen and Nc is a function of microbial biomass C and
nitrogen mineralization capacity combined with three enzyme activities (phosphomonoesterase,b-glucosidase and urease). This ratio, Nc/
Nk, exhibited all the attributes of a good pollution indicator and, in particular, was able to discriminate between the effect of the pollutant and any prior degradation of the sites. It is concluded that quantification of soil degradation can require that information on enzyme activities be
supplemented with information on other biochemical soil properties.q2000 Elsevier Science Ltd. All rights reserved.
Keywords: Soil enzymes; Soil contamination; Soil biochemical properties
1. Introduction
The wide-spread pollution of soils is an increasingly urgent problem because of its contribution to environmental deterioration on a global basis (Bezdicek et al., 1996; Dick, 1997; Lal, 1997; van Beelen and Fleuren-Kamila´, 1997). Until relatively recently, soil was widely regarded as just an environmental filter ensuring the quality of both water and atmosphere. However, in the context of the pursuit of sustainability, it is now recognised that soil is not only an effective de-contaminant of potential pollutants but that its chemical, physical and biological quality must be main-tained (Hornick, 1992; Parr et al., 1992). From the point of view of sustainability, a high-quality soil is a soil that is capable of producing healthy and abundant crops; de-contaminating the water passing through it; not emitting gases in quantities detrimental to the environment; and behaving as a mature, sustainable ecosystem capable of degrading organic input (Doran and Parkin, 1994; Gregor-ich et al., 1994; Brookes, 1995; Pankhurst et al., 1995). This view clearly implies that diagnosis of soil pollution should
be carried out on the basis of observed alterations in the soil properties controlling the behaviours described above, ideally in a way that allows any loss of soil quality to be quantified as well as identified qualitatively (Larson and Pierce, 1991; Doran and Parkin, 1994).
The physical, chemical, biochemical and biological prop-erties of a soil are all important for its behaviour (Arshad and Coen, 1992; Parr et al., 1992). Characterisation of this behaviour should focus on the properties that are most sensi-tive to environmental stress. Since this sensitivity is a feature of many biological and biochemical properties (Dick and Gupta 1994; Vanhala and Ahtiainen 1994), it is these that may be considered as most appropriate for the purpose of soil quality evaluation (Pankhurst et al., 1995; Yakovchenko et al., 1996; Elliott, 1997). Visser and Parkin-son (1992) have suggested that the biological and biochem-ical properties that are most useful for detecting the deterioration of soil quality are those that are most closely related to nutrient cycles, including soil respiration, micro-bial biomass, nitrogen mineralisation capacity and the activ-ities of soil enzymes. In particular, enzyme activactiv-ities are especially significant because of their major contribution to the ability of the soil to degrade organic matter (Franken-berger and Dick, 1983). Furthermore, Kandeler et al. (1996) have indicated that the composition of the microbial Soil Biology & Biochemistry 32 (2000) 1867–1875
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community determines the potential of that community for enzyme synthesis, and thus any modification of microbial community due to environmental factors should be reflected on the level of soil enzymatic activities. In addition, enzyme activities have the further advantage of being easy to deter-mine without expensive, sophisticated instruments (Dick, 1997).
In spite of the above considerations, published findings on the influence of pollutants on soil enzyme activities indicate that polluted soils are a system of great complexity. For example, the behaviour of dehydrogenase activity, which is only present in viable cells (Skujins, 1978; Trevors, 1984) and may therefore be considered as a direct measure of soil microbial activity (Garcı´a and Herna´ndez, 1997), is very variable. Dehydrogenase activity appears to depend on the type of pollutant; for example, it is high in soils polluted with pulp and paper mill effluents (McCarthy et al., 1994) and low in soils polluted with fly ash (Pichtel and Hayes, 1990). Also significant is the concentration of pollutant (higher levels of dehydrogenase at low doses of pesticides and vice versa (Barnah and Mishra, 1986), and the type of soil (Doelman and Haanstra, 1979; Kandeler et al., 1996). Similarly complex behaviour is exhibited by all the hydro-lytic enzymes that have been investigated, which has thrown doubts on the possibility of their use as reliable indicators of soil quality (Dick, 1997).
With a view to determining which of a variety of enzyme activities, if any, might be useful as indicators of the quality of acid soils rich in organic matter, we studied how these parameters were affected in soils that had at some time been subject to pollution by hydrocarbons, tanning effluent or urban waste landfill effluent.
2. Materials and methods
2.1. Soil sample collection
Soil samples were collected from three sites at which different kinds of pollution had been detected by environ-mental protection agencies (see Acknowledgements). At each site, samples were collected from apparently more affected, less affected and unaffected areas, as detailed below. However, the fact that the pollution events were uncontrolled, together with the topographical characteristics of the polluted sites, meant that it was impossible to know exactly the quantity of pollutant to which the soil was initi-ally exposed.
