Water, Air, & Soil Pollution

, Volume 222, Issue 1, pp 315–324

Comparison of In Vitro PBET and Phosphoric Acid Extraction as an Approach to Estimate Selenite and Selenate Bioaccessibility from Soil

Authors

  • Virginia Funes-Collado
    • Department of Analytical Chemistry, Faculty of ChemistryUniversity of Barcelona
    • Department of Analytical Chemistry, Faculty of Chemistry, and Water Research InstituteUniversity of Barcelona
  • José Fermín López-Sánchez
    • Department of Analytical Chemistry, Faculty of Chemistry, and Water Research InstituteUniversity of Barcelona
Article

DOI: 10.1007/s11270-011-0826-5

Cite this article as:
Funes-Collado, V., Rubio, R. & López-Sánchez, J.F. Water Air Soil Pollut (2011) 222: 315. doi:10.1007/s11270-011-0826-5

Abstract

Several approaches have been used to estimate the bioaccessibility of trace metals from soils. Here, we applied phosphoric acid extraction and the in vitro test physiologically based extraction (PBET) to soils containing selenium (Se) and compared their performance in estimating the bioaccessibility of Se. For this purpose, we used two soil samples and two Certified Reference Material soil samples with a range of Se concentrations. The total Se contents were measured in the samples and in the extracts by hydride generation–atomic fluorescence spectroscopy. Moreover, we also measured selenite and selenate in the soil extracts (from phosphoric acid and from the PBET) using the coupled techniques liquid chromatography–UV photooxidation–atomic fluorescence spectroscopy and liquid chromatography–mass spectrometry with inductively coupled plasma. From the results obtained in the present study, the PBET showed that the selenium bioaccessible fraction was mainly attributed to the gastrointestinal step. When comparing the results from PBET with those of the phosphoric acid extraction, similar values of Se (IV) and Se (VI) were obtained for both extraction systems. An estimation of the bioaccessibility percentage of Se is also reported.

Keywords

SeleniteSelenateBioaccessibilitySoilPBETPhosphoric acidLC–UV–HGAFSLC–ICPMS

1 Introduction

Selenium (Se) is an essential trace element for human and animal nutrition. In the human diet, Se is crucial for many body functions, and several regulations have defined the limits for toxicity and deficiency (Scientific Committee on Food 2000; Uden 2005; Canadian Soil Quality Guidelines 2009). Soil Se content is strongly related to its concentration in parent minerals, and it is generally present in the following oxidation states of inorganic forms: selenides [Se (−II)], elemental Se [Se (0)], selenites [Se (IV)], and selenates [Se (VI)] (Kabata-Pendias and Pendias 2001), depending on environmental conditions such as pH, Eh, and biological activity. Furthermore, it is also possible to find some organic Se compounds. Processes of leaching/erosion contribute to the development of seleniferous soils, which in turn promote higher absorption of Se by forage produced for animal consumption. This implies that these animals may then surpass tolerated concentrations (Dhillon and Dhillon 2003; Fessler et al. 2003). The Se content of agricultural soils is relevant for assessing the uptake by the plant to human and animal food chain (Spadoni et al. 2007), and in some countries, the amendment of soils by fortified fertilizers is commonplace (Hartikainen 2005; Hawkesford and Zhao 2006; Finley 2007; Keskinen et al. 2009), and in some countries, the content of this element is regulated. (Environmental Protection, England and Wales 2005). In general, human contact or oral ingestion of soils is described as occasional (Body et al. 1999; Pouschat and Zagury 2006; Lemly 2007; Morman et al. 2009) and in some cases intentional (Smith et al. 2000). Several studies have reported the potential risk caused by oral ingestion of metal-polluted soils (Hansen et al. 2007; Denys et al. 2008; US EPA 2009). The potential risk of ingestion of metal contaminants is currently assessed through bioaccessibility approaches, understanding bioaccessibility as the fraction representing the availability of metal absorption when dissolved in surrogates of body fluids or juices after oral exposure. In this regard, several in vitro tests that simulate gastrointestinal conditions have been proposed to estimate the bioaccessibility of some elements from ingested soils (Carrizales et al. 2006; Intawongse and Dean 2006; Ljung et al. 2007; Hansen et al. 2007; Makris et al. 2008; Bosso and Enzweiler 2008; Madrid et al. 2008; Smith et al. 2009; Zagury et al. 2009; Morman et al. 2009). To date, Se has not been addressed. These protocols are currently under discussion, and a number of studies have reported on their optimization (Drexler and Brattin 2007; Smith et al. 2010). The need for standard methods to assess the bioaccessibility of contaminants in soils has recently been reported in a study on the use of bioaccessibility for the assessment of risk-based regulations for contaminated land (Latawiec et al. 2010). The objective of the present study is twofold. We applied the in vitro test physiologically based extraction (PBET; Ruby et al. 1996), one of the methods widely proposed to assess the bioaccessible fraction of some metals in soils (mainly arsenic) (Wragg and Cave 2002; Meunier et al. 2010), to estimate the bioaccessibility of selenite and selenate from soils. In addition, we compared the results from the PBET with those obtained from the extraction of Se using phosphoric acid in order to ascertain whether a correlation could be established using these two approaches. In both methods, not only was the total Se content in the corresponding extracts determined, but Se (IV) and Se (VI) were also measured, since both species differ in toxicity (Goldberg et al. 2006; Somogyi et al. 2007; Dhillon et al. 2010). Although the significance of metal speciation in bioaccessibility studies has been reported (Reeder et al. 2006), very few studies have addressed the bioaccessibility of metal species (Zagury et al. 2009). Here, we studied two soils and two Certified Reference Material (CRM) soils, all differing in their Se content. The results obtained from these extraction methods are discussed and compared.

