Barbara Proust
email: bproust@pasteur.fr

Ghislaine Lacroix

Franck Robidel

Maryse Marliere

Anthony Lecomte

B Boris

Interference of a short-term exposure to nitrogen dioxide with allergic airways responses to allergenic challenges in BALB/c mice

Keywords: Mouse model of asthma, nitrogen dioxide, air pollutant, bronchopulmonary hyperreactivity, lung permeability, eosinophilia, mucus Bovine serum albumin, BSA Bronchoalveolar lavage fluid, BALF Bronchopulmonary hyperreactivity, BHR Enzyme-linked immunosorbent assay, ELISA Eosinophil peroxidase, EPO Intranasally, i.n. Interleukin, IL Methacholine, Mch Optical density, OD Ovalbumin, OVA Phosphate-buffered saline, PBS Room temperature, RT

Nitrogen dioxide (NO2) is a common indoor and outdoor air pollutant whose role in the Induction of asthma is unclear. We investigated the effects of NO2 on the development of asthma-like responses to allergenic challenge in BALB/c mice. Ovalbumin (OVA)immunized mice were intranasally challenged with OVA or saline solution just before starting a 3 h exposure to 5 or 20 ppm NO2 or air. 20 ppm NO2 induced a significant increase of bronchopulmonary hyperreactivity in OVA-challenged mice and of permeability according to the fibronectin content of the bronchoalveolar lavage fluid (BALF) 24 h after exposure, as compared to air or 5 ppm. Eosinophilia (cell counts in the BALF and eosinophil peroxidase of lung tissue) was detected at 24 and 72 h with similar levels for air and 20 ppm NO2 whereas, unexpectedly, a marked reduction was observed for 5 ppm. At 24 h, IL-5 in the BALF was markedly reduced at 5 ppm compared to 20 ppm NO2 and was also more intense for 20 ppm than for the air group. In contrast to specific IgGl titers, anti-OVA IgE titers and IL-4 in the BALF were not affected by NO2 exposure. Irrespective of the concentration of NO2, OVAchallenged mice did not develop late mucosal metaplasia compared to those exposed to OVAair. These results indicate that a short exposure to NO2 can exacerbate or inhibit some features of the development of allergic disease in mice and may depend to concentration of pollutant.

Introduction

Asthma is an allergic respiratory disease that has captured a great deal of attention for several jrears. One of its perplexing aspects is that its prevalence has increased steadily during this century, doubling in the last 20 years in most industrialized countries [START_REF] Hartert | Epidemiology of asthma: the year in review[END_REF].

Although asthma is familial and genome-wide searches having identified genetic loci predisposing to the disease, it is unlikely that the genetic makeup of stable populations can change significantly in less than one century. The probable cause of the epidemic must therefore relate to the environment [START_REF] Hartert | Epidemiology of asthma: the year in review[END_REF]. Several recent studies have shown an association between air pollution during episodes of smog and asthma exacerbations, and hospital visits for asthma [START_REF] Anderson | Air pollution, pollens, and daily admissions in London 1987-1992[END_REF][START_REF] Morgan | Air pollution and hospital admissions in Sydney, Australia, 1990 to 1994[END_REF][START_REF] Sheppard | Effects of ambient air pollution on nonelderly asthma hospital admissions in Seattle, Washington, 1987-1994[END_REF].

Although this finding does not address causality, it supports that air pollution has an effect on acute asthmatic episodes. Current evidence also suggests that asthmatics are more sensitive to the effects of air pollutants [START_REF] Koren | Associations between criteria air pollutants and asthma[END_REF].

Asthma is characterized by acute bronchoconstriction, late bronchopulmonary hyperreactivity, pulmonary eosinophilic inflammation, excessive mucus production and increased serum IgE titers [START_REF] Djukanovic | Mucosal inflammation in asthma[END_REF], induced by a variety of stimuli. Its pathology seems to be directly linked to the presence in the airways of eosinophils and of Th2 CD4 + lymphocytes [START_REF] Nakajima | CD4 + T-lymphocytes and interleukin-5 mediate antigen-induced eosinophil infiltration into mouse trachea[END_REF]Robinson et al, 1992), which produce IL-4 and IL-5. These Th2 cytokines are said to play a central role in the initiation and perpetuation of asthma (Bochner et al,199A). IL-5 regulates the growth, differentiation and activation of eosinophils and provides an essential signal for their recruitment to the lungs during allergic inflammation [START_REF] Lopez | Recombinant human interleukin 5 is a selective activator of human eosinophil function[END_REF]. IL-4 induces the differential development of ThO into Th2 cells [START_REF] Abehsira-Amar | IL-4 plays a dominant role in the differential development of ThO into Thl and Th2 cells[END_REF], and stimulates B lymphocytes to produce IgE [START_REF] Paul | Interleukin-4: a prototype immunoregulatory lymphokine[END_REF].

Nitrogen dioxide (NO2), a major potent oxidant pollutant, is a well-known airway irritant (American Thoracic Society, 1996). In contrast to other pollutants, NO2 is a widespread contaminant of outdoor and of indoor environment. Its indoor levels can exceed those found outdoor and are provided by gas cooking appliances and tobacco smoke [START_REF] Goldstein | Acute respiratory effects of short-term exposures to nitrogen dioxide[END_REF][START_REF] Norman | Nitrogen oxides in tobacco smoke[END_REF]. By contrast, the main sources of NO2 in outdoor air are motor vehicle emissions and fossil-fuel burning industries (American Thoracic Society, 1996).

