Journal of Environmental Quality 32:466-471 (2003)
© 2003 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
TECHNICAL REPORTS
Ecological Risk Assessment
Cholesterol, ß-Sitosterol, Ergosterol, and Coprostanol in Agricultural Soils
Edoardo Puglisi,
Marco Nicelli,
Ettore Capri,
Marco Trevisan* and
Attilio A. M. Del Re
Istituto di Chimica Agraria ed Ambientale, Università Cattolica del Sacro Cuore, 29100 Piacenza, Italy
* Corresponding author (marco.trevisan{at}unicatt.it)
Received for publication June 24, 2002.
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ABSTRACT
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In this work we analyzed the sterol content of agricultural soils. Three eukaryotic sterols, cholesterol, ß-sitosterol, and ergosterol were chosen as representative of the animal, plant, and fungal kingdoms, while coprostanol was validated as a marker of human fecal matter contamination. Three soils subjected to different treatments (sewage sludge application, irrigation by saline waters, and contamination by industrial and municipal wastes) were sampled and their sterol content was measured and compared with adjacent untreated soils. The effects of time, location, and treatment were evaluated by means of a number of statistical techniques. ß-Sitosterol concentration varied from 0.9 to 30 mg kg-1. Lesser values were measured in Cremona (2.1 mg kg-1) than in Bari (4.0 mg kg-1) and Naples (10.9 mg kg-1) soils. No significant effects were detected for cholesterol and ergosterol. Coprostanol was present after sewage sludge disposal and contamination by industrial and municipal wastes, while it was absent in the soil treated with saline water and in the adjacent untreated soil. Coprostanol concentration did not vary much within site and time of sampling, with a mean value of 0.2 mg kg-1. We confirmed coprostanol as a useful persistent marker of human fecal matter contamination. Multivariate analysis highlighted a clear distinction between the eukaryotic sterols and coprostanol. In addition, a different behavior between ergosterol and cholesterol on one side and ß-sitosterol on the other was detected. This preliminary work suggests that sterols deserve a deeper study of their use as indicators in agricultural soils.
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INTRODUCTION
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STEROLS ARE COMPONENTS of eukaryotic cells. Their detection in soil is an important tool for characterizing biomass (e.g., plant and animal biomass) and other organic pools, as different classes of organisms have different sterols pattern.
Plant sterol composition varies within different species and physiological stage of development. Typical plant sterols include ß-sitosterol, campesterol, stigmasterol, and ß-sitosterol, which has been reported to be the main plant sterol by several researchers (e.g., Ebrahimzadeh et al., 2001). These sterols are also contained in seeds, with ß-sitosterol being the main one, in concentrations that in several species of Astragalus exceed 70% (Knights and Berrie, 1971). ß-Sitosterol increases during development, and in turnip rape [Brassica rapa L. subsp. campestris (L.) A.R. Clapham], reaches its peak in the early phases of floral development (Hobbs et al., 1996). Its fate in agricultural soils has been studied in long-term experiments, and appears to be a labile compound whose concentration diminishes greatly, presumably due to assimilation by soil arthropods (Bull et al., 2000).
Cholesterol is the main animal sterol, and its detection in soil samples is directly related to micro- and mesofauna biomass. Plant matter and organic fertilizer may represent a minor source of cholesterol (Ibanez et al., 2000).
Ergosterol is the main endogenous sterol of fungi and of some microalgae. Ergosterol content in fungal cells varies in different species and under different environmental conditions. Its concentration is an important indicator of fungal growth in organic substrates such as foods (Battilani et al., 1996) and it has been proposed as an indicator of living fungal biomass in soils, because it is easily mineralized at the cell's death (West and Grant, 1987). In addition, a clear correlation between ergosterol and fungal iphae length was shown (Stahl and Parkin, 1996). Some soil fungi are able to degrade a number of organic pollutants (Barr and Aust, 1994), and ergosterol has therefore been proposed as a marker of soil bioremediation potential (Barajas-Aceves et al., 2002).