2 Experimental

Samples and CRMs

Soil samples: two soils, R1 and R3, both from the region of Five Points, California, were supplied by Dr. Zhi-Qing Lin from the Department of Biological Sciences at the Southern Illinois University, Edwardsville. CRMs: CRM023-050 and CRM025-050 soils (Natural Matrix Certified Reference Material) were from the Resource Technology Corporation (USA) and had a reference value for total Se of 105 ± 9 and 518 ± 74 mg Se kg−1, respectively. According to the certification reports, these soils came from a moderately contaminated site located in the Western United States, and they were not spiked or fortified.

Instruments

pH was measured by means of a Crison model Basic 20 pH meter equipped with a combined working electrode. Hydride generation–atomic fluorescence spectroscopy (HGAFS): Atomic Fluorescence Spectrometer, PSA Excalibur, includes the hydride generator Module (model 10.004, P.S. Analytical, Kent, UK) with the Perma Pure drying membrane (Perma Pure Products, Farmingdale, NJ, USA) and equipped with a hollow cathode lamp. Inductively coupled plasma mass spectrometry (ICPMS): A 7500ce series Octopole Reaction System ICPMS with a concentric microflow nebulizer (Agilent Technologies, Waldbronn, Germany) was used for total Se measurements. Hydrogen was used as reaction gas to prevent possible interferences, and Rh was used as internal standard. Se species were measured using the coupled techniques liquid chromatography–UV photooxidation–atomic fluorescence spectroscopy (LC–UV–HGAFS) liquid chromatography-inductively coupled plasma mass spectrometry and LC–ICPMS. For separation, an anion exchange Hamilton PRP-X100 column (Reno, NV, USA) was used in both couplings. LC–UV–HGAFS: A Perkin Elmer 250 LC quaternary pump (CT, USA) equipped with a Rheodyne 7125 injector (Cotati, CA, USA) with a 50-μl loop was used. UV irradiation was generated with a homemade photoreactor from Cole Parmer (Vernen Hills, IL, USA) surrounding a low-pressure Hg vapor lamp, Heraeus TNN 15/32, 15 W (Hanau, Germany). For the LC–ICPMS coupling, an Agilent HPLC 1200 series quaternary pump was used directly connected to the cross-flow nebulizer of the ICPMS. The ion intensity at mass-to-charge ratio 78 (78Se) was monitored by time-resolved analysis software. A Phillips PW 2400 X-ray spectrometer with Rh and Au excitation tubes was used for quantification of the major components of the CRMs and soils.

Apparatus

A thermo-agitator Bath Clifton NE5-28D (37°C ± 0.1) was used for the PBET digestion of the samples and CRMs. A P/Selecta model RAT 4000051 with temperature control was used for aqua regia extraction. An end-over-end shaker was used for phosphoric acid extraction, and a Hettich Universal 30F centrifuge separated the phases.

Standards and Reagents

All the solutions were prepared with double deionized water (18.2 MΩ cm−1 resistivity) obtained from a Millipore system.