Acute exposures to high concentrations of NO2 produce changes in pulmonary function, mcrease airway responsiveness (Mohsenin et al. 1987), and induce pulmonary edema (Hajela et al, 1990). NO2 also may cause release of inflammatory mediators, and induce mast cell and lymphocyte infiltration [START_REF] Sandstrom | Inflammatory cell response in bronchoalveolar lavage fluid nitrogen dioxide exposure of healthy subjects: a dose response study[END_REF]. Animal studies have demonstrated that exposure to NO2 can mcrease susceptibility to infection, presumably through its effect on lung defense mechanisms, mucociliary clearance [START_REF] Hubbard | Acute NO2 exposure alters inflammatory cell activation and particle clearance in silica-injected mice[END_REF], and alveolar macrophage function [START_REF] Davis | Decreased intrapulmonary killing of Mycoplasma pulmonis after short-term exposure to NO2 is associated with damage alveolar macrophages[END_REF][START_REF] Robison | Depression of stimulated arachidonate metabolism and superoxide production in rat alveolar macrophages following in vivo exposure to 0.5 ppm NO2[END_REF]. NO2 is tissue-soluble, unsatured bonds in membrane lipids of the airway and respiratory epithelium, as well lining fluid being its prime targets [START_REF] Postlethwait | Mechanisms of pulmonary NO2 absorption[END_REF]. Several studies have associated morbidity of asthma with elevated concentrations of NO2 [START_REF] Ostro | Indoor air pollution and asthma. Results from a panel study[END_REF]Tunicliffe et al, 1994). Based on epidemiological studies, it has been suggested that NO2 increases the risk for exacerbations of asthma [START_REF] Castellsague | Short-term association between air pollution and emergency room visits for asthma in Barcelona[END_REF][START_REF] Rossi | Association of severe asthma attacks with weather, pollen, and air pollutants[END_REF]. Clinical observations have demonstrated that exposure to NO2 correlates with exacerbations of asthma and with the potentiation of airway reactivity in asthmatics [START_REF] Strand | Nitrogen dioxide exposure enhances asthmatic reaction to inhaled allergen in subjects with asthma[END_REF][START_REF] Strand | Immediate and delayed effects of nitrogen dioxide exposure at an ambient level on bronchial responsiveness to histamine in subjects with asthma[END_REF], in contrast to other studies, which have failed to show an association between exposure to NO2 and increased incidence of asthma [START_REF] Samet | Nitrogen dioxide and respiratory illnesses in infants[END_REF]. These discrepancies show that the role of NO 2 for allergic disease is still unclear.

Since NO2 can increase the bronchial responsiveness to non-specific stimuli such as histamme and methacholine, it may hypothetically affect bronchial responsiveness and other features of asthma to inhaled allergen as well. The existence of such an interaction between NO2 and allergen resulting in an augmented allergic reaction has been suggested [START_REF] Molfino | Effect of low concentrations of ozone on inhaled allergen responses in asthmatic subjects[END_REF][START_REF] Strand | Nitrogen dioxide exposure enhances asthmatic reaction to inhaled allergen in subjects with asthma[END_REF]Tunicliffe et al., 1994). In this study, we investigated the role of NO2 in the exacerbation of asthma in an ovalbumin-immunized mouse model. For that, we studied whether a short-term exposure to low or high concentrations of NO2 can potentiate the responses to allergenic challenge in OVA-immunized mice, in order to define its possible adjuvant role in the development of some features of asthma, such as bronchopulmonary responsiveness, eosinophilic pulmonary inflammation, production of Th2 cytokines, and of specific IgE and mucus secretion.

Materials and Methods

Animals

Antigen immunization and challenge

BALB/c mice were immunized by the subcutaneous injection of 10 ug ovalbumin (OVA, ICN Biomedicals, Inc., Aurora, OH), dispersed in 1.6 mg A1(OH)3 (Merk, Darmstadt, Germany) in 0.4 ml of 0.9 % NaCl (saline) at Days 0 and 7. At Day 14, one week after the second injection, immunized mice were intranasally (i.n.) challenged with 10 (i.g OVA in 50 ul of saline for about 10 s under anesthesia by intravenous injection of ketamine (Imalgene®1000, 35 mg/kg, Merial, France). Control mice were challenged with the same volume of saline solution.

Exposure system

The whole body exposure system used to generate NO2 and expose the animals was developed in the laboratory of INERIS. Unrestrained, and conscious mice challenged with OVA or saline were individually placed in a whole body glass chamber of 0.5 1 and were exposed to 5 or 20 ppm NO2 or to air for 3 h. In each glass chamber, NO2 was delivered with a flow rate of 5 1/min allowing to have a renewal, and was calibrated at the exact desired concentration in synthetic air. The airflow of NO2 in each chamber was monitored by a mass flow-meter during all the period of exposure. The relative pressure of the glass exposure chamber was controlled by a manometer. Concentrations of 5 or 20 ppm of NO2 were obtained from cylinders of NO2 gas prepared and certified by the supplier (Air Liquide, France).

Evaluation of bronchopulmonary hyperreactivity

BHR was evaluated with a barometric plethysmography method. Unrestrained, conscious mice were placed in a whole body plethysmographic chamber (EMKA Technologies, Paris,

Serum sample preparations and bronchoalveolar lavage fluid (BALF)

Mice were anesthetized by the intraperitoneal injection of urethane (ethylcarbamate, 2 g/kg, Sigma, St Louis, MO) and the abdominal cavity was opened. Blood samples were collected from the post vena cava and serum was collected after centrifugation at 500 x g for 10 min and stored at -20°C until used. After exsanguination, the lungs were flushed via the cannulated trachea with 4 x 0.5 ml of a sterile PBS solution (phosphate buffer lOmM, pH 7.4). The total cell numbers were counted automatically (Coulter Counter ZM, Coultronics, Margency, France). BALF was cytocentrifuged for 10 min (Cytospin, Shandon, England).

Slides were stained with a May Griinwald Giemsa-derived method (Diff Quick, Baxter Dade AG, Duedingen, Switzerland), and a total of 200 cells was counted for each sample by light microscopy, the percentage of each cell population being calculated. The BALF was centrifuged for 10 min at 1850 x g, 4°C (Jouan, Saint Herblain, France) and the supernatants were removed and stored at -20°C until used.

Evaluation of lung eosinophil peroxidase activity

To quantify the lung sequestration of eosinophils, eosinophil peroxidase (EPO) activity in the lung was evaluated in 96-well plates by a cytochemical enzyme assay (Van Oosterhoot et al., 1995). Briefly, lungs were removed and homogenized (Potter-Elvejhem glass homogenizer, Thomas, Philadelphia, PA) in 0.05 M Tris-HCl buffer pH 8 containing 0.1% Triton X-100 solution. Lung homogenates were centrifuged for 15 min at 1600 x g, 4°C (Sigma, Bioblock Scientific 2K15, Illkirsh, France). EPO activity was measured in the supernatant, based on the oxidation of o-phenylenediamine (Sigma) by EPO in the presence of peroxide hydrogen.

Incubations in duplicate were carried out in the absence or presence of the peroxidase inhibitor 3-amino-l,2,4-triazole (AMT, Sigma). Plates were read with an automatic microplate reader (Dinatech MR 5000, Dinatech Laboratories, Saint Cloud, France) at 490 nm and results were expressed as optical density (OD).