At each sampling point, 10–15 cores of the top 0–7 cm of soil were taken at random and pooled in the field. The samples were transported to the laboratory in insulated bags and sieved (4 mm mesh). A sub-sample of each sieved soil was air-dried to determine general soil properties and the remainder was stored at 48C. Analyses of biochemical properties (enzyme activities, microbial biomass C, and N
mineralization capacity) were performed within 2 weeks of sample collection.
2.2. Tanning effluent
In November 1993, an area of meadow soils (loamy texture) located in Carballo (A Corun˜a, Spain) was polluted by effluent from a local tanning factory (pH 4.2, suspended solids 252 mg l21, Cr31 21 mg l21, total Pb content 0.34 mg l21, COD 813 mg O2l21). In February 1998 samples were taken at a point adjacent to the effluent outlet (T1), at a nearby point at which the soil was covered with hairs deposited from the effluent (T2), at a point somewhat further removed where the soil surface was hair-free (T3), and at a pollution-free point far from the effluent source (T4).
2.3. Landfill effluent
In April 1997, the meadow soils (loamy texture) adjoin-ing an urban waste landfill near Corun˜a (Spain) were reported to have been polluted by landfill effluent of pH 9.74 with a total C content of 548 mg l21and a total inor-ganic N content of 106 mg l21(mainly as ammonium). In September 1997 samples were taken from two polluted points close to the effluent outlet (L1 and L2), from a shal-low depression in which the effluent had remained stagnant for several days (L3), and from an unaffected point (L4).
2.4. Hydrocarbons
On May 15th 1998 an area of meadow soil (sandy loam texture) near Santiago de Compostela (A Corun˜a, Spain) was polluted by hydrocarbons leaking from a faulty oil pipe-line. On June 10th 1998, samples were collected from a point close to the leak with clear traces of hydrocarbons on the surface (H1), from two rather more distant points with no surface signs of hydrocarbons (H2 and H3), and from an unpolluted point far from the effluent source (H4).
2.5. Soil physical and chemical properties
The methods described by Guitia´n and Carballas (1976) were used to determine the following soil properties: pH in water (1:2.5, soil:water ratio), pH in 1 M KCl (1:2.5, soil:solution ratio), water field capacity (at 33 kPa in a Richard’s membrane-plate extractor); total C content (by potassium dichromate oxidation) and total N content (by Kjeldahl digestion).
2.6. Biochemical properties
(1965). The nitrogen mineralised was estimated from the difference between the inorganic N content before and after incubation. Results are expressed in mg kg21 of oven-dried soil.
Microbial biomass carbon. Microbial biomass C was determined by the chloroform fumigation extraction method, using 0.5 M K2SO4 as extractant (Vance et al., 1987). The organic C of extracts was estimated by oxidation with potassium dichromate. The difference in C content between the fumigated and unfumigated extracts was converted to microbial biomass C (expressed in mg kg21 of oven-dried soil) by applying a factor (Kc) of 0.45 (Jenkin-son, 1988).
Enzymatic activities. Dehydrogenase activity was deter-mined with iodonitrotetrazolium violet (INT) as substrate, incubating at pH 7.5 (1 M Tris–HCl buffer) and 408C for 1 h. The iodonitrotetrazolium formazan (INTF) produced was extracted with a 1:1 (v:v) mixture of ethanol and dimethylformamide and measured spectrophotometrically at 490 nm (Camin˜a et al., 1998). Activity was quantified by reference to a calibration curve constructed using INTF standards incubated with soil under the same conditions described above, and is expressed inmmol INTF g21h21.
Urease activity was determined with urea as substrate, incubating at pH 7.1 (0.2 M phosphate buffer) and 378C for 1.5 h and measuring the NH14 released with an ammo-nia-selective gas electrode (METROHM Ltd., Herisau, Switzerland). The enzymatic activity is expressed inmmol NH3g21h21.
Acid phosphomonoesterase activity was determined with p-nitrophenyl phosphate as substrate, incubating at pH 5.0 (Modified Universal Buffer) and 378C. After 30 min 2 M CaCl2 was added (to stop the reaction and to avoid the colouration caused by organic matter), and the p-nitrophe-nol released was extracted with 0.2 M NaOH and measured spectrophotometrically at 400 nm (Tabatabai and Bremner,
1969; Saa´ et al., 1993). b-glucosidase activity was deter-mined as described for phosphomonoesterase activity except that the substrate was p-nitrophenyl-b-d -glucopyra-noside, the incubation time was 1 h and the p-nitrophenol released was extracted with 0.1 M THAM–NaOH pH 12.0 (Eivazi and Tabatabai, 1988). Both phosphomonoesterase and b-glucosidase activities were quantified by reference to calibration curves constructed using p-nitrophenol stan-dards incubated with soil under the same conditions described above and are expressed in mmol p-nitrophe-nol g21h21.