The 5ß-stanols are transformation compounds produced by microbial hydrogenation of cholesterol, campesterol, ß-sitosterol, and stigmasterol in the intestinal tract of most higher animals. Coprostanol (5ß-cholestan-3ß-ol) is the main sterol (about 60% of total) in human feces (Bull et al., 2002). Several studies focused on coprostanol as an important biomarker of fecal contamination in water, sediments, and soils (see González-Oreja and Saiz-Salinas, 1998). Also, cats and pigs produce coprostanol as the main sterol, but in quantities that are 10 times less than in humans. Concentration of coprostanol is therefore a reliable measure of human fecal pollution. Further information may be given by the relative quantities of other fecal sterols. For example, ruminants produce a higher relative proportion of 5ß-campestanol and 5ß-stigmastanol, produced respectively by biohydrogenation of campesterol and ß-sitosterol (Nichols et al., 1996). In locations where both herbivorous and human sources of fecal pollution are suspected, the ratio of coprostanol and 5ß-stigmastanol has been proposed as a discriminatory tool (Leeming et al., 1996).
There is a growing interest in the determination of chemical and biochemical properties, which can be used to assess the status of agricultural soils (Gianfreda et al., unpublished data, 2002; Trasar-Cepeda et al., 1998). In this work we studied the soil content of the main eukaryotic sterols cholesterol, ß-sitosterol, and ergosterol, representing the three eukaryotic kingdoms, and of coprostanol, a known marker of fecal pollution. Samplings were repeated three times in soils that are different in cultivation and alteration events, to obtain a preliminary description of the effects of time, location, and treatment on sterol content of agricultural soil.
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MATERIALS AND METHODS
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Study Sites
Three sites in different parts of Italy, with different pedological and climatic conditions and subjected to various alteration treatments, were studied. The first site is in Naples, in a volcanic area (50 m above mean sea level). The area has a Mediterranean climate with mild, humid winters and dry, hot summers. Soils are young, quite deep, and well drained, with high field capacity. Texture is sandy-loamy. The area is cultivated with hazel (Corylus avellana L.) and minor vegetable crops, and has been subjected in the last 20 yr to overflowing of Sarno River, which is heavily polluted by municipal and industrial wastes.
Another site is in the coastal area of Bari, which also has a Mediterranean climate. Soils are loamy, 25 cm deep, and have a red color due to a high iron oxide content, hence the name "terre rosse" (red soils). Topsoil is friable, very adhesive, and plastic, while deeper soil has a poor, coarse texture and little drainage. Even if shallow, these soils can sustain intensive horticulture, including tomato (Lycopersicon esculentum Mill.), parsley [Petroselinum crispum (Mill.) Nyman ex A. W. Hill], and several varieties of lettuce (Lactuca sativa L.). Altered soil was irrigated in the past 20 yr with saline waters.
The last site is near Cremona, in the heart of the Po Valley, with continental climate and high annual precipitation (820 mm). Soils are sandy-loamy, alluvial, with hydromorphic conditions due to shallow ground water (<1.5 m). Organic matter content is low, and the structure is degraded with low macroporosity. The area has been cultivated with maize monoculture for at least 20 yr, and was fertilized with sewage sludge in the last 15 yr.
Soil Sampling
In each site four soil samples were collected by means of a spake at a 5- to 15-cm depth in five different positions of the field. Soil samples of each position were then mixed to reach the analytical sample. Following the same sampling scheme, samples were collected as control in adjacent fields, with similar soils, but not subjected to the potential alteration activities considered (industrial and municipal pollution, irrigation with saline waters, and sewage sludge application). The control fields were cultivated as the treated ones, with the exception of Bari, which was cultivated with fig tree (Ficus carica L.). Sampling was repeated three times: May 2000, October 2000, and April 2001. Within three days soil samples were sieved at 2 mm and then stored at 5°C for approximately one month until the analysis.