Evaluation ofcytokines

IL-4 in the BALF was evaluated by ELISA. Briefly, 96-well plates were coated with rat anti-mouse IL-4 (BVD4-1D11, Perbio Sciences, Erembodegem-Aalst, Belgium) at 2 ug/ml diluted in 0.1 M carbonate buffer pH 8.2 and incubated overnight at 4°C. Plates were washed with PBS/0.1% Tween 20 and blocked with PBS containing 1% of bovine serum albumin (BSA) overnight at 4°C. After washing, dilutions of recombinant murine IL-4 (rIL-4, Perbio Sciences) (15.6 to 1,000 pg/ml) or samples were applied overnight at 4°C. Then, biotinylated rat anti-IL-4 antibody (BVD6-24G2, Perbio Sciences) was added at 0.5 ng/ml for 2 h at 4°C.

Plates were incubated with ExtrAvidin® peroxidase conjugate (1:2,000 to each well, Sigma)

for 45 min at room temperature (RT). Plates were developed with tetramethylbenzidine substrate (TMB, Kiregaard Perry Laboratories, Maryland). The reaction was stopped with sulfuric acid 2N and the plates were read at 450 nm with a microplate reader (Dinatech MR 5000, Dinatech Laboratories). The lower limit of detection of this assay is « 10 pg IL-4/ml sample.

IL-5 in the BALF was quantified using an immunometric assay as described previously (Eum et al, 1995). Briefly, 96-well plates were coated with 10 ug/ml of rat anti-mouse IL-5 (TRFK-4). To these, were added dilutions of recombinant IL-5 (rIL-5) standard (7.6 to 1,000 pg/ml) or of the sample was added, followed by an acetylcholinesterase-labelled rat antimouse IL-5 antibody (TRFK-5) at 10 Ellman units/ml. Absorbance was read at 405 nm with a microplate reader (Dinatech MR 5000; Dinatech Laboratories). The lower limit of detection of this assay is « 5 pg IL-5/ml sample.

Evamuation ofanti-OVA specificIgE andIgGl

The specific anti-OVA specific IgE and IgGl in the serum were measured by ELISA.

For the determination of specific IgE, 96-well plates were coated with rat anti-mouse IgE (EM 95-3) at 5 ^ig/ml diluted in 0.5 M carbonate buffer pH 9.6 and incubated overnight at 4°C. The next day, plates were washed with PBS/0.1% Tween 20 and blocked with PBS containing 1% of BSA for 2 h at RT. After blocking, plates were washed and serum samples were added and incubated overnight at 4°C. The plates were then washed and 10 pig/ml of biotinylated OVA was added. The remaining steps were performed exactly as described for IL-4.

For the evaluation of specific

Evaluation offibronectin

In order to evaluate the intensity of exudation through the airways, fibronectin in the BALF was measured by the indirect competitive ELISA method described by [START_REF] Rennard | Enzymelinked Immunoassay (ELISA) for connective tissue components[END_REF].

Briefly, 96-wells plates were coated with murine fibronectin (Anawa, Wangen, Switzerland) at 1 )iig/ml diluted in 0.02 M carbonate buffer pH 9.6 and incubated overnight at 4°C. In another 96-wells plates, BALF samples and standard fibronectin were incubated with a polyclonal rabbit anti-fibronectin antibody (1:10,000, Anawa). The next day, plates coated with fibronectin were washed with PBS/0.1% Tween 20 and blocked with PBS containing 1% of BSA for 2 h at RT. Then, plates were rinsed with PBS/0.1% Tween 20 and 100 ul of antigen-antibody were applied to plates and incubated for 2 h at RT. After washing, the antifibronectin antibody that did not bind with BALF fibronectin content was detected with a biotinylated secondary anti-rabbit antibody (1:1,500, Amersham Pharmacia Biotech, France)

and incubated for 2 h at RT. Plates were washed and incubated with ExtrAvidin® peroxidase conjugate (1:20,000 to each well, Sigma) for 45 min at RT. The remaining steps were performed exactly as described for IL-4. The lower limit detection of this assay is « 0.078 ug fibronectin/ml sample.

Lung histology

After exsanguination, the lungs were removed and fixed by intratracheal instillation with 10 % neutral phosphate-buffer formalin. The whole lung was embedded in paraffin, sectioned at a thickness of 5 um and stained with periodic acid Schiff (PAS) to examine mucus cells in the airway walls.

Statistical analysis

The results are expressed as mean ± standard error of the mean (SEM). Statistical comparisons between groups were made using ANOVA. Multiple comparisons between all groups were performed by Fisher's least-significant difference test. A value of P< 0.05 was considered to be significant.

Results

In order to investigate the influence of NO2 on the responses to the i.n. administration of OVA in immunized mice, two concentrations of NO2 that are relatively high as compared to those encountered in the environment were used, since the actual amounts delivered to the lung are quite below the concentrations inhaled (Hatch et ai, 1994;[START_REF] Kleeberger | Differential susceptibility to oxidant exposure in inbred strains of mice: nitrogen dioxide versus ozone[END_REF]. We have developed a murine model of allergen-induced pulmonary inflammation sharing the essential features of asthma, in which the two time points of 24 and 72 h after allergenic challenge allowed to evaluate the parameters of the asthma phenotype. One week after the booster injection of antigen, OVA-immunized mice were challenged i.n. with 10 jug of OVA or saline just before the exposure to air or to NO2 at 5 or 20 ppm for 3 h. The time points of 24 and 72 h were studied after NO2 exposure. The peak of BHR, the initial phase of pulmonary eosinophil entrapment, and the peak of production of Th2 cytokines were studied at 24 h, whereas at 72 h, the increase of pulmonary eosinophilic inflammation, the late mucosal metaplasia development and specific immunoglobulins in serum were determined.

Bronchopulmonary responsiveness to methacholine

At Day 15, i.e., 24 h after the exposure to 20 ppm NO2 or to air, immunized BALB/c mice challenged with OVA expressed significant BHR as compared to those exposed to 5 ppm NO2

(Figure 1). Mice challenged with saline exposed to air or to NO2 did not develop BHR. In OVA-challenged mice exposed to 20 ppm NO2, BHR was significantly increased as compared to those exposed to air and to 5 ppm. At Day 17, 72 h after exposure, OVA-challenged mice exposed to air, or to NO2 failed to develop BHR (Figure 1).