All determinations of enzymatic activities were performed in triplicate, and all values reported are averages of the three determinations expressed on an oven-dried soil basis (1058C).
2.7. Statistical analysis
All data expressed as percentages were compared by using the test of significance of means (Spiegel, 1969).
3. Results
Table 1 lists general soil properties, microbial biomass C and N mineralisation capacity, and Table 2 the measured enzyme activities. Fig. 1 shows enzyme activities per gram of total C or total N content as percentages of the corre-sponding quotients for the control samples. In the remainder of this section, the trends shown by these results are described for each kind of pollution.
3.1. Tanning effluent
At the site polluted by tanning effluent, the phosphomo-noesterase activities of samples T1, T2 and T3 were 110^ 10;215^20 and 89^8%;respectively, of the activity in C. Trasar-Cepeda et al. / Soil Biology & Biochemistry 32 (2000) 1867–1875 1869 Table 1
General characteristics of soil samples
Soil sample % Total C % Total N C/N pH H2O pH KCl Microbial
biomass C (mg kg21)
N mineralisation capacity (mg kg21)
Tanning effluent
T1 13.93 1.267 11 7.29 6.83 426 43.8
T2 18.91 1.803 11 4.57 4.30 517 16.1
T3 10.50 1.039 10 4.33 3.88 139 6.9
T4 (control) 8.91 0.756 12 6.25 5.31 999 29.9
Landfill effluent
L1 8.56 0.653 13 6.15 5.78 132 50.0
L2 7.26 0.583 13 5.93 5.49 5 26.9
L3 6.85 0.549 13 7.65 7.15 0 73.4
L4 (control) 7.59 0.475 16 5.40 4.31 700 22.9
Hydrocarbons
H1 6.15 0.470 13 6.75 5.75 595 2.9
H2 6.08 0.600 10 5.48 4.51 521 35.7
H3 4.56 0.410 11 6.45 5.48 376 18.6
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Table 2
Activities of four enzymes in the soil samples (mean^S.D.), and the ranges observed in native Galician soils. In parentheses percentage values relative to the control samples^S.D
Soil sample Phosphomonoesterasea b-Glucosidasea Ureaseb Dehydrogenasec
Tanning effluent
T1 6.41^0.12 (110^10) 2.64^0.29 (109^24) 13.76^0.87 (47^5) 0.853^0.094 (61^7) T2 12.49^0.42 (215^20) 3.32^0.20 (137^27) 15.44^0.64 (53^5) 0.616^0.031 (44^3) T3 5.20^0.19 (89^8) 1.29^0.08 (53^10) 8.16^0.39 (28^3) 0.333^0.004 (24^1) T4 (control) 5.85^0.50 (100) 2.42^0.45 (100) 29.28^2.39 (100) 1.395^0.042 (100)
Landfill effluent
L1 4.37^0.36 (121^11) 1.97^0.38 (94^20) 18.94^1.87 (66^9) 0.666^0.100 (89^15) L2 1.72^0.12 (49^4) 0.33^0.05 (16^3) 11.54^0.48 (40^4) 0.310^0.023 (42^4) L3 1.35^0.07 (37^2) 0.38^0.00 (18^2) 23.37^0.83 (81^9) 0.612^0.009 (82^5) L4 (control) 3.62^0.15 (100) 2.10^0.19 (100) 28.91^2.87 (100) 0.745^0.047 (100)
Hydrocarbons
H1 5.61^0.50 (260^25) 2.40^0.04 (250^7) 11.83^1.11 (194^18) 1.391^0.051 (251^13) H2 3.70^0.15 (171^9) 1.57^0.14 (164^15) 11.51^2.34 (188^38) 0.858^0.041 (155^9) H3 1.80^0.07 (83^4) 1.30^0.11 (135^12) 4.87^0.76 (80^12) 0.728^0.049 (132^10) H4 (control) 2.16^0.08 (100) 0.96^0.02 (100) 6.11^0.00 (100) 0.553^0.020 (100)
Native soils 2.33–47.45 0.67–29.63 1.76–66.26 0.10–1.32
a
mmol p-nitrophenol g21h21.
b
mmol NH3g2 1
h21.
c
the control (non-polluted) sample. b-Glucosidase activity rose to 109^24% in T1 and to 137^27% in T2, and declined to 53^10% in T3. Urease and dehydrogenase activities fell in all the samples polluted with tanning effluent: to 47^5% (T1), 53^5% (T2) and 28^3% (T3) of the control for urease and to 61^7% (T1), 44^3% (T2) and 24^1% (T3) of the control for dehydrogenase. All those variations were statistically significant, except in the case of phosphomonoesterase activity in T1 and T3 samples and of b-glucosidase activity in T1 sample.