Standards: 1,000 mg L−1 of Se stock solutions were prepared from selenite 99% Na2SeO3 (Aldrich, Milwaukee, WI, USA) and selenate 99% Na2SeO4 (Aldrich) and then kept at 4°C. Working standard solutions were prepared daily by diluting the stock solutions.

PBET reagents: 0.625 g pepsine [CAS 9001-75-06] (Panreac), 0.25 g citric acid (Fluka), and 0.25 g 99% maleic acid (Aldrich) were dissolved in double deionized water and diluted to 500 ml. Then, 210 μl of DL-lactic acid (Aldrich) and 250 μl of 100% acetic acid (Merck pro-analysis) were added with final dilution with double deionized water. The pH of the resulting solution was adjusted to 1.3 with 37% hydrochloric acid (Panreac Hyperpur). The following reagents were used for the simulated intestinal step: sodium hydrogen carbonate (Merck) porcine bile salts [CAS 8008-63-7] (Sigma-Aldrich) and pancreatin [CAS 8049-47-6] (Sigma-Aldrich). These reagents were added as shown in the flow chart in Fig. 1. Aqua regia extractant was prepared from nitric acid (69% HNO3, Panreac, Hiperpur) and hydrochloric acid (37% HCl, Panreac, Hiperpur). Phosphoric acid extraction reagent was prepared from 1 mol L−1 o-phosphoric acid (85% H3PO4 (PA-ACS Panreac)).
https://static-content.springer.com/image/art%3A10.1007%2Fs11270-011-0826-5/MediaObjects/11270_2011_826_Fig1_HTML.gif
Fig. 1

Flow chart of the modified experimental protocol of the PBET method

The mobile phase for chromatographic separation of Se species was prepared from NH4H2PO4 (Merck Suprapur) and then adjusted to pH 7 with ammonia solution (25% NH3 Panreac PA) and filtered through a 0.22-μm nylon membrane.

Hydride-generating reagents: 6 mol L−1 hydrochloric acid was prepared from 37% HCl (Merck, Pro-analysis). Sodium borohydride at 1% was prepared from NaBH4 tablets (purity >97%, Fluka, Purum) in 0.4% NaOH (Merck, Pro-analysis).

3 Procedures

Sample Pretreatment and Characterization

Soil samples R1 and R3 were air-dried at room temperature, ground to fine powder with a tungsten carbide disk mill (Herzog, Osnabrück, Germany), and sieved through a 90-μm stainless steel mesh. Then, 100–200 g of samples was homogenized for 5 days in a specially designed roller table (Metalitzats Cat, Barcelona) with controlled rotation speed. The homogenized samples were stored in plastic containers until analysis. The results of moisture content, pH, and the major components of CRM023-050, CRM025-050, R1 and R3 soils, determined through X-ray fluorescence, are summarized in Table 1.
Table 1

Characterization of CRMs and soil samples

Sample

Moisture (%)

pH

Major components

[Fe2O3]/%

[CaO]/%

[K2O]/%

[SiO2]/%

[Al2O3]/%

[MgO]/%

[Na2O]/%

CRM 023-050

1.49

7.32

2.6

1.54

3.2

75.2

10.5

0.7

1.8

CRM 025-050

2.76

7.21

2.5

5.51

2.7

64.7

8.7

1

1.4

R1 soil

5.82

8.53

3.8

13.9

1.6

42.5

10.6

2.6

3.2

R3 soil

8.80

8.52

2.7

22.2

1.2

30.6

7.7

2.2

1.8

Extractable Se by Aqua Regia

The extraction was based on the standard ISO (ISO 11466 1995). One gram of soil samples or CRMs (performed in triplicate) was placed in a reflux vessel with the appropriate volume of aqua regia and digested. The soil suspension was maintained at room temperature for 16 h and then heated to 130°C for 2 h. Once at room temperature, the resulting suspension was passed through an ashless filter (Whatman 40), and the solid residue was washed several times in 0.5 mol L−1 HNO3. The resulting filtrate, together with the washings, were diluted to 50 ml, transferred to a polytetrafluoroethylene (PTFE) container, and stored at 4°C until analysis.