Fibronectin exudation into the BALF

To evaluate the exudation through the airways, the concentration of fibronectin was measured in the BALF. OVA-challenged mice exposed to air or to 20 ppm NO2 released significantly increased amounts of fibronectin in the BALF, whereas those levels were very low in animals exposed to 5 ppm NO2 (Figure 2). In contrast, exposure to 20 ppm NO2 augmented by 6-7 fold the fibronectin concentrations in the BALF of OVA-challenged mice after 24 h, as compared to the OVA-air group. NO2 at 20 ppm also induced a marked fibronectin exudation in saline-challenged mice, as compared to the saline-air or saline-NO2 5 ppm groups. Seventy two hours after exposure, BALF fibronectin content persisted in saline-or OVA-challenged mice exposed to 20 ppm NO2. At this time point, in OVA-challenged mice exposed to 5 ppm NO2 or to air, the release of fibronectin was delayed, as compared to mice exposed to 20 ppm NO2 (Figure 2). Indeed, 24 h after exposure to air or to 5 ppm NO2, no or low fibronectin was detected in the BALF of OVA-challenged mice. But, 72 h after exposure, increased amounts of fibronectin were found, contrary to mice exposed to 20 ppm NO2, in which exudation started at 24 h.

BALF cell infiltration and lung sequestration of eosinophils

At 24 h, neutrophil counts were increased in the BALF of OVA-challenged mice exposed to air or to NO2. These counts were found significantly increased and in OVA-challenged mice exposed to 20 ppm, as compared to OVA-air mice. The increase in neutrophil counts in saline-challenged animals exposed to 20 ppm NO2 was also significantly above that found in saline-air or saline-5 ppm NO2 mice (Figure 3A). At 72 h, neutrophil counts were normalized in all groups of mice (Figure 3A). Eosinophils in the BALF were detected at 24 h and increased at 72 h, with similar numbers in OVA-challenged mice exposed to air or to 20 ppm NO2, as compared to those exposed to 5 ppm, in which these counts were markedly reduced after 24 and 72 h (Figure 3B). The EPO activity in the lung increased significantly in OVAchallenged mice exposed to air at 24 and 72 h. OVA-challenged mice exposed to 5 ppm NO2

showed a marked reduction of EPO activity at 24 and 72 h, as compared to the OVA-air group, which correlated with the reduced eosinophil counts in the BALF. Significant reduction of EPO activity was also noted in saline-challenged mice exposed to 5 ppm NO2, as compared to those exposed to air. This contrasts with results in mice exposed to 20 ppm NO2 or to air, in which EPO titers were increased to similar levels (Figure 3C).

Th2 cytokiheproduction in the BALF

OVA-challenged mice exposed to air or to NO 2 released IL-5 and IL-4 in the BALF at 24 h.

In connexion with eosinophilia (cell counts and EPO in the lungs), the production of IL-5 in the BALF was significantly reduced in OVA-challenged mice 24 h after exposure to 5 ppm NO2, as compared to those exposed to 20 ppm NO2 or to air (Figure 4A). However, the production of IL-5 was increased 3-fold in OVA-challenged mice exposed to 20 ppm NO2, as compared to the OVA-air group, and was augmented by 10-fold, as compared to those exposed to 5 ppm (Figure 4A). By contrast, the production of IL-4 in the BALF was increased to the same extent in the three groups 24 h after exposure (Figure 4B). No IL-4 nor IL-5 were detected in the BALF at the 72 h point (Figures 4A and4B).

Production of IgE and IgGl anti-OVA antibody in the serum

Saline-and OVA-challenged mice exposed to air or to NO2 produced specific IgE and IgGl detected in the serum 24 and 72 h later. The anti-OVA IgE titers increased to a similar extend in the three groups after 72 h (Figures 5A), and the both concentrations of NO2 failed to affect their production. Specific IgGl titers were markedly increased as compared to IgE, without significant differences between saline and OVA-challenged mice. Mice exposed to 5 ppm NO2 and challenged with saline or OVA showed a significant increase of IgGl titers as compared to those exposed to air or to NO2 at 20 ppm (Figure 5B).

Histological analysis

As expected, the bronchial epithelium of saline-challenged immunized mice exposed to air was mucus-free (Figure 6A), under conditions where that of immunized and OVA-challenged mice was markedly enriched in mucosal cells (Figure 6B). Saline-or OVA-challenged immunized mice exposed to NO2 at 5 or 20 ppm did not develop mucosal metaplasia as compared to OVA-challenged mice exposed to air (Figures 6D and6F).

Discussion

Studies in asthmatics have shown that air pollutants such as ozone [START_REF] Molfino | Effect of low concentrations of ozone on inhaled allergen responses in asthmatic subjects[END_REF], or NO2 augment the allergic responses [START_REF] Jenkins | The effect of exposure to ozone and nitrogen dioxide on the airway response of atopic asthmatics to inhaled allergen[END_REF][START_REF] Strand | Repeated exposure to an ambient level of NO2 enhances asthmatic response to a non symptomatic allergen dose[END_REF][START_REF] Strand | Nitrogen dioxide exposure enhances asthmatic reaction to inhaled allergen in subjects with asthma[END_REF]Tunicliffe et al, 1994). At high concentrations, NO2 is a well-known airway irritant that can cause bronchial constriction in normal subjects (Hajela et al, 1990;[START_REF] Mohesin | Airway response to 2.0 ppm nitrogen dioxide in normal subjects[END_REF], amd enhance airway responsiveness to histamine [START_REF] Bylin | Ambient nitrogen dioxide concentrations increase bronchial responsiveness in subjects with mild asthma[END_REF] or methacholine [START_REF] Mohesin | Airway responses to nitrogen dioxide in asthmatics subjects[END_REF] in asthmatic patients, which are more sensitive than healthy subjects. By contrast, few studies have addressed to the interaction between NO2 and lung allergy in experimental animals. As shown here, a short-term exposure to NO 2 produces contrasting effects on the development of asthma-related responses in an OVA-immunized mouse model, which depend on the dose of NO2. Thus, the high dose of 20 ppm potentiated BHR, exudation and release of IL-5 in the BALF after OVA challenge, under conditions where the low dose of 5 ppm failed to modify BHR, and reduced significantly pulmonary eosinophilic inflammation and the production of IL-5 in the BALF. Since a single exposure to NO2 was used here, both doses were relatively high as compared to those encountered in the environment. It has been reported nevertheless that the final amounts delivered to the lungs are quite below the concentrations inhaled [START_REF] Hatch | Ozone dose and effect in human and rats. A comparison using oxigen-18 labeling and broncho alveolar lavage[END_REF][START_REF] Kleeberger | Differential susceptibility to oxidant exposure in inbred strains of mice: nitrogen dioxide versus ozone[END_REF].