When the enzyme activities are expressed relative to total C or N content (Fig.1), the polluted soil samples show significantly lower values than the control in all cases but phosphomonoesterase activity in T2 sample. In particular, none of the phosphomonoesterase orb-glucosidase values
exceeds the control value, and the b-glucosidase values exhibit the same trend (T1.T2.T3) as the urease and dehydrogenase values. Both phosphomonoesterase and b -glucosidase values, relative to the control, although not significantly different are lower when expressed with respect to N than when expressed with respect to C (Phos-phomonoesterase: T1/N 65^6%; T1/C 70^6%; T2/N 90^8%; T2/C 100^9%; T3/N 65^6%; T3/C 76^ 7%:b-Glucosidase: T1/N 65^17%;T1/C 70^18%;T2/ N 58^11%;T2/C 67^13%;T3/N 39^8%;T3/C 44^ 9%: However, there is no such difference for urease and dehydrogenase, which for a given sample are also similar one to another when expressed with respect to C or N (T1, between 28^3 and 38^4% for both urease and dehydro-genase; T2 and T3, between 20^2 and 25^2% for urease, and between 17^0 and 19^1% for dehydrogenase). C. Trasar-Cepeda et al. / Soil Biology & Biochemistry 32 (2000) 1867–1875 1871
3.2. Landfill effluent
At the site polluted by landfill effluent (Table 2), the phosphomonoesterase activities of samples L1, L2 and L3 were, respectively, 121^11; 49^4 and 37^2% of the activity in the control sample (L4). All the other activities were lower in polluted samples than in the control, and in all these cases the fall was greatest for sample L2. However, there were marked quantitative differences among the trends displayed: forb-glucosidase, the fall to 94^20% of control in L1 was not significant, but there was a drastic fall to 16^ 3 and 18^2% of control in L2 and L3, respectively; for urease, the fall was moderate in L1 66^9% of control), marked in L2 40^4%and only slight in L3 81^9%; while for dehydrogenase, the marked fall in L2 42^4% of control) was accompanied by only slight falls in L1 89^ 15%and L3 82^5%:
When the activities are expressed relative to C and N contents (Fig. 1) they exhibit patterns similar to those described above. However, in contrast to the absolute values, for L1 the phosphomonoesterase/N value is lower than in the control 88^8%and the phosphomonoester-ase/C value is only slightly higher than in the control 106^ 10%:Theb-glucosidase/C 82^17%andb-glucosidase/ N 68^15values for L1 are lower than those for L4.
3.3. Hydrocarbons
With the sole exceptions of the phosphomonoesterase and urease activities in sample H3, all the enzyme activities were significantly higher than control values in all the hydrocarbon polluted samples (Table 2). The greatest increases were shown by sample H1, which had values ranging from 194^18% of control for urease to 260^ 25% of control for phosphomonoesterase. Urease and phos-phomonoesterase activities were somewhat lower, though
still very high, in sample H2 188^38 and 171^9% of control, respectively), but were both lower than the control values in H3 80^12 and 83^4%; respectively). b -Glucosidase and dehydrogenase behaved quite similarly in all three samples, with values (relative to the control) of, respectively, 250^7 and 251^13% in H1, 164^15 and 155^9% in H2, and 135^12 and 132^10% in H3; like urease and phosphomonoesterase activities, they decreased in the order H1.H2.H3.
When the activities are expressed relative to C and N contents (Fig. 1), the phosphomonoesterase and urease activities of H3 were again the only cases of values lower than the corresponding control. In contrast, the lowest non-control values for b-glucosidase and dehydrogenase were recorded for H2.
4. Discussion
The results, which show that enzyme activities were, in general, altered by all three kinds of pollution, suggests the possibility of using these enzymes as indicators of pollution. However, pollution indicators should possess the following attributes (Doran and Parkin, 1994; Elliott, 1997):
1. sensitivity to the presence of pollutant; 2. ability to reflect different levels of pollution;
3. consistency in the sign of the change undergone in response to any given pollutant (i.e. that independently of the dose the indicator always increases or decreases); and
4. sensitivity to the greatest possible number of pollutants. Furthermore, for quantification of environmental pollution damage, a good indicator should be capable of:
5. discriminating between the effect of the pollutant and any prior degradation of the polluted soil; and
Table 3
Checklist for possession of desirable attributes by potential pollution markers
Enzymatic activity Sensitivity to the presence of pollutant
Consistency in sign of change for a given pollutant
Discrimination between prior and pollution-induced degradation
Differentiation of pollutants by degradation levels
Absolute values
Dehydrogenase Yes Yes No No
Urease Yes No No No
b-glucosidase ^ No No No
Phosphomonoesterase Yes No No No
Values relative to total C and N contents
Dehydrogenase/C Yes Yes No No
Urease/C Yes No No No
b-glucosidase/C Yes Yes No No
Phosphomonoesterase/C No No No No
Dehydrogenase/N No No No No
Urease/N Yes No No No
b-glucosidase/N Yes Yes No No
6. differentiating among pollutants according to the different degrees of soil degradation they cause.