Extraction of Se by the Physiologically Based Extraction Test

The PBET used was based on the method described by Ruby et al. (1996). For this, the following modifications are introduced: the 250-ml polyethylene separator funnel in a specially designed device is replaced by a 100-ml stoppered glass flask because argon purge is not required for the maintenance of anaerobic conditions in the extraction solutions (Wragg and Cave 2002). For the neutralization step, the single addition of 13 ml of a saturated Na2CO3 solution ensures pH 7, thus obviating intermediate pH measurements. Gastric and gastrointestinal extracts are filtered through a 0.20-μm nylon membrane instead of centrifugation, and aliquots of 3 ml instead of 2 ml of the extracts are taken so as to have sufficient volume for the techniques and quantification methods used. The procedure is summarized in Fig. 1.

Phosphoric Acid Extraction

One gram of soil and 25 ml of extracting solution were placed in a 40-ml PTFE tube. The mixture was maintained in an end-over-end shaker for 16 h at room temperature, centrifuged for 15 min at 5,000 rpm, and then filtered (Whatman 40). The resulting solution was transferred to a PTFE container. Se species were measured immediately after extraction.

Total Se Measurement

Se was measured in aqua regia extracts by HGAFS and in PBET extracts by HGAFS and ICPMS. For HGAFS measurements, a prereduction step ensuring the quantitative reduction of Se (VI) to Se (IV) was required. Thus, an aliquot of 5 ml of extract was placed in a sand bath at 170°C with 10 ml of 6 mol L−1 HCl for 30 min. Once at room temperature, the solution was diluted to 25 ml with 6 mol L−1 HCl. Further hydride generation from Se (IV) was achieved with 6 mol L−1 HCl at a flow rate of 8 ml min−1 and 1.5% NaBH4 in 0.4% NaOH at a flow rate of 3 ml min−1. Se in aqua regia extracts was quantified by external curve and Se in PBET extracts by both the external curve and standard addition methods. For Se measurements in PBET extracts by ICPMS, Rh was used as internal standard, and Se was quantified by using the external curve method for calibration.

Se Species Measurement

Se speciation in PBET extracts was performed using LC–UV–HGAFS and LC–ICPMS systems. In both cases, the extracts were first filtered through a nylon membrane of 0.20-μm porosity. A 50-μl aliquot of the extract was injected into the column. Separation was attained at pH 7.0 in isocratic mode using a buffer solution of NH4H2PO4 40 mmol L−1 (adjusted with aqueous NaOH) at a flow rate of 1 ml min−1 as mobile phase. When using LC–UV–HGAFS, the eluate from the column enters the UV reactor (to promote the reduction of Se (VI) to Se (IV)) and then the hydride generation unit with the addition of 6 mol L−1 HCl at a flow rate of 8 ml min−1 and 1% (m/v) NaBH4 at a flow rate of 4 ml min−1. The hydride generated reaches the detector by an argon flow (300 ml min−1) through a gas–liquid separator. An air-based Perma pure drying membrane (air flow 2.5 L min−1) was used to dry the volatile hydride before entering the detector. When using LC–ICPMS, the eluate from the column enters the ICPMS under the conditions described in “Instruments” found in Section 2. For quantifying Se species, standard addition and external curve methods were used for LC–UV–HGAFS and the external curve method for LC–ICPMS. Se speciation in phosphoric acid extracts was carried out using LC–UV–HGAFS, under the conditions described above, and the species were quantified by standard addition calibration method.

4 Results and Discussion

Total Se Determination

The total Se content of aqua regia and PBET extracts of soil samples and CRMs were determined by applying the procedure described in “Total Se measurement” found in Section 3. Total Se in PBET extracts was measured using HGAFS and ICPMS in order to obtain information from two techniques commonly available in analytical laboratories and to compare results (shown in Table 2). There are no significant differences in the results from PBET extracts when using HGAFS or using ICPMS, at 95% confidence level, when applying the statistical t test. Also, when the external curve and standard addition quantification methods was compared, no significant differences at 95% confidence level were found. Then, the external curve is recommended since it is more straightforward to carry out calibration.
Table 2

Results (mean ± SD; n = 3) of total Se, expressed as milligrams of Se per kilogram on dry mass, in the aqua regia and in PBET extracts, by using HGAFS and ICPMS techniques

 

Aqua regia extract

PBET

Extraction efficiency (%)

Extract from gastric solution

Extract from gastrointestinal solution

Technique

HGAFS

HGAFS

ICPMS

HGAFS

ICPMS

Quantification method

Ext. curve

Ext. curve

Std. add.