The potentiation of BHR by 20 ppm NO2 in allergic mice may be accounted for by an increased vascular/epithelial permeability, facilitating the allergen availability and accelerating the inflammatory process. NO2 is a potent tissue-soluble oxidant, which can induce pulmonary edema at high concentrations. In our experiments, immunized animals treated with saline and exposed to 20 ppm NO2 had increased titers of fibronectin in the BALF, which was used as an indicator of permeability, as compared to saline-air or to saline-5 ppm NO2 groups. The high concentration of NO2 potentiated the effects of OVA, since the fibronectin BALF content was significantly increased in OVA-challenged mice exposed to 20 ppm, as compared to OVA-air or to OVA-5 ppm NO2 groups, and correlated with the Increased BHR. Even thought BHR disappeared with time, the increased permeability persisted 72 h after exposure to 20 ppm, whereas a more delayed increase in exudation was observed in OVA-challenged mice exposed to air or to 5 ppm of NO2. The low concentration of NO2 had an effect similar to that of OVA alone. Since our mice underwent a single exposure to NO2, it is possible that such low concentrations of NO2 become effective upon repeated exposures. In this context, the most likely mechanism for NO2-induced increment in bronchial sensitivity to inhaled allergens is the damage of epithelial cells mediated by its oxidative activity, which may increase the cell permeability to the allergen, thus increasing its delivered dose, as occurs in cultured human epithelial cell monolayers exposed to NO2 over short periods [START_REF] Devalia | Human bronchial epithelial cell dysfunction following in vitro exposure to nitrogen dioxide[END_REF]. This would account for the augmented fibronectin titers in the BALF, after its exudation from the plasma. It is also possible that NO2 acts as a permissive agent, by allowing other factors to exacerbate asthma or that underlying factors such as the intensity of allergy or inflammation may be a prerequisite for the expression of the detrimental effects of the gas. Finally, NO2 can reduce the mucociliary activity of the airways in vivo [START_REF] Helleday | Nitrogen dioxide exposure impairs the frequency of the mucociliary activity in healthy subjects[END_REF] and in vitro [START_REF] Devalia | Human bronchial epithelial cell dysfunction following in vitro exposure to nitrogen dioxide[END_REF], further enhancing the accessibility of the allergen to the epithelial cells, owing to its decreased clearance from the airways.

hi our experiments, eosinophilia (cell counts and EPO lung content) and IL-5 in the BALF were significantly reduced in OVA-challenged mice exposed to 5 ppm. By contrast, 20 ppm NO2 did not affect eosinophilia, under conditions where the production of IL-5 in the BALF was significantly increased as compared to the OVA-air group. Recently, [START_REF] Morris | Effect of nitrogen dioxide on ovalbumin-induced allergic airway disease in a murine model[END_REF] reported that the exposure to 0.7 ppm NO2 reduces eosinophilic inflammation in allergic mice, but the IL-5 levels were not measured. The mechanisms of the decreased eosinophilic inflammation and IL-5 production in mice exposed to 5 ppm NO2 are unknown. They may result from alterations in the regional deposition patterns of OVA and of NO2 in the airways, or in absorbance, pulmonary clearance or antioxidant defenses. Indeed, NO2 has a low solubility, and is poorly absorbed by the airway mucosa [START_REF] Chitano | Effect of oxidant air pollutants on the respiratory system insights from experimental animal research[END_REF]. It is also a very reactive molecule whose uptake in the respiratory system is extremely high [START_REF] Chitano | Effect of oxidant air pollutants on the respiratory system insights from experimental animal research[END_REF]. The use of mathematical dosimetry models suggests that the uptake of NO2 between the trachea and the respiratory zone occurs to a similar extent, and peaks at the terminal bronchioles [START_REF] Chitano | Effect of oxidant air pollutants on the respiratory system insights from experimental animal research[END_REF]. It is also possible that the biphasic effect of 5 and 20 ppm NO2 may result from pharmacodynamic alterations. Some studies have indeed demonstrated that NO2 affects lung defense mechanisms, including mucociliary clearance, alveolar macrophages (AM), and the immune system [START_REF] O'neill | Oxidative damage by ozone and nitrogen dioxide: synergistic toxicity in vivo but no evidence of synergistic oxidative damage in an extracellular fluid[END_REF]. The decreased OVA-induced IL-5 production and eosinophilia after an exposure to 5 ppm NO2 may result from an alteration of AM function, including differences in antigen presentation by AM, or from a decreased expression of antigen-derived peptides on their surface, which may be hampered by exposure to NO2. Indeed, [START_REF] Kineast | Modulation of IL-ip, IL-6, IL-8, TNF-a, and TGF-p secretions by alveolar macrophages under NO2 exposure[END_REF] and [START_REF] Erroi | In vivo exposure to NO2 reduces TNF and IL-6 production by endotoxin-stimulated alveolar macrophages[END_REF] have

shown that NO2 exposure of LPS-stimulated human AM results in a functional impairment of AM. Furthermore, [START_REF] Robison | Depression of stimulated arachidonate metabolism and superoxide production in rat alveolar macrophages following in vivo exposure to 0.5 ppm NO2[END_REF] demonstrated that exposure rates to 0.5 ppm NO2 for 0.5-10 days, reduces the arachidonate metabolism and superoxide production in response to external stimuli. Thus, NO2 may reduce the capacity of AM to respond to immunologie stimuli, which might explain the decreased allergic responses in animals exposed to 5 ppm, in particular eosinophilia and production of IL-5.

Airway inflammation is accompanied by mucus secretion, which contributes to airway obstruction. In our experiments, irrespective of the concentration of NO2, OVA-challenged mice did not develop mucosal metaplasia, in contrast to those exposed to air, which is probably related to mucus denaturation by NCVinduced oxidation. The mucous layer forms a protective barrier of the airways against the effects of oxidants, which eliminates and/or scavenges the toxic components of NO2 prior to their diffusion into the airway epithelium.

The lipid content of the mucous layer, in particular esterified unsaturated fatty acids constitutes the primary scavenging oxidants [START_REF] Cavanagh | Mucus protection and airway peroxidation following nitrogen dioxide exposure in the rat[END_REF]. The latter demonstrated that the phospholipids of the mucous layer cannot offer a significant protection against inhaled NO2 and that exposure to 40 ppm NO2 in rats induces in lipid peroxidation correlated with the apparent lack of oxidant scavenging species in the mucous lining the airways.