Analysis
Both free and conjugated sterols were extracted with methanol (Grant and West, 1986). The determination of coprostanol, cholesterol, and ß-sitosterol was made by gas liquid chromatography–mass spectrometry (GLC–MS) after silanization with pyridin, N,O-bis-(trimethylsilyl) trifluoroacetamide, esamethyldisilazane, and trimethyl clorosilane in a 0.2:1:2:1 ratio. The reagent excess was evaporated under a stream of nitrogen, 1 mL of isooctane was added, and the remaining solution was injected. The GLC–MS analysis of the trimethyl silyl esters was performed with an Agilent Technologies (Palo Alto, CA) 5973N gas chromatograph equipped with a Hewlett-Packard (Palo Alto, CA) 30-m length x 0.25-mm i.d. cross-linked methyl silicone (0.25-µm film thickness) HP5-MS capillary column. The chromatographic conditions were: helium as carrier gas at 1.2 mL min-1; injection temperature, 280°C (splitless 1 min); oven temperature gradient, start at 250°C, increase to 290°C at 1°C min-1, hold for 4 min; injection volume, 1 µL; transfer line temperature, 290°C; quadrupol temperature, 150°C; mass electromultiplicator at 1106 eV; mass revelatory delay, 6.2 min; and maximum range from 50 to 500 mw.
Ergosterol analysis was performed by high performance liquid chromatography (HPLC), because for this compound in GLC–MS some interference peaks were found. The HPLC analysis was conducted with a Hewlett-Packard 1100 series HPLC with a diode array detector. Isocratic elution was performed by methyl alcohol (46%), dichloromethane (8%), and acetonitrile (46%) flux of 1 mL for one minute. The column was a Supelco Supelcosil ABZ*Plus (250-mm length x 4.6-mm i.d., 5-µm thickness) (Sigma-Aldrich, St. Louis, MO); the guard column was a Phenomenex (Torrance, CA) ODS (4-mm length x 3.0-mm i.d.); temperature was set to 21 ± 0.2°C; maximum wavelength absorption was 282 nm; all spectra peaks were acquired.
Validation was performed by means of recovery trial from fortified samples. Concentrations of coprostanol, cholesterol, ergosterol, and ß-sitosterol of 5 and 50 mg kg-1 were added to soils whose original concentration of the four sterols were known. Final concentrations were then compared with the original considering how much was added.
Statistical analysis was performed with SAS software (SAS Institute, 1985).
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RESULTS AND DISCUSSION
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Sterol Quantification
Coprostanol was determined at a retention time of 12.81 min, cholesterol at 15.01 min, and ß-sitosterol at 20.95 min (Fig. 1)
. Confirmation was performed with mass spectra fitting. Ergosterol determination was performed by high performance liquid chromatography at a retention time of 6.5 min, and was confirmed with UV spectra fitting. Sterols were quantified from peak areas by linear combination with standards injected every five samples analyzed at concentrations increasing from 0.1 to 10 mg kg-1. In each case, calibration line R2 values were more than 0.98. The quantification limit was 0.001 mg kg-1. Means and standard deviations of the four sterols concentrations in soils are shown in Table 1. Recovery trials performed on the four sterols always gave values of more than 80%.
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Table 1. Means and standard deviations for sterol concentrations. In the second sampling, analyses were repeated three times on three replicates to evaluate the internal variability of samples. No significant differences were found.
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Cholesterol concentrations varied from a minimum of 0.2 to a maximum of 2.4 mg kg-1 (58.5% CV), and ß-sitosterol from 0.9 to 30 mg kg-1 (5.8% CV). Similar quantities of these two compounds were found by Ibanez et al. (2000).