Ext. curve

Ext. curve

Std. add.

Ext. curve

CRM023-050 (certified value 105 ± 9)

93 ± 10

80 ± 11

86 ± 17

87 ± 19

95 ± 15

93 ± 21

87 ± 21

99

CRM025-050 (certified value 518 ± 74)

483 ± 8

341 ± 19

366 ± 16

390 ± 30

374 ± 21

402 ± 24

361 ± 30

78

R1 soil

6.8 ± 0.1

3.3 ± 0.4

3.4 ± 0.3

2.8 ± 0.7

4.0 ± 0.4

4.1 ± 0.4

4.4 ± 0.7

61

R3 soil

7.6 ± 0.2

3.5 ± 0.2

3.3 ± 0.2

3.5 ± 0.1

4.0 ± 0.3

4.2 ± 0.2

4.8 ± 0.3

57

Quantification was made by using both external curve and standard addition methods

When comparing the aqua regia results for the CRMs with certified values, good agreement was found when considering the associated uncertainties of the certified values. This agreement indicates the accuracy of the results. The last column of Table 2 shows the extraction efficiencies of the PBET method, calculated as the ratio of total Se in the gastrointestinal step (as the average of the mean values obtained by HGAFS and ICPMS techniques) to total Se extracted with aqua regia. Extraction efficiencies showed mean values of 99% for CRM023-050 and 78% for CRM025-50. For R1 and R3 soils, which had a much lower Se content than the CRMs, the mean extraction efficiencies were 61% and 57%, respectively. In general, the extraction efficiencies of trace elements from soils are highly dependent on the matrix composition. In the present study, the lower efficiencies obtained for R1 and R3 soils with respect to the two CRMs could be attributed to differences in the Se compounds of the parent material. Thus, the presence of elemental Se or minerals that are not solubilized (Kabata-Pendias and Pendias 2001) with non-oxidizing reagents (PBET) may explain these lower efficiency values. The extraction values from the gastrointestinal fraction were higher than those from the gastric fraction, as expected. These values could be used to estimate bioaccessibility, which is usually calculated as the ratio of the mass of selenium dissolved in the gastrointestinal extract to the mass of selenium in soil sample (Smith et al. 2010).

Taking into account that selenium and arsenic are leached from soils as oxyanions, it is specially relevant to compare data from the present study with those from the few bioaccessibility studies dealing with arsenic since similar behavior could be expected for both elements. For instance, Ruby et al. (1996) estimated relative arsenic bioaccessibility to be 44% and 50% in two residential soil samples, and Carrizales et al. (2006) found bioaccessibility values for arsenic in ten polluted soils ranging from 39% to 66% independently of the total concentration in the samples. More recently, Smith et al. (2010) studied the influence of liquid to soil ratios on arsenic and bioaccessibility in soil Standard Reference Material estimating values ranging from 37% to 60%. These values are in good agreement with the values obtained in the present study for selenium bioaccessibility in the R1 and R3 soil samples. Moreover, Meunier et al. (2010) studied the influence of the mineralogical composition of the soil on arsenic bioaccessibility and found that the oxidized forms yield higher levels of bioaccessibility. This fact would be in agreement with the high values of selenium bioaccessibility observed for the reference materials studied where selenite is the only species extracted. Taking into account these data it could be postulated that arsenic and selenium show a similar behavior when applying PBET method, although more experimental work will be necessary to fully demonstrate this hypothesis.

Se Speciation

Se is concentrated in dry regions where soils tend to be more alkaline, such as soil samples R1 and R3 and CRMs from Western United States (Sharma and Vance 2007). Selenites and selenates can be produced by soil microorganisms from less soluble forms of Se and tend to be adsorbed on clay particles, iron and manganese minerals, and organic matter (Environment Agency U.K. 2009).