In summary, a short-term exposure to NO2 modifies the asthma-like responses to allergenic challenge in BALB/c mice with contrasting effects according to its concentration. Despite the relatively high concentrations of NO2 used in our investigation, as compared to those encountered in the environment, this study provides new informations concerning the subtle interactions between an air pollutant and allergic disease.

  Male strain BALB/c mice (6-7 weeks of age, 23 ± 2 g body weight) purchased from the Centre d'Elevage R. Janvier (Le Genest Saint-Isle, France) were housed (10 mice per cage) in the INERIS animal-care unit, a facility accredited by the Departmental Direction of Veterinary Services. The animals were maintained on a 12 h light/dark cycle, at 20-24°C with relative humidity of 40-70 %, and had free access to conventional laboratory feed and water. Animals were handled in accordance with French State Council guidelines for the care and use of laboratory animals (Decree n°87-849, October the 19 th 1987), and was approved by the Institutional Animal Care and Use Committee at the INERIS.

France) which measured

  the respiratory waveforms. Animals were exposed to an aerosol of methacholine (Mch, Aldrich, Milwaukee, WI) for 20 s at 0.1 M delivered by an aerosolator. The index of airway obstruction was expressed as enhanced pause (Penh), in response to inhaled Mch (Hamelmann et al, 1997). Penh was calculated as : Penh = [Te (expiratory time)/Tr (relaxation time)-l] x [Pef (peak expiratory flow)/Pif (peak inspiratory flow)]. For the graphic representation, each value was expressed every minute and was calculated from the average of three values of Penh recorded every 20 s. In order to simplify the interpretations, the area under the curve (A.U.C) was calculated for 15 min. The graphics in terms of A.U.C represent the quantitative expression of BHR.

Figure 1 .

 1 Figure 1. Bronchopulmonary hyperresponsiveness is significantly augmented in

Figure 2 .

 2 Figure 2. BALF fibronectin content after short exposure to NO2

Figure 3 .

 3 Figure 3. BALF cell infiltration after exposures to N02

Figure 4 .

 4 Figure 4. BALF Th2 cytokine levels after exposures to NO2

Figure 5 .

 5 Figure 5. Specific anti-OVA IgE and IgGl titers in the serum after exposures to NO2

Figure 6 .

 6 Figure 6. PAS-stained histologie sections of lungs from allergic mice exposed to NO2

Acknowledgments

This work has been supported by a grant of the French Ministry of Environment. Tunnicliffe, W.S., Burge, P.S., and Ayres, J.G. (1994). Effect of domestic concentrations of nitrogen dioxide on airway responses to inhaled allergen in asthmatic patients. Lancet 344, 1733-1736.

Van Oosterhout, A.J.M., Fattah, D., Van Ark, I, Hofman, G., Buckley, T.L., and Nijkamp, F.P. (1995). Eosinophil infiltration precedes development of airway hyperreactivity and mucosal exudation after intranasal administration of interleukin-5 to mice. J. Allergy Clin. Immunol. 96,[104][105][106][107][108][109][110][111][112]


L Malherbe
email: laure.malherbe@ineris.fr

DESIGNING A CONTAMINATED SOIL SAMPLING STRATEGY FOR HUMAN HEALTH RISK ASSESSMENT

Keywords: sampling strategy, soil sampling, contaminated sites, risk assessment

Designing a contaminated soil sampling strategy for human health risk assessment

Laure

1 Introduction -Human health risk assessment : stakes of a relevant soil sampling strategy ?

Human health risk assessment is a site-based approach which provides a decision support in the framework of brownfield sites management. It is aimed at determining if an old site contamination may have harmful effects on human health in view of the planned or current use of the site. If need be it can help to formulate a remediation strategy compatible with that use.

In practice human health risk assessment commonly involves four steps [START_REF] Mate | Guide Gestion des Sites Pollués : Diagnostic approfondi -Evaluations détaillées des Risques[END_REF] :

1. Site characterization : data providing appropriate information for exposure assessment are collected (nature and extent of contamination, historical review of the site and its users, knowledge of hydrogeology...). Soil sampling comes at that stage.

2. Exposure assessment : the type and magnitude of exposure to chemicals of potential concern present at or migrating from the site are estimated according to (future) land occupation. This stage should include : characterization of exposure setting, identification of exposure pathways, quantification of exposure.

3. Toxicity assessment : qualitative and quantitative toxicity information about substances being evaluated is gathered. This is accomplished in two steps : hazard identification and doseresponse assessment. [START_REF]Guide pour l'échantillonnage, l'analyse des échantillons et la gestion des données des lieux contaminés[END_REF]. Risk characterization : it brings the toxicity/potency and the exposure data together into an expression of quantitative risk estimation for all receptors.

Obviously the results of risk assessment and the decisions that may ensue are closely dependent on site characterisation. This is why a proper soil sampling is of major importance as it can have consequences in terms of public safety and of costs (remediation expenses).

Therefore soil sampling needs to fit the objectives of the study and to follow a well-defined strategy. This includes the number of sampling points, their location on site, the depth and the type of samples to be taken.

No sampling strategy has been entirely formalised for human health risk assessment yet. National or international guidelines or standards, such as ISO 10381 [START_REF]Soil Quality -Sampling -Part 1 : Guidance on the design of sampling programmes[END_REF], provide guidance which is often dictated by the suspected level of contamination rather than by the aim of the study. Thus the site assessor has to adapt those guidelines to the specific problem he is in charge of.

The following study is focused on the designing of a soil sampling strategy for human health risk assessment. It does not handle the specific matter of sampling techniques, sample preparation and chemical analysis. We propose to review the components of a soil sampling strategy and to comment them from the angle of human health risk assessment. For each component we will compare several alternatives outlining their advantages and drawbacks. A few examples derived from current practice will be given as an illustration and some recommendations be voiced. The question of a multistage sampling strategy and the problem of representativeness will be briefly discussed.

2

Which soil sampling strategy for human health risk assessment ? Besides it must reconcile scientific requirements with resource constraints.