Table 3 is a checklist showing which of the enzyme activity measures satisfied requirements 1, 3, 5 and 6 in this study. The only absolute activity to exhibit a consistent response to pollu-tion was dehydrogenase, which was always reduced by tanning and landfill effluent and always increased by hydro-carbons. However, dehydrogenase failed to discriminate between pollution and prior degradation, or to discriminate between different pollutants. Although prior degradation might in principle be estimated by comparison of the control sample with high-quality reference soils (native soils under climax vegetation), this is in practice prevented by the wide geographic variability of enzyme activities in the latter (Table 2 lists the observed ranges of the activities considered here in Galician soils under climax oakwood). Natural variability within and between soils, and the influence of soil type on the stress induced by any given pollutant, have been pointed out by Nannipieri et al. (1990), Brendecke et al. (1993) and Howard (1993) as decisive impediments to the use of simple parameters such as single enzyme activities for diagnostic purposes. Furthermore, although Barriuso et al. (1988), Kandeler et al. (1996) and other authors have suggested that better results might be obtainable with enzyme activities expressed with respect to C or N (since C and N contents are often significantly altered by pollution), in this study the only improvement so achieved was to makeb-glucosidase activity behave consistently (either increasing or decreasing) for any given pollutant. In short, the individual enzyme activities are of very limited utility as degradation markers.
A possible way to increase the potential of soil enzymes as indicators of soil contamination would be its use in combination with other biochemical properties, developing more complex expressions in the same way that did other authors (Beck, 1984; Stefanic et al., 1984; Perucci, 1992;
Sinsabaugh, 1994; Stefanic, 1994; Yakovchenko et al., 1996). However, these indices, too, have severe limitations, since they have been designed for specific situations or purposes such as the evaluation of alterations in soil ferti-lity, and their general validity is questionable.
Previous experiences of our group (Trasar-Cepeda et al., 1998) have shown that in soils that function correctly, i.e. undisturbed native soils under climax vegetation (Dick, 1994; Doran et al., 1994), a biochemical equilibrium exists represented by a balance between the organic matter content and its biological activity, and this equilibrium is disturbed by distorting agents such as pollutants (Leiro´s et al., 1999). For climax soils of Galicia (NW Spain) this equilibrium can be expressed by an equation which defines the total N content of the soil as a function of microbial biomass C, N mineralisation capacity, and three enzymatic activities: phosphomonoesterase, b-glucosidase, and urease (Trasar-Cepeda et al., 1998):
Total N ×1023 0:38 microbial biomass C11:40
N mineralization capacity113:60 phosphomonoesterase
18:90b-glucosidase11:60 urease
For these climax soils, the Nc/Nk ratio is 100%, Nc being the total N obtained from biochemical properties by using the above equation, and Nk the total N content of the soils measured by Kjeldahl’s method. Furthermore, both labora-tory experiments and the evaluation of soils disturbed by diverse accidents or management practices have shown that the degree of disturbance is reflected by the modifications in the ratio Nc/Nk.
Table 4 lists the values of Nk, Nc and Nc/Nk (the last mentioned as a percentage), for each of the samples studied in this work. The fact that for the control samples Nc/Nk is not 100% but ranges from 56 to 79% reflects pre-pollution degradation of the soil at all three sites, especially the site later polluted with hydrocarbons; these results are not surprising in view of the negative effects of agricultural practices on soil biochemical quality (Leiro´s et al., 1999). For the samples polluted by tanning effluent or landfill efflu-ent, intense degradation is indicated by very low Nc/Nk values, ranging from 15% (T3) to 28% (T1) in the former case, and from 19% (L2) to 35% (L1) in the latter, and significantly different from the Nc/Nk values in their corre-sponding controls. Quite different behaviour is observed among the samples polluted by hydrocarbons: only H1 (the only sample taken at a point with surface traces of hydrocarbons) has a Nc/Nk value differing significantly from the control one, and the value of Nc/Nk for H1 is furthermore greater than the control value, not less. Similar rises in Nc/Nk values have in other situations been inter-preted as reflecting a transitory state of high microbiological and biochemical activity (Leiro´s et al., 1999) which, in the present case, would be due to the hydrocarbon pollutants C. Trasar-Cepeda et al. / Soil Biology & Biochemistry 32 (2000) 1867–1875 1873 Table 4
Kjeldahl N contents (Nk), Nc values and the Nc/Nk ratios (%) of the soil samples
Soil sample Nk Nc Nc/Nk
Tanning effluent
T1 1.267 0.356 28
T2 1.803 0.443 25
T3 1.039 0.159 15
T4 (control) 0.756 0.569 75
Landfill effluent
L1 0.653 0.227 35
L2 0.583 0.111 19
L3 0.549 0.168 31
L4 (control) 0.475 0.376 79
Hydrocarbons
H1 0.470 0.346 74
H2 0.600 0.330 55
H3 0.410 0.213 52
constituting a degradable substrate capable of stimulating the proliferation of part of the soil microflora (Joergensen et al., 1995; Braddock and McCarthy, 1996). By contrast, the values of Nc/Nk for samples H2 and H3 show that at these points the amount of pollution was insufficient to alter the biochemical quality of the soil.