For the extraction of Se species, the approach using phosphoric acid was chosen since this reagent is currently used for the extraction of arsenic and Se species in soils and sediments in an attempt to assess their potential mobility and bioavailability (Orero Iserte et al. 2004; Ruiz-Chancho et al. 2007). Table 3 shows the results obtained on the phosphoric acid extracts of the CRMs and R1 and R3. From the table, it can be observed that in CRMs, only Se (IV) was measured. For R1 and R3 soils, both inorganic selenium species were found, showing Se (VI) with the highest percentage (58% in R1 and 70% in R3) with respect to the extracted selenium. The results obtained from the PBET method are summarized in Table 4. In this case, Se (IV) was also measured in CRMs. For the soil samples, both selenium species were determined in both gastric and gastrointestinal extracts, being Se (VI) the predominant species in gastrointestinal step with respect to the total Se extracted (57% in R1 and 70% in R3). As an example, Fig. 2 shows some of the chromatograms obtained. The slight differences in the retention times in Fig. 2b could be attributed to the differences in the matrix composition of the extracts. In all the chromatograms, always the retention times in LC–UV–HGAFS were slightly higher than LC–ICPMS. These differences are attributed to the different coupling systems since in the LC–UV–HGAFS, the photoreaction step leads to an increase in the dead volume. With respect to the gastric step (first step of the PBET method), 66–118% of selenite was extracted for the CRMs, whereas for R1 and R3 soils, 9–16% of selenite and 29–38% of selenate were extracted. With respect to the gastrointestinal step, 5–15% of selenite and 8% of selenate were extracted in R1 and R3 soils. For the CRMs, the Se (IV) extracted in the gastrointestinal step did not increase the percentage extracted in the gastric step.
Table 3

Results (mean ± SD; n = 3), expressed as milligrams Se per kilogram on dry mass, of Se speciation by using LC–HGAFS in the phosphoric acid extracts

 

Selenium species

Se (IV)

Se (VI)

CRM023-050

106 ± 8

nd

CRM025-050

452 ± 38

nd

R1 soil

1.8 ± 0.2

2.5 ± 0.04

R3 soil

1.2 ± 0.1

2.8 ± 0.6

Quantification was made by standard addition method

nd not detected

Table 4

Results (mean ± SD; n = 3) expressed as milligrams Se per kilogram on dry mass, of Se speciation from gastric and gastrointestinal extracts of the PBET method

 

Selenium species

Se (IV)

Se (VI)

Technique

HGAFS

HGAFS

ICPMS

HGAFS

HGAFS

ICPMS

Quantification method

(Ext. curve)

(Std. add.)

(Ext. curve)

(Ext. curve)

(Std. add.)

(Ext. curve)

PBET method. Extracts from gastric solution

CRM023-050

71 ± 8

110 ± 2

78 ± 2

nd

nd

nd

CRM025-050

317 ± 28

446 ± 23

460 ± 60

nd

nd

nd

R1 soil

0.8 ± 0.1

0.64 ± 0.029

0.74 ± 0.01

2.0 ± 0.4

2.2 ± 0.3

2.1 ± 0.01

R3 soil

0.8 ± 0.1

1.2 ± 0.07

0.79 ± 0.03

2.7 ± 0.1

2.3 ± 0.08

2.9 ± 0.2

PBET method. Extracts from gastrointestinal solution

CRM023-050

83 ± 15

111 ± 9

92 ± 2

nd

nd

nd

CRM025-050

315 ± 43

473 ± 23

471 ± 94

nd

nd

nd

R1 soil

1.4 ± 0.1

1.6 ± 0.4

2.1 ± 0.2

1.8 ± 0.4

2.0 ± 0.3

3.1 ± 0.3

R3 soil

1.1 ± 0.1

1.09 ± 0.07

1.2 ± 0.1

2.5 ± 0.2

2.5 ± 0.1

2.9 ± 0.3

Measurements were done by HGAFS and ICPMS techniques. Quantification was made by using both external curve and standard addition methods

nd not detected

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Fig. 2

Chromatograms of the PBET extracts of soil R1. a Gastrointestinal extract obtained by LC–UV–HGAFS with the standard addition. b Gastric and gastrointestinal extracts obtained by LC–ICPMS

From the results obtained by acid phosphoric extraction and the PBET method, with respect to the gastrointestinal step, we obtained the same chromatographic pattern, since the same Se species were detected and their concentration was similar in both cases. In order to support this observation, the correlation coefficients (at 95% confidence level) have been calculated using the results of Se (IV) and Se (VI) species reported in Tables 3 and 4. In all cases, values higher than 0.99 were obtained. From the experimental point of view, the procedure using phosphoric acid is more straightforward, although it is more time-consuming than PBET.