[FIG.l]

2.1 Sampling pattern :

Statistical (probabilistic) vs non statistical (directed) approaches

Non probabilistic strategies are based on an a priori idea about site pollution. Judgmental sampling in areas assumed representative of the whole site should be banned however because of bias imposed by the investigator's subjectivity [START_REF] Lecomte | Stratégie d'investigation pour les sites industriels pollués par les hydrocarbures aromatiques polycycliques[END_REF], and the resulting uncertainty on exposure. Purposive sampling (fig. 2a) consists in selecting sample locations in regions known to be most contaminated in view of the site history. A few samples may be taken in the remaining part of the site for control only. Before opting for such a strategy the assessor should wonder whether average concentrations, percentiles or extreme values are of concern. The answer depends upon the approach he decides to favour : a reasonably conservative or a maximising one. In the first case a targeted sampling is not sufficient to calculate mean concentrations or percentiles. In the second case it could be appropriate providing that hotspots have already been properly located through a thorough exploratory phase. If not it may provide misleading information about site contamination entailing an incorrect or incomplete risk assessment.

Among the wide range of possible schemes the well-known systematic grid sampling is commonly used in North-America (USA, Canada). A 25 m grid as recommended by CCME 1 [4] ensures a complete site coverage and a homogeneous distribution of samples over the site, hence minimising bias in the estimation of mean concentrations. In a systematic sampling plan every part of the site is equally viewed as a potential exposure area. The likelihood of missing the presence of contamination is thus decreased.

Besides collected data can be processed with statistical and geostatistical tools, the latter leading to a better understanding of contamination spatial variability and providing 2 or 3D-maps of estimated concentrations. The production of such maps rather applies to the devising of soil-cleaning projects. However it can bring out possible high-risk areas or insufficiently sampled areas where the uncertainty on the estimated concentrations might be critical as regards risk evaluation. If the probability of exceeding threshold values is desired then geostatistical techniques such as conditional simulation can be used [START_REF] Nathanail | A geostatistical approach to spatial risk assessment of lead in urban soils[END_REF].

Though easy to implement in the field systematic sampling can require a large number of samples and be relatively costly unless it is reduced to a loose mesh grid. If so highly contaminated areas may be poorly characterised and the advantages of systématisation invalidated.

Another approach designated as stratified random sampling is sometimes advocated (e.g.. in

Germany or the Netherlands [6][7]). It combines a pollution-oriented and a statistical random sampling (fig. 2b). The site is first divided into several areas and/or layers (horizontal and vertical strata respectively) in which pollution is supposed to be relatively homogeneous. Each stratum is then randomly sampled. This approach needs a preliminary detailed study but it helps to evaluate contamination spatial variability within and between strata and to improve the precision of the estimated mean concentrations. Actually this is in keeping with the European trends which is to adapt sampling design and sampling size to the suspected pattern and degree of contamination.

Opting for a sampling design

Some advice can be drawn from this discussion as regards the choice of a sampling pattern. In addition to a purposive sampling scheme a systematic grid sampling (e.g. a square grid) may give a large amount of information about pollution and be rather easily implemented (fig. 2c). To limit costs, sampling density (the mesh size) can be adapted to the suspected level of contamination and exposure, being increased in heavily contaminated or heterogeneous areas or even in cleaner areas likely to be highly frequented. A preliminary stratification of the site seems particularly adequate for large and complex sites where a uniform sampling strategy could either be costly (too many samples) or mask some features of contamination (too loose a grid) and make the conclusions of risk assessment unreliable. A sampling strategy as described above might then be designed for each stratum separately.

[FIG. 2a,2b,2c]

It is also necessary to take background samples in areas thought to be free of contaminants. They can tell whether site concentrations are higher than those observed in natural background conditions and help the assessor to propose realistic clean-up levels if he is asked to. The choice between grab or composite samples can be dictated by soil and contaminants properties and by the analysis costs as well.

A grab sample is an individual sample collected from a single location at a specific time [START_REF] Epa | Manual, Section 5 : Sampling design and quality assurance procedures, Section 12 : Soil sampling[END_REF].

The collection of grab samples is the best way to get information about the spatial (both lateral and vertical) variability of contamination.

A composite sample is made of a series of discrete equal specimens (aliquots) taken at one location and several depths (vertical composite) or several locations and one depth (spatial composite). It is to be representative of the mean composition of either a vertical profile (vertical composite) or an area (spatial composite). Compositing aliquots reduces the intrinsic variability of the final sample and allows the mean concentration of a profile or an area to be estimated with a better precision. Besides analysis costs are diminished. A composite sample should not however represent an area larger than 1 ha (Jones in [START_REF] Tan | Soil Sampling, Preparation and Analysis[END_REF]) and should be made of more than 4 and less than 9

specimens. With more aliquots concentrations might be diluted [START_REF] Epa | Manual, Section 5 : Sampling design and quality assurance procedures, Section 12 : Soil sampling[END_REF]. Other technical difficulties are likely to arise when compositing samples (volatilization of some organic compounds, bad homogénéisation of humid clay soils) and generate large uncertainties on the analytical results.

So that risk is not underestimated grab samples should be preferred in any circumstances to spatial composites unless the absence of volatile compound and the homogeneity of contamination are really well known. The collection of vertical composites should be avoided too (cf. §2.3.).

Sampling depth

The depths at which samples should be taken are linked to the vertical variability of concentrations.

They depend on : the soil stratigraphy ; the soil nature and properties which may have an effect on the chemical form and the mobility of contaminants ;

the physical and chemical properties of contaminants likely to influence their mobility ;

the site use and the potential exposure pathways.

Thus samples can be taken in the upper layer (over the first centimetres) and/or in subsurface (up to one or several meters depth). Since a contamination profile can be quite uneven it is often recommended to collect samples at regular intervals (e.g. every 50 cm or 1 m) and in each stratigraphical unit. For costs reasons only a few specimens may then be selected for laboratory analysis. The most relevant sampling depths are a function of exposure routes as indicated in table 1.

[ TABLE 1] This example highlights the importance of taking deep samples (up to 4 metres in that case) when inhalation of volatile compounds appears to be a potential exposure pathway.

Sampling size

Actually the assessor has to define :

the number of sampling points to be distributed over the site;

the number of samples to be taken at each of those points ;

According to the on-site observations made during the investigations he might then reconsider the total number of samples to be analysed.