Ergosterol concentration fell within the range of 0.3 to 24.2 mg kg-1, with a CV of 101%. These values are similar to those reported by West and Grant (1987). Ergosterol's behavior was similar to cholesterol's, that is, concentrations in the treated and untreated soils were similar for each site at each time; higher concentrations were found in the third sampling, especially in Naples and Bari.
Coprostanol was never detected in Bari soil samples, while it was found in samples contaminated with municipal and industrial wastes from Naples as well as in samples from Cremona treated with sewage sludge, and in only two samples of Cremona's untreated soils. Its presence in the untreated samples may be due to accidental contamination by municipal sludge. Concentration range fell within 0 and 0.455 mg kg-1, with a CV of 158%. Concentrations of coprostanol in Cremona and Naples altered soils were quite similar: 0.3 mg kg-1 at Cremona and 0.2 mg kg-1 at Naples, in good agreement with results of Ibanez et al. (2000) for Spanish soils treated with a single dose of sewage sludge or manure.
Data Analysis
A mixed model analysis of variance (PROC GLM; SAS Institute, 1985), containing fixed and random classification variables, was applied to the data obtained from the sterol determination (Table 2). The three main factors, namely the effects of treatment (classification variable ALTER), location (SITE), and period of sampling (TIME), and their interactions were investigated, for a total of seven classification variables. ALTER was a fixed effect, while SITE and TIME were set as random ones. ALTER is the effect of any treatment, whatever its type. All interactions containing SITE and TIME effects were considered random. With the very presence of fixed and random effects, statistical tests become more restrictive and presumably more realistic (Snedecor and Cochran, 1989).
In this model, the significance of the ALTER effects mainly uses the ALTER x SITE interaction as error estimation. The ALTER x SITE interaction is the effect of the specific treatment applied to a site. When this interaction is high, then a main factor can be significant if all factors are set as fixed and nonsignificant in a mixed model.
All significant effects were confirmed by Duncan's test for comparison of means.
The ALTER x SITE x TIME interaction was significant for cholesterol (P < 0.0001). At the third sampling time, the average concentration of Cremona treated samples was 1.6 mg kg-1 vs. 0.6 mg kg-1 for untreated ones. No other significant effects were detected, and cholesterol appears to be unaffected by treatments, sampling time, and soil type.
SITE (P < 0.01) and SITE x TIME (P < 0.05) are significant for ß-sitosterol. In Cremona lesser values were measured (2.1 mg kg-1) than in Bari (4.0 mg kg-1) and Naples (10.9 mg kg-1) (Fig. 2)
. The Naples third sampling had higher concentrations (14.6 mg kg-1) than the first two (9.8 and 9.5 mg kg-1); the same trend was observed in Bari (6.0 against 3.4 and 3.3 mg kg-1).
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Fig. 2. Site behavior of ergosterol, ß-sitosterol, and cholesterol, showing average values and standard deviations.
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These results are a clear confirmation of the results in Bull et al. (2000), that is, ß-sitosterol is a labile compound, whose concentration is related to cultivation type and decomposing plant matter (SITE effect). Furthermore, its variable content in some sites within time of sampling (SITE x TIME effect) may be explained as a consequence of its utilization in different times of the year by the faunal biomass.
No significant effects were found for ergosterol, even if higher concentrations were observed in the third sampling of Naples and Bari, as for ß-sitosterol. Alteration events did not vary fungal activities in the soil analyzed. This result is supported by evidence from Barajas-Aceves et al. (2002), who recognized how several heavy metals and pesticides, even if reducing fungal biomass, did not affect soil ergosterol content.
Coprostanol was the only sterol to show significance between treated and untreated soils in some sites (SITE x ALTER effect P value < 0.001). This result clearly appears in the raw data obtained from chemical analysis, where coprostanol is found only in treated samples of Cremona and Naples and in two Cremona untreated samples (Fig. 3)
. These results support an expected presence of human feces in sewage sludge and municipal wastes. Furthermore, its concentration did not vary within time of sampling. Coprostanol is confirmed as a persistent (Bull et al., 1998) indicator of human fecal matter (Leeming et al., 1996; Dachs et al., 1999).