Quality Control and Figures of Merit

We used HGAFS and ICPMS for total Se determination and LC–UV–HGAFS and LC–ICPMS for speciation for the PBET extracts. The aim was to comply with Quality Assurance principles, which recommend the use of two independent techniques for comparison purposes. The accuracy of the Se determination was tested by analyzing CRM023-050 and CRM025-050. Our results were comparable with those previously reported (González-Nieto et al. 2006). Good agreement with certified values and the values obtained by aqua regia show that the determination of total Se was quantitative. Detection and quantification limits (LOD and LOQ) were established for total Se as well as for Se (VI) and Se (IV) in gastric and gastrointestinal extracts. Table 5 summarizes the results obtained for the HGAFS and ICPMS techniques. Column recoveries were also calculated. This parameter, which is relevant in analytical speciation, corresponds to the mass balance calculations. It is expressed (in percentages) as the ratios between the sum of the species eluted from the chromatographic column and the total Se in the corresponding extract. The ratios were calculated from the mean values of Se species, obtained from the two techniques and calibration methods, to the mean values of total Se in the extracts (see Table 2). Almost 100% recovery was achieved, thereby indicating the reliability of the chromatographic system applied.
Table 5

Limits of detection (LOD) and limits of quantification (LOQ), expressed as micrograms Se per liter

Total Se in gastric and gastrointestinal reagentsa

 

Gastric blank (n = 10)

Gastrointestinal blank (n = 10)

HGAFS

ICPMS

HGAFS

ICPMS

LOD (μg Se L−1)

0.03

0.06

0.01

0.08

LOQ (μg Se L−1)

0.09

0.19

0.04

0.28

LC–UV–HGAFSb

 

Aqueous matrix (n = 5)

Gastric extract (n = 5)

Gastrointestinal extract (n = 5)

LOD (μg Se L−1)

Se (IV)

1

1

1

Se (VI)

4

3

4

LOQ (μg Se L−1)

Se (IV)

2

3

4

Se (VI)

14

11

12

LC–ICPMSc

LOD (μg Se L−1)

Se (IV)

0.2

0.2

0.2

Se (VI)

0.2

0.2

0.3

LOD (μg Se L−1)

Se (IV)

0.5

0.6

0.6

Se (VI)

0.8

0.8

1.0

aTotal Se in gastric and gastrointestinal reagents. Standard calibration curves range from 1 to 16 μg Se L−1 for HGAFS and from 1 to 24 μg Se L−1 for ICPMS

bSe species by HPLC–UV–HGAFS. Standard calibration curves of Se (IV) and Se (VI) in aqueous matrix and in PBET extracts. Standard calibration curves range from 0.8 to 36 μg Se L−1 and from 15 to 45 μg Se L−1, respectively

cSe species by LC–ICPMS. Standard calibration curves of Se (IV) and Se (VI) in aqueous matrix and PBET extracts. Standard calibration curves range from 15 to 45 μg Se L−1 and from 8 to 40 μg Se L−1, respectively

5 Conclusions

The extraction of Se with phosphoric acid produced similar results to those obtained with the PBET extraction method, and the same Se species were obtained from the CRMs and R1 and R3 soils. This study is the first to report on the use of the in vitro PBET method for total Se, selenite, and selenate from soils. Our results show that, for the soils studied, the estimated bioaccessible fraction can be attributed mainly to the gastrointestinal step in the PBET method and corresponds to 50% of Se, including Se (IV) and Se (VI).

This study provides useful information on the bioaccessibility of the small number of trace elements studied so far in soils. Although in vitro tests require validation with in vivo studies for further bioaccessibility assessment, the data obtained in the present study will be useful for future in vivo studies to assess possible correlation.

Se extracted from CRM025-050 and CRM023-050 by the PBET contributes new and relevant information on these CRMs. Moreover, since the solubility of the contaminants in the gastrointestinal step depends on soil type, source, and age, the results obtained with these CRMs, which are available to many researchers, could facilitate the comparison of results obtained from the application of a range of in vitro bioaccessibility tests.

Acknowledgments

The authors are grateful to the Dirección General de Investigación (DGICyT) for Project number CTQ2010-15377/BQU and the Departament d’Universitats, Recerca i Societat de la Informació for financial support (SGR2009-1188). Virginia Funes thanks the University of Barcelona for a predoctoral grant. The authors also thank Dr. Toni Padró from the Serveis Científico-Tècnics of the University of Barcelona for his valuable help in the ICPMS measurements. The authors thank Dr. Zhi-Qing Lin for supplying R1 and R3 soil samples.

Copyright information

© Springer Science+Business Media B.V. 2011