The number of samples at each point is linked to the number of selected depths for sampling. As for the number of sampling points literature shows many recommendations which will not be detailed here. They are based either on experience or on statistics. In the first case they strongly depend on the type of contamination : non suspected, heterogeneous or diffuse contamination. The use of statistics is more of an Anglo-Saxon approach. Determinant factors are then :

the probability of success in hitting a hot-spot [START_REF] Tucker | A decision support system for designing sampling strategies for potentially contaminated sites[END_REF].;

the precision on the mean that the assessor intends to reach [START_REF] Gilbert | Statistical Methods for Environmental Pollution Monitoring[END_REF][14] [START_REF] Epa | Guidance for Choosing a Sampling Design for Environment Data Collection, Use in the Development of a Quality Assurance Project Plan[END_REF]; the cost not to be exceeded [START_REF] Gilbert | Statistical Methods for Environmental Pollution Monitoring[END_REF].

The first critérium can be relevant when no prior information about the location of past industrial activities and the soil contamination is available. Otherwise the last two criteria should be preferred:

the one allows the site assessor to have a better control over the uncertainties on the calculated risk, the other allows him to integrate cost constraints into the sampling strategy. However the mathematical equations used for determining sampling size assume that concentration variability (expressed by the coefficient of variation) is known, which is scarcely the case unless a first sampling stage has been carried out.

This is why we suggest that the assessor use all or part of those recommendations and formulas as an indicative basis, making assumptions about contamination variability if need be, and then adapt the number of samples to the site-specific constraints.

After samples have been taken and analysed, and variability has been estimated he may appreciate the relevancy a posteriori of his strategy.

Single or multi-stage sampling ? Complementary methods of investigation

The formulation of a suitable sampling strategy is made easier by a prior knowledge of site characteristics and soil contamination. A two-or multi-stage sampling is often recommended so that sampling strategy can be refined and improved at each step. This idea shows through the assessment processes developed in Europe and North-America. In France a simplified risk assessment based on a first site inspection (initial diagnostic) normally precedes a detailed risk assessment which requires a detailed diagnostic. In Germany and in the Netherlands a distinction is made between an oriented investigation which provides a general characterisation of contamination and a further investigation which should give all necessary information for environment and human sampled volume when soil granulometry is heterogeneous and adapting sampling tachniques to the nature of soil.

Conclusion

Soil sampling of a contaminated site is an essential means of collecting qualitative and quantitative data for estimating people exposure. A cost-efficient sampling plan should be developed with respect to the historical, geological and geochemical conditions on the one hand and the current or planned land utilisation on the other hand. Guidance exists on that subject but no sampling strategy has been entirely formalized for human health risk assessment. Given all the constraints the assessor has to cope with, a few recommendations seem more appropriate than the enforcement of a rigid strategy. Fig3 illustrates the kind of procedure that could be followed to design a soil sampling strategy for human health risk assessment.

[FIG. 3] A non probabilistic sampling dictated by an initial knowledge of contamination may fit the actual situation of burden at the site but lead to an under or overestimation of concentrations. A probabilistic approach such as grid sampling ensures a complete coverage of the site and a statistical interpretation of data but it is liable to induce useless sampling. In fact both methods can be combined. The assessor should thoroughly study all available information (site and soil properties, physical characteristics of contaminants, results of previous investigations...) so as to delimit homogeneous contaminated areas and distribute the sampling points at best in each of them (e.g. using a sampling grid and adapting sampling density to the suspected level and the heterogeneity of contamination and to the precision he intends to reach). He should also take the

15 Fig. l :

 15l Fig.l : Use of soil sampling results in exposure assessment (after [1]) Fig.2a, 2b, 2c : purposive, random stratified, and combination of purposive and systematic sampling (after Pellet & al. [8])

Table 1 .

 1 Example of an old industrial site which is to be redeveloped into a residential area

	Soil	Depths to be sampled	Exposure routes
	composition
	coarse-medium	0-5 cm	Soil ingestion, dermal contact with soil and dust, inhalation of contaminated
	sand with sandy		dust, if soil is not to be paved, and inhalation of volatile compounds.
	loam soil
		5-30 cm	Soil ingestion, dermal contact and inhalation of contaminated dust (mostly
			during gardening activities and children games), ingestion of home-grown
			vegetables, if soil is not to be paved, and inhalation of volatile contaminants.
		30-200 cm	Inhalation of volatile compounds
	silt	between 200 and 400 cm	Inhalation of volatile compounds
	clay	> 400 cm	Limited risk, clay does not favour the migration of contaminants towards
			groundwater.

Table 2 .

 2 General guidelines for designing a soil sampling strategy for human health risk assessment

health risk assessment [START_REF] Bosnian | Systematische Probenahmestrategien fur die Untersuchung der Scahdstoffbelastung von Boden und Porenwasser[END_REF] [START_REF] Hortensius | Entwicklung standardisierter Probenahmestrategien fur Bodenuntersuchungen in den Niederlanden[END_REF]. A third stage {remediation investigation) is devoted to site redevelopment. In the same way risk assessment in Québec is the result of three steps : the review of all available information (stage I), a preliminary characterisation (stage II) and an exhaustive characterisation (stage III) [START_REF]Guide de caractérisation des terrains contaminés[END_REF].

As a counterpart a multi-stage sampling strategy requires time and money. To limit sampling to what is strictly needed the assessor should first look into any available information he can have at his disposal : indications about the past activities of the site and possible soil disturbance, aerial photographs, visual signs of contamination {e.g. staining of surface soil), results of less expensive field screening techniques. All those indicators ought to be integrated into a conceptual model of contamination. It must be kept in mind that screening methods may be used as a support for sampling but on no account as a substitute because they are only of a qualitative nature.

4

Sampling error, the problem of representativeness A sample should be representative i.e. reflect the properties of the soil volume from which it has been extracted. However representativeness is spoilt by sampling and preparation errors due to the heterogeneity of the sampled material and to the collection, the transport and the reduction of samples before analysis. The results of a « comparative evaluation of European methods for sampling and sample preparation of soils for inorganic analysis » [START_REF] Wagner | Objectives, concept and design of the CEEM soil project[END_REF] demonstrate that sampling and sample preparation errors can reach about the same order of magnitude as errors caused during chemical analysis. Their relative contribution to the total uncertainty in soil contamination studies (and consequently in the assessed risk) is "more a subject of speculation than of knowledge" even if assurance and control quality (ACQ) sampling, as mostly developed in North America, aids in quantifying them. For example field duplicates are a way of estimating sampling precision. On the other hand the assessor can take some precautions to lower sampling errors, e.g. by increasing the (future) use of the site into account in order to define areas and layers to be sampled as a priority.

Statistical and geostatistical tools can be helpful for constructing a sampling strategy as well as for interpreting the collected data.