The above results illustrate how, unlike the individual enzyme activities discussed previously, the ratio Nc/Nk allows pre-pollution degradation to be evaluated and distin-guishes this from degradation caused by pollution. This facilitates comparison of the effects of different pollutants at different sites. The soils examined in this study, for exam-ple, can be ranked by Nc/Nk ratio (Fig. 2). The tanning and landfill effluents had very similar effects, reducing the Nc/ Nk value by 63–80% in the former case and by 61–76% in the latter, while at the site polluted by hydrocarbons pollu-tion had a negligible effect on soil biochemical quality at the points where samples H2 and H3 were taken, and increased the Nc/Nk value by 32% where H1 was obtained.
5. Conclusions
The four enzymes considered in this study (phosphomo-noesterase, b-glucosidase, urease and dehydrogenase) proved to have serious limitations with respect to their abil-ity to reflect soil degradation caused by the pollution inci-dents that had occurred at the sampling sites. Regardless of whether their activities were expressed as absolute values or relative to total soil C or N contents, they failed to exhibit most of the attributes required of a good pollution indicator. In particular, they are intrinsically incapable of differentiat-ing between degradation due to pollution and prior degrada-tion of the soil at the polludegrada-tion site, which in turn hinders comparison of observations made at different sites. By contrast, differentiation between prior and pollution-induced degradation is straightforward using the Nc/Nk ratio defined above, which relates biological/biochemical properties (three enzyme activities, microbial biomass C and N mineralisation capacity) with total N content. The validity of the Nc/Nk ratio as a pollution marker derives ultimately from the validity of the equation NkNc for
native soils under climax vegetation, which expresses the natural biochemical equilibrium of these soils. The value of the ratio for diagnosis and quantification of pollution has been shown in this study. The ratio proved sensitive to the presence of pollutant, and the alteration of its value always had the same sign for the same pollutant. In addition, as noted above, the ratio was able to differentiate between prior and pollution-induced soil degradation. The ability of Nc/Nk ratio to differentiate between different levels of pollutant is currently being investigated.
Acknowledgements
This research was supported by the Spanish Comisio´n Interministerial de Ciencia y Tecnologı´a. The authors thank Srta. Ana-Isabel Iglesias Tojo and Srta. Isabel Marti-nez Outeda for their help with the analysis of the samples, and the Guardia Civil (Servicio de Proteccio´n de la Natur-aleza, 611 Comandancia de A Corun˜a) and the Policı´a Auto-no´mica de Galicia (Servicio de Medio Ambiente) for their help in the localisation and sampling of the contaminated soils.
References
Arshad, M.A., Coen, G.M., 1992. Characterization of soil quality: physical and chemical criteria. American Journal of Alternative Agriculture 7, 25–33.
Barriuso, E., Pe´rez-Mateos, M., Gonza´lez-Carcedo, S., 1988. Actividad ureasa especı´fica del suelo. Agrochimica 32, 284–294.
Baruah, M., Mishra, R.R., 1986. Effect of herbicides butachlor, 2,4-dand oxyfluorfen on enzyme activities and CO2 evolution in submerged
paddy field soil. Plant and Soil 96, 287–291.
Beck, T., 1984. Methods and application of soil microbiological analysis at the Landensanstalt fu¨r Bodenkultur und Pflanzenbau (LBB) in Munich for the determination of some aspects of soil fertility. In: Nemes, M.P., Kiss, S., Papacostea, P., Stefanic, C¸ ., Rusan, M. (Eds.), Research Concerning a Biological Index of Soil Fertility. Fifth Symposium on Soil Biology, Romanian National Society of Soil Science, Bucharest, pp. 13–20.
Bezdicek, D.F., Papendick, R.I., Lal, R., 1996. Importance of soil quality to health and sustainable land management. In: Doran, J.W., Jones, A.J. (Eds.). Methods for Assessing Soil Quality, Soil Science Society of America, Madison, pp. 1–8.
Braddock, J.F., McCarthy, K.A., 1996. Hydrologic and microbiological factors affecting persistence and migration of petroleum hydrocarbons spilled in a continuous-permafrost region. Environmental Science and Technology 30, 2626–2633.