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Fig. 3. Concentrations of coprostanol in treated and untreated samples from Cremona (CR) and Naples (NA).
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Principal component analysis (PCA) was also performed on the four sterols before (PROC PRINCOMP; SAS Institute, 1985) and after a varimax rotation (PROC FACTOR; SAS Institute, 1985), to investigate sterol relationships and to maximize eventual separations between classes (Table 3). Before varimax rotation, PCA showed a clear distinction between the three eukaryotic sterol groups, heavily weighted on the first axis (55% of variance explained), and coprostanol, on the second (25% of variance). The first three sterols are produced by living eukaryotes to which biomass amount is related, while coprostanol, a digestive transformation compound, is independent from the other three. After varimax rotation a further distinction was obtained: ergosterol and cholesterol weighted on the first axis (44% of variance explained), ß-sitosterol on the second (25% of variance), and coprostanol on the last (25% of variance). Sterols of animals and fungi, who play the most active role in soil metabolic activity, were pooled together, apart from ß-sitosterol, whose behavior can also be related to its lability (Bull et al., 2000).
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Table 3. Principal component analysis performed before and after axis rotation. For each factor the eigenvalues of each sterol, which express the weight on the factor, and the proportion of variance explained by each factor are reported.
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Principal component scores were represented on three-dimensional graphs before and after varimax rotation. Different symbols were assigned to altered and unaltered soils in each site. There was no separation before rotation, while after it altered and control soils in Cremona and Naples were separated along the third axis, the one related to coprostanol (Fig. 4)
. This result is in accordance with the SITE x ALTER effect significant in the mixed model analysis of variance.
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Fig. 4. Principal component analysis performed on soil sterol content after a varimax rotation (Proc FACTOR; SAS Institute, 1985). Different symbols were assigned to altered and unaltered soils in each site: Cremona, pyramids and cubes; Bari, hearts and spades; Naples, flags and squares.
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CONCLUSIONS
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In this preliminary work we analyzed the sterol content of different agricultural soils. The three eukaryotic sterols (cholesterol, ß-sitosterol, and ergosterol) were chosen as representative of the animal, plant, and fungal kingdoms, while coprostanol was surveyed to validate its use as a marker of human fecal matter contamination. By the application of statistical procedures, such as analysis of variance and principal component analysis, we highlighted the fact that ß-sitosterol content was affected by cultivation and time of sampling. Due to its lability, its level in agricultural soils appears to be related to the amount of plant organic matter (Bull et al., 2000). Ergosterol and cholesterol followed a common trend. Their concentrations did not vary in different soils but at different sampling times, even if not in a statistically significant way. The soils surveyed in this work were broadly different in site location, pedology, agronomic utilization, and alteration events. However, soil, animals, and fungi, indirectly measured by cholesterol and ergosterol, did not seem to be affected. We decided to search for coprostanol because two of the three alteration events considered in this work (sewage sludge application and contamination by municipal wastes) deal with human fecal matter. We found coprostanol in concentrations that were almost constant across time and mainly in the soils where these events took place. We confirmed coprostanol's usefulness as a marker of human fecal matter contamination and its persistence (Bull et al., 2002).
No significant effects were found for the other sterols studied. Nevertheless, the concentration variabilities suggest that sterols deserve a deeper study, to better evaluate their potential use as indicators in agricultural soils. It is possible that a broader pattern of sterols may single out differences not detectable considering a few compounds. The same result has been highlighted for phospholipid fatty acids (Saetre and Bååth, 2000; Drijber et al., 2000; Calderon et al., 2000).
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ACKNOWLEDGMENTS
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This research was conducted within the MIUR (COFIN 1999) project "Potential Use of Biological Indicators in Monitoring Soil Quality for a Sustainable Environment."
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ABSTRACT
INTRODUCTION