Brendecke, J.M., Axelson, R.D., Pepper, I.L., 1993. Soil microbial activity as an indicator of soil fertility: long-term effects of municipal sewage sludge on an arid soil. Soil Biology and Biochemistry 25, 751–758. Bremner, J.M., 1965. Inorganic forms of nitrogen. In: Black, C.A., Evans,
D.D., White, J.L., Ensminger, L.L., Clark, F.E. (Eds.). Methods of Soil Analysis, American Society of Agronomy, Canadian Soil Science of agronomy, Soil Science Society of America, Madison, pp. 1179–1237. Brookes, P.C., 1995. The use of microbial parameters in monitoring soil pollution by heavy metals. Biology and Fertility of Soils 19, 269–275. Camin˜a, F., Trasar-Cepeda, C., Gil-Sotres, F., Leiro´s, M.C., 1998. Measurement of dehydrogenase activity in acid soils rich in organic matter. Soil Biology and Biochemistry 30, 1005–1011.
Dick, R.P., 1994. Soil enzyme activities as indicators of soil quality. In: Fig. 2. Classification of the contaminated soil samples considering the value
Doran, J.V., Coleman, D.C., Bezdicek, D.F., Stewart, B.A. (Eds.). Defining Soil Quality for a Sustainable Environment, Soil Science Society of America, American Society of Agriculture, Madison, pp. 107–124.
Dick, R.P., 1997. Soil enzyme activities as integrative indicators of soil health. In: Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R. (Eds.). Biological Indicators of Soil Health, CAB International, Wellingford, pp. 121–156.
Dick, R.P., Gupta, V.V.S.R., 1994. A conceptual model for the role of abiotic soil enzymes in a microbial ecology: A potential analogue for soil quality. In: Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R., Grace, P.R. (Eds.). Soil Biota: Management in Sustainable Farming Systems, Commonwealth Scientific and Industrial Research Organization Infor-mation, Adelaide, pp. 167–168.
Doelman, P., Haanstra, L., 1979. Effect of lead on soil respiration and dehydrogenase activity. Soil Biology and Biochemistry 11, 475–479. Doran, J.W., Parkin, T.B., 1994. Defining and assessing soil quality. In:
Doran, J.V., Coleman, D.C., Bezdicek, D.F., Stewart, B.A. (Eds.). Defining Soil Quality for a Sustainable Environment, Soil Science Society of America, American Society of Agriculture, Madison, pp. 3–21.
Doran, J.W., Sarrantonio, M., Janke, R., 1994. Strategies to promote soil quality and health. In: Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R., Grace, P.R. (Eds.). Soil Biota: Management in Sustainable Farming Systems, Commonwealth Scientific and Industrial Research Organiza-tion InformaOrganiza-tion, Adelaide, pp. 230–237.
Eivazi, F., Tabatabai, M.A., 1988. Glucosidases and galactosidases in soils. Soil Biology and Biochemistry 20, 601–606.
Elliott, E.T., 1997. Rationale for developing bioindicators of soil health. In: Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R. (Eds.). Biological Indi-cators of Soil Health, CAB International, Wellington, pp. 49–78. Frankenberger, W.T., Dick, W.A., 1983. Relationship between enzyme
activities and microbial growth and activity indices in soil. Soil Science Society of America Journal 47, 945–951.
Garcı´a, C., Herna´ndez, T., 1997. Biological and biochemical indicators in derelict soils subject to erosion. Soil Biology and Biochemistry 29, 171–177.
Gregorich, E.C., Carter, M.R., Angers, D.A., Monreal, C.M., Ellert, B.H., 1994. Towards a minimum data set to assess soil organic matter quality in agricultural soils. Canadian Journal of Soil Science 74, 367–385. Guitia´n, F., Carballas, T., 1976. Te´cnicas de ana´lisis de suelos. Pico Sacro
Editorial, Santiago de Compostela.
Hornick, S.B., 1992. Factors affecting the nutritional quality of crops. American Journal of Alternative Agriculture 7, 63–68.
Howard, P.J.A., 1993. Soil protection and soil quality assessment in the EC. The Science of the Total Environment 129, 219–234.
Jenkinson, D.S., 1988. The determination of microbial biomass carbon and nitrogen in soil. In: Wilson, J.R. (Ed.). Advances in Nitrogen Cycling in Agricultural Ecosystems, CAB International, Wellingford, pp. 368–386. Joergensen, R.G., Schmaedeke, F., Windhorst, K., Meyer, B., 1995. Biomass and activity of microorganisms in a fuel oil contaminated soil. Soil Biology and Biochemistry 27, 1137–1143.
Kandeler, E., Kampichler, C., Horak, O., 1996. Influence of heavy metals on the functional diversity of soil microbial communities. Biology and Fertility of Soils 23, 299–306.
Lal, R., 1997. Soil quality and sustainability. In: Lal, R., Blum, W.H., Valentine, C., Stewart, B.A. (Eds.). Methods for Assessment of Soil Degradation, CRC Press, New York, pp. 17–30.
Larson W.E., Pierce, F.J., 1991. Conservation and enhancement of soil quality. In: Evaluation for Sustainable Land Management in the Devel-oping World, vol. 2. International Boarding for Soil Research and Management Proceedings, Bangkok.
Leiro´s, M.C., Trasar-Cepeda, C., Garcı´a-Ferna´ndez, F., Gil-Sotres, F.,
1999. Defining the validity of a biochemical index of soil quality. Biology and Fertility of Soils 30, 140–146.
McCarthy, G.W., Siddaramappa, R., Reight, R.J., Coddling, E.E., Gao, G., 1994. Evaluation of coal combustion byproducts as soil liming materi-als: their influence on soil pH and enzyme activities. Biology and Ferti-lity of Soils 17, 167–172.
Nannipieri, P., Greco, S., Ceccanti, B., 1990. Ecological significance of the biological activity in soil. In: Bollag, J-M., Stotzky, G. (Eds.). Soil Biochemistry, vol. 6. Marcel Dekker, New York, pp. 293–355. Pankhurst, C.E., Hawke, B.G., McDonal, H.J., Kirkby, C.A., Buckerfield,
J.C., Michelsen, P., O’Brien, K.A., Gupta, V.V.S.R., Doube, B.M., 1995. Evaluation of soil biological properties as potential bioindicators of soil health. Australian Journal of Experimental Agriculture 35, 1015–1028.
Parr, J.F., Papendick, R.I., Hornick, S.B., Meyer, R.E., 1992. Soil quality: attributes and relationship to alternative and sustainable agriculture. American Journal of Alternative Agriculture 7, 5–11.
Perucci, P., 1992. Enzyme activity and microbial biomass in a field soil amended with municipal refuse. Biology and Fertility of Soils 1, 111–115. Pichtel, J.R., Hayes, J.M., 1990. Influence of fly-ash on soil microbial activity and populations. Journal of the Environmental Quality 19, 593–597.
Saa´, A., Trasar-Cepeda, C., Gil-Sotres, F., Carballas, T., 1993. Changes in soil phosphorus and acid phosphatase activity immediately following forest fires. Soil Biology and Biochemistry 22, 511–515.
Sinsabaugh, R.L., 1994. Enzymic analysis of microbial pattern and processes. Biology and Fertility of Soils 17, 69–74.
Skujins, J., 1978. History of abiontic soil enzymes. In: Burns, R.G. (Ed.). Soil Enzymes, Academic Press, London, pp. 1–49.
Spiegel, M.R., 1969. Statistics. McGraw-Hill, New York.
Stefanic, G., 1994. Biological definition, quantifying method and agricul-tural interpretation of soil fertility. Romanian Agriculagricul-tural Research 2, 107–116.
Stefanic, F., Eliade, G., Chirnageanu, J., 1984. Researches concerning a biological index of soil fertility. In: Nemes, M.P., Kiss, S., Papacostea, P., Stefanic, C., Rusan, M. (Eds.), Fifth Symposium on Soil Biology, Romanian National Society of Soil Science, Bucharest, pp. 35–45. Tabatabai, M.A., Bremner, J.M., 1969. Use of p-nitrophenyl phosphate for
assay of soil phosphatase activity. Soil Biology and Biochemistry 1, 301–307.
Trasar-Cepeda, C., Leiro´s, M.C., Gil-Sotres, F., Seoane, S., 1998. Towards a biochemical quality index for soils. An expression relating several biological and biochemical properties. Biology and Fertility of Soils 26, 100–106.
Trevors, J.T., 1984. Dehydrogenase activity in soil. A comparison between the INT and TTC assay. Soil Biology and Biochemistry 16, 673–674. van Beelen, P., Fleuren-Kemila´, K., 1997. Influence of pH on the toxic effects of zinc, cadmium, and pentachlorophenol on pure cultures of soil microorganisms. Environmental Toxicology and Chemistry 16, 146–153.
Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707.
Vanhala, P.T., Ahtiainen, J.H., 1994. Soil respiration, ATP content and photobacterium toxicity test as indicators of metal pollution in soil. Environmental Toxicology and Water Quality: An International Journal 9, 115–121.
Visser, S., Parkinson, D., 1992. Soil biological criteria as indicators of soil quality: soil microorganisms. American Journal of Alternative Agricul-ture 7, 33–37.
Yakovchenko, V.I., Sikora, L.J., Rauffman, D.D., 1996. A biologically based indicator of soil quality. Biology and Fertility of Soils 21, 245–251.
